Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition

Abstract

Fibroblasts are key mediators of fibrosis in the kidney and other organs, but their origin during fibrosis is still not completely clear. Activated fibroblasts likely arise from resident quiescent fibroblasts via epithelial-to-mesenchymal transition and from the bone marrow. Here, we demonstrate that endothelial cells also contribute to the emergence of fibroblasts during kidney fibrosis via the process of endothelial-to-mesenchymal transition (EndMT). We examined the contribution of EndMT to renal fibrosis in three mouse models of chronic kidney disease: (1) Unilateral ureteral obstructive nephropathy, (2) streptozotocin-induced diabetic nephropathy, and (3) a model of Alport renal disease. Approximately 30 to 50% of fibroblasts coexpressed the endothelial marker CD31 and markers of fibroblasts and myofibroblasts such as fibroblast specific protein-1 and alpha-smooth muscle actin. Endothelial lineage tracing using Tie2-Cre;R26R-stop-EYFP transgenic mice further confirmed the presence of EndMT-derived fibroblasts. Collectively, our results demonstrate that EndMT contributes to the accumulation of activated fibroblasts and myofibroblasts in kidney fibrosis and suggest that targeting EndMT might have therapeutic potential.

Figure 1.

EndMT in the mouse model of UUO. (A) Kidneys were analyzed after 1 wk of ureter ligation. The pictures display representative photomicrographs of MTS-stained kidney section at an original magnification of ×10 (left) and ×60 (middle). Fibrosis was digitally quantified and is shown as the percentage of MTS-stained blue area in both normal and UUO kidneys (right). (B, left) FSP1 and CD31 double labeling. Frozen kidney sections were double stained with antibodies to FSP1 (green) and CD31 (red). DAPI was used as a nuclear stain (blue). The panels display representative images that were obtained from UUO kidneys (top) and normal kidneys (bottom) using a confocal microscope. The arrows in the merged panel point to CD31⁺FSP1⁺ cells. (B, right) α-SMA and CD31 double labeling. The pictures display representative pictures of kidneys that were labeled with antibodies to α-SMA (green) and CD31 (red). DAPI was used for labeling of nuclei (blue). Yellow color in the merged panel indicates coexpression of α-SMA and CD31. The photomicrographs were obtained by confocal microscopy. (C) Quantification of fibroblasts. The bar graphs summarize average numbers of FSP1⁺ fibroblasts, CD31⁺FSP1⁺ cells, α-SMA⁺ fibroblasts, and CD31⁺α-SMA⁺ cells per visual field in both normal and obstructed kidneys at a magnification of ×63 (n = 3 mice per group, 10 high-power fields [hpf] per mouse, 30 hpf total). (D) Lineage tracing of endothelial cells. UUO was performed in Tie2-Cre;R26R-stop-EYFP double-transgenic mice. In this reporter strain, all cells of endothelial origin are tagged by YFP (shown in green). After UUO, immunostaining was performed for FSP1 (left, red) or α-SMA (middle, red). White arrows indicate fibroblasts of endothelial origin (yellow). For comparison, a representative YFP image from a normal Tie2-Cre;R26R-stop-EYFP kidney is also included (right). Magnification, ×63 in B and D.

Figure 2.

EndMT in the mouse model of STZ-induced diabetic nephropathy. (A) CD1 mice were made diabetic by a single injection of STZ. Kidneys were analyzed after 6 mo. The pictures display representative photomicrographs of MTS-stained kidney sections at an original magnification of ×10 (left) and ×60 (middle). Fibrosis was digitally quantified and is shown as the percentage of MTS-stained blue area in both normal and STZ kidneys (right). (B, left) FSP1 and CD31 double labeling. Frozen kidney sections were double stained with antibodies to FSP1 (green) and CD31 (red). DAPI was used to label the nuclei (blue). The panels display representative images that were obtained from STZ kidneys (top) and normal kidneys (bottom) using a confocal microscope. The arrows in the merged panel point to CD31⁺FSP1⁺ cells. (B, right) α-SMA and CD31 double labeling. The panels display representative images of kidneys that were labeled with antibodies to α-SMA (green) and CD31 (red). Yellow color in the merged panel indicates coexpression of α-SMA and CD31. (C) Quantification of fibroblasts. The bar graphs summarize average number of FSP1⁺ fibroblasts, CD31⁺FSP1⁺ cells, α-SMA⁺ fibroblasts, and CD31⁺α-SMA⁺ cells per visual field in both normal and diabetic kidneys at a magnification of ×63 (n = 3 mice per group, 10 hpf per mouse, 30 hpf total). Magnification, ×63 in B.

Figure 3.

EndMT in COL4A3-deficient mice. (A) Kidneys of COL4A3 KO mice, a mouse model for Alport syndrome, were analyzed at the age of 22 wk. The pictures display representative photomicrographs of MTS-stained kidney sections at an original magnification of ×10 (left) and ×60 (middle). Fibrosis was digitally quantified and is shown as the percentage of MTS-stained blue area in both normal and COL4A3 KO kidneys (right). (B, left) FSP1 and CD31 double labeling. Kidney sections were double stained with antibodies to FSP1 (green) and CD31 (red). DAPI was used as a nuclear stain (blue). The panels display representative images that were obtained from COL4A3 KO kidneys (top) and normal kidneys (bottom). The arrows in the merged panel point to CD31⁺FSP1⁺ cells. (B, right) α-SMA and CD31 double labeling. The pictures display representative photomicrographs of kidneys that were labeled with antibodies to α-SMA (green) and CD31 (red). DAPI was used for labeling of nuclei (blue). Yellow color in the merged panel indicates coexpression of α-SMA and CD31. (C) Quantification of fibroblasts. The bar graphs summarize average number of FSP1⁺ fibroblasts, CD31⁺FSP1⁺ cells, α-SMA⁺ fibroblasts, and CD31⁺α-SMA⁺ cells per visual field in both normal and Alport kidneys at a magnification of ×63 (n = 3 mice per group, 10 hpf per mouse, 30 hpf total). Magnification, ×63 in B.