Expressions of m⁶A methyltransferases and their associations with microR-21 and transforming growth factor-β1 in kidney of rats exposed to cadmium

Background Environmental pollutants can affect N⁶-methyladenosine (m⁶A) level in the body, but the change of m⁶A level in kidney after being exposed to cadmium (Cd) and the molecular mechanism of renal injury need to be further studied.

Objective To analyze the associations of m⁶A modification and methyltransferases/demethylases with microRNA-21 (miR-21) and transforming growth factor- β1 (TGF - β1) in kidney of rats exposed to Cd.

Methods Twenty-four SPF male SD rats were divided into 4 groups, with 6 rats in each group, and were exposed to Cd by subcutaneous injection of 2.0, 1.0, and 0.5 mg·kg⁻¹ cadmium chloride (CdCl₂) and equal volume of normal saline for 2 weeks, 7 d a week, respectively. The levels of N-acetyl-β-D-glucosidase (UNAG) and albumin (UALB) in urine, and the levels of m⁶A methylation and TGF-β1 in kidney were detected by enzyme-linked immunosorbent assay (ELISA). The level of blood urea nitrogen (BUN) was measured by urease method. The levels of renal oxidative stress indicators such as malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were detected by total bile acid method, water-soluble tetrazolium asssay, and colorimetric method respectively. The relative levels of TGF-β1, methyltransferases, and demethylases in kidney were measured by reverse transcription-polymerase chain reaction. The expression of miR-21 in kidney was detected by fluorescent quantitative polymerase chain reaction.

Results After 2 weeks of exposure to Cd, the body weights of rats in the 2.0 and 1.0 mg·kg⁻¹ cadmium chloride groups decreased, and the ratio of kidney/body weight and the levels of BUN, UNAG, and TGF-β1 mRNA and protein increased in the 2.0 mg·kg⁻¹ cadmium chloride group (P<0.05). The expression levels of m⁶A modification, methyltransferases METTL3, METTL14, Wilms’ tumor 1-associated protein (WTAP), and miR-21 were increased both in the 2.0 and 1.0 mg·kg⁻¹ cadmium chloride groups, with significant differences compared with the control group (P<0.05). The results of correlation analysis showed that the m⁶A modification level was negatively correlated with SOD (r=−0.4489, P<0.05) and GSH-Px (r=−0.4874, P<0.05), METTL3 was negatively correlated with MDA (r=−0.5158, P<0.05), while there was a positive correlation between FTO and GSH-Px (r=0.4802, P<0.05). In addition, miR-21 was positively correlated with METTL3 (r=0.7491), METTL14 (r=0.6157), and WTAP (r=0.6660) (P<0.05), TGF-β1 was positively correlated with METTL3 (r=0.5025, P<0.05) but negatively correlated with FTO (r=−0.5634, P<0.05) .

Conclusion Cd can induce m⁶A methylation and up-regulation of METTL3, METTL14, WTAP, and miR-21 expression levels in rat kidney tissues, indicating that m⁶A and miR-21 may be associated with Cd-induced renal fibrosis.