Human ADPKD primary cyst epithelial cells with a novel, single codon deletion in the PKD1 gene...

doi: 10.1152/ajprenal.00285.2006292:F930-F945, 2007. First published 7 November 2006;Am J Physiol Renal Physiol

Angela Wandinger-Ness, Robert Bacallao and Seth L. AlperChang Xu, Sandro Rossetti, Lianwei Jiang, Peter C. Harris, Ursa Brown-Glaberman,

signaling2+ciliary polycystin localization and loss of flow-induced Ca

gene exhibit defectivePKD1single codon deletion in the Human ADPKD primary cyst epithelial cells with a novel,

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Human ADPKD primary cyst epithelial cells with a novel, single codondeletion in the PKD1 gene exhibit defective ciliary polycystin localization andloss of flow-induced Ca2� signaling

Chang Xu,1,2 Sandro Rossetti,3 Lianwei Jiang,1,2 Peter C. Harris,3 Ursa Brown-Glaberman,4

Angela Wandinger-Ness,4 Robert Bacallao,5 and Seth L. Alper1,2

1Molecular and Vascular Medicine Unit and Renal Division, Beth Israel Deaconess Medical Center, and 2Department ofMedicine, Harvard Medical School, Boston, Massachusetts; 3Departments of Medicine and Biochemistry, Mayo MedicalSchool, Rochester, Minnesota; 4Department of Pathology, University of New Mexico School of Medicine,Albuquerque, New Mexico; and 5Department of Medicine, University of Indiana School of Medicine, Indianapolis, Indiana

Submitted 24 July 2006; accepted in final form 1 November 2006

Xu C, Rossetti S, Jiang L, Harris PC, Brown-Glaberman U,Wandinger-Ness A, Bacallao R, Alper SL. Human ADPKDprimary cyst epithelial cells with a novel, single codon deletion inthe PKD1 gene exhibit defective ciliary polycystin localizationand loss of flow-induced Ca2� signaling. Am J Physiol RenalPhysiol 292: F930 –F945, 2007. First published November 8, 2006;doi:10.1152/ajprenal.00285.2006.—Autosomal dominant polycystickidney disease (ADPKD) gene products polycystin-1 (PC1) andpolycystin-2 (PC2) colocalize in the apical monocilia of renal epithe-lial cells. Mouse and human renal cells without PC1 protein showimpaired ciliary mechanosensation, and this impairment has beenproposed to promote cystogenesis. However, most cyst epithelia ofhuman ADPKD kidneys appear to express full-length PC1 and PC2 innormal or increased abundance. We show that confluent primaryADPKD cyst cells with the novel PC1 mutation �L2433 and withnormal abundance of PC1 and PC2 polypeptides lack ciliary PC1 andoften lack ciliary PC2, whereas PC1 and PC2 are both present in ciliaof confluent normal human kidney (NK) epithelial cells in primaryculture. Confluent NK cells respond to shear stress with transientincreases in cytoplasmic Ca2� concentration ([Ca2�]i), dependent onboth extracellular Ca2� and release from intracellular stores. Incontrast, ADPKD cyst cells lack flow-sensitive [Ca2�]i signaling andexhibit reduced endoplasmic reticulum Ca2� stores and store-deple-tion-operated Ca2� entry but retain near-normal [Ca2�]i responses toANG II and to vasopressin. Expression of wild-type and mutantCD16.7-PKD1(115–226) fusion proteins reveals within the COOH-terminal 112 amino acids of PC1 a coiled-coil domain-independentciliary localization signal. However, the coiled-coil domain is re-quired for CD16.7-PKD1(115–226) expression to accelerate decay ofthe flow-induced Ca2� signal in NK cells. These data provide evi-dence for ciliary dysfunction and polycystin mislocalization in humanADPKD cells with normal levels of PC1.

autosomal dominant polycystic kidney disease; monocilium; shearstress; protein trafficking; fura 2

AUTOSOMAL DOMINANT POLYCYSTIC kidney disease (ADPKD) isthe most common life-threatening monogenic human renaldisease, with a prevalence of between 1:400 and 1:1,000. It ischaracterized by progressive development and enlargement offluid-filled cysts originating from only �3% of nephrons,leading ultimately to renal failure in 50% of affected individ-uals. More than 85% of ADPKD cases are caused by mutations

in the PKD1 gene, with almost all remaining cases associatedwith PKD2 gene mutations. The PKD1 polypeptide geneproduct, polycystin-1 (PC1/TRPP1), is a 4,302-amino acid (aa)polypeptide with an NH2-terminal extracellular domain of�3,000 aa, �11 transmembrane domains, and a �200-aaCOOH-terminal cytoplasmic domain interacting with polycys-tin-2 (PC2/TRPP2) (46, 63), heterotrimeric G proteins, and theregulator of G protein signaling RGS7, among many otherproteins. The COOH-terminal tail of PC1 also upregulatesseveral transcriptional pathways, in part by regulated proteol-ysis (24), and activates endogenous Ca2�-permeable cationchannels of 20–30 pS in Xenopus laevis oocytes and HEK-293cells (64, 65). The PKD2 gene product, PC2, is a 968-aapolypeptide believed to function as a Ca2�-permeable cationchannel in the endoplasmic reticulum and/or at the plasmamembrane, independently or in complex with PC1 (11, 26, 33)or other proteins. The cellular functions of PC1, PC2, and thePC1/PC2 complex remain incompletely understood, but likelyinclude roles in epithelial cell proliferation, differentiation andtubulogenesis, matrix interaction, Ca2� signaling, and deter-mination of developmental asymmetry in the embryonic ven-tral node.

Nearly all polarized epithelial cell types express a centralapical monocilium with a “9 � 0” axoneme structure, longconsidered vestigial but periodically proposed to function as amechanosensor. Recent findings from diverse fields have con-verged to suggest a central role for the primary cilia of renaltubular epithelial cells in the cystogenesis of polycystic kidneydisease (8, 12, 41). After the Madin-Darby canine kidney(MDCK) cell cilium was shown to respond to mechanicalbending and to flow by transducing an increase in cytoplasmicCa2� concentration ([Ca2�]i) (42, 43), several genes encodingintraflagellar transport proteins of the green alga Chlamydo-monas were noted to encode cystic kidney disease genes and tolocalize to the renal epithelial cilium (40, 74). These findingspromoted the discovery of altered ciliary morphology in theorpk mouse (40) and provided additional insight into ciliarylocalization of the ADPKD gene homologs lov-1 and pkd2 insensory neurons of Caenorhabditis elegans, and ciliary orbasal body functions of several glomerulocystic disease genesfrom zebrafish.

Address for reprint requests and other correspondence: S. L. Alper, Molec-ular and Vascular Medicine Unit and Renal Div., Beth Israel DeaconessMedical Center E/RW763, 330 Brookline Ave., Boston, MA 02215 (e-mail:salper@bidmc.harvard.edu).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Renal Physiol 292: F930–F945, 2007.First published November 8, 2006; doi:10.1152/ajprenal.00285.2006.

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Nauli et al. (30) soon proposed that the PC1/PC2 complexfunctions as a flow-sensing mechanoreceptor in the primarycilia of primary cultures of mouse embryonic renal epithelialcells and showed that cells from pkd1(�/�) mice are deficientin this proposed sensing function. The partially defectiveflow-sensing in the orpk mouse (21, 55) further supported theproposed central role of defective ciliary sensation of and/orresponse to tubular flow to the cystogenesis of ADPKD (30).The pkd1(�/�) mouse embryonic renal epithelial cells inwhich flow-induced signaling defects were observed com-pletely lacked PC1 and PC2 polypeptides. A recent reportappearing after completion of the work presented here ex-tended this observation to human ADPKD cyst cells expressinglittle or no PC1 polypeptide (31). However, human ADPKD isalmost always characterized by normal or increased renallevels of apparently full-length PC1 polypeptide (32, 36, 37),despite the significantly truncated proteins encoded by mostPKD1 germline mutations (35). Indeed, phenotypically similarmurine polycystic kidney disease (PKD) results from knockoutand from overexpression of the wild-type pkd1 gene (44, 60).

Therefore, we compared ciliary expression of PC1 and PC2and flow-sensitive Ca2� signaling in primary human renalepithelial cells derived from normal kidneys (NK cells) or fromADPKD cysts (PKD cells). We report that NK cells and PKDcells with a novel heterozygous in-frame single codon deletionmutation in the PKD1 gene exhibit equivalent abundance ofPC1 and PC2 polypeptides, but differ in their ciliary localiza-tion of PC1 and PC2. Exposure to low shear stress increased[Ca2�]i in a minority of NK cells, but shear stress at slightlyhigher levels and at the still higher levels achieved duringdiuresis increased [Ca2�]i to higher peak values in most NKcells. In contrast, PKD cyst cells exhibited no flow-sensitiveelevation of [Ca2�]i at any level of shear stress. PKD cyst cellswere also characterized by reduced endoplasmic reticulumCa2� stores, reduced capacitative Ca2� entry, and near-normalhormone-induced Ca2� signaling. We also report that aa 115–226 of the PC1 COOH-terminal tail modulated flow-inducedNK cell Ca2� signaling through its coiled-coil domain andcontain a coiled-coil domain-independent ciliary localizationsequence.

METHODS

Cell culture. Human renal cyst epithelial cells (PKD) were har-vested from multiple superficial cortical cysts of kidneys resectedfrom ADPKD patients PKD 10–27-98 and PKD 3/14/00, in both ofwhom disease progression, family histories, and pathological exami-nation suggested germline PKD1 mutations. Cortical tubules weredissected from one freshly harvested cadaveric human kidney notused for transplant (NK 6–1-99) and from the grossly normal lowerpoles of two human kidneys (NK57M03 and NK 11–7-02) resected forrenal cell carcinoma. The epithelial cells from both sources weregrown in primary culture and passaged as described (4, 5). Cells forexperiments were transferred to glass coverslips coated with Vitrogencollagen (Conhesion Technologies, Palo Alto, CA), fed every otherday with Clonetics Renal Epithelial Cell Media (REBM, Clonetics),and grown to confluence 5–6 days after plating. Only cells betweenpassages 2 and 6 were used for experiments. Experiments presentedin Figs. 3–7 and in Supplemental Fig. 2 were replicated with andinclude cells from all donor lines (the online version of this articlecontains all supplementary material). PKD cyst cell results presentedin Figs. 8–10 and Supplemental Fig. 1 are from donor PKD 10/27/98.All discarded tissue was harvested according to Committee on Clin-

ical Investigations/Institutional Review Board protocols reviewed andapproved at Indiana University School of Medicine and Beth IsraelDeaconess Medical Center.

Human pancreatic adenocarcinoma cells (HPAC), HeLa, and HEK293 human embryonic kidney cells from American Type CultureCollection were grown in DMEM supplemented with 10% fetal calfserum.

Genomic DNA analysis. Genomic DNA was prepared from NKcells from individual NK57M03 and cyst cells from individual PKD10/27/98. All coding exons of the PKD1 and PKD2 genes wereamplified by PCR from genomic DNA as previously reported (51, 52)or with newly developed primers (available on request). Amplicons(300–600 ng) were analyzed by denaturing high-pressure liquidchromatography (DHPLC) performed at two temperatures on aDNASep Cartridge HT with the Wave System 3500HT (Trans-genomic, Omaha, NE) and eluted through a 2.5-min linear gradient ofbuffer A [5% triethylammonium acetate (TEAA)] and buffer B (5%TEAA and 25% acetonitrile). Samples with abnormal elution chro-matograms compared with a normal control were subjected to DNAsequencing. The DNA sequencing allowed identification of heterozy-gous variation in the gene sequence.

Antibodies and other reagents. Rabbit polyclonal anti-PC1 anti-body NM005 raised against the 223-aa recombinant PC1 COOH-terminal cytoplasmic domain aa 4070–4302 (66) was used as an Igfraction, with specificity confirmed by fusion protein antigen pread-sorption as previously described (50) and by small-interference(siRNA) knockdown as described below. Rabbit polyclonal antibodyleucine-rich repeat (LRR) (14) and monoclonal antibody 7e12 (36,37), both raised against the PC1 leucine-rich repeat domain, werepreviously described. Rabbit polyclonal antibody NM002 was raisedagainst PC1 aa 3619–3631, affinity-purified by peptide antigen col-umn, and specificity tested by siRNA knockdown. Anti-PC2 antibody(64) raised against a GST-PC2 fusion protein encoding the PC2COOH-terminal cytoplasmic aa 687–968 was originally obtainedfrom Dr. Oxana Beskrovnaya-Ibraghimova (Genzyme). Anti-CD16monoclonal antibody (mAb) 3G8 was used as an Ig fraction asdescribed (64, 65). Anti-N-acetylated-�-tubulin was obtained fromSigma, anti-calnexin from Stressgen, and anti-GM130 from BD Bio-sciences Pharmingen. FITC-labeled lectins were from Vector Labo-ratories. GsMTx-IV was obtained from Peptides International (Osaka,Japan). All other drugs were from Sigma.

Immunoblots. Cells scraped in ice-cold PBS in the presence ofComplete protease inhibitor (Roche Diagnostics, Mannheim, Ger-many) were pelleted, rinsed in the same medium, then suspended andboiled briefly in 1% SDS, 10 mM Tris �HCl, pH 7.4, or in RIPA buffer(150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris �HCl,pH 7.5). Samples were suspended in Laemmli sample buffer (100 mMTris �HCl, pH 6.8, 4% SDS, 20% glycerol, 0.1% bromophenol blue,5% �-mercaptoethanol) and incubated 30 min at 37°C before loadingonto Nupage 3–8% polyacrylamide-SDS Tris-Acetate gels (Invitro-gen, Carlsbad, CA) or Criterion 5% polyacrylamide-SDS gels (Bio-Rad) for PC1 analysis, and 7.5% acrylamide-SDS gels for PC2analysis. Proteins separated by electrophoresis were transferred at 100V for 1 h to nitrocellulose, blocked with 5% milk in BLOTTO buffer(20 mM Tris, 0.9% NaCl, 0.03% Tween 20, pH 7.4) for 30 min at37°C, and probed with the primary antibody for 1 h at 37°C followedby horseradish peroxidase-conjugated goat anti-rabbit-Ig for 1 h at20°C. Bound secondary antibody was detected by enhanced chemi-luminescence (Boehringer) on SB5 X-ray film (Kodak).

cDNA transfection. Lipofection was ineffective for transfection ofNK and PKD cyst cells. Therefore, cells were trypsinized for 7 min at37°C, pelleted, and resuspended at 1.5–2.5 � 106 cells/100 �l in basicprimary mammalian epithelial cell Nucleofector solution (AmaxaBiosystems, Cologne, Germany). After addition of 10 �g plasmidDNA, the mixture was placed in a 2-mm cuvette and electroporated atroom temperature in the Nucleofector device (Amaxa) with programT-05. Five hundred microliters of prewarmed REBM medium con-

F931ALTERED FLOW SIGNALING IN HUMAN ADPKD

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taining 10% FBS was immediately added to the cuvette, and thesuspension was plated to a 35-mm coverslip in a 60-mm2 culture dish.The transfected cells were incubated at 37°C for 5 days or longer inREBM containing 10% FBS. Apparent transfection efficiency as-sessed cytologically by GFP fluorescence 6 days postelectroporationwas up to 70% in both cell types. Transfection efficiency assessed byexpression of CD16.7-PKD1(115–226) was 15% in NK cells and 5%in PKD cyst cells.

Confocal immunofluorescence microscopy. Cell monolayers grownon coverslips were fixed for 30 min at room temperature with PBScontaining 3% (wt/vol) paraformaldehyde. Fixed cells were exten-sively rinsed with PBS, quenched with 50 mM lysine HCl, pH 8.0,exposed to 1% SDS for 15 min, and blocked for 15 min in PBS with1% bovine serum albumin and 0.05% saponin. After 4°C overnightincubation with the primary antibody against PC1 or PC2 (1:100),coverslips were incubated with Cy3-conjugated donkey anti-rabbit Igsecondary antibody (1:500) for 2 h at room temperature. Somecoverslips immunostained as above for PC1 or PC2 were thencostained with antibodies to the ciliary marker N-acetylated �-tubulin(1:500), the endoplasmic reticulum (ER) marker calnexin (1:500), orthe Golgi marker GM130 (1:200 dilution). On occasional coverslips(as specified), unfixed cells were preextracted for 5 min with 0.01%saponin in “cytoskeletal buffer” [containing (in mM) 138 KCl, 3MgCl2, 2 EGTA, and 10 2-N-morpholino-ethane-sulfonate, pH 6.1]before fixation and immunostaining as above.

Costaining with two rabbit polyclonal antibodies against PC2 andcalnexin (supplemental Fig. 3) was performed by the microwavedenaturation method (62). After completion of PC2 staining, cover-slips were microwaved 10 min in 10 mM citrate, pH 6.0, to denaturebound antibody molecules and prevent cross-reaction during subse-quent calnexin staining with the rabbit polyclonal antibody andFITC-coupled goat anti-rabbit Ig secondary antibody. The same mi-crowave procedure was used to costain with two mAbs against CD16and N-acetylated �-tubulin (see Fig. 9). In both cases, control incu-bations with secondary antibody post-microwave treatment aloneconfirmed successful denaturation of previously bound Ig (notshown). Immunostained cells were imaged with a Bio-Rad MRC-1024 laser-scanning confocal immunofluorescence microscope. Pixelfluorescence intensity of cilia and cell bodies in x-z sections of singlecells was measured with Bio-Rad software.

RNA knockdown. Six human PC1 siRNA sequences of 21 nt inlength complementary to several PC1 domains of the 14,135-pbcoding sequence of PKD1 (GenBank NM000296) were selectedwith Ambion’s “siRNA Target Finder tool” (www.ambion.com).The most effective sequence targeted nt 584 – 605 within the LRRdomain. A BLAST search confirmed the lack of significant homol-ogy with other human genes. Sense and antisense oligodeoxynucle-otides (Integrated DNA Technologies, Coralville, IA) were used togenerate templates and synthesize siRNA according to the manu-facturer’s instructions. PC1 siRNA, GAPDH siRNA, or controlscrambled siRNA (Ambion control template set 4800) were sepa-rately transfected into 50% confluent HPAC (50 –100 pmol/well ofa 6-well plate) using Lipofectamine 2000 (Invitrogen) per themanufacturer’s instructions. Knockdown efficacy was evaluated byimmunoblot of cells lysed in SDS-PAGE sample buffer containing5% 2-mercaptoethanol and resolved on precast 4 –15% gels (Bio-Rad Criterion), using the anti-PC1 antibody NM005 or monoclonalanti-GAPDH antibody (Ambion). Chemiluminescent signal wasquantitated with a PhosphoImager (Molecular Dynamics) usingImageQuant software. The fold-decrease was calculated relative tothe scrambled siRNA control. Maximal knockdown was observed48 h posttransfection.

Measurement of [Ca2�]i. NK and PKD cyst cells cultured toconfluence on collagen-coated 35-mm glass coverslips were loadedwith 5 �M fura 2-AM (Molecular Probes) in HEPES-buffered (pH7.2) at 37°C for 30 min. Extracellular fura 2-AM was removed bywashing twice with HEPES-HBSS. The coverslip was then mounted

into the bottom of a parallel-plate flow chamber (GlycoTech) 0.5 cmin width and 0.0254 cm in depth and perfused at room temperaturewith a Harvard Syringe pump. In some experiments, cells wereperfused at 37°C using a calibrated WPI in-line heater, with monitor-ing of inflow and outflow temperatures. Perfusion medium composi-tion was (in mM) 127 NaCl, 5.4 KCl, 1.27 CaCl2, 1 MgCl2, 5.6glucose, and 11.6 HEPES, final pH 7.2.

[Ca2�]i was measured by fluorescence ratio imaging with aMetafluor digital imaging system (Universal Imaging, West Chester,PA), equipped with an Olympus IMT-2 inverted microscope, and aCoolSNAP CCD camera (Photometrics, Tucson, AZ). Fura 2 emis-sion ratio images were monitored at 510 nm with alternating excita-tion at 340 and 380 nm. Fura 2 fluorescence ratio values determinedby in situ calibration in immortalized epithelial cells did not differfrom values determined by in vitro calibration for [Ca2�]i (29).Therefore, fura 2 fluorescence ratios were calibrated in vitro (29) withthe same experimental settings for the imaging system, using the Ca2�

calibration buffer kit no. 2 (Molecular Probes) with concentrationsbetween 36 nM and 4 �M [Ca2�]. The minimal fluorescence ratio(Rmin) was determined at “zero Ca2�” (free Ca2� 10 nM) and themaximal fluorescence ratio (Rmax) at 4 �M total Ca2�. The equilib-rium constant (Kb) was determined by fitting experimental fluores-cence ratio R values at various free [Ca2�] with the equation[Ca2�]free Kb (Sf2/Sb2)[(R � Rmin)/(Rmax � R)], where the factorSf2/Sb2 corrects for fura 2 ion selectivity at 380 nm.

For each coverslip, one visual field was selected as a region ofinterest, recorded before and during imposition of a uniform rate offluid flow.

Shear stress (�w) was calculated as �w 6�Q/a2b, where � apparent viscosity of superfusate (1.00 for H2O at 20°C; 0.70 at37°C), Q volumetric flow rate (ml/s), and a and b flow chamberdepth and width, respectively.

All cells within the visual field were analyzed as a single region ofinterest. On some coverslips, individual cells were analyzed sepa-rately as noted. Fluorescence ratio emission values were calculated ona pixel-by-pixel basis from ratio images and processed with Meta-morph software (Universal Imaging). The statistical significance ofdifferences in [Ca2�]i between groups was evaluated by Student’spaired or unpaired t-test or by Fisher’s test. These comparisonsbetween mean [Ca2�]i measured from all cells within the visual fieldwere corroborated by application of the Wilcoxon rank order test tocomparisons of means of single-cell [Ca2�]i from all cells within thevisual fields of two experimental groups (not shown).

In addition to analysis of all cells within a coverslip’s visual field,the subset of “responsive” NK cell coverslips was also analyzedseparately. An NK cell coverslip was defined as “unresponsive” if themean fura 2 fluorescence emission ratio of three-to-five arbitrarilyselected subregions of the recorded visual field did not increase duringflow to a peak value significantly higher than the ratio recorded beforeimposition of flow (paired Student’s t-test). Coverslips on which suchsubregions did show significantly increased fluorescence ratio duringflow were deemed responsive coverslips (see Fig. 6, A and B, for ananalysis of all NK coverslips and Fig. 6, C and D, for data from onlyresponsive coverslips).

For each coverslip of fura 2-loaded, cDNA-transfected cells, indi-vidual dsRed-positive and dsRed-negative cells in a visual field werepreselected as regions of interest, then recorded before and duringfluid flow. Pharmacological characterization (see Fig. 8A, C, and D)was carried out in a coverslip chamber of 1-ml volume to which100 � drug stock solution was added by pipette under “no-flowconditions,” which in the absence of drug elicited no increase in[Ca2�]i. Ca2� readdition (see Fig. 8D) was performed by superfusionat 0.75 dyne/cm2, or (as noted) by gentle manual replacement of halfthe cell chamber volume.

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RESULTS

A novel ADPKD mutation. Genomic DNA was preparedfrom cyst cells of individual PKD10–27-98 and from normalcortical tubular epithelial cells of individual NK57M03. Nomutations of the PKD1 or PKD2 genes were detected in theNK cell sample. In contrast, the PKD cyst cell genome re-vealed a heterozygous in-frame deletion of a tgc trinucleotidein exon 18 (7509_7511delTGC), encoding the predicted mu-tant polycystin-1 polypeptide PC1 �L2433 (p.Leu2433del)lacking a leucine in the receptor-for-egg jelly (REJ) domain(Fig. 1). The deleted Leu residue encoded by the human geneis identical in chicken, X. laevis, and Fugu, and is conserved asVal in mouse and rat. Two additional noncoding polymor-phisms were also detected in cis: IVS31–38C-G andIVS44�22delG. Since neither the coding deletion mutationnor the two IVS mutations were present in 100 chromosomesfrom 50 normal control individuals, the deletion is likely thegermline disease mutation. However, the anonymity con-straints under which kidneys were obtained prevented confir-mation by genotyping of family members. No PKD2 mutationswere found in this patient.

PC1 and PC2 polypeptide expression in NK and PKD cystcells. PC1 was detected by immunoblot of confluent NK celllysate with three distinct antibodies (7e12, NM005, and LRR)as a polypeptide of Mr �400 kDa (Fig. 2A). The total cellabundance of PC1 in confluent PKD cells was no less than thatin confluent NK cells (Fig. 2B). Immunoblot specificity of thepreviously characterized NM005 PC1 antibody (50) was con-firmed by the 85% reduction in the �400 kDa PC1 immuno-blot band in PC1 siRNA-treated HPAC cells (Fig. 2C). Asimilar PC1 band of �400 kDa was detected by all three

antibodies also in HEK 293 cells, HeLa cells, and HPAC cells(not shown). PC1 immunolocalization with the NM005 anti-body revealed a predominantly intracellular distribution in bothNK cells and PKD cells, with some PC1 detectable at lateralcell membranes in NK and PKD cells as previously described(50) (Supplemental Fig. 1).

PC2 migrated with Mr �110 kDa and was detected at equalabundance in NK and PKD cyst cells (Fig. 2B). PC2 localizedpredominantly to the ER in NK and PKD cells, as evidenced bycolocalization with calnexin (Supplemental Fig. 2), and con-sistent with previous reports (2, 18).

PKD cyst cells form primary cilia devoid of PC1. PC1 andPC2 have been colocalized, along with other ciliary geneproducts linked to cystic kidney disease, to the primary ciliumin mouse kidney, in primary cultured mouse kidney cells, andin several mammalian kidney cell lines (58, 66, 73). Humankidney cells in culture also exhibit primary cilia (40, 31).Figure 3 shows the presence of cilia in both NK and PKD cystcells as detected by immunostaining of the ciliary axonememarker N-acetylated �-tubulin. -Tubulin is also localized tocilia in both cell types (not shown). Neither cell type expresseda primary cilium at day 1 or 2 after splitting (50% conflu-ence), but by day 3 NK cells within confluent islands expressedshort primary cilia of 2.7 � 0.1 �m in length (n 32). Atconfluence 6 days after splitting, PKD cyst cell ciliary lengthwas 3.1 � 0.1 �m (n 73), slightly shorter than that of NKcells (4.2 � 0.1 �m; n 81; P 0.01).

PC1 polypeptide in NK cells colocalized in the centralcilium with N-acetylated-�-tubulin in all 37 NK cells examinedbut in none of the 38 PKD cyst cells immunostained withanti-PC1 antibody NM005 (Fig. 4). Ciliary immunostaining

Fig. 1. Novel heterozygous missense mutation in the human PKD1 gene. A: aligned nucleotide and predicted polypeptide sequences from exon 18 of wild-type(wt) and mutant PKD1 alleles in primary cyst epithelial cells. The mutant allele has a 3-nucleotide deletion, producing an in-frame deletion of a single codon,and encodes the polycystin 1 mutant polypeptide polycystin-1 (PC1) �L2433. B: aligned genomic DNA sequence traces from a representative wild-type genome(top) and the heterozygous mutant cyst cell genome (bottom). Underscored automated sequence call starts at the deletion site. C: schematic of the PC1 polypeptidelocating the Leu2433 deletion within the receptor-for-egg jelly (REJ) domain. TM, transmembrane.

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intensity exceeded that of the cell body. Use of anti-PC1antibody NM002 similarly revealed ciliary PC1 localization inall 24 NK cells examined, but in none of 17 additionallyexamined PKD cyst cells (not shown). PC2 polypeptide alsocolocalized with the ciliary marker in all 41 cilia examined inNK cells (Fig. 5A), with ciliary PC2 immunofluorescenceintensity again exceeding that of the cell body. However, PKDcyst cells exhibited two patterns of ciliary PC2 expression.Although bright ciliary PC2 staining was evident in 11 of 30PKD cyst cells (Fig. 5C), ciliary PC2 was undetectable in 19 ofthe 30 PKD cyst cells examined (Fig. 5B). The length of

PC2-positive cilia in these 30 PKD cyst cells was 2.8 � 0.2�m, whereas PC2-negative cilia were consistently shorter(2.2 � 0.1 �m; P 0.05), of greater width, and less orthog-onal in the fixed state than PC2-positive cilia (Fig. 5C). Thesedata demonstrate localization of PC1 and PC2 to primary ciliain human NK cells, consistent with the recent observations ofNauli et al. (31). In contrast, PKD cyst cells with normal PC1abundance lack ciliary PC1, and PC2 is absent from most ciliaof cyst cells.

ADPKD cyst cells lack the flow-induced [Ca2�]i increaseobserved in NK cells. Confluent, ciliated MDCK cells (43) andconfluent primary mouse embryonic kidney cells (30) re-sponded to low-level shear stress with increased [Ca2�]i. How-ever, primary mouse embryonic kidney cells from Pkd1(del34/del34) mice lacked this response to fluid flow. Although flow-sensitive Ca2� signaling has been measured in isolated,perfused rabbit (71) and mouse (21) collecting ducts, flow-sensitive Ca2� signaling had until recently (31) not beenexamined in human renal epithelial cells.

In NK cells, room temperature basal [Ca2�]i without flowwas 141 � 4 nM (n 141 coverslips). Abrupt application of0.75 dyne/cm2 shear stress (Fig. 6A) increased NK cell [Ca2�]i

by 19 � 5 nM in 66 coverslips studied (P 0.01). Cells on 43of these 66 coverslips exhibited no flow-induced [Ca2�]i in-crease and were considered unresponsive (see METHODS). Cellson the remaining 23 responsive coverslips (34%), consideredseparately, increased [Ca2�]i 56 � 10 nM above basal levels inresponse to this level of flow (Fig. 6C). An increase in shearstress from 0 to 2.3 dyne/cm2 (Fig. 6A) increased NK cell[Ca2�]i by 14 � 4 nM in 40 coverslips studied (P 0.01).Twenty-seven of these 40 coverslips were unresponsive (seeabove), and the 13 responsive coverslips (33%), consideredseparately, elevated [Ca2�]i 48 � 9 nM above basal levels(Fig. 6C). Cells on responsive coverslips exhibited [Ca2�]elevations which peaked at �10 s and returned to baselinewithin 40 s while flow was maintained (Fig. 6B).

Flow-induced [Ca2�]i increase in NK cells was abolished innominally Ca2�-free medium at both 0.75 and 2.3 dyn/cm2

(Fig. 6A, open symbols; P 0.01 compared with Ca2�-containing medium, Fisher’s exact test applied to all coverslipsstudied). The flow-sensitive and flow-insensitive states of NKcells on responsive and unresponsive coverslips could not beexplained by differences in resting, basal [Ca2�]i since, asshown in Supplemental Table 1, basal NK cell [Ca2�]i did notdiffer between responsive and unresponsive coverslips at anytested shear stress. In 2 of 10 coverslips of NK cells perfusedat 1.6 dyne/cm2 at 37°C, [Ca2�]i increased by 53 and 60 nM,

Fig. 2. Autosomal dominant polycystic kidney disease (ADPKD) cyst cellsexpress normal or elevated levels of PC1 and PC2. A: immunoblot of normalkidney cell (NK) lysates with 3 anti-PC1 antibodies: 7e12, NM005, and LRR.Lanes are from a single blot. Shown is a representative of 6 similar experi-ments. B: immunoblot of NK and ADPKD (PKD) cell lysates containingequivalent amounts of protein were probed with anti-PC1 antibody NM005.Shown is 1 of 2 similar experiments. C: PC1 polypeptide abundance in humanpancreatic adenocarcinoma cells (HPAC) is reduced by transfection of specificPC1 small-interference (si) RNA, whereas GAPDH is unaffected. Transfectionof control siRNA had no effect on either PC1 or GAPDH bands (not shown).Shown is a representative of 6 similar experiments. D: PC2 immunoblot of celllysates separated on 7.5% polyacrylamide gels and probed with anti-PC2antibody. PC2 was detected in cell lysates prepared in RIPA buffer (lanes 1and 3) or in SDS buffer (lanes 2 and 4). The same blot was reprobed withantibody to �-actin as a loading control. Shown is a representative of 3 similarexperiments.

Fig. 3. Identification of primary cilia in human renal epithelial cells in cultureby localization of N-acetylated �-tubulin in NK (left) and PKD cyst cells(right). Note that the primary cilia appear as dots (arrows) in this out-of-focusview from above the apical cell surface. When the dotlike structure was viewedby linescan through the x-z plane, the entire primary cilium can be seen (asshown in Figs. 4 and 5). Scale bar 10 �m.

Fig. 4. Colocalization of PC1 with N-acetylated �-tubulin is altered in PKDcells. Confocal x-z plane reconstructions show immunofluorescence colocal-ization of PC1 with N-acetylated �-tubulin in NK cells (A) but not in PKD cystcells (B). PC1 was detected in NK cell cilia (white arrows) by antibodyNM005. PC1 was absent from the PKD cyst cell cilium but present in the cellbody. Cells were preextracted with saponin before fixation (see METHODS),reducing nuclear staining of PC1. Scale bar 10 �m.

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but cells on the other 8 coverslips were unresponsive (notshown). Thus at these low levels of shear stress, neither themagnitude nor rate of flow-induced [Ca2�] increase, nor theproportion of responsive coverslips, was higher at 37°C than at20°C.

Shear stress values of 20 dyne/cm2 or more have beenestimated in rat collecting ducts during maximal diuresis (sum-marized in Ref. 3). As shown in Fig. 6B (F), increasing shearstress to 10 dyne/cm2 increased [Ca2�]i in NK cells by 160 �53 nM (n 16 coverslips, P 0.01) within 20 s of initiationof flow. After 60 s the falling [Ca2�]i level remained 45 � 17nM above the initial basal level. Among these 16 coverslips, 13were responsive to flow, and 3 were unresponsive. The respon-sive subset increased [Ca2�]i by 207 � 65 nM and after 60 smaintained a plateau [Ca2�]i of 63 � 22 nM above baseline

values (Fig. 6D). An abrupt increase in shear stress to the highlevel of 35 dyne/cm2 increased [Ca2�]i by 200 � 43 nM (n 19 coverslips, P 0.01). The responsive subset of 15 cover-slips exhibited a flow-induced [Ca2�]i of 252 � 43 nM within10 s, and [Ca2�]i remained 64 � 12 nM above the basal levelafter 60 s of perfusion (Fig. 6D). At both these higher shearlevels of 10 and 35 dyn/cm2, 80% of tested NK cell coverslipsexhibited flow-induced [Ca2�]i elevations. High shear stress-induced elevations of [Ca2�]i were similarly abolished in thenominal absence of extracellular Ca2� (Fig. 6B, �; P 10�4

compared with Ca2�-containing medium, Fisher’s exact testapplied to all coverslips studied). The NK cell [Ca2�]i responseto flow at 20°C exhibited a refractory period of �30 minfollowing cessation of flow at all tested shear stress values.

Tests of flow sensitivity at 37°C shortened both the time-to-peak values of [Ca2�]i and the refractory period without anincrease in peak magnitude. Ten of 11 coverslips of NK cellsexposed to 10 dyne/cm2 shear stress at 37°C exhibited flow-stimulated increases in [Ca2�]i to 107 � 6 nM over baseline,peaking between 5 and 10 s in 9 cells, and by 20 s in 1 cell.Rechallenge of these 10 responsive coverslips after 30 min ofstasis elicited identical elevations in [Ca2�]i in response to thesame shear stress. Similar magnitudes and kinetics of [Ca2�]i

increase were elicited in eight additional coverslips by expo-sure to 24 dyne/cm2 shear stress at 37°C. Thus increasing thetemperature from 20 to 37°C increased the rate of rise inflow-induced [Ca2�]i and reduced the refractory period of theCa2� signal to 30 min but did not increase signal magnitude(not shown).

PKD cyst cells exhibited basal (static) [Ca2�]i of 135 � 4nM (n 82), not different from the basal [Ca2�]i level in NKcells (141 � 4 nM, n 141). Imposition of shear stress at 0.75or 2.3 dyne/cm2 produced no significant elevation of [Ca2�]i inPKD cyst cells, with respective values of �[Ca2�]i after 10-sperfusion of 3 � 2 (n 25) and 2 � 2 nM (n 18) (Fig. 6E).These minimal increments differed significantly from the peakflow-induced [Ca2�] elevations in NK cells subjected to thesame shear stress (P 10�4, Fisher’s exact test applied to allcoverslips studied). At the higher shear stress levels of 10 and35 dyne/cm2, PKD cyst cell [Ca2�]i increased �1 � 1 nM(n 14) or 4 � 2 nM (n 25), respectively, (Fig. 6F), andagain differed from flow-induced [Ca2�]i elevations in NKcells at the same shear stress values (P 10�6, Fisher’s exacttest applied to all coverslips studied). Thus [Ca2�]i signaling inPKD cyst cells was uniformly unresponsive to flow across awide range of shear stress in the conditions tested.

Flow-induced Ca2� signaling in NK cells requires bothCa2� influx and Ca2� release from ryanodine-sensitive Ca2�

stores. We characterized the inhibitor pharmacology of flow-induced Ca2� signaling at the high shear stress of 35 dyne/cm2.Praetorius and Spring (42) showed that bending of the MDCKcell primary cilium with a micropipette led to [Ca2�]i eleva-tion, reflecting both Ca2� entry and release from inositol-1,4,5-triphosphate (IP3)-sensitive stores. However, Nauli et al. (30)found that flow-induced, cilium-dependent elevation of [Ca2�]i

in embryonic mouse collecting duct epithelial cells was insen-sitive to inhibitors of phospholipase C and IP3 receptors.

In NK cells, the phospholipase C inhibitor U73122 (10 �M)was without effect on flow-induced elevation of [Ca2�]i (Fig.7A), with a peak flow-induced [Ca2�]i increase of 132 � 20nM (n 5) in U73122-treated cells and 146 � 22 nM (n 6)

Fig. 5. Colocalization of PC2 with N-acetylated �-tubulin is altered in PKDcyst cells. Confocal x-z plane reconstructions showing immunofluorescencecolocalization of PC2 with N-acetylated �-tubulin in NK cells (A) and in PKDcyst cells (B and C). PC2 colocalizes with acetylated �-tubulin in the cilia(white arrows) of NK cells (A) but in only 30% of PKD cyst cells (B). In theother 70% of PKD cyst cells, PC2 was not detected in cilia (C). Scale bar 10 �m.

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in untreated cells. In contrast, 30 �M ryanodine nearly abol-ished the flow response, supporting a role for ryanodine recep-tor-regulated Ca2� stores (Fig. 7C) similar to that proposed forembryonic mouse collecting duct epithelial cells (30). Theflow-induced elevation of [Ca2�]i (224 � 34 nM, n 5) wasalso inhibited nearly completely (to 12 � 8 nM, n 8) by 20�M 2-aminophenylborate (2-APB; Fig. 7B). Since 2-APB

inhibits not only IP3 receptors but also store-operated Ca2�

entry channels, we examined additional inhibitors of Ca2�

entry. The NK cell peak response to initiation of flow (236 �18 nM, n 4) was reduced to 28 � 24 nM (n 9) by 10-minpreincubation with 3 �M GsMTx-IV (Fig. 7D), a potentblocker of mechanosensitive channels isolated from tarantulavenom (57). In contrast, the nonspecific cation channel inhib-

Fig. 6. Graded increases in shear stress induce graded, transient increases in cystosolic Ca2� concentration ([Ca2�]i) in NK cells (A–D) but not in PKD cyst cells(E and F), as measured by fura 2 fluorescence excitation ratio. A and B: [Ca2�]i increase in all the NK coverslips studied during initiation of superfusion withlow calculated shear stresses of 0.75 or 2.3 dyne/cm2 (A) and high shear stress of 10 or 35 dyne/cm2 (B) in the presence (filled symbols) or absence of addedperfusate Ca2� (open symbols). C and D: [Ca2�]i increase in the responsive subset of NK coverslips during initiation of superfusion with low calculated shearstresses of 0.75 or 2.3 dyne/cm2 (C) and with high shear stress of 10 or 35 dyne/cm2 (D). E and F: [Ca2�]i increase in PKD coverslips during initiation ofsuperfusion with low calculated shear stresses of 0.75 or 2.3 dyne/cm2 (E) and high shear stress of 10 or 35 dyne/cm2 (F). Parentheses indicate number ofcoverslips. G: time sequence of fluorescence ratio images of a representative NK coverslip subjected to 10 dyne/cm2 shear stress, as in C. Magnification �20.Pseudocolor scale is at left in all panels.

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itor SKF-96365 (50 �M before and during flow) attenuated anddelayed both activation and inactivation of the flow-inducedCa2� signal (Fig. 7E). After a 30-s lagtime following onset offlow, an increase in [Ca2�]i in a few cells gradually propagatedthroughout the NK population, increasing after �150 s to amodest peak of 67 � 14 nM by �150 s, with a greatlyprolonged decay time (n 6). In contrast, the rapid increase in[Ca2�]i in untreated NK cells (186 � 25 nM, n 4) wasalmost completely reversed by the time [Ca2�]i elevation wasevident in cells exposed to SKF-96365.

Figure 8 shows that PKD cyst cells sustained intact or partialactivities of other Ca2� signaling pathways, despite their lackof flow-responsive Ca2� signaling. The peak response to 10�M thapsigargin was smaller in PKD cyst cells (148 � 29 nM)than in NK cells (648 � 245 nM, n 8, P 0.01) (Fig. 8A),

suggesting that releaseable ER Ca2� stores of PKD cyst cellsare reduced. “Store-operated” or “capacitative Ca2� entry”(CCE) was also evaluated in the two cell types (Fig. 8B). Cellswere pretreated for 10 min with 10 �M thapsigargin in anominally Ca2�-free bath to deplete intracellular Ca2� stores,reducing [Ca2�]i in NK and PKD cells (n 11) to the similarlevels of 111 � 11 nM and 90 � 3 nM, respectively (P �0.05). Superfusion of this Ca2�-free bath at 0.75 dyne/cm2 foran additional 2 min (during which [Ca2�]i was unchanged; seeFig. 6A) was followed at t 0 by addition of 10 mM CaCl2 tothe superfusate (Fig. 8B). The peak CCE response of PKD cystcells was reduced (64 � 9 nM, n 11) compared with thelarger peak in NK cells (180 � 23 nM, n 11; P 0.01). Thepeak [Ca2�]i values in PKD cyst cells exposed (under no-flowconditions) to 1 �M angiotensin II (Fig. 8C) and to 1 �M

Fig. 7. Pharmacology of Ca2� signaling in NKcells exposed to calculated shear stress of 35dyne/cm2. A: fura 2-loaded cells were preincu-bated for 30 min in the absence (control, E) orpresence of 10 �M U73122, then subjected toflow in the continued absence or presence ofdrug. B–E: Flow-sensitive Ca2� signaling wassimilarly tested in the absence or presence of 20�M 2-aminophenylborate (2-APB; B), 30 �Mryanodine (C), or 50 �M SKF96365 (E). D:cells were also preincubated in the absence (con-trol) or presence of 3 �M Grammastola spatu-lata toxin IV (GsMTx) for 30 min, then sub-jected to fluid shear stress in the absence of thetoxin. Numbers in parentheses indicate “respon-sive coverslips” (80% of total studied) in A andE and control coverslips (E) and drug-treatedcoverslips studied (F) in B–D.

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arginine vasopressin (Fig. 8D) were equivalent in magnitude tothose of NK cells but exhibited slightly slower activation andsubstantially slower decay rates. Thus the nearly completeabrogation of flow sensitivity in PKD cyst cells did not reflecta global loss of Ca2� signaling responses.

CD16.7-PKD1(115–226) localizes to cilia of both NK andPKD cyst cells. Heterologous expression of the tripartite fusionprotein CD16.7-PKD1(115–226) increases endogenous Ca2�-permeable cation channel activity in X. laevis oocytes (64) andin EcR-293 cells (65). CD16.7-PKD1(115–226) contains acoiled-coil domain that can bind Ca2�-permeable cation chan-nel PC2. Therefore, we tested the hypothesis that heterologousexpression of CD16.7-PKD1(115–226) in NK and in PKD cystcells might through this mechanism modulate flow-sensitiveCa2� signaling in either cell type. Five days after transfection,unfixed cells were stained for cell surface expression of theCD16 epitope, then fixed. After microwave denaturation ofbound anti-CD16 mAb 3G8, coverslips were stained for N-

acetylated �-tubulin and studied by confocal immunofluores-cence microscopy. CD16.7PKD1-(115–226) was detected notonly on the surface membrane but throughout the cilium in allnine transfected NK cells examined (an example is shown inFig. 9, A–F) and was similarly present throughout the cilium ofall six transfected PKD cyst cells examined (Fig. 9, M–R). Wetested the dependence of this localization on the integrity of thePC1 COOH-terminal tail coiled-coil domain, which is requiredfor interaction with PC2 (63, 46). The PC1 coiled-coil domainmutant CD16.7PKD1-(115–226)L152P (64) was similarly ex-pressed at the cell surface and throughout the cilium in 6 of 6transfected NK cells examined (Fig. 9, G–L) and in 16 of 16transfected PKD cyst cells examined (Fig. 9, S–X). ThusCD16.7PKD1(115–226) can accumulate not only in plasmamembrane but also throughout the primary cilia of both NKand PKD cyst cells. Neither general cell surface localizationnor ciliary localization requires integrity of the PC1 COOH-terminal coiled-coil domain.

Fig. 8. Comparison of other Ca2� signaling pathways in fura 2-loaded NK (E) and PKD cyst cells (F). A: cells were exposed at t 0 to 10 �M thapsigargin(TG), gently added in “no-flow conditions” from a 100-fold concentrated stock. Vehicle alone was without effect (not shown). B: after a 10-min preincubationwith 10 �M TG in the nominal absence of Ca2�, cells were exposed to 10 mM extracellular Ca2� to elicit capacitative Ca2� entry (CCE). C and D: cells wereexposed at t 0 to 10 �M ANG II (C) or 10 �M arginine vasopressin (AVP; D). Numbers in parentheses are total number of coverslips studied.

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Expression of CD16.7-PKD1(115–226) in NK cells shortensthe duration of flow-induced [Ca2�]i increase. Five to six daysafter cotransfection of NK or PKD cyst cells with CD16.7-PKD1(115–226), the consequences to flow-induced Ca2� sig-naling were assessed. Transfected and nontransfected cells onsingle coverslips were identified by the respective presence orabsence of fluorescence from cotransfected dsRed (Fig. 10E,top left) which cosegregated with cell surface CD16 expression(not shown). Expression of DsRed alone in NK cells changedneither resting [Ca2�]i (170 � 6 nM in 21 single transfectedcells vs. 186 � 7 nM in 141 untransfected cells), peak flow-induced [Ca2�]i increase (212 � 36 nM in 21 DsRed cells vs.140 � 12 nM in 141 untransfected cells), nor the rate ofpostpeak decline in [Ca2�]i (Fig. 10C).

Resting [Ca2�]i in CD16.7-PKD1(115–226)-transfected(164 � 9 nM, n 41) and untransfected NK cells (167 � 3nM, n 219) was indistinguishable, as in EcR-293 cellsexpressing this construct (65). The flow-induced peak [Ca2�]i

increase in CD16.7-PKD1(115–226) expressing NK cells(124 � 39 nM) also did not differ from that of untransfectedcells (187 � 21 nM; Fig. 10A). However, 25 s after initiationof flow, [Ca2�]i in NK cells expressing CD16.7-PKD1(115–226) had fallen to lower values (66 � 13 nM) than in untrans-fected cells (116 � 11 nM, P 0.05). In contrast, expressionin NK cells of the coiled-coil domain mutant CD16.7-PKD1(115–226)L152P changed neither the peak flow-induced[Ca2�]i increase (237 � 76 nM vs. 179 � 17 nM in untrans-fected cells) nor its rate of postpeak decrease (Fig. 10B).Expression of CD16.7-PKD1(115–226) failed to rescue flow-sensitive [Ca2�]i signaling in PKD cyst cells (Fig. 10D). Thusheterologous expression of the terminal 112 aa of the PC1COOH-terminal cytoplasmic tail accelerates postpeak decay offlow-induced elevation in [Ca2�]i in NK cells by a mechanismthat requires integrity of the PC1 coiled-coil domain.

DISCUSSION

In the present study, we have shown that confluent primarycultures of normal human renal cortical tubular (NK) cellselevate [Ca2�]i in response to fluid flow. In contrast, confluentprimary cultures of human ADPKD cyst epithelial (PKD) cellsharboring a novel heterozygous in-frame single codon deletionin the PC1 gene completely lack this response. The defect isnot generalized, since PKD cyst cells retain near-normal Ca2�

signaling induced by angiotensin II and by vasopressin, as wellas reduced CCE and a reduced thapsigargin response. Theflow-induced [Ca2�]i elevation in NK cells requires extracel-lular Ca2� and release from ryanodine-sensitive intracellularstores. The NK and PKD cyst cells studied here expressequivalent whole-cell levels of PC1 and PC2, and both developmonocilia on achievement of confluency. However, cilia ofconfluent PKD cyst cells lack detectable PC1, whereas PC1and PC2 are both present in cilia of confluent NK cells. PC2 isexpressed in cilia of only 30% of confluent PKD cyst cells. TheCOOH-terminal PC1 fusion protein CD16.7-PKD1(115–226)localizes to the cilium of both NK and PKD cyst cells by amechanism independent of the PC1 coiled-coil domain. How-ever, accelerated decay of the flow-induced Ca2� signal in NKcells associated with overexpression of CD16.7-PKD1(115–226) requires integrity of that coiled-coil domain. These results

confirm and extend the recent study by Nauli et al. (31)published after completion of these experiments.

A novel in-frame single codon deletion in PC1 is associatedwith loss of ciliary PC1 and (in some cells) of ciliary PC2without reduction in total PC1 or PC2. The novel heterozy-gous PKD1 gene mutation �L2433 reported here is likely agermline mutation, since the epithelial cells from whichgenomic DNA was isolated were harvested from multiple renalcysts of a single ADPKD donor. The failure to detect addi-tional mutation(s) in the PKD1 gene is consistent with thesecond-hit hypothesis of cystogenesis (47). The deleted residueL2433 resides in the REJ domain of PC1. Overexpression inMDCK cells of engineered PC1 missense mutants in the REJdomain has been associated with loss of AP-1 activation and oftubulogenesis induced by wild-type PC1 overexpression (45).

The heterozygous PC1 �L2433 mutant allele is associatedwith normal cellular levels of PC1 and PC2 (Fig. 2). Expres-sion of PC1 and PC2 polypeptides in the cell body of all PKDcyst cells as noted in the present study resembles humanADPKD kidney and Pkd1(�/�) mouse kidney (66, 72) butdiffers from the mosaic (all-or-none) PC2 expression observedin renal cysts of the cy/� rat, and from the mosaic or completeloss of PC2 expression in the cy/cy mouse (34) and in thePkd2(�/�) and Pkd1(�/�)/Pkd2(�/�) mice (71). Thesefindings suggest that the PC1 �L2433 mutant polypeptide maybe present in PKD cyst cells.

PC1 antigen has been detected at tight junctions, adherensjunctions, desmosomes, focal adhesions, intracellular cytoplas-mic vesicles (summarized in Refs. 56 and 50), and nuclei (24),as well as in plasma membrane and cilia. These many PC1localizations may reflect examination of varied cell types anddifferentiation states, as well as posttranslational processing orcovalent modification of PC1, and (at least in rodents) alter-native splicing of PC1 transcripts. PC2 is predominantly local-ized in the ER (2). The presence of the heterozygous PC1�L2433 mutation is associated with complete loss of ciliaryPC1 expression and by loss of ciliary PC2 expression in 70%of cells (Figs. 4 and 5). Our results thus show that ciliarylocalization of PC2 does not require immunocytochemicallydetectable levels of ciliary PC1, consistent with recent findingson ciliary targeting of PC2 (10). The continued ciliary expres-sion of PC2 in 30% of cells with the novel PC1 �L2433mutation contrasts with the reported absence of ciliary PC2 inall SV40 large T-transformed, DBA-positive human 9–12 cystcells and in (genetically uncharacterized) primary human AD-PKD cyst epithelial cells (31). Ciliary localization of PC1 andPC2 was not described in a study of tsSV40LgT-immortalizedhuman ADPKD cells with the E1537X germline mutation,which express normal PC2 levels with very low levels of PC1(56).

Ciliary length was slightly shorter in PKD cyst cells than inNK cells of equivalent confluency, and cyst cilia lacking PC2tended not to be orthogonal in orientation. This ciliary pheno-type of human cyst epithelial cells in primary culture isintermediate in severity between the severely shortened, dys-morphic cilia of cells cultured from the orpk mouse with ahypomorphic polaris mutation (40) and the wild-type ciliarylength in cells from the pkd1del34/del34 mouse (30). Reportedwild-type ciliary length has varied widely: between 8 (42) and2–4 �m (75) in confluent MDCK cells, between 2 and 4 �m inimmortalized juvenile mouse collecting duct cells (75) and 12

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�m in immortalized embryonic mouse collecting duct cells,and between 3 and 5 �m in wild-type mouse and rat renalcortical tubules in situ (39). Thus the ciliary length of NK cellsin the current study falls within the wide range of previouslyreported ciliary lengths and matches the in situ ciliary length inrodent tubules.

The flow-sensitive Ca2� signaling response of NK cells isabsent from PKD cyst cells which retain other Ca2� responses.Flow-sensitive Ca2�-signaling is a property of isolated, per-fused rabbit and mouse collecting ducts (22, 21) and has alsobeen reported in isolated, perfused mouse medullary thickascending limb (19). In the orpk mouse model of recessivePKD, flow-evoked Ca2� signaling in the cortical collectingduct (CCD) remained normal during the first postnatal weekbut was slightly reduced compared with wild-type CCD at age2 wk (21), a difference replicated in CCD cells grown inprimary culture (55).

Our observation of the complete absence of flow-sensitiveCa2� signaling in human primary cultures of PKD cyst cellsresembles those previously reported in immortalized CCD cellsfrom the pkd1del34/del34 mouse and in immortalized and primaryhuman cyst cells (30, 31). Our results extend this earlier workin cyst cells lacking PC1 polypeptide by showing that the lackof flow-induced intracellular Ca2� signaling in human cystcells occurs also in the presence of normal total cell polypep-tide levels of PC1 and PC2, including the 30% of cyst cellswhich retain PC2 expression in the cilium. The results furthershow that human cyst cells fail to elevate [Ca2�]i in responseto flow at all tested levels of shear stress, and at both 20 and37°C. These results comparing adult NK cells with PKD cystcells isolated from end-stage ADPKD kidneys do not allowconclusions about the centrality of defective ciliary flow sens-ing to early cystogenesis or cyst growth in ADPKD.

The current results also present an initial pharmacologicalcharacterization of other Ca2� signaling responses of humanPKD cyst cells, indicating that the absence of flow-inducedelevation in [Ca2�]i in PKD cyst cells does not represent aglobal Ca2� signaling defect. Thus thapsigargin-induced Ca2�

release and CCE are both preserved in PKD cyst cells, al-though decreased in magnitude compared with NK cells (Fig.8). These diminished signals resemble those observed in aorticvascular smooth muscle cells of pkd2(�/�) mice (48). Unlikethe reduced thapsigargin and CCE responses, PKD cyst cellCai

2� elevations in response to angiotensin II and to AVP are ofnormal magnitude, although slightly delayed in onset. Thecomponents of the local renin-angiotensin system are overex-pressed in human ADPKD (23), with possible consequences toflow-induced regulation of apical exocytic insertion of AT1a

receptors in proximal tubular cells (16). The prolonged rate ofCa2� signal decay in PKD cyst cells treated with angiotensin IIor vasopressin recalls that produced in ATP-stimulated M1CCD cells by overexpression of a fusion protein containing the

PC1 COOH-terminal cytoplasmic tail (69), attributed by thoseauthors to prolongation of Ca2� entry. This prolonged rate ofCa2� signal decay may represent the converse of the acceler-ated Ca2� signal decay following ATP stimulation of MDCKcells overexpressing full-length PC1, attributed to enhancedER Ca2� reuptake with inhibition of CCE (13). CCE inhibitionby either overexpression or loss of PC1 function in PKD cystcells parallels the ability of both underexpression and overex-pression of PC1 to cause PKD in mice (60).

Range and uniformity of flow sensitivity of NK cell flow-induced Ca2� signaling. Resting [Ca2�]i measured in theabsence of flow at room temperature and at 37°C was indis-tinguishable in confluent, serum-replete NK and PKD cells andwas equivalent to that reported in confluent embryonic CCDcells from wild-type and pkd1del34/del34 mice and in humannormal and cyst cells by Nauli et al. (30, 31). Both sets of theseresting [Ca2�]i values substantially exceed those reported byYamaguchi et al. (73) in subconfluent primary human epithe-lial cells subjected to 48 h of progressively increasing degreesof serum starvation. In such low-serum conditions, favorablefor testing effects of growth regulators, human cyst cell [Ca2�]i

at 57 nM was significantly lower than the 77 nM measured innormal human cells (73). However, thapsigargin-induced de-pletion of Cai

2� stores in nominally Ca2�-free medium un-masked a similarly lower value of [Ca2�]i in our PKD cystcells compared with NK cells.

The current results with human primary NK cells differsomewhat in flow sensitivity from those reported for immor-talized mouse CCD cells, or for immortalized human cells withlectin-staining properties consistent with collecting duct originand primary human renal cortical epithelial cells of unspecifiedsegment of origin. Mouse CCD cells elevated [Ca2�]i inresponse to shear stress of 0.75 dyne/cm2 but failed to respondto the higher shear stress of 15 dyne/cm2 (30). Immortalizednormal human renal cortical tubular epithelial cells (RCTE)responded optimally to shear stress of 1.2 dyne/cm2 (31),results which we have reproduced with near-uniform respon-siveness of coverslips (Xu and Alper, unpublished observa-tions). However, only 5 of 8 tested coverslips of primarykidney cortical tubular epithelial cells were reported to exhibita flow response (31). NK cells from three individuals in thecurrent study responded to shear stresses of 0.75 or 2.3 dyne/cm2 with modest elevations in [Ca2�]i in only 33% of cover-slips. At 10 dyne/cm2 or at higher values, the robust [Ca2�]i

responses observed in 80% of tested coverslips were of amagnitude comparable to those reported by Nauli et al. (31).Factors distinguishing the NK cells of flow-responsive and offlow-unresponsive coverslips remain unclear and must includemechanisms other than ciliary localization of PC1 and PC2.However, at none of the shear stress values tested did respon-sive and unresponsive NK cell coverslips differ in baseline[Ca2�]i.

Fig. 9. A: COOH-terminal cytoplasmic tail fragment of PC1 localizes to cilia. Transiently transfected CD16.7-PKD1(115–226) is expressed in monocilia of NKcells (A–F) and in monocilia of transiently transfected PKD cyst cells (M–R). Transiently transfected CD16.7-PKD1(115–226)L152P is similarly expressed inmonocilia of both NK cells (G–L) and PKD cyst cells (S–X). Unfixed cells (AD, GJ, MP) were stained for surface expression of CD16 with mAb 3G8 (red),then fixed, microwave-denatured as described in METHODS, and stained for N-acetylated �-tubulin (green). Cells in S–X were fixed before staining withmonoclonal antibody (mAb) 3G8. Large panels show confocal immunofluorescence x-y images of ciliary planes, and small panels immediately below show x-zimages of the cells immediately above. Arrows mark cilia. Insets in A, G, and M: infraciliary x-y plane images of transfected cells (aligned with the higher x-yplane ciliary image of the same cell within each panel) which demonstrate generalized CD16 surface staining not restricted to cilia. Scale bars 10 �m in x-yimages; x-z images are magnified in the z plane.

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Fig. 10. A: COOH-terminal fragment of PC1 modulates flow-induced Ca2� signaling. Transient expression of CD16.7-PKD1(115–226), but not of the mutantCD16.7-PKD1(115–226)L152P, modulates the decay rate of the flow-induced increase in [Ca2�]i elicited by imposition of 35 dyn/cm2 shear stress. A, Timecourse of [Ca2�]i response in CD16.7-PKD1(115–226)-transfected and untransfected NK cells on 8 coverslips. Transfected cells were identified by dsRedexpression. B: time course of [Ca2�]i response in CD16.7-PKD1(115–226)L152P-transfected and untransfected NK cells on 5 coverslips. Transfected cells wereidentified by dsRed expression. C: time course of [Ca2�]i response in dsRed-transfected and untransfected NK cells on 3 coverslips. D: [Ca2�]i response ofCD16.7-PKD1(115–226)-transfected and untransfected PKD cyst cells on 7 coverslips. Numbers in parentheses are total numbers of single cells evaluated. E:fura 2 fluorescence ratio image time course of a representative coverslip of NK cells cotransfected with dsRed and CD16.7-PKD1(115–226). dsRed-expressingcells are outlined in white. Magnification �20. Pseudocolor scale is at left in all panels.

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The NK cell response to graded increases in shear stress withfurther increased elevation of [Ca2�]i, rather than by diminutionor loss of responsiveness, corresponds better to the MDCK cellresponse to flow (42) than to the mouse and human corticalepithelial cell responses to flow reported by Nauli et al. (30, 31).The responses of human NK cells in the current study were notaltered in magnitude or shear sensitivity by increasing temperaturefrom 20 to 37°C. However, the higher temperature accelerated thetime-to-peak [Ca2�]i and shortened the refractory period.

Thus NK cells in the current study were weakly responsiveto low shear stress and exhibited more robust responses toshear values associated with diuresis (3, 68) or with shearstress values close to those present in the central axis of theinitial S1 proximal tubule. These differences in flow sensitivitymay reflect differences in ciliary length (8–12 �m vs. 4 �m inthe current study), in conditions of initial tubule cell outgrowthor of subsequent primary cell culture, and likely reflect heter-ogeneity of the cultured population, and/or in nephron segmentof origin. Nearly all NK cells in the current study were Lotustetragonolobus agglutinin (LTA) positive, whereas only 10%of NK cells expressed Dolichos biflorus agglutinin (DBA).Although all NK cells expressed E-cadherin, strong stainingcharacterized only 10%. Similarly, most ADPKD cyst cells inthe current study were LTA positive, whereas none expressedDBA. Thus proximal tubular markers predominated among boththe NK cells and ADPKD cyst cells used in the present study.

Marker studies and cyst fluid composition have suggestedpossible proximal tubular origin of up to 30–44% of closedcysts in human ADPKD (reviewed in Refs. 61 and 70).Moreover, late-onset renal cysts in the Pkd1(�/�) mouseexpressing LTA were twice as frequently observed as cystsexpressing DBA, although most cysts expressed neither lectinmarker (25). The laminar shear stress predicted for the centralaxis of a human initial S1 proximal tubule of 22 �m diameteris predicted to be 7.6 dyn/cm2 for a 125 ml/min glomerularfiltration rate (with the oversimplifying assumptions of aninelastic tubule without brush border). For a vertical ciliumwith a length of 4.2 �m, the experienced shear stress underparabolic flow in this 22-�m-diameter tubule would be 4.7dyne/cm2 at the ciliary tip. These values might plausiblyincrease twofold in the oligonephronia proposed in essentialhypertension (15) or after uninephrectomy. Thus the shearstresses of 7–10 dyne/cm2 at which our NK cells exhibit amaximal peak elevation of [Ca2�]i are not far outside thephysiological range. Different nephron segments of origin maythus explain most of the difference in flow sensitivity betweenNK cells and the cells expressing collecting duct markersstudied by Nauli et al. (30, 31).

Pharmacological properties of flow-induced elevation of[Ca2�]i in NK cells. The pharmacology of flow-induced Ca2�

signaling in human NK cells resembled mouse CCD andMDCK cells in some ways and differed in others. The flowresponse in all these cell types required both extracellular Ca2�

and Ca2�-induced Ca2� release from internal stores. AlthoughIP3-sensitive Ca2� stores were implicated in MDCK cells (13)and in the perfused rabbit CCD (22), ryanodine-sensitive storesseemed to predominate in the flow response in human NK cellsand in mouse embryonic CCD cells. However, ryanodinereceptor inhibitors can reduce IP3-mediated Ca2� signaling incolonic smooth muscle cells (27). The reported ability of PC2to modulate IP3 receptor function by direct interaction (20)

appeared not to contribute to NK cell flow sensitivity. Whereas2-APB (10 �M, 45 min) was without effect in mouse embry-onic kidney cells, the NK cell flow response in NK cells wascompletely inhibited (20 �M, 30 min). However, interpretationof this inhibition is complicated by the drug’s dual inhibitoryeffects on IP3 receptor and TRP channels, and by its ability toactivate TRPV1–3 channels (6). The novel NK cell response toSKF96365, with reduced peak [Ca2�]i and slowed kinetics ofactivation and decay, may represent weak agonist activity forCa2� store release (7) rather than atypical inhibition of Ca2�

entry.Flow-activated Ca2� entry clearly requires integrity of the

PC1/PC2 complex, but the identity of the Ca2� entry pathwayin NK cells remains unknown. The density and diversity of ionchannels in monocilia may be very high (49). Complete blockof flow-induced Ca2� entry in NK cells by 3 �M GsMTx-IV,an inhibitor of stretch-activated cation channels, may provide apath toward channel identification, but a less specific lipidbilayer intercalation effect remains possible (57). TRPC1,previously shown to bind to PC2 in vitro, has been recentlyimplicated as a mechanosensitive channel in X. laevis oocytes(28). TRPV4 colocalizes with PC2 (59) and may bind andmodulate its activity (17). Since 30% of PKD cyst cells retainnormal ciliary localization of PC2, the presence of PC2 in ciliaapparently does not suffice for normal flow-induced Ca2�

signaling in the absence of ciliary PC1. However, primarycultures of cells isolated from a mature cyst of end-stageADPKD kidney may be dedifferentiated compared with NKcells in ways not specifically related to ADPKD.

The pharmacological properties of flow-induced Ca2� sig-naling in normal human kidney tubular epithelial cells inprimary culture were not reported by Nauli et al. (31). Never-theless, the similarities between our NK cells and the wild-typemouse CCD cells studied by Nauli et al. (30) include arequirement for extracellular Ca2� entry, sensitivity to ryano-dine, the kinetics of Ca2� signal onset and decline, and the timecourse for recovery from the post-flow refractory period at37°C. Thus even between cells expressing markers suggestingdifferent nephron segments of origin, some properties of flow-induced [Ca2�]i elevation are shared.

Overexpression of the PC1 COOH-terminal tail in NK cellsalters the decay kinetics of the flow-induced Ca2� signal.CD16.7-PKD1(115–226) overexpressed in NK cells acceler-ated decay kinetics of the flow-induced Ca2� signal, in contrastto prolongation of the ATP-induced Ca2� signal in M1 cells bya similar fusion protein (69). A disease mutation disrupting thecoiled-coil domain and blocking cation current activation inoocytes and EcR-293 cells prevented the accelerated decay ofthe flow-induced Ca2� signal in NK cells. This result suggeststhat CD16.7-PKD1(115–226) interacts with an endogenousprotein to modulate flow-induced Ca2� signaling. The inhibi-tory effect of CD16.7-PKD1(115–226) might therefore reflectcompetition with mechanosensitive full-length PC1 for bindingto or regulation of PC2 channel activity or signaling, asproposed by Low et al. (24) to explain cystic dilation of thezebrafish pronephric duct. However, the mechanism of mech-anosensation by the ciliary apical PC1/PC2 complex exposedto fluid flow may differ from that by basolateral PC1 or thePC1/PC2 complex exposed to a neighboring cell or to matrix.Moreover, Ca2� entry pathways induced by flow in NK cellsmay differ from those induced by ATP in M1 cells.

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Conclusion. Flow sensing has long been thought to contrib-ute to proximal tubular perfusion-absorption balance, to tubu-loglomerular feedback (9), to CCD K� secretion (71) and Na�

reabsorption (53), and to nitric oxide release by the thickascending limb of Henle’s loop (38) and the inner medullarycollecting duct (3). The cilium, with its apparent concentrationof receptors and signaling molecules, is an attractive candidateto integrate these signals controlling tubular epithelial celldifferentiation and function, perhaps through [Ca2�]i -medi-ated regulation of B-raf (73), regulation of mTOR (54), orother pathways. However, the role of ciliary flow sensing in theprevention of cystogenesis in the normal state remains inquestion, as evidenced in orpk mice by failure of polaristransgene rescue to prevent cystic disease even with the nor-malization of ciliary structure and correction of left-right asym-metry (1). Thus comparative studies of flow sensitivity inhuman renal cells and PKD cyst epithelial cells should play animportant and continuing role in defining the place of defectiveciliary mechanosensation in the pathogenesis of dysregulatedgrowth and secretion in human ADPKD.

ACKNOWLEDGMENTS

We thank Boris E. Shmukler, David H. Vandorpe, and Vince Carone forhelpful discussion, Oxana Ibraghimova-Beskrovnaya for antisera to PC1(LRR) and to PC2, Wayne Lencer for anti-GM130, Carrie Phillips for pathol-ogy support, Genevieve Philips for analytic assistance, and Elsa Romero andAlan Stuart-Tilley for expert technical assistance.

GRANTS

This work was supported by National Institute of Diabetes and Digestiveand Kidney Diseases Grants F32 DK-69049 to C. Xu, R01-DK-57662 to S. L.Alper, R01-DK-58816 to P. C. Harris, R01-DK-50141 to A. Wandinger-Ness,and a Polycystic Kidney Disease Foundation award to R. Bacallao.

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