To investigate the role of fibrinogen-like protein 2 (Fgl2) in macrophage polarization during cisplatin (Cis)-induced acute kidney injury (Cis-AKI).

Twelve male wild-type (Fgl2^+/+) mice and 12 Fgl2 gene knockout (Fgl2^-/-) mice, aged 8~10 weeks and weighing 20~25 g, were selected, and then after being administered with a single intraperitoneal injection of either saline or Cis for 3 d, they were randomly divided into 4 groups (n=6): Fgl2^+/+ Saline group, Fgl2^+/+ Cis group, Fgl2^-/- Saline group and Fgl2^-/- Cis group. Kidney function indicators such as serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured 3 d later, and kidney injury was assessed by HE staining. Western blot analysis was performed to evaluate the expression of Fgl2 and kidney injury molecule 1 (Kim-1) in the renal tissues. RT-qPCR was conducted to assess the expression levels of Fgl2, Kim-1, neutrophil gelatinase-associated lipocalin (NGAL), IL-6, IL-12p40, IL-1β, inducible nitric oxide synthase (iNOS) and TNF-α in the renal tissues. Immunohistochemical assay was employed to detect the expression of Fgl2 and macrophages (F4/80⁺) in the kidneys. Immunofluorescence staining was utilized to examine the expression of macrophages (F4/80⁺) and M1-type macrophages (F4/80⁺CD86⁺) in the renal tissues. Flow cytometry was employed to analyze the expression of macrophages (F4/80⁺) as well as M1-type macrophages (F4/80⁺MHC Ⅱ⁺) and M2-type macrophages (F4/80⁺CD206⁺) in the renal tissues.

Compared with the Fgl2^+/+ Saline group, the Fgl2^+/+ Cis group exhibited a significant decline in renal function (P<0.05), a notable increase in pathological score of renal tubular injury (P<0.05), and an obvious upregulation of renal tissue Fgl2 expression (P<0.05). Compared with the Fgl2^+/+ Cis group, the Fgl2^-/- Cis group demonstrated a significant decline in renal function (P<0.05), an elevation in the expression of renal injury-associated molecules Kim-1 and NGAL (P<0.05), an increase in pathological score of renal tubular injury (P<0.05), increase in macrophage infiltration (P<0.05), and an upregulation in the expression of M1-type macrophage-related molecules IL-6, IL-1β, TNF-α and iNOS (P<0.05), as well as obviously increase in the percentage of M1-type macrophages as indicated by flow cytometry (P<0.05), while there was no significant change in the percentage of M2-type macrophages, and the proportion of M1-type macrophages was significantly higher than that of M2-type macrophages (P<0.05).

Fgl2 gene knockout exacerbates Cis-AKI by promoting macrophage polarization towards the M1 phenotype.

To investigate the effect of hairy and enhancer of split 5 (HES5) on transdifferentiation and apoptosis of renal tubular epithelial cells induced by TGF-β1 and its potential mechanism.

The differentially expressed genes in GSE66494 data were analyzed and screened. The mouse model of unilateral uretera obstruction (UUO) was established, and the expression level of HES5 was detected in the renal tissue. HK-2 cells were treated with 10 ng/mL TGF-β1 for 24 h to establish a tubular epithelial-mesenchymal transition (EMT) model, and then qRT-PCR and Western blotting were performed to detect the expression of HES5 at mRNA and protein levels. After HK-2 cells were transfected with the plasmid overexpressing HES5, the protein levels of fibronectin, collagen Ⅰ, vimentin, apoptosis markers Bax and Bcl2 were detected in 24 h later. Then, HK-2 cells were divided into Control group, siHES5 group, TGF-β1 group, and siHES5+TGF-β1 group. The protein level of fibrosis and apoptosis markers were measured in above groups with Western blotting. TUNEL staining and flow cytometry were employed to detect cell apoptosis. Western blotting was applied to determine the protein levels of AKT, p-AKT, PI3K and p-PI3K. HK-2 cells overexpressing HES5 were treated with PI3K inhibitor LY294002, and the expression of vimentin was detected.

The expression of HES5 was significantly up-regulated in both chronic kidney disease (CKD) and fibrotic kidneys of mice. Overexpression of HES5 promoted the synthesis of fibronectin, collagen Ⅰ, vimentin and Bax in HK-2 cells, and inhibited the expression of Bcl2 (P<0.05). HES5 knockdown not only down-regulated the expression of fibrosis markers, but also inhibited the apoptosis of HK-2 cells. Furthermore, HES5 knockdown inhibited the activation of PI3K/AKT signaling pathway induced by TGF-β1 in HK-2 cells (P<0.05). Inhibitors of the PI3K/AKT signaling pathway inhibitor attenuated the induction of HES5 on vimentin.

HES5 knockdown inhibits the transdifferentiation and apoptosis in TGF-β1-induced renal tubular epithelial cells, which may be related to the decreased activity of the PI3K/AKT signaling pathway.

To investigate the role and mechanism of fibrinogen-like protein 2（Fgl2）in acute kidney injury induced by rhabdomyolysis（RM-AKI）.

Wild-type mice（WT）and Fgl2 gene knockout mice（Fgl2 KO）with the same background, were randomly divided into 4 groups respectively, that is WT+saline, WT+Gly, Fgl2 KO+saline, Fgl2 KO+Gly（n=5）. The mice of the RM-AKI group were given 50% glycerol（10 μL/g）and those of the control groups were given same amount of normal saline into the hind leg by intramuscular injection. In 48 h later, the serum samples were collected to detect the levels of Scr and BUN. HE staining was employed to assess and score the severity of renal injury. qRT-PCR was used to detect the mRNA levels of Fgl2, IL-6, MCP-1, TNF-α, IL-1β and IL-10. The expression of Fgl2 and F4/80 was observed with immunohistochemical staining（IHC）. TUNEL assay and Western blotting were applied to measure cell apoptosis and expression of apoptosis-related proteins, respectively.

Fgl2 expression in renal tissue was significantly elevated in glycerol-treated WT mice compared with saline-treated WT mice. When compared with WT mice, Fgl2 deficiency significantly alleviated renal dysfunction and renal histopathological injury, with statistically lower renal injury score（P<0.05）. Moreover, the expression of pro-inflammatory factors, IL-6, MCP-1, TNF-α and IL-1β in renal tissues was decreased, while that of anti-inflammatory factor IL-10 was increased（P<0.05）. The infiltration of macrophages and apoptosis of renal cells were reduced in the Fgl2 KO mice than the WT mice. The expression of apoptosis-related proteins, Bax, cleaved Caspase-3, and cleaved Caspase-9 was reduced, while that of Bcl2 showed no significant difference.

Knockout of Fgl2 inhibits inflammatory response and reduces apoptosis in renal cells, and exerts a protective effects on RM-AKI mice.