The Role of C- Reactive Protein in Type 2 Diabetes and Diabetic Nephropathy

Abstract

研究背景：大量临床研究表明2型糖尿病患者血清中C-反应蛋白（CRP）水平明显升高, 其严重程度与糖尿病患者肾脏病变的发生发展密切相关。 然而, CRP 在2型糖尿病和糖尿病肾病 (DN)中的具体作用和机制尚不清楚。 因此，在本项研究中，我们应用高表达CRP的自发性2型糖尿病小鼠模型(CRPtg-db/db)和体外实验探讨CRP在2型糖尿病和糖尿病肾脏病变中的具体作用和机制。研究方法：将高表达人类CRP的CRP转基因小鼠与自发性糖尿病的db/db小鼠杂交，产生具有高水平CRP的 CRPtg-db/db 2型糖尿病小鼠模型。 对照组包括同窝和相同年龄的：CRPtg-db/m, db/db 和db/m小鼠。从第四周开始，每隔4周检测各组小鼠空腹血糖、体重、血压、葡萄糖耐量试验（IPGTT），胰岛素耐量试验（IPITT）和24h尿微量白蛋白水平。在36周时处死小鼠并收取血液和肾脏标本，检测肾脏病变情况。此外，通过体外实验进一步研究CRP介导糖尿病和糖尿病肾病的具体信号机制。研究结果：与同窝db/db小鼠比较，CRPtg-db/db小鼠的空腹血糖和胰岛素抵抗明显升高，糖尿病肾脏病变程度进一步加重包括：1）尿微量白蛋白显著增加；2）更多的肾脏炎症因子分泌包括IL-1β、TNF-α和MCP-1表达上调，以及CD3阳性T细胞和 F4/80阳性巨噬细胞浸润；3）肾间质纤维化程度进一步加重，表现为肾脏Ⅰ型和Ⅳ型胶原的过度沉积。通过对CRP在体内、体外的分子病例机制和信号转导通路研究，我们发现CRP介导的糖尿病肾脏病变与CRP受体（CD32b）表达上调以及 NF-κB, TGF-β1/Smad3, mTOR信号通路的过度激活有关， 体外研究进一步表明，CRP与其受体CD32b结合后可以通过TGF-β1和ERK/P38MAPK依赖的信号机制激活Smad3， 激活的Smad3可以与mTOR结合并介导纤维化的发生，而CRP受体（CD32b）抗体和 mTOR 抑制剂（雷帕霉素）可以阻断此病变过程。结论：CRP介导并促进了2型糖尿病和糖尿病肾病的发生发展。CRP与其受体CD32b结合后可以促进NF-κB介导的肾脏炎症和TGF-β1/Smad3以及mTOR 信号通路介导的肾脏纤维化的发生发展。通过干预CRP，可能为糖尿病和糖尿病肾病的防治提供新的靶点和思路。Background: Emerging evidence shows that elevated serum levels of C-reactive protein (CRP) is closely associated with progressive diabetic nephropathy (T2DN) in patients with type-2 diabetes. In this study, we hypothesized that CRP may promote type-2 diabetes and enhance T2DN in a novel mouse model of db/db mice that overexpress human CRP gene (CRPtg-db/db) and in vitro.Methods: Human CRPtg-db/db mice and their littermate controls including db/db, db/m and CRPtg-db/m mice were generated by crossing db/m mouse with CRPtg mouse that expresses human CRP. Fasting blood glucose, intraperitoneal glucose tolerance test (IPGTT), intraperitoneal insulin tolerance test (IPITT), body weight, blood pressure, and 24-h urine microalbumin levels were measured every 4 weeks over the 36-week period and renal fibrosis was examined at 36 weeks. In addition, signaling mechanisms of CRP-mediated DN were investigated in vivo and in vitro.Results: As described in Chapter III and IV, compared with littermate db/db mice, CRPtg-db/db mice developed higher levels of fasting blood glucose and enhanced insulin resistance with more severe diabetic nephropathy including: 1) a marked increase in microalbuminuria; 2) development of more progressive renal inflammation including upregulation of IL-1b, TNF-a, MCP-1 and CD3+ F4/80+ macrophage infiltration; 3) progressive renal fibrosis with excessive deposition of collagen I and IV within the diabetic kidney. To address the possible mechanisms whereby CRP promotes T2DN, we investigated the signaling mechanisms associated with CRP-mediated T2DN in vivo and in vitro. As described in Chapters IV and V, we found that worsen T2DN was associated with a marked upregulation of the CRP receptor CD32b and activation of NF-κB, TGF-b1/Smad3, and mTOR signaling pathways. Further studies in cultured HK-2 cells revealed that addition of CRP induced activation of Smad3 through its receptor CD32b via both TGF-b1-dependent and ERK/p38 MAPK-dependent signaling mechanisms. More importantly, ChIP assays identified that activated Smad3 could bound directly to mTOR and stimulated fibrosis, which was blocked by an anti-CD32b neutralizing antibody or by an mTOR inhibitor (rapamycin).Conclusion: CRP is pathogenic in type-2 diabetes and DN. CRP may enhance CD32b-NF-κB signaling to induce renal inflammation; whereas, CRP may exacerbate renal fibrosis in T2DN via CD32b-Smad3-mTOR signaling.You, Yongke.Thesis Ph.D. Chinese University of Hong Kong 2016.Includes bibliographical references (leaves ).Abstracts also in Chinese.Title from PDF title page (viewed on …).Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only