Objectives: Congenital hepatic fibrosis (CHF) is an important cause of morbidity and mortality in patients with autosomal recessive polycystic kidney disease (ARPKD). The pathogenesis of CHF remains undefined. Several recent studies suggest that the renin-angiotensin system (RAS) is an important mediator of progressive hepatic fibrosis through activation of profibrotic mediators, such as transforming growth factor-β (TGF-β). RAS activation has not previously been studied in patients with CHF or in animal models. The aim of the present study was to characterize RAS expression during the course of CHF in the PCK rat.
Materials and Methods: Studies were conducted in the PCK rat, an orthologous ARPKD/CHF model, and age-matched normal control Sprague-Dawley rats. Expression of the RAS components, renin, angiotensinogen, angiotensin-converting enzyme (ACE), and angiotensin II type 1 receptor (AT1R), as well as the profibrotic mediator TGF-β, was examined in cystic PCK and control rat livers at 2, 4, and 6 months of age by quantitative real-time polymerase chain rection (qRT-PCR). Angiotensin II (ANG II) was examined by immunohistochemistry (IHC). Fibrosis was assessed by IHC using reticulin staining and Masson trichrome. Collagen content was determined by hydroxyproline analysis.
Results: Progressive fibrosis and increased hepatic collagen content occurred in PCK rats with age. In 4- and 6-month-old PCK rat livers, ACE gene expression was markedly increased, 8- and 17-fold, respectively, compared with age-matched control livers. Expression of the other RAS components, renin, angiotensinogen, and AT1R were not significantly different. IHC demonstrated prominent ANG II protein expression in periportal regions in PCK rats. In contrast, no expression was noted in control livers. TGF-β expression was also increased in PCK rat livers with progressive disease.
Conclusions: The present study demonstrates, for the first time, RAS upregulation in an orthologous rat ARPKD/CHF model. Increases in ACE and ANG II, as well as the downstream target, the profibrotic mediator TGF-β, suggest that RAS activation may be an important mediator of CHF disease progression. The findings also suggest that treatment with RAS inhibitors, specifically ACE inhibitors or AT1R blockers, could be therapeutic in slowing disease progression in CHF.
*Department of Research, MetroHealth Medical Center, Case Western Reserve University, Cleveland, OH, USA
‡Division of Gastroenterology, Hepatology, and Nutrition, the Children's Hospital of Philadelphia, Philadelphia, USA
§Departments of Medicine and Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, USA.
Received 4 June, 2009
Accepted 10 November, 2009
Address correspondence and reprint requests to Katherine MacRae Dell, MD, Department of Research, MetroHealth Medical Center, 2500 MetroHealth Dr, R457, Cleveland, OH 44109 (e-mail: KatherineDell@case.edu).
This research was supported by the NIH/NIDDK (DK-058123 to R.G.W.), the National Kidney Foundation of Ohio, MetroHealth Medical Center, and a pilot award from the UAB Recessive PKD Center Core (to K.M.D.). R.G.W. is funded in part by a pilot award from the ARPKD/CHF Alliance. J.W. is supported by NIH training grant T32-DK007006.
The authors report no conflicts of interest.
Congenital hepatic fibrosis (CHF) is an important cause of morbidity and mortality in patients with autosomal recessive polycystic kidney disease (ARPKD) as well as those with other inherited and/or congenital syndromes such as nephronophthisis and Meckel syndrome (1–3). CHF is a biliary ductal plate malformation characterized by bile duct proliferation, dilatation, and periportal fibrosis (2,4). Although it is invariably present histologically at birth, the liver disease may not become clinically evident for many years. More severely affected patients develop portal hypertension and are at risk for potentially life-threatening complications, including gastrointestinal bleeding resulting from varices and recurrent cholangitis (4). Recent reports suggest that CHF is an important cause of mortality and morbidity in patients with ARPKD who have undergone kidney transplantation (5,6). Although the gene for ARPKD, PKHD1, has been identified, the mechanisms by which the gene defect results in CHF are poorly defined (3,7).
The renin-angiotensin system (RAS) is a key mediator of fibrosis in many organs, including the kidney, heart, and vessels. Several recent studies suggest that the RAS is also an important mediator of progressive hepatic fibrosis through activation of profibrotic mediators such as transforming growth factor-β (TGF-β) (8–11). However, RAS expression in ARPKD-associated CHF has not been studied in any ARPKD animal models or human disease.
The goal of the present study was to determine expression of the RAS and its downstream target, TGF-β, in progressive fibrocystic liver disease in the polycystic kidney (PCK) rat, an orthologous model of human ARPKD that develops polycystic kidney disease, as well as significant periportal fibrosis and bile duct dilatation similar to that of human ARPKD-associated CHF.
MATERIALS AND METHODS
The PCK rat model arose out of a spontaneous mutation in a colony of Sprague-Dawley (SD) rats. This model has a spontaneous mutation in the rat ortholog of the human ARPKD gene, PKHD1 (7), and develops not only polycystic kidney disease but also significant biliary abnormalities including bile duct dilatation and fibrosis (12). Liver disease in this model is observed histologically by 19 days' gestation and intrahepatic bile duct dilatation and overgrowth of portal connective tissue progresses with aging. This is similar to the hepatic phenotype of affected patients with ARPKD (13). For the present study, livers from 2-, 4-, 6-, and 7-month-old male PCK rats and age-matched SD rats were used.
All of the experiments were conducted according to the policies of the Institutional Animal Care and Use Committee of Case Western Reserve University School of Medicine.
Clinical and Pathologic Assessments of Disease Severity
PCK and control SD rats were sacrificed at the time points listed above. Liver weight (LW), body weight (BW), and LW/BW were measured at sacrifice. For determination of the fibrotic area, the left lateral lobe of the liver was fixed in 4% paraformaldehyde and embedded in paraffin. Five-micron sections were obtained and stained with Masson trichrome (Richard-Allan Scientific, Waltham, MA). Sections were visualized by light microscopy and 5 random fields per section were imaged at 10× and digitally captured. The percentage of positive staining, corresponding to collagen, was assessed by pixel counting and expressed as the percent of total parenchyma imaged in each section. Means and standard deviations for each section were obtained and then averaged across the 5 sections studied. Reticulin staining, which identifies glycosaminoglycan-bound type III collagen fibrils, was also performed on sections obtained as above from 2- to 7-month-old PCK and control SD rats.
For measurement of hydroxyproline content, livers were flash frozen in liquid nitrogen at sacrifice. One hundred milligrams (wet weight) of each liver was homogenized in 6N HCl and then incubated for 24 hours at 110°C to allow for complete hydrolysis. Aliquots (50 μL) were resuspended in 1 mL of chloramine T solution (Fisher Scientific, Waltham, MA) and incubated at room temperature for 20 minutes. One milliliter of Ehrlich solution was added, and the samples were incubated at 65°C for an additional 15 minutes. Samples were allowed to return to room temperature, and absorbance was measured at 550 nm by spectrophotometry. The hydroxyproline content was determined using a standard curve with known concentrations of hydroxyproline (Sigma, St Louis, MO) and was normalized to milligrams of wet tissue weight.
Serum levels of γ-glutamyl transferase (GGT) were quantified using a commercially available enzymatic kit (Stanbio Laboratory, Boerne, TX), according to the manufacturer's instructions.
Measurement of RAS Components and TGF-β Expression by Quantitative real-time PCR
Renin, angiotensinogen (AGT), angiotensin-converting enzyme (ACE), angiotensin type IA receptor (AT1R), and TGF-β gene expression levels were measured by qRT-PCR of whole liver samples. Samples were flash frozen at sacrifice, and total liver RNA was extracted using TRIZOL reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. cDNA was generated by reverse transcription using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) in accordance with manufacturer's protocols. Real-time PCR was performed using the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Carlsbad, CA). Relative quantification was assessed using the ddCt method with samples standardized to expression of the housekeeping gene, 18s. Samples were run in triplicate and at least 3 livers were studied for each experimental age/group.
Primer sequences used were renin, F-TTC TCT CCC AGA GGG TGC TA; R-CCC TCC TCA CAC AAC AAG GT; AGT, F-CAC GGA CAG CAC CCT ATT TT; R-GCT GTT GTC CAC CCA GAA CT; ACE, F-GAG CCA TCC TTC CCT TTT TC; R-GGC TGC AGC TCC TGG TAT AG; AT1R, F-CGT CAT CCA TGA CTG TAA AAT TTC; R-GGG CAT TAC ATT GCC AGT GTG; TGF-β, F-CCG CAA CAA CGC AAT CTA TG; R-CTC TGC ACG GGA CAG CAA T; 18s, F-CGC GGT TCT ATT TTG TTG; R-AGT CGG CAT CGT TTA TGG TC.
Immunohistochemistry for Angiotensin II
Immunohistochemistry (IHC) was performed on 5-μm serial sections obtained as above from the livers of 2- and 7-month-old PCK rats and age-matched control SD rats. Sections were deparaffinized with xylene and rehydrated with graded ethanol. IHC was performed using the ABC Vectastain kit (Zymed, South San Francisco, CA) for peroxidase-conjugated secondary antibodies according to the manufacturer's instructions. Sections were immersed in 3% H2O2 for inhibition of endogenous peroxidase and blocked with PBS/1% serum from the secondary antibody host, then incubated with primary antibodies overnight at 4°C. Antigen retrieval was performed by heating in 10 mmol/L citrate buffer (pH 6). All of the sections were counterstained with hematoxylin and mounted with Permount (Sigma). Anti-ANG II (Bachem 1:2000) was used as the primary antibody. Negative controls included omission of primary antibody and staining with an irrelevant species-matched antibody.
Data were expressed as mean ± SD. Differences in gene expression between normal and PCK rats were assessed by 2-tailed Student t test, and a P value of <0.05 was considered statistically significant.
Collagen Deposition in Progressive Congenital Hepatic Fibrosis in 2- and 7-month-old PCK and Normal Rat Livers
As illustrated by reticulin and Masson trichrome staining (Fig. 1), PCK rat livers have dilated bile ducts with surrounding periportal fibrosis (collagen shown as black staining for reticulin, blue staining for Masson). Intrahepatic bile duct dilatation and collagen deposition increased with age, illustrating the progressive fibrosis present in this model.
Clinical, Histologic, and Biochemical Features of Progressive CHF in PCK Rats
To further characterize progressive liver disease in the PCK rat, both PCK and normal control SD rats were sacrificed at 2, 4, and 6 months of age. Clinical features (eg, LW) and histologic scoring for biliary duct dilatation and fibrosis (by trichrome staining) were assessed. As shown in Table 1, LW is greater in PCK versus control rats at 4 and 6 months; however, the LW and LW/BW% level off at 4 months and remain constant at that point, despite progressive disease. GGT values are similar in PCK and age-matched SD rats. Published data in this model also demonstrate that alkaline phosphatase and bilirubin levels are similar in PCK rats when compared with normal SD rats (12).
In contrast to the absence of significant changes in these clinical parameters with progressive disease, both the fibrosis score and hydroxyproline content increase significantly during the course of disease (Table 2). Results are similar to reported fibrosis scores using picrosirius red staining (14). These findings mirror the progressive nature of the liver disease in patients with ARPKD.
RAS Gene Expression in PCK and Normal Rat Livers
Renin, AGT, ACE, and AT1R gene expression were studied in PCK and SD rat livers at the ages indicated (Fig. 2). ACE expression in 4-month-old PCK rat livers was significantly increased (8-fold higher) when compared with age-matched SD rat livers. The ACE upregulation in 6-month-old PCK livers was even more dramatic, with a >17-fold higher level of ACE expression than for 6-month-old SD livers. In contrast, renin, AGT, and AT1R expression levels were not significantly different in PCK compared with SD livers at each age studied.
ANG II Expression and Localization in PCK and Normal Rat Livers
IHC for ANG II protein demonstrates increased expression in PCK rat livers, which localizes to the regions around the dilated bile ducts and within the fibrotic portal tracts (Fig. 3). With progressive disease, increased numbers of portal triads show ANG II expression. In contrast, normal rat livers showed no apparent ANG II expression by IHC.
TGF-β Gene Expression in PCK Rat Livers
A major consequence of RAS activation is production of profibrotic mediators, notably TGF-β. In parallel to the ACE gene and ANG II protein upregulation seen in 4- and 6-month-old PCK livers, TGF-β gene expression was also significantly increased in PCK livers at the same ages (7-fold and 3-fold increase in 4- and 6-month-old PCK livers, respectively) (Fig. 4).
CHF is an important cause of morbidity and mortality in patients with ARPKD and can also occur in patients with other inherited and/or congenital syndromes such as Meckel syndrome (1,2). The mediators of disease progression in CHF, as opposed to other forms of biliary fibrosis, however, are poorly delineated. The presence of a “local” (versus systemic) RAS, in which all of the components necessary for RAS activation are present within the tissue of interest, has emerged as an important mechanism for tissue- and organ-specific regulation of RAS (15). A growing body of literature supports the concept of local hepatic RAS activation in the liver. Paizis et al (10) reported that ACE and AT1R gene expression is markedly upregulated in the BDL model of hepatic fibrosis, and increased AT1R binding was seen in fibrotic areas, despite no differences in liver AGT or renin levels. In that same BDL model, systemic ANG II infusion exacerbated hepatic fibrosis (8). Review of the liver fibrosis literature suggests that the key fibrosis mediator TGF-β is downstream of ANG II in fibrosis (8,11,16,17), and that infusion of ANG II induces TGF-β upregulation even in normal rat livers (18). This suggests that upregulation of RAS is sufficient to cause damage even in the absence of an underlying liver disease process. Our results are consistent with a RAS-mediated mechanism of TGF-β upregulation, although additional pathways downstream of ACE may also play an important role, regulating TGF-β more indirectly.
RAS activation has not previously been studied in CHF. The present study demonstrates for the first time RAS upregulation in an orthologous rat ARPKD/CHF model. Increases in ANG II as well as its downstream target the profibrotic mediator TGF-β, suggest that RAS activation may be an important mediator of CHF disease progression. Because systemic levels of ANG I and II were not significantly elevated (data not shown), these findings suggest that it is local, not systemic, RAS activation that is driving the upregulation. CHF is an outlier in the category of biliary fibrosis given the specific mutation, the dilated ducts in the absence of a ductular reaction, and the relatively slow rate of progression. In this context it is particularly interesting that RAS activation is common to ARPKD/CHF and other forms of biliary fibrosis.
IHC demonstrates increased ANG II staining around dilated bile ducts. This pattern suggests that ANG II could be produced by either the biliary epithelial cells (BECs) themselves or surrounding cells such as portal fibroblasts. The fact that BECs produce the abnormal protein fibrocystin/polyduction and the localization of ANG II around bile ducts suggests that at least the earliest RAS components are made in BECs. Abnormal fibrocystin is associated with increased expression of cyclic adenosine monophosphate, which can activate the ACE promoter (19–21). Thus, we speculate that in the presence of abnormal fibrocystin, BECs produce increased ACE. This, in turn, results in increased production of ANG II, which can readily diffuse out of the cell and exert its effects locally on portal fibroblasts and other potentially fibrogenic cells in the portal and periportal regions (22). The primary limitation of the present study is that all of the experiments were performed on whole-liver specimens, and thus conclusions about the role of specific cell types in this newly identified pathogenic RAS activation are speculative. Additional in vitro studies are under way to more fully define these pathways/processes.
Finally, it is important to note that there are no disease-specific therapies for CHF other than standard medical management of complications such as portal hypertension and varices. The data in the present study suggest that RAS inhibition, using ACE inhibitors or angiotensin receptor blockers, may be therapeutic in the treatment of progressive CHF in ARPKD patients.
The authors thank Dr Samir El-Dahr, Tulane University, for helpful suggestions regarding RAS assessments.
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