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Revised immunolocalization of Oat3 in mouse kidney ; Different sex-dependent expression of renal mOat3 and mOat1 proteins

Background: Previous immunocytochemical (IC) studies in mouse kidney have localized the organic anion transporter 3 (mOat3, Slc22a8) to the basolateral membrane (BLM) of proximal tubule (PT), thick ascending limb of Henle, cortical collecting duct, distal tubule, and macula densa (1, 2). However, the specificity of Oat3 antibodies (Ab), used in these studies, has not been properly verified, and the exact renal localization of mOat3 is still controversial. Furthermore, at the level of mRNA, the expression of renal mOat3 was reported to be higher in females (F) than in males (M), whereas the expression of functionally-similar transporter, mOat1 (Slc22a6), was M>F (3). However, at the protein level, the sex-dependent expression of these two transporters is unknown. The aim of this study was to: a) immunolocalize mOat3 in the mouse kidney by using the Oat3 knock-out (Oat3 KO) mice model, b) demonstrate the sex-dependent expression of renal mOat3 and mOat1 at the protein level in wild-type (WT) mice, and c) define the hormone(s) responsible for these sex differences. Methods: Studies were performed in the kidneys of adult M and F WT and Oat3 KO mice. mOat3 expression was studied using polyclonal antibodies specific for the rat (rOat3-Ab) and mouse (mOat3-Ab) proteins, whereas mOat1 expression was studied with an antibody specific for the rat protein (rOat1-Ab). Studied was: a) localization of mOat3 and mOat1 proteins by IC in tissue cryosections, b) abundance of mOat3 and mOat1 proteins by Western blotting (WB) in total cell membranes (TCM) isolated from the whole organs, and c) effect of castration in M mice on the mOat3 and mOat1 protein expression by IC and WB. Results: In accordance with previously-reported data, in WT mice, rOat3-Ab stained the BLM of PT and other nephron segments. However, the same staining was also noted along the nephron of Oat3 KO mice. On the contrary, mOat3-Ab exclusively stained the BLM of S1 and S2 (S1<<S2) segments of PT in the cortex, and weakly S3 in the medullary rays (S3 in the outer stripe and other nephron segments were negative) in WT mice, whereas no staining was noted in Oat3 KO mice. The latter data were confirmed by the absence of mOat3-related protein band of ~70 kDa in TCM from the Oat3 KO mice. The sex-dependent expression of mOat3 and mOat1 proteins was demonstrated with an opposite pattern: the mOat3-related protein band of ~70 kDa and the immunostaining intensity were in F>M, and were up-regulated by castration, whereas the mOat1-related protein band of ~75 kDa and the immunostaining intensity were in M>F, and were down-regulated by castration. Conclusion: The results indicate that: a) rOat3-Ab is not specific for mouse tissues, b) in the mouse kidney, mOat3 is exclusively localized to the BLM of PT in the cortex, c) mOat3 and mOat1 proteins co-localize in the BLM of PT, exhibiting the sex-dependent expression with an opposite pattern ; mOat3 expression is F-dominant due to androgen inhibition, while mOat1 expression is M-dominant due to androgen stimulation. References: 1. Bahn A et al. (2005) Am J Physiol Cell Physiol 289: C1075-C1084 2. Nilwarangkoon S et al. (2007) J Phamacol Sci 103:48-55 3. VanWert AL et al. (2007) Am J Physiol Renal Physiol 293: F1332-F1341 Supported by grant No. 022-0222148-2146 (S.I.).

Kidney; Gender differences; Membrane transporters; Organic anions; Proximal tubule

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