Rho-Kinase Inhibitor Coupled to Peptide-Modified Albumin Carrier Reduces Portal Pressure and Increases Renal Perfusion in Cirrhotic Rats

Abstract

Background: Rho-kinase (ROCK) activation in hepatic stellate cells (HSC) is a key mechanism promoting liver fibrosis and portal hypertension (PTH). Specific delivery of ROCK-inhibitor Y-27632 (Y27) to HSC targeting mannose-6-phosphate-receptors reduces portal pressure and fibrogenesis. In decompensated cirrhosis, presence of ascites is associated with reduced renal perfusion. Since in cirrhosis, platelet-derived growth factor receptor beta (PDGFRβ) is upregulated in the liver as well as the kidney, this study coupled Y27 to human serum albumin (HSA) substituted with PDGFRβ-recognizing peptides (pPB), and investigated its effect on PTH in cirrhotic rats. Methods: In vitro collagen contraction assays tested biological activity on LX2 cells. Hemodynamics were analyzed in BDL and CCl4 cirrhotic rats 3h, 6h and 24h after i.v. administration of Y27pPBHSA (0.5/1mg/kg b.w). Phosphorylation of moesin and myosin light chain (MLC) assessed ROCK activity in liver, femoral muscle, mesenteric artery, kidney and heart. Findings: Three Y27 molecules were coupled to pPBHSA as confirmed by HPLC/MS, which was sufficient to relax LX2 cells. In vivo, Y27pPBHSA-treated rats exhibited lower portal pressure, hepatic vascular resistance without effect on systemic vascular resistance, but a tendency towards lower cardiac output compared to non-treated cirrhotic rats. Y27pPBHSA reduced intrahepatic resistance by reduction of phosphorylation of moesin and MLC in Y27pPBHSA-treated cirrhotic rats. Y27pPBHSA was found in the liver of rats up to 6 hours after its injection, in the HSC demonstrated by double-immunostainings. Interestingly, Y27pPBHSA increased renal arterial flow over time combined with an antifibrotic effect as shown by decreased renal acta2 and col1a1 mRNA expression. Interpretation: Targeting the ROCK inhibitor Y27 to PDGFRI² decreases portal pressure with potential beneficial effects in the kidney. This unique approach should be tested in human cirrhosis. Funding: J. Trebicka is supported by grants from the Deutsche Forschungsgemeinschaft Grant SFB TRR57 P18, European Commission DirectorateGeneral for Research and Innovation Grant 668031, and Cellex Foundation. Declaration of Interest: The authors have no conflict of interest. Ethical Approval: All experiments were performed in accordance with the German Animal Protection Law and the Guidelines of the animal care facility (Haus fur experimentelle Therapie, University Portal and renal effects of targeted Rho-kinase inhibitor Clinics Bonn, Germany), and approved by the North Rhine-Westphalia State Agency for Nature, Environment, and Consumer Protection (LANUV, file reference LANUV NRW, 8402.04.2014.A137).