Abstract
The kidney, and more specifically the proximal tubule, is the main site of elimination of cationic endogenous metabolites and xenobiotics. Although numerous studies exist on renal organic cation transport of rat and rabbit, no information is available from humans. Therefore, we examined organic cation transport and its regulation across the basolateral membrane of isolated human proximal tubules. mRNA for the cation transporters hOCT1 and hOCT2 as well as hOCTN1 and hOCTN2 was detected in these tubules. Organic cation transport across the basolateral membrane of isolated collapsed proximal tubules was recorded with the fluorescent dye 4-(4-dimethylamino)styryl-N-methylpyridinium (ASP(+)). Depolarization of the cells by rising extracellular K(+) concentration to 145 mm reduced ASP(+) uptake by 20 +/- 5% (n = 15), indicating its electrogeneity. The substrates of organic cation transport tetraethylammonium (K(i) = 63 microm) and cimetidine (K(i) = 11 microm) as well as the inhibitor quinine (K(i) = 2.9 microm) reduced ASP(+) uptake concentration dependently. Maximal inhibition reached with these substances was approximately 60%. Stimulation of protein kinase C with 1,2-dioctanoyl-sn-glycerol (DOG, 1 microm) or ATP (100 microm) inhibited ASP(+) uptake by 30 +/- 3 (n = 16) and 38 +/- 13% (n = 6), respectively. The effect of DOG could be reduced with calphostin C (0.1 microm, n = 7). Activation of adenylate cyclase by forskolin (1 microm) decreased ASP(+) uptake by 29 +/- 3% (n = 10). hANP (10 nm) or 8-bromo-cGMP (100 microm) also decreased ASP(+) uptake by 17 +/- 3 (n = 9) or 32 +/- 5% (n = 10), respectively. We show for the first time that organic cation transport across the basolateral membrane of isolated human proximal tubules, most likely mediated via hOCT2, is electrogenic and regulated by protein kinase C, the cAMP- and the cGMP-dependent protein kinases.
Highlights
The proximal tubule is the site of secretion and reabsorption of endogenous metabolites and xenobiotics in the kidney
We show for the first time that organic cation transport across the basolateral membrane of isolated human proximal tubules, most likely mediated via hOCT2, is electrogenic and regulated by protein kinase C, the cAMP- and the cGMP-dependent protein kinases
Our goal was to study for the first time the uptake of organic cations across the basolateral membrane of isolated human proximal tubules which is the first step in the elimination of cationic xenobiotics and endogenous metabolites via the kidney
Summary
The proximal tubule is the site of secretion and reabsorption of endogenous metabolites and xenobiotics in the kidney. Previous functional studies [7, 8] on organic cation transport in the proximal tubule of the rat showed the differences between transport across the luminal or basolateral membrane. Organic cation transporters expressed in the luminal and basolateral membranes of the proximal tubule are molecularly and functionally very different members of this protein family. In the apical membrane of a human proximal tubule cell line (IHKE-1), we found organic cation transport, probably mediated by the OCTNs, to be under the regulation of PKC, cAMPdependent protein kinase (PKA), and cGMP-dependent protein kinase (PKG) [29]. RT-PCR was used to identify cloned transporters in freshly isolated human proximal tubules and to relate those findings to our functional data of organic cation transporters
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