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

Patrick I. Brown

Third Advisor

Lawrence H. Lash

Fourth Advisor

Richard M. Niles

Fifth Advisor

Monica A. Valentovic

Abstract

Glucuronidation is primarily a pathway of detoxification in most species, but many glucuronide conjugates are associated with toxicity. Numerous drugs are excreted in the urine as glucuronide conjugates. Being organic anions, it is likely that glucuronides are secreted into the urine by organic anion transporters found in renal proximal tubule cells (PTCs). Some drugs that are metabolized by glucuronidation have been shown to cause renal toxicity, yet little is known about the renal handling of glucuronide conjugates. It is hypothesized that glucuronides are transported into renal PTCs by an organic anion transporter (OAT) on the basolateral membrane of the cell. A potential human model for examining renal organic anion transport is the HK-2 cell line, an immortalized adult human male proximal tubule cell line. Little evidence exists that this model is capable of transporting organic anions. Therefore, the purpose of this study was to determine which OATs (1-3) exist in this cell line, if the OATs are functional, and finally, the nature of glucuronide transport. HK-2 cells were grown in keratinocyte serum-free media supplemented with 2% fetal bovine serum. Western blot analysis of membrane fractions of HK-2 cells grown for 5-7 days in T-75 flasks revealed the presence of OAT1 and OAT3, but not OAT2 in the cell membranes. In separate studies, HK-2 cells grown on microporous membranes for 4-9 days transported fluorescein (FL) and p-aminohippurate (PAH), prototypical substrates for OAT1 and OAT3 and estrone sulfate (ES), the prototypical substrate for OAT3. Transport of acetaminophen glucuronide (AG) by HK-2 cells demonstrated time-, temperature- and concentration-dependent uptake suggestive of protein-mediated transport. Transport characteristics of AG in the HK-2 model, such as biphasic concentration-dependent transport and lack of inhibition of transepithelial transport by probenecid are consistent with in vivo transport as found in the literature. These results suggest that HK-2 cells possess functional OATs and demonstrate transport of a model glucuronide compound such that HK-2 cells may be useful as a model to study the renal handling of organic anions, including glucuronide conjugates.

Subject(s)

Glucuronides.

Biological transport.