THM1 modulation of GLI2 activation in cystic kidney disease

描述(申请人提供)：囊性肾病(CKD)，肾功能衰竭的主要原因，被认为起源于潜在的睫状体生理缺陷，尽管分子机制尚不清楚。这项拨款提案的目的是阐明Thm1aln/ALN小鼠模型中CKD的分子病因学，该模型显示纤毛蛋白运输缺陷，导致Hedgehog(HH)信号增加。有趣的是，最近在肾炎、Bardet-Biedl综合征和Meckel-Gruber综合征患者中发现了THM1突变，CKD是CKD的主要临床组成部分(N.Katsanis，个人沟通)。重要的是，HH信号的主要转录激活因子GLI2的缺失挽救了Thm1aln/ALN的大部分肾脏表型，Gli2-/-双突变，暗示HH信号过度活跃在Thm1aln/ALN肾囊肿的病因中。在第一个目的中，我们将利用小分子HH拮抗剂来挽救培养的突变肾脏的囊变，以探讨GLI2活性过高在Thm1aln/ALN肾脏囊变中的作用。第二个目的是通过使用这些小分子HH拮抗剂，通过活细胞成像检测GLI2纤毛转运与Thm1缺陷的内髓集合管(IMCD)细胞活性的关系，以及通过评估GLI2蛋白的稳定性和Thm1突变提取液中GLI2蛋白的负调节因子FUSED的状态，来探索GLI2激活及其被THM1调节的分子机制。这些分析将阐明纤毛缺陷与肾脏囊变之间的分子事件，以及纤毛生理和GLI2激活之间的相互联系。这项建议主张HH信号在CKD的病因学中的作用，这在很大程度上还没有被探索，并结合小分子的使用，可能会导致新的治疗方法。 公共卫生相关性：已证实的治疗方法缺乏囊性肾脏疾病(CKD)，这是肾功能衰竭的主要原因，被认为起源于睫状体生理的潜在缺陷，尽管分子机制尚不清楚。最近，在肾炎、Bardet-Biedl综合征和Meckel-Gruber综合征患者中发现了THM1突变，CKD是CKD的主要临床组成部分(N.Katsanis，个人沟通)。这项建议旨在阐明Thm1aln/ALN突变小鼠CKD的分子病因学，该突变小鼠表现出一种独特的纤毛缺陷，导致Hedgehog(HH)信号增强，通过利用小分子HH抑制剂挽救培养的突变肾脏的囊变发生，并利用活细胞成像观察它们对Thm1缺陷的肾脏来源哺乳动物细胞纤毛生理的影响。

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.