Fig 1.
Angpt1 expression was downregulated in UUO kidneys.
(A) Schematic diagram of the inducible whole body knock out system to generate Angpt1 knockout mice. (B) Gene expression analysis of Angpt1 at different time-points (3.5h, 1, 3, 6, and 10 days) after UUO showed a significant down-regulation that started 1 day after UUO in WT mice. Data are expressed as mean ± SEM. Gene expression data in this figure are normalized to Hprt and referenced to WT CL kidney 3.5h after UUO (n = 5–6 for 3.5 h, n = 5 for 1 day, n = 13 for 3 day, n = 3–4 for 6 day, and n = 3–5 for 10 day post UUO). ### p<0.001 compared to WT CL kidney 3.5h after UUO.
Fig 2.
Angpt1 deficiency resulted in increased aSMA area at several time points.
(A) UUO in WT and Angpt1KO mice induced tubulointerstitial fibrosis starting 1 day post UUO as seen from aSMA staining of renal cortex. (B) Quantifications of aSMA-positive areas showed a significant increase in fibrotic area in Angpt1KO mice 3 days after UUO and onward. A minimum of 10 images from renal cortex were used from each mouse (n = 3 for day 1, n = 13 for day 3, n = 3–6 for day 6, and n = 3–4 for day 10 post UUO). Scale bar, 50 μm. Data expressed as mean ± SEM. ### p<0.001 compared to WT CL3.5h, *p<0.05, **p<0.01, ***p0.001 compared to WT at the corresponding time point.
Fig 3.
Angpt1 deficiency increased markers for fibrosis and kidney injury after UUO.
(A) ELISA showed that Col1a1 protein was significantly increased in Angpt1KO mice 3 days after UUO compared to WT UUO kidneys (n = 13–17). (B) Western blotting showed an increase of Vimentin protein after UUO and a further increase in Angpt1KO 3 days after UUO. (C) Fold change densitometry for Vimentin blots corrected for protein loading (GAPDH) (n = 4). (D) Gene expression analysis showed upregulation of markers for fibrosis and mesenchymal transition (Tgfb1, Col1a1, Fn1, and CD44) after UUO and were significantly more increased in Angpt1KO mice 3 days after UUO compared to WT mice (n = 13–17). At the same time point, kidney injury molecule 1 (Kim1/Havcr1) was significantly further upregulated in Angpt1KO mice (n = 13–17). Data are expressed as mean ± SEM *p<0.05, **p<0.01, ***p<0.001 as indicated.
Fig 4.
Angpt1 deficiency increased vascular rarefaction after UUO.
(A) Vessel area in renal cortex was estimated from endomucin staining correlated to nuclei number at all time points after UUO. (B) Vascular rarefaction started 3 days after UUO and vessel density was significantly lower 3, 6, and 10 days after UUO in both WT and Angpt1KO mice. At 6 and 10 days of UUO, Angpt1KO mice showed a decreased capillary density compared to WT UUO mice. A minimum of 10 images from renal cortex were used from each mouse (n = 4–6 for 3.5h, n = 5–7 for day 1, n = 6–7 for day 3, n = 4–5 for day 6, and n = 4 for day 10 post UUO). Data shown as mean ± SEM. Scale bar, 50 μm. ## p<0.01, ### p<0.001 comparing to WT CL3.5h. *p<0.05 compared to WT at corresponding time point.
Fig 5.
Angpt1 deficiency decreased expression of endothelial markers 3 days after UUO.
RNA-seq data at baseline and 3 day UUO kidneys from WT and Angpt1KO mice. Data shown as mean ± SEM. FPKM–Fragments per kilobase million. n = 3 for all groups. *p<0.05, **p<0.01, ***p<0.001 compared to indicated group.
Fig 6.
Angpt1 deficiency did not alter endothelial proliferation and apoptosis.
(A) Proliferation was estimated from Ki67 staining (red) of renal cortex 3 days after UUO. Endomucin staining (green) and Hoechst (not shown) was used to identify endothelial cells and nuclei, respectively. Nuclei positive for Ki67, surrounded by endomucin was identified as an proliferating endothelial cell (arrow). As expected, proliferation was increased after UUO. Angpt1KO showed a significant increase in overall proliferation compared to WT after UUO; however, there was no difference in endothelial proliferation. (B) Apoptosis was estimated by TUNEL staining (red) of renal cortex 3 days after UUO. Endomucin staining (green) and Hoechst (not shown) was used to identify endothelial cells and nuclei, respectively. Nuclei positive for TUNEL, surrounded by endomucin was identified as an apoptotic endothelial cell (arrow). Scale bar, 50 μm. Data shown as mean ± SEM. n = 3–5 for all groups. *p<0.05, ***p<0.001 compared to indicated group.
Fig 7.
Angpt1 deficiency worsens UUO outcome.
A schematic diagram of fibrosis and capillary rarefaction after UUO in Angpt1 deficiency.