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 Ϯ 3,753 labeled glomeruli (n ϭ 5 kidneys). Acid maceration counting of contralateral kidneys yielded an estimate of 30,585 Ϯ 2,053 glomeruli (n ϭ 6 kidneys). Disector/fractionator stereology counting yielded an estimate of 34,963 glomeruli (n ϭ 2). MRI-based measurement of apparent glomerular volume of labeled glomeruli was 4.89 ϫ 10 Ϫ4 mm 3 (n ϭ 5) compared with the average stereological measurement of 4.99 ϫ 10 Ϫ4 mm 3 (n ϭ 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. stereology; nephron; glomerulus count; nanoparticles; magnetic resonance imaging THE PURPOSE OF THIS WORK WAS to measure the number and size of all glomeruli in the entire, intact kidney using magnetic resonance imaging (MRI). Changes in the number and size of glomeruli have been linked to a number of renal and systemic diseases (5, 7). However, current techniques for counting and measuring glomeruli, such as acid maceration (4) and the dissector/fractionator stereology technique (3), require the destruction of the entire kidney. Furthermore, conventional histological techniques extrapolate the total number and size of glomeruli from a selected number of histological sections or isolated glomeruli. Current techniques are thus estimates rather than direct measurements and do not allow for localization of identified functioning glomeruli to specific parts of the kidney. A method for directly and nondestructively measuring the number and size of all glomeruli in the kidney would serve as a useful tool in animal studies and potentially in the clinic.Recently we demonstrated that intravenous injections of the iron-binding protein ferritin, functionalized with cationic amine groups (6), can be used to detect individual glomeruli both in vivo and ex vivo with MRI (1). This method is based on the electrostatic binding of cationic ferritin (CF) to the anionic macromolecules of the glomerular basement membrane (GBM) and subsequent perturbation of the magnetic field around the labeled GBM by ferritin, result...

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.

We evaluated subcutaneous AT oxygen partial pressure (pO 2); liver and whole-body insulin sensitivity; AT expression of genes and pathways involved in inflammation, fibrosis, and branched-chain amino acid (BCAA) catabolism; systemic markers of inflammation; and plasma BCAA concentrations, in 3 groups of participants that were rigorously stratified by adiposity and insulin sensitivity: metabolically healthy lean (MHL; n = 11), metabolically healthy obese (MHO; n = 15), and metabolically unhealthy obese (MUO; n = 20). RESULTS. AT pO 2 progressively declined from the MHL to the MHO to the MUO group, and was positively associated with hepatic and whole-body insulin sensitivity. AT pO 2 was positively associated with the expression of genes involved in BCAA catabolism, in conjunction with an inverse relationship between AT pO 2 and plasma BCAA concentrations. AT pO 2 was negatively associated with AT gene expression of markers of inflammation and fibrosis. Plasma PAI-1 increased from the MHL to the MHO to the MUO group and was negatively correlated with AT pO 2 , whereas the plasma concentrations of other cytokines and chemokines were not different among the MHL and MUO groups. CONCLUSION. These results support the notion that reduced AT oxygenation in individuals with obesity contributes to insulin resistance by increasing plasma PAI-1 concentrations and decreasing AT BCAA catabolism and thereby increasing plasma BCAA concentrations. TRIAL REGISTRATION. ClinicalTrials.gov NCT02706262.

Decreased adipose tissue oxygen tension and increased HIF-1α expression can trigger adipose tissue inflammation and dysfunction in obesity. Our current understanding of obesity-associated decreased adipose tissue oxygen tension is mainly focused on changes in oxygen supply and angiogenesis. Here, we demonstrate that increased adipocyte O2 demand, mediated by ANT2 activity, is the dominant cause of adipocyte hypoxia. Deletion of adipocyte Ant2 improves obesity-induced intracellular adipocyte hypoxia by decreasing obesity-induced adipocyte oxygen demand, without effects on mitochondrial number or mass, or oligomycin-sensitive respiration. This led to decreased adipose tissue HIF-1α expression and inflammation with improved glucose tolerance and insulin resistance in both a preventative or therapeutic setting. Our results suggest that ANT2 may be a target for the development of insulin sensitizing drugs and that ANT2 inhibition might have clinical utility.

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.