The Relationship of miR‐21‐5p and PPARα in CKD‐Related NAFLD and Dyslipidemia
Authors: Nasci Victoria L , Miller Sophie , Chuppa Sandra , Kriegel Alison J. 
Published: November 2023

Chronic Kidney disease (CKD) has been linked to cardiovascular pathologies, such as atherosclerosis and chronic heart failure, as well as dyslipidemia and non‐alcoholic fatty liver disease (NAFLD). These pathologies have also been observed in the 5/6 nephrectomy (5/6Nx) model of CKD. Previous studies in our laboratory have shown miR‐21‐5p, a microRNA known to target peroxisome proliferator‐activated receptor alpha (PPARα), impacts cardiac lipid handling in the 5/6Nx model. PPARα is a regulator of fatty acid up‐take and metabolism and has been shown to be suppressed in CKD, cardiovascular disease, and NAFLD. In this study we evaluated the potential for miR‐21‐5p to regulate serum and liver lipid content and liver PPARα levels in 5/6Nx rats and H4IIE rat hepatoma cells exposed to free‐fatty acids. We performed 5/6Nx (n=13), or sham surgery (n=5), on 10 week old male Sprague‐Dawley rats. LNA‐anti‐miR‐21‐5p (Exiqon), or a scrambled control, was administered to some 5/6Nx animals 6 weeks after surgery (3 daily i.p. injections, 1 mg/kg). Serum and tissues were collected following a 24 hour fast at 10 weeks post‐surgery. Liver lipid content was evaluated by oil red O (ORO) staining. Rats subjected to 5/6Nx developed significantly elevated serum triglyceride (90.4 ± 20.9 mg/dL vs. 47.0 ± 3.7 mg/dL) and cholesterol (154.6 ± 11.8 mg/dL vs. 87.8 ± 3.09 mg/dL) levels and their livers exhibited increased microvesicular lipid accumulation. Anti‐miR‐21‐5p treatment increased lipid vesicle size (1.14 ± 0.02 μm2 vs. 0.78 ± 0.10 μm2; p<0.05) and increased the average ORO stained surface area by nearly 2‐fold, but had no effect on serum lipid levels. In H4IIE cells NAFLD‐like steatosis was induced by 24‐hour serum starvation of H4IIE cells followed by a 24‐hour lipid treatment (LT) of 0.66 mM oleic acid (OA) and 0.33 mM palmitic acid (PA). The regulatory effect of miR‐21‐5p was studied by transfecting pre‐miR‐21‐5p (20 nM), anti‐miR‐21‐5p (40 nM), or scrambled oligonucleotides at the time of serum starvation. Lipid content was measured by AdipoRed assay (Lonza). PPARα and miR‐21‐5p abundance were evaluated by western blot and qRT‐PCR, respectively. We found that LT increased lipid abundance within cells, increased miR‐21‐5p levels and increased PPARα levels. Transfection with either pre‐ or anti‐miR‐21 did not change PPARα expression in the absence of LT. Anti‐miR‐21 + LT cells exhibited no change in lipid content compared to anti‐scrambled + LT treated cells, but surprisingly PPARα expression levels were significantly decreased. Conversely pre‐miR‐21 attenuated the LT induced increase in lipid content and also resulted in a modest reduction in PPARα expression. In conclusion, our studies indicate that the regulatory relationship between miR‐21‐5p and PPARα in the liver is complex and could be modulated by cellular lipid content or fatty acid exposure. Our in vitro studies suggest that there may be additional targets of miR‐21‐5p regulating liver PPARα abundance indirectly. Our study indicates that miR‐21‐5p has a regulatory role in liver lipid uptake and/or processing in 5/6Nx induced dyslipidemia, but is not involved with regulation of dyslipidemia in this model.