Transcriptional regulation of sex-dependently expressed renal organic anion transporter 1 and 3

Abstract

The renal organic anion transporters (OATs) play a pivotal role in the elimination of endogenous and exogenous substrates. In rats, an often used preclinical animal model, the basolateral located organic anion transporter 1 (Oat1) and 3 (Oat3) are known to be male-dominant and testosterone-dependently expressed. They are involved in the secretion of organic anions, including negatively charged drugs such as adefovir, furosemide, or penicillin. Human OAT1 and OAT3 were mentioned as clinically relevant transporters in the kidneys, which have to be investigated during drug development. The antibiotic penicillin has been shown to cause more adverse drug reactions (ADRs) in women compared to men. This higher risk is supposed to be partially due to sex-differences in the OAT1 and OAT3 expression. The aim of my thesis was to identify the molecular mechanism involved in the male-dominant expression of rat Oat1 and Oat3. Activation of rat and human Oat1/OAT1 and Oat3/OAT3 promoters were investigated by using luciferase activity assays. A series of promoter-length-varying rat and human Oat1/OAT1 and Oat3/OAT3 reporter constructs were generated and transiently transfected into OK or LLC-PK1 cells. By co-transfection of the supposed transcriptional regulators, their effects on Oat1/OAT1 and Oat3/OAT3 promoter activities were examined. For the identification of sex-dependently expressed genes in rat proximal tubule cells, RNA from four male and four female rats were investigated by using microarray analyses and real-time PCR. In this thesis, it was shown that the known male-dominant expression of rat Oat1 and Oat3 was not regulated via the testosterone/androgen receptor mediated transcriptional pathway. Similar to rat Oat1 and Oat3, testosterone/androgen receptor complex did not activate human OAT1 and OAT3 promoters. While searching for sex-dependently expressed transcriptional regulators, the transcription factor B-cell CLL / lymphoma 6 (BCL6) was newly identified as a highly male-dominantly expressed gene within the rat kidneys. The known Oats/OATs transcriptional regulators hepatocyte nuclear factor 1α (HNF1α), HNF1β, and HNF4α, revealed not sex-dependent expression. Moreover, BCL6 was shown to activate the promoters of rat and human Oat1/OAT1 and Oat3/OAT3, independent of predicted BCL6 binding sites, but probably via protein-protein interactions with the transcription factors HNF1 or cAMP response element binding protein (CREB). In conclusion, the male-dominantly expressed transcription factor BCL6 is a promising regulator for the sex-dependent rat Oat1 and Oat3 expression, furthermore, it is also assumed to be involved in the regulation of human OAT1 and OAT3 transcription.