Suppression of nF-κB by ro 106-9920 alleviates ischemia/reperfusion-induced renal dysfunction and inflammation via modulation of neutrophil extracellular trap formation in acute kidney injury mice

Abstract

Neutrophil extracellular trap (NET) formation has been proven to take part in the exacerbation of acute kidney injury (AKI). Ro 106-9920, an effective inhibitor of nuclear factor kappa B (NF-κB) signal, could abrogate the formation of NETs. Herein, we explored whether Ro 106-9920 (Ro) exerts a protective role in AKI by repressing NET formation. The AKI model was induced with 30 min-bilateral renal ischemia followed by reperfusion. After finishing the 7-day treatment of Ro or vehicle, blood and the kidney were collected from each mouse for further analysis. Enzyme-linked immunosorbent assay, H&E, and TUNEL staining, immunohistochemistry, as well as Western blot were carried out to observe the kidney function, renal damage, apoptosis, and inflammation, and to preliminarily uncover the underlying mechanism. Administration with Ro effectively protected against AKI in a dose-dependent manner, as proven by the reduction of serum creatinine, serum neutrophil gelatinase-associated lipocalin, blood urea nitrogen, and serum inflammatory cytokine in AKI models after its administration. Moreover, Ro significantly alleviated morphological damage, kidney cells apoptosis, as well as inflammatory cytokine secretion in renal tissues. Mechanically, phosphorylation of NF-κB and myeloperoxidase activity were observed in renal tissues of AKI models, which suggested NF-κB activation and NETosis occurred in AKI. Notably, Ro treatment could significantly repress the nuclear translocation of NF-κB and the increased myeloperoxidase activity. Ro has a protective potential on ischemia/reperfusion-induced AKI by attenuating apoptosis and inflammation, perhaps by suppressing NF-κB activation and is associated with reduced NETosis.

Keywords: Acute kidney injury; Ro 106-9920; ischemia/reperfusion injury; neutrophil extracellular trap formation; nuclear factor kappa B.

Figure 1.

Effects of Ro on the renal function of I/R-induced AKI mice. A. The chemical structure of Ro 106-9920 (Ro). B. Schematic diagram of the experimental design. Mice were subjected to bilateral renal I/R injury and treated with Ro (5, 10, 20 mg/kg/day, i.g.), vehicle, or N-acetylcysteine (150 mg/kg, i.p.). Renal tissue and blood samples were collected at the indicated time points. C. Serum levels of creatine were detected by the commercial kit at Pre-I/R, 24h, 72h, and 168h after reperfusion. Corresponding area under the curve (AUC) analysis is shown on the right. D. Serum levels of cystatin C were detected by the commercial kit at Pre-I/R, 24h, 72h, and 168h after reperfusion. Corresponding AUC analysis is shown on the right. E. Serum levels of BUN were detected by the commercial kit at Pre-I/R, 24h, 72h, and 168h after reperfusion. Corresponding AUC analysis is shown on the right. F. Serum levels of NGAL were detected by the commercial kit at 168h after reperfusion. G. Serum levels of FGF-23 were detected by the commercial kit at 168h after reperfusion. H. Western blot analysis of KIM-1 protein expression in renal tissues. Data are presented as mean ± SEM (n = 8 per group). *** means P < 0.001 when vs. the control group; ^### means P < 0.001 when vs. the I/R group.

Figure 2.

Ro Alleviates renal damage of I/R-induced AKI mice. A. Pathological changes in the kidney were observed by H&E staining. Scale bar = 100 μm. B. Renal cell apoptosis was observed by TUNEL staining. Scale bar = 100 μm. *** means P < 0.001 when vs. the control group; ^### means P < 0.001 when vs. the I/R group.

Figure 3.

Ro Reduces I/R-induced renal inflammation. A–C. Serum levels of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) were assessed by the ELISA kits. D–F. The mRNA expression of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) in the renal tissues was analyzed by qRT-PCR. *** means P < 0.001 when vs. the control group; ^## means P < 0.01 and ^### means P < 0.001 when vs. the I/R group.

Figure 4.

Ro Inhibits NF-κB activation in the renal of I/R-induced AKI mice. A. Expression of NF-κB in the renal sections was observed by IHC analysis. B. Cytosolic p-NF-κB and nuclear NF-κB levels in the renal tissue were detected by WB analysis. *** means P < 0.001 when vs. the control group; ^# means P < 0.05, ^## means P < 0.01 and ^### means P < 0.001 when vs. the I/R group.

Figure 5.

Ro Suppresses NETosis in the renal of I/R-induced AKI mice. A. Expression of MPO in the renal sections was observed by IHC analysis. B. Renal MPO activity was measured using the commercial kit. C. MPO and Cit-H3 protein levels in the renal tissue were detected by WB analysis. *** means P < 0.001 when vs. the control group; ^### means P < 0.001 when vs. the I/R group.