Objective To elucidate the relationship between expressive level alteration of glucose transporter 4(GLUT 4) mRNA,cyclin kinase inhibitor p21 mRNA and glomerular mesangial cell(GMC) hypertrophy in cultured rat GMC.Methods Cultured rat 1097 GMCs were divided into high glucose group, mannitol group, different insulin concentration groups, high glucose plus different insulin concentration groups and control group. Semi quantity RT PCR and flow cytometery were used to detect GLUT 4mRNA and p21mRNA levels and GMC volume. Results A certain expression of GLUT 4mRNA and p21mRNA from GMC was found in control group. High glucose decreased GLUT 4mRNA level and increased p21mRNA level. Insulin up regulated GLUT 4mRNA expression in a dose dependent manner.The more p21mRNA expressed, the stronger forward scatter (FSC) was and the bigger GMC became. Conclusions High glucose can cause GMC hypertrophy. Up regulation of p21mRNA and down regulation of GLUT 4mRNA may be involved in GMC hypertrophy and glomerular hypertrophy in early diabetic nephropathy.

Objective To explore the role and significance of vascular endothelial growth factor(VEGF)and angiopoietin-2(Ang-2)in peritubular angiogenesis of diabetic kidney.Methods diabetic rats were induced by Streptozotocin.The expressions of VEGF and Ang-2 in renal tissue of rats were detected by immunohisochemistry.VEGF and Ang-2 mRNA in kidney was also detected by RT-PCR,and quantified by computerimage analysis.Results From 2-week to 24-week,VEGF mRNA level in diabetic renal upregulated continuously compared with control group,with peak level at 16 and 20 weeks.VEGF immunostaining in diabetic renal tubuli increased apparently compared with control group.Ang-2 mRNA in diabetic renal was only detected at 16 and 20 weeks.Ang-2-expressing peritubular microvessel in diabetic renal cortex was found by immunohistochemistry from 12 -week to 24-week with peak level at 16 week.No detecable Ang-2 mRNA and immunostaining were found in control renal.The changes correlated VEGF with Ang-2 in diabetic renal after 12 weeks.Conclusion There are the formation of Ang-2-staining peritubular microvessels in diabetic renal cortex in middle and later stages.VEGF and Ang-2 take part in angiogenesis in diabetic renal.

Objective To investigate the effects of high glucose and insulin on the expression of glucose transporter 4 (GLUT4), Cbl-associated protein (CAP) and cytoskeleton protein F-actin of glomerular mesangial cells (GMCs), in order to explore the function of GLUT4, Cbl-associated protein and F-actin in the pathogenesis and development of diabetic nephropathy (DN). Methods Cultured 1097 rat glomerular mesangial cells were divided into 8 groups: control, 10-9 mol/L insulin, 10-8 mol/L insulin, 10-6 mol/L insulin, high glucose (30 mmol/L), mannitol (25 mmol/L mannitol+5 mmol/L glucose), high glucose plus 10-6 mol/L insulin, high glucose plus 10-9 mol/L insulin. Expression of CAP mRNA and GLUT4 was measured by RT-PCR and immunohistochemistry method. F-actin was stained by rhodamine-pholloidin and the fluorescent intensity was calculated by image analysis system. Results The expression of GLUT4 mRNA and protein, CAP mRNA was found in normal giomerular mesangial cells (control), and there was no significant difference in 10-9 mol/L insulin group. The expression of GLUT4 mRNA (P<0.05) and protein (P<0.01), CAP mRNA (P<0.01) level was decreased in high glucose group compared with that of control group, but there was no significant difference in mannitol group. The expression of GLUT4 and CAP mRNA up-regulated with the increase of concentration of insulin. The expressions of GLUT4 mRNA in 10-8 mol/L insulin and 10-6 mol/L insulin groups were 2.06-fold and 2.66-fold of 10-9 mol/L insulin group, of GLUT4 protein were 1.93-fold and 2.83-fold of control, and of CAP mRNA were 1.91-fold and 2.15-fold of control, respectively. The expressions of GLUT4 mRNA, GLUT4 protein, CAP mRNA in high glucose plus insulin group were 2.15-fold, 2.08-fold, 2.14-fold of high glucose group respectively. High glucose decreased the fluorescent intensity of F-actin to 44.5% (P<0.01). 10-8 mol/L insulin and 10-6 mol/L insulin groups increased to 1.224-fold (P<0.05), 1.296-fold (P<0.01) in a concentration-dependent manner. The spearman correlation coefficient between GLUT4 and F-actin was 0.929 (P=0.001), between GLUT4 mRNA and CAP mRNA was 0.905 (P=0.002). Conclusions (1) A certain expression of GLUT4 mRNA and protein, CAP mRNA from GMC is found in normal glomerular mesangial cells. (2) High glucose can inhibit the expression of GLUT4 and CAP mRNA significantly, and facilitate the depolymerization of F-aetin. (3) Insulin can reverse down-regnlation of GLUT4 and CAP mRNA caused by high glucose. (4) GLUT4, CAP and F-actin are important factors in the development of DN.

Objective To observe the effects of turicamycin-inhibitor of N-glycosylation of proteins on expression of (?1 integrin,FAK and cyclin D1 in high glucose-induced glomerular mesangial cells (CMC), and to explore the mechanism concerned. Methods Cultured HBZY-1 rat mesangial cells were divided into 5 groups:control group;high glucose group;mannitol group;high glucose plus TM group; TM group.The expression of ?1 integrin was measured by flow cytometry, expression of FAK and cyclin D1 was measured by immunohistochemistry,and proliferation of GMCs was measured by MTT. Results There was a little expression of ?1 integrin, FAK and cyclin D1 on normal mesangial cells. High glucose induced the proliferation and increased the expression of ?1 integrin and cyclin D1.There was no significant difference in mannitol group as compared to control group.The expression of (?1 integrin FAK and cyclin D1 decreased notably by TM.TM could also decrease proliferative abilitiy of cells. All the effects of TM were dose-dependent. Conclusion Through blocking glycosylation of glycoprotein, TM can suppress cellular proliferation and expression of pi integrin induced by high glucose in a dose-dependent manners,then pi integrin affects the expression of FAK and cyclin D1.

Objective To investigate the effects of tunicamycin(TM)-inhibitor of N-glycosylation of proteins on proliferation and apoptosis of glomerular mesangial cells(GMC) and synthesis of FN. Methods Morphology of GMC apoptosis was observed under eletron microscopy, and flowcytometry was used for assessing apoptosis rate. GMC proliferation was examined by MTT assay. The amount of FN was measured by ELISA. Results TM inhibited the proliferation of GMC as compared with untreated controll ( P

AIM: To investigates the role of phosphatidic acid (PA) in the expression of several inflammatory mediators produced by glomerural macrophages (GM?). METHODS: The study was performed on a rat model of accelerated anti-glomerural basement membrane (anti-GBM) glomerulonephritis (GN). GN-GM? were isolated and identified. Peritoneal M? (P-M?) of both normal and GN rats were used as controls. Block and reverse test were investigated with rhIL-1? stimulated, lisofylline (LSF) and phosphatidic acid (PA). Macrophage expression of ICAM-1 and TGF-? 1 were assessed at the level of protein and gene by immunocytochemistry, northern blot and RT-PCR. RESULTS: (1) After stimulated with rhIL-1?, GN-GM? produced much more ICAM-1, MCP-1 and TGF-? 1 than P-M?, and it's gene expression was similar as protein product. (2) mRNA expression of these factors was up-regulated again after the GN-GM? were pretreated with LSF then PA was added. CONCLUSIONS: Since GN-GM? plays an important role for PA in the mediation of glomerular injury, inhibiting of PA production is the keypoint of blocking M? mediated inflammatory effects. LSF may be an effective medicine in therapy for acute inflammatory forms of GN.

In this study, we assessed the effects of shear stress on the expression of plasminogen activator(tPA and uPA) mRNA in cultured NRK-52E cells (a kidney proximal tubular epithelial cell line of normal rat origin) and investigated the mechanism of tubulointerstitial extracellular matrix (ECM) remodeling in the early stage of diabetic nephropathy (DN). The cultured NRK-52E cells were exposed to shear stress of 5 and 10 dyn/cm2 for 1, 3 and 6 hours respectively. Semi-quantity RT-PCR was used to detect the expression of tPA and uPA mRNA. Shear stress down-regulated the expression of tPA and uPA mRNA in cultured NRK-52E cells in a magnitude and time-dependent way. The results suggested that the increased tubular shear stress in the early-stage of DN could decrease the expression of tPA and uPA in renal proximal tubular cells, lead to the reduction of tubulointerstitial fibrinolytic activity and involve in the remodeling of ECM.
Animals ; Cell Line ; Diabetic Nephropathies ; pathology ; Epithelial Cells ; cytology ; metabolism ; pathology ; Extracellular Matrix ; metabolism ; Humans ; Kidney Tubules, Proximal ; cytology ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Shear Strength ; Tissue Plasminogen Activator ; genetics ; metabolism ; Urokinase-Type Plasminogen Activator ; genetics ; metabolism

BACKGROUND: Some studies have presented that vascular endothelial growth factor and its receptor system may take part in onset and development of diabetic nephropathy (DN).OBJECTIVE: To further verify the interventional effects of irbesartan on vascular endothelial growth factor (VEGF) and its Flk-1 receptor expressions in kidney of diabetes mellitus (DM) rat, and the possible mechanism of irbesartan.DESIGN: Randomized control animal study.SETTING: West China Hospital of Sichuan University.MATERIALS: Eighteen male closed colony SD rats weighing 150-200 g were cared in standardization.METHODS: This study was performed at Laboratory of West China Hospital of Sichuan University from August 2006 to April 2007. All rats were randomly divided into a DN group, an irbesartan group and a normal control group, with 6 rats in each group. 10 g/L streptozotocin (55 mg/kg) was intraperitoneally injected to establish models; rats in the control group were administrated with the same dosage of citric acid buffer solution; rats in the irbesartan group were administrated with 3 mg/(kg·d) irbesartan after model establishment. VEGF and FIk-1 expressions were detected using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry technique; while, urine protein level, area and volume of renal glomerulus were detected, and correlations of data were analyzed.MAIN OUTCOME MEASURES: ①Renal pathological indicators, urine protein level, area and volume of renal glomerulus; ②VEGF and Flk-1 expressions;③renal immunohistochemical examination;④ correlation analysis.RESULTS:① Renal pathological examination by HE staining indicated that renal glomerulus was remarkably enlarged in the DN group; mesangial matrix was increased; mesangial cells were also increased; renal tubule was expanded. The lesions in the irbesartan group were milder compared to the DN group. Urine protein level in the DN group was significantly higher compared to the control group (P ＜ 0.01); renal weight/body mass, area and volume of renal glomerulus were significantly higher compared to the control group (P ＜ 0.01); urine protein level, renal weight/body mass, area and volume of renal glomerulus in the irbesartan group were significantly lower compared to the DN group (P ＜ 0.01). ② By the 16th week,VEGF and Flk-1 expressions in the DN group were significantly up-regulated compared to control group (P ＜ 0.05); while,VEGF and Flk-1 expressions in the irbesartan group were also up-regulated compared to the control group by the 16th week (P ＜ 0.05) but down-regulated compared to the DN group (P ＜ 0.05). ③ VEGF staining in the DN group was darker compared to control group (P ＜ 0.01), while the staining in the irbesartan group was also darker compared to the control group (P ＜0.01), but the staining in the irbesartan group was lighter compared to the DN group (P ＜ 0.01). Flk-1 staining was similar to the VEGF. ④ VEGF and Flk-1 were positively correlated, with urine protein level, area and volume of renal glomerulus (P ＜0.05).CONCLUSION: VEGF and its Flk-1 receptor play important roles in DN pathogenesis. Over expressions may cause renal injury, but irbesartan (angiotensin Ⅱ receptor antagonist) has the protective effects on kidney through inhibiting abnormal expression of VEGF and Flk-1.

On May 12,2008,a disastrous earthquake scaled 8.0 Richter hit Wenchuan,Sichuan province in China.Treating the acute kidney injury caused by crush syndrome in survivals of the earthquake has been a big challenge to the nephrologists.In this paper,we shared our experiences on the multi-disciplinary collaboration in management of acute kidney injury caused by crush syndrome.In addition to surgical therapy for crush injury and compartment syndrome and the renal replacement therapy for acute renal injury and its related complications,the early multi-disciplinary collaboration including rehabilitation,mental health care,infection control and ICU also contributed greatly to the successful treatment of the victims of the earthquake.

Objective To observe the change of intima/media thickness ratio and expression of inflammatory factors in renal small artery of diabetic rats, and to explore the correlations of intim/media ratio with inflammatory factors and vascular lesions of diabetic nephropathy (DN) rats. Methods Seventy healthy SD rats were randomly divided into diabetic nephropathy group (DN, n=40) and normal control group (N, n=30). DN rat model was induced by intraperitoneal injection of streptozotocin (STZ). Thirty-five DN rats were successfully established. N group received same dose of citrate buffer. Rats were sacrificed after 4, 12, 24 weeks respectively.The intima/media thickness ratio in renal small artery was detected by immunofluorescence. The monocyte chemoattractant protein-1 (MCP-1) protein and mRNA expression of renal small artery were detected by immunohistochemistry and in-situ hybridization at each time point. Results Blood glucose and urine protein excretion (24 h) at different time points in DN group were significantly higher than those of N group (P＜0.05). From the 12th week, Scr, BUN, serum phosphorus were significantly higher than those of N group (P＜0.05). At the 4th week, renal small artery had the expression of MCP-1 protein and mRNA. The expression increased gradually with time, reached the highest at the 24th week, and was significantly higher than that of N group at each time point (P＜0.05). Immunofluorescence results showed that as compared to N group, in the first 4 weeks, intima/media thickness ratio in DN group was not different, at the 12th week the ratio was higher but without significant difference, at the 24th week the ratio was significantly higher (P＜0.05). Small artery intima/media thickness ratio of DN group was positively correlated with MCP-1, cholesterol, triglyceride, serum phosphate (r=0.742, P＜0.01; r=0.740, P＜0.01; r=0.829, P＜0.01; r=0.580, P＜0.01). Conclusions The arterioles intima/media thickness ratio of early DN is significantly correlated with MCP-1, lipids and phosphorus. MCP-1 may be involved in the DN vascular disease.