A novel KCNJ16 kidney organoid model recapitulates the disease phenotype and shows restoration of lipid accumulation upon treatment with statins

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 mechanistical links between Kir5.1 and a disease phenotype, 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 disease 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. Significance Statement In this study, the use of CRISPR/Cas9 technology resulted in the establishment of a KCNJ16- depleted kidney organoid model, instrumental in elucidating the pathophysiology of the recently reported KCNJ16 -associated kidney tubulopathy. Our study substantiates the role of Kir5.1 (KCNJ16) in kidney disease, confirming already described phenotypes, as well as aiding to gain insight in the causal role of Kir5.1 loss in the disease phenotype. Our approach increases the knowledge on KCNJ16 -related kidney phenotype, and it states the importance of combining CRISPR/Cas9 technology and advanced in vitro models for complex disease modeling and therapy testing. Furthermore, we encourage the application of our approach to the in vitro modeling of rare and/or underrepresented genetic kidney diseases, for which the availability of patient material is limited. ### Competing Interest Statement The authors have declared no competing interest.