Role of P-selectin and anti-P-selectin monoclonal antibody in apoptosis during hepatic/renal ischemia-reperfusion injury

Abstract

AIM: To evaluale the potential role of P-selectin and anti-P-selectin monoclonal antibody (mAb) in apoptosis during -hepatic/renal ischemia-reperfusion injury.

METHODS: Plasma P-selectin level, hepatic/renal P-selectin expression and cell apoptosis were detected in rat model of hepatic/renal ischemia-reperfusion injury. ELISA, immunohist-ochemistry and TUNEL were used. Some ischemia-reperfusion rats were treated with anti-P-selectin mAb.

RESULTS: Hepatic/renal function insuffic-iency, up-regul ated expression of P-selectin in plasma and hepatic/renal tissue, hepatic/renal histopathological damages and cell apoptosis were found in rats with hepatic/renal ischemia-reperfusion injury, while these changes became less conspicuous in animals treated with anti-P-selectin mAb.

CONCLUSION: P-selectin might mediate neutrophil infiltration and cell apoptosis and contribute to hepatic/renal ischemia-reperfusion injury, anti-P-selectin mAb might be an efficient approach for the prevention and treatment of hepatic/renal ischemia-reperfusion injury.

Key Words: hepatic ischemia reperfusion injury; renal ischemia reperfusion injury; P-selectin; antibody, monoclonal; apoptosis

INTRODUCTION

Visceral ischemia-reperfusion injury is very common clinically. Up to now, there has been no effective measure for its prevention and treatment, so this pathological injury process has aroused great attention. Recently, it is proved that neutrophil infiltration, oxygen free radical, some cell factors (TNF-α and IL-β), adhesion molecules and cell apoptosis are related to ischemia-reperfusion injury[1,2]. Depletion or functional inactivation of neutrophils has been demonstrated to prevent effectively reperfusion-induced tissue damage[3,4]. P-selectin is expressed by activated platelets and endothelium, and contributes to the interactions of these activated cells with polymorphonuclear neutrophils (PMN) or monophages. It is also involved in the early stages of inflammation, thrombogenesis and tissue injury induced by these cells[5,6]. In this article, the role of P-selectin in cell apoptosis in hepatic/renal ischemia-reperfusion injury, as well as the preventive and curative effect of P-selectin monoclonal antibody (mAb) were studied with the rat model of ischemia-reperfusion injury.

MATERIALS AND METHODS

Animal model

Ninety male Wistar rats (Shanghai Experimental Animal Center, Chinese Academy of Sciences), weighing 200 g ± 5 g, were given free access to water for three days before experiment. The rats were anesthetized with 2.5% sodium pentobarbital intraperitoneally, and randomly divided into 2 groups. In one group of rats, the ligament linking liver, diaphragm and abdominal wall wereseparated, portal vein and liver artery that drain blood to left hepatic lobe were freed by blunt dissection and blocked with a microvascular clamp for 60 min, then the clamp was removed, and reperfusion was performed; while in another group, the left renal artery was freed, and blood flow was also blocked with a microvascular clamp for 60 min, then the clamp was removed and reperfusion was given, simultaneously, the right kidney was cut off. The two groups of rats were randomly divided into P-selectin monoclonal antibody treatment group (n = 20) and non treatment group (n = 20). They were divided into 4 subgroups according to the indicated time 1, 3, 6, and 24 h after reperfusion. Five minutes before reperfusion, animals were randomly injected with P-selectin mAb (2 mg/kg, Suzhou Medical College), or 0.9% NaCl solution intravenously. Sham-operated group (n = 5) (anesthesia and opening celiac cavity were performed, not blocking hepatic or renal blood flow) served as control.

Collection and measurement methods of specimens

Blood and left hepatic lobe and renal tissues were harvested at the indicated time. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and blood urea nitrogen (BUN) and creatinine (sCr) were measured with a 747 automatic analyzer (Hitachi Boehringer Mannhein, Mannheim, Germany). Plasma P-selectin levels were estimated with ELISA method. Left hepatic lobe and renal tissues were fixed with 10% formalin, and embedded routinely in paraffin. Sections were cut 5 μm thick and stained with hematoxylin and eosin for light microscope examination. Expression of P-selectin in hepatic and renal tissues was assayed by immunohistochemical method with labelled streptavidin biotin (LSAB) kit (Fujian Maixin Biotechnology Co, Products of Biotechnology Co. CA, USA)[7]. Apoptotic cells in hepatic and renal tissues were detected by TUNEL kit (Boehringer Company). Ten HP visual fields (× 400) were selected randomly within each section to count out apoptotic cells, cell with blue- purple nucleolus belonged to positive cell.

Statistical analysis

Data was presented with mean ± SD, and Student’s t test was used to determine changes between different groups.

RESULTS

Hepatic and renal function evaluation

At 24 h after hepatic ischemia-reperfusion, serum AST (667 U/L ± 142 U/L) and ALT (491 U/L ± 94 U/L) levels in saline-treated group were much higher than the sham group (74 U/L ± 19 U/L and 55 U/L ± 12 U/L, respectively), P ＜ 0.05. Treatment with P-selectin mAb resulted in significantly lower levels of AST (271 U/L ± 65 U/L) and ALT (233 U/L ± 44 U/L) compared with saline-treated group (P ＜ 0.05).

At 24 h after renal ischemia-reperfusion, the levels of serum BUN (52.49 mmol/L ± 8.91 mmol/L) and Cr (456.31 μmol/L ± 65.25 μmol/L) in saline-treated group were much higher than the sham group (7.16 mmol/L ± 1.42 mmol/L and 53.65 μmol/L ± 8.91 μmol/L, respectively), P ＜ 0.01. Treatment with P-selectin mAb, significantly lowered the levels of BUN (20.28 mmol/L ± 3.82 mmol/L) and Cr (167.75 μmol/L ± 32.81 μmol/L) as compared with saline-treated group (P ＜ 0.01).

Histopathologic changes of liver and kidney

After 1 h of ischemia of left hepatic lobe, reperfusion was performed. With the observation of naked eye, the left hepatic lobe was more swollen than the right lobe while under light microscope, edema, degradation with different extent and necrosis of hepatic cells, interstitial congestion and infiltration of inflammatory cells were observed. After 1 h of left renal blood flow blocking on rats, reperfusion followed, with the observation of naked eye, the renal cortex was paler, renal medulla displayed blood stagnation and dark colored; under light microscope, swell, degradation with different extent and necrosis of renal tubular epithelial cell were exhibited; simultaneously, interstitial congestion, edema and infiltration of inflammatory cells were also observed. While in the P-selectin mAb treatment group, the outward appearance of liver and kidney was similar to that of normal. Renal cells and tubular cells showed less swelling and no denaturalization or necrosis, and interstitial changes were not obvious.

Plasma P-selectin level changes

At 24 h after hepatic/renal ischemia-reperfusion, plasma P-selectin levels of saline-treated group (5.16 μg/L ± 1.08 μg/L and 8.92 μg/L ± 3.17 μg/L, respectively) were much higher than the sham group (1.59 μg/L ± 0.25 μg/L and 3.57 μg/L ± 0.89 μg/L, respectively), P ＜ 0.05. Treatment with P-selectin mAb, resulted in significantly lower levels of P-selectin (2.89 μg/L ± 0.72 μg/L and 6.02 μg/L ± 2.15 μg/L, respectively) compared with saline-treated group (P ＜ 0.05).

The expression of P-selectin in hepatic and renal tissue

P-selectin expressed widely within hepatic and renal tissues in early stage of ischemia-reperfusion, which was distributed on small vessels of left hepatic lobe, part of hepatic cellular membrane, renal glomerulomesangium, capillary loops, tubules and interstitium, it was especially remarkable on hepatic small vessels and tubular epithelium. After the treatment with P-selectin mAb, the expression of P-selectin was not displayed in essence.

Cell apoptosis in hepatic and renal tissue

It was displayed with TUNEL method that there was few apoptotic bodies in hepatic and renal tissues in the control group. After 1 h of hepatic and renal ischemia and reperfusion, a few apoptotic bodies emerged in hepatic tissues, renal tubules and interstitium; after 3-6 h, a large quantity of apoptotic bodies were observed in the hepatic or renal tissues, especially in renal tubules; and after 24 h apoptotic bodies gathered in flakes, and inclined to necrosis. The quantity of apoptotic bodies in the tissues of P-selectin mAb treated group was smaller than that in the non-treatment group, especially in renal tubules, there was much fewer apoptotic bodies. The examination results of cell apoptosis in hepatic and renal tissues of each group are shown in Table 1.

Table 1 The quantity of apoptotic bodies in hepatic and renal tissues of each group (mean ± SD, number/× 400).

	Renal ischemia-reperfusion group		Hepatic ischemia-reperfusion group
	n	Quantity of apoptotic bodies	n	Quantity of apoptotic bodies
Control group	5	9.0 ± 3.2	5	8.5 ± 3.4
Non-treatment group
1 h of reperfusion	5	23.4 ± 2.9ab	5	18.3 ± 3.5ab
3 h of reperfusion	5	131.6 ± 7.6ab	5	74.9 ± 6.8ab
6 h of reperfusion	5	209.6 ± 8.4ab	5	94.6 ± 8.1ab
24 h of reperfusion	5	427.8 ± 37.1ab	5	397.4 ± 40.2ab
mAb treatment group	20	13.0 ± 3.9a	20	13.1 ± 3.7a

Compared with control group:

^aP ＜ 0.01; compared with mAb treatment group:

^bP ＜ 0.01.

DISCUSSION

Recently, the role of neutrophil and cell adhesio nmolecules in ischemia-reperfusion injury of multiple organs has aroused extensive attention[5]. Investigators have used mAb directed against key adhesion domains[8]. As a potential member of selectin family, P-selectin is mainly found in both weibel-plade body of epithelial cell of middle and small blood vessels and α-granule of platelet. It is expressed rapidly on the surface of these cells after their activation. In the earliest stage of inflammation, P-selectin initiates the adhesion, rolling of neutrophils, and then recruits them to injured site[6]. Blockade of P-selectin expression or interaction with its ligands can attenuate leukocyte adherence and infiltration during ischemia/reperfusion injury or acute inflammation affording impressive functional and morphologic protection[9-11]. Neutrophils participate in pathological injury through releasing oxygen free radicals and some enzymes. As a large number of neutrophils gather in the injured tissues of ischemia-reperfusion, reducing the infiltration of neutrophils can alleviate the ischemia reperfusion injury evidently.

The expression of P-selectin and effect of P-selectin mAb on ischemia-reperfusion injury were observed in this study based on the established rat model of ischemia-reperfusion injury. Hepatic and renal tissue exhibited heavy histopathologic damages after hepatic/renal ischemia-reperfusion, while the serum AST and ALT levels as well as BUN and Cr were increased. It is indicated that ischemia-reperfusion induced hepatic and renal injury which could be significantly attenuated when P-selectin mAb was administrated 5 minutes before reperfusion as shown by improved hepatic/renal function and less pathologic damage. The results suggested that P-selectin mAb has a protective effect on renal ischemia/reperfusion injury by means of inhibiting interaction of neutrophils and endothelium.

Up-regulated expression of P-selectin in plasma and hepatic/renal tissue after rat hepatic/renal ischemia and reperfusion indicated that P-selectin is associated with hepatic/renal ischemia-reperfusion injury. The leukocyte rolling and recruitment was delayed when P or E-selectin deficient mice were infected, suggesting that P-selectin is involved in the early events of inflammation mediated by leukocyte[7,12]. Another study demonstrated that P-selectin might maintain chronic inflammatory state[13]. Increased plasma P-selectin level indicates that: P-selectin was markedly released into blood circulation from activated platelets/endothelial cells by inflammatory mediators during acute hepatic/renal injury, and platelets or endothelial cells were activated in vivo to enhance hepatic/renal injury. P-selectin was widely expressed in hepatic/renal tissue in the early stage of injury, particularly in hepatic small vessels and tubular epithelium, suggesting that P-selectin might play a functional role in the early events of leukocyte adherence, activation and infiltration within the liver and kidney.

Results obtained in this study showed that P-selectin expression in hepatic/renal tissue was inhibited and plasma P-selectin level was significantly lowered in P-selectin mAb-treated group. It is consistent with down-regulated expression of sialyl Lewis X, a ligand for P-selectin located mainly in neutrophils, as with anti-P-selectin therapy (unpublished data). The experiment suggested that in the process of hepatic and renal ischemia-reperfusion injury, P-select in mediated the infiltration of neutrophils in hepatic and renal tissues, neutrophils released a set of inflammatory media, which could aggravate the injury of tissues. When the effect of P-selectin was impeded, less neutrophils infiltration and pathological lesions could be observed in the tissue. This phenomenon showed that P-selectin mAb played a protective role in the injury of hepatic and renal tissues caused by ischemia-reperfusion.

Cell apoptosis, which is also named programmed cell death, is an active death process of cell, its imbalance or changes are relative to the occurrence of many kinds of diseases[14]. Schumer et al[15] studied the death form of cells at different times of renal ischemia-reperfusion in rats, and found that transient renal ischemia mainly led to cell apoptosis, permanent and severe renal ischemia brought about necrosis of cells, so it was proved that cell apoptosis took part in cell injury process of acute renal ischemia. Besides this, Sasaki et al[16] discovered that by 3 h of reperfusion after hepatic ischemia in rats, the quantity of apoptotic cells increased markedly, and reached their peak. Therefore, study of the occurrence rule of cell apoptosis during hepatic and renal ischemia-reperfusion is helpful in the treatment of liver and kidney transplantation as well as the disturbance of renal function in acute renal tubular injury, moreover, it is also beneficial in seeking effective measures to promote the recovery of injured renal function.

We discovered that, after 1 h of reperfusion following 1 h of hepatic and renal ischemia, a few apoptotic bodies emerged, after 3-6 h of reperfusion, large quantities of apoptotic bodies appeared and after 24 h of reperfusion, the number of apoptotic bodies reached their peak and inclined to necrosis. This indicated that the time of reperfusion is closely related to the occurrence of cell apoptosis. After the treatment with P-selectin mAb, the situation of cell apoptosis in hepatic and renal tissues of rat became better than that of the control group, possibly due to the fact that P-selectin mAb impeded the adhesion and infiltration of neutrophils, and alleviated cell apoptosis in hepatic and renal tissues during reperfusion after ischemia.

It is considered currently that the death of renal tubular epithelial cells during reperfusion after transient renal ischemia is not caused by ischemia itself, but because the swollen cells can compress blood vessels during ischemia, which makes the corresponding tissue in a state of less blood flow during reperfusion, the insufficiency of blood flow can lead to a large scale of renal cell apoptosis[17]. Our previous studies displayed that P-selectin expressed not only on the surface of platelet and endothelial cell, but also on that of renal tubular epithelial cells[18]. So we speculate that ischemia may stimulate the up-regulation of the expression of P-selectin on the renal tubular epithelial cells. We discovered in this experiment that inhibiting the effect of P-selectin could reduce the apoptosis of renal tubular epithelial cells, so it was proved that P-selectin is an important factor causing renal tubular injury during the renal ischemia-reperfusion process.

It is suggested that P-selectin mediates the infiltration of neutrophils and the injury of renal tubular epithelial cells in tissues during hepatic and renal ischemia-reperfusion process in rats, inhibiting P-selectin can alleviate the accumulation of neutrophils and cell apoptosis in hepatic and renal tissues, and relieve hepatic and renal ischemia-reperfusion injury. So research into the P-selectin and its inhibitor offers a new thought for the treatment of human liver and kidney transplantation as well as acute renal failure caused by renal tubular injury.