Date Available

10-25-2012

Year of Publication

2012

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Pharmacy

Department/School/Program

Pharmaceutical Sciences

Advisor

Dr. Markos Leggas

Co-Director of Graduate Studies

Dr. Patrick J. McNamara

Abstract

The organic anion transporting polypeptide 4c1 (Oatp4c1) was previously identified as a novel uptake transporter predominantly expressed at the basolateral membrane in the rat kidney proximal tubules. Its functional role was suggested to be a vectorial transport partner of an apically-expressed efflux transporter for the efficient translocation of physiological substrates into urine, some of which were suggested to be uremic toxins. In vitro studies in polarized cell lines showed that upon transfection rat Oatp4c1 localizes at the apical membrane. The objectives of this project were to further validate the subcellular localization of Oatp4c1/OATP4C1 in rat and human tissues as well as their localization and function in polarized cells.

Using several complementary biochemical, molecular and proteomic methods as well as antibodies amenable to immunohistochemistry, immunofluorescence, and immunoblotting, we investigated the expression pattern of Oatp4c1 in epithelial cell lines and in the rat kidney and mammary gland (MG). Collectively, these data demonstrated that rat Oatp4c1 localized at the apical cell surface of polarized epithelium and primarily in the proximal straight tubules, the S3 segment of proximal tubule, in the juxtamedullary cortex.

Drug uptake studies in Oatp4c1-expressing cells demonstrated that Oatp4c1- mediated estrone-3-sulfate (E3S) uptake was ATP-independent and pH-dependent. The increased E3S transport activity at acidic extracellular pH was ascribed to the increased maximum transport rate (Vmax). In addition, E3S transport inhibition by various substrates suggests that Oatp4c1 possesses multiple substrate binding sites.

The apical localization of Oatp4c1 in the rat kidney and MG is a novel finding and implies that this transporter protein plays a role in the reabsorption, not vectorial secretion, of its substrates. In addition, the upregulation of Oatp4c1 expression during lactation indicates that it is involved in reuptake of xenobiotic from the milk, resulting in their reduced exposure to the suckling infants, or that it functions as a scavenger system. Further, studies to identify physiological substrates are needed to better understand the significance of Oatp4c1 function in renal and mammary epithelium.

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