Lipoxin A4 Restores Septic Renal Function via Blocking Crosstalk Between Inflammation and Premature Senescence

Abstract

Acute kidney injury (AKI) occurs in half of patients with septic shock, resulting in unacceptably high mortality. However, effective preventive treatments are still lacking. We hypothesized that pretreatment with lipoxin A4 (LXA4), known to promote inflammation resolution, may attenuate septic AKI via blocking crosstalk between inflammation and cellular senescence. In this study, rats developed AKI following cecal ligation and puncture (CLP), as evidenced by a dynamic increase in serum creatinine, blood urea nitrogen, urinary kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, and pathological injury, accompanied by increased levels of inflammation (IL-6, TNF-α, and HMGB1) and tubular cell senescence. While, on the one hand, inhibition of senescence with rapamycin restored renal function and attenuated septic inflammatory response, on the other hand, LXA4 administration inhibited renal inflammation and tubular epithelial cell senescence after CLP. Ultimately, pretreatment with LXA4 significantly restored renal function and increased the survival rate of rats after CLP. Furthermore, LXA4 inhibited NF-κB-mediated inflammatory response and the p53/p21 senescence pathway in vivo and in vitro. However, the effect was reversed by PPAR-γ siRNA and antagonist. These results indicated that LXA4 exerted its renoprotective effects by blocking the crosstalk between inflammation and premature senescence in a PPAR-γ-dependent manner. Our findings also suggested that premature senescence plays a critical role in septic AKI and that inhibition of the crosstalk between inflammation and premature senescence may represent a new and major mechanism through which LXA4 attenuates septic AKI.

Keywords: acute kidney injury; cellular senescence; inflammation resolution; lipoxin A4; sepsis.

Figure 1

Increased renal damage was accompanied by enhanced tubular cell senescence and inflammation in sepsis induced-AKI. Male Sprague-Dawley rats underwent CLP were sacrificed at the time point of 6-h, 12-h, 18-h, 24-h after surgery. Renal function alternation, renal necrosis and renal tubular epithelial cells senescence were detected with different methods in kidneys. (A–D) levels of SCr, BUN, urinary KIM-1 and NGAL at different surgery time points after CLP. (E) Renal damage of rats at different surgery time points after CLP (H&E; scale bar 100 μm). (F) Kidney histopathology evaluation scores at different surgery time points. (G) Representative images of cellular senescence in kidneys after CLP (β-Gal staining; scale bar 100 μm); Red: β-Gal positive threshold. (H) Senescent tubular areas at different surgery time points after CLP. (I–K) levels of IL-6, TNF-α and HMGB1 at different surgery time points after CLP. (L) levels of lipoxin A4 at different surgery time points after CLP. Data are presented as mean ± SE (n = 8). *p < 0.05; **p < 0.005; ***p < 0.001; ****p < 0.0001; CLP, cecal ligation and puncture; AKI, acute kidney injury; H&E, hematoxylin–eosin staining; SCr, serum creatinine; BUN, blood urea nitrogen; KIM-1, kidney injury molecule-1; NGAL, neutrophil gelatinase-associated lipocalin; IL-6, interlecukin-6; IL-8, interlecukin-8; TNF-α, tumor necrosis factor-alpha; LXA4, lipoxin A4.

Figure 2

Rapamycin restores renal function and attenuates septic inflammatory response. Male Sprague-Dawley rats were treated with or without rapamycin 1 mg/kg i.p. for 1h before CLP and were sacrificed at the time point of 18-h after surgery. Renal function alternation, renal necrosis and inflammatory cytokines were detected. (A–D) levels of SCr, BUN, urinary KIM-1 and NGAL among the four groups. (E) Renal damage and senescence of rats among the four groups (H&E; scale bar 100 μm). (F) Kidney histopathology evaluation scores among the four groups. (G–I) levels of IL-6, TNF-α and HMGB1 in kidneys among the four groups. Data are presented as mean ± SE (n = 8). ****p < 0.0001; CLP, cecal ligation and puncture; H&E, hematoxylin–eosin staining; SCr, serum creatinine; BUN, blood urea nitrogen; KIM-1, kidney injury molecule-1; NGAL, neutrophil gelatinase-associated lipocalin; IL-6, interlecukin-6; IL-8, interlecukin-8; TNF-α, tumor necrosis factor-alpha.

Figure 3

LXA4 alleviates septic AKI. Male Sprague-Dawley rats were treated with or without LXA4 (100 µg/kg, i.p.) for 30-min or BOC-2 (50 mg/kg, i.p.) for 20min before CLP and were sacrificed at the time point of 18-h after surgery. Renal function alternation and renal necrosis were detected. (A–D) levels of SCr, BUN, urinary KIM-1 and NGAL among the four groups. (E) Renal damage of rats among the four groups (H&E; scale bar 100 μm). (F) Kidney histopathology evaluation scores among the four groups. Data are presented as mean ± SE (n = 8). **p < 0.01; ***p < 0.001; ****p < 0.0001; LXA4, Lipoxin A4; CLP, cecal ligation and puncture; H&E, hematoxylin–eosin staining; SCr, serum creatinine; BUN, blood urea nitrogen; KIM-1, kidney injury molecule-1; NGAL, neutrophil gelatinase-associated lipocalin.

Figure 4

LXA4 alleviates renal inflammation and tubular epithelial cell senescence. Levels of inflammatory cytokines and cellular senescence were detected. (A–C) levels of IL-6, TNF-α and HMGB1 at among the four groups. (D) Representative images of cellular senescence in kidneys after CLP (β-Gal staining; scale bar 100 μm); Red: β-Gal positive threshold. (E) quantitative analysis of senescent tubular area among the four groups. (F–H) expression of p53 and p21 protein in kidney tissues among the four groups. (I) Representative images of cellular senescence in NRK52E cells after LPS treatment (β-Gal staining). (J) Expression of p53 and p21 protein in NRK52E cells after LPS treatment. Data are presented as mean ± SE (n = 8). ***p < 0.001; ****p < 0.0001; CLP, cecal ligation and puncture; IL-6, interlecukin-6; IL-8, interlecukin-8; TNF-α, tumor necrosis factor-alpha; LPS, lipopolysaccharide.

Figure 5

LXA4 attenuates AKI and increased rat survival rate in a PPAR-γ-dependent manner. NRK52E cells were treated with 1 μg/ml LPS for 24h with or without 10 nM LXA4 pretreatment for 12h, and PPAR-γ-siRNA transfection for 48h. Male Sprague-Dawley rats were treated with or without LXA4 (100 µg/kg, i.p.) for 30-min, or GW9662 (1 mg/kg, i.v.) or RSG (10 mg/kg, i.v.) for 20min prior CLP and were sacrificed at the time point of 18-h after surgery. Renal function alternation, renal pathological injury and senescence in the neighbored section were detected. (A) expression of PPAR-γ among the four groups. (B) PPAR-γ transcriptional activity among the four groups. (C) Representative images of NRK52E cell senescence among the four groups. (D–G) levels of SCr, BUN, urinary KIM-1 and NGAL among the four groups. (H) Representative images of renal damage and cellular senescence in the neighbored kidney section among the four groups (H&E and β-Gal staining; scale bar 100 μm). (I, J) Kidney histopathology scores and senescent tubular area among the four groups. (K) Survival rate after LXA4 treatment. The survival rate was observed for 24-h after the CLP operation, n=18. (A–G) Data are presented as mean ± SE (n = 8). *p < 0.05; **p < 0.005; ***p < 0.001; ****p < 0.0001; LXA4, Lipoxin A4; RSG, Rosiglitazone; CLP, cecal ligation and puncture; H&E, hematoxylin–eosin staining; SCr, serum creatinine; BUN, blood urea nitrogen; KIM-1, kidney injury molecule-1; NGAL, neutrophil gelatinase-associated lipocalin.

Figure 6

LXA4 attenuates septic AKI via inhibition of PPAR-γ/NF-κB pathway. Expression of p-p65, p53 and p21 in the neighbored section and levels of inflammatory cytokines in NRK52E cells and kidneys were also detected. (A) Expression of p-p65 and p53 in the NRK52E cells were detected by IF. (B) Expression of p-p65, p53 and p21 in the neighbored kidney sections were detected by IF. (C–E) levels of IL-6, TNF-α and HMGB1 at among the four groups. Data are presented as mean ± SE (n = 8). *p < 0.05; **p < 0.005; ***p < 0.001; ****p < 0.0001; LXA4, Lipoxin A4; RSG, Rosiglitazone; CLP, cecal ligation and puncture; IL-6, interlecukin-6; IL-8, interlecukin-8; TNF-α, tumor necrosis factor-alpha.

Figure 7

LXA4 attenuates septic AKI via blocking crosstalk between inflammation and premature senescence in PPAR-γ-dependent manner. Sepsis would cause activation of NF-κB-mediated inflammation response and NF-κB/p53 mediated cellular premature senescence in kidney tissues. The inflammation response interplays with cellular premature senescence in septic AKI. Pretreatment with LXA4 promotes inflammation resolution and blocks the crosstalk between inflammation and premature senescence via PPAR-γ/NF-κB signaling.