Metabolic effects of RUBCN/Rubicon deficiency in kidney proximal tubular epithelial cells.

ABSTRACT

Macroautophagy/autophagy is a lysosomal degradation system which plays a protective role against kidney injury. RUBCN/Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein) inhibits the fusion of autophagosomes and lysosomes. However, its physiological role in kidney proximal tubular epithelial cells (PTECs) remains uncertain. In the current study, we analyzed the phenotype of newly generated PTEC-specific rubcn-deficient (KO) mice. Additionally, we investigated the role of RUBCN in lipid metabolism using isolated rubcn-deficient PTECs. Although KO mice exhibited sustained high autophagic flux in PTECs, they were not protected from acute ischemic kidney injury. Unexpectedly, KO mice exhibited hallmark features of metabolic syndrome accompanied by expanded lysosomes containing multi-layered phospholipids in PTECs. RUBCN deficiency in cultured PTECs promoted the mobilization of phospholipids from cellular membranes to lysosomes via enhanced autophagy. Treatment of KO PTECs with oleic acid accelerated fatty acids transfer to mitochondria. Furthermore, KO PTECs promoted massive triglyceride accumulation in hepatocytes (BNL-CL2 cells) co-cultured in transwell, suggesting accelerated fatty acids efflux from the PTECs contributes to the metabolic syndrome in KO mice. This study shows that sustained high autophagic flux by RUBCN deficiency in PTECs leads to metabolic syndrome concomitantly with an accelerated mobilization of phospholipids from cellular membranes to lysosomes. Abbreviations ABC ATP binding cassette; ACADM acyl-CoA dehydrogenase medium chain; ACTB actin, beta; ATG autophagy related; AUC area under the curve; Baf bafilomycin A1; BAT brown adipose tissue; BODIPY boron-dipyrromethene; BSA bovine serum albumin; BW body weight; CAT chloramphenicol acetyltransferase; CM complete medium; CPT1A carnitine palmitoyltransferase 1a, liver; CQ chloroquine; CTRL control; EGFP enhanced green fluorescent protein; CTSD cathepsin D; EAT epididymal adipose tissue; EGFR epidermal growth factor receptor; EIF4EBP1 eukaryotic translation initiation factor 4E binding protein 1; FA fatty acid; FBS fetal bovine serum; GTT glucose tolerance test; HE hematoxylin and eosin; HFD high-fat diet; I/R ischemia-reperfusion; ITT insulin tolerance test; KAP kidney androgen regulated protein; KO knockout; LAMP1 lysosomal associated membrane protein 1; LD lipid droplet; LRP2 low density lipoprotein receptor related protein 2; MAP1LC3B microtubule associated protein 1 light chain 3 beta; MAT mesenteric adipose tissue; MS mass spectrometry; MTOR mechanistic target of rapamycin kinase; MTORC1 MTOR complex 1; NDRG1 N-myc downstream regulated 1; NDUFB5 NADHubiquinone oxidoreductase subunit B5; NEFA non-esterified fatty acid; OA oleic acid; OCT optimal cutting temperature; ORO Oil Red O; PAS Periodic-acid Schiff; PFA paraformaldehyde; PIK3C3 phosphatidylinositol 3-kinase catalytic subunit type 3; PPARA peroxisome proliferator activated receptor alpha; PPARGC1A PPARG coactivator 1 alpha; PTEC proximal tubular epithelial cell; RAB7A RAB7A, member RAS oncogene family; RPS6 ribosomal protein S6; RPS6KB1 ribosomal protein S6 kinase B1; RT reverse transcription; RUBCN rubicon autophagy regulator; SAT subcutaneous adipose tissue; SFC supercritical fluid chromatography; SQSTM1 sequestosome 1; SREBF1 sterol regulatory element binding transcription factor 1; SV-40 simian virus-40; TFEB transcription factor EB; TG triglyceride; TS tissue specific; TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; UN urea nitrogen; UQCRB ubiquinol-cytochrome c reductase binding protein; UVRAG UV radiation resistance associated; VPS vacuolar protein sorting; WAT white adipose tissue.