Objective Obesity-induced kidney injury contributes to the development of diabetic nephropathy(DN).Here,we identified the functions of ubiquitin-specific peptidase 19(USP19)in HK-2 cells exposed to a combination of hi...Objective Obesity-induced kidney injury contributes to the development of diabetic nephropathy(DN).Here,we identified the functions of ubiquitin-specific peptidase 19(USP19)in HK-2 cells exposed to a combination of high glucose(HG)and free fatty acid(FFA)and determined its association with TGF-beta-activated kinase 1(TAK1).Methods HK-2 cells were exposed to a combination of HG and FFA.USP19 mRNA expression was detected by quantitative RT-PCR(qRT-PCR),and protein analysis was performed by immunoblotting(IB).Cell growth was assessed by Cell Counting Kit-8(CCK-8)viability and 5-ethynyl-2′-deoxyuridine(EdU)proliferation assays.Cell cycle distribution and apoptosis were detected by flow cytometry.The USP19/TAK1 interaction and ubiquitinated TAK1 levels were assayed by coimmunoprecipitation(Co-IP)assays and IB.Results In HG+FFA-challenged HK-2 cells,USP19 was highly expressed.USP19 knockdown attenuated HG+FFA-triggered growth inhibition and apoptosis promotion in HK-2 cells.Moreover,USP19 knockdown alleviated HG+FFA-mediated PTEN-induced putative kinase 1(PINK1)/Parkin pathway inactivation and increased mitochondrial reactive oxygen species(ROS)generation in HK-2 cells.Mechanistically,USP19 stabilized the TAK1 protein through deubiquitination.Importantly,increased TAK1 expression reversed the USP19 knockdown-mediated phenotypic changes and PINK1/Parkin pathway activation in HG+FFA-challenged HK-2 cells.Conclusion The findings revealed that USP19 plays a crucial role in promoting HK-2 cell dysfunction induced by combined stimulation with HG and FFAs by stabilizing TAK1,providing a potential therapeutic strategy for combating DN.展开更多
Objective The goal of this study is to investigate the role and mechanism of endoplasmic reticulum stress and apoptosis regulated by thrombospondin 1(TSP1)in human renal tubular epithelial cells(HK-2 cells).Methods HK...Objective The goal of this study is to investigate the role and mechanism of endoplasmic reticulum stress and apoptosis regulated by thrombospondin 1(TSP1)in human renal tubular epithelial cells(HK-2 cells).Methods HK-2 cells were exposed to high concentrations of glucose(HG).The endoplasmic reticulum stress inhibitor 4-phenylbutyric acid(4-PBA)was administered by transfecting TSP1 or an empty vector to explore the mechanism of the endoplasmic reticulum response regulated by TSP1 and stress in renal cell apoptosis.The effects of TSP1 and 4-PBA on the proliferation and apoptosis of HK-2 cells under HG conditions were assessed using Cell counting kit-8 and flow cytometry.Western blotting was used to detect the apoptosis-and endoplasmic reticulum stress-related protein expression regulated by TSP1 and 4-PBA.Results HG treatment induced high cell apoptosis,abundantly expressed TSP1 level and restrained viability in HK-2 cells.Overexpression of TSP1 significantly inhibited the proliferation of and facilitated apoptosis of HK-2 cells under HG conditions.Administration of endoplasmic reticulum stress inhibitor 4-PBA after overexpression of TSP1 antagonized the inhibitory proliferation and promoted apoptosis rate in HG-triggered HK-2 cells induced by TSP1 overexpression.4-PBA treatment significantly hindered the expression of endoplasmic reticulum stress markers,such as PERK,ATF4,ATF6,p-eIF2α,IRE1,CHOP and XBP1,suggesting that the administration of 4-PBA was successful.Conclusion Overexpression of TSP1 activated endoplasmic reticulum stress by regulating the ATF6-CHOP axis.TSP1 restrained cell proliferation,and promoted apoptosis and endoplasmic reticulum stress by activating the ATF6-CHOP axis.展开更多
We established a monolayer polarized cell model using human kidney 2 (HK-2) cells cultured in a transwell chamber to examine the changes in the morphology and physiological functions of human-derived renal proximal tu...We established a monolayer polarized cell model using human kidney 2 (HK-2) cells cultured in a transwell chamber to examine the changes in the morphology and physiological functions of human-derived renal proximal tubular epithelial cells caused by tripterygium tablet extract (TTE) and triptolide. HK-2 cells were cultured on PCF membranes to form a complete monolayer of cells. A MTT assay was used to select 10, 40, 160, 640 μg·ml-1 TTE or 4, 16, 64, 256 ng·ml-1 triptolide to treat HK-2 monolayer cells. After 24 hours, a FITC permeability assay was performed;GGT, LDH and NAG secretion on the apical (AP) and basolateral (BL) sides of the cells by HK-2 cells were examined. The morphology and the monolayer structure of HK-2 cells was observed via optical microscope and scanning electron microscope, respectively. The effect on the cytoskeleton of HK-2 cells was observed under a fluorescence microscope. The IC50 of TTE was 277.122 μg·ml-1, and the IC50 of triptolide was 148.035 ng·ml-1. Compared with the DMSO group, the FITC leakage rate with TTE 160, 640 μg·ml-1 treated group and 4 - 256 ng·ml-1 triptolide dose group exhibited statistically significant increase. TTE significantly increased secretion of GGT and LDH at 160, 640 μg·ml-1, meanwhile, dramatically increased the AP/BL ratio of LDH at 160 μg·ml-1;triptolide significantly increased secretion and AP/BL ratio of GGT and LDH at 256 ng·ml-1. The morphological observations via optical and electron microscope indicated various degrees of damage to HK-2 cells by TTE and triptolide, and the degree of damage correlated positively with the dosage of the tested articles. Compared with DMSO group, the cellular damage degrees at TTE dosages of 40 - 640 μg·ml-1 and triptolide dose group at 16, 256 ng·ml-1 exhibited statistically significant differences via observation under optical microscope. Both TTE and triptolide caused various degrees of shortening and thickening of intracellular F-actin bundles of HK-2 cells;aggravation of these changes was observed with increasing drug dosage. Thus, we conclude both TTE and triptolide caused damage to human renal proximal tubular epithelial cells at certain dosages;TTE dosages of 40 μg·ml-1 and above and triptolide dose group at 16 ng·ml-1 and above exhibited the changes in the morphology, meanwhile, TTE dosages of 160 μg·ml-1 and above and triptolide dose group at 256 ng·ml-1 exhibited the changes in the physiological functions such as secretion of HK-2 cell.展开更多
Small extracellular vesicles (sEVs) participate in the pathological progression of high glucose (HG)-induced kidney injury, which is closely related to diabetic nephropathy. How sEVs specifically mediate the cell biom...Small extracellular vesicles (sEVs) participate in the pathological progression of high glucose (HG)-induced kidney injury, which is closely related to diabetic nephropathy. How sEVs specifically mediate the cell biomechanics underlying HG injury is unclear. Herein, we utilized a versatile atomic force microscope to determine the contributions of sEVs in HG-induced cellular injury. The sEVs extracted from the culture medium of human proximal tubule kidney (HK-2) cells treated by HG for 72 h (HG-induced sEVs) were verified and analyzed by multiple techniques, and the results indicated the effective production and the effect of dehydration on the shape of HG-induced sEVs. Further investigation on the morphologies of HK-2 cells treated by HG-induced sEVs showed that the surface roughness of the HK-2 cells increased, and their pseudopodia transitioned from lamellipodia to filopodia, with almost doubled mean pseudopodia length. Quantitative analysis of the mechanical responses of the cells revealed that the mean Young’s modulus increased by 26.2%, and the mean adhesion decreased by 36.8%. The indirect mediation of cellular biomechanics guided by HG-induced sEVs was evaluated by comparing it with previously studied direct HG injury. The HG-induced sEVs caused a greater reduction in cell adhesion and an increase in Young’s modulus compared with direct HG stimulation. This work suggested the ability of HG-induced sEVs to elicit specific biomechanical responses during HG injury, advancing the understanding of the injury mechanism caused by HG. The comparison of the cellular biomechanics between direct and indirect HG stimulations through HG-induced sEVs can be beneficial for the diagnosis and treatment of kidney injury.展开更多
Background： Surfactant protein-A （SP-A） contributes to the regulation of sepsis-induced acute kidney injury. In a previous study, we demonstrated that the expression of SP-A in the human renal tubular epithelial （...Background： Surfactant protein-A （SP-A） contributes to the regulation of sepsis-induced acute kidney injury. In a previous study, we demonstrated that the expression of SP-A in the human renal tubular epithelial （HK-2） cells can be stimulated by lipopolysaccharide （LPS）. The present study evaluated the possible signal-transducing mechanisms of LPS-induced SP-A biosynthesis in the HK-2 cells. Methods： Tetrazolium salt colorimetry （MTT） assay was used to detect cell viability of HK-2 cells after LPS stimulation on different time points. HK-2 cells were stimulated with 100 ng/ml of LPS for different durations to determine the effects of LPS on SP-A and toll-like receptor 4 （TLR4） messenger RNA （mRNA） expression, as well as phosphorylation of mitogen-activated/ extracellular signal-regulated kinase （MEK） 1, extracellular signal-regulated kinase 1/2 （ERK1/2）, p38 mitogen-activated protein kinase （p38MAPK）, and nuclear factor-kappa B （NF-KB） inhibitor-alpha （IkB-a）. Then, HK-2 cells were pretreated with CLI-095, a TLR4 inhibitor, to analyze mRNA and protein levels of SP-A and TLR4 and expression of NF-KB ill the cytoplasm and nucleus of HK-2 before LPS exposure. Results： HK-2 cells exposed to 100 ng/ml of LPS for 1,6, and 24 h did not affect cell viability which showed no toxic effect of 100 ng/ml LPS on cells （P = 0.16）; however, the biosynthesis of SP-A mRNA and protein in HK-2 cells was significantly increased （P = 0.02）. As to the mechanism, LPS enhanced transmembrane receptor TLR4 protein expression. Sequentially, LPS time dependently augmented phosphorylation of MEKI, ERKI/2, and p38MAPK. In addition, levels of phospborylatedand nuclear NF-KB were augmented with LPS exposure for 2 h. LPS-induced SP-A and TLR4 mRNA as well as NF-KB expression were significantly inhibited by pretreatment with CLI-095. Conclusions： The present study exhibited that LPS can increase SP-A synthesis in human renal epithelial cells through sequentially activating the TLR4-related MEK1 -ERK 1/2-NF-kB-dependent pathway.展开更多
Objective To investigate the protective effect of Pglycoprotein up-regulated by ulinastatin(UTI)on HK-2cells during paraquat(PQ)-induced injury and its underlying mechanisms.Methods The research was divided into two p...Objective To investigate the protective effect of Pglycoprotein up-regulated by ulinastatin(UTI)on HK-2cells during paraquat(PQ)-induced injury and its underlying mechanisms.Methods The research was divided into two parts.The first part of the research was divided into normal control group,PQ group,UTI+PQ group,UTI control group.The second part of the research was divided into negative virus group(including展开更多
基金supported by Natural Science Foundation of Shaanxi Province(No.2023-JC-YB-743 and No.2021JQ-905).
文摘Objective Obesity-induced kidney injury contributes to the development of diabetic nephropathy(DN).Here,we identified the functions of ubiquitin-specific peptidase 19(USP19)in HK-2 cells exposed to a combination of high glucose(HG)and free fatty acid(FFA)and determined its association with TGF-beta-activated kinase 1(TAK1).Methods HK-2 cells were exposed to a combination of HG and FFA.USP19 mRNA expression was detected by quantitative RT-PCR(qRT-PCR),and protein analysis was performed by immunoblotting(IB).Cell growth was assessed by Cell Counting Kit-8(CCK-8)viability and 5-ethynyl-2′-deoxyuridine(EdU)proliferation assays.Cell cycle distribution and apoptosis were detected by flow cytometry.The USP19/TAK1 interaction and ubiquitinated TAK1 levels were assayed by coimmunoprecipitation(Co-IP)assays and IB.Results In HG+FFA-challenged HK-2 cells,USP19 was highly expressed.USP19 knockdown attenuated HG+FFA-triggered growth inhibition and apoptosis promotion in HK-2 cells.Moreover,USP19 knockdown alleviated HG+FFA-mediated PTEN-induced putative kinase 1(PINK1)/Parkin pathway inactivation and increased mitochondrial reactive oxygen species(ROS)generation in HK-2 cells.Mechanistically,USP19 stabilized the TAK1 protein through deubiquitination.Importantly,increased TAK1 expression reversed the USP19 knockdown-mediated phenotypic changes and PINK1/Parkin pathway activation in HG+FFA-challenged HK-2 cells.Conclusion The findings revealed that USP19 plays a crucial role in promoting HK-2 cell dysfunction induced by combined stimulation with HG and FFAs by stabilizing TAK1,providing a potential therapeutic strategy for combating DN.
基金This study was supported by a grant from Chinese Society of Nephrology(No.14050430580).
文摘Objective The goal of this study is to investigate the role and mechanism of endoplasmic reticulum stress and apoptosis regulated by thrombospondin 1(TSP1)in human renal tubular epithelial cells(HK-2 cells).Methods HK-2 cells were exposed to high concentrations of glucose(HG).The endoplasmic reticulum stress inhibitor 4-phenylbutyric acid(4-PBA)was administered by transfecting TSP1 or an empty vector to explore the mechanism of the endoplasmic reticulum response regulated by TSP1 and stress in renal cell apoptosis.The effects of TSP1 and 4-PBA on the proliferation and apoptosis of HK-2 cells under HG conditions were assessed using Cell counting kit-8 and flow cytometry.Western blotting was used to detect the apoptosis-and endoplasmic reticulum stress-related protein expression regulated by TSP1 and 4-PBA.Results HG treatment induced high cell apoptosis,abundantly expressed TSP1 level and restrained viability in HK-2 cells.Overexpression of TSP1 significantly inhibited the proliferation of and facilitated apoptosis of HK-2 cells under HG conditions.Administration of endoplasmic reticulum stress inhibitor 4-PBA after overexpression of TSP1 antagonized the inhibitory proliferation and promoted apoptosis rate in HG-triggered HK-2 cells induced by TSP1 overexpression.4-PBA treatment significantly hindered the expression of endoplasmic reticulum stress markers,such as PERK,ATF4,ATF6,p-eIF2α,IRE1,CHOP and XBP1,suggesting that the administration of 4-PBA was successful.Conclusion Overexpression of TSP1 activated endoplasmic reticulum stress by regulating the ATF6-CHOP axis.TSP1 restrained cell proliferation,and promoted apoptosis and endoplasmic reticulum stress by activating the ATF6-CHOP axis.
文摘We established a monolayer polarized cell model using human kidney 2 (HK-2) cells cultured in a transwell chamber to examine the changes in the morphology and physiological functions of human-derived renal proximal tubular epithelial cells caused by tripterygium tablet extract (TTE) and triptolide. HK-2 cells were cultured on PCF membranes to form a complete monolayer of cells. A MTT assay was used to select 10, 40, 160, 640 μg·ml-1 TTE or 4, 16, 64, 256 ng·ml-1 triptolide to treat HK-2 monolayer cells. After 24 hours, a FITC permeability assay was performed;GGT, LDH and NAG secretion on the apical (AP) and basolateral (BL) sides of the cells by HK-2 cells were examined. The morphology and the monolayer structure of HK-2 cells was observed via optical microscope and scanning electron microscope, respectively. The effect on the cytoskeleton of HK-2 cells was observed under a fluorescence microscope. The IC50 of TTE was 277.122 μg·ml-1, and the IC50 of triptolide was 148.035 ng·ml-1. Compared with the DMSO group, the FITC leakage rate with TTE 160, 640 μg·ml-1 treated group and 4 - 256 ng·ml-1 triptolide dose group exhibited statistically significant increase. TTE significantly increased secretion of GGT and LDH at 160, 640 μg·ml-1, meanwhile, dramatically increased the AP/BL ratio of LDH at 160 μg·ml-1;triptolide significantly increased secretion and AP/BL ratio of GGT and LDH at 256 ng·ml-1. The morphological observations via optical and electron microscope indicated various degrees of damage to HK-2 cells by TTE and triptolide, and the degree of damage correlated positively with the dosage of the tested articles. Compared with DMSO group, the cellular damage degrees at TTE dosages of 40 - 640 μg·ml-1 and triptolide dose group at 16, 256 ng·ml-1 exhibited statistically significant differences via observation under optical microscope. Both TTE and triptolide caused various degrees of shortening and thickening of intracellular F-actin bundles of HK-2 cells;aggravation of these changes was observed with increasing drug dosage. Thus, we conclude both TTE and triptolide caused damage to human renal proximal tubular epithelial cells at certain dosages;TTE dosages of 40 μg·ml-1 and above and triptolide dose group at 16 ng·ml-1 and above exhibited the changes in the morphology, meanwhile, TTE dosages of 160 μg·ml-1 and above and triptolide dose group at 256 ng·ml-1 exhibited the changes in the physiological functions such as secretion of HK-2 cell.
基金supported by National Natural Science Foundation Program of China(No.62175020)EU H2020 Program(ENSIGN No.101086226)+2 种基金Jilin Provincial Science and Technology Program(Nos.20210101038JC,2020C022-1,20190201287JC and 20190702002GH),Jilin Provincial Education Department(JJKH20220781KJ)“111”Project of China(D17017)This work was also partly supported by Changli Nano Biotechnology(China)and China Scholarship Council(CSC,No.202007585007).
文摘Small extracellular vesicles (sEVs) participate in the pathological progression of high glucose (HG)-induced kidney injury, which is closely related to diabetic nephropathy. How sEVs specifically mediate the cell biomechanics underlying HG injury is unclear. Herein, we utilized a versatile atomic force microscope to determine the contributions of sEVs in HG-induced cellular injury. The sEVs extracted from the culture medium of human proximal tubule kidney (HK-2) cells treated by HG for 72 h (HG-induced sEVs) were verified and analyzed by multiple techniques, and the results indicated the effective production and the effect of dehydration on the shape of HG-induced sEVs. Further investigation on the morphologies of HK-2 cells treated by HG-induced sEVs showed that the surface roughness of the HK-2 cells increased, and their pseudopodia transitioned from lamellipodia to filopodia, with almost doubled mean pseudopodia length. Quantitative analysis of the mechanical responses of the cells revealed that the mean Young’s modulus increased by 26.2%, and the mean adhesion decreased by 36.8%. The indirect mediation of cellular biomechanics guided by HG-induced sEVs was evaluated by comparing it with previously studied direct HG injury. The HG-induced sEVs caused a greater reduction in cell adhesion and an increase in Young’s modulus compared with direct HG stimulation. This work suggested the ability of HG-induced sEVs to elicit specific biomechanical responses during HG injury, advancing the understanding of the injury mechanism caused by HG. The comparison of the cellular biomechanics between direct and indirect HG stimulations through HG-induced sEVs can be beneficial for the diagnosis and treatment of kidney injury.
文摘Background： Surfactant protein-A （SP-A） contributes to the regulation of sepsis-induced acute kidney injury. In a previous study, we demonstrated that the expression of SP-A in the human renal tubular epithelial （HK-2） cells can be stimulated by lipopolysaccharide （LPS）. The present study evaluated the possible signal-transducing mechanisms of LPS-induced SP-A biosynthesis in the HK-2 cells. Methods： Tetrazolium salt colorimetry （MTT） assay was used to detect cell viability of HK-2 cells after LPS stimulation on different time points. HK-2 cells were stimulated with 100 ng/ml of LPS for different durations to determine the effects of LPS on SP-A and toll-like receptor 4 （TLR4） messenger RNA （mRNA） expression, as well as phosphorylation of mitogen-activated/ extracellular signal-regulated kinase （MEK） 1, extracellular signal-regulated kinase 1/2 （ERK1/2）, p38 mitogen-activated protein kinase （p38MAPK）, and nuclear factor-kappa B （NF-KB） inhibitor-alpha （IkB-a）. Then, HK-2 cells were pretreated with CLI-095, a TLR4 inhibitor, to analyze mRNA and protein levels of SP-A and TLR4 and expression of NF-KB ill the cytoplasm and nucleus of HK-2 before LPS exposure. Results： HK-2 cells exposed to 100 ng/ml of LPS for 1,6, and 24 h did not affect cell viability which showed no toxic effect of 100 ng/ml LPS on cells （P = 0.16）; however, the biosynthesis of SP-A mRNA and protein in HK-2 cells was significantly increased （P = 0.02）. As to the mechanism, LPS enhanced transmembrane receptor TLR4 protein expression. Sequentially, LPS time dependently augmented phosphorylation of MEKI, ERKI/2, and p38MAPK. In addition, levels of phospborylatedand nuclear NF-KB were augmented with LPS exposure for 2 h. LPS-induced SP-A and TLR4 mRNA as well as NF-KB expression were significantly inhibited by pretreatment with CLI-095. Conclusions： The present study exhibited that LPS can increase SP-A synthesis in human renal epithelial cells through sequentially activating the TLR4-related MEK1 -ERK 1/2-NF-kB-dependent pathway.
文摘Objective To investigate the protective effect of Pglycoprotein up-regulated by ulinastatin(UTI)on HK-2cells during paraquat(PQ)-induced injury and its underlying mechanisms.Methods The research was divided into two parts.The first part of the research was divided into normal control group,PQ group,UTI+PQ group,UTI control group.The second part of the research was divided into negative virus group(including
