Pre-emptive pharmacological inhibition of fatty acid-binding protein 4 attenuates kidney fibrosis by reprogramming tubular lipid metabolism

Abstract

Kidney fibrosis is a hallmark of chronic kidney disease (CKD) progression that is caused by tubular injury and dysregulated lipid metabolism. Genetic abolition fatty acid-binding protein 4 (FABP4), a key lipid transporter, has been reported to suppress kidney interstitial fibrosis. However, the role and underlying mechanism of chemical inhibition of FABP4 in fibrotic kidney have not been well-documented. Here, we examined preemptive the effect of a FABP4 inhibitor, BMS309403, on lipid metabolism of tubular epithelial cells (TECs) and progression of kidney fibrosis. The expression of FABP4 was significantly elevated, concomitated with the accumulation of lipid droplets in TECs during kidney fibrosis. Treatment with BMS309403 alleviated lipid deposition of TECs, as well as interstitial fibrotic responses both in unilateral ureteral obstruction (UUO)-engaged mice and TGF-β-induced TECs. Moreover, BMS309403 administration enhanced fatty acid oxidation (FAO) in TECs by regulating peroxisome proliferator-activated receptor γ (PPARγ) and restoring FAO-related enzyme activities; In addition, BMS309403 markedly reduced cell lipotoxicity, such as endoplasmic reticulum (ER) stress and apoptosis in fibrotic kidney. Taken together, our results suggest that preemptive pharmacological inhibition of FABP4 by BMS309403 rebalances abnormal lipid metabolism in TECs and attenuates the progression of kidney fibrosis, thus may hold therapeutic potential for the treatment of fibrotic kidney diseases.

Fig. 1. Expression of FABP4 in UUO mice and TGF-β-treated HK-2 cells.

a Fluorescence microscopy revealed that FABP4 was mainly expressed in kidney tubular epithelial, and increased in UUO-treated mice. Quantitative image to depict fluorescence intensity (Right panel). Scale bars: 50 μm. b Western blot showed that FABP4 was increased in UUO mice. Schematic representation of quantitative data of indicated proteins. Representative images from three independent experiments are shown above. n = 6 mice. c Protein expression of FABP4 was detected in HK-2 cells treated with 10 ng/mL TGF-β for 48 h. Schematic representation of quantitative data of indicated proteins. Representative images from three independent experiments are shown above. d In the study of Ju CKD tubules, CKD was associated with significantly increased mRNA value of Fabp4 compared with biopsy samples from health control. e A negative correlation between tubular FABP4 and eGFR was observed in public Ju CKD dataset. Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P <0.001, ns means no statistical significance.

Fig. 2. BMS309403 reduced lipid accumulation in TECs in vivo and in vitro.

a One day before UUO surgery treatment with FABP4 inhibitor BMS309403, and then sacrificed on day 7. b BMS309403 has no significant change in the ratio of body weight to kidney weight (BW/KW). c Cell viability of HK-2 cells was detected by CCK8, which was treated by various doses of BMS309403 for 48 h. d Oil red O staining in frozen section from mice tubulointerstititum. e Oil red O staining in HK-2 cells co-treated with or without BMS309403 for 48 h. f Content of lipid in peripheral blood of mice. g Content of lipid in kidney tissue of mice. Data were presented as mean ± SEM. *P < 0.05, ***P < 0.001, ns means no statistical significance. Scale bars: 50 μm.

Fig. 3. BMS309403 decreased the expression of fibrosis marker in TGF-β-treated HK-2 cells via inhibiting TGF-β/Smad pathway.

a Western blot of HK-2 cell lysates for FABP4, fibronectin, collagen-I, and α-SMA expression. GAPDH sets as loading control. b RT-qPCR for fibronectin, collagen-I, and α-SMA transcripts normalized to Gapdh in HK-2 cells. c Western blot and band intensity quantitation for TGF-β, p-Smad2, and p-Smad3 in HK-2 cells. Blots were stripped and reprobed for GAPDH. d RT-qPCR for TGF-β, CTGF, FGF2, PDGFB transcripts normalized to Gapdh in total RNA extracts of HK-2 cells from DMSO- or BMS309403-treated normal and TGF-β-treated cells. These data were calculated from three independent experiments. ***P < 0.001.

Fig. 4. BMS309403 restored FAO enzymes activity, decreased apoptosis and ER stress in HK-2 cells.

a The changes in PGC1α and PPARγ expression levels were confirmed in HK-2 cells by immunoblotting studies. b RT-qPCR for Ppargc1a, Pparg, Cpt1a, Cpt2, Acox1, and Acox2 transcripts normalized to Gapdh in HK-2 cells. c Western blot of whole-cell lysates for Bcl-2, cleaved caspase-3 expression in HK-2 cells. GAPDH sets as loading control. d Western blot of whole-cell lysates for Grp78 (Bip), CHOP expression in HK-2 cells. GAPDH sets as loading control. e, f ER stress related genes were analyzed by RT-qPCR in HK-2 cells from DMSO- or BMS309403-treated normal and TGF-β-treated cells. The transcript normalized to Gapdh. These data were calculated from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns means no statistical significance.

Fig. 5. BMS309403 improved kidney lesion by UUO.

a Staining with HE, PAS, and MTS of kidney sections from obstructed (UUO) or sham-operated kidneys (control). Animals received BMS309403 (40 mg/kg for 8 days) or vehicle as indicated. n = 6 per group. b Western blot of whole-kidney lysates for FABP4, fibronectin, collagen-I, and α-SMA expression. GAPDH sets as loading control. c Fibronectin, collagen-I, and α-SMA mRNA expression by RT-qPCR. Schematic representation of quantitative data of indicated proteins. The transcripts normalized to Gapdh. Representative images from three independent experiments are shown above. **P < 0.01, ***P < 0.001, ns means no statistical significance. Scale bars: 25 μm.

Fig. 6. BMS309403 reduced the release of profibrotic cytokines by inhibiting TGF-β/Smad3 pathway and restored impaired FAO pathway in fibrotic kidney.

a TGF-β, p-Smad2, and p-Smad3 expression was verified in kidney samples by Western blot. Blots were stripped and reprobed for GAPDH. b RT-qPCR for TGF-β, CTGF, PDGFB, and FGF2 transcripts normalized to Gapdh in kidney tissue. c The changes in PGC1α and PPARγ expression levels were confirmed in mouse kidneys by immunoblotting studies. d Ppargc1a, Pparg, Cpt1a, Cpt2, Acox1, and Acox2 mRNA expression were analyzed by RT-qPCR in total RNA extracts of kidneys from saline- or BMS309403-treated normal and UUO mice. The transcript normalized to Gapdh. Representative images from three independent experiments are shown above. *P < 0.05, **P < 0.01, ***P < 0.001, ns means no statistical significance.

Fig. 7. Apoptosis and ER stress were significantly ameliorated in UUO kidneys after BMS309403 treatment.

a TUNEL staining of kidney tissues (green: TUNEL-positive cells). b Western blot of whole-kidney lysates for Bcl-2, cleaved caspase-3 expression in mice. GAPDH sets as loading control. c Western blot of whole-kidney lysates for Grp78 (Bip), CHOP expression in mice. GAPDH sets as loading control. Representative images from three independent experiments are shown above. ***P < 0.001, ns means no statistical significance. Scale bars: 50 μm.

Fig. 8. Schematic diagram of the mechanisms of FABP4 in kidney fibrosis.

BMS309403 reduces lipotoxicity to improve kidney fibrosis mainly by reducing lipid accumulation and increasing FAO pathway, and also reduce the secretion of a series of profibrotic cytokines by kidney TECs to relieve kidney fibrosis.