Fc 008interdependent Regulation of Polycystin Expression Influences Starvation-Induced Autophagy and Cell Death

Abstract

Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by mutations in either PKD1 (ca. 78%) or PKD2 (ca. 15%), encoding for the proteins polycystin-1 (PC1) or polycystin-2 (PC2), respectively. Mutations in PKD1 generally lead to a more severe disease progression compared to PKD2 patients. The exact function of the polycystins in cyst formation remains unclear, but it is clear that the levels of PC1 and PC2 are inversely correlated to cyst formation. Moreover, renal stress has been proposed to enhance cystogenesis. We therefore aimed to investigate the cellular response towards nutritional stress in mouse inner medullary collecting duct cells (mIMCDs), either wild-type (WT) or lacking PC1 or PC2 (PC1KO or PC2KO), and unique human urine-derived proximal tubular epithelial cells (PTECs) of early-stage ADPKD patients with truncating PKD1 mutations versus healthy individuals, with a focus on cell survival (autophagy) and cell death. Method Cell death was assessed with Cytotox green-based live cell imaging in the Incucyte, by trypane blue exclusion and by analyzing the levels of cleaved Caspase 3. Autophagy was measured by LC3 immunoblotting and by counting GFP-LC3 punctae. Autophagy was blocked with Bafilomycin A1. To modulate the levels of PC1 and PC2, transient overexpression of human PC1 or siRNA-mediated knockdown of PC2 was performed. Results During chronic starvation, cell death was reduced and autophagy was increased in mouse PC1- and PC2-deficient mIMCDs. This was validated in human cells from early-stage ADPKD patients. Autophagy inhibition restored cell death resistance in KO cells, implying that decrease in cell death was caused by autophagy upregulation in PC1- and PC2KO cells. Interestingly, PC2 expression was increased in PC1KO cells, while PC2KO displayed a downregulation of PC1. Although PC2 is known to regulate autophagy, PC2 knockdown did not reduce autophagy in PC1KO cells, while the effect in PC2KO could be reversed by overexpression of PC1. Conclusion These findings indicate that PC1 levels determine the transition from renal cell survival to death, leading to enhanced survival of ADPKD cells during nutritional stress. Moreover, PC1 also indirectly influences this process by regulating PC1 levels during starvation. Our findings imply that in early stage ADPKD, cells with the lowest polycystin levels (which are most prone to form cysts) are more resistant to stress by autophagy upregulation. This is important, as renal stress is inherent to the cystic environment and has been proposed as an additional trigger in cystogenesis.