Fibroblast Growth Factor 21 Attenuates Diabetes-Induced Renal Fibrosis by Negatively Regulating TGF-β-p53-Smad2/3-Mediated Epithelial-to-Mesenchymal Transition via Activation of AKT

Abstract

Background: Epithelial-to-mesenchymal transition (EMT) is required for renal fibrosis, which is a characteristic of diabetic nephropathy (DN). Our previous study demonstrated that fibroblast growth factor 21 (FGF21) prevented DN associated with the suppressing renal connective tissue growth factor expression, a key marker of renal fibrosis. Therefore, the effects of FGF21 on renal fibrosis in a DN mouse model and the underlying mechanisms were investigated in this study.

Methods: Type 1 diabetes mellitus was induced in C57BL/6J mice by intraperitoneal injections of multiple low doses of streptozotocin. Then, diabetic and non-diabetic mice were treated with or without FGF21 in the presence of pifithrin-α (p53 inhibitor) or 10-[4'-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride (10-DEBC) hydrochloride (Akt inhibitor) for 4 months.

Results: DN was diagnosed by renal dysfunction, hypertrophy, tubulointerstitial lesions, and glomerulosclerosis associated with severe fibrosis, all of which were prevented by FGF21. FGF21 also suppressed the diabetes-induced renal EMT in DN mice by negatively regulating transforming growth factor beta (TGF-β)-induced nuclear translocation of Smad2/3, which is required for the transcription of multiple fibrotic genes. The mechanistic studies showed that FGF21 attenuated nuclear translocation of Smad2/3 by inhibiting renal activity of its conjugated protein p53, which carries Smad2/3 into the nucleus. Moreover pifithrin-α inhibited the FGF21-induced preventive effects on the renal EMT and subsequent renal fibrosis in DN mice. In addition, 10-DEBC also blocked FGF21-induced inhibition of renal p53 activity by phosphorylation of mouse double minute-2 homolog (MDM2).

Conclusion: FGF21 prevents renal fibrosis via negative regulation of the TGF-β/Smad2/3-mediated EMT process by activation of the Akt/MDM2/p53 signaling pathway.

Keywords: Epithelial-mesenchymal transition; Fibroblast growth factor 21; Fibrosis; Kidney; Transforming growth factor beta; Tumor suppressor protein p53.

Fig. 1. Effects of fibroblast growth factor 21 (FGF21) supplement on renal extracellular matrix (ECM) accumulation in diabetic nephropathy (DN) mice. Since ECM leads to renal fibrosis, Western blotting was used to examine the expressions of the markers of ECM and fibrosis in the kidneys, including (A) connective tissue growth factor (CTGF), (B) fibronectin 1 (FN1), (C) collagen I (Col I), (D) collagen II (Col II), (E) collagen IV (Col IV), and (F) plasminogen activator inhibitor-1 (PAI-1). The mRNA level of multiple markers of EMC accumulation and renal fibrosis including (G) CTGF, (H) FN1, (I) Col I, (J) Col II, (K) Col IV, (L) and PAI-1 in the kidney with or without FGF21 treatment were examined by quantitative polymerase chain reaction. Data are presented as the mean±standard deviation (n=8/group). ^aP<0.05 vs. the control (Con) group, ^bP<0.05 vs. DN group.

Fig. 2. Effects of fibroblast growth factor 21 (FGF21) supplement on the formation and nuclear translocation of p53/Smad2/3 complex. Western blot analysis was used to examine the expression of (A) renal transforming growth factor beta (TGF-β) and phosphorylation of (B) Smad2, (C) Smad3, and (D) Smad7 as well as (E) renal p53 phosphorylation. (F, G) Immunoprecipitation (IP) was used to examine the protein-protein interaction between p53 and Smad2/3 with or without FGF21 treatment. The nuclear translocation of p53/Smad2/3 complex was examined by changes in the expression of nuclear (H) p53, (I) Smad2, (J) and Smad3, as determined by Western blot analysis. Data are presented as the mean±standard deviation (n=8/group). ^aP<0.05 vs. the control (Con) group, ^bP<0.05 vs. diabetic nephropathy (DN) group.

Fig. 3. Effect of pifithrin-α (PF-α) on fibroblast growth factor 21 (FGF21)-induced suppression of renal p53 phosphorylation and interaction of p53/Smad2/3. (A) The phosphorylation and expression of renal p53 in the presence of FGF21 and/or PF-α were examined by Western blotting. (B, C) The protein-protein interaction between p53 and Smad2/3 in the presence of FGF21 and/or PF-α was examined by immunoprecipitation (IP). Western blot analysis was used to examine the activity and (D) expression of renal AKT and the expression of the extracellular matrix (ECM) and fibrotic markers in the kidneys including (E, F) connective tissue growth factor (CTGF), (E, G) fibronectin 1 (FN1), (E, H) plasminogen activator inhibitor-1 (PAI-1), (E, I) collagen I (Col I), (E, J) collagen II (Col II), and (E, K) collagen IV (Col IV). Data are presented as the mean±standard deviation (n=8/group). ^aP<0.05 vs. the diabetic nephropathy (DN) group.

Fig. 4. Effects of 10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride (10-DEBC) on fibroblast growth factor 21 (FGF21)-induced suppression of renal p53 phosphorylation and complex formation of p53/Smad2/3. The phosphorylation and expression of renal (A, B) AKT, (A, C) p53, and (A, D) mouse double minute-2 homolog (MDM2) in the presence of FGF21 and/or 10-DEBC were examined by Western blotting. (E, F) The protein-protein interaction between p53 and Smad2/3 in the presence of FGF21 and/or 10-DEBC was examined by immunoprecipitation (IP). Data are presented as the mean±standard deviation (n=8/group). DEBC=10-DEBC (10-DEBC hydrochloride). ^aP<0.05 vs. the diabetic nephropathy (DN) group.

Fig. 5. Effects of 10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride (10-DEBC) on fibroblast growth factor 21 (FGF21)-induced prevention of the epithelial-to-mesenchymal transition (EMT) in the diabetic kidney. Western blot analysis was used to examine the markers of EMT and extracellular matrix (ECM) in the kidneys, including (A) E-cadherin, (B) P-cadherin, (C) zonula occludens-1 (ZO-1), (D) α-smooth muscle actin (α-SMA), (E) vimentin, and (F) laminin, (G) connective tissue growth factor (CTGF), (H) fibronectin 1 (FN1), (I) plasminogen activator inhibitor-1 (PAI-1), (J) collagen I (Col I), (K) collagen II (Col II), and (L) collagen IV (Col IV). Data are presented as the mean±standard deviation (n=8/group). ^aP<0.05 vs. the diabetic nephropathy (DN) group.