Urinary adenosine triphosphate and nitric oxide levels in patients with underactive bladder: a preliminary study.

INTRODUCTION: Various in vitro and in vivo animal studies have shown that adenosine triphosphate (ATP) has a stimulatory role and nitric oxide (NO) has an inhibitory role in modulating bladder contractility. However, it is not known what happens to the urinary levels of ATP and NO in humans with underactive bladder (UAB). METHODS: In this prospective case-control study, we compared ATP and NO levels in twenty six male patients of UAB with a bladder contractility index (BCI) of < 100 and 18 healthy male volunteers without any lower urinary tract symptoms (LUTS). RESULTS: The mean urinary ATP levels were significantly lower in cases compared to controls (546.1 ± 37.3 pg/µl vs. 610.7 ± 24.9 pg/µl, p value < 0.001) and the mean NO levels were significantly higher in cases compared to controls (1233.4 ± 91.2 pg/µl vs. 1126.3 ± 91.3.4 pg/µl, p value < 0.001). The mean NO/ATP ratio in cases was significantly higher than that of controls (2.26 ± 0.2 vs. 1.84 ± 0.18, p value < 0.000). Using receiver operating curve (ROC) analysis, we noted the area under the curve (AUC) for NO/ATP ratio to be 0.91 in the diagnosis of cases. A cut-off value of 2.06 for NO/ATP ratio had sensitivity, specificity and diagnostic accuracy of 88.5%, 88.9% and 88.6%, respectively, in diagnosing patients with UAB. CONCLUSION: Patients with UAB have significantly higher levels of urinary NO and decreased levels of urinary ATP. Urinary NO/ATP levels can be considered as a noninvasive alternate test for diagnosing bladder underactivity.

ATP shows more potential as a urinary biomarker than acetylcholine and PGE2 , but its concentration in urine is not a simple function of dilution.

AIMS: To determine whether the amount of ATP, prostaglandin E2 (PGE2 ), and acetylcholine (ACh) in voided urine are influenced enough by that released within the lower urinary tract (LUT) for them to be useful biomarkers of bladder function. METHODS: Participants without LUT symptoms collected total urine voids at 15, 30, 60, and 120 min (20 males/23 females) and 240 min (18 males/26 females) following the previous void. Aliquots of urine were immediately frozen at -20°C and later used to measure ATP (luciferin-luciferase), PGE2 (enzyme-linked immunosorbent assay), ACh (mass spectrometry), creatinine (colorimetric), and lactose dehydrogenase (colorimetric). RESULTS: The amount of ATP in voided urine correlated strongly with the rate of urine production, suggesting that the majority, if not all, the ATP in voided urine has an LUT, and likely bladder, origin. In contrast, there appeared to be no significant net LUTs release of creatinine or ACh into the urine. PGE2 was intermediate with an LUT component that increased with urine production rate and contributed about 25% of the total at 1 ml/min in women but a smaller fraction in men. CONCLUSION: Whereas the majority of the ATP measured within the voided urine originates in the LUT, ACh reflects that extracted from the plasma in the kidneys and PGE2 is a mixture of both sources. ATP has the most potential as a biomarker of benign bladder disorders. Expressing urinary ATP concentration relative to creatinine concentration is questioned in light of these results.

Pannexin-1 mediates fluid shear stress-sensitive purinergic signaling and cyst growth in polycystic kidney disease.

Tubular ATP release is regulated by mechanosensation of fluid shear stress (FSS). Polycystin-1/polycystin-2 (PC1/PC2) functions as a mechanosensory complex in the kidney. Extracellular ATP is implicated in polycystic kidney disease (PKD), where PC1/PC2 is dysfunctional. This study aims to provide new insights into the ATP signaling under physiological conditions and PKD. Microfluidics, pharmacologic inhibition, and loss-of-function approaches were combined to assess the ATP release in mouse distal convoluted tubule 15 (mDCT15) cells. Kidney-specific Pkd1 knockout mice (iKsp-Pkd1-/- ) and zebrafish pkd2 morphants (pkd2-MO) were as models for PKD. FSS-exposed mDCT15 cells displayed increased ATP release. Pannexin-1 inhibition and knockout decreased FSS-modulated ATP release. In iKsp-Pkd1-/- mice, elevated renal pannexin-1 mRNA expression and urinary ATP were observed. In Pkd1-/- mDCT15 cells, elevated ATP release was observed upon the FSS mechanosensation. In these cells, increased pannexin-1 mRNA expression was observed. Importantly, pannexin-1 inhibition in pkd2-MO decreased the renal cyst growth. Our results demonstrate that pannexin-1 channels mediate ATP release into the tubular lumen due to pro-urinary flow. We present pannexin-1 as novel therapeutic target to prevent the renal cyst growth in PKD.

A pilot study to investigate the associations of urinary concentrations of NO, ATP and derivatives with overactive bladder symptom severity.

NEW FINDINGS: What is the central question of this study? Are the urinary concentrations of NO and ATP, and their metabolites, associated with the severity of symptoms of overactive bladder? What is the main finding and its importance? The urinary ratios of [ATP/NO], [ADP/NO] and a combination of these, [ATP/Cr*ADP/Cr]/[NO/Cr], were correlated with overall OAB symptom severity, with the latter combination also being correlated with the severity of urinary frequency and urgency symptoms individually. Together, these data reveal changes in urothelial signalling that accompany the transition from physiology to pathology. ABSTRACT: Overactive bladder (OAB) is a highly prevalent symptom complex characterized by symptoms of urinary urgency and increased frequency and waking to void (nocturia), with or without urge incontinence and in the absence of proven infection or other obvious pathology. The underlying pathophysiology of idiopathic OAB is not clearly known, and the existence of several phenotypes has been proposed. Current diagnostic approaches are based on discordant measures, suffer from subjectivity and are incapable of detecting the proposed OAB phenotypes. Nitric oxide, ATP and their metabolites have previously been shown to underlie the perception of bladder fullness, with their release modifying the pathological perception of urgency. Therefore, in this study we assessed the concentrations of NO, ATP and associated metabolites in the urine of 113 consenting participants recruited from the general population. Recruited participants completed a questionnaire to measure the severity of OAB-associated urinary symptoms and provided a mid-stream urine sample. After identification of infection and haematuria using microbiology and microscopy, 95 samples were subjected to assays to measure NO, NO2- , NO3- , ATP, ADP and creatinine (Cr). There was no correlation between [NO/Cr], [NO2- /Cr] or [NO3- /Cr] and overall OAB symptom severity. In contrast, [ATP/NO], [ADP/NO] and a combination of these, [ATP/Cr*ADP/Cr]/[NO/Cr], were correlated with OAB symptom severity, and [ATP/Cr*ADP/Cr]/[NO/Cr] was also correlated with the severity of urinary frequency and urgency. This study adds to a growing literature that demonstrates the potential of urinary biomarkers and provides a foundation for a larger, longitudinal study.

New participant stratification and combination of urinary biomarkers and confounders could improve diagnostic accuracy for overactive bladder.

Overactive bladder (OAB) is a highly prevalent symptom complex characterised by symptoms of urinary urgency, increased frequency, nocturia, with or without urge incontinence; in the absence of proven infection or other obvious pathology. The underlying pathophysiology of idiopathic OAB is not clearly known and the existence of several phenotypes has been proposed. Current diagnostic approaches are based on discordant measures, suffer from subjectivity and are incapable of detecting the proposed OAB phenotypes. In this study, cluster analysis was used as an objective approach for phenotyping participants based on their OAB characteristic symptoms and led to the identification of a low OAB symptomatic score group (cluster 1) and a high OAB symptomatic score group (cluster 2). Furthermore, the ability of several potential OAB urinary biomarkers including ATP, ACh, nitrite, MCP-1 and IL-5 and participants' confounders, age and gender, in predicting the identified high OAB symptomatic score group was assessed. A combination of urinary ATP and IL-5 plus age and gender was shown to have clinically acceptable and improved diagnostic accuracy compared to urodynamically-observed detrusor overactivity. Therefore, this study provides the foundation for the development of novel non-invasive diagnostic tools for OAB phenotypes that may lead to personalised treatment.

Investigating the associations of mucosal P2Y6 receptor expression and urinary ATP and ADP concentrations, with symptoms of overactive bladder.

AIM: To characterize purinergic signaling in overactive bladder (OAB). METHODS: Mucosal biopsies were taken by flexible cystoscopy from patients with storage symptoms referred to Urology Departments of collaborating hospitals. Immunohistochemistry (n = 12) and Western blot analysis (n = 28) were used to establish the qualitative and quantitative expression profile of P2Y6 in human mucosa. Participants from the general population provided a mid-stream urine sample. Bioluminescent assays were used to quantify adenosine triphosphate (ATP; n = 66) and adenosine diphosphate (ADP; n = 60) concentrations, which were normalized to creatinine (Cr) concentration. All participants completed a questionnaire (International Consultation on Incontinence Questionnaire - Overactive Bladder) to score urinary symptoms of OAB. RESULTS: P2Y6 immunoreactivity, more prominent in the urothelium (colocalized with the uroepithelial marker pan-cytokeratin), was more greatly expressed in OAB compared to age- and sex-matched controls (benign prostatic hyperplasia) without OAB symptoms. Mucosal P2Y6 was positively correlated only with incontinence (P = .009). Both urinary ATP and its hydrolysis product, ADP, an agonist to P2Y6, were positively correlated with total OAB symptom score (P = .010 and P = .042, respectively). CONCLUSIONS: The positive correlation of P2Y6 only with incontinence may indicate a different phenotype in OAB wet and warrants further investigation. Positive correlations of ATP and ADP with total OAB symptom score demonstrate upregulation in purinergic signaling in OAB; shown previously only in animal models. Further research is required to validate whether purinoceptors are indeed new therapeutic targets for this highly prevalent symptom complex.

TRPV4 blockade reduces voiding frequency, ATP release, and pelvic sensitivity in mice with chronic urothelial overexpression of NGF.

Transient receptor potential vanilloid family member 4 (TRPV4) transcript and protein expression increased in the urinary bladder and lumbosacral dorsal root ganglia of transgenic mice with chronic urothelial overexpression of nerve growth factor (NGF-OE). We evaluated the functional role of TRPV4 in bladder function with open-outlet cystometry, void spot assays, and natural voiding (Urovoid) assays with the TRPV4 antagonist HC-067047 (1 µM) or vehicle in NGF-OE and littermate wild-type (WT) mice. Blockade of TRPV4 at the level of the urinary bladder significantly (P ≤ 0.01) increased the intercontraction interval (2.2-fold) and void volume (2.6-fold) and decreased nonvoiding contractions (3.0-fold) in NGF-OE mice, with lesser effects (1.3-fold increase in the intercontraction interval and 1.3-fold increase in the void volume) in WT mice. Similar effects of TRPV4 blockade on bladder function in NGF-OE mice were demonstrated with natural voiding assays. Intravesical administration of HC-067047 (1 µM) significantly (P ≤ 0.01) reduced pelvic sensitivity in NGF-OE mice but was without effect in littermate WT mice. Blockade of urinary bladder TRPV4 or intravesical infusion of brefeldin A significantly (P ≤ 0.01) reduced (2-fold) luminal ATP release from the urinary bladder in NGF-OE and littermate WT mice. The results of the present study suggest that TRPV4 contributes to luminal ATP release from the urinary bladder and increased voiding frequency and pelvic sensitivity in NGF-OE mice.

Knockout of P2rx7 purinergic receptor attenuates cyst growth in a rat model of ARPKD.

The severity of polycystic kidney diseases (PKD) depends on the counterbalancing of genetic predisposition and environmental factors exerting permissive or protective influence on cyst development. One poorly characterized phenomenon in the cystic epithelium is abnormal purinergic signaling. Earlier experimental studies revealed the high importance of the ionotropic P2X receptors (particularly, P2X7) in the pathophysiology of the cyst wall. To study mechanisms of P2X7 involvement in cyst growth and aspects of targeting these receptors in PKD treatment we performed a CRISPR/SpCas9-mediated global knockout of the P2rx7 gene in PCK rats, a model of autosomal recessive PKD (ARPKD). A single base insertion in exon 2 of the P2rx7 gene in the renal tissues of homozygous mutant animals leads to lack of P2X7 protein that did not affect their viability or renal excretory function. However, PCK.P2rx7 rats demonstrated slower cyst growth (but not formation of new cysts) compared with heterozygous and PCK.P2rx7+ littermates. P2X7 receptors are known to activate pannexin-1, a plasma channel capable of releasing ATP, and we found here that pannexin-1 expression in the cystic epithelium is significantly higher than in nondilated tubules. P2X7 deficiency reduces renal pannexin-1 protein expression and daily urinary ATP excretion. Patch-clamp analysis revealed that lack of P2X7 increases epithelial sodium channel activity in renal tissues and restores impaired channel activity in cysts. Interpretation of our current data in the context of earlier studies strongly suggests that P2X7 contributes to cyst growth by increasing pannexin-1-dependent pathogenic ATP release into the lumen and reduction of sodium reabsorption across the cyst walls.

Urinary Biomarkers in Overactive Bladder: Revisiting the Evidence in 2019.

CONTEXT: In overactive bladder (OAB), after an initial outbreak of research, it is more consensual that biomarkers may be better used to phenotype patients. Herein, we revisit this topic, including some of the most promising biomarkers. OBJECTIVE: To provide a comprehensive analysis of the actual role of biomarkers in OAB. EVIDENCE ACQUISITION: A PubMed-based literature search was conducted, including the most relevant articles published in the last 15 yr, on nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), adenosine triphosphate (ATP), genomics, and microbiota as OAB biomarkers. Articles with no full text available or not written in English were excluded. Additional reviews were included. EVIDENCE SYNTHESIS: Urinary NGF, BDNF, and ATP are increased in many OAB patients. These biomarkers can help identify OAB phenotypes and select the ideal candidates for new therapies directed to neurotrophic and purinergic pathways. Circulating urinary miRNA may be useful for establishing the ideal moment for bladder outlet obstruction relief and will eventually lead to the development of therapeutic agents that inhibit or reverse fibrotic pathways in the bladder. Urinary microbiota seems to be related to OAB symptoms, in particular urgency urinary incontinence, and may have strong implications in the prevention, diagnosis, and treatment of OAB. CONCLUSIONS: In the future, physicians may consider the use of biomarkers to identify distinct OAB phenotypes, with distinct causal mechanisms, selecting patients for specific target therapies with expected better outcomes. PATIENT SUMMARY: Overactive bladder biomarkers can be useful for phenotype patients and for selecting more effective target therapies.

Simultaneous detection of ATP metabolites in human plasma and urine based on palladium nanoparticle and poly(bromocresol green) composite sensor.

A sensitive voltammetric sensor based on palladium nanoparticles (PdNPs) and poly-bromocresol green (pBG) composite layer immobilized on amide functionalized single-walled carbon nanotubes (AmSWCNTs) modified pyrolytic graphite (PdNPs:pBG/AmSWCNTs/PG) has been prepared for the simultaneous determination of adenosine triphosphate (ATP) catabolites, inosine (INO), hypoxanthine (HX), xanthine (XT), and uric acid (UA). The modified PdNPs:pBG/AmSWCNTs/PG was characterized by electrochemical experiments and surface analysis, which exhibited exceptional electrocatalytic effects towards the oxidation of INO, HX, XT, and UA with a significant enhanced peak current and well resolved peaks separation for all the analytes. The linear calibration curves were obtained in the concentration range of 0.001-175 µM, 0.001-200 µM, 0.001-150 µM, and 0.001-200 µM and limits of detection were found as 0.95 nM, 1.04 nM, 1.07 nM, and 0.43 nM corresponding to INO, HX, XT, and UA, respectively. The common metabolites present in the biological fluids did not interfere in the determination. The applicability of the proposed sensor was successfully demonstrated by determining INO, HX, XT, and UA in the human plasma and urine and the obtained results were validated by using HPLC.

A Label-Free Fluorescent DNA Calculator Based on Gold Nanoparticles for Sensitive Detection of ATP.

Herein we described a deoxyribonucleic acid (DNA) calculator for sensitive detection of the determination of adenosine triphosphate (ATP) using gold nanoparticles (GNP) and PicoGreen fluorescence dye as signal transducer, and ATP and single-stranded DNA (DNA-M') as activators. The calculator-related performances including linearity, reaction time, logic gate, and selectivity were investigated, respectively. The results revealed that this oligonucleotide sensor was highly sensitive and selective. The detection range was 50⁻500 nmol/L (R² = 0.99391) and the detection limit was 46.5 nmol/L. The AND DNA calculator was successfully used for the ATP detection in human urine. Compared with other methods, this DNA calculator has the characteristics of being label-free, non-enzymic, simple, and highly sensitive.

A Personal Glucose Meter for Label-Free and Washing-Free Biomolecular Detection.

We developed a label-free and washing-free method for biomolecular detection using a personal glucose meter (PGM). ATP was selected as a model target, and cascade enzymatic reactions promoted by hexokinase and pyruvate kinase were adopted to link the amount of ATP to glucose that is detectable by a hand-held PGM. In principle, the presence of target ATP enables hexokinase to catalyze the conversion of glucose to glucose 6-phosphate by providing a phosphate group to glucose, and thus the amount of glucose is decreased in proportion to the amount of ATP. In addition, adenosine 5'-diphosphate (ADP), which is generated after hexokinase-catalyzed enzymatic reaction, is recovered to ATP by a pyruvate kinase enzyme. The regenerated ATP is again supplemented to catalyze multiple rounds of cascade enzymatic reactions, leading to signal amplification. As a result, the change of glucose amount that is inversely proportional to ATP amount is simply measured by a hand-held PGM. By employing this strategy, we successfully determined ATP down to 49 nM with high selectivity even in real samples such as tap water, human serum, and bovine urine. Importantly, the developed system does not require expensive modification and washing steps but is conveniently operated with a commercially available PGM, which would pave the way for the development of a simple and cost-effective sensing platform.

Multiplex detection of quality indicator molecule targets in urine using programmable hairpin probes based on a simple double-T type microchip electrophoresis platform and isothermal polymerase-catalyzed target recycling.

Recently, it has been crucial to be able to detect and quantify small molecular targets simultaneously in biological samples. Herein, a simple and conventional double-T type microchip electrophoresis (MCE) based platform for the multiplex detection of quality indicator molecule targets in urine, using ampicillin (AMPI), adenosine triphosphate (ATP) and estradiol (E2) as models, was developed. Several programmable hairpin probes (PHPs) were designed for detecting different targets and triggering isothermal polymerase-catalyzed target recycling (IPCTR) for signal amplification. Based on the target-responsive aptamer structure of PHP (Domain I), target recognition can induce PHP conformational transition and produce extension duplex DNA (dsDNA), assisted by primers & Bst polymerase. Afterwards, the target can be displaced to react with another PHP and initiate the next cycle. After several rounds of reaction, the dsDNA can be produced in large amounts by IPCTR. Three targets can be simultaneously converted to dsDNA fragments with different lengths, which can be separated and detected using MCE. Thus, a simple double-T type MCE based platform was successfully built for the homogeneous detection of multiplex targets in one channel. Under optimal conditions, the assay exhibited high throughput (48 samples per hour at most, not including reaction time) and sensitivity to three targets in urine with a detection limit of 1 nM (ATP), 0.05 nM (AMPI) and 0.1 nM (E2) respectively. The multiplex assay was successfully employed for the above three targets in several urine samples and combined the advantages of the high specificity of programmable hairpin probes, the excellent signal amplification of IPCTR, and the high through-put of MCE which can be employed for screening in biochemical analysis.

A fluorescent "on-off-on" probe for sensitive detection of ATP based on ATP displacing DNA from nanoceria.

A simple, rapid, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection was developed by using a FAM (carboxyfluorescein) labeled DNA (FAM-DNA). In this strategy, highly fluorescent FAM-DNA was used as a probe, and nanoceria (CeO2 NPs) acted as an efficient quencher. FAM-DNA attached to the surface of nanoceria through the coordination between the phosphate group of DNA and NP surface, which induced complete quenching in the FAM-DNA fluorescence due to a photo induced electron transfer (PET) process. It was found that ATP can readily displace adsorbed DNA from nanoceria surface because of the stronger coordination ability of ATP with nanoceria, and the nanoceria-based competitive binding resulted in over 7-fold fluorescence enhancement. Over a wide range from 0.1nM to 1.5µM, a good linear relationship between the fluorescence intensity and the concentration of ATP was obtained and the detection limit was estimated to be as low as 54pM. This method was successfully used to analyze ATP in a single drop of blood and human urine.

pH detection in biological samples by 1D and 2D 1H-31P NMR.

The chemical shifts of several important endogenous phosphorus compounds under different pH conditions were explored, including adenosine-5'-triphosphate, adenosine-5'-diphosphate, adenosine-5'-monophosphate, phosphorylcholine and phosphorylethanolamine. Their 31P NMR and 1H NMR chemical shifts were all pH-sensitive in the similar pH range. Two dimensional (2D) 1H-31P NMR spectra were found helpful to identify these endogenous phosphorus markers in biological samples from rather complicated NMR spectra. Herein, for the first time, a pH sensor based on 2D 1H-31P NMR was established and applied to biological samples analysis with pH values determined in good agreement with those by potentiometric method. Apart from being simple, green, rapid and less sample-consuming, information concerning both the endogenous phosphorus markers and pH status could be attained in a single NMR run, which demonstrated the great potential of this method in rare sample analysis and even disease diagnosis.

Competitive fluorescence anisotropy/polarization assay for ATP using aptamer as affinity ligand and dye-labeled ATP as fluorescence tracer.

We developed an aptamer-based competitive fluorescence anisotropy (FA)/fluorescence polarization (FP) assay for adenosine triphosphate (ATP). Different from the traditional fluorescence polarization immunoassays for small molecules, here DNA aptamer against ATP was used as affinity ligand, and tetramethylrhodamine (TMR) labeled ATP served as fluorescent tracer. The binding between TMR-labeled ATP and aptamer gave large FA due to molecular volume increase and restricted rotation of the dye-labeled ATP. When ATP was added in solution, ATP competitively displaced the TMR-labeled ATP from aptamer affinity complex, causing decrease of FA of TMR-labeled ATP. The buffer containing MgCl2 and incubation at low temperature were preferred for large FA change in the FA assay. The FA change was further enhanced in this competitive FA assay by increasing the molecular weight of aptamer through extension of aptamer sequences or conjugating streptavidin protein on aptamer. This method allowed for the detection of ATP in the range from 0.5µM to 1mM, generating the maximum FA change about 0.187 (corresponding maximum FP change about 0.242). The detection of ATP spiked in diluted urine or serum sample was achieved, showing capability for analysis in complex sample matrix. This assay also enabled the detection of the analogues of ATP, e.g. adenosine, adenosine monophosphate (AMP), and adenosine diphosphate (ADP) with similar sensitivity. This aptamer-based competitive FA assay takes advantages of aptamer in ease of synthesis, good thermal stability, and facile modulating the molecular mass of aptamer.

ENaC activity in the cortical collecting duct of HKα1 H+,K+-ATPase knockout mice is uncoupled from Na+ intake.

Modulation of the epithelial Na+ channel (ENaC) activity in the collecting duct (CD) is an important mechanism for normal Na+ homeostasis. ENaC activity is inversely related to dietary Na+ intake, in part due to inhibitory paracrine purinergic regulation. Evidence suggests that H+,K+-ATPase activity in the CD also influences Na+ excretion. We hypothesized that renal H+,K+-ATPases affect Na+ reabsorption by the CD by modulating ENaC activity. ENaC activity in HKα1 H+,K+-ATPase knockout (HKα1-/-) mice was uncoupled from Na+ intake. ENaC activity on a high-Na+ diet was greater in the HKα1-/- mice than in WT mice. Moreover, dietary Na+ content did not modulate ENaC activity in the HKα1-/- mice as it did in WT mice. Purinergic regulation of ENaC was abnormal in HKα1-/- mice. In contrast to WT mice, where urinary [ATP] was proportional to dietary Na+ intake, urinary [ATP] did not increase in response to a high-Na+ diet in the HKα1-/- mice and was significantly lower than in the WT mice. HKα1-/- mice fed a high-Na+ diet had greater Na+ retention than WT mice and had an impaired dipsogenic response. These results suggest an important role for the HKα1 subunit in the regulation of purinergic signaling in the CD. They are also consistent with HKα1-containing H+,K+-ATPases as important components for the proper regulation of Na+ balance and the dipsogenic response to a high-salt diet. Such observations suggest a previously unrecognized element in Na+ regulation in the CD.

Increased Urinary Adenosine Triphosphate in Patients With Bladder Outlet Obstruction Due to Benign Prostate Hyperplasia.

BACKGROUND: Diagnosis of bladder outflow obstruction (BOO) in patients with lower urinary tract (LUT) symptoms is challenging without using invasive urodynamic tests. Recently, we showed in vitro that urothelial strips from patients with benign prostatic hyperplasia (BPH) release more ATP than controls. Here, we tested whether urinary ATP can be used as a wall tension transducer non-invasive biomarker to detect BOO in patients with BPH. METHODS: 79 male patients with BOO and 22 asymptomatic controls were recruited prospectively. Patients were asked to complete the International Prostate Symptom Score (IPSS) questionnaire and to void at normal desire into a urinary flowmeter; the postvoid residual volume was determined by suprapubic ultrasonography. Urine samples from all individuals were examined for ATP, creatinine, and lactate dehydrogenase. RESULTS: BOO patients had significantly higher (P < 0.001) urinary ATP normalized by the voided volume (456 ± 36 nmol) than age-matched controls (209 ± 35 nmol). Urinary ATP amounts increased with the voided volume, but the slope of this rise was higher in BOO patients than in controls. A negative correlation was detected between urinary ATP and flow rate parameters, namely maximal flow rate (r = -0.310, P = 0.005), Siroky flow-volume normalization (r = -0.324, P = 0.004), and volume-normalized flow rate index (r = -0.320, P = 0.012). We found no correlation with LUT symptoms IPSS score. Areas under the receiver operator characteristics (ROC) curves were 0.91 (95%CI 0.86-0.96, P < 0.001) for ATP alone and 0.88 (95%CI 0.81-0.94, P < 0,001) when adjusted to urinary creatinine. CONCLUSIONS: Patients with BOO release higher amounts of ATP into the urine than the control group. The high area under the ROC curve suggests that urinary ATP can be a high-sensitive non-invasive biomarker of BOO, which may have a discriminative value of detrusor competence when comparing BPH patients with low urinary flow rates. Prostate 76:1353-1363, 2016. © 2016 Wiley Periodicals, Inc.

An enzyme-free and label-free fluorescent biosensor for small molecules by G-quadruplex based hybridization chain reaction.

An enzyme-free and label-free fluorescent biosensor is developed by G-quadruplex-based hybridization chain reaction (HCR) for small molecules, using adenosine triphosphate (ATP) as the model. Aptamer probes for the recognition of small molecules are hybridized with blocking probes. The G-quadruplex sequences are incorporated into one of the two HCR hairpin probes. In the presence of small molecules (ATP), the formation of aptamer-ATP bioaffinity complexes induces the release of blocking probes; the released blocking probes initiate HCR and numerous G-quadruplexes along DNA nanowires are self-assembled after the HCR process. Using N-methyl mesoporphyrin IX (NMM) as the fluorophore, a "turn-on" fluorescence response can be achieved and detected as low as 15 µmol L(-1) of ATP. This biosensor is applied to detect ATP in biologic samples with satisfactory results.

Urinary ATP as an indicator of infection and inflammation of the urinary tract in patients with lower urinary tract symptoms.

BACKGROUND: Adenosine-5'-triphosphate (ATP) is a neurotransmitter and inflammatory cytokine implicated in the pathophysiology of lower urinary tract disease. ATP additionally reflects microbial biomass thus has potential as a surrogate marker of urinary tract infection (UTI). The optimum clinical sampling method for ATP urinalysis has not been established. We tested the potential of urinary ATP in the assessment of lower urinary tract symptoms, infection and inflammation, and validated sampling methods for clinical practice. METHODS: A prospective, blinded, cross-sectional observational study of adult patients presenting with lower urinary tract symptoms (LUTS) and asymptomatic controls, was conducted between October 2009 and October 2012. Urinary ATP was assayed by a luciferin-luciferase method, pyuria counted by microscopy of fresh unspun urine and symptoms assessed using validated questionnaires. The sample collection, storage and processing methods were also validated. RESULTS: 75 controls and 340 patients with LUTS were grouped as without pyuria (n = 100), pyuria 1-9 wbc µl(-1) (n = 120) and pyuria ≥10 wbc µl(-1) (n = 120). Urinary ATP was higher in association with female gender, voiding symptoms, pyuria greater than 10 wbc µl(-1) and negative MSU culture. ROC curve analysis showed no evidence of diagnostic test potential. The urinary ATP signal decayed with storage at 23°C but was prevented by immediate freezing at ≤ -20°C, without boric acid preservative and without the need to centrifuge urine prior to freezing. CONCLUSIONS: Urinary ATP may have a role as a research tool but is unconvincing as a surrogate, clinical diagnostic marker.