Angiopoietin-1 deficiency increases renal capillary rarefaction and tubulointerstitial fibrosis in mice

Presence of tubulointerstitial fibrosis is predictive of progressive decline in kidney function, independent of its underlying cause. Injury to the renal microvasculature is a major factor in the progression of fibrosis and identification of factors that regulate endothelium in fibrosis is desirable as they might be candidate targets for treatment of kidney diseases. The current study investigates how loss of Angipoietin-1 (Angpt1), a ligand for endothelial tyrosine-kinase receptor Tek (also called Tie2), affects tubulointerstitial fibrosis and renal microvasculature. Inducible Angpt1 knockout mice were subjected to unilateral ureteral obstruction (UUO) to induce fibrosis, and kidneys were collected at different time points up to 10 days after obstruction. Staining for aSMA showed that Angpt1 deficient kidneys had significantly more fibrosis compared to wildtype mice 3, 6, and 10 days after UUO. Further investigation 3 days after UUO showed a significant increase of Col1a1 and vimentin in Angpt1 deficient mice, as well as increased gene expression of Tgfb1, Col1a1, Fn1, and CD44. Kidney injury molecule 1 (Kim1/Havcr1) was significantly more increased in Angpt1 deficient mice 1 and 3 days after UUO, suggesting a more severe injury early in the fibrotic process in Angpt1 deficient mice. Staining for endomucin showed that capillary rarefaction was evident 3 days after UUO and Angpt1 deficient mice had significantly less capillaries 6 and 10 days after UUO compared to UUO kidneys in wildtype mice. RNA sequencing revealed downregulation of several markers for endothelial cells 3 days after UUO, and that Angpt1 deficient mice had a further downregulation of Emcn, Plvap, Pecam1, Erg, and Tek. Our results suggest that loss of Angpt1 is central in capillary rarefaction and fibrogenesis and propose that manipulations to maintain Angpt1 levels may slow down fibrosis progression

Angpt1 deficiency resulted in increased aSMA area at several time points.

<p>(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.</p

Angpt1 deficiency increased vascular rarefaction after UUO.

<p>(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.</p

Angpt1 deficiency worsens UUO outcome.

<p>A schematic diagram of fibrosis and capillary rarefaction after UUO in Angpt1 deficiency.</p

Angpt1 expression was downregulated in UUO kidneys.

<p>(A) Schematic diagram of the inducible whole body knock out system to generate Angpt1 knockout mice. (B) Gene expression analysis of <i>Angpt1</i> 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 <i>Hprt</i> 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.</p

Angpt1 deficiency increased markers for fibrosis and kidney injury after UUO.

<p>(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 (<i>Tgfb1</i>, <i>Col1a1</i>, <i>Fn1</i>, and <i>CD44</i>) 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 (<i>Kim1/Havcr1</i>) 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.</p

Angpt1 deficiency decreased expression of endothelial markers 3 days after UUO.

<p>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.</p