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。结节性硬化症复合物（TSC）是一种常染色体显性遗传疾病，是引起的 通过在TSC1或TSC2基因中灭活突变并影响多个器官，包括肾脏 和肺。该疾病影响了全球超过200万人，并且发展很大 血管瘤和囊肿，最终导致肾衰竭。而TSC的最初遗传事件 已经描绘了，对促进进展的生物过程和因素知之甚少 或囊肿的扩展。我们已经开发了肾脏主（PC）细胞特异性TSC1的小鼠 或TSC2基因（已发表和预定数据），概括了人类TSC囊性肾脏疾病。这 囊肿上皮显示很少的PC细胞，但稳健的Aintscalate（A-IC）细胞的存在。 RNA-seq和确认性表达研究表明，表达显着增加 FOXI1，一种转录因子，对IC细胞的发展至关重要，H+ATPase和Carbonic的激活 TSC1 KO小鼠中的赤道酶II（CAII）。双重免疫荧光标记研究，具有针对的抗体 H+-ATPase和AQP2; H+-ATPase和PCNA（增殖性核细胞抗原，细胞的标记 增殖）和H+-ATPase和CAII表现出PC细胞的逐渐丧失和过化的逐渐丧失 TSC1 KO小鼠中囊肿上皮中的A-IC细胞的。另外，电基质2cl-/h+交换器CLC-5， 在基础条件下，在晚期内体和溶酶体的膜中与H+-ATPase共定位， 与H+-ATPase在TSC1中的囊肿上皮膜的顶膜上表现出显着的共定位 KO老鼠。此外，我们的结果表明pro-Renin受体PRR的共定位，这是一个关键的参与者 Wnt信号通路，在囊肿上皮的顶膜上具有H+-ATPase。这些变化是 与常染色体显性PKD囊肿（初步数据）不同。 TSC1 KO小鼠中FOXI1的删除导致FOXI1/TSC1 Double中的囊肿负担完全消除 突变小鼠。另外，caii的缺失，受FOXI1的调节，对H+-ATPase活性至关重要， 在CAII/TSC1双突变小鼠中显着钝化了囊肿负担。 我们提出，A间隔细胞及其酸/碱/电解质转运的稳健增殖 机械对于TSC1 KO小鼠的肾脏囊肿性至关重要。我们进一步提出，与PKD中的囊肿不同， 通过减少其液体分泌和大小，TSC囊肿，对AVP V2受体拮抗作用响应 几乎没有主要细胞的，将对V2受体拮抗剂具有抗性。 为了检验我们的假设，我们建议：确定A-IC细胞及其酸基碱的运输的作用 TSC疾病中囊肿的生长和扩展，机械（H+-ATPase和CAII）；确定角色 用TSC的小鼠中氯化物分泌的Clc-5和CFTR的氯化物分泌和囊肿扩张；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TSC1同时缺失（在主要细胞中）与FOXI1，CAII， H+-ATPase B亚基，或肾囊肿发生的ClC-5。此外，药理学抑制剂的作用 CFTR激活（V2受体拮抗剂），H+-ATPase/CAII（乙酰唑胺），PRR（手柄区域 将探索肽）或MTORC（Everolimus），以确定其对TSC1 KO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 囊肿上皮的激活将在突变小鼠中确定。我们坚信拟议的研究 是新颖的，可能导致这种毁灭性疾病以及许多其他肾脏的新疗法 囊性疾病。