Copy number gain of ACTN4 is associated with poor prognosis in patients with upper urinary tract urothelial carcinoma.

α-Actinin4 (ACTN4), an isoform of non-muscular α-actinin, is involved in enhancing cell motility and promoting cancer infiltration and metastasis in various cancers. However, information remains limited regarding the pathological significance of ACTN4 expression in upper urinary tract urothelial carcinomas (UUTUCs). We obtained tumor samples from 168 consecutive patients with newly diagnosed UUTUCs (92 with renal pelvic cancers and 76 with ureteral cancers), who were treated with nephroureterectomy or partial ureterectomy, and analyzed the expression of the ACTN4 protein and the amplification of ACTN4 using immunohistochemistry and fluorescence in situ hybridization (FISH), respectively. The median follow-up duration was 65 months. Among 168 cases, 49 (29%) showed ACTN4 protein overexpression and 25 (15%) showed copy number gain (≥4 copies per cell) of ACTN4. The copy number gain of ACTN4 detected using FISH significantly correlated with ACTN4 protein overexpression and several adverse clinicopathological factors, including higher pathological T stage, lymphovascular invasion, lymph node metastasis, positive surgical margin, concomitant subtype histology, and non-papillary gross finding. Cox univariate regression analyses revealed that both copy number gain of ACTN4 and ACTN4 protein overexpression were significant risk factors for extraurothelial recurrence and death (each p < 0.0001), but multivariate analysis revealed that only copy number gain of ACTN4 was an independent risk factor for extraurothelial recurrence and death (p = 0.038 and 0.027, hazard ratio = 2.16 and 2.17, respectively). This is the first study demonstrating the aberrant expression status of ACTN4 in UUTUC and indicating its putative usefulness as a prognostic indicator in patients with UUTUC.

Does the mineral content of tap water correlate with urinary calculus composition?

The association between the mineral content of drinking water and urolithiasis remains elusive. The aim of this study is to investigate whether the mineral composition of tap water correlates with urinary calculus composition. Patients with calculi that underwent biochemical analysis at two urological centres in the North-West of England between November 2015 and December 2020 were included. Calculus composition was reviewed with respect to patient demographics, serum biochemical variables, and water mineral composition data obtained from the local water supply company using patient postcodes. 1711 urinary tract calculi from 1518 patients, living in 87 water supply zones were included. Water sodium concentration was an independent predictor of mixed calcium oxalate/uric acid calculi (OR 1.157, p < 0.001) and a negative independent predictor of calcium oxalate monohydrate (OR 0.896, p = 0.001) and dihydrate (OR 0.742, p = 0.034) calculi. Moreover, the magnesium-to-calcium ratio of tap water was a negative independent predictor of calcium oxalate monohydrate calculi (OR < 0.001, p = < 0.001), while tap water magnesium concentration inversely correlated with the percentage of calcium oxalate within calculi (rs = - 0.054, p = 0.026). Total water hardness did not independently predict calculus type. Many factors are implicated in the formation of urinary calculi. This study is the first to assess calculus composition in relation to tap water mineral content using postcode data on a case-by-case basis. Though total water hardness did not independently predict calculus composition, the interesting findings relating to water sodium and magnesium concentrations are in need of closer scrutiny in larger scale studies.

Comparative functional analysis of the urinary tract microbiome for individuals with or without calcium oxalate calculi.

Individuals with urinary stone disease (USD) exhibit dysbiosis in the urinary tract and the loss of Lactobacillus that promote urinary tract health. However, the microbial metabolic functions that differentiate individuals with USD from healthy individuals are unknown. The objective of the current study was to determine the microbial functions across prokaryotic, viral, fungal, and protozoan domains that are associated with calcium oxalate (CaOx) stone formers through comparative shotgun metagenomics of midstream, voided urine samples for a small number of patients (n = 5 CaOx stone formers, n = 5 healthy controls). Results revealed that CaOx stone formers had reduced levels of genes associated with oxalate metabolism, as well as transmembrane transport, proteolysis, and oxidation-reduction processes. From 17 draft genomes extracted from the data and > 42,000 full length reference genomes, genes enriched in the Control group mapped overwhelming to Lactobacillus crispatus and those associated with CaOx mapped to Pseudomonas aeruginosa and Burkholderia sp. The microbial functions that differentiated the clinical cohorts are associated with known mechanisms of stone formation. While the prokaryotes most differentiated the CaOx and Control groups, a diverse, trans-domain microbiome was apparent. While our sample numbers were small, results corroborate previous studies and suggest specific microbial metabolic pathways in the urinary tract that modulate stone formation. Future studies that target these metabolic pathways as well as the influence of viruses, fungi, and protozoa on urinary tract physiology is warranted.

Dynamics of urinary and respiratory shedding of Severe acute respiratory syndrome virus 2 (SARS-CoV-2) RNA excludes urine as a relevant source of viral transmission.

PURPOSE: To investigate the expression of the receptor protein ACE-2 alongside the urinary tract, urinary shedding and urinary stability of SARS-CoV-2 RNA. METHODS: Immunohistochemical staining was performed on tissue from urological surgery of 10 patients. Further, patients treated for coronavirus disease (COVID-19) at specialized care-units of a university hospital were assessed for detection of SARS-CoV-2 RNA in urinary samples via PCR, disease severity (WHO score), inflammatory response of patients. Finally, the stability of SARS-CoV-2 RNA in urine was analyzed. RESULTS: High ACE-2 expression (3/3) was observed in the tubules of the kidney and prostate glands, moderate expression in urothelial cells of the bladder (0-2/3) and no expression in kidney glomeruli, muscularis of the bladder and stroma of the prostate (0/3). SARS-CoV-2 RNA was detected in 5/199 urine samples from 64 patients. Viral RNA was detected in the first urinary sample of sequential samples. Viral RNA load from other specimen as nasopharyngeal swabs (NPS) or endotracheal aspirates revealed higher levels than from urine. Detection of SARS-CoV-2 RNA in urine was not associated with impaired WHO score (median 5, range 3-8 vs median 4, range 1-8, p = 0.314), peak white blood cell count (median 24.1 × 1000/ml, range 5.19-48.1 versus median 11.9 × 1000/ml, range 2.9-60.3, p = 0.307), peak CRP (median 20.7 mg/dl, 4.2-40.2 versus median 11.9 mg/dl, range 0.1-51.9, p = 0.316) or peak IL-6 levels (median: 1442 ng/ml, range 26.7-3918 versus median 140 ng/ml, range 3.0-11,041, p = 0.099). SARS-CoV-2 RNA was stable under different storage conditions and after freeze-thaw cycles. CONCLUSIONS: SARS-CoV-2 RNA in the urine of COVID-19 patients occurs infrequently. The viral RNA load and dynamics of SARS-CoV-2 RNA shedding suggest no relevant route of transmission through the urinary tract.

Immunohistochemical Localization of Fibrinogen C Domain Containing 1 on Epithelial and Mucosal Surfaces in Human Tissues.

Fibrinogen C domain containing 1 (FIBCD1) is a transmembrane receptor that binds chitin and other acetylated compounds with high affinity. FIBCD1 has previously been shown to be present in the epithelium of the gastrointestinal tract. In the present study, we performed a detailed analysis of normally structured human tissues for the expression of FIBCD1 by quantitative PCR and immunohistochemistry. We find that FIBCD1 is expressed in epithelial cells derived from all three germ layers. Endodermal-derived epithelial cells throughout the gastrointestinal tract and the respiratory system showed high expression of FIBCD1 and also mesodermal-derived cells in the genitourinary system and ectodermal-derived epidermis and sebaceous glands cells expressed FIBCD1. In some columnar epithelial cells, for example, in the salivary gland and gall bladder, the FIBCD1 expression was clearly polarized with strong apical reaction, while other columnar cells, for example, in small and large intestine and in bronchi, the staining was equally strong apically and basolaterally. In keratinocytes in skin, tongue, and oral cavity, the FIBCD1 staining was granular. This expression pattern together with the known binding properties supports that FIBCD1 plays a role in innate immunity in the skin and at mucosal surfaces.

Purification of exosome-like vesicles from urine.

Urinary exosome-like vesicles (ELVs), 20-200nm membrane-bound particles shed by renal epithelium, have been shown to interact with the primary cilia of distant epithelial cells of the nephron. These ELVs are emerging as an important source of protein, mRNA, and miRNA biomarkers to monitor renal disease. However, purification of ELVs is compromised by the presence of large amounts of the urinary protein Tamm-Horsfall Protein (THP). THP molecules oligomerize into long, double-helical strands several microns long. These linear assemblies form a three-dimensional gel which traps and sequesters ELVs in any centrifugation-based protocol. Here, we present a purification protocol that separates ELVs from THP and divides urinary ELVs into three distinct populations.

Immune expression of E-cadherin and α, ß and γ-Catenin adhesion molecules and prognosis for upper urinary tract urothelial carcinomas.

INTRODUCTION: Cell adhesion molecules (CAM) are required for maintaining a normal epithelial phenotype, and abnormalities in CAM expression have been related to cancer progression, including bladder urothelial carcinomas. There is only one study that correlates E-cadherin and Α-, Β- and y-catenin expression with prognosis of upper tract urothelial carcinomas. Our aim is to study the pattern of immune expression of these CAMs in urothelial carcinomas from the renal pelvis and ureter in patients who have been treated surgically. Our goal is to correlate these expression levels and characteristics with well-known prognostic parameters for disease-free survival. MATERIALS AND METHODS: We evaluated specimens from 20 patients with urothelial carcinomas of the renal pelvis and ureter who were treated with nephroureterectomy or ureterectomy between June 1997 and January 2007. CAM expression was evaluated by immunohistochemistry in a tissue microarray and correlated with histopathological characteristics and patient outcomes after a mean follow-up of 55 months. RESULTS: We observed a relationship between E-cadherin expression and disease recurrence. Disease recurrence occurred in 87.5% of patients with strong E-cadherin expression. Only 50.0% of patients with moderate expression and 0% of patients with weak or no expression of E-cadherin had disease recurrence (p = 0.014). There was also a difference in disease-free survival. Patients with strong E-cadherin expression had a mean disease-free survival rate of 49.1 months, compared to 83.9 months for patients with moderate expression (p = 0.011). Additionally, an absence of Α-catenin expression was associated with tumors that were larger than 3 cm (p = 0.003). CONCLUSIONS: We demonstrated for the first time that immune expression of E-cadherin is related to tumor recurrence and disease-free survival rates, and the absence of Α-catenin expression is related to tumor size in upper tract urothelial carcinomas.

Immune expression of E-cadherin and α, β and γ-Catenin adhesion molecules and prognosis for upper urinary tract urothelial carcinomas

INTRODUCTION: Cell adhesion molecules (CAM) are required for maintaining a normal epithelial phenotype, and abnormalities in CAM expression have been related to cancer progression, including bladder urothelial carcinomas. There is only one study that correlates E-cadherin and α-, β- and γ-catenin expression with prognosis of upper tract urothelial carcinomas. Our aim is to study the pattern of immune expression of these CAMs in urothelial carcinomas from the renal pelvis and ureter in patients who have been treated surgically. Our goal is to correlate these expression levels and characteristics with well-known prognostic parameters for disease-free survival. MATERIALS AND METHODS: We evaluated specimens from 20 patients with urothelial carcinomas of the renal pelvis and ureter who were treated with nephroureterectomy or ureterectomy between June 1997 and January 2007. CAM expression was evaluated by immunohistochemistry in a tissue microarray and correlated with histopathological characteristics and patient outcomes after a mean follow-up of 55 months. RESULTS: We observed a relationship between E-cadherin expression and disease recurrence. Disease recurrence occurred in 87.5% of patients with strong E-cadherin expression. Only 50.0% of patients with moderate expression and 0% of patients with weak or no expression of E-cadherin had disease recurrence (p = 0.014). There was also a difference in disease-free survival. Patients with strong E-cadherin expression had a mean disease-free survival rate of 49.1 months, compared to 83.9 months for patients with moderate expression (p = 0.011). Additionally, an absence of α-catenin expression was associated with tumors that were larger than 3 cm (p = 0.003). CONCLUSIONS: We demonstrated for the first time that immune expression of E-cadherin is related to tumor recurrence and disease-free survival rates, and the absence of α-catenin expression is related to tumor size in upper tract urothelial carcinomas.

Human alpha defensin 5 expression in the human kidney and urinary tract.

BACKGROUND: The mechanisms that maintain sterility in the urinary tract are incompletely understood. Recent studies have implicated the importance of antimicrobial peptides (AMP) in protecting the urinary tract from infection. Here, we characterize the expression and relevance of the AMP human alpha-defensin 5 (HD5) in the human kidney and urinary tract in normal and infected subjects. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA isolated from human kidney, ureter, and bladder tissue, we performed quantitative real-time PCR to show that DEFA5, the gene encoding HD5, is constitutively expressed throughout the urinary tract. With pyelonephritis, DEFA5 expression significantly increased in the kidney. Using immunoblot analysis, HD5 production also increased with pyelonephritis. Immunostaining localized HD5 to the urothelium of the bladder and ureter. In the kidney, HD5 was primarily produced in the distal nephron and collecting tubules. Using immunoblot and ELISA assays, HD5 was not routinely detected in non-infected human urine samples while mean urinary HD5 production increased with E.coli urinary tract infection. CONCLUSIONS/SIGNIFICANCE: DEFA5 is expressed throughout the urinary tract in non-infected subjects. Specifically, HD5 is expressed throughout the urothelium of the lower urinary tract and in the collecting tubules of the kidney. With infection, HD5 expression increases in the kidney and levels become detectable in the urine. To our knowledge, our findings represent the first to quantitate HD5 expression and production in the human kidney. Moreover, this is the first report to detect the presence of HD5 in infected urine samples. Our results suggest that HD5 may have an important role in maintaining urinary tract sterility.

Urinary proteomics--a tool for biomarker discovery.

The strong need for the discovery of novel disease markers together with the development of high-throughput techniques that provide highly sensitive analysis of protein content in tissues and bodily fluids, using proteomics, has opened the completely new chapter in biomarker discovery. The detection of biomarkers based on urinary proteome analysis is rapidly advancing and may provide new tools to improve non-invasive diagnostics, prognostics, and therapy enhancement. As a tool for biomarker discovery, urinary proteomics is especially fruitful in the area of early diagnostics and differentiation of renal damage, and it possesses enormous potential for improving and expanding non-invasive cancer diagnostics. An abundance of urinary proteins could provide a wide variety of biomarkers for the diagnosis and follow-up of many systemic diseases as well. This article reviews the utility of urinary proteomics for biomarker discovery from the perspective of clinical application. Despite huge potential and prompt development of urinary proteomics, many challenges are still in front of us. Research effort and financial investment have to be oriented on providing strategies for exceeding current methodological and technical obstacles in a way to ensure the successful validation and implementation of newly discovered urinary biomarkers. The result is expected to be the development of new non-invasive tests and procedures able to guarantee higher efficiency of patient care and provide needed personalized medical approach.

Green tea flavan-3-ols: colonic degradation and urinary excretion of catabolites by humans.

Following the ingestion of green tea, substantial quantities of flavan-3-ols pass from the small to the large intestine (Stalmach et al. Mol. Nutr. Food Res. 2009, 53, S44-S53; Mol. Nutr. Food Res. 2009, doi: 10.1002/mnfr.200900194). To investigate the fate of the flavan-3-ols entering the large intestine, where they are subjected to the action of the colonic microflora, (-)-epicatechin, (-)-epigallocatechin, and (-)-epigallocatechin-3-O-gallate were incubated in vitro with fecal slurries and the production of phenolic acid catabolites was determined by GC-MS. In addition, urinary excretion of phenolic catabolites was investigated over a 24 h period after ingestion of either green tea or water by healthy volunteers with a functioning colon. The green tea was also fed to ileostomists, and 0-24 h urinary excretion of phenolic acid catabolites was monitored. Pathways are proposed for the degradation of green tea flavan-3-ols in the colon and further catabolism of phenolic compounds passing into the circulatory system from the large intestine, prior to urinary excretion in quantities corresponding to ca. 40% of intake compared with ca. 8% absorption of flavan-3-ol methyl, glucuronide, and sulfate metabolites in the small intestine. The data obtained point to the importance of the colonic microflora in the overall bioavailability and potential bioactivity of dietary flavonoids.

Lack of specificity of commercially available antisera against muscarinergic and adrenergic receptors.

Commercially available antisera against five subtypes of muscarinic receptors and nine subtypes of adrenoceptors showed highly distinct immunohistochemical staining patterns in rat ureter and stomach. However, using the M(1-4) muscarinic receptor subtypes and alpha(2B)-, beta(2)-, and beta(3)-adrenoceptors as examples, Western blots with membranes prepared from cell lines stably expressing various subtypes of muscarinic receptors or adrenoceptors revealed that each of the antisera recognized a set of proteins that differed between the cell lines used but lacked specificity for the claimed target receptor. We propose that receptor antibodies need better validation before they can reliably be used.

Pleomorphic extra-renal manifestation of the glomerular podocyte marker podocalyxin in tissues of normal beagle dogs.

Podocalyxin (PC) was initially identified as a major sialoprotein on the apical surface of glomerular podocytes to perform the filtration barrier function. Later, it was reported to be expressed in endothelial cells, megakaryotes/platelets, and hemangioblasts, the common progenitor cells of the hematopoietic and endothelial cells. Recently, increasing numbers of reports have indicated that PC is not merely a molecule restricted at renal glomerulus, angiogenic or hematopoietic system. To further elucidate the expression pattern and address the possible physiological role of PC in adult mammals, we conducted an extensive study by immunohistochemistry and immunofluorescence staining on various tissues of healthy adult beagle dogs. By combinatory usage of two different anti-podocalyxin antibodies recognizing distinct epitopes in PC, we have demonstrated that (1) PC is expressed in renal tubules, mesothelium, myocardium, striated muscles in tongue, esophagus and extraocular region, myoepithelial cells in esophagus and salivary glands, neurons, and ependyma, etc.; (2) there are at least three forms of PC proteins, depending upon the accessibility of two different PC antibodies, expressed in different organs/systems; and (3) a particular form of PC is distributed in a vesicle-like compartment in certain organs/systems, such as the central nervous system.

[Seed loss through the urinary tract and retrieval after prostate seed implant].

This study describes our experience with seed loss and retrieval through the urinary tract following seed implants for prostate cancer, and offers Japanese guidelines for safety and management. Two hundred consecutive patients were analyzed. All patients were preplanned with a modified peripheral loading technique and implanted with a Mick applicator under ultrasound guidance. All patients were instructed to return excreted seeds, if any, to our center. Seed loss occurred in 6% of patients and 0.13% of seeds. Seed loss tended to occur in the early period through either urine or ejaculation.

Identification of kit positive cells in the human urinary tract.

PURPOSE: Analogous to interstitial cells of Cajal in the bowel, functional important networks of interstitial cells could have a role in the complex mechanism of central and peripheral control of urinary tract function. Recently various reports mentioned the presence of interstitial cells in different parts of the urinary tract and in different species. Since important differences among species exist, we performed immunohistochemistry on fresh frozen human tissue to study the presence of interstitial cells in the human urinary tract. MATERIALS AND METHODS: A total of 65 tissue pieces from all levels of the urinary tract were obtained from 44 patients treated at our institution. Tissue was processed for immunohistochemistry immediately after removal. We performed immunohistochemistry for kit, connexin 43 and VRL1/TRPV2. RESULTS: Interstitial cells immunopositive for all 3 antibodies were seen beneath the urothelium and between smooth muscle cells in all tissue pieces with slight topographical differences. CONCLUSIONS: Together with morphological and functional data from other experiments these morphological data suggest that, as in the bowel, networks of interstitial cells might have an important role in the physiology and pathology of the urinary tract. They could be involved in pacemaking or have an integrating role through the modulation of neurotransmission and conduction of electrical impulses. Functional experiments are the next step to study these hypotheses.

Urothelial function reconsidered: a role in urinary protein secretion.

Mammalian bladder epithelium functions as an effective permeability barrier. We demonstrate here that this epithelium can also function as a secretory tissue directly involved in modifying urinary protein composition. Our data indicate that normal bovine urothelium synthesizes, as its major differentiation products, two well-known proteases: tissue-type plasminogen activator and urokinase, as well as a serine protease inhibitor, PP5. Moreover, we demonstrate that the urothelium secretes these proteins in a polarized fashion into the urine via a cAMP- and calcium-regulated pathway. Urinary plasminogen activators of ruminants are therefore urothelium derived rather then kidney derived as in some other species; this heterogeneity may have evolved in response to different physiological or dietary factors. In conjunction with our recent finding that transgenic mouse urothelium can secrete ectopically expressed human growth hormone into the urine, our data establish that normal mammalian urothelium can function not only as a permeability barrier but also as a secretor of urinary proteins that can play physiological or pathological roles in the urinary tract.

Hydrogen peroxide in the human body.

Hydrogen peroxide (H(2)O(2)) is widely regarded as a cytotoxic agent whose levels must be minimized by the action of antioxidant defence enzymes. In fact, H(2)O(2) is poorly reactive in the absence of transition metal ions. Exposure of certain human tissues to H(2)O(2) may be greater than is commonly supposed: substantial amounts of H(2)O(2) can be present in beverages commonly drunk (especially instant coffee), in freshly voided human urine, and in exhaled air. Levels of H(2)O(2) in the human body may be controlled not only by catabolism but also by excretion, and H(2)O(2) could play a role in the regulation of renal function and as an antibacterial agent in the urine. Urinary H(2)O(2) levels are influenced by diet, but under certain conditions might be a valuable biomarker of 'oxidative stress'.

Atypical nephrogenic metaplasia of the urinary tract: a precursor lesion?

BACKGROUND: Nephrogenic metaplasia with cytologic atypia (atypical nephrogenic metaplasia) is occasionally encountered and its biologic potential is uncertain. METHODS: The authors describe 18 cases of atypical nephrogenic metaplasia characterized by the presence of prominent cytologic atypia, including nuclear enlargement, nuclear hyperchromasia, and enlarged nucleoli. DNA ploidy analysis by digital image analysis and immunostaining for high-molecular-weight cytokeratin (34betaE12), cytokeratin 7, cytokeratin 20, carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), p53, and MIB-1 were performed in 9 cases. RESULTS: The mean patient age was 62 years (median, 65 years; range, 39-84 years). The male-to-female ratio was 2.6:1. Two patients had a history of noninvasive papillary urothelial carcinoma. The typical clinical presentation was hematuria (8 patients) and voiding symptoms (5 patients). Cystoscopic findings were suspicious for neoplasm in 7 of 13 cases. The neoplastic cells were positive for high-molecular-weight cytokeratin, cytokeratin 7, and EMA, and were usually negative for cytokeratin 20 and CEA. p53 nuclear accumulation and increased MIB-1 labeling index were seen in 4 cases. DNA ploidy analysis showed aneuploid pattern in 2 of 9 cases. The mean patient follow-up was 3.5 years (range, 0.5-10.6 years); 2 patients had recurrent nephrogenic metaplasia, and the remainder were alive without recurrence or urothelial carcinoma. CONCLUSIONS: Atypical nephrogenic metaplasia is benign; it occasionally displays substantial cytologic abnormalities of no apparent clinical significance. Awareness of the spectrum of cytologic changes within this entity is critical to prevent overdiagnosis of cancer and avoid unnecessary treatment. There is no direct evidence that links atypical nephrogenic metaplasia to cancer.