Mechanism of lysosomal membrane permeabilization in uranyl acetate-induced death of renal proximal tubule epithelial cells / 中华放射医学与防护杂志

Objective:To explore the mechanism of lysosomal membrane permeabilization(LMP)inuranyl acetate-induced death of human kidney proximal tubular epithelial HK-2 cells.Methods:HK-2 cells were exposed to uranyl acetate at concentrations of 100, 300 and 600 μmol/L for 24 h, then in tracellular reactive oxygen species (ROS)and mitochondrial superoxide were measured by DCFH-DA and MitoSOX probe, respectively. HK-2 cells were divided into four groups: blank control group, NAC or CA-074 Me group, uranyl acetate exposure group and uranyl acetate exposure plus NAC or CA-074 Me group. Two-color immune of luorescence staining was used to detect the co-localization of galectin-1 and lysosomal associated membrane protein-1 (LAMP-1) to measure the extent of LMP, and to detect the non- co-localization of cathepsin B and LAMP-1 to reflect the release of cathepsin B in lysosomes. Calcein-AM/PI double staining method was used to detect cell death. One-color immune of luorescence staining of cleaved-caspase-3 expression was used to detect apoptosis. Results:Intracellular ROS and mitochondrial superoxide levels were significantly increased in HK-2 cells after exposure with 100, 300 and 600 μmol/L uranyl acetate for 24 h, about 1.1-2.5 times or 4.0-28 times, respectively( tROS=17.98, 11.84, 11.75, P< 0.05; tmitochondrial superoxide=6.14, 16.02, 13.06, P< 0.05), and they also increased with uranyl acetate concentrations ( tROS=10.10, 10.37, 5.59, P< 0.05; tmitochondrial superoxide=21.50, 15.16, 5.93, P< 0.05). The percentage of co-localization of galectin-1 and LAMP-1 and the percentage of non- co-localization of cathepsin B and LAMP-1 were markedly increased in HK-2 cells after exposure with 600 μmol/L uranyl acetate for 24 h, 5.4-6.7 times or 1.5-2.1 times, respectively ( tGalectin-1=15.85, 12.70, P< 0.05; tCathepsin B=5.95, 6.69, P< 0.05), but these increases were inhibited by NAC ( tGalectin-1=4.74, P<0.05; tCathepsin B=4.51, P< 0.05). Moreover, the cell death rate and the cleaved-caspase-3 expression level were also significantly increased in HK-2 cells after exposure with 600 μmol/L uranyl acetate for 24 h, about 28-47 times or 2.4-6.0 times, respectively( tPI=30.40, 10.34, P<0.05; tCleaved-caspase-3=18.49, 9.52, P<0.05), and these increases were obviously diminished by CA-074 Me ( tPI= 6.76, P<0.05; tCleaved-caspase-3=13.47, P<0.05). Conclusions:Exposure to uranyl acetate induces a burst of intracellular ROSthat leads to LMP and consequently causes leakage of cathepsin B from lysosomes to cytoplasm, in turn triggering the lysosomal-dependent cell death and mitochondrial-regulated apoptosis of HK-2 cells.

Qingshen granule-medicated serum attenuates high glucose-induced epithelial-mesenchymal transition of HK-2 cells via inhibiting oxidative stress-mediated NF-κB signaling pathway / 中国病理生理杂志

AIM:To investigate whether Qingshen granules(QSG)-medicated serum inhibits oxidative stress-mediated NF-κB signaling pathway and attenuates epithelial-mesenchymal transition(EMT)of human proximal tubule epi?thelial HK-2 cells induced by high glucose. METHODS:The active components in QSG were analyzed by HPLC. The HK-2 cells were randomly divided into control group,mannitol group,high glucose group,low-dose QSG group,medium-dose QSG group,high-dose QSG group and pyrrolidine dithiocarbamate(PDTC)group. The morphological changes of the cells were observed by inverted phase contrast microscopy. MTT assay was used to detect the cell viability. Flow cytometry was used to detect the content of reactive oxygen species(ROS)in HK-2 cells. ELISA was used to detect the content of malondi?al dehyde(MDA)and the activity of superoxide dismutase(SOD). The DNA binding activity of nuclear factor-κB(NF-κB) p65 in HK-2 cells was detected by electrophoretic mobility-shift assay(EMSA). The protein expression of NF-κB p65, phosphorylated inhibitor of kappa B alpha(p-IκBα),inhibitor of kappa B kinase alpha(IKKα),monocyte chemoattractant protein-1(MCP-1)and intercellular adhesion molecule-1(ICAM-1)in HK-2 cells was detected by Western blot. Immuno?fluorescence staining was used to detect NF-κB p65 andα-smooth mucle actin(α-SMA)protein expression in HK-2 cells. RESULTS:Chlorogenic acid,berberine hydrochloride,plantamajoside,6,7-dimethoxycoumarin,epiberberine,copti?sine,lithospermicacid B,palmatine,leonurine hydrochloride,rheic acid and tanshinone ⅡA in QSG were preliminarily de?termined by HPLC. Compared with control group,the levels of ROS and MDA in HK-2 cells induced by high glucose in?creased(P<0. 05),while the activity of SOD decreased(P<0. 05). The protein levels of NF-κB p65,p-IκBα,IKKα, MCP-1,ICAM-1 andα-SMA were increased(P<0. 05). After intervened by QSG-medicated serum,the levels of ROS and MDA were decreased(P<0. 05),while the activity of SOD was increased(P<0. 05). The protein levels of NF-κB p65,p-IκBα,IKKα,MCP-1,ICAM-1 andα-SMA were decreased(P<0. 05). CONCLUSION:QSG-medicated serum inhibited oxidative stress-mediated NF-κB signaling pathway,thus attenuating the EMT of HK-2 cells induced by high glucose.

Protective effect of dapagliflozin on renal tubular epithelial cells in diabetic nephropathy and its related mechanism / 中华内分泌外科杂志

Objective:To investigate the protective effect of dapagliflozin on cell damage and in HK-2 cells induced by high concentration of glucose.Methods:HK-2 cells were divided into four groups: control group (NC) , high glucose model group (HG) , dapagliflozin group (CS-179) and metformin group (PC) . After treatment with different drugs for 24 h, CCK-8 assay was applied to determine HK-2 cells viability; ROS, SOD, CAT and MDA levels were measured by a multi-detection reader; The protein expression of Nrf2 was determined by Western blot.Results:The results of CCK8 showed that the cell survival rate of the high glucose model group was 58.0%±0.8%, and that of the dapagliflozin group was 87.0%±0.4%. Dapagliflozin significantly increased the survival rate of HK-2 cells, and the results were statistically significant ( P<0.01) . The microplate reader test found that compared with the high glucose model group (ROS: 3.46 ± 0.05, MDA: 25.37 ± 0.61, SOD: 55.89 ± 4.09, CAT: 10.22 ± 1.67) , dapagliflozin reduced the accumulation of ROS (1.97±0.04) and MDA (9.5±0.4) caused by high glucose in HK-2 cells (both P<0.01) , increasing the vigor of SOD (114.95±4.19) and CAT (32.83±2.01) (both P<0.01) . Compared with the expression of Nrf2 protein in the high glucose model group (0.26±0.03) , the expression of Nrf2 protein (0.48±0.03) in dapagliflozin group was significantly increased ( P<0.01) . Conclusion:Dapagliflozin can alleviate the HK-2 cells damage induced by high concentration of glucose via reducing oxidative damage, and acti-vating Nrf2 antioxidant transcription factor.

Dexmedetomidine Attenuates High Glucose-induced HK-2 Epithelial-mesenchymal Transition by Inhibiting AKT and ERK / 生物医学与环境科学（英文）

Objective@#To explore the protective effects of dexmedetomidine (Dex) against high glucose-induced epithelial-mesenchymal transition in HK-2 cells and relevant mechanisms.@*Methods@#HK-2 cells were exposed to either glucose or glucose+Dex for 6 h. The production of ROS, morphology of HK-2 cells, and cell cycle were detected. Moreover, the expression of AKT, p-AKT, ERK, p-ERK, PI3K, E-Cadherin, Claudin-1, and α-SMA were determined and compared between HK-2 cells exposed to glucose and those exposed to both glucose and Dex with or without PI3K/AKT pathway inhibitor LY294002 and ERK pathway inhibitor U0126.@*Results@#Compared with HK-2 cells exposed to high level of glucose, the HK-2 cells exposed to both high level of glucose and Dex showed: (1) lower level of ROS production; (2) cell morphology was complete; (3) more cells in G1 phase; (4) lower expression of p-AKT, p-ERK and α-SMA, higher expression of E-Cadherin and Claudin-1. PI3K/AKT inhibitor LY294002 and ERK inhibitor U0126 decreased the expression of p-AKT, p-ERK and α-SMA, and increased the expression of E-Cadherin and Claudin-1.@*Conclusion@#Dex can attenuate high glucose-induced HK-2 epithelial-mesenchymal transition by inhibiting AKT and ERK.

Propofol Attenuates Hypoxia/Reoxygenation-Induced Apoptosis and Autophagy in HK-2 Cells by Inhibiting JNK Activation

PURPOSE: The aim of this study was to investigate whether propofol could attenuate hypoxia/reoxygenation-induced apoptosis and autophagy in human renal proximal tubular cells (HK-2) by inhibiting JNK activation. MATERIALS AND METHODS: HK-2 cells were treated with or without propofol or JNK inhibitor SP600125 for 1 hour and then subjected to 15 hours of hypoxia and 2 hours of reoxygenation (H/R). Cell viability and LDH release were measured with commercial kits. Cell apoptosis was evaluated by flow cytometry. The expressions of p-JNK, cleaved-caspase-3, Bcl-2, and autophagy markers LC3 and p62 were measured by Western blot or immunofluorescence. RESULTS: HK-2 cells exposed to H/R insult showed higher cell injury (detected by increased LDH release and decreased cell viability), increased cell apoptosis index and expression of cleaved-caspase-3, a decrease in the expression of Bcl-2 accompanied by increased expression of p-JNK and LC3II, and a decrease in expression of p62. All of these alterations were attenuated by propofol treatment. Similar effects were provoked upon treatment with the JNK inhibitor SP600125. Moreover, the protective effects were more obvious with the combination of propofol and SP600125. CONCLUSION: These results suggest that propofol could attenuate hypoxia/reoxygenation induced apoptosis and autophagy in HK-2 cells, probably through inhibiting JNK activation.

Effect of Slit2 on high glucose-induced renal tubular epithelial-mesenchymal transdifferentiation and its mechanism / 解放军医学杂志

Objective: To explore the effect of Slit2/ROBO1 protein (Slit2/ROBO1) signaling pathway in high glucose-induced epithelial-mesenchymal transdifferentiation (EMT) and its mechanism. Methods: Human renal tubular epithelial cells (HK-2) were cultured in vitro and subjected to high glucose concentration and time gradient experiments. First, for concentration gradient experiment, the sample was randomly divided into normal group, control group 1, control group 2, high glucose group 1, high glucose group 2. While for high glucose time gradient experiment, the sample was randomly divided into normal group, control group, high glucose 24 h group, high glucose 36 h group and high glucose 48 h group. Western blotting was used to detect the expression changes of Slit2, ROBO1, α-smooth muscle actin (α-SMA) and fibronectin in HK-2 cells, and then the optimal high glucose stimulation concentration and time were screened out. Slit2 over-expressed plasmid and negative control plasmid were transfected into HK-2 cells to verify the successful transfection, the cells were then randomly divided into normal group, control group, high glucose group, high glucose empty group and high glucose Slit2 group. The total protein was extracted after stimulation with optimal high glucose concentration and time, and Western blotting was then performed to detect the change in expression of fibronectin and α-SMA. Results: In the high glucose concentration gradient experiment, the expression of Slit2 declined significantly in high glucose group 1(0.647±0.048) and high glucose group 2(0.210±0.023) than in the normal group (1.000±0.050); the expression of ROBO1 declined significantly in high glucose group 1(0.703±0.041) and high glucose group 2(0.303±0.022) than in the normal group (1.000±0.057); while the expression of fibronectin increased significantly in high glucose group 1(1.953±0.042) and high glucose group 2(2.997±0.078) than in the normal group (0.990±0.059), and the expression of α-SMA increased significantly in high glucose group 1(1.767±0.012) and high glucose group 2(2.427±0.059) than in the normal group (1.033±0.067), all the differences were of statistical significance(P<0.05). Compared with the high glucose group 1, the expressions of Slit2 and ROBO1 decreased, and of fibronectin and α-SMA increased significantly in the high glucose group 2(P<0.05). In the high glucose time gradient experiment, compared with the normal group, the expressions of Slit2 in high glucose 36 h group and high glucose 48 h group decreased (0.943±0.032 vs. 0.557±0.020, 0.450±0.055, respectively), and the expression of ROBO1 decreased (1.000±0.058 vs. 0.600±0.023, 0.227±0.028, respectively). Compared with the normal group, the expression of fibronectin increased significantly in high glucose 24 h group, high glucose 36 h group and high glucose 48 h group (0.970±0.040 vs. 1.247±0.052, 1.733±0.084, 2.780±0.090, respectively), and the expression of α-SMA increased significantly in high glucose 24 h group, high glucose 36 h group and high glucose 48 h group (1.033±0.067 vs. 1.277±0.041, 1.767±0.120, 2.537±0.078, respectively), and the difference was statistically significant (P<0.05). Compared with high glucose 24 h group, the expression of Slit2 declined significantly in high glucose 36 h group and high glucose 48 h group(0.893±0.034 vs. 0.557±0.020, 0.450±0.055, respectively), and the expression of ROBO1 declined significantly (0.930±0.025 vs. 0.600±0.023, 0.227±0.028, respectively), the expressions of fibronectin and α-SMA increased significantly with statistical significance (P<0.05). Compared with high glucose 36 h group, the expression of Slit2 and ROBO1 declined significantly, and the expression of fibronectin and α-SMA increased significantly in high glucose 48 h group (P<0.05). In the high glucose environment, and achieving Slit2 overexpression and negative control plasmid transfection, the expression of fibronectin increased significantly in high glucose group, high glucose+empty group and high glucose+Slit2 group (2.760±0.012, 2.667±0.027, 1.460±0.034, respectively) than in normal group (1.000±0.058); the expression of α-SMA increased also in high glucose group, high glucose+empty group and high glucose+Slit2 group (2.487±0.048, 2.557±0.037, 1.270±0.017, respectively) than in normal group (1.000±0.050) with statistical significance (P<0.05). Compared with the high glucose+empty group, the expression of fibronectin and α-SMA declined significantly in the high glucose+Slit2 group(P<0.05). Conclusion: The decreased expression of Slit2 and ROBO1 is involved in the high glucose-induced renal tubular EMT. Overexpression of Slit2 may significantly inhibit the high glucose-induced EMT.

Effects of methylglyoxal on oxidative stress and NLRP3 inflammasome signaling in HK-2 cells / 医学研究生学报

Objective Methylglyoxal can cause the injury of human proximal tubular epithelial cell line(HK-2 cells),but the exact mechanism is still unclear. The present study aimed to explore the influence of oxidative stress and the expression of NLRP3 inflammasome in HK-2 cells induced by methylglyoxal. Methods HK-2 cells at logarithmic phase were divided into six groups:control group and 100,200,400,800,1600 μmol/L methylglyoxal groups (cells were cultured in 100,200,400,800,1600 μmol/L methylg-lyoxal concentration for 24 h). Superoxide dismutase(SOD)levels were assayed by thibabituric acid method. Release of lactate dehydro-genase(LDH)activity was detected by assay kit.ROS production was measured by DCFH-DA staining. The expression levels of NLRP3,caspase-1,IL-1β and NF-κB were evaluated by western blot. Results Compared with control group,different methylglyoxal concen-trations could enhance ROS level and LDH activity in HK-2 cells(P<0.05)and reduce SOD level significantly(P<0.05). The results of western blot showed the protein levels of NLRP3,caspase-1,IL-1β and NF-κB were significant up-regulated after the addition of methylglyoxal(P<0.05). Conclusion Methylglyoxal may induce the injury of HK-2 cells by oxidant stress and activating of NLRP3 inflammasome signaling.

Fructose induces HK-2 ceils to express monocyte chemoattractant protein-1 through uric acid and reactive oxygen species / 上海交通大学学报（医学版）

Objective·To investigate the mechanism of fructose-induced monocyte chemoattratant protein-1(MCP-1) production in HK-2 cells.Methods·The HK-2 cells were divided into fructose incubated (1,5 and 10 mmol/L) group,fructose and ketohexo-kinase inhibitor (KHK-IN) coincubation (fructose 5 mmol/L,KHK-IN was 12,100 and 1 000 nmol/L,respectively) group,uric acid incubation (5,15 and 50 mg/dL) group,fructose and allopurinol co-incubation (fructose 5 mmol/L,allopurinol were 0.01,0.1 and 0.5 mmol/L) group,uric acid and allopurinol co-incubation (uric acid 50 mg/dL,allopttrinol respectively 0.01,0.1and 0.5 mmol/L) group,H2O2 incubation (0.1 and 0.3 mmol/L) group,fructose and N-acetylcysteine (NAC) coincubation (fructose 5 mmol/L,NAC respectively 5,10 and 50 mmol/L) group,and uric acid and NAC co-incubation (uric acid 50 mg/dL,NAC was 5,10and 50 mmol/L,respectively) group.The quantitative PCR method and Western blotting method were used to observe the expression ofMCP-1 mRNA and protein.The effects of fructose and uric acid on the production of ROS in HK-2 cells were observed by using a fluorescent probe.Results·Fructose doseand time-dependently induced MCP-1 gene transcription and protein production in HK-2 cells,which could be blocked by the ketohexo-kinase blockers.Exogenous uric acid induced MCP-1 production in HK-2 cells.Allopurinol inhibited fructose,but not exogenous uric acid-induced MCP-1 expression.Both fructose and uric acid induced ROS generation.Incubation with H2O2promoted MCP-1 production in HK-2 cells.NAC completely inhibited MCP-1production induced by fructose and H2O2.Conclusion·Catalyzed by the ketohexo-kinase,fructose resultes the production of MCP-1 through uric acid and reactive oxygen species.

Fructose induces HK-2 cells to express monocyte chemoattractant protein-1 through uric acid and reactive oxygen species / 上海交通大学学报（医学版）

Objective: To investigate the mechanism of fructose-induced monocyte chemoattratant protein-1(MCP-1) production in HK-2 cells. Methods: The HK-2 cells were divided into fructose incubated (1, 5 and 10 mmol/L) group, fructose and ketohexo-kinase inhibitor (KHK-IN) coincubation (fructose 5 mmol/L, KHK-IN was 12, 100 and 1000 nmol/L, respectively) group, uric acid incubation (5, 15 and 50 mg/dL) group, fructose and allopurinol co-incubation (fructose 5 mmol/L, allopurinol were 0.01, 0.1 and 0.5 mmol/L) group, uric acid and allopurinol co-incubation (uric acid 50 mg/dL, allopurinol respectively 0.01, 0.1and 0.5 mmol/L) group, H2O2 incubation (0.1 and 0.3 mmol/L) group, fructose and N-acetylcysteine (NAC) coincubation (fructose 5 mmol/L, NAC respectively 5, 10 and 50 mmol/L) group, and uric acid and NAC co-incubation (uric acid 50 mg/dL, NAC was 5, 10 and 50 mmol/L, respectively) group. The quantitative PCR method and Western blotting method were used to observe the expression of MCP-1 mRNA and protein. The effects of fructose and uric acid on the production of ROS in HK-2 cells were observed by using a fluorescent probe. Results: Fructose doseand time-dependently induced MCP-1 gene transcription and protein production in HK-2 cells, which could be blocked by the ketohexo-kinase blockers. Exogenous uric acid induced MCP-1 production in HK-2 cells. Allopurinol inhibited fructose, but not exogenous uric acid-induced MCP-1 expression. Both fructose and uric acid induced ROS generation. Incubation with H2O2 promoted MCP-1 production in HK-2 cells. NAC completely inhibited MCP-1 production induced by fructose and H2O2. Conclusion: Catalyzed by the ketohexo-kinase, fructose resultes the production of MCP-1 through uric acid and reactive oxygen species.

Effects of schisandrin B on P21 and Caspase-3 expression in HK-2 cells injuried by CDDP / 中草药

Objective: To explore the effect of schisandrin B (SchB) on P21 and Caspase-3 expression in cisplatin induced human proximal renal tubular epithelial (HK-2) cells injuried by CDDP in vitro. Methods: HK-2 cells were randomly divided into five groups: control group (untreated), CDDP group (treated with 50 μmol/L CDDP for 24 h), SchB pretreat groups (CDDP + SchB, cells were pretreated with 5, 10, and 20 μmol/L SchB for 2 h, and the rest of the operation was the same as CDDP group). CCK-8 kit was used to detect the cell viability of five groups. Flow cytometry was used to detect the apoptotic rate of five groups. The morphology of cells was observed by inverted microscope. The protein expression of P21 and Caspase-3 was assessed by Western blotting assay. Results: Compared with the control group, the cell viability decreased, and the apoptosis increased, the protein expression of P21 and Caspase-3 was up-regulated in HK-2 cells after treated with 50 μmol/L CDDP. Cell volume was reduced, and the mutual connection disappeared. Compared with the CDDP group, the cell viability increased and the apoptosis was decreased, the expression of Caspase-3 was down-regulated and the expression of P21 was up-regulated in HK-2 cells after pretreated with 5, 10, and 20 μmol/L SchB. Cell morphological damage lightened, the volume slightly decreased, and the mutual connection reduced. Conclusion: SchB has a protective effect on HK-2 cells damage induced by cisplatin. The mechanism may be related to up-regulation of P21 and down-regulation of Caspase-3 expression, and its protective effect is dose dependent.

Toxicity screening of andrographolide and its watersoluble derivatives on HK-2 cells and relative toxicity mechanism induced by andrographolide sodium bisulfite / 中国药理学与毒理学杂志

OBJECTIVE To observe and compare the cytotoxicity induced by andrographolide (AD)and its water soluble derivatives:andrographolide sodium bisulfite(ASB),active pharmaceutical ingredients of Chuanhuning and Yanhuning on human renal tubular epithelial cells (HK-2),and to explore the ASB-induced endoplasmic reticulum stress(ERS)mechanism. METHODS HK-2 cells were treated with the above four drugs respectively. The survival rate was examined by methyl thiazolyltetrazolium (MTT) assay and 50% inhibitory concentration (IC50) was calculated. In ASB treated group, Hoechst33342 staining and flow cytometry analysis were used to determine cell apoptosis, intracellular superoxide dismutase(SOD)activity and malondialdehyde(MDA)content were examined, and the protein expressions of binding immunoglobulin protein (Bip),C/EBP-homologous protein (CHOP)and cysteine-containing aspartate-specific protease 4(caspase 4)were detected by Western blotting. RESULTS The four drugs inhibited HK-2 cell growth in a time-dependent and concentration-dependent manner. At 24 h,the IC50 of AD (30.6 μmol · L- 1) was lower than that of others. Active pharmaceutical ingredients of Chuanhuning and Yanhuning (16.2 and 15.6 mmol · L- 1) were very close,ASB was 29.4 mmol · L-1. ASB(0,15,30 and 60 mmol · L-1)increased the apoptotic rate and caused the decrease in SOD activity and the increase in MDA content in a dose-dependent manner. Compared with control group,the protein expression of CHOP increased (P<0.01) at 8 h with ASB (30 and 60 mmol · L-1)treatment,Bip and caspase 4 had no significant change. In addition,at 24 h, ASB(60 mmol·L-1) decreased the expression of Bip(P<0.05),ASB(30 and 60 mmol·L-1)promoted the expression of CHOP(P<0.01),and the protein expression of activated caspase 4 increased in a concentration-dependent manner(P<0.01). CONCLUSION AD and its water soluble derivatives have a toxic effect on HK-2 cells. CHOP and caspase 4 pathway related to ERS is involved in ASB-induced apoptosis.

Preventive Effects of Aspidopterys obcordata on Renal Tubular Epithelial Cells Injury / 医药导报

Objective To explore the preventive effects of different extracts from Aspidopterys obcordata on renal tubu-lar epithelial cells injury induced by sodium oxalate in vitro, and initially identify the effective part for treating urolithiasis. Methods The injury model of HK-2 cells induced by sodium oxalate was established to screen the active parts of Aspidopterys obcordata by testing the protective effects of different polarity extracts on HK-2 injury cells through MTT method. Results Dif-ferent extracts from Aspidopterys obcordata improved the activity of HK-2 injury cells, which were elevated to 86.17% and 95.42%by 0. 5 mg?mL-1 and 1 mg?mL-1 aqueous extract, respectively. And the activity reached to 93. 59% and 84. 77% by 0.5 mg?mL-1 and 1 mg?mL-1 50% alcohol extracts, reached above 81.56% by 95% alcohol extracts,all of which showed sig-nificant difference compared with the model group. The HK-2 cells viability were elevated to 82. 53% and 91. 58% by 0.5 mg?mL-1 and 1 mg?mL-1 95% alcohol extracted ethyl acetate parts,and increased to 77.24% and 87.22% by 0.5 mg?mL-1and 1 mg?mL-1 of 95% alcohol isolated n-butanol extracts, approached to 95.46% and 81.36% by 0.5 mg?mL-1 and 1 mg?mL-1 water extracts, all of which showed significant difference compared with the model control group. Conclusion The aqueous extracts and alcohol extracts from Aspidopterys obcordata have obvious preventive effects on HK-2 cells injury, among which the ethyl acetate extracts, n-butanol extracts and water extracts present the remarkable effects, which are supposed to be the active parts for inhibiting calcium oxalate stone formation in vitro.

Role of JLP on the epithelial to mesenchymal transition in renal tubular epithelial cells / 中华肾脏病杂志

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.

Evaluation of nephrotoxicity induced by total terpenoids from Alismatis Rhizoma on HK-2 cells in vitro and its induction of apoptosis / 中国中药杂志

To evaluate the nephrotoxicity of total terpenoids from Alismatis Rhizoma on human kidney proximal tubular cells (HK-2), explore the iraction in inducing apoptosis of HK-2 cells, and provide reference for the research of controversial nephrotoxicity of total terpenoids from Alismatis Rhizoma, HK-2 cells were used and cells viability was measured by MTT colorimetric method. An assessment of cells apoptosis was also conducted by using flow cytometry. Meanwhile western blot assay was used to detect the protein expressions of caspase-3, Bcl-2, Bcl-xl, Kim-1, clusterin and TFF-3. At last, q-PCR was used to detect the mRNA expressions of caspase-3, Bcl-2, Bcl-xl, Kim-1, clusterin and TFF-3. The flow cytometry results showed that cells apoptosis rate was (37.48±1.76)%, (26.91±1.91)% and (25.61±2.05)% respectively after treating with total terpenoids (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹). Western blot results showed that Bcl-2 and Bcl-xl protein levels were significantly decreased after treating with total terpenoids (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹), while the protein expression of caspase-3 was significantly increased. q-PCR results were the same with western blot results, that mRNA expressions of Bcl-2 and Bcl-xl were significantly decreased while mRNA expression of caspase-3 was significantly increased after treating with total terpenoids (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹). Western blot results and q-PCR results showed that both mRNA and protein expressions of Kim-1, clusterin and TFF-3 were significantly increased after treating with total terpenoids from Alismatis Rhizoma (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹). HK-2 cells in vitro evaluation results showed that, total terpenoids from Alismatis Rhizoma may have nephrotoxicity effect, but further study is still needed for verification; meanwhile, they could induce HK-2 cells apoptosis, providing basis for nephrotoxicity study and safe application of Alismatis Rhizoma.

Protective effects of schisandrin B on hypoxia injury of HK-2 cells / 天津医药

Objective To explore the protective effects of schisandrin B (Sch B) on hypoxia injury induced by cobaltous chloride (CoCl2) in human proximal renal tubular epithelial (HK-2) cells, and the possible mechanism thereof. Methods HK-2 cells were randomly assigned to four groups:control group (Con, cells were untreated), CoCl2 group (CoCl2, cells were treated with 600μmol/L CoCl2 for 24 h), Sch B pretreat group (CoCl2+Sch B, cells were pretreated with 1μmol/L and 10μmol/L Sch B for 2 h) and Sch B group (Sch B, cells were treated with 1μmol/L and 10μmol/L Sch B for 2 h). CCK-8 kit was used to detect the cell viability of four groups. Flow cytometry was used to detect the apoptotic rate of four groups. The protein expression of hypoxia-inducible factor 1α(HIF-1α) was assessed by Western blot assay. The expressions of HIF-1α and inducible nitric oxide synthase (iNOS) mRNA were determined by RT-PCR. Results Compared with the control group, after treated with 600 μmol/L CoCl2, the cell viability was decreased, and the apoptosis was increased, the expressions of HIF-1α and iNOS mRNA were up-regulated in HK-2 cells. There was no significant difference in the expression of HIF-1α mRNA between control group and CoCl2 group. Compared with the CoCl2 group, after pretreated with 1μmol/L and 10μmol/L Sch B, the cell viability was increased and the apoptosis was decreased, the expressions of HIF-1α and iNOS were down-regulated in HK-2 cells. There were no significant differences in the cell viability and apoptotic rate between control group and Sch B group. Conclusion Pretreatment with Sch B can reduce the apoptosis of HK-2 cells by inhibiting the expression of HIF-1α and iNOS mRNA, which shows protective effects on hypoxia injury.

Effects of erythropoietin in high glucose induced proliferation and apoptosis of human kidney proximal tubular epithelial cells and the possible mechanism / 天津医药

Objective To study the effects of erythropoietin (rhEPO) in high glucose induced proliferation and apopto?sis of human kidney proximal tubular epithelial (HK-2) cells, and the possible mechanism thereof. Methods HK-2 cells cultured in vitro were divided into several groups randomly:blank control group, high glucose group, mannitol group, rhEPO control group, different concentrations of rhEPO treatment groups (5, 10, 20 U/mL) and Rho kinase group. The reverse tran?scription polymerase chain reaction (RT-PCR) was used to evaluate the mRNA levels of RhoA and ROCK after 24 hours. Tetrazolium salt method (MTT) was used to determine the cell proliferation. Cell apoptosis was detected by flow cytometry. Results Compared with blank control group the expression levels of RhoA and ROCK1 mRNA were significantly in?creased in high glucose group (P < 0.05). RhoA, ROCK1 mRNA expressions significantly decreased in rhEPO group than those of high glucose group (P<0.05). There was a positive correlation between the expression levels of RhoA mRNA and ROCK1 mRNA in high glucose group and rhEPO group. MTT method showed that rhEPO significantly promoted the prolifer?ation of HK-2 cells (P<0.05). Flow cytometry analysis showed that high glucose induced apoptosis in HK-2 cells, which was significantly inhibited in rhEPO group and Rho kinase group as compared to that of high glucose group in a concentra?tion dependent manner (P<0.05). Conclusion rhEPO can promote HK-2 cell proliferation and inhibit apoptosis, which may be related to RhoA/ROCK signaling pathway.

The influence of rHuEPO on inflammatory factors in the process of renal interstitial fibrosis and its possible mechanism / 医学研究生学报

Objective Clinical treatment can delay the development of renal interstitial fibrosis , but it can not reverse renal dysfuntion.The article was to discuss the influence of recombinant human erythropoietin ( rHuEPO ) on inflammatory factors in the process of renal interstitial fibrosis and its possible mechanism . Methods The vitro cultured HK-2 cells were randomized into 7 groups:the blank control group , rHuEPO control group ( addition of 20U/mL rHuEPO), albumin stimulation group (addition of 5mg/mL albumin), 5mg/mL rHuEPO intervention group (5mg/mL albumin +5U/mL rHuEPO), 10 U/mL rHuEPO intervention group (5mg/mL albumin +10 U/mL rHuEPO), 20U/mL rHuEPO intervention group (5mg/mL albumin +20U/mL rHuEPO), and Rho inhibi-taion group (addition of 5mg/mL albumin 30min after 10μmol/L Y27632), 24 h acting time for each group.We observed the changes of cell morphology in each group .Reverse transcription polymerase chain reaction ( RT-PCR) was used to evaluate the mRNA levels of RhoA, ROCK1 and IL-6 , and ELISA was applied to measure the levels of supernatant TNF-αand IL-6 protein. Results The form of pebbles or paving stone was observed in blank control group and rHuEPO intervention groups , a long and thin spindle change with the appearance of fibre cells in albumin stimulation group , the transformation to pebbles in 5, 10, 20 mg/mL rHuEPO intervention groups , the form of oval and slightly increased intercellular space in Rho inhibitaion group .Compared with the blank control group , the expressions of RhoA mRNA, ROCK1 mRNA and IL-6 mRNA significantly increased in the albumin stimulation group (P<0.05), while significantly reduced in 5, 10, 20 mg/mL rHuEPO intervention groups (P<0.05), which was in negative relation with the rHuEPO concentrations .Compared with the albumin stimulation group , the expressions of ROCK 1 mRNA and IL-6 mRNA reduced in Rho inhibtation group (P<0.05), while there was no significant difference as to the expression of RhoA mRNA .ELISA results showed:compared with blank control group , the expressions of supernatant TNF-α([452.32 ±33.23] ng/L vs [1347.54 ±41.52] ng/L), IL-6 protein([884.62 ±0.73] pg/L vs [95.12 ±0.32]pg/LP<0.05) increased significantly.Compared with albumin stim-ulation group, the expressions of TNF-αin 5, 10, 20 mg/mL rHuEPO intervention groups and Rho inhibitation group reduced signifi-cantly([1003.32 ±3.42] ng/L, [821.32 ±21.32] ng/L, [590.15 ±7.68] ng/L, [488.13 ±65.03] ng/L vs [1 347.54 ± 41.52]ng/L,P<0.05), while the expressions of IL-6 mRNA reduced accordingly in 5, 10, 20 mg/mL rHuEPO intervention groups and Rho inhibitation group reduced significantly ([656.68 ±0.55] pg/L, [422.35 ±0.22] pg/L, [217.32 ±0.35] pg/L, [309.49 ±0.21] pg/L vs [884.62 ±0.73]pg/L,P<0.05).Moreover, there was significant statistical difference among 5, 10, 20 mg/mL rHuEPO intervention groups(P<0.05). Conclusion RHuEPO can inhibit the transdifferentiation process of HK-2 cells in-duced by albumin by suppressing inflammation factors , and the mechanism may be involved in RhoA/ROCK signaling pathway .

HBx modulates apoptosis by activating JAK2/STAT3 signaling pathway in human renal proximal tubular epithelial cells / 中国病理生理杂志

AIM: To investigate the correlation of hepatitis B virus X protein (HBx) with renal tubular epithelialcell apoptosis in hepatitis B virus-associated glomerulonephritis (HBVGN) and the possible signaling mechanism. METHODS: The activation of JAK2/STAT3 signal pathway and the expression of apoptosis -related proteins in humankindey proximal tubular epithelial cells (HK-2 cells) were determined by Western blotting after transfection with HBx eukaryoticexpression vector.The cell proliferation was observed by CCK-8 assay.The cell apoptosis was analyzed by the imagingof HO33342 staining, transmission electron microscopy and flow cytometry with Annexin V /PI double staining.RESULTS:After transfection of the target gene HBx, the expression levels of both p-JAK2 and p-STAT3 were significantly increased.At the same time, the cell proliferation was obviously inhibited, and the apoptotic rate was increased.After incubationwith AG490, the JAK2/STAT3 signal pathway was partially blocked, and the cell apoptosis induced by HBx was reduced. CONCLUSION: HBx up-regulates the activation of JAK2/STAT3 signal pathway to induce renal tubular epithelialcell apoptosis, which is possibly involved in the pathogenic mechanism that HBV directly damages nephridial tissue .

Induction of apoptosis in human renal tubular epithelial HK-2 cells by emodin and mediation of endoplasmic reticulum stress / 中草药

Objective: To investigate the induetion of apoptosis in human renal tubular epithelial HK-2 cells by emodin and whether endoplasmic reticulum (ER) stress is involved in its mechanism. Methods: HK-2 cells were cultured and treated with various concentration of emodin at different time points. The cell viability was determined by MTT assay. The gene expression of glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein-homologous protein (CHOP), activating transcription factor 3 (ATF3), and X-box binding protein 1 splicing (XBP1s) was evaluated by quantitative real-time PCR. The protein expression of caspase-3, GRP78, CHOP, and eukaryotic initiation factor 2 alpha (eIF2α) was detected by Western blotting. Results: The viability of HK-2 cells was decreased by emodin in a dose-dependent manner, and the apoptosis of the cells was associated with caspase-3 shear activation. The treatment with emodin in HK-2 cells caused the increase in eIF2α phosphorylation, XBP1 mRNA splicing, the gene expression of GRP78, CHOP, and ATF3, and the protein expression of GRP78 and CHOP. The pretreatment with 4-phenylbutyric acid and salubrinal significantly increased the viability of HK-2 cells, indicating the role of ER stress in emodin-induced apoptosis. Conclusion: Emodin induces the apoptosis in HK-2 cells and ER stress is involved in emodin-induced apoptosis.

Polyphenols of Mangifera indica modulate arsenite-induced cytotoxicity in a human proximal tubule cell line

Inorganic arsenic is an ubiquitous environmental contaminant able to cause severe pathologies in humans, including kidney disorders. The possible protective effects of Mangifera indica L., Anacardiaceae, stem bark extract (MSBE) and some mango phenols on the cytotoxicity of arsenite (AsIII) in the proximal tubule cell line HK-2 was investigated. In cells cultured for 24 h in presence of AsIII, a dose-dependent loss of cell viability occurred that was significantly alleviated by MSBE, followed by gallic acid, catechin and mangiferin. Mangiferin complexed with Fe+++ proved more efficacious than mangiferin alone. MSBE and pure phenols increased significantly the cell surviving fraction in clonogenic assays. In cells pretreated with MSBE or phenols for 72 h the protection afforded by MSBE resulted decreased in comparison with the shorter experiments. Cells pretreated with a subcytotoxic amount of AsIII or cultured in continuous presence of low concentration of mangiferin proved to be more resistant to AsIII, while cells cultured in presence of albumin resulted more sensitive. Because all the above conditions share changes in expression/activity of P-glycoprotein (P-gp), a transporter potentially involved in arsenic resistance, the capability of M. indica phenols in modulating AsIII-induced cytotoxicity would be at least in part dependent on their interactions with P-gp.