Inhibition of renal fibrosis with a human CXCL9-derived glycosaminoglycan-binding peptide

Abstract

Objectives: Renal fibrosis accompanies all chronic kidney disorders, ultimately leading to end-stage kidney disease and the need for dialysis or even renal replacement. As such, renal fibrosis poses a major threat to global health and the search for effective therapeutic strategies to prevent or treat fibrosis is highly needed. We evaluated the applicability of a highly positively charged human peptide derived from the COOH-terminal domain of the chemokine CXCL9, namely CXCL9(74-103), for therapeutic intervention. Because of its high density of net positive charges at physiological pH, CXCL9(74-103) competes with full-length chemokines for glycosaminoglycan (GAG) binding. Consequently, CXCL9(74-103) prevents recruitment of inflammatory leucocytes to sites of inflammation.

Methods: CXCL9(74-103) was chemically synthesised and tested in vitro for anti-fibrotic properties on human fibroblasts and in vivo in the unilateral ureteral obstruction (UUO) mouse model.

Results: CXCL9(74-103) significantly reduced the mRNA and/or protein expression of connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA) and collagen III by transforming growth factor (TGF)-β1-stimulated human fibroblasts. In addition, administration of CXCL9(74-103) inhibited fibroblast migration towards platelet-derived growth factor (PDGF), without affecting cell viability. In the UUO model, CXCL9(74-103) treatment significantly decreased renal α-SMA, vimentin, and fibronectin mRNA and protein expression. Compared with vehicle, CXCL9(74-103) attenuated mRNA expression of TGF-β1 and the inflammatory markers/mediators MMP-9, F4/80, CCL2, IL-6 and TNF-α. Finally, CXCL9(74-103) treatment resulted in reduced influx of leucocytes in the UUO model and preserved tubular morphology. The anti-fibrotic and anti-inflammatory effects of CXCL9(74-103) were mediated by competition with chemokines and growth factors for GAG binding.

Conclusions: Our findings provide a scientific rationale for targeting GAG-protein interactions in renal fibrotic disease.

Keywords: CXCL9; chemokine‐derived peptides; glycosaminoglycans; inflammation; renal fibrosis.

Figure 1

In vitro anti‐fibrotic effect of intact CXCL9. Human primary fibroblasts were seeded in 6‐well plates and incubated with medium alone (control), intact CXCL9 (1 to 100 nm), TGF‐β1 alone (5 ng mL⁻¹) or TGF‐β1 (5 ng mL⁻¹) in combination with CXCL9 (1 to 100 nm). Relative mRNA expression of the fibrotic markers (a) α‐SMA and (b) collagen III is shown and reported as mean ± SEM (n = 4; the four independent experiments were performed in duplicate, and each cDNA sample was analysed twice). *P < 0.05, **P < 0.01 versus TGF‐β1.

Figure 2

Overview of the synthesised CXCL9 isoforms and effect of CXCL9(74–103) on cell viability. (a) Amino acid sequence and theoretical and experimentally determined relative molecular masses (Mr) of CXCL9(1–78) and CXCL9(74–103). The GAG‐binding motifs are underlined. The residues present in the synthesised NH₂‐terminal chemokine domain and the COOH‐terminal GAG‐binding peptide are indicated in green. (b) The CXCL9‐derived peptides were chemically synthesised based on Fmoc chemistry, and their quality was evaluated by mass spectrometry. The intensities of the detected ions in function of their specific mass/charge (m/z) ratio are shown for folded CXCL9(1–78) and CXCL9(74–103). To calculate the Mr of the peptides from the charged ions, Bruker deconvolution software was used. The experimentally determined Mr of the peptides is shown as an insert on the upper right of the average unprocessed mass spectra. (c) Ball‐and‐stick model structure of full‐length CXCL9(1–103). The residues present in the synthesised NH₂‐terminal chemokine domain and COOH‐terminal GAG‐binding peptide are indicated in green. (d) Human primary fibroblasts were cultured in a 96‐well plate and incubated with CXCL9(74–103) alone or in the presence of TGF‐β1 (5 ng mL⁻¹). Cell viability was measured after 48 h using the alamarBlue assay. Error bars indicate the SEM for n = 4 experiments. In each experiment, all conditions were tested in duplicate.

Figure 3

Effect of CXCL9(74–103) on TGF‐β1 and PDGF. (a) Competition of recombinant human TGF‐β1 with CXCL9(1–78) or CXCL9(74–103) for binding to heparan sulphate‐coated plates was tested. Results are expressed as the percentage inhibition of TGF‐β1 binding to heparan sulphate and shown as mean ± SEM (n = 2). (b) The xCELLigence Real‐Time Cell Analyzer was used to measure fibroblast migration. PDGF‐β (30 ng mL⁻¹) or medium (control) was added to the wells of the lower chamber, and medium with or without CXCL9(74–103) (0.3 or 3 µm) was added with the fibroblasts in the upper chamber. In each experiment, all conditions were tested at least in duplicate. Data represent mean ± SEM (n = 3). *P < 0.05, ***P < 0.001 versus PDGF. (c) To determine the effect of CXCL9(74–103) on TGF‐β1‐induced CTGF production, fibroblasts were seeded in six‐well plates and either left untreated or stimulated with TGF‐β1 in the presence or absence of 100 nm of CXCL9(74–103). After 48 h, an enzyme‐linked immunosorbent sandwich assay (ELISA) was used to measure CTGF production (n = 5; conditions were tested in duplicate in each experiment). *P < 0.05.

Figure 4

In vitro anti‐fibrotic effect of CXCL9(74–103). Relative mRNA expression of (a) α‐SMA and (b) collagen III in human diploid fibroblasts incubated with medium alone (control), TGF‐β1 (5 ng mL⁻¹), CXCL9(74–103) (1–100 nm) or TGF‐β1 (5 ng mL⁻¹) in combination with CXCL9(74–103). Quantitative analysis of (c) α‐SMA and (d) collagen III protein expression as determined by immunocytochemistry. (e) Representative images of cells treated with medium, TGF‐β1 alone or in combination with CXCL9(74–103), stained with antibodies against collagen III or α‐SMA. Data are presented (a–d) as mean ± SEM (n = 4; conditions were tested in duplicate in each experiment, and each sample was analysed twice). *P < 0.05, **P < 0.01, ***P < 0.001 versus TGF‐β1.

Figure 5

In vivo biodistribution of TAMRA‐labelled CXCL9(74–103) in the kidney fibrosis model (UUO). Mice (n = 4 mice per group) were subjected to UUO of the left kidney, and subsequently, osmotic pumps were implanted subcutaneously. On day 7 or 10 after surgery, mice were euthanized. Blood and organs were collected, and the fluorescence intensity was measured. (a) CXCL9(74–103) biodistribution was evaluated as the amount of TAMRA‐labelled peptide fluorescence in UUO kidneys compared to healthy kidneys and kidneys of mice injected with vehicle. (b) Fluorescent peptide biodistribution in plasma and indicated organs (excitation, 535–20 nm and emission, 585–30 nm). Notice the difference in the range of the y‐axis in the two panels.

Figure 6

Inhibition of α‐SMA and vimentin expression by CXCL9(74–103). Osmotic pumps containing 0.4 or 4 mg mL⁻¹ of CXCL9(74–103) peptide (100 µL per pump) were implanted in mice shortly after UUO surgery. Relative gene expression of (a) α‐SMA and (b) vimentin in UUO kidneys at day 7 post‐surgery. Quantitative analysis at day 7 post‐surgery of (c) α‐SMA and (d) vimentin protein expression. (e) Photomicrographs (scale bars are 50 µm) of α‐SMA and vimentin immunohistochemical analysis (evaluated at 200× magnification). Data are presented (a–d) as mean ± SEM of 6 mice per group. Each cDNA sample was analysed in duplicate. Staining was quantified in 5 microscopic fields per kidney. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus vehicle.

Figure 7

Effect of CXCL9(74–103) on extracellular matrix deposition. Relative gene expression of (a) fibronectin and (b) collagen III in UUO kidneys at day 7 post‐surgery. Computerised quantitative analysis at day 7 post‐surgery of (c) fibronectin and (d) collagen III protein expression. (e) Photomicrographs (scale bars are 50 µm) of fibronectin and collagen III immunohistochemical analysis (evaluated at 200× magnification). Data are presented (a–d) as mean ± SEM of six mice per group. Each cDNA sample was analysed in duplicate. Staining was quantified in five microscopic fields per kidney. **P < 0.01 versus vehicle.

Figure 8

Effect of CXCL9(74–103) on MMP‐9 and TGF‐β1 expression and tubular dilation in UUO mice. The relative gene expression of (a) MMP‐9 and (b) TGF‐β1 was evaluated in UUO kidneys 7 days post‐surgery. (c) Computerised quantitative analysis of tubular dilation, which was quantified as the ratio of surface area tubular lumen/surface area tubular lumen + epithelium in UUO mice treated with vehicle (PBS), 0.4 mg mL⁻¹ or 4 mg mL⁻¹ of CXCL9(74–103) (7 days post‐surgery). Data are presented (a–c) as mean ± SEM of six mice per group. Each cDNA sample was analysed in duplicate. Five randomly selected microscopic fields per kidney were analysed *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle. (d) Representative photomicrographs of PAS‐stained kidney (scale bars are 50 µm) sections of UUO mice treated with vehicle (PBS), 0.4 mg mL⁻¹ or 4 mg mL⁻¹ of CXCL9(74–103) (7 days post‐surgery) (200×).

Figure 9

Effect of CXCL9(74–103) on inflammation in UUO mice. The relative gene expression levels (each cDNA sample was analysed in duplicate) of the inflammatory markers (a) TNF‐α, (b) IL‐6, (c) F4/80 and (d) CCL2 were evaluated in UUO kidneys treated with vehicle (PBS), 0.4 mg mL⁻¹ or 4 mg mL⁻¹ CXCL9(74–103) (100 µL per pump) (7 days post‐surgery). The presence of (e) CD45⁺, (f) F4/80⁺ and (g) CD11b⁺/Ly6G⁺ leucocytes was detected by flow cytometry. Data are presented as mean ± SEM of six mice per group. *P < 0.05, **P < 0.01, ****P < 0.001 versus vehicle.

Figure 10

Schematic diagram of the mechanisms that contribute to the anti‐fibrotic effect of CXCL9(74–103). (a) The intact CXCL9(1–103) protein is presented to its G protein‐coupled receptor (GPCR) CXCR3 via GAGs present either on the target cell itself or on a nearby cell. In contrast, CXCL9(1–78) misses the positively charged COOH‐terminus and can still activate CXCR3, but will be washed away quickly by the bloodstream as it cannot be anchored to the endothelial cell layer. The synthetic COOH‐terminal peptide of CXCL9, CXCL9(74–103), however, has a high affinity for GAGs, but cannot activate the GPCR CXCR3. It can displace intact chemokine from the GAGs, forcing detachment of the intact chemokine from the cell membrane, thereby hampering its inflammatory activity [anti‐inflammatory effect of CXCL9(74–103) in b]. (b) Schematic representation of the mechanisms behind the anti‐fibrotic CXCL9(74–103). The anti‐fibrotic effect of CXCL9(74–103) is dual: first by decreasing the influx of leucocytes that locally release pro‐fibrotic factors; and second by competition for GAG binding with pro‐fibrotic factors, for example, TGF‐β. By preventing GAG binding of the pro‐fibrotic factor, the latter can less efficiently activate its cognate receptor.