Src Aggravates Podocyte Injury In Diabetic Nephropathy Via Suppression Of FUNDC1-mediated Mitophagy

Aims: Diabetic nephropathy(DN)is a common diabetic microvascular complication that is rapidly becoming the leading cause of end-stage renal disease(ESRD)around the world.However,the underlying pathological mechanism of DN remains largely unclear.Recent evidence indicates that mitophagy is intimately linked to the pathogenesis of DN.FUN14 domain-containing 1(FUNDC1)is a protein located on the outer mitochondrial membrane that plays a critical regulatory role in mitophagy.However,the role of FUNDC1-mediated mitophagy in DN is still unknown.Therefore,we intend to investigate the role of c-Src(Src)and FUNDC1-associated mitophagy in the development of DN.Methods: The db/db mice were used to establish a DN mice model.The mice were allocated to either the db/m group,the db/db group,or the db/db+PP2 group.The mice accepted PP2(Src inhibitor)treatment to investigate the role of Src in DN.Mice in the db/db+ PP2 group received an intraperitoneal injection of PP2(2 mg/kg)every other day,whereas mice in the db/m and db/db groups received an equal volume of normal saline intraperitoneally.Biochemical testing was used to assess kidney function.Renal histopathology and morphometric analyses were conducted via hematoxylin-eosin(HE),periodic acid-Schiff(PAS),Masson’s staining,and transmission electron microscopy(TEM).Tunel assay was used to determine the degree of apoptosis in the kidney.Mitophagy indexes including LC3 and P62 were evaluated by western blotting(WB).Immunofluorescence(IF)and TEM were also used to evaluate mitophagy.Podocytes given high glucose stimulation were used for the study in vitro.Podocytes were treated with PP2 or FUNDC1 small interfering RNA(siRNA)to evaluate the roles of Src and FUNDC1 in podocytes with high glucose.Flow cytometry was used to detect the apoptotic cells.Mitochondrial function was evaluated by JC-1 staining.Double immunofluorescence labeling of LC3 and TOMM20 and co-immunofluorescence of mitochondrial and lysosomal were used to assess the degree of mitophagy.Results:Increased Src activation was detected in the kidneys of db/db mice,and its expression was positively correlated with mitochondrial damage,podocyte apoptosis,and renal dysfunction.Inhibition of Src activation with PP2 protected against mitochondrial damage and podocyte apoptosis.Experiments in podocytes established that high glucose increased Src activation,promoting FUNDC1 phosphorylation and inhibiting mitophagy.Consistent with the mouse model,inhibiting Src activity protected podocytes against mitochondrial damage.FUNDC1 silencing negated the actions of PP2,indicating that FUNDC1-mediated mitophagy is the downstream pathway of Src.Conclusion: Our data identify Src-activated inhibition of FUNDC1-mediated mitophagy as a novel mechanism promoting DN development,proposing therapeutic opportunities for preventing diabetic renal damage.