Effects of norepinephrine on kidney in a Swine model of cardiopulmonary resuscitation
Original Contribution
Effects of norepinephrine on kidney in a Swine model of cardiopulmonary resuscitation?
Yi Han, Chun-sheng Li?, Zhi-yu Su, Yi Lu, Sheng-qi Wang
Emergency Department of Beijing Chaoyang Hospital affiliated as Capital Medical University, Beijing, China
Received 18 December 2009; revised 29 January 2010; accepted 9 February 2010
Abstract
Background: The aim of this study was to study the effects of norepinephrine (NE)-induced hypertension (HT) on renal biochemistry, enzymology, and morphology after restoration of spontaneous circulation (ROSC) by cardiopulmonary resuscitation (CPR) in swine.
Methods: After 4 minutes of ventricular fibrillation, standard CPR was carried out. The survivors were then divided into 2 groups. The HT group (n = 5) received 0.4 to 1.0 ug kg-1 min-1 of NE continuously to maintain the mean arterial pressure (MAP) at 130% of the baseline (ie, MAP before ventricular fibrillation). The normal pressure (NP) group (n = 5) received 0.2 to 0.5 ug kg-1 min-1 NE continuously to maintain MAP at the baseline level. Hemodynamic status and Oxygen metabolism were monitored, and blood urea nitrogen and creatinine were measured in blood samples obtained at baseline and at 10 minutes, 2 and 4 hours after ROSC. At 24 hours after ROSC, the animals were killed and the kidney was removed to determine Na+-K+-ATPase and Ca2+-ATPase activities and histologic changes under a light and electron microscopy.
Results: mean arterial pressure, cardiac output, and coronary perfusion pressure were significantly higher (P b .01), whereas the Oxygen extraction ratio was lower in the HT group than in the NP group (P b .05). Blood urea nitrogen and creatinine increased in the NP group but did not change in the HT group. Renal ATP enzyme activity was significantly higher in the HT group than the NP group (Na+-K+-ATP enzyme: 4.024 +- 0.740 U versus 3.190 +- 0.789 U, Ca2+-ATP enzyme: 3.615 +- 0.668 versus 2.630 +- 0.816; both P b .05). The HT group showed less cellular edema, necrosis, and fewer damaged mitochondria compared with the NP group.
Conclusion: These data suggest that inducing HT by NE helps to maintain stable hemodynamic status and oxygen metabolism and may protect the kidney in terms of biochemistry, enzymology, and histology after CPR.
(C) 2011
Introduction
Norepinephrine (NE) is regarded as the most commonly used initial vasopressors agent in the United States [1]. The 2005 American Heart Association guidelines for cardiopul-
monary resuscitation (CPR) state that NE is an effective vasoactive drug that can increase the success rate for restoration of spontaneous circulation during CPR. However, these guidelines do not refer to its effect on hemodynamic status and oxygen metabolism after ROSC, or
? Financial support: The study was supported by the National Natural Science Foundation of China (No. 30972863).
* Corresponding author. Beijing Chaoyang Hospital, Chao Yang District, Beijing 10020, China. Tel.:+86 10 85231051; fax: +86 10 85231051.
E-mail address: [email protected] (C. Li).
0735-6757/$ - see front matter (C) 2011 doi:10.1016/j.ajem.2010.02.005
its influence on cardiac, hepatic, or renal ischemic-reperfu- sion injury (IRI) [2].
James and Daniel [3] studied the postresuscitation plasma catecholamines levels and found that the elevated catechol- amine levels may be detrimental to left ventricular function and contribute to cardiac dysfunction. Also, Mills et al [4] also pointed out its harmful effects on the kidney.
Therefore, in this study, we established a swine model of cardiac arrest , and after ROSC, the swine were given NE to increase the mean arterial pressure (MAP) to 130% (hypertension) or 100% (normal blood pressure) of the baseline MAP, and we observed the changes in hemody- namic status and oxygen metabolism, compared renal biochemical function, ATP enzyme activity, pathology, and ultramicrostructure between the 2 groups, and provided evidence for the benefits of NE after ROSC.
Methods
Animal preparation
All trials conformed to the specific guidelines developed by the local government in Beijing for animal experimen- tation. Twelve domestic swine weighing 28 +- 2 kg, of either sex, were used in this study; their care was in accordance with our institutional guidelines. Before the experiment, the swine were fasted but had free access to water for 1 day. Anesthesia was induced by ear vein injection of propofol (0.2 mL/kg) and maintained with intravenous infusion of 3% pentobarbital (30 mg/kg) at a rate of 8 mg kg-1 h-1. A cuffed 6.5-mm endotracheal tube was advanced into the trachea, and the animals were mechanically ventilated with a volume- controlled ventilator (Sero 900c; Siemens, Germany), using a tidal volume of 15 mL/kg, respiratory frequency of 18 breaths/min, and room air. Respiratory frequency was adjusted to maintain end-tidal PCO2 at 35 to 40 mm Hg. A 7F central venous catheter (Arrow, USA) was advanced from the right external jugular vein into the right atrium to measure right arterial pressure. A 7F sheathing canal was inserted to the left internal jugular vein as a Temporary pacemaker conductor. A Swan-Ganz catheter (Edwards, USA) was advanced from the left femoral vein into the pulmonary artery and connected to a Vigilance II CCO monitor (Edwards, USA). A 5F catheter (Terumo, Tokyo, Japan) was inserted from the right femoral artery into the aortic arch to measure aortic pressure.
Experimental protocol
The temporary pacemaker conductor was inserted into the right ventricle through the right sheathing canal and connected to an electrical stimulator (GY-600A, Kaifeng Huanan equipment Ltd. company, China) programmed in the S1S2 mode (300/200 milliseconds), 40 V, 8:1
proportion, and 10-millisecond step length, to provide continuous electrical stimulus until ventricular fibrillation (VF). Ventricular fibrillation was defined as an electrocar- diogram showing waveforms corresponding to VF and a rapid decline in MAP toward zero. Ventilation was stopped while inducing VF. After 4 minutes of VF [5], Manual CPR was carried out at a frequency of 100 compressions/min, with mechanical ventilation at FiO2 100% and a compres- sion-to-ventilation ratio of 30:2. After 2 minutes of CPR, if the spontaneous circulation was not restored, defibrillation was attempted once using a diPhase 150J. If spontaneous circulation was still not achieved, CPR was continued for a further 2 minutes and defibrillation was attempted once more. Restoration of spontaneous circulation was defined as an unassisted pulse with a systolic arterial pressure exceeding 80 mm Hg for more than 5 minutes [5]. If spontaneous circulation was not restored within 15 minutes, we regarded the animal as dead.
The survivors with ROSC were randomly placed into 2
groups (n = 5/group), an HT group, and a normal pressure (NP) group. The HT group was continuously treated with NE at 0.4 to 1.0 ug kg-1 min-1 to maintain MAP at 130% of baseline (ie, MAP before VF). The NP group was continuously treated with NE at 0.2 to 0.5 ug kg-1 min-1 to maintain MAP at a level comparable with that at baseline. Both groups received Fluid replacement (0.9% normal saline) at a rate of 10 mL kg-1 h-1. The animals were monitored for 4 hours and killed at 24 hours after ROSC by pentobarbital overdose.
Measurements
Hemodynamic parameters, including heart rate, cardiac output, aortic systolic pressure, aortic diastolic pressure, right atrial systolic pressure, and right atrial diastolic pressure, were measured continuously, and we recorded the values at baseline and at 10 and 30 min, 1, 2, 3, and 4 hours after ROSC. Blood samples were obtained at baseline and 2 hours,
4 hours after ROSC to analyze blood gas, Blood urea nitrogen , and Creatinine levels.
Other parameters including coronary perfusion pressure , MAP, oxygen supply (DO2), oxygen consumption (VO2), oxygen extraction ratio (ERO2), systemic vascular resistance (SVR), and Pulmonary vascular resistance were calculated using standard formulae.
After the animals were killed at 24 hours after ROSC, both kidneys were excised, and tissue samples were immediately frozen at -70?C until required to measure Na+-K+-ATPase and Ca2+-ATPase enzyme activity. En- zyme activity was assessed by measuring the optical density of Pi decomposed from ATP by the tissue protein using an enzyme-linked immunosorbent assay. Na+-K+- ATPase and Ca2+-ATPase activity were determined using standard formulae. The remaining tissue was preserved in 10% formaldehyde and 4% paraformaldehyde to observe Pathologic changes under a light microscope and changes
in tissue ultramicrostructure under a transmission electron microscope (TEM; Hitachi H-600, Japan). The pathologic data were assessed by reviewers blinded to the experimen- tal groups.
Statistical analysis
Measurement data are expressed as means +- SD and were analyzed by t tests with SPSS version 11.5 (SPSS,
Fig. 1 Mean arterial pressure, CO, CPP, SVR, DO2, VO2, and ERO2 at baseline and at each time-point after ROSC in the HT and NP group. Mean arterial pressure and CO started to increase at 10 minutes after ROSC in the HT group and was significantly higher than NP between 30 minutes and 4 hours after ROSC. Coronary perfusion pressure started to increase at 10 minutes after ROSC and was significantly higher in the HT group than in the NP group at 2 to 3 hours after ROSC. Systemic vascular resistance did not change after ROSC in the HT group but was higher in the NP after ROSC. Oxygen supply was significantly higher in HT after ROSC than at baseline and was comparable with that in the NP group. Oxygen consumption increased in the HT group at 2 hours after ROSC and decreased to the normal level at 4 hours after ROSC. The increase in ERO2 after ROSC was smaller in the HT group than in the NP group and was comparable at 4 hours after ROSC.
Chicago, Ill). Values of P b .05 were considered statistically significant.
Results
Two of the 12 animals died during CPR; 10 survived.
Hemodynamic status and oxygen metabolism
Compared with the NP group, heart rate, MAP, CO, and CPP after ROSC were significantly higher in the HT group, which was particularly noticeable between 30 minutes and 4 hours after ROSC (P b .05). The DO2 in the HT group increased at 2 and 4 hours after ROSC (P b .01) which was much higher than that in the NP group, and VO2 increased at 2 hours after ROSC, which was slightly higher than that in the NP group, and it decreased to the baseline level at 4 hours after ROSC. ERO2 was significantly lower in the HT group than in the NP group at 4 hours after ROSC (P b .05; Fig. 1A-G).
BUN and creatinine
After ROSC, the levels of BUN and creatinine tended to increase in the NP group, which was significant at 2 and 4 hours after ROSC (P b .05). By contrast, the levels of BUN and creatinine in the HT group did not change after ROSC compared with the baseline values (P N .05). Furthermore, there were no significant differences between the 2 groups at each time-point after ROSC (P N .05; Table 1).
Renal ATP enzyme activity
The Na+-K+-ATPase and Ca2+-ATPase enzyme activi- ties were higher in the HT group than in the NP group, which was statistically significant for Ca2+-ATPase activity (P b .01; Table 2).
Renal histology
Under a light microscope, the tubular epithelial cells in the NP group had an abnormal brush border and had marked edema. Furthermore, we noted extensive infiltration and adherence of inflammatory cells in the renal glomerulus. By contrast, the brush border of renal tubular epithelial cells was broadly normal, and there were fewer inflammatory cells in the HT group (Fig. 2). Under TEM, the NP group showed indistinct mitochondria, with increased electron density, and vacuolization could be seen in the glomerular cells. By contrast, the renal tubular epithelial cells in the HT group showed broadly normal mitochondria morphology, with intact capsules, and the ridge structure was clearly visible (Fig. 3).
Discussion
The first aim of treating a patient who experiences CA is to achieve ROSC, and the key factor for good long-term prognosis is to appropriately manage post-CA syndrome (PCAS). Restoration of spontaneous circulation after pro- longed whole body ischemia is an unnatural pathophysio- logic state created by successful CPR. Persistent hyperadrenergic state follows resuscitation from prolong CA and is driven by post-resuscitation ventricular dysfunc- tion and tissue hypoperfusion [3]. Meanwhile, the organs experience IRI, which initiates systemic inflammatory response syndrome and sepsis. This results in dysfunction of some vital organs, leading to Multiple organ dysfunction syndrome. These events significantly decrease the long-term survival rate of patients after CPR [6].
The 2005 American Heart Association guidelines for CPR recommends NE to increase the rate of achieving ROSC during CPR, but these guidelines did not refer to the
Fig. 2 Renal pathology in the NP (A, B) and HT (C, D) groups 24 hours after CPR. A, The brush border of renal tubular epithelial cells is irregular with erosion. B, The number of neutrophils invading the renal glomerulus has increased in the NP group. C, The brush border of renal tubular epithelial cells is broadly normal. D, The number of neutrophil invading the renal glomerulus is relatively low in the HT group.
period after ROSC [2]. Because NE is a strong agonist for ?- receptor, NE causes the visceral vessels to constrict and visceral ischemia may occur; thus, its clinical application is limited [4]. However, in recent years, some researchers have indicated that the use of NE in septic shock could maintain a stable hemodynamic status without causing renal injury [7,8], and some thought the effect of NE to increase renal blood flow (RBF) was more pronounced [9].
Therefore, we investigated the use of NE after ROSC to increase MAP to 130% of the baseline level, to determine its effect on hemodynamic status, oxygen metabolism, and on kidney function and morphology after ROSC, and to support the effective use of this vasoactive drug for treating PCAS.
Our study showed that shortly after achieving ROSC with cardiac dysfunction, low CO, and low MAP, the use of NE to raise MAP to 30% higher than the normal level improve CO and increase CPP, without increasing SVR. This indicates that NE not only significantly increases CO and CPP but also decreases the constriction of Peripheral vessels, and it does
not affect the Blood supply to vital organs. In terms of the hemodynamic status, DO2 increased, whereas VO2 did not change in the HT group; thus, the oxygen extraction ratio decreased in the HT group. Accordingly, this is beneficial for organ perfusion and could improve the low perfusion, ischemia, and hypoxia state caused by CA and ROSC, which is just in line with that found by Lindner et al [10]. Meanwhile, BUN and creatinine increased significantly in the NP group, but not in the HT group. This indicates that HT induced by NE may maintain renal perfusion after ROSC and normalize the renal glomerular filtration rate. We also found that the renal Na+-K+-ATPase and Ca2+-ATPase enzyme activities were much higher in the HT group than in the NP group, which indicates that NE has beneficial effects on Energy metabolism. These effects may be related to the change in RBF characteristics during the pathologic state caused by CPR [8]. Some researchers have documented that NE has a marked influence on organ blood flow, particularly in the kidney. It was proposed that NE would cause renal vascular constriction and aggravate renal ischemia [11].
Fig. 3 Renal ultramicrostructure of the NP (A,B) and HT (C,D) groups after CPR. A, The vacuolization in the renal glomerulus. B, The mitochondria in the renal tubular epithelial cells are irregular with increased electron density. C, The renal tubular epithelial cells are normal, with many normal mitochondria. D, The mitochondria in renal tubular epithelial cells are regular, with mitochondrial crista clear.
Because of the low MAP and low CO shortly after achieving ROSC, RBF decreased, which may lead to acute renal failure. However, our study showed that after ROSC, BUN, and creatinine did not increase compared with the baseline level in the HT group, and the renal ATPase enzyme activity was significantly higher than that in the NP group. ATPase enzymes have marked roles in ionic equilibrium, excitation conduction, transmitter release, and energy metabolism. Decreased ATPase enzyme activity may be caused by 2 pathways. First, because CA leads to systemic ischemia, the renal glomerulus and tubular cells experience ischemia, with inadequate oxygen supply to the mitochondria and decreased ATP synthesis, but not a corresponding reduction in energy consumption, which decreases the ATP concentration and ATPase enzyme activity. The second pathway is that after reperfusion, there is release of oxygen free radicals, intracellular calcium overload, and endotoxin release, resulting in excessive inflammatory responses, which adversely affect the ATPase proteolytic enzymes [9,12]. The HT state induced by NE preserves the renal blood supply after CPR to maintain adequate oxygen supply to the
mitochondria, reduce renal IRI, and increase the activity of renal ATPase enzymes.
To further investigate the effect of NE on the kidney, we observed the renal histology under light microscope and TEM. Under light microscopy, compared with the NP group, the brush border of the renal tubular epithelial cells was broadly normal in the HT group, with less cell degeneration, less edema, and fewer inflammatory cells in the renal glomerulus. Under TEM, the renal morphologic structure was much better in the HT group, and the mitochondrial crista structure was clearer. Furthermore, there were no noticeable abnormalities in the glomerular cells in the HT group. Because the integrity of the ridge structure of mitochondria is necessary for normal activity (ie, ATP production), the mitochondrial structure in the HT group is better preserved than that in the NP group and could maintain greater Na+-K+-ATPase and Ca2+-ATPase enzyme activities. In addition, cell swelling, necrosis, and inflammatory cell infiltration in the HT group were lower than in the NP group, which reflects less severe IRI in the HT group [13]. Collectively, these histologic results
demonstrate that the HT state induced by NE is protective for the kidney.
The dose of NE used in the HT group to maintain MAP at 130% of the baseline was much higher than that in previous studies. We infused NE at a rate of 0.4 to 1.0 ug kg-1 min-1 in the HT group after ROSC. Some studies have indicated that the RBF increased and renal vascular resistance decreased in response to intravenous NE infusion at rates of 0.1, 0.2, and 0.4 ug kg-1 min-1 [9]. Meanwhile, another study showed that as the infusion rate increased, the renal plasma flow and glomerular filtration rate decreased proportionally [14]. However, in our study, NE was infused at 0.4 to 1.0 ug kg-1 min-1 to maintain MAP at 130% of the baseline, which could strengthen the stability of the Hemodynamic state and oxygen metabolism and maintained adequate blood supply to the kidney. Thus, normal mitochondria function was maintained in the glomerular and tubular epithelial cells, with adequate energy supply to the kidney, avoiding acute renal injury or acute renal failure. Therefore, in these swine, NE at 0.4 to 1.0 ug kg-1 min-1 after ROSC prevented PCAS and maintained adequate renal function.
However, there are some limitations in this experiment. First, the sample in this study was small, and there were other control groups that could have been used in the experimental design. The addition of a pure control group, namely, animals that undergo general anesthesia and fluid resuscita- tion only, and a VF/fluid resuscitation only group, without NE, would be appropriate. Second, we did not have an access to equipment to directly measure RBF or renal oxygen metabolism, which should be further investigated.
Conclusions
We evaluated the effect of NE-induced HT on the kidney after ROSC in a CA model and confirmed its favorable effects on the kidney in terms of biochemistry, enzymology, and histology, particularly the ultramicrostructure, by infusing NE at a rate of 0.4 to 1.0 ug kg-1 min-1 after ROSC.
Acknowledgments
We sincerely thank Jun-yuan Wu and Zhao-xia Liu for their help with our experiments.
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