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Objective To investigate the expression of miR-29a in serum of acute kidney injury patients, and whether miR-29a can affect the apoptosis of renal tubular cells by regulating the expressions of phosphatase and tensin homologue deleted on chromosome ten (PTEN).Methods Blood samples were collected from 113 patients with acute kidney injury (AKI) and 110 healthy controls.The expressions of miR-29a in serum were detected by real-fluorescence quantitative polymerase chain reaction (RT-q-PCR).HK-2 cells were cultured in vitro, miR-29a NC or miR-29a mimic was transfected into HK-2 cells, and apoptosis of HK-2 cells was induced by transforming growth factor-β1 (TGF-β1).Flow cytometry was used to investigate the apoptosis of HK-2 cells.Bioinformatics was used to predict potential target genes of miR-29a.Luciferase reporter gene test was used to verify the complementary pairing relationship between miR-29a and PTEN.The expression of PTEN was detected by Western blot.After transfection of plasmids overexpressing PTEN, the apoptosis of HK-2 cells was detected by flow cytometry.Results Compared to the healthy control group, the serum expression of miR-29a was decreased in AKI patients (P ＜ 0.05).Flow cytometry results showed that transfection of miR-29a mimic was significantly decreased HK-2 cells apoptosis compared to miR-29 NC group (P ＜ 0.05).The Luciferase reporter assay results showed that miR-29a and PTEN had complementary relationship.After transfected with overexpression of PTEN, the HK-2 cells apoptosis rate was significantly increased (P ＜ 0.05).Conclusions In the serum of AKI patients, the expression of miR-29a was decreased, overexpression of miR-29a inhibited the apoptosis of renal tubular epithelial cells by inhibiting the expression of PTEN protein.
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ObjectiveTo investigate the effect of Simvastatin on LOX-1 and ROS in NRK52E induced by ox-LDL.MethodsNRK-52E cells were divided into three groups: Control group,ox-LDL group (50 μg/ml ox-LDL) and Simvastatin group (10 μmol/L Simvastatin +50 μg/ml ox-LDL).After incubation for 24 h,the expression of LOX-I was analyzed by Western blotting,and production of reactive oxygen species (ROS) was analyzed with confocal laser scanning microscopy.ResultsNRK-52E expressed LOX-1 at low level,50 μg/ml ox-LDL increased the expression of LOX-1 by 6.80 times.Pre - treatment with Simvastatin decreased LOX-1 expression by 65％.There was little ROS generation in NRK52E cells,50μg/ml ox-LDL promoted the expression of LOX-1 by 4.86.times.Pre - treatment with Simvastatin decreased ROS generation by 60％.ConclusionsSimvastatin upregulate LOX-1 expression and ROS generation induced by Ox-LDL in NRK52E cells.
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Objective To investigate the effect of methylguanidine and 1-methylhydantoin on cells cytotoxicity, apoptosis in human renal tubular epithelial cell line (HK-2). Methods Human PTEC cell line HK-2 was used in this study. HK-2 was cultured and divided into 3 groups: Norma1 control group (A), methylguanidine group(B) and 1-methylhydantoin group (C). The cell inhibitory rate of HK-2 was detected by MTT method. The cytotoxicity of methylguanidine to HK-2 was determined by NAG release test. Cell apoptosis was evaluated by using Hoechst stain and FACS with Annexin-V/PI. Results The OD value and NAG concentration of creatinine, methylguanidine and 1-methylhydantoin group were compared with normal control group. OD value decreased and NAG concentration significantly increased(0.188±0.011, 0.176±0.010 vs 0.545±0.021, F＝1557.74, P＜0.01; 20.488±0.473, 22.225±0.565 vs 5.125±0.198, F＝3848.22, P＜0.01). By Hoechst stain, pycnosis and apoptotic body could be found when HK-2 was cultivated in methylguanidine 1-methylhydantoin group. In methylguanidine, 1-methylhydantoin group apoptotic HK-2 apparently increased, compared with that in control group (18.23±1.1581, 20.22±1.1433 vs 2.473±0.321, F＝526.06, P＜0.01). Compared with group B, the OD value in group C decreased significantly (0.176±0.010 vs 0.188±0.011,t＝2.26, P＜0.05), NAG concentration increased significantly (22.225±0.565 vs 20.488±0.473,t＝-6.67, P＜0.01), and apoptotic rate in-creased significantly (20.22±1.1433 vs 18.23±1.1581,t＝-2.762, P＜0.05). Conclusions 1-methylhydantoin has more powerful cytotoxic effect to renal tubular epithelial cells than that of Methylguanidine.
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Objective To investigate the effect of lectin like oxidized low density lipoprotein receptor 1 ( LOX-1 ) in NRK52E intaking lipid induced by oxidized low density lipoprotein ( ox-LDL). Methods NRK-52E was incubated with ox-LDL (0,25,50, and 100 g/ml ) for 24 hours or pre-treated with the chemical blocker of LOX-1 receptor- polyI or carrageenan, and then exposed to 50 μg/ml of ox-LDL. LOX-Ⅰ mRNA was examined by real-time PGR. LOX-1 protein was assessed by Western blot analysis. Lipid deposit was examined by oil red O. Results LOX-1 mRNA expression in 25,50,100 μg/ml ox-LDL group was 2. 13, 10. 14, 20. 81 times of that in 0 g/ml ox-LDL group ( P <0. 05 ,respectively). LOX-1 protein expression in 25,50,100 μg/ml ox-LDL group was 2. 53,12. 18,21.45 times of that in 0 μg/ml ox-LDL group( P <0. 05 ,respectively). Following the increased LOX-1, lipid intake increased. Pre-treatment with Poly Ⅰ or carrageenan, LOX-1 mRNA expression deceased by 48% or 47%, LOX-1 protein deceased by 72% or 65%, lipid intake induced by 41% or 49% ( P <0.05 ,respectively). Lipid had a close relationship with LOX-1 ( r = 0. 87, P < 0. 05). Conclusion Ox-LDL induced NRK52E to express LOX-1 and promoted NRK52E to intake lipid, and this effect could be partly blocked by LOX-1 blocker.
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Objective To investigate the effect of in vitro high glucose stimulation on the expression of adiponectin receptor (adipoR) in human kidney proximal tubular cells.Methods The HK-2 cells were cultured in the low glucose DMEM culture medium containing 10% fetal bovine serum until the cells were adherent and 80% confluence. After cultured in the serum-free DMEM for 24 hours, these cells were stimulated with glucose-containing 1mg/ml, 2mg/ml, 4mg / ml, 6mg/ml, 8mg/ml serum-free DMEM for 48 hours. Then RT-PCR and western blot were used to analyze adipoR ( R1, R2) expression levels. The HK-2 cells were cultured respectively in high glucose (4mg/ml) , low glucose (1mg/ml) DMEM culture medium containing 10% fetal bovine serum to cultivate 0h, 12h, 24h, 48h, 72h, 96h, then RT - PCR was applied to analyze adipoR (R1, R2) mRNA expression levels semi-quantitatively. Results Two kinds of adiponectin receptor gene were both expressed in HK-2 cells, and the quantity of gene expression of adipoR1 (0. 63 ±0. 12) was 3. 9 times to adipoR2 (0. 16 ±0.03) , the difference was statistically significant ( P<0. 01). The different concentrations of glucose and different time of high glucose on HK-2 cells had no significant effect ( P>0. 05 ) on adipoR gene expression. Expression of adipoR 1 protein in HK-2 cells was detected by western blot, and it was not affected by glucose concentration ( P>0. 05).Conclusion adi-poR1 and adipoR2 gene were both expressed in HK-2 cells, and the adipoR1 was the major one, which suggested that adipoR1 played a more significant role in kidney disease. The expression of adipoRl/R2 of HK-2 cells was not affected by high glucose concentration.