L-Endoglin overexpression increases renal fibrosis after unilateral ureteral obstruction

Abstract

Transforming growth factor-β (TGF-β) plays a pivotal role in renal fibrosis. Endoglin, a 180 KDa membrane glycoprotein, is a TGF-β co-receptor overexpressed in several models of chronic kidney disease, but its function in renal fibrosis remains uncertain. Two membrane isoforms generated by alternative splicing have been described, L-Endoglin (long) and S-Endoglin (short) that differ from each other in their cytoplasmic tails, being L-Endoglin the most abundant isoform. The aim of this study was to assess the effect of L-Endoglin overexpression in renal tubulo-interstitial fibrosis. For this purpose, a transgenic mouse which ubiquitously overexpresses human L-Endoglin (L-ENG+) was generated and unilateral ureteral obstruction (UUO) was performed in L-ENG+ mice and their wild type (WT) littermates. Obstructed kidneys from L-ENG+ mice showed higher amounts of type I collagen and fibronectin but similar levels of α-smooth muscle actin (α-SMA) than obstructed kidneys from WT mice. Smad1 and Smad3 phosphorylation were significantly higher in obstructed kidneys from L-ENG+ than in WT mice. Our results suggest that the higher increase of renal fibrosis observed in L-ENG+ mice is not due to a major abundance of myofibroblasts, as similar levels of α-SMA were observed in both L-ENG+ and WT mice, but to the higher collagen and fibronectin synthesis by these fibroblasts. Furthermore, in vivo L-Endoglin overexpression potentiates Smad1 and Smad3 pathways and this effect is associated with higher renal fibrosis development.

Figure 1. Transgenic mice expressing human L-endoglin.

(a) Schematic representation of the DNA construct used to generate transgenic mice expressing human L-endoglin. A 5.2-kb SalI/KpnI fragment, containing a CMV enhancer, an actin enhancer/promoter, a β-globin (BG) intron (fragment SalI/EcoRI), the endoglin leader sequence-LS, the hemagglutinin epitope-HA, the human L-endoglin cDNA (fragment EcoRI/EcoRI) and a β-globin polyadenylation site-polyA (fragment EcoRI/KpnI), was microinjected in the pronuclei of fertilized oocytes to generate L-ENG ⁺ mice. The transcription start site (TSS) and the translation initiation of the open reading frame (ORF) are indicated. (b) Expression of human L-endoglin protein in different tissues from L-ENG ⁺ mice. Protein extracts were analyzed by western blot using anti-endoglin and anti-HA antibodies. (c) Effects of human L-endoglin overexpression in systolic arterial blood pressure (SABP). N = 12 mice per group.

Figure 2. Effect of L-Endoglin overexpression on interstitial fibrosis after unilateral ureteral obstruction (I).

Representative images of haematoxylin-eosin (a) and Masson’s trichrome (b) staining in sham operated (SO), non-obstructed (NO) and obstructed (O) kidneys from WT and L-ENG ⁺ mice. In O kidneys tubular dilatation, inflammatory cell infiltration and interstitial fibrosis can be observed. Bar = 100 µm.

Figure 3. Effect of L-Endoglin overexpression on interstitial fibrosis after unilateral ureteral obstruction (II).

(a) Representative images of Sirius Red staining in sham operated (SO), non-obstructed (NO) and obstructed (O) kidneys from WT and L-ENG ⁺ mice. Bar = 100 µm. (b) Morphometric quantification (mean ± SEM) of area stained by Sirius Red; SO (n = 3); NO (n = 4); O (n = 4) in each group of mice. *P<0.05 vs. their respective SO kidneys. ^#P<0.05 vs. O kidneys from WT mice.

Figure 4. Effect of L-Endoglin overexpression on fibronectin and collagen I expression following ureteral obstruction.

Representative immunohistochemistry images for fibronectin (a) and collagen I (b) in sham operated (SO), non-obstructed (NO) and obstructed (O) kidneys from WT and L-ENG ⁺ mice. Bar = 100 µm. Western blot analysis of fibronectin (c) and collagen I (d) protein amount in SO, NO and O kidneys from WT and L-ENG⁺ mice. A representative western blot among 5–7 performed in each group is shown on top. Densitometry analysis is represented as the mean ± SEM of the 5–7 western performed per group. *P<0.01 vs. SO kidneys. ^#P<0.05 vs. O kidneys from WT mice.

Figure 5. Effect of L-Endoglin overexpression on myofibroblast abundance following ureteral obstruction.

Representative immunohistochemistry images for α-SMA (a) in sham operated (SO), non-obstructed (NO) and obstructed (O) kidneys from WT and L-ENG ⁺ mice. Bar = 100 µm. (b) Quantification of α-SMA-positive stained area in sham operated (SO), non-obstructed (NO) and obstructed (O) kidneys from WT and L-ENG ⁺ mice, expressed as square microns. (c) Western blot analysis of α-SMA protein amount in SO, NO and O kidneys from WT and L-ENG⁺ mice. A representative western blot among 5–7 performed in each group is shown on top. Densitometry analysis is represented as the mean ± SEM of the 5–7 western performed per group. *P<0.01 vs. SO kidneys. #P<0.05 vs. O kidneys from WT mice.

Figure 6. Effect of L-Endoglin overexpression on Smad1 and phospho-Smad1 expression following ureteral obstruction.

(a) Representative immunohistochemistry images for phospho-Smad1 in sham operated (SO), non-obstructed (NO) and obstructed (O) kidneys from WT and L-ENG ⁺ mice. Bar = 100 µm. (b) Histogram representing the number of phospho-Smad1-positive nuclei per field in SO, NO and O kidneys from WT and L-ENG ⁺ mice. Data is represented as mean ± SEM. Western blot analysis of phospho-Smad1 (c) and total Smad1 (d) protein amount in SO, NO and O kidneys from WT and L-ENG ⁺ mice. A representative western blot among 5–7 performed in each group is shown on top. Densitometry analysis is represented as the mean ± SEM of the 5–7 western performed per group. *P<0.01 vs. SO kidneys. #P<0.05 vs. O kidneys from WT mice.

Figure 7. Effect of L-Endoglin overexpression on Smad2/3 and phospho-Smad3 expression following ureteral obstruction.

(a) Representative immunohistochemistry images for phospho-Smad3 in sham operated (SO), non-obstructed (NO) and obstructed (O) kidneys from WT and L-ENG ⁺ mice. Bar = 100 µm. (b) Histogram representing number of nuclei positively stained for phospho-Smad3 per field in SO, NO and O kidneys from WT and L-ENG ⁺ mice. Data is represented as mean ± SEM. Western blot analysis of phospho-Smad3 (c) and total Smad2/3 (d) protein amount in SO, NO and O kidneys from WT and L-ENG ⁺ mice. A representative western blot among 5–7 performed in each group is shown on top. Densitometry analysis is of the 5–7 western performed per group. *P<0.01 vs. SO kidneys. #P<0.05 vs. O kidneys from WT mice.

Figure 8. Effect of L-Endoglin overexpression on ECM synthesis in renal fibroblasts.

(a) Immunofluorescence of human L-Endoglin and α-SMA in WT and L-ENG ⁺ renal fibroblasts. Magnification, 1,000X. Note the presence of human L-endoglin only in L-ENG⁺ renal fibroblasts. (b) Effect of L-Endoglin on ECM protein levels in renal fibroblasts. Representative western blot analysis of fibronectin, collagen I, human L-Endoglin and GAPDH proteins in WT and L-ENG ⁺ renal fibroblasts under basal conditions and after TGF-β1 (1 ng/mL) treatment for 24 hours. Densitometric analysis is represented as the mean ± SEM. *P<0.01 vs. WT fibroblasts in basal conditions. ^#P<0.05 for TGF-β1 treatments vs. basal conditions. ^σP<0.05 for TGF-β1 treatment in L-ENG ⁺ vs. TGF-β1 treatment in WT fibroblasts.

Figure 9. Effect of L-Endoglin overexpression on TGF-β1/Smad signalling in renal fibroblasts.

(a) Representative western blot analysis of phospho-Smad1, phospho-Smad2, phospho-Smad3, Smad2/3, Smad1 and GAPDH protein expression in WT and L-ENG ⁺ renal fibroblasts under basal conditions and after TGF-β1 (1 ng/mL) treatment for 30 minutes. (b) Densitometric analysis is represented as the mean ± SEM. *P<0.01 vs. WT fibroblasts in basal conditions. ^#P<0.05 for TGF-β1 treatment vs. basal conditions.