ADPKD and ARPKD are important causes of ESRD. In the past cycle, we described a PKD model caused a mutation in Pkhdl (ortholog of the gene mutated in ARPKD), reported that vascular smooth muscle cell from Pkd2+/~ mice have reduced intracellular Ca2+ ([Ca2+]j), sarcoplasmic reticulum Ca2+ stores and capacitative Ca2""""""""1"""""""" entry, and provided evidence for activation of cAMP and Ras/MAPK/ERK signaling and relative inhibition of Akt signaling in PKD. We found that most cysts in PCK rats and Pkd2'/WS25 mice derive from collecting ducts and that administration of antagonists (OPC-31260 and OPC-41061) of the vasopressin V2 receptor (the main adenylyl cyclase agonist in the principal cells) lowers cAMP levels and inhibits PKD development and progression. Hypothesis 1: Alterations in [Ca2+]j homeostasis caused by polycystin or fibrocystin mutations disrupt the functional coordination of cAMP and Ras signaling, hinder negative feedback mechanisms that normally control their activation, and result in a cystic phenotype characterized by abnormal fluid secretion/reabsorption and increased rates of cell proliferation and apoptosis. To address this hypothesis we will determine a) whether reductions in PCI, PC2 or FC expression reduce [Ca2+]j and endoplasmic reticulum stores and inhibit capacitative Ca2+ entry in principal cells; b) what mechanisms are responsible for the increased cAMP levels in PKD, c) what mechanisms are responsible for the activation of Ras/MAPK signaling in PKD; d) whether and how alterations in Ca2+, cAMP or Ras/MAPK signaling disrupt the translocation of AQP2 into the apical membrane of principal cells, and e) what are the mechanism responsible for activation of Rho signaling in PKD. Hypothesis 2: Interventions that correct or enhance the alterations in [Ca2+]j, cAMP, Ras/MAPK or downstream cascades associated with the cystic phenotype will inhibit or aggravate PKD development and progression. To address this hypothesis we will a) generate female Pkd2M325M2R+l' mice and PCK Brattleboro rats to determine whether the protective effect of OPC 31260 and 41061 is due to V2R antagonism; b) compare the effects of selective vasopressin V2 or V1j receptor antagonists or both in combination to determine whether Via antagonism contributes positively o negatively to the observed beneficial effects; c) determine whether calcimimetic drugs inhibit PKD development; and progression d) determine whether drugs acting on effectors of cAMP-PKA, Ras, affect KD development and progression, and e) determine whether L-type calcium channel blockers or a inhibitors have a detrimental effect on PKD development and progression. ? ? ?
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