Morning Void Urine Research Articles

Effect of Avenciguat on Albuminuria in Patients with CKD: Two Randomized Placebo-Controlled Trials.

Avenciguat is a novel, potent soluble guanylate cyclase activator in development for CKD. Two trials investigated avenciguat in diabetic (NCT04750577) and non-diabetic (NCT04736628) CKD. A prespecified pooled analysis of two randomized, double-blind, placebo-controlled trials of identical design. Adults with CKD (eGFR ≥20 and <90 mL/min/1.73 m 2 , urine albumin-to-creatinine ratio [UACR] ≥200 and <3500 mg/g) were randomized to 20 weeks of placebo or avenciguat 1, 2, or 3 mg three times daily (TID; adjunctive to angiotensin-converting-enzyme inhibitor or angiotensin receptor blocker. Primary endpoint: change from baseline in UACR in 10-hour urine at week 20, analyzed per protocol. Secondary endpoint: UACR change from baseline in first morning void urine at week 20. Safety was monitored throughout. Overall 500 patients (mean age 62 years [SD 13]; mean eGFR 44 mL/min/1.73 m 2 [SD 18] and median 10-hour UACR 719 [interquartile range 379-1285] mg/g ) received placebo (n=122) or avenciguat 1 mg (n=125), 2 mg (n=126), or 3 mg (n=127) TID. All 243 patients in study one and 27/261 patients in study two had diabetes. Avenciguat 1, 2, and 3 mg TID reduced UACR in 10-hour and first morning void urine versus placebo throughout the treatment period. At week 20, placebo-corrected geometric mean changes (95% confidence interval) from baseline in UACR in 10-hour urine with avenciguat 1, 2, and 3 mg TID were -15.5% (-26.4, -3.0), -13.2% (-24.6, -0.1), and -21.5% (-31.7, -9.8), respectively, analyzed per protocol. Corresponding changes in first morning void urine were -19.4% (-30.0, -7.3), -15.5% (-26.9, -2.5), and -23.4% (-33.5, -11.8), respectively. Avenciguat was well tolerated, overall frequency of adverse events was low and similar to placebo. The number of patients who discontinued study drug due to adverse events with avenciguat 1, 2, and 3 mg TID were 5 (4%), 11 (9%), and 11 (9%), respectively, compared with 4 (3%) in the placebo group. Avenciguat lowered albuminuria, and was well tolerated in patients with CKD.

Aldosterone synthase inhibitor (BI 690517) therapy for people with diabetes and albuminuric chronic kidney disease: A multicentre, randomized, double-blind, placebo-controlled, Phase I trial.

This Phase I study evaluated the safety and early efficacy of an aldosterone synthase inhibitor (BI 690517) in people with diabetes and albuminuric chronic kidney disease. Double-blind, placebo-controlled study (NCT03165240) at 40 sites across Europe. Eligible participants [estimated glomerular filtration rate ≥20 and <75 ml/min/1.73 m2; urine albumin/creatinine ratio (UACR) ≥200 and <3500 mg/g] were randomized 6:1 to receive once-daily oral BI 690517 3, 10 or 40 mg, or eplerenone 25-50 mg, or placebo, for 28 days. The primary endpoint was the proportion of participants with drug-related adverse events (AEs). Secondary endpoints included changes from baseline in the UACR. Fifty-eight participants were randomized and treated from 27 November 2017 to 16 April 2020 (BI 690517: 3 mg, n = 18; 10 mg, n = 13; 40 mg, n = 14; eplerenone, n = 4; placebo, n = 9) for 28 days. Eight (13.8%) participants experienced drug-related AEs [BI 690517: 3 mg (two of 18); 10 mg (four of 13); 40 mg (two of 14)], most frequently constipation [10 mg (one of 13); 40 mg (one of 14)] and hyperkalaemia [3 mg (one of 18); 10 mg (one of 13)]. Most AEs were mild to moderate; one participant experienced severe hyperkalaemia (serum potassium 6.9 mmol/L; BI 690517 10 mg). UACR responses [≥20% decrease from baseline (first morning void urine) after 28 days] were observed for 80.0% receiving BI 690517 40 mg (eight of 10) versus 37.5% receiving placebo (three of eight). Aldosterone levels were suppressed by BI 690517, but not eplerenone or placebo. BI 690517 was generally well tolerated, reduced plasma aldosterone and may decrease albuminuria in participants with diabetes and albuminuric chronic kidney disease.

Significantly Elevated Urinary OH-PAHs and Oxidative Damage Concentrations Attributable to PM2.5 Exposure: A Panel Study of Preaging Females in Chiang Mai, Thailand

Exposure to ambient particulate matter with an aerodynamic diameter ≤ 2.5 µm (PM2.5) has been associated with oxidative damage. This study investigated the PM2.5-bound polycyclic aromatic hydrocarbon (PAH) exposure levels during elevated PM2.5 levels and the oxidative damage among a preaging females in Chiang Mai, Thailand. The 64 female participants, aged between 50 and 65 years old, were followed for 5 weeks consecutively during the non-haze rainy season as the baseline and haze episodes. The morning void urine sample was collected weekly and analyzed for 8 species of hydroxylated-polycyclic aromatic hydrocarbons (OH-PAHs) and malondialdehyde (MDA). The results showed that the high increase in PM2.5 levels was positively associated with OH-PAH levels. In addition, for every 1 µg/gCre increase in individual urinary OH-PAH concentrations, the MDA concentration increased from 0.073 to 2.071 µg/gCre. Therefore, the present study suggested that detected urinary OH-PAHs were attributable to PM2.5-bound PAH exposure, particularly from inhalation. These study results provide useful information for policy makers and public health agencies to mitigate the chain effects of PM2.5 exposure from haze pollution. Keywords: Air pollution, Hydroxylated-PAHs, Oxidative stress, Urinary biomarkers

Efficacy and safety of aldosterone synthase inhibition with and without empagliflozin for chronic kidney disease: a randomised, controlled, phase 2 trial

Excess aldosterone accelerates chronic kidney disease progression. This phase 2 clinical trial assessed BI 690517, an aldosterone synthase inhibitor, for efficacy, safety, and dose selection. This was a multinational, randomised, controlled, phase 2 trial. People aged 18 years or older with an estimated glomerular filtration rate (eGFR) of 30 to less than 90 mL/min/1·73 m2, a urine albumin to creatinine ratio (UACR) of 200 to less than 5000 mg/g, and serum potassium of 4·8 mmol/L or less, taking an angiotensin-converting enzyme inhibitor or angiotensin receptor blocker, were enrolled. Participants were randomly assigned (1:1) to 8 weeks of empagliflozin or placebo run-in, followed by a second randomisation (1:1:1:1) to 14 weeks of treatment with once per day BI 690517 at doses of 3 mg, 10 mg, or 20 mg, or placebo. Study participants, research coordinators, investigators, and the data coordinating centre were masked to treatment assignment. The primary endpoint was the change in UACR measured in first morning void urine from baseline (second randomisation) to the end of treatment. This study is registered with ClinicalTrials.gov (NCT05182840) and is completed. Between Feb 18 and Dec 30, 2022, of the 714 run-in participants, 586 were randomly assigned to receive BI 690517 or placebo. At baseline, 33% (n=196) were women, 67% (n=390) were men, 42% (n=244) had a racial identity other than White, and mean participant age was 63·8 years (SD 11·3). Mean baseline eGFR was 51·9 mL/min/1·73 m2 (17·7) and median UACR was 426 mg/g (IQR 205 to 889). Percentage change in first morning void UACR from baseline to the end of treatment at week 14 was -3% (95% CI -19 to 17) with placebo, -22% (-36 to -7) with BI 690517 3 mg, -39% (-50 to -26) with BI 690517 10 mg, and -37% (-49 to -22) with BI 690517 20 mg monotherapy. BI 690517 produced similar UACR reductions when added to empagliflozin. Investigator-reported hyperkalaemia occurred in 10% (14/146) of those in the BI 690517 3 mg group, 15% (22/144) in the BI 690517 10 mg group, and 18% (26/146) in the BI 690517 20 mg group, and in 6% (nine of 147) of those receiving placebo, with or without empagliflozin. Most participants with hyperkalaemia did not require intervention (86% [72/84]). Adrenal insufficiency was an adverse event of special interest reported in seven of 436 study participants (2%) receiving BI 690517 and one of 147 participants (1%) receiving matched placebo. No treatment-related deaths occurred during the study. BI 690517 dose-dependently reduced albuminuria with concurrent renin-angiotensin system inhibition and empagliflozin, suggesting an additive efficacy for chronic kidney disease treatment without unexpected safety signals. Boehringer Ingelheim.

Urine andhair drug test results associated with daily consumption ofcodeine-predominant poppy seed food products.

This study examined the urine and hair opiate profiles associated with the daily consumption of presumptive codeine-predominant poppy seed food products. Ten participants consumed one of five food products at breakfast for 10 consecutive days. Baseline urine and hair samples were collected on Day 1. The urine samples were collected 4, 8 and 12 h following poppy seed consumption on Days 1 and 10, and the first morning void urine samples were collected on Days 2-10. A second hair specimen was collected on Day 20 ± 2. Urine drug test results: Three of the food products were associated with opiate-negative urine drug test results at all time points at a 300 ng/mL cut-off. Two of the food products were associated with opiate-positive drug test results at all non-baseline time points at a 300 ng/mL cut-off. Of these, all samples (n = 60) were codeine-positive, and 27 (45%) were morphine-positive. Codeine concentrations exceeded morphine concentrations in every sample and always by multiples. Thirty-nine of the 60 samples (65%) were codeine-positive at a 2,000 ng/mL cut-off, while none of these samples were morphine-positive at this cut-off. None of the 60 samples reached an opiate threshold of 15,000 ng/mL, although one participant produced a maximum codeine concentration of 13,161 ng/mL (13,854 ng/mg creatinine). There was no clear trend toward increasing urinary opiate concentrations over the course of the study. Hair drug test results: The hair samples of two participants produced quantifiable codeine (41 pg/mg and 51 pg/mg), but no sample reached a common reporting threshold of 200 pg/mg for codeine or morphine.

Urinary pesticide mixture patterns and exposure determinants in the adult population from the Netherlands and Switzerland: Application of a suspect screening approach

IntroductionNon-occupational sources of pesticide exposure may include domestic pesticide usage, diet, occupational exposure of household members, and agricultural activities in the residential area. We conducted a study with the ambition to characterize pesticide mixture patterns in a sample of the adult population of the Netherlands and Switzerland, using a suspect screening approach and to identify related exposure determinants. MethodsA total of 105 and 295 adults participated in the Dutch and Swiss studies, respectively. First morning void urine samples were collected and analyzed in the same laboratory. Harmonized questionnaires about personal characteristics, pesticide-related activities, and diet were administered. Detection rates and co-occurrence patterns were calculated to explore internal pesticide exposure patterns. Censored linear and logistic regression models were constructed to investigate the association between exposure and domestic pesticide usage, consumption of homegrown and organic foods, household members’ exposure, and distance to agricultural and forest areas. ResultsFrom the 37 detected biomarkers, 3 (acetamiprid (-CH2), chlorpropham (4-HSA), and flonicamid (-C2HN)) were detected in ≥40% of samples. The most frequent combination of biomarkers (acetamiprid-flonicamid) was detected in 22 (5.5%) samples. Regression models revealed an inverse association between high organic vegetable and fruit consumption and exposure to acetamiprid, chlorpropham, propamocarb (+O), and pyrimethanil (+O + SO3). Within-individual correlations in repeated samples (summer/winter) from the Netherlands were low (≤0.3), and no seasonal differences in average exposures were observed in Switzerland. ConclusionHigh consumption of organic fruit and vegetables was associated with lower pesticide exposure. In the two countries, detection rates and co-occurrence were typically low, and within-person variability was high. Our study results provide an indication for target biomarkers to include in future studies aimed at quantifying urinary exposure levels in European adult populations.

A Syudy on Comparative Evaluation of Four Different Methods to Quantify Proteinuria in Pre-Eclampsia in a Tertiary Care Hospital

Background: Preeclampsia is a progressive, multisystem disorder due to spasm of small blood vessels of all organ systems characterized by new-onset hypertension and end- organ dysfunction in the last half of pregnancy. Objectives: Estimation of proteinuria by sulphosalicylic acid test in women with preeclampsia. Material &amp; Methods: Prospective hospital based clinical study. Study conducted in the Department of Obstetrics and Gynaecology, Murshidabad Medical college, Berhampore, Murshidabad, West Bengal. Sep. 2021 - Aug. 2022. Pregnant women after 20weeks of gestation diagnosed as preeclampsia and admitted in antenatal ward in obstetrics and gynaecology department, Murshidabad Medical college, Berhampore, Murshidabad, West Bengal. Study consisted a total of 100 subjects. Simple Random sampling method. The pregnant women diagnosed as preeclampsia meeting the inclusion and exclusion criteria included in the study. The study comprised of pregnant women who are more than 20 weeks of gestation diagnosed as preeclampsia fulfilling the inclusion and exclusion criteria. Written and informed consent was taken from all pregnant women participating in the study. The pregnant women in the study were subjected to a detailed history and general abdominal and pelvic examination. A first voided morning urine sample was obtained for sulphosalicylic acid test, dipstick test, spot urine PCR. Subsequent urine samples were collected for 24 h, including a next-day first-morning voided sample for the 24-h urine protein estimation. Results: On assessment of the ROC curve to assess the predictability of three methods for spot urine compared against the 24hr urine protein estimation, the AUC for the Sulphosalicylic acid was found to be significantly better (AUC=0.970, p&lt;0.05) compared to the urinary PCR (AUC=0.964, p&lt;0.05) and for urinary dipstick it was AUC=0.784, p&lt;0.05. Conclusion: This study concluded that spot urine sulphosalicylic acid and urine protein creatinine ratio are reliable investigations compared to the dipstick method to assess the proteinuria in hypertensive pregnant women or for diagnosis of pre-eclampsia. So, spot urine Sulphosalicylic acid and urine protein-creatinine ratio can be used for detection of proteinuria in pregnant women with suspected pre-eclampsia with high accuracy, which is more rapid than time consuming 24-hour urine protein estimation

Albuminuria and markers for cardiovascular risk in 12-year-olds from the general Dutch population: a cross-sectional study

In adults, albuminuria represents a risk factor for cardiovascular disease and is associated with hypertension and obesity. Pediatric data from the general population are inconsistent and largely based on randomly collected urine. A possible association between antenatal programming and albuminuria at school age has still to be investigated. The purpose of this study is to assess albuminuria in first morning void urine samples in a population-based pediatric cohort and to investigate cross-sectionally the association with factors related to cardiovascular risk. Moreover, we investigate the possible association of antenatal factors with albuminuria. A first morning void urine sample was collected in the population-based GECKO (Groningen Expert Center for Kids with Obesity) Drenthe cohort at the age of 12 years. We investigated cross-sectionally associations between albuminuria and body mass index (BMI), waist circumference (WC), blood pressure (BP) and antenatal factors. The prevalence of UACR (urinary albumin-creatinine ratio) ≥ 3 mg/mmol was 3.3% (95%CI 2.3–4.2). In a multivariate linear regression model, UAC was negatively associated with z-BMI (β-0.08, p = 0.013) and positively with z-systolic BP (β 0.09, p = 0.006), model significance p = 0.002. UACR was negatively associated with z-BMI (β − 0.13, p < 0.001) and positively with z-diastolic BP (β 0.09, p = 0.003), model significance p = 0.001. Albuminuria was not significantly associated with antenatal factors such as gestational age and standardized birth weight.Conclusions: Albuminuria in first morning void urine in 12-year-olds has a lower prevalence than previously reported by randomly collected samples. A negative association between albuminuria and BMI is confirmed. A positive association with blood pressure, but no association with antenatal factors was found.What is known:• While, in adults, albuminuria is a recognized risk factor for cardiovascular disease and is associated with hypertension and obesity, pediatric data are inconsistent and largely based on randomly collected urine.• A possible association between antenatal programming and albuminuria at school age has still to be investigated.What is new:• In this population study on first morning void urine samples from 12-year-olds of the general population, albuminuria is negatively associated with body mass index, and positively associated with blood pressure, while there is no association with antenatal factors.• The prevalence of albuminuria at 12 years is lower than previously reported in studies based on randomly collected urine samples, probably due to elimination of orthostatic proteinuria.

#4868 MASS-SPECTROMETRY ANALYSIS OF CONGOPHILIC URINE AND ITS DETERGENT RESISTANT FRACTION IN PATIENTS WITH AMYLOID AND NON-AMYLOID NEPHROPATHIES

Abstract Background and Aims Being a well-known intrinsic property of amyloid, сongophilia was recently demonstrated in urine of patients with proteinuria of different etiology including renal amyloidosis (RA) and non-amyloid nephropathies (NANP) [1]. Urine proteins (UPs) responsible for congophilia in RA and NANP are supposed to have another property of amyloid such as resistance to ionic detergents and present in urine as detergent-resistant aggregates (DRA). In the pilot study we performed mass-spectrometry (MS) analysis of congophilic urine samples and its detergent-resistant fraction in patients with RA and NANP to investigate UPs and their specificity to the particular kidney disease. Method We collected first morning void urine samples from patients with RA (n = 4) and NANP (n = 4). Urine congophilia was assessed by Congo red Dot test as described previously [1]. To analyze bulk urine (BU) proteins, 30 μl of supernatant was precipitated with 80% acetone and boiled in 2% sodium dodecyl sulfate (SDS) for 30 minutes. Then 5 μg of UPs was separated in 10% polyacrylamide gel electrophoresis (PAGE) followed by Coomassie blue staining. The ultracentrifugation of 0.5-3 ml of urine at 300,000x g for 16h following by the treatment of precipitate with 3% sarcosyl in phosphate-buffered saline (PBS) for 10 min with subsequent washing of resistant aggregates in PBS were applied to obtain DRA. After boiling in SDS 8 μl of the sample was separated in 10% PAGE. UPs concentration in the DRA was estimated by densitometry regarding to 2 μg of bovine serum albumin. Then 6 μg of UPs was digested with trypsin, followed by purification on silicate (CDS Empore™ C18 Extraction Disks). Prepared samples of BU and DRA were analyzed by electrospray ionization tandem MS. We used MsFragger software to obtain lists of UPs for each sample. Most representative UPs in the sample were selected by the unique spectral count (USC). We considered the protein as having diagnosis-specific potential (DSP) if it was present in every sample of the particular disease group, i.e. AA, AL, NANP in BU or DRA, and was absent in any sample of another disease group. Results The patients had following characteristics: age 51±13 years, 3 male/ 5 female, eGFR = 45 (19; 95) ml/min/1.73 m2; 24 h proteinuria = 6.5 (4.5; 7.9) g. In PAGE analysis, BU proteins appeared to be similar in all 8 samples with 2 major bends in 70 kDa and 50 kDa regions, corresponding to albumin and immunoglobulin (Ig) heavy chains, respectively (Fig. 1A). The amount of protein in DRA was small and comprised 1.02 (0.71; 1.61) % of the total protein in the sample (4.7 (2.3; 5,1) g/l). Compared with BU PAGE analysis of DRA proteins revealed other bends with trace albumin bend, predominance of 45 kDA region bend and more apparent 30 kDa bend (Ig light chains) in the majority of patients (Fig. 1B). Results of MS analysis are shown on the Figure 2. There were more DSP revealed in DRA vs BU: 14 vs 5, 46 vs 2 and 4 vs 2 UPs in samples of AL, AA and NANP, respectively. When compare by a particular disease group, there were no DSP found either in BU or DRA in RA samples. One protein (aminopeptidase N) and 3 proteins (isoform 3 of unconventional myosin-LC, protein S100-A8, elongation factor 1-α 1) were detected as DSP in BU and DRA in AL, respectively. Alpha-1-acid glycoprotein 1 was only DSP for AA in DRA as well as serum paraoxonase 1 for NANP in BU. Venn-diagrams of shared and divergent UPs are present on the Figures 2D-G. Conclusion Although DRA represented a small portion of UPs, its composition significantly differed from BU and could contain more specific disease markers that makes urine detergent-resistant fraction promising for the future research. Understanding the role of DRA proteins in the pathogenesis of amyloid and non-amyloid renal disease and their diagnostic utility requires further studies.

Safety, tolerability, pharmacodynamics and pharmacokinetics of the soluble guanylyl cyclase activator BI 685509 in patients with diabetic kidney disease: A randomized trial.

Albuminuria is associated with abnormalities in the nitric oxide (NO)-soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate pathway. We assessed safety and efficacy of the NO-independent sGC activator BI 685509 in patients with diabetic kidney disease and albuminuria. In this Phase Ib trial (NCT03165227), we randomized patients with type 1 or 2 diabetes, estimated glomerular filtration rate (eGFR) 20-75 mL/min/1.73 m2 and urinary albumin:creatinine ratio (UACR) 200-3500 mg/g to oral BI 685509 (1 mg three times daily, n = 20; 3 mg once daily, n = 19; 3 mg three times daily, n = 20, after final titration) or placebo (n = 15) for 28 days. Changes from baseline in UACR in first morning void (UACRFMV ) and 10-hour (UACR10h ) urine (3 mg once daily/three times daily only) were assessed. Baseline median eGFR and UACR were 47.0 mL/min/1.73 m2 and 641.5 mg/g, respectively. Twelve patients had drug-related adverse events (AEs; 16.2%: BI 685509, n = 9; placebo, n = 3), most frequently hypotension (4.1%: BI 685509, n = 2; placebo, n = 1) and diarrhoea (2.7%: BI 685509, n = 2; placebo, n = 0). Four patients experienced AEs leading to study discontinuation (5.4%: BI 685509, n = 3; placebo, n = 1). Placebo-corrected mean UACRFMV decreased from baseline in the 3-mg once-daily (28.8%, P = 0.23) and three-times-daily groups (10.2%, P = 0.71) and increased in the 1-mg three-times-daily group (6.6%, P = 0.82); changes were not significant. UACR10h decreased by 35.3% (3 mg once daily, P = 0.34) and 56.7% (3 mg three times daily, P = 0.09); ≥50.0% of patients (UACR10h 3 mg once daily/three times daily) responded (≥20% UACR decrease from baseline). BI 685509 was generally well tolerated. Effects on UACR lowering merit further investigation.

The variability of arsenic in blood and urine of humans

BackgroundHumans are exposed to inorganic and organic arsenic. The total arsenic (As) concentration in urine is a commonly used biomarker of exposure. However, little is known about variability of As in biological fluids and the diurnal variation of As excretion. ObjectivesMain objectives were to assess the variability of As in urine, plasma (P-As), whole blood (B-As), and the blood cell fraction (C-As), and to assess diurnal variation of As excretion. MethodsSix urine samples were collected at fixed times during 24 h on two different days around one week apart among 29 men and 31 women. Blood samples were collected when the morning urine samples were delivered. The intra-class correlation coefficient (ICC) was calculated as the ratio of the between-individuals variance to the total observed variance. ResultsGeometric mean (GM) 24 h urinary excretions of As (U-As24 h) were 41 and 39 µg/24 h on the two days of sampling. Concentrations of B-As, P-As and C-As were highly correlated with U-As24 h and As in first void morning urine. No statistically significant differences were observed for the urinary As excretion rate between the different sampling times. A high ICC was observed for As in the cellular blood fraction (0.803), while ICC for first morning urine corrected for creatine was low (0.316). ConclusionsThe study suggests that C-As is the most reliable biomarker for use in exposure assessment of individual exposure. Morning urine samples have low reliability for such use. No apparent diurnal variation was observed in the urinary As excretion rate.

Urine proteomics by mass spectrometry identifies proteins involved in key pathogenic pathways in patients with juvenile dermatomyositis.

To identify and validate biomarkers in JDM patients using a multiplexing tandem mass tag urine proteome profiling approach. First morning void urine samples were collected from JDM patients (n = 20) and healthy control subjects (n = 21) and processed for analysis using a standardized liquid chromatography-tandem mass spectrometry approach. Biomarkers with significantly altered levels were correlated with clinical measures of myositis disease activity and damage. A subset of candidate biomarkers was validated using commercially available ELISA kits. In total, 2348 proteins were detected in the samples, with 275 proteins quantified across all samples. Among the differentially altered proteins, cathepsin D and galectin-3 binding protein were significantly increased in the urine of JDM patients (adjusted P < 0.05), supporting previous findings in myositis patients. These two candidate biomarkers were confirmed with ELISAs. Cathepsin D positively correlated with Myositis Damage Index (r = 0.57, P < 0.05) and negatively correlated with the Childhood Myositis Assessment Scale (r = -0.54, P < 0.05). We also identified novel JDM candidate biomarkers involved with key features of myositis, including extracellular matrix remodelling proteins. This study confirmed the presence of several proteins in the urine of JDM patients that were previously found to be elevated in the blood of myositis patients and identified novel candidate biomarkers that require validation. These results support the use of urine as a source for biomarker development in JDM.

PS-C30-1: URINARY ANGIOTENSINOGEN-MELATONIN RATIO IN GESTATIONAL DIABETES AND PREECLAMPSIA

Objective: A large research portfolio indicates that an activated renal renin-angiotensin system or a deficit on melatonin is associated with several cardiovascular pathologies. In this observational clinical study, we hypothesized that alterations in urinary melatonin or angiotensinogen levels may be altered in two common conditions, preeclampsia and gestational diabetes. Our primary objective was to assess melatonin and angiotensinogen as novel disease biomarkers detectable and quantifiable in the urine of pregnant women with or without pregnancy complications. Design and Methods: This was a concurrent cohort study of pregnant women with selected obstetric pathologies (gestational diabetes, preeclampsia, hypertension and obesity with hypertension). A group of healthy controls was also included. Urinary 6-sulfatoxymelatonin and angiotensinogen were measured by sensitive and specific ELISAs in first morning void urine samples. The patients were included in the cohort consecutively, and the diagnosis was blinded at the level of urine collection. Urinary 6-sulfatoxymelatonin and angiotensinogen levels were investigated in the patients included in the cohort. Results: Urinary levels of angiotensinogen were significantly higher in the gestational diabetes [angiotensinogen/creatinine ratio median (25th, 75th): 0.11 (0.07, 0.18)] and preeclampsia [0.08 (0.06, 0.18)] groups than in those with healthy pregnancy [0.05(0.04, 0.06]; 6-sulfatoxymelatonin levels were significantly lower in the gestational diabetes [ug/h: median (25th, 75th): 0.12(0.08, 0.17)] and preeclampsia [0.12 (0.09, 0.15)] groups than in those with healthy pregnancy [0.20 (0.15, 0.27]. Neither morning void protein/creatinine ratio nor 24-h urine protein estimate were significantly different between the study groups. Conclusion: These results suggest that urinary angiotensinogen levels may indicate an intrarenal RAS activation while melatonin production appears to be defective in gestational diabetes or hypertension. An angiotensinogen/melatonin ratio is suggested as an early biomarker for identification of gestational diabetes or hypertension. This report provides a basis for the potential use of melatonin for the treatment of preeclampsia. A prospective study in a larger number of patients to determine the operative characteristics of these markers as potential diagnostic tests is justified.

Autoimmune gene expression profiling of fingerstick whole blood in Chronic Fatigue Syndrome

BackgroundMyalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating condition that can lead to severe impairment of physical, psychological, cognitive, social, and occupational functions. The cause of ME/CFS remains incompletely understood. There is no clinical diagnostic test for ME/CFS. Although many therapies have been used off-label to manage symptoms of ME/CFS, there are limited, if any, specific therapies or cure for ME/CFS. In this study, we investigated the expression of genes specific to key immune functions, and viral infection status in ME/CFS patients with an aim of identifying biomarkers for characterization and/or treatment of the disease.MethodsIn 2021, one-hundred and sixty-six (166) patients diagnosed with ME/CFS and 83 healthy controls in the US participated in this study via a social media-based application (app). The patients and heathy volunteers consented to the study and provided self-collected finger-stick blood and first morning void urine samples from home. RNA from the fingerstick blood was tested using DxTerity’s 51-gene autoimmune RNA expression panel (AIP). In addition, DNA from the same fingerstick blood sample was extracted to detect viral load of 4 known ME/CFS associated viruses (HHV6, HHV7, CMV and EBV) using a real-time PCR method.ResultsAmong the 166 ME/CFS participants in the study, approximately half (49%) of the ME/CFS patients reported being house-bound or bedridden due to severe symptoms of the disease. From the AIP testing, ME/CFS patients with severe, bedridden conditions displayed significant increases in gene expression of IKZF2, IKZF3, HSPA8, BACH2, ABCE1 and CD3D, as compared to patients with mild to moderate disease conditions. These six aforementioned genes were further upregulated in the 22 bedridden participants who suffer not only from ME/CFS but also from other autoimmune diseases. These genes are involved in T cell, B cell and autoimmunity functions. Furthermore, IKZF3 (Aiolos) and IKZF2 (Helios), and BACH2 have been implicated in other autoimmune diseases such as systemic lupus erythematosus (SLE) and Rheumatoid Arthritis (RA). Among the 240 participants tested with the viral assays, 9 samples showed positive results (including 1 EBV positive and 8 HHV6 positives).ConclusionsOur study indicates that gene expression biomarkers may be used in identifying or differentiating subsets of ME/CFS patients having different levels of disease severity. These gene targets may also represent opportunities for new therapeutic modalities for the treatment of ME/CFS. The use of social media engaged patient recruitment and at-home sample collection represents a novel approach for conducting clinical research which saves cost, time and eliminates travel for office visits.

The dark side of current analytic methods for Bence Jones Proteinuria.

Bence Jones proteinuria (BJP) refers to monoclonal free immunoglobulin light chains detected in urine, deriving from the clonal expansion of plasma cells in the bone marrow in patients with plasma cell dyscrasias, associated with monoclonal gammopathies of uncertain origin. This review summarizes routinely diagnostic procedures to assess BJP highlighting critical steps of pre-analytical, analytical, and post-analytical phases. The best option for BJP detection is the first morning void urine sample and immunofixation electrophoresis detection technique (IFE) the recommended method, with the employment of specific polyvalent antisera. Other qualitative tests for a quick evaluation of BJP are currently available. Densitometric analysis performed on the 24-hour urine is the recommended method to quantify BJP. To overcome the 24-hour collection, it is possible to use morning urine sample and correlate the assessed value of BJP to creatininuria. In addition to the traditional ones, we here reviewed screening methods currently used to avoid false negatives and reduce the time around test (TAT), together with immunochemical quantification methods for increased sensitivity, after checking BJP by IFE. Mass spectrometry emerges as a new challenge in the determination of BJP. The employment of different based-assays methods may be useful for diagnostic purposes to improve the accuracy of BJP monitoring in monoclonal gammopathies.

Long-term stability of several endocrine disruptors in the first morning urine samples and their associations with lifestyle characteristics

Parabens, triclosan (TCS), bisphenols, benzophenones, and phthalates are typical endocrine disruptors (EDs) with short half-lives in the human body. The concentration levels of those EDs in a spot urine sample are frequently used in exposure assessment studies, and the reproducibility of urinary levels of these nonpersistent EDs should be considered. In the present study, we consecutively collected 45-day first morning void (FMV) urine samples, as well as daily questionnaires, in six recruited participants and measured the urinary concentrations of six parabens, TCS, nine bisphenols, five benzophenones, and ten phthalate metabolites by using high-performance liquid chromatography-tandem mass spectrometry. MeP, EtP, PrP, TCS, BPA, BPS, BPF, and most phthalate metabolites were frequently detected (over 62 % of samples). The intraclass correlation coefficients (ICCs) for ED concentrations in FMV urine samples ranged from fair to excellent for MeP (0.683), EtP (0.702), BPA (0.505), BPS (0.908), BPF (0.887), BP-3 (0.712), mMP (0.661), mEP (0.523), mBP (0.500), miBP (0.724), mBzP (0.961) and all metabolites of DEHP (0.867–0.957), whereas they were low for PrP (0.321) and TCS (0.306). After creatinine adjustment, the values of ICCs for most target EDs were increased with mild to significant improvement. The stability of ED concentrations was affected by daily diet (MeP, TCS, BPA, mMP, miBP, mBP and mBzP), food containers (PrP and mECPP), use of personal care products (HMWP metabolites), pharmaceuticals (EtP) and recorded activities (BPS, mEHP, mBzP, mEHHP and mEOHP), as confirmed by a general linear mixed model. Furthermore, extending the FMV sampling period improved the probability of acceptable reproducibility (ICCs > 0.40) of MeP, EtP, BP-3 and mEP concentrations. For BPS, BPF and HMWP metabolite concentrations showed high probabilities (>80 %) of acceptable reproducibility in the last three days, and the increasing sample size slowly improved the ability to discriminate the subjects. The results were exactly the opposite for BPA concentrations.

Post-collection acidification of spot urine sample is not needed before measurement of electrolytes

IntroductionKidney stone formers can have higher oxalate and phosphate salt amounts in their urine than healthy people and we hypothesized that its acidification may be useful. The study aims to compare results of urine concentrations of calcium, magnesium, and inorganic phosphorus in the midstream portion of first voided morning urine samples without (FMU) and with post-collection acidification (FMUa) in kidney stone patients.Materials and methodsThis is a prospective single center study. A total of 138 kidney stone patients with spot urine samples were included in the study. Urine concentrations of calcium, magnesium and inorganic phosphorus were measured with and without post-collection acidification. Acidification was performed by adding 5 µL of 6 mol/L HCl to 1 mL of urine.ResultsThe median age (range) of all participants was 56 (18-87) years. The median paired differences between FMU and FMUa concentrations of calcium, magnesium, and inorganic phosphorus were: - 0.040 mmol/L, 0.035 mmol/L, and 0.060 mmol/L, respectively. They were statistically different: P < 0.001, P < 0.001, P = 0.004, respectively. These differences are not clinically significant because biological variations of these markers are much higher.ConclusionsNo clinically significant differences in urinary calcium, magnesium, and inorganic phosphorus concentrations between FMU and FMUa in patients with kidney stones were found.

FC052: Atrasentan for the Treatment of IGA Nephropathy: Interim Results from the Affinity Study

Abstract BACKGROUND AND AIMS IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the developed world. Up to 40% of patients with IgAN are at risk of progressing to end-stage kidney disease (ESKD), with proteinuria being a strong predictor of disease progression. Treatments that reduce proteinuria in IgAN are accompanied by eGFR preservation and improved kidney outcomes.[1] Endothelin A (ETA) receptor activation is a key driver of proteinuria, inflammation and fibrosis in patients with glomerular diseases.[2] Therefore, ETA receptor blockade has potential to be of therapeutic benefit for patients with proteinuric glomerular diseases, including IgAN. Atrasentan, a potent and selective ETA receptor antagonist that has demonstrated clinically significant reductions in proteinuria and risk of ESKD in a study of over 5300 patients with diabetic kidney disease (DKD), represents a potential therapy to reduce proteinuria and preserve kidney function in patients with IgAN and other glomerular diseases.[3] AFFINITY is a global, phase 2, open-label basket study to evaluate the efficacy and safety of atrasentan in patients with proteinuric glomerular diseases due to IgAN, Alport syndrome, focal segmental glomerulosclerosis and DKD. Here we present interim results from the first 17 patients enrolled in the IgAN cohort of the AFFINITY study, through week 12 of treatment. METHOD Approximately 20 patients will be enrolled in the IgAN cohort. They must have biopsy proven IgAN, be receiving a maximally tolerated and stable dose of renin angiotensin system inhibitor and have a urine protein creatinine ratio [UPCR] between 0.5 and &amp;lt;1.0 g/g from a first morning void urine sample. Patients are treated orally with 0.75 mg atrasentan daily. The primary endpoint is the change in 24-h UPCR from baseline to Week 12. Key exploratory endpoints include changes in eGFR from baseline to Week 56. RESULTS A total of 17 patients have enrolled in the IgAN cohort as of 4 January 2022. A total of 12 and 8 patients have completed visits through Week 6 and Week 12, respectively. The median age is 47 years and 41% are women. The geometric mean (GM) baseline proteinuria is 1.2 g/day, and median eGFR is 41 mL/min/1.73 m2 (Table 1). The 8 patients on treatment through Week 12 had a GM % reduction from baseline in 24-h UPCR of 43.6% [95% confidence interval (95% CI) 29.0–55.2] (Figure 1). Pharmacokinetic data are currently available for nine patients and showed a mean trough plasma atrasentan concentration in the targeted therapeutic range. Adverse events (AE) were observed in nne patients (53%), all mild or moderate in severity, most of which have resolved. One patient experienced an unrelated serious adverse event of traffic accident without long-term injuries. All patients remain on treatment with study drug except for one patient who discontinued drug due to an unrelated AE at week 13. CONCLUSION Treatment of patients with IgAN with atrasentan in addition to standard of care provided a &amp;gt;43% GM reduction in proteinuria after 12 weeks and was well tolerated, strongly supporting a key role of the endothelin pathway in the pathogenesis of IgAN. The ongoing phase 3 ALIGN study for patients with IgAN and proteinuria ≥1g/day will provide further assessment of the proteinuria lowering effects of atrasentan in this high-risk patient population.

Variability in urinary concentrations of primary aromatic amines

Despite their known carcinogenic potential, primary aromatic amines (AAs) continue to be used in various consumer products. Human exposure to AAs is a subject of current concern. Although urinary measurements are used in the assessment of exposure, little is known about within- and between-individual temporal variability in urinary concentrations of AAs. In this study, we determined the concentrations of 30 AAs, nicotine and cotinine in 213 first morning void (FMV) urine samples collected longitudinally for over a five-week period from 15 participants residing in the Albany area of New York State, USA. Eight AAs, namely, aniline, 2-naphthylamine (2-NA), p-cresidine (p-CD), p-toluidine (p-TD), o/m-toluidine (o/m-TD), 4-chloroaniline (4-CA), 4,4′-methylenedi-o-toluidine (4,4’-MDA), and 2,6-dimethylaniline (2,6-DMA) were found in urine at a detection frequency (DF) in the range of 68–100%. Aniline and 2,6-DMA were the predominant compounds found at median concentrations of 6.0 and 3.81 ng/mL, respectively. Intraclass correlation coefficients (ICCs) of all urinary AA concentrations, except for 4-CA, showed moderate to poor predictability (ICC values ranged 0.248–0.697). Gender and ethnicity-related variations in ∑8AA concentrations were significant. Spearman's correlations among AA concentrations suggested that the sources of exposure were not related to tobacco smoke. No significant correlations existed between AAs concentrations and oxidative stress biomarkers (OSBs). The estimated daily intakes of AAs calculated based on urinary concentrations were several orders of magnitude below the tolerable daily intakes.

Urinary cortisol metabolites are reduced in MDR1 mutant dogs in a pilot targeted GC-MS urinary steroid hormone metabolome analysis.

P-glycoprotein (P-gp) is the gene product of the multidrug resistance gene (MDR1, syn. ABCB1) that normally restricts the transfer of cortisol across the blood-brain barrier. In the absence of P-gp, cortisol access to the hypothalamus is increased and, by feedback inhibition, this finally leads to lower endogenous plasma cortisol levels in dogs with homozygous nt230(del4) MDR1mutation (MDR1-/- mutant dogs). While a previous study only focused on plasma cortisol levels, the present study used urinary steroid hormone metabolites to analyze cortisol metabolism in MDR1-/- mutant dogs. Morning void urine was collected from 23MDR1-/- mutant and 16MDR1+/+ normal dogs and was subjected to targeted GC-MS steroid hormone metabolome analysis. Seven cortisol metabolites, cortisol itself, and 13 other steroid metabolites were detected. In general, all cortisol metabolites were lower in the urine of the MDR1-/- mutant dogs, with allo-tetrahydro-cortisol and β-cortol reaching the level of significance. In addition, 11-keto-pregnanetriol levels were significantly lower in the urine of the MDR1-/- mutant dogs, indicating that also the 17alpha-OH-progesterone-derived metabolism was altered. In conclusion, the present study provides the first steroid hormone metabolome analysis in the urine of MDR1-/- mutant dogs. Significant differences in the steroid metabolome of MDR1-/- mutant dogs point to a significant role of P-gp for cortisol metabolism and excretion and so indirectly also for hypothalamic-pituitary-adrenal axis regulation in dogs.