L-Arginine-enriched preservation solution decreases ischaemia/reperfusion injury in canine kidneys after long-term cold storage

Serdar Erkasap and Ersin Ates

Department of General Surgery, University of Osmangazi, Eskisehir, Turkey



   Abstract
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
Background. Kidneys stored hypothermically for transplantation show varying degrees of tissue injury, depending on the duration of preservation. The causes of injury are not entirely clear. We investigated the quality of renal functional recovery in canine kidneys after 72 h hypothermic preservation in custodiol solution or custodiol solution plus L-arginine.

Methods. Kidneys obtained from mongrel dogs, weighing 18–25 kg, were subjected to 72-h cold ischaemia after flushing. Animals were divided into two groups (n=18/group). A flush solution of either custodiol solution or custodiol solution plus L-arginine 1 mmol/l was used for each group. After 72-h cold storage all animals had a contralateral nephrectomy, and autotransplantation was performed to external iliac artery and vein. Survivals were evaluated at 3 days.

Results. Renal damage was assessed by kidney function tests, serum creatinine (SCr), blood urea nitrogen (BUN) and light histology. Malondialdehyde (MDA) was measured as an index of lipid peroxidation. SCr and BUN (24, 48 and 72 h) were significantly different from the control and L-arginine groups. Histological damage was less in the L-arginine group. MDA levels were significantly different with the lower levels in the L-arginine group.

Conclusions.On the basis of these data, we concluded that exogenous L-arginine (a substrate for NO synthesis) has a beneficial and protective effect on long-term (72 h) hypothermic ischaemical damage in canine kidneys.

Keywords: canine kidney; cold ischaemia; ischaemia/reperfusion; L-arginine; nitric oxide



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
It has been shown that kidneys may be preserved for 30 h in cold storage and for as long as 72 h by continuous perfusion. Many investigators have tested a variety of cold-storage solutions and some have claimed successful preservation for 48 to 72 h [1,2]. It has also been shown, however, that graft survival rates differ significantly between kidneys stored for 36 h and >36 h [2]. Hypothermia does not stop metabolism, rather it simply slows reaction rates and cell death until the organ ultimately ceases to function and loses viability. There are many theories pertaining to the effectiveness of hypothermia.

Recent studies report that reperfusion after ischaemic injury causes tissue damage so that suppression of reperfusion prevents organ function disturbance more effectively [3]. Reperfusion injury is related to reactive oxygen species, breakdown of cellular calcium metabolism, loss of high-energy substrates, capillary blockage that results from preformed blood elements and reduced blood flow [4,5].

There is evidence that nitric oxide (NO) decreases kidney allograft reperfusion injury [6]. NO has a variety of biologic properties that might affect ischaemia/reperfusion injury in allografts; it is a potent vasodilator and it has been shown to play a critical role in the maintenance of vascular integrity through its interaction with neutrophils [7,8], platelets [9], and vascular endothelial cells [10].

NO is synthesized, via L-arginine oxidation, by a family of NO synthases (NOS) [11]. Multiple experiments in different species have confirmed that NOS blockade increases systemic blood pressure and modifies renal function [12,13]. NOS blockade decreases glomerular filtration rate (GFR), renal plasma flow (RPF), and urinary sodium excretion, in different species both when awake and when under anaesthesia [12,13].

The role of NO in the various pathophysiologic conditions depends therefore not only on decreased generation of NO but also on its excess production. Multiple factors in the NO milieu affect and modulate NO activity, such as conversion of an anti-inflammatory factor to a pro-inflammatory factor and transformation of a cytoprotective agent into a major cytotoxic factor [12]. L-Arginine has also been reported to have beneficial effects on long-term continuous perfusion of canine kidneys [14].

Based on the above information, we designed a study to examine the potential beneficial effects of an L-arginine-enriched preservation solution on prolonged cold-induced renal ischaemia and subsequent reperfusion injury in canine kidneys.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In this experimental study, 36 mongrel dogs of either sex, weighing 18–25 kg (21±0.7 kg) were used. The dogs were provided by Eskisehir Environmental Health Institute. Local Ethical Committee approval was obtained in accordance with institutional requirements. Following 14 days of observation, the dogs were deprived of chow, but allowed to have free access to water. Initial anaesthesia was achieved by an i.m. injection of ketamine hydrochloride (Ketalar, Parke Davis) at 10 mg/kg body weight. This was followed by induction of general anaesthesia by an i.v. injection of an 8 mg/kg dose of thiopental sodium (Pentothal Sodium, Abbott) via a catheter placed in the right femoral vein, with subsequent supplemental doses given as needed. To ensure adequate hydration, an i.v. infusion of 5% dextrose in 0.9% saline solution was administered at the rate of 75 ml/h throughout the procedure.

Left nephrectomy was performed on all the dogs through a midline incision. Immediately after nephrectomy, the left renal artery was cannulated (warm ischaemia for 1–2 min) and the blood was flushed from the kidney under aseptic conditions. All the dogs were randomly assigned to one of two groups. In group I (control group, n=18); kidneys were flushed with cold (4°C) custodiol solution (Dr F. Kohler, Chemie GmbH, Germany), suspended 100 cm above the kidney. Flush-out volume was 150–200 ml. The kidneys were stored at 4°C for 72 h in the same preservation solution. In group II (L-arginine group, n=18); kidneys were treated identically except that the Custodiol flush solution was enriched with L-arginine (1 mmol L-arginine/l, Sigma Chemical Company, St Louis, MO). The concentration of L-arginine was similar to that used by Saunder et al. [14]. The kidneys were kept in storage for 72 h.

The animals were anaesthetised once again at 72 h and the right kidneys were removed. The preserved left kidneys were subsequently autotransplanted, establishing end-to-side anastomoses for both arteries and veins to the external iliac vessels (average time to anastomoses: 26±5 min). The distal ureter was implanted into the bladder using a simple fishmouth type of technique.

A total of 12 dogs (six dogs randomly selected from each group) were taken back to the operating room exactly 1 h after the re-anastomoses of the left kidneys and tissue specimens were obtained and stored immediately at -70°C for future tissue malondialdehyde (MDA) level determinations.

An additional 12 dogs (six per group) were taken for histological examination. Tissue specimens were obtained from these 12 animals at 24 h after reperfusion and processed in 10% formalin.

Peripheral blood specimens were obtained from 12 more dogs (six per group) for 0, 24, 48, and 72 h following re-anastomosis for serial blood urea nitrogen (BUN) and serum creatinine (SCr) level determinations.

MDAs
Determination of kidney tissue MDA level was made using an assay described by Van Ye et al. [15]. The kidney tissue was homogenized in a buffer (100 mg/ml) at pH 7.4. Artifactual production of MDA during processing was eliminated by adding 2% butylated hydroxytoluene to homogenized tissue. After this step, 20% trichloroacetic acid in 0.6 N HCl was added. The mixture was centrifuged at 10 000 g for 10 min at 4°C. Thiobarbituric acid (0.12 M) was added to the supernatant prior to cooling at room temperature. Colour reaction was measured spectrophotometrically at 532 nm.

Histology
Kidney tissue specimens obtained 24 h after ischaemia/reperfusion were fixed in 10% formalin and embedded in paraffin. Four samples taken from each kidney were sectioned at 5–6-µm thickness and stained with haematoxylin–eosin. These were then submitted for blinded histopathologic examination and grading. Light-microscopic sections were examined (10 random fields with objective of x40) for tubular vacuolization, tubular dilatation, tubular necrosis, intratubular detachment, tubular cell brush border integrity and interstitial oedema. The tubulointerstitial damage score was calculated by averaging the mean score of each of the following pathological characteristics [16]: 1, no abnormality; 2, mild lesions affecting 10% of the kidney samples; 3, lesions affecting 25% of the kidney samples; 4, lesions affecting 50% of the kidney samples; and 5, lesions affecting 75% of the kidney samples.

Biochemical determinations
The peripheral blood specimens for BUN and SCr determinations were processed by using a Hitachi automatic multi analyser.

Statistical analysis
Most data were analysed using Student's t-test. Histology scores were performed by analysis of variance. Statistical significance was set at P<0.05. Data are presented as mean±standard deviation (SD) of the mean.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Lipid peroxidation
MDA levels in transplanted kidney tissue specimens from control subjects were significantly higher than those from subjects treated with L-arginine-enriched custodiol solution (P<0.001) (Figure 1Go).



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Fig. 1. Kidney tissue MDA levels were performed after 72-h cold storage and 1-h reperfusion in custodiol-flushed and custodiol plus L-arginine-flushed kidneys. Values are mean±SD (*P<0.001).

 

Histology
The tubulointerstitial damage in transplanted kidneys was significantly decreased by L-arginine (P<0.001). The results of this histological study are presented in Table 1Go.


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Table 1. Histopathological evaluation of kidneys after 24-h reperfusion

 

Biochemical determinations
BUN and SCr levels were determined on multiple specimens obtained from 12 animals (six from the control group; six from the L-arginine group) at 0, 24, 48, and 72 h. BUN and SCr levels were not significantly different between control and L-arginine groups at 0 h. However BUN levels were significantly higher in the control group than the L-arginine group at 24 (P<0.01), 48 (P<0.001), and 72 h (P<0.001) (Figure 2Go). SCr levels were significantly higher in the control group than the L-arginine group at 24, 48, and 72 h (P<0.01, P<0.01, P<0.05 respectively) (Figure 3Go). The addition of L-arginine to the perfusion solution in this study resulted in a significantly faster improvement in the abnormal BUN and SCr values to near normal levels compared with controls (Figures 2Go and 3Go).



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Fig. 2. BUN levels of all subjects following the 72-h cold-storage period (‘0’ is the time of re-anastomosis). Values are mean±SD (*P<0.01; #P<0.001).

 


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Fig. 3. Serum creatinine (SCr) levels of all subjects following 72-h cold storage (‘0’ is the time of re-anastomosis). Values are mean±SD (*P<0.01; #P<0.05).

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Kidneys stored hypothermically for transplantation show varying degrees of tissue injury, depending on the duration of preservation. The causes of this injury are not entirely clear. It is clear however that organs preserved for a shorter period have better long-term survival [17]. The prevention or reversal of damage to organs stored for long periods may be possible by suppressing the factors affecting delayed graft function or the loss of organ viability. This may be accomplished either by the development of improved storage methods or by the use of a combination of pharmacologic agents during reperfusion to suppress reperfusion-induced injury [18].

Several hypotheses have been put forward that attempt to outline the mechanism of ischaemic damage. Satoh et al. suggested that post-ischaemic renal failure is a result of injury to the endothelium and suppression of synthesis of nitric oxide [19]. Furthermore, Pinsky et al. demonstrated that NO levels sharply decrease after preservation and reperfusion [20]. Others have suggested that the endothelial injury suffered during hypothermic preservation with the resultant decrease in NO production could lead to further vascular injury by way of platelet adhesion to vascular walls, vascular smooth muscle contraction, and increased renal vascular resistance [14]. Yamashita and colleagues proposed that NO administered at harvest in the flush solution might enhance vascular homeostasis and integrity of the donor organs during ischaemia [21].

NO inhibits cytokine-stimulated expression of vascular cell adhesion molecule-1, selectins, and intercellular adhesion molecule-1. NO also exerts an inhibitory effect on platelet adhesion and aggregation and this results in increasing levels of platelet cGMP [22]. Because of the above properties, NO may potentially improve the allograft kidney function by a variety of mechanisms, so in the present study, we added L-arginine to the preservation solution as a substrate for NO synthesis.

Tissue MDA activity is an index of lipid peroxidation. Lipid-based cell membranes are a primary target of oxygen-derived radicals [24]. End-products of lipid peroxidations include MDA, other aldehydes, hydrocarbon gases, and conjugated dienes [25]. We found that tissue MDA levels were lower in autografts treated with L-arginine, suggesting diminished lipid peroxidation and tissue injury. Our results are consistent with other reports describing increased levels of MDA as an index of reperfusion injury [21,26].

We also found that addition of L-arginine to the preservation solution resulted in a significant reduction in ischaemic damage to autograft kidneys, which were transplanted following 72-h cold ischaemia, as evidenced by the histopathologic evaluation of kidney tissue obtained 24-h post-transplant.

In this study, serial BUN and SCr level determinations performed during the first 3 days post-transplant were also consistently lower in the L-arginine group. Saunder et al. produced similar results in a study using a long-term machine perfusion technique [14]. Our study also showed that with an L-arginine concentration of 1 mmol/l, a single flush is as effective as long-term machine perfusion.

Saunder et al. claimed that during long-term machine perfusion endogenous renal arginine may either leak into the perfusate or is metabolized. Our study suggests that using a single flush method might cause metabolization of L-arginine rather than a leak into the perfusate.

We conclude that L-arginine improved long-term hypothermic-storaged autograft kidney function in this study. Our findings include reduced tissue MDA levels, limited tissue damage, and a more rapid recovery of abnormal BUN and SCr values.



   Acknowledgments
 
This work was supported by Osmangazi University Research Institute.



   Notes
 
Correspondence and offprint requests to: Serdar Erkasap, Hasan Polatkan Bulvari, Akin Sitesi, No. 122/13, Eskisehir, Turkey. Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 4.10.99
Revision received 7. 4.00.



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