Lipophagy deficiency exacerbates ectopic lipid accumulation and tubular cells injury in diabetic nephropathy

Abstract

Autophagy-mediated lipotoxicity plays a critical role in the progression of diabetic nephropathy (DN), but the precise mechanism is not fully understood. Whether lipophagy, a selective type of autophagy participates in renal ectopic lipid deposition (ELD) and lipotoxicity in the kidney of DN is unknown. Here, decreased lipophagy, increased ELD and lipotoxcity were observed in tubular cells of patients with DN, which were accompanied with reduced expression of AdipoR1 and p-AMPK. Similar results were found in db/db mice, these changes were reversed by AdipoRon, an adiponectin receptor activator that promotes autophagy. Additionally, a significantly decreased level of lipophagy was observed in HK-2 cells, a human proximal tubular cell line treated with high glucose, which was consistent with increased lipid deposition, apoptosis and fibrosis, while were partially alleviated by AdipoRon. However, these effects were abolished by pretreatment with ULK1 inhibitor SBI-0206965, autophagy inhibitor chloroquine and enhanced by AMPK activator AICAR. These data suggested by the first time that autophagy-mediated lipophagy deficiency plays a critical role in the ELD and lipid-related renal injury of DN.

Fig. 1. Decreased lipophagy and pathological changes in the kidney tissues of patients with DN.

HE, PAS, PASM, Masson trichrome, Oil-red O staining and immunohistochemical analysis of ADRP in the renal biopsy tissues of patients with DN and control (magnification, ×200) (A). Tubular interstitial damage score (B). Quantification of Oil-Red O density (C) and ADRP expression (D). Ultrastructural analysis of lipid deposition in the kidney tissues of patients with DN and control (magnification, ×10,000) * indicates LDs, red arrows indicate double-membrane autophagic vacuoles containing lipids (E). IF analysis and semiquantification of AdipoR1, p-AMPK and LC3B in the kidney tissues of patients with DN and control individuals (F, G). Values are the mean ± SD; *p < 0.05 versus control. n = 15.

Fig. 2. Analysis of AdipoR1 genes.

GO analysis of genes coexpressed with AdipoR1 (positive correlation coefficient ≥0.6) according to KEGG pathway (A) and Biology process (B). Detection of AdipoR1, AdipoR2 and AdipoQ by PCR amplification of DNA from HK-2 cells, BUMPT cells and mouse podocytes (C). Correlation of AdipoR1 expression and GFR (D) and serum creatinine level (E) in patients with DN and control individuals from the Nephroseq database. Values are the mean ± SD; r: correlation coefficient; *p < 0.05 versus control.

Fig. 3. AdipoRon increases lipophagy, ameliorated lipid deposition and renal pathological injury of db/db mice.

Kidney sections stained with HE, PAS, Masson trichrome, TUNEL (magnification, ×400) and DHE (magnification, ×200) (A, B). Glomerular damage score and tubular interstitial damage score (C). TUNEL-positive cells in the kidney tissues and quantitative analysis of intrarenal DHE (D). Renal non-esterified fatty acids in the kidney tissues (E). Renal triglycerides (F). Renal total cholesterol (G). IHC analysis of ADRP and Oil-Red O staining in the kidneys (H). Semiquantification of the mean density of Oil-Red O and ADRP expression in the kidney (I). TEM analysis of lipid deposition in the kidney tissues of three groups of mice (magnification, ×10,000). * indicates the LDs, and red arrows indicate double-membrane autophagic vacuoles containing lipidsnamely, lipophagy (J). Average LD area in the kidney tissues of the three mouse groups (K). Values are the mean ± SD; *p < 0.05 versus control group; #p < 0.05 versus db/db group. n = 12.

Fig. 4. AdipoRon increased lipophagy, activated AdipoR1/AMPK pathway and alleviated fibrosis in the kidney of db/db mice.

Representative IF images of AdipoR1, p-AMPK, and LC3B in the kidney tissues (magnification, ×400) (A). Semiquantification of the IF analysis of AdipoR1, p-AMPK and LC3B (B). WB analysis and quantification of AdipoR1, p-mTOR, p-AMPK and p-ULK1 expression in the kidneys (C, D). WB analysis of the indicated molecules using homogenates (HOM) and isolated LD fractions from the kidneys (E). Representative IF images and semiquantification of FN and Collagen I in the kidneys (F, G). WB analysis and quantification of the ADRP, SREBP-1, FN and Collagen I levels (H, I). Values are the mean ± SD; *p < 0.05 versus control, ^#p < 0.05 versus db/db group. n = 12.

Fig. 5. AdipoRon increased lipophagy in HK-2 cells exposed to HG environment.

Representative images of intracellular autophagosome (MCD), lysosomes (Lyso-tracker) and lipid (Bodipy) co-localization in HK-2 cells in an HG environment with or without AdipoRon, CQ pretreatment (A). WB analysis of LC3II expression using HOM and isolated LD fractions from HK-2 cells exposed to HG conditions with or without different AdipoRon concentrations (B). WB and densitometric analysis of TFEB expression using nuclear protein from HK-2 cells exposed to HG conditions with or without AdipoRon (C, D). WB analysis of LC3II expression using isolated LD fractions from HK-2 cells exposed to HG conditions with or without AdipoRon and CQ pretreatment (E). Values are the mean ± SD; *p < 0.05 versus LG group, ^#p < 0.05 versus HG group. n = 3.

Fig. 6. AdipoRon increased lipophagy through AdipoR1/AMPK pathway.

WB and densitometric analysis of AdipoR1, p-mTOR, p-AMPK and p-ULK1 expression in HK-2 cells exposed to HG conditions with or without different AdipoRon concentrations (A, B). WB and densitometric analysis of AdipoR1, p-mTOR, p-AMPK and p-ULK1 expression in HK-2 cells exposed to an HG environment pretreated with AdipoRon, AdipoR1 siRNA, AICAR or SBI-0206965 (C, D). WB analysis of BECN1, ATG5, LC3 and SQSTM1 using isolated LD fractions from HK-2 cells exposed to HG conditions with or without AdipoRon, AdipoR1 siRNA, AICAR and SBI-0206965 (E). Values are the mean ± SD; *p < 0.05 versus LG group, ^#p < 0.05 versus HG group, ^&p < 0.05 versus HG + AdipoRon group. n = 3.

Fig. 7. The effects of AdipoRon on increasing lipohagy and alleviating lipid deposition and fibrosis were partially blocked by ULK1 inhibitor, while enhance by AMPK activator.

Confocal IF images showing the co-localization of RAB7 and LC3B and the co-localization of BECN1 and LDs (Bodipy) in HK-2 cells exposed to HG conditions and pretreated with or without AdipoRon, AdipoR1 siRNA, AICAR and SBI-0206965 (A, B). Representative images and semiquantification of the lipid deposition in HK-2 cells exposed to an HG environment pretreated with AdipoRon, AdipoR1 siRNA, AICAR and SBI-0206965 (C, D). WB and densitometric analysis of ADRP, SREBP-1, FN and Collagen I expressions in HK-2 cells exposed to HG conditions with or without AdipoRon, AdipoR1 siRNA, AICAR and SBI-0206965 (E, F). Values are the mean ± SD; *p < 0.05 versus LG group, ^#p < 0.05 versus HG group. n = 3.