Change in Oxidative Stress and Mitochondrial Dynamics in Response to Elevated Cold-Inducible RNA-Binding Protein in Cardiac Surgery-Associated Acute Kidney Injury

Abstract

Cardiac surgery-associated acute kidney injury (CSA-AKI) is a common yet serious complication that is closely related to cardiopulmonary bypass (CPB). Extracellular cold-inducible RNA-binding protein (eCIRP) can mediate aseptic inflammation and trigger intracellular oxidative stress. In the present study, expression of serum CIRP was significantly elevated post-CPB (785.0 ± 640.5 pg/mL vs. 149.5 ± 289.1 pg/mL, P < 0.001) and was positively correlated with CPB duration (r = 0.502, P < 0.001). Patients with high expression of CIRP had higher risks of postoperative AKI than patients with low CIRP expression (OR: 1.67, 95% CI 1.04-2.68). In a rat CPB model, the serum CIRP concentration increased significantly after CPB. Similarly, the levels of Scr and BUN significantly increased 4 hours after CPB. KIM-1 and NGAL mRNA levels in the CPB group were 8.2 and 4.3 times higher than the sham group, respectively. In addition, the levels of inflammatory cell infiltration, oxidative stress, and apoptosis in the renal tissue of the CPB group were significantly higher compared to the sham group. The expression levels of serum inflammatory factors at 4 hours post-CPB were also increased. Administration of recombinant human CIRP protein promoted the expression of NADPH oxidase via the TLR-4/MyD88 pathway, aggravated intracellular oxidative stress, mediated mitochondrial dynamics disorder, and eventually increased apoptosis in HK-2 cells. However, the CIRP inhibitor C23 improved the CIRP-mediated oxidative stress and mitochondrial dysfunction in both rat and cell models. In summary, elevated CIRP could mediate oxidative stress and mitochondrial dynamics in the kidney to promote CSA-AKI.

Figure 1

Elevated CIRP after CPB was associated with increased incidence of AKI. (a) The ΔCIRP levels in patients who experienced CPB were 4.3-fold higher than those who did not. (b) The ΔCIRP levels were positively correlated with the CPB time (n = 292, r = 0.502, and P < 0.001). (c) Incidence of AKI stratified by median serum ΔCIRP concentration. CIRP: cold-inducible RNA-binding protein; CPB: cardiopulmonary bypass. ^∗P < 0.05; ^∗∗P < 0.001.

Figure 2

CPB promoted CIRP secretion and aggravated renal injury. (a) H&E staining showing more dilated renal tubules and tube casts in the CPB group compared to the sham group and CPB+C23 group. (b, c) Expression levels of Scr at 0 and 4 hours after CPB. (d, e) Expression levels of BUN at 0 and 4 hours after CPB. (f, g) Expression levels of CIRP at 0 and 4 hours after CPB. (h, i) TUNEL staining indicating the increased number of apoptotic cells in the renal tissue after CPB; C23 administration effectively reduced apoptosis (magnification 200x). (j, k) The mRNA levels of renal injury markers KIM-1 and NGAL in renal tissues. CIRP: cold-inducible RNA-binding protein; CPB: cardiopulmonary bypass; Scr: serum creatinine; BUN: blood urea nitrogen. ^∗P < 0.05 vs. the sham group; ^#P < 0.05 vs. CPB. (a, h, top) Scale bar: 200 μm and (a, bottom) scale bar: 100 μm.

Figure 3

CPB induced mitochondrial dynamics disorder and increased inflammatory factor secretion. (a–f) Expression of IL-6, IL-1β, and TNF-α in the serum at 0 and 4 hours after CPB. Compared to the CPB group, C23 administration decreased the expression of IL-6, IL-1β, and TNF-α by 54.0%, 67.5%, and 45.2% at 4 hours, respectively. (g) Western blot analysis of renal Bax, Bcl-2, and cleaved-caspase-3 expression. (h) CPB upregulated the NADPH oxidase via the TLR-4/MyD88 pathway, which promoted the expressions of mitochondrial fission-related proteins Fis1 and Drp1 and reduced the expression of fusion-related protein Mfn2. ^∗P < 0.05 vs. sham; ^#P < 0.05 vs. CPB.

Figure 4

CPB aggravated inflammatory cell infiltration and renal oxidative stress. (a) Immune cell infiltration of macrophages and neutrophils in rat renal tissue at 4 hours after CPB (magnification 400x); quantification of inflammatory cells in renal tissue after CPB; CPB aggravated immune cell infiltration in both the outer medulla and cortex. (b) F4/80-positive macrophages in the outer medulla. (c) F4/80-positive macrophages in the cortex. (d) MPO-positive neutrophils in the outer medulla. (e) MPO-positive neutrophils in the cortex. (f–h) MDA, GSH, and SOD levels in the renal tissue of each group. CPB: cardiopulmonary bypass; MDA: malonaldehyde; GSH: glutathione; SOD: superoxide dismutase. ^∗P < 0.05 vs. sham; ^#P < 0.05 vs. CPB. (a) Scale bars: 200 μm.

Figure 5

CIRP mediated oxidative stress and mitochondrial dynamics disorder in HK-2 cells. (a) Renal DHE staining (red) and counterstaining DAPI (blue), magnification 400x. (b) Renal DHE fluorescence intensity in each group. (c–e) Expression of inflammatory factors IL-6, IL-1β, and TNF-α in the medium. (f, g) Flow cytometry analysis of HK-2 cell apoptosis. (h, i) The mRNA levels of KIM-1 and NGAL. (j) Western blot analysis of NADPH oxidase and mitochondrial proteins, rhCIRP, and recombinant human CIRP protein. DHE: dihydroethidium; NADPH: nicotinamide adenine dinucleotide phosphate. ^∗P < 0.05 vs. control; ^#P < 0.05 vs. high rhCIRP. (a) Scale bars: 100 μm.

Figure 6

Putative mechanism of CIRP in cardiac surgery-associated acute kidney injury. During cardiac surgery, CIRP is secreted into the circulation in response to hypothermia and hemodynamics change. Extracellular CIRP promotes the expression of NADPH oxidase in renal tubular epithelial cells via the TLR-4/MyD88 pathway and aggravates intracellular oxidative stress. ROS accumulation induces mitochondrial dynamics disorder, which ultimately increases apoptosis and promotes AKI. CIR: cold-inducible RNA-binding protein; NADPH: nicotinamide adenine dinucleotide phosphate; ROS: reactive oxygen species.