背景:KCNJ16基因与一种新的肾小管病表型相关,viz.酸碱稳态受干扰,低钾血症和肾盐转运改变。KCNJ16编码Kir5.1,与Kir4.1一起构成位于肾小管细胞基底外侧膜的钾通道。临床前研究提供了Kir5.1和肾小管病之间的机制联系,然而,该疾病的病理学仍然知之甚少。这里,我们的目的是产生和表征一种新的先进的体外人类肾脏模型,该模型概括了疾病表型,以进一步研究肾小管病的病理生理机制和潜在的治疗干预措施。
方法:我们使用CRISPR/Cas9从健康人诱导多能干细胞(iPSC)KCNJ16对照(KCNJ16WT)产生KCNJ16突变体(KCNJ16+/-和KCNJ16-/-)细胞系。iPSC按照优化方案在气-液界面中分化为肾脏类器官。
结果:KCNJ16耗尽的肾脏类器官显示关键电压依赖性电解质和水平衡转运蛋白的转录组和潜在功能损害。我们观察到囊肿形成,Kir5.1功能丧失后的脂滴积累和纤维化。此外,大规模,谷氨酰胺示踪通量代谢组学分析表明,KCNJ16-/-类器官表现出TCA循环和脂质代谢障碍。药物筛选显示他汀类药物治疗,特别是辛伐他汀和C75的联合使用可防止KCNJ16-/-肾类器官中的脂滴积累和胶原蛋白I沉积。
结论:成熟的肾脏类器官代表了研究Kir5.1功能的相关体外模型。我们发现了这种遗传性肾小管病的新分子靶标,并将他汀类药物确定为肾脏KCNJ16缺陷的潜在治疗策略。
BACKGROUND: The KCNJ16 gene has been associated with a novel kidney
tubulopathy phenotype, viz. disturbed acid-base homeostasis, hypokalemia and altered renal salt transport. KCNJ16 encodes for Kir5.1, which together with Kir4.1 constitutes a potassium channel located at kidney tubular cell basolateral membranes. Preclinical studies provided mechanistic links between Kir5.1 and
tubulopathy, however, the disease pathology remains poorly understood. Here, we aimed at generating and characterizing a novel advanced in vitro human kidney model that recapitulates the disease phenotype to investigate further the pathophysiological mechanisms underlying the
tubulopathy and potential therapeutic interventions.
METHODS: We used CRISPR/Cas9 to generate KCNJ16 mutant (KCNJ16+/- and KCNJ16-/-) cell lines from healthy human induced pluripotent stem cells (iPSC) KCNJ16 control (KCNJ16WT). The iPSCs were differentiated following an optimized protocol into kidney organoids in an air-liquid interface.
RESULTS: KCNJ16-depleted kidney organoids showed transcriptomic and potential functional impairment of key voltage-dependent electrolyte and water-balance transporters. We observed cysts formation, lipid droplet accumulation and fibrosis upon Kir5.1 function loss. Furthermore, a large scale, glutamine tracer flux metabolomics analysis demonstrated that KCNJ16-/- organoids display TCA cycle and lipid metabolism impairments. Drug screening revealed that treatment with statins, particularly the combination of simvastatin and C75, prevented lipid droplet accumulation and collagen-I deposition in KCNJ16-/- kidney organoids.
CONCLUSIONS: Mature kidney organoids represent a relevant in vitro model for investigating the function of Kir5.1. We discovered novel molecular targets for this genetic
tubulopathy and identified statins as a potential therapeutic strategy for KCNJ16 defects in the kidney.