Investigation of protein kinase C activity in a polycystic kidney disease model

Academic Article

Abstract

  • Polycystic kidney disease (PKD) is characterized by cyst formation that eventually leads to renal failure. Recent work has shown that PKD is associated with the loss of the structure/function of the primary cilia in cells. Cilia are long, narrow structures projecting from the apical membrane of polarized epithelial cells. Studies will be performed using duct cells from the Oak Ridge polycystic kidney mouse which lacks primary cilia cells (ciliaf-]) vs control cells that possess normal cilia (cilia[+]). In cilia (-) cells, there is increased apical Ca 2+ entry, resulting in an increased cytosolic Ca 2+ concentration. It is generally known that elevations in Ca 2+ concentration activate the enzyme protein kinase C (PKC), and that results in its translocation from the cytosol to the plasma membrane. This investigation tests the hypothesis that there is chronic activation of PKC in cilia (-) cells, and that elevated PKC activity results in a higher rate of cell proliferation in cilia (-) cells. Differences in cytosolic PKC protein levels between cilia (+) and cilia (-) cells will be determined using the protein kinase non-radioactive assay (Calbiochem). Variations in PKC activity will be determined after treatments with PKC activator phorbol 12-myristate 13-acetate (PMA), intracellular calcium chelator Bapta-AM, and protein kinase inhibitor Ro-32-0432. The effects of PKC on the rate of cell proliferation of cilia (+) and cilia (-) cells will be ascertained by cell counting with a hemacytometer during activation or inhibition of PKC activity. Immunofluorescence will be performed with a monoclonal antibody raised against conventional PKC isozymes.
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    Author List

  • Simmons C; Komlosi P; Bell PD
  • Volume

  • 18
  • Issue

  • 2 SUPPL. 1