An atlas of healthy and injured cell states and niches in the human kidney.

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.

Removal of Small, Asymptomatic Kidney Stones and Incidence of Relapse.

BACKGROUND: The benefits of removing small (≤6 mm), asymptomatic kidney stones endoscopically is unknown. Current guidelines leave such decisions to the urologist and the patient. A prospective study involving older, nonendoscopic technology and some retrospective studies favor observation. However, published data indicate that about half of small renal stones left in place at the time that larger stones were removed caused other symptomatic events within 5 years after surgery. METHODS: We conducted a multicenter, randomized, controlled trial in which, during the endoscopic removal of ureteral or contralateral kidney stones, remaining small, asymptomatic stones were removed in 38 patients (treatment group) and were not removed in 35 patients (control group). The primary outcome was relapse as measured by future emergency department visits, surgeries, or growth of secondary stones. RESULTS: After a mean follow-up of 4.2 years, the treatment group had a longer time to relapse than the control group (P<0.001 by log-rank test). The restricted mean (±SE) time to relapse was 75% longer in the treatment group than in the control group (1631.6±72.8 days vs. 934.2±121.8 days). The risk of relapse was 82% lower in the treatment group than the control group (hazard ratio, 0.18; 95% confidence interval, 0.07 to 0.44), with 16% of patients in the treatment group having a relapse as compared with 63% of those in the control group. Treatment added a median of 25.6 minutes (interquartile range, 18.5 to 35.2) to the surgery time. Five patients in the treatment group and four in the control group had emergency department visits within 2 weeks after surgery. Eight patients in the treatment group and 10 in the control group reported passing kidney stones. CONCLUSIONS: The removal of small, asymptomatic kidney stones during surgery to remove ureteral or contralateral kidney stones resulted in a lower incidence of relapse than nonremoval and in a similar number of emergency department visits related to the surgery. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases and the Veterans Affairs Puget Sound Health Care System; ClinicalTrials.gov number, NCT02210650.).

A crystallin mutant cataract with mineral deposits.

Connexin mutant mice develop cataracts containing calcium precipitates. To test whether pathologic mineralization is a general mechanism contributing to the disease, we characterized the lenses from a nonconnexin mutant mouse cataract model. By cosegregation of the phenotype with a satellite marker and genomic sequencing, we identified the mutant as a 5-bp duplication in the γC-crystallin gene (Crygcdup). Homozygous mice developed severe cataracts early, and heterozygous animals developed small cataracts later in life. Immunoblotting studies showed that the mutant lenses contained decreased levels of crystallins, connexin46, and connexin50 but increased levels of resident proteins of the nucleus, endoplasmic reticulum, and mitochondria. The reductions in fiber cell connexins were associated with a scarcity of gap junction punctae as detected by immunofluorescence and significant reductions in gap junction-mediated coupling between fiber cells in Crygcdup lenses. Particles that stained with the calcium deposit dye, Alizarin red, were abundant in the insoluble fraction from homozygous lenses but nearly absent in wild-type and heterozygous lens preparations. Whole-mount homozygous lenses were stained with Alizarin red in the cataract region. Mineralized material with a regional distribution similar to the cataract was detected in homozygous lenses (but not wild-type lenses) by micro-computed tomography. Attenuated total internal reflection Fourier-transform infrared microspectroscopy identified the mineral as apatite. These results are consistent with previous findings that loss of lens fiber cell gap junctional coupling leads to the formation of calcium precipitates. They also support the hypothesis that pathologic mineralization contributes to the formation of cataracts of different etiologies.

Integrated Cytometry With Machine Learning Applied to High-Content Imaging of Human Kidney Tissue for In Situ Cell Classification and Neighborhood Analysis.

The human kidney is a complex organ with various cell types that are intricately organized to perform key physiological functions and maintain homeostasis. New imaging modalities, such as mesoscale and highly multiplexed fluorescence microscopy, are increasingly being applied to human kidney tissue to create single-cell resolution data sets that are both spatially large and multidimensional. These single-cell resolution high-content imaging data sets have great potential to uncover the complex spatial organization and cellular makeup of the human kidney. Tissue cytometry is a novel approach used for the quantitative analysis of imaging data; however, the scale and complexity of such data sets pose unique challenges for processing and analysis. We have developed the Volumetric Tissue Exploration and Analysis (VTEA) software, a unique tool that integrates image processing, segmentation, and interactive cytometry analysis into a single framework on desktop computers. Supported by an extensible and open-source framework, VTEA's integrated pipeline now includes enhanced analytical tools, such as machine learning, data visualization, and neighborhood analyses, for hyperdimensional large-scale imaging data sets. These novel capabilities enable the analysis of mesoscale 2- and 3-dimensional multiplexed human kidney imaging data sets (such as co-detection by indexing and 3-dimensional confocal multiplexed fluorescence imaging). We demonstrate the utility of this approach in identifying cell subtypes in the kidney on the basis of labels, spatial association, and their microenvironment or neighborhood membership. VTEA provides an integrated and intuitive approach to decipher the cellular and spatial complexity of the human kidney and complements other transcriptomics and epigenetic efforts to define the landscape of kidney cell types.

Kidney stone formation in a novel murine model of polycystic kidney disease.

Individuals with autosomal dominant polycystic kidney disease have a higher incidence of stone formation than the general population. However, there are no cystic animal models known to develop stones. Cystic mice compound heterozygous for hypomorphic Pkd1V and Pkd1RC alleles develop cystic kidneys within a few weeks of birth but live beyond 20 wk of age, allowing for the study of cystic comorbidities including stone formation. Cystic Pkd1V/RC mice were euthanized at 3, 13, or 26 wk of age, and their kidneys were analyzed by microcomputed tomography (µCT) for stone formation. Mice had occasional mineral aggregates that could be detected by µCT analysis at 3 wk of age. At 13 or 26 wk of age, numerous white masses were visible beneath the kidney surface. µCT analysis confirmed the masses to be large mineral stone deposits throughout the renal cortex, with mineral content increasing with age. Staining of histological sections with alizarin red and von Kossa suggested that the stone deposits were composed primarily of calcium and phosphate. Microdissection confirmed stones localized within cyst lumens. Analysis of individual stones by µCT and infrared spectroscopy confirmed apatite mineral composition. Urinalysis revealed elevated levels of phosphate and citrate at 3 wk of age and lower pH and elevated levels of calcium and citrate at 13 wk of age, suggesting altered phosphate and calcium homeostasis as a potential cause of mineralization and renal stone formation. This is the first animal model exhibiting overt kidney stone formation in the context of cystic kidney disease.NEW & NOTEWORTHY Compound heterozygous Pkd1V/RC mice were found to form calcium phosphate-containing stones within cysts of the renal cortex by 13 wk of age. This is the first polycystic kidney disease animal model exhibiting spontaneous stone formation. A growing body of evidence suggests a link between renal stone formation and cystic kidney disease. This mouse model may be useful for studying the interplay between stone and cyst formation and the functional role of polycystins in mineral homeostasis.

Fragmentation of Stones by Burst Wave Lithotripsy in the First 19 Humans.

PURPOSE: We report stone comminution in the first 19 human subjects by burst wave lithotripsy (BWL), which is the transcutaneous application of focused, cyclic ultrasound pulses. MATERIALS AND METHODS: This was a prospective multi-institutional feasibility study recruiting subjects undergoing clinical ureteroscopy (URS) for at least 1 stone ≤12 mm as measured on computerized tomography. During the planned URS, either before or after ureteroscope insertion, BWL was administered with a handheld transducer, and any stone fragmentation and tissue injury were observed. Up to 3 stones per subject were targeted, each for a maximum of 10 minutes. The primary effectiveness outcome was the volume percent comminution of the stone into fragments ≤2 mm. The primary safety outcome was the independent, blinded visual scoring of tissue injury from the URS video. RESULTS: Overall, median stone comminution was 90% (IQR 20, 100) of stone volume with 21 of 23 (91%) stones fragmented. Complete fragmentation (all fragments ≤2 mm) within 10 minutes of BWL occurred in 9 of 23 stones (39%). Of the 6 least comminuted stones, likely causative factors for decreased effectiveness included stones that were larger than the BWL beamwidth, smaller than the BWL wavelength or the introduction of air bubbles from the ureteroscope. Mild reddening of the papilla and hematuria emanating from the papilla were observed ureteroscopically. CONCLUSIONS: The first study of BWL in human subjects resulted in a median of 90% comminution of the total stone volume into fragments ≤2 mm within 10 minutes of BWL exposure with only mild tissue injury.

Determining the threshold of acute renal parenchymal damage for intrarenal pressure during flexible ureteroscopy using an in vivo pig model.

PURPOSE: To identify a threshold for intrarenal pressure (IRP), that if exceeded, will result in renal parenchymal damage. Herein, we attempt to identify an IRP threshold by subjecting in vivo porcine kidneys to various levels of extreme pressurized irrigation. Our objective was not to simulate ureteroscopy treatment, but to attempt identify a threshold of IRP injury. METHODS: Ten female pigs were intubated and sedated. The abdomen was opened; the ureters were isolated and incised. A LithoVue™ (Boston Scientific) ureteroscope was inserted. A 0-silk tie was then used to tie the ureter around the scope to create a closed system (to achieve a constant level of pressure). Real-time IRPs were measured using the Comet™ Pressure guidewire (Boston Scientific). Kidneys were exposed to pressurized, saline for 36 min (at control, 50, 100, 150 mmHg and higher pressures). Kidneys were then immediately harvested. Two expert histologists independently analyzed kidney slides to identify areas of renal damage. RESULTS: The two kidneys exposed to IRPs > 185 mmHg resulted in forniceal rupture and large areas of hematoma. The other IRP groups (control, 50, 100, and 150 mmHg) had no identifiable gross or histologic renal parenchymal damage. CONCLUSIONS: No differences in renal parenchymal morphology were identified between pressure groups of control, 50, 100, or 150 mmHg. However, IRPs > 185 mmHg did result in forniceal rupture in this closed-system in vivo porcine model. Further study is required to elucidate the damage threshold.

Consultation on kidney stones, Copenhagen 2019: lithotripsy in percutaneous nephrolithotomy.

PURPOSE: To evaluate the balance between existing evidence and expert opinions on the safety and efficacy of new technological improvements in lithotripsy techniques for percutaneous nephrolithotomy (PCNL). METHODS: A scoping review approach was applied to search literature in Pubmed, Embase, and Web of Science. Consensus by key opinion leaders was reached at a 2-day meeting entitled "Consultation on Kidney Stones: Aspects of Intracorporeal Lithotripsy" held in Copenhagen, Denmark, in September 2019. RESULTS: New-generation dual-mode single-probe lithotripsy devices have shown favourable results compared with use of ballistic or ultrasonic lithotripters only. However, ballistic and ultrasonic lithotripters are also highly effective and safe and have been the backbone of PCNL for many years. Compared with standard PCNL, it seems that mini PCNL is associated with fewer bleeding complications and shorter hospital admissions, but also with longer operating room (OR) time and higher intrarenal pressure. Use of laser lithotripsy combined with suction in mini PCNL is a promising alternative that may improve such PCNL by shortening OR times. Furthermore, supine PCNL is a good alternative, especially in cases with complex renal stones and large proximal ureteric stones; in addition, it facilitates endoscopic combined intrarenal surgery (ECIRS). CONCLUSION: Recent technological improvements in PCNL techniques are promising, but there is a lack of high-level evidence on safety and efficacy. Different techniques suit different types of stones and patients. The evolution of diverse methods has given urologists the possibility of a personalized stone approach, in other words, the right approach for the right patient.

Using micro computed tomographic imaging for analyzing kidney stones.

Stone analysis is a critical part of the clinical characterization of urolithiasis. This article reviews the strengths and limitations of micro CT in the analysis of stones. Using micro CT alone in a series of 757 stone specimens, micro CT identified the 458 majority calcium oxalate specimens with a sensitivity of 99.6% and specificity of 95.3%. Micro CT alone was also successful in identifying majority apatite, brushite, uric acid, and struvite stones. For some minor minerals-such as apatite in calcium oxalate or calcium salts in uric acid stones-micro CT enables the detection of minute quantities well below 1%. The addition of a standard for calibrating X-ray attenuation values improves the ability of micro CT to identify common stone minerals. The three-dimensional nature of micro CT also allows for the visualization of surface features in stones, which is valuable for the study of stone formation.

Papillary Ductal Plugging is a Mechanism for Early Stone Retention in Brushite Stone Disease.

PURPOSE: Mechanisms of early stone retention in the kidney are under studied and poorly understood. To date attachment via Randall's plaque is the only widely accepted theory in this regard, which is best described in idiopathic calcium oxalate stone formers. Brushite stone formers are known to have distinct papillary morphology relative to calcium oxalate stone formers. As such we sought to determine whether stone attachment mechanisms in such patients may be similarly unique. MATERIALS AND METHODS: Patients undergoing percutaneous and or ureteroscopic procedures for stone removal consented to endoscopic renal papillary examination and individual stone collection. Each removed stone was processed using micro computerized tomography to assess the 3-dimensional microstructure and the minerals contained, and search for common structural features indicative of novel mechanisms of early growth and attachment to renal tissue. RESULTS: A total of 25 intact brushite stones were removed from 8 patients and analyzed. Video confirmed attachment of 13 of the 25 stones with the remainder believed to have been accidently dislodged during the procedure. Microscopic examination by light and computerized tomography failed to show evidence of Randall's plaque associated with any stone containing brushite. Conversely each brushite stone demonstrated microstructural evidence of having grown attached to a ductal plug formed of apatite. CONCLUSIONS: Three-dimensional analysis of small brushite stones suggests overgrowth on ductal apatite plugs as a mechanism of early stone growth and retention. Such findings represent what is to our knowledge the initial supporting evidence for a novel mechanism of stone formation which has previously been hypothesized but never verified.

Preoperative Bladder Urine Culture as a Predictor of Intraoperative Stone Culture Results: Clinical Implications and Relationship to Stone Composition.

PURPOSE: We examine the relationship between urine and stone cultures in a large cohort of patients undergoing percutaneous stone removal and compare the findings in infectious vs metabolic calculi. MATERIALS AND METHODS: A total of 776 patients treated with percutaneous nephrolithotomy who had preoperative urine cultures and intraoperative stone cultures were included in the study. Statistical analysis used chi-square or logistic fit analysis as appropriate. RESULTS: Preoperative urine culture was positive in 352 patients (45.4%) and stone cultures were positive in 300 patients (38.7%). There were 75 patients (9.7%) with negative preoperative cultures who had positive stone cultures, and in patients with both cultures positive the organisms differed in 103 (13.3%). Gram-positive organisms predominated in preoperative urine and stone cultures. CONCLUSIONS: Preoperative urine cultures in patients undergoing percutaneous nephrolithotomy are unreliable as there is a discordance with intraoperative stone cultures in almost a quarter of cases. There has been a notable shift toward gram-positive organisms in this cohort of patients.

Label-free proteomic methodology for the analysis of human kidney stone matrix composition.

BACKGROUND: Kidney stone matrix protein composition is an important yet poorly understood aspect of nephrolithiasis. We hypothesized that this proteome is considerably more complex than previous reports have indicated and that comprehensive proteomic profiling of the kidney stone matrix may demonstrate relevant constitutive differences between stones. We have analyzed the matrices of two unique human calcium oxalate stones (CaOx-Ia and CaOx-Id) using a simple but effective chaotropic reducing solution for extraction/solubilization combined with label-free quantitative mass spectrometry to generate a comprehensive profile of their proteomes, including physicochemical and bioinformatic analysis.`. RESULTS: We identified and quantified 1,059 unique protein database entries in the two human kidney stone samples, revealing a more complex proteome than previously reported. Protein composition reflects a common range of proteins related to immune response, inflammation, injury, and tissue repair, along with a more diverse set of proteins unique to each stone. CONCLUSION: The use of a simple chaotropic reducing solution and moderate sonication for extraction and solubilization of kidney stone powders combined with label-free quantitative mass spectrometry has yielded the most comprehensive list to date of the proteins that constitute the human kidney stone proteome.

Dual-Energy CT for Quantification of Urinary Stone Composition in Mixed Stones: A Phantom Study.

OBJECTIVE: The purpose of this study was to assess the feasibility of using dual-energy CT to accurately quantify uric acid and non-uric acid components in urinary stones of mixed composition. MATERIALS AND METHODS: A total of 24 urinary stones were analyzed with micro CT to serve as the reference standard for uric acid and non-uric acid composition. These stones were placed in water phantoms to simulate body attenuation of slim to obese adults and scanned with a third-generation dual-source CT scanner by use of dual-energy modes adaptively selected on the basis of phantom size. CT number ratio, which is distinct for different materials, was calculated for each pixel of the stones. Each pixel was then classified as uric acid and non-uric acid by comparison of the CT number ratio with preset thresholds ranging from 1.10 to 1.70. Minimal, maximal, and root-mean-square errors were calculated by comparing composition with the reference standard, and the threshold with the minimal root-mean-square error was determined. A paired t test was performed to compare the stone composition determined with dual-energy CT with the reference standard obtained with micro CT. RESULTS: The optimal CT number ratio threshold ranged from 1.27 to 1.55, dependent on phantom size. The root-mean-square error ranged from 9.60% to 12.87% across all phantom sizes. Minimal absolute error ranged from 0.04% to 1.24% and maximal absolute error from 22.05% to 35.46%. Dual-energy CT and the reference micro CT did not differ significantly on uric acid and non-uric acid composition (paired t test, p = 0.20-0.96). CONCLUSION: Accurate quantification of uric acid and non-uric acid composition in mixed stones is possible with dual-energy CT.

Lithotripter outcomes in a community practice setting: comparison of an electromagnetic and an electrohydraulic lithotripter.

PURPOSE: We assessed patient outcomes using 2 widely different contemporary lithotripters. MATERIALS AND METHODS: We performed a consecutive case series study of 355 patients in a large private practice group using a Modulith® SLX electromagnetic lithotripter in 200 patients and a LithoGold LG-380 electrohydraulic lithotripter (TRT, Woodstock, Georgia) in 155. Patients were followed at approximately 2 weeks. All preoperative and postoperative films were reviewed blindly by a dedicated genitourinary radiologist. The stone-free rate was defined as no residual fragments remaining after a single session of shock wave lithotripsy without an ancillary procedure. RESULTS: Patients with multiple stones were excluded from analysis, leaving 76 and 142 treated with electrohydraulic and electromagnetic lithotripsy, respectively. The stone-free rate was similar for the electrohydraulic and electromagnetic lithotripters (29 of 76 patients or 38.2% and 69 of 142 or 48.6%, p = 0.15) with no difference in the stone-free outcome for renal stones (20 of 45 or 44.4% and 33 of 66 or 50%, p = 0.70) or ureteral stones (9 of 31 or 29% and 36 of 76 or 47.4%, respectively, p = 0.08). The percent of stones that did not break was similar for the electrohydraulic and electromagnetic devices (10 of 76 patients or 13.2% and 23 of 142 or 16.2%) and ureteroscopy was the most common ancillary procedure (18 of 22 or 81.8% and 30 of 40 or 75%, respectively). The overall mean number of procedures performed in patients in the 2 groups was similar (1.7 and 1.5, respectively). CONCLUSIONS: We present lithotripsy outcomes in the setting of a suburban urology practice. Stone-free rates were modest using shock wave lithotripsy alone but access to ureteroscopy provided satisfactory outcomes overall. Although the acoustic characteristics of the electrohydraulic and electromagnetic lithotripters differ substantially, outcomes with these 2 machines were similar.

Motion artifacts in kidney stone imaging using single-source and dual-source dual-energy CT scanners: a phantom study.

PURPOSE: Dual-energy computed tomography (DECT) has shown the capability of differentiating uric acid (UA) from non-UA stones with 90-100% accuracy. With the invention of dual-source (DS) scanners, both low- and high-energy images are acquired simultaneously. However, DECT can also be performed by sequential acquisition of both images on single-source (SS) scanners. The objective of this study is to investigate the effects of motion artifacts on stone classification using both SS-DECT and DS-DECT. METHODS: 114 kidney stones of different types and sizes were imaged on both DS-DECT and SS-DECT scanners with tube voltages of 80 and 140 kVp with and without induced motion. Postprocessing was conducted to create material-specific images from corresponding low- and high-energy images. The dual-energy ratio (DER) and stone material were determined and compared among different scans. RESULTS: For the motionless scans, all stones were correctly classified with SS-DECT, while two cystine stones were misclassified with DS-DECT. When motion was induced, 94% of the stones were misclassified with SS-DECT versus 11% with DS-DECT (P < 0.0001). Stone size was not a factor in stone misclassification under motion. Stone type was not a factor in stone misclassification under motion with SS-DECT, although with DS-DECT, cystine showed higher number of stone misclassification. CONCLUSIONS: Motion artifacts could result in stone misclassification in DECT. This effect is more pronounced in SS-DECT versus DS-DECT, especially if stones of different types lie in close proximity to each other. Further, possible misinterpretation of the number of stones (i.e., missing one, or thinking that there are two) in DS-DECT could be a potentially significant problem.

A four-grating interferometer for x-ray multi-contrast imaging.

BACKGROUND: X-ray multi-contrast imaging with gratings provides a practical method to detect differential phase and dark-field contrast images in addition to the x-ray absorption image traditionally obtained in laboratory or hospital environments. Systems have been developed for preclinical applications in areas including breast imaging, lung imaging, rheumatoid arthritis hand imaging and kidney stone imaging. PURPOSE: Prevailing x-ray interferometers for multi-contrast imaging include Talbot-Lau interferometers and universal moiré effect-based phase-grating interferometers. Talbot-Lau interferometers suffer from conflict between high interferometer sensitivity and large field of view (FOV) of the object being imaged. A small period analyzer grating is necessary to simultaneously achieve high sensitivity and large FOV within a compact imaging system but is technically challenging to produce for high x-ray energies. Phase-grating interferometers suffer from an intrinsic fringe period ranging from a few micrometers to several hundred micrometers that can hardly be resolved by large area flat panel x-ray detectors. The purpose of this work is to introduce a four-grating x-ray interferometer that simultaneously allows high sensitivity and large FOV, without the need for a small period analyzer grating. METHODS: The four-grating interferometer consists of a source grating placed downstream of and close to the x-ray source, a pair of phase gratings separated by a fixed distance placed downstream of the source grating, and an analyzer grating placed upstream of and close to the x-ray detector. The object to be imaged is placed upstream of and close to the phase-grating pair. The distance between the source grating and the phase-grating pair is designed to be far larger than that between the phase-grating pair and the analyzer grating to promote simultaneously high sensitivity and large FOV. The method was evaluated by constructing a four-grating interferometer with an 8 µm period source grating, a pair of phase gratings of 2.4 µm period, and an 8 µm period analyzer grating. RESULTS: The fringe visibility of the four-grating interferometer was measured to be ≈24% at 40 kV and ≈18% at 50 kV x-ray tube operating voltage. A quartz bead of 6 mm diameter was imaged to compare the theoretical and experimental phase contrast signal with good agreement. Kidney stone specimens were imaged to demonstrate the potential of such a system for classification of kidney stones. CONCLUSIONS: The proposed four-grating interferometer geometry enables a compact x-ray multi-contrast imaging system with simultaneously high sensitivity and large FOV. Relaxation of the requirement for a small period analyzer grating makes it particularly suitable for high x-ray energy applications such as abdomen and chest imaging.

Beet supplementation mitigates post-exercise inflammation.

Objectives: This study investigated the efficacy of a mixed beet-based supplement (BEET) versus placebo (PL) in countering inflammation during recovery from 2.25 h of intensive cycling in 20 male and female cyclists. A multi-omics approach was used that included untargeted proteomics and a targeted oxylipin panel. Methods: A randomized, placebo-controlled, double-blind, crossover design was used with two 2-week supplementation periods and a 2-week washout period. Supplementation periods were followed by a 2.25 h cycling bout at close to 70%VO2max. The BEET supplement provided 212 mg of nitrates per day, 200 mg caffeine from green tea extract, 44 mg vitamin C from Camu Camu berry, B-vitamins from quinoa sprouts (40% Daily Value for thiamin, riboflavin, niacin, and vitamin B6), and 2.5 g of a mushroom blend containing Cordyceps sinensis and Inonotus obliquus. Six blood samples were collected before and after supplementation (overnight fasted state), immediately post-exercise, and at 1.5 h-, 3 h-, and 24 h-post-exercise. Results: The 2.25 h cycling bout increased plasma levels of 41 of 67 oxylipins detected. BEET supplementation significantly increased plasma nitrate (NO3 -) and nitrite (NO2 -) (sum, NO3 - + NO2 -) concentrations (interaction effect, p < 0.001) and two anti-inflammatory oxylipins [18-hydroxyeicosapentaenoic acid (18-HEPE) and 4-hydroxy-docosahexanoic acid (4-HDoHE)]. The untargeted proteomics analysis identified 616 proteins (458 across all times points), and 2-way ANOVA revealed a cluster of 45 proteins that were decreased and a cluster of 21 that were increased in the BEET versus PL trials. Functional enrichment supported significant BEET-related reductions in inflammation-related proteins including several proteins related to complement activation, the acute phase response, and immune cell adhesion, migration, and differentiation. Discussion: Intake of a BEET-based supplement during a 2-week period was linked to higher plasma levels of NO3 - + NO2 -, elevated post-exercise levels of two anti-inflammatory oxylipins, and a significant decrease in a cluster of proteins involved in complement activation and inflammation. These data support that 2-weeks intake of nitrate from a mixed beet-based supplement moderated protein biomarkers of exercise-induced inflammation in athletes.

Phenotypic characterization of kidney stone formers by endoscopic and histological quantification of intrarenal calcification.

Interstitial Randall's plaques and collecting duct plugs are distinct forms of renal calcification thought to provide sites for stone retention within the kidney. Here we assessed kidney stone precursor lesions in a random cohort of stone formers undergoing percutaneous nephrolithotomy. Each accessible papilla was endoscopically mapped following stone removal. The percent papillary surface area covered by plaque and plug were digitally measured using image analysis software. Stone composition was determined by micro-computed tomography and infrared analysis. A representative papillary tip was biopsied. The 24-h urine collections were used to measure supersaturation and crystal growth inhibition. The vast majority (99%) of stone formers had Randall's plaque on at least 1 papilla, while significant tubular plugging (over 1% of surface area) was present in about one-fifth of patients. Among calcium oxalate stone formers the amount of Randall's plaque correlated with higher urinary citrate levels. Tubular plugging correlated positively with pH and brushite supersaturation but negatively with citrate excretion. Lower urinary crystal growth inhibition predicted the presence of tubular plugging but not plaque. Thus, tubular plugging may be more common than previously recognized among patients with all types of stones, including some with idiopathic calcium oxalate stones.

Differentiation of calcium oxalate monohydrate and calcium oxalate dihydrate stones using quantitative morphological information from micro-computerized and clinical computerized tomography.

PURPOSE: We differentiated calcium oxalate monohydrate and calcium oxalate dihydrate kidney stones using micro and clinical computerized tomography images. MATERIALS AND METHODS: A total of 22 calcium oxalate monohydrate and 15 calcium oxalate dihydrate human kidney stones were scanned using a commercial micro-computerized tomography scanner with a pixel size of 7 to 23 µm. Under an institutional review board approved protocol, image data on 10 calcium oxalate monohydrate and 9 calcium oxalate dihydrate stones greater than 5 mm were retrieved from a total of 80 patients who underwent clinical dual energy computerized tomography for clinical indications and had stones available for infrared spectroscopic compositional analysis. Micro and clinical computerized tomography images were processed using in-house software, which quantified stone surface morphology with curvature based calculations. A shape index was generated as a quantitative shape metric to differentiate calcium oxalate monohydrate from calcium oxalate dihydrate stones. Statistical tests were used to test the performance of the shape index. RESULTS: On micro-computerized tomography images the shape index of calcium oxalate monohydrate and calcium oxalate dihydrate stones significantly differed (ROC curve AUC 0.92, p <0.0001). At the optimal cutoff sensitivity was 0.93 and specificity was 0.91. On clinical computerized tomography images a significant morphological difference was also detected (p = 0.007). AUC, sensitivity and specificity were 0.90, 1 and 0.73, respectively. CONCLUSIONS: On micro and clinical computerized tomography images a morphological difference was detectable in calcium oxalate monohydrate and calcium oxalate dihydrate stones larger than 5 mm. The shape index is a highly promising method that can distinguish calcium oxalate monohydrate and calcium oxalate dihydrate stones with reasonable accuracy.