Platelet Inhibition Prevents NLRP3 Inflammasome Activation and Sepsis-Induced Kidney Injury

Abstract

Platelets, cellular mediators of thrombosis, are activated during sepsis and are increasingly recognized as mediators of the immune response. Platelet activation is significantly increased in sepsis patients compared to ICU control patients. Despite this correlation, the role of activated platelets in contributing to sepsis pathophysiology remains unclear. We previously demonstrated NOD-like receptor protein 3 inflammasome (NLRP3) inflammasome activation in sepsis-induced platelets from cecal-ligation puncture (CLP) rats. Activated platelets were associated with increased pulmonary edema and glomerular injury in CLP vs. SHAM controls. In this study, we investigated whether inhibition of platelet activation would attenuate NLRP3 activation and renal and pulmonary injury in response to CLP. CLP was performed in male and female Sprague Dawley (SD) rats (n = 10/group) to induce abdominal sepsis and SHAM rats served as controls. A subset of CLP animals was treated with Clopidogrel (10 mg/kg/day, CLP + CLOP) to inhibit platelet activation. At 72 h post-CLP, platelet activation and NLRP3 inflammasome assembly were evaluated, IL-1β and IL-18 were measured in plasma, and tissues, renal and pulmonary pathology, and renal function were assessed. Activated platelets were 7.8 ± 3.6% in Sham, 22 ± 6% in CLP and significantly decreased to 14.5 ± 0.6% in CLP + CLOP (n = 8-10/group, p < 0.05). NLRP3 inflammasome assembly was inhibited in platelets of CLP + CLOP animals vs. CLP. Significant increases in plasma and kidney IL-1β and IL-18 in response to CLP were decreased with Clopidogrel treatment. Renal injury, but not lung histology or renal function was improved in CLP + CLOP vs. CLP. These data provide evidence that activated platelets may contribute to sepsis-induced renal injury, possibly via NLRP3 activation in platelets. Platelets may be a therapeutic target to decrease renal injury in septic patients.

Keywords: Clopidogrel; NLRP3; endothelial activation; inflammation; multi-organ injury; platelets; sepsis.

Figure 1

Platelet Activation. Platelet activation was measured by flow cytometry. Platelets were isolated form Sham, Cecal ligation and puncture (CLP) and CLP + Clopidogrel (CLP + CLOP). Activated platelets were identified as CD41⁺/CD62P⁺. * p < 0.05 vs. Sham; # p > 0.05 vs. CLP.

Figure 2

NLRP3 Inflammasome. (A–C) NLRP3 inflammasome activation was evaluated by visualizing the co-localization of NLRP3 and apoptosis-associated speck-like protein (ASC) using immunocytochemistry. * p < 0.05 vs. Sham, # p < 0.05 vs. CLP.

Figure 3

Inflammasome-associated cytokines. Plasma (A,D), kidney (B,E), and lung (C,F) IL-1β and IL-18 were measured in Sham, CLP, and CLP rats treated with Clopidogrel (n = 12/group) via ELISA. * p < 0.05 vs. Sham, # p < 0.05 vs. CLP, n.d. = not detected.

Figure 4

Endothelial activation. Plasma levels of endocan (n = 10/group) (A) and angiopoietin-2 (n = 7–8/group) (B), soluble markers of endothelial activation were measured in Sham, CLP and CLP + CLOP via ELISA. * p < 0.05 vs. Sham, # p < 0.05 vs. CLP.

Figure 5

Lung injury. Neutrophil Infiltration (A) and alveolar wall thickness (B) into pulmonary tissues were assessed in Sham, CLP, and CLP + CLOP. H&E staining was performed on paraffin-embedded lungs from Sham, CLP, and CLP + CLOP rats (Representative images) (n = 6/group). Black arrows indicate neutrophil infiltration. * p < 0.05 vs. Sham.

Figure 6

Renal function and injury. Glomerular filtration rate was assessed via FITC sinistrin clearance in Sham, CLP, and CLP + CLOP rats (A; n = 6). Periodic acid Schiff (PAS) staining was performed on paraffin-embedded kidneys from Sham, CLP, and CLP + CLOP rats (representative images). Glomerular injury (B; n = 6/group) was scored from PAS-stained sections. * p < 0.05 vs. Sham, # p < 0.05 vs. CLP.