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The aimof 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 fromkidney cortex revealed that Sglt1-2, Oat1-3, and Oct1 were higher expressed in kidneys of lean females. Oct2 and Mrp2 were higher expressed in obesemales. Renal mRNA levels of transporters were reduced with diabetic nephropathy in females and the expression of transcription factors Hnf1𝛽 andHnf4𝛼 in both sexes.Thehighest 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. Open-Access-Publikationsfonds 2015 peerReviewed

In human kidneys, endogenous and exogenous organic anions (OA) are removed by organic anion transporters (OAT) that belong to the SLC family of transporters and reside in the basolateral membrane (BLM) of epithelial cells along the nephron, pedominantly in proximal tubules. The active contributors to this process are the primary-active sodium-potassium adenosine triphosphatase (Na/K-ATPase), secondary-active sodium-dicarboxylate cotransporter 3 (NaDC3/SLC13A3) and tertiary-active OA exchangers OAT1/SLC22A6, OAT2/SLC22A7, and OAT3/SLC22A8. Several previous studies showed that the expression of renal OAT1-3 was sex-dependent in rodents (rats and mice), but similar investigations in human kidneys have not been reported. Accordingly, here we studied the expression and distribution of various BLM transporters that contribute to OA secretion along the human nephron by immunofluorescence cytochemistry (IC) in tissue cryosections and by Western blotting (WB) of total cell membranes (TCM) isolated from the kidneys of men (M ; n = 7 ) and women (W ; n = 9). Virtually healthy kidney tissue was obtained following surgical removal of the carcinoma-diseased organs. Specificity of the used polyclonal antibodies for human NaDC3 and OAT1-3 was confirmed in the transporters-transfected HEK293 cells by IC and WB, whereas the proper transfection was validated in uptake experiments with radiolabeled succinate (NaDC3), p-aminohippurate (OAT1), cGMP (OAT2) and estrone-3-sulfate (OAT3). In IC studies, in human kidneys we confirmed colocalization of Na/K-ATPase, NaDC3 and OAT1-3 in the proximal tubule BLM, whereas Na/K-ATPase and NaDC3 were also colocalized in the BLM of collecting ducts. In WB of TCM, the protein bands of ~85, ~80, ~75 and ~110 kDa were detected for OAT1, OAT2, OAT3 and Na/K-ATPase, respectively, whereas two protein bands of 70 and 90 kDa were detected for NaDC3. The density of protein bands in WB studies, and the staining intensity of various transporters in IC studies were similar in M and W kidneys. Therefore, various primary-, secondary- and tertiary-active transporters that contribute to OA secretion along the human nephron exhibit fair colocalization in the proximal tubules, but in more distal nephron segments, the co-expression is restricted to Na/K-ATPase and NaDC3. Sex differences in the expression of tested transporters were not observed, thus indicating that the sex-dependent expression of some renal transporters is species-related.

Background: The epidemiological incidence of oxalate nephrolithiasis is higher in men than in women. Oxalate is predominantly produced in the liver and then released into the systemic circulation by sat1, a sulfate anion transporter, which is localized in the sinusoidal membrane of hepatocytes, where it mediates exit of oxalate in exchange for sulfate. In renal proximal tubules, sat1 is responsible for basolateral uptake of oxalate into the cell and release of sulfate into systemic circulation. Finally, oxalate is extruded into the urine by the luminal chloride-formate exchanger, CFEX, which also accepts oxalate as a substrate. Methods: The model of ethylene glycol (EG)-induced oxalate nephrolithiasis in adult male and female rats was used to monitor changes in sat1, CFEX, and the rate-limiting enzymes of oxalate synthesis, alcohol dehydrogenase (Adh1) and hydroxy acid oxidase (Hao1). Protein and mRNA expression was studied by immunochemistry and real time RT-PCR. Results: As compared to controls, EG-treated animals exhibited higher concentrations of oxalate in plasma and urine, and a higher abundance of oxalate crystals in urine with male dominance. Sat1 protein in liver and kidneys was male-dominant in controls, increased only in EG-treated female rats, while sat1 mRNA stayed unchanged. CFEX mRNA in both organs was sex-independent and unaffected by EG treatment. Adh1 and Hao1 mRNA in both organs exhibited distinct sex dependency which remained unchanged upon EG treatment. A general male-dominant Hao1 expression in kidneys of untreated rats was also shown by microarray analysis. Conclusions: In conclusion, despite hyperoxaluria in EG-treated animals, the expression of sat1 in males and of CFEX in both sexes was sufficient to handle the EG-induced production and secretion of oxalate.

BACKGROUND: Organic anion transporters 1 (Oat1) and 3 (Oat3) mediate the transport of organic anions, including frequently prescribed drugs, across cell membranes in kidney proximal tubule cells. In rats, these transporters are known to be male-dominant and testosterone-dependently expressed. The molecular mechanisms that are involved in the sex-dependent expression are unknown. Our aim was to identify genes that show a sex-dependent expression and could be involved in male-dominant regulation of Oat1 and Oat3. METHODOLOGY/PRINCIPAL FINDINGS: Promoter activities of Oat1 and Oat3 were analyzed using luciferase assays. Expression profiling was done using a SurePrint G3 rat GE 8 × 60K microarray. RNA was isolated from renal cortical slices of four adult rats per sex. To filter the achieved microarray data for genes expressed in proximal tubule cells, transcription database alignment was carried out. We demonstrate that predicted androgen response elements in the promoters of Oat1 and Oat3 are not functional when the promoters were expressed in OK cells. Using microarray analyses we analyzed 17,406 different genes. Out of these genes, 56 exhibit a sex-dependent expression in rat proximal tubule cells. As genes potentially involved in the regulation of Oat1 and Oat3 expression, we identified, amongst others, the male-dominant hydroxysteroid (17-beta) dehydrogenase 1 (Hsd17b1), B-cell CLL/lymphoma 6 (BCL6), and polymerase (RNA) III (DNA directed) polypeptide G (Polr3g). Moreover, our results revealed that the transcription factor BCL6 activates promoter constructs of Oat1 and Oat3. CONCLUSION: The results indicate that the male-dominant expression of both transporters, Oat1 and Oat3, is possibly not directly regulated by the classical androgen receptor mediated transcriptional pathway but appears to be regulated by the transcription factor BCL6.

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