The Protective Effect of A Short Peptide Derived From Cold-Inducible RNA-Binding Protein in Renal Ischemia-Reperfusion Injury

Abstract

Extracellular cold-inducible RNA-binding protein (CIRP) functions as damage-associated molecular pattern and has been demonstrated to be responsible in part for the damage occurring after renal ischemia-reperfusion (I/R). A short peptide derived from CIRP, named C23, binds to myeloid differentiation factor 2, a Toll-like receptor 4 coreceptor. We hypothesize that C23 reduces renal ischemia-reperfusion (RIR) injury by blocking CIRP. We observed that pretreatment with C23 significantly decreased the levels of recombinant mouse CIRP-induced tumor necrosis factor-α (TNF-α) in a dose-dependent fashion in cultured macrophages. C57BL/6 mice were subjected to bilateral renal pedicle clamps for 35 min to induce ischemia, followed by reperfusion for 24 h and harvest of blood and renal tissue. C23 peptide (8 mg/kg) or vehicle was injected intraperitoneally at the beginning of reperfusion. Plasma TNF-α, interleukin 1 beta (IL-1β), and IL-6 levels were decreased in C23-treated RIR mice as compared with vehicle-treated mice by 74%, 85%, and 68%, respectively. Expressions of TNF-α and keratinocyte chemoattractant in the kidneys from C23-treated mice were decreased by 55% and 60%, respectively. Expression of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin in the kidney of C23-treated mice were significantly reduced by 46% and 55%, respectively. Renal tissue histological assessments revealed significant reduction in damage score by 44% in C23-treated mice. Finally, a survival study revealed a significant survival advantage with a 70% survival rate in C23 group vs. 37% in vehicle group. Thus, C23 has potential as a novel therapy for the patients suffering from I/R-induced renal injury.

Figure 1. C23 inhibits TNF-α production in macrophages stimulated with CIRP

A total of 0.8 × 10⁶ differentiated THP-1 cells were treated with either PBS or C23 at 25, 50, 100, 250, or 450 ng/ml doses. After 1 h, cells were then stimulated with rmCIRP at 300 ng/ml for 4 h, followed by the assessment of TNF-α levels in the culture supernatant by ELISA. We used triplicate cell subcultures for each experimental arm and repeated our process in 3 separate experiments. Data are expressed as mean ± SEM and compared by one-way analysis of variance and Student-Newman-Keuls method. *P < 0.05 versus no rmCIRP; ^#P < 0.05 versus rmCIRP without C23. rmCIRP, recombinant murine cold-inducible RNA-binding protein; TNF-α, tumor necrosis factor-α; ELISA, enzyme-linked immunosorbent assay.

Figure 2. Treatment with C23 attenuates systemic inflammation in the mice after RIR

Serum was collected 24 h after RIR and used to measure (A) TNF-α, (B) IL-6, (C) IL-1β using ELISA. Data are expressed as mean ± SEM (sham: n = 4 mice; RIR + vehicle: n = 5 mice; RIR + vehicle: n = 5 mice) and compared by one-way analysis of variance and Student-Newman-Keuls method. *P < 0.05 versus sham; ^#P < 0.05 versus vehicle. RIR, renal ischemia-reperfusion; SEM, standard error of the mean; TNF-α, tumor necrosis factor-α; ELISA, enzyme-linked immunosorbent assay.

Figure 3. Treatment with C23 attenuates the expression of TNF-α and KC in the mice after RIR

Kidney tissue was collected 24 h after RIR and analyzed for (A) TNF-α and (B) KC gene expression by quantitative RT-PCR. Data are expressed as mean ± SEM (sham: n = 4 mice; RIR + vehicle: n = 5 mice; RIR + vehicle: n = 5 mice) and compared by one-way analysis of variance and Student-Newman-Keuls method. *P < 0.05 versus sham; ^#P < 0.05 versus vehicle. RIR, renal ischemia-reperfusion; SEM, standard error of the mean; TNF-α, tumor necrosis factor-α; KC, keratinocyte chemoattractant; RT-PCR, real time polymerase chain reaction.

Figure 4. Treatment with C23 improves renal histology injury in the mice after RIR

(A) Kidney tissue was collected 24 h after RIR and analyzed for histological assessment (H&E) of outer medulla for each experimental group at 200× magnification. (B) Averaged semi-quantitative score of experimental groups. Data are expressed as mean ± SEM (sham: n = 4 mice; RIR + vehicle: n = 5 mice; RIR + vehicle: n = 5 mice) and compared by one-way analysis of variance and Student-Newman-Keuls method. *P < 0.05 versus sham; ^#P < 0.05 versus vehicle. RIR, renal ischemia- reperfusion; H&E, hematoxylin and eosin; SEM, standard error of the mean.

Figure 5. C23 treatment attenuates the expression of KIM-1 and NGAL in the kidneys of the mice after RIR

After 24 h of RIR, kidney tissue from each experimental group was harvested to assess mRNA expression of (A) NGAL and (B) KIM-1 by quantitative RT-PCR. Data are expressed as mean ± SEM (sham: n = 4 mice; RIR + vehicle: n = 5 mice; RIR + vehicle: n = 5 mice) and compared by one-way analysis of variance and Student-Newman-Keuls method. *P < 0.05 versus sham; ^#P < 0.05 versus vehicle. RIR, renal ischemia- reperfusion; SEM, standard error of the mean; NGAL, neutrophil gelatinase-associated lipocalin; KIM-1, kidney injury molecule 1.

Figure 6. C23 treatment reduces apoptotic cells in the kidneys of the mice after RIR

Kidney tissue was harvested 24 h after RIR and the tissue sections from all experimental groups were subjected to TUNEL assay. (A) The sections were visualized under fluorescent microscope at 100× magnifications. (B) Averaged quantification of TUNEL-positive cells per section per group by computer counting software. Data are expressed as mean ± SEM (sham: n = 4 mice; RIR + vehicle: n = 5 mice; RIR + vehicle: n = 5 mice) and compared by one-way analysis of variance and Student-Newman-Keuls method. *P < 0.05 versus sham; ^#P < 0.05 versus vehicle. RIR, renal ischemia- reperfusion; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; SEM, standard error of the mean.

Figure 7. C23 treatment improves overall survival rate of the mice after RIR

Mice were subjected to 30 min of ischemia followed by reperfusion with intraperitoneal injection of either vehicle (normal saline) or C23 (8 mg/kg BW). A Kaplan-Meier survival curve generated from RIR + vehicle (n = 19 mice) and RIR + C23 (n = 20 mice) after an 8-day monitoring period is shown. *P < 0.05 versus vehicle. RIR, Renal Ischemia Reperfusion.