A mouse model of autosomal recessive polycystic kidney disease with biliary duct and proximal tubule dilatation
2007; Elsevier BV; Volume: 72; Issue: 3 Linguagem: Inglês
10.1038/sj.ki.5002294
ISSN1523-1755
AutoresJohn R. Woollard, R. Punyashtiti, Samantha J. Richardson, Tatyana V. Masyuk, Shelly Whelan, Bing Huang, Donna J. Lager, J. vanDeursen, Vicente E. Torres, Vincent H. Gattone, Nicholas F. LaRusso, Peter C. Harris, Christopher J. Ward,
Tópico(s)Hedgehog Signaling Pathway Studies
ResumoAutosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in the polycystic kidney and hepatic disease (PKHD1) gene encoding the protein fibrocystin/polyductin. The aim of our study was to produce a mouse model of ARPKD in which there was no functional fibrocystin/polyductin to study the pathophysiology of cystic and fibrocystic disease in renal and non-renal tissues. Exon 2 of the gene was deleted and replaced with a neomycin resistance cassette flanked by loxP sites, which could be subsequently removed by Cre-lox recombinase. Homozygous Pkhd1del2/del2 mice were viable, fertile and exhibited hepatic, pancreatic, and renal abnormalities. The biliary phenotype displayed progressive bile duct dilatation, resulting in grossly cystic and fibrotic livers in all animals. The primary cilia in the bile ducts of these mutant mice had structural abnormalities and were significantly shorter than those of wild-type (WT) animals. The Pkhd1del2/del2 mice often developed pancreatic cysts and some exhibited gross pancreatic enlargement. In the kidneys of affected female mice, there was tubular dilatation of the S3 segment of the proximal tubule (PT) starting at about 9 months of age, whereas male mice had normal kidneys up to 18 months of age. Inbreeding the mutation onto BALBc/J or C57BL/6J background mice resulted in females developing PT dilatation by 3 months of age. These inbred mice will be useful resources for studying the mechanisms underlying the pathogenesis of ARPKD. Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in the polycystic kidney and hepatic disease (PKHD1) gene encoding the protein fibrocystin/polyductin. The aim of our study was to produce a mouse model of ARPKD in which there was no functional fibrocystin/polyductin to study the pathophysiology of cystic and fibrocystic disease in renal and non-renal tissues. Exon 2 of the gene was deleted and replaced with a neomycin resistance cassette flanked by loxP sites, which could be subsequently removed by Cre-lox recombinase. Homozygous Pkhd1del2/del2 mice were viable, fertile and exhibited hepatic, pancreatic, and renal abnormalities. The biliary phenotype displayed progressive bile duct dilatation, resulting in grossly cystic and fibrotic livers in all animals. The primary cilia in the bile ducts of these mutant mice had structural abnormalities and were significantly shorter than those of wild-type (WT) animals. The Pkhd1del2/del2 mice often developed pancreatic cysts and some exhibited gross pancreatic enlargement. In the kidneys of affected female mice, there was tubular dilatation of the S3 segment of the proximal tubule (PT) starting at about 9 months of age, whereas male mice had normal kidneys up to 18 months of age. Inbreeding the mutation onto BALBc/J or C57BL/6J background mice resulted in females developing PT dilatation by 3 months of age. These inbred mice will be useful resources for studying the mechanisms underlying the pathogenesis of ARPKD. Mutations in the polycystic kidney and hepatic disease (PKHD1) gene are responsible for autosomal recessive polycystic kidney disease (ARPKD) (MIM 263200).1.Zerres K. Rudnik-Schöneborn S. Steinkamm C. et al.Autosomal recessive polycystic kidney disease.J Mol Med. 1998; 76: 303-309Crossref PubMed Scopus (102) Google Scholar, 2.Zerres K. Hansmann M. Knopfle G. et al.Prenatal diagnosis of genetically determined early manifestation of autosomal dominant polycystic kidney disease?.Human Genet. 1985; 71: 368-369Crossref PubMed Scopus (30) Google Scholar, 3.Jorgensen M.J. The ductal plate malformation: a study of the intrahepatic bile duct lesion in infantile polycystic disease and congenital hepatic fibrosis.Acta Pathol Microbiol Scand Suppl. 1977; 257: 1-87PubMed Google Scholar ARPKD is characterized by various combinations of bilateral renal cystic disease and congenital hepatic fibrosis with or without non-obstructive intrahepatic bile duct dilatation (Caroli disease). Most patients present in utero or at birth with marked renal enlargement and biliary dysgenesis on liver biopsy, whereas a minority of patients present as older children, teenagers, or adults, usually with manifestations of portal hypertension or cholangitis and renal involvement ranging from non-existent to medullary ectasia of the collecting ducts (CD) and cystic disease. Severe ARPKD develops in utero, starting with the dilatation of both proximal tubules (PT) and CD.4.Nakanishi K. Sweeney Jr, W.E. Zerres K. et al.Proximal tubular cysts in fetal human autosomal recessive polycystic kidney disease.J Am Soc Nephrol. 2000; 11: 760-763PubMed Google Scholar After 34 weeks of fetal age the CD dilatation predominates and is responsible for the massive enlargement of the kidney. The PT dilatation appears to be an early and transient feature of both ARPKD and multiple animal models such as the orpk,5.Moyer J.H. Lee-Tischler M.J. Kwon H.-Y. et al.Candidate gene associated with a mutation causing recessive polycystic kidney disease in mice.Science. 1994; 263: 1329-1333Crossref Scopus (303) Google Scholarcpk,6.Avner E.D. Studnicki F.E. Young M.C. et al.Congenital murine polycystic kidney disease. I. The ontogeny of tubular cyst formation.Pediatr Nephrol. 1987; 1: 587-596Crossref PubMed Scopus (45) Google Scholar and bpk7.Nauta J. Ozawa Y. Sweeney Jr, W.E. et al.Renal and biliary abnormalities in a new murine model of autosomal recessive polycystic kidney disease.Pediatr Nephrol. 1993; 7: 163-172Crossref PubMed Scopus (96) Google Scholar mice. PKHD1 is a large (67 exon) gene extending over ∼0.5 Mb of human chromosome 6p12 that encodes an mRNA of 16 kb.8.Ward C.J. Hogan M.C. Rossetti S. et al.The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein.Nat Genet. 2002; 30: 259-269Crossref PubMed Scopus (550) Google Scholar,9.Onuchic L.F. Furu L. Nagasawa Y. et al.PKHD1, the polycystic kidney and hepatic disease 1 gene, encodes a novel large protein containing multiple immunoglobulin-like plexin-transcription-factor domains and parallel beta-helix 1 repeats.Am J Hum Genet. 2002; 70: 1305-1317Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar The protein product, fibrocystin (FC), is 4074 amino acids long and is predicted to be a large type 1 membrane protein that has been shown to be present in the basal body and primary cilium.10.Wang S. Luo Y. Wilson P.D. et al.The autosomal recessive polycystic kidney disease protein is localized to primary cilia, with concentration in the basal body area.J Am Soc Nephrol. 2004; 15: 592-602Crossref PubMed Scopus (117) Google Scholar, 11.Menezes L.F. Cai Y. Nagasawa Y. et al.Polyductin, the PKHD1 gene product, comprises isoforms expressed in plasma membrane, primary cilium, and cytoplasm.Kidney Int. 2004; 66: 1345-1355Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 12.Ward C.J. Yuan D. Masyuk T.V. et al.Cellular and subcellular localization of the ARPKD protein; fibrocystin is expressed on primary cilia.Hum Mol Genet. 2003; 12: 2703-2710Crossref PubMed Scopus (248) Google Scholar, 13.Zhang M.Z. Mai W. Li C. et al.PKHD1 protein encoded by the gene for autosomal recessive polycystic kidney disease associates with basal bodies and primary cilia in renal epithelial cells.Proc Natl Acad Sci USA. 2004; 101: 2311-2316Crossref PubMed Scopus (129) Google Scholar The cilium/centriolar localization makes it a member of a growing list of ciliary proteins involved in cystic kidney disease, which include: polycystin-1 and -2, cystin, polaris/IFT88,14.Yoder B.K. Hou X. Guay-Woodford L.M. The polycystic kidney disease proteins, polycystin-1, polycystin-2, polaris, and cystin, are co-localized in renal cilia.J Am Soc Nephrol. 2002; 13: 2508-2516Crossref PubMed Scopus (688) Google Scholar and inversin,15.Yoder B.K. Tousson A. Millican L. et al.Polaris, a protein disrupted in orpk mutant mice, is required for assembly of renal cilium.Am J Physiol Renal Physiol. 2002; 282: F541-F552Crossref PubMed Scopus (198) Google Scholar,16.Watanabe D. Saijoh Y. Nonaka S. et al.The left-right determinant Inversin is a component of node monocilia and other 9+0 cilia.Development. 2003; 130: 1725-1734Crossref PubMed Scopus (168) Google Scholar as well as a range of other proteins associated with nephronophthisis and Bardet–Biedl syndrome.17.Beales P.L. Lifting the lid on Pandora's box: the Bardet–Biedl syndrome.Curr Opin Genet Dev. 2005; 15: 315-323Crossref PubMed Scopus (95) Google Scholar The large size of the gene makes it a significant mutational target and a wide range of mainly private mutations have been described. Phenotype/genotype correlations indicate that two truncating mutations are associated with the severe, lethal phenotype, whereas one or two missense changes are often associated with milder disease.18.Rossetti S. Torra R. Coto E. et al.A complete mutation screen of PKHD1 in autosomal-recessive polycystic kidney disease (ARPKD) pedigrees.Kidney Int. 2003; 64: 391-403Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar This phenotypic spread is also reflected in the available animal models of ARPKD with Pkhd1 mutations.19.Lager D.J. Qian Q. Bengal R.J. et al.The pck rat: a new model that resembles human autosomal dominant polycystic kidney and liver disease.Kidney Int. 2001; 59: 126-136Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar,20.Moser M. Matthiesen S. Kirfel J. et al.A mouse model for cystic biliary dysgenesis in autosomal recessive polycystic kidney disease (ARPKD).Hepatology. 2005; 41: 1113-1121Crossref PubMed Scopus (66) Google Scholar The polycystic kidney (PCK) rat with a frame-shifting exon 36 skipping mutation8.Ward C.J. Hogan M.C. Rossetti S. et al.The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein.Nat Genet. 2002; 30: 259-269Crossref PubMed Scopus (550) Google Scholar and the ex40 mouse with an in-frame deletion of exon 4020.Moser M. Matthiesen S. Kirfel J. et al.A mouse model for cystic biliary dysgenesis in autosomal recessive polycystic kidney disease (ARPKD).Hepatology. 2005; 41: 1113-1121Crossref PubMed Scopus (66) Google Scholar develop liver cysts and fibrosis. In the kidney the PCK rat has distal tubule and CD cysts,19.Lager D.J. Qian Q. Bengal R.J. et al.The pck rat: a new model that resembles human autosomal dominant polycystic kidney and liver disease.Kidney Int. 2001; 59: 126-136Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar but the ex40 mouse has been reported to have a histologically normal kidney up to 14 months of age.20.Moser M. Matthiesen S. Kirfel J. et al.A mouse model for cystic biliary dysgenesis in autosomal recessive polycystic kidney disease (ARPKD).Hepatology. 2005; 41: 1113-1121Crossref PubMed Scopus (66) Google Scholar The ex40 mouse has been suggested to be a hypomorphic allele with an in-frame deletion, and because the PCK rat has a splicing change, it is possible that it could also produce a shortened but functional protein, leading to a hypomorphic phenotype. To overcome this potential problem, we targeted the first coding exon, exon 2, which contains the start methionine and the signal peptide of FC. Here we report a mouse model with an ARPKD gene mutation, which develops PT dilatation in the adult female mouse without the transition to CD dilatation. This animal develops liver disease similar to that observed in Caroli disease and so may be a valuable resource for studying the development of fibrocystic liver disease as well as the PT and CD disease transition in the development of human ARPKD. We targeted exon 2 of the mouse Pkhd1 gene with a conventional LoxP flanked phosphoglycerate kinase (PGK) neomycin cassette (Figure 1a) and screened by Southern blotting (Figure 1b). Mice were derived from three separate embryonic stem cell clones to generate Pkhd1neo2 animals and these were in turn crossed to mouse mammary tumor virus-Cre animals to allow for the germline removal of the PGK neomycin cassette yielding the Pkhd1del2 allele.21.Wagner K.U. McAllister K. Ward T. et al.Spatial and temporal expression of the Cre gene under the control of the MMTV-LTR in different lines of transgenic mice.Transgenic Res. 2001; 10: 545-553Crossref PubMed Scopus (237) Google Scholar Wild type (WT), Pkhd1neo2, and Pkhd1del2 were easily distinguished by polymerase chain reaction (PCR) (Figure 1b). 5′ RACE and directed reverse transcription-PCR showed that there was a transcript produced from the mutant allele, which skips exon 2 using the splice donor of exon 1 and the splice acceptor of exon 3 in both Pkhd1neo2 and Pkhd1del2 homozygous mice (Figure 1c). No significant phenotypic differences were seen between the animals derived from the three different clones, with or without the neo cassette. The number and genotypes of the mice used in this study are shown in Table 1. Mice were born in normal Mendelian ratios (28 WT, 59 Pkhd1neo2/+, 17 Pkhd1neo2/neo2χ2=4.2, 2 d.f., P=0.121). Pkhd1del2/+ animals had no phenotype up to 18 months of age in line with the observation that ARPKD carriers are healthy.Table 1Number of outbred animals used for phenotypic evaluationAge (months)Wild typePkhd1del2/del212646315396825915341261915281825 Open table in a new tab Mice harboring the homozygous Pkhd1neo2 or Pkhd1del2 mutation were fertile. The initial Pkhd1del2/del2 mice had a significant and variable 129Sve contribution and are referred to as outbred. We inbred these animals onto BALBc/J and C57BL/6J backgrounds to generation F10, referred to as inbred. Pkhd1del2/del2 mice developed fibrocystic disease of the liver so that by 9 months there was a significant increase in the liver body weight ratio (Figure 2a), which was maintained out to 18 months of age. Often the livers were massively enlarged occupying much of the abdominal cavity and were macroscopically cystic (Figure 3a).Figure 3Pkhd1del2/del2 liver and pancreas gross appearance and Mallory trichrome stained sections. (a) Gross appearance of 11 months outbred Pkhd1del2/del2 liver. (b–g) Sections stained with Mallory trichome, (b) 1, (c) 3, (d) 6, (e) 9, (f) 12 (g) 15 months. The livers become more cystic and fibrotic with age. (h) Severely affected grossly cystic pancreas 16 days mouse. (i) Trichrome-stained section of 16 days severely affected cystic pancreas. (j) and (k) Hematoxylin and eosin- stained pancreas of mildly affected 9-month-old Pkhd1del2/del2 animals, note the thickened and enlarged pancreatic ducts versus (l) the 9 month WT animal. Original magnification of (b–g) and (i–l) is × 10.View Large Image Figure ViewerDownload (PPT) Histologically, biliary dilatation was observed at 1 month of age in 100% of outbred Pkhd1del2/del2 mice (Figure 3b) and it worsened with time (Figure 3c–g) so that by 12–15 months most of the liver was replaced by cysts. At early stages, bile duct proliferation was evident in the portal areas with primitive tortuous bile ducts and reactive stroma. By 6–9 months progressive portal fibrosis was evident (Figure 3e and g). In 11 mice, the pancreas was enlarged and markedly cystic. Up to 20 ml of cystic fluid could be aspirated from these pancreas (Figure 3h and Table 2). Dilated pancreatic ducts and cysts were lined with a hyperplastic columnar epithelium surrounded by a fibrotic stroma (Figure 3i). Microscopic cysts were detected in 44 of 92 Pkhd1del2/del2 pancreas examined (Figure 3j and k) when compared to the WT (Figure 3l). Duct walls were thickened and the ducts were dilated and lined with hyperplastic columnar epithelium.Table 2Number of grossly cystic pancreas observed in Pkhd1del2/del2 animalsAge (months)1369121518Pkhd1del2/del21333100Percent of total2.27.712.08.85.300Pkhd1, polycystic kidney and hepatic disease. Open table in a new tab Pkhd1, polycystic kidney and hepatic disease. The kidneys of the Pkhd1del2/del2 mice appeared normal until approximately 9 months of age, when outbred females began to develop cystic dilatation in the outer stripe of the outer medulla and inner cortex (Figure 4). To determine the tubular segment dilated, kidneys were stained with Lotus tetragonolobus agglutinin (LTA) for the PT and Dolichos biflous agglutinin (DBA) for the CD as well as Calbindin D28k for the distal tubule and Tamm Horsfall protein for the thick ascending limb of the loop of Henle. The CD and the distal tubule were never dilated in the 159 Pkhd1del2/del2 outbred animals. Tubular dilatation was always restricted to LTA-positive tubule segments in females analyzed at 9–15 months (Figures 5, 6b, e, and h). Outbred male kidneys were not affected even at 18 months (Figure 4a–c, Table 3). Female outbred animals exhibited an increase in PT dilation from 9 to 12 months in both the WT and Pkhd1del2/del2 (Table 4). The increase in the WT PT was small yet significant (2.5 μm, P<0.05), whereas the increase in the Pkhd1del2/del2 PTs was much greater (11.2 μm; P<0.0005). On inbreeding on a BALBc/J or C57BL/6J background, the female PTs become dilated by 3 months of age (n=3) BALBc/J and (n=3) C57BL/6J (Figure 6b, e, and h). Kidney body mass ratios did not significantly differ from WT animals (Figure 2b).Figure 6Fluorescent histochemistry of WT and Pkhd1del2/del2 animals stained with phalloidin (PHA) (red), LTA (green), and DAPI (blue). (a, d, and g) Three month normal female, (b, e, and h) 3 month Pkhd1del2/del2 female inbred, F 10, BALBc/J background, (c, f, and i) 15 month Pkhd1del2/del2 male. PHA binds to F-actin of the brush border in the PT. Loss of PHA staining is evident in the dilated proximal S3 segments in the Pkhd1del2/del2 animals (indicated by yellow arrowheads) similar to the loss, and disorganized pattern, of periodic acid shift staining (data not shown). PHA staining is present in S3 tubules that are not dilated in WT and Pkhd1del2/del2 animals (indicated by white arrowheads). This shows that differentiation and polarity are maintained in the PT until very late in the male's life. (Original magnification × 40).View Large Image Figure ViewerDownload (PPT)Table 3Number of outbred animals where dilated proximal tubules were observedGenotypeTotalDilated% DilatedWild type (9 months)600Wild type (12 months)600Wild type (15 months)200Wild type (18 months)200Pkhd1del2/del2 (9 months, male)800Pkhd1del2/del2 (9 months, female)10770Pkhd1del2/del2 (12 months, male)700Pkhd1del2/del2 (12 months, female)111091Pkhd1del2/del2 (15 months, male)400Pkhd1del2/del2 (15 months, female)66100Pkhd1del2/del2 (18 months, male)200Pkhd1del2/del2 (18 months, female)33100 Open table in a new tab Table 4Tubule diameter measurementsAnimalAge (months)GenotypeDiameteraValues with the same symbol are significantly different. Only the group averages were tested. (s.d.) (μm)No. of tubules19WT29.51 (9.12)10629WT26.99 (6.69)14039WT25.71 (8.18)112Average27.34 (8.06)*†35849Pkhd1del2/del238.78 (11.89)10959Pkhd1del2/del251.07 (13.63)7569Pkhd1del2/del253.35 (12.70)91Average46.98 (14.27)†**276712WT30.60 (8.61)133812WT26.30 (8.54)104912WT32.07 (8.30)131Average29.91 (8.78)*‡3681012Pkhd1del2/del251.23 (18.67)1471112Pkhd1del2/del264.95 (16.03)1331212Pkhd1del2/del258.56 (18.00)181Average58.19 (18.26)‡**461WT, wild type*P<0.05; **P<0.0005; †P<0.0001; ‡P<0.00001.a Values with the same symbol are significantly different. Only the group averages were tested. Open table in a new tab WT, wild type *P<0.05; **P<0.0005; †P<0.0001; ‡P<0.00001. In the older Pkhd1del2/del2 animals (15 month), the brush border of the dilated tubules was attenuated and in extreme cases was lost as assessed by periodic acid shift (data not shown). To assess the state of brush border in non-dilated PT, we used fluorescent LTA (green) and fluorescent Phalloidin (Red), which binds F-actin in the brush border. The presence of a brush border and apical LTA signal was used as a proxy for differentiation. Female mice inbred on the BALBc/J background that developed cystic changes at 3 months clearly showed an attenuation of the brush border and an enhancement of the LTA staining, which appeared to be cytoplasmic and apical as opposed to purely apical (Figure 6a, b, d, e, g, and h). Outbred male mice had a normal brush border and did not become cystic up to the age of 15 months (Figure 6c, f, and i), implying strong resistance to PT disease. In females of these ages, all the S3 segments were dilated as observed by LTA staining. Ciliary morphology was assessed by scanning electron microscopy (SEM), as there are conflicting reports about ciliary morphology in models of ARPKD.20.Moser M. Matthiesen S. Kirfel J. et al.A mouse model for cystic biliary dysgenesis in autosomal recessive polycystic kidney disease (ARPKD).Hepatology. 2005; 41: 1113-1121Crossref PubMed Scopus (66) Google Scholar,22.Masyuk T.V. Huang B.Q. Ward C.J. et al.Defects in cholangiocyte fibrocystin expression and ciliary structure in the PCK rat.Gastroenterology. 2003; 125: 1303-1310Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar SEMs on cystic liver showed that the primary cilia in the affected biliary tree were shorter 0.82±0.37 mm (n=112) versus WT cilia length 2.93±0.86 mm (n=46, P<0.0001) (Figure 7a and b). The cilia were often misshapen with multiple blebs (Figure 7b insert). Transmission Electron Microscopy (TEM) of primary cilia in the biliary tree showed that they were always surrounded by clumps of small 50–80 nm vesicular structures (Figure 7e and f). In the kidney, cilia were seen above the PT brush border (Figure 7c and d). In 9 months old female animals, surface blebs, not present on normal cilia, were observed and some cilia appeared as branched clumps (Figure 7d). We confirmed that these 'star burst' structures were cilia by staining frozen sections with anti-acetylated tubulin (Figure 7g and h). In animals with more advanced cystic disease (15 months), the cilia from cyst lining epithelia were usually stunted and highly branched or 'star-burst'. The cilia in the CD of affected and WT female mice were not different in length (1.47±0.40 mm, n=39, and 1.55±0.48 mm, n=41, respectively) and appeared normal (not shown). Therefore, cilia are abnormal in the PT and biliary tree but not in the CD in this model of ARPKD. We generated a mouse model of ARPKD by targeting exon 2, the first coding exon of the Pkhd1 gene. This removes the start codon and the signal peptide from the open reading frame of the cDNA. Analysis of 5′ RACE products and directed PCR shows that exon 2 is skipped and no possible alternative start site was detected, indicating that Pkhd1del2 cannot make full-length FC. Western analysis with both rabbit anti-mouse FC and chicken anti-mouse FC affinity purified antibodies failed to detect FC in the WT kidney membrane preparation, so we have not been able to verify loss of the protein in the knockout mice. We are now generating an animal with an epitope-tagged form of FC to overcome this important problem. The Pkhd1del2/del2 mouse is a faithful model of the fibrocystic liver disease associated with human ARPKD. The hepatic phenotype of Pkhd1del2/del2 is similar to the other rodent models with mutations in Pkhd1, the ex40 mouse and PCK rat. The disease starts at 1 m of age with the development of tortuous bile ducts and enlargement of the portal triads. At between 3 and 9 months both portal fibrosis and cystogenesis become more prominent so that by 12–15 months the liver is very cystic and fibrotic. The pancreatic disease is an important feature of the Pkhd1del2/del2 mouse with histologically evident cysts budding from the pancreatic duct by 6 month of age in 48% of the animals. The aggressive pancreatic disease appears to be a unique feature of the model, which has not been observed in the PCK or ex40 mouse. This severe phenotype only occurred in animals with a significant 129Sve contribution and as we inbred the animals it has become rarer. This could be because 129Sve modifier genes are important in the generation of this phenotype. There is one report of a series of nine human patients with ARPKD, six of whom were known to have pancreatic fibrosis, probably secondary to cystogenesis.23.Kugler A. Stuhler G. Walden P. et al.Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids (see comments).Nat Med. 2000; 6: 332-336Crossref PubMed Scopus (595) Google Scholar We observed an unexpected and novel renal phenotype consisting of PT dilatation in female homozygotes; male homozygotes appear to be protected from PT dilatation. Inbreeding onto either the BALBc/J or C57BL/6J backgrounds exacerbates the kidney disease so that all females develop PT cysts by 3 months. The kidney phenotype in the Pkhd1del2/del2 mouse is different from the two other animal models of rodent ARPKD as the animals develop PT cysts in adulthood. The ex40 mouse never develops renal tubular dilatation up to 14 months of age,20.Moser M. Matthiesen S. Kirfel J. et al.A mouse model for cystic biliary dysgenesis in autosomal recessive polycystic kidney disease (ARPKD).Hepatology. 2005; 41: 1113-1121Crossref PubMed Scopus (66) Google Scholar whereas the PCK rat develops cysts in the CD and the distal tubule.19.Lager D.J. Qian Q. Bengal R.J. et al.The pck rat: a new model that resembles human autosomal dominant polycystic kidney and liver disease.Kidney Int. 2001; 59: 126-136Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar However, PT dilatation is a feature of early ARPKD in the human fetus and at least two models of mouse polycystic kidney disease with defects in ciliary proteins. These models, orpk (IFT88)5.Moyer J.H. Lee-Tischler M.J. Kwon H.-Y. et al.Candidate gene associated with a mutation causing recessive polycystic kidney disease in mice.Science. 1994; 263: 1329-1333Crossref Scopus (303) Google Scholar and cpk (cystin)6.Avner E.D. Studnicki F.E. Young M.C. et al.Congenital murine polycystic kidney disease. I. The ontogeny of tubular cyst formation.Pediatr Nephrol. 1987; 1: 587-596Crossref PubMed Scopus (45) Google Scholar,24.Hou X. Mrug M. Yoder B.K. et al.Cystin, a novel cilia-associated protein, is disrupted in the cpk mouse model of polycystic kidney disease.J Clin Investig. 2002; 109: 533-540Crossref PubMed Scopus (203) Google Scholar mice, have a transient phase of PT dilatation, which has been studied in the cpk mouse. In the cpk mouse, 81% of the PTs are cystic at birth but by 3 weeks of age; there is a significant decrease in the number of dilated PTs with only 31% of PT dilated, whereas all CDs are dilated.5.Moyer J.H. Lee-Tischler M.J. Kwon H.-Y. et al.Candidate gene associated with a mutation causing recessive polycystic kidney disease in mice.Science. 1994; 263: 1329-1333Crossref Scopus (303) Google Scholar In the early ARPKD fetus, PT dilatation is approximately as common as CT dilatation but after 34 weeks of gestation the CT disease predominates and there is no visible PT disease. This regression of PT dilatation and subsequent shift to CD dilatation does not appear to occur in the Pkhd1del2/del2 mouse implying that it may be a stalled form of ARPKD. One important aspect of this animal model is that the renal disease is restricted to the female and the male appears to be protected. This is unusual as there are several models of polycystic kidney disease in which the males are more severely affected than the females, such as the jck mouse25.Smith L.A. Bukanov N.O. Husson H. et al.Development of polycystic kidney disease in juvenile cystic kidney mice: insights into pathogenesis, ciliary abnormalities, and common features with human disease.J Am Soc Nephrol. 2006; 17: 2821-2831Crossref PubMed Scopus (143) Google Scholar and the Han:SPRD, Cy/+ rat.26.Cowley Jr, B.D. Rupp J.C. Muessel M.J. et al.Gender and the effect of gonadal hormones on the progression of inherited polycystic kidney disease in rats.Am J Kidney Dis. 1997; 29: 265-272Abstract Full Text PDF PubMed Scopus (61) Google Scholar,27.Nagao S. Kusaka M. Nishii K. et al.Androgen receptor pathway in rats with autosomal dominant polycystic kidney disease.J Am Soc Nephrol. 2005; 16: 2052-2062Crossref PubMed Scopus (25) Google Scholar Male patients appear to have more severe disease in PKD228.Magistroni R. He N. Wang K. et al.Genotype-renal function correlation in type 2 autosomal dominant polycystic kidney disease.J Am Soc Nephrol. 2003; 14: 1164-1174Crossref PubMed Scopus (101) Google Scholar and perhaps PKD1.29.Harris P.C. Bae K.T. Rossetti S. et al.Cyst number but not the rate of cystic growth is associated with the mutated gene in autosomal dominant polycystic kidney disease.J Am Soc Nephrol. 2006; 17: 3013-3019Crossref PubMed Scopus (171) Google Scholar The sex difference in renal disease in mice could be due to sexual dimorphism in the S3 segment of the PT. In the male, the S3 segment has larger and more electron lucent mitochondria and more and larger lysosomes than the female; a feature controlled by androgens.30.Koenig H. Goldstone A. Blume G. et al.Testosterone-mediated sexual dimorphism of mitochondria and lysosomes in mouse kidney proximal tubules.Science. 1980; 209: 1023-1026Crossref PubMed Scopus (62) Google Scholar This segment of the nephron secretes lysosomal enzymes, a function better developed in males and also under the control of androgens. Females have a more prominent brush than males.31.Yabuki A. Suzuki S. Matsumoto M. et al.Sex and strain differences in the brush border and PAS-positive granules and giant bodies of the mouse renal S3 segment cells.Exp Anim. 2001; 50: 59-66Crossref PubMed Scopus (17) Google Scholar,32.Yabuki A. Suzuki S. Matsumoto M. et al.Sexual dimorphism of proximal straight tubular cells in mouse kidney.Anat Rec. 1999; 255: 316-323Crossref PubMed Scopus (16) Google Scholar These hormonal effects on PT morphology raise the possibility that the PT disease seen in the human fetus with ARPKD and in embryos of many rodent models of ARPKD could be driven by the high circulating levels of maternal estrogen. This would be consistent with development of PT cysts restricted to females and at a later age in our model of ARPKD. There is debate over whether there is a primary cilia defect in animals with mutations in Pkhd1. Moser et al. showed by TEM that the cilia were normal in ex40 mice. However, TEM is probably not the best tool to study ciliary morphology as the entire length of the cilium is rarely visualized. We used SEM and showed that, in the bile ducts of 3-month-old mice, the cilia are shorter and have multiple blebs on their surface. In the early PT cysts in the female (9 months), cilia with multiple blebs and in some cases branched morphologies are seen penetrating the brush border. In other cysts of the same age, cilia are difficult to visualize possibly because they are small and hidden in the brush border. TEM showed that the biliary cilia were always surrounded by clumps of vesicular structures of 50–80 nm in diameter of unknown origin. They may be fragments of ciliary plasma membrane that are being shed from the cilium itself or vesicles similar to 'nodal vesicular parcels'33.Tanaka Y. Okada Y. Hirokawa N. FGF-induced vesicular release of Sonic hedgehog and retinoic acid in leftward nodal flow is critical for left-right determination.Nature. 2005; 435: 172-177Crossref PubMed Scopus (387) Google Scholar thought to interact with the primary cilium in the embryonic node. In summary the Pkhd1del2/del2, mouse model of ARPKD develops both cystic liver and pancreatic disease in both sexes. The PT dilation in the kidney is only seen in the female mouse relatively late in the animals life, 9 months in the outbred background and around 3 months in the inbred lines. The proximal dilatation seen in these animals may mimic the very early stages of human ARPKD making the Pkhd1del2/del2 mouse a useful tool for dissecting out the role of sex hormones and genetic background in the initiation of the disease state. All animal experiments were conducted under protocols approved by the Mayo Institutional Animal Care and Use Committee (IACUC). The knockout construct contains genomic DNA from 3.9 kb 5′ to the start codon of exon 2 to 2.2 kb 3′ to exon 4. A conventional loxP flanked PGK neomycin cassette replaced exon 2 and at the 5′ end there is a PGK-thymidine kinase cassette for negative selection (Figure 1a). Embyronic stem cell targeting and chimera production were performed with a standard protocol.34.van Deursen J. Gene targeting in mouse embryonic stem cells.Methods Mol Biol. 2003; 209: 145-158PubMed Google Scholar To remove the germline floxed neomycin cassette, heterozygous mice, Pkhd1neo2/+, were crossed to mice transgenic for mouse mammary tumor virus-Cre.21.Wagner K.U. McAllister K. Ward T. et al.Spatial and temporal expression of the Cre gene under the control of the MMTV-LTR in different lines of transgenic mice.Transgenic Res. 2001; 10: 545-553Crossref PubMed Scopus (237) Google Scholar We inbred both Pkhd1neo2 and Pkhd1del2 mice on a BALBc/J and C57BL/6J background to generation 10. A multiplex PCR assay was developed to genotype the animals. The WT product is 1028 bp, the Pkhd1neo2 is 794 bp and the Cre recombined Pkhd1del2 is 900 bp. Three primers were used: F1 5′-GGACCTTACAATCTTTTTGCCCC-3′, R1 5′-GCTTCCTCGTGCTTTACGGTATC-3′ and R2 5′-CATCATACAGTTCTCAGACCCCG-3′. The reaction conditions were 30 cycles of 30 s at 94°C (denaturing), 30 s at 56°C (annealing) and 90 s at 72°C (extension) using a QIAGEN® (Valencia, CA, USA) multiplex PCR kit mix. Reverse transcription-PCR was carried out on total cellular RNA from liver and kidney that has reverse transcribed and amplified with an exon 1 primer 5′-CGGCTTCGTTTAGAAGGGGAC-3′ and an exon 7 primer 5′-TCCAGCCAGGTAGTGATGACCC-3′. The reaction conditions were 35 cycles of 60 s 94°C, 30 s 58°C, and 60 s of 72°C, using Taq polymerase (AmpliTaq Gold) Perkin Elmer (Wellesley, MA, USA). Animals were euthanized and the organs dissected, weighed, and fixed in 4% paraformaldehyde. Tissues were then embedded in parafin and 5 μm sections cut from each tissue. Hematoxylin/eosin and Mallory trichrome staining was performed according to standard procedures by the Mayo Clinic Histology laboratory. Kidney tubules were measured using MetaMorph software package version 6.3r7 (Molecular Devices, Downingtown, PA, USA). Number of tubules, measured from basement lamina to basement lamina, ranged from 75 to 181 from female kidneys at 9 and 12 months of age and from both WT and Pkhd1del2/del2 genotypes. Student's t-test was performed to test differences between age-genotype groups. SEM of liver and kidney tissue employed the protocol of Masyuk et al.35.Masyuk T.V. Huang B.Q. Masyuk A.I. et al.Biliary dysgenesis in the PCK rat, an orthologous model of autosomal recessive polycystic kidney disease.Am J Pathol. 2004; 165: 1719-1730Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar Briefly, liver or kidney tissue was fixed in 4% paraformaldehyde/2% glutaraldehyde for 2 h, incubated in 1% osmium tetroxide for 30 min, dehydrated, dried in a critical point drying device and mounted. SEM was performed with a Hitachi S-4700 microscope. For TEM, samples were postfixed in 1% osmium tetroxide for 1 h, dehydrated, embedded in Spurrs resin, sectioned at 80 nm, and visualized with a Joel 1200 electron microscope. Mice were anesthetized with 50 mg/kg sodium pentobarbital. The left ventricle was cannulated and the inferior vena cava cut. The mice were perfused with phosphate-buffered saline (PBS) followed by a similar volume of 4% paraformaldehyde in PBS. Finally, the animals were perfused with 10% sucrose/20 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (pH 7.2). Kidney, liver, pancreas, and gall bladder were removed, embedded in Optimum Cutting Temperature (OCT), snap frozen in liquid nitrogen cooled methyl butane and then stored at -80°C until use. Five micron sections were taken mounted on positively charged slides and dried for 20 min at 60°C. For LTA36.Schulte B.A. Spicer S.S. Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates.Am J Anat. 1983; 168: 345-362Crossref PubMed Scopus (99) Google Scholar or DBA36.Schulte B.A. Spicer S.S. Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates.Am J Anat. 1983; 168: 345-362Crossref PubMed Scopus (99) Google Scholar,37.Watanabe M. Muramatsu T. Shirane H. et al.Discrete Distribution of binding sites for Dolichos biflorus agglutinin (DBA) and for peanut agglutinin (PNA) in mouse organ tissues.J Histochem Cytochem. 1981; 29: 779-780Crossref PubMed Scopus (111) Google Scholar (Vector Laboratories, Burlingame, CA, USA) staining, biotinylated LTA or DBA was applied at 5 μg/ml in 1% bovine serum albumin PBS (30 min; 20°C), to sections permeabilized with 0.1% Triton-X in PBS. Slides were washed three times in PBS then detected with 5 μg/ml Alexa Fluor® 488 labeled streptavidin (Invitrogen, Carlsbad, CA, USA) for 20 min and 100 nM Alexa Fluor® Phalloidin 594 (Invitrogen). Slides were washed with PBS three times then exposed to 3 μM 4′-diamidino-2-phenylindole (DAPI) for 1 min, washed then mounted with vector shield (Vector Laboratories). Kidney morphology was accessed by a trained, blinded pathologist (DL). We thank the Indiana University School of Medicine EM Center and to the generous support of that facility by the Polycystic Kidney Disease Foundation. We also thank the technical support of Mark Consugar. This work was funded by NIDDK Grants DK65056 (CJW), DK59597 (PCH), and DK24031 (NFL), the Polycystic Kidney Disease Foundation Postdoctoral Fellowship (TVM), and the Mayo Foundation.
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