Open Access Austin Journal of Anesthesia and Analgesia

Special Article - Pediatric Anesthesiology Possible Protective Effect or Harmful of on Isquemia-Induced Acute Kidney Injury in a Pediatric Murine Model

Acosta-Murillo NR and Dueñas Gómez Z* Department of Physiological Sciences, National Abstract University of Colombia, Colombia The association between Ketamine and renal function begins in animal *Corresponding author: Dueñas Gómez Z, models, since the 1970s, particularly in relation to the effects on renal blood Department of Physiological Sciences, Division of flow [1,2]. Factors on renal hemodynamics such as decreased cardiac output Physiology, National University of Colombia, Bogotá, and blood pressure, sympathetic nerve stimulation and catecholamine release Colombia and, increased renin, angiotensin and vasopressin were involved [3]. From this century, the presence of N-Methyl-D-Aspartate Receptors (NMDA-R) outside Received: April 26, 2016; Accepted: June 01, 2016; the Central Nervous System (CNS) [4] where they had been initially identified Published: June 06, 2016 has been proposed, whereby the existence of the NMDA-R in the kidney and its functional role becomes important [5,6] in this way, the possible effects of NMDA-R antagonists, such as ketamine.

Keywords: L-Glutamate; NMDA receptors; Renal function, Ketamine; Acute kidney injury; Ischemia/Reperfusion

Abbreviations Α-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionic Acid (AMPA) receptor [8,9]. NMDA-R is large heterotetrameric membrane protein L-Glu: L-Glutamate; NMDA-R: N-Methyl-D-Aspartate complexes with a high permeability to calcium, which triggers a series Receptors; iGluRs: Ionotropic Glutamate Receptors; mGluRs: of calcium mediated intracellular events that have an outstanding Metabotropic Glutamate Receptors; AMPA: Α-Amino-3-Hydroxy- role in many physiological and pathological processes. Over the 5-Methyl-4-Isoxazolepropionic Acid, Gly: ; IMCD: Inner past decade, a variety of NMDA-R subunits have been recognized: Medullary Collecting Duct, APV: D-Amino phosphono valeric Acid; the ubiquitously expressed NR1 subunit; a family of four distinct 7CK: 7-Chlorokynurenic Acid; Con: -G and –T; PCP: NR2 subunits (A,B,C & D); and two NR3 subunits (A & B) [5]. ; PICU: Pediatric Intensive Care Unit; EPC: Epithelial These subunits have different isoforms and several splicing variants, Phenotypic Changes; AJC: Apical Junctional Complex; RBF: Renal of which the most studied are the NR1subunit, while the functional Blood Flow; AKI: Acute Kidney Injury; IRI: Ischemia-Reperfusion relevance of the different splicing forms of the NR2 and NR3 subunits Injury; MPO: Myeloperoxidase; LPS: lipopolysaccharide; TLR4: Toll- remains uncertain [5,7]. All NMDA receptors have NR1subunit; most Like Receptor; IL: Interleukin of them have a combination of NR2 and NR3 subunits. Activation of Introduction NMDA-R requires simultaneous binding of two different agonists, the L-Glu and Glycine (Gly), for this reason they are referred to as L-Glutamate (L-Glu) it is an α-amino acid abundant in the human co-agonists NMDA-R [10]. body and is a key compound in cellular metabolism, particularly in the human brain, where it is the most prominent neurotransmitter, NMDA-R and its distribution outside the CNS have emerged the main excitatory neurotransmitter, and also the precursor as an interesting research topic in the past decade. Recent studies for GABA, the main inhibitory neurotransmitter. L-Glu is a key show that the NMDA-R expressed in a variety of tissues, where it intermediate in metabolic pathways related to energy production, play an important role in numerous processes (such as proliferation, nitrogen metabolism and responses to oxidative stress. L-Glu exerts apoptosis, cell adhesion, and migration, actin rearrangement, cell its effects by acting on a large number of ionotropic (iGluRs) and growth and differentiation and regulation of hormone secretion), metabotropic (mGluRs) receptors, according to the mechanism by and may play an important role as a potential therapeutic target which their activation gives rise to a postsynaptic current, either for kidney and cardiovascular diseases, as well as bone diseases [4]. directly through the formation of ion channel pore or indirectly via NR1, the main subunit of the NMDA receptor, is located in several activation of ion channels through signaling cascades that involves extra neuronal tissues, including the rat kidney and heart. There is protein G, respectively. Glutamate receptors are expressed mainly also some expression of the NR1 subunit in the lung, thymus, and in the central nervous system and are involved in a number of stomach, suggesting that the NMDA receptor may play a much wider neurological conditions [5,7]. role than previously speculated [11]. Of the many specific subtypes of iGluRs, N-Methyl-D-Aspartate The functional relevance of NMDA-R for normal kidney Receptors (NMDA-R) are part of the most important group of physiology is not well understood although have been most receptors, together with , delta receptor and extensively studied in podocytes. It is also quite likely that NMDA-R

Austin J Anesthesia and Analgesia - Volume 4 Issue 1 - 2016 Citation: Acosta-Murillo NR and Dueñas Gómez Z. Possible Protective Effect or Harmful of Ketamine on ISSN : 2381-893X | www.austinpublishinggroup.com Isquemia-Induced Acute Kidney Injury in a Pediatric Murine Model. Austin J Anesthesia and Analgesia. 2016; Dueñas Gómez et al. © All rights are reserved 4(1): 1045. Dueñas Gómez Z Austin Publishing Group in other parts of the kidney have different properties. NMDA-R is Of the above medications, the most widely used is the ketamine, a expressed in the renal cortex and medulla, and appears to play a phencyclidine derived. In the 1950s, Parke-Davis industries developed role in the regulation of renal blood flow, glomerular filtration, CI-395 (Phencyclidine or N-1-Phenyl-Cyclohexylpiperidine and proximal tubule reabsorption and urine concentration within (PCP) chlorhydrate) and CI-400 (N-ethyl-1-phenylcyclohexamine medullary collecting ducts [12]. The NR1 and NR2C subunits are chlorhydrate), among cyclohexylamine drugs. In 1978 its production present in the rat renal cortex and medulla. stopped because of the severe psychodysleptic effects and its abusive use as a recreational drug. Further research in the 1960s led to the The other NR2 subunit proteins are not expressed in the kidney. synthesis and development of ketamine (CI-581, 2-phenyl-Ochloro- NR1 subunit protein increases during renal development and NR2C is 2-methylamino-cyclohexanone) [15]. The first clinical study in also present but in a non-significant amount. Immunohistochemistry humans was conducted in 1964, and the drug was introduced into studies reveal that the NR1 subunit is abundant in the apical region clinical use in 1970. In the central nervous system, ketamine has of the proximal tubule (S1-S3) [11] and in the basolateral surface affinity for various receptors. Unlike many other anesthetics, it of the proximal convoluted tubule [13]. Until recently, there was does not affect gamma-aminobutyric acid receptors at clinically no evidence for expression of NR3 subunits (NR3a&NR3b) in the relevant concentrations. The effects are mediated mainly kidney. NR3 is highly expressed in the neonate with expression levels through blockade of NMDA-R and possibly by enhancement of substantially decreasing shortly after birth. It is not, however, entirely descending inhibition in the spinal cord in chronic pain conditions absent in the adult: although NR3a decreases with age; there are [16]. Even though there are extensive research and descriptions of examples of sustained NR3a levels, for instance, in discrete regions of ketamine effects in the nervous system, few studies have evaluated the central nervous system including the amygdala, layer V pyramidal the physiological, pharmacological and toxic effects of ketamine in neurons, mesencephalic trigeminal neurons and retina [13]. tissues outside the CNS, despite the recognition of the presence of The importance of the NMDA receptors in the kidney and NMDA-R almost ubiquitously. its functional role has been of great interest as research area, The effects of ketamine, as a blocker NMDA-R in different although experimental data are scarce. The results of the study by tissues to the nervous system, have not been established in a pediatric Sproul and colleagues and their main finding are related with the murine model. In this study, we wanted to identify in three different expression of the NR3a subunit of the NMDA-R in the kidney. By age groups, if the renal ischemia-reperfusion injury (assessed by immunofluorescence, it was possible to determine that there was very serum creatinine and histopathologic findings) is related to the dose little expression of NR3a in the adult mouse renal cortex, but this was of ketamine used as an anesthetic. not the case, in the medullary/papillary region, where they detected a high level of expression of this protein. In addition, they were able Materials and Methods to show in Inner Medullary Collecting Ductcells (IMCD),that NR3a Animals protein was upregulated by hypoxia and, when the mice were on The study was approved by the Ethics Committee of our College water restriction, a condition that induce an elevated renal medullary of Medicine and the procedures were carried out according to the osmolality. Thus, the decreased of oxygen level and high osmolality routine animal-care guidelines. All experimental procedures were mayhelp to selectively drive expression of NR3a in this region [13]. complied with the Guide for the Care and Use of Laboratory Animals. There is now considerable evidence that excessive NMDA-R Male Wistar rats obtained from the Faculty of Veterinary Medicine activation is toxic for renal cells in vivo and in vitro. It is certainly of the National University of Colombia were selected and assigned to possible that metabolic disturbances, especially alterations in energy one of 3 groups: Group 1=10 days old, Group 2=20 days of age, Group metabolism, could make renal cells at risk to excitotoxic effects of 3=30 days of age, corresponding approximately to the equivalent of NMDA agonists and co-agonists [12], which supports the use of age in humans for infants, pre-school and pre-pubescent respectively. NMDA blockers as renal protective. They were maintained at approximately 21ºC on a 12 h light/dark cycle with free access to food and water. In the case of the 10 infants rats Blockers NMDA-R have been widely used for pain management (groups 10 days old) were guaranteed breastfeeding on demand. The and some diseases of the nervous system, however, is well known that rats were randomly divided into the Sham group (n=15, n=5 in each the potential of targeting NMDA receptors outside the CNS, have age group), and the model group (n=15, n=5 in each age group). emerged other possible pharmacological applications of NMDA-R antagonists. There are 3 types of NMDA antagonists: Competitive Preparation for experiment antagonists including both glycine site antagonists and glutamate The equipment, surgical tools and other materials (number needed site antagonists (D-Aminophosphonovaleric Acid (APV), the low in parentheses) were: a homeothermic monitor system (1), animal efficacy agonist HA966, CPP, 7-Chlorokynurenic Acid (7CK), hair clipper (1), chronometer (1), tissue forceps with blunt points , CPPene, Conantokins-G and -T (Con), -R, (2), tweezers with ultra-sharppoints (1), dissecting and operating NVP-AAM077 and UBP141; Channel blockers (acting at the ion scissors with sharp points (2), 5-0 Vicryl suture with curved needle channel pore) (Phencyclidine (PCP), ketamine, , (1), hemostats (4), skin separators (2), needle holder (1), syringes 1 MK801, , argiotoxin, polyamines, arcaine, ) and ml, 5 ml syringes, swabs, cotton swabs, gauze sponges, paper Negative allosteric modulators (acting at the amino terminal domain towels, red bags for anatomical and pathological waste, heparinized of GluN2B-containg receptors) (, , dynorphin, capillary blood collection, eppendorf tubes, centrifugelaboratory, neurosteroids, CP101606, Ro25-6981, UBP141, TCN201, QNZ46, micropipettes, disposable tips and surgical gloves. The solutions used DQP) [14]. were saline (0.9% sodium chloride), 10% Ketamine, and 2% Xylazine,

Submit your Manuscript | www.austinpublishinggroup.com Austin J Anesthesia and Analgesia 4(1): id1045 (2016) - Page - 02 Dueñas Gómez Z Austin Publishing Group and 10% buffered formalin, 70% alcohol, urine bottles collectors, and examination of renal tissues with hematoxylin and eosin (H&E) was conical tubes 15 ml, veterinary artificial tears. All of the surgical tools, performed. Histopathology studies and light microscopy were done materials, and solutions are sterilized. in the Department of Pathology, Faculty of Medicine of the National University of Colombia. Surgery and experimental protocol All procedures of individuals, were performed using strict sterile Statistical analysis techniques under general anesthesia with ketamine (40-90 mg / A descriptive analysis of the variables study was conducted. kg intraperitoneally) and xylazine(0,5 – 1,5 mg/kg). The solution Frequencies and percentages for the qualitative variables were was prepared with ketamine/xylazine in 1ml syringe in 9:1 ratio obtained, measures of central tendency (median and mean) and respectively. After injections of ketamine and xylazine, hair on one dispersion measures (standard deviation and inter-quartile range) for side of the rat was removed with hair cutter. The skin in the surgical the quantitative variables were found according to their distribution, area was cleaned with a cotton swab with 70% alcohol. which was known by the Shapiro-Wilk statistical test. Then we evaluated in each experimental (case) and control the relationship Immediately after the preparation of the skin, the rat was placed with histopathological variables and renal function. Finally the on a heating system and covered with sterile gauze. Surgery will not relationship according to the age group of subjects was obtained. The be started until the rat was in deep anesthesia and thus does not relationship between variables was performed using f Fisher test for respond to pain induced by tail pinch. The time was approximately qualitative variables and mean differences by Student t test and Mann 10 minutes after injection of ketamine + xylazine for deep anesthesia. Whitney U. For quantitative analysis between values preoperatively The rat was placed in the thermostatic station on the right side. The and postoperatively, student t test was used for paired samples and skin and muscle in the left flank were cut along the back to expose the Wilcoxon signed-ranks. Kruskal Wallis and ANOVA were used for left kidney. The incision is performed in a third body from the back of categorical variables and for pre- and post-analysis were used paired the rat and the size of the incision was 1-1.5 cm along. The kidney is techniques. A statistically significant p values <0.05 were considered. then pushed out from the cut with sterile cotton swabs to expose the renal pedicle. Dissection of the pedicle tissue was done with ultra-fine- Results point tweezers to remove the tissue around the renal pedicle in order A total of 30 rats were analyzed, which were distributed in three to expose the blood vessels for renal pedicle clamping. Hemostats groups according to age, 10 days (n=10), 20 days (n=10) and 30 days were used to block blood flow to the kidney inducing renal ischemia. (n=10). They were taken from each group 5 cases and 5 controls. The period of renal ischemia started from the time of clamping. Pathological analysis consisted of 45 specimens of kidney, 30 Complete ischemia was detected by color change of the kidney from intervention group (15 clamping kidneys, 15 not clamping kidneys) red to dark purple in a few seconds. After ischemia, the clamp was and 15 kidneys in the control group. Pre- and post-intervention released at desired times of 30 minutes to initiate reperfusion, which median for weight in the group 10 days old days was equal. was indicated by the change of kidney color to red. A 5-0 Vicryl The median pre and post-intervention weight in group 10 days suture was used to close the muscle layer of the incision followed by was equal, in the group 20 days post-intervention weight was slightly skin closure. Immediately after wound closure, 0.5 ml of warm sterile higher and the group 30 days an increase was evident in the post saline intraperitoneally to each rat was administered. The animal was intervention weight (Table 1). kept on a heating pad immediately after, until it had full recovery of consciousness before being returned to its home cage, to continue Anesthetic ketamine requirements in the first and second their previous overall food and habitat. During the time of anesthesia intervention are presented in (Table 2). and while waiting for full recovery of consciousness, artificial tears Median serum creatinine preoperatively was 0.46 (± 0.04), were placed to need. minimum value of 0.37 and maximum value of 0.56; after the At 72 hours of the initial surgical procedure, a second surgery intervention there was a significant increase in values with a global was carried on in the same conditions as the previous one, under average of 0.51 (± 0.07), minimum of 0.37 and maximum of 0.71 the same combination of anesthetic for the removal of the injured (p=0,002). We found that initial creatinine values were equal between kidney and contralateral uninjured in experimental rats. In control cases and controls in both the global population and by age group. rat’s only one kidney was extract under the same conditions that Creatinine values post intervention showed no differences between experimental group. The removed kidneys were immediately placed groups (Table 3). For values between pre and post creatinine found in urine collectors vials with 30ml, 20ml of 10% buffered formalin for Table 1: Distribution pre and post intervention weight according to ages. tissue fixation. Age Group Weight in grams N min p25 p50 p75 max The success of renal IR was monitored at several stages. First, after Ischemia surgery 10 22 27 30 31 32 clamping, the kidney color should change from red to dark purple, 10 days Nephrectomy 10 25 28 30 31 35 an indicative of a successful renal ischemia. Second, serum creatinine Ischemia surgery 10 35 40 40,5 43 50 was taken before starting the procedure and then at 72 hours of 20 days reperfusion. The samples were obtained from the retro-orbital venous Nephrectomy 10 35 42 42,5 45 60 plexus, frozen and sent to the Clinical Laboratory of Department Ischemia surgery 10 65 73 81 85 91 for Sciences Animal Health of the Faculty of Veterinary Medicine 30 days Nephrectomy 10 80 83 87,5 92 100 of the National University of Colombia. Finally, histopathological Min: Minimum; Max: Maximum

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Table 2: Ketamine dose distribution in first and second intervention according was significant. Confidence intervals of 95% were performed for to the ages. quantitative variables. Doses of Ketamine (mg/ Age Group N min p25 p50 p75 max dl) Discussion Ischemia surgery 10 24 29,0 38,4 45 60 10 days Ketamine has been used in clinical for over half a century and its Nephrectomy 10 25,7 29,0 31,0 45 54 role in the management of critically ill children has focused primarily Ischemia surgery 10 22,5 45 47,5 52,3 71,0 20 days on sedation and analgesia [16,18], withdrawal and bronchospasm. Nephrectomy 10 22,5 30 43,9 60 64,2 Ketamine provides bronchodilation by increasing catecholamine Ischemia surgery 10 37,0 49,4 60,6 65,8 87,6 transmission and stimulation of β2 adrenergic receptors and as a 30 days Nephrectomy 10 36 45 51,4 58,2 61,8 result of ketamine’s NMDA activity, it may decrease development of tolerance, making it a reasonable adjunct for sedation and Min: Minimum; Max: Maximum analgesia in the Pediatric Intensive Care Unit (PICU) [18]. Table 3: Values pre and post creatinine according to case-control group in the general population and according to the days of old. Ketamine elimination clearance is high (12-20ml/kg/min), Not clamping Clamping elimination half-life is 2-3 hand clearance may be 20% higher in kidney kidney women than in men. Intramuscular (IM) absorption is faster in Creatinine N Average SD Average SD p children than in adults. Distribution volume is slightly lower (1.9 l/ Ischemia 15 0,47 0,04 0,45 0,13 0,51 kg) but plasma clearance is more important (16.8ml/kg/min) than in General surgery population adults. The average dose of removal / elimination half-life of Ketamine Nephrectomy 15 0,51 0,08 0,51 0,06 0,77 is also shorter in children: 100 min after an identical dose expressed Ischemia 5 0,47 0,05 0,45 0,07 0,60 in mg/kg; plasma concentrations of the major active metabolite of 10 days surgery Nephrectomy 5 0,51 0,05 0,45 0,05 0,13 ketamine, , are higher in children. However, in children Ischemia from 4 to 10 years old and after an Intravenous (IV) 2mg/kg or IM 5 0,48 0,03 0,44 0,03 0,13 20 days surgery 6mg/kg injection, ketamine plasma concentrations are similar to Nephrectomy 5 0,55 0,10 0,55 0,06 0,98 those observed in adults. On the other hand, in the first 3 months of Ischemia 5 0,45 0,05 0,46 0,05 0,57 life, ketamine plasma clearance is shorter, probably due to decrease of 30 days surgery transformation in the liver and excretion in the kidney. In this case, Nephrectomy 5 0,46 0,06 0,51 0,05 0,16 there is an increase in the ketamine elimination half-life in newborns Table 4: Distribution of surgical specimens according to the scale of Goujon [17]. and infants. Distribution volume seems to be comparable to older N Escala de Goujon 100% children [15]. (45) 1. Normal 18 40 Related to the binding of the NMDA antagonistic drug with 2. PCT and DCT with intracellular edema, renal inner medulla the NMDA-R, the mechanisms by which exert an impairment of 20 44,4 and outer with intracellular edema NMDA receptor function, are quite complex. A substantial reason 3. Extensive coagulation necrosis compromising cortex and 3 6,6 for the variation in effect lies in the off-rate of the compound due medulla 4. Coagulation necrosis compromising cortex and medulla (50% to a phenomenon has been termed “trapping block”. Ketamine is an 3 6,6 of the renal parenchyma) example of high-trapping antagonist (86% trapping),) as when the 5. Necrosis and recent thrombosis of an artery and focal 1 2,2 dystrophic calcification glutamate has dissociated from its binding site on the NMDA-R, PCT: Proximal Convoluted Tubule; DCT: Distal Convoluted Tubule the ketamine remains trapped in the, now closed, ion channel thus causing a prolonged tonic blockade which disrupts both physiological in the control group a median of 0.46 (interquartile range = 0.43 to and pathological functions [19]. The antagonism is more important 0.50) in the value of pre creatinine and 0.49 (interquartile range = if the NMDA channel has been previously opened by the glutamate 0.44 to 0.56) in the value of creatinine post-intervention (p=0.09). fixation [15]. In the case group, the pre-intervention median creatinine was 0.44 ((interquartile range = 0.41 to 0.49) and 0.52 (interquartile range = It is controversial the effect of ketamine on the renal tissue and 0.46 to 0.56) for the value of creatinine post-intervention (p=0, 03). little is known about the effects it could have on the acute kidney injury of critical patients in part because its precise mechanisms of Pathological analysis consisted of 45 specimens, 30 intervention action remain unknown. First documented in 2007 [20,21], Ketamine group (15 clamping kidneys, 15 not clamping) and 15 kidneys in has been shown to injure the bladder, causing ulcers (wounds) and the control group. By classifying the histopathologic features using fibrosis (stiffening of the bladder walls and shrinkage). Patients the scale of Goujon, we found that 44.4% had TCP and TCD with present with multiple symptoms including incontinence, bleeding, intracellular edema, spinal internal and external with intracellular overactive bladder and bladder shrinkage, as well as damage to both edema (Table 4). the kidneys and the ureter. In 2009, Hills CE and colleagues consider Because rats were chosen homogeneously in terms of age, classified that Ketamine alters epithelial cell-to-cell adhesion and cell-coupling into three groups respectively according to their days old, of the same in the proximal kidney via a non-classical pro-fibrotic mechanism race and litter, a design of completely randomized experimental and the data provides the first indication that this illicit substance can classification balanced by fixed effect was used, complemented have major implications on renal function [21]. with T students test at 5% significance when analysis of variance In the study of Lee TH et al propose that some volatile anesthetics

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Table 5: Ketamine vs creatinine in the group of 10 days old two-sample t test In 2002, Deng and colleagues published the first report of assuming unequal variances. functioning NMDA-R in the kidney. The data document the Ketamine (mg/kg) Creatinine* presence of NMDA-R mRNA and protein in rat kidney cortex. Mean 743,453,062 492,172,579 These data confirm a role for renal NMDA-R in maintaining normal Variance 23,754,455 18,788,746 renal function. The co-agonist requirement of the NMDA-R for glycine gives credibility to the latter suggestion as does the ability of Observations 10 10 systemically administered NMDA-R inhibitors to selectively alter Hypothetical mean difference 0 renal hemodynamics, independently of their effects on baseline Renal Degrees of freedom 18 Blood Flow (RBF) [6]. In 2004, Leung JC and colleagues, in a rat Statistical t 106,436,829 model of short-term, high dose gentamicin nephrotoxicity, showed that there was an increase expression of both renal NMDA subunits, P (T <= t) one-tail 1.70E-05 NR1 and NR2C. They speculate that following glomerular filtration of Critical value of t (one-tail) 173,406,361 gentamicin, the drug interacts with the NMDA receptor, stimulating P (T <= t) two-tail 3.39E-06 entry of calcium into the cell through the NMDA calcium channel. Critical value of t (two-tail) 210,092,204 Finally, the amelioration of renal damage in rats exposed to short- *Expressed in percentage change between pre and post intervention value. term gentamicin that are pretreated with the NMDA receptor blocker MK-801 provides further proof of the important role of this receptor Table 6: Ketamine vs pathology score in the group of 10 days old (not clamping kidneys) two-sample t test assuming unequal variances. in this model of renal toxicity [24]. Ketamine (mg/kg) Pathology score** Expression of both renal NR1 protein and mRNA is upregulated Mean 743,453,062 1,8 during ischemia-reperfusion insult [25] and by short-term treatment

Variance 23,754,455 0,17777778 with gentamicin [23], as well as in the endotoxemic model reported by Lin CS et al [26]. Several studies have shown the involvement Observations 10 10 of NMDA-R in Acute Kidney Injury (AKI) induced by ischemia- Hypothetical mean difference 0 reperfusion and have tried to elucidate the causes. It has been seen that Degrees of freedom 9 NMDA agonists significantly aggravated renal Ischemia-Reperfusion Statistical t 148,790,201 Injury (IRI) -induced oxidative stress and AKI. The NMDA-R has various sites within it that regulate channel function. Major sites on P (T <= t) one-tail 6.05E-04 NMDA-R include the NMDA or glutamate binding site, glycine, and Critical value of t (one-tail) 183,311,293 polyamine binding site. Ketamine is an antagonist of NMDA-R that P (T <= t) two-tail 1.21E-03 binds at the polyamine binding site. Unlike the study of Dimer Leffa Critical value of t (two-tail) 226,215,716 D et al [23], the study of Pundir M et al found a significant protection with ketamine in renal IRI at a significantly higher dose (180mg/kg, **Value 1-5 according to score Goujon. intraperitoneally) than is used for anesthesia, while the anesthetic confer profound protection against renal ischemia–reperfusion injury dose of ketamine had no protective effect on kidney. Treatment with compared with or ketamine anesthesia by attenuating ketamine reduced Myeloperoxidase (MPO) activity and neutrophil inflammation. They anesthetize rats with equipotent doses of volatile accumulation and the renoprotection observed with NMDA-R anesthetics (, , , or ) or antagonists along with restoration of endogenous antioxidants, is injectable anesthetics (pentobarbital or ketamine) and subjected to supported by other reports claiming restoration of catalase activity 45min of renal ischemia and 3h of reperfusion during anesthesia, and stores as a significant therapeutic strategy in the and conclude that volatile anesthetic treatment reduced renal cortex management of renal disorders [25]. myeloperoxidase activity and reduced nuclear translocation of proinflammatory nuclear factor κB [22]. Nevertheless Dimer Leffa It is also known that NMDA receptor hyperfunction D et al demonstrated [23] that after the anesthesia procedure in contributes to acute renal failure during LPS induced endotoxemia. mice, the ketamine (doses between 80-140mg/kg in different groups) Lipopolysaccharide (LPS) binds to Toll-Like Receptor 4 (TLR4) and showed DNA damage after 1 and 12h for blood collection in all doses induces Interleukin (IL)-1β release from tubular cells. TLR4 and IL-1 receptor (IL-1R) signaling intubular cells increase expression of the tested. Studies with inhalator anesthetic sevoflurane, halotano, and NMDA receptor NR1 subunit and racemase. Upregulation of desflurane also demonstrated DNA damage after 2h of exposure. NR1 and serine racemase, together with increased D-serine levels, After 24h, the results in blood cells demonstrate that DNA damage results in NMDA receptor hyper function. NMDA receptor hyper persists only in the highest dose of ketamine, demonstrating that DNA function in LPS causes tubular cell damage and poor renal perfusion damage was not totally repaired. This probably happened because the through vasoconstriction leading to acute renal failure. Inhibition of DNA repair system was effective against DNA damage induced by NMDA receptors attenuated LPS-induced functional deterioration ketamine, but this system is dose limited. After 24h of the anesthesia, and tubular cell damage in in vivo and in vitro. In this study [26], mice were killed and the liver, kidney, brain, and bone marrow were NMDA-R inhibition prevented reductions in renal perfusion and analyzed to verify the extent of DNA damage in these organs. The ameliorated decline in ultra filtration but not NR1 expression, results demonstrated that only the highest dose of ketamine (140 mg/ indicating that NMDA-R hyperfunction contributes to renal kg, alone or in combination with xylazine) has damaged DNA in the vasoconstriction in endotoxemic kidneys. brain cortex and no damage was found in the liver and kidney.

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Table 7: Ketamine vs pathology score in the group of 10 days old (clamping Table 10: Ketamine vs pathology score in the group of 20 days old (clamping kidneys) two-sample t test assuming unequal variances. kidneys) two-sample t test assuming unequal variances. Ketamine (mg/kg) Pathology score** Ketamine (mg/kg) Pathology score**

Mean 723,330,087 1,6 Mean 98,348,157 2,6

Variance 214,484,669 0,8 Variance 428,484,692 1,3

Observations 5 5 Observations 5 5

Hypothetical mean difference 0 Hypothetical mean difference 0

Degrees of freedom 4 Degrees of freedom 4

Statistical t 107,795,488 Statistical t 103,273,752

P (T <= t) one-tail 0,00020999 P (T <= t) one-tail 0,00024802

Critical value of t (one-tail) 213,184,679 Critical value of t (one-tail) 213,184,679

P (T <= t) two-tail 0,00041999 P (T <= t) two-tail 0,00049605

Critical value of t (two-tail) 277,644,511 Critical value of t (two-tail) 277,644,511 **Value 1-5 according to score Goujon. ** Value 1-5 according to score Goujon. Table 8: Ketamine vs creatinine in the group of 20 days old Two-sample t test Table 11: Ketamine vs creatinine in the group of 30 days old Two-sample t test assuming unequal variances. assuming unequal variances. Ketamine (mg/kg) Creatinine* Ketamine (mg/kg) Creatinine*

Mean 926,247,429 201,998,312 Mean 111,133,687 753,835,361

Variance 396,029,221 542,952,407 Variance 222,567,377 270,602,296

Observations 10 10 Observations 10 10

Hypothetical mean difference 0 Hypothetical mean difference 0 753,835,361

Degrees of freedom 18 Degrees of freedom 18 270,602,296

Statistical t 7,474,109 Statistical t 147,516,979

P (T <= t) one-tail 3.18E-03 P (T <= t) one-tail 8.52E-09

Critical value of t (one-tail) 173,406,361 Critical value of t (one-tail) 173,406,361

P (T <= t) two-tail 6.37E-03 P (T <= t) two-tail 1.70E-07

Critical value of t (two-tail) 210,092,204 Critical value of t (two-tail) 210,092,204 *Expressed in percentage change between pre and post intervention value. *Expressed in percentage change between pre and post intervention value. Table 9: Ketamine vs pathology score in the group of 20 days old (not clamping Table 12: Ketamine vs pathology score in the group of 30 days old (not clamping kidneys) two-sample t test assuming unequal variances. kidneys) two-sample t test assuming unequal variances. Ketamine (mg/kg) Pathology score** Ketamine (mg/kg) Pathology score**

Mean 926,247,429 1,5 Mean 111,133,687 1,4

Variance 396,029,221 0,27777778 Variance 222,567,377 0,26666667

Observations 10 10 Observations 10 10

Hypothetical mean difference 0 Hypothetical mean difference 0

Degrees of freedom 9 Degrees of freedom 9

Statistical t 144,750,624 Statistical t 232,460,518

P (T <= t) one-tail 7.68E-04 P (T <= t) one-tail 1.20E-05

Critical value of t (one-tail) 183,311,293 Critical value of t (one-tail) 183,311,293

P (T <= t) two-tail 1.54E-03 P (T <= t) two-tail 2.40E-05

Critical value of t (two-tail) 226,215,716 Critical value of t (two-tail) 226,215,716 ** Value 1-5 according to score Goujon. ** Value 1-5 according to score Goujon. Another important observation of the study of Leung JC and duct principal cells in the adult kidney a further step in understanding colleagues [24] is the finding that NR3a regulates Ca2+ permeability of the complex regulation of urinary concentrating capacity [13,27]. 2+ but it is not able to respond to changes in [Ca ]i. This may suggest In the study of Yang in 2008, ischemia-reperfusion injury enhances that NR3a has other effects on cell function and regulation of [Ca2+] NR1 expression in the outer medulla, a major damaged area after i that may be independent of the classic role of NMDAR subunits. In ischemia-reperfusion. An intra-renal NMDA agonist significantly addition, potentially NR3a could play a role for nephron development reduces glomerular and tubular function. Glomerular and tubular considering the abundant expression in the neonatal kidney and dysfunction, after exposure to ischemia-reperfusion injury, can be decreases with age. The novel finding that NR3a is located to collecting enriched by pretreatment with the NMDA-R blocker D-AP-5, which

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Table 13: Ketamine vs pathology score in the group of 30 days old (clamping children that the NR3 subunit of NMDA-R is highly expressed in the kidneys) Two-sample t test assuming unequal variances. neonate with expression levels substantially decreasing shortly after Ketamine (mg/kg) Pathology score** birth and that the participation of this subunit may play a role so far Mean 109,532,211 3,2 unknown NMDA-R in kidney and pathophysiology of acute kidney Variance 977,623,646 2,7 injury in children. Observations 5 5 The mechanisms by which NMDA-R may affect renal function Hypothetical mean difference 0 are diverse and, additional studies are needed to establish whether the effect of ketamine in the kidney depends on the dose, the mechanism Degrees of freedom 4 of induction of acute kidney injury, the different ways in where Statistical t 237,218,277 antagonism of NMDA-R occurs and the relationship or interaction P (T <= t) one-tail 9.36E-02 of NMDA-R with factors understudied in children as Klotho protein, Critical value of t (one-tail) 213,184,679 highly expressed in the kidney and recognized as “anti-aging” protein

P (T <= t) two-tail 1.87E-01 because its levels and protective action could have a different impact on the AKI at different ages [34-36]. Critical value of t (two-tail) 277,644,511 ** Value 1-5 according to score Goujon. Acknowledgement is suggestive of a definitive role for the NMDA receptor in renal This work was supported by the Medical School at the National injury [28]. University of Colombia, HERMES Code: 28075. Although most authors concluded that NMDA-Rs exist in the References kidney to mediate injury. In that sense Deng proposes that inhibiting 1. Fischer D, Omlor D, Kreuscher D. Influence of ketamine anaesthesia on renal NMDA-R with a channel blocker or glycine antagonist causes renal and cardiovascular functions in mongrel dogs. Int Urol Nephrol. 1979; 11: 271-277. vasoconstriction and explains that the effects of NMDA-R on the kidney must be dichotomous as there is precedent for this in the 2. Bailie MD, Alward CT, Sawyer DC, Hook JB. 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Austin J Anesthesia and Analgesia - Volume 4 Issue 1 - 2016 Citation: Acosta-Murillo NR and Dueñas Gómez Z. Possible Protective Effect or Harmful of Ketamine on ISSN : 2381-893X | www.austinpublishinggroup.com Isquemia-Induced Acute Kidney Injury in a Pediatric Murine Model. Austin J Anesthesia and Analgesia. 2016; Dueñas Gómez et al. © All rights are reserved 4(1): 1045.

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