The Relationship Between ET,TGF-β And Diabetic Nephropathy, And The Intervention Effect Of Cordycepic Hypha

Objective and Background:In recent years, the incidence of diabetes mellitus (DM), especially the type-2 diabetes mellitus (T2DM), is on the tendency of rising. Diabetic nephropathy (DN) is one of the most common complications of DM, which involves about 30%-35% of DM patients. Once the kidney is affected, diagnosed by continuous proteinuria, the damaged renal function can not be restored and finally the DN patients will enter the end stage of renal disease (ESRD) in a short period. The number of ESRD patients caused by DN who need maintenance dialysis increases every year, and therefore, leads to the raise of the therapeutic expenditure on ESRD patients every year. All this demonstrated that it is very important to elucidate the pathogenetic mechanism and hence provide effective prevention strategy for DN.Besides the well-known mechanisms of metabolic disorder and hemodynamic disorder, many new hypotheses are under investigation about the p athogenesis of DN. But at present, it is commonly regarded that the hemodynamic disorder and the consequences of metabolic disorder caused by high glucose are the basic foundation of renal pathogenesis; numerous growth factors(GF) and cytokines(CK) are direct mediators of renal pathogenesis. Among them, the endothelin (ET) and transforming growth factor (TGF) and their relationship to DN, have attracted more attention from researchers than any other factors.The main patho-changes of DN are the enlargement of kidney, the thickness of glomerular and tubular basement membrane, and the progressive accumulation of extracelluar matrix (ECM) between glomerulus and renal tubule. Many factors are believed to contribute to the genesis of those patho-changes. ET is now generally regarded as an ? important somatodedin(SM), which has strong effect of vasoconstriction, stimulates the proliferation of vascular smooth muscle cells, glomerular endothelial cells and mesengial cells. It can also promote the accumulation of ECM; hence accelerate the glomerular sclerosis and interstitial fibrosis. The persistent state of hyperglycemia stimulates renal overproduction of ET, especially in the mesangial cells, renal tubular epithelial cells and renal interstitium fibroblasts. The overexpressed ET, through paracrine and autocrine, up-regulates the expression of TGF-pl, CTGF and PAI-1 which can promote the renal fibrosis, and down-regulates the expression of catabolic enzymes and BMP-7 (bone morphogenetic protein), thus breaks the balance of synthesis and degradation of ECM and results in the accumulation of ECM. The overexpression of TGF-pi is the most important pathophysiological feature of DN, which can accelerate the speed of DN progression through stimulation of collagen oversynthesis. TGF-J31 pathway is the final step of renal tubular interstitial fibrosis.In this research, we will detect the level of ET and TGF-pi in blood and urine, before and after the treatment of Cordycepic Hypha of DM patient, DN patient in every stage and DN rat mould. Through those indexes combined with urinary microalbumin(UMA), urinary albumin excretion rate (UAER), creatinine clearance rate (Ccr), serum creatinine(Scr), blood urea nitrogen(BUN) and glycosylated hemoglobin(GHbAlc), we study the relationship between ET, TGF-|3iand DN, and its effect on renal function. By studying the effect of CordycepicHypha on urinary and blood level of ET, TGF-Pi in every stage of DN, we study the renal protection effect of Cordycepic Hypha, then search for a new promising therapeutic approach for DN, and provide theoretical foundation for clinical treatment and prognosis improvement. Methods:1. Divide the selected 141 T2DM patient into DM group and DN group according to their UAER. Then divide the DN group into five groups according to Mogensen's staging method of DN: DN1, DN2, DN3, DN4, DN5. Choose 20 healthy individuals as control group(HC). Information obtained here can be used to study the relationship between ET, TGF-Pj and DN, and its influence on renal function.2. Divide randomatically the groups of DN1, DN2, DN3 and DN4 into treatment group A and treatment group B respectively: DN1A, DN1B, DN2A, DN2B, DN3A, DN3B, DN4A, DN4B; and divide group DN5 into maintenance hemodialysis(MHD) group and non-hemodialysis(NHD) group. Each group contains 10. Information obtained here can be used to study the influencing effect of different intervention strategy on the level of ET and TGF-j3i in DN patients, and search for new therapeutic strategy for renal protection.3. detect sETl, sTGF-pl, Scr, BUN, TP, ALB, blood-fat series, HB, FPG and GHbAlc of venous blood in the morning with an empty stomach. Collect 24h urine the day before blood sampling to detect the level of UET, UTGF-pi, Upro/24h, urine creatinine(Ucr) and urinary micro-albumin(UMA), then calculate UAER. Record the blood pressure regularly.4. select 30 healthy male Wistar rats, divide them at random into control group(RC, 10 rats) and experiment group(RE, 20 rats). After the successful moulding of DN rats, the RE group is divided into treatment group(RET) and control group(REC), each group has 10 rats. Measure theblood level of ET and TGF-p! before and after the treatment of Cordycepic Hypha.5. detect method of SET and RET: radioimmunoassay technique. The ET RIA kit is provided by Beijing Kemei Dongya Biotechnique Company, the procedure is strictly coherent with its instruction manual.6.detect method of STGF-pl, UTGF-pl: We use enzyme linked immunosorbent assay(ELISA) technique to test the concentration of TGF-P 1, this anti-human TGF-P 1 kit is provided by Jingmei Bioengineering Company Limited. The procedure is strictly coherent with its instruction manual. The enzyme-label machine is ANTHOS2010 made in Austria. Results:1. the relationship between ET, TGF-pland DN, and its influencing effect on renal function1.1 the urinary and blood level of ET and TGF-p 1 in all DM and DN groups are higher than HC group significantly (P<0.01); the urinary and blood level of ET and TGF-p 1 in all DN group are higher than all DM group significantly (P<0.01); DN5 group is higher than all other DN group significantly (P<0.01); DN4 group is higher than DNl-3 groups significantly (P<0.01); DN3 group is higher than DNl-2 groups significantly (P<0.01); DN2 group is higher than DNl group significantly1.2 the urinary and blood level of ET and TGF-pi in all DN groups are in positive correlation with Scr, BUN, GHbAlc, UAER/Upro/24h, and systolic arterial pressure (P<0.01), negative correlation with Ccr (P<0.01)and no relation with TP, ALB, blood fat series, FPQ urinary production, age, sex and course of DN (P>0.05).1.3 In DN, the urinary and blood level of ET, TGF-p 1 has no relation withHB and HCT (P>0.05), which shows that ET, TGF-pl is not the maincause of renal anemia.1.4 the level of urinary ET and TGF-J31 in groups DM and early stage DNrises more than the level of UMA significantly (P<0.01), has no relationwith S ET, S TGF-pi and C cr(P>0.05), and has positive correlation withUAER.2. the influencing effect of Cordycepic Hypha on urinary and blood level of ET, TGF-pi in DN patient2.1 the urinary and blood level of ET, TGF-pl andUAER/Upro/24h, Scr, BUN after treatment of Cordycepic Hypha is significantly lower than before treatment(DNl-3P<0.01, DN4PO.05) and group HC (PO.05). Ccr after treatment is augmented significantly than before (PO.05). All this demonstrates that Cordycepic Hypha has the renal protection effect. 2.2The urinary and blood level of ET, TGF-pl in group MHD has no significant difference between before and after the treatment(P>0.05), These data show that MHD is not significant in late stage of DN.3. the influencing effect of Cordycepic Hypha on the blood level of ET, TGF-pl in DN rat mould3.1 The blood level of ET, TGF-pi in DN rat is significantly higher than DM rat and normal rat(P<0.01), has positive correlation with UAER, FBG and GHbAlc (P<0.05) and has no relation with Scr, BUN, Ccr(P>0.05).3.2 The blood level of ET, TGF-pl and UAER in group RET after treatment is significantly lower than group REC and before treatment(P<0.001, P<0.05), and has no significant deviation with DM rat(P>0.05), but still higher than healthy rat before DN moulding(P<0.05). Conclusion:1. The urinary and blood level of ET, TGF-pi in group DN is significantly higher than group DM and group HC, which shows that ET and TGF-piis related with the genesis and development of DN.2. The urinary and blood level of ET, TGF-pl in group DN increases progressively from DN1 to DN5 and reach the peak at uremia stage. The correlation analysis shows that the level of ET, TGF-pi has the positive correlation with Scr, BUN, GHbAlc, UAER/Upro/24h, and systolic' arterial pressure(SBP), has negative correlation with Ccr, GFR, and has no relation with TP, ALB, blood fat series, FPG, urinary production, age, sex, and course of DN. This demonstrates that the level of ET, TGF-J31 is in close correlation with renal lesion and renal hypofunction.3. In groups DM and early stage DN, the level of UET and UTGF-pl increases more than the level of UMA does, has no significant correlation with SET, STGF-pi, Ccr, and has positive correlation with UAER. In group DM when UAER<30mg/24h, the excretion of UET and UTGF-pl is significantly higher than group HC. This shows that UET and UTGF-pi may be a more sensitive index for early diagnosis of DN.4. 12 weeks after the treatment of Cordycepic Hypha, the urinary and blood level of ET, TGF-pl and UAER/Upro/24h in groups DN1-4 is significantly lower than control group and before treatment. The therapeutic effect is best in group DN1-2, better in group DN3, good in DN4. All this shows that Cordycepic Hypha has the renal protection effect, and this protection effect is much better if used earlier.5. In group DN5, the treatment of hematodialysis(HD) has no influence on the urinary and blood level of ET, TGF-pi. This shows that although HD can prolong the life span of DN patient, it can not restore the damaged renal function when DN enters the ESRD stage.6. The urinary .and blood level of ET, TGF-pi in group DN3-5 has no significant correlation with HB and HCT and shows that ET, TGF-pl may not be one of the main causes of renal anemia.7. The blood level of ET, TGF-pl in group RE is significantly higher than DM rat and HC rat, has positive correlation with UAER, FPG, GHbAic, and has no relation with Scr, BUN> Ccr. All this shows that the level of ET and TGF-J31 increases as early as DM stage and early stage of DN, and can be affected by the level of blood sugar. So the prevention strategy should begin at early stage.8. 12 weeks after the treatment of Cordycepic Hypha, the blood level of ET, TGF-pi and UAER in group RET is significantly lower than control group and before treatment. This shows that Cordycepic Hypha has renal protection effect on DN rat.9 The data of the above-mentioned DN patients and DN mould rats show:the level of ET and TGF-pi in DN patients is higher than control groupand is significantly in positive correlation with renalfunctional parameters;in early stage of DN patients and DN mould rats, the level of ET andTGF-pi is also higher than control, but has no positive correlation withrenalfunctional parameters significantly. It only has positive correlationwith parameters of renal injury such as UAER. This demonstrates that ETand TGF-pi participate in the renal injury at the initial stage of DN, risewith and accelerate the development of DN and deterioration of renalfunction, and thus build a vicious cycle. That is to say, the level of ET andTGF-pi can represents the severity of DN to some extent, so we candiagnose DN in early stage and judge the severity of renal damage bymeasuring urinary and blood level of ET and TGF-pi in DM and DNpatients. In conclusion, the anti-ET and anti-TGF-pi method is the keytherapy of DN treatment. The future study should follow the direction offinding a specific way to inclusively antagonize ET and TGF-pi in kidneyto prevent the happening and progress of DN.10. The data of our experiment show: the therapeutic effect of the allianceof Cordycepic Hypha, ACEI and ARB is significantly better than the effect of ACEI and ARB, especially to the decrease of proteinuria and improvement of renal function in early stage of DN. So we can deduce that Cordycepic Hypha has synergistic effect with ACEI and ARB in the treatment of DN patients. Among them, ACEI and ARB are commonly used medicine in clinic, while Cordyceps sinensis Sacc is one of our famouse traditional Chinese herbs which has no significant adverse side effects and may has a more brighter future in clinical application.