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Background/aims: The PCK rat model of polycystic kidney disease is characterized by the progressive development of renal medullary cysts. Here, we evaluated the suitability of high resolution ultrasonography (HRU) to assess the kidney and cyst volume in PCK rats, testing three different ultrasound image analysis methods, and correlating them with kidneys weights and histological examinations., Methods: After inducing anesthesia, PCK rats (n=18) were subjected to HRU to visualize the kidneys, to perform numeric and volumetric measurements of the kidney and any cysts observed, and to generate 3-dimensional images of the cysts within the kidney parenchyma., Results: HRU provided superior information in comparison to microscopic analysis of stained kidney sections. HRU-based kidney volumes correlated strongly with kidney weights (R2=0.809; P<0.0001)., Conclusion: HRU represents a useful diagnostic tool for kidney and cyst volume measurements in PCK rats. Sequential HRU examinations may be useful to study the effect of drugs on cyst growth without the need to euthanize experimental animals., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

The rat Pck gene is orthologous to the human PKHD1 gene responsible for autosomal recessive polycystic kidney disease (ARPKD). Both renal and hepatic fibrocystic pathology occur in ARPKD. Affected humans have a variable rate of progression, from morbidly affected infants to those surviving into adulthood. This study evaluated the PCK rat, a model of slowly progressive ARPKD. This model originated in Japan and was rederived to be offered commercially by Charles River Laboratories (Wilmington, MA). Previous studies have described the basic aspects of PCK pathology from privately held colonies. This study provides a comprehensive characterization of rats from those commercially available. Rats were bred, maintained on a 12:12 hr light/dark cycle, fed (7002 Teklad), and water provided ad libitum. Male and female rats were evaluated from 4 through 35 weeks of age with histology and serum chemistry. As the hepatorenal fibrocystic disease progressed beyond 18 weeks, the renal pathology (kidney weight, total cyst volume) and renal dysfunction (BUN and serum creatinine) tended to be more severe in males, whereas liver pathology (liver weight as % of body weight and hepatic fibrocystic volume) tended to be more severe in females. Hyperlipidemia was evident in both genders after 18 weeks. Bile secretion was increased in PCK rats compared with age-matched Sprague Dawley rats. The PCK is an increasingly used orthologous rodent model of human ARPKD. This characterization study of hepatorenal fibrocystic pathology in PCK rats should help researchers select stages of pathology to study and/or monitor disease progression during their longitudinal studies., ((c) 2010 Wiley-Liss, Inc.)

Polycystic kidney (PCK) rats are a spontaneous model of autosomal recessive polycystic kidney disease that exhibit cholangiocyte-derived liver cysts. We have previously reported that in normal cholangiocytes a subset of vesicles contain three proteins (ie, the water channel AQP1, the chloride channel CFTR, and the anion exchanger AE2) that account for ion-driven water transport. Thus, we hypothesized that altered expression and location of these functionally related proteins contribute to hepatic cystogenesis. We show here that under basal conditions and in response to secretin and hypotonicity, cysts from PCK rats expanded to a greater degree than cysts formed by normal bile ducts. Quantitative reverse transcriptase-polymerase chain reaction, immunoblot analysis, and confocal and immunoelectron microscopy all indicated increased expression of these three proteins in PCK cholangiocytes versus normal cholangiocytes. AQP1, CFTR, and AE2 were localized preferentially to the apical membrane in normal rats while overexpressed at the basolateral membrane in PCK rats. Exposure of the cholangiocyte basolateral membrane to CFTR inhibitors [5-nitro-2-(3-phenylpropylamino)-benzoic acid and CFTRinh172], or Cl(-)/HCO(3)(-) exchange inhibitors (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate and 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid disodium salt hydrate) blocked secretin-stimulated fluid accumulation in PCK but not in normal cysts. Our data suggest that hepatic cystogenesis in autosomal recessive polycystic kidney disease may involve increased fluid accumulation because of overexpression and abnormal location of AQP1, CFTR, and AE2 in cystic cholangiocytes. Therapeutic interventions that block the activation of these proteins might inhibit cyst expansion in polycystic liver disease.

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 Pkhd1(del2/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 Pkhd1(del2/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.

Background/methods: We have examined the hypothesis that cyst formation is key in the pathogenesis of cardiovascular disease in a Lewis polycystic kidney (LPK) model of autosomal-recessive polycystic kidney disease (ARPKD), by determining the relationship between cyst development and indices of renal function and cardiovascular disease., Results: In the LPK (n = 35), cysts appear at week 3 (1.1 +/- 0.1 mm) increasing to week 24 (2.8 +/- 2 mm). Immunostaining for nephron-specific segments indicate cysts develop predominantly from the collecting duct. Cyst formation preceded hypertension (160 +/- 22 vs. Lewis control 105 +/- 20 mm Hg systolic blood pressure (BP), n = 12) at week 6, elevated creatinine (109 +/- 63 vs. 59 +/- 6 micromol/l, n = 16) and cardiac mass (0.7 vs. 0.4% bodyweight, n = 15) at week 12, and left ventricular hypertrophy (2,898 +/- 207 vs. 1,808 +/- 192 mum, n = 14) at week 24 (all p < or = 0.05). Plasma-renin activity and angiotensin II were reduced in 10- to 12-week LPK (2.2 +/- 2.9 vs. Lewis 11.9 +/- 4.9 ng/ml/h, and 25.0 +/- 19.1 vs. 94.9 +/- 64.4 pg/ml, respectively, n = 26, p < or = 0.05). Ganglionic blockade (hexamethonium 3.3 mg/kg) significantly reduced mean BP in the LPK (52 vs. Lewis 4%, n = 9, p < or = 0.05)., Conclusion: Cyst formation is a key event in the genesis of hypertension while the sympathetic nervous system is important in the maintenance of hypertension in this model of ARPKD., (Copyright 2007 S. Karger AG, Basel.)

In the PCK rat, a rodent model of Autosomal Recessive Polycystic Kidney Disease (ARPKD), a spontaneous splicing mutation of Pkhd1 initiates hepatic cyst development. Cystic cholangiocytes possess short and malformed cilia that do not express fibrocystin, the Pkhd1 protein. During the disease course, cysts continue to grow; however, the mechanisms underlying cyst progression are unclear due in part to the lack of suitable cell lines to study cystogenesis. Here, we describe the development of a PCK-derived cholangiocyte cell line (PCK-CCL). Normal rat cholangiocytes (NRCs) were used as a control. The PCK-CCL maintained a cholangiocyte phenotype as assessed by the expression of the CK-19, CK-7 and GGT. PCK-CCL grown on collagen formed a polarized monolayer with well-developed junctional complexes, and distinct apical and basolateral membranes. Compared to NRCs, cilia in the PCK-CCL were short and malformed and did not express fibrocystin. The PCK-CCL exhibited a higher rate of proliferation (P<0.05) with a doubling time approximately half that of NRCs. By RT-PCR analysis of exons 33-37, an approximately 800 bp product of Pkhd1 was amplified in NRCs. In contrast and as expected, in the PCK-CCL, the Pkhd1 amplicon was smaller ( approximately 630 bp) reflecting the IVS35-2A --> T mutation. PCK-CCL and NRCs seeded in 3-D cultures formed cystic structures; however, the PCK cysts expanded progressively up to day 21 while cysts formed by NRCs remained the same size after day 9. In summary, we have developed a cholangiocyte cell line from the PCK rat that retains properties of the cholangiocytes lining hepatic cysts in vivo. The cells have been grown continuously for approximately 18 month and 45 passages without crisis or senescence. The morphology and growth characteristics of the PCK-CCL are consistent with those seen in vivo in the PCK rat, suggesting that this cell line will be useful in dissecting the mechanisms of hepatic cyst formation.

Autosomal recessive polycystic kidney disease (ARPKD) is an important cause of liver- and renal-related morbidity and mortality in childhood. Recently, PKHD1, the gene encoding the transmembrane protein polyductin, was shown to be mutated in ARPKD patients. We here describe the first mouse strain, generated by targeted mutation of Pkhd1. Due to exon skipping, Pkhd1ex40 mice express a modified Pkhd1 transcript and develop severe malformations of intrahepatic bile ducts. Cholangiocytes maintain a proliferative phenotype and continuously synthesize TGF-beta1. Subsequently, mesenchymal cells within the hepatic portal tracts continue to synthesize collagen, resulting in progressive portal fibrosis and portal hypertension. Fibrosis did not involve the hepatic lobules, and we did not observe any pathological changes in morphology or function of hepatocytes. Surprisingly and in contrast to human ARPKD individuals, Pkhd1ex40 mice develop morphologically and functionally normal kidneys. In conclusion,our data indicate that subsequent to formation of the embryonic ductal plate, dysgenesis of terminally differentiated bile ducts occurs in response to the Pkhd1ex40 mutation. The role of polyductin in liver and kidney may be functionally divergent, because protein domains essential for bile duct development do not affect nephrogenesis in our mouse model.

Background: Advances in the understanding of cystogenesis, identification of the PKHD1 gene and availability of a rat model (the PCK rat) caused by a Pkhd1 mutation facilitate testing of therapies for autosomal-recessive polycystic kidney disease (ARPKD). Considerable support exists for the importance of the epidermal growth factor (EGF)/transforming growth factor-alpha (TGF-alpha)/EGF receptor (EGFR) axis and of the adenylyl cyclase-adenosine 3',5'-cyclic monophosphate (cAMP) pathway in the pathogenesis of cyst formation and progressive enlargement., Methods: To determine whether EGFR tyrosine kinase inhibition is protective in the PCK rat, male and female animals were treated with EKI-785 or EKB-569 or with vehicle alone between 3 and 10 weeks of age. Biochemical and histomorphometric analysis, immunohistochemistry, immunoblotting, enzyme immunoassay, and quantitative reverse transcription-polymerase chain reaction (RT-PCR) were used to ascertain the effects of treatment., Results: Contrary to other murine models of ARPKD, overexpression and apical mislocalization of EGFR were not detected in the PCK rats. Consistent with these expression results, EKI-785 or EKB-569 administration had no effect or worsened PKD, and had no effect on the development of fibrocystic liver disease. Increased renal cAMP and vasopressin V2 receptor expression were observed in the EKI-785-treated animals., Conclusion: EGFR tyrosine kinase inhibition did not protect PCK rats from the development of PKD. This may be due to effects on collecting duct cAMP that counteract possible beneficial effects on the extracellular-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway, particularly in the absence of EGFR overexpression or mislocalization. The relevance of these observations to the treatment of human cystic kidney diseases deserves further study.

Rodent models of polycystic kidney disease (PKD) have provided valuable insight into the cellular changes associated with cystogenesis in humans. The present study characterizes the morphology of renal and extrarenal pathology of autosomal recessive PKD induced by the wpk gene in Wistar rats. In wpk(-/-) rats, proximal tubule and collecting duct cysts develop in utero and eventually consume the kidney. Increased apoptosis, mitosis, and extracellular tenascin deposition parallel cyst development. Extrarenal pathology occurs in the immune system (thymic and splenic hypoplasia) and central nervous system (CNS; hypoplasia to agenesis of the corpus callosum with severe hydrocephalus). Severity of hydrocephalus varied inversely with size of the corpus callosum. In wpk(-/-) rats, the corpus callosum exhibits relatively few axons that cross the midline. This CNS pathology is similar to that described in three human renal cystic syndromes: orofaciodigital, genitopatellar, and cerebrorenal-digital syndromes. Collecting duct and ventricular ependymal cilia appear morphologically normal. To determine if rodent background strain and the presence of modifier genes affect severity of the disease, we crossed the Wistar-wpk rat with Brown Norway (BN) and Long Evan (LE) rats and found the degree of renal and cerebral pathology was diminished as evidenced by lower kidney weight as a percent of body weight and serum urea nitrogen concentration in cystic rats on LE or BN strains as well as less prominent cranial enlargement. Crosses with BN rats allowed us to localize the wpk gene on chromosome 5 very close to the D5Rat73 marker. The wpk gene lies within a chromosomal region known to harbor a PKD modifier locus. In summary, the types of renal and cerebral pathology seen in the Wistar wpk rat are a unique combination seen only in this rodent model., (Copyright 2004 Wiley-Liss, Inc.)

Ke 6 is a 17beta-hydroxysteroid dehydrogenase (17betaHSD) that is expressed in the kidneys and gonads. The expression of this gene is markedly reduced in three murine models of recessive polycystic kidney disease, a developmental disorder, where some nephrons within the affected kidneys develop into huge fluid-filled cysts while the non-cystic nephrons atrophies by apoptosis. Here, we show that in the cpk/cpk mouse, which have polycystic kidneys, the female reproductive organs also fail to mature properly and remain arrested at an early stage of development. Direct measurement of 17betaHSD activity showed a severe reduction in estrogen and androgen metabolism within gonadal and non-gonadal tissues of the cpk/cpk mouse. Using immunofluorescent staining we localized the expression of the Ke 6 protein within the female mouse reproductive organs. Our findings suggest that estrogen/androgen metabolism may play an important role in the development of the urogenital systems.

Numerous murine models of polycystic kidney disease (PKD) have been described. While mouse models are particularly well suited for investigating the molecular pathogenesis of PKD, rats are well established as an experimental model of renal physiologic processes. Han:SPRD-CY: rats have been proposed as a model for human autosomal dominant PKD. A new spontaneous rat mutation, designated wpk, has now been identified. In the mutants, the renal cystic phenotype resembles human autosomal recessive PKD (ARPKD). This study was designed to characterize the clinical and histopathologic features of wpk/wpk mutants and to map the wpk locus. Homozygous mutants developed nephromegaly, hypertension, proteinuria, impaired urine-concentrating capacity, and uremia, resulting in death at 4 wk of age. Early cysts were present in the nephrogenic zone at embryonic day 19. These were localized, by specific staining and electron microscopy, to differentiated proximal tubules, thick limbs, distal tubules, and collecting ducts. In later stages, the cysts were largely confined to collecting ducts. Although the renal histopathologic features are strikingly similar to those of human ARPKD, wpk/wpk mutants exhibited no evidence of biliary tract abnormalities. The wpk locus maps just proximal to the CY: locus on rat chromosome 5, and complementation studies demonstrated that these loci are not allelic. It is concluded that the clinical and renal histopathologic features of this new rat model strongly resemble those of human ARPKD. Although homology mapping indicates that rat wpk and human ARPKD involve distinct genes, this new rat mutation provides an excellent experimental model to study the molecular pathogenesis and renal pathophysiologic features of recessive PKD.

Current models of autosomal recessive polycystic kidney disease (ARPKD) fail to demonstrate biliary abnormalities in association with renal cysts. We therefore studied a new murine model of ARPKD in which dual renal tubular and biliary epithelial abnormalities are present. Affected homozygous animals typically die 1 month postnatally in renal failure with progressively enlarged kidneys. Renal cysts shift in site from inner cortical proximal tubules at birth to collecting tubules 20 days later, as determined by segment-specific lectin binding. Increased numbers of mitosis were demonstrated in proximal and collecting tubular cysts. In addition, epithelial hyperplasia was demonstrated morphometrically in the intra- and extrahepatic biliary tract of affected animals. The number of intrahepatic biliary epithelial cells was increased by 50% on postnatal day 5 and by 100% on postnatal day 25 (P < 0.01). Despite an increased frequency of "chaotic" portal areas in mice with renal cysts, no intrahepatic cysts or shape abnormalities of the biliary lumen were detected using biliary casts and morphometry. Additionally there was nonobstructive hyperplastic dilatation of the extrahepatic biliary tract which was linked in all animals to the presence of renal cysts. The hyperplastic abnormalities in both renal and biliary epithelium make this new mouse strain a good model for the study of the dual organ cellular pathophysiology of ARPKD.

We describe the renal cystic disease occurring in a new inbred strain of mice which developed as a spontaneous mutation in otherwise healthy Balb/c mice. The disease displays characteristics of an autosomal recessive polycystic kidney disease. Affected animals develop massive cystic enlargement of the kidneys and die of renal failure at the age of 4 weeks. During postnatal development, there is a gradual shift in site of the lesions. At birth, cystic dilations are localized almost exclusively in proximal tubular segments, whereas in the terminal stages of the disease, 80% of the cysts are localized in collecting tubular segments as defined by segment specific lectin binding. The composition of the basement membrane of the cystic tubular walls during postnatal development as analyzed by immunocytochemistry is essentially normal during the earliest stage of cyst formation. However, with disease progression, the cystic tubular basement membrane demonstrates a decreased immunoreactivity to anti-laminin and anti-entactin antibodies. This indicates a shift in cyst localization during disease progression in this model, and suggests that basement membrane abnormalities are not a primary feature of the early cyst formation and progressive enlargement.