Differential impacts of mineralocorticoid receptor antagonist potassium canrenoate on liver and renal changes in high fat diet-mediated early hepatocarcinogenesis model rats

Abstract

Mineralocorticoid receptor (MR)/NADPH oxidase (NOX) signaling is involved in the development of obesity, insulin resistance, and renal diseases; however, the role of this signaling on steatotic preneoplastic liver lesions is not fully elucidated. We determined the effects of the MR antagonist potassium canrenoate (PC) on MR/NOX signaling in hepatic steatosis and preneoplastic glutathione S-transferase placental form (GST-P)-positive liver foci. Rats were subjected to a two-stage hepatocarcinogenesis model and fed with basal diet or high fat diet (HFD) that was co-administered with PC alone or in combination with the antioxidant alpha-glycosyl isoquercitrin (AGIQ). PC reduced obesity and renal changes (basophilic tubules that expressed MR and p22phox) but did not affect blood glucose tolerance and non-alcoholic fatty liver disease activity score (NAS) in HFD-fed rats. However, the drug increased the area of GST-P-positive liver foci that expressed MR and p22phox as well as increased expression of NOX genes (p22phox, Poldip2, and NOX4). PC in combination with AGIQ had the potential of inhibiting the effects of PC on the area of GST-P-positive liver foci and the effects were associated with increasing expression of an anti-oxidative enzyme (Catalase). The results suggested that MR/NOX signaling might be involved in development of preneoplastic liver foci and renal basophilic changes in HFD-fed rats; however, the impacts of PC were different in each organ.

Immunohistochemical expression of autophagosome markers LC3 and p62 in preneoplastic liver foci in high fat diet-fed rats

“…Effects of the HFD on GST-P-positive preneoplastic liver foci were variable in our studies, probably reflecting variations due to the study condition and diet lot. Both the area and number of foci were significantly increased in the HDF group compared with the CTL group in our previous study (Murayama et al, 2018) or were both weakly increased without a significant difference in another study (Nakamura et al, 2018). The present study revealed only a minor effect on the number of GST-P-positive preneoplastic liver foci.…”

“…To elucidate the effects of the HFD feeding, we initially examined the histopathology of liver tissue using the NAS scoring system (Kleiner et al, 2005) with gene expression analysis for lipid metabolism and oxidative stress. NAS is suitable to evaluate NAFLD and NASH in the liver obtained from patients and rodent models (Itagaki et al, 2013;Nakamura et al, 2018). The HFD increased NAS with ballooning changes in the liver sections, although there was no significant change in steatosis.…”

“…Liver sections were routinely dehydrated in graded ethanol, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for histopathological examination. The pathological changes, including the presence of steatotic cells, ballooning cells, and lobular inflammation, were graded by the NAFLD activity score (NAS) (Kleiner et al, 2005;Itagaki et al, 2013;Nakamura et al, 2018). Immunohistochemical staining of adipophilin (ADFP), glutathione S-transferase placental form (GST-P), Ki-67, MAP1LC3A (LC3), and p62/SQSTM1 (p62) was performed under the conditions shown in Supplemental Table 1.…”

“…Recently, our group (Yoshida et al, 2017) developed a steatosis-related early hepatocarcinogenesis model in HFD-fed rats based on a medium-term liver assay for 8 weeks (Ito et al, 2003). The HFD-induced hyperlipidemia and steatosis and preneoplastic lesions in the liver, and the effects on the liver were related to altered lipid metabolism and oxidative stress (Murayama et al, 2018;Nakamura et al, 2018).…”

“…In this study, to elucidate the roles of autophagy in NAFLD/NASH-related hepatocarcinogenesis, we determined the expression of the autophagosome markers LC3 and p62 in preneoplastic liver foci, together with gene expression analysis for several autophagy-related genes, including Atg5 and Atg7, and the effects of an autophagy inducer or repressor were assessed in a steatosis-related hepatocarcinogenesis model (Yoshida et al, 2017;Murayama et al, 2018;Nakamura et al, 2018).…”