Ischemia-reperfusion injury (IRI) is an inevitable phenomenon during transplantation [
AKI incidence continues to increase despite the use of new biomarkers in clinical care to anticipate diagnosis and improve treatment [
Cell injury induced by reactive oxygen species (ROS) is a determinant of IRI. Ketamine infusion appears to inhibit lipid peroxidation and the amount of ROS [
The aim of this study was to investigate the effects of IP and IP associated with subanesthetic S(+)-ketamine on renal function and histology in rats.
The rats received care that complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and with Brazilian law regarding animal experimentation. Following approval from the Research Ethics Committee for Animal Experimentation (protocol number 946/2012) of Botucatu Medical School, São Paulo State University (UNESP), 41 male Wistar rats weighing 300–500 g were allocated into four groups: CG, control group,
Experimental algorithm. ISO: isoflurane; RIJV: right internal jugular vein cannulation; RL: ringer lactate; LCA: left carotid artery; MAP: mean arterial pressure; RN; right kidney nephrectomy; LN: left kidney nephrectomy; KI: S(+)-ketamine infusion; LCI: left artery clamping; IP: ischemia-reperfusion cycles (12 min total); REP: full reperfusion; CG: control group; KG: S(+)-ketamine group; IPG: ischemic postconditioning group; KIPG: S(+)-ketamine ischemic postconditioning group.
The temperature of the rats was maintained between 35.5°C and 37.5°C using a thermal blanket and was monitored with a rectal thermometer. The right internal jugular vein (RIJV) was dissected and cannulated with a 24 GA venocath for infusion of Ringer lactate solution (RL) (3 mL·kg−1·h−1) in all groups and subanesthetic (1.25 mg·kg−1·h−1) S(+)-ketamine continuous infusion only in KG and KIPG, in compliance with the Food and Drug Administration (FDA) publication Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers [
Serum NGAL (Rat NGAL ELISA Kit, Bioporto Diagnostics, Gentofte, Denmark), creatinine, and BUN (standard kits) were estimated to evaluate renal function. Serum sodium was used to evaluate hemodilution among the groups.
The left kidneys were processed for histological analysis. Once removed, the kidneys were sectioned longitudinally and stored in separate vials. They were maintained in Duboscq-Brazil solution for the first 48 h and were then preserved in 70% ethanol. Histological sections were stained with hematoxylin-eosin. Histological analysis was performed by a single pathologist blinded to the origin of the groups studied. The scale described by Park et al. [
All data are expressed as the means ± standard deviations (SDs). Repeated measures analysis of variance (ANOVA) and the contrast test were used to assess the behavior of the biochemical and monitored variables for three periods within each experimental group. One-way repeated measures ANOVA was used to determine whether the evolution of the variables was different among the groups (interaction effect). In these analyses, logarithmic transformation (natural log) was applied to the data due to the lack of normality, according to the Kolmogorov-Smirnov test. The nonparametric Kruskal-Wallis ANOVA was used to compare body weight among the four groups. The nonparametric histological scores were analyzed between IPG and KIPG using the Mann-Whitney test. In all instances, values of
The body weight values were similar for all groups (
Descriptive and longitudinal analysis of the biochemical variables within each group.
Collect |
|
|
|
Time effecta | Contrast analysis | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Group | Mean | SD | Mean | SD | Mean | SD | |||||
Cr | |||||||||||
CG | 0.430 | ± | 0.142 | 0.620 | ± | 0.193 | 0.550 | ± | 0.108 | 0.021 |
|
KG | 0.350 | ± | 0.053 | 0.520 | ± | 0.079 | 0.570 | ± | 0.082 | 0.0001 |
|
IPG | 0.390 | ± | 0.074 | 0.820 | ± | 0.274 | 2.60 | ± | 1.67 | 0.0001 |
|
KIPG | 0.418 | ± | 0.117 | 0.964 | ± | 0.112 | 3.26 | ± | 1.83 | 0.0001 |
|
NGAL | |||||||||||
CG | 5.8 | ± | 4.9 | 20.0 | ± | 23.0 | 217.2 | ± | 65.4 | 0.0001 |
|
KG | 5.3 | ± | 4.5 | 5.2 | ± | 2.7 | 226.5 | ± | 96.6 | 0.0001 |
|
IPG | 10.1 | ± | 10.4 | 37.7 | ± | 41.3 | 343.6 | ± | 50.6 | 0.0001 |
|
KIPG | 20.8 | ± | 19.9 | 66.0 | ± | 104.9 | 374.0 | ± | 17.7 | 0.0001 |
|
Na+ | |||||||||||
CG | 132.4 | ± | 3.2 | 130.9 | ± | 4.1 | 134.4 | ± | 2.2 | 0.091 | |
KG | 130.1 | ± | 4.0 | 132.9 | ± | 1.7 | 133.8 | ± | 3.3 | 0.025 |
|
IPG | 128.5 | ± | 2.8 | 128.2 | ± | 2.4 | 128.6 | ± | 1.9 | 0.90 | |
KIPG | 130.7 | ± | 3.3 | 129.4 | ± | 2.7 | 131.4 | ± | 3.6 | 0.32 | |
BUN | |||||||||||
CG | 55.0 | ± | 14.8 | 63.7 | ± | 16.3 | 56.3 | ± | 11.6 | 0.17 | |
KG | 50.6 | ± | 3.1 | 58.0 | ± | 3.9 | 60.1 | ± | 8.5 | 0.004 |
|
IPG | 45.4 | ± | 5.3 | 60.8 | ± | 7.2 | 174.5 | ± | 70.1 | 0.0001 |
|
KIPG | 46.4 | ± | 5.9 | 62.9 | ± | 4.2 | 206.0 | ± | 76.2 | 0.0001 |
|
SD: standard deviation; Cr: creatinine; NGAL: neutrophil gelatinase-associated lipocalin; Na+: sodium; BUN: blood urea nitrogen; CG: control group; KG: subanesthetic S(+)-ketamine continuous infusion group; IPG: ischemic postconditioning (IP) group; KIPG: subanesthetic S(+)-ketamine continuous infusion + IP group.
aANOVA for repeated measures within each experimental group.
A subanesthetic dose of S(+)-ketamine alone did not alter the functional parameters evaluated or promote any detectable injury in the histological analysis (grade 0, Park et al.). Repeated measures ANOVA showed a significant variation for sodium only in KG. Despite performing nephrectomy, ketamine infusion did not worsen the evolution of the biochemical variables.
IP alone (PG) promoted progressive increases in all renal function parameters (creatinine, NGAL, and BUN), which exhibited statistically significant differences from the basal values (
Repeated measures ANOVA one factor and contrast analysis among the groups.
Main effect | Interaction | Contrast analysis among groups | ||||
---|---|---|---|---|---|---|
Group | Time | Time point match |
|
Commentaries | ||
Cr | 0.0001 | 0.0001 | 0.0001 |
|
0.034 | (CG = KG) < KIPG, IPG |
|
0.0001 | (CG = KG) < (IPG = KIPG) | ||||
|
0.0001 | (CG = KG) < (IPG = KIPG) | ||||
|
||||||
NGAL | 0.0008 | 0.0001 | 0.081 |
|
0.041 | KG < (CG = IPG = KIPG) |
|
0.54 | CG = KG = IPG = KIPG | ||||
|
0.11 | CG = KG = IPG = KIPG | ||||
|
||||||
Na | 0.0001 | 0.014 | 0.13 |
|
0.096 | CG = KG = IPG = KIPG |
|
0.21 | CG = KG = IPG = KIPG | ||||
|
0.30 | CG = KG = IPG = KIPG | ||||
|
||||||
Ur | 0.0001 | 0.0001 | 0.0001 |
|
0.004 | (CG = KG) < (IPG = KIPG) |
|
0.0001 | (CG = KG) < (IPG = KIPG) | ||||
|
0.0001 | (CG = KG) < (IPG = KIPG) |
Cr: creatinine; NGAL: neutrophil gelatinase-associated lipocalin; Na+: sodium; BUN: blood urea nitrogen; CG: control group; KG: subanesthetic S(+)-ketamine continuous infusion; IPG: ischemic postconditioning (IP) group; KIPG: subanesthetic S(+)-ketamine continuous infusion + IP group.
The values of renal function parameters obtained for KIPG were greater than those obtained for IPG; however, longitudinal contrast analysis showed similar progressions in these values between these groups. Repeated measures ANOVA (time effect) for KIPG showed statistically significant values for creatinine, NGAL, and BUN. One-way repeated measures ANOVA showed that only creatinine and BUN were statistically significant regarding the interaction effect; contrast analysis confirmed that IPG and KIPG showed very similar progressions in values. As observed for the IPG rats, all KIPG rats demonstrated tubular injury during the histological analysis.
Since no lesions were detected in CG or KG, only IPG and KIPG were analyzed regarding differences in the degree of renal injury. No differences were observed between these groups (
Representative light micrographs of rat kidneys, magnification 200x. Hematoxylin-eosin stain of kidney sections, graded for severity of tubular injury, according to Park et al. [
In our study, IP was unable to prevent structural renal tubular damage. Subanesthetic S(+)-ketamine showed no additional beneficial effects for IP but was also not responsible for worsening lesion scores.
The best animal model for IP is controversial. In 2007, using a mouse model, Szwarc et al. were the first to demonstrate that IP could prevent ischemic AKI [
In the context of renal transplantation, cold ischemia and the concomitant use of immunosuppressive medications could impair positive strategies for successful reperfusion. McCafferty et al. [
The N-methyl-D-aspartate receptor (NMDAR) is a heterotetrameric amino acid, part of the glutamate receptor family, originally described in the central nervous system, where it functions as a membrane calcium channel. These receptors definitely participate in various parts of the nephron, including the collecting ducts, glomerulus, and podocytes; therefore, it is not difficult to associate the receptors with a type of renal function regulation. In the neonatal kidney, NMDAR subunits NR3a and NR3b are expressed; Sproul et al. reported the upregulation of NR3a secondary to hypoxia and hypertonicity in a mouse model [
Ketamine is a noncompetitive antagonist of the NMDAR that binds at the polyamine-binding site. The protective role of ketamine through NMDAR blockade has been studied in the brain with additional regenerative effects for S(+)-ketamine in cultured neurons [
Serum creatinine, BUN, and NGAL are widely accepted for assessing renal function and renal histology reveals any parenchymal injuries. Creatinine is the most commonly used biomarker in clinical practice, even though NGAL presents greater sensitivity for the early detection of AKI [
Evaluations of the weight of the rats and the control of blood pressure, temperature, and hydration were all adequately performed in our model; the groups exhibited homogeneous behaviors.
The majority of murine studies focus on AKI following bilateral warm ischemia [
The mechanisms involved in pharmacological preconditioning and IP require experimental research that corroborates and improves our current understanding of the intervention models. Reflections concerning the results obtained accrue from trying to determine the optimal algorithm, from control of the intervention and from safe and favorable reproducibility, which permit clear extrapolation to clinical practice.
In our study, the effect of postconditioning itself was unable to prevent severe structural tubular injury, probably due to the prolonged ischemia time in the algorithm. We conclude that a subanesthetic dose of S(+)-ketamine provided no additional beneficial effects for the postconditioning model, but neither was it responsible for the worst injury scores. The distinction in lesion progression between functional injury and permanent structural damage and how to anticipate and disrupt this complex process remains the subject of future research.
Acute kidney injury
Analysis of variance
Acute tubular necrosis
Blood urea nitrogen
Control group
Food and Drug Administration
Ischemic postconditioning
Ischemic postconditioning group
Ischemia-reperfusion injury
Subanesthetic S(+)-ketamine group
Subanesthetic S(+)-ketamine ischemic postconditioning group
Left carotid artery
15 minutes after the beginning of the anesthesia
After 30 min of left renal artery clamping
After 30 min of reperfusion and cycles of ischemic postconditioning
After 24 h of reperfusion
Neutrophil gelatinase-associated lipocalin
N-Methyl-D-aspartate
N-Methyl-D-aspartate receptor
Right internal jugular vein
Ringer lactate
Reactive oxygen species.
Marco A. C. de Resende was responsible for acquisition and interpretation of data and was involved in experimental procedures and paper writing. Alberto V. Pantoja and Bruno M. Barcellos were responsible for statistical analysis. Eduardo P. Reis, Thays D. Consolo, and Renata P. Módolo were responsible for acquisition of data and helped with technical procedures. Maria A. C. Domingues was responsible for histopathological examinations. Alexandra R. Assad and Ismar L. Cavalcanti were responsible for paper preparation and critical revision. Yara M. M. Castiglia was responsible for study design and critical revision. Norma S. P. Módolo was responsible for study design, data analysis, and paper preparation and supervised all phases of study.
The authors declare no conflict of interests.
This study was supported by the São Paulo Research Foundation (FAPESP), 2012/13606-1.