The main objective of this proposal is to understand the cellular and molecular mechanisms that mediate the adaptive responses to alterations in acid/base balance in intercalated cells (ICCs) of the renal cortical collecting duct (CCD). Depending on the acid/base status of the organism, the CCD either secrets or reabsorbs HCO3. HCO3 secretion and reabsorption take place in two subtypes of ICCs with opposing functional polarity. Adaptation to acidosis seems to involve both up- and down-regulation of the transporters mediating these opposing functions and conversion of HCO3 secreting cells to H+ secretors. The extracellular signals of adaptation and the underlying molecular mechanisms are poorly understood. Our observations during the previous grant period indicate that adaptation to acidosis involves opposite changes in mRNA levels of H-ATPase vs. the novel colonic isoform of H/K-ATPase and in mRNA levels of anion exchangers 1 vs. 2. Several of these in vivo changes can be mimicked in cultured cells by altering extracellular pH or by increasing the concentration of NH4+. In addition, we demonstrated that adaptation is accompanied by significant changes in the expression of other genes among them a novel EP3 prostaglandin receptor.
With Aim (l) we will explore the role of the colonic H,K-ATPase in HCO3 transport in the CCD. By measuring mRNA and protein levels in isolated ICC subtypes and by subcellular localization with immunohistochemistry, we will determine in which cell type and in which membrane the colonic H,K- ATPase resides, and how it is regulated by acid/base balance.
With Aim (2) we will test the hypothesis that signals generated as a result of adaptation in more proximal nephron segments influence CCD function. In particular, we will examine the effects of chronic changes in NH4+ concentrations and apical fluid composition on H+/HCO3 transport and the expression of key transporters. The goal of Aim (3a) is to define the role of the novel pH-regulated EP3 receptor in adaptation to acidosis/alkalosis. We will determine the signalling pathway associated with this receptor, the functional consequences of its acid/base-dependent regulation and the mechanisms by which acid/base balance regulates its expression.
Under Aim (3b) we will continue our systematic search for additional regulatory genes involved in adaptation to acidosis. Such genes will be identified using differential display, and their regulatory potential will be tested by determining the consequences their overexpression and suppression. It is anticipated that the new information acquired by these studies will help to clarify the mechanisms underlying adaptation to acidosis/alkalosis occurring in the distal nephron and consequently will lead to a better understanding of the etiology of renal disorders which result in disturbances of acid/base homeostasis, like type l renal tubular acidosis or Banter's syndrome.
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