Search

The kidneys maintain fluid-electrolyte balance and excrete waste in the presence of constant fluctuations in plasma volume and systemic blood pressure. The kidneys perform these functions to control capillary perfusion and glomerular filtration by modulating the mechanisms of autoregulation. An effect of these modulations are spontaneous, natural fluctuations in nephron perfusion. Numerous other mechanisms can lead to fluctuations in perfusion and flow. The ability to monitor these spontaneous physiological fluctuations in vivocould facilitate the early detection of kidney disease. The goal of this work was to investigate the use of resting- state magnetic resonance imaging (rsMRI) to detect spontaneous physiological fluctuations in the kidney. We performed rsMRI of rat kidneys in vivoover 10 minutes, applying motion correction to resolve time series in each voxel. We observed spatially variable, spontaneous fluctuations in rsMRI signal between 0-0.3 Hz, in frequency bands also associated with autoregulatory mechanisms. We further applied rsMRI to investigate changes in these fluctuations in a rat model of diabetic nephropathy. Spectral analysis was performed on time series of rsMRI signal in kidney cortex and medulla. Power from spectra in specific frequency bands from kidney cortex correlated with severity of glomerular pathology caused by diabetic nephropathy. Finally, we investigated the feasibility of using rsMRI of the human kidney in two participants, observing the presence of similar, spatially variable fluctuations. This approach may enable a range of preclinical and clinical investigations of kidney function, and facilitate the development of new therapies to improve outcomes in patients with kidney disease.

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 Nglomand aVglomby high-resolution ex vivo MRI. CFE-MRI measured a mean Nglomof 12,220 ± 2,028 and 6,848 ± 1,676 (means ± SD) for WT and Os/+mouse kidneys in vivo, respectively. Nglommeasured in all mice in vivo using CFE-MRI varied by an average 15% from Nglommeasured 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 Nglommeasured in vivo in the same kidney varied within ~3%. Values of aVglomcalculated 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 Nglomand aVglomin healthy and diseased mice in vivo.

Nephron number is highly variable in humans and is thought to play an important role in renal health. Chronic kidney disease (CKD) is the result of too few nephrons to maintain homeostasis. Currently, nephron number can only be determined invasively or as a terminal assessment. Due to a lack of tools to measure and track nephron number in the living, the early stages of CKD often go unrecognized, preventing early intervention that might halt the progression of CKD. In this work, we present a technique to directly measure glomerular number (Nglom) and volume in vivo in the rat kidney (n= 8) using MRI enhanced with the novel contrast agent cationized ferritin (CFE-MRI). Adult male rats were administered intravenous cationized ferritin (CF) and imaged in vivo with MRI. Glomerular number was measured and each glomerulus was spatially mapped in 3D in the image. Mean apparent glomerular volume (aVglom) and intrarenal distribution of the individual glomerular volume (IGV), were also measured. These metrics were compared between images of the same kidneys scanned in vivo and ex vivo with CFE-MRI. In vivo Nglomand aVglomcorrelated to ex vivo metrics within the same kidneys and were within 10% of Nglomand aVglompreviously validated by stereologic methods. This is the first report of direct in vivo measurements of Nglomand aVglom, introducing an opportunity to investigate mechanisms of renal disease progression and therapeutic response over time.

Glomerular volume is an important metric reflecting glomerular filtration surface area within the kidney. Glomerular hypertrophy, or increased glomerular volume, may be an important marker for renal stress. Current stereological techniques report the average glomerular volume (AVglom) within the kidney. These techniques cannot assess the spatial or regional heterogeneity common in developing renal pathology. Here, we report a novel “unfolding” technique to measure the actual distribution of individual glomerular volumes in a kidney from the two-dimensional glomerulus profiles observed by optical microscopy. The unfolding technique was first developed and tested for accuracy with simulations and then applied to measure the number of glomeruli (Nglom), AVglom, and intrarenal distribution of individual glomerular volume (IVglom) in the oligosyndactyl (Os/+) mouse model compared with wild-type (WT) controls. The Os/+mice had fewer and larger glomeruli than WT mice: Nglomwas 12,126 ± 1,658 (glomeruli/kidney) in the WT mice and 5,516 ± 899 in the Os/+mice; AVglomwas 2.01 ± 0.28 × 10−4mm3for the WT mice and 3.47 ± 0.35 × 10−4mm3for the Os/+mice. Comparing the glomerular volume distributions in Os/+and WT kidneys, we observed that the Os/+distribution peaked at a higher value of IVglomthan the WT distribution peak, and glomeruli with a radius greater than 55 μm were more prevalent in the Os/+mice (3.4 ± 1.6% of total glomeruli vs. 0.6 ± 1.2% in WT). Finally, the largest profiles were more commonly found in the juxtamedullary region. Unfolding is a novel stereological technique that provides a new quantitative view of glomerular volume distribution in the individual kidney.

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 Nglomand Vglomin intact kidneys. This work demonstrates the use of cationized ferritin (CF) as a magnetic resonance imaging (MRI) contrast agent to measure Nglomand Vglomin 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 kidney has an extraordinary ability to maintain glomerular filtration despite natural fluctuations in blood pressure or nephron loss. This is partly due to local coordination between single nephron filtration and vascular perfusion. An improved understanding of the three-dimensional 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 develop magnetic resonance imaging (MRI) tools to visualize the dynamics of single nephron function in three dimensions throughout the isolated perfused rat kidney. We used a intravenous slow perfusion of a glomerulus- targeted imaging tracer (cationized ferritin, CF), to map macromolecular dynamics and to identify glomeruli in 3D, followed by a bolus of a freely-filtered tracer, gadolinium diethylenetriamine penta-acetic acid (Gd-DTPA), to map filtration kinetics. We observed a wide intra-kidney distribution of CF binding rates and snGFR between nephrons. snGFR and CF uptake rates did not vary significantly by distance from the kidney surface. snGFR varied from ~10 to ~100 nL/min throughout the kidney. Whole single kidney GFR (skGFR) was similar across all kidneys, despite differences in the distributions of estimated snGFR and glomerular number, indicating a robust adaptive regulation of individual nephrons to maintain constant skGFR in the presence of a natural variation in nephron number. This work provides a framework for 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 heterogenous. To comprehensively understand kidney structure and pathology, it is critical to develop tool to map tissue microstructure in the context of the whole, intact organ. Magnetic resonance imaging (MRI) can provide a unique, three-dimensional (3D) view of the kidney and allows for measurement of multiple pathologic features. Here, we develop a platform to systematically render and map gross and microstructural features of the human kidney based on 3D MRI. These features include pyramid number and morphology, and associated medulla and cortex. in a subset of these kidneys, we also map individual glomeruli and glomerular volumes using cationic ferritin enhance-MRI to report intra-renal heterogeneity in glomerular density and size. Finally, we render and measure 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 anatomical 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.