Simple urine storage protocol for extracellular vesicle proteomics compatible with at-home self-sampling

Urinary extracellular vesicles (EVs) have gained increased interest as a biomarker source. Clinical implementation on a daily basis requires protocols that inevitably includes short-term storage of the clinical samples, especially when collected at home. However, little is known about the effect of delayed processing on the urinary EVs concentration and proteome. We evaluated two storage protocols. First, urine stored at 4 °C. Secondly a protocol compatible with at-home collection, in which urine was stored with the preservative EDTA at room temperature (RT). EVs were isolated using the ME-kit (VN96-peptide). For both conditions we explored the effect of storage duration (0, 2, 4 and 8 days) on EV concentration and proteome using EVQuant and data-independent acquisition mass spectrometry, respectively. The urinary EV concentration and proteome was highly stable using both protocols, in terms of protein number and quantitative changes. Furthermore, EDTA does not affect the urinary EV concentration or global proteome. In conclusion, urine can be stored either at 4 °C or with EDTA at RT for up to 8 days without any significant decay in EV concentration or a notable effect on the EV-proteome. These findings open up biomarker studies in urine collected via self-sampling at home.

Pendampingan Usaha Produksi dan Pemasaran Pupuk Organik Cair (POC) Berbasis Urin Manusia Pada Kelompok Usaha Rajawali Desa Wunduwatu Kecamatan Andoolo Kabupaten Konawe Selatan

AbstrakKelompok usaha POC Rajawali Desa Wunduwatu merupak kelompok usaha yang mengolah urin manusia dari warga sekitar sejak tahun 2017. Pengumpulan urin dilakukan dengan cara mengharapkan warga sekitar datang menukarkan urinnya dilokasi usaha ini. Kelompok Rajawali masih menggunakan cara-cara yang konvensional sederhana sehingga tahapan proses produksi belum optimal, demikian pula metode pemasaran masih dari mulut ke mulut warga. Tujuan pendampingan ini guna meningkatkan produktifitas POC dan meningkatkan keuntungan usaha melalui strategi pemasaran yang efektif. Metode pendampingan dilaksanakan melalui pelatihan selama 2 (dua) hari) pada aspek peningkatan produksi dengan metode pembuatan mikroba khusus campuran urin manusia, melalui dan pembuatan 200 buah wadah penyimpanan urin kapasitas 1 liter dan mengurangi waktu penghancuran sabuk kelapa (80%). Pada kegiatan pelatihan teknologi produksi yakni mengurangi waktu produksi melalui penghancuran sabuk kelapa menggunakan mesin parut sabut kelapa. Selanjutnya dilakukan pendampingan modal produksi yakni inisiasi kerjasama donatur keuangan pengembangan modal kerjasama dengan Bank Bahteramas Provinsi Sulawesi Tenggara Cabang Unaaha. Sedangkan pada aspek pemasaran dilakukan pelatihan pendampingan promosi produk, menjajakan langsung produk didistributor/Toko pertanian, pasar tradisional walaupun penjualan dilakukan tidak resmi oleh karena produk ini belum mengantongi ijin edar. Hasil kegiatan pendampingan menunjukkan peningkatan produksi stok urin naik 200 liter/hari) atau naik 40%, kemudian peningkatan produksi POC 600 liter/6 hari atau naik 25% serta hasil kegiatan pendampingan pemasaran keuntungan usaha awalnya omset ini 4-5 juta/bulan setelah kegiatan pendampingan omset meningkat 9 juta/bulan. Hasil kegiatan pendampingan ini mampu pula mengurangi jumlah pengangguran disekitar lokasi mampu menyerap pekerja baru khususnya dari kalangan ibu rumah tangga.. Kata Kunci : Pendampingan, Pelatihan Produksi, Pemasaran, POC. AbstractThe POC Rajawali business group in Wunduwatu Village is a business group that has been processing human urine from local residents since 2017. Urine collection is done by expecting local residents to exchange their urine at this business location. The Rajawali group still uses conventional methods so that the stages of the production process are not optimal, as well as marketing methods are still word of mouth. The purpose of this assistance is to increase POC productivity and increase profits through effective marketing strategies. The mentoring method is carried out through training for 2 (two) days on the aspect of increasing production with the method of making special microbes mixed with human urine, through and making 200 urine storage units with a capacity of 1 liter and reducing coconut belt time (80%). In production technology activities, namely reducing production time through a coconut belt using a coconut grater machine. Furthermore, production capital assistance was carried out, namely the initiation of cooperation with donors for the development of cooperative capital with Bank Bahteramas, Southeast Sulawesi Province, Unaaha Branch. Meanwhile, in the marketing aspect, training on product promotion assistance is carried out, direct selling of products to agricultural distributors/shops, traditional markets even though sales are not official because this product has not yet obtained a distribution permit. The results of the activity show an increase in urine stock production (200 liters/day) or up 40%, then an increase in POC production of 600 liters/6 days or an increase of 25% and the results of this initial marketing assistance activity turn out 4-5 million/month after the mentoring activity increased turnover. 9 million/month. The results of this mentoring activity were able to reduce the number of movements around the location and were able to absorb new workers, especially from housewives.Keywords: Assistance, Production Training, Marketing, POC.

Lateral Preoptic Area Neurons Activated by Angiotensin-(1–7) Increase Intravesical Pressure: A Novel Feature in Central Micturition Control

Central micturition control and urine storage involve a multisynaptic neuronal circuit for the efferent control of the urinary bladder. Electrical stimulation of the lateral preoptic area (LPA) at the level of the decussation of the anterior commissure in cats evokes relaxation of the bladder, whereas ventral stimulation of LPA evokes vigorous contraction. Endogenous Angiotensin-(1–7) [(Ang-(1–7)] synthesis depends on ACE-2, and its actions on binding to Mas receptors, which were found in LPA neurons. We aimed to investigate the Ang-(1–7) actions into the LPA on intravesical pressure (IP) and cardiovascular parameters. The gene and protein expressions of Mas receptors and ACE-2 were also evaluated in the LPA. Angiotensin-(1–7) (5 nmol/μL) or A-779 (Mas receptor antagonist, 50 nmol/μL) was injected into the LPA in anesthetized female Wistar rats; and the IP, mean arterial pressure (MAP), heart rate (HR), and renal conductance (RC) were recorded for 30 min. Unilateral injection of Ang-(1–7) into the LPA increased IP (187.46 ± 37.23%) with peak response at ∼23–25-min post-injection and yielded no changes in MAP, HR, and RC. Unilateral or bilateral injections of A-779 into the LPA decreased IP (−15.88 ± 2.76 and −27.30 ± 3.40%, respectively) and elicited no changes in MAP, HR, and RC. The genes and the protein expression of Mas receptors and ACE-2 were found in the LPA. Therefore, the LPA is an important part of the circuit involved in the urinary bladder control, in which the Ang-(1–7) synthetized into the LPA activates Mas receptors for increasing the IP independent on changes in RC and cardiovascular parameters.

X-ray videocystometry for high-speed monitoring of urinary tract function in mice

Lower urinary tract dysfunction (LUTd) represents a major health care problem with a high, unmet medical need. Design of additional therapies for LUTd requires precise tools to study bladder storage and voiding (dys)function in animal models. We developed videocystometry in mice, combining intravesical pressure measurements with high-speed fluoroscopy of the urinary tract. Videocystometry substantially outperforms current state-of-the-art methods to monitor the urine storage and voiding process, by enabling quantitative analysis of voiding efficiency, urethral flow, vesicoureteral reflux, and the relation between intravesical pressure and flow, in both anesthetized and awake, nonrestrained mice. Using videocystometry, we identified localized bladder wall micromotions correlated with different states of the filling/voiding cycle, revealed an acute effect of TRPV1 channel activation on voiding efficiency, and pinpointed the effects of urethane anesthesia on urine storage and urethral flow. Videocystometry has broad applications, ranging from the elucidation of molecular mechanisms of bladder control to drug development for LUTd.

Solifenacin/Mirabegron Induces an Acute Compliance Increase in the Filling Phase of the Capacity-Reduced Urinary Bladder: A Pressure-Volume Analysis in Rats

Aims: Pressure in the bladder, which is a high compliance organ, is only slightly elevated to a considerable filling volume during storage. Although cystometry off-line offers mean compliance, no protocol is available for real-time assays of the dynamics of bladder compliance, and the potential impact of solifenacin and mirabegron on dynamic bladder compliance has not been established.Methods: Along with constantly infused cystometry, a pressure-volume analysis (PVA) was performed by plotting intra-vesical volume against pressure in Sprague-Dawley rats. The instant compliance was assayed as the slope of the trajectory, and the mean compliance (Cm) was determined by the slope of the line produced by regression of the data points at the end of the first, second, and third quarters of the filling phase.Results: Under a steady-state, the PVA trajectory moved clockwise which shaped coincident enclosed loops with stable compliance. Though administering to naïve animals solifenacin, but not mirabegron (both 1 × 10−5−1 × 10−1 mg/kg, i.a.) decreased the peak pressure, both of these reagents exhibited acute increments in the trajectory slope and Cm of the filling phase in a dose-dependent manner (ED50 = 1.4 × 10−4 and 2.2 × 10−5 mg/kg, respectively). Resembling urine frequency/urgency in OAB patients, the voiding frequency of a capacity-reduced bladder was increased in association with decreased compliance which was ameliorated by both acute solifenacin and mirabegron injections (both 1 × 10−1 mg/kg).Conclusion: In addition to their well-known anti-inotropic/relaxative effects, solifenacin, and mirabegron induce an acute increase in bladder compliance to ameliorate OAB-like syndromes. Together with time-domain cystometry, PVA offers a platform for investigating the physiology/pathophysiology/pharmacology of bladder compliance which is crucial for urine storage.

Dietary glycine improves urine storage symptoms in urology outpatients

Objectives Spinal glycinergic mechanisms inhibit the micturition reflex, and administration of glycine inhibits bladder activity in rats. Therefore, we examined whether dietary glycine would improve storage symptoms in urological outpatients. Methods We enrolled 20 participants (16 men and four women) with an overactive bladder symptom score (OABSS) ≥ 3. All participants took 3 g of glucose (placebo) twice a day for the first four weeks, then 3 g of glycine twice a day for the next four weeks. We evaluated blood pressure, international prostate symptom score (IPSS), nocturia quality of life (N-QOL) score, OABSS, frequency of urination, sleep latency, time to first nighttime void, bladder pain, global self-assessment (GSA) evaluated urinary symptom improvement, and adverse events. Results Glucose administered as a placebo improved urinary frequency, urine force on the IPSS, and five of the 13 items on the N-QOL. However, compared to the results before and after glucose administration, glycine treatment decreased the number of nocturnal voids, urgency, and total score for urine storage items on the IPSS. It also reduced blood pressure and improved IPSS-QOL. For the OABSS, improvements with glycine were noted in the number of nocturnal urinations, urinary urgency, urge incontinence, and total score. For the N-QOL, eight of 13 items, and the total score, improved. The actual number of nighttime urinations, sleep latency, latency to first nighttime urination, bladder pain, and GSA also improved. There were no adverse events. Conclusions Glycine might improve urine storage symptoms, cardiovascular function, pain, and sleep.

Abnormal Degree Centrality in the Right Inferior Frontal Gyrus During Urine Holding in Children With Primary Monosymptomatic Nocturnal Enuresis

Background: Primary monosymptomatic nocturnal enuresis (PMNE) is a common disorder among school-age children. Previous research has suggested that the prefrontal cortex (PFC) is essential to maintain urine storage in bladder control. We hypothesized that children with PMNE have functional deficits in several brain regions, especially the PFC, during urine storage. In this work, we investigated 30 children with PMNE and 28 controls in a state of natural urine holding to evaluate dysfunction in the bladder control network by applying degree centrality (DC) analysis methods based on resting-state functional magnetic resonance imaging. And seed-based functional connectivity (FC) analysis was used to investigate whether the dysfunctional areas exhibited altered FC with other brain regions.Results: Compared with the typical healthy children, the children with PMNE showed increased DC in the right inferior frontal gyrus (IFG). Also, the right IFG showed increased connectivity with the left middle and inferior frontal gyri and the right precuneus extending to the cuneus in the children with PMNE.Conclusion: The children with PMNE showed abnormal neural activity during urine storage and exhibited increased DC in the right IFG and increased connectivity with the left PFC and right precuneus during urine storage. These results suggest that compensatory effects may be associated with the right IFG combined with the precuneus and left PFC working together to maintain high vigilance and improve micturition's inhibition function to preserve the state of urine holding in children with PMNE.

NephroCheck® Quality Test

<b><i>Background:</i></b> The acute kidney injury (AKI) risk score helps detect moderate and severe AKI in the next 12–24 h. However, inappropriate urine collection may impact its results. <b><i>Aim:</i></b> The aim of this study was to evaluate the stability of NephroCheck® after urine storage at different temperatures. <b><i>Methods:</i></b> The urine sample was centrifuged and split into 3 tubes. One was tested as soon as possible by the laboratory. The other 2 samples were frozen at −20 and −80°C, and the NephroCheck® test was performed 8 weeks later. <b><i>Results:</i></b> The mean values of the AKI risk score were 1.19 ± 0.93, 1.15 ± 1.14, and 1.20 ± 1.11 (ng/mL)²/1,000 for fresh urine, −20, and −80°C, respectively (<i>p</i> = 0.70). Spearman’s rank correlation for −20 and −80°C versus immediate processing was strong with a rho of 0.82 and 0.98, respectively. <b><i>Conclusion:</i></b> The AKI risk score was relatively stable. Urine could be collected without dry ice or liquid nitrogen and kept for up to 8 weeks with either −20 or −80°C freezing with stable NephroCheck® results.

Detrusor Underactivity as an HCN-Mediated Failure of Resilience in Aging

Sympathetic relaxation of the bladder wall permits low pressure urine storage and allows central regulation of afferent sensitivity to volume. Impaired regulation of volume sensitivity has been linked to symptoms of underactive bladder and cystometric detrusor underactivity. Hyperpolarization-activated cyclic nucleotide-gated channels (HCN channels) are mediators of sympathomimetic-induced detrusor relaxation in young mouse bladder tissue, however in bladder strips from old female mice, HCN blockade enhanced age-diminished isoproterenol-induced relaxation. We therefore hypothesized that loss of HCN would compromise cystometric function and enhance sympathomimetic responses in old mice. Male HCN1 KO mice (20-22 mo) and their WT littermates underwent pressure-flow cystometry under urethane anesthesia to assess urinary performance at the level of the autonomic nervous system in the absence of cortical control. Following cystometry, bladders were harvested and pharmacomyography was performed on bladder strips to determine tissue-level changes in the absence of CNS input. All mice responded to continuous-fill cystometry by establishing regular filling/voiding cycles. HCN KO mice function showed discrete changes in volume sensitivity vs. WT. Bladder strip studies showed minimal response to isoproterenol regardless of HCN status, and no significant differences in response to carbachol based on HCN status. We conclude that HCN status impacts the brainstem-bladder reflexic control over urine storage/voiding, but not by regulating bladder wall tensions during filling. The absence of HCN influence on the loss of end-organ responsiveness to sympathetic control in old mice points to an increasing dependency on central control mechanisms with aging.