- Email: [email protected]
In Vivo 31P MR Spectroscopy of Human Kidney Grafts Using the 2D-Chemical Shift Imaging Method
In Vivo 31P MR Spectroscopy of Human Kidney Grafts Using the 2D-Chemical Shift Imaging Method P. Vyhnanovská, M. Dezortová, V. Herynek, P. Táborský, O. Viklický, and M. Hájek ABSTRACT The aim of this work was to evaluate the possibility to use in vivo 31P magnetic resonance spectroscopy (MRS) for the diagnosis of kidney graft dysfunction after transplantation. We examined 68 kidney grafted patients using a 1.5 T MR scanner. 31P MRS was performed using the 2D-chemical shift imaging method. The patients were divided into 4 groups: acute rejection episode; acute tubular necrosis; late graft dysfunction; or good renal function. We measured the signal intensities of phosphomonoesters (PME), inorganic phosphate (Pi), phosphodiesters (PDE), and ␣-, -, ␥-adenosine triphosphate (ATP; with contributions of ␣- and -adenosine diphosphate) and their ratios. Patients with an acute rejection episodes showed a significantly elevated PME/-ATP and PDE/-ATP, PME/Pi, and PDE/Pi signal ratios compared with the control group. The group with acute tubular necrosis had decreased ratios. Patients with late graft dysfunction revealed only an insignificant decrease in PME/Pi and PDE/Pi ratios. We concluded that 31 P MRS was capable of distinguishing the two main causes of graft dysfunction early after transplantation. HE MOST common renal complications, after kidney transplantation, are acute rejection episodes (ARE), acute tubular necrosis (ATN) and cyclosporine toxicity. These complications are observed in 50%– 60% of patients in the initial postoperative period. Correct assessment of the cause of dysfunction is important, because each diagnosis requires a different therapy. Renal ARE are defined as an immune response to the presence of a foreign organ. The process most frequently occurs between the end of week 1 and 3 months after transplantation. ATN implies cellular damage to renal tissue as a consequence of ischemia/reperfusion injury. The ischemia reduces return of kidney function; often, hemodialysis treatments are required. ARE and ATN are evidenced as failure of a fall in serum creatinine levels.1 Various causes may also lead to late kidney graft dysfunction (LGD), which was formerly termed “chronic allograft nephropathy” (CAN); however, this term was abandoned in 2005 because it is not specific and includes assimilated various pathological conditions leading to graft dysfunction.2 The algorithm for diagnosis of early graft dysfunction involves histologic examination, which is regarded as the gold standard. Unfortunately, occasional errors and invasive interventions are sometimes associated with this method. Nephrologists therefore concentrate on using im-
T
aging methods, which offer noninvasively acquired information about the status of the kidney graft. Sonographic Doppler examinations show a high sensitivity to recognize an ARE, but have a low index of specificity.3 Early detection of ARE as the cause of graft dysfunction is difficult. In contrast, ultrasonography is the method of choice to diagnose kidney grafts with ureteral obstruction. Performing noninvasive studies of kidney metabolism is possible using phosphorus magnetic resonance spectroscopy (31P MRS), which describes posttransplantation changes in the kidney in vivo.4,5 Several metabolites that participate in energy and membrane metabolism can be detected in the kidney: ␣-, -, and ␥-adenosine triphosFrom the MR Unit, Department of Diagnostic and Interventional Radiology (P.V., M.D., V.H., M.H.), the Department of Nephrology, Transplant Center, Institute for Clinical and Experimental Medicine (O.V.), and the Dialysis Center, Fresenius Medical Care (P.T.), Prague, Czech Republic. Supported by MZ0IKEM2005, Czech Republic, and IGA no. NT11275, Czech Republic. Address reprint requests to Mgr. Vít Herynek, PhD, MR-unit, ZRIR, Institute for Clinical and Experimental Medicine, Videnska 1958/9, 140 21 Prague 4, Czech Republic. E-mail: vit.herynek@ medicon.cz
0041-1345/11/$–see front matter doi:10.1016/j.transproceed.2010.11.027
© 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
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Transplantation Proceedings, 43, 1570 –1575 (2011)
KIDNEY GRAFT SPECTROSCOPY
phate (ATP), ␣- and - adenosine diphosphate (ADP) as well as signals of phosphomonoesters (PME; mainly phosphoethanolamine and phosphocholine), phosphodiesters (PDE; mainly glycerol-3-phosphocholine and glycerol3-phosphoethanolamine), and inorganic phosphate (Pi); see Fig 1. The phosphocreatine (PCr) signal should not
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occur in the kidney; nevertheless, it can often be observed in MR spectra owing to a partial volume effect from surrounding tissue. The aim of this study was to use the method of 2D chemical shift imaging (2D-CSI) 31P MRS for the discrimination among the common causes of kidney graft dysfunction.
Fig 1. 31P spectra of transplanted kidneys. (A) ARE, 20 days posttransplantation. (B) ATN, 4 days posttransplantation. (C) LGD, 65 months posttransplantation. (D) Normal renal function, 8 months posttransplantation. PME, phosphomonoesters; Pi, inorganic phosphate; PDE, phosphodiesters; PCr, phosphocreatine; ATP, adenosine triphosphate.
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VYHNANOVSKÁ, DEZORTOVÁ, HERYNEK ET AL
MATERIALS AND METHODS Subjects We examined 87 kidney graft recipients. We eliminated 12 spectra owing to excessive depth of the graft (n ⫽ 5), technical errors (n ⫽ 7), or an unknown diagnosis owing to nonspecific histology findings (n ⫽ 7), resulting in a cohort of 68 patients. There were 47 male and 21 female recipients of mean age 47.1 ⫾ 11.9 years. The subjects were divided into 4 groups: ATN, patients with delayed graft function (n ⫽ 6); ARE (n ⫽ 20); LGD (n ⫽ 11) which while heterogenous, was not subdivided due to its small size; and controls with stable graft function (n ⫽ 31). Written consent with information about the protocol and the reasons for the examinations, as approved by the local ethics committee, was obtained from all subjects.
Clinical Tests Patient clinical data are summarized in Table 1, including the duration after renal transplantation (months), actual serum creatinine level at the time of 31P MRS examination, glomerular filtration rate, and cyclosporine levels since this immunosuppression was used in 77% of all patients within 24 hours of the MR examination. Doppler ultrasound, and ultrasound-guided biopsy of the renal grafts was performed in 37 patients but not the control group because it is an invasive intervention that was not indicated for these patients. Eleven patients received kidneys from living related donors; others, from donors after cardiac death.
MR Imaging and MRS The MR examination was performed using a Siemens Vision whole-body MR scanner operating at 1.5 Tesla. A commercial dual 1 H/31P surface coil was used for 31P MRS. Multi-slice sagittal, coronal, and transversal MR images (repetition time TR ⫽ 15 ms, echo time TE ⫽ 6 ms, flip angle 30°, 450 mm field-of-view [FOV], 10-mm slice thickness, acquisition time 1 min 2 sec) were used to determine the exact position for localization of the volume of interest (VOI) for MR spectroscopy (Fig. 2). The patients were examined in the supine position. The surface coil was fixed in the right or left iliac fossa underneath the kidney graft.
MRS Measurement. 31P MR spectra were obtained using the 2D-CSI technique in the coronal plane: 8 ⫻ 8 phase encoding steps, TR ⫽ 323 ms, TE ⫽ 3 ms, FOV ⫽ 320 mm, flip angle 90°, slice thickness of 30 or 40 mm, 32 acquisitions, and total scanning time 11 minutes 1 second. Automatic shim for optimizing the magnetic field homogeneity was used in the 2D-CSI technique. The VOI was chosen according to the basic images and the nominal voxel volume, namely, either 48 or 64 mL, depending on organ size and shape. The total examination time was about 20 minutes.
Evaluation of Spectra. Spectra were evaluated manually in the range of ⫺20.0 to 15.5 ppm using the standard Siemens Numaris software. The following steps of spectral evaluation were performed for all measurements: Gauss apodization with halfwidth ⫽ 30 ms, Fourier transformation, phase, and spline baseline correction and curve fitting with the assumption of Gaussian single lines. Signal intensities of PME (⬃6.5 ppm), Pi (⬃5.1 ppm), PDE (⬃2.6 ppm), PCr (0.0 ppm), and ATP (␣ ⬃⫺7.8 ppm,  ⬃⫺18.3 ppm, ␥ ⬃⫺2.7 ppm) superimposed with ADP (␣ ⬃⫺7.5 ppm,  ⬃⫺3.0) were obtained using the deconvolution method. Signals of ␣-ATP and ␣-ADP were not separated as well as ␥-ATP and -ADP. We considered spectra with signal (-ATP) to noise ratios (S/N) over 3 to be suitable for further analysis. Spectra with lower S/N were considered as technical errors. Data on all subjects were evaluated twice; mean values were used to minimize subjective errors. Partial Volume Effect. The PCr signal, which has its origin in surrounding muscles, was observed in almost all spectra as a result of a partial volume effect and a point spread function effect6 (Fig 1). The contribution of metabolite signals from surrounding tissue was calculated by comparison of PCr signal intensity in the VOI placed into kidney and in pure muscle. Even if the PCr signal in the MR spectra originates only in the surrounding tissue, because of its low intensity the contribution of other metabolites from the surrounding tissue was negligible (⬍6%) with respect to the experimental error. Conversely, the PCr signal was used as an internal standard to determine the chemical shifts that were used to calculate intracellular pH according to an equation derived from the classical Henderson-Hasselbalch formula:7 pH ⫽ pK ⫹ log [(␦ ⫺ ␦HA) ⁄ (␦A ⫺ ␦)] where pK is the logarithm of the equilibrium constant for the proton exchange H2P04⫺ ↔ HP042⫺ ⫹ H⫹; ␦ ⫽ to the chemical shift of the Pi peak relative to PCr in ppm; and ␦HA and ␦A are, chemical shifts of protonated and unprotonated fractions. For pH calculations we used values of pK ⫽ 6.77, ␦HA ⫽ 3.23, ␦A ⫽ 5.70 as determined elsewhere.7
Statistical Analysis The results of 31P MRS were evaluated using analysis of variance. The Dunnet test was used to compare all groups with the control group; the Duncan multiple range test, to compare groups with various causes of kidney dysfunction. Zero hypotheses with P ⬍ .05 (as well as P ⬍ .01) were tested for patient group comparisons. The probability level .05 ⬍ P ⬍ .08 was considered to be only a trend.
RESULTS
The results of biochemical tests characterizing kidney function in each patient group at the time of our study are
Table 1. Results of Biochemical Tests of Patients and Control Groups (Mean Values and Standard Deviations)
n Age of patients (y) Duration after Tx (mos) Creatinine (mol/L) Glomerular filtration (mL/sec/1.73 m2) Cyclosporine (ng/mL)
Control
ATN
ARE
LGD
31 48.7 ⫾ 11.9 15.0 ⫾ 43.1 134 ⫾ 32 1.00 ⫾ 0.38 203 ⫾ 91
6 56.4 ⫾ 10.6 0.4 ⫾ 0.3 901 ⫾ 325 0.13 ⫾ 0.13 182 ⫾ 23
20 41.7 ⫾ 9.7 0.9 ⫾ 1.3 249 ⫾ 195 0.69 ⫾ 0.30 218 ⫾ 100
11 47.5 ⫾ 11.8 55.7 ⫾ 70.8 228 ⫾ 62 0.53 ⫾ 0.24 179 ⫾ 57
Abbreviations: ARE, acute rejection episode; ATN, acute tubular necrosis; LGD, late graft dysfunction.
KIDNEY GRAFT SPECTROSCOPY
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Fig 2. T1-weighted images of kidney grafts in coronal (A), sagittal (B), and transversal (C) planes were used for estimation of the VOI for 2D-CSI. The typical slice thickness was 30 mm, and the outlined box positioned in the center of the kidney in the matrix shows the nominal VOI selected for spectral evaluation.
summarized in Table 1. We obtained spectra with sufficient quality to determine the peak integrals from 68 patients. Examples of 31P MR spectra of kidney grafts with ARE, ATN, LGD, and good stable function are shown in Figure 1. We performed methodologic tests before the statistical comparisons of the patient groups. The reproducibility of data processing was calculated from the mean values of data from 13 patients evaluated 3 times by 1 operator. The relative error was 7.3%. Reproducibility, as tested on a group of 8 subjects who were measured twice using the same protocol, showed a calculated mean value of 14.4%. The average distance from the surface coil to the transplanted kidney was 5.8 ⫾ 3.5 cm. Table 2 shows the results of relative signal intensity ratios of metabolites in the 4 groups. We evaluated the metabolites Pi, PME, and PDE related to -ATP, because this signal represents the pure contribution from ATP. ␣-ATP or ␥-ATP signals were contaminated by contributions of phosphates from ADP and possibly other metabolites. We also compared ratios of PME/Pi, PDE/Pi, and PME/PDE. The ATN group showed a significant decrease in PDE/-ATP, PDE/Pi, and PME/Pi
and a trend in the PME/-ATP ratio compared with the controls. There was no change in Pi/-ATP. The group of ARE patients showed a significant increase in PME/ATP and PME/Pi compared with the control group. Also, individual signal intensities of PME were visibly increased. The ratio of PDE/Pi was significantly higher as well; however, the increase of PDE/-ATP was not significant. No difference was observed in the case of the Pi/-ATP ratio among ARE and control groups. CAN patients did not differ from the control group in Pi/ATP, PDE/-ATP, and PME/-ATP ratios; however, we observed a significant decrease in PME/Pi and a trend toward a decrease in PDE/Pi compared with the control group. The group of ARE patients displayed higher ratios of PDE/Pi, PME/Pi (significant differences) and PDE/-ATP, PME/-ATP (trend) compared with the ATN group. LGE patients showed a significantly higher Pi/-ATP ratios and significantly lower PDE/Pi and PME/Pi ratios compared with ARE subjects. Significant changes in pH values were not noted among any patient group (Table 2).
Table 2. Relative Signal Intensity Ratios of Metabolites in 4 Groups of Patients and Controls (Mean Values and Standard Deviation)
PME/-ATP Pi/-ATP PDE/-ATP PME/PDE PME/Pi PDE/Pi pH
Control
ATN
ARE
LGD
0.85 ⫾ 0.20 0.52 ⫾ 0.14 1.21 ⫾ 0.38 0.75 ⫾ 0.27 1.78 ⫾ 0.57 2.44 ⫾ 0.87 7.29 ⫾ 0.23
0.68 ⫾ 0.24 (*) 0.54 ⫾ 0.22 0.80 ⫾ 0.28* 0.94 ⫾ 0.33 1.36 ⫾ 0.57 1.38 ⫾ 0.46* 7.27 ⫾ 0.34
1.11 ⫾ 0.54*( ) 0.44 ⫾ 0.18 1.35 ⫾ 0.69(†) 0.88 ⫾ 0.32 2.74 ⫾ 1.19*† 3.23 ⫾ 1.34*† 7.31 ⫾ 0.47 †
0.82 ⫾ 0.30 0.63 ⫾ 0.30 1.29 ⫾ 0.43‡ 0.69 ⫾ 0.34 1.36 ⫾ 0.61*§ 2.06 ⫾ 0.68(*)§ 7.22 ⫾ 0.21
Abbreviations: PME, phosphomonoesters; PDE, phosphodiesters; Pi, inorganic phosphate; -ATP, adenosine triphosphate; ATN, acute tubular necrosis; ARE, acute rejection episode; LGD, late graft dysfunction. *P ⬍ .05 from the control group; (*) P ⬍ .08 trend. † P ⬍ .05 between the groups ATN and ARE (†) P ⬍ .08 trend. ‡ P ⬍ .05 between the groups ATN and LGD; (‡) P ⬍ .08 trend. § P ⬍ .05 between the groups ARE and LGD, (§) P ⬍ .08 trend.
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DISCUSSION
Understanding the metabolic processes and differences in pathologic conditions among transplanted organs can significantly distinguish rejection from other complications. In vivo phosphorus spectroscopy seems to be a proper tool to assess energy metabolites. Several studies have been published using in vivo phosphorus MRS to evaluate pretransplant assessment of renal viability9 –11 and to monitor altered kidney status after transplantation.5,8,12–15 Although there were attempts to quantify the absolute concentrations of phosphorus metabolites in human transplanted kidneys,16 most authors favor evaluation of relative concentrations owing to technical difficulties. Nevertheless, there is a huge dispersion among the published data. Although the basic trends are similar, several factors render the metabolic ratios not comparable. We used CSI, instead of ISIS as used by other authors,4,5,14,16 or DRESS,6 which can improve localization of the VOI in the kidney tissue. However, the method is time consuming. Therefore, we used short repetition times in the sequence. This substantially affects the signal intensity, because relaxation times of phosphorus compounds are in the range from 0.4 (PDE signals) to 2 seconds (Pi signals).16 Spectra localization may represent another reason for the huge data dispersion. In general, the resolution of 31P MR spectra at 1.5 T was not sufficient to distinguish the cortex from the medulla or hilum of the kidney. The reason may be the partial volume effects owing to an excessively large VOI. Nevertheless, it may not be so important for processes that occur diffusely, involving all kidney parenchyma as in the case of ARE or ATN. Several parameters have been proposed to differentiate patients with ARE versus ATN or controls. Patients with ARE have been characterized to show a significant decrease in the intracellular amounts of -ATP4 and significant changes in the signal ratios of Pi/⌺ATP and PDE/ PME.6 Heindel et al5 described the group of ARE patients with an increased ratio of Pi/␣-ATP compared with controls and by different pH values than those with tubular necrosis. The present study also observed that kidneys with ATN showed lower PME/PDE ratios than controls. Although we evaluated only relative concentrations and not the absolute values of metabolites, the ratios indicated substantial global changes, such as the increased PME during ARE and decreased PME and PDE content during ATN. Biopsies from patients with ARE are characterized by mononuclear cell infiltration in the interstitium between the renal tubules and focal interstitial cell infiltration around smaller veins. Although the pathogenesis of the alterations of high-energy phosphate compounds during ARE not entirely clear, we hypothesized that the increased PME was due to accelerated membrane synthesis and more intensive cell growth. ATN is among the most common causes of kidney failure after transplantation. It is caused by a lack of oxygen to the kidney tissues (ischemia). The internal structures of the
VYHNANOVSKÁ, DEZORTOVÁ, HERYNEK ET AL
kidney, particularly the tubules, gradually become damaged or destroyed. ATN is among the most common structural changes that lead to acute renal failure; however, if diagnosed early, it can be reversible. ATN is characterized by suppressed metabolism, which was confirmed by our data. We observed a decrease in PME and PDE signals compared with the control or the ARE group. This observation agrees with the results of Möller et al,17 who noted a decreased PME/Pi ratio in kidney grafts during hypothermic ischemia before transplantation and used this parameter to predict kidney dysfunction in recipients. Changes among the group of patients with LGD showed moderate (but significant) decrease in PME/Pi, and a trend to decrease PDE/Pi. These changes were not dramatic, but may represent slow functional deterioration of the kidney, corresponding with long-term chronic fibrosis. whereas ARE or ATN occur relatively shortly after transplantation (see Table 1). Several factors may be responsible for LGD. Besides the time factor and biological age of the graft, we must also consider the influence of cyclosporine, which changes cellular energy metabolism at the mitochondrial level.18 We also monitored the pH changes in the patients, observing that the pH value of the control group (pH ⫽ 7.29 ⫾ 0.24) was similar to that of 5 healthy volunteers (pH ⫽ 7.22 ⫾ 0.29). This observation agrees with the Heindel study.5 Nevertheless, we did not confirm a significant reduction of pH among ARE group as observed by Heindel.5 In conclusion although a biopsy remains the gold standard to assess kidney graft status, 31P MRS may be a useful, noninvasive method to monitor the status and metabolism of transplanted organs. We characterized various pathologic conditions distinguishing them by the relative concentrations of phosphorus metabolites. The CSI method offers a noninvasive, promising possibility to obtain spatially resolved 31P MR spectra. The relative concentrations of PME or PDE seem to be crucial to distinguish the two main causes of allograft dysfunction during the critical period shortly after transplantation. ACKNOWLEDGMENTS The authors thank Jelena Skibová (Institute for Clinical and Experimental Medicine, Prague, Czech Republic) for statistical evaluation of the MRS data.
REFERENCES 1. Allen R, Chapman JR: Early renal transplant dysfunction: In: Allen R, Chapman JR Eds. A manual of renal transplantation. London: Edward Arnold; 1994, P144 2. Solez K, Colvin RB, Racusen LC, et al: Banff’05 Meeting Report: differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy (‘CAN’). Am J Transplant, 7:518, 2007 3. Hollenbeck M: New diagnostic techniques in clinical nephrology. Colour coded duplex sonography for evaluation of renal transplants-tool or toy for the nephrologist? Nephrol Dial Transplant 9:1822, 1994
KIDNEY GRAFT SPECTROSCOPY 4. Klemm A, Rzanny R, Funfstuck R, et al: 31P-magnetic resonance spectroscopy (31P-MRS) of human allografts after renal transplantation. Nephrol Dial Transplant 13:3147, 1998 5. Heindel W, Kugel H, Wenzel F: Localized 31P MR spectroscopy of the transplanted human kidney in situ shows altered metabolism in rejection and acute tubular necrosis. J Magn Reson Imaging 7:858, 1997 6. Tosner Z, Dezortova M, Tintera J, et al: Application of two-dimensional CSI for absolute quantification of phosphorus metabolites in the human liver. MAGMA 13:40, 2001 7. DeGraaf RA: In vivo NMR spectroscopy. Principles and techniques. Chichester: John Wiley and Sons; 1998 8. Grist TM, Charles HC, Sostman HD: Renal transplant rejection: diagnosis with 31P MR spectroscopy. Am J Roentgenol 156:105, 1991 9. Ota K, Fuchinoue S, Nakamura M, et al: Transplantation of kidneys donated from the USA: long-term results and viability testing using 31P-MRS. Transplant Int 10:7, 1997 10. Kurkova D, Herynek V, Gintelova J, et al: Potential of 31P magnetic resonance spectroscopy in monitoring the viability of human renal grafts stored in Euro-Collins perfusion solution. Physiol Res 44:327, 1995 11. Niekisch MB, Von Elverfeldt D, Saman AE, et al: Improved pretransplant assessment of renal quality by means of phosphorus-31 magnetic resonance spectroscopy using chemical shift imaging. Transplantation 77:1041, 2004
1575 12. Vallee JP, Lazeyras F, Sostman HD, et al: Proton-decoupled phosphorus-31 magnetic resonance spectroscopy in the evaluation of native and well-functioning transplanted kidneys. Acad Radiol 3:1030, 1996 13. Hricak H: Phosphorus-31 MRS of the kidney. Invest Radiol 24:993, 1989 14. Boska MD, Meyerhoff DJ, Twieg DB, et al: Image-guided 31P magnetic resonance spectroscopy of normal and transplanted human kidneys. Kidney Int 38:294, 1990 15. Fiorina P, Perseghin G, De Cobelli F, et al: Altered kidney graft high-energy phosphate metabolism in kidney-transplanted end-stage renal disease type 1 diabetic patients: a cross-sectional analysis of the effect of kidney alone and kidney-pancreas transplantation. Diabetes Care 30:597, 2007 16. Kugel H, Wittsack HJ, Wenzel F, et al: Non-invasive determination of metabolite concentrations in human transplanted kidney in vivo by 31P MR spectroscopy. Acta Radiologica 41:634, 2000 17. Moller HE, Gaupp A, Dietl K, et al: Tissue pH in human kidney transplants during hypothermic ischemia. Magn Res Imaging 18:743, 2000 18. Serkova N, Klawitter J, Niemann CU: Organ-specific response to inhibition of mitochondrial metabolism by cyclosporine in the rat. Transplant Int 16:748, 2003
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aging methods, which offer noninvasively acquired information about the status of the kidney graft. Sonographic Doppler examinations show a high sensitivity to recognize an ARE, but have a low index of specificity.3 Early detection of ARE as the cause of graft dysfunction is difficult. In contrast, ultrasonography is the method of choice to diagnose kidney grafts with ureteral obstruction. Performing noninvasive studies of kidney metabolism is possible using phosphorus magnetic resonance spectroscopy (31P MRS), which describes posttransplantation changes in the kidney in vivo.4,5 Several metabolites that participate in energy and membrane metabolism can be detected in the kidney: ␣-, -, and ␥-adenosine triphosFrom the MR Unit, Department of Diagnostic and Interventional Radiology (P.V., M.D., V.H., M.H.), the Department of Nephrology, Transplant Center, Institute for Clinical and Experimental Medicine (O.V.), and the Dialysis Center, Fresenius Medical Care (P.T.), Prague, Czech Republic. Supported by MZ0IKEM2005, Czech Republic, and IGA no. NT11275, Czech Republic. Address reprint requests to Mgr. Vít Herynek, PhD, MR-unit, ZRIR, Institute for Clinical and Experimental Medicine, Videnska 1958/9, 140 21 Prague 4, Czech Republic. E-mail: vit.herynek@ medicon.cz
0041-1345/11/$–see front matter doi:10.1016/j.transproceed.2010.11.027
© 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1570
Transplantation Proceedings, 43, 1570 –1575 (2011)
KIDNEY GRAFT SPECTROSCOPY
phate (ATP), ␣- and - adenosine diphosphate (ADP) as well as signals of phosphomonoesters (PME; mainly phosphoethanolamine and phosphocholine), phosphodiesters (PDE; mainly glycerol-3-phosphocholine and glycerol3-phosphoethanolamine), and inorganic phosphate (Pi); see Fig 1. The phosphocreatine (PCr) signal should not
1571
occur in the kidney; nevertheless, it can often be observed in MR spectra owing to a partial volume effect from surrounding tissue. The aim of this study was to use the method of 2D chemical shift imaging (2D-CSI) 31P MRS for the discrimination among the common causes of kidney graft dysfunction.
Fig 1. 31P spectra of transplanted kidneys. (A) ARE, 20 days posttransplantation. (B) ATN, 4 days posttransplantation. (C) LGD, 65 months posttransplantation. (D) Normal renal function, 8 months posttransplantation. PME, phosphomonoesters; Pi, inorganic phosphate; PDE, phosphodiesters; PCr, phosphocreatine; ATP, adenosine triphosphate.
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VYHNANOVSKÁ, DEZORTOVÁ, HERYNEK ET AL
MATERIALS AND METHODS Subjects We examined 87 kidney graft recipients. We eliminated 12 spectra owing to excessive depth of the graft (n ⫽ 5), technical errors (n ⫽ 7), or an unknown diagnosis owing to nonspecific histology findings (n ⫽ 7), resulting in a cohort of 68 patients. There were 47 male and 21 female recipients of mean age 47.1 ⫾ 11.9 years. The subjects were divided into 4 groups: ATN, patients with delayed graft function (n ⫽ 6); ARE (n ⫽ 20); LGD (n ⫽ 11) which while heterogenous, was not subdivided due to its small size; and controls with stable graft function (n ⫽ 31). Written consent with information about the protocol and the reasons for the examinations, as approved by the local ethics committee, was obtained from all subjects.
Clinical Tests Patient clinical data are summarized in Table 1, including the duration after renal transplantation (months), actual serum creatinine level at the time of 31P MRS examination, glomerular filtration rate, and cyclosporine levels since this immunosuppression was used in 77% of all patients within 24 hours of the MR examination. Doppler ultrasound, and ultrasound-guided biopsy of the renal grafts was performed in 37 patients but not the control group because it is an invasive intervention that was not indicated for these patients. Eleven patients received kidneys from living related donors; others, from donors after cardiac death.
MR Imaging and MRS The MR examination was performed using a Siemens Vision whole-body MR scanner operating at 1.5 Tesla. A commercial dual 1 H/31P surface coil was used for 31P MRS. Multi-slice sagittal, coronal, and transversal MR images (repetition time TR ⫽ 15 ms, echo time TE ⫽ 6 ms, flip angle 30°, 450 mm field-of-view [FOV], 10-mm slice thickness, acquisition time 1 min 2 sec) were used to determine the exact position for localization of the volume of interest (VOI) for MR spectroscopy (Fig. 2). The patients were examined in the supine position. The surface coil was fixed in the right or left iliac fossa underneath the kidney graft.
MRS Measurement. 31P MR spectra were obtained using the 2D-CSI technique in the coronal plane: 8 ⫻ 8 phase encoding steps, TR ⫽ 323 ms, TE ⫽ 3 ms, FOV ⫽ 320 mm, flip angle 90°, slice thickness of 30 or 40 mm, 32 acquisitions, and total scanning time 11 minutes 1 second. Automatic shim for optimizing the magnetic field homogeneity was used in the 2D-CSI technique. The VOI was chosen according to the basic images and the nominal voxel volume, namely, either 48 or 64 mL, depending on organ size and shape. The total examination time was about 20 minutes.
Evaluation of Spectra. Spectra were evaluated manually in the range of ⫺20.0 to 15.5 ppm using the standard Siemens Numaris software. The following steps of spectral evaluation were performed for all measurements: Gauss apodization with halfwidth ⫽ 30 ms, Fourier transformation, phase, and spline baseline correction and curve fitting with the assumption of Gaussian single lines. Signal intensities of PME (⬃6.5 ppm), Pi (⬃5.1 ppm), PDE (⬃2.6 ppm), PCr (0.0 ppm), and ATP (␣ ⬃⫺7.8 ppm,  ⬃⫺18.3 ppm, ␥ ⬃⫺2.7 ppm) superimposed with ADP (␣ ⬃⫺7.5 ppm,  ⬃⫺3.0) were obtained using the deconvolution method. Signals of ␣-ATP and ␣-ADP were not separated as well as ␥-ATP and -ADP. We considered spectra with signal (-ATP) to noise ratios (S/N) over 3 to be suitable for further analysis. Spectra with lower S/N were considered as technical errors. Data on all subjects were evaluated twice; mean values were used to minimize subjective errors. Partial Volume Effect. The PCr signal, which has its origin in surrounding muscles, was observed in almost all spectra as a result of a partial volume effect and a point spread function effect6 (Fig 1). The contribution of metabolite signals from surrounding tissue was calculated by comparison of PCr signal intensity in the VOI placed into kidney and in pure muscle. Even if the PCr signal in the MR spectra originates only in the surrounding tissue, because of its low intensity the contribution of other metabolites from the surrounding tissue was negligible (⬍6%) with respect to the experimental error. Conversely, the PCr signal was used as an internal standard to determine the chemical shifts that were used to calculate intracellular pH according to an equation derived from the classical Henderson-Hasselbalch formula:7 pH ⫽ pK ⫹ log [(␦ ⫺ ␦HA) ⁄ (␦A ⫺ ␦)] where pK is the logarithm of the equilibrium constant for the proton exchange H2P04⫺ ↔ HP042⫺ ⫹ H⫹; ␦ ⫽ to the chemical shift of the Pi peak relative to PCr in ppm; and ␦HA and ␦A are, chemical shifts of protonated and unprotonated fractions. For pH calculations we used values of pK ⫽ 6.77, ␦HA ⫽ 3.23, ␦A ⫽ 5.70 as determined elsewhere.7
Statistical Analysis The results of 31P MRS were evaluated using analysis of variance. The Dunnet test was used to compare all groups with the control group; the Duncan multiple range test, to compare groups with various causes of kidney dysfunction. Zero hypotheses with P ⬍ .05 (as well as P ⬍ .01) were tested for patient group comparisons. The probability level .05 ⬍ P ⬍ .08 was considered to be only a trend.
RESULTS
The results of biochemical tests characterizing kidney function in each patient group at the time of our study are
Table 1. Results of Biochemical Tests of Patients and Control Groups (Mean Values and Standard Deviations)
n Age of patients (y) Duration after Tx (mos) Creatinine (mol/L) Glomerular filtration (mL/sec/1.73 m2) Cyclosporine (ng/mL)
Control
ATN
ARE
LGD
31 48.7 ⫾ 11.9 15.0 ⫾ 43.1 134 ⫾ 32 1.00 ⫾ 0.38 203 ⫾ 91
6 56.4 ⫾ 10.6 0.4 ⫾ 0.3 901 ⫾ 325 0.13 ⫾ 0.13 182 ⫾ 23
20 41.7 ⫾ 9.7 0.9 ⫾ 1.3 249 ⫾ 195 0.69 ⫾ 0.30 218 ⫾ 100
11 47.5 ⫾ 11.8 55.7 ⫾ 70.8 228 ⫾ 62 0.53 ⫾ 0.24 179 ⫾ 57
Abbreviations: ARE, acute rejection episode; ATN, acute tubular necrosis; LGD, late graft dysfunction.
KIDNEY GRAFT SPECTROSCOPY
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Fig 2. T1-weighted images of kidney grafts in coronal (A), sagittal (B), and transversal (C) planes were used for estimation of the VOI for 2D-CSI. The typical slice thickness was 30 mm, and the outlined box positioned in the center of the kidney in the matrix shows the nominal VOI selected for spectral evaluation.
summarized in Table 1. We obtained spectra with sufficient quality to determine the peak integrals from 68 patients. Examples of 31P MR spectra of kidney grafts with ARE, ATN, LGD, and good stable function are shown in Figure 1. We performed methodologic tests before the statistical comparisons of the patient groups. The reproducibility of data processing was calculated from the mean values of data from 13 patients evaluated 3 times by 1 operator. The relative error was 7.3%. Reproducibility, as tested on a group of 8 subjects who were measured twice using the same protocol, showed a calculated mean value of 14.4%. The average distance from the surface coil to the transplanted kidney was 5.8 ⫾ 3.5 cm. Table 2 shows the results of relative signal intensity ratios of metabolites in the 4 groups. We evaluated the metabolites Pi, PME, and PDE related to -ATP, because this signal represents the pure contribution from ATP. ␣-ATP or ␥-ATP signals were contaminated by contributions of phosphates from ADP and possibly other metabolites. We also compared ratios of PME/Pi, PDE/Pi, and PME/PDE. The ATN group showed a significant decrease in PDE/-ATP, PDE/Pi, and PME/Pi
and a trend in the PME/-ATP ratio compared with the controls. There was no change in Pi/-ATP. The group of ARE patients showed a significant increase in PME/ATP and PME/Pi compared with the control group. Also, individual signal intensities of PME were visibly increased. The ratio of PDE/Pi was significantly higher as well; however, the increase of PDE/-ATP was not significant. No difference was observed in the case of the Pi/-ATP ratio among ARE and control groups. CAN patients did not differ from the control group in Pi/ATP, PDE/-ATP, and PME/-ATP ratios; however, we observed a significant decrease in PME/Pi and a trend toward a decrease in PDE/Pi compared with the control group. The group of ARE patients displayed higher ratios of PDE/Pi, PME/Pi (significant differences) and PDE/-ATP, PME/-ATP (trend) compared with the ATN group. LGE patients showed a significantly higher Pi/-ATP ratios and significantly lower PDE/Pi and PME/Pi ratios compared with ARE subjects. Significant changes in pH values were not noted among any patient group (Table 2).
Table 2. Relative Signal Intensity Ratios of Metabolites in 4 Groups of Patients and Controls (Mean Values and Standard Deviation)
PME/-ATP Pi/-ATP PDE/-ATP PME/PDE PME/Pi PDE/Pi pH
Control
ATN
ARE
LGD
0.85 ⫾ 0.20 0.52 ⫾ 0.14 1.21 ⫾ 0.38 0.75 ⫾ 0.27 1.78 ⫾ 0.57 2.44 ⫾ 0.87 7.29 ⫾ 0.23
0.68 ⫾ 0.24 (*) 0.54 ⫾ 0.22 0.80 ⫾ 0.28* 0.94 ⫾ 0.33 1.36 ⫾ 0.57 1.38 ⫾ 0.46* 7.27 ⫾ 0.34
1.11 ⫾ 0.54*( ) 0.44 ⫾ 0.18 1.35 ⫾ 0.69(†) 0.88 ⫾ 0.32 2.74 ⫾ 1.19*† 3.23 ⫾ 1.34*† 7.31 ⫾ 0.47 †
0.82 ⫾ 0.30 0.63 ⫾ 0.30 1.29 ⫾ 0.43‡ 0.69 ⫾ 0.34 1.36 ⫾ 0.61*§ 2.06 ⫾ 0.68(*)§ 7.22 ⫾ 0.21
Abbreviations: PME, phosphomonoesters; PDE, phosphodiesters; Pi, inorganic phosphate; -ATP, adenosine triphosphate; ATN, acute tubular necrosis; ARE, acute rejection episode; LGD, late graft dysfunction. *P ⬍ .05 from the control group; (*) P ⬍ .08 trend. † P ⬍ .05 between the groups ATN and ARE (†) P ⬍ .08 trend. ‡ P ⬍ .05 between the groups ATN and LGD; (‡) P ⬍ .08 trend. § P ⬍ .05 between the groups ARE and LGD, (§) P ⬍ .08 trend.
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DISCUSSION
Understanding the metabolic processes and differences in pathologic conditions among transplanted organs can significantly distinguish rejection from other complications. In vivo phosphorus spectroscopy seems to be a proper tool to assess energy metabolites. Several studies have been published using in vivo phosphorus MRS to evaluate pretransplant assessment of renal viability9 –11 and to monitor altered kidney status after transplantation.5,8,12–15 Although there were attempts to quantify the absolute concentrations of phosphorus metabolites in human transplanted kidneys,16 most authors favor evaluation of relative concentrations owing to technical difficulties. Nevertheless, there is a huge dispersion among the published data. Although the basic trends are similar, several factors render the metabolic ratios not comparable. We used CSI, instead of ISIS as used by other authors,4,5,14,16 or DRESS,6 which can improve localization of the VOI in the kidney tissue. However, the method is time consuming. Therefore, we used short repetition times in the sequence. This substantially affects the signal intensity, because relaxation times of phosphorus compounds are in the range from 0.4 (PDE signals) to 2 seconds (Pi signals).16 Spectra localization may represent another reason for the huge data dispersion. In general, the resolution of 31P MR spectra at 1.5 T was not sufficient to distinguish the cortex from the medulla or hilum of the kidney. The reason may be the partial volume effects owing to an excessively large VOI. Nevertheless, it may not be so important for processes that occur diffusely, involving all kidney parenchyma as in the case of ARE or ATN. Several parameters have been proposed to differentiate patients with ARE versus ATN or controls. Patients with ARE have been characterized to show a significant decrease in the intracellular amounts of -ATP4 and significant changes in the signal ratios of Pi/⌺ATP and PDE/ PME.6 Heindel et al5 described the group of ARE patients with an increased ratio of Pi/␣-ATP compared with controls and by different pH values than those with tubular necrosis. The present study also observed that kidneys with ATN showed lower PME/PDE ratios than controls. Although we evaluated only relative concentrations and not the absolute values of metabolites, the ratios indicated substantial global changes, such as the increased PME during ARE and decreased PME and PDE content during ATN. Biopsies from patients with ARE are characterized by mononuclear cell infiltration in the interstitium between the renal tubules and focal interstitial cell infiltration around smaller veins. Although the pathogenesis of the alterations of high-energy phosphate compounds during ARE not entirely clear, we hypothesized that the increased PME was due to accelerated membrane synthesis and more intensive cell growth. ATN is among the most common causes of kidney failure after transplantation. It is caused by a lack of oxygen to the kidney tissues (ischemia). The internal structures of the
VYHNANOVSKÁ, DEZORTOVÁ, HERYNEK ET AL
kidney, particularly the tubules, gradually become damaged or destroyed. ATN is among the most common structural changes that lead to acute renal failure; however, if diagnosed early, it can be reversible. ATN is characterized by suppressed metabolism, which was confirmed by our data. We observed a decrease in PME and PDE signals compared with the control or the ARE group. This observation agrees with the results of Möller et al,17 who noted a decreased PME/Pi ratio in kidney grafts during hypothermic ischemia before transplantation and used this parameter to predict kidney dysfunction in recipients. Changes among the group of patients with LGD showed moderate (but significant) decrease in PME/Pi, and a trend to decrease PDE/Pi. These changes were not dramatic, but may represent slow functional deterioration of the kidney, corresponding with long-term chronic fibrosis. whereas ARE or ATN occur relatively shortly after transplantation (see Table 1). Several factors may be responsible for LGD. Besides the time factor and biological age of the graft, we must also consider the influence of cyclosporine, which changes cellular energy metabolism at the mitochondrial level.18 We also monitored the pH changes in the patients, observing that the pH value of the control group (pH ⫽ 7.29 ⫾ 0.24) was similar to that of 5 healthy volunteers (pH ⫽ 7.22 ⫾ 0.29). This observation agrees with the Heindel study.5 Nevertheless, we did not confirm a significant reduction of pH among ARE group as observed by Heindel.5 In conclusion although a biopsy remains the gold standard to assess kidney graft status, 31P MRS may be a useful, noninvasive method to monitor the status and metabolism of transplanted organs. We characterized various pathologic conditions distinguishing them by the relative concentrations of phosphorus metabolites. The CSI method offers a noninvasive, promising possibility to obtain spatially resolved 31P MR spectra. The relative concentrations of PME or PDE seem to be crucial to distinguish the two main causes of allograft dysfunction during the critical period shortly after transplantation. ACKNOWLEDGMENTS The authors thank Jelena Skibová (Institute for Clinical and Experimental Medicine, Prague, Czech Republic) for statistical evaluation of the MRS data.
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1575 12. Vallee JP, Lazeyras F, Sostman HD, et al: Proton-decoupled phosphorus-31 magnetic resonance spectroscopy in the evaluation of native and well-functioning transplanted kidneys. Acad Radiol 3:1030, 1996 13. Hricak H: Phosphorus-31 MRS of the kidney. Invest Radiol 24:993, 1989 14. Boska MD, Meyerhoff DJ, Twieg DB, et al: Image-guided 31P magnetic resonance spectroscopy of normal and transplanted human kidneys. Kidney Int 38:294, 1990 15. Fiorina P, Perseghin G, De Cobelli F, et al: Altered kidney graft high-energy phosphate metabolism in kidney-transplanted end-stage renal disease type 1 diabetic patients: a cross-sectional analysis of the effect of kidney alone and kidney-pancreas transplantation. Diabetes Care 30:597, 2007 16. Kugel H, Wittsack HJ, Wenzel F, et al: Non-invasive determination of metabolite concentrations in human transplanted kidney in vivo by 31P MR spectroscopy. Acta Radiologica 41:634, 2000 17. Moller HE, Gaupp A, Dietl K, et al: Tissue pH in human kidney transplants during hypothermic ischemia. Magn Res Imaging 18:743, 2000 18. Serkova N, Klawitter J, Niemann CU: Organ-specific response to inhibition of mitochondrial metabolism by cyclosporine in the rat. Transplant Int 16:748, 2003