Polycystic Kidney Rat Research Articles

Angiotensin-converting enzyme inhibitor limits pulse-wave velocity and aortic calcification in a rat model of cystic renal disease

The effect of angiotensin-converting enzyme inhibition on function and structure of the aorta was studied in the Lewis polycystic kidney (LPK) rat model of cystic renal disease and Lewis controls. Pulse-wave velocity (PWV) was recorded under urethane anesthesia (1.3 g/kg ip) in mixed-sex animals aged 6 and 12 wk and in 12-wk-old animals treated with perindopril (3 mg·kg(-1)·day(-1) po) from age 6-12 wk. Tail-cuff systolic pressures were recorded over the treatment period. After PWV measurements, animals were euthanized and the aorta was removed for histomorphological and calcium analysis. Hypertension in LPK at 6 and 12 wk was associated with a shift of the PWV curve upward and to the right, indicating a decrease in aortic compliance, which was significantly reduced by perindopril. LPK demonstrated greater aortic calcification (6 wk: 123 ± 19 vs. 65 ± 7 and 12 wk: 406 ± 6 vs. 67 ± 6 μmol/g, P < 0.001, LPK vs. Lewis, respectively). This was reduced by treatment with perindopril (172 ± 48 μmol/g, 12 wk LPK P < 0.001). Medial cross-sectional area and elastic modulus/wall stress of the aorta were greater in LPK vs. Lewis control animals at 6 and 12 wk of age and showed an age-related increase that was prevented by treatment with perindopril (P < 0.001). Perindopril also ameliorated the degradation of elastin, increase in collagen content, and medial elastocalcinosis seen in 12-wk LPK. Overall, perindopril improved the structural and functional indices of aortic stiffness in the LPK rats, demonstrating a capacity for angiotensin-converting enzyme inhibition to limit vascular remodeling in chronic kidney disease.

Caroli's Disease: Current Knowledge of Its Biliary Pathogenesis Obtained from an Orthologous Rat Model.

Caroli's disease belongs to a group of hepatic fibropolycystic diseases and is a hepatic manifestation of autosomal recessive polycystic kidney disease (ARPKD). It is a congenital disorder characterized by segmental saccular dilatations of the large intrahepatic bile duct and is frequently associated with congenital hepatic fibrosis (CHF). The most viable theory explaining its pathogenesis suggests that it is related to ductal plate malformation. The development of the polycystic kidney (PCK) rat, an orthologous rodent model of Caroli's disease with CHF as well as ARPKD, has allowed the molecular pathogenesis of the disease and the therapeutic options for its treatment to be examined. The relevance of the findings of studies using PCK rats and/or the cholangiocyte cell line derived from them to the pathogenesis of human Caroli's disease is currently being analyzed. Fibrocystin/polyductin, the gene product responsible for ARPKD, is normally localized to primary cilia, and defects in the fibrocystin from primary cilia are observed in PCK cholangiocytes. Ciliopathies involving PCK cholangiocytes (cholangiociliopathies) appear to be associated with decreased intracellular calcium levels and increased cAMP concentrations, causing cholangiocyte hyperproliferation, abnormal cell matrix interactions, and altered fluid secretion, which ultimately result in bile duct dilatation. This article reviews the current knowledge about the pathogenesis of Caroli's disease with CHF, particularly focusing on studies of the mechanism responsible for the biliary dysgenesis observed in PCK rats.

Aortic stiffness is associated with vascular calcification and remodeling in a chronic kidney disease rat model

Increased aortic pulse-wave velocity (PWV) reflects increased arterial stiffness and is a strong predictor of cardiovascular risk in chronic kidney disease (CKD). We examined functional and structural correlations among PWV, aortic calcification, and vascular remodeling in a rodent model of CKD, the Lewis polycystic kidney (LPK) rat. Hemodynamic parameters and beat-to-beat aortic PWV were recorded in urethane-anesthetized animals [12-wk-old hypertensive female LPK rats (n = 5)] before the onset of end-stage renal disease and their age- and sex-matched normotensive controls (Lewis, n = 6). Animals were euthanized, and the aorta was collected to measure calcium content by atomic absorption spectrophotometry. A separate cohort of animals (n = 5/group) were anesthetized with pentobarbitone sodium and pressure perfused with formalin, and the aorta was collected for histomorphometry, which allowed calculation of aortic wall thickness, medial cross-sectional area (MCSA), elastic modulus (EM), and wall stress (WS), size and density of smooth muscle nuclei, and relative content of lamellae, interlamellae elastin, and collagen. Mean arterial pressure (MAP) and PWV were significantly greater in the LPK compared with Lewis (72 and 33%, respectively) animals. The LPK group had 6.8-fold greater aortic calcification, 38% greater aortic MCSA, 56% greater EM/WS, 13% greater aortic wall thickness, 21% smaller smooth muscle cell area, and 20% less elastin density with no difference in collagen fiber density. These findings demonstrate vascular remodeling and increased calcification with a functional increase in PWV and therefore aortic stiffness in hypertensive LPK rats.

PPAR-γ agonist ameliorates kidney and liver disease in an orthologous rat model of human autosomal recessive polycystic kidney disease

In autosomal recessive polycystic kidney disease (ARPKD), progressive enlargement of fluid-filled cysts is due to aberrant proliferation of tubule epithelial cells and transepithelial fluid secretion leading to extensive nephron loss and interstitial fibrosis. Congenital hepatic fibrosis associated with biliary cysts/dilatations is the most common extrarenal manifestation in ARPKD and can lead to massive liver enlargement. Peroxisome proliferator-activated receptor γ (PPAR-γ), a member of the ligand-dependent nuclear receptor superfamily, is expressed in a variety of tissues, including the kidneys and liver, and plays important roles in cell proliferation, fibrosis, and inflammation. In the current study, we determined that pioglitazone (PIO), a PPAR-γ agonist, decreases polycystic kidney and liver disease progression in the polycystic kidney rat, an orthologous model of human ARPKD. Daily treatment with 10 mg/kg PIO for 16 wk decreased kidney weight (% of body weight), renal cystic area, serum urea nitrogen, and the number of Ki67-, pERK1/2-, and pS6-positive cells in the kidney. There was also a decrease in liver weight (% of body weight), liver cystic area, fibrotic index, and the number of Ki67-, pERK1/2-, pERK5-, and TGF-β-positive cells in the liver. Taken together, these data suggest that PIO inhibits the progression of polycystic kidney and liver disease in a model of human ARPKD by inhibiting cell proliferation and fibrosis. These findings suggest that PPAR-γ agonists may have therapeutic value in the treatment of the renal and hepatic manifestations of ARPKD.

Sirolimus attenuates disease progression in an orthologous mouse model of human autosomal dominant polycystic kidney disease

In autosomal dominant polycystic kidney disease (ADPKD), abnormal proliferation of tubular cells drives cyst development and growth. Sirolimus, an inhibitor of the protein kinase mammalian target of rapamycin (mTOR) and a potent anti-proliferative agent, decreases cyst growth in several genetically distinct rodent models of polycystic kidney disease (PKD). We determined here the effect of sirolimus on renal cyst growth in Pkd2WS25/- mice; an ortholog of human ADPKD involving mutation of the Pkd2 gene. In Pkd2WS25/- mice treated with sirolimus, both the two kidney/total body weight (2K/TBW) ratio and the cyst volume density (CVD) were significantly decreased by over half compared with untreated mice suffering with PKD. However, there was no effect on the increased blood urea nitrogen (BUN) levels as an index of kidney function. There are two distinct complexes containing mTOR depending on its binding partners: mTORC1 and mTORC2. Western blot analysis of whole kidney lysates and immunohistochemistry of the cysts found that phospho-S6 ribosomal protein, a marker of mTORC1 activity, was increased in Pkd2WS25/- mice and its phosphorylation was decreased by sirolimus treatment. Phospho-Akt at serine 473, a marker associated with mTORC2 activity, was not different between Pkd2WS25/- mice and normal littermate controls. Hence, our study found that inhibition of mTORC1 by sirolimus correlated with decreased renal cyst growth in this model of human ADPKD but had no effect on the decline in renal function.

The renin-angiotensin system and hypertension in autosomal recessive polycystic kidney disease

Hypertension is a well-recognized complication of autosomal recessive polycystic kidney disease (ARPKD). The renin-angiotensin system (RAS) is a key regulator of blood pressure; however, data on the RAS in ARPKD are limited and conflicting, showing both up- and down-regulation. In the current study, we characterized intrarenal and systemic RAS activation in relationship to hypertension and progressive cystic kidney disease in the ARPKD orthologous polycystic kidney (PCK) rat. Clinical and histological measures of kidney disease, kidney RAS gene expression by quantitative real-time PCR, angiotensin II (Ang II) immunohistochemistry, and systemic Ang I and II levels were assessed in 2-, 4-, and 6-month-old cystic PCK and age-matched normal rats. PCK rats developed hypertension and progressive cystic kidney disease without significant worsening of renal function or relative kidney size. Intrarenal renin, ACE and Ang II expression was increased significantly in cystic kidneys; angiotensinogen and Ang II Type I receptor were unchanged. Systemic Ang I and II levels did not differ. This study demonstrates that intrarenal, but not systemic, RAS activation is a prominent feature of ARPKD. These findings help reconcile previous conflicting reports and suggest that intrarenal renin and ACE gene upregulation may represent a novel mechanism for hypertension development or exacerbation in ARPKD.

Activation of Trpv4 Reduces the Hyperproliferative Phenotype of Cystic Cholangiocytes From an Animal Model of ARPKD

In polycystic liver diseases, cyst formation involves cholangiocyte hyperproliferation. In polycystic kidney (PCK) rats, an animal model of autosomal-recessive polycystic kidney disease (ARPKD), decreased intracellular calcium [Ca(2+)](i) in cholangiocytes is associated with hyperproliferation. We recently showed transient receptor potential vanilloid 4 (Trpv4), a calcium-entry channel, is expressed in normal cholangiocytes and its activation leads to [Ca(2+)](i) increase. Thus, we hypothesized that pharmacologic activation of Trpv4 might reverse the hyperproliferative phenotype of PCK cholangiocytes. Trpv4 expression was examined in liver of normal and PCK rats, normal human beings, and patients with autosomal-dominant polycystic kidney disease or ARPKD. Trpv4 activation effect on cell proliferation and cyst formation was assessed in cholangiocytes derived from normal and PCK rats. The in vivo effects of Trpv4 activation on kidney and liver cysts was analyzed in PCK rats. Trpv4 was overexpressed both at messenger RNA (8-fold) and protein (3-fold) levels in PCK cholangiocytes. Confocal and immunogold electron microscopy supported Trpv4 overexpression in the livers of PCK rats and ARPKD or autosomal-dominant polycystic kidney disease patients. Trpv4 activation in PCK cholangiocytes increased [Ca(2+)](i) by 30%, inhibiting cell proliferation by approximately 25%-50% and cyst growth in 3-dimensional culture (3-fold). Trpv4-small interfering RNA silencing blocked effects of Trpv4 activators by 70%. Trpv4 activation was associated with Akt phosphorylation and beta-Raf and Erk1/2 inhibition. In vivo, Trpv4 activation induced a significant decrease in renal cystic area and a nonsignificant decrease in liver cysts. Taken together, our in vitro and in vivo data suggest that increasing intracellular calcium by Trpv4 activation may represent a potential therapeutic approach in PKD.

More on Clinical Renal Genetics

Massive polycystic liver may be the source of severe discomfort, early satiety, pain, dyspnea, and finally denutrition. This is in part due to the increase with age in the number of hepatic cysts, but also because the individual cyst volume grows from 0.25 to 22.8 ml over 20 years. Polycystic liver is found in two inherited disorders—autosomal dominant polycystic kidney disease (ADPKD) and autosomal dominant polycystic liver disease (PCLD), an infrequent disorder in which no kidney cysts are detected caused by mutations in PKRCSH or SEC63 genes. Studies in ADPKD have indicated that total liver cyst volume and the prevalence of massive polycystic liver are greater in women than in men. Liver cysts arise from cholangiocytes. Secretin is a potent stimulator of adenylate cyclase and thus of cAMP generation. cAMP acts by two major mechanisms: increased electrolyte and water secretion and stimulation of cholangiocyte proliferation. Conversely, somatostatin inhibits cAMP production and thus fluid secretion and proliferation. Additional experimental and clinical data supported the project of initiating a clinical trial of somatostatin analogues on liver cyst progression. Octreotide, a somatostatin analogue, prevented outgrowth of liver and kidney cysts in polycystic kidney (PCK) rats. In contrast, antagonists of the V2 vasopressin receptor have no effect on liver cysts that do not express this receptor, whereas they prevent or slow the progression of renal cysts in various experimental models. In two patients with polycystic livers, a 3- to 6-month treatment with somatostatin analogues led to impressive reductions in liver volume (1). Finally, a randomized, placebo-controlled trial in ADPKD patients using slow-release octreotide (40 mg) showed that kidney volume increase could be reduced by 60% at 6 months compared with placebo but there was an ongoing increase of kidney volume—2.2% in the octreotide group versus 5.9% in the placebo group (2). Van Keimpema et al. (1) designed a multicenter, randomized, double-blind, placebo-controlled trial to evaluate the effects of the long-acting somatostatin analogue, lanreotide autosolution (120 mg), or placebo administered subcutaneously every 28 days for 24 weeks in patients with polycystic liver due to ADPKD (32 patients) or PLCD (22 patients). The high percentage of PLCD is explained by the fact that the initiators of and participants in this trial were gastroenterologists and hepatologists. The primary end point was change in liver volume measured on computed tomography (CT) scan at baseline and after 24 weeks of treatment. Health-related quality of life was a secondary end point. Of 113 consecutive patients assessed during the study period, 54 were included in the trial. Relatively more PCLD patients (68%) than ADPKD patients (38%) received lanreotide. The mean liver volume decreased from 4606 to 4471 ml (i.e., an average reduction of 2.9%) whereas there was an average increase of 1.6% in patients receiving lanreotide and in those in the placebo group, respectively. Eighty-five percent of patients on lanreotide showed a decrease of liver volume, compared with 27% of those receiving placebo. Larger livers had proportionally more volume reduction than smaller livers. In the subgroup of patients with ADPKD, mean kidney volume decreased by 1.5% with lanreotide, whereas it increased in the placebo group (P = 0.02). Current health perception improved in lanreotide-treated patients. The most common adverse effect of lanreotide consisted of loose, pale, and fatty stools (19 patients), which were relieved by pancreatic enzymes. Gamma-glutamyl transferase levels increased in the lanreotide group, possibly reflecting increased cyst epithelium degeneration. In this trial, no correlation was found between lanreotide serum levels and response to treatment. Little is known about the optimal dosage of somatostatin analogues. The authors chose for the highest licensed dose of lanreotide. Lanreotide at 120 mg is equivalent to octreotide at 60 mg. However, somatostatin receptor affinity of octreotide is higher compared with that of lanreotide. The results of this trial are very encouraging. Aspiration and sclerosis, laparoscopic fenestration, or hepatic resection have led to disappointing or limited results in women with massive polycystic liver. Combined liver and kidney transplantation has been mandatory in some of them. Noninvasive effective therapy is eagerly awaited. Another trial with a somatostatin analogue is in progress.

Chronic treatment with lisinopril decreases proliferative and apoptotic pathways in autosomal recessive polycystic kidney disease

Angiotensin converting enzyme (ACE) inhibition is a common therapeutic modality in the treatment of autosomal recessive polycystic kidney disease (ARPKD). This study was designed to investigate whether chronic inhibition of ACE would have a therapeutic effect in attenuating the progression of renal cystogenesis in an orthologous rat model of ARPKD, the polycystic kidney (PCK) rat. Lisinopril (3 mg/kg per day) was administered orally for a period of 12 weeks, beginning at post-natal week 4. Lisinopril treatment resulted in an approximately 30% improvement in the collecting duct cystic indices (CT CI) of PCK animals. Activation of extracellular signal-regulated kinase 1 (ERK1) and 2 (ERK2), proliferative signaling markers, and proliferating cell nuclear antigen (PCNA), an end-point marker for proliferation, was reduced following chronic treatment with lisinopril compared to that in vehicle-treated PCK rats. To assess whether apoptotic pathways were altered due to chronic ACE inhibition, we examined p38 mitogen activated protein kinase (MAPK) and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), which are markers of apoptotic signaling cascades. p38 MAPK was significantly reduced (P < 0.0001) following chronic treatment with lisinopril, but no change in the activation of SAPK/JNK could be detected by immunoblot analysis. Lisinopril treatment resulted in a significant reduction (P < 0.01) in cleaved caspase-7 levels, but not caspase-3 activity, in PCK rat kidneys compared to the vehicle-treated PCK rat kidneys. Proteinuria was completely ameliorated in the presence of chronic ACE inhibition in the lisinopril-treated rats compared with the vehicle-treated PCK rats. In all, these findings demonstrated that chronic ACE inhibition can beneficially alter proliferative and apoptotic pathways to promote therapeutic reductions in renal cyst development in ARPKD.

Cyclic nucleotide signaling in polycystic kidney disease

Increased levels of 3'-5'-cyclic adenosine monophosphate (cAMP) stimulate cell proliferation and fluid secretion in polycystic kidney disease. Levels of this molecule are more sensitive to inhibition of phosphodiesterases (PDEs), whose activity far exceeds the rate of cAMP synthesis by adenylyl cyclase. Several PDEs exist, and here we measured the activity and expression of PDE families, their isoforms, and the expression of downstream effectors of cAMP signaling in the kidneys of rodents with polycystic kidney disease. We found a higher overall PDE activity in kidneys from mice as compared with rats, as well as a higher contribution of PDE1, relative to PDE4 and PDE3, to total PDE activity of kidney lysates and lower PDE1, PDE3, and PDE4 activities in the kidneys of cystic as compared with wild-type mice. There were reduced amounts of several PDE1, PDE3, and PDE4 proteins, possibly due to increased protein degradation despite an upregulation of their mRNA. Increased levels of cGMP were found in the kidneys of cystic animals, suggesting in vivo downregulation of PDE1 activity. We found an additive stimulatory effect of cAMP and cGMP on cystogenesis in vitro. Cyclic AMP-dependent protein kinase subunits Ialpha and IIbeta, PKare, the transcription factor CREB-1 mRNA, and CREM, ATF-1, and ICER proteins were upregulated in the kidneys of cystic as compared with wild-type animals. Our study suggests that alterations in cyclic nucleotide catabolism may render cystic epithelium particularly susceptible to factors acting on Gs-coupled receptors. This may account, in part, for increased cyclic nucleotide signaling in polycystic kidney disease and contribute substantially to disease progression.

Corrigendum

CORRIGENDACorrigendumPublished Online:01 Oct 2009https://doi.org/10.1152/ajprenal.zh2-5691-corr.2009Original articleMoreSectionsPDF (556 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Volume 296, March 2009Volume 65, March 2009Pages F575–F582. Park F, Sweeney WE Jr, Jia G, Akbulut T, Mueller B, Falck JR, Birudaraju S, Roman RJ, Avner ED. Chronic blockade of 20-HETE synthesis reduces polycystic kidney disease in an orthologous rat model of ARPKD. Am J Physiol Renal Physiol 296: , 2009 (doi:10.1152/ajprenal.90705.2008). On p. F579, Fig. 2C is a histological section from the same vehicle-treated polycystic kidney disease (PCK) rat kidney as shown in Fig. 2A. This inadvertant duplication has now been removed and replaced with a new image from another vehicle-treated PCK rat kidney at the same magnification. The results and conclusions in the manuscript were not affected by this error, and the authors sincerely apologize for the error. The revised version of Fig. 2 and the legend are shown below.Fig. 2.Effect of 20-HETE synthesis inhibition on kidney mass in a rat model of autosomal recessive polycystic kidney disease (ARPKD). HET-0016 (10 mg·kg–1·day–1), a specific inhibitor of 20-HETE synthesis, was administered daily to polycystic kidney (PCK) rats into the intraperitoneal space, and the animals were killed between postnatal days 56 and 63. The kidneys were harvested, fixed in plastic, and sectioned. Representative kidney sections from four different rats treated with either vehicle (A and C) or HET-0016 (B and D) are shown. Sections were stained with hematoxylin; n = 4 kidneys/group. Magnification ×20.Download figureDownload PowerPointThis article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformationRelated ArticlesChronic blockade of 20-HETE synthesis reduces polycystic kidney disease in an orthologous rat model of ARPKD 01 Mar 2009American Journal of Physiology-Renal Physiology More from this issue > Volume 297Issue 4October 2009Pages F1135-F1135 Copyright & PermissionsCopyright © 2009 American Physiological Societyhttps://doi.org/10.1152/ajprenal.zh2-5691-corr.2009History Published online 1 October 2009 Published in print 1 October 2009 Metrics

Hepatic Cystogenesis Is Associated with Abnormal Expression and Location of Ion Transporters and Water Channels in an Animal Model of Autosomal Recessive Polycystic Kidney Disease

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.

Matrix proteins of basement membrane of intrahepatic bile ducts are degraded in congenital hepatic fibrosis and Caroli's disease

Congenital hepatic fibrosis (CHF) and Caroli's disease are though to result from ductal plate malformation, and the basal laminar components play important roles in biliary differentiation during development. To clarify the involvement of basal laminar components in the ductal plate malformation, this study examined the immunohistochemical expression of laminin and type IV collagen in the livers of CHF and Caroli's disease. Using the polycystic kidney (PCK) rat, an animal model of Caroli's disease with CHF, in vivo and in vitro experiments were also performed. Immunostaining showed that basement membrane expression of laminin and type IV collagen around intrahepatic bile ducts was degraded in CHF, Caroli's disease, and the PCK rats. The degradation of laminin and type IV collagen around bile ducts was also observed in foci of cholangiocarcinoma in situ of Caroli's disease. In vitro, PCK cholangiocytes were found to overexpress plasminogen and a serine proteinase, the tissue-type plasminogen activator (tPA). When PCK cholangiocytes were cultured in Matrigel, the amounts of laminin and collagen in the gel were significantly reduced, and addition of alpha2-antiplasmin in the culture medium inhibited the degradation of laminin and collagen in Matrigel. These results suggest that biliary overexpression of plasminogen and tPA leads to the generation of excessive amounts of plasmin, and subsequent plasmin-dependent lysis of the extracellular matrix molecules may contribute to the biliary dysgenesis in CHF and Caroli's disease, including progressive cystic dilatation of the intrahepatic bile ducts in Caroli's disease. In addition, it is suggested that once cholangiocarcinoma in situ develops in the biliary epithelium of CHF and Caroli's disease, it tends to transform into invasive carcinoma, due to instability of the basement membrane of the bile ducts.

Tumor necrosis factor‐α induces the aberrant expression of mucus core protein‐2 in non‐neoplastic biliary epithelial cells via the upregulation of CDX2 in chronic cholangitis

Chronic cholangitis, such as hepatolithiasis, is frequently associated with goblet cell metaplasia and the aberrant expression of mucus core protein-2 (MUC2). In this study, we clarified the role of inflammatory cytokines in the expression of MUC2 in lining biliary epithelial cells (BEC) in chronic cholangitis with an emphasis on CDX2, an intestine-specific transcription factor. We used human hepatolithiatic livers and polycystic kidney (PCK) rats, an animal model of Caroli's disease, and cultured BEC from PCK rats. As a control, extrahepatic biliary obstruction and histologically normal livers and Crj:CD rats were used. Immunohistochemically, tumor necrosis factor-alpha (TNF-alpha) was expressed in periductal inflammatory cells and BEC of the affected intrahepatic bile ducts with an aberrant expression of MUC2 and CDX2 in hepatolithiasis and the PCK rats. In cultured BEC, TNF-alpha, interleukin (IL)-1beta, IL-6, and interferon-gamma induced the expression of CDX2 mRNA, though only TNF-alpha additionally induced the expression of MUC2 mRNA. The expression of CDX2 mRNA and the MUC2 protein induced in BEC by TNF-alpha were abolished by pretreatment of nuclear factor-kappaB inhibitors. The aberrant expression of CDX2 and MUC2 in the affected bile ducts showing goblet cell metaplasia was closely associated with TNF-alpha expressed in periductal infiltrating inflammatory cells and BEC. TNF-alpha induced the expression of CDX2 and MUC2 in cultured BEC. Taken together, it seems likely that TNF-alpha plays a role in MUC2 expression via CDX2 upregulation in the bile ducts with chronic cholangitis and goblet cell metaplasia.

Cholangiocytes with Mesenchymal Features Contribute to Progressive Hepatic Fibrosis of the Polycystic Kidney Rat

The polycystic kidney (PCK) rat is an animal model of Caroli's disease with congenital hepatic fibrosis, in which the mechanism of progressive hepatic fibrosis remains unknown. This study aimed to clarify the mechanism of hepatic fibrosis of the PCK rat from the viewpoint of the contribution of pathological cholangiocytes. In liver sections of the PCK rats, intrahepatic bile ducts were constituted by two different phenotypes: bile ducts lined by cuboidal-shaped and flat-shaped cholangiocytes. The flat-shaped cholangiocytes showed reduced immunohistochemical expression of the biliary epithelial marker cytokeratin 19 and positive immunoreactivity for vimentin and fibronectin. When cultured cholangiocytes of the PCK rat were treated with transforming growth factor (TGF)-beta1, a potent inducer of epithelial-mesenchymal transition, induction of vimentin, fibronectin, and collagen expression occurred in the PCK cholangiocytes. Although the TGF-beta1 treatment reduced cytokeratin 19 expression, the epithelial cell features characterized by the expression of E-cadherin and zonula occludens-1 was maintained, and alpha-smooth muscle actin expression was not induced in the cholangiocytes. Cholangiocytes of the PCK rat may acquire mesenchymal features in response to TGF-beta1 and participate in progressive hepatic fibrosis by producing extracellular matrix molecules, which seems to be a different event from epithelial-mesenchymal transition.

Interaction of Toll-like receptors with bacterial components induces expression of CDX2 and MUC2 in rat biliary epithelium in vivo and in culture

The mechanism of transformation of biliary epithelium leading to intestinal metaplasia, which is significantly involved in biliary diseases, remains unclear. CDX2, an intestine-specific transcription factor, is thought to regulate intestinal mucin MUC2 (mucus core protein) expression. We took advantage of polycystic kidney (PCK) rats as a model of chronic suppurative cholangitis with intestinal metaplasia and of cultured biliary epithelial cells (BECs) from PCK rats to clarify the causal relation between bacterial components such as pathogen-associated molecular patterns (PAMPs) and the development of intestinal metaplasia of bile ducts. Histological, immunohistochemical, and in situ hybridization studies were conducted in PCK rat livers. In cultured BECs, CDX2 and MUC2 were expressed following treatment with PAMPs and inhibitors (anti-Toll-like receptor (TLR)2/TLR4 antibody, nuclear factor-κB (NF-κB) inhibitor MG132). Chronic suppurative cholangitis with intestinal metaplasia developed as the PCK rats aged, and intestinal metaplasia and aberrant CDX2 and MUC2 expression developed in parallel. Intraluminal bacteria and the expression of TLR2 and TLR4 in BECs were demonstrated in the bile ducts, showing chronic suppurative cholangitis. In cultured BECs, treatment with PAMPs induced upregulation of CDX2 and MUC2 expression, and this effect was abolished by pretreatment with anti-TLR2 and anti-TLR4 antibody and MG132. A knockdown of CDX2 by CDX2 small interfering RNA inhibited MUC2 expression in cultured BECs induced by PAMPs, and transfection of CDX2 expression vector induced MUC2 expression. In conclusion, bacterial components may induce upregulation of the CDX2 expression followed by MUC2 expression via TLR and the NF-κB system in cultured BECs, and could be related to the development of intestinal metaplasia of the bile ducts.

Octreotide Inhibits Hepatic Cystogenesis in a Rodent Model of Polycystic Liver Disease by Reducing Cholangiocyte Adenosine 3′,5′-Cyclic Monophosphate

In polycystic liver diseases (PCLDs), increased cholangiocyte proliferation and fluid secretion are key features and cholangiocyte adenosine 3',5'-cyclic monophosphate (cAMP) is an important regulator of these processes. Thus, we assessed cAMP levels and evaluated octreotide (an analogue of somatostatin known to inhibit cAMP) in hepatic cyst growth using an in vitro model of cystogenesis and an in vivo animal model of autosomal recessive polycystic kidney disease (ARPKD), one of the PCLDs. Expression of somatostatin receptors (SSTRs) was assessed by reverse-transcription polymerase chain reaction and confocal microscopy in cholangiocytes from normal and polycystic kidney (PCK) rats, the ARPKD model of autosomal recessive polycystic kidney disease. Effects of octreotide on cAMP levels and cyst expansion were studied in vitro using PCK bile ducts grown in 3-dimensional culture. The effects of octreotide on hepatic and renal cystogenesis were investigated in PCK rats in vivo. In cholangiocytes and serum of PCK rats, cAMP concentrations were approximately 2 times higher than in normal rats. SSTR subtypes that bind octreotide (ie, SSTR2, SSTR3, and SSTR5) were expressed in both normal and PCK cholangiocytes. In vitro, octreotide inhibited cAMP levels by 35% and reduced cyst growth by 44%. In vivo, octreotide lowered cAMP content in cholangiocytes and serum by 32%-39% and inhibited hepatic disease progression, leading to 22%-60% reductions in liver weight, cyst volume, hepatic fibrosis, and mitotic indices. Similar effects were observed in kidneys of PCK rats. This preclinical study provides a strong rationale for assessing the potential value of octreotide in the treatment of PCLDs.

Inhibition of Intrahepatic Bile Duct Dilation of the Polycystic Kidney Rat with a Novel Tyrosine Kinase Inhibitor Gefitinib

The polycystic kidney (PCK) rat represents a liver and kidney cyst pathology corresponding to Caroli's disease with congenital hepatic fibrosis and autosomal recessive polycystic kidney disease. We previously reported that an epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib (Iressa), significantly inhibited the abnormal growth of biliary epithelial cells of PCK rats in vitro. This study investigated the effects of gefitinib on cyst pathogenesis of the PCK rat both in vitro and in vivo. A three-dimensional culture model of biliary epithelial cells in the collagen gel matrix was used for in vitro analysis. For in vivo experiments, PCK and control rats were treated with gefitinib between 3 and 10 weeks of age. In vitro, gefitinib had strong inhibitory effects on biliary cyst formation of PCK rats. In vivo, treatment with gefitinib significantly inhibited the cystic dilatation of the intrahepatic bile ducts of PCK rats, which was accompanied by improvement of liver fibrosis. By contrast, no beneficial effects were observed on renal cyst development because of the treatment. These results suggest that signaling pathways mediated by epidermal growth factor receptor are involved in biliary dysgenesis of the PCK rat, with the mechanisms of cyst progression being different between the liver and kidney.

Two different phenotypes of cholangiocytes constitute the pathologic intrahepatic bile ducts of the PCK rat: Possible contribution of epithelial‐mesenchymal transition to progressive hepatic fibrosis

Background The polycystic kidney (PCK) rat exhibits cystic dilations of intrahepatic bile ducts and progressive portal fibrosis, and is an animal model of Caroli□fs disease with congenital hepatic fibrosis. In liver sections of the PCK rat, two different types of intrahepatic bile ducts can be clearly recognized. That is, bile ducts lined by cuboidal-shaped cholangiocytes (C-type) and spindle-shaped cholangiocytes (S-type). Aim This study was perfomed to determine whether cholangiocytes of the PCK rat undergo epithelial-mesenchymal transition (EMT), whereby contributes progressive portal fibrosis. Methods An immuonostaining was performed for paraffin-embedded liver sections using primary antibodies against epithelial markers [cytokeratin (CK)19, E-cadherin] and mesenchymal markers (vimentin, fibronectin). For in vitro experiments, cultured cholangiocytes of the PCK rat were incubated with transforming growth factor (TGF)-beta1, and RT-PCR analysis was performed for the markers. Results Immunohistochemically, CK19 and E-cadherin were diffusely expressed in both C- and S-types with reduced signal intensity in S-type. S-type showed positive immunoreactivity for vimentin and fibronectin, while C-type was negative. In vitro, TGF-beta1 strongly induced vimentin and fibronectin expression in the cholangiocytes. Conclusions This study provided evidence of possible occurrence of EMT in cholangiocytes of the PCK rat, which presumably participates in pregressive hepatic fibrosis.

Activation of the MEK5/ERK5 Cascade Is Responsible for Biliary Dysgenesis in a Rat Model of Caroli's Disease

Polycystic kidney (PCK) rats exhibit a multiorgan cyst pathology similar to human autosomal recessive polycystic kidney disease, and are proposed as an animal model of Caroli's disease with congenital hepatic fibrosis (CHF). This study investigated the expression and function of selected components of the mitogen activated protein kinase (MAPK) pathway in cultured intrahepatic biliary epithelial cells (BECs) of PCK rats. Compared to the proliferative activity of cultured BECs of control rats, those of the PCK rats were hyperresponsive to epidermal growth factor (EGF). The increase in BEC proliferation was accompanied by overexpression of MAPK/extracellular signal-regulated protein kinase (ERK) kinase 5 (MEK5), and subsequent phosphorylation of ERK5 in vitro. The increased proliferative activity was significantly inhibited by the transfection of short interfering RNA against MEK5 mRNA. An EGF receptor tyrosine kinase inhibitor, gefitinib ("Iressa", ZD1839), also significantly inhibited the abnormal growth of cultured BECs of PCK rats. By contrast, treatment with PD98059 and U0126, inhibitors for MEK1/2, was less effective. These results suggest that the activation of the MEK5-ERK5 cascade plays a pivotal role in the biliary dysgenesis of PCK rats, and also provide insights into the pathogenesis of Caroli's disease with CHF. As the MEK5-ERK5 interaction is highly specific, it may represent a potential target of therapy.