Salvianolic Acid F Regulates Bax/Caspase-3/GSDME Signaling Pathway to Inhibit Pyroptosis of HK-2 Cells / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of salvianolic acid F （Sal F） in repairing the high glucose-induced injury in human kidney-2 （HK-2） cells via the B-cell lymphoma-2 （Bcl-2）-associated X protein （Bax）/cysteinyl aspartate-specific proteinase 3 （Caspase-3）/gasdermin-E （GSDME） pathway. MethodThe cell counting kit-8 （CCK-8） was used to measure the relative viability of HK-2 cells exposed to high glucose and different concentrations （2.5， 5， 10， 20 μmol·L-1） of Sal F and the relative viability of HK-2 cells treated with Sal F for different time periods. The levels of lactate dehydrogenase （LDH） and interleukin-1β （IL-1β） in the supernatant of the cell culture were measured by the LDH assay kit and enzyme-linked immunosorbent assay （ELISA） kit， respectively. Flow cytometry combined with Annexin V-FITC/propidium iodide （PI） and Hoechst 33342/PI staining was employed to reveal the proportion of PI-positive HK-2 cells exposed to high glucose. Western blotting was employed to determine the protein levels of Bax， Bcl-2， cytochrome C， cysteinyl aspartate-specific proteinase （Caspase）-9， Caspase-3， and GSDME in the HK-2 cells exposed to high glucose and treated with Sal F. The 2，7-dichlorodihydrofluorescein diacetate fluorescence probe （DCFH-DA） and mitochondrial membrane potential assay kit （JC-1） were used to determine the production of reactive oxygen species （ROS） and the mitochondrial membrane potential in the HK-2 cells exposed to high glucose and treated with Sal F. ResultCompared with the blank group， the model group showed decreased cell viability （P<0.01）， elevated levels LDH and IL-1β， increased proportion of PI-positive cells （P<0.01）， up-regulated protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME （P<0.01）， down-regulated protein level of Bcl-2 （P<0.01）， decreased mitochondrial membrane potential， and excessive ROS accumulation. Compared with the model group， Sal F repaired the high glucose-induced injury in HK-2 cells （P<0.05）， lowered the levels of LDH and IL-1β （P<0.05， P<0.01）， and decreased the proportion of PI-positive cells （P<0.01）. In addition， Sal F down-regulated the protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME and up-regulated the protein level of Bcl-2 （P<0.05， P<0.01）， increased the mitochondrial membrane potential， and decreased the accumulation of ROS in HK-2 cells. ConclusionSal F can reduce the production of ROS， restore the balance of mitochondrial membrane potential， and inhibit pyroptosis via the Bax/Caspase-3/GSDME signaling pathway to repair the high glucose-induced injury 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.

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.

A Cadaveric Study Of Variations In The Urological System

Introduction: Knowledge of anatomical variations of the urinary system is important for urological surgeriesinvolving renal transplant and radiological interpretations. When urologists and clinicians have a soundknowledge of anatomical variations, it eases management, surgical interventions and helps to reducecomplications.Advanced imaging technology is the boon for the patients requiring minimally invasive approaches for variouskidney disorders. These approaches require precise knowledge of normal and variant anatomy of the kidneys,ureters and vascular structures at the hilum of the kidney. Therefore, the objective of this study was to furnish theclinicians with the major anatomical variations of urological system.Method: Ninety adult human cadavers were examined for number, shape and position of kidneys and the uretersover a period of 5 years. Out of these, 85 were males and 15 were female cadavers.Results: Kidneys were bilaterally present in all the cadavers. Hypoplastic kidneys were seen bilaterally in 3.3%cadavers. Out of 90 cadavers, 3 showed bilateral and 6 showed unilateral lobulated kidneys. 2 cadavers showedunilateral (1 L, 1 R) incomplete double ureter. One showed bilateral and 5 showed unilateral accessory renalartery amongst 90 cadavers. Ectopic kidney was seen in one cadaver.Conclusions: Morphological variations in the kidney are very common and are clinically important for urosurgeons

Effect of cadmium chloride on the expression of KIM-1 in in vitro HK-2 cell culture / 中国职业医学

OBJECTIVE: To investigate the effect of cadmium chloride on the expression of kidney injury molecule-1( KIM-1)in human renal tubular epithelial cells( HK-2 cells). METHODS: HK-2 cells at logarithmic phase were divided into a control group and 5 treatment groups that were treated with 5. 0,10. 0,20. 0,50. 0 and 100. 0 μmol / L of cadmium chloride dissolved in phosphate buffer solution. Cell pathology observation was carried out after 24 hours of cultivation. The methyl thiazolyl tetrazolium assay was used to calculate the survival rate of HK-2 cells. The expression of KIM-1 mRNA and protein were detected by the reverse transcription-polymerase chain reaction and Western blotting analysis respectively.RESULTS: There were no cellular morphologic change in HK-2 cells in the control group,the 5. 0 and 10. 0 μmol / L groups;the HK-2 cells showed different degree of swellings or vacuoles in the 20. 0 and 50. 0 μmol / L groups; a large number of cells were found dead in the 100. 0 μmol / L group. The cell survival rates of HK-2 cells in the 20. 0,50. 0 and 100. 0μmol /L groups were lower than those of control group,the 5. 0 and 10. 0 μmol /L groups( P < 0. 05). The pairwise comparison among survival rates of the 20. 0,50. 0 and 100. 0 μmol / L groups showed significant difference( P < 0. 05).The expression levels of KIM-1 mRNA and protein in the 20. 0 and 50. 0 μmol / L groups were higher than those of control group,the 5. 0 and 10. 0 μmol / L groups( P < 0. 05). The levels of KIM-1 mRNA and protein in the 50. 0 μmol / L group were higher than those of the 20. 0 μmol / L group( P < 0. 05). CONCLUSION: Cadmium chloride at certain concentration can increase the expression of KIM-1 mRNA and protein in HK-2 cells. Therefore,the expression of KIM-1 could be used as one of the effect biomarkers for cadmium induced kidney tubule injury.

Histomorphometric Study of the Glomeruli of the Kidney in Bangladeshi Population.

Background: Controversies still prevail on glomerular changes of kidney whether due to normal aging or its association with diseases. Objective: The aim of the present study was to see the variation in number and size of the glomeruli of kidney with increasing age in a Bangladeshi population based on autopsy. Methods: This cross-sectional, descriptive study was done in the Department of Anatomy, Dhaka Medical College, Dhaka, from July 2008 to June 2009, based on collection of 140 post mortem human kidneys collected from 70 unclaimed dead bodies from the morgue. All the samples were divided into three age-groups: 10-19 years, 20-39 years and 40-59 years. Histological slides were prepared by using routine Harris’ Haematoxylin and Eosin (H & E) stain. The number of glomeruli was measured by point counting technique, while the size (diameter) was measured by using ocular and stage micrometer. Results: The mean ± SE number of glomeruli per sq. mm found in the right and left kidney were 8.45±0.52 and 8.67±0.80 in group 10-19 years, 9.90±0.42 and 9.92±0.47 in 20-39 years, and 8.52±0.18 and 8.55±0.16 in 40-59 years respectively. Besides, the size (mean ± SE diameter) of glomeruli was found in the right and left kidney were 43.96±3.01ìm and 143.92±2.90ìm in group 10-19 years, 153.69±5.18ìm and 153.61±5.24ìm in 20-39 years, and 140.48±0.95ìm and 140.78±0.88ìm in 40-59 years respectively. Conclusion: No difference was found in number and size of glomeruli between right and left kidney in any group. Similarly, no difference was also evident among different age groups.

Up-regulation effect of cyclosporine A on level of kidney inj ury molecule-1 in culture supernatant of human kidney cells and its mechanism / 吉林大学学报(医学版)

Objective To investigate the mechanism of increasing of the level of kidney injury molecule-1(KIM-1)in culture supernatant of human kidney cells(HKC)induced by cyclosporine A(CsA),and to clarify the relationships between the expression levels of KIM-1 and p38 MAPK pathway and ERK1/2MAPK pathway in HKC. Methods The HKC at logarithmic growth phase were randomly divided into control group, CsA control group, CsA + p38 kinase inhibitor group, p38 kinase inhibitor group, CsA + ERK1/2 inhibitor group and ERK1/2 kinase inhibitor group.The inhibitory rates of proliferation of HKC in various groups were detected by MTT assay, and the expression levels of KIM-1 in HKC supernatant in various groups were detected by ELISA;the survival rates,apopototic rates and necrotic rates of the HKC in various groups were detected by flow cytometry. Results Compared with control group,the expression level of KIM-1 protein in the supernatant of HKC in CsA control group was significantly increased (P0.05).Compared with CsA control group,the expression levels of KIM-1 protein in CsA+ p38 kinase inhibitor group and CsA+ ERK1/2 kinase inhibitor group were significantly decreased (P<0.05),and the survival rate was significantly increased (P<0.05),while the apoptotic rate and the necrotic rate were significantly decreased (P<0.05).Conclusion p38 MAPK pathway and ERK1/2MAPK pathway are involved in the process of up-regulation of the KIM-1 level in HKC culture supernatant induced by CsA,and the expression of KIM-1 may become the biochemical marker of clinical monitoring of CsA nephrotoxicity.

Expression of an Anion Exchanger Pendrin in Human Kidney / 대한해부학회지

It has been reported that new apical anion exchanger perndrin, encoded by the pendred syndrome (PDS/pds, Slc26A4) gene, was expressed in the AE1-negative intercalated cells of rat and mouse kidneys. The purpose of this study was performed that expression of pendrin in the subtypes of intercalated cells in human kidney. The normal human renal tissues obtained from nephrotomized kidneys for renal cell carcinoma were fixed in periodate-lysine-paraformalde-hyde, and processed for immunohistochemistry. Subtypes of intercalated cells were identified by using antibodies for H(+)-ATPase and AE1, and connecting tubule cells and principal cells of collecting duct were identified using antibodies for calbindin D28K and AQP2, respectively. In human kidney, pendrin was expressed in the apical domain of AE1-negative intercalated cells including type B cells with diffuse and/or basolateal H(+)-ATPase, non A-non B (non -A/B) type intercalated cells with apical H(+)-ATPase and bipolar type of intercalated cells with apical and basolateral H(+)-ATPase. The AQP2-positive principal cells of cortical collecting duct were also had apical pendrin immunoreactivity. However, there was no pendrin immunoreactivity in AE1-positive type A intercalated cells, calbindin D28K-positive connecting tubule cells, and AQP2-positive medullary collecting duct. These results suggest that pendrin is an apical anion exchanger not only in the AE1-negative intercalated cells (type B, non-A/B and bipolar cells) but also in the principal cells of cortical collecting duct, and has an essential role in HCO3-secretion in human kidney.

Effect of valsartan on expression of TGF-?/Smads signaling pathway in human kidney proximal tubular epithelial cell / 中国药理学通报

Aim To observe the effect of valsartan on expression of Smads of HKCs stimulated by TGF-?1.Methods HKCs were divided into three groups:control group,TGF-?1 group and valsartan group.Total protein was firstly abstracted at 30 min after stimulation to detect p-Smad2/3 protein by Western blot.Total RNA and protein were abstracted at 48 hours after stimulation.The expressions of Smad2/3,Smad7 were measured by Western blot.Smad2mRNA and Smad7mRNA were measured by RT-PCR.Meanwhile, The protein synthesis of Col Ⅰ in the supernatants of the HKCs was detected by Western blot.MTT assay was used to investigate the effect of valsartan on proliferation of HKCs stimulated by 5 ?g?L-1 TGF-?1 at different time and different drug concentration.Results Western blot analysis indicated that TGF-?1 stimulation could increase the expression of Smad2/3 and activate phosphorylation of Smad2/3 at 48 hour.At the same time,Smad7 decreased.Valsartan could reduce the level of Smad2/3 and p-Smad2/3 but it did not have any effect on the expression of Smad7 protein.RT-PCR analysis also showed that the expression of Smad2mRNA or Smad7mRNA was higher after TGF-?1 stimulation than control group,while valsartan could down-regulate the expression of Smad2mRNA.There was no difference of the expression of Smad7mRNA between the TGF-?1 stimulation group and valsartan treatment group.Valsartan could also greatly ameliorate the secretion of Col Ⅰ in the supernatants of the HKCs stimulated by TGF-?1 detected by Western blot.Compared with control group,TGF-?1 supressed the proliferation of HKCs,While 1,10 and 100 ?mol?L-1 valsartan ameliorated it and the effect was dependent of dose.Conclusion Valsartan has some renal protective effects on renal interstitial fibrosis,partly through inhibiting TGF-?/Smads signaling pathway.

Human kidney proximal tubular epithelial cell line expresses paxillin induced by TGF-?_1 / 第三军医大学学报

Objective To investigate the expression of Paxillin (Pax) in human kidney proximal tubular epithelial cell line (HKC) induced by transforming growth factor ?1(TGF?1). Methods HKC cells cultured in vitro were divided into three groups at random: control group(C): cultured with free serum medium(FSM), TGF?1-treated groups (T1 and T2): cultured with FSM containing different concentrations of TGF?1 (T1: 5 ng/ml, T2: 10 ng/ml). After 48-hour treatment, the expression of Pax mRNA and protein was detected by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining in HKC cells respectively. Results In Group C, the expression of Pax mRNA and protein in HKC cells was elementary. However, in T1 and T2 groups , the expression of Pax mRNA and protein in HKC cells was greatly increased, especially in T2 group which was more significantly increased than T1 group (P

Protection in vitro of aFGF on human liver cell L-02 and human kidney cell 293 caused by nitidine chloride / 中成药

AIM: To detect the inhibition effect of nitidine chloride on proliferation of human liver cell L-02 and human kidney cell 293 and the protective effect of acidic fibroblast growth factor(aFGF) on human liver cell L-02 and human kidney cell 293 damaged by nitidine chloride in vitro. METHODS: The MTT assay was used to assess the proliferation of human liver cell L-02 and human kidney cell 293 treated with nitidine chloride.The contents of SOD and MDA and LDH in cultural supernate were measured by ultraviolet spectrophotometry. RESULTS: Nitidine chloride inhibited the proliferation of human liver cell L-02 and human kidney cell 293 in a dose-dependent manner. CONCLUSION: Nitidine chloride has certain toxicity on human liver cell L-02 and human kidney cell 293.aFGF could protect human liver cell L-02 and human kidney cell 293 damaged by nitidine chloride.

The effect of TGF-?_1 on expression of paxillin in human renal proximal tubular epithelial cell line / 解放军医学杂志

Objective To explore the effect of transforming growth factor ?_1 (TGF-?_1) on expression of paxillin(Pax) in human kidney proximal tubular epithelial cell line (HKC cells) in different phases. Methods HKC cells cultured in vitro were divided into four groups at random. Control group(C group): HKC cells were cultured with serum free medium (FSM), and TGF-?_1-treated groups (T1, T2, T3 group): HKC cells were cultured with FSM containing 10ng/ml TGF-?_1. In the latter groups, duration of treatment varied as follows: T1 group for 24h, T2 group for 48h, T3 group for 72h. The proliferation of HKC cells induced by TGF-?_1 was assessed by MTT and the expression of Pax was determined. The gene expression of Pax was determined by reverse transcripton polymerase chain reaction (RT-PCR) and the protein expression of Pax was assessed by immunohistochemical staining and Western blot. Results TGF-?_1 could induce the proliferation of HKC cells in a time-dependent manner. In C group there was expression of Pax mRNA and protein in HKC cells. The expression of Pax mRNA and protein in HKC cells was positive in all the T groups, especially in T2 group and T3 group they were significantly increased compared with T1 group (P

Effect of activated PI3-K on epithelial-mesenchymal trans-differentiation of HK cells induced by TGF-?_1 in vitro / 解放军医学杂志

Objective To investigate the effect of activated PI3-K on epithelial-mesenchymal transdifferentiation of HKCs induced by transforming growth factor?1 (TGF-?1). Methods The human kidney proximal tubular epithelial cells (HKCs) cultured on plastic plates were divided into following groups: cultured with free serum medium (FSM); culture in the different concentrations of TGF-?1; cultured in the presence of recombinant human TGF-?1 and PI3-K inhibitor Wortmannin. The expression of total and phosphor-Akt (t-Akt and p-Akt) was assessed at different time points by Western blot,? smooth muscle actin(?-SMA) and E-cadherin were detected by Western blot. Results A marked increase in p-Akt was seen in HKC at 1h after being induced by TGF-?1, and its protein level was enhanced in a TGF-?1 concentration-dependent manner. Protein level of ?-SMA was increased markedly at 48 hours after the treatment of TGF-?1, but protein level of E-cadherin was decreased 48 hours after treatment of TGF-?1. Addition of PI3-K inhibitor Wortmannin (10nmol/L) largely abrogated the effect of TGF-?1. Wortmannin was showed to down-regulate ?-SMA and p-Akt expression and in response to TGF-?1 and up-regulate E-cadherin expression. Conclusion PI3-K was activated in epithelial-mesenchymal trans-differentiation of HKCs promoted by TGF-?1, and PI3-K inhibitor Wortmannin can significantly inhibit TGF-?1 induction of EMT.