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 .

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.

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.

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.

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.

Effect of Compound Tangshenning on Proliferation and RhoA/ROCK Signaling Pathway of High Glucose-induced Epithelial Cells of Renal Tubules / 世界科学技术-中医药现代化

Objective:To study the effect of the prescription Tangshenning on the proliferation of the high glucoseinduced cells of near-end kidney tubules.Method:To prepare durg-contained serum from mice to enter into in vitor reaction system,the cells were randomly divided into 4 groups:the normal group,the model group,the,the irbesartan group,the tangshenning group and then detect the effects of all serum sections on the proliferation of high glucoseinduced epithelial cells of kidney tubules by the MTT colorimetric method,and the expression of RhoA,ROCK 1,Ecadherin and α-SMA in each group were detected by Western blotting.Result:The high glucose group of renal tubular epithelial cells form from the flat irregular polygon into long fusiform;After adding the drug-containing serum corresponding intervention into the cells into a flat in irregular polygon.The high glucose group of renal tubular epithelial cells show obvious proliferation condition,In the condition of 24 h,48 h,The high glucose group proliferation is better than the normal group (P＜0.01),in 60 h,The high glucose group proliferation is better than the normal group (P＜0.05);In 24 h,compared with the irbesartan,Tangshenning has better effect of inhibiting the cell proliferation(P＜0.05);In 48 h,Tangshenning has the better effect than irbesartan and Y27632 (P＜0.01);In 60 h,Tangshenning has the better effect than irbesartan and Y27632 (P＜0.05);Western blotting:Western blotting analysis showed that compared with the normal group,the expression of RhoA protein in high glucose group and Y27632 decreased (P＜0.01);The high glucose group and Tangshenning have significant difference (P＜0.01);Y27632 and Tangshenning have difference (P＜0.05).compared with the normal group,the expression of ROCK1 protein in high glucose group decreased (P＜0.01);The high glucose group,Tangshenning,the irbesartan and Y27632 have difference (P＜0.05).Compared with the normal group,the expression of a-SMA protein in high glucose group decreased (P＜0.01);The high glucose group,Tangshenning,the irbesartan and Y27632 have difference (P＜0.05).Compared with the normal group,the expression of E-Cadherin protein in high glucose group increased (P＜0.05).Y27632 and Tangshenning have difference (P＜0.05).The high glucose group,Tangshenning,the irbesartan and Y27632 have difference (P＜0.05).Conclusion:The prescription Tangshenning is able to inhibit the proliferation of high glucose-induced epithelial cells of kidney tubules and and can reverse renal tubularepithelial cell transdifferentiation via regulating RhoA/ROCK signaling pathway,and restrain renal interstitial fibrosis,thereby delaying the pathogenesis of diabetic kidney disease.

Effect of Raloxifene on Cadmium Chloride-Contaminated Renal Tubular Epithelial Cells / 医药导报

Objective To investigate the effect of raloxifene on ERα, ERβ and MT-1a expression in renal tubular epithelial cells contaminated with cadmium chloride,for exploring the protective effect and action mechanism of raloxifene in renal injury induced by cadmium. Methods Renal tubule cells were isolated from kidneys of new born SD rats and purified by Percoll. The cells of the second generation were selected for experiment. The cells were divided into 3 groups:blank control group,cadmium chloride group and raloxifene group. After 4 h,cell viability was detected by MTT,and after 24 h,mRNA and protein expression levels of ERα, ERβ and MT-1a in renal tissues were determined by RT-PCR and immunohistochemistry technology,respectively. Results Compared with the blank control group,morphology of plenty cells were changed in cadmium chloride group,a number of cells were dead,and the survival rate was only 55.3%;at the same time,mRNA and protein expression levels of ERβ and MT-1a significantly increased (P0.05) . Conclusion Raloxifene can bond with ERβcompetively,down-regulate MT-1a and decrease Cd-MT synthesis,so as to reduce cadmium-induced damage of renal tubular epithelial cells.

Effects of New Tangshenkang on α-SMA and E-cadherin of Human Renal Tubular Epithelial Cell HK-2 in High Concentrations of Glucose / 中国中医药信息杂志

Objective To observe he effects of new Tangshenkang on α-SMA and E-cadherin of human renal tubular epithelial cell HK-2 in high concentrations of glucose; To explore the mechanism of new Tangshenkang on the prevention and treatment of diabetic renal fibrosis. Methods The HK-2 cells were cultured and divided into control group, high glucose group, animal serum control group, new Tangshenkang low-, medium-, and high-dosage group. After medicine intervention, cell proliferation was tested by MTT assay, and contents of α-SMA and E-cadherin were observed by ELISA assay. Results Compared with control group, α-SMA of HK-2 cultured with high glucose was much notable, but the content of E-cadherin significantly decreased, with statistical significance (P<0.05). The content of α-SMA of HK-2 cultured with new Tangshenkang decreased, and the content of E-cadherin increased; cell proliferation was markedly inhibited in culture medium supernatant of HK-2 cells cultured with high glucose plus new Tangshenkang compared with only high glucose, with statistical significance. Conclusion New Tangshenkang can inhibit cell proliferation and epithelial-myofibroblast transdifferentiation of HK-2 cell induced by high glucose, and prevent the development of diabetic renal fibrosis to a certain extent.

Construction of a C3aR expres sion vector and infection in tubular epithelial cells / 医学研究生学报

Objective Our previous study showed that the expression of C 3aR was increased in renal tubular epithelial cells (RTEC).However, the role of C3aR in diabetic nephropathy remains unknown, and so does the exact physiological and pathological signifi-cance of C3aR in relevant renal tissues.In the present study, we investigated the physiological and pathological significance of C3aR in RTECs using a lentivirus expression vector and anRTEC strain overexpressing C3aR. Methods Based on the sequence of C3aR mRNA, the human C3aR gene was synthesized and cloned into the multi-clonal site of the lentivirus expression vector pLenti6.3-MCS-IRES2-EGFP to construct a C3aR expression vector pLenti6.3-C3aR-IRES2-EGFP.After identification by sequencing, the recombinant lentivirus expressing C3aR was packaged by cotransfecting293T cellswith the recombinant lentivirusexpression vector and packaged plasmid .Then,the recombinant lentivirus was used to infect the human RTECline HK 2.After screening in the medium with blasticidin, blasticidinr-esistant cell clones were obtained, followed by identification ofthe human RTECline stably overexpressing C 3aR by real-time PCR and immunochemical staining. Results TheC3aR expression vector pLenti6.3-C3aR-IRES2-EGFP was successfully constructed and the sequence was proved to be correct. C3aR expression recombinant lentivirus was successfully packaged with a titer of 5×108/mL.Blasticidin-resistant HK2 cell clones were ob-tained and the expression of HK2-C3aR mRNA was significantly higherin the HK2-C3aR cells than in the non-transfected HK2 cells (2.33± 0.45 sv 1.00±0.09, P<0.05). Conclusion We successfully constructed a C3aR expression lentivirus vector pLenti6.3-C3aR-IRES 2-EGFP and a C3aR overexpression renal tubular epithelial cell strain HK2-C3aR, which is very contributive to further studies of the roles of C3aR in renal tubular epithelial cells and other types of cells.

Role of homeobox A13 in epithelial - to - mesenchymal transition induced by serum albumin overload in hu-man renal tubular epithelial cells / 中华实用儿科临床杂志

Objective To explore the role of nuclear translational factor homeobox A13(HOXA13)gene in epithelial - to - mesenchymal transition(EMT)induced by human serum albumin(HSA)overload in human renal tu-bular epithelial(HKC)cells. Methods HKC cells were treated with different concentrations of HSA(ranging from 0 - 30 g/ L)for 48 h or 20 g/ L HSA for different times(ranging from 0 - 72 h)in vitro. The protein expressions of cy-tokeratin(CK),Vimentin,and HOXA13 protein in HKC cells were detected by using Western blot respectively. Mean-while,liposome - mediated DNA transfection was used to transfect the HOXA13 gene into HKC cells before HSA treat-ment,and the expressions of CK,Vimentin and HOXA13 protein in HKC cells were also detected by using Western blot. Results (1)The protein expression of CK decreased but Vimentin increased after HKC cells were exposed to HSA,which was in a concentration - and time - dependent manner.(2)Expression of HOXA13 was down - regulated by HSA in a dose - and time - dependent manner,and the expressions of HOXA13 protein in HKC in 5 g/ L,10 g/ L, 20 g/ L,30 g/ L group were 58. 24%(P = 0. 005),44. 73%(P = 0. 003),38. 40%(P = 0. 033)and 24. 83%(P =0. 011)respectively as compared with 0 g/ L group. Likewise,the protein expressions of HOXA13 in 24 h,48 h,72 h group were 52. 00%(P = 0. 023),46. 83%(P = 0. 008)and 35. 10%(P = 0. 034)respectively as compared with 0 h group.(3)There was a positive correlation between the levels of HOXA13 protein expression and CK protein expression (r = 0. 86,P = 0. 005),while the relationship between the levels of HOXA13 protein expression and Vimentin protein expression was negative(r = - 0. 94,P = 0. 002).(4)Up - regulated expression of HOXA13 in HKC cells by lipo-fectamine transfection alleviated the degree of EMT induced by HSA significantly. The expression of Vimentin decreased by 35. 34%(P = 0. 005)while the expression of CK increased 360. 00% - fold(P = 0. 005),compared with that of untransfected HKC cells. Conclusion EMT induced by HSA in HKC cells may play a role through HOXA13.

Effects ofSimiaosan on CTGF, BMP-7 Expression in Human Renal Tubular Epithelial Cell Induced by Uric Acid / 中国中医药信息杂志

Objective;To explore the mechanism ofSimiaosan drug-containing serum on prevention and treatment of renal fibrosis.Methods Human renal tubular epithelial cells (HK-2) were divided into blank group, model group, control group, and drug-containing serum high dosage group, medium dosage group and low dosage group. The expressions of CTGF and BMP-7 were determined by real-time PCR assay and immunocytochemistry.Results After the HK-2 cell was induced by uric acid (UA), the expression of CTGF significantly increased (P<0.05), the expression of BMP-7 significantly decreased (P<0.05). After intervened bySimiaosan, the expression of CTGF significantly decreased (P<0.05), the expression of BMP-7 significantly increased (P<0.05) and present dosage dependent.Conclusion Reducing the expression of CTGF and up-regulation the expression of BMP-7 and controlling epithelial-myofibroblast transdifferentiation may be the mechanism ofSimiaosan on prevention and treatment of renal fibrosis.

EIF6 delays the kidney interstitial tissue fibrosis process by regulating the expression of TGF-β1 / 重庆医学

Objective To investigate the possible role of eukaryotic initiation factor 6(eIF6)in the development of kidney inter-stitial tissue fibrosis in a mouse model of unilateral ureteral obstruction(UUO).Methods (1)UUO model was set up in eIF6+/-and eIF6+/+ mice.(2)HE and Masson staining were used to histologically assess the interstitial deposition of collagen in mouse kidney.Immunohistochemistry and image analysis were applied to analyze the expression of α-SMA and TGF-β1 in mouse kidney as well as eIF6 in human kidney samples.Results 10 days after unilateral ureteral obstruction,more kidney interstitial fibrosis tissues were found in eIF6+/- mice than that in eIF6+/+ mice.TGF-β1 andα-SMA were expressed more intensive in tubular epithelial cells of eIF6+/- mice than that of eIF6+/+ mice in the UUO model.Expression of eIF6 is lower in the region of fibrosis than that in non-fibrotic area.Conclusion eIF6 might be involved in the development of kidney interstitial tissue fibrosis by regulating the expres-sion of TGF-β1 .