Background Renal ischemia-reperfusion (RIR) injury, commonly caused by major surgery and shock, leads to acute kidney injury, and is associated with high morbidity and mortality. Cold-inducible RNA-binding protein (CIRP), a cold shock protein, has been recently identified as a damage-associated molecular pattern (DAMP). We hypothesized that CIRP exacerbates severity of injury in RIR. Methods Renal ischemia was induced in 8-week-old male C57BL/6 wild-type (WT) mice and Cirp−/− mice via bilateral clamping of renal pedicles for 30 min, followed by reperfusion for 5 h or 24 h and harvest of blood and renal tissue for analysis. Anti-CIRP antibody or non-immunized IgG was injected intravenously (10 mg/kg body weight) at time of reperfusion. Results After RIR, Cirp−/− mice demonstrated a reduction of BUN and creatinine of 53% and 60%, respectively, compared to WT mice. Serum IL-6 levels were significantly reduced 70% in Cirp−/− mice compared to WT mice after RIR. Levels of nitrotyrosine, an oxidatively-modified protein marker, and cyclooxygenase-2, an inflammatory mediator, were also significantly decreased in the kidneys of the Cirp−/− mice compared to WT mice after RIR. Renal caspase-3 activity was decreased in Cirp−/− mice compared to WT mice after RIR, which corresponded to the reduction of apoptotic cells determined by TUNEL assay. Injection of neutralizing anti-CIRP antibody into WT mice led to an 82% reduction in BUN compared to the vehicle after RIR. Conclusions Deficiency of CIRP results in less renal injury after RIR by attenuating inflammation and oxidative stress. Furthermore, blockade of CIRP shows a protective effect, indicating CIRP as a target in the treatment of RIR.

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Renal ischemia-reperfusion (RIR) injury is a common occurrence after major surgery and shock, leading to acute kidney injury (AKI). Osteopontin (OPN) is a secreted glycoprotein that acts as a proinflammatory cytokine and activator of T lymphocytes. We hypothesized that blockade of OPN reduces the severity of inflammation and injury in RIR. Renal ischemia was induced in adult C57BL/6 mice via bilateral clamping of renal pedicles for 35 min, followed by reperfusion for 24 h. Anti-OPN antibody (Ab), non-immunized isotype IgG, or normal saline was injected intravenously at time of reperfusion. Blood and kidneys were collected for analysis. At 24 h after RIR, OPN mRNA and protein levels were significantly increased in renal tissue compared to sham mice. In serum, elevated levels of BUN and creatinine were reduced in anti-OPN Ab-treated mice compared to vehicle. Anti-OPN Ab-treated mice also had decreased mRNA levels of injury markers neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) compared to the vehicle. The histologic architecture and apoptosis of renal tissue were improved in the anti-OPN Ab-treated mice. In renal tissue, inflammatory cytokines IL-6 and TNF-α protein levels were reduced in the Ab-treated mice. NK cell infiltration was decreased after anti-OPN Ab treatment, as was neutrophil infiltration, shown by reduced chemokine expression and Gr1 renal immunohistochemical staining. These findings demonstrate a beneficial role of OPN blockade in RIR associated with NK cell-mediated downregulation of inflammatory cytokines and chemokines. Administration of anti-OPN Ab may therefore serve as an immunomodulatory adjunct in the treatment of RIR-induced AKI.

Introduction Acute kidney injury (AKI) is most commonly caused by sepsis in critically ill patients, and it is associated with high morbidity and mortality. The pathophysiology of sepsis-induced AKI is generally accepted to include direct inflammatory injury, endothelial cell dysfunction, and apoptosis. Milk fat globule-epidermal growth factor-factor VIII (MFG-E8) is a secretory glycoprotein with a known role in the enhancement of apoptotic cell clearance and regulation of inflammation. We hypothesize that administration of recombinant mouse MFG-E8 (rmMFG-E8) can protect mice from kidney injuries caused by sepsis. Methods Sepsis was induced in 8-week-old male C57BL/6 mice by cecal ligation and puncture (CLP). rmMFG-E8 or PBS (vehicle) was injected intravenously at a dosage of 20 μg/kg body weight at time of CLP (n=5–8 mice/group). After 20 h, serum and renal tissue were harvested for various analyses. The renal injury markers blood urea nitrogen (BUN) and creatinine were determined by enzymatic and chemical reactions, respectively. The gene expression analysis was carried-out by real-time qPCR. Results At 20 h after CLP, serum levels of BUN and creatinine were both significantly increased in the vehicle group compared to the sham group, while the mice treated with rmMFG-E8 had a significant reduction in BUN and creatinine levels by 28% and 24.1%, respectively (BUN: 197.7 ± 23.6 vs. 142.3 ± 20.7 mg/dL; creatinine: 0.83 ± 0.12 vs. 0.63 ± 0.06 mg/dL; p<0.05). Expression of novel biomarkers of renal tissue injury neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) were also significantly downregulated by 58.2% and 95%, respectively after treatment with rmMFG-E8. Pro-inflammatory cytokine IL-6 and TNF-α mRNA were significantly reduced by 50.8% and 50.3%, respectively, in rmMFG-E8-treated mice compared to vehicle-treated mice. The mRNA levels of the chemokines keratinocyte chemoattractant (KC) and macrophage inhibitory protein-2 (MIP-2) were reduced by 85.1% and 78%, respectively in mice treated with rmMFG-E8 compared to the vehicle mice. In addition, the expression of inter-cellular cell adhesion molecule-1 (ICAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) mRNA was downregulated by 35.6% and 77.8%, respectively in rmMFG-E8-treated mice compared to the vehicle animals (p<0.05). Conclusions Treatment with rmMFG-E8 reduces renal tissue injury induced by sepsis through inhibiting the production of pro-inflammatory cytokines and chemokine, as well as through the activation of endothelial cells. Thus, MFG-E8 may have a therapeutic potential for treating AKI induced by sepsis.

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