This applicant proposes a program to prepare him for a career in academic basic science studying renal function, addressing the regulation of proton secretion in the kidney under physiological and patho- physiological conditions. The research will be conducted in the laboratory of Dr. Dennis Brown in the Program in Membrane Biology and Renal Unit, MGH. Renal H+ secretion is mainly mediated by the vacuolar proton-pumping ATPase (V-ATPase), an enzyme that also acidifies some intracellular organelles. However, when expressed on the plasma membrane, as in collecting duct A-type intercalated cells (1C),the V-ATPase mediates transepithelial H+ secretion. Defects in H+ secretion cause distal renal tubular acidosis (dRTA), associated with sensorineural deafness in humans. dRTA is caused by mutations in the gene encoding the 56 kDa B1 subunit isoform of the V-ATPase. We hypothesize that an alternative V-ATPase B subunit, the B2 isoform might serve as a replacement back-up that functionally replaces the B1 in renal H+ secretion under some conditions, because our available B1 subunit knockout mice are not acidotic and they express more B2 subunit in the apical membrane of 1C than do normal mice. Understanding the ways in which B1 and B2 assemble into V-ATPase complexes and the role of these subunits in V-ATPase targeting and trafficking processes could suggest novel treatment strategies by """"""""isoform replacement therapy"""""""" in cells in which B1- mediated H+ secretion is defective. The main goal of this proposal and training program is use novel techniques and animal models to determine the relative role of the V-ATPase B1 and B2 isoforms in H+ secretion and V-ATPase trafficking in renal epithelial cells. How does the membrane expression of the B2 subunit increase in mice lacking B1, and how is this expression/trafficking process regulated? We will examine regulation of V-ATPase mRNA and protein expression in 1C from unique B1-knockout mice under different acid-base conditions. mRNA from 1C will be isolated by laser capture microdissection using new transgenic mice that express EGFP in 1C.Compensatory B2-V-ATPase function in 1C will be examined by pH ratio imaging. The role of the B1-interacting protein NHE-RF1 in V-ATPase trafficking will be addressed, and a role for the bicarbonate-stimulated soluble adenylate cyclase (sAC) in the membrane insertion of V- ATPases containing the B1 and B2 subunit will be examined using a multidisciplinary approach.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Scientist Development Award - Research & Training (K01)
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Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
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Rankin, Tracy L
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Massachusetts General Hospital
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P?unescu, Teodor G; Shum, Winnie W C; Huynh, Chuong et al. (2014) High-resolution helium ion microscopy of epididymal epithelial cells and their interaction with spermatozoa. Mol Hum Reprod 20:929-37
P?unescu, Teodor G; Lu, Hua A J; Russo, Leileata M et al. (2013) Vasopressin induces apical expression of caveolin in rat kidney collecting duct principal cells. Am J Physiol Renal Physiol 305:F1783-95
Vedovelli, Luca; Rothermel, John T; Finberg, Karin E et al. (2013) Altered V-ATPase expression in renal intercalated cells isolated from B1 subunit-deficient mice by fluorescence-activated cell sorting. Am J Physiol Renal Physiol 304:F522-32
P?unescu, Teodor G; Rodriguez, Steven; Benz, Eric et al. (2012) Loss of the V-ATPase B1 subunit isoform expressed in non-neuronal cells of the mouse olfactory epithelium impairs olfactory function. PLoS One 7:e45395
Brown, Dennis; Bouley, Richard; P?unescu, Teodor G et al. (2012) New insights into the dynamic regulation of water and acid-base balance by renal epithelial cells. Am J Physiol Cell Physiol 302:C1421-33
Winter, Christian; Kampik, Nicole B; Vedovelli, Luca et al. (2011) Aldosterone stimulates vacuolar H(+)-ATPase activity in renal acid-secretory intercalated cells mainly via a protein kinase C-dependent pathway. Am J Physiol Cell Physiol 301:C1251-61
Gao, Xiaobo; Eladari, Dominique; Leviel, Francoise et al. (2010) Deletion of hensin/DMBT1 blocks conversion of beta- to alpha-intercalated cells and induces distal renal tubular acidosis. Proc Natl Acad Sci U S A 107:21872-7
P?unescu, Teodor G; Ljubojevic, Marija; Russo, Leileata M et al. (2010) cAMP stimulates apical V-ATPase accumulation, microvillar elongation, and proton extrusion in kidney collecting duct A-intercalated cells. Am J Physiol Renal Physiol 298:F643-54
Miranda, Kevin C; Bond, Daniel T; McKee, Mary et al. (2010) Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease. Kidney Int 78:191-9
Brown, Dennis; Paunescu, Teodor G; Breton, Sylvie et al. (2009) Regulation of the V-ATPase in kidney epithelial cells: dual role in acid-base homeostasis and vesicle trafficking. J Exp Biol 212:1762-72

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