R ram...” 55,. ., . a" I, . J,|'>’§‘J; «st-Join. bu. ..fl. "M .. , . a: ': :At'. ; Vub‘dL'nsdu .-. a " .'-‘ ’1‘ £555.} J h ”A. 1 “ _ .‘ ‘1' 3.5" 1’31 r“- .u ~ 5J4! 4d: . dd \ln . ..l «flu-trimm- ~~-- -- filing-:r; ' «may V u "71:“. - 4': '-_ ‘t.’.4¢ ¢;.3.'4- .2334; _ LIBRARY ' Michigan State Duh-emit)! This is to certify that the , thesis entitled BIOCHEMICAL CORRELATES OF ORGANIC ANION TRANSPORT IN DEVELOPING KIDNEY presented by DAVID GLENN PEGG has been accepted towards fulfillment of the requirements for Ph . D. degree in Pharmacology ’ / / f, ,./’ X/ I. ”(an 5 N ,./'// / / xix/4; - /E—«+~ - ’/ Major professor Date .June_3_0_._L925_ ; 0-7639 l , ”Milt. IMI‘II AA ‘ E'; l ABSTRACT BIOCHEMICAL CORRELATES OF ORGANIC ANION TRANSPORT IN DEVELOPING KIDNEY BY David Glenn Pegg Renal organic anion transport capacity is less in the newborn than in the adult of most animal species. A primary stimulus to development of transport is substrate availability. Pretreatment of newborn animals with substrates for the transport system such as penicillin increases the rate of transport maturation. The pur- pose of this investigation was, therefore: 1) to formulate a maximally stimulating penicillin dose and dosage regimen; 2) to investigate the mechanisms of substrate stimulation of renal organic anion transport; 3) to evaluate substrate stimulation as a tool to facilitate isolation and characterization of components of the transport system. Penicillin pretreatment of two-week-old rabbits significantly increased the ability of renal cortical slices to transport p-amino- hippuric acid (PAH). The PAH slice to medium ratio (S/M) increased with dose to a maximum at 90,000 1.0. procaine penicillin G per animal. Increasing the dose to 180,000 1.0. with sodium penicillin G pro- duced no greater increase. Stimulation was fully developed after 4 penicillin injections at 12-hour intervals. The maximal response David Glenn Pegg was observed 24 hours after the final injection, while after 72 hours the capacity of slices from treated animals to transport PAH was no different than control. To determine the effect of a maximal substrate challenge on the development of transport capacity, preg- nant does were treated with 90,000 I.U. procaine penicillin G i.m. twice daily through the last half of gestation. Young animals received 2 penicillin injections prior to sacrifice. PAH S/M from pups 3 days, 1 and 2 weeks old were not significantly different from that normally observed at 4 weeks. Thus, intrinsic transport capacity for PAH is mature at 4 weeks of age. Only prior to this age may transport be enhanced by substrate. Maximal enhancement of PAH transport capacity occurs when 4 injections of 90,000 I.U. procaine penicillin G are administered at 12-hour intervals fol- lowed by sacrifice 24 hours after the final injection. The effect of penicillin on PAH transport was investigated in rabbit renal cortical slices and separated proximal tubules. Slices were preincubated for 30 minutes, then PAH was added to produce con- centrations in the medium of l, 2 and 4 x 10'.4 M and the slices incubated for 15 minutes. The effect of penicillin pretreatment on runout was observed by preloading slices in 6.3 x 10-4 M PAH for 90 minutes. Slices were transferred at 1-minute intervals through a series of 20 beakers plus an initial rinse beaker. Results were expressed as ug PAH per 9 tissue remaining in the slice and runout constants calculated. Penicillin pretreatment increased the rate of PAH uptake at each concentration in the medium. Passive diffusion of PAH into the cells and efflux from the cells was not significantly different from control. In separated proximal tubules PAH uptake David Glenn Pegg was analyzed using a double reciprocal plot. Tubule suspensions were preincubated for 15 minutes and incubated in medium containing 1, 4 or 8 x 10-4 M PAH. The maximal velocity of transport was increased following penicillin, but the apparent binding affinity of the system for substrate was unaffected. Furthermore, since substrate stimulation of PAH transport was blocked by cycloheximide, these kinetic data suggest that the effect of penicillin on the active transport system is quantitative (theoretical maximal velocity of transport) rather than qualitative (apparent affinity). Proximal tubular ultrastructure was not changed by penicillin pretreatment. Either the effect was too subtle to be observed or it involved soluble rather than particulate proteins. The correla- tion between glutathione S-transferases, cytosolic drug metabolizing enzymes implicated as transport carriers, and organic anion trans- port was therefore investigated. Glutathione (GSH) S-aryltransferase activity in 100,000 x g supernatant of renal homogenates, an estimate of GSH S-transferase concentration in the tissue, was less in newborn rats and rabbits than adults. Enzyme activity increased to adult values by 1 week ,of age in rats, prior to maturation of transport capacity. Enzyme activity in rabbit kidney was not different at 1 day and 2 weeks but was increased by 4 weeks coincident with transport maturation. In rats, 25 mg/kg 3-methy1cholanthrene (3-MC) administered once a day for 3 days significantly increased enzyme activity but had no effect on transport capacity. Chronic ammonium chloride acidosis increased enzyme activity 8-fold but decreased transport capacity. Forty-eight hours following unilateral nephrectomy in rats, transport EOE 3-3: a _' v... An. (I) t) David Glenn Pegg capacity was significantly increased with little effect on enzyme activity. L-Methionine-SR—sulfoximine (1.85 mmoles/kg) signifi- cantly reduced glutathione concentration in renal cortex but had no effect on transport capacity. Organic anion transport was greater in male mice than female, yet there was no difference in enzyme activity between sexes. 3-MC (10, 20, 30, 40 mg/kg) admin- istered to 2-week-old rabbits twice daily for 3 days increased transport in a dose dependent manner. GSH S-transferase activity was also increased. Penicillin (90,000 I.U. twice daily for 2 days) similarly increased transport but had no stimulating effect on enzyme activity. The apparent lack of correlation between transport capacity and GSH S-transferase in several instances suggests that GSH S-transferase concentration is probably not the rate limiting step in renal organic anion transport. Penicillin pretreatment of newborn rabbits had no effect on DNA/protein, RNA/protein, nor RNA/DNA ratios in kidney cortex. Incorporation of 14C L-leucine into renal cortical slice protein was similarly unaffected by substrate stimulation. Using suspensions of separated proximal tubules, the incorporation of label into pro- tein was slightly increased 24 hours after termination of penicillin treatment. In vivo, after 3 injections of 90,000 I.U. penicillin, uptake of label into 100,000 x g pellet protein was enhanced com- pared with control but the difference was not statistically signifi- cant. Penicillin had no apparent effect when total renal cortical protein was assayed. PAH S/M ratio was significantly enhanced 8 hours after a single injection of 90,000 I.U. penicillin. Stimulation was maximal after H- be David Glenn Pegg 24 hours and had returned to control 48 hours after treatment. l4C L-leucine incorporation into 100,000 x g pellet protein was increased 16 hours after a single injection of penicillin, but the effect was variable. There was no consistent effect of penicillin on cytosolic proteins. The specific activity of intracellular leucine precursor pools was not altered by penicillin treatment. Sephadex filtration and gel electrophoresis of soluble and particu- late proteins, respectively, following in vivo labeling, did not indicate localized uptake of radioactivity in protein fractions. Thus, either substrate stimulation of organic anion transport involves slight alterations in protein biochemistry that may not be detected by these methods, or penicillin acts through a mechanism not involving increased synthesis of a protein component of the transport system. BIOCHEMICAL CORRELATES OF ORGANIC ANION TRANSPORT IN DEVELOPING KIDNEY BY David Glenn Pegg A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology 1976 ACKNOWLEDGEMENTS I would like to express my sincere appreciation to Dr. J. B. Hook for his continued guidance and constructive criticism and to Dr. T. M. Brody, Dr. J. Goodman, and Dr. D. Rovner for their assistance in preparation of this dissertation. Especially I would like to thank my wife, Christine, for the patience and encouragement she has given me throughout my graduate career. Dr. J. Bernstein conducted the ultrastructural studies and Dr. W. Bergen the amino acid analysis described in this dissertation. The technical assistance of Mrs. Peggy Wagner and Mr. Daniel Pietryga is gratefully acknowledged. ii TABLE OF CONTENTS INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . Organic Anion Secretion in the Kidney. . . . . . . . . Characteristics of the Renal Organic Anion Transport System . . . . . . . . . . . . . . . . . . . . . . . Localization of Transport and Identification of Possible Transport Carriers. . . . . . . . . . . . . Use of the Slice Technique . . . . . . . . . . . . . . Development and Substrate Stimulation of Renal Organic Anion Transport. . . . . . . . . . . . . . . Substrate Stimulation as a Tool in the Investigation of Renal Organic Anion Transport Mechanisms. . . . . METHODS O O O I O O I O I O O O O O O O O O O O O O O O O O 0 General. . . . . . . . . . . . . . . . . . . . . . . . 1. Animals . . . . . . . . . . . . . . . . . 2. Description of the in vitro slice technique 3. Description of the in vitro separated proximal tubule technique . . . . . . . . . Pharmacodynamic Analysis of Substrate Stimulation of Renal Organic Anion Transport by Penicillin. . . . . 1. Effect of dosage and treatment schedule . . 2. Substrate stimulation during development. . Investigation of Mechanisms of Penicillin Stimulation of PAH Transport . . . . . . . . . . . . . . . . . . 1. Uptake and efflux o PAH by slices. . . . . 2. Kinetic analysis of PAH uptake by separated proximal tubules. . . . . . . . . . . . . . 3. Inhibitory effect of cycloheximide. . . . . 4. Proximal tubular ultrastructure . . . . . . 5. Na, K-ATPase. . . . . . . . . . . . . . . . Evaluation of the Role of Glutathione (GSH) S- Transferases as Determinants of Renal Organic Anion Transport. . . . . . . . . . . . . . . . . . . 1. Description of GSH S-aryltransferase assay technique . . . . . . . . . . . . . . 2. Studies on possible correlations between PAH transport capacity and GSH S-transferases. . . . . . . . . . . . . . . 3. PAH transport capacity following GS depletion . . . . . . . . . . . . . . . . . iii Page 13 17 19 19 19 19 21 22 22 23 24 24 25 25 26 26 27 27 29 31 Page 4. Effect of age and penicillin pretreatment on penicillin binding to GSH S-transferases. . . . . . . . . . . . . . . . 31 Incorporation of Amino Acids and Protein Synthesis Following Substrate Stimulation by Penicillin. . . . . 33 1. Description of techniques for partial purification and quantification of DNA, RNA and protein . . . . . . . . . . . . . . 33 2. Incorporation of 14C L-leucine in vitro . . . 34 3.’ Incorporation of 14C L-leucine in vivo.. . . 35 4. Leucine pool size . . . . . . . . . . . . . . 36 Separation of Protein Fractions of Renal Cortical Homogenates Following Substrate Stimulation by Penicillin and in vivo Labeling with 14C L-leucine . . 37 l. Labeling of renal cortical protein and subcellular fractionation . . . . . . . . . . 37 2. Sephadex filtration . . . . . . . . . . . . . 37 3. Gel electrophoresis . . . . . . . . . . . . . 38 Statistical Analyses . . . . . . . . . . . . . . . . . . 39 RESULTS 0 O O O O O O O O O O O I O O O O O O O O I O O O O O O 40 Pharmacodynamic Analysis of Substrate Stimulation. . . . 40 1. Effect of dosage and treatment schedule . . . 40 2. Substrate stimulation during development. . . 49 Investigation of Mechanisms in Penicillin Stimulation of PAH Transport . . . . . . . . . . . . . . . . . . 54 l. Uptake and efflux of PAH by slices. . . . . . 54 2. Kinetic analysis of PAH uptake in sepa- rated proximal tubules. . . . . . . . . . . . 57 3. Inhibitory effect of cycloheximide. . . . . . 57 4. Proximal tubular ultrastructure . . . . . . . 57 5. Na, K-ATPase. . . . . . . . . . . . . . . . . 68 Studies on Possible Correlations Between PAH Transport Capacity and GSH S-Transferase . . . . . . . . . . . . 68 . Quantification of enzyme activity . . . . . . 68 . Effect of age . . . . . . . . . . . . . 71 . Effect of 3-methylcholanthrene (3-MC) . . . . 76 . Effect of 2, 3, 7 ,8-tetrachlorodibenzo-p- dioxin (TCDD) . . . . . . . . . . . . . . . . 76 . Effect of chronic metabolic acidosis. . . . . 84 Effect of substrate stimulation by penicillin 84 Sex difference. . . . . . . . . . . . . . . . 84 Effect of uninephrectomy. . . . . . . . . . 84 . PAH transport capacity following GSH depletion . . . . . . . . . . . . . . . . . . 93 10. Effect of age and penicillin pretreatment on penicillin binding to soluble proteins. . . . 93 Incorporation of Amino Acids and Protein Synthesis Following Substrate Stimulation by Penicillin. . . . . 102 1. Incorporation of 14C L-leucine in vitro . . . 102 2. Incorporation of 14C L-leucine in vivo. . . . 102 3. Leucine pool size . . . . . . . . . . . . . . 107 bWNI-J \DCDQOU'I iv b-I Page Separation of Protein Fractions of Renal Cortical Homogenates Following Substrate Stimulation by Penicillin and in vivo Labeling. . . . . . . . . . . . 107 l. Sephadex filtration . . . . . . . . . . . . . 107 2. Gel electrophoresis . . . . . . . . . . . . . 110 DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . . . 113 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 REFERENCES. 0 O O O O O O O O O O O O O O I O O O O O O O O I O 150 LIST OF TABLES Table Page 1 Effect of TCDD on accumulation of PAH by slices of renal cortex from adult rats . . . . . . . . . . . . . . 83 2 Effect of TCDD on accumulation of PAH and NMN by slices of renal cortex from adult rats . . . . . . . . . 83 3 Effect of penicillin treatment of 2-week-old rabbits on incorporation of 14C L-leucine by renal cortical Slices O O O O O O O O O O O O O O O O O O O 0 O O O O I 103 4 Effect of age and substrate stimulation of organic anion transport by penicillin on renal cortical pro- tein and nucleic acid composition in rabbits . . . . ... 103 5 Effect of penicillin treatment of 2-week-old rabbits on incorporation of 14C L—leucine by separated proximal tubules . . . . . . . . . . . . . . . . . . . . 104 6 Effect of penicillin treatment of 2-week-old rabbits on incorporation of 14C L-leucine in vivo. . . . . . . . 104 7 Effect of penicillin on leucine pool size in renal cortex from 2-week-old rabbits . . . . . . . . . . . . . 107 vi LI ST OF FIGURES Figure Page 1 Effect of procaine penicillin dose on accumulation of PAH by rabbit renal cortical slices . . . . . . . . . 42 2 Effect of sodium penicillin dose on accumulation of PAH by rabbit renal cortical slices. . . . . . . . . . . 44 3 Effect of treatment duration on enhancement of PAH 8/" ratio. 0 O O O O O I O O O O O I O I I O O O O O O O 46 4 Accumulation of PAH by rabbit renal cortical slices 12, 24, 36 and 72 hours after termination of treatment. . . . . . . . . . . . . . . . . . . . . . . . 48 5 Accumulation of PAH by rabbit renal cortical slices 2, 4, 8, 12, 16, 24 and 48 hours following penicillin treatment. . . . . . . . . . . . . . . . . . . . . . . . 51 6 Effect of penicillin on development of PAH S/M by rabbit renal cortical slices . . . . . . . . . . . . . . 53 7 p-Aminohippurate (PAH) uptake in 2-week control and penicillin-pretreated rabbit renal cortical slices . . . 56 8 Runout of p-aminohippurate (PAH) from renal cortical slices from control and treated littermates. . . . . . . 59 9 Double reciprocal plot of p-aminohippuric acid (PAH) uptake in separated proximal tubules from 2—week control and penicillin-pretreated rabbits. . . . . . . . 61 10 Effect of cycloheximide on the penicillin-induced enhancement of PAH transport by rabbit renal cortical Slices O I O O O O O O O I O O O O O O I O O O O O O O O 63 11 Electron micrograph of separated proximal tubules from (a) adult and (b) 2-week rabbit . . . . . . . . . . 65 12 Electron micrograph of separated proximal tubules from 2-week rabbits. . . . . . . . . . . . . . . . . . . 67 13 Effect of penicillin pretreatment and maturation on renal cortical Na+, K+-activated ATPase determined from a crude cortical homogenate . . . . . . . . . . . . 70 vii Figure Page 14 Effect of age on accumulation of PAH by rat renal cortical slices and on GSH S-aryltransferase activity in 100,000 x g supernatant of kidney homogenates . . . . 73 15 Effect of age on accumulation of PAH by rabbit renal cortical slices and on GSH S-aryltransferase activity in 100,000 x g supernatant of kidney homogenates . . . . 75 16 Effect of 3-MC on accumulation of PAH by rat renal cortical slices and on GSH S-aryltransferase activity in 100,000 x g supernatant of kidney homogenates . . . . 78 17 Effect of 3-MC on PAH accumulation by rabbit renal cortical slices and kidney wt/body wt ratio. . . . . . . 80 18 Effect of 3-MC on GSH S-aryltransferase activity in 100,000 x g supernatant of rabbit kidney homogenates . . 82 19 Effect of chronic ammonium chloride acidosis on accumulation of PAH by rat renal cortical slices and GSH S-aryltransferase activity in 100,000 x g supernatant of kidney homogenates. . . . . . . . . . . . 86 20 Effect of penicillin on PAH accumulation by rabbit renal cortical slices and GSH S—aryltransferase activity in 100,000 x g supernatant of kidney homogenates. . . . . . . . . . . . . . . . . . . . . . . 88 21 Accumulation of PAH by renal cortical slices and GSH S-aryltransferase activity in 100,000 x 9 super- natant from adult male and female mouse kidneys. . . . . 90 22 Effect of uninephrectomy on accumulation of PAH by rat renal cortical slices and on GSH S-aryltransferase activity in 100,000 x g supernatant of kidney homogenates. . . . . . . . . . . . . . . . . . . . . . . 92 23 Effect of L-methionine-SR—sulfoximine treatment on GSH concentration and accumulation of PAH by renal cortical slices. . . . . . . . . . . . . . . . . . . . . 95 24 Binding of 14C-benzyl penicillin to 100,000 x g supernatant protein from adult female rabbit renal cortical homogenates . . . . . . . . . . . . . . . . . . 97 25 Binding of 14C-benzyl penicillin to 100,000 x g supernatant protein from 2-week saline control rabbits. . . . . . . . . . . . . . . . . . . . . . . . . 99 26 Binding of l4C-benzyl penicillin to 100,000 x g supernatant protein from 2-week penicillin treated rabbits. 0 O O O O O O O O O O O I O O O O O O O O O O O 101 viii Figure 27 28 29 Page 14 . . . Uptake of C L-leuc1ne in soluble and m1crosoma1 protein of renal cortical homogenates from control and penicillin treated 2-week rabbits. . . . . . . . . . 106 Sephadex G-100 column chromatography of 100,000 x g supernatant protein from 2-week control and penicillin treated rabbits following in vivo pulse labeling with 14C L-leucine . . . . . . . . . . . . . . . . . . . 109 SDS polyacrylamide gel electrophoresis of renal cortical m1crosoma1 proteins from control and penicillin treated 2—week rabbits following in vivo pulse labeling with 14C L-leucine. . . . . . . . . . . . 112 ix H 0.1 INTRODUCTION Organic Anion Secretion in the Kidney Active secretory systems in renal excretory function were first hypothesized by Heidenhain (1880) following the observation that injection of certain highly colored dyes resulted in localization of color within proximal tubular cells. Marshall and Vickers (1923) provided additional evidence for active secretion when they determined that the rate of excretion of phenolsulfonphthalein (PSP) was greater than could be accounted for by glomerular filtration alone. Subse- quently, renal organic anion secretory processes were anatomically localized by stop flow analysis (Malvin et al., 1958) and micro- puncture (Edwards and Marshall, 1924) to the proximal segment of the nephron. In early studies emphasis was placed on compounds such as p-aminohippurate (PAH) and PSP because of uncomplicated handling by tubular cells (i.e., unmetabolized) and because of a relative lack of the analytical technology required for quantification of other substances. As a result, as late as 1939, Shannon (1939) suggested that PSP secretion in the kidney might "be considered to be the result of incidents in the cell's genetic history" and lack a normal function because no endogenous analogue to PSP could be identified. Since then it has become increasingly apparent that animals and man produce or ingest large amounts of compounds which are not extensively metabolized and in many cases are pharmacologically 1 b. an. .; d.- 2 active. Metabolic alterations which do occur usually produce forms which are more easily eliminated from body fluids. Those compounds are then ideal substrates for the relatively rapid rates of elimina- tion which may be attained through active secretion in the kidney. The capacity of renal tubules to secrete organic anions and cations is nearly universal in occurrence among vertebrates. Simi- larly, the structural specificity for substrate appears to be common among species though the efficacy of transport may vary. In mammals, the primary function of the renal organic anion secretory system may be the elimination of various conjugates. The excretion of a large number of organic compounds of either exogenous or endogenous origin is facilitated by conjugation in the liver and kidney with glycine, glucuronate and sulfate (Sperber, 1959; Wesson, 1969). Conjugation, however, is not a prerequisite for transport. Barac- Nieto and Cohen (1968) studied renal extraction of non-esterified fatty acids and determined that probenecid, an effective competi- tive inhibitor of organic anion transport, decreased renal uptake. Subsequently, Barac-Nieto (1971) observed that palmitate competi- tively inhibited PAH uptake. These data suggest transport by a common mechanism. Selleck and Cohen (1965) hypothesized that the organic anion transport system was primarily involved in transfer of metabolic intermediates such as non-esterified fatty acids, citrate and a=-ketoglutarate to sites of dissimilation in the kidney. The resulting pool of rapidly convertible intermediates may then provide a source of energy for other proximal tubular functions such as sodium and nutrient reabsorption. 3 ad. «.3. F» \u A)» no» Although a role for the organic anion secretory system in transport of endogenous compounds has been demonstrated, greater emphasis has been placed on excretion of exogenous compounds. In addition to the acidic dyes such as PSP and substituted hippurates, many drugs are eliminated by the renal secretory system. Penicillin was studied extensively after its discovery as an anti-infective agent. The renal clearance of penicillin is of the same magnitude as PAH and therefore plasma elimination is rapid (Rammelkamp and Bradley, 1943; Rantz and Kirby, 1944). Limited availability of drug following its introduction necessitated the use of other organic anions to competitively inhibit excretion and prolong plasma half life. This led to the development of probenicid, which remains a classical inhibitor of organic anion secretion (Beyer, 1947). Other drugs as well, such as thiazide diuretics and furosemide and salicylates, are actively secreted by the kidney at rates approach- ing that of PAH (Baer et al., 1959; Bowman, 1975; Hirsch et al., 1975; Schachter and Manis, 1958). Therefore, far from the view of Shannon (1939), organic anion secretion may be considered an impor- tant mechanism in drug elimination as well as regulation of body fluid concentrations of various endogenous compounds and their metabolites. Characteristics of the Renal Organic Anion Transport System Active transport is defined as carrier mediated translocation of substances across biological membranes against electrochemical concentration gradients (Wesson, 1969). The process is therefore dependent upon a constant supply of energy derived from cell a». :4 .u o .\y .7 v. I. ‘- I- u q . V y. nu .. e . a e v T. u! p c. .5 S A . s . :4 a . s 1.. .1 .2 4.. Ad is 4 metabolism. In the kidney, PAH and related compounds are trans- ferred from peritubular blood to tubular lumen by a mechanism which satisfies the requirements set forth in the definition of active transport. The net movement of PAH and related substances is "uphill", into the cell, against a concentration gradient (Foulkes and Miller, 1959; Foulkes, 1963). The rate of transport increases with substrate concentration until a maximum is reached at which the system becomes saturated (Weiner, 1973). Furthermore, secretion of one anion is competitively inhibited by the simultaneous admin- istration of a second_CWesson, 1969). Though secretion is substrate selective, specific structural requirements have not been determined due to the diverse nature of transported substances. In this area, Taggart (1958) hypothesized that the determining factor in transport was the net negative charge on the carboxyl group of anionic com- pounds. Several years later, Despopolous (1965) concluded that all secreted ions were characterized by three oxygen or equivalent atoms spatially related to approximate the configuration of the PAH side chain. Both theories appear to be oversimplifications in that many transported substances do not meet these criteria. The data, however, are consistent with a process mediated by specific and discrete transport carriers. Uncouplers of oxidative phosphorylation, hypoxia, cold and metabolic poisons decrease organic anion transport capacity in vivo and in vitro, suggesting strict dependence of the system on adequate availability of oxidative energy (Maxild and Moller, 1969; Mudge and Taggart, 1950a; Shideman and Rene, 1951; Taggart and Forster, 1950). The mechanisms of energy transfer, however, are unclear. Va: ,4... m.“ ..‘ i d or- vao 5A,. Ma'- D! (I! n. 8‘, i‘-‘ t" ‘n V sf I 0—4 - “ll 4 n 11' 5 Maxild (1973) observed that inhibitors of mitochondrial ATP pro- duction such as 2,4-dinitrophenol and carbonylcyanide-M-chloropheny1- hydrazone decreased PAH transport. Concentrations of inhibitor which produced 50% depression in transport capacity had no effect on ATP concentration. Similarly, anaerobic conditions decreased PAH transport and ATP in the cell, but upon return to aerobiosis, transport capacity was regained prior to regeneration of a full complement of ATP. Though transport appears to be intimately linked to respiratory electron transfer, endogenous ATP may not function as the primary energy donor. Therefore, it was suggested that decreased transport following treatment with metabolic inhibitors did not result from lower ATP concentration but was due to altera- tions in metabolic patterns (Weiner, 1973). Fluoroacetate, for example, inhibits tarnsport and results in the accumulation of Krebs cycle intermediates (Farah et al., 1953, 1955) which have been shown to be inhibitory to the PAH transport mechanism (Cross and Taggart, 1950). In any regard, the route and intermediates of energy transfer from the respiratory chain to the transport mechanism have not been identified. Localization of Transport and Identification of Possible Transport Carriers Compounds which are secreted in vivo must cross the peritubular membrane, cell cytoplasm and luminal membrane before entering the tubular lumen. The first step in the secretory process appears to be active transport into the cell (Cross and Taggart, 1950; Forster and Copenhaver, 1956; Foulkes and Miller, 1959). Tune et a1. (1969) used isolated microperfused rabbit proximal tubules and determined u 311‘ u..‘ . v‘( (I) '1’) "I" "1 6 that PAH concentration in proximal tubular cells was higher than in either the bathing medium or tubular perfusate. From these data a mechanism was proposed whereby PAH was actively transported across the peritubular membrane, accumulated intracellularly, and passively diffused down a concentration gradient from cell to lumen. The transport mechanism would then most likely be located within or in close proximity to the peritubular membrane. Renal organic anion secretory rate is governed in part by the structure and conformation of substrate molecules (Wiener, 1973; Wesson, 1969). Proteins possess the structural complexity necessary for discrimination between closely related substances and are there- fore implicated as transport carriers. A prerequisite for active transport is that a specific, reversible interaction occur between substrate and carrier. Measurement of binding affinity is then a useful tool in characterization of transport mechanisms. A carrier mediated mechanism has been proposed for the system which actively secretes N-methylnicotinamide (NMN) and other organic bases (Ross et al., 1975).. Competitive binding studies utilizing organic base substrates and dibenamine, an irreversible inhibitor of base trans- port (Ross et al., 1968), suggest that a protein carrier exists in proximal tubular cells (Holohan et al., 1973; Magour et al., 1969; Ross et al., 1969). The renal transport system for organic bases possesses many of the same functional characteristics as that for organic anions (Peters, 1960; Rennick et al., 1954; Rennick and Moe, 1960). The process is energy dependent and substrate saturable. Transport of one organic cation is competitively inhibited by simul- taneous administration of a second but not by organic anions (Farah 7 et al., 1959; Kandel and Peters, 1957; Rennick and Farah, 1956). Therefore, renal secretion of organic anions and cations comprise two similar but distinct systems. Investigations of substrate binding to components of the organic acid secretory system have been hindered by failure to develop an irreversible inhibitor of transport. Recently, however, a ligand binding assay for organic acids in the kidney was developed. The first conclusive evidence of binding capacity for trans- ported organic anions in renal subcellular fractions was obtained by Holohan et a1. (1975) using Sephadex and Millipore filtration techniques. These investigators determined that of the fractions studied the 100,000 x g pellet of renal cortical homogenates possessed the highest binding capacity for PAH and NMN. There was no binding to proteins in the soluble fraction. The binding affinity constants in 100,000 x g pellet material were 25 and 20 mM for PAH and NMN, respectively, indicating relatively low affinity systems. The bind- ing component was released from membranes by treatment with non— ionic detergents and was heat labile and substrate specific. Though there was competition for binding among anionic and cationic com- pounds, there was no interaction between the groups, further demon- strating independence of the mechanisms. From the proposed model, that is, active transport across peritubular membranes, one would anticipate that the carrier protein would be associated with the membranous fraction of kidney homogenates as has been observed. However, a great deal of evidence has accumulated which suggests that cytosolic proteins may also be involved in organic anion transport. 8 In the liver, organic anions are actively cleared from sinu- soidal blood and transported into bile by hepatic cells. Arias and co-workers (Levi et al., 1969; Reyes et al., 1969, 1971) isolated two cytosolic proteins, Y and Z, from liver homogenates which com- petitively bound organic anions. The protein present in highest concentration, protein Y, was designated ligandin. The concentra- tion of ligandin in liver, as well as the rate of anion excretion, is increased after administration of various microsomal enzyme inducers (Klaasen, 1975; Reyes et al., 1969). A role for ligandin in biliary excretion of organic anions was proposed based on this evidence. A protein immunologically identical to ligandin was isolated from renal homogenates and a similar function in organic anion transport hypothesized (Kirsch et al., 1975a,b). Renal and hepatic ligandin are immunologicallyidentical to glutathione S-transferase B (Habig et al., 1974a), one of a class of drug metabolizing enzymes which conjugate various compounds to reduced glutathione (Habig et al., 1974b). In the kidney, gluta- thione S-transferases are located only in cells of the proximal tubules, the only area of the nephron which secretes organic anions (Fine et al., 1975; Kirsch et al., 1975a,b). Several lines of evidence support the hypothesis that these enzymes function as transport carriers in the kidney. Glutathione S-transferase activity was competitively inhibited by transported organic anions (Clifton et al., 1974, 1975a; Kaplowitz et al., 1975). After injection of labeled penicillin, a substrate of the transport system, 88% of the radioactivity was bound to the glutathione S-transferase containing fraction of renal cell homogenates (Kirsch 9 et al., 1975a,b). Probenecid, a competitive inhibitor of organic anion transport, reduced binding of penicillin to the renal enzyme (Kirsch et al., 1975a,b). Inducers of drug metabolizing enzymes in the kidney such as 3-methylcholanthrene (3-MC) and 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) increased renal concentration of GSH S-transferases as well as urinary excretion, plasma disappear- ance and binding of organic anions (Clifton et al., 1975b; Kirsch et al., 1975b). Therefore, two plasible explanations exist to describe the mechanism of PAH transport in the kidney. The first involves active transport at the peritubular membrane (Holohan et al., 1975; Tune et al., 1969); and the second, specific binding within the cell as the primary events in intracellular accumulation of organic anions (Kirsch et al., 1975a,b). The transport model proposed by Tune et a1. (1969) does not include intracellular binding of PAH. Farah et a1. (1963) and Foulkes and Miller (1959) suggested that two pools of PAH existed within the proximal tubular cell, one in rapid equili- bration with extracellular fluid and the second, less readily exchangeable with the extracellular fluid and responsible for the high conCentration of PAH within the cell. Whether these data are related to the observations of Arias and co-workers (Kirsch et al., 1975b) is unknown. The data, however, might suggest that renal organic anion transport is mediated by both membrane transport and intracellular binding. Use of the Slice Technique Kidney slices were first used to quantify transport when Forster (1948) determined that thin slices of frog kidney accumulated phenol 10 red within the lumen by a process which was dependent upon meta- bolic energy. Cross and Taggart (1950) utilized thin slices of rabbit renal cortex as a means to study organic anion transport in the mammalian kidney. This technique permitted investigation of transport phenomenon in the absence of complications due to renal blood flow, blood flow distribution and glomerular filtration which might alter availability of metabolites or substrate. In addition, the study of renal transport in vitro allows investigation of a wider range of experimental variables, some of which might be dele- terious or impossible to produce in the intact animal. The ability to precisely control experimental conditions over long periods of time and to rapidly change these conditions is a further asset of the slice preparation. Major criticisms of the slice technique have involved objection to the artificial conditions imposed by in vitro studies. The first step in renal organic anion transport involves active uptake of substrate into the tubular cell (Foulkes and Miller, 1959). Therefore, slices of renal cortex incubated in physiological, oxygen- ated medium accumulate transported organic anions resulting in high intracellular concentrations. Foulkes and Miller (1959) determined, in slices, that the movement of PAH across the luminal membrane of proximal tubular cells was very slow compared to flux across the peritubular membrane, probably due to collapse of the lumen in the absence of glomerular filtration. Net uptake studies then constitute an evaluation of active transport across the peritubular membrane. Uptake data are generally represented as a slice to medium (S/M) concentration ratio where 5 equals concentration in the slice and 11 M equals concentration in the medium. An S/M ratio greater than 1 is assumed to be indicative of active transport when non-specific cellular binding of substrate is negligible. Autoradiographic studies have localized PAH in tubular cells to the cytoplasm (Miatello et al., 1966). In addition, incubation under nitrogen, which effectively inhibits active transport, results in S/M ratios of unity (Cross and Taggart, 1950). Therefore, PAH enters the cell and is apparently not bound to an appreciable extent to cell mem- branes or intracellular organelles. During incubation of tissue slices, substrate is accumulated until a steady state is reached, whereupon rates of active influx and passive efflux are equal. The final S/M ratio is a function of the relative rates of substrate flux across the membrane. An increase in the active uptake rate of substrate would be reflected as a higher S/M ratio and vice versa. Slice to medium ratio can then be used as an indication of transport capacity. A large amount of data are available which suggest that organic anion S/M ratio in kidney slices is closely representative of tubular transport in vivo (Berndt, 1976). Acetate and lactate enhance slice uptake of PAH (Cross and Taggart, 1950) and tubular transport in intact animals (Mudge and Taggart, 1950b), while succinate and fumarate block transport in both preparations. Metabolic inhibitors such as 2,4-dinitrophenol and fluoroacetate, as well as competitive inhibitors such as probenecid and penicillin decrease transport capacity measured both in vitro and in vivo (Beyer et al., 1944; Cross and Taggart, 1950; Farah et al., 1953; Mudge and Taggart, 1950a; Weiner, 1973). Furthermore, organic anionic compounds which are accumulated in slices are secreted in the 12 intact animal while no slice accumulation is observed in species which are unable to secrete the agent (Mudge et al., 1971). There- fore, steady state uptake of organic anions in slices appears to serve as an adequate representation of intact organ function. How- ever, slices may not be the best estimate of renal function in vivo. Slices of renal cortex consist of several cell layers. There— fore, cells on the interior of the slice are dependent upon dif- fusion for contact with medium. If oxygen penetration becomes rate limiting, function through the slice may be altered and measure- ments reflect an average value for a heterogeneous population of cells. Burg and Orloff (1962) described a technique for enzymic digestion of renal cortex resulting in a free suspension of separated proximal tubules. In this preparation each cell is in constant contact with bathing medium. Separated proximal tubules consume oxygen, maintain electrolyte concentration gradients, and accumulate PAH (Burg and Orloff, 1962; Burg and Orloff, 1969; Ecker and Hook, 1974a; Guder et al., 1971; Huang and Lin, 1965). Barriers to dif- fusion and much of the non-transporting tissue present in slices (distal tubules, connective tissue, glomeruli) are eliminated when using separated tubules. In addition, PAH uptake is more rapid and greater tissue to medium concentration ratios are attained (Huang and Lin, 1965). Therefore, though uptake of organic anions in renal cortical slices is indicative of in vivo secretion, perhaps a better estimate may be obtained using separated proximal tubules. As esti— mates of tubular function other than transport (i.e., metabolism, protein synthesis), the separated proximal tubule preparation may be a superior model. 13 Development and Substrate Stimulation of Renal Organic Anion Transport At birth the kidneys of most animal species are structurally and functionally immature. The ability of the newborn kidney to excrete a large load of sodium and water is less than would be pre- dicted on size alone (Dean and McCance, 1949; Edelmann and Spitzer, 1969). In addition, mechanisms for tubular secretion of drugs such as penicillin and sulfas are quantitatively immature. Several investigators observed that clearance and extraction of PAH in the newborn was less than in the adult (Alexander and Nixon, 1962; Calcagno and Rubin, 1963; Hook et al., 1970; Horster and Lewy, 1970; Levine and Levine, 1958). Transport capacity measured as the accumu- lation of PAH in renal cortical slices or separated proximal tubules was also less in the newborn (Ecker and Hook, 1974a; Hirsch and Hook, 1970a; Hirsch and Pakuts, 1974; Kim et al., 1972; Rennick et al., 1961). The relatively low functional capacity in young animals has been attributed to structural immaturity and to the presence of nephrogenic and incompletely differentiated tissue in the outer cortex of the kidney. In humans the full complement of glomeruli and tubules is present at birth and the major changes observed are increases in size and differentiation (Edelmann, 1969), whereas in rats at birth nephrogenesis is a prominent characteristic (Baxter and Yoffey, 1948; Bogomolova, 1966). Cortical tissue is not fully developed in rats until 28 days of age. Up to one year is required for the cortex to reach its adult thickness and for the brush border to reach adult heights (Bogomolova, 1966). Rennick et a1. (1961) have shown greater in vitro ability of inner, more mature, juxta- medullary tissue to transport PAH than the histologically immature l4 outer cortex. Though renal tubular cells in young animals appear by light microscopy to be small (Hirsch et al., 1971), electron micro- scopic studies in newborn and fetal animals have shown complete ultrastructural differentiation with only small quantitative dif— ferences from mature kidney (Bernstein, 1971). Structural develop- ment does not appear to be the sole or principal determinant of transport capacity. Transport capacity exhibits a characteristic pattern of develop- ment in vitro (Hirsch and Hook, 1970a). In rabbits PAH S/M ratio developed slowly with age until at 2 to 3 weeks a rapid increase was observed. Maximal PAH S/M ratio occurred at 4 weeks of age and this was followed by a decline to adult values. Similar patterns of development in vitro were observed using rats, dogs and pigs (Kim et al., 1972; Rennick et al., 1961). The rapid increase in trans- port capacity occurring during early development is apparently a result of an increased availability of substrate (Ecker and Hook, 1974b; Hook, 1974). During the neonatal period renal vascular resistance falls and renal blood flow increases, presumably pre- senting a larger substrate load to the tubules (Edelmann, 1969). Several examples are available in the literature of substrate induced adaptations in enzyme activity and transport function (Heppel, 1969; Knox et al., 1956; Tepperman and Tepperman, 1963). To investigate the effect of increased substrate load on renal organic anion trans- port development, Hirsch and Hook (1969, 1970a,b,c) challenged the system with large doses of penicillin, an organic anion rapidly transported in the kidney, and observed a significantly increased PAH S/M ratio. The effect of penicillin on transport was not unique 15 in that other transported substances, such as PAH and probenecid, also increased the rate of maturation (Hirsch and Hook, 1970a; Hook and Bostwick, 1973). If animals were sacrificed several days after the termination of substrate treatment, no stimulation of transport was apparent, providing further evidence for the substrate dependence of transport rate (Hirsch and Hook, 1969, 1970c). If precautions were taken to prevent young rabbits from contacting solid food, the rate of transport maturation was decreased (Ecker and Hook, 1974b). During normal development, then, environmental substances such as derived from food probably contribute to func- tional maturation. Several investigators have since determined that PAH transport in vivo, measured as elimination from plasma, extrac- tion or clearance in several animal species, was also increased by penicillin pretreatment (Bond et al., 1976; Kaplan et al., 1975; Lewy and Grosser, 1974). Thus, by several methods the data demon- strate that organic anion transport maturation is dependent upon substrate availability and may be increased by substrate pretreatment. The stimulating effect of penicillin pretreatment on organic anion transport in young animals is in several ways analogous to induction of drug metabolizing enzymes in the liver. Agents such as phenobarbital and 3-methy1cholanthrene enhance microsomal drug metabolizing capacity in liver through increased synthesis of enzyme protein (Chiesara et al., 1967; Conney, 1967; Fouts and Rogers, 1965; Funcke and Timmerman, 1973; Hayes and Campbell, 1974; Pantuck et al., 1968). The effect of substrate stimulation on renal protein biochemistry was investigated by Hirsch and Hook (1970b). Penicillin pretreatment of newborn rats was associated with an increased kidney 16 weight to body weight ratio. Concomitant administration of cyclo- heximide, a potent inhibitor of protein synthesis (Ennis and Lubin, 1964), blocked the stimulating effect of penicillin. The protein content of the 100,000 x g pellet of renal cortical homogenates was increased following penicillin, suggesting that the effect was con— fined to microsomal proteins and that the transport mechanism was also localized to membranes. Interpretation of these data was difficult, however, because the protein content of all subcellular fractions was slightly increased, though the effect was only sig— nificant in the 100,000 x g pellet. In addition, cycloheximide decreased transport capacity in controls. Therefore, to obtain a more specific estimate of protein synthesis, rates of incorporation of labeled amino acids into protein were determined. The uptake of 14C L-leucine and 14C L-glutamine into the trichloroacetic acid insoluble fraction of renal cortical homogenates was increased following penicillin (Hirsch and Hook, 1970b). There was no stimu- lation of amino acid uptake in medullary tissue consistent with the inability of medullary slices to transport PAH (Cross and Taggart, 1950). Ammonium chloride acidosis increased kidney weight as well as labeled amino acid uptake (Bignall et al., 1968) but had no stimulating effect on PAH transport (Hirsch and Hook, 1970b). Therefore, the effect of penicillin is probably not the result of non-specific increases in protein content. From these data Hirsch and Hook (1970b) concluded that the stimulating effect of penicillin on PAH transport was the result of increased synthesis of specific transport proteins. 17 Substrate Stimulation as a Tool in the Investigation of Renal Organic Anion Transport Mechanisms Isolation and characterization of components of the renal organic anion transport system have been hindered by an apparent low binding affinity of substrate (Holohan et al., 1975) and failure to develop an irreversible inhibitor of transport. Substrate stimu- lation may provide an alternate method. Enhancement of organic anion transport capacity by penicillin is specific; that is, organic base transport is unaffected (Hirsch and Hook, 1970a,c), and is apparently mediated through an increased synthesis of protein components of the system. Therefore, it should be possible to stimulate transport and label the induced protein by supplying radioactive precursors. The incorporation of label may then provide a marker to facilitate further investigation. In earlier studies rats were used as the test animal (Hirsch and Hook, 1970b). However, rabbits are more susceptible to substrate stimulation and may similarly exhibit more pronounced changes in renal cortical biochemistry following substrate stimula- tion. Therefore, in the present investigation, rabbits were used exclusively in studies of substrate stimulation. The specific objectives of this study were first to reevaluate substrate stimulation in order that maximal enhancement of transport capacity might be produced. Once an optimal treatment regimen was determined, the next objective was to investigate the mechanism of penicillin enhancement of transport, including the effect of peni- cillin on membrane flux of PAH and related alterations in proximal tubule ultrastructure. Several investigators suggested that cytosolic drug metabolizing enzymes in the kidney functioned as primary acceptor proteins for l8 transport (Clifton et al., 1975a; Kirsch et al., 1975b). Conclusive evidence, however, was not obtained. Therefore, another objective of this study was to determine the relationship between glutathione S-transferase concentration in kidney cortex and organic anion transport capacity. Finally, the effect of substrate stimulation on amino acid incorporation and protein synthesis in vivo and in vitro was to be investigated. Gel electrophoresis and Sephadex filtration were to be used to partially purify labeled proteins from kidney homogenates. From these data the feasibility of using substrate stimulation as a tool to isolate components of the trans- port system was to be evaluated. METHODS General 1. Animals New Zealand White rabbits, Sprague-Dawley rats and Swiss Webster mice were purchased from local breeders. Animals were main- tained in departmental animal quarters. Rabbits and rats were bred, or litters were purchased with lactating females. Young animals remained with their mothers during treatment and until the time of experimentation. Within each litter, half the pups were treated with drugs and the remainder received drug vehicle or saline as control. Unless otherwise noted, all animals were male; littermates were of both sexes. 2. Description of the in vitro slice technique Animals were killed by a blow to the head and the kidneys removed, weighed and placed in ice cold isotonic saline. Thin slices of renal cortex were prepared free hand and briefly kept in a further aliquot of ice cold saline until incubation. Slices of renal cortex (100-200 mg wet weight) were incubated in 2.7 m1 of the phosphate buffer medium1 described by Cross and Taggart (1950) which contained 197 mM NaCl, 40 mM KCl, 0.7 mM CaClz, 7 mM NaH2P04-Na2HPO4 (pH 7.4). 19 20 7.4 x 10.5 M p—aminohippuric acid (PAH) and/or 6.0 x 10-6 M 14C N-methyl-nicotinamide (NMN). In some cases 10 mM sodium acetate was included in the medium. The incubation period was 90 minutes at 25°C under 100% oxygen. After incubation slices were removed from the medium, blotted and weighed. Tissue and a 2 m1 aliquot of medium were homogenized in a 3 ml volume of 10% trichloroacetic acid (TCA). Water was added to a final volume of 10 ml and samples centrifuged. Aliquots of tissue and medium supernatant were assayed for PAH by the method of Smith et a1. (1945). 14C NMN was quantified by adding 1 ml tissue or medium supernatant to scintillation vials containing 10 ml modified Bray's solution (6 g 2,5—diphenyloxazole and 100 g naphthalene per liter of dioxane). Radioactivity was determined in a Beckman LS-100 liquid scintillation spectrometer using external standardization. Results were expressed as a slice to medium (S/M) concentration ratio where S equals milligrams per gram tissue (wet weight) and M equals milligrams per milliliter medium. L-leucine S/M ratios were determined by incubating slices in Krebs Henseleit bicarbonate buffer2 containing 55 uM 14C L-leucine (sp. act. 1.45 uCi/um) for 60 minutes at 37°C under 100% oxygen. Slices were homogenized and assayed as described for NMN and S/M ratios calculated as the ratio of DPM per milligram tissue divided by DPM per milliliter medium. 2118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4-7H20, 10 mM glucose, 1 mM Cac12, 25 mM NaHCO3, 1.2 mM KH2P04 (pH 7.4) . 21 3. Description of the in vitro separated proximal tubule technique Separated proximal tubules were prepared by a technique similar to that of Burg and Orloff (1962) with some of the modifications employed by Huang and Lin (1965). Animals were killed by a blow to the head and the kidneys exposed through a midline incision. The abdominal aorta was clamped rostral and cannulated caudal to the renal arteries. The kidneys were perfused with ice cold physio- logical saline until clear of blood. The infusion was continued using 0.3% collagenase (Nutritional Biochemical Corp., Cleveland, OH) dissolved in Ringer's solution.3 The renal vein was clamped and the perfusion continued until the kidney became turgid. The infusion was stopped and the renal vein clamped. After approxi— mately 3 minutes the kidney was removed and cortical tissue dissected free. Tissue was finely minced and incubated for 45 minutes at 25°C while being slowly stirred and oxygenated by bubbling 100% oxygen through the suspension. After digestion the suspension was centri- fuged for 60 seconds at 500 rpm and the supernatant discarded. The pellet was rinsed three times by resuspension in 5% calf serum- Ringer's solution. During the third wash the tissue suspension was filtered through two layers of surgical gauze prior to centri- fugation. The pellet of tubules was resuspended in sufficient Ringer's-acetate medium containing PAH to produce a 2 to 4% (w/v) suspension. The suspension was incubated at 25°C while being con- tinually gassed and mixed by bubbling with 100% oxygen. Following 3120 mM NaCl, 16.2 mM xc1, 1.2 mM M9804, 1.0 mM CaClz, 5.5 mM dextrose, 10 mM Na acetate, 10.0 mM KH2P04-Na2HPO4 (pH 7.4). 22 incubation the samples were centrifuged at 0°C for ten minutes at 10,000 x g in the special centrifuge tubes described by Burg and Orloff (1962). To estimate trapped medium in the pellet 0.1 m1 5% inulin per 4 m1 incubation medium was added immediately prior to centrifugation. Tissue water content was determined by drying the tissue pellet to constant weight. The pellet was homogenized in 5 m1 3% TCA and the protein precipitate removed by centrifugation. The supernatant and a sample of medium treated in a similar manner were assayed for PAH (Smith et al., 1945) and inulin (Schreiner, 1950). Pharmacodynamic Analysis of Substrate Stimulation of Renal Organic Anion Transport by Penicillin 1. Effect of dosage and treatment schedule The relationship between penicillin dose and PAH S/M in rabbits was determined using both procaine penicillin G and sodium penicillin G. Treatment was begun on the 10th day of life and continued at 12-hour intervals for a total of 6 injections. In all cases, animals were sacrificed 24 hours after the final injection. Procaine peni- cillin G (15,000, 30,000 and 90,000 I.U.) was given subcutaneously. Sodium penicillin G (30,000, 100,000 and 180,000 I.U.) was injected intraperitoneally. PAH S/M ratios were determined after 2, 4, 6 or 8 injections of 90,000 I.U. of procaine penicillin G, subcutaneously, to newborn rabbits. Injections were given at 12-hour intervals beginning on the 10th day of life. Animals were sacrificed 24 hours after final injection and run in pairs, control and treated together. 23 PAH S/M was determined at several intervals after 90,000 I.U. of procaine penicillin G, given subcutaneously, twice daily for 2 days. Treatment was begun on the 11th day of life. Pairs of control and treated animals together were sacrificed and PAH S/M determined 12, 24, 36 and 72 hours after the final injection. The effect of a single injection of penicillin on PAH S/M was determined by administering 90,000 I.U. procaine penicillin G sub- cutaneously on the 12th day of life. Animals were sacrificed and transport capacity determined 2, 4, 8, 12, 16, 24 and 48 hours following treatment. Residual penicillin in various tissues 24 hours after a subcu- taneous injection was determined by combining l uc of l4C-benzyl penicillin (penicillin G) with the final injection of procaine penicillin. Samples of bladder urine, blood, fat, kidney cortex, liver and muscle were obtained, solubilized in 1 ml of Soluene at 37°C and counted in 10 m1 of toluene counting solution containing 5 g of 2,5-diphenyloxazole (PPO) and 200 mg of dimethyl 1'4"E$§f 2(5-phenyloxazolyl)-benzene (POPOP) per liter. Results were expressed as disintegrations per minute per gram of tissue. 2. Substrate stimulation during development To elucidate the effect of a maximal penicillin challenge on PAH S/M, pregnant does were injected intramuscularly with 90,000 I.U. of procaine penicillin G twice daily from day 14 of gestation to delivery. Within a litter, pups were sampled at 3 days, 1, 2 and 4 weeks. Two days before experimentation, a pair of pups was ran- domly selected. One member of the pair received two injections of 24 90,000 I.U. of procaine penicillin G at 12-hour intervals, and the other served as a saline control. Animals were sacrificed 24 hours after the second injection. Investigation of Mechanisms of Penicillin Stimulation of PAH Transport 1. Uptake and efflux of PAH by slices Rabbits 10 days old received 30,000 I.U. procaine penicillin G subcutaneously twice daily for 3 days. Control littermates received saline. Animals were killed 24 hours after the final injection. To estimate the maximal rate of uptake into slices, tissue was pre- incubated for 30 minutes. PAH was then added to achieve medium concentrations of 1.0, 2.0, and 4.0 x 10"4 M and the slices incubated for another 15 min. Duplicate tissue samples were incubated simul- taneously in a two-chambered Dubnoff metabolic shaker, one under a gaseous phase of 100% oxygen and the other under 100% nitrogen. The oxygen-requiring component of PAH transport was determined by calculating the difference between PAH uptake under oxygen and nitrogen. The rate of transport was expressed as micrograms PAH taken up per gram of tissue per minute of incubation. Runout of PAH was determined using the method of Farah et al. (1963) with some of the modifications devised by Berndt (1965). Slices were preloaded by incubating 300-600 mg of tissue in 6 m1 of medium containing 6.3 x 10-4 M PAH for 90 minutes. Tissue was removed from the incubation medium, rinsed, and placed in a net fashioned of nylon mesh. The tissue was transferred at l-minute intervals with continuous shaking through a series of 20 beakers each containing 4.0 ml of PAH-free medium. At the conclusion of 25 the runout experiment, the tissue was removed from the net, blotted, weighed, and treated as before. Tissue and runout beakers were assayed for PAH and the results expressed as micrograms PAH remaining in the slices per 100 mg tissue. 2. Kinetic analysis of PAH uptake by separated proximal tubules At 11 days of age treatment was begun. Procaine penicillin G was administered to rabbits in a dose of 90,000 I.U. twice daily for 2 days. Animals were killed 24 hours after the final injection. PAH uptake in separated proximal tubules was determined by preincu- bating an aliquot of suspension under 100% oxygen at 25°C for 15 minutes. PAH was then added to produce concentrations in the medium of 1, 4, and 8 x 10“4 M and the incubation continued for another 15 minutes. The tubule suspension was then centrifuged at 2°C for 10 minutes at 10,000 g in the special centrifuge tubes described by Burg and Orloff (1962) and the tissue pellet assayed for PAH. To estimate trapped medium in the pellet, inulin was added to the suspension immediately prior to centrifugation. Results were expressed as micrograms PAH per gram of tissue (dry weight). 3. Inhibitory effect of cycloheximide The effect of cycloheximide on penicillin enhancement of PAH S/M was determined in rabbits by administering 0.18 mg cycloheximide (approx. 0.4 mg/kg) dissolved in saline, intraperitoneally, followed by 90,000 I.U. procaine penicillin G, subcutaneously, or saline for control. Two injections (lZ-hour intervals) of each drug were administered beginning on the 12th day of life. Animals were tested 24 hours after the final injection. 26 4. Proximal tubular ultrastructure Adult female and 2-week littermate rabbits were used. Adults were not treated. Beginning on the 11th day of life, 90,000 I.U. procaine penicillin G was administered twice daily for 2 days. Animals were sacrificed 24 hours after the final injection. Ali- quots of tubule suspension were prepared and shipped to the microscopy laboratory in a double blind fashion.4 The code was not broken until all analyses were complete. Tissue was prepared for electron microscopy by fixing the suspension of tubules in ice cold 1% osmium tetroxide in Millionig's buffer at pH 7.3 before dehydration in alcohol. After fixation, tubules were rinsed in buffer and embedded in epoxy resin by standard techniques. Sections for electron microscopy were stained with uranyl acetate and lead citrate by a modified Reynolds technique.) Several sections of each tissue sample were taken and multiple photographs were developed from each section. 5. Na, K-ATPase 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 after the final injection. Na, K-ATPase activity was determined in a crude homogenate of sepa- rated tubules. Freshly prepared tubules were homogenized in a 4Electron micrographs were prepared and analyzed by Dr. Jay Bernstein, Director, Department of Anatomic Pathology, William Beaumont Hospital, Royal Oak, MI 48072. Electron microscopic investigations were supported in part by the William Beaumont Hospital Research Institute and the Kidney Foundation of Michigan. 27 solution containing 0.25 M sucrose, 5 mM EDTA, and 30 mM histidine (pH 6.8). Enzyme activity was measured in medium containing 5 mM ATP, 30 mM Tris, and 5 mM MgCl at pH 7.4. Half the beakers con- 2 tained 115 mM NaCl and 10 mM KCl (total ATPase) and half contained neither NaCl nor KCl (Mg-ATPase) in a total volume of 3 ml. The incubation mixtures were preincubated for 5 minutes and the reaction was started by adding ATP. The reaction was allowed to proceed for 15 minutes and then stopped by adding 1.0 ml 10% TCA. After centri- fugation, the supernatant was analyzed for phosphate (Pi) (Chen et al., 1956) and an aliquot of homogenate was analyzed for protein (Lowry et al., 1951). Na, K-dependent ATPase was represented as the difference between total and Mg-dependent ATPase. Results were expressed as micromoles of Pi released per milligram of protein in 10 minutes. Evaluation of the Role of Glutathione (GSH) S-Transferases As Determinants of Renal Organic Anion Transport 1. Description of GSH S-aryltransferase assay technique Animals were killed by a blow to the head and the kidneys removed. One kidney was minced in ice cold 0.25 M sucrose -0.01 M KHZPO4 (pH 7.4) buffer, rinsed and homogenized in 2 volumes of the same buffer. The second kidney was used for PAH S/M ratio determina- tion as previously described. In very young animals kidneys from several pups were pooled. GSH S-transferase concentration in renal tissue was estimated as GSH S-aryltransferase activity by the method of Booth et a1. (1961) or Habig et al. (1974b). Whole kidney homogenates were 28 centrifuged at 2°C for 90 minutes at 100,000 x g in a Beckman L3-50 ultracentrifuge. The 100,000 x g supernatant was assayed for enzyme activity. Enzyme incubation medium contained 0.1 M pyrophosphate buffer (pH 8.0), 5 mM reduced glutathione and 1 mM 1,2-dichloro-4- nitrobenzene (dichloronitrobenzene) as substrate. Alternatively, l-chloro-2,4-dinitrobenzene (dinitrochlorobenzene) was used as substrate in a medium of 0.1 M potassium phosphate (pH 6.5), 1 mM reduced glutathione and 1.0 mM dinitrochlorobenzene. Three milli- liter aliquots of medium were preincubated for 3 minutes. One hundred microliters of enzyme preparation was added and initial optical density readings recorded. Enzyme activity was measured at 37°C and 25°C and optical density measured at 344 and 340 nm for dichloronitrobenzene and dinitrochlorobenzene substrate, respec- tively. Dilutions of 100,000 x g supernatant using sucrose-phosphate buffer were required when dinitrochlorobenzene was used as substrate to maintain OD changes within measurable limits. After 15 minutes incubation final optical density was determined. Sucrose-phosphate buffer was used as blank. Product formation was estimated from the change in optical density during incubation minus non-enzymatic formation determined in the blank. Protein concentration in the 100,000 x g supernatant or a dilution of the supernatant was esti- mated by the method of Lowry et a1. (1951). Data were expressed as net change in optical density per milligram protein per 15 minute incubation. 29 2. Studies on_possib1e correlations between PAH transport capacity and GSH S-transferases a. Effect of age. The ability of renal cortical slices to accumulate PAH and GSH S-transferase concentration in renal cortical homogenates was quantified using rat and rabbit pups randomly selected from litters at the designated ages. PAH S/M ratio and enzyme activity were determined in kidneys from the same animal except in l-day rats where kidneys from 4 pups were pooled. Rabbits 1 day, 2 weeks and 4 weeks old and rats 1 day, 1 week and adult were used. b. Effect of 3-methy1cholanthrene. Weanling rats weighing approximately 50 g and 2-week-old rabbits were used. 3-Methyl- cholanthrene (3-MC) was dissolved in corn oil and injected intra- peritoneally. Controls received a comparable volume of corn oil. Rats were administered 25 mg/kg once daily for 3 days. Rabbits were treated with 10, 20, 30 or 40 mg/kg twice daily for 3 days beginning on the 11th day of life. All animals were killed 24 hours after the final injection. c. Effect of 2,3,7,8 tetrachlorodibenzo-p-dioxin. Adult rats weighing approximately 150 g were injected intraperitoneally with 10 or 25 ug/kg 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) dissolved in acetone-corn 011 (1:9). Alternatively, 1 or 5 ug TCDD was admin- istered intragrastrically using a gavage needle. Volume was never greater than 1 m1 when administered orally. Controls received vehicle alone. Animals were allowed free access to food and water. S/M ratios were determined 3 or 7 days after treatment using renal 30 cortical slices taken in a plane both parallel and perpendicular to the major renal axis. Extracellular water in the slices was esti- mated by inulin space. Inulin in tissue and medium supernatant was assayed by the method of Schreiner (1950). d. Effect of chronic metabolic acidosis. Adult rats weighing approximately 150 g were made acidotic by supplying 0.28 M ammonium chloride as sole drinking fluid for 7 days. Controls were allowed tap water. Animals had free access to food and were killed on the 7th day of treatment. e. Effect of substrate stimulation by penicillin. Rabbits were injected subcutaneously with 90,000 I.U. procaine penicillin twice a day for 2 days. Animals were sacrificed 24 hours after the final injection. f. Sex difference. PAH S/M ratio in adult male and female mice was determined by incubating slices in medium containing 7.4 x 10-5 M 14C-PAH (sp. act. 1.4 uCi/umole) and measuring radioactivity in tissue and medium supernatants using liquid scintillation spectrometry. 9. Effect of uninephrectomy. Adult rats weighing approxi- mately 150 g were lightly anesthetized with ether and the left kidney removed through a flank incision. Caution was exerted not to disturb the adrenal. The abdominal wall was sutured using 00 silk and the skin incision closed with wound clips. Control animals were sham operated and the kidney left undisturbed. The animals were killed 48 hours after surgery. Either PAH S/M ratio or GSH 31 S—aryltransferase activity were determined in the remaining kidney. Only the unoperated kidney was used in sham operated animals. 3. PAH transport capacity following GSH depletion Adult rats weighing approximately 150 g were used. L-methionine- SR-sulfoximine was dissolved in distilled water and administered intraperitoneally in a dose of 1.85 mmoles/kg. Controls received saline. After 2 hours animals were killed by a blow to the head and the kidneys removed. Renal cortical slices from both kidneys were pooled and divided into 3 portions. One was assayed for gluta- thione and the others incubated for 60 minutes at 25°C under 100% oxygen in phosphate buffer medium as described earlier. Medium in one incubation vessel contained 7.4 x 10'.5 M PAH for determination of PAH S/M ratio. The other contained no PAH and was used for determination of tissue glutathione concentration after incubation. Glutathione was estimated by the method of Hewitt et a1. (1974). A 5% homogenate of renal cortex in 6% TCA was spun for 10 minutes at 3000 rpm in an International centrifuge. Aliquots of supernatant were diluted to 2.0 ml with 6% TCA and 8.0 ml 0.3 M Na HPO 2 4' 0.04% 5,5'-dithio-bis-(2-nitrobenzoic acid) (DNTB) in 10% sodium 1.0 m1 citrate added. GSH was quantified as the increase in absorbance at 412 nm. 4. Effect of age and penicillin pretreatment on penicillin binding to GSH S-transferases Binding of penicillin to soluble proteins was estimated in adults and 2-week—old rabbits. The effect of substrate stimulation on binding was determined in 2-week-old pups treated with 90,000 I.U. 32 procaine penicillin G subcutaneously twice daily for 2 days. Animals were sacrificed 24 hours after the final injection. Controls received saline. Kidneys were minced in ice cold 0.25 M sucrose-0.01 M KH2P04 (pH 7.4) and rinsed several times. Tissue was homogenized in a further aliquot of buffer. Homogenate was centrifuged at 100,000 x g for 90 minutes at 2°C. An aliquot of 100,000 x g supernatant containing 50 mg soluble protein was taken, to which was added 0.88 umole penicillin (0.57 uCi/umole) and 1 mM reduced glutathione. Cold sucrose-phosphate buffer was added to make the final volume 5 ml. The mixture was allowed to stand in ice for 4 hours and layered on a 3 x 100 cm Sephadex G-100 column equilibrated wtih 0.01 M KH2P04 (pH 7.4). Flow rate was adjusted to 15 ml/hr by varying the height of the buffer reservoir and 3 m1 samples of eluate collected. Samples were assayed for protein by the method of Lowry (1951). Radioactivity was determined in 1 m1 fractions of eluant samples which were added to 10 m1 modified Bray's and counted in a Beckman LS-100 liquid scintillation spectrometer. Data were represented as mg/ml protein and CPM/ml 14C-penicillin in each sample. GSH S-transferase concentration in eluant fractions was estimated as GSH S—aryltransferase activity using dinitrochloro- benzene as enzyme substrate. Data were expressed as AOD/mg protein/ 15 minutes. 33 Incorporation of Amino Acids and Protein Synthesis Followipg Substrate Stimulation by Penicillin 1. Description of techniques for partial purification and quanti- fication of DNA, RNA and protein Lipid, RNA and DNA were extracted from TCA precipitates of tissue by a modified Schneider procedure (Fleck and Munro, 1962; Schneider, 1957). Renal cortical tissue was homogenized in 10% TCA and the insoluble pellet resuspended in 10 ml 95% ethanol. The suspension was allowed to stand at room temperature for 30 minutes and centrifuged at 2°C in a Beckman J-21 centrifuge at 10,000 rpm. After centrifugation the ethanol extraction was repeated. The pellet was resuspended in 10 m1 ethanol:ether (3:1) and heated in a 60°C water bath for 30 minutes. After heating the suspension was allowed to stand in ice for 20 minutes and centrifuged. The pellet was solubilized in 2 ml 1 N KOH and incubated at 37°C for approximately 14 hours. After incubation 0.4 m1 6 N HCl and 2 ml 5% TCA were added. The suspension stood in ice for 30 minutes. After centrifugation the supernatant was assayed for riboses by the orcinol procedure (Keleti and Lederer, 1974). The acid insoluble precipitate was resuspended in 10 m1 5% TCA and heated in a 90°C water bath for 20 minutes. The suspension stood in ice for 30 minutes after heating and the supernatant was assayed for 2-deoxy- sugars by the diphenylamine reaction (Burton, 1956; Keleti and Lederer, 1974). The final pellet was solubilized in l N KOH and aliquots taken for protein determination by the method of Lowry (1951). When amino acid incorporation into protein was determined, 200 pl solubilized material was neutralized with 1 N HCl and counted 34 in 10 ml PCS liquid scintillation cocktail (Amersham Searle). Data were represented as DPM/mg protein or DPM/pg DNA. The effect of age and penicillin pretreatment on DNA/protein, RNA protein and RNA/DNA ratios in rabbits was also determined. Adults were used without treatment. Young rabbits were treated with 90,000 I.U. procaine penicillin G twice daily for 2 days beginning on the 11th day of life and killed 24 hours after the final injection. . 4 . . . 2. Incorporation of l C L—leuc1ne 1n v1tro Littermate rabbits were treated with 90,000 I.U. procaine penicillin G subcutaneously twice daily for 2 days beginning on the 11th day of life. Animals were killed and renal cortical slices prepared 24 hours after the final injection. Incorporation of amino acids into separated proximal tubular protein from control and penicillin treated rabbits was determined 3 and 24 hours after treatment. Tissue slices were incubated in phosphate buffer medium con- taining 2.7 x 10—4 M L-leucine (sp. act. 0.14 uCi/umole) for 60 minutes at 25°C under 100% oxygen. Separated proximal tubules were incubated in medium containing 1.26 x 10-4 M L-leucine (sp. act. 0.27 uCi/umole) for 60 minutes at 25°C while being constantly gassed and mixed by bubbling with 100% oxygen. After incubation tissue was homogenized in 10% TCA and lipid, RNA and DNA extracted. The final protein pellet was solubilized and assayed for protein and radioactivity. 35 3. Incorporation of 14C L-leucine in vivo Rabbits were treated with either 1 or 3 injections of 90,000 I.U. procaine penicillin G at 12-hour intervals. Treatment was begun such that the experiment was run on the 14th day of life. All animals were sacrificed 16 hours after the final injection. Amino acid uptake was quantified by anesthetizing animals with 2 g/kg urethane, i.p. The left femoral vein was exposed and 8 uCi/kg 14C L-leucine (sp. act. 302 uCi/umole) in normal saline injected. Fifteen minutes after injection of label the animals were killed, the kidneys quickly removed, and cortex dissected free. Cortical tissue was minced in ice cold 0.25 M sucrose, washed with several changes of 0.25 M sucrose and homogenized. The homogenate was centrifuged at 10,000 x g for 20 minutes at 2°C and the pellet discarded. The supernatant was centrifuged at 100,000 x g for 60 minutes at 2°C in a Beckman L3-50 ultracentrifuge. Following cen- trifugation a sample of supernatant was precipitated with an equal volume of cold 10% TCA, allowed to stand in ice for 30 minutes and the pellet waShed three times by resuspension with TCA. The 100,000 x g pellet was resuspended in 0.25 M sucrose and centrifuged at 100,000 x g for 60 minutes at 2°C. Following centrifugation the supernatant was discarded and the pellet solubilized in 2 ml 1 N KOH. The precipitate from the soluble fraction was treated similarly. Solubilized material was incubated at 37°C for approximately 20 hours and reprecipitated by addition of 0.4 m1 6 N HCl and 2 m1 5% TCA. The resulting pellets were washed 3 times with 3 m1 cold 10% TCA and solubilized in 1 m1 1 N KOH. Aliquots were taken for assay of protein by the method of Lowry (1951) and radioactivity by liquid 36 scintillation spectrometry. Results were represented as DPM/mg protein. In an alternate experiment animals were injected i.v. with 14C L-leucine as described and sacrificed after 60 minutes. Cortex was dissected free and homogenized in 10% TCA. The acid insoluble pellet was extracted for lipid, RNA and DNA as described earlier. Results were represented as DPM/mg protein. 4. Leucine pool size The specific activity of amino acid pools was determined in animals treated with 1, 2 or 3 injections of penicillin. Sixteen hours after the final injection 8 UCi/kg l4C L-leucine (sp. act. 302 HCi/Umole) were injected i.v. and the animals sacrificed after 15 minutes. Kidneys were perfused with 20 m1 ice cold physiological saline and the cortex dissected free. One gram cortical tissue was homogenized in 10 m1 5% sulfosalicylic acid and kept in ice for 30 minutes. The suspension was centrifuged at 10,000 x g for 20 minutes at 2°C and the supernatant was dried. The residue was dissolved in 2 ml lithium citrate buffer, pH 2.0. Aliquots were taken for 14C determination using liquid scintillation spectrometry. Leucine was determined using a Technicon Autoanalyzer. Results were represented as DPM/nmole leucine. SLeucine determinations were conducted by Dr. W. Bergen, Department of Animal Husbandry, Michigan State University, East Lansing, MI 48824. 37 Separation of Protein Fractions of Renal Cortical Homogenates Following Substrate Stimulation by Penicillin and in vivo Labeling with 14C L-leucine l. Labeling of renal cortical_protein and subcellular fractionation Rabbits were treated with 90,000 I.U. procaine penicillin G subcutaneously on the 13th day of life and sacrificed 16 hours after the injection. Animals were anesthetized with 2 g/kg urethane and the left femoral vein exposed. Three injections of 8 uCi/kg 14C L-leucine (sp. act. 302 uCi/umole) were administered intravenously at 20-minute intervals. Twenty minutes after the third pulse animals were killed, kidneys quickly removed and cortex dissected free. Cortical tissue was minced and washed in 0.25 M sucrose. Minced tissue was homogenized in 0.25 M sucrose and the homogenate centri- fuged at 10,000 x g for 20 minutes at 2°C. The supernatant was centrifuged at 100,000 x g for 90 minutes at 2°C. Supernatant and pellet proteins were analyzed by Sephadex filtration and gel electro- phoresis, respectively. 2. Sephadex filtration A 5 ml aliquot of 100,000 x g supernatant was layered on a 3 x 100 cm Sephadex G-100 column equilibrated with 0.01 M KH2P04 (pH 7.4). Elution rate was 15 ml/hr and 3 m1 samples were collected. Protein and radioactivity concentrations in eluant fractions were determined by standard procedures and data plotted as CPM and mg/ml protein versus sample number. 38 3. Gel electrophoresis The 100,000 x g pellet was resuspended in sufficient 10 mM Tris 1 mM EDTA (pH 8.0) to result in a final concentration of from 3 to 8 mg/ml protein. The suspension was dialyzed against 10 mM Tris 1 mM EDTA pH 7.5 overnight to remove K+. The final electro- phoresis sample was 1% sodium dodecyl sulfate (SDS), 7% sucrose, 10 mM Tris, 1 mM EDTA and contained from 2-5 mg/ml protein. Immediately prior to electrophoresis sufficient concentrated mer- captoethanol was added to result in 2% final concentration and the sample heated in boiling water for 15 minutes. After cooling, bromphenol blue was added as tracking dye. Polyacrylamide gels (5-6% acrylamide) containing 1% SDS at pH 7.4 were prepared by the method of Fairbanks et a1. (1972) in glass tubes 6 mm inner diameter by 75 mm in length. Gels were electrophoresed for 30 minutes in buffer containing 40 mM Tris, 20 mM sodium acetate, 2 mM EDTA, and 1% SDS (pH 7.4) prior to addi- tion of sample. A 20—30 ul sample was layered on the gel and electrophoresed at 5 mA/gel. Usually 4 hours were required for the tracking dye to migrate the length of the gel. Gels were removed from the tubes, fixed in 10% TCA overnight, and stained in a solution of 0.4% Coomassie Brilliant Blue and 5% TCA. Gels were destained using a Bio-Rad diffusion destainer in 10% TCA, 33% methanol solution. Alternatively, gels were sliced using a Bio-Rad gel slicer immediately following TCA fixation. Gel slices were heated at 50°C in 1 m1 Soluene 100 for 3 hours and allowed to stand at room temperature overnight. Radioactivity was determined by 39 liquid scintillation spectrometry after addition of 10 ml PCS liquid scintillation cocktail (Amersham-Searle). Statistical Analyses Data were analyzed statistically by either Student's Eftest, paired or group comparison, or randomized complete block analysis of variance. Treatment means were tested using the Student Newman Keul's test (Steels and Torrie, 1961). The 0.05 level of proba- bility was used as the criterion of significance. RESULTS Pharmacodynamic Analysis of Substrate Stimulation 1. Effect of dosage and treatment schedule PAH S/M ratio in 2-week rabbit kidney cortex was enhanced by the administration of both procaine penicillin G and sodium penicillin G. A dose-dependent relationship existed between penicillin dose and PAH S/M ratio in both cases. Maximal accumulation of PAH by kidney slices occurred after treatment with 90,000 I.U. of procaine penicillin in the presence of 10 mM sodium acetate. The presence of acetate in the incubation medium increased the S/M ratio in slices from treated animals but did not alter the dose-response relation- ship (Figure l). Increasing the dose of penicillin to 180,000 I.U. with the soluble form increased the PAH S/M ratio above control (Figure 2), but the magnitude of the stimulation was no greater than that observed after 90,000 I.U. of procaine penicillin G (Figure 1). The effect of penicillin on PAH S/M ratio appeared maximal after 2 injections of 90,000 I.U. of procaine penicillin G (Figure 3). Continuing treatment for 4 days (8 injections) produced no greater effect. Enhancement of PAH S/M ratio after penicillin was greatest 24 hours after the final injection, although the effect was significant at both 12 and 36 hours (Figure 4). After 72 hours, 40 41 Figure 1. Effect of procaine penicillin dose on accumulation of PAH by rabbit renal cortical slices. Procaine penicillin G was administered twice daily for 3 days. Rabbits were 10 days old at the beginning of treatment and were killed 24 hours after the last injection. Each bar represents the mean (:_S.E.) obtained from 3 litters. _ A 1%.... Iwa/////////////////a 1 1... ..... m .. w . 03¢: 2\m 2(a— 0 15000 I. U. 30000 U. 00000 I. U. DOS PENICILLIN Cont. 43 Figure 2. Effect of sodium penicillin dose on accumulation of PAH by rabbit renal cortical slices. Sodium penicillin G was administered intraperitoneally following the same regimen as in Figure 1. Each bar represents the mean (:_S.E.) obtained from 3 litters. I: “/0 Acetate w. Acetate IT gig/Z 1%W//////////M//////////. m 5 0 5 0 1 1 Ave—(~— 23 :5— 30000 LU. 100000 I.U. 180000 I. U. PENICILLIN DOSE Cont. 45 Figure 3. Effect of treatment duration on enhancement of PAH S/M ratio. Beginning on the 10th day of life, rabbits were administered 2, 4, 6 or 8 injections of 90,000 I.U. procaine penicillin G at 12-hour intervals. Animals were sacrificed 24 hours after the final injection. Each bar represents mean (:_S.E.) obtained from 3 litters. D “I A H W w.Acotlto \ W5- §§§\\§§\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ =- [ W3. ® 2 Cont. I r—- 2ln|.-—I r—- 4h|.——1 [—— 61nl.“‘1 r—' Sin].- ‘ + a a 2 9 ° ° OIIVU W/S HVcl 30- Cont. 47 Figure 4. Accumulation of PAH by rabbit renal cortical slices 12, 24, 36 and 72 hours after termination of treatment. Beginning on the 11th day of life, rabbits were treated with 90,000 I.U. of procaine penicillin G subcutaneously twice daily for 2 days. Each bar represents mean (:_S.E.) obtained from 3 litters. D '/0 Acetate r—12hr.—1 r——24|Ir.—'1 r——36|Ir.—1 [—— 72hr.——1 48 W in. loot“. \§\F\\\\\\\\\\\\a- m. . 7\\\\\\\\\‘- L fl»- 8 g 9 2 o o ouva ms HVJ 49 there was no significant difference between control and penicillin— treated tissue with or without acetate. PAH S/M ratio was significantly increased 8 and 24 hours after a single 90,000 I.U. injection of procaine penicillin (Figure 5). Though PAH S/M in penicillin treated slices was greater than control after 12 and 16 hours, the stimulation was not significant due to high variability and low number of replications. Maximal stimula- tion following a single injection occurred 24 hours after treatment. There was no significant difference between control and treated after 48 hours. No radioactivity over background was observed in renal cortical slices 24 hours after the injection of l uCi of 14C—benzyl penicillin. All of the tissues except bladder urine were similarly devoid of any activity. Samples from bladder urine contained measurable, although physiologically insignificant, traces of radioactivity 24 hours after injection. 2. Substrate stimulation during development The effect of acute administration of penicillin on PAH S/M ratio was determined in animals whose mothers had received chronic penicillin treatment during gestation. S/M ratio in the pups born of these mothers was significantly greater (P<.05) than the S/M ratio normally observed at birth (Hirsch and Hook, 1970c). When littermates were treated acutely with procaine penicillin G the S/M ratio was significantly elevated at 3 days, 1 and 2 weeks of age (Figure 6). There was no significant difference in the S/M ratio of the treated animals. That is, the PAH S/M ratio was as high at 50 Figure 5. Accumulation of PAH by rabbit renal cortical slices 2, 4, 8, 12, 16, 24 and 48 hours following penicillin treatment. On the 12th day of life, rabbits received a single injection of 90,000 I.U. procaine penicillin G or saline for control. The upper panel represents PAH S/M ratio versus time for control and penicillin treated animals. The difference in mean value of S/M ratios from control and treated animals is plotted versus time in the lower panel. Each S/M value repre- sents the mean (:_S.E.) of at least 3 determinations. MEAN DIFFERENCE 2° 1 T r I I 'fl’ L JF‘..—~‘~‘..~“‘—.“.“‘-ml I“) P 5 .. O POII'CI'IIII O Conlrol o i i I s 1‘ 9 l. ‘3 _ 1 v- 6 >— 5 n— 4 - 3 — 2 l- 1 - () l l 1 1 1 O 10 20 3O 4O 50 51 HOURS P051 INJECTION Figure 5 52 Figure 6. Effect of penicillin on development of PAH S/M by rabbit renal cortical slices. Pregnant does were injected intramuscularly with 90,000 I.U. of procaine penicillin G twice daily from day 14 of gestation to delivery. Within a litter, pups were sampled at 3 days, 1, 2 and 4 weeks. Two days before experi- mentation, a pair of pups was randomly selected. One member of the pair received 2 injections of 90,000 I.U. procaine penicillin G at lZ-hour intervals and the other served as control. Animals were sacrificed 24 hours after the second injection. 0, accumulation of PAH in cortical tissue from pups receiving prenatal and post- natal penicillin; 0, animals receiving prenatal penicillin and postnatal saline. Each point represents the mean (: S.E.) obtained for 3 litters. 53 m museum augmuga uO< n N _. - u - 2.3.222 O .6523 O L CD “7 C) N v- '- ouvu w/s HVd IO N on 54 3 days of age as at 4 weeks. Animals 4 weeks of age did not respond to acute treatment with penicillin. Investigation of Mechanisms in Penicillin Stimulation of PAH Transport 1. Uptake and efflux of PAH by slices The rate of PAH uptake increased with increasing substrate concentration. Renal cortical slices from penicillin-treated 2-week rabbits took up 2.50 i.0'14' 3.65 :_0.10, and 5.51 i 0.09 ug PAH g-1 min.1 at medium concentrations of 1.0, 2.0, and 4.0 x 10.4 M, respectively (Figure 7). The rate of PAH uptake in these slices was significantly greater at each of the medium concentrations than in slices from control animals (1.90 :_0.10, 3.02 :_0.l7, and 4.87 t 0.17 pg 9'.1 min-1). The rate of PAH uptake under nitrogen was not affected by penicillin pretreatment (Figure 7). The difference in uptake between oxygen and nitrogen incubation, the best estimate of the oxygen-requiring, active component of uptake, was significantly enhanced by penicillin at each concentration (Figure 7). The runout of PAH from preloaded slices exhibited an initial fast component followed by a slower component. The data were linear when plotted on a semilogarithmic scale (Figure 8). First-order rate constants were calculated for the linear portion of the curve. The runout constant for control tissue (0.022 :_0.002 min-1) was not significantly different from that for penicillin-treated tissue (0.023 :_0.003 min-1). Farah et a1. (1963) suggested that the rapid component of PAH runout was due to a loosely bound intracellular pool. Contributions to enhanced PAH accumulation following penicillin might be made by either decreased runout or enhanced intracellular 55 Figure 7. p-Aminohippurate (PAH) uptake in 2-week control and penicillin-pretreated rabbit renal cortical slices. Beginning on the 10th day of life, animals received 30,000 I.U. procaine penicillin G twice daily for 3 days. Animals were killed 24 hours after the final injection. In each experiment, slices from control and treated animals within a litter were pooled and distri- buted into 12 beakers, 4 at each concentration of PAH and 2 under each gaseous phase. Duplicate values were averaged. Each bar represents means (: S.E.) of pups from 8 litters. Vertical bars represent S.E. of OZ-N2 uptake. PAH UPTAKE (pg/g/min) 56 CONTROL PENICILLIN ale E E E 1.0 2.0 4.0 PAH CONCENTRATION (M‘4) Figure 7 57 binding. Both phenomena would result in a slower rate of PAH runout. These possibilities may be excluded since an alteration in runout was not observed (Figure 8). 2. Kinetic analysis of PAH uptake in separated proximal tubules Penicillin treatment of newborn rabbits significantly increased the rate of PAH uptake at each PAH concentration in the medium when compared to saline control. A double reciprocal plot of the data (Figure 9) suggested that there was an increase in the apparent maximal rate of uptake from 3.03 in the control to 6.25 ug/g/min after penicillin treatment. There appeared to be, however, no change in the apparent affinity of the carrier for substrate (2.10 x 10-4 M). 3. Inhibitory effect of cycloheximide Administration of 0.18 mg of cycloheximide concurrently with 90,000 I.U. of procaine penicillin effectively blocked the stimu- lating effect of penicillin on PAH accumulation. Cycloheximide had no effect on control PAH S/M (Figure 10). 4. Proximal tubular ultrastructure Treatment of 2-week rabbit kidneys with collagenase produced a homogeneous suspension of tubules, with no significant contamina- tion with connective tissue or undifferentiated cells. It was evident from electron microscopy that proximal tubular cells from 2-week animals were differentiated to a degree comparable to adult tissues (Figures 11 and 12). Brush border, apical vacuoles, and intracellular organelles were well formed in the young animals. The brush border was less dense, however, than in the adult, and the basilar plasma membrane was less infolded. The basement membranes, 58 Figure 8. Runout of p-aminohippurate (PAH) from renal cortical slices from control and treated littermates. Beginning on the 10th day of life, animals received 30,000 I.U. procaine penicillin G twice daily for 3 days. Animals were killed 24 hours after the final injection. Slices were preloaded with PAH for 90 minutes, rinsed, and transferred through a series of beakers containing no PAH at l-minute intervals. Concentration of PAH in the slices as a function of runout time was determined and a first-order rate constant (K) calculated. Points and regression lines represent means from 4 litters. Constants represent means :_S.E. and are not significantly different from each other (P<0.05). 59 ON «006 mm «0.0 u v. ._O~:ZOU Q. mood H mwod u v. 23...!me m wusowm mwhDZE‘ m— 0— V— N— 0.. Go. . a 0.0.9 99$» .6 0 ON av 00 on § 6m OOl/HVd 6d 60 Figure 9. Double reciprocal plot of p-aminohippuric acid (PAH) uptake in separated proximal tubules from 2-week control and penicillin-pretreated rabbits. At 11 days of age, treatment was begun. Ninety thousand International Units procaine penicillin G was administered twice daily for 2 days. Animals were killed 24 hours after the final injection. In each experiment, tubules were preincubated for 15 minutes without PAH. PAH was then added to produce concentrations in the medium of 1, 4, or 8 x 10'4 M and the tubules further incubated for 15 minutes. Each point represents mean :_S.E. of pups from 3 litters. 61 o $ng :15: 2053:23on :35 0.— «.0 0.0 v.0 «.0 0 u q u d d 23:0.me ._O~=ZOU m.0 0.— (NIw/B/Bd) axvun HVd/l 62 Figure 10. Effect of cycloheximide on the penicillin- induced enhancement of PAH transport by rabbit renal cortical slices. Animals were treated with penicillin (90,000 I.U. s.c.) and/or cycloheximide (0.18 mg i.p.) twice over a 24-hour period. Beginning on the 12th day of life, rabbits were killed 24 hours after the second injection. Each bar represents the mean (:_S.E.) obtained from 3 litters. PAH S/M RATIO 30 25* N O l 15- 10" D Saline I///A Cycloheximide F iiiiiii 64 Figure 11. Electron micrograph of separated proximal tubules from (a) adult and (b) 2-week rabbit (x6020). Both tissues are structurally similar. Differences in structure include a more convoluted brush border (BB), a greater com- plexity and interdigitation of basilar infoldings (BI) and a thicker basement membrane (BM) in the adult. Due to the lesser complexity of BI, less compartmentation and alignment of mitochondria are observed in tissue from young animals. Other structures are comparable. Figure 11 in... 66 Figure 12. Electron micrograph of separated proximal tubules from 2-week rabbits (x6400). (a) Saline control, (b) penicillin treated. Animals received 90,000 I.U. of procaine penicillin twice daily for 2 days and were sacri- ficed 24 hours after the final injection. PAH transport capacity was enhanced with no alterations in tubular ultrastructure. Figure 12 68 which were irregularly preserved in both groups, were not as thick as in adult animals. There was no difference between newborn and adult kidney in the degree of disruption of basement membranes due to the enzymic digestion. Because basilar membrane infoldings were less complex in the newborn, mitochondria were not aligned in basilar compartments as in the adult tubule; they appeared to be unaltered in size. Other structures, including endoplasmic reticulum, free ribosomes, and dense bodies were comparable in both newborns and adults. Electron micrographs of specimens from control and penicillin— treated animals showed no difference in cellular structure (Figure 12). The density of brush border and of apical vacuoles, the size and number of lysosomal vacuoles, and the configuration of basilar membranes were comparable in proximal tubules of both groups. 5. Na, K-ATPase Na, K—ATPase activity in crude homogenates of renal cortex (Figure 13) was less in the newborn than the adult (0.32 :_0.08 and 0.69 :_0.08 umole PO4/mg protein/10 min, respectively). Treat- ment of 2-week animals with penicillin had no effect on enzyme activity (0.30 :_0.05 umole PO4/mg protein/10 min). Similarly, treatment had no effect on magnesium-dependent ATPase. Studies on Possible Correlations Between PAH Transport Capacity_and GSH S-Transferase l. Quantification of enzyme activity The concentration of aryltransferase substrates in the incuba- tion medium was limited by low water solubility. Dinitrochlorobenzene 69 Figure 13. Effect of penicillin pretreatment and matura- tion on renal cortical Na+, K+-activated ATPase determined from a crude cortical homogenate. Beginning on day 11, 90,000 I.U. procaine penicillin G was administered twice daily for 2 days. Animals were sacrificed 24 hours after the final injection. Each bar represents mean :_S.E. of 4 determinations. 7O -1, -. n p n n b n n a. 7. a. s. 4. a. 2. m. 0 O O O O O O 22.2 o_\z.u§: os\¢o._ $52 3 33:4 322572.67.on control penicillin ADULT I4 DAY Figure 13 71 concentration in the medium exceeded Km values for each of the glutathione S-transferase enzymes. Dichloronitrobenzene concentra- tion was slightly less than the reported Km values for the trans- ferase enzymes (Habig et al., 1974b). Increased optical density of the enzyme incubation mixture was linearly related to time during the 15 minute incubation period using both substrates. The rate of increase in optical density was directly proportional to protein concentration. Addition of TCA or boiling destroyed enzyme activity and reduced changes in optical density to values observed in buffer blanks. 2. Effect of age PAH S/M ratio was less in renal cortical slices from newborn rats and rabbits than in adults and increased with age. GSH S—aryl- transferase activity using both dichloronitrobenzene (Figures 14 and 15) and dinitrochlorobenzene was also less in the newborn of both species, but development of enzyme activity and transport capacity were asynchronous. At 1 week of age GSH S—aryltransferase activity using dichloronitrobenzene as substrate was not significantly dif- ferent than in kidney from adult rat while transport capacity was less (Figure 14). In rabbits, dichloronitrobenzene conjugating capacity in kidney cytosol from 3-day and 2-week pups was not dif- ferent, though transport was significantly less in the 3-day animal (Figure 15). Enzyme activity and transport capacity were greater in 4-week animals than in either 3-day or 2-week pups. 72 Figure 14. Effect of age on accumulation of PAH by rat renal cortical slices and on GSH S-aryltransferase activity in 100,000 x g supernatant of kidney homogenates. Each bar repre- sents the mean :_S.E. from at least 4 experiments. 73 (NM Sl/Nlaioaa Btu/we '00 V) All/\IIDV 3wxz~a ;%‘%°.%%%° W , 7W OIIVEI W/S HVd I WEEK ADULT I DAY AGE Figure 14 74 Figure 15. Effect of age on accumulation of PAH by rabbit renal cortical slices and on GSH S-aryltransferase activity in 100,000 x g supernatant of kidney homogenates. Each bar represents the mean :_S.E. determined in 4 litters. 75 (NM Sl/Nlaioad Bus/we gov) All/\IIDV SWAZNEI I :II/ / Z l >_ -—////////////// °‘ “‘ LL [:1 OIIVH W/S HVd 2 WEEK 4 WEEK I DAY AGE Figure 15 76 3. Effect of 3-methylcholanthrene (3-MC) PAH transport capacity was slightly, though not significantly, reduced in weanling rats pretreated with 25 mg/kg 3-MC when compared to corn oil controls (Figure 16). Kidney weight to body weight ratios, a measure of toxicity, were not altered by treatment. Dini- trochlorobenzene conjugation in kidney cytosol from treated animals was significantly increased following 3—MC (Figure 16). 3-MC pretreatment of 2-week rabbits resulted in a dose dependent increase in PAH S/M ratio. Maximal enhancement of S/M ratio was observed following 40 mg/kg 3—MC (Figure 17). Kidney weight to body weight ratio, one measure of toxicity, was not altered by 3-MC (Figure 17). GSH S-aryltransferase activity in kidneys from treated animals was similarly increased with dose. Maximal enhancement of enzyme activity was observed following 20 mg/kg 3-MC (Figure 18). 4. Effect of 2,3,7,8-tetrachlorodibenzofp-dioxin (TCDD) In preliminary studies PAH S/M was decreased when compared to control after 7 days in animals which had been treated with 1 or 5 ug TCDD intragastrically (Table 1). Ten micrograms per kilogram TCDD had no effect on PAH S/M 3 or 7 days after treatment (Table 2). PAH S/M ratio in animals which had received 25 ug/kg TCDD intra— peritoneally 7 days earlier was decreased (Table 2). There was no significant difference in percent extracellular water between control and treated at either dose and, therefore, the effect of TCDD reflected a true decrease in transport capacity. Though the maximal accumulation of PAH in all cases was less in perpendicular slices than in parallel, the relationship between control and treated within Eliza‘s-um 77 Figure 16. Effect of 3-MC on accumulation of PAH by rat renal cortical slices and on GSH S-aryltransferase activity in 100,000 x g supernatant of kidney homogenates. Weanling rats were administered 25 mg/kg 3-MC intraperitoneally once a day for 3 days and sacrificed 24 hours after the final injec- tion. Each bar represents the mean :_S.E. of 5 determinations. 78 we museum 2:2 2299... 9.51% 5.03 Z_>:u< £232“ n u m. a 2 m m o. o. o. m. m. o. o. o A q _ _ _ _ q Y s \\\ m + M u v. m m +5s\\\\\\\\\\\\\\\a IR 1 — _ r F _ b P M n w 8 6 4 2 O O_._.<¢ <<\m Is. 3- METHYLCHOLANTHRENE CONTROL 79 Figure 17. Effect of 3-MC on PAH accumulation by rabbit renal cortical slices and kidney wt/body wt ratio. Beginning on day 11, animals were treated with 10, 20, 30 or 40 mg/kg 3-MC twice daily for 3 days and sacrificed 24 hours after the final injection. Controls received vehicle. Each bar repre— sents the mean :_S.E. of 4 or 5 litters. 80 KIDNEY WT/BODY WT X IOO v.0 a .0 N.— 0.— 0.N Va 0* ha musmwm A333 2352502912252-" 2 0— 0 3 0.2:. <<\m :5. D 3 00— x ._.>> >000 \._.>> >m20§ 3 7/2 of 0.0 0&— 0.0— 0.0m 0.?“ OIIVII W/ S HVd le 81 Figure 18. Effect of 3-MC on GSH S-aryltransferase activity in 100,000 x g supernatant of rabbit kidney homo- genates. See Figure 8 for treatment details. Each bar represents the mean : S.E. of 3 litters. 82 ma musofim Aniaev “235230231552-..” 00.0 00.0 N —.0 2.0 0N.0 0N.0 0N.0 (le Sl/Nlaioaa Btu/'a'o v) AllAllDV SWAIN! 83 Table 1. Effect of TCDD on accumulation of PAH by slices of renal cortex from adult ratsa PAH S/M Ratio Treatment n Control TCDD Treated 1 pg 4 11.0 :_0.3 9.6 :_0.4 5 pg 2 11.0 + 0.9 9.2 + 0.8 aAnimals were treated with l or 5 ug TCDD, intragastrically. Controls received vehicle. After 7 days animals were sacrificed and PAH transport capacity in slices of renal cortex determined. Each value represents the mean (:_S.E.). bn = number of animals. Table 2. Effect of TCDD on accumulation of PAH and NMN by slices of renal cortex from adult ratsa SZM Ratio PAH NMN Duration Perpen- Perpen- Treatment (days) Parallel dicular Parallel dicular; Control 3 13.8 8.5 9.1 5.3 10 ug/kg TCDD 14.2 9.3 7.5 4.5 Control 7 13.7 7.8 8.6 5.4 10 HQ/kg TCDD 13.2 6.4 6.5 3.8 Control 7 12.8 6.3 7.9 4.3 25 HQ/kg TCDD 11.4 5.6 5.1 3.9 aAnimals were treated with 10 or 25 ug/kg TCDD, intraperi- toneally. Controls received vehicle. Three and 7 days later animals were sacrificed. Slices of renal cortex were taken in both a hori- zontal and parallel plane to the major renal axis. Slices from 3 control and 3 treated rats were pooled before incubation. Therefore, each number represents the mean value of a duplicate incubation of tissue from its respective pool. 84 a type of slice remained. NMN accumulation appeared to be decreased at all doses of TCDD (Table 2). 5. Effect of chronic metabolic acidosis Chronic ammonium chloride acidosis increased kidney weight to body weight ratio. PAH S/M ratio in slices from acidotic animals was significantly decreased whereas GSH S-aryltransferase activity was increased at least 8-fold using both enzyme substrates (Figure 19). 6. Effect of substrate stimulation by penicillin Penicillin pretreatment significantly increased PAH S/M ratios as did 3-MC but had no effect on GSH S-aryltransferase activity using either enzyme substrate (Figure 20). 7. Sex difference GSH S-aryltransferase activity was lO-fold higher in kidneys from adult mice than adult rats. PAH S/M ratio was significantly greater in male mice than in females. Enzyme activity determined using dichloronitrobenzene was not significantly different between sexes (Figure 21). 8. Effect of uninephrectomyfi Accumulation of PAH by renal cortical slices was significantly increased in the remaining kidney of uninephrectomized rats 48 hours following surgery. GSH S-aryltransferase activity was not signifi- cantly increased using either dichloronitrobenzene (Figure 22) or dinitrochlorobenzene substrates. r" 'J‘m ' . 85 Figure 19. Effect of chronic ammonium chloride acidosis on accumulation of PAH by rat renal cortical slices and GSH S-aryltransferase activity in 100,000 x g supernatant of kidney homogenates. Rats were maintained for 7 days on 0.28 M NH4C1 as sole drinking fluid. Controls were allowed tap water. Each bar represents the mean :_S.E. of 8 determinations. 86 2.2 £2.39: 9531” ads I.>:o< 32:5 ACIDOTIC 1.1 .1... u. m m. m. m“ m. m. 0 Y NW\\\\\\\\\\\\\\\\\\\\\\\\\ M m . . IT 3 as c a“ A“ .41 0“ nu _ _ _ O_._.<~_ <<\m I- : 8 * \§ 010 E P- -4-\\\\ ‘+ ' gi \ nu \ E 4 _ \ .. :2 2 Lu 0 \ o CONTROL PENICILLIN Figure 20 89 Figure 21. Accumulation of PAH by renal cortical slices and GSH S-aryltransferase activity in 100,000 x g supernatant from adult male and female mouse kidneys. Each bar represents the mean :_S.E. of at least 6 determinations. “2:2 52.39: as}; do 3 55:3 ”2»sz 2. 3 4. o. 3 2 2 2 _ _ _ _ O O O _ _ 6 2 3 4. _ _ §\\\\\\\\\\\\\\\\\\. + FEMALE 7_ PAH S/M RATIO § 8L— O:<~_ <<\m I:>=u< 52>sz u u u m M u. m m. 0. O. 0. O. O O O O 0 q _ _ _ d A _ _ _ ..\\\\\\\ \\ m m .1 T V - M n m M .v m \\\\ H H ..\ \ \ M m + V - Im _ _ _ L _ _ _ p _ M 14: W. .0: 8 6 4 2 O 18 0:22. <<\m I (a. UNINEPHRECTOMY SHAM Figure 22 93 9. PAH transport capacity following GSH depletion L—Methionine-SR-sulfoximine significantly decreased GSH concen- trations in the kidneys of adult male rats (Figure 23). There was no regeneration of GSH in slices from sulfoximine treated rats during incubation in vitro. GSH concentration in renal cortical slices taken from control animals was not significantly decreased during the incubation period. PAH S/M ratio was not significantly decreased by sulfoximine pretreatment (Figure 23). 10. Effect of age and penicillin pretreatment on penicillin binding to soluble proteins Sephadex filtration of soluble proteins from adult renal corti- cal homogenates resulted in elution of 4 major protein peaks (Figure 24). GSH S-aryltransferase specific activity was greatest in protein eluted with the third peak (sample number 70-80).A Two peaks of radioactivity were observed, one associated with the protein peak not containing enzyme activity and the second corresponding to the GSH S-transferase containing peak (Figure 24). Protein profiles of 2-week-old rabbit renal cortical soluble proteins consisted of only 3 major peaks which eluted in similar volumes compared to the adult (Figure 25). Two radioactivity peaks were observed, one correspond— ing to samples containing no enzyme activity and the second to the major enzyme containing samples. Penicillin pretreatment did not alter either protein or radioactivity elution profiles (Figure 26). Binding was estimated as CPM/mg protein using maximal protein and radioactivity values within the GSH S-transferase containing peak. In the adult, binding was approximately 600 CPM/mg protein (Figure 24), whereas in young animals binding was approximately 400 CPM/mg 94 Figure 23. Effect of L-methionine-SR—sulfoximine treatment on GSH concentration and accumulation of PAH by renal cortical slices. Rats were treated with 1.85 mmoles/kg sulfoximine and sacrificed 2 hours after injection. Reduced glutathione was assayed in slices before and after 60-minute incubation. Each bar represents the mean :_S.E. of 6 determinations. 95 OIlVU W/S HVd 0d 0.? 0.0 0.0 0.0— 0.“— 0.: mm magmas m2_$:x0n_.5ml._0 I uZ_ZO_I.pm< o- -