Para-aminohippurate Transport Research Articles

Comparative Analysis of the Physiological and Transport Functions of Various Sources of Renal Proximal Tubule Cells Under Static and Fluidic Conditions in PhysioMimix{trade mark, serif} T12 Platform.

In vitro models that can faithfully replicate critical aspects of kidney tubule function such as directional drug transport are in high demand in pharmacology and toxicology. Accordingly, development and validation of new models is underway. The objective of this study was to characterize physiological and transport functions of various sources of human renal proximal tubule epithelial cells (RPTECs). We tested TERT1-immortalized RPTEC, including OAT1-, OCT2- or OAT3-overexpressing variants, and primary RPTECs. Cells were cultured on transwell membranes in static (24-well transwells) and fluidic (transwells in PhysioMimix{trade mark, serif} T12 organ-on-chip with 2 mL/s flow) conditions. Barrier formation, transport, and gene expression were evaluated. We show that two commercially available primary RPTECs were not suitable for studies of directional transport on transwells because they formed a substandard barrier even though they exhibited higher expression of transporters, especially under flow. TERT1-parent, -OAT1 and -OAT3 cells formed robust barriers, but were unaffected by flow. TERT1-OAT1 cells exhibited inhibitable para-aminohippurate transport, it was enhanced by flow. However, efficient tenofovir secretion and perfluorooctanoic acid reabsorption by TERT1-OAT1 cells were not modulated by flow. Gene expression showed that TERT1 and TERT1-OAT1 cells were most correlated with human kidney than other cell lines, but that flow did not have noticeable effects. Overall, our data show that addition of flow to in vitro studies of the renal proximal tubule may afford benefits in some aspects of modeling kidney function, but that careful consideration of the impact such adaptations would have on the cost and throughput of the experiments is needed. Significance Statement The topic of reproducibility and robustness of the complex microphysiological systems is looming large in the field of biomedical research; therefore, the uptake of these new models by the end-users is slow. This study systematically compared various RPTEC sources and experimental conditions, aiming to identify the level of model complexity needed for testing renal tubule transport. We demonstrate that while tissue chips may afford some benefits, their throughput and complexity need careful consideration in each context of use.

Impaired renal organic anion transport 1 (SLC22A6) and its regulation following acute myocardial infarction and reperfusion injury in rats

Acute kidney injury (AKI) is a high frequent and common complication following acute myocardial infarction (AMI). This study examined and identified the effect of AMI-induced AKI on organic anion transporter 1 (Oat1) and Oat3 transport using clinical setting of pre-renal AKI in vivo. Cardiac ischaemia (CI) and cardiac ischaemia and reperfusion (CIR) were induced in rats by 30-min left anterior descending coronary artery occlusion and 30-min occlusion followed by 120-min reperfusion, respectively. Renal hemodynamic parameters, mitochondrial function and Oat1/Oat3 expression and function were determined along with biochemical markers. Results showed that CI markedly reduced renal blood flow and pressure by approximately 40%, while these parameters were recovered during reperfusion. CI and CIR progressively attenuated renal function and induced oxidative stress by increasing plasma BUN, creatinine and malondialdehyde levels. Correspondingly, SOD, GPx, CAT mRNAs were decreased, while TNFα, IL1β, COX2, iNOS, NOX2, NOX4, and xanthine oxidase were increased. Mitochondrial dysfunction as indicated by increasing ROS, membrane depolarisation, swelling and caspase3 activation were shown. Early significant detection of AKI; KIM1, IL18, was found. All of which deteriorated para-aminohippurate transport by down-regulating Oat1 during sudden ischaemia. This consequent blunted the trafficking rate of Oat1/Oat3 transport via down-regulating PKCζ/Akt and up-regulating PKCα/NFκB during CI and CIR. Thus, this promising study indicates that CI and CIR abruptly impaired renal Oat1 and regulatory proteins of Oat1/Oat3, which supports dysregulation of remote sensing and signalling and inter-organ/organismal communication. Oat1, therefore, could potentially worsen AKI and might be a potential therapeutic target for early reversal of such injury.

Organic Anion Transporter 1 Is Inhibited by Multiple Mechanisms and Shows a Transport Mode Independent of Exchange

The mechanism by which drugs inhibit organic anion transporter 1 (OAT1) was examined. OAT1 was stably expressed in Chinese hamster ovary (CHO) cells, and para-aminohippurate (PAH) and 6-carboxyfluorescein were the substrates. Most compounds (10 of 14) inhibited competitively, increasing the Michaelis constant (Km) without affecting the maximal transport rate (Jmax). Others were mixed-type (lowering Jmax and increasing Km) or noncompetitive (lowering Jmax only) inhibitors. The interaction of a noncompetitive inhibitor (telmisartan) with OAT1 was examined further. Binding of telmisartan to OAT1 was observed, but translocation was not. Telmisartan did not alter the plasma membrane expression of OAT1, indicating that it lowers Jmax by reducing the turnover number. PAH transport after telmisartan treatment and its washout recovered faster in the presence of 10% fetal bovine serum in the washout buffer, indicating that binding of telmisartan to OAT1 and its inhibitory effect are reversible. Together, these data suggest that telmisartan binds reversibly to a site distinct from substrate and stabilizes the transporter in a conformation unfavorable for translocation. In the absence of an exchangeable extracellular substrate, PAH efflux from CHO-OAT1 cells was relatively rapid. Telmisartan slowed PAH efflux, suggesting that some transporter-mediated efflux occurs independent of exchange. Although drug-drug interaction predictions at OAT1 assume competitive inhibition, these data show that OAT1 can be inhibited by other mechanisms, which could influence the accuracy of drug-drug interaction predictions at the transporter. Telmisartan was useful for examining how a noncompetitive inhibitor can alter OAT1 transport activity and for uncovering a transport mode independent of exchange.

A renal-like organic anion transport system in the ciliary epithelium of the bovine and human eye.

The purpose of this study was to determine the direction of organic anion (OA) transport across the ciliary body and the transport proteins that may contribute. Transport of several OAs across the bovine ciliary body was examined using ciliary body sections mounted in Ussing chambers and a perfused eye preparation. Microarray, reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemistry were used to examine OA transporter expression in human ocular tissues. Microarray analysis showed that many OA transporters common to other barrier epithelia are expressed in ocular tissues. mRNA (RT-PCR) and protein (immunoblotting) for OAT1, OAT3, NaDC3, and MRP4 were detected in extracts of the human ciliary body from several donors. OAT1 and OAT3 localized to basolateral membranes of nonpigmented epithelial cells and MRP4 to basolateral membranes of pigmented cells in the human eye. Para-aminohippurate (PAH) and estrone-3-sulfate transport across the bovine ciliary body in the Ussing chambers was greater in the aqueous humor-to-blood direction than in the blood-to-aqueous humor direction, and active. There was little net directional movement of cidofovir. Probenecid (0.1 mM) or novobiocin (0.1 mM) added to the aqueous humor side of the tissue, or MK571 (5-(3-(2-(7-chloroquinolin-2-yl)ethenyl)phenyl)-8-dimethylcarbamyl-4,6-dithiaoctanoic acid; 0.1 mM) added to the blood side significantly reduced net active PAH transport. The rate of 6-carboxyfluorescein elimination from the aqueous humor of the perfused eye was reduced 80% when novobiocin (0.1 mM) was present in the aqueous humor. These data indicate that the ciliary body expresses a variety of OA transporters, including those common to the kidney. They are likely involved in clearing potentially harmful endobiotic and xenobiotic OAs from the eye.

A renal‐like organic anion transport system in the ciliary body epithelium of the eye (1064.16)

The ciliary body epithelium (CE) is a bilayer consisting of a pigmented cell layer and a non‐pigmented cell layer, which together act as a physical barrier between the circulation and the eye interior. Previous studies show that clearance of organic anions from the eye is slowed by the well‐established organic anion transport inhibitor, probenecid. The purpose of this study was to determine the direction of organic anion transport across the CE and the transport proteins that may contribute. Paired unidirectional fluxes (aqueous humor‐to‐blood and blood‐to‐aqueous humor) and net flux (the difference between the unidirectional fluxes) of the organic anion para‐aminohippurate (PAH) across the freshly dissected rabbit CE was determined using Ussing chambers. RT‐PCR, immunoblotting and immunohistochemistry were used to examine organic anion transporter expression in the human eye. Net active PAH transport across the rabbit CE was in the aqueous humor‐to‐blood direction, with flux ratios of ~5. Probenecid (0.1 mM) inhibited net active PAH transport by reducing the unidirectional aqueous humor‐to‐blood flux. The organic anion transporters OAT1, OAT3, NaDC3 and MRP4 were detected in extracts of human ciliary body by RT‐PCR. The efflux transporter MRP4 was also detected in human ciliary body extracts by immunoblotting, where it localized to the basolateral membrane (blood‐facing side) of pigmented cells (determined by immunohistochemistry). These data suggest that the CE contains an organic anion transport system similar to that found in the renal proximal tubule. We speculate that this system is functionally similar in that it is involved in clearing potentially harmful endobiotic and xenobiotic organic anions from the eye interior. Supported by NSERC, NSHRF and CIHR.Grant Funding Source: Supported by NSERC, NSHRF and CIHR

Functional Expression of MRP4 in the Ciliary Epithelium of the Eye

The multidrug‐resistance associated protein 4 (MRP4) is an efflux pump that transports organic anions, including para‐aminohippurate. Transepithelial transport of para‐aminohippurate by the ciliary epithelium was previously demonstrated (Pelis and Delamere, The FASEB Journal 23, Abstract #797.9, 2009). This led to the hypothesis that MRP4 contributes to organic anion transport by the ciliary epithelium. MRP4 expression was examined using reverse transcriptase PCR, Western blotting and functional assays. MRP4 mRNA and protein were detected in the freshly dissected human ciliary body and in a simian virus transformed cell line derived from human ciliary body epithelial cells (SVHCE cells). MRP4 protein levels (relative to total protein) in the native tissue and cultured cells were comparable. In SVHCE cells, the intracellular accumulation of the fluorescent MRP4 substrate bimane glutathione increased in a dose‐dependent manner in the presence of the MRP4 inhibitors, probenecid and indomethacin. These data indicate that MRP4 contributes to organic anion transport by the human ciliary epithelium. The functional expression of MRP4 in SVHCE cells makes them a potentially useful system for examining MRP4‐mediated organic anion transport by ciliary epithelia. Supported by NIH.

Functional Maturation of Drug Transporters in the Developing, Neonatal, and Postnatal Kidney

Because renal function in newborns is immature, the pharmacokinetics of drugs administered to neonates vary significantly from adult patients. The establishment of drug transport systems is a key process in the functional maturation of the nephron. However, a thorough examination of the expression of the main drug transporters in the kidney throughout all stages of development (embryonic, postnatal, and mature) has yet to be carried out, and the functional (physiological) impact is not well understood. Using time-series microarray data, we analyzed the temporal behavior of mRNA levels for a wide range of SLC and ABC transporters in the rodent kidney throughout a developmental time series. We find dynamic increases between the postnatal and mature stages of development for a number of transporters, including the proximal tubule-specific drug and organic anion transporters (OATs) OAT1 (SLC22a6) and OAT3 (SLC22a8). The OATs are the major multispecific basolateral drug, toxin, and metabolite transporters in the proximal tubule responsible for handling of many drugs, as well as the prototypical OAT substrate para-aminohippurate (PAH). We therefore performed specific in vivo pharmacokinetic analysis of the transport of PAH in postnatal and maturing rodent kidney. We show that there is a 4-fold increase in PAH clearance during this period. Clearance studies in Oat1 and Oat3 knockouts confirm that, as in the adult, Oat1 is the principle transporter of PAH in the postnatal kidney. The substantial differences observed supports the need for better understanding of pharmacokinetics in the newborn and juvenile kidney compared with the adult kidney at the basic and clinical level.

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.

Interactions of Stevioside and Steviol with Renal Organic Anion Transporters in S2 Cells and Mouse Renal Cortical Slices

Our previous studies have shown that both stevioside and steviol inhibited transepithelial transport of para-aminohippurate (PAH) in isolated rabbit renal proximal tubules by interfering with organic anion transport system. The current study examined the direct interactions of stevioside and steviol with specific organic anion transporters. S2 cells expressing human organic anion transporters (hOAT1, hOAT2, hOAT3, and hOAT4) and an intact renal epithelium were used to determine the inhibitory effect of stevioside and steviol on organic anion transport. Stevioside at 0.5-1 mM showed no interaction with any OAT. In contrast, steviol markedly inhibited substrate uptake in all S2hOAT cells. Steviol had low IC50 for hOAT1 (11.4 microM) and hOAT3 (36.5 microM) similar to that of probenecid, whereas IC50 for hOAT2 (1000 microM) and hOAT4 (285 microM) was much higher. Results obtained in mouse renal cortical slices were very similar; that is, stevioside was without inhibitory effect and steviol was a potent inhibitor of PAH and estrone sulfate (ES) transport. Stevioside has no interaction with human or mouse OATs. In contrast, steviol interacts directly with human OATs, in particular, hOAT1 and hOAT3, with a potency approximating probenecid, suggesting that the inhibition of OAT-mediated transport by steviol could alter renal drug clearance.

Transport of the Natural Sweetener Stevioside and Its Aglycone Steviol by Human Organic Anion Transporter (hOAT1; SLC22A6) and hOAT3 (SLC22A8)

The natural sweetening agent stevioside and its aglycone metabolite, steviol, have been shown to inhibit transepithelial transport of para-aminohippurate (PAH) in isolated rabbit renal proximal tubules by interfering with basolateral entry. The aim of the present study was to determine which of the cloned basolateral organic anion transporters were involved in the renal transport of stevioside and steviol. This question was addressed in Xenopus laevis oocytes expressing human organic anion transporter 1 (hOAT1), 3 (hOAT3), and winter flounder OAT (fOat1). The parent compound, stevioside, had no inhibitory effect on either PAH (hOAT1) or ES (estrone sulfate; hOAT3) uptake. In contrast, steviol showed significant, dose-dependent inhibition of PAH and ES uptake in hOAT1- or hOAT3-expressing oocytes, respectively. The IC(50) of steviol for hOAT1-mediated PAH transport was 11.1 microM compared with 62.6 microM for hOAT3-mediated ES uptake. The Michaelis-Menten inhibition constants (K(i)) for steviol transport mediated by hOAT1 and hOAT3 were 2.0 +/- 0.3 and 5.4 +/- 2.0 microM, respectively. Trans-stimulation of PAH efflux by steviol was assessed to determine whether steviol itself was transported by hOAT1 or hOAT3. A low concentration of 1 microM steviol increased the efflux of [(3)H]PAH (trans-stimulated) via both hOAT1 and hOAT3. In addition, it was shown by electrophysiology that steviol entry induced inward current in fOat1-expressing oocytes. In conclusion, stevioside had no interaction with either hOAT1 or hOAT3, whereas hOAT1, hOAT3, and fOat1 were all shown to be capable of steviol transport and thus, can play a role in its renal transport and excretion.

ATP-dependent para-aminohippurate transport by apical multidrug resistance protein MRP2

Para-aminohippurate (PAH), a widely used model substrate for organic anion transport in proximal tubule epithelia, was investigated as a substrate for the apical multidrug resistance protein MRP2 (symbol ABCC2). This ATP-dependent export pump for anionic conjugates and additional amphiphilic anions was cloned recently and localized to the apical membrane of proximal tubules in human and rat kidney. Membrane vesicles from HEK-MRP2 cells containing recombinant human MRP2 and from control vector-transfected HEK-Co cells were incubated with various concentrations of [3H]PAH, and the net ATP-dependent transport into inside-out vesicles was determined. Comparative studies were performed with membrane vesicles containing recombinant human MRP1. Transport rates at 10 micromol/L PAH were 21.9 +/- 1.9 and 1.6 +/- 0.4 pmol x mg protein-1 x min-1 (means +/- SEM, N = 10) with membrane vesicles from HEK-MRP2 and HEK-Co cells, respectively. The Km value for PAH was 880 micromol/L. The high-affinity substrate leukotriene C4 and the inhibitor of MRP-mediated transport, MK571, inhibited MRP2-mediated transport of PAH (100 nmol/L) with IC50 values of 3.3 and 4.0 micromol/L, respectively. The nephrotoxic mycotoxin ochratoxin A inhibited MRP2-mediated PAH transport with an IC50 value of 58 micromol/L. Ochratoxin A was itself a substrate for MRP2. PAH is a good substrate for the ATP-dependent export pump MRP2. The localization and function of MRP2 indicate that this unidirectional transport protein contributes to the secretion of PAH and other amphiphilic anions into the lumen of kidney proximal tubules.

Removal of digoxin and doxorubicin by multidrug resistance protein-overexpressed cell culture in hollow fiber

Drug removal by hemoperfusion is not effective because of its lower capacity and nonspecificity. We invented a new hybrid type of hemodialysis system. An immortalized proximal tubular cell line (PCTL) overexpressing human multidrug resistance protein-1 (MDR-1) was cultured either on polus filter membranes or on hollow fiber modules. The modules were incubated in an incubator conditioned with 95% O2/5% CO2 that was kept at 37 degrees C. At 10 days on culture, the drug-transporting capacity of these systems was examined. MDR was successfully expressed in the PCTL as evaluated by Western blot. Basolateral to apical transport of 3H-digoxin, a substrate of MDR, was examined by using the cells cultured on a microporous membrane. PCTL-MDR showed a 10-fold increase in MDR protein and a 12-fold increase of 3H-digoxin transport through a cell layer on a microporous membrane. The increase of the transport was abolished by the addition of 5 microM verapamil, an inhibitor of MDR, to the apical side. When digoxin or doxorubicin was infused in the capillary side of the hollow fiber modules after 10 days on culture, the largest portion of the drugs was transported to the pericapillary side (P < 0.001). This transport was also abolished by an addition of verapamil to the pericapillary side. Transport of para-aminohippurate was not different between two cells, and inulin was not transported in this system. The hybrid hollow fiber system can selectively remove a significant amount of drugs that have an affinity to MDR from the medium, and perfuse them to the capillary side in vitro.

Luminal transport step of para-aminohippurate (PAH): transport from PAH-loaded proximal tubular cells into the tubular lumen of the rat kidney in vivo.

Proximal tubular cells were loaded for 10 s with [3H]para-aminohippurate ([3H]PAH) by microperfusing the peritubular capillaries with Ringer solution containing 0.05 mmol/l PAH. Immediately thereafter [3H]PAH influx from cells into a column of equilibrium solution injected into the oil-filled tubular lumen was measured by re-aspirating the fluid after 1-10 s of contact time. The rise of luminal PAH concentration within 2 s of contact time was almost linear, reaching a luminal/capillary concentration ratio of 1.6 after 2 s and of 3.2 after 5 s. The 2-s PAH concentration ratio was not changed when different manoeuvres were applied to depolarize proximal tubular cells. Also, the 2-s PAH concentration ratio was not influenced by varying the luminal pH from 6.0 to 8.0 or the luminal Cl- concentration from zero to 134 mmol/l or when either 5 mmol/l urate or 25 mmol/l lactate was in the luminal perfusate. A decrease in the 2-s PAH concentration ratio, i.e. trans-inhibition, was observed when 25 or 50 mmol/l HCO3- (-50%) was in the luminal perfusate. Trans-inhibition was also seen with 5 mmol/l of the following substituted benzoates: 2-hydroxy-benzoate (-58%), 2-methoxy-benzoate (-46%), 2-hydroxy-benzoate-acetyl ester (-36%), 2-hydroxy-3,5-dinitro-benzoate (-48%), 3,5-dichloro-benzoate (-49%), and 2,3,5-trichloro-benzoate (-45%). No effect was seen with benzoate, 3-hydroxy-benzoate, 2-chloro-benzoate, 2-nitro-benzoate, 2,5-dinitro-benzoate, 3-sulfamoyl-benzoate and 4-sulfamoyl-benzoate. However, analogues of the latter two compounds possessing two additional side groups, such as furosemide and piretanide, or a hydrophobic moiety, such as probenecid, were inhibitory (by -62, -41 and -49% respectively). Phenoxyacetate had no effect; however, it inhibited if in addition it had three chloro groups, as in 2,4,5-trichlorophenoxyacetate (-71%) or a hydrophobic carbamoyl side group, as in mersalylic acid (salyrgan, -75%). Benzene-sulfonate trans-inhibited (-33%), as did phenolsulfonphthalein (phenol red, -39%) and sulfofluorescein (-55%). However, the trans-inhibitory effect of the corresponding carboxy-compounds was absent (phenolphthalein) or weaker (fluorescein, -42%). The trans-inhibitory effect of the uricosurics ethacrynic acid (-53%), tienilic acid (-55%) indacrinone (-72%) and benzbromarone (-42%) could be attributed to two chloro or bromo side groups on the benzene ring. Other trans-inhibiting uricosuric substances were indomethacin (-42%), sulfinpyrazone (-38%), losartan (-80%) its metabolite EXP 3174 (-55%), and AA 193 (-65%). These organic acids, with pKa values between 2.8 and 4.9, possess chloro and sulfin groups, as well as heterocyclic 5-ring and hydrophobic ring or chain areas. No significant effect was seen with 5 mmol/l PAH, 2-oxo-glutarate, DIDS, cGMP, prostaglandin E2, cortisol, benzylamiloride, pyrazinoic acid and 25 mmol/l lactate. Our data indicate that in situ the secretory luminal PAH transport proceeds in a non-rheogenic fashion, per exclusionem by anion exchange. The observed trans-inhibition of PAH secretion seems to correlate with the affinity for the luminal PAH transporter and, for uricosuric substances, with their uricosuric potency.

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.

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.

Differential toxicity as a result of apical and basolateral treatment of LLC-PK1 monolayers with S-(1,2,3,4,4-pentachlorobutadienyl)glutathione and N-acetyl- S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine

Monolayers of LLC-PK1 cells, a cell line with features typical of proximal tubular epithelial cells, were treated at the apical and basolateral side with S-(1,2,3,4,4-pentachlorobutadienyl)glutathione (PCBD-GSH) and N-acetyl- S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBD-NAC). Apical treatment with PCBD-GSH (>20 μM) resulted in cytotoxicity, which could be inhibited by acivicin and aminooxyacetic acid (AOAA), inhibitors of γ-glutamyltranspeptidase (γGT) and β-lyase respectively. In contrast apical treatment with PCBD-NAC was only toxic at high concentrations (>850 μM), and this effect could hardly be inhibited by AOAA. Basolateral treatment of confluent LLC-PK1 monolayers, grown on porous membranes, with PCBD-GSH gave a much smaller response than apical treatment, consistent with the fact that γGT is predominantly present at the apical side. Basolateral treatment even with high concentrations of PCBD-NAC (1.1 mM) did not show an increase in cytotoxicity when compared to the effect after apical treatment. These results suggest the absence of an organic anion transporter, by which these conjugates in vivo are transported into the cells from the basolateral side. This supposition was substantiated in a study of transcellular transport of the model ions tetraethyl ammonium (TEA) and para-aminohippurate (PAH), in LLC-PK1 monolayers, grown as indicated above. No active PAH transport could be demonstrated, whereas an active TEA transport was present. The absence of an organic anion transporter limits the usefulness of LLC-PK1 cells for the study of nephrotoxicity of compounds, like PCBD-NAC, needing this transport to enter the cells. However, the finding of an active basolateral organic cation transporter, together with the presence of γGT, dipeptidase and β-lyase, makes this system especially interesting for testing all compounds that use this transporter or these enzymes in order to elicit toxicity.

Contraluminal para-aminohippurate transport in the proximal tubule of the rat kidney. III. Specificity: monocarboxylic acids.

In order to study the specificity of the contraluminal para-aminohippurate (PAH) transport system, the inhibitory potency of monocarboxylates on the 3H-PAH influx from the interstitium into cortical tubular cells in situ has been determined. The following was found: if a homologous series of fatty acids with increasing chain length is tested, inhibition of contraluminal PAH influx is first seen with valerate (app. Ki 1.4 mmol/l), increasing up to nonanoate (app. Ki 0.06 mmol/l) and remaining in this range up to duodecanoate, the last compound of this series which is sufficiently water-soluble. Similarly, the inhibitory potency of aromatic monocarboxylates increases with increasing hydrophobicity. If the fatty acids are esterified, their inhibitory potency is lost. If they are transformed to the respective aldehydes their inhibitory potency is preserved at a reduced degree. Introduction of a hydrophobic methyl-, ethyl-, or propyl-group increases the inhibitory potency. A beta-, but not an alpha-oxo-group augments the inhibitory potency of phenylpropionate analogs, an OH group diminishes it, and a NH2 group abolishes it. Among phenyl-fatty acids an increase in affinity is observed from phenyl- less than benzoylamine- less than phenoxy- less than benzoyl-acetate and -propionate. All monocarboxylate compounds, so far tested, do not inhibit contraluminal sulfate and Na+/succinate influx. The data indicate that the PAH transporter interacts with monocarboxylates and also with aldehydes which have a hydrophobic moiety. An additional oxo-group facilitates the interaction. Thus, the benzoyl compounds show the highest affinity observed.

Contraluminal para-aminohippurate (PAH) transport in the proximal tubule of the rat kidney. I. Kinetics, influence of cations, anions, and capillary preperfusion.

In order to study the characteristics of contraluminal para-aminohippurate transport into proximal tubular cells the stopped flow capillary perfusion method was applied. The disappearance of 3H-para-aminohippurate from the capillary perfusate at different concentrations and contact times was measured and saturation type behaviour was found with a Km of 0.08 +/- 0.01 (SE) mmol/l, Jmax of 1.1 +/- 0.1 pmol X s-1 X cm-1 and r, the final extracellular/intracellular distribution ratio of 0.93 +/- 0.03. Omission of Na+ from the capillary test perfusate caused a small reduction of contraluminal PAH uptake at small transport rates (0.1 mmol/l PAH in the test perfusate) but not at high transport rates (1.0 mmol/l PAH in the test perfusate). Change of K+ between 0 and 40 mmol/l and pH between 6.0 and 8.0 did not influence contraluminal PAH uptake. Isotonic replacement of chloride by gluconate, nitrate, sulfate, phosphate, methanesulfonate or increase in bicarbonate to 50 mmol/l did not influence PAH uptake at small transport rates. But isotonic sulfate and phosphate, as well as 50 mmol/l HCO3- and 25 mmol/l Hepes in isotonic solutions reduced PAH uptake at high transport rates. Addition of 5 mmol/l Ca2+, Mg2+, Mn2+, Ba2+, Cd2+ to isotonic Na+-gluconate solution did not influence PAH uptake except for Mg2+ and Mn2+ which inhibited uptake at small transport rates only. Preperfusion of the peritubular capillaries with rat serum, Na+ gluconate (Ca2+- + Mg2+-free), Na+ gluconate (Ca2+- + Mg2+-free) plus 10 mmol/l lactate or pyruvate or 0.1 mmol/l 2-oxoglutarate did not influence PAH uptake at small PAH transport rates, but inhibited at high transport rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Contraluminal para-aminohippurate (PAH) transport in the proximal tubule of the rat kidney. II. Specificity: aliphatic dicarboxylic acids.

In order to study the specificity for contraluminal para-aminohippurate (PAH) transport, the inhibitory potency of aliphatic dicarboxylates on 3H-PAH influx, as well as the inhibitory effect on 35SO4(2-)- and 3H-succinate influx, from the interstitium into cortical tubular cells in situ has been determined. The following was found: Testing a homologous series of dicarboxylates--ranging from the 2 C oxalate to the 10 C sebacate--PAH transport was inhibited by succinate (app. Ki 1.35 mmol/l), and all longer dicarboxylates, with high potency (app. Ki 0.05--0.35 mmol/l). Sulfate transport was inhibited only by oxalate (app. Ki 1.1 mmol/l), while dicarboxylate transport was inhibited by succinate, glutarate, adipate and pimelate with decreasing potency (app. Ki 0.04, 0.24, 0.91, 4.0 mmol/l, respectively). PAH transport was inhibited by succinate and glutarate with high potency (app. Ki 1.35 and 0.05 mmol/l), by the correspondent monomethylester to a lesser extent (app. Ki 1.7 and 0.74 mmol/l), but not by the dimethylester. On the other hand, the semialdehyde of succinate with a Ki-value of 1.2 mmol/l, had the same inhibitory potency as succinate itself, while the dialdehyde of glutarate (app. Ki 1.4 mmol/l) was much less potent as glutarate. Introduction of an oxo-, methyl- or sulfhydroxyl-group group onto the 2-position of succinate, or of an oxo-group onto the 2-position of glutarate moderately augmented the inhibitory potency against PAH-uptake. However, introduction of a 2-hydroxy group onto succinate or glutarate in the L-position reduced the inhibitory potency more than in the D-position. Introduction of two methyl-, sulfhydryl- or hydroxyl-groups in the 2-3 position of succinate reduced or abolished its inhibitory potency. The introduction of a 2-amino group onto succinate or glutarate abolished its effect on PAH transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of Para-aminohippurate Transport in the Isolated Perfused Kidney by an Inhibitor of Protein Binding in Uremia

Serum from patients with advanced renal failure contains substances that inhibit binding of small ligands to albumin. The extractable inhibitors of binding to albumin (lx), which were previously shown in rat kidney slices to inhibit para-aminohippurate (PAH) transport, were added to the in vitro perfused rat kidney in an attempt to investigate effects on organic acid transport in the intact organ. Following addition of extract to the perfusate there was an immediate and profound decrease in whole kidney PAH secretion. Mean PAH clearance fell an average of 60% +/- 7% after addition of extract from human uremic pleural exudate and an average of 62% +/- 6% after addition of similarly prepared extract from normal human urine. There was no change in inulin clearance, vascular resistance, urine flow, or fractional reabsorption of sodium. The effect was shown to be as marked when bovine serum albumin (4.0 g/dL) was added to the perfusate, clearances falling 66% +/- 7%. Controls showed no change in PAH or inulin clearance during 90 minutes of in vitro perfusion. An increase in ultraviolet (UV) absorbance by the urine within minutes after addition of inhibitor to the perfusate suggested tubular secretion of a major portion of the extract. Partial recovery of PAH clearance was observed after 20 to 40 minutes in most kidneys and near complete recovery occurred in some kidneys. The inhibitory effect was duplicated by addition of hippurate at 24 mg/dL (1.2 mmol/L) to the perfusate.(ABSTRACT TRUNCATED AT 250 WORDS)