Protective Effects Of Beraprost Sodium On Mouse Podocytes Apoptosis Induced By Angiotensin Ⅱ And Its Potential Mechanism

BACKGROUND:Diabetic kidney disease (DKD) is one of the most common microvascular complications of diabetes mellitus (DM). The prevalence of DM increased significantly throughout the world in the past decades. According to the International Diabetes Federation (IDF) in 2010,285 million adults suffer from diabetes, and they predicted the number would climb to 439 million in 2030. It’s worth noting that the count in developing country grows faster than that in developed country. In 2011, it was reported that DKD is the cause of 50% of all cases of end stage renal disease (ESRD) in the United States, as well as the leading cause of dialysis and kidney transplantation. In China, with our rapid economic development, individuals’dietary habit and lifestyle have changed greatly, resulting in the great increase of diabetes prevalence. As one of the major microvascular complications, DKD prevalence was also increased rapidly due to the expanding size of the diabetes population, seriously affecting the quality of life of a large amount of DM patients.DKD was defined by the National Kidney Foundation-Kidney Disease Outcomes Quality Initiative(NKF-KDOQI) as diabetes with albuminuria(ratio of albumin to creatinine)>30mg/g), or with impaired glomerular filtration rate(<60ml/min/1.73m(2)). The pathogenesis of DKD is quite complicated and not clear still, involving several factors including the effect of genetic susceptibility, high glucose. kidney hemodynamic changes, increased advanced glycation end products(AGEs), polyol pathway activation, reactive oxygen species(ROS), activation of the protein kinase C signaling pathway. It is believed that proteinuria is one of the earliest detectable clinical marker of diabetes-induced kidney injury. As a part of glomerular filtration barrier, podocyte gradually becomes the focus of research related to DKD. It has been demonstrated that podocytopathy plays an important role in the progression of DKD. Podocytes undergo apoptosis in the early stage of DKD, indicating podocyte apoptosis one of the earliest incidence of DKD. Therefore, protecting podocytes from apoptosis has an important value on the control and therapy of DKD.In diabetic condition, production of angiontensin II increases in an abnormal way, resulting from activation of rennin-angiotensin-aldosterone system, further contributing to progressive kidney damage. Several researches indicated that AngⅡ can promote apoptosis in time-dependent and doze-dependent manners. It was also demonstrated that AngⅡ could activate p38MAPK signaling pathway to promote apoptosis in podocytes. Foreign researchers found in experimental rats that AngⅡ infusion contributed to hypertension and proteinuria, correlated with the foot process fusion, slit pore narrowed and even podocyte apoptosis.Mitogen active protein kinase (MAPK) consists of p38MAPK, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK)/ stress-activated protein kinase (SAPK), participating in cell growth, differentiation and apoptosis. More and more researches indicated that p38MAPK signaling pathway is interrelated to DKD. In diabetic condition, MAPK can be activated by inflammatory factors and phosphorylate their downstream transcription factor, participating in the regulation of target genes expression. It was demonstrated in foreign research that aldosterone could activate p38MAPK and promote apoptosis in podocytes. It is believed that p38MAPK plays an important role in podocyte apoptosis.Beraprost sodium (BPS) is a stable oral analogue of prostaglandin Ⅰ(2) (PGI2), playing a role in vasodilatation, antiplatelet aggregation and anti-inflammation. Researchers found that BPS can ameliorate OLETF rat proteinuria by inhabiting AngⅡ-induced glomerular efferent arteriolar vasoconstriction and the production of inflammatory factors, such as TGF-β1, tumor necrosis factor α(TNF-α), implying its protection for kidney. In vivo study, BPS can protect diabetic myocardial cells against apoptosis through the inhibition of p38MAPK signaling pathway. However, it remains unknown that whether BPS can restore podocyte apoptosis or not.In our study, we investigated effect of BPS on apoptosis in podocyte and its underlying mechanism, aiming to find a new therapy to DKD.Chapter oneEffects of Beraprost sodium on apoptosis in mouse podocyte induced by Angiotensin ⅡObjectives:1.To observe the biological characteristics of cultured mouse podocyte in different groups, and master the basic process of cell culture.2.To explore the effect of Beraprost Sodium(BPS) on cell viability and apoptosis in podocytes induced by Angiotensin Ⅱ (AngⅡ).3. To expore the effect of BPS on expression of cleaved caspase3 in podocytes induced by AngⅡ.Methods:1. Cell culture:Undifferentiated podocyte were cultured in 5% CO2,33℃ incubator with RPMI 1640 containing 10% fetal bovine serum (FBS),100 units/ml Pen/Strep,10 units/ml y-interferon(Y-IFN). Podocytes were cultured in 5% CO2, 37℃ incubator with RPMI 1640 containing 10% FBS,100 units/ml Pen/Strep to differentiate for 2 weeks.2. Podocytes were randomly divided into 5 groups, according to different intervention.Control (Con) group, RPMI 1640Angiotensin (Ang) Ⅱ group, the AngⅡ concentration was 1×10-7mol/LAngll combined with different concentrations of BPS groups, the BPS concentrations were 1 μmol/L(uM),μM and 5μM, respectively.3. CCK-8 assay to detect cell viability in podocytes with different interventions.4. Detect apoptosis in podocyte with Annexin V-FITC staining and flow cytometry.5. Relative expressions of cleaved caspase-3 were detected by Western blots.6. The SPSS version 19.0 software was used to analyze the data.All values are expressed as the means±standard deviation. Differences between groups were examined for statistical significance using one-way ANOVA followed by Least-significant difference test. A P value<0.05 was considered statistically significant.Results:1. Cell viability of MPC5 cells by CCK-8 assay. Compared with control group, O.D. value of MPC5 cells significantly decreased in Angll group after stimulating for 24h and 48h, respectively. Compared with Angll group, O.D. value of MPC5 cells increased significantly in different concentrations of BPS groups after 24h and 48h culture, respectively.2. Podocyte apoptosis by Annexin V-FITC assay. Compared with control group, apoptotic rate in Angll group was significantly higher. Compared with Angll group, all the apoptotic rates in different concentrations of BPS groups were significantly lower.3.Cleaved caspase3 expression by Western blots. Compared with control group, Angll significantly increased level of cleaved caspase3 in podocytes, while BPS decreased level of cleaved caspase3.Conclusion:BPS can effectively increase cell viability and attenuate apoptosis in podocytes induced by Angll, modulating relative expression of cleaved caspase3.Chapter TwoBeraprost sodium protects podocytes from apoptosis induced by Angiotensin Ⅱ via activating p38MAPK signaling pathwayObjectives:To explore effects of SB203580 and BPS on apoptosis in podocytes induced by Angll and its underlying mechanism.Methods:1. Podocytes were grouped according to different interventions: Control (Con), Angiotensin Ⅱ (AngⅡ), AngII+5μMBPS (5μMBPS), AngII+SB203580(SB)2. Expression of p38MAPK phosphorylated protein are analysed by Western blots.3. CCK-8 assay to detect cell viability in podocytes in different groups.4. Annexin V-FITC assay and flow cytometry to detect apoptosis in podocytes in different groups.5. Expression of cleaved caspase3 protein are analysed by Western blots.6. The SPSS version 19.0 software was used to analyze the data.All values are expressed as the means±standard deviation. Differences between groups were examined for statistical significance using one-way ANOVA followed by Least-significant difference test. A P value<0.05 was considered statistically significant.Results:1. Expression of p38MAPK phosphorylated protein. Little phosphorylated p38MAPK protein expressed in AngⅡ-induced podocytes after Omin stimulation. After 15min stimulation, phosphorylated p38MAPK protein expressed the most among all the AngⅡ groups. Therefore, we chose the 15min time point to step to the next experiments. After 15min stimulation, expression of phosphorylated p38MAPK protein in Angll group increased significantly compared with the control group(P=0.002). Compared with Angll group, expression of phosphorylated p38MAPK protein in all BPS group(except 1μMBPS group) decreased significantly (2uMBPS:P=0.045; 5μMBPS:?=0.017). Expression of phosphorylated p38MAPK protein in SB group significantly decreased than the other groups (except the normal control group).2. Cell viability of podocytes in different groups by CCK-8 assay. Compared with Angll group, O.D. value of podocytes in SB203580 group were significantly decreased after 24h and 48h culture, respectively(.P<0.001).3. Podocyte apoptosis by Annexin V-FITC assay. Compared with Angll group, apoptotic rate in SB203580 group was significantly lower(P=0.005).4. Cleaved caspase3 expression by Western blots. Compared with AngⅡ group, SB203580 significantly decreased level of cleaved caspase3 in podocytes(P=0.008).Conclusion:BPS can effectively protect mouse podocytes from apoptosis partially via inhabiting p38MAPK signaling pathway.