Scott C. Beeman

Measuring glomerular number and size in perfused kidneys using MRI

The goal of this work was to nondestructively measure glomerular (and thereby nephron) number in the whole kidney. Variations in the number and size of glomeruli have been linked to many renal and systemic diseases. Here, we develop a robust magnetic resonance imaging (MRI) technique based on injection of cationic ferritin (CF) to produce an accurate measurement of number and size of individual glomeruli. High-field (19 Tesla) gradient-echo MR images of perfused rat kidneys after in vivo intravenous injection of CF showed specific labeling of individual glomeruli with CF throughout the kidney. We developed a three-dimensional image-processing algorithm to count every labeled glomerulus. MRI-based counts yielded 33,786 ± 3,753 labeled glomeruli (n = 5 kidneys). Acid maceration counting of contralateral kidneys yielded an estimate of 30,585 ± 2,053 glomeruli (n = 6 kidneys). Disector/fractionator stereology counting yielded an estimate of 34,963 glomeruli (n = 2). MRI-based measurement of apparent glomerular volume of labeled glomeruli was 4.89 × 10(-4) mm(3) (n = 5) compared with the average stereological measurement of 4.99 × 10(-4) mm(3) (n = 2). The MRI-based technique also yielded the intrarenal distribution of apparent glomerular volume, a measurement previously unobtainable in histology. This work makes it possible to nondestructively measure whole-kidney glomerular number and apparent glomerular volumes to study susceptibility to renal diseases and opens the door to similar in vivo measurements in animals and humans.

MRI-based glomerular morphology and pathology in whole human kidneys

Nephron number (N(glom)) and size (V(glom)) are correlated with risk for chronic cardiovascular and kidney disease and may be predictive of renal allograft viability. Unfortunately, there are no techniques to assess N(glom) and V(glom) in intact kidneys. This work demonstrates the use of cationized ferritin (CF) as a magnetic resonance imaging (MRI) contrast agent to measure N(glom) and V(glom) in viable human kidneys donated to science. The kidneys were obtained from patients with varying levels of cardiovascular and renal disease. CF was intravenously injected into three viable human kidneys. A fourth control kidney was perfused with saline. After fixation, immunofluorescence and electron microscopy confirmed binding of CF to the glomerulus. The intact kidneys were imaged with three-dimensional MRI and CF-labeled glomeruli appeared as punctate spots. Custom software identified, counted, and measured the apparent volumes of CF-labeled glomeruli, with an ~6% false positive rate. These measurements were comparable to stereological estimates. The MRI-based technique yielded a novel whole kidney distribution of glomerular volumes. Histopathology demonstrated that the distribution of CF-labeled glomeruli may be predictive of glomerular and vascular disease. Variations in CF distribution were quantified using image texture analyses, which be a useful marker of glomerular sclerosis. This is the first report of direct measurement of glomerular number and volume in intact human kidneys.

The emerging role of MRI in quantitative renal glomerular morphology

Techniques to measure morphological parameters, such as glomerular (and thereby nephron) number, glomerular size, and kidney volume, have been vital to understanding factors contributing to chronic kidney disease (CKD). These techniques have also been important to understanding the associations between CKD and other systemic and cardiovascular diseases and have led to the identification of developmental risk factors for these pathologies. However, existing techniques in quantitative kidney morphology are resource- and time-consuming and are destructive to the organ. This review discusses the emerging generation of techniques to study kidney morphology quantitatively using magnetic resonance imaging (MRI) using the intravenous injection of the superparamagnetic nanoparticle cationic ferritin, which binds to the glomerular basement membrane. A primary advantage of MRI over previously established techniques is the ability to quantify morphology in the intact organ with minimal sample preparation. We highlight areas of research where MRI-based morphological measurements will be helpful in animal models and possibly diagnostic clinical nephrology, discuss technical challenges in light of the progress in MRI techniques to date, and identify novel measurements that may be possible using MRI, both ex vivo and in vivo.

Phenotyping by magnetic resonance imaging nondestructively measures glomerular number and volume distribution in mice with and without nephron reduction

Reduced nephron mass is strongly linked to susceptibility to chronic renal and cardiovascular diseases. There are currently no tools to identify nephropenia in clinical or preclinical diagnostics. Such new methods could uncover novel mechanisms and therapies for chronic kidney disease (CKD) and reveal how variation among traits can affect renal function and morphology. Here we used cationized ferritin (CF) enhanced-MRI (CFE-MRI) to investigate the relationship between glomerular number (Nglom) and volume (Vglom) in kidneys of healthy wild type mice and mice with oligosyndactylism (Os/+), a model of congenital nephron reduction. Mice were injected with cationic ferritin and perfused and the resected kidneys imaged with 7T MRI to detect CF-labeled glomeruli. CFE-MRI was used to measure the intrarenal distribution of individual glomerular volumes and revealed two major populations of glomeruli distinguished by size. Spatial mapping revealed that the largest glomeruli were located in the juxtamedullary region in both wild type and Os/+ mice and the smallest population located in the cortex. Os/+ mice had about a 50% reduction and 35% increase of Nglom and Vglom, respectively, in both glomerular populations compared to wild type, consistent with glomerular hypertrophy in the Os/+ mice. Thus, we provide a foundation for whole-kidney, MRI-based phenotyping of mouse renal glomerular morphology and provide new potential for quantitative human renal diagnostics.

Wireless Amplified Nuclear MR Detector (WAND) for High-Spatial-Resolution MR Imaging of Internal Organs: Preclinical Demonstration in a Rodent Model

PURPOSE To assess the feasibility of imaging deep-lying internal organs at high spatial resolution by imaging kidney glomeruli in a rodent model with use of a newly developed, wireless amplified nuclear magnetic resonance (MR) detector. MATERIALS AND METHODS This study was approved by the Animal Care and Use Committee at the National Institutes of Health/National Institute of Neurologic Disorder and Stroke. As a preclinical demonstration of this new detection technology, five different millimeter-scale wireless amplified nuclear MR detectors configured as double frequency resonators were chronically implanted on the medial surface of the kidney in five Sprague-Dawley rats for MR imaging at 11.7 T. Among these rats, two were administered gadopentetate dimeglumine to visualize renal tubules on T1-weighted gradient-refocused echo (GRE) images, two were administered cationized ferritin to visualize glomeruli on T2*-weighted GRE images, and the remaining rat was administered both gadopentetate dimeglumine and cationized ferritin to visualize the interleaved pattern of renal tubules and glomeruli. The image intensity in each pixel was compared with the local tissue signal intensity average to identify regions of hyper- or hypointensity. RESULTS T1-weighted images with 70-μm in-plane resolution and 200-μm section thickness were obtained within 3.2 minutes to image renal tubules, and T2*-weighted images of the same resolution were obtained within 5.8 minutes to image the glomeruli. Hyperintensity from gadopentetate dimeglumine enabled visualization of renal tubules, and hypointensity from cationic ferritin enabled visualization of the glomeruli. CONCLUSION High-spatial-resolution images have been obtained to observe kidney microstructures in vivo with a wireless amplified nuclear MR detector.

Toxicity, biodistribution, and ex vivo MRI detection of intravenously injected cationized ferritin

The goal of the work was to establish the toxicity and biodistribution of the superparamagnetic protein cationized ferritin (CF) after intravenous injection. Intravenously injected CF has been used to target the extracellular matrix with high specificity in the kidney glomerulus, allowing measurements of individual glomeruli using T  2* ‐weighted MRI. For the routine use of CF as an extracellular matrix‐specific tracer, it is important to determine whether CF is toxic. In this work, we investigated the renal and hepatic toxicity, leukocyte count, and clearance of intravenously injected CF. Furthermore, we studied CF labeling in several organs using MRI and immunohistochemistry. Serum measurements of biomarkers suggest that intravenous injection of CF is neither nephrotoxic nor hepatotoxic and does not increase leukocyte counts in healthy rats at a dose of 5.75 mg/100 g. In addition to known glomerular labeling, confocal and MRI suggest that intravenously injected CF labels the extracellular matrix of the hepatic sinusoid, extracellular glycocalyx of alveolar endothelial cells, and macrophages in the spleen. Liver T  2* values suggest that CF is cleared by 7 days after injection. These results suggest that CF may serve as a useful contrast agent for detection of a number of structures and functions with minimal toxicity. Magn Reson Med, 2013.

Principles and emerging applications of nanomagnetic materials in medicine.

The development of nanometer-scale magnetic materials for biomedical applications spans the interface between the physical sciences and biology. Applications of these materials are rapidly becoming important in medicine and enable targeted therapies and diagnostics. At the same time, specific applications add focus to the development of novel magnetic materials and facilitate a deeper understanding of the physical mechanisms behind their function. This review presents a broad, nontechnical overview of the basis of magnetism in materials at the nanometer scale and describes how these materials are created, characterized, and used. Specific emerging applications in medical diagnostics and therapies are discussed, including cancer cell targeting for thermal ablation, tissue engineering, and three-dimensional noninvasive molecular imaging. Challenges in these fields are discussed, including the toxicity and delivery of magnetic nanomaterials and the sensitivity of imaging and therapeutic techniques. The development of novel nanomagnetic nanomaterials should continue to accelerate as new applications are identified and researchers uncover new mechanisms to increase and modulate magnetism at the nanometer scale.

O2‐sensitive MRI distinguishes brain tumor versus radiation necrosis in murine models

The goal of this study was to quantify the relationship between the 1H longitudinal relaxation rate constant, R1, and oxygen (O2) concentration (relaxivity, r1) in tissue and to quantify O2‐driven changes in R1 (ΔR1) during a breathing gas challenge in normal brain, radiation‐induced lesions, and tumor lesions.

Disruptive chemical doping in a ferritin‐based iron oxide nanoparticle to decrease r2 and enhance detection with T1‐weighted MRI

Inorganic doping was used to create flexible, paramagnetic nanoparticle contrast agents for in vivo molecular magnetic resonance imaging (MRI) with low transverse relaxivity (r2). Most nanoparticle contrast agents formed from superparamagnetic metal oxides are developed with high r2. While sensitive, they can have limited in vivo detection due to a number of constraints with T2 or T2*-weighted imaging. T1-weighted imaging is often preferred for molecular MRI, but most T1-shortening agents are small chelates with low metal payload or are nanoparticles that also shorten T2 and limit the range of concentrations detectable with T1-weighting. Here we used tungsten and iron deposition to form doped iron oxide crystals inside the apoferritin cavity to form a WFe nanoparticle with a disordered crystal and un-coupled atomic magnetic moments. The atomic magnetic moments were thus localized, resulting in a principally paramagnetic nanoparticle. The WFe nanoparticles had no coercivity or saturation magnetization at 5 K and sweeping up to ± 20,000 Oe, while native ferritin had a coercivity of 3000 Oe and saturation at ± 20,000 Oe. This tungsten-iron crystal paramagnetism resulted in an increased WFe particle longitudinal relaxivity (r1) of 4870 mm(-1) s(-1) and a reduced transverse relaxivity (r2) of 9076 mm(-1) s(-1) compared with native ferritin. The accumulation of the particles was detected with T1-weighted MRI in concentrations from 20 to 400 nm in vivo, both injected in the rat brain and targeted to the rat kidney glomerulus. The WFe apoferritin nanoparticles were not cytotoxic up to 700 nm particle concentrations, making them potentially important for targeted molecular MRI.

Efficient Small Blob Detection Based on Local Convexity, Intensity and Shape Information

The identification of small structures (blobs) from medical images to quantify clinically relevant features, such as size and shape, is important in many medical applications. One particular application explored here is the automated detection of kidney glomeruli after targeted contrast enhancement and magnetic resonance imaging. We propose a computationally efficient algorithm, termed the Hessian-based Difference of Gaussians (HDoG), to segment small blobs (e.g. glomeruli from kidney) from 3D medical images based on local convexity, intensity and shape information. The image is first smoothed and pre-segmented into small blob candidate regions based on local convexity. Two novel 3D regional features (regional blobness and regional flatness) are then extracted from the candidate regions. Together with regional intensity, the three features are used in an unsupervised learning algorithm for auto post-pruning. HDoG is first validated in a 2D form and compared with other three blob detectors from literature, which are generally for 2D images only. To test the detectability of blobs from 3D images, 240 sets of simulated images are rendered for scenarios mimicking the renal nephron distribution observed in contrast-enhanced, 3D MRI. The results show a satisfactory performance of HDoG in detecting large numbers of small blobs. Two sets of real kidney 3D MR images (6 rats, 3 human) are then used to validate the applicability of HDoG for glomeruli detection. By comparing MRI to stereological measurements, we verify that HDoG is a robust and efficient unsupervised technique for 3D blobs segmentation.