Objective To explore the role and mechanism of tumor necrosis factor-α (TNF-α) in the formation of chronic transplant renal interstitial fibrosis and to study Smurf2 expression change and role played in this process.Methods We collected 26 cases of normal renal tissues and 26 cases of renal allograft specimens from chronic allograft dysfunction patients to observe the degree of renal interstitial fibrosis by Hematoxylin and eosin (HE) staining and Masson staining.Immunohistochemical staining was applied to detect the expression and distribution of E-cadherin,α-smooth muscle actin (α-SMA) and TNF-α,Smad ubiquitination regulatory factor 2 (Smurf2).Furthermore,The HK2 cells were divided into five groups depending on different concentrations of TNF-α (0,10,20,50,and 100 ng/mL).After 48 h,we collected the cells to analyze the expression changes of Smurf2,E-cad,α-SMA by Western blotting.Results As compared with normal group,the degree of renal interstitial fibrosis in CAD group was aggravated according to the results of HE and Masson staining (P＜0.01).Immunohistochemical staining results showed that the positive expression of E-cadherin was reduced,and that of α-SMA,TNF-α and Smurf2 increased greatly in CAD group as compared with normal group (P＜0.01).The results of Western blotting also revealed that after treatment with different concentrations of TNF-α for 48 h,the expression of E-cadherin was downregulated,and that of α-SMA and smurf2 was up-regulated in HK2 cells (P＜0.05).Conclusion TNF-α may promote the formation of allograft renal interstitial fibrosis via transdifferentiation of human tubular epithelial cells to mesenchymal cells by up-regulating the expression of Smurf2.

Objective: To investigate the awareness of and participation in Healthy City construction among residents in Hangzhou City, so as to provide insights into promotion of participation in Healthy City construction. Methods: Residents at ages of 15 to 75 years were sampled using the multi-stage stratified random sampling method, from 30 townships in Jianggan, Xiaoshan and Tonglu counties of Hangzhou City from November 2019 to July 2020, and a questionnaire survey of 10 representative projects pertaining to Healthy City construction in Hangzhou City was performed to investigate the awareness of and participation in Healthy City construction. Results: A total of 5 559 questionnaires were allocated, and 5 211 valid questionnaires were recovered, with an effective recovery rate of 93.74%. The respondents had a mean age of ( 43.82±17.25 ) years, and included 2 280 males ( 43.75% ) and 2 931 females ( 56.25% ). The overall standardized awareness and participation rates of Healthy City construction were 81.73% and 48.58% among the respondents. The projects with the three highest awareness included healthy environment improvements ( 92.67% ), travelling by public transportation ( 92.22% ) and tobacco control action ( 91.04% ), while the projects with the three lowest awareness included chronic disease management ( 75.57% ), maternal and child healthcare ( 72.73% ) and “Healthy Cell” Program ( 45.56% ). The projects with the three highest participation rates included travelling by public transportation ( 74.59% ), healthy environment improvements ( 65.17% ), tobacco control action ( 61.52% ), while the projects with the three lowest participation rate included chronic disease management ( 35.92% ), “Healthy Cell” Program ( 34.96% ) and maternal and child healthcare ( 33.20% ). Conclusions The overall proportion of participation in Healthy City construction is low among residents in Hangzhou City, and notably, the awareness rate of and the proportion of participation in chronic disease management, maternal and child healthcare and “Healthy cell” Program are both low.

Objective To investigate the possible mechanism that hepatocyte growth factor (HGF) inhibits renal tubular epithelial-mesenchymal transition (EMT),and to determine whether Smurf2 expression induced by TGF-β1 can be reversed by HGF in normal rat kidney epithelial cells (NRK-52E).Methods Using rat NRK-52E cell line as an in vitro system,NRK-52E cells were incubated with 5 μg/L TGF-β1 for 0-24 h.Part of cells were pretreated with 20 μg/L HGF for 30 min or not,then incubated with or without 5 μg/L TGF-β1 for 1 h or 48 h.The other cells were transfected with pFlag-Smurf2 or Smurf2 siRNA for 24 h,then treated with or without 20 μg/L HGF for 24 h.The expressions of Smurf2,SnoN,E-cadherin,alpha-smooth muscle actin (α-SMA) and fibronectin (FN) were detected by Western blotting and indirect immunofluorescence staining assays.Results Compared to normal control,TGF-β1 could rapidly induce Smurf2 protein expression in a short time (P＜0.01).Meanwhile,the expressions of FN and α-SMA were significantly induced,and the expression of E-cadherin was reduced in NRK-52E cells by TGF-β1.In contrast,in the NRK-52E cells pretreated with HGF,HGF could obviously inhibit Smurf2 expression induced by TGF-β1,and reversed the down-regulation of SnoN (P＜0.01) and E-cadherin (P＜0.05),the up-regulation of α-SMA (P＜0.01) and FN (P＜0.01) induced by TGF-β1.Moreover,overexpression of Smurf2 in NRK-52E cells could partly inhibit the up-regulation of SnoN protein by HGF,while down-regulation of Smurf2 could up-regulate the expression of SnoN induced by HGF.Conclusions HGF can abolish EMT induced by TGF-β1 in renal tubular epithelial cells through down-regulating Smurf2 expression and suppressing ubiquitin-proteasome dependent degradation of SnoN.

Objective To investigate the effect of high glucose on renal tubular epithelial-mesenchymal transition,and to analyze the relationship between high glucose and transforming growth factor β1(TGF-β1)and the mechanism of renal interstitial fibrosis. Methods HKC and Smad7-overexpression HKC cells were grown in DMEM/F12 medium containing 5%～10%newborn calf serum.They were cultured for 16 h in free serum medium after 80%cells were adhered onto the surface of the flask.Afterwards,they were stimulated by high glucose(glucose concentration:25 mmol/L and 50 mmol/L).The expression of α-SMA,E-cadherin and fibronectin was detected by Western blot while the supernatant level of TGF-β1 was detected by ELISA.Cell motility and migration was evaluated using Boyden chamber motogenicity assay. Results In HKC induced by high glucose,the expression of α-SMA and fibronectin protein was highly upregulated while the expression of E-cadhefin protein was down-regulated.The expression of TGF-β1was up-regulated in a dose-dependent manner.These above-mentioned effects could be obviously inhibited by anti-TGF-β1 antibody.The protein expression of α-SMA,fibronectin and E-cadherin had no obvious change in Smad7-overexpression HKC induced by high glucose.HKC exhibited enhanced motility and invasive capacity in high glucose groups,compared to that in control group.Migrated cell counting was(12.4±3.7)and(18.6±4.4)cell/HP in 25 and 50 mmol/L glucose groups respectively. Conclusion High glucose may induce renal tubular epithelialmesenchymal transition through TGF-β1 pathway,which can be inhibited by blocking the Smad signal pathway.

Objective To investigate the protective effects of purified effective component group in extract from Xiaoshuan Tongluo(CGXT)formula on chronic brain ischemia in rats.Methods CGXT 75,150,and 300 mg/kg or vehicle were ig administered daily for four weeks to rats with bilateral common carotid arteries ligation(BCCAL).From the day 24 to 28 after BCCAL,Morris water maze was performed to assess the learning and memory impairment of rats.Four weeks after BCCAL,brain gray and white matter damage were assessed.Results In Morris test,the mean escape latency of rats in the CGXT(150 and 300 mg/kg)groups was significantly shorter than that in the vehicle group.CGXT also attenuated the neuronal damage in hippocampus and cortex and reduced the pathological damage in the optic tract and corpus callosum.Conclusion CGXT could improve learning and memory impairment resulted from BCCAL in rats.These results provide the experimental basis for the clinical use of CGXT in stroke treatment and may help in investigation of multimodal therapy strategies in ischemic cerebrovascular diseases including stroke.

Objective To investigate the relationship between high-glucose-induced fibronectin(FN) expression and up-regulation of integrin-linked kinase(ILK) in human kidney tubular epithelial cells (HKC) and kidney of CD-1 mice. Methods Cultured human kidney tubular epithelial cells and streptozotocin (STZ)-indueed diabetic model of CD-1 mice were enrolled in this study.Western blot was used to detect the expression of FN and ILK.The kinase dead ILK plasmid (pCMV-kdlLK) were transferred to HKC. Results Four weeks after injection of STZ,CD-1 mice had higher blood glucose level as compared to the control [(20.3±2.7) mmol/L vs (6.1±1.4) mmol/L,P＜0.01].Meanwhile,expression of FN and ILK was significantly increased in diabetic mice as compared to the control (P＜0.01).There was positive correlation between the expression of FN and ILK (r=0.899,P＜0.01).High-glucose could up-regulate FN and ILK expression in cultured HKC in a time- and dose-dependent manner.Blockage of ILK activation by pCMV-kdILK abrogated high-glucose-incuced FN expression in HKC. Conclusions Highglucose can induce FN expression through up-regulating ILK expression.Blockage of ILK activation abrogates this effect.

Objective To investigate the possible mechanism of glomerular injury in diabetes mellitus by determining whether epithelial-mesenchymal transition (EMT) is caused by high glucose in mice podocytes. Methods Using mice glomerular podocyte cell line as an in vitro system, podocytes were incubated with glucose(12.5 mmol/L, 25 mmol/L, 50 mmol/L) and mannitol (50 mmol/L) for 36 hours. Then the cells were collected and expression of alpha-smooth muscle actin(α-SMA), fibronectin (FN), CD2 associated protein (CD2AP) and Wilms' tumor 1 gene (WT-1) was detected by Western blot and indirect immunofluorescence staining. Results Under low glucose (5.6 mmol/L) and mannitol (50 mmol/L) condition, there were high expression of CD2AP and WT-1, and low expression of α-SMA and FN in mice podocytes. After 36 hours treatment with high glucose (12.5 mmol/L), the expression of α-SMA and FN in podocytes was significantly increased, and the expression of α-SMA and FN was further up-regulated with the increase of glucose dosage (25, 50 mmol/L). The indirect immunofluorescence staining revealed the similar result, and the percentage of positive α-SMA cells was also increased compared with low glucose and mannital group (P<0.05). Meanwhile, Western blot showed that high glucose could down-regulate the expressions of CD2AP and WT-1 in a dose-dependent manner. Conclusion EMT may be a potential pathway leading to podocyte dysfunction and glomerular injury under high glucose conditions.

Objective: To investigate the effect and related mechanisms of RTA-408 on rat vascular smooth muscle cells (VSMCs) calcification induced by advanced glycation end products(AGE). Methods: VSMCs were isolated from the aorta of Sprague Dawley rats and cultured in vitro. The fifth generation of VSMCs were randomly divided into 4 groups with random number table including control group(cells were incubated with normal medium for 2 days, then incubated with bovine serum albumin for 5 days),AGE group (cells were incubated with normal medium for 2 days, then incubated with 200 mg/L AGE for 5 days), experimental group(cells were incubated with 100 nmol/L RTA-408 for 2 days,then incubated with 200 mg/L AGE for 5 days),and RTA group(cells were incubated with 100 nmol/L RTA-408 for 2 days,then incubated with bovine serum albumin for 5 days). Cytosolic calciumin VSMC was measured using arsenazo Ⅲ assay. Von Kossa staining was utilized to detect the calcium deposition.The contents of malondialdehyde(MDA) and superoxide dismutase(SOD) in VSMCs were tested by appropriate kits.The protein expressions of osteopontin (OPN), alkaline phosphatase (ALP), nuclear factor E2 related factor 2(Nrf2), and NAD(P)H: quinone oxidoreductase 1(NQO1) were examined using Western blot. Results: (1) Cytosolic calciumconcentration was significantly higher in AGE group than in control group((2.43±0.15) mmol/L vs. (1.23±0.09) mmol/L, P<0.01), which was significantly reduced in experimental group((1.62±0.18) mmol/L,P<0.01 vs. AGE group). (2) Calcium deposition in VSMCs was significantly upregulated in AGE group than in control group(3.64±0.50 vs. 1.00±0.12, P<0.01), and was downregulated in experimental group (1.56±0.37, P<0.01 vs. AGE group). (3) The MDA contents were higher((3.79±0.27) nmol/mg prot vs.(1.99±0.15) nmol/mg prot, P<0.01), while the SOD activities were lower((308.45±14.28) U/mg prot vs. (428.58±11.00) U/mg prot, P<0.01) in AGE group than in control group. The MDA contents were lower((2.37±0.19) nmol/mg prot vs. (3.79±0.27) nmol/mg prot, P<0.01),while the SOD activities were higher((391.03±22.92) U/mg prot vs. (308.45±14.28) U/mg prot, P<0.05)in experimental group compared with AGE group. (4) The relative expressions of OPN and ALP were higher in AGE group than in control group(3.06±0.21 vs. 1.00±0.07, and 2.89±0.29 vs. 1.00±0.10,both P<0.01), both (OPN(1.15±0.12) and ALP(1.45±0.15)) were downregulated in experimental group (both P<0.01 vs. AGE group). (5) The relative protein expressions of Nrf2 and NQO1 in experimental group were higher than AGE group(2.37±0.17 vs. 1.17±0.09, and 3.91±0.18 vs. 1.05±0.08, both P<0.01). Conclusion Activation of nrf2/NQO1 signaling pathway by RTA-408 can reduce the AGE-induced VSMC calcification through attenuating oxidative injury.

Objective To compare the accuracy of dynamic contrast-enhanced magnetic resonance (DCE-MRI) and SPECT in the measurement of glomerular filtration rate (GFR) in renal allografts.Methods Sixty renal transplant recipients were enrolled in this study.DCE-MRI and SPECT were used to measure the GFR of the transplanted kidneys,and compared with the endogenous creatinine clearance rate (Ccr).Bias,precision,correlation and Bland-Altman agreement were calculated for each modality compared with the endogenous Ccr.Results In 60 renal transplant recipients,the corrected Ccr was (60.63 ± 24.83) ml · min-1 · 1.73 m-2.The GFR measured by SPECT was (65.31 ± 17.08) ml · min-1 · 1.73 m-2,and (50.44 ± 22.78) ml · min-1 · 1.73 m-2 by MRI,respectively.The bias of GFR-SPECT was 4.69 ml·min-1 · 1.73 m-2,and the precision was 23.76 ml·min-1 1.73 m-2.The bias of GFR-MRI was-10.18 ml·min-1 ·1.73 m-2,and the precision was 13.87 ml·min-1 · 1.73 m-2.Correlation analysis showed that GFR-MRI and the endogenous Ccr had a good correlation (r=0.833,P＜0.01),GFR-SPECT and the endogenous Ccr had a moderate correlation (r=0.406,P＜0.01),and GFR-MRI and GFR-MRI had a poor correlation (r=0.342,P ＜0.01).Bland-Altman analysis showed a confidence interval of 95.3 ml·min-1 ·1.73 m-2 for GFR-SPECT and 62.3 ml· min-1 · 1.73 m-2 for GFR-MRI.Conclusion DCE-MRI can be used as confidently as SPECT to evaluate the renal function of transplanted kidneys in the same time of determining anatomical information.

Objective To investigate the effect of Bortezomib on renal tubular epithelialmesenchymal transition (EMT),and to determine whether Smad ubiquitin regulatory factor 2 (Smurf2) expression induced by tumor necrosis factor-α (TNF-α) can be reversed by Bortezomib in human renal tubular epithelial cells (HK-2).Methods The HK-2 cells were divided into control group (cultured with fetal bovine serum),TNF-α group (cultured with TNF-α),Bortezomib group (cultured with Bortezomib) and experimental group (treated with Bortezomib and TNF-α together).Each group of cells was observed under an inverted microscope,and then the cells of each group were collected.RT-PCR and Western blotting were performed to detect the expression levels of fibronectin (FN),Smurf2,E-cadherin and α-smooth muscle actin (α-SMA).Results As compared with the TNF-α-treated group,the morphology of HK-2 cells was still cobblestone-like after intervention with bortezomib;however,the cellular morphology did not change significantly in the bortezomib-treated group as compared with the control group.As compared with the control group,the expression of FN mRNA and protein in the TNF-α group was significantly increased (P＜0.05).As compared with the TNF-α group,bortezomib inhibited the expression of FN induced by TNF-α (P＜0.05) There was no significant difference in the expression of FN between bortezomib-treated group and control group (P ＞0.05).As compared with the control group,E cadherin was significantly decreased in HK-2 cellsafter treatment with TNF-α,and α-SMA was significantly increased (P＜0.05).As compared with TNFα group,co-stimulation with bortezomib reversed the expression of E cadherin and α-SMA induced by TNF-α (P ＜ 0.05),but the expression of E-cadherin and α-SMA did not change significantly in the bortezomib-treated group as compared with the control group (P ＞ 0.05).As compared with the control group,TNF-α could increase the expression of Smurf2 mRNA and protein (P＜0.05),and bortezomib could inhibit the increase in Smurf2 induced by TNFα (P＜0.05).As compared with the control group,the expression of Smurf2 did not change significantly in the bortezomib-treated group (P＞0.05).Conclusion Bortezomib can antagonize the expression of Smurf2 and EMT induced by TNF-α in HK-2 cells.