Macura, Slobodan

Background The urine metabolites and chemistries that contribute to kidney stone formation are not fully understood. This study examined differences between the urine metabolic and chemistries profiles of first-time stone formers and controls. Methods High-resolution 1 H-nuclear magnetic resonance (NMR) spectroscopy-based metabolomic analysis was performed in 24-hour urine samples from a prospective cohort of 418 first-time symptomatic kidney stone formers and 440 controls. In total, 48 NMR-quantified metabolites in addition to 12 standard urine chemistries were assayed. Analysis of covariance was used to determine the association of stone former status with urine metabolites or chemistries after adjusting for age and sex and correcting for the false discovery rate. Gradient-boosted machine methods with nested cross-validation were applied to predict stone former status. Results Among the standard urine chemistries, stone formers had lower urine oxalate and potassium and higher urine calcium, phosphate, and creatinine. Among NMR urine metabolites, stone formers had lower hippuric acid, trigonelline, 2-furoylglycine, imidazole, and citrate and higher creatine and alanine. A cross-validated model using urine chemistries, age, and sex yielded a mean AUC of 0.76 (95% CI, 0.73 to 0.79). A cross-validated model using urine chemistries, NMR-quantified metabolites, age, and sex did not meaningfully improve the discrimination (mean AUC, 0.78; 95% CI, 0.75 to 0.81). In this combined model, among the top ten discriminating features, four were urine chemistries and six NMR-quantified metabolites. Conclusions Although NMR-quantified metabolites did not improve discrimination, several urine metabolic profiles were identified that may improve understanding of kidney stone pathogenesis.

To describe transverse relaxation of water in fixed tissue, we propose a model of transverse relaxation accelerated by diffusion and exchange (TRADE) that assumes exchange between free (visible) and bound (invisible) water, which relax by the dipole–dipole interaction, chemical exchange, and translation in the field gradient. Depending on the prevailing mechanism, transverse relaxation time (T2) of water in fixed tissue could increase (when dipole–dipole interaction prevails) or decrease with temperature (when diffusion in the field gradient prevails). Chemical exchange can make T2 even temperature independent. Also, variation of resolution from 100 to 15 μm/pxl (or less) affects effective transverse relaxation. T2 steadily decreases with increased resolution ( , is the read direction resolution). TRADE can describe all of these observations (semi)quantitatively. The model has been experimentally verified on water phantoms and on formalin‐fixed zebrafish, mouse brain, and rabbit larynx tissues. TRADE could help predict optimal scanning parameters for high‐resolution MRM from much faster measurements at lower resolution.

AbstractObjectivesTo examine the degree of agreement between MRI and histologically generated volumetric measurements of residual injection laryngoplasty material.MethodsFollowing left recurrent laryngeal nerve transection, rabbit vocal cords were injected with jellyfish collagen, Cymetra®, or Restylane®. Laryngeal tissue was harvested 4 or 12 weeks post injection followed by MRI imaging and histologic cross‐sectioning. Two raters estimated the volume of remaining injection material in specimens within MRI and histologic axial cross sections. Wilcoxon signed rank tests were employed to detect gross differences between inter‐rater measurements and between imaging modalities across time. Agreement between rater measurements and imaging (histology and MRI) was assessed using intra‐class correlation coefficients.ResultsData was available from 16 rabbits sacrificed at 4 weeks (n = 8) and 12 weeks (n = 8). Inter‐rater testing of MRI imaging revealed no significant differences (p > .05) between rater measurements across time points, and excellent agreement (0.93; 95% confidence interval 0.80–0.98) while histologically estimated volumes demonstrated a significant difference at 4 weeks (p < .05) and overall good agreement (0.89; 95% confidence interval 0.59–0.97). Comparison of MRI and histologically estimated volume measurements revealed significant differences at the 4‐week time point (p < .05) but not at 12 weeks (p > .05). Overall, there is only moderate agreement between MRI and histology estimates (0.72; 95% confidence interval 0.22–0.90).ConclusionsMRI imaging demonstrates good reliability and similar estimates of volume to histologically estimated measurements of residual injection laryngoplasty material at time points clinically relevant for future injection laryngoplasty experiments.Level of EvidenceNA.

Abstract INTRODUCTION Glioblastoma (GBM) is a diffusely invasive primary brain tumor with significant spread of tumor cells to the periphery of visible image abnormality. Enhancement of Gadolinium (Gd) contrast agent on magnetic resonance imaging (MRI) has historically been considered a confirmation of local breakdown of the blood brain barrier (BBB) and sufficient drug delivery to the bulk of tumors. In this work, we used GBM-derived xenograft (PDX) models to compare drug delivery in GBM brain for high and low BBB-permeable drugs. MATERIALS AND METHODS Five patient-derived orthotopic xenograft models from two GBM cell lines (GBM39 and GBM12) were co-dosed with erlotinib and osimertinib, two drugs with low and high BBB-permeability, respectively. T1Gd and T2-weighted MRIs were acquired from all animals prior to model sacrifice. Tumors were manually segmented on denoised and standardized MRIs and intensity patterns were captured using first and second order statistical features in the moving 3x3 kernel. We compared drug levels found in Matrix Assisted Laser Desorption Ionization (MALDI) in T1Gd enhancement, T2 enhancement, and normal brain. We also performed linear regression modeling to predict drug levels using MRI features. Model performance was measured using root mean squared error (RMSE). RESULTS Our analysis showed correlations between imaging features and MALDI drug levels. Osimertinib had a uniform distribution across the brain for all animals and all cell lines, consistent with our expectation for a high BBB-penetrant drug. Erlotinib showed the highest drug levels in T2 for GBM39 and in T1Gd for GBM12. Regression models showed promising results for predicting Erlotinib with a low RMSE of 0.037. CONCLUSION Our preliminary results suggest MRI can be predictive of drug levels for low-BBB penetrant drugs. Understanding the relationship between MRIs and drug distribution in diffuse tumors can be beneficial to developing effective treatment.

Polycystic kidney disease (PKD) is the most common monogenic kidney disorder and an important cause of end stage renal disease. Vascular abnormalities are the most important non‐cystic complication and contribute to renal disease progression. Homocysteine (Hcy)‐mediated endothelial dysfunction (ED) and oxidative stress are evident early on, but the underlying mechanisms remain unknown. We hypothesized that abnormalities in Hcy metabolism are responsible for the increased levels, leading to Hcy‐induced ED from early stages of PKD. Methods We investigated the Hcy metabolic pathway (transulfuration/remethylation fig. 1) and downstream effects ‐main sources of renal tubular (RT) and endothelial cell (EC) reactive oxygen species (ROS), NADPH oxidase 4, (NOX4) and mitochondria‐ in 4‐week‐old PCK and Sprague‐Dawley (SD) control rats (n=12 each). Kidneys were harvested, frozen in liquid nitrogen, or preserved in formalin for metabolomics and ex‐vivo studies. Twenty‐four‐hour urine and terminal blood samples were collected for metabolite analysis and chemistries. Plasma folic acid, vitamin B12, and Hcy were determined by ELISA. Hcy pathway metabolites (betaine, dimethylglycine) were determined by 1 HNMR. Endothelial NO (eNOS) and oxidative stress were assessed by double immunofluorescence staining for CD31/eNOS and CD31/NOX4, respectively. RT and EC mitochondrial structure was assessed by electron microscopy. Results Serum creatinine and BUN remained unchanged, yet kidney weight/body weight ratio were increased in PCK. eNOS immunoreactivity was lower in PCK vs SD. Plasma Hcy and urine excretion of methyl donor betaine were elevated in PCK, and Hcy positively correlated with urine betaine. Plasma betaine was similar between the groups, but its tissue concentration was lower in PCK, which may lead to betaine insufficiency, impaired betaine dependent remethylation, and hyperhomocysteinemia. Contrarily, tissue glutathione concentration was higher in PCK vs SD, and plasma folate and vitamin B12 were similar, arguing against a defect in the transsulfuration or folate‐dependent remethylation pathways. RT NOX4 expression and immunoreactivity were increased in PCK compared to SD, in association with renal oxidative stress. PCK had increased number of CD31/NOX4 positive cells in peritubular capillaries, indicating increased EC ROS production. Collecting duct and EC mitochondria were swollen and showed cristae remodeling. Conclusion In PKD, early elevation in Hcy is likely related to betaine insufficiency and impaired betaine‐dependent remethylation may be responsible for the early elevation of Hcy. NOX4 and mitochondria abnormalities may be important contributors to oxidative stress and disease severity. Our data suggests these may not only be early vascular disease markers, but also play an important pathogenic role in vascular complications associated with PKD, and contribute to renal disease progression. Support or Funding Information R25‐DK101405, NIDDK P30 DK090728, NIDDK P30 DK 106912 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

AbstractObjectives/HypothesisTest a new jellyfish collagen biomaterial aimed to increase duration of injection medialization laryngoplasty (IL) against two products in clinical practice.Study DesignAnimal model.MethodsLeft recurrent laryngeal nerve sectioning and IL were performed in New Zealand White rabbits (N = 6/group). Group 1 received micronized cross‐linked jellyfish collagen (MX‐JC) and adipose derived stem cells (ADSCs), Group 2, MX‐JC alone, Group 3, cross‐linked hyaluronic acid (X‐HA), and Group 4, micronized acellular dermis (MACD). Animals were sacrificed at 4 and 12 weeks. Major outcomes were MRI tissue volumes and histopathology.ResultsAfter 100 μL IL MRI volumes (means ± STD) at 4 and 12 weeks were: Group 1: 27.2 ± 15.6 and 13.1 ± 5.2 μL, Group 2: 60.8 ± 18 and 27.8 ± 2.47 μL, Group 3: 27.4 ± 12 and 10.6 ± 8 μL, and Group 4: 37.5 ± 11 and 9.85 ± 1 μL. Group 2 volumes were largest and Group 3 were smallest in all comparisons (P < .05). Histologically, low grade inflammatory responses were observed in Group 1, mild histiocytic infiltration in Group 2, widespread muscle fiber loss in Group 3, and plasmocytic infiltration in Group 4.ConclusionsMX‐JC showed the least resorption at 4 and 12 weeks among all groups. T cell inflammatory responses were observed with MX‐JC but were reduced by 12 weeks while B cell immune responses, indicative of antibody priming, were predominantly noted with MACD. MX‐JC + ADSC showed low grade immunity while the XHA showed greater myocyte loss compared to the other groups.Level of EvidenceNA Laryngoscope, 131:E2452–E2460, 2021

BackgroundRenal fibrosis is a common pathway in tubulointerstitial injury and a major determinant of renal insufficiency. Collagen deposition, a key feature of renal fibrosis, may serve as an imaging biomarker to differentiate scarred from healthy kidneys.PurposeTo test the feasibility of using quantitative magnetization transfer (qMT), which assesses tissue macromolecule content, to measure renal fibrosis.Study TypeProspective.Animal ModelFifteen 129S1 mice were studied 4 weeks after either sham (n = 7) or unilateral renal artery stenosis (RAS, n = 8) surgeries.Field Strength/SequenceMagnetization transfer (MT)‐weighted images were acquired at 16.4T using an MT‐prepared fast‐low‐angle‐shot sequence. Renal B0, B1, and T1 maps were also acquired, using a dual‐echo gradient echo, an actual flip angle, and inversion recovery method, respectively.AssessmentA two‐pool model was used to estimate the bound water fraction (f) and other tissue imaging biomarkers. Masson's trichrome staining was subsequently performed ex vivo to evaluate renal fibrosis.Statistical TestsComparisons of renal parameters between sham and RAS were performed using independent samples t‐tests. Pearson's correlation was conducted to investigate the relationship between renal fibrosis by histology and the qMT‐derived bound pool fraction f.ResultsThe two‐pool model provided accurate fittings of measured MT signal. The qMT‐derived f of RAS kidneys was significantly increased compared to sham in all kidney zones (renal cortex [CO], 7.6 ± 2.4% vs. 4.6 ± 0.6%; outer medulla [OM], 8.2 ± 4.2% vs. 4.2 ± 0.9%; inner medulla [IM] + P, 5.8 ± 1.6% vs. 2.9 ± 0.6%, all P < 0.05). Measured f correlated well with histological fibrosis in all kidney zones (CO, Pearson's correlation coefficient r = 0.95; OM, r = 0.93; IM + P, r = 0.94, all P < 0.05).Data ConclusionThe bound pool fraction f can be quantified using qMT at 16.4T in murine kidneys, increases significantly in fibrotic RAS kidneys, and correlates well with fibrosis by histology. Therefore, qMT may constitute a valuable tool for measuring renal fibrosis in RAS.Level of Evidence1Technical Efficacy Stage3

AbstractOmega-3 polyunsaturated fatty acids (n3-PUFA) are well recognized for their potent triglyceride-lowering effects, but the potential influence of these bioactive lipids on other biological processes, particularly in the context of healthy aging, remains unknown. With the goal of gaining new insight into some less well-characterized biological effects of n3-PUFAs in healthy older adults, we performed metabolomics of fasting peripheral blood plasma collected from 12 young adults and 12 older adults before and after an open-label intervention of n3-PUFA (3.9 g/day, 2.7 g eicosapentaenoic [EPA], 1.2 g docosahexaenoic [DHA]). Proton nuclear magnetic resonance (1H-NMR) based lipoprotein subclass analysis revealed the expected reduction in total triglyceride (TG), but also demonstrated that n3-PUFA supplementation reduced very low-density lipoprotein (VLDL) particle number, modestly increased high-density lipoprotein (HDL) cholesterol, and shifted the composition of HDL subclasses. Further metabolite profiling by 1H-NMR and mass spectrometry revealed pronounced changes in phospholipids, cholesterol esters, diglycerides, and triglycerides following n3-PUFA supplementation. Furthermore, significant changes in hydroxyproline, kynurenine, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) following n3-PUFA supplementation provide further insight into some less well-recognized biological effects of n3-PUFA supplementation, including possible effects on protein metabolism, the kynurenine pathway, and glucose metabolism.

While zebrafish embryos in the first five days after fertilization are clear and amenable to optical analysis, older juveniles and adults are not, due to pigmentation development and tissue growth. Thus other imaging methods are needed to image adult specimens. NMR is a versatile tool for studies of biological systems and has been successfully used for in vivo zebrafish microscopy. In this work we use NMR microscopy (MRM) for assessment of zebrafish specimens, which includes imaging of formalin fixed (FF), formalin fixed and paraffin embedded (FFPE), fresh (unfixed), and FF gadolinium doped specimens. To delineate the size and shape of various organs we concentrated on 3D MRM. We have shown that at 7 T a 3D NMR image can be obtained with isotropic resolution of 50 μm/pxl within 10 min and 25 μm/pxl within 4 h. Also, we have analyzed sources of contrast and have found that in FF specimens the best contrast is obtained by T 1 weighting (3D FLASH, 3D FISP), whereas in FFPE specimens T 2 weighting (3D RARE) is the best. We highlight an approach to perform segmentation of the organs in order to study morphological changes associated with mutations. The broader implication of this work is development of NMR methodology for high contrast and high resolution serial imaging and automated analysis of morphology of various zebrafish mutants.