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

闰细胞及其酸碱和电解质转运机制在结节性硬化症肾囊肿发生中的作用

• 批准号: 10253492
• 负责人: MANOOCHER SOLEIMANI
• 金额: --
• 依托单位: ALBUQUERQUE VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10253492
• 关键词: Acetazolamide; Acids; Affect; Angiomyolipoma; Animal Model; Animals; Antibodies; Antigens; Apical; Biological Factors; Biological Process; Blood Pressure; CLC Gene; Carbonic Anhydrase II; Carbonic Anhydrase Inhibitors; Cell Proliferation; Cells; Chlorides; Cyst; Cystic Fibrosis Transmembrane Conductance Regulator; Cystic Kidney Diseases; Cystic kidney; Data; Development; Disease; Dominant Genetic Conditions; Electrolytes; Epithelial cyst; Event; FRAP1 gene; Fluids and Secretions; Functional disorder; Gene Deletion; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Diseases; Genotype; Growth; Histology; Human; Image; Immunofluorescence Microscopy; Intercalated Cell; Kidney; Kidney Diseases; Kidney Failure; Knockout Mice; Label; Lead; Life Expectancy; Lung; Lysosomes; Magnetic Resonance Imaging; Membrane; Modeling; Mus; Mutant Strains Mice; Mutation; Nuclear; Organ; Outcome; Pathway interactions; Patients; Peptides; Pericytes; Persons; Pharmacology; Play; Population; Property; Proton-Translocating ATPases; Publishing; Regulation; Renal function; Reporting; Resistance; Role; SDZ RAD; Systemic blood pressure; TSC1 gene; TSC2 gene; Testing; Tuberous Sclerosis; V2 Receptors; WNT Signaling Pathway; antagonist; apical membrane; base; cell type; disease-causing mutation; inhibitor; late endosome; novel; novel therapeutic intervention; novel therapeutics; prorenin receptor; transcription factor; transcriptome sequencing; tumor

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.

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多个器官，包括肾脏 和肺。该疾病影响全球超过 200 万人，其中很大一部分正在发展 血管平滑肌脂肪瘤和囊肿，最终导致肾衰竭。虽然 TSC 的最初遗传事件 已经被描绘出来，但对促进进展的生物过程和因素知之甚少 或囊肿扩大。我们开发了 Tsc1 肾主细胞 (PC) 特异性失活的小鼠 或 Tsc2 基因（已发表和初步数据），概括了人类 TSC 囊性肾病。这 囊肿上皮显示很少的 PC 细胞，但大量存在 A 嵌入 (A-IC) 细胞。 RNA-seq 和验证性表达研究表明， Foxi1，一种对 IC 细胞发育以及 H+-ATPase 和碳酸激活至关重要的转录因子 Tsc1 KO 小鼠中的脱水酶 II (CAII)。使用抗体进行双重免疫荧光标记研究 H+-ATP酶和AQP2； H+-ATPase 和 PCNA（增殖核细胞抗原，细胞增殖标志物） 增殖）和 H+-ATPase 和 CAII 表现出 PC 细胞进行性损失和过度增殖 Tsc1 KO 小鼠囊肿上皮中的 A-IC 细胞。此外，生电2Cl-/H+交换器CLC-5， 在基础条件下与晚期内涵体和溶酶体的膜中的 H+-ATPase 共定位， 在 Tsc1 中表现出与囊肿上皮顶膜上的 H+-ATPase 显着共定位 KO老鼠。此外，我们的结果表明肾素原受体 PRR 的共定位，PRR 在 Wnt 信号通路，囊肿上皮顶膜上有 H+-ATPase。这些变化是 与常染色体显性 PKD 囊肿不同（初步数据）。 Tsc1 KO 小鼠中 Foxi1 的缺失导致 Foxi1/Tsc1 双基因中的囊肿负担完全消除 突变小鼠。此外，CAII 的缺失受 Foxi1 调节，对 H+-ATPase 活性至关重要， 显着减轻了 CAII/Tsc1 双突变小鼠的囊肿负担。 我们认为 A 嵌入细胞的强劲增殖及其酸/碱/电解质运输 机械对于 Tsc1 KO 小鼠的肾囊肿发生至关重要。我们进一步提出，与 PKD 中的囊肿不同， 通过减少液体分泌和体积、TSC 囊肿来响应 AVP V2 受体拮抗作用， 主细胞很少，会对 V2 受体拮抗剂产生耐药性。 为了检验我们的假设，我们建议： 确定 A-IC 细胞及其酸碱转运的作用 TSC 疾病中包囊生长和扩张的机制（H+-ATPase 和 CAII）；确定角色 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；并检查 CAII/H+-ATPase、PRR 和 mTOR 抑制剂对 TSC 小鼠囊肿生长的影响。为此，我们 将检查同时删除 Tsc1（在主细胞中）与 Foxi1、CAII 结合的效果， H+-ATPase B 亚基或 CLC-5 对肾囊肿发生的影响。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） 将探索它们对 Tsc1 KO 中囊肿扩张的影响 老鼠。囊肿生长和大小、肾功能、全身血压、预期寿命以及 mTORC1 将在突变小鼠中测定囊肿上皮的激活。我们坚信拟议的研究 是新颖的，可能会导致针对这种破坏性疾病以及许多其他肾脏疾病的新疗法 囊性疾病。