International Journal of Surgery 18 (2015) 88e94
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Original research
Is microdialysis useful for early detection of acute rejection after kidney transplantation? Hamidreza Fonouni a, 1, Mohammad Golriz a, 1, Ali Majlesara a, Alireza Faridar a, Majid Esmaeilzadeh a, Parvin Jarahian a, Morva Tahmasbi Rad a, Mohammadreza Hafezi a, Camelia Garoussi a, Stephan Macher-Goeppinger b, Thomas Longerich b, Berk Orakcioglu c, Oliver W. Sakowitz c, Arianeb Mehrabi a, * a
Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany Department of Pathology, University of Heidelberg, Heidelberg, Germany c Department of Neurosurgery, University of Heidelberg, Heidelberg, Germany b
h i g h l i g h t s Kidney transplantation (KTx) is a curative treatment for patients with end-stage renal diseases. Acute rejection is still one of the challenging complications of KTx. The gold standard for diagnosis of acute rejection is renal biopsy which is invasive and time-consuming. Microdialysis (MD) has the capability in monitoring the graft function during the stages of KTx. MD may help to identify the need to immunosuppression adjustment in the early KTx phase.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 February 2015 Accepted 25 March 2015 Available online 9 April 2015
Introduction: Acute rejection following kidney transplantation (KTx) is still one of the challenging complications leading to chronic allograft failure. The aim of this study was to investigate the role of microdialysis (MD) in the early detection of acute graft rejection factor following KTx in porcine model. Methods: Sixteen pigs were randomized after KTx into case (n ¼ 8, without immunosuppressant) and control groups (n ¼ 8, with immunosuppressant). The rejection diagnosis in our groups was confirmed by histopathological evidences as “acute borderline rejection”. Using MD, we monitored the interstitial concentrations of glucose, lactate, pyruvate, glutamate and glycerol in the transplanted grafts after reperfusion. Results: In the early post-reperfusion phase the lactate level in our case group was significantly higher comparing to the control group and remained in higher levels until the end of monitoring. The lactate to pyruvate ratio showed a considerable increase in the case group during the post-reperfusion phase. The other metabolites (glucose, glycerol, glutamate) were nearly at the same levels at the end of our monitoring in both study groups. Conclusion: The increase in lactate and lactate to pyruvate ratios seems to be an indicator for early detection of acute rejection after KTx. Therefore, MD as a minimally invasive measurement tool may help to identify the need to immunosuppression adjustment in the early KTx phase before the clinical manifestation of the rejection. © 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
Keywords: Kidney transplantation Microdialysis Metabolic changes Acute rejection
1. Introduction * Corresponding author. Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany. E-mail address: [email protected] (A. Mehrabi). 1 Both authors contributed equally to this work.
Kidney transplantation (KTx) has nowadays been considered as a curative treatment for patients with end-stage renal diseases. Despite the improvements in histocompatibility testing methods including cross-match as well as progression in
http://dx.doi.org/10.1016/j.ijsu.2015.03.024 1743-9191/© 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
H. Fonouni et al. / International Journal of Surgery 18 (2015) 88e94
immunosuppressive agents and induction therapy, acute rejection is still one of the challenging complications of KTx [1]. The incidence of acute rejection of kidney allograft is approximately 10% [2] which has consistently been reported as the most important risk factor leading to chronic allograft failure [3,4]. Therefore, early detection and immediate treatment can prevent irreversible graft damage and deterioration of long-term graft survival [5,6]. An important, routine and diagnostic sign of acute rejection is the increase in serum creatinine over baseline in an asymptomatic patient but it is a delayed rejection marker [7]. The gold standard for the diagnosis of acute rejection is renal biopsy. This method is an invasive and time-consuming diagnostic method which may delay the initiation of the treatment. Thus, developing of a minimally invasive on-line and on-time method for early detection of rejection seems mandatory for decreasing the risk of graft loss as well as for improving the long term results. Microdialysis (MD) is a technique based on the passive diffusion of substances according to their concentration gradients from the extracellular fluid to the dialysate, by which the biochemical composition of the interstitial fluid in all parenchymatous organs (central nervous system, livers, kidney, etc.) can be detected [8]. It was initially developed in experimental studies, but over the past years found the way to the clinic as well [7,9]. Today, MD is applied almost everywhere in research and clinic and there are several clinical and experimental studies about the application of microdialysis in neurosurgery, reconstructive surgery and liver transplantation [10,11]. It is a safe and easy sampling tool which has been used in pre-clinical as well as clinical studies for continuous monitoring of free, protein-unbound concentrations in extracellular fluids by means of a microdialysis catheter [12e14]. We have reported its capability in monitoring the kidney graft function during the different stages of KTx [15]. The aim of this experimental study is to investigate, if MD could be used for early detection of the acute graft rejection in KTx in porcine model.
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Cl: 156 mmol/L) at a flow rate of 2 ml per minute into the catheter. The outlet tube extends to a microdialysis collector CMA 142 (CMA microdialysis, Sweden) holding the microvials. After collecting the samples, our microvials were stored at 80 C for further assessment. The samples were then analysed by a CMA 600 microdialysis analyser. The measured parameters included: Glucose (mmol/l), Lactate (mmol/l), Pyruvate (mmol/l), Glycerol (mmol/l) and Glutamate (mmol/l). 2.3. Measurement protocol Different samples were collected from both case and control groups during the monitoring period. The Microdialysis samples were collected in 20 min intervals during the 3 h post-reperfusion phase. In each group, in order to standardize our experimental study, blood samples were obtained from donors (1 sample during procurement), and recipients (1 sample before and another sample at the end of the reperfusion phase) to determine haemoglobin, haematocrit, glucose, blood urea nitrogen and creatinine values. This analysis was performed in the central laboratory of our clinic. 2.4. Histopathological evaluations Biopsy samples were taken before and 180 min after reperfusion to discriminate the tissue injury. All biopsy samples were fixed in formaldehyde 5% and analysed by one experienced nephropathologist who was blinded to the randomization. The samples were evaluated to detect the histopathological evidences of “acute rejection” in the grafts based on the Banff classification [17]. Special emphasis was placed on detection of “rejection” in early stages with patchy interstitial infiltration, with or without tubulitis in less than 3 tubular cross-sections [18]. 2.5. Animal rights
2. Materials and methods 2.1. Study design In our study, a group of 16 Landrace pigs with a mean body weight of 29.06 kg (27e32 kg, SD ¼ ±1.97) was used. The animals were divided into two groups based on receiving or not receiving immunosuppressive treatment with methylprednisolone (Urbason, Sanofi-Aventis, Frankfurt, Germany) with the dosage of 250 mg i.v. intraoperatively, which was administrated in our control group (n ¼ 8) before reperfusion but not in the case group (n ¼ 8). During the explantation the organs were perfused with the standard histidineetryptophaneketoglutarate (HTK) solution (Custodiol, Dr. F. Kohler Chemie GmbH, Alsbach-Hahnlein, Germany). After a cold ischaemia time (CIT) of 24 h, the grafts were implanted using a standardized KTx technique as previously published [15,16]. The metabolic changes were monitored within 180 min after reperfusion, using MD (Fig. 1).
The Study protocol was approved by the German Committee for Animal Care, Karlsruhe, Germany (AZ: 35-9185.81/G-113/05). Animals received human care according to the institutional guidelines established for the Animal Care Facility at the University of Heidelberg. Following completion of the experimental protocol, the animals were sacrificed with an intravenous injection of kaliumchloride (2 mmol/kg), in deep anaesthesia. 2.6. Statistics The statistical analysis was performed using SPSS 14.0 (Stata Corp, College Station, Texas, USA) for windows. The variable analysis was performed using T-test when needed. All metabolic parameters were expressed as mean ± standard error of mean (SEM). The laboratory results were expressed as mean ± standard deviation (SD). The P-value of less than 0.05 was considered statistically significant.
2.2. Collecting and analysing of the microdialysis samples 3. Results The MD method has been described completely by the authors [15]. Briefly, A CMA 20 microdialysis catheter (CMA microdialysis, Sweden) was used to collect the samples. The dialysing membrane was 10 mm in length and the catheter membrane cut-off was 20,000 Da. Insertion into the tissue was achieved with the help of a unique slit cannula introducer that left the catheter in place when it was withdrawn. This catheter was connected to a CMA 102 pump (CMA microdialysis, Sweden) by inlet tubes perfused with isotonic, sterile solution T1 (Na: 147 mmol/L, K: 4 mmol/L, Ca: 2.3 mmol/L,
In both study groups the monitored cardiovascular parameters during the implantation and post-reperfusion phase including MAP, CVP heart rate, and body temperature did not show any significant differences. The mean values of the laboratory results are summarized in Table 1. As it is shown, in both study groups during different phases [explantation, warm ischaemia time (WIT) and post-reperfusion] no significant fluctuations could be seen in any of the above mentioned variables.
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Fig. 1. Study design: kidney procurement, preservation, implantation and interstitial metabolites monitoring after reperfusion, using microdialysis in both case and control groups. (CIT: cold ischaemia time, WIT: warm ischaemia time).
Table 1 The mean values of laboratory results in case and control groups during organ procurement and kidney transplantation. Haemoglobin (g/dl) Explantation Warm ischaemia time Post-reperfusion
Control group Case group Control group Case group Control group Case group
9.9 9.6 9.6 9.6 10.4 10.7
± ± ± ± ± ±
0.4 1.0 0.5 1.2 1.2 1.7
3.1. Histopathological analysis In all samples which had been taken 180 min after reperfusion, histopathological evidences of acute rejection were evaluated. In all of our case group samples, patchy interstitial infiltrations and neutrophil margination were reported which represented “acute borderline rejection”, as the early stage of the rejection process. None of these evidences were observed in our control group. 3.2. Monitoring of metabolic changes The glucose (reflecting local blood flow), lactate and pyruvate (representing anaerobic and aerobic metabolism, respectively), glutamate (indicator of cell ischaemia) and glycerol (reflecting cell membrane injury) levels in graft interstitium were monitored after reperfusion and the values were compared in both study groups (the control and case groups) as below:
Haematocrit (%) 31 30 29 30 32 33
± ± ± ± ± ±
2 3 1 4 4 6
Glucose (mg/dl) 106.5 132.4 125.6 153.3 138.8 146.5
± ± ± ± ± ±
Blood urea nitrogen (mg/dl) 33.1 36.9 27.1 62.1 34.4 44.1
32 24.0 16.5 22.7 21.2 29.4
± ± ± ± ± ±
7.4 10.5 5.8 10.5 6.6 8.8
Creatinine (mg/dl) 1.2 1.2 1.1 1.0 1.0 1.0
± ± ± ± ± ±
6.2 0.2 0.1 0.1 0.1 0.1
reperfusion the lactate value in the case group remained significantly higher in comparison to the control group (1.53 ± 0.32 vs. 0.6 ± 0.07 mmol/L; p-value ¼ 0.02). From this point until the end of monitoring, the control group showed nearly a steady state while the case group demonstrated a slight decline. At the end of monitoring, case group had a higher trend of lactate level comparing to the control one without reaching any significance (1.18 ± 0.26 vs. 0.62 ± 0.09 mmol/L; p-value ¼ 0.2) (Fig. 3). 3.2.3. Lactate to pyruvate ratio For both study groups we calculated the lactate to pyruvate ratio. During the first 60 min after reperfusion lactate to pyruvate ratios in both study groups were almost in the same levels (30.78 ± 8.41 vs. 24.39 ± 3.89; p-value ¼ 0.61). Thereafter, this ratio in the case group started to increase sharply in comparison to the control group with no significance (62.08 ± 29.45 vs. 23.41 ± 2.52;
3.2.1. Glucose After reperfusion, a relative higher level of glucose was seen in the case group which demonstrated no significant difference from the control one (Fig. 2). Both groups followed an increasing trend during the next 20 min which was sharper in the case group. This ended to the peaks in different levels in both groups as shown in Fig. 2. During the next 60 min decreasing trends were observed in both study groups which was sharper in the case group. Thereafter, until the end of monitoring the mentioned decreasing trend continued and the levels in two study groups approached each other. In the late reperfusion phase, the glucose levels were approximately the same in both groups (Fig. 2). 3.2.2. Lactate Early after reperfusion the lactate was significantly higher in the case group comparing to the control group (1.92 ± 0.34 vs. 0.84 ± 0.15 mmol/L; p-value ¼ 0.01). Eighty minutes after
Fig. 2. Glucose values measured after reperfusion in our case and control groups using microdialysis.
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2,5
Lactate (mmol/l)
2
1,5
1
0,5
0
20
100
180
Postreperfusion period (180 min) Fig. 3. Lactate values measured after reperfusion in our case and control groups using microdialysis.
p-value ¼ 0.42). Until the end of monitoring, the lactate to pyruvate ratio in the case group remained higher than the control group (75.08 ± 30.78 vs. 18.75 ± 2.78; p-value ¼ 0.2) (Fig. 4).
3.2.4. Glycerol After reperfusion the glycerol level in the case group was lower comparing to the control group (Fig. 5). In the next 20 min, glycerol levels dropped sharply in both groups, still the case group showed lower level comparing to the control one which was not statistically significant. Thereafter both groups followed a steady state near each other and at the end of 180 min monitoring, two groups demonstrated similar levels (Fig. 5).
3.2.5. Glutamate Early after reperfusion the glutamate level in the case group showed a higher level comparing to control group (p-value ¼ 0.36). Both groups decreased sharply in the next 20 min. Still the case group showed a higher level comparing to control one (92.29 ± 7.71 vs. 72.55 ± 3.73 mmol/L; p-value ¼ 0.03). In the next 140 min both groups demonstrated some small fluctuations in approximately the same levels (p-value ¼ 0.68) (Fig. 6).
Fig. 4. Lactate to pyruvate ratios measured after reperfusion in our case and control groups using microdialysis.
Fig. 5. Glycerol values measured after reperfusion in our case and control groups using microdialysis.
4. Discussion Acute rejection with an incidence of 10% after KTx [2] and its potential to end stage renal failure plays an important role in the outcome of graft function. Therefore, early detection and immediate management of acute rejection can prevent irreparable graft damage and deterioration of long-term graft survival [5,6]. We could demonstrate in our previous studies [15,19], that MD can effectively be used for monitoring of the kidney graft and for early detection of graft damages induced by prolonged cold ischaemia time (CIT) as well as vascular thrombosis in early stages of KTx. After transplantation, the microcirculatory impairment besides mitochondrial disturbance, as an early manifestation of acute graft rejection, results in an ischaemic status which can lead to graft metabolic changes [20e23].The present study evaluates the effect of early acute rejection on interstitial metabolites in an experimental KTx model using MD. We use HTK routinely as our preservation solution in the Eurotransplant area. In contrast to some arguments that the kidney transplantation especially with long term cold ischaemia time has a better outcome with University of Wisconsin solution in comparison with HTK, we could show a comparable result to UW solution in maintaining graft function in a long preservation period (>24 h) [24]. Recently, the capability of MD to characterize local metabolic changes in renal grafts and as a
Fig. 6. Glutamate values measured after reperfusion in our case and control groups using microdialysis.
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tool for early ischaemia detection has been demonstrated as well [25,26].According to our knowledge, this study is the first one in evaluating the role of MD in early detection of acute rejection in KTx. In all samples that had been taken from our case group patchy interstitial infiltrations as well as neutrophil margination were seen. These histopathologic evidences were considered as an acute borderline rejection based on Banff classification [17,18,27]. In our study, glucose levels increased considerably after reperfusion in both study groups. This increase depends on the occurrence of hyperperfusion and ‘flow-metabolism’ mismatch [28].Only in our control group corticosteroid was administrated, so the higher level of glucose was expected in this group due to the induced gluconeogenesis and subsequent higher glucose production by glucocorticoids [29e31].Unexpectedly, the glucose level after reperfusion was higher in our case group with no acceptable reason to this event. In the late post-reperfusion phase there were no considerable differences between the glucose levels in both groups. Deteriorated glycolytic enzymatic function in addition to mitochondrial disturbance during acute rejection declined glucose consumption in the graft, leading to higher interstitial glucose values [21,23,32]. On the other hand, the microvascular circulation deficit following the acute graft rejection [33] by decreased glucose supply in the graft interstitium can be an explanation. Hence, decreased glucose consumption as well as lower glucose supply in the graft leads to the unchanged glucose value in the interstitium of rejected grafts. In summary, interstitial glucose values could not be considered as an appropriate marker for early detection of acute rejection using MD. The high lactate value was observed in the both study groups early after reperfusion which was expected due to hypermetabolism [34]. The lactate levels were significantly different between these 2 groups. The lower lactate value in the control group was due to uncoupling effect of glucocorticoid on the mitochondria, reducing the efficacy of oxidative phosphorylation [35]. This will result in increased oxidation rate leading to more pyruvate consumption by oxidative phosphorylation and subsequently less lactate production [36]. The lactate value in our case group was considerably higher until late reperfusion phase comparing to the control group. This could be justified in the presence of ischaemia induced by graft microcirculation impairment, as an early manifestation of acute graft rejection [20], in addition to the disturbance of mitochondrial oxidative metabolism following the rejection process [22,23]. The lactate values may not only be related to hypoxia and ischaemia but also to the hypermetabolism [37]. In a series of studies Haugaa et al. have demonstrated that an increase in lactate levels as well as an increase in the lactate/pyruvate ratio can distinguish acute rejection from ischaemia in liver transplantation [38e40]. Therefore, we determined the lactate to pyruvate ratio as a marker of cell ischaemia [10]. In our study, shortly after reperfusion the lactate to pyruvate ratios were in similar levels in both study groups but in our case group increased considerably in comparison with the control one during the monitoring phase. It could support the theory that increased lactate level early after reperfusion is induced by hypermetabolism and its higher preserved levels in our case group until the end of monitoring, due to the ischaemia following the rejection process. In addition, increased lactate dehydrogenase activity following acute rejection, augments lactate production from pyruvate via anaerobic glycolysis [41]. This event ends to further increase in lactate to pyruvate ratio at the end of monitoring in our case group. Waelgaard et al. [42] have reported the increased lactate level in the liver grafts rejection which was compatible to our findings in the kidney grafts. It could be concluded that lactate level have the potential to be considered as markers for early detection of graft rejection. Glycerol, an integral component of the cell membrane, has been
recognized as one of the important markers of cell injury [42,43]. Corticosteroid reduces the ischaemia reperfusion injury [44] which could result in reduced liberation of the glycerol to the interstitium after reperfusion [44,45]. In our study, glycerol levels in both case and control groups were not significantly different in early postreperfusion phase. Forty minutes after reperfusion, glycerol levels in both groups showed a very sharp decline. This may be caused by the washout effect, in which disintegrated cell membrane compounds are transported away by the systemic circulation [46]. Thereafter, until the end of monitoring, the glycerol levels in interstitium did not increase during the early rejection process. According to previous studies, glycerol is a delayed marker of ischaemic injury [47,48], so its changes during rejection process might be revealed in a longer monitoring time. Glutamate has been considered as a marker of bioenergetic insufficiency [34]. In our study the level of glutamate was higher in our case group which had not received corticosteroids. Steroids have anti-inflammatory functions, such as inhibiting phospholipase A2, and preventing synthesis of arachidonic acid [49]. It has been stated that even a brief exposure to arachidonic acid produces a prolonged inhibition of glutamate uptake [50]. Therefore, the lower glutamate level in control group early after reperfusion could be due to lower arachidonic acid production following corticosteroid administration. Forty minutes after reperfusion, the glutamate level declined sharply in our both study groups. This could be explained by a reactivated glutamine synthetase thanks to increased oxygen delivery and ATP production [51,52] as well as the recovered function of the glutamate carriers [52]. From this point until the end of monitoring the glutamate level were in near steady state in both study groups. Thus in our study, glutamate has not showed a characteristic to be considered as a marker for rejection process. In conclusion, MD as a minimally invasive measurement method has the potential to monitor interstitial metabolic changes in transplanted kidney graft and could be appropriate for early detection of acute graft rejection in KTx. The increase in lactate values looks to be an indicator for in-time detection of the early rejection process after KTx. Therefore, using MD may help us to identify the patients who will most likely need an immunosuppression adjustment before the episode of clinical rejection. Further studies to evaluate the potency of MD in detection of acute rejection after KTx for example by the assessment of cytokines in a prolonged monitoring period have a great worth. Disclosure All the authors declared no potential conflicts of interests with respect to the authorship and/or publication of this article. They had no personal relationships with other people or organizations that could potentially and inappropriately influence their work and conclusions. Ethical approval The Study protocol was approved by the German Committee for Animal Care, Karlsruhe, Germany (AZ: 35-9185.81/G-113/05). Funding The study has been supported by a grant of “Heidelberger Stiftung Chirurgie”. Author contribution H. Fonouni: conception and design, data collection, writing the
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article, critical revision of the article. M. Golriz: conception and design, data collection, writing the article, critical revision of the article. A. Majlesara: data collection, analysis and interpretation, writing the article, critical revision of the article. A. Faridar: data collection, analysis and interpretation, drafting the manuscript. M. Esmaeilzadeh: data collection, analysis and interpretation, drafting the manuscript. P. Jarahian: data collection, drafting the manuscript, drafting the manuscript. M. Tahmasbi Rad: data collection, analysis and interpretation, drafting the manuscript. M. Hafezi: data collection, analysis and interpretation. C. Garoussi: data collection, analysis and interpretation. S. Macher-Goeppinger: performing pathological assessment, data collection and interpretation. T. Longerich: performing pathological assessment, data collection and interpretation. B. Orakcioglu: involving in study concept and revising the manuscript for critically intellectual content. O. W. Sakowitz: involving in study concept and revising the manuscript for critically intellectual content. A. Mehrabi: conception and design, critical revision of the article, giving the final approval for publication. Guarantor Dr. med. A. Mehrabi, FEBS, FICS. Attending General and Transplant Surgeon. Department of General, Visceral and Transplantation Surgery. University of Heidelberg, Heidelberg, Germany. Tel.: 0049 6221 5636223. Fax: 0049 6221 567470. E-Mail: [email protected]. Research registration unique identifying number (UIN) This is an experimental study and doesn't have any unique identifying number. Acknowledgements We are very grateful that this publication has been supported by a grant of “Heidelberger Stiftung Chirurgie”. References [1] A. Ishikawa, S.M. Flechner, D.A. Goldfarb, J.L. Myles, C.S. Modlin, N. Boparai, et al., Quantitative assessment of the first acute rejection as a predictor of renal transplant outcome, Transplantation 68 (Nov 15 1999) 1318e1324. [2] United States Renal Data System, 2009. Available: WWW.USRDS.org (accessed on May, 2009). [3] S.M. Flechner, C.S. Modlin, D.P. Serrano, D.A. Goldfarb, D. Papajcik, B. Mastroianni, et al., Determinants of chronic renal allograft rejection in cyclosporine-treated recipients, Transplantation 62 (Nov 15 1996) 1235e1241. [4] A.J. McLaren, S.V. Fuggle, K.I. Welsh, D.W. Gray, P.J. Morris, Chronic allograft failure in human renal transplantation: a multivariate risk factor analysis, Ann Surg 232 (Jul 2000) 98e103. [5] R.L. Madden, J.G. Mulhern, B.J. Benedetto, M.H. O'Shea, M.J. Germain, G.L. Braden, et al., Completely reversed acute rejection is not a significant risk factor for the development of chronic rejection in renal allograft recipients, Transpl. Int. 13 (2000) 344e350. [6] P. Vereerstraeten, D. Abramowicz, L. de Pauw, P. Kinnaert, Absence of deleterious effect on long-term kidney graft survival of rejection episodes with complete functional recovery, Transplantation 63 (Jun 27 1997) 1739e1743. [7] K.J. Weld, C. Montiglio, A.C. Bush, H.H. Harroff, R.D. Cespedes, Real-time analysis of renal interstitial metabolites during induced renal ischemia, J. Endourol. 22 (Mar 2008) 571e574.
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Chakkera, E.J. Weil, J. Castro, R.L. Heilman, K.S. Reddy, M.J. Mazur, et al., Hyperglycemia during the immediate period after kidney transplantation, Clin. J. Am. Soc. Nephrol. 4 (Apr 2009) 853e859. [30] N. Kraus-Friedmann, Hormonal regulation of hepatic gluconeogenesis, Physiol. Rev. 64 (Jan 1984) 170e259. [31] J. Wyzgal, L. Paczek, J. Ziolkowski, M. Pawlowska, W. Rowinski, M. Durlik, Early hyperglycemia after allogenic kidney transplantation, Ann. Transpl. 12 (2007) 40e45. [32] E.B. Maneus, J.L. Pomar-Moya, F. Climent, P.P. de la Ossa, Glycolytic enzyme activities are decreased during acute rejection in transplanted rat hearts, Transpl. Proc. 37 (Nov 2005) 4122e4123. [33] K. Matsuo, S. Takebayashi, Ultrastructure of microcirculation in renal grafts in patients with acute or chronic rejections, Acta Pathol. Jpn. 35 (Sep 1985) 1123e1134.
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[34] J.C. Goodman, C.S. Robertson, Microdialysis: is it ready for prime time? Curr. Opin. Crit. Care 15 (Apr 2009) 110e117. [35] V. Desquiret, N. Gueguen, Y. Malthiery, P. Ritz, G. Simard, Mitochondrial effects of dexamethasone imply both membrane and cytosolic-initiated pathways in HepG2 cells, Int. J. Biochem. Cell Biol. 40 (2008) 1629e1641. [36] B. Sibille, C. Filippi, M.A. Piquet, P. Leclercq, E. Fontaine, X. Ronot, et al., The mitochondrial consequences of uncoupling intact cells depend on the nature of the exogenous substrate, Biochem. J. 355 (Apr 1 2001) 231e235. [37] A.H. Harken, Lactic acidosis, Surg. Gynecol. Obstet. 142 (Apr 1976) 593e606. [38] H. Haugaa, R. Almaas, E.B. Thorgersen, A. Foss, P.D. Line, T. Sanengen, et al., Clinical experience with microdialysis catheters in pediatric liver transplants, Liver Transpl. 19 (Mar 2013) 305e314. [39] H. Haugaa, E.B. Thorgersen, A. Pharo, K.M. Boberg, A. Foss, P.D. Line, et al., Early bedside detection of ischemia and rejection in liver transplants by microdialysis, Liver Transpl. 18 (Jul 2012) 839e849. [40] H. Haugaa, E.B. Thorgersen, A. Pharo, K.M. Boberg, A. Foss, P.D. Line, et al., Inflammatory markers sampled by microdialysis catheters distinguish rejection from ischemia in liver grafts, Liver Transpl. 18 (Dec 2012) 1421e1429. [41] N.A. Lopatkin, Y.M. Lopukhin, V.D. Shemetov, Activity of certain enzymes in cadaver renal transplants, Int. Urol. Nephrol. 3 (1971) 61e65. [42] L. Waelgaard, E.B. Thorgersen, P.D. Line, A. Foss, T.E. Mollnes, T.I. Tonnessen, Microdialysis monitoring of liver grafts by metabolic parameters, cytokine production, and complement activation, Transplantation 86 (Oct 27 2008) 1096e1103. [43] O.G. Nilsson, L. Brandt, U. Ungerstedt, H. Saveland, Bedside detection of brain ischemia using intracerebral microdialysis: subarachnoid hemorrhage and delayed ischemic deterioration, Neurosurgery 45 (Nov 1999) 1176e1184 discussion 1184-5.
[44] R.F. Saidi, J. Chang, S. Verb, S. Brooks, I. Nalbantoglu, V. Adsay, et al., The effect of methylprednisolone on warm ischemia-reperfusion injury in the liver, Am. J. Surg. 193 (Mar 2007) 345e347 discussion 347-8. [45] P.H. Chan, R.A. Fishman, The role of arachidonic acid in vasogenic brain edema, Fed. Proc. 43 (Feb 1984) 210e213. [46] G. Nowak, J. Ungerstedt, A. Wernerson, U. Ungerstedt, B.G. Ericzon, Hepatic cell membrane damage during cold preservation sensitizes liver grafts to rewarming injury, J. Hepatobiliary Pancreat. Surg. 10 (2003) 200e205. [47] A.K. Keller, T.M. Jorgensen, L.H. Olsen, L.B. Stolle, Early detection of renal ischemia by in situ microdialysis: an experimental study, J. Urol. 179 (Jan 2008) 371e375. [48] A.K. Keller, T.M. Jorgensen, L.H. Olsen, L.B. Stolle, Detection of local metabolic changes after progressive and stepwise reduction of renal blood flow in pigs, Transpl. Proc. 41 (Jan-Feb 2009) 44e48. [49] G. Willemart, K.R. Knight, M. Ayad, M. Wagh, W.A. Morrison, The beneficial antiinflammatory effect of dexamethasone administration prior to reperfusion on the viability of cold-stored skin flaps, Int. J. Tissue React. 21 (1999) 71e78. [50] H. Hagberg, A. Lehmann, M. Sandberg, B. Nystrom, I. Jacobson, A. Hamberger, Ischemia-induced shift of inhibitory and excitatory amino acids from intra- to extracellular compartments, J. Cereb. Blood Flow. Metab. 5 (Sep 1985) 413e419. [51] A. Mehrabi, M. Golling, C. Busch, B. Hashemi, R. Ahmadi, A. Volkl, et al., Experimental monitoring of hepatic glucose, lactate, and glutamate metabolism by microdialysis during surgical preparation of the liver hilus, J. Surg. Res. 105 (Jun 15 2002) 128e135. [52] E.A. Newsholme, J.R. Arch, B. Brooks, B. Surholt, The role of substrate cycles in metabolic regulation, Biochem. Soc. Trans. 11 (Jan 1983) 52e56.
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Original research
Is microdialysis useful for early detection of acute rejection after kidney transplantation? Hamidreza Fonouni a, 1, Mohammad Golriz a, 1, Ali Majlesara a, Alireza Faridar a, Majid Esmaeilzadeh a, Parvin Jarahian a, Morva Tahmasbi Rad a, Mohammadreza Hafezi a, Camelia Garoussi a, Stephan Macher-Goeppinger b, Thomas Longerich b, Berk Orakcioglu c, Oliver W. Sakowitz c, Arianeb Mehrabi a, * a
Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany Department of Pathology, University of Heidelberg, Heidelberg, Germany c Department of Neurosurgery, University of Heidelberg, Heidelberg, Germany b
h i g h l i g h t s Kidney transplantation (KTx) is a curative treatment for patients with end-stage renal diseases. Acute rejection is still one of the challenging complications of KTx. The gold standard for diagnosis of acute rejection is renal biopsy which is invasive and time-consuming. Microdialysis (MD) has the capability in monitoring the graft function during the stages of KTx. MD may help to identify the need to immunosuppression adjustment in the early KTx phase.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 February 2015 Accepted 25 March 2015 Available online 9 April 2015
Introduction: Acute rejection following kidney transplantation (KTx) is still one of the challenging complications leading to chronic allograft failure. The aim of this study was to investigate the role of microdialysis (MD) in the early detection of acute graft rejection factor following KTx in porcine model. Methods: Sixteen pigs were randomized after KTx into case (n ¼ 8, without immunosuppressant) and control groups (n ¼ 8, with immunosuppressant). The rejection diagnosis in our groups was confirmed by histopathological evidences as “acute borderline rejection”. Using MD, we monitored the interstitial concentrations of glucose, lactate, pyruvate, glutamate and glycerol in the transplanted grafts after reperfusion. Results: In the early post-reperfusion phase the lactate level in our case group was significantly higher comparing to the control group and remained in higher levels until the end of monitoring. The lactate to pyruvate ratio showed a considerable increase in the case group during the post-reperfusion phase. The other metabolites (glucose, glycerol, glutamate) were nearly at the same levels at the end of our monitoring in both study groups. Conclusion: The increase in lactate and lactate to pyruvate ratios seems to be an indicator for early detection of acute rejection after KTx. Therefore, MD as a minimally invasive measurement tool may help to identify the need to immunosuppression adjustment in the early KTx phase before the clinical manifestation of the rejection. © 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
Keywords: Kidney transplantation Microdialysis Metabolic changes Acute rejection
1. Introduction * Corresponding author. Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany. E-mail address: [email protected] (A. Mehrabi). 1 Both authors contributed equally to this work.
Kidney transplantation (KTx) has nowadays been considered as a curative treatment for patients with end-stage renal diseases. Despite the improvements in histocompatibility testing methods including cross-match as well as progression in
http://dx.doi.org/10.1016/j.ijsu.2015.03.024 1743-9191/© 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
H. Fonouni et al. / International Journal of Surgery 18 (2015) 88e94
immunosuppressive agents and induction therapy, acute rejection is still one of the challenging complications of KTx [1]. The incidence of acute rejection of kidney allograft is approximately 10% [2] which has consistently been reported as the most important risk factor leading to chronic allograft failure [3,4]. Therefore, early detection and immediate treatment can prevent irreversible graft damage and deterioration of long-term graft survival [5,6]. An important, routine and diagnostic sign of acute rejection is the increase in serum creatinine over baseline in an asymptomatic patient but it is a delayed rejection marker [7]. The gold standard for the diagnosis of acute rejection is renal biopsy. This method is an invasive and time-consuming diagnostic method which may delay the initiation of the treatment. Thus, developing of a minimally invasive on-line and on-time method for early detection of rejection seems mandatory for decreasing the risk of graft loss as well as for improving the long term results. Microdialysis (MD) is a technique based on the passive diffusion of substances according to their concentration gradients from the extracellular fluid to the dialysate, by which the biochemical composition of the interstitial fluid in all parenchymatous organs (central nervous system, livers, kidney, etc.) can be detected [8]. It was initially developed in experimental studies, but over the past years found the way to the clinic as well [7,9]. Today, MD is applied almost everywhere in research and clinic and there are several clinical and experimental studies about the application of microdialysis in neurosurgery, reconstructive surgery and liver transplantation [10,11]. It is a safe and easy sampling tool which has been used in pre-clinical as well as clinical studies for continuous monitoring of free, protein-unbound concentrations in extracellular fluids by means of a microdialysis catheter [12e14]. We have reported its capability in monitoring the kidney graft function during the different stages of KTx [15]. The aim of this experimental study is to investigate, if MD could be used for early detection of the acute graft rejection in KTx in porcine model.
89
Cl: 156 mmol/L) at a flow rate of 2 ml per minute into the catheter. The outlet tube extends to a microdialysis collector CMA 142 (CMA microdialysis, Sweden) holding the microvials. After collecting the samples, our microvials were stored at 80 C for further assessment. The samples were then analysed by a CMA 600 microdialysis analyser. The measured parameters included: Glucose (mmol/l), Lactate (mmol/l), Pyruvate (mmol/l), Glycerol (mmol/l) and Glutamate (mmol/l). 2.3. Measurement protocol Different samples were collected from both case and control groups during the monitoring period. The Microdialysis samples were collected in 20 min intervals during the 3 h post-reperfusion phase. In each group, in order to standardize our experimental study, blood samples were obtained from donors (1 sample during procurement), and recipients (1 sample before and another sample at the end of the reperfusion phase) to determine haemoglobin, haematocrit, glucose, blood urea nitrogen and creatinine values. This analysis was performed in the central laboratory of our clinic. 2.4. Histopathological evaluations Biopsy samples were taken before and 180 min after reperfusion to discriminate the tissue injury. All biopsy samples were fixed in formaldehyde 5% and analysed by one experienced nephropathologist who was blinded to the randomization. The samples were evaluated to detect the histopathological evidences of “acute rejection” in the grafts based on the Banff classification [17]. Special emphasis was placed on detection of “rejection” in early stages with patchy interstitial infiltration, with or without tubulitis in less than 3 tubular cross-sections [18]. 2.5. Animal rights
2. Materials and methods 2.1. Study design In our study, a group of 16 Landrace pigs with a mean body weight of 29.06 kg (27e32 kg, SD ¼ ±1.97) was used. The animals were divided into two groups based on receiving or not receiving immunosuppressive treatment with methylprednisolone (Urbason, Sanofi-Aventis, Frankfurt, Germany) with the dosage of 250 mg i.v. intraoperatively, which was administrated in our control group (n ¼ 8) before reperfusion but not in the case group (n ¼ 8). During the explantation the organs were perfused with the standard histidineetryptophaneketoglutarate (HTK) solution (Custodiol, Dr. F. Kohler Chemie GmbH, Alsbach-Hahnlein, Germany). After a cold ischaemia time (CIT) of 24 h, the grafts were implanted using a standardized KTx technique as previously published [15,16]. The metabolic changes were monitored within 180 min after reperfusion, using MD (Fig. 1).
The Study protocol was approved by the German Committee for Animal Care, Karlsruhe, Germany (AZ: 35-9185.81/G-113/05). Animals received human care according to the institutional guidelines established for the Animal Care Facility at the University of Heidelberg. Following completion of the experimental protocol, the animals were sacrificed with an intravenous injection of kaliumchloride (2 mmol/kg), in deep anaesthesia. 2.6. Statistics The statistical analysis was performed using SPSS 14.0 (Stata Corp, College Station, Texas, USA) for windows. The variable analysis was performed using T-test when needed. All metabolic parameters were expressed as mean ± standard error of mean (SEM). The laboratory results were expressed as mean ± standard deviation (SD). The P-value of less than 0.05 was considered statistically significant.
2.2. Collecting and analysing of the microdialysis samples 3. Results The MD method has been described completely by the authors [15]. Briefly, A CMA 20 microdialysis catheter (CMA microdialysis, Sweden) was used to collect the samples. The dialysing membrane was 10 mm in length and the catheter membrane cut-off was 20,000 Da. Insertion into the tissue was achieved with the help of a unique slit cannula introducer that left the catheter in place when it was withdrawn. This catheter was connected to a CMA 102 pump (CMA microdialysis, Sweden) by inlet tubes perfused with isotonic, sterile solution T1 (Na: 147 mmol/L, K: 4 mmol/L, Ca: 2.3 mmol/L,
In both study groups the monitored cardiovascular parameters during the implantation and post-reperfusion phase including MAP, CVP heart rate, and body temperature did not show any significant differences. The mean values of the laboratory results are summarized in Table 1. As it is shown, in both study groups during different phases [explantation, warm ischaemia time (WIT) and post-reperfusion] no significant fluctuations could be seen in any of the above mentioned variables.
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Fig. 1. Study design: kidney procurement, preservation, implantation and interstitial metabolites monitoring after reperfusion, using microdialysis in both case and control groups. (CIT: cold ischaemia time, WIT: warm ischaemia time).
Table 1 The mean values of laboratory results in case and control groups during organ procurement and kidney transplantation. Haemoglobin (g/dl) Explantation Warm ischaemia time Post-reperfusion
Control group Case group Control group Case group Control group Case group
9.9 9.6 9.6 9.6 10.4 10.7
± ± ± ± ± ±
0.4 1.0 0.5 1.2 1.2 1.7
3.1. Histopathological analysis In all samples which had been taken 180 min after reperfusion, histopathological evidences of acute rejection were evaluated. In all of our case group samples, patchy interstitial infiltrations and neutrophil margination were reported which represented “acute borderline rejection”, as the early stage of the rejection process. None of these evidences were observed in our control group. 3.2. Monitoring of metabolic changes The glucose (reflecting local blood flow), lactate and pyruvate (representing anaerobic and aerobic metabolism, respectively), glutamate (indicator of cell ischaemia) and glycerol (reflecting cell membrane injury) levels in graft interstitium were monitored after reperfusion and the values were compared in both study groups (the control and case groups) as below:
Haematocrit (%) 31 30 29 30 32 33
± ± ± ± ± ±
2 3 1 4 4 6
Glucose (mg/dl) 106.5 132.4 125.6 153.3 138.8 146.5
± ± ± ± ± ±
Blood urea nitrogen (mg/dl) 33.1 36.9 27.1 62.1 34.4 44.1
32 24.0 16.5 22.7 21.2 29.4
± ± ± ± ± ±
7.4 10.5 5.8 10.5 6.6 8.8
Creatinine (mg/dl) 1.2 1.2 1.1 1.0 1.0 1.0
± ± ± ± ± ±
6.2 0.2 0.1 0.1 0.1 0.1
reperfusion the lactate value in the case group remained significantly higher in comparison to the control group (1.53 ± 0.32 vs. 0.6 ± 0.07 mmol/L; p-value ¼ 0.02). From this point until the end of monitoring, the control group showed nearly a steady state while the case group demonstrated a slight decline. At the end of monitoring, case group had a higher trend of lactate level comparing to the control one without reaching any significance (1.18 ± 0.26 vs. 0.62 ± 0.09 mmol/L; p-value ¼ 0.2) (Fig. 3). 3.2.3. Lactate to pyruvate ratio For both study groups we calculated the lactate to pyruvate ratio. During the first 60 min after reperfusion lactate to pyruvate ratios in both study groups were almost in the same levels (30.78 ± 8.41 vs. 24.39 ± 3.89; p-value ¼ 0.61). Thereafter, this ratio in the case group started to increase sharply in comparison to the control group with no significance (62.08 ± 29.45 vs. 23.41 ± 2.52;
3.2.1. Glucose After reperfusion, a relative higher level of glucose was seen in the case group which demonstrated no significant difference from the control one (Fig. 2). Both groups followed an increasing trend during the next 20 min which was sharper in the case group. This ended to the peaks in different levels in both groups as shown in Fig. 2. During the next 60 min decreasing trends were observed in both study groups which was sharper in the case group. Thereafter, until the end of monitoring the mentioned decreasing trend continued and the levels in two study groups approached each other. In the late reperfusion phase, the glucose levels were approximately the same in both groups (Fig. 2). 3.2.2. Lactate Early after reperfusion the lactate was significantly higher in the case group comparing to the control group (1.92 ± 0.34 vs. 0.84 ± 0.15 mmol/L; p-value ¼ 0.01). Eighty minutes after
Fig. 2. Glucose values measured after reperfusion in our case and control groups using microdialysis.
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2,5
Lactate (mmol/l)
2
1,5
1
0,5
0
20
100
180
Postreperfusion period (180 min) Fig. 3. Lactate values measured after reperfusion in our case and control groups using microdialysis.
p-value ¼ 0.42). Until the end of monitoring, the lactate to pyruvate ratio in the case group remained higher than the control group (75.08 ± 30.78 vs. 18.75 ± 2.78; p-value ¼ 0.2) (Fig. 4).
3.2.4. Glycerol After reperfusion the glycerol level in the case group was lower comparing to the control group (Fig. 5). In the next 20 min, glycerol levels dropped sharply in both groups, still the case group showed lower level comparing to the control one which was not statistically significant. Thereafter both groups followed a steady state near each other and at the end of 180 min monitoring, two groups demonstrated similar levels (Fig. 5).
3.2.5. Glutamate Early after reperfusion the glutamate level in the case group showed a higher level comparing to control group (p-value ¼ 0.36). Both groups decreased sharply in the next 20 min. Still the case group showed a higher level comparing to control one (92.29 ± 7.71 vs. 72.55 ± 3.73 mmol/L; p-value ¼ 0.03). In the next 140 min both groups demonstrated some small fluctuations in approximately the same levels (p-value ¼ 0.68) (Fig. 6).
Fig. 4. Lactate to pyruvate ratios measured after reperfusion in our case and control groups using microdialysis.
Fig. 5. Glycerol values measured after reperfusion in our case and control groups using microdialysis.
4. Discussion Acute rejection with an incidence of 10% after KTx [2] and its potential to end stage renal failure plays an important role in the outcome of graft function. Therefore, early detection and immediate management of acute rejection can prevent irreparable graft damage and deterioration of long-term graft survival [5,6]. We could demonstrate in our previous studies [15,19], that MD can effectively be used for monitoring of the kidney graft and for early detection of graft damages induced by prolonged cold ischaemia time (CIT) as well as vascular thrombosis in early stages of KTx. After transplantation, the microcirculatory impairment besides mitochondrial disturbance, as an early manifestation of acute graft rejection, results in an ischaemic status which can lead to graft metabolic changes [20e23].The present study evaluates the effect of early acute rejection on interstitial metabolites in an experimental KTx model using MD. We use HTK routinely as our preservation solution in the Eurotransplant area. In contrast to some arguments that the kidney transplantation especially with long term cold ischaemia time has a better outcome with University of Wisconsin solution in comparison with HTK, we could show a comparable result to UW solution in maintaining graft function in a long preservation period (>24 h) [24]. Recently, the capability of MD to characterize local metabolic changes in renal grafts and as a
Fig. 6. Glutamate values measured after reperfusion in our case and control groups using microdialysis.
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tool for early ischaemia detection has been demonstrated as well [25,26].According to our knowledge, this study is the first one in evaluating the role of MD in early detection of acute rejection in KTx. In all samples that had been taken from our case group patchy interstitial infiltrations as well as neutrophil margination were seen. These histopathologic evidences were considered as an acute borderline rejection based on Banff classification [17,18,27]. In our study, glucose levels increased considerably after reperfusion in both study groups. This increase depends on the occurrence of hyperperfusion and ‘flow-metabolism’ mismatch [28].Only in our control group corticosteroid was administrated, so the higher level of glucose was expected in this group due to the induced gluconeogenesis and subsequent higher glucose production by glucocorticoids [29e31].Unexpectedly, the glucose level after reperfusion was higher in our case group with no acceptable reason to this event. In the late post-reperfusion phase there were no considerable differences between the glucose levels in both groups. Deteriorated glycolytic enzymatic function in addition to mitochondrial disturbance during acute rejection declined glucose consumption in the graft, leading to higher interstitial glucose values [21,23,32]. On the other hand, the microvascular circulation deficit following the acute graft rejection [33] by decreased glucose supply in the graft interstitium can be an explanation. Hence, decreased glucose consumption as well as lower glucose supply in the graft leads to the unchanged glucose value in the interstitium of rejected grafts. In summary, interstitial glucose values could not be considered as an appropriate marker for early detection of acute rejection using MD. The high lactate value was observed in the both study groups early after reperfusion which was expected due to hypermetabolism [34]. The lactate levels were significantly different between these 2 groups. The lower lactate value in the control group was due to uncoupling effect of glucocorticoid on the mitochondria, reducing the efficacy of oxidative phosphorylation [35]. This will result in increased oxidation rate leading to more pyruvate consumption by oxidative phosphorylation and subsequently less lactate production [36]. The lactate value in our case group was considerably higher until late reperfusion phase comparing to the control group. This could be justified in the presence of ischaemia induced by graft microcirculation impairment, as an early manifestation of acute graft rejection [20], in addition to the disturbance of mitochondrial oxidative metabolism following the rejection process [22,23]. The lactate values may not only be related to hypoxia and ischaemia but also to the hypermetabolism [37]. In a series of studies Haugaa et al. have demonstrated that an increase in lactate levels as well as an increase in the lactate/pyruvate ratio can distinguish acute rejection from ischaemia in liver transplantation [38e40]. Therefore, we determined the lactate to pyruvate ratio as a marker of cell ischaemia [10]. In our study, shortly after reperfusion the lactate to pyruvate ratios were in similar levels in both study groups but in our case group increased considerably in comparison with the control one during the monitoring phase. It could support the theory that increased lactate level early after reperfusion is induced by hypermetabolism and its higher preserved levels in our case group until the end of monitoring, due to the ischaemia following the rejection process. In addition, increased lactate dehydrogenase activity following acute rejection, augments lactate production from pyruvate via anaerobic glycolysis [41]. This event ends to further increase in lactate to pyruvate ratio at the end of monitoring in our case group. Waelgaard et al. [42] have reported the increased lactate level in the liver grafts rejection which was compatible to our findings in the kidney grafts. It could be concluded that lactate level have the potential to be considered as markers for early detection of graft rejection. Glycerol, an integral component of the cell membrane, has been
recognized as one of the important markers of cell injury [42,43]. Corticosteroid reduces the ischaemia reperfusion injury [44] which could result in reduced liberation of the glycerol to the interstitium after reperfusion [44,45]. In our study, glycerol levels in both case and control groups were not significantly different in early postreperfusion phase. Forty minutes after reperfusion, glycerol levels in both groups showed a very sharp decline. This may be caused by the washout effect, in which disintegrated cell membrane compounds are transported away by the systemic circulation [46]. Thereafter, until the end of monitoring, the glycerol levels in interstitium did not increase during the early rejection process. According to previous studies, glycerol is a delayed marker of ischaemic injury [47,48], so its changes during rejection process might be revealed in a longer monitoring time. Glutamate has been considered as a marker of bioenergetic insufficiency [34]. In our study the level of glutamate was higher in our case group which had not received corticosteroids. Steroids have anti-inflammatory functions, such as inhibiting phospholipase A2, and preventing synthesis of arachidonic acid [49]. It has been stated that even a brief exposure to arachidonic acid produces a prolonged inhibition of glutamate uptake [50]. Therefore, the lower glutamate level in control group early after reperfusion could be due to lower arachidonic acid production following corticosteroid administration. Forty minutes after reperfusion, the glutamate level declined sharply in our both study groups. This could be explained by a reactivated glutamine synthetase thanks to increased oxygen delivery and ATP production [51,52] as well as the recovered function of the glutamate carriers [52]. From this point until the end of monitoring the glutamate level were in near steady state in both study groups. Thus in our study, glutamate has not showed a characteristic to be considered as a marker for rejection process. In conclusion, MD as a minimally invasive measurement method has the potential to monitor interstitial metabolic changes in transplanted kidney graft and could be appropriate for early detection of acute graft rejection in KTx. The increase in lactate values looks to be an indicator for in-time detection of the early rejection process after KTx. Therefore, using MD may help us to identify the patients who will most likely need an immunosuppression adjustment before the episode of clinical rejection. Further studies to evaluate the potency of MD in detection of acute rejection after KTx for example by the assessment of cytokines in a prolonged monitoring period have a great worth. Disclosure All the authors declared no potential conflicts of interests with respect to the authorship and/or publication of this article. They had no personal relationships with other people or organizations that could potentially and inappropriately influence their work and conclusions. Ethical approval The Study protocol was approved by the German Committee for Animal Care, Karlsruhe, Germany (AZ: 35-9185.81/G-113/05). Funding The study has been supported by a grant of “Heidelberger Stiftung Chirurgie”. Author contribution H. Fonouni: conception and design, data collection, writing the
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article, critical revision of the article. M. Golriz: conception and design, data collection, writing the article, critical revision of the article. A. Majlesara: data collection, analysis and interpretation, writing the article, critical revision of the article. A. Faridar: data collection, analysis and interpretation, drafting the manuscript. M. Esmaeilzadeh: data collection, analysis and interpretation, drafting the manuscript. P. Jarahian: data collection, drafting the manuscript, drafting the manuscript. M. Tahmasbi Rad: data collection, analysis and interpretation, drafting the manuscript. M. Hafezi: data collection, analysis and interpretation. C. Garoussi: data collection, analysis and interpretation. S. Macher-Goeppinger: performing pathological assessment, data collection and interpretation. T. Longerich: performing pathological assessment, data collection and interpretation. B. Orakcioglu: involving in study concept and revising the manuscript for critically intellectual content. O. W. Sakowitz: involving in study concept and revising the manuscript for critically intellectual content. A. Mehrabi: conception and design, critical revision of the article, giving the final approval for publication. Guarantor Dr. med. A. Mehrabi, FEBS, FICS. Attending General and Transplant Surgeon. Department of General, Visceral and Transplantation Surgery. University of Heidelberg, Heidelberg, Germany. Tel.: 0049 6221 5636223. Fax: 0049 6221 567470. E-Mail: [email protected]. Research registration unique identifying number (UIN) This is an experimental study and doesn't have any unique identifying number. Acknowledgements We are very grateful that this publication has been supported by a grant of “Heidelberger Stiftung Chirurgie”. References [1] A. Ishikawa, S.M. Flechner, D.A. Goldfarb, J.L. Myles, C.S. Modlin, N. Boparai, et al., Quantitative assessment of the first acute rejection as a predictor of renal transplant outcome, Transplantation 68 (Nov 15 1999) 1318e1324. [2] United States Renal Data System, 2009. Available: WWW.USRDS.org (accessed on May, 2009). [3] S.M. Flechner, C.S. Modlin, D.P. Serrano, D.A. Goldfarb, D. Papajcik, B. Mastroianni, et al., Determinants of chronic renal allograft rejection in cyclosporine-treated recipients, Transplantation 62 (Nov 15 1996) 1235e1241. [4] A.J. McLaren, S.V. Fuggle, K.I. Welsh, D.W. Gray, P.J. Morris, Chronic allograft failure in human renal transplantation: a multivariate risk factor analysis, Ann Surg 232 (Jul 2000) 98e103. [5] R.L. Madden, J.G. Mulhern, B.J. Benedetto, M.H. O'Shea, M.J. Germain, G.L. Braden, et al., Completely reversed acute rejection is not a significant risk factor for the development of chronic rejection in renal allograft recipients, Transpl. Int. 13 (2000) 344e350. [6] P. Vereerstraeten, D. Abramowicz, L. de Pauw, P. Kinnaert, Absence of deleterious effect on long-term kidney graft survival of rejection episodes with complete functional recovery, Transplantation 63 (Jun 27 1997) 1739e1743. [7] K.J. Weld, C. Montiglio, A.C. Bush, H.H. Harroff, R.D. Cespedes, Real-time analysis of renal interstitial metabolites during induced renal ischemia, J. Endourol. 22 (Mar 2008) 571e574.
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![[Color-coded Doppler ultrasonography in early detection of rejection reactions after allogeneic kidney transplantation].](/assets/img/hold.png)
