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Objective To investigate the effect and possible mechanism of microRNA-26a(miR-26a)on the syn-thesis of extracellular matrix(ECM)induced by high glucose(HG)in renal tubular epithelial cells(RTECs).Methods A model of diabetic kidney disease(DKD)was constructed by inducing RTECs with HG.MiR-26a was overexpressed in HG-induced RTECs,and RT-qPCR and Western blot were used to assess the effects of miR-26a on ECM synthesis and ferroptosis-related markers in HG-treated RTECs.Ferrostatin(Fer-1)was used to inhibit ferroptosis in the DKD model,and its impact on ECM synthesis was evaluated.RT-qPCR and Western blot were performed to measure ferroptosis-related markers,and fluorescence microscopy was used to observe the intensity of reactive oxygen species(ROS).Results Compared with the control group,the expression of miR-26a decreased in HG-treated cells,while the expression levels of ECM synthesis-related indexes fibronectin and collagen Ⅰ in-creased.After overexpressing miR-26a,the HG+miR-26a group showed a significant increase in miR-26a expres-sion and a decrease in fibronectin and collagen Ⅰ expression compared to the HG group.In terms of ferroptosis,the protein and mRNA expression of SLC7A11 and GPX4 significantly decreased,the expression of TFR-1 and AC-SL4 significantly increased,and the fluorescence intensity of ROS was significantly enhanced in the HG group com-pared with the control group.Inhibition of ferroptosis in the HG+Fer-1 group resulted in significant changes in fer-roptosis and ECM synthesis-related indicators expression levels compared to the HG group.Furthermore,re-expres-sion of miR-26a in the HG+miR-26a led to significant changes in ferroptosis-related indicators expression levels and decreased ROS fluorescence intensity compared to the HG group.Conclusions In HG-induced RTECs,miR-26a inhibits the occurrence of ferroptosis,thus reducing ECM synthesis.
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Objective @#To establish an in vitro renal injury model of amikacin (AKN) and investigate the protective effect and mechanism of vaccarin (VA) in the AKN-induced in vitro renal injury model .@*Methods @#Human renal tubular epithelial cells (HK-2) were cultured in vitro and incubated with different drugs of AKN or/and VA to de- termine the optimal drug concentration based on cell viability tested by MTT. The changes in intracellular oxidative stress were assessed using the dihydroethidium ( DHE) probe and malondialdehyde ( MDA) /glutathione ( GSH) assay kits at different time points . Total RNA was extracted , and RT-qPCR was performed to detect the changes in the gene expression of kidney injury molecule-1 ( KIM-1) and neutropil gelatinase-associated lipocalin ( NGAL) . Western blot analysis was performed to detect the levels of ferroptosis-related markers solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) in HK-2 cell lysis .@*Results @#High concentrations of AKN significantly decreased the viability of HK-2 cells in vitro , with a half maximal inhibitory concentration (IC50) of (5 . 74 ±0. 47) mmol/L. VA at concentrations of 25 - 100 μmol/L increased the viability of AKN-stimulated HK- 2 cells (P < 0. 05) . After treatment with AKN (4 mmol/L) , the mRNA expression levels of KIM-1 and NGAL were significantly higher than those of the negative control (NC) group ( P < 0. 001) . VA (50 μmol/L) significantly reduced the mRNA expression levels of KIM-1 (P < 0. 01) and NGAL (P < 0. 05) . The intensity of DHE staining increased after 3 hours of AKN treatment , but the difference was not statistically significant. However , the intensity of DHE staining was significantly higher in the 6 - 24 hours group compared to the 0 - hour group (P < 0. 01) . Furthermore , MDA levels significantly increased , while GSH levels significantly decreased after 6 - 24 hours of AKN treatment , with statistically significant differences (P < 0. 05) . After 6 - 24 hours of AKN stimula- tion , the ferroptosis-related proteins SLC7A11 and GPX4 both significantly decreased (P < 0. 001) . Co-incubation with VA for 24 hours effectively reversed the changes in DHE staining , MDA and GSH levels , as well as the chan- ges of SLC7A11 and GPX4 protein levels (P < 0. 001) .@*Conclusion @#In this study , an in vitro renal injury model was established by stimulating HK-2 cells with high concentrations of AKN , and it was found that VA might allevi- ate the damage to renal tubular cells caused by AKN via inhibiting oxidative stress related ferroptosis .
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@#Objective To explore the effect of microRNA-138(miR-138)on injury of ischemia/reperfusion(I/R)induced human renal tubular epithelium(HK-2)cells through neutrophil gelatinase-associated lipocalin(NGAL).Methods HK-2 cells were used to construct I/R model cells,and transfected with miR-138 mimic,miR-138 inhibitor,NGAL,NGAL + miR-138 mimic plasmids,respectively.qRT-PCR determined the expression of miR-138 or NGAL mRNA in different cells to identify the transfection results.Cell counting kit-8(CCK-8)method and flow cytometry were used to detected the activities and apoptosis of cells.ELISA and western blot were used to determine the effects of miR-138 mimic or miR-138 inhibitor on levels of interleukin(IL)-6,IL-1β,tumor necrosis factor(TNF-α)and protein expression of toll like receptor 4(TLR4),nuclear factor kappa-B(NF-κB),inhibitor of NF-κB(IκBα),pho-IκBα(p-IκBα),NGAL of cells.Results miR-138 mRNA expression and cell activity were decreased,while apoptosis increased in I/R cells(P<0.01).Plasmid transfected well,miR-138 mimic increased activity while decreased apoptosis and NGAL mRNA expression of I/R cell.miR-138 inhibitor or NGAL mimic inhibited activity and increased apoptosis and NGAL mRNA expression of I/R cell.The negative effects of NGAL mimic on I/R cell were reversed by miR-138 mimic.miR-138 inhibitor increased levels of IL-6,IL-1β,TNF-α of I/R cell,and increased TLR4,NF-κB,p-IκBα,NGAL protein expression and decreased IκBα protein expression(P<0.05).While miR-138 mimic decreased levels of IL-6,IL-1β,TNF-α of I/R cell,and decreased TLR4,NF-κB,p-IκBα,NGAL protein expression and increased IκBα protein expression(P<0.05).Conclusion miR-138 reduced apoptosis and inflammation factor levels to play a protective role on I/R induced HK-2 cells may through regulating NGAL and TLR4/NF-κB pathway.
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Chronic kidney disease (CKD) has become a significant global public health problem. It is defined as chronic renal structural and functional dysfunction caused by various reasons. The prevalence of obesity and diabetes has increased dramatically in developing countries, which substantially affected the patterns of CKD observed in these regions. It’s inevitable that the disease spectrum of CKD is converting to metabolic diseases. CKD is also considered an independent risk factor for renal aging and cardiovascular disease in the elderly, which usually progresses to end-stage renal disease (ESRD). Renal interstitial fibrosis is the pathological basis of ESRD and is a microscopic manifestation of renal aging. Conversely, renal aging is a risk factor for interstitial fibrosis. Although the healthy kidney has a relatively low lipid level, CKD-associated dyslipidemia has been extensively studied. Nevertheless, less is known about the contribution of lipid disorders to the development of renal senescence and interstitial fibrosis. Recent studies have demonstrated that lipid metabolism disorders occur in the progress of renal aging and interstitial fibrosis. Renal lipids accumulate once lipid uptake and synthesis exceed the balance with lipolysis, which is mainly characterized by increased levels of triglyceride (TG) and oxidized low-density lipoprotein, and decreased levels of high-density lipoprotein. Excessive lipid accumulation in the kidney not only induces lipotoxicity and endoplasmic reticulum stress but also increases intracellular and mitochondrial reactive oxygen species, which induce stress injury and senescence in renal tubular epithelial cells. Pro-inflammatory and pro-fibrotic cytokines in a senescence-associated secretory phenotype secreted by senescent renal tubular epithelial cells further accelerate their senescence as well as the occurrence of inflammation and pericyte loss, promoting secretion of extracellular matrix (ECM) and subsequent fibrosis in the tubulointerstitial compartment. In addition, podocyte hypertrophy also leads to glomerulosclerosis. Currently, most of the studies on inhibiting or even reversing renal interstitial fibrosis are still in the experimental stage. What’s more, effective drugs to slow down renal aging have not been reported. Many inflammatory and fibrotic factors are both components of the senescence-associated secretory phenotype (SASP), nevertheless, they are not sufficient to recognize cellular senescence. Given that indicators of senescence may vary from disease to disease and organ to organ, there is a need for more sensitive and specific senescence assays. Crucial enzymes and regulatory proteins of lipid metabolic pathways are expected to be potential targets for ameliorating renal aging and interstitial fibrosis. Lipid-lowering approach might represent another therapeutic in the management of kidney injury associated with metabolic dysfunction. Thus, clarifying the molecular regulatory mechanisms of lipid metabolism in kidney is extremely important for the delay of renal aging and the treatment of interstitial fibrosis. This review outlines the effects of lipid metabolism disorders on renal aging and renal fibrosis, analyses the role of lipid metabolism disorders in the development of renal diseases, and summarizes the potential targets and strategies for the prevention of renal aging and renal fibrosis based on lipid metabolism regulation, which will provide a reference for the discovery of new targets for the treatment of renal fibrosis.
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To investigate the protective effect and the potential mechanism of leonurine(Leo) against erastin-induced ferroptosis in human renal tubular epithelial cells(HK-2 cells), an in vitro erastin-induced ferroptosis model was constructed to detect the cell viability as well as the expressions of ferroptosis-related indexes and signaling pathway-related proteins. HK-2 cells were cultured in vitro, and the effects of Leo on the viability of HK-2 cells at 10, 20, 40, 60, 80 and 100 μmol·L~(-1) were examined by CCK-8 assay to determine the safe dose range of Leo administration. A ferroptosis cell model was induced by erastin, a common ferroptosis inducer, and the appropriate concentrations were screened. CCK-8 assay was used to detect the effects of Leo(20, 40, 80 μmol·L~(-1)) and positive drug ferrostatin-1(Fer-1, 1, 2 μmol·L~(-1)) on the viability of ferroptosis model cells, and the changes of cell morphology were observed by phase contrast microscopy. Then, the optimal concentration of Leo was obtained by Western blot for nuclear factor erythroid 2-related factor 2(Nrf2) activation, and transmission electron microscope was further used to detect the characteristic microscopic morphological changes during ferroptosis. Flow cytometry was performed to detect reactive oxygen species(ROS), and the level of glutathione(GSH) was measured using a GSH assay kit. The expressions of glutathione peroxidase 4(GPX4), p62, and heme oxygenase 1(HO-1) in each group were quantified by Western blot. RESULTS:: showed that Leo had no side effects on the viability of normal HK-2 cells in the concentration range of 10-100 μmol·L~(-1). The viability of HK-2 cells decreased as the concentration of erastin increased, and 5 μmol·L~(-1) erastin significantly induced ferroptosis in the cells. Compared with the model group, Leo dose-dependently increased cell via-bility and improved cell morphology, and 80 μmol·L~(-1) Leo promoted the translocation of Nrf2 from the cytoplasm to the nucleus. Further studies revealed that Leo remarkably alleviated the characteristic microstructural damage of ferroptosis cells caused by erastin, inhibited the release of intracellular ROS, elevated GSH and GPX4, promoted the nuclear translocation of Nrf2, and significantly upregulated the expression of p62 and HO-1 proteins. In conclusion, Leo exerted a protective effect on erastin-induced ferroptosis in HK-2 cells, which might be associated with its anti-oxidative stress by activating p62/Nrf2/HO-1 signaling pathway.
Subject(s)
Humans , Ferroptosis , Reactive Oxygen Species/metabolism , NF-E2-Related Factor 2/metabolism , Sincalide/pharmacology , Signal Transduction , Epithelial Cells/metabolism , GlutathioneABSTRACT
Objective To investigate the impact of daidzein(DAI)on the pyroptosis of renal tubular epithelial cells induced by high glucose by regulating NOD-like receptor protein 3(NLRP3)/caspase-1 signal pathway.Methods HK-2 cells were divided into control group(NC group)(5.5 mmol/L D-glucose),HG group(30 mmol/L D-glucose),DAI-L,DAI-M,DAI-H groups(HK-2 cells were incubated with 30 mmol/L D-glucose and 25,50,100 μmol/L DAI,respectively),and DAI-H+LPS group(HK-2 cells were incubated with 30 mmol/L D-glucose,100 μmol/L DAI and 1 μg/mL LPS).MTT assay was applied to detect the cytotoxicity and proliferation of HK-2 cells;the apoptosis of HK-2 cells was detected by flow cytometry.The level of interleukin-1β(IL-1β)and inter-leukin-18(IL-18)in HK-2 cells was detected by ELISA.The morphology of pyroptosis cells was ob-served by scanning electron microscope.Immunofluorescence staining was applied to detect pyroptosis related pro-teins.The expression of NLRP3,cleaved casase-1 and GSDMD-N was detected by Western blot.Results In NC group,the cells were spherical with regular boundaries,while in HG group,the cells swelled and became larger with irregular boundaries;the OD value(490 nm)of HK-2 cells in HG group was obviously lower than that in NC group(P＜0.05),the apoptosis rate,IL-1β,IL-18 contents,NLRP3,cleaved casase-1,GSDMD-N protein level of HK-2 cells were obviously higher(P＜0.05);After DAI treatment,the swelling of cells was alleviated,the A value(490 nm)of HK-2 cells increased significantly(P＜0.05),the apoptosis rate of HK-2 cells,IL-1 β,IL-18 content,NLRP3,cleaved-caspase-1 and GSDMD-N protein levels were significantly decreased(P＜0.05)and the therapeutic effect of DAI was dose-dependent;LPS eliminated the beneficial effect of DAI-H on high glucose in-duced apoptosis of renal tubular epithelial cells.Conclusions DAI may alleviate pyroptosis of renal tubular epithe-lial cells induced by high glucose through inhibition of NLRP3/caspase-1 signaling pathway.
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Objective:To investigate the role of ferroptosis in calcium oxalate (Calcium Oxalate, CaOx) crystal-induced injury of human renal tubular epithelial cells (HK-2 cells).Methods:From March 2021 to September 2021, I used calcium oxalate crystal suspension to intervene HK-2 cells to build a HK-2-CaOx reaction model. Set the concentration gradient group and time gradient of calcium oxalate crystal intervention in HK-2 cells: 7 groups of calcium oxalate crystals with different concentrations (0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 mmol/L) were used to intervene HK-2 cells24 hours, the HK-2 cell protein was extracted after the intervention; HK-2 cells were intervened with calcium oxalate crystals at optimum concentration, and extract proteins at different time points (0, 3, 6, 9, 12, 24, 48 h) after intervention, the expression of intracellular ferroptosis marker protein glutathione peroxidase 4 (GPX4) was detected by Western blot. Intervention of HK-2 cells with ferroptosis inducer Erastin and ferroptosis inhibitor ethyl 3-amino-4-cyclohexylaminobenzoate (Ferrostatin-1, Fer-1) to regulate intracellular ferroptosis Level. HK-2 cells were divided into 4 groups: normal control group (NC; no intervention treatment, cultured in complete medium only); calcium oxalate crystal stimulation group (CaOx; cultured in complete medium containing 4.0 mmol/L CaOx crystals); calcium oxalate crystals + erastine treatment group (CaOx+ Erastin; cultured in complete medium containing 10.0 μmol/L erastine and 4.0 mmol/L calcium oxalate crystals); calcium oxalate crystals + Fer-1 Treatment group (CaOx+ Fer-1; cultured in complete medium containing 1.0 μmol/L Fer-1 and 4.0 mmol/L calcium oxalate crystals). After 24 hours, the expression of ferroptosis-related protein GPX4, long-chain fatty acyl-CoA synthase 4 (ACSL4) and solute carrier family 7 member 11 (SLC7A11) in HK-2 cells was analyzed by western blot and immunofluorescence techniques; the content of glutathione in HK-2 cells was detected; DCFH-DA fluorescence staining was used to observe the expression of reactive oxygen species (ROS) in HK-2 cells. The adhesion of calcium oxalate in HK-2 cells in each group was observed by light microscope, and the nuclear damage of HK-2 cells was detected by DAPI staining.Results:The expression levels of GPX4 in cells in the concentration gradient of 0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 mmol/L were5.67±1.05, 5.60±0.02, 4.99±0.94, 4.82±0.93, 4.50±0.70, 4.14± 0.53, 0.97±0.53. The expression difference of GPX4 between the 4.0 mmol/L group and the 0 mmol/L group was statistically significant ( P=0.026). 4.0 mmol/L was selected as the optimal concentration to intervene the cells. The expression levels of GPX4 in the time gradient (0, 3, 6, 9, 12, 24, 48 h) cells were 11.73±1.29, 11.68±1.32, 11.72±1.30, 10.97±1.28, 10.63±1.21, 8.79±1.10, 8.03±1.06. The expression difference of GPX4 between the 24h intervention group and the 0h intervention group was statistically significant( P=0.090), so 24h was chosen as the optimal intervention time for calcium oxalate crystals. Compared with the NC group, the CaOx+ Erastin group had higher expression of ACSL4 (9.71±0.68 vs. 3.96±0.17, P<0.01); SLC7A11 (5.76±1.31 vs. 9.18±1.54, P=0.001) and GPX4 (3.61±0.25 vs. 9.26±0.13, P<0.01) the expression level decreased. Compared with the CaOx group, the CaOx+ Fer-1 group had higher protein expression levels of GPX4 (7.52±0.23 vs. 3.61±0.25, P<0.01), SLC7A11 (7.85±1.34 vs. 5.76±1.31, P=0.012), ACSL4 (5.84 ±0.62 vs. 9.71±0.68, P=0.002) protein expression was significantly decreased. Compared with CaOx group, CaOx+ Erastin group had significantly lower protein expression of GPX4 (2.71±0.18 vs. 3.61±0.25, P=0.001), SLC7A11 (3.82±1.60 vs. 5.75±1.31, P=0.017), ACSL4(11.15±0.44 vs.9.71±0.68, P<0.01) protein expression increased. The results of glutathione determination showed that compared with the NC group, the glutathione content in the CaOx group was significantly reduced [(53.38±3.53) mmol/L vs. (81.88±4.02) mmol/L, P<0.01]. Compared with the CaOx group, the CaOx+ Fer-1 group had significantly higher glutathione content [(68.26±4.55)mmol/L vs. (53.38±3.53)mmol/L, P=0.001]. Compared with the CaOx group, the glutathione content was decreased [(38.22±2.95)mmol/L vs.(53.38±3.53)mmol/L, P=0.01]. The results of DCFH-DA fluorescence staining showed that compared with the NC group (63.36±5.17 vs. 22.72±3.73, P<0.01), the CaOx group had a significantly higher fluorescence intensity, Compared with the CaOx group (45.32±4.33 vs. 63.36±5.17, P=0.002), the fluorescence intensity of cells in the CaOx+ Fer-1 group was significantly weakened, Compared with the CaOx group (82.38±6.25 vs.63.36±5.17, P=0.002), the fluorescence intensity of the cells in the CaOx+ Erastin group was significantly increased. The results of immunofluorescence showed that the CaOx group was significantly weakened compared with the NC group (31.63±2.86 vs. 50.36±4.23, P<0.01), and the CaOx+ Fer-1 group was significantly weakened compared with the CaOx group (39.89±3.35 vs. 31.63±2.86), P=0.038), the fluorescence intensity of cells in the CaOx+ Fer-1 group was significantly enhanced, the CaOx+ Erastin group was compared with the CaOx group (23.36±3.74 vs. 31.63±2.86, P=0.022), the cell fluorescence in the CaOx+ Erastin group was The intensity is significantly reduced. DAPI staining to calculate the damage ratio of each group of nuclei: NC group (2.85%), CaOx group (11.96%), CaOx+ Fer-1 group (8.76%), CaOx+ Erastin group (16.27%). Conclusion:CaOx crystals can induce ferroptosis in HK-2 cells by increasing the level of oxidative stress in HK-2 cells.
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Objective @#To investigate the molecular mechanisms underlying the feedback regulation of single immunoglobin interleukin-1 related receptor ( SIGIRR) expression by the nuclear factor kappa-κB ( NF-κB) in human renal tubular epithelial cells ( HKC) ．@*Methods @#The pLNCX2-G418-SIGIRR overexpression vector was constructed by molecular cloning，and the SIGIRR overexpression cells and control cells were constructed by infecting HKC cells after packaging with PT67 cells．Using IL-1 β induction，Western blot verified that overexpression of SIGIRR inhibited NF-κB activation．After using NF-κB blocker and interfering with NF-κB activity ，immunofluorescence assay verified that activated NF-κB regulated SIGIRR expression． Online tools predicted the presence of NF-κB binding sites in the SIGIRR promoter region．The SIGIRR promoter sequence containing the binding site was obtained from within human genomic DNA by molecular cloning，ligated to the luciferase vector pGL3-Luc，constructed pGL3-Luc-SIGIRR , and mutated the binding site．The luciferase reporter gene assay and chromatin immunopre- cipitation technique ( ChIP) were used to jointly verify that activated NF-κB could bind to the SIGIRR promoter region to regulate SIGIRR gene expression. @*Results @#The results showed that the constructed pLNCX2-G418-SIGIRR retroviral vector was verified by enzymatic digestion and sequencing to be identical to the coding sequence of the SIGIRR gene for comparison，the recombinant and control vectors were transferred into HKC cells after viral packaging，and the HKC / SIGIRR experimental and HKC / Co control cell lines were successfully constructed at the mRNA and protein levels of SIGIRR expression differences were statistically significant (P＜0．05，P＜0. 001) ．Overexpression of SIGIRR cell groups reduced IL-1 β-induced NF-κB activation compared to control cells (P＜0. 001) . SIGIRR expression was downregulated after inhibition of NF-κB activation and interference with NF-κB expression. After extracting human genomic DNA ，the SIGIRR target promoter sequence was obtained by molecular cloning method and linked to the vector，and the pGL3-Luc-SIGIRR luciferase vector was successfully constructed and targeted to mutate the vector，which was verified to be identical to the target sequence by digestion and sequencing. The luciferase reporter gene assay and CHIP assay confirmed that NF-κB could bind to SIGIRR promoter region and regulate SIGIRR expression．@*Conclusion@#It has been verified that SIGIRR can influence the activation of NF-κB in HKC cells，and activated NF-κB can bind to the promoter region of SIGIRR and regulate the gene expression changes of SIGIRR , forming a feedback system to control the over-activation of NF-κB.
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Objective @#To investigate the expression of Ca2 + -independent phospholipase A2 β (iPLA2 β) in human renal tubular epithelial cells (HK-2) induced by high glucose(HG) ，the relationship between iPLA2 β and ferroptosis and the protective mechanism of HG treated HK-2 cells．@*Methods@#The HK-2 cells were treated with 30 mmol /L glucose，the overexpression model was constructed by transfection of iPLA2 β plasmid．Ferrostatin-1 ( Fer1) (an inhibitor of ferroptosis) and erastin (an activator of ferroptosis) were used as controls．After 36 hours of intervention，the kit detected the levels of superoxide (SOD) ，malonaldehyde(MDA) and iron in HK-2 cells．DCF immunofluorescence was used to detect intracellular reactive oxygen species ( ROS) ．The expression of ACSL4， GPX4，LPCAT3，TFR1 in HK-2 cells were measured by Western blot. @*Results @#The expression of iPLA2 β downregulated in HG-induced injury of HK-2 cells．The levels of ROS and MDA in HK-2 cells increased，while the levels of GSH and SOD decreased．The expression of ACSL4，LPCAT3 and TFR1 decreased，and the expression of GPX4 increased in HK-2 cells．However，these indexes were improved after Fer-1 intervention．iPLA2 β overexpression could reduce the injury of HK-2 cells via attenuation of KIM-1． Further research revealed that iPLA2 β overexpression inhibited oxidative stress and ferroptosis in HK-2 cells injury induced by high glucose．Meanwhile， the improvement effect of iPLA2 β on HG-induced HK-2 cells damage could be eliminated by erastin．@*Conclusion@#iPLA2 β prevents HG-induced injury of HK-2 cells via regulating ferroptosis.
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Objective:To investigate the effect of L-carnitine on calcium oxalate-induced ferroptosis in renal tubular epithelial cells (HK-2).Methods:The effects of calcium oxalate(0, 2, 4 and 8 mmol/L) on the expression of ferroptosis-related protein long chain fatty acyl-CoA synthetase 4 (ACSL4), cystine/glutamate transporter(XCT) and glutathione peroxidase 4 (GPX4) in HK-2 cells were detected by Western blotting. The experiment was then divided into four groups: ①control group, cells were cultured in normal medium for 12 hours, then continued to use normal medium; ②L-carnitine group, cells were pretreated with medium containing 5mmol/L L-carnitine for 12 hours, then changed to medium containing 5mmol/L L-carnitine; ③calcium oxalate group, cells were cultured in normal medium for 12 hours, and then replaced with medium containing 4 mmol/L calcium oxalate; ④calcium oxalate+ L-carnitine group, the cells were pretreated with medium containing 5mmol/L L-carnitine for 12 h, and then replaced with 5mmol/L L-carnitine and 4mmol/L calcium oxalate medium. After changing the culture medium for 24 hours, the cells or supernatants were collected, and the expression levels of ferroptosis-related protein quinone oxidoreductase (NQO1), ACSL4, XCT and GPX4 were detected by Western blotting. The levels of superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde were detected by corresponding kit, and the level of reactive oxygen species in cells was detected by reactive oxygen species kit.Results:The results of Western blotting showed that the expression of ACSL4 protein in 0, 2, 4, 8 mmol/L calcium oxalate was 0.37±0.16, 0.68±0.16, 0.73±0.09, 0.89±0.03 respectively. The expression of XCT protein was 1.11±0.10, 0.91±0.14, 0.83±0.09, 0.80±0.07, respectively. The expression of GPX4 protein was 1.23±0.13, 0.99±0.17, 0.81±0.05, 0.72±0.06, respectively. Compared with 0mmol/L group, the expression of ACSL4 protein increased and the expression of XCT and GPX4 decreased in 2, 4 and 8 mmol/L groups, and the difference was more significant between 4 mmol/L group and 0 mmol/L group. So 4 mmol/L was taken as the optimal concentration for follow-up experiment. The levels of NQO1 in control group, L-carnitine group, calcium oxalate group and calcium oxalate+ L-carnitine group were (0.36±0.06, 0.54±0.05, 0.76±0.07, 0.90±0.03) respectively. There was significant difference between L-carnitine group and control group ( P<0.05). There was significant difference between calcium oxalate group and control group ( P<0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of ACSL4 in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (0.66±0.10, 0.58±0.08, 0.99±0.03, 0.77±0.09) respectively. There was no significant difference between L-carnitine group and control group(P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of XCT in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (0.93±0.08, 0.85±0.07, 0.76±0.06, 0.99±0.05). There was no significant difference between L-carnitine group and control group (P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of GPX4 in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (1.10±0.09, 1.09±0.09, 0.85±0.03, 0.99±0.02) respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of LDH in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine were (100.00±5.37)%, (99.50±6.38)%, (153.77±6.06)% and (132.50±5.58)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The SOD levels in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (100.00±5.79)%, (105.80±3.26)%, (44.74±7.60)% and (85.01±5.15)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of GSH in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (100.00±4.73)%, (107.10±5.48)%, (53.49±3.98)% and (85.18±5.48)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.01). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.01). The levels of MDA in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (100.00±2.36)%, (98.00±11.10)%, (129.11±2.59)% and (113.35±5.79)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.01). The fluorescence intensity of ferrous ion in control group, calcium oxalate group and calcium oxalate + L-carnitine group was (39.77±0.68) AU, (68.40±3.14) AU and (48.60±4.30) AU, respectively. There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The fluorescence intensity of reactive oxygen species in control group, calcium oxalate group and calcium oxalate + L-carnitine group was (63.98±9.41) AU, (145.41±8.39) AU and (85.37±4.51) AU, respectively. There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.01). Transmission electron microscopy results showed that mitochondria were wrinkled, cristae were broken or disappeared in the calcium oxalate group compared to the control group, and a double-layer membrane structure was evident. DAPI staining showed that compared with the control group, some of the nuclei in the calcium oxalate group were significantly more damaged, while compared with the calcium oxalate group, the nuclei in the calcium oxalate + L-carnitine were significantly less damaged. The results of crystal adhesion test showed that compared with the control group, calcium oxalate crystals in the calcium oxalate group adhered to the cells in black-like particles and formed clusters. Compared with the calcium oxalate group, the calcium oxalate + L-carnitine showed less black particles adhering to the cells. Conclusions:L-carnitine may reduce the effects of oxidative stress and ferroptosis induced by calcium oxalate, thus reducing cell damage and crystal adhesion.
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Aim To investigate the effect of aloin, an aloe extract,on fibrosis of renal tubular epithelial cells (HK-2) induced by TGF-β and the underlying molecular mechanism. Methods The experiment included a control group,TGF-β induced group,TGF-β + Aloin 50 or 100 μmol • L
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OBJECTIVE To study the effects of ginsenoside Rb 1（G-Rb1）on epithelial-mesenchymal transition （EMT）of renal tubular epithelial cells and its potential mechanism. METHODS The growth factor β1（TGF-β1）10 ng/mL was used to induce EMT of human renal tubular epithelial cells HK- 2. The morphological changes of HK- 2 cells were observed after treated with 10， 20，30 μmol/L G-Rb1 for 48 h. The transcriptional activities of biovector SBE in human embryonic kidney cell HEK 293 were determined after 24 h treatment with 1.0，2.5，5.0，10，20，30 μmol/L G-Rb1. Effects of above concentration of G-Rb 1 on the viability of HK- 2 cells were determined after 24 h of treatment. mRNA expressions of α-smooth muscle actin （α-SMA），collagen Ⅰ （COL-Ⅰ）and fibronectin （FN）in HK- 2 cells were detected after treated with 10，20，30 μmol/L G-Rb1 for 24 h. The expressions of α-SMA，Smad3，p-Smad3，COL-Ⅰ，FN and E-cadherin were detected after treated with 10，20，30 μmol/L G-Rb1 for 24 h. RESULTS G-Rb1 of 10-30 μmol/L significantly inhibited TGF-β1-induced EMT in HK- 2 cells and the increase of transcriptional activities of biovector SBE induced by TGF-β1（P＜0.05），but had no effects on relative activities of HK- 2 cells（P＞0.05）. The protein and mRNA expressions of α-SMA，COL-Ⅰ and FN ， the protein expressions of Smad 3 and p-Smad 3 were significantly up-regulated induced by TGF-β1（P＜0.05），while the protein expression of E-cadherin was significantly down- regulated（P＜0.05）；G-Rb1 could effectively reverse aboveprotein or mRNA expressions. CONCLUSIONS G-Rb1 can protect renal tubular epithelial cells from EMT induced byxiezhishen TGF-β1 to a certain extent ，which may be related to inhibiting the activation of TGF-β1/Smad3 signaling pathway.
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Objective:To explore the role of the fat mass and obesity-associated protein (FTO) in human renal tubular epithelial cells (HK-2) suffering ischemia-reperfusion injury (IRI).Methods:The in vitro IRI mo-del was established in HK-2 cells by induction with antimycin A, A23187 and 2-deoxy-D-glucose.The cells were divided into control group and ischemia-reperfusion group (I/R group). The mRNA and protein expressions of FTO, B-cell lymphoma / leukemia 2(Bcl-2)-associated X(Bax), Bcl-2 and cleaved cysteinyl aspartate specific proteinase(cleaved Caspase-3) in HK-2 cells before and after IRI were detected by real-time fluorescent quantitative PCR(qPCR) and Western blot, respectively.Cell apoptosis was measured using flow cytometry.The level ofe N 6-methy-ladenosine (m 6A) RNA was detected by colorimetry. Results:(1) The mRNA expressions of FTO (0.15±0.05 vs.1.00±0.23) and Bcl-2 (0.14±0.07 vs.1.02±0.25) in I/R group were significantly lower than those in control group; While those of Bax (3.10±0.35 vs.1.00±0.13) and cleaved Caspase-3 (4.21±0.56 vs.1.00±0.09) were significantly higher ( t=6.28, 5.84, -9.83, and -9.84, respectively, all P<0.01). (2) The protein expressions of FTO (0.69±0.14 vs.1.37±0.02) and Bcl-2 (0.50±0.12 vs.1.25±0.21) were significantly lower in I/R group than those of control group; While those of Bax (1.04±0.08 vs.0.57±0.06) and cleaved Caspase-3 (0.99±0.05 vs.0.36±0.07) were significantly higher ( t=8.10, 5.49, -8.22, and -12.09, respectively, all P<0.05). (3) Compared with the control group, the apoptosis rate of HK-2 cells in I/R group was significantly higher [(61.70±1.01)% vs.(0.16±0.10)%, t=63.80, P<0.01]. (4) Compared with the control group, the percentage of m 6A modification level in total RNA in I/R group was significantly higher [(3.13±0.21)% vs.(1.10±0.26)%, t=-10.61, P<0.01]. Conclusions:FTO-mediated RNA m 6A modification may affect renal IRI by regulating the apoptosis of HK-2 cells.
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ObjectiveTo investigate the role and mechanism of Panax notoginseng saponins （PNS） in inhibiting transforming growth factor-β1 （TGF-β1）-induced renal tubular epithelial cell injury. MethodNRK-52E renal tubular epithelial cells were cultured and divided into control group， TGF-β1 group，TGF-β1+12.5 mg·L-1 PNS group，TGF-β1+25 mg·L-1 PNS group and TGF-β1+50 mg·L-1 PNS group. After 48 hours of PNS intervention， the cells and the supernatant were collected， and cell morphology was observed by inverted microscope. Western blot was used to detect the expression of epithelial-mesenchymal transition （EMT）-related proteins and autophagy-related proteins. Flow liquid chromatography for multiple protein quantification and flow cytometry were employed to determine the content of inflammatory factors and apoptosis rate， respectively. ResultCompared with the conditions in the control group， after TGF-β1 induction， the cells showed a spindle-shaped change and the expression of E-cadherin was down-regulated （P<0.05）， while the expression of α-smooth muscle actin （α-SMA） was up-regulated （P<0.05）. After PNS treatment， most of the cells tended to be normal and reversed the occurrence of EMT. In addition， compared with the conditions in the control group， the level of TNF-α was increased while that of IL-10 was decreased， with elevated apoptosis rate （P<0.05） in the TGF-β1 group. After PNS treatment， the level of TNF-α was lowered while that of IL-10 was boosted with the increase of the dose， with reduced apoptosis rate （P<0.05）. Moreover， after TGF-β1 induction， the expression of autophagy-related proteins Beclin 1 and LC3Ⅱ/Ⅰ in renal tubular epithelial cells were up-regulated， while PNS inhibited their expression（P<0.05，P<0.01）. ConclusionPNS had a protective effect on TGF-β1-induced renal tubular epithelial cells， and the mechanism might be that it reduced inflammation and apoptosis by inhibiting autophagy， thus alleviating TGF-β1-induced injury.
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Aim To study the protective effect of Mon¬golian medicine rhubarb-3 decoction on renal function of CRF model rats and explore its mechanism.Meth¬ods SD rats were randomly divided into normal group and model group.The model group was established by adenine gavage.After successful modeling, they were randomly divided into model, positive, rhubarb-3 de¬coction low, medium and high dose groups, which were administered at intervals for 12 weeks.The bio¬chemical detection of CSF, BUN and Scr was per¬formed.The pathological changes of renal tissues were observed by HE and Masson staining.The expression of PCNA and a-SMA in renal tissues was detected by immunohistochemistry.The mRNA expressions of CK18, Vimentin, TGF-pi and FN in kidney tissues were detected by RT-PCR.The expressions of a-SMA, E-cadherin, PCNA, Smad2, Smad3 protein in kidney tissue were detected by Western blot.TGF-pi induced abnormal proliferation of I IMC and interstitial transfor¬ mation of HK-2, and at the same time, it was treated with serum containing rhubarb-3 decoction.The prolif¬eration of HMC was detected by CCK-8.Interstitial transformation of HK-2 was detected by RT-PCR.Re- suits Compared with the model group, BUN and Scr in CRF rats decreased, and the expression of fibrosis- related proteins and genes in renal tissue decreased af¬ter rhubarb-3 decoction treatment.The serum contai¬ning rhubarb-3 decoction significantly inhibited HMC proliferation and HK-2 interstitial transformation.Con¬clusions Rhubarb-3 Decoction can improve renal function in CRF model rats, and its mechanism may be related to inhibiting HMC proliferation and HK-2 inter¬stitial transformation and probably regulating TGF-f}/ Smad signaling pathway.
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OBJECTIVE：To study the effects of sulforaphane on the prolifera tion and apoptosis of human renal tubular epithelial cells HK- 2 induced by high glucose ，and to investigate its mechanism primarily. METHODS ：HK-2 cells were divided into normal group ，high glucose group ，irbesartan group （positive control ，1 μmol/L），sulforaphane low ，medium and high concentration groups （10，20，40 μmol/L）. The cells in normal group were cultured in DMEM medium for 96 hours. T he cells in other groups were cultured in high glucose DMEM medium （containing 40 mmol/L glucose ）for 48 hours. After inducing cell injury，the cells were added with corresponding drugs for 48 hours. Survival rate and apoptotic rate of cells were detected. mRNA expression of cyclin D 1，caspase-3，Bcl-2 and Bax as well as protein expression of p-mTOR ，p-AMPK，p-Akt and p-PI 3K were also determined. In addition ，HK-2 cells were divided into normal group ，high glucose group ，sulforaphane high concentration group（40 μmol/L），acardicin group （AMPK agonist ，1 mmol/L），sulforaphane high concentration+compound C group （sulforaphane 40 μmol/L+AMPK inhibitor compound C 40 μmol/L），perifoxine group （Akt inhibitor ，19.95 μmol/L）、sulforaphane high concentration+SC 79 group（sulforaphane 40 μmol/L+Akt agonist SC79 4 μmol/L）. After cultured with the same method ， protein expression of p-mTOR ，p-AMPK，p-Akt and p-PI 3K were detected in HK- 2 cells. RESULTS ：Compared with normal group，survival rate of HK- 2 cells，mRNA expression of cyclin D 1 and Bcl- 2 as well as protein expression of p-AMPK were decreased significantly in high glucose group （P＜0.05）；apoptotic rate ，mRNA expression of caspase- 3 and Bax ，protein expression of p-mTOR ，p-Akt and p-PI 3K in HK- 2 cells were increased significantly （P＜0.05）. Compared with high glucose group，above indexes of sulforaphane low ，medium and high concentration groups ，irbesartan group were all improved significantly （P＜0.05）；the improvement of above indexes in sulforaphane medium and high concentration groups were significantly better those of sulforaphane low concentration group （P＜0.05）. There was no significant difference in above indexes between sulforaphane high concentration group and irbesartan group （P＞0.05）. Compared with sulforaphane high concentration group，there were no significant difference in the protein expression of p-AMPK ，p-mTOR in acardicin group and p-mTOR ，p-Akt and p-PI 3K in perifoxine group （P＞0.05）；the protein expression of p-AMPK in sulforaphane high concentration+compound C group was decreased significantly （P＜0.05），while the protein expression of p-mTOR was increased significantly （P＜0.05）；the protein expression of p-mTOR 、p-Akt、p-PI3K in sulforaphane high concentration+SC 79 group were increased significantly （P＜ 0.05）. CONCLUSIONS ：Sulforaphane can promote the proliferation of renal tubular epithelial cells and inhibit its apoptosis ；its mechanism may be associated with up-regulating the expression of p-AMPK and down-regulating the expression of p-mTOR ，p-Akt and p-PI 3K.
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Objective:To investigate the effect of hydroxysafflor yellow A(HSYA) preconditioning group on apoptosis induced by cold hypoxia/reoxygenation (cold H/R) injury in human renal tubular epithelial cells (HK2 cells).Methods:After digestion and passage, HK2 cell lines were divided into Sham group (control group), cold hypoxia and reoxygenation group (cold H/R group, cells cold hypoxia for 4 h, reoxygenation for 4 h), and HSYA preconditioning group (each HSYA subgroup was given different doses of HSYA 0.5 h before hypoxia, and the other operations were the same as the cold H/R group). The cell survival rate was measured by CCK-8 method.The expression of Bcl-2, Bax and Caspase-3 proteins in HK-2 cells were detected by immunocytochemistry and Western blotting.Results:(1) Compared with cold H/R group, different doses of HSYA could improve cell survival rate in different degrees, but only HSYA25 μmol/L group had the most significant effect (74.000±5.500 vs.59.000±3.800, P<0.05). (2) Immunocytochemistry semi-quantitative score: Compared with cold H/R group, the expression of Bax and Caspase-3 in HK2 cells of HSYA25 μmol/L group was significantly decreased(0(0, 1) vs. 8(6, 8), Z=2.041, P<0.05 and (3.400±0.548) vs.(7.800±1.095), t=11.000, P<0.01). The expression of Bcl-2 protein was increased significantly ((6.800±1.095) vs.(1.400±0.548), t=10.590, P<0.01). The ratio of Bcl-2/Bax increased significantly.(3)Western blot was used to detect protein: Compared with the cold H/R group, the protein levels of Bax, Cleaved-Caspase-3 and Pro-caspase-3 of HK2 cells in the HSYA25 μmol/L group were significantly decreased ((0.707±0.012) vs.(0.968±0.117), (0.480±0.009)vs.(0.735±0.005), (0.992±0.008)vs.(1.197±0.005), all P<0.01). The expression of Bcl-2 protein was significantly increased, and the ratio of Bcl-2/Bax was significantly increased ((0.410±0.009) vs.(0.273±0.008), (0.582±0.016) vs (0.282±0.080), all P<0.01). The experimental results were consistent with the immunocytochemistry. Conclusion:HSYA can effectively reduce the damage of HK2 cells after cold hypoxia and reoxygenation.
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Fucoidan(FPS) is an effective component of the Chinese patent medicine named Haikun Shenxi, which treats schronic renal failure in clinics, and has the potential anti-aging effects. However, it is still unclear whether FPS can improve renal aging, especially the molecular mechanism of its anti-aging. The human proximal renal tubular epithelial cells(HK-2) in vitro were divided into normal group(N), D-gal model group(D), low dose of FPS group(L-FPS), high dose of FPS group(H-FPS) and vitamin E group(VE), and treated by the different measures, respectively. More specifically, the HK-2 cells in each group were separately treated by 1 mL of 1% fetal bovine serum(FBS) or D-galactose(D-gal, 75 mmol·L~(-1)) or D-gal(75 mmol·L~(-1))+FPS(25 μg·mL~(-1)) or D-gal(75 mmol·L~(-1))+FPS(50 μg·mL~(-1)) or D-gal(75 mmol·L~(-1))+VE(50 μg·mL~(-1)). After the treatment for 24 h, firstly, the effects of D-gal on senescence-associated β-galactosidase(SA-β-gal) staining characteristics and klotho, P53 and P21 protein expression le-vels, as well as adenosine monophosphate activated protein kinase(AMPK)-uncoordinated 51-like kinase 1(ULK1) signaling pathway activation in the HK-2 cells were detected, respectively. Secondly, the effects of FPS and VE on SA-β-gal staining characteristics and klotho, P53 and P21 protein expression levels in the HK-2 cells exposed to D-gal were investigated, respectively. Finally, the effects of FPS and VE on microtubule-associated protein 1 light chain 3(LC3) protein expression level and AMPK-ULK1 signaling pathway activation in the HK-2 cells exposed to D-gal were examined severally. The results indicated that, for the HK-2 cells, the dose of 75 mmol·L~(-1) D-gal could induce the changes of SA-β-gal staining characteristics and klotho, P53 and P21 protein expression levels. That is causing cells aging. FPS and VE could both ameliorate the changes of SA-β-gal staining characteristics and klotho, P53 and P21 protein expression levels in the HK-2 cells exposed to D-gal. That is anti-cells aging, here, the functions of FPS and VE are similar. D-gal could not only induce cell aging but also increase LC3Ⅱ, phosphorylated-AMPK(p-AMPK) and phosphorylated-ULK1(p-ULK1) protein expressions, and activate autophagy-related AMPK-ULK1 signaling pathway. FPS and VE could both improve the changes of LC3Ⅱ, p-AMPK and p-ULK1 protein expression levels in the HK-2 cells exposed to D-gal. That is inhibiting autophagy-related AMPK-ULK1 signaling pathway activation. On the whole, for the human proximal renal tubular epithelial cells aging models induced by D-gal, FPS similar to VE, can ameliorate renal cells aging by possibly inhibiting autophagy-related AMPK-ULK1 signaling pathway activation. This finding provides the preliminary pharmacologic evidences for FPS protecting against renal aging.
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Humans , Aging , Autophagy , Epithelial Cells , Polysaccharides , Signal TransductionABSTRACT
BACKGROUND: Canine kidney injury is characterized by the apoptosis and necrosis of renal tubular epithelial cells. Recent developments in mesenchymal stem cells and their exosomes research have shown great promise for the treatment of kidney injury in humans, rats and mice, but little research has been done on dogs. OBJECTIVE: To investigate the effects of canine adipose-derived mesenchymal stem cells and their exosomes on canine renal tubular epithelial cell injury induced by gentamicin in vitro. METHODS: Canine renal tubular epithelial cells were treated by 5 mmol/L gentamicin sulfate. Subsequently, canine adipose-derived mesenchymal stem cells and their conditional medium and exosomes were co-cultured with damaged canine renal tubular epithelial cells respectively. After 24 and 48 hours, the cell proliferation activity of each group was measured by cell counting kit-8 method, and the apoptosis rate of each group was detected by flow cytometry. Finally, Q-PCR was used to further reveal the effects of canine adipose-derived mesenchymal stem cell exosomes on PCNA, Bcl-2 and Bax genes in these cells. RESULTS AND CONCLUSION: Canine adipose-derived mesenchymal stem cells, their conditioned media, and exosomes could significantly promote proliferation and reduce apoptosis in damaged canine renal tubular epithelial cells (P < 0.05). Among them, canine adipose-derived mesenchymal stem cell exosomes worked best, which could significantly increase the expression of PCNA and Bcl-2 genes in damaged canine renal tubular epithelial cells (P < 0.05). These results suggest that canine adipose-derived mesenchymal stem cells can repair the canine renal tubular epithelial cell damage induced by gentamicin through their exosomes.
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Aim To investigate the effect of CMHX008, a novel peroxisome proliferator-activated receptor γ (PPARγ) partial agonist, on fibrogenic pathways and its potential mechanism in renal tubular epithelial HK-2 cells in comparison with rosiglitazone. Methods HK-2 cells were cultured with 30 mmol . L 1 D-(+)-glucose, and then treated with rosiglitazone and CMHX008. Cell counting kit-8(CCK-8) was used to detect cell viability; immunoblotting was used to detect protein expression fibrogenic markers and p-PPARγ(ser273) in HK-2 cells; quantitative real time PCR (qRT-PCR) was used to detect fibrosis-related mRNA levels; wound healing assay and transwell assay were used to detect the migration and invasion ability of HK-2 cells. Results CCK-8 analysis showed that 3 (imol . L 1 rosiglitazone and 3 μmol . L 1 CMHX008 had no obvious cytotoxicity to HK-2 cells; immunoblotting revealed that rosiglitazone and CMHX008 could reverse the up-regulation of p-PPARγ(ser273), reduce transforming growth factor β1 (TGF-β1) and α-smooth muscle actin(α-SMA) protein levels, and up-regulate E-cadherin protein expression levels in HK-2 cells in high glucose conditions; wound healing assay and transwell assay showed that rosiglitazone and CMHX008 could inhibit the increase of migration and invasion ability of hyperglycemia-cultured HK-2 cells. Conclusions The novel PPAR7 agonist CMHX008 can improve hyperglycemia-induced renal tubular fibrosis, which may be possibly related to the inhibition of p-PPARγ(Ser273) phosphorylation.