The role of Cullin 3- mediated protein ubiquitination and degradation in the initiation of kidney fibrosis

慢性肾脏病（CKD）的特点是随着时间的推移肾功能逐渐丧失，并可能最终导致肾衰竭，这需要透析或肾移植来维持生命。CKD影响全球普通人群中十分之一的成年人，主要由高血压或糖尿病引起。纤维化是几乎所有慢性肾脏损伤的最终途径。因此，抑制纤维化代表了抑制CKD进展至其结论的逻辑策略。Cullin在调节多种生物学过程中发挥关键作用，包括细胞周期、肿瘤发生和纤维化。Cullin 3（Cul 3）属于Cullin-RING连接酶（CRL）家族，其将泛素部分添加到蛋白质，从而通过向其发出降解信号来控制蛋白质的表达水平。大卫埃里森和吉姆麦考密克最近报告说，肾小管特异性Cul 3删除成年小鼠（KS-Cul 3-/-）导致肾功能在几周内进行性丧失，这意味着Cul 3在肾脏中的重要生理作用。在合作的基础上，我在这些小鼠中观察到进行性肾小管间质纤维化的公认特征，如细胞外基质积累增加、细胞凋亡、细胞增殖和T细胞浸润。然而，目前还不清楚Cul 3如何介导其抗纤维化作用。到目前为止，我们只知道Cul 3的破坏与主要的细胞周期调节因子cyclin E的表达增加有关。本研究的目的是探讨Cul 3在肾纤维化和CKD发病机制中的作用。在大卫埃里森（医学博士，医学教授）和吉姆麦考密克（博士，医学助理教授），两位都是美国俄勒冈州健康与科学大学的主要研究者，我将描述KS-Cul 3-/-小鼠肾脏纤维化的时间过程。我将测试KS-Cul 3-/-小鼠是否可以作为新的肾小管间质纤维化模型，因为其他小鼠模型是不完善的。为了确定在体内的适应不良的过程中，这个建议将集中在细胞周期，细胞因子和活性氧的释放，肌成纤维细胞的激活，和肾小管间质胶原蛋白的生产后，肾Cul 3删除。除了KS-Cul 3-/-模型，我还将分析Cul 3泛素化途径在三种不同的小鼠模型的肾纤维化和肾脏组织中的各种病因的CKD患者。在体外，我将测试CRISPR/Cas9系统在人原代肾近端小管上皮细胞中Cul 3失活导致细胞周期停滞和旁分泌肌成纤维细胞活化的假设。了解Cul 3在纤维化中的作用可以为靶向CKD患者的促纤维化机制开辟新的治疗机会。此外，计划研究的结果可能不仅适用于肾脏，也适用于其他器官。

Chronic kidney disease (CKD) is characterized by gradual loss of kidney function over time, and may eventually lead to kidney failure, which requires dialysis or a kidney transplant to maintain life. CKD affects one in ten adults in the general population worldwide, and is primarily caused by hypertension or diabetes. Fibrosis is the final pathway of virtually all chronic injury to the kidney. Inhibiting fibrosis therefore represents a logical strategy to inhibit progression of CKD to its conclusion. However, the initiating causes are poorly understood and importantly, no specific therapy exists.Cullins play critical roles in regulating a variety of biological processes, including the cell cycle, tumorigenesis, and fibrosis. Cullin 3 (Cul3) belongs to the Cullin-RING ligase (CRL) family, which adds ubiquitin moieties to proteins, thus controlling expression level of proteins by signaling it for degradation. David Ellison and Jim McCormick recently reported that kidney tubule-specific Cul3 deletion in adult mice (KS-Cul3-/-) led to progressive loss of kidney function within weeks, implying an essential physiological role of Cul3 in the kidney. On a collaborative basis, I observed in these mice well-accepted features of progressive tubulointerstitial fibrosis, such as increased extracellular matrix accumulation, apoptosis, cell proliferation and T-cell infiltration. However, it is unclear how Cul3 mediates its anti-fibrotic effects. So far, we only know that Cul3 disruption is associated with increased expression of the major cell cycle regulator cyclin E. The aim of this proposal is to investigate the role of Cul3 in the pathogenesis of renal fibrosis and CKD. In the labs of David Ellison (M.D., Professor of Medicine) and Jim McCormick (Ph.D., Assistant Professor of Medicine), both principal investigators at Oregon Health and Science University, USA, I will characterize the time-course of kidney fibrosis in KS-Cul3-/- mice. I will test if KS-Cul3-/- mice could serve as new tubulointerstitial fibrosis model since other mouse models are imperfect. In order to determine the maladaptive processes in vivo, this proposal will focus on the cell cycle, release of cytokines and reactive oxygen species, activation of myofibroblasts, and production of tubulointerstitial collagen upon renal Cul3 deletion. In addition to the KS-Cul3-/- model, I will also analyze the Cul3 ubiquitination pathway in three different mouse models of kidney fibrosis and in kidney tissue of patients with CKD of various etiologies. In vitro, I will test the hypothesis that Cul3 inactivation by CRISPR/Cas9 system in human primary renal proximal tubule epithelial cells causes cell-cycle arrest and paracrine myofibroblast activation. Understanding the role of Cul3 in fibrosis could open new therapeutic opportunities for targeting pro-fibrotic mechanisms in patients with CKD. In addition, the results of the planned studies may not only apply to kidney, but also to other organs.