Objective    To analyze cases diagnosed with glomerular minor lesion (GML) by light microscopy and immunofluorescence, uncover their final pathology diagnosis by electron microscopy, and thereby clarify the pathological and clinical meaning of GML.    Methods    One hundred and forty?eight patients receiving renal biopsy between 2003 and 2008 in Peking Union Medical College Hospital, with diagnosis of GML described by light microscopy and immunofluorescence examination were retrospectively studied. All the clinical data and pathological observation were collected and analyzed, including intact results of electron microscopic examination which were considered as golden standards of pathological diagnosis.    Results    The 148 patients with GML had heterogenous clinical features, with isolated hematuria as the most common presentation. Electron microscopy revealed various pathological presentations: thin basement membrane nephropathy (TBMN, 66.2%), mesangial proliferative glomerulonephritis (MsPGN, 20.3%), Alport syndrome (2.7%), membranous nephropathy (MN, 3.4%), normal tissue (4.7%). Among GML patients with isolated hematuria, TBMN ranked as the most common pathology (76.9%).    Conclusions    GML is only an equivocal description of pathological manifestation by light microscopy and immunofluorescence examination. And electron microscopy is necessary to obtain accurate pathology diagnosis for patients undergoing renal biopsies.

Objective    To explore the effects of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] on memory CD4+ T cells of focal proliferative IgA nephropathy (IgAN）patients.    Methods    (1) Total of twenty incipient focal proliferative IgAN patients (Lee classification: Ⅲ level) were chosen as IgAN group and 20 healthy volunteers were chosen as healthy control group. The level of serum 1,25(OH)2D3 was measured by radioimmunoassay (RIA). Peripheral blood mononuclear cells (PBMCs) were separated by the method of Ficoll density gradient centrifugation and were stimulated with anti-CD3/anti-CD28 in the absence or presence of various concentrations of 1,25(OH)2D3, Dexamethasone(DEX) and 1,25(OH)2D3 and DEX combined. PBMCs were cultured for 72 hours and the levels of IFN-γ, IL-4, IL-17A, Foxp3 were measured by flow cytometry(FCM), standing for the levels of Th1, Th2, Th17, Treg. (2) IgAN group was divided into two subgroups (proteinuria＜1 g/24 h subgroup, proteinuria≥1 g/24 h subgroup), then the serum levels of 1,25(OH)2D3, IFN-γ, IL-4, IL-17A, Foxp3 were compared.    Results    Compared with healthy control group, serum 1,25(OH)2D3 level of IgAN group was significantly lower (P＜0.05). Serum 1,25(OH)2D3 level in proteinuria≥1 g/24 h subgroup was significantly lower than proteinuria＜1 g/24 h subgroup and healthy control group (P＜0.05). The level in proteinuria＜1 g/24 h subgroup was lower than healthy control group, but the difference was not statistically significant (P＞0.05). (2) The levels of IFN-γ and IL-17A and the ratios of IFN-γ/IL-4, IL-17A/Foxp3 in IgAN group increased significantly compared with healthy control group (all P＜0.05), and the level of Foxp3 decreased significantly (P＜0.05). The level of IL-4 also increased, but the difference was not statistically significant (P＞0.05). The levels of IFN-γ and IL-17A and the ratio of IL-17A/Foxp3 in proteinuria≥1 g/24 h subgroup increased significantly, and the level of Foxp3 decreased significantly, compared with urinary protein＜1 g/24 h subgroup and healthy control group (P＜0.05). The ratio of IFN-γ/IL-4 in proteinuria≥1 g/24 h subgroup and proteinuria＜1 g/24 h subgroup all increased, compared with healthy control group, and the ratio in proteinuria≥1 g/24 h subgroup increased significantly (P＜0.05). There was no significant difference in the level of IL-4 among all groups. (3) After treatment with 1,25(OH)2D3, the levels of IFN-γ and IL-17A and the ratios of IFN-γ/IL-4 and IL-17A/Foxp3 decreased significantly, and the level of Foxp3 increased significantly (P＜0.05), and these effects were more obvious as the increase of the drug concentration. The level of IL-4 did not change significantly. The combination of 1,25(OH)2D3 and DEX had a synergistic inhibition on the production of IFN-γ, IL-4, IL-17A, and the ratios of IFN-γ/IL-4 and IL-17A/Foxp3, and had a synergistic promotion on the production of Foxp3.    Conclusions    There is a certain extent of vitamin D deficiency in focal proliferative IgAN patients, which may be associated with the severity of proteinuria. The disorder of immunomodulatory effects of memory CD4+ T cell might exist in the patients of focal proliferative IgAN. 1α,25-dihydroxyvitamin D3 might have beneficial effects on the immunoregulation of memory CD4+T cells of focal proliferative IgAN patients. 

Objective     To investigate the relationship of the serum anti-lysosome associated membrane protein 2 (LAMP-2) antibody levels and anti-neutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis.    Methods    Thirty-three patients with new onset ANCA-associated glomerulonephritis and thirty healthy controls were enrolled. ANCA detection was performed using indirect immunofluorescence (IIF). Enzyme-linked immunosorbent assay (ELISA) was used to detect myeloperoxidase (MPO), proteinase-3 (PR3) and other ANCA-associated antibodies including LAMP-2. The cut-off value of the serum anti-LAMP-2 antibody was determined by a receiver operating characteristic (ROC) curve.    Results    The serum levels of anti-LAMP-2 antibody in new onset ANCA-associated glomerulonephritis patients were significantly higher than remission stage ANCA-associated glomerulonephritis patients and healthy controls (P＜0.05). The serum levels of anti-LAMP-2 antibody showed no visible difference between the remission ANCA-associated glomerulonephritis patients and healthy controls (P＞0.05). The levels of anti-LAMP-2 antibody showed a strong positive correlation with ESR, Scr, BUN, proteinuria, crescent proportion and Birmingham vasculitis activity score (BVAS) and a negative correlation with Ccr, Hb and Alb.    Conclusions    Anti-LAMP-2 antibody is correlated with the activity of ANCA-associated glomerulonephritis and the severity of renal damage. It may be a useful indicator on the activity of ANCA-associated glomerulonephritis.  

Objective    To investigate the associated factors of 6-minute walk test (6MWT) and its prediction of outcome in patients undergoing peritoneal dialysis (PD).    Methods    Stable patients who could tolerant 6MWT in Renji Hospital of Shanghai Jiaotong University School of Medicine between May 1, 2010 and April 30, 2011 were enrolled in present study. All included subjects performed 6MWT, and 6-minute walk distances (6MWDs) were recorded. Patients were divided into two groups according to the median of 6MWD. The laboratory parameters and echocardiography of patients were also tested. Patients were prospectively followed up till death, cessation of PD or the end of the study. Logistic regression was used to determine the associated factors of 6MWD, and multivariate Cox proportional hazards model was used to examine the predicting effect of 6MWD on patients’ outcome.    Results    A total of 145 patients were enrolled. Compared to the patients with long 6MWD, the patients had short 6MWD were older, had lower systolic blood pressure, diastolic blood pressure, as well as lower left ventricular ejection fraction (LVEF) and normalized protein catabolic rate (nPCR) (P＜0.01). Higher high-sensitivity C-reactive protein (hs-CRP) and low density lipoprotein were noted in short 6MWD group (P＜0.05). Logistic regression showed that older age (OR＝1.040, P＝0.018), lower diastolic blood pressure (OR＝0.948, P＝0.007), lower LVEF (OR＝0.927, P＝0.042) were independent associated factors of shorter 6MWD. By the end of the study, 6 cases (8.3%) died in long 6MWD group while 15 cases (20.5%) in short 6MWD group, a significantly lower patient survival was observed in short 6MWD group (Log-rank＝4.983, P＝0.026). Cardiovascular disease [95% CI (1.098-8.952), P＝0.033] and 6MWD [95%CI(0.104-0.964）, P＝0.043] were independent predictors of patient survival. Patients with long 6MWD showed superior technique survival (Log-rank＝4.838, P＝0.028). There was no significant difference in peritonitis-free survival between two groups. However, more patients in the short 6MWD group had been transferred to hemodialysis due to peritonitis (25.0% vs 4.2%, P＝0.013).    Conclusions    Age, diastolic blood pressure, LVEF are independent associated factors of 6MWD in patients undergoing PD. Having the advantages of easy applicability and safety, 6MWT may be proposed as an important predictor of outcome in PD patients.

Objective    To research the relationship between the serum level of cystatin C (Cys-C), N-terminal pro brain natriuretic peptide (NT-proBNP) and the cardiovascular (CV) events in maintenance hemodialysis (MHD) patients, looking for a new and effective biological prediction method for cardiovascular disease (CVD).    Methods    According to the excluded criteria and included criteria, a total of 126 patients [male 67(53.2%), female 59 (46.8%)] were included in this study, screening out of 452 MHD patients from 3 blood purification centre, no secondary hyperparathyroidism, blood pressure controlled, hemoglobin standard, no lipid abnormalities, and without history of coronary heart disease, heart failure and arrhythmia. Participants adopted 3 dialysis treatment, including hemodialysis, hemoperfusion and hemodiafiltration. Every 3 months before the dialysis, the Cys-C, NT-proBNP, serum phosphorus, serum intact parathyroid hormone (iPTH), hemoglobin and electrocardiogram were detected. The heartbeat ultrasound was examined every 6 months, observed for 24 months and followed up for 3 years, recording the incidence and the inspection results. The correlation and the occurrence of CVD were analyzed by conducting a multiple factor logistic regression analysis. The forecast performance of Cys-C, NT-proBNP was evaluated by using receiver operating characteristic (ROC) curves and area under curves (AUC).    Results    Eighteen episodes of CV events occurred in 126 patients during the experiment and follow-up, including 8 episodes of heart failure, 4 episodes of myocardial infarction, 6 episodes of arrhythmia. Detection indexes had no statistically significant correlation (P＞0.05), and the results of ECG and ultrasound heartbeat graph showed that no significant difference in cardiac structure and function before treatment (P＞0.05). After 24 months duration, the research showed that the level of serum calcemia was lower, and the levels of phosphorus and iPTH were higher in hemodialysis group compared with that in the other 2 groups, and the differences had statistical significance (P＜0.05). The median levels of Cys-C and NT-proBNP were 8.59 (9.74, 7.10) mg/L and 7 739 (9 887, 6 736) ng/L in the patients CV events occurred. Non conditional multivariate logistic regression analysis demonstrated that the increasing interdialytic weight, Cys-C, NT-proBNP, iPTH, dialysis hypotension were the independent risk factors of CV occurrence. AUCs to predict CVD occurrence in MHD patients was 0.64 (95%CI 0.53-0.71, P＜0.05) and 0.79 (95%CI 0.72-0.89, P＜0.01) using Cys-C and NT-proBNP respectively. The cut-off values of serum Cys-C and NT-proBNP for CVD occurrence were 8.59 mg/L and 7 739 ng/L, with a sensitivity of 84.3% and a specificity of 92.7%.    Conclusions    Cys-C, NT-proBNP can be used to predict the risk of CV events in dialysis patients.

Objective    To observe the expression of tristetraprolin (TTP) and vascular endothelial growth factor (VEGF) family, to test and verify whether lymphangiogenesis was involved in the occurrence of ultrafiltration failure (UFF) as well as angiogenesis.    Methods    Forty male SD rats of clean grade were selected (180-200 g). These rats were divided into five groups randomly: normal group (n=8), sham operation group (n=8), uremia group (n=8), peritoneal dialysis (PD) 2-week group (n=8), PD 4-week group (n=8). The uremic rats model was established by 5/6 nephrectomy, and of which the PD rats model was established on the basis. The rats of PD2-week group and PD4-week group were given regular PD with 4.25% peritoneal dialysis fluid in dose of 3 ml/100 g body weight. PD2-week group received peritoneal dialysis for 2 weeks, PD4-week group for 4 weeks. Before the rats were sacrificed, peritoneal equilibration test (PET) was applied to calculate the mass transfer of glucose and peritoneal ultrafiltration volume. The protein expressions of VEGF, VEGF–C in each group of rats’ parietal peritoneum were detected by immunohistochemical staining. Microvessel density (MVD) and lymphatic vessel density (LVD) of peritoneal tissue were marked and quantified with anti-CD31 antibody, anti-LYVE-1 antibody. RT-PCR was applied to detect the mRNA expressions of VEGF-A, VEGF-B, VEGF-C, VEGF-D, TTP. Western blotting was used to detect the protein expression of TTP.     Results    (1)PET revealed that, compared with normal group, the mass transport of glucose and the peritoneal ultrafiltration volume of both PD 2-week group and PD 4-week group elevated (P＜0.05); and compared with PD 2-week group, PD 4-week group’s elevated (P＜0.05). (2) Compared with normal group, the protein expression of CD31, LYVE-1, the count of MVD and LVD were increased in uremia group and PD4-week group (P＜0.05). Those of PD4-week groups likewise were increased compared to uremia group (P＜0.05). (3) Compared with normal group, the mRNA expressions of VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D were significantly increased in uremia group (P＜0.05); Compared with uremia group, the expressions in PD4-week group were significantly increased (P＜0.05). Compared with normal group, the mRNA and protein expressions of VEGF, VEGF-C were increased in PD 2-week group (P＜0.05); Compared with PD 2-week group, the expressions were increased in PD 4-week group (P＜0.05). (4) Compared with normal group, the expressions of TTP protein was decreased in uremia group and PD 2-week group (P＜0.05). Compared with uremia and PD2-week group, the expressions of TTP protein was significantly decreased in PD4-week group (P＜0.05).    Conclusions    High glucose peritoneal dialysis fluid and uremic circumstance result in the expression changes of TTP and VEGF family in a PD time-dependent manner. High glucose peritoneal dialysis liquid gives rise to angiogenesis and lymphangiogenesis, both of which lead to UFF.

Objective    To investigate the protective effect of resveratrol (RSV) on renal damage in spontaneously hypertensive rats (SHR) and the related mechanisms on interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1).    Methods    Twelve male spontaneously hypertensive rats were randomly divided into two groups: model group (SHR, n＝6）and RSV group (RSV, n＝6). Six male Wistar-Kyoto rats served as control group (WKY, n＝6). RSV (20 mg·kg-1·d-1) or vehicle were gavaged for 20 weeks. Microalbuminuria and urinary β2-microglobulin were determined by urine collection from 8:00 to 16:00 at 20th week. Scr, BUN and the renal pathological changes were measured after 20 weeks. Immunohistochemistry staining of fibronectin, collagenⅠ, IL-6 and ICAM-1 were used to analysis the changes of renal fibrosis and inflammation. Real-time PCR and Western blotting were used to measure the expression of IL-6 and ICAM-1 in kidneys.    Results    Compared with the control group, SHR significantly increased the level of microalbuminuria, urinary β2-microglobulin (P＜0.05), but they were diminished in RSV group (P＜0.05). The expressions of fibronectin, collagenⅠ, IL-6 and ICAM-1 by immunohistochemistry staining were augmented in SHR group, and were significantly inhibited in RSV group. Compared with the control group, the expressions of renal IL-6, ICAM-1 mRNA and protein were significantly increased in SHR group (P＜0.05), and RSV treatment significantly inhibited the up-regulation (P＜0.05).    Conclusions    RSV treatment can attenuate microalbuminuria, urinary β2-microglobulin and renal fibrosis in SHR rats. This renal protective effect is associated with the inhibition of IL-6, ICAM-1 expression, which suggesting that inflammation may be a potential therapeutic target of hypertensive renal damage.

Objective    To observe the impact of heparanase on glomerular endothelium glycocalyx during sepsis and to investigate the prevention of glycocalyx injury.    Methods    C57/BL6 mice were injected with lipopolysaccharide (LPS) or tumor necrosis factor-α(TNF-α) and sacrificed one hour later. Glomerular endothelium glycocalyx traced with lanthanum was observed by transmission electron microscope(TEM). Western blotting was used to observe heparanse protein expression of renal cortex tissue. Human renal glomerular endothelial cells (HRGECs) were stimulated with TNF-α and active heparanase protien expression was detected by Western blotting. Mice were administrated with heparin sodium or heparinase Ⅲ and renal endothelium glycocalyx was observed by TEM. Urine during twenty-four hours was collected to measure urinary albumin and creatinine. The ratio of albumin to creatinine was calculated and compared among groups.    Results    The glomerular endothelium glycocalyx of LPS group and TNF-α group was degradated and the one of podocyte was integrated. Renal cortex tissue heparanase protein expression was significantly increased since one hour after LPS injection (P＜0.01). The protein expression of activited heparanase of HRGECs which were stimulated with TNF-α was increased (P＜0.05). Administration of heparin sodium which could inhibit the activity of heparanase could prevent the glycocalyx form degradation. The ratio of urine albumin to creatinine of heparin sodium group was decreased compared with LPS group (P＜0.05) and the ratio of heparinase Ⅲ group was higher than control group(P＜0.01) as a result of degradation of glomerular endothelium glycocalyx.    Conclusions    During the early stage of sepsis, TNF-α can induce glomerular endothelium heparanase to increase and active, and consequently the glycocalyx is degradated which leads to albuminuria. Inhibition of heparanase can protect glomerular endothelium glycocalyx and prevent albuminuria.

Objective    To investigate the effect and mechanism of cysteine-rich protein 61(Cyr61) on oxidative stress in human kidney tubular epithelial cell line after anoxia.    Methods    Human kidney tubular epithelial cell line (HK-2 cells) were divided into 5 groups：control group, Cyr61 group, MAPK inhibitor group (Cyr61+PD98059), p38 inhibitor group (Cyr61+SB203580) and PI3K inhibitor group (Cyr61+Wortmannin). Each group was pretreated for 12 h and then injured by anoxia．The cell viability was determined by MTT assay and the apoptosis rate of HK-2 cells was determined by flow-cytometry. The cellular ROS level was measured by spectro-fluorometry. The cellular superoxide dismutase (SOD) and catalase (CAT) were measured by nephelometry test. The expression of Nrf2 in HK-2 cells was detected by Western blotting.    Results    Anoxia enhanced the expression of ROS and Nrf2, decreased the expression of SOD and CAT significantly，meanwhile decreased HK-2 viability and increased HK-2 apoptosis (all P＜0.05). Cyr61 increased the expression of p-Akt, Nrf2, SOD and CAT in HK-2, and decreased the expression of ROS, at the same time increased HK-2 viability and decreased HK-2 apoptosis (all P＜0.05). Wortmannin inhibited the expression of p-Akt，Nrf2, SOD and CAT, meanwhile decreased HK-2 viability and increased HK-2 apoptosis (P＜0.05). PD98059 and SB203580 had no affect on HK-2 compared to Cyr61 group (P＞0.05).    Conclusions    Cyr61 promotes the expression of Nrf2 through PI3K pathway in HK-2, which enhances the expression of SOD and CAT, and decreases the expression of ROS. Cyr61 exhibits protective effects on HK-2 cells injured by oxidative stress after anoxia.