Quantitative arterial spin labelling perfusion measurements in rat models of renal transplantation and acute kidney injury at 3 T

ZEMEDI-10657; No. of Pages 10

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ORIGINALARBEIT

Quantitative arterial spin labelling perfusion measurements in rat models of renal transplantation and acute kidney injury at 3 T Fabian Zimmer a,∗ , Sarah Klotz b , Simone Hoeger b , Benito A. Yard b , Bernhard K. Krämer b , Lothar R. Schad a , Frank G. Zöllner a a b

Computer Assisted Clinical Medicine, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Department of Medicine V, University Medical Centre Mannheim, Heidelberg University, Mannheim, Germany

Received 27 October 2015; accepted 22 February 2016

Abstract Objectives: To employ ASL for the measurement of renal cortical perfusion in particular renal disorders typically associated with graft loss and to investigate its potential to detect and differentiate the related functional deterioration i.e., in a setting of acute kidney injury (AKI) as well as in renal grafts showing acute and chronic transplant rejection. Materials and Methods: 14 Lewis rats with unilateral ischaemic AKI and 43 Lewis rats with renal grafts showing acute or chronic rejections were used. All ASL measurements in this study were performed on a 3 T MR scanner using a FAIR True-FISP approach to assess renal blood flow (RBF). Perfusion maps were calculated and the cortical blood flow was determined using a region-of-interest based analysis. RBF of healthy and AKI kidneys as well as of both rejection models, were compared. In a subsample of 20 rats, creatinine clearance was measured and correlated with cortical perfusion. Results: RBF differs significantly between healthy and AKI kidneys (P < 0.001) with a mean difference of 213 ± 80 ml/100 g/min. Renal grafts with chronic rejections show a significantly higher (P < 0.001) mean cortical perfusion (346 ± 112 ml/100 g/min) than grafts with acute rejection (240 ± 66 ml/100 g/min). Both transplantation models have a significantly (P < 0.001) lower perfusion than healthy kidneys. Renal creatinine clearance is

Quantitative Perfusionsmessungen mittels Arterial Spin Labelling in Rattenmodellen mit Transplantatnieren und Akutem Nierenversagen bei 3T Zusammenfassung Ziele: Die Anwendung sowie die Untersuchung des Leistungsvermögens von Arterial Spin Labelling (ASL) zur quantitativen Messung von renalem Blutfluss in spezifischen Störungen der Nierenfunktion, die typischerweise mit einem Transplantatverlust verbunden sind, d.h. im akuten Nierenversagen (ANV) sowie bei chronischer und akuter Transplantatabstoßung. Material und Methoden: Es wurden 14 Lewis-Ratten mit einseitigem ischämischem ANV und 43 LewisRatten mit Transplantatnieren, die akute oder chronische Abstoßungsreaktionen aufweisen, verwendet. Alle ASLMessungen in dieser Studie wurden an einem 3T-MRT durchgeführt. Eine FAIR True-FISP Methode wurde verwendet, um den renalen Blutfluss (RBF) zu bestimmen. Perfusionskarten wurden berechnet und die kortikale Durchblutung mit Hilfe einer ,,region-of-interest“-basierten Analyse, bestimmt. Der RBF von gesunden und ANVNieren, sowie von beiden Abstoßungsmodellen wurde verglichen. Zusätzlich wurde in 20 Ratten die Kreatininclearance bestimmt und mit der kortikalen Perfusion korreliert.

∗ Corresponding author: Fabian Zimmer, Computer Assisted Clinical Medicine, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. Tel.: +49 621 383 5118; fax: +49 621 383 5123. E-mail: [email protected] (F. Zimmer).

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significantly correlated (R = 0.85, P < 0.001) with cortical blood flow. Conclusion: Perfusion measurements with ASL have the potential to become a valuable diagnostic tool, regarding the detection of renal impairment and the differentiation of disorders that lead to a loss of renal function and that are typically associated with graft loss.

Keywords: Arterial Spin Labelling (ASL), perfusion, acute kidney injury, transplant rejection, renal functional imaging, quantitation

Introduction The incidence of acute kidney injury (AKI) in North America and in Europe has rapidly increased over the past decades [1]. AKI is associated with a significantly increased mortality, a longer hospital stay and a higher risk for developing end stage renal disease (ESRD) [2,3]. Since dialysis therapy may be associated with technical problems and inferior quality of life and patient survival [4], kidney transplantation is considered the therapy of choice for patients with ESRD [5]. Although the preservation of renal grafts, the choice of appropriate donors and the usage of immunosuppression has improved, graft loss is still a problem [6,7]. Such losses are mainly driven by immunological reactions which lead to acute and chronic graft rejections or non-immunological factors like ischaemia-reperfusion injury (IRI). The fact that ischaemia is a major cause for AKI, and that AKI (delayed graft function DGF) may trigger acute renal allograft rejection shows that graft rejection and AKI are directly related. Both are major obstacles in modern nephrology. With regard to the increasing numbers of AKI, Siew and Davenport [1] clearly state that a reduction of this growing burden for the public health and health-care funds requires alternative biomarkers for the diagnosis that can complement the limitations of serum creatinine. Microvascular renal cortical perfusion can be such a “biomarker” because of the functional deterioration of the kidney accompanying AKI [8] and graft rejection [9].

Ergebnisse: Die Nierenperfusion von gesunden und ANV-Nieren ist signifikant unterschiedlich (P < 0.001) mit einer mittleren Differenz von 213 ± 80 ml/100 g/min. Transplantatnieren mit chronischer Abstoßung weisen eine signifikant (P < 0.001) höhere mittlere Perfusion auf (346 ± 112 ml/100 g/min) als Transplantate mit akuter Abstoßung (240 ± 66 ml/100 g/min). Beide Abstoßungsmodelle zeigen eine signifikant (P < 0.001) niedrigere Durchblutung als die gesunden Nieren. Die Kreatininclearance ist signifikant (R = 0.85, P < 0.001) mit dem kortikalen Blutfluss korreliert. Schlussfolgerung: Die Perfusionsmessung mit ASL hat das Potential, eine wertvolle, diagnostische Methode zu werden, um Funktionsminderungen in Nieren zu detektieren und zu unterscheiden, welche Störungen unterschiedlicher Ätiologie aufweisen, die typischerweise mit einem Transplantatverlust verbunden sind. Schlüsselwörter: Arterial Spin Labelling (ASL), Perfusion, Akutes Nierenversagen, Transplantatabstoßung, Funktionale Nierenbildgebung, Quantifizierung

The occurrence of a graft rejection is an immune response against donor alloantigens [9]. Depending on their histological appearance and the time of occurrence after transplantation, different types of rejection are distinguished. An acute rejection occurs within the first days and weeks [10] while chronic rejections can appear several months or years [11] after surgery. The acute rejection is predominantly characterised by an infiltration of inflammatory cells, e.g. macrophages, monocytes and lymphocytes in the interstitium as a first reaction of the recipient’s immune system [12]. Further, an inflammation of the tubuli (tubulitis) and the vascular system (vasculitis) are histologically evident [9,13]. Chronic graft rejection is characterised by its progressive etiopathology. Its major hallmarks are the atrophy of tubuli and interstitial fibrosis, i.e. the occurrence of scar formations that replace renal tissue [9,14]. Hence, it has an essential impact on the long-term graft survival. In this study acute and chronic graft rejection were investigated. Both types of rejection deteriorate graft function [15] and hence have an impact on renal perfusion. MRI allows to measure renal perfusion with both dynamic contrast-enhanced MRI (DCE-MRI) and arterial spin labelling (ASL) [16]. Although DCE-MRI allows to derive different physiological parameters [17], it involves the injection of contrast agent which can be contraindicated in patients with an impaired renal function or in elderly and paediatric populations. Further, cases of nephrogenic systemic fibrosis (NSF) and contrast-induced nephropathy (CIN) in connection with the administration of gadolinium based contrast agents have been reported [18–20]. ASL, in comparison, is

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a completely non-invasive technique that uses magnetically labelled arterial blood as an endogenous tracer. This makes ASL an interesting candidate for the detection and diagnosis of impaired kidney function. Although, ASL has already progressed to clinical research, most of the studies focus on investigating its application in the brain, mainly as a biomarker for vascular disorders and neurodegenerative diseases [21]. Preclinical studies on organs outside the brain are limited and only a very small number exists where ASL was used in specific disease models like AKI [22,23] or in renal grafts [24–26]. Naturally, those studies employing ASL in renal allografts, put their main focus on the assessment of renal perfusion in transplants with different degrees of functional deterioration. Unfortunately, serum creatinine served as the sole measure for renal function, and thus deterioration, leading to a large heterogeneity of underlying pathologies pooled within the same groups of functional impairment. So far, specific disorders leading to graft rejection, i.e. in terms of a model system, have not been separately studied employing ASL perfusion measurements. The aim of this study was to employ ASL for the measurement of renal cortical perfusion in particular renal disorders typically associated with graft loss and to investigate its potential to detect and differentiate the related functional deterioration. Therefore, renal perfusion was measured in rat models of unilateral AKI as well as in models of acute and chronic graft rejection. To simplify the transfer to clinical research or routine, the study has been conducted on a wholebody MR scanner rather than on a dedicated small animal scanner.

Materials and Methods A 3 T whole-body MR scanner (Magnetom Tim Trio, Siemens Healthcare Sector, Erlangen, Germany) was used for all measurements. The system operates at a maximum gradient strength of 45 mT/m and a maximum slew rate of 200 T/m/s. The MR signal was received with a dedicated eight channel receive-only volumetric rat array (RAPID Biomedical GmbH, Rimpar, Germany). A homogeneous excitation was ensured by using the body coil as RF transmitter. A total of 57 Lewis rats were employed in this study. Fourteen rats had a unilateral ischaemic acute kidney injury. 43 rats with an allogeneic renal transplant were used. All animals underwent perfusion MRI. They were placed supine and head first into the rat array with the kidneys in the RF centre. Animals Inbred male Lewis (LEW, RTI1) rats were used for the AKI model and as organ recipients in the transplantation model. Male Fisher (F344/DuCrl) rats served as organ donors. All animals were obtained from Charles River (Sulzfeld, Germany) and had an initial weight of 250–300 g. They were kept under standard conditions with standard food and water

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ad libitum. All procedures were performed according to the Guide of the Care and Use of Laboratory Animals published by the National Academy of Sciences and were approved by the local authorities (Regierungspräsidium Karlsruhe AZ: 35-9185.81/G – 19/12). Acute kidney injury (AKI) For the induction of AKI, a well-standardised warm ischaemia model was used which has been described previously [8,17,22,27]. The model is known to reduce renal perfusion due to endothelial lesion, interstitial oedema, tubulus dilatation and necrosis [28,29]. AKI was induced in the left kidney of 14 animals following the protocol described in Zimmer et al. [22]. In short, renal vessels are clamped for 45 min by using nontraumatic clamps. After opening the clamps again, renal perfusion was evaluated macroscopically. The right kidney served as native control. During all inductions of AKI, a general anaesthesia using ketamine (100 mg/ml; Ketamin® , Intervet Deutschland GmbH, Unterschleißheim, Germany) and xylazine (6 mg/ml; Rompun® , Bayer Health Care, Leverkusen, Germany) was used intraperitoneal. To keep their body temperature constant during surgery, animals were placed on a heating table (38 ◦ C). Renal transplantation model (Tx) For renal transplantation an allogenic model (Fisher to Lewis) was used which was described several times before [30–32]. After explantation, donor kidneys were stored at 4 ◦ C in University of Wisconsin solution (ViaSpan® , BristolMyers Squibb GmbH & Co. KG, Munich, Germany) for 20 h before transplantation into bilaterally nephrectomised recipients. No immunosuppressive agents were administered in this model, therefore graft rejections were expected in transplant recipients. During all renal transplantations an initial anaesthesia with ketamine (100 mg/ml; Ketamin® , Intervet Deutschland GmbH, Unterschleißheim, Germany) and xylazine (6 mg/ml; Rompun® , Bayer Health Care, Leverkusen, Germany) was used intraperitoneal. To obtain anaesthesia during renal implantation an inhalation with isoflurane (Isofluran, Forene® , Abbott GmbH & Co. KG, Wiesbaden, Deutschland) was used. To evaluate early and late graft function as well as renal perfusion, the recipients were observed for 7 days and for 24 weeks, respectively. Renal function In the model of chronic graft rejection, serum creatinine (SCr ), urine creatinine (UCr ) and urine volume accumulated during t = 24 h were measured prior to perfusion MRI, i.e.

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14 weeks after surgery. Creatinine clearance CCr was then calculated according to: CCr =

UCr · VU SCr · t

(1)

and served as an additional marker for renal function. The values are expressed in ml/min. Serum creatinine could not be determined in rats with acute graft rejection or AKI. Additional anaesthesia required for taking blood samples shortly after surgery and the necessity to keep the animals in a metabolic cage were not conformable with animal welfare. During all surgeries, buprenorphine hydrochloride (0.05 mg per kg body weight; Temgesic® , Reckitt Benckiser Healthcare Ltd., Berkshire, United Kingdom) was injected subcutaneously for analgesic treatment. MRI For MRI measurements animals were anaesthetised with thiobutabarbital sodium (Inactin® , Sigma–Aldrich Chemie GmbH, Steinheim, Germany). AKI rats were measured five days after surgery. To investigate acute and chronic graft rejection, organ recipients were imaged seven days and fourteen weeks after transplantation, respectively. Arterial spin labelling The employed ASL sequence uses the combination of a FAIR labelling scheme and a 2D true fast imaging with steady-state precession (TrueFISP) [33] readout. A single axial slice with a thickness of 4 mm was imaged. Both left and right kidneys were imaged at the same time. Images following global inversion (control, ns-IR), sliceselective inversion (tag, ss-IR) and proton density images without magnetic preparation (M0 ), were recorded in an interleaved manner. Overall, 90 images were acquired resulting in 30 tag-control pairs and 30 M0 images. For both global and slice-selective inversion, a VERSE [34] transformed adiabatic hyperbolic secant pulse [35] was used. The inflow time TI was set to 1.2 s. The slice thickness of the selective inversion was set to 8 mm its inversion slab was placed symmetrically around the imaging slice. TrueFISP imaging parameters were: TR/TE = 5.44 ms/2.72 ms, flip angle = 70◦ , FOV = 140 mm × 140 mm, matrix = 256 × 256, bandwidth = 651 Hz/px and GRAPPA factor = 3. To minimise signal oscillations in the transient phase, an α/2 pulse plus ten additional TR-intervals without data recording were used before the acquisition. Centric reordering of phase-encoding was used to maximise perfusion contrast. A single image, excluding the inversion time, was recorded in 0.6 s. The inter image time was 6 s to ensure sufficient T1 -relaxation which resulted in an overall measurement time of 9 min.

Morphological MRI In addition to ASL perfusion imaging, high-resolution morphological data was recorded in selected animals. For this purpose, a T2 -weighted spin-echo sequence was used with the following parameters: TR/TE = 9770 ms/80 ms, flip angle = 140◦ , FOV = 65.25 mm × 87 mm, matrix = 480 × 640 resulting in a resolution of 0.14 mm × 0.14 mm. 25 slices centrered around the kidneys were imaged. A GRAPPA factor of 2 was used to accelerate the image acquisition. Data evaluation To calculate ASL perfusion maps, the images were averaged according to their magnetic preparation (ns-IR, ss-IR and M0 ). To obtain a perfusion-weighted difference image M at the time TI = 1.2 s the averaged ns-IR image was subtracted from the averaged ss-IR image. Perfusion maps were then calculated by applying the following equation [36,37] on a pixel-wise basis: f =

λ M exp 2TI M0



TI T1

 (2)

The longitudinal relaxation time of the tissue T1 was set to 1.14 s as proposed by de Bazelaire et al. [38]. λ is the blood-tissue water partition coefficient and was assumed to have a constant value of 0.8 ml/g [39]. Transit delays were neglected. All calculations were performed with an in-house written MATLAB (Version 7.10, The MathWorks, Natick, MA) script. To determine the mean cortical perfusion, a region of interest (ROI) was drawn in the perfusion maps delineating the renal cortex. The mean cortical RBF of a kidney was then computed as the mean of all pixels inside the ROI. To check for significant RBF differences between models, different statistical tests were applied to the data. A one-way analysis of variance (ANOVA) was used to test the applicability of a two-sample t-test [40,41] in order to investigate whether RBF samples from rejected renal grafts and healthy kidneys are significantly different. As samples from healthy and AKI kidneys are dependent, AKI kidneys were excluded from the ANOVA. Instead, a paired-sample t-test [42] was employed to compare RBF of healthy and AKI kidneys. Additionally, Levene’s test [43] was used to check for homoscedasticity in different groups. For the AKI model, a Bland–Altman plot [44] was drawn to visualise the pair-wise differences. A box plot is used to illustrate differences in cortical perfusion between RBF samples of different disease models.

Results Perfusion MRI of each rat was successfully conducted without any incidents. ASL data was obtained from each rat and was individually checked for imaging artefacts and

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Figure 1. Axial slices of a rat with unilateral acute kidney injury. (A) A T2 -weighted high resolution spin-echo image with an in-plane resolution of 0.14 mm × 0.14 mm is shown. (B) Perfusion map calculated from ASL data of the same slice. When comparing to the healthy kidney, the impaired left kidney can be well identified in the morphological image as well as in the perfusion map.

Figure 2. Morphology (A and C) and perfusion maps (B and D) of rats with chronic (A and B) and acute (C and D) transplant rejection. The morphological images are high-resolution T2 -weighted spin-echo images that show the graft as the only kidney of the animals. The according perfusion maps show a higher cortical perfusion for the organ with chronic rejection.

distortions. No images were corrupted but all showed diagnostic quality allowing for further post-processing and a good delineation of the kidney cortex. Exemplary images of a rat with AKI can be found in Figure 1A showing a high-resolution coronal slice. Both kidneys can be seen. The impaired left kidney can be well identified by its hypertrophic appearance caused by an oedema, one of the major hallmarks of AKI [45]. Figure 2 shows representative high-resolution morphological data of a rat with chronic (A) and acute graft rejection (C), respectively. The mean cortical RBF in the healthy kidneys was 500 ± 91 ml/100 g/min compared to 287 ± 83 ml/100 g/min in the AKI kidneys. The error represents the sample standard deviation. The application of a paired sample t-test rejected the null hypothesis which states that the pairwise differences are samples of a normal distribution with mean zero. The probability was calculated to be smaller than 0.1% (P < 0.001). Hence, AKI kidneys show a significantly lower

cortical perfusion than healthy kidneys with a mean difference of 213 ± 80 ml/100 g/min. The data is illustrated in a Bland–Altman plot in Figure 3 which visualises the systematically lower RBF in the diseased kidneys. The fact that a difference of zero does not lie within the limits of agreement (±1.96 standard deviations of the average difference), strongly supports the outcome of the t-test. Further, no systematic errors are visible. RBF ratios between left and right kidneys were calculated and are listed Table 1. They are displayed in Figure 4. The mean ratio was found to be 0.58 ± 0.15. The evaluation of the transplantation models resulted in a mean cortical blood flow of 240 ± 66 ml/100 g/min and 346 ± 112 ml/100 g/min for samples from acute and chronic graft rejection, respectively. The ANOVA confirmed that the perfusion values of at least one disease model is significantly (P < 0.001) different from another model. Levene’s test failed to reject the null hypothesis claiming equal variances amongst

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400

1.96 sd

700

300 mean

200 100

−1.96 sd 0 200 300 400 500 mean RBF healthy and diseased [ml/100g/min]

Figure 3. Bland–Altman plot to visualise differences of renal cortical perfusion between healthy and diseased kidneys in the AKI model. The mean difference (black line) is significantly different from zero and not included in the limits of agreement (green dashed lines). No systematic effects correlated with the mean RBF are visible.

cortical RBF [ml/min/100g]

RBF difference healthy − diseased [ml/100g/min]

6

600

500

400

300

200

100

healthy

ANV

acute

chronic

model

Table 1 Ratios of cortical RBF between healthy and diseased kidneys in ml/100 g/min as estimated from the AKI models. Rat

RBF ratio diseased/healthy

1 2 3 4 5 6 7 Mean

0.73 0.24 0.48 0.61 0.74 0.70 0.50

± ± ± ± ± ± ±

0.13 0.11 0.11 0.19 0.27 0.15 0.14

Rat

RBF ratio diseased/healthy

8 9 10 11 12 13 14

0.72 0.66 0.69 0.62 0.49 0.37 0.54 0.58

± ± ± ± ± ± ± ±

0.15 0.16 0.17 0.14 0.13 0.20 0.18 0.15

relative perfusion AKI/healthy

healthy samples and both transplantation models. Consequently, a two-sample t-test was used to investigate whether cortical blood flow in both groups of rejected grafts is significantly different to that in healthy kidneys. Both tests rejected

1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

Figure 5. Boxplots of the perfusion data measured in different rat models. The red lines indicate the median RBF of the respective group, the blue box delineates the first and third quartiles of the data. Minimal and maximal RBF of each model are depicted by the whiskers. The RBF value of one healthy kidney was considered as an outlier and is plotted as a red cross. Significant differences (*** ↔P < 0.001) between different models are indicated.

the null hypothesis of equal means and revealed that RBF in acutely as well as chronically rejected transplants is significantly (P < 0.001) lower than in healthy kidneys. A boxplot of the perfusion data of all models is shown in Figure 5. The significant difference in cortical perfusion between healthy and diseased kidneys is clearly visible. The optical impression of a significant RBF difference between the two rejection models is confirmed by Welch’s t-test (P < 0.001) which is appropriate because of the unequal variances of the two samples. All quantities necessary to estimate creatinine clearance were successfully determined and renal function was calculated. Renal clearance was ranging from 0.13 ml/min to 1.64 ml/min with a mean of 0.63 ± 0.45 ml/min. A comparison against renal cortical blood flow values of the same rats revealed a significant correlation (R2 = 0.72, P < 0.001) of both parameters. A scatter plot of the data is shown in Figure 6 including a linear fit to the data.

Discussion

1

2

3

4

5

6

7

8

rat no.

9

10 11 12 13 14

Figure 4. Ratios of renal perfusion between left (diseased) and right (healthy) kidneys in the AKI model. The decreased cortical blood flow in the acutely injured kidneys is clearly visible. The according values are listed in Table 1.

The aim of this work was to investigate the ability of ASL to detect and differentiate the impaired renal function in disorders which typically accompany graft loss. Therefore, rat models were employed and renal cortical perfusion was noninvasively measured in kidneys with AKI as well as in renal grafts with acute and chronic rejections using ASL. We showed that ASL provides significantly different perfusion estimates when comparing healthy kidneys and kidneys

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creatinine clearance [ml/min]

2 2

R = 0.72 1.5

1

0.5

0 100

200

300

400

500

600

cortical perfusion [ml/min/100g]

Figure 6. Scatter plot showing the renal cortical perfusion of rats with chronic graft rejection plotted against the creatinine clearance. The blue line shows the best linear fit to the data. Renal function is significantly correlated with cortical blood flow.

with AKI. Therefore, perfusion MRI with ASL is capable to differentiate between the RBF of healthy kidneys and the decreased RBF expected from a diseased kidney. The RBF differences are well reflected in the Bland–Altman plot in Figure 2. The result of the paired t-test is supported by the fact that the difference of zero is not included in the limits of agreement. Further, no dependency of the differences on the mean values is visible in the plot. All RBF ratios between AKI and healthy kidneys are smaller than one. Besides animal number five, a value of one is not even included in the standard deviation of the ratios. Figure 1 not only shows the reduced RBF in the AKI kidneys via the perfusion map but also the morphological changes of the AKI kidney which are readily visible in the high-resolution image. Regarding the detection of renal deterioration with noninvasive MRI, our results are in good agreement with the findings of other studies that investigate the impact of IRI on renal function. Oostendorp et al. [46] used blood-oxygenation level dependent (BOLD) and DCE-MRI to monitor renal oxygenation in a murine model of IRI. Oxygenation was measured before, during as well as 1 h and 24 h after unilateral renal ischaemia and the authors could show that cortical oxygenation was significantly lower in the injured kidney at all times during and after clamping the renal artery. Perfusion was measured 24 h after the induction of IRI and yielded significantly lower perfusion in the injured kidney. While perfusion values in the healthy kidneys are comparable (481 ± 26 ml/100 g/min) to our findings, the observed blood flow in the injured kidney is higher (451 ± 24 ml/100 g/min) than in our study. This can be explained by a different ischaemia time, a different time point after IRI or the very low temporal resolution of their contrast dynamic measurement. A study investigating IRI with diffusion tensor imaging was published by Cheung et al. [47]. Eight Sprague-Dawley

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rats with unilateral IRI underwent diffusion tensor imaging (DTI) on a 7 T small animal scanner. Besides the calculation of the apparent diffusion coefficient (ADC) and the fractional anisotropy (FA), a two-compartment model was used to estimate the perfusion fraction (Pfrac ). The authors report that 5 h after the induction of IRI, all parameters were significantly different when comparing the injured with the contralateral healthy kidney. Literature reports on the usage of ASL in AKI are limited. Hueper et al. [23] assessed renal perfusion in mice with unilateral AKI using a 7 T small-animal MR scanner. Two different ischaemia times where used to investigate models with moderate (35 min) and severe (45 min) AKI. The authors compare renal perfusion before and at five time points after the induction of AKI. The ischaemia time used to induce severe AKI equals the time used in our study. Seven days after surgery, which represents the time point closest to the five days used in our study, the authors found significantly reduced RBF values for both AKI models when comparing RBF to a baseline measurement before surgery. The reported values for the eleven mice with severe AKI lie within the range of 113–259 ml/100 g/min for the injured kidney and 456–663 ml/100 g/min for the contralateral organ. Taking into account that mice were used instead of rats and that the longitudinal design of the study did not include a measurement on day five after surgery, the reported results are in good agreement with our findings. In our study, the time required for the ASL measurement is significantly lower (9 min compared to approximately 20 min), which can be advantageous for elderly, paediatric, or sick patients. The thereby necessary transfer of the ASL measurement to an application in humans is facilitated by the usage of a whole-body MR scanner compared the small-animal scanner used by Hüper et al. Another directly related study was published by Zimmer et al. [22] who conducted perfusion measurements in rats with unilateral AKI five days after IRI was induced. Their findings where strengthened by using both ASL and DCEMRI to determine renal cortical perfusion and the authors report significantly different perfusion estimates between healthy and diseased kidneys for both methods. They conclude that ASL is sufficient to detect the abnormal perfusion of AKI kidneys. The authors found a mean RBF difference of 147 ± 47 ml/100 g/min, which is in good agreement with our results, although slightly lower. The mean RBF in the diseased kidneys (316 ± 102 ml/100 g/min) is in excellent agreement to our findings. However, their work is limited by small number of only five rats compared to the eleven mice used by Hüper et al. or the 14 rats employed in this study. Chen et al. [20] employed the injection of iodinated contrast agent to induce AKI in seven rats. In comparison to the RBF estimated from six healthy control animals, cortical RBF was significantly reduced in a time span from 12 h to 48 h after the injection. Afterwards, RBF returned to baseline level, suggesting that the damage caused by the contrast agent was less severe than that induced by IRI.

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To investigate ASL in kidneys impaired by immunological reactions, RBF was measured in rats with a renal graft. Animals were either imaged seven days or six months after surgery, when an acute and a chronic graft rejection was present, respectively. Our study revealed that absolute perfusion in both rejection models is significantly decreased compared to the RBF sample of healthy kidneys. Further, the mean cortical blood flow in kidneys six month after surgery was found to be higher than in the models of acute rejection. The RBF differences between each model are well depicted in the presented boxplot (Figure 5). As the rats were nephrectomised prior to receiving the donor organ, no contralateral control organs were available. Due to ethical reasons and animal welfare it was not possible to gather data from a healthy control group. Instead, the native kidneys in the AKI rats served as a reference. These kidneys might have an increased perfusion to compensate for the functional loss of the contralateral kidney. Hence, the comparison against these kidneys reflects the clinical setting where a present contralateral organ will show the same compensation. Wang et al. [24] were the first to apply ASL in an animal model of renal transplantation. Renal blood flow was measured three and five days after the rats had received either a syngeneic or an allogeneic kidney transplant. Similar to our findings, renal perfusion was significantly reduced in the allogeneic models seven days after surgery when the grafts showed hallmarks of acute rejection. However, in contrast to our study, blood flow in the affected kidneys was lower than 30 ml/100 g/min while perfusion in the healthy kidneys of the same group was exceeding 800 ml/100 g/min, most likely compensating the almost complete functional loss of the renal graft. Blood flow in renal transplants later than seven days after surgery was not investigated. In humans, first ASL measurements in transplanted kidneys were presented by Lanzman et al. [48]. The authors investigate renal blood flow in three different groups. The first two groups comprised organ recipients with stable or good (defined by serum creatinine level and its temporal variation, respectively) allograft function and no signs of rejection, renal artery stenosis (RAS) or ureteral obstruction. Patients were divided in good long-term function (group a) or short-term function (group b). A third group consisted of patients with an acute decrease in renal function comprising acute and chronic rejection. Absolute perfusion values showed significant differences between the first two groups and group c. The study confirms the finding of Wang et al. that ASL has the potential to monitor the function of kidney transplants. However, no healthy control group was available to compare against. In a similar study, Heusch et al. [26] used ASL to investigate renal perfusion in 98 allograft recipients. Based on the estimated glomerular filtration rate (eGFR) patients were divided in two groups. Allograft recipients with a heavily impaired renal function were found to have a significantly lower perfusion than patients with good or moderate function. Cortical renal blood flow and eGFR showed a significant correlation

(R = 0.59) which is in agreement with our findings (R = 0.85), although less pronounced. Artz et al. [25] measured the reproducibility of ASL RBF measurements in native and transplanted kidneys. Cortical perfusion in the healthy kidneys was found to be higher than in renal grafts. Using a FAIR-TrueFISP approach, similar to the one used in this study, the authors report a high agreement of cortical perfusion for both intra- and inter-day measurements. The recruited subjects showed a stable but wide range of renal function that was estimated up front via serum creatinine levels. The main difference between the study presented here and the lastly discussed studies is the specificity of the investigated underlying pathology. The three studies stand out due to the application of ASL in renal grafts of humans. However, serum creatinine served as the main criterion for recruitment and group allocation, respectively. Although, Lanzman and co-authors include cases of acute and chronic rejection in their work, they were not analysed separately but pooled within the generalised and relatively small (n = 6) group of patients with decreased renal function. Similar, the two confirmed cases of acute rejection in the work of Heusch and co-authors were not analysed separately but where included in the group with heavily impaired renal function. In both studies, the authors state that future studies should include higher number of subjects that all have the same underlying pathology, e.g. acute allograft rejection, to decrease the pathological heterogeneity. The study presented here, includes three large groups with well-defined pathologies due to the employment of dedicated and well-established animal models. All of them are renal disorders typically associated with graft loss. Still, future studies might include further pathologies that can lead to graft loss, e.g. ureteral obstruction, ideally with human subjects. Generally, a rather large sample standard deviation of the cortical perfusion is present in all rat models. Because of this, the cortical perfusion of the diseased kidney of some animals may be higher than the RBF in the healthy kidney of a different rat. The main reason for this may be the different physiological conditions of the animals at the time of the measurement. Although, the induction of AKI and the transplantation model were standardised for all animals, each rat responds individually to the treatment which leads to a different functional impairment of the kidney. Further, anaesthesia during MRI has an impact on the animals’ kidney function. Rieg et al. [49] report that the anaesthetic used in our study, i.e. thiobutabarbital sodium, can possibly affect kidney function. Another potential influence on renal activity is the fluid intake prior to MRI due to the ad libitum approach. Further, renal blood flow in small animals is sensitive to the body temperature and although animals were kept on a hot plate until immediately before MRI, different body temperatures during the measurement could not be avoided. Non-physiological aspects, leading to the observed intramodel variations, are extensively discussed in Zimmer et al. [22] and include challenges like respiratory motion, low SNR,

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inversion efficiency and precise longitudinal relaxation times of the renal tissue. Especially in diseased or transplanted kidneys the spin-lattice relaxation time might differ from that measured in healthy kidneys. For example, Wang et al. [24] found a noticeable increase in the observed cortical T1 in allogeneic transplants within seven days after surgery. This result is most likely explained by the fact that the observed or apparent T1 is the relaxation time in the presence of perfusion with blood flow leading to an accelerated relaxation. Nevertheless, the true T1 of renal tissue in diseased kidneys might differ from that found in healthy kidneys, e.g. because of inflammations that lead to an increase in tissue water content. Therefore, in future studies, it would be beneficial to obtain T1 -maps. Not only to minimise the potential of an erroneous calculation of blood flow values but also to allow for an individual T1 in each pixel. Although the rats’ renal cortices could be well differentiated from the medulla and surrounding tissue, partial volume effects cannot be fully excluded when taking into account the size of the kidneys relative to the in-plane resolution. Therefore, this is another potential source of variations. However, the ROIs drawn to delineate the cortices have been approved by an experienced vet. Further, size and position have been verified using the high-resolution morphological images, if at hand. Although, a slice thickness of 4 mm was used, through plane partial volume effects are negligible due to the axial orientation of the kidneys. Here, the conclusions drawn from the values obtained from the AKI rats are less affected because all but one of the discussed limitations result in global changes within one animal and are not specific to one laterality. Regardless of systematic errors, the difference between healthy and diseased organs within one animal would still be given. Still, the conclusions involving the models of graft rejection are reliable. On the one hand, the increased number of animals builds a strong basis for the statistically derived claims. On the other hand, the significant correlation of RBF and creatinine clearance strongly supports the reliability and the reproducibility of the obtained perfusion values. Hence, the observed variations are related to the points discussed above and not to instabilities in the measurement technique. Further, our results are in good comparison to other studies and the reliability of ASL has been shown in repeated measurements and in comparison to other methods [22,50–52]. In this study, the ability of non-invasive ASL perfusion measurements to detect and differentiate abnormal kidney function in three specific disorders typically associated with graft loss was investigated. Animal models covering immunological and non-immunological factors for an impairment of renal function were used. In all models, ASL was capable of distinguishing the hypoperfusion in the disease models from the cortical blood flow in healthy kidneys. Further, RBF in kidneys with chronic and acute rejection was significantly different. Therefore, the perfusion measurement with ASL has the potential to become a valuable diagnostic tool,

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regarding the early detection of an impaired kidney function and the differentiation of the underlying disorders. Especially for patients who are at risk, e.g. after receiving a renal graft, the non-invasive measurement of renal blood flow with ASL can be an excellent monitoring tool. Although, further studies investigating its exact sensitivity, e.g. measurements earlier after the induction of AKI, are necessary, the study has shown that ASL can potentially complement and considerably extend the information provided by clinical parameters such as serum creatinine.

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