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Objective:To investigate the effect of interleukin (IL)-7 receptor α (IL-7Rα) antibody on the immune inflammation and renal injury in MRL/lpr lupus mice.Methods:Fifteen 3-4-week-old female MRL/lpr lupus mice (specific pathogen free) weighing 15-16 g were bred to 14-week-old and randomly divided into three groups: IL-7Rα antibody intervention group, isotype antibody (positive control) group and normal saline (negative control) group. The mice in the threc groups were intraperitoneally injected with IL-7Rα antibody, isotype antibody and normal saline respectively, with 100 μg three times a week for 4 weeks. At the age of 18-week old, the mice were sacrificed. Twenty-four-hour urinary protein was detected by Coomassie brilliant blue method, serum creatinine was detected by peroxidase method, and the expression of autoantibody (anti-double strand DNA antibody) and inflammatory factors such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-21 was detected by enzyme-linked immunosorbent assay method. Renal pathology was detected by PAS and Sirius red staining, and CD3 and F4/80 in renal tissues were detected by immunohistochemistry method. Regulatory T cells, follicullar helper T cells (Tfh) and follicular regulatory T cells (Tfr) were detected by flow cytometry.Results:The 24-hour urinary protein, serum creatinine, serum anti-double strand DNA antibody and serum IFN-γ and IL-21 in the IL-7Rα antibody intervention group were significantly lower than those in the control groups (all P<0.01). However, there was no significant difference in serum TNF-α among the three groups ( F=0.39, P>0.05). The positive infiltrating cells of CD3 and F4/F80, and the ratio of type Ⅰ/Ⅲ collagen fibers ( F=41.11, P<0.01) of renal tissues in the IL-7Rα antibody intervention group were lower than those in the other two groups. Compared with the control groups, the ratio of regulatory T cells (CD4 +CD25 +Foxp3 +)/effector T cells (CD4 +CD25 +) in blood of IL-7Rα antibody intervention group increased ( F=21.64, P<0.01), while the ratio of Tfr (CD4 +CXCR5 +Foxp3 +)/Tfh (CD4 +CXCR5 +) in peripheral blood and spleen increased ( F=38.95, P<0.01; F=12.90, P<0.01). Conclusion:IL-7Rα antibody can reduce the production of autoantibodies such as anti-double strand DNA antibody and inflammatory factors by increasing the ratio of regulatory T cells and Tfr/Tfh, thus alleviating immune inflammation and renal damage in MRL/lpr lupus mice.
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Objective:To observe the expression of sirtuin 3 (Sirt3) and mitochondrial damage-associated proteins in lipopolysaccharide (LPS)-induced acute kidney injury mouse model and renal tubular epithelial cells, and to explore the role of Sirt3 in LPS-induced abnormal mitochondrial dynamics in renal tubular epithelial cells.Methods:Eighteen specific pathogen free (SPF) male C57BL/6 mice were randomly assigned to control group, LPS 24 h group and LPS 48 h group. The control group was intraperitoneally injected with physiological saline (0.1 ml/10 g), and LPS 24 h group and LPS 48 h group were intraperitoneally injected with LPS (10 mg/kg) solution. Renal functional indexes of mice were analyzed by automatic biochemical analyzer. The pathological change of the kidney was observed by HE staining, and the expressions of dynamin-related protein-1 (Drp1), optic atrophy type 1 (Opa1) and Sirt3 were evaluated by Western blotting. Expression and distribution of Sirt3 in kidney was assessed by immunohistochemistry. Human renal tubular epithelial cells (HK-2) were exposed to 10 μg/ml LPS for 24 h, and the expression of Drp1, Opa1 and Sirt3 were detected by Western blotting. Cell apoptosis was assessed by Hoechst-33342 staining. After transfection to HK-2 cells with pcDNA3.1-Sirt3 recombinant plasmid, the expressions of Sirt3, Drp1, Opa1 and cell apoptosis were detected by the same methods as above.Results:(1) The levels of blood urea nitrogen and serum creatinine in LPS group were significantly higher than those in control group (both P<0.05), and the pathological changes of kidney were obvious. (2) Compared with the control group, the expression of mitochondrial fission-associated protein Drp1 in renal tissue of LPS group was significantly higher ( P<0.05), and the expression of mitochondrial fusion associated protein Opa1 was significantly lower ( P<0.05). (3) Compared with the control group, the expression of Sirt3 in LPS group was significantly lower ( P<0.05), and immunohistochemistry results showed that Sirt3 was mainly expressed in glomerular vascular endothelial cells and renal tubular epithelial cells. (4) In vitro, LPS stimulation induced increased Drp1 expression in HK-2 cells ( P<0.05), decreased Opa1 and Sirt3 expression (both P<0.05), and increased apoptosis ( P<0.05). (5) LPS-induced mitochondrial dynamics disturbance and apoptosis were alleviated by pcDNA3.1-Sirt3 recombinant plasmid transfection. Conclusions:LPS can induce down-regulation of Sirt3 expression and disturbance of mitochondrial dynamics, and Sirt3 may play a protective role in LPS-induced acute kidney injury by regulating mitochondrial dynamics.
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Objective:To investigate the role of mitofusion 2 (Mfn2) in high glucose (HG)-induced endoplasmic reticulum stress (ERS) and apoptosis of podocytes.Methods:(1) Streptozocin was used to induce a diabetes mellitus (DM) rat model. Renal histopathological changes in rats were observed by HE staining. Expression of Mfn2 and CCAAT/enhancer-binding protein homologous protein (CHOP) in glomeruli was observed by immunohistochemistry. Protein levels of Mfn2, protein kinase RNA-like ER kinase (PERK), phospho(p)-PERK, and CHOP in glomeruli were analyzed by Western blotting. (2) Conditionally immortalized human podocytes (HPC) cultured in vitro were divided into control, mannitol (MA) and HG groups. Expression of Mfn2 was observed by immunofluorescence. Protein levels of Mfn2, p-PERK, PERK and CHOP in HPC were analyzed by Western blotting. Podocyte apoptosis in each group was evaluated by flow cytometry with AnnexinⅤ-PE/7AAD double staining method. (3) HPC were divided into control, HG, HG+Mfn2-Myc plasmid-transfected and HG+control plasmid-transfected groups. Protein levels of Mfn2, p-PERK, PERK and CHOP in HPC were analyzed by Western blotting. Expression of CHOP was observed by immunofluorescence. Mitochondrial membrane potential in each group was observed by mitochondrial membrane potential assay kit with JC-1. Podocyte apoptosis in each group was evaluated by flow cytometry with AnnexinⅤ-PE/7AAD double staining method. Results:(1) Compared with the control group, the glomerular mesangial matrix of the DM group rats was significantly proliferated, and the expression of Mfn2 was down-regulated with the expression of ERS-related proteins p-PERK/PERK and CHOP up-regulated (all P<0.05). (2) Compared with the control group, Mfn2 was down-regulated and p-PERK/PERK and CHOP were up-regulated in HPC of HG group (all P<0.05). Apoptosis of HPC was also increased in HG group. There was no significant difference in the above indicators between the control group and the mannitol group (all P>0.05). (3) Compared with the HG group, mitochondrial membrane potential of HPC was alleviated and apoptosis of HPC was decreased in HG+Mfn2-Myc plasmid-transfected group ( P<0.05). P-PERK/PERK and CHOP were down-regulated in HG+Mfn2-Myc plasmid-transfected group (both P<0.05). There was no significant difference in the above indicators between the HG group and the HG+control plasmid-transfected group (all P>0.05). Conclusions:Mfn2 down-regulation in HG-stimulated podocytes may induce ERS to increase apoptosis of podocytes. Up-regulation of Mfn2 can alleviate the HG-induced ERS and apoptosis in podocytes.
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Objective To investigate the role of autophagy in high glucose-induced podocyte lipid droplet metabolism.Methods (1) Cultured,conditionally immortalized human podocytes (HPC)were divided into normal control group,high glucose group and mannitol group.Oil red O staining and oil red O staining extraction assay was used to observe the degree of lipid accumulation;Protein level of SREBP-1 was analyzed by Western blotting.(2) HPC were cultured and divided into normal control group,high glucose group,high glucose+3-methyladenine (3-MA) group,and mannitol group.Acridine orange staining was used to observe the formation of autophagosomes.Western blotting was used to detect the protein levels of beclin-1 and LC3-Ⅱ/LC3-Ⅰ.Oil red O staining and oil red O staining extraction assay was used to observe the degree of lipid accumulation;Western blotting was used to analyze the expression of SREBP-1.Results (1) Compared with the normal control group,the lipid accumulation in the high glucose group was increased and the lipid metabolism related molecule SREBP-1 was up-regulated (P ＜ 0.05);There was no significant difference between the normal control group and the mannitol group in lipid accumulation (P ＞ 0.05).(2) Compared with the normal group,the number of autophagosomes was increased and autophagy-related proteins beclin-1 and LC3-Ⅱ/LC3-Ⅰ were up-regulated in high glucose group (all P ＜ 0.05).After intervened with 3-methyladenine,a significant decrease in autophagosomes was observed;Protein levels of autophagy-related proteinsbeclin-1 and LC3-Ⅱ/LC3-Ⅰ were decreased (all P ＜ 0.05);The lipid droplets in the high glucose+3-MA group was decreased and lipid metabolism related molecule SREBP-1 was down-regulated (all P ＜0.05).Conclusion Autophagy may be involved in the process of high-glucose-induced podocyte lipid accumulation by affecting SREBP-1 expression,and inhibition of autophagy can alleviate the high-glucose-induced podocyte lipid accumulation.
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Objective To investigate the roles of A kinase anchoring protein1(AKAP1)in high-glucose induced mitochondrial fission in podocytes.Methods Conditionally immortalized human podocytes were cultured in serum-free medium for 24 hours,and then exposed to different glucose concentration conditions in different time periods.The protein expressions of AKAP1 were observed by immunofluorescence,and AKAP1,dynamin related protein1 (Drp1) and phospho Ser 637-Drp1 (p-Drp1)were analyzed by Western blotting.AKAP1 siRNA was transfected to block AKAP1 expression.Podocytes were then divided into normal control group (5 mmol/L glucose),hypertonic group (30 mmol/L mannitol+5 mmol/L glucose),high glucose group (35 mmol/L glucose),and high glucose+AKAP1 siRNA group.Mitochondrial morphological changes were assessed by mitotracker red staining.Podocyte apoptosis was assessed by flow cytometry.Results Compared with normal group,high-glucose induced more podocytes apoptosis (P ＜ 0.05),more mitochondrial fission with decreased aspect ratio and form factor (all P ＜ 0.05).Upregulated AKAP1 protein level,and increased ratio of p-Drp1/Drp1 (all P ＜ 0.05) in time and concentration dependent manners were also obscrvcd.Compared with high glucose group,transfection of AKAP1 siRNA showed less apoptosis (P ＜ 0.05),less mitochondrial fission with increased aspect ratio and form factor (all P ＜ 0.05),and down-regulated AKAP1 protein level as well as p-Drp1/Drp1 ratio (all P ＜ 0.05).Conclusion High glucose induced mitochondrial fission might be induced through AKAP1-Drp1 pathway.
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Objective To investigate the effects of angiotensin Ⅱ (Ang Ⅱ) on the expression of ATP-binding cassette transporter A1(ABCA1) in Ang Ⅱ-infused rat model and cultured human podocytes,and to explore the role of ABCA1 in Ang Ⅱ-induced cholesterol accumulation of podocytes.Methods Twelve Wistar rats were randomly subjected to normal saline infusion,or Ang Ⅱ infusion at 400 ng· kg-1 · min-1 (via subcutaneous osmotic minipumps) for 8 weeks.The expression of glomerular ABCA1 was analyzed by Western blotting and real-time fluorescent quantitative PCR.In vitro,conditionally immortalized human podocytes were divided into normal group,Ang Ⅱ group,Ang Ⅱ + scrambled siRNA group,Ang Ⅱ + ABCA1 siRNA group.The expression of podocyte ABCA1 was assessed by immunofluorescence,Western blotting and real-time fluorescent quantitative PCR.Oil Red 0 staining was used to observe the lipid droplets in podocytes and cholesterol efflux assay kit was used to measure the cholesterol efflux rate of podocytes.Fluorescein isothiocyanate (FITC)-conjugated phalloidin was used to observe the podocyte cytoskeleton.Results In vivo,compared with normal group,the protein and mRNA expression of glomerular ABCA1 in Ang Ⅱ-infused rats were decreased (P ＜ 0.05).In vitro,ABCA1 was distributed in the cytomembrane of podocytes,and compared with normal group,Ang Ⅱ treatment down-regulated the expression of ABCA1 (P ＜ 0.05).Increased lipid droplets,decreased cholesterol efflux and cytoskeletal rearrangement were observed in Ang Ⅱ-treated podocytes.When compared to Ang Ⅱ group,podocytes stimulated by Ang Ⅱ and then transfected with ABCA1 siRNA had lower expression level of ABCA1 mRNA and protein (all P ＜ 0.05).More lipid droplets and lower cholesterol efflux rate could be observed in Ang Ⅱ +ABCA1 siRNA group (P＜ 0.05).Conclusion The reduced expression of ABCA1 may be involved in Ang Ⅱ-induced cholesterol accumulation in podocytes.
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Objective To investigate the risk factors of heart failure in patients with non-dialysis chronic renal failure,to provide a reference for the prevention and treatment of heart failure.Methods A total of 289 patients in Renmin Hospital of Wuhan University from January 2015 to January 2017 were enrolled in this retrospective study.They were divided into two groups according to the occurrence of heart failure General information,relevant auxili-ary examination and laboratory tests were collected to analyze.Multivariate Logistic regression was used to analyze the risk factors of heart failure in patients with chronic renal failure.Results Univa-riate analysis showed that diabetes,blood pressure,anemia,myocardial damage,albumin,renal function,myocardial hypertrophy,pericardial effusion in the two groups were statistically significant(P ＜ 0.05).Logistic regression analysis showed that diabetes (OR =2.308,P =0.002),hypertension (OR =2.464,P =0.000),elevated creatine kinase (OR =3.065,P =0.000),hypoalbuminaemia (OR =0.845,P =0.000),hypocalcemia (OR =0.062,P =0.000),hyperphosphatemia (OR =2.868,P =0.000) were independently associated with heart failure in patients of chronic renal failure.Conclusion Diabetes mellitus,hypertension,myocardial in-jury,low albumin,imbalance of calcium-phosphorus metabolism are risk factors for heart failure.Paying attention to these factors is of great significance to the prevention and treatment of heart failure.
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Objective To investigate the characteristics and outcome of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) in patients with renal injury. Methods AAV patients with renal injury diagnosed in the Department of Nephrology, Renmin Hospital of Wuhan University, from January 2012 to January 2017 were included into this study. Patients were divided into MPO-ANCA positive and PR3-ANCA positive groups for further study. The clinical characteristics, pathological and laboratory indexes, treatment and prognosis were retrospectively analyzed. Results A total of 68 cases were enrolled, among which 52 cases (76.5%) were MPO-ANCA positive and 16 cases (23.5%) were PR3-ANCA positive, and 41 patients (60.3%) were over 65 years old. The incidences of interstitial lung disease, digestive and nervous system damage in PR3-ANCA positive group were significantly higher than those MPO - ANCA positive group (P<0.05). There were significant differences of hemoglobin, complement C3, complement C1q, IgE, 24 h urinary protein,erythrocyte sedimentation rate, procalcitonin, BVAS score and eGFR in two groups (P<0.05). 19 cases had done renal biopsy ,among them 14 cases were MPO-ANCA positive and 5 cases were PR3-ANCA positive. Incidence of crescentic necrotizing glomerulonephritis in PR3-ANCA positive group was significantly higher than that in MPO - ANCA positive group, and incidence of diffuse global glomerulosclerosis in MPO-ANCA positive group was significantly higher than that in PR3-ANCA positive group (all P<0.05). At the median follow-up time of 32 months, the relapse rate at 6 month of MPO-ANCA-positive and PR3-ANCA-positive patients were 46.2% and 75.0%, respectively (P<0.05). Multivariate logistic regression analysis showed that PR3-ANCA positive, age≥65 years old, baseline eGFR<30 ml·min-1·(1.73 m2)-1, and combined with pulmonary interstitial lesions were all independent risk factors for relapse. And the incidence of ESRD were 42.3%and 75.0%during the follow-up period and 10 patients (14.7%) died. COX regression analysis showed that patients older than 65 years old, BVAS score≥18 points, eGFR<30 ml·min-1·(1.73 m2)-1 and complicated with pulmonary interstitial disorders at the onset were independent risk factors causing ESRD or death. Conclusion The PR3-ANCA-positive patients had more severe renal injury than those with MPO-ANCA-positive patients, and the injury of extrarenal organs was more serious, recurrence rate was higher, and the prognosis was worse.
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Objective To investigate the effects of hyperglycemia on ubiquitination and endoplasmic reticulum stress in renal intrinsic cells (podocytes and proximal tubular epithelial cells)and its role in pathogenesis of diabetic nephropathy.Methods Diabetic mice were induced by streptozotocin injection.After 16 weeks of hyperglycemia,immunofluorescence was used to detect the expressions of ubiquitination and glucose-regulating protein 94 (GRP94) in renal cortex and medulla area of kidney sections.Primary mouse podocyte and proximal tubular epithelial cells were isolated by flow cytometry,and exposed to 30 mmol/L glucose for indicated time (1 d,3 d and 7 d).Their ubiquitination and GRP94 expressions were evaluated by Western blotting.Results Diabetic mice presented microalbuminuria and slightly widened mesangium was found in glomerular area.Ubiquitinated proteins,mainly localized in podocytes and tubular epithelial cells,exhibited an apparently higher expression in diabetic mice than control mice (all P ＜ 0.05).Hyperglycemia promoted the ubiquitination in a time-dependent manner.Compared with their normal cells,primary mouse podocyte and primary tubular epithilial cells treated with high glucose for 3 d and 7 d showed increased ubiquitinated protein (all P ＜ 0.05).GRP94 was interspersed in podocytes and proximal tubular epithelial cells.Expression of GRP94 was significantly increased in glomerular area of diabetic mice and podocyte with 3 and 7 day-high glucose as compared with those in their control groups (all P ＜ 0.05).GRP94 expression had no significant change in tubular area and tubular epithilial cells treated with high glucose.Conclusions Hyperglycemia may lead to accumulation of ubiquitinated proteins in intrinsic kidney cells.The imbalance of protein homeostasis in podocyte may contribute to podocyte injury during the onset of diabetic nephropathy.
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Objective To observe the influence of renal sympathetic denervation (RSD) on renal interstitial fibrosis and transforming growth factor beta 1(TGF-β1) and microRNA-21 (miR-21) in rats with unilateral ureteral obstruction(UUO).Methods 40 male Wistar rats were randomly divided into UUO group (A group,n=10),sham UUO group (B group,n=10),RSD+UUO group (C group,n=1O) and RSD + sham UUO group (D group,n=10).Rats in A group and C group underwent unilateral ureteral ligation,while those in B group and D group underwent sham operation.Rats in C group and D group were followed by RSD.Rats were sacrificed at 21 days after the operation to evaluate the fibrosis by Masson staining.Immunohistochemical staining and Western blotting were used to detect the expressions of collagen I (COL-Ⅰ),collagen Ⅲ (COL-Ⅲ) and TGF-β1 in four groups.The expression of miR-21 was detected by fluorescence in situ hybridization (FISH) and quantitative real-time PCR (RT-qPCR).Results A large amount of collagen deposition was observed in the renal interstitial area in A and C group compared to either B or D group (P ＜ 0.05),but the change in C group was decreased significantly than that in A group (P ＜ 0.05).Similarly,the expressions of COL-Ⅰ,COL-Ⅲ,TGF-β1and miR-21 were obviously higher in A and C group compared to either B or D group (P ＜ 0.05),but those change in C group were decreased significantly than those in A group (P ＜ 0.05).The above indexes were not significantly different between B group and D group (P ＞ 0.05).Conclusion RSD may relieve the renal interstitial fibrosis in UUO rats,and down-regulate the expression of TGF-β1 and miR-21.
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Objective To investigate the ABO blood type and sepsis complicated with acute kidney injury,to provide the basis for prevention and treatment.Methods A total of 130 patients with sepsis from renmin hospital of Wuhan University intensive care unit from january 2015 to december 2015 were enrolled in this study,divided into complicated AKI group (64 patients in the observation group) and non-complicated AKI group (66 patients in the control group),analyzed two groups of general data,laboratory indicators,multivariate logistic regression analysis was used to screen out the risk factors for AKI in patients with sepsis.Results A total of 64 patients with AKI were collected from the observation group and 66 patients with non-AKI in the control group,the age of the observation group was higher than that of the control group (65.7 ± 13.1 years old vs 58.5 ± 15.4 years old,P =0.005),male proportion was higher than control group (76.6％ vs 56.1％,P =0.014),drop calcium pigment original quantitative was higher than control group (28.1 ± 21.0pg/L vs 21.1 ± 13.61μg/L,P =0.026),positive blood culture rate was higher than in the control group (30.2％ vs 15.3％,P =0.006).There was no significant difference in ABO blood group distribution between the two groups (P =0.825).The levels of white blood cell count,C-reactive protein and partially activated thrombin were higher in the observation group than in the control group,the platelet count and albumin level were lower than those in the control group,the difference was not statistically significant.The risk factors associated with the incidence of sepsis with AKI were analyzed by multivariate analysis of the logistic regression model:age(P =0.021,OR =0.965),gender (P =0.003,OR =5.321),calcitonin-original(P =0.047,OR =0.975),positive blood culture (P =0.002,OR =1.009),comparison of type A blood and type O blood (P =0.037,OR =5.409) were associated with sepsis complicated with AKI.Conlusion Type A blood and sepsis with AKI associated with the existence of independent correlation,type A blood may increase the risk of sepsis with AKI.
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Objective To observe the effect of costimulatory molecule B7-1 on cytoskeleton rearrangement in mouse podocytes induced by angiotensin Ⅱ (Ang Ⅱ),and to study the underlying molecular mechanism of B7-1 in the pathological changes of podocytes.Methods All cultivation of conditionally immortalized mouse podocytes (MPC) in vitro were divided into the following groups:normal control group,CTLA-4 group,Ang Ⅱ group (10-6 mmol/L 12 h,24 h;10-8 mmol/L 12 h,24 h) and CTLA-4 with Ang Ⅱ group.Transfect B7-1 RNA interference fragment (siRNA) to the mature podocytes,and then restimulated by Ang Ⅱ (10-6 mmol/L 12 h),the change of podocyte cytoskeleton after Ang Ⅱ stimulation were observed.The expression of B7-1 in each group was assayed by flow cytometry and Western blotting.The nephrin and p-nephrin protein levels in the four groups were also analyzed by Western blotting.At the same time,the podocyte cytoskeleton distribution as indicated by F-actin was observed by fluorescence microscopy.Results Flow cytometry and Western blotting showed that B7-1 was not expressed in the normal control group.Ang Ⅱ showed a concentration and time dependent induction of B7-1 expression in mouse podocytes (P ＜ 0.05).Western blotting indicated that Ang Ⅱ induced B7-1 protein expression (P ＜ 0.05).Expression of nephrin and p-nephrin was significantly down-regulated by Ang Ⅱ (P ＜ 0.05).Compared with the normal control group,the expression of podocyte protein nephrin and p-nephrin in Ang Ⅱ stimulation group was significantly reduced (P ＜ 0.05).Using FITC phalloidin fluorescence staining showed that CTLA-4+Ang Ⅱ stimulation group cytoskeleton rearrangement was improved significantly and F-actin recombinant score (mCFS) decreased compared with Ang Ⅱ group (P ＜ 0.05),suggesting that Ang Ⅱ led to the disorder of the podocytes cytoskeleton and the destruction of the cytoskeleton of podocytes by Ang Ⅱ could be improved after B7-1 blocking.Compared with the Ang Ⅱ stimulation,transfection of B7-1 siRNA + Ang Ⅱ stimulation group improved F-actin cytoskeletal rearrangement,and mCFS also decreased significantly (P ＜ 0.05),suggesting that transfection of B7-1 siRNA might improve the damage of Ang Ⅱ on podocytes cytoskeleton.Conclusions B7-1 participates in the process of cytoskeleton reconstruction and plays an important role in the pathological changes of podocytes.
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Objective To observe the effect of JLP deficiency on the progression of renal interstitial fibrosis in mice model of unilateral ureteral obstruction (UUO),and to investigate the role and underlying mechanism of JLP in the development of renal fibrosis in obstructive nephropathy.Methods jlp Wild type (jlp+/+) and jlp deficient (jlp-/-) mice were divided into four groups:jlp+/+-and jlp-/--sham-operated groups(jlp-/--sham group and jlp+/+-sham group),jlp+/+-and jlp-/--unilateral ureteral obstruction (UUO)-operated groups (jlp/--UUO group and jlp+/+-UUO group).Mice were sacrificed at 7 days and 14 days after the operation respectively to evaluate the fibrosis by Masson staining.The expression of JLP in jlp +/+ renal tissue was assayed by immunohistochemistry staining,immunofluorescence and Western blotting.Immunohistochemical staining was used to detect the expression of α-smooth muscle actin (α-SMA),collagen Ⅰ (COL-Ⅰ),collagen Ⅲ (COL-Ⅲ) and transforming growth factor-β1 (TGF-β1) in sham and UUO groups.Besides,the α-SMA,COL-Ⅰ,COL-Ⅲ,TGF-β1,p-Smad2 and p-Smad3 protein levels were also analyzed by Western blotting in four groups.Results The expression of JLP was mainly demonstrated in the renal tubules of mice.A large amount of collagen deposition was observed in the renal interstitial area in jlp-/--UUO group compared to jlp+/+-UUO group.Similarly,the expression of α-SMA,COL-Ⅰ,COL-Ⅲ and TGF-β1 was significantly increased in the kidney cortices in jlp-/--UUO-operated groups.Meanwhile,Western blotting showed that the expression of α-SMA,COL-Ⅰ,COL-Ⅲ,and TGF-β1 protein was obviously higher in jlp-/--UUO group.Moreover,the expression of p-Smad2 and p-Smad3 protein was markedly higher in jlp-/--UUO group.Conclusion Scaffolding protein JLP is critical in preventing renal fibrosis through the inhibition of TGF-β1 expression and myo-fibroblast production.
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Objective To observe the effect of JLP on transdifferentiation of human renal proximal tubular epithelial cells (HK-2),and to investigate the role of p38 MAPK signaling pathway in this process.Methods The knock-down plasmids of JLP were constructed.HK-2 cells were randomly divided into four groups:negative control cells (Ctrl-shRNA group),knock-down jlp cells (jlpshRNA group),negative control cells with FGF-2 treatment (FGF-2 group) and knock-down jlp cells with FGF-2 treatment(jlp-shRNA +FGF-2 group).The expressions of JLP,E-cadherin,TGF-β1,α-SMA,p-p38 MAPK protein were detected by Western blotting.After the induction of FGF-2 for 24 hours,the expressions of α-SMA,COL-Ⅰ,FN were detected by immunocytochemistry.Results Compared with Ctrl-shRNA group,the expression of JLP protein was significantly down-regulated in FGF-2 group.Compared with FGF-2 group,the expressions of TGF-β1,α-SMA,p-p38 MAPK protein were significantly up-regulated,while E-cadherin protein was significantly down-regulated (P ＜ 0.05).Compared with FGF-2 group,the expressions of α-SMA,COL-Ⅰ,FN immunostaining increased markedly in jlp-shRNA+FGF-2 group.Conclusion Scaffolding protein JLP is critical in preventing EMT in the course of fibrosis through the inhibition of p-p38 activation in HK-2 cells.
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Objective To investigate the effects of angiotensin Ⅱ (Ang Ⅱ) on the expression of C-terminal Src kinase (Csk) in Ang Ⅱ-infused rat model and cultured podocytes,and to explore the role of Csk in Ang Ⅱ-induced cytoskeletal rearrangement of podocytes.Methods Twenty-four Wista rats were randomly subjected to normal saline infusion,or Ang Ⅱ infusion at 400 ng · kg1 · min-1 (via subcutaneous osmotic minipumps) for 2 or 4 weeks.Renal histomorphology was evaluated through electron microscopy.The expression of glomerular Csk was analyzed by immunofluorescence and Western blotting.In vitro,conditionally immortalized mouse podocytes were cultured and treated with Ang Ⅱ doses ranging from 10-9 mol/L to 10-5 mol/L and for different hours.The expression of podocytes Csk was assessed by Western blotting.After transfection to podocytes with Csk siRNA,FITC-conjugated phalloidin was used to stain F-actin,to investigate the role of Csk in Ang Ⅱ-induced or cytochalasin D-induced cytoskeletal rearrangement.Results (1) Examination of Ang Ⅱ infusion rats glomerular and podocyte ultrastructure by electron microscopy revealed foot process effacement and fusion; (2) In Ang Ⅱ infusion rats,the expression of glomerular Csk was increased (P ＜ 0.05); (3) In vitro,Ang Ⅱ-stimuli up-regulated the expression of Csk (P ＜ 0.05),and the effects of Ang Ⅱ were on dose-dependent and time-dependent manner; (4) Ang Ⅱ-induced disruption of F-actin was alleviated by Csk siRNA transfection in cultured podocytes; furthermore,cytochalasin D depolymerized the F-actin cytoskeleton,while Csk siRNA stabilized the actin filaments.Conclusion The enhanced expression of Csk may be involved in Ang II-induced podocytes cytoskeletal rearrangement and foot process fusion.
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Objective To observe functional changes of renal tubular epithelial cells stimulated by high mobility group protein box 1 (HMGB1) and associated mechanism.Methods Renal tubular epithelial cells (NRK52E) were divided into control group,HMGB1 group and HMGB1+ lipopolysaccharide from Rhodobactersphaeroides (LPS RS) group.Toll-like receptor 4 (TLR4) expression was detected by immunofluorescence and Western blotting.Apoptosis rate and cell cycle arrest were identified with flow cytometry.The activation of MAPK signaling pathway and NF-κB were detected by Western blotting.The IL-1,IL-6 and tissue inhibitor of metalloproteinases 2 (TIMP2) mRNA levels were measured by real-time PCR.The secretion levels of IL-1,IL-6 and TIMP2 were measured by protein chips assay.Results TLR4 was expressed by NRK52E cells.Compared with the control group,there were increased cell cycle G1 arrest,MAPK signaling pathway and NF-κB activation in HMGB1 group.Furthermore,IL-1,IL-6 and TIMP2 mRNA levels were increased and IL-1,IL-6 and TIMP2 were secreted by NRK52E when stimulated with HMGB1 (all P ＜0.05).However,effects mediated by HMGB1 stimulation could be inhibited by LPS RS (all P＜0.05).Conclusions Inflammatory activation of NRK52E cells can be mediated by the interaction of HMGB1 and TLR4.
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<p><b>BACKGROUND</b>Surfactant protein A (SP-A) contributes to the regulation of sepsis-induced acute lung injury. In a previous study, we demonstrated the expression and localization of SP-A in the kidneys. The present study evaluated the effect of SP-A on lipopolysaccharide (LPS)-induced tumor necrosis factor-a (TNF-α) expression and its underlying mechanisms in the human renal tubular epithelial (HK-2) cells.</p><p><b>METHODS</b>Indirect immunofluorescence assay was used to detect SP-A distribution and expression in HK-2 cells. HK-2 cells were treated with various concentrations of LPS (0, 0.1, 1, 2, 5, and 10 mg/L) for 8 hours and with 5 mg/L LPS for different times (0, 2, 4, 8, 16, and 24 hours) to determine the effects of LPS on SP-A and TNF-α expression. Then, HK-2 cells were transfected with SP-A siRNA to analyze nuclear factor κB (NF-κB) P65 and TNF-α expression of HK-2 cells after LPS-treatment.</p><p><b>RESULTS</b>Indirect immunofluorescence assay revealed that SP-A is localized to the membrane and cytoplasm of HK-2 cells. Interestingly, SP-A1/SP-A2 and TNF-a expression were found to be significantly increased in HK-2 cells upon LPS treatment. Transfection of LPS-treated HK-2 cells with SP-A siRNA resulted in significant increases in the levels of NF-κB P65 protein and TNF-α mRNA and protein compared to those in non-transfected LPS-treated HK-2 cells.</p><p><b>CONCLUSION</b>SP-A plays an important role in protecting cells against sepsis-induced acute kidney injury by inhibiting NF-κB activity to modulate LPS-induced increase in TNF-α expression.</p>
Subject(s)
Humans , Cell Line , Epithelial Cells , Cell Biology , Metabolism , Fluorescent Antibody Technique, Indirect , Kidney Tubules, Proximal , Cell Biology , Lipopolysaccharides , Pharmacology , Pulmonary Surfactant-Associated Protein A , Metabolism , Pharmacology , Tumor Necrosis Factor-alpha , MetabolismABSTRACT
Objective To evaluate the effects of autophagy on oxidative stress induced by contrast media in podocytes.Methods The differentiated mouse podocytes were exposed to contrast media (Iopromide,50 mg/L)、rapamycin (Rap,autophagy enhancer,1 ng/L),3-methyladenine (3-MA,autophagy inhibitor,2 mmol/L) for 2 hours.The expression of autophagy protein LC3-Ⅱ and Beclin-1 as well as oxidative stress-related proteins Catalase,MnSOD were detected by Western blot.The formations of autophagy were observed by MDC staining,and the levels of reactive oxygen species (ROS) by CM-H2DCFDA staining.Cell activity was evaluated by CCK8 assay.Results Both the levels of oxidative stress and autophagy in podocytes increased when stimulated by contrast media,the expression of LC3-Ⅱ and Beclin-1 were enhanced,Catalase and MnSOD were inhibited (all P ＜ 0.05).Rapamycin increased the expression of Catalase,MnSOD and cell activity of podocytes,reduced the generation of ROS (all P ＜ 0.05),but in Rap group,cell activity showed no significant difference (P ＞ 0.05).3-MA decreased the expression of Catalase 、MnSOD and inhibited the cell activity of podocyte,increased the generation of ROS (all P ＜ 0.05).Conclusion Autophagy protects podocyte from contrast media by the means of reducing oxidative stress.
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Objective To investigate association between serum uric acid (SUA),albuminuria and glomerular filtration rates (eGFR) in type 2 diabetic patients.Methods A total of 220 patients were enrolled in this cross-sectional study.According to urinary albumin excretion rates,patients were divided into 3 groups:normoalbuminuria (NAU) group,microalbuminuria (MAU) group,and macroalbumnuria group (MAAU).The first two groups were subdivided at SUA ＞ 420 μmol/L (＞ 357 μmol/L,female) into normouricemia group and hyperuricemia group,at eGFR ＞ 90 ml/min into high and low renal function groups.General information,blood biochemical results were collected to analyze the association between serum uric acid,eGFR,UAER and urine albumin quantification among different groups.Results The difference of SBP,duration of diabetes (DD),Scr,SUA and eGFR between every two groups were significant (P ＜ 0.05).SBP,DD,Scr and SUA were highest in subjects with macroalbumnuria,second in microalbuminuria group,and lowest in normoalbuminuria group,while eGFR was lowest in macroalbumnuria group and highest in normoalbuminuria group.Prevalence of hyperuricemia in macroalbumnuria group (56.9％) and microalbuminuria group (51.2％) were also significantly higher than that in normoalbuminuria group (17.5％) (all P ＜ 0.01).The difference of UAER in the subgroups of normouricemia and hyperuricemia was more significant in microalbuminuria group than in normoalbuminuria group.eGFR was significantly lower in hyperuricemia subgroups (P ＜0.01).Age and SUA were significantlg higher in subjects with low renal function compared with high eGFR (P ＜ 0.05).Linear regression analysis indicated SUA was negatively correlated with eGFR after adjusted age,DD and UAER (β =-0.430,P ＜ 0.01).Binary logistic regression analysis found that increased age,DD and SUA were risk factors of microalbuminuria β =1.092,95％ CI(1.025,1.163),P ＜ 0.01;β =1.005,95％CI(1.001,1.009),P ＜ 0.05;β =1.407,95％ CI(1.052,1.881),P ＜ 0.05)] and SUA,age were risk factors of early renal function decline [β =1.015,95 ％ CI(1.00,1.023),P ＜ 0.01;β =1.098,95％ CI(1.006,1.199),P ＜ 0.05].Conclusion SUA is independently associated with albumnuria and renal function decline in type 2 DM patients.
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Objective To investigate the role of IQ domain GTPase-activating protein 1 (IQGAP1) in angiotensin Ⅱ (Ang Ⅱ)-induced podocyte apoptosis and the underlying mechanism.Methods Differentiated mouse podocytes were exposed to Ang Ⅱ at different concentrations for 6 h or at 10-8 mol/L for variable incubation time.Podocyte apoptosis was assessed by flow cytometry.Expression of IQGAP1 was analyzed by immunofluorescence and Western blotting.IQGAP1 siRNA and MAPK pathway inhibitors(10 μmol/L SB202190,25 μmol/L SP600125,10 μmol/L U0126) were further introduced to investigate the role of IQGAP1 and MAPK signalings in the process.And coimmunoprecipitation was used to evaluate the interaction between ERK1/2 and IQGAP1.Results (1)Ang]] promoted podocyte apoptosis in a dose-and time-dependent manner.(2) IQGAP1 was located in celluar membrane and cytoplasm of cultured podocytes.Exposure to Ang Ⅱ stimulated IQGAP1expression in a dose-and time-dependent manner,and elevated phosphorylation of p38,JNK,and ERK1/2 simultaneously.(3) Pretreatment with SB202190,SP600125,or U0126 dramatically prevented Ang Ⅱ-promoted podocyte apoptosis respectively (P ＜ 0.05).However,the protein level of IQGAP1 was not altered.(4) Knockdown of IQGAP1 with siRNA obviously prevented Ang]Ⅱ-induced apoptosis of podocytes(P ＜ 0.05) and reduced Ang Ⅱ-induced phosphorylation of ERK1/2(P ＜ 0.05),but not that of p38,JNK.This was accompanied by a reduced interaction between ERK1/2 and IQGAP1(P ＜ 0.05).Conclusion IQGAP1 contributes to Ang Ⅱ-induced podocyte apoptosis by interacting with the ERK1/2 signaling protein.