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PKD1 Haploinsufficiency Causes a Syndrome of Inappropriate Antidiuresis in Mice

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PKD1 Haploinsufficiency Causes a Syndrome of Inappropriate Antidiuresis in Mice JASN Express. Published on May 2, 2007 as doi: 10.1681/ASN.2006010052 PKD1 Haploinsufficiency Causes a Syndrome of Inappropriate Antidiuresis in Mice Ali K. Ahrabi,* Sara Terryn,† Giovanna Valenti,‡ Nathalie Caron,§ ʈ ʈ Claudine Serradeil-Le Gal, Danielle Raufaste, Soren Nielsen,¶ Shigeo Horie,** Jean-Marc Verbavatz,†† and Olivier Devuyst* *Division of Nephrology, Universite´catholique de Louvain Medical School, Brussels, Belgium; †Laboratory of Cell Physiology, Center for Environmental Sciences, Hasselt University, Diepenbeek, Belgium; ‡Department of Physiology, University of Bari, Bari, Italy; §Department of Physiology and Pharmacology, University of Mons-Hainaut, Mons, ʈ Belgium; Sanofi-Aventis, Toulouse, France; ¶The Water and Salt Research Center, University of Aarhus, Aarhus, Denmark; **Department of Urology, Teikyo University, Tokyo, Japan; and ††Cell and Molecular Imaging, CEA/Saclay, Gif-sur-Yvette, France Mutations in PKD1 are associated with autosomal dominant polycystic kidney disease. Studies in mouse models suggest that the vasopressin (AVP) V2 receptor (V2R) pathway is involved in renal cyst progression, but potential changes before cystogenesis are unknown. This study used a noncystic mouse model to investigate the effect of Pkd1 haploinsufficiency on water handling and AVP signaling in the collecting duct (CD). In comparison with wild-type littermates, Pkd1ϩ/Ϫ mice showed inappropriate antidiuresis with higher urine osmolality and lower plasma osmolality at baseline, despite similar renal function and water intake. The Pkd1ϩ/Ϫ mice had a decreased aquaretic response to both a water load and a selective V2R antagonist, despite similar V2R distribution and affinity. They showed an inappropriate expression of AVP in brain, irrespective of the hypo-osmolality. The cAMP levels in kidney and urine were unchanged, as were the mRNA levels of aquaporin-2 (AQP2), V2R, and cAMP-dependent mediators in kidney. However, the (Ser256) phosphorylated AQP2 was upregulated in Pkd1ϩ/Ϫ kidneys, with AQP2 recruitment to the apical plasma membrane of CD principal cells. The basal intracellular Ca2؉ concentration was significantly lower in isolated Pkd1ϩ/Ϫ CD, with downregulated phosphorylated extracellular signal–regulated kinase 1/2 and decreased RhoA activity. Thus, in absence of cystic changes, reduced Pkd1 gene dosage is associated with a syndrome of inappropriate antidiuresis (positive water balance) reflecting decreased intracellular Ca2؉ concentration, decreased activity of RhoA, recruitment of AQP2 in the CD, and inappropriate expression of AVP in the brain. These data give new insights in the potential roles of polycystin-1 in the AVP and Ca2؉ signaling and the trafficking of AQP2 in the CD. J Am Soc Nephrol ●●: ●●●–●●●, ●●●●. doi: 10.1681/ASN.2006010052 utosomal dominant polycystic kidney disease (AD- tion, and dedifferentiation (1,3). All nephron segments may be PKD) is the most frequent inherited nephropathy and involved in cyst formation in ADPKD, but an important frac- A an important cause of ESRD (1). Mutations in two tion of the cysts is derived from the collecting ducts (CD) (4,5). genes, PKD1 and PKD2, have been associated with ADPKD. In vitro studies have shown that cAMP plays a major role in Mutations in PKD1 account for approximately 85% of the af- cystogenesis. Exposure to cAMP agonists stimulates fluid se- fected families, and they are associated with a renal disease that cretion across monolayers of ADPKD cyst-lining epithelial cells progresses more rapidly than in families with PKD2 (2). PKD1 (6), as well as the proliferation of these cells (7). Furthermore, and PKD2 encode integral membrane proteins, polycystin-1 increased levels of cAMP resulting from the activation of vaso- and polycystin-2, that interact in renal primary cilia and regu- pressin (AVP) V2 receptor (V2R) pathway in CD cells may late the proliferation and differentiation of renal tubular cells contribute to the progression of cystogenesis. In two cystic via different signaling pathways (3). Mutations in PKD1/PKD2 models that are orthologous to human autosomal recessive disrupt these pathways, leading to cystogenesis by a combina- PKD (PCK rat) and nephronophthisis (pcy mouse) and one tion of increased cellular proliferation, abnormal fluid secre- cystic model that is orthologous to human ADPKD type 2 Ϫ (Pkd2 /tm1Som mouse), increased renal cAMP levels compared with normal mice, paralleled with higher expression of aqua- Received January 18, 2006. Accepted March 9, 2007. porin-2 (AQP2) and V2R, have been reported (8–10). The ad- Published online ahead of print. Publication date available at www.jasn.org. ministration of V2R antagonists to these models lowered renal cAMP and inhibited the development and progression of es- Address correspondence to: Dr. Olivier Devuyst, Division of Nephrology, UCL Medical School, 10 Avenue Hippocrate, B-1200 Brussels, Belgium. Phone: ϩ32-2- tablished renal cystic disease (8–10), motivating trials to test the 764-5450; Fax: ϩ32-2-764-5455; E-mail: [email protected] efficacy of V2R antagonists in patients with ADPKD (11). It is Copyright © ●●●● by the American Society of Nephrology ISSN: 1046-6673/●●●●-0001 2 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: ●●●–●●●, ●●●● important to note that all rodent models tested so far develop privation. The capacity to excrete a water load was tested after intra- renal cysts (and subsequent renal failure) within a few weeks of peritoneal injection of 2 ml of sterile water; urine was collected under age. a plastic-wrapped container on an hourly basis for the next 6 h. The In normal CD cells, the stimulation of V2R by AVP leads to aquaretic effect of the V2R antagonist SR121463B (Sanofi-Aventis, Chilly-Mazarin, France), which has a high affinity for renal V2R from the phosphorylation of AQP2 on the Ser256 residue and its several species, including rat, mouse, and human (K ϭ 0.26 Ϯ 0.04 nM) subsequent insertion in the apical plasma membrane, an essen- i (23), was tested after intraperitoneal administration of dosages that tial step to mediate final urine concentration (12,13). A mild ranged from 0.1 to 30 mg/kg and hourly determination of diuresis for impairment in urinary concentrating ability, with increased thenext6hasdescribed above. circulating AVP levels, has been described in patients with ADPKD and cystic kidneys (11,14,15). However, this urinary V2R Binding Assays and Autoradiography ϩ ϩ ϩ Ϫ concentrating abnormality is probably not specific, because any Renomedullary preparations from Pkd1 / and Pkd1 / mice (or modification of the medullary architecture (e.g., cystic changes) CHO membranes that expressed human V2R used as positive controls) impairs the constitution of the corticomedullary osmotic gradi- were incubated in a 50-mM Tris-HCl buffer (pH 8.1) that contained 2 3 ent, resulting in nephrogenic diabetes insipidus (16). Consider- mM MgCl2, 1 mM EDTA, 0.1% BSA, 0.1% bacitracin, and [ H]SR121463 ing that an activation of the V2R pathway has been involved in (0.8 to 28 nM for saturation experiments or 2 nM for binding studies). ␮ PKD mouse models with cysts originating from the CD (8–10), The reaction was started by the addition of membranes (7.5 g/assay for CHO and 100 to 130 ␮g/assay for mouse renal tissue) and incuba- we hypothesized that the complex chain of events that mediates tion for 45 min at 25°C, stopped by filtration through Whatman GF/B urinary concentration in the CD could be modified early, before filters as described previously (24). Nonspecific binding was deter- cystogenesis. mined in the presence of 1 ␮M SR121463B. Data for equilibrium bind- In this study, we used a well-established mouse model with ing (apparent equilibrium dissociation constant [K ] and maximum ϩ Ϫ d a targeted deletion of Pkd1 (Pkd1 / ) (17) to test whether Pkd1 binding density [Bmax]) were calculated using an interactive nonlinear haploinsufficiency causes abnormal water handling and AVP regression program (25). signaling in the CD before cystogenesis and renal failure. Like For performance of autoradiography, kidneys from Pkd1 mice were Ϫ Ϫ other Pkd1-null mutants, the homozygous Pkd1 / mice die in frozen at Ϫ40°C in isopentane and further stored at Ϫ80°C. Serial utero with massive cystic kidneys, hydrops fetalis, and cardio- sections (15 ␮m) were mounted onto gelatin chrome-alum slides, rinsed vascular defects (17–20). By contrast, there is no consistent to eliminate endogenous AVP, and incubated with 1.5 nM ϩ Ϫ 3 ␮ phenotype in heterozygous Pkd1 / mice that do not develop [ H]SR121463 alone (total binding) and in the presence of 1 M unla- beled SR121463B or AVP (nonspecific binding) as described previously renal cysts until (in a few individuals) a very old age (21,22). (24). After incubation, the sections were washed three times for 10 min Our investigations reveal for the first time that reduced Pkd1 each in ice-cold binding buffer, dipped in distilled water, and dried gene dosage results in inappropriate antidiuresis and positive under a stream of cold air. Rinsed labeled sections were placed on a water balance, reflecting decreased intracellular calcium phosphor-imaging plate for 4 d and further analyzed with a BAS5000 2ϩ ([Ca ]i) levels with lowered RhoA activity and recruitment of Bio-Image Analyser (Fuji, Tokyo, Japan). SR121463B, monophosphate AQP2 in the apical membrane of CD principal cells and inap- salt, and [3H]SR121463 (47.5 Ci/mmol) were synthesized at Sanofi- propriate expression of AVP in the brain. Aventis, whereas AVP was obtained from Sigma Chemical Co. (L’Isle d’Abeau, France). Materials and Methods Pkd1 Mice and Sampling Plasma and Urine Analyses Experiments were conducted on age- and gender-matched adult Sodium, urea, creatinine, and calcium were measured using a Kodak mice (aged 20 to 35 wk) with a targeted deletion of the exons 2 to 5 and Ektachem DT60II Analyzer (Johnson & Johnson, New Brunswick, NJ), part of exon 6 of Pkd1, resulting in a null allele (17). The mice were and osmolality was measured using a Fiske Osmometer (Needham maintained on a mixed 129/sv/C57BL/6J background.
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