| Tubulointerstitial fibrosis is the inevitable path and ending shared by the occurrence and development of all kinds of chronic renal disease, characterized with the overdeposition of extracellular matrix, tubular atrophy and fibroblast proliferation, its pathological process consists of multiple segments as follows: oxidative stress reaction, inflammatory stimulation, unbalance of cytokines regulating the interstitial fibrosis and so on. A variety of primary or secondary renal diseases such as:primary glomerulonephritis, benign arteriolar nephrosclerosis, diabetic nephropathy, obstructive nephropathy and so on, all of which could lead to a decline in renal function to a varying degree, and gradual development into uremic syndrome in the absence of an effective and timely diagnosis and treatment.However, EMT can not be stopped automatically when there is a persistent presence of inflammatory reaction, and finally an organ fibrosis process could be caused. When EMT occurred to the epithelial cell due to the influence of multiple factors, the adhesion between the differentiated and matured epithelial cells is lost, the connection with the peripheral cellular matrix is decreased, the cells obtain a stronger function of migration and movement, the marker protein of epithelial cell is lost, such as E-cadherin, keratin and p-catenin and so on, while the expression of marker protein of mesenchymal cell is elevated, such as fibrous vimentin, α-smooth muscle actin, fibro-speeific protein-1 and so on, finally, the cell phenotype has been transformed into myofibroblast. The following factors can equally further the occurrence and development of EMT, transforming growth factor-β1 is generally accepted to be the most important inducing cytokine, connective tissue growth factor, complement C3, angiotensin II(AngII) and so on can equally further the development of EMT.Advanced oxidation protein products (advanced oxidation protein products, AOPP) is formed by the crosslinking products of plasma albumin and hypochlorite oxidation reaction in the body, containing double tyrosine carbonyl groups.It is found to not only rise in the body of patients with chronic renal insufficiency and further the occurrence and development of related long-term complications, but also be closely associated with the onset of other diseases like atherosis, diabetes, IgA renal disease, senile cataract and so on. Meanwhile, AOPP also participated in the cycle of inflammatory reaction, it can trigger the respiratory burst of mononuclear-macrophage, mediate the massive synthesis and release of proinflammatory cytokine predominated by tumor necrosis factor, allowing the inflammatory reaction to be cascadingly amplified and leading to a systemic micro-inflammatory state, it is an important link for the pathogenesis of abovementioned diseases.CTGF is a polypeptide containing cysteine, it is capable of inducing and adhering, promoting differentiation and cellular proliferation, it plays a role in the injury repair of human body. It has been proved that, CTGF is an important growth factor of progression of chronic renal disease, TGF-β1 can significantly increase the quantity of myofibroblast in many ways, it is a generally admitted fibrosis-inducing factor. When the kidney is injured, the generation of TGF-β1 will be increased due to the stimulation of a variety of inflammatory factors, TGF-β1 acted on the fibroblast and induced it to synthesize and secrete CTGF, while CTGF can also mediate the fibrosis effect of TGF-β1, and expedite the cell proliferation and the formation of extra-cellular matrix, and in turn expedite the fibrosis of renal mesenchymeMultiple signal pathways participated in the mediation of renal fibrosis, such as the ILK (intergrin-linked kinase) signal pathway, which could lead to the change in phenotype of sertoli cell and the transdifferentiation of sertoli cell into mesenchymal cell; while TGF-β/Smad interacted with the ILK signal pathway to further the expression of ILK and enhance the mesenchymal transdifferentiation; RhoA/ROCK, receptor tyrosine kinase-Ras/mitogenactivated protein kinases (MAPK), Wnt and other signal pathways are all involved in the transdifferentiation of renal tubular epithelial cell, in addition, JNK signal pathway has also been proved to be involved in the progression of renal fibrosis, cardiomegaly and other diseases.In this experiment, AOPPs was prepared by the oxidation of BSA with hypochloric acid solution, the human proximal renal tubular epithelial cell (HK-2 cell line) had been stimulated by AOPPs as a target cell, the transdifferentiation of renal tubular epithelial cell had been induced by oxidative stress in order to observe the expression of cytokine CTGF protein and mRNA; and the expression of cell phenotype protein of E-cadherin and vimentin and its according mRNA and the cell hypertrophy index of HK-2 cell after the transfection of HK-2 cell into CTGF siRNA had been observed; the occurrence of oxidative stress and change in EMT in the HK-2 cell induced by AOPPs after being processed with JNK inhibitor (SP600125) had been observed, so as to discuss the effect and mechanism of action on EMT of renal tubular epithelial cell by AOPPs, and provide a new theoretical basis for the further inhibition against the EMT of renal tubular epithelial cell and prevention of renal interstitial fibrosis.Research ObjectivesIn this study, we investigated whether AOPPs increased the expression of CTGF in HK-2 cells (a human proximal tubular epithelial cell line), and whether CTGF is involved in AOPPs-induced hypertrophy and EMT in HK-2 cells. Furthermore, we determined the role of the JNK signaling pathway in this process.Research methods1.AOPPs-BSA preparation and content determinationBriefly,100 mg/mL of BSA was exposed to 200 mmol/L of HOCl for 30 min at room temperature and then dialyzed overnight against phosphate-buffered saline (PBS) in order to remove free HOCl. The AOPPs preparation was passed through a Detoxi-Gel column (Pierce, Rockford, IL, USA) to remove the contaminating endotoxins. The levels of endotoxin in the preparations were assessed using the Amebocyte lysate assay kit (Sigma, St. Louis, MO, USA) and were examined to be <0.025 EU/mL.2.Culture of HK-2 cellsThe HK-2 cells were obtained from American Type Culture Collection (Rockville, MD, USA) and cultured in Dulbecco modified Eagle medium/F12 (DMEM/F12, Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum,100 μg/mL streptomycin, and 100 U/mL penicillin. The HK-2 cells were maintained at 37℃ in a humidified incubator containing a 5% CO2 atmosphere.3.Small interfering RNA (siRNA) transfectionThe non-specific siRNA and siRNA for CTGF were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China). The cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s protocols. The sequence of CTGF-specific siRNA was CCGACTGGAAGACACGTTT. We used the non-specific siRNA as a negative control in the experiments. The silencing effect of CTGF siRNA was assessed using western-blot analysis.4.Real-time PCRTotal RNA was extracted from cells by using TRIzol reagent (Invitrogen) according to the manufacturer’s protocols. Total RNA was reverse transcribed into cDNA by using an MMLV reverse transcriptase (Invitrogen, USA). Real-time PCR was performed using a PCR-system-9700 kit and the SYBR Green assay (TaKaRa, Japan). The quantitative real-time PCR was performed using the following conditions: 95℃ for 2 min, followed by 40 cycles of 95℃ for 30 s and 60℃ for 35 s. All data were normalized using the internal control P-actin, and the expression levels were analyzed using the 2-DDCt method.5.Western-blot analysisThe cells were lysed in RIPA buffer, and the lysates were separated using SDS-polyacrylamide gel electrophoresis. Then the separated proteins were transferred to polyvinylidene fluoride membranes. Subsequently, the membranes were blocked in 5% non-fat milk in TBST for 2 h and incubated overnight at 4℃ with primary antibodies against these molecules:CTGF, E-cadherin, vimentin, JNK, and Phospho-JNK. The immunoblots were washed with TBST and then incubated with appropriate HRP-conjugated secondary antibodies at room temperature for 1 h. and the band densities were quantified using the Image J software.(三) ResultsLAOPPs induced the overexpression of CTGF in HK-2 cellsThe expression of CTGF of AOPP-induced HK-2 cell was elevated, the level of CTGF protein and expression of mRNA of HK-2+AOPP (200 μg/mL) group were equally higher than that of control group, a peak was reached at hour 12, after a duration of 24 hours it still was significantly higher than that of control group.2.CTGF was involved in AOPP-induced hypertrophy and EMT in HK-2 cellsThe transfection of CTGF siRNA into HK-2 cell can clearly reduce the protein expression level of CTGF. It was equally shown by the western-blot detection and RT-PCR that, compared with the interference control group of non-specific siRNA, the transfection of specificity CTGF siRNA can significantly reverse expression inhibition of cadherin of HK-2 cell induced by AOPP and the overexpression of vimentin. The blocking of CTGF can clearly down-regulate the hypertrophy index of HK-2 cell induced by AOPP.3.CTGF was involved in the regulation of AOPP-induced hypertrophy of human-proximal renal tubular epithelial cell and EMT through JNK signal pathwayThe HK-2 cell was induced by the stimulation of AOPP to raise its JNK level of phosphorylation, indicating the involvement of AOPPs in the activation of JNK signal pathway in the HK-2 cell. The JNK inhibitor clearly blocked the JNK phosphorylation induced by AOPP. Meanwhile, JNK inhibitor significantly inhibited the AOPP-induced expression reduction of cadherin of HK-2 cell and the expression elevation of CTGF, vimentin filament protein on the mRNA and protein level. In addition, the AOPP-induced hypertrophy index of HK-2 cell was also significantly inhibited by SP600125.(四) Conclusions1. This experiment re-confirmed that AOPPs was capable of inducing the hypertrophy of human-proximal renal tubular epithelial cell and EMT;2. This experiment confirmed for the first time that, AOPPs caused the expression of CTGF to be elevated at human-proximal renal tubular epithelial cell, CTGF was involved in the regulation of AOPPs-induced hypertrophy of human-proximal renal tubular epithelial cell and EMT;3.It was simultaneously proved that, CTGF mediated the process of induction of hypertrophy of human-proximal renal tubular epithelial cell and EMT by AOPPs through the JNK signal pathway.All in all, it was proved by the data of this experiment that, CTGF is a newly found, advanced oxidation protein product-induced regulator of the mastocyte and transdifferentiation of human proximal renal tubular epithelial cell, and simultaneously indicating that, blocking the pathway of CTGF and JNK may become a new treating and research direction. |
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