In the kidney, endothelins (ETs) are important regulators of blood flow, glomerular hemodynamics, and sodium and water homeostasis. They have been implicated in the pathophysiology of acute ischemic renal failure, diabetic nephropathy, vascular rejection of renal transplants and nephrotoxicity by cyclosporin, cisplatin and radiocontrast agents. Recently, we revealed a new role for renal ET: regulation of ATP-driven drug transport in renal proximal tubule. This segment of the nephron transports a wide variety of potentially toxic xenobiotics, xenobiotic metabolites, and metabolic wastes from blood to urine. Among the proteins implicated in this process are two ATP-dependent transporters: P-glycoprotein (substrates are uncharged and cationic compounds) and the multidrug resistance protein 2 (Mrp2, anionic substrates), both of which are present at high levels in the luminal membrane of proximal tubular cells. We found that ET-1, acting through a basolateral B-type receptor (ETBR) and through PKC, rapidly reduced cell-to-tubular lumen drug excretion by decreasing the transport activity of Mrp2 and P-gp. Furthermore, various different nephrotoxic agents, including aminoglycosides, radio contrast agents and heavy metals, reduced transport activity via the same pathway through ET-1 release from proximal tubule, triggered by an increase in intracellular calcium. These studies strongly indicated a new common autocrine/paracrine mechanism in the early response to renal injury, but its pathophysiological context is still unclear. One possible role of ET release may be to reduce certain ATP-consuming processes in order to conserve ATP for more important cellular functions. On the other hand, a reduction of transport activity may contribute to the progression of cellular damage as a result of higher intracellular concentrations of toxic compounds and metabolites. The objective of this project is to resolve this issue by investigating the mechanisms underlying the crucial steps in this common pathway. The following objectives will be addressed:
1. Do ETBR antagonists protect against drug-induced tubular toxicity or do they aggravate cell injury?
2. What is the role of nitric oxide (NO)? Presurnably NO signals the calcium-induced ET release and an increased tubular NO production has been associated with cell injury.
3. Does PKC play a direct or indirect role in the regulation of ATP-dependent transporters, and how do the expression levels progress after prolonged exposure to nephrotoxins?
4. What happens to the expression levels of the ATP-independent transport systems? Do they compensate for the decrease in P-gp and Mrp2 activity, or are they also down-regulated?
Experiments will be performed in isolated perfused rat kidney, isolated rat proximal tubular cells, isolated killifish proximal tubules and in proximal tubule derived cell lines transfected with cDNAs of specific transporters. The outcome of these studies will advance our understanding of the role of ET in tubular drug transport and toxicity, and may have important practical implications for renal protection. |
