BACKGROUND: To explore the role of protein phosphatase 2A (PP2A) in renal interstitial fibrosis by using rat model of unilateral ureteral obstructive (UUO) or cell model of human kidney proximal tubular epithelial (HK)-2 cells treated with transforming growth factor-β1 (TGF-β1).
 METHODS: 1) A total of 15 Sprague-Dawley rats were randomly divided into a sham group, a UUO group and an okadaic acid (OA) treated group (OA group) (n=5 in each group). The OA 
[30 μg/(kg·d)], diluted with 1.8% alcohol, was given to the rats in the OA group through gastric tube after at 72 h after the surgery, while the equal volume of 1.8% alcohol was given to the rats in the sham group and the UUO group. After sacrificing rats, the blood and kidney were collected to detect the renal function and the expression of PP2Ac, fibronectin (FN), collagen-I (Col-I), E-cadherin (E-cad) and α-smooth muscle actin (α-SMA) by immunohistochemistry, Western blot and RT-PCR, respectively; 2) The likely concentration of OA was determined by Trypan blue dye exclusive assay and methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The HK-2 cells were incubated with serum-free Dulbecco's modified eagle medium (DMEM) for 24 h; then they were divided into a control group, a TGF-β1 group (treated with 5 ng/mL TGF-β1 for 24 h) and a TGF-β1+OA group (treated with 5 ng/mL TGF-β1 and 40 nmol/L OA for 24 h). The HK-2 cells were collected and the expression of PP2Ac, FN, Col-I, E-cad and α-SMA were detected by Western blot.
 RESULTS: 1) Compared with the sham group, the BUN and Scr in the UUO group increased (both P<0.05); compared with the UUO group, the BUN and Scr in the OA group decreased (both P<0.05); the expression of PP2Ac, FN, Col-I and α-SMA was up-regulated while the expression of E-cad was down-regulated in the UUO group compared with those in the sham group (all P<0.05). The expression of PP2Ac, FN, Col-I and α-SMA was down-regulated while the expressions of E-cad was up-regulated in the OA group compared with those in the UUO group (all P<0.05); 2) The likely concentration of OA was 40 nmol/L. Western blot showed that the expression of PP2Ac, FN, Col-I and α-SMA was up-regulated while the expressions of E-cad was down-regulated in the TGF-β1 group compared with those in the control group (all P<0.05); the expression of PP2Ac, FN, Col-I and α-SMA were down-regulated while the expression of E-cad was up-regulated in the TGF-β1+OA group compared with those in the TGF-β1 group (all P<0.05).
 CONCLUSIONS PP2A might be able to promote the renal interstitial fibrosis.
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Actins ; metabolism ; Animals ; Cadherins ; metabolism ; Cell Line ; Collagen Type I ; metabolism ; Drugs, Chinese Herbal ; Fibronectins ; metabolism ; Fibrosis ; Humans ; Kidney ; metabolism ; pathology ; Kidney Diseases ; enzymology ; Protein Phosphatase 2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; pharmacology

Objective To establish mouse models of intracerebral hemorrhage using autologous arterial blood,to study the physiological property of hippocampal neurons,brain edema changes and learning ability in the mouse models after intracerebral hemorrhage.Methods Eighty male C57/BL6 mice were randomly divided into intracerebral hemorrhage group and control group (n=40); 20 μL arterial blood from the tail arteries or normal saline were injected into the caudate nucleus of intracerebral hemorrhage group and control group by stereotactic technique,respectively.One,three,five and seven d after injection,the neurological impairment was scored; the behavioral changes of the mice in the Morris water maze (navigation test and space exploration experiment) were observed; brain edema was measured by wet and dry weight method and electrophysiological differences of hippocampal neurons were recorded by whole-cell patch-clamp technique and computer software.Results As compared with those in the control group,significantly increased neurological deficit scores one,three,five and seven d after injection,statistically decreased residence time in the platform on the fifth d of training,obviously increased water content around the brain edema one,three,five and seven d after injection,and significantly decreased resting membrane potential and input resistance in the hippocampal CA1 pyramidal cells five d after injection of mice in the intracerebral hemorrhage group were noted (P＜0.05).Conclusion The hippocampus-dependent spatial leaming ability of intracerebral hemorrhage mice is decreased,and the permeability of potassium channels is enhanced.

Objective:To evaluate the mortality and risk factors for acute kidney injury (AKI) in hospitalized patients by the risk, injury, failure, loss, end stage kidney disease (RIFLE) and acute kidney injury network (AKIN).
Methods:We constructed a retrospective study of all AKI patients in the Second Xiangya Hospital of Central South University between February 2006 and January 2011. The diagnosis and classiifcation of AKI were reconifrmed and categorized by RIFLE and AKIN criteria. To compare the clinical characteristics, mortality and associated risk factors in AKI patients by the RIFLE and AKIN stage, univariate analysis and multivariate logistic regression analysis were performed. Results:The patients were diagnosed as AKI by AKIN (n=1027) or by RIFLE criteria (n=1020). There was no signiifcant difference in the hospital mortality, hospital length stay (days), or the proportion of complete recovery in each stage of AKI patients by RIFLE and AKIN (P>0.05). In the univariate analysis, age, pre-renal causes, proportion of hospital acquired AKI, mechanical ventilation, hypotension, the number of failed organs, acute tubular necrosis-index severity score (ATN-ISS), and the peak of serum potassium ion concentration were signiifcantly higher in the non-survivors than in the survivors (P<0.05). Logistic regression analysis revealed that age older than 65, hospital acquired AKI, hypotension, number of failed organs, ATN-ISS scores, and the peak of serum potassium ion concentration were independent risk factors for hospital mortality. Conclusion:Both RIFLE and AKIN criteria have similar scientiifc value in assessing hospital mortality. AKI stage is associated with the recent prognosis of AKI patients.

Objective To determine the effect of smad anchor for receptor activation (SARA) on renal tubular epithelial to mesenchymal transtion (EMT) induced by high glucose and to investigate the associated mechanism.Methods HK-2 cells were exposed to high glucose (30 mmol/L).HK-2 cells were transfected with the plasmids of wild-type SARA [SARA (WT)] or SARA mutant [SARA with SBD deletion,called SARA (dSBD)] and then was stimulated by high glucose.The gene expression was assayed by real-time PCR and the protein expression was detected by Western blotting.Results During the process of high glucose-induced EMT of HK-2 cells,the gene and protein expression of SARA were down-regulated.The expression of TGF-β1 and Smad3 increased after stimulation of high glucose in HK-2.However,the Smad2 mRNA expression increased while its protein expression was down-regulated in a time-dependent manner.Smad2 and Smad3 were activated by high glucose stimulation and Smad3 kept activation for longer time than Smad2.Compared with high glucose group,over-expression of SARA by transfection of SARA (WT) up-regulated the expression of zona occludens(ZO)1 and down-regulated the expression of vimentin (P＜0.05).However,SARA (dSBD) had no such effects on above expressions.The Smad2 protein expression increased along with the over-expression of SARA.Meanwhile,over-expression of SARA prolonged the activation time of Smad2 and shortened the activation time of Smad3.Conclusions TGF-β1 signaling is activated and SARA expression is down-regulated during the process of high glucose-induced EMT in HK-2 cells.Over-expression of SARA can inhibit the EMT via increase of Smad2 protein expression and longer activation time of Smad2.

OBJECTIVE: To observe the effect of norcantharidin (NCTD) on the expression of mRNA and protein of fibronectin (FN), collagen IV(Col IV) and transforming growth factor-β1(TGF-β1) in human kidney proximal tubular epithelial (HK)-2 cells induced by high glucose. METHODS: HK-2 cells were incubated with serum-free DMEM for 24 h to synchronize cell growth, and then the cells were divided into 4 groups: Group C (5.5 mmol/L D-glucose), Group M (5.5 mmol/L D-glucose + 24.5 mmol/L-mannitol), Group HG (30 mmol/L D-glucose), and Group HG + NCTD (30 mmol/L D-glucose + 0.5-40 mg/L NCTD). Cytotoxicity of HK-2 cells induced by high glucose of NCTD was detected by Trypan blue dye exclusive assay. The effect of NCTD on the proliferation of HK-2 cells in high glucose was determined by MTT. The cells were collected to extract total RNA and protein at 6, 24 and 48 h after the incubation. The expression of FN, Col IV and TGF-β1 mRNA was examined by RT-PCR, and FN, Col IV and TGF-β1 protein was analyzed by Western blot. RESULTS: Trypan blue dye exclusive assay showed NCTD concentrations over 5 mg/L were rather toxic in HK-2 cells. The proliferation of HK-2 cells in high glucose was interrupted by interfered with 5 mg/L NCTD as measured by MTT (P<0.05). NCTD at 5 mg/L had a stronger inhibitory effect than NCTD at 2.5 mg/L. Real-time PCR and Western blot showed that the mRNA and protein expression of FN, collagen IV and TGF-β1 increased in HK-2 cells treated with high glucose (P<0.05), while that in cells treated by NCTD was dramatically inhibited (P<0.05). No change in these parameters was detected in the 30 mmol/L D-mannitol control group (P>0.05). CONCLUSION NCTD can downregulate FN, collagen IV and TGF-β1 mRNA and protein expression in HK-2 cells stimulated by 30 mmol/L D-glucose.
Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Line ; Collagen Type IV ; genetics ; metabolism ; Down-Regulation ; drug effects ; Epithelial Cells ; cytology ; Fibronectins ; genetics ; metabolism ; Glucose ; pharmacology ; Humans ; Kidney Tubules, Proximal ; cytology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism

Objective To compare the efficiency of original neuromyelitis optica(NMO)-IgG assay of detecting NMO-IgG with a new anti-aquaporin-4(AQP4)assay of detecting AQP4,and to explore the accuracy of the method in the diagnosis of NMO and multiple sclerosis(MS).Methods The sera were obtained from 44 patients with NMO and 46 patients with MS and were tested by both NMO-IgG and antiAOP4 assays.NMO-IgG was identified by original NMO-IgG assay with a substrate from mouse brain.AntiAQIP4 was detected by anti-AQP4 antibody assay.The results from the two assays were statistically analyzed to compare accuracy and specificity of the methods.Results The results of the two assays were concordant in 45 testing negative cases and 36 positive cases(Kappa=0.798.P=0.000).The McNemar test showed that the positive rate of the two assays were not significantly different(P=1.000).The NMO-IgG assay showed 77.3% sensitivity,87% specificity,82.2% diagnosis accuracy,85%positive predictive value,87% negative predictive value.and 74.3%Younden index. The anti-AOP4 antibody assay showed 88.6% sensitivity,95.7%specificity,92.2% diagnosis accuracy,98.1% positive predictive value,89.8% negative predictive value.and 84.3% Younden index.Conclusions This study demonstrated that NMO-IgG and AQP4 antibody detection have high sensitivity and specificity to detect NMO and MS.Anti-AQP4 detected by anti-AQP4 antibody assay may be more useful for NMO diagnosis.

Objective To investigate the prevalence of chronic kidney disease (CKD) and associated factors in Changsha county of Hunan province. Methods Using a stratified, multistage sampling, 1950 residents (older than 20 years old) from 3 towns of Changsha county were randomly selected to be interviewed and tested for the kidney damage indicators and the associated factors with CKD. Results Eligible data of 1727 subjects were enrolled in the study. After the adjustment of age and gender compenent, the prevalence of albuminuria was 8.5%, hematuria 5.1%, and reduced eGFR 1.5%. Approximately 14.6% subjects had at least one indicator of kidney damage, and the awareness rate was 16.5%. Age, hypercholesteremia, hypertriglyceridemia, hypertension and diabetes were independently correlated with albuminuria. Female, age, hypertriglyceridemia and hyperuricemia were independently correlated with reduced renal function. Female was independently correlated with hematuria. Conclusions The prevalence of chronic kidney disease is 14.6% and the awareness rate is 16.5% in suburban adult population of the central south area of China. The spectrum and correlated factors of CKD in this county undergoing fast economic development are close to those of Guangzhou and developed countries.

Objective To study the clinical characteristics and HBeAg status in HBV liver cirrhosis patients with different HBV DNA levels, Method Three hundred and thirty-seven patients with liver cirrhosis caused by chronic HBV infection were investigated. HBV DNA levels were detected by PCR, and HBV markers were detected by MEIA. The ratio of patients with HBeAg positive or negative in groups with different HBV DNA levels was compared, and the clinical characteristics in patients with different HBV DNA levels and HBeAg status were evaluated. Results The positive ratio of HBV DNA and HBeAg were 80.4% (271/337) and 31.5% (106/337). The negative ratio of HBeAg was 68.5% (231/337). The proportion of patients with Child-Pugh grade A, B or C and hepatocellular carcinoma (HCC) in different groups of HBV DNA levels and in different HBeAg status showed no significant difference, but the ratio of HCC in patients with HBV DNA 3-4 lg copies/ml was higher than that in patients with HBV DNA <3 lg copies/ml (P=0.014) and ≥7 lg eopies/ml (P =0.009). No significant difference of HBV DNA levels was found in different age groups, but the negative ratio of HBeAg increased with the increasing of the age. Conclusions More than 80% of patients with liver cirrhosis caused by chronic HBV infection axe HBV DNA positive, and 2/3 of them are HBeAg negative. Suppressing HBV replication may improve the prognosis of HBV related cirrhosis and HCC.

Objective To determine the effect of 2 transforming growth factor β1 (TGF-β1) short hairpin RNA (shRNA) expression plasmids (pcDU6-A1-A2 and pcDU6-B1-B2) on proliferation, TGF-β1, connective tissue growth factor (CTGF), and fibronectin (FN) expression induced by human serum albumin (HAS) in HK2 cells. Methods A vector plasmid containing the TGF-β1 shRNA was generated. An HK2 cell line was used in the study. The 2 TGF-β1 shRNA expression plasmids were transfected into cultured HK2 cells by lipofectamine 2000. Cellular proliferation was assessed by tetrazolium salt colorimetry. The semi-quantitative reverse transcriptive PCR was performed to detect the expression of TGF-β1,CTGF, and FN mRNA. Levels of TGF-β1 and FN protein were measured with a sandwich enzyme-linked immunosorbent assay. Results After treating with 5 g/L HAS for 24 hours in HK2 cells, cellular proliferating capacity increased significantly (P＜0.05). The expression levels of TGF-β1, CTGF, and FN mRNA were upregulated in HK2 cells stimulated by 5 g/L HAS, and levels of TGF-β1 and FN protein in the culture supernatant increased (P＜0.05). The introduction of pcDU6-A1-A2 and pcDU6-B1-B2 resulted in significant reduction of cellular proliferation activity, and the expression levels of TGF-β1, CTGF, and FN mRNA were downregulated (P＜0.05). Levels of TGF-β1 and FN protein in the culture supernatant decreased (P＜0.05) after 12 or 24 hours of TGF-β1 shRNA transfection into HK2 cells There was no significant difference in the expression levels of TGF-β1, CTGF, and FN mRNA between the 2 pcDU6 vector plasmid mediated TGF-β1 shRNA groups (P＞0.05). Conclusion pcDU6 vector plasmid mediated TGF-β1 shRNAs could obviously inhibit the expression levels of TGF-β1, CTGF, FN and cellular proliferation stimulated by HAS in HK2 cells, which may be related to the mediation of TGF-β1.

OBJECTIVE: To determine the incidence and risk factors associated with acute kidney injury (AKI) in patients after cardiac surgery with extracorporeal circulation. METHODS: A retrospective case control study was done in patients who underwent cardiac surgery from 2003 to 2007 in Second Xiangya Hospital, with 340 patients in an AKI group and the other 4 760 patients without AKI as a control group. All variables were analyzed by univariate analysis, Mann-Whitney U test and logistic regression. RESULTS: AKI occurred in the 340 patients (6.7% incidence). Univariate analysis revealed that age, preoperative serum creatinine, preoperative ejection fraction (EF), preoperative beta2-microglobulin, preoperative blood albumin, preoperative blood uric acid, intraoperative cardiopulmonary bypass time, intraoperative aortic cross-clamp time, and dosage of mannitol were significantly related to AKI following cardiac surgery with extracorporeal circulation. Logistic multivariate regression analysis showed that preoperative serum creatinine (P<0.001), preoperative ejection fraction (EF) (P<0.001), preoperative beta2-microglobulin (P=0.002), preoperative blood uric acid (P=0.015), intraoperative cardiopulmonary bypass time (P<0.001), and intraoperative aortic cross-clamp time (P<0.001) were independent risk factors for AKI. CONCLUSION The incidence of AKI after cardiac surgery with extracorporeal circulation is closely related with a variety of perioperative risk factors. Our data suggest that patients planning to accept cardiac surgery with extracorporeal circulation should be more comprehensively assessed and monitored, thereby preventing the occurrence of AKI.
Acute Kidney Injury ; epidemiology ; etiology ; Adolescent ; Adult ; Cardiac Surgical Procedures ; adverse effects ; Cardiopulmonary Bypass ; adverse effects ; Case-Control Studies ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Incidence ; Infant ; Logistic Models ; Male ; Middle Aged ; Retrospective Studies ; Risk Factors ; Young Adult