Causal connections between axon guidance proteins and early progressive kidney function decline in diabetes

项目摘要/摘要 尽管血糖控制有所改善，但糖尿病引起的终末期肾病(ESKD)仍在继续上升 并广泛使用肾炎保护性药物进行治疗。对机制有了更全面的了解 对肾功能进行性下降负有责任的人迫切需要找到新的、更有效的 糖尿病并发ESKD的治疗和预防方法。通过研究患有晚期肾脏的糖尿病患者 与蛋白尿和慢性肾脏病≥3相关的疾病，我们获得了两个与两个T1D患者进展为ESKD相关的新发现 和T2D。使用来自两个独立队列和全球miRNAs平台的跟踪数据，我们发现了一个特定的 血浆miRNAs谱与进展为ESKD密切相关。这些miRNAs是专门针对 轴突引导途径(AGP)蛋白。值得注意的是，循环中6种AGP蛋白(两个配体EFNA4， EFN5和4受体EphA2、EPHB2、EPHB6、unc5C)与肾脏的范围有很强的相关性 T2D PIMA印度队列中的结构性损害和在四个独立队列中进展为ESKD。我们的 这是首次发现循环中的AGP蛋白与糖尿病发展为ESKD有关。 为了跟进这些发现，我们建议进行一项多学科研究，以评估AGP蛋白在 早期进行性肾功能下降(PKFD)的发生发展并了解其机制 循环中的AGP可能会损害肾脏。本文提出了三项互补性研究。克罗夫斯基博士的实验室将 检查AGP蛋白EFNA4和EphA2的两个样本水平升高是否相关/因果关系 与早期PKFD的发生发展有关。Kretzler博士的实验室将检查AGP循环水平如何 蛋白质可能会影响肾脏细胞的候选致病途径。科斯坦杰博士的实验室将检查 遗传操作对糖尿病小鼠循环中EFNA4和EphA2水平的影响 糖尿病肾病。拟议的研究有以下具体目标： 目的#1：我们将检查候选miRNAs和AGP蛋白循环水平的变化的影响 在两项嵌套病例对照研究中进行基线检查：T1D患者中有正常白蛋白(n=390)和微量白蛋白(n=390) 白蛋白(n=430)对7~15年随访的早期PKFD风险。目标2：我们将检查两者之间的联系 候选AGP蛋白的循环水平和候选致病途径的失调 细胞RNA测序结果从研究肾活检获得的皮马印第安人与T2D。目标3：我们 将测试EphA2和EFNA4在糖尿病小鼠模型中改变肾脏表型的能力，并测试 是否miR-328-5p和miR-339-5p在向ESKD进展过程中下调，并具有 在小鼠中，可以影响这些AGP蛋白对糖尿病肾脏疾病的影响。 这项拟议的研究极有可能产生新的发现。它的成功是由 共同调查人员有合作的记录，并研究样本的队列和生物库 乔斯林肾脏研究和皮马印度肾脏研究的所有成员都可以获得。

PROJECT SUMMARY/ABSTRACT End stage kidney disease (ESKD) due to diabetes continues to rise despite improvements in glycemic control and widespread treatment with reno-protective drugs. A more complete understanding of the mechanisms responsible for the progressive decline in kidney function is urgently needed to identify new, more efficacious methods of treatment and prevention of ESKD in diabetes. By studying diabetic patients with advanced kidney disease with proteinuria and CKD≥3, we obtained two novel findings related to progression to ESKD in both T1D and T2D. Using follow-up data from two independent cohorts and global miRNAs platform, we found a specific profile of plasma miRNAs strongly associated with progression to ESKD. These miRNAs specifically targeted axon guidance pathway (AGP) proteins. Strikingly, circulating levels of 6 AGP proteins (two ligands EFNA4, EFN5 and 4 receptors EPHA2, EPHB2, EPHB6, UNC5C) were very strongly associated with the extent of kidney structural lesions in the T2D Pima Indian cohort and progression to ESKD in four independent cohorts. Our findings are the first to implicate circulating AGP proteins in the progression to ESKD in diabetes. To follow-up these findings, we propose a multi-disciplinary study to assess the role of the AGP proteins in the development of early progressive kidney functional decline (PKFD) and learn about mechanisms through which circulating AGP may damage the kidney. Three complementary studies are proposed. Dr. Krolewski’s lab will examine whether elevated levels of two exemplars of AGP proteins EFNA4 and EPHA2 are associated/causally related to the development of early PKFD. Dr. Kretzler’s lab will examine how circulating levels of the AGP proteins may impact candidate etiological pathways in kidney cells. Dr. Korstanje’s lab will examine whether genetic manipulation of circulating levels of EFNA4 and EPHA2 in mice models of diabetes lead to onset of diabetic kidney disease. The proposed research has the following specific aims: Aim #1: We will examine the effect of variation of circulating levels of the candidate miRNAs and AGP proteins examined at baseline in the two nested cases-control studies of T1D patients with Normo-Alb (n=390) and Micro- Alb (n=430) on risk of early PKFD during 7-15 years of follow-up. Aim #2: We will examine the link between circulating levels of the candidate AGP proteins and dysregulation of candidate etiological pathways using single cell RNA sequencing results obtained from research kidney biopsies from Pima Indians with T2D. Aim #3: We will test EPHA2 and EFNA4 for their ability to modify renal phenotypes in a diabetic mouse model and test whether miR-328-5p and miR-339-5p, which are both downregulated during progression to ESKD and have orthologs in the mouse, can affect the impact of these AGP proteins on diabetic kidney disease. The proposed research has a very high probability of generating novel findings. Its success is assured by the fact that the co-investigators have a track record of collaboration, and study cohorts and biobanks of specimens from all members of the Joslin Kidney Study and Pima Indian Kidney Study are available.