THM1 modulation of GLI2 activation in cystic kidney disease

描述（由申请人提供）：囊性肾脏病（CKD）是肾衰竭的主要原因，被认为起源于睫状体生理的潜在缺陷，尽管其分子机制尚不清楚。这项拨款提案的目标是阐明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）信号传导增加。有趣的是，最近在肾脏病、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，个人交流）。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、GLI2 -/-双突变体的大部分肾脏表型，暗示了Thm1aln/aln肾囊肿病因中Hh信号过度活跃。在第一个目标中，我们将通过使用小分子Hh拮抗剂来挽救培养的突变肾脏的膀胱发生，研究过度GLI2活性在Thm1aln/aln肾膀胱发生中的作用。在第二个目标中，将通过使用这些相同的小分子Hh拮抗剂，使用活细胞成像检查GLI2睫状体运输与THM1缺陷内髓集管（IMCD）细胞活性之间的关系，以及通过评估GLI2蛋白稳定性和THM1突变提取物中GLI蛋白的负调节因子Suppressor of Fused的状态，来探索GLI2激活及其被THM1调节的分子机制。这些分析将阐明睫状体缺陷与肾膀胱形成之间的分子事件，以及睫状体生理与GLI2激活之间的相互联系。该建议提倡Hh信号在CKD病因学中的作用，这在很大程度上尚未被探索，并且与小分子的使用相结合，可能会导致新的治疗方法。

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.