PKD1 Haploinsufficiency Causes a Syndrome of Inappropriate Antidiuresis in Mice

JASN Express. Published on May 2, 2007 as doi: 10.1681/ASN.2006010052

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

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 unlabeled 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. They were Heights, MA). The nitrite/nitrate (NOx) concentrations were measured housed in light- and temperature-controlled room with ad libitum access in urine and plasma using a colorimetric assay (Cayman Chemical, Ann to tap water and standard chow (Pavan, Oud-Turnhout, Belgium). Arbor, MI). Because sevoflurane may induce the release of AVP, Previous experiments showed that the Pkd1 mice had similar heart rate thereby increasing plasma values in our protocols, we measured urine and BP (N. Morel, et al., unpublished data, 2007). Water handling at AVP levels, which were not obtained under sevoflurane anesthesia, baseline and during various protocols was assessed in individual met- using RIA (Peninsula Laboratories, San Carlos, CA). For cAMP deter- abolic cages, after appropriate training. Blood and tissue samples were minations, whole kidneys were ground under liquid nitrogen and obtained at time of killing, after anesthesia with Sevoflurane (Abbott, homogenized in 10 volumes of 0.1 M HCl. The homogenate was cen- Ottignies, Belgium) and exsanguination. Blood was collected by venous trifuged at 600 ϫ g for 10 min, and the supernatant was collected, puncture, and plasma samples were kept at Ϫ20°C. The sampling diluted (1:10) in 0.1 M HCl, and processed with acetylation using an procedures were exactly similar in both groups. Tissue samples were enzyme immunoassay kit (Sigma-Aldrich, St. Louis, MO). The urine immediately processed for fixation and mRNA/protein extraction. The samples were diluted (1:5000) in 0.1 M HCl and were processed with-

experiments were conducted in accordance with the National Research out acetylation. The urinary prostaglandin E2 (PGE2) was measured by Council Guide for the Care and Use of Laboratory Animals and were EIA (Amersham Biosciences, Piscataway, NJ). approved by the local Animal Ethics Committee. Reverse Transcription–PCR and Real-Time Reverse Water Handling Protocols Transcription–PCR Plasma samples and 24-h urine collections were obtained at baseline, Total RNA from mouse kidney and brain (26) was extracted with and the urinary concentrating ability was tested after 24-h water de- Trizol (Invitrogen, Merelbeke, Belgium), treated with DNase I, and J Am Soc Nephrol ●●: ●●●–●●●, ●●●● PKD1 Haploinsufficiency Causes SIAD 3

reverse-transcribed into cDNA. The primers (Supplementary Table 1) contained a 1:100 dilution of anti-AQP2 polyclonal antibodies. Sections were designed using Beacon Designer 2.0 (Premier Biosoft Interna- were washed three times in buffer-T, then incubated in a 1:25 dilution tional, Palo Alto, CA). Changes in target gene mRNA levels were of 10 nm of gold-conjugated secondary antibodies for 45 min. After determined by semiquantitative real-time reverse transcription–PCR washing in Tris, sections were stained with uranyl-acetate and lead (RT-PCR) with an iCycler IQ System (Bio-Rad Laboratories, Hercules, citrate and photographed on a Philips EM 400 microscope (FEI, Eind- CA) using SYBR Green I. Real-time semiquantitative PCR analyses hoven, Netherlands). Three samples from three pairs of mice were were performed in duplicate as described previously (27). The PCR processed, and at least 10 pictures of outer medullary CD principal cells conditions were 94°C for 3 min followed by 31 cycles of 30 s at 95°C, were randomly taken for each sample. The data are expressed as 30 s at 61°C and 1 min at 72°C. Negative controls excluded amplifica- number of gold particles per micron of apical membrane length. Ultra- tion from genomic DNA. For each assay, standard curves were pre- structural examination of the vasa recta was performed on three pairs pared by serial four-fold dilutions of cDNA samples. The efficiency of of kidney slices and fixed overnight in 2% glutaraldehyde before wash- the reactions was calculated from the slope of the standard curve ing in PBS. Ϫ [efficiency ϭ (10 1/slope) Ϫ 1] (27). Measurement of RhoA Activity Antibodies Quantification of active RhoA (GTP-bound) was measured by selec- Rabbit polyclonal antibodies against AQP2 (Sigma-Aldrich), Ser256 tive affinity precipitation of GTP-Rho (Upstate, Temecula, CA), follow- phosphorylated AQP2 (p-AQP2) (12), AQP1 (Chemicon, Temecula, ing the procedure described in detail previously (28,29). The kidneys CA), AQP3 (a gift from J.-M. Verbavatz, CEA Saclay), extracellular were ground under liquid nitrogen and homogenized in ice-cold 1ϫ ϩ signal–regulated kinase 1/2 (ERK1/2; C16) and Tyr204 p-ERK1/2 Mg2 lysis/wash buffer according to the manufacturer’s instructions (Santa Cruz Biotechnologies, Santa Cruz, CA), mouse monoclonal (Upstate, Temecula, CA). The homogenates were centrifuged at RhoA and Ser188 p-RhoA (Santa Cruz Biotechnologies), and mouse 15,000 ϫ g for 30 min, and the supernatant of each sample was col- mAb against ␤-actin (Sigma-Aldrich) were used. lected. A total of 30 ␮g of GST-tagged fusion protein, corresponding to residues 7 to 89 of mouse Rhotekin Rho Binding Domain, bound to Immunoblotting glutathione-agarose beads, was added to the supernatant of each sam- ␮ Kidneys were ground under liquid nitrogen and homogenized as ple (500- l exact) and were rotated overnight at 4°C. Beads were 2ϩ described previously (27). The homogenate was centrifuged at 1000 ϫ washed three times with Mg lysis/wash buffer, and bound proteins g for 15 min at 4°C. The resulting supernatant was either kept at Ϫ80°C were separated by SDS-PAGE and detected by Western blotting using (as the “total extract” fraction) or centrifuged at 100,000 ϫ g for 120 min a monoclonal RhoA antibody (28,29). at 4°C. The pellet (“membrane” fraction) was suspended in homogeni- ϩ zation buffer before determination of protein concentration and storage Measurement of Intracellular Ca2 Concentration Ϫ at 80°C. SDS-PAGE was performed under reduced (kidney) or non- Inner medullary collecting ducts (IMCD) were isolated from collag- ϩ ϩ ϩ Ϫ reduced (urine) conditions. After blotting on nitrocellulose, the mem- enase-digested medulla from three pairs of Pkd1 / and Pkd1 / mouse branes were incubated overnight at 4°C with primary antibodies, kidneys. The tubule segments were seeded onto glass coverslips and washed, incubated for1hatroom temperature with peroxidase-labeled examined using an inverted Nikon TMD35 epifluorescence microscope antibodies (Dako, Glostrup, Denmark), and visualized with enhanced (Analis, Namur, Belgium) in a thermostated chamber at 37°C. The chemiluminescence. Normalization for ␤-actin was obtained after strip- 2ϩ 2ϩ intracellular Ca concentration ([Ca ]i) was measured as described ping and reprobing. Densitometry analysis was performed with a previously (30). Briefly, after measurement of the background signal, StudioStar Scanner (Agfa-Gevaert, Mortsel, Belgium) using the NIH- isolated tubules were loaded with Fura-2 by incubation with the mem- Image V1–57 software. brane-permeant acetoxymethyl (AM) ester form of the dye (10 ␮M) for 1 h at 37°C. Tubules were excited at 340 and 380 nm, and the fluores- Immunohistochemistry cence emission was recorded at 510 nm. Data collection time for an Kidney samples were fixed in 4% paraformaldehyde (Boehringer image was 2 s. Fura-2 was calibrated in vivo at the end of each exper- 2ϩ ϭ Ϫ Ingelheim, Heidelberg, Germany) in 0.1 mol/L phosphate buffer (pH iment, according to the equation derived by [Ca ]i Kd Rbf [(r Ϫ 7.4) before embedding in paraffin. The 6-␮m sections were stained with rmin)/(rmax r)], where Kd is the dissociation constant of Fura-2 for 2ϩ hemalum-eosin or incubated for 30 min with 0.3% H2O2, followed by 20 Ca (135 nM), Rbf is the maximum fluorescence intensity as a result of ϩ min with 10% normal serum, and 45 min with the primary antibodies excitation at 380 nm (in the absence of Ca2 ) divided by the minimum ϩ diluted in PBS that contained 2% BSA. After washing, sections were fluorescence intensity at 380 nm (in the presence of saturating Ca2 ), r

successively incubated with biotinylated secondary anti-IgG antibod- is the F340/F380 fluorescence ratio, rmax and rmin are the F340/F380 fluo- ϩ ies, avidin-biotin peroxidase, and aminoethylcarbazole (Vectastain rescence ratios in the presence of saturating Ca2 and in the absence of 2ϩ Elite; Vector Laboratories, Burlingame, CA). Sections were viewed un- Ca , respectively. rmax was obtained by permeabilization of the tu- ϩ der a Leica DMR coupled to a Leica DC300 digital camera (Leica, bules with Ca2 ionophore ionomycin (10 ␮M), in the presence of 5 mM 2ϩ Heerbrugg, Switzerland). extracellular Ca . For obtaining subsequently the minimum ratio rmin, ϩ the tubules were exposed to a Ca2 free solution (containing 10 mM ␮ Immunoelectron Microscopy EGTA) with 10 M ionomycin and 1,2-bis(o-aminophenoxy)ethane- N,N,NЈ,NЈ-tetraacetic acid (BAPTA)-acetoxymethyl (10 ␮M) to buffer For electron microscopy, kidney samples from Pkd1 mice were fixed ϩ intracellular Ca2 . overnight in 4% paraformaldehyde and 0.1% glutaraldehyde in PBS and washed in PBS. Small samples, including the outer medulla and the top of inner medulla, were embedded in unicryl, and 80-nm-thick Statistical Analyses Ϯ sections were cut. Sections were preincubated in 20 mM Tris buffer (pH Data are means SEM. LogED50 (the dosage of agonist that pro- 7.5) that contained 0.1% BSA, 0.1% fish gelatin, and 0.05% Tween 20 vokes 50% of the maximum response) values were calculated by non- (buffer-T), followed by a 90-min incubation in the same buffer-T that linear curve fitting of the individual concentration-effect curve data 4 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: ●●●–●●●, ●●●●

ϩ Ϫ (GraphPad, San Diego, CA). Comparisons between groups were per- Confirming the previous observations, the Pkd1 / mice had a formed using two-tailed unpaired t test. Significance level was P Ͻ 0.05. lower urine output and higher urine osmolality at baseline. Water deprivation resulted in a similar weight loss (averaging ϩ ϩ ϩ Ϫ Results 14 Ϯ 0.7% in Pkd1 / and 13 Ϯ 0.2% in Pkd1 / ; n ϭ 4 pairs), Kidney Structure and Baseline Parameters but the urinary concentrating ability was significantly higher in ϩ Ϫ Macroscopic and histology analyses (Supplementary Figure Pkd1 / mice, as indicated by lower volume and higher urine 1) confirmed the normal kidney structure in heterozygous osmolality at the end of the test. ϩ/Ϫ ϩ/ϩ Pkd1 versus Pkd1 mice (20 to 35 wk old). In particular, no A test of acute water loading (2 ml intraperitoneally [approx- cysts or tubular dilations were observed in any segment of the ϩ Ϫ imately 70 ml/kg]) was performed to investigate the capacity of Pkd1 / kidneys. The baseline clinical and biologic parameters ϩ Ϫ Pkd1 mice to eliminate water during a 6-h period (Figure 1C). In / ϩ Ϫ are shown in Table 1. The Pkd1 mice had similar body and comparison with WT littermates, Pkd1 / mice showed a sig- kidney weight as wild-type (WT) littermates and similar ϩ Ϫ nificant decrease in their ability to excrete water up to 3 h after / ϩ Ϫ plasma urea and creatinine values. The Pkd1 mice showed a the water load. Although Pkd1 / mice were able to excrete three-fold decrease in urinary output, associated with a higher more water than the WT mice during the last 3 h of the test, the urine sodium/osmolality and a lower plasma sodium/osmola- total excreted volume of water after 6 h was slightly lower (total ϩ Ϫ lity in comparison with WT littermates, despite similar water 6-h urine output: 1720 Ϯ 70 ␮linPkd1 / mice versus 1894 Ϯ 88 ϩ ϩ ϩ Ϫ intake. These observations were confirmed in other sets of adult ␮linPkd1 / mice; n ϭ 9 pairs; P ϭ 0.88). Thus, the Pkd1 / ϩ/Ϫ mice, irrespective of gender. The Pkd1 mice were also char- mice have an inappropriate antidiuresis at baseline, a higher acterized by significantly lower cumulative urinary excretion of ability to concentrate urine when challenged by water depriva- AVP, calcium, and NOx and a trend for lower urinary PGE2, tion, and a decreased ability to excrete a water load. whereas the cAMP levels in kidney and urine and the plasma NOx were unchanged. The urinary concentrations of calcium ϩ Ϫ and AVP both were higher in Pkd1 / mice, reflecting the net Characterization of Renal V2R and Response to V2R water reabsorption. These data show that, in the absence of Antagonist ϩ Ϫ cystic changes and renal failure, the Pkd1 / mice are in posi- To characterize the distribution and affinity of V2R, we per- ϩ ϩ ϩ Ϫ tive water balance, with similar water intake but lower plasma formed autoradiography of Pkd1 / and Pkd1 / kidneys that sodium and osmolality. were incubated with a highly selective V2R ligand, alone or with an excess of unlabeled ligand or AVP (Figure 2A). We Urinary Concentrating Ability and Water Handling observed a dense specific labeling confined in the inner/outer A 24-h water deprivation was performed to assess the uri- medulla and papilla area, corresponding to the main localiza- nary concentrating ability in the Pkd1 mice (Figure 1, A and B). tion of the V2R in rodent CD. The binding pattern, as well as

Table 1. Baseline parameters, renal function, and water metabolisma

ϩ ϩ ϩ Ϫ Parameter Pkd1 / Pkd1 / P

Body weight (g) 28.7 Ϯ 0.12 (7) 27.9 Ϯ 0.43 (7) NS Kidney weight (% body wt) 1.38 Ϯ 0.05 (7) 1.23 Ϯ 0.05 (7) NS Plasma urea (mg/dl) 27.4 Ϯ 1.91 (7) 28.4 Ϯ 1.17 (7) NS Plasma creatinine (mg/dl) 0.27 Ϯ 0.02 (7) 0.29 Ϯ 0.01 (7) NS Water intake (ml/24 h) 5.7 Ϯ 0.3 (15) 5.5 Ϯ 0.4 (15) NS Urine volume (␮l/24 h) 1315 Ϯ 140 (15) 413 Ϯ 26 (15) Ͻ0.0001 Ϯ Ϯ Ͻ Urine osmolality (mosmol/kgH2O) 3034 227 (15) 4880 179 (15) 0.0001 Urine sodium (mM) 128.0 Ϯ 13.4 (7) 260.0 Ϯ 20.6 (7) 0.0002 Ϯ Ϯ Plasma osmolality (mosmol/kgH2O) 377 9 (12) 351 6 (13) 0.02 Plasma sodium (mM) 151.0 Ϯ 0.86 (7) 147.0 Ϯ 1.17 (7) 0.01 Urine AVP (pg/24 h) 1022 Ϯ 83 (15) 619 Ϯ 52 (15) 0.0003 Urine AVP (pg/ml) 930 Ϯ 121 (15) 1567 Ϯ 154 (15) 0.003 Renal cAMP (pmol/mg protein) 3.8 Ϯ 0.2 (8) 4.1 Ϯ 0.5 (8) NS Urine cAMP (nmol/24 h) 11.6 Ϯ 2.2 (5) 12.8 Ϯ 2.9 (5) NS Plasma NOx (␮M) 96 Ϯ 12 (9) 90 Ϯ 12 (9) NS Urine NOx (␮M/24 h) 4.2 Ϯ 0.4 (9) 1.8 Ϯ 0.2 (9) 0.0004 ␮ Ϯ Ϯ Urine PGE2 ( g/24 h) 7.1 1.1 (13) 4.6 0.8 (13) 0.07 Urine calcium (␮mol/24 h) 32 Ϯ 5 (15) 19 Ϯ 2 (15) 0.02 Urine calcium (␮M) 30 Ϯ 5 (15) 48 Ϯ 5 (15) 0.01

aData are means Ϯ SEM. Numbers in parentheses refer to number of mice. All urinary data are expressed in cumulative values per 24 h, obtained in metabolic cages. The urinary concentrations of AVP and calcium are also indicated since they may interact with luminal receptors. AVP, Arginine vasopressin; NOx, nitric oxide metabolites; PGE2, prostaglandin E2. J Am Soc Nephrol ●●: ●●●–●●●, ●●●● PKD1 Haploinsufficiency Causes SIAD 5

Figure 1. Response to water deprivation and water loading in Pkd1 mice. (A) Urine output was measured during 24-h baseline (BL) ϩ ϩ ϩ Ϫ ϩ Ϫ and 24-h water deprivation (WD) in four pairs of Pkd1 / and Pkd1 / mice. The Pkd1 / mice had a significantly lower urine output at BL and after 24-h WD. The WD resulted in a similar weight loss in both groups. (B) Urine osmolality (Uosm) was higher ϩ Ϫ ϩ ϩ ϩ Ϫ ϩ ϩ at BL and after WD in the Pkd1 / group versus Pkd1 / group. *P Ͻ 0.05, Pkd1 / versus Pkd1 / . (C) Nine pairs of mice were ϩ Ϫ administered an intraperitoneal injection of 2 ml of sterile water. In comparison with wild-type (WT) littermates, Pkd1 / mice ϩ Ϫ ϩ ϩ showed a significantly delayed ability to excrete water up to 3 h after water load. *P Ͻ 0.035, #P Ͻ 0.005, Pkd1 / versus Pkd1 / .

␤ the binding parameters (Kd and Bmax) were similar in both AQP3, the epithelial sodium channel (ENaC) -subunit, and the groups. We next tested the aquaretic response of Pkd1 mice to urea transporter 1 (UTA1) were measured (Figure 3). The ex- the V2R antagonist SR121463B. Using incremental dosages, we pression levels of these mediators were similar in both groups, showed a dosage-dependent increase in the aquaretic effect in except for a slight but significant decrease (average Ϫ21%) in ϩ Ϫ WT mice, whereas Pkd1 / mice showed a decreased sensitiv- the expression of endothelin 1 (ET1). The expression levels of ϩ ity to the V2R antagonist (lower diuresis during the 6-h period) transcripts related to intracellular Ca2 , such as adenylate cy- at dosages that ranged from 0.1 to 10 mg/kg (Figure 2B). The clase isoforms 3 and 6 (AC3, AC6), calmodulin (CaM), parval- decreased response to SR121463B was confirmed by a right bumin, and calcineurin A␣ or ␤ (PPP3CA, PP3CB) were similar. ϩ Ϫ shift of the dosage-response curve (Figure 2C), with Pkd1 / The mRNA levels of endothelial nitric oxide synthase (eNOS) mice showing a significantly higher ED50 in comparison with and neuronal nitric oxide synthase (nNOS), two mediators in ϩ ϩ ϩ Ϫ Pkd1 / mice (Log ED50: 0.723 Ϯ 0.03 in Pkd1 / versus 0.507 Ϯ cAMP-independent cell-surface expression of AQP2, were also ϩ ϩ 0.07 in Pkd1 / ; n ϭ 5 pairs; P ϭ 0.02). These data demonstrate similar in both groups. There was no upregulation of Pkd2 that, despite similar distribution and binding parameters of expression in kidney and brain. Importantly, the brain AVP ϩ Ϫ ϩ Ϫ V2R, the Pkd1 / mice have a decreased sensitivity to a V2R expression was unchanged in the Pkd1 / mice (average 108%) antagonist. despite chronic hypo-osmolality.

ϩ Ϫ Mechanism of Antidiuresis in Pkd1 / Mice: Real-Time Mechanism of Antidiuresis: Phosphorylation and RT-PCR Analyses Recruitment of AQP2 The potential mechanism for the inappropriate antidiuresis We next investigated whether the antidiuresis that was ob- ϩ Ϫ in the Pkd1 / mice was further investigated. We used real- served in the heterozygous Pkd1 mice could be related to mod- time RT-PCR to test for the differential expression of transcripts ifications in the AQP2 trafficking in the CD (Figure 4). Immu- that primarily are involved in the AVP signaling pathway, noblotting showed a significant upregulation of AQP2 and ϩ Ϫ including the V1a and V2 receptors, the calcium-sensing recep- p-AQP2 in membrane fractions from Pkd1 / kidneys, contrast- tor (CaR), endothelin-1 (ET1), AQP2, the cAMP-responsive el- ing with stable AQP1 levels (Figure 4A). Densitometry analysis ement binding protein (CREB, a mediator in gene transcription confirmed the significant increase in AQP2 (approximately 0.6- of AQP2) in the kidney, and AVP in the brain. In addition, fold) and p-AQP2 (approximately 1.15-fold) in the membrane ϩ Ϫ mRNA levels of other cAMP-dependent molecules, such as fractions that were obtained from Pkd1 / kidneys (Figure 4B). 6 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: ●●●–●●●, ●●●●

Figure 2. Autoradiography and binding properties of renal V2 receptors and efficacy of the vasopressin (AVP) V2 receptor (V2R) ϩ ϩ ϩ Ϫ antagonist SR121463B in Pkd1 mice. (A) Autoradiography of Pkd1 / and Pkd1 / kidneys that were incubated with the highly selective V2R ligand [3H]SR121463 alone (1.5 nM, total binding; a and b) and in the presence of 1 ␮M unlabeled SR121463B (c and d) or AVP (e and f; nonspecific binding). The obtained autoradiograms show a dense specific labeling confined in the inner/outer medulla and papilla area, corresponding to the main localization of the V2R in rodent collecting ducts (CD). Binding studies ϩ ϩ ϩ Ϫ showed that [3H]SR121463 binds with high affinity to renal V2R. The distribution pattern is similar in Pkd1 / and Pkd1 / kidneys. The saturation binding experiments revealed similar binding parameters of [3H]SR121463 for renal V2R in Pkd1 mice (NS; i.e., apparent equilibrium dissociation constant [Kd] and maximum binding density [Bmax]). (B) Hourly urine output after injection ϩ Ϫ of various dosages (0.1 to 30 mg/kg) of SR121463B in five pairs of mice. In comparison with WT littermates (solid trait), Pkd1 / mice (dashed trait) showed a systematically lower urine excretion during the 6-h period for dosages that ranged from 0.1 to 10 mg/kg. Each point is the mean of five mice in each group. (C) Dosage-response curves and cumulative ED50 determination. A ϩ Ϫ ϩ ϩ significant higher ED50 is observed in Pkd1 / mice versus WT littermates (Log ED50 mean 0.507 Ϯ 0.07 in Pkd1 / , 0.723 Ϯ 0.03 ϩ Ϫ ϩ ϩ in Pkd1 / ;nϭ 5 pairs). The Log ED50 value corresponds to dosage concentrations of 3.2 mg/kg in Pkd1 / and 5.3 mg/kg in ϩ Ϫ ϩ Ϫ ϩ ϩ Pkd1 / mice. Diuresis values (␮l per 6-h period) were significantly lower in Pkd1 / versus Pkd1 / at dosage concentrations of 1, 3, and 10 mg/kg. *P Ͻ 0.05.

The recruitment of AQP2 to the apical membrane of CD cells plasma membrane demonstrated a two-fold increase in the ϩ Ϫ was also reflected by its increased excretion in urine (Figure density of AQP2 labeling in the Pkd1 / mice (Figure 5C). Of 4C). note, there were no modifications in the structure or diameter ϩ Ϫ In strictly controlled conditions of incubation (Figure 5), the of the vasa recta in the Pkd1 / mice (data not shown). staining for both AQP2 and p-AQP2 was upregulated in the In view of the role of Rho signaling in regulating the cy- apical membrane of the principal CD cells in the medulla of toskeletal dynamics and AQP2 translocation in CD cells, we ϩ Ϫ kidneys from Pkd1 / mice (Figure 5A). In contrast, the AQP3 investigated the expression and phosphorylation of RhoA (Fig- labeling was restricted to the basolateral plasma membrane, ure 6A) and activity level of RhoA (Figure 6B) in the Pkd1 with no difference in staining intensity or distribution. Immu- kidneys. Western blotting demonstrated a significant upregu- nogold staining at the EM level (Figure 5B) showed a significant lation of p-RhoA, with downregulation of RhoA in kidney ϩ Ϫ labeling for AQP2 at the apical plasma membrane in most CD extracts from Pkd1 / mice. Furthermore, affinity precipitation ϩ ϩ principal cells in Pkd1 / mice (Figure 5B, top) but an even of GTP-Rho followed by Western blotting confirmed that the more abundant apical membrane labeling in the cells of amount of active RhoA was significantly decreased in the ϩ Ϫ ϩ Ϫ Pkd1 / mice (Figure 5B, bottom). Of interest, the principal cells Pkd1 / kidney extracts. The ERK1/2 signaling, another regu- ϩ Ϫ in the CD of Pkd1 / mice often exhibited extensive infoldings lator of Rho activity, was also downregulated as evidenced by (small microvilli or more probably microplicae as a result of the significant decrease of p-ERK1/2 over total ERK1/2 ratio in ϩ Ϫ apical plasma membrane infoldings), which were not observed homogenates from Pkd1 / kidneys (Figure 6C). These data ϩ ϩ as often in the Pkd1 / cells (Figure 5B, top versus bottom). demonstrate that the inappropriate water reabsorption that was ϩ Ϫ Morphometry analysis of the gold particles that localized at the observed in Pkd1 / mice reflects an increased phosphorylation J Am Soc Nephrol ●●: ●●●–●●●, ●●●● PKD1 Haploinsufficiency Causes SIAD 7

modifications in heterozygous Pkd1 mice, emphasize the im- ϩ portance of abnormal AVP and Ca2 signaling in ADPKD and ϩ Ϫ give insights in the potential roles of PKD1. Also, the Pkd1 / mice represent a model of inappropriate antidiuresis that may be useful to decipher the mechanisms that are involved in AQP2 trafficking. In contrast with the Pkd1-null mouse models, which are embryonically lethal, heterozygous Pkd1 mice have a normal growth and no detectable abnormalities at birth and during adulthood (17–21). With the exception of a limited number of renal and liver cysts in a minority of old mice (17,21,22), no detailed functional phenotype has been associated with Pkd1 Ϫ haploinsufficiency. Recent studies in cystic mouse (Pkd2 /tm1Som and pcy) and rat (PCK) models with various degree of renal failure pointed out that increased cAMP levels, secondary to abnormal V2R signaling in CD cells, could play a role in cyst progression (8–10). However, the effects of such an abnormal signaling could be masked by nonspecific structural changes that alter the osmotic water handling by the CD (16). Thus, ϩ Ϫ adult Pkd1 / mice offer the opportunity to test the functional consequences of a Pkd1 haploinsufficient state on AVP signaling and water handling in the absence of intercurrent mecha- Figure 3. Real-time reverse transcription–PCR quantification of the nisms. ϩ Ϫ mRNA expression of mediators that are involved in AVP signaling in Several lines of evidence show that Pkd1 / mice have a ϩ Ϫ the kidney and brain of Pkd1 mice. The mRNA levels were first SIAD. At baseline, the Pkd1 / mice have a decreased urinary adjusted to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), output with higher urine sodium/osmolality and lower plasma then normalized to the WT level set at 100% using the following for- ϩ/Ϫ ⌬ Ϫ ϩ Ϫ Ϫ⌬ Ϫ ϩ ϩ sodium/osmolality. After water deprivation, the Pkd1 mice mula: Ratio ϭ 2 [ Ct (GAPDH Target / ) Ct (GAPDH Target / ). are able to concentrate urine to a greater extent than WT litter- These normalized values (mean Ϯ SEM) are shown in the right mates. Conversely, they have an impaired ability to excrete a column. In addition to the haploinsufficiency in Pkd1, there was a significant decrease in the expression of endothelin-1 (ET1) in water load. All of these elements indicate that haploinsuffi- ϩ Ϫ Pkd1 / kidneys. Nine pairs of kidneys and 11 pairs of brains ciency in Pkd1 is associated with abnormal osmoregulation and were analyzed. a positive water balance. That both the water intake and the expression of AVP in the brain are unchanged, irrespective of the chronic hypo-osmolality, does suggest a central defect in ϩ Ϫ Pkd1 / mice. Such a central defect could reflect high expres- of AQP2 and RhoA and decreased activity of RhoA, promoting sion levels of polycystins in the brain (20) and a potential role the recruitment of AQP2 at the apical plasma membrane of the in the pathways that regulate AVP secretion. Of note, two principal cells. potential mechanisms may explain the significantly lower val- ϩ/Ϫ 2ϩ ues of urine AVP excretion in the Pkd1 mice: (1) The water [Ca ]i in IMCD from Pkd1 Mice For investigation of whether the heterozygous loss of Pkd1 is retention, causing a decrease in the urinary output, and (2) that 2ϩ urinary AVP excretion is influenced by the osmolar clearance sufficient to alter resting [Ca ]i in the principal cells of the CD, isolated IMCD that were dissected from three pairs of age- and (Cosm), as a result of interference with the reabsorption/deg- gender-matched Pkd1 mice were loaded with Fura-2 to measure radation of filtered AVP in the proximal tubule (31). Accord- 2ϩ 2ϩ ingly, the lower urinary AVP excretion could reflect the signif- [Ca ]i levels. As shown in Figure 7, [Ca ]i values were sig- ϩ Ϫ ϩ ϩ ϩ/Ϫ Ϯ nificantly lower in Pkd1 / versus Pkd1 / cells (146 Ϯ 3.0 icant decrease in Cosm in Pkd1 versus WT mice (3.9 0.2 Ϯ ␮ ϭ Ͻ versus 186 Ϯ 3.5 nM, respectively; P Ͻ 0.0001). versus 6.2 0.3 l/min; n 15 pairs; P 0.0001), leading to accelerated tubular degradation of AVP. Recent studies have Discussion shown an increased reactivity of the aortic and renal vascula- ϩ/Ϫ In this study, we show that reduced Pkd1 gene dosage in ture in Pkd1 mice (32), and a reduced renal blood flow could mouse leads to a syndrome of inappropriate antidiuresis explain such a reduced Cosm. At any rate, the positive water ϩ Ϫ (SIAD), in the absence of cystic changes and renal failure. The balance of noncystic Pkd1 / mice contrasts with the mild ϩ Ϫ heterozygous Pkd1 / mice are characterized by the inappro- concentrating defect reported in patients with ADPKD (14). The priate expression of AVP in brain and the recruitment of AQP2 existence of nephrogenic diabetes insipidus in conditions that in the apical plasma membrane of the CD principal cells, re- are associated with structural changes in the medulla (16) and 2ϩ flecting decreased [Ca ]i levels and decreased activity of the correlation between the number of renal cysts and the RhoA in these cells. These data, the first to document functional extent of the concentrating defect (33) suggest that the latter 8 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: ●●●–●●●, ●●●●

Figure 4. Expression of aquaporin 1 (AQP1), AQP2, and phosphorylated AQP2 (p-AQP2): Immunoblotting. (A) Representative immunoblots for AQP1, AQP2, and p-AQP2 in homogenate (H) and membrane (M) fractions that were prepared from Pkd1 mouse kidneys. Equal loads (20 ␮g) were compared, as verified by similar ␤-actin expression. Although there is no difference in AQP2 ϩ Ϫ expression in the H fractions, there is an upregulation of AQP2 and p-AQP2 in the M fractions from Pkd1 / kidneys. There is no difference in AQP1 expression in these very M fractions. (B) Densitometry analysis (core and glycosylated bands) confirms that there is a significant increase of AQP2 (relative OD 161 Ϯ 2%; P ϭ 0.01) and p-AQP2 (relative OD 214 Ϯ 6%; P ϭ 0.0002) in the ϩ Ϫ M fractions of Pkd1 / kidneys. (C) Representative immunoblot for AQP2 and p-AQP2 in Pkd1 mouse urine. Samples (15 ␮l) were ϩ Ϫ ϩ ϩ loaded and analyzed by Western blot under nonreducing condition. *P Ͻ 0.05, Pkd1 / versus Pkd1 / .

primarily reflects cystic changes in the medulla of patients with (EP3R)-mediated activation of RhoA, resulting in F-actin forma- ADPKD (11). tion and reduced insertion of AQP2 into the apical plasma Kidney-specific mechanisms are also involved in the inap- membrane (28,36,37). The stimulation of CaR may also activate ϩ/Ϫ propriate antidiuresis phenotype of the Pkd1 mice (Figure PKC isoforms that mediate AQP2 endocytosis (34,38). Several 8). In normal conditions, the binding of AVP to V2R at the abnormalities in these signaling pathways could potentially ϩ Ϫ basolateral pole of CD principal cells triggers a heterotrimeric lead to the inappropriate water retention in the Pkd1 / mice, G-protein–coupled cascade, activating AC6 and increasing as discussed next. cAMP levels, which leads to the phosphorylation of AQP2 at First, there is a consistent and highly significant decrease in Ser256 by protein kinase A (PKA), followed by the trafficking of 2ϩ ϩ/Ϫ [Ca ]i levels in isolated CD from Pkd1 mice. It is increas- p-AQP2 to the apical plasma membrane and the increase in the ingly recognized that the functional interaction between poly- osmotic water permeability of the cells. The cAMP-induced cystins 1 and 2 in primary cilia plays an important role in translocation of AQP2 is facilitated by PKA-mediated phos- ϩ luminal flow sensing and regulation of [Ca2 ] homeostasis in phorylation (Ser188) of the small GTP-binding protein RhoA, i response to mechanosensation in tubular cells (11,39–41). Sev- causing Rho inactivation and depolymerization of F-actin (28). eral lines of evidence suggest that disruption of the polycystins In some conditions, the V2R-mediated antidiuretic actions of ϩ pathway leads to reduced [Ca2 ] . For instance, decreased rest- AVP may be balanced by the apical V1aR and CaR in the i 2ϩ principal cells (31,34,35). Binding of luminal AVP to V1aR ing [Ca ]i levels have been observed in cultured cells that stimulates phospholipase C (PLC), leading to inositol trisphos- were derived from human ADPKD cysts (42) and vascular ϩ/Ϫ phate receptor (IP3R)-mediated release of Ca2ϩ smooth muscle cells from heterozygous Pkd2 mice (43). A from the endo- ϩ ϩ 2 2 significant decrease in [Ca ]i was also observed in vascular plasmic reticulum. In turn, increased [Ca ]i activates phos- ϩ/Ϫ phodiesterase-1 (PDE1) and inhibits AC6, leading to decreased smooth muscle cells from the Pkd1 mice that were used in 2ϩ cAMP. However, activation of CaR by high luminal calcium this study (32). By analogy, the lower [Ca ]i levels in IMCD ϩ Ϫ 2ϩ / concentrations leads to (1) increased [Ca ]i via PLC and IP3R that were isolated from Pkd1 mice could reflect the reduced 2ϩ and (2) activation of protein kinase C (PKC) and phosphoryla- Pkd1 dosage. Alternatively, the lower [Ca ]i may reflect a tion of ERK1/2, followed by activation of phospholipase A2 decreased activity of the apical V1aR and/or CaR signaling ϩ/Ϫ (PLA2), release of PGE2, and prostaglandin EP3 receptor (31,34,35). However, the Pkd1 mice showed significantly J Am Soc Nephrol ●●: ●●●–●●●, ●●●● PKD1 Haploinsufficiency Causes SIAD 9

Figure 5. Immunostaining and electron microscopy immunogold labeling for AQP2 in the kidneys of Pkd1 mice. (A) In comparison ϩ ϩ ϩ Ϫ with Pkd1 / , there is a strong increase in the apical signal for AQP2 (a and b) and p-AQP2 (c and d) in the kidneys of Pkd1 / mice. The typical staining for AQP3 (e and f) in the basolateral membrane of the principal cells (PC) is similar in both groups. Several unlabeled CD cells correspond to intercalated cells. Bar ϭ 50 ␮m. (B) Representative electron microscopy gold labeling of ϩ ϩ ϩ Ϫ AQP2 in the outer medullary CD of Pkd1 / (a) and Pkd1 / (b) mouse kidney. The labeling was restricted to PC and particularly ϩ Ϫ ϩ ϩ abundant at the plasma membrane. However, AQP2 labeling was remarkably more intense in Pkd1 / than in Pkd1 / kidneys. IC, intercalated cell; N, nucleus; J, tight junction; L, collecting duct lumen. Bar ϭ 0.5 ␮m. (C) Morphometric analysis of the density ϩ ϩ ϩ Ϫ of gold particles at the apical plasma membrane of Pkd1 / and Pkd1 / PC confirmed an approximately two-fold increase (*P Ͻ Ϫ ϩ Ϫ ϩ ϩ 1.10 6) in the apical AQP2 in the Pkd1 / versus Pkd1 / kidney (number of gold particles per millimeter of membrane length: 11.44 Ϯ 0.87 versus 5.18 Ϯ 0.58). The total number of particles and total membrane length were 725 particles over 140 mm (29 cells ϩ ϩ ϩ Ϫ from 3 Pkd1 / mice) and 1633 particles over 142 mm (27 cells from 3 Pkd1 / mice).

higher urinary concentration of calcium and AVP, with un- converted to a cystic-like phenotype, with cAMP-dependent 2ϩ changed expression of both V1aR and CaR in the kidney. activation of ERK and proliferation. Of note, lowering [Ca ]i ϩ Ϫ Second, we documented an increased p-AQP2 and recruit- alone (a situation that is similar to that observed in Pkd1 / ment of AQP2 in the apical plasma membrane of CD cells and kidneys) was not sufficient to activate ERK and proliferation in increased p-RhoA coupled to decreased activity of RhoA in the these cells (45). Many elements contribute to the differences ϩ Ϫ ϩ Ϫ Pkd1 / kidneys. These data suggest that inactivation of RhoA, between the native, noncystic Pkd1 / kidneys and cultured 2ϩ causing the depolymerization of F-actin, facilitates the AVP- cells, including time course and magnitude of [Ca ]i modifi- elicited insertion of AQP2 into the apical plasma membrane of cations, residual levels of polycystin-1, and adaptation mecha- ϩ/Ϫ 2ϩ the Pkd1 mice. Several factors could contribute to these nisms, yet the lower [Ca ]i levels that were observed in the 2ϩ ϩ/Ϫ modifications, including the decreased [Ca ]i ; the increased Pkd1 CD cells may represent an intermediate state, in which PKA-mediated phosphorylation of AQP2 and RhoA (28); and a further loss of polycystin-1 or changes in cAMP levels may the less active ERK-PLA2 pathway, as indicated by the lower lead to a proliferative or cystic phenotype. Conversely, a low 2ϩ 2ϩ p-ERK1/2 over total ERK1/2 ratio and a trend for lower uri- [Ca ]i could decrease the activity of Ca -dependent protein ␣ ␤ nary PGE2 excretion (28,36). These modifications of the ERK phosphatases and calcineurin-A and , resulting in higher pathway are different from observations in cultured renal cells. levels of phosphorylated AQP2 and a reduced recycling from Yamaguchi et al. (44) showed that the cAMP-dependent prolif- the apical plasma membrane (46). eration of cultured human ADPKD cyst-lining cells is mediated Third, the apical V1aR and CaR pathways seem to be inactive ϩ/Ϫ 2ϩ through phosphorylation/activation of ERK. By contrast, in Pkd1 mice, as evidenced by lower [Ca ]i in isolated CD, cAMP inhibits ERK activity and slows proliferation in normal decreased activity of the ERK-PLA2 pathway, and decreased epithelial cells from human kidney cortex. However, when RhoA activity. As discussed, the apical CaR may sense urinary immortalized mouse M1 cortical CD cells were treated with calcium levels and influence AQP2 targeting to adjust water 2ϩ calcium channel blockers or EGTA to lower [Ca ]i, the cells homeostasis. The experiments of Sands et al. (38), performed on 10 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: ●●●–●●●, ●●●●

Figure 6. Activity of RhoA and extracellular signal–regulated kinase (ERK) signaling in Pkd1 kidneys: Immunoblotting and affinity precipitation. (A) Representative immunoblots for p-RhoA and RhoA in homogenates from Pkd1 mouse kidneys. Equal loads (20 ␮g per lane) were compared, as verified by similar ␤-actin expression. After probing for p-RhoA, the membrane was stripped and ϩ Ϫ reprobed for RhoA. The upregulation of p-RhoA in the Pkd1 / group is reflected by the downregulation of RhoA. Densitometry ϩ Ϫ ϩ ϩ analysis confirms the significant increase of the p-RhoA over RhoA ratio in Pkd1 / kidneys versus 100% of the Pkd1 / group (749 Ϯ 165%; P Ͻ 0.001). (B) Representative immunoblot for quantification of active RhoA (GTP-bound) in Pkd1 mouse kidneys. Equal loads (20 ␮l per lane) were compared. Affinity precipitation followed by Western blotting confirmed that the amount of ϩ Ϫ active RhoA was decreased in the Pkd1 / kidneys. Densitometry analysis confirms that there is a significantly decreased amount ϩ Ϫ of active RhoA in Pkd1 / kidneys (relative OD 35 Ϯ 13%; P ϭ 0.01). (C) Representative immunoblot for phosphorylated and total ERK1/2 in homogenates from Pkd1 mouse kidneys. Equal loads (20 ␮g per lane) were compared and verified by similar ␤-actin ϩ Ϫ expression. There is a significant decrease of the p-ERK1/2 over ERK1/2 ratio in Pkd1 / kidneys, as confirmed by densitometry ϩ ϩ (46 Ϯ 7% versus Pkd1 / taken as 100%; P ϭ 0.02).

isolated rat IMCD, showed that increasing luminal calcium the mRNA level of targets of the V2R signaling pathway (in- from 1 to 5 mM causes a 30% decrease in the AVP-elicited cluding urea transporter 1 and the ␤ subunit of epithelial so- ϩ ϩ ϩ Ϫ osmotic water permeability but no change in the basal perme- dium channel) were similar in Pkd1 / and Pkd1 / mice. ability. Similar calcium concentrations were used to show the These data contrast with the elevation of cAMP, paralleled by link between CaR activation and AQP2 trafficking in cultured the upregulation of AQP2 and V2R mRNA that was observed cells (34). These calcium concentrations are 100-fold higher than in other PKD mouse models (8,9). However, all of these models ϩ Ϫ those observed in Pkd1 / mice (50 ␮M, only 1.5-fold higher show renal cysts, with a positive correlation between cAMP than in WT mice). A significant polyuria has also been observed level and the magnitude of cystic changes, suggesting a causal in hypercalciuric mouse models in vivo. For instance, the diure- link between the two (10). Although we did not detect an sis is increased two-fold in the Trpv5-null mice, characterized increase in cAMP in whole tissue, we cannot exclude that a ␮ 2ϩ by very high calciuresis (averaging 250 mol/24 h, six-fold lower [Ca ]i could favor a local increase in cAMP by a dual higher than in WT littermates) (47). Again, these conditions are effect on AC6 and phosphodiesterase E1 in the principal cells of ϩ Ϫ ϩ Ϫ very distinct from the Pkd1 / mice, which have no real hyper- the Pkd1 / mice. Indeed, recent studies suggested that com- calciuria (average 20 ␮mol/24 h). Regarding the luminal V1a partmentalization of cAMP signaling in microdomains may receptors, their antagonistic action has been demonstrated only participate in the regulation of AQP2 trafficking (48). in rabbit CD, with no evidence in rat or mouse kidney (31). Fifth, in addition to the aforementioned mechanisms, the Therefore, the increased luminal concentrations of calcium and trafficking of AQP2 in CD cells can be stimulated by cGMP via ϩ Ϫ ϩ Ϫ AVP in Pkd1 / mice versus WT probably have limited biologic activation of NOS (49). The Pkd1 / mice showed no difference relevance. in the renal expression of eNOS and nNOS isoforms but a Fourth, the unchanged cAMP levels in kidney and urine and significant decrease in the urinary excretion of NO metabolites. the stable expression of AQP2 and V2R mRNA expression in These data, which confirm previous reports of impaired NO ϩ Ϫ the kidney of Pkd1 / mice argue against a direct role of synthesis in this mouse model (17) and patients with ADPKD chronically increased cAMP levels in this model. Furthermore, (50,51), suggest that the cGMP-mediated cascade is probably J Am Soc Nephrol ●●: ●●●–●●●, ●●●● PKD1 Haploinsufficiency Causes SIAD 11

Figure 8. Model for the effects of PKD1 haploinsufficiency on 2ϩ 2ϩ Figure 7. Baseline intracellular Ca concentrations ([Ca ]i)in AVP signaling and AQP2 trafficking in the PC of CD. Repre- inner medullary CD (IMCD) tubules of Pkd1 mice. Different sentation of a typical PC of the CD showing the influence of ϩ/ϩ 2ϩ regions (n ϭ 24 per group) from six Pkd1 (filled symbols) [Ca ]i levels and various signaling pathways on the trafficking ϩ Ϫ ϩ Ϫ and six Pkd1 / (open symbols) IMCD tubules that originated of AQP2 and the modifications in Pkd1 / mice. In the normal 2ϩ from three pairs of mice were analyzed. [Ca ]i were lower in state, the binding of AVP to the basolateral V2R activates ϩ Ϫ the Pkd1 / group (open symbols) compared with WT group adenylyl cyclase 6 (AC6), resulting in cAMP-dependent activa- (filled symbols; 146 Ϯ 3.0 versus 186 Ϯ 3.5 nM, respectively; P Ͻ tion of protein kinase A (PKA) and phosphorylation of AQP2 0.0001). (Ser256) and its insertion into the apical membrane. The process is facilitated by PKA-mediated phosphorylation of RhoA (Ser188), which inactivates RhoA and causes the depolymerization of F-actin. The V2R-mediated effects of AVP could be not involved in the recruitment of AQP2. Furthermore, the ϩ ϩ Ϫ balanced by the apical vasopressin-1a (V1aR) and calcium- decreased [Ca2 ] in Pkd1 / CD could participate in the de- i sensing (CaR) receptors. Luminal AVP activates V1aR, which creased renal NOS activity, because eNOS and nNOS both are 2ϩ ϩ increases [Ca ]i via phospholipase C (PLC) and inositol 2 ϩ Ca dependent. Therefore, a reduced generation of NO could trisphosphate receptor (IP3R). In turn, the increased Ca2 acti- ϩ/Ϫ decrease the medullary blood flow, sensitizing the Pkd1 vates phosphodiesterase-1 (PDE1) and inhibits AC6, leading to ϩ mice to the sympathetic tone and contributing to the antidiure- decreased cAMP. High extracellular, urinary Ca2 levels can 2ϩ sis phenotype by a positive effect on medullary hypertonicity. activate CaR, leading to (1) increased [Ca ]i via PLC and IP3R Another factor is medullary ET1, which antagonizes the AVP- and (2) activation of PKC and dual phosphorylation of ERK1/2 induced cAMP accumulation in CD cells and increases medul- via PLC and diacylglycerol (DAG), leading to activation of phos- lary blood flow in vivo (52). The ET1 action is mediated by the pholipase A2 (PLA2) and release of PGE2, activation of prostaglan- ETB receptor, and it involves PLC, PLA2, and PKC, as well as din EP3 receptor, and downstream activation of RhoA with sub- 2ϩ sequent F-actin formation, which reduces the insertion of AQP2 [Ca ] , NO, and PGE2. Mice that lack ET1 in the CD show no i into the apical plasma membrane. The polycystin-1 and -2 abnormalities at baseline but a reduced ability to excrete urine interact in the primary cilium located in the apical plasma during acute water loading (53). Together with the reduced ϩ membrane of CD cells. In response to luminal flow, the poly- 2 ϩ ϩ [Ca ]i and decreased NO production, the mild but significant 2 2 cystin-1/2 complex regulates [Ca ]i by mediating a Ca entry ϩ/Ϫ ϩ decrease in the mRNA expression of ET1 evidenced in Pkd1 into the cell, which releases Ca2 stores via the ryanodine kidneys could thus contribute both to increased AVP signaling receptors (RyR) on the endoplasmic reticulum (ER). The hap- and to decreased medullary blood flow, leading to water re- loinsufficient Pkd1 state is characterized by the increased retention. cruitment of AQP2 into the apical plasma membrane, reflecting ϩ ϩ/Ϫ 2 The Pkd1 mice show a significant resistance to V2R an- decreased [Ca ]i, decreased ERK-PLA2 activity, increased tagonism, despite similar distribution and affinity of the V2R PKA-mediated phosphorylation of AQP2 and RhoA, and de- 2ϩ and unchanged mRNA expression of AVP. This observation creased activity of RhoA. The decreased [Ca ]i may also result in increased efficiency of the cAMP-mediated signaling in mi- may be relevant for the use of V2R antagonists in patients with crodomains of the cell. These events increase the efficiency of ADPKD, with the aim to interfere with the cystogenic effect of V2R-mediated signaling, leading to the recruitment of AQP2 in cAMP (11). In the sole orthologous model of ADPKD that has the apical plasma membrane and inappropriate reabsorption of Ϫ/tm1Som been investigated thus far (Pkd2 mouse), treatment with water by the PC. The large arrows indicate the changes that ϩ Ϫ the V2R antagonist OPC31260 has not been associated with were documented in Pkd1 / mice. The increased urinary con- significant changes in urine output and osmolality, and a com- centrations of calcium and AVP are between parentheses be- parison of its efficacy in WT and Pkd2 mice has not been cause there is no evidence of stimulated apical receptors. reported (9). Of interest, it has been shown recently that a Adapted from references (31,34–36). 12 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: ●●●–●●●, ●●●● partial loss of Pkd2 attenuated the polyuria and increased the Wilson PD: Expression of aquaporin-1 and -2 during urine osmolality in a mutant V2R mouse model of nephrogenic nephrogenesis and in autosomal dominant polycystic kid- diabetes insipidus (54), suggesting that an alteration of the ney disease. Am J Physiol 271: F169–F183, 1996 polycystin pathway may indeed cause water retention by CD 6. Mangoo-Karim R, Uchic M, Grant M, Shumate WA, Calvet principal cells. Another interesting observation is that hypona- JP, Park CH, Grantham JJ: Renal epithelial fluid secretion and cyst growth: The role of cyclic AMP. FASEB J 3: tremia is frequently observed in neonates with autosomal re- 2629–2632, 1989 cessive polycystic kidney disease (55). ϩ/Ϫ 7. Belibi FA, Reif G, Wallace DP, Yamaguchi T, Olsen L, Li H, Finally, it should be pointed out that the Pkd1 mice that Helmkamp GM Jr, Grantham JJ: Cyclic AMP promotes were studied here represent a potentially interesting model to growth and secretion in human polycystic kidney epithe- investigate water handling in the CD. A new disease entity, the lial cells. Kidney Int 66: 964–973, 2004 nephrogenic SIAD, has recently been attributed to activating 8. Gattone VH, Xiaofang W, Harris PC, Torres VE: Inhibition (gain-of-function) missense mutations in AVPR2, the gene that of renal cystic disease development and progression by a encodes V2R in humans (56). To the best of our knowledge, vasopressin V2 receptor antagonist. Nat Med 9: 1323–1326, there are no genetically modified mice with such an antidiure- 2003 sis phenotype at baseline. An impaired ability to lower urine 9. Torres VE, Xiaofang W, Qian Q, Somlo S, Harris PC, Gat- osmolality and increase urinary water excretion was recently tone VH: Effective treatment of an orthologous model of reported in mice that lack the taurine transporter gene Taut (57), autosomal dominant polycystic kidney disease. Nat Med 10: 363–364, 2004 which could play a role in primary cilia (58). Thus, in addition 10. Wang X, Gattone V 2nd, Harris PC, Torres VE: Effective- to the interactions between polycystins and calcium signaling ϩ/Ϫ ness of vasopressin V2 receptor antagonists OPC-31260 pathways, the Pkd1 mice could give insights into the mech- and OPC-41061 on polycystic kidney disease development anisms that govern osmoregulation, AVP signaling, and traf- in the PCK rat. J Am Soc Nephrol 16: 846–851, 2005 ficking of AQP2 in the CD. 11. Torres VE: Vasopressin antagonists in polycystic kidney disease. Kidney Int 68: 2405–2418, 2005 Acknowledgments 12. Christensen BM, Zelenina M, Aperia A, Nielsen S: Local- These studies were supported in part by the Fonds National de la ization and regulation of PKA-phosphorylated AQP2 in Recherche Scientifique, the Fonds de la Recherche Scientifique Me´di- response to V2-receptor agonist/antagonist treatment. cale, an Action de Recherche Concerte´e (ARC 05/10-328), an IAP VI, Am J Physiol Renal Physiol 278: F29–F42, 2000 and the EuReGene integrated project (FP6). 13. Nielsen S, Frokiaer J, Marples D, Kwon TH, Agre P, Knep- Some of these data were presented during the 39th annual meeting of per MA: Aquaporins in the kidney: From molecules to the American Society of Nephrology; November 16 through 19, 2006; medicine. Physiol Rev 82: 205–244, 2002 San Diego, CA; and published in abstract form (J Am Soc Nephrol 17: 14. Gabow PA, Kaehny WD, Johnson AM, Duley IT, Manco- 513A, 2006). Johnson M, Lezotte DC, Schrier RW: The clinical utility of We are grateful to V. Beaujean, Y. Cnops, H. Debaix, F. 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