Pathophysiological Role of K2P Channels in Human Diseases.

The family of two-pore domain potassium (K2P) channels is critically involved in central cellular functions such as ion homeostasis, cell development, and excitability. K2P channels are widely expressed in different human cell types and organs. It is therefore not surprising that aberrant expression and function of K2P channels are related to a spectrum of human diseases, including cancer, autoimmune, CNS, cardiovascular, and urinary tract disorders. Despite homologies in structure, expression, and stimulus, the functional diversity of K2P channels leads to heterogeneous influences on human diseases. The role of individual K2P channels in different disorders depends on expression patterns and modulation in cellular functions. However, an imbalance of potassium homeostasis and action potentials contributes to most disease pathologies. In this review, we provide an overview of current knowledge on the role of K2P channels in human diseases. We look at altered channel expression and function, the potential underlying molecular mechanisms, and prospective research directions in the field of K2P channels.

TRPM8 Channels: Advances in Structural Studies and Pharmacological Modulation.

The transient receptor potential melastatin subtype 8 (TRPM8) is a cold sensor in humans, activated by low temperatures (>10, <28 °C), but also a polymodal ion channel, stimulated by voltage, pressure, cooling compounds (menthol, icilin), and hyperosmolarity. An increased number of experimental results indicate the implication of TRPM8 channels in cold thermal transduction and pain detection, transmission, and maintenance in different tissues and organs. These channels also have a repercussion on different kinds of life-threatening tumors and other pathologies, which include urinary and respiratory tract dysfunctions, dry eye disease, and obesity. This compendium firstly covers newly described papers on the expression of TRPM8 channels and their correlation with pathological states. An overview on the structural knowledge, after cryo-electron microscopy success in solving different TRPM8 structures, as well as some insights obtained from mutagenesis studies, will follow. Most recently described families of TRPM8 modulators are also covered, along with a section of molecules that have reached clinical trials. To finalize, authors provide an outline of the potential prospects in the TRPM8 field.

Urological implications of SARS CoV-19.

INTRODUCTION: The novel coronavirus disease 2019 (COVID-19), pandemic has afflicted > 3.3 million people around the world since December 2019. Though, more than 1000 publications have appeared in scientific journals addressing a plethora of questions, there is a considerable hiatus in understanding of the behavior and natural history of the virus and its impact on urology. Also, a modified approach is the need of hour in taking care of patients as urologists should safeguard their teams, families, and patients. MATERIAL AND METHODS: The authors have used guidelines from USA, Canada, UK, Europe and India for making recommendations to help urologist define their own policies that may have to be fine-tuned on the basis of continued and evolving challenges they would encounter and the local resources at their disposal. RESULTS: COVID-19 do effect genitourinary system from kidney to testis. The authors provide scientific basis to urologists to help identify patients by remote consultation who are likely to be harmed by coming to the hospital, and not to miss those who need hospitalization for diagnostic or therapeutic interventions. There is uncompromised need of specific precautions during surgery to safe guard the surgeon and his team along with the patient. CONCLUSIONS: Urological operations during COVID-19 pandemic should be limited to emergency cases during the acute phase with an exit strategy planned in a staggered manner, based on the scientific risk stratification. Telemedicine (e-clinics or virtual clinics) would help achieve the goal of risk stratification.

Bioinformatics for Renal and Urinary Proteomics: Call for Aggrandization.

The clinical sampling of urine is noninvasive and unrestricted, whereby huge volumes can be easily obtained. This makes urine a valuable resource for the diagnoses of diseases. Urinary and renal proteomics have resulted in considerable progress in kidney-based disease diagnosis through biomarker discovery and treatment. This review summarizes the bioinformatics tools available for this area of proteomics and the milestones reached using these tools in clinical research. The scant research publications and the even more limited bioinformatic tool options available for urinary and renal proteomics are highlighted in this review. The need for more attention and input from bioinformaticians is highlighted, so that progressive achievements and releases can be made. With just a handful of existing tools for renal and urinary proteomic research available, this review identifies a gap worth targeting by protein chemists and bioinformaticians. The probable causes for the lack of enthusiasm in this area are also speculated upon in this review. This is the first review that consolidates the bioinformatics applications specifically for renal and urinary proteomics.

Nerve growth factor-dependent hyperexcitability of capsaicin-sensitive bladder afferent neurones in mice with spinal cord injury.

NEW FINDINGS: What is the central question of this study? Nerve growth factor (NGF) is reportedly a mediator inducing urinary bladder dysfunction. Is NGF directly involved in hyperexcitability of capsaicin-sensitive C-fibre bladder afferent pathways after spinal cord injury (SCI)? What is the main finding and its importance? Neutralization of NGF by anti-NGF antibody treatment reversed the SCI-induced increase in the number of action potentials and the reduction in spike thresholds and A-type K+ current density in mouse capsaicin-sensitive bladder afferent neurones. Thus, NGF plays an important and direct role in hyperexcitability of capsaicin-sensitive C-fibre bladder afferent neurones attributable to the reduction in A-type K+ channel activity in SCI. ABSTRACT: Nerve growth factor (NGF) has been implicated as an important mediator in the induction of C-fibre bladder afferent hyperexcitability, which contributes to the emergence of neurogenic lower urinary tract dysfunction after spinal cord injury (SCI). In this study, we determined whether NGF immunoneutralization using an anti-NGF antibody (NGF-Ab) normalizes the SCI-induced changes in electrophysiological properties of capsaicin-sensitive C-fibre bladder afferent neurones in female C57BL/6 mice. The spinal cord was transected at the Th8/Th9 level. Two weeks later, continuous administration of NGF-Ab (10 µg kg-1  h-1 , s.c. for 2 weeks) was started. Bladder afferent neurones were labelled with Fast-Blue (FB), a fluorescent retrograde tracer, injected into the bladder wall 3 weeks after SCI. Four weeks after SCI, freshly dissociated L6-S1 dorsal root ganglion neurones were prepared. Whole-cell patch-clamp recordings were then performed in FB-labelled neurones. After recording action potentials or voltage-gated K+ currents, the sensitivity of each neurone to capsaicin was evaluated. In capsaicin-sensitive FB-labelled neurones, SCI significantly reduced the spike threshold and increased the number of action potentials during membrane depolarization for 800 ms. These SCI-induced changes were reversed by NGF-Ab. Densities of slow-decaying A-type K+ (KA ) and sustained delayed rectifier-type K+ currents were significantly reduced by SCI. The NGF-Ab treatment reversed the SCI-induced reduction in the KA current density. These results indicate that NGF plays an important role in hyperexcitability of mouse capsaicin-sensitive C-fibre bladder afferent neurones attributable to a reduction in KA channel activity. Thus, NGF-targeting therapies could be effective for treatment of afferent hyperexcitability and neurogenic lower urinary tract dysfunction after SCI.

Tamm-Horsfall Protein is a Potent Immunomodulatory Molecule and a Disease Biomarker in the Urinary System.

Tamm-Horsfall protein (THP), or uromodulin (UMOD), is an 80-90-kDa phosphatidylinositol-anchored glycoprotein produced exclusively by the renal tubular cells in the thick ascending limb of the loop of Henle. Physiologically, THP is implicated in renal countercurrent gradient formation, sodium homeostasis, blood pressure regulation, and a defense molecule against infections in the urinary system. Investigations have also revealed that THP is an effective binding ligand for serum albumin, immunoglobulin G light chains, complement components C1 and C1q, interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α, and interferon-γ through its carbohydrate side chains for maintaining circulatory and renal immune homeostasis. Thus, THP can be regarded as part of the innate immune system. UMOD mutations play crucial roles in congenital urolithiasis, hereditary hyperuricemia/gout, and medullary cystic kidney diseases. Recent investigations have focused on the immunomodulatory effects of THP on immune cells and on THP as a disease biomarker of acute and chronic kidney diseases. Our studies have suggested that normal urinary THP, through its epidermal growth factor (EGF)-like domains, binds to the surface-expressed EGF-like receptors, cathepsin G, or lactoferrin to enhance polymorphonuclear leukocyte phagocytosis, proinflammatory cytokine production by monocytes/macrophages, and lymphocyte proliferation by activating the Rho family and mitogen-activated protein kinase signaling pathways. Furthermore, our data support both an intact protein core structure and carbohydrate side chains are important for the different protein-binding capacities of THP. Prospectively, parts of the whole THP molecule may be used for anti-TNF-α therapy in inflammatory diseases, autoantibody-depleting therapy in autoimmune disorders, and immune intensification in immunocompromised hosts.

A mouse model of urofacial syndrome with dysfunctional urination.

Urofacial syndrome (UFS) is an autosomal recessive disease with severe dysfunctional urination including urinary incontinence (UI). Biallelic mutations of HPSE2 are discovered from UFS patients, suggesting that HPSE2 is a candidate disease gene. Here, we show that deletion of Hpse2 is sufficient to cause the UFS-like phenotype in mice. Hpse2 knockout mutants display a distended bladder (megacystis) phenotype and abnormal voiding behavior similar to that found in patients. While Hpse2 is largely dispensable for detrusor smooth muscle and urothelial cell fate determination, the mutants have significantly lower rates of cell proliferation than wild-type littermate controls. All Hpse2 mutants have a growth retardation phenotype and die within a month after birth. Comprehensive blood chemistry and urinalysis indicate that Hpse2 mutants have renal dysfunction and malnutrition. We provide evidence that transforming growth factor beta (Tgfß) signaling is attenuated at birth. However, Tgfß activity is significantly enhanced at later stages when the urological phenotype is severe, and the mutant bladders have accumulated excessive amount of fibrotic tissue. Together, these findings strongly suggest that Hpse2 is a causative gene of human UFS and further uncover unexpected roles of Hpse2 in bladder physiology, tissue remodeling and Tgfß signaling.

SOCS3 Immunohistochemical Expression Seems to Support the 2005 and 2014 International Society of Urological Pathology (ISUP) Modified Gleason Grading System.

BACKGROUND: In the 2014, The International Society of Urological Pathology (ISUP) consensus conference update the grading of prostate, last revised in 2005. In this study we evaluate the SOCS3 immunohistochemical protein expression in different Gleason prostatic adenocarcinoma: classical Gleason grade 3, classical Gleason grade 3 upgraded to Gleason grade 4 according to the ISUP modifications and classical and modified Gleason grade 4. The major conclusions were: (i) Cribriform glands should be assigned a Gleason pattern 4, regardless of morphology; (ii) Glomeruloid glands should be assigned a Gleason pattern 4, regardless of morphology; (iii) Grading of mucinous carcinoma of the prostate should be based on its underlying growth pattern rather than all as pattern 4; and (iv) Intraductal carcinoma of the prostate without invasive carcinoma should not assigned Gleason grade and a comment about aggressive carcinoma probably associated should be made. In a recent report we analyzed the methylathion status of cytokine signaling (SOCS) proteins 3 (SOCS3) gene and the consequences of promoter hypermethylation on mRNA and protein expression in a collection of prostate cancer and benign prostate hyperplasia (BPH) and for the first time we demonstrated that a hypermethylation of SOCS3 with a significant reduction of its mRNA and protein expression identifies a subgroup of prostate cancer with a more aggressive behavior. Moreover we demonstrated that the immunohystochemical analysis of SOCS3 protein expression in prostatic cancer biopsies may provide a useful and easier method than SOCS3 methylation analysis to individuate in cancer with intermediate-high grade Gleason score a subgroup of prostate cancer with a more aggressive behavior. METHODS: A total of 148 radical prostatectomy with diagnosis of prostatic acinar adenocarcinoma were stratified into three different categories on the basis of Gleason grade: (i) Twenty-six prostatic adenocarcinoma with classical and modified Gleason grade 3; (ii) Fifty seven prostatic adenocarcinoma with classical Gleason grade 3 upgraded to Gleason grade 4 by 2005 and 2014 ISUP Consensus Conference; and (iii) Sixty five prostatic adenocarcinoma with classical and modified Gleason grade 4. Immunohistochemical analysis for SOCS3 was performed and SOCS3 staining intensity were evaluated by two pathologists in three different ways on the basis of the intensity of cytoplasmatic staining: positive (intense cytoplasmatic staining in more than 50% of neoplastic cells) (+), negative (absence of cytoplasmatic staining in more than 50% of neoplastic cells) (-), weakly positive (weak cytoplasmatic staining in more than 50% of neoplastic cells (+/-). RESULTS: In the group of prostatic adenocarcinoma Gleason grade 3 we found that SOCS3 positivity (+) were observed in 19 out of 26 cases (73.1%); in 5 out of 26 prostatic adenocarcinoma the neoplastic glands showed weak intensity SOCS3 staining (+/-) (19.2%), while in only two cases we found SOCS-3 negativity (-) (7.7%); in the group of cases with prostatic adenocarcinoma with Gleason grade 4, 16 out 65 cases (24.6%) showed SOCS3 positivity (+); 18 out 65 cases (27.7%) SOCS3 weakly positive (+/-), and in 31 cases (47.7%) SOCS3 negative staining (-) were observed. Interestingly, the group of prostatic adenocarcinoma with histological Gleason 3 pattern upgraded to Gleason 4 pattern according to the 2005 and 2014 ISUP modified grading system, showed SOCS3 positivity (+) in 16 out of 57 cases (28%), in 16 out 57 cases (28%) a weakly positive for SOCS3 (+/-) were observed, while 25 cases (44%) showed negative SOCS3 staining (-). CONCLUSIONS: In this study we demonstrated a significant association of SOCS3 positivity (+) with prostatic carcinoma classical Gleason pattern 3 (P < 0.0001), while SOCS3 negative pattern (-) or SOCS3 weakly positive pattern (+/-) were associated to prostatic carcinomas with Gleason pattern 3 upgraded to Gleason pattern 4 (P = 0.0002) and with classical Gleason pattern 4. The significant difference of SOCS3 immunohistochemical expression between classical Gleason grade 3 and Gleason grade 4 upgraded to grade 4 seems to support the definitions and the modifications of Gleason grade 4 of the 2005 and the 2014 International Society of Urological Pathology (ISUP). The hypoexpression of SOCS3 protein in glomeruloid glands could support the hypothesis that from molecular point of view this growth pattern could be different from classical Gleason pattern 3 and biologically more closely to Gleason pattern 4, confirming the conclusions of the 2014 ISUP Conference assigning a Gleason pattern 4 to glomeruloid glands regardless of morphology. Prostate 77: 597-603, 2017. © 2017 Wiley Periodicals, Inc.

Identification of novel non-invasive biomarkers of urinary chronic pelvic pain syndrome: findings from the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network.

OBJECTIVE: To examine a series of candidate markers for urological chronic pelvic pain syndrome (UCPPS), selected based on their proposed involvement in underlying biological processes so as to provide new insights into pathophysiology and suggest targets for expanded clinical and mechanistic studies. METHODS: Baseline urine samples from Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network study participants with UCPPS (n = 259), positive controls (PCs; chronic pain without pelvic pain, n = 107) and healthy controls (HCs, n = 125) were analysed for the presence of proteins that are suggested in the literature to be associated with UCPPS. Matrix metalloproteinase (MMP)-2, MMP-9, MMP-9/neutrophil gelatinase-associated lipocalin (NGAL) complex (also known as Lipocalin 2), vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGF-R1) and NGAL were assayed and quantitated using mono-specific enzyme-linked immunosorbent assays for each protein. Log-transformed concentration (pg/mL or ng/mL) and concentration normalized to total protein (pg/µg) values were compared among the UCPPS, PC and HC groups within sex using the Student's t-test, with P values adjusted for multiple comparisons. Multivariable logistic regression and receiver-operating characteristic curves assessed the utility of the biomarkers in distinguishing participants with UCPPS and control participants. Associations of protein with symptom severity were assessed by linear regression. RESULTS: Significantly higher normalized concentrations (pg/µg) of VEGF, VEGF-R1 and MMP-9 in men and VEGF concentration (pg/mL) in women were associated with UCPPS vs HC. These proteins provided only marginal discrimination between UCPPS participants and HCs. In men with UCCPS, pain severity was significantly positively associated with concentrations of MMP-9 and MMP-9/NGAL complex, and urinary severity was significantly positively associated with MMP-9, MMP-9/NGAL complex and VEGF-R1. In women with UCPPS, pain and urinary symptom severity were associated with increased normalized concentrations of MMP-9/NGAL complex, while pain severity alone was associated with increased normalized concentrations of VEGF, and urinary severity alone was associated with increased normalized concentrations of MMP-2. Pain severity in women with UCPPS was significantly positively associated with concentrations of all biomarkers except NGAL, and urinary severity with all concentrations except VEGF-R1. CONCLUSION: Altered levels of MMP-9, MMP-9/NGAL complex and VEGF-R1 in men, and all biomarkers in women, were associated with clinical symptoms of UCPPS. None of the evaluated candidate markers usefully discriminated UCPPS patients from controls. Elevated VEGF, MMP-9 and VEGF-R1 levels in men and VEGF levels in women may provide potential new insights into the pathophysiology of UCPPS.

Clinical Application of Human Urinary Extracellular Vesicles in Kidney and Urologic Diseases.

Extracellular vesicles (EVs) have been isolated in different body fluids, including urine. The cargo of urinary EVs is composed of nucleic acids and proteins reflecting the physiological and possibly pathophysiological state of cells lining the nephron and the urinary tract. Urinary EVs have been confirmed to contain low amounts of various types of RNA that play a role in intercellular communication by transferring genetic information. This communication through EV RNAs includes both continuation of normal physiological processes and conditioning in disease mechanisms. Although proteins included in urinary EVs represent only 3% of the whole-urine proteome, urinary EVs can influence cells in the renal epithelia not only by delivering RNA cargo, but also by delivering a wide range of proteins. Since urine is a readily available biofluid, the discovery of EVs has opened a new field of biomarker research. The potential use of urinary EV RNAs and proteins as diagnostic biomarkers for various kidney and urologic diseases is currently being explored. Here, we review recent studies that deal in identifying biomarker candidates for human kidney and urologic diseases using urinary EVs and might help to understand the pathophysiology.

[VValidation of the combined medical therapy for LUTS using 1-adrenergic blockers and M-cholinoblockers].

In the majority of patients, urination disorders are caused by both mechanical and functional factors. In patients with BPH timely determination of the type and nature of urinary dysfunction, especially bladder outlet obstruction and detrusor overactivity is of great practical importance. Without accounting for this factor, functional results of surgical treatment may be significantly impaired. 1-adrenergic blockers are the first-line therapy for men with moderate to severe lower urinary tract symptoms (LUTS). In selecting the appropriate therapy, more selective 1-adrenergic blockers should be chosen. The selectivity of the 1- adrenergic blockers provides high performance along with a favorable side effect profile especially regarding cardiovascular event rate. Like 1-adrenergic blockers, M-cholinoblockers have varying degrees of selectivity regarding the impact on the bladder. Solifenacin has greater selectivity for the bladder receptors than tolterodine and oxybutynin. The selectivity of this medication regarding the bladder manifests itself in the relatively low incidence of side effects, such as dry mouth, which enables the possibility of long-term therapy. Combined therapy with 1- adrenergic blockers and M-cholinoblockers may be considered as a treatment modality in patients with moderate to severe LUTS with a predominance of filling symptoms, especially if monotherapy proves not effective enough.

Biochemical analysis of ascites fluid as an aid to etiological diagnosis: a series of 100 cases of nonimmune fetal ascites.

OBJECTIVE: The aim of this study is to analyze the contribution of biochemistry and cytology of fetal ascites fluid to the etiological diagnosis of ascites after ultrasonographic scan, maternal blood sampling, and fetal karyotyping. METHOD: This is a retrospective study of 100 consecutive cases of nonimmune fetal ascites in which ascites fluid was sampled. All women underwent referral ultrasound scan and fetal karyotyping. All cases of fetal ascites were studied by biochemistry (total protein, ß2 -microglobulin, IgM, gamma-glutamyl transpeptidase, aspartate aminotransferase, aminopeptidase M, and intestinal isoform of alkaline phosphatase) and cytology (lymphocyte count and vacuolated cells). RESULTS: The etiology of ascites was diagnosed at ultrasound scan in only 50% of cases. We observed significantly (P < 0.001) low levels of total protein in ascites of urinary origin, high levels of digestive enzymes in ascites of digestive origin, and high ß2 -microglobulin in infectious ascites. Vacuolated cells were observed in all ten storage metabolic diseases. CONCLUSION: Sampling of fetal ascites fluid for biochemical and cytological examination provides important additional information. We propose a two-step management: (1) detailed ultrasound scan examination, maternal blood analysis, and fetal karyotyping and (2) biochemical and cytological analyses. On the basis of such an approach, 63% and 96% of etiologies would have been identified in our series after the first and second steps, respectively. © 2014 John Wiley & Sons, Ltd.

Elucidation of the etiology and characteristics of pink urine in young healthy subjects.

BACKGROUND: Pink urine syndrome (PUS) is attributed to the precipitation of uric acid caused by low urinary pH (U-pH). However, the reasons for the lower U-pH are unclear. OBJECTIVES: To investigate the occurrence of PUS and verified the cause of U-pH reduction. METHODS: Participants comprised 4,940 students who had undergone a physical examination. Data on the presence [PUS (+)] or absence [PUS (-)] of PUS, as well as age, gender, body mass index (BMI), blood pressure (BP), heart rate (HR), and U-pH were collected. Of these participants, 300 randomly selected individuals were evaluated for their waist circumference, as well as their levels of urinary C-peptide, angiotensinogen, methylglyoxal, thiobarbituric acid-reactive substances (TBARS), and Na(+) excretion. Independent risk factors of lower U-pH were decided by a multiple-regression analysis. RESULTS: PUS was observed in 216 students (4.4 %). A greater number of men comprised the PUS (+) group compared with the PUS (-) group, and subjects in this group had high BMI, BP, and HR values, as well as low U-pH. A logistic regression analysis revealed that the BMI and U-pH were independent risk factors for PUS (+). The decrease of U-pH was closely related to the progress of chronic kidney disease (CKD). BMI value was related to PUS (+) in the CKD (-) subjects. On the other hand, low U-pH was related to PUS (+) in the CKD (+) subjects. All factors other than HR showed a significant negative correlation with U-pH. However, multiple-regression analysis revealed that TBARS and angiotensinogen were independent risk factors. CONCLUSION: Obesity and lower U-pH were each independently related to PUS, whereas increased intrarenal oxidative stress and exacerbation of the renin-angiotensin system activation were associated with the lowering of U-pH. U-pH low value is related to potential CKD.

[Uroplakins as markers of diseases of the urinary system]. / Uroplakiny jako markery chorób ukladu moczowego

An unique element of bladder urothelium is a multilayer membrane, which extends from the renal pelvis to the urethra. Urotelial membrane covers more than 90% of the inner portion of the bladder and is in direct contact with urine. Urothelium is composed of characteristic two-dimensional, asymmetric plaques, composed of uroplakins (UP), differentiated, hexagonally arranged proteins. The unique structure of the urothelial plaques determines the tightness, integrity and strength of the urothelium, prevent rupture of the walls of the bladder during the build-up of urine in the bladder and protects against the toxic ingredients. Uroplakins are tissue-specific, heterogeneous glycoproteins whose oligosaccharide part plays a specific role in the structure and function of urothelium. Disorders of normal expression of uroplakins are highly associated with the pathogenesis in infection and urinary tract malignancies, primary vesico-urinary reflux, hydronephrosis and renal impairment. The emergence of uroplakins in urine and / or plasma may have a potential role in the early detection of bladder tumors. In this paper, the structure and function of uroplakins types Ia, Ib, II, IIIa, their natural oligomerization into heterodimers, tetramers and hexamers, and the role in the construction of asymmetric and flexible urothelial epithelium is presented. We discuss the potential role of uroplakins in laboratory diagnosis of umbrella cell differentiation and in the screening analysis of urinary bladder disorders. The possibilities of using the knowledge of uroplakins in clinical settings as well as in modern strategies for treatment of infectious diseases and cancer of the urinary tract are highlighted.

Glycosylation of uroplakins. Implications for bladder physiopathology.

Urothelium, a specialized epithelium, covers the urinary tract and act not only as a barrier separating its light from the surrounding tissues, but fulfills an important role in maintaining the homeostasis of the urothelial tract and well-being of the whole organism. Proper function of urothelium is dependent on the precise assemble of highly specialized glycoproteins called uroplakins, the end products and differentiation markers of the urothelial cells. Glycosylation changes in uroplakins correlate with and might reflect progressive stages of pathological conditions of the urothelium such as cancer, urinary tract infections, interstitial cystitis and others. In this review we focus on sugar components of uroplakins, their emerging role in urothelial biology and disease implications. The advances in our understanding of uroplakins changes in glycan moieties composition, structure, assembly and expression of their glycovariants could potentially lead to the development of targeted therapies and discoveries of novel urine and plasma markers for the benefit of patients with urinary tract diseases.

Purinergic signalling in the urinary tract in health and disease.

Purinergic signalling is involved in a number of physiological and pathophysiological activities in the lower urinary tract. In the bladder of laboratory animals there is parasympathetic excitatory cotransmission with the purinergic and cholinergic components being approximately equal, acting via P2X1 and muscarinic receptors, respectively. Purinergic mechanosensory transduction occurs where ATP, released from urothelial cells during distension of bladder and ureter, acts on P2X3 and P2X2/3 receptors on suburothelial sensory nerves to initiate the voiding reflex, via low threshold fibres, and nociception, via high threshold fibres. In human bladder the purinergic component of parasympathetic cotransmission is less than 3 %, but in pathological conditions, such as interstitial cystitis, obstructed and neuropathic bladder, the purinergic component is increased to 40 %. Other pathological conditions of the bladder have been shown to involve purinoceptor-mediated activities, including multiple sclerosis, ischaemia, diabetes, cancer and bacterial infections. In the ureter, P2X7 receptors have been implicated in inflammation and fibrosis. Purinergic therapeutic strategies are being explored that hopefully will be developed and bring benefit and relief to many patients with urinary tract disorders.

The different molecular forms of urine neutrophil gelatinase-associated lipocalin present in dogs with urinary diseases.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is a useful biomarker for the early prediction of renal diseases. NGAL may exist as monomer, dimer and/or NGAL/MMP-9 complex forms in humans. In this study, the existence of various forms of NGAL in urine (uNGAL) was determined and whether these forms are related to the different urinary diseases found in dogs is further discussed. RESULTS: Eighty-one urine samples from dogs with different forms of renal disease (41), pyuria (19) and a number of non-renal related diseases (10), as well as healthy dogs (11), were collected. uNGAL concentrations and their molecular forms in dogs were measured by ELISA and Western blot analysis, respectively. The uNGAL concentrations of dogs with pyuria (median: 15.35 ng/mL) were significantly higher than those of the healthy control animals (median: 3.92 ng/mL) (p < 0.01), but lower than those of dogs with renal diseases (median: 23.77 ng/mL). Each NGAL molecular form could be detected in dog urine. In particular, monomer was detected more frequently in patients with renal disease than those with non-renal diseases; while the dimer form appeared in a significantly higher percentage of cases with pyuria compared to those without pyuria. The NGAL/MMP-9 complex was found to exist not only in the patients with cystitis, but also in the cases with renal injury. CONCLUSION: Different molecular forms of uNGAL can indicate different origins of the urinary abnormalities. Determining the molecular forms of uNGAL present in diseased dogs may provide clinical workers with a tool that will help the early and more precise detection of different urinary diseases.

Epigenetic insights to pediatric uropathology: Celebrating the fundamental biology vision of Tony Khoury.

INTRODUCTION: Many pediatric urology conditions affect putatively normal tissues or appear too commonly to be based solely on specific DNA mutations. Understanding epigenetic mechanisms in pediatric urology, therefore, has many implications that can impact cell and tissue responses to settings, such as environmental and hormonal influences on urethral development, uropathogenic infections, obstructive stimuli, all of which originate externally or extracellularly. Indeed, the cell's response to external stimuli is often mediated epigenetically. In this commentary, we highlight work on the critical role that epigenetic machinery, such as DNA methyltransferases (DNMTs), Enhancer of Zeste Polycomb Repressive Complex 2 Subunit (EZH2), and others play in regulating gene expression and cellular functions in three urological contexts. DESIGN: Animal and cellular constructs were used to model clinical pediatric uropathology. The hypertrophy, trabeculation, and fibrosis of the chronically obstructed bladder was explored using smooth muscle cell models employing disorganised vs. normal extracellular matrix (ECM), as well as a new animal model of chronic obstructive bladder disease (COBD) which retains its pathologic features even after bladder de-obstruction. Cell models from human and murine hypospadias or genital tubercles (GT) were used to illustrate developmental responses and epigenetic dependency of key developmental genes. Finally, using bladder urothelial and organoid culture systems, we examined activity of epigenetic machinery in response to non uropathogenic vs. uropathogenic E.coli (UPEC). DNMT and EZH2 expression and function were interrogated in these model systems. RESULTS: Disordered ECM exerted a principal mitogenic and epigenetic role for on bladder smooth muscle both in vitro and in CODB in vivo. Key genes, e.g., BDNF and KCNB2 were under epigenetic regulation in actively evolving obstruction and COBD, though each condition showed distinct epigenetic responses. In models of hypospadias, estrogen strongly dysregulated WNT and Hox expression, which was normalized by epigenetic inhibition. Finally, DNA methylation machinery in the urothelium showed specific activation when challenged by uropathogenic E.coli. Similarly, UPEC induces hypermethylation and downregulation of the growth suppressor p16INK4A. Moreover, host cells exposed to UPEC produced secreted factors inducing epigenetic responses transmissible from one affected cell to another without ongoing bacterial presence. DISCUSSION: Microenvironmental influences altered epigenetic activity in the three described urologic contexts. Considering that many obstructed bladders continue to display abnormal architecture and dysfunction despite relief of obstruction similar to after resection of posterior valves or BPH, the epigenetic mechanisms described highlight novel approaches for understanding the underlying smooth muscle myopathy of this crucial clinical problem. Similarly, there is evidence for an epigenetic basis of xenoestrogen on development of hypospadias, and UTI-induced pan-urothelial alteration of epigenetic marks and propensity for subsequent (recurrent) UTI. The impact of mechanical, hormonal, infectious triggers on genitourinary epigenetic machinery activity invite novel avenues for targeting epigenetic modifications associated with these non-cancer diseases in urology. This includes the use of deactivated CRISPR-based technologies for precise epigenome targeting and editing. Overall, we underscore the importance of understanding epigenetic regulation in pediatric urology for the development of innovative therapeutic and management strategies.

Congenital urinary tract obstruction: defining markers of developmental kidney injury.

Congenital urinary tract obstruction (diagnosed antenatally by ultrasound screening) is one of the main causes of end-stage kidney disease in children. The extent of kidney injury in early gestation and the resultant abnormality in kidney development determine fetal outcome and postnatal renal function. Unfortunately, the current approach to diagnostic evaluation of the severity of injury has inherently poor diagnostic and prognostic value because it is based on the assessment of fetal tubular function from fetal urine samples rather than on estimates of the dysplastic changes in the injured developing kidney. To improve the outcome in children with congenital urinary tract obstruction, new biomarkers reflecting these structural changes are needed. Genomic and proteomic techniques that have emerged in the past decade can help identify the key genes and proteins from biological fluids, including amniotic fluid, that might reflect the extent of injury to the developing kidney.

Exosomes in urological diseases - Biological functions and clinical applications.

Exosomes are extracellular vesicles with a variety of biological functions that exist in various biological body fluids and exert their functions through proteins, nucleic acids, lipids, and metabolites. Recent discoveries have revealed the functional and biomarker roles of miRNAs in urological diseases, including benign diseases and malignancies. Exosomes have several uses in the diagnosis, treatment, and monitoring of urological diseases, especially cancer. Proteins and nucleic acids can be used as alternative biomarkers for detecting urological diseases. Additionally, exosomes can be detected in most body fluids, thereby avoiding pathogenesis. More importantly, for urological tumors, exosomes display a higher sensitivity than circulating tumor cells and tumor-derived DNA in body fluid biopsies because of their low immunogenicity and high stability. These advantages have made it a research hotspot in recent years. In this review, we focus on the biological characteristics and functions of exosomes and summarize their advantages and the latest progress in the diagnosis and treatment of urological diseases.