纤维蛋白肽Bβ15～42对大鼠缺血再灌注损伤肾脏局部炎性反应的影响

目的 观察纤维蛋白肽Bβ15～42(the fibrin-derived peptide Bβ15-42,FgBβ15～42肽)对大鼠肾脏缺血再灌注损伤(IRI)后肾脏局部炎性反应的影响并探讨其机制.方法 将SD大鼠随机分成假手术组(Sham组)、IRI组、阴性治疗组和FgBβ15 ～ 42肽治疗组.Sham组:分离肾动脉后关闭腹腔;IRI组:采用双侧肾动脉夹闭的方法制作肾脏IRI模型;阴性治疗组:于肾脏再灌注后立即尾静脉注射随机肽段3.6 mg/kg; FgBβ15～42肽治疗组:于肾脏再灌注后立即尾静脉注射FgBβ15～ 42肽3.6 mg/kg.后3组按照再灌注24h、48 h分为两个亚组,Sham组与各亚组均为8只大鼠.常规生化法检测肾功能;HE、PAS染色观察肾脏组织学改变;免疫组化、实时荧光定量PCR法及Western印迹检测肾组织白细胞介素1β(IL-1β)、细胞间黏附分子1(ICAM-1)的mRNA及蛋白表达.结果 与Sham组相比,IRI组的Scr和BUN水平均显著增加(均P ＜0.05),肾小管及间质病理损伤显著,以再灌注48 h更为明显;与IRI组相比,FgBβ15～ 42肽治疗组Scr和BUN显著下降(均P＜0.05),小管间质损伤程度明显减轻(P＜0.05).与Sham组相比,IRI组IL-1β和ICA M-1的mRNA和蛋白水平于再灌注24h显著上升,48 h稍微下降,但仍维持在较高水平;FgBβ15～ 42肽治疗组大鼠肾组织IL-1β和ICAM-1的表达于再灌注24h、48 h显著低于同时间点的IRI组(均P＜0.05),但仍明显高于Sham组.上述各指标在阴性治疗组和IRI组之间的表达差异无统计学意义.结论 FgBβ15～42肽对肾脏IRI具有保护作用,其作用机制可能与其减少炎性因子IL-1β、黏附分子ICAM-1的表达有关.     

Serum uric acid level is positively related to albuminuria in type 2 diabetic patients

Objective To investigate association between serum uric acid (SUA), albuminuria and glomerular filtration rates (eGFR) in type 2 diabetic patients. Methods A total of 220 patients were enrolled in this cross-sectional study. According to urinary albumin excretion rates, patients were divided into 3 groups: normoalbuminuria (NAU) group, microalbuminuria (MAU) group, and macroalbumnuria group (MAAU). The first two groups were subdivided at SUA＞420 μmol/L (＞357 μmol/L, female) into normouricemia group and hyperuricemia group, at eGFR＞90 ml/min into high and low renal function groups. General information, blood biochemical results were collected to analyze the association between serum uric acid, eGFR, UAER and urine albumin quantification among different groups. Results The difference of SBP, duration of diabetes (DD), Scr, SUA and eGFR between every two groups were significant (P＜0.05). SBP, DD, Scr and SUA were highest in subjects with macroalbumnuria, second in microalbuminuria group, and lowest in normoalbuminuria group, while eGFR was lowest in macroalbumnuria group and highest in normoalbuminuria group. Prevalence of hyperuricemia in macroalbumnuria group (56.9%) and microalbuminuria group (51.2%) were also significantly higher than that in normoalbuminuria group (17.5%) (all P＜0.01). The difference of UAER in the subgroups of normouricemia and hyperuricemia was more significant in microalbuminuria group than in normoalbuminuria group. eGFR was significantly lower in hyperuricemia subgroups (P＜0.01). Age and SUA were significantlg higher in subjects with low renal function compared with high eGFR (P＜0.05). Linear regression analysis indicated SUA was negatively correlated with eGFR after adjusted age, DD and UAER (β＝-0.430, P＜0.01). Binary logistic regression analysis found that increased age, DD and SUA were risk factors of microalbuminuria [β＝1.092, 95％CI(1.025, 1.163), P＜0.01; β＝1.005, 95%CI(1.001, 1.009), P＜0.05; β＝1.407, 95％CI(1.052, 1.881), P＜0.05)] andSUA, age were risk factors of early renal function decline [β＝1.015, 95％CI(1.00, 1.023), P＜0.01; β＝1.098, 95％CI(1.006, 1.199), P＜0.05]. Conclusion SUA is independently associated with albumnuria and renal function decline in type 2 DM patients.     

Protective effect of resveratrol on 5/6 nephrectomized rats and its mechanism

Objective To investigate the protective effect of resveratrol (RSV) on 5/6 nephrectomized rats and its mechanism. Methods Fifty male SD rats were randomly divided into three groups: sham operated (Sham, n＝10）, 5/6 nephrectomy (Nx, n＝20), and 5/6 nephrectomy＋RSV 20 mg/kg (Nx＋RSV, n＝20). RSV or normal saline was administered one week after 5/6 nephrectomy. Proteinuria was detected every 4 weeks. Serum creatinine and the renal pathological changes were measured after 12 weeks. Immunohistochemisty staining of fibronectin (FN), collagenⅠ, transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) were used to analyze the changes of renal fibrosis. Western blotting was used to measure the expression of Smad3, phospho-Smad3, and acety-Smad3. Immunoprecipitation was used to detect the interaction between Sirt1 and Smad3. Results Compared with the sham operated rats, subtotal nephrectomy significantly increased proteinuria [(152.14±30.49) mg/24 h vs (25.34±7.54) mg/24 h], serum creatinine[(111.60±21.50) μmol/L vs (53.90±11.59) μmol/L], glomerular sclerosis index (1.56±0.34 vs 0.35±0.08) and the expressions of fibronectin, collagenⅠ, TGF-β and CTGF in renal tissue at 12 weeks after operation (all P＜0.01), and RSV treatment significantly inhibited the above up-regulations (all P＜0.01). Compared with the sham operated rats, subtotal nephrectomy increased the expression of phospholylation and acetylation of Smad3. RSV treatment significantly reduced the expression of acety-Smad3, but had no effect on the phospho-Smad3. Immunoprecipitation revealed a binding effect of Smad3 with Sirt1. Conclusions RSV treatment can attenuate proteinuria, protect renal function and inhibit renal fibrosis in 5/6 nephrectomized rats. This renal protective effect is associated with reduced Smad3 acetylation and activation of Sirt1, which suggesting that Sirt1 may be a potential therapeutic target of CKD.     

IL-6强化的肠外营养对肝硬化大鼠肝切除术后残肝的影响

目的 探讨白细胞介素-6(IL-6)强化的肠外营养(PN)对肝硬化大鼠肝切除术后残肝的影响.方法 6只正常大鼠为正常对照组(A组),24只肝硬化大鼠随机分为4组(B～E组,n＝6);B组:肝硬化术前组,C组:肝切除+颈内静脉插管术后1 d组,D组:肝切除术后行PN 5 d组,E组:肝切除术后行PN+IL-6 5d组.测大鼠肝功能,炎症反应和脂质过氧化指标.肝组织ALB mRNA的表达.结果 与D组比较,E组血清AST、ALT、ALP显著下降(P＜0.05),血清ALB显著升高(P＜0.05);血清IFN-γ、TNF-α、MDA显著下降(P＜0.05),血清SOD活性显著升高(P＜0.05);肝组织ALB mRNA表达显著升高(P＜0.05).结论 PN+IL-6可以加快肝硬化大鼠肝切除术后肝功能的恢复,减轻炎症反应和脂质过氧化损伤,促进肝脏蛋白合成.     

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.     

肝硬化大鼠肝切除术后不同营养支持途径的对比研究

目的 探讨肝硬化大鼠肝切除术后不同营养支持途径的作用.方法 6只正常大鼠作为正常对照组,30只肝硬化大鼠随机分为术前组6只,肝切除+颈内静脉插管术后1d组6只,肝切除+颈内静脉插管术后行PN 5 d组6只,肝切除+胃造瘘术后1 d组6只,肝切除+胃造瘘术后行EN 5 d组6只.测大鼠肝功能、免疫功能、炎症反应和脂质过氧化指标.结果 与PN 5 d组比较,EN 5 d组血清AST、ALT、ALP显著下降(P＜0.05),血清ALB、IGF-1显著升高(P＜0.05),外周血CD3+、CD4+、CD4+/CD8+显著升高(P＜0.05),血清IL-6、IFN-γ、TNF-α、MDA显著下降(P＜0.05),血清SOD活性显著升高(P＜0.05).结论 EN可以加快肝硬化大鼠肝切除术后肝功能的恢复,避免胆汁淤积,促进肝脏蛋白合成,改善术后免疫功能低下,减轻炎症反应和脂质过氧化损伤.     

Expression of NLRP3 inflammasome in the BSA-overloaded rats kidney

Objective To observe NLRP3 inflammasome expression and inflammatory cells infiltration in the BSA-overloaded rats kidney, and to investigate the potential mechanism of renal injury induced by proteinuria. Methods After unilateral right nephrectomy, eighteen healthy male Wistar rats were randomly divided into two groups: protein overload nephropathy model group (n=10), treated with intraperitoneal injections of bovine serum albumin (BSA); control group (n=8), treated with intraperitoneal injections of 0.9% saline for 9 weeks. Body weigh were measured every week and 24 h urine were collected in 0, 2, 5, 7, 9 week. The plasma levels of blood total protein (TP), albumin (Alb), serum creatinine (Scr) and blood urea nitrogen (BUN) were determined by automatic analyzers. Renal pathological changes were evaluated by PAS and Masson stains. Immunohistochemical staining was used to detect the expression of NLRP3, caspase-1, IL-1β, and IL-18, as well as the types of inflammatory cells. The NLRP3, caspase-1, IL-1β, and IL-18 protein and mRNA levels were also analyzed by Western blot and real-time PCR in two groups. Results It was found that there was a significant increase of proteinuria and BUN in model group compare to that in control group (all P＜0.05). However, there were no significant changes in body weight, TP, Alb and Scr between the two groups. Morphological study demonstrated that renal tubular epithelial cell injury, proteinaceous casts in tubular lumen, accompanying with the dominant macrophages and lymphocytes infiltration in interstitium in model group. The immunohistochemistry showed that there were more T (CD3+), B cells (CD20+) and macrophages (CD68+) in renal interstitium in model group than that in control group (P＜0.05). Tubulointerstitial injury score was higher than that of the control group (P＜0.05). Immunohistochemistry, Western blot and real-time PCR all showed that the expression of NLRP3, caspase-1, IL-18 and IL-1 β were significantly increased compared to those in control group (P＜0.05). Furthermore, there were significant correlations between proteinuria and IL-1β/IL-18 expression (P＜0.05). Conclusion NLRP3 inflammasome activation is involved in tubulointerstitial inflammation caused by proteinuria.     

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.     

Relationship between islet α - cell function and glomerular filtration rate in type 2 diabetic patients

Objective To analyze the islet α-cell function in type 2 diabetic patients with different levels of glomerular filtration rate(eGFR). Methods Three hundred and eighty-eight cases of type 2 diabetic patients were classified into four groups according to eGFR: glomerular hyperfiltration group, normal renal function group, mild renal dysfunction group and moderate-severe renal dysfunction group. Oral glucose tolerance test, insulin releasing test and glucagon releasing test were conducted to compare the changes of glucagon(GLA), insulin/glucagon ratio（INS/GLA）, glucose/glucagon ratio（GLU/GLA）, the area under the curve of glucagon(AUCGLA) among the groups and correlation analysis were performed among glucagon and glomerular filtration rate and other indicators. Results With the decline of eGFR, the change curve of glucagon level was“J”shaped; the level of fasting glucagon in each group were（82.21±15.06）ng/L,（74.25±15.34）ng/L,（81.16±20.18）ng/L, （100.21 ± 24.73）ng/L, respectively. Compared with normal renal function group, GLA, AUCGLA in glomerular hyperfiltration group and renal dysfunction group increased significantly(P＜0.05), GLU/GLA, INS/GLA decreased significantly. Pearson correlation coefficient analysis showed that fasting glucagon had a negative correlation with eGFR（r＝-0.360,P＜0.01）, plasma albumin (ALB)（r＝ -0.170,P＜0.01）and high - density lipoprotein cholesterol (HDL - C)（r＝-0.128,P＜0.05）, had a positive correlation with fasting venous blood glucose (FPG)（r＝0.339,P＜0.01）, postprandial 2 hours venous blood glucose(2hPG)（r＝0.443,P＜0.01）, the area under the curve of blood glucose (AUCG)（r＝0.475,P＜0.01）, duration（r＝0.257,P＜0.01）and glycosylated hemoglobin(HbA1c) （r＝0.202,P＜0.01）. Multiple stepwise regression analysis showed that fasting glucagon was negatively correlated with eGFR（β ＝-0.290,t＝-5.393,P＜0.01） and HDL - C（β ＝-0.157,t＝ -3.026,P＜0.01）. Conclusions Glucagon level is influenced by eGFR in type 2 diabetic patients. Glucagon in patients with glomerular hyperfiltration or renal dysfunction is significantly higher than those with normal renal function. The inhibition effect of blood glucose and insulin to glucagon are both weakened.     

Effects of Intercellular Adhesion Molecule-1 on Renal Damage in Spontaneously Hypertensive Rats

Aims: Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the inflammatory process and immune response. The aim of the study was to investigate ICAM-1 expression in the kidneys of spontaneously hypertensive rats (SHRs) and the relationship between the level of ICAM-1 and renal damage. Methods: Male Wistar–Kyoto (WKY) rat and SHR models were employed. Blood pressure (BP) was recorded using tail cuff method. Twenty-four hour proteinuria and β₂-microglobulin (β₂-MG) were measured using biuret method and radioimmunity kits, respectively. Biochemical parameters were measured after the animals were killed. ICAM-1 expression in renal tissue was assessed using western blot and real-time polymerase chain reaction (PCR). Results: It was observed that BP, proteinuria, and urine β₂-MG were more increased in SHR groups than that in the same age WKY groups. In SHR groups, BP, proteinuria, and urine β₂-MG at the 56th week were significantly higher than that at the 28th week. ICAM-1 protein and mRNA expression in SHR renal tissues was significantly increased in SHR groups compared with the same age in WKY rats. ICAM-l expression was positively correlated with proteinuria and urine β₂-MG. Conclusion: This study demonstrated that the renal expression of ICAM-1 was increased in SHR with renal damage, and inflammation may be involved in the hypertensive renal damage.     

Energy metabolism changes of renal cortex in a rat model of progressive kidney disease and its mechanisms

Objective To explore the changes of renal cortical energy metabolism and its related molecular mechanisms in rats with progressive kidney disease. Methods A rat model of 5/6 nephrectomy was established as the model of progressive nephropathy. Rats were divided into surgical group (5/6Nx group) and sham-operated group (Sham group). Respectively, the rats were sacrificed at 1 week and 12 weeks after completing the model, and their blood, urine sample and kidney specimens were collected. Blood urea nitrogen, serum creatinine and 24 h urine protein were used to evaluate the renal function. Pathological changes in renal tissue were detected by PAS staining and Sirius red staining. The renal cortical energy metabolites were made quantitative analysis by liquid chromatography-mass spectrometry-based targeted metabolomics. The mRNA expressions of inflammatory cytokines (IL-6, IL-1β), fibrosis factors (fibronectin, collagen-1), glycolytic and tricarboxylic acid (TCA) cycle related enzymes were confirmed by real-time PCR. The protein expressions of fibrotic proteins (fibronectin, collagen-1), silent information regulator 1 (SIRT-1) and liver kinase B1 (LKB1) were tested by Western blotting. Results Compared with those in Sham group, the renal function indexes increased, the renal tissue pathological damage was obvious, the mRNA expressions of renal cortical inflammatory and fibrosis factors increased, and fibrotic proteins also increased in 5/6Nx group rats at 1 week and 12 weeks (all P＜0.05), meanwhile, kidney damage worsened over time. Compared with those in Sham group, in the renal cortex of 5/6Nx group glycolytic metabolite lactate, the TCA cycle metabolites (citrate, isocitrate, oxaloacetate) and the oxidized phosphorylation metabolite reduced coenzymeⅠ were up-regulated (all P＜0.05), but adenosine triphosphate (ATP) was no change at 1 week, then the abnormal metabolites increased further at 12 weeks, such as the down-regulation of pyruvate, oxidized coenzyme Ⅰ and ATP (all P＜0.05). The pentose phosphate pathway metabolites (reduction and oxidized coenzyme Ⅱ) shows no statistical significant difference in the two group (all P＞0.05). Compared with those in Sham group, in the 5/6Nx group the mRNA expressions of glycolytic enzyme hexokinase 2 and lactate dehydrogenase a were up-regulated in the renal cortex at 1 week, whereas the mRNA expressions of pyruvate dehydrogenase α, pyruvate dehydrogenase β and succinate dehydrogenase of the TCA cycle related enzymes were down-regulated (all P＜0.05). Meanwhile, renal abnormal metabolic enzyme mRNA expressions were further increased in the 5/6Nx group at 12 weeks. The protein levels of SIRT-1 and LKB1 were not significantly different in the renal cortex of two group rats at 1 week, while SIRT-1 and LKB1 levels decreased in 5/6Nx group than those in Sham group at 12 weeks (all P＜0.05). Conclusions During the progression of nephropathy, rats accompanied with renal fibrosis and inflammatory have energy metabolism changes in the renal cortex which accompanies. The features of metabolic changes are manifested as enhanced glycolysis and decreased oxidative phosphorylation, which is aggravated gradually. Its mechanism is related to the inhibition of energy-regulating proteins LKB1 and SIRT-1.     

Mitochondrial dysfunction in the process of cisplatin-induced acute kidney injury in mice

Objective To assess the characteristics of different doses of cisplatin-induced acute kidney injury, further to understand mitochondrial dysfunction and its role in acute kidney injury (AKI). Methods Male C57BL/6J mice were first randomly divided into two groups: control group (n＝6) and AKI group (n＝12). Then, AKI group was subsequently divided into other two groups according to different dose of cisplatin (10 mg/kg or 20 mg/kg). AKI group received intraperitoneal injection of cisplatin. All mice were sacrificed after 72 h of injection. Renal biochemical function, renal pathological changes, renal injury markers, kidney mitochondrial function and structural changes were observed. Results (1) After 72 hours of injection, the AKI group performed significant kidney injury changes compared to control group, thereinto 20 mg/kg group was more serious than 10 mg/kg group. With the cisplatin dose increasing, renal function markers such as serum creatinine, urine protein gradually increased. (2)Kidney biopsy showed tubular structural damage, the formation of protein casts, kidney injury molecule-1 (KIM-1) gradually increased(P＜0.05). (3)Electron microscopy found tubular mitochondrial structural damage, mtDNA copy number decreased, the level of peroxisome proliferator-activated receptor -gamma coactivator-1alpha (PGC-1α), ATP synthase β decreased(P＜0.05), and Western blotting manifested cytochrome C was released from mitochondria to the cytoplasm. These data all exhibited significant difference between different groups(P＜0.05). Conclusions Cisplatininduces acute kidney injury in dose-dependent manner. Mitochondrial dysfunction participates in kidney injury, and is also related to the kidney pathological damage.     

冬虫夏草菌粉对5/6肾大部切除大鼠肾脏纤维化的抑制作用及机制

目的 探讨冬虫夏草菌粉对5/6肾大部切除术大鼠肾脏纤维化的抑制作用及其可能机制.方法 30只雄性SD大鼠随机分为3组:假手术组(Sham组,n=10)、5/6肾大部切除模型组(SNx组,n=10)以及5/6肾大部切除+冬虫夏草菌粉干预组(CS组,n=10).术前及术后4、8、12周分别检测大鼠体质量、尿蛋白量变化,并于术后第12周末处死大鼠,检测血尿素氮、肌酐变化,取肾组织切片行HE、Masson染色观察肾脏病理变化,免疫组化观察转化生长因子β1(TGF-β1)及其Ⅰ型受体(TβR Ⅰ)、Ⅱ型受体(TβR Ⅱ)的表达,免疫荧光观察E-c adherin、α-SMA的表达,Western印迹法检测肾脏组织TGF-β1、TβR Ⅰ、TβR Ⅱ、磷酸化(p)Smad2/3、Smad7、E钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA)的表达.结果 术后CS组大鼠的体质量高于SNx组,尿蛋白量及血尿素氮、血肌酐低于SNx组.肾脏组织病理分析显示,CS组肾小球硬化、肾小管间质损伤程度均显著低于SNx组(均P＜0.01).CS组TGF-β1、TβR Ⅰ、TβR Ⅱ、p-Smad2/3蛋白表达量均显著低于SNx组(均P＜0.05),E-cadherin蛋白表达量显著高于SNx组(P＜0.05),α-SMA蛋白表达量显著低于SNx组(P＜0.05),Smad7蛋白表达量显著高于SNx组(P＜0.05).结论 冬虫夏草菌粉对5/6肾大部切除大鼠肾脏纤维化具有明显的抑制作用,其机制可能是与其抑制TGF-β1及其下游信号通路以及抑制EMT的发生有关.     

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.     

Activation of CXCL16 pathway by inflammation accelerates the progression of diabetic nephropathy

Objective To investigate the potential role of CXC chemokine ligand 16 (CXCL16)/CXC chemokine receptor 6 (CXCR6) pathway in the progression of diabetic nephropathy (DN). Methods 8 - week old male db/db mice were randomly divided into DN group and DN inflamed group. 10% casein was subcutaneously injected to induce the DN mouse model with inflammation. In vitro, HK-2 cells were treated with high glucose (HG), and IL-1β + HG to investigate the effect of inflammatory stress on HK-2 cells. Further knockdown CXCL16 was mediated by RNA interference to determine the effects of CXCl16, then cells were divided into HG + IL-1β group, HG + IL-1β + siCXCL16 group and HG + IL-1β + vehicle group. Changes of renal function in mice were assessed by 24 h proteinuria and N-acetyl-β-D-glucosaminidase (NAG) during 8 weeks. The ultra- microstructure was checked by electron microscopy at 8th week. Lipid accumulation in kidneys and HK - 2 were observed by Filipin staining and quantitative assay of intracellular free cholesterol. The protein expressions of CXCl16, CXCR6, a disintegrin and metalloproteinase-10 (ADAM10), fibronectin and α smooth muscle actin (α - SMA) in renal tissue were detected by immunohistochemistry and Western blotting. The mRNA and protein expressions of CXCl16, CXCR6, ADAM10, fibronectin and α-SMA in HK-2 cells were detected by real-time PCR and Western blotting, and protein expressions of CXCl16, CXCR6 and ADAM10 in HK - 2 cells were also tested by cell immunofluorescence. Results Mice in DN inflamed group had higher 24 h proteinuria and NAG than those in DN group, and the differences between two groups shown statistical significance at 8th week (all P＜0.05). Compared with DN mice, DN inflamed mice had more vacuoles within renal tubular cells, with mitochondrial swelling, deformation and decrease. Lipid accumulation and protein expressions of fibronectin and α-SMA were increased in DN inflamed group when compared with DN group (all P＜ 0.05). Further, the expressions of CXCL16, CXCR6, ADAM10 were significantly increased in DN inflamed group (all P＜0.05). In vitro, the mRNA and protein expressions of CXCL16, CXCR6, ADAM10, fibronectin and α-SMA, and lipid accumulation were increased in high glucose plus IL-1β group when compared with high glucose group (all P＜0.05). However, after siRNA of CXCL16 transfection, the mRNA and protein expressions of CXCL16, CXCR6, ADAM10, fibronectin and α-SMA were down-regulated in HG+IL-1β+siCXCL16 group as compared with high glucose+IL-1β group (all P＜0.05). Furthermore, lipid accumulation was decreased (P＜0.05). Conclusion Inflammation accelerates tubulointerstitial injury in DN partly through the activation of CXCL16 pathway, which may facilitate the lipid accumulation in tubular epithelial cells.     

AMPK attenuates inflammation to reduce fibrosis induced by acute ischemia reperfusion injury in mice

Objective To observe the effect of adenosine monophosphate activated protein kinase (AMPK) on attenuating inflammation in fibrosis induced by acute ischemia reperfusion injury (IRI) in mice. Methods Forty eight male C57BL/6 mice were randomly divided into four groups: sham operation group (sham group), IRI group, AMPK inhibitor+IRI group (AMPK/IRI group) and normal saline+IRI group (NS/IRI group), 12 mice each group. The mice with renal IRI were occluded for 30 min through clipping bilateral renal pedicle, then released renal perfusion. Mice in sham group were performed the separation of renal pedicle without clipping. Mice in AMPK/IRI group and NS/IRI group were respectively intraperitoneal injected AMPK inhibitor and normal saline before IRI. At the 2 d after operation, 6 randomly-selected mice from each group were blooded by extraction eyeball to detect BUN and Scr. The renal histopathological changes were observed through HE staining. The mRNA expression of IL-1β, IL-6 and TNF-α was detected by real time PCR, and the level of AMPK phosphorylation was detected by Western blotting. At the 14 d after operation, Collagen 1 (COL1), α-SMA and fibronectin (FN) were detected by immunofluorescence and Western blotting in 6 remained mice from each group. The degree of kidney fibrosis was observed through sirus red staining. Results Compared with those in sham group, tubular interstitial damage was aggravated (P＜0.05), BUN and Scr were increased (P＜0.05), the mRNA expression of IL-1β, IL-6 and TNF-α was increased at the 2 d after operation (all P＜0.05), and the level of AMPK phosphorylation was activated in IRI group and NS/IRI group (all P＜0.05); the degree of kidney fibrosis and the expression of COL1, α-SMA and FN were increased obviously at the 14 d (all P＜0.05). Compared with those in IRI group, in AMPK/IRI group tubular interstitial damage was aggravated (P＜0.05), BUN and Scr were increased (all P＜0.05), the mRNA expression of IL-1β, IL-6 and TNF-α was increased at the 2 d (all P＜0.05), and the level of AMPK phosphorylation was decreased (P＜0.05). Moreover, the degree of kidney fibrosis and the expression of COL1, α-SMA and FN were increased obviously at the 14 d in AMPK/IRI group (all P＜0.05). Conclusions AMPK can ameliorate the acute renal ischemia reperfusion injury induce fibrosis in mice, and the mechanism may be related to the decrease of inflammatory reaction.     

Effect of long-term low-dose 1α, 25-dihydroxy vitamin D3 on the expression of aquaporin 2 in kidneys of uremic rats

Objective To investigate the effect of long-term low-dose 1α, 25-dihydroxy vitamin D3 [1,25(OH)2D3] on rat kidney aquaporin (AQP) 2 expression in 5/6 nephrectomized rats. Methods Twelve Sprague-Dawley rats underwent 5/6 nephrectomy surgery were divided into model group (n＝6) and 1,25(OH)2D3 group (n＝6) randomly; sham-operated rats only received the renal capsule stripping (control group, n＝6). Rats in 1,25(OH)2D3 group received 1,25(OH)2D3 (3 ng•100 g-1•d-1, ip) for 24 weeks. Serum and 24-hour urine specimens were collected for measurement of serum creatinine, arginine vasopressin (AVP) and urine protein. Animals were sacrificed at week 24 and kidneys were removed for routine pathological, immunohistochemistry and immunoblotting analysis. Results At week 24, plasma AVP level in 1,25(OH)2D3 and model group was much higher than that in control group (all P＜0.05), with no significant differences between the former two groups (P＞0.05). Lower serum creatinine and urinary protein were presented in 1,25(OH)2D3 group compared with the model group rats at week 24 (P＜0.05). Renal medullar fibrosis and inflammatory cell infiltration were improved significantly in 1,25(OH)2D3 group compared with model group (P＜0.01, P＜0.05). Immunohistochemistry analysis revealed abundant AQP2 and p-AQP2 expressed in the renal medulla of sham group, mainly in apical membrane of collecting duct cells. AQP2 expression in model group was down-regulated (P＜0.05) and p-AQP2 expression in apical membrane was reduced. AQP2 expression in 1,25(OH)2D3 group increased compared with model group, with increased p-AQP2 expression in apical membrane. Western blotting revealed same results of these expressions (all P＜0.05). Correlation analysis showed a negative correlation of AQP2 expression with urine volume, medullary fibrosis, and inflammatory cell infiltration (P＜0.05). Conclusion Long-term low-dose 1,25(OH)2D3 improves AQP2 expression and response to AVP in collecting duct, which may involve in the anti-polyuric effect of 1,25(OH)2D3 in uremic rat.     

Effect of NADPH oxidases inhibition on the development of interstitial fibrosis in unilateral ureter obstruction rats

ObjectiveTo clarify whether the NADPH oxidases (NOXs) family contributed to the reactive oxygen species (ROS) production and subsequent interstitial fibrosis in unilateral ureter obstruction (UUO) rats. MethodsMale Wistar rats were randomly divided into sham operation group (n＝8), sham operation + apocynin treatment group (n＝8), UUO operation group (n＝8) and UUO operation+apocynin treatment group (n＝8). Either vehicle or apocynin (100 mg/kg per day) were given by gavage for 7 days after surgery. Rats were sacrificed at 7th day. ELISA was used to detect the activity of superoxide dismutase (SOD) and catalase (CAT), and the level of 8-iso-prostaglandin F2alpha (8-iso- PGF2α) in renal tissue. Western blotting was used to detect the protein expressions of NADPH oxidase subunit NOX2 and NOX4, α- smooth muscle actin(α-SMA), collagen I (COL-I) and the level of ERK1/ 2 phosphorylation (p-ERK1/2). ResultsUUO rats with vehicle displayed increased oxidative stress, as measured by renal tissue 8-iso-PGF2α, accompanied with increased renal expression of NADPH oxidases (NOX2, 1.5-fold and NOX4, 1.7-fold, respectively), compared with sham-operated rats (P＜ 0.05). Furthermore, vehicle treated UUO rats showed increased renal COL - I and α - SMA levels, compared with sham-operated rats (P＜0.05). ERK1/2 was also activated as detected by p-ERK1/2 expression in UUO rats with vehicle (P＜0.05). Apocynin treatment significantly decreased renal tissue 8-iso-PGF2α level and expressions of NOX2 (-28.7%) and NOX4 (-31.0%) in UUO rats, respectively, compared with vehicle treated rats (P＜0.05). And significant decrease of COL-I (-26.4%) and α-SMA expression (-80.0%) were also observed (P＜0.05). The activation of ERK1/2 in UUO rats was greatly inhibited by apocynin treatment (P＜0.01). Despite the pronounced dysregulation of pro – oxidative NOXs family, no compensatory increase of antioxidative enzyme activities occurred. ConclusionThe NOXs family contributes largely to the production of ROS and subsequent interstitial fibrosis after ureter ligation, and inhibition of the NOXs family may be a choice for preventing interstitial fibrosis.     

细胞间黏附分子-1在自发性高血压大鼠肾损害作用的研究

目的：研究细胞间黏附分子-1（ICAM-1）在自发性高血压大鼠（SHR）肾组织的表达及其与肾损害的关系。方法：以同龄雄性正常血压大鼠（WKY）和SHR为研究对象,分别于10周龄和28周龄检测两种大鼠尾动脉压、24h尿蛋白定量、肾功能等;留取肾组织行HE染色、免疫组织化学及RT-PCR法检测ICAM-1蛋白及mRNA表达情况,并作分析。结果：与同龄WKY大鼠比较,SHR尾动脉压和24h尿蛋白定量明显增高（P〈0.05和P〈0.05）;与12周龄SHR比较,28周龄SHR尾动脉压和24h尿蛋白定量明显增加（P〈0.05和P〈0.05）。SHR组肾组织ICAM-1蛋白及mRNA表达较同龄WKY增强（P〈0.05）,28周龄SHR较10周龄SHR表达增强（P〈0.05）,ICAM-1表达与24h尿蛋白定量呈正相关（P〈0.05）。结论：SHR出现蛋白尿时,肾组织ICAM-1表达增强,炎症参与了高血压肾损害的发生。