Research Of The Role And Mechanism Of HSP47 In Renal Fibrosis

Renal fibrosis refers to the increase of interstitial cells and intercellular matrix due to pathogenic factors such as inflammation, injury, etc, particularly, the increase of matrix protein synthesis, the inhibition of matrix degradation, leading to a huge accumulation of extracellular matrix (ECM), and leading to glomerular sclerosis and tubulointerstitial fibrosis. Ultimately, it is the main pathological changes and common pathways leading to renal failure in a number of chronic kidney diseases. Prevention and delaying of renal fibrosis is the key to preventing and arresting the progression of chronic kidney disease. Collagen is the main component of ECM, it increases significantly in various renal fibrosis diseases, and its increased synthesis and deposition are closely related to renal fibrosis.Heat shock protein 47 is a collagen-specific "molecular chaperone". During the biosynthesis of collagen, it helps in modification, folding and assembly of procollagen. It is the necessary condition for the biosynthesis of collagen, and its expression is closely related to the collagen production.The expression of HSP47 is increased significantly in a variety of animal studies and clinical diseases of renal fibrosis, including hypertensive nephrosclerosis in rats,5/6 nephrectomy rat model, human IgA nephropathy and diabetic nephropathy, etc, and the increase is directly proportional with that of collagen. Some researches preliminarily confirmed that inhibiting its expression in animals could reduce the degree of fibrosis, for example, transfecting HSP47 antisense oligonucleotides to anti-Thy-1 glomerulonephritis rats and HSP47siRNA to UUO rats could improve renal fibrosis, suggesting that its role in renal fibrosis may not be limited to regulating collagen synthesis.Mitogen-activated protein kinase (Mitogen-activated protein kinase, MAPK) signaling pathway is an important signaling system mediated cell response, which can transmit extracellular signals to the cell and its nucleus through the conservative cascade 3 (MAPKKK-MAPKK-MAPK) activated transcription factors to regulate gene expression. Extracellular regulated protein kinase (extracellular signal-regulated kinase, ERK1/2) and c-Jun N-terminal kinase (c-Jun NH2-terminal kinase, JNK) are two major members of the MAPK family, in which ERK1/2 is known as the mitogen-activated MAPK pathway. Its main function is to mediate the promotion of cell proliferation and differentiation; and the main function of JNK is to participate and regulate body stress, inflammation reaction. In-vitro researches show that the MAPK signaling pathway can mediate the synthesis of heat shock protein induced by external stimuli.The analysis above indicated that HSP47 is a collagen-specific "molecular chaperone", its expression is increased in renal fibrosis, and it could promote renal fibrosis, its role and mechanism in renal fibrosis may not be limited to the regulation of collagen synthesis alone. MAPK signaling pathway may also be involved in the regulation of synthesis of HSP47. To this end, the following research was carried out. Objective:To observe the expression of HSP47 in renal tissues of UUO rats and investigate its role in renal interstitial fibrosis.Method:Sprague-Dawley rats were randomly divided into control and UUO groups (each group:n=8). A unilateral ureter obstruction (UUO) model was induced in male rats by ligation of the left ureter as described. Rats were sacrificed at day 14 post-surgery and the obstructed kidneys were harvested and studied. In both groups, the proteinuria, serum creatinine levels were determined, renal morphological changes were observed by HE, masson staining and the location and expression levels of HSP47, collagenlV and fibronectin(FN) were determined using immunohi stochemistry.Results:1. Compared to the sham-operated kidneys, serum creatinine levels increased in the rats of UUO model group, but there was no significant difference in terms of proteinuria levels.2. Compared to the sham-operated kidneys, varying degrees of tubular dilatation or atrophy, infiltration of inflammatory cells in interstitial areas, expansion of interstitial space, and bubble-like degeneration of tubular epithelial cells were observed in the rats of UUO model group under light microscopy.3. The results of Masson staining demonstrated that collagen significantly increased around the renal tubule and in interstitial areas in the rats of UUO model group compared to sham-operated kidneys.4. Compared to the sham-operated kidneys, the results of immunohistochemistry demonstrated that the expression of HSP47 significantly increased in the renal tissue of UUO rats model, especially around the renal tubule and in interstitial areas; The expression of collagenⅣsignificantly increased in the renal tissue of UUO rats model, especially in renal tubular basement membrane and interstitial areas; The expression of FN significantly increased, especially in interstitial areas.Conclusion:The expression of HSP47 increased in the kidneys of UUO rats; The location of expression of HSP47 was also the same as that of expressed collagen. These hint HSP47 could probably promote renal interstitial fibrosis, and the effect was related to promote biosynthesis of collagen. Objective:To study the role of HSP47 in the synthesis of collagen, FN and other ECM proteins by HK-2 cells induced by TGF-β1, and determine its effect on PAI-1 expression, to explore its role in the balance of ECM synthesis and degradation.Method:Human proximal tubular epithelial cells (HK-2) were divided into three groups:control (only culture medium), TGF-β1 (treated with TGF-β1) and HSP47siRNA (treated with TGF-β1 and HSP47siRNA). The expressions of HSP47,collagenⅣ,FN,PAI-1 mRNA and HSP47,collagenⅣ,FN protein were detected by RT-PCR and Western blot respectively.PAI-1 protein was detected by ELISA.Results:1. HK-2 can express HSP47 under normal medium. The expression of HSP47 gene and protein gradually increased with different concentrations of TGF-β1 (0,2.5,5,10ng/ml) intervening with HK-2 at different times(12h,24h,48h), the gene and protein level of HSP47 were highest with 10ng/ml TGF-β1 intervening at 48h on HK-2 cells. 2. Due to the effect of different concentrations of TGF-β1 (0,2.5,5,10ng/ml)at different time points(12h,24h,48h), the levels of gene and protein of collagenⅣ, FN, PAI-1 gradually increased, the gene and protein levels of collagenⅣ, FN, PAI-1 were strongest with TGF-β1 10ng/ml intervening at 48h on HK-2 cells.3. HSP47siRNA Group (HSP47siRNA intervenes at 24h, then TGF-β1 10ng/ml intervenes at 48h) can significantly reduce the expression of HSP47, collagenⅣ, FN, PAI-1 mRNA and protein compared to the intervention only by TGF-β1 on HK-2 cells.Conclusion:l.TGF-β1 can upregulate the expression of HSP47 in a concentration and time dependent mode on HK-2 cell.2. TGF-β1 can upregulate the expression of collagenⅣ, FN, PAI-1 in a concentration and time dependent mode on HK-2 cell.3. HSP47siRNA can reduce the expression of collagenⅣ, FN, PAI-1 mRNA and protein induced by TGF-β1 on HK-2. Objective:To investigate whether mitogen-activated protein kinase (MAPK) channels mediate the up-regulation of expression of HSP47 in HK-2 cells induced by TGF-β1.Methods:MAPK inhibitor was applied to observe the impact of up-regulation of expression of HSP47 in HK-2 cells induced by TGF-β1.Results:PD98059 and SP600125 can specifically inhibit the MAPK pathways:extracellular signal-regulated protein kinase (ERK1/2) and c-Jun-N-terminal kinase (JNK) respectively, and have different effects on the expression of HSP47 up-regulated by TGF-β1.1. PD98059 can inhibit the activation of ERK1/2 signaling pathway and the up-regulation of expression of HSP47 induced by TGF-β1 on HK-2 cells, and it was different among the effect with different concentrations of PD98059 (25uM,50uM,75uM)2. SP600125 can inhibit the activation of JNK signaling pathway and the up-regulation of expression of HSP47 induced by TGF-β1 on HK-2 cells, and there was no difference among the effect with different concentrations of SP600125 (10uM,20uM,30uM)Conclusion:ERK1/2,JNK signaling pathway may be involved in mediating the up-regulation of expression of HSP47 induced by TGF-β1 in HK-2 cells.