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Effects of Sugammadex and Neostigmine on Renal Biomarkers

Background Neostigmine, the currently commonly used agent for reversal of neuromuscular blockade. Sugammadex is a novel and unique compound designed as an antagonist of steroidal neuromuscular blockers. In this study, we evaluated the effects of sugammadex or neostigmine on kidney functions in patients scheduled for elective surgery. Material/Methods Patients scheduled for a surgical procedure under desflurane/opioid anesthesia received an intubating dose rocuronium. Patients were divided into 2 groups receiving either sugammadex or neostigmine atropine to reverse neuromuscular blockade. Cystatin C, creatinine, urea, blood urea nitrogen, sodium, potassium, and calcium levels in the blood and α1microglobulin, β2microglobulin, and microalbumin levels in the urine were measured. Results There was no significant difference between the groups with regard to the demographic data. In the Neostigmine Group, although β2microglobulin and microalbumin were similar, a significant increase was found in the postoperative α1microglobulin and cystatin C values. In the Sugammadex Group, although β2-microglobulin and cystatin C were similar, a significant increase was found in the postoperative α1-microglobulin and microalbumin values. The only significant difference was cystatin C value variation in the Neostigmine Group compared to the Sugammadex Group. Conclusions We believe that the use of more specific and sensitive new-generation markers like cystatin C to evaluate kidney function will provide a better understanding and interpretation of our results. Sugammadex has more tolerable effects on kidney function in patients than does neostigmine. However, when compared to preoperative values, there is a negative alteration of postoperative values. Neostigmine and sugammadex do not cause renal failure but they may affect kidney function.


Background
The reversal of the effects of muscle relaxant agents is an important issue in general anesthesia practice. The conventional cholinesterase-inhibiting agents, called anti-cholinesterase, exert their effect by indirectly inactivating cholinesterase in the neuromuscular junction. They are metabolized in the liver by acetyl cholinesterase and plasma esterase. Approximately 50% of these agents are excreted unchanged by the kidneys [1][2][3]. Neostigmine is the most potent and selective drug [4]. It is secreted from the tubular lumen, and its clearance is higher than the glomerular filtration rate (GFR). Additionally, its clearance is substantially decreased and its half-life is prolonged in patients with renal failure [5].
Cyclodextrins confine the muscle-relaxing agents via "molecular encapsulation" and render them water soluble. Sugammadex firmly binds one-to-one with steroid muscle relaxants and provides disposal through urine. The cavity of the sugammadex molecule encloses the 4 hydrophobic steroid rings through encapsulation [6][7][8]. The reason for the popularity of sugammadex is its reversal effect of blocking at any depth, both effectively and quickly, without waiting for a spontaneous reversal [9,10]. No metabolite of sugammadex has been found, and it has been determined to be excreted unchanged by the kidney. In healthy people, 48-86% of sugammadex is excreted unchanged in urine within 24 h [11].
It is well known that Cr is an indicator of renal function. However, as long as the GFR decreases it to less than 50 ml/min/1.73 m 2 , the serum Cr concentration does not change. Furthermore, its value is affected by age, sex, muscle mass, and dietary proteins. Unlike Cr, cystatin C (Cys C), a novel renal biomarker (cysteine protease inhibitor), is not affected by these factors. Moreover, it is freely filtered by the glomerulus, completely reabsorbed by the proximal tubule, and not secreted [12,13]. In evaluating renal function, the sensitivity of Cys C is 70%, and its specificity is 100%. It has been demonstrated that Cys C is a better parameter than serum Cr for detecting the smallest changes in GFR [15,16].
There are studies in the literature comparing the effects of sugammadex and neostigmine [8,13,18,19]. However, few of these studies were primarily designed to evaluate the effects of sugammadex and neostigmine on renal function. Therefore, we aimed to compare the short-term effects of sugammadex and neostigmine on renal glomerular filtration and tubular functions using new more sensitive biomarkers in patients with normal renal function.

Study design and patient selection
This study, which was supported by Yuzuncu Yil University, Department of Scientific Research Projects, with the Project Number 2010-TF-U123, was carried out with the approval of the Yuzuncu Yil University Local Ethics Committee (no. 07/11.04.2011), and it was conducted in an operating room at Yuzuncu Yil University, Dursun Odabas Medical Center. This study was conducted with written informed consent from the study subjects.
This study involved a total of 50 patients between the ages of 18 and 65 years who were scheduled for elective surgery under general anesthesia. For inclusion, the patients had normal renal function (serum Cr <1.5 mg/dL) and were classified as American Society of Anesthesiologists (ASA) Class I-II. Exclusion criteria were: liver failure, kidney failure, neuromuscular disorders, pregnant or breastfeeding, being treating with corticosteroids or oral contraceptives, a contraindication to the study drugs, an allergy to the drugs, a body mass index over 30 kg/m 2 , receiving medication known to interfere with the action of rocuronium (e.g., amino glycoside antibiotics and anticonvulsants), or did not wish to participate.

Anesthesia protocol
Premedication was not given to the patients. The patients were taken into the operating room and were monitored for heart rate (HR), mean arterial blood pressure (MAP), and peripheral oxygen saturation (SpO 2 ). The neuromuscular blockade was monitored by acceleromyography at the adductor pollicis muscle in response to ulnar nerve stimulation using the TOF-Watch SX device (Schering-Plough, Dublin, Ireland). The forearm was immobilized and surface skin electrodes were placed over the ulnar nerve proximal to the wrist. A train-of-four (TOF) stimulation was initiated. Following the initiation of 0.9% sodium chloride (NaCl) infusion, routine anesthesia induction was performed with 2 µg/kg fentanyl (fentanyl, Janssen-Cilag, Beerse, Belgium), 2 mg/kg propofol (propofol 1%, Fresenius Kabi, Austria), and 0.6 mg/kg rocuronium (Esmeron, N.V. Organon, Oss, Holland intubation was performed, and the patients were ventilated with anesthesia equipment with 4-6 L/min fresh gas flow, with the tidal volume maintained at 8 mL/kg and a frequency of 12/min. For the continuation of anesthesia, 60% N 2 O-O 2 and 4-6% desflurane (Suprane, Baxter Healthcare, Puerto Rico, USA) were administered. For the continuation of muscle relaxation, an additional dose of rocuronium 0.15 mg/kg was administered at the reappearance of a second twitch of TOF, as needed. The HR, MAP, and SpO 2 levels of the patients were recorded preoperatively, at every 10 min during anesthesia, and at 5 and 10 min after the drugs were administered. Patients were randomized preoperatively into 2 groups: the Neostigmine group (Group N, n: 25) and the Sugammadex group (Group S, n: 25). Randomization was performed by one of the authors (OP) using previously prepared, sealed, opaque envelopes. Randomization sequence was generated by using computer-generated random numbers. At the end of the surgical procedure, Group S was given 4 mg/kg sugammadex (Bridion, N.V. Organon, Oss, Holland) intravenously upon the reappearance of a post-tetanic count 1-2 or a second twitch of TOF after the last dose of rocuronium, because sugammadex had sufficient efficiency to reverse a deep and moderate neuromuscular blockade. Group N was administered a combination of 0.04 mg/kg neostigmine (neostigmine, Adeka, Samsun, Turkey) with 0.01 mg/kg atropine (Atropinsulfat, Drogsan, Ankara, Turkey) intravenously at the reappearance of a second twitch of TOF after the last dose of rocuronium, because neostigmine does not have adequate efficiency to reverse a deep neuromuscular blockade.

Sample collection and analysis
Preoperative blood and urine samples were collected from the patients before they were taken into the operating room. At 12 h following decurarization, blood and urine samples were collected postoperatively. The serum Cys C, Cr, urea, blood urea nitrogen (BUN), sodium (Na), potassium (K), and calcium (Ca) levels and the urine a1µg, b2µg, and µA levels were preoperatively and postoperatively determined. Routine biochemical parameters were measured by the colorimetric method and serum Cys C, urine a1µg, b2µg, and µA were measured using a nephelometric method. The primary endpoint was the acute effects of sugammadex or neostigmine on renal function as determined with more specific and sensitive tests.
To avoid affecting renal functions, we tried to keep the type of surgery, fluid resuscitation, and duration of anesthesia similar in both groups. Central body temperature was measured continuously during anesthesia and normothermia was maintained.

Sample size calculation and statistical analysis
According to the power analysis, sample size was calculated as a minimum 23 patients, based on our results to detect a minimum difference of 10% in the values of cystatin C between the 2 groups with a power of 80%, an a of 0.05, and a b of 0.2. Taking into account that approximately 10% of the enrolled patients might be excluded from the intent-to-treat evaluation, we decided to enroll 25 patients in each group [20].
Data are expressed as the means ± standard deviation (SD) or n, as appropriate. The Shapiro-Wilk test were used for a normality test of the data. The t test was used for comparisons of parameters that showed normal distribution, and the Mann-Whitney U test was used for comparisons of parameters that did not show a normal distribution. A one-way variance analysis with repeated measures was used to determine the differences in continuous variables between the groups. A P value of less than 0.05 was accepted as statistically significant. All analyses were performed using the Statistical Package for Social Sciences for Windows (SPSS 20.0.1; SPSS Inc.; Chicago, IL).

Results
The baseline characteristics data and total rocuronium doses were comparable in both groups and are presented in Table 1.
There was no statistically difference between the 2 groups with regard to the MAP or HR (Figures 1, 2).
There was no significant difference between the 2 groups with regard to Cr, urea, Na, or K values (p>0.05). When Group N was compared with Group S, both the preoperative and postoperative BUN values were increased (p=0.026 and p=0.012, respectively), but their value variation was similar (p>0.05). These values were not clinically significant. In both groups, the postoperative Ca values were decreased compared with the preoperative values when they were analyzed within the groups (p=0.015 and p=0.032, respectively; Table 2).
In both groups, the b2µg values and their value variations were similar (p>0.05), and the postoperative a1µg values were increased compared with preoperative values (p=0.008 and p=0.022, respectively; Table 2). For intra-group comparisons, the postoperative µA value was increased in Group S (p=0.001) but it was similar in Group N (p>0.05; Table 2). Additionally, their value variation was similar when Group N was compared with Group S (p>0.05). Although there was no difference between the preoperative and postoperative Cys C values in Group S, the postoperative Cys C value was increased relative to the preoperative value in Group N (p=0.008; Table 2). Additionally, the value variation was increased when Group N was compared with Group S (p=0.033).

Discussion
This study revealed that sugammadex and neostigmine affected renal glomerular filtration and tubular functions minimally, which was unexpected, because they are excreted unchanged. However, this effect was greater with neostigmine than sugammadex.
There were contradictions between the obtained results and the hemodynamic changes reported in the literature when sugammadex and neostigmine were compared. Two studies reported no differences in the MAP of patients administered neostigmine and sugammadex; however, this was only in the neostigmine group, whereas the HR was lower at first and subsequently became higher [18,21]. Other studies determined that there were no significant differences in the HR and MAP for both agents [11,19,22].
In this study, the hemodynamic changes were similar in both groups. There was no hemodynamic instability that could have affected renal function.
b2µg has a molecular weight of 11.000 Daltons and is found in all nucleated cells. a1µg is completely reabsorbed in the tubules. Both b2µg and a1µg are important indicators of renal tubular dysfunction. However, µA is a medium-sized protein.
An increase in its level in the urine is an indicator of damage in the glomerular barrier [12,17].
Sugammadex is biologically inactive and has no effect on animal tissues in vitro [8]. Although urinary excretion times of rocuronium and sugammadex are less than 2 h, the rocuronium sugammadex complex is excreted in urine within 24 h [5,23]. With respect to the renal biomarkers, because most of the studies were primarily designed to compare the reversal effects of sugammadex and neostigmine, the literature presents limited data. The routine biochemistry and urine analyses were similar in the reported studies [24,25]. In patients with mild or moderate renal failure, a change in dosage is not recommended. In patients with severe renal failure, the excretion of sugammadex or the sugammadex-rocuronium complex is somewhat delayed; however, no sign of recurrence of neuromuscular block has been observed [26]. Therefore, in the present study, to evaluate the glomerular function, in addition to microalbumin and Cr, we used Cys C, which is a more specific marker of glomerular function.
Bostan et al. reported that application of 1 mg/kg rocuronium +96 mg/kg sugammadex caused a histopathological degeneration in the kidneys. They determined glomerular vacuolation, tubular dilatation, vascular vacuolation and hypertrophy, lymphocyte infiltration, and tubular cell sloughing. However, they suggested that these histopathological changes did not cause any deterioration in renal function [27].
Sparr et al. [11] administered a placebo and different doses of sugammadex to 99 patients; 4 patients displayed an abnormality in µA, 2 patients were determined to have an abnormality of NAG, and 3 patients were determined to have an abnormality in b 2 µg values. Furthermore, they reported that in 1 patient, the Cr level was increased on the first postoperative day but returned to normal on the 20th postoperative day.  Sorgenfrei et al. [28] determined that 5 of the 27 patients receiving sugammadex had increased NAG levels, which is another renal tubular damage marker, and observed an increase in urinary albumin levels in 1 patient.
Ghoneim and El Beltagy [29] reported that the levels of serum Na, K, and kidney enzymes were similar in their study that compared the reversal effects of sugammadex and neostigmine in neurosurgical pediatric patients.
A study that compared the effects of sugammadex and neostigmine on vecuronium-induced neuromuscular blockade reported that the routine biochemistry and urine analyses, including the µA, b 2 µg, and NAG levels, were similar in both groups [30].
Another study compared the effects of sugammadex and neostigmine on rocuronium-induced neuromuscular blockade under sevoflurane anesthesia and reported that in the neostigmine group, albumin was presented in the urine and the urine b 2 µg values were increased [31]. Flocton et al. [22] detected increased NAG levels in 7 of the 34 patients in the sugammadex group and 1 of the 39 patients in the neostigmine group.
In a study that evaluated the effectiveness and safety of sugammadex in patients with normal renal function and terminal-stage renal failure, the researchers reported that the postoperative serum BUN and Cr, as well as urinary NAG, b 2 µg, and µA levels, of terminal-stage renal failure patients were higher than the preoperative values.
In the present study, in terms of sensitive biomarkers, because the postoperative values were higher than preoperative values, both agents also affected the kidneys. Additionally, the Cys C values were higher with neostigmine than with sugammadex. Although all the values in both groups were within the reference values, in the neostigmine group, a significant increase was found in the postoperative a1µg levels and the µA and Cys C levels. In the sugammadex group, there was a significant increase in the b2µg and µA levels. In the sugammadex group, the Cys C level was not changed. Therefore, we decided that sugammadex has more tolerable effects on the glomerular and tubular systems in patients than neostigmine does.

Conclusions
We found that when neostigmine and sugammadex were compared, neither affected hemodynamic parameters. Although neither neostigmine nor sugammadex affected the tubular and glomerular functions in the kidney, it must be remembered that both medications changed the renal parameters in patients who have normal kidney function.
Accordingly, we do not suggest that these medications should be used seamlessly, even in patients with mild renal dysfunctions. There is a need to study greater numbers of patients in evaluating long-term renal functions. A study limitation is that although patients with impaired or borderline renal function may be good choice as subjects in this type of study, we did not include these patients due to recommendations for use of sugammadex. Unfortunately, there was no control group of patients who reversed NMB spontaneously.