KCNJ16-depleted kidney organoids recapitulate tubulopathy and lipid recovery upon statins treatment

Abstract

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.

1. Introduction

Renal tubulopathies are a broad cluster of individual rare diseases that are mostly characterized by disruptions in cell homeostasis due to genetic defects in key renal transport proteins [1]. The KCNJ16 gene has recently been associated with a novel kidney tubulopathy phenotype. Patients carrying biallelic loss-of-function mutations in KCNJ16 exhibit disturbed acid–base homeostasis, severe hypokalemia, polyuria, and salt wasting [2]. KCNJ16 encodes for the inward rectifier potassium channel Kir5.1, which forms functional heterodimers with Kir4.2 (encoded by KCNJ15) and Kir4.1 (encoded by KCNJ10) [3]. These proteins function as potassium channels that regulate the basolateral membrane potential of several kidney epithelial cells [4, 5]. Altered membrane potential through dysfunctions of Kir4.1/Kir5.1 or Kir4.2/Kir5.1 heteromeric channels leads to dysregulation of several membrane potential-dependent transport processes and protein activities [6, 7], resulting in a tubulopathy phenotype as observed in patients with KCNJ16 loss-of-function [2, 3].

In the proximal tubule (PT), Kir4.2/Kir5.1 channels regulate bicarbonate reabsorption from the PT cells into circulation via a direct feedback loop between the membrane potential in response to changes in intracellular pH, and the activation/inhibition of the Na-bicarbonate cotransporter 1 (NBC1; SLC4A4) [8–10]. Dysfunction of Kir4.2/Kir5.1 leads to metabolic acidosis, accompanied by an increase in intracellular pH, membrane depolarization and reduced NBC1 activity in the PT [9, 11]. In the distal convoluted tubule (DCT), Kir5.1 acts both as a potassium transporter as well as an extracellular potassium sensor involved in the potassium conductance of the basolateral membrane of the cells by adjusting the intracellular chloride concentration [12, 13]. Furthermore, Kir4.1/Kir5.1 channels showed to be involved in sodium reabsorption (via the epithelial Na+ channel (ENaC) [14]) and potassium secretion (facilitated by the outer medullary potassium channel (ROMK) [15]) in the collecting duct (CD).

Previous evidence suggests that defects in potassium transporters such as KCNJ10 and KCNJ15 is associated with a kidney phenotype, there is no precedent for KCNJ16 defects alone ca