Trametinib Ameliorated Adriamy-Induced Podocyte Injury In Vivo and In Vitro by Inhibiting METTL3-Mediated RCAN1 m6A RNA Methylation

Background: Trametinib can protects mice from adriamycin(ADR)-induced focal segmental glomerulosclerosis (FSGS). Recently, METTL3, a m6A writer, is reported to involve in pathogenesis of kidney diseases. We aimed to investigate whether Trametinib can attenuate podocyte injury through regulating METTL3 and its modified downstream gene in ADR-induced FSGS mice and injured podocytes. Methods: TREW database was used to find regulator calcineurin 1(RCAN1) as METTL3 modulated mRNA. First we examined WT-1, METTL3, and RCAN1 in biopsied kidney tissue from FSGS patients. Second, FSGS was induced by ADR injection in BALB/c mice in both normal and pretreated with trametinib. Urinary albumin to creatinine ratio (UACR) and serum albumin (sALB) were measured. Kidney damages were evaluated by PAS, Masson and EM. Third, overexpression and knockdown of METTL3 were performed in human podocytes in vitro. Podocyte specific gene WT-1and synaptopodin (SYNPO); Profibrotic COL-1, α-SMA, and fibronectin (FN); and RCAN1 were assessed at mRNA by qPCR and protein levels by western blotting and immunostaining both in vivo and in vitro. The level of total m6A was measured by the m6A colorimetric qPCR and m6A methylated mRNA of RCAN1 wwa examined by SELECT qPCR. Results: WT-1 was downregulated, METTL3 was increased, and RCAN1 was decreased in biopsied kidney sections compared with normal control kidney sections. Trametinib alleviated ADR-induced proteinuria, hypoalbuminuria, hyperlipidemia, and renal dysfunction; podocyte foot process effacement, and sclerotic glomeruli. It also reverted the downregulation of WT-1 and SYNPO as well as reduced production of profibrotic COL-1,α-SMA, and FN protein in FSGS mice. Importantly, Trametinib inhibited the increment of METTL3 and decrease of RCAN1. In vitro study, overexpression of METTL3 in normal podocyte or knockdown of METTL3 by siRNA or Trametinib in ADR-treated podocyte, the changes of podocyte markers and profibrotic proteins displayed similar to those in vivo. The change directions of total level of m6A RNA methylation and relative level of METTL3 methylated mRNA of RCAN1 in podocytes were same as the expressiom changes in vivo. Conclusion: Trametinib attenuated podocyte injury in FSGS mice and ADR-treated podocyte by regulating METTL3/RCAN1 axis. Trametinib might serve as a potential agent to protect podocyte injury Funding: Government Support – Non-U.S.