The overall goal of this proposal is to elucidate the role of Ca-permeable TRPV channel isoforms in regulating mechanically-induced intracellular Ca, [Ca]i, dynamics and Ca reabsorption in renal cortical collecting duct. Mechanical stresses, such as fluid shear stress or hypoosmotic stress, vary widely in late distal tubule and cortical collecting duct (CCD) especially during pathophysiological states (e.g., hyponatremia, diabetes insipidus, diabetes mellitus, diuretic administration), which will contribute to hypocaliuric and/or hypercaliuric states that lead to altered Ca balance. We have implicated two TRPV channels as being mechanosensitive and central to regulating mechano-stimulated Ca entry and reabsorption in mouse CCD. The following four specific aims are proposed to achieve the overall goal: 1. To determine the relative role and pharmacological/ electrophysiological properties of mechanoregulated Ca entry pathways (Ca imaging, patch clamp) in control of [Ca]i dynamics in mouse CCD cells;2. To identify TRPV channel isoforms expressed in mouse CCD cells (RT-PCR, Northern blot, immunofluorescence, immunoblots) and to determine which specific isoforms participate in mechanoregulated [Ca] dynamics;3. To delineate the relative contribution of specific mechanoregulated TRPV channels in Ca reabsorption in mouse CCD cells (overexpression/siRNA knockdown) and to examine the mechanism of regulation of the channels (phospholipases, protein kinases);and 4. To evaluate the role of membrane localization/trafficking of TRPV isoforms (confocal microscopy/immunofluorescence, biotinylation-streptavidin purification) and the association with accessory proteins (immunoprecipitation/pull-downs), including other TRPs, on mechanoregulated [Ca]i dynamics and Ca reabsorption. The outcome of the study has important health relatedness as it will provide new insights into the poorly understood mechanisms of calcium signaling and reabsorption in renal tubules and provide a critical new understanding of the mechanism by which altered pathophysiological conditions regulate calcium reabsorption in renal cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK070950-05
Application #
7590356
Study Section
Cellular and Molecular Biology of the Kidney Study Section (CMBK)
Program Officer
Ketchum, Christian J
Project Start
2005-04-01
Project End
2011-03-31
Budget Start
2009-04-01
Budget End
2011-03-31
Support Year
5
Fiscal Year
2009
Total Cost
$273,218
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Biology
Type
Schools of Medicine
DUNS #
800771594
City
Houston
State
TX
Country
United States
Zip Code
77225
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