Opioid Preconditioning Modulates Repair Responses to Prevent Renal Ischemia-Reperfusion Injury

Abstract

Progression to renal damage by ischemia-reperfusion injury (IRI) is the result of the dysregulation of various tissue damage repair mechanisms. Anesthetic preconditioning with opioids has been shown to be beneficial in myocardial IRI models. Our main objective was to analyze the influence of pharmacological preconditioning with opioids in renal function and expression of molecules involved in tissue repair and angiogenesis. Experimental protocol includes male rats with 45 min ischemia occluding the left renal hilum followed by 24 h of reperfusion with or without 60 min preconditioning with morphine/fentanyl. We analyzed serum creatinine and renal KIM-1 expression. We measured circulating and intrarenal VEGF. Immunohistochemistry for HIF-1 and Cathepsin D (CTD) and real-time PCR for angiogenic genes HIF-1α, VEGF, VEGF Receptor 2 (VEGF-R2), CTD, CD31 and IL-6 were performed. These molecules are considered important effectors of tissue repair responses mediated by the development of new blood vessels. We observed a decrease in acute renal injury mediated by pharmacological preconditioning with opioids. Renal function in opioid preconditioning groups was like in the sham control group. Both anesthetics modulated the expression of HIF-1, VEGF, VEGF-R2 and CD31. Preconditioning negatively regulated CTD. Opioid preconditioning decreased injury through modulation of angiogenic molecule expression. These are factors to consider when establishing strategies in pathophysiological and surgical processes.

Keywords: angiogenesis; fentanyl; morphine; opioids; renal ischemia-reperfusion; wound healing.

Figure 1

Experimental scheme. Graph illustrating experimental conditions in the study. Six male Wistar rats were treated with 0.5 mg/kg morphine (dark gray bar) or 10 µg/kg fentanyl (light gray bar) for 60 min before the left renal pedicle was occluded for 45 min to promote ischemia and a 24 h reperfusion period. As controls we performed a sham group (white bar) and ischemia-reperfusion injury (IRI) without preconditioning (black bar). The gray scale identification is preserved in all figures. Blood and kidney tissue samples were collected at the end of the reperfusion period.

Figure 2

Effects of opioid preconditioning on serum creatinine and KIM-1 mRNA levels. (A) Serum creatinine was evaluated using a dry chemistry technique. (B) Renal expression of KIM-1 was evaluated by quantitative RT-PCR in the ischemia-reperfusion treated kidneys. HPRT was used as a housekeeping gene for normalization. The sham group is represented by the white bars, IRI by the black bars, IRI + morphine by the dark gray bars, and IRI + fentanyl by the light gray bars. Values are means ± S.E.M. (n = 6). # p < 0.05 vs. sham, * p < 0.05 vs. IRI, ° p < 0.05 vs. fentanyl.

Figure 3

Opioid preconditioning promoted HIF-1 expression. (A) HIF-1α mRNA expression was analyzed by real-time PCR in the ischemia-reperfusion treated kidneys. Immunohistochemistry was performed in the ischemia-reperfusion treated kidney slides and the number of positive signal nuclei was quantified at 100× (B) from sham group (C), IRI (D), morphine- (E) and fentanyl-preconditioned animals (F). The average count of positive nuclei in the sham group was set as 100%. Sham group is represented by the white bars, IRI by the black bars, IRI + morphine by the dark gray bars, and IRI + fentanyl by the light gray bars. Scale bars represent 20 µm. Values are means ± S.E.M. (n = 6). # p < 0.05 vs. sham, * p < 0.05 vs. IRI.

Figure 4

Opioid preconditioning modulated VEGF an VEGF-R2 expression. Quantitative RT-PCR evaluation of mRNA levels of VEGF (A) and VEGF-R2 (B) in the ischemia-reperfusion treated left kidneys. Circulating (C) and intrarenal (D) levels of VEGF in the ischemia-reperfusion treated left kidneys (L) and the nontreated right ones (R). Levels were normalized using total protein quantification. Sham group is represented by the white bars, IRI by the black bars, IRI + morphine by the dark gray bars, and IRI + fentanyl by the light gray bars. Values are means ± S.E.M. (n = 6). # p < 0.05 vs. sham left (L) kidney. * p < 0.05 vs. IRI left kidney.

Figure 5

IL-6 and CD31 expression levels after IRI injury. Quantitative RT-PCR evaluation of renal expression of IL-6 (A) and CD31 (B) after ischemia-reperfusion with or without opioid preconditioning. HPRT was used as a housekeeping gene for normalization. Sham group is represented by the white bars, IRI by the black bars, IRI + morphine by the dark gray bars and IRI + fentanyl by the light gray bars. Values are means ± S.E.M. (n = 6). * p < 0.05 vs. IRI.

Figure 6

Opioid preconditioning reduced CTD expression. (A) CTD mRNA expression was examined by real-time PCR. Immunohistochemistry was executed in the ischemia-reperfusion treated left kidney and areas with positive staining (B) from the sham group (C), IRI (D), morphine- (E) and fentanyl-preconditioned rats (F) were quantified. Sham group is represented by the white bars, IRI by the black bars, IRI + morphine by the dark gray bars and IRI + fentanyl by the light gray bars. The average count of positive staining areas in the sham group was set as 100%. Scale bars represent 50 µm. Values are means ± S.E.M. (n = 6). # p < 0.05 vs. sham, * p < 0.05 vs. IRI.