Collecting duct cells that lack normal cilia have mislocalized vasopressin-2 receptors
ABSTRACT 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.
SourceAvailable from: Erica A Golemis[Show abstract] [Hide abstract]
ABSTRACT: Mutations inactivating the cilia-localized Pkd1 protein result in autosomal dominant polycystic kidney disease (ADPKD), a serious inherited syndrome affecting ∼1 in 500 people, in which accumulation of renal cysts eventually destroys kidney function. Severity of ADPKD varies throughout the population, for reasons thought to involve differences both in intragenic Pkd1 mutations and in modifier alleles. The scaffolding protein NEDD9, commonly dysregulated during cancer progression, interacts with Aurora-A (AURKA) kinase to control ciliary resorption, and with Src and other partners to influence proliferative signaling pathways often activated in ADPKD. We here demonstrate Nedd9 expression is deregulated in human ADPKD and a mouse ADPKD model. Although genetic ablation of Nedd9 does not independently influence cystogenesis, constitutive absence of Nedd9 strongly promotes cyst formation in the tamoxifen-inducible Pkd1fl/fl;Cre/Esr1(+) mouse model of ADPKD. This cystogenic effect is associated with striking morphological defects in the cilia of Pkd1(-/-);Nedd9(-/-) mice, associated with specific loss of ciliary localization of adenylase cyclase III in the doubly mutant genotype. Ciliary phenotypes imply a failure of Aurora-A activation: Compatible with this idea, Pkd1(-/-);Nedd9(-/-) mice had ciliary resorption defects, and treatment of Pkd1(-/-) mice with a clinical Aurora-A kinase inhibitor exacerbated cystogenesis. In addition, activation of the ADPKD-associated signaling effectors Src, Erk, and the mTOR effector S6 was enhanced, and Ca(2+) response to external stimuli was reduced, in Pkd1(-/-);Nedd9(-/-) versus Pkd1(-/-) mice. Together, these results indicated an important modifier action of Nedd9 on ADPKD pathogenesis involving failure to activate Aurora-A.Proceedings of the National Academy of Sciences 08/2014; 111(35). DOI:10.1073/pnas.1405362111 · 9.81 Impact Factor
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ABSTRACT: Epidermal growth factor (EGF) is linked to the pathogenesis of polycystic kidney disease (PKD). We explored signaling pathways activated by EGF in orpk cilia (-) collecting duct cell line derived from a mouse model of PKD (hypomorph of the Tg737/Ift88 gene) with severely stunted cilia, and in a control orpk cilia (+) cell line with normal cilia. RT-PCR demonstrated mRNAs for EGF receptor subunits ErbB1, ErbB2, ErbB3, ErbB4, and mRNAs for Na(+)/H(+) exchangers, NHE-1, NHE-2, NHE-3, NHE-4, and NHE-5 in both cell lines. EGF stimulated proton efflux in both cell lines. This effect was significantly attenuated by MIA, 5-(n-methyl-N-isobutyl) amiloride, a selective inhibitor of NHE-1 and NHE-2, and orpk cilia (-) cells were more sensitive to MIA than control cells (P<0.01). EGF significantly induced extracellular signal regulated kinase (ERK) phosphorylation in both cilia (+) and cilia (-) cells (63.3% and 123.6%, respectively) but the effect was more pronounced in orpk cilia (-) cells (P<0.01). MIA significantly attenuated EGF-induced ERK phosphorylation only in orpk cilia (-) cells (P<0.01). EGF increased proliferation of orpk cilia (+) cells and orpk cilia (-) cells by 19.3% and 35% respectively, and MIA at 1-5 µM attenuated EGF-induced proliferation in orpk cilia (-) cells without affecting proliferation of orpk cilia (+) cells. EGF-induced proliferation of both cell lines was significantly decreased by the EGFR tyrosine kinase inhibitor AG1478 and the MEK inhibitor PD98059. These results suggest that EGF exerts mitogenic effects in the orpk cilia (-) cells via activation of growth-associated amiloride-sensitive NHEs and ERK.AJP Cell Physiology 07/2014; 307(6). DOI:10.1152/ajpcell.00188.2014 · 3.67 Impact Factor
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ABSTRACT: Polycystic kidney disease (PKD) is a common genetic disorder leading to cyst formation in the kidneys and other organs that ultimately results in kidney failure and death. Currently, there is no therapy for slowing down or stopping the progression of PKD. In this study we identified the disintegrin metalloenzyme 17 (ADAM17) as a key regulator of cell proliferation in kidney tissues of conditional knockout Ift88-/- mice and collecting duct epithelia cell from Ift88orpk mice, animal models of autosomal recessive polycystic kidney disease (ARPKD). Using Western blotting, an enzyme activity assay and a growth factor shedding assay in the presence or absence of the specific ADAM17 inhibitor TMI-005, we show that increased expression and activation of ADAM17 in cystic kidney and in collecting duct epithelial cells originating from the Ift88orpk mice (designated as PKD cells) leads to constitutive shedding of several growth factors including heparin-binding EGF-like growth factor (HB-EGF), amphiregulin and transforming growth factor-alpha (TGFα). Increased growth factor shedding induces activation of the EGFR/MAPK/ERK pathway and maintains higher cell proliferation rate in PKD cells compared to control cells. PKD cells also displayed increased lactate formation and extracellular acidification indicative of aerobic glycolysis (Warburg effect) that was blocked by ADAM17 inhibition. We propose that ADAM17 is a key promoter of cellular proliferation in PKD cells by activating the EGFR/ERK axis and a pro-proliferative glycolytic phenotype.American journal of physiology. Renal physiology 06/2014; 307(5). DOI:10.1152/ajprenal.00218.2014 · 3.30 Impact Factor