AMPK protects proximal tubular epithelial cells from lysosomal dysfunction and dedifferentiation induced by lipotoxicity

Abstract

Renal proximal tubules are a primary site of injury in metabolic diseases. In obese patients and animal models, proximal tubular epithelial cells (PTECs) display dysregulated lipid metabolism, organelle dysfunctions, and oxidative stress that contribute to interstitial inflammation, fibrosis and ultimately end-stage renal failure. Our research group previously pointed out AMP-activated protein kinase (AMPK) decline as a driver of obesity-induced renal disease. Because PTECs display high macroautophagic/autophagic activity and rely heavily on their endo-lysosomal system, we investigated the effect of lipid stress on autophagic flux and lysosomes in these cells. Using a model of highly differentiated primary PTECs challenged with palmitate, our data placed lysosomes at the cornerstone of the lipotoxic phenotype. As soon as 6 h after palmitate exposure, cells displayed impaired lysosomal acidification subsequently leading to autophagosome accumulation and activation of lysosomal biogenesis. We also showed the inability of lysosomal quality control to restore acidic pH which finally drove PTECs dedifferentiation. When palmitate-induced AMPK activity decline was prevented by AMPK activators, lysosomal acidification and the differentiation profile of PTECs were preserved. Our work provided key insights on the importance of lysosomes in PTECs homeostasis and lipotoxicity and demonstrated the potential of AMPK in protecting the organelle from lipid stress.Abbreviation: ACAC: acetyl-CoA carboxylase; ACTB: actin beta; AICAR: 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; AMPK: AMP-activated protein kinase; APQ1: aquaporin 1 (Colton blood group); BSA: bovine serum albumin; CDH16: cadherin 16; CKD: chronic kidney disease; CTSB: cathepsin B; CTSD: cathepsin D; EPB41L5: erythrocyte membrane protein band 4.1 like 5; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; EMT: epithelial-to-mesenchymal transition; FA: fatty acid; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GFP: green fluorescent protein; GUSB: glucuronidase beta; HEXB: hexosaminidase subunit beta; LAMP: lysosomal associated membrane protein; LD: lipid droplet; LGALS3: galectin 3; LLOMe: L-leucyl-L-leucine methyl ester hydrobromide; LMP: lysosomal membrane permeabilization; LRP2: LDL receptor related protein 2; LSD: lysosomal storage disorder; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCOLN1: mucolipin TRP cation channel 1; MG132: N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal; MmPTECs: Mus musculus (mouse) proximal tubular epithelial cells; MTORC1: mechanistic target of rapamycin kinase complex 1; OA: oleate; PA: palmitate; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PTs: proximal tubules; PTECs: proximal tubular epithelial cells; PRKAA: protein kinase AMP-activated catalytic subunit alpha; RFP: red fluorescent protein; RPS6KB: ribosomal protein S6 kinase B; SLC5A2: solute carrier family 5 member 2; SOX9: SRY-box transcription factor 9; SQSTM1: sequestosome 1; TFEB: transcription factor EB; Ub: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; VIM: vimentin.

Keywords: AMPK; Autophagy; chronic kidney disease; lipid accumulation; obesity; proximal tubules.

Figure 1.

PA induces the accumulation of autophagosomes in Mus musculus (mouse) proximal tubular epithelial cells (MmPTECs). (A) MmPTECs were treated with 300 µM PA or 0.4% BSA for 6 or 24 h and stained with BODIPY^TM 493/503 for 15 min. (B, C) Quantifications of lipid droplet (B) number and (C) size on 100 cells per condition by lipid droplets MRI tool. (D) Representative western blot and (E,F) quantitative densitometry analysis of LC3, SQSTM1 and ACTB/β-actin in MmPTECs treated with 300 µM PA or 0.4% BSA for 6 or 24 with or without 2 nM bafilomycin A₁ for last 6 h. (G) Representative micrographs of MmPTECs expressing mRFP-GFP-LC3B and treated with 300 µM PA or 0.4% BSA for 6 or 24 h with or without 2 nM bafilomycin A₁ for the last 6 h. GFP⁺ RFP⁺ (yellow) puncta indicate autophagosomes (neutral pH), and GFP⁻ RFP⁺ (red) ones indicate acidic pH. Quantifications of the number of (H) GFP⁺ RFP⁺ and (I) GFP⁻ RFP⁺ puncta on 30 cells per group. Data are represented as (B, C) means and quarters or as (E, F, H, I) means ± SEM of three independent biological experiments. Statistical analyses were performed by two-way ANOVA followed by (F, H, I) Dunnett’s or (B, C, E) Tukey’s post-hoc test.

Figure 2.

PA alters AMPK activity and signalization after 24 h in MmPTECs. (A, E) AMPK was immunoprecipitated by anti-PRKAA1/AMPKα1 and anti-PRKAA2/AMPKα2 antibodies in lysates from MmPTECs previously treated for (A) 6 or (E) 24 h with 300 µM PA or 0.4% BSA with or without 100 µM A769662 or 2 mM AICAR. Phosphotransferase activity was assessed toward the AMARA peptide. Data are presented as the means of pi (pmol) incorporated per µg of proteins per min ± SEM. (B, F) Representative western blot of p-ACAC (Ser79), p-PRKAA (Thr172), ACAC, AMPK and ACTB in MmPTECs treated with 300 µM PA or 0.4% BSA with or without 100 µM A769662 or 2 mM AICAR for (B) 6 or (F) 24 h. (C, D, G, H) Quantitative densitometry analysis of the p-PRKAA:PRKAA (C for 6 h, G for 24 h) and the p-ACAC:ACAC (D for 6 h, H for 24 h) ratios. Data are presented as means ± SEM of five independent biological experiments. Statistical analyses were performed by two-way ANOVA followed by Tukey’s post-hoc test.

Figure 3.

AMPK activation prevents PA-induced autophagosome accumulation in MmPTECs. (A) Representative western blot and (B, C) quantitative densitometry analysis of LC3, SQSTM1 and ACTB in MmPTECs treated for 24 h with 300 µM PA or 0.4% BSA, in the presence of 100 µM A769662 or 2 mM AICAR and with or without 2 nM bafilomycin A₁ for last 6 h. (D) Representative micrographs of MmPTECs expressing mRFP-GFP-LC3B and treated for 24 h in the same conditions. GFP⁺ RFP⁺ (yellow) puncta indicate autophagosomes (neutral pH), and GFP⁻ RFP⁺ (red) ones indicate acidic pH. (E, F) Quantifications of the number of (E) GFP⁺ RFP⁺ and (F) GFP⁻ RFP⁺ puncta on 30 cells per group. (G) Representative western blot and (H) quantitative densitometry analysis of p-ULK1 (Ser555), ULK1 and ACTB in MmPTECs treated for 24 h with 300 µM PA or 0.4% BSA in the presence of 100 µM A769662 or 2 mM AICAR. Data are presented as means ± SEM of three or five independent biological experiments as indicated on the charts. Statistical analyses were performed by two-way ANOVA and Tukey’s post-hoc test.

Figure 4.

Autophagosomes in response to PA contain ubiquitin-positive aggregates in MmPTECs. (A) Representative micrographs of mPTECs treated for 24 h with 300 µM PA, 0.4% BSA or for 6 h with 10 µM MG132 and 2 nM bafilomycin A₁ (positive control), fixed and immuno-stained for ubiquitin (red) and SQSTM1 (green). (B) Quantifications of the number of ubiquitin puncta per nucleus on 30 cells per group. (C) Representative western blot and (D) quantitative densitometry analysis of ubiquitinylated proteins normalized by total proteins in MmPTECs treated with 300 µM PA or 0.4% BSA for 24 h. (D) Quantifications of the percentages of colocalization of ub with SQSTM1 calculated by Mander’s correlation coefficients on 30 cells per group. (F) Representative micrographs of MmPTECs treated for 24 h with 300 µM PA or 0.4% BSA in the presence of 100 µM A769662 or 2 mM AICAR with and without and 2 nM bafilomycin A₁ for last 6 h, fixed and immuno-stained for ubiquitin (red) and SQSTM1 (green). (G, H) Quantifications of the number of (G) ubiquitin puncta and (H) ubiquitin- and SQSTM1-double-positive puncta per nucleus calculated with the objects-based method on 30 cells per group. Data are presented as (B, D, G, H) means ± SEM or as (E) means and quarters of three or four independent biological experiments as indicated on the charts. Statistical analyses were performed by (D) Student’s unpaired t-test, (B, E) one-way ANOVA followed by Dunnett’s post-hoc test or (G, H) two-way ANOVA followed by Tukey’s post-hoc test.

Figure 5.

PA does not alter the fusion between autophagosomes and lysosomes in MmPTECs. (A) Representative micrographs of MmPTECs treated for 6 or 24 h (only 24 h is represented) with 300 µM PA or 0.4% BSA with and without 2 nM bafilomycin A₁ for last 6 h, fixed and immuno-stained for LAMP2 (red) and LC3 (green). (B) Quantification of the colocalization percentages of LC3 with LAMP2 calculated by Mander’s correlation coefficients on 30 cells per group. (C) Representative micrographs of MmPTECs treated for 24 h with 300 µM PA or 0.4% BSA, fixed and immuno-stained for ubiquitin (red) and LAMP1 (green). (D) Quantification of the number of ub puncta surrounded by LAMP1 staining on 30 cells per group. Data are presented as (B) means and quarters or as (D) means ± SEM of three independent biological experiments. Statistical analyses were performed by (D) Student’s unpaired t-test or by (B) two-way ANOVA followed by Dunnett’s post-hoc test.

Figure 6.

PA impairs lysosomal acidification in MmPTECs which is prevented by AMPK activation. (A) Blue (neutral pH):yellow (acidic pH) fluorescence ratios from MmPTECs treated for 6 or 24 h with 300 µM PA or 0.4% BSA with or without 2 nM bafilomycin A₁ for last 6 h and incubated with 5 µM LysoSensor^TM yellow/blue DND-160 for 3 min. (B) Representative micrographs of MmPTECs treated for 6 h with 300 µM PA or 0.4% BSA and incubated with 2.5 µM acridine orange for 15 min. (C) Quantification of the red:green fluorescence ratios on 30 cells per group. (D, E) Blue (neutral pH):yellow (acidic pH) fluorescence ratios from MmPTECs treated for (D) 6 or (E) 24 h with 300 µM PA or 0.4% BSA in the presence of 100 µM A769662 or 2 mM AICAR with or without 2 nM bafilomycin A₁ for last 6 h and stained with LysoSensor^TM yellow/blue DND-160. Data are presented as means ± SEM of three independent biological experiments. Statistical analyses were performed by (C) Student’s unpaired t-test or by (A, D, E) two-way ANOVA followed by (A) Dunnett’s or (D, E) Tukey’s post-hoc test.

Figure 7.

PA alters lysosomal membrane permeability in MmPTECs while pharmacological AMPK activation is protective. (A) Representative micrographs of cells treated for 6 or 24 h with 300 µM PA, 0.4% BSA or with 1 mM LLOMe for 1 h (positive control), fixed and immuno-stained for LGALS3 (red) and LC3 (green). (B, C) Quantifications of (B) the number of LGALS3 puncta per nucleus and (C) their percentages of colocalization with LC3 on 30 cells per group. (D) Free enzymatic activities of CTSB, GUSB and HEXB (expressed as percentages of total activities) in MmPTECs treated for 24 h with 300 µM PA, 0.4% BSA or with 1 mM LLOMe for 1 h (positive control). (E) Representative micrographs of cells treated for 24 h with 300 µM PA in the presence of 100 µM A769662 or 2 mM AICAR, fixed and immuno-stained for LGALS3 (red). (F) Quantifications of the number of LGALS3 puncta per nucleus on 30 cells per group. Data are presented as (B, D, F) means ± SEM or as (C) means and quarters of three independent biological experiments. Statistical analyses were performed by (B, D) Student’s unpaired t-test (comparison between BSA and PA only) or by (C, F) one-way ANOVA followed by Dunnett’s post-hoc test.

Figure 8.

Lysosomal biogenesis is activated in response to pa-induced lysosomal stress in MmPTECs. (A) Representative micrographs of cells treated with 300 µM PA or 0.4% BSA for 6 or 24 h, fixed and immuno-stained for TFEB (green). (B) Quantification of the percentages of cells showing nuclear TFEB staining on more than 100 cells per group. (C) Relative mRNA expression of tfeb-targeted genes on MmPTECs treated for 24 h with 300 µM PA or 0.4% BSA. (D) Quantification of LAMP1-fluorescence intensities per nucleus by the integrated density tool on 30 cells per group (micrographs in Figure 5C). Data are presented as means ± SEM of three independent biological experiments. Statistical analyses were performed by (C) Student’s unpaired t-test, or two-way ANOVA followed by (B) Sidak’s or (D) Tukey’s post-hoc test.

Figure 9.

PA-induced lysosomal dysfunction drives MmPTEC dedifferentiation. (A) Representative micrographs of MmPTECs treated with 300 µM PA or 0.4% BSA for 6 or 24 h, incubated with 100 µg/mL BSA-488 for 30 min at 37°C and fixed. (B) Quantification of BSA fluorescence intensities per nucleus by the integrated density tool on more than 100 cells per group. (C, D) Relative mRNA expression of (C) differentiation and (D) dedifferentiation markers on MmPTECs treated with 300 µM PA or 0.4% BSA for 24 h. (E) Representative micrographs of MmPTECs treated for 6 or 24 h with 300 µM PA or 0.4% BSA with or without 2 nM bafilomycin A₁ for last 6 h, incubated with 100 µg/mL BSA-488 for 30 min at 37°C and fixed. (F) Quantification of BSA fluorescence intensities per nucleus by the integrated density tool on more than 100 cells per group. Data are presented as means ± SEM in three or four independent biological experiments as indicated on the charts. Statistical analyses were performed by (C, D) Student’s unpaired t-test or by (B, F) two-way ANOVA followed by Dunnett’s post-hoc test.

Figure 10.

AMPK activation protects MmPTECs from pa-induced dedifferentiation. (A) Representative micrographs of cells treated for 6 or 24 h with 300 µM PA or 0.4% BSA in the presence of 100 µM A769662 or 2 mM AICAR with and without 2 nM bafilomycin A₁ for last 6 h, incubated with 100 µg/mL BSA-488 for 30 min at 37°C and fixed. (B, C) Quantification of BSA fluorescence intensities per nucleus by the integrated density tool after (B) 6 and (C) 24 h on more than 100 cells per group. (D) Relative mRNA expression of differentiation markers (Cdh16, Lrp2, Aqp1 and Slc5a2) on MmPTECs treated for 24 h with 300 µM PA in the presence of 100 µM A769662 or 2 mM AICAR. Data are presented as means ± SEM in three independent biological experiments. Statistical analyses were performed by (D) one-way ANOVA followed by Dunnett’s post-hoc test or by (B, C) two-way ANOVA followed by Tukey’s post-hoc test.