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AIM: To study the toxicity of genipin-a kind of geniposide metabolites induced human tubular epithelial cells HK-2 and its effect on NLRP3 pathway. METHODS: The dose of GP on HK-2 cells were preliminarily determined by CCK8 method, the apoptosis or necrosis rate of HK-2 cells was detected by Hoechst 33342 / PI, the level of LDH release and reactive oxygen species was detected by Kits, and mitochondrial membrane potential and intracellular calcium ion concentration were detected by high content imaging. Real-time PCR detected mRNA levels of kindey injury factor-1, osteopontin, NLRP3, Caspase-1, interleukin 1β, and interleukin 18. RESULTS: Compared with the 0 μg / mL group, GP>50 μg/mL significantly reduced cell viability (P< 0.05, P<0.01), and the IC50 value was 110.50 μg/mL. Set the control group, the low, medium and high dose groups of GP (50, 100, 200 μg/mL); Compared with the control group, the cell density decreased in the medium and high dose groups of GP, and the PI positivity, LDH release, ROS, Ca
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This study aimed to investigate the effect of methyl eugenol(ME) on hypoxia/reoxygenation(H/R)-induced injury of human renal tubular epithelial HK-2 cells and its mechanism. The viability of HK-2 cells cultured with different concentrations of ME and exposed to H/R was detected by cell counting kit-8(CCK-8) assay. The effect of ME on the morphology of HK-2 cells was observed under an inverted microscope. The content of intracellular reactive oxygen species in different groups was detected after 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA) fluorescence staining. Cell apoptosis was determined by flow cytometry. Changes in mitochondrial membrane potential were monitored by JC-1 dye. The concentrations of nuclear factor erythroid 2 related factor 2(Nrf2), heme oxygenase-1(HO-1), and nicotinamide adenine dinucleotide phosphatase oxidase 4(Nox4) were measured by Western blot, followed by the assay of Nrf2 concentration changes in cytoplasm and nucleus by confocal fluorescence staining. The results showed that when the concentration of ME was 0-40 μmol·L~(-1), the activity of HK-2 cells was not affected. Compared with the model group, ME enhanced the activity of HK-2 cells and the cell morphology was normal. As revealed by further experiments, ME inhibited the release of reactive oxygen species and the decline in mitochondrial membrane potential of HK-2 cells after H/R injury, promoted Nrf2/HO-1 expression and Nrf2 translocation to the nucleus, and down-regulated the expression of Nox4, thereby significantly reducing apoptosis. This protective effect of ME could be reversed by the specific Nrf2 inhibitor ML385. These findings have preliminarily proved that ME effectively protected HK-2 cells against H/R injury, which might be related to its promotion of Nrf2/HO-1 signaling pathway and inhibition of Nox4. Such exploration on the possible mechanism of ME in the treatment of renal ischemia-reperfusion injury(IRI) and protection of organ function from the perspective of antioxidant stress has provided reference for related research on the treatment of acute kidney injury with traditional Chinese medicine.
Subject(s)
Humans , Apoptosis , Epithelial Cells/metabolism , Eugenol/pharmacology , Heme Oxygenase-1/metabolism , Hypoxia , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Reactive Oxygen Species , Reperfusion Injury/drug therapyABSTRACT
ObjectiveIopromide can induce injury to HK-2 cells, but its exact mechanism remains poorly understood. This study aimed to explore the influence of iopromide on ROS-NLRP3 inflammasome signaling in HK-2 cells.MethodsHK-2 human renal tubular epithelial cells were divided into six groups: control and iopromide at 37, 74, 111, 148 and 185 mgI/mL. The HK-2 cells in the latter five groups were treated with different concentrations of iopromide for 24 hours. Then the ROS level in the cells was detected by 2′,7′-Dichlorodihydrofluorescein diacetate staining and flow cytometry and the protein expressions of NLRP3, ASC, caspase-1, IL-1β, NF-κB and TNF-α determined by Western blot.ResultsThe ROS level was significantly increased in the HK-2 cells treated with iopromide at 37 mgI/ml (4103.89±98.89), 74 mgI/mL (4450.12±108.90), 111 mgI/mL (5050.85±606.76), 148 mgI/mL (6210.57±145.74) and 185 mgI/ml (7105.13±426.63) as compared with that in the control group (2551.71±84.00) (P<0.05). Western blot showed markedly upregulated expressions of NLRP3, ASC, caspase-1, IL-1β and TNF-α in the HK-2 cells in all the latter five groups in comparison with the control (P<0.05) and an increased level of NF-κB after treated with iopromide at ≥111 mgI/ml (P<0.05).ConclusionIopromide may induce injury to HK-2 cells by activating the ROS-NLRP3 inflammasome signaling pathway.
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Objective To investigate the effects of glycogen synthase kinase-3β (GSK-3β) and β-catenin signaling on the human renal proximal tubular epithelial HK-2 cell injury induced by depleted uranium(DU),and provide a new enlightenment for the development of DU antidotes.Methods H K-2 cells were exposed to different concentrations of DU for 3-24 h,then the protein expressions of kidney injury molecule 1 (KIM-1),neutrophil gelatinase-associated lipocalin (NGAL) and nuclear β-catenin were detected by immunofluorescence staining.The protein expressions of p-GSK-3 β(S9),GSK-3β and cmyc were detected by Western blot assay.HK-2 cells were transiently transfected by GSK-3β (KD) plasmid or treated by TDZD-8 to inhibit the activity of GSK-3β specifically.Other HK-2 cells were transiently transfected by β-catenin plasmid to overexpress the β-catenin protein.Results The percentages of KIM-1 and NGAL-positive cells increased with DU exposure time and concentrations from 300 and 600 μmol/L,and they were significantly higher than those of the blank control at 6-24 h of DU exposure (KIM-1-positive cells:t =11.06,18.97,30.49,P ＜0.05;t =6.79,16.02,85.45,P ＜ 0.05;NGAL-positive cells:t =11.78,11.37,34.29,P ＜0.05;t =7.34,21.63,36.84,P ＜0.05).In contrast,the ratio of p-GSK-3β (S9) to GSK-3β and percentage of nuclear β-catenin-positive cells were significantly higher than that of the blank control at 3-24 h of DU exposure (p-GSK-3β(S9)/GSK-3β:t =3.95,4.69,5.40,3.34,P ＜ 0.05;nuclear β-catenin-positive cells:t =4.61,6.52,36.64,14.93,P ＜ 0.05) with a maximum response at 9 h of DU exposure accompanied with corresponding increase of protein level of c-myc,a downstream target gene of β-catenin.Transient transfection of HK-2 cells with GSK-3β (KD) plasmid significantly inhibited the activity of GSK-3β (t =8.07,P ＜ 0.05) and reduced the DU-increased percentage of KIM-1-positive cells (t =24.77,P ＜ 0.05).Treatment cells with TDZD-8 inhibited the activity of GSK-3β and enhanced the percentage of nuclear β-catenin-positive cells,and it also significantly reduced the percentage of KIM-1-positive cells in HK-2 cells exposed to DU (t =6.25,6.73,P ＜ 0.05).Moreover,overexpression of β-catenin significantly reduced DU-induced cell injury (t =7.48,P ＜ 0.05).Conclusions GSK-3β/β-catenin signaling plays a key role in regulating the DU-induced cytotoxicity of HK-2 cells.Inhibition of GSK-3β activity and overexpression of β-catenin can protect the HK-2 cells from DU-induced damage.
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Objective:To study the effects of panax notoginsenosides on the proliferation and oxidation indices of cisplatin-induced nephroxicity in HK-2 cells. Methods:HK-2 cells were cultured in vitro till the number was up to 1 × 106/ml. The cells were inoculated in 96-well culture plate and randomly divided into six groups:normal saline ( NS) group,the model group, the positive control group and the high dose group , medium dose group and low dose group of panax notoginsenosides ( PNS) . The nephroxicity model was dupli-cated with the addition of cisplatin (the final concentration was 6. 25μg·L-1). The model group, positive control group and the three panax notoginsenosides groups was treated with saline solutions, amifostine, panax notoginsenosides at the dose of 100,50 and 25 mg· L-1 , respectively. The cell viability was detected with an MTT method, the content of MDA and the activity of SOD, GSH-PX and LDH were measured and the cell structure was observed. DCFH-DA was used as the fluorescence probe to detect the level of ROS by a fluorescence microplate reader. Results:Compared with those in the model group, the cell viability and the activity of SOD and GSH-PX in the three PNS groups and the positive control group significantly increased (P<0. 05);the content of MDA, the level of ROS and the activity of LDH significantly decreased (P<0. 05); the cell structure was significantly improved. Conclusion: PNS can pro-mote the proliferation of HK-2 cells in vitro, and improve the biochemical parameters and enzyme levels. The results suggest that PNS has a protective effect on HK-2 cell,and the protective mechanisms may be related with its antioxidant effect.
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@#To investigate the effect and mechanism of cytotoxicity by rhein in human renal tubular epithelial HK-2 cells, HgCl2 was choosen as positive control. Cell viability was determined by MTT assay. LDH release assay was used to evaluate cell membrane damage. The activity of caspase-3, and -8 was measured by assay kit. Real-Time qPCR was employed to determine the mRNA expressions of Fas, FasL, FADD, caspase-3 and caspase-8. The protein expressions of Fas, FasL, cytoplasmic cytochrome C, caspase-3, caspase-8, caspase-9 were detected by Western blot. The results demonstrated that rhein inhibited cell viability and increased LDH release in a dose-dependent manner. The activity of caspase-3 and caspase-8 was significantly enhanced by rhein. The mRNA expression of Fas, FasL, FADD, caspase-3, caspase-8 was remarkably up-regulated by rhein. Rhein also elevated protein expressions of Fas, FasL, cytoplasmic cytochrome C, cleaved caspase-3, caspase-8 and reduced expressios of Pro caspase-8. There was no significant difference in caspase-9 expression. These results indicate that rhein has a cytotoxic effect and apoptosis-inducing effect in HK-2 cells. The Fas-dependent pathway is involved in rhein-induced apoptosis.
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Objective To investigate the protective effect and mechanism of taurine on the cytotoxicity of iohexol on HK-2 cells. Methods HK-2 cells were exposed to iohexol at different dosage (25, 50, 100, 125 gI/L) for 6 h and at the dose of 100 gl/L for different time(2 h, 4 h, 6 h). Then taurine (3,12,24 mmol/L) was coincubated with iohexol (100 gI/L) for 6 h.Cell viability was assessed by CCK-8 assay. Cell apoptosis was determined by Hoechest 33342 flurescence stains,flow cytometry with Annexin V-FITC/PI double stains and caspase-3 activity by colorimetric assay. Bcl-2 and Bax expression were examined by Western blot. Intracellular ROS was detected by flow cytometry with fluorescent probe DCFH-DA. Results Iohexol decreased HK-2 cell viability and induced apoptosis in concentration-dependant and time-dependant manner (all P＜0.05). ROS was increased following iohexol (100 gI/L for 6 h) treatment (P＜0.05). Taurine increased cell viability and attenuated apoptosis in dose-dependant manner. The cell viability levels in taurine intervention (3,12,24 mmol/L) group were significantly increased compared with that in iohexol treated group respectively [(88.00±1.00)%, (91.33±0.58)%, (95.67±1.52) % vs (76.67±1.53)%, all P＜0.05]. Apoptosis rate by flow cytometry were decreased respectively [(8.84±1.75)%,(7.86±1.82)%, (6.30±1.50)% vs (11.98±0.39)%, all P＜0.05]. Caspase-3 activities were decreased respectively [(1.33±0.10), (1.27±0.06), (1.10±0.04) vs (1.42±0.13), all P＜0.05].Taurine up-regulated the expression of Bcl-2, and decreased the intracellular ROS (all P＜0.05).Conclusions Iohexol induces cell apoptosis and oxidative stress. Taurine attenuates direct cytotoxic effect induced by iohexol. The anti-oxidative stress effect and up-regulated Bcl-2 expression may partly account for the protection of taurine.
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Objective To investigate the influence of albumin on the expression of angiotensin-eonverting enzyme (ACE) in cultured human proximal tubular ceils (HK-2). Methods HK-2 cells were exposed to 2.5, 5, 10 g/L bovine serum albumin (BSA) for 6 h and 12 h respectively. The expression of ACE in HK-2 cells was detected by real-time RT-PCR and Western blot. Results Compared to the control group, the expression of HK-2 cells ACE mRNA treated for 12 h with different concentrations of BSA (2.5, 5, 10 g/L) significantly increased (P<O.05). Furthermore, Western blot analysis showed that the expression of ACE protein induced by BSA significantly increased (P<0.05). Treated with BSA (10 g/L) for 6 h and 12 h, the expression of ACE mRNA significantly increased in a time-dependent manner (P <0.05, respectively), and the ACE protein expression significantly increased (P<0.05, respectively). Conclusion BSA can induce ACE up-regulation in proximal tubular cells, which may lead to the increased production of local Ang Ⅱ and finally contributes to the intrarenal activation of renin angiotensin system (RAS).
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Objective To investigate the influence of Cordyceps polysaceharide (Cp) on epithelial-to-mesenchymal transition (EMT) induced by transforming growth factor-β1 (TGF-β1)in proximal tubular epithelial cells (PTEC). Methods HK-2 cell proliferation was determined by MTT assay. After incubation of HK2 cells with increasing concentrations of TGF-β1 (0, 0.1, 0.5, 1, 5, 10 μg/L) at 48 h and with TGF-β1 (5 μ/L) at different time points, E-cadherin, α-SMA, FN expression at transcriptional and protein levels were detected by real-time PCR and Western blotting respectively. The cells were pretreated with 1,5, 10 g/L Cp respectively for 24 h before adding TGF-β1 (5μg/L), then the cells were incubated for additional 48 h, mRNA and proteinexpression of above 3 cytokines was examined by real-time PCR and Western blotting as well. Results CP alone (0.01, 0.1, 1,5, 10 g/L) induced HK-2 cell proliferation in a dose-dependent manner. TGF-β1 enhanced α-SMA, FN expression while inhibited E-cedherin expression at both transcriptional and" protein level in HK-2 cell. At transcriptional level, compared to single TGF-β1 (5 μg/L) stimulating group, after Cp (1,5, 10 g/L) pretreatment for 24 h, the inhibition rate of a-SMA mRNA was 37.98%, 68.08% and 84.36%, respectively; FN mRNA was 46.97%, 63.82% and 81.85%, respectively; E-cadherin was up-regulated by 0.67 fold, 2.69 folds and 5.43 folds, respectively (P＜0.05). At protein level, the inhibition rate of α-SMA was 33.40%, 47.75% and 68.50%, respectively; FN was 16.26%, 65.92% and 80.30%, respectively; E-cadherin was up-regulated by 1.33 folds, 3.19 folds and 4.29 folds, respectively (all P＜0.05). Under Light microscopy, the Cp reversed cell shape from spindle-shape induced by TGF-β1 to nearly normal shape. Conclusion Cp may exert its inhibitive effects on TGF-β1-induced EMT.
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Objective To determine the inhibitory effect of a synthetic hexa-peptide GGWSHW (G peptide) derived from thrombospondin-1 (TSP1) on TGF-? activation induced by angiotensin Ⅱ (Ang Ⅱ) in cultured human renal tubular epithelial cells. Methods Human proximal tubular epithelial cell line (HK-2) was cultured in vitro, untreated HK-2 cells were acted as normal control group. HK-2 cells were then stimulated by Ang Ⅱ for 1-24 hours (Ang Ⅱ stimulation group),so that optimal dosage and duration could be chosen.One hour prior to induction,HK-2 cells were pretreated with 10 ?mol/L peptide G (G peptide treated group)or losartan (losartan treated group), the blocker of I type receptor of Ang Ⅱ was acted as inhibitory control.The mRNA and protein levels of TSP1,TGF-?1,FN and PAI-1 were measured by RT-PCR and Western blot. Confocal microscopy and flow cytometry were performed to detect the presence of TSP1, TGF-?1 and co-positive expression of two protein, respectively.The concentrations of total and active TGF-?1 as well as FN and PAI-1 in cell culture supematants were measured by ELISA. Additionally, the expression of Smad2 and p-Smad2 was also examined for the bioactivity of TGF-?1 signaling protein. Results Ang Ⅱ enhanced the expression of TSP1 and TGF-?1 in a temporal-spatial dependent manner. The optimal dosage and duration were 1 ?mol/L and 6 hours,for TSP1,and 0.1 ?mol/L and 12 hours for TGF-?1 respectively.Comparing with untreated HK-2,the co-expression of TSP1 and TGF-?1 induced by A Ⅱ showed a increase of 5.4 folds.In addition,the protein level of p-Smad2 was elevated remarkedly, the mRNA level of FN and PAI-1 was up-regulated by 3 and 1.5 folds, and the concentration was increased by 2.0 and 1.9 folds respectively. Peptide G had less effect on the expression of TSP1 and TGF-?1,whereas it significantly reduced the secretion of active TGF-?1,though total level of TGF-?1 remained up-regulated. Furthermore,comparing with losartan treated group, p-Smad2 expression was reduced by 28.9%, the mRNA level of FN and PAI-1 was decreased by 34.5% and 26% respectively,and the protein levels were reduced by 11.0% and 8.9% respectively. Conclusion The inhibitory peptide derived from TSP1 effectively suppresses TGF-?1 activation through a competitive mechanism and also reduces the secretion of FN and PAI-1 associated with fibrosis.