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

抽象的。多发性硬化症（TSC）是一种常染色体显性遗传病， 通过灭活TSC 1或TSC 2基因中的突变，并影响多个器官，包括肾脏 和肺这种疾病影响了全世界200多万人，其中很大一部分是发展中国家。 血管平滑肌脂肪瘤和囊肿，最终导致肾衰竭。虽然TSC的初始遗传事件 虽然已经描述了这些疾病，但对促进疾病进展的生物学过程和因素知之甚少 或囊肿扩张。我们已经开发了具有Tsc 1的肾主（PC）细胞特异性失活的小鼠 或Tsc 2基因（Published and Approprim.数据），其概括了人TSC囊性肾病。的 囊肿上皮显示很少的PC细胞，但大量存在A-插入（A-IC）细胞。 RNA-seq和确证性表达研究表明， Foxi 1是一种对IC细胞发育和H ~+-ATP酶和碳源性磷酸酶激活至关重要的转录因子 脱水酶II（CAII）。用抗-HLA-IgM抗体进行免疫荧光双标记研究 H+-ATP酶和AQP 2; H+-ATP酶和PCNA（增殖核细胞抗原，细胞增殖的标志物） H+-ATP酶和CAII显示PC细胞进行性丢失和过度增殖 Tsc 1基因敲除小鼠囊肿上皮中A-IC细胞的数量。此外，产电2Cl-/H+交换剂CLC-5， 其在基础条件下与晚期内体和溶酶体的膜中的H+-ATP酶共定位， 在Tsc 1细胞中，H-ATPase与H+-ATPase共定位于囊上皮细胞顶膜上 KO小鼠。此外，我们的研究结果表明，共定位的原肾素受体PRR，一个关键的球员， Wnt信号通路，H+-ATP酶位于囊上皮细胞顶膜。这些变化是 与常染色体显性PKD囊肿中的那些不同（Ambrim Data）。 Tsc 1 KO小鼠中Foxi 1的缺失导致Foxi 1/Tsc 1双细胞中囊肿负荷的完全消除 突变小鼠此外，缺失由Foxi 1调节并对H+-ATP酶活性至关重要的CAII， 显著减弱CAII/Tsc 1双突变小鼠的囊肿负荷。 我们认为，A-嵌入细胞的稳健增殖及其酸/碱/电解质转运 在Tsc 1基因敲除小鼠中，肾脏细胞机械对肾脏囊肿形成至关重要。我们进一步提出，与PKD中的囊肿不同， 其通过减少其液体分泌和大小，TSC囊肿， 其具有很少的主细胞，将对V2受体拮抗剂具有抗性。 为了验证我们的假设，我们提出：确定A-IC细胞及其酸碱转运的作用 TSC疾病中囊肿生长和扩张中的机械（H+-ATP酶和CAII）;确定 CLC-5和CFTR在TSC小鼠中氯化物分泌到囊肿腔和囊肿扩张中的作用; CAII/H+-ATP酶、PRR和mTOR抑制剂对TSC小鼠包囊生长的影响。为此我们 将检查同时缺失Tsc 1（在主细胞中）与Foxi 1，CAII， H+-ATP酶B亚单位或CLC-5对肾囊肿形成的影响。此外，药物抑制剂的作用， CFTR激活（V2受体拮抗剂），H+-ATP酶/CAII（乙酰唑胺），PRR（手柄区 肽）或mTORC（依维莫司）来确定它们对Tsc 1 KO中的囊肿扩张的作用 小鼠囊肿生长和大小、肾功能、全身血压、预期寿命以及mTORC 1 将在突变小鼠中测定囊肿上皮中的活化。我们坚信， 是新的，可能导致这种毁灭性疾病的新疗法，以及其他一些肾脏疾病， 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.