THM1 modulation of GLI2 activation in cystic kidney disease

描述（由申请人提供）：囊性肾病（CKD）是肾衰竭的主要原因，被认为是源于睫状体生理学的潜在缺陷，但分子机制尚不清楚。这项资助提案的目标是阐明Thm 1aln/aln小鼠模型中CKD的分子病因学，该模型表现出纤毛蛋白运输缺陷，导致Hedgehog（Hh）信号传导增加。有趣的是，THM 1的突变最近已经在患有肾单位营养不良、Bardet-Biedl综合征和Meckel-Gruber综合征的患者中被鉴定，其特征在于CKD作为主要临床组分（N. Katsanis，个人通信）。重要的是，Hh信号传导的主要转录激活因子GLI 2的缺失挽救了Thm 1aln/aln、Gli 2-/-双突变体中的大多数肾脏表型，这暗示了Thm 1aln/aln肾囊肿病因学中Hh信号传导过度活跃。在第一个目标中，将通过使用小分子Hh拮抗剂来挽救培养的突变肾脏中的囊肿形成来研究过度GLI 2活性在Thm 1aln/aln肾脏囊肿形成中的作用。在第二个目标中，将通过使用这些相同的小分子Hh拮抗剂来探索GLI 2激活及其通过THM 1调节的分子机制，以使用活细胞成像来检查GLI 2纤毛运输与Thm 1缺陷的内髓集合管（IMCD）细胞中的活性之间的关系，并通过评估GLI 2蛋白稳定性和融合抑制因子（GLI蛋白的负调节因子）的状态，在Thm 1突变体提取物中。这些分析将阐明连接睫状体缺陷与肾囊肿形成的分子事件，以及睫状体生理学与GLI 2激活之间的相互联系。该提案主张Hh信号传导在CKD病因学中的作用，这在很大程度上尚未探索，并且与小分子的使用相结合，可能导致新的治疗方法。 公共卫生相关性：慢性肾脏病（CKD）是肾功能衰竭的主要原因，被认为是源于睫状体生理学的潜在缺陷，尽管分子机制尚不清楚。最近，THM 1突变已在肾单位结核、Bardet-Biedl综合征和Meckel-Gruber综合征患者中被鉴定，其特征在于CKD作为主要临床组分（N. Katsanis，个人通信）。该提案旨在阐明Thm 1aln/aln突变小鼠CKD的分子病因学，其表现出独特的纤毛缺陷，导致刺猬（Hh）信号传导增加，通过利用小分子Hh抑制剂来拯救培养的突变肾脏中的囊性形成，并使用活细胞成像来可视化它们对Thm 1缺陷的肾脏衍生哺乳动物细胞的纤毛生理学的影响。

DESCRIPTION (provided by applicant): Cystic kidney disease (CKD), a leading cause of renal failure, is proposed to originate from an underlying defect in ciliary physiology, though the molecular mechanisms are unclear. The goal of this grant proposal is to elucidate the molecular etiology of CKD in the Thm1aln/aln mouse model, which exhibits a defect in ciliary protein trafficking that leads to increased Hedgehog (Hh) signaling. Interestingly, mutations in THM1 have recently been identified in patients with nephronophthisis, Bardet-Biedl Syndrome and Meckel-Gruber Syndrome, which feature CKD as a major clinical component (N. Katsanis, personal communication). Importantly, the loss of GLI2, the primary transcriptional activator of Hh signaling, rescues most of the kidney phenotype in Thm1aln/aln,Gli2-/- double mutants, implicating overactive Hh signaling in the etiology of Thm1aln/aln renal cysts. In the first aim, the role of excessive GLI2 activity in Thm1aln/aln renal cystogenesis will be investigated by using small molecule Hh antagonists to rescue cystogenesis in cultured mutant kidneys. In the second aim, the molecular mechanism of GLI2 activation and its modulation by THM1 will be explored by using these same small molecule Hh antagonists to examine the relationship between GLI2 ciliary trafficking and activity in Thm1-deficient inner medullary collecting duct (IMCD) cells using live-cell imaging, and by assessing GLI2 protein stability and the status of Suppressor of Fused, a negative regulator of the GLI proteins, in Thm1-mutant extracts. These analyses will elucidate the molecular events connecting a ciliary defect to renal cystogenesis, as well as the interconnection between ciliary physiology and GLI2 activation. This proposal advocates a role for Hh signaling in the etiology of CKD, which has been largely unexplored, and combined with the use of small molecules, can potentially lead to novel therapeutic approaches. PUBLIC HEALTH RELEVANCE: Proven therapies lack for cystic kidney disease (CKD), a leading cause of renal failure, which is proposed to originate from an underlying defect in ciliary physiology, though the molecular mechanisms are unclear. Recently mutations in THM1 have been identified in patients with nephronophthisis, Bardet-Biedl Syndrome and Meckel-Gruber Syndrome, which feature CKD as a major clinical component (N. Katsanis, personal communication). This proposal aims to elucidate the molecular etiology of CKD in the Thm1aln/aln mutant mouse, which exhibits a unique ciliary defect leading to increased Hedgehog (Hh) signaling, by utilizing small molecule Hh inhibitors to rescue cystogenesis in cultured mutant kidneys and to visualize their effects on ciliary physiology of Thm1-deficient, kidney-derived mammalian cells using live-cell imaging.