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Role of DC-SIGN in renal tubulointerstitial lesions of immune-mediated experimental nephritis and intervention regulation by anti-P-selectin domain monoclonal antibody

Minchao CAI ; Jie ZOU ; Tong ZHOU ; Xiao LI ; Jing CHEN ; Chaoming MAO ; Yanyun ZHANG ; Nan CHEN ; Chundi XU

Chinese Journal of Nephrology.2010;26(5):376-383. doi:10.3760/cma.j.issn.1001-7097.2010.05.015

Objective To explore the role of dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) in the tubulointerstitial lesions of immune-mediated nephrotoxic nephritis (NTN) and the intervention regulation by anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb). Methods WKY rats were randomly divided into control,NTN and PsL-EGFmAb-treated groups. The mrs in NTN group were injected with 1 ml nephrotoxic rabbit serum per kilogram of rat body weight; the ones in PsL-EGFmAb-treated group were injected with 2 mg PsL-EGFmAb per kilogram of rat body weight simultaneously and 2 h later after nephrotoxic rabbit serum injection; and those in control group were injected with equal volume of 0.9% saline. Renal function and pathology were observed at day 4, 7 and 14 after the induction of NTN. Distribution of DC-SIGN + dendritic cells (DCs) in renal tissues was measured by immunofluorescence. Real-time PCR was performed to examine the expression of P-selectin,RANTES, TNF-α, IL-10, IFN-γ and IL-4. Expression of MHC Ⅱ , CD80 and DC-SIGN on dendritic cells was analyzed by flow cytometry. Transendothelial migration was used to detect the ability of DCs migration. DCs ability to activate T cells was determined by mixed lymphocyte reaction (MLR). ELISA was used to detect the concentration of IFN-γ and IL-4 in the supernatant of MLR. Results At day 4, immature DC-SIGN+ DCs infiltrated the rat renal tubulointerstium of NTN group, matured at day 14, and enhanced the ability to migrate and activate T cells. The distribution of DC-SIGN + DCs was significantly related to the form of crescent, tubulointerstial lesions and renal function. In addition, expression of chemokine RANTES and proinflammatory cytokine TNF-α continuously augmented since day 4, while anti-inflammatory eytokine IL-10 decreased after markedly increased at day 4. At day 14, IFN-γ/IL-4 mRNA increased, which was obviously related to DCs maturation. The intervention of PsL-EGFmAb supressed the expression of DC-SIGN and CD80 on DCs, depressed DCs maturation, migration and ability to activate T cells,down-regulated proinflammatory cytokines and up-regulated anti-inflammatory cytokines in kidney,and thus regulated Th1/Th2 bias. At the same time, kidneys showed the decrease of crescents,improvement of tnbulointerstium damage and renal function. Conclusions DC-SIGN may mediate DCs tubulointerstitial infiltration. It may be also a potent regulator of local immune reaction imbalance and pathology of tubulointerstium. PsL-EGFmAb may depress DCs migration and downregulate DCs maturation and function through DC-SIGN, and thus having a role in prevention and treatment.

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Protective effect of N-acetyl-L-cysteine on the cytotoxicity induced by iohexol in HK-2 cells

Ning DAI ; Hua WU ; Tianhui LI ; Ban ZHAO ; Huan CHEN ; Zhan LI

Chinese Journal of Nephrology.2010;26(5):370-375. doi:10.3760/cma.j.issn.1001-7097.2010.05.013

Objective To explore the protective effect and mechanism of antioxidant N-acetyl-L-cysteine (NAC)on the cytotoxicity induced by iohexol in HK-2 cells. Methods The incubated HK-2 cells were divided into four groups:control group,iohexol group,NAC group,and NAC+iohexol group(pre-incubated with NAC and then co-incubated with iohexol).The cell viability was tested by CCK-8 assay;cell apoptosis was determined by Hoechst 33342 fluorescence staining and flow cytometry with Annexin V-FITC/PI double staining.Intracelluar ROS waft detected by flow cytometry with DCFH-DA fluorescence staining.The signaling transduction pathways were investigated by Western blotting and immunofluorescence staining. Results Iohexol decreased cell viability,and increased apoptosis in a dose-and time-dependent manner.In iohexol(100 gl/L,6 h)group,ROS was increased by 1.30-fold of control(P＜0.05).In NAC(5,10,15 mmol/L)+iohexol groups,the cell viability was increased by 104%,118%,130%respectively,and iohexol group was 63% (P＜0.05, respectively); apoptosis rate was decreased by 13.51%, 13.46%, 12.23% respectively, and iohexol group was 24.41% (P＜0.05, respectively); ROS was decreased by 1.05-fold, 0.93-fold, 0.86-fold respectively, and iohexol group was 1.3-fold (P＜0.05, respectively).Iohexol induced the increase of p53 phosphorylatian and activity, then up-regulation of Bax and down-regulation of Bcl-2 protein expression. Iohexol induced the release of cytochrome C from mitochondria to cytoplasm, all of which caused final activation of caspase-3. The expression levels of p53, Bax and caspase-3 were decreased, while Bcl-2 protein expression level was increased by NAC. Conclusions Iohexol induces the increase of apeptosis rate and ROS generation in HK-2 cells. NAC attenuates this iohexol-induced cytotoxicity by decreasing intracelluar ROS, which is mairdy through the intrinsic pathway.

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Effect of local aldosterone on renal epithelial-mesenchymal transition in diabetic nephropathy rats

Qiaoling ZHOU ; Kanghan LIU ; Pouranan VEERARAGOO ; Huiyi HUO ; Mingxia YUAN ; Zhou XIAO ; Weisheng PENG

Chinese Journal of Nephrology.2010;26(5):364-369. doi:10.3760/cma.j.issn.1001-7097.2010.05.011

Objective To explore the effect of aldosterone on renal epithelialmesenchymal transition in streptozocin(STZ)-induced diabetic nephropathy rats. Methods Wistar rats were intraperitoneally injected with STZ(60 mg/kg)for the preparation of diabetic model.After 4 weeks,the rats with urinary protein＞30 mg/d were regarded as successful diabetic nephropathy(n=16),and were randomly divided into diabetic nephropathy(DN group,n=8)and spironolactone group(SP group,n=8).Then eight healthy rats were selected randomly as control group(N group,n=8).SP group rats were treated with spironolactone 40 mg·kg-1·d-1,and N group and DN group rats were given equal water.After 8 weeks,rats were sacrificed to collect urine,blood plasma,kidney tissue for detection of 24 h urinary protein,creatinine and renal pathological changes.Aldosterone concentration in plasma and kidney tissue was detected by mdioimmunoassay;E-cadherin,α-SMA protein expression by immunohistochemistry,Western blotting; E-cadherin,α-SMA mRNA expression by RT-PCR. Results Compared with N group,serum creatinine, urinary protein excretion in the DN rats were significantly higher (P＜0.01,respectively), E-cadhefin protein and mRNA were significantly reduced (P＜0.01, respectively),α-SMA protein and mRNA expression was up-regulated (P＜0.01, respectively). Aldosterone level of kidney tissue in DN rats was increased obviously [(24.71±5.30) ng/g vs (16.38±2.85) ng/g, P＜0.01], which was positively correlated with urinary protein excretion, serum creatinine and α-SMA protein (r=0.737, 0.574, 0.688, P＜0.01, respectively), and negatively correlated with E-cadherin protein (r=-0.659, P＜0.O1). While no significant difference was found in serum aldosterone among three groups. Compared with DN rats, urinary protein excretion, serum creatinine were reduced (P＜0.01, respectively), E-cadherin protein and mRNA were increased (P＜0.01, respectively), α-SMA protein and mRNA expression were decreased (P ＜0.01, respectively) in SP group rats.Conclusions Local aldosterone involves in renal epithelial-mesenchymal transition in diabetic nephropathy rat. Spironolactone can block the effect of aldosterone and play a role in renal protection.

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Suppressor of cytokine signaling-1 inhibits high glucose-induced expression of monocyte chemoattractant protein-1 in glomerular mesangial cells

Yonghong SHI ; Chunyang DU ; Yunzhuo REN ; Song ZHAO ; Jun HAO ; Huijun DUAN

Chinese Journal of Nephrology.2010;26(5):352-357. doi:10.3760/cma.j.issn.1001-7097.2010.05.008

Objective To investigate the effect of suppressor of cytokine signaling-1 (SOCS-1)on expression of monocyte chemoattractant protein-1 (MCP-1)in human glomerular mesangial cells (HMCs) under high concentration of glucose. Methods Stable transfections of HMC with pCR3.1 vector and pCR3. 1-SOCS-1 were performed with hpofectamine 2000, and cells were selected with geneticin. Cells were stimulated with low glucose (LG, 5.5 mmol/L), high glucose (HG, 30 mmol/L), LG plus mannitol (24.5 mmol/L) and AG490 (10 μmol/L). The protein expression levels of SOCS-1, signal transducer and activators of transcription 1,3 (STAT1, STAT3),p-STAT1 and p-STAT3 were observed by Western blotting. The protein synthesis of MCP-1, FN and type Ⅳ collagen in the supernatants of the HMCs were detected by ELISA and radioimmunoassay. The expression level of SOCS-1 and MCP-1 mRNA was measured by BT-PCR.Results HG induced the expression of SOCS-1 protein and mRNA in HMCs in time-dependant manner, peaked at 4 h, and gradually decreased to baseline at 24 h. Compared with low glucose control group, the phosphorylation levels of STAT1 and STAT3 and the expression of MCP-1 mRNA [(0.39±0.05) vs (0.16±0.02)]were significantly increased in HMCs under high glucose medium (P ＜0.01 ). Exposure of HMCs to high glucose conditions showed high concentration of MCP-1 [(459±67) ng/L vs (241±19) ng/L], fibronectin [(5.84±0.61) mg/L vs (3.41±0.31) mg/L]and type Ⅳ collagen [(16.45±2.30) μg/L vs (9.56±1.52) μg/L]in the supernatants (all P＜0.01).Overexpression of SOCS-1 inhibited the phosphorylation levels of STAT1 and STAT3 and the expression of MCP-1 mRNA [(0.34±0.04) vs (0.42±0.05)]in HMCs under high glucose condition (all P＜0.05). Compared with vector control group, the concentration of MCP-1 [(387±47) ng/L vs (463±56) ng/L], fibronectin [(4.61±0.57) mg/L vs (5.76±0.74) mg/L]and type Ⅳ collagen [(13.4±2.32) μg/L vs (17.1±2.57) μg/L]was decreased in supernatants of HMCs with SOCS-1 overexpression (all P＜0.05). Compared with HG group, the expression of MCP-1 mRNA (0.31±0.04) and the concentration of MCP-1 [(361±53) ng/L], FN [(5.46±0.71) mg/L]and type Ⅳ collagen [(15.2±1.97) μg/L]in supernatants were decreased in HMCs treated with AG490.Conclusion Overexpression of SOCS-1 inhibits overproduction of MCP-1 and ECM proteins in HMCs under high glucose conditions, which may be partly by regulating the phosphoryhtion of STAT1 and STAT3.

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Effect of serum uric acid on clinicopathology and prognosis of IgA nephropathy

Genyang CHENG ; Dongwei LIU ; Zhangsuo LIU

Chinese Journal of Nephrology.2010;26(5):343-345. doi:10.3760/cma.j.issn.1001-7097.2010.05.006

Objective To explore the effect of serum uric acid (SUA) on the clinicopathological manifestation and prognosis of IgA nephropathy(IgAN)patients. Methods A total of 348 patients with renal biopsy-proven IgAN in our hospital were enrolled in this study.The data were retrospectively analyzed to examine the association of SUA level with clinicopathological manifestation and prognosis of IgA nephropathy(IgAN)patients. Results There were no significant differences of 24 hour proteinuria,BUN and Scr between patients of high SUA level with various GFR and those of normal SUA level.While differences of glomerular sclerosis,tubulointerstitial scores and vascular injury between these two groups were significant (P＜0.05).At the end of follow-up,prevalence of GFR decline and ESRD was significantly higher in patients with high SUA as compared to those with normal SUA(40.82%vs 15.70%,64.71% vs 35.00%,respectively,P＜0.05). Conclusions Patients with different SUA levels have similar clinical manifestations,but different pathological findings and prognosis.It is important to pay attention to the follow-up of SUA level in IgAN patients.

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Interrelationship among urinary albumin elimination rate, serum adiponectin and blood glucose level in patients with type 2 diabetic nephropathy

Xiaoling ZHOU ; Menghua CHEN

Chinese Journal of Nephrology.2010;26(5):339-342. doi:10.3760/cma.j.issn.1001-7097.2010.05.005

Objective To investigate the interrelationship among urinary albumin elimination rate(UAER),serum adiponectin(ADPN)and blood glucose level in patients with type 2 diabetic nephropathy(DN). Methods A total of 89 type 2 diabetic patients and 30 healthy people as control were enrolled in the study.According to UAER,the diabetic patients were divided into three groups:normal group(35 cases,UAER＜30 mg/24 h),microalbuminura group (32 cases,UAER 30-300 mg/24 h)and macroalbuminura group(22 cases,UAER＞300 mg/24 h).Serum adiponectin was measured by ELISA.Body mass index (BMI),waist hip radio(WHR),fasting plasma glucose(FPG),2-hour plasma glucose(2hPG),glycated haemoglobin(HbAlc),blood lipid and UAER were measured routinely.Data were compared among groups. Results Serum adiponectin level was lower in diabetic patients than that in healthy control group(P＜0.05).Among diabetic patients.serum adiponectin of macroalbuminura group was significantly higher than that of normal and microalbuminura groups[(67.74±14.89)vs(39.36±13.92),(54.38±10.14)ng/L,P＜0.051.Multiple regression analysis showed 2hPG,HbAlc,BMI,disease course,UAER and DBP were closely associated with the level of serum adiponectin. Correlation analysis indicated serum adiponectin was positively correlated with age (r=0.255), disease course (r=0.405), HDL (r=0.501)and UAER (r=0.843); while negatively with HbAlc (r=-0.479), FPG (r=-0.436), 2hPG(r=-0.418),BMI (r=-0.479) and WHR (r= -0.531), all P＜0.01. Conclusions Serum adiponectin is positively correlated with UAER and negatively correlated with FPG. Blood glucose level is one of the main factors affecting serum adiponectin. Strict controlling of blood glucose level is beneficial to higher level of ADPN.

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Effect of 4-phenyibutyric acid on diabetic nephropathy rats

Zhifeng LUO ; Jiao MU ; Wei QI ; Wei ZENG ; Yanhong GUO ; Bing FENG

Chinese Journal of Nephrology.2010;26(5):358-363. doi:10.3760/cma.j.issn.1001-7097.2010.05.009

Objective To investigate the effect of 4-phenylbutyric acid(4-PBA)on the renal pathogenesis of rats with streptozotocin-induced diabetes and its mechanism. Methods Fifty-four male SD rats were randomly divided into three groups:normal control group(NC group,n=18),diabetic nephropathy group(DN group,n=18),diabetic nephropathy plus 4-PBA treatment group(4-PBA group,n=18).At the end of 4,8 and 12 weeks,index of kidney weight/body weight ratio(KI)were measured and calculated.Serum creatinine (Scr),blood urea nitrogen(BUN),urinary MDA levels,urinary SOD activity,and 24 hour urinary protein excretion ram(UAER)were detected by HITACHI automatically.Morphology of kidney wag examined by special staining of periodic acid-schitt (PAS).The p47phox and nitrotyrosine (NT) expression in kidney were determined by real-time fluorescence PCR and Western blotting. Results Compared with the NC group, the DN group rats showed a significant increase of KI(P＜0.05), UAER(mg/24 h) (4.92±0.70 vs 0.26±0.07, 5.29±0.83 vs 0.28±0.08, 5.54±0.81 vs 0.29±0.04,respectively, P＜0.05]for indicated time, mesangial cells proliferation and mesangial matrix expansion at 12 week. However,4-PBA treatment could significantly inhibit the increase of KI (P＜0.05), decrease UAER (mg/24 h) (3.71±0.37, 3.47±0.36, 3.28±0.40, respectively, P＜0.05]for indicated time, and prevent the glomeruler pathological alteration induced by diabetes. Moreover, the mRNA expression of p47phox in the kidney of DN group was 154.72%, 148.60% and 91.95% more than that of NC group (all P＜0.05) for indicated time. The protein expression of p47phox was 118.00%, 140.10% and 177.82% more than that of NC group (all P＜0.05), and the protein expression of NT was 45.29%,59.13% and 89.28% more than that of NC group (all P＜0.05). In addition, urinary MDA levels in DN group were 2.05-, 2.26- and 2.43- folds of NC group, and urinary SOD activities were decreased by 64.78%, 71.29% and 79.32% of NC group. Compared with the DN group, the mRNA and protein expression of p47phox, and protein expression of NT in 4-PBA group were decreased markedly (all P＜0.05) at the end of 8 and 12 weeks. The urinary MDA level was decreased, and the urinary SOD activity was increased significantly in rats with diabetes after 4-PBA treatment for indicated time (all P＜0.05). Conclusion 4-PBA treatment can significantly inhibit the renal pathogenesis of rats with diabetes through inhibition of oxidative stress.

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NG2 proteoglycan promotes mesangial cells proliferation and extracellular matrix production

Jing XIONG ; Yang WANG ; Jianshe LIU ; Zhonghua ZHU

Chinese Journal of Nephrology.2010;26(1):43-47. doi:10.3760/cma.j.issn.1001-7097.2010.01.012

Objective To explore the role of NG2 proteoglycan in the pathogenesis of glomerulosclerosis. Methods Eukaryotic expression vectors carrying the small hairpin RNA (shRNA) for NG2 mRNA , named as Psilencer-NG2, was constructed. Then, rat mesangial cells (RMC) were transfeeted with Psilencer-NG2, Psilencer-NC (negative control), pcDNA/NG2 (NG2 over-expressive vector) and empty vector pcDNA 1 respectively. The expression of endogenous NG2 in RMCs was examined by real-time PCR and Western blotting. Cell proliferation was analyzed by MTT assay and flow cytometry. The expression of laminin was detected by real-time PCR. Results Transfection of pcDNA/NG2 into HBZY-1 cells resulted in over-expression of NG2 mRNA and protein (P<0.05, P<0.05). Transfection of Psilencer-NG2 led to reduced expression of NG2 mRNA and protein (P<0.01, P<0.01). The expression of laminin β1 significantly increased due to overexpression of NG2 and decreased by treating with NG2 siRNA. According to MTT assay, overexpreasion of NG2 significantly stimulated the proliferation of mesangial cells while NG2 silencing inhibited it. NG2 increased the cell number in S phase and decreased the cell number in G0/G1 phase, while silencing NG2 induced the decrease of cell number in S phase and the increase of cell number in G0/G1 phase. Conclusion NG2 actively participates in the pathogenesis of glomerulosclerosis by stimulating proliferation of RMCs and increasing the deposition of ECM.

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Expression and regulation of aquaporins in cystic epithelial cells of mice with polycystic kidney disease

Xiufang KAN ; Hong ZHOU ; Baoxue YANG

Chinese Journal of Nephrology.2010;26(1):39-42. doi:10.3760/cma.j.issn.1001-7097.2010.01.011

Objective To study the expression and regulation of aquapofins (AQP) in cystic epithelial cells of jck mice with polycystic kidney disease. Methods Localization and regulation of AQP1, AQP2, AQP3 and AQP4 protein were analyzed by using the immunofluorescence and Western blotting. Results Kidneys of jck homozygous mice were 4 folds larger than those of litter matched wild-type mice. There were multiple cysts and fibrosis in the renal tissue of jck mice. The epithelial cells in cysts were flat in shape. Blood urea level in jck mice was (42.6 ± 6.7) mmol/L, which was 5 folds higher than that in wild-type mice [(8.4±1.9) mmol/L] (P<0.01). Immunofluorescence analysis showed that AQP1 was expressed in the apical and hasolatend membranes of epithelial cells in proximal tubules, as well as in the thin descending limb of Henle and endothelial cells of descending vasa recta. There was no AQP1 expression in epithelial cells of cysts. AQP2 was expressed in the apical membranes of collecting ducts and renal cysts. AQP3 and AQP4 were expressed in basolateral membranes of collecting duct and renal cystic epithelial cells of jck mice. Western blot analysis showed the same protein sizes of AQP1, AQP2, AQP3 and AQP4 in both jck and wild-type kidneys. However, AQP1 expression was down-regulated in jck kidneys (P<0.01). Conclusion The renal cystic epithelia expresses AQP2, AQP3 and AQP4, which indicates that epithelial cells in renal cysts are derived from renal collecting ducts in jck mice and aquaporins may play an important role in renal cyst development.

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Establishment of type 2 diabetes mellitus with early renal injury on spontaneously hypertensive rats

Houyong DAI ; Rining TANG ; Kunling MA ; Min ZHENG ; Jie NI ; Qing LI ; Xiaoliang ZHANG ; Bicheng LIU

Chinese Journal of Nephrology.2010;26(1):48-52. doi:10.3760/cma.j.issn.1001-7097.2010.01.013

Objective To develop a model of type 2 diabetes with early renal injury on spontaneously hypertensive rats (SHR). Methods The 6-week old SHR were fed with the diets enriched with sucrose (20%, W/W), lard (10%, W/W), cholesterol (2.5%, W/W) and chleolate (1%, W/W) to induce insulin resistance. Hyperglycemia was developed by intraperitoneal injection of streptozotocin (STZ, 35 mg/kg). Wistar-Kyoto rats (WKY) were used as normal controls. Rats with plasma glucose (PGL) ≥ 16.7 mmol/L were diagnosed as diabetes. Eight weeks after the induction of diabetes, plasma triglyceride (TG), cholesterol (CHO), glucose, systolic pressure(SP), 24-h urine protein excretion (Upro) were examined in all the rats, and the homeostasis model assessment of insulin resistance (HOMA-IR) was analyzed. Renal pathological changes were studied by immunohistochemical staining and electron microscope. Results After 2 weeks on the high sucrose and fat diets, the model rats exhibited significant increase in basal PGL, TG and CHO levels as compared to control rats (P<0.05, respectively). The insulin resistance was developed in model rats demonstrated by the higher HOMA-IR (5.03±0.38 vs 2.61±0.34, P<0.05). At the end of the experiment, model rats were associated with hypertension. Upro level was significantly increased in model rats compared with that in controls [(57.58±16.54) mg/24 h vs (5.35±1.90) mg/24 h, P<0.01]. The kidney hypertrophy index (KWI) was significantly increased in the model rats compared to controls (P <0.05). Moreover, the diabetic model rats showed glomerular hypertrophy, foot process effacement, micro villous transformation, glomerular basement membrane (GBM) thickening. Conclusion A rat model is successfully established, which presents typical features of human type 2 diabetes and can be served as an ideal model to study the diabetic nephropathy.

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