Active H+ Transport in Urinary Acidification

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DESCRIPTION (provided by applicant): Development of epithelia in organs like the kidney proceeds in two steps; stem cells are first converted to a proto-epithelium, which then undergo terminal differentiation into the 14 types of cells of the nephron. In the latter process, epithelia develop regulated exocytosis, apical endocytosis, and apical microvilli composed of newly induced actin binding proteins, a subapical network of actin and cytokeratins, as well as changes in cell shape. An identical phenomenon occurs in the HCO3 secreting beta-intercalated cell of the cortical collecting duct when it changes to an acid-secreting alpha-phenotype in response to an acid diet. This change is induced by the deposition of a new extracellular matrix protein, which we termed hensin. Hensin is secreted as a soluble monomer but gets polymerized by cyclophilin C, and later converted to fibers by galectin 3. Only the fiber form can bind and activate (by tyrosine phosphorylation) its receptor, which we recently identified as alpha-v beta-1 integrins. We generated a knockout of hensin and found that it is lethal at the time of implantation of the blastocyst. The trophectoderm, which is the first terminally differentiated epithelium of the embryo, expresses hensin. Hensin is expressed in all epithelia, and the recent findings that it is deleted in many human cancers suggest that it is a new tumor suppressor gene. We wish to pursue these studies by examining the locus of action of hensin in the blastocyst, in particular to examine whether its effect is due to a defect in the trophectoderm (the epithelium) or in the inner cell mass. In our second aim we will examine whether hensin induces terminal differentiation in the collecting tubule and other epithelia. We will generate tissue specific knockouts of hensin using the Cre/LoxP system deleting it from the intestine, the prostate, and the collecting duct in general and from the intercalated cell in particular. In a third aim, we will examine the signal transduction pathway induced by hensin by activation of integrin receptors with emphasis on discovering molecules that get activated by tyrosine phosphorylation during terminal differentiation. These studies should define the function of this critical new protein in differentiation of the kidney tubule, in acid base balance and in early embryonic development.