Organic cation transporters in the kidney play key roles in the body's defense against foreign substances including drugs and other xenobiotics. Many clinically used drugs (e.g., metformin ), hormones (e.g., dopamine) as well as toxic substances (e.g., MPP+ (1-methyl-4-phenylpyridinium)) are organic amines and are eliminated in the kidney by secretory transporters. The working hypothesis of the proposed studies is that the organic cation transporter, OCT2, is localized to the basolateral membrane of the proximal tubule and is the key transporter responsible for the first step in the renal secretion of many organic cations. To test this hypothesis, comprehensive studies, ranging from molecular analysis of specificity through studies of an OCT2 knockout mouse and genetic studies in humans, are proposed.
The specific aims are to: (1) Determine the specificity of OCT2 in heterologous expression systems; (2) Construct a humanized cell culture model to localize the transporter to the apical or basolateral membrane and localize the transporter along the nephron; (3) Determine the role of OCT2 in renal secretion by studying renal elimination and accumulation in an OCT2 knockout mouse that will be provided by our collaborator, A. Schinkel; and (4) Determine whether genetic variation in OCT2 contributes to variation in renal clearance of the model organic cation, metformin, an anti-diabetic drug, in humans. Methods used in analysis of specificity of OCT2 include two-electrode voltage clamp methods in oocytes. Confocal microscopy, in situ hybridization techniques and immunohistochemistry will be used to localize OCT2 to the apical or basolateral membrane and to ascertain the regional specific distribution of the transporter along the nephron. Pharmacokinetic studies in mice will be used to determine the role of the transporter in in vivo renal elimination. Finally, clinical studies in normal volunteers with particular OCT2 genotypes and haplotypes including OCT2*1, OCT*3D and three missense mutations (M1651, R400C, K432Q) will be performed to determine whether genetic variation in OCT2 contributes to variation in renal clearance of metformin. These variants have been shown in cells to have altered function in comparison to the common form of OCT2. These studies are significant to our basic understanding of the mechanisms involved in renal drug elimination and will enhance our ability to predict renal clearance, toxicities and drug response.
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