The chemokine receptor CXCR6 contributes to recruitment of bone marrow-derived fibroblast precursors in renal fibrosis

Abstract

Bone marrow-derived fibroblasts in circulation are of hematopoietic origin, and they proliferate, differentiate into myofibroblasts, and express the chemokine receptor CXCR6. As chemokines mediate the trafficking of circulating cells to sites of injury, we studied the role of CXCR6 in mouse models of renal injury. Significantly, the kidney of CXCR6 knockout mice accumulated fewer bone marrow-derived fibroblasts in response to injury, expressed less profibrotic chemokines and cytokines, displayed fewer myofibroblasts, and expressed less α-smooth muscle actin in the obstructed kidneys compared with wild-type (WT) mice. CXCR6 deficiency inhibited total collagen deposition and suppressed the expression of collagen I and fibronectin in the obstructed kidneys. Furthermore, WT mice engrafted with CXCR6(-/-) bone marrow cells displayed fewer bone marrow-derived fibroblasts in the kidneys with obstructive injury and showed less severe renal fibrosis compared with WT mice engrafted with CXCR6(+/+) bone marrow cells. Transplant of WT bone marrow into CXCR6(-/-) recipients restored recruitment of myeloid fibroblasts and susceptibility to fibrosis. Hematopoietic fibroblasts migrate into injured kidney and proliferate and differentiate into myofibroblasts. Thus, CXCR6, together with other chemokines and their receptors, may have important roles in the recruitment of bone marrow-derived fibroblast precursors into the kidney and contribute to the pathogenesis of renal fibrosis.

Figure 1. Characterization of bone marrow-derived fibroblasts

A. Representative photomicrographs of kidney sections stained for CD45 (red) and counter stained with DAPI (blue). B. Representative photomicrographs of kidney sections stained for CD11b (red) and counter stained with DAPI (blue). C. Representative photomicrographs of kidney sections stained for Ki-67 (red) and counter stained with DAPI (blue). D. Representative photomicrographs of kidney sections stained for α-SMA (red) and counter stained with DAPI (blue). E. Representative photomicrographs of kidney sections stained for CXCR6 (red) and counter stained with DAPI (blue). Scale bar: 25 µm.

Figure 2. CXCR6 deficiency suppresses the accumulation of bone marrow-derived fibroblasts in the kidney in response to obstructive injury

A. Representative photomicrographs of kidney sections from WT and CXCR6-KO mice 7 days after UUO stained for CD45 (red), procollagen I (green), and DAPI (blue). Scale bar: 25 µm. B. Quantitative analysis of CD45⁺ and procollagen I⁺ cells in kidneys of WT and CXCR6-KO mice 7 days after UUO. ** P < 0.01 versus WT controls, ⁺ P < 0.05 versus KO UUO, and ^# P < 0.05 versus WT UUO. n=6 per group. C. Representative photomicrographs of kidney sections from WT and CXCR6-KO mice 7 days after UUO stained for CD45 (red), PDGFR-β (green), and DAPI (blue). Scale bar: 25 µm. D. Quantitative analysis of CD45⁺ and PDGFR-β⁺ cells in kidneys of WT and CXCR6-KO mice 7 days after UUO. ** P < 0.01 versus WT controls, ⁺ P < 0.05 versus KO UUO, and ^# P < 0.05 versus WT UUO. n=6 per group. E. Representative cytometric diagrams showing the effect of CXCR6 deficiency on the accumulation of CD45⁺ and collagen I⁺ cells in the kidneys. F. Quantitative analysis of CD45⁺ and collagen I⁺ cells in the kidneys. ** P < 0.01 versus WT controls, ⁺⁺ P < 0.01 versus KO UUO, and ^## P < 0.01 versus WT UUO. n=3 per group.

Figure 3. CXCR6 deficiency attenuates profibrotic chemokine and cytokine expression

A. Quantitative analysis of CXCL16 mRNA expression in the kidney. B. Quantitative analysis of MCP-1 mRNA expression in the kidney. C. Quantitative analysis of TNF-α mRNA expression in the kidney. D. Quantitative analysis of TGF-β1 mRNA expression in the kidney. ** P <0.01 versus WT controls, ⁺ P <0.05 versus KO UUO, ⁺⁺ P <0.01 versus KO UUO, and ^# P <0.05 versus WT UUO. n=6 per group.

Figure 4. CXCR6 deficiency inhibits myofibroblast activation and α-SMA expression in obstructive nephropathy

A. Representative photomicrographs of α-SMA immunofluorescent staining in kidneys of WT and CXCR6-KO mice 2 weeks after UUO. Scale bar: 25 µm. B. Quantitative analysis of α-SMA protein expression in kidneys of WT and CXCR6-KO mice 2 weeks after UUO. ** P <0.01 versus WT controls, ⁺ P <0.05 versus KO UUO, and ^# P <0.05 versus WT UUO. n=6 per group. C. Representative Western blots show the levels of α-SMA protein expression in kidneys of WT and CXCR6-KO mice. D. Quantitative analysis of α-SMA protein expression in kidneys of WT and CXCR6-KO mice. ** P <0.01 versus WT controls, ⁺ P <0.05 versus KO UUO, and ^# P <0.05 versus WT UUO. n=6 per group.

Figure 5. CXCR6 deficiency suppresses renal fibrosis and collagen deposition in the kidney

A. Representative photomicrographs of kidney sections from WT and CXCR6-KO mice 2 weeks after UUO stained with Sirius red. Scale bar: 50 µm. B. Quantitative analysis of interstitial collagen content in kidneys. ** P < 0.01 versus WT controls, ⁺ P < 0.05 versus KO UUO, and ^# P < 0.05 versus WT UUO. n= 6 per group.

Figure 6. CXCR6 deficiency inhibits fibronectin and collagen I expression in the kidney

A. Representative photomicrographs of fibronectin immunofluorescence staining of kidneys 2 weeks after UUO. Scale bar: 25 µm. B. Quantitative analysis of fibronectin positive area of kidneys 2 weeks after UUO. ** P <0.01 versus WT controls, ⁺ P <0.05 versus KO UUO, and ^# P <0.05 versus WT UUO. n=6 per group. C. Representative photomicrographs of collagen I immunofluorescence staining of kidneys 2 weeks after UUO. Scale bar: 25 µm. D. Quantitative analysis of collagen I positive area of kidneys 2 weeks after UUO. ** P <0.01 versus WT controls, ⁺ P <0.05 versus KO UUO, and ^# P <0.05 versus WT UUO. n=6 per group. E. Representative Western blots show the protein levels of fibronectin and collagen I of kidneys 2 weeks after UUO. F. Quantitative analysis of fibronectin and collagen I protein expression of kidneys 2 weeks after UUO. * P < 0.05 versus WT controls, ⁺ P < 0.05 versus s KO UUO, and ^# P < 0.05 versus WT UUO. n=6 per group.

Figure 7. CXCR6 deficiency in bone marrow-derived cells inhibits myeloid fibroblast accumulation and collagen deposition in the kidney

A. Representative photomicrographs of kidney sections from WT or KO mice transplanted with CXCR6^+/+ or CXCR6^−/− bone marrow cells 1 week after UUO stained for CD45 (red), PDGFR-β (green), and DAPI (blue). Scale bar: 25 µm. B. Quantitative analysis of CD45⁺ and PDGFR-β⁺ fibroblasts in the kidneys. **p<0.01 versus WT → WT CON; ^#p<0.05 versus WT → WT UUO; ⁺p<0.05 versus KO → WT UUO; ^{^}p<0.05 versus KO→KO CON; ^$ p<0.05 versus KO→KO UUO; ^&& p<0.01 versus WT→KO UUO. n=5–6 per group. C. Representative photomicrographs of kidney sections stained with Sirius red. Scale bar: 50 µm. D. Quantitative analysis of renal interstitial collagen content in different groups as indicated. **p<0.01 versus WT → WT CON; ^#p<0.05 versus WT → WT UUO; ⁺p<0.05 versus KO → WT UUO; ^{^}p<0.05 versus KO→KO CON; $ p<0.05 versus KO→KO UUO; ^&& p<0.01 versus WT→KO UUO. n=5–6 per group.

Figure 8. CXCR6 deficiency suppresses bone marrow-derived fibroblast accumulation and collagen deposition in the kidneys in response to IRI

A. CXCL16 is induced in the kidney after IRI. * indicate p<0.05 versus sham controls. n=5. B. Representative photomicrographs of kidney sections from WT and CXCR6-KO mice 7 days after IRI stained for CD45 (red), PDGFR-β (green), and DAPI (blue). Scale bar: 25 µm. C. Quantitative analysis of CD45⁺ and PDGFR-β⁺ cells in the kidneys 7 days after IRI. ** P < 0.01 versus WT controls, ⁺ P < 0.05 versus KO IRI, and ^# P < 0.05 versus WT IRI. n=6 per group. D. Representative photomicrographs of kidney sections from WT and CXCR6-KO mice 2 weeks after IRI stained with Sirius red. Scale bar: 50 µm. E. Quantitative analysis of interstitial collagen content in the kidneys. ** P < 0.01 versus WT controls, ⁺ P < 0.05 versus KO IRI, and ^# P < 0.05 versus WT IRI. n= 6 per group.