Defects in V-ATPase-dependent acidification in the kidney collecting duct results in distal tubular acidosis and nephrolithiasis. This MERIT award focuses on the role of intercalated cells (IC) in modulating proton secretion via the V-ATPase. Since the start of this award in 2008, we have published 20 original papers and 4 review articles on V- ATPase function and protein trafficking. Our work, summarized in the progress report, includes identifying signal transduction and trafficking pathways regulating proton- secretion by IC; identifying new V-ATPase interacting proteins; revealing many new splice variants of V-ATPase subunits that may have cell specific roles; performing an exhaustive proteomic analysis of 10; characterizing accessory proteins involved in trafficking pathways in different kidney tubules; generating new knockout and transgenic mice that open new avenues for understanding IC development and V-ATPase function; identifying multiple phosphorylation sites on V-ATPase subunits by mass spectrometry. Importantly, we have developed techniques to maintain differentiated 10 in vitro for several days. This now allows us unprecedented access to their proteome, RNA expression, and their acidification function in direct response to physiological/hormonal stimuli. In the extension period, we will: 1) Exterid our ongoing studies to identify the sensing and signal transduction cascade that leads to modulation of acid/base transport by 10 in the kidneys of normal and V-ATPase subunit-depJeted mice, and in our isolated EGFP-IC in vitro. We will use V-ATPase-Cre mice to selectively delete critical proteins including the soluble adenylyl cyclase (sAO), cytoskeletal proteins and accessory trafficking proteins from ICs. In vitro real time and in vivo intravital microscopy as well as functional measurements of proton secretion will be applied to follow 10 responses to stimuli. 2) We will dissect protein interactions and phosphorylation events that regulate V-ATPase trafficking and function a) by continuing to characterize cytoskeletal protein interactions (including FRET and Biacore assays) and b) by identifying phosphorylation events necessary for V-ATPase activation using expression of phosphomutant subunits in our cell culture systems. We will continue producing anti-phospho-V-ATPase antibodies. 3) We will characterize the expression, distribution and function of newly- identified V-ATPase a4 subunit splice variants that could explain differential membrane targeting of V-ATPase in 10 and other cells. In summary, our proposed studies are focused on the application-of new tools that will ultimately allow us to therapeutically modify renal acidification process by understanding acid/base sensing and V-ATPase trafficking mechanisms.
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