Effect of overexpression of protein phosphatase 2Cm on transcriptome of human renal tubular epithelial cells / 中国组织工程研究

BACKGROUND:A previous study by our group found that protein phosphatase 2Cm(PP2Cm)null mice developed significantly fewer symptoms of renal failure relative to wild-type mice,and thus it was speculated that PP2Cm may play an important protective role in the development of renal fibrosis,however,the molecular mechanisms remain undefined. OBJECTIVE:To investigate the effect of the PP2Cm gene on the transcriptome of human renal tubular epithelial cells. METHODS:Cultured human renal tubular epithelial cells were transfected with the PP2Cm gene into human renal tubular epithelial cells using plasmids.The expression of PP2Cm in the cells was detected by fluorescence quantitative PCR assay and western blot assay,and subsequently,cell RNA was separately extracted for transcriptome sequencing to look for differentially expressed genes between transfected and control groups.The resulting differential genes were further subjected to GO analysis and KEGG analysis using bioinformatics methods. RESULTS AND CONCLUSION:There were 796 differentially expressed genes,553 of which were downregulated genes and 243 upregulated genes,in human renal tubular epithelial cells transfected with the PP2Cm gene compared with untransfected blank cells by sequencing analysis.GO analysis results showed that the upregulated genes were significantly enriched in cellular biosynthetic processes,protein translation,intrinsic apoptotic signaling pathways,and so on.The downregulated expressed genes were significantly enriched in endothelial cell proliferation,cell adhesion and other signaling pathways.KEGG analysis results showed that the significantly up-regulated genes were enriched in metabolism-related signaling pathways such as amino acid metabolism and biosynthesis.The downregulated expressed genes were significantly enriched in signaling pathways such as pantothenate and coenzyme A biosynthesis.Our results show that PP2Cm overexpression can affect a number of signaling pathways related to a range of biological processes in renal tubular epithelial cells,which may be important in metabolism-related signaling pathways such as amino acid metabolism and biosynthesis.

Regulation of the TGF-β type I Receptor/Smad Pathway Inhibits Renal Fibrosis by Shenkang Injection / 世界科学技术-中医药现代化

Objective Chinese herbal injections called Shenkang injections(SKI)have become widely used for treating chronic kidney disease in the clinic.An investigation into the underlying mechanisms of SKI inhibition of renal tubular epithelial cell trans differentiation treated with TGF-β1 was carried out in this study.Methods To create an in vitro model of RF,HK-2 cells were treated with TGF-1(10 ng·mL-1)at 37℃for 48 h.After the cells were treated with SKI for 48 h,the morphology of the cells was observed by electron microscope.And Western blot,RT-PCR and immunofluorescence techniques were used to detect α-Smooth muscle actin(α-SMA),type III collagen(COI-Ⅲ),TGF-β1,Smad3,Smad7 and TβR-I expression changes of proteins and genes.Results SKI can significantly reduce expressed proteins and genes related to renal fibrosis,such as α-Smooth muscle actin(α-SMA)and type Ⅲ collagen(COI-Ⅲ).SKI control the production of proteins associated with the TGF-β/Smad signaling pathway.By downregulating TGF-β1,Smad3,and TβR-I protein expression,as well as upregulating Smad7 protein expression,it is able to prevent cell trans differentiation.Conclusions SKI can inhibit renal tubular epithelial cell mesenchymal trans differentiation.In addition,this drug may prevent chronic kidney disease by downregulating the expression of TβR-I and regulating the TGF-β/Smad pathway-related molecules.

High glucose inhibition of miR-126-5p promotes renal tubular epithelial cell injury / 中国医师杂志

Objective:To explore the differential expression profile of miRN in the development of diabetes nephropathy (DN), and further explore the mechanism of miR-126-5p involved in high glucose induced injury of renal tubular epithelial cells.Methods:Firstly, we downloaded existing chip data from the Gene Expression Integrated Database (GEO) and used GEO2R, miRanda, gene ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to mine differential miRNAs. Subsequently, a high glucose induced HK-2 cell injury model was used and divided into three groups: high glucose model group, si-HOTAIR group, and si HOTAIR+ miR-126-5p inhibitor group. The three groups of cells were sequentially transfected with siRNA-NC, siRNA-HOTAIR, and siRNA-HOTAIR+ miR-126-5p mimic, and cultured in a medium containing 60 mmol/L glucose. Flow cytometry was used to detect changes in apoptosis levels in each group, while cell counting kit-8 (CCK-8) was used to detect changes in cell proliferation.Results:Through data mining analysis using GEO, it was found that compared to ordinary mice, DN mice had 74 upregulated miRNAs and 80 downregulated miRNAs in their kidney tissue. Enrichment analysis results showed that miRNAs could target signaling pathways such as Wnt, PKG, MAPK, and Rap1, and miR-126-5p was significantly downregulated. In the high glucose induced HK-2 cell injury model, the experimental results showed that the inhibitory effect on cell proliferation activity was more significant at a high glucose concentration of 60 mmol/L ( P<0.05); High glucose stimulation significantly reduced the expression of miR-126-5p ( P<0.05). The results of flow cytometry showed that compared with the high glucose model group, the apoptosis rate of the si-HOTAIR group significantly decreased ( P<0.05), while the apoptosis rate of the si-HOTAIR+ miR-126-5p inhibitor group significantly increased ( P<0.05). The CCK-8 experiment showed that compared with the high glucose model group, the cell viability of the si-HOTAIR group significantly increased ( P<0.05); The cell viability of the si-HOTAIR+ miR-126-5p inhibitor group was inhibited ( P<0.05). Conclusions:miR-126-5p can inhibit high glucose induced apoptosis in HK-2 cells and protect them.

Transcriptome changes upon Toll-like receptor 9 pathway activation in primary renal tubular epithelial cells / 中华危重病急救医学

Objective:To explore the effect of Toll-like receptor 9 (TLR9) signaling pathway activation on the transcriptome in the renal tubular cells.Methods:Mouse primary renal tubular epithelial cells were extracted and cultured. When the degree of cell fusion reached 80%, they were divided into two groups, which were added with 10 μL phosphate buffered saline (PBS, PBS control group) and TLR9 activator cytosine phosphate guanidine oligodeoxynucleotide (CpG-ODN) with a final concentration of 5 μmol/L (CpG-ODN treatment group). The RNA sequencing was performed on the Illumina platform after extraction. DEGseq software was used to analyze the differential expression of genes between the two groups. Goatools and KOBAS online software were used to analyze the differential genes involved signal pathways. Homer software was used to predict transcription factors.Results:Compared with the PBS control group, there were a total of 584 differentially expressed genes in the CpG-ODN treatment group, of which 102 were up-regulated and 482 were down-regulated. The most significantly enriched gene ontology (GO) terms of differentially expressed genes included response to interferon-β, defense response to virus and other inflammatory pathway. The most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways included 2'-5'-oligoadenylate synthase activity, regulation of ribonuclease activity, negative regulation of virus life cycle, cellular response to interferon-βand defense response to protozoan. The results of transcription factor prediction showed that interferon regulatory factor 3 (IRF3) was the most significantly enriched transcription factor in the promoter sequence of differential genes; the most significant transcription factor downstream of TLR9 was IRF3, and other predicted transcription factors such as transcription factor 21 (TCF21), zinc finger protein 135 (ZNF135), and PR domain containing 4 (PRDM4) might be new candidates for TLR9 signaling pathway.Conclusion:CpG-ODN activates TLR9 signaling pathway, and primary renal tubular epithelial cells can directly respond to CpG-ODN stimulation and undergo transcriptome changes, which provides a basis for further research on the molecular mechanism of TLR9 pathway in sepsis induced acute kidney injury.

Effect of resveratrol on PTEN expression and fibrosis of renal tubular epithelial cells in a high-glucose environment / 中国中药杂志

This study explored the effects of resveratrol(Res) on the expression of phosphatase and tensin homolog deleted on chromosome ten(PTEN) and the fibrosis of rat renal tubular epithelial cells in a high-glucose environment and the possible mechanism underlying the fibrosis reduction. After the pretreatment of rat renal tubular epithelial cells(NRK-52 E) cultured in a high-glucose condition with Res or PTEN inhibitor SF1670, they were divided into several groups, i.e., normal glucose(NG), normal glucose + SF1670(NS), high glucose(HG), high glucose + SF1670(HS), high glucose + Res at different concentrations(5, 10, 25 μmol·L~(-1)). The expression and distribution of E-cadherin and α-SMA in renal tubular epithelial cells were observed by immunofluorescence cytochemistry. The protein expression levels of PTEN, E-cadherin, α-SMA, p-Akt~((Thr308)) and collagen Ⅳ were determined by Western blot. Real-time PCR was employed to detect the expression of PTEN mRNA. Compared with the NG group, the HG group witnessed the reduced expression of PTEN mRNA, PTEN protein and E-cadherin protein, but saw the increased expression of α-SMA, p-Akt~((Thr308)) and collagen Ⅳ proteins. Besides, with the increase in Res concentration, the expression levels of PTEN mRNA, PTEN protein and E-cadherin protein gradually increased, while those of α-SMA, collagen Ⅳ, p-Akt~((Thr308)) proteins gradually decreased in the Res groups, showing a dose-effect dependence, compared with the HG group. No distinct difference was found between the NS group and the NG group. The expression level of E-cadherin was even lower and those of α-SMA, p-Akt~((Thr308)), and collagen Ⅳ were higher in the HS group than in the HG group, with no marked difference shown in the two groups in terms of PTEN mRNA and protein. Although the PTEN inhibitor did not affect PTEN, the expression changes of the other proteins were opposite to the results after Res treatment and the fibrosis was aggravated, which suggested that SF1670 promoted the fibrosis by inhibiting PTEN, activating Akt and increasing the synthesis of collagen Ⅳ and other extracellular matrix. The results show that Res can antagonize the high glucose-mediated fibrosis of renal tubular epithelial cells. This may be achieved via the up-regulation of PTEN and the inhibition of PI3 K/Akt signaling pathway.

Effects and Mechanism Study of Pioglitazone on High Glucose-induced Epithelial-mesenchymal Transition in Renal Tubular Epithelial Cells of Rat / 中国药房

OBJECTIVE：To investigate the effects o f pioglitazone （PIO）on high glucose-induced epithelial-mesenchymal transition（EMT）in renal tubular epithelial cells of rat and its possible mechanism ，and to provide theoretic reference and new target for the prevention and treatment of diabetic nephropathy. METHODS ：The rat renal tubular epithelial NRK- 52E cells were randomly divided into control group （5.5 mmol/L glucose ），high-glucose group （30 mmol/L glucose ），PIO intervention group （30 mmol/L glucose+ 5.0 μmol/L PIO），GW9662 intervention group （30 mmol/L glucose+ 5.0 μmol/L PIO+5.0 μmol/L specific anta- gonist GW 9662）. The cells of the first 3 groups were detected at 6，12，24，48 h of culture ，while those in GW 9662 intervention group were detected at 48 h of culture. mRNA expression of PTEN and PPARγ were detected by real-time PCR. The protein expression of PTEN ，PPARγ，α-SMA and E-cadherin as well as the changes of PI 3K/AKT signaling pathway were determined by Western blotting assay. RESULTS ：With the extension of culture time ，compared with control group ，the mRNA and protein expression of PPARγ（except for protein expression at 6 h）and PTEN in high-glucose group reduced significantly ，while the protein expression of α-SMA and p-AKT （Thr308）increased significantly ，and the protein expression of E-cadherin reduced significantly （P＜0.05），showing time-dependent trend. Compared with high-glucose group ，the mRNA and the protein expression （except for 6 h） of PPAR γ and PTEN were increased significantly in PIO intervention group ， while the protein expression of α-SMA and p-AKT （Thr308） were decreased significantly，and the protein expression of E-cadherin wasincreased significantly （P＜0.05）， showing time-dependent trend. There was no statistical significance in mRNA and protein expression of PPAR γ and PTEN，protein expression of E-cadherin ，α-SMA and p-AKT （Thr308） between GW 9662 intervention group and high-glucose group ；the effect of PIO was blocked by PPAR γ antagonist GW9662. CONCLUSIONS ：PIO may up-regulate the expression of PTEN by activating PPARγ，inhibit PI 3K/AKT signaling pathway so as to inhibit the occurrence of EMT of renal tubular epithelial cells .

The inhibitory effect of calcium dobesilate on cisplatin induced HK-2 cell apoptosis and its mechanism / 药学学报

We observed the effect of calcium dobesilate (CaD) on apoptosis induced by cisplatin in human proximal tubular epithelial cells (HK-2) and explored the possible mechanism. Based on HK-2 cells apoptosis model induced by cisplatin, CCK-8 method was used to detect the effect of CaD on the proliferation of HK-2. Apoptosis was detected by flow cytometry. Reactive oxygen species (ROS) assay was used to evaluate the level of oxidative stress. The mitochondrial membrane potential was measured by JC-1 method. The expression levels of p53, caspase-3, bcl-2 and bax in cisplatin-induced HK-2 were detected by Western blot. The expression of renal injury factor 1(KIM-1) and neutrophil gelatin-related apolipoprotein (NGAL), markers of acute kidney injury, were detected by ELISA. The results showed that CaD could reduce the oxidative stress level induced by cisplatin and inhibit apoptosis in renal tubular epithelial cells. Cisplatin can up-regulate the protein expressions of p53, caspase-3, bax, KIM-1 and NGAL, and reduce the expression of bcl-2. After using CaD, the protein levels of KIM-1, NGAL, p53, caspase-3 and bax were significantly reduced, while the levels of bcl-2 were increased. This study has shown that CaD can alleviate cisplatin-induced HK-2 injury and inhibit HK-2 apoptosis, which may be related to the regulation of bax/bcl-2/caspase-3 apoptosis signaling pathway.

Paricalcitol attenuates indoxyl sulfate-induced apoptosis through the inhibition of MAPK, Akt, and NF-κB activation in HK-2 cells

BACKGROUND/AIMS: Indoxyl sulfate (IS) is a uremic toxin and an important causative factor in the progression of chronic kidney disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to exhibit protective effects in kidney injury. Here, we investigated the effects of paricalcitol treatment on IS-induced renal tubular injury. METHODS: The fluorescent dye 2ʹ,7ʹ-dichlorofluorescein diacetate was used to measure intracellular reactive oxygen species (ROS) following IS administration in human renal proximal tubular epithelial (HK-2) cells. The effects of IS on cell viability were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and levels of apoptosis-related proteins (Bcl-2-associated protein X [Bax] and B-cell lymphoma 2 [Bcl-2]), nuclear factor-κB (NF-κB) p65, and phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) were determined by semiquantitative immunoblotting. The promoter activity of NF-κB was measured by luciferase assays and apoptosis was determined by f low cytometry of cells stained with f luorescein isothiocyanate-conjugated Annexin V protein. RESULTS: IS treatment increased ROS production, decreased cell viability and induced apoptosis in HK-2 cells. IS treatment increased the expression of apoptosis-related protein Bax, decreased Bcl-2 expression, and activated phosphorylation of MAPK, NF-κB p65, and Akt. In contrast, paricalcitol treatment decreased Bax expression, increased Bcl-2 expression, and inhibited phosphorylation of MAPK, NF-κB p65, and Akt in HK-2 cells. NF-κB promoter activity was increased following IS, administration and was counteracted by pretreatment with paricalcitol. Additionally, flow cytometry analysis revealed that IS-induced apoptosis was attenuated by paricalcitol treatment, which resulted in decreased numbers of fluorescein isothiocyanate-conjugated Annexin V positive cells. CONCLUSIONS: Treatment with paricalcitol inhibited IS-induced apoptosis by regulating MAPK, NF-κB, and Akt signaling pathway in HK-2 cells.

Effect of Xuebijing on paraquat-induced cell oxidative stress and inflammation level in human renal proximal convoluted tubular epithelial cell-2 / 中国中西医结合急救杂志

Objective To investigate the effect of Xuebijing on paraquat-induced oxidative stress and inflammation level in human kidney cell line-2 (HK-2) cells. Methods The HK-2 cells were routinely cultured and divided into blank control group, paraquat poisoning model group, Xuebijing injection pretreatment group and Xuebijing control group according to random number table method. The concentration of paraquat in HK-2 cells were measured by high performance liquid chromatography (HPLC); the activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) were measured by chemical colorimetry method; the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected by enzyme linked immunosorbent assay (ELISA). Results With prolonged treatment, paraquat concentration gradually increased in HK-2 cells in the paraquat poisoning model group and the Xuebijing injection pretreatment group, reaching the peak value after treatment for 48 hours, moreover, the concentration of paraquat in HK-2 in the Xuebijing injection pretreatment group was significantly lower than that in the paraquat poisoning model group (mg/L: 4.26±0.20 vs. 5.77±0.18, P < 0.05). Compared with the blank control group, the contents of MDA, TNF-α and IL-6 of paraquat poisoning model group were obviously elevated [MDA (nmol/mg): 4.47±0.10 vs. 2.21±0.08, TNF-α (ng/L): 206.91±13.22 vs. 98.14±5.67, IL-6 (ng/L): 253.33±5.22 vs. 116.97±13.54, all P <0.05], and the activity of SOD was significantly reduced (U/mg: 33.30±0.62 vs. 41.58±0.17, P < 0.05). Compared with paraquat poisoning model group, the contents of MDA, TNF-α and IL-6 in Xuebijing injection pretreatment group were obviously decreased [MDA (nmol/mg): 2.92±0.17 vs. 4.47±0.10, TNF-α (ng/L): 166.29±15.47 vs. 206.91±13.22, IL-6 (ng/L): 209.39±3.18 vs. 253.33±5.22, all P < 0.05], and the activity of SOD was significantly increased (U/mg:38.10±0.67 vs. 33.30±0.62, P < 0.05) in the paraquat pretreatment group. Conclusion Xuebijing could reduce the concentration of paraquat in HK-2 cells, and decrease oxidative stress and inflammation factor levels in HK-2 cells.

Effect of ethyl pyruvate on mitochondrial dynamics of lipopolysaccharide-induced human kidney-2 cells / 中华危重病急救医学

Objective To examine the effects of ethyl pyruvate (EP) on mitochondrial dynamics and cell apoptosis in lipopolysaccharide (LPS)-induced human kidney-2 (HK-2) cells. Methods HK-2 cells were divided into three groups: HK-2 cells were challenged with LPS (800 μg/L) for 24 hours as LPS group, or LPS mixed with EP (0.25 mmol/L) for 24 hours as EP group. Cells were incubated with normal saline for 24 hours as control group. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and intracellular adenosine triphosphate (ATP) were detected by enzyme linked immunosorbent assay (ELISA). JC-1 staining and Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) assays were used to evaluate mitochondrial membrane potential and cell apoptosis, respectively. Western Blot was used to evaluate the protein expressions of mitochondrial dynamics, including death-associated protein kinase 2 (DAPK-2), mitofusin (Mfn-1 and Mfn-2), and apoptotic associated biomarkers, including caspase-3, caspase-9, Bcl-2, Bcl-xL, cytochrome C (Cyt C), and DNA repair enzyme poly ADP-ribose polymerase (PARP). Results Compared with the NC group, MDA, IL-6, TNF-α of LPS group were significantly increased, the expression of SOD, mitochondrial membrane potential and ATP level were significantly decreased, the expression of mitochondrial fission protein DAPK-2 was significantly increased, and mitochondrial fusion proteins Mfn-1 and Mfn-2 were significantly decreased, cell apoptosis and apoptotic protein caspase-3, caspase-9 and Cyt C were increased, and anti-apoptotic protein Bcl-2, Bcl-xL, PARP were significantly decreased. Compared with the LPS group, the oxidative activities and inflammatory factors above were inhibited in EP group [MDA (μmol/L):12.35±2.21 vs. 45.95±1.76, SOD (kU/L): 54.68±1.42 vs. 40.73±1.60, IL-6 (ng/L): 67.87±2.61 vs. 338.92±20.91, TNF-α (ng/L): 19.23±1.80 vs. 180.69±6.51], mitochondrial membrane potential and ATP level were significantly increased [mitochondrial membrane potential: (99.43±0.25)％ vs. (69.40±0.75)％, ATP (×106 RLU): 0.19±0.01 vs. 0.12±0.05], the expression of mitochondrial fission protein was significantly decreased (DAPK-2/β-actin:0.03±0.01 vs. 0.61±0.02), mitochondrial fusion proteins were significantly increased (Mfn-1/β-actin: 0.43±0.04 vs. 0.17±0.01, Mfn-2/β-actin: 0.201±0.004 vs. 0.001±0.001), percentage of cell apoptosis was significantly decreased [(5.25±0.17)％ vs. (34.42±0.64)％], the expressions of apoptotic proteins were significantly decreased (caspase-3/β-actin: 0.25±0.15 vs. 1.76±0.01, caspase-9/β-actin: 0.09±0.02 vs. 1.52±0.12, Cyt C/β-actin: 0.001± 0.001 vs. 0.350±0.030), and the expressions of anti-apoptotic proteins and PARP were significantly increased (Bcl-2/β-actin: 0.500±0.010 vs. 0.009±0.004, Bcl-xL/β-actin: 0.550±0.010 vs. 0.009±0.001, PARP/β-actin:0.94±0.01 vs. 0.16±0.13), with statistically significant differences (all P < 0.05). Conclusions There are enhanced mitochondrial fission and diminished mitochondrial fusion in LPS-induced HK-2 cells. EP can protect mitochondria functions by regulate mitochondrial dynamics, and reducethe apoptosis of LPS-induced HK-2 cells.

Effect of ethyl pyruvate on mitochondrial dynamics of lipopolysaccharide-induced human kidney-2 cells / 中华危重病急救医学

Objective@#To examine the effects of ethyl pyruvate (EP) on mitochondrial dynamics and cell apoptosis in lipopolysaccharide (LPS)-induced human kidney-2 (HK-2) cells.@*Methods@#HK-2 cells were divided into three groups: HK-2 cells were challenged with LPS (800 μg/L) for 24 hours as LPS group, or LPS mixed with EP (0.25 mmol/L) for 24 hours as EP group. Cells were incubated with normal saline for 24 hours as control group. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and intracellular adenosine triphosphate (ATP) were detected by enzyme linked immunosorbent assay (ELISA). JC-1 staining and Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) assays were used to evaluate mitochondrial membrane potential and cell apoptosis, respectively. Western Blot was used to evaluate the protein expressions of mitochondrial dynamics, including death-associated protein kinase 2 (DAPK-2), mitofusin (Mfn-1 and Mfn-2), and apoptotic associated biomarkers, including caspase-3, caspase-9, Bcl-2, Bcl-xL, cytochrome C (Cyt C), and DNA repair enzyme poly ADP-ribose polymerase (PARP).@*Results@#Compared with the NC group, MDA, IL-6, TNF-α of LPS group were significantly increased, the expression of SOD, mitochondrial membrane potential and ATP level were significantly decreased, the expression of mitochondrial fission protein DAPK-2 was significantly increased, and mitochondrial fusion proteins Mfn-1 and Mfn-2 were significantly decreased, cell apoptosis and apoptotic protein caspase-3, caspase-9 and Cyt C were increased, and anti-apoptotic protein Bcl-2, Bcl-xL, PARP were significantly decreased. Compared with the LPS group, the oxidative activities and inflammatory factors above were inhibited in EP group [MDA (μmol/L): 12.35±2.21 vs. 45.95±1.76, SOD (kU/L): 54.68±1.42 vs. 40.73±1.60, IL-6 (ng/L): 67.87±2.61 vs. 338.92±20.91, TNF-α (ng/L): 19.23±1.80 vs. 180.69±6.51], mitochondrial membrane potential and ATP level were significantly increased [mitochondrial membrane potential: (99.43±0.25)% vs. (69.40±0.75)%, ATP (×106 RLU): 0.19±0.01 vs. 0.12±0.05], the expression of mitochondrial fission protein was significantly decreased (DAPK-2/β-actin: 0.03±0.01 vs. 0.61±0.02), mitochondrial fusion proteins were significantly increased (Mfn-1/β-actin: 0.43±0.04 vs. 0.17±0.01, Mfn-2/β-actin: 0.201±0.004 vs. 0.001±0.001), percentage of cell apoptosis was significantly decreased [(5.25±0.17)% vs. (34.42±0.64)%], the expressions of apoptotic proteins were significantly decreased (caspase-3/β-actin: 0.25±0.15 vs. 1.76±0.01, caspase-9/β-actin: 0.09±0.02 vs. 1.52±0.12, Cyt C/β-actin: 0.001±0.001 vs. 0.350±0.030), and the expressions of anti-apoptotic proteins and PARP were significantly increased (Bcl-2/β-actin: 0.500±0.010 vs. 0.009±0.004, Bcl-xL/β-actin: 0.550±0.010 vs. 0.009±0.001, PARP/β-actin: 0.94±0.01 vs. 0.16±0.13), with statistically significant differences (all P < 0.05).@*Conclusions@#There are enhanced mitochondrial fission and diminished mitochondrial fusion in LPS-induced HK-2 cells. EP can protect mitochondria functions by regulate mitochondrial dynamics, and reducethe apoptosis of LPS-induced HK-2 cells.

Renal Protective Effect of Tangshiping Capsule on Diabetic Nephropathy Kkay Mice and Its Effect on Wnt/β-catenin Signal Transduction Pathway / 中国实验方剂学杂志

Objective:To investigate the renal protective effect of Tangshenping capsule (Tangshenping) on diabetic nephropathy (DN) KKAy mice and its effect on Wnt/β-catenin signaling pathway. Method:Sixty female Sprague-Dawley KKAy mice aged 10 weeks old were induced with KKAy rat feed for 10 weeks. The DN animal model was successfully determined with blood glucose (>16.7 mmol·L-1) and 24 hour urine protein (>0.4 mg). The model mice were randomly divided into a model group, an irbesartan group, and low, medium and high-dose Tangshenping group, with 10 female C57BL/6J mice as a control group. The treatment groups were given the corresponding drugs by gavage. The normal group and the model group were given an equal volume of deionized water by gavage. The intragastric dose was 0.01 mL·g-1 body weight coefficient once a day. The general conditions of the mice were observed, the body mass was weighed every 4 weeks, and 24 h urine protein was quantified. At the 26th week, the blood was collected from eyeballs, and the mice were put to death. The quality of the kidneys, serum blood urea nitrogen (BUN), serum creatinine (SCr), triglyceride (TG), malondialdehyde (MDA), nitric oxide (NO) and superoxide dismutase (SOD) content were measured. In situ hybridization and immunohistochemistry were used to detect the expressions of Wnt4, glycogen synthase kinase 3β(GSK3β) and β-catenin in kidney tissues. Result:Compared with model group, body mass, kidney mass/body mass, and 24 h urine protein were significantly lower in high-dose Tangshenping group (PPPβ and β-catenin were decreased (PConclusion:Tangshenping may inhibit the activation of Wnt/β-catenin signaling pathway, reverse the transdifferentiation of renal tubular epithelial cells in DN KKAy mice, delay the progression of renal interstitial fibrosis, and then exert renal protection.

miR-384-5p promotes renal fibrosis through blocking post-transcriptional regulation of BMP7 / 西安交通大学学报(医学版)

Objective: To investigate whether microRNA-384-5p can interfere with renal fibrosis by regulating BMP7 transcription in renal tubular epithelial cells. Methods: Unilateral ureteral obstructive (UUO) model was established, and fluorescence activated cell sorter (FACS) was used to isolate renal tubular epithelial cells for culture. Plasmid expressing miR-384-5p, antisense miR-384-5p, and blank plasmid were transfected by liposome and injected in UUO mice. Masson staining was used to detect renal fibrosis, and Western blot and real-time PCR were used to detect the expressions of target-genes of miRNA (miR-384-5p, miR-30, miR-92, and miR-128). Results: There was obvious renal fibrosis at 14 day in UUO model group; the expression of BMP7 mRNA in the renal tissue increased obviously while the expression of BMP7 protein was not increased. Although miR-384-5p could not change BMP7 mRNA in the renal tubular epithelial cells, BMP7 protein in the renal tubular epithelial cells that overexpressed miR-384-5p decreased significantly, and BMP7 protein in these cells overexpressing antisense miR-384-5p increased significantly. Immunohistochemistry and quantitative PCR showed that renal tissue fibrosis in UUO mice increased significantly in 14 days, while antisense miR-384-5p could significantly reduce the renal tissue fibrosis. Although antisense-miR-384-5p treatment did not affect the level of BMP7 mRNA, it could significantly increase the expression of BMP7 protein in renal tissue, suggesting that inhibiting miR-384-5p could lessen renal injury in UUO mice. Conclusion: As a key factor regulating the mechanism of renal fibrosis after renal injury, targeting miR-384-5p may become a new method to prevent and treat renal fibrosis.

Research progress of calcium oxalate stones / 医学研究生学报

Calcium oxalate stones are the main components of the urinary calculi .The reasons for the formation of calcium ox-alate stones, the methods of researching calcium oxalate stones in vivo and in vitro , the relationship between renal tubular epithelial cell injury and calcium oxalate stones , the relationship between renal calcium oxalate stones and oxidative stress , the source of reactive oxygen species in renal calcium oxalate stones and renal calcium oxalate stones with antioxidant therapy were summarized in this paper . A new viewpoint was also provided for the treatment of renal calcium oxalate stones .Oxalate degradation enzymes can degrade oxalic acid into hydrogen peroxide and carbon dioxide to reduce the absorption of oxalic acid in the gastrointestinal and then limit the formation of calcium oxalate stones.Therefore, it will become a research hotspot in the future to screen oxalate degradation enzymes with high effi -ciency and low toxicity for calcium oxalate stones prevention .

Effects of Emodin against Renal Tubular Epithelial Cell Fibrosis via the Inhibition of Akt/mTOR Signal Pathway / 中国药师

Objective: To investigate the mechanism of emodin ( EM) in the expression of related protein for the fibrosis of the transforming growth factor-β1(TGF-β1)-stimulated human renal tubular epithelial (HK-2) cells. Methods: HK-2 cells were randomly divided into the normal control group, TGF-β1-stimulated model control group and emodin ( TGF-β1 +EM) group. The contents of Collage Ⅰ and fibronectin in the culture supernatant were determined by ELISA. After HK-2 cells were stimulated with TGF-β1 for 24 h, the cells were collected for immunofluorescence, Western blot and RT-PCR analysis. RT-PCR was used to detect PI3K, p-Akt and mTOR. The protein expressions of PI3K, p-Akt and mTOR were detected by Western blot. Immunofluorescence was used to detect PI3K. Results: Compared with those in the model control group, the contents of CollageⅠand fibronectin in the supernatant of emod-in group significantly decreased (P<0. 05), the expression of PI3K protein was inhibited, the expression of downstream p-Akt protein decreased, and the downstream mTOR decreased (P<0. 05), the expression levels of PI3K, p-Akt and mTOR mRNA decreased, the differences were statistically significant (P<0. 05), and the expression of PI3K decreased. Conclusion: Emodin can alleviate fibrosis of HK-2 cells stimulated by TGF-β1 through the classical Akt/mTOR pathway of autophagy.

Matrix stiffness impacts the morphology and the levels of glucose transporter proteins in human renal tubular epithelial cells / 药学学报

As the common pathway of chronic renal diseases leading to end-stage renal failure, renal tubulointerstitial fibrosis is characterized by the deposition of extracellular matrix and scar hardening. Our study aimed to construct an in vitro cell culture platform to explore the impact of matrix stiffness on cell morphology and function of renal tubular epithelial cells. Photopolymerized polyacrylamide gels (PAA gel) with varying stiffnesses as model substrates was selected to simulate the matrix stiffness of normal and fibrotic renal tissues with elastic moduli ranging from 1 to 40 kPa. The human renal tubular epithelial cells (HK-2) were seeded on the surface of PAA gels. The impact of matrix stiffness on the morphology of HK-2 were investigated via immunofluorescence staining and confocal microscopy. The expression levels of glucose transporter 1 (GLUT1), glucose transporter 2 (GLUT2), glucose transporter 5 (GLUT5) were semi-quantitatively analyzed. With increasing matrix stiffness, both the levels of GLUT1 and GLUT5 in HK-2 cells were significantly decreased, whereas the expression level and the distribution pattern of GLUT2 in HK-2 remained unchanged with stiffness variation.

Mitochondrial oxidative damage and apoptosis induced by high glucose through Rho kinase signal pathway in renal tubular epithelial cells

Objective:To investigate the role of oxidative stress in human renal tubular epithelial cells (HK-2) induced by high glucose and the underlying signal pathway in vitro.Methods:MYPT1, pro-caspase-3, PGC-1α, and Drp1 protein expressions were measured by Western blot. MnSOD2, Drp1 and PGC-1α mRNA expressions were detected by real time PCR.Results:Results showed that high glucose significantly up-regulated the protein expressions of MYPT1, pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells; while Rho kinase inhibitor fasudil and ROCK1 siRNA inhibited protein expressions of pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells induced by high glucose. Importantly, fasudil and ROCK1 siRNA markedly inhibited the expressions of mitochondrial motor proteins Drp1 and mitochondrial gene PGC-1α in HK-2 cells induced by high glucose.Conclusions:Our findings suggest that Rho kinase signal pathway is involved in mitochondrial oxidative damage and apoptosis in high glucose-induced renal tubular epithelial cells by regulating mitochondrial motor proteins Drp1 and mitochondrial gene PGC-1α. Targeting Rho kinase signal pathway might be a potential strategy for the treatment of diabetic nephropathy.

Mitochondrial oxidative damage and apoptosis induced by high glucose through Rho kinase signal pathway in renal tubular epithelial cells

Objective: To investigate the role of oxidative stress in human renal tubular epithelial cells (HK-2) induced by high glucose and the underlying signal pathway in vitro. Methods: MYPT1, pro-caspase-3, PGC-1α, and Drp1 protein expressions were measured by Western blot. MnSOD2, Drp1 and PGC-1α mRNA expressions were detected by real time PCR. Results: Results showed that high glucose significantly up-regulated the protein expressions of MYPT1, pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells; while Rho kinase inhibitor fasudil and ROCK1 siRNA inhibited protein expressions of pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells induced by high glucose. Importantly, fasudil and ROCK1 siRNA markedly inhibited the expressions of mitochondrial motor proteins Drp1 and mitochondrial gene PGC-1α in HK-2 cells induced by high glucose. Conclusions: Our findings suggest that Rho kinase signal pathway is involved in mitochondrial oxidative damage and apoptosis in high glucose-induced renal tubular epithelial cells by regulating mitochondrial motor proteins Drp1 and mitochondrial gene PGC-1α. Targeting Rho kinase signal pathway might be a potential strategy for the treatment of diabetic nephropathy. http://www.apjtm.org/article.asp?issn=1995-7645;year=2018;volume=11;issue=6;spage=399;epage=404;aulast=Li;type=2.

Effect of long non-coding RNA TapSAKI on the renal tubular epithelial cells induced by hypoxia / 中华实用儿科临床杂志

Objective To study the effect of long non-coding(LncRNA)TapSAKI on HK-2 cells of renal tubular epithelial cells induced by hypoxia.Methods The cultured HK-2 cells were divided into the normal oxygen group(group A),hypoxia injury group(group B),hypoxia injury+control siRNA group(group C),and hypoxia injury+TapSAKI siRNA group(group D).The real-time quantitative polymerase chain reaction(qPCR)was used to detect the change in TapSAKI expression;proliferation was measured by CCK-8 assay;cell cycle and cell apoptosis were measured by flow cytometry;Western blot was used to detect cycle related CyclinD1 protein,CDK2 and apoptosis related protein Bcl-2 and Bax.Results From the result of qPCR,compared with group A,the expression of TapSAKI gene in group B increased significantly(48.92 ± 0.31 vs.1.00 ± 0.01,P<0.05).Compared with group C,the expression of TapSAKI gene in group D decreased significantly(4.70 ± 0.60 vs.48.31 ± 0.29,P<0.05).The result of CCK-8 showed that the proliferation in group B significantly decreased compared with group A(P<0.05).Compared with group C,the proliferation in group D significantly increased(P<0.05).The flow cytometer test results showed that the apoptosis rate in group B was higher than that in group A[(26.38 ± 1.21)% vs.(6.45 ± 0.46)%,P<0.05].Com-paring with group C,the apoptosis rate in group D decreased significantly[(10.98 ± 0.88)% vs.(21.59 ± 1.30)%, P<0.05].Compared with group A,Bax expression in group B increased,and Bcl-2 expression decreased(1.304 ± 0.082 vs.0.411 ± 0.002,0.390 ± 0.007 vs.1.027 ± 0.022,all P<0.05).In group D,the expression of Bax was lower than that in group C,and the expression of Bcl-2 increased,both of which were statistically significant(0.655 ± 0.819 vs.1.419 ± 0.087,0.819 ± 0.034 vs.0.437 ± 0.014,all P<0.05).Compared with group A,the proportion of G0/G1 phase cells in group B significantly increased(69.82 ± 1.14 vs.34.46 ± 0.82,P<0.05),and the proportion of S phase cells decreased significantly(11.6 ± 0.60 vs.42.23 ± 1.46,P<0.05).Compared with group A,CyclinD1 and CDK2 protein expressions in group B decreased(0.659 ± 0.062 vs.1.723 ± 0.084,0.414 ± 0.015 vs.0.87 ± 0.031, all P<0.05).Compared with group C,group D G0/G1 phase cells significantly decreased(30.77 ± 0.33 vs.61.81 ±1.50,P<0.05),the proportion of S phase cells significantly increased(40.32 ± 0.72 vs.17.92 ± 0.71,P<0.05), CyclinD1 and CDK2 protein expressions increased(2.049 ± 0.027 vs.0.626 ± 0.024,0.89 ± 0.104 vs.0.424 ± 0.012,all P<0.05).Conclusions Under hypoxic conditions,LncRNA TapSAKI in renal tubular epithelial cells was abundant,which may inhibit renal tubular epithelial cell proliferation and accelerate apoptosis.It is suggested that Ln-cRNA TapSAKI may play a role in the deterioration of cell proliferation and apoptosis.

Changes of neutrophil gelatinase associated lipocalin after endoplasmic reticulum stress by thapsigargin in renal tubular epithelial cells / 国际儿科学杂志

Objective To observe the changes of neutrophil gelatinase associated lipocalin,NGAL after endoplasmic reticulum stress by thapsigargin in renal tubular epithelial cells.Methods The renal tubular epithelial cells were treated with thapsigargin to establish the model of endoplasmic reticulum stress (ER group).CHOP and GRP78 expression were detected by Western-blot to confirm endoplasmic reticulum stress in renal tubular epithelial cells by thapsigargin.NGAL expression were detected by Western-blot in ER group and control group(normal renal tubular epithelial cells).Results After treated with 2.5 μmol/L and 5 μmol/L thapsigargin for 4 hours and 8 hours respectively,endoplasmic reticulum stress in the renal tubular epithelial cells were significantly increased comparing with control group (0.585 ± 0.045 to 0.523 ± 0.030;0.785 ± 0.049 to 0.728 ± 0.064),which was approved by the increased expression of CHOP and GRP78 (P ＜ 0.05).The expression of NGAL was significantly increased in ER group (0.567 ± 0.024 to 0.826 ± 0.057,P ＜ 0.05) and it was corresponded with the changes of CHOP and GRP78,which increased significantly with the severity of endoplasmic reticulum stress.Conclusion Endoplasmic reticulum stress can be induced by thapsigargin in renal tubular epithelial cells.The expression of CHOP,GRP78 and NGAL were significantly increased correspondingly with the severity of endoplasmic reticulum stress.It is speculated that the expression of NGAL may be related with endoplasmic reticulum stress in tubular epithelial cells.