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Objective:To explore the correlation between socioeconomic status (SES) and diabetic kidney disease (DKD) in patients with type 2 diabetes (T2D).Methods:A total of 276 T2D patients admitted to the Second Affiliated Hospital of Nanjing Medical University from January to June 2020 were enrolled in this cross-sectional study. The estimated glomerular filtration rate (eGFR) was calculated according to the urinary albumin/creatinine ratio (UACR) and the chronic kidney disease epidemiology collaboration equation(CKD-EPI formula) based on serum creatinine. The patients were divided into simple T2D group (184 cases) and DKD group (92 cases). Collect demographic and laboratory examination data, record education, income and occupation, and calculate standardized SES scores. According to SES scores, subjects were divided in three levels: SES≤9, SES≥10-≤12, and SES≥13. Student's t test was used for comparison of measurement data with normal distribution between two groups, and one-way ANOVA was used for comparison between multiple groups. Non-normal distribution was represented by M( Q1, Q3), and rank-sum test was used for comparison between groups. Counting data were expressed as frequency or percentage, and chi-square test was used for comparison between groups. Bofferoni test was further used for pairwise comparison of indicators with statistical significance among multiple groups. Spearman correlation analysis was used to analyze the correlation between variables. The risk factors were analyzed by binary Logistic regression. Results:The age of the subjects was (53.37±10.68) years, men accounted for 55.8% (154/276), the duration of diabetes was 60.00 (12.00, 134.00) months, and eGFR was (97.56±21.15) mL/(min·1.73 m 2). In simple T2D group and DKD group, prevalence of hypertension were 39.7% (73/184) and 57.6% (53/92), systolic blood pressure were (129.43±14.92) mmHg and (139.29±17.61) mmHg, diastolic blood pressure were (81.86±10.06) mmHg and (87.74±11.19) mmHg, serum albumin were (45.74±4.15) g/L and (43.99±5.05) g/L, triglycerides were (1.82±1.24) mmol/L and (2.64±2.92) mmol/L, high density lipoprotein cholesterol were (1.17±0.37) mmol/L and (1.07±0.26) mmol/L, serum uric acid were (298.44±90.73) μmol/L and (336.22±94.01) μmol/L, serum creatinine were (62.83±14.45) μmol/L and (87.75±57.37) μmol/L, eGFR were (102.6±14.28) mL/(min·1.73 m 2) and (87.47±28.04) mL/(min·1.73 m 2), UACR were (7.60 (4.63, 13.15)) mg/g and (93.95 (47.25, 310.25)) mg/g. Prevalence of hypertension, systolic blood pressure, diastolic blood pressure, triglycerides, serum uric acid, serum creatinine, UACR in DKD group were higher than those in simple T2D group. Serum albumin, high density lipoprotein cholesterol and eGFR in DKD group were lower than those in simple T2D group. There was significant difference between the two groups ( χ2=7.95, t values were 4.87, 4.40, 3.04, 3.26, 2.30, 3.22, 5.56, 5.95, Z=13.07, P values were 0.005, <0.001, <0.001, 0.003, 0.001, 0.022, 0.001, <0.001, <0.001, and <0.001, respectively). The number of males in the three groups with SES ≥13 group, SES≥10-≤12 group, SES ≤9 group were 61 (81.3%, 61/75), 55 (59.8%, 55/92), 38 (34.9%, 38/109), respectively. The number of cases with smoking history were 42 (56.0%, 42/75), 41 (44.6%, 41/92), 35 (32.1%, 35/109), respectively. The number of cases with drinking history were 38 (50.7%, 38/75), 32 (34.8%, 32/92), 26 (23.9%, 26/109), respectively. The ages were (47.77±10.76), (52.76±11.22), (57.74±7.96) years old, respectively. Body mass index (BMI) were (26.17±3.87), (24.96±3.93), (24.27±4.89) kg/m 2, respectively. High density lipoprotein cholesterol (HDL) were (1.03±1.03), (1.16±0.41), (1.21±0.32) mmol/L, respectively. Serum uric acid were (336.56±82.05), (293.78±94.78), (307.99±96.53) μmol/L, respectively. EGFR were (105.03±19.72), (99.77±19.44), (90.57±21.49) mL/(min·1.73 m 2),respectively.The difference between groups were statistically significant (χ 2=39.79, 10.55, 14.08, F=22.69, 4.03, 6.20, 4.53, 12.02, P values were <0.001, 0.005, 0.001, <0.001, 0.019, 0.002, 0.012, and <0.001, respectively). Pairwise comparison shows that male and eGFR in SES ≤9 group were lower than those in SES ≥13 group and SES≥10-≤12 group, age in SES ≤9 group was higher than that in SES ≥13 group and SES≥10-≤12 group. The difference was statistically significant (all P<0.05). Smoking history, alcohol history and BMI in SES ≤9 group were lower than those in SES ≥13 group, and the high density lipoprotein cholesterol in SES ≤9 were higher than that in SES ≥13 group. The difference was statistically significant (all P<0.05). Male, alcohol history and serum uric acid in SES≥10-≤12 group were lower than those in SES ≥13 group, and age and high density lipoprotein cholesterol in SES≥10-≤12 group were higher than those in SES ≥13 group. The difference was statistically significant (all P<0.05). Spearman correlation analysis showed that SES in T2D was positively correlated with male, smoking history, alcohol history, BMI, serum uric acid and eGFR ( r values were 0.38, 0.20, 0.24, 0.16, 0.13 and 0.31, P values were <0.001, 0.001, <0.001, 0.008, 0.028, and <0.001, respectively), and negatively correlated with age, high density lipoprotein cholesterol and UACR ( r values were -0.35, -0.24 and -0.14, P values were <0.001, <0.001, and 0.017, respectively). Logistic regression analysis showed that SES (OR=2.71,95% CI:1.10-6.68, P=0.031) was associated with T2DM combined with DKD. The risk of developing DKD increased when the SES was ≤9. Conclusion:The SES in patients with type 2 diabetes is closely related to DKD. Low SES may be a new risk factor for DKD in type 2 diabetic patients.
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Objectives:To investigate the epidemiological features and associated factors of chronic renal insufficiency (CRI) in Binhai county from Jiangsu province.Methods:This is a cross-sectional study including individuals aged≥18 years old and participating in health examinations of Binhai county from January to December 2018. Medical records were collected to analyze the epidemiology of CRI [estimated glomerular filtration rate <60 ml·min -1·(1.73 m 2) -1]. Multivariate logistic regression was used to analyze the associated influencing factors of CRI. Results:A total of 395 541 individuals residing in Binhai county were enrolled, with 190 258 males (48.1%) and age of (55.34±15.12) years old. The overall crude prevalence of CRI was 2.04% (8 065/395 541, 95% CI 2.00%-2.08%) in this adult population. Furthermore, the age- and gender-standardized overall prevalence of CRI was 1.22% (95% CI 1.18%-1.25%), with a rate of 1.47% (4 676/205 283, 95% CI 1.42%-1.52%) in women and a rate 0.95% (3 389/190 258, 95% CI 0.91%-1.00%) in men. There was a strong positive correlation between the risk of CRI and age (per 10-year increase, OR=2.449, 95% CI 2.402-2.497). Compared with individuals <30 years old, the OR of CRI in individuals aged 60-69, 70-79 and ≥80 years old were 3.827 (95% CI 3.010-4.864), 12.004 (95% CI 9.457-15.239) and 44.636 (95% CI 35.187-56.622) respectively. Females ( OR=1.142, 95% CI 1.083-1.203), increasing systolic blood pressure (per 10 mmHg increase, OR=1.062, 95% CI 1.048-1.076), increasing heart rate (per 10-beat/min increase, OR=1.071, 95% CI 1.044-1.098), elevating triglyceride (per 1.33 mmol/L increase, OR=1.140, 95% CI 1.119-1.162), elevating fasting blood glucose (5.6-6.9 mmol/L/<5.6 mmol/L, OR=1.158, 95% CI 1.086-1.233; ≥7 mmol/L/<5.6 mmol/L, OR=1.387, 95% CI 1.296-1.484) and central obesity ( OR=1.126, 95% CI 1.068-1.187) were independent risk factors for CRI. Conclusions:The age- and gender-adjusted prevalence of CRI in adults in Binhai county is 1.22%. Older age, females, central obesity, and high levels of triglyceride, systolic blood pressure, heart rate and fasting glucose are independent associated factors of CRI.
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Objective:To study the relationship between the expression of carnitine palmitoyltransferase 1α (CPT1α) and progression of renal interstitial fibrosis and chronic kidney disease (CKD), and to evaluate the value of CPT1α as a biomarker in pathological diagnosis of renal interstitial fibrosis and CKD.Methods:As a retrospective cohort study, information of CKD patients dignosed with tubulointerstitial fibrosis by renal biopsy and receiving follow-up from March 1, 2010 to July 30, 2017 in the Second Affiliated Hospital of Nanjing Medical University were collected. Renal tissues were stained by immunohistochemistry to detect the expression of CPT1α protein and then divided into three groups according to the quartile of proportion of CPT1α positive staining cells, including group Q1(>67.89%), group Q2(49.84%-67.89%) and group Q3(<49.84%). The degree of renal interstitial fibrosis was measured by Masson staining and lipid deposition was represented by Bodipy staining. Messenger RNA of CPT1α and collagen as well as other extracellular matrix genes were detected by real time-PCR. Relationships between proportion of CPT1α positive staining cells and renal interstitial fibrosis and renal function were analyzed by linear regression analysis. The relationship between CPT1α positive cell number ratio and renal function progression was measured by Pearson correlation analysis and generalized linear model. The effect of lipid-lowering medicine on renal function of CKD patients was analyzed by paired comparative analysis.Results:Ninety patients with CKD were included in this study. Renal interstitial fibrosis and lipid droplets deposition area increased in Q2/Q3 group compared with Q1 group by Masson and Bodipy staining (all P<0.05). Messenger RNA level of extracellular matrix-related proteins increased in Q2/Q3 group by real time-PCR than those of Q1 group (all P<0.05). Linear regression analysis showed that fibrosis area was negatively correlated with the proportion of CPT1α positive staining cells ( r=-0.309, P<0.01). The baseline expression of CPT1α in renal issues was negatively related with serum creatinine (Scr) ( r=-2.801, P<0.001), positively related with estimated glomerular filtration rate (eGFR) ( r=1.240, P<0.001). After a medium follow-up of 3.47 years, CPT1α positive cell number ratio was positively correlated with eGFR change rate by Pearson analysis ( r=0.220, P=0.038). Paired stratified analysis showed that taking lipid-lowering medicines attenuated the decrease of eGFR in Q2 group and Q3 group but not in Q1 group (both P<0.05). Conclusions:The decline of CPT1α in renal tissues of CKD patients is associated with the increase of Scr, the decrease of eGFR and renal interstitial fibrosis. CPT1α is a promising molecular marker to evaluate the degree of renal fibrosis and the progression of CKD.
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Objective To establish a UPLC-MS/MS method for the determination of dexmedetomidine in human plasma and investigate the effect of obstructive jaundice on pharmacokinetics of dexmedetomidine in vivo. Methods Samples were obtained by liquid-liquid extraction. Agilent Eclipse Plus C18 column was used for chromatograph with methanol and 0.1% formic acid-water solution as mobile phase. Flow rate was 0.2 ml/min. The column temperature was 35 ℃, and the MS detection was selected in MRM mode. Results The calibration curves of dexmedetomidine showed good linearity in the ranges of 0.01−10.00 ng/ml. The results of intra and inter-day precisions were both within 15%. The recovery rate was 85.5%−93.1%. Matrix effect was 91.2%−95.6%. Samples remained stable during analysis. Compared with the control group, cmax、AUC(0−t)、AUC(0−∞) and Vz of dexmedetomidine in the patients with obstructive jaundice were increased by 63.4%, 78.9, 66.4%, 82.5%, respectively (P<0.01). CLz was decreased by 42.1%. Conclusion This method is accurate, sensitive and reproducible. It is suitable for dexmedetomidine assay in human plasma. The elimination rate of dexmedetomidine is slower in obstructive jaundice.
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Objective To analyze the incidence and risk factors of hypocalcemia after total parathyroidectomy without autotransplantation. Methods A total of 783 maintenance hemodialysis patients who underwent TPTX in the Second Affiliated Hospital of Nanjing Medical University from September 2008 to September 2017 were included in the study. The preoperative blood biochemical examination, preoperative iPTH, total mass of parathyroid gland (M) and postoperative iPTH and electrolyte results were collected. The incidence of severe hypocalcemia after TPTX were analyzed retrospectively. Binary logistic regression model was used to analyze the risk factors of severe hypocalcemia after TPTX. Results The age of 783 patients with TPTX was (46.90±10.78) years old, and the average dialysis age was (91.36±41.75) months. Postoperative severe hypocalcemia occurred in 235 cases (30.01％). Binary logistic regression analysis showed that higher preoperative blood iPTH (OR=7.56, 95％CI: 1.55-36.79, P=0.01), higher blood alkaline phosphatase (OR=36.71, 95％CI:14.75-91.36, P<0.01), blood phosphorus (OR=1.74, 95％CI: 1.11-2.71, P=0.02) and greater mass of resected glands (OR=1.18, 95％ CI: 1.06-1.31, P<0.01) were the risk factors for post-hypocalcemia. The higher preoperative serum calcium can reduce the risk of postoperative hypocalcemia (OR=0.02,95％CI: 0.01-0.07, P<0.01). Conclusions The incidence of hypocalcemia after TPTX treatment for SHPT is very high. Blood iPTH, alkaline phosphatase, phosphorus, and total mass of intraoperative parathyroid gland excision are the independent risk factors for severe hypocalcemia after surgery.
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Objective To analysis the post-dialysis fatigue status of maintenance hemodialysis patients,explore the influencing factors in these patients and propose effective interventions.Methods One hundred and twenty maintenance hemodialysis patients in Department of Nephrology,Second Affiliated Hospital of Nanjing Medical University were enrolled.Clinical data were obtained by questionnaires.Biochemical changes before and post hemodialysis were recorded.The serum concentrations of hemoglobin,albumin,electrolyte,bicarbonate and lactic acid were collected for analysis.Results One hundred and nine(90.8％)effective questionnaires were collected,in which more than half of patients claimed to experience post-dialysis fatigue.Time to recover from hemodialysis(TIRD)was different:the median(interquartile range)time was 2.00(0.00,3.00)hours.In the study,30.3％patients reported no fatigue after hemodialysis.Recovery time in 35.8％patients was more than 30 minutes to 2 hours,22.0％was 3 to 4 hours,11.0％was 5 to 12 hours,0.9％patients took longer time to recover from a dialysis session.According to the recovery time,these patients were divided into three groups.Among the three groups,the ultrafiltration,the serum sodium and lactic acid after dialysis showed significant difference.It was showed by the unconditional logistic regression analysis that ultrafiltration(OR=2.35,95％CI 1.44-3.83),serum sodium(OR=0.75,95％CI 0.65-0.88),lactic acid(OR=3.16,95％CI 1.32-7.55)were associated of TIRD.Conclusions The incidence of post-dialysis fatigue is high.Most of the patients require more rest or sleep immediately after dialysis.The level of lactic acid is a significant influencing factor of the fatigue of patients.TIRD is correlated with the elevation of lactic acid during the dialysis process,and more attention should be paid to postdialysis fatigue.
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Objective@#To explore the impact of weight management and related medication intervention based on body weight changes on cardiac function among patients with chronic congestive heart failure (CHF).@*Methods@#Using prospective, randomized, controlled study methods, consecutive CHF patients, who hospitalized in our department from June 2014 to June 2016 (n=350), were randomly divided into intervention group (n=175) and control group (n=175). Patients in the intervention group received weight management guidance and the post discharge diuretic drugs regimen was adjusted based on body weight changes. The control group received routine medical care post discharge. Left ventricular ejection fraction (LVEF), B type natriuretic peptide precursor (NT-proBNP), 6 minutes walk distance and NYHA classification at one day before discharge and after 6 months were compared between the two groups respectively.@*Results@#Follow-up visit data were not available from 6 patients in the control and intervention group respectively. NYHA classification, LVEF, NT-proBNP and 6 minutes walk distance were similar between the two groups at one day before discharge (all P>0.05). After 6 months, the LVEF and 6 minutes walk distance were significantly higher while NT-proBNP level was significantly lower in the intervention group compared to the control group (all P<0.01). Meanwhile, the LVEF and 6 minutes walk distance were significantly increased, while NT-proBNP was significantly reduced at 6 months post discharge compared to one day before discharge in the intervention group (all P<0.01). The LVEF was also significantly improved (P=0.035), but the NT-proBNP and 6 minutes walk distance were similar (P were 0.328 and 0.807 respectively) at 6 months after discharge compared to one day before discharge in the control group. The NYHA classification was significantly lower in intervention group and in control group at 6 months after discharge compared to one day before discharge (Z=5.154, P<0.01 and Z=10.497, P<0.01), and the NYHA classification improved more in the intervention group than in control group at 6 months after discharge (Z=9.235, P<0.01). The re-hospitalization rate of CHF patients in intervention group was 11.83% (20/169), which was significantly lower than the control group (33.14% (56/169), χ2=21.99, P<0.01). At 6 months follow up, body weight remained unchanged in the intervention group, while body weight tended to be higher in the control group compared to one day before discharge.@*Conclusion@#The weight management and diuretic drug regimen adjudgment intervention based on body weight changes can improve cardiac function and reduced re-hospitalization rate in CHF patients.
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Objective To investigate the compound donkey-hide gelatin syrup in reducing adverse reactions of qi-blood weakness patients caused by clozapine.Methods 132 patients from Psychiatric Hospital of Yunnan Province between January 2010 and June 2010 were randomly divided into a treatment group and a control group.Both groups were taken clozapine orally.On this basis,the treatment group was taken compound donkey-hide gelatin syrup and the control group was taken placebo syrup.After 8 weeks treatment for both groups,the PANSS,TESS,physical examination and experiment examination were observed to evaluate the clinical efficacy and safety.Results ① the total curative effect:the treatment group was 73.53％,the control group was 65.63％,showing statistical difference (x2=2.543,P＜0.05).② PANSS scores changes before and after the treatment:PANSS score at 2,4,6,8 weeks after the treatment of both groups were [(72.51 ±27.55),(60.54±24.03),(53.12± 15.27),(48.15± 11.88) in treatment group respectively,and (70.71 ±23.90),(58.89± 18.95),(53.06± 14.38),(48.98 ± 9.78) in the control group,respectively],both showing significant difference than the same group before the treatment [(103.99±39.12) in the treatment group,(99.78±34.35) in the control group] (P＜0.05).But there was no statistical significance between two groups (F=2.413,P＞0.05).③ adverse reactions:during the treatment liver function,blood cell analysis,dystonia,Parkinson's obstacle,akathisia,abnormal gastrointestinal reaction,heart rate,heart rate variability and blood pressure in the treatment group was significantly lower than the control group (x2=4.562,P＜0.05).Conclusion Compound donkey-hide gelatin syrup can effectively relieve adverse reactions in qi-blood weak psychosis patients after clozapine treatment and improve their drug tolerance.
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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.
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Objective To evaluate the effect of pediatric renal transplantation using donation after cardiac death (DCD).Methods The male DCD meeting Chinese standard Ⅲ (C-Ⅲ) was 49 years old,and the recipient with chronic renal failure was 14 years old.The right kidney of the donor was transplanted to the recipient.The renal artery and renal vein of the donor were end-to-side anastomoscd to the common iliac artery and common iliac vein of the recipient,respectively.The graft was transplanted into the fight iliac fosse.Warm ischemia time was 12 min,and cold ischemia time was 2 h.Immunity induction therapy was performed with basiliximab.Tacrolimus + mycophnolate mofetil + Pred were used as immunosuppressive regimen.Results The transplantation was done successfully.One day after operation,ALT was increased dramatically.The recipient was diagnosed as acute drug-induced liver injury.There was no occurrence of acute rejection and delayed graft function.The recipient was discharged one month after the operation,and followed up for 6 months with normal graft function.Conclusion Pediatric renal transplantation using DCD is effective and safe,even though the long-term effect still needs to be further observed.
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Objective To observe the steps of vascular smooth muscle cells (VSMCs) calcification induced by high phosphate enviroment in vitro. Methods VSMCs were incubated with high phosphate (2.5 mmol/L or 3.5 mmool/L) medium for different times. Expression of core binding factor α1(Cbfα1), osteopontin(OP), collagen type Ⅰ(Col Ⅰ), osteocalcin(OC) and α-smooth muscle actin (α-SMA) was investigated by Western blot, immunofluorcscencc staining and real time PCR. Mineral deposition was assessed by von Kossa aad Alizarin red staining. Ultrastructure of VSMCs calcification was observed by electron microscopy (EM). Results Up-regulated expression of osteoblast-specific transcription factor Cbfα1 in the nuclei oceured at as early as 12 hours. The protein of Col Ⅰ and OP was up-regalated when VSMCs were incubated in high phosphate medium for 3 days, and content of OC increased at the time of 6 days. When cultured in 2.5 mmol/L phosphate medium for 15 days, VSMCs lost their lineage marker α-SMA, developed granular calcium deposits. Moreover, the results of real time PCR indicated mRNA level of OP and Col Ⅰ increased at day 1, OC increased at day 5 and α-SMA level decreased at day 10, respectively. Ultrastructural analysis also confirmed the presence of collagen and matrix vesicles in the cells. Conclusion VSMCs phenotype transformation induced by high phosphate enviroment is an orchestrated, highly regulated process.
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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.
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Objective To construct and purify the recombinant protein of platelet-activating factor acetylhydrolase (PAF-AH) isoform I , and study the enzyme activity by different substrates. Methods The (3 subunit of PAF-AH isoform I was cloned and expressed in E. coli. Exogenously expressed recombinant protein was purified to SDS-PAGE homogeneity, and its activity was identified by arylesterase detection. Phenylacetate, 1-O-hexadecyl-2-deoxy-2-thioacetyl-sn-glycero-3-phosphocholine ( 2-Thio PAF) and l-myristoy1-2-( 4-nitrophenylsuccinyl) phosphatidylcholine (the latter two were commercial plasma PAF-AH substrates) were used for the substrate identification. The plasma type PAF-AH was served as positive control. Results Recombinant protein of β subunit of PAF-AH isoform I was successfully constructed and expressed in E. coli after purification. Compared with positive control, the recombinant protein could hydrolyze phenylacetate and 2-Thio PAF, but could not hydrolyze l-myristoyl-2-( 4-nitrophenylsuccinyl) phosphatidylcholine. Conclusion Recombinant protein of β subunit of PAF-AH isoform I can be successfully constructed. There are differences in the substrate specification to the two commercial PAF substrates for PAF-AH isoform I and plasma type PAF-AH, which provides a quick method to differentiate PAF-AH isoform I from plasma type PAF-AH.
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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.
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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.