Curtailing endothelial TGF-β signaling is sufficient to reduce endothelial-mesenchymal transition and fibrosis in CKD

Abstract

Excessive TGF-β signaling in epithelial cells, pericytes, or fibroblasts has been implicated in CKD. This list has recently been joined by endothelial cells (ECs) undergoing mesenchymal transition. Although several studies focused on the effects of ablating epithelial or fibroblast TGF-β signaling on development of fibrosis, there is a lack of information on ablating TGF-β signaling in the endothelium because this ablation causes embryonic lethality. We generated endothelium-specific heterozygous TGF-β receptor knockout (TβRII(endo+/-)) mice to explore whether curtailed TGF-β signaling significantly modifies nephrosclerosis. These mice developed normally, but showed enhanced angiogenic potential compared with TβRII(endo+/+) mice under basal conditions. After induction of folic acid nephropathy or unilateral ureteral obstruction, TβRII(endo+/-) mice exhibited less tubulointerstitial fibrosis, enhanced preservation of renal microvasculature, improvement in renal blood flow, and less tissue hypoxia than TβRII(endo+/+) counterparts. In addition, partial deletion of TβRII in the endothelium reduced endothelial-to-mesenchymal transition (EndoMT). TGF-β-induced canonical Smad2 signaling was reduced in TβRII(+/-) ECs; however, activin receptor-like kinase 1 (ALK1)-mediated Smad1/5 phosphorylation in TβRII(+/-) ECs remained unaffected. Furthermore, the S-endoglin/L-endoglin mRNA expression ratio was significantly lower in TβRII(+/-) ECs compared with TβRII(+/+) ECs. These observations support the hypothesis that EndoMT contributes to renal fibrosis and curtailing endothelial TGF-β signals favors Smad1/5 proangiogenic programs and dictates increased angiogenic responses. Our data implicate endothelial TGF-β signaling and EndoMT in regulating angiogenic and fibrotic responses to injury.

Keywords: ALK1; Endoglin; Smad1/5; TGF-β receptor type II; microvasculature; renal fibrosis.

Figure 1.

Characterization of endothelial TβRII heterozygote mice. (A) Genotyping of pups from TGF-βRII^Flox/Flox and Tie2-Cre: TGF-βRII^Flox/WT matings. Tail PCR genotyping analysis. The top panel shows detection of floxed and WT fragments. The bottom panel shows detection of Cre-transgene. (B) Real-time PCR analysis for TβRII mRNA expression in kidney ECs isolated from TβRII^endo+/+ and TβRII^endo+/− mice (n=4). *P<0.05, TβRII^+/+ versus TβRII^+/−. (C) Western blot for Smad2 signaling in kidney ECs. WT, wild type; Cont, control.

Figure 2.

Functional characterization of TβRII heterozygote mice. (A) Images and quantification of ex vivo angiogenesis assays in three-dimensional matrigel using explant cultures of aortic ring segments at day 6 from TβRII^endo+/+ and TβRII^endo+/− mice (n=5). (B) Western blot for Smad1/5 signaling in kidney ECs isolated from TβRII^endo+/+ and TβRII^endo+/− mice. (C) Endoglin S/L mRNA expression ratios in kidney ECs. *P<0.05, TβRII^+/+ versus TβRII^+/− and TGF-β1 treated TβRII^+/+ versus TβRII^+/−. Cont, control.

Figure 3.

Fibrosis is reduced in TβRII^endo+/− mice with FA toxicity. (A) Representative images of Masson’s trichrome–stained kidney sections from 12-week-old TβRII^endo+/+ and TβRII^endo+/− mice treated with vehicle or FA 6 weeks after injection (n=5). (B) Quantification of fibrotic area (color quantification method). (C) Expression of collagen I and collagen III examined by quantitative real-time PCR. *P<0.05, treated TβRII^endo+/+ versus treated TβRII^endo+/−. Original magnification, ×4 and ×10.

Figure 4.

Fibrosis is reduced in TβRII^endo+/− UUO mice. (A) Representative images of Masson’s trichrome–stained UUO and contralateral kidney sections from TβRII^endo+/+ and TβRII^endo+/− mice (n=4). (B) Quantification of fibrotic area (color quantification method) *P<0.01, TβRII^endo+/+ UUO versus TβRII^endo+/− UUO. (C) Representative images for α-SMA staining in UUO and contralateral kidney sections from TβRII^endo+/+ and TβRII^endo+/− UUO mice. Original magnification, ×4 and ×10.

Figure 5.

Ex vivo angiogenesis assays in mice with FA nephrotoxicity. (A) Representative images of sprouting capillary cords in aortic explants at day 13. (B) Quantitative angiogenesis analysis in TβRII^endo+/+ or TβRII^endo+/− vehicle or FA-treated animals. *P<0.01 untreated versus treated TβRII^endo+/+ or untreated versus treated TβRII^endo+/−.

Figure 6.

Microvascular density (CD31) and patency (lectin) in mice with FA nephrotoxicity. (A) Representative images for CD31 (green) and lectin (red) staining (n=5). (B) Average lengths (in micrometers) of CD31- or lectin-positive peritubular capillaries per image. (C) Ratios of average lengths of lectin- to CD31-positive capillaries (percentage) per image. *P<0.01, treated TβRII^endo+/+ versus TβRII^endo+/− lectin-positive capillary length; **P<0.001, control versus FA-treated TβRII^endo+/+ mice; ^#P<0.01, treated TβRII^endo+/+ versus TβRII^endo+/−; N.S., P=NS for control versus FA-treated TβRII^endo+/− mice. Original magnification, ×40. Per the journal style, P values were rounded to two decimal places.

Figure 7.

Renal blood flow measurement and pimonidazole staining in kidneys of TβRII^endo+/+ and TβRII^endo+/− UUO mice. (A) Representative image scans from laser-Doppler flowmetry analysis of contralateral and UUO kidneys from TβRII^endo+/+ and TβRII^endo+/− mice (n=3). (B) Quantification of cortex and medullary renal blood flow in kidneys. *P<0.001, TβRII^endo+/+ and TβRII^endo+/− UUO mice. (C) Immunohistochemical staining for pimonidazole adducts in UUO kidneys of TβRII^endo+/+ and TβRII^endo+/− mice. Original magnification, ×10 and ×40.

Figure 8.

EndoMT in mice with FA nephrotoxicity. (A) Representative images for CD31 (green) and α-SMA (red) staining (n=5). Wild-type control kidneys shows costaining for CD31 and α-SMA only in vessels. These areas were excluded during quantitative analysis. (B) Quantitative analysis of CD31 plus α-SMA double-positive cells in TβRII^endo+/+ or TβRII^endo+/− vehicle- or FA-treated mice. *P<0.01; ^#P<0.01. Original magnification, ×60.

Figure 9.

EndoMT in UUO mice. (A) Representative images for CD31 (green) and α-SMA (red) staining (n=4). No primary antibody controls from CD31 staining and α-SMA staining are also shown. (B) Quantitative analysis of CD31 plus α-SMA double-positive cells in UUO kidneys of TβRII^endo+/+ and TβRII^endo+/− mice. *P<0.01. Original magnification, ×60.

Figure 10.

EndoMT in TβRII^endo+/− mice with FA nephrotoxicity. (A) Representative images for Cre (red) and α-SMA (green) staining (n=5). Arrows indicate α-SMA–stained areas with and without Cre costaining. (B) Quantitative analysis of Cre plus α-SMA double-positive cells in TβRII^endo+/− vehicle or FA-treated mice. *P<0.01. Original magnification, ×40.

Figure 11.

Modulation of EndoMT in TβRII^+/+ and TβRII^+/− kidney ECs. (A) Immunofluorescence images for CD31 or α-SMA in cultured TβRII^+/+ and TβRII^+/− kidney ECs treated with TGF-β1 (5 ng/ml) for 6 days (n=5). The nuclei are stained with 4′,6-diamidino-2-phenylindole. (B) Quantitative analysis of α-SMA–positive cells per 100 cells. *P<0.002. Original magnification, ×10 for α-SMA; ×40 for CD31 and α-SMA.