Mechanism Of Renal Injury And Screening And Evaluation Of Active Compounds Against Injury

Kidney is one of the organs that was subjected easily to be damaged, besides mechanical injury, chemical and biologic and immunity factors could also induce kidney impairment. Meanwhile kidney had redundant capillary network, so vascular function could easily effect renal function.In the recent years, it was found that hyperuricacidemia was an important risk factor that could damage vessel and kidney, The kidney was the major target organ of diethylene glycol toxicity, but their impairment action and mechanism had studied very little. In this study, hyperuricacidemia and diethylene glycol were represented as endogenous and exogenous material that damaged kidney, their pathological role on vessel and kidney was explored and active compounds which inhibited detriment material production, promoted excretion and protected them from injury were screened and evaluated, the action mechanism of active compound were studied also.Chaptar 1 The pathological effect of hyperuricacidemia on the vessel and kidney and the screening and evaluation of the active compound against hyperuricacidemia impairmentHyperuricacidemia may damage the vascular and kidney, result in cardiovascular and kidney disease . The hyperuricacidemia model and mesenteric vascular bed perfusion model ex vivo of rat were established to reveal the pathological effect and possible mechanism of hyperuricacidemia on the vessel and kidney. A high-throughput assay for xanthine oxidase inhibitor screening was established, the pathological effect of Hyperuricacidemia on vascular cells and renal tubular epithelial cell were observed. Active compounds with good protection were screened out and evaluated also. The results were as follows:1 Uric acid level in serum of rats induced by oxonic acid inceased significantly and blood pressure rised slightly. The perfused mesenteric vascular bed ex vivo of hyperuricacidemia rat had a sensitive contractile response to KCL and NE at various concentrations, and the pre-constricted perfused mesenteric vascular bed showed weakened vasodilation response to SNP and Ach at various concentrations, the obvious weakened vasodilation response to Ach was observed. The NO level and T-NOS activity in serum and expression of eNOS protein in mesenteric vascular bed in hyperuricemia rat diminished significantly, and the vWF level in plasma, activity of iNOS in serum, expression of CRP and MCP-1 protein in mesenteric vascular bed in hyperuricemia rat increased significantly. Those results showed that the vascular impairment in hyperuricemia rat was related to change of NOS activity and CRP and MCP-1 and eNOS protein expression.2. The contents of creatinine, urea nitrogen, NAG and MDA, activity of iNOS, expression of CRP and MCP-1 protein in serum and kindey of hyperuricacidemia rats increased significantly, and activity of T-NOS, expression of eNOS protein decreased obviously. But these changes were improved by allopurinol. The result showed that the kindey impairment in hyperuricemia rat was related to lipid peroxidation reaction, change of NOS activity, CRP and MCP-1 and eNOS expression.3. the Screening method for xanthine oxidase inhibitor in vitro was effective and reliable, 27 compounds were identified from 71760 compounds, the hits was 0.038%, among which 17 compounds were confirmed by subsequent testing at multiple concentrations.4. The effect of active compounds on the renal function and uric acid excretion was evaluated by rat kidney perfuse model ex vivo, it was found that code name of J11222( 24.8ug/ml)and DF2956 could obviously lessen glomerular filtration rate and urine flow rate, it was indicated that J11222 and DF2956 could damage glomerulus function; code name of J11222 could obviously hold back the tubular reabsorption of Na, it was indicated that J11222 could damage nephric tubule function. Code name of J11222( 8.27 and 2.48ug/ml) and J11225 could obviously increase the uric acid excretion fraction and total amount of uric acid excretion during 90 min, it was indicated that J11222 and J11225 could promote uric acid excretion. 5. Cell proliferation were inhibited effectively in EA.hy926 cell lines cultured with 1.5% fetal calf serum and treated with UA 500μmol/L for 48 h. Among 30 selected traditional Chinese medicine compound , the effect of Curcumin, Notoginsenoside R1 and TanshinoneⅠat concentration of 1-00μM against EA.hy926 cell line proliferation inhibited by UA was presented obviously at dose-dependent effect. Those compounds significantly improved the NO generation and inhibited CRP and MCP-1 and increased eNOS protein expression .6. Vascular smooth muscle cell proliferation was induced by UA 500μmol/L treatment cell for 48 h. Among 30 screened traditional Chinese medicine compound, the Salvianolic acid B, TanshinoneⅠ, Rutin, Curcumin, Ferulic Acid, Notoginsenoside R1 and Ligustrazine hydrochloride at concentration of 100μM could inhibited the vascular smooth muscle cell proliferation induced by uric acid, the ratio of cell proliferation was less than 15%.7. Vascular adventitial fibroblast proliferation was induced by UA 500μmol/L treatment cell for 48 h. Among 30 screened traditional Chinese medicine compound, the Salvianolic acid B, TanshinoneⅠ, Curcumin, Ferulic Acid, Notoginsenoside R1 and Ligustrazine hydrochloride at concentration of 100μM could inhibited the vascular adventitial fibroblast proliferation induced by uric acid, the ratio of cell proliferation was less than 15%. 8. HK-2 cell line proliferation was induced by UA 500μmol/L treatment cell for 48 h. Among 30 screened traditional Chinese medicine compound, the Rutin, Curcumin, Ferulic Acid, Ginsenoside Rg1, Notoginsenoside R1 and Peoniflorin at concentration of 100μM could inhibited the renal proximal tubule cell proliferation induced by uric acid, the ratio of cell proliferation was less than 25%.It was concluded that hyperuricacidemia could induce vessel and kidney impairment, some monomer compounds from traditional chinese medicine could improve the function of kidney and blood vessel in hyperuricacidemia rats, the protective mechanism may result from its ability to improve NO production, increase the activity of NOS and the expression of eNOS, as well as decrease the expression of CRP and MCP-1 in kidney and vessel.Chapter 2 the impairment effect of diethylene glycol on the kidney and the screening and evaluation of the active compound against diethylene glycol injuryDiethylene glycol(DEG) has distinct physical and chemical fuction, it was widely used in industry and daily chemical expenses. The human beings are subjected to diethylene glycol intoxication, its major clinical manifestation are nephrotoxicity (acute renal failure; ARF). The experiments were to explore nephrotoxicity of diethylene glycol, observe the toxicity of diethylene glycol in mice and injury to kidney ex vivo, vascular endothelial cell and kidney proximal tubule cell, the drug intervention was observed also.The study is helpful to reveal the feature of diethylene glycol nephrotoxicity, to find out prevention and therapy drugs, and to provid experiment model for kidney exogenous impairment and drug intervention in diethylene glycol poisoning.The results were as follows:1. In isolated perfused kidney experiment with diethylene glycol, It was found that DEG could increase perfusion pressure, urine protein level, urine flow rate and MDA contents in renal tissues, it had no effect on glomerular filtration rate. It was indicated that diethylene glycol could impair kidney vessel and nephric tubular, this impairment may be related to lipid peroxidation induced by DEG.2. After poisoned by diethylene glycol, The hepatic index of mice damaged by DEG decreased, renal index increased; urea and creatinine in serum increased, the activity of SOD and GSH-PX in the hepatic and renal tissues decreased, the contents of MDA increased. Extract from rhubarb and alismae rhizome could improve mice diethylene glycol poisoning, the protection role was related to improve antioxidation and suppress lipid peroxidation in poisoned mice.3. There had relationship of dose and time dependence in the vascular endothelial cell damaged by DEG. Vascular endothelial cell was obviously damaged after cultured with DEG 400μmol/L for 24 h. Salvianolic acid A, TanshinoneⅡA, Astragoloside, Hyperoside, Quercitin and Ginsenoside Rb1 at concentration of 100μM could improve the vascular endothelial cell viability damaged by DEG in the 30 monomer compounds from traditional chinese medicine that were screened, the protection ratio was more than 10%.4. There had relationship of dose and time dependence in the renal proximal tubule cell damaged by DEG. Renal proximal tubule cell was obviously damaged after cultured with DEG 400μmol/L for 24 h. Salvianolic acid B, Cryptotanshinone Astragoloside, Quercitin, Curcumin and procyanidin at concentration of 100μM could improve the renal proximal tubule cell viability which was damage by DEG in the 30 monomer compounds from traditional chinese medicine that were screened, the protection ratio was more than 60%.It was concluded that DEG could damage kidney obviously, the target of toxicity may be renal vessel and tubule, some monomer compounds from Traditional Chinese Medicine could protect the kindey vascular cell and tubule cell from the damage of DEG.