Cloning of the human kidney pah transporter: narrow substrate specificity and regulation by protein kinase c.. Lu, Run, Brenda Chan, and Victor L. Schuster. Departments of Medicine, Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, N.Y. (U.S.A.)
APStracts 5:0190F, 1998.
Conserved from fish to mammals, renal proximal tubule organic anion secretion plays an important role in drug and xenobiotic elimination. Studies with the model substrate p-aminohippurate (PAH) have suggested that a basolateral PAH/[alpha]-ketoglutarate exchanger imports diverse organic substrates into the proximal tubule prior to apical secretion. cDNAs encoding PAH transporters have been cloned recently from rat and flounder. Here we report the cloning of a highly similar human PAH transporter (hPAHT) from human kidney. By northern blot analysis and EST database searching, hPAHT mRNA was detected in kidney and brain. PCR-based monochromosomal somatic cell hybrid mapping placed the hPAHT gene on chromosome 11. When expressed transiently in vitro, hPAHT catalyzed time-dependent and saturable [3H]-PAH uptake (Km [sim]5 (M). Pre-incubation with unlabeled [alpha]-ketoglutaric or with glutaric acid stimulated tracer PAH uptake, and pre-incubation with unlabeled PAH stimulated tracer [alpha]-ketoglutarate uptake, results that are consistent with PAH/[alpha]-ketoglutarate exchange. Several structurally diverse organic anions cis-inhibited PAH uptake. Like rat OAT1, hPAHT was inhibited by furosemide, indomethacin, probenecid, and [alpha] -ketoglutarate. Unlike OAT1, hPAHT was not inhibited by prostaglandins or methotrexate. Moreover, tracer PGE2 and methotrexate (MTX) were not transported, indicating that the substrate specificity for transport by hPAHT is not broad. PAH uptake was inhibited by phorbol 12-myristate 13-acetate (PMA) in a dose- and time-dependent fashion, but not by the inactive 4 [alpha]-phorbol-12,13 didecanoate. PMA -induced inhibition was blocked by staurosporine. Thus, the protein kinase C-mediated inhibition of basolateral organic anion entry previously reported in intact tubules is likely due, at least in part, to direct modulation of the PAH/[alpha]-ketoglutarate exchanger. 

Received 2 June 1998; accepted in final form 22 October 1998
APS Manuscript Number F137-8.
Article publication pending Am. J. Physiol. (Renal Physiology).
ISSN 1080-4757 Copyright 1998 The American Physiological Society.
Published in APStracts on 10 November 1998