铜离子内流增加促进ECM过度交联在AngII-β-arrestin2偏向性信号通路所致肾间质纤维化中的作用及机制研究

Copper associating organ fibrosis has been noticed recently，but the underlying mechanism is unknown. Our previous study found that: 1) in both renal fibrosis tissues of CKD patients and kidneys of unilateral ureteral obstruction (UUO) rats, the expression of copper transporter 1(CTR1) and the copper influx were increased. the activities of lysine oxidase, copper as the active auxiliary base, was increased significantly, extracellular matrix (ECM) was over-crosslinking; 2) In UUO model, Inhibiting CTR1 or copper chelate reduced the activity of LOX significantly, alleviated the ECM crosslinking; 3）Angiotensin II upregulated CTR1 expression in NRK-49F cells; 4) We have previously confirmed that AngII results in renal interstitial fibrosis through AT1R-β-arrestin2 pathway. Accordingly we propose the hypothesis that AngII could upregulate CTR1 expression through AT1R-β-arrestin2-STAT3 pathway, increase copper intracellular flow, activate LOX, induce ECM over-crosslinking, and promote renal interstitial fibrosis finally. Conditional CTR1 gene knockout and Ang II micropump perfusion model combining in vitro experiment will be used to clarify the regulating mechanism of AngII-AT1R-β-arrestin2 pathway on regulation of CTR1, reveal the important role of copper and ECM over crosslinking in renal fibrosis.

铜与脏器纤维化的关联在近年得到关注，但具体机制不清。我们前期研究发现：1）CKD患者肾纤维化组织及单侧输尿管结扎（UUO）大鼠肾组织中铜转运蛋白（CTR1）表达增加，铜细胞内流增加，以铜为辅基的赖氨酸氧化酶（LOX）活性增加，细胞外基质（ECM）交联增加；2）在UUO中，抑制CTR1或螯合铜显著降低LOX活性，减少ECM交联；3）AngII处理肾成纤维细胞上调CTR1表达；4）我们以往已证实AngII是通过AT1R-β-arrestin2通路导致肾间质纤维化。据此我们提出：AngII通过AT1R-β-arrestin2通路上调CTR1表达，增加铜内流，增加LOX活性，导致ECM过度交联，促进肾间质纤维化。本课题将利用CTR1条件性敲除及AngII微泵灌注模型，结合体外实验，阐明AngII-AT1R-β-arrestin2通路对CTR1的调控，揭示铜和ECM过度交联在肾间质纤维化中的作用。

铜离子在哺乳动物细胞中扮演着不同的角色，可能是因为它们参与了不同的酶促反应。一些研究表明，血清铜与肝和肺等器官的纤维化相关。赖氨酰氧化酶（LOX）是一种铜依赖性单胺氧化酶，其主要功能是细胞外基质（ECM）中胶原的共价交联。有证据表明，LOX与癌症和某些纤维化疾病有关。在此，我们探讨了铜在肾纤维化发展中的作用以及可能的潜在机制。我们发现，（1）在纤维化模型和肾纤维化患者的肾组织中，铜转运蛋白1（CTR1）与LOX表达增加；（2）CTR1的上调需要Smads依赖性TGF-β信号通路，升高的CTR1诱导铜细胞内流增加；（3）在肾纤维化中，β-arrestin-ERK-STAT3途径的调节 LOX，诱导胶原沉积和交联增加；（4） 体内BAPN对LOX的药理学抑制抑制了LOX的上调，减少了胶原过度交联，并改善了缺血损伤后的肾纤维化。总之，细胞内铜积累通过激活LOX介导的胶原和弹性蛋白交联在肾纤维化中起着独特的作用。抑制细胞内铜超载或LOX活性可能是缓解肾纤维化的潜在途径。