? This application is for supplementation of a K08 grant.
The aims of the K08 grant, progress to date on those aims, and proposed future studies if the R03 is funded are set forth below. ? Organic anion transporters (OATs), membrane proteins expressed in the proximal tubule of the kidney, have been implicated in the renal excretion (as well as nephrotoxicity) of a large number of clinically important compounds, including many commonly prescribed drugs (e.g., beta-lactam antibiotics, NSAIDs, diuretics). Our overall goals are to characterize the molecular mechanisms underlying the transcriptional and developmental regulation of the OATs and to investigate their function in vivo by assessing the renal excretion of presumptive substrates in OAT knockout mice.
Aim 1 : To identify the cis-regulatory elements and trans-acting factors governing OAT gene expression. We have identified potential regulatory elements using computational sequence analyses. We will next test the activity of these elements and their cognate factors in an OAT-expressing cell-line (OK cells).
Aim 2 : To characterize the developmental regulation of OAT expression. We have found that OAT expression is induced relatively early in kidney development, and that this induction also occurs in organ culture models of nephrogenesis such as whole embryonic kidney (WEK) culture. Thus WEK culture might serve as a convenient in vitro model for experimental investigation of OAT induction during development. We will perform transfections of promoter-reporter constructs into WEKs as a novel approach to identification of the regulatory elements mediating this induction.
Aim 3 : To determine the in vivo function of OATs by assessing transport in OAT knockout (KO) mice. We have assessed renal clearance of organic anions in an OAT1 knockout mouse, elucidating the role of OAT1 in vivo. We find a pronounced decrease in clearance of the prototypic OAT-substrate, PAH, indicating that the bulk of renal secretion of this compound is mediated by OAT1. We will extend these findings to other presumptive substrates (focusing on clinically significant compounds) as well as assess the relative in vivo contribution of OAT3 by determining clearance in an OATS KO mouse. ? These studies will shed light on the means by which the kidney excretes many important drugs (including those used to treat infections, cancer, and high blood pressure), and therefore will contribute to the development of safer and more effective drug therapy. ? ?
