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N,N-dimethyltryptamine Prevents Renal Ischemia-Reperfusion Injury in a Rat Model
N,N-dimethyltryptamine Prevents Renal Ischemia-Reperfusion Injury in a Rat Model } b, Norbert Némethb, Anita Mesterb, Zsuzsanna Magyarb, Balázs Nemesa,*, Katalin Peto b Souleiman Ghanem , Viktória Sógorb, Bence Tánczosb, Ádám Deákb, Márk Kállaya, László Bidigac, and Ede Frecskad a
Department of Organ Transplantation, Institute of Surgery, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; Department of Operative Techniques and Surgical Research, Institute of Surgery, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; cDepartment of Pathology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; and dDepartment of Psychiatry, Faculty of Medicine, University of Debrecen, Debrecen, Hungary b
ABSTRACT Background. Ischemia reperfusion (I/R) injury remains one of the most challenging fields of organ transplantation. It is highly associated with the use of expanded criteria donors that might conclude to delayed graft function or early or late graft failure. Objective. To investigate the metabolic, microcirculatory parameters, and histologic changes under the effect of N,N-dimethyltryptamine (DMT) in a renal I/R model in rats. Method. In 26 anesthetized rats both kidneys were exposed. In the control group (n ¼ 6) no other intervention happened. In 20 other animals, the right renal vessels were ligated, and after 60 minutes the right kidney was removed. The left renal vessels were clamped for 60 minutes then released, followed by 120 minutes of reperfusion. In the I/R group (n ¼ 10), there was no additive treatment, while in I/R þ DMT group (n ¼ 10) DMT was administered 15 minutes before ischemia. Blood samples were taken, laser Doppler measurement was performed, and both kidneys were evaluated histologically. Results. Microcirculation (blood flux units [BFU]) diminished in all groups, but remarkably so in the I/R þ DMT group. This group compensated better after the 30th minute of reperfusion. The control and I/R þ DMT groups had similar BFUs after 120 minutes of reperfusion, but in the I/R group BFU was higher. Tubular necrosis developed in the I/R and I/R þ DMT groups too; it was moderated under DMT effect, and severe without. Histologic injuries were less in I/R þ DMT Group compared to non-treated animals. Conclusion. Histologic changes characteristic to I/R injuries were reversible and microcirculation recovered at the end of 120 minutes reperfusion under the administration of DMT. DMT can be used for renoprotection in kidney transplantation.
M
ORE and more people are in need of transplants worldwide, but the number of donor organs and living donors cannot keep up with this growing demand [1]. The average waiting time has greatly increased and adversely affects the survival of both patients and transplanted organs [2]. Acceptance of older and more seriously injured marginal and non-heart beating donor organs experiencing different injuries could bridge the gap between demand and availability; however, it is a challenge in the 0041-1345/19 https://doi.org/10.1016/j.transproceed.2019.04.005
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context of organ preservation [3]. Ischemia reperfusion (I/R) injury remains one of the most challenging fields of
*Address correspondence to Balázs Nemes, MD, PhD, Division of Organ Transplantation, Institute of Surgery, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Nagyerdei krt. 98., Hungary. Tel: þ36-30-983.4764. E-mail: nemes.balazs@ med.unideb.hu ª 2019 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 51, 1268e1275 (2019)
DMT PREVENTS RENAL ISCHEMIA REPERFUSION
transplantation. Even the United States Food and Drug Administration held a meeting on this topic in 2011 [4]. I/R injury is a multicausal and multifactorial phenomenon. It is highly associated with the use of expanded criteria donors (ECDs), which might conclude to delayed graft function and/or early graft loss, or late graft failure [5]. On the other hand, there is a need to accept ECDs in the age of donororgan shortage. Therefore, the magnitude of the problem is that I/R injuries have become a regular clinical issue instead of a marginal field of interest. The pathophysiology is still under debate. During ischemia the core process is the decrease in cellular oxidative phosphorylation, when adenosine triphosphate (ATP) is further dephosphorylated to adenosine diphosphate, adenosine monophosphate, and purines, leading to an accumulation of hypoxanthine [6,7]. The drop in cellular ATP leads to the dysfunction of ATPdependent membrane ion-pumps, resulting in calcium, sodium, and water influx to the cells. The swelling results in disruption of the cell membrane (opening stretch-activated channels, further modulating the conductance) and intracellular membranes, such as of the endoplasmic reticulum, the Golgi apparatus, the mitochondrial membrane and the cytoskeletal microtubule [8e10]. Hypoxia leads to microcirculatory, micro-rheological, inflammatory, innate, and adaptive immune responses, and alterations. The presence of oxidative stress and the worsening rheological properties of the blood are to be highlighted. During the complex hemodynamic changes under I/R, NO plays an important role in local flow regulation [11,12]. I/R injury is a main target for therapeutic issues. Many attempts have been made to reduce cold ischemia time [13,14], to develop better perfusion fluid [15], and to create new a perfusion technique [16e18]. It has already been published that the intravenous administration of the sigma-1 receptor agonist N,N-dimethyltryptamine (DMT) protects kidneys from I/R injury [19]. However, the detailed mechanism of action is not well known. The sigma-1 receptor is a ligand-regulated molecular chaperone with various ion channels and G-protein-coupled membrane receptors as clients [20]. It is located at the mitochondrion-associated membrane of the endoplasmic reticulum and regulates the cross-talk between endoplasmic reticulum stress and oxidative stress [21]. The sigma-1 receptor protects against cellular oxidative stress and activates antioxidant response elements [22]. The recent discovery that DMT is an endogenous ligand of the sigma-1 receptor [23] may shed light on yet undiscovered physiological mechanisms of DMT activity and reveal some of its putative biological functions, such as a role in tissue protection [24], immunoregulation [25], and alleviation of ischemic injury [26]. Other studies have already proved that part of the neuroprotective effects of sigma-1 receptor agonists is reduced levels of pro-inflammatory cytokines and enhanced anti-inflammatory cytokines following stroke [27]. It has also been found that fluvoxamine stimulation in rat kidney activated nitric oxide production, which was blocked by s1-receptor knockdown or Akt inhibition. The sigma-1 receptor activation by fluvoxamine triggered the Akt-nitric
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oxide synthase signaling pathway, resulting in time- and isoform-specific endothelial and neuronal nitric oxide synthase activation and NO production. A prompt peritubular vasodilation was seen after fluvoxamine treatment [19]. DMT does not inhibited CYP2D6 activity, in contrast to diallyl-tryptamines. Diallyl-tryptamines also inhibited CYP1A2 activity comparable to fluvoxamine [28]. The protective effect of some other sigma-1 receptors has also been proven. For example, cutamesine (SA4503) enhanced functional recovery after experimental stroke with a treatment initiation window of 48 hours and chronic treatment for 28 days [29]. The following questions arise: Will the administration of DMT protect the kidney against ischemia, what changes are present in the different structures of the kidney, and to what extent? Will there be any beneficial changes in the microcirculation of the kidney caused by the protective agent? MATERIALS AND METHODS Our group settled the following animal experiment to answer these questions. The animal experiment was approved and registered by the University of Debrecen Committee of Animal Welfare (permission no.:20/2011), in accordance with national and European Union regulations (the Hungarian Animal Protection Act [Law XVIII/1998] and Edict 63/2010).
Operative Protocol Twenty-seven male CD-outbred rats (343 29 g) were involved in the experiment, divided into 3 experimental groups: control (C), I/R, and treated (T). Anesthesia was introduced with 62.5 mg/kg thiopental I.P., then, the following operating procedures were performed on the experimental groups. The C group had only anesthesia, cannulation, abdominal opening, blood sampling, laser measurement, kidney removal, and termination of the animal. The I/R and I/R þ DMTs groups had the same operating procedure: anesthesia, cannulation, opening the abdomen, preparing the aorta and renal vessels, ligating the right renal artery and vein and clamping the left renal vessels with a plastic clip (to induce ischemia) for 60 minutes, then removal of the clip from the left renal vessels, removal of the right kidney for histology (ischemia injury only), and keep the left one for a further 120 minutes. The left kidney was then removed and the animal was terminated. The I/ R group was not treated, while the I/R þ DMT group received DMT; 2.95 mL/bwkg using 2.45 mg/mL solution was given i.m. first 15 minutes before ligating/clamping the renal vessels and again 60 minutes later, 15 minutes before the start of reperfusion. Blood sampling (150 uL, K3-EDTA each) was performed via the femoral artery immediately after cannulation (base), further at the 60th minute after ischemia was induced (I-60), then at the 30th (R-30), 60th (R-60), and 120th minutes after reperfusion (R-120). An equal volume of physiological saline was administered via the cannula. The blood samples were sent to the laboratory for basic analysis.
Laboratory Measurements Hematological parameters were determined by Sysmex K-4500 automated hematology analyzer (TOA Medical Electronics Corp., South San Francisco, Calif, United States). In this paper only white blood cell count (WBC [g/L]) and platelet count (Plt [g/L]) are shown. Electrolytes were tested by an EPOC blood analysis device
} NÉMETH ET AL NEMES, PETO,
1270 (Alere, Waltham, Mass, United States). We have reported the sodium, potassium and serum Caþþ levels [mmol/L].
Laser-Doppler Flowmetry/Laser-Doppler Data Acquisition The microcirculation of the left kidney, liver, and intestine was monitored by laser-Doppler technique (LD-01 Laser Doppler Flowmeter, Experimetria, Budapest, Hungary), using a standard pencil probe (Oxford Optronix, Abingdon, England) at every sampling point of the experiment (Base, I-60, R-30, R-60, and R-120). The device determines blood flux unit (BFU) based on the number of moving red blood cells and their mean velocity in the tested tissue volume (1e1.5 mm3). The probe was gently placed consecutively on the surface of the anterior middle surface of the left kidney, on the middle region of the right liver lobe, and on the intestine. The signal was recorded by S.P.E.L. Advanced Kymograph software (Experimetria) at a 1 kHz sampling rate for 30e60 seconds. During offline data analysis, the average value of a noisefree 10-second-long representative section of each recorded graph was calculated.
Histology Histologic examination was performed on both kidneys. Samples were identified by a combination of characters and numbers; however, to keep the process blinded, decoding was not available for the pathologists, so hematoxylin and eosin and periodic acid-Schiff staining were carried out. The glomerular, vascular, and tubular structures of the kidney were examined and scored by a pathologist.
Statistical Analysis Data are presented as means standard deviation. One-way and repeated measure analysis of variance tests were used for intra- and inter-group comparisons (Bonferroni/Dunn methods). For simple comparison of inter-group differences at single time points, the Student t test, and Mann-Whitney rank-sum tests were applied as well, depending on the normality of data distribution. A P < .05 value was considered statistically significant.
RESULTS
Sodium level [mmol/L] did not change in any groups. Potassium level [mmol/L] increased significantly in all groups from the base level to R-120. The value at the 120th minute after reperfusion was 150% of basal in the I/R group
(6.3 1.86 vs 4.2 .33, respectively), while it was 130% in I/R þ DMT group (5.4 2.12 vs 4.15 .33). The serum Caþþ [mmol/L] level decreased significantly in all study groups from the base to R-120. Serum Ca level at the 120th minutes was 88% of base level in the I/R group (1.23 .08 vs 1.40 .04), while it was 81% in the I/R þ DMT group (1.08 .16 vs 1.33 .04). There were no significant differences between the I/R and I/E þ DMT groups regarding serum Kþ and Caþþ. WBC [x103/mL] increased to 153% of base at the 60th minute of ischemia in the I/R group (8.1 4.68 vs 5.43 1.6), while it was 94% of base in the I/R þ DMT group (4.71 1.5 vs 5.05 .86). This difference was significant between the I/R and I/R þ DMT groups. By the end of 120 minutes of reperfusion, WBC was 65% of base in the I/R group (3.51 1.75 vs 5.43 1.6) and 126% of base in the I/R þ DMT group (5.43 1.6 vs 5.43 1.6). This difference was significant between the I/R and I/R þ DMT groups. The platelet count [x103/mL] increased to 150% of base in the I/R group at the end of 60 minutes of ischemia (958.7 138.5 vs 646.1 317.1), but remained unchanged in the DMT-treated group (729 92 vs 794.8 125.6). The difference was significant. Platelet count decreased back to base level at the end of 120 minutes of reperfusion in the I/R group, and was the same at base and I-60 levels in the I/R þ DMT group. Results of the microcirculation parameters are shown in Table 1 and Fig 1. The initial BFU was lower in Group I/R compared to the C and I/R þ DMT groups in both left and right kidneys; however, it did not disturb the estimation of the changing trends. When kidneys (both left and right) were exposed to ischemia (60 minutes in the I/R and I/R þ DMT groups), BFU decreased in both groups (left kidney, 23.9 2.4e22.9 1.3 in the I/R group and 29.8 4.1e19.7 4.4 in the I/R þ DMT group). The decrease was significant in the I/R þ DMT group. However, if we consider both right kidneys in the I/R and I/R þ DMT groups, the decrease was the same (27.3 3.2e17.2 0.1). After 30 minutes both the I/R and I/R þ DMT groups showed an compensational increase in BFU; however, this trend was more prominent
Table 1. Laser Doppler Measurements
Left Kidney Control I/R Treated Right Kidney Control I/R Treated
Base
I-60
R-30
R-60
R-120
32.7 3.7 23.9 2.4† 29.8 4.1‡
26.2 6.2 22.9 1.3 19.7 4.4*
27.3 3.1* 27.7 1.8 27.4 6.7
24.4 4.3* 28.4 1.6 26.5 7.3
21.2 6.1 28.2 1.4 23.9 4.4*
34.4 5.6 27.3 3.2 30.7 4.9
24.4 5.1 17.2 .1
24.1 4.7
26.2 3.0
29.0 1.2
Results are in BFU. Abbreviations: BFU, blood flux unit; I-60, 60th minute after induction of ischemia; R-30, 30 minutes of reperfusion; R-60, 60 minutes of reperfusion; R-120, 120 minutes of reperfusion. *P < .05 vs base. † P < .05 vs control. ‡ P < .05 vs I/R.
DMT PREVENTS RENAL ISCHEMIA REPERFUSION
1271 LEFT KIDNEY
35 33 31
Tengelycím
29 27 25 23 21 19 17
Control I/R Treated i.m.
base 32.66685799 23.95662048 29.79226689
I-60 26.16157092 22.94351963 19.66905003
R-30 27.31680933 27.74166475 27.39338585
R-60 24.36945329 28.40528512 26.55107548
R-120 27.21486057 28.1550753 23.9374008
Fig 1. Laser Doppler results of the left kidney in the different study groups. BFU, blood flux unit.
compared to I-60 in the C group (19.7 4.4e27.4 6.7) than it was in the I/R group (22.9 1.3e27.7 1.8). This increase continued in the I/R group until the end of the observation period. The R-120 BFU exceeded its base value in the I/R group (23.9 2.4 vs 28.2 1.4), while R-120 results were lower compared to the initial value in the I/R þ DMT group (29.8 4.1 vs 23.9 4.4). When the control group is compared to the I/R group regarding base BFU vs R-120 BFU, the trend is the opposite (32.7 3.7 vs 21.2 6.1 and 23.9 2.4 vs 28.2 1.4, respectively). When the control group is compared to the I/R þ DMT group, they are similar (there is a slight decrease in BFU). Both kidneys were sent to pathology. The results are shown in Table 2 and Figs 2e6. Because the right kidney was removed before reperfusion it only experienced an ischemic injury. The left kidneys were left until the end of the entire procedure. Therefore, histologic changes of the left kidneys represent both ischemic and reperfusion injury. Tubular changes were equally developed on both right and left kidneys. Ischemic-only changes were extensive dilation and stasis in the periglomerular capillaries and vasa recta. However, it almost completely disappeared after reperfusion in the I/R þ DMT group (Figs 5 and 6). The scores given for epithelial nuclear staining, tubular necrosis, and hydropic degeneration increased both in the I/R and Treated groups compared to controls. However, these signs were less serious when the animals were treated with DMT (I/R þ DMT group) compared to simple I/R. This difference was more extensive in the left kidneys than in the right ones. Regarding vascular and glomerular changes, stasis was the main sign of ischemia that increased in all structures. Surprisingly, it was more extensive in the I/R þ DMT group
compared to the I/R group in the right kidneys. On the other hand, stasis was less extensive in the I/R þ DMT group compared to the I/R group in the left kidneys. It looks as though DMT worsened the stasis during ischemia and enhanced its spontaneous recovery in the reperfusion phase. Total scores for the histologic changes of ischemiareperfusion are shown in Table 2. There was no difference in scores between the I/R and I/R þ DMT groups regarding the right kidneys. The scores reduced by tubular recovery was neutralized by the additive scores given for the worsening stasis. However, the I/R þ DMTs group had significantly lower scores compared to the I/R group (6.82 1.78 vs 10.36 4.12; P ¼ .0009) at the end of the 120 minutes of reperfusion. The results of the I/R þ DMT group was still statistically higher compared to the control group (6.82 1.78 vs 3.9 4.89; P ¼ .01). DISCUSSION
It is well-known that serum potassium is a marker of ischemic renal injuries. An increase is obviously expected after 60 minutes of renal ischemia, as seen in the I/R group. Recovery was better in the I/R þ DMT group compared to non-treated animals (I/R group). The impairment of microcirculation has a significant role in the pathophysiology of many diseases, such as cardiovascular diseases, diabetes mellitus, sepsis, compartment syndrome, and ischemia-reperfusion injuries [30]. Therefore, the investigation of microcirculation could be useful in diagnoses, particularly in determining the severity of and in following up on diseases. It is very important in predicting the viability of tissue, postoperative organ function, and complications.
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1272 Table 2. Histologic Findings of the Retrieved Kidneys Control
Right Kidney Tubular Epithelial nuclear staining Necrosis Epithelial hyaline deposit Hydropic degeneration Brush Glomerular Stasis Vascular Vasa recta stasis Peritubular capillary stasis Scores together Left Kidney Tubular Epithelial nuclear staining Necrosis Epithelial hyaline deposit Hydropic degeneration Brush Glomerular Stasis Vascular Vasa recta stasis Peritubular capillary stasis Scores together
.57 .57 1.43 .57 1.57
.97 .97 .53 0.79 1.13
I/R
2.1 2.1 1.2 1.2 1.9
.57 .57 1.03 .63 .57
Treated
P
.005 C vs I/R NS T vs I/R .005 C vs I/R; .03 T vs I/R NS .005 T vs I/R NS
1.5 1.3 1.2 .5 1.5
.85 .82 .42 .7 .85
.28 0.49
1.0 .67
1.8 .79
.003 C vs T; .03 T vs I/R
.44 1.28 .16 0.37 3.9 4.89
1.5 .85 .30 .48 11.3 3.16
2.4 .97 1.4 1.07 11.6 2.27
.001 C vs T; .02 C vs I/R; .047 T vs I/R .001 C vs T; .01 T vs I/R .001 C vs T; .002 C vs I/R; NS T vs I/R
.0001 C vs I/R; .08 C vs T; .02 T vs I/R .0001 C vs I/R; .08 C vs T; .002 T vs I/R NS .03 C vs I/R; NS C vs I/R; NS T vs I/R NS
.57 .57 1.43 .57 1.57
.97 .97 .53 .79 1.13
2.4 2.4 1.2 1.2 2.1
.5 .5 .6 .4 .31
1.3 1.2 1 1.2 1.9
.8 .8 .5 1.03 .87
.28 .49
.7 .5
.4 .5
.03 C vs I/R; NS C T vs I/R
.44 1.28 .16 .37 3.9 4.89
1.2 .9 .2 .42 10.36 4.12
.3 .67 .016 .04 6.82 1.78
.008 C vs I/R; .05 T vs I/R NS .002 C vs I/R; .0009 T vs I/R
Abbreviations: C, control; I/R, ischemia-reperfusion; NS, not significant; T, treated.
During I/R several mechanisms may occur, such as intravascular thrombosis, leukocyte plugging, hemoconcentration, endothelial cell swelling, vasomotor dysfunction, and interstitial edema, causing the absence of blood flow in the micro-vessels (also called the “no-reflow” phenomenon). In addition, impaired deformability and enhanced aggregation of red blood cells also contribute to microcirculatory disturbance [31]. There are numerous ways to measure microcirculation, including invasive and non-invasive techniques such as hand-held Doppler ultrasonography, laser Doppler flowmetry, implantable Doppler, pulse oximetry, near-infrared spectroscopy, tissue pH, transcutaneous oxygen tension, and bioelectrical impendence measurements. We chose laser Doppler flowmetry, which is a non-invasive, widely used method that can easily detect changes in the microcirculation. The technique is based on the assessment of the Doppler’s low-power laser light, which is scattered by moving red blood cells; shifts in the light are then plotted on a computer screen [32]. The number and velocity of moving red blood cells influence the measurement. It can be expressed in BFUs. Several factors may influence the results of the laser Doppler flowmetry such as temperature, instability of the probe, or the movement, drying, and cooling of the tissue. Therefore, it is very important to provide standard circumstances [33]. Our results are presented in Table 1 and Fig 1. Both the I/ R and I/R þ DMT groups experienced a decrease in BFU
during the 60 minutes of ischemia. The extent of this change was more extensive in the I/R þ DMT group. Control animals also showed some extent of constant stepwise decrease
Fig 2. Prominent histological changes in the control group after complete I/R. Periodic acid-Schiff staining revealed prominent stasis in the cortical dilated veins; the periodic acidSchiff-positive mesangium epithelium of the cortical tubules are intact, w/o brush rupture, there are no signs of necrosis, only 10% nuclear loss, some hyaline globules in the lumen, some hydropic degeneration in the distal tubules, almost no signs of peritubular capillaries, and the structures of interlobar arteries are intact. I/R, ischemia-reperfusion.
DMT PREVENTS RENAL ISCHEMIA REPERFUSION
Fig 3. Prominent histological changes in the I/R group after complete I/R. Glomeruli without mesangial changes; 90% nuclear loss in the epithelium of tubules, ruptured brush; no coagulation necrosis. There are eosinophil hyaline globules, edematous tubules with compressed lumen; cortical interlobar arteries are intact, vasa recta dilated. I/R, ischemia-reperfusion.
in BFU, which is explained by the necrosis and opened abdomen itself. When reperfusion started all groups started to compensate, which resulted in a tendentious increase in BFU through all measurement time points. After 120 minutes of reperfusion BFU was reached and exceeded its original value in the I/R group, while it was lower both in groups C and Group I/R þ DMT. Together with the histopathological findings, we observed that a spontaneous course of ischemia resulted in a decrease in microcirculation, stasis in the glomeruli, and peritubular
Fig 4. Prominent histological changes in the I/R þ DMT group after complete I/R. Periodic acid-Schiff staining revealed that the glomeruli and mesangium are intact, no signs of necrosis; there are some ruptured brushes and shrunken epithelium in the proximal tubules, and 50% nuclear loss in the epithelial cells. The interlobar arteries are intact, with minimal ischemic changes present.
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Fig 5. Histology of the right (ischemia only) kidney, within the I/R þ DMT group. Glomerular capillaries are full of red blood cells, nuclear staining is intact, the epithelium of the proximal tubules is intact, with periodic acid-Schiff staining þ graining, with no signs of necrosis. Distal tubules are intact. Peritubular capillaries are full with blood cells and no vascular changes; the pyelic epithelium is intact.
capillaries (Figs 5 and 6). First, it was worse in the presence of DMT compared to non-treated animals. When reperfusion started, the stasis automatically began to recover, more extensively in the I/R þ DMT group than in the others. Finally, after 120 minutes of reperfusion, structural injuries were less extensive in the DMT-treated group (I/R þ DMT) than in the non-treated (I/R) animals (Figs 3 and 4).
Fig 6. Histology of the left kidney (ischemia and reperfusion) within the I/R þ DMT group. Glomeruli are intact, capillaries are emptied, no signs of stasis, and dilatation is present. There is 5% nuclear loss, in the subcapsular tubular epithelium. The brush is ruptured focally. Cytoplasmic periodic acid-Schiff staining þ graining is more prominent, some periodic acid-Schiff staining þ area in the distal tubular structures. The vasa recta is still dilated, and moderate acute tubular necrosis is present.
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It is not yet completely clear whether the much higher rate of microcirculation (BFU) observed in the non-treated group compared to both the control and DMT-treated animals at 120 minutes is a consequence of circulatory overcompensation after an ischemic exposuredis it a cause or a causative factor? According to Conger et al, renal ischemia caused by total arterial clamping concluded in smooth muscle necrosis after 48 hours of reperfusion, while endothelial injury was predominant when I/R was induced by norepinephrine [34]. It is also known that, in contrast to normoxia, nitrite exerts potent vasorelaxation during ischemic conditions already at physiological concentrations [35], and it is proven that NO-dependent adenosine 5’-o-(2-thiodiphosphate) (ADP beta S)-induced dilation is reduced after I/R [36]. The cadaveric donor population has changed in recent decades since ECDs became common [37]. Since there is a growing gap between the number of donors and the need for cadaveric organs, ECDs must be involved in transplantation in an increasing number. Machine perfusion is one answer, also called organ recovery after retrieval of the cadaveric organ. However, machine perfusion has its limits, as irreversible changes cannot be recovered [5]. The other approach is organ preconditioning. The components of different organ-preserving solutions indicate that many targets have been evaluated since the first ones produced by Koyama [38] and Starzl [39]. The most vulnerable part of the kidney is the tubular system. Acute tubular necrosis is a well-known phenomenon that is caused mainly by I/R injury and is responsible for delayed kidney graft function. It is characterized by numerous patterns, including the activation of transcriptional factors, endothelial injury of peritubular small vessels, immune responses, and inflammatory processes associated with necrosis and apoptosis of the renal tubular epithelium. The tubular epithelium may regenerate in weeks [40]; however, any additional damage will expose the graft-kidney to acute rejection or cytomegalovirus or other severe infection in the long-term, and the remnant tubular and partly glomerular structures will not be enough anymore to maintain kidney function, causing rapid deterioration. Soon after transplantation, tubulointerstitial damage is predominantly related to ischemia reperfusion injury, acute tubular necrosis, acute and subclinical rejection, and/or calcineurin inhibitor nephrotoxicity, superimposed on pre-existing donor disease [41]. Therefore, acute tubular necrotic kidneys are often exposed to early chronic rejection. The half-life of these kidneys might be shorter [42]. I/R injury is a challenge in organ transplantation. Several attempts have been made to increase the tolerance of different tissues to I/R injury. The protective effects of tNKAb against renal I/R injury was concluded to occur via stimulation of PKC3 pathways [43]. Type I interferon signaling might also play a role in the mechanism of kidney I/R injury. Type I interferon may thus serve as a novel target for the therapy against renal I/R injury [44]. Besides its psychedelic activity, DMT, as endogenous ligand of the sigma-1 receptor, has a proven role in
protecting tissues from oxidative stress [22], immunoregulation [24], ischemic injury [26], and neuroprotection [27]. It also increases urinary normetanephrine excretion [45], and some DMT derivates, as serotonin analogues, play a role in the body’s thermoregulation [46]. Tryptamines such as fluvoxamine were also proved to be protective factors for kidney I/R injury [19]. Based on our results, we concluded that DMT administration prevented the tubular necrosis and hydropic degeneration during ischemia-reperfusion in rat model. It was clearly observed that circulatory stasis around the glomeruli was more extensive when DMT was administered; however, the recovery phase was found to be more rapid, extensive, and complete. Histologic changes characteristic of I/R injury were proved to be reversible following the administration of DMT. The reconstruction of microcirculation was closed to the control animals because DMT was used as pre-treatment. We concluded that DMT can be used for renoprotection. As metabolism differs among tryptamines, with regard to CYP-450 subgroups, DMT might be even more effective in preventing I/R injury than other tryptamines. REFERENCES [1] Shafran D, Kodish E, Tzakis A. Organ shortage: the greatest challenge facing transplant medicine. World J Surg 2014;38:1650e7. [2] Jay CL, Washburn K, Dean PG, et al. Survival benefit in older patients associated with earlier transplant with high KDPI kidneys. Transplantation 2017;101:867e72. [3] Thornton SR, Hamilton N, Evans D, et al. Outcome of kidney transplantation from elderly donors after cardiac death. Transplant Proc 2011;43:3686e9. [4] Cavaillé-Coll M, Bala S, Velidedeoglu E, et al. Summary of FDA workshop on ischemia reperfusion injury in kidney transplantation. Am J Transplant 2013;13:1134e48. [5] Nemes B, Gámán G, Polak WG, et al. Extended-criteria donors in liver transplantation part II: reviewing the impact of extended-criteria donors on the complications and outcomes of liver transplantation. Expert Rev Gastroenterol Hepatol 2016;10: 841e59. [6] Doma nski L, Safranow K, Dołegowska B, et al. Hypoxanthine as a graft ischemia marker simulates catalase activity in the renal vein during reperfusion in humans. Transplant Proc 2006;38: 35e8. [7] Wijermars LG, Schaapherder AF, de Vries DK, et al. Defective postreperfusion metabolic recovery directly associates with incident delayed graft function. Kidney Int 2016;90:181e91. [8] Fan Y, Zhang C, Li T, et al. A new approach of short wave protection against middle cerebral artery occlusion/reperfusion injury via attenuation of Golgi apparatus stress by inhibition of downregulation of secretory pathway Ca(2þ)-ATPase isoform 1 in rats. J Stroke Cerebrovasc Dis 2016;25:1813e22. [9] Kosieradzki M, Rowinski W. Ischemia/reperfusion injury in kidney transplantation: mechanisms and prevention. Transplant Proc 2008;40:3279e88. [10] Mangino MJ, Tian T, Ametani M, et al. Cytoskeletal involvement in hypothermic renal preservation injury. Transplantation 2008;85:427e36. [11] He SQ, Zhang YH, Venugopal SK, et al. Delivery of antioxidative enzyme genes protects against ischemia/reperfusioninduced liver injury in mice. Liver Transpl 2006;12:1869e79. [12] Collard CD, Gelman S. Pathophysiology, clinical manifestations, and prevention of Ischemia-reperfusion injury. Anesthesiology 2001;94:1133e8.
DMT PREVENTS RENAL ISCHEMIA REPERFUSION [13] Kim SM, Ahn S, Min SI, et al. Cold ischemic time is critical in outcomes of expanded criteria donor renal transplantation. Clin Transplant 2013;27:132e9. [14] Haneya A, Haake N, Diez C, et al. Impact of the Eurotransplant high-urgency heart allocation system on the outcome of transplant candidates in Germany. Thorac Cardiovasc Surg 2011;59:93e7. [15] Gallinat A, Luer B, Swoboda S, et al. Use of the new preservation solution custodiol-N supplemented with dextran for hypothermic machine perfusion of the kidney. Cryobiology 2013;66:131e5. [16] Matsuno N, Uchida K, Furukawa H. Impact of machine perfusion preservation of liver grafts from donation after cardiac death. Transplant Proc 2014;46:1099e103. [17] Lindell SL, Muir H, Brassil J, et al. Hypothermic machine perfusion preservation of the DCD kidney: machine effects. J Transplant 2013;2013:802618. https://doi.org/10.1155/2013/802618. [18] Schlegel A, Graf R, Clavien PA, et al. Hypothermic oxygenated perfusion (HOPE) protects from biliary injury in a rodent model of DCD liver transplantation. J Hepatol 2013;59: 984e91. [19] Hosszu A, Antal Z, Lenart L, et al. s1-Receptor agonism protects against renal ischemia-reperfusion injury. J Am Soc Nephrol 2017;28:152e65. [20] van Waarde A, Rybczynska AA, Ramakrishnan NK, et al. Potential applications for sigma receptor ligands in cancer diagnosis and therapy. Biochim Biophys Acta 2015;1848:2703e14. [21] Penke B, Fülöp L, Sz} ucs M, Frecska E. The role of sigma-1 receptor, an intracellular chaperone in neurodegenerative diseases. Curr Neuropharmacol 2018;16:97e116. [22] Pal A, Fontanilla D, Gopalakrishnan A, Chae YK, et al. The sigma-1 receptor protects against cellular oxidative stress and activates antioxidant response elements. Eur J Pharmacol 2012;682:12e20. [23] Fontanilla D, Johannessen M, Hajipour AR, et al. The hallucinogen N,N-dimethyltryptamine (DMT) is an endogenous sigma-1 receptor regulator. Science 2009;323:934e7. [24] Frecska E, Szabo A, Winkelman MJ, et al. A possibly sigma1 receptor mediated role of dimethyltryptamine in tissue protection, regeneration, and immunity. J Neural Transm 2013;120:1295e303. [25] Szabo A, Kovacs A, Frecska E, Rajnavolgyi E. Psychedelic N,N-dimethyltryptamine and 5-methoxy-N,N-dimethyltryptamine modulate innate and adaptive inflammatory responses through the sigma-1 receptor of human monocyte-derived dendritic cells. PLoS One 2014;9:e106533. [26] Szabo A, Kovacs A, Riba J, Djurovic S, Rajnavolgyi E, Frecska E. The endogenous hallucinogen and trace amine N,Ndimethyltryptamine (DMT) displays potent protective effects against hypoxia via sigma-1 receptor activation in human primary iPSC-derived cortical neurons and microglia-like immune cells. Front Neurosci 2016;10:423. [27] Allahtavakoli M, Jarrott B. Sigma-1 receptor ligand PRE084 reduced infarct volume, neurological deficits, proinflammatory cytokines and enhanced anti-inflammatory cytokines after embolic stroke in Rats. Brain Res Bull 2011;85:219e24. [28] Dinger J, Woods C, Brandt SD, et al. Cytochrome P450 inhibition potential of new psychoactive substances of the tryptamine class. Toxicol Lett 2016;241:82e94.
1275 [29] Urfer R, Moebius HJ, Skoloudik D, et al. Cutamesine stroke recovery study group: Phase II trial of the Sigma-1 receptor agonist cutamesine (SA4503) for recovery enhancement after acute ischemic stroke. Stroke 2014;45:3304e10. [30] Németh N, Furka I, Mikó I. Hemorheological changes in ischemia-reperfusion: an overview on our experimental surgical data. Clin Hemorheol Microcirc 2014;57:215e25. [31] Reffelmann T, Kloner RA. The no-reflow phenomenon: a basic mechanism of myocardial ischemia and reperfusion. Basic Res Cardiol 2006;101:359e72. [32] Frerichs KU, Feuerstein GZ. Laser-Doppler flowmetry: a review of its application for measuring cerebral and spinal cord blood flow. Mol Chem Neuropathol 1990;12:55e70. [33] Rajan V, Varghese B, van Leeuwen TG, et al. Review of methodological developments in laser Doppler flowmetry. Lasers Med Sci 2009;24:269e83. [34] Conger JD, Hammond WS. Renal vasculature and ischemic injury. Ren Fail 1992;14:307e10. [35] Liu M, Zollbrecht C, Peleli M, et al. Nitrite-mediated renal vasodilatation is increased during ischemic conditions via cGMPindependent signaling. Free Radic Biol Med 2015;84:154e60. [36] Kristiansen SB, Sheykhzade M, Edvinsson L, et al. Changes in vasodilation following myocardial ischemia/reperfusion in rats. Nitric Oxide 2017;70:68e75. [37] Zádori G, Kovács DÁ, Fedor R, et al. Results of expandedcriteria donor kidneys: a single-center experience in Hungary. Transplant Proc 2015;47:2189e91. [38] Koyama I, Bulkley GB, Williams GM, et al. The role of oxygen free radicals in mediating the reperfusion injury of cold preserved ischemic kidneys. Transplantation 1985;40:590e5. [39] Starzl TE, Miller C, Broznick B, et al. An improved technique for multiple organ harvesting. Surg Gynecol Obstet 1987;165: 343e8. [40] Yoshida M, Honma S. Regeneration of injured renal tubules. J Pharmacol Sci 2014;124:117e22. [41] Nankivell BJ, Chapman JR. Chronic allograft nephropathy: current concepts and future directions. Transplantation 2006;81: 643e54. [42] Masutani K, Kitada H, Yamada S, et al. Progressive interstitial fibrosis of kidney allograft early after transplantation from a non-heart-beating donor: possible role of persistent ischemic injury. Clin Transplant 2010;24(Suppl 22):70e4. [43] Gong H, Sun J, Xue W, et al. Protective effect of truncated Naþ/Kþ-ATPase b on ischemia/reperfusion-induced renal injury in rats. Exp Biol Med 2014;39:677e85. [44] Freitas MC, Uchida Y, Lassman C, et al. Type I interferon pathway mediates renal ischemia/reperfusion injury. Transplantation 2011;92:131e8. [45] Riba J, Valle M, Urbano G, et al. Human pharmacology of ayahuasca: subjective and cardiovascular effects, monoamine metabolite excretion, and pharmacokinetics. J Pharmacol Exp Ther 2003;306:73e83. [46] Jiang XL, Shen HW, Yu AM. Potentiation of 5-methoxy-N, N-dimethyltryptamine-induced hyperthermia by harmaline and the involvement of activation of 5-HT1A and 5-HT2A receptors. Neuropharmacology 2015;89:342e51.
Department of Organ Transplantation, Institute of Surgery, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; Department of Operative Techniques and Surgical Research, Institute of Surgery, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; cDepartment of Pathology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; and dDepartment of Psychiatry, Faculty of Medicine, University of Debrecen, Debrecen, Hungary b
ABSTRACT Background. Ischemia reperfusion (I/R) injury remains one of the most challenging fields of organ transplantation. It is highly associated with the use of expanded criteria donors that might conclude to delayed graft function or early or late graft failure. Objective. To investigate the metabolic, microcirculatory parameters, and histologic changes under the effect of N,N-dimethyltryptamine (DMT) in a renal I/R model in rats. Method. In 26 anesthetized rats both kidneys were exposed. In the control group (n ¼ 6) no other intervention happened. In 20 other animals, the right renal vessels were ligated, and after 60 minutes the right kidney was removed. The left renal vessels were clamped for 60 minutes then released, followed by 120 minutes of reperfusion. In the I/R group (n ¼ 10), there was no additive treatment, while in I/R þ DMT group (n ¼ 10) DMT was administered 15 minutes before ischemia. Blood samples were taken, laser Doppler measurement was performed, and both kidneys were evaluated histologically. Results. Microcirculation (blood flux units [BFU]) diminished in all groups, but remarkably so in the I/R þ DMT group. This group compensated better after the 30th minute of reperfusion. The control and I/R þ DMT groups had similar BFUs after 120 minutes of reperfusion, but in the I/R group BFU was higher. Tubular necrosis developed in the I/R and I/R þ DMT groups too; it was moderated under DMT effect, and severe without. Histologic injuries were less in I/R þ DMT Group compared to non-treated animals. Conclusion. Histologic changes characteristic to I/R injuries were reversible and microcirculation recovered at the end of 120 minutes reperfusion under the administration of DMT. DMT can be used for renoprotection in kidney transplantation.
M
ORE and more people are in need of transplants worldwide, but the number of donor organs and living donors cannot keep up with this growing demand [1]. The average waiting time has greatly increased and adversely affects the survival of both patients and transplanted organs [2]. Acceptance of older and more seriously injured marginal and non-heart beating donor organs experiencing different injuries could bridge the gap between demand and availability; however, it is a challenge in the 0041-1345/19 https://doi.org/10.1016/j.transproceed.2019.04.005
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context of organ preservation [3]. Ischemia reperfusion (I/R) injury remains one of the most challenging fields of
*Address correspondence to Balázs Nemes, MD, PhD, Division of Organ Transplantation, Institute of Surgery, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Nagyerdei krt. 98., Hungary. Tel: þ36-30-983.4764. E-mail: nemes.balazs@ med.unideb.hu ª 2019 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 51, 1268e1275 (2019)
DMT PREVENTS RENAL ISCHEMIA REPERFUSION
transplantation. Even the United States Food and Drug Administration held a meeting on this topic in 2011 [4]. I/R injury is a multicausal and multifactorial phenomenon. It is highly associated with the use of expanded criteria donors (ECDs), which might conclude to delayed graft function and/or early graft loss, or late graft failure [5]. On the other hand, there is a need to accept ECDs in the age of donororgan shortage. Therefore, the magnitude of the problem is that I/R injuries have become a regular clinical issue instead of a marginal field of interest. The pathophysiology is still under debate. During ischemia the core process is the decrease in cellular oxidative phosphorylation, when adenosine triphosphate (ATP) is further dephosphorylated to adenosine diphosphate, adenosine monophosphate, and purines, leading to an accumulation of hypoxanthine [6,7]. The drop in cellular ATP leads to the dysfunction of ATPdependent membrane ion-pumps, resulting in calcium, sodium, and water influx to the cells. The swelling results in disruption of the cell membrane (opening stretch-activated channels, further modulating the conductance) and intracellular membranes, such as of the endoplasmic reticulum, the Golgi apparatus, the mitochondrial membrane and the cytoskeletal microtubule [8e10]. Hypoxia leads to microcirculatory, micro-rheological, inflammatory, innate, and adaptive immune responses, and alterations. The presence of oxidative stress and the worsening rheological properties of the blood are to be highlighted. During the complex hemodynamic changes under I/R, NO plays an important role in local flow regulation [11,12]. I/R injury is a main target for therapeutic issues. Many attempts have been made to reduce cold ischemia time [13,14], to develop better perfusion fluid [15], and to create new a perfusion technique [16e18]. It has already been published that the intravenous administration of the sigma-1 receptor agonist N,N-dimethyltryptamine (DMT) protects kidneys from I/R injury [19]. However, the detailed mechanism of action is not well known. The sigma-1 receptor is a ligand-regulated molecular chaperone with various ion channels and G-protein-coupled membrane receptors as clients [20]. It is located at the mitochondrion-associated membrane of the endoplasmic reticulum and regulates the cross-talk between endoplasmic reticulum stress and oxidative stress [21]. The sigma-1 receptor protects against cellular oxidative stress and activates antioxidant response elements [22]. The recent discovery that DMT is an endogenous ligand of the sigma-1 receptor [23] may shed light on yet undiscovered physiological mechanisms of DMT activity and reveal some of its putative biological functions, such as a role in tissue protection [24], immunoregulation [25], and alleviation of ischemic injury [26]. Other studies have already proved that part of the neuroprotective effects of sigma-1 receptor agonists is reduced levels of pro-inflammatory cytokines and enhanced anti-inflammatory cytokines following stroke [27]. It has also been found that fluvoxamine stimulation in rat kidney activated nitric oxide production, which was blocked by s1-receptor knockdown or Akt inhibition. The sigma-1 receptor activation by fluvoxamine triggered the Akt-nitric
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oxide synthase signaling pathway, resulting in time- and isoform-specific endothelial and neuronal nitric oxide synthase activation and NO production. A prompt peritubular vasodilation was seen after fluvoxamine treatment [19]. DMT does not inhibited CYP2D6 activity, in contrast to diallyl-tryptamines. Diallyl-tryptamines also inhibited CYP1A2 activity comparable to fluvoxamine [28]. The protective effect of some other sigma-1 receptors has also been proven. For example, cutamesine (SA4503) enhanced functional recovery after experimental stroke with a treatment initiation window of 48 hours and chronic treatment for 28 days [29]. The following questions arise: Will the administration of DMT protect the kidney against ischemia, what changes are present in the different structures of the kidney, and to what extent? Will there be any beneficial changes in the microcirculation of the kidney caused by the protective agent? MATERIALS AND METHODS Our group settled the following animal experiment to answer these questions. The animal experiment was approved and registered by the University of Debrecen Committee of Animal Welfare (permission no.:20/2011), in accordance with national and European Union regulations (the Hungarian Animal Protection Act [Law XVIII/1998] and Edict 63/2010).
Operative Protocol Twenty-seven male CD-outbred rats (343 29 g) were involved in the experiment, divided into 3 experimental groups: control (C), I/R, and treated (T). Anesthesia was introduced with 62.5 mg/kg thiopental I.P., then, the following operating procedures were performed on the experimental groups. The C group had only anesthesia, cannulation, abdominal opening, blood sampling, laser measurement, kidney removal, and termination of the animal. The I/R and I/R þ DMTs groups had the same operating procedure: anesthesia, cannulation, opening the abdomen, preparing the aorta and renal vessels, ligating the right renal artery and vein and clamping the left renal vessels with a plastic clip (to induce ischemia) for 60 minutes, then removal of the clip from the left renal vessels, removal of the right kidney for histology (ischemia injury only), and keep the left one for a further 120 minutes. The left kidney was then removed and the animal was terminated. The I/ R group was not treated, while the I/R þ DMT group received DMT; 2.95 mL/bwkg using 2.45 mg/mL solution was given i.m. first 15 minutes before ligating/clamping the renal vessels and again 60 minutes later, 15 minutes before the start of reperfusion. Blood sampling (150 uL, K3-EDTA each) was performed via the femoral artery immediately after cannulation (base), further at the 60th minute after ischemia was induced (I-60), then at the 30th (R-30), 60th (R-60), and 120th minutes after reperfusion (R-120). An equal volume of physiological saline was administered via the cannula. The blood samples were sent to the laboratory for basic analysis.
Laboratory Measurements Hematological parameters were determined by Sysmex K-4500 automated hematology analyzer (TOA Medical Electronics Corp., South San Francisco, Calif, United States). In this paper only white blood cell count (WBC [g/L]) and platelet count (Plt [g/L]) are shown. Electrolytes were tested by an EPOC blood analysis device
} NÉMETH ET AL NEMES, PETO,
1270 (Alere, Waltham, Mass, United States). We have reported the sodium, potassium and serum Caþþ levels [mmol/L].
Laser-Doppler Flowmetry/Laser-Doppler Data Acquisition The microcirculation of the left kidney, liver, and intestine was monitored by laser-Doppler technique (LD-01 Laser Doppler Flowmeter, Experimetria, Budapest, Hungary), using a standard pencil probe (Oxford Optronix, Abingdon, England) at every sampling point of the experiment (Base, I-60, R-30, R-60, and R-120). The device determines blood flux unit (BFU) based on the number of moving red blood cells and their mean velocity in the tested tissue volume (1e1.5 mm3). The probe was gently placed consecutively on the surface of the anterior middle surface of the left kidney, on the middle region of the right liver lobe, and on the intestine. The signal was recorded by S.P.E.L. Advanced Kymograph software (Experimetria) at a 1 kHz sampling rate for 30e60 seconds. During offline data analysis, the average value of a noisefree 10-second-long representative section of each recorded graph was calculated.
Histology Histologic examination was performed on both kidneys. Samples were identified by a combination of characters and numbers; however, to keep the process blinded, decoding was not available for the pathologists, so hematoxylin and eosin and periodic acid-Schiff staining were carried out. The glomerular, vascular, and tubular structures of the kidney were examined and scored by a pathologist.
Statistical Analysis Data are presented as means standard deviation. One-way and repeated measure analysis of variance tests were used for intra- and inter-group comparisons (Bonferroni/Dunn methods). For simple comparison of inter-group differences at single time points, the Student t test, and Mann-Whitney rank-sum tests were applied as well, depending on the normality of data distribution. A P < .05 value was considered statistically significant.
RESULTS
Sodium level [mmol/L] did not change in any groups. Potassium level [mmol/L] increased significantly in all groups from the base level to R-120. The value at the 120th minute after reperfusion was 150% of basal in the I/R group
(6.3 1.86 vs 4.2 .33, respectively), while it was 130% in I/R þ DMT group (5.4 2.12 vs 4.15 .33). The serum Caþþ [mmol/L] level decreased significantly in all study groups from the base to R-120. Serum Ca level at the 120th minutes was 88% of base level in the I/R group (1.23 .08 vs 1.40 .04), while it was 81% in the I/R þ DMT group (1.08 .16 vs 1.33 .04). There were no significant differences between the I/R and I/E þ DMT groups regarding serum Kþ and Caþþ. WBC [x103/mL] increased to 153% of base at the 60th minute of ischemia in the I/R group (8.1 4.68 vs 5.43 1.6), while it was 94% of base in the I/R þ DMT group (4.71 1.5 vs 5.05 .86). This difference was significant between the I/R and I/R þ DMT groups. By the end of 120 minutes of reperfusion, WBC was 65% of base in the I/R group (3.51 1.75 vs 5.43 1.6) and 126% of base in the I/R þ DMT group (5.43 1.6 vs 5.43 1.6). This difference was significant between the I/R and I/R þ DMT groups. The platelet count [x103/mL] increased to 150% of base in the I/R group at the end of 60 minutes of ischemia (958.7 138.5 vs 646.1 317.1), but remained unchanged in the DMT-treated group (729 92 vs 794.8 125.6). The difference was significant. Platelet count decreased back to base level at the end of 120 minutes of reperfusion in the I/R group, and was the same at base and I-60 levels in the I/R þ DMT group. Results of the microcirculation parameters are shown in Table 1 and Fig 1. The initial BFU was lower in Group I/R compared to the C and I/R þ DMT groups in both left and right kidneys; however, it did not disturb the estimation of the changing trends. When kidneys (both left and right) were exposed to ischemia (60 minutes in the I/R and I/R þ DMT groups), BFU decreased in both groups (left kidney, 23.9 2.4e22.9 1.3 in the I/R group and 29.8 4.1e19.7 4.4 in the I/R þ DMT group). The decrease was significant in the I/R þ DMT group. However, if we consider both right kidneys in the I/R and I/R þ DMT groups, the decrease was the same (27.3 3.2e17.2 0.1). After 30 minutes both the I/R and I/R þ DMT groups showed an compensational increase in BFU; however, this trend was more prominent
Table 1. Laser Doppler Measurements
Left Kidney Control I/R Treated Right Kidney Control I/R Treated
Base
I-60
R-30
R-60
R-120
32.7 3.7 23.9 2.4† 29.8 4.1‡
26.2 6.2 22.9 1.3 19.7 4.4*
27.3 3.1* 27.7 1.8 27.4 6.7
24.4 4.3* 28.4 1.6 26.5 7.3
21.2 6.1 28.2 1.4 23.9 4.4*
34.4 5.6 27.3 3.2 30.7 4.9
24.4 5.1 17.2 .1
24.1 4.7
26.2 3.0
29.0 1.2
Results are in BFU. Abbreviations: BFU, blood flux unit; I-60, 60th minute after induction of ischemia; R-30, 30 minutes of reperfusion; R-60, 60 minutes of reperfusion; R-120, 120 minutes of reperfusion. *P < .05 vs base. † P < .05 vs control. ‡ P < .05 vs I/R.
DMT PREVENTS RENAL ISCHEMIA REPERFUSION
1271 LEFT KIDNEY
35 33 31
Tengelycím
29 27 25 23 21 19 17
Control I/R Treated i.m.
base 32.66685799 23.95662048 29.79226689
I-60 26.16157092 22.94351963 19.66905003
R-30 27.31680933 27.74166475 27.39338585
R-60 24.36945329 28.40528512 26.55107548
R-120 27.21486057 28.1550753 23.9374008
Fig 1. Laser Doppler results of the left kidney in the different study groups. BFU, blood flux unit.
compared to I-60 in the C group (19.7 4.4e27.4 6.7) than it was in the I/R group (22.9 1.3e27.7 1.8). This increase continued in the I/R group until the end of the observation period. The R-120 BFU exceeded its base value in the I/R group (23.9 2.4 vs 28.2 1.4), while R-120 results were lower compared to the initial value in the I/R þ DMT group (29.8 4.1 vs 23.9 4.4). When the control group is compared to the I/R group regarding base BFU vs R-120 BFU, the trend is the opposite (32.7 3.7 vs 21.2 6.1 and 23.9 2.4 vs 28.2 1.4, respectively). When the control group is compared to the I/R þ DMT group, they are similar (there is a slight decrease in BFU). Both kidneys were sent to pathology. The results are shown in Table 2 and Figs 2e6. Because the right kidney was removed before reperfusion it only experienced an ischemic injury. The left kidneys were left until the end of the entire procedure. Therefore, histologic changes of the left kidneys represent both ischemic and reperfusion injury. Tubular changes were equally developed on both right and left kidneys. Ischemic-only changes were extensive dilation and stasis in the periglomerular capillaries and vasa recta. However, it almost completely disappeared after reperfusion in the I/R þ DMT group (Figs 5 and 6). The scores given for epithelial nuclear staining, tubular necrosis, and hydropic degeneration increased both in the I/R and Treated groups compared to controls. However, these signs were less serious when the animals were treated with DMT (I/R þ DMT group) compared to simple I/R. This difference was more extensive in the left kidneys than in the right ones. Regarding vascular and glomerular changes, stasis was the main sign of ischemia that increased in all structures. Surprisingly, it was more extensive in the I/R þ DMT group
compared to the I/R group in the right kidneys. On the other hand, stasis was less extensive in the I/R þ DMT group compared to the I/R group in the left kidneys. It looks as though DMT worsened the stasis during ischemia and enhanced its spontaneous recovery in the reperfusion phase. Total scores for the histologic changes of ischemiareperfusion are shown in Table 2. There was no difference in scores between the I/R and I/R þ DMT groups regarding the right kidneys. The scores reduced by tubular recovery was neutralized by the additive scores given for the worsening stasis. However, the I/R þ DMTs group had significantly lower scores compared to the I/R group (6.82 1.78 vs 10.36 4.12; P ¼ .0009) at the end of the 120 minutes of reperfusion. The results of the I/R þ DMT group was still statistically higher compared to the control group (6.82 1.78 vs 3.9 4.89; P ¼ .01). DISCUSSION
It is well-known that serum potassium is a marker of ischemic renal injuries. An increase is obviously expected after 60 minutes of renal ischemia, as seen in the I/R group. Recovery was better in the I/R þ DMT group compared to non-treated animals (I/R group). The impairment of microcirculation has a significant role in the pathophysiology of many diseases, such as cardiovascular diseases, diabetes mellitus, sepsis, compartment syndrome, and ischemia-reperfusion injuries [30]. Therefore, the investigation of microcirculation could be useful in diagnoses, particularly in determining the severity of and in following up on diseases. It is very important in predicting the viability of tissue, postoperative organ function, and complications.
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1272 Table 2. Histologic Findings of the Retrieved Kidneys Control
Right Kidney Tubular Epithelial nuclear staining Necrosis Epithelial hyaline deposit Hydropic degeneration Brush Glomerular Stasis Vascular Vasa recta stasis Peritubular capillary stasis Scores together Left Kidney Tubular Epithelial nuclear staining Necrosis Epithelial hyaline deposit Hydropic degeneration Brush Glomerular Stasis Vascular Vasa recta stasis Peritubular capillary stasis Scores together
.57 .57 1.43 .57 1.57
.97 .97 .53 0.79 1.13
I/R
2.1 2.1 1.2 1.2 1.9
.57 .57 1.03 .63 .57
Treated
P
.005 C vs I/R NS T vs I/R .005 C vs I/R; .03 T vs I/R NS .005 T vs I/R NS
1.5 1.3 1.2 .5 1.5
.85 .82 .42 .7 .85
.28 0.49
1.0 .67
1.8 .79
.003 C vs T; .03 T vs I/R
.44 1.28 .16 0.37 3.9 4.89
1.5 .85 .30 .48 11.3 3.16
2.4 .97 1.4 1.07 11.6 2.27
.001 C vs T; .02 C vs I/R; .047 T vs I/R .001 C vs T; .01 T vs I/R .001 C vs T; .002 C vs I/R; NS T vs I/R
.0001 C vs I/R; .08 C vs T; .02 T vs I/R .0001 C vs I/R; .08 C vs T; .002 T vs I/R NS .03 C vs I/R; NS C vs I/R; NS T vs I/R NS
.57 .57 1.43 .57 1.57
.97 .97 .53 .79 1.13
2.4 2.4 1.2 1.2 2.1
.5 .5 .6 .4 .31
1.3 1.2 1 1.2 1.9
.8 .8 .5 1.03 .87
.28 .49
.7 .5
.4 .5
.03 C vs I/R; NS C T vs I/R
.44 1.28 .16 .37 3.9 4.89
1.2 .9 .2 .42 10.36 4.12
.3 .67 .016 .04 6.82 1.78
.008 C vs I/R; .05 T vs I/R NS .002 C vs I/R; .0009 T vs I/R
Abbreviations: C, control; I/R, ischemia-reperfusion; NS, not significant; T, treated.
During I/R several mechanisms may occur, such as intravascular thrombosis, leukocyte plugging, hemoconcentration, endothelial cell swelling, vasomotor dysfunction, and interstitial edema, causing the absence of blood flow in the micro-vessels (also called the “no-reflow” phenomenon). In addition, impaired deformability and enhanced aggregation of red blood cells also contribute to microcirculatory disturbance [31]. There are numerous ways to measure microcirculation, including invasive and non-invasive techniques such as hand-held Doppler ultrasonography, laser Doppler flowmetry, implantable Doppler, pulse oximetry, near-infrared spectroscopy, tissue pH, transcutaneous oxygen tension, and bioelectrical impendence measurements. We chose laser Doppler flowmetry, which is a non-invasive, widely used method that can easily detect changes in the microcirculation. The technique is based on the assessment of the Doppler’s low-power laser light, which is scattered by moving red blood cells; shifts in the light are then plotted on a computer screen [32]. The number and velocity of moving red blood cells influence the measurement. It can be expressed in BFUs. Several factors may influence the results of the laser Doppler flowmetry such as temperature, instability of the probe, or the movement, drying, and cooling of the tissue. Therefore, it is very important to provide standard circumstances [33]. Our results are presented in Table 1 and Fig 1. Both the I/ R and I/R þ DMT groups experienced a decrease in BFU
during the 60 minutes of ischemia. The extent of this change was more extensive in the I/R þ DMT group. Control animals also showed some extent of constant stepwise decrease
Fig 2. Prominent histological changes in the control group after complete I/R. Periodic acid-Schiff staining revealed prominent stasis in the cortical dilated veins; the periodic acidSchiff-positive mesangium epithelium of the cortical tubules are intact, w/o brush rupture, there are no signs of necrosis, only 10% nuclear loss, some hyaline globules in the lumen, some hydropic degeneration in the distal tubules, almost no signs of peritubular capillaries, and the structures of interlobar arteries are intact. I/R, ischemia-reperfusion.
DMT PREVENTS RENAL ISCHEMIA REPERFUSION
Fig 3. Prominent histological changes in the I/R group after complete I/R. Glomeruli without mesangial changes; 90% nuclear loss in the epithelium of tubules, ruptured brush; no coagulation necrosis. There are eosinophil hyaline globules, edematous tubules with compressed lumen; cortical interlobar arteries are intact, vasa recta dilated. I/R, ischemia-reperfusion.
in BFU, which is explained by the necrosis and opened abdomen itself. When reperfusion started all groups started to compensate, which resulted in a tendentious increase in BFU through all measurement time points. After 120 minutes of reperfusion BFU was reached and exceeded its original value in the I/R group, while it was lower both in groups C and Group I/R þ DMT. Together with the histopathological findings, we observed that a spontaneous course of ischemia resulted in a decrease in microcirculation, stasis in the glomeruli, and peritubular
Fig 4. Prominent histological changes in the I/R þ DMT group after complete I/R. Periodic acid-Schiff staining revealed that the glomeruli and mesangium are intact, no signs of necrosis; there are some ruptured brushes and shrunken epithelium in the proximal tubules, and 50% nuclear loss in the epithelial cells. The interlobar arteries are intact, with minimal ischemic changes present.
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Fig 5. Histology of the right (ischemia only) kidney, within the I/R þ DMT group. Glomerular capillaries are full of red blood cells, nuclear staining is intact, the epithelium of the proximal tubules is intact, with periodic acid-Schiff staining þ graining, with no signs of necrosis. Distal tubules are intact. Peritubular capillaries are full with blood cells and no vascular changes; the pyelic epithelium is intact.
capillaries (Figs 5 and 6). First, it was worse in the presence of DMT compared to non-treated animals. When reperfusion started, the stasis automatically began to recover, more extensively in the I/R þ DMT group than in the others. Finally, after 120 minutes of reperfusion, structural injuries were less extensive in the DMT-treated group (I/R þ DMT) than in the non-treated (I/R) animals (Figs 3 and 4).
Fig 6. Histology of the left kidney (ischemia and reperfusion) within the I/R þ DMT group. Glomeruli are intact, capillaries are emptied, no signs of stasis, and dilatation is present. There is 5% nuclear loss, in the subcapsular tubular epithelium. The brush is ruptured focally. Cytoplasmic periodic acid-Schiff staining þ graining is more prominent, some periodic acid-Schiff staining þ area in the distal tubular structures. The vasa recta is still dilated, and moderate acute tubular necrosis is present.
} NÉMETH ET AL NEMES, PETO,
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It is not yet completely clear whether the much higher rate of microcirculation (BFU) observed in the non-treated group compared to both the control and DMT-treated animals at 120 minutes is a consequence of circulatory overcompensation after an ischemic exposuredis it a cause or a causative factor? According to Conger et al, renal ischemia caused by total arterial clamping concluded in smooth muscle necrosis after 48 hours of reperfusion, while endothelial injury was predominant when I/R was induced by norepinephrine [34]. It is also known that, in contrast to normoxia, nitrite exerts potent vasorelaxation during ischemic conditions already at physiological concentrations [35], and it is proven that NO-dependent adenosine 5’-o-(2-thiodiphosphate) (ADP beta S)-induced dilation is reduced after I/R [36]. The cadaveric donor population has changed in recent decades since ECDs became common [37]. Since there is a growing gap between the number of donors and the need for cadaveric organs, ECDs must be involved in transplantation in an increasing number. Machine perfusion is one answer, also called organ recovery after retrieval of the cadaveric organ. However, machine perfusion has its limits, as irreversible changes cannot be recovered [5]. The other approach is organ preconditioning. The components of different organ-preserving solutions indicate that many targets have been evaluated since the first ones produced by Koyama [38] and Starzl [39]. The most vulnerable part of the kidney is the tubular system. Acute tubular necrosis is a well-known phenomenon that is caused mainly by I/R injury and is responsible for delayed kidney graft function. It is characterized by numerous patterns, including the activation of transcriptional factors, endothelial injury of peritubular small vessels, immune responses, and inflammatory processes associated with necrosis and apoptosis of the renal tubular epithelium. The tubular epithelium may regenerate in weeks [40]; however, any additional damage will expose the graft-kidney to acute rejection or cytomegalovirus or other severe infection in the long-term, and the remnant tubular and partly glomerular structures will not be enough anymore to maintain kidney function, causing rapid deterioration. Soon after transplantation, tubulointerstitial damage is predominantly related to ischemia reperfusion injury, acute tubular necrosis, acute and subclinical rejection, and/or calcineurin inhibitor nephrotoxicity, superimposed on pre-existing donor disease [41]. Therefore, acute tubular necrotic kidneys are often exposed to early chronic rejection. The half-life of these kidneys might be shorter [42]. I/R injury is a challenge in organ transplantation. Several attempts have been made to increase the tolerance of different tissues to I/R injury. The protective effects of tNKAb against renal I/R injury was concluded to occur via stimulation of PKC3 pathways [43]. Type I interferon signaling might also play a role in the mechanism of kidney I/R injury. Type I interferon may thus serve as a novel target for the therapy against renal I/R injury [44]. Besides its psychedelic activity, DMT, as endogenous ligand of the sigma-1 receptor, has a proven role in
protecting tissues from oxidative stress [22], immunoregulation [24], ischemic injury [26], and neuroprotection [27]. It also increases urinary normetanephrine excretion [45], and some DMT derivates, as serotonin analogues, play a role in the body’s thermoregulation [46]. Tryptamines such as fluvoxamine were also proved to be protective factors for kidney I/R injury [19]. Based on our results, we concluded that DMT administration prevented the tubular necrosis and hydropic degeneration during ischemia-reperfusion in rat model. It was clearly observed that circulatory stasis around the glomeruli was more extensive when DMT was administered; however, the recovery phase was found to be more rapid, extensive, and complete. Histologic changes characteristic of I/R injury were proved to be reversible following the administration of DMT. The reconstruction of microcirculation was closed to the control animals because DMT was used as pre-treatment. We concluded that DMT can be used for renoprotection. As metabolism differs among tryptamines, with regard to CYP-450 subgroups, DMT might be even more effective in preventing I/R injury than other tryptamines. REFERENCES [1] Shafran D, Kodish E, Tzakis A. Organ shortage: the greatest challenge facing transplant medicine. World J Surg 2014;38:1650e7. [2] Jay CL, Washburn K, Dean PG, et al. Survival benefit in older patients associated with earlier transplant with high KDPI kidneys. Transplantation 2017;101:867e72. [3] Thornton SR, Hamilton N, Evans D, et al. Outcome of kidney transplantation from elderly donors after cardiac death. Transplant Proc 2011;43:3686e9. [4] Cavaillé-Coll M, Bala S, Velidedeoglu E, et al. Summary of FDA workshop on ischemia reperfusion injury in kidney transplantation. Am J Transplant 2013;13:1134e48. [5] Nemes B, Gámán G, Polak WG, et al. Extended-criteria donors in liver transplantation part II: reviewing the impact of extended-criteria donors on the complications and outcomes of liver transplantation. Expert Rev Gastroenterol Hepatol 2016;10: 841e59. [6] Doma nski L, Safranow K, Dołegowska B, et al. Hypoxanthine as a graft ischemia marker simulates catalase activity in the renal vein during reperfusion in humans. Transplant Proc 2006;38: 35e8. [7] Wijermars LG, Schaapherder AF, de Vries DK, et al. Defective postreperfusion metabolic recovery directly associates with incident delayed graft function. Kidney Int 2016;90:181e91. [8] Fan Y, Zhang C, Li T, et al. A new approach of short wave protection against middle cerebral artery occlusion/reperfusion injury via attenuation of Golgi apparatus stress by inhibition of downregulation of secretory pathway Ca(2þ)-ATPase isoform 1 in rats. J Stroke Cerebrovasc Dis 2016;25:1813e22. [9] Kosieradzki M, Rowinski W. Ischemia/reperfusion injury in kidney transplantation: mechanisms and prevention. Transplant Proc 2008;40:3279e88. [10] Mangino MJ, Tian T, Ametani M, et al. Cytoskeletal involvement in hypothermic renal preservation injury. Transplantation 2008;85:427e36. [11] He SQ, Zhang YH, Venugopal SK, et al. Delivery of antioxidative enzyme genes protects against ischemia/reperfusioninduced liver injury in mice. Liver Transpl 2006;12:1869e79. [12] Collard CD, Gelman S. Pathophysiology, clinical manifestations, and prevention of Ischemia-reperfusion injury. Anesthesiology 2001;94:1133e8.
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