Exploring the pathogenesis of classic Bartter syndrome

Bartter综合征（BS）是一种先天性肾小管病变，由Henle袢（TAL）升支粗段转运蛋白突变导致NaCl重吸收受损所致。胎儿BS是由TAL顶膜中的NKCC2或ROMK突变引起的。经典型Bartter's（cBS）是由于基底外侧氯离子通道ClC-Kb的突变，表现出高度可变的表型和肾脏结局。与普遍认为BS中的盐消耗是由于成熟TAL中转运蛋白功能丧失的观点相反，我们最近报道了Clc-k2-/-（ClC-Kb的小鼠直系同源物）小鼠中的cBS表型主要是由于内髓质的发育缺陷和TAL发育不全。Clc-k2缺乏如何导致肾小管发育不全尚不清楚。肾小管的生长源于细胞增殖和细胞死亡之间的正平衡。初步数据显示，Clc-k2-/-肾小管细胞比WT细胞增殖性更低，凋亡更多。使用原代培养的TAL细胞的细胞周期分析揭示了比WT细胞更多的Clc-k2-/-细胞驻留在G1期，表明Clc-k2缺陷损害TAL细胞的增殖和细胞周期。是什么原因导致Clc-k2-/-肾小管细胞中的细胞发育不良和细胞周期停滞尚不清楚。线粒体为运输提供能量，线粒体功能障碍与细胞周期停滞有关。我们推测，降低运输活动和线粒体功能障碍的基础肾小管发育不全的cBS。为了支持这一假设，Specific Aim-1将研究Clc-k2缺陷通过降低转运活性导致肾小管细胞中的细胞周期停滞和线粒体功能障碍。将在原代TAL和DCT细胞或小管中进行细胞增殖、细胞周期分析和线粒体生物能量学试验。将在Clc-k2-/-小鼠肾脏切片的小管中检查线粒体形态。通过表达PGC1 α（过氧化物酶体增殖物激活受体共激活因子-1 α，一种线粒体生物发生的激活因子）转基因或Nrf2（核因子-红细胞因子2相关因子2，PGC1 α下游的一种转录因子）激动剂直接增强线粒体功能将用于拯救Clc-k2-/-小鼠。Specific Aim-2将使用两种具有功能获得（GOF）转运活性的小鼠模型进一步检验假设。将研究GOF小鼠对由Clc-k2缺乏引起的细胞增殖和线粒体功能障碍的拯救作用。传统观点认为BS的发病机制是成熟肾小管中的盐消耗，导致治疗集中在盐饱。然而，许多患者尽管容量充盈，但仍进展为慢性肾病。我们的研究将为BS的发病机制提供新的见解，并提供针对线粒体功能恢复的潜在治疗考虑。

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.