The long term objectives of this proposal are: 1) To elucidate the cellular and molecular mechanisms responsible for increased renal acid excretion during chronic administration, and 2) To investigate the cellular and molecular mechanisms responsible for the distal acidification defect following unilateral ureteral obstruction. The first specific aim is to investigate the basis for adaptational increase in H+ secretion in acid loading. We will examine whether changes in H+-ATPase content occur in acid loading by analyzing the expression of an intercalated cell-specific subunit isoform of the vacuolar H+-ATPase using immunoassays and RNA blots in rat kidneys after acid loading. We study several aspects of the cellular basis for H+ - ATPase polarization in acid loading. We will endeavor to isolate and characterize a newly identified serum factor that induces H+ ATPase polarization in chronic acid loading; we will attempt to develop a model for polarization in tubular suspensions or primary cultures of intercalated cells to allow changes in H+ -ATPase distribution to be examined in vitro; we will examine whether changes in cytoskeletal organization occur in the H+-ATPase polarization response; and we will examine whether the rab low molecular weight GTP-binding proteins in kidney participate in H+-ATPase targeting to plasma membrane and regulated polarization. We will examine whether changes in levels of H+- ATPase regulatory proteins (inhibitor and activator) may have a role in altering kinetic properties of the H+-ATPase during the adaptational response to acid loading. The second specific aims is to investigate the mechanisms responsible for the acidification defect in unilateral ureteral obstruction. We will determined whether abnormalities in H+-ATPase polarization are present using immunocytochemistry; we will determined if changes in H+-ATPase quantity occur by examining the expression of an intercalated cell- specific subunit isoform of the vacuolar H+-ATPase using immunoassays and RNA blots; and we investigate whether kinetic changes in the H+-ATPase contribute to the defect by examining whether changes in level of H+- ATPase regulatory protein (inhibitor and activator) occur in obstruction. These studies will advance our understanding of the physiologic and cellular mechanisms operating in renal adaptation and the cellular and molecular basis for ion transport defects in obstructive renal disease.
