The Effect Of Autophagy On Chronic Kidney Disease Muscle Atrophy And Its Intervention

Objectives: Previous study of this research team found that autophagy was hyperactive in atrophic muscles of chronic kidney disease(CKD)patients,and for autophagy plays important roles in muscle atrophy of other diseases,this research aims to study the role of autophagy in CKD muscle atrophy and its potential mechanisms.We aims to set leucine amino acid and unconventional nonproteolytic calpain,calpain-6(CAPN6)as the point,to study the effect of CAPN6 on autophagy and its role in leucine inhibiting autophagy.Finally we would study the role of low protein diet plus ketoacids on muscle mass maintenance and its potential mechanism.Methods: Rats with 5/6 nephrectomy were randomly divided into three groups: CKD group(vehicle i.p.),Wort group(Wortmannin i.p.)and 3-MA group(3-Methyladenine i.p.),gastrocnemius muscle were obtained after 8 weeks treatment.Rats with 5/6 nephrectomy were randomly divided into three groups,and fed with either 22% protein(normal-protein diet;NPD),6% protein(LPD)or 5% protein plus 1% KA(LPD+KA).Sham-operated rats with NPD intake were used as control.24 weeks after treatment gastrocnemius muscle were obtained for experiments.Finally,CAPN6 plasmid were transfected into rat L6 myoblast,and autophagy related LC3 and p62 were tested,and m RFP-GFP-LC3 AAV were transfected to analyze autophagy flux.We deprived L6 myoblast with leucine or re-supplemented it with leucine,transfected CAPN6 plasmid to study LC3 and p62 expression,as well as m TOR,raptor,p-p70s6k1/p70s6k1 and p-4EBP1/4EBP1.Results: Authophagy inhibition,especially by 3-MA could significantly improve decreased cross-sectional area of muscle fibers,improve gastrocnemius muscle mass and body weights loss of CKD.NALP3(NACHT-PYD-containing protein 3)inflammasome and found they were significantly decreased in authophagy inhibitor treated Wort and 3-MA group,when compared to the CKD group,as well as inflammation cytokines IL-1β(interleukin-1-beta),IL-18,tumor necrosis factor-α(TNF-α),IL-6.Furthermore,we found that although there was no significant changes of total mt DNA in authophagy inhibitor treated group when compared to CKD group and control group,authophagy inhibitor could alleviated mt DNA released into cytosol.Although LPD could not ameliorate the lower serum albumin level,it improved body weight and gastrocnemius muscle mass,with the effect being more obvious in the LPD+KA group.LPD+KA could further attenuate autophagy as indicated by LC3,p62,Parkin and Pink1 than LPD which were significantly increased in NPD rats,but its effect on ROS,NALP3 inflammasome,Caspase-1,and mt DNA were not that obvious,as well as LPD.In rat L6 myoblast,CAPN6 inhibited autophagy related LC3 and p62 expression,LC3 red immunoflurence through associates with the m TORC1 in the cytoplasm.Leucine could increase CAPN6 expression and inhibit autophagy.Conclusions: Hyperactive autophagy and mitophagy may play important roles in CKD muscle atrophy.Autophagy disruption could decrease mt DNA released into cytoplasm which maybe a contribution of muscle atrophy and inflammation alleviation.Although LPD+KA could further ameliorate activated autophagy in NPD,its effect on the activated inflammation state was not consistent between different cytokines.Leucine could inhibit autophagy by increasing CAPN6 expression,which may be related with promoting autophagy through association with the m TORC1.Further study is still required to explore the method of ameliorating inflammation to provide new therapeutic approaches for CKD protein energy wasting.