Lipid Peroxidation Drives Renal Cyst Growth In Vitro through Activation of TMEM16A

Abstract

The chloride channels cystic fibrosis transmembrane conductance regulator (CFTR) and TMEM16A (anoctamin 1) drive cyst enlargement in polycystic kidney disease (PKD), ultimately leading to kidney failure. The mechanisms for channel activation, however, are poorly understood. The authors show for the first time that peroxidation of plasma membrane phospholipids activates renal TMEM16A; this facilitates calcium signaling and activation of the calcium-sensitive adenylate cyclase ADCY1, which further stimulates CFTR. The antioxidant idebenone significantly delays cyst enlargement as does ferrostatin-1, suggesting activation of ferroptosis, an apoptosis-independent regulated cell death pathway during PKD. These findings show a strong effect of reactive oxygen species on cyst progression via lipid peroxidation. Inhibition of lipid peroxidation and blockade of TMEM16A are potential novel therapeutic approaches to delay cyst development in PKD. Transepithelial chloride − secretion, through the chloride channels cystic fibrosis transmembrane conductance regulator (CFTR) and TMEM16A (anoctamin 1), drives cyst enlargement in polycystic kidney disease (PKD). Polycystic kidneys are hypoxic, and oxidative stress activates TMEM16A. However, mechanisms for channel activation in PKD remain obscure. Using tissue samples from patients with autosomal dominant PKD, embryonic kidney cultures, and an MDCK in vitro cyst model, we assessed peroxidation of plasma membrane phospholipids in human and mouse polycystic kidneys. We also used electrophysiologic Ussing chamber and patch clamp experiments to analyze activation of TMEM16A and growth of renal cysts. Peroxidation of phospholipids in human and mouse kidneys as well as MDCK cysts in vitro is probably due to enhanced levels of reactive oxygen species. Lipid peroxidation correlated with increased cyst volume as shown in renal cultures and MDCK cysts in three-dimensional cultures. Reactive oxygen species and lipid peroxidation strongly activated TMEM16A, leading to depletion of calcium ion stores and store-operated calcium influx. Activation of TMEM16A- and CFTR-dependent chloride secretion strongly augmented cyst growth. Exposure to scavengers of reactive oxygen species, such as glutathione, coenzyme Q10, or idebenone (a synthetic coenzyme Q10 homolog), as well as inhibition of oxidative lipid damage by ferrostatin-1 largely reduced activation of TMEM16A. Inhibition of TMEM16A reduced proliferation and fluid secretion in vitro . These findings indicate that activation of TMEM16A by lipid peroxidation drives growth of renal cysts. We propose direct inhibition of TMEM16A or inhibition of lipid peroxidation as potentially powerful therapeutic approaches to delay cyst development in PKD.