Objective To investigate the relationship between serum renalase and disease activity in lupus nephritis (LN). Methods Total of 70 patients with LN and 35 healthy volunteers admitted in Renji Hospital of Shanghai Jiao Tong University from March 2012 to March 2013 were enrolled in the study. LN patients were classified into two groups according to their systemic lupus erythematosus disease activity index (SLEDAI) scores: patients with SELDAI score lower than 8 were defined as inactive LN while others were defined as active LN. Serum samples were collected after an overnight fast and serum renalase level was determined by ELISA. Twenty active LN patients were followed up for six-months, and serum renalase was also determined before and after treatment. The differences in serum renalase level between LN patients and healthy controls were assessed, as well as the association of serum renalase with disease activity in LN. Results In 70 LN patients, 35 were classified active LN while others were inactive LN. Serum renalase level was significantly higher in LN patients than than in healthy controls (P﹤0.01). Moreover, active LN patients had higher serum renalase level compared to patients with inactive LN (P﹤0.01). Active LN patients had higher 24-hour urine protein excretion, erythrocyte sedimentation rate and anti-dsDNA antibody titers than inactive LN patients. Serum albumin was lower in inactive LN patients compared to active LN patients. There were no differences in gender, age, blood pressure and C-reactive protein between the two groups. Serum renalase levels were positively correlated with SLEDAI, 24-hour urine protein excretion, ds-DNA and ESR but inversely correlated with serum albumin and C3. Renalase amounts decreased significantly after six-months of standard therapy. The performance of renalase as a marker for diagnosis of active LN was 0.894 with a cutoff value of 66.67 mg/L. Logistic regression showed that serum renalase (OR=1.078, 95%CI 1.031-1.120, P=0.001) and complement C3 (OR=0.022, 95%CI 0.002-0.326, P=0.005) is independent indicators for disease activity in LN. Conclusions Serum renalase level was correlated with disease activity in LN. Serum renalase may serve as a potential indicator for disease activity in LN.

Objective To explore a new pathogenic gene mutationin in COL4A3 and COL4A4 genes of a family with thin-basement-membrane nephropathy (TBMN), and explain its mechanism. Methods Genomic DNA was extracted from blood samples. Mutation screening for all the exons in COL4A3 and COL4A4 of the proband was carried out by direct PCR sequencing. The sequences of the proband were compared with standard sequences in GenBank. After identifying the mutation in COL4A4, screening for the mutation site in 200 healthy controls and the rest of family members were conducted. RNA sequence of the proband was analyzed by reverse transcription PCR and TA cloning. The positive clones were sequenced for RNA screening. Results There was a G to A mutation in the 1459 site of COL4A4 (c.1459+G＞A) in the proband, her mother, and the elder sister, whereas the mutation was not found in other family members and healthy people. RNA screening showed that the COL4A4 (c.1459+G＞A) mutation was a heterozygous substitution in position +1 of exon 21, in the splicing region. This mutation leaded to eliminating of exon 21 from the COL4A4 mRNA, causing the exon 21 deletion and frameshift mutation following the exon 20 in its amino acids sequence. Conclusions It is described that COL4A4 (c.1459+G＞A) is a new pathogenic mutation in TBMN, which further help understanding the pathogenesis and clinical diagnosis of TBMN.

Objective To investigate the role of DNA methylation changes in the regulatory region of TGFB1 gene in patients with diabetic nephropathy (DN). Methods According to the WHO 1999 guideline for diabetes mellitus diagnosis and classification standard, 91 patients who were hospitalized in June 2013 to May 2015 and diagnosed as diabetes mellitus were selected, including 42 patients with diabetes mellitus (DM group) and 49 with diabetic nephropathy (DN group). Thirty cases with health examination were selected as healthy control group (Con group). DNA was extracted from all the subjects' peripheral blood and modified by sodium bisulfite. DNA methylation status of TGFB1 gene regulatory region was screened by methylation specific PCR and the DNA methylation level was detected by bisulfite sequencing PCR. ELISA was used to test serum TGF-β1. Blood urea nitrogen, creatinine, fasting blood sugar, postprandial blood sugar, glycosylated hemoglobin and urinary albumin to creatinine ratio (UACR) were detected by automatic biochemistry analyzer. Pearson correlation analysis and multiple stepwise regression were used to analysis TGF-β1-related factors, the correlation between the level of serum TGF-β1 and the pathological grade was analyzed in DN patients. Results There were 12.2% patients in DN group with DNA methylation of TGFB1 gene regulation region, lower than those in DM group (42.8%) with Con group (73.3%) (all P＜0.05). The methylation level of TGFB1 regulatory region was 12.5%±8.1% in DN group, significantly lower than those in DM group (35.6%±6.0%) and Con group (66.7%±9.1%) (all P＜0.05). Moreover, compared with that in DM group (1367.22±126.13 ng/L) and Con group [(296.38±74.37) ng/L], TGF-β1 expression was increased significantly in DN group [(2885.73±411.36 ng/L] (all P＜0.01). In DN patients serum TGF-β1 was correlated with eGFR (β=-0.690, P＜0.01) and the methylation (β=-0.302, P＜0.01), and the serum TGF-β1 was negatively correlated with DNA methylation level (r=-0.925, P＜0.01), but positively correlated with the pathological scores among glomerulus, tubulus and arterioles (rs=0.847, P＜0.01). Meanwhile the methylation level related to the pathological grade (χ2=23.667, P=0.04). Conclusion Demethylation in the regulatory region of TGFB1 may play an important role in the activation of TGFB1 induced by high glucose in mesangial cells, so as to participate in the occurrence and development of DN.

Objective To analysis the relationship between anemia and clinic outcomes retrospectively in maintenance hemodialysis patients for Renji Hospital, Shanghai Jiao Tong University School of Medicine, China. Methods This study enrolled all maintenance hemodialysis（MHD）patients between 1 January, 2007 and 31 December, 2014 at the Renji Hospital. They were followed up until death, cessation of hemodialysis, transfer to other centers or to the end of the study (31 December, 2014). Laboratory parameters, including hemoglobin concentrations, transferrin saturation, ferritin, serum albumin, were measured every 3 months. According to the Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines, the patients were divided into target-hemoglobin group (110≤Hb ≤120 g/L) and non target-hemoglobin group (< 110 g/L or > 120 g/L), and then the compliance rate of Hb, Ferritin, transferrin saturation, and the influence factors of compliance rate of Hb as well as its relationship with the prognosis were analyzed. Results Total 517 maintenance hemodialysis patients were involved in this study. The mean age was (63.76±14.78) years and 59.96% patients were male. Only 35.20%, 91.26% and 31.18% of them met the K/DOQI defined targets for hemoglobin, transferrin saturation and ferritin levels. The average levels of TSAT and Ferritin had no significant difference between the target-hemoglobin group and the non target-hemoglobin group. Compared with patients in non target-hemoglobin group, the target-hemoglobin group had significantly higher qualified rate of transferrin saturation (94.97% vs 89.41%, P=0.045) and Ferritin (37.22% vs 28.13%, P=0.036). Multivariate logistic regression analysis showed that the serum albumin, blood intact parathyroid hormone (iPTH) and dialysis vintage were independent risk factors that affected whether hemoglobin was up to the target. Kaplan-Meier analysis showed that the 8-year survival rate and cardiovascular survival rate in target-hemoglobin group were obviously higher than that in the non target-hemoglobin group (86.70% vs 75.30%, χ2Log rank=7.134, P=0.008; 93.80% vs 85.30%, χ2Log rank=6.134, P=0.013, respectively). Dialysis frequency, age and ferritin were independent risk factors of all-cause mortality for non target-hemoglobin group, and Dialysis frequency was independent risk factors of cardio-cerebral vascular disease mortality for non target-hemoglobin group. Conclusions The compliance rate of hemoglobin in MHD patients is still not steady controlled. Blood iPTH, serum albumin and dialysis vintage are independent risk factors that affect whether hemoglobin is up to the target in MHD patients. Sub-standard hemoglobin increases both all-cause mortality and cardio-cerebral vascular disease mortality in MHD patients.

Objective To investigate whether the T-helper cell 17 (Th17)/regulatory T cell (Treg) of patients on maintenance hemodialysis (MHD) present imbalance in terms of proportion and function, and if so, the relationship between such imbalance and atherosclerosis cardiovascular disease (ASCVD). Methods Fifty-seven MHD patients, included 25 with ASCVD and 32 without ASCVD, and 24 healthy volunteers were enrolled. The common carotid artery intima media thickness (CCA-IMT), carotid plaque and plaque area were determined with high-resolution B-mode ultrasonography. Treg (CD4+CD25+Foxp3+) and Th17 (CD4+IL-17+) were measured by flow cytometry. The Foxp3 and RAR-related orphan receptor γt (RORγt) mRNA expressions were measured by real-time PCR. TGF-β1, IL-10, IL-17 and IL-6 in serum were detected by ELISA. Results There were decreased Treg proportion, Foxp3 mRNA, TGF-β1 and IL-10, and increased Th17 proportion, RORγt mRNA, IL-17, IL-6 and Th17/Treg in the ASCVD group compared with that in the non-ASCVD group and healthy group (all P＜0.01). No correlation was observed between Treg and CCA-IMT, but IL-10 were negatively correlated with CCA-IMT (P＜0.05). Th17, IL-17 and ratio of Th17/Treg were positively correlated with CCA-IMT (all P＜0.05). MHD patients with carotid plaques had lower Treg, TGF-β1 and IL-10, higher Th17, IL-17 and ratio of Th17/Treg than those without carotid plaques (all P＜0.05). Moreover, Treg proportion was negatively correlated with carotid plaque area in MHD patients with carotid plaques (P＜0.01). Conclusions The Th17/Treg numerical and functional imbalance exists in MHD patients, especially in patients with ASCVD. This might act synergistically with micro-inflammation on immune-mediated atherosclerosis and contribute to the high incidence of ASCVD.

Objective To investigate the effect of hypoxia inducible factor 1α (HIF1α)-kidney injury molecule 1 (KIM1) pathway on extracellular matrix degradation in human tubular epithelial cells under high glucose, and to explore the possible mechanism of this pathway participated in renal interstitial fibrosis of DN. Methods The human tubular epithelial cells (HK2) were cultured in vitro and divided into the following groups: (1)Normal control Group (D-glucose 5.6 mmol/L); (2) Mannitol group (D-glucose 5.6 mmol/L+D-mannitol 24.4 mmol/L); (3) High glucose group (D-glucose 30 mmol/L); (4) Control siRNA group; (5) HIF1α siRNA group; (6) KIM1 siRNA group. The corresponding indexes were measured at 12th, 24th and 36th hours. Western blotting, immunofluorescence and qRT-PCR were used to examine the expression of HIF1α, KIM1, matrix metalloproteinase-9 (MMP9), tissue inhibitor of metalloproteinases-1 (TIMP1), fibronectin (FN) and type I collage (COL-I) in protein and mRNA. Results Compared with the control group, the protein and mRNA expression of HIF1α, KIM1, TIMP1, FN and COL-I in the high glucose group were increased in a time-dependent manner (P＜0.01), and MMP9 was decreased in time-dependent manner (P＜0.01). Compared with the high glucose group, the protein and mRNA expression of HIF1α, KIM1, TIMP1, FN and COL-I in HIF1α siRNA group was decreased (P＜0.01), and MMP9 was increased (P＜0.01); However, the protein and mRNA expressions of KIM1, TIMP1, FN and COL-I in KIM1 siRNA group was decreased (P＜0.01), MMP9 was increased (P＜0.01), and the change of HIF1α was of no significance. Conclusions Down-regulation of HIF1α can significantly inhibit the expression of KIM1 in HK2 and decrease the expression of extracellular matrix, and down-regulation of KIM1 can also decrease the extracellular matrix under high glucose, which suggests that HIF1α may regulate the expression of KIM1 in human tubular epithelial cells under high glucose condition, and this pathway may participate in renal interstitial fibrosis of DN.

Objective To investigate the expression of Notch signaling molecules, transforming growth factor-β (TGF-β) and fibronectin (FN) in mesangial cell induced by high glucose, and the underlying mechanism of cordyceps sinensis. Methods Rat glomerular mesangial cells were divided into following groups: normal control group (5.5 mmol/L glucose), hypertonic control group (5.5 mmol/L glucose+19.5 mmol/L mannitol), high glucose group (25.0 mmol/L glucose), DAPT inhibitor group (25.0 mmol/L glucose+1 μmol/L DAPT), cordyceps sinensis intervention group (25.0 mmol/L glucose+10 mg/L cordyceps sinensis). Cell proliferation was detected by MTT. The protein and mRNA expression of Notch signaling molecules (Notch1, Jagged1 and Hes1), TGF-β and FN was detected by Western blotting and real time PCR. Results Compared with normal control group, high glucose induced mesangial cell proliferation, as well as the mRNA and protein expression of Notch1, Jagged1, Hes1, TGF-β1 and FN was up-regulated in high glucose group (all P＜0.05). Compared with that in high-glucose group, DAPT and cordyceps sinensis inhibited high glucose-induced mesangial cell proliferation and down-regulated the mRNA and protein expression of Notch pathway, TGF-β1 and FN (all P＜0.05). Conclusion By inhibiting the abnormal activation of Notch signaling pathway and TGF-β signaling pathway, cordyceps sinensis may alleviate high glucose-induced mesangial cell proliferation and reduce extracellular matrix accumulation, thus protecting kidney.

Objective To investigate the possible role of oxidative stress in the protection of hydrogen sulfide during renal ischemia reperfusion. Methods Male Wistar rats were randomly divided into 3 groups: sham operation (Sham) group, renal ischemia reperfusion (IR) group subject to occlusion of left renal pedicle for 45 min then reperfusion for 24 h, and sodium hydrosulfide (NaHS) preconditioning group with continuous infusion of NaHS (450 nmol/min) by left renal artery for 10 min before ischemia reperfusion. Renal injuries were evaluated by PAS staining. The protein levels of NADPH oxidase (NOX) 4, NOX2 were analyzed by Western blotting. The reactive oxygen species (ROS) level of renal tissue was determined by dihydroethidium (DHE) staining assay. Renal superoxide dismutase (SOD), malonic dialdehyde (MDA) and Scr, BUN were evaluated by chromatometry assay. Cell apoptosis were evaluated by TdT-mediated dUTP nick end labeling (TUNEL) staining. Results Compared with Sham group, in IR group the renal NOX4 and NOX2 protein expressions, the existence of acute tubular necrosis and ROS expression were up-regulated (all P＜0.01); MDA, Scr, and BUN were increased and SOD was decreased significantly in IR-treated kidney (all P＜0.01); Moreover, more apoptotic cells presented in the risk zone of IR-treated kindey (P＜0.01). The effects induced by IR were inhibited by NaHS. Compared to that in IR group, NaHS precondition reversed IR-induced damages of renal function and renal tissue, increased SOD activity and decreased MDA expression (all P＜0.05), as well as reduced the expression of NOX4, NOX2 and ROS (all P＜0.05). Moreover, NaHS precondition reduced apoptosis after IR (P＜0.05). Conclusions NaHS alleviates renal ischemia reperfusion injury through inhibiting oxidative stress. Hydrogen sulfide can decrease ROS by inhibiting the activation of NOX, further inhibit the activation of NOD-like receptor, and alleviate kidney damage.

Objective To study the effect of interleukin (IL)-10 knockout (IL-10-/-) on renal repair after renal ischemia-reperfusion injury in mice. Methods Eighteen IL-10-/- mice (KO) aged 8-10 weeks and 18 C57BL/6 wild type mice (WT) aged 8-10 weeks were divided into control group (Sham) and renal ischemia-reperfusion injury (IRI) group. The renal tissue morphology change was observed by Hematoxylin and eosin (HE) staining and Masson staining. The expressions of IL-18, Ki67 and TGF-β1 were detected by immunohistochemistry. The expression of TGF-beta1 and IL-18 were detected by Western blotting. Results Compared with that in WT-IRI group, in KO-IRI group renal pathological damage was more severe, renal interstitial fibrosis was visible, Ki67 expression of renal tubular epithelial cells decreased distinctly (P＜0.01), the expression of TGF-beta1 increased significantly (P＜0.01). Conclusion Repair slows down significantly after kidney ischemia-reperfusion injury and fibrosis occurs gradually in IL-10-/- mice, eventually progressing to chronic kidney disease.