Abstract
The "classical" organic anion secretory pathway of the renal proximal tubule is critical for the renal excretion of the prototypic organic anion, para-aminohippurate, as well as of a large number of commonly prescribed drugs among other significant substrates. Organic anion transporter 1 (OAT1), originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J. G., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478), has physiological properties consistent with a role in this pathway. However, several other transporters (e.g. OAT2, OAT3, and MRP1) have also been proposed as important PAH transporters on the basis of in vitro studies; therefore, the relative contribution of OAT1 has remained unclear. We have now generated a colony of OAT1 knock-out mice, permitting elucidation of the role of OAT1 in the context of these other potentially functionally redundant transporters. We find that the knock-out mice manifest a profound loss of organic anion transport (e.g. para-aminohippurate) both ex vivo (in isolated renal slices) as well as in vivo (as indicated by loss of renal secretion). In the case of the organic anion, furosemide, loss of renal secretion in the knock-out results in impaired diuretic responsiveness to this drug. These results indicate a critical role for OAT1 in the functioning of the classical pathway. In addition, we have determined the levels of approximately 60 endogenous organic anions in the plasma and urine of wild-type and knock-out mice. This has led to identification of several compounds with significantly higher plasma concentrations and/or lower urinary concentrations in knock-out mice, suggesting the involvement of OAT1 in their renal secretion. We have also demonstrated in xenopus oocytes that some of these compounds interact with OAT1 in vitro. Thus, these latter compounds might represent physiological substrates of OAT1.
Highlights
Organic anion transporter 1 (OAT1) is the prototypic member of a family of phylogenetically related transmembrane proteins principally expressed in barrier epithelia, such as renal tubules
We have demonstrated in xenopus oocytes that some of these compounds interact with OAT1 in vitro
We have demonstrated that some of these compounds interact with OAT1 in vitro
Summary
Animals—Homologous recombination was used to replace part of the first exon of the OAT1 gene with a cassette encoding -galactosidase and a neomycin-selectable marker (LacZ-Neo), resulting effectively in the loss of the ability to either transcribe or translate OAT1 (for details, see “Results”). Since PAH clearance differed between genotypes, it is important to note that in both knock-out and wild-type mice, the ED50 values of the natriuretic response were independent of the PAH dose applied in the initial clearance experiment This is consistent with the assumption that any PAH remaining in the circulation (after letting the kidneys clear PAH for 30 min before application of diuretic) did not exert a significant effect on the response to furosemide. Sufficient urine for colorimetric detection of furosemide in both genotypes was obtained after the 3- and 10-mg/kg doses of the diuretic At these time points, plasma PAH (and probably urinary PAH as well) was estimated to be reduced to less than 1% of initial levels in the knock-out mice (and to ϳ10Ϫ10 of initial levels in wild-type mice). The S.E. of the difference between two mean values (e.g. the difference between uptake in probenecid-treated and control renal slices) was estimated to be the square root of the sum of the squares of the S.E. of those values
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