Disruption of low-density lipoprotein receptor pathway induced by inflammation contributes to podocyte injury in diabetic nephropathy

Objective To investigate the effects of low density lipoprotein receptor (LDLr) pathway on podocyte injury in diabetic nephropathy (DN) under inflammatory stress. Methods Male db/db mice and db/m mice were randomly divided into four groups (8 mice in each group): db/m group (control), casein injected db/m group (db/m＋casein), db/db group (db/db), and casein injected db/db group (db/db＋casein). An inflamed model of DN was established according to our previous study. 24-hour urinary protein was measured every week. The plasma lipid profile was detected by clinical biochemistry assay. Podocyte changes were evaluated by electron microscope and immunofluorescent staining. Lipid accumulation in the kidney was evaluated by oil red O staining and intracellular cholesterol quantitative assay. The protein expression of Wilm's tumor-1 (WT-1), nephrin, α-smooth muscle actin (α-SMA), and molecules correlated with LDLr pathway were examined by immunohistochemical staining or Western blotting. The colocalized protein expression of LDLr with WT-1 was examined by immunofluorescent staining and laser confocal microscopy. Results There were no differences in plasma levels of LDL and HDLamong four groups. Compared with db/db group, the db/db+casein group showed markedly increased 24-hour urinary protein, more significant podocyte foot process effacement and podocyte damage, increased lipid droplet accumulation in kidneys, increased protein expressions of LDLr, SCAP and SREBP-2 in kidneys (all P＜0.05). Interestingly, increased LDLr protein expression in kidneys of db/db mice was negatively correlated with decreased nephrin protein expression (r＝-0.855, P＜0.01) and positively correlated with increased α-SMA protein expression (r＝0.768, P＜0.01). Conclusions The disruption of LDLr pathway induced by inflammation contributes to podocyte injuries in diabetic nephropathy.     

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.     

Effects and the possible mechanism of histone deacetylase inhibitor SAHA on the interstitial fibrosis induced by diabetes

Objective To explore the effects and possible mechanism of histone deacetylase inhibitor SAHA on the interstitial fibrosis induced by diabetes. Methods The SD rats were divided into three groups: control group (Con, n=9), diabetes mellitus (DM) group (n=9) and SAHA treatment group (n=9). The diabetic rat model was established by injecting streptozotocin (STZ) through tail vein. After 8 weeks, the SAHA treatment group rats were fed with a SAHA solution (25 mg?kg-1?d-1) by gastric gavage. After 16 weeks, all rats were sacrificed to detect relevant biochemical parameters, and observe the changes of pathomorphology in kidney. In addition, immunohistochemistry staining and Western blotting were employed to detect the protein expressions of transforming growth factor-β1 (TGF-β1), Smad2, Smad3, p-Smad2, p-Smad3, Smad7, collagen-Ⅰ and collagen-Ⅲ, respectively. Results Compared with Con group, the levels of blood glucose (BG), urinary trace albumin/urinary creatinine (ACR), triglyceride (TG) and total cholesterol (TC) in the diabetic group were all increased significantly (all P＜0.05), the protein expressions of TGF-β1, p-Smad2, p-Smad3, collagen-Ⅰ and collagen-Ⅲ in kidney were all increased in diabetic group (all P＜0.05), and the expression of Smad7 was significantly reduced (P＜0.05). Compared with DM group, the levels of ACR was reduced, the renal fibrosis was alleviated, the protein expressions of TGF-β1, p-Smad2, p-Smad3, collagen-Ⅰ and collagen-Ⅲ in SAHA group were all decreased (all P＜0.05), and the expression of Smad7 was increased significantly (P＜0.05). Conclusion SAHA may restore the protein level of Smad7 by enhancing protein stability, then promote the moderate transduction of TGF-β1/Smads signaling pathway, which reduce the fibrosis of renal tubules in diabetic rats.     

Tacrolimus protects podocytes by up-regulating autophagy in type 2 diabetic model rats

Objective To assess the effects of tacrolimus (FK506) on podocyte in type 2 diabetic model rats and to explore the potential mechanism. Methods The model rats were fed with high fat and high sugar food and combining with a low-dose of streptozotocin (STZ). They were then randomly divided into a diabetic mellitus group (DM group) and a FK506 group. A normal control group (NC group) was also set. The rats in FK506 group were given with 0.5 mg?kg-1?d-1 FK506 for 8 weeks. The biochemical parameters were measured. The changes of renal pathology and ultrastructure of podocyte were observed by the light and electron microscopy. The expression of nephrin and LC3-Ⅱ was determined by immunohistochemistry and Western blotting. Results (1) Compared with those in NC group, KW/BW, systolic blood pressure (SBP), fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC), urinary albumin excretion rate (UAE) and creatinine clearance rate (Ccr) in DM group were significantly increased (all P＜0.05). And the KW/BW, UAE and Ccr were decreased in FK506 group compared to those in DM group (all P＜0.05), while other parameters had no significant difference (all P＞0.05). (2) Compared with those in NC group, the glomerular volume, mesangial cell proliferation and accumulation of mesangial matrix were increased, and the foot process became disorder and fusion in DM group, while these changes were significantly reduced in FK506 group. (3) Compared with that in NC group, the expression of nephrin and LC3-Ⅱ was decreased in DM group (all P＜0.05), and both of parameters were higher in FK506 group than those in DM group (all P＜0.05). Conclusion FK506 may enhance podocyte autophagy in type 2 diabetic model rats and attenuate podocyte injury.     

维生素D受体在糖尿病肾病足细胞损伤及蛋白尿缓解中的作用

目的探讨维生素D受体(VDR)在糖尿病肾病(DKD)足细胞中的表达水平及在足细胞损伤及蛋白尿缓解中的作用。方法(1)本研究纳入了65例诊断患有2型糖尿病(伴或不伴蛋白尿)的患者,并纳入了25例年龄和性别相匹配的健康体检者为对照组。根据白蛋白/肌酐(ACR)的尿排泄比例对2型糖尿病患者进行分组,分别为无蛋白尿(ACR<30 mg/g,n=24)、微量白蛋白尿(ACR 30~300 mg/g,n=18)和临床蛋白尿(ACR>300 mg/g,n=23)。另选择25例经肾活检确诊的DKD患者作为DKD组。正常肾脏组织标本均取自泌尿外科同一时期肾脏肿瘤切除患者10例。将各组检测指标进行对比,同时采用实时定量PCR、ELISA法和免疫组化法检测VDR在各组患者的血液、尿液样本和肾脏组织中的表达情况,以及使用Pearson相关分析分析VDR与尿蛋白的相关性。(2)在2型糖尿病肾病小鼠模型中对上述结果进行验证,将遗传背景均为C57BLKs/J的雄性db/db小鼠及同窝出生的db/m小鼠,随机分为正常对照组(A组)、DKD对照组(B组)、DKD二甲基亚砜处理组(C组)、DKD帕立骨化醇(VDR激动剂)处理组(D组),C、D组连续腹腔注射处理8周,对照组不做任何处理。小鼠10周龄时开始连续干预8周,在小鼠22周龄(开始干预后12周)检测各组小鼠体重、血、尿生化指标对比;Western印迹法检测β⁃catenin、VDR的变化;免疫荧光观察足细胞标志蛋白podocin及足细胞损伤蛋白α⁃SMA的表达变化。结果(1)与正常健康对照组相比,无蛋白尿组、微量白蛋白尿组和临床蛋白尿组的糖尿病患者血浆中VDR的mRNA和蛋白水平均较低(均P<0.05);与无蛋白尿组的糖尿病患者相比,微量白蛋白尿组和临床蛋白尿组的糖尿病患者血浆中VDR的mRNA和蛋白水平均较低(均P<0.05)。(2)与正常健康对照组相比,无蛋白尿糖尿病组和DKD组患者血浆中VDR的mRNA和蛋白水平均较低(均P<0.05);与无蛋白尿糖尿病组患者相比,DKD组患者血浆中VDR的mRNA和蛋白水平亦较低(均P<0.05)。(3)免疫组化结果显示,DKD组肾组织中VDR的表达明显少于正常对照组。(4)DKD患者血浆中VDR mRNA相对水平与ACR呈负相关(r=-0.342,P<0.05)。(5)各组尿液上清液中VDR的水平与血浆中的水平呈相反趋势。(6)Western印迹结果显示,B组、C组肾小球足细胞β⁃catenin蛋白表达高于D组(均P<0.05),VDR蛋白的表达低于D组(均P<0.05);免疫荧光结果显示,B组、C组肾小球足细胞podocin的表达低于D组(均P<0.05),α⁃SMA的表达高于D组(均P<0.05)。结论VDR高表达缓解DKD足细胞损伤及蛋白尿。     

cAMP/PKA signal activation prevents chemical-induced podocyte injury

Objective To investigate the role of activated cylic AMP(cAMP) signaling in chemical-induced podocyte injury. Methods Eight-weeks-old male BalB/C mice were randomly divided into three groups: control group, Adriamycin (ADR) group and Forskolin+ADR group. ADR nephropathy models were established by tail intravenous injection,and part of them were injected Forskolin, an agonist of adenylate cyclase, intraperitoneally. Phosphorylation of cAMP response element binding protein (CREB) was detected by laser confocal microscopy,morphology of foot processes were determined with transmission electron microscope, and WT-1 expression in glomeruli were detected by immunohistochemistry. Conditionally immortalized podocytes were treated with puromycin aminonucleoside (PAN), Exchange protein directly activated by cAMP (Epac) agonist 8-pCPT-2-O-Me-cAMP (2Me),protein kinase A (PKA) antagonist H89 and its agonist pCPT-cAMP(pCPT). Western blot was used to detect the expression levels of Epac, caspase3 and cleaved caspase3. PKA activity was assayed using cAMP-dependent protein kinase detection system. Cell viability was determined by a cell count kit and podocyte apoptosis was estimated by TUNEL staining. Mitochondrial membrane potential was evaluated by JC-1 staining. Results （1）Compared with ADR group, the urine albumin decreased significantly (P＜0.05) among Forskolin+ADR group and the WT-1 positive cells per glomerulus increased obviously (P＜0.05). （2）PAN decreased podocyte number in a time-dependent manner (P＜0.05), pre-treatment with pCPT obviously inhibited PAN induced podocyte decrease (P＜0.05), but H89 prevented the effect of pCPT in a dose-dependent manner (P＜0.05). （3）JC-1 staining showed that the percentage of podocyte with green fluorescence for control, PAN and pCPT+PAN group were (12.67±2.15)%, (31.35±4.60)% and (16.96 ± 2.51)%respectively (P＜0.05), and pretreatment with H89 inhibited the effect of pCPT (P＜0.05). （4）PAN promoted podocyte apoptosis and cleaved caspase3 expression (P＜0.05), and pretreatment with pCPT significantly prevented PAN-induced podocyte apoptosis and cleaved caspase3 expression (P＜0.05). Conclusions cAMP signaling activation ameliorated podocyte injury in ADR mice and PAN-induced podocyte apoptosis, and cAMP/PKA pathway may mediate these processes.     

Role of autophagy in high glucose-induced podocyte lipid droplet accumulation

Objective To investigate the role of autophagy in high glucose-induced podocyte lipid droplet metabolism. Methods (1) Cultured, conditionally immortalized human podocytes (HPC) were divided into normal control group, high glucose group and mannitol group. Oil red O staining and oil red O staining extraction assay was used to observe the degree of lipid accumulation; Protein level of SREBP-1 was analyzed by Western blotting. (2) HPC were cultured and divided into normal control group, high glucose group, high glucose+3-methyladenine (3-MA) group, and mannitol group. Acridine orange staining was used to observe the formation of autophagosomes. Western blotting was used to detect the protein levels of beclin-1 and LC3-II/LC3-I. Oil red O staining and oil red O staining extraction assay was used to observe the degree of lipid accumulation; Western blotting was used to analyze the expression of SREBP-1. Results (1) Compared with the normal control group, the lipid accumulation in the high glucose group was increased and the lipid metabolism related molecule SREBP-1 was up-regulated (P＜0.05); There was no significant difference between the normal control group and the mannitol group in lipid accumulation (P＞0.05). (2) Compared with the normal group, the number of autophagosomes was increased and autophagy-related proteins beclin-1 and LC3-II/LC3-I were up-regulated in high glucose group (all P＜0.05). After intervened with 3-methyladenine, a significant decrease in autophagosomes was observed; Protein levels of autophagy-related proteins beclin-1 and LC3-II/LC3-I were decreased (all P＜0.05); The lipid droplets in the high glucose+3-MA group was decreased and lipid metabolism related molecule SREBP-1 was down-regulated (all P＜0.05). Conclusion Autophagy may be involved in the process of high-glucose-induced podocyte lipid accumulation by affecting SREBP-1 expression, and inhibition of autophagy can alleviate the high-glucose-induced podocyte lipid accumulation.     

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.     

Podocyte COX-2 exacerbates diabetic nephropathy by increasing podocyte (pro)renin receptor expression

Diabetic nephropathy (DN) increases podocyte cyclooxygenase-2 (COX-2) expression, and COX-2 inhibition reduces proteinuria and glomerular injury in animal models of diabetes. To investigate the role of podocyte COX-2 in development of diabetic nephropathy, we employed a streptozotocin model of diabetic mellitus in wild-type and transgenic mice expressing COX-2 selectively in podocytes. Progressive albuminuria developed only in diabetic COX-2 transgenic mice despite hyperglycemia, BP, and GFR being similar to those in wild-type mice. Transgenic mice also manifested significant foot-process effacement, moderate mesangial expansion, and segmental thickening of the glomerular basement membrane. In cultured podocytes overexpressing COX-2, high glucose induced cell injury and increased both expression of the pro(renin) receptor and activation of the renin-angiotensin system. Downregulation of the (pro)renin receptor attenuated the injury induced by high glucose. In vivo, podocyte pro(renin) receptor expression increased in diabetic COX-2-transgenic mice, and treatment with a COX-2 inhibitor abrogated the upregulation of (pro)renin receptor and reduced albuminuria, foot-process effacement, and mesangial matrix expansion. In summary, these results demonstrate that increased expression of podocyte COX-2 predisposes to diabetic glomerular injury and that the (pro)renin receptor may be one mediator for this increased susceptibility to injury.     

糖尿病大鼠甩尾潜伏期升高早期心脏感觉神经神经生长因子的表达变化

目的 观察糖尿病大鼠甩尾潜伏期(tail flick latency,TFL)升高早期上胸段背根神经节(dorsal root ganglion,DRG)、血清神经生长因子(nerve growth factor,NGF)含量变化. 方法 雄性SD大鼠18只,体重200～250 g,按随机数字表法分为对照组(C组,6只)和糖尿病组(DM组,12只).DM组大鼠经腹腔注射链脲佐菌素(streptozotocin,STZ)50 mg/kg.用疼痛甩尾仪每周测定大鼠TFL,监测躯体感觉神经病变的形成及变化.当DM组大鼠TFL显著升高时,取T1～T5节段DRG和血液样本,采用ELISA法测定标本中NGF水平. 结果 糖尿病造模5周末时,DM组大鼠TFL较C组显著升高.将TFL变化率＜100％的DM组大鼠设为DM1组(6只),TFL变化率≥100％的DM组大鼠设为DM2组(6只).NGF检测结果显示:与C组比较,DM1组DRG、血清中的NGF含量分别升高18％(P＜0.05)和16％(P＜0.05),DM2组DRG、血清中的NGF含量分别降低40％(P＜0.01)和14％(P＜0.05). 结论 糖尿病神经病变早期,随神经病变的加重,DRG、血清NGF含量表现为过表达和低表达,其机制和生物学意义值得进一步探讨.     

乙酰肝素酶在糖尿病肾病大鼠蛋白尿发生中的作用

目的 观察糖尿病肾病大鼠肾组织乙酰肝素酶（HPA）的表达，探讨其在糖尿病肾病大鼠蛋白尿发生中的作用。 方法 SD健康大鼠被随机分为健康对照组（n = 6）、糖尿病6周组(DM6w, n = 10)和糖尿病12周组(DM12w, n = 10)，采用一次性腹腔注射链脲菌素（STZ）的方法建立糖尿病大鼠模型。分别于造模后6周和12周末测定各组大鼠相对肾质量、血糖、尿素氮、血肌酐、24 h尿量及尿蛋白量(24 h)等指标，并观察肾脏病理改变。RT-PCR和免疫组织化学法检测各组大鼠肾组织HPA mRNA和蛋白表达变化，并分析其与蛋白尿发生的相关性。 结果 （1）DM6w和DM12w组大鼠的相对肾质量、血糖、尿素氮、24 h尿量及尿蛋白量(24 h)与健康对照组相比明显升高, 差异均有统计学意义(P < 0.05或P < 0.01)。（2）DM6w和DM12w组大鼠HPA mRNA和蛋白表达比健康对照组均显著增高(P < 0.01)。（3）大鼠肾组织HPA mRNA和蛋白表达与尿蛋白量(24 h)之间均呈正相关 (r = 0.783，P < 0.01；r = 0.793，P < 0.01)。 结论 HPA在糖尿病肾病中的表达升高可能参与了糖尿病肾病蛋白尿的发生。     

Expression and significance of Pdlim2 in the glomerular podocyte of hyperlipidemic rats

Objective To investigate the expression changes and significance of Pdlim2 in theglomerular podocyte of hyperlipidemic rats. Methods Forty-five individuals of SD rats were divided randomly into 3 groups (n＝15 in each group). The control group was fed with normal diet. The high fat group was fed with high fat diet. The simvastatin group was fed with high fat diet plus with simvastatin gavage (10 mg·kg-1·d-1). Five rats were sampled randomly from each group at week 4, 6, and 10 and the urinary protein excretion, the concentration of serum cholesterol, and the concentration of low density lipoprotein cholestorol were determined, the glomerular podocyte damage in rats was detected by electron microscope, the expression of Pdlim2 protein was determined by immunohistochemistry and by Western blotting. Results The levels of serum cholesterol and low density lipoprotein cholestorol increased significantly in high fat group and simvastatin group at week 4 compared to that in control group(P＜0.05), and the level in simvastatin group was significantly decreased compared with that in high fat group(P＜0.05). The urinary protein levels of high fat group and simvastatin group were significantly higher than that in control group at week 10, and the level in simvastatin group was significantly decreased in high fat group, there was significant difference in each group of comparison(P＜0.05). Podocyte injury was detected by electronic microscopy in high fat group at week 4, and the injury became more serious as the treatment time increased. Podocyte injury in the simvastatin group was significantly less than that in the high fat group and the control group at week 10. The positive staining of Pdlim2 was mainly in the glomeruli and the expression of Pdlim2 of the high fat group was lower than the simvastatin group, and both were lower than the control group at week 10. The expression of Pdlim2 protein of high fat group was lower than that in the control group since week 4(P＜0.05). The expression of Pdlim2 protein of high fat group was lower than that in the simvastatin group(P＜0.05), and both were lower than the control group at week 10(P＜0.05). ConclusionsHypedipidemia induces podocyte injury before urinary protein, which is suggested to be associated with the decrease of Pdlim2 protein. Simvastatin reduces podocyte foot processes of high fat induced fusion, which may be through protecting the expression of glomerular Pdlim2.     

Protective effect of chenodeoxycholic acid against lipid kidney injury induced by high-fructose-feeding in rats and the underlying mechanism

Objective To study the intervention of chenodeoxycholic acid (CDCA) on kidney of high-fructose-fed rats, and investigate the mechanism of CDCA on lipid kidney injury. Methods Forty-eight healthy male Wistar rats were randomly divided into three groups: normal control group (n＝16), high fructose group (n＝16) and CDCA group (n＝16). Eight rats were sacrificed at the end of 8 and 16 weeks in each group. BUN, Scr, uric acid (UA), fast glucose, serum lipid concentration, urinary albumin were measured. The triglyceride content of renal cortices was detected. The change of renal histopathology was observed by Periodic acid Schiff staining and electron microscopy. The mRNA expressions of farnesoid X receptor (FXR), small heterodimer partner (SHP), sterol regulatory element-binding protein 1c (SREBP-1c), stearoyl-CoA carboxylase (SCD-1), peroxisome proliferator-activated receptor α (PPARα), acyl coenzyme A oxidase (ACO), transforming growth factor β1 (TGF-β1), type 1 plasminogen activator inhibitor (PAI-1), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and NADPH oxidase 2 (Nox2) were measured by real-time PCR, and the protein expressions of which were analyzed by Western blotting. Results Left kidney weight/body weight, triglyceride, very-low- density-lipoprotein and UA in blood were significantly increased in high fructose group (all P＜0.05). Renal function and fast glucose did not change much (P＞0.05). The urinary albumin significantly increased in high fructose group (P＜0.01). The triglyceride content in renal cortex was much more abundant than that in control group (P＜0.01). Renal injuries including mesangial expansion, glomerular basement membrane thickening and podocyte foot process effacement were found in fructose-fed Wistar rats. The gene and protein expressions of FXR and SHP in kidneys of rats fed with high fructose were significantly down-regulated (all P＜0.01). The gene and protein expressions of SREBP-1c and SCD-1 in kidneys of rats fed with high fructose were significantly up-regulated (all P＜0.01). The gene and protein expressions of PPARα and ACO in kidneys of rats fed with high fructose were significantly down-regulated (all P＜0.01). TGF-β1, PAI-1, TNF-α, IL-6 and Nox2 in kidneys of rats fed with high fructose were significantly up-regulated (all P＜0.01). The levels of these changes were prominent with the extention of time. CDCA treatment could reverse these changes (all P＜0.05). Conclusions High-fructose feeding can lead to kidney injury in rats. CDCA enhances lipid anabolism, attenuates lipid catabolism, by activating FXR, down-regulating SREBP-1c and SCD-1, up-regulating PPARα and ACO, subsequently decreases profibrotic growth factors，improves renal inflammation, and protects kidney against oxidative stress.     

α-actinin-4在糖尿病肾病大鼠的表达及其与足细胞数目的相关性

目的：探讨糖尿病肾病发展过程中α-actinin-4的表达变化及其与足细胞数目的关系。方法：建立STZ诱导的糖尿病大鼠模型,应用免疫组化及RT-PCR方法观察在糖尿病肾病发展过程中α-actinin-4的蛋白及mRNA表达变化,以及应用WT-1来标记足细胞核,观察足细胞数目的变化。结果：8周时,糖尿病组染色强度较正常组明显增高（P〈0．01）,12周时变化更为明显（P〈0．01）。α-actinin-4的mRNA的表达水平在各时间点与组化结果相同。8周时,糖尿病组足细胞数目较正常组明显降低（P〈0．05）,12周时变化更为明显（P〈0．01）。α-actinin-4与足细胞数目呈负相关关系（r=-0．957,P〈0．01）,足细胞数目与尿蛋白呈负相关（r=-0．95,P〈0．01）。结论：α-actinin-4在糖尿病肾病发展过程中表达增高,并与足细胞数目呈负相关α-actinin-4的表达增高与尿蛋白的发生密切相关。     

Hydrogen sulfide reduce renal ischemia/reperfusion injury by NOD-like receptor pathway

Objective To investigate whether the nod-like receptor (NLR) pathway is involved in protection of hydrogen sulfide (H2S) preconditioning during renal ischemia reperfusion. Methods Male Wistar rats were randomly divided into 3 groups: sham operation (Sham) group, renal ischemia/reperfusion (I/R) group subjected to occlusion of left renal pedicle for 45 min then reperfusion for 24 hours, and sodium hydrosulfide (NaHS) preconditioning group with continuous infusion of NaHS (300 nmol/min) by left renal artery for 15 min before I/R treatment. Renal injuries were evaluated by HE staining. The protein levels of NOD1, NOD2, nuclear NF-κB P65 and caspase-1 were analyzed by Western blot assay. The protein level of MCP-1 and IL-1β expressions was determined by immunohistochemical staining assay. Cell apoptosis were evaluated by Tunel staining assay. Results In I/R group, the renal NOD1 and NOD2 protein expressions were upregulated. Moreover, the nuclear NF-κB P65 expression was also elevated with an increase in its target genes-MCP-1 and IL-1β (All P＜0.01). HE staining revealed the existence of acute tubular necrosis in I/R kidney. TUNEL staining revealed more apoptotic cells in risk zone with the activation of caspase-1 of I/R-treated kidney(P＜0.01). NaHS preconditioning reversed I/R-induced increase in the expression of NOD1 and NOD2(P＜0.05). NaHS preconditioning also reduced I/R-induced activation of NF-κB P65 (P＜0.05) and upregulation of MCP-1 and IL-1β (P＜0.01). Moreover, NaHS preconditioning attenuated inflammation, repressed caspase-1 activation and reduced apoptotic cells after I/R. Conclusion Hydrogen sulfide preconditioning can alleviate renal ischemia/reperfusion injury by Nod-like receptor dependent on inflammatory pathway.     

洛沙坦对糖尿病大鼠肾脏炎症反应及足细胞损伤的影响

目的 探讨洛沙坦对糖尿病大鼠模型肾组织炎症反应及足细胞损伤的影响。方法 Wistar大鼠随机分为3组：对照组（NC）、糖尿病组（DM）、洛沙坦治疗组（DL）。链脲菌素（STZ）注射建立糖尿病动物模型，洛沙坦治疗组在大鼠建立糖尿病同时即开始以洛沙坦（20mg·kg·d^-1）灌胃治疗。分别于2周和12周杀鼠，观察体重、肾重、肾重／体重指数、尿蛋白量（24h）、Scr、Ccr以及肾组织病理改变；免疫组化染色观察肾小球细胞间黏附分子（ICAM-1）、白细胞共同抗原（CD45）、nephrin、血管内皮生长因子（VEGF）表达的改变；免疫印迹观察肾组织nephrin、VEGF表达的改变。结果 （1）DL组大鼠肾重／体重指数、尿蛋白量（24h）、Ccr则均低于DM组，差异均有统计学意义（P均〈0．05），其病理改变程度也比DM组轻。（2）免疫组化及免疫印迹显示，DM组和DL组肾小球ICAM-1、CD45、VEGF表达均高于对照组，而DL组ICAM-1、CD45、VEGF表达均低于DM组；DM组和DL组大鼠肾小球nephrin表达低于对照组；DL组肾小球nephrin表达高于DM组，差异均有统计学意义（P均〈0．01）。结论 洛沙坦可能通过抑制肾组织白细胞浸润、炎症反应及减轻足细胞损伤，达到肾脏保护作用。     

Relationship of angiotensin II type 1 receptor autoantibody and renal cell apoptosis induced by caspase-12 in diabetic nephropathy rats

Objective To study the relationship of angiotensin II type 1 receptor (AT1R) autoantibody (AT1-AA) and renal cell apoptosis induced by caspase-12 in diabetic nephropathy (DN) rats. Methods High-sucrose and high-fat diet and intraperitoneal injection of streptozotocin (35 mg/kg) were utilized to establish DN rat model. Serum AT1-AA was detected by enzyme-linked immunosorbent assay (ELISA) and renal cell apoptosis was detected by TUNEL staining. Furthermore, the mRNA levels of the endoplasmic reticulum stress (ERS) chaperone protein glucose regulated protein 78 (GRP78) and ERS-associated apoptosis protein caspase-12 were measured by real-time quantitative PCR. Additionally, the levels of GRP78 and caspase-12 protein were measured by Western blotting. Results The renal cell apoptosis rate in DN group was increased significantly (P＜0.01), and the renal cells apoptosis rate in AT1-AA positive DN group was higher than that in AT1-AA negative DN group [(20.05±1.71)% vs (13.24±4.93)%, P＜0.01]. The mRNA expressions of GRP78 and caspase-12 in DN group, in comparison to NC group, were increased significantly (P＜0.01), as well as the proteins (P＜0.01). And the expression of these mRNA and proteins had significant increment in AT1-AA positive DN rats when compared with AT1-AA negative DN rats (P＜0.05). Conclusions AT1-AA can induce ERS in the renal tissue of DN rats, and promote renal cell apoptosis likely via the modulation of caspase-12 signaling pathway.     

1,25(OH)2D3抑制嘌呤霉素氨基核苷酸肾病大鼠足细胞凋亡

目的 观察1，25（OH）2D3对嘌呤霉素氨基核苷酸（PAN）肾病大鼠足细胞凋亡的影响。 方法 72只雄性SD大鼠随机分为健康对照组（NC）、PAN组和1，25（OH）2D3治疗组 [1，25（OH）2D3 0.2 μg·kg-1·d-1灌胃]。一次性尾静脉注射PAN 100 mg/kg体质量建立足细胞损伤的PAN肾病动物模型。于3、7、14、21 d分批处死动物，分别检测不同时间点尿蛋白量（24 h）和肾功能。光镜和透射电镜观察肾组织学改变。TUNEL法检测足细胞凋亡。RT-PCR、免疫荧光、免疫组化分别检测nephrin、TGF-β1 mRNA和蛋白的表达。Western印迹检测磷酸化（p）-Smad2/3的表达。 结果 （1）PAN组各时间点BUN、Scr、尿蛋白量（24 h）[7 d时，（20.26±4.87） mg比（1.01±0.41） mg，P < 0.01]均高于同期的NC组，而肾小球足细胞显著减少[14 d时，(10.9±4.2) 个/肾小球切面比(31.9±6.2)个/肾小球切面，P ＜ 0.01]，且足突增宽融合。1，25（OH）2D3治疗组各时间点尿蛋白量（24 h）[7 d时（9.95±3.82） mg]和BUN、Scr显著低于PAN组（P < 0.05），且肾脏病理改变减轻。（2）PAN组7 d时nephrin mRNA和蛋白的表达显著降低，nephrin由正常的沿毛细血管襻线状分布向颗粒状、团快状改变，足细胞凋亡数显著增加[14 d时，（37.4±7.9）个/肾小球切面]。与PAN组相比，1，25（OH）2D3治疗组各时间段nephrin mRNA和蛋白的表达显著增加，且保持着正常的沿毛细血管襻线状分布，足细胞凋亡数显著减少[14 d时，(21.9±6.2) 个/肾小球切面，P < 0.01]。（3）PAN组TGF-β1 mRNA和蛋白的表达以及p-Smad2/3蛋白的表达均高于NC组（P < 0.01），1，25（OH）2D3治疗组TGF-β1 mRNA和蛋白的表达以及p-Smad2/3蛋白的表达低于PAN组（P < 0.01）。 结论 1，25（OH）2D3能有效地抑制PAN诱导的足细胞凋亡，减少尿蛋白，其对足细胞损伤的保护作用可能与抑制TGF-β1信号通路有关。