The aims of this proposal are to define the cellular pathophysiology of nephrotoxic and ischemic renal tubular cell injury which is the key pathologic event in acute tubular necrosis (ATN) and to find and investigate the mechanisms of maneuvers which ameliorate renal tubular cell injury and acute excretory failure. Much of this proposal will utilize an in vitro preparation of proximal tubule suspensions to investigate these aims. This in vitro system provides a direct approach to understand the critical cellular and biochemical events involved in renal tubular cell injury wihout the interfering nephronal and hemodynamic events occuring in vivo in ATN. Critical metabolic parameters including oxygen consumption measurements, cation levels measured with atomic absorption spectroscopy, adenine nucleotide levels assessed with HPLC techniques, CA++ compartmentation determined by dual wavelength spectroscopy, lipid turnover and peroxidation identified by isotopic labelling, thin layer chromatography, and spectroscopic methods, will be followed in these preparations to quantitate important cellular processes and cell viability. This preparation will be used to assess the role of phopholipase activation and phospholipid breakdown products, extracellular and intracellular Ca++, lipid peroxidation and free radical generation, adenine nucleotide pool size, pH and phosphate in mediating ischemic renal tubular cell injury. Additional studies with this system will define precise sites of action and cellular mechanisms of several maneuvers which ameliorate ATN in vivo, including calcium channel blockers, exogenous adenine nucleotides, hypertonic agents, and loop diuretics. Further experiments with this preparation will examine the direct tubule cell toxicity of heme pigments, myeloma light chains, and radiocontrast agents. Additional in vivo and in vitro experiments utilizing isolated membrane preparations, tubule suspensions, and whole animal protocols are planned to characterize nephrotoxinmembrane interactions as a pathogenetic basis of toxicity and to determine cellular mechanisms of protective maneuvers ameliorating nephrotoxicity. Finally, both in vitro and in vivo studies utilizing techniques to study mitrochondrial Ca++ metabolism are planned to determine the role alterations in mitochondrial Ca++ handling and overload play in hypercalcemia-induced or potentiated renal tubular cell injury in order to clarify better the role of Ca++ in cell injury.
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