The role of DNA damage response in chronic kidney disease

摘要 慢性肾脏病（CKD）是一种破坏性的肾脏疾病，可导致肾功能衰竭， 其他系统并发症。据估计，CKD影响美国超过2500万人， 人口中的发病率正在上升。虽然CKD的病因多种多样，包括糖尿病、急性肾功能衰竭、 损伤、遗传和环境因素，导致CKD进展的机制较少 明白越来越多的证据表明，尿毒症毒素和其他应激介质造成的反复损伤 导致CKD患者的基因组不稳定性和细胞周期异常。尽管，对基因的破坏 材料，如果未修复，对肾小管细胞是有害的，因为它干扰DNA复制和肾小管损伤。 再生，DNA损伤反应在调节肾小管修复和细胞衰老中的作用 方案尚未建立。 最近，我们鉴定了范可尼贫血相关DNA修复酶FAN 1的突变， 导致受影响个体的CKD。FAN 1在DNA复制中起作用，并调节DNA交联 修复.然而，它在预防CKD中的作用尚不清楚。为了研究Fan 1在肾脏中的作用，我们制作了一个 Fan 1基因敲除小鼠，重现人FAN 1缺陷型CKD表型。我们现在证明， Fan 1缺失的肾脏对遗传毒性和非遗传毒性肾小管损伤的敏感性增加， 修复激活肾小管衰老和纤维化程序。根据初步结果，我们 假设肾小管细胞中受损的DNA损伤反应导致肾小管修复缺陷， 是慢性肾病发病机制的基础。在具体目标1中，我们将研究Fan 1在以下方面的作用： 顺铂和单侧输尿管梗阻损伤（UUO）后肾小管细胞衰老，使用各种 免疫组化和分子方法对新产生的Fan 1-null小鼠模型的研究。最近 我们实验室的研究表明，Fan 1的缺失影响细胞周期检查点的激活，并导致有丝分裂， 肾小管细胞异常。基于这些观察，具体目标2将解决风扇1的作用 在有丝分裂调控，并将检查疗效的治疗策略，旨在调节细胞周期 Fan 1缺失肾的进展。在具体目标3中，我们将描述 FAN 1和儿童CKD基因SDCCAG 8在肾脏中。我们还将使用BioID邻近标记 一种鉴定细胞中与Fan 1相关的蛋白质的方法，以深入了解其功能 途径。 总之，Fan 1小鼠模型为我们提供了一个新的机会来研究分子生物学。 与慢性肾脏疾病发展相关的途径，以及评估DNA的作用 在这种人类疾病的发展过程中损害反应途径，希望开发新的治疗方法。 !

ABSTRACT Chronic kidney disease (CKD) is a devastating renal condition that leads to kidney failure and causes other systemic complications. It is estimated that CKD affects more than 25 million people in the US and its incidence in the population is growing. While various etiologies underlie CKD, including diabetes, acute kidney injury, genetic and environmental factors, the mechanisms that contribute to the progression of CKD are less understood. Accumulating evidence suggests that recurrent injury by uremic toxins and other stress mediators leads to genomic instability and cell cycle abnormalities in CKD patients. Although, damage to the genetic material, if unrepaired, is detrimental to the tubular cell, since it interferes with DNA replication and tubular regeneration, the role of DNA damage response in regulating renal tubular repair and cellular senescence programs has not been established. Recently, we identified mutations in a Fanconi anemia-associated DNA repair enzyme, FAN1, as causing CKD in affected individuals. FAN1 has functions in DNA replication and regulates DNA cross-link repair. However, its role in preventing CKD is not understood. To study Fan1 role in the kidney we generated a Fan1-null mouse that recapitulates the human FAN1-deficient CKD phenotype. We now demonstrate that Fan1-null kidneys display increased sensitivity to genotoxic and non-genotoxic tubular injury, whose defective repair activates renal tubular senescence and fibrogenic programs. Based on our preliminary results, we hypothesize that impaired DNA damage response in the renal tubular cells leads to defective tubular repair and underlies the pathogenesis of chronic kidney disease. In Specific Aim 1, we will investigate the role of Fan1 in renal tubular cell senescence after cisplatin- and unilateral ureteral obstruction injury (UUO), using a variety of immunohistochemical and molecular approaches on the newly generated Fan1-null mouse model. Recent studies in our lab indicate that loss of Fan1 affects cell cycle checkpoint activation and causes mitotic abnormalities in kidney tubular cells. Based on these observations Specific Aim 2 will address the role of Fan1 in mitotic regulation and will examine the efficacy of therapeutic strategies designed to modulate cell cycle progression in Fan1-null kidneys. In Specific Aim 3, we will characterize the functional interaction between FAN1 and a pediatric CKD gene, SDCCAG8, in the kidney. We will also use the BioID proximity labeling method to identify proteins that are associated with Fan1 in the cell, in order to gain insights into its functional pathways. In summary, the Fan1-mouse model provides us with a novel opportunity to examine the molecular pathways associated with the development of chronic kidney disease, as well as to assess the role of DNA damage response pathway in the progression of this human disease in hopes of developing new treatments. !