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The kidney has an extraordinary ability to maintain glomerular filtration despite natural fluctuations in blood pressure and nephron loss. This is partly due to local coordination between single-nephron filtration and vascular perfusion. An improved understanding of the three-dimensional (3-D) functional coordination between nephrons and the vasculature may provide a new perspective of the heterogeneity of kidney function and could inform targeted therapies and timed interventions to slow or prevent the progression of kidney disease. Here, we developed magnetic resonance imaging (MRI) tools to visualize single-nephron function in 3-D throughout the isolated perfused rat kidney. We used an intravenous slow perfusion of a glomerulus-targeted imaging tracer [cationized ferritin (CF)] to map macromolecular dynamics and to identify glomeruli in 3-D, followed by a bolus of a freely filtered tracer (gadolinium diethylenetriamine penta-acetic acid) to map filtration kinetics. There was a wide intrakidney distribution of CF binding rates and estimated single-nephron glomerular filtration rate (eSNGFR) between nephrons. eSNGFR and CF uptake rates did not vary significantly by distance from the kidney surface. eSNGFR varied from ∼10 to ∼100 nL/min throughout the kidney. Whole single-kidney GFR was similar across all kidneys, despite differences in the distributions eSNGFR of and glomerular number, indicating a robust adaptive regulation of individual nephrons to maintain constant single-kidney GFR in the presence of a natural variation in nephron number. This work provides a framework for future studies of single-nephron function in the whole isolated perfused kidney and experiments of single-nephron function in vivo using MRI.

Kidney pathologies are often highly heterogeneous. To comprehensively understand kidney structure and pathology, it is critical to develop tools to map tissue microstructure in the context of the whole, intact organ. Magnetic resonance imaging (MRI) can provide a unique, three-dimensional view of the kidney and allows for measurements of multiple pathological features. Here, we developed a platform to systematically render and map gross and microstructural features of the human kidney based on three-dimensional MRI. These features include pyramid number and morphology as well as the associated medulla and cortex. In a subset of these kidneys, we also mapped individual glomeruli and glomerular volumes using cationic ferritin-enhanced MRI to report intrarenal heterogeneity in glomerular density and size. Finally, we rendered and measured regions of nephron loss due to pathology and individual glomerular volumes in each pyramidal unit. This work provides new tools to comprehensively evaluate the kidney across scales, with potential applications in anatomic and physiological research, transplant allograft evaluation, biomarker development, biopsy guidance, and therapeutic monitoring. These image rendering and analysis tools could eventually impact the field of transplantation medicine to improve longevity matching of donor allografts and recipients and reduce discard rates through the direct assessment of donor kidneys. NEW & NOTEWORTHY We report the application of cutting-edge image analysis approaches to characterize the pyramidal geometry, glomerular microstructure, and heterogeneity of the whole human kidney imaged using MRI. This work establishes a framework to improve the detection of microstructural pathology to potentially facilitate disease monitoring or transplant evaluation in the individual kidney.

Acute kidney injury (AKI) increases the risk for chronic kidney disease (CKD). However, there are few tools to detect microstructural changes after AKI. Here, cationic ferritin-enhanced magnetic resonance imaging (CFE-MRI) was applied to examine the heterogeneity of kidney pathology in the transition from AKI to CKD. Adult male mice received folic acid followed by cationic ferritin and were euthanized at four days (AKI), four weeks (CKD-4) or 12 weeks (CKD-12). Kidneys were examined by histologic methods and CFE-MRI. In the CKD-4 and CKD-12 groups, glomerular number was reduced and atubular cortical lesions were observed. Apparent glomerular volume was larger in the AKI, CKD-4 and CKD-12 groups compared to controls. Glomerular hypertrophy occurred with ageing. Interglomerular distance and glomerular density were combined with other MRI metrics to distinguish the AKI and CKD groups from controls. Despite significant heterogeneity, the noninvasive (MRI-based) metrics were as accurate as invasive (histological) metrics at distinguishing AKI and CKD from controls. To assess the toxicity of cationic ferritin in a CKD model, CKD-4 mice received cationic ferritin and were examined one week later. The CKD-4 groups with and without cationic ferritin were similar, except the iron content of the kidney, liver, and spleen was greater in the CKD-4 plus cationic ferritin group. Thus, our study demonstrates the accuracy and safety of CFE-MRI to detect whole kidney pathology allowing for the development of novel biomarkers of kidney disease and providing a foundation for future in vivo longitudinal studies in mouse models of AKI and CKD to track nephron fate.

Acute kidney injury (AKI) in premature neonates is common due to the administration of life-saving therapies. The impact of AKI on renal morphology and susceptibility to further renal damage is poorly understood. Recent advances in radiological imaging have allowed integration of soft tissue morphology in the intact organ, facilitating a more complete understanding of changes in tissue microstructure associated with pathology. Here, we applied magnetic resonance imaging (MRI) to detect both glomerular and vascular changes in a rabbit model of neonatal AKI, induced by indomethacin and gentamicin. Using combined spin-echo MRI and cationic ferritin enhanced gradient-echo MRI (CFE-MRI), we observed (a) an increased cortical arterial diameter in the AKI cohort compared to healthy controls, and (b) focal loss of vascular density and glomerular loss in a circumferential band ~1 mm from the cortical surface. This combined use of vascular and glomerular imaging may give insight into the etiology of AKI and its impact on renal health later in life.

Preterm birth is a leading cause of neonatal morbidity. Survivors have a greater risk for kidney dysfunction and hypertension. Little is known about the molecular changes that occur in the kidney of individuals born preterm. Here, we demonstrate that mice delivered two days prior to full term gestation undergo premature cessation of nephrogenesis, resulting in a lower glomerular density. Kidneys from preterm and term groups exhibited differences in gene expression profiles at 20- and 27-days post-conception, including significant differences in the expression of fat-soluble vitamin-related genes. Kidneys of the preterm mice exhibited decreased proportions of endothelial cells and a lower expression of genes promoting angiogenesis compared to the term group. Kidneys from the preterm mice also had altered nephron progenitor subpopulations, early Six2 depletion, and altered Jag1 expression in the nephrogenic zone, consistent with premature differentiation of nephron progenitor cells. In conclusion, preterm birth alone was sufficient to shorten the duration of nephrogenesis and cause premature differentiation of nephron progenitor cells. These candidate genes and pathways may provide targets to improve kidney health in preterm infants.

The mammalian kidney is a complex organ, requiring the concerted function of up to millions of nephrons. The number of nephrons is constant after nephrogenesis during development, and nephron loss over a life span can lead to susceptibility to acute or chronic kidney disease. New technologies are under development to count individual nephrons in the kidney in vivo. This review outlines these technologies and highlights their relevance to studies of human renal development and disease.

Imaging biomarkers are being rapidly developed for early diagnosis and staging of disease. The development of these biomarkers requires advances in both image acquisition and analysis. Detecting and segmenting objects from images are often the first steps in quantitative measurement of these biomarkers. The challenges of detecting objects in images, particularly small objects known as blobs, include low image resolution, image noise and overlap between the blobs. The Difference of Gaussian (DoG) detector has been used to overcome these challenges in blob detection. However, the DoG detector is susceptible to over-detection and must be refined for robust, reproducible detection in a wide range of medical images. In this research, we propose a joint constraint blob detector from U-Net, a deep learning model, and Hessian analysis, to overcome these problems and identify true blobs from noisy medical images. We evaluate this approach, UH-DoG, using a public 2D fluorescent dataset for cell nucleus detection and a 3D kidney magnetic resonance imaging dataset for glomerulus detection. We then compare this approach to methods in the literature. While comparable to the other four comparing methods on recall, the UH-DoG outperforms them on both precision and F-score.

The development of chronic kidney disease (CKD) is associated with the loss of functional nephrons. However, there are no methods to directly measure nephron number in living subjects. Thus, there are no methods to track the early stages of progressive CKD before changes in total renal function. In this work, we used cationic ferritin-enhanced magnetic resonance imaging (CFE-MRI) to enable measurements of glomerular number ( Nglom) and apparent glomerular volume (aVglom) in vivo in healthy wild-type (WT) mice ( n = 4) and mice with oligosyndactylism (Os/+; n = 4), a model of congenital renal hypoplasia leading to nephron reduction. We validated in vivo measurements of Nglom and aVglom by high-resolution ex vivo MRI. CFE-MRI measured a mean Nglom of 12,220 ± 2,028 and 6,848 ± 1,676 (means ± SD) for WT and Os/+ mouse kidneys in vivo, respectively. Nglom measured in all mice in vivo using CFE-MRI varied by an average 15% from Nglom measured ex vivo in the same kidney (α = 0.05, P = 0.67). To confirm that CFE-MRI can also be used to track nephron endowment longitudinally, a WT mouse was imaged three times by CFE-MRI over 2 wk. Values of Nglom measured in vivo in the same kidney varied within ~3%. Values of aVglom calculated from CFE-MRI in vivo were significantly different (~15% on average, P < 0.01) from those measured ex vivo, warranting further investigation. This is the first report of direct measurements of Nglom and aVglom in healthy and diseased mice in vivo.

Background: Accumulating evidence supports an association between nephron number and susceptibility to kidney disease. However, it is not currently possible to directly measure nephron number in a clinical setting. Recent clinical studies have used glomerular density from a single biopsy and whole kidney cortical volume from imaging to estimate both nephron number and single nephron glomerular filtration rate. However, the accuracy of these estimates from individual subjects is unknown. Furthermore, it is not clear how sample size or biopsy location may influence these estimates. These questions are critical to study design and to the potential translation of these tools to estimate nephron number in individual subjects. Methods: We measured the variability in estimated nephron number derived from needle or virtual biopsies and cortical volume in human kidneys declined for transplantation. We performed multiple needle biopsies in the same kidney, and examined the three-dimensional spatial distribution of nephron density by magnetic resonance imaging. We determined the accuracy of a single kidney biopsy to predict the mean nephron number estimated from multiple biopsies from the same kidney. Results: A single needle biopsy had a 15% chance and virtual biopsy had a 60% chance of being within 20% of whole kidney nephron number. Single needle biopsies could be used to detect differences in nephron number between large cohorts of several hundred subjects. Conclusions: The number of subjects required to accurately detect differences in nephron number between populations can be predicted based on natural intra-kidney variability in glomerular density. A single biopsy is insufficient to accurately predict nephron number in individual subjects.

Dynamic contrast-enhanced (DCE) MRI monitors the transit of contrast agents, typically gadolinium chelates, through the intrarenal regions, the renal cortex, the medulla, and the collecting system. In this way, DCE-MRI reveals the renal uptake and excretion of the contrast agent. An optimal DCE-MRI acquisition protocol involves finding a good compromise between whole-kidney coverage (i.e., 3D imaging), spatial and temporal resolution, and contrast resolution. By analyzing the enhancement of the renal tissues as a function of time, one can determine indirect measures of clinically important single-kidney parameters as the renal blood flow, glomerular filtration rate, and intrarenal blood volumes. Gadolinium-containing contrast agents may be nephrotoxic in patients suffering from severe renal dysfunction, but otherwise DCE-MRI is clearly useful for diagnosis of renal functions and for assessing treatment response and posttransplant rejection.Here we introduce the concept of renal DCE-MRI, describe the existing methods, and provide an overview of preclinical DCE-MRI applications to illustrate the utility of this technique to measure renal perfusion and glomerular filtration rate in animal models.This publication is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This introduction is complemented by two separate publications describing the experimental procedure and data analysis.

Tubular pathologies are a common feature of kidney disease. Current metrics to assess kidney health, in vivo or in transplant, are generally based on urinary or serum biomarkers and pathological findings from kidney biopsies. Biopsies, usually taken from the kidney cortex, are invasive and prone to sampling error. Tools to directly and noninvasively measure tubular pathology could provide a new approach to assess kidney health. This study used diffusion magnetic resonance imaging (dMRI) as a noninvasive tool to measure the size of the tubular lumen in ex vivo, perfused kidneys. We first used Monte Carlo simulations to demonstrate that dMRI is sensitive to restricted tissue water diffusion at the scale of the kidney tubule. We applied dMRI and biophysical modeling to examine the distribution of tubular diameters in ex vivo, fixed kidneys from mice, rats, and a human donor. The biophysical model to fit the dMRI signal was based on a superposition of freely diffusing water and water diffusing inside infinitely long cylinders of different diameters. Tubular diameters measured by dMRI were within 10% of those measured by histology within the same tissue. Finally, we applied dMRI to investigate kidney pathology in a mouse model of folic-acid-induced acute kidney injury. dMRI detected heterogeneity in the distribution of tubules within the kidney cortex of mice with acute kidney injury compared with control mice. We conclude that dMRI can be used to measure the distribution of tubule diameters in the kidney cortex ex vivo and that dMRI may provide a new noninvasive biomarker of tubular pathology. NEW & NOTEWORTHY Tubular pathologies are a common feature of kidney disease. Current metrics to assess kidney health, in vivo or in transplant, are generally based on urinary or serum biomarkers and pathological findings from kidney biopsies. Diffusion MRI can be used to measure the distribution of tubule diameters in the kidney cortex ex vivo and may provide a new noninvasive biomarker of tubular pathology.

Here we present an analysis protocol for dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) data of the kidneys. It covers comprehensive steps to facilitate signal to contrast agent concentration mapping via T1 mapping and the calculation of renal perfusion and filtration parametric maps using model-free approaches, model free analysis using deconvolution, the Toft’s model and a Bayesian approach.This chapter is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This analysis protocol chapter is complemented by two separate chapters describing the basic concept and experimental procedure.

Recent advances in medical imaging technology bring great promises for medicine practices. Imaging biomarkers are discovered to inform disease diagnosis, prognosis, and treatment assessment. Detecting and segmenting objects from images are often the first steps in quantitative measurement of these biomarkers. The challenges of detecting objects in images, particularly small objects known as blobs, include low image resolution, image noise and overlap among the blobs. This research proposes a Bi-Threshold Constrained Adaptive Scale (BTCAS) blob detector to uncover the relationship between the U-Net threshold and the Difference of Gaussian (DoG) scale to derive a multi-threshold, multi-scale small blob detector. With lower and upper bounds on the probability thresholds from U-Net, two binarized maps of the distance are rendered between blob centers. Each blob is transformed to a DoG space with an adaptively identified local optimum scale. A Hessian convexity map is rendered using the adaptive scale, and the under-segmentation typical of the U-Net is resolved. To validate the performance of the proposed BTCAS, a 3D simulated dataset (n = 20) of blobs, a 3D MRI dataset of human kidneys and a 3D MRI dataset of mouse kidneys, are studied. BTCAS is compared against four state-of-the-art methods: HDoG, U-Net with standard thresholding, U-Net with optimal thresholding, and UH-DoG using precision, recall, F-score, Dice and IoU. We conclude that BTCAS statistically outperforms the compared detectors.

Nephron number varies widely in humans. A low nephron endowment at birth or a loss of functioning nephrons is strongly linked to increased susceptibility to chronic kidney disease. In this work, we developed a contrast agent, radiolabeled cationic ferritin (RadioCF), to map functioning glomeruli in vivo in the kidney using positron emission tomography (PET). PET radiotracers can be detected in trace doses (/+), a model of congenital hypoplasia and low nephron mass. The average standardized uptake value (SUV) measured by PET 90 min after injection was 21% higher in WT mice than in Os/+ mice, consistent with the higher glomerular density in WT mice. The difference in peak SUV from SUV at 90 min correlated with glomerular density in male mice from both WT and Os/+ cohorts ( R2 = 0.98). Finally, we used RadioCF-PET to map functioning glomeruli in a donated human kidney. SUV within the kidney correlated with glomerular number ( R2= 0.78) measured by CF-enhanced magnetic resonance imaging in the same locations. This work suggests that RadioCF-PET appears to accurately detect nephron mass and has the potential for clinical translation.

Studies of human nephron number have been conducted for well over a century and have uncovered the large variability in nephron number. However, the mechanisms influencing nephron endowment and loss along with the etiology for the wide range amongst individuals is largely unknown. Advances in imaging technology have allowed investigators to revisit the principles of renal structure and physiology and their roles in the progression of kidney disease. Here, we will review the latest data on the influences impacting nephron number, innovations made over the last six years to understand and integrate renal structure and function, and new developments in the tools used to count nephrons in vivo.

Background: Acute kidney injury affects nearly 30% of preterm neonates in the intensive care unit. We aimed to determine if nephrotoxin-induced AKI disrupted renal development assessed by imaging (CFE-MRI). Methods: Neonatal New Zealand rabbits received indomethacin and gentamicin (AKI) or saline (control) for four days followed by cationic ferritin (CF) at six weeks. Ex vivo images were acquired using a gradient echo pulse sequence on 7T MRI. Glomerular number (Nglom) and apparent glomerular volume (aVglom) were determined. CF toxicity was assessed at 2 and 28 days in healthy rabbits. Results: Nglom was lower in the AKI group as compared to controls (74,034 vs 198,722, p

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.

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.

Nephron number ( Nglom) and size (Vglom) 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 Nglom and Vglom in intact kidneys. This work demonstrates the use of cationized ferritin (CF) as a magnetic resonance imaging (MRI) contrast agent to measure Nglom and Vglom 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.

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, as there are few noninvasive clinical techniques and markers to assess early kidney dysfunction. Noninvasive imaging techniques are being developed to quantitatively measure renal morphology and function, both in preclinical research and in clinical trials. Magnetic resonance imaging (MRI) techniques in particular have the potential to provide both 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 patient’s 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.

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.

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 T2*-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 T2* 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.

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 × 10−4mm3( n = 5) compared with the average stereological measurement of 4.99 × 10−4mm3( 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.

The integrity of the basement membrane is essential for tissue cellular growth and is often altered in disease. In this work a method for noninvasively detecting the structural integrity of the basement membrane, based on the delivery of cationic iron-oxide nanoparticles, was developed. Cationic particles accumulate due to the highly negative charge of proteoglycans in the basement membrane. The kidney was used to test this technique because of its highly fenestrated endothelia and well-established disease models to manipulate the basement membrane charge barrier. After systemic injection of cationic or native ferritin (CF or NF) in rats, ex vivo and in vivo MRI showed selective accumulation of CF, but not NF, causing a 60% reduction in signal intensity in cortex at the location of individual glomeruli. Immunofluorescence and electron microscopy demonstrated that this CF accumulation was localized to the glomerular basement membrane (GBM). In a model of GBM breakdown during focal and segmental glomerulosclerosis, MRI showed reduced single glomerular accumulation of CF, but a diffuse accumulation of CF in the kidney tubules caused by leakage of CF through the glomerulus. Cationic contrast agents can be used to target the basement membrane and detect the breakdown of the basement membrane in disease.

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.

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 3 weeks. Transient weight loss occurred in the ferritin groups with no difference in renal function parameters. Ferritin-injected mice demonstrated a lower serum iron 3 weeks 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.

The glomeruli of the kidney perform the key role of blood filtration and the number of glomeruli in a kidney is correlated with susceptibility to chronic kidney disease and chronic cardiovascular disease. This motivates the development of new technology using magnetic resonance imaging (MRI) to measure the number of glomeruli and nephrons in vivo. However, there is currently a lack of computationally efficient techniques to perform fast, reliable and accurate counts of glomeruli in MR images due to the issues inherent in MRI, such as acquisition noise, partial volume effects (the mixture of several tissue signals in a voxel) and bias field (spatial intensity inhomogeneity). Such challenges are particularly severe because the glomeruli are very small, (in our case, a MRI image is ~16 million voxels, each glomerulus is in the size of 8~20 voxels), and the number of glomeruli is very large. To address this, we have developed an efficient Hessian based Difference of Gaussians (HDoG) detector to identify the glomeruli on 3D rat MR images. The image is first smoothed via DoG followed by the Hessian process to pre-segment and delineate the boundary of the glomerulus candidates. This then provides a basis to extract regional features used in an unsupervised clustering algorithm, completing segmentation by removing the false identifications occurred in the pre-segmentation. The experimental results show that Hessian based DoG has the potential to automatically detect glomeruli,from MRI in 3D, enabling new measurements of renal microstructure and pathology in preclinical and clinical studies.

there is no organ in which structure and function are more directly coupled than in the kidney. Physiological abnormalities in the kidney can be a direct reflection of microstructural pathology, a fact long recognized by clinicians who use biopsy to assess disease state. New technology in small

The total number of glomeruli (nephrons) in a kidney is an important microanatomical parameter for at least three reasons: it provides an index of the success/extent of nephrogenesis and can thereby provide insights into the roles of specific genes and feto-maternal environmental factors in nephrogenesis; low nephron number has been linked to an increased risk of cardiovascular and renal disease in adulthood; and knowledge of quantitative kidney microanatomy can illuminate our understanding of physiological mechanisms in health and disease. A range of methods has been used to count glomeruli in kidneys over the past 100 years, with design-based stereology (the physical disector/fractionator combination) considered the gold standard. However, this approach is labor-intensive and expensive, and therefore is not utilized by most laboratories. A new method for counting and sizing every glomerulus in the kidney has recently been described. This method involves in vivo labeling of glomeruli with cationic ferritin, and then magnetic resonance imaging (MRI) of the ex vivo kidney. Values are obtained in one sixth of the time of disector-based approaches. This new MRI method holds great promise for studies of glomerular number and size ex vivo and in vivo.