In patients with relapsing remitting multiple sclerosis (RRMS), interferons reduce the attack frequency of the disease, the mean attack duration, the annual lesion burden, and the frequency of gadolinium (Gd) enhancement on MRI. However the mechanism of action of interferons in patients with MS and the effect of interferons on axonal injury remain undefined. We questioned whether treatment with interferon beta-1a (INF-1a) would enable the recovery of injured axons. To determine this, we used MRSI to examine changes in the metabolic peaks of neuronal markers NAA, Cho(Choline), and Cr(Creatine) after treatment with INF-1a(Rebif 44 g) in a small group of patients with RRMS. An untreated

We studied 10 patients (7 women 3 men) with clinically definite RRMS (median age, 39 years; age range 22-50 years). The median expanded status scale score (EDSS) was 2.1 (range 1.0-3.0), and the median duration of disease was 4.7 years (range, 1-20 years). In the 2 years before their participation in the study, all patients had had at least one relapse. None of the patients had undergone immunosuppressant therapy ever before, or corticosteroid treatment within 2 months before the initiation of the study. Five patients elected not to be

group was studied for comparison, and healthy volunteers were used as controls.

**2. Materials and methods 2.1 Patients and methods**

1994; Ferguson, Matyszak, Esiri, & Perry, 1997; Fernando et al., 2004).

Fig. 1. a-f: Axial fluid-attenuated, inversion recovery (FLAIR) MR image of a 50-year-old woman receiving INF-1a treatment shows a voxel placed over a WM lesion in the right centrum semiovale (1a). The multivoxel CSI MRI spectrum of this lesion is obtained during study months zero, 1, 3, 6, and 12 (b-f). The results of proton MRSI show that there is a statistically significant increment in absolute NAA and Cho values.

MRS in MS, With and Without Interferon Beta 1a Treatment,

between the untreated and treated MS patients (Table 2).

NAA: N-acetyl aspartate, Cho: Choline, Cr: Creatine

and untreated patients and controls

therapy once daily for 5 days.

throughout the study.

**3.2 MRSI results**

to Define the Dynamic Changes of Metabolites in the Brain, and to Monitor Disease Progression 97

clinical MS attack at the initiation of the study in 3 patients during the sixth month of the study. The results of serial MR examinations of another patient revealed new T2 lesions without Gd-enhancement that were present without evidence of a clinical MS attack from the initiation of the study until the twelfth study month. However, during the twelfth month, that patient experienced a clinical MS attack (moderate paresis [4/5] of the left leg), and a new Gd-enhancing lesion was identified. And only that patient received pulse steroid

Five other patients received INF-1a treatment (subcutaneous INF-1a 44 µg –Rebif-3 times per week). Among the subjects who received INF, two patients had Gd-enhancing new T2 lesions on almost every MRI examination, another patient had a Gd-enhancing lesion only on the last MRI examination of the study, and the remaning patients exhibited no new lesions. None of those patients experienced a clinical MS attack accompanying the MRI activities. There was no significant change in the EDSS scores of the patients in either group

There was no statistically significant difference between the basal metabolite levels in the NAWM of patients with MS and the healthy control subjects (*P*> .005) (Table 1). There was also no statistically significant difference between the basal metabolite peaks in the WML

**NAWM metabolic peaks NAA Basal CHO Basal CR Basal**  Untreated Mean 6,5±4,6 4,3±1,9 3,9±1,8 Median 4,8950 3,4650 3,1650 Treated Mean 7,1±2,3 4,5±1,4 4,3±1,3 Median 7,8100 4,7550 4,5200 Control Mean 6,8±0,4 4,1±0,7 4,0±0,2 Median 6,9250 4,2900 3,9800 **WML metabolic peaks NAA Basal CHO Basal CR Basal**  Untreated Mean 6,03,7 4,41,9 3,81,8 Median 4,8900 4,6700 3,1900 Treated Mean 6,82,5 4,81,6 3,91,2 Median 5,4800 5,0950 3,7900

Table 1. Basal normal-appearing white matter and white matter metabolite levels in treated

We analyzed whether any difference emerged in the NAWM metabolite peaks between the treated and untreated patients throughout the study period and found that a significant difference existed in NAWM NAA peaks (P = .003); Cho peaks (P = .001); and Cr peaks (P = .006) beginning from the first month of the study. When we evaluated the groups separately to assess when that difference emerged between the basal and the following months' NAA, Cho and Cr peaks we noted that in the treatment group, the NAA peaks began to increase during the sixth month (P = .000); Cho peaks increased during the sixth and twelfth months (P = .000); and an increment in the Cr peak was found in the third (P< .04), sixth (P = .001), and twelfth

treated. Five other patients received INF-1a treatment. All patients were evaluated at the beginning of the study and during the first, third, sixth, and twelfth month. During their follow-up evaluations, neurological examinations that included EDSS assessment were performed in addition to MR studies with gadopentetate dimegluminelinium (Gd) 0,1 mmol/kg intravenously, and MRSI (Figure 1a-f). A relapse was defined as the appearance of new neurological symptoms or worsening of prior neurological symptoms of 48 hours' duration in addition to objective evidence of a change in neurological status of a patient who has been clinically stable for the 4 prior weeks. All patients suspected of having an attack were examined within 7 days of the onset of symptoms and were treated with intravenous methyprednisolone (IVMP) at a dose of 1 g/day for 5 days. At the beginning of the study, MRI and MRSI studies were also performed on 6 healthy, age-matched and sex-matched control subjects (4 women, 2 men; median age, 33.6 years, age range, 27-48 years) who had neither systemic nor neurologic disease. The local ethics committee approved the study.

#### **2.1.1 MRI studies**

All MRI and MRSI studies were performed with a 1.5 Tesla MR unit (Siemens Magnetom Symphony, Erlangen, Germany) with a standard quadrature head coil. To detect focal WM lesions, we used conventional MRI, including axial T1 weighted (TR 586 ms, TE 15 ms, 5 mm slice thickness), fluid-attenuated, inversion recovery (FLAIR) (TR 9000 ms, TE 98 ms, 5 mm slice thickness), axial and sagittal proton density (TR 2200 ms, TE 30 ms, 5 mm slice thickness), T2-weighted spin echo sequences (TR 2200 ms, TE 80 ms, 5 mm slice thickness) and post-gadolinium axial and sagittal T1 weighted (TR 779 ms, TE 15 ms, 5 mm slice thickness) spin echo sequences. MRI images were acquired with a matrix size of 145x256 mm and FOVof 230x230 mm. During the same session, water-suppressed proton MRSI was performed by means of multivoxel chemical shift imaging (TR/TE: 1500/135, voxel volume 1 cm3). Metabolite peaks of NAA, Cho, and Cr were assessed in both WML -without Gd enhancement- and in NAWM.

#### **2.1.2 Statistics**

SPSS 11 for Windows was used for statistical analysis. The Kruskal-Wallis Test was used to compare the basal NAWM metabolite peaks among treated, untreated and healthy control subjects. The Mann-Whitney U test was used to compare the basal WML metabolite peaks in treated and untreated patients. Then, multivariate analysis of variance (MANOVA) was used to compare the NAWM and WML metabolite peaks in treated and untreated patients throughout the study period. The Wilcoxon signed rank test was used to compare changes in the NAWM and WML metabolite peaks during study months zero, 1, 3, 6, and 12. Results with p< .05 were considered as significant.

#### **3. Results**

#### **3.1 Treatment effect as assessed by relapse rate, and new T2 lesions with and without GD enhancement**

Five patients (3 women and 2 men) elected not to be treated. Four of those 5 patients experienced no attacks during the 12 months of the study. However, the results of MRI examination revealed new Gd-enhancing lesions without a trace of an accompanying clinical MS attack at the initiation of the study in 3 patients during the sixth month of the study. The results of serial MR examinations of another patient revealed new T2 lesions without Gd-enhancement that were present without evidence of a clinical MS attack from the initiation of the study until the twelfth study month. However, during the twelfth month, that patient experienced a clinical MS attack (moderate paresis [4/5] of the left leg), and a new Gd-enhancing lesion was identified. And only that patient received pulse steroid therapy once daily for 5 days.

Five other patients received INF-1a treatment (subcutaneous INF-1a 44 µg –Rebif-3 times per week). Among the subjects who received INF, two patients had Gd-enhancing new T2 lesions on almost every MRI examination, another patient had a Gd-enhancing lesion only on the last MRI examination of the study, and the remaning patients exhibited no new lesions. None of those patients experienced a clinical MS attack accompanying the MRI activities. There was no significant change in the EDSS scores of the patients in either group throughout the study.

#### **3.2 MRSI results**

96 Magnetic Resonance Spectroscopy

treated. Five other patients received INF-1a treatment. All patients were evaluated at the beginning of the study and during the first, third, sixth, and twelfth month. During their follow-up evaluations, neurological examinations that included EDSS assessment were performed in addition to MR studies with gadopentetate dimegluminelinium (Gd) 0,1 mmol/kg intravenously, and MRSI (Figure 1a-f). A relapse was defined as the appearance of new neurological symptoms or worsening of prior neurological symptoms of 48 hours' duration in addition to objective evidence of a change in neurological status of a patient who has been clinically stable for the 4 prior weeks. All patients suspected of having an attack were examined within 7 days of the onset of symptoms and were treated with intravenous methyprednisolone (IVMP) at a dose of 1 g/day for 5 days. At the beginning of the study, MRI and MRSI studies were also performed on 6 healthy, age-matched and sex-matched control subjects (4 women, 2 men; median age, 33.6 years, age range, 27-48 years) who had neither systemic nor neurologic disease. The local ethics committee approved the study.

All MRI and MRSI studies were performed with a 1.5 Tesla MR unit (Siemens Magnetom Symphony, Erlangen, Germany) with a standard quadrature head coil. To detect focal WM lesions, we used conventional MRI, including axial T1 weighted (TR 586 ms, TE 15 ms, 5 mm slice thickness), fluid-attenuated, inversion recovery (FLAIR) (TR 9000 ms, TE 98 ms, 5 mm slice thickness), axial and sagittal proton density (TR 2200 ms, TE 30 ms, 5 mm slice thickness), T2-weighted spin echo sequences (TR 2200 ms, TE 80 ms, 5 mm slice thickness) and post-gadolinium axial and sagittal T1 weighted (TR 779 ms, TE 15 ms, 5 mm slice thickness) spin echo sequences. MRI images were acquired with a matrix size of 145x256 mm and FOVof 230x230 mm. During the same session, water-suppressed proton MRSI was performed by means of multivoxel chemical shift imaging (TR/TE: 1500/135, voxel volume 1 cm3). Metabolite peaks of NAA, Cho, and Cr were assessed in both WML -without Gd

SPSS 11 for Windows was used for statistical analysis. The Kruskal-Wallis Test was used to compare the basal NAWM metabolite peaks among treated, untreated and healthy control subjects. The Mann-Whitney U test was used to compare the basal WML metabolite peaks in treated and untreated patients. Then, multivariate analysis of variance (MANOVA) was used to compare the NAWM and WML metabolite peaks in treated and untreated patients throughout the study period. The Wilcoxon signed rank test was used to compare changes in the NAWM and WML metabolite peaks during study months zero, 1, 3, 6, and 12. Results

**3.1 Treatment effect as assessed by relapse rate, and new T2 lesions with and without**

Five patients (3 women and 2 men) elected not to be treated. Four of those 5 patients experienced no attacks during the 12 months of the study. However, the results of MRI examination revealed new Gd-enhancing lesions without a trace of an accompanying

**2.1.1 MRI studies**

enhancement- and in NAWM.

with p< .05 were considered as significant.

**2.1.2 Statistics**

**3. Results**

**GD enhancement**

There was no statistically significant difference between the basal metabolite levels in the NAWM of patients with MS and the healthy control subjects (*P*> .005) (Table 1). There was also no statistically significant difference between the basal metabolite peaks in the WML between the untreated and treated MS patients (Table 2).

NAA: N-acetyl aspartate, Cho: Choline, Cr: Creatine

Table 1. Basal normal-appearing white matter and white matter metabolite levels in treated and untreated patients and controls

We analyzed whether any difference emerged in the NAWM metabolite peaks between the treated and untreated patients throughout the study period and found that a significant difference existed in NAWM NAA peaks (P = .003); Cho peaks (P = .001); and Cr peaks (P = .006) beginning from the first month of the study. When we evaluated the groups separately to assess when that difference emerged between the basal and the following months' NAA, Cho and Cr peaks we noted that in the treatment group, the NAA peaks began to increase during the sixth month (P = .000); Cho peaks increased during the sixth and twelfth months (P = .000); and an increment in the Cr peak was found in the third (P< .04), sixth (P = .001), and twelfth

MRS in MS, With and Without Interferon Beta 1a Treatment,

untreated group (*P* > .05) (Graphic 2).

**4. Discussion**

concentrations.

to Define the Dynamic Changes of Metabolites in the Brain, and to Monitor Disease Progression 99

We found a significant difference between the changes in WML NAA (*P*= .001), Cho (*P* = .025); and Cr (*P* = .031) peaks of untreated and treated patients, beginning from the first month. When we evaluated the groups separately to determine when the difference emerged between the basal WML NAA peaks and the following months' NAA peaks, we noted that the NAA peaks started to increase during the sixth month in the treatment group (*P* = .001); the Cho peak started to increase during the first month (*P* = .03); and the Cr peak began to increase during the third month in the treatment group (*P* = .04). However, NAA, Cho or Cr peaks did not vary significantly in WML over the entire period of study in the

In this study, variations in cerebral metabolites were assessed during treatment with INF- 1a in a small group of patients with RRMS, and the results were compared with those in untreated patients matched for age and disability. We used the absolute quantification of the metabolites. The use of ratios is potentially less sensitive to the effects of disease in MS than is the use of absolute values; measurement error may be increased when two measures rather than one are required(Fernando et al., 2004). The use of the ratio to Cr also requires the assumption that Cr is normal in the patients' NAWM. However, there have been reports of abnormalities of Cr levels in demyelinating lesions and in the NAWM in patients with MS(Brex et al., 1999; Pan et al., 1996; Rooney et al., 1997; Suhy et al., 2000; van Walderveen, Truyen et al., 1999). Pan and colleagues found an isolated increase in Cr without a change in the level of Cho or NAA in the NAWM(Pan et al., 1996)of patients with MS. An increase in Cr was also found in a clinically isolated syndrome (CIS) group(Fernando et al., 2004). In that study, an increase in Cr was found in a subgroup of subjects with abnormal T2 weighted MRI results and in those with MS defined according to the McDonald criteria(Fernando et al., 2004). Those studies suggest that Cr may not be a reliable internal standard for use in the investigation of the effects of demylinating disease upon metabolite

We have found no significant difference between the basal metabolite peak areas of NAA, Cho and Cr in the NAWM of patients with MS and that of healthy controls (*P* >.005). This was attributed to the patients' having been on relatively early stages of the disease (primarily 2-5 years after diagnosis) and their low EDSS levels. Those factors indicate that widespread axonal damage either had not occurred or was not detectactable at this stage and that the reduced NAA found in established MS is acquired later in the disease course. In a study, when 15 clinically isolated syndrome patients with T2 abnormalities were considered separately, their NAWM NAA concentrations were not found to be significantly different from those in controls(Brex et al., 1999). In our study, there was no significant difference between NAWM metabolite peaks of the treated and untreated groups in the results of the initial MRSI (*P*> .005) showing that the 2 patient groups were homogenous. We found that in the treatment group, NAWM NAA and Cho peaks began to increase during the sixth month, and that Cr peaks began to increase during the third month. No change was observed in the untreated group. When we evaluated WML metabolite changes, we found that in the treatment group, NAA and Cr peaks began to increase during the third month and Cho peaks began to increase during the first month. No changes in the

metabolite peak areas of WML were observed in the untreated group.

study months (P = .00). However, in the untreated group, none of the NAA, Cho or Cr peaks (in NAWM) varied significantly during the entire study period (P> .05) (Graphic 1).

Graphic 1.

Graphic 2.

We found a significant difference between the changes in WML NAA (*P*= .001), Cho (*P* = .025); and Cr (*P* = .031) peaks of untreated and treated patients, beginning from the first month. When we evaluated the groups separately to determine when the difference emerged between the basal WML NAA peaks and the following months' NAA peaks, we noted that the NAA peaks started to increase during the sixth month in the treatment group (*P* = .001); the Cho peak started to increase during the first month (*P* = .03); and the Cr peak began to increase during the third month in the treatment group (*P* = .04). However, NAA, Cho or Cr peaks did not vary significantly in WML over the entire period of study in the untreated group (*P* > .05) (Graphic 2).

#### **4. Discussion**

98 Magnetic Resonance Spectroscopy

study months (P = .00). However, in the untreated group, none of the NAA, Cho or Cr peaks

(in NAWM) varied significantly during the entire study period (P> .05) (Graphic 1).

Graphic 1.

Graphic 2.

In this study, variations in cerebral metabolites were assessed during treatment with INF- 1a in a small group of patients with RRMS, and the results were compared with those in untreated patients matched for age and disability. We used the absolute quantification of the metabolites. The use of ratios is potentially less sensitive to the effects of disease in MS than is the use of absolute values; measurement error may be increased when two measures rather than one are required(Fernando et al., 2004). The use of the ratio to Cr also requires the assumption that Cr is normal in the patients' NAWM. However, there have been reports of abnormalities of Cr levels in demyelinating lesions and in the NAWM in patients with MS(Brex et al., 1999; Pan et al., 1996; Rooney et al., 1997; Suhy et al., 2000; van Walderveen, Truyen et al., 1999). Pan and colleagues found an isolated increase in Cr without a change in the level of Cho or NAA in the NAWM(Pan et al., 1996)of patients with MS. An increase in Cr was also found in a clinically isolated syndrome (CIS) group(Fernando et al., 2004). In that study, an increase in Cr was found in a subgroup of subjects with abnormal T2 weighted MRI results and in those with MS defined according to the McDonald criteria(Fernando et al., 2004). Those studies suggest that Cr may not be a reliable internal standard for use in the investigation of the effects of demylinating disease upon metabolite concentrations.

We have found no significant difference between the basal metabolite peak areas of NAA, Cho and Cr in the NAWM of patients with MS and that of healthy controls (*P* >.005). This was attributed to the patients' having been on relatively early stages of the disease (primarily 2-5 years after diagnosis) and their low EDSS levels. Those factors indicate that widespread axonal damage either had not occurred or was not detectactable at this stage and that the reduced NAA found in established MS is acquired later in the disease course. In a study, when 15 clinically isolated syndrome patients with T2 abnormalities were considered separately, their NAWM NAA concentrations were not found to be significantly different from those in controls(Brex et al., 1999). In our study, there was no significant difference between NAWM metabolite peaks of the treated and untreated groups in the results of the initial MRSI (*P*> .005) showing that the 2 patient groups were homogenous. We found that in the treatment group, NAWM NAA and Cho peaks began to increase during the sixth month, and that Cr peaks began to increase during the third month. No change was observed in the untreated group. When we evaluated WML metabolite changes, we found that in the treatment group, NAA and Cr peaks began to increase during the third month and Cho peaks began to increase during the first month. No changes in the metabolite peak areas of WML were observed in the untreated group.

MRS in MS, With and Without Interferon Beta 1a Treatment,

reactive features like those in the NAWM of patients with MS.

design because of ethical problems.

monitor the disease progression in MS.

**5. Conclusions**

**6. References**

81-91.

to Define the Dynamic Changes of Metabolites in the Brain, and to Monitor Disease Progression 101

Although an increase in the Cr level has been found in the NAWM of patients with MS(Brex et al., 1999; Rooney et al., 1997; Suhy et al., 2000; van Walderveen, Barkhof et al., 1999), some experts do not agree on the fluctuations of that metabolite in MS lesions. However, some authors have reported a decrease in total Cr(van Walderveen, Barkhof et al., 1999); others have reported an increase(Kapeller et al., 2001; Suhy et al., 2000). It has been suggested that apart from the axonal damage occurring in the NAWM of patients with MS, there is a proliferation of glial cells that produces an increase in the Cr level(Pan et al., 1996; van Walderveen, Barkhof et al., 1999). When working at 4.1tesla with 0.5 voxels, Pan and colleagues found an isolated increase in the Cr level without concomitant changes in Cho or NAA levels in the WM far from the WM lesions and suggested that in NAWM, there may have been reactive astrocytosis without inflammatory changes. It has been postulated that an increase in the Cr level may reflect an astrocytic (gliotic) or oligodendrocytic remyelinating process. Histological evidence confirms that gliosis is the most common abnormality in macroscopically normal WM of patients with MS. Thus the MRSI abnormalities observed in such patients may be due to the effect of microscopic lesions below the resolution of the images(Rooney et al., 1997; Suhy et al., 2000). However, the increase in Cr along with Cho levels in NAWM and WM may also be accounted for an increase in metabolism, which might be expected to occur with inflammatory or glial

There are some limitations of our study: The small number of patients, the short duration of study, an open label design, and the patients' being either in relatively early stages of disease or experiencing a relatively benign course of MS. However the numbers of patients in relatively early stages of the disease are more or less similar between the groups, and again the ones with a relatively benign form are again similarly distributed between the groups. Therefore we do not consider that this would have caused a bias in our results. It would have been better if the study were performed with a greater number of patients along with a longer study period. However we could not perform such a more desirable study

Our results showed that NAA, Cho and Cr in a VOI that predominantly contains NAWM increased in patients with MS after 1 year of treatment with INF-1a. The same effect was also observed in WML after treatment. This evidence supports the theory that although axonal pathology begins early in the course of MS, INF-1a therapy may reverse part of that early axonal and metabolic dysfunction, and thus inhibit permanent axonal loss. Follow-up studies could help to define the dynamic changes of metabolites in MS plaques and to

Allen, I. V., & McKeown, S. R. (1979). A histological, histochemical and biochemical study of

the macroscopically normal white matter in multiple sclerosis. *J Neurol Sci, 41*(1),

There are a number of possible explanations for the increase in NAA that occurred between the sixth and twelfth months of treatment. Chronic low-grade encephalitis is probably present in patients with MS. There is pathological evidence of an increase in inflammatory cells in the NAWM of such patients(Allen & McKeown, 1979). The results of studies using Gd-enhanced MRI suggest that the blood-brain barrier is diffusely impaired in patients with MS. This chronic, low-level inflammation could result in widespread, partially reversible axonal damage and loss, and INF could induce its reversal.

A number of tissue injury mediating factors have been implicated in the production of the symptoms of MS(Moreau et al., 1996). INF directly or indirectly inhibits both the production of a number of those associated inflammatory factors (including interferon gamma) and the ability of those factors to stimulate nitric oxide release from astrocytes(Stewart et al., 1997). Elevated nitric oxide production and the consequent damage to mitochondria in surrounding neurons may be mitigated by INF and this could be a mechanism of action of INF in patients with MS(Stewart, Land, Clark, & Heales, 1998). NAA is produced by neuronal mitochondria(Patel & Clark, 1979), this could explain the recovery of NAA in patients with MS who are treated with INF. The increase in NAA could also be associated with the reversal of axonal metabolic dysfunction that is associated with in apparent myelin pathology in the NAWM.

In this study, one of the principal effects, which seems to be due to INF-1a, was an increase in the total Cho peak, which in the NAWM became evident during the sixth month of treatment and persisted through the twelfth month, and in the WML became evident during the first month of treatment then persisted throughout the study period. Choline-containing compounds are considered to be turnover products of cell membranes, and an increase in the level of Cho seems to be related primarily to inflammatory and glial cell proliferation rather than to myelin destruction. This observation was confirmed in a histopathological and spectroscopic correlative study in EAE, the results of which showed that a high Cho was associated with inflammation and not with demyelination. Furthermore, glial proliferation in bioptic samples from patients with MS was consistent with an in vivo increase in Cho(Brenner et al., 1993). An increased level of Cho has been described in MS lesions in a variety of studies(Bitsch et al., 1999; Davie et al., 1994; Kapeller et al., 2001; Narayana et al., 1998). Myelin and membrane constituents make a major contribution to the constituent of Cho; therefore, increased lesion cellularity and turn over may account for the observed increase in the level of Cho. However, an increase in Cho has been interpreted as an index of active or recent demyelination by some authors(Confort-Gouny et al., 1993; Davie et al., 1994; Kapeller et al., 2001; Larsson et al., 1991), an association suggested by the finding of an abundance of Cho-containing compounds in myelin and in all cell membranes, including those of inflammatory cells, in patients with MS(Brenner et al., 1993). This increase in the level of Cho may occur without significant clinical deterioration or disease activity. In a recent study, an increase in metabolite concentrations of Cho and Cr was found in CIS NAWM(Fernando et al., 2004). H-MRS-visible Cho containing compounds are derived primarily from cell membranes and may be elevated when there is increased cell turnover. Because the relation between the variations in Cho levels and the pathological changes in underlying lesions is not very well known at the moment, it cannot be excluded that the rise in Cho peak could express an increased turnover of myelin in the plaques and NAWM examined, a turnover that may not be the result of new demyelination, but the consequence of the remodeling of plaques or the effects of a remyelination process.

Although an increase in the Cr level has been found in the NAWM of patients with MS(Brex et al., 1999; Rooney et al., 1997; Suhy et al., 2000; van Walderveen, Barkhof et al., 1999), some experts do not agree on the fluctuations of that metabolite in MS lesions. However, some authors have reported a decrease in total Cr(van Walderveen, Barkhof et al., 1999); others have reported an increase(Kapeller et al., 2001; Suhy et al., 2000). It has been suggested that apart from the axonal damage occurring in the NAWM of patients with MS, there is a proliferation of glial cells that produces an increase in the Cr level(Pan et al., 1996; van Walderveen, Barkhof et al., 1999). When working at 4.1tesla with 0.5 voxels, Pan and colleagues found an isolated increase in the Cr level without concomitant changes in Cho or NAA levels in the WM far from the WM lesions and suggested that in NAWM, there may have been reactive astrocytosis without inflammatory changes. It has been postulated that an increase in the Cr level may reflect an astrocytic (gliotic) or oligodendrocytic remyelinating process. Histological evidence confirms that gliosis is the most common abnormality in macroscopically normal WM of patients with MS. Thus the MRSI abnormalities observed in such patients may be due to the effect of microscopic lesions below the resolution of the images(Rooney et al., 1997; Suhy et al., 2000). However, the increase in Cr along with Cho levels in NAWM and WM may also be accounted for an increase in metabolism, which might be expected to occur with inflammatory or glial reactive features like those in the NAWM of patients with MS.

There are some limitations of our study: The small number of patients, the short duration of study, an open label design, and the patients' being either in relatively early stages of disease or experiencing a relatively benign course of MS. However the numbers of patients in relatively early stages of the disease are more or less similar between the groups, and again the ones with a relatively benign form are again similarly distributed between the groups. Therefore we do not consider that this would have caused a bias in our results. It would have been better if the study were performed with a greater number of patients along with a longer study period. However we could not perform such a more desirable study design because of ethical problems.

#### **5. Conclusions**

100 Magnetic Resonance Spectroscopy

There are a number of possible explanations for the increase in NAA that occurred between the sixth and twelfth months of treatment. Chronic low-grade encephalitis is probably present in patients with MS. There is pathological evidence of an increase in inflammatory cells in the NAWM of such patients(Allen & McKeown, 1979). The results of studies using Gd-enhanced MRI suggest that the blood-brain barrier is diffusely impaired in patients with MS. This chronic, low-level inflammation could result in widespread, partially reversible

A number of tissue injury mediating factors have been implicated in the production of the symptoms of MS(Moreau et al., 1996). INF directly or indirectly inhibits both the production of a number of those associated inflammatory factors (including interferon gamma) and the ability of those factors to stimulate nitric oxide release from astrocytes(Stewart et al., 1997). Elevated nitric oxide production and the consequent damage to mitochondria in surrounding neurons may be mitigated by INF and this could be a mechanism of action of INF in patients with MS(Stewart, Land, Clark, & Heales, 1998). NAA is produced by neuronal mitochondria(Patel & Clark, 1979), this could explain the recovery of NAA in patients with MS who are treated with INF. The increase in NAA could also be associated with the reversal of axonal metabolic dysfunction that is associated

In this study, one of the principal effects, which seems to be due to INF-1a, was an increase in the total Cho peak, which in the NAWM became evident during the sixth month of treatment and persisted through the twelfth month, and in the WML became evident during the first month of treatment then persisted throughout the study period. Choline-containing compounds are considered to be turnover products of cell membranes, and an increase in the level of Cho seems to be related primarily to inflammatory and glial cell proliferation rather than to myelin destruction. This observation was confirmed in a histopathological and spectroscopic correlative study in EAE, the results of which showed that a high Cho was associated with inflammation and not with demyelination. Furthermore, glial proliferation in bioptic samples from patients with MS was consistent with an in vivo increase in Cho(Brenner et al., 1993). An increased level of Cho has been described in MS lesions in a variety of studies(Bitsch et al., 1999; Davie et al., 1994; Kapeller et al., 2001; Narayana et al., 1998). Myelin and membrane constituents make a major contribution to the constituent of Cho; therefore, increased lesion cellularity and turn over may account for the observed increase in the level of Cho. However, an increase in Cho has been interpreted as an index of active or recent demyelination by some authors(Confort-Gouny et al., 1993; Davie et al., 1994; Kapeller et al., 2001; Larsson et al., 1991), an association suggested by the finding of an abundance of Cho-containing compounds in myelin and in all cell membranes, including those of inflammatory cells, in patients with MS(Brenner et al., 1993). This increase in the level of Cho may occur without significant clinical deterioration or disease activity. In a recent study, an increase in metabolite concentrations of Cho and Cr was found in CIS NAWM(Fernando et al., 2004). H-MRS-visible Cho containing compounds are derived primarily from cell membranes and may be elevated when there is increased cell turnover. Because the relation between the variations in Cho levels and the pathological changes in underlying lesions is not very well known at the moment, it cannot be excluded that the rise in Cho peak could express an increased turnover of myelin in the plaques and NAWM examined, a turnover that may not be the result of new demyelination, but the consequence

axonal damage and loss, and INF could induce its reversal.

with in apparent myelin pathology in the NAWM.

of the remodeling of plaques or the effects of a remyelination process.

Our results showed that NAA, Cho and Cr in a VOI that predominantly contains NAWM increased in patients with MS after 1 year of treatment with INF-1a. The same effect was also observed in WML after treatment. This evidence supports the theory that although axonal pathology begins early in the course of MS, INF-1a therapy may reverse part of that early axonal and metabolic dysfunction, and thus inhibit permanent axonal loss. Follow-up studies could help to define the dynamic changes of metabolites in MS plaques and to monitor the disease progression in MS.

#### **6. References**

Allen, I. V., & McKeown, S. R. (1979). A histological, histochemical and biochemical study of the macroscopically normal white matter in multiple sclerosis. *J Neurol Sci, 41*(1), 81-91.

MRS in MS, With and Without Interferon Beta 1a Treatment,

sclerosis. *Magn Reson Med, 22*(1), 23-31.

measurements. *Radiology, 196*(2), 511-515.

sclerosis. *Brain, 119 ( Pt 1)*, 225-237.

*Reson Med, 36*(1), 72-77.

*Brain, 122 ( Pt 3)*, 513-521.

*36*(2), 157-165.

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539-546.

*3*(4), 231-237.

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Gasperini, C., Horsfield, M. A., Thorpe, J. W., Kidd, D., Barker, G. J., Tofts, P. S., et al. (1996).

Kapeller, P., McLean, M. A., Griffin, C. M., Chard, D., Parker, G. J., Barker, G. J., et al. (2001).

Loevner, L. A., Grossman, R. I., Cohen, J. A., Lexa, F. J., Kessler, D., & Kolson, D. L. (1995).

Matthews, P. M., Pioro, E., Narayanan, S., De Stefano, N., Fu, L., Francis, G., et al. (1996).

Moreau, T., Coles, A., Wing, M., Isaacs, J., Hale, G., Waldmann, H., et al. (1996). Transient

Narayana, P. A., Doyle, T. J., Lai, D., & Wolinsky, J. S. (1998). Serial proton magnetic

Patel, T. B., & Clark, J. B. (1979). Synthesis of N-acetyl-L-aspartate by rat brain mitochondria

Rooney, W. D., Goodkin, D. E., Schuff, N., Meyerhoff, D. J., Norman, D., & Weiner, M. W.

Sarchielli, P., Presciutti, O., Pelliccioli, G. P., Tarducci, R., Gobbi, G., Chiarini, P., et al. (1999).

quantitative MR spectroscopic imaging study. *J Neurol, 248*(2), 131-138. Larsson, H. B., Christiansen, P., Jensen, M., Frederiksen, J., Heltberg, A., Olesen, J., et al.

Macroscopic and microscopic assessments of disease burden by MRI in multiple sclerosis: relationship to clinical parameters. *J Magn Reson Imaging, 6*(4), 580-584. Gonen, O., Catalaa, I., Babb, J. S., Ge, Y., Mannon, L. J., Kolson, D. L., et al. (2000). Total brain N-acetylaspartate: a new measure of disease load in MS. *Neurology, 54*(1), 15-19. Husted, C. A., Goodin, D. S., Hugg, J. W., Maudsley, A. A., Tsuruda, J. S., de Bie, S. H., et al.

(1994). Biochemical alterations in multiple sclerosis lesions and normal-appearing white matter detected by in vivo 31P and 1H spectroscopic imaging. *Ann Neurol,*

Preliminary evidence for neuronal damage in cortical grey matter and normal appearing white matter in short duration relapsing-remitting multiple sclerosis: a

(1991). Localized in vivo proton spectroscopy in the brain of patients with multiple

Microscopic disease in normal-appearing white matter on conventional MR images in patients with multiple sclerosis: assessment with magnetization-transfer

Assessment of lesion pathology in multiple sclerosis using quantitative MRI morphometry and magnetic resonance spectroscopy. *Brain, 119 ( Pt 3)*, 715-722. McDonald, W. I., Compston, A., Edan, G., Goodkin, D., Hartung, H. P., Lublin, F. D., et al.

(2001). Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis. *Ann Neurol, 50*(1), 121-

increase in symptoms associated with cytokine release in patients with multiple

resonance spectroscopic imaging, contrast-enhanced magnetic resonance imaging, and quantitative lesion volumetry in multiple sclerosis. *Ann Neurol, 43*(1), 56-71. Pan, J. W., Hetherington, H. P., Vaughan, J. T., Mitchell, G., Pohost, G. M., & Whitaker, J. N.

(1996). Evaluation of multiple sclerosis by 1H spectroscopic imaging at 4.1 T. *Magn*

and its involvement in mitochondrial/cytosolic carbon transport. *Biochem J, 184*(3),

(1997). 1H MRSI of normal appearing white matter in multiple sclerosis. *Mult Scler,*

Absolute quantification of brain metabolites by proton magnetic resonance spectroscopy in normal-appearing white matter of multiple sclerosis patients.

Armspach, J. P., Gounot, D., Rumbach, L., & Chambron, J. (1991). In vivo determination of

Arnold, D. L., Matthews, P. M., Francis, G. S., O'Connor, J., & Antel, J. P. (1992). Proton

Arnold, D. L., Riess, G. T., Matthews, P. M., Francis, G. S., Collins, D. L., Wolfson, C., et al.

Bitsch, A., Bruhn, H., Vougioukas, V., Stringaris, A., Lassmann, H., Frahm, J., et al. (1999).

Brex, P. A., Gomez-Anson, B., Parker, G. J., Molyneux, P. D., Miszkiel, K. A., Barker, G. J., et

Confort-Gouny, S., Vion-Dury, J., Nicoli, F., Dano, P., Donnet, A., Grazziani, N., et al. (1993).

Davie, C. A., Barker, G. J., Thompson, A. J., Tofts, P. S., McDonald, W. I., & Miller, D. H.

Davie, C. A., Hawkins, C. P., Barker, G. J., Brennan, A., Tofts, P. S., Miller, D. H., et al. (1994).

Davies, S. E., Newcombe, J., Williams, S. R., McDonald, W. I., & Clark, J. B. (1995). High

De Stefano, N., Matthews, P. M., Antel, J. P., Preul, M., Francis, G., & Arnold, D. L. (1995).

Ferguson, B., Matyszak, M. K., Esiri, M. M., & Perry, V. H. (1997). Axonal damage in acute

Fernando, K. T., McLean, M. A., Chard, D. T., MacManus, D. G., Dalton, C. M., Miszkiel, K.

Filippi, M., Campi, A., Dousset, V., Baratti, C., Martinelli, V., Canal, N., et al. (1995). A

Fu, L., Matthews, P. M., De Stefano, N., Worsley, K. J., Narayanan, S., Francis, G. S., et al.

*Magn Reson Imaging, 9*(1), 107-113.

demyelinating plaques. *Ann Neurol, 31*(3), 235-241.

the Cho:Cr ratio. *Magn Reson Med, 29*(6), 737-745.

multiple sclerosis. *J Neurol Sci, 166*(1), 16-22.

*J Neurol Sci, 118*(2), 123-133.

*Psychiatry, 63*(6), 736-742.

*Brain, 117 ( Pt 1)*, 49-58.

disability. *Ann Neurol, 38*(6), 901-909.

sclerosis. *Brain, 121 ( Pt 1)*, 103-113.

multiple sclerosis lesions. *Brain, 120 ( Pt 3)*, 393-399.

multiple sclerosis. *Neurology, 45*(3 Pt 1), 478-482.

suggestive of multiple sclerosis. *Brain, 127*(Pt 6), 1361-1369.

*64*(2), 742-748.

progression in multiple sclerosis. *Ann Neurol, 36*(1), 76-82.

multiexponential T2 relaxation in the brain of patients with multiple sclerosis.

magnetic resonance spectroscopic imaging for metabolic characterization of

(1994). Use of proton magnetic resonance spectroscopy for monitoring disease

Inflammatory CNS demyelination: histopathologic correlation with in vivo quantitative proton MR spectroscopy. *AJNR Am J Neuroradiol, 20*(9), 1619-1627. Brenner, R. E., Munro, P. M., Williams, S. C., Bell, J. D., Barker, G. J., Hawkins, C. P., et al.

(1993). The proton NMR spectrum in acute EAE: the significance of the change in

al. (1999). Proton MR spectroscopy in clinically isolated syndromes suggestive of

A multiparametric data analysis showing the potential of localized proton MR spectroscopy of the brain in the metabolic characterization of neurological diseases.

(1997). 1H magnetic resonance spectroscopy of chronic cerebral white matter lesions and normal appearing white matter in multiple sclerosis. *J Neurol Neurosurg*

Serial proton magnetic resonance spectroscopy in acute multiple sclerosis lesions.

resolution proton NMR spectroscopy of multiple sclerosis lesions. *J Neurochem,*

Chemical pathology of acute demyelinating lesions and its correlation with

A., et al. (2004). Elevated white matter myo-inositol in clinically isolated syndromes

magnetization transfer imaging study of normal-appearing white matter in

(1998). Imaging axonal damage of normal-appearing white matter in multiple

**6**

**Magnetic Resonance Spectroscopy**

**(MRS) in Kidney Transplantation:**

Bon Delphine, Seguin François and Hauet Thierry

*CHU Poitiers, Pole UBM, Service de Biochimie, Poitiers IBISA, Domaine Expérimental du Magneraud, Surgères*

*Université de Poitiers, Faculté de Médecine et de Pharmacie, Poitiers*

Currently, in biology, Magnetic Resonance Spectroscopy (MRS) is widely used in metabonomics for diagnosis and prognostication in a wide range of studies from brain (Blasco et al., 2010) to leg (Borel et al., 2009) pathologies. Between both, the topic of interest in this section: the kidney. We will especially focus on kidney transplantation. Indeed, in France as well as in Europe, the number of patients awaiting transplant is still rising while the number of transplantations performed remains stable and even decreases (Figure 1.).

Patient awaiting transplant and transplants performed between 2003 and 2010 in France Awaiting transplant Transplant performed

2003 2004 2005 2006 2007 2008 2009 2010

Fig. 1. Evolution of the number of patients on the waiting list for transplant and the number

of transplantations performed between 2003 and 2010 in France (from Agence de la

**1. Introduction**

0

Biomédecine Rapport annuel 2011).

**Interest and Perspectives**

*Inserm U927, Poitiers*

*France*

### **Magnetic Resonance Spectroscopy (MRS) in Kidney Transplantation: Interest and Perspectives**

Bon Delphine, Seguin François and Hauet Thierry *Inserm U927, Poitiers Université de Poitiers, Faculté de Médecine et de Pharmacie, Poitiers CHU Poitiers, Pole UBM, Service de Biochimie, Poitiers IBISA, Domaine Expérimental du Magneraud, Surgères France*

#### **1. Introduction**

104 Magnetic Resonance Spectroscopy

Stewart, V. C., Giovannoni, G., Land, J. M., McDonald, W. I., Clark, J. B., & Heales, S. J.

Suhy, J., Rooney, W. D., Goodkin, D. E., Capizzano, A. A., Soher, B. J., Maudsley, A. A., et al.

Tourbah, A., Stievenart, J. L., Gout, O., Fontaine, B., Liblau, R., Lubetzki, C., et al. (1999).

van Walderveen, M. A., Truyen, L., van Oosten, B. W., Castelijns, J. A., Lycklama a Nijeholt,

secondary progressive multiple sclerosis. *Neurology, 53*(5), 1091-1097. van Walderveen, M. A., Barkhof, F., Pouwels, P. J., van Schijndel, R. A., Polman, C. H., &

and relapsing-remitting MS. *Mult Scler, 6*(3), 148-155.

inflammatory activity. *Arch Neurol, 56*(3), 345-351.

gamma induction of nitric oxide synthase. *J Neurochem, 68*(6), 2547-2551. Stewart, V. C., Land, J. M., Clark, J. B., & Heales, S. J. (1998). Pretreatment of astrocytes with

*Neurochem, 70*(1), 432-434.

*Neurol, 46*(1), 79-87.

(1997). Pretreatment of astrocytes with interferon-alpha/beta impairs interferon-

interferon-alpha/beta prevents neuronal mitochondrial respiratory chain damage. *J*

(2000). 1H MRSI comparison of white matter and lesions in primary progressive

Localized proton magnetic resonance spectroscopy in relapsing remitting versus

Castelijns, J. A. (1999). Neuronal damage in T1-hypointense multiple sclerosis lesions demonstrated in vivo using proton magnetic resonance spectroscopy. *Ann*

G. J., van Waesberghe, J. H., et al. (1999). Development of hypointense lesions on T1-weighted spin-echo magnetic resonance images in multiple sclerosis: relation to

> Currently, in biology, Magnetic Resonance Spectroscopy (MRS) is widely used in metabonomics for diagnosis and prognostication in a wide range of studies from brain (Blasco et al., 2010) to leg (Borel et al., 2009) pathologies. Between both, the topic of interest in this section: the kidney. We will especially focus on kidney transplantation. Indeed, in France as well as in Europe, the number of patients awaiting transplant is still rising while the number of transplantations performed remains stable and even decreases (Figure 1.).

Patient awaiting transplant and transplants performed between 2003 and 2010 in France

Fig. 1. Evolution of the number of patients on the waiting list for transplant and the number of transplantations performed between 2003 and 2010 in France (from Agence de la Biomédecine Rapport annuel 2011).

Magnetic Resonance Spectroscopy (MRS) in Kidney Transplantation: Interest and Perspectives 107

diabetic nephropathy induced by streptozotocin, the authors performed a holistic metabolic analysis in order to elucidate the mechanism of this disease. For that purpose, they realised 1H liquid NMR spectra of urine on one hand and kidney extracts on the other. Elevated level of glucose in diabetic rats affected the ketone pathway, fatty acid oxidation, tricarboxylic acid cycle and glycolosis. Those metabolic changes led to decrease of energy production, hence aggravating kidney damage. Moreover, in this study the authors assessed osmolyte

Another cause of kidney failure is autosomal dominant polycystic kidney diseases (ADPKD). Some candidate urinary protein biomarkers such as KIM1 or NGAL have already been evaluated for this nephropathology. However, to improve the quality of diagnosis for ADPKD, complete 1H and 13C NMR fingerprint of urine were put in place (Gronwald et al., 2011). Elevated levels of proteins and methanol were found in patients with ADPKD

NMR is well suited for the study of neprotoxicity. In 1998, the team of Nicholson reported urine metabolic profiles in 15 groups of animal treated with nephrotoxic molecules and compared spectra with control. They thus highlighted endogenous biomarkers of nephrotoxic insult (Holmes et al., 1998). Citrate, 2-oxoglutarate and hippurate were the most discriminating metabolites between treated group and control group whereas formate and dimethylamine showed almost not change. Other studies on administration of nephrotoxic compounds realised with urine and plasma of Sprague-Dawley rats focused on lanthanum, a rare earth compound used as fertilizer with a higher accumulation rate and a lower metabolic rate (Feng et al., 2002) or thioacetamide (Waters et al., 2005). In the first study, the authors showed that a 6-months long ingestion of La3+-induced nephro- and hepato-toxicity which could be highlighting by using NMR. From a dose of 10 mg/kg of La(NO3)3+, all the evolving quantified metabolites involved Krebs cycle intermediates or amino acids that are potential NMR markers for La3+-induced proximal tubular lesions. Moreover, decrease of creatinine in urine highlighted a low glomerular filtration. A second study focused on energy intermediairy (citrate, lactate, succinate) and lipid metabolisms. The work also realised on intact kidney tissue using hrmas NMR spectroscopy (Garrod et al., 2001; Wang et al., 2006) established changes in the spectral profile of renal papilla involved the marked depletion of several renal

Finally, the effects of cyclosporine A, the basis for most immunosuppressive protocols and used for allograft recipients on kidneys were evaluated (Lenz et al., 2004; Serkova et al., 2003). This treatment is well known for its nephrotoxicity. Cyclosporine A induced a decrease of poly unsaturated fatty acid and an increase of lipid peroxidation which suggests the use of an alternative pathway for energy production when the oxidative mitochondrial pathway is inhibited. This lipid peroxidation is responsive for kidney damage especially in the cortex and medulla. Cyclosporine A also leads to osmolyte regulation (taurine, betaine

The isolated perfused pig kidney (IPK) was used to mimic the ischemia reperfusion episode occurring during transplantation. This model was destined to assess initial renal function

osmolytes such as glycerophosphocholine, betaine, and myo-inositol.

metabolism.

receiving medication against hypertension.

and trimethylamine-N-oxide (TMAO)).

**2.2 Kidney transplantation analysis**

**2.2.1 Isolated Perfused Kidney (IPK) of pig**

Although campaigns for organ donation increase the number of donors, donated organs are most often in marginal conditions because the donors often present co-morbidity factors (diabetes, obesity, cardiovascular disease, hypertension, poor renal function). In the case of the kidney, the percentage of successful grafts tends to decrease. These factors weaken the organ and render it more susceptible to develop lesions following ischemia reperfusion, the unavoidable syndrome encountered during organ preservation. As the extend of these lesions is correlated to graft outcome, it is necessary to improve the entire sequence of ischemia-reperfusion and in particular to find ways to improve storage conditions and decrease the rate of delayed graft function after kidney transplantation.

In kidney transplantation, delayed graft function is mainly due to the intensity of ischemia reperfusion lesions associated with the hypothermic and hypoxic conditions during preservation. These lesions are exacerbated at the moment of transplantation with reoxygenation that occurs during the reperfusion. In this light, tissue damages identified by the detection of endogenous metabolic changes in plasma and urine would permit a better estimation of graft quality and improved management of the graft, to the direct benefit of the patient. High Resolution Magnetic Resonance Spectroscopy (HR MRS) in these fluids could permit the identification of biomarkers indicative of specific kidney lesions occurring during an ischemia-reperfusion sequence.

The purpose of the chapter is to make a mini-review about thematics involving kidney transplantation from animals, mainly large white pig, to humans in order to highlight the potential of MRS, high resolution or imaging, and what perspectives could be envisioned to improve comprehension of kidney transplantation.

#### **2. High resolution NMR**

#### **2.1 Kidney analysis**

Before considering transplantation, it is important to review nephrologic situations and nephropathologies that have been widely analyzed by MRS to find biomarkers of pathologies associated to kidney.

In 1999, Garrod and al. established the biochemical composition of normal inner and outer renal cortex and renal papilla from rats (Garrod et al., 1999). This work realised with 1H liquid NMR spectroscopy on tissue extracts and with 1H hrmas (high resolution magic angle spining) NMR spectroscopy on crude tissue allowed the direct observation of metabolites into kidney and showed the metabolic composition of the 3 main areas of the kidney. They established the 1H (hydrogen-1) and 13C (carbon-13) chemical shift assignments of metabolites found in cortex and/or papilla. Still with 1H hrmas NMR spectroscopy, a team realised the metabolic profiling of normal and hypertensive rat kidney (Huhn et al., 2004). They showed that better differentiation between both groups occurred in the kidney cortex. It has also been shown that NMR can be used to compare intact kidney, blood plasma and urine of 4 species of rodent. They thus showed that metabolic data acquired on laboratory animals can only be extended to wild species with the use of a lot of precaution (Griffin et al., 2000).

On those bases, and among pathologies affecting the kidney, diabetic nephropathy is one of the lethal manifestations of diabetic systemic disease (Zhao et al., 2011). Using a rat model of diabetic nephropathy induced by streptozotocin, the authors performed a holistic metabolic analysis in order to elucidate the mechanism of this disease. For that purpose, they realised 1H liquid NMR spectra of urine on one hand and kidney extracts on the other. Elevated level of glucose in diabetic rats affected the ketone pathway, fatty acid oxidation, tricarboxylic acid cycle and glycolosis. Those metabolic changes led to decrease of energy production, hence aggravating kidney damage. Moreover, in this study the authors assessed osmolyte metabolism.

Another cause of kidney failure is autosomal dominant polycystic kidney diseases (ADPKD). Some candidate urinary protein biomarkers such as KIM1 or NGAL have already been evaluated for this nephropathology. However, to improve the quality of diagnosis for ADPKD, complete 1H and 13C NMR fingerprint of urine were put in place (Gronwald et al., 2011). Elevated levels of proteins and methanol were found in patients with ADPKD receiving medication against hypertension.

NMR is well suited for the study of neprotoxicity. In 1998, the team of Nicholson reported urine metabolic profiles in 15 groups of animal treated with nephrotoxic molecules and compared spectra with control. They thus highlighted endogenous biomarkers of nephrotoxic insult (Holmes et al., 1998). Citrate, 2-oxoglutarate and hippurate were the most discriminating metabolites between treated group and control group whereas formate and dimethylamine showed almost not change. Other studies on administration of nephrotoxic compounds realised with urine and plasma of Sprague-Dawley rats focused on lanthanum, a rare earth compound used as fertilizer with a higher accumulation rate and a lower metabolic rate (Feng et al., 2002) or thioacetamide (Waters et al., 2005). In the first study, the authors showed that a 6-months long ingestion of La3+-induced nephro- and hepato-toxicity which could be highlighting by using NMR. From a dose of 10 mg/kg of La(NO3)3+, all the evolving quantified metabolites involved Krebs cycle intermediates or amino acids that are potential NMR markers for La3+-induced proximal tubular lesions. Moreover, decrease of creatinine in urine highlighted a low glomerular filtration. A second study focused on energy intermediairy (citrate, lactate, succinate) and lipid metabolisms. The work also realised on intact kidney tissue using hrmas NMR spectroscopy (Garrod et al., 2001; Wang et al., 2006) established changes in the spectral profile of renal papilla involved the marked depletion of several renal osmolytes such as glycerophosphocholine, betaine, and myo-inositol.

Finally, the effects of cyclosporine A, the basis for most immunosuppressive protocols and used for allograft recipients on kidneys were evaluated (Lenz et al., 2004; Serkova et al., 2003). This treatment is well known for its nephrotoxicity. Cyclosporine A induced a decrease of poly unsaturated fatty acid and an increase of lipid peroxidation which suggests the use of an alternative pathway for energy production when the oxidative mitochondrial pathway is inhibited. This lipid peroxidation is responsive for kidney damage especially in the cortex and medulla. Cyclosporine A also leads to osmolyte regulation (taurine, betaine and trimethylamine-N-oxide (TMAO)).

#### **2.2 Kidney transplantation analysis**

106 Magnetic Resonance Spectroscopy

Although campaigns for organ donation increase the number of donors, donated organs are most often in marginal conditions because the donors often present co-morbidity factors (diabetes, obesity, cardiovascular disease, hypertension, poor renal function). In the case of the kidney, the percentage of successful grafts tends to decrease. These factors weaken the organ and render it more susceptible to develop lesions following ischemia reperfusion, the unavoidable syndrome encountered during organ preservation. As the extend of these lesions is correlated to graft outcome, it is necessary to improve the entire sequence of ischemia-reperfusion and in particular to find ways to improve storage conditions and

In kidney transplantation, delayed graft function is mainly due to the intensity of ischemia reperfusion lesions associated with the hypothermic and hypoxic conditions during preservation. These lesions are exacerbated at the moment of transplantation with reoxygenation that occurs during the reperfusion. In this light, tissue damages identified by the detection of endogenous metabolic changes in plasma and urine would permit a better estimation of graft quality and improved management of the graft, to the direct benefit of the patient. High Resolution Magnetic Resonance Spectroscopy (HR MRS) in these fluids could permit the identification of biomarkers indicative of specific kidney lesions occurring

The purpose of the chapter is to make a mini-review about thematics involving kidney transplantation from animals, mainly large white pig, to humans in order to highlight the potential of MRS, high resolution or imaging, and what perspectives could be envisioned to

Before considering transplantation, it is important to review nephrologic situations and nephropathologies that have been widely analyzed by MRS to find biomarkers of

In 1999, Garrod and al. established the biochemical composition of normal inner and outer renal cortex and renal papilla from rats (Garrod et al., 1999). This work realised with 1H liquid NMR spectroscopy on tissue extracts and with 1H hrmas (high resolution magic angle spining) NMR spectroscopy on crude tissue allowed the direct observation of metabolites into kidney and showed the metabolic composition of the 3 main areas of the kidney. They established the 1H (hydrogen-1) and 13C (carbon-13) chemical shift assignments of metabolites found in cortex and/or papilla. Still with 1H hrmas NMR spectroscopy, a team realised the metabolic profiling of normal and hypertensive rat kidney (Huhn et al., 2004). They showed that better differentiation between both groups occurred in the kidney cortex. It has also been shown that NMR can be used to compare intact kidney, blood plasma and urine of 4 species of rodent. They thus showed that metabolic data acquired on laboratory animals can only be extended to wild species with the use of a lot

On those bases, and among pathologies affecting the kidney, diabetic nephropathy is one of the lethal manifestations of diabetic systemic disease (Zhao et al., 2011). Using a rat model of

decrease the rate of delayed graft function after kidney transplantation.

during an ischemia-reperfusion sequence.

**2. High resolution NMR**

pathologies associated to kidney.

of precaution (Griffin et al., 2000).

**2.1 Kidney analysis**

improve comprehension of kidney transplantation.

#### **2.2.1 Isolated Perfused Kidney (IPK) of pig**

The isolated perfused pig kidney (IPK) was used to mimic the ischemia reperfusion episode occurring during transplantation. This model was destined to assess initial renal function

Magnetic Resonance Spectroscopy (MRS) in Kidney Transplantation: Interest and Perspectives 109

glomerular filtration rate (GFR), tubular reabsortion of sodium and lactate dehydrogenase

The IPK model, which provided ex vivo information, was converted to a preclinical model with 3-months old large white pig. Briefly, the basic model was to remove the kidney, preserve it 24 hours and then transplant it in the same animal with a contralateral nephrectomy. We followed the animal for 3 months after transplantation and we studied

In this model, damage biomarkers appearing after an ischemia reperfusion episode were analyzed in order to confirm their pertinence with the follow up of the recovery of function. To summarize, metabolites quantified with liquid NMR were TMAO, correlated to medullar lesions, acetate correlated to cortical lesions and lactate linked to global ischemia. Amino acids such as alanine or valine could be associated with proximal tubules dysfunction. Creatinine measured in both plasma and urine allowed the determination of GFR. All these

Fig. 3. Levels of trimethylamine-N-oxide (A) and dimethylamine (B) in urine and changes of trimethylamine-N-oxide in plasma (C). Levels of citrate (D), acetate (E), and lactate (F) excretion in urine after autotransplantation. °P , 0.05, °° P , 0.01 (Hauet et al., 2000b).

levels (Hauet et al., 2000d).

**2.2.2 In vivo autotransplantation pig model**

biochemical parameter in order to correlate them with MRS data.

metabolites were highlighted in different studies summarized below.

after different preservation condition. The schematic diagram of the isolated perfusion method is illustrated in the Figure 2. Based on an isolated perfused kidney, HR MRS injury biomarkers of an ischemia-reperfusion sequence were identified in the urine. Spectra modifications compared to histological or biochemical analysis showed changes of some metabolites excretion.

Fig. 2. Schematic diagram illustrating the isolated perfusion method in which A is the peristaltic pump, B, the oxygenator, C, the filter, D, the pressure transducer, E, the flow meter and electromagnetic flow probe (Hauet et al., 2000d).

One of the first studies using HR MRS on this model was performed to mimic the non-heartbeating donor situation. The kidney underwent a warm ischemic period before the hypothermic period of conservation and reperfusion (Hauet et al., 1997b). In this study, 3 groups of pigs were used. In the control group, kidneys were flushed with cold heparinized saline and immediately perfused. Kidney of a second group underwent a cold flush and 24 hours of cold-storage preservation (CSP) and finally reperfusion. The last group was composed of kidneys with 30 minutes of warm ischemia, 24 hours of CSP and reperfusion. The reperfusion occurred at 37°C and was performed with Kreb's solution supplemented with 22 amino acids. Among the interesting metabolites, TMAO and lactate were higher in the group with warm ischemia and related to damage of medullar cells and to tubular dysfunction respectively. Another study performed with the IPK was performed to assess the effect of cold storage conservation time with EuroCollins solution (EC) (Hauet et al., 1997a). Perfusion time was pushed to 72 hours and the degree of proximal tubule cell damage was increased with prolonged cold ischemia (Goujon et al., 1999). Urine content after a delay of 24 hours of perfusion was not significantly different from urine content at the beginning of the perfusion. After 48 hours, urine profile was significantly different. Rises of TMAO/creatinine and lactate/creatinine ratio measured with NMR were in accordance with elevated levels of beta-N-acetylglucosaminidase and lactate dehydrogenase. The same protocol was used to compare EC to University of Wisconsin solution (UW) (Hauet et al., 2000c; Hauet et al., 1999). Metabolites released into the conservation solution analyzed with NMR also confirmed classical biochemical parameters such as perfusion flow rate, glomerular filtration rate (GFR), tubular reabsortion of sodium and lactate dehydrogenase levels (Hauet et al., 2000d).

#### **2.2.2 In vivo autotransplantation pig model**

108 Magnetic Resonance Spectroscopy

after different preservation condition. The schematic diagram of the isolated perfusion method is illustrated in the Figure 2. Based on an isolated perfused kidney, HR MRS injury biomarkers of an ischemia-reperfusion sequence were identified in the urine. Spectra modifications compared to histological or biochemical analysis showed changes of some

Fig. 2. Schematic diagram illustrating the isolated perfusion method in which A is the peristaltic pump, B, the oxygenator, C, the filter, D, the pressure transducer, E, the flow

One of the first studies using HR MRS on this model was performed to mimic the non-heartbeating donor situation. The kidney underwent a warm ischemic period before the hypothermic period of conservation and reperfusion (Hauet et al., 1997b). In this study, 3 groups of pigs were used. In the control group, kidneys were flushed with cold heparinized saline and immediately perfused. Kidney of a second group underwent a cold flush and 24 hours of cold-storage preservation (CSP) and finally reperfusion. The last group was composed of kidneys with 30 minutes of warm ischemia, 24 hours of CSP and reperfusion. The reperfusion occurred at 37°C and was performed with Kreb's solution supplemented with 22 amino acids. Among the interesting metabolites, TMAO and lactate were higher in the group with warm ischemia and related to damage of medullar cells and to tubular dysfunction respectively. Another study performed with the IPK was performed to assess the effect of cold storage conservation time with EuroCollins solution (EC) (Hauet et al., 1997a). Perfusion time was pushed to 72 hours and the degree of proximal tubule cell damage was increased with prolonged cold ischemia (Goujon et al., 1999). Urine content after a delay of 24 hours of perfusion was not significantly different from urine content at the beginning of the perfusion. After 48 hours, urine profile was significantly different. Rises of TMAO/creatinine and lactate/creatinine ratio measured with NMR were in accordance with elevated levels of beta-N-acetylglucosaminidase and lactate dehydrogenase. The same protocol was used to compare EC to University of Wisconsin solution (UW) (Hauet et al., 2000c; Hauet et al., 1999). Metabolites released into the conservation solution analyzed with NMR also confirmed classical biochemical parameters such as perfusion flow rate,

meter and electromagnetic flow probe (Hauet et al., 2000d).

metabolites excretion.

The IPK model, which provided ex vivo information, was converted to a preclinical model with 3-months old large white pig. Briefly, the basic model was to remove the kidney, preserve it 24 hours and then transplant it in the same animal with a contralateral nephrectomy. We followed the animal for 3 months after transplantation and we studied biochemical parameter in order to correlate them with MRS data.

In this model, damage biomarkers appearing after an ischemia reperfusion episode were analyzed in order to confirm their pertinence with the follow up of the recovery of function. To summarize, metabolites quantified with liquid NMR were TMAO, correlated to medullar lesions, acetate correlated to cortical lesions and lactate linked to global ischemia. Amino acids such as alanine or valine could be associated with proximal tubules dysfunction. Creatinine measured in both plasma and urine allowed the determination of GFR. All these metabolites were highlighted in different studies summarized below.

Fig. 3. Levels of trimethylamine-N-oxide (A) and dimethylamine (B) in urine and changes of trimethylamine-N-oxide in plasma (C). Levels of citrate (D), acetate (E), and lactate (F) excretion in urine after autotransplantation. °P , 0.05, °° P , 0.01 (Hauet et al., 2000b).

Magnetic Resonance Spectroscopy (MRS) in Kidney Transplantation: Interest and Perspectives 111

with perchloric acid of kidney tissue samples 24 hours after transplantation were performed and hydrosoluble metabolites were analyzed with 1H liquid NMR spectroscopy. In parallel, blood samples were analyzed. TMAO again gave the best results. Its decreased levels in the blood of NAC group compared to control group were associated with a decrease of allantoin in the blood and kidney tissue. This latter metabolite results from the free radical action of urate and its presence indicates free radical activities. Pretreatment with NAC reduced radical excretion, hence reduced free radical activity and thus tissue damages (Fuller et al., 2004).

Use of HR MRS in Human to follow transplanted kidney recovery of function highlighted several issues. The main problem came from the drug treatment received by patients after transplantation. We observed overlapping between the signals of the metabolites of interest and of the drugs. In these conditions it is often difficult to extract information about the kidney function. Moreover a high variability, in addition of the individual variability, is often observed. This variability, mainly due to diet**,** may cause urine profile modifications which can hide variations of the metabolite of interest in relation with the kidney injuries. One solution could be to introduce a 2-days controlled diet before each sampling to minimise those environmental variation. However, an older study performed on 33 patients who underwent primary renal allograft transplantation, using the same immunosuppressive regiment and of which 57.6% achieve immediate graft function, showed that patients with good graft function and normal patients had urinary TMAO/creatinine level inferior to 200 µmol/mM while patients with graft dysfunction had urinary TMAO/creatinine level of 410

Hypothermic machine perfusion (MP) is increasingly being preferred to CSP. Indeed, MP has been showed to offer protection to the organ since the 80's (Alijani et al., 1985; Kwiatkowski et al., 2007; Wight et al., 2003) with benefits such as reduction of delayed graft function and increased survival. However, the mechanisms involved in these improvements are still unclear and need to be elucidated. Biomarkers such as lactate dehydrogenase, total glutathione-S-transferase or N-acetyl--D-glucosaminidase measured at the end of the perfusion in the machine perfusates are used by clinicians. Even if those markers are independently efficient to predict graft outcome, they are not efficient enough to be taken into account in the decision to transplant or discard a donation after brain death or controlled donation after cardiac death (Moers et al., 2010; Siew et al., 2011). Again the potential of multivariate analysis offered by NMR could be used to predict graft outcome. In a recent study in a pig model of autotransplantation of livers, the effect of warm ischemia on

Currently, our team is attempting to answer the question: can 1H HR NMR metabonomic analysis during machine perfusion be used to predict graft outcome? We thus work on perfusates from different protocols of kidney transplantation using MP. We evaluate the effect of warm ischemia, duration of ischemia, types of solution and temperatures of preservation and finally effect of adjuvant in the perfusion solution. We showed, in a small

metabolites contents into perfusats was measured (Liu et al., 2009).

**2.2.3 Application to Human**

± 102 µmol/mM (Foxall et al., 1993).

**2.3 Perspectives**

**2.3.1 Perfusate analysis**

In 2000, comparison of EC and UW solution with CSP, already performed on IPK of pig, was performed on this preclinical model of kidney autotransplantation (Hauet et al., 2000b). The control group, undergoing uninephrectomy, presented 100% survival at day 14 whereas only 75% and 66 % of UW and EC pigs survived, respectively. Glutathione-S-transferase and creatinine clearance were significantly different between EC and UW groups with a light advantages for UW compared to control. Results obtained with MRS on urine and plasma are presented in figure 3. Delayed graft function was associated with the release of osmolytes such as TMAO in urine and in plasma. Moreover, dimethylamine, acetate, citrate and lactate level in the UW group were always closer to control group levels compared to levels in the EC group. These data outline the effect of UW preservation against renal medullar injury and impairment of oxidative metabolism. Here, strength of MRS was highlighted: in one experiment, with a 200 to 500 µL volume of sample, detection and quantitation of large number of metabolites can be made. This allows for a multivariable analysis which is an important factor for diagnostic and prognostic purpose.

Using the same experimental design, the impact of the preservation solution was demonstrated and the addition of colloids (polyethylene glycol (PEG)) into an extracellular solution showed better results (Faure et al., 2002). Indeed, in the urine spectra, TMAO/creatine ratio were significantly higher in kidney preserved with EC, UW or a solution containing PEG-50 than in kidney preserved with a solution containing PEG-30. This demonstrated that PEG reduced damages to the renal medulla during conservation, in a dose-dependent manner. Citrate was not detected during the first postoperative week in urine from kidneys preserved in EC. Its excretion was detected significantly earlier in urine from the ICPEG30 group than from the UW and ICPEG50 groups. This suggested that PEG associated to an intracellular solution improved oxidative metabolism and reduced renal medulla injury, also in a concentration-dependent manner. Furthermore, the addition of antioxidative (Baumert et al., 1999) or anticoagulant (Favreau et al., 2010) molecules, trimetazidine and melagatran respectively, into the preservation solution increased the performance of the hypothermic conservation. The effect of trimetazidine during conservation with EC or UW showed interesting results. The early excretion of acetate in the trimetazidine supplemented group demonstrated an efficient function recovery of the citric acid cycle compared with standard solutions (Hauet et al., 2000a). Combination of PEG and trimetazidine in conservation solution was the next step of long term time study which included 18 groups also exploring cold ischemia time (Doucet et al., 2004). Biological parameters indicated that reduced delayed graft was found when combining PEG and trimetazidine during CSP. Histological staining for CD4+ positive cells indicated that PEG reduced CD4+ positive cells infiltration and trimetazidine was efficient in reducing the inflammatory reaction. The NMR analysis was again in accordance with these results. Acetate excretion between day 1 and day 14 was improved in all experimental group preserved with trimetazidine while urinary TMAO was reduced in all trimetazidine group and we noticed presence of TMAO in plasma in all group except from control. Combination of trimetazidine with PEG seemed to be the best option for kidney conservation and MRS brought supplementary information to classical biochemical parameters such as GFR or sodium excretion. In addition, this study underlined that MRS permits the performance of a multivariate analysis in a single experiment.

Use of conditioning drugs before conservation was also investigated. Donor pretreatment with N-acetylcysteine (NAC) has been shown to ameliorate acute renal failure. Extractions with perchloric acid of kidney tissue samples 24 hours after transplantation were performed and hydrosoluble metabolites were analyzed with 1H liquid NMR spectroscopy. In parallel, blood samples were analyzed. TMAO again gave the best results. Its decreased levels in the blood of NAC group compared to control group were associated with a decrease of allantoin in the blood and kidney tissue. This latter metabolite results from the free radical action of urate and its presence indicates free radical activities. Pretreatment with NAC reduced radical excretion, hence reduced free radical activity and thus tissue damages (Fuller et al., 2004).

#### **2.2.3 Application to Human**

110 Magnetic Resonance Spectroscopy

In 2000, comparison of EC and UW solution with CSP, already performed on IPK of pig, was performed on this preclinical model of kidney autotransplantation (Hauet et al., 2000b). The control group, undergoing uninephrectomy, presented 100% survival at day 14 whereas only 75% and 66 % of UW and EC pigs survived, respectively. Glutathione-S-transferase and creatinine clearance were significantly different between EC and UW groups with a light advantages for UW compared to control. Results obtained with MRS on urine and plasma are presented in figure 3. Delayed graft function was associated with the release of osmolytes such as TMAO in urine and in plasma. Moreover, dimethylamine, acetate, citrate and lactate level in the UW group were always closer to control group levels compared to levels in the EC group. These data outline the effect of UW preservation against renal medullar injury and impairment of oxidative metabolism. Here, strength of MRS was highlighted: in one experiment, with a 200 to 500 µL volume of sample, detection and quantitation of large number of metabolites can be made. This allows for a multivariable

Using the same experimental design, the impact of the preservation solution was demonstrated and the addition of colloids (polyethylene glycol (PEG)) into an extracellular solution showed better results (Faure et al., 2002). Indeed, in the urine spectra, TMAO/creatine ratio were significantly higher in kidney preserved with EC, UW or a solution containing PEG-50 than in kidney preserved with a solution containing PEG-30. This demonstrated that PEG reduced damages to the renal medulla during conservation, in a dose-dependent manner. Citrate was not detected during the first postoperative week in urine from kidneys preserved in EC. Its excretion was detected significantly earlier in urine from the ICPEG30 group than from the UW and ICPEG50 groups. This suggested that PEG associated to an intracellular solution improved oxidative metabolism and reduced renal medulla injury, also in a concentration-dependent manner. Furthermore, the addition of antioxidative (Baumert et al., 1999) or anticoagulant (Favreau et al., 2010) molecules, trimetazidine and melagatran respectively, into the preservation solution increased the performance of the hypothermic conservation. The effect of trimetazidine during conservation with EC or UW showed interesting results. The early excretion of acetate in the trimetazidine supplemented group demonstrated an efficient function recovery of the citric acid cycle compared with standard solutions (Hauet et al., 2000a). Combination of PEG and trimetazidine in conservation solution was the next step of long term time study which included 18 groups also exploring cold ischemia time (Doucet et al., 2004). Biological parameters indicated that reduced delayed graft was found when combining PEG and trimetazidine during CSP. Histological staining for CD4+ positive cells indicated that PEG reduced CD4+ positive cells infiltration and trimetazidine was efficient in reducing the inflammatory reaction. The NMR analysis was again in accordance with these results. Acetate excretion between day 1 and day 14 was improved in all experimental group preserved with trimetazidine while urinary TMAO was reduced in all trimetazidine group and we noticed presence of TMAO in plasma in all group except from control. Combination of trimetazidine with PEG seemed to be the best option for kidney conservation and MRS brought supplementary information to classical biochemical parameters such as GFR or sodium excretion. In addition, this study underlined that MRS permits the performance of a

Use of conditioning drugs before conservation was also investigated. Donor pretreatment with N-acetylcysteine (NAC) has been shown to ameliorate acute renal failure. Extractions

analysis which is an important factor for diagnostic and prognostic purpose.

multivariate analysis in a single experiment.

Use of HR MRS in Human to follow transplanted kidney recovery of function highlighted several issues. The main problem came from the drug treatment received by patients after transplantation. We observed overlapping between the signals of the metabolites of interest and of the drugs. In these conditions it is often difficult to extract information about the kidney function. Moreover a high variability, in addition of the individual variability, is often observed. This variability, mainly due to diet**,** may cause urine profile modifications which can hide variations of the metabolite of interest in relation with the kidney injuries. One solution could be to introduce a 2-days controlled diet before each sampling to minimise those environmental variation. However, an older study performed on 33 patients who underwent primary renal allograft transplantation, using the same immunosuppressive regiment and of which 57.6% achieve immediate graft function, showed that patients with good graft function and normal patients had urinary TMAO/creatinine level inferior to 200 µmol/mM while patients with graft dysfunction had urinary TMAO/creatinine level of 410 ± 102 µmol/mM (Foxall et al., 1993).

#### **2.3 Perspectives**

#### **2.3.1 Perfusate analysis**

Hypothermic machine perfusion (MP) is increasingly being preferred to CSP. Indeed, MP has been showed to offer protection to the organ since the 80's (Alijani et al., 1985; Kwiatkowski et al., 2007; Wight et al., 2003) with benefits such as reduction of delayed graft function and increased survival. However, the mechanisms involved in these improvements are still unclear and need to be elucidated. Biomarkers such as lactate dehydrogenase, total glutathione-S-transferase or N-acetyl--D-glucosaminidase measured at the end of the perfusion in the machine perfusates are used by clinicians. Even if those markers are independently efficient to predict graft outcome, they are not efficient enough to be taken into account in the decision to transplant or discard a donation after brain death or controlled donation after cardiac death (Moers et al., 2010; Siew et al., 2011). Again the potential of multivariate analysis offered by NMR could be used to predict graft outcome. In a recent study in a pig model of autotransplantation of livers, the effect of warm ischemia on metabolites contents into perfusats was measured (Liu et al., 2009).

Currently, our team is attempting to answer the question: can 1H HR NMR metabonomic analysis during machine perfusion be used to predict graft outcome? We thus work on perfusates from different protocols of kidney transplantation using MP. We evaluate the effect of warm ischemia, duration of ischemia, types of solution and temperatures of preservation and finally effect of adjuvant in the perfusion solution. We showed, in a small

Magnetic Resonance Spectroscopy (MRS) in Kidney Transplantation: Interest and Perspectives 113

Magnetic resonance imaging (MRI) is a non invasive technique bringing essential information in the diagnostic of kidney transplant rejection since it can provide data on the anatomic and functional status of the transplanted kidney (Beckmann et al., 2000). Briefly, anatomic MRI is applied to evaluate the transplanted organ by assessing structural and anatomical changes or by measuring the graft volume. The most popular imaging sequences are the T1- and T2-weighted spin echo sequences and the T1-weighted gradient echo sequence. However, it would really be informative to add other acquisition sequences to obtain functional information about the transplanted kidney, if the magnet system is available during enough time to perform these exams. Indeed, further information can be

In addition of the magnetic resonance angiography to image blood vessels, quantification of the renal blood flow can be performed with dynamic MRI (Montet et al., 2003). Using a contrast agent, an association of a lower MRI perfusion with chronic allograft nephropathy severity was found (Pereira et al., 2011). Perfusion was determined in chosen area of the kidney, by measuring enhanced signal in the region of interest during the injection of the contrast agent. These results led to an homogeneous estimation of the perfusion, and the ability to evaluate the entire kidney with perfusion MRI may have benefits in establishing which areas of the transplant have a reduction in perfusion. The limitation in obtaining an entire kidney image is the necessity to use a cardiac gating to avoid pulsatility in the arterial curve. Another problem is the potential nephrotoxicity of the gadolinium which is generally used as contrast agent in a chelated form. Using low dose three dimensional magnetic resonance renography, the mean transit time of the tracer for the different compartment of the kidney (vascular, tubular and collecting system) was measured different in normal kidneys, transplanted kidneys with acute tubular necrosis and transplanted kidney with acute rejection (Yamamoto et al., 2011) indicating that a multicompartimental tracer kinetic renal model may

obtained on perfusion and functionality of the graft and the integrity of the tissue.

help to differentiate acute rejection from acute tubular necrosis in transplanted kidney.

Ultra-small superparamagnetic particles of iron oxides (USPIO) could be used as contrast agent in clinical medicine for in vivo MRI due to their properties to locally perturb magnetic field and novel particles are in development (Mills et al., 2011). Their toxicity is not well known and liquiq 1H NMR analysis of plasma and urine demonstrated metabolic changes (Feng et al., 2010). Energenetic and lipidic pathways such as glucose and amino acid metabolisms were affected by USPIO administration. Whereas no change has been found in histopathological analysis of tissue between two types of USPIO administration, coated with dextran or uncoated, coated-USPIO administration led to acetate, unsaturated fatty acids and some amino acids elevation in the 1H hrmas kidney profile. It also led to decrease of triacylglycerol, myo-niositol or taurine, for instance (Feng et al., 2011). Hence, this sort of

A noninvasive magnetic resonance arterial spin labeling (ASL) technique has been performed to evaluate kidney perfusion without contrast agents (Artz et al., 2011). ASL uses the blood as an endogenous contrast agent allowing perfusion measurements without gadolinium injection. Using a flow-sensitive alternating inversion recovery (FAIR) sequence, inflowing blood is selectively labeled to have an opposite magnetization compared to the

**3. Magnetic resonance in vivo**

tracer has to be used with precaution.

**3.1 MRI**

cohort of pig with two distinct graft outcomes, that a correlation between creatininemia at day 7 after transplantation and choline concentration after 24 hours of perfusion existed. Other metabolites like TMAO, lactate or glutathione showed the same correlation (Figure 4.). Metabonomic NMR analysis of machine perfusates could thus be use to predict graft outcome. A larger study is required to validate this finding, especially since this technique is easily transposable to the clinic.

Fig. 4. 1H HR NMR spectra of kidney perfusion solution (a) at the beginning of the perfusion and (b) after 24 hours of perfusion. (Ace: acetate, Ala: alanine, Cho: choline, Cr: creatine, Crine: creatinine, Glu: glutamate, GSH: reduced glutathione, GSSG: oxidized glutathione, TMAO: trimethylamine-N-oxide, Val: valine).

#### **2.3.2 <sup>1</sup> H hrmas NMR on kidney tissue or cells**

In an hrmas probe, adapted for classical spectrometer, samples are spun with high speed (2000 – 5000 Hz) at a 54.7° angle. This rotation leads to decreased heterogeneity of samples and allows direct sample analysis. 1H hrmas NMR is thus particularly adapted for tissue or cells without sample pre-treatment (Desmoulin et al., 2008). Addition of deuterated water into phosphate buffer solution is the only required modification of the samples.

In regards to the in vitro analysis of ischemia reperfusion injuries, our team developed an endothelial cell model mimicking transplantation through a hypoxia-reoxygenation episode in which conditions of hypoxia and/or reoxygenation can be modulated by changing temperature of hypoxia, atmosphere or conservation solutions. 1H hrmas NMR is a precious tool for this application but needs improvement for cell analysis. Indeed, the main problem resides on cell quantity, especially after hypoxia when cell number is inferior to 1 million as it is the case in our experiments. Use of the rotor allows for limitation of volume inside the tube and is one of the solutions we are examining. On the other hand, after 30 minutes spinning at 2500 Hz at 4°C, when cells were replaced into culture medium, 40 to 60 % of cells were still alive and restarted growing normally after 24 hours. Thus, we could imagine the use of 1H hrmas NMR to study all hypoxia-reoxygenation sequence on the same cells. In parallel, supernatant can also been analyzed in order to follow the cell metabolism.

Concerning tissue analysis, care has to be taken during tissue collection. Indeed, as already mentioned, because of its histological heterogeneity the kidney has different metabolites composition depending on the area (Garrod et al., 1999). For autotransplantation models as in clinical studies, a defined protocol for biopsy must be respected by all involved parties.

#### **3. Magnetic resonance in vivo**

#### **3.1 MRI**

112 Magnetic Resonance Spectroscopy

cohort of pig with two distinct graft outcomes, that a correlation between creatininemia at day 7 after transplantation and choline concentration after 24 hours of perfusion existed. Other metabolites like TMAO, lactate or glutathione showed the same correlation (Figure 4.). Metabonomic NMR analysis of machine perfusates could thus be use to predict graft outcome. A larger study is required to validate this finding, especially since this technique is

Fig. 4. 1H HR NMR spectra of kidney perfusion solution (a) at the beginning of the perfusion and (b) after 24 hours of perfusion. (Ace: acetate, Ala: alanine, Cho: choline, Cr: creatine, Crine: creatinine, Glu: glutamate, GSH: reduced glutathione, GSSG: oxidized glutathione,

In an hrmas probe, adapted for classical spectrometer, samples are spun with high speed (2000 – 5000 Hz) at a 54.7° angle. This rotation leads to decreased heterogeneity of samples and allows direct sample analysis. 1H hrmas NMR is thus particularly adapted for tissue or cells without sample pre-treatment (Desmoulin et al., 2008). Addition of deuterated water

In regards to the in vitro analysis of ischemia reperfusion injuries, our team developed an endothelial cell model mimicking transplantation through a hypoxia-reoxygenation episode in which conditions of hypoxia and/or reoxygenation can be modulated by changing temperature of hypoxia, atmosphere or conservation solutions. 1H hrmas NMR is a precious tool for this application but needs improvement for cell analysis. Indeed, the main problem resides on cell quantity, especially after hypoxia when cell number is inferior to 1 million as it is the case in our experiments. Use of the rotor allows for limitation of volume inside the tube and is one of the solutions we are examining. On the other hand, after 30 minutes spinning at 2500 Hz at 4°C, when cells were replaced into culture medium, 40 to 60 % of cells were still alive and restarted growing normally after 24 hours. Thus, we could imagine the use of 1H hrmas NMR to study all hypoxia-reoxygenation sequence on the same cells. In

into phosphate buffer solution is the only required modification of the samples.

parallel, supernatant can also been analyzed in order to follow the cell metabolism.

Concerning tissue analysis, care has to be taken during tissue collection. Indeed, as already mentioned, because of its histological heterogeneity the kidney has different metabolites composition depending on the area (Garrod et al., 1999). For autotransplantation models as in clinical studies, a defined protocol for biopsy must be respected by all involved parties.

easily transposable to the clinic.

TMAO: trimethylamine-N-oxide, Val: valine).

**H hrmas NMR on kidney tissue or cells**

**2.3.2 <sup>1</sup>**

b

a

Magnetic resonance imaging (MRI) is a non invasive technique bringing essential information in the diagnostic of kidney transplant rejection since it can provide data on the anatomic and functional status of the transplanted kidney (Beckmann et al., 2000). Briefly, anatomic MRI is applied to evaluate the transplanted organ by assessing structural and anatomical changes or by measuring the graft volume. The most popular imaging sequences are the T1- and T2-weighted spin echo sequences and the T1-weighted gradient echo sequence. However, it would really be informative to add other acquisition sequences to obtain functional information about the transplanted kidney, if the magnet system is available during enough time to perform these exams. Indeed, further information can be obtained on perfusion and functionality of the graft and the integrity of the tissue.

In addition of the magnetic resonance angiography to image blood vessels, quantification of the renal blood flow can be performed with dynamic MRI (Montet et al., 2003). Using a contrast agent, an association of a lower MRI perfusion with chronic allograft nephropathy severity was found (Pereira et al., 2011). Perfusion was determined in chosen area of the kidney, by measuring enhanced signal in the region of interest during the injection of the contrast agent. These results led to an homogeneous estimation of the perfusion, and the ability to evaluate the entire kidney with perfusion MRI may have benefits in establishing which areas of the transplant have a reduction in perfusion. The limitation in obtaining an entire kidney image is the necessity to use a cardiac gating to avoid pulsatility in the arterial curve. Another problem is the potential nephrotoxicity of the gadolinium which is generally used as contrast agent in a chelated form. Using low dose three dimensional magnetic resonance renography, the mean transit time of the tracer for the different compartment of the kidney (vascular, tubular and collecting system) was measured different in normal kidneys, transplanted kidneys with acute tubular necrosis and transplanted kidney with acute rejection (Yamamoto et al., 2011) indicating that a multicompartimental tracer kinetic renal model may help to differentiate acute rejection from acute tubular necrosis in transplanted kidney.

Ultra-small superparamagnetic particles of iron oxides (USPIO) could be used as contrast agent in clinical medicine for in vivo MRI due to their properties to locally perturb magnetic field and novel particles are in development (Mills et al., 2011). Their toxicity is not well known and liquiq 1H NMR analysis of plasma and urine demonstrated metabolic changes (Feng et al., 2010). Energenetic and lipidic pathways such as glucose and amino acid metabolisms were affected by USPIO administration. Whereas no change has been found in histopathological analysis of tissue between two types of USPIO administration, coated with dextran or uncoated, coated-USPIO administration led to acetate, unsaturated fatty acids and some amino acids elevation in the 1H hrmas kidney profile. It also led to decrease of triacylglycerol, myo-niositol or taurine, for instance (Feng et al., 2011). Hence, this sort of tracer has to be used with precaution.

A noninvasive magnetic resonance arterial spin labeling (ASL) technique has been performed to evaluate kidney perfusion without contrast agents (Artz et al., 2011). ASL uses the blood as an endogenous contrast agent allowing perfusion measurements without gadolinium injection. Using a flow-sensitive alternating inversion recovery (FAIR) sequence, inflowing blood is selectively labeled to have an opposite magnetization compared to the

Magnetic Resonance Spectroscopy (MRS) in Kidney Transplantation: Interest and Perspectives 115

relation with the oxygen consumption in the tissue. In a murine model, BOLD MRI of a clamped kidney showed the decreased oxygenation of all regions. 24 hours after reperfusion, a lower reoxygenation of the outer medulla than control was found, and a higher reoxygenation of the cortex and an identical reoxygenation of the inner medulla were determined (Oostendorp et al., 2011). These results highlighted the potential of BOLD MRI for the detection of change in kidney tissue oxygenation. Non invasive exploration of human kidneys showed that oxygenation was lower in medulla than in the cortex, confirming a physiological medullar hypoxia (Malvezzi et al., 2009; Thoeny et al., 2006). A few days post transplantation, this technique showed an increase of T2 relaxation times in the cortex and medulla of the transplanted kidney, compared to the donated kidney of living donor before collection, demonstrating increased oxygenation (Malvezzi et al., 2009). A few months after transplantation, an increased oxygen content in the medulla was observed despite an unchanged perfusion fraction (Thoeny et al., 2006). Comparing allografts with normal function or acute tubular necrosis, a decreased blood flow and an increased oxygen bioavailability in the medulla of acutely rejecting kidneys were observed, suggesting a greater decrease in oxygen use (Sadowski et al., 2010). No significant modification of BOLD and DW MRI were observed in patients with acute tubular necrosis compared to normal functioning allografts. These results indicate the potential of these MRI techniques to longitudinally follow transplanted kidneys and

As an anecdote, MRI was tested for the counting the kidney glomerules number and size distribution in normal rat kidneys using ferritin as labeling and compared to standard stereological evaluation (Heilmann et al., 2011). This study showed that this ex vivo analysis

Magnetic resonance spectroscopy of Phosphorus-31 (31P) can be performed in kidney during the preservation period and in vivo after transplantation. In these two cases, 31P MRS brings information about the energetic metabolism. This non invasive methods allows the observation of a limited number of metabolites which are mainly: adenosine triphospate (ATP) which gives three signals corresponding to the three phosphorus nuclei in the ATP molecule ( and phosphorus corresponding to the position of the phosphorus nucleus in the molecule); inorganic phosphate (Pi) which has a chemical shift varying with the intracellular pH; phosphomonoesters (PME), mainly including phosphocholine and phosphoethanolamine that is highly concentrated in the renal cortex; phosphodiesters (PDE), corresponding to the resonances of glycerophosphocholine (GPC) and glycerolphosphoethanolamine (GPE) which can be used as an indicator of the physiological integrity of the organ (Wolff &Balaban, 1988). Phosphocreatine is not seen in NMR spectrum

Ex vivo or in vivo, 31P MRS does not allow an absolute quantification thus only signal ratios can be used. These ratios were studied in the preservation period and after the transplantation of the kidney, and compared with the kidney graft function. During the preservation period, 31P MRS was performed ex vivo with the kidney in the preservation solution and in the apparatus of conservation. The ATP disappeared quickly after the collection and perfusion with a cold preservation solution, in spite of the fact that the kidney was immediately cooled to 4°C and maintained to this temperature. The most significant

of entire kidney was less time consuming than stereological method.

of kidney, and when it appears in vivo, it comes from the surrounding tissues.

obtain information on function.

**3.2 31P MRS**

destination tissue. The difference between a labeled image and a non labeled image can be used to calculate tissue perfusion. The first results obtained indicated that medullar perfusion was systematically lower in transplanted versus native kidneys irrespectively of estimated glomerular filtration rate (eGFR). Cortical perfusion was lower for transplanted kidneys when eGFR was > 60 ml/min per 1.73 m² and correlated with eGFR in both native and transplanted kidneys. These results were obtained with a one model compartment requiring rapid water exchange assumption between the intravascular and extravascular space. A two compartments model would allow more accurate perfusion quantification, but would require measurements which are not possible in a clinically feasible scan time.

Diffusion weighted (DW) MRI, which was established for the tissue characterization and lesion detection (Thoeny &De Keyzer, 2011), is particularly interesting for kidney function exploration because of its high blood flow and water transport functions. Simple acquisition is performed with a bipolar gradient (diffusion gradient) dedicated to the assessment of fluid movements. When molecules of water moves, the absence of rephasement of the nuclear spin appears as a loss of signal intensity on the image. If diffusion is restricted, molecules of water reduce their movement and may be refocused by the second gradient impulse (Palmucci et al., 2011). Different intensities of diffusion gradient can be used to determine the Apparent Diffusion Coefficient (ADC). The DW MRI techniques yields a total ADC in each voxel explored that provides information on diffusion properties of water, including contribution from micro-circulation (Le Bihan et al., 1988). Thus image processing allows to separate the micro-circulation information, quantified with the perfusion fraction (FP) which reflects micro-circulation of blood and movement of fluids in predefined structure such as tubules and glomeruli, and the primarily pure diffusion with the perfusion-free diffusion (ADCD) (Eisenberger et al., 2010). The potential of ADC has been studied in kidney transplantation to determined modification in case of graft rejection or acute tubular necrosis. In patients with stable allograft function (posttransplant time between 8 and 10 months), total ADC and ADCD were found identical in the cortex and medulla of the transplanted kidney while values were higher in cortex than in medulla of healthy volunteers. Cortical total ADC and ADCD were higher in native kidneys than in transplanted kidneys (Thoeny et al., 2006), and a difference in ADCD was observed between patients with normal clearance (>60 mL/min) and low clearance (>30 mL/min) where ADCD were the lowest (Palmucci et al., 2011). In allografts with stable function early after transplantation (posttransplantation time of 10 days) ADCD and FP were also found identical in cortex and medulla, but the FP was strongly reduced in the cortex and medulla of renal transplants with allograft rejection and acute tubular necrosis. FP correlated with eGFR, while no correlation were found between eGFR and ADCD or total ADC (Eisenberger et al., 2010). These results show that DW MRI is a promising non-invasive method for detection or monitoring of functional derangements early after kidney transplantation.

The blood oxygen level dependent (BOLD) MRI is a functional MRI technique allowing assessment of the tissue oxygenation. When the kidney is the explored organ, it allows the differentiation of specific anatomical regions (I.E cortex, inner and outer medulla). BOLD MRI is based on susceptibility differences between oxyhemoglobin and deoxyhemoglobin which induce image contrast differences, generally obtained with a gradient echoes sequence. Oxyhemoglogin is diamagnetic, and has no effect on the NMR signal of the surrounding water, by contrast deoxyhemoglobin is paramagnetic, increasing its magnetic properties and modifying the signal relaxation of the surrounding water molecules. Higher concentrations of deoxyhemoglobin induces shorter apparent T2 relaxation time, which is in relation with the oxygen consumption in the tissue. In a murine model, BOLD MRI of a clamped kidney showed the decreased oxygenation of all regions. 24 hours after reperfusion, a lower reoxygenation of the outer medulla than control was found, and a higher reoxygenation of the cortex and an identical reoxygenation of the inner medulla were determined (Oostendorp et al., 2011). These results highlighted the potential of BOLD MRI for the detection of change in kidney tissue oxygenation. Non invasive exploration of human kidneys showed that oxygenation was lower in medulla than in the cortex, confirming a physiological medullar hypoxia (Malvezzi et al., 2009; Thoeny et al., 2006). A few days post transplantation, this technique showed an increase of T2 relaxation times in the cortex and medulla of the transplanted kidney, compared to the donated kidney of living donor before collection, demonstrating increased oxygenation (Malvezzi et al., 2009). A few months after transplantation, an increased oxygen content in the medulla was observed despite an unchanged perfusion fraction (Thoeny et al., 2006). Comparing allografts with normal function or acute tubular necrosis, a decreased blood flow and an increased oxygen bioavailability in the medulla of acutely rejecting kidneys were observed, suggesting a greater decrease in oxygen use (Sadowski et al., 2010). No significant modification of BOLD and DW MRI were observed in patients with acute tubular necrosis compared to normal functioning allografts. These results indicate the potential of these MRI techniques to longitudinally follow transplanted kidneys and obtain information on function.

As an anecdote, MRI was tested for the counting the kidney glomerules number and size distribution in normal rat kidneys using ferritin as labeling and compared to standard stereological evaluation (Heilmann et al., 2011). This study showed that this ex vivo analysis of entire kidney was less time consuming than stereological method.

### **3.2 31P MRS**

114 Magnetic Resonance Spectroscopy

destination tissue. The difference between a labeled image and a non labeled image can be used to calculate tissue perfusion. The first results obtained indicated that medullar perfusion was systematically lower in transplanted versus native kidneys irrespectively of estimated glomerular filtration rate (eGFR). Cortical perfusion was lower for transplanted kidneys when eGFR was > 60 ml/min per 1.73 m² and correlated with eGFR in both native and transplanted kidneys. These results were obtained with a one model compartment requiring rapid water exchange assumption between the intravascular and extravascular space. A two compartments model would allow more accurate perfusion quantification, but would require measurements which are not possible in a clinically feasible scan time.

Diffusion weighted (DW) MRI, which was established for the tissue characterization and lesion detection (Thoeny &De Keyzer, 2011), is particularly interesting for kidney function exploration because of its high blood flow and water transport functions. Simple acquisition is performed with a bipolar gradient (diffusion gradient) dedicated to the assessment of fluid movements. When molecules of water moves, the absence of rephasement of the nuclear spin appears as a loss of signal intensity on the image. If diffusion is restricted, molecules of water reduce their movement and may be refocused by the second gradient impulse (Palmucci et al., 2011). Different intensities of diffusion gradient can be used to determine the Apparent Diffusion Coefficient (ADC). The DW MRI techniques yields a total ADC in each voxel explored that provides information on diffusion properties of water, including contribution from micro-circulation (Le Bihan et al., 1988). Thus image processing allows to separate the micro-circulation information, quantified with the perfusion fraction (FP) which reflects micro-circulation of blood and movement of fluids in predefined structure such as tubules and glomeruli, and the primarily pure diffusion with the perfusion-free diffusion (ADCD) (Eisenberger et al., 2010). The potential of ADC has been studied in kidney transplantation to determined modification in case of graft rejection or acute tubular necrosis. In patients with stable allograft function (posttransplant time between 8 and 10 months), total ADC and ADCD were found identical in the cortex and medulla of the transplanted kidney while values were higher in cortex than in medulla of healthy volunteers. Cortical total ADC and ADCD were higher in native kidneys than in transplanted kidneys (Thoeny et al., 2006), and a difference in ADCD was observed between patients with normal clearance (>60 mL/min) and low clearance (>30 mL/min) where ADCD were the lowest (Palmucci et al., 2011). In allografts with stable function early after transplantation (posttransplantation time of 10 days) ADCD and FP were also found identical in cortex and medulla, but the FP was strongly reduced in the cortex and medulla of renal transplants with allograft rejection and acute tubular necrosis. FP correlated with eGFR, while no correlation were found between eGFR and ADCD or total ADC (Eisenberger et al., 2010). These results show that DW MRI is a promising non-invasive method for detection or

monitoring of functional derangements early after kidney transplantation.

The blood oxygen level dependent (BOLD) MRI is a functional MRI technique allowing assessment of the tissue oxygenation. When the kidney is the explored organ, it allows the differentiation of specific anatomical regions (I.E cortex, inner and outer medulla). BOLD MRI is based on susceptibility differences between oxyhemoglobin and deoxyhemoglobin which induce image contrast differences, generally obtained with a gradient echoes sequence. Oxyhemoglogin is diamagnetic, and has no effect on the NMR signal of the surrounding water, by contrast deoxyhemoglobin is paramagnetic, increasing its magnetic properties and modifying the signal relaxation of the surrounding water molecules. Higher concentrations of deoxyhemoglobin induces shorter apparent T2 relaxation time, which is in Magnetic resonance spectroscopy of Phosphorus-31 (31P) can be performed in kidney during the preservation period and in vivo after transplantation. In these two cases, 31P MRS brings information about the energetic metabolism. This non invasive methods allows the observation of a limited number of metabolites which are mainly: adenosine triphospate (ATP) which gives three signals corresponding to the three phosphorus nuclei in the ATP molecule ( and phosphorus corresponding to the position of the phosphorus nucleus in the molecule); inorganic phosphate (Pi) which has a chemical shift varying with the intracellular pH; phosphomonoesters (PME), mainly including phosphocholine and phosphoethanolamine that is highly concentrated in the renal cortex; phosphodiesters (PDE), corresponding to the resonances of glycerophosphocholine (GPC) and glycerolphosphoethanolamine (GPE) which can be used as an indicator of the physiological integrity of the organ (Wolff &Balaban, 1988). Phosphocreatine is not seen in NMR spectrum of kidney, and when it appears in vivo, it comes from the surrounding tissues.

Ex vivo or in vivo, 31P MRS does not allow an absolute quantification thus only signal ratios can be used. These ratios were studied in the preservation period and after the transplantation of the kidney, and compared with the kidney graft function. During the preservation period, 31P MRS was performed ex vivo with the kidney in the preservation solution and in the apparatus of conservation. The ATP disappeared quickly after the collection and perfusion with a cold preservation solution, in spite of the fact that the kidney was immediately cooled to 4°C and maintained to this temperature. The most significant

Magnetic Resonance Spectroscopy (MRS) in Kidney Transplantation: Interest and Perspectives 117

*in vivo* **et** *in vitro models.*

**Metabolomic analysis Quantitation**

Fig. 5. Summary of samples for liquid or hrmas MRS analysis to study ischemia-reperfusion

From liquid NMR analysis of urine in IPK or perfusate in an autotransplantation model of large white pig, to hrmas analysis of cell culture or biopsy from Human (abstracted in the figure 5.) through imaging, MRS, combined with metabolomic analysis is a powerful tool for the study of transplantation. Despite all the difficulties in regards to kidney heterogeneity or low detection limit for cell analysis, MRS is still an interesting implement deserving of amelioration. Advances concerning imaging and contrast agents as well as transposition of hrmas to imaging are also the key for better diagnosis, prognosis and transplantation

We thank all people working in the INSERM U927 unit and allow us to access in vitro or in vivo samples: William Hebrard, Sébastien Giraud, Frédéric Favreau, Nicolas Chatauret and Raphael Thuillier, for his grammatical help especially. Thank to Nadège Boildieu, technician

 **Hrmas NMR** ✔ Kidney tissue ✔Endothelial cells

**Liquid NMR :** ✔ Plasma ✔ Urine ✔ Preservation solution ✔ Tissue extract ✔ Cellular extract ✔ Culture supernatant

phenomena occurring during kidney transplantation.

performed almost all the NMR acquisition in the team.

ADC Apparent Diffusion Coefficient ADCD Perfusion-free diffusion

ADPKD Autosomal dominant polycystic kidney diseases

**4. Conclusion**

comprehension.

**5. Acknowledgment**

**6. Abbreviations**

1H Hydrogen-1 13C Carbon-13 31P Phosphorus-31

indicator of graft quality was the ratio of PME to Pi (PME/Pi), determined in human cadaveric kidneys prior to planned transplantation, which has been correlated with tubular necrosis and delayed graft function (Bretan et al., 1989; Hene et al., 1994). A high PME/Pi ratio was associated with the best renal function after transplantation; a decrease of this ratio (decrease of PME and/or increase of Pi) correlated with prolonged acute tubular necrosis. In regards to the determination of this ratio in the preserved kidney, the difficulty was the presence of a phosphate buffer in the preservation solution, this Pi signal overlapping the intracellular inorganic phosphate. Determination of this ratio can be obtained using Chemical Shift Imaging (CSI). This technique uses spatial resolution to obtain a 2D-CSI spectra, where each spectrum comes from a voxel. The interest is to minimize the Pi signal of the preservation solution in the voxel's content. In these conditions, a better correlation was obtained between PME/Pi ratio and serum creatinine 14 days after transplantation (Niekisch et al., 2004). A decrease of the PME/Pi ratio was shown as a monoexponential time dependent function in pig kidneys (von Elverfeldt et al., 2007). This decrease shows the influence of preservation time on transplantation outcome, and the authors determined a different decay constant with the preservation solution (comparison between two solutions) suggesting a predictive indicator of the preservation quality with time. This PME/Pi ratio indicator can be obtained with the kidney staying in its preservation container, at preservation temperature. No injection or biopsy is necessary, allowing easy integration of this exam in the period between collection and transplantation; the only condition being a non-magnetic container to be introduced in the magnet of a magnetic resonance apparatus (whole body scanner MR for this exam).

After transplantation it is possible to perform an in vivo 31P MRS of the kidney, which remains fully non invasive as it does not require injection nor anesthesia. More information can be obtained from the 31P spectrum of kidney since in addition to the PME and PDE signals, ATP signals can be observed and intracellular pH can be determined with the chemical shift of the Pi. When spectrum is obtained from the whole kidney, -ATP to Pi ratio measured 2 or 3 years after transplantation has been shown to be an excellent indicator of long term survival of the transplanted kidney (3 years when ratio > 1.2 and 5 years when ration > 2.5) (Seto et al., 2001). When MRS was performed in the 4 weeks after transplantation, the PME to PDE ratio seemed to be an indicator of allograft function (Klemm et al., 1998), showing a good correlation with serum creatinine levels. High PME/PDE ratio reflects the cell membrane regeneration process occurring during the first weeks after ischemia; PME level is considered as a reflection of the membrane phospholipid metabolism with the degree of cell growth and regeneration, while in contrast PDE level correspond to intermediate metabolites formed during cell membrane degradation. A decreased PME/PDE ratio was found in patients with delayed graft function due to acute tubular necrosis, but was not observed in patient with allograft rejection. A better localization of the voxel for 31P MRS could be obtained with CSI technique (Vyhnanovska et al., 2011), allowing the discard of surrounding tissue from the kidney signal in the spectra and the reduction in result dispersion, even if resolution did not permit to distinguish cortex from medulla in the spectra. In these conditions it is possible to distinguish different types of kidney failure after transplantation: (i) acute tubular necrosis, characterized by a decrease of PDE/-ATP, PDE/Pi and PME/Pi compared to controls; (ii) acute rejection episodes (ARE), characterized by an increase of PME/-ATP and PME/Pi ratio compare to controls. This method permitted the demonstration that late kidney graft dysfunction patients exhibited a higher Pi/-ATP and lower PDE/Pi and PME/Pi ratio compared with ARE patients.

Fig. 5. Summary of samples for liquid or hrmas MRS analysis to study ischemia-reperfusion phenomena occurring during kidney transplantation.

#### **4. Conclusion**

116 Magnetic Resonance Spectroscopy

indicator of graft quality was the ratio of PME to Pi (PME/Pi), determined in human cadaveric kidneys prior to planned transplantation, which has been correlated with tubular necrosis and delayed graft function (Bretan et al., 1989; Hene et al., 1994). A high PME/Pi ratio was associated with the best renal function after transplantation; a decrease of this ratio (decrease of PME and/or increase of Pi) correlated with prolonged acute tubular necrosis. In regards to the determination of this ratio in the preserved kidney, the difficulty was the presence of a phosphate buffer in the preservation solution, this Pi signal overlapping the intracellular inorganic phosphate. Determination of this ratio can be obtained using Chemical Shift Imaging (CSI). This technique uses spatial resolution to obtain a 2D-CSI spectra, where each spectrum comes from a voxel. The interest is to minimize the Pi signal of the preservation solution in the voxel's content. In these conditions, a better correlation was obtained between PME/Pi ratio and serum creatinine 14 days after transplantation (Niekisch et al., 2004). A decrease of the PME/Pi ratio was shown as a monoexponential time dependent function in pig kidneys (von Elverfeldt et al., 2007). This decrease shows the influence of preservation time on transplantation outcome, and the authors determined a different decay constant with the preservation solution (comparison between two solutions) suggesting a predictive indicator of the preservation quality with time. This PME/Pi ratio indicator can be obtained with the kidney staying in its preservation container, at preservation temperature. No injection or biopsy is necessary, allowing easy integration of this exam in the period between collection and transplantation; the only condition being a non-magnetic container to be introduced in the magnet of a magnetic resonance apparatus

After transplantation it is possible to perform an in vivo 31P MRS of the kidney, which remains fully non invasive as it does not require injection nor anesthesia. More information can be obtained from the 31P spectrum of kidney since in addition to the PME and PDE signals, ATP signals can be observed and intracellular pH can be determined with the chemical shift of the Pi. When spectrum is obtained from the whole kidney, -ATP to Pi ratio measured 2 or 3 years after transplantation has been shown to be an excellent indicator of long term survival of the transplanted kidney (3 years when ratio > 1.2 and 5 years when ration > 2.5) (Seto et al., 2001). When MRS was performed in the 4 weeks after transplantation, the PME to PDE ratio seemed to be an indicator of allograft function (Klemm et al., 1998), showing a good correlation with serum creatinine levels. High PME/PDE ratio reflects the cell membrane regeneration process occurring during the first weeks after ischemia; PME level is considered as a reflection of the membrane phospholipid metabolism with the degree of cell growth and regeneration, while in contrast PDE level correspond to intermediate metabolites formed during cell membrane degradation. A decreased PME/PDE ratio was found in patients with delayed graft function due to acute tubular necrosis, but was not observed in patient with allograft rejection. A better localization of the voxel for 31P MRS could be obtained with CSI technique (Vyhnanovska et al., 2011), allowing the discard of surrounding tissue from the kidney signal in the spectra and the reduction in result dispersion, even if resolution did not permit to distinguish cortex from medulla in the spectra. In these conditions it is possible to distinguish different types of kidney failure after transplantation: (i) acute tubular necrosis, characterized by a decrease of PDE/-ATP, PDE/Pi and PME/Pi compared to controls; (ii) acute rejection episodes (ARE), characterized by an increase of PME/-ATP and PME/Pi ratio compare to controls. This method permitted the demonstration that late kidney graft dysfunction patients exhibited a higher Pi/-ATP

and lower PDE/Pi and PME/Pi ratio compared with ARE patients.

(whole body scanner MR for this exam).

From liquid NMR analysis of urine in IPK or perfusate in an autotransplantation model of large white pig, to hrmas analysis of cell culture or biopsy from Human (abstracted in the figure 5.) through imaging, MRS, combined with metabolomic analysis is a powerful tool for the study of transplantation. Despite all the difficulties in regards to kidney heterogeneity or low detection limit for cell analysis, MRS is still an interesting implement deserving of amelioration. Advances concerning imaging and contrast agents as well as transposition of hrmas to imaging are also the key for better diagnosis, prognosis and transplantation comprehension.

#### **5. Acknowledgment**

We thank all people working in the INSERM U927 unit and allow us to access in vitro or in vivo samples: William Hebrard, Sébastien Giraud, Frédéric Favreau, Nicolas Chatauret and Raphael Thuillier, for his grammatical help especially. Thank to Nadège Boildieu, technician performed almost all the NMR acquisition in the team.

#### **6. Abbreviations**

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