Kevin M. Bennett

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.

Small Blob Identification in Medical Images Using Regional Features From Optimum Scale

Recent advances in medical imaging technology have greatly enhanced imaging-based diagnosis which requires computational effective and accurate algorithms to process the images (e.g., measure the objects) for quantitative assessment. In this research, we are interested in one type of imaging objects: small blobs. Examples of small blob objects are cells in histopathology images, glomeruli in MR images, etc. This problem is particularly challenging because the small blobs often have inhomogeneous intensity distribution and an indistinct boundary against the background. Yet, in general, these blobs have similar sizes. Motivated by this finding, we propose a novel detector termed Hessian-based Laplacian of Gaussian (HLoG) using scale space theory as the foundation. Like most imaging detectors, an image is first smoothed via LoG. Hessian analysis is then launched to identify the single optimal scale on which a presegmentation is conducted. The advantage of the Hessian process is that it is capable of delineating the blobs. As a result, regional features can be retrieved. These features enable an unsupervised clustering algorithm for postpruning which should be more robust and sensitive than the traditional threshold-based postpruning commonly used in most imaging detectors. To test the performance of the proposed HLoG, two sets of 2-D grey medical images are studied. HLoG is compared against three state-of-the-art detectors: generalized LoG, Radial-Symmetry and LoG using precision, recall, and F-score metrics. We observe that HLoG statistically outperforms the compared detectors.

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.

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.

Live nephron imaging by MRI

The local sensitivity of MRI can be improved with small MR detectors placed close to regions of interest. However, to maintain such sensitivity advantage, local detectors normally need to communicate with the external amplifier through cable connections, which prevent the use of local detectors as implantable devices. Recently, an integrated wireless amplifier was developed that can efficiently amplify and broadcast locally detected signals, so that the local sensitivity was enhanced without the need for cable connections. This integrated detector enabled the live imaging of individual glomeruli using negative contrast introduced by cationized ferritin, and the live imaging of renal tubules using positive contrast introduced by gadopentetate dimeglumine. Here, we utilized the high blood flow to image individual glomeruli as hyperintense regions without any contrast agent. These hyperintense regions were identified for pixels with signal intensities higher than the local average. Addition of Mn(2+) allowed the simultaneous detection of both glomeruli and renal tubules: Mn(2+) was primarily reabsorbed by renal tubules, which would be distinguished from glomeruli due to higher enhancement in T1-weighted MRI. Dynamic studies of Mn(2+) absorption confirmed the differential absorption affinity of glomeruli and renal tubules, potentially enabling the in vivo observation of nephron function.

Biocompatibility of ferritin-based nanoparticles as targeted MRI contrast agents

Ferritin is a naturally occurring iron storage protein, proposed as a clinically relevant nanoparticle with applications as a diagnostic and therapeutic agent. Cationic ferritin is a targeted, injectable contrast agent to measure kidney microstructure with MRI. Here, the toxicity of horse spleen ferritin is assessed as a step to clinical translation. Adult male mice received cationic, native and high dose cationic ferritin (CF, NF, or HDCF) or saline and were monitored for 3weeks. Transient weight loss occurred in the ferritin groups with no difference in renal function parameters. Ferritin-injected mice demonstrated a lower serum iron 3weeks after administration. In ferritin-injected animals pre-treated with hydrocortisone, there were no structural or weight differences in the kidneys, liver, lung, heart, or spleen. This study demonstrates a lack of significant detrimental effects of horse-derived ferritin-based nanoparticles at MRI-detectable doses, allowing further exploration of these agents in basic research and clinical diagnostics.

MRI tools for assessment of microstructure and nephron function of the kidney.

MRI can provide excellent detail of renal structure and function. Recently, novel MR contrast mechanisms and imaging tools have been developed to evaluate microscopic kidney structures including the tubules and glomeruli. Quantitative MRI can assess local tubular function and is able to determine the concentrating mechanism of the kidney noninvasively in real time. Measuring single nephron function is now a near possibility. In parallel to advancing imaging techniques for kidney microstructure is a need to carefully understand the relationship between the local source of MRI contrast and the underlying physiological change. The development of these imaging markers can impact the accurate diagnosis and treatment of kidney disease. This study reviews the novel tools to examine kidney microstructure and local function and demonstrates the application of these methods in renal pathophysiology.

MRI-Detectable Nanoparticles: The Potential Role in the Diagnosis of and Therapy for Chronic Kidney Disease

Chronic kidney disease (CKD) is a common, deadly, and expensive threat to public health. Patients susceptible to the development of CKD are difficult to identify because there are few noninvasive clinical techniques and markers to assess early kidney dysfunction. Noninvasive imaging techniques are being developed to quantitatively measure kidney morphology and function in preclinical research and in clinical trials. Magnetic resonance imaging (MRI) techniques in particular have the potential to provide structural and functional information in the kidney. Novel molecular imaging techniques, using targeted magnetic nanoparticles that exploit the characteristics of the endogenous protein, ferritin, have been developed in conjunction with MRI to count every perfused glomerulus in the kidney and measure their individual volumes. This technique could open the door to the possibility of prospectively assessing and eventually reducing a patients risk for progression to CKD. This review highlights the potential clinical benefits of early detection in patients predisposed to CKD and discusses technologic and regulatory hurdles to the translation of these molecular MRI techniques to provide early diagnosis of CKD.

Use of Cationized Ferritin Nanoparticles to Measure Renal Glomerular Microstructure with MRI.

Magnetic resonance imaging (MRI) is becoming important for whole-kidney assessment of glomerular morphology, both in vivo and ex vivo. MRI-based renal morphological measurements can be made in intact organs and allow direct measurements of every perfused glomerulus. Cationic ferritin (CF) is used as a superparamagnetic contrast agent for MRI. CF binds to the glomerular basement membrane after intravenous injection, allowing direct, whole-kidney measurements of glomerular number, volume, and volume distribution. Here we describe the production, testing, and use of CF as an MRI contrast agent for quantitative glomerular morphology in intact mouse, rat, and human kidneys.