Dexmedetomidine preconditioning inhibits the long term inflammation induced by renal ischemia / reperfusion injury in rats 1

PURPOSE: To investigate the protective effects of dexmedetomidine (Dex) against renal ischemia/reperfusion injury (IRI). METHODS: Sprague-Dawley rats were randomly divided to sham group, IRI group and Dex group. The SD rats were subjected to 45 min of ischemia followed by eight weeks of reperfusion. Prior to ischemia, rats were either treated with Dex or not. Blood samples were collected for the detection of blood urea nitrogen (BUN) and creatinine (Cr) levels. Immunohistochemistry was performed for CD3 T-cell infiltrates. Real-time PCR and western blot were detected for the expression of TNF-α, IL-1β, ICAM-1, HMGB1 and TLR4. RESULTS: Compared with sham group, renal IRI significantly increased the serum levels of BUN and Cr. The H&E staining indicated that renal IRI resulted in obvious renal injury and immunohistochemistry found that there were more CD3 T-cell infiltrates in IRI group. Also, renal IRI upregulated the expression of TNF-α, IL-1β, ICAM-1, HMGB1 and TLR4. However, all these changes were alleviated by the treatment with Dex. CONCLUSIONS: Dexmedetomidine has beneficial effects on long term inflammation induced by renal ischemia/reperfusion injury. Its mechanisms may be achieved through inhibiting the HMGB1/TLR4 pathway to exert protective effects.


Introduction
Renal ischemia and reperfusion injury (IRI), which usually occurs in patients undergoing kidney transplantation, can increase the rates of acute kidney failure, delayed graft function, and early mortality 1 .In the pathogenesis of renal IRI, there has some factors implicated in it, such as vasoconstriction, oxygenderived free radicals, loss of proximal tubular cell polarity 2 .The acute inflammation caused by renal IRI is characterized by the induction of an inflammatory cytokine cascade, expression of adhesion molecules, and cellular infiltration 2 .Therefore, the strategy to inhibit inflammatory response in IRI will afford effective protection to reduce tissue injury and improve prognosis.
Dexmedetomidine (Dex) is a highly selective α2adrenoreceptor agonist, with sedative, analgesic, sympatholytic and hemodynamic stabilizing effects 3 .There is accumulating evidence to suggest that Dex has organoprotective effects, including kidney, brain, heart and intestine [4][5][6][7] .It has been reported that Dex can increase urine output through the inhibition of vasopressin secretion and the enhancement of renal glomerular filtration.Also, it has been found that Dex can alleviate renal IRI through the effect of cytoprotection 8 .However, the protective mechanism of Dex against renal IRI still remains unclear.
Recently, the effect of HMGB1 and TLR4 has been investigated in organic IRI, including heart, liver and brain [9][10][11] .High mobility group box 1 (HMGB1) can be released from necrotic cells to the extracellular or secreted by the immune cells after proinflammatory stimulation 12 .As a alarmin, HMGB1 in the extracellular can initiate inflammatory reaction caused by IRI, which has been demonstrated by the model of hepatic IRI in mice 13 .Toll-like receptors are a family of transmembrane proteins.Its activation leads to over-expressions of inflammatory cytokine gene leading to pro-inflammatory response 14 .The previous study was found that Dex could have beneficial effect against intestinal IRI through the reduction of inflammation 15 .However, whether Dex could inhibit inflammation caused by renal IRI still remains unknown.Therefore, the aim of this study was to determine whether Dex could attenuate long term inflammation induced by renal IRI and its possible mechanism.

Animal model of I/R
This project was approved by the committee of experimental animals of Wuhan University, and the procedures were carried out according to the routine animal-care guidelines.
All adult male SD rats (220-250g) were from the Center of Experimental Animals in Medical College, Wuhan University.
Briefly, rats were anesthetized with pentobarbital (45 mg/kg) and placed on a homeothermic table in order to maintain core body temperature at 37°C.The midline laparotomy was made and right nephrectomy was performed.After it, the left kidney was subjected to 45 min of ischemia followed by eight weeks of reperfusion.
All animals were divided into three different group: sham group, IRI group and Dex group.Each group had 6 rats.In sham group, only the right kidneys were removed.In IRI and Dex group, the left kidney vessels were clamped for 45min followed by eight weeks of reperfusion.In Dex group, dexmedetomidine(100μg / kg, i.p., based on the previous study 16 ) was administrated 30 min before ischemia.While rats in IRI group and sham group were simultaneously injected the equal volume of PBS.At eight weeks of reperfusion period, the left kidneys were removed for the following experiments and the blood samples were collected for the detection of blood urea nitrogen (BUN) and creatinine (Cr) levels.

Sampling
The left kidney was removed under fully maintained anesthesia.After removal, the kidney was fixed in 10% phosphatebuffered formalin or immediately frozen, and stored at -80°C for following experiments.
At eight weeks after IRI in every group, 1 ml blood samples were taken and performed according to directions of the commercial kits (Nanjing Jiancheng Co., China).The absorbance was measured by spectrophotometer and then the concentrations of BUN and Cr were calculated.
Proteinuria was determined at the end of eight weeks from 24-h urine collections according to directions of the commercial kits (Nanjing Jiancheng Co., China).

Histologic examinations
After the kidney fixed in 10% phosphate -buffered formalin, it was embedded with paraffin and sectioned at 4-μm thick.The sections were deparaffinized and hydrated gradually, and stained with hematoxylin and eosin (H&E).Morphologic assessments were observed by an experienced renal pathologist who was unaware of the treatments.

Masson's trichrome staining
After the kidney fixed in 10% phosphate -buffered formalin, it was embedded with paraffin and sectioned at 4-μm thick.The sections were deparaffinized and hydrated gradually, and stained with Masson's trichrome.Morphologic assessments were observed by an experienced renal pathologist who was unaware of the groups and treatments.

Immunohistochemistry
The immunohistochemical staining of CD3 was performed using UltraVision™ Quanto Detection System HRP DAB sample kit (Thermo Scientific).The CD3 antibody (1:100 dilution; SantaCruz Biotechnology, Santa Cruz, CA) was incubated at 4ºC overnight.The steps were following the manufacturer's instruction.

Western Blot analysis
Total proteins were extracted, and quantified using Bicinchoninic acid method.Then, equivalent weights of protein (40 μg/lane) was separated on 10% SDS-PAGE gels and then transferred to nitrocellulose membrane.The membranes were blocked with 5% non-fat milk in TBST buffer and then incubated with the following primary antibodies: HMGB1 (1:1000 dilution; Abcam, Cambridge, MA) and TLR4 (1:1000 dilution; SantaCruz Biotechnology, Santa Cruz, CA).Subsequently, after being washed twice with TBST, the membranes were incubated with secondary antibody conjugated with horseradish peroxidase at 1:2000 dilution.Specific bands were visualized by using an enhanced chemiluminescence detection kit.

Statistical analysis
Data were presented as mean±SEM.The means of the different groups were compared using one-way ANOVA 3 Student-Newman-Keuls test.Differences were considered statistically significant when p<0.05.

Functional parameters
It was manifest from the results that the levels of BUN and Cr had no significant difference in three groups.But Dex could decrease the level of proteinuria caused by renal IRI (Figure 1).However, the treatment with Dex could reduce these severe renal damages (Figure 2).

Immunohistochemistry
In our study, the expression of CD3 (Figure 2) was detected by immunohistochemical staining.The results revealed that CD3 positive cells were rarely found in sham group.But in IRI group, there were more CD3 T-cell infiltrates.Compared with IRI group, this change was ameliorated in Dex group.

Masson's trichrome staining
The result revealed that IRI could lead to renal fibrosis and rats treated with Dex had less fibrosis compared with rats in IRI group (Figure 2).

Realtime PCR analysis
The relative mRNA expression of TNF-α, IL-1β and ICAM-1 to β-actin were shown (Figure 3).These mRNA levels were significantly greater in IRI group than those in sham group.
However, the treatment with Dex could significantly reduce the mRNA expression of TNF-α, IL-1β and ICAM-1 after IRI.

Western Blot analysis
To investigate the different levels of protein expression, we measured HMGB1 and TLR4 by Western blot (Figure 4).It was obvious from the results that the expressions of HMGB1 and TLR4 were increased after subjected to IRI when compared with sham group.But Dex could attenuate these expression induced by renal IRI.

Discussion
IRI has elicited an increasing interest of the researchers in its impact on such organs as kidney, liver and heart in recent years.Inflammation represents key factor in the occurrence and development of ischemic damage, which is thought to occur secondary to an intense inflammatory response initiated by the infiltration of leukocytes and the production of proinflammatory cytokines after IRI 17 .Previous studies has demonstrated that renal ischemic damage can lead to severe inflammation after 24 hours of reperfusion 18 .In addition, recent study also found that after 12 weeks of reperfusion, renal IRI could result in long term inflammation 19 .
In this study, a 45-min rat renal ischemia model was established to investigate the protective effect of Dex against renal IRI, which was induced by unilateral clamping of renal vascular structures.Renal injury was examined by the renal function and H&E staining after 8 weeks of reperfusion.At current study, the levels of BUN and Cr had no significant difference in three groups.
But histopathology result showed that renal IRI resulted in severe renal injury, as evidenced by inflammatory infiltration and loss of renal parenchyma characterized by tubular atrophy, rarefaction of peritubular capillaries, and podocyte depletion 20 .However, Dex can significantly reduce these severe renal damage.These results were in accordance with prior study which has shown that although renal function at the end of 12 weeks showed no significant differences among all groups, their results showed that IRI induced severe renal injury 19 .
Dexmedetomidine (Dex) is a highly selective α2adrenoreceptor agonist, with sedative, analgesic, sympatholytic and hemodynamic stabilizing effects 3 .In addition, it has been reported that Dex has organoprotective effects against ischemic damage, including celebral, hepatic, cardiac, renal and testicular IRI 4,6,7,21,22 .Gonullu et al. 16 showed that the preconditioning or postconditioning with the administration of Dex could both reduce the renal IRI histomorphologically, as the renal histopathological score in the IRI group was significantly higher than the groups with Dex.Other studies also demonstrated that the administration with Dex could reduce the number of apoptotic tubular epithelial cells and attenuate renal tissue necrosis and histological lesions in a rat IRI model 7,8 .Besides, wang et al. 23 showed that innormoglycemic condition Dex could attenuate renal damage caused by IRI, but in hyperglycemic condition there was no difference between Dex and IRI group.These studies just investigate the protective effect of Dex after 24 hours of reperfusion under ischemic stimuli and our data showed that the model of 45 minutes' ischemia and 8 weeks' reperfusion could result in long term tissue damage, including inflammation, as the evidence of H&E staining and immunohistochemical staining with CD3.However, the treatment with Dex prior to ischemia could significantly reduce renal damages caused by IRI and attenuate the CD3 T-cell infiltrates.
A growing body of evidence indicates that the inflammatory response, associated with pro-inflammatory cytokines IL-1β, TNF-α and chemotactic cytokine MCP-1, plays a key role in renal dysfunction following IRI 24 .ICAM, as an adhesion molecule, could facilitate leukocytes infiltration and adhesion to

Conclusion
Dexmedetomidine has the long term protective effect against renal IRI through the alleviation of inflammation.

FIGURE 1 -
FIGURE 1 -(A) Effects of Dex on the serum Cr concentrations after 45 min of ischemia followed by 8 weeks of reperfusion.(B) Effects of Dex on the serum BUN concentrations after 45 min of ischemia followed by 8 weeks of reperfusion.(C) Effects of Dex on the proteinuria after 45 min of ischemia followed by 8 weeks of reperfusion.Bars means ± SEM (n=4).

FIGURE 2 -
FIGURE 2 -Histologic features were evaluated by H&E, immunohistochemical and Masson staining.(A, B, C) Representative kidney sections stained with H&E.(D, E, F) The expression spots of CD3 in kidneys at the end of reperfusion 8 weeks.(G, H, I) The fibrosis caused by ischemic damage in kidneys at the end of reperfusion 8 weeks.Sham group (A, D, G): Section from sham-operated rat.IRI (B, E, H): Section from rat subjected to IRI .Dex (C, F, I): Section from rat treated to Dex.H&E staining, original magnification ×200; Immunohistochemical staining, original magnification ×200; Masson staining, original magnification ×200.

FIGURE 3 -
FIGURE 3 -The mRNA level of TNF-α, IL-1β and ICAM-1 in the kidney.(A) Effects of Dex on the mRNA level of TNF-α after 45 min of ischemia followed by 8 weeks of reperfusion.(B) Effects of Dex on the mRNA level of IL-1β after 45 min of ischemia followed by 8 weeks of reperfusion.(C) Effects of Dex on the mRNA level of ICAM-1 after 45 min of ischemia followed by 8 weeks of reperfusion.mRNA was standardized for β-actin mRNA.Bars represent means ± SEM (n=3); *p<0.05versus Sham, # p<0.05 versus IRI.

FIGURE 4 -
FIGURE 4 -Representative Western blots showed the effects of Dex on HMGB1 and TLR4 expression in the kidney after 45 min of ischemia followed by 8 weeks of reperfusion.β-actin was used to show equal amounts of protein loading in each lane.(A) Representative Western blots showing the effects of Dex on HMGB1 and TLR4 expression.(B) Relative band densities of HMGB1 to the mean value of the control.(C) Relative band densities of TLR4 to the mean value of the control.Bars represent means ± SEM (n=3); *p<0.05versus Sham, # p<0.05 versus IRI.