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: NADH:ubiquinone 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.

Keywords: Autophagic flux; RUBCN/Rubicon; autophagy; lipid efflux; lysosome; metabolic syndrome.

Figure 1.

RUBCN deficiency increases autophagic flux in the PTECs. (A and B) In vivo evaluation of the autophagic flux in PTECs during the fed state. (A) Representative images of immunofluorescence analysis for LRP2 (red) and 4ʹ, 6-diamidino-2-phenylindole (DAPI; blue) as counterstaining on the kidney sections from 3-month-old Rubcn^F/F-CTRL;GFP-MAP1LC3B and rubcn^F/F-TSKO;GFP-MAP1LC3B mice with or without CQ administration 6 h before euthanasia. The right panels show a magnification of the indicated areas (white squares) in the left panels. (B) Quantification of the number of GFP-positive puncta per proximal tubule. (C) Representative images of western blot analysis for LRP2, RUBCN, LAMP1, BECN1, and RAB7A in the cell lysate from cultured RUBCN CTRL and KO PTECs are shown. (D) Densitometric quantification of the protein levels in (C). (E) The mRNA expression of Rubcn in the cell lysate from cultured CTRL and KO PTECs was analyzed. (F-H) Evaluation of the autophagic flux in cultured PTECs. Representative images of western blot analysis (F) and immunofluorescence analysis for MAP1LC3B (red) and DAPI (blue) (H) of CTRL and KO PTECs with or without Baf. (C and F) ACTB (actin, beta) was used as a loading control. (G) Densitometric quantification of the MAP1LC3B-II protein levels in (F). n = 3 to 5 (B); 3 (D); 4 or 5 (E); 5 (G) in each group. Data are provided as the mean ± SD. ns, not significant. Statistically significant differences (*P < 0.05, **P < 0.01) are indicated. Bars: 20 µm (A) and 5 µm (H).

Figure 2.

Phospholipids accumulation in PTEC lysosomes of rubcn^F/F-TSKO mice. (A-E) Representative images of PAS (A) and ORO (B) staining, immunofluorescence analysis for LAMP1 (green), LRP2 (red), and DAPI (blue) as counterstaining (C), toluidine blue staining (D), and electron microscopic analysis (E) on the kidney sections from 4-month-old- (A) and 12-month-old- (A-E) Rubcn^F/F-CTRL and rubcn^F/F-TSKO mice are shown. (F) The plasma UN, plasma creatinine, and the ratio of urinary albumin to creatinine were analyzed. (A, C, and E) The right panels show a magnification of the indicated areas (black or white squares) in the left panels. n = 12 or 13 in each group. Data are provided as the mean ± SD. ns, not significant. Bars: 20 µm (A, B, and D), 5 µm (C), and 1 µm (E).

Figure 3.

Fat accumulation and BW gain in rubcn^F/F-TSKO mice. Metabolic effects of RUBCN deficiency in PTECs were analyzed. Appearance (A), BW curves (B), the weight and ratio relative to BW of liver, MAT, EAT, SAT, BAT, kidney, heart, and soleus (C), images of hematoxylin and eosin (HE) (left) and ORO (right) staining on the liver sections (D), the ratio of hepatic TG levels to weight (E), and HE staining on MAT (left) and SAT (right) sections (F), the mRNA expression of Srebf1 in the liver and MAT (G), blood glucose levels during GTT (H), and occasional plasma concentrations of total cholesterol (I) in Rubcn^F/F-CTRL and rubcn^F/F-TSKO mice are shown. (A, D, and F) Representative images are presented. (H) AUCs were calculated by summation of trapezoids (right). (A, C-I) Data from 12-month-old mice (n = 10 to 13 in each group). Data are provided as the mean ± SD. ns, not significant. Statistically significant differences (*P < 0.05, **P < 0.01) are indicated. Bars: 20 µm (D) and 40 µm (F). MAT, mesenteric adipose tissue; EAT, epididymal adipose tissue; SAT, subcutaneous adipose tissue; BAT, brown adipose tissue; BW, body weight.

Figure 4.

RUBCN deficiency activates MTOR pathway in PTECs. (A) Representative images of immunohistochemical analysis for p-RPS6 in the kidney sections from 12-month-old Rubcn^F/F-CTRL and rubcn^F/F-TSKO mice are shown. Sections were co-immunostained for the proximal tubule marker, LRP2 in blue. The right panels show a magnification of the indicated areas (black squares) in the left panels. (B) The protein levels of p-RPS6KB1, p-EIF4EBP1, and total RPS6KB1 and EIF4EBP1 in the cell lysate from cultured CTRL and KO PTECs are shown. ACTB was used as a loading control. (C) Densitometric quantification of protein levels in (B). (D) The mRNA expression of Tfeb in the cell lysate from cultured CTRL and KO PTECs was analyzed. n = 3 (C); 4 or 5 (D) in each group. Data are provided as the mean ± SD. ns, not significant. Statistically significant differences (*P < 0.05, **P < 0.01) are indicated. Bars: 20 µm.

Figure 5.

RUBCN deficiency promotes the mobilization of phospholipids from cellular membranes to lysosomes in cultured PTECs. (A) Schematic representation of the FA pulse-chase assay. PTECs were incubated with CM (DMEM with 5% FBS) containing 2 μM FL HPC for 16 h, and then chased for the indicated times. (B) Representative images of fluorescence analysis for LysoTracker Red (red) and DAPI (blue) as counterstaining in CTRL and KO PTECs. (C) Colocalization of FL HPC and LysoTracker Red in (B) was assessed by the Pearson correlation coefficient. (D) The average size of LysoTracker Red in (B) was quantified. n = 3 in each group. Data are provided as the mean ± SD. ns, not significant. Statistically significant differences (*P < 0.05) are indicated. Bar: 5 µm.

Figure 6.

RUBCN deficiency promotes LD degradation in cultured PTECs. (A) Schematic representation of the FA pulse-chase assay. PTECs were incubated with CM containing 1 μM Red C12 and 250 μM OA for 36 h, and then chased for the indicated times. (B) Representative images of fluorescence analysis for BODIPY-LD (green) or MitoTracker Deep Red (green) and DAPI (blue) as counterstaining in chased CTRL and KO PTECs at 0 and 24 h. (C) Colocalization of Red C12 and MitoTracker Deep Red in (B) was assessed by the Pearson correlation coefficient. (D) The ratio of TG levels to protein in chased CTRL and KO PTECs was analyzed. (E) Representative images of ORO staining in chased CTRL and KO PTECs. n = 3 in each group. Data are provided as the mean ± SD. ns, not significant. Statistically significant differences (*P < 0.05) are indicated. Bars: 5 µm (B) and 200 µm (E).

Figure 7.

RUBCN deficiency promotes FA expulsion from the PTECs. (A) Schematic representation of the co-culture assay. PTECs treated with 500 µM OA for 36 h on cell culture inserts were co-cultured with BNL-CL2 cells in OA-free CM. (B and C) Representative images of ORO staining (B) and fluorescence analysis for BODIPY-LD (green) (C) of BNL-CL2 cells co-cultured for 18 h (C) or 36 h (B and C) with CTRL and KO PTECs. (D) The number and the average size of BODIPY-LD-positive puncta per BNL-CL2 cell co-cultured for 36 h in (C) were quantified. (E) Schematic representation of another co-culture assay. PTECs treated with 500 µM OA and 1 μM Red C12 for 36 h on cell culture inserts were co-cultured with BNL-CL2 cells in OA-free CM. (F) Representative images of fluorescence analysis for BODIPY-LD (green) in BNL-CL2 cells co-cultured for 36 h with Red C12-prelabeled CTRL and KO PTECs. n = 3 in each group. Data are provided as the mean ± SD. Statistically significant differences (*P < 0.05, **P < 0.01) are indicated. Bars: 200 µm (B) and 5 µm (C and F).

Figure 8.

Model of FA mobilization in RUBCN CTRL and KO PTECs. In KO PTECs, the mobilization of phospholipids to lysosomes is promoted by enhanced autophagy, followed by increased FA release (1). Excessive FAs distribute into mitochondria for β-oxidation, otherwise FAs are exported from PTECs (2).