Normal Human Urine Research Articles

Research progress of hyperuricemia on kidney damage

Urea is the main product secreted in normal human urine, which will contain a small amount of uric acid.Uric acid is the end product of purines known as trioxypurines, which have a weakly acidic alcoholic form, and if purines are metabolized, the risk of hyperuricemia will be increased. It has been shown that the risk of developing CKD in patients with increased serum uric acid levels is 2.21 times that of normal patients. There are experimental data to divide patients into two groups, one is patients with hyperuricemia and the other is patients with normal serum uric acid, the results of the study show that patients with hyperuricemia have a significantly greater probability of developing acute kidney injury than patients with normal serum uric acid, and most of them need kidney replacement therapy. This review focuses on the development of kidney damage in hyperuricemia.

INHIBITION OF CALCIUM OXALATE CRYSTALLIZATION BY AN EXTRACT OF OCIMUM BASILICUM SEEDS: AN IN-VITRO STUDY

Objective: Ocimum basilicum (OB) has been used to treat diverse illnesses which include urinary stone disorder for a reason that historical time in India. We investigated OB seeds for antiurolithic activity.
 Methods: Calcium oxalate crystallization becomes triggered by the addition of 0.01 M sodium oxalate answers in normal human urine and nucleation was done.
 Results: OB seeds were discovered to be robust and promising antiurolithiatic agents which are in accordance with its use in traditional medication.
 Conclusion: An extract of the traditional herb OB has super inhibitory activity on crystalluria and therefore might be useful in dissolving urinary stone; however, in addition, a study in animal fashions of urolithiasis is needed to assess its capability antiurolithiatic interest.

Development of Low-Cost AuNP-Based Aptasensors with Truncated Aptamer for Highly Sensitive Detection of 8-Oxo-dG in Urine.

8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG), an oxidized form of guanosine residues, is a critical biomarker for various cancers. Herein, a sensitive citrate-capped gold nanoparticle-based aptasensor device has been developed for the detection of 8-oxo-dG in urine. We previously designed a 38-nt anti-8-oxo-dG-aptamer by a computer simulation and the experimental validation has been performed in the present work. The analytical performance of the 38-nt aptamer from the in silico design was compared with the parent 66-nt aptamer. This assay is based on the principle of salt-induced aggregation of citrate-capped gold nanoparticles. Based on this sensing mechanism, the difference between the absorbance in the presence and absence of 8-oxo-dG at λ = 525 nm (ΔA525) increased linearly as a function of 8-oxo-dG concentrations in the ranges of 10–100 and 15–100 nM for 38-nt and 66-nt aptasensors, respectively. This method can provide detection limits of 6.4 nM for 8-oxo-dG in the 38-nt aptasensor and 13.2 nM in the 66-nt aptasensor. Similar to the 66-nt aptamer, the shortened aptamer, 38-nt long, can provide high sensitivity and selectivity with rapid detection time. In addition, using the 38-nt aptamer as a recognition component in the developed portable low-cost device showed high sensitivity in the detection range of 15–100 nM with a detection limit of 12.9 nM, which is much lower than the threshold value (280 nM) for normal human urine. This easy-to-use device could effectively and economically be utilized for monitoring 8-oxo-dG in real urine samples and potentially serve as a prototype for a commercial device.

In vitro activity of omadacycline and levofloxacin against Escherichia coli, Klebsiella pneumoniae and Staphylococcus saprophyticus in human urine supplemented with calcium and magnesium.

BackgroundOmadacycline, an aminomethylcycline, was approved in 2018 for the treatment of acute bacterial skin and skin structure infections and community-acquired bacterial pneumonia. In a Phase Ib study, around 34% of the absorbed dose of omadacycline was shown to be excreted in urine—an important property for urinary tract infection (UTI) treatment. Therefore, omadacycline has been studied in two Phase II trials for the treatment of uncomplicated UTIs and acute pyelonephritis. The activity of omadacycline against UTI pathogens in human urine is important to understand in this context.ObjectivesTo study the in vitro activity of omadacycline against UTI pathogens in human urine supplemented with calcium and magnesium.MethodsOmadacycline activity was compared with that of levofloxacin against the urinary pathogens Escherichia coli, Klebsiella pneumoniae and Staphylococcus saprophyticus in standard medium, pooled normal human urine and neutral pH-adjusted pooled normal human urine spiked with calcium or magnesium at concentrations consistent with hypercalcaemia and hypermagnesaemia.ResultsThe activities of omadacycline and levofloxacin against these urinary pathogens were lower in urine relative to standard medium; addition of Mg2+ to broth and urine had a further negative impact on omadacycline activity, whereas the addition of Ca2+ had less of an impact. Levofloxacin activity was not substantially reduced in either broth or urine by the addition of divalent cations.ConclusionsThe activity of omadacycline against UTI organisms was lower in urine relative to standard medium and was negatively impacted by magnesium. Omadacycline displayed slightly reduced activity when excess calcium was present, but, overall, the differences were ≤2-fold. These observations should be considered along with the pharmacokinetics of the agent for clinical context.

Nanoporous Anodic Alumina As a Three-dimensional Nanostructured material for the Remote Optical Sensing of Urea

Millions of people in the world suffer from some form of kidney disease [1]. Due to reduce the economic losses caused by this disease and also to improve the public health, the fabrication of a device to diagnose kidney disease is important for every government. Up to now, several biosensors have been reported for the determination of urea [2,3]. However, no of them is remote biosensor. The most advantage of the remote sensor is, analyzing the real samples without getting any fungal/virus/bacterial infection that can be in it. Infection. The interferometric reflectance spectroscopy (IRS) is one of the remote sensing devices. IRS is a physical method based on the interference of white light at porous thin substrates, which is used to investigate molecular interaction. This investigation is detected by the charge-coupled device (CCD) detector. Macroporous silicon (MPS) and nanoporous anodic alumina (NAA) are two of the interesting porous thin substrates for the fabrication of biosensor based on IRS [4-6]. However, NAA has become gradually influential in the IRS biosensors due to its high surface area, biocompatibility, easy functional ability, versatile, durable and low-cost substrate.Herein, we fabricated a remote optical biosensor for the determination of urea. For this purpose first, the self-orderednanoporous anodic alumina (NAA) was fabricated by electrochemical anodization of aluminum substrates in a two-step anodization process [7, 8]. The morphological characterization of the NAA was examined by SEM. The surface of NAA has a hexagonal multi-pore structure. The average diameter pore size of NAA was 41 nm. Then, the NAA pore walls were functionalized with 3-aminopropyl tri-methoxy silane (NAA-NH2). After that, the urease enzyme was immobilized to the pore walls of NAA-NH2 by the use of glutaraldehyde as cross-linker. After that, fluorescein 5(6)-isothiocyanate (FLITC) as the pH-sensitive optical probe was attached to urease enzyme to fabricate a novel urea and trypsin biosensor. The operation principle of the proposed biosensor is based on a change in the pH of the solution during the reaction of urease and urea and then change in the absorption properties of the FLITC. The reaction of urease with urea lets to increase the pH of its environment because of producing ammonia, which is basic. So, the absorption property of FLITC that is a pH-sensitive opt-probe will increase by increasing the urea in solution due to the increase of pH solution during urease/urea reaction. Consequently, the residence of light intensity that will be detected by the CCD detector in the presence of urea is less than in the absence of it. The proposed biosensor exhibited a good response to the concentration of urea in the range of 0.12 to 3.0 mM. The limit of detection (LOD) for urea was 0.06 mM (Fig.1).To examine its applicability in real sample analysis, the urease biosensor was used for the determination of urea in normal human urine with the standard addition method. Briefly, 1.0 mL of the filtered urine solution added to 9.0 mL of NaAC solution (0.1 M, pH 6.4) and then the solution was pumped into the analytical cell to be analyzed by NAA-NH-GLA-urease-FLITC. The concentration of urea was determined to be 192 mM. The recovery of the analysis was 92 %, considering the value determined by the fluorescent-based sensor [9].The NAA-NH-GLA-urease-FLITC exhibited good reproducibility in the detection of urea. The effect interfering compounds were also studied on the response of biosensor. No interference was observed with 0.1 mM of L-cysteine, dopamine, glucose, and nicotinamide adenine dinucleotide and common cations and anions (0.1 mM of Na+, Al+3, K+, Cl-, NO3 -, SO4 -2).The proposed biosensor exhibited good selectivity, linear range responsibility, and stability.References(1) https://www.worldkidneyday.org/2019-campaign/2019-wkd-theme/.(2) Mishra, G.K., Mishra, R.K., Bhand, S., 2010. Biosens Bioelectron.26, 1560-1564.(3) Parashar, U.K., Nirala, N.R., Upadhyay, C., Saxena, P.S., Srivastava, A., 2015. Appl Biochem Biotechnol. 176, 480-492.(4) Amouzadeh Tabrizi, M., Ferré-Borrull, J, Marsal, L. F., 2019. Biosens Bioelectron 137, 279-286.(5) Amouzadeh Tabrizi, M., Ferré-Borrull, J, Marsal, L. F., 2020. Biosens Bioelectron 137, 111828.(6) Ferré-Borrull, J, Pallarès, J, Macías, G, Marsal, L. F., 2014 Materials 7, 5225-5253.(7) Santos, A., Vojkuvka, L., Alba, M., Balderrama, V. S., Ferré‐Borrull, J, Pallarès, J., Marsal, L.F., Physica Status Solidi (a) 209, 2045-2048.(8) Santos, A., Macías, G., Ferré-Borrull, J., Pallarès, J., Marsal, L.F., 2012. Appl. Mater. Interfaces 4, 3584-3588.(9) Deng, H.-H., Li, K.-L., Zhuang, Q.-Q., Peng, H.-P., Zhuang, Q.-Q., Liu, A.-L., Xia, X.-H., Chen, W., 2018. Nanoscale 10, 6467-6473. Figure 1

Albuminuria detection using graphene oxide-mediated fluorescence quenching aptasensor

A simple and sensitive graphene oxide-mediated fluorescence quenching aptasensor is developed to quantify albuminuria in urine samples. The developed aptasensor used the specific target binding property of aptamer and fluorescence quenching property of graphene oxide to determine the concentration of human serum albumin in urine. The limit of detection of the developed platform is 0.05 µg.mL−1 and the detection range is 0.1–600 µg.mL−1, which covers the albuminuria concentration range present in normal human urine and the urine of the patient with chronic kidney disease. This approach can be modified to measure albuminuria using a high-throughput quantification platform and portable point of care testing. In addition, the production cost for one reaction is cheaper than those for the standard automated method. Therefore, this aptasensor has significant potential for commercialization and public use.•Our protocol is customized by using the fluorescence quenching property of graphene oxide and specific binding property of human serum albumin aptamer to detect human serum albumin in urine sample•The limit of detection of our developed platform is 0.05 µg.mL−1•The detection range of our aptasensor is 0.1–600 µg.mL−1

Development of a Urine Recycling Chemostat System Coupled to an Ammonia Fuel Cell Using Urease-Positive P. vulgaris Bacteria for Urea Removal

Current urine recycling treatments are unable to completely recover clean water free of toxic metabolic products such as urea. Urea is a small and uncharged molecule, diffulting its removal from wastewater. Previous publications from our research group reported the use of immobilized urease enzyme on carbon matrix as a urea bioreactor to convert urea to ammonia. The stability of the enzyme was limitation for long time use. For this, a microbial system has been selected. An ureolysis system in a microbial chemostat using robust Proteus vulgaris bacteria that can transform urea to ammonia is being proposed. The chemostat will be coupled to an ammonia fuel cell to further oxidize ammonia to nitrogen and generate energy. The first goal of this work is to establish the growth parameters for P. vulgaris in synthetic human urine (SHU) by testing different formulations of synthetic urine similar to real urine. Our preliminary results indicate that P. vulgaris achieve exponential growth in Enriched Synthetic Human Urine during a 12 h Growth Assay using Genesys UV-Vis Spectrophotometer, and the Colony-forming Units counting technique. Future bacterial growth experiments will be done with a more chemically consistent synthetic human urine with normal human urine components such as amino acids.

Profiling of lysine-acetylated proteins in human urine.

A biomarker is a measurable indicator associated with changes in physiological state or disease. In contrast to the blood which is under homeostatic controls, urine reflects changes in the body earlier and more sensitively, and is therefore a better biomarker source. Lysine acetylation is an abundant and highly regulated post-translational modification. It plays a pivotal role in modulating diverse biological processes and is associated with various important diseases. Enrichment or visualization of proteins with specific post-translational modifications provides a method for sampling the urinary proteome and reducing sample complexity. In this study, we used anti-acetyllysine antibody-based immunoaffinity enrichment combined with high-resolution mass spectrometry to profile lysine-acetylated proteins in normal human urine. A total of 629 acetylation sites on 315 proteins were identified, including some very low-abundance proteins. This is the first proteome-wide characterization of lysine acetylation proteins in normal human urine. Our dataset provides a useful resource for the further discovery of lysine-acetylated proteins as biomarkers in urine.

Urinary proteome analysis of acute hypercoagulable state in rat model induced by ε-aminocaproic acid

The hypercoagulable state occurs in a group of prothrombotic disorders associated with an increased risk for thromboembolic events, but it is difficult to diagnose due to the lack of available biomarkers. This study aimed to investigate systematic changes of urinary proteome in acute hypercoagulable state induced by certain antifibrinolytics. To reduce the effects of both genetic and environmental factors on the urinary proteome, we used a rat model of acute hypercoagulable state induced by an antifibrinolytic agent ε-aminocaproic acid, resembling human hypercoagulable state. Urine samples were collected during acute hypercoagulable state for analysis by liquid chromatography-tandem mass spectrometry (LCMS/MS). Of 65 significantly changed proteins in acute hypercoagulable state, 38 proteins had human orthologs, and 18 proteins were identified as stable in normal human urine. None of the identified proteins have been found to be clotting factors, but 4 proteins are known to be involved in the regulation of blood coagulation factors. Two proteins were verified as the markers associated with acute hypercoagulable state by Western blot analysis. In addition, four common differential urinary proteins have been found in acute hypercoagulable state induced by another antifibrinolytics tranexamic acid. These four proteins are potential biomarkers for early diagnosis of hypercoagulable state to prevent the development of thrombotic diseases.

A sequential separation strategy for facile isolation and comprehensive analysis of human urine N-glycoproteome.

Urine is an attractive and non-invasive alternative source to tissue, blood or other biofluids for biomarker screening in clinical research. In normal human adult urine, 48% of the total urinary protein is in the sediment, 49% is soluble and the remaining 3% is contained in urinary extracellular vesicles (EVs). The soluble proteins and EV proteins in urine have attracted particular attention in recent years as cancer diagnostics. Furthermore, considering the important role of N-glycoproteins in practically all physiological processes, including regulating receptor-ligand binding, cell-cell interactions, inflammatory response and tumour progression, N-glycoproteome in human urine is an invaluable target for monitoring the physiological status and pathological changes of the kidney and urinary tract. Given the different origins of the soluble proteins and EV proteins in the urine, different N-glycoproteome patterns exist. Therefore, isolating the soluble N-glycoproteins and EV N-glycoproteins for separate analysis will provide a more specific and comprehensive view and provide a deeper understanding of human urinary N-glycoproteome. In this work, we developed a sequential separation method that isolates urinary soluble proteins and EV proteins via stepwise ultrafiltration based on their obvious size difference. A facile and reproducible protein isolation was achieved using this strategy. Subsequent N-glycoproteome enrichment and identification revealed distinct patterns in the two sub-proteomes of urine with more than 60% differential N-glycopeptides. A more comprehensive picture of the urinary N-glycoproteome with close to 1800 identified N-glycopeptides was obtained by this new analysis strategy, therefore making it advantageous for urinary biomarker screening. Graphical abstract A sequential separation method that isolates urinary soluble proteins and EV proteins via stepwise ultrafiltration was developed in this work. Subsequent N-glycopeptides enrichment and mass spectrometry analysis reveals distinct N-glycoproteome patterns in the two sub-proteomes of urine and a deep mapping of close to 1800 N-glycopeptides.

Transcription factor-based biosensor for detection of phenylalanine and tyrosine in urine for diagnosis of phenylketonuria

Clinicians require a simple quantitative method for the detection of both phenylalanine and tyrosine to facilitate the diagnosis of phenylketonuria, a common inherited disorder of amino acid metabolism. In this study, we developed a novel whole-cell biosensor for the quantification of phenylalanine and tyrosine through the expression of red and green fluorescent proteins. The proposed system responds specifically and sensitively to phenylalanine/tyrosine without interferences from other amino acids. Furthermore, the precision of the biosensor was evaluated using specimens of normal human urine by LC-MS.

Proteomic profile‑based screening of potential protein biomarkers in the urine of patients with nephrotic syndrome.

Nephrotic syndrome is not a single disease; rather, it is a term for numerous diseases and pathological types. Renal biopsy is of use in determining the diagnosis and prognosis, and for guiding treatment; however, the use of this intervention is limited due to its invasive nature. Abnormal kidney‑derived proteins in the urine of patients provide useful information regarding numerous pathological processes that occur in the kidneys, and may be considered a potential non‑invasive biomarker for kidney disease. Proteomic analysis exhibits the advantage of being high‑throughput and has previously been used to identify biomarkers of disease. The present study aimed to identify abnormal kidney‑derived proteins in the urine of patients with nephrotic syndrome using a novel proteomic strategy. Urine samples from 5patients with nephrotic syndrome were subjected to acetone precipitation and albumin/immunoglobulin G depletion prior to analysis by two‑dimensional liquid chromatography tandem mass spectrometry. The resulting data were compared to a publicly available proteomic database of normal human plasma/urine and normal human kidney in PeptideAtlas, and of normal human kidney in the Human Protein Atlas. Candidate biomarkers were validated using ELISA analysis in 60patients with nephrotic syndrome: 30with focal segmental glomerulosclerosis (FSGS) and 30with minimal change disease (MCD), as well as in 30healthy controls. The initial screening identified 809 proteins in the urine of patients with nephrotic syndrome. A total of 13/809 proteins were additionally present in the kidney proteome of PeptideAtlas and the Human Protein Atlas, although not in normal human urine and normal human plasma according to PeptideAtlas; these were referred to as 'kidney‑derived disease‑associated proteins'. One of the kidney‑derived disease‑associated proteins, ubiquitin‑60S ribosomal protein L40 (UBA52) was observed to be increased in the urine of patients compared with normal controls [Creatinine, 637ng/mg (216‑1,851) vs.1.89ng/mg (1.37‑3.33), P<0.001; and 18.58ng/mg (11.11‑46.25) vs. 1.89ng/mg (1.37‑3.33), P<0.001)], and the urinary UBA52 levels were significantly increased in patients with FSGS compared with in patients with MCD (P<0.001). In conclusion, the present study identified potential novel urinary protein biomarkers for nephrotic syndrome, in addition to an extensive urinary proteomic profile of patients with nephrotic syndrome.

Binding induced colocalization activated hybridization chain reaction on the surface of magnetic nanobead for sensitive detection of adenosine

Herein, a sensitive and enzyme-free assay for adenosine detection has been developed on the basis of binding induced colocalization activated hybridization chain reaction (HCR) strategy on the surface of magnetic nanobead. First, the recognition probe was fabricated and divided into two parts: the Apt-1 that composed a part of adenosine aptamer and toehold domain, and the Apt-2 that consisted of another part of adenosine aptamer and branch migration domain. The Apt-1 was immobilized on a streptavidin-magnetic nanobead (streptavidin-MNBs) that played the roles of enrichment and separation. Then the recognition event of adenosine could bring the two parts of aptamer together and induce the colocalization of toehold domain and branch migration domain, which could serve as an integrated initiator to trigger the HCR, producing a long nicked double-stranded polymer. Finally, the intercalating dye SYBR Green I was inserted into the polymer, generating an enhanced fluorescence signal. In this strategy, the initiator was divided into two parts and could be suppressed effectively in the absence of adenosine. Utilizing the separated function, the spontaneous hybridization of H1 and H2 could be avoided, and a low background could be acquired. Moreover, through the double amplification of HCR and multimolecules binding of SYBR Green I, highly sensitive and enzyme-free detection were achieved. The detection limit for adenosine detection was 2.0×10−7mol/L, which was comparable or superior to the previous aptasensors. Importantly, adenosine analysis in human urines has been performed, and this strategy could significantly distinguish the adenosine content in normal human urines and cancer patient urines, suggesting that this proposed assay will become a reliable and sensitive adenosine detection method in early clinical diagnosis and medical research.

An integrated proteomic and peptidomic assessment of the normal human urinome.

Urine represents an ideal source of clinically relevant biomarkers as it contains a large number of proteins and low molecular weight peptides. The comprehensive characterization of the normal urinary proteome and peptidome can serve as a reference for future biomarker discovery. Proteomic and peptidomic analysis of urine can also provide insight into normal physiology and disease pathology, especially for urogenital diseases. We developed an integrated proteomic and peptidomic analytical protocol in normal urine. We employed ultrafiltration to separate protein and peptide fractions, which were analyzed separately using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on the Q-Exactive mass spectrometer. By analyzing six urines from healthy individuals with advanced age, we identified 1754 proteins by proteomic analysis and 4543 endogenous peptides, arising from 566 proteins by peptidomic analysis. Overall, we identified 2091 non-redundant proteins by this integrated approach. In silico protease activity analysis indicated that metalloproteases are predominantly involved in the generation of the endogenous peptide signature. In addition, a number of proteins that were detected in normal urine have previously been implicated in various urological malignancies, including bladder cancer and renal cell carcinoma (RCC). We utilized a highly sensitive proteomics approach that enabled us to identify one of the largest sets of protein identifications documented in normal human urine. The raw proteomics and peptidomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD003595.

Evaluation of the in vitro growth of urinary tract infection-causing gram-negative and gram-positive bacteria in a proposed synthetic human urine (SHU) medium

Bacteriuria is a hallmark of urinary tract infection (UTI) and asymptomatic bacteriuria (ABU), which are among the most frequent infections in humans. A variety of gram-negative and gram-positive bacteria are associated with these infections but Escherichia coli contributes up to 80% of cases. Multiple bacterial species including E. coli can grow in human urine as a means to maintain colonization during infections. In vitro bacteriuria studies aimed at modeling microbial growth in urine have utilized various compositions of synthetic human urine (SHU) and a Composite SHU formulation was recently proposed. In this study, we sought to validate the recently proposed Composite SHU as a medium that supports the growth of several bacterial species that are known to grow in normal human urine and/or artificial urine. Comparative growth assays of gram-negative and gram-positive bacteria E. coli, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus agalactiae, Staphylococcus saprophyticus and Enterococcus faecalis were undertaken using viable bacterial count and optical density measurements over a 48h culture period. Three different SHU formulations were tested in various culture vessels, shaking conditions and volumes and showed that Composite SHU can support the robust growth of gram-negative bacteria but requires supplementation with 0.2% yeast extract to support the growth of gram-positive bacteria. Experiments are also presented that show an unexpected but major influence of P. mirabilis towards the ability to measure bacterial growth in generally accepted multiwell assays using absorbance readings, predicted to have a basis in the release of volatile organic compound(s) from P. mirabilis during growth in Composite SHU medium. This study represents an essential methodological validation of a more chemically defined type of synthetic urine that can be applied to study mechanisms of bacteriuria and we conclude will offer a useful in vitro model to investigate the basis of some of the most common infections of humans.

Characterizations of heparin-binding proteins in human urine by affinity purification-mass spectrometry and defining “L-x(2,3)-A-x(0,1)-L” as a novel heparin-binding motif

Heparin-binding proteins (HBPs) are considered as potential modulators of kidney stone formation. However, HBPs had not been characterized in the urine previously. In this study, we applied affinity purification-mass spectrometry (AP-MS) using cellufine sulfate column chromatography and liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-Q-TOF MS/MS) to identify HBPs in normal human urine. Using this approach, 83 HBPs were identified, including those involved in metabolic process, cellular process, immune system, developmental process, response to stimuli, cell communication, transport, cell adhesion and others. The AP-MS data were confirmed by Western blot analysis and chemico-protein interactions analysis using STITCH tool. In addition, 59, 55 and 51 identified HBPs had the known heparin-binding motifs “XBBXnBX”, “XBXnBBX” and “XBBBnX”, respectively. Moreover, a novel heparin-binding motif “L-x(2,3)-A-x(0,1)-L” was found in 58 identified HBPs using PRATT tool. The sensitivity and specificity of this novel motif were 85% and 100%, respectively, by validation using 20 known HBPs and 11 non-HBPs. We report herein for the first time a large number of HBPs in normal human urine and defined “L-x(2,3)-A-x(0,1)-L” as a novel heparin-binding motif. These findings will be useful to further understand the renal physiology and may also lead to identification of novel modulators of kidney stone formation. Biological significanceHeparin-binding proteins (HBPs) have several important roles in various biological processes, including kidney stone formation. However, HBPs had not been characterized in the urine. Our present work using affinity purification coupled to mass spectrometry (AP-MS) is the first large-scale study on HBPs in human urine. In addition to the three known heparin-binding motifs, “XBBXnBX”, “XBXnBBX”, and “XBBBnX”, we successfully defined the amino acid pattern “L-x(2,3)-A-x(0,1)-L” as a novel heparin-binding motif. These findings will be useful to further understand the renal physiology and may also lead to identification of novel modulators of kidney stone formation.

Human urinary exosome proteome unveils its aerobic respiratory ability

Exosomes are 40–100-nm vesicles released by most cell types after fusion of multivesicular endosomes with the plasma membrane. Exosomes, ubiquitary in body fluids including urines, contain proteins and RNA species specific of the tissue of origin. Exosomes from urine have been extensively studied as a promising reservoir for disease biomarkers.Here, we report the proteome analysis of urinary exosomes compared to urinoma, studied by Orbitrap mass spectrometry. A discovery approach was utilized on the sample. 3429 proteins were present, with minimal overlapping among exosome and urinoma. 959 proteins (28%) in exosome and 1478 proteins (43%) in urinoma were exclusively present in only one group. By cytoscape analysis, the biological process gene ontology was correlated to their probability (P≤0.05) to be functional. This was never studied before and showed a significant clustering around metabolic functions, in particular to aerobic ATP production. Urinary exosomes carry out oxidative phosphorylation, being able to synthesize ATP and consume oxygen.A previously unsuspected function emerges for human urinary exosomes as bioactive vesicles that consume oxygen to aerobically synthesize ATP. Determination of normal human urine proteome can help generate the healthy urinary protein database for comparison, useful for various renal diseases. Biological significanceThe findings reported represent a significant advance in the understanding of the healthy human urinary proteome. The methodology utilized to analyze the collection of proteomic data allowed the assessment of the unique composition of urinary exosomes with respect to urinoma and to elucidate the presence in the former of molecular pathways previously unknown. The paper has the potential to impact its field of research, due to the biological relevance of the metabolic capacity of urinary exosomes, which may represent their important general feature.

Discovery and Characterization of Human-Urine Utilization by Asymptomatic-Bacteriuria-Causing Streptococcus agalactiae.

Streptococcus agalactiae causes both symptomatic cystitis and asymptomatic bacteriuria (ABU); however, growth characteristics of S. agalactiae in human urine have not previously been reported. Here, we describe a phenotype of robust growth in human urine observed in ABU-causing S. agalactiae (ABSA) that was not seen among uropathogenic S. agalactiae (UPSA) strains isolated from patients with acute cystitis. In direct competition assays using pooled human urine inoculated with equal numbers of a prototype ABSA strain, designated ABSA 1014, and any one of several UPSA strains, measurement of the percentage of each strain recovered over time showed a markedly superior fitness of ABSA 1014 for urine growth. Comparative phenotype profiling of ABSA 1014 and UPSA strain 807, isolated from a patient with acute cystitis, using metabolic arrays of >2,500 substrates and conditions revealed unique and specific l-malic acid catabolism in ABSA 1014 that was absent in UPSA 807. Whole-genome sequencing also revealed divergence in malic enzyme-encoding genes between the strains predicted to impact the activity of the malate metabolic pathway. Comparative growth assays in urine comparing wild-type ABSA and gene-deficient mutants that were functionally inactivated for the malic enzyme metabolic pathway by targeted disruption of the maeE or maeK gene in ABSA demonstrated attenuated growth of the mutants in normal human urine as well as synthetic human urine containing malic acid. We conclude that some S. agalactiae strains can grow in human urine, and this relates in part to malic acid metabolism, which may affect the persistence or progression of S. agalactiae ABU.

Graphene/Cu nanoparticle hybrids fabricated by chemical vapor deposition as surface-enhanced Raman scattering substrate for label-free detection of adenosine.

We present a graphene/Cu nanoparticle hybrids (G/CuNPs) system as a surface-enhanced Raman scattering (SERS) substrate for adenosine detection. The Cu nanoparticles wrapped by a monolayer graphene shell were directly synthesized on flat quartz by chemical vapor deposition in a mixture of methane and hydrogen. The G/CuNPs showed an excellent SERS enhancement activity for adenosine. The minimum detected concentration of the adenosine in serum was demonstrated as low as 5 nM, and the calibration curve showed a good linear response from 5 to 500 nM. The capability of SERS detection of adenosine in real normal human urine samples based on G/CuNPs was also investigated and the characteristic peaks of adenosine were still recognizable. The reproducible and the ultrasensitive enhanced Raman signals could be due to the presence of an ultrathin graphene layer. The graphene shell was able to enrich and fix the adenosine molecules, which could also efficiently maintain chemical and optical stability of G/CuNPs. Based on the G/CuNPs system, the ultrasensitive SERS detection of adenosine in varied matrices was expected for the practical applications in medicine and biotechnology.

A study examining the bias of albumin and albumin/creatinine ratio measurements in urine.

The objective of the study was to examine the bias of albumin and albumin/creatinine (ACR) measurements in urine. Pools of normal human urine were augmented with purified human serum albumin to generate a series of 12 samples covering the clinical range of interest for the measurement of ACR. Albumin and creatinine concentrations in these samples were analyzed three times on each of 3 days by 24 accredited laboratories in Canada and the USA. Reference values (RV) for albumin measurements were assigned by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) comparative method and gravimetrically. Ten random urine samples (check samples) were analyzed as singlets and albumin and ACR values reported according to the routine practices of each laboratory. Augmented urine pools were shown to be commutable. Gravimetrically assigned target values were corrected for the presence of endogenous albumin using the LC-MS/MS comparative method. There was excellent agreement between the RVs as assigned by these two methods. All laboratory medians demonstrated a negative bias for the measurement of albumin in urine over the concentration range examined. The magnitude of this bias tended to decrease with increasing albumin concentrations. At baseline, only 10% of the patient ACR values met a performance limit of RV ± 15%. This increased to 84% and 86% following post-analytical correction for albumin and creatinine calibration bias, respectively. International organizations should take a leading role in the standardization of albumin measurements in urine. In the interim, accuracy based urine quality control samples may be used by clinical laboratories for monitoring the accuracy of their urinary albumin measurements.