The salient feature of both ischemic and nephrotoxic acute renal failure is renal tubular injury. Acute renal failure is a frequent and serious complication of endotoxemia and in vitro, endotoxin is cytotoxic to renal tubules. However, the biochemical events that lead ultimately to endotoxin-induced cytotoxicity to renal tubular cells have not been previously examined. My hypothesis is endotoxin-induced cytotoxicity to renal tubules is mediated through derangements in calcium homeostasis and can be modulated by the concomitant release of proposed mediators. This hypothesis is based on the following: a. There is compelling recent evidence which suggests that derangements in calcium homeostasis may lead to the progression of sublethal to lethal cell injury; b. Several in vivo studies have indicated that the effects of endotoxin may be mediated through the release of mediators including thromboxane A2 (TXA2), platelet- activating factor (PAF), and tumor necrosis factor (TNF); c. In in vitro studies endotoxin has been shown to cause release of these mediators in other cell types, and d. Some of these mediators have been shown to cause a rise in [Ca2+]i. In additions, I have obtained preliminary data demonstrating that endotoxin, lipid-A, TXA2 and PAF induce a rise in intracellular calcium [Ca2+]i in the LLC-PK1 renal tubular cell line.
The specific aims are: 1. To examine the cytotoxic effects of endotoxin on rat proximal tubules and LLC-PK1 cells. The role of calcium in the cytotoxic effects of endotoxin will be evaluated by inhibiting intracellular release and extracellular influx. The direct effects of endotoxin on [Ca2+]i will be monitored using the fluorescent probe fura-2, evaluating changes in the Ca2+ uptake and release potential of the endoplasmic reticulum and mitochondrial membrane, and by monitoring changes in the activity of plasma membrane Ca2+-ATPase. The temporal relationship between changes in Ca2+ and cytotoxicity will be examined. 3. To examine the roles of TXA2, PAF and TNF in the endotoxin-induced derangement of [Ca2+]i homeostasis and cytotoxicity. Given that TXA2, PAF and TNF are proposed mediators of many of the effects of endotoxin in vivo, their direct effects on calcium homeostasis as well as their potential for mediating the effects of endotoxin will be examined. 4. To further characterize the putative receptors for endotoxin, TXA2, and PAF proximal tubules and LLC-PK1 cells using radioligand binding assays.
| Proksch, J W; Traylor, L A; Mayeux, P R (1996) Effects of lipid A on calcium homeostasis in renal proximal tubules. J Pharmacol Exp Ther 276:555-60 |
| Mayeux, P R; Shah, S V (1993) Glomerular thromboxane A2/prostaglandin H2 receptors: characterization and effect of adriamycin-induced nephrotic syndrome. Biochim Biophys Acta 1181:148-54 |
| Mayeux, P R; Shah, S V (1993) Intracellular calcium mediates the cytotoxicity of lipid A in LLC-PK1 cells. J Pharmacol Exp Ther 266:47-51 |
