Research Of Niban In Renal Interstitial Fibrosis

Part1Differential protein screening in renal interstitial fibrosis.Background:Renal interstitial fibrosis characters by renal tubular hypertrophy and interstitial fibrosis. Renal interstitial fibrosis is an important factor in predicting rising prevalence of chronic kidney disease. Researches of nowadays suggest that degree of renal interstitial fibrosis indicates the degree of kidney damage and has important guiding significance to the prognosis of patients with kidney diseases. Studying mechanisms, searching biomarkers and exploring effective methods of prevention and cure of renal interstitial fibrosis are meaningful to delay chronic kidney diseases. And it is also the hot and difficult points of researches of kidney diseases in the world. Occurrence and development of renal interstitial fibrosis are affect by many factors. Apoptosis of renal tubular cells, inflammation, oxidative stress, proliferation and activation of fibroblast cells, renal tubular cells transdifferentiation to myofibroblasts, production of cytokines and vasoactive substance, imbalance of synthesis and degradation of extracellular matrix all take part in progress of renal interstitial fibrosis. As complex mechanisms of renal interstitial fibrosis, we insist that there is still other unknown and important new biomarkers and mechanisms in progress of renal interstitial fibrosis. In recent years, methods of Omics research such as proteomics and genomics have made remarkable progress. These methods provide a new technical platform for science research. We will joint analysis of proteomics and gene chip to select differential proteins in renal tissue of unilateral ureteral obstruction(UUO) rats to find biomarkers in process of renal interstitial fibrosis.Objective:To investigate differential proteins in progress of renal interstitial fibrosis through joint analysis of proteomics and gene chip in renal tissue of days3,7,14and21UUO rats.Methods:25SD rats were divided randomly into five groups:Sham operation group (SHM), UUO groups of3,7,14and21days. SD rats were submitted to unilateral ureteral obstruction (UUO) and sacrificed at day3,7,14and21. One half of renal tissue after ureteral obstruction of different time were dyed by HE and Masson staining for general histology, collagen detection and immunohistochemistry. The other part of renal tissue was studied by technics of isobaric tags for relative and absolute quantitation (iTRAQ), gene chip, western blot and real time-PCR to study differential proteins in progression of renal interstitial fibrosis.Result:1) HE, MASSON staining and immunohistochemistry of collagen Ⅲ revealed that there is obvious interstitial pathological damage and great collagen deposition in renal tissue of UUO rat (p<0.05). The tubulointerstitial damage index and relative area of renal interstitial collagen significantly increased in UUO models (p<0.05). 2) Joint analysis with iTRAQ and gene chip to study differential proteins in renal tissue of differential time of UUO rats. As we focus on finding early biomarkers in renal interstitial fibrosis, we screened differential proteins in renal tissue from days3to days14UUO rats. Compared to SHM, there are15proteins keep changing. In them,9proteins continue up regulating, and6proteins continue down regulating(including Niban). These15proteins changing>1.2or<0.8, at the same time, their gene expression changing>2.0or<0.5.3) Niban has unclear relationship with renal interstitial fibrosis, so we chose Niban for our target protein of follow-up study. We focus research on expression and possible mechanism of Niban in renal interstitial fibrosis. Part2Expression of Niban in renal interstitial fibrosisBackground:Niban was firstly found by Majima in2000. Niban express mainly in cytoplasm. Niban was found in Eker rat (a model of hereditary renal tumors). Niban also expressed in other renal tumor of human and animal models, in tissue of head and neck squamous cell carcinoma, head and neck dysplasia, intemperant, heavy smokers and patients of thyroid damage. Niban, as an early tumor biomarker, may play an effect in promoting the proliferation and inhibiting apoptosis of cells. Based on results of Niban in our research of joint analysis of proteomics and gene chip, we aimed to verifying relationship between Niban and renal interstitial fibrosis.Objectives:Verifying expression of protein and mRNA of Niban in renal interstitial fibrosis of human, animal and cells.Methods:Expression of Niban in renal tissue of patients of obstructive nephropathy was detected by immunohistochemistry. Expression of Niban in renal tissue of UUO rats were detected by immunohistochemistry. mRNA of Niban was detected by real time-PCR. TGF-β1(10ng/ml) stimulated HK-2cells for48hours to induce apoptosis of HK-2cells and create model of renal interstitial fibrosis in vitro. Expression of Niban and fibronectin were detected in TGF-β1stimulated HK-2cells.Results:1) Expression of Niban decreased obviously in renal tissue of patients of obstructive nephropathy compare to the control.2) Protein Niban decreased obviously in renal tissue of UUO rats compare to the SHM. mRNA of Niban, however, increased in renal tissue of UUO rats compare to the SHM.3) After stimulating by TGF-β1(10ng/ml) for48hours, expression of fibronectin increased and Niban decreased in HK-2cells. Conclusion:1) Niban decreased in renal tissue of UUO rats and patients of obstructive nephropathy.2) Niban decreased in renal interstitial fibrosis induced by TGF-β1in HK-2cells. Part3Exploring mechanism of Niban in renal interstitial fibrosisBackgroud:Renal interstitial fibrosis, which is closely related with progress of chronic kidney diseases, was charactered by renal tubular hypertrophy and interstitial fibrosis. Degree of renal interstitial fibrosis indicates the one of kidney damage and has an important guiding significance to the progress of patients with kidney diseases. Mechanisms of renal interstitial fibrosis are complex and relate to multiple steps. Apoptosis of renal tubular cells, inflammation, oxidative stress, proliferation and activation of fibroblast cells, renal tubular cells transdifferentiation to myofibroblasts, production of cytokines and vasoactive substance, imbalance of synthesis and degradation of extracellular matrix all take part in progress of renal interstitial fibrosis. In them, apoptosis plays an important role in inducing atrophy of renal tubular cells and formation of renal interstitial fibrosis. Apoptois, also called programmed cell death or cell suicide, is a genetic control of physical death. In normal circumstance, apoptosis plays the role of removing aged cells or lymphocyte which was not involved in the immune response. Pathological circumstance, however, apoptosis plays a role to many pathologic condition including tumorigenesis. Nowadays, researchers have observed apoptosis in renal tissue in many models of kidney diseases and clinical renal pathology detection.Functional studies of Niban show that it is a functional protein. Niban can inhibites apoptosis through regulating cell death signals in the transcription. Niban also indirectly increases free MDM2(a functional substrate in downstream of the pathway of AKT) through competitive antagonism. This effect leads to increasing degradation of P53and plays the anti-apoptosis role finally. The relationship between Niban and renal fibrosis is still unclear. Based on effect of Niban in regulating apoptosis of carcinoma cells and verification of Niban in renal interstitial fibrosis before, we will next explore whether Niban can regulate apoptosis in renal tubular cells to study mechanism of Niban in renal interstitial fibrosis.Objectives:Observe apoptosis of renal tubular cells in UUO rats and TGF-β1(10ng/ml) stimulated HK-2cells. Observe apoptosis of HK-2cells after silencing Niban with siRNA. Observe apoptosis of HK-2cells after transfecting plasmid of Niban, which can increase expression of Niban in cells. Exploring role of Niban in regulating apoptosis in renal tubular cells. Methods:In vivo experiment, apoptosis of renal tubular cells of UUO rats was detected by method of TUNEL. In vitro experiments, secondly, apoptosis of HK-2cells which were stimulated for48hours by TGF-β1(10ng/ml) were detected by methods of TUNEL and flow cytometry. To observe role of Niban in regulating apoptosis of HK-2cells, we used transfection to change expression of Niban. After silencing Niban with siRNA, apoptosis of HK-2cells were detected by methods of TUNEL and flow cytometry. After increasing expression of Niban through transfecting Niban with plasmid, apoptosis of HK-2cells was detected by flow cytometry.Results:1) Compare to SHM, apoptosis of renal tubular cells in UUO rats obviously increased.2) Compare to the control, apoptosis of HK-2cells significantly increased after stimulated by TGF-β1.3) Compare to the control, apoptosis of HK-2cells increased after silencing Niban with siRNA (p<0.05).4) Compare to the control, apoptosis of HK-2cells stimulated by TGF-β1nchanged after transfecting plasmid of Niban.Conclusions:1) Express of Niban decreased and apoptosis of renal tubular cells increased in renal interstitial fibrosis.2) Silencing expression of Niban can increase apoptosis of HK-2cells. Niban may participate renal interstitial fibrosis induced by apoptosis of renal tubular cells.3) Role of Niban in regulating apoptosis in HK-2cells does not take place through pathway of TGF-β1.