engleski

New insight in cell localization of Oat3 in the mouse kidney : different sex-dependent expression of Oat3 and Oat1

Immunolocalization of the organic anion transporter 3 in the mouse kidney (mOAT3, Slc22a8) was previously studied with antibodies against the rat (rOAT3-Ab) and mouse (mOAT3-Ab) proteins, and detected in both species in the basolateral membrane (BLM) of various nephron segments, including proximal tubule (PT), thick ascending limb of Henle, cortical collecting duct, and distal tubule. The mOat3 mRNA, and mRNA of the functionally-similar transporter mOAT1 (Slc22a6), were detected in the whole mouse kidney, where they exhibited the female (F)-dominant and male (M)-dominant expression, respectively. The sex-dependent expression of these transporters at the protein level is poorly documented. In our preliminary experiments we observed some discrepancies in immunolocalization of OAT3 in the mouse kidney with previously used antibodies. Here we performed detailed studies with both antibodies in order to define correct localization of mOAT3 and sex-dependency of mOAT3 and mOAT1 proteins. In these experiments we used adult wild-type (WT) intact and gonadectomised mice and mOat3 knockout (KO) mice of both sexes, and studied: a) cell localization of mOAT3 (with rOAT3-Ab and mOAT3-Ab) and mOAT1 (with rOAT1-Ab) proteins by immunocytochemistry (IC) in kidney cryosections, b) abundance of both proteins by Western blotting (WB) of total cell membranes (TCM) isolated from the whole kidney, and c) effect of castration in M mice on protein expression by IC and WB. In accordance with previous studies, rOAT3-Ab stained the BLM of various nephron segments in WT mice, but the same staining was noted along the nephron of Oat3 KO mice, proving the nonspecificity of rOAT3-Ab in mouse organs. On the contrary, mOAT3-Ab exclusively stained the BLM of cortical PT, and no staining was noted in the kidneys of mOat3 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. In addition, the expression in WT mice of mOAT3 protein was F>M, and upregulated by castration, while the expression of mOAT1 protein was M>F, and downregulated by castration. In conclusion, our results indicate that in the mouse nephron: a) mOAT3 and mOAT1 proteins are exclusively localized in the PT BLM, and b) both proteins exhibit the sex-dependent expression, however, with an opposite pattern ; the mOAT3 expression is F-dominant due to androgen inhibition, while the expression of mOAT1 is M-dominant due to androgen stimulation.

kidney; proximal tubules; organic anion transporters; sexual differences; Oat3 knock-out

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