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. 2022 Aug 1;1868(8):166414.
doi: 10.1016/j.bbadis.2022.166414. Epub 2022 Apr 18.

FGF1ΔHBS delays the progression of diabetic nephropathy in late-stage type 2 diabetes mouse model by alleviating renal inflammation, fibrosis, and apoptosis

Qian Lin  1 , Oscar Chen  2 , John P Wise Jr  2 , HongXue Shi  3 , Kupper A Wintergerst  4 , Lu Cai  5 , Yi Tan  6
Affiliations

FGF1ΔHBS delays the progression of diabetic nephropathy in late-stage type 2 diabetes mouse model by alleviating renal inflammation, fibrosis, and apoptosis

Qian Lin et al. Biochim Biophys Acta Mol Basis Dis. .

Abstract

Elderly adults are at higher risk for developing diabetic complications including diabetic nephropathy (DN), contributing to excess morbidity and mortality in elderly individuals. A non-mitogenic variant of fibroblast growth factor 1 (FGF1ΔHBS) was demonstrated to prevent DN in an early-stage (2-month-old) type 2 diabetes (T2D) mouse model. The present study aimed to investigate the potential therapeutic effects of FGF1ΔHBS against the progression of renal dysfunction in a late-stage T2D mouse model with established DN. Nine-month-old db/db mice were administered FGF1ΔHBS every other day for 3 months. db/db mice at 12-month-old without FGF1ΔHBS treatment exhibited high blood glucose level and elevated urine albumin-to-creatinine ratio. FGF1ΔHBS treatment effectively reversed hyperglycemia, delayed the development of renal dysfunction, and reduced kidney size and weight. Furthermore, FGF1ΔHBS treatment significantly prevented the progression of renal morphologic impairment. FGF1ΔHBS treatment demonstrated anti-inflammatory and anti-fibrotic effects, with significantly decreased protein levels of key pro-inflammatory cytokines and pro-fibrotic factors in kidney. Moreover, FGF1ΔHBS treatment greatly decreased apoptosis of renal tubular cells, accompanied by significant downregulation of the proapoptotic protein and upregulation of the antiapoptotic protein and peroxisome proliferator-activated receptor α (PPARα) expression in kidney. Mechanistically, FGF1ΔHBS treatment directly protected mouse proximal tubule cells against palmitate-induced apoptosis, which was abolished by PPARα inhibition. In conclusion, this study demonstrated that FGF1ΔHBS delays the progression of renal dysfunction likely through activating PPARα to prevent renal tubule cell death in late-stage T2D, exhibiting a promising translational potential in treating DN in elderly T2D individuals by ameliorating renal inflammation, fibrosis and apoptosis.

Keywords: Apoptosis; Diabetic nephropathy; FGF1(ΔHBS); Fibrosis; Inflammation; Type 2 diabetes.

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Conflict of interest statement

Declaration of competing interest

No potential conflicts of interest relevant to this article are reported.

Figures

Fig. 1.
Fig. 1.
Features of established diabetic nephropathy (DN) in 9-month-old db/db diabetic mice. (A) The ratio of kidney weight to tibia length. (B) Urine albumin-to-creatinine ratio (UACR) in spot urine. (C) Representative images of H&E-stained kidney sections and (D) glomerular size quantified from images of H&E staining. (E) Representative images of PAS-stained kidney sections and (F) mesangial matrix expansion (% glomerular area) from images of PAS staining. (G) Representative images of Sirius Red staining and (H) quantified Sirius Red positive-stained area of kidney sections. Quantitative data are normalized to those of db/m control mice and expressed as mean ± SEM, n = 3 for db/m mice, n = 4 for db/db mice. *P < 0.05, **P < 0.01, vs. db/m littermate controls.
Fig. 2.
Fig. 2.
FGF1ΔHBS delays the progression of DN in late-stage db/db mice. 9-month-old db/db mice were administered with FGF1ΔHBS (0.5 mg/kg body weight) or PBS vehicle via intraperitoneal injection every other day for 3 months. (A) Blood glucose level. (B) UACR in spot urine. Quantitative data are expressed as mean ± SEM, n = 7 for PBS group, n = 5 for FGF1ΔHBS group. #P < 0.01 and &P < 0.01 vs. db/db mice at 9-month-old and 10-month-old, respectively. (C) The kidney size and (D) the ratio of kidney weight to tibia length. (E) Representative images of H&E staining of glomeruli and renal tubules in kidney sections. Blue arrows indicate dilated glomerular blood capillaries; yellow arrows indicate exfoliation of epithelial cells in the renal tubular lumen; black arrows indicate renal tubular vacuolar lesions. (F) Glomerular size quantified from images of H&E staining. (G) Representative images of PAS staining of glomeruli and renal tubules in kidney sections and (H) mesangial matrix expansion (% glomerular area) quantified from images of PAS staining, normalized to PBS group. (I) Representative images of Sirius Red staining of glomeruli and renal tubules in kidney sections and (J) quantified Sirius Red positive-stained area of kidney sections, normalized to PBS group. Scale bars = 50 μm. Quantitative data are expressed as mean ± SEM, n = 7 for PBS group, n = 5 for FGF1ΔHBS group. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. PBS group,
Fig. 3.
Fig. 3.
The effect of FGF1ΔHBS on the expression of renal inflammatory factors in late-stage diabetic mice. (A) The inflammatory protein expressions of ICAM-1, VCAM-1, TNFα and CD68 were detected by western blot and β-actin was used as a loading control. The expression abundance of (B) ICAM-1, (C) VCAM-1, (D) TNFα and (E) CD68 was quantified by densitometry analysis. Quantitative data are expressed as mean ± SEM, n = 5 for PBS group, n = 4 for FGF1ΔHBS. *P < 0.05 and **P < 0.01 vs PBS group.
Fig. 4.
Fig. 4.
Effect of FGF1ΔHBS on protein expression and localization of renal inflammatory factors in late-stage diabetic mice. (A) Representative images of immunohistochemical staining of inflammatory markers (ICAM-1, VCAM-1, TNFα and CD68) in kidney sections. Relative expression abundance of (B) ICAM-1, (C) VCAM-1, (D) TNFα and (E) CD68 on images of immunohistochemical staining was quantified using Image-Pro Plus 6.0 and normalized to PBS group. Scale bars = 50 μm. Quantitative data are expressed as mean ± SEM, n = 7 for PBS group, n = 5 for FGF1ΔHBS group. **P < 0.01 vs PBS group.
Fig. 5.
Fig. 5.
Effect of FGF1ΔHBS on the expression of renal fibrotic factors in late-stage diabetic mice. (A) The fibrotic protein expressions of TGFβ, CTGF, PAI-1, fibronectin and collagen I were detected by western blot and β-actin was used as a loading control. The expression abundance of (B) TGFβ, (C) CTGF, (D) PAI-1, (E) fibronectin, and (F) collagen I were quantified by densitometry analysis, respectively. Quantitative data are expressed as mean ± SEM, n = 5 for PBS group, n = 4 for FGF1ΔHBS group. *P < 0.05 and **P < 0.01 vs PBS group.
Fig. 6.
Fig. 6.
The effect of FGF1ΔHBS on the protein expression and localization of renal fibrotic factors in late-stage diabetic mice. (A) The representative images of immunohistochemical staining of fibrotic markers (TGFβ, CTGF, fibronectin, collagen I and collagen IV) in kidney sections. The relative expression abundance of (B) TGFβ, (C) CTGF, (D) fibronectin, (E) collagen I, and (F) collagen IV on image of immunohistochemical staining was quantified using Image-Pro Plus 6.0 and normalized to PBS group. Scale bars = 50 μm. Quantitative data are expressed as mean ± SEM, n = 7 for PBS group, n = 5 for FGF1ΔHBS group. *P < 0.05 vs PBS group.
Fig. 7.
Fig. 7.
The effect of FGF1ΔHBS on renal apoptosis in late-stage diabetic mice. (A) The expressions of Bcl-2, Bax and cleaved-caspase 3 were detected by western blot and β-actin was used as a loading control. The expression abundance of (B) Bcl-2, (C) Bax, and (D) cleaved-caspase 3 was quantified by densitometry analysis. Quantitative data are expressed as mean ± SEM, n = 5 for PBS group, n = 4 for FGF1ΔHBS group. *P < 0.05 and ***P < 0.001 vs PBS group. (E) Apoptotic cell death was detected by TUNEL staining of kidney sections. The sections were counterstained with DAPI and images were merged with the TUNEL staining (Merged). Glomerulus was circled in red. (F) Quantitative analysis of TUNEL positive nuclear number per field. Scale bars = 50 μm. Quantitative data are expressed as mean ± SEM, n = 7 for PBS group, n = 5 for FGF1ΔHBS group. *P < 0.05 and ***P < 0.001 vs PBS group.
Fig. 8.
Fig. 8.
FGF1ΔHBS upregulates PPARα in kidney and prevents palmitate (Pal)-induced apoptosis in BUMPT cells mediated by PPARα. (A) The protein expression of PPARα was detected by western blot and β-actin was used as a loading control. The expression abundance of (B) PPARα was quantified by densitometry analysis. Quantitative data are expressed as mean ± SEM, n = 5 for PBS group, n = 4 for FGF1ΔHBS. **P < 0.01 vs PBS group. The BUMPT cells were exposed to PPARα inhibitor GW6471 (10 μmol/L) or DMSO control for 8 h, then pretreated with FGF1ΔHBS (100 nmol/L) for 1 h and followed by exposure to Pal (90 μmol/L) for additional 16 h in the presence or absence of FGF1ΔHBS. (C) The protein expression of PPARα and caspase 3 cleavage was detected by western blot and β-actin was used as a loading control. (D) The expression abundance of PPARα and (E) caspase 3 cleavage (cleaved/pro caspase 3 ratio) was quantified by densitometry analysis. Quantitative data were normalized to “DMSO + BSA + PBS” control group and expressed as mean ± SEM, and three independent experiments were performed. *P < 0.05, **P < 0.01, vs. indicated groups.
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