Sex-independent expression of chloride/formate exchanger Cfex (Slc26a6) in rat pancreas, small intestine, and liver, and male-dominant expression in kidneys.

Chloride/formate exchanger (CFEX; SLC26A6) mediates oxalate transport in various mammalian organs. Studies in Cfex knockout mice indicated its possible role in development of male-dominant hyperoxaluria and oxalate urolithiasis. Rats provide an important model for studying this pathophysiological condition, but data on Cfex (rCfex) localisation and regulation in their organs are limited. Here we applied the RT-PCR and immunochemical methods to investigate rCfex mRNA and protein expression and regulation by sex hormones in the pancreas, small intestine, liver, and kidneys from intact prepubertal and adult as well as gonadectomised adult rats treated with sex hormones. rCfex cDNA-transfected HEK293 cells were used to confirm the specificity of the commercial anti-CFEX antibody. Various biochemical parameters were measured in 24-h urine collected in metabolic cages. rCfex mRNA and related protein expression varied in all tested organs. Sex-independent expression of the rCfex protein was detected in pancreatic intercalated ducts (apical domain), small intestinal enterocytes (brush-border membrane; duodenum > jejunum > ileum), and hepatocytes (canalicular membrane). In kidneys, the rCfex protein was immunolocalised to the proximal tubule brush-border with segment-specific pattern (S1=S2

Interaction of Excitatory Amino Acid Transporters 1 - 3 (EAAT1, EAAT2, EAAT3) with N-Carbamoylglutamate and N-Acetylglutamate.

BACKGROUND/AIMS: Inborn deficiency of the N-acetylglutamate synthase (NAGS) impairs the urea cycle and causes neurotoxic hyperammonemia. Oral administration of N-carbamoylglutamate (NCG), a synthetic analog of N-acetylglutamate (NAG), successfully decreases plasma ammonia levels in the affected children. Due to structural similarities to glutamate, NCG may be absorbed in the intestine and taken up into the liver by excitatory amino acid transporters (EAATs). METHODS: Using Xenopus laevis oocytes expressing either human EAAT1, 2, or 3, or human sodium-dependent dicarboxylate transporter 3 (NaDC3), transport-associated currents of NAG, NCG, and related dicarboxylates were assayed. RESULTS: L-aspartate and L-glutamate produced saturable inward currents with Km values below 30 µM. Whereas NCG induced a small inward current only in EAAT3 expressing oocytes, NAG was accepted by all EAATs. With EAAT3, the NAG-induced current was sodium-dependent and saturable (Km 409 µM). Oxaloacetate was found as an additional substrate of EAAT3. In NaDC3-expressing oocytes, all dicarboxylates induced much larger inward currents than did L-aspartate and L-glutamate. CONCLUSION: EAAT3 may contribute to intestinal absorption and hepatic uptake of NCG. With respect to transport of amino acids and dicarboxylates, EAAT3 and NaDC3 can complement each other.

Differential Interaction of Dantrolene, Glafenine, Nalidixic Acid, and Prazosin with Human Organic Anion Transporters 1 and 3.

In renal proximal tubule cells, the organic anion transporters 1 and 3 (OAT1 and OAT3) in the basolateral membrane and the multidrug resistance-associated protein 4 (MRP4) in the apical membrane share substrates and co-operate in renal drug secretion. We hypothesized that recently identified MRP4 inhibitors dantrolene, glafenine, nalidixic acid, and prazosin also interact with human OAT1 and/or OAT3 stably transfected in human embryonic kidney 293 cells. These four drugs were tested as possible inhibitors of p-[3H]aminohippurate (PAH) and [14C]glutarate uptake by OAT1, and of [3H]estrone-3-sulfate (ES) uptake by OAT3. In addition, we explored whether these drugs decrease the equilibrium distribution of radiolabeled PAH, glutarate, or ES, an approach intended to indirectly suggest drug/substrate exchange through OAT1 and OAT3. With OAT3, a dose-dependent inhibition of [3H]ES uptake and a downward shift in [3H]ES equilibrium were observed, indicating that all four drugs bind to OAT3 and may possibly be translocated. In contrast, the interaction with OAT1 was more complex. With [14C]glutarate as substrate, all four drugs inhibited uptake but only glafenine and nalidixic acid shifted glutarate equilibrium. Using [3H]PAH as a substrate of OAT1, nalidixic acid inhibited but dantrolene, glafenine, and prazosin stimulated uptake. Nalidixic acid decreased equilibrium content of [3H]PAH, suggesting that it may possibly be exchanged by OAT1. Taken together, OAT1 and OAT3 interact with the MRP4 inhibitors dantrolene, glafenine, nalidixic acid, and prazosin, indicating overlapping specificities. At OAT1, more than one binding site must be assumed to explain substrate and drug-dependent stimulation and inhibition of transport activity.

Counter-flow suggests transport of dantrolene and 5-OH dantrolene by the organic anion transporters 2 (OAT2) and 3 (OAT3).

Dantrolene is the only available drug for the treatment of malignant hyperthermia, a life-threatening inborn sensitivity of the ryanodine receptor (RyR1) in skeletal muscles to volatile anesthetics. Dantrolene is metabolized in the liver to 5-OH dantrolene. Both compounds are zwitterions or net negatively charged. Here, we investigated interactions of dantrolene and 5-OH dantrolene with solute carrier (SLC) family members occurring in skeletal muscle cells, hepatocytes, and renal proximal tubule cells. SLC22A8 (organic anion transporter 3, OAT3) was very sensitive to both compounds exhibiting IC50 values of 0.35 ± 0.03 and 1.84 ± 0.34 µM, respectively. These IC50 concentrations are well below the plasma concentration in patients treated with dantrolene (3-28 µM). SLC22A7 (OAT2) was less sensitive to dantrolene and 5-OH dantrolene with IC50 values of 15.6 ± 2.1 and 15.8 ± 3.2 µM, respectively. SLCO1B1 (OATP1B1), SLCO1B3 (OATP1B3), and SLCO2B1 (OATP2B1) mainly interacted with 5-OH dantrolene albeit with higher IC50 values than those observed for OAT2 and OAT3. Dantrolene and 5-OH dantrolene failed to inhibit uptake of 1-methyl-4-phenylpyrimidinium (MPP) by OCT1 and of carnitine by OCTN2. In counter-flow experiments on OAT3, dantrolene and 5-OH dantrolene decreased pre-equilibrated cellular [3H]estrone-3-sulfate (ES) content as did the transported substrates glutarate, furosemide, and bumetanide. With OAT2, dantrolene and 5-OH dantrolene slightly decreased the pre-equilibrated [3H]cGMP content. If no other transporter markedly contributes to uptake or release of ES or cGMP, respectively, these data suggest that OAT3 and OAT2 may be involved in absorption, metabolism, and excretion of dantrolene and its metabolite 5-OH dantrolene.

Distribution of organic anion transporters NaDC3 and OAT1-3 along the human nephron.

The initial step in renal secretion of organic anions (OAs) is mediated by transporters in the basolateral membrane (BLM). Contributors to this process are primary active Na(+)-K(+)-ATPase (EC 3.6.3.9), secondary active Na(+)-dicarboxylate cotransporter 3 (NaDC3/SLC13A3), and tertiary active OA transporters (OATs) OAT1/SLC22A6, OAT2/SLC22A7, and OAT3/SLC22A8. In human kidneys, we analyzed the localization of these transporters by immunochemical methods in tissue cryosections and isolated membranes. The specificity of antibodies was validated with human embryonic kidney-293 cells stably transfected with functional OATs. Na(+)-K(+)-ATPase was immunolocalized to the BLM along the entire human nephron. NaDC3-related immunostaining was detected in the BLM of proximal tubules and in the BLM and/or luminal membrane of principal cells in connecting segments and collecting ducts. The thin and thick ascending limbs, macula densa, and distal tubules exhibited no reactivity with the anti-NaDC3 antibody. OAT1-OAT3-related immunostaining in human kidneys was detected only in the BLM of cortical proximal tubules; all three OATs were stained more intensely in S1/S2 segments compared with S3 segment in medullary rays, whereas the S3 segment in the outer stripe remained unstained. Expression of NaDC3, OAT1, OAT2, and OAT3 proteins exhibited considerable interindividual variability in both male and female kidneys, and sex differences in their expression could not be detected. Our experiments provide a side-by-side comparison of basolateral transporters cooperating in renal OA secretion in the human kidney.

The Isoquinolone Derived Prolyl Hydroxylase Inhibitor ICA Is a Potent Substrate of the Organic Anion Transporters 1 and 3.

OBJECTIVE: Many cellular responses to hypoxia are mediated by the transcription factor complex hypoxia-inducible factor (HIF). HIF stability is governed by a family of dioxygenases called HIF prolyl hydroxylases (PHDs). Isoquinolone-derived PHD inhibitors, like 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA), which stabilize the intracellular HIF-α have been suggested as a potentially beneficial therapeutic strategy for the treatment of disorders associated with ischemia. To stabilize HIF-α, ICA has to be taken up into proximal tubule cells (PCTs) across the basolateral membrane by one of the organic anion transporters 1, 2 or 3 (OAT1, OAT2 or OAT3). The release into the urine across the luminal membrane may be mediated by OAT4. METHOD: To demonstrate interaction of ICA with human OAT1, OAT2, OAT3 and OAT4, ICA was tested on these transporters stably transfected in HEK293 cells by using p-aminohippurate (PAH), cGMP and estrone-3-sulfate (ES) as reference substrates, respectively. RESULTS: Uptakes of PAH and ES in OAT1- and OAT3-transfected HEK293 cells were inhibited by ICA with half-maximal inhibition values of 0.29 ± 0.05 and 2.58 ± 0.16 µM, respectively. OAT2 was less sensitive to ICA. Efflux experiments identified ICA as an OAT1 and OAT3 substrate. Preloading OAT4-transfected HEK293 cells with ICA stimulated ES uptake by 18.3 ± 3.8%. CONCLUSION: The uptake of ICA across the basolateral membrane of PCTs occurs mainly by OAT1 and the efflux into the tubular lumen by OAT4.

In female rats, ethylene glycol treatment elevates protein expression of hepatic and renal oxalate transporter sat-1 (Slc26a1) without inducing hyperoxaluria.

AIM: To investigate whether the sex-dependent expression of hepatic and renal oxalate transporter sat-1 (Slc26a1) changes in a rat model of ethylene glycol (EG)-induced hyperoxaluria. METHODS: Rats were given tap water (12 males and 12 females; controls) or EG (12 males and 12 females; 0.75% v/v in tap water) for one month. Oxaluric state was confirmed by biochemical parameters in blood plasma, urine, and tissues. Expression of sat-1 and rate-limiting enzymes of oxalate synthesis, alcohol dehydrogenase 1 (Adh1) and hydroxy-acid oxidase 1 (Hao1), was determined by immunocytochemistry (protein) and/or real time reverse transcription polymerase chain reaction (mRNA). RESULTS: EG-treated males had significantly higher (in µmol/L; mean±standard deviation) plasma (59.7±27.2 vs 12.9±4.1, P<0.001) and urine (3716±1726 vs 241±204, P<0.001) oxalate levels, and more abundant oxalate crystaluria than controls, while the liver and kidney sat-1 protein and mRNA expression did not differ significantly between these groups. EG-treated females, in comparison with controls had significantly higher (in µmol/L) serum oxalate levels (18.8±2.9 vs 11.6±4.9, P<0.001), unchanged urine oxalate levels, low oxalate crystaluria, and significantly higher expression (in relative fluorescence units) of the liver (1.59±0.61 vs 0.56±0.39, P=0.006) and kidney (1.77±0.42 vs 0.69±0.27, P<0.001) sat-1 protein, but not mRNA. The mRNA expression of Adh1 was female-dominant and that of Hao1 male-dominant, but both were unaffected by EG treatment. CONCLUSIONS: An increased expression of hepatic and renal oxalate transporting protein sat-1 in EG-treated female rats could protect from hyperoxaluria and oxalate urolithiasis.

Role of N-glycosylation in renal betaine transport.

The osmolyte and folding chaperone betaine is transported by the renal Na(+)-coupled GABA (γ-aminobutyric acid) symporter BGT-1 (betaine/GABA transporter 1), a member of the SLC6 (solute carrier 6) family. Under hypertonic conditions, the transcription, translation and plasma membrane (PM) insertion of BGT-1 in kidney cells are significantly increased, resulting in elevated betaine and GABA transport. Re-establishing isotonicity involves PM depletion of BGT-1. The molecular mechanism of the regulated PM insertion of BGT-1 during changes in osmotic stress is unknown. In the present study, we reveal a link between regulated PM insertion and N-glycosylation. Based on homology modelling, we identified two sites (Asn(171) and Asn(183)) in the extracellular loop 2 (EL2) of BGT-1, which were investigated with respect to trafficking, insertion and transport by immunogold-labelling, electron microscopy (EM), mutagenesis and two-electrode voltage clamp measurements in Xenopus laevis oocytes and uptake of radiolabelled substrate into MDCK (Madin-Darby canine kidney) and HEK293 (human embryonic kidney) cells. Trafficking and PM insertion of BGT-1 was clearly promoted by N-glycosylation in both oocytes and MDCK cells. Moreover, association with N-glycans at Asn(171) and Asn(183) contributed equally to protein activity and substrate affinity. Substitution of Asn(171) and Asn(183) by aspartate individually caused no loss of BGT-1 activity, whereas the double mutant was inactive, suggesting that N-glycosylation of at least one of the sites is required for function. Substitution by alanine or valine at either site caused a dramatic loss in transport activity. Furthermore, in MDCK cells PM insertion of N183D was no longer regulated by osmotic stress, highlighting the impact of N-glycosylation in regulation of this SLC6 transporter.

Human organic anion transporter 2 is distinct from organic anion transporters 1 and 3 with respect to transport function.

Phylogentically, organic anion transporter (OAT)1 and OAT3 are closely related, whereas OAT2 is more distant. Experiments with human embryonic kidney-293 cells stably transfected with human OAT1, OAT2, or OAT3 were performed to compare selected transport properties. Common to OAT1, OAT2, and OAT3 is their ability to transport cGMP. OAT2 interacted with prostaglandins, and cGMP uptake was inhibited by PGE2 and PGF2α with IC50 values of 40.8 and 12.7 µM, respectively. OAT1 (IC50: 23.7 µM), OAT2 (IC50: 9.5 µM), and OAT3 (IC50: 1.6 µM) were potently inhibited by MK571, an established multidrug resistance protein inhibitor. OAT2-mediated cGMP uptake was not inhibited by short-chain monocarboxylates and, as opposed to OAT1 and OAT3, not by dicarboxylates. Consequently, OAT2 showed no cGMP/glutarate exchange. OAT1 and OAT3 exhibited a pH and a Cl- dependence with higher substrate uptake at acidic pH and lower substrate uptake in the absence of Cl-, respectively. Such pH and Cl- dependencies were not observed with OAT2. Depolarization of membrane potential by high K+ concentrations in the presence of the K+ ionophore valinomycin left cGMP uptake unaffected. In addition to cGMP, OAT2 transported urate and glutamate, but cGMP/glutamate exchange could not be demonstrated. These experiments suggest that OAT2-mediated cGMP uptake does not occur via exchange with monocarboxylates, dicarboxylates, and hydroxyl ions. The counter anion for electroneutral cGMP uptake remains to be identified.

Epithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis.

Kidney fibrosis is marked by an epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells (TECs). Here we find that, during renal fibrosis, TECs acquire a partial EMT program during which they remain associated with their basement membrane and express markers of both epithelial and mesenchymal cells. The functional consequence of the EMT program during fibrotic injury is an arrest in the G2 phase of the cell cycle and lower expression of several solute and solvent transporters in TECs. We also found that transgenic expression of either Twist1 (encoding twist family bHLH transcription factor 1, known as Twist) or Snai1 (encoding snail family zinc finger 1, known as Snail) expression is sufficient to promote prolonged TGF-ß1-induced G2 arrest of TECs, limiting the cells' potential for repair and regeneration. In mouse models of experimentally induced renal fibrosis, conditional deletion of Twist1 or Snai1 in proximal TECs resulted in inhibition of the EMT program and the maintenance of TEC integrity, while also restoring cell proliferation, dedifferentiation-associated repair and regeneration of the kidney parenchyma and attenuating interstitial fibrosis. Thus, inhibition of the EMT program in TECs during chronic renal injury represents a potential anti-fibrosis therapy.

Next generation sequencing of sex-specific genes in the livers of obese ZSF1 rats.

Type 2 diabetes induces pathophysiological changes in the liver. The aim of this study was to identify differently expressed genes in the livers of male and female ZSF1 rats (ZDFxSHHF-hybrid, generation F1), a model for type 2 diabetes. Gene expression was investigated using next-generation sequencing (NGS). Selected candidate genes were verified by real-time PCR in the livers of obese and lean rats. 103 sex-different genes, associated to pathways "response to chemical stimulus", "lipid metabolism", and "response to organic substance", were identified. Male-specific genes were involved in hepatic metabolism, detoxification, and secretion, e.g. cytochrome P450 2c11 (Cyp2c11), Cyp4a2, glutathione S-transferases mu 2 (Gstm2), and Slc22a8 (organic anion transporter 3, Oat3). Most female-specific genes were associated to lipid metabolism (e.g. glycerol-3-phosphate acyltransferase 1, Gpam) or glycolysis (e.g. glucokinase, Gck). Our data suggest the necessity to pay attention to sex- and diabetes-dependent changes in pre-clinical testing of hepatic metabolized and secreted drugs.

Sex-differences in renal expression of selected transporters and transcription factors in lean and obese Zucker spontaneously hypertensive fatty rats.

The aim of this study was to identify sex-dependent expression of renal transporter mRNA in lean and obese Zucker spontaneously hypertensive fatty (ZSF1) rats and to investigate the interaction of the most altered transporter, organic anion transporter 2 (Oat2), with diabetes-relevant metabolites and drugs. Higher incidence of glomerulosclerosis, tubulointerstitial fibrosis, and protein casts in Bowman's space and tubular lumen was detected by PAS staining in obese male compared to female ZSF1 rats. Real-time PCR on RNA isolated from kidney cortex revealed that Sglt1-2, Oat1-3, and Oct1 were higher expressed in kidneys of lean females. Oct2 and Mrp2 were higher expressed in obese males. Renal mRNA levels of transporters were reduced with diabetic nephropathy in females and the expression of transcription factors Hnf1ß and Hnf4α in both sexes. The highest difference between lean and obese ZSF1 rats was found for Oat2. Therefore, we have tested the interaction of human OAT2 with various substances using tritium-labeled cGMP. Human OAT2 showed no interaction with diabetes-related metabolites, diabetic drugs, and ACE-inhibitors. However, OAT2-dependent uptake of cGMP was inhibited by furosemide. The strongly decreased expression of Oat2 and other transporters in female diabetic ZSF1 rats could possibly impair renal drug excretion, for example, of furosemide.

Transporters involved in renal excretion of N-carbamoylglutamate, an orphan drug to treat inborn n-acetylglutamate synthase deficiency.

Inborn defects in N-acetylglutamate (NAG) synthase (NAGS) cause a reduction of NAG, an essential cofactor for the initiation of the urea cycle. As a consequence, blood ammonium concentrations are elevated, leading to severe neurological disorders. The orphan drug N-carbamoylglutamate (NCG; Carbaglu), efficiently overcomes NAGS deficiency. However, not much is known about the transporters involved in the uptake, distribution, and elimination of the divalent organic anion NCG. Organic anion-transporting polypeptides (OATPs) as well as organic anion transporters (OATs) working in cooperation with sodium dicarboxylate cotransporter 3 (NaDC3) accept a wide variety of structurally unrelated drugs. To test for possible interactions with OATPs and OATs, the impact of NCG on these transporters in stably transfected human embryonic kidney-293 cells was measured. The two-electrode voltage-clamp technique was used to monitor NCG-mediated currents in Xenopus laevis oocytes that expressed NaDC3. Neither OATPs nor OAT2 and OAT3 interacted with NCG, but OAT1 transported NCG. In addition, NCG was identified as a high-affinity substrate of NaDC3. Preincubation of OAT4-transfected human embryonic kidney-293 cells with NCG showed an increased uptake of estrone sulfate, the reference substrate of OAT4, indicating efflux of NCG by OAT4. In summary, NaDC3 and, to a lesser extent, OAT1 are likely to be responsible for the uptake of NCG from the blood. Efflux of NCG across the luminal membrane into the tubular lumen probably occurs by OAT4 completing renal secretion of this drug.

Mutation of a single threonine in the cytoplasmic NH2 terminus disrupts trafficking of renal betaine-GABA transporter 1 during hypertonic stress.

Betaine is an important osmolyte and is, compared with other organs, much more abundant in the kidneys, where it enters cells in the medulla by betaine-GABA transporter 1 (BGT1) to balance osmoregulation in the countercurrent system. In wild-type (wt-)BGT1-expressing oocytes, GABA-mediated currents were diminished by preincubation of oocytes with 100 nM PMA or 5 µM dioctanoyl-sn-glycerol, activators of PKC, whereas the application of staurosporine before the application of dioctanoyl-sn-glycerol restored the response to GABA. Four potential phosphorylation sites on BGT1 were mutated to alanine by site-directed mutagenesis. Three mutants (T235A, S428A, and S564A) evoked GABA currents comparable in magnitude to currents observed in wt-BGT1-expressing oocytes, whereas GABA currents in T40A were barely detectable. Uptake of [(3)H]GABA was also determined in human embryonic kidney-293 cells expressing enhanced green fluorescent protein (EGFP)-tagged BGT1 with the same mutations. T235A, S428A, and S564A showed upregulation of GABA uptake after hypertonic stress and downregulation by PMA similar to EGFP-wt-BGT1. In contrast, T40A did not respond to either hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in Madin-Darby canine kidney cells revealed that T40A was present in the cytoplasm after 24 h of hypertonic stress. whereas the other mutants and EGFP-wt-BGT1 were in the plasma membrane. All mutants, including T40A, comigrated with wt-BGT1 on Western blots, suggesting that they are full-length proteins. T40A, however, cannot be phosphorylated, as revealed using a specific anti-phosphoantibody, and, therefore, T40 may be important for the trafficking and insertion of BGT1 in the plasma membrane.

Glutathione is a low-affinity substrate of the human sodium-dependent dicarboxylate transporter.

BACKGROUND/AIMS: During a single pass through the kidneys, more than 80% of glutathione (GSH) is excreted, indicating not only glomerular filtration, but also tubular secretion. The first step in tubular secretion is the uptake of a substance across the basolateral membrane of proximal tubule cells by sodium-dependent and -independent transporters. Due to the dicarboxylate-like structure, we postulated that GSH uptake across the basolateral membrane is mediated by the sodium-dependent dicarboxylate transporter 3 (NaDC3). METHODS: Tracer uptake and electrophysiologic measurements using a two-electrode voltage clamp device were performed in Xenopus laevis oocytes expressing the human (h)NaDC3. RESULTS: Uptake of succinate, the reference substrate of hNaDC3, was inhibited by GSH in a dose-dependent manner with an IC50 of 1.88 mM. GSH evoked potential-dependent inward currents, which were abolished under sodium-free conditions. At -60 mV, GSH currents showed saturation kinetics with a KM of 1.65 mM. CONCLUSION: hNaDC3 present at the basolateral membrane of proximal tubule cells mediates sodium-dependent GSH uptake. The kinetic data show that NaDC3 is a low-affinity GSH transporter.

Oxalate: from the environment to kidney stones.

Oxalate urolithiasis (nephrolithiasis) is the most frequent type of kidney stone disease. Epidemiological research has shown that urolithiasis is approximately twice as common in men as in women, but the underlying mechanism of this sex-related prevalence is unclear. Oxalate in the organism partially originate from food (exogenous oxalate) and largely as a metabolic end-product from numerous precursors generated mainly in the liver (endogenous oxalate). Oxalate concentrations in plasma and urine can be modified by various foodstuffs, which can interact in positively or negatively by affecting oxalate absorption, excretion, and/or its metabolic pathways. Oxalate is mostly removed from blood by kidneys and partially via bile and intestinal excretion. In the kidneys, after reaching certain conditions, such as high tubular concentration and damaged integrity of the tubule epithelium, oxalate can precipitate and initiate the formation of stones. Recent studies have indicated the importance of the SoLute Carrier 26 (SLC26) family of membrane transporters for handling oxalate. Two members of this family [Sulfate Anion Transporter 1 (SAT-1; SLC26A1) and Chloride/Formate EXchanger (CFEX; SLC26A6)] may contribute to oxalate transport in the intestine, liver, and kidneys. Malfunction or absence of SAT-1 or CFEX has been associated with hyperoxaluria and urolithiasis. However, numerous questions regarding their roles in oxalate transport in the respective organs and male-prevalent urolithiasis, as well as the role of sex hormones in the expression of these transporters at the level of mRNA and protein, still remain to be answered.

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.

Selective stabilization of HIF-1α in renal tubular cells by 2-oxoglutarate analogues.

The role of proximal versus distal tubular injury in the pathogenesis of acute kidney injury (AKI) is debatable. Inhibition of prolyl hydroxylases that regulate the degradation of hypoxia-inducible transcription factors (HIFs) is a promising therapeutic approach to optimize energy preservation under hypoxia and has successfully been applied to protect kidney structure and function in AKI models. Presently used prolyl hydroxylase inhibitors are lipophilic 2-oxoglutarate analogues (2OGAs) that are widely taken up in cells of most organs. Given the selective expression of organic anion transporters (OATs) in renal proximal tubular cells, we hypothesized that hydrophilic 2OGAs can specifically target proximal tubular cells. We found that cellular hydrophilic 2OGAs uptake depended on OATs and largely confined to the kidney, where it resulted in activation of HIF target genes only in proximal tubular cells. When applied in ischemia-reperfusion experiments, systemically active 2OGA preserved kidney structure and function, but OAT1-transported 2OGA was not protective, suggesting that HIF stabilization in distal tubular rather than proximal tubular cells and/or nontubular cells mediates protective effects. This study provides proof of concept for selective drug targeting of proximal tubular cells on the basis of specific transporters, gives insights into the role of different nephron segments in AKI pathophysiology, and may offer options for long-term HIF stabilization in proximal tubules without confounding effects of erythropoietin induction in peritubular cells and unwarranted extrarenal effects.

α-Ketoglutarate-related inhibitors of HIF prolyl hydroxylases are substrates of renal organic anion transporters 1 (OAT1) and 4 (OAT4).

2-Oxoglutarate or α-ketoglutarate (αKG) is a substrate of HIF prolyl hydroxylases 1-3 that decrease cellular levels of the hypoxia-inducible factor 1α (HIF-1α) in the presence of oxygen. αKG analogs are applied to stabilize HIF-1α even in the presence of oxygen and thus provide a novel therapeutic option in treating kidney diseases. In the kidneys, the organic anion transporters 1 and 3 (OAT1 and OAT3, respectively) in cooperation with the sodium-dependent dicarboxylate transporter 3 (NaDC3) and the OAT4 might be responsible for the uptake of αKG analogs into and the efflux out of the tubular cells. Using the radiolabelled substrates p-aminohippurate (PAH, OAT1), estrone-3-sulfate (ES; OAT3, OAT4), and succinate (NaDC3), N-oxalylglycine (NOG), dimethyloxalyl glycine (DMOG), 2,4-diethylpyridine dicarboxylate (2,4-DPD), and pyridine-2,4-dicarboxylic acid (PDCA) were tested in cis-inhibition and trans-stimulation experiments. None of these αKG analogs interacted with NaDC3. 2,4-DPD and PDCA inhibited ES uptake by OAT3 moderately. NOG, 2,4-DPD and PDCA, but not DMOG, inhibited PAH uptake by OAT1 significantly. trans-Stimulation experiments and experiments demonstrating stabilization of HIF-1α revealed that NOG and PDCA, but not 2,4-DPD, are translocated by OAT1. All compounds trans-stimulated ES uptake by OAT4, but only PDCA stabilized HIF-1α. The data suggest that OAT1 is involved in the uptake of NOG and PDCA across the basolateral membrane of proximal tubule cells, whereas OAT4 may release these compounds into the primary urine.