Protective Effect of Infliximab on Ischemia/Reperfusion-Induced Damage in Rat Kidney

Abstract

Objective: To investigate the protective effect of infliximab on ischemia–reperfusion (I/R) injury of the rat kidney. Methods: Twenty-eight male Wistar albino rats were divided into four groups: sham-operated, I/R, I/R with infliximab administered before ischemia [I/R + infliximab (bi)], and I/R with infliximab administered before reperfusion [I/R + infliximab (br)]. After a right nephrectomy to produce damage, the left renal vessels were occluded for 60 min, followed by 24-h reperfusion in rats. Changes in the rat kidney were observed by measuring the tissue levels of malondialdehyde (MDA), myeloperoxidase (MPO), glutathione (GSH), and superoxide dismutase (SOD) and by evaluating hematoxylin–eosin (H&E)-stained and periodic acid–Schiff (PAS) sections. Results: The MDA and MPO levels in the I/R group were significantly higher than in the other groups (p < 0.05), and the SOD and GSH levels in the I/R + infliximab (bi) and I/R + infliximab (br) groups were significantly higher than in the I/R group (p < 0.05). However, histological examination revealed that the I/R + infliximab (bi) group and the I/R + infliximab (br) group had significantly fewer tubular changes and interstitial inflammatory cell infiltration than the I/R group. Conclusion: These results show that infliximab may protect against I/R injury in the rat I/R model.

• ischemia–reperfusion
• renal
• infliximab

INTRODUCTION

Renal ischemia–reperfusion (I/R) injury is a major problem in urological operations, such as renal transplantation, surgical revascularization of the renal artery, and partial nephrectomy.^1–3 I/R injury is clinically important because it results in acute renal failure and high mortality.⁴ In ischemia, renal blood flow is reduced in the kidney by the occluded vessels that supply oxygen to the kidney. Reperfusion has also been reported to aggravate cell damage.⁵ Reactive oxygen species (ROS) and proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), play a key role in the pathophysiology of renal I/R injury.^6,7 Free radicals trigger the accumulation of leukocytes in the tissues. Activated neutrophils secrete enzymes, such as myeloperoxidase (MPO), and liberate more free radicals.⁸

Infliximab, a novel immunomodulatory agent, has a high affinity for TNF-α. It is being used for refractory Crohn’ s disease, rheumatoid arthritis, and also ulcerative colitis^9,10 Recently, researchers demonstrated using human fibroblasts, endothelial cells, neutrophils, lymphocytes, and epithelial cells that infliximab inhibits functional TNF-α activity in various in vitro bioassays.⁹ Marioni et al. showed that infliximab has a therapeutic effect in treating corticosteroid-resistant Crohn’s disease via a blockade of TNF-α.^9,10 In addition, in a recent clinical trial, infliximab—which is used to manage diseases such as rheumatoid arthritis and Crohn’s disease and dermatologic, chronic ocular and neurological diseases—was indicated as an alternative conservative therapy.^11–13

However, no study has investigated the effects of infliximab on I/R damage in rat kidneys. Therefore, this study was undertaken to examine whether infliximab shows a protective effect in experimental rat models with I/R-induced renal damage. We evaluated tissue biochemical analysis and histopathological changes in these I/R rat models.

METHODS

Experimental Groups

In this study, we used 28 male Wistar albino rats weighing 250–300 g. The rats were obtained from the Production Center of Experimental Animals at Inonu University, Malatya, Turkey. Throughout the experimental period, all animals were provided, ad libitum, with tap water and standard rat pellet food. The 28 animals were randomly divided into four groups: (I) sham-operated (n = 7), sham-operated animals underwent right nephrectomy only, but the left renal pedicle was not occluded. Renal I/R (n = 7), I/R with infliximab administration before ischemia [I/R + infliximab (bi)] (n = 7), and I/R with infliximab administration before reperfusion [I/R + infliximab (br)] (n = 7). Renal damage measurements were carried out in seven animals for each group. Infliximab (Remicade, Schering Plough Co., Innihannon, County Cork, Ireland) was administered intraperitoneally at a dose of 5 mg/kg.¹⁴

All experimental procedures conform to the National Research Council guidelines¹⁵ for the handling and care of laboratory animals. Moreover, all experimental procedures and protocols used in this investigation were reviewed and approved by the Animal Research Committee of Inonu University.

Anesthesia and Ischemia–Reperfusion Procedure

Anesthesia was induced by intramuscular administration of 50 mg/kg ketamine hydrochloride (Ketalar, Pfizer, Istanbul, Turkey) and 10 mg/kg xylazine (Rompun, Bayer, Istanbul, Turkey) before the operation. Right nephrectomy was performed through dorsolateral incisions in all rats. I/R was induced by the occlusion of the left renal vessels for 60 min, followed by 24-h reperfusion. Sham-operated animals underwent right nephrectomy only; the left renal pedicle was not occluded.

At the end of each in vivo study, the rats were killed and the kidneys were quickly removed, decapsulated, and divided equally into two longitudinal sections. One section was placed in formaldehyde solution for a routine histopathological examination using light microscopy. The other section of the kidney was placed in liquid nitrogen and stored at −70°C until the section was assayed for biochemical evaluation.

Histological Analysis

The kidney tissue was fixed in 10% formalin and was embedded in paraffin. Sections of the tissue were cut into 5 μm, mounted on slides, and stained with hematoxylin–eosin (H&E) and periodic acid–Schiff (PAS). These sections were evaluated for the presence of tubular-glomerular injury and interstitial changes. Renal damage was described in terms of the detachment of tubular epithelial cells, loss of brush borders, tubular necrosis, interstitial congestion, glomerular shrinkage, and increased Bowman’s space. The microscopic score for each tissue was calculated as the sum of the scores given to each criterion. The scores given for each criterion were 0 (absent), 1 (slight), 2 (moderate), and 3 (severe).¹⁶ For each specimen, 10 microscopic fields were analyzed under a 20× objective per animal. A histopathologist who was unaware of the animal treatment groups examined the tissue sections using a Leica DFC 280 light microscope (Leica Micros Imaging Solutions Ltd., Cambridge, UK).

Biochemical Analysis

Tissues were homogenized (PCV Kinematica Status Homogenizator) in ice-cold phosphate-buffered saline (pH 7.4). The homogenate was sonified with an ultrasonifier (Bronson Sonifier 450) with three cycles (20 s sonications and 40 s pause on ice). The homogenate was centrifuged (15,000×g, 10 min, 4°C), and cell-free supernatant was immediately subjected to enzyme assay.

Malondialdehyde (MDA) level analysis was performed as described with a minor modification.¹⁷ The reaction mixture was prepared by adding 250 μL homogenate into 2 mL reaction solution (15% trichloroacetic acid: 0.375% thiobarbituric acid: 0.25 N HCl. 1:1:1. w/v) and heated at 1000°C for 15 min. The mixture was cooled to room temperature, centrifuged (10,000×g for 10 min), and the absorbance of the supernatant was recorded at 532 nm. 1,1,3,3-Tetramethoxypropane was used as MDA standard.¹⁷ The values were expressed as nmol/mg protein in the homogenate. Tissue-associated MPO activity was measured using a procedure similar to that documented by Hillegas et al.¹⁸ MPO activity was expressed as U/g tissue. Tissue glutathione (GSH) levels were determined using a modified Ellman method.¹⁹ The values were expressed as nmol/mg protein in the homogenate. Superoxide dismutase (SOD) activity in the supernatant fraction was measured using the xanthine oxidase/cytochrome c method.²⁰ The amount of SOD in the extract was determined as U/mg protein, utilizing a commercial SOD as the standard.

Statistical Analysis

Data are expressed as means ± SEM. One-way analysis of variance (ANOVA), followed by the Bonferroni test, was applied to calculate the statistical significance between various groups; when p < 0.05, the difference was considered statistically significant.

RESULTS

Biochemical Results

The MDA, MPO, and GSH levels and the SOD activity in the renal tissue of each experimental group are shown in Table 1. Renal I/R increased the MDA levels in rats. The MDA levels in the renal I/R (1.33 ± 0.16 nmol/mg) group were significantly higher than those in the sham-operated group (0.52 ± 0.05 nmol/mg). Administering infliximab in rats before ischemia produced a significant reduction in MDA levels compared with those obtained from the I/R group kidneys. In addition, administering infliximab in rats immediately before reperfusion caused significantly decreased MDA levels. However, this reduction was not statistically significant when compared with the results from the I/R + infliximab (bi) group.

Table 1. Effects of infliximab on MDA, MPO, GSH and SOD levels in rats exposed to renal I/R.

	MDA	MPO	GSH	SOD
	(nmol/mg protein)	(U/mg protein)	(nmol/mg protein)	(U/mg protein)
Sham	0.52 ± 0.05	0.47 ± 0.07	24 ± 2.56	15.18 ± 1.76
I/R	1.33 ± 0.16^a	4.30 ± 0.39^a	12.56 ± 0.73^b	8.59 ± 0.69^b
I/R + infliximab (bi)	0.53 ± 0.11^c	1.5 ± 0.16^c	18.25 ± 1.33^d	12.02 ± 1.10^d
I/R+ infliximab (br)	0.55 ± 0.03^c	3.22 ± 0.30^c,e	17.95 ± 1.57^d	10.70 ± 0.91^d

Notes: GSH, glutathione; I/R, ischemia–reperfusion; MDA, malondialdehyde; MPO, myeloperoxidase; SOD, superoxide dismutase. Values are expressed as the mean ± SEM.

(bi): before ischemia, (br): before reperfusion.

^a,bp < 0.05 versus Sham group.

^c,d p < 0.05 versus I/R group.

^ep < 0.05 versus I/R + infliximab (bi) group.

Table 2. The comparison of severity of renal damage among groups.

Groups	Renal damage
Sham	0.5 ± 0.3
I/R	14.3 ± 0.6^a
I/R + infliximab (bi)	7.8 ± 0.3^a,b
I/R+ infliximab (br)	8.14 ± 0.5^a,b,c

Notes: (bi): before ischemia, (br): before reperfusion.

^aMean value was significantly increased compared with that of the Sham group (p < 0.05).

^bMean value was significantly decreased compared with that of the I/R group (p < 0.05)

^cNot significant when compared with group I/R + infliximab (bi) (p > 0.05).

Similarly, renal I/R increased the MPO levels in rats. The MPO levels in the renal I/R (4.30 ± 0.39 U/mg) group were significantly higher than those in the sham-operated group (0.47 ± 0.07 U/mg). Administering infliximab before ischemia produced a significant reduction in MPO levels compared with the levels obtained from the I/R group kidneys. However, administering infliximab immediately before reperfusion also caused significantly decreased MDA levels. This reduction was not noticeable relative to the I/R + infliximab (bi) group.

I/R injury in the kidney tissue caused a significant decrease in the tissue GSH level compared with the levels in other groups. The GSH levels were significantly lower in the renal I/R group (12.56 ± 0.73 nmol/mg) compared with the sham-operated group (24 ± 2.56 nmol/mg). In the I/R+ infliximab (br) group, the GSH concentration was preserved and was not significantly different from that in the sham-operated group. SOD activity was significantly lower in the kidney tissue of the I/R group than in the kidney tissue of the sham-operated group. However, the group administered infliximab before ischemia showed significantly increased SOD levels in the renal tissue.

Microscopy Results

The sham-operated group did not show any detectable histological abnormalities, except slight epithelial desquamation (Figure 1A and B). However, in the I/R group, extensive cortical and medullar damage was present. Tubular desquamation, tubular necrosis (Figure 1C), and loss of brush borders (Figure 1D) were observed in this group. In addition to these changes, most of the glomeruli were damaged. The affected glomeruli had shrunk and Bowman’s space increased (Figure 1D). Glomerular alterations were detected only in the I/R group. In the I/R, I/R + infliximab (bi), and I/R+ infliximab (br) groups, light microscopic investigation revealed moderate degeneration and tubular necrosis as patchy areas in the renal cortex (Figure 1E and G). Interstitial congestion was evident in the I/R+ infliximab (br) group (Figure 1G). Although tubular desquamation and loss of brush borders were present, the glomeruli were intact in these groups (Figure 1F and H). The results of the semiquantitative histologic analyzes are shown in Table 2.

Figure 1. (A) Sham; renal tubules and glomeruli are intact. H&E ×66. (B) Sham; appearance of brush border (arrows). PAS ×132. (C) I/R group; tubules show diffuse and marked necrosis (*). H&E ×66. (D) I/R group; notice lost of the brush border and glomerular changes. PAS ×132. (E) I/R + infliximab (bi); despite tubular degeneration are visible in some area, glomeruli are seen intact. H&E ×66. (F) I/R + infliximab (bi); the loss of brush border was marked in affected tubules. PAS ×132. (G) I/R+ infliximab (br); moderate tubular damage and interstitial congestion are observed. H&E ×66. (H) I/R+ infliximab (br); the view of brush border is similar to ischemia group. PAS ×132.

DISCUSSION

In this study, we found that I/R caused an increase in MDA and MPO levels and a decrease in SOD activity and GSH levels in renal tissue. However, administering infliximab to the I/R group reduced the MDA and MPO values while increasing the SOD activity and GSH levels. I/R caused structural changes in the kidney, but infliximab reversed these changes. These findings indicate that infliximab, along with the TNF-α inhibitor, is important in protecting the kidney from I/R-induced damage in I/R rat models.

After renal surgeries, such as renal transplantation, surgical revascularization of the renal artery, and partial nephrectomy, I/R injury may result from the interruption of the renal arterial circulation.²¹ As a result, I/R injury may cause acute renal failure. Moreover, ROS play a key role in the pathophysiology of renal IR injury.^6,7,22 ROS have many deleterious effects on renal tissue because they react with proteins, membrane lipids, and nucleic acids in the kidney cells.^23,24 ROS are generated by mitochondrial cytochrome oxidase, nicotinamide adenine dinucleotide phosphate oxidase, xanthine oxidase, lipoxygenase, cyclooxygenase, hemeoxygenase, cytochrome P-450 enzymes, nitric oxide synthase (NOS), and various other oxidase enzymes. Additionally, ROS may lead to severe injury to the cell membrane by lipid peroxidation reactions. Membrane lipid peroxidation may generate reactive carbonyl compounds such as MDA, one of the reliable indicators of ROS-induced I/R tissue damage.²⁵ MDA is the most abundant aldehyde resulting from lipid peroxidation.²⁶ Among various antioxidant systems equipped within aerobic cells, the key antioxidant enzymes (SOD, GSH) are major mechanisms to reduce local levels of ROS. These enzymes abase primary ROS, such as superoxide anion and hydrogen peroxide, before they can interact to form more reactive cytotoxic metabolites.²⁷ Previous studies have shown that I/R causes an increase in MDA levels and a decrease in GSH levels and SOD activity.^28,29 Similarly, we observed a significant decrease in GSH levels and SOD activity and an increase in MDA content during I/R-induced renal injury, making our findings agree with results from previous studies. In our study, infliximab tended to increase SOD activity and GSH levels. These effects were statistically significant. We also observed histopathologic changes in ischemic rat kidneys as well as alterations in oxidative stress markers. These changes are tubular cell swelling, tubular dilatation, cellular vacuolation, medullary congestion, and moderate to severe necrosis.

Proinflammatory factors also play an important role in the pathogenesis of renal I/R injury through leukocyte activation and expression of adhesion molecules and cytokines.^14,30,31 Similarly, the increased production of TNF-α has a role in the pathophysiology of I/R injury. MPO, an abundant enzyme in phagocytes, has been implicated in the pathogenesis of various inflammatory diseases.^32,33 Thus, MPO is known as an inflammation marker. This enzyme shows an increase in inflammatory conditions. We observed a significant increase in MPO after I/R-induced renal injury.

The literature includes many studies demonstrating that many free radical scavengers and antioxidant agents, such as flavonoids and melatonin, have beneficial effects on I/R-induced damage.^23,31,34,35 However, no study has investigated the effects of an immunomodulatory agent, such as infliximab, on I/R damage in rat kidneys. Infliximab, a humanized mouse monoclonal antibody to TNF-α, is a new immunomodulatory agent. Infliximab exerts beneficial effects in several autoimmune diseases, including rheumatoid arthritis, Crohn’s disease, and psoriasis.³⁶ Kurt et al. reported that spinal cord ischemic injury (SCI) reportedly increases MDA levels and that treatment with infliximab decreases the MDA levels in the spinal cord tissue of rats with SCI.³⁷ Furthermore, Guven et al. demonstrated that treatment of I/R injury with infliximab decreased SOD activity and increased GSH levels in spinal cord tissue. In our study, infliximab tended to increase SOD activity and GSH levels. In addition, histologic damage was considerably improved with infliximab treatment.¹⁴ Although the results of our study cannot explain the whole mechanism of the attenuation of renal I/R injury, oxidative stress markers and histologic changes suggest that infliximab helps to decrease I/R-induced injury in rat kidneys.

In conclusion, the findings of this study indicate that infliximab has a protective effect on I/R damage in the kidney. Therefore, this agent may be used to prevent renal I/R in clinical practice, particularly in transplantation and renal surgery. However, further studies are necessary to improve our understanding of the role of infliximab in renal I/R.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.