Date of Award

2004

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Gary O. Rankin

Second Advisor

Carl A. Gruetter

Third Advisor

Lawrence H. Lash

Fourth Advisor

Richard M. Niles

Fifth Advisor

Monica A. Valentovic

Abstract

Glucuronidation is a common Phase II biotransformation reaction that increases the hydrophilicity, and thus elimination, of toxins, xenobiotics, and endogenous compounds. Previous studies suggest that the kidney can secrete glucuronide conjugates, but the renal transport mechanisms for glucuronide secretion have not been determined. Based on the chemical nature of glucuronide metabolites, it is hypothesized that organic anion transporter (OAT) proteins along the basolateral membrane of the renal proximal tubule promote renal accumulation of glucuronide conjugates. The purpose of this study was to develop a rat renal proximal tubule model which demonstrates OAT activity and by which the contribution of OAT in the renal accumulation of glucuronide metabolites could be assessed. In the current study two in vitro models were established; freshly isolated renal proximal tubules (IRPTs), and renal cortical slices (RCSs) from the male Fischer 344 rat. These models demonstrate time-, temperature- and probenecid-sensitive uptake of prototypical OAT1 and OAT3 substrates fluorescein (FL) and 14C- p-aminohippuric acid (PAH), and the prototypical OAT 3 selective substrate 3H-estrone sulfate (ES). Accumulation of 14C-4-acetamidophenyl-β-D-glucuronide (AG) was found to be time-dependent, but not temperature- or probenecid-sensitive in IRPTs. In the RCSs, AG uptake was time-dependent, but only minimally temperature- and probenecid-sensitive. Accumulation/inhibition studies with FL, PAH, ES and AG indicate very limited interaction between AG and OAT. PAH uptake was not affected by other glucuronides (i.e. testosterone glucuronide, methylumbelliferyl glucuronide) in IRPTs or RCSs. Studies using RCSs from Sprague-Dawley rats yielded comparable results to those found in Fischer 344 rat. In all models, accumulation of 14C-AG was not inhibited by excess unlabeled AG. These results suggest that in these rat models, AG does not appear to be a substrate for renal basolateral membrane OAT proteins; that the contribution of OAT in the basolateral membrane transport of AG (and potentially other glucuronide metabolites) is minimal; and accumulation of AG does not appear to be a protein carrier-mediated process. Thus, rat renal proximal tubular cells do not appear to facilitate the accumulation of glucuronide conjugates, and thus do not appear to contribute to the renal secretion of glucuronide conjugates.

Subject(s)

Glucuronides.

Glucuronides -- Secretion.

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