The Interaction of Apelin and FGFR1 Ameliorated the Kidney Fibrosis through Suppression of TGFβ-Induced Endothelial-to-Mesenchymal Transition

Figure 1. Proximity between apelin and FGFR1 suppresses the TGFβ/Smad signaling pathway in HMVECs. (a) HMVECs were treated with TGFβ2 (5 ng/mL) or N-FGFR1 (1.5 μg/mL) for 48 h with or without preincubation with apelin (100 nM) for 2 h. The proximity between apelin and FGFR1 was then analyzed by the Duolink In Situ Assay. For each slide, images at a ×400 original magnification were obtained from six different areas. The scale bar is 60 μm in each panel. (b) HMVECs were treated with TGFβ2 (5 ng/mL) or with apelin (100 nM) for 48 h, and the p-Smad3, TGFβR1, TGFβR2, and FGFR1 levels were analyzed by western blot. Densitometric analysis of the p-Smad3/Smad3, TGFβR1/β-actin, TGFβR2/β-actin, and FGFR1/β-actin levels from each group (n = 5) was analyzed. (c) HMVECs were treated with or without apelin (100 nM) for 48 h, and the FGFR1 levels were analyzed by western blot. Densitometric analysis of the FGFR1/GADPH levels from each group (n = 5) was analyzed.

Figure 2. Apelin inhibits TGFβ/Smad signaling and EndMT via regulating FGFR1 expression. (a) HMVECs were treated with TGFβ2 (5 ng/mL) for 15 min or 48 h with or without preincubation with apelin for 2 h, and the VE-cadherin, CD31, α-SMA, SM22α, and FSP1 levels were analyzed by western blot. Densitometric analysis of the VE-cadherin/β-actin, CD31/β-actin, α-SMA/β-actin, SM22α/β-actin, and FSP1/β-actin levels from each group (n = 5) was analyzed. (b) HMVECs were incubated with N-FGFR1 (1.5 μg/mL) in the presence or absence of apelin for 48 h, and the p-Smad3, VE-cadherin, CD31, vimentin, and α-SMA levels were analyzed by western blot. Densitometric analysis of the p-Smad3/Smad3, VE-cadherin/β-actin, CD31/β-actin, vimentin/β-actin, and α-SMA/β-actin levels from each group (n = 5) was analyzed. (c) HMVECs were transfected with or without apelin siRNA for 48 h in the presence or absence of TGFβ2, and the p-smad3, FGFR1, VE-cadherin, α-SMA, vimentin, and SM22α levels were analyzed by western blot. Densitometric analysis of the p-Smad3/Smad3, FGFR1/GADPH, VE-cadherin/GADPH, α-SMA/GADPH, vimentin/GADPH, and SM22α/GADPH levels from each group (n = 5) was analyzed. (d) HMVECs were incubated with or…

Figure 3. Apelin deficiency stimulates neighboring epithelial cells to transition into mesenchymal cells. (a) Design of the conditional medium experiment. HUVECs were transfected with scramble or apelin siRNA, and after 6 h, the medium was replaced with fresh medium and incubated for 48 h. After incubation of fresh medium for 48 h, the experimental media of HUVECs were harvested and transferred to cultured HK2 cells. (b) HK2 cells incubated with conditioned medium from apelin-knockdown HMVECs for 48 h, and the E-cadherin, vimentin, and α-SMA levels were analyzed by western blot. Densitometric analysis of the E-cadherin/GADPH, α-SMA/GADPH, and vimentin/GADPH levels from each group (n = 5) was analyzed. (c) ELISA analysis of TGFβ1 levels from conditioned medium.

Figure 4. CEBPA knockdown promotes TGFβ-mediated EndMT. (a) HMVECs were transfected with or without CEBPA siRNA for 48 h in the presence or absence of TGFβ2, and the VE-cadherin, α-SMA, vimentin, and SM22α levels were analyzed by western blot. Densitometric analysis of the VE-cadherin/GADPH, α-SMA/GADPH, vimentin/GADPH, and SM22α/GADPH levels from each group (n = 5) was analyzed. (b) HMVECs were transfected with CEBPA siRNA for 48 h in the presence or absence of TGFβ2 and apelin, and the VE-cadherin, α-SMA, vimentin, and SM22α levels were analyzed by western blot. Densitometric analysis of the VE-cadherin/GADPH, α-SMA/GADPH, vimentin/GADPH, and SM22α/GADPH levels from each group (n = 5) was analyzed. (c) HMVECs were treated with TGFβ2 for 15 min or 48 h with or without preincubation with apelin for 2 h, and the CEBPA levels were analyzed by western blot. Densitometric analysis of the CEBPA/GADPH level from each group (n = 5) was analyzed. (d) HMVECs were treated with N-FGFR1 for 48 h or 15 min in the presence or absence of apelin, and the CEBPA levels was analyzed by western blot. Densitometric analysis of the CEBPA/GADPH level from each group (n = 5) was analyzed. Immunofluorescence…

Figure 5. Apelin suppresses organ fibrosis and ameliorates diabetes-induced glomerular damage. Masson's trichrome staining (MTS) in the control (a), diabetic (b), and apelin-treated diabetic kidney (c). Scale bar: 40 μm. Quantification of relative fibrosis areas was calculated using the ImageJ software (d). MTS in the control (e), diabetic (f), and apelin-treated diabetic heart (g). Scale bar: 40 μm. Quantification of relative fibrosis areas was calculated using the ImageJ software (h). Sirius red staining in the control (i), diabetic (j), and apelin-treated diabetic kidney (k). Scale bar: 40 μm. Quantification of relative fibrosis areas was calculated using the ImageJ software (l). Periodic acid-Schiff (PAS) staining in the control (m), diabetic (n), and apelin-treated diabetic kidney (o). Scale bar: 60 μm. Quantification of the relative areas of glomeruli by the ImageJ software (p). For each section, images from six different fields of view at ×400 original magnification were obtained.

Figure 6. Apelin inhibits EndMT in diabetic kidneys. Immunofluorescence microscopy analysis of CD31/FSP1 (a) and CD31/α-SMA (b) in the kidney tissues from each group of mice. The scale bar is 40 μm in each panel. The CD31 and FSP1 double-positive cells, the CD31 and α-SMA double-positive cells, and the CD31 and vimentin double-positive cells were recognized as the cells undergoing EndMT. For each section, images from six different fields of view at ×400 original magnification were obtained. And the cells undergoing the EndMT were counted and quantified by the ImageJ software.