1 Introduction

Renal dysfunction is recognized as a significant health problem originating from a variety of causes leading to acute or chronic kidney diseases. Predisposition to acute kidney injury and premature mortality are frequent outcomes for chronic kidney diseases [1]. Moreover, according to the Global Burden of Disease 2013 study, the age-standardized death rates for chronic kidney diseases showed one of the highest increases in the last two decades [2]. Therefore, reliable and early diagnosis of acute and chronic kidney diseases is needed to preserve renal functionality and improve patients’ outcome. Despite these premises, current clinical evaluation of renal function is still based on measurement of serum creatinine, which is well known to have several limitations. In fact, an elevated serum creatinine concentration is only discovered at late stages of the disease when renal functionality is already compromised. Therefore, novel noninvasive imaging approaches are needed for a more accurate and early diagnosis of renal physiology. Magnetic resonance imaging (MRI) has been exploited for tissue anatomic imaging, owing to the high spatial resolution and lack of ionizing radiation. In research, novel MRI techniques have been investigated for assessing multiple functional parameters of the kidneys, including perfusion, filtration, oxygenation and tissue elasticity [3,4,5,6,7,8,9,10,11]. Several reviews covering the role of these MRI approaches for assessing renal functionality have been published [12,13,14,15]. This additional information should enable a better characterization of acute and chronic kidney diseases in comparison to standard urine and serum-based assays.

Recently, Chemical Exchange Saturation Transfer (CEST) MRI has emerged as a novel approach for functional and molecular imaging with great promise for clinical translation [16,17,18]. In addition, CEST MRI pH imaging has emerged as a valuable approach for assessing extracellular pH values in several tissues, including kidneys and tumors, providing the highest accuracy and spatial resolution achievable so far [19]. Owing to the key role of the kidneys in maintaining the acid-base homeostasis, CEST MRI pH mapping has emerged as a novel and promising approach in monitoring kidney functionality.

In this chapter, we will address the basic concept and the developments of renal CEST pH imaging with the emphasis on kidney disease models in rodents.

This introduction chapter is complemented by two separate chapters describing the experimental procedure and data analysis, which are part of this book.

This chapter is part of the book Pohlmann A, Niendorf T (eds) (2020) Preclinical MRI of the Kidney—Methods and Protocols. Springer, New York.

2 Measurement Concept

2.1 Basic Concept of Chemical Exchange Saturation Transfer (CEST) Imaging

CEST is a new technique that enables the indirect detection of molecules possessing mobile protons in exchange with water [20]. Because of this, CEST makes MRI sensitive to endogenous or exogenous molecules that possess suitable protons [21,22,23,24,25,26,27,28,29,30,31,32]. The generation of contrast is based on a selective irradiation with a radiofrequency pulse at the specific absorption frequency of the exchanging proton, followed by a subsequent transfer, due to chemical exchange with bulk water, of the saturated signal. Upon the application of a long saturation RF (radiofrequency) pulse, the water signal decreases, allowing the amplification of the labeled protons from the molecule to water. As a consequence, low concentration molecules can be specifically and indirectly detected, owing to the frequency selective irradiation of their mobile proton pool(s) [33,34,35,36,37,38].

2.2 CEST Contrast Agents for Imaging Kidneys and Mapping pH

Several classes of CEST contrast agents can be used as pH responsive agents and exploited for in vivo experiments.

2.2.1 Diamagnetic CEST Agents

Several molecules, including natural occurring molecules, possess exchangeable protons that can be indirectly detected within the CEST approach [39]. Most of these labile protons have chemical exchange rates that show a strong pH dependence. Hence differences in CEST contrast can be exploited for assessing the pH of the solution where they are dissolved. Due to the concomitant contribution of concentration and exchange rate to the observed CEST contrast, several approaches have been proposed to rule out the concentration term, based on the ratiometric method, that is, taking the ratio of the observed contrast, providing accurate pH readouts [40, 41]. Most of the pH responsive diamagnetic molecules investigated so far in vivo are iodinated contrast agents used for radiographic procedures, since they have been used in the last 40 years in clinical examinations owing to their high safety profiles [42, 43]. The presence of amide groups in their chemical structure allow them to be exploited as CEST MRI contrast agents, upon selective irradiation of the mobile proton pool [44].

Among the radiographic contrast media, Iopamidol (Isovue®, Bracco Imaging, Italy) (Fig. 1) was the first agent exploited for mapping pH thanks to the presence of two amide groups with different resonance frequencies, at 4.2 ppm and at 5.5 ppm, respectively, that can be selectively irradiated [45,46,47]. This chemical peculiarity led to the development of a ratiometric approach based on the ratio of the CEST effects at these two frequencies to provide accurate pH measurements. In vitro experiments showed a high pH responsiveness within the physiological range and accurate pH measurements (Fig. 2a, b). Following intravenous administration, it can provide selective contrast at specific frequency offsets and from the ratio of these two parametric maps it is possible to measure renal pH map (Fig. 2c–f). To validate the approach and test the pH-responsiveness of the agent, induced alkalinisation or acidification of urine was obtained in mice upon providing acidic or alkaline drinking water for a week. For control mice, mean pH values calculated for cortex, medulla and calyx regions were: 7.0 ± 0.11; 6.85 ± 0.15 and 6.6 ± 0.20, respectively. In mice drinking acidic water a significant acidification of renal pH values was obtained. Conversely, a marked increase of renal pH values was observed upon alkaline water administration. Both observations demonstrate the capability to measure in vivo renal pH changes with CEST MRI.

Fig. 1
figure 1

Chemical structures of the reported pH-responsive CEST agents investigated for renal pH mapping

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Fig. 2
figure 2

Iopamidol ratiometric curve obtained from the rations of the CEST contrast upon irradiating at 4.2 and 5.5 ppm, respectively, showing the high pH responsiveness of Iopamidol. Irradiation power levels of 3 μT, Iopamidol 30 mM, 7.05 T, 310 K, irradiation time 5 s (a). Calculated pH values obtained by the ratiometric method (Iopamidol 30 mM, 7.05 T, 310 K, irradiation time 5 s, B1 3 μT) are compared with the values read on the pH-meter (b). Representative images of in vivo renal pH mapping showing the anatomical image (c) and the CEST contrast parametric maps overlaid to the anatomical image at 4.2 ppm (d) and 5.5 ppm (e) and the observed pH map (f) obtained by ratioing maps (d) and (e) and using the calibration curve in (a) for calculating the pH values. (Adapted with permission from Magnetic Resonance in Medicine 2011 (Iopamidol as a responsive MRI-chemical exchange saturation transfer contrast agent for pH mapping of kidneys: in vivo studies in mice at 7 T. Volume: 65, Issue: 1, Pages: 202–211, DOI: https://doi.org/10.1002/mrm.22608))

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Since the accuracy in measuring pH depends on several factors, including chemical exchange rate, irradiation conditions (saturation power and duration), and main magnetic field, quantification of multisite pH-dependent chemical exchange properties is needed to improve pH accuracy [48]. This characterization of chemical exchange rates and optimal irradiation RF pulses led to the development of an optimized saturation for each single amide proton pools, hence resulting in a higher pH sensitivity. As a result, the capability to measure pH was demonstrated also at magnetic field strengths of 4.7 T, where amide resonances partially overlap. In the study of Wu and coworkers, they demonstrated an improved pH sensitivity, extending the pH detection range from 5.5 to 7.5, with high resolution pH maps of the kidneys in healthy rats (Fig. 3) [49]. More recently, the exploitation of the same ratiometric approach for measuring in vivo pH was also demonstrated at magnetic fields as low as 3 T, which still preserved good pH accuracy [50].

Fig. 3
figure 3

Extension of pH detection range using the modified ratiometric analysis (red circles) versus the standard ratiometric approach (blue squares) (a). Representative in vivo ratiometric map for the proposed approach (b). pH map shows the renal pH gradually decreasing from the cortex, medulla to calyx (c). Maps are overlaid on the corresponding T2-weighted image. (Adapted with permission from Magnetic Resonance in Medicine 2018 (A generalized ratiometric chemical exchange saturation transfer (CEST) MRI approach for mapping renal pH using Iopamidol, Volume: 79, Issue: 3, Pages: 1553–1558, DOI: https://doi.org/10.1002/mrm.26817))

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Another similar iodinated contrast media, Iopromide (Ultravist®, Bayer Healthcare, Germany), was also demonstrated and compared to Iopamidol for measuring pH, with a pH sensitivity that was not substantially different, although Iopamidol allows for more precise pH measurements [51].

The exploitation of the ratiometric approach requires two distinguishable protons pools on the same molecule, therefore it is limited to a select group of compounds possessing this feature. To overcome this limitation, Longo and Sun proposed a novel approach based on the irradiation of a single pool under different radiofrequency powers [52]. The proposed approach, called ratio of RF power mismatch or RPM, was demonstrated by using Iobitridol (Omnipaque®, GE Healthcare, USA), a radiographic contrast medium possessing only one amide proton pool resonating at 5.5 ppm (Fig. 1). Since the measured CEST contrast is dependent on both pH and irradiation power (B1), a strong pH dependence was demonstrated by calculating the ratio of the CEST contrast at two different B1 power levels. In comparison to the conventional ratiometric approach, a good pH accuracy and an even higher pH sensitivity were demonstrated. When investigated in healthy kidneys, the measured pH values upon iobitridol injection strongly correlated with the pH values obtained following iopamidol injection. Recently, to overcome the specific absorption rate (SAR) limitations when using a continuous wave (CW) irradiation of the mobile proton pools, a pulsed ratiometric approach has been exploited and tested on the Iodixanol (Visipaque®, GE Healthcare, USA) X-ray contrast medium for CEST pH imaging [53].

Diamagnetic contrast agents possess resonances that are close in frequency to the bulk water signal resulting in a reduction of sensitivity (due to an inefficient labeling selectivity) when moving from high to low magnetic fields. Following previous investigations, McMahon and colleagues developed new diamagnetic systems based on the imidazole, salicylate or anthranilate moieties which possess mobile protons shifted very far from the bulk water peak [54,55,56]. In particular, candidate compounds derived from imidazole-4,5-dicarboxamides (#5 and #8, Fig. 1) show a CEST signal shifted up to 7.8 ppm upfield by exploiting the presence of intramolecular bond shifted hydrogens. Besides the large chemical shift, a good pH sensitivity and a high-water solubility made compound #5 suitable as a pH sensor and so it has been tested in vivo [57]. pH imaging of the kidneys at 11.7 T resulted in average pH values for the whole kidneys of 6.5 ± 0.1, consistent with renal pH values reported by radiographic contrast agents (Fig. 4).

Fig. 4
figure 4

pH calibration curve of compound #5 (a) and calibration plot using all tubes in the phantom showing experimental versus calculated pH (b). Experimental conditions: CEST data were obtained at 6.25 mM, 25 mM, or 50 mM concentration, saturation time = 3 s, saturation power = 5.9 μT and 37 °C. pH measurements were made with a precision of ±0.1 unit. T2-weighted image (c) and pH map (d) following administration of compound #5. (Adapted with permission from Contrast Media Molecular Imaging 2016 (Developing imidazoles as CEST MRI pH sensors, Volume: 11, Issue: 4, Pages: 304–312, DOI: https://doi.org/10.1002/cmmi.1693))

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2.2.2 Paramagnetic CEST Agents

Paramagnetic chemical exchange saturation transfer (paraCEST) agents typically consist of a paramagnetic metal ion and an organic chelate based on a macrocyclic cage due to the high kinetic stability of these metal chelates to prevent the release of the toxic free lanthanide cation [58]. The CEST signal is therefore generated by selective irradiation of the bound water molecule, or of the slowly exchanging ligand protons, such as hydroxyl, amine, or amide groups [59,60,61,62]. The main advantage of paraCEST agents in comparison to diamagnetic ones relies in the exceptionally large chemical shifts (MR frequency relative to the water frequency) due to the hypershift contribution of the lanthanide metal ion. This large range of chemical shifts increases the specificity of the exchanging proton pools, hence reducing the adverse contributions of direct saturation and of the endogenous semisolid macromolecular effects [63]. Since the chemical exchange rate of the paraCEST agent can be altered by environmental factors such as pH, changes in CEST amplitudes can be used as well for deriving pH values.

The first demonstration of CEST detection in kidneys using a paraCEST agent (TmDOTA-4AmC, Fig. 1) was provided by Vinogradov et al., showing good detectability in the whole kidney regions [64]. Later on, a Europium paraCEST pH responsive agent (europium(III) DO3A-tris(amide) complex, Fig. 1) was exploited to measure pH in mouse kidneys [65]. Owing to its chemical structure, a quite large frequency shift of the ion-bound water molecule due to the delocalization of negative charge coming from deprotonation of phenolic residue was observed. This shift is pH dependent, therefore an alkalinisation from 6.0 to 7.6 at 298 K leads to a 4 ppm shift in frequencies, from 50.5 to 54.5 ppm, respectively, that can be exploited for assessing pH. By exploiting a 9.4 T MR scanner and ensuring stable temperature homogeneity, in vivo pH measurements were feasible in kidneys upon the administration of a dose of 0.4 mmol/kg [66].

2.3 Imaging Readout

CEST MRI techniques includes continuous wave (CW) or pulsed train RF saturation to prepare the magnetization followed by a fast image readout such as echo planar imaging (EPI), Rapid Imaging with Refocused Echoes (RARE) and/or fast imaging with steady-state precession (FISP) [45, 67, 68]. More advanced methods now provide more sophisticated sampling schemes, such as 3D radial or spiral acquisitions, but those are currently available only on clinical scanners and not on preclinical scanners, thus limiting CEST acquisition in mice and rats to single slice based approaches [69, 70]. However, recent studies have utilized multislice acquisition schemes on preclinical scanners as well [67, 71, 72].

2.3.1 Rapid Imaging with Refocused Echoes (RARE)

Fast spin echo (FSE) or rapid imaging with refocused echoes (RARE) are commonly used because of the strong SNR (signal-to-noise ratio), high tolerance to B0 inhomogeneities and moderate to short acquisition times. These methods allow acquisitions of several lines for full sampling the k-space for a single slice within a single TR, which greatly reduces the acquisition time and still preserves SNR. With single shot acquisitions, usually centric encoding is exploited to maximize the SNR. Further reductions in acquisition time can be achieved by adjusting the bandwidth (hence reducing the echo times or the distance between each refocused echo time) or by partial Fourier approaches (i.e., by acquiring only a portion of the k-space).

2.3.2 Fast Imaging with Steady-State Precession (FISP)

FISP readout has been used with CEST MRI that provides robust image readout with little distortion, although more sensitive to B0 inhomogeneity than RARE. B0 inhomogeneity might be an important issue particularly for body applications like the kidneys.

2.3.3 Spin-Echo Echo Planar Imaging (EPI)

CEST MRI is often combined with EPI acquisition, which provides fast image readout after a relatively long RF saturation. Whereas both gradient echo and spin echo EPI have been used, SE EPI is often preferred for body application because it is less susceptible to mild magnetic field inhomogeneity distortions that are common in body applications. The use of an EPI readout also enables multislice acquisitions in reasonable times.

3 Overview of Applications

CEST imaging has been exploited for assessing renal pH values in healthy and in several models of renal damage, including either bilateral or unilateral acute kidney injury models. Both endogenous CEST approaches and exogenous CEST approaches have been proposed and validated in vivo.

3.1 Endogenous CEST Methods for Assessing Renal Diseases

Since diabetic nephropathies (DNs) are associated with changes in renal metabolites, the utility of CEST MRI to detect changes in glucose/glycogen hydroxylic protons was investigated in murine models of diabetic nephropathies. The study was conducted longitudinally from 8 to 24 weeks on two groups of diabetic mice and on nondiabetic mice as control. Based on the variation of glucose/glycogen composition and the consequent CEST effects measured in kidney regions, a significantly increased CEST of hydroxyl metabolites was observed in diabetic mice during the progression of DN [73].

3.2 CEST pH Imaging for Assessing Renal Diseases

Most of the studies reported so far have exploited the pH-responsiveness of Iopamidol for investigating the changes in pH homeostasis following renal injuries. In one of these studies, the pH evolution in an acute kidney injury model induced by intramuscular glycerol injection and consequent rhabdomyolysis was monitored [74]. Renal pH maps acquired at 1, 3, 7, 14, 21 days after the injury reported a marked increase of pH values during the damage evolution up to 3–7 days, followed by recovery of pH toward baseline values at 14 and 21 days. These results were in good agreement with morphological and Blood Urean Nitrogen (BUN) quantification supporting this CEST MRI pH mapping approach for investigating renal function. Furthermore, along with the progression of the damage, a reduction of pixels where Iopamidol was detectable was observed, suggesting that also the percentage of CEST-detected pixels can be used as an imaging biomarker of renal injury.

In another study, Longo and coworkers investigated a unilateral kidney ischemia reperfusion injury (KIRI) model to validate MRI-CEST pH mapping for assessing single kidney functionality [75]. Two different times of ischemia duration, 20 and 40 min, were applied to model moderate or severe KIRI, respectively. Following the damage evolution at days 0, 1, 2, 7, a significant increase in renal pH values was observed even at day 1 in both cases. Furthermore, in the following MRI acquisitions a clear distinction between moderate and severe AKI is possible since a recovery of normal acid-base balance was observed only in the moderate KIRI mice whereas in severe KIRI mice the increased pH values did not restore to baseline values (Fig. 5a, b). In additions, as in the previous study, the percentage of CEST detected pixels, representing a marker of the filtration fraction, showed significant differences between the injured kidneys and the contralateral ones, reflecting the different evolution of moderate-to-severe damage (Fig. 5c, d).

Fig. 5
figure 5

MRI-CEST pH mapping detects renal pH changes and regional distribution of damage after moderate and severe unilateral kidney ischemia reperfusion injury (KIRI) showing clamped (right) and contralateral normal kidney (left). Representative MRI-CEST pH maps overimposed onto anatomical images before and after moderate (a) and severe (b) KIRI at different time points (day 1, day 2, and day 7) showing pronounced alkalinization and reduced filtration (noncolored pixels within the renal region, c, d) of the pH-responsive contrast agent in clamped kidney in comparison to contralateral kidney. (Adapted with permission from: NMR in Biomedicine 2017 (Noninvasive evaluation of renal pH homeostasis after ischemia reperfusion injury by CEST-MRI, Volume: 30, Issue: 7, DOI: https://doi.org/10.1002/nbm.3720))

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3.3 GlucoCEST Imaging for Assessing Renal Diseases

Since the capability of CEST imaging to detect hydroxyl protons, native glucose has also been proposed as a tracer for MRI GlucoCEST imaging [76,77,78]. Besides its exploitation in oncological applications, it has been applied for monitoring allograft rejection [79]. In this study, Brown Norway rat kidneys were transplanted into Lewis rats and imaged 4 days following the surgery, before and after glucose administration. By calculating the cortex-to-medulla CEST ratio (CESTR), dependent on the accumulation of the administered glucose, rats that underwent allogeneic transplant showed the highest CESTR values compared to syngeneic transplant group and to control mice.