| Objective:1. To evaluate the effectiveness and safety of spironolactone in thetreatment process of diabetic nephropathy through systematic reviews.2. To builddiabetic nephropathy rat model by combination with different diet and left-nephrectomyand intraperitoneal injection of STZ.3. To explore the relationship between aldosteroneand renal aging mechanism in diabetic nephropathy and the value of spironolactoneintervention on it. Methods: Part one: According to Cochrane Systematic Review,literature was retrieved in database including Cochrane Library, MEDLINE(1974-Dec.2012), Embase (1974-Dec.2012), CNKI (1989-Dec.2012), and Wanfangdata-base (1989-Dec.2012); Manual retrievals including related conference proceedingsand all references of retrieved literatures, were brought into all randomized orquasi-randomized control trial concerning treatment of DN by spironolactone, andevaluated methodology quality of above controlled clinical trials. Using unified qualityevaluation independently on each article in accordance with its standard literature withrandom allocation hidden, blinded, intentional analysis and follow-up four sorts ofquality evaluation, finally summarizes the evaluation data. Part two:36clean grade maleSD rats were selected.After excision of the left kidney, different diet which was high saltand fat feed (group A), high sugar and high fat feed (group B) and normal feed group (C)were given.When insulin resistance was induced, they were intraperitoneal injected withstreptozotocin. After72hours blood sugar was tested, if it was over16.7mmol/L, thediabetic rat models were established successfully, continue to offer the feed after12weeks.Every4weeks blood pressure and blood sugar were measured. After16weeks,rats were sacrificed to collect urine, blood plasma, kidney tissue for detection of24hurinary microalbuminuria, creatinine, aldosterone and renal pathological changes. Part three:48SPF SD male rats were provided by the animal experiment center of XinJiangMedical University, weigh ted about180±20g, after adapted for1week, the left kidneysof them were removed, then they were fed with high fat and high sugar feed to induceinsulin resistance. After4weeks, these rats were given an intraperitoneal injection of25mg/kg STZ. After72hours blood sugar was tested, if it was over16.7mmol/L, thediabetic rat models were established successfully. Then those rats were divided into4groups, group a, b, c and d, respectively given intervention of water, spironolactone,benazapril, benazapril+spironolactone. Every4weeks blood pressure and blood sugarwere measured. After16weeks, rats were sacrificed to collect urine, blood plasma,kidney tissue for detection of24h urinary microalbuminuria, creatinine, aldosterone andrenal pathological changes. Part Four NRK52E was divided into five groups, whichwere a, b, c, d and e. Cells in group a grow only in high glucose culture. Group B wasinterfered with aldosterone, while the other groups treated by different doses ofspironolactone before aldosterone was added into high glucose culture. After theseintervention for72hours, cell aging will be tested by β-galactosidase while mRNA andprotein expression of klotho, P53, p21and β-actin will be detected by qPCR and WesternBlot. Results: Part one1. The difference between the effects of spironolactone andplacebo on the effectiveness in DN patients.1) The evaluation of the quality of theincluded literature showed that all studies were randomized, but none described whetherblinded assessment and allocation concealment were used. In the four literature, therewere not patients lost to follow-up, but there were some discontinued the researchbecause of hyperkalemia, but the ratio bellows10%. Four studies were all class B. Thus,the quality of the literature was not high, but the baselines were comparable without theneed for sensitivity analysis.2) A total of162relevant articles were retrieved using thepreviously mentioned search strategy and data collection methods. The studies thatcompared the effects of spironolactone and placebo on DN patients were screened, andthe17studies that met the criteria were all RCTs. After papers were read in their entiretyand further screened according to the inclusion criteria and data integrity, four RCTs、thetotal of196patients were included in this analysis. The included studies reported thechanges of the patients’ urinary albumin-creatinin ratio (ACR), systolic and diastolicblood pressure, serum creatinine levels and hyperkalemia after treatment.3) The resultsof literature heterogeneity analysis and meta-analysis revealed literature heterogeneity(Q=19.95, P＜0.001), and thus, the random effects model was adopted for furtheranalysis. The post-treatment ACR data were merged into-1.27[95％CI (-2.10,-0.45)], The results of the Z-test were Z=3.03and P=0.002(P＜0.01), suggesting a significantdifference. Thus, there was a significant difference between the effects of the two classesof drugs on ACR, and spironolactone was superior to placebo. The effects ofspironolactone on renal function, four experiments respectively from the angle of theglomerular filtration rate and creatinine levels were analyzed, the conclusion is different.The antihypertensive effects of spironolactone were not superior to the otherhypertensive drugs in lowering both systolic blood pressure-1.23[95％CI (-6.32,3.85)],P=0.71(P＞0.05) and diastolic blood pressure-0.44[95％CI (-3.84,4.71)], P=0.84(P＞0.05).4) As to side-effect, two literature refers to hyperkalemia and results of literatureheterogeneity analysis and meta-analysis revealed literature homogeneity (Q=0.06,P=0.81＞0.05), and thus, the fixed effects model was adopted for further analysis. Theresults suggested that the relative ratio of hyperkalemia in spironolactone group was6.4times than placebo group (RR=6.40,95%CI (2.3,20.2), P=0.002＜0.01)。From thesearticles it can be concluded that spironolactone might decrease ACR, but it could scarcelyplay an important role on kidney function and blood pressure. Part two Different groupwere all built successful diabetic nephropathy rat model.After12weeks, the bloodpressure of each group was152.00±11.06mmHg,128.10±11.73mmHg,114.43±14.67mmHg (F=14.764, P＜0.001) respectively. urine microalbumin of24hoursof each group was3.67±1.26,1.51±0.62,0.22±0.20(F=12.666, P＜0.001). Theglomerular sclerosis and renal tubular damage index of A and B group were higher thanC group (P＜0.05). Group A had more protein cast and its walls of renal smallartery-arteriole were more thicken than group B and C. Part three The main effect ofspironolactone group reducing urinary protein in treatment of DN rat models wasF=8.418, P＜0.05.The main effect of benazepril reducing urinary protein was F=11.71, P＜0.01,whie the interaction effect of spironolactone combined benazepril group wasF=6.569, P＜0.05.In terms of increasing blood albumin, the main effect ofspironolactone group was F=13.419, P＜0.01and the main effect of benazepril groupwas F=5.255, P＜0.05, the interaction effect of combined group was F=9.295, P＜0.01,But for blood glucose, serum creatinine, systolic blood pressure, kidney index, nosignificant statistical differences among these groups were found(P＞0.05). Theglomerular sclerosis and renal tubular damage index of spironolactone and benazeprilgroup were improved significantly than the control group (P＜0.05). The expression ofKLotho gene and protein has the significant difference among different groups,spironolactone and benazepril can both raise the level of KLotho. Part Four (1) After different exposure as above for72hours, the senescent cells proportion of group a, b, c, dand e were (47.9±4.7)%,(49.0±5.2)%,(38.2±4.1)%,(39.7±3.8)%,(42.1±3.9)%,respectively. Comparing the aging cells positive rate of each group in72hour, thechi-square was45.850. The aging cells ratio increased despending the time. Comparedwith group a and b, the ratio of group c, d and e decreased followed by different doses ofspironolactone (P＜0.01).(2) Comparedwith group a, the Klotho mRNA expression ofgroup b decreased while the p53mRNA and p21mRNA increased obviously (P＜0.05).The Klotho mRNA levels of each group were1.13±0.09,0.85±0.02,1.42±0.11,1.37±0.08and1.14±0.06, respectively (F=8.134, P＜0.01). The expression of p53mRNA of each group were0.62±0.13,0.93±0.15,0.45±0.06,0.51±0.09and0.61±0.09respectively (F=9.629, P＜0.05).As p21mRNA, they were0.74±0.06,1.05±0.05,0.42±0.02,0.61±0.07,0.72±0.03, respectively (F=5.450, P＜0.05).(3) Comparison withgroup b, Klotho of group c, d and e, Whatever mRNA or protein expression, increased atdifferent level, while p53, p21had the opposite results. Klotho protein expression hadthenegative correlation with p53and p21(r=-0.744, r=-0.627, P＜0.05).Conclusion:1.Spironolactone in treatment of diabetic nephropathy can lower the urine protein of DNpatients, but whether it can improve renal function has not been concluded.Because thesample size is too small to evaluate completely the effectiveness and safety ofspironolactone in treating DN. In the future, its curative effect needs to be confirmed byenlarging sample size and high-quality randomized control trials. At the same time it alsoneeds to have more basic and clinical trials to study further the role of aldosterone indiabetic nephropathy mechanisms and intervention mechanism of spironolactone.2.Spironolactone can reduce proteinuria of DN rats without relying on its antihypertensiveeffect, and has synergistic effect with benazepril.It can also decrease renal fibrosis, whichmay related to its function of up-regulating of anti-aging factor Klotho. The expression ofKlotho and TGFβ1presents negative correlation, it showed that Klotho’s antifibrosiseffect might be related to its inhibition of TGFβ1.3. Aldosterone can accelerate renaltubular epithelial cell aging in high sugar environment.However, spironolactone, a kindofmineralcorticoid receptor antagonist can intervene in it with a dose-dependent manner.Its interventional mechanism may be through MR/Klotho/P53/p21signaling pathways. |
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