Ischaemia-reperfusion injury (IRI) is a common clinical problem, and therefore avoiding and attenuating the influ- ence of IRI on organ function is of great significance. Recently, some researchers found that advance inducing the expression of heat shock protein (HSP), an auto-protective mechanism of the organism, would alleviate IRI. However, most methods (including heat induction, ischaemic pre- treatment and chemicals) being used at present for inducing HSP are clinically ineligible, and most of the inductiondrugs are toxic. It is an urgent task to find a safe, atoxic and effective HSP inducer for clinical use. Some researchers found in their recent studies that glutamine is such a poten- tial HSP inducer.

        The correlation between glutamine and HSP was first reported by Sanders et al.¹ in their study of fruit fly Kc, where they found that the greatest cell expression occurred only when glutamine was added. Under thermal stress, addi- tion of glutamine was able to increase HSP expression by 10 times. To see whether glutamine could induce HSP in animals, Wischmeyer et al.² used Sprague–Dawley (SD) rats to establish an animal model, where the animals were administered i.v. with lipopolysaccharide and 0.75 g/kg glutamine. They found that administration of glutamine greatly reduced mortality and end-organ injury arising from endotoxin-induced sepsis in the animals, and that this pro- tective effect was related to increased expression of HSP72 and HSP27 in the heart, lungs, liver, kidneys and colon of animals. In their study of cardiomyocytes, Wischmeyer et al.³ also found that glutamine induced cardiomyocytes to produce HSP72 to raise cell viability and help recover their function of contraction after IRI stress. In their study of the protective effect of glutamine on lungs, Singleton et al.^4,5 established a rat sepsis model, where they injected glutamine or normal saline (NS) by tail vein into the animals and used quercetin (Que; a HSP inhibitor) as control. They found that glutamine promoted HSP expres- sion of epithelia and macrophages of the lungs, and allevi- ated the pulmonary injury and metabolic dysfunction of the lungs. An overview of these data seems to suggest that glutamine is an inducer that can induce high HSP expres- sion of almost all organs.

          Studies showed that expressions of HSP70, heme oxyge- nase (HO-1) and small molecular weight HSP were all upregulated in the presence of renal ischaemia.^6,7 Either sodium arsenite,⁸ or small dosage of cyclosporin A,⁹ or tran- sient ureteral ligation¹⁰ may induce upregulation of HSP 70 expression, exerting a significant protective effect against IRI to renal tissues. Neuhofer et al.¹¹ found that transfection of human (inducing) HSP72 clone to renal cells of Madin- Darby dogs to induce large amounts of HSP72 expression may significantly increase 24 h viability of cells in highly concentrated urea solution, suggesting that high expression of HSP72 in the medulla of kidney may play an important role in resisting the high concentration of urea in the medulla. The above findings suggest that pre-induction of HSP may alleviate IRI to the kidneys. The question is whether we can use glutamine to induce renal HSP in advance to alleviate IRI to the kidneys.

          Fuller ¹² administered glutamine (0.75 g/kg) or saline i.p. to male donor rats 24 h and 6 h before donor nephrectomy. Kidneys (n = 6/group) were cold-stored in University of Wisconsin (UW) solution for 40 h and transplanted into bilaterally nephrectomized syngeneic recipients. Within
24 h, these transplants were examined for serum creatinine, histology, apoptosis and expression of HSP. HSP were elevated after glutamine i.p treatment, histology improved and apoptosis reduced.
          In Fuller et al.’s study,¹² glutamine was administered i.p. to male donor rats 24 h in advance. Could renal IRI also be relieved by i.v. administration of a single dose of glutamine? In their experiment, the kidneys (n = 6/group) were cryo- preserved in UW solution for 24 h. Could the same conclu- sion be drawn when the kidneys experienced warm ischaemia-reperfusion?

         In the present experiment, we used a single dose of glutamine i.v. to study renal IRI, as i.v. drug administration is more convenient than i.p. drug administration in clinical practice. Furthermore, most clinical cases of renal ischaemia occur at normal temperatures such as severe trauma, haemorrhagic shock and dehydration, and therefore warm ischaemia injury seems to be more commonly seen than cold ischaemia injury. If glutamine could be proved to attenuate normothermic renal ischaemia-reperfusion injury, it would be of some value in reducing acute renal injury in clinical cases. Thus, a warm renal IRI (RIRI) rat model was established to see whether pre-treatment of glutamine i.v. (0.75 g/kg) could alleviate RIRI.


METHODS

The study was approved by the animal ethics committee of the Second  Military Medical University (Shanghai, P.R. China).

Animal grouping

Forty-eight SD rats of either sex weighing approximately 250 g were equally randomized into four groups: control (untreated) group, glutamine group (Gln group), quercetin (HSP inhibitor) plus glutamine (Gln + Que) group and quercetin + NS group. All animals were housed at 22°C, exposed to a light–dark cycle and given water ad libitum.


Materials

Glutamine was purchased from the High-Tech Company of the Second Military Medical University. Quercetin was a product of Sigma Chemi- cal (St Louis, MO, USA). HSP70 (W27) sc24 type primary antibody (rabbit antirat) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Western Electrotransfer System was a product of Bio-Rad (Richmond, CA, USA). A terminal deoxynucleotidyl trans- ferase dUTP nick end labelling (TUNEL) apoptosis assay kit was pur- chased from Shenzhen Jing Mei Bioengineering Company (Shenzhen, P.R. China). An immunohistochemical universal EnVision kit (HRP- labelled secondary antibody) was purchased from Zymed Laboratory (Seattle, WA, USA).

Medical intervention of the four groups

In the Gln group, 3% glutamine was administered at 0.75 g/kg at a rate of 0.5 mL/min i.v. by tail vein; in the control group, NS was adminis- tered at the same dose and rate as the Gln group; in the Gln + Que group, quercetin as a HSP inhibitor was administered 400 mg/kg i.p. 6 h in advance, followed by glutamine administration as the Gln group; in the Que + NS group, quercetin was administrated as the Gln + Que group, followed by NS as the control group. The RIRI rat model was established within 1 h of medical intervention. The animals were anaesthetized and the kidneys were exposed by laparotomy. The renal pedicles were isolated, making sure that the ureters were well protected. The bilateral renal arteries and veins were occluded by silk thread for 45 min and then released. Macroscopic evidence of the kidneys turning from dark red to bright red indicated successful reperfusion. Finally, the incision was sutured. The animals were killed 6 and 24 h after reperfu- sion, and blood sample and kidneys were taken for use.


Examination indexes

Blood specimens were centrifuged and tested for serum creatinine (Cr) and blood urea nitrogen (BUN) with an auto-biochemical analyzer. The kidney specimens were HE stained and observed by light micros- copy. HSP was assayed by immunohistochemical EnVision and western blot. Apoptotic index (AI) was assessed by TUNEL.
         For Paller et al. scoring,¹³ 10 HE stained tubules were selected for
each field and observed under a light microscope (¥400) to count five fields, totalling 50 tubules. By the Paller method, evidence of signifi- cantly dilated tubules and flattened cells was expressed as a score of 1, injury or exfoliation of brush border as 1 or 2, casts as 2, and presence of exfoliated necrotic cells in empty tubules without forming casts or fragments as 1.

        For immunohistochemical EnVision result scoring, 10 fields were selected randomly and observed under a light microscope (¥400). The number of positive cells and the total number of cells in each field were counted. The positive rate was calculated and scored as follows with reference to the Riera criteria:¹⁴ absence of positive cells was considered as negative (0), positive cells less than 25% as weakly positive, (2) 25–50% as positive (4) and more than 50% as strongly positive (6). Finally, the overall score was calculated.
         For TUNEL AI scoring, the number of positive cells was calculated in 10 high-power fields under an ordinary light microscope, and con- verted to the mean number of apoptotic cells in each mm² as AI. The apoptotic rate was then calculated.


Statistical treatment

         All resultant data were treated with SPSS ver. 10.0 software (SPSS, Chicago, IL, USA). Measurement data were by analyzed by anova. Enumeration data were analyzed by  x²-test and the histological data were analyzed by the Kruskal–Wallis test. P < 0.05 was considered sta- tistically significant.

RESULTS

Results of kidney specimen HE staining

         The results of HE staining (Fig. 1) and Paller scoring (Table 1) indicated that injuries in the Gln group were alleviated significantly compared with those of the control group (P < 0.01). Injury in the Gln + Que group and the Que + NS group was more severe than that in the control group (P < 0.01), showing that use of glutamine played a role in alleviating RIRI, and this effect disappeared after using HSP inhibitor quercetin. The differences within the Gln group were statistically significant (injury at 6 h of ischaemia-reperfusion was more severe than that at 24 h), indicating that injury became less severe gradually with time lapse of ischaemia-reperfusion (P < 0.05).


Results of immunohistochemical EnVision staining

The results of immunohistochemical EnVision staining (Fig. 2) and Riera scoring (Table 2) indicated that HSP expression in the Gln group was significantly stronger than that in the control group (P < 0.01), while HSP expression in the Gln + Que group and Que + NS group was signifi- cantly weaker than that in the control group (P < 0.01), indicating that glutamine had an effect of promoting HSP expression, and that this effect disappeared after use of HSP inhibitor quercetin.

        In the control group, HSP expression at 24 h (n = 6) was higher than that at 6 h (n = 6) (P < 0.05), indicating that  HSP expression was enhanced at 24 h as compared with that at 6 h in normal rats undergoing RIRI. There was no time-phase difference at 6 and 24 h (n = 6) in the Gln group, Gln + Que group and Que + NS group.

Results of western blot 

Figure 3 shows that HSP expression was the strongest at 6 and 24 h in the Gln group, and that HSP expression was weaker in the control group than that in Gln group, but stronger than that in the Gln + Que group and Que + NS group. 


 

TUNEL results of apoptosis

TUNEL results of apoptosis were shown in figure 4. The AI of the Gln group was significantly lower than that of the control group (P < 0.01). There was no significant differ- ence in AI within all groups (P > 0.05). These results suggest that glutamine-induced HSP was able to alleviate apoptosis, and this effect disappeared after the use of HSP inhibitor quercetin.


CR and BUN changes at 6 and 24 h of renal ischaemia-reperfusion

The results in Tables 3 and 4 show that CR and BUN levels at 24 h after renal ischaemia-reperfusion were significantly higher than those at 6 h (P < 0.05). In the Gln group, both CR and BUN decreased significantly (P < 0.05), while CR and BUN levels in the Gln + Que group and Que + NS group were significantly higher than those in the other two groups (P < 0.05).


DISCUSSION

The results of the studies by Uehara et al.¹⁵ and Singleton et al.⁵ not only offered a preliminary explanation of the important role of HSP in the protective effect of glutamine but put forward a disputable idea about the pharmacology and administration of glutamine. In previous human and animal studies, glutamine was administered enterally or parenterally at a dose of 0.2–0.3 g/kg for 12–24 h, but they recommended i.v. administration of glutamine by a single dose of 0.75 g/kg. Ex vivo studies ^16–18 showed that glutamine at a plasma concentration of 4–8 mmol/L could promote HSP expression best. In the studies of Wischmeyer et al.,² Uehara et al.¹⁵ and Singleton et al.,⁵ where they used i.v. injection of glutamine at a dose of 0.75 g/kg in an endotoxin-induced sepsis rat model, the plasma concentra- tion of glutamine was 3–7 mmol/L. This dose was able to increase HSP expression in multiple organs, reduce release of inflammatory cytokines and improve the survival of post endotoxin-induced sepsis.
          In the present study, we pre-treated the animals with a single dose of glutamine i.v. (0.75 g/kg) 1 h in advance and then observed IRI at normal temperature. The results of HE staining and TUNEL assay showed that the injury and apo- ptosis were alleviated significantly in the Gln group as com- pared with the control group, indicating that glutamine pre-treatment was effective in alleviating IRI in rats. The results of immunohistochemistry and western blot showed that HSP expression was significantly higher in the Gln group than that in the control group. In the Gln group and Gln + Que group, with the use of HSP inhibitor quercetin,¹⁹ HSP was blocked, its expression was suppressed, and there- fore injury and apoptosis became worse. These findings sug- gests that the mechanism of glutamine in reducing renal IRI is partially due to the induction of HSP expression, through which anti-inflammatory, anti-injurious and anti-apoptotic actions work. Pertinent research shows that Gln depletion potentiates acute inflammation, possibly by increasing neu- trophil migration through resident cell activation and pro- duction of interleukin (IL)-1b and tumour necrosis factor (TNF)-a.²⁰ Gln supplementation reverses these effects and may be useful during inflammatory catabolic stress. Addi- tionally, Gln attenuates oxidant stress.²¹ In-depth research on the mechanism of the protective effect of Gln on renal IRI, such as leukocyte aggregation and oxidant stress, is necessary.

        In addition, the results of HE staining revealed that the injury at 6 h was more severe than that at 24 h in the Gln group, and this phenomenon was not observed in the control group. We therefore assume that glutamine- induced HSP not only protects against IRI but plays a pre- liminary repairing role as a molecule-based mate during the period between 6 and 24 h. But this result was not obtainable by the TUNEL method where no difference was found. This may be because apoptosis is a programmed and irreversible process. Although glutamine-induced HSP is anti-apoptotic, it is unable to repair cells that have apoptosed.
         The results of immunohistochemistry and western blot showed that HSP expression in the control group at 6 h was significantly lower than that at 24 h, suggesting that the peak of HSP expression in normal rats was later than that at 6 h after IRI, while in the Gln group, there was no signifi- cant difference in HSP expression between 6 and 24 h, seemingly suggesting that HSP expression had reached the maximum at 6 h post-IRI and lasted for 24 h. It can be concluded that glutamine-induced HSP expression in the kidneys of rats was not only greater than that of the control group, but the peak of HSP expression was much earlier, thus ensuring the repairing effect to occur at an earlier time.
         There is no statistical difference between the Que + Gln group and Que+NS group. The underlying mechanism might be that the effect of Gln was offset by Que for the latter could inhibit HSP to a great extent. The injury was more severe in the Que+NS group than in the control group, which shows that pre-treatment of Que in RIRI might be harmful.
          In conclusion, the present study used the same methods as those of Wischmeyer et al.,² Uehara et al.¹⁵ and Singleton et al.⁵ to observe the effect of glutamine in inducing HSP expression, but in an animal model different from theirs. The results showed that glutamine not only induced the protective effect of HSP on different organs in septic shock, but on the kidneys sustaining IRI.
          Fuller et al.¹² investigated peritoneal injection of glutamine in advance to prevent renal IRI, in which study kidneys experienced a cold ischaemic process. In this study, i.v. injection of glutamine 1 h before renal warm IRI was used. The results showed that i.v. injection of glutamine in advance can protect the kidneys from experiencing the warm ischaemic process.
          The above conclusion was based on the results obtained from the study of HSP70, the changes and effects of other HSP, such as HSP25, in renal IRI awaits further experimen- tal studies.

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