Effect Of Apoptosis Signal-Regulating Kinase 1 Inhibitor And Enalapril In The Kidneys On Rats With 5/6 Nephrectomy And Its Mechanism

Organisms have multiple stress response signaling pathways in the body to adapt to the environment and plays a crutiall role. Among them, the Mitogen activated protein kinase(MAPK) kinase kinase pathways, as one of the most important signaling pathways regulating cellular responses, is closely related to the stress response and signaling pathways regulating cell response, widely exists in eukaryotic cells. Apoptosis signal regulating kinase(Apoptosis signal- regulating kinase 1, ASK1), as a member of the MAPK family, is located in the JNK(c- JUN N- terminal kinase) and upstream of P38 MAPK and activated by phosphorylation reaction. A variety of stress injury and proinflammatory factor(H202, TNF-α, ischemia-reperfusion and chemotherapy drugs) may activate ASK1, and activated ASK1 in turn activate MKK- 4 /MKK7-JNK and MKK3 / MKK6- P38 lightning kinase pathway, make responses to stress or induce cell apoptosis. Studies in vitro showed that activated ASK1 can activate a variety of biological reaction process, including the different types of cells of apoptosis, inflammation, differentiation and survival. Wild type of ASK1 excessive expression or mutation can induce various cells apoptosis. Studies have found that damage of ASK1 genes in mice can obviously reduce the level of TNF-α and various cell apoptosis induced by oxidative stress, suggesting that ASK1 is a key factor of apoptosis induced by stress.As we all know that the activation of ASK1 play an important role in many diseases. Selective inhibition of the expression of ASK1 can reduce the necrosis and apoptosis of myocardial cells caused by ischemia-reperfusion injury. MPTP(1-Methyl- 4-phenyl- 1, 2, 3, 6-tetrahydropyridine, MPTP) may be one of the causes of Parkinson’s disease, the study found that MPTP in the environment may be one of the causes of Parkinson’s disease, MPTP may activate ASK1 M and promote the phosphorylation of downstream with MEE4 and JNK kinase, finally damage to cells. Studies in kidney diseases showed that in obstructive nephropathy rats model, ASK1 / P38 lightning signaling pathways of renal tubular epithelial cells may be activated and play an important role in the process in kidney inflammation, apoptosis and fibrosis. ASK1 in acute kidney injury caused by ischemiareperfusion model was also activated, blocking ASK1 can reduce the apoptosis of renal tubular epithelial cells and reduce the expression levels of MCP- 1 and improve renal function. In diabetic nephropathy model, and the published research thinks that blocking the activation of ASK1 and phosphorylated P38 lightning, can reduce glomerular damage. Professor FOGO’s prophase study found, in 5/6 nephrectomy of rat model of chronic kidney disease(CKD), double amount of using ACEI/ARB can obviously improve the glomerular sclerosis, but the long-term follow-up did not find the RAS blockers can significantly slow or reverse the progress of kidney disease. We still haven’t found in the published study that RAS blockers used alone can delay the progress of kidney disease. it is necessary to explore multiple combined metheods to slow the progress of CKD.PART 1 Study of ASK 1 inhibitor and Enalapril in 5/6 nephrectomy rat in renal protectionObjective:To compare the renal protection of combined apoptosis signal regulating kinase1 inhibitor(ASK-1I) with angiotensin-converting-enzyme inhibitor(Enalapril) on 5/6 nephrectomy rats.Methods:Adult male Sprague Dawley rats underwent 5/6 nephrectomy(5/6 Nx) at week 0 and open renal biopsy at week 8. Rats were randomized to four groups with equal glomerulosclerosis: control group, ASK1 inhibitor group, Enalapril group and combination group. After four weeks treatment, animals were sacrificed at week 12. Systolic blood pressure(SBP) 、 albumin to creatinine ratio(ACR) 、 serum creatinine and creatinine clearance rate(Ccr) were measured at intervals. At week 8 and 12, we evaluated the degree of glomerulosclerosis.Results: All rats developed hypertension, proteinuria and glomerulosclerosis from week 0 to 8. At 12 th week, BP of Enalapril group was significantly lower than control group(P < 0.05). ACR growth rate of Enalapril group and combination group were both significantly lower than control group(57.8±24.4%, 50.2±27.5%, 244.9±69.4% respectively, P < 0.05). Creatinine growth rate of ASK1 inhibitor group and combination group were both lower than the control group(19.1±17.5%, 22.3±17.7%, 89.8±24.7% respectively, P < 0.05). Simultaneously, Combination group can effectively reverse the glomerular sclerosis(P < 0.05).Conclusion: Combined ASK1 inhibitor with Enalapril can reduce blood pressure, proteinuria and glomerulosclerosis significantly, and can improve renal function.PART2 Study of ASK1 inhibitor combined Enalapril in 5/6 nephrectomy rat on renal protection mechanism Chapter1 The effect of ASK1 inhibitor combined Enalapril on kidney inflammation in 5/6 nephrectomy ratsObjective:To explore the underlying mechanism of oxidative stress approach by combing apoptosis signal regulating kinase inhibitors( ASK1 inhibitor) with angiotensin converting enzyme inhibitors(Enalapril) on 5/6 nephrectomy ratsMethods:Immunohistochemical method to detect each group WT1, ED1 level, Western-blot to detect the level of GAPDH of each group.Results:1. Compared with the control group, Enalapril and ASK1 inhibitor single treatment group were not improved WT1 positive cell density, but the comination treatment group can obviously increase the WT1 positive cell density, the difference was statistically significant(ASK-1I+ Enalapril group(17.04 + 42.31) % vs the control group(30.55 + 25.24) % vs group ASK1 inhibitor(40.01 + 40.01) %, p < 0.05).2. At 12 th week in Enalapril group can obviously reduce glomerular ED1 positive area, a reduction in the macrophages in glomerular infiltration, with ASK-1I group increasing. comparing the two groups, the difference was statistically significant(Enalapril group 0.41 + /- 0.08% vs ASK1 inhibitor 1.12 + /- 0.25%, p < 0.05).3. Compared to the control group, ASK-1I+Enalapril therapy group can obviously decrease the the level of CTGF.Conclusion:The combination therapy of ASK1 inhibitors and Enalapril in 5/6 nephrectomy rat can reduce oxidative stress to protect podocyte, reduce apoptosis, reduce macrophage infiltration and has better renal protective effect.Chapter2 The effect of ASK1 inhibitor combined Enalapril on MAPK-pathway in 5/6 nephrectomy ratObjective: To explore the related mechanism on MAPK-pathway by detecting the PP38/JNK levels, apoptosis cells in glomerular and renal interstitium.Methods:TUNEL method to compare groups of apoptotic cells and Western-blot to detect P38/JNK.Results: Each treatment groups of glomerular apoptosis cells did not decrease respectivly, but the combined treatment group and the ASK1 inhibitor compared with the Enalapril group and control group, have significantly reduced the apoptotic cells of renal tubule interstitial level with obvious statistical significance(ASK1 inhibitor 1. 25 + /- 0.22, ASK1 inhibitor + Enalapril 1.28 + /- 0.2 x 10-5 /(including 2.07 + /- 0.21 m2 vs Cont, Enalapril 2.74 + /- 0.37 x- 5 /(including 10 m2, P < 0.05).Conclusion:The combination therapy of ASK1 inhibitors and Enalapril in 5/6 nephrectomy rat can inhibit MAPK downstream P38 lightning, JNK phosphorylation, reduce apoptosis and finally has better renal protective effect. Chapter3 The expression of TGF-?/Smad-3 with ASK1 inhibitor and Enalapril on 5/6 nephrectomy ratsObjective: To investigate the effect of ASK-1I with Enalapril on renal fibrosis in 5/6 nephrectomy rat.Methods: Western blot to detect every groups of CTGF、PAI-1 and Smad3.Results: 1. Compared to the control group, ASK-1I+ Enalapril therapy group can not decrease the the level of PAI-1 and Smad3. 2. Compared with the control group, Enalapril and ASK-1I single treatment group in the kidney tissues of CTGF expression level has no statistical significance(Enalapril group 0.50 + /- 0.11, ASK-1I set of 0.49 + /- 0.08 vs. 0.64 + /- 0.13 in the control group, p > 0.05), and the combination group in renal tissue CTGF expression level was lower,the difference was statistically significant(Enalapril + 0.32 + /- 0.04 ASK-1I group vs control group, p < 0.05).Conclusion:Combination group showed reduced expression of fibrosis factors by inhibiting the TGF-?/Smad3 pathway and PAI-1 to protect podcyte to delay the fibrosis of CKDSummary:from all above research, we found that the combination therapy of ASK1 inhibitors and Enalapril in 5/6 nephrectomy rat can inhibit MAPK downstream P38 lightning, JNK phosphorylation and oxidative stress, reduce apoptosis and macrophage infiltration and podocyte damage, decrease the expression of firbrosis factors,which in turn, reverse levels of glomerulosclerosis and has better renal protective effect.