Functional analysis reveals calcium-sensing receptor gene regulating cell-cell junction in renal tubular epithelial cells.

PURPOSE: Calcium-sensing receptor (CASR) influences the expression pattern of multiple genes in renal tubular epithelial cells. The objective of this inquiry was to explore the molecular mechanisms of CASR in renal tubular epithelial cells and nephrolithiasis. METHODS: HK-2 cells were transfected with lentiviruses carrying either CASR (named CASR) or an empty vector negative control (named NC), as well as shRNA intended to target CASR (named shCASR) or its corresponding negative control (named shNC). CCK-8 assay was used to detect the effect of CASR on the proliferation of HK-2 cells. RNA-Sequencing was applied to explore potential pathways regulated by CASR in HK-2 cells. RESULTS: PCR and western blot results showed that CASR expression was significantly increased in CASR cells and was decreased in shCASR cells when compared to their corresponding negative control, respectively. CCK-8 assay revealed that CASR inhibited the proliferation of HK-2 cells. RNA-Sequencing results suggested that the shCASR HK-2 cells exhibited a significant up-regulation of 345 genes and a down-regulation of 366 genes. These differentially expressed genes (DEGs) were related to cell apoptosis and cell development. In CASR HK-2 cells, 1103 DEGs primarily functioned in mitochondrial energy metabolism, and amino acid metabolism. With the Venn diagram, 4 DEGs (Clorf116, ENPP3, IL20RB, and CLDN2) were selected as the hub genes regulated by CASR. Enrichment analysis revealed that these hub genes were involved in cell-cell junction, and epithelial cell development. CONCLUSIONS: In summary, our investigation has the potential to offer novel perspectives on CASR regulating cell-cell junction in HK-2 cells.

Effect of L-carnitine on ferroptosis in renal tubular epithelial cells induced by calcium oxalate / 中华泌尿外科杂志

Objective:To investigate the effect of L-carnitine on calcium oxalate-induced ferroptosis in renal tubular epithelial cells (HK-2).Methods:The effects of calcium oxalate(0, 2, 4 and 8 mmol/L) on the expression of ferroptosis-related protein long chain fatty acyl-CoA synthetase 4 (ACSL4), cystine/glutamate transporter(XCT) and glutathione peroxidase 4 (GPX4) in HK-2 cells were detected by Western blotting. The experiment was then divided into four groups: ①control group, cells were cultured in normal medium for 12 hours, then continued to use normal medium; ②L-carnitine group, cells were pretreated with medium containing 5mmol/L L-carnitine for 12 hours, then changed to medium containing 5mmol/L L-carnitine; ③calcium oxalate group, cells were cultured in normal medium for 12 hours, and then replaced with medium containing 4 mmol/L calcium oxalate; ④calcium oxalate+ L-carnitine group, the cells were pretreated with medium containing 5mmol/L L-carnitine for 12 h, and then replaced with 5mmol/L L-carnitine and 4mmol/L calcium oxalate medium. After changing the culture medium for 24 hours, the cells or supernatants were collected, and the expression levels of ferroptosis-related protein quinone oxidoreductase (NQO1), ACSL4, XCT and GPX4 were detected by Western blotting. The levels of superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde were detected by corresponding kit, and the level of reactive oxygen species in cells was detected by reactive oxygen species kit.Results:The results of Western blotting showed that the expression of ACSL4 protein in 0, 2, 4, 8 mmol/L calcium oxalate was 0.37±0.16, 0.68±0.16, 0.73±0.09, 0.89±0.03 respectively. The expression of XCT protein was 1.11±0.10, 0.91±0.14, 0.83±0.09, 0.80±0.07, respectively. The expression of GPX4 protein was 1.23±0.13, 0.99±0.17, 0.81±0.05, 0.72±0.06, respectively. Compared with 0mmol/L group, the expression of ACSL4 protein increased and the expression of XCT and GPX4 decreased in 2, 4 and 8 mmol/L groups, and the difference was more significant between 4 mmol/L group and 0 mmol/L group. So 4 mmol/L was taken as the optimal concentration for follow-up experiment. The levels of NQO1 in control group, L-carnitine group, calcium oxalate group and calcium oxalate+ L-carnitine group were (0.36±0.06, 0.54±0.05, 0.76±0.07, 0.90±0.03) respectively. There was significant difference between L-carnitine group and control group ( P<0.05). There was significant difference between calcium oxalate group and control group ( P<0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of ACSL4 in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (0.66±0.10, 0.58±0.08, 0.99±0.03, 0.77±0.09) respectively. There was no significant difference between L-carnitine group and control group(P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of XCT in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (0.93±0.08, 0.85±0.07, 0.76±0.06, 0.99±0.05). There was no significant difference between L-carnitine group and control group (P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of GPX4 in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (1.10±0.09, 1.09±0.09, 0.85±0.03, 0.99±0.02) respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of LDH in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine were (100.00±5.37)%, (99.50±6.38)%, (153.77±6.06)% and (132.50±5.58)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The SOD levels in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (100.00±5.79)%, (105.80±3.26)%, (44.74±7.60)% and (85.01±5.15)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of GSH in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (100.00±4.73)%, (107.10±5.48)%, (53.49±3.98)% and (85.18±5.48)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.01). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.01). The levels of MDA in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (100.00±2.36)%, (98.00±11.10)%, (129.11±2.59)% and (113.35±5.79)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.01). The fluorescence intensity of ferrous ion in control group, calcium oxalate group and calcium oxalate + L-carnitine group was (39.77±0.68) AU, (68.40±3.14) AU and (48.60±4.30) AU, respectively. There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The fluorescence intensity of reactive oxygen species in control group, calcium oxalate group and calcium oxalate + L-carnitine group was (63.98±9.41) AU, (145.41±8.39) AU and (85.37±4.51) AU, respectively. There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.01). Transmission electron microscopy results showed that mitochondria were wrinkled, cristae were broken or disappeared in the calcium oxalate group compared to the control group, and a double-layer membrane structure was evident. DAPI staining showed that compared with the control group, some of the nuclei in the calcium oxalate group were significantly more damaged, while compared with the calcium oxalate group, the nuclei in the calcium oxalate + L-carnitine were significantly less damaged. The results of crystal adhesion test showed that compared with the control group, calcium oxalate crystals in the calcium oxalate group adhered to the cells in black-like particles and formed clusters. Compared with the calcium oxalate group, the calcium oxalate + L-carnitine showed less black particles adhering to the cells. Conclusions:L-carnitine may reduce the effects of oxidative stress and ferroptosis induced by calcium oxalate, thus reducing cell damage and crystal adhesion.

Determining the influence of high glucose on exosomal lncRNAs, mRNAs, circRNAs and miRNAs derived from human renal tubular epithelial cells.

Diabetic nephropathy is a lethal disease that can lead to chronic kidney disease and end-stage kidney disease. Exosomes, which are nanosized extracellular vesicles, are closely involved in intercellular communication. Most importantly, exosomes play critical roles in disease occurrence and development. However, the function of exosomes in diabetic nephropathy progression has not been fully elucidated. In the present study, we determined the expression profiles and differences of lncRNAs, mRNAs, circRNAs and miRNAs in exosomes derived from human renal tubular epithelial cells with or without high glucose (HG) treatment. A total of 169 lncRNAs, 885 mRNAs, 3 circRNAs and 152 miRNAs were differentially expressed in exosomes secreted by HG-challenged HK-2 cells (HG group) compared with controls (NC group). The functions of differentially expressed mRNAs, mRNAs colocalized or coexpressed with differentially expressed lncRNAs (DElncRNAs), potential target genes of miRNAs and source genes of circRNAs were investigated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. According to these differentially expressed RNAs, we established an integrated circRNA-lncRNA-miRNA-mRNA regulatory network. In conclusion, our study suggested that exosomal lncRNAs, mRNAs, circRNAs and miRNAs participate in the progression of diabetic nephropathy and may be possible biomarkers and therapeutic targets in diabetic nephropathy.

Potential signaling pathway through which Notch regulates oxidative damage and apoptosis in renal tubular epithelial cells induced by high glucose.

Diabetic nephropathy (DN) is one of the most common and serious complications of diabetes mellitus, and glomerular sclerosis and renal tubular interstitial fibrosis are the main pathological features. Current evidence indicates that the Notch pathway can mediate the impairment of renal tubular function and induce angiogenesis and renal interstitial fibrosis. This study was conducted to explore the potential signaling pathway through which Notch regulates oxidative damage and apoptosis in renal tubular epithelial cells induced by high glucose. mRNA and protein expression levels were assessed using real-time PCR and Western blot, respectively. The protein expression levels of Jaggedl, Notchl, pro-caspase-3, Drpl, and PGC-1α were increased by high glucose, but N-[N-(3,5-difluorohenacetyl)-l-alanyl]-S-phenylglycine tert-butyl ester (DAPT; an inhibitor of the Notch signaling pathway) reversed these effects. Furthermore, DAPT reduced the mRNA expression of Jaggedl, Notchl, MnSOD2, Drpl, and PGC-1α in renal tubular epithelial cells induced by high glucose. In conclusion, the Notch signaling pathway may regulate oxidative damage and apoptosis in renal tubular epithelial cells induced by high glucose by regulating mitochondrial dynein and biogenesis genes, which can accelerate renal interstitial fibrosis in DN. The Notch signaling pathway might be a potential therapeutic target for DN, and DAPT might become a potential drug for the treatment of DN.

Effect of hydroxysafflor yellow A on apoptosis of human renal tubular epithelial cells under cold hypoxia and reoxygenation / 中国综合临床

Objective:To investigate the effect of hydroxysafflor yellow A(HSYA) preconditioning group on apoptosis induced by cold hypoxia/reoxygenation (cold H/R) injury in human renal tubular epithelial cells (HK2 cells).Methods:After digestion and passage, HK2 cell lines were divided into Sham group (control group), cold hypoxia and reoxygenation group (cold H/R group, cells cold hypoxia for 4 h, reoxygenation for 4 h), and HSYA preconditioning group (each HSYA subgroup was given different doses of HSYA 0.5 h before hypoxia, and the other operations were the same as the cold H/R group). The cell survival rate was measured by CCK-8 method.The expression of Bcl-2, Bax and Caspase-3 proteins in HK-2 cells were detected by immunocytochemistry and Western blotting.Results:(1) Compared with cold H/R group, different doses of HSYA could improve cell survival rate in different degrees, but only HSYA25 μmol/L group had the most significant effect (74.000±5.500 vs.59.000±3.800, P<0.05). (2) Immunocytochemistry semi-quantitative score: Compared with cold H/R group, the expression of Bax and Caspase-3 in HK2 cells of HSYA25 μmol/L group was significantly decreased(0(0, 1) vs. 8(6, 8), Z=2.041, P<0.05 and (3.400±0.548) vs.(7.800±1.095), t=11.000, P<0.01). The expression of Bcl-2 protein was increased significantly ((6.800±1.095) vs.(1.400±0.548), t=10.590, P<0.01). The ratio of Bcl-2/Bax increased significantly.(3)Western blot was used to detect protein: Compared with the cold H/R group, the protein levels of Bax, Cleaved-Caspase-3 and Pro-caspase-3 of HK2 cells in the HSYA25 μmol/L group were significantly decreased ((0.707±0.012) vs.(0.968±0.117), (0.480±0.009)vs.(0.735±0.005), (0.992±0.008)vs.(1.197±0.005), all P<0.01). The expression of Bcl-2 protein was significantly increased, and the ratio of Bcl-2/Bax was significantly increased ((0.410±0.009) vs.(0.273±0.008), (0.582±0.016) vs (0.282±0.080), all P<0.01). The experimental results were consistent with the immunocytochemistry. Conclusion:HSYA can effectively reduce the damage of HK2 cells after cold hypoxia and reoxygenation.

Effects of Sulforaphane on the Proliferation and Apoptosis of Human Renal Tubular Epithelial Cells Induced by High Glucose and Its Mechanism / 中国药房

OBJECTIVE：To study the effects of sulforaphane on the prolifera tion and apoptosis of human renal tubular epithelial cells HK- 2 induced by high glucose ，and to investigate its mechanism primarily. METHODS ：HK-2 cells were divided into normal group ，high glucose group ，irbesartan group （positive control ，1 μmol/L），sulforaphane low ，medium and high concentration groups （10，20，40 μmol/L）. The cells in normal group were cultured in DMEM medium for 96 hours. T he cells in other groups were cultured in high glucose DMEM medium （containing 40 mmol/L glucose ）for 48 hours. After inducing cell injury，the cells were added with corresponding drugs for 48 hours. Survival rate and apoptotic rate of cells were detected. mRNA expression of cyclin D 1，caspase-3，Bcl-2 and Bax as well as protein expression of p-mTOR ，p-AMPK，p-Akt and p-PI 3K were also determined. In addition ，HK-2 cells were divided into normal group ，high glucose group ，sulforaphane high concentration group（40 μmol/L），acardicin group （AMPK agonist ，1 mmol/L），sulforaphane high concentration+compound C group （sulforaphane 40 μmol/L+AMPK inhibitor compound C 40 μmol/L），perifoxine group （Akt inhibitor ，19.95 μmol/L）、sulforaphane high concentration+SC 79 group（sulforaphane 40 μmol/L+Akt agonist SC79 4 μmol/L）. After cultured with the same method ， protein expression of p-mTOR ，p-AMPK，p-Akt and p-PI 3K were detected in HK- 2 cells. RESULTS ：Compared with normal group，survival rate of HK- 2 cells，mRNA expression of cyclin D 1 and Bcl- 2 as well as protein expression of p-AMPK were decreased significantly in high glucose group （P＜0.05）；apoptotic rate ，mRNA expression of caspase- 3 and Bax ，protein expression of p-mTOR ，p-Akt and p-PI 3K in HK- 2 cells were increased significantly （P＜0.05）. Compared with high glucose group，above indexes of sulforaphane low ，medium and high concentration groups ，irbesartan group were all improved significantly （P＜0.05）；the improvement of above indexes in sulforaphane medium and high concentration groups were significantly better those of sulforaphane low concentration group （P＜0.05）. There was no significant difference in above indexes between sulforaphane high concentration group and irbesartan group （P＞0.05）. Compared with sulforaphane high concentration group，there were no significant difference in the protein expression of p-AMPK ，p-mTOR in acardicin group and p-mTOR ，p-Akt and p-PI 3K in perifoxine group （P＞0.05）；the protein expression of p-AMPK in sulforaphane high concentration+compound C group was decreased significantly （P＜0.05），while the protein expression of p-mTOR was increased significantly （P＜0.05）；the protein expression of p-mTOR 、p-Akt、p-PI3K in sulforaphane high concentration+SC 79 group were increased significantly （P＜ 0.05）. CONCLUSIONS ：Sulforaphane can promote the proliferation of renal tubular epithelial cells and inhibit its apoptosis ；its mechanism may be associated with up-regulating the expression of p-AMPK and down-regulating the expression of p-mTOR ，p-Akt and p-PI 3K.

Effect of Tongfengning Serum Containing on Expression of Urate Transporter in HK-2 Induced by Uric Acid / 中国实验方剂学杂志

Objective: Study on the mechanism of Tongfengning in reducing serum uric acid from the perspective of renal urate transporter. Method: The human renal tubular epithelial cells(HK-2)was randomly divided into normal group, model group, Tongfengning low, medium and high dose group (7.65,15.3,30.6 g·kg-1) and benzbromarone group (50 μmo1·L-1),different culture media were given for intervention.HK-2 and cell supernatant were collected after 24 h of intervention. The expressions of urate transporter 1 (URAT1), glucose transporter 9 (GLUT9), organic anion transporter 1(OAT1), organic anion transporter 3(OAT3), and ATP-binding cassette superfamily G member 2 (ABCG2) protein and mRNA were detected in HK-2 of all groups by Western blot and Real-time PCR. Result: Compared with normal group, the expression of URAT1, GLUT9 protein and mRNA was significantly increased(PPPPPConclusion: Tongfengning can regulate the reabsorption and secretion of uric acid in renal tubules, promote the excretion of uric acid in kidney and reduce the level of serum uric acid by down-regulating the expression of URAT1, GLUT9 protein and mRNA in HK-2 and up-regulating the expression of ABCG2 protein and mRNA. It is suggested that the regulation of renal uric acid transporter protein may be one of the specific mechanisms of Tongfengning to reduce serum uric acid by promoting dampness and turbid removal. OAT1, OAT3 protein and mRNA were not expressed in HK-2 cultured in vitro.

Effect and mechanism of atorvastatin on cellular inflammatory response induced by calcium oxalate crystals / 中华泌尿外科杂志

Objective@#To investigate the effect and mechanism of atorvastatin (ATV) on the inflammatory response of human renal tubular epithelial cells (HK-2 cells) induced by calcium oxalate crystals.@*Methods@#HK-2 cells were divided into control group (normal medium), ATV group (after 3 h pretreatment with 40 μmol/L ATV, replaced with normal medium), calcium oxalate crystal stimulation group (4 mmol/L calcium oxalate crystal) and ATV treatment group (after 3 h pretreatment with 40 μmol/L ATV, replaced with 4 mmol/L calcium oxalate crystals). After 12 h, the cells were collected, and the expression levels of NLRP3 and Cleaved caspase-1 were detected by immunohistochemical staining and Western blotting. The expression level of NF-κB was detected by immunofluorescence and Western blotting. The cell culture supernatant was collected to detecte the concentrations of interleukin-1β (IL-1β) and interleukin-18 (IL-18) by enzyme linked immunosorbent assay (ELISA).@*Results@#Western blot analysis showed that the relative expression of NLRP3 (0.125±0.013 vs. 0.135±0.007) and Cleaved caspase-1 (0.090±0.014 vs. 0.095±0.006) was decreased in the ATV group compared with the control group, but the difference was not statistically significant (P>0.05). The relative expression of NLRP3 (0.315±0.021 vs. 0.135±0.007, P<0.001) and Cleaved caspase-1 (0.235±0.008 vs. 0.095±0.006, P<0.001) was significantly increased in the calcium oxalate crystal stimulation group compared with the control group. While the relative expression of NLRP3 (0.245±0.007 vs. 0.315±0.021, P<0.05) and Cleaved caspase-1 (0.170±0.017 vs. 0.235±0.008, P<0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group. The results of immunohistochemical staining showed that the expression trends of NLRP3 and Cleaved caspase-1 in each group were consistent with those obtained by Western blotting. The ELISA results showed that the concentration of inflammatory factors IL-1β [(162.00±21.21)pg/ml vs. (183.50±7.78) pg/ml, P>0.05] and IL-18 [(176.50±24.12)pg/ml vs.(182.50±20.51)pg/ml, P>0.05] in the ATV group was lower than that in the control group, but the difference were not statistically significant (P>0.05). The concentrations of IL-1β[(850.50±48.79)pg/ml vs. (183.50±7.78)pg/ml, P<0.001] and IL-18 [(526.00±39.61)pg/ml vs. (182.50±20.51)pg/ml, P<0.001] were significantly increased in the cell culture medium of the calcium oxalate crystal stimulation group compared with the control group, while the concentrations of IL-1β [(452.50±36.06)pg/ml vs. (850.50±48.79) pg/ml, P<0.01] and IL-18 [(403.50±23.33)pg/ml vs. (526.00±39.61)pg/ml, P<0.05] was significantly reduced in the cell culture medium of the ATV treatment group compared with the calcium oxalate crystal stimulation group. Western blot analysis showed that the relative expression of NF-κB (0.105±0.021 vs. 0.100±0.014) in the ATV group was decreased compared with the control group, but the difference was not statistically significant (P>0.05). The relative expression of NF-κB (0.295±0.035 vs. 0.100±0.014, P<0.001) in the calcium oxalate crystal stimulation group was significantly increased compared with the control group. While the relative expression of NF-κB (0.160±0.012 vs. 0.295±0.035, P<0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group. The expression of NF-κB by immunofluorescence staining was consistent with the results of Western blotting.@*Conclusions@#Calcium oxalate crystals can induce the inflammatory response of HK-2 cells, while ATV can exert anti-inflammatory effects by inhibiting the activation of NLRP3 inflammasome and decreasing the secretion of inflammatory factors IL-1β, IL-18 and the expression of NF-κB.

Effect and mechanism of atorvastatin on cellular inflammatory response induced by calcium oxalate crystals / 中华泌尿外科杂志

Objective To investigate the effect and mechanism of atorvastatin (ATV) on the inflammatory response of human renal tubular epithelial cells (HK-2 cells) induced by calcium oxalate crystals.Methods HK-2 cells were divided into control group (normal medium),ATV group (after 3 h pretreatment with 40 μmol/L ATV,replaced with normal medium),calcium oxalate crystal stimulation group (4 mmol/L calcium oxalate crystal) and ATV treatment group (after 3 h pretreatment with 40 μmol/L ATV,replaced with 4 mmol/L calcium oxalate crystals).After 12 h,the cells were collected,and the expression levels of NLRP3 and Cleaved caspase-1 were detected by immunohistochemical staining and Western blotting.The expression level of NF-κB was detected by immunofluorescence and Western blotting.The cell culture supernatant was collected to detecte the concentrations of interleukin-1 β (IL-1β) and intedeukin-18 (IL-18) by enzyme linked immunosorbent assay (ELISA).Results Western blot analysis showed that the relative expression of NLRP3 (0.125 ±0.013 vs.0.135 ±0.007) and Cleaved caspase-1 (0.090 ±0.014 vs.0.095±0.006) was decreased in the ATV group compared with the control group,but the difference was not statistically significant (P ＞ 0.05).The relative expression of NLRP3 (0.315 ±0.021 vs.0.135 ± 0.007,P ＜ 0.001) and Cleaved caspase-1 (0.235 ± 0.008 vs.0.095 ± 0.006,P ＜0.001) was significantly increased in the calcium oxalate crystal stimulation group compared with the control group.While the relative expression of NLRP3 (0.245 ±0.007 vs.0.315 ±0.021,P ＜0.05) and Cleaved caspase-1 (0.170 ±0.017 vs.0.235 ±0.008,P ＜0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group.The results of immunohistochemical staining showed that the expression trends of NLRP3 and Cleaved caspase-1 in each group were consistent with those obtained by Western blotting.The ELISA results showed that the concentration of inflammatory factors IL-1 β [(162.00±21.21)pg/ml vs.(183.50±7.78) pg/ml,P＞0.05] and IL-18 [(176.50±24.12)pg/ml vs.(182.50 ± 20.51) pg/ml,P ＞ 0.05] in the ATV group was lower than that in the control group,but the difference were not statistically significant (P ＞ 0.05).The concentrations of IL-1β [(850.50 ± 48.79)pg/ml vs.(183.50 ± 7.78) pg/ml,P ＜ 0.001] and IL-18 [(526.00 ± 39.61) pg/ml vs.(182.50 ±20.51)pg/ml,P ＜0.001] were significantly increased in the cell culture medium of the calcium oxalate crystal stimulation group compared with the control group,while the concentrations of IL-1 β [(452.50 ±36.06)pg/ml vs.(850.50±48.79) pg/ml,P＜0.01] and IL-18 [(403.50 ±23.33)pg/ml vs.(526.00 ±39.61)pg/ml,P ＜0.05] was significantly reduced in the cell culture medium of the ATV treatment group compared with the calcium oxalate crystal stimulation group.Western blot analysis showed that the relative expression of NF-κB (0.105 ±0.021 vs.0.100 ±0.014) in the ATV group was decreased compared with the control group,but the difference was not statistically significant (P ＞ 0.05).The relative expression of NF-κB (0.295 ±0.035 vs.0.100 ±0.014,P ＜0.001) in the calcium oxalate crystal stimulation group was significantly increased compared with the control group.While the relative expression of NF-κB (0.160 ± 0.012 vs.0.295 ± 0.035,P ＜ 0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group.The expression of NF-κB by immunofluorescence staining was consistent with the results of Western blotting.Conclusions Calcium oxalate crystals can induce the inflammatory response of HK-2 cells,while ATV can exert anti-inflammatory effects by inhibiting the activation of NLRP3 inflammasome and decreasing the secretion of inflammatory factors IL-1β,IL-18 and the expression of NF-κB.

Astragalosides IV protected the renal tubular epithelial cells from free fatty acids-induced injury by reducing oxidative stress and apoptosis.

Renal tubular injury is associated with the development of diabetic nephropathy (DN) and the end-stage renal disease (ESRD). Free fatty acids (FFAs)-associated lipotoxicity contributes to injury of proximal renal tubular epithelial (HK-2) cells in diabetes. Palmitic acid (PA) which is the most abundant saturated fatty acid in FFAs is closely associated with the gradual decline of renal function. Astragalosides IV (AS-IV) has a variety of pharmacological effects such as anti-inflammation and anti-oxidation. In the current study, we investigated the effects of AS-IV on PA-induced apoptosis of HK-2 cells and the underlying mechanisms. The results showed that AS-IV (10, 20, 40 µmol/L) could alleviate PA-induced apoptosis of HK-2 cells. We found that AS-IV reduced the expression of Bax and cleaved-caspase3, but increased the expression of Bcl-2 and phosphorylated Nrf2 in HK-2 cells. Moreover, AS-IV reduced the level of reactive oxygen species (ROS) in the cells. Our study suggests that AS-IV could protect against PA-induced apoptosis in HK-2 cells by inhibiting ROS generation and apoptotic protein expression. This study may provide a new theoretical option for the patients with type 2 diabetes.

[Effects of cadmium chloride on mitochondrial function and PGC-1α expression in HK-2 cells].

OBJECTIVE: To observe the effects of cadmium chloride on mitochondrial function and the expression of PGC-1α in HK-2 cells, and explore the possible role of cadmium chloride in the process of mitochondrial oxidative damage of HK-2 cells. METHODS: HK-2 cells were exposed to 0, 10, 20, 30, 40, 50 and 60 µmol/L of CdCl_2 respectively for 24 h in vitro. MTT assay was used to observe the viability of HK-2 cells. The mitochondrial ROS was detected by Mito SOXTMRed staining. Mitochondrial membrane potential was measured by JC-1 staining flow cytometry. The activity of respiratory chain complex III was determined by Multi-functional microplate reader and PGC-1α expression was detected by Western blot analysis. RESULTS: Compared with control, CdCl_2 treatment at 20 and 60 µmol/L for 24 h the survival rate of HK-2 cells was decreased from( 77. 60 ± 0. 82) % ( 41. 97 ± 1. 22) %( P < 0. 01). The activity ofrespiratory chain complex III was decreased in a concentration-dependent manner( P <0. 05), while the mitochondrial ROS production was increased at the same time( P <0. 05), the JC-1 monomers positive ratio by the percentage of the treated cells was 1. 51, 1. 58, 1. 71, 2. 41 3. 47 times higher than untreated control( P < 0. 01). In addition, the expression of PGC-1α was decreased at 24 h( P < 0. 05). CONCLUSION: Cadmium chloride may inhibit the expression of PGC-1α and the activity of respiratory chain complex III, and by inducing the mitochondrial ROS production and reducing mitochondrial membrane potential, leading to the damage of HK-2 cells.

Glioma-associated oncogene homolog 1 promotes epithelial-mesenchymal transition in human renal tubular epithelial cell.

Sonic hedgehog (Shh) signaling critically regulates embryogenesis and tissue homeostasis. Here, we investigated the role of Shh signaling in mediating epithelial-mesenchymal transition (EMT) in human renal tubular epithelial cells HKC-8. Our RT-PCR assays demonstrated that TGF-ß1 induced time-dependent changes in the mRNA transcript levels of Shh, with a steady rise from one hour post TGF-ß1 treatment and a peak at four hours post TGF-ß1 treatment. Furthermore, TGF-ß1 induced a time-dependent increase in the mRNA transcript levels of Gli1. Pre-treatment with 2 or 5 µM cyclopamine significantly attenuated TGF-ß1-induced rise in the mRNA transcript levels of Gli1, but failed to attenuate TGF-ß1-induced rise in Shh mRNA transcript levels. Additionally, immunoblotting assays and immunofluorescence staining demonstrated that inhibition of Shh signaling by cyclopamine significantly attenuated TGF-ß1-induced increase in the mRNA transcript levels of α-SMA, collagen I, and fibronectin. Gli1 overexpression induced Snail1 expression. Moreover, Gli(-/-) mice that had undergone unilateral ureteral obstruction for seven days showed significant reduction in the mRNA transcript levels of Snail1 compared to the wildtype controls. In conclusion, the current study provides novel insight into the regulation of EMT by the Shh/Gli1 signaling pathway, suggesting a critical role of Shh/Gli1 signaling in EMT of human renal tubular epithelial cells.

Human renal tubular epithelial cells suppress alloreactive T cell proliferation.

Renal tubular epithelial cells (TECs) are one of the main targets of alloreactive T cells during acute rejection. We hypothesize that TECs modulate the outcome of alloimmunity by executing immunosuppressive effects in order to dampen the local inflammation. We studied whether TECs possess immunosuppressive capacities and if indoleamine 2,3-dioxygenase (IDO) might play a role in suppressing T cell alloreactivity. Next, we studied the role of programmed death ligand 1 (PD-L1) and intercellular adhesion molecule-1 (ICAM-1 with regard to TEC-related immunomodulatory effects. CD3/CD28 and alloactivated peripheral blood mononuclear cells were co-cultured with activated TECs. We analysed CD4(+) and CD8(+) T cell proliferation and apoptosis in the absence or presence of IDO inhibitor 1-methyl-L-tryptophan (1-L-MT), anti-PD-L1 and anti-ICAM-1. Further, we examined whether inhibition of T cell proliferation was cell-cell contact-dependent. We found that TECs dose-dependently inhibited CD4(+) and CD8(+) T cell proliferation (P<0.05). Activated TECs showed significantly increased IDO activity and up-regulated PD-L1 and ICAM-1 expression. Suppressed CD4(+) and CD8(+) T cell proliferation was only partially restored or failed to restore using 1-L-MT. Activated TECs increased early and late apoptosis of proliferating CD4(+) and CD8(+) T cells; only CD4(+) T cell apoptosis was statistically affected by 1-L-MT. Transwell experiments revealed that TEC-mediated immunosuppression is cell-cell contact-dependent. We found that anti-ICAM-1 affected only CD4(+) T cell apoptosis and not T cell proliferation. Our data show that TECs suppress both CD4(+) and CD8(+) T cell proliferation contact dependently. Interestingly, inhibition of proliferation and enhancement of apoptosis of T cell subsets is differentially regulated by indoleamine 2,3-dioxygenase and ICAM-1, with no evidence for the involvement of PD-L1 in our system.

Effects of 1,25-dihydroxyvitamin D3 on high glucose-induced expression of uncoupling protein 2 and oxidative stress in human renal tubular epithelial cells / 中华内分泌代谢杂志

[Summary] The HK-2 cells with different culture media were divided into normal glucose group (NG group,5.5 mmol/L D-glucose) ; high glucose group (HG group,30 mmol/L D-glucose) ; mannitol group (MG group,5.5mmol/L D-glucose+24.5 mmol/L mannitol) ; 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] groups (V1-V3 group)which were exposed to medium containing 30 mmol/L D-glucose and different concentrations of 1,25-(OH)2D3 ;Nethyl-cysteim control group (NAC group,30 mmoL/L D-glucose + 1.0 mmol/L N-Nethyl-cysteim) ; and ethanol control group(SG group,30 mmol/L D-glucose+6.86 × 10-2 mol/L ethanol).The level of intracellular reactive oxygen species,mitochondrial membrane potential,activity of total-superoxide dismutase (T-SOD),level of malondialdehyde,expression of UCP2 mRNA and protein in HK-2 cells were detected.Compared with NG group,the mitochondrial membrane potential significantly decreased in HG group (P＜0.01),and the mitochondrial membrane potential in V group was lower than that in HG group(P＜0.01).The activity ofT-SOD in HG group was significantly lower than that in NG group(P＜0.01),while its level of malondialdehyde was significantly higher than that in NG group(P＜0.01).Compared with HG group,the activity of T-SOD in V groups was significantly increased (P＜0.05)and the level of malondialdehyde in these groups significantly decreased (P＜0.01).The mRNA expression of UCP2 in HG group was increased significantly in comparison with NG group (P ＜ 0.05) and the expression in V groups was significantly decreased in comparison with HG group (P＜0.01).The results suggest that 1,25-(OH)2D3 could reduce the mitochondrial membrane potential,the production of reactive oxygen species,and regulate the expression of UCP2 in order to suppress the oxidative stress induced by high glucose.

Effect of cysteine-rich protein 61 on proliferation and cell cycle in human renal tubular epithelial cells / 中华肾脏病杂志

Objective To investigate the effect of cysteine-rich protein 61 (Cyr61) on proliferation and cell cycle in human renal tubular epithelial cells (HK-2).Methods Cyr61 cDNA was cloned into pEGFP-N2,then HK-2 cells were transfected with the recombinant plasmid pEGFP-N2-Cyr61 by Lipofectamine.The cell proliferation was measured by MTT.The expression level of Cyr61,p-FAK and cyclin dependent cyclin-dependent kinase 2 (CDK2) protein were detected by Western blotting.The cell cycle and cell apoptosis were analyzed by flow cytometry.Results The recombinant plasmid pEGFP-N,-Cyr61 could be transfected into HK-2 efficiently.After transfection,the proliferative activity was significantly increased,the proportion of HK-2 cells in G1 phase decreased and in S-phase increased significantly,the level of cell apoptosis decreased markedly (all P ＜ 0.01).The expressions of Cyr61,p-FAK and CDK2 in Cyr61-transfected group were all amplified significantly (all P ＜ 0.01).Conclusions Cyr61 protein over-expressed in HK-2 cells can increase CDK2 expression throngh FAK pathway,resulting in the promotion of HK-2 cells entering into S phase,cell proliferation and the reduction of cell apoptosis.

A preliminary study on the interaction of calcifying nanoparticles with human renal tubular epithelial cells / 中华泌尿外科杂志

ObjectiveTo observe the interaction of the calcifying nanoparticles (CNP) with human renal tubular epithelial cells (HK-2) in vitro,to observe and investigate the mechanisms of HK-2 injury induced by CNP,and to explore the potential role of CNP in the formation of Calcium oxalate kidney stones.MethodsHuman renal tubular epithelial cells were cultured in vitro and CNP was then added to the culture medium,the cell-crystal reaction was detected by light microscopy and transmission electron microscopy (TEM).To investigate the oxidative stress,NADPH oxidase inhibitor apocynin was chosen as the intervener.The levels of LDH,MDA,HA in the mediums after 24 h were assessed. ResultsCNP could induce changes of the HK-2.Adhesion and phagocytosis of CNP by the HK-2 were observed under TEM.After 24 h,the levels of LDH,MDA,HA were significantly different among the 4 groups ( P ＜ 0.05 ). ConclusionsHK-2 has abilities of adhering and phagocyting with CNP.CNP can cause damage induced by oxidative stress of HK-2.

Role of ERK1/2 kinase system in the expression of the type-1 plasminogen activator inhibitor induced by parathormone in human renal tubular epithelial cells / 中华肾脏病杂志

ObjectiveTo explore the role of ERK1/2 in the expression of the type-1 plasminogen activator inhibitor(PAI-1) induced by parathonnone (PTH) in human renal tubular epithelial cell line HK-2 cells.MethodsVarious concentrentions of PTH and manifold durations were applied in the test.The expression of PAI-1 mRNA and protein in HK-2 cells was measured by RT-PCR and Western blotting,respectively.Besides,ERK1/2 protein was detected by Western blotting before the ERK1/2 inhibitor incubated with the HK-2 cells or after.Results The expression of PAI-1mRNA and protein was gradually up-regulatad along with the increasing concentrations of PTH(10-12-10-10 mol/L).The maximum level of PAI-1 mRNA and protein was detected in 10-10 mol/L PTH and was 4.01 and 3.81 times of control group.Otherwise,the decreased expression of PAI-1 was found while the concentrations of PTH were beyond 10-10 mol/L.The levels of PAI-1 mRNA and protein were increased in pace withtime from 12 to 72 hour,in time-dependent manner,which was 4.06 (12 h) and 4.03 (72 h) times of 0 hour group.The levels of ERK1/2 and PAI-1 were ascended after 10-10 mol/L PTH incubated with the HK-2 cells (all P＜0.01).Howerver,both of them decended after cells were pretreated by the ERK1/2 inhibitor (all P＜0.01),but were still higher than those of control group(all P＜0.05).ConclusionERK1/2 kinase system partly participates in the regulation of PAI-1 induced by PTH in HK-2 cells.

Mechanism of aristolochic acid induced injury in human renal tubular epithelial cells in vitro / 第三军医大学学报

Objective To investigate the possible injury mechanism of human kidney proximal tubular epithelial cell-2 (HK-2) induced by aristolochic acid (AA). Methods Cultured HK-2 cells were divided into 4 groups:normal control,treated by AA at the concentration of 30,60 and 120 ?mol/L for 48 h respectively. The morphological changes were observed by inverted phase contract microscopy. The cell viability was measured by the Cell Counting Kit-8 (CCK8) assay. Apoptotic cells were identified by flow cytometry. Expression of active Caspase-3 was measured by Western blot analysis. Automatic biochemical analyzer was used to detect the contents of LDH and ?-N-Acetylglucosaminidase (NAG) in the supernatant. The expression of E-cadherin and a-SMA was detected with laser scanning confocal microscope (LSCM). Enzyme linked immunosorbent assay (ELISA) was used to measure the levels of TGF-?1 and collagen Ⅲ in the supernatant quantitatively. Results AA inhibited HK-2 cells proliferation,induced cell apoptosis and activated Caspase-3 expression,and increased the LDH and NAG levels. All of these were in a concentration-dependent manner. AA at the concentration of 60 ?mol/L inhibited E-cadherin expression,increased ?-SMA expression and TGF-?1 and collagen Ⅲ secretion. Conclusion AA inhibits cell proliferation,induces apoptosis and epithelial-mesenchymal transition (EMT) in HK-2 cells. AA at relatively low concentration (≤60 ?mol/L) mainly induces EMT in HK-2 cells,while,that at high concentration (≥120 ?mol/L) causes apoptosis and cytotoxicity.

Study on inhibition effect of small interfering RNA on monocyte chemotacite protein-1 in HKC / 中国实用内科杂志

Objective To investigate the inhibition effect of plasma-mediated small interfering RNA (siRNA) on the expression of monocyte chemotacite protein-1 (MCP-1) gene in human renal tubular epithelial cells (HKC).Methods Three pairs of siRNAs directed human's MCP-1 mRNA 67,116,142 targets were designed and synthesized.Eukaryotic expression vector special for MCP-1,pRNAT-MCP-1-Ⅰ、pRNAT-MCP-1-Ⅱ、pRNAT-MCP-1-Ⅲ were constructed and transfected into HKC by lipofectamine.At 24 hour after transfection,the expression of MCP-1 in the levels of mRNA was detected by Real Time RT-PCR,and the expression of MCP-1 in the levels of protein was detected by Western blot.Results Transfection efficiency of siRNA expression vector was 60%;the expression of MCP-1 in the levels of mRNA and protein of three pairs of plasma-mediated siRNA group were markedly decreased compared with normal control group(P

Effect of over-expressed Cyr61 on the expression of extracellular matrix of human renal tubular epithelial cells / 解放军医学杂志

Objective To investigate the effect of over-expressed Cyr61 on the expression of extracellular matrix of human renal tubular epithelial cells(HKC), and explore the role of Cyr61 in the pathogenesis and development of autosomal dominant polycystic kidney disease(ADPKD). Methods Cyr61 gene cDNA was amplified by RT-PCR. A recombinant plasmid pcDNA3.1+ Cyr61 was constructed by cloning Cyr61 gene into pcDNA3.1. HKC cells were transfected with wild-type Cyr61 plasmid vector (pcDNA3.1+Cyr61). Then the fusion of Cyr61 gene and expression of its protein were detected by RT-PCR and Western blot. The mRNA expression of Cyr61, collagen Ⅰ, collagen Ⅳ, and laminin were determined in four groups (the untransfected cells, pcDNA3.1 transfected cells, pcDNA3.1+Cyr61 -transfected cells and cyst-lining epithelial cells) by fluorescence quantum PCR. Results The amount of Cyr61 protein in Cyr61-transfected cells and cyst-lining epithelial cells were much greater than the control. The mRNA expression of Cyr61, collagen Ⅰ, collagen Ⅳ and laminin in Cyr61-transfected cells were all amplified significantly, and the level of collagen Ⅳwas much higher than collagen I(P