The proposed studies plan to characterize channels in cell membranes of renal collecting duct and proximal tubule cells, from transgenic mouse, rat, rabbit, or salamander kidney, using a combination of preparations: cell cultures, isolated perfused and non-perfused renal tubules, and separated single cells which have preserved their epithelial polarity. Patch-clamp, optical and molecular biology techniques will be used. The objective are: 1. To study the physiological role and regulation of a cGMP-sensitive non-selective cation channel in the apical membrane of the M-1-cells line, derived from mouse cortical collecting duct. Incomplete cDNA sequences derived from M-1-cell line are extremely similar to that of the cGMP-gated non-selective channel of vertebrate photoreceptor. Full length kidney channel clones will be isolated and used to explore which genes are expressed in renal cells. Those genes will be studied further by expression in oocytes and their electrophysiology properties assessed. 2. To study the physiological role and regulation of single chloride channels in the basolateral membrane of mammalian proximal tubule cells using patch-clamp recordings, as well as of ensembles of channels through whole-patch and whole-cell current recording. In the basolateral membrane of single, isolated amphibian proximal tubule cells small-conductance chloride channels were detected, which are activated by forskolin and cAMP, and inhibited by the chloride channels will be explored by patch-clamping in unperfused mammalian proximal tubules, in controlled microperfusions of the lumen of cannulated proximal tubules, and during modulation of transepithelial chloride transport. The biophysics and kinetics of chloride channels in mammalian proximal tubule will be studied as well as the intracellular signal transduction pathways involved in regulation. In parallel, the effect of these regulator mechanisms will be tested on transepithelial chloride transport in perfused tubules. 3. To study a simple model of proximal tubule cell injury induced by angiotensin II, which is accompanied by elevation of intracellular calcium and plasma membrane blebbing. Using confocal vital microscopy and ion-sensitive fluorescent dyes on single isolated salamander proximal tubule cells, the mechanism of bleb formation will be studied as a direct response to increases in intracellular calcium, as a function of extracellular Na and of intracellular acidosis. Signal in order to develop possible interventions of cyto-protection. Cell membrane effects of angiotensin- induced cell injury will be monitored by conductance and capacitance determination in whole-cell patch-clamping. The overall scope of the project is the understand transepithelial solute movement by the kidney at the single cell membrane and single channel protein level and to contribute to the understanding of clinical disorders such as hypertension, metabolic alkalosis/acidosis, acute renal failure and potassium balance.
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