Urinary Markers Research Articles

Abstract 6962: Shed Trop2 drives prostate cancer progression and Trop2 is a novel tissue prognostic biomarker and a candidate urinary marker for prostate cancer

Abstract Distinguishing indolent from clinically significant localized prostate cancer and treatment of metastatic prostate cancer are two major clinical challenges in prostate cancer. The development of novel predictive biomarkers will help with risk stratification, and influence clinical decision-making between treatment and active surveillance, leading to a decrease in over or under-treatment of patients with prostate cancer. Here, we report that Trop2, an oncogenic transmembrane surface protein, is a prognostic tissue biomarker for clinically significant prostate cancer by utilizing the Canary Prostate Cancer Tissue Microarray (CPCTA) cohort composed of over 1100 patients from a multi-institutional study. We demonstrate that higher Trop2 expression is correlated with worse clinical features and elevated Trop2 expression at radical prostatectomy predicts worse overall survival in men undergoing radical prostatectomy. Additionally, we detected shed Trop2 in urine from men with clinically significant prostate cancer. We further define the functional role of shed Trop2 on metastasis in prostate cancer and identify that shed Trop2 increases cell migration, invasion, metastatic colonization, and spontaneous metastasis in vitro and in vivo. Proteomic profiling reveals that shed Trop2 modulates a set of proteins associated with invasion, migration, mTOR signaling, and epithelial-to-mesenchymal transition. shed Trop2 binds to EGFR and results in the activation of the EGFR-PI3K-AKT-mTOR pathway in prostate cancer. Our study reveals the new function of shed Trop2 in driving prostate cancer progression and identifies Trop2 as a novel tissue prognostic biomarker and a candidate non-invasive marker for prostate cancer that could be used to optimize treatment decision-making. Citation Format: Shiqin Liu, Sarah Hawley, Christian Kunder, En-Chi Hsu, Michelle Shen, Merve Aslan, Fernando J. Marques, Chung S. Lee, Abel Bermudez, Lennart Westphalen, Heidi Auman, Lisa F. Newcomb, Daniel W. Lin, Peter S. Nelson, Ziding Feng, Maria S. Tretiakova, Lawrence D. True, Funda Vakar-Lopez, Peter R. Carroll, Jeffry Simko, Martin E. Gleave, Dean A. Troyer, Jesse K. McKenney, Donna Peehl, Sharon J. Pitteri, James D. Brooks, Michael A. Liss, Tanya Stoyanova. Shed Trop2 drives prostate cancer progression and Trop2 is a novel tissue prognostic biomarker and a candidate urinary marker for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6962.

Cross-sectional and longitudinal analyses of urinary extracellular vesicle mRNA markers in urothelial bladder cancer patients

We designed this multi-center prospective study with the following objectives: (1) the cross-sectional validation of extracellular vesicles (EV) mRNA markers to detect urothelial bladder cancer (UBC) before transurethral resection of bladder cancer (TURBT), and (2) the longitudinal validation of EV mRNA markers to monitor non-muscle invasive bladder cancer (NMIBC) recurrence after TURBT. EV mRNA markers evaluated in this study were KRT17, GPRC5A, and SLC2A1 in addition to two additional markers from literatures, MDK and CXCR2, and measured by quantitative RT-PCR with normalization by a reference gene (ALDOB). Diagnostic performances of EV mRNA markers were compared to conventional markers. Regarding the first objective, we confirmed that EV mRNA biomarkers in urine were higher in UBC patients, particularly those with higher stage/grade tumors, than in those without UBC (n = 278 in total) and the diagnostic performance of EV mRNA MDK and KRT17 outperformed conventional biomarkers with AUC 0.760 and 0.730, respectively. Concerning the second objective, we prospectively analyzed the time courses of EV mRNA markers while NMIBC patients (n = 189) (median follow-up 19 months). The expression of EV mRNA KRT17 was significantly high in patients with recurrence, while it gradually decreased over time in those without recurrence (p < 0.01).

Kenneth Dawson Bagshawe. 17 August 1925—27 December 2022

Kenneth Bagshawe was a physician, a scientist and an architect of modern oncology. He was first to show that combination chemotherapy can cure a non-haematological cancer, choriocarcinoma. He demonstrated how a tumour marker in blood and urine can give a quantitative representation of the total amount of tumour, providing a monitor of response to treatment or relapse and an indicator of prognosis. He enhanced understanding and treatment of gestational trophoblastic diseases (GTD), a group of which choriocarcinoma is the most lethal member. The most common form of GTD is hydatidiform mole and he developed a national system for management, resulting in a cure rate of virtually 100%. For choriocarcinoma, the figure is today approaching 98%. His system is used in many countries, and over several decades has contributed to the cure and preservation of fertility in hundreds of thousands of women who previously had little or no hope. He used the same principles to treat testicular and ovarian germ cell tumours, again achieving high levels of cure in patients whose disease had spread and would previously have been fatal. He innovated throughout his career, seeking improved tumour specificity in diagnosis and treatment of GTD and inventing antibody-directed enzyme prodrug therapy for more common cancers. He founded the first department of oncology in the UK and devoted much time to developing the speciality, directing research funding organizations and supporting the development of the speciality worldwide. He was a dedicated, original and hugely productive pioneer of oncology.

Abstract P417: A Cumulative Score Approach to Evaluating the Impact of Five Modifiable Lifestyle Factors on Kidney Function

Background: Data from the Gubbio population study were used to investigate the impact of a cumulative score combining physical activity, intake of alcohol, sodium, potassium and protein on the decline of kidney function. Methods: The Gubbio Study is a longitudinal study on the population of Gubbio, a town in central Italy; it started in the year 1983, and addressed the resident population aged 5 +. Three exams were performed: Exam 1 (baseline) Exam 2 (6-year follow-up) and Exam 3 (20-year follow-up); this analysis is on the 3,039 adult individuals (Age 18+ at baseline) who participated in Exam 2 (1989-92) and Exam 3 (2001-07). Kidney function was estimated as glomerular filtration rate (eGFR) with the CKD-Epi equation (mL/min x 1.73 m 2 ). Habitual physical activity and alcohol intake were assessed by standard questionnaires. Dietary intake of protein, sodium and potassium were measured by urinary markers. Each factor received score 0 for the tertile associated with greater eGFR decline, score 1 for the tertile associated with intermediate decline and score 2 for the tertile associated with greater decline. A cumulative score (CS) was then calculated for each individual and used as main independent variable. Results: After correction to avoid low-n bias, the CS ranged from 1 (low risk for all factors) to 9 (high risk for all factors). The mean was 5+/-2, with skewness = 0.016. Multiple linear regression showed a relation with the annualized decline in eGFR (slope = - 0,027, 95% CI = - 0.039/ - 0.014, p &lt; 0.001). Incidence of low eGFR was higher in the groups with higher CS, ranging from 2% in the group with CS = 1 to 12.9% in the group with CS = 9. Multivariate logistic regression analysis confirmed the correlation (p &lt; 0.001). Conclusion: These data indicate a significant independent association between a score comprising five modifiable lifestyle factors and the long-term decline of kidney function, providing a tool for the quantification of its impact on the long-term incidence of low eGFR.

Inhibition of Interleukin-33 to Reduce Glomerular Endothelial Inflammation in Diabetic Kidney Disease

Inflammation is a significant contributor to cardiorenal morbidity and mortality in diabetic kidney disease (DKD). The pathophysiological mechanisms linking systemic, subacute inflammation and local, kidney injury-initiated immune maladaptation is partially understood. Here, we explored the expression of proinflammatory cytokines in patients with DKD; investigated mouse models of type 1 and type 2 diabetes (T2D); evaluated glomerular signaling invitro; performed post hoc analyses of systemic and urinary markers of inflammation; and initiated a phase 2b clinical study (FRONTIER-1; NCT04170543). Transcriptomic profiling of kidney biopsies from patients with DKD revealed significant glomerular upregulation of interleukin-33 (IL-33). Inhibition of IL-33 signaling reduced glomerular damage and albuminuria in the uninephrectomized db/db mouse model (T2D/DKD). On a cellular level, inhibiting IL-33 improved glomerular endothelial health by decreasing cellular inflammation and reducing release of proinflammatory cytokines. Therefore, FRONTIER-1 was designed to test the safety and efficacy of the IL-33-targeted monoclonal antibody tozorakimab in patients with DKD. So far, 578 patients are enrolled in FRONTIER-1. The baseline inflammation status of participants (N > 146) was assessed in blood and urine. Comparison to independent reference cohorts (N > 200) validated the distribution of urinary tumor necrosis factor receptor 1 (TNFR1) and C-C motif chemokine ligand 2 (CCL2). Treatment with dapagliflozin for 6 weeks did not alter these biomarkers significantly. We show that blocking the IL-33 pathway may mitigate glomerular endothelial inflammation in DKD. The findings from the FRONTIER-1 study will provide valuable insights into the therapeutic potential of IL-33 inhibition in DKD.

Longitudinal assessment of oxidative stress markers and their relationship with exposure to PM2.5 and its bound metals in healthy participants

ObjectiveExposure to ambient PM2.5 and its bound metals poses a risk to health and disease, via, in part, oxidative stress response. A variety of oxidative stress markers have been used as markers of response, but their relevance to environmental exposure remains to be established. We evaluated, longitudinally, a battery of oxidative stress markers and their relationship with the exposure of PM2.5 and its bound metals in a panel of healthy participants. Material and methodsLevels of residence- and personal-based ambient air PM2.5 and its bound metals, as well as of lung function parameters, were assessed in a total of 58 questionnaire-administered healthy never smoker participants (male, 39.7%). Levels of urinary oxidative stress markers, including Nε-(hexanoyl)-lysine (HEL; an early lipid peroxidation product), 4-hydroxynonenal (4-HNE), N7-methylguanine (N7-meG), and 8-hydroxy-2-deoxyguanosine (8-OHdG), plasma antioxidants [superoxide dismutase (SOD) and glutathione peroxidase (GPx), and urinary metals were measured by ELISA, LC-MS, and ICP-MS, respectively. The results of three repeated measurements at two-month intervals were analyzed using the Generalized Estimating Equation (GEE). ResultsAfter adjusting for confounders, residence- and personal-based PM2.5 levels were positively associated with HEL (β = 0.22 and 0.18) and N7-meG (β = 0.39 and 0.13). Significant correlations were observed between personal air PM2.5-Pb and urinary Pb with HEL (β = 0.08 and 0.26). While FVC, FEV1, FEV1/FVC, MMF, and PEFR predicted% were normal, a negative interaction (pollutant*time, P < 0.05) was noted for PM2.5-V, Mn, Co, Ni, Zn, As, and Pb. Additionally, a negative interaction was found for N7-meG (β = −21.35, −18.77, −23.86) and SOD (β = −26.56, −26.18, −16.48) with FEV1, FVC, and PEFR predicted%, respectively. ConclusionThese findings emphasize potential links between environmental exposure, internal dose, and health effects, thereby offering valuable markers for future research on metal exposure, oxidative stress, and health outcomes.

Urinary markers of Mycobacterium tuberculosis and dysbiosis in paediatric tuberculous meningitis cases undergoing treatment

BackgroundThe pathogenesis of tuberculous meningitis (TBM) involves infection by Mycobacterium tuberculosis in the meninges and brain. However, recent studies have shown that the immune response and inflammatory processes triggered by TBM can have significant effects on gut microbiota. Disruptions in the gut microbiome have been linked to various systemic consequences, including altered immunity and metabolic dysregulation. Inflammation caused by TBM, antibiotic treatment, and changes in host immunity can all influence the composition of gut microbes. This complex relationship between TBM and the gut microbiome is of great importance in clinical settings. To gain a deeper understanding of the intricate interactions between TBM and the gut microbiome, we report innovative insights into the development of the disease in response to treatment. Ultimately, this could lead to improved outcomes, management strategies and quality of life for individuals affected by TBM.MethodWe used a targeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) approach to investigate metabolites associated with gut metabolism in paediatric participants by analysing the urine samples collected from a control group (n = 40), and an experimental group (n = 35) with confirmed TBM, which were subdivided into TBM stage 1 (n = 8), stage 2 (n = 11) and stage 3 (n = 16).FindingsOur metabolomics investigation showed that, of the 78 initially selected compounds of microbiome origin, eight unique urinary metabolites were identified: 2-methylbutyrlglycine, 3-hydroxypropionic acid, 3-methylcrotonylglycine, 4-hydroxyhippuric acid, 5-hydroxyindoleacetic acid, 5-hydroxyhexanoic acid, isobutyrylglycine, and phenylacetylglutamine as urinary markers of dysbiosis in TBM.ConclusionThese results – which are supported by previous urinary studies of tuberculosis – highlight the importance of gut metabolism and of identifying corresponding microbial metabolites as novel points for the foundation of improved management of TBM patients.

Sex Differences in Hydration Biomarkers and Test-Retest Reliability Following Passive Dehydration.

This study investigated (a)differences between males and females for changes in serum, tear, and urine osmolality, hematocrit, and urine specific gravity following acute passive dehydration and (b)assessed the reliability of these biomarkers separately for each sex. Fifteen males (age: 26.3 ± 3.5years, body mass: 76 ± 7kg) and 15 females (age: 28.8 ± 6.4years, body mass: 63 ± 7kg) completed a sauna protocol twice (5-28days apart), aiming for 4% body mass loss (BML). Urine, blood, and tear markers were collected pre- and postdehydration, and change scores were calculated. Male BML was significantly greater than that of females in Trial 1 (3.53% ± 0.55% vs. 2.53% ± 0.43%, p < .001) and Trial 2 (3.36% ± 0.66% vs. 2.53% ± 0.44%, p = .01). Despite significant differences in BML, change in hematocrit was the only change marker that displayed a significant difference in Trial 1 (males: 3% ± 1%, females: 2% ± 1%, p = .004) and Trial 2 (males: 3% ± 1%, females: 1% ± 1%, p = .008). Regression analysis showed a significant effect for sex (male) predicting change in hematocrit (β = 0.8, p = .032) and change in serum osmolality (β = -3.3, p = .005) when controlling for BML but not for urinary or tear measures. The intraclass correlation coefficients for females (ICC 2, 1) were highest for change in urine specific gravity (ICC = .62, p = .006) and lowest for change in tear osmolarity (ICC = -.14, p = .689), whereas for males, it was posthematocrit (ICC = .65, p = .003) and post tear osmolarity (ICC = .18, p = .256). Generally, biomarkers showed lower test-retest reliability in males compared with females but, overall, were classified as poor-moderate in both sexes. These findings suggest that the response and reliability of hydration biomarkers are sex specific and highlight the importance of accounting for BML differences.

Prospective evaluation of the RT-PCR based urinary marker Bladder Epicheck® as a diagnostic tool in upper urinary tract tumor.

Upper-tract-urothelial-carcinoma (UTUC) represents 5-10% of all urothelial-neoplasms with increasing incidence in the last decades. Current standard tools for diagnosis of UTUC include cytology, computed tomography (CT) urography and ureterorenoscopy (URS). The aim of this study was to evaluate the impact of Bladder Epicheck® Test as diagnostic tool for UTUC diagnosis and recurrence. Overall, 136 urine samples, selective collected from upper-urinary-tract before URS for suspicion of UTUC were analyzed with cytology and Bladder Epicheck® Test. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of both markers were calculated and compared to URS and/or histology as reference. UTUC was detected in 40 cases (33.3%), among them 30 were classified as low-grade (LG) and 10 as high-grade (HG). Overall sensitivity of Bladder Epicheck® for UTUC detection was 65% compared to 42.5% for cytology, increasing to 100% for Bladder Epicheck® and 90% for cytology if considering only HG tumors. Overall specificity of Bladder Epicheck® was 81.2% and of cytology 93.7%. PPV and NPV were 63.4% and 82.2% for Bladder Epicheck® and 77.2% and 76.5% for cytology. Considering an EpiScore cut-off >75, instead of 60, specificity of Bladder Epicheck® improves to 89% and PPV to 74.2%. Limitations include the use of a marker validated only for bladder-cancer and the relatively small number of cases. Due to its high sensitivity for HG tumors, the Bladder Epicheck® Test can be used in diagnosis and treatment decision-making of UTUC. Furthermore, it could be very useful in follow-up of UTUC, after endoscopic treatment to postpone or avoid unnecessary endoscopic exploration. Even if further studies are needed to validate these findings, Bladder Epicheck® could be a promising clinical tool for detection of UTUC.

Identification of Endothelial Cell Protein C Receptor by Urinary Proteomics as Novel Prognostic Marker in Non-Recovery Kidney Injury.

Acute kidney injury is a common and complex complication that has high morality and the risk for chronic kidney disease among survivors. The accuracy of current AKI biomarkers can be affected by water retention and diuretics. Therefore, we aimed to identify a urinary non-recovery marker of acute kidney injury in patients with acute decompensated heart failure. We used the isobaric tag for relative and absolute quantification technology to find a relevant marker protein that could divide patients into control, acute kidney injury with recovery, and acute kidney injury without recovery groups. An enzyme-linked immunosorbent assay of the endothelial cell protein C receptor (EPCR) was used to verify the results. We found that the EPCR was a usable marker for non-recovery renal failure in our setting with the area under the receiver operating characteristics 0.776 ± 0.065; 95%CI: 0.648-0.905, (p < 0.001). Further validation is needed to explore this possibility in different situations.

Scoring system for diagnosis and pretreatment risk assessment of neuroblastoma using urinary biomarker combinations.

The urinary catecholamine metabolites, homovanillic acid (HVA) and vanillylmandelic acid (VMA), are used for the adjunctive diagnosis of neuroblastomas. We aimed to develop a scoring system for the diagnosis and pretreatment risk assessment of neuroblastoma, incorporating age and other urinary catecholamine metabolite combinations. Urine samples from 227 controls (227 samples) and 68 patients with neuroblastoma (228 samples) were evaluated. First, the catecholamine metabolites vanillactic acid (VLA) and 3-methoxytyramine sulfate (MTS) were identified as urinary marker candidates through comprehensive analysis using liquid chromatography-mass spectrometry. The concentrations of these marker candidates and conventional markers were then compared among controls, patients, and numerous risk groups to develop a scoring system. Participants were classified into four groups: control, low risk, intermediate risk, and high risk, and the proportional odds model was fitted using the L2-penalized maximum likelihood method, incorporating age on a monthly scale for adjustment. This scoring model using the novel urine catecholamine metabolite combinations, VLA and MTS, had greater area under the curve values than the model using HVA and VMA for diagnosis (0.978 vs. 0.964), pretreatment risk assessment (low and intermediate risk vs. high risk: 0.866 vs. 0.724; low risk vs. intermediate and high risk: 0.871 vs. 0.680), and prognostic factors (MYCN status: 0.741 vs. 0.369, histology: 0.932 vs. 0.747). The new system also had greater accuracy in detecting missing high-risk neuroblastomas, and in predicting the pretreatment risk at the time of screening. The new scoring system employing VLA and MTS has the potential to replace the conventional adjunctive diagnostic method using HVA and VMA.

Effect of urine alkalization on urinary inflammatory markers in cystinuric patients.

Cystinuria is associated with a high prevalence of chronic kidney disease (CKD). We previously described a urinary inflammatory-protein signature (UIS), including 38 upregulated proteins, in cystinuric patients (Cys-patients), compared with healthy controls (HC). This UIS was higher in Cys-patients with CKD. In the present observational study, we aimed to investigate the UIS in Cys-patients without CKD and patients with calcium nephrolithiasis (Lith-patients), versus HC and the effect of urine alkalization on the UIS of Cys-patients. UIS was evaluated by nano-liquid chromatography coupled to high-resolution mass spectrometry in adult HC, Lith-patients and non-treated Cys-patients with an estimated glomerular filtration rate >60mL/min/1.73 m2, and after a 3-month conventional alkalizing treatment in Cys-patients. Twenty-one Cys-patients [12 men, median age (interquartile range) 30.0 (25.0-44.0) years], 12 Lith-patients [8 men, 46.2 (39.5-54.2) years] and 7 HC [2 men, 43.1 (31.0-53.9) years] were included. Among the 38 proteins upregulated in our previous work, 11 proteins were also upregulated in Cys-patients compared with HC in this study (5 circulating inflammatory proteins and 6 neutrophil-derived proteins). This UIS was also found in some Lith-patients. Using this UIS, we identified two subclusters of Cys-patients (5 with a very high/high UIS and 16 with a moderate/low UIS). In the Cys-patients with very high/high UIS, urine alkalization induced a significant decrease in urinary neutrophil-derived proteins. A high UIS is present in some Cys-patients without CKD and decreases under alkalizing treatment. This UIS could be a prognostic marker to predict the evolution towards CKD in cystinuria.

Influential factors on urine EV DNA methylation detection and its diagnostic potential in prostate cancer.

The value of Extracellular vesicles (EVs) diagnostic markers is widely recognized. However, current research on EV DNA remains limited. This study investigates the biological properties, preprocessing factors, and diagnostic potential of EV DNA. We found that DNA positive vesicles account for 23.3% ± 6.7% of the urine total EV, with a large amount of DNA attached to the outside. EV DNA fragments are large, there is no significant effect on uEV DNA when store urine less than 6h at 4°C. In addition, the influence of different EV extraction methods on methylation detection is also minor. More importantly, RASSF1A methylation in urine total EV DNA can distinguish between PCa and BPH, with an AUC of 0.874. Our results suggest the potential of urine EV DNA as a novel marker for PCa diagnosis. This provides a new idea for the study of urinary tumor markers.

Utility of urine dipstick in predicting urinary tract infection in pediatric outpatients

Objectives: Children are most frequently affected by urinary tract infections (UTIs). Urine dipsticks along with use in urine routine testing can help in predicting UTI. In this study we aim to evaluate the utility of individual and combination of urine dipstick parameters, including nitrite and leukocyte esterase (LE), against gold standard urine culture for detection of UTI among pediatric outpatients. Materials and Methods: Nine hundred and forty-nine pediatric urine samples from January 2021 to December 2021 were cultured on Cysteine Lactose Electrolyte Deficient Agar and incubated at 37°C overnight. Siemens Multistix 10SG strips were used to perform a urine dipstick for urine markers. Results from the urine dipstick and culture were contrasted. Statistical Analysis: Data obtained in the study was analyzed statistically using stata version 15. Diagnostic performance of urine dipstick parameters was assessed taking urine culture as gold standard. Receiver operating characteristics (ROC) curve was plotted. Results: About 153 of the 949 urine samples received for culture were positive. Males comprised 102 of the 153 positive patients. The most common organism isolated was Escherichia coli, followed by Klebsiella, Pseudomonas, and Proteus. LE, nitrite, red blood cells (RBC), and protein had a sensitivity of 77.1%, 64.7%, 45.1%, and 46.4%, respectively, whereas their specificities were 94.5%, 97.2%, 95.9%, and 93.1%, respectively. When taken together, “LE and Nitrite and RBC and Protein” had a lower area under the curve (AUC) of 0.641, whereas “LE/Nitrite/RBC/Protein” had the highest AUC of 0.914. Conclusions: The urine dipstick test may be utilized as a screening tool and a positive predictor for UTI. When evaluated simultaneously, nitrite and LE can be used as point-of-care tests preceding the culture examination for decision-making. They can aid in the optimized management of children by minimizing UTI-related complications and inappropriate antibiotic use.

Oxygen Vacancies-Induced Antifouling Photoelectrochemical Aptasensor for Highly Sensitive and Selective Determination of α-Fetoprotein.

Accurate measurement of cancer markers in urine is a convenient method for tumor monitoring. However, the concentration of cancer markers in urine is so low that it is difficult to achieve their measurement. Photoelectrochemical (PEC) sensors are a promising technology to realize the detection of trace cancer markers due to their high sensitivity. Currently, the interference of nonspecific biomolecules in urine is the main reason affecting the high sensitivity and selectivity of PEC sensors in detecting cancer markers. In this work, a strategy of oxygen vacancy (OV) modulation is proposed to construct a fouling-resistant PEC aptamer sensing platform for the detection of α-fetoprotein (AFP), a liver cancer marker. The introduction of OVs induces the formation of intermediate localized states in the photoelectric material, which not only facilitates the separation of photogenerated carriers but also leads to the redshift of the light absorption edge. More importantly, OVs with positive electrical properties can be employed to modify the antifouling layer (C-PEG) with negatively charged groups through an electrostatic interaction. The synergistic effect of OVs, antifouling layer, and aptamer resulted in a TiO2/OVs/C-PEG-based PEC sensor achieves a wide linear range from 1 pg/mL to 100 ng/mL and a low detection limit of 0.3 pg/mL for AFP. In addition, the sensor successfully realized the determination of AFP in urine samples and accurately differentiated between normal people and liver cancer patients in the early and advanced stages. This project is of great significance in advancing the application of photoelectrochemical bioanalytical technology to achieve the detection of cancer markers in urine by investigating the construction of an OVs-regulated fouling-resistant sensing interface.

Rationale and Design of a Phase 2, Double-blind, Placebo-Controlled, Randomized Trial Evaluating AMP Kinase-Activation by Metformin in Focal Segmental Glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS), the most common primary glomerular disease leading to end-stage kidney disease (ESKD), is characterized by podocyte injury and depletion, whereas minimal change disease (MCD) has better outcomes despite podocyte injury. Identifying mechanisms capable of preventing podocytopenia during injury could transform FSGS to an "MCD-like" state. Preclinical data have reported conversion of an MCD-like injury to one with podocytopenia and FSGS by inhibition of AMP-kinase (AMPK) in podocytes. Conversely, in FSGS, AMPK-activation using metformin (MF) mitigated podocytopenia and azotemia. Observational studies also support beneficial effects of MF on proteinuria and chronic kidney disease (CKD) outcomes in diabetes. A randomized controlled trial (RCT) to test MF in podocyte injury with FSGS has not yet been conducted. We report the rationale and design of phase 2, double-blind, placebo-controlled RCT evaluating the efficacy and safety of MF as adjunctive therapy in FSGS. By randomizing 30 patients with biopsy-confirmed FSGS to MF or placebo (along with standard immunosuppression), we will study mechanistic biomarkers that correlate with podocyte injury or depletion and evaluate outcomes after 6 months. We specifically integrate novel urine, blood, and tissue markers as surrogates for FSGS progression along with unbiased profiling strategies. Our phase 2 trial will provide insight into the potential efficacy and safety of MF as adjunctive therapy in FSGS-a crucial step to developing a larger phase 3 study. The mechanistic assays here will guide the design of other FSGS trials and contribute to understanding AMPK activation as a potential therapeutic target in FSGS. By repurposing an inexpensive agent, our results will have implications for FSGS treatment in resource-poor settings.

ADB-HEXINACA-a novel synthetic cannabinoid with a hexyl substituent: phase I metabolism in authentic urine samples, a case report and prevalence on the German market.

Synthetic cannabinoid receptor agonists (SCRAs) are one of the largest groups of new psychoactive substances (NPS). Yet, another novel analog started spreading on the NPS market around 2021. Soon after, the substance could be analytically characterized in herbal material as ADB-HEXINACA, an SCRA containing a hexyl-substituted tail on the indazole core. Here, we present suitable urinary markers to prove the consumption of this analog, a case report of acute polydrug intoxication and data on its prevalence in Germany. Anticipated phase I metabolites were detected in 12 authentic urine samples that were collected for abstinence control and analyzed by ultra-performance liquid chromatography coupled to a time-of-flight mass spectrometer (UPLC-qToF-MS). The results of in vivo samples were confirmed by analysis of in vitro incubates with pooled human liver microsomes (pHLMs). Forensic samples were used to assess the prevalence of ADB-HEXINACA. Thirty-two phase I metabolites were detected in the authentic urine samples. The main metabolites resulted from amide hydrolysis in combination with either monohydroxylation or ketone formation at the hexyl moiety (M15 and M26), the monitoring of which is recommended as a proof of consumption. ADB-HEXINACA was detected in 3.5% of SCRA positive urine samples collected for abstinence control in Freiburg up to December 2022 and in 5.5% of the SCRA positive blood/serum samples. The hexyl substituent of ADB-HEXINACA allows for the detection of specific urinary biomarkers suggested as analytical targets to confirm its prior intake. ADB-HEXINACA had a rather low prevalence in Germany, alternating months of higher prevalence with periods of total absence.

Urinary podocyte markers in diabetic kidney disease.

Podocytes are involved in maintaining kidney function and are a major focus of research on diabetic kidney disease (DKD). Urinary biomarkers derived from podocyte fragments and molecules have been proposed for the diagnosis and monitoring of DKD. Various methods have been used to detect intact podocytes and podocyte-derived microvesicles in urine, including centrifugation, visualization, and molecular quantification. Quantification of podocyte-specific protein targets and messenger RNA levels can be performed by Western blotting or enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, respectively. At present, many of these techniques are expensive and labor-intensive, all limiting their widespread use in routine clinical tests. While the potential of urinary podocyte markers for monitoring and risk stratification of DKD has been explored, systematic studies and external validation are lacking in the current literature. Standardization and automation of laboratory methods should be a priority for future research, and the added value of these methods to routine clinical tests should be defined.

Research Progress of New Urine Markers in the Diagnosis of Bladder Cancer

Bladder cancer is the tenth most commonly diagnosed cancer worldwide and poses a great threat to human health. It has a high recurrence rate and requires long-term close monitoring and follow-up after surgery. At present, the most reliable method for the clinical diagnosis of bladder cancer is still cystoscopy and urine exfoliative cytology. However, cystoscopy is an invasive examination, which is often accompanied by complications such as infection, bleeding, pain and discomfort, and is cost ineffective. At the same time, the sensitivity of urine cytology for low-grade tumors is low, and the subjective factors of the examiners have a great impact on the test results. Urinary biomarkers have the advantages of non-invasive, safe, and simple detection, possessing clinical diagnostic value. At present, it has been found that many urine markers show higher sensitivity than urine exfoliative cytology in the detection of bladder cancer, but due to their poor specificity, they are not widely used in clinical practice. Therefore, there is an urgent need to find novel noninvasive and reliable method for the diagnosis of bladder cancer with high specificity and sensitivity. This article reviews the recent research progress of some new urine biomarkers in the diagnosis of bladder cancer.

Fentanyl as a marker of illicit drug use in morphine-positive urine specimens from workplace drug testing.

Total morphine is an important urinary marker of heroin use but can also be present from prescriptions or poppy seed ingestion. In specimens with morphine concentrations consistent with poppy seed ingestion (<4,000 ng/mL), 6-acetylmorphine has served as an important marker of illicit drug use. However, as illicit fentanyl has become increasingly prevalent as a contaminant in the drug supply, fentanyl might be an alternative marker of illicit opioid use instead of or in combination with 6-acetylmorphine. The aim of this study was to quantify opiates, 6-acetylmorphine, fentanyl and fentanyl analogs in 504 morphine-positive (immunoassay 2,000 ng/mL cutoff) urine specimens from workplace drug testing. Almost half (43%) of morphine-positive specimens had morphine concentrations below 4,000 ng/mL, illustrating the need for markers to differentiate illicit drug use. In these specimens, fentanyl (22% co-positivity) was more prevalent than 6-acetylmorphine (12%). Co-positivity of 6-acetylmorphine and semi-synthetic opioids increased with morphine concentration, while fentanyl prevalence did not. In 110 fentanyl-positive specimens, the median norfentanyl concentration (1,520 ng/mL) was 9.6× higher than the median fentanyl concentration (159 ng/mL), illustrating the possibility of using norfentanyl as a urinary marker of fentanyl use. The only fentanyl analog identified was para-fluorofentanyl (n = 50), with results from most specimens consistent with para-fluorofentanyl contamination in illicit fentanyl. The results confirm the use of fentanyl by employees subject to workplace drug testing and highlight the potential of fentanyl and/or norfentanyl as important markers of illicit drug use.