Objectives    To investigate demographic and clinical factors which could be predictive of clinical remission or relapse in patients with idiopathic membranous nephropathy treated with oral steroids and iv cyclophosphamide therapy.    Methods    This was a retrospective cohort study where a total of 83 patients with biopsy-proven IMN who had received oral prednisone and iv cyclophosphamide for at least 6 months were enrolled. Demographic and clinical factors of these patients were analyzed at baseline and three months after the initiation of therapy to evaluate their respective effects upon clinical remission and relapse.    Results    Median follow-up duration was 20 (12-30) months. At the end of follow-up, 80.7% (67/83) of the patients attained remission and 47.0% (39/83) attained complete remission (CR). The amount of proteinuria at baseline and the reduction rate of proteinuria three months after treatment predicted clinical remission and CR. 54 patients in the remission group were followed up to the endpoint and 14 of them relapsed with nephroticsyndrome. The presence of a PR versus CR significantly predicted relapse (HR: 40.198, 95%CI: 5.023-333.355, P=0.001). Infection was the most common adverse event. Two patients developed malignancies after onset of cyclophosphamide treatment.    Conclusions    A high rate of good response and a relatively low rate of adverse events were observed in this study. Baseline proteinuria and reduction rate of proteinuria 3 months after onset of cyclophosphamide regimen were independent predictive factors of response. Compared with CR, PR was predictive of a high probability of relapse.

Objective    To investigate clinical and pathological features of idiopathic membranous nephropathy (IMN) accompanied by mesangial dense deposit.    Methods    Clinical data of 46 patients who were diagnosed as IMN accompanied by mesangial dense deposit admitted to Zhejiang Provincial People's Hospital from January 2013 to December 2014 were retrospectively analyzed. They were compared with those of 29 patients who were diagnosed as IMN without mesangial dense deposit during the same period in the hospital. Analysis of their clinical and pathological features was conducted.    Results    The IMN accompanied by mesangial dense deposit accounted for IMN 61.3%, and had more hyaline changes of arteriole (43.5% vs 6.9%, P=0.001) and more obvious arteriolar wall thickening (78.2% vs 51.7%, P=0.016) than IMN without mesangial dense deposit. Furthermore, the positive rate of IgA deposition in IMN accompanied by mesangial dense deposit was greatly higher than that in IMN without mesangial dense deposit (21.7% vs 0, P=0.007). In other index, such as serum biochemical parameters, urine protein, glomerular lesion, tubulointerstitium pathological damage and other immunopathologic changes, no statistically significant differences were found between these two groups.    Conclusions    IMN patients accompanied by mesangial dense deposit have severe intrarenal artery lesions，and high positive rate of IgA deposition.

Objective    To explore the blood pressure variability (BPV) in pediatric patients undergoing maintenance hemodialysis (MHD) and to assess the factors associated with pre-dialysis BPV(pre-HD BPV).    Methods    The pediatric patients who undergone regular dialysis for more than twelve months from Oct 2005 to Oct 2011 in hemo dialysis center of Guizhou Provincial People's Hospital were divided into high pre-HD BPV group and low pre-HD BPV group. Baseline characteristics, biochemical indexes and cardiac function parameters measured by echocardiography were collected in both groups and multiple linear regression analysis was performed.    Results    Pediatric patients in high pre-HD BPV group demonstrated significantly higher inter-dialytic weight growth rate (IDWG), pre-dialysis systolic blood pressure and average amount of dehydration  than those in low pre-HD BPV group (P＜0.05), while significantly lower hemoglobin and albumin levels than those in low pre-HD BPV group (P＜0.05). Comparison among laboratory indicators, serum phosphorus and parathyroid hormone demonstrated significant difference between groups (P＜0.05). For all pediatric patients,  pre-HD BPV was positively correlated with IDWG (β=0.165), pre-dialysis systolic blood pressure (β=0.259), and iPTH (β=0.187), while negatively correlated with hemoglobin level (β=-0.199).    Conclusions    Increasing IDWG, higher pre-dialysis systolic blood pressure, anemia and secondary hyperparathyroidism influence BPV in pediatric patients on MHD.

Objective    To investigate the effects of ursolic acid (UA) on autophagy and podocyte injury induced by high glucose.    Methods    Conditionally immortalized murine podocyte were cultured in high glucose, the effect of PI3K inhibitor LY294002 and ursolic acid treatment were observed. The miR-21 expression was detected using RT-qPCR. The activation of PTEN-PI3K/Akt/mTOR pathway, expression of autophagy-related protein and podocyte marker protein were determined by Western blot. Immunofluorescence staining showed the expression of podocyte marker protein and endogenous accumulation of LC3. Autophagosomes were observed using electron microscopy.    Results  Compared with normal control group，the cells exposed to high glucose condition showed down-regulated synaptopodin, podocin and nephrin expression (P＜0.01), up-regulated miR-21 expression (P＜0.01), down-regulated PTEN expression (P＜0.01), up-regulated p85-P13K, phospho(p)-Akt, p-mTOR，p62/SQSTMI, expression and down-regulated LC3II and Beclin1 expression (all P＜0.01). Ursolic acid and LY294002 promoted synaptopodin, podocin and nephrin expression (all P＜0.01), up-regulated LC3II, Beclin1 expression and down-regulated p62/SQSTM1 expression (all P＜0.01), down-regulated p85-PI3K, p-Akt, p-mTOR expression (all P＜0.01). However, LY294002 did not affect the expression of miR-21 and PTEN. Ursolic acid inhibited miR-21 expression and upregulated PTEN level.    Conclusions    The podocyte injury is associated with defective autophagy level under high glucose condition. Ursolic acid could reduce podocyte injury by increasing autophagy level via inhibition of miR-21 expression and PTEN/Akt/mTOR pathway.

Objective    To observe functional changes of renal tubular epithelial cells stimulated by high mobility group protein box 1 (HMGB1) and associated mechanism.    Methods    Renal tubular epithelial cells (NRK52E) were divided into control group, HMGB1 group and HMGB1+lipopolysaccharide from Rhodobactersphaeroides (LPS RS) group. Toll-like receptor 4 (TLR4) expression was detected by immunofluorescence and Western blotting. Apoptosis rate and cell cycle arrest were identified with flow cytometry. The activation of MAPK signaling pathway and NF-κB were detected by Western blotting. The IL-1, IL-6 and tissue inhibitor of metalloproteinases 2 (TIMP2) mRNA levels were measured by real-time PCR. The secretion levels of IL-1, IL-6 and TIMP2 were measured by protein chips assay.    Results    TLR4 was expressed by NRK52E cells. Compared with the control group, there were increased cell cycle G1 arrest, MAPK signaling pathway and NF-κB activation in HMGB1 group. Furthermore, IL-1, IL-6 and TIMP2 mRNA levels were increased and IL-1, IL-6 and TIMP2 were secreted by NRK52E when stimulated with HMGB1 (all P＜0.05). However, effects mediated by HMGB1 stimulation could be inhibited by LPS RS (all P＜0.05).    Conclusions    Inflammatory activation of NRK52E cells can be mediated by the interaction of HMGB1 and TLR4.

Objective    To investigate the protective effect and mechanism of MST1 inhibition on kidney tissue in diabetic rats, and to find a new therapeutic target for diabetic nephropathy. Methods    Total of 54 male SD rats enrolled in this study were divided into 3 groups including normal control (group A, n=18), MST1 inhibition group (Group B, n=18) and diabetes group (group C, n=18). Diabetes was induced by a single streptozotocin (STZ, 50 mg/kg) injection in group B and group C.  rats in group B received lentiviral vector contain Mst1 interference RNA (shRNA) and the rats in group C received empty vector. The end of 4th, 8th and 12th week after modeling were considered as time points in this study. At each time point, the level of 24 hours urine protein (24-HUP), blood glucose and serum creatinine were examined. Pathological changes were observed with HE stain; Injury of podocyte and glomerular basement membrane (GBM) were examined with transmission electron microscope (TEM). The intensity and location of MST1 in kidney tissue were detected by immunohistochemistry. The level of MST1, Phosphorylated-MST1, nephrin, Caspase-3 and FasL were detected by western bloting.    Results    (1) At the starting point, there were no significant differences among groups in terms of weight, activity, eating and drinking. Since the end of 72nd hour after modeling, the levels of glucose in both group B and group C, compared to those in group A, significantly increased (P＜0.05). There was no significant difference between group B and group C for glucose level at each time point (P＞0.05); the level of 24-HUP increased significantly since the end of 4th week after modeling, and the level in group C was higher than its counterpart in group B at the same point (P＜0.05); (2) There was no significant pathological lesion observed in group A. Without obvious K-W nodular changes, mesangial proliferation was observed in group B and group C. It was shown by TEM that podocyte fusion and thickening of the GBM could be found in group B and group C. The pathological change in group B was better than that in group C; (3) Compared to group A, it was shown by western blot that the levels of MST1, Phosphorylated-MST1, Caspase-3 and FasL in group B and group C were significantly higher (P＜0.05), and the levels of nephrin in group B and group C were significantly lower (P＜0.05) since the end of 4th week after modeling. Meanwhile, the levels of MST1, Phosphorylated-MST1, Caspase-3 and FasL in group B were significantly lower than that in group C at each time point (P＜0.05), the level of nephrin in group B was significantly higher than the one in group C; (4) It was shown by immunohistochemistry that there was low MST1 expression in normal condition, especially in cytoplasm of tubular epithelial cells. The level of MST1 in group B and group C significantly increased after modeling, and the change could be the same as Western blot shown. Conclusions    MST1 pathway could be involved in kidney injury induced by diabetes. MST1 inhibition could alleviate the kidney injury in STZ-induced diabetes animal model.

Objective    To investigate the effects of angiotensin II (Ang II) on the expression of C-terminal Src kinase (Csk) in Ang II-infused rat model and cultured podocytes, and to explore the role of Csk in Ang II-induced cytoskeletal rearrangement of podocytes.    Methods    Twenty-four Wista rats were randomly subjected to normal saline infusion, or Ang II infusion at 400 ng•kg^-1•min^-1 (via subcutaneous osmotic minipumps) for 2 or 4 weeks. Renal histomorphology was evaluated through electron microscopy. The expression of glomerular Csk was analyzed by immunofluorescence and Western blotting. In vitro, conditionally immortalized mouse podocytes were cultured and treated with Ang II doses ranging from 10^-9 mol/L to 10^-5 mol/L and for different hours. The expression of podocytes Csk was assessed by Western blotting. After transfection to podocytes with Csk siRNA, FITC-conjugated phalloidin was used to stain F-actin, to investigate the role of Csk in Ang II-induced or cytochalasin D-induced cytoskeletal rearrangement.    Results    (1) Examination of Ang II infusion rats glomerular and podocyte ultrastructure by electron microscopy revealed foot process effacement and fusion; (2) In Ang II infusion rats, the expression of glomerular Csk was increased (P＜0.05); (3) In vitro, Ang II-stimuli up-regulated the expression of Csk (P＜0.05), and the effects of Ang II were on dose-dependent and time-dependent manner; (4) Ang II-induced disruption of F-actin was alleviated by Csk siRNA transfection in cultured podocytes; furthermore, cytochalasin D depolymerized the F-actin cytoskeleton, while Csk siRNA stabilized the actin filaments.    Conclusion    The enhanced expression of Csk may be involved in Ang II-induced podocytes cytoskeletal rearrangement and foot process fusion.

Objective    To investigate the renoprotective effect of transforming growth factor beta activator kinase 1 (TAK1) inhibitor 5Z-7-oxozeaenol (OZ) in diabetic db/db mice and the mechanism.    Methods    Twenty-four male db/db mice were randomly divided into two groups: db/db mice (db/db, n=12) and db/db mice with 5Z-7-oxozeaenol treatment (db/db+OZ, n=12). Another group of wild type mice (n=12) was held as the control group. OZ 2 mg/kg was administrated by intraperitoneal injection every other day. At week 8 and 12 after 5Z-7-oxozeaenol treatment, blood glucose (BG), body weight (BW), kidney weight (KW) and urinary albumin excretion rate (UAER) were evaluated. Kidney pathological lesions were detected by light and electron microscopy. NF-κB p65, monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) were detected by immunohistochemistry. Western blotting was used to detect p-TAK1, TAB1, p-p38MAPK and IL-1β expression, while ICAM-1 and MCP-1 mRNA levels were evaluated by real-time PCR.    Results    Compared with control group, the levels of BG, BW, KW and UAER were higher (P＜0.01) in db/db mice group, while BW, KW and UAER levels were significantly decreased in db/db+OZ group compared with that in db/db mice group (P＜0.05). In week 8 and 12 db/db mice, glomerular volume and extracellular matrix were increased, while pathological lesions in kidney tissue were positively improved by TAK1 inhibitor.  Immunohistochemistry showed that NF-κB p65, MCP-1 and TNF-α expression levels were apparently increased in db/db mice group compared with that in control group (P＜0.05) and were significantly inhibited by TAK1 inhibitor  (P＜0.05). Western blotting showed that p-TAK1, TAB1, p-p38MAPK and IL-1β expression levels were higher in db/db mice group than that in control group (P＜0.05) and lower in db/db+OZ group than that in db/db mice group (P＜0.05). Moreover, real-time PCR showed that the expressions of ICAM-1 and MCP-1 mRNA were higher in db/db mice group than that in control group and lower in db/db+OZ group than that in db/db mice group (P＜0.05).    Conclusions    TAK1 Inhibitor can down-regulate MAPK and NF-κB pathway to restrain the reaction of inflammation and alleviate kidney injury in diabetic db/db mice.