Exploring the pathogenesis of classic Bartter syndrome

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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.