Oxymatrine ameliorates renal ischemia-reperfusion injury from oxidative stress through Nrf 2 / HO-1 pathway 1

PURPOSE: To investigate if oxymatrine pretreatment could ameliorate renal I/R injury induced in rats and explore the possible role of oxymatrine in Nrf2/HO-1 pathway. METHODS: Unilaterally nephrectomized rats were insulted by I/R in their left kidney. Twenty four rats were randomly divided into three groups: sham group, I/R + saline-treated group, I/R + OMT-treated group. Oxymatrine or vehicle solution was administered intraperitoneally injected 60 min before renal ischemia, respectively. Renal function, histology, makers of oxidative stress, cell apoptosis and Nrf2/HO-1 expressions were assessed. RESULTS: Oxymatrine pretreatment exhibited an improved renal functional recovery, alleviated histological injury and oxidative stress, inhibiting tubular apoptosis, and accompanied by upregulated the expression of Nrf2/HO-1 proteins. CONCLUSION: Oxymatrine may attenuate renal ischemia/reperfusion injury, and this renoprotective effect may be through activating the Nrf2/HO-1 pathway.


Introduction
Ischemia-reperfusion (I/R) injury of the kidney, a serious clinical condition, is a commonly encountered problem in renal transplantation, hemorrhagic shock, vascular surgery, partial nephrectomy, and accidental or iatrogenic trauma, which causes a serious injury to tissues and organs 1 .The mechanisms underlying renal I/R injury are complex and not completely understood.One important pathway that contributes to the pathogenesis of renal I/R injury is oxidative stress 2 .Interruption of blood flow to the kidney and the subsequent reperfusion lead to an acute oxidative stress response that may cause the generation of reactive oxygen species (ROS).Consequently, the overproduction of ROS causes lipid peroxidation, DNA mutation, and induced apoptotic and necrotic cascades, ultimately resulting in cell death in various ways 3,4 .
The nuclear factor erythroid-2-related factor 2 (Nrf2)/ heme oxygenase-1 (HO-1) pathway was found to be related to antioxidative stress and scavenging of ROS under conditions of oxidative stress.Nrf2 (a transcription factor) responsible for the expression of phase II enzymes the primary function of which is to reduce redox stress 5 .Especially, Nrf2 binds to anti-oxidant response element (ARE) localized in the promoter regions of a battery anti-oxidant and detoxifying genes including HO-1 6 .
Previous studies have demonstrated the therapeutic potential of targeting the Nrf2/Ho-1 pathway in renal ischemia-reperfusion 7 .
Oxymatrine (OMT), extracted from a traditional Chinese herb, Sophora flavescens Ait, has a variety of pharmacological properties such as anti-inflammatory, anti-oxidative, antivirus effects and immunological regulation, and has been used for the treatment of chronic hepatitis 8 , bronchial asthma 9 , myocardial ischemic injuries 10 , and lung 11 , liver 12 , intestinal 13 and brain [14][15][16] ischemia/reperfusion injury in animal models.OMT also have a protective role in adriamycin-induced chronic renal fibrosis 17 .
More recently, OMT has been reported to have neuroprotection in cerebral ischemia-reperfusion injury in rats, which is related to Nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant response 18 .However, the relationship between OMTinduced renoprotection and signal transduction pathway has not been elucidated, Thus, the purpose of the current study was to investigate whether OMT can protect the kidney against ischemic injury through Nrf2/HO-1 signal pathway.

Methods
The experimental protocol used in this study was approved by the Animal Ethics Review Committee of Wuhan University, and the procedures were carried out accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
Male Sprague-Dawley rats (220-250g) were purchased from the center of Experimental Animals in Medical College, Wuhan University.The animals were kept under standard conditions and allowed free access to feed and tap water.

Experimental design
The I/R injury rat model was induced as previously described 19 .Briefly, after one week of acclimation, rats were fasted for 12 h before surgery with free access to tap water.24 rats were randomly divided into three groups: sham group (n=8), I/R + saline-treated group (I/R group, n=8), I/R + OMTtreated group (n=8).Under the pentobarbital sodium (50mg/kg, intraperitoneally) anesthesia, rat underwent a median laparotomy to expose kidneys, followed by a right nephrectomy.The left renal hilus was occluded using a non-traumatic microvascular clamp for 45 min to effect complete cessation of renal blood flow, followed by 24 h reperfusion.The sham operated rat underwent the same procedure without vessel occlusion.At the end of the reperfusion period, Blood samples were collected by cardiac puncture for detection of blood urea nitrogen (BUN), serum creatinine (SCr) and lactate dehydrogenase (LDH) levels.Left nephrectomy was performed and renal tissue samples were fixed in 4% paraformaldehyde or snap-froze them in liquid nitrogen, and stored at -80°C for the subsequent measurement.

Drug administration
OMT was purchased from Nanjing Spring & Autumn Biological Engineering Co., Ltd (CAS 16837-52-8, purity ≥ 98%, molecular formula: C15H24N2O2).OMT was dissolved in 0.9% sodium chloride and stored at 4°C.We intraperitoneally injected OMT (150mg/kg) 60 min before renal ischemia.We chose the dose on the basis of previous studies and our preliminary experiment 11 .
For control purposes, I/R groups were injected with an equal volume of 0.9% sodium chloride.

Assessment of renal function
The SCr, BUN and LDH levels were measured with an automated analyzer (Siemens ADVIA 2400).

Renal tissues SOD and MDA assay
The activity of superoxide dismutase (SOD) was detected using commercialized assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions.The malondialdehyde (MDA) concentrations were determined with commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in accordance with the manufacturer's instrument, and the optical densities were read at 535nm.The results are expressed as nanomoles per milligram protein.

Histologic examination
We embedded kidneys in paraffin, 4-μm thickness sections were stained with hematoxylin-eosin and examined under the microscope (Olympus, Tokyo, Japan) in a blinded manner by pathologist who was unaware of the treatment.The degree of tubular damage was evaluated using a semiquantitative scale according to the criteria described previously 20 : 0 = normal kidney; 1 = minimal damage (< 5% area, outer medulla or the cortex); 2 = mild damage (5%-25% area, outer medulla or cortex); 3 = moderate damage (25%-75% area outer medulla or cortex); and 4 = severe damage (>75% area, outer medulla or cortex).

TUNEL staining
Paraffin-embedded sections were deparaffinized in xylene and rehydrated in a graded series of ethanol.TUNEL staining were performed using an situ terminal deoxynucleotidyl tansferase mediated-dUTP nick end labeling (TUNEL) assay with Cell Death Detection kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturers' instructions.The nucleus was stained using 3,3-diaminobenzidine (DAB) as a substrate for the peroxidase.For each paraffin section, ten areas were randomly selected and the numbers of TUNEL-positive cells were counted at × 400 magnification in a blinder manner.

Renal Nrf2 and HO-1 immunohistochemical assays
Immunohistochemical staining for Nrf2 and HO-1 detection was performed on formalin-fixed paraffin sections using the streptavidin-biotin-peroxidase method.Under a light microscope (Olympus, Tokyo, Japan), the yellowish-brown color were accounted as positive staining.

Renal tissues Nrf2 and HO-1 Western Blot analysis
Samples (50μg/lane) were separated on a 10% SDS-PAGE gels and then electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane.The membranes were blocked with 5% nonfat milk solution for 2 h at room temperature with gently shaking, and then incubated with primary antibodies overnight at 4°C.Subsequently, the membranes were washed and incubated for 2 h at room temperature with a secondary antibody.The protein bands were detected by chemiluminescence technology.

Statistical analyses
Data were expressed as mean ± standard deviation values.Statistical analysis of the results was carried out by oneway analysis of variance (GraphPad Prism TM 5.0, San Diego, USA).p<0.05 were considered significant.

Effects of OMT on renal function induced by reperfusion after renal ischemia
BUN and SCr levels, two important indexes of kidney function, were significantly increased in the saline-treated I/R group after 24 h of reperfusion, while in the OMT-treated group, these levels of BUN and SCr were markedly decreased (p < 0.05, Figure 1A and B).We also measured lactate dehydrogenase (LDH) levels in these experimental groups.LDH levels in the saline-treated I/R group were obviously increased after 24 h of reperfusion.However, the LDH levels in the OMT-treated group were significantly decreased by 24 h of reperfusion (p<0.05, Figure 1C).

Effect of OMT on I/R-induced histopathological damage
OMT could alleviate renal histology injury at 24 h after renal reperfusion.In Figure 3, the renal tubules in the saline-treated I/R group showed pathological changes, including loss of brush border, congestion, tubular cell swelling, tubular dilation, and inflammatory cell infiltration.However, a significant amelioration of histological damage was seen in the OMTtreated I/R groups.

Effect of OMT on apoptosis of tubular epithelial cells
We investigated the ability of OMT pretreatment to mediate protection against ischemia induced apoptotic cell death.After 24 h reperfusion, TUNEL assay was performed on renal tissue sections.As shown in Figure 4 2015 -425 Effects of OMT on SOD and MDA levels in renal tissuesUsually antioxidative ability of tissue is estimated by SOD activity and MDA content, which were the sensitive indicators of oxidative stress21 .SOD is the most important endogenous antioxidant enzymes that scavenges oxygen free radicals and protects mitochondria against damage caused by cytotoxic reactions.MDA is one of the products of lipid peroxidation which is induced by the attack of reactive oxygen species on polyunsaturated fatty acid.SOD activity and MDA concentration in kidney tissue was measured as shown in Figure2, following 45 min of ischemia, reperfusion obviously decreased SOD activity and increased MDA concentration in the saline-treated I/R group compared with those in sham group (p<0.05).In the OMT-treated group, the SOD levels increased and the MDA levels declined compared to the saline-treated I/R group (p<0.05).

FIGURE 1 -
FIGURE 1 -OMT attenuates renal injury after renal I/R.Blood were collected 24h after reperfusion.BUN (A), SCr (B) and LDH (C) were significantly higher in the saline-treated I/R group than sham-operated group.OMT treatment inhibited renal dysfunction after renal I/R injury.*p<0.05 compared with the sham-operated group and # p<0.05 versus the I/R group.

FIGURE 2 -
FIGURE 2 -OMT attenuates I/R-induced oxidative stress.We measured SOD levels (A) and MDA (B) activity in kidneys at 24h after reperfusion.*p<0.05 compared with the sham-operated group and # p<0.05 versus the I/R group.
, a significant number of apoptosis cells in saline-treated I/R group were observed compared with the sham group.Few apoptosis cells were observed in the sham group.Pretreatment with OMT significantly decreased apoptosis cells as compared to the salinetreated I/R group (p<0.05).

FIGURE 3 -
FIGURE 3 -OMT attenuates I/R-induced histopathologic renal damage.The sham group did not observed morphological changes; In the I/R group, kidney section shows tubular cell swelling, vacuolization, cast formation and tubular necrosis; and less damage was observed in the OMT+I/R group compared to the I/R group.(A) Representative hematoxylin-eosin stained kidney sections (magnification ×400).(B) Histopathologic score measure at 24h after reperfusion.Scale bar = 100 μm.*p<0.05 compared with the sham-operated group and # p<0.05 versus the I/R group.

FIGURE 4 -
FIGURE 4 -OMT attenuates I/R-induced tubular cell apoptosis at 24h after reperfusion by TUNEL staining.At 24h after reperfusion, the numbers of apoptotic positive cells dramatically increased when compared with the sham group.In contrast to the I/R group, an increase significantly reduced by treatment with OMT.(A) Representative photomicrographs of TUNEL assay (magnification ×400).(B) Quantitative analyses of apoptotic positive cells per field.Scale bar = 100 μm.*p<0.05 compared with the sham-operated group and # p<0.05 versus the I/R group.