Objective    To analyze the risk factors of mortality among patients treated by maintenance hemodialysis (MHD), and identify whether handgrip strength (HGS) or other nutrient markers could predict the mortality independently.    Methods    One hundred and eight patients receiving regular MHD in Peking Union Medical College Hospital from July to September, 2008 were involved. Baseline data including clinical data, nutrient data such as subjective global assessment, anthropometrics and biochemical measurement were collected. After being followed for 72 months, the patients' mortality and morbidity of cardiovascular event were recorded. Cox regression model was used to estimate the risk factors of mortality.    Results    The average age of 108 MHD patients was (57.6±13.0) years. During the 6-years following up, 35 patients died (32.4%), of whom 62.9% died of cardiovascular events. Among variables, patients’ age, residual urine volume, serum creatinine level, prealbumin level and mean leg circumference were risk factors for all-cause mortality. The patient with lower HGS bore higher risk for all-cause mortality (HR=2.842, 95%CI 1.390-5.811) and cardiovascular death (HR=2.826, 95%CI 1.150-6.947). After adjusting gender, age, history of cardiovascular disease and diabetes, body mass index (BMI), dialysis vintage, Kt/V, nPCR and prealbumin, lower handgrip strength was still an independent risk factor of all-cause mortality (HR=2.505, 95%CI 1.112-5.642). In prediction for all-cause mortality by HGS, the area under the receiver operating characteristic curve(ROC) were 0.705 and 0.682 among men and women respectively.    Conclusion    Lower handgrip strength can predict mortality of maintenance hemodialysis patients independently.

Objective    To evaluate cardiac biomarkers as biological risk factors for cardiovascular and all-cause motality in HD patients. In addition, a multimarker approach including inflammatory index was performed to improve the cardiovascular and all-cause risk assessment of these patients.    Methods    The author measured Troponin-T (TnT), N-terminal pro brain natriuretic peptide (NT-proBNP) and high-sensitivity C-reactive protein (HsCRP), collected the clinical data at baseline(January 2012) in 229 HD patients in three hemodialysis centers in Haidian District of Beijing, recorded time and cause of death in the next 1000 days. Kaplan-Meier was used to calculate survival rate and impact factors of prognosis. Cox proportional hazard model was used to estimate significance of TnT, NT-proBNP and HsCRP and adjusted hazard ratios (HRs) of death.    Results    During the follow-up, 37 patients died, mainly from cardiac cause (54.05%, 20/37). Univariate analysis found old age, diabetes, cardiovascular disease, low serum albumin, CRP≥3 mg/L, TnT≥0.1 mg/L, NT-proBNP≥4381 ng/L were associated with prognosis. Elevated cTnT, NT-proBNP or HsCRP were all associated with increased cardiovascular and all-cause motality. Moreover, the combination of all parameters (NT-proBNP≥4381 ng/L and TnT≥0.1 mg/L and HsCRP≥3 mg/L) were dramatically associated with increased cardiovascular cause mortality (HR=25.25, P＜0.01) and all-cause mortality (HR=27.33, P＜0.01). The association were significant even after full adjustment for cardiovascular (HR=14.33, P＜0.01) and all-cause mortality (HR=11.54, P＜0.01) respectively.    Conclusions    A combined index of cardiovascular risk factors could provide supplementary risk stratification in HD patients for cardiovascular mortality and all-cause mortality, strongly supporting the annual routine determination of these biomarkers.

Objective    To assess the value of multislice spiral computed tomography (MSCT) in diagnosing pulmonary hypertension.    Methods    One hundred and forty-two patients on hemodialysis were divided into the group with pulmonary artery hypertension and the group without pulmonary artery hypertension. The diagnosis of pulmonary artery hypertension (pulmonary artery systolic pressure, PASP＞35 mmHg) was according to the guideline from the American Society of Echocardiography. All patients were received the check of MSCT and the diameters of the main pulmonary artery, ascending aorta and descending aorta were recorded. PASP and left ventricular ejection fraction were assessed by echocardiography. High sensitivity C-reactive protein and tumor necrosis factor were measured by automatic analyzer and enzyme linked immunosorbent assay.    Results    There were significant differences between the two groups in systolic blood pressure, hemoglobin, serum albumin, high sensitivity C-reactive protein and TNF-α (P＜0.05); There were significant differences between the two groups in diameters of the maim pulmonary artery, ratio of the diameter of the main pulmonary artery to the diameter of ascending aorta and ratio of the diameter of the main pulmonary artery to the diameter of descending aorta (P＜0.05). In different heart function groups, there were significant differences in diameters of the main pulmonary artery, ratio of the diameter of the main pulmonary artery to the diameter of ascending aorta, and ratio of the diameter of the main pulmonary artery to the diameter of descending aorta, and left ventricular ejection fraction (P＜0.05). Ratio of the diameter of the maim pulmonary artery to the diameter of ascending aorta was positively related to PASP (r=48.77, P＜0.01), and left ventricular ejection fraction was negatively related to PASP (r=-0.40, P＜0.01). In multivariate linear regression, TNF-α, ratio of the diameter of the maim pulmonary artery to the diameter of ascending aorta and ejection fraction were independent factors of PASP (P＜0.01).    Conclusions    MSCT measurements play an important role in diagnosis of pulmonary hypertension and in evaluation of clinical prognosis in patients on hemodialysis.

Objective    To investigate the relationship between indoxyl sulfate (IS) and left ventricular hypertrophy (LVH) in hemodialysis patients.    Methods    For the eligible patients (age ≥18 years, dialysis duration > 6 months, without history of congestive heart failure within 3 months and comorbidity of cardiac aneurysm), clinical data were collected, biochemical measurements were completed, and echocardiographic examinations were performed. Plasma IS concentration was determined by high performance liquid chromatography electrospray tandem spectrometry (HPLC-ESI-MS/MS). Linear and Logistic regression models were employed to assess the associations of plasma IS and left ventricular mass index (LVMI) and LVH, respectively.    Results     Two hundred and ten hemodialysis patients (117 males) with mean age of（57.2 ± 14.3）years were enrolled. The prevalence of LVH was up to 64.0%. Univariate linear regression showed that plasma IS was positively correlated with LVMI (β=7.09, P=0.02). The result persisted after adjustment for all kinds of risk factors (β=4.16, P=0.03). Patients were categorized into two groups: LVH and non-LVH group. Logistic regression models were employed to assess the relationship of plasma IS and LVH. The result showed that plasma IS was independently associated with LVH after adjustment for other confounding risk factors (β=6.54, OR=1.13, 95%CI 1.09-1.44, P=0.03).    Conclusions    LVH is prevalent in hemodialysis patients. Plasma IS is significantly correlated with LVMI and the independent risk factor for LVH.

Objective    To compare the detection ratio of bacterial colony count in dialysis water, dialysate and raw water with different media and different incubation conditions, searching for suitable methods to detect bacteria from dialysis water.    Methods    Between Jan 2012 and Dec 2012, 176 samples of hemodialysis water and dialysate, 88 samples of raw water were collected by hemodialysis unit, Renji hospital. Samples were inoculated in duplicate on spread plates with blood agar, Reasoner's 2A(R2A), tryptone glucose extract agar(TGEA) respectively, at different temperature and time. After incubation, the numbers of colonies were quantified.    Results    The bacterial colony counts in dialysis water, dialysate and raw water were the lowest in blood agar and the difference had statistical significance with R2A or TGEA culture media (P＜0.01). The detection rate of dialysis water and dialysate were 61.9% in R2A at 20℃ for 168 hours. According to the standard of the association for the advancement of medical instrumentation(AAMI), the detection rate had no difference among these media with different conditions. Combined with medium of R2A and TGEA, the detection rate of bacteria was 77.6% at 20℃ for 168 hours and the bacteria detection rate was significantly increased than that in culture R2A and TGEA under the same conditions (77.6% vs 61.9%, 50.6%, P value was 0.003 and 0.001).    Conclusions    The method of R2A or TGEA agar culture can improve the detection rate of bacterial colony count compared with the method of blood agar. The method of R2A combined with TGEA at 20℃ for 168 hours can improve the detection rate of bacteria in dialysis water and dialysate.

Objective    To discuss the relationship between serum anti-Phosphalipase A2 receptor (PLA2R) antibodies and glomerular IgG4 subclass in patients with membranous nephropathy and evaluate the diagnostic value of the two markers.    Methods    Patients diagnosed as membranous nephropathy from October 2011 to April 2014 in Peking Union Medical College Hospital were included and divided into IMN and SMN groups accoding to their clinical diagnosis. Serum anti-PLA2R antibodies and glomerular IgG subclasses were both detected by indirect immunofluorescence assay. Receiver operator characteristic curves were used to evaluate the diagnostic efficiency of anti-PLA2R antibodies and glomerular IgG4.    Results    Prevalence of serum anti-PLA2R antibodies of IMN patients was 69.5% (41/59); prevalence of MLN patients was 4.8% (1/21). Within the IMN group, thirty-five patients showed positive results of both serum anti-PLA2R antibodies and glomerular IgG4; Six patients were positive for serum anti-PLA2R antibodies but negative for glomerular IgG4; Seventeen patients were positive for glomerular IgG4 but negative for serum anti-PLA2R antibodies; one patient was negative for both tests. The sensitivity of serum anti-PLA2R antibody was 69.5% and the specificity was 95.2%; the sensitivity of glomerular IgG4 was 89.8% and the specificity was 52.3%. The sensitivity of the combined marker consisting of serum anti-PLA2R antibody and glomerular IgG4 was 59.3% and the specificity was 100%. Four out of the six patients secondary to HBV infection, one out of the three patients secondary to Sjögren syndrome, one out of the three patients secondary to malignant tumor showed positive results of serum anti-PLA2R antibodies.    Conclusions    Serum anti-PLA2R antibodies were of high prevalence among IMN patients; the prevalence among SMN patients varied with etiologies. Results of serum anti-PLA2R antibodies and glomerular IgG4 were helpful to rule out secondary etiologies in the diagnosis of membrnous nephropathy.

Objective    To establish an uric acid associated nephropathy (UAN) animal model and explore the mechanisms involved.    Methods    Eighteen (6-8 weeks old) male Sprague-Dawley rats weighed 200-220 g were randomly assigned into 2 groups: the control group (n=9), the uric acid associated nephropathy group (n=9). UAN rat model was established by oral administration of adenine (0.1g/kg) and potassium oxonate (1.5 g/kg) mixture daily for 3 weeks. After 3 weeks, the rats were sacrificed and blood and kidney samples were collected. Serum uric acid, creatinine and other biochemistry index were measured weekly. PAS and Masson staining were conducted to evaluate renal pathology and renal fibrosis. Serum activity of xanthine oxidase (XOD) was examined. Expression of p-EGFR and EGFR were detected by western blot and α-SMA expression was detected by immunohistochemical staining.    Results    After 3 weeks, the model group rats got 1.5 folds increased  serum creatinine and significantly elevated serum uric acid. PAS and Masson staining showed that the UAN kidney developed glomerulosclerosis, tubulointerstitial damage and inflammatory cell infiltration. Serum activity of xanthine oxidase (XOD) was significantly upregulated in UAN group [(52.68±9.79) μmol/L vs (32.23±6.72) μmol/L, P＜0.05]. Western blot showed that EGFR was activated and α-SMA expression increased remarkably in renal interstitial area of UAN rats.    Conclusions    The mixture of adenine and potassium oxonate can successfully establish UAN model. Phosphorylation of EGFR may mediate the activation of renal interstitial fibroblasts and accelerate the development and progression of UAN.

Objective    To investigate the effect of macrophage polarization on tubulointerstitial fibrosis of mouse unilateral ureteral obstruction(UUO) model.    Methods    Twelve male C57BL/6J mice were employed, each of which with an age of 8 to 10 weeks. UUO model was established with these mice with the method of unilateral ureteral ligation. Mice were then sacrificed on the 7th and 14th day respectively after operation, and renal tissue specimens were obtained. The authors detected collagen deposition by Masson staining, and alpha smooth muscle actin (alpha SMA) as well as collagen type I (Coll-1) mRNA by real-time quantitative PCR. The authors also detected the degree of renal interstitial macrophages infiltration and expression changes of polarization by immunofluorescence staining.    Results    Compared with the mice that were observed on the 7th day after operation, the degree of renal interstitial fibrosis in mice observed on the 14th day after operation was comparatively serious, the difference shown by semi-quantitative results was statistically significant (P＜0.05). Moreover, mice observed on the 14th day after operation have more M2 macrophages, the difference between two groups of mice was statistically significant (P＜0.05). On the contrary, there was no statistically significant difference in the degree of M1 macrophages infiltration between these two groups of mice.    Conclusions    In the renal interstitial fibrosis model induced by UUO, the degree of macrophage infiltration increased significantly, mainly resulted from M2 macrophage infiltration, suggesting that M2 macrophages were involved in the formation of renal fibrosis.

Objective    To test the hypothesis of autophagy that silencing  PHD2 gene could increase hypoxia inducible factor (HIF)-1α levels in the renal medulla and attenuate hypoxia injury in cultured human renal proximal tubular epithelial cell (HK-2) under cobalt dichloride (CoCl2) exposure.  Methods  HK-2 cells were harvested at hour 0, 6, 12, 24, 36 and 48 after exposure to CoCl2 (200 μmol/L). The role of HIF/PHD pathway in CoCl2-induced cell apoptosis/autophagy was studied by employing small-interfering RNA (siRNA).  Dynamic profiles of apoptosis markers (Bax, Bcl-xl) and autophagy marker (LC3) of HK-2 cells within 48 h after exposing to CoCl2 were recorded. Alamar Blue assay was used for quantitative analysis of cellular growth and viability. Electron microscopy analysis was employed to evaluate the changes in autophagic structures.  Results The protein expressions of PHD2 were gradually increased after exposing to CoCl2 (200 μmol/L), with statistics significance at 24 h and reached the peak at 48 h (both P＜0.01). PHD2 siRNA reduced PHD2 levels by＞60% and significantly increased HIF-1α protein levels (P＜0.01), but had little effect on HIF-2α. The protein expression of Bcl-xl was significantly up-regulated, while the level of Bax and LC3-Ⅱ/LC3-Ⅰ were down-regulated in PHD2 siRNA group (all P＜0.01), compared with the negative control group. Meanwhile, either 3-Methyladenine (an autophagy inhibitor) treatment or PHD2 knockdown rescued cell death and increased cell viability through autophagy inactivation. The ratio of LC3-Ⅱ/LC3-Ⅰand the quantity of autophagosomes were decreased, and the cell ultrastructure was also relatively intacter than the negative control group. Of interest, co-administration of HIF-1α siRNA with PHD2 siRNA abrogated renoprotective effect conveyed by PHD2 siRNA alone, suggesting that activation of endogenous HIF-1α-dependent pathways mediated the autophagy inactivation effects of PHD2 silencing.    Conclusions    Direct inhibition of PHD2 promotes renal epithelia cell survival against CoCl2-induced cell apoptosis/autophagy. Activation of the HIF-1α signaling pathway is required to reduce apoptosis and autophagy via up-regulating the expression of Bcl-xl protein.

Objective    To explore the protective effect and underlying mechanism of telmisartan on hyperuricemic nephropathy.    Methods    (1)High level of uric acid (600 μmol/L) and telmisartan in different concentrations (10nmol/L, 100 nmol/L, 1000 nmol/L, 10000 nmol/L) were added to renal tubule epithelial cells and cultured for 48 h, the expression of UAT, TGF-β1 and α-SMA were detected by Real-time PCR, RT-PCR, Western blotting or cell immunofluorescence. (2) Wister rats were randomly divided into normal control group(Con), high uric acid group (HU), and telmisartan treatment group (Tel). Four weeks later, Scr, BUN and serum uric acid of the rats were detected. The expression of UAT in rat kidney was detected by Western blotting.    Results    (1)In vitro, compared to control group, high uric acid (600 μmol/L) inhibited the expression of UAT (P＜0.01), and the inhibition could be alleviated by telmisartan; Telmisartan inhibited the upregulation of TGF-β1 and α-SMA induced by high uric acid(all P＜0.05); (2)In vivo, compared to high uric acid group rats, telmisartan  group rats had significantly reduced serum uric acid levels (189.9 μmol/L vs 204.5 μmol/L, P＜0.05), upregulated UAT and downregulated TGF-β1 expression in rat kidney (all P＜0.05).    Conclusion    Telmisartan significantly inhibits the upregulation of TGF-β1 and α-SMA induced by uricemia,  which may prevent kidney from fibrosis. The protect effect of telmisartan may be related to the upregulation of UAT.

Objective    To explore the role of Hippo pathway in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD), and find potential targets for drug therapy.    Methods    By means of immunofluorescence staining, Western blotting, Real-time PCR, the differences of sublocalization, expression and phosphorylation level about Hippo pathway molecules in Han:SPRD（cy/+）and ADPKD patients compared with the control were observed. Knockdown Yes kinase-associated protein (YAP), transcriptional coactivator with PDZ binding motif (TAZ) and large tumor suppressor kinase1 (LATS1) in cystic lining epithelium cell line WT9-12 were took by siRNA interference, and then their effects on cell proliferation, apoptosis and cell cycle were assessed.    Results    In cystic lining epithelium of Han:SPRD(cy/+), decreased expression of LATS1 and increased expression of YAP were found compared with the control, and the immunofluorescence of YAP was distributed both in cytoplasm and nucleus, while distribution and expression level of TAZ were without significant variance. Abnormal mRNA expressions of Hippo pathway components in ADPKD patients were found (P＜0.05). Down-regulation of LATS1 in WT9-12 cells could prohibit phosphorylation of YAP, and prompted proliferation and cell division. Knockdown YAP in WT9-12 cells could inhibited cell proliferation by arresting cell cycle in G0/G1 phase, but down-regulating TAZ showed no significant differences in proliferation and cell cycle.    Conclusions    Altered Hippo signaling exists in ADPKD, and YAP activation may be one leading cause of autosomal dominant polycystic kidney disease onset. In vitro, knockdown YAP in WT9-12 cells can inhibit cell proliferation by arresting cell cycle and depressing cell division, suggesting the expression level and activity of YAP are potential targets for ADPKD treatment.