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Objective To investigate the effects of WNK3 kinase on the regulation of large-conductance calcium-activated potassium channels (Maxi K channels) on African green monkey kidney fibroblast-like cells (Cos-7 cells) and its mechanisms.Methods (1) Cos-7 cells were transfected with 0,0.6,1.2,1.8 μg WNK3 plasmid+0.5 μg Maxi K plasmid.The total protein expression of Maxi K channel and the phosphorylation of mitogen-activated protein kinase (MAPK) extracellular regulated kinase-1 and-2 (ERK1/2) were detected by Western blotting.(2) Cos-7 cells were divided into the control group (2.5 μg Maxi K plasmid) and the experimental group (2.5 μg WNK3 plasmid+2.5 μg Maxi K plasmid).Cell surface biotinylation was used to investigate the cell surface protein expression of Maxi K channel in Cos-7 cells.Immunoprecipitation and Western blotting were used to detect the ubiquitination of Maxi K channel protein.(3) WNK3 kinase was knocked down by WNK3 siRNA.The lysosomal degradation pathway was blocked by the proton pump inhibitor (Baf-A1).Cos-7 cells were divided into Maxi K+negative control siRNA group,Maxi K+WNK3 siRNA group and Maxi K+WNK3 siRNA+Baf-A1 group.The protein expression of Maxi K channel protein was detected by Western blotting.Results (1) Compared with those in 0 μg WNK3 plasmid groups,in 0.6,1.2,1.8 μg WNK3 plasmid groups the total protein expression of the Maxi K channel increased and the phosphorylation level of MAPK ERK1/2 reduced on a dose-dependent manner (all P ＜ 0.01).(2)Compared with those in the control group,the total protein expression and cell surface membrane protein expression of the Maxi K channel increased in the experimental group (P ＜ 0.01),while the ubiquitination of the Maxi K channel protein reduced (P ＜ 0.01).(3) Compared with the Maxi K +negative control siRNA group,the expression of Maxi K protein reduced in the Maxi K+WNK3 siRNA group (P ＜ 0.01),but did not change in the Maxi K+WNK3 siRNA + Bar-A1 group (P ＞ 0.05).The expression of Maxi K protein in Maxi K+WNK3 siRNA+Baf-A1 group was higher than that in Maxi K+WNK3 siRNA group (P ＜ 0.01).Conclusions WNK3 kinase inhibits the lysosomal degradation pathway of Maxi K channel protein by reducing the ubiquitination of Maxi K channel,and promotes the expression of Maxi K channel protein in cells and on cell membrane.These effects may be achieved by suppressing MAPK ERK1/2 signal transduction pathway.
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Objective To investigate the effects of protein expressions and the urea transport activity of aldosterone on urea transporter A1 (UT-A1) and urea transporter A3 (UT-A3) in HEK293 cells and Xenopus laevis oocytes.Methods (1) Western Blot was used to investigate the protein expressions of UT-A1 and UT-A3.(2) Cell surface biotinylation was used to investigate the protein expressions of UT-A1 and UT-A3 on the cell surface of Xenopus laevis oocytes.(3) 14C-urea transport experiment was conducted to investigate the transport activity of UT-A1 and UT-A3 in Xenopus laevis oocytes.Results (1) Compared with UT-A1 or UT-A3 high expression groups,the total protein levels of UT-A 1 and UT-A3 were all significantly reduced in aldosterone treatment groups (all P ＜ 0.01).(2) Compared with UT-A1 or UT-A3 high expression groups,the levels of protein expression on cell surface were all significantly reduced in aldosterone groups (all P ＜ 0.01).(3) Compared with UT-A1 or UT-A3 high expression groups,14C-urea transport experiment results showed that aldosterone treatment groups had significantly reduced the urea transporter activity of UT-A1 (1 min:94.32±9.044vs 40.68±4.274,P＜0.01,n=6;3 min:165.0±4.7 vs 80.3±0.6,P＜0.01,n=6),and UT-A3 (1 min:204.6± 3.1 vs 176.7± 9.1,P＜0.05,n=6;3 min:371.4 ± 14.9 vs 318.8 ± 12.0,P＜0.05,n=6).Conclusion Aldosterone can directly down-regulate the protein expressions of UT-A1 and UT-A3 in both total protein and cell surface level,which reduces their urea transport activity.
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Objective To assess the associations of anti-Smith antibodies with clinical manifestations and disease activity in children with systemic lupus erythematosus (SLE).Methods According to SLE disease activity index (SLEDAI) score,72 children with SLE were divided into the active group and inactive group.An immunoblotting method was used to detect serum anti-Smith antibodies in these subjects.Chi-square test was conducted to assess the associations of anti-Smith antibodies with clinical manifestations and disease activity in these patients.Results Of these patients,28 (38.9％) were assigned into the inactive group,and 44 (61.1％) to the active group.Anti-Smith antibodies were detected in 17 (23.6％) patients,but not in the other 55 (76.4％) patients.Elevated incidence rate of kidney injury was observed in anti-Smith antibody-positive patients compared with anti-Smith antibody-negative patients (70.6％ (12/17) vs.41.8％ (23/55),P ＜ 0.05).Meanwhile,the positivity rate of anti-Smith antibodies was 31.8％ (14/44) in the active group,significantly higher than that in the inactive group (10.7％,3/28,P ＜ 0.05).Conclusions Anti-Smith antibodies are not only an important indicator for the diagnosis of SLE,but also a risk factor for disease exacerbation and kidney injury in children with SLE.
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Objective To analyze the clinical characteristics and prognosis of acute pyelonephritis (APN) in children.Methods A retrospective analysis was performed in 230 pediatric patients with APN admitted from January 2009 to December 2010.The clinical characteristics,etiology,drug-resistant and prognosis were reviewed,and logistic regression analysis was performed to identify the risk factors of renal scarring after APN.Results Among 230 patients with APN,93 were boys and 137 were girls with a malefemale ratio of 1∶ 1.47.Ninety-nine patients were younger than 1 year with a male-female ratio of 1.30∶1 ;75 patients were 1 to 5 year with a male-female ratioof 1 ∶ 2.75 ; 56 patients were older than 5 year with a male-female ratio of 1∶2.29.A total of 106 strains were detected,in which 91 strains were Gram-negative bacteria,13 strains were Gram-positive bacteria and 2 strains were fungus.The most frequent bacteria were Escherichia coli (65 strains,61.32％),Klebsiella pneumonia (13 strains,12.6％) and Enterococcus faecium (9 strains,8.49％).Forty-four strains of Escherichia coli produced ESBLs,and all of them were resistant to ampicillin,part of them resistant to cephalosporin,compound sulfamethoxazole and aztreonam,but all were sensitive to amikacin,amoxicillin/clavulanic acid,nitrofurantoin,and imipenem.Renal emission computed tomography (ECT) was performed again in 52 children who were followed up for 6-12 months,in which 31 cases (59.62％) developed renal scar,and 21 cases (40.38％) were recovered.Abnormalities in urinary system or vesicoureteric reflux were identified as the risk factors for renal scarring after APN (OR =6.89,P ＜ 0.05).Conclusions The incidence of APN in children drops with age,which is frequently in the males younger than 1 year,and in the females older than 1 year.Escherichia coli is the most frequent pathogen of APN in children,and most strains are multidrug resistant.Children with abnormalities in urinary system or vesicoureteric reflux are prone to develop renal scarring.
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Objective To investigate the mechanism underlying the WNK4 kinasemediated inhibitory effect on BK channel. Methods Cos-7 cells were cotransfected with BK in combination with either CD4 (control group) or wild type WNK4 (WNK4-WT).Immunostaining and confocal microscopy,chemiluminescence,Western blotting analysis were then employed to determine the BK localization in cells,BK surface expression and total protein level,respectively.To further investigate whether the reduction of BK protein expression is due to an increase in degradation through a lysosomal pathway,BK protein level was determined after treated with bafilomycin A1(Baf A1),a proton pump inhibitor affecting lysosomal degradation. Results Immunostaining and confocal microscopic study showed that BK was localized both in plasma membrane and cytosol in the control group.After cells transfected with WNK4-WT,BK expression was markedly reduced.Chemiluminescent assay found that BK surface expression level was 299.9±18.6 in the control group,whereas it was significantly reduced (148.4±13.7,P＜0.01) in the WNK4-WT group.Western blotting analysis showed that total BK protein level was markedly reduced in the presence of WNK4-WT compared to the control group.WNK4-WT was shown to significantly reduce the BK total protein level (42.3％±15.2％) compared to the control group (100％) (P＜0.01).When the cells was treated with Bafilomycin A1 (Baf A1,0.5 μmol/L),WNK4-mediated reduction in BK protein was reversed (82.2％±12.1％,P＜0.05). Conclusions WNK4 inhibits total and surface protein expression of BK in Cos-7 cells whick is likely due to an increase in BK degradation through a lysosomal pathway.