替米沙坦对糖尿病肾脏疾病患者尿足细胞排泄的影响

目的 探讨糖尿病肾脏疾病（diabetic kidney disease,DKD）患者尿足细胞排泄特征并研究替米沙坦对其干预作用.方法 前瞻性研究入选72例2型DKD患者,依据尿白蛋白与尿肌酐比值（albumin to creatinine ratio,ACR）将其分为2组.DKD 1组,30 μg/mg≤ACR＜300 μg/mg; DKD2组,ACR≥300 μg/mg,分别给予替米沙坦40 mg/d,治疗12周.选取20名健康志愿者作为正常对照组.采用免疫酶细胞化学镜检法检测单克隆抗体podocalyxin标记的尿足细胞,比较替米沙坦治疗前、后DKD患者与健康人群尿足细胞数的变化.结果 2组DKD患者收缩压、舒张压、空腹血糖、糖化血红蛋白、血肌酐、ACR和尿足细胞数差异均显著高于正常对照组（P＜0.05）,且DKD 2组患者血肌酐、ACR和尿足细胞数高于DKD 1组（P＜0.05）.替米沙坦治疗12周后,DKD 1组患者ACR下降,尿足细胞数减少;DKD 2组患者ACR和尿足细胞数无明显变化.结论 尿足细胞检测可预测早期DKD,替米沙坦可能通过保护足细胞延缓DKD进展.     

自噬相关分子Beclin1和LC3在糖尿病肾脏疾病患者血清中的表达及意义

目的探究自噬相关分子Beclin1、LC3在2型糖尿病肾脏疾病(DKD)患者血清中的表达水平及意义。方法纳入2016年1月至2017年6月期间于宜昌市第二人民医院就诊的2型DKD患者123例,按照尿白蛋白/肌酐比值(ACR)进行分组,分为尿白蛋白正常组(ACR30 mg/g)、微量白蛋白尿组(30 mg/gACR300 mg/g)、大量白蛋白尿组(ACR300 mg/g)。同时纳入48例体检健康者作为对照组。收集4组研究对象的一般资料,并且采集静脉血,检测ACR、尿微量白蛋白(UAlb)、Beclin1、LC3的水平。对各指标进行组间差异比较和相关性分析。结果对照组与尿白蛋白正常组的ACR比较,无统计学差异(P0.05);微量白蛋白尿组、大量白蛋白尿组ACR与尿白蛋白正常组比较显著升高,有统计学差异(P0.05)。4组间的UAlb比较,差异均有统计学意义(P0.05)。在Beclin1和LC3水平方面,4组间两两比较显示,差异均具有显著性(P0.05),其中对照组最高,大量白蛋白尿组最低。在相关性分析方面,不同组别的DKD患者血清中Beclin1与ACR、UAlb均呈反比,LC3与ACR、UAlb均呈反比。结论在DKD的发病过程中存在细胞自噬反应,自噬反应的增强可能会对DKD蛋白尿起到一定的抑制作用。     

尿NGAL和血、尿胱抑素C对早期糖尿病肾脏疾病诊断价值的研究

目的:探讨中性粒细胞明胶酶相关脂质运载蛋白(NGAL)和胱抑素C(Cys C)对糖尿病肾脏疾病(DKD)的早期诊断价值和临床意义。方法:选取2型糖尿病(T2DM)患者60例,根据尿白蛋白/肌酐比(ACR)分为3组,正常白蛋白尿(NA)组20例,ACR≤30 mg/g;微量白蛋白尿(MA)组20例,30 mg/gACR300 mg/g;大量白蛋白尿(CA)组20例,ACR≥300 mg/g;选择同期的健康查体者20例作为对照组(NC)。留取晨尿和空腹血液标本,用ELISA法检测尿NGAL、血和尿Cys C水平,分析其与肾小球滤过率(e GFR)之间的相关关系,应用受试者操作特征(ROC)曲线评价其对DKD早期诊断的敏感性和特异性。结果:(1)DKD各组的尿NGAL、血和尿Cys C水平,除NA组与NC组比较血Cys C差异无统计学意义(P0.05)、NA组与MA组比较尿Cys C差异无统计学意义(P0.05)外,其他各组间差异均有统计学意义(P0.05)。(2)糖尿病患者尿NGAL、血和尿Cys C水平与e GFR均呈负相关(r值分别为-0.82,-0.787,-0.716,P0.05)。(3)DKD患者尿NGAL、血和尿Cys C的ROC曲线下面积分别为0.821,0.79和0.734。结论:DKD患者尿NGAL与血和尿Cys C,与e GFR均具有相关性,且尿NGAL较血和尿Cys C更为敏感,尿NGAL可作为早期诊断DKD的敏感指标。     

胰岛素样生长因子1受体抑制剂可减轻糖尿病肾病小鼠肾小管病变

目的探讨胰岛素样生长因子1受体(IGF-1R)抑制剂在缓解糖尿病肾病(DKD)小鼠肾小管病变中的作用。方法C57BL/6J雄性小鼠被随机分为正常对照组(n=10)和DKD模型组(n=30)。用单次腹腔注射链脲佐菌素(STZ,150 mg/kg)的方法建立糖尿病肾病模型。建模成功后,DKD模型组小鼠被随机分为DKD组(n=10)、贝那普利组(n=10)和IGF-1R抑制剂组(n=10)。IGF-1R抑制剂组给予30 mg·kg-1·d-1 IGF-1R抑制剂灌胃;贝那普利组给予30 mg·kg-1·d-1贝那普利灌胃;正常对照组和DKD组分别给予等量生理盐水灌胃。8周后处死小鼠,记录小鼠体重、肾重。收集小鼠血清、尿液和肾脏样本。自动生化仪上测定血糖、尿白蛋白等生化指标,计算尿白蛋白排泄率。HE、PAS染色法观察小鼠肾脏病理改变。免疫组化、Western印迹法测定肾组织磷酸化(p)IGF-1R的表达。结果与正常对照组比较,DKD组小鼠的随机血糖、肾重/体重、尿白蛋白排泄率明显升高(均P<0.01)。DKD组小鼠肾脏病理表现为肾小球增大,肾小管管腔狭窄,肾小管肿胀、萎缩;近端小管上皮(PTE)细胞数目减少,肾小管损伤评分增加,平均肾小球体积增大,肾组织pIGF-1R表达增加(均P<0.05)。与DKD组相比,IGF-1R抑制剂组和贝那普利组小鼠尿白蛋白排泄率显著降低(P<0.01),肾脏病理改变减轻,且IGF-1R抑制剂组的作用效果更加显著。与DKD组相比,IGF-1R抑制剂组肾组织pIGF-1R蛋白表达减少(P<0.05);与贝那普利组相比,IGF-1R抑制剂组肾组织pIGF-1R蛋白表达减少(P<0.05)。结论IGF-1R抑制剂能减轻DKD小鼠肾小管病变,且效果优于贝那普利。     

血清SAA、CysC、ACR及IL-19在2型糖尿病肾脏病诊断中的应用

目的分析血清淀粉样蛋白A(serum amyloid A,SAA)、胱抑素C(Cystatin C,CysC)、尿微量白蛋白与肌酐比值(albumin to creatinine ratio, ACR)及白细胞介素-19(interleukin, IL-19)在2型糖尿病肾脏病(diabetic kidney disease, DKD)诊断中的应用价值。方法本研究于2017年10月至2020年10月选取院内接受治疗的108例2型糖尿病患者作为研究对象,根据患者是否合并DKD将其分为DKD组和糖尿病组,同期选取于院内接受体检的50例相似年龄段健康体检者作为对照组,对比各组的SAA、CysC、ACR及IL-19的表达水平。结果 3组的SAA、CysC、ACR及IL-19比较,差异有统计学意义(P0.05)。组间比较显示,DKD组和糖尿病患者的SAA、CysC、ACR及IL-19表达水平均高于对照组(P0.05),且DKD组各项血清学指标均高于糖尿病组(P0.05);DKD组不同肾损伤分期患者的血清学指标比较,差异有统计学意义(P0.05)。随着患者肾损伤分期的升高,患者的SAA、CysC、ACR及IL-19表达水平也逐渐升高(P0.05);通过对各血清学指标之间的相关性分析发现,各指标之间呈显著的正相关(P0.05);SAA、CysC、ACR及IL-19单独检测的诊断准确率分别为65.48%、76.58%、73.81%和69.62%,均明显低于联合检测的92.41%(P0.05)。结论血清SAA、CysC、ACR及IL-19均可以有效实现对2型DKD的早期诊断,与患者的肾损害程度密切相关,且各指标的联合检测有较高的诊断准确率。     

阿霉素诱导的足细胞转分化中β-catenin及TGF-β的变化及活性维生素D_3的干预作用

目的:探讨阿霉素诱导的足细胞转分化(EMT)中β-catenin及TGF-β的变化及活性维生素D3干预作用。方法:以体外培养的小鼠足细胞系为研究对象,分6组进行实验:正常组(C组)、阿霉素组(即模型组,ADR组)、低剂量活性维生素D3组(LVD组)、高剂量活性维生素D3组(HVD组)、缬沙坦组(ARB组)、缬沙坦+高剂量活性维生素D3组(AHVD组)。各实验组干预24 h、48 h时通过RT-PCR检测β-catenin、TGF-βmRNA的表达,Western Bolt检测β-catenin蛋白的表达。结果:用药物干预24 h及48 h时,与正常对照组相比,ADR组足细胞β-catenin mRNA及蛋白表达均上调(P<0.01);与ADR组相比,四个药物干预组(ARB组、LVD组、HVD组、AHVD组)β-catenin mRNA及蛋白表达均下调(P<0.05)。24 h时与正常对照组相比,ADR组TGF-βmRNA表达上调(P<0.01);与ADR组相比,四个药物干预组TGF-βmRNA表达明显下调(P<0.01)。48 h时与正常对照组相比,ADR组TGF-βmRNA表达上调(P<0.01);与ADR组相比,HVD组与AHVD组TGF-βmRNA表达明显下调(P<0.01),而LVD组及ARB组无显著下降(P>0.05)。结论:活性维生素D3通过抑制信号转导通路中β-catenin、TGF-β的表达而抑制足细胞发生EMT。     

microRNA-215在糖尿病肾病小鼠肾组织中的表达及意义

目的 探讨microRNA-215( miR-215)在糖尿病肾病(DN)小鼠肾组织中的表达变化规律及在DN发病中的作用.方法 选择4周龄的2型糖尿病肾病db/db小鼠(实验组)和db/m小鼠(对照组),采用实时荧光定量PCR法动态检测8、12及16周龄时肾组织miR-215的表达变化;实时荧光定量PCR和Western印迹法、免疫组化法测定连环蛋白β互动蛋白1( CTNNBIP1)的mRNA及蛋白的表达;双荧光素酶报告法确证miR-215对CTNNBIP1表达的直接调控作用.结果 (1)随着周龄的增加,db/db小鼠肾小球逐渐肥大、节段性系膜细胞增生和系膜基质积聚.(2)与同周龄的db/m小鼠比较,8、12及16周龄的db/db小鼠体质量(BW)、血糖( Glu)及24 h尿白蛋白排泄量(UAE)均显著增加(均P＜0.05).(3)随着周龄的增加,db/db小鼠肾脏组织miR-215表达显著高于同周龄的db/m小鼠(P＜0.05).(4)与同周龄的db/m小鼠比较,db/db小鼠肾脏组织CTNNBIP1 mRNA和蛋白均显著降低(均P＜0.05).(5)应用双荧光素酶报告法证实,miR-215可显著抑制CTNNBIP1的表达(P＜0.01).结论 miR-215表达上调可能通过抑制CTNNBIP1的表达参与DN的发生、发展.     

阿利吉仑对2型糖尿病db/db小鼠肾脏损伤的保护作用

目的 观察肾素抑制剂阿利吉仑对2型糖尿病db/db小鼠肾脏损伤的保护作用。 方法 8周龄db/db和db/m小鼠行单侧肾切除,16周进入实验,分为4组：db/m小鼠对照组（db/m组）、db/db糖尿病小鼠对照组（db/db组）、db/db糖尿病小鼠阿利吉仑3 mg&#8226;kg-1&#8226;d-1治疗组（db/db+A3组）和db/db糖尿病小鼠阿利吉仑25 mg&#8226;kg-1&#8226;d-1治疗组(db/db+A25组)。阿利吉仑溶于磷酸盐缓冲液（PBS,350 mg/L）皮下泵入（0.25 μl/h）给药,疗程４周。治疗前后检测体质量、血糖、糖化血红蛋白、尿蛋白量、血压水平;PAS染色观察肾脏组织学变化;ELISA法检测肾皮质转化生长因子β1（TGF-β1）和纤溶酶原激活抑制因子1（PAI-1）含量;间接免疫荧光检测肾小球Ⅳ型胶原（ColⅣ）和纤连蛋白（FN）表达;实时定量PCR检测TGF-β1、PAI-1、ColⅣ、FN和肾素mRNA表达;放射免疫法检测肾皮质肾素活性和血管紧张素Ⅱ（AngⅡ）水平。 结果 与db/m组小鼠比较,db/db组小鼠有大量蛋白尿,肾小球细胞外基质沉积增加,TGF-β1、PAI-1、ColⅣ和FN蛋白及mRNA表达增加,同时肾皮质肾素mRNA、肾素活性和AngⅡ水平增高（均P < 0.05）。阿利吉仑25 mg&#8226;kg-1&#8226;d-1治疗在没有影响血压情况下,显著减少db/db小鼠24 h尿蛋白量,减少肾小球细胞外基质沉积,减少TGF-β1、PAI-1、ColⅣ、FN蛋白和mRNA表达,同时降低肾皮质肾素活性和AngⅡ水平（均P < 0.05）。 结论 阿利吉仑对2型糖尿病db/db小鼠肾脏损伤有保护作用。     

糖尿病肾病患者血清Beclin1、ATG7与蛋白尿相关性研究

目的:探讨糖尿病肾病(DKD)患者血清Beclin1、ATG7水平与蛋白尿的相关性.方法:收集2018年12月—2019年10月在本院门诊或住院部诊断为DKD共108例患者的临床资料,按ACR分为正常白蛋白尿组、微量白蛋白尿组和大量白蛋白尿组.纳入同期我院体检健康者共30例为对照组.检测血清Beclin1、ATG7水平...     

血清Klotho水平与早期糖尿病肾病足细胞损伤的相关研究

目的:观察早期糖尿病肾病(DN)患者血清Klotho水平与足细胞损伤的相关性。方法:选取2018年01月～2018年10月入住本院明确诊断为2型糖尿病的患者60例,依据其尿白蛋白肌酐比值(UACR)分为正常蛋白尿组、微量白蛋白尿组,每组30例,同时选取同期健康体检者30例作为对照组,采用RT-PCR法检测各组血清Klotho、Nephrin和Podocin mRNA水平,分析血清Klotho与足细胞损伤的相关性。结果:正常蛋白尿组和微量白蛋白尿组患者血清Klotho、Nephrin及Podocin mRNA水平均低于对照组,差异均有统计学意义(均P 0. 01);而与正常蛋白尿组比较,微量白蛋白尿组患者血清Klotho、Nephrin、Podocin mRNA水平也均明显降低,差异均有统计学意义(分别为P 0. 05、P 0. 05和P 0. 01);进一步Pearson相关分析显示,血清Klotho水平与Nephrin、Podocin均呈正相关;多元回归分析显示,早期DN患者血清Klotho水平与Nephrin关系密切。结论:早期DN患者血清Klotho水平降低可能参与了足细胞损伤。     

Bufalin alleviates adriamycin-induced podocyte injury by up-regulating the expression of vitamin D receptor

Objective To investigate the role of vitamin D receptor (VDR) in the protection of bufalin on podocyte injury induced by adriamycin (ADR). Methods (1) In vitro: the toxic effect of different concentrations of bufalin (10-9, 10-8, 10-7, 10-6 mol/L) on podocyte was evaluated by lactate dehydrogenase (LDH) test;Annexin V-FITC and RT-PCR were utilized for podocyte apoptosis and VDR mRNA level respectively. Western blotting was used to analyze the protein expression of VDR and nephrin. SiRNA intervene was also applied to evaluate the role of VDR in bufalin's protective effect on podocyte injury induced by ADR. (2) In vitro: 24 SD rats were randomly divided into three groups: control group, ADR group and ADR+bufalin group. TUNEL assay was applied to detect the apoptosis of podocytes in the kidney. Immunofluorescence and transmission electron microscope (TEM) were applied to analyze the expression of VDR and the ultrastructure of the glomerulus. Results Bufalin concentration lower than 10-7 mol/L had no toxicity on normal podocyte. Bufalin reduced the urinary protein excretion (P＜0.05), alleviated the removal of podocyte foot processes and attenuated the changes in nephrin expression in the glomerulus of the adriamycin (ADR) rats (P＜0.05). Bufalin notably inhibited the down-regulation of VDR in protein levels on the glomerulus of the ADR rats. Additionally, bufalin inhibited the down-regulation of VDR in both mRNA levels and protein levels (P＜0.05）, nephrin protein expression (P＜0.05), and apoptosis induced by ADR in cultured podocytes. Additionally, VDR specific siRNA intervene abolished the protective effect of bufalin in ADR-induced podocyte injury. Conclusion Bufalin can alleviate ADR-induced podocyte injury via enhancing VDR expression.     

Long-term outcomes of peritoneal dialysis patients with diabetes as a comorbid condition

Objective To investigate the long-term outcomes of peritoneal dialysis (PD) patients with diabetes as a comorbid condition. Methods All diabetic patients who commenced PD between January 1, 1995 and June 30, 2012 at Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine were included in the present study. Patients were divided into diabetic kidney disease group (DKD group) and non-diabetic kidney disease group (NDKD group) according to their diagnosis of primary renal disease at the initiation of PD. They were followed until death, cessation of PD, transferred to other centers or to the end of study (June 30, 2013). Outcomes were analyzed by Kaplan-Meier method. Cox proportional hazards models were utilized to determine the predictors of outcomes. Results A total of 163 diabetic patients were enrolled in the study. Compared with patients in DKD group, patients in NDKD group had a significantly lower fasting plasma glucose, a higher serum C-reactive protein level, a higher normalized protein nitrogen appearance, a lower dialysate glucose exposure, a lower peritoneal creatinine clearance and were treated with lower dialysate dose (all P＜0.05). Kaplan-Meier analysis showed that patients in NDKD group had a worse patient survive compared to those in DKD group (log rank Chi-square=4.830, P=0.028). Patients in NDKD group had a marginally shorter peritonitis-free period (log rank Chi-square=3.297, P=0.069), however, there was no significant difference in technique survival between these two groups. Multivariate Cox regression analysis showed that older age (HR 1.047, 95%CI 1.022~1.073, P＜0.001) and cardiovascular disease comorbidity (HR 2.200, 95%CI 0.1.269~3.814, P=0.005) and diabetes as a comorbid condition (HR 1.806, 95%CI 1.003~3.158, P=0.038) were the independent predictors for increased mortality. While higher serum C-reactive protein level (HR 1.023, 95% CI 1.008~1.036, P=0.003) was the independent predictor for shorter peritonitis-free period. Conclusion PD patients with diabetes as a comorbid condition had a higher mortality compared to those with diabetic kidney disease, and closer monitoring and extra attention in the former subgroup of patients are therefore warranted.     

Inhibition of cyclooxygenase-2 expression contributes to podocyte injury in streptozotocin-induced diabetic rats

Objective To investigate the role of cyclooxygenase-2 (COX-2) in podocyte injury in diabetic rats mediated by the disruption of low-density lipoprotein receptor (LDLr) pathway. Methods Eight-week old male Sprague-Dawley (SD) rats were treated for 12 weeks by dividing into three groups: control rats, streptozotocin (STZ) induced diabetic rats (DM), and diabetic rats treated with aspirin (DM+Aspirin). The plasma lipid profile was checked by clinical biochemistry assay. The ratio of urinary microalbumin to creatinine (ACR) was detected by enzyme-linked immunosorbent assay. Intracellular lipid accumulation was evaluated by Oil Red O staining and a free cholesterol quantitative assay. The glomerular podocyte injury and the expression of molecules related with LDLr pathway were evaluated by electron microscope, immunohistochemical staining, immunofluorescent staining, and Western blotting. Results There were increased levels of urinary ACR (P＜0.01) and podocyte injury(P＜0.01) in DM rats compared with the controls. Additionally, lipid accumulation in kidneys of DM rats were significantly increased (P＜0.01), due to increased protein expressions of COX-2, LDLr, sterol regulatory element–binding protein (SREBP) cleavage activating protein (SCAP), and SREBP-2 (P＜0.01). However, these changes were significantly inhibited by an inhibitor of COX-2, Aspirin (P＜0.05). It's worth noting that, COX-2 protein expression was closely correlated with LDLr protein expression (r=0.85, P＜0.01). Conclusion Dysregulation of LDLr pathway contributes to podocyte injury in diabetic nephropathy, which may be mediated through the increased COX-2 expression.     

Influence of albumin on expression of NLRP3 inflammasome in renal tubular epithelial cells

Objective To investigate the effect of albumin on expression of NLRP3 inflammasome and its downstream cytokines IL-1β and IL-18 in tubular epithelial cells. Methods Thirty mesangioproliferative glomerulonephritis (MsPGN) patients with different levels of proteinuria were selected, and their renal biopsy samples were stained by PAS and Masson to observe tubular epithelial cells injury and inflammatory cells infiltration. NLRP3, caspase-1, IL-1β and IL-18, as well as different inflammatory cells, were detected by immunohistostaining. In vitro, Western blotting and real-time PCR were employed to detect NLRP3, caspase-1, IL-1β and IL-18 protein and mRNA in HK-2 cells stimulated by bovine serum albumin (BSA) (20 g/L). Results In MsPGN patients with high levels of proteinuria, there were obvious renal tubular epithelial cell injury and inflammatory cells infiltration (all P＜0.05), and the expressions of NLRP3, caspase-1, IL-1β and IL-18 were up-regulated compared to patients with low levels of proteinuria (all P＜0.05). Furthermore, IL-1β and IL-18 expressions were positively correlated with the degree of proteinuria (r=0.836, P＜0.05; r=0.901, P＜0.05). NLRP3, caspase-1, IL-1β and IL-18 protein and mRNA were significantly increased in HK-2 cells stimulated by BSA compared to the control group (all P＜0.05). Conclusions Albumin is able to induce NLRP3 inflammasome activation in tubular epithelial cells, which may be the mechanism of tubulointerstitial injury and inflammation caused by proteinuria.     

Comparation of the effects of insulin-like growth factor-1 receptor inhibitor and insulin on renal interstitial macrophage infiltration in mice with type 2 diabetic kidney disease

Objective To compare the effect of insulin-like growth factor-1 receptor (IGF-1R) inhibitor and insulin on renal interstitial macrophage infiltration in mice with type 2 diabetic kidney disease (DKD) mice. Methods Twenty-four male C57BL/6 mice were selected. After 1 week of adaptive feeding, 6 rats were randomly selected as the control group. The other mice were intraperitoneally injected with streptozotocin (30 mg/kg) after 8 weeks of high-fat and high-sugar feeding. After 72 h, the type 2 diabetes mellitus (DM) models were successfully established if random blood glucose was greater than 16.7 mmol/L. After 8 weeks, if the proteinuria of DM mice increased, the DKD models were successful. DKD mice were divided into 3 groups by random number remainder method: DKD group (n=6), DKD+insulin group (insulin group, n=6, subcutaneous injection of 1-2 U/d insulin) and DKD+IGF-1R inhibitor (IGF-1R inhibitor group, n=6, administered with 30 mg?kg-1?d-1 IGF-1R inhibitor). They were continuously treated for 8 weeks. Random blood glucose was tested by glucometer. Blood and urine were collected, and biochemical indicators, such as serum creatinine, urea nitrogen and urine protein were measured by biochemical analyzer. Renal pathological changes were detected by hematoxylin-eosin staining (HE) and periodic acid-schiff staining (PAS). Suppressor of cytokine signaling 2 (SOCS2) mRNA and insulin-like growth factor-1 (IGF-1) mRNA were detected by in situ hybridization. The protein expressions of SOCS2, F4/80, Toll-like receptor 4 (TLR4) and CD68 were detected by immunohistochemistry. Results Compared with the control group, blood glucose, serum creatinine, serum urea nitrogen and urinary protein excretion rate were significantly higher in DKD mice (all P＜0.05), and CD68+ cells number, F4/80+ cells number and the expression of TLR4 in the tubulointerstitial of DKD mice were significantly higher (all P＜0.05). After intervention with insulin or IGF-1R inhibitor, serum creatinine, serum urea nitrogen and urinary protein excretion rate of DKD mice were significantly reduced (all P＜0.05). Insulin intervention could significantly reduce blood glucose in mice (P＜0.05), but had no significant effect on macrophages. Although IGF-1R inhibitor did not significantly reduce blood glucose, it could significantly reduce the number of CD68, F4/80 positive cells and the expression of TLR4 protein in renal interstitium of DKD mice (all P＜0.05). Compared with the DKD group, insulin intervention significantly reduced the expression of IGF-1 protein and mRNA (both P＜0.01), and increased the expression of SOCS2 mRNA and protein (both P＜0.01). And the expression of SOCS2 protein was correlated with the number of F4/80+ cells in insulin group (R2=0.8461, P=0.005). However, IGF-1R inhibitors had no significant effect on SOCS2 expression, but had better inhibition of macrophage infiltration. Conclusion IGF-1R inhibitor has a better inhibitory effect on DKD renal interstitial macrophage infiltration than insulin. The mechanism may be related to the fact that IGF-1R inhibitor does not up-regulate SOCS2 expression, whereas insulin up-regulates SOCS2 expression to activate some potential pathways.     

Attenuation of high glucose induced podocyte injury by ursolic acid up-regulating autophagy level

Objective To investigate the effects of ursolic acid (UA) on autophagy and podocyte injury induced by high glucose. Methods Conditionally immortalized murine podocyte were cultured in high glucose, the effect of PI3K inhibitor LY294002 and ursolic acid treatment were observed. The miR-21 expression was detected using RT-qPCR. The activation of PTEN-PI3K/Akt/mTOR pathway, expression of autophagy-related protein and podocyte marker protein were determined by Western blot. Immunofluorescence staining showed the expression of podocyte marker protein and endogenous accumulation of LC3. Autophagosomes were observed using electron microscopy. Results Compared with normal control group,the cells exposed to high glucose condition showed down-regulated synaptopodin, podocin and nephrin expression (P＜0.01), up-regulated miR-21 expression (P＜0.01), down-regulated PTEN expression (P＜0.01), up-regulated p85-P13K, phospho(p)-Akt, p-mTOR,p62/SQSTMI, expression and down-regulated LC3II and Beclin1 expression (all P＜0.01). Ursolic acid and LY294002 promoted synaptopodin, podocin and nephrin expression (all P＜0.01), up-regulated LC3II, Beclin1 expression and down-regulated p62/SQSTM1 expression (all P＜0.01), down-regulated p85-PI3K, p-Akt, p-mTOR expression (all P＜0.01). However, LY294002 did not affect the expression of miR-21 and PTEN. Ursolic acid inhibited miR-21 expression and upregulated PTEN level. Conclusions The podocyte injury is associated with defective autophagy level under high glucose condition. Ursolic acid could reduce podocyte injury by increasing autophagy level via inhibition of miR-21 expression and PTEN/Akt/mTOR pathway.     

帕立骨化醇对糖尿病肾病大鼠蛋白尿的影响

目的 研究维生素D类似物帕立骨化醇对糖尿病肾病(DN)大鼠蛋白尿的影响,并探讨其可能机制.方法 用链脲菌素(STZ)腹腔注射法构建DN大鼠动物模型,将造模成功的大鼠随机分为帕立骨化醇组(P组)、DN组(D组),并设置健康对照组(N组).给药12周后检测24 h尿蛋白量及血生化指标.用ELISA法检测肾组织肾素和血管紧张素Ⅱ(AngⅡ)水平.免疫组化和实时PCR检测肾小球基底膜乙酰肝素酶(HPA)、足细胞podocin蛋白及mRNA的表达.结果 D组和P组24h尿蛋白量、Scr、肾素及AngⅡ水平均显著高于N组,而D组显著高于P组,差异均有统计学意义(均P＜ 0.05).D组和P组HPA蛋白及mRNA表达均显著高于N组,而D组显著高于P组；D组和P组podocin蛋白及mRNA表达较低,D组显著低于P组,差异均有统计学意义(均P＜ 0.05).肾素水平与HPA蛋白表达呈正相关(r=0.78,P＜0.05)；与podocin蛋白表达呈负相关(r=-0.63,P＜0.05)；而与两者mRNA表达无相关.结论 帕立骨化醇可显著减少DN大鼠早期蛋白尿,其机制可能与通过抑制肾组织肾素表达,下调肾小球基底膜HPA,上调足细胞podocin蛋白表达有关.     

Effect of aldosterone receptor antagonist on obesity-related glomerulonephropathy

Objective To examine whether aldosterone contribute to obesity related glomerular disease. Methods C57BL/6J mice were randomly divided into three groups: a control group (low-fat-diet, n=10), a model group (high-fat-diet, n=10) and a intervention group (high-fat-diet, n=12). After 8 weeks intervention group were treated with a mineralocorticoid receptor antagonist, spirolactone (SPL).The physicochemical indexes and the renal pathology of the three groups were all detected. The mRNA and protein expressions of podocyte marker protein were determined by real-time PCR and Western blotting, respectively. Results Compared with the control group, body weight, kidney weight, Lee’s index, fat index, blood cholesterol, blood triglyceride, creatinine clearance rate, urinary protein excretion, glomerular average diameter, glomerular foot process average width were significantly up-regulated (P＜0.05); The mRNA and protein expression of nephrin, podocin, podoplanin and podocalyxin were significantly down-regulated in model group (P＜0.05). Meanwhile, these changes were attenuated by SPL. Conclusion Aldosterone can participate in the process of obesity- related renal injury, and these can be attenuated by mineralocorticoid receptor antagonist, spirolactone. This gives us preliminary clues to treat ORG.     

Lisinopril suppresses the endoplasmic reticulum stress associated tubular cell apoptosis induced by proteinuria in adriamycin nephropathy

Objective To investigate the role of endoplasmic reticulum stress in tubular epithelial cell apoptosis in chronic proteinuria rat model and the effect of lisinopril intervention. Methods Adriamycin nephropathy was induced in male Wistar rats (n=12) by a single injection of adriamycin at 2 mg/kg body weight. Rats were then randomly assigned to model group or treatment group, to which distilled water or lisinopril were administered respectively for 12 weeks. Six normal rats serving as controls were administered distilled water. 24 h urine samples were collected at week 4, 8, 12 and the urine protein was measured. At the end of study, serum was obtained and physiological parameters (serum creatinine, urea, total protein and albumin) were measured. Renal tubular epithelial cell apoptosis was assessed by TUNEL. GRP78, CHOP protein expression in kidney was quantified by immunohistochemistry and Western blotting. Results Compared to control group rats, increased proteinuria was observed in model group rats at week 4, 8, 12 (P＜0.05). Lisinopril treatment attenuated urine protein excretion significantly (P＜0.05). At week 12, hypoalbuminemia was detected in model group rats (P＜0.05), whereas the condition was alleviated by lisinopril (P＜0.05). There were no significant differences of serum creatinine, urea and total protein in each group (P＞0.05). Compared to control group rats, increased TUNEL positive tubular epithelial cells and tubular GRP78 and CHOP expression were also observed in model group rats (P＜0.05); however, these conditions in the kidney were significantly decreased in treatment group (P＜0.05). Conclusions Endoplasmic reticulum stress may be involved in the process of tubular epithelial cell apoptosis induced by proteinuria. Lisinopril may attenuate tubular epithelial cell apoptosis through regulating this signal pathway.     

Early autophagy activation inhibits podocytes from apoptosis induced by aldosterone

Objective To explore the protection of early autophagy activation on podocyte injury induced by aldosterone. Methods In vitro cultured mouse podocyte clones (MPC5) were treated with aldosterone for 6, 12, 24, 48 h respectively. Apoptosis of podocytes was detected by Annexin V combined with flow cytometry. After 24 h treatment with aldosterone, the existence of apoptotic body and autophagosome was observed by electron microscopy. The protein expressions of LC3, caspase?3 and nephrin were examined by Western blotting. The mRNA expression of Beclin?1 was detected by real?time PCR. Results The induction of apoptosis and autophagy by aldosterone in podocytes was in time?dependent mannner. After 24 h treatment with aldosterone, the apoptosis was increased by 26.5% (P＜0.05) and the expression of nephrin was decreased by 28.0% (P＜0.05) compared to control group. Aldosterone remarkably induced the expression of Beclin?1 at 6 h and promoted the transformation of LC3?Ⅰto LC3?Ⅱ at 12 h (P＜0.05). Compared to simple aldosterone treatment, the apoptosis rate of podocyte was increased by 39.0%（P＜0.05）and the expression of nephrin was declined by 19.5%（P＜0.05) after 3?methyladenine (3?MA) pre?treatment. Conclusions Aldosterone can induce autophagy and apoptosis in podocytes. Autophagy occurs earlier (12 h) than apoptosis (24 h). The occurrence of autophagy can inhibit the apoptosis,so the autophagy pathway may be a new research topic of glomerular disease treatment.