Supplementary Material for: miR-499 Ameliorates Podocyte Injury by Targeting Calcineurin in Minimal Change Disease

Background: Podocyte injury is a hallmark of minimal change disease (MCD). Calcineurin inhibitors have been widely used in the current treatment of MCD, and miR-499 may target calcineurin. We aimed to study the function of miR-499 in MCD and test whether miR-499 delivery can improve MCD. Methods: An MCD mouse model was generated using puromycin aminonucleoside (PAN). MiR-499 was delivered using lentiviruses. Biochemical indicators including serum albumin, triglyceride, cholesterol, and 24-h urine protein were determined. Targets of miR-499 were confirmed using reporter gene activity assays. The ultrastructure of podocytes was analyzed using transmission electron microscopy. Results: MiR-499 significantly improved MCD-related symptoms and signs. Foot-process effacement was caused by PAN and partially reversed by miR-499. We identified that both CnAα and CnAβ were targets of miR-499, and were overexpressed in the presence of PAN. However, miR-499 reduced the expression of CnAα and CnAβ, leading to a decreased activity of calcineurin signaling in mouse podocytes in vitro and in vivo. In addition, miR-499 recovered PAN-induced reduction of cell viability. Conclusions: MiR-499 ameliorated podocyte injury by targeting CnAα and CnAβ in a PAN-induced MCD mouse model. Delivery of miR-499 can be a novel strategy for MCD treatment.