Exploring the pathogenesis of classic Bartter syndrome

探索经典巴特综合征的发病机制

• 批准号: 10708041
• 负责人: Chih-Jen Cheng
• 金额: $ 31.1万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-21 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10708041
• 关键词: Affect; Agonist; Apical; Apoptotic; Arrhythmia; Bartter Disease; Bioenergetics; Biogenesis; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Cycle Regulation; Cell Death; Cell Proliferation; Cells; Cellular Assay; Chloride Channels; Chlorides; Chronic Kidney Failure; Coupled; DNA Sequence Alteration; Data; Defect; Development; Disease; Distal convoluted renal tubule structure; Disuse Atrophy; Diuretics; Electrolytes; Embryo; Equilibrium; Erythroid; Furosemide; G1 Phase; G1/S Transition; Gene Deletion; Genetic; Genotype; Growth; Heterogeneity; Human; Hypertrophy; Impairment; In Vitro; Inherited; Ions; KCNJ1 gene; Kidney; Kidney Diseases; Knock-out; Light; Link; Microscopy; Mitochondria; Morphology; Mus; Mutation; Nuclear; Orthologous Gene; Outcome; Oxidative Phosphorylation; Pathogenesis; Patients; Peroxisome Proliferator-Activated Receptors; Phenotype; Proliferating; Regulation; Renal tubule structure; Reporting; Role; Side; Sodium Chloride; Structure of ascending limb of Henle' s loop; Testing; Therapeutic; Thick; Three-Dimensional Imaging; Transgenes; Tubular formation; Type II Pseudohypoaldosteronism; absorption; antenatal; apical membrane; cell type; emerging adult; gain of function; in vivo; insight; kidney medulla; loss of function; mitochondrial dysfunction; mouse model; mutant; overexpression; restoration; thiazide; transcription factor; transcriptome; wasting

Bartter syndrome (BS) is a congenital renal tubulopathy caused by mutations of transporters impairing NaCl reabsorption in the thick ascending limb of Henle's loop (TAL). Antenatal BS is caused by mutations of NKCC2 or ROMK in the apical membrane of TAL. Classic Bartter’s (cBS) is due to mutations of the basolateral chloride channel ClC-Kb, presenting highly variable phenotypes and renal outcomes. As opposed to the prevailing view that salt wasting in BS is due to loss of function of transporters in mature TAL, we recently reported that the phenotype of cBS in Clc-k2-/- (mouse ortholog of ClC-Kb) mice is mainly due to developmental defects in the inner medulla and TAL hypoplasia. How Clc-k2 deficiency leads to renal tubule hypoplasia is unknown. The growth of renal tubules arises from a positive balance between cell proliferation and cell death. Preliminary data reveal Clc-k2-/- tubular cells are less proliferative and more apoptotic than WT cells. Cell cycle analysis using primary cultured TAL cells reveals more Clc-k2-/- cells reside in the G1 phase than WT cells, suggesting that Clc-k2 deficiency impairs the proliferation and cell cycle of TAL cells. What causes cellular hypoplasia and cell cycle arrest in Clc-k2-/- renal tubular cells is unknown. Mitochondria provide energetics for transport, and mitochondria dysfunction is linked to cell cycle arrest. We hypothesize that decreased transport activity and mitochondrial dysfunction underlies tubular hypoplasia in cBS. To support the hypothesis, Specific Aim-1 will examine that Clc-k2 deficiency causes cell cycle arrest and mitochondrial dysfunction in renal tubular cells via decreasing transport activity. Assays for cell proliferation, cell cycle analysis, and mitochondria bioenergetics will be performed in primary TAL and DCT cells or tubules. Mitochondrial morphology will be examined in tubules of the kidney section of Clc-k2-/- mice. Direct enhancement of mitochondrial functions by expressing PGC1α (peroxisome proliferator-activated receptor coactivator-1α, an activator of mitochondrial biogenesis) transgene or Nrf2 (Nuclear factor-erythroid factor 2-related factor 2, a transcription factor downstream of PGC1α) agonists will be used to rescue Clc-k2-/- mice. Specific Aim-2 will further test the hypothesis using two mouse models with gain-of-function (GOF) transport activity. The effect of GOF mice to rescue cell proliferation and mitochondrial dysfunction caused by Clc-k2 deficiency will be studied. The traditional view of the pathogenesis of BS as salt-wasting in mature renal tubules has led to treatment focused on salt repletion. However, many patients progress to chronic kidney disease despite volume repletion. Our studies will provide new insights into the pathogenesis of BS and provide potential therapeutic considerations targeting mitochondrial function restoration.

易货综合征（BS）是一种先天性肾小管病，是由转运蛋白突变引起的，损害了Henle Loop（TAL）厚的NaCl重吸收的NaCl重吸收。产前BS是由TAL的顶膜中NKCC2或ROMK突变引起的。经典易货（CBS）是由于基体氯化物通道CLC-KB的突变，其呈现高度可变的表型和肾脏结果。与普遍的观点相比，BS中的盐浪费是由于成熟TAL中转运蛋白功能的丧失所致，我们最近报道说CLC-K2 - / - （CLC-KB的小鼠直立性）小鼠的CBS的表型主要是由于内部髓质和TAL滞后性肿瘤中出现缺陷所致。 CLC-K2缺乏导致肾小管发育不全是未知的。肾小管的生长源于细胞增殖和细胞死亡之间的正平衡。初步数据揭示了CLC-K2 - / - 核细胞比WT细胞更少增殖，凋亡更大。使用原发性培养的TAL细胞进行细胞周期分析，揭示了与WT细胞相比，G1相位的CLC-K2 - / - 细胞更多，这表明CLC-K2缺乏症会损害TAL细胞的增殖和细胞周期。导致细胞性发育不全和CLC-K2 - / - 肾结核细胞中细胞周期停滞的原因尚不清楚。线粒体为运输提供了能量，线粒体功能障碍与细胞周期停滞有关。我们假设降低的运输活性和线粒体功能障碍是CBS中的管状发育不全。为了支持该假设，特定的目标1将检查CLC-K2缺乏会导致肾小管细胞中的细胞周期停滞和线粒体功能障碍通过降低转运活性。细胞增殖，细胞周期分析和线粒体生物能学的测定将在原发性TAL和DCT细胞或试管中进行。线粒体形态将在CLC-K2 - / - 小鼠的肾脏部分的管中检查。 Direct enhancement of mitochondrial functions by expressing PGC1α (peroxysome proliferator-activated receptor coactivator-1α, an activator of mitochondrial biogenesis) transgene or Nrf2 (Nuclear factor-erythroid factor 2-related factor 2, a transcription factor downstream of PGC1α) agonists will be used to rescue Clc-k2-/- mice.具体的AIM-2将使用具有功能获得（GOF）运输活性的两个小鼠模型进一步检验假设。 GOF小鼠挽救CLC-K2缺乏引起的细胞增殖和线粒体功能障碍的影响将被研究。 BS发病机理作为成熟肾小管中盐的传统观点导致了重点是替代盐的治疗方法。但是，许多患者发展为慢性肾脏疾病目的地量补充。我们的研究将提供有关BS发病机理的新见解，并提供针对线粒体功能恢复的潜在治疗考虑因素。