Renin-angiotensin system activation in congenital hepatic fibrosis in the PCK rat model of autosomal recessive polycystic kidney disease

Abstract

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-beta (TGF-beta). 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-beta, was examined in cystic PCK and control rat livers at 2, 4, and 6 months of age by quantitative real-time polymerase chain reaction (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-beta 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-beta, 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.

Figure 1. Collagen deposition in progressive congenital hepatic fibrosis (CHF) in 2 and 7 month-old PCK and normal rat livers

(A) Reticulin (black=collagen) staining of PCK rat livers (lower panels) at 2 months (left panel) and 7 months (right panel) illustrates the increasing intrahepatic bile duct dilatation and collagen deposition with progressive liver disease in this model. Normal rat livers at both ages (upper panels) are shown as comparison. (B) Masson’s trichrome (blue = collagen) staining of PCK rat livers highlights the progressive worsening of periportal fibrosis and bile duct dilatation with advancing disease. 10× original magnification.

Figure 2. RAS gene expression in PCK and normal rat livers

Livers were obtained at sacrifice at the ages indicated and flash frozen in liquid nitrogen. RNA was extracted using TRIZOL and cDNA generated. qRT-PCR for renin, AGT, ACE and AT₁R gene expression was performed using the ABI Prism system 7700 and normalized to the housekeeping gene, 18s. Expression of renin, AGT and AT₁R did not differ significantly in the groups studied. However, ACE gene expression was markedly increased in PCK rat livers compared to control livers at 4 months and rose further by 6 months. (* p<0.05)

Figure 3. Ang II expression in PCK and normal SD rat livers

IHC was performed on paraformaldehyde-fixed, paraffin-embedded liver sections obtained from PCK and Normal SD rats at 2 and 7 months of age as described in the text. In the normal SD liver (upper panels), minimal to no ANG II protein (indicated by brown staining) is seen at either 2 or 7 months. In contrast, in PCK rat livers at both ages (lower panels), there is a marked increase in ANG II expression in areas surrounding dilated bile ducts within fibrotic portal areas. Original magnification 20×.

Figure 4. TGF-β gene expression in PCK and normal rat livers

Livers were obtained at sacrifice at the ages indicated and cDNA generated as described in Figure 2. qRT-PCR for TGF-β gene expression was performed using the ABI Prism system 7700 and normalized to the housekeeping gene, 18s. TGF-β gene expression is increased in PCK rat livers compared to control livers at 4 and 6 months (* p<0.05).