Based On The Imbalance Of IRS-1 Ser/Tyr Phosphorylation, The Mechanism Of The Treatment Of Diabetic Nephropathy Proteinuria With Qidi Tangshen Granules Was Discussed

Background and objective:Proteinuria is an independent risk factor for the progression of diabetic nephropathy(DN).Podocyte injury plays an important role in the development of DN proteinuria and glomerulosclerosis,and insulin resistance is one of the vital mechanisms for the injury of podocytes.Insulin receptor substrate-1(IRS-1)/PI3K/Akt is the most classical pathway for insulin signal transduction.Normally,the phosphorylation of IRS-1 tyrosine(Tyr)residues further activates the downstream PI3K/Akt signaling pathway and then plays a series of biological effects.Under the stimulation of insulin,phosphorylation of IRS-1 serine(Ser)residues follows a sensitive mechanism in the regulation of insulin signaling.Basal level phosphorylation of IRS-1 Ser plays an important positive regulatory role in the secondary phosphorylation of IRS-1 Tyr induced by insulin receptor kinase.However,the enhanced phosphorylation of IRS-1 Ser will weaken the interaction between Tyr and insulin receptor.In the state of type 2 diabetes mellitus(DM),the excessive activation of the inhibitory pathway of insulin signal transduction,such as p38MAPK,increases the phosphorylation level of IRS-1 Ser,inhibits the phosphoiylation of its tyrosine residues,and causes the imbalance of IRS-1 Ser/Tyr phosphorylation,then affects the insulin signal transduction in the downstream.Chinese herbal medicine is effective in the treatment of DN albuminuria.Various Chinese herbal extracts or compound preparations have been proved to have a clear kidney protective effect.Professor Liu Hongfang suggested that the core Traditional Chinese Medicine(TCM)pathogenesis of DN is kidney essence deficiency along with stagnation of collaterials,and the principle of treatment should be supplementing kidney essence and dredging the collaterals,and created Qi Di Tang Shen granules(QDTS),a special prescription for DN treatment.And it formed a complete theoretical system of principle-method-recipe-medicines.Previous clinical study had confirmed that QDTS can effectively reduce the level of albuminuria in DKD patients.In TCM theory,kidney essence is stored in the life-gate.It is mentioned in the Jingyue Quanshu that the life-gate is the foundation of the promordial Qi,and it's also the house of water and fire.And the Yin and Yang of the five zang organs all depend on the kidney essence stored in the life-gate.Thus it can be seen that the kidney essence contains kidney yin and kidney yang,and the kidney Yin and Yang is the root of whole-body Yin and Yang.Therefore,the deficiency of kidney essence makes both Yin and Yang deficiency,and supplementing kidney essence and dredging the collaterals is the prerequisite of coordinating Yin and Yang.The current views hold that the positive or negative feedback mechanism or the concept of a regulatory network in molecular biology is very similar to the concept of "Yin and Yang" used in the theory of TCM.Therefore,the hypothesis that Qi Di Tang Shen Granule can improve the IRS-1 Tyr/Ser phosphorylation balance of podocytes,improve insulin resistance and protect podocytes in kidney is suggested.By observing the effect of QDTS on the phosphorylation balance of IRS-1 Ser/Tyr in renal tissue of db/db mice,and its effect on the PI3K/Akt signaling pathway and the inhibition pathway of insulin signal,p38MAPK,the possible target and therapeutic mechanism of QDTS are discussed in order to further clarify the core TCM pathogenesis of DN by molecular biology.Methods:1 Literature review and meta analysisThe research progress of insulin resistance and DN podocyte injury was reviewed by traditional review.Then the meta analysis was used to evaluate the clinical efficacy of the decoction of Liuwei Dihuang Wan(LDW)prescriptions in the treatment of DN albuminuria,so as to clarify the effect of kidney tonifying therapy in DN treatment.The targeted search strategies were formulated respectively,and all the clinical trials about LDW prescriptions used for DN treatment were searched from the CNKI,Wanfang data,Chinese Scientific Journal Database(VIP),Chinese Biomedical Literature Database(CBM),PubMed,Embase and Cochrane library.The retrieved prescriptions included LDW,Zhibai Dihuang Wan,Maiwei Dihuang Wan,Guishao Dihuang Wan.Qiju Dihuang Wan,Guifu Dihuang Wan,Jingui Shenqi Wan and Jisheng Shenqi Wan.After the inclusion of eligible randomized controlled trials,two researchers independently extracted information and evaluated the quality of the included literatures.RevMan 5.3 software was used to analyze the data by meta.2 Clinical research40 cases eligible DKD microalbuminuria patients and 26 simple DM patients were enrolled in the study.The basic information and laboratory examination results were collected;the TCM syndrome was evaluated by the "TCM syndromes determination scale" previously formulated by the study group;the content of urinary podocytes specific protein nephrin,podocalyxin and WTlwere detected by ELISA.The laboratory indicators,TCM syndrome characteristics and urine podocyte specific protein contents of two groups were compared,and the correlation between TCM syndromes,laboratory indicators and podocyte specific proteins in DKD microalbuminuria patients were analyzed.3 Experimental research30 eight-week-old male db/db mice were used as DN animal models,and 8 C57BL/6 mice were used as normal control.After one weeks of adaptive feeding,urine was collected for 24 hours,urinary albumin was measured by ELISA,and urinary albumin excretion rate(UAER)was calculated.The db/db mice whose UAER was higher than the normal group were randomly divided into 3 groups according to body weight,blood glucose and UAER,DN model group(db/db group),valsartan group(db/db+v group),QDTS group(db/db+Q group),and 8 in each group.They were given equal volume of corresponding drugs or carboxymethyl cellulose sodium(CMC)solution for 12 weeks.Kidney injury was evaluated by UAER,serum urea nitrogen(BUN),serum creatinine(Cr),serum uric acid(UA),creatinine clearance rate(Ccr)and pathological examination;insulin sensitivity was evaluated by blood glucose,serum insulin and insulin resistance index(HOMA-IR).The expression of podocyte specific protein nephrin,podocalyxin and WT1 were detected by western blot,the ultrastructure of podocyte was observed under electron microscope and the fusion rate of foot process(FRFP)was calculated in order to evaluate the injury of podocyte.The protein expression and phosphorylation level of IRS-1,PI3K.Akt and p38MAPK were detected by western blot,so as to evaluate the activation level of insulin signaling pathway.Results:1 Literature review and meta analysisA total of 14 literatures were included in the meta analysis.The results showed that the total effective rate of the decoction of LDW prescriptions was higher than that of the control group(OR 2.87,CI 1.98-4.15,P<0.00001);It's alleviating effect on 24 hour urine protein and UAER were better than the control group(MD 0.12,CI 0.06-0.17,P<0.0001,SMD 0.87,CI 0.41-1.32,P<0.0002).In addition,the effect of reducing fasting blood glucose(FGB)and glycosylated hemoglobin in the Jisheng Shenqi wan group was better than that of the control group(MD 1.96,CI 1.08-2.84,P<0.0001;MD 1.87,Cl 1.50-2.23,P<0.00001).2 Clinical researchCompared with the DM group,the urine microalbuminuria/creatinine ratio(ACR)in the DKD microalbuminuria group was significantly higher(P<0.01),the Ccr was significantly decreased(P<0.05),and the urinary content of podocyte specific protein nephrin and podocalyxin were significantly increased(P<0.05).Meanwhile,the syndrome scores of spleen kidney Yang deficiency,Yin and Yang deficiency and dampness heat syndrome in DKD microalbuminuria group were significantly higher than those in DM group(P<0.05).The results of correlation analysis showed that in the DKD microalbuminuria group,the urinary nephrin content was negatively correlated with the syndrome score of Qi and Yin deficiency(P<0.05),and there was a positive correlation between the urinary WT1 content and the syndrome score of spleen kidney Yang deficiency(P<0.05).3 Experimental researchCompared with db/db group,the UAER level of db/db+Q group was significantly decreased(P<0.01),and the pathological damage of kidney was significantly reduced.In ultrastructure,compared with db/db group,the thickness of GBM and the FRFP were significantly decreased in db/db+Q group(P<0.01,P=0.026).Meanwhile,the HOMA-IR of db/db+Q group was significantly decreased(P<0.05),and the expression of nephrin protein in renal tissue of db/db+Q were significantly increased(P=0.028),compared with the db/db group.In the study of mechanism,the protein expression of p-PI3K,p-Akt and p-p38MAPK in the renal tissue of db/db+Q group was significantly higher than that of the db/db group(P=0.03,P=0.049,P=0.021).Although the phosphorylation level of the three amino acid residues(ser302,Ser307,tyr896)of IRS-1 in renal tissue of db/db+Q group was not significantly changed,conpared with the db/db group(P>0.05),the proportion of ser307/tyr896 in the renal tissue of db/db+Q group was significantly decreased(P=0.032).Conclusion:Podocyte injury is one of the most important causes of albuminuria in DKD patients.Insulin resistance can lead to the occurrence of podocyte injury.The level of urinary albumin and podocyte injury are closely related to the core TCM pathogenesis of kidney essence deficiency along with stagnation of collaterials.Therefore,the treatment of DN albuminuria should be based on the principle of supplementing kidney essence and dredging the collaterals.The experimental research showed that QDTS could restore the phosphorylation balance of IRS-1 Ser/Tyr residues by decreasing the activity of insulin signal suppressor p38MAPK,and regain the normal transduction of insulin signal,so as to reduce the podocyte injury and urinary albumin.