Role of NO in endothelin-regulated drug transport in the renal proximal tubule

Abstract

. Role of NO in endothelin-regulated drug transport in the renal proximal tubule. Am J Physiol Renal Physiol 282: F458-F464, 2002; 10.1152/ajprenal.00173.2001.-We previously demonstrated in intact killifish renal proximal tubules that endothelin (ET), acting through an ETB receptor and protein kinase C (PKC), reduced transport mediated by multidrug resistance-associated protein 2 (Mrp2), i.e., luminal accumulation of fluorescein methotrexate (FL-MTX) (Masereeuw R, Terlouw SA, Van Aubel RAMH, Russel FGM, and Miller DS. Mol Pharmacol 57: 59-67, 2000). In the present study, we used confocal microscopy and quantitative image analysis to measure Mrp2-mediated transport of FL-MTX in killifish tubules as an indicator of the status of this ET-fired, intracellular signaling pathway. Exposing tubules to sodium nitroprusside (SNP), a nitric oxide (NO) donor, signaled a reduction in luminal accumulation of FL-MTX, which suggested pathway activation. N G -monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor, blocked the action of ET-1 on transport. Because SNP effects on transport were blocked by bisindoylmaleide, a PKC-selective inhibitor, but not by RES-701-1, an ET B-receptor antagonist, generation of NO occurred after ET B receptor signaling but before PKC activation. NO generation was implicated in the actions of several nephrotoxicants, i.e., diatrizoate, gentamicin, amikacin, HgCl 2, and CdCl 2, each of which decreased Mrp2-mediated transport by activating ET signaling. For each nephrotoxicant, decreased FL-MTX transport was prevented when tubules were exposed to L-NMMA. ET-1 and each nephrotoxicant stimulated NO production by the tubules, as determined by a fluorescencebased assay. Together, the data show that NO generation follows ET binding to the basolateral ET B receptor and that, in activating the ET-signaling pathway, nephrotoxicants produce NO, a molecule that could contribute to subsequent toxic effects. multidrug resistance-associated protein 2; calcium; endothelin signaling; protein kinase C; xenobiotic transport; nitric oxide METABOLISM AND EXCRETION provide a first line of defense against the wide variety of potentially toxic chemicals to which we are continually exposed. In the kidney, the proximal tubule is responsible for the excretory transport from blood to urine of xenobiotics, xenobiotic metabolites, and waste products of metabolism. As a result of its rich transport function, the proximal tubule is also an important target for toxic effects (2, 27).To accomplish this excretory function, epithelial cells in the proximal tubule possess multiple plasma membrane transporters that use ATP and transmembrane ion gradients to drive active drug secretion into urine. Among the proteins implicated in this process is a member of the ATP-binding cassette superfamily of membrane transporters: multidrug resistance protein 2 (Mrp2). This transport protein is present at high levels in the luminal membrane of proximal tubule cells and handles a wide range of chemicals, from large, lipophilic organic anions to...

Involvement of guanylyl cyclase and cGMP in the regulation of Mrp2-mediated transport in the proximal tubule

“…Since we previously demonstrated that several nephrotoxicants also fired ET signaling in the tubules (33), we also determined whether nephrotoxicant effects on signaling and FL-MTX transport could be blunted by ODQ. Consistent with previous results (23,33), exposing tubules to gentamicin, amikacin, diatrizoate, HgCl 2 , and CdCl 2 significantly reduced luminal accumulation of FL-MTX (Table 1); cellular accumulation was not affected (not shown). The concentrations of nephrotoxicants used here do not reduce transport of FL by the classic Na-dependent organic anion system (33) and do not reduce mitochondrial membrane potential measured using a fluorescent indicator dye (Notenboom S, Miller DS, Russel FGM, and Masereeuw R, unpublished observations).…”

“…Here, we provide evidence that NOdependent guanylyl cyclase and cGMP are involved in the regulation of Mrp2-mediated transport in the renal proximal tubule. We previously showed that ET, acting through a basolateral ET B receptor, NOS, and PKC, decreases cell-to-lumen organic anion transport mediated by Mrp2 (18,23). Figure 6 summarizes this sequence of events.…”

“…We previously used intact killifish (Fundulus heteroclitus) renal proximal tubules to demonstrate that Mrp2 function is regulated by the vasoactive peptide endothelin (ET) working through a basolateral ET B receptor, nitric oxide synthase (NOS), nitric oxide (NO), and PKC (18,23,33). Firing this signaling pathway rapidly reduced transport mediated by Mrp2.…”

Fractional urinary excretion of endothelin-1 is reduced by acute ETB receptor blockade

“…This effect was inhibited by ETB receptor antagonists (12) and appears to be nitric oxide dependent (13), suggesting a role for ET-1 acting via ETB receptors in tubular transport mechanisms. Additionally, ETB receptor-deficient rats demonstrate an attenuated increase in urinary excretion of ET-1 on a high-salt diet compared with wild-type rats, suggesting a role for ETB receptors specifically in the excretion of renal ET-1 (21).…”

Endothelin-1 Inhibits Thick Ascending Limb Transport via Akt-stimulated Nitric Oxide Production

“…We showed that endothelin-1 stimulates NOS3 expression and NO production in primary cultures of thick ascending limbs (16). Nakano et al (7) reported that endothelin-1 enhances NO production in the renal inner medulla, and Notenboom et al (34) have shown that it stimulates NO production by isolated proximal tubules. More recently, Stricklett et al (35) reported that endothelin-1 increases NO production in rat inner medullary collecting duct suspensions.…”