TNF-α induces PIP3-mediated necroptosis in MLO-Y4 cells

AIM: To explore whether tumor necrosis factor-α (TNF-α) induces necroptosis in murine long bone osteocyte-like cell line MLO-Y4 and the possible mechanism. METHODS: The MLO-Y4 cells were divided into control group, TNF-α group, TNF-α+necrostatin-1 (Nec-1) group, TNF-α+Z-VAD group and TNF-α+receptor-interacting protein 3 (RIP3)-siRNA group. The death rate of MLO-Y4 cells was assessed by flow cytometry with Annexin V-FITC/PI staining. The morphological features of the cells were observed under transmission electron microscope (TEM). The protein levels of RIP1, RIP3 and cleaved caspase-3 were determined by Western blot. Finally, the numbers of total cells and RIP1-RIP3-positive cells were observed under laser scanning confocal microscope. The production of reactive oxygen species (ROS) in the cells was measured by DCFH-DA staining. RESULTS: Compared with control group, the apoptotic or necroptotic rate of the cells induced by TNF-α was increased significantly (P<0.01). The increased apoptotic or necroptotic rate was dramatically reduced by treating with Nec-1, Z-VAD or RIP3-siRNA transfection (P<0.01). In TNF-α group and TNF-α+Z-VAD group, a lot of MLO-Y4 cells with typical necroptotic morphological features were observed under TEM. However, obvious necroptotic cells were not found in Nec-1 or RIP3-siRNA treatment group. The protein level of RIP1 in the cells treated with Nec-1 was sharply lower than that in TNF-α group (P<0.01). However, Z-VAD did not reduce the elevated levels of RIP1 and RIP3. RIP3-siRNA effectively down-regulated the protein level of RIP3 compared with TNF-α group (P<0.01). Nec-1 effectively down-regulated the protein levels of RIP1 colocalized with RIP3 compared with TNF-α group (P<0.01). However, Z-VAD did not reduce the levels of RIP1 colocalized with RIP3. Nec-1, Z-VAD and RIP3 siRNA significantly decreased the ROS levels (P<0.01). CONCLUSION: TNF-α induces the necroptosis of MLO-Y4 cells. RIP3 play vital roles in the cell necroptotic signal pathway. ROS may be the executor of necroptosis of MLO-Y4 cells.     

Activation of Rip1 promotes necroptosis in LNCaP-AI cells via inhibiting SHARPIN

AIM: To explore the role of SHARPIN in regulation of Rip1 in castration-resistant prostate cancer LNCaP-AI cells. METHODS: The LNCaP-AI cells were treated with TNF-α+Z-VAD(an inhibitor of pan-caspase) to activate necroptosis, which were compared to the cells treated with TNF-α+Z-VAD+Nec-1(an inhibitor of Rip1). A blank group and a TNF-α-treated group were set up as controls. The cell viability in each group was measured by MTS assay. In addition, SHARPIN was knocked down by siRNA, and the inhibitory efficiency was evaluated by RT-qPCR. The expression of Rip1 at mRNA and protein levels after knocking down SHARPIN was determined by RT-qPCR and Western blot to explore the underlying mechanism of regulatory network of necroptosis in prostate cancer. RESULTS: Compared with blank control group and TNF-α-treated group, the viability of LNCaP-AI cells treated with TNF-α+Z-VAD decreased by 28%(P<0.05). After treated with TNF-α+Z-VAD+Nec-1, the LNCaP-AI cells showed no significant difference in the viability compared with blank control and TNF-α-treated groups. Taken together, necroptosis may be an important way of cell death in LNCaP-AI cells. Besides, the expression of Rip1 at protein level was up-regulated following the inhibition of SHARPIN using siRNA, indicating that down-regulation of SHARPIN enhanced necroptosis via activating Rip1 in LNCaP-AI cells. CONCLUSION: Necroptosis is an important way of cell death.Inhibition of oncogenic factor SHARPIN enhances necroptosis via activating Rip1 in LNCaP-AI cells.     

Effect of autophagy on necroptosis of renal tubular epithelial cells in subtotal nephrectomy rats

AIM: To explore whether autophagy is involved in the excessive death of renal tubular epithelial cells in subtotal nephrectomy(SNx) rats and the relationship between autophagy and necroptosis in the kidney of SNx rats. METHODS: Male Sprague-Dawley rats were randomly assigned to control group(n=6) and SNx group(n=42). The rats in SNx group were subjected to SNx. Sham surgery was performed in the rats in control group. The rats in SNx group were divided into subgroups at 0, 4, 8 and 12 weeks(n=6) and the other rats in SNx group were divided into SNx+vehicle group, SNx+necrostatin-1(Nec-1) group and SNx+3-methyladenine(3-MA) group. The expression of RIP1, RIP3, LC3 and beclin-1 at mRNA and protein levels was measured at 0, 4, 8 and 12 weeks by qPCR and immunohistochemistry. The effects of Nec-1 or 3-MA on the protein expression of LC3-I, LC3-II and beclin-1, and production of reactive oxygen species(ROS) in the rat kidney were determined by Western blot and DCFH-DA staining. The death of renal tubular epithelial cells in the SNx rats was observed by TUNEL staining and electron microscopy. Finally, the effects of Nec-1 and 3-MA on blood urea nitrogen(BUN), serum creatinine(SCr) and the pathological changes of the renal tissues were analyzed. RESULTS: The highest mRNA and protein levels of RIP1, RIP3, LC3 and beclin-1 appeared at the 8th week after SNx(P<0.01). Compared with the rats in SNx+vehicle group, the protein over-expression of LC3-II/I and beclin-1, renal tubular epithelial cells with typical morphological features of necroptotic cell death and TUNEL-positive renal tubular cells were decreased in the SNx rats treated with Nec-1 and 3-MA(P<0.01), but 3-MA did not reduce the increased concentration of ROS. In addition, treatment with Nec-1 and 3-MA obviously reduced BUN, SCr(P<0.05), glomerulosclerosis index and tubulointerstitial injury score(P<0.01). CONCLUSION: Autophagy participates in the excessive death of renal tubular epithelial cells in SNx rats. Inhibition of autograph prevents necroptotic cell death of renal tubular cells, and alleviates chronic renal injury in SNx rats.     

Effect of netrin-1 on high glucose induced injury of renal tubular epithe-lial cells

AIM:To study the effect of netrin-1 on the damage of renal tubular epithelial cells induced by high glucose. METHODS:Human renal tubular epithelial HK-2 cells were treated with high glucose. Real-time PCR and Western blot were used to detect the expression level of netrin-1 in the cells. HK-2 cells were infected with netrin-1-over-expressing lentivirus, and the effect of netrin-1 over-expression on the HK-2 cells treated with high glucose was observed. The apoptosis rate was analyzed by flow cytometry. The protein level of cleaved caspase-3 was determined by Western blot. lactate dehydrogenase (LDH) activity in the culture medium was measured by 2,4-binitrobenzene hydrazine method. The content of malondialdehyde (MDA) in the culture medium was detected by thiobarbituric acid method. The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the culture medium were measured by ELISA. RESULTS:The expression of netrin-1 at mRNA and protein levels in the HK-2 cells after high glucose treatment was significantly lower than that in the control cells (P<0.05). Infection with netrin-1-over-expressing lentivirus up-regulated the expression of netrin-1 in the HK-2 cells treated with high glucose. High glucose promoted the secretion of IL-1β and TNF-α, decreased the levels of LDH and MDA in the cell culture supernatant, and induced apoptosis and activation of caspase-3 in renal tubular epithelial cells (P<0.05). After the HK-2 cells with up-regulation of netrin-1 were induced by high glucose, the IL-1β and TNF-α secretion, the levels of LDH and MDA in the culture medium, the apoptosis, and the level of activated caspase-3 protein in the cells were all decreased, as compared with the control cells (P<0.05). CONCLUSION:Up-regulation of netrin-1 expression attenuates oxidative damage and inflammatory injury, and reduces apoptosis induced by high glucose in renal tubular epithelial cells.     

Effect of VDUP-1 on apoptosis of renal tubular epithelial cells induced by high glucose

AIM: To investigate the effect of vitamin D3 up-regulated protein 1 (VDUP-1) on apoptosis of renal tubular epithelial cells induced by high glucose and its mechanism. METHODS: Human renal proximal tubular epithelial cell line HK-2 was treated with high glucose. The mRNA and protein levels of VDUP-1 in HK-2 cells were detected by real-time PCR and Western blot. HK-2 cells were transfected with VDUP-1 small interfering RNA (siRNA). Real-time PCR and Western blot were used to detect the inhibitory effect. The HK-2 cells were treated with high glucose, and the change of VDUP-1 expression was detected. The apoptosis was analyzed by flow cytometry. The activities of caspase-3 and caspase-9 in the cells were measured. The tumor necrosis factor-α (TNF-α) content in the culture supernatant was examined by ELISA. The key proteins of Sonic hedgehog (Shh) signaling pathway, Patched 1 (Ptch1), Smoothened (Smo), zinc finger protein Gli2 and Shh, were determined by Western blot. The HK-2 cells were treated with exogenous Shh, and the levels of Ptch1, Smo and Gli2 were detected by Western blot. After the HK-2 cells with VDUP-1 silencing were treated with exogenous Shh and high glucose, the apoptosis was analyzed by flow cytometry, the activities of caspase-3 and caspase-9 in the cells were examined, and the TNF-α content in culture supernatant was measured by ELISA. RESULTS: High levels of VDUP-1 mRNA and protein were observed in the HK-2 cells treated with high glucose. The mRNA and protein levels of VDUP-1 were decreased in the HK-2 cells transfected with VDUP-1 siRNA(P<0.05). Compared with the normally cultured cells, the apoptotic rate of HK-2 cells was increased after high glucose treatment, and the activities of caspase-3 and caspase-9 and the content of TNF-α were also significantly increased (P<0.05). After down-regulation of VDUP-1 expression by siRNA transfection, the apoptotic rate of HK-2 cells decreased after high glucose treatment, and the activities of caspase-3 and caspase-9, and the content of TNF-α were also significantly decreased (P<0.05). The protein levels of Ptch1, Smo, Gli2 and Shh were decreased after high glucose culture, while down-regulation of VDUP-1 partly antagonized the effect of high glucose on the expression of Ptch1, Smo, Gli2 and Shh in the HK-2 cells. Exogenous Shh promoted the expression of Ptch1, Smo and Gli2, and inhibited the apoptosis of the HK-2 cells induced by high glucose. Exogenous Shh and down-regulation of VDUP-1 synergistically inhibited high glucose-induced apoptosis of the HK-2 cells. CONCLUSION: Down-regulation of VDUP-1 expression inhibits high glucose-induced apoptosis and release of TNF-α in renal tubular epithelial cells by activating Shh signaling pathway.     

Angiotensin-(1-7) protects H9c2 cardiac cells against high glucose-induced injury and inflammation by inhibiting the interaction between TLR4 activation and necroptosis

AIM: To investigate whether angiotensin-(1-7)[Ang-(1-7)] protects H9c2 cardiac cells against high glucose (HG)-induced injury and inflammation by inhibiting the interaction between Toll-like receptor 4 (TLR4) activation and necroptosis. METHODS: The expression levels of receptor-interacting protein 3 (RIP3; an indicator of necroptosis) and TLR4 were determined by Western blot. Cell viability was measured by CCK-8 assay. The activity of lactate dehydrogenase (LDH) in the culture medium was measured with a commercial kit. The releases of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were measured by ELISA. The intracellular level of reactive oxygen species (ROS) was analyzed by 2', 7'-dichlorfluorescein-diacetate (DCFH-DA) stating followed by photofluorography. Mitochondrial membrane potential (MMP) was examined by rhodamine 123 staining followed by photofluorography. RESULTS: After the H9c2 cardiac cells were treated with HG (35 mmol/L glucose) for 24 h, the expression of RIP3 was obviously increased. Co-treatment of the cells with 30 μmol/L TAK-242 (an inhibitor of TLR4) attenuated the up-regulation of RIP3 induced by HG. Furthermore, the expression of TLR4 was significantly increased after the cells were exposed to HG for 24 h, and co-treatment of the cells with 100 μmol/L necrostatin-1 (Nec-1; a specific inhibitor of necroptosis) and HG for 24 h attenuated the up-regulation of TLR4 expression induced by HG. Moreover, 1 μmol/L Ang-(1-7) simultaneously blocked the up-regulation of the RIP3 and TLR4 induced by HG. On the other hand, co-treatment of the cells with 1 μmol/L Ang-(1-7), 30 μmol/L TAK-242 or 100 μmol/L Nec-1 and HG for 24 h attenuated HG-induced injuries and inflammatory response, leading to the increase in the cell viability, and the decreases in the activity of LDH, ROS generation, MMP loss as well as the releases of IL-1β and TNF-α. CONCLUSION: Ang-(1-7) protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting the interaction between TLR4 activation and necroptosis.     

RIP1-mediated necroptosis regulates inflammatory response of renal tubular epithelial cells via NF-κB signaling pathway

AIM To investigate the effect of receptor-interacting protein 1 (RIP1)-mediated necroptosis on human kidney proximal tubular cell inflammation and its related mechanisms. METHODS Human kidney proximal tubular HK-2 cells were cultured in vitro, and stimulated with tumot tumor necrosis factor-α (TNF-α) and Z-VAD-FMK for 24 h. Lactate dehydrogenase (LDH) cytotoxicity assay was used to detect the percentage of necrosis. Western blot was used to detect the protein expression of RIP1, IKK-α and NF-κB p65. The protein levels of interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were determined by Western blot and ELISA. Real-time PCR was used to detect the mRNA expression level of NF-κB p65. Furthermore, the RIP1 inhibitor necrostatin-1 (Nec-1) and the NF-κB specific inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) were used, and the above indicators were also detected. RESULTS Compared with control group, the protein level of RIP1 was increased in TNF-α combined with Z-VAD-FMK stimulation group (T/Z group). The protein levels of IKK-α and NF-κB p65 were obviously increased, and the release of LDH was increased (P<0.01). Western blot and ELISA showed that the expression levels of IL-1β and MCP-1 were significantly increased (P<0.01). Real-time PCR showed that the mRNA expression level of NF-κB p65 was also obviously increased. After Nec-1 or PDTC stimulation (T/Z+N group or T/Z+P group), the release of LDH, and the expression levels of inflammation-related indicators IL-1β and MCP-1 were significantly decreased. The protein expression levels of IL-1β and MCP-1 were further reduced after treatment with the above 2 stimulati (T/Z+P/N group). CONCLUSION Under T/Z condition, RIP1-mediated necroptosis plays an important role in renal tubular inflammatory response, which may be partly achieved by regulating the activation of NF-κB signaling pathway.     

Protective effect of astragaloside Ⅳ on acute tubular injury induced by aristolochic acid I

AIM: To study the effect of astragaloside Ⅳ (AS Ⅳ) on acute aristolochic acid nephropathy (AAN). METHODS: MTT assay was used to observe the viability of human proximal tubule epithelial cell line HK-2 in vitro. In in vivoexperiments, Kunming mice were intra peritoneally injected with aristolochic acid I (AAⅠ) for 6 d to induce acute AAN model.AS Ⅳ at dose of 50 mg·kg^-1·d^-1 was gavaged for 6 d, and the levels of urine protein, urine γ-glutamyltransferase (γ-GT), serum creatinine (SCr) and blood urea nitrogen (BUN) were measured. The histological changes of the kidneys were observed under microscope by HE and periodic acid-silver methenamine (PASM) staining at the 7th day. RESULTS: The cell viability was significantly inhibited by AA I. However, the cell viability increased when AA I combined with AS Ⅳ was given as compared with control group, indicating that AS Ⅳ plays a dose-dependent protective role in HK-2 cells against the injury of AA I. The results of in vivo experiments showed that the levels of urine protein, urine γ-GT, SCr and BUN were decreased in AA I combined with AS Ⅳ group compared with AA I renal injury group. Histological study showed that AA I-induced kidney injury was improved with the decrease in the area of tubule necrosis and nude tubular basement membrane. CONCLUSION: AS Ⅳ has a protective effect on renal tubular damage induced by AA I.     

Over-expression of P21 attenuates cisplatin-induced HK-2 cells injury

AIM:To investigate the effect of P21 on cisplatin-induced renal tubular epithelial cells injury.METHODS:The expression of P21 at mRNA and protein levels in cisplatin treated human renal tubular epithelial cells (HK-2) cells was determined by RT-qPCR and Western blot. Over-expression of P21in the HK-2 cells was induced by the transfection of pcDNA3-P21. The cell viability and cell apoptosis were detected by CCK-8 assay and flow cytometry, respectively. Furthermore, the protein expression of kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), caspase-3, glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP), and phosphorylation level of eucaryotic translation initiation factor 2α (eIF2α) and protein kinase R-like endoplasmic reticulum kinase (PERK) were detected by Western blot.RESULTS:Cisplatin increased the mRNA and protein levels of P21 in a time-and concentration-dependent manner in the HK-2 cells. Over-expression of P21 inhibited cisplatin-induced cell apoptosis, and down-regulated the expression of KIM-1 and NGAL. Furthermore, Over-expression of P21 decreased the protein levels of GRP78, p-PERK, p-eIF2α, CHOP and cleaved caspase-3.CONCLUSION:Over-expression of P21 attenuates cisplatin-induced HK-2 cells injury, and the mechanism may be related to the modulation of endoplasmic reticulum stress pathway and inhibition of cell apoptosis.     

TAK1 regulates fibrosis of renal tubular epithelial cells by regulating p38 MAPK signaling pathway

AIM:To investigate the effect of transforming growth factor-β (TGF-β) activated kinase 1(TAK1) on renal tubular epithelial fibrosis. METHODS:The renal tubular epithelial cell line HK-2 was used as the research object. After induced by TGF-β1, real-time PCR and Western blot were used to detect the expression of TAK1 in the HK-2 cells. TAK1 shRNA lentivirus was used to infect HK-2 cells, real-time PCR and Western blot were used to determine the interference effect on TAK1 expression in the HK-2 cells with TGF-β1 stimulation. Under the condition of treating with p38 MAPK activator anisomycin, the levels of type I collagen and type Ⅲ collagen in the supernatant, and the protein levels of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) and p-p38MAPK^{Thr 180/Tyr 182} in the HK-2 cells with TAK1 knock-down were determined by ELISA and Western blot, respectively. RESULTS:TGF-β1 significantly increased the expression of TAK1 in the HK-2 cells(P<0.05). TAK1 shRNA significantly decreased the expression of TAK1 in the HK-2 cells with TGF-β1 stimulation. Type I collagen and type Ⅲ collagen secreted by the HK-2 cells after treatment with TGF-β1 were increased, the protein levels of α-SMA, CTGF and p-p38MAPK^{Thr 180/Tyr 182} were also increased(P<0.05). Knock-down of TAK1 expression significantly inhibited the secretion of type I and type Ⅲ collagen, reduced the protein levels of α-SMA, CTGF and p-p38MAPK^{Thr 180/Tyr 182} in the TGF-β1-induced HK-2 cells(P<0.05). Treatment with p38 MAPK activator reversed the inhibitory effect of TAK1 knock-down on the secretion of type I and type Ⅲ collagens, and the protein levels of α-SMA, CTGF and p-p38 MAPK^{Thr 180/Tyr 182} in the HK-2 cells(P<0.05). CONCLUSION:Knock-down of TAK1 expression attenuates the TGF-β1 induced fibrosis of renal tubular epithelial cells by inhibiting p38 MAPK signaling pathway.     

miR-10b promotes high glucose-stimulated epithelial-mesenchymal transition of renal tubular epithelial cells by repressing KLF10 expression

AIM:To explore the effect and the underlying mechanisms of microRNA-10b (miR-10b) on high glucose-stimulated epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells. METHODS:The expression level of miR-10b was examined by RT-qPCR in the kidney tissues of the type 2 diabetes patients with kidney fibrosis. The EMT model of HK-2 cells was induced by high glucose stimulation and the miR-10b expression in the process was detected by RT-qPCR. The morphological changes of the HK-2 cells were observed using a microscope. EMT markers, such as fibronectin and N-cadherin, were examined by Western blot. The online database predicted that the 3'-UTR of KLF10 bound to miR-10b and their direct interaction was confirmed by dual luciferase report assay. RESULTS:Compared with the para-carcinoma normal tissues, the expression level of miR-10b was up-regulated in the tissues of type 2 diabetes patients with kidney fibrosis (P<0.01). In high glucose-stimulated HK-2 cells, the expression level of miR-10b was increased in a time-dependent manner (P<0.01). miR-10b inhibitor reversed the morphological changes and the increases expression of the EMT markers including fibronectin, SLUG, N-cadherin and SNAI1 induced by high glucose stimulation. Online database showed miR-10b was able to bind with the 3'-UTR in the promoter region of KLF10, thus negatively regulating its expression. Meanwhile, over-expression of KLF10 inhibited the EMT induced by high glucose. Inhibition of TGF-β/Smad3 activation was observed during the process of KLF10-repressed EMT. CONCLUSION:miR-10b promotes high glucose-stimulated epithelial-mesenchymal transition of renal tubular epithelial cells may through repressing KLF10 expression.     

Hydrogen sulfide protects H9c2 cardiomyocytes against high glucose-induced injury by inhibiting necroptosis

AIM: To study whether hydrogen sulfide(H₂S) protects H9c2 cardiomyocytes against high glucose(HG)-induced injury by inhibiting necroptosis. METHODS: The protein levels of RIP3(an indicator of necroptosis) and cleaved caspase-3 were determined by Western blot. The cell viability was measured by CCK-8 assay. The intracellular le-vels of reactive oxygen species(ROS) were detected by 2', 7'-dichlorfluorescein diacetate staining followed by photofluorography. Mitochondrial membrane potential(MMP) was examined by rhodamine 123 staining followed by photofluorography. The number of apoptotic cells was observed by Hoechst 33258 nuclear staining followed by photofluorography. RESULTS: After the H9c2 cells were treated with HG(35 mmol/L glucose) for 0~24 h, the protein expression of RIP3 in the H9c2 cells was significantly increased at 3 h, 6 h, 9 h, 12 h and 24 h, reaching the maximum level at 24 h. Pretreatment of the cells with 400μmol/L NaHS(a donor of H₂S) or co-treatment of the cells with necrostatin-1(Nec-1; a speci-fic inhibitor of necroptosis) considerably blocked the up-regulation of RIP3 protein induced by HG. Moreover, pretreatment with NaHS or co-treatment with Nec-1 obviously inhibited HG-induced injuries, leading to an increase in the cell viability, and decreases in the generation of ROS and MMP loss. On the other hand, pretreatment with NaHS also reduced the number of apoptotic cells and the protein level of cleaved caspase-3 in the HG-treated H9c2 cardiomyocytes. CONCLUSION: H₂S protects H9c2 cardiomyocytes against HG-induced injury by inhibiting necroptosis.     

Effect of HMGA2 gene on high glucose-induced apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway

AIM:To investigate the effect of HMGA2 down-regulation on apoptosis and Notch signaling pathway in renal tubular epithelial cells exposed to high glucose (HG). METHODS:D-glucose at 5, 10, 20 and 30 mmol/L was used to stimulate human renal tubular epithelial HK-2 cells for 2 h, and D-glucose at 30 mmol/L was used to stimulate the HK-2 cells for 10 min, 60 min and 120 min. The protein expression of HMGA2 was determined by Western blot. The HK-2 cells were divided into normal glucose (NG) group, HG group, HG+si-HMGA2 group and HG+NC group, in which siRNA was transfected by Lipofectamine^TM 2000 for 48 h. Flow cytometry was used to analyze the apoptotic rate, reactive oxygen species (ROS) assay kit was used to detect ROS content, and Western blot was used to detect the protein levels of Notch1, Hes1 and Bcl-2. The HK-2 cells were treated with the Notch signaling pathway inhibitor DAPT, and then the cells were divided into HG group, HG+DAPT group and HG+si-HMGA2+DAPT group. The apoptotic rate was analyzed by flow cytometry. RESULTS:Exposure of the HK-2 cells to D-glucose at different concentrations for different time significantly increased the expression of HMGA2 (P<0.05). Compared with NG group, the protein expression of HMGA2, Notch1 and Hes1 in HG group was increased, the expression of Bcl-2/Bax was decreased, the apoptotic rate was increased, and the content of ROS was increased obviously (P<0.05). Compared with HG group, the protein expression of HMGA2, Notch1 and Hes1 of HG+si-HMGA2 group was decreased, the expression of Bcl-2/Bax was increased, the apoptotic rate was decreased, and the content of ROS was decreased significantly (P<0.05). The apoptotic rate in HG+DAPT group was significantly lower than that in HG group, while the apoptotic rate in HG+si-HMGA2+DAPT group was significantly lower than that in HG+DAPT group (P<0.05). CONCLUSION:Down-regulation of HMGA2 expression inhibits the apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway and decreasing ROS production.     

Blockage of core fucosylation contributes to inhibition of epithelial mesenchymal transition of renal tubular epithelial cells

AIM: To investigate the role of inhibiting core fucosylation in the process of epithelial mesenchymal transition (EMT) in HK-2 cells.METHODS: An EMT cell model with transforming growth factor-β₁(TGF-β₁) was established and RNAi technique was used to silence the expression of α-1,6-fucosyltransferase ( FUT8) gene which is responsible to catalysation of core fucose. The morphological changes of HK-2 cells were observed under light microscope. The epithelial cell marker E-cadherin and fibrotic cell markers N-cadherin, fibroblast-specific protein-1(FSP-1) and α-smooth muscle actin(α-SMA) were detected by Western blotting and immunocytochemistry. The apoptosis induced by TGF-β₁ was determined by flow cytometry. RESULTS: After incubated with TGF-β₁ at the concentration of 5 μg/L for 48 h, HK-2 cells lost epithelial morphology and showed fibrotic morphology. The expression of α-SMA, FSP-1 and N-cadherin was markedly increased, while E-cadherin was decreased. Meanwhile, the expression of FUT8 was up-regulated, and the apoptosis of the cells increased. However, pre-incubation of the cells with FUT8 siRNA inhibited these changes above.CONCLUSION: The core fucosylation involves in the process of EMT in HK-2 cells. Blockage of core fucosylation results in the inhibition of EMT in HK-2 cells.     

Fasudil hydrochloride prevents cisplatin-induced renal tubular epithelial cell apoptosis via Akt activation and PTEN inhibition

AIM: To explore the protective effect of fasudil hydrochloride against cisplatin(CP)-induced renal tubular epithelial cell apoptosis via Akt activation and PTEN inhibition. METHODS: Healthy male Sprague-Dawley (SD) rats were randomly divided into control group, CP group and CP+ fasudil group. All animals were sacrificed 96 h after injection of 0.9% saline or CP. Blood samples and kidney tissues were collected to evaluate levels of blood urea nitrogen (BUN), serum creatinine (sCr) and morphological alteration of the kidneys, respectively. The apoptosis of renal tubular epithelium cells was detected by TUNEL. Protein levels of Rho-associated protein kinase 1 (ROCK1), PTEN and Akt were measured by Western blotting and immunohistochemistry. The protein level of p-Akt was analyzed by Western blotting. RESULTS: Compared with control group, the sCr and BUN levels, the expression of ROCK1 and PTEN and TUNEL-positive cells were increased, while the level of p-Akt was decreased in CP group and CP+fasudil group. The histological structure of the kidneys observed by PAS staining was developed marked structural damage in CP group(P<0.05). Compared with CP group, sCr level, the expression of ROCK1 and PTEN and TUNEL-positive cells were decreased, while the level of p-Akt was increased in CP+fasudil group (P<0.05). Very little structural damage was detected in fasudil-treated groups. CONCLUSION: Fasudil hydrochloride has a protective effect on CP-induced renal tubular epithelial cell apoptosis via Akt activation and PTEN inhibition 1.     

Activation of renal tubular epithelial cells by monocytes/macrophages and the relative mechanisms

AIM: To observe the direct effects of peripheral blood monocytes/macrophages (MO/MAC) on renal tubular epithelial cells (RTEC),and further probe into the possible mechanisms. METHODS: Conditioned medium(M-CM) of human peripheral blood MO/MAC was collected and added to HK-2,a human renal proximal tubular cell line.After incubation with M-CM for 24 hours,HK-2 cells were detected for DNA synthesis by [³H]-TdR incorporation,osteopontin (OPN) and α-smooth muscle actin (α-SMA) expression by Western blot,and fibronectin(FN) secretion by ELISA.Furthermore,anti-TGFβ₁ neutralizing antibody and interlukin-10(IL-10) were used separately to antagonize the effects of M-CM on HK-2 cells. RESULTS: ①DNA synthesis,α-SMA expression and FN secretion were all increased in HK-2 cells when incubated with M-CM.②When adding with anti-TGFβ₁ neutralizing antibody (5 mg/L) in the M-CM,the degree of upregulation of α-SMA and FN in HK-2 cells was much lower than that stimulated by M-CM alone.③M-CM added with IL-10 (20 μg/L) had a weaker ability to induce the increasing in α-SMA expression and FN excretion in HK-2 cells, compared with M-CM itself alone.M-CM from MO/MAC preincubated with IL-10 caused a lower upregulation of α-SMA expression in HK-2 cells than M-CM from non-preincubated MO/MAC. CONCLUSION: MO/MAC can directly induce proliferation,transdifferentiation and extracellular matrix secretion in RTEC.TGFβ₁ and proinflammatory cytokines secreted by MO/MAC might be involved in the aboveeffects.     

Sedum sarmentosum Bunge extract inhibits epithelial-mesenchymal transition and collagen accumulation in aristolochic acid-treated rat renal tubular epithelial cells

AIM:To investigate the effect of Sedum sarmentosum Bunge (SSB) extract on epithelial-mesenchymal transition (EMT) and collagen accumulation induced by aristolochic acid (AA) in renal tubular epithelial cells. METHODS:Rat renal tubular epithelial NRK-52E cells were randomly divided into 3 groups, including control group (only treated with solvent), AA group (treated with AA at concentrations ranging from 1 to 100 mg/L) and SSB group (treated with AA at a concentration of 10 mg/L plus SSB extract at concentrations ranging from 10 to 2 000 mg/L). After cultured for 24 h, the morphology of the NRK-52E cells was observed under inverted phase-contrast microscope. The level of transforming growth factor β1 (TGF-β1) in the culture supernatant was measured by ELISA. Immunofluorescent analysis was performed to detect the expression of epithelial marker α-smooth muscle actin (α-SMA), mesenchymal marker E-cadherin, and extracellular cell matrix component type III collagen. The mRNA expression of E-cadherin, α-SMA, bone morphogenetic protein 7 (BMP-7) and type I collagen was also quantified by real-time PCR. RESULTS: Fibrosis-like reaction observed under microscope was obviously increased in AA-treated NRK-52E cells, and aggravated as the increase in the concentration of AA. AA at concentrations of 1 and 10 mg/L increased the expression of α-SMA, type I and type III collagens, and decreased the expression of E-cadherin. With SSB extract treatment, fibrosis in NRK-52E cells was alleviated, accompanied with the decreasing expression of α-SMA, type I and type III collagen, and the enhancing expression of E-cadherin and BMP-7.Moreover, SSB extract down-regulated TGF-β1 level in a concentration-dependent manner. CONCLUSION: AA-induced fibrosis-like reaction in renal tubular epithelial cells is reduced by the treatment with SSB extract. The possible mechanism is that SSB extract decreases TGF-β1 level, and inhibits renal EMT and collagen accumulation induced by AA.［KEY WORDS］Sedum sarmentosum Bunge|Aristolochic acid|Transforming growth factor β1|Epithelial-mesenchymal transition|Collagen     

Inflammatory cytokines up-regulate FAT/CD36 expression in renal cells loaded by fatty acids

AIM: To investigate the effects of inflammatory cytokines on the expression of fatty acid transporter (FAT/CD36) in renal cells loaded by fatty acids. METHODS: Human mesangial cells (HMCs) and renal tubular epithelial HK-2 cells were treated with palmitate at concentrations of 0 mmol/L, 0.02 mmol/L, 0.04 mmol/L, 0.08 mmol/L, 0.16 mmol/L and 0.32 mmol/L for 24 h. The expression of FAT/CD36 at mRNA and protein levels was detected by real-time PCR and Western blotting,respectively. The renal cells were treated with palmitate at concentration of 0.04 mmol/L combined with TNF-α (25 μg/L) or IL-6 (20 μg/L) for 24 h. The effect of inflammatory cytokines on the mRNA and protein levels of FAT/CD36 in the renal cells was also investigated. Oil red O staining was used to determine the intracellular lipid droplet formation. The intracellular triglyceride (TG) and free fatty acid (FFA) were measured by enzymic assay and ELISA, respectively. RESULTS: Palmitate loading dose-dependently increased the expression of FAT/CD36 at mRNA and protein levels in both HMCs and HK-2 cells. The inflammatory cytokines further increased the expression of FAT/CD36 at mRNA and protein levels in both cells loaded by palmitate. Oil red O staining, TG detection and FFA assay showed that the inflammatory cytokines increased intracellular lipid levels in both HMCs and HK-2 cells. CONCLUSION: Inflammatory cytokines up-regulate the expression of FAT/CD36 in renal cells loaded by fatty acids and exacerbate the intracellular lipid accumulation.     

C-reactive protein induces apoptosis in human renal tubular epithelial cells

ATM: To explore whether the C-reactive protein (CRP) level in microinflammation state induces the apoptosis of renal tubular epithelial cells. METHODS: HK-2 cells were stimulated with recombinant human CRP. Annexin-FITC-PI staining and flow cytometry were used to detect the percentage of apoptotic cells. Morphology observation of apoptosis was assessed by Hoechst 33258 staining. Caspase-3 activity was measured by a colorimetric assay. The expression of apoptotic gene bax and anti-apoptotic gene bcl-2 at mRNA levels was determined by real-time PCR. RESULTS: CRP induced apoptosis of HK-2 cells in a time- and dose-dependent manner. The maximal apoptotic effect of CRP concentration was 10 mg/L CRP at concentration of 20 mg/L. CRP treatment was associated with the characteristic morphological features of apoptosis such as condensation, fragmentation or margination of nuclear chromatin. CRP exposure increased caspase-3 activity, up-regulated the mRNA expression of Bax and down-regulated the mRNA expression of Bcl-2. CONCLUSION: Slightly increased CRP level has the potential to induce apoptosis of renal tubular cells.