Development of Polycystic Kidney Disease in Juvenile Cystic Kidney Mice: Insights into Pathogenesis, Ciliary Abnormalities, and Common Features with Human Disease
Laurie A. Smith*,
Nikolay O. Bukanov*,
Hervé Husson*,
Ryan J. Russo*,
Tiffany C. Barry*,
Ava L. Taylor*,
David R. Beier and
Oxana Ibraghimov-Beskrovnaya*
* Cell Biology, Genzyme Corporation, Framingham, and Division of Genetics, Brigham and Women’s Hospital, Boston, Massachusetts
Address correspondence to: Dr. Oxana Ibraghimov-Beskrovnaya, Genzyme Corporation, 5 Mountain Road, Framingham, MA 01701-9322. Phone: 508-270-2134; Fax: 508-620-1203; oxana.beskrovnaya{at}genzyme.com
Received for publication February 10, 2006.
Accepted for publication July 18, 2006.
Significant progress in understanding the molecular mechanismsof polycystic kidney disease (PKD) has been made in recent years.Translating this understanding into effective therapeutics willrequire testing in animal models that closely resemble humanPKD by multiple parameters. Similar to autosomal dominant PKD,juvenile cystic kidney (jck) mice develop cysts in multiplenephron segments, including cortical collecting ducts, distaltubules, and loop of Henle. The jck mice display gender dimorphismin kidney disease progression with more aggressive disease inmale mice. Gonadectomy experiments show that testosterone aggravatesthe severity of the disease in jck male mice, while female gonadalhormones have protective effects. EGF receptor is overexpressedand mislocalized in jck cystic epithelia, a hallmark of humandisease. Increased cAMP levels in jck kidneys and activationof the B-Raf/extracellular signal–regulated kinase pathwayare demonstrated. The effect of jck mutation on the expressionof Nek8, a NIMA-related (never in mitosis A) kinase, and polycystinsin jck cilia is shown for the first time. Nek8 overexpressionand loss of ciliary localization in jck epithelia are accompaniedby enhanced expression of polycystins along the cilia. The primarycilia in jck kidneys are significantly more lengthened thanthe cilia in wild-type mice, suggesting a role for Nek8 in controllingciliary length. Collectively, these data demonstrate that thejck mice should be useful for testing potential therapies andfor studying the molecular mechanisms that link ciliary structure/functionand cystogenesis.
Autosomal dominant polycystic kidney disease (ADPKD) is characterizedby formation and progressive enlargement of cysts in the kidneyand other organs (reviewed in references [1–5]). Mutationsin either PKD1 gene (approximately 85% of cases) or PKD2 gene(approximately 15% of cases) lead to the development of cystsin multiple segments of the nephron (6,7). Cystogenesis is characterizedby increased epithelial cell proliferation and apoptosis, lossof cellular polarity, and fluid secretion (2,8,9). Molecularpathways that lead to these abnormalities have been a subjectof extensive research by us and others in recent years (10–15).
Animal models of PKD have been instrumental in supporting studiesthat aim to understand the molecular mechanisms of cystogenesisand direct assessment of potential therapies. Recently, noveltherapeutic approaches have emerged (4,16–18). A numberof preclinical PKD models are available with mutations in differentgenes, including Pkd1 and Pkd2 genes (19). No single animalmodel perfectly recapitulates human ADPKD in a reasonable timeframe for therapeutic testing; however, each model representsa subset of human pathology. The usefulness of a particulartherapy for human PKD has been determined largely by studiesthat were done in more than one model (20–22). Therefore,models that more closely resemble human PKD by multiple parametersare highly desirable.
Cystic disease, whether it is caused by mutations in the PKD1gene or by mutations in a number of other genes, seems to besimilar. Common features include abnormal proliferation, proteinsorting, and intracystic fluid secretion. Recent data suggestthat ciliary dysfunction may represent one possible common abnormalityfound in different cystic diseases (23–25). The gene Nek8,encoding a member of NIMA-related (never in mitosis A) kinasefamily protein that is responsible for the juvenile cystic kidney(jck) mouse, was mapped recently to cilia (26–29). Unlikecpk and pcy lines, some of the first models with pathobiologybeing studied for more than two decades, the jck model is relativelynew and has not been characterized fully. Although the jck mutationis transmitted in autosomal recessive mode, it resembles humanADPKD phenotypically (19).
We set out to characterize phenotypic, cellular, and molecularaspects of cystogenesis in the jck mouse to determine its utilityfor testing of potential therapies for PKD. Here we report forthe first time that cysts in jck kidneys are formed from multiplesegments of the nephron, similar to ADPKD. We show that EGFreceptor (EGFR) is overexpressed and mislocalized to the apicalmembranes of cystic epithelia, a hallmark of human disease.In addition to the role of the EGFR pathway in jck cystogenesis,we demonstrate increased cAMP levels in jck kidneys, which maycontribute to increased proliferation and secretion of cysticepithelia, another resemblance of ADPKD. Importantly, similarto human disease and rat models of PKD and unlike many mousemodels, jck mice show gender dimorphism in the progression ofcystic disease, with gonadal hormones playing a role in mediatingdifferences in disease progression in male and female mice.Finally, we demonstrate ciliary lengthening in jck kidney epitheliawith abnormal ciliary expression of mutated Nek8 and polycystins.Taken together, our data suggest that the jck model should beextremely useful in further dissection of molecular mechanismsof cystogenesis and provide a foundation for testing novel therapiesin this model.
C57BL/6J jck Mouse Colony and Genotyping Assay
C57BL/6J jck/+ mice were obtained from The Jackson Laboratory(Bar Harbor, ME) and used to establish a breeding colony thatis maintained at Biomedical Research Models (Worcester, MA)and Genzyme (Framingham, MA). Genzyme and Biomedical ResearchModels Institutional Animal Care and Use Committees approvedall studies. Genotyping of jck mice was performed using a customTaqMan SNP genotyping assay (Applied Biosystems, Foster City,CA) to monitor the mutations described for jck Nek8 gene (26)(forward primer 5'-AGCCAGCCCACCATTGTAGA-3', reverse primer 5'-ACAGGGCCAGCACATGAGAG-3',wild-type [WT] probe 5'-VIC-CCTTGCTGGGCTATG-MGBNFQ-3', and MTprobe 5'-6FAM-CCTTGCTTGTCTATGAGATG-MGBNFQ-3'; Applied Biosystems).After denaturing at 95°C for 10 min, 40 cycles of 95°Cfor 15 s and 60°C for 1 min were performed. PCR productswere analyzed on ABI Prism 7700 sequence detector (Applied Biosystems).
Surgeries and Biochemical Studies
Mice were anesthetized with 3 to 5% isoflurane/oxygen beforecastration, ovariectomy, or sham operation. Vehicle or dihydrotestosterone(DHT) 50 mg/kg in 90% PEG 300/10% ethanol (vol/vol; Sigma-Aldrich,St Louis, MO) was administered subcutaneously from 26 to 64d. Mice were killed by CO2 asphyxiation, and kidneys were removedfor histologic examination. Serum urea nitrogen levels weremeasured using a VetACE analyzer (Alfa Wasserman, West Coldwell,NJ).
Histology and Quantitative Analysis of Cystogenesis
Longitudinal and cross-sections were cut at 4 µm and stainedwith hematoxylin and eosin with a Tissue Tek 2000 processor(Sakura-Finetek, Torrance, CA). Slides were digitized with anACIS system (Clarient, San Juan Capistrano, CA) and processedwith the Metamorph Imaging Series software (Molecular DevicesCorp., Dowington, PA). The severity of cystogenesis was quantifiedfrom both longitudinal and transverse sections. Cystic percentagewas measured as a ratio of cystic area to a total section area.
Immunostaining
Proliferating cell nuclear antigen (PCNA) staining was performedas described in the manufacturer’s protocol for the M.O.M.kit (Vector Laboratories, Burlingame, CA). Terminal deoxynucleotidyltransferase–mediated dUTP nick-end labeling (TUNEL) stainingwas performed using ApopTag Apoptosis Detection Kit (Chemicon,Temecula, CA). Lectins Dolichos Biflorus Agglutinin, Lotus TetragonolobusLectin (Vector Laboratories), anti-calbindin (Sigma-Aldrich),and anti-Tamm-Horsfall glycoprotein (US Biological, Swampscott,MA) antibodies were used as recommended by the manufacturers.Primary kidney epithelial cells were isolated from WT or jckkidney cortex as described previously (30). All primary cultureswere used between passages 2 and 5. Epithelial origin of cellswas identified using mouse anti-cytokeratin pan mAb (ChemiconInternational). Jck and WT cell cultures were stained with anti–polycystin-1(PC-1; leucine rich repeats [LRR], AA 27-360) or anti–PC-2(AA 687-968) rabbit antisera at 1:100 dilution as describedpreviously (11,31,32). Anti-Nek8 rabbit antibody was used at1:200 (26). Anti-acetylated tubulin (clone 6-11B-1; Sigma-Aldrich)was used at 1:400. Secondary antibodies used were goat anti-mouseAlexa fluor 488 and goat anti-rabbit Alexa fluor 546 (MolecularProbes, Eugene, OR). Cell nuclei were stained with DAPI (VectorLaboratories). Staining was visualized on an Olympus IX70 microscope(Olympus, Melville, NY). Images were captured with QED CameraPlug-In imaging system (Pittsburgh, PA).
Scanning Electron Microscopy
Kidney vibratome sections (200 µm) were fixed in 2.5%glutaraldehyde and 0.1 M cacodylate buffer, cryoprotected in70% ethanol, and freeze-fractured in liquid nitrogen. Afterfixation with 1% OsO4, sections were dehydrated with ethanoland viewed with a JEOL scanning electron microscope. The analysiswas performed on kidney cortex, where cortical collecting ductscan be identified by the presence of morphologically distinctintercalated cells.
SDS-PAGE and Immunoblotting
Proteins were resolved by SDS-PAGE using 3 to 12% gradient gelsand blotted as described previously (33). Membranes were blockedwith 3% BSA and incubated overnight with anti–extracellularsignal–regulated kinase (anti-ERK), anti–phosphorylatedERK 1/2 (Thr202/Tyr204), anti–B-Raf (Cell Signaling, Danvers,MA), and anti-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA)antibodies. Equal loading of protein was controlled by anti-glyceraldehyde-3-phosphatedehydrogenase staining (US Biologic). Membranes were washedin TBST and incubated with horseradish peroxidase (HRP)-labeledsecondary antibodies at 1:10,000 dilution (anti-mouse IgG-HRP,anti-rabbit IgG-HRP, or anti-goat-HRP; Promega, Madison, WI).Immunoreactive proteins were detected using enhanced chemiluminescence(Amersham Pharmacia Biotech, Little Chalfont Buckinghamshire,England).
cAMP Measurement
Frozen kidneys were homogenized in 10 vol/wt 0.1 N HCl. Aftercentrifugation at 600 x g, supernatants were collected and assayedby competitive ELISA as recommended by the manufacturer (R&DSystems, Minneapolis, MN). Protein concentrations were determinedwith BCA protein assay kit (Pierce, Rockford, IL).
Statistical Analyses
Data are expressed as means ± SD. Comparisons were madeby two-tailed t test, and significance was accepted at P <0.05.
Sex Dimorphism in the Course of Disease Progression in C57BL/6J jck Mice
To assess the development of polycystic kidney disease in jckanimals of C57BL/6J genetic background, we analyzed mutant miceat 26, 38, 45, 50, 64, and 100 d of age. The disease was progressivewith enlargement of kidneys over time, particularly in malemice, evident by initial assessment of kidney weights relativeto body weights (Figure 1A). To quantify the percentage of cystictissue, we used MetaMorph analysis of hematoxylin- and eosin-stainedkidney sections. This analysis confirmed that PKD was more severein male than in female mice (Figure 1A). Kidneys of jck micewere significantly enlarged by day 26 in both male and femalemice as compared with the age-matched WT mice. Sex differencesin cystogenesis were evident later in the course of the disease,between 38 and 64 d (Figure 1A and Supplementary Table 1). Also,analysis of renal function showed progressive elevation in serumcreatinine and blood urea nitrogen over time, again, to a largerdegree in male than in female mice (Figure 1A and SupplementaryTable 1).
Figure 1. Disease progression in C57BL/6J juvenile cystic kidney (jck) mice. (A) Quantitative analysis of disease progression. Shown are percentage of kidney to body weight ratio (K/BW ratio), percentage of cystic tissue (Cyst %), and blood urea nitrogen (BUN; mg/dl) relative to age. Note more aggressive increase in all of the parameters in male versus female mice during the course of the disease. *P < 0.05 male versus female mice at a given age. (B) Renal pathology of jck mice. Representative scans of hematoxylin and eosin (H&E)-stained kidney sections of jck male and female mice of 26, 45, and 64 d of age are shown. (C) Kaplan-Meier survival analysis of jck male and female mice. Percentage survival of jck male (n = 80) and female mice (n = 138) is plotted against age in weeks.
Microscopic examination of the jck kidney sections revealedmultiple cysts in the cortex and medulla at day 26, althoughthe majority of kidney tissue was not affected (Figure 1B).Cysts increased in number and size over time, and disease progressedsignificantly by day 45. At 64 d of age, little normal tissueremained, and large cysts were seen in both cortex and medulla(Figure 1B). No extrarenal pathology was identified in the jckline as described previously (27). Kaplan-Meier analysis wasused to estimate survival in jck male and female mice (Figure 1C).The median average survival for jck female mice was 19 wk comparedwith male mice at 16 wk of age, showing a statistical difference(P < 0.05) in survival between the sexes.
Cysts Are Formed in Multiple Nephron Segments of jck Kidneys
We examined the origin of nephron segments that were affectedby cystogenesis in jck mice early and late in the course ofthe disease. Immunohistochemical staining of jck kidney sectionsfrom 26-d-old (early stage) and 50-d-old (late stage) mice wasperformed using markers for different parts of the nephron:proximal tubule (PT), loop of Henle (LH), distal tubule (DT),and cortical collecting duct (CD) as indicated in Figure 2.At the early stage of disease, the majority of cysts were formedin cortical CD, whereas small cysts were identified in DT (Figure 2A).At the late stage of the disease (50 d), large DT and CD cystswere evident. Medullary cysts were formed in LH, but no medullaryCD cysts were detected. Proximal and glomerular cysts were notdetected in jck mice. Analysis of the dynamics of cystogenesisshowed that CD cysts contributed to cystogenesis early in thedisease, and cysts from DT and LH continued to develop duringthe course of the disease and contributed most to the totalcystic tissue at day 64 (Figure 2B). Such detailed understandingof jck kidney cystogenesis should be useful for testing potentialtherapies, because it is possible that therapeutic targets mayexpress differentially in different nephron segments.
Figure 2. Multiple segments of the nephron are affected by cystogenesis in jck mouse. (A) Immunofluorescence staining of jck female kidney sections with nephron-specific markers. Kidneys from 26- and 50-d-old animals were used as indicated. Collecting ducts (CD) were stained with Dolichos Biflorus Agglutinin lectin (DBA); proximal tubules (PT) were stained with Lotus Tetragonolobus Lectin (LTL); distal tubules (DT) were stained with anti-calbindin antibodies; thick ascending limbs of Henle (LH) were stained with Tamm-Horsfall glycoprotein (THG) antibody. Note that in the early stage of disease (26 d), the majority of cortical cysts are of CD origin (top, left), cysts of DT origin are small (top, center). Later in the disease (50 d), fast-growing DT cysts are seen (bottom, center). Medullary cysts are from LH (bottom, right). PT are not affected (top, right). (B) Dynamic of cystogenesis in jck mice. Kidney sections that were stained with nephron-specific markers were processed with MetaMorph to calculate total cystic tissue (Total) and contribution of cysts of different origin. Each time point group is represented by two mice; for each mouse, two complete kidney sections were processed (longitudinal and cross-section).
Similarities between Pathways of Human and jck Cystogenesis
In human PKD and some but not all animal models of cystic disease,EGFR was found to be abnormally expressed at the apical membraneof cystic cells, indicating loss of epithelial polarity (34,35).We examined the status of EGFR expression in jck kidneys. AbnormalEGFR expression was detected in apical membranes of cystic epitheliaboth early and late in the disease by immunofluorescence andWestern blot analysis (Figure 3, A and B). Therefore, similarto human PKD, EGFR was abnormally expressed in jck cystic epithelia.
Figure 3. EGF receptor (EGFR) axis and cAMP-activated pathways in jck model. (A) EGFR is mislocalized to the apical membranes in cystic epithelia. Shown are kidney sections from wild-type (WT) mice (left) and jck mice of 26 (center) and 50 d of age (right) stained with anti-EGFR antibody (red). (B) Upregulation of EGFR expression in jck kidney. Shown is Western blot analysis of EGFR from kidney lysates of WT and jck mice of 26 and 50 d of age (200 µg/lane). Note the increased expression of EGFR as disease progresses. (C) Activation of cAMP pathway in jck kidneys. The level of cAMP was normalized by total amount of protein. WT kidney corresponds to 64-d-old mouse. (D) Activation of extracellular signal–regulated kinase (ERK) and B-Raf pathways in jck kidneys. Note upregulation of B-Raf, ERK 1/2, and phosphorylated ERK 1/2 (p-ERK 1/2) in jck kidney extracts (50 µg/lane).
We also examined cAMP levels in jck kidney because recent evidencesuggests that cAMP is critical in specifically promoting proliferationin cystic cells (36,37). cAMP content was measured by ELISAin jck kidneys of 26- and 50-d-old mice (Figure 3C). Significantupregulation of cAMP levels was detected in cystic samples,and the level of cAMP increased with progression of the disease.It has been shown that cAMP activates the B-Raf/ERK pathwayin human ADPKD cells; therefore, we studied the possible activationof this pathway in jck mice. Western blot analysis showed thatERK 1/2 protein expression levels are increased in jck kidneys,in addition to increased levels of ERK phosphorylation (Figure 3D).We also detected increased levels of B-Raf (both 90- and 68-kDisoforms) in jck kidneys compared with WT controls (Figure 3D).Therefore, it is likely that, similar to ADPKD, increased levelsof cAMP in jck kidney play a role in activating the ERK pathway.
Increased Proliferation and Apoptosis of jck Cystic Epithelia
To investigate whether increased proliferation and apoptosiscontribute to jck cystogenesis, as has been shown for humanPKD (38), we performed PCNA immunostaining and TUNEL labelingof WT and jck kidneys. PCNA-positive nuclei and TUNEL-positivenuclei commonly were seen in cyst-lining epithelial cells, whereasvery few reactive cells were found in normal mouse kidney. Likewise,apoptotic nuclei were very common in cystic epithelia, but fewwere seen in normal tubular epithelial cells (Figure 4). Therefore,jck cyst enlargement was accompanied by a high rate of epithelialcell proliferation and increased apoptosis.
Figure 4. Increased proliferation and apoptosis in jck kidney. Kidney sections from 50-d-old WT and jck mice were stained with anti–proliferating cell nuclear antigen (anti-PCNA) antibodies (green). Bars = 400 µm as indicated. An elevated level of apoptosis in jck cystic kidney is evident by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling staining of apoptotic cells (green).
Sex Hormones Influence Disease Progression in jck Mice
To test whether sex hormones are responsible for differencesin the rate of disease progression in jck male versus jck femalemice, we studied the effect of testosterone and estrogen oncystogenesis. Jck male mice were castrated or underwent shamoperation at 26 d of age and were treated with testosteroneanalogue (DHT) until day 64. Castration slowed PKD progressionin jck male mice, showing statistically significant reductionin kidney to body weight ratio, percentage of cystic tissue,and blood urea nitrogen as compared with their sham-operatedcounterparts (Figure 5A). Administration of DHT to castratedmale mice reversed this effect (Figure 5A). Kidney histologyfrom castrated male mice showed less cystic tissue as comparedwith sham-operated or DHT-treated mice, as illustrated in Figure 5C.WT mice were used as control for each surgery plus treatmentgroup (Figure 5B). Castrated WT male mice showed no significantchanges in kidney to body weight ratio compared with the sham-operatedcohort. Administration of DHT to jck female mice resulted insignificant aggravation of PKD (Figure 6). Therefore, testosteroneseems to exacerbate cystic disease in jck mice. Ovariectomyof jck female mice resulted in acceleration of cystic disease,supporting the notion that female hormones may have a protectiveeffect (Figure 6). We also detected an increase in jck femalebody weights in groups that were either ovariectomized or treatedwith DHT (Supplementary Table 2). In WT female mice, increasedkidney to body weight ratio was detected in ovariectomized andDHT-treated groups (Figure 6B).
Figure 5. Effect of castration and dihydrotestosterone (DHT) on cystogenesis in jck male mice. (A) Disease progression in three groups of mice: Sham-operated (Sham), castrated (Cx), or castrated and treated with DHT (Cx+DHT). *P < 0.05 versus sham operated; #P < 0.05 versus castrated. (B) Castration and DHT treatment have no effect on kidney/body weight ratio in WT male mice. (C) Representative H&E sections from Sham, Cx, and Cx+DHT male mice (64 d old).
Figure 6. Effect of ovariectomy and DHT treatment on cystogenesis in jck female mice. (A) Disease progression in four groups of animals: Sham operated (sham), ovariectomized (Ox), sham-operated and treated with DHT (sham+DHT), and Ox with DHT treatment (Ox+DHT). Note that both removal of estrogen (Ox) and addition of testosterone (DHT treatment) aggravate the disease. *P < 0.05 versus Sham. (B) Ovariectomy and DHT treatment increase kidney/body weight ratio in control WT female mice. *P < 0.05 versus Sham; #P < 0.05 versus ovariectomized. (C) Representative H&E sections from Sham, Ox, and DHT-treated female mice (64 d old). Note that Ox and DHT-treated mice have larger cysts.
Abnormal Expression of Nek8 and Polycystins and Lengthening of Cilia in jck Kidney Epithelia
To determine whether Nek8 mutation in jck mice affects the mRNAexpression levels of Nek8 kinase, PC-1, and PC-2, we performedreal-time PCR analysis in cystic versus WT kidneys (Figure 7A).A significant upregulation of mRNA levels for Nek8, PC-1, andPC-2 was detected in jck kidneys as compared with WT. The overallexpression pattern of Nek8 protein in kidney sections of jckand normal mice was described previously (26). Specifically,in WT kidneys, Nek8 was found mainly in apical location of tubularepithelial cells, whereas enhanced cytoplasmic pattern of Nek8was seen in jck epithelia. To be able to examine ciliary localizationpatterns of Nek8 in WT and jck epithelia, we analyzed primarykidney epithelial cells that were derived from eight WT andeight jck mice. Nek8 showed punctate pattern of expression inprimary cilia of WT cells (Figure 7B, top). In contrast, Nek8was not detected in cilia of jck cells, where only cytoplasmic/perinuclearstaining was seen (Figure 7B, top). It is interesting that ciliaryexpression of PC-1 and PC-2 in jck epithelia also was changed(Figure 7B, middle). Whereas PC-1 and PC-2 were localized mainlyto the basal body of the cilium in WT cells, enhanced accumulationalong the length of the cilium was seen clearly in jck epithelia.We also noticed that jck cilia seemed longer than WT cilia (Figure 7B,acetylated tubulin staining). Therefore, we performed ultrastructuralscanning electron microscopy analysis of WT and cystic kidneys(Figure 7C). We observed clear lengthening of cilia (8.5 ±2.5 µM) in jck mutants as compared with WT controls (2.5± 1.5 µM). Therefore, mutation in the Nek8 genein jck mice results in a loss of Nek8 from cilium and leadsto redistribution and enhanced expression of polycystins alongthe lengthened cilium.
Figure 7. Ciliary abnormalities of jck renal epithelia. (A) Real-time PCR analysis of Nek8, Pkd1, and Pkd2 mRNA expression in WT and jck kidney. The expression levels were normalized to Rps12 housekeeping gene. The results are shown as the mean and SD of three different animals. (B) Differential ciliary distribution of Nek8 and polycystins in jck and WT renal epithelia. The cilia are marked with antibody to acetylated tubulin (ac. tubulin). Note punctate ciliary expression of Nek8 in WT epithelia and loss of ciliary localization in cystic epithelial cells. Polycystin-1 (PC-1) and PC-2 are expressed mainly at the base of cilia in WT cells, whereas their expression is enhanced along the cilia in jck cells. (C) Scanning electron micrographs of renal epithelial cells in WT and jck kidney. Primary cilia are significantly lengthened in cystic kidneys.
The development of renal tubular cysts in human ADPKD and autosomalrecessive PKD is characterized by several common features: increasedproliferation of cyst-lining epithelial cells, alterations incellular polarity and protein sorting, changes in extracellularmatrix composition, and fluid secretion into the cyst cavity(2,39). Cysts are formed in all segments of the nephron in ADPKD,whereas collecting ducts predominantly are affected in autosomalrecessive PKD. Efforts to understand the molecular pathwaysof cystic disease have led to identification of possible newtargets for ADPKD therapeutic interventions (16). For elucidationof the in vivo relevance of newly identified pathways of cystogenesis,studies in animal models of PKD are crucial. Multiple murinePKD models with mutations in different genes have been usedin such studies (19). Although every model characterized sofar mimics a subset of human disease, no single model fullyrecapitulates all aspects of human PKD pathogenesis. Severalspontaneous mouse models of PKD, such as cpk, bpk,and orpk,resemble human recessive disease with respect to cyst localizationand rapid rate of disease progression, whereas cystic diseasein pcy mice is slowly progressive with focal cyst formationsimilar to ADPKD.
Rapidly accumulating evidence implicates defects in the primarycilium as a major cause of cystogenesis (40). Importantly, genesthat are responsible for spontaneous murine models as well asgenes that cause human PKD are found to be expressed at leastpartially in cilia, suggesting that a unifying pathogenic mechanismunderlying all cystic epithelial disorders exists (41). We setout to characterize fully the jck mouse model of PKD becausethe gene Nek8, which is responsible for this disease, was mappedrecently to the primary cilia (26–29). It is interestingthat we detected overexpression of Nek8 mRNA in jck kidneysrelative to WT and loss of ciliary localization of Nek8 proteinin jck kidney cells. Overexpression and mislocalization of Nek8in jck cells was accompanied by increased expression of polycystinsalong the jck cilia, whereas expression in normal cells wasdetected mainly at the base of the cilium. NIMA-related kinaseswere implicated recently in regulating cilia length in Tetrahymena(42). We therefore analyzed the kidneys from jck and WT miceby scanning electron microscopy. Remarkably, the cilia in jckkidneys were significantly lengthened, suggesting that Nek8may play an important role in controlling ciliary length. Itis possible that Nek8 might regulate disassembly of cilia, becauseprevious work demonstrated that Nek8 knockdown does not affectciliogenesis (28).
In addition, the jck mutation resembles human ADPKD phenotypically,despite its autosomal recessive mode of inheritance (19). Severalfeatures make the jck mouse an attractive and unique model forPKD. The disease progresses relatively slowly, thereby offeringa convenient time frame of 3 to 5 wk for potential therapeutictesting. The jck mouse model is strikingly different from othermouse models of PKD in that it clearly displays gender dimorphism.Male gender in ADPKD is a recognized risk factor for a moreaggressive course of the disease. Therefore, males with ADPKDprogress faster to ESRD than females (43). Gender as a riskfactor was demonstrated recently only in rat models of PKD:Han:SPRD and PCK (44,45). To define the cause of sex dimorphismthat is seen in jck mice, we addressed whether gonadal hormonesmight be responsible. Castration of jck male mice significantlyslowed the disease progression, whereas exogenous administrationof testosterone reversed the castration effect. Therefore, testosteroneis likely to be responsible for a more aggressive course ofPKD in jck male mice, whereas female hormones may have a protectiveeffect. Further studies will be necessary to determine how exactlygonadal hormones modulate cystic growth.
We also found similarities between human dominant PKD and jckcystic disease on the basis of the analysis of specific nephronsegments that are affected by cystic transformation. Human ADPKDis characterized by formation of cysts in all segments of thenephron (46,47). Unlike cpk, bpk, and orpk mouse models, whichdevelop cysts in collecting ducts, jck mice develop cysts inmultiple segments of the nephron: DT, LH, and CD but not inPT and glomerulus. Moreover, we performed quantitative analysisof cystic disease dynamic and found that cysts from CD are formedearly in the course of the disease, and their contribution isnot significantly changed between 3 and 5 wk of age. Cysts fromDT and LH, however, continue to develop during the course ofthe disease. Such detailed analysis of cyst progression in thejck model should be useful for dissecting pathobiology of thedisease and, most important, to set out expectations for testingpotential target-specific therapies because expression of someproteins may be limited to a particular segment of the nephron.
Evidence from multiple groups suggests an important role forEGF/TGF-/EGFR in the progression of human PKD. EGFR has beenshown to be overexpressed and mislocalized to apical membranesof cyst-lining epithelia (48,49). Great promise has been shownin modulating EGFR tyrosine kinase activity for effective PKDtreatment using the bpk mouse and Han:SPRD rat; however, nobenefit was seen in PCK rat (21,35). In contrast with otheranimal models, no abnormal expression of EGFR was detected inthe PCK rat cystic epithelia, which may explain the lack oftherapeutic effect in this model. We analyzed expression andlocalization of EGFR in jck kidney and found that similar tohuman ADPKD, EGFR was overexpressed and mislocalized to theapical membranes of cystic epithelial cells. These data furtherhighlight parallels between jck cystogenesis and human disease.
Because the rate of cyst proliferation is controlled by growthfactors and hormones that result in activation of cAMP pathways,we also measured the levels of cAMP in jck kidneys. Indeed,we found increased levels of cAMP in jck kidneys, correlatingwith the severity of the disease. Recent data demonstrated thatcAMP is capable of activating B-Raf and ERK in human ADPKD-derivedepithelia (36), leading to increased cellular proliferation.We also detected activation of this pathway in jck kidneys,resulting in ERK activation and increased levels of B-Raf. Thisobservation is consistent with a recent report that describedincreased levels of phosphorylated ERK in PCK rats (50).
We provide in-depth characterization of disease progressionin the jck mouse model of PKD with initial characterizationof cellular and molecular defects. We show for the first timeabnormal expression of Nek8 and polycystins and lengtheningof cilia in jck kidney tubular epithelia. The jck disease progressionis more severe in male than in female mice, similar to ADPKDand rat models, but uncommon for other mouse models described.Significant similarities between human ADPKD and jck mouse werefound in molecular pathways that are affected by cystogenesis,including EGFR axis and cAMP-activated pathways, leading toincreased proliferation and apoptosis in jck cystic cells. Takentogether, our data strongly suggest that the jck model resemblesmultiple facets of human ADPKD and therefore is suitable fordissecting further molecular mechanisms of cystic disease aswell as for serving as a convenient model to evaluate potentialtherapies directly.
Acknowledgments
This study was supported in part by the National Institutesof Health grant RO1 DK66370 to D.R.B.
Part of this work was presented at the 38th annual meeting ofthe American Society of Nephrology; November 8 through 13 2005;Philadelphia, PA; and has been published in abstract form (JAm Soc Nephrol 16: 137A, 2005).
We thank the staff of the Department of Comparative Medicineand Histology unit for excellent assistance. We thank R. Serriello,S. Scull-Lopez, K. Norton, S. Savage, J. Serriello, M. Callahan,and W. Weber for expert technical help. Scanning electron microscopyanalysis was performed by T. Pepper (Iowa State University).We are grateful to T. Natoli and K. Klinger for helpful discussionsand comments on this manuscript.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
L.A.S. and N.O.B. contributed equally to this work.
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Top
Abstract
Introduction
/EGFR in the progression of human PKD. EGFR has been shown to be overexpressed and mislocalized to apical membranes of cyst-lining epithelia (48,49). Great promise has been shown in modulating EGFR tyrosine kinase activity for effective PKD treatment using the bpk mouse and Han:SPRD rat; however, no benefit was seen in PCK rat (21,35). In contrast with other animal models, no abnormal expression of EGFR was detected in the PCK rat cystic epithelia, which may explain the lack of therapeutic effect in this model. We analyzed expression and localization of EGFR in jck kidney and found that similar to human ADPKD, EGFR was overexpressed and mislocalized to the apical membranes of cystic epithelial cells. These data further highlight parallels between jck cystogenesis and human disease. 
