肾小管基底膜增厚的新机制--缺氧对MMP-2活性调控影响的机制探讨

Accumulating evidence emphasizes chronic hypoxia in the tubulointerstitium as the final common pathway to end stage kidney disease. One of the characteristic appearances of the tubulointerstitial fibrosis is the tubules atrophy and thickened basement membranes. Matrix metalloproteinase -2(MMP-2) is able to digest various components of the extracellular matrix including collogen Ⅳ in the basement membrane. For this reason, it suggests that tubular basement membrane thickening results from low activity of the enzyme. We found that hypoxia inhibited the activity of MMP-2 in the supernatant of the tubular cells (HK-2). However, it is unclear for the mechanism. In addition, it was found that hypoxia increased the autophagy and endocytosis in the cells by our group. We hypothesized that the autophagy and endocytosis in the tubular cells under hypoxia may be involved in the regulation of the MMP-2 activation at the surface of the cells, and not only highten MMP-2 activity, but also delay the progression of renal fibrosis through inhibiting autophagy and endocytosis. To test this hypothesis, at first, we will use tubular cell line and renal biopsy tissues to investigate the relation among the autophagy, endocytosis level and the MMP-2 activity, and to clarify the regulating mechanism. And then, we will use transgene animals to build renal fibrosis model, to inhibit autophagy and endocytosis by targeted intervention and observe the MMP-2 expression and activity as well as the fibrosis degree of the renal tissues in situ, to reveal the correlation among autophagy, endocytosis, enzyme and fibrosis. The results will be useful to verify a new mechanism of the enzyme activity regulation and search new targets for the treatment of the chronic kidney diseases.

小管间质缺氧与各种慢性肾脏病持续进展有关，肾小管萎缩、基底膜增厚是小管间质纤维化的重要特征。MMP-2能降解包括基底膜Ⅳ型胶原在内的多种细胞外基质，基底膜增厚提示其活性不足。本组发现缺氧抑制肾小管上皮细胞MMP-2活性，但调节机制不清；还发现缺氧使细胞自噬、内吞活动增强。我们假设在缺氧时，自噬和内吞影响了MMP-2在胞膜处的活化；抑制自噬和内吞，可能使MMP-2活性升高，延缓肾纤维化发展。基于该假说，我们拟利用培养的细胞和肾穿刺组织，研究缺氧状况下肾小管上皮细胞自噬、内吞水平和MMP-2活性之间的关系并探讨其内在机制；建立转基因动物肾纤维化模型，靶向干预肾小管上皮细胞自噬、内吞，原位观察肾内缺氧状况、MMP-2 表达及活性水平、纤维化程度，在整体水平了解肾小管上皮细胞自噬和内吞对MMP-2活性的影响。本研究有助于阐明一种新的MMP-2活性调控机制，为临床治疗慢性肾脏病提供新的治疗靶点。

肾小管萎缩、基底膜增厚是各种慢性肾脏病的重要形态特征，但形成机制不清。小管间质缺氧与疾病持续进展有关。基于上述事实及本组前期工作基础，提出缺氧肾小管上皮细胞由于自噬内吞增强，影响基质金属蛋白酶-2（matrix metalloproteinase-2, MMP-2）在细胞膜处的活性调节，使包括基底膜Ⅳ型胶原在内的多种细胞外基质降解减少，从而促进纤维化的假说。为此进行了以下研究：①通过二代测序，了解缺氧肾小管上皮细胞基因表达谱变化规律；②缺氧肾小管上皮细胞自噬、内吞与MMP-2相关性分析；③肾小管上皮细胞自噬、内吞调节MMP-2活性的机制探索；④建立动物肾纤维化模型进一步验证。结果发现：①肾小管上皮细胞在缺氧状态下的表达谱模式发生了变化。表现为损伤修复相关基因活动明显增强，而与抗损伤有关的抗原处理和递呈相关基因、内质网蛋白处理相关基因、能量产生相关基因、谷胱甘肽生成相关基因下调。②在缺氧状态下，细胞自噬内吞活动增强，从不同途径影响MMP-2表达和活性；自噬主要抑制MMP-2表达，内吞明显抑制MMP-2活性。③自噬内吞共享分子RAB7和内吞相关蛋白caveolin-1通过酶活性调节相关分子RECK、MMP-14、TIMP-2而发挥作用。④缺氧可激活肾小管上皮细胞Src激酶促进内吞使MMP-2活性下降而加重肾间质纤维化。⑤Rab7激活自噬减轻白蛋白诱导的肾小管上皮细胞损伤。⑥成功建立了缺氧性肾纤维化动物模型。⑦利用CRISPR/Cas技术在ROSA26位点敲入RAB7基因，获得F1代转基因C57BL/6小鼠。本研究结果表明：缺氧不仅改变肾小管上皮细胞基因表达模式，而且使细胞自噬内吞增强，导致MMP-2活性调节相关分子的表达异常，酶活性因此受抑。而RAB7、小窝蛋白Caveolin-1、Src激酶等为重要的介导分子。此工作对于深入认识肾纤维化机制、寻找干预靶点具有重要意义，也为后续研究打下了坚实基础。