The (pro)renin receptor antagonist, PRO20, attenuates high fat diet induced hepatic steatosis and fibrosis in non‐alcoholic fatty liver disease (NAFLD)

Abstract

Non‐alcoholic fatty liver disease (NAFLD) comprises a spectrum of liver damage directly related to diabetes, metabolic syndrome, and obesity. The (Pro)renin receptor (PRR) in the liver recently has been shown to play an important role in hepatic steatosis. In this study, we test our hypothesis that PRR antagonist, PRO20, reduces hepatic steatosis and fibrosis. Wild‐type mice in C57Bl/6J background (n=9‐10/group) were fed with either HFD (60% calories from fat) or normal fat chow (NFD, 10% calories from fat) with matching calories for 6 weeks. Methionine choline deficient (MCD) diet was used to study fibrosis development in C57BL/6J mice for 8 weeks. Two weeks following respective diet modifications, mice were implanted with a subcutaneous osmotic pump (4 or 6 week release model) containing either PRO20 (700ug/kg/d) or saline. Liver tissues were processed for Oil Red O (ORO), H&E, and Masson’s trichrome histology staining for lipid and collagen accumulation. The presence of lipids, indicative of steatosis, was quantified using ImageJ/FIJI software and presented as % of ORO staining to the total area. We found that 6 weeks of HFD (15.90 ± 1.49 %) induced a significant elevation on liver ORO staining compared with the liver from mice fed with NFD (3.60 ± 1.61 %, p<0.0001). More importantly, PRO20 treatment (9.91 ± 1.46 %; p = 0.001) significantly reduced the ORO staining in mice treated with HFD. Hepatic triglyceride concentrations were quantified with colorimetric assay and shown to be significantly lowered in PRO20 treated mice (2.110 ± 0.6175) compared with scramble peptide treated (6.470 ± 0.3553, p<0.0001) mice under HFD. Collagen area was also found to be reduced in MCD PRO20 (4.970 ± 0.9096 %) treated mice compared with the control mice (8.901 ± 0.2782, p = 0.0033). Moreover, PRO20 treatment (258 ± 53.12 U/L) decreased liver ALT levels in MCD‐fed PRO20 mice compared with the control mice (682 ± 116.4 U/L). De novo lipogenesis genes thought to contribute to the development and progression of NAFLD were evaluated by real‐time PCR and presented as fold‐change expression normalized to β‐actin. The glycerol‐3‐phosphate acetyltransferase 3 (GPAT3) (1.835 ± 0.1798, p = 0.0183) expression was found to be downregulated in the livers of HFD PRO20 treated mice. Our findings indicate that PRO20 reduces HFD‐induced hepatic steatosis and MCD‐induced liver fibrosis and liver dysfunction. We conclude that PRR antagonism using PRO20 might be an effective therapeutic approach for treatment of NAFLD.