Study on expressions of collagen Ⅰ and Ⅱ in aldosterone-induced human kidney tubular epithelial cells / 重庆医学

Objective To investigate the role of Rho kinase on collagen(COL)Ⅰ and Ⅲ expression of human renal tubular epithelial cells .Methods Human kidney tubular epithelial (HK‐2) cells were cultured in the RPMI‐1640 culture solution containing 15% fetal bovine serum .After 30 min pretreatment by the ALD receptor antagonist eplerenone(10 μmol/L) and Rho kinase inhibi‐tor Y27632(1 μmol/L) ,100 nmol/L ALD acted the HK‐2 cells for 24 h .The expressions of collagenⅠ and Ⅲ mRNA in each group were detected by real time PCR and the expression levels of COL Ⅰ ,Ⅲ and Rho kinase protein were detected by ELISA .Results ALD could up‐regulate the expressions of COLⅠ ,Ⅲ mRNA in HK‐2 cells ,and increased the levels of Rho kinase ,COLⅠ and Ⅲprotein ,while the Rho kinase inhibitor Y27632 and ALD receptor inhibitor eplerenone could antagonize these effects .Conclusion ALD could activate Rho kinase signal transduction pathway in HK‐2 cells and accelerate the progression of tubular interstitial fibro‐sis via Rho kinase induced expression of COL Ⅰ and Ⅲ .

The time-dependent effect of insulin on the expression of SREBP-1,FAS and lipid droplet formation in HKC cells / 中国药理学通报

Aim To investigate the time-dependent effect of insulin on the expression of SREBP-1(sterol regulatory element binding protein-1),FAS(fat acid synthase)and lipid droplet formation in HKC cells(human renal proximal tubular epithelial cells line).MethodsHKC cells were respectively treated with 100 nmol·L~(-1) insulin for 0,2,4,6,12 h and 24 h.The analysis of SREBP-1 and FAS mRNA was performed by RT-PCR and the expression of SREBP-1 protein was detected by Western blot and immunocytochemistry.Furthermore,Oil Red O staining was used to determine cellular lipid droplet formation.ResultsCompared with HKC cells of 0 h group,there was no difference of SREBP-1 and FAS mRNA in HKC cells of 2 h group.However,the expression of SREBP-1 and FAS mRNA was significantly increased in HKC cells of 4,6 h and 12 h group.Further,the most expression of SREBP-1 and FAS mRNA was at 6 h group and was respectively increased by 3.578 and 4.272 times compared with 0 h group.The results of Western blot showed that the precursor and mature segments of SREBP-1 protein in 4,6 h and 12 h group HKC cells were increased and those of 6 h group HKC cells were the highest and about 4.106 and 5.167 times than those of 0 h group HKC cells.Immunocytochemistry presented the result that SREBP-1 protein was located in the plasma and the expression of 4,6 h and 12 h group HKC cells was significantly higher than that of 0,2 h and 24 h group HKC cells.The result of Oil Red O staining showed that lipid droplet markedly deposited in 6 h group HKC cells,contrarily,no lipid droplet was found in HKC cells of other groups.ConclusionAbove results suggested that insulin up-regulated SREBP-1 and FAS in time-dependent manner that led to cellular lipid droplet deposit,which may play an important role in the pathogenesis of renal lipid accumulation in metabolism syndrome.

Effects of Mycophenolic Acid and Rapamycin on Toll-like Receptor Expression in Hypoxic Human Proximal Tubular Epithelial Cells / 대한이식학회지

BACKGROUND: Toll like receptor (TLR), an element of innate immunity, is upregulated by Ischemia/reperfusion (IR) injury and may be involved in adaptive immune response. Immunosuppressive agents may increase or attenuate IR injury and TLR expression. To explore the involvement of TLRs in hypoxic tubular injury and modification by mycophenolic acid (MPA) rapamycin (RAP), this study examined TLR expression in hypoxia-induced human renal proximal tubular epithelial cells (HK-2). METHODS: HK-2 cells were cultured in keratinocyte-SFM media supplemented with epidermal growth factor and bovine pituitary extract. The Induction of hypoxia was achieved using GasPak pouch system. TLR 2, 3, and 4 mRNA expression was analyzed by real time RT-PCR using SYBR green and TLR 4 protein expression was evaluated by Western blot analysis. MPA at concentration of 100 nM and 1uM and RAP at concentration of 20, 50, and 100 nM were added to culture medium. RESULTS: TLR4 but noTLR2 or TLR3 mRNA expressions increased in hypoxic HK-2 cells at 24 and 48 hrs. TLR4 protein expression also increased in hypoxic HK-2 cells at 24 and 48 hrs. MPA (100 nM and 1uM) and RAP (20, 50, and 100 nM) decreased hypoxia-induced TLR4 mRNA expression in HK-2 cells compared to normoxia at 24 hrs. However, TLR4 protein expression was decreased only by RAP at 20 and 50 nM. CONCLUSIONS: The results suggest that RAP may modify hypoxic renal tubular damage by decreasing TLR4-mediated inflammatory and immune reactions.

Effect of Endothelin-1 on the Expression of Monocyte Chemoattractant Protein-1 in Cultured Human Proximal Tubular Epithelial Cells / 대한신장학회잡지

BACKGROUND: Monocyte chemoattractant protein- 1 (MCP-1) is produced by renal cells and an important mediator for monocyte/macrophage infiltration in various inflammatory renal diseases. In the process of renal disease, endothelin-1 is known to play an active role in cell growth, inflammation and fibrosis. The aim of this study was to investigate whether endothelin-1 regulates MCP-1 expression in cultured human proximal tubular epithelial cells. METHODS: Primary cultured human proximal tubular epithelial cells (PTEC) were incubated with or without various dose of endothelin-1. MCP-1 concentration in PTEC conditioned medium was measured by sandwich ELISA. MCP-1 mRNA expression was analyzed by Northern blotting. The NF-kB or AP-1 activity in response to endothelin-1 was measured by electrophoretic mobility shift assay. RESULTS: Endothelin-1 (10(-7) M) stimulated MCP- 1 production in PTEC, which was significant at 48 hours and various doses of endothelin-1 (10(-8)-10(-6) M) increased MCP-1 production from PTEC in a dose-dependent manner. Northern blot analysis revealed that endothelin-1 stimulated MCP-1 mRNA expression. Endothelin-1 (10(-7) M) stimulated both AP-1 binding activity and NF-kB binding activity up to 8 hour. Supershift analysis showed that p65 and p50 are major NF-kB subunit bound to the DNA probe and that c-Fos and c-Jun are major AP-1 subunit bound to the DNA probe. CONCLUSION: Our results suggest that endothelin- 1 may stimulate MCP-1 expression in proximal tubular epithelial cells through the activation of NF- kB and AP-1 binding activity.

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

Induced expression of indoleamine 2,3-dioxygenase in epithelial cells of human proximal renal tubule / 解放军医学杂志

Objective To investigate the induced expression of indoleamine 2,3-dioxygenase in epithelial cells of cultured human proximal renal tubule under the stimulation of interferon-?(IFN-?)in vitro.Methods The expression of IDOmRNA in epithelial cells of human proximal renal tubule under normal and different dosage or time of IFN-? stimulation were assayed by semi-quantitative RT-PCR,the expression of IDO protein under normal and 2000U/ml IFN-? stimulation for 24h were assayed by immunocytochemistry,and the activity of IDO under different dosage of IFN-? stimulation was assessed by the special kynurenine metabolizable ratio in culture medium detected by high performance liquid chromatography(HPLC).Results No expression of IDO mRNA or IDO was found in the epithelial cells of human proximal renal tubule under normal culture conditions.IDO mRNA expression increased significantly after IFN-? stimulation in a dose dependent manner compared with that of control group(P