Inhibition of the mineralocorticoid Receptor reduces Cirrhosis associated changes of hepatocyte Glucose and Lipid Metabolism in hepatocytes

Background & aims: Recent studies have suggested that mineralocorticoid receptor (MR) activation may contribute to the progression of liver cirrhosis. Hypoxia, a common feature in liver cirrhosis, has been found to induce MR activation hepatocytes. However, the impact of non-physiological MR activation remains poorly understood. In this study, we investigate the effect of cirrhosis-induced MR activation in hepatocytes. Methods: RNA sequencing followed by gene ontology term enrichment analysis was performed on liver samples from rats treated for 12 weeks with or without CCl4 and for the last four weeks with or without eplerenone (MR antagonist). We investigated if these changes can be mimicked by hypoxia and if those changes lead to alterations of glucose and lipid metabolism in primary rat hepatocytes (pRH) and human HepG2 cells. All animal experiments were conducted in accordance with the german law and the directive 2010/63/EU. They were ethically and legally approved by the governmental, local animal committees (42502-2-1123 MLU, Landesverwaltungsamt Sachsen-Anhalt or TWZ 16-2021, Jena University hospital). All animal studies were conducted in compliance with the ARRIVE guidelines and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The rats were kept in a specific pathogen-free environment, with ad libitum access to water and standard rodent diet at constant temperature of 22±2°C with a 12/12 h light-dark cycle. Cirrhosis was induced by inhalation of carbon tetrachloride (CCl4) and ingestion of phenobarbital via the drinking water (0.35 g/L) three times a week for 12 weeks in age-matched, male Wistar rats (purchased from Charles river) with an initial weight of 75-100 g. Eplerenone treatment (Inspra, 100 mg kg 1 BW) per os (hazelnut spread, 5g/day) was initiated after eight weeks of CCl4 inhalation, and continued until 12 weeks of CCl4 inhalation. Livers were isolated 7-10 days after the last dose of CCl4. At the end of the experiment, the animals were anesthetized with ketamine/xylazine (100/5 mg/kg i.p.; Ketaset 100 mg/ml, Zoetis, 2% Rompun, Bayer Healthcare) and euthanized by exsanguination while in deep narcosis. RNA sequencing from liver samples was performed. The quality of the RNA samples was assessed using a 2100 Bioanalyzer (Agilent Technologies). All samples had an RNA Integrity Number (RIN) above 7 (with 10 as the maximal possible value). Novogene Co., Ltd. (Cambridge, UK) carried out the sequencing, libraries preparation (poly(A) enrichment), and the paired-end sequencing (2 × 150 bp) runs on a NovaSeq6000 Illumina system. Adaptor clipping and data quality control were provided by the service company as well. Read mapping to the rat genome rn6 was performed with HISAT2 (v. 2.1.0), and featureCounts (2.0.0, –M –t exon) was used to count the mapped reads. Gene annotation was performed using BiomaRt (v.2.44.4) to access the Ensembl archive v101. Three treatment groups were defined: a) control: animals received no treatment, b)CCl4: liver cirrhosis was induced by 12 week of treatment with CCl4 & phenobarbital, c) CCl4 + eplerenone: liver cirrhosis was induced by 12 weeks of treatment with CCl4 & phenobarbital, after 8 weeks rats were supplemented with eplerenone per os in hazelnut spread.