Inhibition of Axl attenuates acute kidney injury by alleviating inflammation via SOCS3 downregulation in tubular epithelial cells

Abstract

Background: In acute kidney injury (AKI), inflammatory crosstalk between tubular epithelial cells (TECs) and immune cells drives disease progression. Although the Axl-SOCS3 axis in myeloid cells typically suppresses inflammation, TEC-specific SOCS3 deletion paradoxically protects against AKI, suggesting a cell type-specific pro-inflammatory role.

Methods: We induced AKI via bilateral ischemia/reperfusion (IRI) in mice. The Axl-specific pharmacological inhibitor R428 was administered via subcutaneous injection immediately post-IRI, with plasma and kidney samples collected 24 h later. To assess the effects of SOCS3 in TECs, small interfering RNA was used to silence SOCS3 in cisplatin injured HK2 cells. Axl/SOCS3 expression levels were assessed in human AKI biopsies.

Results: In AKI patients and IRI mice, Axl was upregulated in interstitial immune cells, while SOCS3 increased in TECs. Axl inhibition by R428 attenuated renal injury, reducing inflammatory infiltration, NF-κB p65 phosphorylation, and TEC SOCS3 expression. Notably, SOCS3 knockdown in TECs suppressed NF-κB activation and IL-1β/IL-6 production, implicating Axl-SOCS3 as a pro-inflammatory amplifier in AKI.

Conclusion: The Axl-SOCS3 axis exacerbates AKI by reinforcing NF-κB-driven inflammation in TECs, creating a vicious cycle between immune cells and TECs. Targeting this cross-cellular pro-inflammatory pathway offers a promising therapeutic strategy for AKI.

Keywords: Acute kidney injury; Axl; Inflammation; Suppressor of cytokine signaling 3; Tubular epithelial cells.

Fig. 1

Expression patterns of Axl and SOCS3 in kidneys following IRI-induced AKI. (A-B) Representative immunohistochemical staining of Axl (A) and SOCS3 (B) in renal tissues. Arrows indicate positively stained cells/tubules. Scale bars: 200 μm (A), 100 μm (B). (C) Quantitative analysis of Axl-positive cells/100× (n = 4 mice/group). (D) Quantitative analysis of SOCS3-positive tubules/200× (n = 4). Data are shown as mean ± SEM. ** P < 0.01

Fig. 2

Effects of Axl inhibitor on mice with IRI-induced AKI. (A) PAS-stained kidney sections showing tubular injury. Scale bar, 200 μm. (B) Immunofluorescence co-staining of Axl (green) and E-cadherin (red). The arrows indicate the positive staining of Axl. Scale bar, 100 μm. (C) Semiquantitative renal pathological scores (n = 5). (D-E) Plasma Cr (D) and BUN (E) levels (n = 4). (F) Quantification of Axl-positive cells/200× (n = 4). (G) Representative Western blot of Axl expression. (H) Densitometric analysis of Axl/β-actin ratio (n = 6). Data are shown as mean ± SEM or median ± interquartile range (IQR). *P < 0.05, **p < 0.01, and P****Р<0.0001

Fig. 3

Effects of Axl inhibitor on renal inflammation in mice with IRI-induced AKI. (A) Immunofluorescence co-staining of F4/80 (red) and Axl (green). Arrows indicate F4/80-positive macrophages. (B) Ly6G immunohistochemistry showing neutrophil infiltration. (C-D) Quantification of F4/80- (C) and Ly6G- (D) positive cells/400× (n = 4). (E-F) Western blot analysis (E) and quantification (F) of Arg-1 expression. (G-H) Western blot analysis (G) and quantification (G) of p-p65 expression (n = 4). Scale bar, 100 μm. Data are shown as mean ± SEM. **P < 0.01

Fig. 4

Reduction of SOCS3 expression in TECs of IRI-induced AKI mice by Axl inhibition. (A) Immunofluorescence co-staining of SOCS3 (green) and Ecadherin (red). The arrows indicate the positive staining of SOCS3. Scale bar, 100 μm. (B) Quantification of SOCS3-positive cells/400× (n = 4). (C-D) Western blot analysis (C) and quantification (D) of SOCS3 expression (n = 4). Data are shown as mean ± SEM or median ± interquartile range (IQR). *P < 0.05

Fig. 5

Impact of SOCS3 knockdown in cisplatin-treated HK2 cells. (A) Cell viability assessed by CCK-8 assay. (B) Representative Ki67 immunofluorescence (green). (C) Quantification of Ki67-positive cells/400× (n = 4). Scale bar, 100 μm. (D) The relative mRNA expression of SOCS3, IL-1β, and IL-6 determined by qRT-PCR (n = 5). (E-F) Western blot analysis (E) and quantification (F) of p-p65 levels (n = 6). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01

Fig. 6

Expressions of Axl and SOCS3 in kidneys of patients with AKI. (A) Representative Axl immunohistochemistry (400×). (B) SOCS3 immunofluorescence (400×). The arrows indicate the positive staining of Axl and SOCS3. Scar bar, 100 μm. (C-D) Quantification of Axl-positive cells (C) and SOCS3-positive tubules/400× (n = 1–3 patients). Data are shown as mean ± SEM. *P < 0.05