Objective     To assess the relationship between levels of Klotho hypermethylation of intrarenal and peripheral blood mononuclear cells(PBMC) and clinical and histological severity of the disease. Methods     Intrarenal and PBMC levels of Klotho promoter methylation were quantified in 47 CKD patients by pyroseqencing. The results were compared with 47 nephrectomy specimens and PBMC of 48 healthy volunteers. Results     Higher levels of Klotho promoter methylation were observed in both renal biopsy and PBMC at specific CpG sites in CKD patients compared to control[(17.04±6.42) % vs (9.34±2.43) %, P＜0.01; (14.19±5.86) % vs (6.90±2.39)%, P＜0.01]. The level of Klotho promoter methylation on PBMC was positively correlated with intrarenal level (r＝0.811，P＜0.01). Using receiver operator characteristic curve analysis, PBMC level of Klotho promoter methylation had an area under the curve (AUC) of 0.958 (P＜0.01) in predicting intralrenal Klotho promoter hypermethylation.Estimated glomerular filtration rate inversely correlated with intrarenal and PBMC levels of Klotho hypermethylation (r=－ 0.827, P＜0.01; r=－ 0.626, P＜0.01). Tubulointerstitial scarring positively correlated with intrarenal level of Klotho hypermethylation, as well as PBMC level of Klotho hypermethylation (r＝0.865, P＜0.01; r＝0.748, P＜0.01). There was no correlation between proteinuria and the level of Klotho promoter methylation of renal tissue or PBMC. Conclusions     Klotho promoter methylation levels of intrarenal and PBMC are significantly elevated in CKD patients and correlated with clinical and histological severity. Klotho promoter hypermethylation may play animportant role in renal fibrosis. The level of Klotho promoter methylation of PBMC may be a potential biomarker of intrarenal Klotho promoter hypermethylation.

Objective    To investigate the genetic association of complement factor B (CFB) gene polymorphisms with the susceptibility and prognosis of IgA nephropathy (IgAN). Methods    Four hundreds and sixty-three Northern Han Chinese patients with IgAN and two hundreds and ninty-six geographically and ethnic matched healthy volunteers were recruited. Peripheral blood was collected from recruited individuals for DNA extracting. After amplified by plymerase chain reaction (PCR), genotyping of the four single nucleotide polymorphisms (SNPs) in CFB gene, which were rs4151667, rs12614, rs641153 and rs117314762, were performed by sequencing. Differences of allele and genotype frequencies were analyzed between IgAN patients and healthy controls. Moreover, the association between these SNPs and disease clinical manifestation, pathological features and long term renal outcome in IgAN patients were further analyzed. Results    The G allele and GG genotype frequencies of rs117314762 in CFB gene were significantly higher in IgAN patients than that in healthy controls. No difference in allele and genotype frequencies of rs4151667, rs12614, rs641153 between IgAN patients and healthy controls was observed. Furthermore, no association was found between these SNPs in CFB gene and clinical manifestation, pathological features and long term renal outcome of IgAN. Conclusion    Association between rs117314762 in CFB gene and IgAN susceptibility suggests that there may be functional variants in CFB gene or its linked genetic region, which needs further exploration.

Objective     To discuss the changes of neutrophil gelatinase - associated lipocalin (NGAL) in patients with primary nephrotic syndrome（PNS）and the correlation with renal pathological type, renal tubulointerstitial lesions and the clinical indicators.   Methods       Forty patients with PNS were divided into acute kidney injury (AKI) group and non - AKI group according to whether renal tubular necrosis（ATN） occurred in renal pathology. Moreover, on the basis of pathological type they were divided into minimal change disease (MCD) group, mesangial proliferative glomerulonephritis (MsPGN) group, focal segmental glomerulosclerosis (FSGS) group, membrane proliferative glomerulonephritis (MPGN) group and membranous nephropathy (MN) group. Twenty healthy subjects and normal kidney tissues which came from 20 patients with renal tumor nephrectomy and were distant from the tumor sites were the control groups. Enzyme-linked immunosorbent assay (ELISA) was applied to detect the serum and urine level of NGAL, and immunohistochemical staining was used to observe the expression of NGAL in the renal tissue.   Results    (1)The serum and urine level of NGAL and the expression of NGAL in the renal tissue in the PNS complicated with AKI group were significantly higher than that in the PNS without AKI group and in the control group(P＜0.05). (2)The serum and urine level of NGAL and the expression of NGAL in the renal tissue were enhanced in MPGN group and FSGS group than that in the other three groups(P＜0.05). (3) Before developing to severe tubulointerstitial lesions, with the aggravation of tubulointerstitial damage, the serum and urine level of NGAL and the expression of NGAL in the renal tissue were increased. But when renal tubular interstitial lesions developed to severe disease, serum level of NGAL and the expression of NGAL in the renal tissue were decreased(P＜0.05). (4)The serum and urine level of NGAL and the expression of NGAL in the renal tissue were positively correlated with serum creatinine(r values were 0.198, 0.352, 0.146 respectively, P values were 0.048, 0.000, 0.028 respectively), were positively correlated with blood urea nitrogen(r values were 0.199, 0.278, 0.325 respectively, P values were 0.043, 0.000, 0.019 respectively), were negatively correlated with serum albumin(r values were -0.384, -0.318, -0.259 respectively, P values were 0.028, 0.024, 0.020 respectively) and were negatively correlated with urine osmotic pressure(r values were -0.250, -0.256, -0.277 respectively, P values were 0.012, 0.027, 0.002 respectively).   Conclusion      NGAL is a sensitive biological parameter for predicting AKI in the patients with PNS, and it can be used to evaluate the degree of tubulointerstitial lesions and renal function to a certain extent.

Objective      To investigate the clinical effectiveness of balloon angioplasty with covered - stent placement for arteriovenous graft stenosis.   Methods      A total of 15 patients with arteriovenous graft stenosis which all had poor therapeutic effect by percutaneous transluminal angioplasty (PTA) were enrolled. All the patients had either one or more stenotic lesion, which had below features: 7 cm or less in length and more stenosis (＞50%); stenosis recurred two or more times within 3 months after PTA; residual stenosis above 30% or elastic recoil immediately after expansion. Polytetrafluoroethylene-covered stents with different diameter were placed after balloon angioplasty by digital subtraction angiography (DSA).   Results       Patients were three men and twelve women with mean age (66±12) years. The mean duration of arteriovenous graft was (19.5±15.0) months at the time of stent insertion. Total 16 stents were placed. The technical success rate was 100%. Nine patients (9/15) were vein-graft anastomosis stenosis and 6 patients (6/15) were outflow stenosis with 3 at cephalic vein, 2 at brachial vein and 1 at axillary vein. The primary access patency rates were 40% at 3 months, 19% at 6 months and 13% at 12 months. The secondary access patency rates were 93% at 3 months, 88% at 6 months and 87% at 12 months. The mean follow-up time was (14.9±5.3) months. The restenosis rate was 87%(13/15). PTA was done 36 times after stent placemen. The reasons for primary patency failure were in-stent stenosis 36% (13/36), inflow stenosis 8% (3/36), outflow stenosis 22% (8/36) and stenosis unrelated to stent placement 33% (12/36). The median survival time of AVG was 25 months.  Conclusion      Balloon angioplasty with covered - stent placement for arteriovenous graft stenosis has high rates of technical success and less complication. The primary access patency is low but secondary access patency is satisfied.

Objective     To investigate the effect of renal tubular epithelial cells with hepatitis B virus x (HBx) gene on mediating T-cell activation and differentiation.   Methods     The eukaryotic vector pcDNA3.1-myc-HBx containing HBx gene was transiently transfected into HK-2 cells by lipofectamine mediation. The expression of HBx was confirmed by real-time PCR and Western blotting. CD4+ T cell in peripheral blood of health volunteer was isolated and purified by immunomagnetic beads. Subsequently, purified CD4+ T cells were co-cultured with HK-2 cells. Flow cytometry was used to detecte the expression of CD40 of HK-2 and the expression of CD40L and cell cycle of CD4+ T cells. ELISA assay was used to quantify the IFN - γ and IL - 4 in co - culture supernatant. The experiment was divided into four groups: experimental group(HK - 2 cells with HBx + CD4 + T cells), negative control group (HK - 2 cells with pcDNA3.1-myc +CD4+ T cells), blank control group (HK-2 cells +CD4+ T cells) and separate culture group (CD4+ T cells).      Results     Real-time PCR and Western blotting results verified that HBx was successfully expressed in HK - 2 cells after transfection. After transfection of HBx gene, the expression of CD40 was significantly increased on HK - 2 cells(P＜0.01). Compared with control groups, the expression level of CD40L and the percentage of cells at S and G2/M phase increased(all P＜0.01). Meanwhile, the level of IFN-γ in the supernatant was higher(P＜0.05), but the level of IL-4 was lower in experimental group(P＜0.05).   Conclusions     Over-expression of HBx gene may up-regulate the expression of costimulatory molecules CD40 of renal tubular epithelial cells, which may active CD4+ T cells, promote the proliferation of CD4+ T cells and up-regulate Th1-type cytokines. These events may induce immune injury of renal in hepatitis B virus-associated glomerulonephritis.

Objective     To observe the expression of glycoprotein non-metastatic melanoma protein B (Gpnmb) in the kidney and urine after ischemic - reperfusion injury (IRI), and explore the relationship between Gpnmb and macrophage phenotypes in the IRI kidney. Methods     Male C57BL/6J mice were randomly divided into control group (n＝4), sham group (n＝4) and IRI group (n＝12). Both renal pedieles of mice in IRI group were identified and occluded with microvascular clamps for 30 min. Renal pathological injury was observed by PAS staining. The expression of Gpnmb was examined by real-time PCR and immunofluoresence staining. The location of Gpnmb was observed by flow cytometry and double immunofluoresence staining with F4/80. The mRNA expressions of Gpnmb, CD40, CRR7, CD163 and MMR were examined by real - time PCR. The expression of Gpnmb in the urine was examined by Western blotting and ELISA. Results     PAS - stained IRI kidney section showed desquamative epithelia, necrosis debris and a large number of inflammatory cell infiltration. Real-time PCR results showed that there was little expression of Gpnmb in the kidney of control group and sham group. However, the Gpnmb mRNA level in IRI kidneys was highly up-regulated at day 1 and day 2 (both P＜0.01) and followed by a decrease that was similar to the control level at day 3. Double immunofluoresence staining of kidney sections from IRI mice revealed that Gpnmb was predominantly detected in F4/80 positive macrophages. The mRNA expression of Gpnmb was not correlated with M1 macrophage phenotypes CD40 and CCR7, but positively correlated with M2 macrophages phenotypes CD163 and MMR. Western blotting and ELISA result showed that there was significant increase of Gpnmb expression in the urine from IRI mice compared to those of the control group and the sham group (P＜0.01). Conclusions    Gpnmb expression is up-regulated in IRI kidney and is associated to M2 macrophages. It may play a role in the process of acute kidney injury. Gpnmb expression is also increased in urine after IR injury and it may be a new biomarker to diagnose AKI.

Objective      To evaluate the effects of high glucose on autophagy and apoptosis of podocyte and explore the signaling pathway in high glucose - induce podocyte autophagy.    Methods       Differentiated mouse podocytes were exposed to high glucose(30 mmol/L) or rapamycin (autophagy enhancer, 1 μg/L) or LY294002 (a selective PI3K inhibitor, 50 mmol/L) for 24 h. The formations of autophagy were observed by electron microscopy and acridine orange staining. Apoptosis was evaluated by flow cytometry. The expression of autophagy protein LC3 - II/I and Beclin - 1 as well as the phosphorylation of AKT and mTOR were examined by Western blotting analysis.   Results       High glucose induced podocytes apoptosis，increased autophagy and the expression of autophagy-associated proteins (all P＜0.05). Rapamycin further increased the expression of LC3-II and Beclin-1 protein (all P＜0.05)，but LY294002 inhibited partialiy the protein expression of LC3-II and Beclin-1 induced by high glucose (both P＜0.05). Treatment with rapamycin increased the phosphorylation of AKT, but reduced that of mTOR in podocytes. Moreover, LY294002 inhibited phosphorylation of both AKT and mTOR (both P＜0.05).   Conclusions       High glucose promotes podocyte autophagy and apoptosis. High glucose-induced autophagy is mediated partly through PI3K-AKT-mTOR signaling pathway.

Objective       To investigate the role of human adipose-derived mesenchymal stem cells (ADSC) cultured with astragaloside IV(Ast) on renal tubular epithelial cells line (HKC) induced by cisplatin in vitro.   Methods       HKC was divided into four groups: ⑴ normal control group；⑵ cisplatin group；⑶ ADSC and HKC group；⑷ ADSC cultured with Ast and HKC group. After coincubated with ADSC,the apoptosis index and proliferation of HKC were detected respectively. The levels of interleukin - 6 (IL - 6), regulated upon activation normal T cell expressed and secreted (RANTES), erythropoietin (EPO) and insulin like growth factor 1 (IGF - 1) were detected by ELISA. Transwell culture system were used to test the migration effect of ADSC.   Results       Compared with cisplatin group, the apoptotic rates of HKC were decreased and the cell number increased obviously (P＜0.05) in ADSC and HKC group and ADSC cultured with Ast and HKC group． Compared with cisplatin group, the expression of IL-6 and RANTES were lower (P＜0.05)，and the level of EPO and IGF -1 increased significantly (P＜0.05), in ADSC cultured with Ast and HKC group. Crystal violet staining showed that ADSC cultured with Ast and HKC group migration index was higher than the other three groups (P＜0.05).   Conclusion       The intervention of Ast may stimulate paracrine effect and migration of ADSC, so as to reduce apoptosis of HKC.

Objective     To investigate the expression and significance of related substances of coagulation and fibrinolysisintheratkidneyswithischemiareperfusion(IR)injury.   Methods       Forty-eight male Wistar rats were randomly divided into six groups: Sham, IR 0 h, IR 2 h, IR 6 h, IR 12 h and IR 24 h. IR rats were made by occlusion of bilateral renal arteries by bulldog clamp for 45 minutes. Renal pathology was observed by HE staining. The expression of endothelin-1（ET-1）, tissue plasminogen activator (t - PA) and fibrinogen - like protein 2 (Fgl - 2) of renal tissue was detected by immunohistochemical method.   Results       Scr and BUN increased significantly in IR 24 h group compared with sham group(P＜0.05). Renal pathological injury was significantly aggravated with prolonged reperfusion. The semi- quantitative scores of renal tubular injury were increased in all IR group compared with sham group(P＜0.05). The expression of ET-1 was gradually increased before the first six hour after IR and decreased after twelve hour. Compared with sham group, ET-1 was increased significantly in all IR group. The expression of Fgl-2 fibroleukin in renal tissue increased progressively in the first six hour after IR, weaken in IR 12 h group and trended to normal in IR 24 h group. The expression of t-PA increased progressively during the first six hour after IR, maintained this level in IR 12 h group and decreased in IR 24 h group.   Conclusion       The abnormal expression of related substances of coagulation and fibrinolysis of rats may participate in renal IR injury.