AIM To study effects of ACE inhibitor on the ex pression of GluT4 mRNA and protein in diabetic rat heart. METHODS The following groups of rats were randomly designed: control rats, diabetic ra ts and diabetic rats treated with benazepril (an ACE inhibitor). After 4 weeks o f treatment, ACE activities were determined by fluorimetric assay in plasma and heart; Myocardial GluT4 mRNA expression were measured by Northern blot analysis; GluT4 protein expression were measured by Western blot analysis. RESULT S After 4 weeks of treatment, there was a significant increase in myocard ial ACE activities despite a trend toward reduce in plasma activitics, ACE activ ities were inhibited by 92 1%, 89 0% in plasma and heart, respectively; Northe rn blot analysis revealed that the expression of GluT4 mRNA was reduced in diabe tic rat heart by 40%, and treatment with benazepril did not prevent the diabetes -induced reduction of GluT4 mRNA; However, Western blot analysis revealed that benazepril did prevent the diabetes-induced reduction of GluT4 protein in cell membrane of diabetic rat heart. CONCLUSION Benazepril could significantly suppress ACE activities in diabetic rat heart, bu t it did not influence the expression of the myocardial GluT4 mRNA. However, it did prevent the diabetes-induced reduction of GluT4 protein in cell membrane of rat heart.

ObjectiveTo investigate the effect of all-trans retinoic acid (atRA) on the renin-angiotensin system (RAS) in 5/6 renal ablation model. MethodsAtRA was administered to 5/6 renal ablation rats by three dosages: 5 mg·kg-1·d-1 (n=8), 10 mg·kg-1d-1 (n=8) and 20 mg·kg-1 d-1 (n=8) and vehicle (vehicle group, n=8) for 10 weeks. Healthy rats consisted of shamoperation group (n =8). The level of renin and angiotensin Ⅱ in renal tissues were measured by radioimmunoassary. The level of angiotensin type 1 receptor (AT1R) in remnant renal cortex was measured by Western blot. The mRNA expression levels of two subunits of activative protein 1(AP-1),c-jun and c-fos was quantitated by real-time PCR. ResultsAfter 10 weeks of atRA treatment by gavarge, artery blood pressure decreased (P＜0.05). AtRA reduced the levels of renin (P＜0.05) and angiotensin Ⅱ (P＜0.05) in kidney and down-regulated the expression of AT1R protein in renal cortex. Larger dose of atRA (20 mg·kg-1·d-1) performed higher activity in inhibiting renin and AT1R. Compared with vehicle group, atRA could significantly inhibit the expression of renal c-jun and c-fos mRNA (P＜0.05). Conclusion atRA can decrease the over-expression of main components of RAS.

Objective To study whether the uremic toxins accumulated long-term in uremia patients may be involved in oxidation of protein by forming advanced oxidative protein products (AOPPs). Methods Malonylaldehyde (MDA), hippuric acid (HA) and p-cresol were used as the representatives of uremic toxins. Human albumin serum (HSA), plasma specimens from normal or uremia patients were incubated respectively with MDA (10 retool/L), HA (20 mmol/L) and p-cresol (10 retool/L) or PBS (20 retool/L, pH 7.4, as control groups) at 37℃ for 30 minutes or 24 hours, respectively. Those indices such as AOPPs, protein thiol groups (Pt-SH) and dityrosine were used as biomarkers of protein injury. High performance liquid chromatography (HPLC) was employed to identify the aggregation and cross-links of modified proteins. Results AOPPs levels in all groups containing poison compounds were significantly increased by 121.5%(P<0.05) compared to that in control groups. Uremic toxins also resulted in over 14.7% loss in Pt-SH (P< 0.05) and 119.2% increment in dityrosine, respectively (P<0.05). Meanwhile, the formation of HMW-AOPPs in a time-dependent manner was observed by HPLC and cross-linked protein levels were significantly increased by 148.45%～333.3% in comparison with control groups. Conclusion Uremic toxins can directly mediate the damage of proteins by inducing the formation of HMW- AOPPs in a time-dependent manner, which is also one of the mechanism of AOPPs production in vivo besides the activation of the myeloperoxidase-H2O2-Cl pathway.

Objective To observe the influence of low protein diet with α-keto acid on kidney sclerosis and renin-angiotensin system in renal ablation rats. Methods Chronic renal failure rat model was established by renal ablation in 30 male SD rats,then the animals were randomly assigned to the following diet groups:normal protein group (NPD:18%casein protein),low protein group (LPD:6%casein protein) and supplemented low protein group (LK:5%casein protein+1%α-keto acids).Ten male SD sham-operated rats received 18%casein protein as control.All the rats were killed at the end of the 12th week.Pathologic changes were assessed by PAS staining.Ang II in homogenate and plasma were measured by radioimmunoassay and ELISA respectively.Immunohistochemistry and Western blotting were used to detect the protein expression of TGF-β1,renin and AT1R.Real-time PCR was used to detect the gene expression of renin and ATla,the main subtype of AT1 receptor. Results Body weight,total protein and serum albumin had not significant difference among the four groups(all P>0.05).Serum creatinine and proteinuria of nephrectomized rats were significantly higher compared to the control group (all P<0.05).Proteinuria of the LK group was lower than that of NPD and LPD groups (all P<0.05).Pathological results indicated fibrosis indices were significantly improved after LPD and LK intervention.Expressions of renin,Ang II and AT1R in LK group were significantly lower than those in NPD group (all P<0.05). Conclusions Low protein diet with α-keto acids supplement therapy exhibits renal protective effects of reducing urine protein excretion and improving renal fibrosis,which might be related to the attenuation of local renin-angiotensin system in activity nephrectomized rats.

Objective To investigate the removal effect of immunoadsorption (IA) on associated antibodies and the efficacy in late-onset myasthenia gravis (MG). Methods A total of 25 late-onset MG patients were randomly selected to enroll in this study. IA therapy was given to 10 patients (IA group), while immunoglobin (0.4 g·kg-1·d-1) was administrated to the other 15 patients for 5 days(Ig group). The titers of Titin antibody (Titin-ab), acetylcholine receptor antibody (AchR-ab) and presynaptic membrane antibody (PrsmR-ab) were detected before and after the treatment. Quantitive MG (QMG) score was assessed before and immediately after the entire course of treatment. The clinical efficacy, the duration of respiratory support and in-hospital were compared between two groups. The correlation between three antibodies and QMG score was also analyzed. Results Compared with that before treatment, the Titin-ab PIN values, the AchR-ab PIN values, and the PrsmR-ab P/N values of IA group were all decreased significantly after treatment (P<0.05, respectively). The P/N value of Titin-ab in IA group was decreased by 54.7%～3.5%, which was significantly higher than that in Ig group(19.9%±3.1%) (P<0.01). QMG score reduced by 42.4%± 4.2% and 23.8%±3.7% in IA group and Ig group respectively (P<0.01, respectively). Symptoms were effectively ameliorated by both treatments, but the effective power of IA group was higher than that of Ig group (70% vs 40%, P<0.05). Remission time of IA group was significantly shorter than that of Ig group [(5.38±0.42) d vs (8.4±1.54) d, P=0.008), so was the duration of in-hospital [(13.50±0.50) d vs (16.50±0.50) d, P<0.05). The number of respiratory support in IA group was less than that in Ig group (1/10 vs 6/15, P<0.05). By the Pearson correlation analysis, the decrease of Titin-ab showed a better longitudinal correlation with the decrease of QMG score than the other two antibodies (r=0.6315, P<0.01). Conclusion IA can rapidly and effectively clear the pathogenic antibodies of late-onset MG patients and its short-term clinical efficacy is better than immunoglobin.

Objective To investigate the injury of podocyte and its association with proteinuria in IgA nephropathy (IgAN). Method Thirty-five patients of IgA nephropathy with proteinuria more than 1.0 g/24 h were enrolled in the study, and eight cases of renal harmatomaeetomy or renal cancinomaectomy were as controls. Cell cycle regulatory proteins (p21, p27), podocyte-associated molecules (integrin-β1, nephrin, α-actinin 4, nestin), foot process width (FPW) and the amount of podocyte were examined by immunohistochemistry and real-time PCR, respectively. Patients were divided into two groups according to podocyte number per volume (Nv): podocytopenia group (n=15, Nv<52.49×106/μm3) and normal number group (n=20, Nv≥52.49× 106/μm3). Proteinuria was followed up for eighteen months. Results Compared with the controls, podocyte p21 was re-expressed, while the expression of p27 was decreased (0.71±0.12 vs 0.91±0.07, P<0.05) in IgAN. The nestin protein level was markedly decreased in IgAN (13.4%± 0.04% vs 17.6%±0.04%, P<0.05). The mRNA expression of integrin-β1 was significantly increased (12.54±5.20 vs 1.02±0.30, P<0.05), while the amount of nephrin, α-actinin4 remained unchanged. Effacement of foot processes and podocyte detachment from the glomerulax basement membrane were observed in some cases. Nv was significantly less than that of controls (161.27± 225.92 vs 323.22±138.12, P<0.05), which was associated with the Lee's grade of IgA nephropathy. The integrin-β1 mRNA expression and Nv were negatively correlated with baseline proteinuria by univariate analysis (r=-0.840, P=0.034; r =-0.4483, P=0.014, respectively). Proteinuria in podocytopenia group was decreased more slowly than that in normal number group. Conclusions Podocyte injury exsists in IgAN with proteinuria, which manifests alterations in cell cycle regulatory protein and some podocyte-associated molecules, as well as foot process effacement and loss of pedocyte. Podocyte injury may be involved in proteinuria by affecting the progression of proteinuria in IgAN.

Objective:To determine whether ox-LDL (oxdized low-density lipoprotein) is highly deposited on the uremic vessel wall and its influence on the vascular remodeling. Methods: Segments of radial arteries were obtained from 21 uremic subjects during the operation of A-V fistula prior to hemodialysis. Segments of internal thoracic arteries of similar diameter were obtained from patients with benign chest tumors as control.The vascular lesions and ox-LDL, CD68,MCP-1, eNOS,ET-1, PCNA,FN on the vessel wall were determined by means of H-E stain and immunohistochemistry. Results： With H-E stain,atherosclerotic plaques were found in the radial arteries of 4 uremic patients. The middle layer of the arteries in uremic patients were obviously thickened, and the T/D　(thickness of the wall/external diameter) ratio was significantly higher than those in control group(P<0.01). ox-LDL,CD68,MCP-1, ET-1, PCNA,FN on the vessel wall in uremic patients were much higher than those in control group (P<0.01). Moreover, ox-LDL on the vessel wall was positively related to the expression of other above mentioned substances on the vessel wall (P<0.01). Whereas the expression of eNOS on the vessel wall was lower than control group (P<0.01),and was negatively related to ox-LDL on the vessel wall(P<0.01). Conclusion: ox-LDL is an important factor contributing to uremic vascular remodeling by increasing the migration,adhesion and infiltration of monocyte,the proliferation of vascular smooth muscle cell and dysfunction of endothelia.

Objective To observe the growing shape and function of Madin-Darby canine kidney (MDCK) cells implanted on the polysilicon nanopore membrane by micro-electro-mechanical system (MEMS). Methods The polysilicon nanomembrane was made by silicon film processed via whirling photoresist, wet etching, electroplating and so on, and then it was coated by extracellular matrix and implanted with MDCK cells. The cell growth shape and function was observed or examined by scanning electron microscope or MTT test and Trypan Blue staining.Results The nanomembrane with regular slit pores was successfully fabricated. Extracellular matrix-coated nanomembrane, especially for collagen Ⅳ coating, was more suitable for MDCK cells to adhere and proliferate without membrane injury. The polysilicon nanomembrane coated with extracellular matrix did not induce the cell death and also not stimulate cells releasing the cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α). Under scanning electron microscope, MDCK cells formed a flat single-cell fusion with tight junction on the surface of polysilicon nanomembrane and there was a large number of microvillus on the top of cells.Conclusion The collagen-coated polysilicon nanomembrane made by MEMS techniques, with no cytotoxicity and good biocompatibility, is valuable to frame the artificial glomerular filtration membrane

Objective To observe the changes of renin-angiotensin system (RAS) in cultured mesangial cells by serum from 3/4 nephrectomized rats feeding with low protein diet and α-keto acid. Methods Thirty male SD rats received 3/4 nephrectomy (Nx) were placed on 18%normal protein diet (NPD,n=10),6% low protein diet(LPD,n=10) or 5% low protein plus 1%α-keto acid diet (LK,n=10) flor 12 weeks.Ten male SD sham-operated rats fed with 18% normal protein diet were used as control (sham group).In addition,mesangial cells were cultured in sera (10%) collected from above animals treated with or without losartan (0.02 mmol/L)for 48 hours.ELISA was applied to detect the level of Ang II,TGF-β1 and fibronectin (FN) in cell medium.Westem blotting was used to determine the protein level of ATI receptor (AT1R)and real-time PCR was used to detect the mRNA level of AT1R,TGF-β1 and FN. Results (1) Nutritional indices including body weight,total protein and albumin had no significant difference in each group. (2) Serum creatinine and 24 h pruteinuria were significantly inceased in nephrectomized groups compared to sham group(P<0.05,respectively).24 h proteinuria was greatly lower in LK group than that in NPD and LPD groups(P<0.05,respectively).(3)LK greatly decteased the level of Ang II[NPD(12.70±0.12)mg/g protein;sham(8.04±0.62)mg/g protein]in supernatant as well as the protein and mRNA expression of AT1R in cultured mesangial cells (P<0.05).(4)NPD serum directly induced higher secretion[FN:sham(20.58±0.46)g/g protein,NPD (39.84±0.06)g/g protein;TGF-β1:sham(10.12±O.56)mg/g protein,NPD(83.85±3.58)mg/g protein] and mRNA expression of FN and TGF-β1 compared with sham group (P<0.05).LPD decreased these increment (P<0.05) and LK showed stronger inhibitory effect (P<0.05). (5)Losartan application sharply reduced FN and TGF-β1 production both in supematant and in mRNA expression in NPD serum treated cells (P<0.05,respectively). Conclusion Low protein diet with α-keto acids supplement directly inhibits the RAS in mesangial cells which may contribute to its beneficial effect on the kidney.

Objective To elucidate the malnutrition in patients with hospital-acquired acute kidney injury(AKI), and to examine the association betweensubjective global assessment (SGA) and prognosis.Methods Adult patients with hospital-acquired AKI were prospectively enrolled in this cohort study.Nutritional evaluations, including SGA, anthropometric and serum nutritional markers were conducted at enrollment.Overall survival at 90 days among different SGA scores was analyzed using Kaplan-Meier methods, and differences were tested using the log-rank test.The Cox model was used to analyze the relationship between SGA scores and all-cause mortality after adjusting for confounders.Results A total of 170 patients were enrolled.The prevalence of moderate malnutrition(SGA B) and severe malnutrition(SGA C) was 51.8％ and 22.9％ respectively, while patients with normal nutrition(SGA A) accounted for 25.3％.After 90 days follow-up, all-cause mortality was 9.8％ in SGA A group, 34.9％ in SGA B group and 56.8％inSGACgrouprespectively. Afteradjustingforage,sex,dialysis,ventilation, hemoglobin, platelets and bilirubin, the hazard ratio(HR) of 90 days all-cause mortality was 4.0(95％ CI 1.42-11.22, P=0.008) in malnutrition group (SGA B group and SGA C group) compared with SGA A group.The Kaplan-Meier curve also revealed that the worse the SGA score was, the lower the cumulative survival became (P＜0.01).Conclusion SGA score is an independent risk factor for all-cause mortality within 90 days in patients with hospital-acquired acute kidney injury.