HDAC4 Promotes The Activation Of CaN By Inhibiting Gap43 Expression Mediates Podocyte Injury In Diabetic Nephropathy

BackgroundDiabetic nephropathy(DN)is a severe chronic complication of diabetes mellitus[1,2].Podocyte injury is one of the most important pathogenesis of DN[3,4],and calcium/calcineurin(CaN)signal pathway plays a key role in podocyte injury and apoptosis[5,6].The growth-associated protein 43(Gap43)is a neuronal membrane protein involved in axonal growth and regeneration as well as in the modulation of synaptic plasticity[7,8].Gap43 also play a role in the regulation of CaN activation to modulate the release of synaptic transmission by interacting with calmodulin(CaM)[9].Recent studies have shown that histone deacetylase family(HDACs)are associated to the occurrence and development of kidney diseases.HDAC4 has been confirmed to increase expression in podocyte of DN and is one of critical components of a signal transduction pathway that links podocyte injury and apoptosis to autophagy in DN[10-17].ObjectThis study aimed to verify the expression of Gap43 and its protective effect on podocyte injury of DN.We further investigated whether HDAC4/Gap43 signal pathway is involved in mediating podocyte injury by promoting the activation of CaN in DN.MethodsIn this study,we used immunoblotting and immunofluorescence to investigate the expression of Gap43 in the kidney tissue from DN patients,db/db mice and podocytes cultured with high glucose.Electron microscopy and tissue staining were used to detect the protective effect of Gap43 on podocytes and kidney structures in db/db mice.In vitro,with the simulation of high glucose,cultured podocytes were treated with adenovirus(silence or overexpress Gap43)to observe podocyte injury,including the expression of synaptopodin,cell mobility and apoptosis.AAV was used to establish DN models with the overexpression of Gap43 in db/db mice.The transfection of silence and overexpression adenovirus were used to interfere with the expression of Gap43 in podocytes to observe the activation of CaN after high glucose treatment in vitro.The interaction of Gap43 and CaM were explored by co-immunoprecipitation(Co-IP)and Pull down to clarify the mechanism of CaN activation.High glucose cultured podocytes interfere with the expression of HDAC4,and observe the expression of Gap43.The chromatin immunoprecipitation-qPCR(ChIP-qPCR)was used to verify the regulation of HDAC4 on Gap43 histone H3 acetylation.In vitro,silencing HDAC4 under high glucose stimulation or interfering with the expression of HDAC4 and Gap43 together to observe the effect on the activation of CaN and the function of podocytes.ResultsGap43 is specifically expressed in human,mouse glomerulus and podocytes.The overexpression of Gap43 in db/db mice and cultured podocytes were used and observed that Gap43 had a protective effect on podocytes in DN.In high glucose condition,the decrease in the interaction between Gap43 and CaM and increase in the activation of CaN were observed in vivo and vitro.Overexpression of HDAC4 reduce the expression of Gap43,and silencing HDAC4 restore the expression of Gap43 induced by high glucose.In addition,high glucose decreased the expression of Gap43 through increasing the nuclear protein expression of HDAC4,which inhibits Gap43 histone H3 acetylation and decreases the transcription level of Gap43.In vitro,silencing HDAC4 under high glucose stimulation decreased the activation of CaN and podocyte injury,and overexpress Gap43 can reduce the activation of CaN,which induced by HDAC4 overexpression,and reduce podocyte injury.ConclusionsThis study clarified the expression of Gap43 in the kidney and found the role of HDAC4/Gap43 signal pathway is involved in the regulation of podocyte injury by promoting the activation of CaN,which provide a new theories and therapeutic targets for prevention and treatment of podocyte injury in DN.