The effect of polymerized placenta hemoglobin on renal ischemia/reperfusion injury

Abstract

The goal of this study was to investigate whether hemoglobin-based oxygen carrier (HBOC) attenuated ischemia/reperfusion (I/R)-induced kidney injury. Male SD rats were randomly divided into a sham group, I/R group, and HBOC group (injection of 0.1 gHb/kg PolyPHb). The ischemia was induced by bilateral renal pedicle cross-clamping for 45min. Then the clamp was released to allow 24h reperfusion. Without increasing blood pressure, PolyPHb reduced the blood urea nitrogen and creatinine in plasma and attenuated the tumor necrosis factor-α and interleukin-8 in kidney tissue. Therefore, our findings suggest that PolyPHb could reduce kidney injury after I/R injury, and this effect was probably associated with the depressed inflammatory response.

Keywords::

• Hemoglobin-based oxygen carrier
• ischemia/reperfusion injury
• kidney
• inflammation

Introduction

Renal ischemia/reperfusion (I/R) is a major cause of acute kidney injury (AKI), which is unavoidable in major vascular surgeries, such as thoraco-abdominal aortic aneurysm repair and renal transplantation. The alterations of renal medullary blood flow in these surgeries contribute to the pathogenesis of renal I/R injury. Also, due to the increased production of oxygen free radicals and macrophage/neutrophil infiltration during I/R injury, renal parenchyma was progressively damaged and finally developed renal failure. Despite considerable advances in medical services, the postoperative renal dysfunction is still common and remains the leading cause of morbidity and mortality of these patients (Schrier and Wang 2004, Thadhani et al. 1996).

The development of hemoglobin-based oxygen carrier (HBOC) is particularly required in the case of urgent need of oxygen delivery to tissues and to solve the problems related to blood transfusions. Polymerized human placenta hemoglobin (PolyPHb) is one type of HBOC developed in China (Li et al. 2006a, Li et al. 2006b). The applications of PolyPHb are manifold, including in hemorrhagic shock, organ I/R injury, anaerobic metabolism, and so on (Li et al. 2011, McNeil et al. 2001, Standl et al. 2003). Except for its excellent oxygen-transporting capacity, the mechanisms of the protective effect of this product have been demonstrated to be related to attenuation of NO-mediated myocardial apoptosis, restoration of nitroso-redox balance, and preservation of mitochondria function (Li et al. 2009a, Li et al. 2009b, Li et al. 2010). Therefore, the present study was designed to investigate whether PolyPHb could also protect kidney from I/R injury.

Materials and Methods

This study was approved by the Institutional Animal Care and Use Committee of Sichuan University, and all animals received human care in compliance with The Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Sixty-five adult male Sprague-Dawley (SD) rats, approximately 10 weeks old and weighing 200–250g, were housed at constant temperature (22 ± 3^°C) on a 12:12-h light/dark cycle and given free access to food and water.

Preparation of hemoglobin-based oxygen carrier

The HBOC used in this study was PolyPHb, which was prepared as previously described (Li et al. 2009a, Li et al. 2006a, Li et al. 2006b). Briefly, purified and viral inactivated fresh human placenta hemoglobin (Tianjin Union Stem Cell Genetic Engineering Ltd., Tianjin, China) were modified with pyridoxal phosphate to achieve optimal O₂ affinity. After crosslinkage with glutaraldehyde, ultrafiltration and molecular sieve chromatography were employed to harvest the PolyPHb solution with molecular weight of 64-600 kDa. Before use, the PolyPHb was quickly mixed with Lactated Ringer's solution (L-RS) to a final concentration of 3 gHb/dL.

Non-invasive blood pressure measurement

Systolic blood pressure (SBP) and heart rate (HR) were measured in restrained awake animals by means of the tail-cuff method (Blood Pressure Analysis System, BP-2000, Visitech System, Physiological Research Instruments, Apex, NC, USA). Twenty rats were accustomed to the blood pressure measurement device for 1 h. HBOC solution (0.1 gHb/kg, n = 10) or a similar amount of L-RS (n = 10) was injected via a polyethylene catheter pre-cannulated in tail vein. The measurements were undertaken before and after the injection. The dosage of HBOC was chosen on the basis of a previous in vivo study (Li et al. 2011).

Experimental protocol

Forty-five SD rats were randomly divided into three groups (n = 15 in each group): a sham group, I/R group (injection of L-RS), and HBOC group (injection of 0.1 gHb/kg PolyPHb). All the rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg) and mechanically ventilated with 95% oxygen at respiratory rate (RR) of 30 bpm and tidal volume (Vt) of 6 ml/kg (DH-150, Medical Instrument Company of Zhejiang University, Hangzhou, Zhejiang, China). After midline laparotomy and L-RS or HBOC administration, the renal ischemia was induced by bilateral renal pedicle cross-clamping for 45 min. Body temperature was maintained at 36.5-37.5^°C by a heating blanket during the experiment. The clamp was then released and the wound was closed with sutures. Rats with the same procedure of tail cannulation and surgery to expose the kidney with no obstruction of the renal artery were used as sham controls. After the study, the blood samples were collected via tail vein, and then all the rats were sacrificed with a bolus of I.V. sodium pentobarbital (120 mg/kg). The experimental protocol is schematically illustrated in Figure 1.

Figure 1. The experimental protocol of this study. I/R: ischemia/reperfusion; L-RS: Lactated Ringer's solution; HBOC: hemoglobin-based oxygen carrier.

Determination of creatinine and blood urea nitrogen (BUN) levels in plasma

After centrifugation of the blood sample, the plasma was isolated. The blood urea nitrogen (BUN) and creatinine levels in the plasma were measured with an autoanalyzer (Cobas C501, Roche, Basel, Switzerland).

Measurement of Tumor Necrosis Factor (TNF)-α and Interleukin (IL)-8 in kidney

The left renal cortex was homogenized and centrifuged. The protein concentration of its supernatant was determined by the BCA method (Pierce, Rockford, IL, USA). Then the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-8 were determined by commercial ELISA kits (R&D, Minneapolis, MN).

Statistical analysis

All values in the text and figures were presented as mean ± SD. The values of SBP and HR were analyzed by repeated measures of ANOVA, and use of a post-hoc t test with Bonferroni correction for multiple comparisons. Other data in this study were subjected to one-way ANOVA followed by LSD correction for post-hoc t test (SPSS 13.0 software). P values < 0.05 were considered statistically significant.

Results

Changes of SBP and HR after polyPHb injection

In order to observe the vasoactive effect of 0.1 gHb/kg of HBOC and minimize the interference from anesthesia, we measured the SBP and HR of awake rats before and after injection of HBOC and vehicle. The results indicated that there were no significant changes of both SBP and HR after injection of 0.1 gHb/kg HBOC when compared with the respective baseline or vehicle (Figure 2).

Figure 2. The SBP (A) and HR (B) before and after HBOC injection. Values were presented as mean ± SD (n = 15). HBOC: hemoglobin based oxygen carrier; HR: heart rate; L-RS: Lactated Ringer's solution; SBP: systolic blood pressure.

PolyPHb improved renal function

Twenty-four h after reperfusion, the levels of creatinine and BUN were greatly increased in the I/R group (P< 0.01 and P< 0.01 vs the sham group, respectively). HBOC pretreatment markedly depressed the elevation of creatinine (P< 0.05 vs the I/R group, Figure 3A). Similarly, the increased release of BUN was also significantly reduced by HBOC (P< 0.05 vs the I/R group, Figure 3B). However, it is noteworthy that, when compared to the sham group, the levels of creatinine and BUN were still higher (P< 0.05 and P< 0.01), indicating that HBOC could not totally reverse the renal functional damage caused by I/R injury.

Figure 3. The releases of creatinine (A) and BUN (B) in the plasma after renal I/R injury. Values were presented as mean ± SD (n = 15). *P< 0.05 and **P< 0.01 vs. the sham group; †P< 0.05 vs. the I/R group. BUN: blood urea nitrogen.

PolyPHb attenuated renal inflammation

Renal I/R injury greatly increased the level of TNF-α in the I/R group (P< 0.01 vs the sham group, respectively). Such an elevation was significantly decreased by HBOC treatment (P< 0.05, Figure 4A). Similarly, the increased release of IL-8 from renal I/R injury was also greatly lessened in the HBOC group (P< 0.05 vs the I/R group, Figure 4B), even though it was still higher than that of the sham group (P< 0.05).

Figure 4. The levels of TNF-α (A) and IL-8 (B) in the renal tissue after I/R injury. Values were presented as mean ± SD (n = 15). **P< 0.01 and *P< 0.05 vs. the sham group; †P< 0.05 vs. the I/R group. TNF-α: tumor necrosis factor-α; IL-8: interleukin-8.

Discussion

The current study provided three main findings: 1) venous injection of HBOC with amount of 0.1gHb/kg would not significantly alter the hemodynamic values; 2) pretreatment with HBOC could protect kidney from I/R injury; 3) HBOC inhibited I/R injury-induced inflammatory response.

As a promising oxygen carrier, HBOC is able to freely diffuse in microcirculation and transport oxygen to hypoxia tissues, owing to its high oxygen affinity, low viscosity, and small mean diameter. These properties made HBOC an attractive agent for treatment of organ ischemia injury or I/R injury. During the last several years, a series of studies have been initiated to investigate the organ-protective effect of HBOC, especially for the organ I/R injury. The results of these studies provided distinct evidence that HBOC could attenuate I/R injury in major solid organs, such as the heart and lung (Li et al. 2011, McNeil et al. 2001, Standl et al. 2003, Wu et al. 2009). In addition, these studies indicated that the organ-protective effect of HBOC should not be totally attributed to its oxygen-transporting capacity, because HBOC could provide more profound protection to the I/R heart than whole blood, which could also transport oxygen. Moreover, we found that HBOC could attenuate nitric oxide-mediated myocardial apoptosis, restoration of nitroso-redox balance, and reduce mitochondrial oxidative damage (Li et al. 2009a, Li et al. 2009b).

In the present experiment, we tested our hypothesis that HBOC could also protect the kidney from I/R injury. The results confirmed our hypothesis and demonstrated that the inflammatory response was down-regulated by HBOC. As we know, reperfusion is associated with a distinct inflammatory response characterized by an increase in inflammatory cytokine production and neutrophil accumulation in the reperfused tissue, which contributed to cell death and apoptosis and associated with postischemic renal failure (Eldaif et al. 2010, Schrier and Wang 2004, Thadhani et al. 1996). Thus, attenuation of inflammatory response may alleviate renal cell death and apoptosis and ultimately reduce the renal injury during I/R. In our study, we showed that HBOC significantly reduced the releases of inflammatory cytokine TNF-α and IL-8, suggesting inflammatory response induced by I/R injury was attenuated. Consistently, the renal function was preserved by HBOC, as evidenced by depressed BUN and creatinine levels. Therefore, we believe that the observed protective effect of HBOC on I/R kidney was (or partially) related to the down-regulation of inflammation.

It has been reported that patients treated with an HBOC had a 30% increased risk of mortality and a 2.7-fold increase for myocardial infarction, which was probably related to nitric oxide scavenging by HBOC (Natanson et al. 2008). In order to avoid these complications, we need to improve the quality of present HBOC product. Apart from that, reduction of the dosage seems to also be important, because administration of HBOC with low dosage, like 0.1 gHb/kg in this study, might not be accompanied by severe complications, such as sudden hypertension.

In conclusion, by use of the rat renal I/R injury model, we demonstrated that PolyPHb provided a protective effect on the kidney, and this effect was probably related to the depressed inflammatory response.

Declaration of interest

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

Acknowledegments

This study was supported by grants from the National Nature Science Foundation of China (81100180 and 81070117), the China Postdoctoral Specialized Science Foundation (201003700), the Specialized Research Fund for the Doctoral Program of Higher Education (20100181120090), the Major Program of the Clinical High and New Technology of PLA (2010gxjs039), and the Scientific Research Staring Foundation for Young Teachers of Sichuan University (2010SCU11022).