Current Research: My research focuses on the role of the cytosolic protein Pyruvate tyrosine kinase-2, Pyk2, and its involvement in the distal tubule’s response to metabolic acidosis, or decreased systemic pH. Renal epithelial cells respond to an acid-load by increasing the transport of ammonium and hydrogen ions through transporters in the apical membrane. It is believed that Pyk2 is directly activated by acid pH in OKP cells, an opossum-derived proximal tubule cell line, as demonstrated by Preisig and Li et al. Evidence of Pyk2 functioning as an intracellular pH sensor in the proximal tubule has led to the examination of its function in other segments of the nephron. My research aims to show that Pyk2 is also acid-activated in the collecting duct. Using immortalized outer and inner medullary collecting duct (OMCD and IMCD) cells derived from mice, we simulate acidosis in vitro and measure changes in protein expression the cells’ response using western blot. Acid-stimulated include increased Pyk2 auto-phosphorylation at Tyrosine 402. A cell-free assay using Pyk2 immunoprecipitated from mOMCD1 and m-IMCD-3 cells tests whether Pyk2 is directly activated by acid and if any upstream signaling molecules are involved in Pyk2’s response to acidosis. Additional studies are designed to determine Pyk2’s downstream targets, which may include c-Src, ERK, JNK, and hydrogen ion transporters, H+-ATPases and H+,K+-ATPases. Understanding Pyk2 activation and signaling in the collecting duct is important because as a putative intracellular pH sensor, it may play a role in the pathogenesis of diseases such as metabolic acidosis or distal renal tubular acidosis (dRTA). |