Mechanism of growth arrest-specific protein 6/Axl receptor tyrosine kinase signaling pathway in promoting macrophage polarization and contributing to interstitial fibrosis in IgA nephropathy
Abstract
ObjectiveTo investigate the regulatory role of the growth arrest-specific protein 6 （Gas6）/Axl receptor tyrosine kinase （Axl） signaling pathway in macrophage polarization， and to further elucidate its role and underlying mechanisms during the progression of interstitial fibrosis in immunoglobulin A nephropathy （IgAN）.MethodsM0-type macrophages were transfected with Axl-targeting small interfering RNA （siRNA） and negative control （NC） siRNA， respectively. Quantitative real-time polymerase chain reaction （qPCR） and Western blotting were used to detect the expression of Axl and M2-type macrophage-specific factors including cluster of differentiation 206 （CD206）， arginase-1 （Arg-1）， transforming growth factor-β （TGF-β）， and interleukin-10 （IL-10）. Flow cytometry was used to detect the proportions of M1- and M2-type macrophages. A co-culture system of human kidney-2 （HK-2） cells and M0-type macrophages was established and divided into a control group， a TGF-β1 group， a TGF-β1＋siRNA-NC group， and a TGF-β1＋siRNA-Axl group. Enzyme-linked immunosorbent assay （ELISA） was used to measure collagen （COL）1 and COL3 levels in culture supernatants. Immunofluorescence staining was used to observe α-smooth muscle actin （α-SMA） expression in HK-2 cells. Western blotting was used to detect α-SMA， fibronectin （FN）， Gas6， phosphorylated protein kinase B （p-Akt）， and protein kinase B （Akt） protein expression， and the p-Akt/Akt ratio was calculated. For the animal experiment， 10 wild type （WT） mice and 10 Axl gene knockout （Axl-/-） mice were randomly assigned （5 mice per group） into four groups： WT control group， WT IgAN group， Axl-/- control group， and Axl-/- IgAN group. Hematoxylin and eosin （HE） and Masson staining were used to assess renal pathological injury and fibrosis. Western blotting was used to detect α-SMA and FN protein expression. Immunofluorescence double staining was used to evaluate M2-type macrophage infiltration.ResultsIn vitro experiments showed that compared with the control group and siRNA-NC group， the siRNA-Axl group had lower relative Axl mRNA expression （0.28±0.02 vs 1.00±0.06， 1.02±0.08） and lower relative Axl protein expression （0.07±0.01 vs 0.35±0.03， 0.32±0.04）. The proportion of M1-type macrophages increased （［73.50±7.65］% vs ［50.00±6.02］%， ［51.00±4.98］%）， while the proportion of M2-type macrophages decreased （［25.50±2.71］% vs ［49.70±5.13］%， ［49.10±5.01］%）. The mRNA and protein expression of CD206， Arg-1， TGF-β， and IL-10 decreased （all P<0.05）. After co-culture of HK-2 cells with macrophages， compared with the control group， the TGF-β1 group showed increased COL1 （［2.07±0.21］ μg/L vs ［0.54±0.04］ μg/L） and COL3 （［1.53±0.14］ μg/L vs ［0.42±0.04］ μg/L）， enhanced α-SMA immunofluorescence expression， and increased relative expression of α-SMA （0.72±0.06 vs 0.11±0.01）， FN （0.70±0.08 vs 0.09±0.01）， and Gas6 （0.68±0.07 vs 0.06±0.01）， as well as an increased p-Akt/Akt ratio （0.34±0.03 vs 0.12±0.01）. Compared with the TGF-β1＋siRNA-NC group， the TGF-β1＋siRNA-Axl group showed decreased COL1 （［1.01±0.08］ μg/L vs ［2.06±0.21］ μg/L） and COL3 （［0.73±0.06］ μg/L vs ［1.53±0.14］ μg/L）， weakened α-SMA immunofluorescence expression， and decreased relative protein expression of α-SMA （0.15±0.02 vs 0.74±0.08）， FN （0.17±0.02 vs 0.73±0.08）， and Gas6 （0.15±0.01 vs 0.66±0.07）， as well as a decreased p-Akt/Akt ratio （0.21±0.02 vs 0.32±0.03） （all P<0.05）. In animal experiments， compared with the WT control group， the WT IgAN group showed increased renal interstitial fibrosis area （［12.46±1.34］% vs ［1.23±0.14］%）， increased relative protein expression of α-SMA （1.27±0.13 vs 0.43±0.04） and FN （0.93±0.10 vs 0.14±0.01）， and increased M2-type macrophage proportion （［21.50±2.24］% vs ［6.34±0.65］%）. Compared with the Axl-/- control group， the Axl-/- IgAN group showed increased renal interstitial fibrosis area （［7.54±0.80］% vs ［1.01±0.11］%）， increased relative protein expression of α-SMA （0.63±0.06 vs 0.42±0.04） and FN （0.47±0.05 vs 0.13±0.01）， and increased M2-type macrophage proportion （［17.83±1.84］% vs ［6.81±0.67］%）. Compared with the WT IgAN group， the Axl-/- IgAN group showed decreased renal interstitial fibrosis area （［7.54±0.80］% vs ［12.46±1.34］%）， decreased relative protein expression of α-SMA （0.63±0.06 vs 1.27±0.13） and FN （0.47±0.05 vs 0.93±0.10）， and decreased M2-type macrophage proportion （［17.83±1.84］% vs ［21.50±2.24］%） （P<0.05）.ConclusionThe Gas6/Axl signaling pathway participates in the progression of interstitial fibrosis in IgAN by regulating macrophage polarization. Axl silencing or knockout can alleviate renal interstitial fibrosis injury， providing a potential target for IgAN treatment.
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BibTeX
@article{shufen2026mechanism,
title = {{Mechanism of growth arrest-specific protein 6/Axl receptor tyrosine kinase signaling pathway in promoting macrophage polarization and contributing to interstitial fibrosis in IgA nephropathy}},
author = {Yang Shu-fen and Zhu Guo-qiang and Meng Zhao-xi and Jiang Hong and Yalikun Dilina and Bai Lu and Ha Xiao-wen},
journal = {Linchuang shenzangbing zazhi},
year = {2026},
month = oct,
url = {http://www.lcszb.com/thesisDetails#10.3969/j.issn.1671-2390.2026.10.009},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Mechanism of growth arrest-specific protein 6/Axl receptor tyrosine kinase signaling pathway in promoting macrophage polarization and contributing to interstitial fibrosis in IgA nephropathy
AU - Yang Shu-fen
AU - Zhu Guo-qiang
AU - Meng Zhao-xi
AU - Jiang Hong
AU - Yalikun Dilina
AU - Bai Lu
AU - Ha Xiao-wen
PY - 2026
DA - 2026/10/24
JO - Linchuang shenzangbing zazhi
UR - http://www.lcszb.com/thesisDetails#10.3969/j.issn.1671-2390.2026.10.009
AB - ObjectiveTo investigate the regulatory role of the growth arrest-specific protein 6 （Gas6）/Axl receptor tyrosine kinase （Axl） signaling pathway in macrophage polarization， and to further elucidate its role and underlying mechanisms during the progression of interstitial fibrosis in immunoglobulin A nephropathy （IgAN）.MethodsM0-type macrophages were transfected with Axl-targeting small interfering RNA （siRNA） and negative control （NC） siRNA， respectively. Quantitative real-time polymerase chain reaction （qPCR） and Western blotting were used to detect the expression of Axl and M2-type macrophage-specific factors including cluster of differentiation 206 （CD206）， arginase-1 （Arg-1）， transforming growth factor-β （TGF-β）， and interleukin-10 （IL-10）. Flow cytometry was used to detect the proportions of M1- and M2-type macrophages. A co-culture system of human kidney-2 （HK-2） cells and M0-type macrophages was established and divided into a control group， a TGF-β1 group， a TGF-β1＋siRNA-NC group， and a TGF-β1＋siRNA-Axl group. Enzyme-linked immunosorbent assay （ELISA） was used to measure collagen （COL）1 and COL3 levels in culture supernatants. Immunofluorescence staining was used to observe α-smooth muscle actin （α-SMA） expression in HK-2 cells. Western blotting was used to detect α-SMA， fibronectin （FN）， Gas6， phosphorylated protein kinase B （p-Akt）， and protein kinase B （Akt） protein expression， and the p-Akt/Akt ratio was calculated. For the animal experiment， 10 wild type （WT） mice and 10 Axl gene knockout （Axl-/-） mice were randomly assigned （5 mice per group） into four groups： WT control group， WT IgAN group， Axl-/- control group， and Axl-/- IgAN group. Hematoxylin and eosin （HE） and Masson staining were used to assess renal pathological injury and fibrosis. Western blotting was used to detect α-SMA and FN protein expression. Immunofluorescence double staining was used to evaluate M2-type macrophage infiltration.ResultsIn vitro experiments showed that compared with the control group and siRNA-NC group， the siRNA-Axl group had lower relative Axl mRNA expression （0.28±0.02 vs 1.00±0.06， 1.02±0.08） and lower relative Axl protein expression （0.07±0.01 vs 0.35±0.03， 0.32±0.04）. The proportion of M1-type macrophages increased （［73.50±7.65］% vs ［50.00±6.02］%， ［51.00±4.98］%）， while the proportion of M2-type macrophages decreased （［25.50±2.71］% vs ［49.70±5.13］%， ［49.10±5.01］%）. The mRNA and protein expression of CD206， Arg-1， TGF-β， and IL-10 decreased （all P<0.05）. After co-culture of HK-2 cells with macrophages， compared with the control group， the TGF-β1 group showed increased COL1 （［2.07±0.21］ μg/L vs ［0.54±0.04］ μg/L） and COL3 （［1.53±0.14］ μg/L vs ［0.42±0.04］ μg/L）， enhanced α-SMA immunofluorescence expression， and increased relative expression of α-SMA （0.72±0.06 vs 0.11±0.01）， FN （0.70±0.08 vs 0.09±0.01）， and Gas6 （0.68±0.07 vs 0.06±0.01）， as well as an increased p-Akt/Akt ratio （0.34±0.03 vs 0.12±0.01）. Compared with the TGF-β1＋siRNA-NC group， the TGF-β1＋siRNA-Axl group showed decreased COL1 （［1.01±0.08］ μg/L vs ［2.06±0.21］ μg/L） and COL3 （［0.73±0.06］ μg/L vs ［1.53±0.14］ μg/L）， weakened α-SMA immunofluorescence expression， and decreased relative protein expression of α-SMA （0.15±0.02 vs 0.74±0.08）， FN （0.17±0.02 vs 0.73±0.08）， and Gas6 （0.15±0.01 vs 0.66±0.07）， as well as a decreased p-Akt/Akt ratio （0.21±0.02 vs 0.32±0.03） （all P<0.05）. In animal experiments， compared with the WT control group， the WT IgAN group showed increased renal interstitial fibrosis area （［12.46±1.34］% vs ［1.23±0.14］%）， increased relative protein expression of α-SMA （1.27±0.13 vs 0.43±0.04） and FN （0.93±0.10 vs 0.14±0.01）， and increased M2-type macrophage proportion （［21.50±2.24］% vs ［6.34±0.65］%）. Compared with the Axl-/- control group， the Axl-/- IgAN group showed increased renal interstitial fibrosis area （［7.54±0.80］% vs ［1.01±0.11］%）， increased relative protein expression of α-SMA （0.63±0.06 vs 0.42±0.04） and FN （0.47±0.05 vs 0.13±0.01）， and increased M2-type macrophage proportion （［17.83±1.84］% vs ［6.81±0.67］%）. Compared with the WT IgAN group， the Axl-/- IgAN group showed decreased renal interstitial fibrosis area （［7.54±0.80］% vs ［12.46±1.34］%）， decreased relative protein expression of α-SMA （0.63±0.06 vs 1.27±0.13） and FN （0.47±0.05 vs 0.93±0.10）， and decreased M2-type macrophage proportion （［17.83±1.84］% vs ［21.50±2.24］%） （P<0.05）.ConclusionThe Gas6/Axl signaling pathway participates in the progression of interstitial fibrosis in IgAN by regulating macrophage polarization. Axl silencing or knockout can alleviate renal interstitial fibrosis injury， providing a potential target for IgAN treatment.
SN - 1671-2390
ER -