Endocrinol Metab.  2023 Apr;38(2):226-244. 10.3803/EnM.2022.1604.

Inhibition of Fatty Acid β-Oxidation by Fatty Acid Binding Protein 4 Induces Ferroptosis in HK2 Cells Under High Glucose Conditions

• Chen J
• Wu K
• Lei Y
• Huang M
• Cheng L
• Guan H
• Lin J
• Zhong M
• Wang X
• Zheng Z

• KMID: 2541879
• DOI: http://doi.org/10.3803/EnM.2022.1604

Abstract

Background
Ferroptosis, which is caused by an iron-dependent accumulation of lipid hydroperoxides, is a type of cell death linked to diabetic kidney disease (DKD). Previous research has shown that fatty acid binding protein 4 (FABP4) is involved in the regulation of ferroptosis in diabetic retinopathy. The present study was constructed to explore the role of FABP4 in the regulation of ferroptosis in DKD.
Methods
We first detected the expression of FABP4 and proteins related to ferroptosis in renal biopsies of patients with DKD. Then, we used a FABP4 inhibitor and small interfering RNA to investigate the role of FABP4 in ferroptosis induced by high glucose in human renal proximal tubular epithelial (HG-HK2) cells.
Results
In kidney biopsies of DKD patients, the expression of FABP4 was elevated, whereas carnitine palmitoyltransferase-1A (CP-T1A), glutathione peroxidase 4, ferritin heavy chain, and ferritin light chain showed reduced expression. In HG-HK2 cells, the induction of ferroptosis was accompanied by an increase in FABP4. Inhibition of FABP4 in HG-HK2 cells changed the redox state, sup-pressing the production of reactive oxygen species, ferrous iron (Fe²⁺), and malondialdehyde, increasing superoxide dismutase, and reversing ferroptosis-associated mitochondrial damage. The inhibition of FABP4 also increased the expression of CPT1A, reversed lipid deposition, and restored impaired fatty acid β-oxidation. In addition, the inhibition of CPT1A could induce ferroptosis in HK2 cells.
Conclusion
Our results suggest that FABP4 mediates ferroptosis in HG-HK2 cells by inhibiting fatty acid β-oxidation.

Keyword

Diabetic kidney disease; Ferroptosis; FABP4 protein, human; Fatty acid β-oxidation; Lipid accumulation; Reactive oxygen species

Figure

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  Fig. 1. Changes related to ferroptosis in biopsies of diabetic kidney disease (DKD) patients. (A) Prussian blue staining of kidney biopsy tissue. The red arrows indicate iron deposition. Control refers to patients with glomerular minor lesion. (B) Transmission electron microscopy images of mitochondria in renal proximal tubular epithelial cells. Control refers to patients with early-stage DKD.

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  Fig. 2. Expression of fatty acid binding protein 4 (FABP4) and ferroptosis-associated proteins in diabetic kidney disease (DKD) patients. Representative immunofluorescence images of kidney tissue sections in negative controls and DKD patients for (A) glutathione peroxidase 4 (GPX4), ferritin heavy chain (FTH), and ferritin light chain (FTL), (B) FABP4, and (C) carnitine palmitoyltransferase-1A (CPT1A). 4′,6-Diamidino-2-phenylindole (DAPI) indicates nuclear staining.

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  Fig. 3. Ferroptosis-related changes induced by high glucose in human renal proximal tubular epithelial (HG-HK2) cells. (A) Viability of HK2 cells detected by cell counting kit-8 (CCK-8) in the three groups indicated. Levels of (B) ferrous iron (Fe2+), (C) malondialdehyde (MDA), (D) superoxide dismutase (SOD), and (E) glutathione (GSH) in the control, HG, and HG+ferrostatin-1 (Fer-1) groups. Protein expression of (F) glutathione peroxidase 4 (GPX4), (G) ferritin heavy chain (FTH), ferritin light chain (FTL), and (H) phospho-AMP-activated protein kinase (p-AMPK) in the three groups. (I) Intracellular reactive oxygen species (ROS) production stained with DCFDA/H2DCFDA fluorescent probes. (J) Mitochondrial morphology detected by transmission electron microscopy. The red arrows indicate the damaged mitochondria (mitochondria cristae vanish and membrane rupture). NS, no statistical significance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CTL, control. aP<0.05, bP<0.01, cP<0.001 when compared with the control group; dP<0.05, eP<0.01, fP<0.001 when compared with the HG group.

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  Fig. 4. Inhibition of fatty acid binding protein 4 (FABP4) by BMS309403 (BMS) attenuated ferroptosis induced by high glucose in human renal proximal tubular epithelial (HG-HK2) cells. (A) Expression of FABP4 in the three groups indicated. (B) Viability of cells in the three groups indicated. (C) Representative images of cell death measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (Tunel) staining (positive=red), where the nucleus is labeled with 4′,6-diamidino-2-phenylindole (DAPI; blue). Concentrations of (D) ferrous iron (Fe2+), (E) malondialdehyde (MDA), (F) superoxide dismutase (SOD), (G) glutathione (GSH), (H) expression of glutathione peroxidase 4 (GPX4), (I) ferritin heavy chain (FTH), ferritin light chain (FTL), and (J) phospho-AMP-activated protein kinase (p-AMPK) in each group. (K) Content of reactive oxygen species (ROS) stained with DCFDA/H2DCFDA fluorescent probes. (L) Mitochondrial morphology detected by transmission electron microscopy. The red arrows indicate the damaged mitochondria (mitochondria cristae vanish and membrane rupture). NS, no statistical significance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CTL, control. aP<0.01, bP<0.001 when compared with the control group; cP<0.05, dP<0.01, eP<0.001 when compared with the HG group.

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  Fig. 5. Silencing fatty acid binding protein 4 (FABP4) inhibited ferroptosis induced by high glucose in human renal proximal tubular epithelial (HG-HK2) cells. (A) Expression of FABP4 after transfection with FABP4-small interfering RNA (siRNA). (B) Effect of silencing FABP4 on cell viability. Concentrations of (C) ferrous iron (Fe2+), (D) malondialdehyde (MDA), (E) superoxide dismutase (SOD), and (F) glutathione (GSH). (G) Reactive oxygen species (ROS) stained with DCFDA/H2DCFDA fluorescent probes. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS, no statistical significance. aP<0.001 compared with the negative control (NC)-siRNA group; bP<0.01, cP<0.001 compared with the HG+NC-siRNA group.

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  Fig. 6. Inhibition of fatty acid binding protein 4 (FABP4) restores impaired fatty acid β-oxidation in human renal proximal tubular epithelial (HK2) cells. (A) Protein expression of carnitine palmitoyltransferase-1A (CPT1A) in HK2 cells for the three groups indicated. (B) Representative immunofluorescence images of HK2 cells for long chain 3-hydroxyl-coenzyme A (CoA) dehydrogenase (HADHA), acyl-CoA dehydrogenase very long chain (ACADVL), and acyl-CoA dehydrogenase, medium chain specific (ACADM). (C) Lipid accumulation after high glucose (HG) and palmitic acid (PA) stimulation, with and without BMS309403 (BMS), as detected by Oil Red O staining. The red arrows indicate lipid droplets. (D) Mitochondrial morphology in HG, HG+PA, and HG+PA+BMS groups. The red arrows indicate the damaged mitochondria (mitochondria cristae vanish and membrane rupture). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CTL, control. aP<0.001 compared with the control group; bP<0.05 compared with the HG group.

• 

  Fig. 7. Silencing carnitine palmitoyltransferase-1A (CPT1A) induced ferroptosis in human renal proximal tubular epithelial (HK2) cells. (A) Expression of CPT1A after transfection with CPT1A-small interfering RNA (siRNA). (B) Effect of silencing CPT1A on cell viability. Concentrations of (C) ferrous iron (Fe2+), (D) malondialdehyde (MDA), (E) superoxide dismutase (SOD), and (F) glutathione (GSH). (G) Reactive oxygen species (ROS) content stained with DCFDA/H2DCFDA fluorescent probes. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Fer-1, ferrostatin-1; NS, no statistical significance. aP<0.01, bP<0.001 compared with the negative control (NC)-siRNA group; cP<0.01, dP<0.001 compared with the CPT1A-siRNA group.

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  Fig. 8. Potential mechanism of fatty acid binding protein 4 (FABP4) involvement in ferroptosis-mediated renal tubule injury induced by high glucose. Physiologically, fatty acids are taken up from the basolateral membrane by proximal renal tubular epithelial cells through the cluster of differentiation 36 (CD36) receptor; and from the apical membrane through endocytosis in albumin-bound form. In the cell, they are converted to fatty acyl-coenzyme A (CoA) and are transported to mitochondria for fatty acid β-oxidation (FAO). In diabetes (red arrow), high glucose upregulates FABP4 expression in renal tubular cells, which inhibits FAO through downregulating carnitine palmitoyltransferase-1 (CPT1) and leads to lipid accumulation, providing more substrate for lipid peroxidation. Polyunsaturated fatty acids (PUFAs) are converted to PUFA-CoA and are incorporated into PUFA-phospholipids, which react with iron-dependent reactive oxygen species (ROS) and induce lipid peroxidation and ferroptosis. ALB, albumin; FA, fatty acid; Fe3+, ferric iron; TfR1, transferrin receptor 1; TG, triglycerides; Fe2+, ferrous iron; O2−, superoxide anion; SOD, superoxide dismutase; H2O2, hydrogen peroxide; PL, phospholipid.

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