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

慢性肾脏疾病（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。慢性肾病影响全球十分之一的成年人，主要由高血压或糖尿病引起。纤维化是几乎所有肾脏慢性损伤的最终途径。因此，抑制纤维化是一种抑制CKD进展的合理策略。然而，人们对发病原因知之甚少，重要的是，目前还没有专门的治疗方法。Cullins在调节多种生物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 （Cul3）属于Cullin- ring连接酶（CRL）家族，它将泛素片段添加到蛋白质上，从而通过信号传导降解来控制蛋白质的表达水平。David Ellison和Jim McCormick最近报道，成年小鼠肾小管特异性Cul3缺失（KS-Cul3-/-）导致肾脏功能在数周内进行性丧失，这表明Cul3在肾脏中具有重要的生理作用。在合作的基础上，我在这些小鼠中观察到公认的进行性小管间质纤维化的特征，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，Cul3如何介导其抗纤维化作用尚不清楚。到目前为止，我们只知道Cul3的破坏与主要细胞周期调节因子cyclin e的表达增加有关。本提案的目的是研究Cul3在肾纤维化和CKD发病机制中的作用。在美国俄勒冈健康与科学大学的首席研究员David Ellison（医学博士，医学教授）和Jim McCormick（医学博士，医学助理教授）的实验室中，我将描述KS-Cul3-/-小鼠肾纤维化的时间过程。由于其他小鼠模型不完善，我将测试KS-Cul3-/-小鼠是否可以作为新的小管间质纤维化模型。为了确定体内的不适应过程，本研究将重点关注肾Cul3缺失时的细胞周期、细胞因子和活性氧的释放、肌成纤维细胞的激活和小管间质胶原的产生。除了KS-Cul3-/-模型外，我还将分析三种不同肾纤维化小鼠模型和不同病因CKD患者肾组织中的Cul3泛素化途径。在体外，我将验证CRISPR/Cas9系统在人原发性肾近端小管上皮细胞中灭活Cul3导致细胞周期阻滞和旁分泌肌成纤维细胞激活的假设。了解Cul3在纤维化中的作用可以为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，还适用于其他器官。

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.