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
Abstract
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
Abbreviations
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
Introduction
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
Animals
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.
Results
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).
Age Group
Weight in grams
N
min
p25
p50
p75
max
10 days
Ischemia surgery
10
22
27
30
31
32
Nephrectomy
10
25
28
30
31
35
20 days
Ischemia surgery
10
35
40
40,5
43
50
Nephrectomy
10
35
42
42,5
45
60
30 days
Ischemia surgery
10
65
73
81
85
91
Nephrectomy
10
80
83
87,5
92
100
Table 1: Distribution pre and post intervention weight according to ages.
Anesthetic ketamine requirements in the first and second intervention are presented in (Table 2).
Age Group
Doses of Ketamine (mg/ dl)
N
min
p25
p50
p75
max
10 days
Ischemia surgery
10
24
29,0
38,4
45
60
Nephrectomy
10
25,7
29,0
31,0
45
54
20 days
Ischemia surgery
10
22,5
45
47,5
52,3
71,0
Nephrectomy
10
22,5
30
43,9
60
64,2
30 days
Ischemia surgery
10
37,0
49,4
60,6
65,8
87,6
Nephrectomy
10
36
45
51,4
58,2
61,8
Table 2: Ketamine dose distribution in first and second intervention according to the ages.
Median serum creatinine preoperatively was 0.46 (± 0.04), minimum value of 0.37 and maximum value of 0.56; after the intervention there was a significant increase in values with a global average of 0.51 (± 0.07), minimum of 0.37 and maximum of 0.71 (p=0,002). We found that initial creatinine values were equal between cases and controls in both the global population and by age group. Creatinine values post intervention showed no differences between groups (Table 3). For values between pre and post creatinine found in the control group a median of 0.46 (interquartile range = 0.43 to 0.50) in the value of pre creatinine and 0.49 (interquartile range = 0.44 to 0.56) in the value of creatinine post-intervention (p=0.09). In the case group, the pre-intervention median creatinine was 0.44 ((interquartile range = 0.41 to 0.49) and 0.52 (interquartile range = 0.46 to 0.56) for the value of creatinine post-intervention (p=0, 03).
Not clamping kidney
Clamping kidney
Creatinine
N
Average
SD
Average
SD
p
General population
Ischemia surgery
15
0,47
0,04
0,45
0,13
0,51
Nephrectomy
15
0,51
0,08
0,51
0,06
0,77
10 days
Ischemia surgery
5
0,47
0,05
0,45
0,07
0,60
Nephrectomy
5
0,51
0,05
0,45
0,05
0,13
20 days
Ischemia surgery
5
0,48
0,03
0,44
0,03
0,13
Nephrectomy
5
0,55
0,10
0,55
0,06
0,98
30 days
Ischemia surgery
5
0,45
0,05
0,46
0,05
0,57
Nephrectomy
5
0,46
0,06
0,51
0,05
0,16
Table 3: Values pre and post creatinine according to case-control group in the general population and according to the days of old.
Pathological analysis consisted of 45 specimens, 30 intervention group (15 clamping kidneys, 15 not clamping) and 15 kidneys in the control group. By classifying the histopathologic features using the scale of Goujon, we found that 44.4% had TCP and TCD with intracellular edema, spinal internal and external with intracellular edema (Table 4).
Escala de Goujon
N (45)
100%
1. Normal
18
40
2. PCT and DCT with intracellular edema, renal inner medulla and outer with intracellular edema
20
44,4
3. Extensive coagulation necrosis compromising cortex and medulla
3
6,6
4. Coagulation necrosis compromising cortex and medulla (50% of the renal parenchyma)
3
6,6
5. Necrosis and recent thrombosis of an artery and focal dystrophic calcification
1
2,2
PCT: Proximal Convoluted Tubule; DCT: Distal Convoluted Tubule
Table 4: Distribution of surgical specimens according to the scale of Goujon [17].
Ketamine (mg/kg)
Creatinine*
Mean
743,453,062
492,172,579
Variance
23,754,455
18,788,746
Observations
10
10
Hypothetical mean difference
0
Degrees of freedom
18
Statistical t
106,436,829
P (T <= t) one-tail
1.70E-05
Critical value of t (one-tail)
173,406,361
P (T <= t) two-tail
3.39E-06
Critical value of t (two-tail)
210,092,204
*Expressed in percentage change between pre and post intervention value.
Table 5: Ketamine vs creatinine in the group of 10 days old two-sample t test assuming unequal variances.
Ketamine (mg/kg)
Pathology score**
Mean
743,453,062
1,8
Variance
23,754,455
0,17777778
Observations
10
10
Hypothetical mean difference
0
Degrees of freedom
9
Statistical t
148,790,201
P (T <= t) one-tail
6.05E-04
Critical value of t (one-tail)
183,311,293
P (T <= t) two-tail
1.21E-03
Critical value of t (two-tail)
226,215,716
**Value 1-5 according to score Goujon.
Table 6: Ketamine vs pathology score in the group of 10 days old (not clamping kidneys) two-sample t test assuming unequal variances.
Ketamine (mg/kg)
Pathology score**
Mean
723,330,087
1,6
Variance
214,484,669
0,8
Observations
5
5
Hypothetical mean difference
0
Degrees of freedom
4
Statistical t
107,795,488
P (T <= t) one-tail
0,00020999
Critical value of t (one-tail)
213,184,679
P (T <= t) two-tail
0,00041999
Critical value of t (two-tail)
277,644,511
**Value 1-5 according to score Goujon.
Table 7: Ketamine vs pathology score in the group of 10 days old (clamping kidneys) two-sample t test assuming unequal variances.
Ketamine (mg/kg)
Creatinine*
Mean
926,247,429
201,998,312
Variance
396,029,221
542,952,407
Observations
10
10
Hypothetical mean difference
0
Degrees of freedom
18
Statistical t
7,474,109
P (T <= t) one-tail
3.18E-03
Critical value of t (one-tail)
173,406,361
P (T <= t) two-tail
6.37E-03
Critical value of t (two-tail)
210,092,204
*Expressed in percentage change between pre and post intervention value.
Table 8: Ketamine vs creatinine in the group of 20 days old Two-sample t test assuming unequal variances.
Ketamine (mg/kg)
Pathology score**
Mean
926,247,429
1,5
Variance
396,029,221
0,27777778
Observations
10
10
Hypothetical mean difference
0
Degrees of freedom
9
Statistical t
144,750,624
P (T <= t) one-tail
7.68E-04
Critical value of t (one-tail)
183,311,293
P (T <= t) two-tail
1.54E-03
Critical value of t (two-tail)
226,215,716
** Value 1-5 according to score Goujon.
Table 9: Ketamine vs pathology score in the group of 20 days old (not clamping kidneys) two-sample t test assuming unequal variances.
Ketamine (mg/kg)
Pathology score**
Mean
98,348,157
2,6
Variance
428,484,692
1,3
Observations
5
5
Hypothetical mean difference
0
Degrees of freedom
4
Statistical t
103,273,752
P (T <= t) one-tail
0,00024802
Critical value of t (one-tail)
213,184,679
P (T <= t) two-tail
0,00049605
Critical value of t (two-tail)
277,644,511
** Value 1-5 according to score Goujon.
Table 10: Ketamine vs pathology score in the group of 20 days old (clamping kidneys) two-sample t test assuming unequal variances.
Ketamine (mg/kg)
Creatinine*
Mean
111,133,687
753,835,361
Variance
222,567,377
270,602,296
Observations
10
10
Hypothetical mean difference
0
753,835,361
Degrees of freedom
18
270,602,296
Statistical t
147,516,979
P (T <= t) one-tail
8.52E-09
Critical value of t (one-tail)
173,406,361
P (T <= t) two-tail
1.70E-07
Critical value of t (two-tail)
210,092,204
*Expressed in percentage change between pre and post intervention value.
Table 11: Ketamine vs creatinine in the group of 30 days old Two-sample t test assuming unequal variances.
Ketamine (mg/kg)
Pathology score**
Mean
111,133,687
1,4
Variance
222,567,377
0,26666667
Observations
10
10
Hypothetical mean difference
0
Degrees of freedom
9
Statistical t
232,460,518
P (T <= t) one-tail
1.20E-05
Critical value of t (one-tail)
183,311,293
P (T <= t) two-tail
2.40E-05
Critical value of t (two-tail)
226,215,716
** Value 1-5 according to score Goujon.
Table 12: Ketamine vs pathology score in the group of 30 days old (not clamping kidneys) two-sample t test assuming unequal variances.
Ketamine (mg/kg)
Pathology score**
Mean
109,532,211
3,2
Variance
977,623,646
2,7
Observations
5
5
Hypothetical mean difference
0
Degrees of freedom
4
Statistical t
237,218,277
P (T <= t) one-tail
9.36E-02
Critical value of t (one-tail)
213,184,679
P (T <= t) two-tail
1.87E-01
Critical value of t (two-tail)
277,644,511
** Value 1-5 according to score Goujon.
Table 13: Laboratory values at hospital admission.
Because rats were chosen homogeneously in terms of age, classified into three groups respectively according to their days old, of the same race and litter, a design of completely randomized experimental classification balanced by fixed effect was used, complemented with T students test at 5% significance when analysis of variance was significant. Confidence intervals of 95% were performed for quantitative variables.
Discussion
Ketamine has been used in clinical for over half a century and its role in the management of critically ill children has focused primarily on sedation and analgesia [16,18], withdrawal and bronchospasm. Ketamine provides bronchodilation by increasing catecholamine transmission and stimulation of β2 adrenergic receptors and as a result of ketamine’s NMDA activity, it may decrease development of opioid tolerance, making it a reasonable adjunct for sedation and analgesia in the Pediatric Intensive Care Unit (PICU) [18].
Ketamine elimination clearance is high (12-20ml/kg/min), elimination half-life is 2-3 hand clearance may be 20% higher in women than in men. Intramuscular (IM) absorption is faster in children than in adults. Distribution volume is slightly lower (1.9 l/ kg) but plasma clearance is more important (16.8ml/kg/min) than in adults. The average dose of removal / elimination half-life of Ketamine is also shorter in children: 100 min after an identical dose expressed in mg/kg; plasma concentrations of the major active metabolite of ketamine, norketamine, are higher in children. However, in children from 4 to 10 years old and after an Intravenous (IV) 2mg/kg or IM 6mg/kg injection, ketamine plasma concentrations are similar to those observed in adults. On the other hand, in the first 3 months of life, ketamine plasma clearance is shorter, probably due to decrease of transformation in the liver and excretion in the kidney. In this case, there is an increase in the ketamine elimination half-life in newborns and infants. Distribution volume seems to be comparable to older children [15].
Related to the binding of the NMDA antagonistic drug with the NMDA-R, the mechanisms by which exert an impairment of NMDA receptor function, are quite complex. A substantial reason for the variation in effect lies in the off-rate of the compound due to a phenomenon has been termed “trapping block”. Ketamine is an example of high-trapping antagonist (86% trapping),) as when the glutamate has dissociated from its binding site on the NMDA-R, the ketamine remains trapped in the, now closed, ion channel thus causing a prolonged tonic blockade which disrupts both physiological and pathological functions [19]. The antagonism is more important if the NMDA channel has been previously opened by the glutamate fixation [15].
It is controversial the effect of ketamine on the renal tissue and little is known about the effects it could have on the acute kidney injury of critical patients in part because its precise mechanisms of action remain unknown. First documented in 2007 [20,21], Ketamine has been shown to injure the bladder, causing ulcers (wounds) and fibrosis (stiffening of the bladder walls and shrinkage). Patients present with multiple symptoms including incontinence, bleeding, overactive bladder and bladder shrinkage, as well as damage to both the kidneys and the ureter. In 2009, Hills CE and colleagues consider that Ketamine alters epithelial cell-to-cell adhesion and cell-coupling in the proximal kidney via a non-classical pro-fibrotic mechanism and the data provides the first indication that this illicit substance can have major implications on renal function [21].
In the study of Lee TH et al propose that some volatile anesthetics confer profound protection against renal ischemia–reperfusion injury compared with pentobarbital or ketamine anesthesia by attenuating inflammation. They anesthetize rats with equipotent doses of volatile anesthetics (desflurane, halothane, isoflurane, or sevoflurane) or injectable anesthetics (pentobarbital or ketamine) and subjected to 45min of renal ischemia and 3h of reperfusion during anesthesia, and conclude that volatile anesthetic treatment reduced renal cortex myeloperoxidase activity and reduced nuclear translocation of proinflammatory nuclear factor κB [22]. Nevertheless Dimer Leffa D et al demonstrated [23] that after the anesthesia procedure in mice, the ketamine (doses between 80-140mg/kg in different groups) showed DNA damage after 1 and 12h for blood collection in all doses tested. Studies with inhalator anesthetic sevoflurane, halotano, and desflurane also demonstrated DNA damage after 2h of exposure. After 24h, the results in blood cells demonstrate that DNA damage persists only in the highest dose of ketamine, demonstrating that DNA damage was not totally repaired. This probably happened because the DNA repair system was effective against DNA damage induced by ketamine, but this system is dose limited. After 24h of the anesthesia, mice were killed and the liver, kidney, brain, and bone marrow were analyzed to verify the extent of DNA damage in these organs. The results demonstrated that only the highest dose of ketamine (140 mg/ kg, alone or in combination with xylazine) has damaged DNA in the brain cortex and no damage was found in the liver and kidney.
In 2002, Deng and colleagues published the first report of functioning NMDA-R in the kidney. The data document the presence of NMDA-R mRNA and protein in rat kidney cortex. These data confirm a role for renal NMDA-R in maintaining normal renal function. The co-agonist requirement of the NMDA-R for glycine gives credibility to the latter suggestion as does the ability of systemically administered NMDA-R inhibitors to selectively alter renal hemodynamics, independently of their effects on baseline Renal Blood Flow (RBF) [6]. In 2004, Leung JC and colleagues, in a rat model of short-term, high dose gentamicin nephrotoxicity, showed that there was an increase expression of both renal NMDA subunits, NR1 and NR2C. They speculate that following glomerular filtration of gentamicin, the drug interacts with the NMDA receptor, stimulating entry of calcium into the cell through the NMDA calcium channel. Finally, the amelioration of renal damage in rats exposed to shortterm gentamicin that are pretreated with the NMDA receptor blocker MK-801 provides further proof of the important role of this receptor in this model of renal toxicity [24].
Expression of both renal NR1 protein and mRNA is upregulated during ischemia-reperfusion insult [25] and by short-term treatment with gentamicin [23], as well as in the endotoxemic model reported by Lin CS et al [26]. Several studies have shown the involvement of NMDA-R in Acute Kidney Injury (AKI) induced by ischemiareperfusion and have tried to elucidate the causes. It has been seen that NMDA agonists significantly aggravated renal Ischemia-Reperfusion Injury (IRI) -induced oxidative stress and AKI. The NMDA-R has various sites within it that regulate channel function. Major sites on NMDA-R include the NMDA or glutamate binding site, glycine, and polyamine binding site. Ketamine is an antagonist of NMDA-R that binds at the polyamine binding site. Unlike the study of Dimer Leffa 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, intraperitoneally) than is used for anesthesia, while the anesthetic dose of ketamine had no protective effect on kidney. Treatment with ketamine reduced Myeloperoxidase (MPO) activity and neutrophil accumulation and the renoprotection observed with NMDA-R antagonists along with restoration of endogenous antioxidants, is supported by other reports claiming restoration of catalase activity and glutathione stores as a significant therapeutic strategy in the management of renal disorders [25].
It is also known that NMDA receptor hyperfunction contributes to acute renal failure during LPS induced endotoxemia. Lipopolysaccharide (LPS) binds to Toll-Like Receptor 4 (TLR4) and induces Interleukin (IL)-1β release from tubular cells. TLR4 and IL-1 receptor (IL-1R) signaling intubular cells increase expression of the NMDA receptor NR1 subunit and serine racemase. Upregulation of NR1 and serine racemase, together with increased D-serine levels, results in NMDA receptor hyper function. NMDA receptor hyper function in LPS causes tubular cell damage and poor renal perfusion through vasoconstriction leading to acute renal failure. Inhibition of NMDA receptors attenuated LPS-induced functional deterioration and tubular cell damage in in vivo and in vitro. In this study [26], NMDA-R inhibition prevented reductions in renal perfusion and ameliorated decline in ultra filtration but not NR1 expression, indicating that NMDA-R hyperfunction contributes to renal vasoconstriction in endotoxemic kidneys.
is suggestive of a definitive role for the NMDA receptor in renal injury [28].
Although most authors concluded that NMDA-Rs exist in the kidney to mediate injury. In that sense Deng proposes that inhibiting NMDA-R with a channel blocker or glycine antagonist causes renal vasoconstriction and explains that the effects of NMDA-R on the kidney must be dichotomous as there is precedent for this in the central nervous system. A reason for these phenomena in the brain is that low-level NMDA-R activity provides calcium entry that drives nitric oxide synthase to produce nitric oxide needed for neuronal plasticity or vasodilation, whereas high-level NMDA-R activity floods the cell with toxic amounts of calcium leading to injury and death [28]. In cases of chronic use and addiction, ketamine is associated with significant bladder and renal toxicity in animal models and human case studies. Ketamine can increase the expression of Snail, Slug, Twist, and ZEB1, which further suppress E-cadherin expression, and induce Epithelial Phenotypic Changes (EPC) in renal distal tubular cells. These EPCs interrupt the formation of Apical Junctional Complex (AJC) and increase permeability and cell motility, consequently leading to renal impairment [30]. These mechanisms could explain that prolonged ketamine addiction resulted in the animals prone to urinary infection as shown by the study of Yeung LY et al [31] and inflammatory cystitis and reversible hydronephrosis reported by Selby NM et al [32]. Another aspect against NMDA-R blockers is the fact that podocytes possess the complete machinery for glutamatergic signaling, raising the possibility that neuron-like signaling contributes to glomerular function. The results of the study of Giardino L et al [33] suggest that glutamatergic signaling driven by podocytes contributes to the integrity of the glomerular filtration barrier and that derangements in this signaling may lead to protein uric renal diseases.
Our data seems to provide evidence that supports the hypothesis that blocking NMDA-R receptors by ketamine could be harmful in children (pediatrics) and it could be worse with increasing age. Analyzes shown in (Tables 5-13) indicating that as the age of the rats progresses, there is a highest score of acute kidney injury, in connection with an increased need for anesthetic dose of ketamine in the group 30 days old despite the largest increase in creatinine is evident in the group 20 days old. It is important to consider in children that the NR3 subunit of NMDA-R is highly expressed in the neonate with expression levels substantially decreasing shortly after birth and that the participation of this subunit may play a role so far unknown NMDA-R in kidney and pathophysiology of acute kidney injury in children.
The mechanisms by which NMDA-R may affect renal function are diverse and, additional studies are needed to establish whether the effect of ketamine in the kidney depends on the dose, the mechanism of induction of acute kidney injury, the different ways in where antagonism of NMDA-R occurs and the relationship or interaction of NMDA-R with factors understudied in children as Klotho protein, highly expressed in the kidney and recognized as “anti-aging” protein because its levels and protective action could have a different impact on the AKI at different ages [34-36].
Acknowledgement
This work was supported by the Medical School at the National University of Colombia, HERMES Code: 28075.
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