The overall objective of this project is to elucidate the subcellular reaction to injury in the kidney. The mechanisms of renal cell injury in vivo remain incompletely understood due to the complexity of the nephron and the technical difficulties involved in studying key cellular events in the intact kidney. Our in vitro and in vivo experiments on the many structural and functional events that follow lethal and sublethal injury in proximal tubular epithelium clearly indicate a major role for ion deregulation, especially that of free ionized cytosolic calcium ((Ca2+)i), in this process. In this proposal, new methodologies such as digital imaging microscopy combined with recently developed specific fluorescent probes, coupled with video intensification microscopy and other computer-assisted morphologic techniques (image analysis and processing) will be used to permit detailed studies of the mechanisms involved with ion deregulation in cell injury. Cell injury may proceed by many postulated pathways. For our investigation, we have chosen four injury models involving different primary mechanisms: HgC12, anoxia without substrate, paraquat or the addition of xanthine plus xanthine oxidase, and cisplatin. The present proposal represents a definitive approach to studies on the role of (Ca2+)i in the initiation and progression of cell injury. This project will emphasize two major specific aims: Project I will examine the temporal changes in (Ca2+)i in relationship to other parameters of cell injury to determine if the change in (Ca2+)i is a primary or secondary event. In each model, changes in (Ca2+)i will be studied and related to alterations in: cell killing, changes in morphology such as blebbing; cytosolic pH; cellular energy status; thiols; calmodulin function; and activation of Ca-dependent phospholipases and proteases. Project II will investigate specific aspects of (Ca2+)i deregulation and will include: the effect of injury on Ca2+ compartmentation and transport; the plasma membrane involvement in Ca2+ and Na+ deregulation; and alterations in mitochondrial and/or ER release or retention of Ca2+. Investigation into the complex relationships being proposed requires the use of an in vitro system. Primary culture of proximal tubular cells isolated from both Fischer 344 rats and from human kidneys will be used. The human cell model will permit us to test and compare important findings and conclusions derived from the rat model. Such an approach will provide valuable information concerning the applicability of animal models for the study of human disease processes.
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