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

Special Article - Pediatric Anesthesiology

Austin J Anesthesia and Analgesia. 2016; 4(1): 1045.

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

Acosta-Murillo NR and Dueñas Gómez Z*

Department of Physiological Sciences, National University of Colombia, Colombia

*Corresponding author: Dueñas Gómez Z, Department of Physiological Sciences, Division of Physiology, National University of Colombia, Bogotá, Colombia

Received: April 26, 2016; Accepted: June 01, 2016; Published: June 06, 2016


The association between Ketamine and renal function begins in animal models, since the 1970s, particularly in relation to the effects on renal blood flow [1,2]. Factors on renal hemodynamics such as decreased cardiac output and blood pressure, sympathetic nerve stimulation and catecholamine release and, increased renin, angiotensin and vasopressin were involved [3]. From this century, the presence of N-Methyl-D-Aspartate Receptors (NMDA-R) outside the Central Nervous System (CNS) [4] where they had been initially identified 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


L-Glu: L-Glutamate; NMDA-R: N-Methyl-D-Aspartate Receptors; iGluRs: Ionotropic Glutamate Receptors; mGluRs: Metabotropic Glutamate Receptors; AMPA: Α-Amino-3-Hydroxy- 5-Methyl-4-Isoxazolepropionic Acid, Gly: Glycine; IMCD: Inner Medullary Collecting Duct, APV: D-Amino phosphono valeric Acid; 7CK: 7-Chlorokynurenic Acid; Con: Conantokins-G and –T; PCP: Phencyclidine; PICU: Pediatric Intensive Care Unit; EPC: Epithelial Phenotypic Changes; AJC: Apical Junctional Complex; RBF: Renal Blood Flow; AKI: Acute Kidney Injury; IRI: Ischemia-Reperfusion Injury; MPO: Myeloperoxidase; LPS: lipopolysaccharide; TLR4: Toll- Like Receptor; IL: Interleukin


L-Glutamate (L-Glu) it is an α-amino acid abundant in the human body and is a key compound in cellular metabolism, particularly in the human brain, where it is the most prominent neurotransmitter, the main excitatory neurotransmitter, and also the precursor for GABA, the main inhibitory neurotransmitter. L-Glu is a key intermediate in metabolic pathways related to energy production, nitrogen metabolism and responses to oxidative stress. L-Glu exerts its effects by acting on a large number of ionotropic (iGluRs) and metabotropic (mGluRs) receptors, according to the mechanism by which their activation gives rise to a postsynaptic current, either directly through the formation of ion channel pore or indirectly via activation of ion channels through signaling cascades that involves protein G, respectively. Glutamate receptors are expressed mainly in the central nervous system and are involved in a number of neurological conditions [5,7].

Of the many specific subtypes of iGluRs, N-Methyl-D-Aspartate Receptors (NMDA-R) are part of the most important group of receptors, together with kainate receptor, delta receptor and Α-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionic Acid (AMPA) receptor [8,9]. NMDA-R is large heterotetrameric membrane protein complexes with a high permeability to calcium, which triggers a series of calcium mediated intracellular events that have an outstanding role in many physiological and pathological processes. Over the past decade, a variety of NMDA-R subunits have been recognized: the ubiquitously expressed NR1 subunit; a family of four distinct NR2 subunits (A,B,C & D); and two NR3 subunits (A & B) [5]. These subunits have different isoforms and several splicing variants, of which the most studied are the NR1subunit, while the functional relevance of the different splicing forms of the NR2 and NR3 subunits remains uncertain [5,7]. All NMDA receptors have NR1subunit; most of them have a combination of NR2 and NR3 subunits. Activation of NMDA-R requires simultaneous binding of two different agonists, the L-Glu and Glycine (Gly), for this reason they are referred to as co-agonists NMDA-R [10].

NMDA-R and its distribution outside the CNS have emerged as an interesting research topic in the past decade. Recent studies show that the NMDA-R expressed in a variety of tissues, where it play an important role in numerous processes (such as proliferation, apoptosis, cell adhesion, and migration, actin rearrangement, cell growth and differentiation and regulation of hormone secretion), and may play an important role as a potential therapeutic target for kidney and cardiovascular diseases, as well as bone diseases [4]. NR1, the main subunit of the NMDA receptor, is located in several extra neuronal tissues, including the rat kidney and heart. There is also some expression of the NR1 subunit in the lung, thymus, and stomach, suggesting that the NMDA receptor may play a much wider role than previously speculated [11].

The functional relevance of NMDA-R for normal kidney physiology is not well understood although have been most extensively studied in podocytes. It is also quite likely that NMDA-R in other parts of the kidney have different properties. NMDA-R is expressed in the renal cortex and medulla, and appears to play a role in the regulation of renal blood flow, glomerular filtration, and proximal tubule reabsorption and urine concentration within medullary collecting ducts [12]. The NR1 and NR2C subunits are present in the rat renal cortex and medulla.

The other NR2 subunit proteins are not expressed in the kidney. NR1 subunit protein increases during renal development and NR2C is also present but in a non-significant amount. Immunohistochemistry studies reveal that the NR1 subunit is abundant in the apical region of the proximal tubule (S1-S3) [11] and in the basolateral surface of the proximal convoluted tubule [13]. Until recently, there was no evidence for expression of NR3 subunits (NR3a&NR3b) in the kidney. NR3 is highly expressed in the neonate with expression levels substantially decreasing shortly after birth. It is not, however, entirely absent in the adult: although NR3a decreases with age; there are examples of sustained NR3a levels, for instance, in discrete regions of the central nervous system including the amygdala, layer V pyramidal neurons, mesencephalic trigeminal neurons and retina [13].

The importance of the NMDA receptors in the kidney and its functional role has been of great interest as research area, although experimental data are scarce. The results of the study by Sproul and colleagues and their main finding are related with the expression of the NR3a subunit of the NMDA-R in the kidney. By immunofluorescence, it was possible to determine that there was very little expression of NR3a in the adult mouse renal cortex, but this was not the case, in the medullary/papillary region, where they detected a high level of expression of this protein. In addition, they were able to show in Inner Medullary Collecting Ductcells (IMCD),that NR3a protein was upregulated by hypoxia and, when the mice were on water restriction, a condition that induce an elevated renal medullary osmolality. Thus, the decreased of oxygen level and high osmolality mayhelp to selectively drive expression of NR3a in this region [13].

There is now considerable evidence that excessive NMDA-R activation is toxic for renal cells in vivo and in vitro. It is certainly possible that metabolic disturbances, especially alterations in energy metabolism, could make renal cells at risk to excitotoxic effects of NMDA agonists and co-agonists [12], which supports the use of NMDA blockers as renal protective.

Blockers NMDA-R have been widely used for pain management and some diseases of the nervous system, however, is well known that the potential of targeting NMDA receptors outside the CNS, have emerged other possible pharmacological applications of NMDA-R antagonists. There are 3 types of NMDA antagonists: Competitive antagonists including both glycine site antagonists and glutamate site antagonists (D-Aminophosphonovaleric Acid (APV), the low efficacy agonist HA966, CPP, 7-Chlorokynurenic Acid (7CK), Selfotel, CPPene, Conantokins-G and -T (Con), Conantokin-R, NVP-AAM077 and UBP141; Channel blockers (acting at the ion channel pore) (Phencyclidine (PCP), ketamine, dextrorphan, MK801, memantine, argiotoxin, polyamines, arcaine, aptiganel) and Negative allosteric modulators (acting at the amino terminal domain of GluN2B-containg receptors) (ifenprodil, eliprodil, dynorphin, neurosteroids, CP101606, Ro25-6981, UBP141, TCN201, QNZ46, DQP) [14].

Of the above medications, the most widely used is the ketamine, a phencyclidine derived. In the 1950s, Parke-Davis industries developed CI-395 (Phencyclidine or N-1-Phenyl-Cyclohexylpiperidine (PCP) chlorhydrate) and CI-400 (N-ethyl-1-phenylcyclohexamine chlorhydrate), among cyclohexylamine drugs. In 1978 its production stopped because of the severe psychodysleptic effects and its abusive use as a recreational drug. Further research in the 1960s led to the synthesis and development of ketamine (CI-581, 2-phenyl-Ochloro- 2-methylamino-cyclohexanone) [15]. The first clinical study in humans was conducted in 1964, and the drug was introduced into clinical use in 1970. In the central nervous system, ketamine has affinity for various receptors. Unlike many other anesthetics, it does not affect gamma-aminobutyric acid receptors at clinically relevant concentrations. The analgesic effects are mediated mainly through blockade of NMDA-R and possibly by enhancement of descending inhibition in the spinal cord in chronic pain conditions [16]. Even though there are extensive research and descriptions of ketamine effects in the nervous system, few studies have evaluated the physiological, pharmacological and toxic effects of ketamine in tissues outside the CNS, despite the recognition of the presence of NMDA-R almost ubiquitously.

The effects of ketamine, as a blocker NMDA-R in different tissues to the nervous system, have not been established in a pediatric murine model. In this study, we wanted to identify in three different age groups, if the renal ischemia-reperfusion injury (assessed by serum creatinine and histopathologic findings) is related to the dose of ketamine used as an anesthetic.

Materials and Methods


The study was approved by the Ethics Committee of our College of Medicine and the procedures were carried out according to the routine animal-care guidelines. All experimental procedures were complied with the Guide for the Care and Use of Laboratory Animals. Male Wistar rats obtained from the Faculty of Veterinary Medicine of the National University of Colombia were selected and assigned to one of 3 groups: Group 1=10 days old, Group 2=20 days of age, Group 3=30 days of age, corresponding approximately to the equivalent of age in humans for infants, pre-school and pre-pubescent respectively. They were maintained at approximately 21°C on a 12 h light/dark cycle with free access to food and water. In the case of the 10 infants rats (groups 10 days old) were guaranteed breastfeeding on demand. The rats were randomly divided into the Sham group (n=15, n=5 in each age group), and the model group (n=15, n=5 in each age group).

Preparation for experiment

The equipment, surgical tools and other materials (number needed in parentheses) were: a homeothermic monitor system (1), animal hair clipper (1), chronometer (1), tissue forceps with blunt points (2), tweezers with ultra-sharppoints (1), dissecting and operating scissors with sharp points (2), 5-0 Vicryl suture with curved needle (1), hemostats (4), skin separators (2), needle holder (1), syringes 1 ml, 5 ml syringes, alcohol swabs, cotton swabs, gauze sponges, paper towels, red bags for anatomical and pathological waste, heparinized capillary blood collection, eppendorf tubes, centrifugelaboratory, micropipettes, disposable tips and surgical gloves. The solutions used were saline (0.9% sodium chloride), 10% Ketamine, and 2% Xylazine, and 10% buffered formalin, 70% alcohol, urine bottles collectors, and conical tubes 15 ml, veterinary artificial tears. All of the surgical tools, materials, and solutions are sterilized.

Surgery and experimental protocol

All procedures of individuals, were performed using strict sterile techniques under general anesthesia with ketamine (40-90 mg / kg intraperitoneally) and xylazine(0,5 – 1,5 mg/kg). The solution was prepared with ketamine/xylazine in 1ml syringe in 9:1 ratio respectively. After injections of ketamine and xylazine, hair on one side of the rat was removed with hair cutter. The skin in the surgical area was cleaned with a cotton swab with 70% alcohol.

Immediately after the preparation of the skin, the rat was placed on a heating system and covered with sterile gauze. Surgery will not be started until the rat was in deep anesthesia and thus does not respond to pain induced by tail pinch. The time was approximately 10 minutes after injection of ketamine + xylazine for deep anesthesia. The rat was placed in the thermostatic station on the right side. The skin and muscle in the left flank were cut along the back to expose the left kidney. The incision is performed in a third body from the back of the rat and the size of the incision was 1-1.5 cm along. The kidney is then pushed out from the cut with sterile cotton swabs to expose the renal pedicle. Dissection of the pedicle tissue was done with ultra-finepoint tweezers to remove the tissue around the renal pedicle in order to expose the blood vessels for renal pedicle clamping. Hemostats were used to block blood flow to the kidney inducing renal ischemia.

The period of renal ischemia started from the time of clamping. Complete ischemia was detected by color change of the kidney from red to dark purple in a few seconds. After ischemia, the clamp was released at desired times of 30 minutes to initiate reperfusion, which was indicated by the change of kidney color to red. A 5-0 Vicryl suture was used to close the muscle layer of the incision followed by skin closure. Immediately after wound closure, 0.5 ml of warm sterile saline intraperitoneally to each rat was administered. The animal was kept on a heating pad immediately after, until it had full recovery of consciousness before being returned to its home cage, to continue their previous overall food and habitat. During the time of anesthesia and while waiting for full recovery of consciousness, artificial tears were placed to need.

At 72 hours of the initial surgical procedure, a second surgery was carried on in the same conditions as the previous one, under the same combination of anesthetic for the removal of the injured kidney and contralateral uninjured in experimental rats. In control rat’s only one kidney was extract under the same conditions that experimental group. The removed kidneys were immediately placed in urine collectors vials with 30ml, 20ml of 10% buffered formalin for tissue fixation.

The success of renal IR was monitored at several stages. First, after clamping, the kidney color should change from red to dark purple, an indicative of a successful renal ischemia. Second, serum creatinine was taken before starting the procedure and then at 72 hours of reperfusion. The samples were obtained from the retro-orbital venous plexus, frozen and sent to the Clinical Laboratory of Department for Sciences Animal Health of the Faculty of Veterinary Medicine of the National University of Colombia. Finally, histopathological examination of renal tissues with hematoxylin and eosin (H&E) was performed. Histopathology studies and light microscopy were done in the Department of Pathology, Faculty of Medicine of the National University of Colombia.

Statistical analysis

A descriptive analysis of the variables study was conducted. Frequencies and percentages for the qualitative variables were obtained, measures of central tendency (median and mean) and dispersion measures (standard deviation and inter-quartile range) for the quantitative variables were found according to their distribution, which was known by the Shapiro-Wilk statistical test. Then we evaluated in each experimental (case) and control the relationship with histopathological variables and renal function. Finally the relationship according to the age group of subjects was obtained. The relationship between variables was performed using f Fisher test for qualitative variables and mean differences by Student t test and Mann Whitney U. For quantitative analysis between values preoperatively and postoperatively, student t test was used for paired samples and Wilcoxon signed-ranks. Kruskal Wallis and ANOVA were used for categorical variables and for pre- and post-analysis were used paired techniques. A statistically significant p values <0.05 were considered.


A total of 30 rats were analyzed, which were distributed in three groups according to age, 10 days (n=10), 20 days (n=10) and 30 days (n=10). They were taken from each group 5 cases and 5 controls. Pathological analysis consisted of 45 specimens of kidney, 30 intervention group (15 clamping kidneys, 15 not clamping kidneys) and 15 kidneys in the control group. Pre- and post-intervention median for weight in the group 10 days old days was equal.

The median pre and post-intervention weight in group 10 days was equal, in the group 20 days post-intervention weight was slightly higher and the group 30 days an increase was evident in the post intervention weight (Table 1).