Transporters in the kidney are critical in the body's defense against foreign substances including drugs and other xenobiotics. Recently, the first polyspecific (i.e., broadly selective) organic cation transporters (OCT1 and OCT2) were cloned and found to belong to a single gene family, termed OCT. These transporters were cloned from various species including rat (rOCT1, rOCT1A (this laboratory) and rOTC2), rabbit (rbOTC1, this laboratory), and human (hOCT1 (this laboratory) and hOCT2). Although the mRNA transcripts of the OCT transporters are expressed to variable degrees in mammalian kidney, the specific role of OCT1 and OCT2 in renal transport and particularly in the site specific transport of xenobiotics including clinically important drugs has not been established. The primary goal of the proposed research in this continuing application is to understand the fundamental mechanism of organic cation transport in the kidney with particular emphasis on ascertaining the functional roles of OCT1 and OCT2 in renal transport. The studies will focus primarily on mechanisms of transport in the human kidney; however, some studies of the cloned rabbit transporters, rbOCT1 and rbOCT2, will be carried out.
The aims of the proposed studies are to 1. Clone and sequence the organic cation transporter (rbOCT2) in rabbit kidney. 2. Elucidate the functional characteristics of the cloned renal organic cation transporters, OCT1 and OCT2. 3. Determine the mechanisms of transport of model organic cations across the basolateral membrane of the human kidney. 4. Determine the segmental distribution of OCT1 and OCT2 in the nephron and the sorting to the brush border or basolateral membrane. Briefly, the PI will employ PCR based methods to clone the transporters and then perform functional studies in heterologous expression systems. Specifically, the PI will determine the substrate selectivity, saturability and driving forces of the transporters. Parallel studies in isolated basolateral membrane vesicles from human kidney will be carried out to determine the biological relevance of the cloned transporters. Polyclonal antibodies will be used to localize the transporters along the nephron and determine their intracellular sorting. These studies will greatly advance our understanding of the mechanisms involved in organic cation transport in the human kidney as well as our understanding of tissue-specific elimination and toxicities of drugs, environmental toxins and other xenobiotics.
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