Human organic anion transporter OAT1 is not responsible for glutathione transport but mediates transport of glutamate derivatives.

Due to their clearance function, the kidneys are exposed to high concentrations of oxidants and potentially toxic substances. To maintain cellular integrity, renal cells have to be protected by sufficient concentrations of the antioxidant glutathione (GSH). We tested whether GSH or its precursors are taken up by human organic anion transporters 1 (OAT1) and 3 (OAT3) stably expressed in HEK293 cells. GSH did not inhibit uptake of p-aminohippurate (PAH) or of estrone sulfate (ES) in OAT3-transfected HEK293 cells. In OAT1-transfected cells, GSH reduced the uptake of PAH marginally. Among the GSH constituent amino acids, glutamate, cysteine, and glycine, only glutamate inhibited OAT1, but labeled glutamate was not taken up by a probenecid-inhibitable transport system. Thus OAT1 binds glutamate but is unable to translocate it. The GSH precursor dipeptide, cysteinyl glycine (cysgly), and the glutamate derivative N-acetyl glutamate (NAG), inhibited uptake of PAH when present in the medium and trans-stimulated uptake of PAH from the intracellular side, indicating that they are hitherto unrecognized transported substrates of OAT1. N-acetyl aspartate weakly interacted with OAT1, but aspartate did not. NAG inhibited also OAT3, albeit with much lower affinity compared with OAT1, and glutamate did not interact with OAT3 at all. Taken together, human OAT3 and OAT1 cannot be involved in renal GSH extraction from the blood. However, OAT1 could support intracellular GSH synthesis by taking up cysteinyl glycine.

Effects of radical scavengers and antioxidant on ischemic acute renal failure in rabbits.

This study was undertaken to determine whether reactive oxygen species (ROS) are involved in the pathogenesis of ischemic acute renal failure (IARF) in rabbits. Renal ischemia was induced by clamping bilateral renal arteries for 60 min. Animals were pretreated with combination of xanthine oxidase inhibitor (allopurinol), hydrogen peroxide scavenger (catalase), and hydroxyl radical scavenger (sodium benzoate). Serum creatinine level significantly increased 24 h after ischemia and remained higher to 72 h. Ischemia caused a reduction of GFR and an increase of FENa. Such changes were significantly attenuated by scavenger pretreatment. The uptake of p-aminohippurate in cortical slices and microsomal Na(+)-K(+)-ATPase activity were depressed in kidneys subjected to 72 h of reflow following ischemia, indicating impairment of tubular transport function, which were significantly attenuated by scavenger treatment. Renal blood flow 72 h after reflow was markedly reduced and it was restored by scavenger pretreatment. When animals were pretreated with a potent antioxidant DPPD, lipid peroxidation in cortex and medulla was significantly inhibited. However, ischemia-induced impairment of renal function was not attenuated by pretreatment of the antioxidant. These results suggest that radical scavengers may exert a protective effect against ischemia acute renal failure by other actions rather than ROS scavenging. Thus, the data do not support involvement of ROS in IARF in rabbits.

The cytotoxic effect of paraquat to isolated renal proximal tubular segments from rabbits.

Paraquat (PQ) induces lung, liver and kidney damage. Since PQ mainly is eliminated by the kidney, the kidney damage is of particular importance to the outcome of PQ poisoning. The exact toxic mechanism of PQ is still unclear but it is assumed to involve redox cycling and formation of reactive oxygen species. In this study, further investigations on the toxic mechanism and metabolic effects of PQ were performed using isolated renal proximal tubules from rabbits. Proximal tubules were isolated using a combined iron perfusion and collagenase method. Suspended tubules were incubated for varying periods and concentrations of PQ at 25 or 37 degrees C in Krebs-Ringer phosphate buffer or HCO3-/CO2 buffer. The cytotoxic effect of PQ was evaluated by (1) markers of oxidative stress: status of glutathione (GSH/GSSG) and formation of malondialdehyde (MDA); and (2) markers of tubular metabolism: oxygen consumption (QO2), transport of 14C-p-aminohippuric acid (PAH) and 14C-tetraethylammonium (TEA). Using 0.5 and 5 mM PQ, the GSH/GSSG ratio decreased whereas formation of MDA increased indicating oxidative stress. PQ reduced the accumulation of PAH and TEA, the basal QO2 and the ouabain sensitive QO2 indicating inhibition of the Na/K-ATPase. Nystatin-stimulated QO2 was reduced by PQ, excluding inhibition of Na+ entry as a possible cytotoxic mechanism and suggesting mitochondrial injury. This was confirmed by measuring FCCP-uncoupled QO2. Thus high concentrations of PQ appear to disrupt mitochondrial electron chain transfer resulting in reduction of metabolic functions.

Lack of correlation between para-aminophenol toxicity in vivo and in vitro in female Sprague-Dawley rats.

The present study was designed to test the hypothesis that para-aminophenol (PAP) nephrotoxicity is due to autooxidation. We compared renal functional responses following PAP administration to female Sprague-Dawley rats and following incubation of renal proximal tubules with PAP. The concentrations of PAP selected for in vitro incubations produced cytotoxicity (for example, a decrease in oxygen consumption or adenine nucleotide concentration) in rat renal epithelial cells or rabbit proximal tubule suspensions. In rats, PAP (300 mg/kg i.p.) caused proximal tubular necrosis within 24 hr. Changes in renal function 24 hr following PAP administration included increased kidney weight and blood urea nitrogen concentration and decreased renal glutathione (GSH) content and adenine nucleotide concentrations. PAP did not cause hepatic damage. Within 2-4 hr following PAP administration, renal GSH content and adenine nucleotide concentrations were significantly decreased. In renal cortical slices prepared from PAP-treated rats, oxygen consumption and accumulation of organic ions (para-aminohippurate and tetraethylammonium) were significantly decreased compared with renal cortical slices prepared from control rats. In liver, GSH content was significantly decreased from 1 to 4 hr following PAP administration. In contrast to the effects of PAP in vivo, renal proximal tubules showed little evidence of injury when incubated with 0.1 or 0.5 mM PAP for up to 4 hr in the presence or absence of amino acids in the incubation medium. When tubules were incubated with 1 mM PAP for 4 hr in the presence of amino acids, GSH content, AMP concentration, and TEA uptake were significantly decreased. When amino acids were removed from the incubation medium, 1 mM PAP caused decreases in oxygen consumption and ATP concentration after 4 hr of incubation. Functional changes observed during incubation with PAP in vitro were not consistent with functional changes observed in vivo. The discrepancy between PAP toxicity in vivo and in vitro suggests that autooxidation is unlikely to be responsible for PAP nephrotoxicity and that nephrotoxicity in vivo is primarily mediated by extrarenal bioactivation. Further, depletion of hepatic GSH content prior to changes in renal function suggests that PAP or a PAP metabolite may conjugate with hepatic GSH. These observations suggest that PAP nephrotoxicity may be mediated by PAP-GSH conjugates rather than autooxidation of PAP in the kidney.

In vitro and in vivo effects of quinidine on the kidneys in Fischer-344 rats.

Quinidine in vitro significantly reduced accumulation of TEA (tetraethyl ammonium) and PAH (p-amino hippurate) and inhibited oxygen consumption in renal cortical slices. Mitochondrial respiratory control index (RCI) and ADP/O ratio were decreased. Intraperitoneal administration of quinidine at 75 mg/kg twice a day for four days inhibited TEA transport in renal cortical slices and decreased oxygen consumption. Mitochondria showed a reduction in ADP/O ratio but no change in RCI. Serum biochemical measurements indicated a significant elevation in serum creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST).

Radiation target sizes of the Na,K-ATPase and p-aminohippurate transport system in the basolateral membrane of renal proximal tubule.

Basolateral membrane vesicles made from rabbit kidney proximal tubules were frozen and irradiated with a high energy electron beam and the effects of irradiation on Na,K-ATPase activity, p-aminohippurate (PAH) transport, the membrane diffusion barrier and vesicle volume were measured. The vesicle volume and diffusion barrier were not significantly changed by radiation exposure. Na,K-ATPase activity was inactivated as a simple exponential function of radiation dose. Target size analysis of the data yielded a molecular size of 267 +/- 17 kDa, consistent with its existence as a (alpha beta)2 dimer. The carrier-mediated PAH uptake by basolateral membrane vesicles was also inactivated as a function of radiation dose. A target molecular size of 74 +/- 16 kDa was calculated for the PAH transport system. This study is the first measurement of the functional size of the organic acid transport system based directly on flux measurements.

Transport of urate and p-aminohippurate in rabbit renal brush-border membranes.

The mechanisms involved in urate and p-aminohippurate (PAH) transport in the rabbit renal brush-border membrane were investigated through study of membrane vesicles. Transport of [14C]urate and [3H]PAH was measured by a rapid filtration method. As previously reported by others, no OH(-)-PAH exchanger could be demonstrated by imposing an outwardly directed OH- gradient (pHin 7.4, pHout 6). In contrast, an OH(-)-lactate exchanger (or H(+)-lactate cotransport) was demonstrated. In the presence of valinomycin and an inwardly directed K+ gradient, both [14C]urate and [3H]PAH vesicle uptake were stimulated, demonstrating a potential-driven transport of these two anions. Probenecid, PAH, or cold urate decreased potential-driven urate uptake, suggesting that this transport was facilitated by a specific transport mechanism. The potential-driven urate transport described here may play a role in the second step of urate secretion in rabbits, because rate (or PAH) is transported across the brush-border membrane from the negative interior of the cell to the more positive omen.

Moment analysis of drug disposition in kidney. III: Transport of p-aminohippurate and tetraethylammonium in the perfused kidney isolated from uranyl nitrate-induced acute renal failure rats.

A different manner of insufficiency of renal epithelial cell transport between the organic anion and cation, p-aminohippurate and tetraethylammonium, respectively, was observed in the perfused kidney isolated from uranyl nitrate-induced acute renal failure (ARF) rats. The single-pass outflow pattern of the perfused kidney was analyzed by noncompartmental moment analysis. The active tubular secretion was impaired faster than the reduction of glomerular filtration, and the tetraethylammonium secretion decreased at an earlier stage of ARF than p-aminohippurate. The apparent uptake rate constant from blood to cells of p-aminohippurate was reduced with the progress of ARF and associated with the amount of this drug secreted, whereas the uptake rate constant of tetraethylammonium did not change until the late stage of ARF. The mean residence time in renal epithelial cells of tetraethylammonium was prolonged with reduction of the amount to be secreted, while that of p-aminohippurate remained unchanged. Therefore, the uptake of p-aminohippurate across the basolateral membranes decreased gradually, and the transport across the brush border membranes was still unchanged after uranyl nitrate treatment. On the other hand, the secretion of tetraethylammonium from cells to lumen was impaired at first, and then the uptake from blood to cells was impaired. These results suggest that impairment by uranyl nitrate-induced ARF appears at the carrier-mediated transport process of the epithelial cell membranes for both organic anions and cations.

Contraluminal para-aminohippurate (PAH) transport in the proximal tubule of the rat kidney. VI. Specificity: amino acids, their N-methyl-, N-acetyl- and N-benzoylderivatives; glutathione- and cysteine conjugates, di- and oligopeptides.

In order to evaluate the specificity of the renal contraluminal PAH transport system for amino acids, oligopeptides and their conjugates, the inhibitory potency of these substances against contraluminal [3H] PAH influx has been determined. For this, inhibition of 3H-PAH flux from the interstitium into cortical tubular cells of the rat kidney in situ has been measured. Apparent Ki values were evaluated by a computer program assuming competitive inhibition. Unconjugated amino acids (glycine, cysteine, alanine, leucine, phenylalanine, tyrosine, aspartate, glutamate, arginine, ornithine and lysine) do not inhibit [3H] PAH influx. The very hydrophobic tryptophan, however, does. N-alpha-methylation does not change this behaviour. N-alpha-acetylation does not evoke interaction with the PAH transporter when it occurs with glycine, cysteine (to yield mercapturic acid), arginine, ornithine and lysine. However, it renders alanine, leucine, phenylalanine, tryptophan, L-aspartate moderately, and L-glutamate strongly, inhibitory. The acetylated D-isomers of alanine, leucine and phenylalanine exert a higher inhibitory potency compared with the respective L-isomers. N-alpha-benzoylation of L-lysine is ineffective. N-alpha-benzoylation, however, evokes interaction with the PAH transporter, when it occurs with ornithine less than arginine less than histidine less than glycine = leucine less than alanine = phenylalanine = aspartate = glutamate. Dipeptides interact with the PAH transporter according to their hydrophobicity (Nozaki scale down to 0.9, Fauchère scale up to 1.0). N-acetylation does not change this behaviour. Hydrophobicity also renders oligopeptides, as angiotensin II, inhibitory against PAH transport. Similarly the anionic angiotensin I converting enzyme inhibitors Captopril, Enalapril and Ramipril inhibit contraluminal PAH influx.

Size selected mRNA induces expression of P-aminohippurate transport in Xenopus oocytes.

Xenopus oocytes were injected with size-fractionated mRNA isolated from the renal cortex of rabbit kidney and after 4 days incubation, PAH uptake in oocytes injected with mRNA (0.7-1.3 kb) was 8 to 45 fold that of the water injected controls. The oocyte to medium ratio of accumulated PAH was 1.95. The Km and Vmax for transport were 333 microM and 66.6 nmoles.oocyte-1.min-1, respectively. This Km is similar to that reported for PAH transport in intact kidneys and slices. The uptake of PAH was unaffected by the absence of Na+ or the presence of probenecid. Expression of the transport represents the first step in an effort to clone and identify the gene for PAH transport.

Effect of diatrizoate on renal extraction of PAH in man.

The effect of diatrizoate on the renal extraction of para-amino hippurate (EPAH) was studied in 8 healthy male volunteers. The contrast medium was injected into an antecubital vein and into a renal vein in each individual. A single-injection technique for the determination of EPAH was used and EPAH was measured before and over a period of 30 min after each contrast medium injection. In addition, the renal extraction of diatrizoate was measured simultaneously. Small but significant and similar decreases in EPAH were observed after both antecubital and renal venous administrations of the contrast medium, with a duration of less than 30 min after the injection. The renal extraction ratio for the diatrizoate was 0.20. It is concluded that diatrizoate should not be used before the determination of EPAH, at least not until 30 min after the administration of the contrast medium. The decrease in EPAH caused by diatrizoate seems to be due to a direct tubular depressant effect.

Coupled transport of p-aminohippurate by rat kidney basolateral membrane vesicles.

p-Aminohippuric acid (PAH) transport by basolateral membrane (BLM) vesicles isolated from rat renal cortex was stimulated very little by a Na+ gradient (out greater than in). However, when micromolar concentrations of glutaric acid or alpha-ketoglutaric acid were added in the presence of a out greater than in Na+ gradient, PAH uptake was accelerated greater than 20-fold and an overshoot of greater than fivefold was produced. Other anions, e.g., fumarate, stimulated PAH uptake very modestly under these conditions (approximately 2-fold), and that stimulation was totally prevented by short circuiting, i.e., with K+ (in = out) and valinomycin. Glutarate-stimulated uptake was inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and probenecid and was slightly stimulated by the imposition of an inside-negative membrane potential. Furthermore, even in the absence of a Na+ gradient, glutarate-loaded vesicles exhibited a marked acceleration of PAH uptake (5-fold) and a modest overshoot (2.5-fold). These results suggest an indirect coupling of BLM PAH uptake to the Na+ gradient by a cyclic accumulation (Na+-dependent) of glutarate followed by its efflux from the vesicle in exchange for PAH. This coupled system was absent in apical membranes. Thus net secretory transport of PAH may entail Na+-dependent, glutarate-driven PAH uptake at the BLM, followed by the exit of PAH into the lumen down its electrochemical gradient, probably in exchange for other anions, e.g., Cl-, HCO3-, or OH-.

Renal handling of drugs in renal failure. I: Differential effects of uranyl nitrate- and glycerol-induced acute renal failure on renal excretion of TEAB and PAH in rats.

Two etiologically different models of experimental acute renal failure were induced in rats by administration of either glycerol or uranyl nitrate. Both compounds caused a substantial decrease in the glomerular filtration rate (GFR) and the net tubular secretion of tetraethylammonium bromide (TEAB) and para-aminohippuric acid (PAH). The degree of renal impairment induced by uranyl nitrate and glycerol appeared to be dose related. Deprivation of drinking water 24 hr before the administration of glycerol potentiated the renal damage. In uranyl nitrate-induced renal failure, the decline of the net tubular secretion for TEAB and PAH was not proportional to the decrease in GFR; the secretion process deteriorated faster than the GFR. For example, when 0.5 mg/kg uranyl nitrate was administered, GFR fell to approximately 65% of normal, whereas the net tubular secretion was decreased to 30% of normal. These results suggest that the tubular transport was preferentially affected by uranyl nitrate. In contrast, in glycerol-induced renal failure, the decline of TEAB secretion fell in a parallel fashion with the GFR, suggesting that the glomeruli and the proximal tubules were equally damaged by glycerol. However, in this latter model, the decline of PAH secretion did not parallel the decrease in GFR, contradicting the proposal that glycerol affects equally the glomeruli and the proximal tubules. This discrepancy may be due to the selective competitive inhibition of PAH secretion by the accumulation of naturally occurring organic acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Na+ and H+ gradient-dependent transport of p-aminohippurate in membrane vesicles from dog kidney cortex.

The transport of p-aminohippurate (PAH) was studied in basolateral (BLMV) and brush border membrane vesicles (BBMV) isolated from dog kidney cortex. Imposition of an inwardly directed 100 mN Na+ gradient stimulated the uptake of 50 microM [3H]PAH into BLMV, whereas a pH gradient (pHout = 6.0, pHin = 7.4) only slightly enhanced uptake. The Na+ gradient-dependent uptake of PAH was electroneutral, saturable and sensitive to inhibition by probenecid and several anionic drugs, with (apparent) Km = 0.79 +/- 0.16 mM, Vmax = 0.80 +/- 0.05 nmol/mg protein, 15 sec and Ki for probenecid = 0.08 +/- 0.01 mM. Simultaneous imposition of the pH gradient (outward OH- gradient) and inward Na+ gradient stimulated PAH uptake significantly over that with an Na+ gradient alone. These results are consistent with an Na+ gradient-stimulated PAH/OH- exchange mechanism in the basolateral membrane. In BBMV, PAH uptake could be stimulated by an outwardly directed OH- gradient as well as an inward Na+ gradient. Both gradients could drive PAH transport via a mediated probenecid-sensitive pathway. Na+ gradient-stimulated uptake was electrogenic with a (apparent) Km = 4.93 +/- 0.57 mM, Vmax = 6.71 +/- 0.36 nmol/mg protein, 15 sec and Ki,prob = 0.13 +/- 0.01 mM. The kinetic parameters for PAH/OH- exchange were virtually the same, (apparent) Km = 5.72 +/- 0.49 mM, Vmax = 7.87 +/- 0.33 nmol/mg protein, 15 sec and Ki,prob = 0.16 +/- 0.02 mM. When both the Na+ and pH (outward OH-) gradient were simultaneously imposed an almost twofold stimulation in uptake was observed over that with either an Na+ or pH gradient alone. These results suggested that both gradients stimulate PAH transport in BBMV via the same pathway. However, inhibition experiments with various organic anions showed that the specificities of Na+ and pH gradient-stimulated PAH uptake do not entirely overlap. Thus, our results support a simple transport in BBMV, but it cannot be excluded that two separate pathways are involved.

Preparation of basolateral membranes that transport p-aminohippurate from primary cultures of rabbit kidney proximal tubule cells.

The organic anion p-aminohippurate (PAH) is specifically secreted by the renal proximal tubule. The possibility was examined that the probenecid sensitive PAH transport system (which is involved in this secretory process in renal proximal tubule cells in vivo) is retained in primary cultures of rabbit kidney proximal tubule cells. Significant 3H-PAH uptake into primary cultures of proximal tubule cells was observed. After 10 min, 150 pmole PAH/mg protein had accumulated intracellularly. Given an intracellular fluid volume of 10 microliter/mg protein, the intracellular PAH concentration was estimated to be 15 microM. The initial rate of PAH uptake (when 50 microM PAH was in the uptake buffer) was inhibited 50% by 2 mM probenecid. Intact monolayers also exhibited Na+-dependent alpha methyl-D-glucoside uptake (an apical marker). Basolateral membranes were purified from primary rabbit kidney proximal tubule cell cultures. Probenecid sensitive PAH uptake into the membrane vesicles derived from the primary cultures was observed. The rate of PAH uptake was equivalent to that obtained with vesicles obtained from the rabbit renal cortex. No significant Na+-dependent D-glucose uptake into the vesicles was observed, indicating that primarily basolateral membrane vesicles had indeed been obtained.

Moment analysis of drug disposition in kidney: transcellular transport kinetics of p-aminohippurate in the isolated perfused rat kidney.

The mean renal epithelial cell residence time, Tcell, was defined as a model-independent characteristic of the transcellular transport process in the isolated perfused kidney. The transcellular transport process includes transport at the basolateral membrane, diffusion in the cytosol, and transport at the brush border membrane. The parameter Tcell represents the mean time for the drug secreted from the tubules to pass through the renal epithelial cells, and is calculated as the difference of the mean urinary transit time between secreted drug and inulin in the single-pass perfusion system. Therefore, the urinary excretion rate-time course is indispensable to evaluate Tcell. p-Amino-hippuric acid was used as a model compound. The bovine erythrocytes in the perfusate kept the isolated kidney in an almost constant physiological condition, including secretion function. The renal vein outflow curves were also analyzed by the use of moments. The dispersion in the catheter was corrected by a deconvolution. The apparent secretion intrinsic clearance and the apparent volume of distribution were calculated from the moments. The present method will be useful for analysis of the transcellular transport mechanism and the effect of disease states on renal transport of drugs.

In vitro assessment of cephaloridine nephrotoxicity: comparison of renal cortical slice and renal tubule fragment techniques.

Renal cortical slices and a suspension of renal tubule fragments were prepared from male Wistar rats that had received a single s.c. dose of either cephaloridine (100 mg/kg) or normal saline (1 ml/kg) 48 h previously. The comparative sensitivity of these tissue preparations as in vitro models to assess nephrotoxin-induced changes in renal function was investigated by measuring the ability of the preparations to undertake the active accumulation of [3H]para-aminohippuric acid and to undertake gluconeogenesis from sodium pyruvate. Through the use of the cortical slice technique, [3H]para-aminohippuric acid tissue accumulation and glucose production in the cephaloridine-treated group were not significantly different from control. In contrast, using the tubule fragment technique, significant (p less than 0.05) reductions in the accumulation of [3H]para-aminohippuric acid and in the production of glucose via gluconeogenesis, between cephaloridine and normal saline control treatments were observed. Control values in the tubule fragment technique, for para-aminohippuric acid transport and glucose production via gluconeogenesis, were observed to be much greater than control values obtained from the cortical slice technique. It is suggested that the tubule fragment technique may be a more valuable in vitro preparation to assess the effects of potential nephrotoxins on tubule transport function than the still widely used cortical slice technique. The use of specific metabolic substrates, such as acetate, that will stimulate cellular metabolism and substrate transport, will also enhance the value of the technique.

[Physicochemical mechanisms of organic acid transport in the apical membrane vesicles of the cells of proximal kidney tubules in rats. II. Kinetic parameters of transport and phase state of the lipid bilayer in mutant Campbell strain rats]. / Fiziko-khimicheskie mekhanizmy transporta organicheskikh kislot v vezikulakh apikal'noi membrany kletok proksimal'nykh kanal'tsev pochki krys. II. Kineticheskie parametry transporta i fazovoe sostoianie lipidnogo bisloia u mutantnykh krys linii Kempbell.

The kinetics of 14C-para-aminohippuric acid (PAH) transport in the vesicles and the influence of the temperature on the initial rate of this transport were studied using a purified fraction of the apical membrane isolated from the kidney cortex of the Campbell strain rats with an autosomic recessive gene. The transport was brought about owing to the facilitate diffusion mechanism. At 36 degrees C the apparent Michaelis constant was equal to 29 mM, the maximum rate--62 nmol/min on 1 mg of protein, the inhibition constant for the PAH-transport by probenecid--1.5 mM. The temperature dependence of the initial rate of PAH-transport in vesicles and that of the rate of substrate splitting by alkaline phosphatase show the break point on the Arrhenius plot at 36 degrees C-38 degrees C. The analysis of electron magnetic resonance reveals the thermotropic transition at temperatures near 30 degrees-35 degrees C. Therefore the affinity of the carrier to its substrates in vesicles of the Campbell strain rats is strongly reduced and the lipid layer is more viscous than in the normal rats. We decide therefore that the mutation taking place in the Campbell strain leads to pleotropic membrane reconstructions in different organs (eye, kidney). The discovery of such a mutation is of considerable biological interest and promotes bases for development of the membrane biochemical genetics.

Excretion of para-aminohippurate in the isolated perfused rat kidney: net secretion and net reabsorption.

1. The excretion of para-aminohippurate (PAH) in the isolated perfused rat kidney was examined over a wide range of perfusate PAH concentrations (15 microM to 6 mM). PAH excretion increased steadily over the range of perfusate concentrations, reaching a maximal excretion rate of 3.28 mumol/min at a free-PAH concentration of 6 mM. 2. Tubular transport of PAH was evaluated from the difference between ultrafiltered PAH and excreted PAH. Net PAH secretion was observed at low perfusate free PAH concentrations. Net PAH transport was zero at a perfusate free PAH concentration of 2.1 mM. Above this level there was progressive net reabsorption. 3. Probenecid (2.5 mM) decreased PAH secretion to 18% of the initial value at 129 microM-free PAH (P less than 0.05). Probenecid had no effect on net reabsorption of PAH at high perfusate levels of the anion. 4. Alanine (5 mM) decreased net PAH secretion by 50% at low free PAH concentrations (P less than 0.05) and decreased net PAH reabsorption by 50% at at a free PAH concentration of 6 mM (P less than 0.05). These effects could not be related to effects of PAH, probenecid or alanine on glomerular filtration rate (GFR), vascular resistance or electrolyte excretion. 5. The results confirm the existence and integrity of the proximal tubular organic anion secretory system in the isolated kidney. In addition, net PAH reabsorption occurs at high perfusate levels.