Effects And The Underlying Mechanisms Of CQ8 Antibody In Diabetic Nephropathy

Objective:Diabetic nephropathy is the leading cause of end stage renal disease,and the underlying molecular mechanisms relevant to its pathogenesis are considerably complicated,among which the activation of RAS is believed to play a vital role.In our preliminary work,our group had successfully developed the therapeutic vaccine-ATRQ?-001,which is against the second extracellular loop of AT1 R.In addition to its function of significantly lowering of blood pressure,it showed to be protective for target organs at the same time.What is more,ATRQ ?-001 vaccine has been proved to be effective in alleviating diabetic nephropathy in STZ rats.CQ8 antibody is a specific monoclonal antibody derived from the AT1RQ?-001 vaccine.The purpose of this experiment is to prove that the protective effect of the ATRQ?-001 vaccine in diabetic nephropathy is mainly achieved by its CQ8 monoclonal antibody,and furtherly to explore the effects of CQ8 antibody in diabetic nephropathy and its possible mechanisms.Method: In animal experiments,db/db mice and their normal control db/m mice were selected as experimental subjects.When their blood glucose reached the standard of diabetes at 8 weeks old,the db/db mice were randomly divided into 3 groups,and the total 4 groups were designed as follows:(1)db/db control group(2)db/db 20 ug CQ8 antibody group: mice were given intravenous injection of 20 ug CQ8 antibody once a week starting from 8 weeks old;(3)db/db 100 ug CQ8 antibody group: mice were given intravenous injection of 100 ug CQ8 antibody once a week starting from 8 weeks old;(4)db/m group: db/m mice of the same age were set as normal control group.All mice were monitored for body weight,blood glucose and blood pressure during the experiment,and were put in metabolic cage to collect 24 h urine for analysis at 17-week-old and 27-week-old respectively.At 28 weeks old,mice were sacrificed.Serum creatinine and blood urea nitrogen were measured,and renal samples were saved for pathological examinations.q RT-PCR was used to detect the expression of fatty acid oxidation and autophagy related indicators(CPT1,CPT2,Acox1,ppar?,beclin-1,LC3,Atg5).In vitro,HK-2 cells(human proximal tubular epithelial cells)were stimulated with 30 m M D-glucose,5 groups were set as follows:(1)normal glucose group:normal control group;(2)mannitol group:30m M mannitol was given as an isotonic control;(3)High glucose group:cells were stimulated with 30 m M D-glucose;(4)High glucose+CQ8 antibody group:cells were preincubated with 5*10-3mg/ml CQ8 antibody for 1 hour,and then were given 30 m M D-glucose;(5)High glucose +losartan group:cells were preincubated with 5*10-6 mol/l losartan for 1 hour,and then were given 30 m M D-glucose.CCK-8 assay was used to detect cell viability,oil red O staining to observe intracellular lipid formation and q RT-PCR to detect the expression of fatty acid oxidation and autophagy related factors.Result: In vitro,CQ8 antibody effectively reduced the body weight,blood pressure and urinary protein levels in db/db mice,and improved renal function to some extent,it also showed to be renoprotective by alleviating kidney pathological changes and improving fatty acid oxidation and autophagy function.In vivo,cell viability decreased with the time as HK-2 cells were stimulated with high glucose,however,with CQ8 antibody,cell viability was effectively enhanced,high glucose-induced fatty acid oxidation and autophagy defects were significantly improved,and lipid formation was apparently reduced in cells.Conclusion: CQ8 antibody can effectively alleviate renal pathological changes and some biochemical parameters in db/db mouse,which may be partly explained by the improvement of impaired fatty acid oxidation and autophagy in proximal tubular epithelial cells.