The Role of Intercalated Cells and Their Acid Base and Electrolyte Transport Machinery in Kidney Cystogenesis by Tuberous Sclerosis

抽象的。结节性硬化症(TSC)是一种常染色体显性遗传性疾病，由 通过使TSC1或TSC2基因突变失活并影响包括肾脏在内的多个器官 还有肺。这种疾病影响着全球200多万人，其中很大一部分人患上了 血管肌脂肪瘤和囊肿，最终导致肾功能衰竭。而TSC的初始遗传事件 对于促进进展的生物过程和因素，人们知之甚少 或囊肿胀大。我们已经建立了肾主体(PC)细胞特异性TSC1失活的小鼠 或TSC2基因(已发表并出版)。数据)，这些数据概括了人类TSC囊性肾病。这个 囊上皮细胞很少，但可见大量的A-IC细胞。 Rna-seq和验证性表达研究表明， Foxi1--IC细胞发育及H-ATPase和Carbon激活的关键转录因子 TSC1-KO小鼠的脱氢酶II(CAII)活性。抗体的双重免疫荧光标记研究 H-ATPase和AQP2；H-ATPase和增殖细胞核抗原(增殖细胞核抗原，细胞标志) 增殖)和H-ATPase和CAII显示PC细胞进行性丧失和过度增殖 TSC1KO小鼠囊性上皮中A-IC细胞的研究此外，电生2Cl-/H交换剂CLC-5， 在基础条件下与H-ATPase共定位于晚期内切体膜和溶酶体膜， 与H-ATPase在TSC1囊上皮细胞顶膜上的显著共定位 Ko老鼠。此外，我们的结果表明，前肾素受体的共同定位，一个关键的参与者 WNT信号通路，H-ATPase位于囊性上皮细胞顶膜上。这些变化是 与常染色体显性遗传性PKD囊性病变不同(原始数据)。 TSC1KO小鼠Foxi1基因缺失导致Foxi1/TSC1双重囊虫负担完全消除 突变的小鼠。此外，受Foxi1调控的对H-ATPase活性至关重要的CAII的缺失， 显著降低CAII/TSC1双突变小鼠的包囊负担。 我们认为A-插层细胞的强劲增殖及其酸/碱/电解质转运 机械在TSC1KO小鼠肾囊变过程中起着至关重要的作用。我们进一步提出，与PKD中的包囊不同， 它们对AVP V2受体拮抗的反应是通过减少其液体分泌和大小，TSC囊， 主细胞少，对V2受体拮抗剂有抗药性。 为了验证我们的假设，我们建议：确定A-IC细胞的作用及其酸碱运输 机械(H-ATPase和CAII)在TSC病中的囊性生长和扩张；确定其作用 TSC小鼠囊腔和囊腔扩张过程中ClC-5和CFTR的表达 CAII/H-ATPase、PRR和mTOR抑制剂对TSC小鼠包囊生长的影响为此，我们 将研究同时缺失TSC1(在主细胞中)与Foxi1，CAII， H-ATPase B亚单位，或ClC-5对肾囊变的影响。此外，药物抑制剂的作用 CFTR激活(V2受体拮抗剂)、H-ATPase/CAII(乙酰唑胺)、PRR(手柄区域 为了确定它们对TSC1 KO囊性扩张的影响，将探索它们对TSC1 KO囊性扩张的影响。 老鼠。囊性生长和大小、肾功能、全身血压、预期寿命以及mTORC1 将在突变小鼠身上确定囊上皮细胞的激活情况。我们坚信拟议中的研究 是新颖的，并可能导致这种毁灭性的疾病以及其他一些肾脏疾病的新疗法 囊性疾病。

Abstract. Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder, which is caused by inactivating mutations in either the TSC1 or TSC2 gene and affects multiple organs, including kidney and lung. The disease affects over two million people worldwide, with a large proportion developing angiomyolipomata and cysts, which eventually lead to renal failure. While the initial genetic events of TSC have been delineated, little is known about the biological processes and factors that facilitate progression or expansion of cysts. We have developed mice with kidney principal (PC) cell-specific inactivation of Tsc1 or Tsc2 genes (Published and Prelim. Data) which recapitulate the human TSC cystic kidney disease. The cyst epithelia display few PC cells, but robust presence of A-intercalated (A-IC) cells. RNA-seq and confirmatory expression studies demonstrated a significant increase in the expression of Foxi1, a transcription factor critical to the development of IC cells and activation of H+-ATPase and carbonic anhydrase II (CAII) in Tsc1 KO mice. Double immunofluorescent labeling studies with antibodies against H+-ATPase and AQP2; H+-ATPase and PCNA (proliferative nuclear cell antigen, a marker of cell proliferation) and H+-ATPase and CAII demonstrated progressive loss of PC cells and hyperproliferation of A-IC cells in cyst epithelium in Tsc1 KO mice. In addition, the electrogenic 2Cl-/H+ exchanger CLC-5, which colocalizes with H+-ATPase in membranes of late endosomes and lysosomes under basal conditions, demonstrated remarkable co-localization with H+-ATPase on the apical membrane of cyst epithelia in Tsc1 KO mice. Further, our results indicated the co-localization of pro-renin receptor PRR, a critical player in Wnt signaling pathway, with H+-ATPase on the apical membrane of cyst epithelia. These changes are distinct from those in autosomal dominant PKD cysts (Prelim Data). Deletion of Foxi1 in Tsc1 KO mice resulted in complete abrogation of cyst burden in Foxi1/Tsc1 double mutant mice. In addition, deletion of CAII, which is regulated by Foxi1 and is critical to H+-ATPase activity, significantly blunted the cyst burden in CAII/Tsc1 double mutant mice. We propose that the robust proliferation of A-intercalated cells and their acid/base/electrolyte transport machinery are crucial to kidney cystogenesis in Tsc1 KO mice. We further propose that unlike cysts in PKD, which respond to AVP V2 receptor antagonism by reduction in their fluid secretion and size, TSC cysts, which have few principal cells, will be resistant to V2 receptor antagonists. To test our hypotheses, we propose to: Ascertain the role of A-IC cells and their acid base transport machinery (H+-ATPase and CAII) in the growth and expansion of cysts in TSC disease; Determine the role of CLC-5 and CFTR in chloride secretion into cyst lumen and cyst expansion in mice with TSC; and Examine the effect of inhibitors of CAII/H+-ATPase, PRR and mTOR on cyst growth in TSC mice. To this end, we will examine the effect of simultaneous deletion of Tsc1 (in principal cells) in combination with Foxi1, CAII, H+-ATPase B subunit, or CLC-5 on renal cystogenesis. Further, the effect of pharmacologic inhibitors of CFTR activation (V2 receptor antagonists), H+-ATPase/CAII (acetazolamide), PRR (the handle region peptide) or mTORC (everolimus) will be explored to determine their effect on cyst expansion in Tsc1 KO mice. Cyst growth and size, kidney function, systemic blood pressure, life expectancy, as well as mTORC1 activation in cyst epithelium will be determined in mutant mice. We strongly believe the proposed studies are novel and could lead to new therapies for this devastating disease as well as a number of other renal cystic diseases.