1mm» ' 11.. m; 1.5“ "VI!!! Y .«u. . I _ v’.3:j~\..'c4~{x_:- W: 25¢ per day per item RETURNIMS LIBRARY MATERIALS: Place in book return to remve charge from circulation records ENDOGENOUS FATTY ACIDS DO NOT INHIBIT RENAL TRANSPORT OF ORGANIC ACIDS By William Edward Stroo A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology & Toxicology 1979 ABSTRACT ENDOGENOUS FATTY ACIDS DO NOT INHIBIT RENAL TRANSPORT OF ORGANIC ACIDS William Edward Stroo A large number of observations suggest that a link_exists between renal nonesterified fatty acids (NEFA) and the transport of organic anions. Recently, the mech- anism of enhanced renal transport of PAH after penicillin treatment of the neonate was postulated to be reduced inhibition of transport subsequent to reduction of the cellular content of NEFA by penicillin. Analysis of this problem required the resolution of three questions. First, are NEFA transported into the cell of the proximal tubule by the organic anion transport system? Second, do NEFA, in physiological concentrations, act as inhibitors of organic anion transport? Third, is the alteration in lipid metabolism observed after penicillin treatment responsible for the alteration in transport capac- ity? Palmitic acid effectively inhibited PAH transport in vitro. However, enhanced organic anion transport William Edward Stroo capacity was not associated either with increased incorpor— ation of labeled fatty acid into tissue total lipid nor was it associated with increased extraction of label from the medium. Thus it appears that NEFA do not enter the renal cell primarily by the organic anion transport system. Alterations in transport capacity were not associ- ated with alterations in tissue NEFA. Penicillin treat- ment enhanced transport but did not decrease cellular NEFA and, in fact, increased serum NEFA. Fasting did not sig- nificantly alter renal NEFA content although transport was decreased. Renal NEFA content was higher in adult kidneys than it was in immature rabbit kidneys although adults had a greater transport capacity. Finally incubation conditions under which fatty acids decrease transport were associated with tissue NEFA concentration markedly above the endogenous concentration of NEFA in the kidney. Thus, NEFA are not likely to exert an inhibitory effect on organic anion trans- port at physiological concentrations. Penicillin induced alterations in lipid metabolism were not observed in_glomeruli, indicating the dependence of altered metabolism on organic anion transport. However, penicillin treatment enhanced triglyceride synthesis and decreased incorporation of labeled fatty acid into tissue William Edward Stroo NEFA without a concommitant change in transport capacity. Nicotinic acid treatment of the adult rabbit produced a similar change in metabolism but failed to enhance trans- port capacity. PAH, probenecid, and nicotinic acid pro- duced similar in vitro effects. Furthermore changes in renal triglyceride and serum NEFA produced by penicillin are similar to the reported effects of nicotinic acid. These results suggest that penicillin induced changes in renal metabolism of NEFA are not related to alterations in transport and that penicillin may be acting in a manner similar to nicotinic acid in producing altered lipid meta- bolism. These results, therefore, are not consistent with the conclusion that penicillin pretreatment of the neonate enhances organic anion transport by decreasing the endogenous inhibition of NEFA. ACKNOWLEDGEMENTS I would like to express my sincere appreciation to Dr. J. B. Hook.for his counsel, encouragement and construe- tive criticism during the course of this investigation. I would also like to thankars. T. M. Brody, J. Bennett and M. Bailie, M.D. for their assistance in preparing this thesis. My wife, Ellen, deserves special recognition for her patience, love, encouragement and motivation without which I would not have been successful. I have also received excellent technical assist— ance and would like to express my appreciation to Mr. T. Steele and Ms. S. Mehl whose efforts greatly furthered the research. H! H! TABLE OF CONTENTS ACMOWLEDGEMENTS O O O O O O I O O I O O O O O O O O O TAB LE OF CONTENT S O O O O O O O O O O O O O O O O O O 0 LIST OF TABLES O O O O O O O O O O O O I O O O O O O 0 LIST OF FIGURES O O O O O O O O O O O O O O O O O I O 0 INTRODUCTION 0 O I O O O O O O O O O O O O O O O O O 0 An overview of the renal organic anion transport system . . . . . . . . . . . . . . . . . . . Cellular receptor sites. . . . . . . . . . . . . Peritubular membrane carriers. . . . . . . . . . Energy coupling to transport . . . . . . . . . . Luminal membrane carriers. . . . . . . . . . . . Mechanisms of penicillin enhanced transport. . . Kinetic Analysis . . . . . . . . . . . . . . . . Protein synthesis. . . . . . . . . . . . . . . . Intracellular binding. . . . . . . . . . . . . . Triglyceride biosynthesis. . . . . . . . . . . . Objectives . . . . . . . . . . . . . . . . . . . METHODS MD MATERIALS 0 I O O O O O O I O O O O O O O 0 Media. . . . . . . . . . . . . . . . . . . . . . Preparation of tubules and glomeruli . . . . . . Determinations of organic anion transport. . . . Palmitate esterification . . . . . . . . . . . . Glomerular metabolism. . . . . . . . . . . . . . Palmitate extraction from the medium . . . . . . Triglyceride and NEFA assay. . . . . . . . . . . iii Page ii iii vi vii 12 14 16 21 23 24 25 31 32 34 35 38 39 41 43 43 44 TABLE OF CONTENTS (Cont'd) Page Diacyl glycerol acyl transferase (DAGAT) assay . . . . . . . . . . . . . . . . . . . 46 Statistical analysis. . . . . . . . . . . . . . 48 RESULTS 0 O O O O O O O O O O O O O O O O O O O O C 0 50 Effect of palmitate in the medium on PAH accumulation. . . . . . . . . . . . . . . . 50 Effect of penicillin treatment of utilization of palmitate. . . . . . . . . . . . . . . . 53 Effect of penicillin treatment on endogenous NEFA and triglyceride . . . . . . . . . . . 57 Effect of fasting on endogenous lipids. . . . . 57 Effect of organic anions on NEFA content of tubules incubated in a medium containing palmitate acid. . . . . . . . . . . . . . . 63 Effect of fasting on renal transport. . . . . . 70 Effect of penicillin treatment of glomerular lipid metabolism. . . . . . . . . . . . . . 75 Effect of various penicillin dosing regimens on tubular metabolism of palmitic acid. . . 75 Effect of fasting on transport and palmitate metabolism. . . . . . . . . . . . . . . . . 84 Effect of nicotinic acid on accumulation of PAH and lipid metabolism in the fasted rabbit. . . . . . . . . . . . . . . . . . . 84 Effect of probenecid, PAH and nicotinic acid in vitro on the metabolism of palmitic acid . . . . . . . . . . . . . . . 89 Effect of penicillin treatment and organic anions on diacyl glycerol acyl transferase (DAGAT) . . . . . . . . . . . . 98 DISCUSSION. 0 O O O O O O O O O O O C O O O O O O O C 108 Renal fatty acid utilization and organic anion transport . . . . . . . . . . . . . . . . . 109 NEFA as endogenous inhibitors of the renal organic anion transport system. . . . . . . 117 iv TABLE OF CONTENTS (Cont'd) Altered renal metabolism of NEFA and altered transport . Penicillin — hypolipidemic drug?. Conclusions . SPECULATION . SUMMARY . . . BIBLIOGRAPHY. O Page 122 125 128 129 131 135 Table LIST OF TABLES Page Penicillin Effect on PAH T/M and Palmitate Incorporation into Tissue Total Lipid in 2 week Old Rabbits O O O O O O O I O O O O O O O 5 6 Effect of Penicillin Treatment on Endogenous Serum and Kidney NEFA Content of 2 Week Old Rabbits O O O O O O O O O O O O O O O O O O O O 60 Effect of Various Penicillin Treatment Regimens on NEFA Metabolism and Organic Anion Transport in the 2 Week Old Rabbit . . . 83 vi Figure 10 LIST OF FIGURE LEGENDS Accumulation of PAH by suspensions of renal cortical tubules in the presence or absence of added palmitate . . . . . . . . . . . . . Effect of penicillin treatment on transport of PAH and on palmitate extraction from the med ium O O O O O O O O O O O O O O O O O O 0 Effect of penicillin treatment on renal and serum concentrations of NEFA . . . . . . . . Effect of penicillin treatment on renal, liver and serum triglyceride . . . . . . . . Effect of fasting on renal, liver and serum NEFA concentrations. . . . . . . . . . . . . Effect of fasting on renal, liver and serum triglyceride O O O O O C O O O O O O O O O 0 Effect of probenecid and PAH on tubule content of NEFA in vitro . . . . . . . . . . Effect of fasting, nicotinic acid and bicarbonate on the transport of PAH by the rat kidney 0 O O O O O O O O O O C O O O O 0 Effect of fasting, nicotinic acid and bicarbonate on the transport of TEA in the rat kidney 0 O O O O O O O O O O O O O O O 0 Effect of penicillin treatment on glomerular incorporation of C14—palmitate into tri— glyceride. . . . . . . . . . . . . . . . . . vii Page 52 55 59 62 65 67 69 72 74 77 LIST OF FIGURE LEGENDS (Cont.‘d) Figure 11 12 13 14 15 l6 17 18 19 20 Page Effect of penicillin treatment on glomerular incorporation of Cl4—pa1mitate into tissue NEFA O O O O O O O O O O O O O O O O O O O C O 7 9 Effect of various penicillin treatment regimens on tubular transport capacity and tubular metabolism of palmitate. . . . . . 81 Effect of fasting on accumulation of PAH into tubules and the incorporation of labeled palmitate into tissue total lipid. . . . . . . 86 Effect of fasting on the distribution of labeled palmitate into tissue lipids . . . . . 88 Effect of nicotinic acid on renal accumu- 1ation of PAH and incorporation of label into tissue total lipid in the fasted rabbit . . . . . . . . . . . . . . . . . . . . 91 Effect of nicotinic acid on the distribu- tion of labeled palmitate into tissue lipid. O O O O O O O O O O O O O O O O O O O C 93 In vitro effect of probenecid or PAH on incorporation of label into tissue total lipid. O C O O O O O O O O O O C O O O O O O O 95 In vitro effect of probenecid or PAH on incorporation of label into tissue tri— glyceride. O O O O O I O O O O O O O O O O O O 97 In vitro effect of probenecid or PAH on incorporation of label into tissue NEFA. . . . lOl ‘Eg;zi§rg_effect of 2 mM nicotinic acid or 2'mM PAH on incorporation of label into tissue total lipid . . . . . . . . . . . . . . 103 LIST OF FIGURE LEGENDS (Cont.'d) Figure 21 22 Page In vitro effect of 2 mM nicotinic acid or 2 mM PAH on incorporation of label into various lipid classes . . . . . . . . . . . . 105 Effect of penicillin treatment and added organic anions on the activity of DAGAT . . . 107 INTRODUCT ION The report by Marshall and Vickers (1923) of secre- tion of phenolsulphonephthalein by the dog kidney was one of the earliest demonstrations of the secretion of an organic anion (Weiner, 1973). It was, however, the discovery of the secretion of penicillin, coupled with its limited supply and its clinical value, which.provided the initial impetus for intensive study of the renal organic anion transport system (Beyer et al., 1944; Beyer 1950). The subsequent realiza- tion that a wide.variety of organic anions are secreted by the mammalian kidney has sustained interest in this system as a determinant of organic anion pharmakokinetics. The renal organic anion transport system may be involved in processes in addition to excretion of drugs and chemicals. For instance, the anion secretory system may play a role in the renal utilization of metabolic fuels such.as alpha-ketoglutarate and nonesterified fatty acids (Cross and Taggart, 1950; Cohen and Wittman, 1963; Barac- Nieto and Cohen, 1968; Selleck and Cohen, 1965). The action of several diuretics upon the kidney appears to depend upon their secretion (Bowman, 1975; Duggan, 1966). Similarly, the nephrotoxicity of cephaloridine is also dependent upon its cellular accumulation by the organic anion transport system (Tune and Fernholt, 1973; Tune, 1972). The apparent secretion of prostaglandins, coupled with their potent renal actions, suggests that their effects may, in part, be dependent upon secretion by the transport system (Bito and Baroody, 1978). Recent studies also indicate that a fre- quent early sign of renal toxicity is enhanced or decreased organic anion transport (Hirsch, 1976). While the mech- anisms underlying these alterations remain obscure, it seems apparent that an understanding of the mechanism of transport would also yield significant insight into the mechanisms of renal toxicity. These observations indicate that an increased understanding of the mechanism of renal organic anion transport will be of broad interest to clinical, industrial, and regulatory pharmacologists. Extensive study of renal organic anion transport has led to an extensive body of knowledge and several different hypotheses have been presented concerning the.mechanism of transport. A relatively recent approach to this problem has been to use the immature animal as a tool to evaluate the mechanisms of transport. Use of the immature animal as a model for the analysis of renal mechanisms is based on the observation that the functional capacity of the neonate is lees than in adults. Renal blood flow (Rakusan and Marcinek, 1973; Gruskin et al., 1970; Jose et al., 1971; Aschinberg et al., 1975), glomerular filtration rate (Horster and Valtin, 1971; Spitzer and Edelmann, 1971; Aperia and Kerin, 1975), concen- trating ability (Sakai and Endov, 1971; Stanier, 1972), and several biochemical measures including ammoniagenesis (Cort and McCance, 1954; Goldstein, 1970), Na+ - K+ - ATP ase (Burich, 1974; Schmidt and Horster, 1977), and the transport of organic anions and cations (Horster and Valtin, 1971; Horster and Lewy, 1970; Lewy and Grosser, 1974; Rennick, et al., 1961) are not fully developed in the newborn of most species. Various investigators have quantified alterations in specific renal functions with maturation in attempts to elucidate basic mechanisms of several renal functions CUllrich+ 1976; Schriver et al., 1976; Reynolds and Segal, 1976). Unfortunately, analysis of the biochemical corre- lates of a functional change during development is frequently complicated by biochemical changes associated with the ‘meturation of other functional parameters. Other investi- gators have used the immaturity of the kidney to evaluate the role of a specific renal mechanism in the response to func- tional loading. For example, saline loading of the immature animal has been used to evaluate the role of alterations in renal blood flow in the renal excretion of excess salt and water (Bengele and Solomon, 1974; Aperia et al., 1975; Arandt,l978). Finally other investigators have attempted to selectively alter the rate of maturation of specific renal functions by stressing the function physiologically or pharmacologically. This approach can be a powerful tool in the analysis of the biochemical correlates of a specific renal function. Extensive studies have elucidated three basic types of renal response to functional or pharmacological stimula- tion. Several physiological functions are resistant to stimulation because of the structural immaturity of the kidney (McCrory, 1972; McCance and Widdowson, 1958). Other functions such as ammoniagenesis (Benyajati and Goldstein, 1975), and some drug metabolizing enzymes (Kluwe et al., 1978) are inducible in the immature animal and are also inducible in the adult. Finally, some functions appear to be inducible only in the immature animal. The administra- tion of cortisol to neonatal rats evoked premature increases of renal glucose-G—phosphatase, and renal aspartate amino transferase to adult levels, while adults were unresponsive to cortisol treatment (Greengard, 1975). Similarly, and of particular interest to this discussion, renal organic anion transport was inducible in neonates but not in adults by treatment with penicillin or 3 methylcholanthrene (Hirsch and Hook, 1969; Kluwe et al., 1978). While it is unclear why the adult is unresponsive, these results suggest that the neonatal animal offers unique advantages for the analy- sis of the biochemical correlates of altered organic anion transport. ‘An‘overview of the renal organic anion "transport system The excretion of a compound in excess of that attributable to glomerular filtration, implies the net transfer (secretion) of that compound across the tubule from the plasma into the urine. Traditionally, the secre- tory transport of organic anions has been visualized as a two step process: accumulation into the cell followed by passive diffusion into the urine. The primary role of accumulation into the cell in the transport process allows the use of in vitro preparations for investigations of the mechanism by which organic anions are transported. Indeed, numerous investigators have demonstrated that the ability of renal cortical tissue to accumulate organic anions in vitro under a variety of conditions is qualitatively analogous to similar in 2339 determinations of renal secre- tion (Weiner, 1973). The overall secretion of organic anions occurs against a concentration gradient (Cross and Taggart, 1950; Foulkes and Miller, 1959; Tune et al., 1969; Carrasquer and Wilczewski, 1971). Transport is dependent on metabolic energy since cold and a nitrogen atmosphere effec- tively inhibit transport (Cross and Taggart, 1950; Farah and Rennick, 1956). A wide variety of metabolic inhibitors are also effective inhibitors of transport (Maxild, 1973; Mudge and Taggart, 1950; Cross and Taggart, 1950; Shideman and i Rene, 1957; Shideman et al., 1952; Farah et al., 1953; Farah and Rennick, 1956; Maxild and M¢ller; 1969). The transport system is not only dependent on metabolic energy but is also sensitive to metabolic modula— tion. Transport is sensitive to pH, K+ and Na+ and to the presence of a wide variety of metabolic substrates, such as acetate (Cross and Taggart, 1950; Taggart, et al., 1953; Gerencser et al., 1973; COpenhaver, et a1., 1965). Unfor— tunately, the mechanisms by which modulation occurs remain, for the most part unresolved (Podevin and Boumendil-Podevin, 1977; Chung et al., 1970; Weiner, 1973; Kippen and Klinenberg, 1978; Hewitt and Hook, 1978). The secretion of organic anions has been localized within the kidney to the proximal tubule by a wide variety of techniques including micropuncture (Baines et al., 1968; Cortney et al., 1965; Richards et al., 1938), perfusion of isolated nephron segments (Tune et al., 1969), stop flow (Kessler et al., 1959; Malvin et al., 1958), and autoradiog- raphy (Kinter et al., 1960; Wedeen and Jernow, 1968). Most recently, the transport of organic anions has been localized primarily to the 82 segment of the proximal tubule by a com— bined ultrastructural and isolated perfused segment proce- dure (WOOdhall et al., 1978). Significantly, both the $1 and 83 segments were also capable of secretion but at a rate 23% of that observed in the $2 segment, indicating that while transport was substantially localized in the 82 seg- ment, the transport system is a function of the entire proximal tubule. A cellular receptor has been implicated in the transport mechanism since the transport system appears to be saturable, susceptible to competitive inhibition, and shows some structural selectivity. £3 yi!g_the secretion of organic anions is characterized by a tubular maximum implying saturation (Weiner, 1973). Competition is indi— cated since similarly secreted anions such as probenecid, p—aminohippurate or penicillin can reduce the secretion of each other (Beyer et al., 1944; Beyer et al., 1947). Simi- larly, evidence for saturation and inhibition can also be obtained in vitro (Weiner, 1973). Analysis based upon ’Michaelissnenten kinetics suggests that the inhibition is competitive (Huang and Lin, 1965; Park et al., 1971). The transport system also appears to demonstrate some selectiv— ity since organic cations do not affect transport, nor are all anions secreted (Weiner, 1973; Despopoulas, 1965; Nielsen and Rassmussen, 1975). While these results clearly imply the presence of a cellular receptor, the site of the receptor remains obscure. Two prominent hypothesis have been advanced. Foulkes and ”Miller (1959) visualized an intracellular receptor while Tune et al., (1969) have emphasized the presence of a peritubular membrane carrier. ‘Ceilular receptor sites The demonstration by Foulkes and Miller (1959) of two kinetic components in the efflux of the prototype organic anion p-aminohippurate (PAH) from renal cortical slices led to the suggestion that two intracellular pools existed for transported organic anions. They suggested that the rapid component of efflux represented freely dif- fusable intracellular anion, while the slower component represented a subsequent intracellular concentrating or binding step. Several investigators have confirmed that two kinetic components exist in the renal influx or efflux of organic anions, but have differed in their interpretation of the meaning of the two components. Sheikh and M¢ller (1970) determined that the influx of PAH into isolated tubule suspensions had two kinetic components. They inter- preted the initial rapid component as the concentrating step since concentrations greater than the medium were rapidly achieved. The second component was suggested to represent subsequent passive intracellular binding or com- partmentalization. Anderson et a1. (1969) demonstrated that the accumulation of nitrofurantoin also had an active and passive component but concluded that the two com— ponents were arranged in parallel rather than in series. Burg and Orloff (1969) suggested that the two components of efflux were also arranged in parallel and that the rapid 10 component reflected efflux from injured cells while the slow component reflected efflux from uninjured cells. In support of the possibility of intracellular bind— ing several investigators have suggeSted that intracellular binding does occur for a variety of transported organic anions (Duggan, 1966; Berndt, 1966; Berndt, 1967; Berndt and Grote, 1968; Despopolous, 1965; Despopolous and Segerfeldt, 1964; Forster and Hong, 1958). Furthermore, Kirsch et a1. (1975) have also demonstrated the presence of low molecular weight binding proteins which.can bind organic anions spe- cifically and reversibly. Moreover, the failure to demon- strate intracellular binding of PAH (weiner, 1973) could be due to the inadequacy of current methodology (Farah et al., 1963). These results suggest that intracellular binding could be involved in organic anion transport. However, the failure to demonstrate intracellular binding of PAH could also imply that intracellular binding is not a component of the transport system, a position which has been taken by several investigators (Foulkes, 1963; Tune et al., 1969). An alternative to intracellular binding was proposed based upon measurements of transport in the isolated per- fused tubule (Tune et al., 1969). When PAH was supplied to the tubule in the.bathing media it was concentrated in the 11 cell. If, however, PAH was supplied from the tubule lumen, it was not concentrated in the tubule. Tune et al., (1969) proposed a model of PAH transport in which intracellular binding was not involved. They suggested that the energy dependent concentration step occurred at a peritubular site via a membrane carrier. Subsequent efflux out of the cell occurred via passive diffusion along a concentration gradient. Furthermore, since the peritubular membrane was less permeable to efflux than the luminal membrane, net secretion was assured once the organic anion was concentra- ted in the cell. They, however, did not suggest a mechanism to account for the difference in permeability. Furthermore, while a peritubular membrane carrier probably does exist, the Tune model is subject to other criticisms. First, several studies have failed to elucidate a mechanism by which transport is linked to cellular metabolism at a peri- tubular membrane site. Second, the conclusion that intra- cellular binding does not occur is based upon the observa- tion that PAH was not concentrated inside the cell when it was supplied from the lumen. The use of back flow to test for intracellular binding assumes that the luminal membrane is permeable to anions presented from the luminal surface. The back flow test also assumes that intracellular binding 12 will occur without prior interaction with a peritubular site. Thus if the luminal membrane were relatively impermeable to PAH or if the binding of PAH in the cell required a prior, perhaps catalytic, interaction with.the peritubular carrier, then despite the involvement of intracellular binding in the transport of organic anions, luminally presented organic anions would not be concentrated. ‘Peritubular membrane carriers Confirmation of the existence of a peritubular mem- brane carrier has been provided by several investigators. Fujimoto and his colleagues (watrous et al., 1970; Hakim et al., 1970) demonstrated that the secretion of infused mor— phine ethereal sulphate (MES) and 5-hydroxyindolacetic acid (5—HIAA) could be inhibited by probenecid, indicating that these organic anions were secreted by the renal organic anion transport system. However, excretion of 5-HIAA and MES generated intrarenally via the metabolism of the organic cations serotonin and morphine, was not affected by probenecid. These results suggest that the interaction between the organic anions occurred prior to their entry into cellular metabolic pools. The authors concluded that a 13 peritubular membrane interaction consistent with a membrane carrier was responsible for the effect of probenecid on the excretion of MES and 5-HIAA. In further support of the con- cept of a peritubular carrier, Hong et a1. (1978) observed that the "nonpermeating" organic anion, isothiocyano-Z, Z‘edisulfonic stilbene (SITS), inhibited PAH transport in an apparently competitive manner. Berne and Kinne (1976) demonstrated that PAH pene- tration into basal-lateral membrane vesicles was susceptible to probenecid inhibition. Ross and his colleagues (Holohan et al., 1976; Kinsella et al., 1977) demonstrated that pene- tration of PAH into basal—lateral membrane vesicles was both saturable and susceptible to competitive inhibition and was not sensitive to the presence of organic cations. Thus, it appears that a peritubular membrane carrier, as suggested by Tune et a1. (1969), does exist. If, however, the rest of the Tune model is correct, then it is difficult to defend the earlier concept of the involvement of intracellular binding in the transport of organic anions. Several obser- vations, however, indicate that the Tune model may have to be reVised, since it appears that the coupling of cellular energy with.the transport system may not occur at the l4 peritubular membrane, and that a carrier mechanism may be involved in the efflux of organic anions from the cell. Energy coupling to transport Transport of organic anions does not appear to be directly dependent on the concentration of cellular adenosine triphosphate (ATP). Maxild (1973) used the uncoupling agents, 2, 4—dinitrophenol and carbonylcyanide-m— chlorophenylhydrazone and found that at concentrations which decreased transport 50%, little of any change in cellular ATP was observed. If, however, transport was significantly inhibited, the addition of 10 mM acetate would enhance transport to values comparable to those observed in the absence of the uncoupling agent and the presence of acetate. Moreover, the addition of acetate rather than increasing cellular ATP actually appears to decrease cellular ATP (Gerencser et al., 1977). These results suggest that cell- ular transport of organic anions is not directly driven by cellular ATP and that the decrease in transport is not due to competition for the peritubular carrier between the uncoupling agent and PAH. 15 Numerous investigators have demonstrated that the transport of organic anions is dependent on the cation sodium (Chung et al., 1970; Gerencser et al., 1973). SOdium.dependent carrier mediated transport, as has been deScribed for the renal transport of sugars and amino acids, can be regenerated by the application of a sodium gradient across brush.border vesicles (Kinne, 1976; Ullrich, 1976). Podevin et a1. (1978) have recently demonstrated that PAH can be concentrated in sodium depleted renal cortical slices by application of a NaCl gradient, suggesting that organic anion transport is, in part, dependent on a sodium gradient. However, neither Berne and Kinne (1976) nor Kinsella et a1. (1977) were able to demonstrate sodium linked co-transport in brush border or basal-lateral membrane vesicles. These results suggest that the dependency of transport on sodium is not explained as simple sodium driven co-transport. Hewitt (1977) has suggested that the lack of "energized" transport in vesicles was consistent with the transport pro- cess being coupled with an intracellular step. In any case, the relationship between the peritubular carrier, the sodium dependency of transport and the coupling of transport with cellular energy remains unresolved. l6 Luminal membrane carriers The efflux of organic anions from the cell cannot be explained as simple passive diffusion as suggested in the Tune model. Several lines of evidence suggest that a membrane carrier exists in the luminal membrane for the efflux of organic anions from the cell. The mechanism by which organic anions leave the cell is clearly different from the mechanism by which they enter the cell. Devel- Opmental studies of transport in renal cortical slices demonstrate that a different developmental pattern exists for efflux and influx. Measurements of initial uptake rates show a peak uptake rate at 4 weeks in rabbit kidneys which subsequently declines to adult values. Efflux exhibits a gradual increase in the rate of efflux as the animal matures (Ecker and Hook, 1974; Cole et al., 1978). Furthermore, penicillin induced stimulation of PAH transport selectively affects the initial uptake rate without altering efflux time constants (Pegg et al., 1975). Other investigators confirm the distinction between efflux and influx and fur- ther imply that efflux is carrier mediated. Early studies of the effect of organic anions on the efflux or runout of PAH from renal cortical slices 17 preloaded with PAH demonstrated that low concentrations of PAH in the bathing medium enhanced the runout of PAH from the slice. Higher concentrations of PAH in the medium, however, decreased the runout of intracellular PAH (Farah et al., 1963; Ross and Farah, 1966). Runout may represent the sum of diffusion out of the cell plus a component of active reuptake of organic anion which had diffused out of the cell (Farah.et al., 1963). The increase in runout at low concen— trations of PAH in the medium is thus consistent with com— petitive inhibition of a high affinity, carrier mediated uptake mechanism. Inhibition of runout at higher medium concentrations of PAH is similarly suggestive of inhibition of a carrier mediated efflux mechanism. Use of the renal cortical slice as a model of the efflux of organic anions from the cell across the luminal ‘membrane into the urine has been criticized since the tubular lumens appear to be for the most part collapsed (Bojesen and Leyssac, 1965). This result suggests that organic anion could escape from the slice across the luminal “membrane only with difficulty. Efflux from the slice is thus likely to represent actions occurring primarily at the peritubular membrane. Several studies, however, suggest that similar considerations apply to the luminal membrane. 18 A peritubular efflux carrier is suggested by the observation of an apparent secretion of organic anions across the luminal membrane in the tubule of certain teleost fishes (Forster and Heng, 1958; Heng and Forster, 1959; Puck et al., 1952). Similarly, autoradiographic studies of renal cortical slices incubated in the presence of H3-hippuran, demonstrated high.luminal concentrations of label (Wedeen and Weiner, 1973; wedeen and Vyas, 1978). If phlorizin is added to the incubation medium, the steady state concentra- tions of PAH (PAH S/M) are increased, but the distribution of label to the lumens is inhibited. This observation sug- gests a selective effect on efflux. Foulkes (1977) found that low doses of probenecid increased the time required for a pulse of PAH to reach the urine. Furthermore, the pulse occurred after a pulse of simultaneously injected inulin. TheSe results indicate that secretion of PAH had been blocked and that the filtered PAH had distributed to a cell— ular compartment after filtration. If a larger dose of probenecid was given, the transit time was reduced to that of inulin indicating that the intracellular distribution of filtered PAH across the.luminal membrane had been blocked by inhibition of a luminal carrier. Phenolsulphonthalein binding to a microsomal fraction indicates the presence of 19 two binding sites, one sensitive to competitive inhibition by 2, 4—dinitr0phenol and a second, higher affinity site sensitive to 2,4-dinitrophenol and probenecid (Evelhoff et al., 1976). While the high affinity site appears to corre--~ late with.the peritubular carrier in basal-lateral membrane vesicles (Bernet and Kinne, 1976; Evelhoff et al., 1976), the role of the low affinity site remains obscure. Evelhoff et a1. (1976) suggested that it represented a weak intra- cellular binding site. But clearly, a second binding site is also consistent with the concept of two membrane carriers. Kinsella et al. (1977) have confirmed that saturable and inhibitable uptake of PAH into brush border vesicles exists. Berne and Kinne (1976) also observed a weak.probenecid effeCt on PAH influx into brush.border vesicles at a concen- tration of probenecid whichgmarkedly reduced influx into basal-lateral vesicles, indicating that if a carrier is involved, it is of a lower affinity than the carrier in the peritubular membrane or that it is similar to the low affinitybinding site observed by Evelhoff et a1. (1976) which.was inhibitable by 2,4—dinitrophenol but not by probenecid. The possibility of carrier mediated diffusion across the luminal membrane suggeSts that the membrane could 20 exhibit differential permeability. A carrier could have a higher affinity for organic anions when it is orientated toward the inside of the cell than it would have when ori- entated toward the outside of the cell. This effect would make the luminal membrane relatively more permeable to anions inside the cell compared to the anions outside the cell. This possibility suggests that measurement of permeability of the luminal membrane based on efflux from the cell as per- formed by Tune et a1. (1969) may not reflect the permee ability of the brush border membrane to influx of PAH into the cell across the luminal membrane as was assumed by Tune et a1. (1969). The failure to observe significant intra- cellular concentration of PAH when PAH was supplied from the luminal surface may therefore be a manifestation of low per- meability of the luminal membrane to influx into the cell. Unfortunately, comparative measurements of influx and efflux from brush border vesicles have not been reported. The Tune model for organic anion transport does not rule out the pos- sibility for the involvement of intracellular binding in the transport of organic anions and may have to be expanded to account for a carrier for efflux of anions as well as an influx carrier. 21 Mechanisms of penicillin enhanced transport Renal organic anion transport can be enhanced by two principle manipulations: Acetate in the medium and penicil- 1in pretreatment of neonates. Several mechanisms have been proposed to account for the enhanced transport observed after acetate (Cross and Taggart, 1950; Schachter et al., 1955; Cohen and Randall, 1964; Weiner, 1973; Hewitt et al., 1976). It appears that the mechanism for penicillin enhanced transport is distinct from the acetate enhanced transport. Acetate enhanced transport is effective in neonates and in adults while penicillin is effective only in neonates (Kim et al., 1972; Kim and Hook, 1972; Pegg and Hook, 1975). Furthermore, acetate enhances transport in naive and penicillin pretreated neonates alike (Hirsch and Hook, 1970; Pegg and Hook, 1975). The addition of acetate produces an immediate in vitro effect (Hewitt et al., 1976). In contrast penicillin induced enhancement of transport requires pretreatment several hours prior to evaluation for expression of enhanced transport (Pegg and Hook, 1975). Finally, while penicillin specifically enhances PAH trans- port and does not enhance urate transport (Hewitt and Hook, 22 1978), acetate and other metabolites enhance urate and PAH transport (Kippen and Klinenberg, 1978). Penicillin treatment, despite its ability to increase kidney weight, does not appear to increase trans- port by a nonspecific enhancement of growth. Acid loading increased kidney weight without enhancing anion transport (Pegg and Hook, 1977). Penicillin treatment also failed to enhance cation transport (Hirsch and Hook, 1970a), or the transport of several organic anions whichappear to be secreted by the kidney (Hewitt et al., 1977). Penicillin treatment also failed to alter the histological (Hirsch et al., 1971) or ultrastructural (Pegg et al., 1976) appearance of renal tissue. Recently, the transport of PAH in control and penicillin pretreated neonates was evaluated with the isolated perfused tubule technique pioneered by Tune et a1. (1969). In this preparation, transport was increased per mm of tubule confirming that penicillin enhanced transport by an intrinsic change in the transport system (Schwartz et al., 1978). The lack.of effect on cation transport or urate transport also suggests that penicillin treatment does not increase transport by a generalized increase in cellular energy. 23 ‘Kinetic analysis Several attempts to define the phenomenon of peni— cillin stimulation of organic anion transport by the use of kinetic analysis have been made and have met with mixed suc- cess. Analysis based on Michaelis-Menten kinetics of the initial uptake of PAH into isolated proximal tubule suspen— sions indicated that stimulation was associated with a change in the apparent maximal velocity of transport without a change in the apparent affinity of the transport system for the organic anion (Pegg et al., 1975). If, however, the transport was evaluated using renal cortical slices, then a change in the apparent affinity was observed instead of a change in maximal velocity (Pegg, 1976). The complex- ity of the renal cortical slice or the tubule preparations makes kinetic analysis of transport a hazardous approach (Christensen, 1969; Weiner, 1973). The reason for the different results with these two methods remain unresolved, but their variance serves to underscore the hazard of ana- 1yzing the biochemical mechanism of transport using kinetic analysis in complex systems such.as the slice or isolated tubule. 24 Protein'synthesis Several studies have implicated the involvement of protein synthesis in the stimulation of transport by peni- cillin. Inhibition of protein synthesis with.cycloheximide appeared to block penicillin stimulated transport (Hirsch and Hook, 1970b; Pegg and Hook, 1975). However, cyclo- heximide has several effects other than inhibition of pro- tein synthesis (Jomain—Baum and Hanson, 1975). Furthermore, subsequent investigators have been unable to repeat the blocking effects of cycloheximide without significant signs of toxicity (Hewitt, unpublished observation; Stroo, unpub- lished observation). These results suggest that the earlier observations with cycloheximide may have been mani- festations of renal toxicity. Unfortunately, the previous studies did not include simultaneous determinations of oxy— _gen consumption nor of the function of other cellular transport processes. Hirsch and Hook (1970b) observed an increase in the incorporation of amino acids into TCA pre— cipitable protein after penicillin treatment, an observa- tion which.is consistent with.increased protein synthesis. However, the increase in kidney weight associated with penicillin treatment suggests that the increased synthesis 25 might be due to enhanced growth rather than a manifestation of the increased synthesis of a specific protein of the transport system. In contrast, Pegg (1976) failed to observe a significant increase in protein synthesis although a trend toward increased incorporation into microsomal pro- tein was observed. These results are thus suggestive of the involvement of protein synthesis in the mechanism of penicillin enhanced transport, but fail to conclusively demonstrate its involvement. Intracellular binding The demonstration of an organic anion binding pro- tein in the renal cortical cytosol (Kirsch et al., 1975), and the apparent correlation of hepatic organic anion trans- port with the binding protein, ligandin or Y protein (Levi et al., 1969; Reyes et al., 1969; Reyes et al., 1971), sug- gested that penicillin might produce enhanced transport by increasing intracellular binding to ligandin (Pegg and Hook, 1977). However, penicillin treatment failed to alter ligandin content (Pegg and Hook, 1977). Furthermore, a recent developmental study revealed no clear correlation between development of the renal organic anion transport 26 system and renal ligandin activity (Cole et al., 1978). It is interesting to note that the role of ligandin in the hepatic transport of organic anions has also been questioned (Klassen, 1975). These results thus fail to demonstrate a role for intracellular binding to ligandin in the renal transport of organic anions. An alternative mechanism involving intracellular binding visualized penicillin treatment as disinhibiting the renal organic anion transport system. Hewitt and Hook (1978) suggested that nonesterified fatty acids (NEFA) inhibited organic anion tranSport by binding to intracellu— lar binding sites which could bind PAH and which are involved in the transport of PAH. The binding of the NEFA thus inhib— ited renal organic anion transport. Several observations support this hypothesis. Several subtrates of the renal organic anion transport system including probenecid, inhibit the renal utilization of exogenous NEFA in vivo (Barac-Nieto and Cohen, 1968; Dies et al., 1970) and decrease oxidation of palmitate in vitro (Pakarinen, 1970; Heinemann et al., 1975). Barac—Nieto (1970) demonstrated that palmitate could inhibit transport of PAH in an apparently competitive man- ner. The addition of carnitine relieved this inhibition, suggesting that the interaction involved increased 27 mitochondrial use of palmitate, since the primary action of carnitine is to enhance the permeability of the mitochondria to fatty acids (Bressler, 1970). Pakarinen (1970) similarly suggested that probenecid altered lipid metabolism from an intracellular site since he failed to observe any change in incorporation of a labeled fatty acid into the tissue total lipid fraction. Consistent with the suggestion that fatty acids may be important modulators of transport is the report that fasting increases renal fatty acid content and plasma fatty acid concentration, and also decreases renal organic anion transport (Dahlquist et al., 1972; Chorvathova et al., 1974; Hook and Munro, 1968; Fox et al., 1976). It is thus interesting to note that the newborn animal is also hyperlipemic in comparison to the adult (Palkovic et al., 1976; Battaglia and Mesckin, 1978). These results suggest that the decreased transport in the fasted animal and in the neonate may be the result of inhibition of transport by intracellular NEFA. Metabolic studies further support this hypothesis. Alpha-bromopalmitate inhibits fatty acid oxidation in_ ' 33339 and decreases the incorporation of labeled fatty acid into tissue triglyceride while increasing the incor- poration of label into the tissue NEFA fraction (Trimble 28 et al., 1977; Ockner and Manning, 1976). Alpha—brompalmitate also markedly inhibits organic anion transport at a concen— tration which produces minimal effects on oxygen consumption (Maxild, 1970). In contrast, the enhanced transport associ— ated with penicillin treatment of neonates is associated with decreased incorporation of label into tissue NEFA and increased incorporation into tissue triglyceride (Hewitt and Hook, 1978). These results suggest that transport can be modulated by the renal content of NEFA and the content of NEFA can be regulated by renal metabolism. Hewitt and Hook (1978) supported the hypothesis that the altered metabolism of fatty acids was related to the enhanced transport of PAH by demonstrating that penicillin treatment of adults and iodipamide treatment of neonates, failed to alter transport and also failed to alter metabolism. These results suggest that the alteration in metabolism is a specific manifesta- tion of altered organic anion transport, and not a non— specific effect of organic anion treatment. While altered lipid metabolism does appear to be associated with altered organic anion transport, contrary to earlier expectations, NEFA do not appear to be extracted from the plasma by the renal organic anion transport system. Hewitt and Hook (1978) observed that penicillin stimulation 29 of transport failed to significantly increase incorporation of labeled NEFA into CO2 or into tissue total lipid. This would appear to indicate that NEFA are not substrates of the renal organic anion transport system and is consistent with the suggestion that the interaction of the transport system with lipid metabolism is at an intracellular site. Several problems remain to be resolved before the relationship between renal lipid metabolism and renal organic anion transport can be evaluated. Several invest- igators have reported that they failed to observe signifi- cant inhibition of PAH transport by NEFA (Maxild, 1970; Kippen and Klinenberg, 1978). This result suggests that the failure of Hewitt and Hook (1978) to observe increased utilization of palmitate may have been due to the use of inappropriate conditions for demonstrating palmitate uptake by the transport system. Alternatively, the failure to observe increased utilization may have been due to the several metabolic fates available to NEFA. While Hewitt and Hook (1978) failed to observe increased oxidation or incorporation of labeled fatty acid into total lipid they did observe a tendency for both parameters to increase. This observation suggests that while the individual fates for NEFA metabolism were not increased, the possibility exists 30 that the sum of those fates would indicate a significant increase. The relationship between altered metabolism and altered transport is also subjeCt to criticism. Close eXami- nation of the effects of iodipamide on lipid metabolism indi- cates that while metabolism was not significantly altered, the incorporation of labeled palmitate into triglyceride was quantitatively similar to that observed after penicillin treatment. This result suggests that the failure to achieve significant alterations in metabolism may be due to the use of an inadequate treatment dose. Similarly, since the adult secretory capacity and renal blood flow is greater than the newborn's, a given dose will have a shorter duration of action in the adult. This suggests that the failure to observe an effect in adults may also be due to an inadequate treatment dose. Finally, critical to the hypothesis of dis- inhibition of transport by reduced renal NEFA content is the demonstration that penicillin treatment reduces renal NEFA content. However, no evidence to support this hypothesis has been obtained. 31 Triglyceride biosynthesis The central role played by triglyceride synthesis in the Hewitt and Hook model suggests that it would be perti— nent to review the biochemistry of triglyceride synthesis. Triglycerides are synthesized directly from diglycerides by the action of the microsomal enzyme, diacylglycerol acyl transferase (DAGAT) (O'Doherty, 1978). Recent studies using therepatic enzyme indicate that this reaction is dependent on the presence of an intracellular binding protein; fatty acid binding protein or Z protein (O'Doherty and Kuksis, 1978). Cellular diglycerides are derived from two pathways. The pathway which predominates in the liver but which may also be active in the kidney, converts monoacylglycerol to the diglyceride via the action of a soluble enzyme mono- acylglycerol acyltransferase (O'Doherty, 1978; Hubscher, 1961). Diglycerides can also be derived from phosphatidic acid by the action of phosphitidate phosphohydrolase. This pathway is of central importance to the biosynthesis of phospholipids since phosphatidic acid is a common precursor to the phospholipids. The failure to observe any altera- tion in phospholipid metabolism suggests that the penicillin 32 induced alteration in metabolism is not in the phosphatidic acid pathway. An interaction with Z protein is indicated since alpha-bromopalmitate binds to Z protein (Ockner and Manning, 1976), decreases incorporation into triglyceride and increased incorporation into NEFA (Trimble et al., 1977; Ockner and Manning, 1976). Furthermore, the addition of Z protein enhances the synthesis of di-, and triglycerides (Mishkin and Turcotte, 1974; O'Doherty and Kuksis, 1978). These results suggest that penicillin treatment may alter renal lipid metabolism by interaction in the monoacyl- glycerol pathway or by an interaction with the intracellular binding protein; Z protein. Objectives The objective of this study was to clarify the rela- tionship between penicillin enhanced transport and altered lipid metabolism. Several specific questions were evaluated. First, does penicillin enhance renal utilization of NEFA? Second, is the altered metabolism an indication of a specific interaction of lipid metabolism with the transport system? Third, is the altered metabolism a specific indication of 33 transport? Finally, we sought preliminary evidence for the site at which penicillin alters renal lipid metabolism. METHODS AND MATERIALS Litters of 2 week old New Zealand White Rabbits were bred in the departmental animal quarters or purchased from local suppliers. Young animals remained with their mothers until immediately prior to use. Adult female New Zealand White Rabbits (2-4kg) and adult male Sprague—Dawley Rats (150-300g) were purchased from local suppliers and acclimated to the animal facility prior to use. Neonates (13-15 days old) were treated with 30,000 I.U. procaine penicillin G (Crystacillin, E.R. Squibb and Sons) by subcutaneous injection. In multiple dose treatment regimens, injections were given at 12 hour intervals. A total of 2 or 4 doses was given. Animals were killed 24 hours after the last dose except as noted in some treatment _groups where the animals were killed 12 hours after the last dose. Nicotinic acid (Sigma Chem. Co.) was dissolved in 0.9% NaCl and the pH adjusted to 7.4 before being injected intraperitoneally. Fasted adult rabbits were killed 24 34 35 hours after a single 250 mg/kg dose of nicotinic acid. Fasted adult male Sprague-Dawley rats were treated with 250 mg/kg nicotinic acid at 6 hour intervals for four treatments. Animals were then killed 6—8 hours after the last treatment. Controls were treated with equivalent volumes of 0.9% NaCl. The in vitro effect of 2 mM PAH, probenecid and nicotinic acid on lipid metabolism was determined following a 15 minute preincubation of tubules in the presence of a 2 mM concentration of the organic anion followed by incubation in the presence of the anion and the standard palmitate con- taining KRP-BSA medium. Fasted animals were allowed free access to water but food was removed 48 hours prior to use. Media Several media were used to evaluate organic anion transport or lipid metabolism. Transport in slices was determined in the phosphate based medium originally described by Cross and Taggart (1950) containing 7.4 x 10..5 M p—amino- hippurate (PAH) and l x 10“4 M tetraethylammonium (TEA) to evaluate organic anion and cation transport, respectively. Isolated tubules were prepared in a Krebs—Ringer Phosphate 36 Buffer (KRP-buffer) medium containing 2.15 g/l NaZHPO4.7H20, 0.296 g/l MgSO .7H 0, 7.614 9/1 NaCl, 1.2076 0.267 g/1 KH PO 4 2 2 4' g/l, and 0.147 g/l CaCl Anhydrous sodium acetate was also 2. added to yield a concentration of 10 mM. A second medium had an identical electrolyte composition but included approxi- mately 10 mg/ml magnetic iron oxide. The third medium had an identical electrolyte composition as the KRP-buffer medium but also contained 1% defatted Bovine Serum Albumin (BSA). The final medium was identical in electrolyte composition to the KRP-buffer, but also contained 1% BSA and 1 mM sodium palmitate with sufficient l-C14-palmitic acid to yield a con- centration of 0.4uCi/m1 and a specific activity of 0.4uCi/u mole. Magnetic iron oxide was prepared as described by Cook and Pickering (1958) except that it was stored in 0.9% NaCl. Sodium hydroxide (2.6 gm) and potassium nitrate (20 gm) were dissolved in 100 m1 of oxygen-saturated distilled water and then added to 100 m1 of oxygen—saturated water containing 0.9% ferrous sulphate. The mixture formed a green-black slurry which.was boiled for 20 minutes. The.magnetic iron oxide was then separated from the reaction mixture with a strong magnet and the resulting precipitate was washed and precipitated several times by suspension in distilled water 37 followed by precipitation with the magnet. The precipitate was then stored in 0.9% NaCl. Defatted BSA was prepared from Sigma fraction V BSA (Sigma Chem. Co.) as described by Chen (1967). A 10% BSA solution was prepared. Activated charcoal (Norit A) was then added to yield a 4% by weight suspension and the mix- ture adjusted to a pH of 3.00. The mixture was cooled to 0°C in an ice bath and stirred for 30 minutes. Then the mix- ture was transferred to centrifuge tubes and spun at 18,000 rpm. The resulting supernate was separated and recentrifuged at 18,000 rpm. Then, the supernate was adjusted to a pH of 7.4 and the protein content assayed with Biuret reageants. Sodium palmitate was prepared from palmitic acid (Sigma Chem. Co.) by dissolving the free acid in absolute ethanol followed by the addition of 2.5 mM NaOH. The ethanol was evaporated and the sodium palmitate dissolved in 100 m1 of freshly boiled distilled water. Media contain- ing palmitate were prepared by adding warm stock palmitate to the media containing the electrolyte and BSA but without calcium. The mixture was then equilibrated for 30 minutes prior to the addition of labeled palmitic acid. The mixture was allowed to equilibrate for a further 15 minutes. Calcium was then added and the media pH adjusted to 7.4. 38 Preparation of tubules and glomeruli The preparation used was essentially that described by Hewitt and Hook (1978) as modified from the method described by Brendel and Meezan (1974). h The animals were killed by a blow to the head. Kid- neys in adult animals were perfused via the renal artery. Kidneys in neonates were perfused via the abdominal aorta from below the renal arteries after clamping the abdominal aorta above the renal arteries. The kidneys were initially perfused with KRP-buffer. Subsequently, they were perfused with a medium containing magnetic iron oxide. The kidneys were removed and the renal cortex dissected out and minced. The mince was then homogenized by gentle hand homogenization (5-7 strokes, teflon on glass). The homogenate was filtered through a 253p nylon sieve and the retained material thoroughly washed with KRP—buffer. The filtrate was saved and subjected to a second filtration through a 63p nylon sieve. The retained solute was saved and resuspended in KRP—buffer. The glomeruli, containing iron oxide particles trapped in the glomerular capillaries, were then separated from the supernate by exposure to a strong magnet which pre- cipitated the iron containing tissue. The precipitate was 39 resuspended in KRP-buffer, reprecipitated with the.magnet to wash the glomeruli and then resuspended in KRP-buffer con- taining 1% BSA. The tubules suspended in the supernate were reexposed to the magnet. Any resulting precipitate was dis- carded and the supernate was saved. The supernate was centrifuged at 1,000 x g for 1 minute. The resulting tissue pellet was then suspended in KRP-buffer containing 1% BSA to a concentration of 1 g/10 ml. Microsc0pic inspection indi- cated that tubule preparations were 95-99% pure while glomerular preparations were enriched to 85—90% purity. 'Determinations of organic anion transport Transport in renal cortical slices was determined essentially as described by Hook and Munro (1968). Renal cortical slices were incubated in a phosphate buffer medium containing PAH and C14- TEA, under an oxygen atmosphere for 90 minutes. The slices were removed, weighed, and minced in 3 ml of 10% TCA. A 2 ml aliquot of medium was added to 3 ml of 10% TCA. Both the tissue and the medium sample were adjusted to a final volume of 10 ml. The resulting mixtures were then assayed for PAH by the.method of Smith et al., (1945) or for TEA by liquid scintillation spectrometry. The 40 results were expressed as a slice concentration (mg or dpm / g) to medium concentration (mg or dpm / ml) ratio (S/M ratio). The ability of proximal tubule suspensions to trans- port organic anions was determined as described by Hewitt and Hook (1978). Five m1 of a KRP—buffer medium containing 1% BSA and l g/lOml medium tubules was placed in a 25 ml erlenmeyer flask and incubated under an oxygen atmosphere for 15 minutes at 37 degrees C. Subsequent to this pre- incubation, 50 pl of a 7.4 x 10.3 M PAH stock was added and incubation continued for 30 minutes. One minute prior to termination of incubation 120 ul of 5% inulin was added to the reaction mixture in order to estimate extracellular water in the tubules. The incubation mixture was then transferred to a special centrifuge tube described by Burg and Orloff (1962) and centrifuged at 10,000 rpm for 10 minutes. The resulting supernate was separated from the pellet and a 2 ml aliquot added to 3 ml of 10% TCA and 5 ml of water. The pellet was removed and a wet weight obtained. The pellet was then dried overnight at 50 degrees C and a dry weight subsequently obtained. The pellet was removed and pulverized in a graduated cylinder and the powder was suspended in 5 m1 of 3% TCA. The PAH in the medium and pellet was assayed by 41 the method of Smith et a1. (1945). Inulin was assayed by the method of Schreiner (1950) and was used to estimate the contribution of the medium trapped in the pellet to the tissue wet weight and tissue PAH content. The results were expressed as a T/M ratio where T was the concentration of PAH in the intracellular water (mg/ml) and M was the concen- tration of PAH in the medium (mg/ml). Palmitate Esterification The capacity of the tissue to esterify palmitate was determined as described by Hinemann, et a1. (1975) as modi- fied by Hewitt and Hook (1978). An aliquot (5-10 m1) of the KRP-buffer — 1% BSA tubule suspension was preincubated in an Erlenmeyer flask for 15 minutes at 37°C under an oxygen atmosphere. The tubule suspension was then centrifuged for 1 minute at 1,000xgyin a Sorvall desk top laboratory centri- fuge. The pellet was resuspended to a concentration of lg/lOml in a KRP-buffer containing 1% BSA and 1 mM l-C14- palmitate (sp. act. 0.4%UCi/ UMole). This mixture was incu- bated for 30 minutes under oxygen at 37°C. Upon completion of incubation, the mixture was centrifuged (1 min., 1,000 x g) and resuspended in 2—5 ml of .2N H2504 and recentrifuged 42 for one minute at 1,000 x g. The resulting pellet was resus- pended and washed in ice cold KRP—buffer containing 1% BSA, and then centrifuged 1 minute at 1,000 x g. The tubule pellet was weighed and homogenized in chloroform:methanol (2:1) in a ratio of 1 gm tissue per 20 m1 of solvent as described in the Folch procedure (Folch, et al., 1957). Methanol (0.2 x the volume of chloroform:methanol added) was then added and the mixture centrifuged. The supernate was separated and saved. A volume of 0.05 M KCl 0.2 times the volume of chloroform:methanol was added to induce phase separation. The upper phase was discarded and the lower phase washed 1 time with previously prepared upper phase. The lower phase was saved and an aliquot plated on a 250 u Silica Gel G thin layer chromatography plate. The thin layer plate was then developed in a solvent system of 80 parts hexane, 20 parts diethyl ether and 1 part glacial acetic acid. The triglyceride and NEFA fractions were identified by comparison to a triglyceride and NEFA standard. The triglyceride, NEFA, and origin (phospholipid fractions were scraped into scintillation vials and counted in an Omniflour-toluene cocktail (New England Nuclear) containing 4% Cab-O—Sil (Eastman Kodak Co.). A second aliquot was taken and added to an Omniflour-toluene cocktail and counted 43 by liquid scintillation spectrometry. Efficiency was deter- mined by internal standards or by the sample channels ratio. Glomerular metabolism Glomeruli were suspended in 5 m1 of KRP-buffer con- taining 1% BSA. The suspension was incubated for 15 minutes at 37°C. The glomeruli were then separated from the medium with a magnet and resuspended in 5 m1 of KRP-buffer contain- ing 1% BSA and lmM-Cl4—palmitate (spe. act. 0.4u Ci/u Mole). This mixture was incubated for 30 minutes at 37°C. Next, the mixture was centrifuged and the pellet resuspended in 1 ml of .2N H2804. The mixture was centrifuged and the resulting pellet saved. The lipid was extracted by adding 0.5 ml of 0.5 N KCl followed by 10 ml of 1:2 chloroform: methanol. Phase separation was induced by adding 2 m1 of methanol and 2 ml of .05 N KCl. The bottom, organic layer was saved and plated on a thin layer chromatography plate and label determined as described for tubule esterification. Palmitate extraction from.medium Tubular extraction of NEFA acid was determined by comparing label content of incubation media before and after 44 incubation. An aliquot of medium containing Cl4 labeled palmitic acid was extracted by the Folch_procedure and sub- jected to thin layer chromatography. The C14 content of the NEFA fraction was determined by liquid scintillation Spectrophotometry as described for tubular esterification. Triglyceride and NEFA assay Animals were killed by a blow to the head and blood removed from the heart and centrifuged immediately. The supernate was separated and allowed to clot. The resulting serum was used for triglyceride and NEFA analysis. Liver tissue was snap frozen in liquid nitrogen. Kidneys were flushed with ice cold KRP-buffer, and the cortex dissected away prior to being snap frozen in liquid nitrogen. Triglyceride was analyzed as described by Van Handel and Zilversmit (1957) as modified by Butler, et a1. (1961). Tristearin (Sigma, St. Louis) was dissolved in chloroform and used as the triglyceride standard. Tissue was homoge- nized in a 0.33 M pH 7.0 phosphate buffer (1 g / 9 ml). Triglyceride was extracted from 1 m1 of tissue homogenate or 1 m1 of serum with 20 ml of chloroform. Phospholipids were absorbed out with 4 g of chloroform-moistened, 45 pulverized zeolite (Doucil). Aliquots of the chloroform extract were then saponified (0.5 ml of 0.2% Ethanolic KOH at 60°C for 30 minutes) or not saponified (0.5 ml of ethanol at 60°C for 30 minutes) and the triglyceride content evalu- ated by analysis of the difference in glycerol content between saponified and nonsaponified samples. Glycerol was estimated chemically with the metaperiodateoarsenate- chromatropic acid reaction described by Lambert and Neish (1950) and as modified by Butler et a1. (1961). NEFA were extracted from 1 g of tissue by the method of Folch, et a1. (1957). The lipid extract was plated on a 250p or 500p Silica Gel H thin layer chromatography plate and the plate developed in a solvent system consisting of 70 parts hexane, 30 parts diethyl ether and 1 part glacial acetic acid. Lipid fractions were identified by comparison to appropriate standards after exposure to iodine vapors. Care was taken to avoid exposure of NEFA samples to iodine by covering the Spot with a plass plate. The tentatively identified NEFA fraction was then scraped from the plate and the NEFA extracted from the silica gel by the method of Elphick and Lawlor (1976). Briefly, the NEFA acids were extracted by incuba- tion of the Silica Gel H in 2.62 N KOH (l-3ml). The 46 resulting slurry was suspended in 2.17 M glycerol (3—9ml) and acidified by adding 2.8 N HCl (l-3ml). The lipid was then extracted with 4 m1 of chloroform. The extraction was repeated and the chloroform layers saved and evaporated. The lipid was dissolved in 1 m1 of diethyl ether and the fatty acids methylated by exposure to diazo methane as described by Levitt (1973). The fatty acid methyl esters were then injected onto a gas chromatograph using a carrier gas (nitrogen) flow rate of 40 ml, operating at a tempera- ture of 165°C. A six foot column packed with 100 / 120 mesh chromasorb W A W with a liquid phase of 10% SP-2330 (Supelco) was used to separate the various fatty acids. Fatty acids were quantitated by measuring peak height. An internal standard of lOug heptadecanoic acid (C 17) was added prior to the Folch extraction to correct for loss of sample. The quantities of individual fatty acids were estimated by com- parison to known standards. Diagyl glycerol acyl transferase (DAGAT) Assay [AGED is a microsomal enzyme which is dependent upon intracellular proteins for activity (Manley et al., 1974; O'Doherty and Kuksis, 1978). Microsomes and cytosol were 47 prepared as described by Manley et a1. (1974). Tissue was minced in a 0.02 M pH 7.4 phosphate buffer containing 0.1 mM EDTA. The mince was then homogenized in the same medium at a ratio of 1 part tissue per 2 parts medium using a motor driven teflon pestle in glass homogenizer. The homogenate was centrifuged at 1,000 g for 10 minutes and the supernate saved. The supernate was then centrifuged at 20,000 g for 15 minutes. The supernate was saved and centrifuged at 100,000>ugfor 60 minutes. The middle layer of the supernate was saved and the microsomal pellet resuspended in the EDTA phosphate buffer. Both fractions were centrifuged again at 100,000 g for 60 minutes. The pellet was resuspended and recentrifuged then resuspended. Cytosol and microsomal fractions were then frozen and stored in a -80°C freezer. Protein content of each fraction was evaluated by the Lowry method (Lowry et al., 1951). DAGAT was assayed as described by Manly et a1. (1974) as modified by O'Doherty and Kuksis (1978). The incubation mixture included 1 mg microsomal protein, 7.5 or 10 mg supernate protein, 5 nM of 1-2 dioleayl - 5 n-glycerol, 1 nM Cl4 palmitoyl - S - Coenzyme A (.2uCi), 4 n Moles MgCl2 and 4 n Moles of ATP in a volume of 1.05 ml. 48 Diolein was dissolved in 2:1 dioxane:proply1ene glycol and added in a 10 ml aliquot. Palmitoyl Coenzyme A was dissolved in 0.1 M pH 6 acetate buffer and added in a 10 m1 aliquot. The incubation mixture was incubated for l or 2 hours in a gyrorotary incubator at 37°C. The reaction was stopped by the addition of 3.75 ml of chloroform:methanol (1:2). Triglyceride was extracted and phase separation induced by the addition of 1.25 ml of chloroform and 1.25 ml water. The bottom organic layer was saved and plated on a silica gel G thin layer chromatography plate. The plate was developed in a solvent consisting of 80 parts hexane, 20 parts diethyl ether and 1 part glacial acetic acid. The triglyceride fraction was identified by comparison to a triglyceride standard. The triglyceride fraction was then scraped into a scintillation vial and suspended in an omni- flour-toulene 4% cabosil cocktail. Statistical analysis Where appropriate, data were analyzed using analysis of variance, completely random design or randomized complete block design, or student's "t" test for paired or group com- parisons. Treatment means were compared by Student-Newman 49 Keul's procedure (Steele and Torrie, 1960). The 0.05 level of probability was used as the criterion of significance. RESULTS Effect of palmitate in the medium ‘of PAH accumulation Tubules from control and penicillin treated 2 week old rabbits were incubated in a KRP-buffer medium contain- ing 1% BSA in the absence of palmitate (-pa1m) and in the presence of 1 mM palmitic acid (+palm) (Figure 1). Animals were pretreated with 90,000 I.U. of procaine penicillin G 60, 48, 36 and 24 hours before use. Such treatment enhanced the capacity of tubules to accumulate PAH from a T/M of 3.17 i 0.85 to a T/M of 8.17 i 1.44 when transport was measured 24 hours after the last treatment dose. Addi- tion of palmitate to the medium consistently decreased transport (Figure l). The average T/M for all individual determinations of transport in the absence of palmitate was 5.94 t 1.24. The addition of palmitate decreased the T/M 40% to an average value of 3.57 i 0.67. 50 51 Figure 1. Accumulation of PAH by suspensions of renal cortical tubules in the presence or absence of added palmitate. Tubules were prepared from litters that had been pretreated with 4 doses of saline (open circles) or 90,000 I.U. of procaine penicillin G (closed circles). One aliquot of tubules was incubated in a KRP-buffer- 1% BSA medium (-pa1m); a paired aliquot of tissues was incubated in a KRP-buffer- 1% BSA medium containing 1 mM palmitic acid (+palm). Individual data points are displayed. Values determined in the absence of palmitate are connected to corresponding values determined in the presence of palmitate in tubules from the same pool of tubules. PAH T/M 16 I2 C PEN 0 CON - PALM +PA|.M Figure l 53 Effect of penicillin treatment on utilization of palmitate Two week_old rabbit litters were treated with saline or 90,000 I.U. of procaine penicillin G at 12 hour intervals for a total of 4 treatments. Twenty-four hours after the last injection transport was determined in a KRP—buffer medium containing 1% BSA and 7.4 x 10'.5 M PAH. Penicillin treatment increased transport 100% from a control T/M of 3.04 i 0.71 to a T/M of 6.22 i 1.12 (Figure 2). Paired aliquots of tissue were also incubated in a KRP—buffer - 1% BSA medium containing 1 mM palmitic acid. Tubules from con- trol animals extracted 1.08 t 0.20 micromoles of palmitic acid from the medium during a 30 minute incubation. Peni- cillin treatment did not significantly increase the extrac- tion of palmitate from the medium (Figure 2). In a separate series of experiments, using an identical treatment regimen, penicillin treatment increased T/M 138% from 2.97 i 0.50 to a value of 7.07 i 0.86 (Table 1). However, penicillin treatment did not increase incor- poration of label into tissue total lipid (Table l). 54 Figure 2. Effect of penicillin treatment on transport of PAH and on palmitate extraction from the medium. Rabbit litters were treated with saline or procaine penicillin G (90,000 I.U.) at 12 hour intervals to a total of 4 treatments. Twenty- four hours after the last treatment, the capacity of one aliquot to transport PAH was determined while the capacity of a second aliquot, from the same pool of tubules, to extract palmitate from a medium was determined. Values are means i S.E. *Significantly different from control (P<0.05). p mole PALMITATE EXTRACTED /30 min 1.5 1.0 CON PEN Ix CON PEN W/l HVd 56 TABLE I. Penicillin Effect on PAH T/M and Palmitate Incorporation into Tissue Total Lipid in 2 Week Old Rabbits CON PEN % CHANGE PAH T/M 2.97 i .50 7.07 i .86b 138 TissueC Lipid 2.3lzt.23 2.44zt.20 6 aN = 9. Values are means i S.E. bSignificant p<.05. Cu mol/g/30 min. 57 Effect of penicillin treatment of endpgenous NEFA and triglyceride Two week old rabbits were treated with 90,000 I.U. procaine penicillin G at 12 hour intervals for a total of 2 doses. Twenty-four hours after the last dose animals were killed and the serum, kidney and liver content of triglyceride determined. Serum and kidney content of several fatty acids was determined by gas—liquid chromato- graphy. The individual fatty acids were summed to estimate total NEFA. Penicillin treatment increased the serum con- tent of several individual NEFA (Table 2) and increased the overall serum content of measured NEFA from 87.7 i 3.4 to 159 i 6.8 mg/ml (Figure 3). Penicillin treatment significantly decreased renal triglyceride from 0.264 i 0.035 mg/g to 0.177i 0.032 mg/g (Figure 4). Serum triglyceride and hepatic triglyceride were not significantly affected by penicillin treatment (Figure 4). Effect of fasting on endogenous lipids Adult female rabbits were fasted for 48 hours and endogenous lipid content of serum, liver and kidney 58 Figure 3. Effect of penicillin treatment on renal and serum concen- trations of NEFA. Animals were treated with two doses of 90,000 I.U. of penicillin G with a 12 hour interval, and killed 24 hours after the last dose. Individual fatty acids were quantitated with gas-liquid chromatography and select fatty acids summed to estimate the total NEFA content. Values are means i S.E. *Significantly different from control (p<0.05) 15 O 15 O x N § C O :1 E R U .5. E N C O .30 N EP YE N p 9 N E F A /m I S E R U M A 3 N 0 I)! 6/ v :I 3 N 6 d 60 .mo.ovm Houusoo Eoum Hcmnmmwfip kHHGMOHMHcmHm AU .m.m H memos mum mosam> Au .Amuma UV oHom OHmHosHH tam ”Huma UV OHmHo .Ama 0v OHHmmHm Aauma UV OHOHOHHEHMQ .Ama UV OHHHEHMQ .Ava UV owumwuxfi mums ownsmmma moHom muumm one in .mms 0H0mmn mason mm new vm Acmmv w sHHHHOHcmm mo .D.H ooo.om Ho Asoov mcHHmm nuwz ompmmuu mums mHmEHcm Am mm.o H va.a mm.o H vm.m m~.o H ~«.h ma.o H om.o w.v H H.mm m>.o H mm.m zmm NmZQHx om.o H mh.a om.o H ve.m vm.H H ma.m mm.o H om.H mv.~ H N.hm om.o H m~.m zoo oo.a H mm.m N.H H o.ma mm.a H va.aa.mm.o H om.m ha.> H o.Hm mm.a H Ha.m zmm m o o o c sommm mo.a H mm.¢ mo.H H mm.m m~.H H vm.m v~.o H HN.H Nv.m H ~.om mm.o H vm.v zoo mama o Huma 0 ma 0 Huoa 0 ma 0 «a U nmoHom mupmm mmHHnnmm oHo x003 m H0 Hcmucoo «mmz wmcon ocm Esumm msocmmoocm so usmEpmmuB GHHHHoHcmm mo Hommwm .N mqmfifi 61 Figure 4. Effect of penicillin treat- ment on renal, liver and serum tri- glyceride. Two week old litters were treated with two doses of 90,000 I.U. penicillin G with a 12 hour interval. Triglyceride was quantified 24 hours after the last dose. Values are means i S.E. *Significantly different from control (p<0.05) 62 _._ .omcHsHmumc chHH HmHoH oucH Hmnma mo coaumnomuoocfl may on w>wumawu moflumowamfluu ousw mumuwfiamm owamnma wumuomuoosfl on muflommmo HHOSH was omummmum mnm3 HHSHmanm usmfiummuu puma mnu kumm muses Macmlmusm39 .mam>umucH Hsoa NH Hm AucmspmmHH\.b.H ooo.omv o :HHHHoHamm Ho mmmoc 4 Ho N nqu ompmmuumum muck muwbnmn oao x003 03¢ mo mnmuuflq .O©HHOO>HmHHH once mHmuHEHmm M:0 mo coaumuomHOOCH HMHsHmEOHm so Haws lummnu cHHHHOHcmm mo Hommmm .oa musmfim 2m: 28 2m... 28. ///A W / % W / // NuZ OuZ $8 a 309. o ‘7‘ N 0. Q Q 11/91 awnuawow co 0. 9. 78 .m.m H momma mum mmsHm> .owswe lumump UHQHH Hmuou oucw sowumuomuoosw on m>wumamu smmz wsmmwu ousw wumuwsamm «Ho wumuomuoocw ou Muwowmwo Hausa ocm omummmum mums HHSHmEqu Hcmsummuu puma map umumm mason H50mlmpcmze .mHm>HmucH Hoot NH Hm iuamsum0HH\.o.H ooo.cmc o cHHHHOstm mswmooum duwz mafia» v no N omummuumum mums muwnamu pHo x003 03H mo mnmuqu .smmz mammwu ousw muwuHEme «Ho Ho cowumuomuoocw umHusEon co Hams lummuu sHHHHOHsmm mo Hommmm .HH madman 2mm ZOU 2mm ZOU “I o //////// 11 / VdiN avomaawow W N u 2 o u 2 300 N meo v 0... 80 Tubules from litters treated with a single dose of penicillin did not exhibit altered metabolism or altered transport when evaluated 12 hours after treatment (Figure 12; Table 3). Treatment with two doses of penicillin at a 12 hour interval enhanced incorporation of label into trigly- ceride and decreased incorporation of label into NEFA but did not enhance PAH transport capacity when evaluated 12 hours after the last treatment (Figure 12; Table 3). Treatment with 2 doses of penicillin G signifi- cantly enhanced PAH transport 24 hours after treatment (Figure 12; Table 3). Incorporation of labeled palmitate into tissue NEFA was slightly, but significantly decreased (Figure 12; Table 3). Treatment with four doses of penicillin G was associated with increased transport 24 and 48 hours after the last treatment dose. (Figure 12; Table 3). The four dose regimen was also associated with a significant increase in incorporation of label into triglyceride 24 hours after the last dose (Figure 12; Table 3). Incorporation of labeled palmitate into tissue NEFA was decreased 24 hours after treatment but the effect appeared to have decreased markedly 48 hours after the last dose (Figure 12; Table 3). 81 Figure 12. Effect of various penicillin treatment regimens on tubular transport capacity and tubular metabolism of palmitate. Litters were treated with 1, 2 or 4 doses of penicillin (90,000 I.U./dose), at 12 hour intervals and the effect of treatment determined 12, 24 or 48 hours after the last treatment. Values are mean differences (treated- control). Values represent 3 or 9 individual determinations. (Treated - Control) RELATIVE INCORPORATION 82 Q PAH TIM El TGITI. I NEFAITI. r 2.- U! 5 I -.I5 t. I dose 2 do” 2 do“ I2 hr I2 hr 24 hr Figure 12 4 do» 24 I" 4 do“ 48 hr N O (gonna) - puma“ Wll HVd 83 .mo.o v a .onHH Hmuou oucH sowumuomnoocfi op m>HuonH ommmmumxm mm3 iaaxHmHaH Hue: NH Hm umummHH mums mmumaomz in .mcoHHmsHEHmumo m MD m «o .m.m + AHOHHcOOIomHmmHHV mmocmHNMMHo some mum mosam> Aw ohm.OH we.~ ohm.OH ma.v 0mm.HH HN.m cm. H mH.o mm.0H 5N.o E\B mmm mmo. H mom.o HNH. H vma.o Nho. H who.l Hmo. H moo.| mm. H mm.o was Nmo. H hoo.l mmo. H HHo.o mac. H wmo.o ovo. H Nmo.o NNo. H moo.o oA9\Am mmao. H omo.o mmao. H omo.o mmNo. H mmo.o mmao. H HNH.o vmo. H MNo.o UAB\ mo Hommwm 34 Effect of fasting on transport and palmitate metabolism Adult rabbits were fasted for 48 hours or allowed free access to food. Tubule suspensions were then prepared and the effect of fasting on transport or upon the metabolism of C14 — palmitic acid determined. Fasting significantly decreased the accumulation of PAH into tubules (Figure 13). Fasting did not affect the incorporation of palmitate into tissue total lipid (Figure 13). While fasting did not sig- nificantly alter incorporation of palmitate into tissue triglyceride, tissue NEFA, or tissue phospholipid, incor- poration of label into triglyceride and phospholipid tended to be decreased, and the incorporation of label tended to be increased in tissue NEFA (Figure 14). Effect of nicotinic acid on accumulation of PAH and lipid metabolism in the fasted rabbit Adult rabbits were fasted for 48 hours before use. Twenty-four hours before use 250 mg/kg nicotinic acid or saline was injected intraperitoneally. The ability of tubule suspensions to accumulate PAH and to metabolize palmitate was then determined. Nicotinic acid did not 85 Figure 13. Effect of fasting on accu— mulation of PAH into tubules and the incorporation of labeled palmitate into tissue total lipid. Adult rabbits were fasted for 48 hours and the ability of tubules to metabolize palmitate and accumulate PAH were determined. Values are means : S.E. *Significantly different from control (p<0.05). PAH T/M I2 N=3 10 D FED NN FASTED Z N //////// N PAH T/M TOTAL LIPID :0 .w U! 0 N o (ugw 03/6] agow rI) (IIle “1101 OlNI NOIlVZIOdHODNI 1"" 7" 0 UI .0 u: 87 Figure 14. Effect of fasting on the distribution of labeled palmitate into tissue lipids. Adult rabbits were fasted 48 hours and tubule suspensions prepared. Tubules were incubated in a medium containing Cl4 palmitate and the distribution of label in various lipid classes determined relative to incorp- oration of label into tissue lipid. Values are means i S.E. RELATIVE INCORPORATION .0 .0 .0 .— uh an O 7 AD.- 71 I'l'l o O O: .2 I T 89 affect PAH transport capacity nor the incorporation of label into tissue total lipid (Figure 15). Nicotinic acid did, however, significantly alter the renal utilization of Cl4 — palmitate. Nicotinic acid treatment increased incorporation of labeled palmitate into triglyceride about 60% compared to control from a relative incorporation of 0.144 t 0.034 to a relative incorporation of 0.241 i 0.040 (Figure 16). Nico- tinic acid also decreased incorporation into tissue NEFA about 20% from a relative incorporation of 0.752 i 0.026 to a relative incorporation of 0.599 t 0.066 (Figure 16). Effect of probenecid, PAH and nicotinic acid in vitro on the metabolism of palmitic acid Incubation of tubules in the presence of 2 mM pro— benecid, 2 mM PAH or 2 mM nicotinic acid did not affect incorporation of label into tissue total lipid (Figure 17; Figure 20). In fed rabbits, probenecid and PAH signifi- cantly decreased the relative incorporation of label into tissue triglyceride (25 and 20% respectively compared to control) (Figure 18). In fasted rabbits, PAH and nicotinic acid decreased the relative incorporation of labeled palmitic acid into 90 Figure 15. Effect of nicotinic acid on renal accumulation of PAH and incorporation of label into tissue total lipid in the fasted rabbit. Rabbits were fasted 48 hours before use. Rabbits were treated with nicotinic acid (250 mg/kg) or saline 24 hours before use. Values are means : S.E. PAH T/M 12 N = 3 I o CONTROL N\\\\‘ NICOTINIC ACID N PAH T/M F iiiiiiii =4 TOTAL LIPID /////////////////////2 N (ugw 08 [6/ 9pm rI) NOIlVlIOdUODNI CIIdI'I TVLOl d 92 Figure 16. Effect of nicotinic acid on the distribution of labeled palmitate into tissue lipid. Adult rabbits were fasted 48 hours before use. Nicotinic acid (250 mg/kg) or saline was injected intraperitoneally 24 hours before use. Incorporation of label into lipid classes by tubule suspensions was deter- mined relative to the incorporation of label into total lipid. Values are means i S.E. *Significantly different from control (p<0.05). ZO_._.<~_On_ mOuZ. m>_.—<._m~_ NE FA 94 .mcowuosHEHoumo m No n no .m.m H momma mum mosam> .NNN 28 N no owomsoaoum 28 N mo woodman map No .wo mosmmmum may cw Hmnuwm osm muouHEHom I «Ho SE H mswsfloucoo Edwoms m CH omuoasocw oco muwnoon pom Eoum ooummmum mum? mmasasa .onHH HMHOH msmmwu oudH Honda mo sowumuomnoosw so mdm Ho owomsmnonm mo Hommmm_mmmmwjmm_ .NH gunman 95 NH musmwm ZOU ZOU /§ I“. C) Q In ,_' Q N m.N (ugw 09 / 6/ paImodJoaul slow d) OIdI'I 'IVlOl NI 31V1IW1V¢I 96 .imo.ovmo Houuaoo scum HamumuuHa HHHcmoHHH: ImHmH .UHQHH Houou oucH Hmowa mo sowumuom IHoocH OH m>wuoawn mowumomamwuu ousw HmamH mo COHHmHomHoocH mm oOmmmmew mum mmaam> .mcowumswsumumo m no N no .m.m H momma mum mosam> .25 N no cowumuusmocoo m 0H omoom mums mmm Ho oHomcoooum mcowco owsmmuo one .cowso owcmmuo omoom mo mocomao Ho wosmmmum on» CH ocm owom OHHHEHMQ I vao SE A mawawousoo Edwome m cw oouon IsocH ocm muwoamu pom Scum omuommum mum? mcowmsmmmsm masons .mowumowaowuu msmmHH ousH Hmnoa mo sowumuomnoosw so use no owooconoum mo Hommmm ONHH> CH...mH musmwm 97 ma musowm ZOU 1(a— ZOU ON ca 00— JOIUBDA'IOIIIL OlNI NOILVIIOdlIODNI 3AI1V13II 98 triglyceride, (27 and 39% respectively compared to control) (Figure 21). PAH and probenecid tended to increase incorporation of label into tissue NEFA in fed rabbits (Figure 19). Simi- larly, PAH and nicotinic acid slightly, but significantly, enhanced incorporation of label into tissue NEFA (Figure 21). Effect of penicillin treatment and organic anions on diacyl glycerol acyl transferase (DAGAT) Two week old litters were pretreated with 90,000 I.U. of procaine penicillin G 24 and 36 hours prior to preparation of hepatic and renal microsomes and a 100,000 x g supernate. Supernates prepared from control and penicillin treated rabbit kidneys were added to a standard amount of hepatic microsomes and the synthesis of triglyceride from diolein and palmitoyl coenzyme A determined. Supernate dependent syn- thesis of triglyceride from penicillin treated rabbits was not significantly different from synthesis supported by supernate from control rabbits (Figure 22). Similarly, microsomes from control rabbit kidneys were not different from microsomes from penicillin treated rabbits in their capacity to support triglyceride formation (Figure 22). 99 Finally, the addition of 2.mM PAH or 2 mM nicotinic acid did not affect the synthesis of triglyceride by DAGAT (Figure 22). 100 .cHaHH Houou mummflu OHGH Honda mo GOHHNHOQHOOGH ou 0>Huoamu «mmz OHGH Honda «0 GOHHMHOQHOOGH mo ommmmumxm who mosam> .mGOHHosHEHmumo o No b m0 .m.m H momma mum mmsHm> .GOHH Imuuswocoo 28 N m UHOH» on omoom muck omooo cmn3 mdm Ho oflomsmnoum macaco ownomuo one .GOHcm owcmmuo owoom mo mosmmnm Ho mocmmmum on» Ca osm oflom OHHHEHMQ I vHU SE A msflsflmu Isoo Edwowfi m cH omumndocw osm muHQQMH pom EOHM owummmum mum3 mCOHmsmmmsm OHDQSB . .COHCM OHCmeo oooom mo NUCOmnm Ho OOCOmmHm OCH CH oCo owom OHHHEHCQ I HAD 2E H mCHCHoHCOO ECHomE m CH COHMQCOCH oCM “Hmmm HCOC mvv muwanou omumom scum omuommum mum? mCOHmCommCm masons .onHH Houop osmmHH ouCH Momma mo CowumuomHOOCH Co CNN 2E N no owom OHCHHOOHC 2E N no Hummum OHHH> CH .ON musowm 103 Z -0 k) /§ 4 - CON N II- (ugw 08/ 6/ slow r1) CIIdI'I 111101 OlNI NOIlVEIOdlIODNI BALLV'IJH Figure 20 104 Figure 21. £2 vitro effect of 2 mM nicotinic acid or 2 mM PAH on incorporation of label into various lipid classes. Tubule suspensions were prepared from fasted (48 hour fast) rabbits and were incubated in a medium containing 1 mM Cl4 palmitic acid. Tubules were incubated in the presence or absence of the organic anions probenecid or PAH. Values are expressed as a % of control and are means : S.E. *Significantly different from control (p<0.05). ‘73 OF CONTROL TG PL PAH NEFA F iiiiiiii //////////% ////// § 5555 '6 Pl- NEFA NIc 106 .HOHHCOO no N o no oommmumxm mum oCo .m.m H mCmmE mum mmCHm> .COHHMHMQOHQ OHMCHomCm OHHMQOC onoCmum m Cqu omuon ICOCH mm3 mmmsowx HHCCCH scum mmfiomouows .NHHMHHEHm .Coflumuommum mEOmOHOHE owuommn onoCoum m CHH3 COHMCCOCH no? nmeoHM COMM OHMCHmmsm .COHCM OHComHo mConmH mHCHNHE mCHoCommouuoo OCH How meHm> ou oouomsoo oCm nmmEOmOHOHE + OHMCHmmCmV stuxHE Cowuommu wumamaoo m on oooom was Ass NV mam Ho Ass NV oHom OHcHHoon .OHMCHomCm m ooo.ooa oCm moEOmOHOHE OCH mo COHumummmHm on HOHHQ mHCOC mm oCm vN oonoumflsweom moz A.D.Hooo.omv CHHHHOHCOC .Aoao mxmoz NV mOHMCOOC HHQQCH omuomuu CHHHHOHCOQ mo osmmwu Hm>HH oCm NOCCHM Eoum omummonm mumz Auomsmv muosummsm m x ooo.ooa cam iaamdv mmsomoHon .Hmoaa Ho NHH>HHOM oCH Co mCOHCm OHCmmHo omoom oCm HCOEHomHu CHHHHOHCOQ mo Hommmm .NN mHCmHm 107 NN musmwm «was; muZ ON 0* 00 Om 00. ON— TOHlNOD :IO % DISCUSSION The hypothesis that penicillin stimulation of organic anion transport in the immature rabbit is due to disinhibition of the transport system by removal of NEFA is based on the hypothesis that NEFA interact competitively with the organic anion transport system (Hewitt and Hook, 1978). Analysis of the relationship between the renal organic anion transport system and NEFA requires the reso— lution of three separate, but related questions: First, are NEFA transported by the renal organic anion transport system? Second, can NEFA act as endogenous inhibitors of the renal organic anion transport system? Third, is the altered renal metabolism of palmitate produced by penicillin treatment of the neonate a specific manifestation of altered transport? The results of this study support the conclusion that NEFA are not transported by the organic anion transport system and do not, in physiological concentrations, act as inhibitors of the transport system. The data demonstrate that altered metabolism of palmitate is not specifically related to transport. Consequently, penicillin apparently 108 109 does not act to increase transport in the neonate by decreas- ing NEFA—mediated inhibition. Renal fatty acid utilization and organic anion transport The kidney readily utilizes NEFA to support renal work in vivo and in vitro (Cohen and Barac-Nieto, 1973). Since NEFA are extensively bound to plasma proteins in vivo (Goodman, 1958; Goodman, 1961), Cohen and Kamm (1976) con— cluded that the principle pathway for the uptake of fatty acids into renal cells is across the peritubular membrane. However, the mechanism by which NEFA enter the renal cell remains obscure. The uptake of fatty acids into a variety of tissues has been characterized as a two step process, since there appear to be two cellular pools of NEFA (Spector et al., 1965). A pool which is readily accessible to extracellular fluid is indicated since tubules which are washed with an albumin containing medium readily releases 45 to 75% of the cellular NEFA (Spector, 1968; Spector et al., 1965). Uptake into and release from this pool occurs at low temperature and in the presence of metabolic inhibitors (Spector, 1971), indicating that flux into this pool is essentially passive. 110 Human erythrocytes appear to contain a lipoprotein binding site for NEFA (Spector, 1971; Goodman, 1958). Bind— ing is readily reversible and not dependent on cellular metabolism, which suggests that the readily reversible pool corresponds to those fatty acids bound to membrane sites (Spector et al., 1965; Weismann and Korn, 1966). A second tissue pool of NEFA is indicated since 25 to 55% of tissue NEFA is not readily removed by washing (Spector, 1968; Spector et al., 1965). Entry into this pool can occur passively (Spector, et al., 1965). However, it appears that entry into the second cellular pool is also in part enzymatically mediated and energy dependent (Shohet et al., 1968). Spector (1971) reviewed three possible enzymatically mediated mechanisms for the penetration of fatty acids into the cell. Each of the mechanisms shared an initial activa- tion step in which the fatty acid is esterified with coenzyme A (CO A) by an ATP dependent thiokinase. In the first mech- anism the fatty acleXDZxdiffuses into the cellular water after being formed. Fatty acyl CO A, however, cannot pene- trate mitochondrial membranes. Penetration into the mito- chondrial matrix is mediated via the synthesis of a fatty acyl carnitine, from fatty acyl CO A, which can penetrate 111 the mitochondrial membrane (Fritz and Yue, 1968). The addi— tion of carnitine thus increases mitochondrial utilization of NEFA. The third mechanism has been postulated to involve the cyclic acylation of l—acyl glycerol-3-phorphory1 choline to form a phosphatidyl choline. Intracellular NEFA can then be formed by deacylation of the phosphatidyl choline to regenerate l-acyl glycerol-B—phosphoryl choline. The demonstration that probenecid, a classic inhibitor of renal organic anion transport, significantly decreased renal utilization of NEFA suggests that in the kidney, fatty acids may penetrate into the renal cell via the renal organic anion transport system (Barac-Nieto and Cohen, 1968). Several observations support this suggestion. First, palmitate can inhibit renal organic anion transport in an apparently competitive manner (Barac-Nieto, 1971). Second, the dog kidney preferentially extracts palmitic acid from the blood (Geyer et al., 1949) and third, NEFA exists primarily as organic anions at physiolOgical pH (Goodman, 1958b). In the context of the previous discussion of the mechanism of NEFA penetration into the cell, it is interest- ing to note that carnitine not only increases mitochondrial use of NEFA, it also stimulates transport of PAH into renal 112 cortical slices (Barac-Nieto, 1971). Furthermore, Holohan et al. (1976) have reported a dependency of the renal organic anion transport system for phosphatidyl choline. Hewitt and Hook (1978) attempted to clarify the relationship between renal organic anion transport and the renal uptake of NEFA by using the phenomenon of enhanced PAH transport produced by penicillin pretreatment of the immature rabbit. Penicillin treatment of the two week old rabbit enhances the renal capacity for the transport of the organic anions PAH, penicillin, phenosulfonphthalein and cephalori- dine (Hewitt et al., 1977; W01d et al., 1977). These results suggest that if NEFA are transported into the cell by the same mechanism which transports PAH, then penicillin treat- ment should enhance NEFA utilization. Penicillin treatment, however, failed to significantly increase incorporation of palmitate into tissue total lipid or to increase renal oxi- dation of palmitate. These results suggest that palmitate is not taken up into renal tissue via the renal organic anion transport system. However, careful examination of their (Hewitt and Hook, 1978) data indicates that while neither oxidation nor incorporation into total lipid was significantly increased, both tended to increase in conjunction with 113 penicillin, enhanced transport of PAH. Since metabolism and not actual transport of palmitate was evaluated, the possi— bility should be considered that the failure to observe increased oxidation or incorporation into tissue lipid may be due to factors apart from the primary interaction with the transport system. Three possibilities exist. First, the measurement of the several fates of the labeled palmitate introduces a very significant source of variance. This sug- gests that if total utilization were determined by summing the various fates, overall NEFA utilization may have been enhanced without a significant increase in the various indi- vidual metabolic fates. Second, since oxidation of fatty acids is a saturable process (Spector and Steinberg, 1965; Spector and Steinberg, 1967; Barac-Nieto, 1976) the failure to observe increased utilization of NEFA.may be due to satu- ration of the metabolic pathways by excess substrate in the incubation medium. Third, the failure of several investiga- tors (Maxild, 1971; Kippen and Klinenberg, 1971) to observe palmitate inhibition of organic anion transport, an observa- tion which is critical to the concept of an endogenous inhibition of organic anion transport, suggests the inhibi- tory effects of palmitate on the organic anion transport system should be confirmed in this model. 114 Incubation of tubule suspensions in the presence of lmM palmitate significantly decreased the capacity of the tissue to accumulate PAH (Figure 1). This result confirms the inhibitory character of palmitate and demonstrates that incubation conditions were apprOpriate for demonstrating accumulation of NEFA by the organic anion transport system. The failure to observe increased utilization does not however, appear to be due to the multiple metabolic fates of NEFA. Consistent with the results reported by Hewitt and Hook (1978), penicillin treatment failed to enhance the incorporation of labeled palmitate into tissue total lipid despite a significant increase in the capacity to accumulate PAH (Table 1; Table 3). Furthermore, in a separate series of experiments, penicillin treatment enhanced the accumulation of PAH but did not affect the extraction of labeled palmitate from the medium (Figure 2). Finally, the failure to observe increased utiliza- tion of NEFA related to the increased transport does not appear to be due to saturation of the metabolic pathways. If at the palmitate concentration used in the incubation medium, the metabolic pathways had been saturated, then increased uptake of NEFA should be observed as an increase 115 in tissue NEFA. Penicillin treatment did not, however, appear to increase renal NEFA content. Incubation of tissue in the presence of labeled fatty acids leads to the rapid accumulation of fatty acid and achievement of maximum values within a matter of minutes (Spector, 1971). Content of label present as NEFA remains constant during prolonged incubation (Spector, 1971). This result suggests that an evaluation of the uptake of fatty acids into the cell can be estimated from the tissue content of labeled NEFA. If palmitate were being accumulated by the transport system then penicillin enhanced transport should increase the tissue content of labeled NEFA. However, peni— cillin treatment decreased incorporation of label into tissue NEFA (Figure 12; Table 3; Hewitt and Hook, 1978). Conversely, inhibition of transport with competitive inhibi— tors would be expected to decrease label incorporation into tissue NEFA if fatty acids were being transported into the cell by the anion transport system. ‘However, the organic anions probenecid, PAH and nicotinic acid tend to increase label incorporation into tissue NEFA (Figure 19; Figure 21). The failure to correlate tissue NEFA with altered transport has been confirmed by direct measurement of fatty acids in renal tissue. Incubation of tubules in the presence 116 of palmitate increased tissue content compared to endogenous levels (Figure 7; Figure 3; Figure 5). However, probenecid 2 mM or PAH 2 mM not only failed to decrease tissue NEFA but, instead, tended to increase the tissue NEFA (Figure 7). Furthermore, penicillin treatment failed to increase endogenous tissue NEFA at a time when transport was enhanced in vitro (Figure 3). While the possibility exists that an organic anion transport system other than the one responsible for the secretion of PAH may be responsible for uptake of NEFA (Hewitt et al., 1978) the failure of probenecid to decrease NEFA content suggests that the alternate transport systems are not involved, since 2 mM probenecid would be expected to effectively inhibit all organic anion transport. Thus, these results are inconsistent with the con- cept that the renal organic anion transport system functions in the renal uptake of NEFA. These results are thus con— sistent with the speculations of Pakarinen (1970) and Cohen and Kamm (1976) that any interactions which occur between NEFA and the organic anion transport system must occur at some point subsequent to the penetration of fatty acids into the cell. 117 NEFA as endoqenous inhibitors of the renal organic anion transport system While it is apparent that the renal organic anion transport system is not responsible for the penetration of NEFA into the cell, the possibility remains that endogenous NEFA could be acting as endogenous modulators of the renal organic anion transport system. Extensive study of a variety of organic anions has demonstrated, in general, a positive correlation between the potency of a compound as an inhibitor of transport with its molecular weight, lipid solubility, accumulation in the renal cell and binding to plasma proteins (Weiner, 1973). Significantly, there is a negative correlation between potency as an inhibitor and rapidity of net secretion (Weiner, 1973). It is thus of interest that NEFA are exten- sively bound to plasma proteins (Goodman, 1958; Goodman, 1961). Similarly, fatty acids are bound to intracellular proteins (fatty acid binding protein; Z protein) (Kamisaka et al., 1975a, b; Listowsky et al., 1976). Furthermore, fatty acids appear to be more tightly bound to these proteins than do organic anions such as PAH.(Kamisaka et al., 1975a; Listowsky et al., 1976). NEFA reach maximal concentrations in tissue very rapidly (Spector, 1971) and are soluble in 118 nonpolar organic solvents which suggests they are readily soluble in membranes. Furthermore, NEFA exist at cellular pH primarily as organic anions (Goodman, 1958b) and, as discussed in the previous section, are not extensively trans— ported by the renal organic anion transport system even though they can inhibit the transport of PAH (Figure l; Barac-Nieto, 1971). These observations suggest that NEFA could act as effective inhibitors of renal organic anion transport. Several observations suggest physiological relevance of the concept that fatty acids act as inhibitors of renal organic anion transport. Fasting markedly increases plasma NEFA (Cahill et al., 1966; Goldrick and Hirsch 1964; Dahlquist et al., 1972) and significantly decreases the clearance of uric acid (Fox et al., 1976). Feeding rats a high fat diet resulted in rapid and specific inhibition of PAH transport (Johnson et al., 1973a, b). Finally, the neonatal animal is relatively hyperlipemic in comparison to the adult (Hardmann and Hull, 1969; Persson et al., 1966; Dahlquist et al., 1972), and also exhibits decreased capac— ity to transport organic anions (Rennick et al., 1961; Horster and Lewy, 1970; Lewy-and Grosser, 1974). 119 Investigations of the renal metabolism of NEFA support the concept that NEFA act as endogenous inhibitors of transport. Hewitt and Hook (1978) observed that a penicillin treatment regimen which enhanced organic anion transport decreased the incorporation of labeled palmitate into tissue NEFA. As discussed above the decreased incorporation of label into NEFA presumably indicates a decrease in the “steady state" level of tissue NEFA since labeled palmitate achieved maximum, stable levels of radioactive NEFA within minutes (Spector, 1971). Hewitt and Hook (1978) also observed increased triglyceride synthesis from labeled palmitate which suggested to them that the increased triglyceride synthesis was decreasing cellular NEFA content and thus disinhibiting transport. Consistent with this speculation, Barac-Nieto (1971) observed that the addition of carnitine disinhibited the decrease in transport produced by added palmitate. These results support the suggestion that penicillin— enhanced transport of PAH in the neonate, the development of transport with maturation and decreased transport of PAH with fasting are all due to alterations in NEFArproduced inhibi- tion of transport. This hypothesis suggests that increased transport should be correlated with decreased tissue or serum 120 NEFA and decreased transport should be correlated with increased serum and tissue NEFA concentrations. Consistent with this speculation and consistent with the results of others, the decreased transport of fasting was associated with elevated serum concentrations of NEFA (Figure 5; Figure 13). Furthermore, consistent with previous observations the increase in PAH transport capacity in adults (compared to newborns) was associated with decreased serum NEFA concentrations (Figure 3; Figure 5). However, peni— cillin-enhanced PAH transport in the neonate was associated not with a decrease in serum NEFA; but, rather, penicillin treatment increased serum NEFA (Figure 3). This result suggests that altered transport is not the result of altera- tions in serum NEFA. Analysis of renal NEFA content further supports the suggestion that NEFA are not responsible for altered trans- port. While fasting decreased transport (Figure 13) renal NEFA content did not decrease (Figure 5). Furthermore, penicillin enhanced transport (Table 3) was not associated with.decreased renal NEFA content (Figure 3). Increased transport capacity in the adult was associated with, if any- thing, higher NEFA concentrations (Figure 5) than those observed in the neonate (Figure 3). These results clearly 121 suggest that alterations in transport are not directly related to alterations in tissue NEFA. The failure of the hypolipidemic agent, nicotinic acid, to alter fasting-induced decreased PAH transport further supports the contention that altered NEFA is not responsible for altered transport. Nicotinic acid reduced tissue and plasma concentrations of NEFA and triglyceride (Carlson 1967; Trout et al., 1967; Carlson et al., 1966). However, treatment with nicotinic acid according to a treat- ment regimen that could be expected to decrease tissue and serum NEFA failed to enhance renal capacity to accumulate PAH (Figure 8). Finally, in the experiment reported here (Figure 1) and by Barac—Nieto (1971) in which palmitate decreased PAH transport, the tissue NEFA.measured concommitantly was 10 times that found in the fasted adult (Figure 7; Figure 5). This result coupled with the failure of other investigators to observe an effect of palmitate on PAH transport at physio— logical palmitate concentrations (Maxild, 1971, Kippen and Klinenberg, 1978) suggests that inhibition of transport by palmitate occurs only at pharmacological concentrations of NEFA and not at levels expected under physiological condi- tions. 122 These results support the conclusion that NEFA do not act as physiologically effective endOgenous inhibitors of organic anion tranSport. Altered renal metabolism of NEFA and altered transport The demonstration in the previous section that generalized alterations in NEFA content are not responsible for alterations in organic anion transport in the kidney leaves unresolved the relationship between altered transport in the neonate and altered metabolism. Conceivably, renal organic anion transport could be linked to lipid metabolism by a mechanism other than by a generalized reduction in NEFA. If, for example, renal metabolism was associated with a change in a small specific pool of tissue NEFA, then measure- ments of overall tissue NEFA might not detect a change in the NEFA content of the effective pool. However, the observation that the kidney readily oxidizes and esterifies fatty acids (Cohen and Barac—Nieto, 1973) and the observation that incu- bation of tissue in the presence of fatty acids yields stabile maximal levels in minutes (Spector, 1971) suggests that fatty acids rapidly equilibrate between pools. Thus, if metabolism were selectively disinhibiting the transport 123 system by removal of inhibiting NEFA then one would expect that altered transport and altered metabolism would be intimately and specifically integrated. Alternatively, if the enzymes of NEFA metabolism were actual components of the transport system, then altered metabolism and altered trans- port would also be expected to be tightly integrated. Several observations suggest that fatty acid metab- olism and organic anion transport in the kidney are inte- grated. Treatment of neonates with iodipamide or treatment of adults with penicillin failed to alter metabolism and failed to alter transport (Hewitt and Hook, 1978). However, the possibility must be considered that the failure to observe altered metabolism could have been due to inadequate dosage (see Introduction). A relationship of altered metabolism with the organic anion transport system is confirmed by the failure to observe altered glomerular metabolism at a time when tubular metab- olism and transport was altered (Figure 10; Figure 11; Figure 12; Table 3). The localization of the organic anion transport system to the proximal tubule is well known (Weiner, 1973); consequently the failure to observe altered glomerular metabolism supports the suggestion that altered metabolism is related to the transport system. However, the 124 possibility should be considered that tubules were affected and glomeruli were not, simply because the organic anion transport system concentrated penicillin in the tubular cells. Finally, fasting did decrease organic anion transport and did tend to indicate that the increased transport capac- ity of the fed animal was associated with an increased triglyceride synthesis and decreased incorporation of label into tissue NEFA. This observation is consistent with the suggestion that in the penicillin treated neonate, increased transport capacity was associated with increased triglycerice synthesis and decreased incorporation into NEFA. However, other observations suggest that altered metabolism and altered transport are not tightly integrated. In the penicillin treated neonate, lipid metabolism was affected before any change in transport was observed (Figure 12; Table 3). Similarly, nicotinic acid treatment of the fasted adult did not affect transport (Figure 15) but did enhance incorporation of labeled palmitate into triglyceride and decreased incorporation of label into tissue NEFA (Figure 16). Clearly, the failure to observe altered trans- port at a time when metabolism was altered demonstrates that altered metabolism and altered transport were not tightly 125 integrated. This observation suggests that altered metab— olism does not cause altered transport. The lack of correlation between transport and metabolism suggests that penicillin treatment of neonates alters lipid metabolism by a mechanism unrelated to its effects on organic anion transport. Penicillin — hypolipidemic drug? Penicillin treatment of neonates clearly affects lipid metabolism in vivo since treatment significantly decreased renal triglyceride (Figure 4) and produced a sig— nificant increase in serum NEFA (Figure 3). A number of organic anions are effective hypolipidemic agents. Analysis of a number of nicotinic acid derivatives indicates that the effective chemical is nicotinic acid (Bailey et al., 1972). Similarly, clofibrate is rapidly metabolized to an organic anion which may be the effective agent (Thorp and waring, 1962). These observations suggest that penicillin may act in a manner similar to that of other hypolipidemic agents to produce altered lipid metabolism. Several observations support the suggestion that penicillin and hypolipidemic agent, nicotinic acid, share a 126 similar mechanism of action on renal lipid metabolism. Nicotinic acid treatment decreases the triglyceride content of several tissues including kidney (Carlson et al., 1966; Trout et al., 1967). Furthermore, subsequent to nicotinic acid treatment plasma NEFA are first decreased. Following excretion of the drug, plasma NEFA increased (Pereira, 1967). Similarly, penicillin treatment decreases renal triglyceride (Figure 4) and 24 hours after the last dose increased serum NEFA (Figure 3). Nicotinic acid treatment decreased tri- glyceride synthesis in the liver (Solyom and Puglisi, 1967) and similarly decreased triglyceride synthesis in the kidney (Figure 21) an action which it shared with probenecid and PAH (Figure 18). Furthermore, probenecid, PAH and nicotinic acid all tend to increase the incorporation of labeled palmitate into tissue NEFA (Figure 19; Figure 21). These results suggest a common mechanism of action. The mechanism by which nicotinic acid alters lipid metabolism remains obscure (Howe, 1973; Kudchodkar et al., 1978; VekrMo andle¢s, 1978). Several possibilities exist: lipid metabolism.might be altered by an interaction with the triglyceride synthesizing enzymes. The in vitro effects of probenecid, PAH and nicotinic acid (Figure 18; Figure 19; Figure 21) are similar to those observed with 127 u-bromopalmitate in the small intestine (Ockner and Manning, 1976) and in the kidney (Trimble et al., 1977). Ockner and Manning (1976) suggested that the effects of a-bromopalmitate to decrease triglyceride synthesis were mediated by its bind- ing to Z protein. Triglyceride synthesis via diacyl gly- cerol acyl transferase is dependent on Z protein (O'Doherty and Kuksis, 1978); consequently, the effect of penicillin pretreatment on this reaction and the in vitro effect of nicotinic acid and PAH were determined. Penicillin pretreat- ment failed to alter either DAGAT activity or the stimulatory effectiveness of 100,000 x g supernate, indicating that neither Z protein nor enzyme activity were altered (Figure 22). Nicotinic acid and PAH similarly failed to alter tri— glyceride synthesis (Figure 22) indicating that this meta- bolic step was not the site of action. Consistent with this observation, Brindly (1977) demonstrated that altered DAGAT activity correlates poorly with altered triglyceride syn— thesis in the liver. The failure to observe an effect directly on DAGAT does not, however, rule out an action on other enzymes responsible for lipid synthesis. These results thus leave unresolved the mechanism by which penicillin alters lipid metabolism, but the results do suggest an action which is similar to that of nicotinic acid. 128 Conclusions The data support four conclusions. First, NEFA do not penetrate into the renal proximal tubule cell via the renal organic anion transport system. Second, NEFA do not act as endogenous inhibitors of the transport of PAH at physiological levels. Third, altered lipid metabolism observed after penicillin treatment of neonatal animals is not specifically linked to altered transport, and may be acting in a manner similar to the hypolipidemic agent, nicotinic acid. Fourth, alterations in lipid metabolism do not appear to be due to an action on DAGAT. These conclusions are not consistent with the hypothesis that penicillin enhanced transport in the neonate is the result of disinhibition of transport by the metabo- lism of inhibiting NEFA. SPECULATION The mechanism by which penicillin treatment enhances transport of PAH in the immature rabbit kidney remains obscure. While it is clear that NEFA do not act as endogenous inhibitors of transport, the possibility remains that other endogenous organic anions may be acting as inhibitors. Thus, penicillin enhanced tranSport may be, in fact, a process of disinhibition of latent "transporters". Alternatively, the involvement of (protein) synthesis of new transporters cannot be ruled out. It is not surprising that increased labeling of a single protein can not be observed against the background of synthesis of the myriad of other cellular proteins. The failure of approaches taken to elucidate the mechanism of penicillin enhanced transport suggests that a fresh approach will be required to eluci- date these effects. The recent demonstration of increased transport capacity in isolated hepalocytes produced by a number of enzyme inducers, (Eaton and Klaasen, 1978) suggeSts that such an approach might be useful with kidney tissue. The 129 130 capacity to induce transport in vitro would allow for a more precise analysis of the role of protein synthesis in the mechanism of penicillin induced transport. Alternatively the demonstration of Hong et a1. (1978) of an interaction of SITS with.the anion transport system suggests that the membrane transporter may be identifiable as was the anion transporter in the red blood cell membrane (Rothstein et al., 1976). Thus, while the mechanism remains obscure, fresh approaches promise to broaden our understand- ing of the biochemical mechanisms of organic anion transport. S UMMARY The purpose of this investigation was to evaluate the hypothesis that altered transport of organic anions in the neonatal kidney observed after penicillin treatment, was related to altered renal metabolism of NEFA. Hewitt and Hook (1978) observed that penicillin treatment decreased incorporation of labelled palmitate into tissue NEFA ip yippp. They suggested that NEFA acted as endogenous inhibitors of transport of organic anions and that altered transport in the neonate was due to a disinhibition of transport by the removal of inhibitory NEFA. The evaluation of this hypothesis requires the resolution of three basic questions: First, are NEFA transported into the renal cell via the renal organic anion transport system? Second, do NEFA act as endogenous inhibitors of the organic anion transport system? Third, is the altered renal metabolism of NEFA observed after penicillin treatment integrally related to altered transport? Penicillin treatment failed to increase the incor- poration of labelled palmitate into tissue total lipid ip 131 132 yippp, failed to increase the extraction of labelled palmitate from the medium in vitro and also failed to increase the endogenous renal content of NEFA. Further— more, inhibitors of the organic anion transport system failed to decrease the renal content of NEFA in vitro. These results are not consistent with the suggestion that NEFA enter the renal cell via the organic anion transport system. Penicillin treatment of the neonatal rabbit did not decrease renal NEFA content and in fact increased serum levels of NEFA. This result suggests that altered serum levels of NEFA are not responsible for altered transport of organic anions in vitro and that the increased transport of organic anions observed in the kidney is not related to a decrease in inhibition produced by tissue NEFA. This sug- gests that NEFA do not act as endogenous inhibitors of the organic anion transport system. Several observations sup- port this suggestion. Fasting decreased transport in vitro and increased serum NEFA but did not increase tissue NEFA content. Furthermore, while transport increased with age and serum levels of NEFA tended to decrease, increased transport with aging was associated with, if anything, an increase in renal NEFA. Finally, NEFA decreased transport 133 in vitro was associated with tissue levels of NEFA 10 fold greater than the endogenous content of NEFA observed in the adult or neonate. These results are consistent with the conclusions that NEFA do not act as endogenous inhibitors of the transport system. Penicillin treatment altered tubular transport of organic anions and altered tubular metabolism of NEFA but did not affect metabolism of NEFA in glomeruli. Since glomeruli do not transport organic anions, this observation indicates that the expression of altered metabolism was dependent on the organic anion transport system. However, altered metabolism preceded altered transport in the peni- cillin treated neonate. Furthermore, an identical pattern of altered lipid metabolism was observed in the adult rabbit after treatment with nicotinic acid, but transport of organic anions by the kidney was not altered. 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