engleski

The expression of organic anion transporter OAT2 in the rat kidney exhibits gender differences due to androgen inhibition and estrogen stimulation

Elimination of various organic anions (OA) in the mammalian liver and kidney is mediated by the members of organic anion transporters (OAT ; subfamily of SLC22 drug transporters) that reside in the epithelial cell basolateral or apical membrane. In humans and experimental animals the rate of excretion of many OA exhibit gender differences, which, at least in rats, rabbits and mice, can be correlated with gender-dependent expression of relevant OATs at the level of mRNAs and/or proteins. OAT2 (SLC22A7) has been identified in rat kidney by Northern blotting, where the expression of its mRNA was: a) 6-8 times higher in females (F) than in males (M), b) upregulated by castration in M, and c) downregulated by testosterone treatment in castrated M. However, the exact localization of the OAT2 protein in the mammalian kidney is not known. In some studies the protein was immunolocalized to the apical membrane of the thick ascending limb of Henle and cortical and medullary collecting ducts in the rat kidney, and to the basolateral membrane of proximal tubules in the human kidney. In this work we re-investigated localization of OAT2 in the rat kidney in more details by using an affinity-purified, polyclonal anti-peptide antibody. The experiments were performed in intact and gonadectomized F and M, and in castrated M rats treated with testosterone (T), estradiol (E) or progesterone (P). Western blot (WB) experiments were performed with brush-border (BBM) and total cell membranes (TCM) isolated from the rat kidney cortex (C) and outer stripe (OS), whereas immunocytochemistry (IC) was performed in tissue cryosections using an antigen retreival technique. Some related studies were performed in kidneys from F and M pigs and mice. By WB in isolated membranes from the rat kidney OS, the antibody labeled a single, immunizing peptide-blocable protein band at ~66 kDa, that was 12 times stronger in F than in M. This was confirmed by IC, where brush-border of the proximal tubule S3 segments in the OS and medullary rays was exclusively and brightly stained in F, whereas no significant staining was observed in M. The density of the specific protein band in TCM from the OS increased by ~110% following castration in M and decreased by ~40% following ovariectomy in F rats, whereas the treatment of castrated M with T, E or P, resulted in decreased (20%) and increased (44% and 36%) abundances of the 66 kDa protein, respectively. However, the overall changes were too small to be visible by IC. Similar gender differences in the expression of OAT2 in the renal OS (F > M) were detected in pig kidney by IC and in mouse kidney by WB and IC. We conclude that OAT2 in the rat (and pig and mouse) kidney is exclusively localized to the brush-border of the proximal tubule S3 segments, where it exhibits strong gender differences (F > M) that are caused by both androgen inhibition and estrogen stimulation.

immunocytochemistry; nephron; sex differences; steroid hormones

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