Discussion
Macrophages could promote renal fibrosis, a major driver of progression to end-stage kidney disease, and M2 macrophages is strongly associated with kidney fibrosis in both human and experimental diseases. In this study, we report that macrophage ITGAM contributes to kidney fibrosis in hyperuricemia-related CKD. Mechanistically, ITGAM promotes macrophage M2 polarization through activating FAK/Akt1/β-catenin pathway.
ITGAM, also known as CD11b, was commonly used as a monocyte/macrophage surface marker(Ross & Lambris, 1982; Wolf et al., 2018). Gradually, ITGAM diverse functions were reflected by its rapid confirmational change that increased affinity for its more than 40 ligands, including ICAM-1(Dunne et al., 2002), fibrinogen(Altieri et al., 1990), fibronectin(Kanse et al., 2004), GPIbα(Ehlers et al., 2003), RAGE(Chavakis et al., 2003), JAM-c(Lange-Sperandio et al., 2006), and others(Fan & Ley, 2015). We examined main ligands frequently reported, and found ICAM-1 and fibronectin were significantly upregulated (Figure 1E and Supplementary Figure 4A-B ), which indicated potential role of ITGAM in cell-ECM and cell-cell interactions in kidneys of hyperuricemia-related CKD. Further studies are needed to clarify how ITGAM mediates cross-talk between macrophage and other cells or compartments in kidney tissue. ITGAM was reported to be involved in various immune responses but with bidirectional effects(Rosetti & Mayadas, 2016). Negative regulation of ITGAM could be observed in systemic lupus erythematous and acute infectious diseases(Hu et al., 2016; Villanueva et al., 2022). On the contrary, ITGAM positively regulated chronic inflammatory diseases(Zirlik et al., 2007). Lange-Sperandio et al reported that high expression of Mac-1 and its ligands ICAM-1 and JAM-3 in murine unilateral ureteric obstruction (UUO) model, and knockout of Mac-1 greatly attenuated kidney fibrosis(Lange-Sperandio et al., 2006). Taking our results together, the functional role of ITGAM in contributing to kidney fibrosis through promoting M2 polarization could be inferred.
Both of integrin α and β subunits are single membrane-spanning segments with short cytoplasmic tails, thus need to interact with downstream tyrosine kinases to enable “outside-in” signal transduction(Kornberg et al., 1991; Takada et al., 2007). Binding to ligands (e.g extracellular matrix) induces integrins clustering at focal adhesions and connecting to intracellular molecules. FAK is one of the earliest identified central integrin signaling. The activated FAK undergoes autophosphorylation and then stimulates difference molecules corresponding to various pathways, including Src, Akt, Grb7, and others(Guan, 2010). Numerous studies reported FAK/Akt pathway participated in chronic inflammation and fibrosis, such as atherosclerosis(Yamaura et al., 2019), and lung and liver fibrosis(Gimenez et al., 2017; Lv et al., 2018). Of notes, we additionally observed the change in Src phosphorylation and found p-Src were both attenuated after silencing Itgam and inhibiting p-FAK. Our finding indicated that Src participated in ITGAM/FAK signaling and might act as the downstream molecular. It’s to be further explored whether Src together with FAK stimulates Akt1, or acts through other pathways, such as Stat3 and Erk (Bolos et al., 2010; Jang et al., 2022; Pan et al., 2019). β-catenin activation, as the canonical pathway of Wnt signaling, is associated with macrophage M2 polarization in tumor malignant behaviors and renal fibrosis(Feng et al., 2018; Yang et al., 2018). Several studies have shown that Akt activation and phosphorylation of GSK-3β led to β-catenin stabilization. Accumulated β-catenin in cytosol then translocates into nucleus and stimulates transcription of target genes that participates in tissue fibrosis(MacDonald et al., 2009; Niehrs, 2012).
To our best acknowledgement, this is the first paper investigating the role of integrin and how it regulates macrophage M2 polarization in kidneys of hyperuricemia-related CKD. Hyperuricemia-related, especially hyperuricemia-induced CKD is relatively less studied, due to long time and difficulty when modeling. We firstly fed mice with potassium oxonate only at high dose (4.8g/kg) for up to 14 weeks with the purpose of establishing uric acid-induced CKD, but only found moderately increased serum uric acid (1.5-2 times of baseline) and almost unchanged kidney tissue histology. Urate oxidase-knockout mouse is an optimal option, but low survival greatly limited experiment feasibility and reproducibility(Lu et al., 2018; Zhao et al., 2022). Not only signal pathway ITGAM regulates, how ITGAM mediates cell-cell or ECM-cell cross-talk is also potential mechanism of great meaning to be further explored.
In summary, macrophage integrin ITGAM might play the vital role in mediating kidney fibrosis in hyperuricemia-related CKD through activating its downstream pathway FAK/Akt1/β-catenin and promoting macrophage M2 polarization. This finding provides new insight into the prevention and treatment of kidney fibrosis induced by uric acid, and acts as potential therapeutic target, although more well-designed studies are needed to verify further mechanisms.