Excreted urine,bladder urine,and the delay of sexual maturation in female house mice

A series of experiments tested effects of excreted and bladder urine of group- and singly-caged female house mice (Mus musculus) on sexual maturation of young female mice caged alone or in groups of eight. Puberty was determined by vaginal smears. A maturation-delaying pherompne was present in bladder urine of all female mice and excreted urine of group-caged females, but not in excreted urine from singly-caged females. Test mice treated with bladder urine homogenized with urethras from singly-caged females matured at the same age as controls. Apparently the urethras or associated glands of singly-caged females produce a substance which deactivates the maturation-delaying pheromone contained in bladder urine.     

Functional and Molecular Characterization of Hyposensitive Underactive Bladder Tissue and Urine in Streptozotocin-Induced Diabetic Rat

Background

The functional and molecular alterations of nerve growth factor (NGF) and Prostaglandin E2 (PGE2) and its receptors were studied in bladder and urine in streptozotocin (STZ)-induced diabetic rats.

Methodology/Principal Findings

Diabetes mellitus was induced with a single dose of 45 mg/kg STZ Intraperitoneally (i.p) in female Sprague-Dawley rats. Continuous cystometrogram were performed on control rats and STZ treated rats at week 4 or 12 under urethane anesthesia. Bladder was then harvested for histology, expression of EP receptors and NGF by western blotting, PGE2 levels by ELISA, and detection of apoptosis by TUNEL staining. In addition, 4-hr urine was collected from all groups for urine levels of PGE2, and NGF assay. DM induced progressive increase of bladder weight, urine production, intercontraction interval (ICI) and residual urine in a time dependent fashion. Upregulation of Prostaglandin E receptor (EP)1 and EP3 receptors and downregulation of NGF expression, increase in urine NGF and decrease levels of urine PGE2 at week 12 was observed. The decrease in ICI by intravesical instillation of PGE2 was by 51% in control rats and 31.4% in DM group at week 12.

Conclusions/Significance

DM induced hyposensitive underactive bladder which is characterized by increased inflammatory reaction, apoptosis, urine NGF levels, upregulation of EP1 and EP3 receptors and decreased bladder NGF and urine PGE2. The data suggest that EP3 receptor are potential targets in the treatment of diabetes induced underactive bladder.     

Free DNA in urine: a new marker for bladder cancer? Preliminary data

The aim of the present preliminary study was to investigate the presence of free DNA (FDNA) in urine as a possible marker for the diagnosis of bladder cancer. Naturally voided morning urine specimens were collected from 57 patients with suspected bladder cancer before cystoscopy. A standard urine test was performed; the specimens were then processed in order to obtain a quantitative evaluation of the presence of free DNA in the urine. Twenty-two patients were excluded from the study because they had leukocyturia and/or bacteriuria. Free DNA concentrations higher than 250 ng/mL were found in all 16 patients showing bladder cancer at cystoscopy and in seven (36.8%) of the 19 patients with negative cystoscopy. Urinary FDNA seems to have an excellent sensitivity: we observed no false negative cases and 36.8% false positive cases. By contrast, only 6.25% of the bladder cancer patients had positive urine cytology. Our results seem promising, although further studies and larger numbers are needed to define urinary free DNA as a reliable marker of bladder cancer.     

Structural and ultrastructural features of the inlet and outlet regions of the urinary bladders of the leech,Hirudo medicinalis L.

Each of the 34 nephridia in the leech, Hirudo medicinalis, has its own separate bladder. Urine flows from the last portion of the nephridium, the final canal, into the bladder through a special inlet which prevents backflow of urine. This inlet consists of a vestibule formed by two serially arranged septa, each with a small pore. As no muscles or cilia are associated with either the nephridia or the bladder inlet, urine flow into the bladder is passive. Urine leaves the bladder through an outlet that consists of a urethra with sphincters at both ends and an opening, the nephridiopore, in the ventral skin. The sphincter muscles are distinct from the body wall muscles and receive double innervation: urine retention and release is therefore active.     

Water economy in a terrestrial toad (Bufo bufo), with special reference to cutaneous drinking and urinary bladder function

Undisturbed toads, acclimated to a simulated terrestrial habitat with access to water, generally visited the water resource for cutaneous drinking before evaporative water losses had resulted in dehydration of the body, and often the bladder still contained ample amounts of urine. The toads did not urinate when they stayed out of water, but exposure to water in the terrestrially-acclimated state facilitated urination, even when the bladder contained only insignificant amounts of urine. Daily emptying of the bladder often resulted in substantial water deficits prior to drinking, but the severity and frequency of the deficits declined with time, concurrently with an increase in the frequency of cutaneous drinking. Volumes of urine stored in the bladder when the toads suspended cutaneous drinking varied from negligible to large, corresponding to up to 20% of the body mass. Daily emptying of the bladder tended to increase the volume of urine stored at the end of drinking episodes. It is concluded that toads and other terrestrial amphibians primarily maintain normal water balance by anticipatory cutaneous drinking; emergency drinking in response to dehydration plays a secondary role.     

miR-126 在膀胱癌患者尿液中的表达及临床意义

目的:探讨miR-126在膀胱癌患者尿液中的表达与临床病理特征的关系,评估miR-126的肿瘤标志物诊断价值。方法:收集48例初发膀胱尿路上皮癌患者与32例健康对照者晨尿,提取尿液总RNA,通过实时荧光定量PCR技术检测各样本中的miR-126的表达水平,并经受试者工作曲线(ROC)分析其诊断价值。结果:膀胱癌患者尿液中的miR-126表达水平相对健康对照组明显上调(P0.01),其表达水平在不同病理级别之间存在显著差异(P均0.05),且低级别组表达水平略高于高级别组,与肿瘤大小、数目以及淋巴转移也有一定的相关性(P0.05),而与患者的年龄、性别、TNM分期等均无相关性(P0.05)。通过ROC曲线分析尿液中miR-126诊断膀胱肿瘤的曲线下面积(AUC)为0.861,当最佳切点定在7.475时,miR-126诊断膀胱肿瘤的敏感性和特异性分别为75.0%、81.2%。结论:膀胱癌患者尿液中miR-126的表达差异能够反映病情进展程度,其表达水平对膀胱肿瘤的早期诊断及病情评估具有一定的价值。     

近红外荧光染料IR-783在膀胱癌中的成像研究

目的:观察研究近红外荧光染料IR-783在膀胱癌中的特异性成像。方法:通过染料与膀胱癌细胞及正常细胞共孵育,观察近红外荧光染料IR-783是否能够实现膀胱癌细胞的选择性成像。利用细胞器示踪剂观察近红外荧光染料在膀胱癌细胞内的共定位;使用IR-783检测循环血液中及尿液中的膀胱癌细胞。结果:近红外荧光染料IR-783可被膀胱癌细胞选择性摄取。IR-783可选择性聚集在膜性细胞器如线粒体和溶酶体内,这种选择性聚集作用使IR-783可以保持较长的染色效果。近红外染料可以检测到血液或尿液极少量的膀胱癌细胞。结论:近红外荧光染料IR-783能够被膀胱癌细胞特异性吸收,可用于血液和尿液中膀胱肿瘤细胞的特异性诊断,具有重要的临床应用前景。     

The development of the bladder trigone, the center of the anti-reflux mechanism

The urinary tract is an outflow system that conducts urine from the kidneys to the bladder via the ureters that propel urine to the bladder via peristalsis. Once in the bladder, the ureteral valve, a mechanism that is not well understood, prevents backflow of urine to the kidney that can cause severe damage and induce end-stage renal disease. The upper and lower urinary tract compartments form independently, connecting at mid-gestation when the ureters move from their primary insertion site in the Wolffian ducts to the trigone, a muscular structure comprising the bladder floor just above the urethra. Precise connections between the ureters and the trigone are crucial for proper function of the ureteral valve mechanism; however, the developmental events underlying these connections and trigone formation are not well understood. According to established models, the trigone develops independently of the bladder, from the ureters, Wolffian ducts or a combination of both; however, these models have not been tested experimentally. Using the Cre-lox recombination system in lineage studies in mice, we find, unexpectedly, that the trigone is formed mostly from bladder smooth muscle with a more minor contribution from the ureter, and that trigone formation depends at least in part on intercalation of ureteral and bladder muscle. These studies suggest that urinary tract development occurs differently than previously thought, providing new insights into the mechanisms underlying normal and abnormal development.     

Non-Proteasomal Urine Activity in Bladder Cancer

Bladder cancer (BC) is the sixth common cancer in the world, characterized by high recurrent rate and poor prognosis. In most cases is asymptomatic and it can take years until symptoms develop. What is more, diagnosed patients need regular re-examinations which are invasive and expensive. Here, we used chromogenic substrates for the qualitative determination of specific activity of urine enzymes in healthy and bladder cancer patients. The peptide ABZ-Met-Lys-Val-Trp-ANB-NH₂ appears at low absorbance at 410 nm. During the hydrolysis, a free ANB-NH₂ is released which has a maximum absorbance at 410 nm. Using the peptide, we identified proteolytic activity in the majority of urine samples collected from patients with diagnosed bladder cancer, while the proteolytic activity in urine samples from healthy volunteers was not detected.     

Proteomic analysis of urinary biomarker candidates for nonmuscle invasive bladder cancer

Nonmuscle invasive tumors of the bladder often recur and thereby bladder cancer patients need regular re-examinations which are invasive, unpleasant, and expensive. A noninvasive and less expensive method, e.g. a urine dipstick test, for monitoring recurrence would thus be advantageous. In this study, the complementary techniques mass spectrometry (MS) and Western blotting (WB)/dot blot (DB) were used to screen the urine samples from bladder cancer patients. High resolving MS was used to analyze and quantify the urinary proteome and 29 proteins had a significantly higher abundance (p<0.05) in bladder cancer samples compared with control urine samples. The increased abundance found in urine from bladder cancer patients compared with controls was confirmed with Western blot for four selected proteins; fibrinogen β chain precursor, apolipoprotein E, α-1-antitrypsin, and leucine-rich α-2-glycoprotein 1. Dot blot analysis of an independent urine sample set pointed out fibrinogen β chain and α-1-antitrypsin as most interesting biomarkers having sensitivity and specificity values in the range of 66-85%. Exploring the Human Protein Atlas (HPA) also revealed that bladder cancer tumors are the likely source of these proteins. They have the potential of being useful in diagnosis, monitoring of recurrence and thus may improve the treatment of bladder tumors, especially nonmuscle invasive tumors.     

Discovery of Apo-A1 as a potential bladder cancer biomarker by urine proteomics and analysis

Bladder cancer is clinically characterized by high recurrent rate and poor prognosis and thereby patients need regular re-examinations which are invasive, unpleasant, and expensive. A noninvasive and less expensive method for detecting and monitoring bladder cancer would thus be advantageous. In this study, by using the two-dimensional electrophoresis (2-DE) approach with subsequent mass spectrometry (MS), we demonstrated the increased expression of apolipoprotein-A1 (Apo-A1) in individual urine from patients with bladder cancer, which was confirmed by Western blot results. A further analysis of the urinary Apo-A1 levels by an enzyme-linked immunosorbent assay yielded results that were consistent with the Western blot, and suggested Apo-A1 could provide diagnostic utility to distinguish patients with bladder cancer from healthy controls at 19.21 ng/ml. Further validation assay in a larger number of urine samples (n = 379) showed that Apo-A1 could be used as a biomarker to diagnosis bladder cancer with a sensitivity and specificity of 89.2% and 84.6% respectively. Moreover, the application of exfoliative urinary cytology in combination with the urine Apo-A1 detection could significantly increased the sensitivity in detecting bladder cancer. Our data showed a significant relationship of expressed Apo-A1 was established between bladder cancer and normal controls. Apo-A1 could be a potential biomarker for the diagnosis of bladder cancer.     

Size-Based Enrichment of Exfoliated Tumor Cells in Urine Increases the Sensitivity for DNA-Based Detection of Bladder Cancer

Bladder cancer is diagnosed by cystoscopy, a costly and invasive procedure that is associated with patient discomfort. Analysis of tumor-specific markers in DNA from sediments of voided urine has the potential for non-invasive detection of bladder cancer; however, the sensitivity is limited by low fractions and small numbers of tumor cells exfoliated into the urine from low-grade tumors. The purpose of this study was to improve the sensitivity for non-invasive detection of bladder cancer by size-based capture and enrichment of tumor cells in urine. In a split-sample set-up, urine from a consecutive series of patients with primary or recurrent bladder tumors (N = 189) was processed by microfiltration using a membrane filter with a defined pore-size, and sedimentation by centrifugation, respectively. DNA from the samples was analyzed for seven bladder tumor-associated methylation markers using MethyLight and pyrosequencing assays. The fraction of tumor-derived DNA was higher in the filter samples than in the corresponding sediments for all markers (p<0.000001). Across all tumor stages, the number of cases positive for one or more markers was 87% in filter samples compared to 80% in the corresponding sediments. The largest increase in sensitivity was achieved in low-grade Ta tumors, with 82 out of 98 cases positive in the filter samples (84%) versus 74 out of 98 in the sediments (75%). Our results show that pre-analytic processing of voided urine by size-based filtration can increase the sensitivity for DNA-based detection of bladder cancer.     

Levels of tissue inhibitor of metalloproteinases 1 in plasma and urine from patients with bladder cancer

AIM: To assess the potential use of plasma and urine levels of tissue inhibitor of metalloproteinases 1 (TIMP-1) in urothelial cancer. METHODS: TIMP-1 levels were determined in urine and plasma from healthy donors (n=26), patients with bacterial bladder infection (n=24), urothelial bladder adenoma (n=3) or adenocarcinoma (n=7). RESULTS: Free and total TIMP-1 in plasma were weakly but significantly correlated with age; urinary TIMP-1 was not. A strong correlation between free and total TIMP-1 in plasma was observed, with an average ratio of 0.85. No correlation between total TIMP-1 in urine and plasma was found (p=0.55). No significant differences in free or total TIMP-1 in plasma were found between healthy individuals, patients with cystitis or bladder cancer (p=0.4). Urinary TIMP-1 levels were significantly increased in patients with cystitis (p=0.001). No apparent differences in TIMP-1 levels were found in patients with bladder cancer at different stages. CONCLUSION: Our previous observation of a weak but significant correlation between plasma TIMP-1 and age was confirmed. Likewise, an association between free and total TIMP-1 in plasma with a ratio of 0.85 was established. No correlation between plasma and urine TIMP-1 was found. Measurement of TIMP-1 in plasma and/or urine is apparently not useful for the identification of bladder cancer.     

6-keto-prostaglandin F1α is not added to urine during passage through the rat urinary bladder

Studies were conducted to determine whether prostaglandins are added to the urine during its passage through the rat urinary blader . Control rats and rats with chronic streptozotocin-induced diabetes were anesthetized with Inactin, 100 mg/kg i.p., and urine was collected simultaneously from both kidneys. Urine from the left kidney was collected directly from the renal pelvis via a ureteral cannula, while urine from the right kidney was collected via a cannula in the urinary bladder. Prostaglandins in the urine were measured by radioimmunoassay. No difference in urinary concentration or rate of excretion of 6-keto-PGF_1α or PGE₂ was seen between ureteral urine and bladder urine from either normal or diabetic rats. The results of this study indicate that there is no intralumenal addition of either 6-keto-PGF_1α or PGE₂ to the urine by the ureteral bladder of rats.     

ELISA定量分析尿液BLCA-1/-4水平诊断膀胱癌的临床价值分析

目的:探讨膀胱癌患者尿液膀胱特异性核基质蛋白(bladder cancer specific nuclear matrix proteins,BLCA)-1/-4水平及其临床应用价值。方法:本研究纳入38例膀胱癌患者、40例正常对照组。采集受试者尿液样本,通过竞争性酶联免疫吸附法(enzymelinked immunosorbent assay,ELISA)定量分析尿液中BLCA-1和BLCA-4的水平,绘制受试者工作曲线,确定cut-off值。结果:膀胱癌患者尿液BLCA-1/-4水平均显著高于对照组(P0.001);当cut-off值取0.859 ng/mL时,BLCA-1诊断膀胱癌的敏感性和特异性分别为71%(27/38)、90%(36/40)。肌层浸润性膀胱癌患者尿液BLCA-1较非肌层浸润性膀胱癌患者水平显著升高(P0.001),但不同分级膀胱癌患者尿液BLCA-4水平无显著差异(P0.05)。高级别膀胱癌患者尿液BLCA-4水平较低级别膀胱癌患者显著升高(P0.05),但不同分期膀胱癌患者尿液BLCA-4水平无显著差异(P0.05)。以cut-off为0.859 ng/mL时,BLCA-1诊断膀胱癌的敏感性和特异性分别为71%(27/38)、90%(36/40)。以cut-off为0.620 ng/mL时,BLCA-4诊断膀胱癌的敏感性和特异性分别为76.3%(29/38)、97.5%(39/40)。联合检测尿液BLCA-1和BLCA-4诊断膀胱癌的敏感性和特异性分别为84.2%(32/38)和100%(40/40),准确度为0.923(77/78),阳性预测值为100%(32/32),阴性预测值为86.9%(40/46)以及YOUDEN指数分别为0.842。结论:膀胱癌患者尿液BLCA-1和BLCA-4水平显著升高,且敏感性和特异性均较高。联合检测尿液BLCA-1和BLCA-4较单一检测用于诊断膀胱癌的临床应用价值更高。     

低频电刺激联合间歇导尿及Motomed运动训练对脊髓损伤致神经源性膀胱患者膀胱内压力及膀胱容量的影响

目的:探讨低频电刺激联合间歇导尿及Motomed运动训练对脊髓损伤致神经源性膀胱患者膀胱内压力及膀胱容量的影响。方法:选取我院2015年12月～2018年2月收治的脊髓损伤致神经源性膀胱患者92例,根据随机数字表法将其分为对照组(n=46)与研究组(n=46)。两组均给予间歇性导尿、盆底肌训练、膀胱功能训练等常规干预,对照组在此基础上采取低频电刺激,研究组于对照组基础上采取Motomed运动训练,两组均干预2个月。比较两组的临床疗效、治疗前及治疗2个月后的排尿情况(日均单次排尿量、日单次最大排尿量、日均排尿次数)、尿动力学情况(最大尿流率、膀胱容量、残余尿量、膀胱内压力)、LUTS(国际下尿路症状评分)及USDS(泌尿症状困扰评分)。结果:治疗后,研究组总有效率(93.48%)显著高于对照组(78.26%)(P0.05);治疗2个月后,两组日均单次排尿量、日单次最大排尿量、日均排尿次数、最大尿流率、膀胱容量、膀胱内压力均较治疗前显著增多,且研究组以上指标均明显高于对照组(P0.05);两组LUTS及USDS分值均较治疗前显著降低,且研究组以上指标均显著低于对照组(P0.05)。结论:间歇导尿联合低频电刺激与Motomed运动训练可有效改善脊髓损伤致神经源性膀胱患者尿动力学状态及排尿情况,增大膀胱容量及膀胱内压力等,缓解下尿路症状及泌尿症状困扰程度,提高治疗效果。     

Proteomic analysis of urinary fibrinogen degradation products in patients with urothelial carcinomas

Despite many years of research efforts and continued progress in the identification of urine markers for detection of bladder cancer, none of the markers described to date has been able to replace cystoscopy and urine cytology, the current gold standards for diagnosis. Here, we present a comprehensive gel-based proteomic study in which we have analyzed the presence and origin of fibrinogen (FG) and its degradation products (FDPs) in the urine of patients with and without urothelial carcinoma (UCs), with the aim of evaluating the potential of two-dimensional (2D) gel FDP profiling for detecting bladder cancer. A total of 151 urine samples collected from patients with Ucs of varying degrees of atypia and stages of invasion were compared with a control group consisting of 34 healthy volunteers with no clinical history of bladder cancer. The level and degree of degradation of FG in the urine were determined by 2D gel Western blotting in combination with enhanced chemilumenscence detection. Elevated levels of urine FG/FDPs were found in 99% of patients bearing superficial tumors, in 97% of the cases with early invasive disease, and in 96% of patients with highly invasive tumors. 2D gel profiling of urine FG/FDPs showed that the FG chains exhibited differential susceptibility to in situ proteolysis, with the α-chain being the most susceptible and the γ-chain the most resistant. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified peptide sequence regions in several of the most representative and common FDPs, which can be of value for producing novel specific antibodies to detect FG/FDPs in the urine. In addition, we present evidence indicating that FG is not produced by normal or malignant urothelium, although it is present both in the stroma of malignant tissue and tumor lesions. Taken together, the data indicate that increased levels of FG/FDPs amounts in the urine are a characteristic feature of bladder cancer, and emphasize the value of 2D gel profiling of urine FG/FDPs for detecting low-grade, noninvasive UCs.     

Detection of bladder cancer using proteomic profiling of urine sediments

We used protein expression profiles to develop a classification rule for the detection and prognostic assessment of bladder cancer in voided urine samples. Using the Ciphergen PBS II ProteinChip Reader, we analyzed the protein profiles of 18 pairs of samples of bladder tumor and adjacent urothelium tissue, a training set of 85 voided urine samples (32 controls and 53 bladder cancer), and a blinded testing set of 68 voided urine samples (33 controls and 35 bladder cancer). Using t-tests, we identified 473 peaks showing significant differential expression across different categories of paired bladder tumor and adjacent urothelial samples compared to normal urothelium. Then the intensities of those 473 peaks were examined in a training set of voided urine samples. Using this approach, we identified 41 protein peaks that were differentially expressed in both sets of samples. The expression pattern of the 41 protein peaks was used to classify the voided urine samples as malignant or benign. This approach yielded a sensitivity and specificity of 59% and 90%, respectively, on the training set and 80% and 100%, respectively, on the testing set. The proteomic classification rule performed with similar accuracy in low- and high-grade bladder carcinomas. In addition, we used hierarchical clustering with all 473 protein peaks on 65 benign voided urine samples, 88 samples from patients with clinically evident bladder cancer, and 127 samples from patients with a history of bladder cancer to classify the samples into Cluster A or B. The tumors in Cluster B were characterized by clinically aggressive behavior with significantly shorter metastasis-free and disease-specific survival.     

6-Keto-prostaglandin F1 alpha is not added to urine during passage through the rat urinary bladder in vivo

Studies were conducted to determine whether prostaglandins are added to the urine during its passage through the rat urinary bladder in vivo. Control rats and rats with chronic streptozotocin-induced diabetes were anesthetized with Inactin, 100 mg/kg i.p., and urine was collected simultaneously from both kidneys. Urine from the left kidney was collected directly from the renal pelvis via a ureteral cannula, while urine from the right kidney was collected via a cannula in the urinary bladder. Prostaglandins in the urine were measured by radioimmunoassay. No difference in urinary concentration or rate of excretion of 6-keto-PGF1 alpha or PGE2 was seen between ureteral urine and bladder urine from either normal or diabetic rats. The results of this study indicate that in vivo there is no intralumenal addition of either 6-keto-PGF1 alpha or PGE2 to the urine by the ureteral bladder of rats.     

Upregulation of macrophage migration inhibitory factor (MIF) and CD74, receptor for MIF, in rat bladder during persistent cyclophosphamide-induced inflammation

The objective of this study was to determine if macrophage migration inhibitory factor (MIF) is upregulated in the bladder during persistent cystitis. MIF is a pro-inflammatory cytokine found pre-formed in the urothelium. Previous findings showed that acute bladder inflammation increased MIF release into the bladder lumen while upregulating MIF and CD74 (MIF receptor) in the bladder. Because the effects of persistent cystitis on MIF and CD74 are not known, MIF and CD74 changes in the bladder were examined after short-term (1-day) or persistent (8-day) cyclophosphamide (CYP)-induced bladder inflammation. Anesthetized male Sprague-Dawley rats received either a single CYP treatment (150 mg/kg, ip; saline, control) and examined 1 day after treatment (short-term), or repeated CYP doses (20-75 mg/ kg, ip; saline, control; every third day for 8 days) and examined after 8 days of treatment (persistent). MIF protein levels in urine and bladder were determined. In addition, Mif, CD74, and cox-2 expression in the bladder was determined. Histology verified cystitis and MIF and CD74 immunoreactivity in the bladder. Repeated CYP doses were decreased to avoid toxicity. Short-term or repeated low CYP doses (40 mg/kg; 8 days) increased urinary MIF and decreased bladder MIF amounts while upregulating bladder Mif and CD74 mRNA expression. Persistent CYP-induced bladder inflammation (even at 40 mg/kg; 8-day treatment) also upregulated other inflammatory cytokines (CCL5, IL-11, iNOS) in the bladder. Short-term and persistent (low dose) CYP cystitis are associated with markedly increased MIF release into the urine and upregulation of Mif and CD74 in bladder. This supports the hypothesis that MIF and CD74 play a significant role in both acute and persistent stages of bladder inflammation.