Cellular senescence promotes macrophage-to-myofibroblast transition in chronic ischemic renal disease

Abstract

Cellular senescence participates in the pathophysiology of post-stenotic kidney damage, but how it regulates tissue remodeling is incompletely understood. Macrophage-myofibroblast transition (MMT) contributes to the development of tissue fibrosis. We hypothesized that cellular senescence contributes to MMT and renal fibrosis in mice with renal artery stenosis (RAS). INK-ATTAC mice expressing p16^INK-4a and green fluorescent protein in senescent cells were assigned to control or unilateral RAS, untreated or treated with AP20187 (an apoptosis inducer in p16^INK-4a-expressing cells) for 4 weeks. Renal perfusion was studied in vivo using micro-MRI, and kidney morphology, senescence, and MMT ex vivo. Cellular senescence was induced in human renal proximal tubular epithelial cells (HRPTEpiC) in vitro, and interferon-induced transmembrane protein-3 (IFITM3), a cellular senescence vector, was silenced (siRNA) or over-expressed (plasmid). HRPTEpiC were then co-incubated with macrophages with silenced integrin-3 (ITGB3), a regulator of mesenchymal transitions. CD68/p16^INK-4a/α-SMA co-expression and senescence markers were studied. Murine RAS kidneys showed increased expression of p16^INK-4a and MMT markers (F4/80, α-SMA) vs. controls, which decreased after AP20187, as did renal fibrosis and plasma creatinine, whereas renal perfusion increased. IFITM3 and ITGB3 expression were upregulated in senescent HRPTEpiC or co-cultured macrophages, respectively. MMT markers and TGF-β/Smad3 expression also rose in these macrophages and decreased after IFITM3 or ITGB3 silencing. p16^INK-4a-expressing macrophages may regulate interstitial fibrosis in RAS via MMT. This process is associated with elevated expression of ITGB3 and TGF-β/Smad3 pathway activation through neighboring senescent cell-derived IFITM3. These findings may implicate MMT as a therapeutic target in ischemic kidneys.

Fig. 1. Chronic ischemia induces fibrosis in the stenotic kidney (STK), while AP20187 (AP) improves renal function and perfusion.

A Schematic representation of the experimental protocol. B, D Masson’s Trichrome staining revealed increased fibrosis in the STK of renal artery stenosis (RAS) mice, which was significantly attenuated by AP treatment. Scale bar: 50 µm. C Renal perfusion maps generated by arterial spin-labeling MRI (mL/100 g/min; brighter red indicates higher perfusion) demonstrated reduced STK perfusion in RAS mice, which was restored in RAS + AP20187 (E). Plasma creatinine levels, elevated in RAS mice, were reduced following AP treatment (F). Additionally, renal gene expression of proinflammatory and profibrotic factors was blunted in RAS + AP20187 (G). The expression of senescence-associated secretory phenotype (SASP) genes, including IL-6, MMP3, and TNF-α, was markedly elevated in RAS compared to normal kidneys but significantly decreased with AP treatment (H). Data are mean ± SD (n = 6/group). *P < 0.05 vs. Normal; ^#P < 0.05 vs. RAS.

Fig. 2. Senescent cell clearance decreases MMT.

Triple-immunofluorescence images (A) and quantification (B) identified MMT cells in RAS kidneys that co-express macrophage (F4/80, red), senescence activation (p16-GFP, green), and myofibroblast (α-SMA, pink) markers, compared to controls (Normal). These MMT cell populations were significantly reduced in RAS kidneys treated with the p16^INK-4a+ cell apoptosis inducer, AP20187 (RAS + AP20187). A positive correlation was observed between MMT cell populations and the number of α-SMA⁺ cells (D). The senescence marker SA-β-gal activity, which was elevated in RAS compared to normal kidneys, showed a significant reduction following AP20187 treatment (A, C). Scale bar: 20 µm (immunofluorescence), 50 µm (SA-β-gal). E, F The numbers of F4/80⁺ macrophages correlated positively with the numbers of p16^INK-4a+ and SA-β-gal⁺ cells in both normal and RAS kidneys. G, H Similarly, renal fibrosis correlated strongly with the numbers of p16^INK-4a+ and SA-β-gal⁺ cells in the mouse kidneys. Data are mean ± SD (n = 6 per group). *P < 0.05 vs. Normal; #P < 0.05 vs. RAS. p16-GFP: p16^INK-4a+ labeled by green fluorescent protein (GFP).

Fig. 3. TNF-α and TGF-β induce senescence in HRPTEpiC.

SA-β-gal staining (A) and the mRNA expression of senescence markers (PAI-1, p16^INK-4a, p21^Cip1/Waf1, p53, IL-6, MCP-1, TNF-α) (B–H) were significantly increased in human renal proximal tubular epithelial cells (HRPTEpiC) following TNF-α and TGF-β treatment. Similarly, the protein levels of IFITM3 were elevated under the same conditions (I, J). Data are mean ± SD (n = 3/group). *P < 0.05 vs. Normal. PAI-1: plasminogen activator inhibitor-1. Scale bar: 200 µm (A).

Fig. 4. Senescent cells (SC) induce senescence in macrophages.

A Schematic of the in vitro experimental protocol. B, C: Immunofluorescence staining of Ki67 and quantitative analysis of Ki67⁺ cells. The number of Ki67⁺ cells decreased after co-culture with senescent human renal proximal tubular epithelial cells (HRPTEpiC) (senescent cells, SC). Scale bar: 100 µm (B). The data are mean ± SD (n = 3/group). D–H: The effects of IFITM-3 and ITGB-3 on macrophage senescence. IFITM3 and ITGB3 were manipulated in SC HRPTEpiC and macrophages, respectively. Macrophages were subsequently collected for Western blot analysis of senescence markers p21^Cip1/Waf1, p53, p-p53.S15, and γ-H2AX. Silencing ITGB3 or IFITM3 individually using siRNA reduced the expression of these senescence markers. However, this blunting effect was mitigated when IFITM3 was overexpressed in HRPTEpiC, indicating that IFITM3 plays a critical role in maintaining senescence signaling despite ITGB3 knockdown. Data are mean ± SD (n = 3/group). *P < 0.05 vs. Normal control (NC) and Non-SC groups; ^#P < 0.05 vs. SC group; and P < 0.05 vs. SC + IFITM3 over-expressing groups. I–K: Relative mRNA expression of the senescence markers p16^INK-4a, p21^Cip1/Waf1, and p53 increased in macrophages co-incubated with senescent cells. β-actin was used as loading control. Data are mean ± SD (n = 3/group). *P < 0.05 vs. Normal.

Fig. 5. IFITM3 and ITGB3 regulate MMT in macrophages.

A Triple immunofluorescence analysis identified MMT cells co-expressing macrophage (CD68, red), senescence activation (p16^INK-4a, green), and myofibroblast (α-SMA, pink) markers under different experimental conditions. Scale bar: 20 µm. B Semi-quantitative analysis of average fluorescence intensity for CD68, p16^INK-4a, and α-SMA-positive cells demonstrated that silencing ITGB3 or IFITM3 using siRNA significantly reduced the colocalization of these markers. In contrast, treatment with an IFITM3 plasmid increased the immunofluorescent colocalization of CD68, p16^INK-4a, and α-SMA, suggesting a role for IFITM3 in promoting MMT marker expression. Data are mean ± SD (n = 3/group). *P < 0.05 vs. Normal control (NC) and Non-SC groups; #P < 0.05 vs. SC group; and P < 0.05 vs. SC + IFITM3 over-expressing groups.

Fig. 6. The effects of IFITM-3 and ITGB-3 on MMT in macrophages.

A IFITM3 and ITGB3 were manipulated in senescent human renal proximal tubular epithelial cells (SC) and macrophages, respectively. Macrophages were subsequently collected for Western blot analysis to assess markers of mesenchymal transition (MMT) like Collagen-I, and pathway-related proteins (p-Smad2/3 and TGF-β). Knockdown of ITGB3 or IFITM3 using siRNA individually reduced the expression of Collagen I (B), TGF-β (C), and p-Smad2/3 (D). However, the blunting effects of siRNA treatment were attenuated when IFITM3 and ITGB3 were overexpressed, highlighting their roles in MMT and pathway activation in macrophages. Data are mean ± SD (n = 3/group). *P < 0.05 vs. Normal control (NC) and Non-SC groups; ^#P < 0.05 vs. SC group; and ^&P < 0.05 vs. SC + IFITM3 over-expressing groups; ^§p < 0.05 vs. SC IFITM3 plasmid+M ITGB3 siRNA.

Fig. 7. Schematic diagram of senescent cell induction of Macrophage-Myofibroblast Transition (MMT), which may result in tissue fibrosis.

We identified a critical role for macrophages in giving rise to myofibroblasts, contributing to interstitial fibrosis in RAS through mesenchymal transition (MMT). This process appears to be mediated, at least in part, by senescent cell-derived IFITM3, which promotes ITGB3 expression and activates the TGF-β/Smad3 signaling pathway.