MULTIPLE RENAL ANION TRANSPORT SYSTEMS: SEPARATION OF EXCRETORY AND METABOLIC FUNCTIONS Dissertation for the Degree of Ph. D. MICHIGAN STATE UNNERSITY WILLIAM ROHL stm ‘ 1977 LIFP I h v Michiga..; . ,3 Ilhfivexsflay This is to certify that the thesis entitled Multiple Renal Anion Transport Systems: Separation of Excretory and Metabolic Functions presented by William Rohl Hewitt has been accepted towards fulfillment of the requirements for Ph.D. degree in Pharmacology $52 fiéé r / Major professor Date 8-12-77 0-7 639 ABSTRACT Multiple Renal Anion Transport Systems: Separation of Excretory and Metabolic Functions BY William Rohl Hewitt The structural diversity and characteristic tubular handling of actively secreted organic anions suggested that several discrete anion transport systems exist in the kidney. This study was designed to define renal anion secretory systems, identify endogenous substrates and examine modulation of anion transport. The ability of renal proximal tubular tissue to accumulate anions in_yi£rg_is low in the newborn and matures in a characteristic pattern. A primary stimulus to development of transport is availability of substrate to be secreted. Transport can be selectively enhanced by challenging immature rabbits with exogenous substrates such as penicillin. Multiple transport systems for ogranic anions could be identified if they mature at different rates or respond differently to substrate challenge during the newborn period. [Anions were chosen to represent three proposed transport systems: I) Hippurate-system (p-aminohippurate, PAH); II) urate system (urate, UA); III) L-system (chenodeoxycholate, CDC) or to represent various structural categories of secreted anions. Anions were tentatively placed into three groups based on similar patterns of maturation. PAH, penicillin G (PEN) and phenosulfonphthalein comprised William Rohl Hewitt group I. UA, sulfisoxazole (SULFI) and acetylsalicylate (ASA) were designated group II. CDC was relegated to a third group. In theory, pretreatment with a substrate from one transport system should enhance accumulation of that anion group only. PEN pretreatment markedly enhanced maturation of group I anion accumulation (slice-to—medium (S/M) concentration ratio) without altering maturation of renal corti- cal slice UA, ASA or CDC accumulation. PEN stimulation of SULFI accumulation was minimal and probably related to accumulation by the overlapping group I system. Pretreatment of neonatal rabbits with most group II or group III anions did not alter group I (PAH) accumula- ation. ASA stimulation of PAH accumulation was probably secondary to the marked toxicity observed following ASA pretreatment. Kinetic analysis of the rate of PAH accumulation by adult kidney slices suggested that two-energy requiring modes of accumulation existed. These results supported the concept of multiple renal secretory systems for organic anions. The PAH transport system has been linked to the selective extrac- tion of nonesterified fatty acids (NEFA) from arterial blood. PAH and NEFA may compete for a common.intracellular binding site or may be handled by a common enzymatic pathway. Selective maturation of the group I transport system was used to identify interactions between PAH transport and palmitate metabolism. Penicillin treatment increased PAH accumulation by suspensions of proximal tubules and altered distribution of incorporated palmitate-IAC within tubule lipid Classes. Penicillin increased palmitate-lac esterified to triglycerides (TG) and decreased 14C recovered as NEFA. Age related changes in palmitate esterification to TG correlated with similar changes in tubule PAH William Rohl Hewitt accumulation. Administration of iodipamide (group III) had no effect on PAH accumulation and did not alter palmitate utilization. Peni- cillin treatment of mature rabbits did not alter either tubule PAH accumulation or palmitate esterification. These results suggested that palmitate and PAH share a common intracellular binding site and that penicillin enhanced PAH accumulation by removing endogenous inhibitors (NEFA). Acetate increased PAH accumulation by kidney cortex slices from several species. The PAH transport capacity of slices from various species was correlated with the endogenous renal cortical citrate concentration. Preparation and incubation of slices depleted tissue citrate concentration. Acetate increased citrate concentration and PAH accumulation by kidney cortex slices. Physiological concentra- tions of citrate increased slice PAH accumulation. These data sug- gested that citrate may act as an intracellular modulator of organic anion transport. Physiological concentrations of a-ketoglutarate or succinate increased slice PAH accumulation. a-Ketoglutarate increased PAH accumulation but had no effect on slice citrate concentration. Slices incubated without substrate were depleted of citrate but not a— ketoglutarate. Acetate significantly increased slice concentration of both a—ketoglutarate and citrate. Thus, the group I anion transport system could be modulated by several metabolic intermediates acting through similar, but separate, mechanisms. MULTIPLE RENAL ANION TRANSPORT SYSTEMS: SEPARATION OF EXCRETORY AND METABOLIC FUNCTIONS By William Rohl Hewitt A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology 1977 ACKNOWLEDGEMENTS I would like to express my sincere appreciation to Dr. J.B. Hook for his counsel, encouragement and constructive criticism during the course of this investigation. I would also like to thank Drs. TgM. Brody, J.I. Goodman and L.L. Bieber for their helpful assistance in the preparation of this thesis. My wife, Carmen Slider, deserves special mention and thanks both for her continued support and under- standing and for her skillful preparation of the figures which appear in this thesis. Mrs. June P. Mack of the Center for Electron Optics, Michigan State University prepared the electron micrographs contained in this dissertation. The assistance of Ms. Vicki Stygles, Ms. Peggy Wagner, Ms. Eileen Bostwick, Ms. Harriette Sherman, Mr. Robert Clark and Ms. Cathy Contardi is gratefully acknowledged. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS — TABLE OF CONTENTc — LIST OF TABLES _ * — LIST OF FIGURES _____ INTRODUCTION — Characteristics of the Renal Organi Anion Trans- port System Evidence Supporting the Concept of Multiple Transport Systems for Organic Anion Secre- tion in the Kidney Relationship Between Transport of PAH and Trans- Objectives port and Utilization of Endogenous Anionic Metabolic Substrates - bflflHDDS General 1) 2) 3) 4) 5) 6) 7) Experimental Animals - Determination of Slice Anion Accumulation--— Preparation of Suspensions of Isolated Proximal Tubules — Determination of Proximal Tubule PAH Accumu- lation — Determination of Proximal Tubule Palmitate Oxidation Determination of Palmitate Esterification by Suspensions of Proximal Tubules — Preparation and Incubation of Suspensions of Renal Cortical Mitochondria Use of Transport Ontogeny and Selective Substrate Stimulation as Models for Identification of Mul- tiple Renal Organic Anion Transport Systens --------- 1) Effect of Age 2) Effect of Substrate Stimulation —_ 3) Slice Anion Accumulation _ iii Page ii iii vi viii ll 24 35 36 36 36 36 37 38 39 41 43 46 46 46 47 TABLE OF CONTENTS (continued) Page Investigations on the Metabolic Medulation of p— Amdnohippurate Accumulation by Rabbit Renal Corti— cal Slices 87 1) Effect of Acetate Washout * 87 2) Onset of Acetate Stimulation 87 3) Effect of 100% N on PAH Accumulation------- 87 4) Effect of Acetate on the Rate of PAH Uptake- 95 5) Effect of Acetate on Medium pH 100 6) Effect of Acetate (10 mM) on Slice PAH Accu— mulation and Citrate Concentration - 105 7) Citrate Dose-Response Curve 105 8) Interspecies Variation in Acetate-induced Stimulation of Slice PAH Accumulation and Citrate Concentration 112 9) Effect of Potassium Concentration 114 10) Effect of Chronic Metabolic Acidosis -------- 114 11) Effect of Preincubation in Citrate on Stimu- lation of Slice PAH Accumulation by Acetate- 117 12) a-Ketoglutarate and Succinate Dose Response Curves 121 13) Effect of a—Ketoglutarate and Glucose on Slice PAH Accumulation and Citrate Concen- trations — 126 14) Effect of Acetate (1.0 mM) on Slice PAH Accu- mulation, Citrate and a-Ketoglutarate Con- centration — 126 15) Mitochondrial Accumulation of PAH 131 16) Effect of Penicillin Treatment of 2-wk-old Rabbits on the Endogenous Citrate Concentra- tion of Renal Cortex 131 Effect of Age and Substrate Stimulation on Rabbit Proximal Tubule PAH Accumulation and Palmitate Meta- bolism 131 1) Effect of Age 135 2) Effect of Penicillin Pretreatment of 2-wk- old Rabbits 147 3) Effect of Iodipamide Pretreatment of 2-week- Old Rabbits 153 4) Effect of Penicillin Pretreatment of Adult Rabbits 153 5) Effect of Penicillin Pretreatment of 2-Week- 01d Rabbits Palmitate Metabolism in an Acetate-free Medium. 160 DISCUSSION — ~ 169 SPECULATION — 192 SUMMARY — 199 BIBLIOGRAPHY — 203 Table 10 LIST OF TABLES Effect of Acetate Washout on PAH S/M Ratio- Relationship Between Acetate-Induced Stimu- lation of PAH Accumulation and Citrate Con- centration in Renal Cortex Slices from Several Species —- Effect of Citrate on PAH Accumulation by Slices of Renal Cortex Obtained from Seve— ral Species Effect of Potassium Concentration on Stimu- lation of PAH Accumulation and Citrate Con- centration in Slices of Rat Renal Cortex--- Effect of Chronic Ammonium Chloride Acidosis on the Endogenous Citrate Concentration of Rat Renal Cortex and PAH Accumulation by Slices of Rat Kidney Cortex Accumulation of p-Aminohippurate (PAH) by Mitochondria Isolated from Rabbit Kidney Cortex Accumulation of p-Aminohippurate (PAH) by Mitochondria Isolated from Rabbit Kidney Cortex Effect of Penicillin Treatment of 2-Week- Old Rabbits on the Endogenous Citrate Con- centration of Kidney Cortex Effect of Age on PAH Accumulation and Pal- ‘mitate Metabolism by Separated Rabbit Proxi- mal Tubules Effect of Penicillin Pretreatment of 2-Week- Old Rabbits on PAH Accumulation and Palmi- tate Metabolism vi Page 90 113 115 116 118 132 133 134 146 150 LIST OF TABLES (continued) Table 11 12 Page Effect of Penicillin Pretreatment of 2—Week- Old Rabbits on the Concentration of NOn- esterefied Fatty Acids in Separated Proximal Tubules and Incorporation of Palmitate-14C into Tubule Nonesterified Fatty Acids ----- 154 Effect of Penicillin Pretreatment of 2-Week- 01d Rabbits on Plasma Concentration of Non- esterified Fatty Acids 155 vii Figure 10 ll 12 LIST OF FIGURE LEGENDS Relationship between age and accumulation (S/M) ratio of PAH, PSP, and UA by rabbit renal cortical slices Relationship between age and accumulation (S/M ratio) of PAH, CDC and SULFI by rabbit renal cortical slices Effect of reduced incubation temperature (0°C) on CDC accumulation by renal cortical slices prepared from rabbits of various ages Relationship between age and accumulation (S/M ratio) of PAH, PEN, and ASA Effect of penicillin and probenecid admini- stration of PAH, PSP and UA by rabbit renal cortical slices Effect of penicillin administration on slice accumulation of PAH, PEN, and ASA Effect of penicillin administration on slice accumulation of PAH and SULFI Effect of penicillin administration and incu- bation temperature on accumulation of PAH and CDC by rabbit renal cortical slices Effect of sulfisoxazole administration on PAH accumulation and kidney weight/body weight ratio Effect of uric acid administration on slice accumulation of PAH, PSP and UA Effect of acetylsalicylic acid administra- tion on slice PAH accumulation and kidney weight/body weight ratio Effect of iodipamide administration on accu- mulation of PAH and CDC by rabbit renal cor- tical slices viii Page 61 63 66 68 70 72 75 77 79 81 83 85 LIST OF FIGURES (continued) Figure Page 13 Effect of p—aminobenzoic acid administra- tion on slice PAH accumulation and kidney weight/body weight ratios 88 14 Onset of effect of acetate on steady—state PAH concentration (Hg PAH/g tissue) -------- 91 15 Accumulation (S/M ratio) of PAH by rabbit renal cortical slices under oxygen or nitro- gen 93 16 Effect of 10.0 mM Na Acetate on the rate of the oxygen-requiring component of PAH uptake in rabbit kidney slices (15 min incubation)- 96 17 Eadie-Hofstee plot representing the effect of 10.0 mM Na Acetate on the 0 -dependent PAH uptake by rabbit kidney cortical slices- 98 18 Effect of 10.0 mM Na Acetate on final media pH and on PAH accumulation by rabbit kidney slices 101 19 Effect of 10.0 mM Na Acetate on steady state final media pH and PAH accumulation (S/M ratio) in rabbit kidney slices 103 20 Effect of 10.0 mM Acetate on slice concen- tration of citrate and PAH accumulation----- 106 21 Effect of citrate on final media pH and on PAH accumulation (SIM ratio) by rabbit kid- ney slices 108 22 Effect of medium concentration of citrate on accumulation (S/M ratio) of PAH ( , ) and final media pH ( a ) 110 23 Effect of pre-incubation with citrate on acetate—induced increase in final media pH and on accumulation (SIM ratio) of PAH.by rabbit kidney slices 119 24 Effect of medium concentration of a—ketoglu- tarate on PAH accumulation (S/M ratio) and final media pH 122 LIST OF FIGURE LEGENDS (continued) Figure 25 26 27 28 29 30 31 32 33 34 35 Page Effect of medium concentration of succinate on PAH accumulation (S/M ratio) and final media pH 124 Effect of 0.5 mM a-ketoglutarate and 10 mM glucose on slice citrate concentration and PAH accumulation 127 Effect of 1.0 mM acetate on slice citrate and a-ketoglutarate concentration and PAH accumu- lation— 129 Light micrograph of separated proximal tu- bules from adult rabbit kidneys 136 Electron micrograph of a separated proximal tubule prepared from adult rabbit kidney---- 138 Electron micrograph of separated proximal tubules prepared from adult rabbit kidney--— 140 Electron micrograph of separated proximal tubules prepared from kidney of young (4 week old) rabbits 142 Electron micrograph of separated proximal tubules prepared from rabbits treated with 90,000 I.U. procaine penicillin G (twice daily for 2 days) ~ 144 Effect of penicillin pretreatment on the ability of proximal tubule suspensions to accumulate PAH (T/M) and oxidize or esteri- ify palmitic acid 148 Incorporation of palmitate-146 into nonesteri- fied fatty acid (NEFA) and triglyceride (TG) fractions from control and treated rabbit tubule suspensions — 151 Effect of iodipamide pretreatment on PAH accumulation (TIM) and palmitate oxidation and esterification by suspensions of renal proximal tubules 156 LIST OF FIGURE LEGENDS (continued) Figure Page 36 Effect of penicillin administration to adult rabbits on PAH accumulation (T/M) and palmiO tate oxidation and esterification by suspen- sions of renal proximal tubules 158 37 Effect of penicillin pretreatment on the ability of proximal tubule suspensions to esterify palmitate in an acetate free medium 161 38 Incorporation of palmitate-1-14C into non- esterified fatty acid (NEFA) and triglyceride (TG) fractions from control and treated rabbit tubule suspensions — 163 39 Effect of acetate on palmitate esterifica- tion by tubule suSpensions prepared from young (2 week) rabbits 165 40 Effect of acetate on palmitate esterifica- tion by tubule suspensions prepared from neonatal (2 week) rabbits 167 xi INTRODUCTION Introduction of penicillin into clinical medicine stimulated the first major interest in renal organic anion secretion. Two outstanding obstacles, limited supply and rapid renal excretion, restricted the extensive or intensive clinical use of penicillin. Beyer and his colleagues began intensive studies into the mechanisms of renal anion secretion in order to devise stratagems that would modify the rate of penicillin excretion by the kidney and thereby improve the physiologi- cal economy of penicillin in man (Beyer g£_al., 1944; Beyer, 1950). These investigations rapidly led to the clinical use of p-aminohippu— rate (PAH), and subsequently to the development and use of carinamide and probenecid, as competitive inhibitors of renal penicillin excretion (Beyer SE a1,, 1944; Beyer, 1950). Although the mass production of sufficient quantitites of various penicillins eventually eliminated the need for concurrent administration of competing organic anions, Pharmacological interest in the mechanisms of renal anion secretion has been maintained. Cells of renal proximal tubules in animals and man daily secrete large amounts of anionic compounds produced by normal physiological Processes. Included among these compounds are uric acid, glucuronide conjugates of several endogenous steroids and degradation products of cerebral metabolism such as homovanillic acid and 5-hydroxyindolacetic acid. Man is also exposed to a multitude of organic acids in his diet 2 (e.g., benzoates, quinic acid, butylated hydroxyanisole, saccharin) and his environment (e.g., 2,4,5-trichlorophenoxyacetate; 2,2-bis (chlorophenyl)acetic acid) which are actively secreted by the kidney. In addition, the pharmacologic and/or toxic potential of several anionic drugs used therapeutically (e.g., penicillins, cephalosporins, methotrexate) is determined, in part, by the rate of their renal secretion into the urine. Additionally, diuretic agents such as furosemide, ethacrynic acid and several thiazides require secretion by the anion transport system to attain effective concentrations at their site of action along the luminal aspect of the ascending limb of the 100p of Henle. The activity of the renal organic anion transport system in newborn infants and children is of particular relevance. Renal excretion of drugs is less in the newborn than what would be predicted in terms of body weight. This is due, in part, to an imma- ture ability in the kidney of the newborn to transport drugs. Thus, administration of drugs to infants and children based on regimens appropriate for adults, would be expected to produce inappropriate results. The continued impetus provided by these considerations has resulted in a large fund of information pertaining to the mechanisms of renal organic anion secretion. Characteristics of the Renal Organic Anion Transport System In general, active transport in biological systems may be regarded as substrate penetration through a membrane against an electrochemical gradient, caused by a process which requires cellular metabolic energy. Interaction of the tranSported substrate with a cellular 3 receptor molecule(s) is implicit in this concept of active transport (Deepopoulos and Callahan, 1962). The interaction of substrates with finite numbers of receptor molecules confers characteristics upon active transport systems which may be used, in part, to define sub- strate movement as active. Thus, active transport systems would be expected to exhibit saturation kinetics, substrate specificity and competition of substrates for tranSport (Despopoulos, 1965; Torretti and Heiner, 1976). Renal organic anion secretion has been localized anatomically to the proximal tubule by micropuncture (Edwards and Marshall, 1924), stop flow analysis (Malvin g£_a1,, 1958) and in_gitrg perfusion of isolated nephron segments (Tune E£.§l:a 1969). Secretion of organic anions by the proximal tubule (as exemplified by PAH transport) ful- fills all of the criteria for classification as active transport. Transport of PAH across the peritubular membrane in_!i££9_proceeds uphill, against large concentration gradients (Cross and Taggart, 1950; Foulkes and'Miller, 1959; Tune 35 El-: 1969; Carrasquer and Wilczewski, 1971) and follows a kinetic pattern similar to the Mi- chaelisAMenten model of enzyme reactions (Huang and Lin, 1965; Tune 35 31,, 1969). Transport of PAH in 1132 exhibits characteristics of saturation, since as plasma PAH concentration increased a maximal PAH transport capacity (Tm) of the renal tubules is reached (Weiner, 1973). Competition between anions for transport and substrate selec- tivity have also been demonstrated. For example, high plasma con— centrations of PAH and probenecid inhibited the secretion of peni- cillin (Beyer ££.§l:s 1944; Beyer, 1950). Similarly, probenecid 4 penicillin, phenosulfonphthalein (PSP) and numerous other organic acids have been shown to inhibit PAH secretion ig_yigg_and PAH accu- mulation i3 gitgg (Weiner, 1973). Kinetic analysis of PAH inhibition by several organic anions has yielded double reciprocal Plots con— sistent with competitive inhibition (Huang and Lin, 1965; Park g£_al,, 1971). substrate selectivity has been inferred from observations that not all organic anions (e.g., p-aminobenzoic acid, p-nitrobenzylglycine) are transported by the kidney (Despopoulos, 1965; Nielsen and Rassmussen, 1975). In addition, organic cations do not alter secretion of anions, and competitive inhibitors of anion secretion do not interfere with organic cation secretion (Weiner, 1973). Oxidative metabolism has also been found to be essential for the maintenance of the tubular transport process responsible for organic anion secretion. Incubation of renal cortical slices under anaerobic conditions or at reduced temperature (0°C) markedly depressed PAH accumulation (Cross and Taggart, 1950; Farah and Rennick, 1956). Uncoupling phosphorylation from respiration by addition of dinitrophe— nol (DNP) or carbonylcyanide-m—chlorophenylhydrazone to incubation medium or by infusion of DNPpin_yizg_resulted in a marked depression of PAH transport (Maxild, 1973; Farah and Rennick, 1956; Mudge and Taggart, 1950b). Similarly, organic anion transport is inhibited by a long list of metabolic poisons including cyanide, azide, arsenite, fluoride, dehydroacetate, malonate, monofluoroacetate and oligomycin (Cross and Taggart, 1950; Shideman and Rene, 1951; Shideman g£_§l,, 1952; Farah 25 El}, 1953; Farah §£_al,, 1955; Farah and Rennick, 1956; Maxild and'Mdller, 1969; Maxild, 1973). 5 Although the organic anion transport system as a whole exhibits characteristics of active transport, one or more of its components may operate passively. Sperber (1959) suggested that the movement of an anionic substrate through a secreting cell may be logically divided into three major components: 1) uptake (passage from extracellular to intracellular fluid across the peritubular membrane), 2) accumulation and/or transcellular movement, and 3) passage from the cell to the tubular lumen. In accordance with this suggestion, Foulkes and Miller (1959) demonstrated the existence of two intracellular compartments for PAH; one formed by facilitated diffusion across the peritubular membrane, the second formed by an energy-dependent intracellular concentrating mechanism, possibly involving binding to cellular com- ponents. The two compartments were considered to be linked in series, that is, facilitated diffusion of PAH provided an intracellular sub- strate pool for the concentrating mechanism (Foulkes and Miller, 1959). These conclusions were the result of analyses of PAH flux into and efflux from rabbit renal cortical slices. The first compartment rapidly equilibrated with extracellular PAH but contained PAH in excess of that found in the interstitial Space. The amount of PAH accumulated into this compartment approximated that expected, assuming distribution of PAH throughout the intracellular water at a concentra— tion equal to that of the incubation medium. The second compartment equilibrated at a slower rate and was responsible for the high slice- t0~medium PAH ratios (Foulkes and Miller, 1959). Subsequent investi— gations (Foulkes, 1963; Farah g£_al,, 1963; Anderson ggflal., 1969; WElch and Bush, 1970) confirmed the existence of multiple compartments for intracellular PAH (and other organic anions) and extended this 6 observation to isolated proximal tubule suspensions as well (Sheikh and Mallet, 1970). However, some controversy as to the energy re- quirements of each compartment and the link (if any) between compart- ments has arisen. Sheikh and Mdller (1970) concluded that PAH was actively transported across the peritubular membrane (in contrast to facilitated diffusion) since the rapid PAH influx in their experiments reached concentrations 3—4 fold greater than the medium PAH concentra- tion. The slow phase of PAH influx (compartment 2) was thought to represent passive diffusion (binding) to subcellular components (Sheikh and Mdller, 1970). Nitrofurantoin accumulation by rabbit kidney slices also proceeds by formation of two intracellular compartments, one by passive diffusion and one by an energy—requiring mechanism (Anderson §£_§1,, 1969). However, the compartments were apparently arranged in parallel (1,3,, both had access to extracellular nitrofuran- toin) rather than existing in series as had been postulated by Foulkes and Miller (1959) and Sheikh and Heller (1970). Recently, transport of PAH into basal-lateral plasma membrane vesicles derived from rat proximal tubule cells has been demonstrated (Berner and Kinne, 1976). Vesicle PAH uptake exhibited saturation kinetics and was competitively inhibited by probenecid. PAH uptake varied inversely with medium osmolality and destruction of vesicles (heat inactivation, osmotic shock) markedly reduced PAH uptake thus demonstrating that PAH uptake primarily represented transport into an osmotically reactive intravesicular space rather than simple adsorp- tion by vesicle membranes (Berner and Kinne, 1976). Ross and his colleagues (Holohan §£_§1,, 1975; 1976) have reconstitited a solubi- lized, PAH specific binding protein(s) derived from dog kidney plasma 7 membrane proteins in phospholipid vesicles and obtained results similar to those of Berner and Kinne (1976). TranSport of PAH was saturable and was inhibited by known competitors of PAH secretion (Holohan §£“§1., 1976). Since both vesicle preparations are free of metabolism and devoid of intracellular components, PAH transport across vesicular membranes likely represents facilitated diffusion across the peritubu- lar membrane of proximal tubules (Berner and Kinne, 1976; Holohan 35 gl,, 1975; 1976). Although the possibility of active transport of PAH across the peritubular membrane cannot be eliminated on the basis of these experiments, the model of PAH transport proposed by Foulkes and Miller (1959) is strengthened. Binding of organic anions to intracellular organelles and/or cytoplasmic proteins has often been considered an integral component of the organic anion transport system. Foulkes and Miller (1959) and Farah gghal. (1963) initially postulated that the slow component of PAH diffusion observed during efflux experiments represented binding to intracellular components which constituted the PAH concentrating mechanism. Competitive inhibitors of transport were believed to act, in part, by prohibiting access of PAH to the trapping mechanism thereby increasing efflux by increasing PAH content in the freely diffusible pool (compartment 1) (Foulkes and Miller, 1959; Farah 25 31., 1963). Acceptance of this hypothesis has been hindered by the failure to demonstrate evidence of quantitatively important tissue binding of PAH (Berndt, 1976). One group of investigators have attributed this failure to use of methods inadequate for demonstration of the relatively weak tissue binding of PAH (Farah gE_§1,, 1963). However, a second group including Foulkes (1963) and Tune eg_a1, 8 (1969) have accepted the alternate hypothesis. That is, these investi- gators believe that intracellular binding is not a requisite step in the accumulation and transcellular movement of PAH. Foulkes (1963) determined that secretion of PAH into the urine occurred from an intracellular pool coextensive with total cellular content. When a known inhibitor of PAH secretion (Diodrast) was infused, no effect on the size of the PAH secretory pool was observed. If Diodrast competed for the intracellular binding sites responsible for PAH accumulation as suggested earlier (Foulkes and Miller, 1959), then Diodrast infu- sion should have decreased the PAH secretory pool. The absence of such competition was believed to contradict the concept of intracellu- alr binding of these anions (Foulkes, 1963). Tune g£_§1, (1969) studied PAH tranSport using single segments of proximal tubule dissected from a rabbit kidney. Each tubule segment was immersed in a bath Which represented the peritubular blood and perfused via micropipets with an artifical "urine" of any desired composition. Tubules actively accumulated PAH when it was added to the surrounding medium. The intracellular PAH concentration was found to be greater than that of either the surrounding medium or the luminal perfusate. The movement of PAH from the cell into the luminal fluid was therefore consistent with a process of simple diffusion (Tune gt_§1,, 1969). In addition, Tune 25 El: (1969) estimated the relative permeability of the luminal and peritubular membranes to PAH. The luminal membrane was estimated to be at least 10-fold more permeable to PAH than the peritubular membrane (Tune 5531., 1969). Tune and his colleagues also conducted experiments in which PAH was present only in the luminal perfusate. When PAH was added only to 9 the perfusate, it moved across the luminal membrane into the cell and eventually appeared in the surrounding bath. However, the intracellu- lar PAH concentration determined in these tubules was not different than the PAH concentration of the luminal perfusate. If intracellular binding of PAH occurred, the tissue concentration should have been greater than that in the perfusing fluid. Thus, Tune 25 31. (1969) concluded that PAH was not bound significantly within the tubule cell, and that the measured tissue PAH concentrations were representative of a free transport pool. In spite of the persuasive negative argument pro- vided by these data, a causative relationship between intracellular binding and kidney anion accumulation has continued to be stressed. The continued emphasis on intracellular binding is due, in part, to recent examinations of the binding of phenolsulfonphthalein (phenol red, PSP) to homogenates of renal cortical tissue (Sheikh, 1972; Eveloff 25 a1., 1976). Extensive binding of PSP to microsomal and mitochondrial fractions of rabbit kidney homogenates has been observed (Sheikh, 1972). However, only that fraction bound to microsomes was depressed by competitive inhibitors (e.g., probenecid) and thus could be considered as representing binding to a specific protein(s). Phenol red found in the cytosolic fraction was completely ultrafil- terable. Thus, cytoplasmic proteins were apparently incapable of binding phenol red (Sheikh, 1972). More detailed analyses of PSP binding to the microsomal fraction of rabbit kidney cortex demonstrated the existence of two independent papulations of binding sites (Eveloff §$p§1., 1976). Probenecid inhibited binding to only one population (the high affinity papulation) of binding sites whereas a Scatchard analysis suggested that dinitrophenol competitively inhibited PSP 10 binding to both high and low affinity sites. Dual populations of binding sites are of particular relevance to the model of PAH trans- port proposed by Foulkes and Miller (1959). Thus, it is attractive to speculate that the high affinity population of binding sites represents a carrier system for the initial facilitated diffusion step across the peritubular membrane (Eveloff g£_§l,, 1976). The near identity of association constants calculated for the high affinity PSP binding sites and for PAH uptake into basal—lateral membrane vesicles (Berner and Kinne, 1976) adds further credence to this hypothesis (Eveloff 33 $1., 1976). The low affinity population of PSP binding sites might then participate in the intracellular concentrating mechanism proposed by Foulkes and Miller (Eveloff 25.21:: 1976). However, this last speculation must be tempered with the observation that cytoplasmic proteins bind significant amounts of organic anions. Using different techniques to study anion binding, Arias and his colleagues have postulated that the cytoplasmic protein, ligandin (GSH—S-transferase B, Y-protein), serves as the major organic anion acceptor in renal proximal tubule cells (Kirsch g£_§l,, 1975; Arias g£_§l,, 1976). Several lines of evidence support the hypothesis that ligandin (and related proteins) functions as a transport carrier within the cell. The GSH—S-transferase fraction isolated from homogenates of renal cortex binds several transported organic anions including PAH and penicillin (Kirsch g£_§l,, 1975). Probenecid administration can inhibit the binding of penicillin to renal ligandin following ig_21!2 injection (Kirsch £5 31,, 1976). Administration of inducers of renal drug metabolizing enzymes increased the concentration of renal GSH—S- transferases and concomitantly increased renal binding, plasma dis— appearance and urinary excretion of organic anions (Kirsch g£_§1,, 11 1975; Arias gg_§l,, 1976; Kaplowitz and Clifton, 1976). These obser- vations appear to support the hypothesis that ligandin is a component of the renal organic anion transport system. However, Pegg and Hook (1977) were unable to demonstrate that binding of PAH to ligandin was the rate-limiting step in PAH transport. In general, the contradic- tory nature of the observations and conclusions presented by various investigators do not permit a definitive conclusion regarding the contribution of intracellular binding to the overall transport process. Evidence Supporting the Concept of Multiple Transport Systems for Organic Anion Secretion in the Kidney The structural requirements for transport by the renal organic anion secretory system have not as yet been elucidated (Rennick, 1972; Weiner, 1973). Several classes of endogenous and exogenous organic acids, including mono- and dicarboxylic acids, sulfonic acids, substi- tuted benzoic acids, ethereal sulfates and sulfonamides are actively secreted by the cells of the renal proximal tubule (Despopoulos, 1965; Weiner, 1973; Nielsen and Rasmussen, 1976). Two proposals regarding the structural requirements necessary for transport have been based on the structure of PAH, a model substrate of the anion transport system. Taggart (1958), concluded that the carboxyl moiety alone was respon— sible for substrate specificity since occlusion of the carboxyl by formation of a hydroxamate resulted in a non-transported compound. However, the carboxyl group above cannot account for substrate specifi- city since several carboxylic anions (e.g., paminobenzoic acid) are not actively transported by the system (DeSpopoulos, 1965). subse- quently, Despopoulos (1965) suggested that transport specificity 12 relied on a 3 point drug-receptor attachment involving a reinforced ionic bond between the two carboxyl oxygens and a supporting hydrogen bond formed with the carbonyl oxygen. Sulfonic acid and sulfonamide groups were considered to be equally reactive in these transport reactions (Despopoulos, 1965). This theory explained the transport of a large number of organic anions, but could not satisfactorily account for the active transport of several anions such as diodrast and phenoxyacetate (Weiner, 1973). Although additional proposals have been advanced, the structural heterogeneity of these transported anions has made it impossible to delineate the structural requirements for transport (assuming substrate interaction with a single receptor molecule). This has led to the suggestion that several different anionic transport systems with differing specificities exist within the cells of the proximal tubule (Weiner, 1973). The characteristics of tubular secretion of two well studied organic anions, uric acid (UA) and PAH are consistent with the hypo— thesis of multiple anion transport systems (Weiner and Fanelli, 1974). Uric acid is the major end product in the degradative metabolism of purines in man and higher apes (Rastegern and Their, 1972). In most other species, uric acid is of quantitatively less importance due to formation of allantoin by the uricolytic enzyme, uricase (Mudge at 31., 1973). Renal handling of uric acid includes glomerular filtra— tion, active tubular reabsorption and secretion, the relative magni— tude of each varying from one species to another (Mmdge 25 31., 1973). Both reabsorption and secretion of urate occur along the proximal tubule, with transport in either direction being carrier mediated (Nudge gt al,, 1973). Proximal tubule secretion of uric acid has 13 several characteristics in common with secretion of PAH (Weiner and Fanelli, 1975). Secretion of both anions is remarkably potassium dependent and several substances that inhibit PAH transport also inhibit urate secretion (Weiner and Fanelli, 1975). However, several investigations with various species suggested that uric acid and PAH are secreted by separate, overlapping transport mechanisms. May and Weiner (1971) demonstrated that the hyperuricemic agent, m—hydroxybenzoic acid (m—HBA), inhibited excretion of uric acid by Cebus kidney. Since m-HBA was actively secreted, competitive inhibi- tion of urate secretion was suggested as the mechanism of action (May and Weiner, 1971). These investigators also demonstrated that m-HBA was a more powerful inhibitor of uric acid secretion than PAH. These observations could have resulted from differing affinity of m—HBA and PAH for a single transport system (May and Weiner, 1971). However, m- HBA completely suppressed urate excretion at concentrations that caused only slight inhibition of PAH transport, results which were not consistent with a single secretory mechanism for PAH and uric acid. Therefore, May and Weiner (1971) postulated that two overlapping transport systems were involved, one system with high affinity for m- HBA was also the anion secretory system utilized by uric acid, whereas the second system was the major secretory system for PAH and had little affinity for mrHBA. Weiner and Tinker (1972) and Fanelli and Weiner (1973) subsequently demonstrated that pyrazinoic acid, the active metabolite of pyrazinamide, effectively blocked urate secretion by monkey (Cebus albifrons) and chimpanzee (Pan troglodytes verus) kidney at doses which did not influence the clearance or the Tm of PAH. The authors concluded that these findings supported the previous 14 suggestion that two secretory systems with different affinity for PAH and urate exist in the mammalian renal tubule. Tubular secretion of PAH and uric acid also appear to be separate in man. Boner and Steele (1973) demonstrated that infusion of suffi— cient PAH to produce plasma concentrations of 10 mg/100 ml did not significantly decrease urate secretion. Suppression of urate secretion with pyrazinamide did not inhibit tubular secretion of PAH nor did PAH inhibit the antiuricosuric effect of pyrazinamide (Boner and Steele, 1973). Meisel and Diamond (1977) demonstrated that both PAH and pyrazinamide inhibited the uricosuric response to probenecid administra- tion in man by separate mechanisms. Induction of uricosuria by pro- benecid is dependent upon secretion of probenecid into the renal tubule in concentrations sufficient to competitively inhibit the urate reabsorptive mechanism (Kippen gt al., 1974). PAH infusion signifi— cantly decreased probenecid secretion without altering urate secretion. The decreased uricosuric response to probenecid was proportionate to the reduction in probenecid excretion. In contrast, pyrazinamide had no effect on probenecid excretion but did significantly inhibit urate secretion, thus reducing the concentration of urate available for inhibition of reabsorption by probenecid (Meisel and Diamond, 1977). Evidence of separate secretory mechanisms for PAH and urate exist in lower species as well. Dantzler (1970) observed no effect of PAH infusion on urate clearance in water snakes. Subsequently, Dantzler (1974) demonstrated that high bath urate concentrations ([urate] = ZOIPAfll) had no effect on PAH.secretion by isolated perfused garter snake tubules. Although both the PAH and urate transport systems were saturable, PAH secretion reached saturation at a bath concentration 15 approximately 85% lower than the concentration needed to saturate the urate secretory system (Dantzler, 1974). Zmuda and Quebbeman (1975) demonstrated that tubular secretion of urate in chickens genetically selected for a high incidence of articular gout and hyperuricemia was approximately 40% that of nongouty controls. In contrast, secretion of PAH in gouty chickens was not different from control. Foulkes (1975) and Kramp and Lenoir (1975) demonstrated little interaction between urate and PAH tranSport at the peritubular membrane of rat kidney. Barany (1972; l973a,b; 1974a,b; 1975) postulated the existence of a group of separate, but overlapping, transport systems to explain the effects of various anions on the slice accumulation of iodipamide. One system (H-system), sensitive to iodohippurate inhibition, was assumed to be the classical hippurate transport system (Barany, 1972). The second, hippurate insensitive system (L-system), was inhibited by anions normally excreted by the liver (Barény, l9733,b). Thus, bile acids (e.g., chenodeoxycholate), cholographic agents and flavaspidic acid produced marked inhibition of iodipamide accumulation by the L- system (Bérany, 1973b; 1974b; 1975). Benzylpenicillin and PAH had little effect on iodipamide accumulation by the L-system. In addi- tion, pyrazinoic acid, mrhydroxybenzoic acid and salicylic acid also had little effect on iodipamide accumulation via the L-system, sug— gesting that the L-system was not involved in uric acid accumulation in rabbit kidney cortex (Barany, 1973b). Several straight chain, dicarboxylic TCA cycle intermediates (e.g., a-ketoglutarate, succi- nate, malate) had little or no inhibitory effect on iodipamide 16 accumulation indicating that transport of these metabolic intermediates was not mediated by the L—system.(Barany, l973b). Comparative studies of iodipamide secretion in rabbit and dog also support the concept of multiple organic anion transport systems. Berndt gt 31. (1968) used both ig_vigg_(iodipamide/inulin clearance ratio = 3.7) and in gi££g_(iodipamide S/M ratio = 32.3:2.06) techni- ques to demonstrate the existence of an active secretory system for iodipamide in rabbit kidney cortex. Iodipamide accumulation was oxygen and pH dependent, saturable and was depressed by both metabolic poisons and competitive inhibitors. In contrast, iodipamide handling by dog kidneys was not consistent with the concept of an active secre- tory system. Renal clearance ratios were not different from unity nor did stop-flow techniques demonstrate a secretory peak for iodipamide. 12,31532 accumulation of iodipamide by dog renal cortex slices was negligible (S/M = 1.21:0.06). Dinitrophenol, cyanide and iodoaceta- mide had no effect on iodipamide accumulation by dog kidney slices. In contrast, kidney slices from the same dogs accumulated PAH against a concentration gradient (PAH S/M = 3.5 without acetate; 9.8 with ‘ acetate). Thus, the lack of iodipamide accumulation by dog slices could not be ascribed to nonviability (Berndt gt al., 1968). Barany (1972, l973a) subsequently confirmed these results and concluded that dog kidney completely lacked the L-transport system for iodipamide while retaining the classical hippurate (H—system) transport system. These observations support the hypothesis that there exists within the renal proximal tubule at least 3 separate, overlapping transport systems for organic anions: 1) a system with high relative l7 affinity for hippurates; 2) a system with high relative affinity for anions like iodipamide; and, 3) a third system with high relative affinity for uric acid. The ability of rabbit renal cortical tissue to accumulate PAH in y}££g_is low in the newborn and matures in a characteristic pattern. PAH transport can be selectively stimulated by challenging newborn rabbits with large doses of substrates of the transport system (Hirsch and Hook, 1969, 1970). If multiple transport systems for organic anions exist in the kidney they might mature at different rates or respond differently to substrate challenge during the newborn period. Therefore, an analysis of the factors modulating the in_!i££g handling of several organic anions by neonatal and adult rabbit renal cortical slices was undertaken to provide evidence which would support or refute this hypothesis. Three criteria were utilized to identify transport systems and classify member substrates: 1) The pattern of maturation of intrinsic transport capacity measured as accumulation of Organic anions into renal cortical slices; 2) changes in anion accumu~ lation by renal cortical slices after pretreatment of neonatal rabbits with large doses of penicillin; and, 3) changes in PAH accumulation by renal cortical slices following pretreatment of neonatal rabbits with various anions. Accumulation of PAH by renal cortical slices was used as the standard of reference during each phase of the investigation. Evidence SupportinggMetabolic‘Modulation of PAH Secretion The net PAH secretory capacity of the organic anion transport system may be influenced by alterations in several physiological variables. An analysis of the effects produced by altering these 18 variables may serve to illuminate the mechanism(s) controlling renal PAH tranSport. For example, the profound enhancement of renal PAH secretion produced by acetate, and its precursors, lactate and pyru— vate has suggested that the PAH transport system normally operates at less than maximal capacity (Cross and Taggart, 1950; Mudge and Taggart, 1950; Schachter g£_al,, 1955; Cohen and Randall, 1964). These metabo- lic intermediates (and, in particular, acetate) have been shown to increase the intrinsic slice capacity for PAH accumulation in_3i££g_and to increase the maximum tubular transport capacity for PAH (Tm PAH) when infused in_gigg. Similarly, renal cortical slices from alkalotic rats accumulate more hippurate under normal incubation conditions (i.g., pH 7.40) than slices from nontreated controls (Weiss and Preuss, 1970). Production of acute metabolic alkalosis i§_xigg_has also been found to increase Tm PAH in dogs (Mudge and Taggart, 1950). The ability of the kidney to secrete PAH can also be reduced below normal. Weiss and Preuss (1970) observed that chronic metabolic acidosis depressed the ability of rat cortical slices to accumulate hippurate. Marked depression of in_gi££9_PAH accumulation and/or Tm PAH can also be produced by various dicarboxylic TCA—cycle intermediates (e.g., a—ketoglutarate, succinate, fumarate), fatty acids of interme- diate carbon chain length (C ) and by certain amino acids (e.g., s’clo alanine, glutamate). In addition, Hirsch (1974) found that renal organic anion transport was markedly depressed during the chronic potassium depletion produced by feeding rats a low potassium diet. Renal cortical slices from.potassium repleted rats exhibited a normal ability to accumulate PAH- 19 The relationship between these random observations and a mecha- nism(s) for control of renal PAH secretion is not well understood. Disturbances in normal acid—base balance or in Rf homeostasis are known to alter several metabolic parameters, including plasma and renal cortical concentrations of several of the aforementioned metabo- lic intermediates (Relman, 1972; Adler gt_§l,, 1974). Thus, an exami- nation of the effect(s) of one or more of these metabolites might aid in the characterization of a common mechanism(s) by which any of the variables mentioned alter PAH secretion. The two carbon fragment, acetate has been the substrate most extensively studied in relation- ship to PAH tranSport. Several mechanisms have been proposed for the acetate-induced stimulation of PAH accumulation. Cross and Taggart (1950) suggested that acetate might play a unique role in PAH transport since only acetate and certain of its precursors to a lesser degree, uniformly stimulated PAH accumulation by rabbit kidney cortex slices. Since PAH accumulation was not correlated with the respiratory stimulation obtained with several substrates, Cross and Taggart (1950) further postulated that acetate acted as one of the rate-limiting cellular components of the transport system, rather than by producing general acceleration of oxidative metabolism. An alternate mechanism arose from investigations regarding the conjugation of benzoic acid derivatives with glycine to form hippu- rates and related N—acylglycines. After demonstrating the presence of enzymes capable of synthesizing and degrading aromatic and aliphatic acylglycines in rabbit kidney cortex, Schachter gt_§l, (1955) evaluated the ability of several aliphatic acylglycines to inhibit slice 20 accumulation of PAH, an aromatic acylglycine. Acylglycines formed from fatty acids of intermediate carbon-chain length (C ) de- s'clo pressed PAH accumulation to variable degrees. The inhibition was a function of the affinity of the acylglycine for the PAH transport system and the rate of hydrolysis of the compound to a non-competitive metabolite. Although the appropriate acylglycines were formed from acetate, propionate and butyrate, the rapid hydrolysis and/or low affinity of the transport system for these compounds prevented inhi- bition of PAH accumulation. The lack of a competitive interaction between acetylglycine and PAH is the basis for the mechanism of acetate—induced stimulation of PAH secretion proposed by Schachter e£_al, (1955). Schachter e£_al, (1955) concluded that addition of exogenous acetate would yield acetyl-CoA as the most abundant acyl-CoA available to glycine acyltrans- ferase, thereby reducing the amount of inhibitory acylglycines (e.g., octanoylglycine) by competitive formation of acetylglycine. This hypothesis was strengthened when a comparative study of acetate stimula- tion demonstrated enhancement of PAH accumulation only in those species containing a glycine acyltransferase enzyme system. Interestingly, the converse relationship was not valid since PAH accumulation by rat renal cortical slice was not responsive to exogenous acetate although an acylglycine synthetic mechanism.was present (Schachter gt 31., 1955). Based on the proposed mechanism Schachter et_al, (1955) inter- Preted the acetate stimulation of PAH transport observed in in_zi££2_ and ighgigg_preparations devoid of exogenous fatty acid as indicating that PAH transport was partially inhibited at all times by acylgly- cines formed from endogenous fatty acids. 21 Although the mechanism proposed by Schachter et_al, (1955) was attractive, sufficient contradictory evidence exists to make this hypothesis unacceptable. For example, renal acylglycine synthesis was thought to be limited by cortical glycine concentration. Acetate stimulation would therefore be related to reduction of the rate- limiting component. However, Murdaugh and Elliot (1969) demonstrated that acetate stimulation of slice PAH accumulation persists in the presence of excess glycine. These results could be interpreted as an indication that the competitive site occurred earlier in the reaction sequence for acylglycine formation (1,3,, competition for coenzyme A or the acylating enzyme) (Weiner, 1973). However, addition of excess acetate to rat renal cortex homogenates did not influence acylglycine synthesis (Kim and Hook, 1972). Acetate also had no effect on acylgly- cine synthesis in experiments where glycine concentration had been made rate-limiting (Kim and Hook, 1972). During the course of this study, Kim and Hook (1972) also observed acetate-induced stimulation of PAH accumulation by rat and chicken slices although only rat tissue was capable of acylglycine synthesis. Kim and Hook (1972) concluded that there was no relationship between the effect of acetate on p- aminohippurate transport and the acetylglycine synthesis system. What, then, is the mechanism of the stimulatory effect of acetate on PAH transport? Although PAH transport is dependent on oxidative energy metabolism, neither acetate, lactate or pyruvate alter slice ATP concentration despite a pronounced stimulation of PAH accumulation (Maxild, 1973). Indeed, recent studies by Gerencser gt_§l, (1977) demonstrated a significant reduction of ATP concentration in renal cortical slices incubated with acetate. These and other (weiner, 22 1973), observations indicate a lack of correlation between the magni- tude of renal PAH accumulation and the absolute amount of ATP available in renal cortical tissue (Maxild, 1973; Weiner, 1973). Cohen and Randall (1964) have postulated that alterations in renal metabolism.produced by disturbances in acid-base balance might affect PAH transport if specific substrates (either stimulatory or inhibitory) were to accumulate or disappear from within the kidney. Similarly, Weiner (1973) suggested that metabolic inhibitors cause a shift in patterns of metabolic products resulting in accumulation of specific inhibitors of PAH transport. In this respect, organic anion transport appears to be remarkably sensitive to inhibition of TCA cycle activity. Depression of citrate (monofluoroacetate) and succi- nate (dehydroacetate, malonate, chlorguanide) oxidation markedly decreased PAH transport in zitrg_and in gigg_(8hideman and Rene, 1951; Shideman st 21., 1952; Farah gt_§l,, 1953; Farah gt al., 1955; Farah and Rennick, 1956). The effect of these metabolic poisons was speci- fic since organic cation accumulation was not altered at inhibitor concentrations that essentially eliminate PAH accumulation. In addi— tion, dehydroacetate has also been shown to selectively inhibit PAH secretion without altering the energy dependent reabsorption of glucose or phosphate (Seevers £3 31., 1950). As might be expected, acetate protects against inhibition of PAH transport produced by these inhi- bitors (Shideman 95 al,, 1952; Farah gt 21., 1953). The specific inhibition of PAH transport resulting from decreased TCA cycle activity and acetate stimulation of PAH secretion can be interpreted in several ways, each consistent with the hypotheses of 23 Cohen and Randall (1964) and Weiner (1973). However, one hypothesis, capable of accounting for both stimulation and inhibition of PAH tranSport, deserves particular attention. If a modulator (with concen- tration dependent stimulatory and inhibitory properties) was maintained at a low, substimulatory concentration by normal TCA cycle activity, acetate could stimulate PAH transport by increasing modulator concen- tration. Alternatively, TCA cycle inhibition at a point subsequent to modulator synthesis could elevate the concentration into the inhibitory range and thus specifically inhibit PAH transport. Inhibition might also occur if sufficient acetate was metabolized to elevate modulator concentration into the inhibitory range. This model for metabolic regulation of PAH transport was chosen for further study for several reasons. Cross and Taggart (1950) demonstrated that several TCA cycle intermediates markedly inhibited PAH accumulation when present at high concentration in the incubation medium. In contrast, low concentra- tions of these same intermediates significantly stimulated PAH accumu- lation (Cross and Taggart, 1950; Farah gt_§l,, 1955). Finally, incubation of rat renal cortical slices with acetate (10 mM) at 37°C, or incubation of dog kidney slices with acetate (100 mM) at 25°C resulted in a small but significant depression of PAH accumulation (Koishi, 1959; Despopoulos, 1956). Teleologically, it seems unlikely that the primary function of an elaborate metabolic regulatory mechanism such as that proposed above would exist merely to control the excretion rates of exogenous organic acids (Forster, 1967). Systems of this complexity, if they do indeed exist, seem better adapted to serve as a mechanism that may control 24 delivery rates or maintain intracellular levels of oxidative interme— diates or metabolites (Forster, 1967). Relationship Between Transport of PAH and Transport and Utilization of Endogenous Anionic Metabolic Substrates The kidney is capable of considerable substrate specificity in that it selectively extracts specific metabolic substrates from arte- rial blood (Cohen and Barac-Nieto, 1973). The substrates present in normal arterial blood which the kidney extracts and utilizes at signi- ficant rates iphzigp are: free fatty acids (in particular, palmitate), lactate, glutamine, glucose and citrate (Cohen and Kamm, 1976). In addition, the kidney extracts several substrates, including inositol, pyruvate, a-ketoglutarate, fructose glycerol, acetoacetate and B-OH butyrate, which are present at such low concentration in arterial blood that they could not make a substantial contribution to the energy requirements of the kidney even if they were completely extracted and oxidized (Cohen and Kamm, 1976). That several of these metabolic substrates are organic anions suggested the possibility that the organic anion transport system serves as a mechanism to extract anionic metabolites from arterial blood and deliver them to sites of intracellular dissimilation (Barac- Nieto and Cohen, 1973). Indeed, the literature contains experimental evidence suggesting that lactate, citrate, a-ketoglutarate and palmi- tate enter the proximal tubule cell from the peritubular blood by a mechanism(s) other than simple passive diffusion. Dies g5 31. (1969) demonstrated that lactate was filtered at the Slomerulus and subsequently was completely reabsorbed (<1% of the 25 filtered load was excreted) along the proximal tubule. The lactate reabsorptive process operated against an electrochemical gradient and exhibited a maximal rate, characteristics consistent with transport by a saturable, energy-requiring mechanism (Dies e£_§1,, 1969). Barac- Nieto and Cohen (1973) recalculated data from experiments performed by Dies g£_§l, (1970) and concluded that renal uptake of lactate exceeded the filtered load when GFR was reduced by partial uretral occlusion. These results suggested that some lactate must also be obtained from the peritubular capillary aspects of the cell (Barac-Nieto and Cohen, 1973). Subsequently Dies gE_al, (1974) observed instances of net renal lactate uptake exceeding total lactate reabsorption when dogs were in metabolic or respiratory alkalosis and during glycine infusion. These investigators calculated an equilibrium potential of —39.5 mV for lactate or approximately 50% of the known electrical gradient (-70 mV) between renal cortical tubular cells and extracellular fluid. Therefore, Dies and co-workers (1974) concluded that peritubular entry of lactate was not mediated by a simple diffusion process. However, probenecid infusion had no effect on the renal uptake of lactate, thus, obscuring any direct relationship between lactate uptake and the PAH transport mechanism (Dies epflal., 1974). In the dog, uptake and utilization of a-ketoglutarate (a-KG) and citrate is limitied to the liver and kidney (Martensson, 1940; Selleck and Cohen, 1965). This organ specificity parallels that of PAH secretion and led Selleck and Cohen (1965) to postulate that the major function of the PAH transport system was to deliver these and other organic acids into the intracellular fluid. Although filtration and reabsorption appear to be the primary mechanisms for tubular handling 26 of these acids, evidence for peritubular entry of citrate and a- ketOglutarate exist. Net renal uptake of citrate was proportional to the arterial plasma concentration (Herndon and Freeman, 1958; Baruch gp”§1., 1973, 1975). If the plasma citrate concentration was elevated without altering extracellular fluid pH, the renal citrate uptake rate exceeded the rate at which citrate was filtered, indicating that a considerable fraction of citrate entered renal tubule cells across the peritubular membrane, probably by the organic acid transport system (Baruch 35 al., 1973, 1975; Cohen and Kamm, 1976). Baruch e£_al, (1973, 1975) estimated that peritubular citrate transport was approximately 13% of the peritubular load at endogenous concentrations but only 6% of the peritubular load at high plasma citrate levels. Net renal citrate uptake increased seven-fold at high plasma citrate concentration and the fraction of citrate entering the kidney by peritubular transport did not vary with plasma citrate concentration. Baruch g£_al, (1973) concluded that the limited rate of citrate uptake at high plasma concentrations could be due to approaching saturation of membrane transport systems for citrate. Net secretion of u-KG by dog kidney has been observed during citrate infusion and following induction of metabolic alkalosis (Balagura and Stone, 1967). Cohen and Wittman (1963) reported that probenecid administration inhibited net utilization of u-KG by the dog kidney ip_gi!p. Therefore, these authors postulated that a-KG entered proximal tubule cells by the PAH transport system and that the rate of a-KG utilization was limited by the rate of a—KG transport 27 (Cohen and Wittman, 1963). However, Cohen 35 E1, (1969) later demon— strated that u-KG was not accumulated against a concentration gradient (S/M ratio = 0.65) by dog kidney slices. Although probenecid inhi- bited o-KG utilizationby dog kidney slices, no significant decrease in the o—KG S/M ratio was produced. Thus, probenecid did not inhibit o-KG utilization by depressing entry of a-KG into the slice but apparent- ly altered a-KG metabolism at some intracellular locus (Cohen ggflgl., 1969). In contrast, Pakarinen and Runeberg (1969) demonstrated that guinea pig kidney cortical slices accumulated a-KC from the incubation medium against a concentration gradient. Accumulation of a-KC was inhibited by decreased temperature (0°C) and probenecid. These results were consistent with accumulation of a-KG by the PAH transport system. In addition o-KG can depress the activity of the organic acid transport system. Balagura-Baruch and Stone (1969) demonstrated that infusion of u-KG depressed PAH Tm to 50% of control, results which were similar to those of Knoefel and Huang (1959). Cross and Taggart (1950) and Koishi (1959) demonstrated that a—KG inhibited uptake of PAH by renal cortical slices. Thus, both the iprigg_and 12.33522 observations are consistent with an interaction (possibly competitive) between peritubular transport of PAH and a-KG. Nonesterified fatty acids (NEFA) are transported in plasma as tightly-bound, noncovalent complexes with albumin and lipoproteins. At normal physiological concentrations only 1-6% of plasma NEFA remains unbound, presumably in the form of acyl carboxylate anions (Nikkila, 1971; Spector, 1971). Due to the tight binding of NEFA to plasma proteins, virtually none of the plasma NEFA is filtered at the 28 glomerulus (Cohen and Kamm, 1976). Therefore, all of the NEFA uti- lized by the kidney must enter the renal tubule cell across the peri- tubular cell membrane (Cohen and Kamm, 1976). The mechanism involved in the transfer of NEFA into the renal tubule has not as yet been elucidated. However, the preferential extraction of palmitate by the kidney over other long-chain NEFA suggested that palmitate penetrates the cells by a mechanism other than by simple diffusion (Pakarinen, 1970). Several investigators have provided evidence to suggest that palmitate is transported into proximal tubule cells by a mechanism similar to that for PAH. Cross and Taggart (1950) demonstrated that addition of medium chain fatty acids (e.g., octanoate) inhibited accumulation of PAH by rabbit renal cortical slices. Barac-Nieto and Cohen (1968) demonstrated that the lflzxixg uptake of NEFA by dog kidney could be blocked by substrates of the PAH transport system. Probenecid produced a reversible blockade of net renal NEFA uptake while chlorothiazide inhibited renal NEFA uptake irreversibly (Barac-Nieto and Cohen, 1968). Similarily, Dies g$_§l, (1970) found that infusion of furosemide depressed the uptake of NEFA by dog kidney. Cohen (1964) also demonstrated that a-KG, which inhibits PAH transport _i_n_ _wl_tr_g_ (Cross and Taggart, 1950; Koishi, 1959) and ip_!igg_(Balagura-Baruch and Stone, 1968) depressed net uptake of NEFA by dog kidney. Interpretation of these investiga- tions was hampered by possible intracellular effects of probenecid, chlorothiazide and furosemide. However, Barac-Nieto and Cohen (1968) concluded that NEFA entry into renal tubular cells occurred via a discrete transport system inhibitable by substrates of the PAH trans- POrt system or that substrates of the PAH system inhibited the 29 intracellular utilization of NEFA. Barac—Nieto (1971) subsequently demonstrated that palmitate competitively inhibited PAH accumulation by rat renal cortical slices. In addition, PAH accumulation by rabbit cortical slices was depressed when intracellular NEFA concentration was increased following blockade of NEFA oxidation by (+)-pa1mitylcarni- tine or 2-bromopalmitate (Maxild, 1971). These results supported the earlier suggestion that the PAH transport system participates in the uptake of palmitate by the renal proximal tubule (Barac-Nieto, 1971). However, several investigators have presented what appear to be conflicting data. Pakarinen (1970) investigated the effects of pro— benecid, PAH and PSP on the uptake and oxidation of palmitate by guinea pig kidney cortical slices. Low concentrations of probenecid (3-6 mM) inhibited slice oxygen consumption without affecting the uptake and oxidation of exogenous palmitate. Increased probenecid concentration (12 mM) decreased oxygen consumption further and inhi— bited palmitate uptake and oxidation. Addition of PAH (1-9 mM) stimulated palmitate oxidation by cortical slices whereas added PSP (0.4-3.6 mM) had no significant effect on palmitate oxidation or cellular respiration although 3.6 mM PSP produced a slight, but sig- nificant decrease in palmitate uptake. Pakarinen (1970) concluded that probenecid inhibited the intracellular metabolism of palmitate, not its entry into renal cells. Heineman 35 31. (1975) supported these observations, demonstrating that probenecid limited the oxidation and esterification of palmitate without altering palmitate uptake by rat renal cortical slices. Various experimental maneuvers that depress slice PAH accumulation (i,§,, addition of ethacrynate, merallu- ride, ouabain, oligomycin and reduction of medium sodium.concentration) 30 routinely altered the degree of oxidation and/or esterification of palmitate without altering total slice palmitate uptake (Heinemann 33 3;. , 1975) . Barac-Nieto (1971) demonstrated that inclusion of carnitine in incubation medium stimulated PAH accumulation by rat kidney cortex slices. Carnitine enhancement of PAH accumulation was eliminated when exogenous palmitate was added, suggesting that enhanced PAH accumulation was secondary to reduction of intracellular NEFA concentration (Barac- Nieto, 1971). Based on these results Cohen and Kamm (1976) concluded that a component(s) of the renal organic acid (PAH) transport mecha- nism participated either in the cellular entry or intracellular binding of NEFA, resulting in delivery of NEFA to their intracellular sites of dissimilation (rather than secretion into the urine). Development and Substrate Stimulation of Renal Organic Anion Transport The kidneys of newborn humans and animals are markedly undeveloped in their ability to excrete foreign organic anions such as penicillin (Barnett §£_§l,, 1949) and phenolsulfonphthalein (Williamson and Hiatt, 1947). Undoubtedly the anatomic and hemodynamic immaturity of the neonatal kidney is at least partially responsible for this pheno- menon. However, the reduced extraction of organic anions by the kidney suggests that the transport capacity (e.g., secretion) of proximal tubule cells is immature as well (Sereni and Principi, 1968). The renal clearance and extraction of PAH by the newborn has been reported to be low in human subjects (Calcagno and Rubin, 1963), Puppies (Horster and Valtin, 1971; Hook gpq§1., 1970), rats (Horster and Lewy, 1970), rabbits (Lewy and Grosser, 1974) and sheep (Phelps pp .El-: 1976). In addition, slices of renal cortex from newborn animals 31 of several species exhibited a reduced transport capacity for PAH when compared to adult or fetal tissue (New 35 al., 1959; Rennick g£_§l., 1961; McIsaac, 1965; Hirsch and Hook, 1969a, 1970b). Maturation of tissue transport capacity proceeds in a characteristic pattern. Accumulation of PAH by renal tissue increases from birth to two weeks of age. Accumulation then increases rapidly to a peak at four weeks and declines to an intermediate value (Hook, 1974). Since kidney weight develops more linearly, these data suggest that increased accumulation of PAH represents more than simple organ growth and development. That is, there appears to be specific maturation of the transport system. A question arises as to the triggering stimuli responsible for initiating the maturation of PAH transport capacity. The decreased transport capacity observed in renal tissue from newborn as compared to fetal rabbits could be interpreted as an indication that the func- tional load presented to the kidney had increased (Hirsch and Hook, . 1970b). At birth, the sudden change in supply of energy yielding substrates from maternal blood glucose to a milk diet high in lipid (including medium and long chain NEFA) would increase the circulating concentration of organic anion substrates (Smith and Abraham, 1975; Aprille and Rulfs, 1976; Aprille, 1976). In addition, renal vascular resistance falls and renal blood flow increases during the immediate newborn period (Edelmann and Spitzer, 1969). The combination of these- factors would serve, presumably, to increase the functional load 1mPosed upon the transport system (Hook, 1974). The functional capacity of many developing enzyme systems increase in response to an increased functional load (Yaffe and Juchau, 1974). 32 Similarly, when Hirsch and Hook (l969a,b; 1970a,b) treated newborn rabbits prior to maturation with large exogenous loads of penicillin, the ability of renal tissue to accumulate PAH was more than doubled. However, upon reaching maturation (1,3,, four weeks), exogenous substrate loads produced no effect (Hirsch and Hook, 1969a). Sub- strate stimulation of PAH transport has been produced with a variety of secreted organic anions. In addition to penicillin, transport was stimulated with PAH and triiodothyronine (Hirsch and Hook, 1969b,c; 1970a). This phenomenon has been observed in species other than rabbit, including rat and dog (Hirsch and Hook, 1970; Johnson ES 31., 1974). Substrate stimulation also altered the excretion of drugs in the intact animal. Lewy and Grosser (1974) used modified micropuncture techniques to document the low renal clearance and extraction of PAH by newborn rabbits. Following penicillin treatment, PAH extraction was significantly increased. Noordewier and Withrow (1976) demon- strated that the low clearance and transport maximum of PAH in newborn rats was increased after penicillin. Extraction of PAH across the newborn dog kidney was also increased by substrate stimulation (Bond EE_§1,, 1976). A recent report of Schwartz 3; pl. (1976) sug- gested that the phenomenon of substrate stimulation of anion transport occurs in human neonates. These investigators were treating a human neonate with oxacillin and phenobarbital. Subsequently, they were unable to obtain therapeutic concentrations of dicloxacillin even with supratherapeutic doses. Intestinal absorption appeared to be high, but plasma levels were low and were correlated with a high urinary excretion rate. Therefore, these investigators postulated that the renal tubular transport of dicloxacillin was stimulated by the admini- Stration of oxacillin (and/or phenobarbital). 33 The enhanced PAH transport capacity following administration of penicillin occurred without any change in organic cation transport (Hirsch and Hook, 1970) and did not result from an alteration in the morphologic (Hirsch g£_§1,, 1971) or ultrastructural (Pegg ggugl., 1976), characteristics of the proximal tubule cell. Although substrate stimulation enhanced the ability of renal tissue to accumulate PAH, it did not alter the oxygen consumption of proximal tubules (Ecker and Hook, 1974b). In addition, penicillin and acetate stimulated PAH accumulation via separate mechanisms, since the acetate-induced stimula- tion of PAH accumulation was not blunted in renal tissue from treated neonates (Hirsch and Hook, 1970b; Pegg and Hook, 1975b). The degree of stimulation produced by penicillin pretreatment was found to be dose—dependent (Pegg and Hook, 1975b). Maximal stimula- tion of PAH transport capacity was observed 24 hours after the last of four injections of 90,000 I.U. of procaine penicillin C were admini- stered to young rabbits at 12 hour intervals (Pegg and Hook, 1975b). Penicillin excretion was essentially completed during the intervening 24 hour period (Pegg and Hook, 1975b). Therefore, the enhanced PAH transport capacity could not be attributed to a mechanism (e.g., counter-transport) dependent upon the immediate presence of penicillin in kidney tissue during estimation of PAH accumulation. Indeed, when penicillin was added directly to incubation beakers containing PAH and renal cortical slices, PAH accumulation was markedly depressed (Hirsch and Hook, 1970b). Interestingly, if estimation of transport capacity was delayed more than 36 hours after the final penicillin injection, no stimulation of PAH accumulation could be detected (Hirsch and Hook, 1970b; Pegg and Hook, 1975b). The transitory nature of this effect Suggested that the proximal tubule cells responded to the increased 34 functional load produced through penicillin injection by increasing the number of specific transport proteins available for anion trans— port (Hirsch and Hook, 1970c; Pegg and Hook, 1975b). This hypothesis appeared reasonable since both normal (Ecker and Hook, 1974a) and penicillin-induced (Pegg §£_§1,, 1976) maturation of transport capacity were associated with an increase in the apparent maximum velocity (vmax) for PAH accumulation. In addition, Hirsch and Hook (1970c) and Pegg and Hook (1975b) demonstrated that an intact protein synthetic mechanism was required for substrate stimulation since concomitant administration of cycloheximide prevented penicillin enhancement of PAH transport capacity. The effect of penicillin administration on renal protein biochemistry was further analyzed by Hirsch and Hook (1970c). Substrate stimulation was associated with increased uptake of 14C-L-leucine and 14C-L—glutamine into the trichloroacetic acid insoluble fractions of rat renal cortical homogenates. However, no effect was observed on amino acid uptake into the corresponding frac- tion from rat medulla. In addition, a non-specific stimulus (chronic NH4C1 acidosis) of renal protein synthesis increased labelled amino acid uptake but had no stimulating effect on PAH transport (Hirsch and Hook, 1970c). Therefore, the effect of penicillin was probably not the result of nonspecific increases in protein content. From these observations, Hirsch and Hook (1970c) concluded that the stimulatory effect of penicillin on PAH transport was the result of increased Synthesis of specific transport proteins. 35 Objectives The unifying purpose of this investigation was to further define the specific biochemical sequences and the physiological functions involved in the translocation of organic anions across the proximal tubule cell. Three primary objectives were identified: 1) to demon- strate the existence of multiple transport systems for the secretion of organic acids within the kidney; 2) to provide additional evidence that palmitate is an endogenous substrate of the PAH transport system; 3) to provide evidence that an intracellular modulator of the PAH transport system exists and to elucidate the role of this modulator in acetate-induced stimulation of PAH accumulation by renal cortical slices. The maturing kidney was used as an experimental model during several segments of this investigation because of its selective physiological response to exogenously administered functional loads. METHODS GENERAL 1) Experimental Animals Litters of New Zealand white rabbits were bred and raised in departmental animal quarters or purchased from a local breeder. Young animals remained with their mothers during treatment or until the time of experimentation. Rabbits used for determination of developmental patterns of anion transport and metabolism were weaned at 4 weeks of age. Adult female New Zealand white rabbits (2.5—4.0 kg), male Sprague-Dawley rats (150-250 gm) and mongrel dogs of either sex were purchased from local suppliers and used after a suitable period of acclimation. Male bullfrogs (Rana Catesbiana, 500-750 gm) were pur- chased from Southwestern Scientific Co., Tucson, Arizona. 2) Determination of Slice Anion Accumulation Animals were killed by a blow to the head, the kidneys removed and placed in ice-cold saline (0.13 M NaCl - 0.02 M KCl). Thin renal cortical slices were prepared freehand and incubated in 3.0 ml of the phosphate buffered medium1 described by Cross and Taggart (1950) which contained the anion under study. Metabolic substrates (333,, acetate, lactate, citrate, o-ketoglutarate, succinate l 97 mM NaCl, 40 mM KCl, 0.7 mM CaClZ, 10 mM NaH2 4 2 4 36 37 glucose) were added to the media as sodium salts and equimolar amounts of NaCl were added to control media. Incubations were performed in duplicate (150—200 mg slices/beaker) in a Dubnoff metabolic shaking incubator at 25°C under an atmosphere of 100% 02. Duration of incu- bation varied with the study undertaken. After incubation slices were removed from the medium, blotted and weighed. Tissue and a 2.0 ml aliquot of medium were extracted in 3.0 ml of 10% trichloroacetic acid (TCA). Water was added to a final volume of 10.0 ml and the samples were centrifuged. Aliquots of tissue and medium extracts were taken for assay and results expressed as slice-to-medium (SIM) ratios where S = mg (DPM) of anion/gm wet weight of tissue and M = mg (DPM of anion/ml of medium. 3) Preparation of Suspensions of Isolated Proximal Tubules The technique of preparing separated renal proximal tubules was similar to the method of Brendel and Meezan (1975) and Meezan pp El, (1973) with modifications drawn from the work of Burg and Orloff (1962) and Huang and Lin (1965). Rabbits were stunned by a blow to the head and rapidly exsanguinated. In young (2 week) rabbits, the abdominal aorta was clamped superior to the branching of the renal arteries. A cannula (blunted, 20 ga. stainless steel needle) was inserted inferior to the kidneys, both renal branches of the vena cava slit, and the kidneys perfused simultaneously with chilled, oxygenated, Ringer's solution2 until clear of blood. The infusion was continued with a suspension of magnetic iron oxide (M10, S‘mg/ml) in 2120 mM NaCl, 16.2 mM KCl, 1.2 mMLMgSO4, 1.0 mM CaClz, 10 mM NaAcetate, 10 mM KHZPOA-NaZHPO4, pH 7.40. 38 Ringer's solution until the kidneys were a uniform gray-black in color (25-30 mls were usually sufficient). The kidneys were removed and the cortex rapidly dissected and finely minced in 40-50 ml (=1 gram cortex/5 m1 Ringer's) cold Ringer's solution. The cortical mince was gently homogenized with 6-8 vertical strokes in a hand held Potter- Elvehjem homogenizer with teflon pestle. The resulting homogenate was poured over a 253 u nylon sieve and thoroughly washed with a large volume of chilled, oxygenated Ringer's solution (l-l.5 liters). The resulting filtrate was poured through an 83 u nylon sieve and washed with buffer. The material remaining on this sieve (tubules and glome— ruli) was suspended in Ringer's solution and placed on t0p of a power— ful, permanent magnet. The iron-containing glomeruli were attracted to the magnet allowing the supernatant, containing the proximal tubules, to be decanted. This procedure was repeated three times to ensure removal of iron-containing glomeruli. The tubule suspension was centrifuged at room temperature for 60 seconds at 1000 rpm and the supernatant decanted. The pellet was resuspended and washed three times with 50 ml of oxygenated 5% calf serueringer's solution. The tubules were again pelleted and resuspended in the appropriate incu— bation medium at a concentration of 90-120 mg tubule/m1 medium. The procedure was similar when proximal tubules from older rabbits (4 week-adult) were prepared except that each renal artery was cannulated and the kidneys perfused separately. 4) Determination of Proximal Tubule PAH Accumulation Proximal tubules were suspended (=lOO mg tubule/ml) in Ringer's medium containing 10 mg/ml defatted (Chen, 1967) bovine serum 39 albumin (BSA) and 10 mM Na acetate. Six to 8 ml samples of the tubule suspension were added to 25 ml Erlenmeyer flasks in a New Brunswick Scientific Gyrotory Shaker and preincubated for 15 minutes at 37°C under a gas phase of 100% oxygen. Following preincubation, PAH was added to attain a final medium concentration of 7.4x10-5M and incuba- tion continued for a further 30 minutes. Two samples (3-4 ml/sample) were taken at the end of incubation and rapidly centrifuged for 10 minutes, 10,000 x g at 0°C in the special centrifuge tubes designed by Burg and Orloff (1962). Inulin was added to a concentration of 0.09% prior to centrifugation to estimate trapped medium in the tubule pellets. Tissue water content was determined by drying the pellets to a constant weight. Pellets were homogenized in 5.0 m1 of 3% trichloro- acetic acid and precipitated protein removed by centrifugation. Duplicate 2 ml aliquots of the medium were deproteinized with 3.0 ml of 10% trichloroacetic acid, centrifuged and brought to a final volume of 10.0 ml with water. Aliquots of the trichloroacetic acid extracts of tubule pellets and media samples were taken for assay of PAH (Smith 55;§1,, 1945) and inulin (Schreiner, 1950). PAH accumulation was expressed as the tubule—to-medium (T/M) ratio where T equals milli- grams of PAH per milliliter of intracellular water and M equals milligrams of PAH per milliliter of medium. 5) Determination of Proximal Tubule Palmitate Oxidation Proximal tubules were suspended in Ringer's medium containing 10 mg/ml defatted (Chen, 1967) bovine serum albumin and 10 mM Na acetate. An aliquot of the tubule suspension was transferred to 25 ml erlenmeyer flask and pre-incubated for 15 minutes at 37°C under a gas phase of 100% oxygen. Following pre—incubation, the tubules were pelleted by centrifugation (1,000 rpm, 1 minute, room temperature) and 40 resuspended in an equivalent volume of temperature and gas equili- brated Ringers medium containing 1 mM palmitate-1-14C (0.4 uCi/umole) complexed to 10 mg/ml defatted bovine serum albumin ([NEFA]/[BSA] = 6) and 10 mM Na acetate. The tubule suspension was transferred to a 25 ml erlenmeyer flask (Kontes Glass Co. #K-882300), gassed for 1 minute with 100% 02 and the flask sealed with a serum rubber stopper (Kontes Glass Co. #K882310) to which an empty plastic cup for CO2 collection was attached (Kontes Glass Co. #K882320). The tubules were incubated for a further 30 minutes. Incubation was terminated by addition of 0.2N H2804 (0.2 ml/ml suSpension) to the incubation mixture (Heinemann gp_sl,, 1975). Liberated CO2 was trapped in 0.4 ml of l M hyamine hydroxide (New England Nuclear Co.). Hyamine was added with a syringe and needle to the plastic cup suspended from the stopper of each flask, immediately after the addition of H2S04. Incubation was continued for an additional hour at 37°C with constant shaking to achieve complete trapping of C02 in the hyamine hydroxide. The cup containing hyamine carbonate was then transferred to a scin- tillation vial containing 10 ml of Omniflour (New England Nuclear Co.) in toluene. The radioactivity in 14CO2 was determined by liquid scin- tillation spectrometry using a Beckman LS—lOO liquid scintillation counter. Counting efficiency was determined by internal standardiza— tion with l4C-toluene. Aliquots of the incubation medium containing palmitate-l-14C, equivalent to the volume of the tubule suSpension were handled in a similar fashion in each experiment to correct for nonspecific evolution of 14C02. Liberation of 1('C02 by medium blanks represented less than 1% of the 14C02 produced by tubule suspensions in each experiment. 41 6) Determination of Palmitate Esterification by Suspensions of Proximal Tubules Incubation of proximal tubules was essentially the same as that outlined for the palmitate oxidation studies. Incubation was terminated by centrifugation (3,500 rpm, 1 minute, room temperature). The medium supernatant was decanted and the tubule pellet suspended in 2.0 ml of 0.2N H2504. attached to the tissue pellet was removed by resuspending the pellet Following centrifugation palmitate loosely in 10.0 ml cold Ringer's medium containing 10 mg/ml defatted BSA (Heinemann g£_§l,, 1975). The suspension was centrifuged and the pellet weighed. All calculations were based on the wet weight of this pellet. Esterification of palmitate was established by homogenization and extraction of the tissue pellet according to the method of Folch sfihgl. (1957). Homogenization was performed on ice with 2:1 CHC13: CH30H (20:1 volume:pe11et wet weight) and left overnight for extraction. Insoluble material was pelleted by centrifugation following addition of 0.2 volumes CHBOH. The resulting supernatant was returned to the correct solvent ratio and the extract partitioned with 0.2 volumes of 0.05 M KCl and washed with equilibrated upper phase to remove water soluble products (Heinemann et al., 1975; Barac-Nieto, 1976; Folch pp 21., 1957). The lower phase was taken to dryness under 100% N2, a known volume of CH013:CH30H (2:1) added and the sample stored at —20°C until analyzed. An aliquot of the pooled upper phase and washings was added to scintillation vials containing 10 ml of Omniflour in toluene for determination of l['C-radioactivity in water soluble compounds. Insoluble material contained in the original homogenate was solubilized in a known volume of SHT (8% sodium hydroxide, 1% Triton X405, 3% 42 methanol) by heating at 80°C for 2 hrs with continuous shaking (J. Dent, personal communication). An aliquot was neutralized with 4.4 M nitric acid and added to scintillation vials containing 15 m1 of Aquasol (New England Nuclear Co.). Duplicate aliquots of the lower phase of the Folch extract were added to scintillation vials con- taining 10 m1 of Omniflour in toluene for determination of the 14C- radioactivity present in the total tissue lipids (TL). Individual lipid classes were separated by thin layer chromatography on Silica Gel C, using as solvent, n-hexane, diethylether, glacial acetic acid (80:20:l, v/v) (Blank and Snyder, 1975). Duplicate aliquots of the lower phase were spotted on heat activated (30 min, 110—120°C), 250 u Silica Gel G TLC plates (Analtech Inc., Wilmington, Del.) and developed in closed glass chambers lined with filter paper. Component spots were identified by comparison with known standard mixtures (TLC Mix A and C, Supelco Inc.; Mixture TLC-8, Applied Science Laboratories, Inc.) included on each plate. Spots were visualized with iodine vapor, circumscribed, and when the iodine had disappeared, scraped and transferred to scintillation vials. Carbon—l4 activity was determined by suspending the scrapings in 4% Cab-O-Sil (Eastman Kodak Co.) in Omniflour-toluene. Five fractions were obtained (in descending order from the solvent front): 1) cholesterol esters (CHE), 2) trigly— cerides (TC), 3) nonesterified fatty acids (NEFA), 4) cholesterol (CH; di- and mono-glycerides migrated with this fraction), and 5) phospho— lipids (PL). Carbon—l4 activity was determined by liquid scintilla- tion spectrometry using a Beckman LS-lOO liquid scintillation counter. Counting efficiency was determined by internal standardization with 4 C-toluene. Carbon-l4 activity in each fraction was converted to 43 palmitate equivalents by comparing it with the specific activity of the palmitate initially placed in the incubation medium. Results were expressed as umoles of palmitate incorporated per gram wet weight of tubule. The NEFA concentration (Ho, 1970) and the concentration of radioactivity in the incubation medium before and after incubation with proximal tubule suspensions was determined in several experiments to verify constancy of medium NEFA specific activity (Barac-Nieto,. 1976). The ratio of initial-to-final medium NEFA specific activities approximated 1 in each case, suggesting that medium NEFA concentration was not diluted by endogenous tubule NEFA. These results were similar to those of Barac-Nieto (1976). In certain experiments, total nonesterified fatty acids present in the lower phase of the Folch extract was determined by a modification of the method of Ho (1970; Bieber gp_sl,, 1975). The plasma NEFA concentration was also determined in several experiments. 7) Preparation and Incubation of Suspensions of Renal Cortical Mitochondria Adult, female New Zeland white rabbits were stunned by a blow to the head, exsanguinated and the kidneys removed and rinsed in an ice cold saline (0.13 N NaCl—0.02 N KCl). The cortex was removed by scissor dissection, minced and homogenized (1.0 gm cortex/5.0 ml solution) in 0.3 M sucrose, 5 mM Hepes3 , 1 mM EDTA, pH.7.4 with six strokes of a motorized (600 rpm) Potter-Elvehjem homogenizer with Teflon pestle (Simpson and Angielski, 1973; Adam.and Simpson. 1974. 1975; Simpson and Adam, 1975). The homogenate was centrifuged for 5 3Hepest N_2-hydroxyethylpiperazine-N—Z-ethane sulfonic acid. 44 min, 700 x g at 2°C in a Beckman J-21 centrifuge. The supernatant was decanted and saved, and the pellet rehomogenized in an equivalent volume of the sucrose-Hepes-EDTA solution. Following centrifugation, the two supernatants were combined and centrifuged for 10 min, 8,000 x g (max) at 2°C. The supernatant was discarded and the mitochondrial pellet resuspended and washed twice in 0.3 M sucrose, 5 mM Hepes, pH 7.4 and centrifuged in the same manner. The final pellet was sus- pended in sufficient 0.3 M sucrose to yield a mitochondrial protein concentration of 30-60 mg/ml. Mitochondrial protein concentration was determined by the Biuret method (Layne, 1957). Mitochondrial oxygen consumption and respiratory control ratios were determined by a modification of the methods of Simpson and Adam (1975) and Sordahl et al. (1971). Aliquots (0.2 ml/4-6 mg protein) of the mitochondrial suspension were added to 2.8 ml of incubation mediumé and oxygen consumption determined at 25°C using a Yellow Springs Instrument model 53 oxygen monitor employing a Clark- type oxygen electrode. State 3 oxygen consumption was determined following addition of sufficient adenosine-5'-diphosphate to achieve a concentration of 0.3 mM. State 4 oxygen consumption was determined after the state 3 burst had subsided. Mitochondrial PAH accumulation was estimated by a modifica- tion of the methods of Simpson and Angielski (1973) and Adam and Simpson (1974, 1975). For measurement of PAH uptake, incubations were Performed in 25 m1 Erlenmeyer flasks containing 0.5 m1 of the mitochon- drial suspension and 4.5 ml of incubation mediumfi containing either 1 g 4 60‘mM‘KCl, 10 mM potassium phosphate, 5 mM.MgSOA, 142 mM.sucrose, 5 m8/m1 bovine serum.albumin, 10 thmalate and 10 mM pyruvate, pH 7.4. 585 mM K01, 25 mM moo 5 mM Hepes, 5 mM MgSO 2.5 mM KH P0 9 , 10 EM acetate, pH 7.4. 3 4 2 A, 45 mM (sp.ac. = 0.5 uCi/umole) or 10 mM (sp.ac. = 0.05 uCi/umole) 14C— PAH. Mannitol3H (0.5 uCi/ml) was added to the medium.to determine concurrently the outer space of the mitochondria. The relationship of the outer mitochondrial space to total mitochondrial water was deter- mined in parallel flasks containing 14C-sucrose (0.5 uCi/ml) and 3H- HZO (0.5 uCi/ml). Incubations were performed at 25°C under a gas phase of 95% 0 -5% C0 . Duration of incubation varied from 5-20 2 2 minutes. Mitochondria and medium were separated by the method of Harris and Van Dam (1968) as adapted by Simpson and Angielski (1973) and Simpson and Adam (1975). Perchloric acid (500 pl, 1 N) was placed in a 1.5 m1 polyprOpylene microcentrifuge tube (Arthur H. Thomas Co.) and a layer of silicone 011 (0.5 m1 of Versilube F—50, General Elec- tric Co.) was introduced on tOp. At each time point, duplicate 200 pl aliquots of the incubation mixture were layered on top of silicone oil and the tubes immediately centrifuged in an Eppendorf 5412 micro- centrifuge (12,000 rpm, 8,000 x g) for 1.0 minute. A second pair of 200 pl aliquots were added directly to 500 pl of 1 N HCl)4 for deter- mination of medium.radioactivity. Neutralized samples were counted in 10.0 ml Aquasol (New England Nuclear) in a Packard Tri-Carb liquid scintillation spectrometer. Samples were corrected for quench by the channel ratio technique. The volume of a space was determined by dividing the total dpm of a labelled compound present in the mitochon- drial pellet by the dpm/ul of that substance in the medium. The matrix space was calculated by subtracting the volume of the outer (sucrose-impermeable) space from the total mitochondrial water space 3 ( H-HZO-permeable) (Simpson and Adam, 1975). The outer space was also 46 calculated in the presence of 14C—PAH by the distribution of 3H- mannitol and the matrix space in these samples calculated from the ratio of matrix to outer space in those samples containing 3H-H20 and 14C-sucrose (Adam and Simpson, 1974). The l4C-PAH dpm in the outer space was calculated from the PAH concentration (dpm/pl) in the medium and the measured outer space (3Hdmannitol) in each sample. This quantity was subtracted from the total 1(”G-PAH dpm in the mitochondrial extract to give the total 14C-PAH dpm in the matrix space. A ratio of matrix 14C-PAH dpm per ul of matrix space to the 14C—PAH concentration (dpm/pl) in the medium greater than 1.0 was defined as mitochondrial PAH accumulation. Mitochondrial PAH uptake was also calculated as nmole matrix PAH per mg mitochondrial protein following conversion of l4C—PAH dpm by comparison to the PAH specific activity initially placed in the medium. USE OF TRANSPORT ONTOGENY AND SELECTIVE SUBSTRATE STIMULATION AS MODELS FOR IDENTIFICATION OF MULTIPLE RENAL ORGANIC ANION TRANSPORT SYSTEMS 1) Effect of Age The ability of renal cortical slices to accumulate PAH and several other organic anions was quantified using rabbit pups randomly selected from litters at the designated ages. Rabbit pups ranging from 1-8 weeks of age were used. The values obtained were compared to those obtained from unrelated adult rabbits. 2) Effect of Substrate Stimulation Within each litter animals were proportioned equally among the several dosages (treatments) employed with the appropriate control being included for each treatment. Procaine penicillin G was administered 47 subcutaneously as a suspension while suspensions of sulfisoxazole and uric acid were prepared in 1% methylcellulose and administered intra- peritoneally. The sodium salts of iodipamide, probenecid, acetyl- salicylic acid and para-aminobenzoic acid were dissolved in 0.9% saline brought to pH 7.4 and injected intraperitoneally. In each treatment group control animals received a volume of the appropriate vehicle equivalent to that required to deliver the maximum dose of the corresponding drug. Treatment was begun on day 10 or 11 of age. Procaine penicillin G was administered either in a dose of 30,000 I.U. twice daily (60,000 I.U./day) for 3 days or in a dose of 90,000 I.U. twice daily (180,000 I.U./day) for 2 days. All other drugs were administered twice daily for 3 days beginning on day 10 of age. Animals were sacrificed 24 hours after the final drug injection, on day 14 of age. 3) Slice Anion Accumulation Renal cortical slices from rabbits in each treatment group (control or drug) were pooled and then randomly distributed among beakers containing the anions under study. Each pair of duplicate beakers contained a single organic anion substrate in 3.0 ml of the standard incubation medium. In this manner, accumulation of 3 anions (PAH and 2 others) could be determined in a single litter of rabbits. Alternatively, accumulation of a single anion (PAH) could be deter- mined at four drug dosage levels. Anionic transport substrates added to the various incubation media included: p-aminohippurate (PAH), 7.4xlO-5M; phenolsulfonphthalein (PSP), 7.4x10-5M; sulfisoxazole (SULFI), 7.4x10-5M; benzyl (14C) penicillin G (PEN), 7.4x10_5M; acetyl 1 (carboxyl-14C) salicylic acid (ASA), 7.4x10-5M; uric acid-Z- 4C (HA), 48 5.0x10—4M; chenodeoxycholate (carboxyl-14C) (CDC), 1.0x10_6M. In some instances 10 mM sodium acetate was also included in the incubation medium. Duration of incubation was 90 min. After incubation the slices were removed from the beakers, blotted and weighed. Trichloro- acetic acid extracts of slices and media were prepared as outlined for assays of PAH, PSP, PEN, UA and SULFI. ASA accumulation was deter- mined following extraction of slice and medium samples in 0.1 N NaOH (Putney and Borzelleca, 1973). Slices incubated with CDC were digested by incubating (37°C) with Soluene-lOO (Packard) Barany, 1975). Aliquots of medium containing CDC were treated with 10% TCA. PAH and SULFI were estimated by the method of Bratton and Marshall (1939) as modified by Smith e£_§l, (1945). PSP was deter- mined with minor modification of the method of Forster and Copenhaver (1956). Carbon-14 activity was determined by adding aliquots of tissue and medium digest to scintillation vials containing 10 m1 of modified Bray's solution (Hirsch and Hook, 1970) or 10 ml of Omniflour (New England Nuclear) in toluene and counted in a Beckman LS-lOO liquid scintillation counter. Counting efficiency was determined by internal standardization with toluene-14C. INVESTIGATIONS ON THE METABOLIC MODULATION 0F p—AMINOHIPPURATE ACCU— MULATION BY RABBIT RENAL CORTICAL SLICES 1) Effect of Acetate Washout Rabbit renal cortical slices were prepared as outlined. Three groups of slices were incubated for a total of 90 minutes, the final 30 minute period in the presence of PAH. The control group was Dre-incubated for 60 minutes in the normal Cross and Taggart buffer 49 with PAH omitted. The slices were then transferred to beakers con— taining medium.+ PAH for an additional 30 minutes. Acetate-incubated slices were handled in a similar fashion except sodium acetate (10 mM) was included in the final incubation. Acetate-washed slices were preincubated for 30 minutes with acetate (10 mM) and washed without acetate in 3 separate 10 minute washings. The final incubation was conducted as in the control group. 2) Onset of Acetate Stimulation Slices from the cortices of kidneys of two rabbits were pooled. Approximately one half of the slices were incubated in 7.4x10‘5 M PAH for intervals from O to 90 minutes. The second group of slices was incubated in PAH for 60 minutes. Sodium acetate (10 mM) was then added and PAH accumulation measured at 0.5, 1, 2, 3, 5, 10, 15, 20 and 30 minutes. PAH accumulation was expressed as pg PAH/g wet weight of tissue. 3) Effect of 100% N%;on PAH Accumulation Rabbit renal cortical slices were pooled. Approximately one half of the slices were preincubated under 100% 02 for 30 minutes while the remaining slices were preincubated under 100% N2. PAH was added at the end of the pre-incubation period to a concentration of 7.4x10-5M.and incubation continued for durations of 15—60 minutes. 4) Effect of Acetate on the Rate of PAH Uptake Renal cortical slices from the kidneys of two rabbits were pooled. Half the slices were pre-incubated under 100% O and half 2 under 100% N2 for 30 minutes. Within each group Na acetate (10 mM) was present in one-half of the beakers. PAH was added at the end of 50 the pre-incubation period to attain final medium concentrations of 1, 2, 4, 8, 16, 32, 64, 128 and 256x10-4M. Slices were removed from the incubation beakers after 15 minutes and handled as before. PAH uptake under 100% N2 was subtracted from uptake under 100% 02 to estimate the rate of uptake of the oxygen-requiring transport of PAH. 5) Effect of Acetate on Medium pH Cortical slices were pre-incubated in medium (initial pH adjusted to 7.40) containing either 10 mM Na Acetate or 10 mM NaCl. PAH was added at the end of pre-incubation to a final concentration of 7.4xlO-5M. Slices were incubated for 15, 30, 60 or 90 minute intervals. After incubation, the final pH of the medium was determined. Final pH was measured within one (1.0) minute of removal from the shaker with an Instrumentation Laboratory, Inc. pH meter (Model 205) and an H+- sensitive glass electrode (I.L. Cat. No. 14043). Hydrogen ion con- centration was calculated from the measured pH under the assumption that the activity for hydrogen ion is 1.0. Mean hydrogen ion concen- tration i S.E. was calculated from replicated experiments. For illustrative purposes, the data were then converted back to pH. 6) Effect of Acetate (10 mM) on Slice PAH Accumulation and Citrate Concentration The kidneys from two rabbits were pooled for each experiment. One-quarter of the cortex from each kidney was removed for determination of the endogenous renal cortical concentration of citrate. The corti- cal samples were placed immediately in pre—weighed beakers containing 2.0 ml of ice—cold 10% TCA (w/v). The remaining cortex was placed in ice-cold saline and sliced. 250-500 mg of slices were incubated for 90 min in 3.0 ml of media containing either 10 mM Na acetate or 10 mM 51 NaCl. PAH was not present in the incubation media. The slices from two beakers (total sample wt = BOO-1,000 mg) were combined at the end of incubation and placed immediately in pre—weighed beakers containing 2.0 m1 of ice-cold 10% TCA. Both the cortical samples and the slice samples were weighed and homogenized in cold TCA using a Polytron homogenizer. The samples were centrifuged and the proteinfree filtrates were analyzed for citrate content by the pentabromacetone method of McArdle (1955). The results from duplicate samples were expressed as umoles citrate/gram wet weight tissue. PAH accumulation in control and acetate media was determined in each experiment during a 60 minute incubation period after an initial 30 minute pre-incubation without PAH. 7) Citrate Dose-response Curve Renal cortical slices were pre-incubated for 30 minutes in Cross and Taggart buffer (pH 7.40) containing 0.3, 0.5, 1.0, 2.5, 5.0, 10.0 and 20.0 mM Na citrate (Citric Acid, A Grade, Calbiochem.; 3 adjusted to pH 7.40 with NaOH). Control slices were pre-incubated in media containing the appropriate concentrations of NaCl ([NaCl] = 3[Na3 Citrate]). PAH (pH 7.40) was added at the end of pre-incubation to a final concentration of 7.4xlO-5M. Slices were removed from the incubation beakers at the end of a 60 minute incubation period and handled as before. Final medium pH of the.incubation beakers was obtained as described above. 8) Effect of Altered Potassium Concentration Rat renal cortical slices were incubated in 3.0 ml of normal Cross and Taggart buffer ([Kf] = 40 mM) or in 3.0 ml of a reduced potassium medium ([Kf] = 5 mM). In some beakers, Na acetate (10 mM) 52 or Na lactate (10 mM) were added as metabolic substrates. PAH accu- mulation and slice citrate concentration were estimated as outlined previously. 9) Effect of Chronic Metabolic Acidosis Metabolic acidosis was induced in groups of rats by ingestion of 0.28 M ammonium chloride as sole drinking fluid for 7 days (Adam and Simpson, 1974). Control rats had free access to tap water. The pH and pCO of arterial blood (drawn from the abdominal aorta of 2 ether-anesthetized rats) were measured at 37°C with an Instrumentation Laboratory Model 27 pH/gas analyzer. The bicarbonate concentration of blood was calculated from the Henderson-Hasselbalch equation. A pK of 6.1 was used for blood. The solubility coefficient used to correct C02 tensions to H2C03 was 0.0306 (Alguire ep_gl,, 1974). The endoge- nous renal cortical citrate concentration and slice PAH accumulation were determined as before. 10) Effect of Pre-incubation in Citrate on Stimulation of PAH Accumulation (S/M Ratio) by Acetate Renal cortical slices were pre-incubated in medium containing either 10.0 mM Na3 citrate or 30.0 mM NaCl for 30 minutes. After pre- incubation the slices were transferred to beakers containing Na acetate (0.5 mM or 10 mM) or NaCl in phosphate buffer and incubated with 7.4x10-5M PAH for 60 minutes. All media were adjusted to pH 7.40 before the addition of tissue. PAH S/M ratios and final media pH were determined as described above. 53 11) Effect of Acetate (1 mM) on Slice PAH Accumulation, Citrate and a-Ketoglutarate Concentration Preparation of slices, rabbit kidney cortex samples and slice incubation studies were performed essentially as outlined above. The concentration of Na acetate in the incubation medium was reduced to 1 mM. Samples taken for a—ketoglutarate analysis were placed in preweighed beakers containing 3.0 ml cold 3% (w/w) HC104, weighed and homogenized with a motor driven Potter-Elvehjem homogenizer with Teflon pestle. The homogenates were centrifuged at 11,000 x g for 30 minutes at 2°C. Supernatants were brought to pH 7.4 with 1 N NaOH (final volume = 15.0 ml) and assayed by the glutamic dehydrogenase method of Selleck g£_§l, (1964). The results of duplicate samples were expressed as pmoles a-ketoglutarate/gm wet weight of tissue. EFFECT OF AGE AND SUBSTRATE STIMULATION 0N RABBIT PROXIMAL TUBULE PAH ACCUMULATION AND PALMITATE METABOLISM 1) Effect of Ages The ability of renal proximal tubule suspensions to accumu- late PAH and to oxidize and esterify palmitate—l—lac was quantified using rabbit pups randomly selected from litters at the designated ages. Rabbit pups ranging from 1-6 weeks of age were used. In some instances, a mixed arterial and venous blood sample was collected during exsanguination in heparinized tubes. The samples were centri- fuged and the plasma stored at -20°C until analysis. Plasma concen- tration of NEFA was estimated by a modification of the method of Ho (1970). Total NEFA present in the lower phase of the Folch extracts was also determined in several experiments. The values obtained were compared to those obtained from.unrelated adult rabbits. 54 2) Effect of Penicillin Pretreatment of 2-Week-Old Rabbits Procaine penicillin G was administered to rabbits in a dose of 90,000 I.U. twice daily for 2 days beginning on the 11th day of life. Animals were sacrificed 24 hours following the final injection. SuSpension of proximal tubules prepared from control and penicillin treated littermates were incubated in a paired fashion for determina- tion of PAH accumulation and oxidation and esterification of palmitate- 1-14C. The NEFA concentration of plasma and Folch extracts was deter- mined in several experiments as discussed previously. Results were expressed as the mean difference i standard error for the difference between functions measured in control and treated tubule Suspensions. 3) Effect of Iodipamide Pretreatment of 2-Week-Old Rabbits The sodium salt of iodipamide was administered intraperito- neally in a dose of 100 mg/kg body weight. Rabbits were treated twice daily for 3 days beginning on the 10th day of life. Animals were sacrificed 24 hours following the final injection. Proximal tubular PAH accumulation and palmitate oxidation and esterification.were determined as before. Results were expressed as the mean difference i standard error for the difference between functions measured in control and treated tubule suspensions. 4) Effect of Pencillin Pretreatment of Adult Rabbits Pairs of adult, female New Zealand white rabbits were matched by body weight for use in these experiments. Rabbit body weight ranged from 2.5 kg to 4.0 kg. Procaine penicillin G was admini- stered in a dose of 450,000 I.U./kg body weight twice daily for 2 days. Animals were sacrificed 24 hours after the last injection. Proximal tubular PAH accumulation, palmitate oxidation and palmitate 55 esterification were determined as described. Results were expressed as the mean difference i standard error for the difference between functions measured in control and treated tubule suspensions. CHEMICAL AND ENZYMATIC ASSAYS 1) Citrate Asssy Tissue citrate concentration was determined spectrophotometri- cally following oxidation and bromination of citrate in an acid medium to form pentabromoacetone (McArdle, 1955). Aliquots of the trichloro- acetic acid tissue extracts (containing 10-50 ug citrate) were added to tubes containing 5.0 ml of 33 N H and thoroughly mixed. After 2304 10 minutes, 5.0 ml of a 2% potassium bromide-0.5% potassium bromate- 1.2% sodium vanadate solution was added, the tubes capped and mixed and incubated for 20 minutes at 30°C. Following incubation, 3.0 ml of a 22% ferrous sulphate solution was added, the contents mixed and the tubes left for at least 5 minutes to collect excess bromine generated. The pentabromoacetone formed was extracted with 7.0 ml of petroleum ether and the aqueous layer removed. The petroleum.ether extract was washed by shaking with 20.0 ml water and the aqueous layer removed. A small amount of anhydrous NaZSO4 was added to remove the last traces Of water. An aliquot (5.0 ml) of the petroleum ether extract was transferred to a second tube and mixed with 4.0 ml of a 0.2% sodium sulfide (Ventron Alfa Products, Beverly, Mass.); 4% thiourea, 2% borate solution by gentle bubbling with 100% N2. A colored, water- soluble complex of pentabromoacetone in thiourea-borate-sulfide was formed which had a maximum absorption at 435 nm. The absorption of unknown samples was compared to that of known standards of anhydrous citric acid treated in a similar faShion. The citrate concentration 56 of tissue samples was calculated assuming a molecular weight of 192.1 for citric acid. The standard curve was linear over a range of 5-25 ug citrate/ml. The metabolic substrates used in the slice incubation sudies did not interfere with the assay procedure. Recovery of known amounts of citrate added to trichloroacetic acid tissue extracts ranges from 95-101%. 2) a-Ketoglutarate Assay Tissue u-ketoglutarate (uKG) concentration was determined by measuring the decrease in absorbance at 340 nm produced by the oxida- tion of NADH coupled to the enzymatic reduction of a-KG by glutamic dehydrogenase (Selleck g£_sl,, 1964). Approximately 0.05-0.10 ml of an NADH2 (Calbiochem, A grade) solution (30 mg/ml in 1% NaHCOB) was added to 15 ml of a neutralized perchloric acid tissue extract ([a- KG] = 0.02-0.10 mM). Duplicate aliquots (5.0 ml) were added to test tubes. One aliquot (blank) received 0.05 ml of water while 0.05 ml of glutamic dehydrogenase (Calbiochem, A grade; 2 mg protein/ml in 2 M (NH4)2804) was added to the second aliquot. The change in absorbance between the tubes was measured after 20 minutes at 340 nm using a Hitachi-Perkin Elmer spectrophotometer. The change in absorbance of unknown samples was compared to that produced by known amounts of u- KG. The standard curve was linear over a range of 0.02-0.10 mM a- KG. Recovery of added a-KG varied from 93-102%. 3) Determination of Nonesterified Fatpy Acids (NEFA) Using 63Ni Nonesterified fatty acids were determined by liquid scintilla- tion spectrometry following formation of a chloroform soluble 63Ni- NEFA complex as described by Ho (1970). Duplicate plasma samples (50- 200 ul) were brought to a known volume (200 pl) with 0.9% NaCl, mixed 57 with 1.0 ml of Dole extraction mixture6 and placed on ice. After 10 minutes, 200 pl of heptane and 200 pl of distilled water were added, the tubes mixed and placed on ice. After the phases had separated (5- 10 minutes) 200 pl of the heptane layer (upper phase) was withdrawn and placed in polypropylene microcentrifuge tubes. Chloroform (800 63 ul) and 100 pl of a Ni-solution (20 uCi/ml; 1 mg/ml NiCl in l M 2 triethanolamine) were added to each tube, the tubes thoroughly mixed and centrifuged for 10 minutes at 500 x g. The remaining 63Ni-trietha- nolamine was aspirated and discarded. A 500 pl aliquot of the chloro- 63Ni-NEFA complex was added to a scintilla- form layer containing the tion vial and the chloroform evaporated. Omniflour—toluene (10.0 mls) was added to each scintillation vial and the samples counted in a Beckman LS-lOO liquid scintillation counter using a channel Optimized for 63Ni. The NEFA content of unknowns was determined by comparison to a standard curve following correction for 63Ni-cpm in blank samples. Palmitic acid was used as standard. The standard curve was linear over a range of 10—100 nmole palmitic acid. Recovery of known amounts 0f palmitic acid was essentially complete. Total nonesterified fatty acids present in the lower phase of Folch extracts of proximal tubules was determined by a modification of the method of Ho (1970), similar to that described by Bieber e; 31. (1975). Quadruplicate aliquots of the Folch extract were added to tubes containing approximately 50 mg silicic acid. The chloroform: methanol was evaporated and NEFA extracted as outlined above. This procedure did not alter the recovery of known amounts of palmitic acid. 6Dole extraction mixture - isopropanolzheptanezl N H SO (40:40: 1 V/v). 2 A 58 In several experiments an aliquot of the upper heptane layer was subjected to thin layer chromatography on silica Gel G with hexane: diethyl ether:acetic acid (80:30:l v/v) as solvent. All of the 14C— label recovered from the TLC plates was associated with the NEFA fraction. No contamination by phospholipids was detected following visualization of TLC plates with iodine vapor or rhodamine-6—G. Following extraction of NEFA, two of the aliquots were assayed with 63Ni as described while the remaining pair of aliquots was assayed with solution containing an equivalent amount of unla- 14 belled NiClz. The C-activity appearing in the 63Ni—channel from these samples was subtracted from the total counts appearing in those samples assayed with 63Ni. This served to correct for the counts added by 14C-NEFA. MATERIALS 1) Magnetic iron oxide was prepared as described by Cook and Pickering (1958), except that it was stored as a suspension in 0.9% NaCl and diluted before use with Ringers medium. Sodium.hydroxide (2.6 gm) and potassium nitrate (20 gm) were dissolved in 100 ml of oxygen saturated water and added to a solution of ferrous sulphate (9 gm) dissolved in 100 ml of oxygen saturated water. The mixture was boiled for approximately 20 min. The precipitate of magnetic iron oxide was washed by resuspending in water and attraction to a perma- nent magnet. The wash water containing any nonmagnetic substance was decanted. This procedure was repeated 5—10 times. The iron oxide was dried, weighed and suspended in 0.9% NaCl at a concentration of 50 Ins/ml. 59 2) Contaminating NEFA were removed from bovine serum albumin (Fraction V, Sigma Chemical Co.) by the method of Chen (1967). Norit A charcoal (3.0 gm) was added to a solution of 7.0 gm BSA in 70 ml distilled water. The pH of the solution was adjusted to 3.0 by addition of HCl and stirred continuously for 1 hr at 0°C. The solu- tion was centrifuged at 20,200 x g, 20 min at 2°C. The supernatant was brought to pH 7.4 with NaOH assayed for protein content and stored at -20°C. 3) Probenecid was the kind gift of Dr. John Baer, Merck, Sharpe and Dohme. Dr. Michael Cohen, Hoffman LaRoche, Inc. generously provided the sulfisoxazole used in these experiments. Iodipamide was the gracious gift of Dr. S.J. Lucania, E.R. Squibb and Sons, Inc. Procaine penicillin G (Crystacyllin) was purchased from E.R. Squibb and Sons. STATISTICAL ANALYSES Where appropriate, data were analyzed using Student's "t" test, paired or group comparison, or analysis of variance, completely random or randomized complete block design. Treatment means were tested using Tukey's-whprocedure (Sokal and Rohlf, 1969). The 0.05 level of probability was used as the criterion of significance. RESULTS USE OF TRANSPORT ONTOGENY AND SELECTIVE SUBSTRATE STIMULATION AS MODELS FOR IDENTIFICATION OF MULTIPLE RENAL ORGANIC ANION TRANSPORT SYSTEMS 1) Effect of Age Three distinct developmental patterns of organic anion accumulation by rabbit renal cortical slices were observed (Figures 1,2,3,4). Active accumulation of UA into slices (defined as S/M ratio greater than 1.0) was not evident until two weeks of age (one week S/M = 1.21:0.06) (Figure l)l. Transport capacity for UA in adult tissue (S/M = 3.38:0.24) was observed by 4 weeks of age (S/M = 3.08:0.30) (Figure 1). In contrast, active accumulation of PAH occurred in slices from one week rabbits (PAH S/M = 3.54:0.01) (Figure l). PAH accumulation increased rapidly with age reaching a peak at 4 weeks (S/M = 15.27il.40) and then declining to adult values (S/M = 10.071152) (Figure 1). Accumulation of PSP followed a developmental pattern similar to that of PAH (Figure l). Accumulation of CDC produced a third pattern of development (Figure 2)- CDC accumulation at two weeks (S/M = 37.58i1.78) was approximately 5-fold greater than that of PAH and continued to increase throughout development, being greatest in slices from adults (CDC S/M = 127.17: 12.79). When renal cortical slices were incubated at 0°C accumulation 1Identity of the radiolabelled carbon present in several tissue digests was confirmed as uric acid by thin layer chromatography (Orsulak EEHEL-: 1968). 60 61 H amps ecu mueommudmu .muouuaa m Eoum mufioomu a on m CH omewauoumo A.m.mv uouum mummcmum ucfion zoom .mwwoa seasonsoefi ecu Boom ueomom mos oumuoo< .mooHHm Hmofiuuoo Hmcmu uHoomu mo <3 mom .mmm .m on muonz 62 H mmswwm 1.85:5 w0< lbw—30¢ a o — — _ 90¢ U35 0 mm.— 0 IS. . l 0.3 I 0.0— m 63 .uoHon one mo mafimmu ecu amen mmoa ma .m.m one neonm ma mesa Hmofluum> H some one musomounou usflon oe muonz .muouuHH q aouw mufloomu mum ea mosflauouom .m.m comm .mHmmE sowumoooefi onu :H uemmoud mp3 A28 oHV oumuoo< .mooHHm Hmofiuwou Honey ufioomu mo qubm mom one .m mo mufioomu Boom moumdoun mooHHm Hmowuuou .m muswfim Isoom one wofiueomoudou o>uso ocH Hmcou ho coaumHsesom 090 so AUoOV monumuonEmu coeumosoefi mooswou mo uoommm 67 ~93va m mudwfim w0< has; o v.3 I b D ._.......J.-..__L-..-_L__-_L_.___L___J___- J____J q 8 C) q o o 0' ' O. C) a g; OllVll W/S 303 68 .uefion osu mo mswmmu ecu ammo mmmH we .m.m ozu B3osm ma mafia Hmofiuuo> o: mumcz .muouuHH q Boom mufloomu elm :H omeHBuouoo .m.m n smoB was musomoudmu uBHon zoom .mwmoB coaumosoce msu BH ueommun mos AzB OHV mumuoom + .omm mm>wooou mumnomu Houueoo .mmmm mouse pom mammv mum3u mmumummcmamm ouo3 wa\wB OOHV wmuoemooun mam A.D.H ooo.omV 0 BmHHHofiBon mammooum .mmvoB comumoooem onu ea wmvsaoew uoc mmB monuoo< .mmomam Hmomuuoo Hmeou ufioomu mo <5 new mmm .mxonoozmzo w shaman is: misinfozifn o.o — . a“. _ _ _ 1 _ _ . _ 1 . _ - _ - 1 _ _ _ . 1 . . _ _ 1 . . . . “I 0 " 2.3.6.22 _ .1 5528 . . _ l 0.0 0.0 0&— 0.0— .. 0.0a 1 0.0“ 0.0a OIiVI W/S HVd 79 .BsfimoB eomumosoem onu BH anemone mmB AZB OHV monsoo< meson «N moaamx mums mumonmm .oaomno> mo mussosm omBsHo>omH mo>Hmoou maouucoo .mmmm m mom mammm momsu momom wmumommem onu BH monoummemBmm mp3 oHommxomHmHBm .omumu unmask hmoo \ustoB xmemmx mom Bomumasesoom m mo mussoBm loosen .m.m + mo «H mom so BOHuomnsH ummH ocu noumm muso: «N mmHHHx mums muHoomm .mhmm m pom mHHmm MUHBu monoumHeHawm mp3 wa\wB OOHV mumps .OH musmHm oHBBHo>omH 0m>Hmomu mHouueoo .U_._hwu< 00— 00 mm 0 1.0 0.0 N.— 0.— 0.0 l 1N l 5 E I 3 is O_»<~_ «(\m 15. E A 00— x 25 >00m\ ._.>> 5200. % I 0&— 1 0.0— .. 0.0m 0.0a OIiVII W/S HVd 85 .ABV muouuHH mo umoBse moumUHweH one Boom moBHmuoo .m.m H BmmB use odommueou mumm .Aw mustmV muomon mm woBuomuon meme mueoBHuonxm .mmoHHm HmoHuuoo Hmemu uHoomu mp 000 mam mmm mo BOHumHsBsoum so BOHumuumHBHamm omHedevOH mo uoommm .NH ousmHh 86 000 NH oustm 3qu. $92530; sz 02in S\» 000 1].. I J 0:5. is :1 U 0.0— 0.0a 0.00 0.0? 0.0m 0.00 011V! W/S 87 Slice accumulation of PAH was not enhanced after low doses of the nontransported organic anion para-aminobenzoic acid (Figure 13). However, after 200 mg/kg of PABA, PAH accumulation was signi- ficantly increased. At no dose did PABA change kidney weight/body weight ratio (Figure 13). INVESTIGATIONS ON THE METABOLIC MODULATION 0F p-AMINOHIPPURATE ACCUMULATION BY RABBIT RENAL CORTICAL SLICES 1) Effect of Acetate Washout The immediate presence of acetate was required for stimula- tion of PAH accumulation. No stimulation of PAH accumulation was observed when the slices were pre-incubated in a medium containing acetate, washed and finally incubated with PAH (PAH S/M = 3.23:0.36) (Table 1). However, incubation of slices in 10 mM acetate during the final 30 minute incubation period significantly increased the PAH S/M ratio from 3.54:0.48 to 6.22: 0.60. 2) Onset of Acetate Stimulation In control experiments, tissue concentration of PAH in- creased with duration of incubation. However, the rate of rise in tissue PAH concentration decreased after approximately 30 minutes of incubation (Figure 14). Addition of acetate after 60 minutes imme— diately increased the rate of PAH accumulation, producing concentra- tions 40-50% higher than control within 10 minutes (Figure 14). 3) Effect of 100% N“ on PAH Accumulation To determine the effect of acetate on the oxygen-requiring component of PAH uptake the optimal duration of incubation was first established by incubating slices under 100% 02 or 100% N2 (Figure 15). 88 whom .muouuHH m BH mmeHBuouom .m.m H emmB msu ueomounou .Am mostmv whomoo mm 0oBH0muom some muBoBHuonxm .moHumu ucmHos mvoo\uawH03 xmsme mom BOHumHBBBoom mHm mo emoB was usomoonou mueHom .mmm mo mGOHumuusooeoo oBHB BH mV oxmuns m can. an o.« 3 3 ad [A O O l) 32:28 O mange oz 2... o. O nun .y0 (l-NlW-inSS|l l-B-HVd 5") ZN-‘o A 100 that more than one mode of active uptake was used for PAH accumula- tion. One uptake mode had an apparent Km of approximately 1.7x10-4M but only a limited capacity for PAH (apparent Vmax = 3.7 pg PAH ° g”1 tissue - min-1). The second mode of uptake had an apparent Km of 22x10~4M but possessed a relatively large capacity for PAH (apparent vmax - 13.6 pg PAH - g"1 tissue - min-1). Addition of 10 mM Na acetate to the medium markedly increased the capacity of both the high (V'max = 51.6 pg PAH - g-l tissue - min-1) and low (V'max = 23.8 pg PAH g-1 tissue - min—1) capacity uptake modes (Figure 17). Acetate did not appear to change the apparent K.In of either mode for PAH. 5) Effect of Acetate on Mediumng Slices incubated in a control medium containing no acetate acidified the medium from an initial pH of 7.40 to a final pH of approximately 7.25 after 15 minutes of incubation (Figure 18). The pH of the control medium did not change significantly with further incu- bation. PAH S/M ratio in control slices reached 7.36i1.42 by 90 minutes. In the presence of 10 mM Na Acetate medium pH initially fell to pH 7.33 and then increased with time, reaching a final pH of approxi- mately 7.54 after 90 minutes. In the presence of acetate the PAH S/M ratio was significantly elevated at all incubation times (Figure 18). Addition of acetate to slices that had been incubated with PAH for 60 minutes resulted in an immediate increase in final medium pH. The rise in pH was temporally correlated with the increase in accumulation of PAH (Figure 19). The pH of medium bathing control slices was not significantly different at the times studied. 101 Figure 18. Effect of 10.0 mM Na Acetate on final media pH and on PAH accumulation by rabbit kidney slices. Initial medium pH adjusted to 7.40 (dashed line). Final media pH determined (methods) within one minute of termination of incubation. Hydrogen ion concentration in the media was calculated from the experimentally measured pH. Mean i S.E. of hydrogen ion concentration was calculated from dupli- cate determinations of 3 experiments and reconverted to pH. PAH concentration in the media was 7.4x10'5M PAH. PAH s/M “no FINAL MEDIA pH I6.0 14.0 12.0 10.0 8.0 6.0 4.0 2.0 7.60 7.50 7.40 7.30 7.20 7.10 15 102 CONTROI. 10 mM Na ACETATE N'3 30 4s 60 TIME (MIN) Figure 18 75 90 103 .muBmBHHmnxm usom mo .m.m H emoB unommudmu muBHom .AQH oustmv ouomoo mm ooBuomuoa mueoBHuodxm .mooHHm moson uHoomu BH AOHumu z\mv BOHuMHoBBooo m .mSmmHu ucwfiofiwwsm casuno ou wwaooa was massage Hmum>mm Scum xouuoo .mmoddumda meow :H .woawauomm nouns kkumav Ioaafi cmaflmuno was coaumpuaooaou oumuufiu msoaowovam mo coauma waumuov you nouuoo Hosea .ucweaummxo 50mm aw wwHooa meB mmfiommm am>Hw m mo mamaaam old mo w%mswfix mnu Scum mwofiam u Amv amoo.oumam.o Amv ooo.owomo.o va ammo.oummo.o Aqv ama.OH-.qH Aev «5.0Hoo.aa umm Amv 0Nqwedwmqmd Amv ouo.oawma.o II: Amv QHN.OHH¢.NH AmV m~.owom.o woum Aqv ammo.oumoq.o AqV woo.owowo.o Aqv quo.oao¢m.o Aqv @ms.oam¢.aa Aqv No.0Hmm.m panama Amy amoc.onomo.o Amv moo.oaamo.o Asv QHNO.OHmQH.o Ame amm.onmn.oa Amv mm.¢ams.q won A28 oav oumumod Hopucoo msoaomovam A26 oav oumuoo< Houuaoo mmwuoam Amsmmau w\waoenv coaumuunmuaou mumuufio OHuMM Exm mmm scum moofiam xmuuoo Hmsmm cg nowumuusoocoo oumuufio was coaumasasoo< m .voumaa msoeufiuaou dofiuwnsuna msoaum> onu mauamsuumnsm was voaooa mums muwu elm mo assumes an» scum mouuam Hmowu [Humane mo weapon some GH .m>onm kumfia evsum man a“ dofiumanasuom mHm mo .m.m H momma ucomouaou mumm .m4m unonuas coauwnsuafilmua use on Hmauficw am .umumm woauon coaumnsosfi was 00 m wsausw voafishoumu was coaumasssuom =;>:.>._.m _ZnO_~v0_~>._._OZ A: 2.0—.33 qcmcrmv O 5 0. 5. 0. 5 O. 5 O 5 am an an «4 1a a; a; In 1“ nm .1 _ a L m 1 _ _ _ _ . .\:\.V.\i\.. .3. \ . a N" _ L p b r a _ P _ L 0. 3 A 4. 2. o. 3 6 4. 2. 2 II I II .I ll 0 O o 0 $52: 63302 3 20:29:82. $3.22.. PEN A AC CONT & AC PEN 8. AC CONT 81 AC Figure 34 153 examined, penicillin pretreatment decreased both, the tubule NEFA concentration and the amount of palmitate-140 present as NEFA (Table 11). However, due to the large variability the observed differences were not statistically significant (Table 11). The specific activity of NEFA present in Folch extracts of control and penicillin treated tubules were not significantly different (Table 11). The penicillin pretreatment of neonatal rabbits had no effect on the plasma NEFA concentration (Table 12). 3) Effect of Iodipamide Pretreatment of 2—Week-01d Rabbits 14 Incorporation of palmitate-l- C into tubule lipid fractions was not altered by prior treatment of rabbits with iodipamide (Figure 35). Palmitate-14C incorporation into NEFA fractions of control (0.92:0.09 nmole/g tubule) and iodipamide treated (0.89:0.18 nmole/g tubule) tubule suspension was not significantly different (BiS.E. = -0.03i0.19 nmole/g tubule) (Figure 35). Iodipamide pretreatment did not significantly alter the amount of label recovered as T0 from control (1.57:0.13 nmole/g tubule) or treated (1.87:0.36 nmole/g tubule) tubules (BiS.E. = 0.30:0.27 nmole/g tubule). PAH accumulation by iodipamide pretreated tubules (T/M = 3.60i0.39) was not signifi- cantly different than control (T/M = 3.97:0.50) (Figure 35). 4) Effect of Penicillin Pretreatment of Adult Rabbits Palmitate-14C incorporation into lipid fractions of adult tubules was not altered by prior penicillin pretreatment (Figure 36). Recovery of label present as NEFA was not different in control (1.02:0.39 nmole/g tubule) or pretreated (1.41:0.42 nmole/g tubules) tubule suspensions (EES.E. = 0.3910.38 nmole/g tubule) (Figure 36). 154 TABLE 11 Effect of Penicillin Pretreatment of 2-Week-01d Rabbits on the Concentration of Nonesterified Fatty Acids in Separated Proximal Tubules and Incorporation of Palmitate-14C into Tubule Nonesterified Fatty Acidsa Treatment [NEFA] [NEFA-14C] Sp. AC. (nmole/g tubule) (uCi/g tubule) (uCi/ mole) Control 3.98 0.32 0.08 Penicillin 3.73 0.29 0.06 b" -0.24 —0.09 —0.02 i i: i' i S.E. 0.18 0.05 0.01 a Four injections of 90,000 I.U. procaine penicillin G were admini- stered (twice daily for 2 days). Animals were sacrificed 24 hrs after the last injection. Paired, control and treated tubule suspensions were incubated in a medium containing 1 mM palmitate- l-1 C (sp.ac. 0.4 uCi/umole) + 10 mM acetate. Values represent the means and mean difference 1 standard error obtained from 5 litters. 155 Table 12 Effect of Penicillin Pretreatment of 2-Week-Old Rabbigs on Plasma Concentration of Nonesterified Fatty Acids Treatment (n)b Plasma [NEFA] (nmole/ml) Control 16 443 i 47 Penicillin 19 449 i 49 aFour injections of 90,000 I.U. procaine penicillin C were administered (twice daily for 2 days). Animals were stunned 24 hrs after the last injection, imme- diately exsanguinated and a mixed arterial and venous blood sample collected in a heparinized tube. Assays were performed on frozen plasma samples. b(n) = number of animals. 156 Figure 35. Effect of iodipamide pretreatment on PAH accumulation (T/M) and palmitate oxidation and esterification by suspensions of renal proximal tubules. Iodipamide was administered twice daily for 3 days beginning on day 10 of age. Animals were sacrificed 24 hours after the last injection. Experiments were Similar to those of Figure 33. Results were expressed as the mean difference + standard error for the difference between functions measured in control and treated tubule suspensions. 157 0.80 16.0 122:3: 23.2.3262 C. 2.053. 2ch mums. O O 0 0 6 4. 2. o m w . w 0 O O O O. O. O. 8.0 0. 0. 0 4 0 4 12.0 8.0 12.0 mane. 2: :3. 0.80 16.0 PL CH NEFA 76 TL PAH T/M CO2 IODIPAMIDE 81 AC Figure 35 158 Figure 36. Effect of penicillin administration to adult rabbits on PAH accumulation (T/M) and palmitate oxidation and esterification by suspensions of renal proximal tubules. Pairs of adult, female, New Zealand white rabbits were used. Procaine penicillin G, 450,000 I.U./kg body wt. was administered to one rabbit twice daily for 2 days. Controls received isovolumnic amounts of 0.9% NaCl. Animals were sacrificed 24 hr after the last injection. Experiments were similar to those in Figure 33. Results were expressed as the mean difference 1 standard error for the difference between functions measured in control and treated tubule suspensions. PAH T/M D!S.E. 159 r N36 16.0 .- 12.0 .— 8.0 _. v ., 0“. 3'3: 0.11.0 4 O — 0" ’0 . 00 :4»: 03 ’6 I 0.190 0.0 7 if 1 a a 4.0 .— 8.0 ._ 12.0 _. 16.0 _ PAH T/M C02 Tl PL CH NEFA TG PENICILLIN 81 AC ADULT Figure 36 0.80 0.60 0.40 0.20 0. 00 0. 20 0.40 0.60 0.80 '3'530 (31mm I5/5310111 11) nouvaoaaoam ilVilW'lVd 160 Similarly, palmitate-14C incorporated into TG fractions from peni- cillin treated (2.05i0.54 nmole/g tubule) and control (2.32i0.61 nmole/g tubule) tubule suspension was not different (BiS.E. = -0.26i 0.6l nmole/g tubule). Penicillin pretreatment of adult rabbits also did not alter tubule PAH accumulation (cont. T/M = l4.l6i2.70; treat. T/M = 18.72i6.25) (Figure 36). 5) Effect of Penicillin Pretreatment of 2-Week-Old Rabbits Palmitate Metabolism in an Acetate-free Medium Incubation of tubule suspensions from control and penicillin pretreated rabbits in an acetate free incubation medium produced a pattern of palmitate-14C distribution (Figure 37,38) similar to that observed in previous studies (Figure 33,34). In each experiment label recovered as NEFA was less in tubules from treated rabbits than in paired controls (Figure 37,38). Similarly, palmitate-14C recovered as T0 was greater in treated tubule suspensions than impaired control tubule suspensions (Figure 37,38). However, due to variability, these differences were not statistically significant. Addition of acetate (10 mM) to the incubation medium appeared to increase palmitate-14C incorporation into total lipid of control tubule suspensions by increasing label recoverable as NEFA and TG (Figure 39). The observed differences were not statistically significant (Figure 39). Acetate had no effect on incorporation of label into lipid fractions from penicillin treated tubules (Figure 40). 161 Figure 37. Effect of penicillin pretreatment on the ability of proximal tubule suspensions to esterify palmitate in an acetate free medium. Beginning on day 11, 90,000 I.U. of procaine penicillin G was administered twice daily for 2 days. Animals were sacrificed 24 hr after the last injection. Tubules were incubated in medium con- taining 1 mM palmitate-l-lAC (sp.ac. 0.4 uCi/umole). Results were expressed as mean difference i standard error of the difference between functions measured in control and treated tubule suspensions. PALMITATE INCORPORATION (p MOLES/g 1030113) ‘15: S.E. 0 .80 0.60 0.40 0.20 0.00 0.20 0.40 _ 0.60 .. 0.80 ._ TL 162 PL CH NEFA PENICILLIN - AC Figure 37 TG 163 Figure 38. Incorporation of palmitate-l-IAC into nonesterified fatty acid (NEFA) and triglyceride (TG) fractions from control and treated rabbit tubule suspensions. Procaine penicillin G was admini- stered twice daily for 2 days, beginning on day 11 of age. Animals were sacrificed 24 hr after the last injection. Tubules were incu— bated in an acetate—free medium containing 1 mM palmitate-l-1 C (sp.ac. 0.4 uCi/umole). Individual data points from 4 litters were plotted. Each pair of points represents the average value obtained for control and penicillin treated tubules within a litter. 164 TRIGLYCERIDE NEFA .. 5.0 5 4 _ 153.9: 282525.02 it 30:30 ESE 0. 5. 0. 5. 0. 5 0 4 3 3 2 2 .I. .1. _ _ _ _ _ _ _ 1.6 ,_ _ _ _ _ _ _ A 2. O. 8. b 4. .1 .1 .1 O O 0 $52: 63392 3 205205.002. 34:22.. _. 0.5 0.2 .— PEN 'AC PEN CONT 'AC CONT 'AC 'AC Figure 38 165 Figure 39. Effect of acetate on palmitate esterification by tubule suspensions prepared from young (2 week) rabbits. Rabbits were the saline-treated controls used in experiments described in Figure 37 and 38. One-half of the tubules prepared were incubated in medium con- taining 1 mM palmitate whereas the remaining tubules were incubated with 1 mM palmitate + 10 mM acetate. Results were expressed as the mean difference i standard error of the difference between functions measured in tubules incubated with and without acetate. PALMITATE INCORPORATION (p MOLES/g 1013015) 615.15. 0 .80 0.60 0.40 0.20 0.00 0.20 0.40 0.60 0.80 TL 166 PL CH NEFA CONTROL 2 AC Figure 39 TG 167 Figure 40. Effect of acetate on palmitate esterification by tubule suspensions prepared from neonatal (2 week) rabbits. Rabbits were the penicillin treated animals described in Figure 37 and 38. Experiments were handled in the same fashion as Figure 39. Results were expressed as mean difference i standard error of the difference between functions measured in tubules incubated with and without acetate. 168 0. _ _ _ _ r u nu Au 3 a 4. m m m. w. w m. nu nu nu nu nu nu nu nu nu .usnm :52: 6330.2 3 20:59:82. 35.22.. TG NEFA PL CH TL PENICILLIN 1 AC Figure 40 DISCUSSION Analysis of renal proximal tubular transport capabilities in developing animals has been successfully used to demonstrate the existence of functionally separate transport systems for sugars and amino acids (Segal g£_al,, 1971) and for organic anions and cations (Rennick gt al., 1961; Hook, 1974). Determination of the patterns of development of amino acid reabsorption demonstrated multiple transport systems within a single class of transported solutes. For example, Segal and Smith (l969a; 1969b) used patterns of develOpment to deli- neate differences between transport systems for cystine and lysine reabsorption in neonatal rat kidney cortex. In addition, Baerlocher .ggial. (1971a; 1971b) demonstrated that the separate high affinity transport systems for proline and glycine reabsorption were absent in newborn rats and developed independently, with proline transport appearing at one week of age and glycine transport capacity observed not until 3 weeks of age. These investigations suggested that analysis of the developemtnal patterns of renal organic anion secretion might aid in the discrimination of multiple organic anion transport systems. Hirsch and Hook (1969; 1970) and Johnson g£_§l, (1974) demon- strated that treatment of neonatal rabbits, rats and dogs with sub— strates of the organic acid transport system (e.g., PAH, penicillin) selectively enhanced maturation of slice PAH accumulation without altering accumulation of organic cations. Selective substrate 169 170 stimulation of the organic anion transport system following admini- stration of exogenous anionic substrate loads has been subsequently confirmed in several ig_yi££g and in_yigg_preparations (Ecker and Hook, 1974; Pegg and Hook, 1975; Bond gt_§l,, 1976; Lewy and Grosser, 1974). These investigations suggested that multiple anion transport systems might be defined by differential response to administration of exogenous substrates of each suspected transport system. Theoreti- cally, pretreatment with a substrate from one transport system should enhance accumulation of that anion group only. The literature contains a vast amount of experimental evidence supporting the dissimilarity of secretory transport systems for PAH and HA in man and lower animals. The delay in appearance of active accumulation of UA by renal cortical slices compared to PAH accumula- tion (Figure 1) coupled with the marked differences in developmental patterns for these anions supports the possibility of separate secre- tory systems for these anions in rabbit proximal tubules. Additional support for this concept can be derived from the failure of PEN (group I) and probenecid to alter slice accumulation of UA (group II). Similarly, the lack of effect of DA pretreatment on PAH accumulation (Figure 9) is in accordance with this hypothesis. However, other investigators have reached different conclusions. Based on inhibitor studies in rabbit tissue Mdller (l967a,b) and Sheikh and Mdller (1971) concluded that a common transport system for HA and PAH was involved in tubular secretion of these anions and that the affinity of HA for the system was less than the affinity of PAH. Nevertheless, suffi- cient differences in the characteristics of uptake of PAH and UA exist to suggest that the transport processes are not absolutely identical (Mudge et_al,, 1973). Optimal in_vitro incubation conditions for UA 171 and PAH accumulation differed with respect to potassium concentration (Berndt and Beechwood, 1965), medium pH (Berndt and Beechwood, 1965; Copenhaver and Davis, 1965) and metabolic substrates (Platts and Mudge, 1961). Berndt (1965) also demonstrated that efflux of UA from preloaded rabbit cortex slices occurred through a process substan- tially different from the process mediating PAH efflux. Different affinities for a single common transport system could not be responsible for differences in optimal incubation medium conditions for maximal UA or PAH accumulation or for the differences in developmental accumulation patterns or response to substrate stimulation. Addition of acetate to the incubation medium (Kim and Hook, 1972), normal maturation of renal cortical slice anion accumu— lation (Ecker and Hook, 1974) and penicillin stimulation of immature tubule anion accumulation (Pegg 35 al., 1976a) are all processes which alter the maximum velocity of transport rather than affinity of the carrier system for anionic substrates. Thus, accumulation of UA and PAH should be affected in a similar fashion by these treatments if only a single transport system exists. The differences in response of slice UA and PAH accumulation might therefore be better explained by postulating the existence of two separate, but overlapping, transport systems for PAH and HA in rabbit renal proximal tubules as has been done for kidneys of other species. An alternative explanation for the above observations (Figures. 1 and 4) is that cortical slice UA accumulation resulted from the reabsorptive transport of UA (Berndt and Beechwood, 1965). However, Shiekh and Mdller (1971) demonstrated that at high medium UA concen- trations (greater than 0.1 mM UA) UA was accumulated intracellularly 172 as a consequence of the secretory process. Therefore, under the experimental conditions used in these investigations (0.5 mM) de- velopment of UA accumulation should reflect maturation of the UA secretory system. BarAny (1972; 1973a; 1973b; l974a,b; 1975) described a similar group of separate, but overlapping, transport systems to explain the effects of various anions on the slice accumulation of iodipamide. One system (H-system), sensitive to iodohippurate inhibition, was assumed to be the classical hippurate transport system (Barany, 1972). The second, hippurate insensitive system (L-system), was inhibited by anions normally excreted by the liver (Bdrany, l973a,b). Thus, bile acids (e.g., chenodeoxycholate), cholographic agents and flavaspidic acid produced marked inhibition of iodipamide accumulation by the L- system (Barany, l973b; 1974b; 1975). Benzylpenicillin and PAH had little effect on iodipamide accumulation by the L-system. In addition, pyrazinoic acid, m-hydroxybenzoic acid and salicylic acid also had little effect on iodipamide accumulation via the L-system, suggesting that the L-system was not involved in UA accumulation in rabbit kidney cortex (BArany, 1973b). The independent pattern of maturation for CDC (group III) accumulation as compared to PAH or UA (Figures 1, 2) supports this hypothesis. Selective enhancement of PAH accumulation without alteration of CDC accumulation after PEN pretreatment (Figure 7) was consistent with separate H.and L transport systems. This position is strengthened by the lack of stimulation of PAH by iodi- pamide (Figure 11). 173 Several aspects of CDC accumulation by renal cortical slices evident during the course of these investigations deserve comment. Barany (1975) observed considerable nonactive slice accumulation of CDC at 0° or following incubation under 100% nitrogen. Similar re— sults were obtained in the present studies (Figure 7). However, the nonactive portion of CDC accumulation did not appear to participate in the age-related changes of L-system transport capacity since no differences in CDC S/M ratios were observed at 0°. As expected, penicillin administration to neonatal rabbits had no effect on the nonactive component of CDC accumulation (Figure 7). Finally, the marked large CDC S/M ratios obtained were apparently not caused by sequestration of CDC, preventing egress from the tissues, since runout of CDC from preloaded adult rabbit slices was not different than runout of PAH (unpublished results). Similarly, Berndt g£_al, (1968) demonstrated that reduced efflux was not responsible for the large tissue—to~medium iodipamide gradients generated in rabbit cortical slices. In general, accumulation of the remaining anions utilized in this investigation paralleled the responses of PAH, UA and CDC and were consistent with the basic hypothesis. That is, Group I anion accumu- lation (PSP, Figure 4; PEN, Figure 5) was enhanced by prior admini- stration of Group I substrates but was not altered by group II (UA, Figure 9, SULFI, Figure 8) substrate pretreatment. Similarly PEN administration did not enhance ASA (group II) accumulation (Figure 5). 174 Three exceptions to the general response pattern were noted during these investigations. Penicillin pretreatment significantly increased slice accumulation of sulfisoxazole (group II, Figure 6). Enhanced slice accumulation of SULFI was probably related to a small increase in the fraction of SULFI accumulated by the PEN activated group I transport system. Barény (1973b,l974a) observed considerable overlap between the H- and L- transport systems (as much as 20% of iodipamide accumulation occurred via the hippurate system). Stimulation of PAH transport capacity subsequent to ASA (Figure 10) and high dose (200 mg/kg) PABA (Figure 12) administration were the second and third apparent inconsistencies. A major route of PABA biotransformation in rabbits resulted in formation of PAH (Wan g£H§1., 1972). Hirsch and Hook (1969) demonstrated that administration of PAH to neonatal rabbits stimulated slice PAH accumulation. Thus, the substrate stimulation observed at high PABA doses most likely resulted from a secondary response to an increased PAH load. The frank toxicity exhibited by ASA suggested a possible mecha— nism for enhanced PAH accumulation following ASA treatment. ASA has been found to be nephrotoxic in several species (Abel, 1971) and this toxic effect was greater in newborn rats (Yeary g£_§l,, 1966). Several nephrotoxic agents including gentamicin (Cohen g£_§l,, 1975), neomycin (Lapkin g£_§1,, 1975), and potassium dichromate (Hirsch, 1973) increased 32 gig g PAH accumulation either specifically or secondarily following renal compensation to incurred damage. Possibly ASA induced alterations in PAH accumulation occurred as part of a toxic response rather than by substrate stimulation. Alternatively, biotransformation of ASA to anionic metabolites transported by the 175 group I system could result in substrate stimulation in a fashion similar to PABA. Following administration, ASA was rapidly hydrolyzed to salicylate (Mandel, 1971) which has been shown to be actively accumulated by rat renal cortical slices (Putney and Borzelleca, 1973). Interestingly, the glycine and glucuronic acid metabolites of salicylate (Hollister and Levy, 1965) were also observed to be acti- vely transported by a mechanism qualitatively similar to that for PAH (Despopoulos, 1960,1965). The rapid hydrolysis of ASA by plasma and tissue esterases also suggested that slice ASA accumulation and the effects of ASA pretreatment reflect, at least in part, active sali- cylate transport by renal cortex. Enhancement of transport system maturation as a response to exogenous substrate loads was apparently a function of the group I system alone. Administration of urate (Figure 9) or iodipamide (Figure 11) did not alter the rate of maturation of their respective transport systems. This lack of response could be attributed to inadequate (or excessive) drug administration, resulting in an insufficient stimulus for induction of extra transport capacity (or in competitive suppres- sion of slice anion accumulation). Alternatively, duration of drug administration may have been inappropriate for optimal enhancement of maturation. However, the group II and group III transport systems may be in- herently resistant to potential inducing stimuli such as exogenous substrate loads. This appears to be the case for renal organic cation secretion. Challenge of neonatal rats and rabbits (unpublished results) and adult rats (Pegg g£_§l,, 1976b) with large doses of cationic substrates had no effect on intrinsic slice capability to 176 accumulate actively secreted organic cations. Similar observations have been obtained during investigations on induction of hepatic xenobiotic biotransformation pathways (Conney, 1967). Thus, while repeated administration of drugs such as diphenylhydantoin, phenyl- butazone, tolbutamide and aminopyrine were capable of inducing enzymes that catalyzed their own biotransformation, repeated administration of aspirin, trimethadione and sulfanilamide had no subsequent effect on their metabolic fate within the body (Conney, 1967). These results suggest that while further evaluation of the group II and III trans— port systems is required with regard to induction potential and member substrates, the highly selective response of group I anion accumula- tion to substrate stimulation provides an exquisitely sensitive tool for determination of physiologic substrates, functions and biochemical correlates of transport. The graphical representation (Figure 17) of the rate of oxygen- dependent slice PAH uptake (Figure 16) was qualitatively consistent with the existence of two active modes of PAH transport. However, the validity of using Michaelis-Menten kinetics to describe PAH accumula- tion in this slice system must be considered (Weiner, 1973; Christen- sen, 1960). The kinetic analysis of PAH transport was performed on data obtained in experiments designed to achieve a maximum rate of active uptake while minimizing the passive diffusion component of PAH uptake. Ross and Weiner (1972) demonstrated that freshly prepared renal cortical slices exhibit decreased adenylate energy charges, probably due to hypoxia during preparation. Aerobic incubation for 30 minutes prior to estimating uptake rate allowed the slices to regain normal adenylate energy charges and presumably normal levels of tissue 177 constiuents necessary for maximal rates of transport. The kinetic analysis of slice PAH accumulation is complicated by the presence of nonsecretory tissue in slices and the PAH content of interstitial space. Therefore, PAH accumulation under nitrogen was determined to partially correct for diffusional processes. Since the observed Km and Vmax of the low capacity PAH uptake mode were within the range of values found by other investigators for PAH secretion (Gerencser ggngl., 1973; Park ggmgl., 1971), this portion of the curve may represent the group I transport system described above. Bareny (1972,1973b) found little competitive interaction between PAH and the L—transport system (Hippurate-insensitive system) even at medium PAH concentrations greater than those used in this study (Figure 16). Thus, PAH transport by the L-system would be negligible at best. Therefore the high capacity mode of PAH uptake (Figure 17) likely represents PAH transport by the overlapping group II substrate system. Initial interest in the biochemical mechanisms mediating renal extraction of nonesterified fatty acids (NEFA) was derived from in- vestigations (Lee g£_§l,, 1962; Nieth and Schollmeyer, 1966; Barac- Nieto and Cohen, 1968; Weidemann and Krebs, 1969) which suggested that NEFA were the major substrate whose oxidation supported most renal work functions. Although subsequent studies (Barac—Nieto and Cohen, 1971; Park g£_§l,, 1973; Bertermann g; Elf: 1975) have demonstrated that oxidation of palmitate to supply energy was not the major fate of palmitate in the renal cortex, net renal NEFA uptake was directly proportional to renal tubular sodium reabsorption (Barac-Nieto and Cohen, 1968). Therefore the nonoxidative fates of NEFA have been 178 suggested to play a significant role in support of renal sodium reabsorption (Cohen, 1975). The organic acid secretory system has been suggested to parti- cipate either in the entry of NEFA into proximal tubule cells or in the intracellular binding of fatty acids prior to activation and dissimilation (Cohen and Kamm, 1976). Support for this hypothesis has been derived primarily from analyses of NEFA uptake and metabolism by kidney preparations in the presence of inhibitors of organic acid transport (e.g., probenecid, PSP, chlorothiazide, furosemide) (Barac- Nieto and Cohen, 1968; Dies g£_gl,, 1970; Pakarinen, 1970; Heinemann 23 31-9 1975). However, interpretation of these experiments has been complicated by actions of these compounds unrelated to their specific inhibitory action on renal PAH transport. In contrast, administration of penicillin to immature rabbits specifically enhanced the PAH transport capacity of the kidney (Hirsch and Hook, 1970a,b; Ecker and Hook, l974b; Pegg and Hook, 1975b; 1977; Pegg g£_§l., 1976). There- fore, an alteration in proximal tubule palmitate accumulation or metabolism following induction of PAH transport capacity via substrate stimulation would be expected if palmitate interacts with (i.e., is a substrate of) the PAH tranSport system. The experiments were designed to maximize the possibility of detecting a penicillin induced alteration in palmitate utilization. Brendel and Meezan (1975) demonstrated that proximal tubules isolated nonenzymatically were morphologically intact and had intact basement membranes in contrast to tubules isolated with the use of collagenase treatment. This preparation of tubules equalled or surpassed those isolated with the use of collagenase in metabolic functions such as 179 oxidative metabolism and gluconeongenesis and in transport functions such as PAH and 2-deoxyg1ucose uptake (Brendel and Meezan, 1975). Electron micrographs of randomly selected proximal tubule suspensions (Figures 29—32) confirmed the observations of Brendel and Meezan (1975). That is, tubules prepared by this procedure had intact brush border and basement membranes. Therefore, suspensions of proximal tubules prepared by the method of Brendel and Meezan were used in this series of experiments. Weidemann and Krebs (1969) demonstrated that oleate utilization by rat kidney cortex slices incubated in a phos- phate buffered medium occurred at a high rate only at a fatty acid-to- albumin molar ratio of 7. Even at such a high oleate-to-albumin molar ratio, endogenous lipids still provided half the fuel for oxidation (weidemann and Krebs, 1969). Similarly, Barac-Nieto (1976) found that both the rates of esterification and oxidation of palmitate by kidney slices were limited when a low fatty acid-to-albumin molar ratio ( palmitate]/[BSA] = 1.25) was used. Both the esterification and oxidation rate were increased when the molar ratio in the medium.was increased (Barac-Neito, 1976). Therefore, in order to insure a high rate of palmitate-1-14C utilization a palmitate—to-albumin ratio of approximately 6 was used in these experiments. Since this ratio was much higher than those existing ig_yiyg, extrapolation of these results to the ig_yigg_situation should be made cautiously. These experiments were designed to explore only potential capabilities of the metabolic systems so that an interaction between renal palmitate utilization and PAH transport might be further defined (Barac-Nieto, 1976). 180 Proximal tubular palmitate esterification to triglyceride was significantly enhanced when tubule PAH transport capacity was in- creased by penicillin (Figures 33, 34, Table 10). This observation was consistent with the hypothesis that the metabolic fate of palmi- tate within the kidney was linked to transport by (or at least, inter— action with) the PAH secretory system. The inability of iodipamide pretreatment to alter either tubule PAH accumulation or palmitate metabolism (Figure 35) strengthened this position. Similarly, since penicillin pretreatment does not alter organic anion secretion in mature rabbits (Hirsch and Hook, 1969; Pegg and Hook, 1975b) no effect on tubular capability to accumulate PAH and metabolize palmitate was expected, or observed (Figure 36). When data from inhibitor studies were examined (Heinemann g£_§1,, 1975) an analagous response was observed. That is, esterification of palmitate—l-IAC to triglycerides was inhibited under conditions that would be expected to completely inhibit PAH accumulation (Heinemann §£_al,, 1975). Thus, inhibitors of PAH accumulation increased palmitate-14C recovered as NEFA and decreased radiolabel present as triglyceride relative to the amounts present in uninhibited slices (Heinemann g£_§1,, 1975). Recently wagner and Heinemann (1977) demonstrated that increasing medium po— tassium (and thereby tissue potassium) concentration promoted the accumulation and subsequent esterification and oxidation of palmitic acid. When medium potassium concentrations was increased from 6 mM Rf to 50 mM KI, esterification of palmitate~14C to triglyceride was significantly increased (wagner and Heinemann, 1977). Interestingly, the accumulation of PAH by renal cortical slices is modulated by medium potassium concentration (Cross and Taggart, 1950; Taggart 35 181 31,, 1953). Cross and Taggart (1950) demonstrated that the optimal medium potassium concentration for maximal PAH accumulation was 40 mM K+, or similar to the 50 mM K+ at which Wagner and Heinemann (1977) observed the maximal rate of palmitate esterification to triglyceride. These results are completely consistent with the hypothesis that palmitate interacts with the PAH transport system. The increased rate of palmitate esterification to triglyceride (Table 9) observed in tubules from 4 weeks as compared to 2 week old rabbits corresponded with the marked increase in PAH transport capacity between 2 and 4 weeks of age (Table 9). The mechanism by which substrate stimulation with penicillin enhanced palmitate esterification remains obscure. Several investiga— tors have demonstrated that addition of metabolic substrate such as glucose, lactate and glutamine to incubation medium could alter both the rate and eventual disposition of palmitate by kidney cortex slices (Weidemann and Krebs, 1969; Chorvathova g; 31., 1975; Barac-Nieto, 1976). Since acetate was routinely added to the palmitate medium in these experiments, penicillin treatment might have altered acetate metabolism by renal tubules. Secondary products of acetate metabolism could have then altered palmitate metabolism nonspecifically. How- ever, the same pattern of alterations in palmitate esterification was produced when tubules from penicillin treated and control rabbits were incubated in an acetate-free medium (Figures 37, 38). Therefore, penicillin did not appear to alter palmitate esterification in this fashion. Since substrate stimulation with penicillin had no effect on body weight (Hirsch and Hook, 1969) and did not change plasma NEFA concentration (Table 12) alterations in diet or an extrarenal effect(s) 182 of penicillin (e.g., increased lipolysis of adipose tissue triglyce- ride) were not indicated. Penicillin stimulation of PAH transport capacity in neonatal rabbits has been shown to be dependent on an intact protein synthetic mechanism (Hirsch and Hook, 19700; Pegg and Hook, 1975b). Hirsch and Hook (1970c) also demonstrated that peni- cillin pretreatment significantly increased the protein content of the 100,000 x g pellet from renal cortical homogenates of nursing rats. In addition, penicillin treatment significantly increased the incorpor- ation of 14C-amino acid into the acid-insoluble fraction of similar homogenates (Hirsch and Hook, 1970c). These observations coupled with the effect of penicillin on palmitate esterification to triglyceride suggest that penicillin might increase the synthesis of one or more enzymes involved in triglyceride formation (e.g., acyl-GOA: glycerophos- phate acyl-transferase, acyl—GOA: 1-acylglycerophosphate acyltransferase; phosphatidate phosphohydrolase) (Hubscher, 1970; Kupke, 1972; Bremer g£_§l,, 1976). Alternatively, substrate stimulation with penicillin might increase the activity of existing enzyme(s) by stimulating synthesis of a peptide activator (Gelehrter, 1976). Palmitic acid has been shown to competitively inhibit PAH uptake by rat renal cortical slices (Barac—Nieto, 1971). Penicillin pre- treatment of immature rabbits increased PAH transport capacity of proximal tubules, enhanced esterification of palmitate—14C to trigly- ceride and appeared to reduce the intracellular concentration of nonesterified fatty acids in proximal tubules (Table 11). These observations could indicate that the stimulation of PAH transport capacity by substrate stimulation resulted from an increased transfer of an inhibitor (NEFA) from an intracellular binding site Shared with 183 PAH to a storage depot (TG) with which PAH did not interact. Barac- Nieto (1971) postulated a similar mechanism, that is, removal of NEFA from a common intracellular binding site, to explain the stimulation of slice PAH accumulation in the presence of exogenous carnitine. These results might suggest that agents or treatment regimens which result in a reduction of the intracellular concentration of NEFA, regardless of the mechanism, would enhance PAH transport capacity. The lack of effect of acetate on palmitate esterification and recovery of label associated with NEFA (Figures 39, 40) was consistent with the proposal (Hirsch and Hook, 1970b; Pegg and Hook, 1975b) that acetate and substrate stimulation enhanced PAH transport capacity by separate mechanisms. Addition of sodium acetate to the incubation medium increased final medium pH (Figure 18). Several investigators (Copenhaver and Davis, 1965; Bowman ggugl., 1973) demonstrated that maximal PAH accumulation by rabbit renal cortical slices occurred at pH 8. However, Mudge and Taggart (1950) concluded that enhanced PAH transport could not be explained solely by the alkalinizing effect of acetate. A similar conclusion was obtained when the effect of various TCA cycle intermediates on PAH accumulation was examined (Figures 22,24,25). Low concentrations of citrate, a-ketoglutarate and succinate signifi- cantly increased PAH accumulation without appreciably altering the final medium pH (Figures 22,24,25). This marked dissociation between medium pH and PAH accumulation suggests that these intermediates do not stimulate PAH accumulation merely by alkalinizing the incubation medium. The parallel changes in medium pH and PAH accumulation with acetate (Figures 14,18,19) suggested that stimulation of PAH accumulation 184 resulted from the metabolism of acetate rather than from a direct effect of acetate on the transport system. Maxild (1973) and Ger- encser 35 El- (1977) demonstrated that acetate stimulation of PAH accumulation did not occur by increasing renal cortical tissue ATP but was nevertheless, dependent on oxidative energy metabolism. Cohen and Randall (1964) postulated that renal metabolic changes could affect PAH transport if specific substances were to accumulate or disappear from within the kidney. Therefore, it would appear reasonable to suspect that acetate stimulated PAH accumulation by formation of a positive modulator of anion transport. Dixit and Stern (1974) demonstrated that formation of citrate was a likely intermediate step in renal metabolism of acetate both igugigg_ and inhyiggg, Preparation and incubation of rabbit renal cortical slices significantly reduced tissue citrate concentration (Figure 20). Acetate (10 mM) significantly elevated both citrate concentration and the ability of rabbit slices to accumulate PAH (Figure 20). Although Cross and Taggart (1950) demonstrated that 0.01 M citrate slightly depressed PAH accumulation, a more physiological concentration of citrate (Cohen and Barac—Nieto, 1973) significantly increased PAH S/M ratios (Figure 21). A dose-response curve (Figure 22) described a biphasic relationship between PAH accumulation and medium citrate concentration. Low concentrations of citrate in the medium.stimu1ated while higher concentrations depressed PAH accumulation. The changes in slice citrate concentration and PAH accumulation produced by acetate coupled with the stimulating effect of physiolo- gical concentrations of citrate on PAH accumulation suggested that 185 transport of organic anions is regulated by the renal cortical con- centration of citrate. This hypothesis was supported by the obser- vation that the PAH transport capacity of a given species was corre- lated with the endogenous renal cortical citrate contraction (Table 2). Similar correlations between acetate stimulation of slice PAH accumulation and citrate concentration (Table 2) coupled with enhanced PAH accumulation in the presence of added citrate (Table 3) strengthened the hypothesis. Alterations in organic anion tranSport (Weiss and Preuss, 1970) and renal citrate metabolism (Simpson, 1967; Simpson and Sherrard, 1969; Simpson and Angielski, 1973; Pahsley and Cohen, 1973) in various acid-base states also support the hypothesis that citrate could act as an intracellular modulator of PAH transport. Maintenance of systemic acid-base homeostasis is one of the major regulatory functions of the kidney. Several excretory and nonexcre- tory (i.e., metabolic) approaches are utilized to fulfill this func- tion. For example, the enhanced excretion of ammonium (NH4+) ions following increased production of ammonia (NH3) by deamination and deamination of glutamine in renal cortical mitochondria is an example of a major excretory mechanism operating in systemic acidosis. The product of renal glutamine metabolism is a-KG, a low pKa dicarboxylic acid. If each mole of u-KG produced from glutamine were released into the renal venous blood it would neutralize 2 moles of bicarbonate (HCO3—), thus reducing the efficiency of renal HCOB- regeneration (Cohen and Kamm, 1976). However, renal cortical metabolism undergoes several alterations in order to eliminate this problem. Renal cortical 186 tissue exhibits a markedly increased rate of gluconeogenesis and C02 production from u-KG and other organic acids (e.g., citrate, lactate) in response to the reduction in intracellular fluid pH (ICF pH) and/or bicarbonate concentration (ICF [HC03-]) produced by metabolic acidosis, respiratory acidosis or Kf-depletion (Cohen and Kamm, 1976). The enhanced rate of conversion of these organic acids to neutral products (glucose, C02) during acidosis increases the efficiency of renal NH 3 regeneration and thus acts as a major nonexcre— 3 tory (metabolic) mechanism in the kidney for pH regulation (Cohen and production and HCO Kamm, 1976). However, these metabolic adaptations also offer an explanation for the reduced PAH transport capacity observed in chro— nically acidotic or Kfedepleted rats. Both metabolic acidosis (Hams g£_§l,, 1971; Adler g£_§l,, 1971; Relman, 1972; Cohen and Kamm, 1976) and Kfedepletion (Adler gg_gl., 1974; Cohen and Kamm, 1976) signifi- cantly depressed ICF pH and/or ICF[HC03-] and significantly reduced renal citrate concentration. The concentration of all other TCA cycle intermediates was also reduced. The experiments described above (Table 5) confirmed the relationship between reduced cortical citrate concentration and reduced PAH transport capacity in chronic metabolic acidosis and provide additional evidence that citrate could act as an, intracellular modulator of PAH accumulation. The inability of acetate to stimulate PAH accumulation in slices from acidotic rats was parti- cularly intriguing (Table 5). Metabolic acidosis reduced renal cor- tical oxaloacetate concentration below the limit of detection (Relman, 1972). Since oxaloacetate is necessary for citrate synthesis, addi- tion of exogenous acetate would not be expected to elevate citrate concentration in slices from acidotic rats and therefore would not be 187 expected to elevate slice PAH accumulation. Conversely, acetate did elevate both citrate concentration and PAH accumulation in slices from normal rats (Table 2). Thus, it would appear that either sufficient endogenous oxaloacetate was present in kidney cortex to permit citrate synthesis from exogenous acetate or that endogenous precursors sup- plied the requisite oxaloacetate. Interestingly, PAH accumulation by slices from acidotic rats was significantly stimulated by addition of citrate or lactate (Hewitt and Hook, unpublished results). Since lactate increased both slice citrate concentration and PAH accumulation to a greater extent than acetate (Table 4), the slice oxaloacetate concentration may limit acetate-induced stimulation of PAH accumulation. However, Cross and Taggart (1950) suggested that acetate rather than oxaloacetate was one of the rate limiting components of the PAH trans— port system.since oxaloacetate had only 25% of the stimulatory ability of an equimolar concentration of acetate._ In contrast to acidosis, the kidney conserves acidic substrates during metabolic alkalosis (Cohen and Kamm, 1976). Gluconeongensis from organic acids was depressed whereas conversion of glucose and glycerol to lactic acid was enhanced. The renal utilization of citrate appeared to be particularly sensitive to alterations in ICF[HCO3-] whether produced igwyiyg_or ig_yiggg_(Simpson, 1967). For example, citrate oxidation and utilization by renal cortical slices and/or mitochondria was decreased when medium [HCO3-] was elevated (Simpson, 1967; Pashley and Cohen, 1973). The 2-3 fold increase in renal cortical citrate concentration observed during metabolic alka- 10313 was consistent with decreased citrate metabolism (Simpson, 1963; Adler g£_§l,, 1971; Relman, 1972). The rise in tissue citrate (and 188 other organic acids) concentration during alkalosis would tend to correct the metabolic alkalosis by neutralizing a portion of the HCOB- generated in the kidney and thus would serve as a nonexcretory mecha- nism for regulating systemic acid—base balance (Cohen and Kamm, 1976). The increased hippurate accumulation by kidney cortex slices from alkalotic rats (Weiss and Preuss, 1970) is consistent with the sug— gestion that enhanced tissue citrate concentration, arising from either renal metabolic adaptations or exogenous substrate, modulates PAH transport capacity. Additional evidence for citrate involvement in acetate stimulation is provided in Figure 23. Pre-loading of slices with citrate increased PAH accumulation above NaCl pre-incubated controls while reducing stimulation of accumulation produced by acetate. These data suggested that acetate stimulation of PAH accumulation has a maximal value determined by cortical citrate concentration or possibly by a limited capacity of the slices to contain higher concentrations of PAH. Although the data presented above support the concept that citrate concentration functions as a positive modulator of PAH accumulation, they did not permit the elimination of other possible metabolites (e.g., a—ketoglutarate) which follow citrate in the TCA cycle from consideration as possible regulators of PAH transport. Various investigators (Cross and Taggart, 1950; Farah g; gl,, 1955; Koishi, 1959) demonstrated that several renal metabolic sub— strates (e.g., a—ketoglutarate, octanoate) enhanced PAH accumulation by cortical slices when present at low medium concentrations. Dose— response curves for a—ketoglutarate and succinate (Figures 24, 25) confirmed the biphasic relationship between PAH accumulation and medium concentration of various TCA cycle intermediates. 189 Progressive alkalinization of the incubation medium occurred as medium substrate (citrate, a—ketoglutarate or succinate) concentration was increased to 5.0 mM (Figures 22,24,25). However, substrate con- centrations greater than 5.0 mM (Figure 22) did not produce additional increases in final medium pH. The relationship between substrate concentration and final medium pH cannot be adequately explained. Accummlation of these substances EEHXEEEQ is, at least in part, via a peritubular transport system(s) similar to that for PAH (Selleck and Cohen, 1965: Balagura—Baruch et al., 1973). Maximal alkalinization of the medium could conceivably be reflecting maximal transport or meta— bolism of these compounds. Although medium pH reached optimal levels for PAH accumulation at high medium substrate concentrations (Figures 22,24,25), PAH S/M ratios were markedly depressed. Competition for a limited number of transport sites could explain the observed inhi- bition of PAH accumulation. Acetate (10.0 mM) stimulated PAH accumulation and increased slice citrate concentration suggesting that restoration of slice citrate to endogenous levels (or moderately increasing citrate stores above endo— genous concentrations) was the key step in PAH accumulation. Citrate might act as a positive modulator of PAH accumulation. However, citrate could stimulate accumulation of PAH by acting as precursor for a subsequent, positive modulator of anion transport. This possibility was examined by determining slice PAH accumulation and citrate con- centration when a TCA cycle intermediately downstream from citrate was present as substrate. a—Ketoglutarate was chosen as a representative intermediate. In addition, the effect of glucose on these parameters 190 was determined. a—Ketoglutarate (0.5 mM) significantly stimulated PAH accumulation but did not alter slice citrate concentration (Figure 26). Glucose, a substrate not extensively utilized by kidney cortex (Cohen and Barac-Nieto, 1973), had no effect on either PAH accumula- tion or slice citrate concentration. These results demonstrated that intermediates following citrate in the TCA cycle could serve as modulators of PAH accumulation through a mechanism independent of replacement of depleted cellular citrate stores. Slices were incubated with acetate as substrate (Figure 27) to determine if citrate and a-ketoglutarate could act as modulators through two, similar, but distinct mechanisms or if citrate stimulated PAH accumulation solely by increasing slice a-ketoglutarate concentra- tion. Incubation of slices in 10.0 mM acetate increased slice PAH accumulation, citrate concentration and a—ketoglutarate concentration (unpublished results). However, Kim and Hook (1972) demonstrated that maximal stimulation (100%) of PAH accumulation by rabbit renal cor— tical slices was achieved at a medium concentration of 2.0 mM acetate. Thus, total tissue capacity for PAH, rather than modulator concentra- tion could have been the limiting factor in PAH accumulation when 10.0 mM acetate was present as substrate. Therefore, slices were incubated with 1.0 mM acetate in order that tissue metabolite concentrations could be determined under conditions where the intrinsic slice capa- city for PAH was not the limiting factor in PAH accumulation. Tissue citrate concentration was depleted in control slices (Figure 27) whereas a-ketoglutarate concentration was not different than the endogenous concentration. Acetate (1.0 mM) produced submaximal 191 stimulation of PAH accumulation and increased both tissue citrate and d-ketoglutarate. Although with acetate, the a-ketoglutarate concen- tration was greater than the endogenous concentration, acetate did not completely restore slice citrate concentration. The increase in PAH accumulation (51%) appeared to correlate better with the submaximal increase in slice citrate concentration (42%) than the increase in slice a-ketoglutarate content concentration (220%) (Figure 27). These investigations suggested that a single mechanism for modulation of PAH accumulation probably does not exist. Citrate, a-ketoglutarate, and most likely other TCA cycle intermediates, apparently modulate PAH accumulation through at least two similar, but separate, mechanisms. SPECULATION Separate mechanisms for stimulation of PAH accumulation by TCA cycle intermediates could be envisioned if these intermediates served as counter—anions for PAH accumulation by their respective mitochondrial translocating systems. Three different carriers which catalyze the permeation of TCA cycle intermediates through the inner mitochondrial membrane have been characterized: (a) for dicarboxylates (malate, succinate); (b) for a-ketoglutarate; (c) for tricarboxylates (citrate, isocitrate) (Klingen- berg, 1970). These anion translocators bring about an energy-dependent, one-to-one exchange of an intramitochondrial for an extramitochondrial anion (Klingberg, 1970; Meijer and Van Dam, 1974). Therefore, a major modulating factor for the transport of one anion is the concentration of the appropriate counter—anion (Meijer and Van Dam, 1974). Theo- retically then, citrate or u-ketoglutarate could serve as counter- anions for PAH, first accumulating within the mitochondrial matrix space and then exchanging with cytosolic PAH. Compartmentalization of PAH within the mitochondrial matrix space would represent the energy~ dependent intracellular concentrating mechanism in the model of PAH transport proposed by Foulkes and Miller (1959). Enhanced accumu- lation of PAH in the presence of acetate or low concentrations of citrate, u-KG and succinate (Figures 22, 24, 25) would result from an increased matrix space concentration of counter-anion(s) available for 192 193 exchange with PAH. Conversely, inhibition of PAH accumulation at high medium concentrations of TCA cycle intermediates (Figures 22,24,25) might represent competition with PAH for the available carrier sites on the inner mitochondrial membrane. Some support for such a hypothesis can be derived from experi— mental data present in the literature. Simpson and Angielski (1973) demonstrated the existence of a citrate translocating system in mito- chondria isolated from rabbit renal cortex. The tricarboxylate carrier system, at least in hepatic mitochondria, was extremely un- specific (Kleineke §£_§1,, 1973). In addition to citrate, threo-D3- isocitrate, L-malate, phosphoenolpyruvate (PEP), several structurally related anions including phenyl—PEP, D-2-phosphoglycerate (D-Z-PGA), ”“ D-3-PGA and L-2—PGA were able to exchange with intramitochondrial tricarboxylates via the tricarboxylate carrier system (Kleineke gg gl,, 1973). An interaction of secreted organic anions with renal anion translocators can also be inferred from the data of Pakarinen and Runeberg (1969). PSP and probenecid inhibited the oxidation of citrate, a—ketoglutarate and succinate by mitochondria prepared from guinea pig kidney cortex. Inhibition of a-KG and succinate by PSP was concentration dependent, with 50% inhibition occurring at a medium concentration of 1.5 mM PSP. Citrate oxidation was relatively resis- tant to PSP, with a 40% inhibition achieved with 3.0 mM PSP in the medium, Interestingly, at lower medium PSP concentrations (0.75 mM and 1.5 mM PSP) citrate oxidation was stimulated (Pakarinen and Runeberg, 1969). The effects of probenecid on oxidation of these intermediates differed slightly. The concentration of probenecid (0.3 mM) required to inhibit a-KG oxidation 50% was much less than that of 194 PSP (1.5 mM). However, at this probenecid concentration, citrate and succinate oxidation were only slightly depressed (lo-20%). Probene— cid, at a concentration (0.75 mM) which inhibited a-KG oxidation by 90% did not produce a further reduction of either citrate or succinate oxidation (Pakarinen and Runeberg, 1969). These observations are consistent with (but do not prove) the concept that secreted organic anions inhibit the translocation of TCA cycle intermediates into the mitochondria and thereby reduce the oxidation. The concentration related effects discussed might be interpreted as demonstrating a much higher affinity of the a—ketoglutarate translocator (as opposed to the citrate and dicarboxylate translocators) for probenecid and PSP. However, the inhibition of citrate and succinate oxidation suggested that all of the translocators were subject to inhibition. The obser- vation that rabbit kidney cortex mitochondria accumulated PAH (Tables 7,8), against a concentration gradient, was also consistent with an interaction of secreted organic anions with mitochondrial anion trans- locating systems. In this model, mitochondrial PAH accumulation has been postulated to depend on the concentration of citrate (and other counter-anions) within the matrix space. As mentioned above, weiss and Preuss (1970) demonstrated that PAH transport capacity was increased in alkalosis. This observation appeared to be consistent with the proposed model of PAH accumulation since renal cortical citrate concentration was also found to be elevated in alkalosis (Simpson, 1963; Adler g£_al., 1971; Relman, 1972). However, the matrix space citrate concentration of rabbit kidney cortex mitochondria has been shown to be significantly decreased under alkalotic conditions (Simpson and Angielski, 1973). 195 Simpson and Angielski (1973) demonstrated that the steady-state matrix space citrate concentration was regulated by alterations in medium [HCO3-]. Increased medium [HCOB-1 decreased matrix space citrate concentration whereas lowering medium [HCOB-J below normal increased the concentration of citrate in the matrix space. Since regulation occurred in the presence or absence of inhibition of TCA cycle acti- vity, Simpson and Angielski (1973) concluded that bicarbonate altered citrate transport by the tricarboxylate translocator of the inner mitochondrial membrane. These investigators suggested that the increased [HC03-] of alkalosis enhanced the rate of citrate transport out of the matrix space. No evidence for inhibition of citrate entry into the matrix space by [HC03-] was presented. Thus, PAH accumu1a~ tion by the citrate translocator might be modulated by the rate of citrate efflux from the mitochondria rather than by the steady-state matrix space citrate concentration. The movement of citrate from the matrix space to the medium (cytoplasm) has also been shown to be modulated by long chain fatty acyl-CoA. The hepatic mitochondrial tricarboxylate carrier was re-‘ versibly inhibited by long-chain fatty acyl—CoA when added ig_git£g_or in states where these intermediates were elevated 13 zigg_(Ha1perin 35 _§1., 1972; Cheema-Dhaldi and Halperin, 1976; WOjtczak, 1976). Inhi- bition was concentration-related, competitive in nature and dependent on the chain length of the fatty acid moiety (WOjtczak, 1976). If fatty acyl-CoA inhibit citrate transport from.mitochondria in kidney as they do in liver, then mitochondrial PAH accumulation would pre- sumably, be inhibited by fatty acyl-00A. The degree of inhibition would be determined by the concentration of fatty acyl—00A. As 196 mentioned above, palmitate competitively inhibits slice PAH accumu- lation (Barac-Nieto, 1971). Since palmitate is rapidly activated and metabolized by kidney tissue (Heinemann g£_§l., 1970; Cohen and Kamm, 1976), addition of palmitate to incubation media would be expected to increase tissue fatty acyl-CoA concentration and thereby inhibit PAH accumulation. Similarly, factors which depress the concentration of this intermediate by enhancing catabolism (carnitine) or esterifica- tion (penicillin substrate stimulation) would be expected to secon- darily increase the rate of citrate exchange and thereby enhance mitochondrial PAH accumulation. An interaction of PAH with mitochondrial anion translocators need not be postulated to explain substrate stimulation by penicillin. The first step in NEFA utilization by cells is probably transfer of NEFA from albumin, the intravascular carrier, to binding sites on the cell surface (Spector, 1971; Heinemann ggugl., 1975). Following binding, the water-insoluble NEFA must cross an aqueous medium (cytoplasm), in which they would be expected to exhibit only limited solubility, to reach their intracellular sites of dissimilation. Intracellular movement of NEFA is apparently facillated by binding of NEFA to specific cyt0plasmic proteins which reduce their hydrophobicity (Ockner g£_§l,, 1972; Ockner and Manning, 1974,1976; Mishkin 25 al., 1972,1975). Two such proteins, Y protein (GSH—S-transferase B; Ligandin) and Z-protein (FABP = fatty acid binding protein) have been isolated from the cytoplasm of several tissues, including kidney cortex (Ockner gE_§l,, 1972; Ockner and Manning, 1974, 1976; Mishkin Ig§_§l,, 1972,1975). Analysis of binding properties has demonstrated an approximately equal affinity of these proteins for NEFA (association 197 constant = 106M.1 for oleate) (Kamisaka g£_§l,, 1975a,b; Listowsky, 1976). As mentioned in the introduction, Y—protein has been suggested to be a major component of the renal organic anion transport system. As such, Y-Protein (and Z-protein) binds numerous small organic anions, including PAH (Kirsch g£_§l., 1975; Arias §£_§l,, 1976). However, the affinity of Y-protein for these organic acids was found to be approxi- mately lOOO—fold less than the affinity for NEFA (association constant = 103 M71) (Kamisaka g£_§l,, 1975a; Listowsky gghgl., 1976). At the PAH concentrations normally observed ig_gi££g_and ig_gigg, NEFA would act essentially as an irreversible inhibitor of PAH binding. If binding of PAH to Y- (or Z-) protein was a requisite step in PAH accumulation, the transport capacity of the system would be determined by the amount of NEFA bound to these proteins whereas the rate of PAH accumulation would be determined by the rate at which the NEFA was removed. Thus, stimulation of reaction sequences utilizing NEFA either for oxidation (carnitine) or esterification (penicillin sub- strate stimulation) would result in an increased PAH transport capa- city and an increased rate of PAH accumulation. This model of PAH transport would also be consistent with Foulkes and Miller's proposed model of PAH accumulation. In this example, PAH binding to Y— (or Z—) protein would represent the intracellular concentrating mechanism. The reaction sequences responsible for the ATP dependent activation of NEFA prior to metabolism would serve as the energy-requiring component and as the rate-limiting step in PAH accumulation since they would catalyze the removal of an otherwise irreversible inhibitor of trans- port. 198 The two models proposed here are obviously based on a number of assumptions for which little or no experimental data are available. These hypotheses have been discussed primarily because they represent two, among several, possible mechanisms for PAH accumulation which fit the majority of the experimental data, can be tested experimen- tally and therefore be used to further characterize the biochemical sequences responsible for secretion of organic anions. SUMMARY The purpose of this investigation was three—fold: 1) to demon- strate the existence of multiple tranSport systems for the secretion of organic acids within the kidney; 2) to provide additional evidence that palmitate is an endogenous substrate of the PAH transport system; 3) to provide evidence that an intracellular modulator of the PAH transport system exists and to elucidate the role of this modulator in acetate-induced stimulation of PAH accumulation by renal cortical slices. The newborn rabbit kidney was used as an experimental model because maturation of anion transport can be selectively accelerated by administration of exogenous functional loads. Three distinct developmental patterns of organic anion accumula— tion by rabbit renal cortical slices were observed. Anions were ten- tatively placed into three groups based on similarities in patterns of development of slice accumulation. Group I contained PAH, PSP and PEN; group II contained UA, ASA and SULFI; and CDC was placed alone in group III. The effect of administering various agents to newborn rabbits was evaluated according to this tentative classifi- cation. PEN pretreatment markedly enhanced maturation of group I anion accumulation without altering slice accumulation of UA, ASA or CDC. PEN stimulation of SULFI accumulation was minimal and probably related to accumulation by the overlapping group I system. 199 200 Pretreatment of neonatal rabbits with most group II or group III anions did not alter group I anion accumulation. ASA stimulation of PAH accumulation was probably secondary to the marked toxicity ob- served following ASA pretreatment. Group II and group III anion admini- stration had no effect on maturation of their respective transport systems. Graphical representation of the rate of slice PAH uptake was consistent with the existence of two active modes of PAH transport. The low capacity PAH uptake mode represented the group I transport system whereas the high capacity uptake mode probably represented PAH transport by the overlapping group II substrate system. These results supported the concept of multiple renal secretory systems for organic anions. Analysis of renal NEFA extraction and metabolism in the presence of inhibitors of organic acid and transport suggested that the PAH secretory system participated either in the entry of NEFA into proximal tubule cells or in the intracellular binding of the fatty acids prior to activation and dissimilation. Palmitate metabolism by proximal tubule suSpensions prepared from penicillin treated newborn rabbits supported this suggestion. Penicillin pretreatment increased PAH accumulation by suspensions of proximal tubules and enhanced palmi- tate esterification to triglycerides. Palmitate-140 recovered as NEFA in penicillin treated tubules was decreased. Age—related changes in palmitate esterification to TG correlated with similar changes in tubule PAH accumulation. Neither iodipamide administra- tion to immature, or penicillin treatment of mature, rabbits altered tubule PAH accumulation or palmitate esterification. These results 201 suggested that PAH and palmitate shared, at least in part, a common transport system. In addition, penicillin apparently enhanced PAH accumulation by enhancing maturation of an enzymatic pathway(s), resulting in increased transfer of endogenous inhibitors (NEFA) from an intracellular binding site shared with PAH. Accumulation of PAH by renal cortical slices from several species was increased when acetate was included in the incubation medium. The parallel changes in medium pH with acetate suggested that stimulation of PAH accumulation resulted from metabolism of acetate to a positive modulator of transport rather than a direct effect of acetate on the transport system. The PAH transport capacity of slices from various species was correlated with the endogenous renal cortical citrate concentration. Preparation and incubation of slices depleted tissue citrate concentration. Acetate increased citrate concentration and PAH accumulation by kidney cortex slices. Physiological concentra- tions of citrate increased PAH accumulation and final medium pH. Increasing concentrations of citrate produced a biphasic effect on PAH accumulation. These data suggested that citrate may act as an intra- cellular modulator of organic anion transport. This hypothesis was tested with other stimulators of PAH accumulation. Physiologic con- centration of a-ketoglutarate or succinate increased slice PAH accumu- lation. Higher concentrations of either substrate inhibited PAH accumulation. Final medium pH increased with increased medium concen- tration of both substrates. a-Ketoglutarate increased PAH accumula- tion but had no effect on slice citrate concentration. Slices incu- bated without substrate were depleted of citrate but not a-ketoglutarate. 202 Acetate significantly increased slice concentration of both a-keto- glutarate and citrate. Thus, the group I anion transport system could be modulated by several metabolic intermediates acting through similar, but separate, mechanisms. Rabbit kidney cortex mitochondria accumulated PAH against a concentration gradient. This observation suggested that TCA cycle intermediates might serve as counter-anions for PAH accumulation by their respective mitochondrial translocating systems. BIBLIOGRAPHY Abel, J.A.: Analgesic Nephropathy - A review of the literature, 1967- 1970. Clin. Pharmacol. Ther. 12: 583-598, 1971. Adam, W.R. and Simpson, D.P.: Glutamine transport in rat kidney mito- chondria in metabolic acidosis. J. Clin. Invest. 55: 165-174, 1974. Adler, S., Anderson, B. and Zemotel, L.: Metabolic acid-base effects on tissue citrate content and metabolism in the rat. Am. J. Physiol. 220: 986-992, 1971. Alguire, P.C., Bailie, M.C., Weaver, W.J., Taylor, D.G. and Hook, J.B.: Differential effects of furosemide and ethacrynic acid on elec- trolyte excretion in anesthetized dogs. J. Pharmacol. Exp. Ther. 1129: 515-522, 1974. Anderson, H.M. and Mudge, G.H.: The effect of potassium on intra- cellular bicarbonate in slices of kidney cortex. J. Clin. Invest. 34: 1691-1697, 1955. Anderson, J., Kopko, F., Nohle, E.G. and Siedler, A.J.: Intracellular accumulation of nitrofurantoin by rabbit renal cortical slices. Am. J. Physiol. 217: 1435-1440, 1969. Aprille, J.R.: Dietary lipid and postnatal development. 11. Palmityl coenzyme A oxidation in heart and liver. Pediat. Res. 19: 982—985, 1976. Aprille, J.R. and Rulfs, J.: Dietary lipid and postnatal development. I. A model for neonatal studies requiring high and low fat diets from birth. Pediat. Res. 19: 978—981, 1976. Arias, I.M., Fleischner, G., Kirsch, R., Mishkin, S. and Gatmaitain, 2.: 0n the structure, regulation and function of ligandin. In Glutathione: Metabolism and Function, ed. I.M. Arias and W. B. Jakoby, pp. 175—188. Raven Press, New York, 1976. Baerlocher, K.E., Scriver, C.R. and Mohyuddin, F.: The ontogeny of amino acid transport in rat kidney. I. Effect of distribution ratios and intracellular metabolism of proline and glycine. Biochim. Biophys. Acta'gflg: 353-363, 1971a. 203 BIBLIOGRAPHY 204 Baerlocher, K.E., Scriver, C.R. and Mohyuddin, F.: The ontogeny of amino acid transport in rat kidney. 11. Kinetics of uptake and effect of anoxia. Biochim. Biophys. Acta 249: 364-372, 1971b. Balagura-Baruch, S. and Stone, W.J.: Renal tubular secretion of p- aminohippurate in the dog: Effects of a-ketoglutarate. Nephron 6; 633-642, 1969. Balagura—Baruch, S., Burich, R.L. and King, V.F.: Effects of alkalosis on renal citrate metabolism in dogs infused with citrate. Am. J. Physiol. 225: 385-388, 1973. Barac-Nieto, M.: Renal uptake of p-aminohippuric acid ig_vitro. Biochim. Biophys. Acta 233: 446-452, 1971. Barac-Nieto, M.: Effects of lactate and glutamine on palmitate metabo- lism in rat kidney cortex. Am. J. Physiol. 231: 14-19, 1976. Barac-Nieto, M. and Cohen, J.J.: Nonesterified fatty acid uptake by dog kidney: Effects of probenecid and chlorothiazide. Am. J. Physiol. 215: 98-107, 1968. Barac-Nieto, M. and Cohen, J.J.: The metabolic fates of palmitate in the dog kidney ig_vivo; evidence for incomplete oxidation. Nephron 8: 488-499, 1971. Barany, E.H.: Inhibition by hippurate and probenecid of ig_vitro uptake of iodipamide and o-iodohippurate. A composite uptake system of iodipamide in choroid plexus, kidney cortex and anterior uvea of several species. Acta Physiol. Scand. 86: 12-27, 1972. Barany, E.H.: The liver—like anion transport system in rabbit kidney, uvea and choroid plexus. I. Selectivity of some inhibitors, direction of transport, possible physiological substrates. Acta Physiol. Scand. 88: 412—429, l973a. Barany, E.H.: The liver-like anion transport system in rabbit kidney, uvea and choroid plexus. II. Efficiency of acidic drugs and other anions as inhibitors. Acta Physiol. Scand. 88: 491-504, 1973b. Bérény, E.H.: Selectivity of probenecid congeners for different organic acid transport systems in rabbit renal cortex. Acta Pharmacol. et Toxicol. 35: 309-316, 1974a. Barany, E.H.: Bile acids as inhibitors of the liver-like anion trans— port system in the rabbit kidney, uvea and choroid plexus. Acta Physiol. Scand. 22: 195-203, 1974b. Barany, E.H.: Ig_vitro uptake of bile acids by choroid plexus, kidney cortex and anterior uvea. I. The iodipamide-sensitive transport systems in the rabbit. Acta Physiol. Scand. 23; 250-268, 1975. 205 Barnett, H.L.,.McNamara, H., Schultz, S. and Tomsett, R.: Renal clearances of sodium penicillin G, procaine penicillin G and inulin in infants and children. Pediatrics 8: 418-422, 1949. Barach, S.E., Burich, R.L., Eun, C.K. and King, V.F.: Renal metabo- lism of citrate. Med. Clin. N. Amer. 82: 569-582, 1975. Berndt, W.O.: The efflux of urate from rabbit renal cortex slices. J. Pharmacol. Exp. Ther. 150: 414-426, 1965. Berndt, W.O.: Use of the tissue slice technique for evaluation of renal transport processes. Environ. Health Perspec. 18: 73-88, 1976. Berndt, W.0. and Beechwood, E.G.: Influence of inorganic electrolytes and ouabain on uric acid transport. Am. J. Physiol. 208: 642- 648, 1965. Berndt, W.0., Mudge, G.H., James, W., Grote, D. and Akerman, L.: Renal excretion of iodipamide. Comparative study in the dog and rabbit. Invest. Radiol. 8: 414-426, 1968. Berner, W. and Kinne, R.: Transport of p—aminohippuric acid by plasma membrane vesicles isolated from rat kidney cortex. Pflugers Arch. 361: 269-277, 1976. Bertermann, H., Gronow, G., Schirmer, A. and Weiss, C.: Contribution of long chain fatty acids to the energy supply of the rat kidney cortex. Pflugers Arch. 356: 9-17, 1975. Beyer, K.H.: Functional characteristics of renal transport mechanisms. Pharmacol. Rev. 8: 227-280, 1950. Beyer, K.H., Peters, L., Woodward, R. and Verwey, W.F.: The enhance- ment of the physiological economy of penicillin in dogs by the simultaneous administration of para-aminohippuric acid. J. Pharmacol. Exp. Ther. 88: 310-323, 1944. Bieber, L.L., Pettersson, B. and Lindberg, 0.: Studies on norepine— phrine induced efflux of free-fatty acid from hamster brown adipose tissue cells. Eur. J. Biochem. 88: 375-381, 1975. Blank, M.L. and Snyder, F.: Qualitative and Quantitative Aspects of Thin Layer Chromatography in the Analysis of Phosphorous—Free Lipids, In: Analysis g§_Lipids and Lipoproteins, ed. E.G. Perkins, pp. 6369, Am. Oil Chemists Soc., Champaign, 1975. Bond, J.T., Bailie, M.D. and Hook, J.B.: Maturation of renal organic acid transport i3 vivo: Substrate stimulation by penicillin. J. Pharmacol. Exp. Ther. 199: 25-31, 1976. Boner, G. and Steele, T.H.: Relationship of urate and p-aminohippurate secretion in man. Am. J. Physiol. 225: 100-104, 1973. 206 Bowman, H.M., Hirsch, G.H. and Hook, J.B.: Effect of medium pH on p- aminohippurate accumulation by slices of rat renal cortex. Experientia 88: 955-956, 1973. Bratton, A.C. and Marshall, E.R., JR.: A new coupling component for sulfanilamide determination. J. Biol. Chem. 128: 537—550, 1939. Bremer, J., Bjerve, K.S., Borrebaek, B. and Christiansen, R.: The glycerophosphateacyltransferases and their function in the meta— bolism of fatty acids. Mol. Cell Biochem. 88: 113-125, 1976. Brendel, K. and Meezan, E.: Isolation and prOperties of apure pre- paration of proximal kidney tubules obtained without collagenase treatment. Fed. Proc. 83: 803, 1974. Calcagno, P.L. and Rubin, M.I.: Renal extraction of para-aminohippurate in infants and children. J. Clin. Invest. 88: 1632-1639, 1963. Burg, M.B. and Orloff, J.: Oxygen consumption and active transport in separated renal tubules. Am. J. Physiol. 203: 327-330, 1962. Carrasquer, G. and Wilczewski, T.W.: Effect of uretral stop flow on PAH in lumen and cortex homogenate in the rat kidney. Proc. Soc. Exp. Biol. Med. 137: 284-291, 1971. Cheema-Dhadli, S. and Halperin, M.L.: Effect of palmitoyl-CoA and B-oxidation of fatty acids on the kinetics of mitochondrial citrate transporter. Can. J. Biochem. 23: 171-177, 1976. Chorvathova, V., Dzfirick, R., Ondreicka, R., Cerven, J. and Nemec, R.: Effect of glucose on the utilization of palmitate-l-14C by rat kidney cortex slices. Physiol. Bohemoslovaca 83: 217-220, 1975. Chen, R.F.: Removal of fatty acids from serum albumin by charcoal treatment. J. Biol. Chem. 242: 173—181, 1967. Christensen, H.N.: Some special kinetic problems of transport. Adv. Enzymol. 88: 120, 1969. Chung, S.T., Park, Y.S. and Hong, S.K.: Effect of cations on trans— port of weak organic acids in rabbit kidney slices. Am. J. Physiol. 219: 30—33, 1970. Cohen, J.J.: Metabolic support for renal sodium reabsorption. Med. Clin. N. Amer. 82: 523—538, 1975. Cohen, J.J. and Kamm, D.E.: Renal metabolism: Relation to renal function. In: The Kidney, Vol. 1, ed. B.M. Brenner and F.C. Rector, Jr., pp. 126-214, W.B. Saunders Co., Philadelphia, 1976. Cohen, J.J. and Randall, E.W.: Alkalosis and renal p—aminohippurate transport in dog: Relation to lactate uptake. Am. J. Physiol. 206: 383-390, 1964. 207 Cohen, J.J. and Wittman, E.: Renal utilization and excretion of u- ketoglutarate in dogs: Effects of alkalosis. Am. J. Physiol. 204: 795-811, 1963. Cohen, J.J. and Barac-Nieto, M.: Renal metabolism of subsrates in relation to renal function. In: Handbook 88_Physiology. Section 8: Renal Physiology, edited by J. Orloff, R.W. Berliner, and S.R. Geiger, Baltimore: Williams and Wilkins, 1973, pp. 909—1002. Cohen, J.J., Chesney, R.W., Brand, P.H., Neville, H.F. and Blanchard, C.F.: a—Ketoglutarate metabolism and K+ uptake by dog kidney slices. Am. J. Physiol. 217: 161-169, 1969. Cohen, L., Lapkin, R. and Kaloyanides, G.J.: Effect of gentamicin on renal function in the rat. J. Pharmacol. Exp. Ther. 193: 264- 273, 1975. Conney, A.H.: Pharmacological implications of microsomal enzyme induction. Pharmacol. Rev._18: 317-366, 1967. Cook, W.F. and Pickering, G.W.: A rapid method for separating glo- meruli from rabbit kidney. Nature 182: 1103-1104, 1958. Copenhaver, J.H., JR. and Davis, J.R.: Effects of hydrogen ion concentration on transport characteristics of p-aminohippurate by rabbit kidney slices. Proc. Soc. Exp. Biol. Med. 119: 611-614, 1965. Cross, R.J. and Taggart, J.V.: Renal tubular transport: Accumulation of p-aminohippurate by rabbit kidney slices. Am. J. Physiol. 161: 181-190, 1950. Dantzler, W.H.: Comparison of renal tubular transport of urate and PAH in water snakes: Evidence for differences in mechanisms and sites of transport. Comp. Biochem. Physiol. 84: 609—623, 1970. Dantzler, W.H.: PAH transport by snake proximal renal tubules: Differences from urate transport. Am. J. Physiol. 226: 634—641, 1974. Despopoulos, A.: 88_vitro effects of acetate ion on renal metabolism of p-aminohippurate. Am. J. Physiol. 184: 396-399, 1950. DespOpoulos, A.: Renal metabolism of salicylate and salicylurate. Am. J. Physiol. 198: 230-232, 1960. Despopoulos, A.: A definition of substrate specificity in renal transport of organic anions. J. Theoret. Biol. 8: 163-192, 1965. Despopoulos, A. and Callahan, P.X.: Molecular features of sulfonamide transport in renal excretory processes. Am. J. Physiol. 203: 19- 26, 1962. 208 Dies, F., Herrera, J., Matos, M., Avelar, E. and Ramos, C.: substrate uptake by dog kidney 8§_vivo. Am. J. Physiol. 218: 405-410, 1970a. Dies, F., Ramos, G., Avelar, E. and Lenahoff, M.: Renal excretion of lactic acid in the dog. Am. J. Physiol. 216: 106-111, 1969. Dies, F., Ramos, G., Avelar, E. and Matos, M.: Relationship between renal substrate uptake and tubular sodium reabsorption in the dog. Am. J. Physiol. 218: 411-416, 1970b. Dies, F., Sandoval, G., Martinez, R., Garza, R. and Ordéfiez, A.: Effects of probenecid, alkalosis and glycine on net renal uptake and on tubular reabsorption of lactate in dogs. Rev. Invest. Clin. (Mex.) 88: 111-123, 1974. Dixit, P.K. and Stern, A.M.: Method for determining incorporation of acetate into citrate. Proc. Soc. Exp. Biol. Med. 146: 176-185, 1974. Ecker, J.L. and Hook, J.B.: Analysis of factors influencing the EB. vitro developmental pattern of p-aminohippurate transport by rabbit kidney. Biochim. Biophys. Acta 339: 210-217, 19743. Ecker, J.L. and Hook, J.B.: Accumulation of p-aminohippuric acid by separated renal tubules from newborn and adult rabbits. J. Pharmacol. Exp. Ther. 190: 325-357, 1974b. Edelmann, C.M. and Spitzer, A.: The maturing kidney. J. Pediat. 88: 509-519, 1969. Edwards, J.G. and Marshall, E.K., Jr.: Microscopic observations of the living kidney after injection of phenolsulfonphthalein. Am. J. Physiol. 88: 489-495, 1924. Eveloff, J., Morishige, W.K. and Hong, S.R.: The binding of phenol red to rabbit renal cortex. Biochim. Biophys. Acta 448: 167- 180, 1976. Farah, A. and Rennick, 3.: Studies on the renal tubular transport of tetraethylammonium ion in renal slices of the dog. J. Pharmacol. Farah, A., Frazer, M. and Stoffel, M.: Studies on the run out of p-aminohippurate from renal slices. J. Pharmacol. Exp. Ther. 139: 120-128, 1963. Farah, A., Koda, F. and Frazier, M.: The action of fluoroacetate on the p—aminohippurate uptake of renal slices of the dog. J. Pharmacol. Exp. Ther. 113: 169-177, 1955. Farah, A., Graham, G. and Koda, F.: The action of sodium fluoro- acetate on the renal tubular transport of para-aminohippurate and glucose in the dog. J. Pharmacol. Exp. Ther. 108: 410-423, 1953. 209 Folch, J., Lee, M. and Sloane-Stanley, G.H.: A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 266: 497-509, 1957. Forster, R.P.: Renal transport mechanisms. Fed. Proc. 88: 1008- 1019, 1967. Forster, R.P. and Copenhaver, J.H., JR.: Intracellular accumulation as an active process in a mammalian renal transport system 88_ vitro. Am. J. Physiol. 186: 167-175, 1956. Foulkes, E.C.: Kinetics of p-aminohippurate secretion in the rabbit. Am. J. Physiol. 205: 1019-1024, 1963. Foulkes, E.C.: Peritubular transport of urate in rat kidneys. Pflfigers Arch. 360: 1-6, 1975. Foulkes, E.C. and Miller, B.F.: Steps in p-aminohippurate transport by kidney slices. Am. J. Physiol. 196: 86-92, 1959. Foulkes, E.C. and Miller, B.F.: The role of potassium in renal trans- port of p-aminohippurate. In: Membrane Transpprt and Metabolism, ed. A. Kleinzeller and A. Kotyk, pp. 559-565, Academic Press, London, 19691. Gelehrter, T.D.: Enzyme induction. N. Eng. J. Med. 294: 589-595, 1976. Gerencser, G.A., Chaisetseree, C. and Hong, S.K.: Acetate influence upon the transport kinetics of p-aminohippurate at 37°C in rabbit kidney slices. Proc. Soc. Exp. Biol. Med. 154: 397-400, 1977. Gerencser, G.A., Park, Y.S. and Hong, S.K.: Sodium influence upon the transport kinetics of p-aminohippurate in rabbit kidney slices. Proc. Soc. Exp. Biol. Med. 144: 440-444, 1973. Halperin, M.L., Robinson, B.H. and Fritz, I.B.: Effects of palmitoyl CoA on citrate and malate transport by rat liver mitochondria. Proc. Nat. Acad. Sci. U.S.A. 88: 1003-1007, 1972. Harris, E.J. and Van Dam, K.: Changes of total water and sucrose space accompanying induced ion uptake or phosphate swelling of rat liver mitochondrion. Biochem. J. 106: 758-766, 1968. Heinemann, H.O., wagner, M. and Frederiksen, A.: Palmitic acid uti- lization by the renal cortex of the rat. Med. Clin. N. Amer. 88: 699-711, 1975. Hems, D.A. and Brosnan, J.T.: Effects of metabolic acidosis and starvation on the content of intermediary metabolites in rat kidney. Biochem. J. 123: 391-397, 1971. 210 Herndon, R.P. and Freeman, 8.: Renal citric acid utilization in the dog. Am. J. Physiol. 888: 369—372, 1958. Hirsch, G.H.: Differential effects of nephrotoxic agents on renal organic ion transport and metabolism. J. Pharmacol. Exp. Ther. 1139 593-599, 1973. Hirsch, G.H.: Effects of potassium depletion in rats on renal organic ion transport. Can. J. Biochem. 88: 90-92, 1974. Hirsch, G.H. and Hook, J.B.: Maturation of renal organic acid trans- port: Substrate stimulation by penicillin. Science 165: 909- 910, 1969a. Hirsch, G.H. and Hook, J.B.: Stimulation of PAH transport by slices of rat renal cortex following 83_vivo administration of triiodo- thyronine. Proc. Soc. Exp. Biol. Med. 131: 513-517, 1969b. Hirsch, G.H. and Hook, J.B.: Maturation of renal organic acid trans- port: Substrate stimulation by penicillin and p-aminohippurate (PAH). J. Pharmacol. Exp. Ther. 171: 103-105, 1970a. Hirsch, G.H. and Hook, J.B.: Additional studies on penicillin-induced stimulation of renal PAH transport. Can. J. Physiol. Pharmacol. _4_1_3_: 550-556, 1970b. Hirsch, G.H. and Hook, J.B.: Stimulation of renal organic acid transport and protein synthesis by penicillin. J. Pharmacol. Exp. Ther. 174: 152-158, 1970c. Hirsch, G.H., Cowan, D.F. and Hook, J.B.: Histological changes in normal and drug-induced development of renal PAH transport. Proc. Soc. Exp. Biol. Med. 137: 116-121, 1971. Ho, R.J.: Radiochemical assay of long-chain fatty acids using 63Ni as tracer. Anal. Biochem. 88: 105-113, 1970. Hollister, L. and Levy, G.: Some aspects of salicylate distribution and metabolism in man. J. Pharm. Sci. 88: 1126-1129, 1965. Holohan, P.D., Pessah, N.I. and Ross, C.R.: Binding of N'-methyl- nicotinamide and p-aminohippuric acid to a particulate fraction from dog kidney. J. Pharmacol. Exp. Ther. 195: 22-33, 1975. Holohan, P.D., Pessah, N.I. and Ross, C.R.: Reconstitution of N'- methylnicotinamide (NMN) and p-aminohippurate (PAH) transport in phospholipid vesicles with a protein fraction isolated from dog kidney microsomal membranes. Kid. Int. 88: 585, 1976. Hook, J.B.: Postnatal development of renal transport systems in the maturing kidney. Proc. 5th Int. Cong. Nephrol., Mexico 8: 6-13, 1974. 211 Hook, J.B., Williamson, H.E. and Hirsch, G.H.: Functional maturation of renal PAH transport in the dog. Can. J. Physiol. Pharmacol. £§= 169-175, 1970. Horster, M. and Lewy, J.B.: Filtration fraction and extraction of PAH during neonatal period in the rat. Am. J. Physiol. 219: 1061- 1065, 1970. Horster, M. and Valtin, H.: Postnatal development of renal function: micropuncture and clearance studies in the dog. J. Clin. Invest. 88: 779-795, 1971. Huang, K.C. and Lin, O.S.T.: Kinetic studies on transport of PAH and other organic acids in isolated renal tubules. Am. J. Physiol. 208: 391-396, 1965. Hfibscher, C.: Glyceride Metabolism. In: Lipid Metabolism, ed. S.J. wakil, pp. 279-370, Academic Press, New York, 1970. Johnson, J.T., Holloway, L.S., Heisey, S.R. and Hook, J.B.: Substrate stimulation of organic anion transport in newborn dog kidney and choroid plexus. Biochem. Pharmacol. 88: 754-758, 1974. Jones, C.T. and Ashton, I.K.: The appearance, properties and functions of gluconeogenic enzymes in the liver and kidney of the guinea pig during fetal and early neonatal development. Arch. Biochem. Biophys. 888: 506-422, 1976. Kamisaka, K., Listowsky, I., Gatmaitan, Z. and Arias, I.M.: Inter- actions of bilirubin and other ligands with ligandin. Biochem. _1_4_: 2175-2180, 1975a. Kamisaka, K., Listowsky, 1., Gatmaitan, Z. and Arias, I.M.: Circular dichroism analysis of the secondary structure of 2 protein and its complexes with bilirubin and other organic anions. Biochim. Biophys. Acta 888: 24—30, 1975b. Kaplowitz, N. and Clifton, C.: The glutathione S-transferases in rat liver and kidney: Drug induction, hormonal influences and organic anion-binding. In: Glutathione: Metabolism and Function, ed. I.M. Arias and W.B. Jakoby, pp. 301-308, Raven Press, New York, 1976. ’ Kim, J.K. and Hook, J.B.: On the mechanism of acetate enhancement of renal p-aminohippurate transport. Biochim. Biophys. Acta 290: 368-374, 1972. Kim, J.K., Hirsch, G.H. and Hook, J.B.: 88_vitro analysis of organic ion transport in renal cortex of the newborn rat. Pediat. Res. 8: 600-605, 1972. 212 Kippen, I., Whitehouse, M.W. and Klineberg, J.R.: Pharmacology of uricosuric drugs. Ann. Rheum. Dis._88: 391-396, 1974. Kirsch, R., Fleischner, G., Kamisaka, K. and Arias, I.M.: Structural and functional studies of ligandin, a major renal organic anion-binding protein. J. Clin. Invest. 88: 1009-1019, 1975. Klahr, S., Robson, A.M., Guggenheim, S.J., Tateishi, S., Bourgoignie, J.J. and Hwang, K.H.: Ammonium induced alterations in PAH up- take and cation composition of kidney slices. Am. J. Physiol. 219: 994-1000, 1970. Kleineke, J., Sauer, H. and Soling, H.D.: On the specificity of the tricarboxylate carrier system in rat liver mitochondria. F.E.B.S. Letters 88: 82-86, 1973. Klingenberg, M.: Metabolite transport in mitochondria: An example for intracellular membrane function. Essays Biochem. 8: 119- 159, 1970. Knoefel, P.K. and Huang, K.C.: Biochemorphology of renal tubular transport: Hippuric acid and related substances. J. Pharmacol. Exp. Ther. 126: 296-303, 1959. Koishi, T.: Studies on renal tubular transport. 1. Accumulation of p- aminohippurate by kidney slices. Jap. J. Pharmacol. 8: 101-123, 1959. Kramp, R.A. and Lenoir, R.H.: Characteristics of urate influx in the rat nephron. Am. J. Physiol. 229: 1654-1661, 1975. Kfipke, I.R.: Lipid Biosynthesis. In: Progress 18_Biochemical Pharmacolqu, Vol. 8, ed. W.L. Holmes and W.M. Bortz, pp. 57-124, Karger, Basel, 1973. Lapkin, R., Enser, H. and Kaloyanides, G.J.: Neomycin stimulates PAH transport in rat kidney slices. Fed. Proc. 88: 408, 1975. Layne, E.: Spectrophotometric and turbidimetric methods for measuring protein. Methods Enzymol. 8: 447-454, 1957. Lee, J.B., Vance, V.K. and Cahill, G.F., Jr.: Metabolism of Cla- labelled substrates by rabbit kidney cortex and medulla. Am. J. Physiol. 203: 27-36, 1962. Lewy, J.B., and Grosser, D.: Stimulation of PAH transport in the maturing rabbit kidney. Third Intl. Symp. on Ped. Nephrol., Abstracts, p. 7, 1974. Listowsky, 1., Kamisaka, Kl, Ishitani, K. and Arias, I.M.: Structure and prOperties of ligandin. In: Glutathione: Metabolism and Function, ed. I.M. Arias and W.B. Jakoby, pp. 233-242, Raven Press, New York, 1976. 213 Lowry, G.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.: Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951. Malvin, R.L., Wilde, W.S. and Sullivan, L.P.: Localization of nephron transport by stop flow analysis. Am. J. Physiol. 194: 135-141, 1958. Mandel, H.G.: Pathways of drug biotransformation: Biochemical conjugations. In: Fundamentals of Drug Metabolism and Drug Disposition, ed. B.N. LaDu, H.G. Mandel and E.L. Way, pp. 149- 186, The Williams and Wilkins Company, Baltimore, 1971. Martensson, J.: On the citric acid metabolism in mammals. Acta Physiol. Scand. (Suppl.) 88: 1-96, 1940. Maxild, J.: Role of fatty acid metabolism on renal transport of p- aminohippurate 12 vitro. Biochim. Biophys. Acta 233: 434-445, 1971. Maxild, J.: Energy requirements for active transport of p—amino- hippurate in renal cortical slices. Arch. Int. Physiol. Biochim. 88: 501-521, 1973. Maxild, J. and Muller, J.V.: Metabolic studies on renal transport of p-aminohippurate 88_vitro. Biochim. Biophys. Acta 184: 614-624, 1969. May, D.G. and Weiner, I.M.: The renal mechanisms for the excretion of m-Hydroxybenzoic acid in cebus monkeys: Relationship to urate transport. J. Pharmacol. Exp. Ther. 176: 407-417, 1971. McArdle, B.: A modified method for the microdetermination of citric acid. Biochem. J._88: 647-649, 1955. McIsaac, R.J.: The uptake of hexamethonium-C14 by kidney slices. J. Pharmacol. Exp. Ther. 150: 92-98, 1965. Meezan, E., Brendel, K., Ulreich, J. and Carlson, E.C.: Properties of a pure metabolically active glomerular preparation from rat kidneys. 1. Isolation. J. Pharmacol. Exp. Ther. 187: 332-341, 1973. Meijer, A.J. and Van Dam, K.: The metabolic significance of anion transport in mitochondria. Biochim. Biophys. Acta 346: 213- 244, 1974. Meisel, A.D. and Diamond, H.S.: Inhibition of probenecid uricosuria by pyrazinamide and para-aminohippurate. Am. J. Physiol. 232: F222—F226, 1977. 214 Mishkin, S., Stein, L., Fleischner, G., Gatmaitan, Z. and Arias, I.M.: Z protein in hepatic uptake and esterification of long-chain fatty acids. Am. J. Physiol. 228: 1634-1640, 1975. Mishkin, S., Stein, L., Gatmaitan, Z. and Arias, I.M.: The binding of fatty acids to cytoplasmic proteins: Binding to Z protein in liver and other tissues of the rat. Biochem. Biophys. Res. Comm. 88: 997-1003, 1972. Mdller, J.V.: The relation between secretion of urate and p-amino- hippurate in the rabbit kidney. J. Physiol. 192: 505-517, 1967a. Mdller, J.V.: The renal accumulation of urate and p-aminohippurate in the rabbit. J. Physiol. 192: 519-527, 1967b. Mudge, G.H. and Taggart, J.V.: Effect of acetate on the renal excre- tion of p-aminohippurate in the dog. Am. J. Physiol. 161: 191- 197, 1950a. Mudge, G.H. and Taggart, J.V.: Effect of 2,4-dinitrophenol on renal transport mechanisms in the dog. Am. J. Physiol. 161: 173-180, 1950b. Mudge, G.H., Berndt, W.O. and Valtin, H.: Tubular transport of urea, glucose, phosphate, uric acid, sulfate and thiosulfate. In Handbook 88_Physiolggy, Section 8: Renal Physiology, ed. by J. Orloff, R.W. Berliner and S.R. Geiger, pp. 587-652, The Williams and Wilkins Company, Baltimore, 1973. Murdaugh, H.V., Jr. and Elliot, H.C.: Effect of glycine excess on para-aminohippurate uptake by the kidney. Proc. Soc. Exp. Biol. Med. 130: 1181-1182, 1969. New, M., McNamara, H. and Kretchmer, N.: Accumulation of para-amino- hippurate by slices of kidney from rabbits of different ages. Proc. Soc. Exp. Biol. Med. 102: 558-560, 1959. Nielsen, P. and Rasmussen, F.: Relationships between molecular struc- ture and excretion of drugs. Life Sci. 88: 1495-1512, 1975. Nieth, H. and Schollmeyer, P.: Substrate utilization of the human kidney. Nature 209: 1244-1245, 1966. Nikkila, E.A.: Transport of free fatty acids. In: Progress $3 Biochemical Pharmacolqu, Vol. 6, ed. W.L. Holmes and WQM. Bortz, pp. 102-129, Karger, Basel, 1971. Noordewier, B. and Withrow, C.D.: Effects of substrate on the matu- ration of PAH transport. Fed. Proc. 88: 224, 1976. 215 Ockner, R.K. and Manning, J.A.: Fatty acid-binding protein in small intestine: Identification, isolation, and evidence for its role in cellular fatty acid transport. J. Clin. Invest. 88: 326-338, 1974. Ockner, R. K. and Manning, J.A.: Fatty acid binding protein: Role in esterification of absorbed long chain fatty acid in rat intestine. J. Clin. Invest. 88: 632—641, 1976. Ockner, R.K., Manning, J.A., Poppenhausen, R.B. and Ho, W.K.L.: A binding protein for fatty acids in cytosol of intestinal mucosa, liver, myocardium, and other tissues. Science 177: 56-58, 1972. Orsulak, P.J., Haab, W. and Appleton, M.D.: Quantitative estimation of uric acid, xanthine and hypoxanthine in plasma using thin- layer chromatography. Anal. Biochem. 88: 156-162, 1968. Pakarinen, A.: Palmitate uptake and oxidation by kidney cortex slices. Effects of probenecid, p-aminohippurate and phenolsul- fonphthalein. Biochem. Pharmacol. 88: 2707-2718, 1970. Pakarinen, A. and Runeberg, L.: Effects of phenolsulfonphthalein and probenecid on the uptake and utilization of citrate and a- ketoglutarate by kidney 88_vitro. Biochem. Pharmacol. 88: 2439- 2452, 1969. Park, H.G., Leal-Pinto, E., MacLeod, M.B. and Pitts, R.F.: CO production from plasma free fatty acids by the intact functioning kidney of the dog. Am. J. Physiol. 227: 1192-1198, 1974. Park, Y.S., Yoo, H.S. and Hong, S.K.: Kinetic studies on transport of organic acids in rabbit kidney slices. Am. J. Physiol. 220: 95- 99, 1971. Pashley, D.H. and Cohen, J.J.: Substrate interconversion in dog kidney cortex slices: regulation by ECF-pH. Am. J. Physiol. 225: 1519-1528, 1973. Pegg, D.G. and Hook, J.B.: Pharmacodynamic analysis of substrate stimulation of p-aminohippurate transport by newborn rabbit kidney. J. Pharmacol. Exp. Ther. 195: 16-21, 1975b. Pegg, D.G. and Hook, J.B.: Glutathione S-transferases: An evaluation of their role in renal organic anion transport. J. Pharmacol. Pegg, D.G., Bernstein, J. and Book, J.B.: Biochemical and ultrastruc- tural correlates of substrate stimulation of renal organic anion transport. Proc. Soc. Exp. Biol. Med. 151: 720-725, 1976a. Pegg, D.G., McCormack, K.M. and Hook, J.B.: Effect of substrate pre- treatment on renal organic ion transport in the adult rat. Experientia 88: 1315-1316, 1976b. 216 Phelps, D.L., Omori, K. and 0h, W.: PAH clearance, sodium excretion and PAH extraction ratio in acidotic near-term lambs treated with hypertonic sodium bicarbonate. Biol. Neon. 88: 57—62, 1976. Platts, M.M. and Mudge, G.H.: Accumulation of uric acid by slices of kidney cortex. Am. J. Physiol. 200: 387-391, 1961. Putney, J.W., JR., and Borzelleca, J.F.: Active accumulation of 14C- salicylic acid by rat kidney cortex 88_vitro. J. Pharmacol. Exp. Rastegar, A. and Thier, 8.0.: The physiologic approach to hyperuri- cemia. N. Eng. J. Med. 286: 470-476, 1972. Relman, A.S.: Metabolic consequences of acid-base disorders. Kid. Int. 8: 347-359, 1972. Rennick, B.R.: Renal excretion of drugs: tubular transport and meta- bolism. In: Annual Review 88_Pharmac018gy: Edited by H.W. Elliot, R. Okun and R. George. Palo Alto: Annual Reviews, Vol. 12, pp. 141-156, 1972. Rennick, B., Hamilton, B. and Evans, R.: Development of renal tubular transports of TEA and PAH in the puppy and piglet. Am. J. Physiol. 201: 743-746, 1961. Ross, C.R. and Farah, A.: p-Aminohippurate and N-methylnicotinamide transport in dog renal slices - an evaluation of the counter- transport hypothesis. J. Pharmacol. Exp. Ther. 151: 159-167, 1966. Ross, C.R. and Weiner, I.M.: Adenine nucleotides agd PAH transport in slices of renal cortex: Effects of DNP and ON . Am. J. Physiol. 222: 356-359, 1972. Ross, C.R., Pessah, N.I. and Farah, A.: Studies of uptake and runout of p-aminohippurate and N-methylnicotinamide in dog renal slices. Schachter, D., Manis, J.G. and Taggart, J.V.: Renal synthesis, degra- dation and active transport of aliphatic acyl amino acid. Relationship to p-aminohippurate transport. Am. J. Physiol. 888: 537-544, 1955. Schreiner, G.E.: Determination of inulin by means of resorcinol. Proc. Soc. Exp. Biol. Med. 88: 117-120, 1950. Schwartz, G.J., Hegyi, T., and Spitzer, A.: Sub-therapeutic di- cloxacillin levels in a neonate. Possible mechanisms. J. Pediat. 88: 310—316, 1976. 217 Seevers, M.H., Shideman, F.E., Whods, L.A., Weeks, J.R. and Kruse, W.T.: Dehydroacetic acid (DHA). II. General pharmacology and mechanism of action. J. Pharmacol. Exp. Ther. 88: 69-83, 1950. Segal, S. and Smith, 1.: Delineation of cystine and cysteine trans- port systems in rat kidney cortex by developmental patterns. Proc. Natl. Acad. Sci. US 88: 926-933, 1969a. Segal, S. and Smith, 1.: Delineation of separate transport systems in rat kidney cortex for L-lysine and L-cystine by developmental patterns. Biochem. Biophys. Res. Comm. 88: 771-777, 1969b. Segal, S., Rea, C. and Smith, 1.: Separate transport systems for sugars and amino acids in developing rat kidney cortex. Proc. Natl. Acad. Sci. US 88: 372-376, 1971. Selleck, B.H. and Cohen, J.J.: Specific localization of a-ketogluta- rate uptake to dog kidney and liver 88 vivo. Am. J. Physiol. 208: 24-37, 1965. Selleck, B., Cohen, J.J. and Randall, B.M., Jr.: Enzymic assay of a- ketoglutarate in dog blood, plasma urine, and tissue. Anal. Biochem. 8: 178-188, 1964. Sereni, F. and Princip, N.: Developmental Pharmacology. Ann. Rev. Pharmacol. 8: 453-466, 1968. Sheikh, M.I.: Renal handling of phenol red. I. A comparative study on the accumulation of phenol red and p-aminohippurate in rabbit kidney tubules 88 vitro. J. Physiol. 277: 565-590, 1972. Sheikh, M.I. and Mdller, J.V.: The kinetic parameters of renal trans- port of p-aminohippurate 88_vitro. Biochim. Biophys. Acta 196: 305-319, 1970. Sheikh, M.I. and Mdller, J.V.: The mechagism of urate transport in rabbit kidney tubules 88 vitro. Pflugers Arch. 325: 235-246, 1971. Shideman, F.E. and Rene, R.M.: Succinate oxidation and Krebs cycle as an energy source for renal tubular transport mechanisms. Am. J. Physiol. 166: 104-112, 1951. Shideman, F.E., Rathbun, R.C. and Stoneman, F.: Inhibition of the renal tubular transport of p—aminohippurate (PAH) and phenol- sulfonphthalein (PSP) as affected by acetate. Am. J. Physiol. 888: 31-37, 1952. Shideman, F.E., woods, L.A. and Seevers, M.B.: Dehydroacetic acid (DHA). IV. Detoxification and effects on renal function. J. Pharmacol. Exp. Ther. 88: 98-111, 1950. 218 Simpson, D.P.: Tissue citrate levels and citrate utilization after sodium bicarbonate administration. Proc. Soc. Exp. Biol. Med. 114: 263—265, 1963. Simpson, D.P.: Regulation of renal citrate metabolism by bicarbonate ion and pH: observations in tissue slices and mitochondria. J. Clin. Invest. 88: 225-238, 1967. Simpson, D.P. and Adam, W.: Glutamine transport and metabolism.by mitochondria from.dog renal cortex. J. Biol. Chem. 250: 8148- 8158, 1975. Simpson, D.P. and Angielski, 8.: Regulation by bicarbonate ion of intramitochondrial citrate concentration in kidney mitochondria. Biochem. BiOphys. Acta 298: 115-123, 1973. Simpson, D.P. and Sherrard, D.J.: Regulation of glutamine metabolism 88 vitro by bicarbonate ion and pH. J. Clin. Invest. 88: 1088- 1096, 1969. Smith, S. and Abraham, 8.: The composition and biosynthesis of milk lipids. Adv. Lipid Res. 88: 195-236, 1975. Smith, H.W., Finkelstein, H., Aliminosa, L., Crawford, B. and Graber, M.: The renal clearances of substituted hippuric acid deriva- tives and other aromatic acids in dog and man. J. Clin. Invest. 88: 388-404, 1945. Sokal, R.R. and Rohlf, F.J.: Biometry, pp. 175-539, W.H. Freeman and Co., San Francisco, 1969. Sordahl, L.A., Johnson, C., Blailock, Z.R. and Schwartz, A.: The mitochondrion. In: Methods 88_Pharmacology, Vol. 1, ed. A. Schwartz, Chap. 8, pp. 247-286, Appleton-Century-Crofts, New York, 1971. Spector, A.: Metabolism of free fatty acids. In: Progress 12. Biochemical Pharmacology, Vol. 6, ed. W.L. Holmes and W.M. Bortz, pp. 130—176, Karger, Basel, 1971. Sperber, 1.: Secretion of organic acids in the formation of urine and bile. Pharmacol. Rev. 88: 109-134, 1959. Strickler, J.G. and Kessler, R.H.: Effects of certain inhibitors on renal excretion of salt and water. Am. J. Physiol. 205: 117-122, 1963. "“" Taggart, J.V.: Mechanisms of renal tubular transport. Am. J. Med. ‘88: 774-784, 1958. Taggart, J.V., Silverman, L. and Trayner, B.M.: Influence of renal electrolyte composition on the tubular excretion of p-amino- hippurate. Am. J. Physiol. 173: 345-350, 1953. 219 Torretti, J. and Weiner, I.M.: The renal excretion of drugs. In: Methods in_Pharmacology, Vol. 4A, Renal Pharmacology, ed. M. Martinez-Maldonado, Chap. 12, pp. 357-379, Plenum Press, New York, 1976. Trimble, M.B. and Bowman, RJH.: Renal Na+ and K+ transport: Effects of glucose, palmitate, and a-bromopalmitate. Am. J. Physiol. 225: 1057-1062, 1973. Tune, B.M., Burg, M.B. and Patlak, C.S.: Characteristics of p-amino— hippurate transport in proximal renal tubules. Am. J. Physiol. g_1_7: 1057-1063, 1969. Wagner, M.B. and Heinemann, H.G.: Effect of potassium on utilization of [1-14C1palmitic acid in renal cortex of the rat. Am. J. Physiol. 232: F254-F259, 1977. Walter, C.: Graphical procedures for the detection of deviations from the classical model of enzyme kinetics. J. Biol. Chem. 249: 699-703, 1974. Wan, S.E., Von Lehmann, B. and Riegelman, 8.: Renal contribution to overall metabolism of drugs III: Metabolism of p-aminobenzoic acid. J. Pharm. Sci. Q1: 1288—1292, 1972. Weidemann, M.J. and Krebs, R.A.: The fuel of respiration of rat Kidney cortex. Biochem. J. 112: 149-166, 1969. Weiner, I.M.: Transport of weak acids and bases. In: Handbook of Physiology, Section g, Renal Physiology, ed. J. Orloff, R.W. Berliner and S.R. Geiger, Baltimore, Williams and Wilkins, Weiner, I.M. and Fanelli, C.M., Jr.: Bidirectional transport: Urate and other organic anions. In: Recent Advances ig_Renal Physiology and Pharmacology, ed. L.G. Wesson and C.Mw Fanelli, Jr., University Park Press, Baltimore, pp. 53—68, 1974. weiner, I.MQ and Tinker, J.P.: Pharmacology of pyrazinamide: Meta- bolic and renal function studies related to the mechanisms of drug-induced urate retention. J. Pharmacol. Exp. Ther. 189: 411—434, 1972. Weiss, E.R. and Preuss, H.G.: Influence of extracellular and intra- cellular factors in hippurate uptake by rat kidney cortex: acid—base effects. Proc. Soc. Exp. Biol. Med. 135: 30-32, 1970. Welsh, L.T. and Bush, M.T.: Intracellular distribution and runout of p-aminohippurate in rabbit kidney slices. Am. J. Physiol. 218: 1751-1756, 1970. 220 Williamson, R.C. and Hiatt, E.P.: Development of renal function in fetal and neonatal rabbits using phenolsulfonphthalein. Proc. Soc. Exp. Biol. Med. gg; 554-557, 1947. Wojtczak, L.: Effect of long-chain fatty acids and acyl-CoA on mito- chondrial permeability, transport, and energy~coupling processes. J. Bioenerget. Biomemb. 8; 293-311, 1976. Yaffe, S.J. and Juchau, M.R.: Perinatal pharmacology. Ann. Rev. Pharmacol. 13; 219-238, 1974. Yeary, R.A., Benish, R.A. and Finkelstein, M.: Acute toxicity of drugs in newborn animals. J. Pediat. g2: 663-667, 1966. Zmuda, M.J. and Quebbemann, A.J.: Localization of renal tubular uric acid transport defect in gouty chickens. Am. J. Physiol. 229: 820-825, 1975. Zorzoli, A.: Gluconeogenesis in mouse kidney cortex. II. Glucose production and enzyme activities in newborn and early postnatal animals. Devel. Biol. 11; 400-412, 1968. INHIWHNH 0 5 1 2 5 8 o 3 0 3 9 1WWIIIUWIIIHIH1H SH 3 12 1 ElilliHHIH’ :I ll!» ,IL