Introduction:
The mineralocorticoid receptor (MR) is a pivotal regulator of water and electrolyte balance and has been implicated in various physiological processes, including blood pressure regulation and renal function. However, aberrant MR activation has been associated with detrimental effects such as promoting fibrosis in the cardiovascular system. Recent studies have indicated a potential role of MR activation in the progression of liver cirrhosis, particularly under conditions of hypoxia commonly observed in cirrhotic livers. Nevertheless, the precise impact of non-physiological MR activation in hepatocytes remains poorly understood.
Objective:
This study aimed to elucidate the consequences of hypoxia-induced MR activation in hepatocytes.
Methods:
We conducted RNA sequencing analysis on rat livers from control animals, cirrhotic animals, and those treated with eplerenone, an MR antagonist (N= 5 animals per group). Animal experiments complied with German law (Tierschutzgesetz, 42502-2-1123 MLU, Landesverwaltungsamt Sachsen-Anhalt), directive 2010/63/EU, and ethical guidelines (ARRIVE guidelines and NIH standards). With respect to the results of the Gene ontology (GO )term enrichment analysis from rat livers, we investigated the impact of MR activation on metabolic processes in HepG2 cells . To induce non-physiological MR activation, we exposed the cells to hypoxia, using an oxygen concentration of 0.2% for at least 24h. mRNA and protein levels of key metabolic genes was determined using quantitative RT-PCR and Western blotting. Glucose consumption, lactate production, and lipid accumulation were assessed as well with or without eplerenone treatment. The data are presented as mean ± standard error of the mean (SEM) % of control. Statistical significance was determined using a two-tailed t-test, with a significance threshold set at p < 0.05. Each experiment was conducted with N = 5-10 replicates, and there were n = 15-24 petri dishes per group.
Results:
GO term enrichment analysis revealed that in a rat model for liver cirrhosis (CCl4 treatment) eplerenone, an MR antagonist, reverses the downregulation of genes annotated to the GOterm “Monocarboxylic acid metabolic process”. We already demonstrated that under hypoxic conditions that the MR-induced transcriptional activity shifts to other response elements on the DNA (Schreier, 2018). Therefore, we incubated HepG2 cells with or whithout hypoxia in the presence of eplerenone. We have demonstrated that hypoxia reduced the mRNA levels of PPARα (46.3 ± 8.4%), PDK4 (11,4 ± 3.3%), AMACR (39.2 ± 6.6%), ABCC2 (67.4 ± 10.5%), and Lipin 1(49.1 ± 15.8 %; as well as the protein. This downregulation can be partially attenuated by eplerenone treatment for PPARα (73.7 ± 12.7 %), PDK4 (66.3 ± 17.9 %), and ABCC2 (99.3 ±12. 2 %), suggesting a mineralocorticoid receptor (MR)-dependent mechanism. Hypoxia augments glucose uptake (282,7± 33.2%), and lactate production (202,7± 11.8%) in HepG2 cells. This effect was partially reversed upon eplerenone administration (glucose: 169,2± 10.2% and lactate: 153,5± 10.2; p<0.05, two-tailed t-test) respectively. Additionally, hypoxia-associated lipid accumulation (257,9 ± 26,7%) in hepatocytes is partially mitigated by MR blocker(120.3 ± 9,8%,).
Conclusion
Our findings suggest role of MR in dysregulating glucose and lipid metabolism in hepatocytes, potentially contributing to liver cirrhosis development. Therefore, MR antagonism may hold therapeutic promise in the management of liver cirrhosis.