The proposed studies plan to characterize channels, in particular chloride channels, in cell membranes of proximal tubule cells, from kidneys of rabbit, salamander, or CFTR knockout mouse, using a combination of isolated perfused tubules, non-perfused renal tubules, and separated single cells that have preserved their epithelial polarity. Patch-clamp and optical techniques will be used.
The specific aims are: 1. To test the hypothesis that the chloride channel in the basolateral membrane of the proximal tubule cells share many of the intrinsic properties of the cystic fibrosis transmembrane conductance regulator CI- channel, we will study the biophysics and kinetics of chloride channels in mammalian proximal tubule and the intracellular signal transduction pathways involved in their regulation. 2. To test whether the gating of the basolateral chloride channel in proximal tubule cells by hydrolytic and non-hydrolytic interactions of ATP is analogous to that of the CFTR CI-channel. 3. To test the hypothesis that the basolateral chloride channel in proximal tubule cells regulates transcellular chloride reabsorption by the proximal tubule, and that alterations of apical chloride uptake indirect modulate the function of the basolateral chloride channel. 4. To explore the alternative pathways for transcellular chloride movement in proximal tubules of a CFTR-knockout mouse. Although the kidney expresses CFTR, there appears to be no impaired ion transport in patients suffering from cystic fibrosis. We will use a CFTR-knockout mouse, in order to establish how chloride channels contribute to transcellular chloride movement, and whether there is a regulatory relationship between CFTR and other chloride pathways. 5. To test the hypothesis that epithelial polarity in dissociated cells is not maintained by intrinsic proteins associated with the tight junction, we will study a model of a proximal tubule cells, that maintains epithelial polarity for us to ten days, and examine the cytoskeletal interactions that are essential for preserving polarity. The overall scope of the project is to 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 cystic fibrosis, hypertension, metabolic alkalosis, acidosis, and acute renal failure.
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