Ischemia in vivo and ATP in vitro have been shown to result in extensive alterations of the actin cytoskeleton and surface membrane of proximal tubule cells. We hypothesize that correction of these alterations is essential for the cellular restitution process. Furthermore, we postulate the injured epithelial cell has the ability to reutilize, with high efficiency and fidelity, the internalized surface membrane components and disrupted and highly polymerized actin cytoskeletal constituents during cellular ATP repletion. Finally, we hypothesize that reorganization of the cortical actin cytoskeleton occurs prior to and is essential for the efficient and polarized reutilization of internalized surface membrane components during the restitution process. To test these different but interrelated hypotheses we have developed and extensively characterized a reversible model of ATP depletion in LLCPK1 cells. Furthermore, we have developed, characterized and are presently using a myriad of immunofluorescent, fluorescent analog, digital confocal, videoimaging, microinjection immunocytochemical, immunogold and quantitative biochemical techniques to study cellular repair at the single cell level. Specifically, we will use selective surface membrane domain specific biotinylation and avidin Texas-red fluorescent techniques in combination with digital confocal, immunogold an quantitative biochemical- spectrofluorimetric techniques to follow surface membrane internalization, intracellular trafficking and reutilization during ATP depletion and repletion. We will then use fluorescent analog, microinjection, digital confocal and videoimaging techniques to evaluate both G and F-actin during ATP depletion and repletion. Finally, we will combine these two approaches to observe morphologically and quantify biochemically and spectrofluorometrically the interaction between the actin cytoskeleton and apical and basolateral surface membrane domains.
| Molitoris, Bruce A; Sutton, Timothy A (2004) Endothelial injury and dysfunction: role in the extension phase of acute renal failure. Kidney Int 66:496-9 |
| Sutton, Timothy A; Mang, Henry E; Campos, Silvia B et al. (2003) Injury of the renal microvascular endothelium alters barrier function after ischemia. Am J Physiol Renal Physiol 285:F191-8 |
| Dunn, Kenneth W; Sandoval, Ruben M; Molitoris, Bruce A (2003) Intravital imaging of the kidney using multiparameter multiphoton microscopy. Nephron Exp Nephrol 94:e7-11 |
| Kwon, Osun; Phillips, Carrie L; Molitoris, Bruce A (2002) Ischemia induces alterations in actin filaments in renal vascular smooth muscle cells. Am J Physiol Renal Physiol 282:F1012-9 |
| Sutton, Timothy A; Fisher, Charles J; Molitoris, Bruce A (2002) Microvascular endothelial injury and dysfunction during ischemic acute renal failure. Kidney Int 62:1539-49 |
| Molitoris, Bruce A; Sandoval, Ruben; Sutton, Timothy A (2002) Endothelial injury and dysfunction in ischemic acute renal failure. Crit Care Med 30:S235-40 |
| Sandoval, Ruben M; Bacallao, Robert L; Dunn, Kenneth W et al. (2002) Nucleotide depletion increases trafficking of gentamicin to the Golgi complex in LLC-PK1 cells. Am J Physiol Renal Physiol 283:F1422-9 |
| Sundin, D P; Sandoval, R; Molitoris, B A (2001) Gentamicin inhibits renal protein and phospholipid metabolism in rats: implications involving intracellular trafficking. J Am Soc Nephrol 12:114-23 |
| Ashworth, S L; Sandoval, R M; Hosford, M et al. (2001) Ischemic injury induces ADF relocalization to the apical domain of rat proximal tubule cells. Am J Physiol Renal Physiol 280:F886-94 |
| Sandoval, R M; Dunn, K W; Molitoris, B A (2000) Gentamicin traffics rapidly and directly to the Golgi complex in LLC-PK(1) cells. Am J Physiol Renal Physiol 279:F884-90 |
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