h-89 and Polycystic-Kidney-Diseases

Polycystic kidney disease (PKD) is a ciliopathy characterized by renal cysts and hypertension. These changes are presumably due to altered fluid and electrolyte transport in the collecting duct (CD). This is the site where vasopressin (AVP) stimulates vasopressin-2 receptor (V2R)-mediated aquaporin-2 (AQP2) insertion into the apical membrane. Since cysts frequently occur in the CD, we studied V2R and AQP2 trafficking and function in CD cell lines with stunted and normal cilia [cilia (-), cilia (+)] derived from the orpk mouse (hypomorph of the Tg737/Ift88 gene). Interestingly, only cilia (-) cells grown on culture dishes formed domes after apical AVP treatment. This observation led to our hypothesis that V2R mislocalizes to the apical membrane in the absence of a full-length cilium. Immunofluorescence indicated that AQP2 localizes to cilia and in a subapical compartment in cilia (+) cells, but AQP2 levels were elevated in both apical and basolateral membranes in cilia (-) cells after apical AVP treatment. Western blot analysis revealed V2R and glycosylated AQP2 in biotinylated apical membranes of cilia (-) but not in cilia (+) cells. In addition, apical V2R was functional upon apical desmopressin (DDAVP) treatment by demonstrating increased cAMP, water transport, and benzamil-sensitive equivalent short-circuit current (I(sc)) in cilia (-) cells but not in cilia (+) cells. Moreover, pretreatment with a PKA inhibitor abolished DDAVP stimulation of I(sc) in cilia (-) cells. Thus we propose that structural or functional loss of cilia leads to abnormal trafficking of AQP2/V2R leading to enhanced salt and water absorption. Whether such apical localization contributes to enhanced fluid retention and hypertension in PKD remains to be determined.

Topics: Animals; Aquaporin 2; Cell Line; Cilia; Cyclic AMP-Dependent Protein Kinases; Isoquinolines; Kidney Tubules, Collecting; Mice; Polycystic Kidney Diseases; Receptors, Vasopressin; Sulfonamides; Vasopressins

Developmental control of cell proliferation is crucial, and abnormal principal cell proliferation may contribute to cystogenesis in polycystic kidney disease. This study investigates roles of cAMP and its primary effector, cAMP-dependent protein kinase (protein kinase A; PKA), in control of cell proliferation in filter-grown noncystic (NC) and cystic (CY)-derived principal cell cultures. These cultures had similar cAMP pathway characteristics upstream of PKA subunit distribution but differed in predicted PKA subtype distribution. Functionally, cultures were proliferative before polarization, with constitutively higher proliferation in CY cultures. NC cultures achieved levels similar to those of CY cultures on pharmacological manipulation of cAMP production or PKA activation or inhibition of PKA subtype I activity. Inhibition of overall PKA activity, or of PKA subtype II anchoring, diminished cAMP/PKA-mediated proliferation in NC cultures but had no effect on CY cultures. Polarized CY monolayers remained proliferative, but NC monolayers lost responsiveness. No large proliferation changes resulted from treatments of polarized cultures; however, polarized NC and CY cultures differed in poststimulation handling of PKA catalytic and type IIalpha regulatory subunits. Our results support PKA subtype regulation of prepolarization proliferation in NC principal cells and altered regulation of PKA in CY cells and suggest that differences at or downstream of PKA can contribute to altered proliferation in a developmental renal disease.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Cell Division; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Epithelial Cells; ErbB Receptors; Isoquinolines; Kidney; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Phenotype; Polycystic Kidney Diseases; Quinazolines; Signal Transduction; Sulfonamides

Polycystic kidney disease (PKD) is characterized by the abnormal proliferation of tubular epithelial cells. It was recently shown that the growth of PKD cyst-lining cells is stimulated by cyclic adenosine monophosphate (cAMP), whereas the growth of normal human kidney cortex cells is inhibited.. We have examined the effects of overexpressing the C-terminal cytosolic tail of mouse polycystin-1, as a membrane-targeted fusion protein, on cAMP-responsive cell proliferation in stably transfected M-1 cortical collecting duct cells. Two cell lines that express high levels of the polycystin-1 fusion protein and two control cell lines that do not express the fusion protein were tested.. Growth of parental M-1 cells and the control cell lines was inhibited by 8-Br-cAMP and by a variety of cAMP agonists. In contrast, growth of the polycystin-1-expressing clones was stimulated by cAMP. Consistent with this, the protein kinase A (PKA) inhibitor H-89 caused either a positive or a negative growth effect depending on the primary response to cAMP. PD98059 blocked the cAMP stimulation of cell proliferation, indicating that the pathway is MEK1 dependent.. Expression of the polycystin-1 C-terminal tail disrupts normal cellular signaling and transforms the stably transfected M-1 cells to an abnormal PKD cell proliferation phenotype.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Cell Division; Cell Line; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Flavonoids; Gene Expression; Isoquinolines; Kidney Tubules, Collecting; Mice; Phenotype; Polycystic Kidney Diseases; Proteins; Recombinant Fusion Proteins; RNA, Messenger; Signal Transduction; Sulfonamides; Transfection; TRPP Cation Channels