腎臟纖維化與基質金屬蛋白酶-9與氧化壓力相關性的研究

The Relationships between Renal Fibrosis and Matrix Metalloproteinase-9 and Oxidative Stress

蔡任弼(Jen-Pi Tsai)

指導教授 ： 張浤榮

中山醫學大學/醫學院/醫學研究所/博士(2014年)

https://doi.org/10.6834/CSMU.2014.00090

摘要

背景
細胞外基質的週轉失衡與活性氧化物質（reactive oxygen species）的過度產生在腎臟纖維化扮演一定的角色。基質金屬蛋白酶（MMP）與許多腎絲球疾病相關。另外有研究指出活性氧化物質與諸多疾病相關，比如在移植後腎臟功能退化、移植腎失能、病患死亡或是糖尿病生成等疾病中，也顯示活性氧化物質是危險因子之一。在氧化壓力與抗氧化壓力控制失衡的研究上，穀胱甘肽轉移酶（Glutathione S-transferases；GST）這個與抗氧化壓力相關的物質，己被提出在糖尿病的生成上佔有其重要的角色；而8-羥基去氧鳥糞核苷（8-OHdG）是白血球DNA因氧化傷害而形成的代謝物，己被探討是一個慢性腎臟病病患受到氧化壓力傷害的標記之一。但是基質金屬蛋白酶與8-OHdG與人類腎臟纖維化的組織相關性仍不清楚。故執行此實驗。
方法
在申請人體實驗委員會同意執行本實驗後，執行此回溯性之研究。此研究包括回顧病患的醫療記錄、生化資料與取得病理切片。共有74位因疾病切除腎臟病患的腎臟切片，利用免疫組織化學染色（IHC）檢驗基質金屬蛋白酶-9（MMP-9）與8-OHdG。進而分析染色後，腎臟所表現的腎臟纖維化嚴重度與MMP-9與8-OHdG的相關性。MMP-9與8-OHdG在個別腎臟的結構與纖維化分級為高或低的MMP-9與8-OHdG與纖維化的分數。
結果
在MMP-9與腎臟纖維化的分析後發現，有高間質纖維化分數（IFS）的病患有較高的血漿肌酸酐（high vs low：3.59 ± 3.93 mg/dl vs. 1.04 ± 0.32 mg/dl; p = 0.001）、較低的腎絲球過濾率（high vs. low：42.2 ± 27.5 ml/min vs. 74.2 ± 22.7; p = 0.001）、較高的可能罹患慢性腎臟病（CKD）（high vs. low：72.2% vs. 35.7%; p = 0.016）與泌尿上皮細胞癌（UCC）（high vs. low：55.6% vs. 14.3%）；高腎絲球纖維化分數（GFS）的病患有較高的血漿肌酸酐（high vs. low： 4.78 ± 4.38 mg/dl vs. 1.07 ± 0.32 mg/dl; p < 0.001）、較低的腎絲球過濾率（high vs. low：32.1 ± 25.8 ml/min vs. 72.2 ± 22.3 ml/min; p < 0.001）、較高的可能罹患慢性腎臟病（high vs. low：83.3% vs. 38.2%; p = 0.007）與泌尿上皮細胞癌（high vs. low：50% vs. 23.5%; p < 0.001）。單變項分析發現間質纖維化分數與正常（r = -0.455; p = 0.001）與萎縮（r = -0.410; p = 0.005）的腎小管細胞質MMP-9的表現呈現負相關；腎絲球纖維化分數與正常（r = -0.642; p < 0.001）與萎縮（r = -0.324; p = 0.03）的腎小管細胞質MMP-9的表現呈現負相關；而間質纖維化分數與萎縮腎小管細胞核MMP-9的表現呈現正相關（r = 0.333; p = 0.024）。多變項逐步迴歸分析顯示出在萎縮腎小管細胞核中MMP-9的表現是間質纖維化的獨立預測因子（r = 0.4, p = 0.002）；而在正常腎小管細胞質MMP-9的表現是腎絲球纖維化的獨立預測因子（r = -0.465, p < 0.001）。
在8-OHdG與腎臟纖維化的分析後發現，有高間質纖維化的病患有較低的腎絲球過濾率（high vs. low：41.1 ± 27.6 ml/min vs. 72.5 ± 23.9；p = 0.001）、較高的可能罹患慢性腎臟病（high vs. low：75% vs. 37.9%；p = 0.024）與泌尿上皮細胞癌（high vs. low：50% vs. 10.3%；p < 0.001）；高GFS分數的病患有較低的腎絲球過濾率（high vs. low：31.9 ± 25.8 ml/min vs. 70.8 ± 23.2 ml/min；p < 0.001）、較高的可能罹患慢性腎臟病（high vs. low：85.7% vs. 40%；p = 0.005）與UCC（high vs. low：50% vs. 17.1%；p = 0.001）。腎臟表現較高的間質纖維化的組織中，在正常腎小管細胞質（35.7% vs. 64.3%；p = 0.011）與正常腎小管細胞核（28.6% vs. 71.4%, p = 0.023）有較低的8-OHdG的染色強度。
結論
研究顯示腎臟纖維化程度與正常腎小管細胞質或是與萎縮腎小管細胞核MMP-9的表現相關。另外在正常腎小管細胞質中8-OHdG表現的強度與血清肌酸酐與腎臟纖維化的嚴重程度相關。

關鍵字

腎臟纖維化 ； 基質金屬蛋白酶-9 ； 8-羥基去氧鳥糞核苷免疫組織化學染色

並列摘要

Introduction
Abnormal balance turnover of extracellular matrix and overproduction of reactive oxygen species （ROS）have roles in renal fibrosis. Matrix metalloproteinases （MMPs）is associated with many glomerular diseases. Reactive oxygen species have been proposed to be associated with multiple diseases and also a risk factor at the development of post-transplant complications, including renal dysfunction, graft failure, patient death, and diabetes mellitus. Glutathione S-transferases（GST）is reported to be associated with effects against oxidative stress and was reported to be associated with the occurrence of diabetes mellitus. 8-hydroxy-2’ deoxyguanosine （8-OHdG）, a metabolite of oxidative damage to leukocyte DNA, has been identified as a marker of oxidative stress in patients with chronic renal failure. Because the histological association of MMPs and 8-OHdG and human renal fibrosis is unclear, we conducted this study to evaluate their relationship
Materials and Methods
This is a retrospective study. Institutional Review Board approval was obtained for the review of patients’ medical records, data analysis and pathological specimens staining with waiver of informed consents. Specimens of seventy-four patients were examined by immunohistochemical stain of MMP-9 and 8-OHdG in nephrectomized kidneys, and the association between renal expression of MMP-9 and 8-OHdG and renal fibrosis were determined. MMP-9 and 8-OHdG expression in individual renal components and fibrosis were graded as high or low based on MMP-9 and 8-OHdG staining and fibrotic scores.
Results
After evaluation the relationship between renal fibrosis and MMP-9, we found that patients with high interstitial fibrosis scores (IFS) and glomerular fibrosis scores (GFS) had significantly higher serum creatinine, lower estimated glomerular filtration rate (eGFR), and were more likely to have chronic kidney disease (CKD) and urothelial cell carcinoma. Univariate analysis showed that IFS and GFS were negatively associated with normal and atrophic tubular cytoplasmic MMP-9 expression and IFS was positively correlated with atrophic tubular nuclear MMP-9 expression. Multivariate stepwise regression indicated that MMP-9 expression in atrophic tubular nuclei（r = 0.4；p = 0.002）was an independent predictor of IFS, and that MMP-9 expression in normal tubular cytoplasm（r = -0.465；p < 0.001）was an independent predictor of GFS.
After evaluation the relationship between renal fibrosis and MMP-9, we found that Patients with higher IFS and GFS had significantly higher serum creatinine, lower eGFR, increased percentage of CKD and urothelial cell carcinoma. The renal tissues with higher IFS had lower expressions of 8-OHdG in normal tubular cytoplasm (NTc) (35.7% vs. 64.3%, p = 0.011) and normal tubular nuclei (NTn) (28.6% vs. 71.4%, p = 0.023). Univariate analysis showed that IFS and GFS correlated with the NTc 8-OHdG expression and IFS negatively correlated with NTn 8-OHdG expression. Multivariate stepwise regression revealed that serum creatinine (r = 0.351 for IFS, p = 0.021; r = 0.563 for GFS, p < 0.001) and intensity of 8-OHdG expression in NTc (r = 0.397 for IFS, p = 0.01; r = 0.278 for GFS, p = 0.043) were the independent factors predicting IFS or GFS.
Conclusion
Our results indicate that severity of renal fibrosis is associated with a decline of MMP-9 expression in the cytoplasm of normal tubular cells, increased expression of MMP-9 in the nuclei of tubular atrophic renal tubules, and increased expression of 8-OHdG intensity in normal tubular cytoplasm.

並列關鍵字

Renal fibrosis ； Matrix metalloproteinase-9 ； 8-hydroxy-2’ deoxyguanosine ； Immunohistochemical stain

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