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Urinary tract infections (UTIs) commonly afflict people with diabetes. To better understand the mechanisms that predispose diabetics to UTIs, we employ diabetic mouse models and altered insulin signaling to show that insulin receptor (IR) shapes UTI defenses. Our findings are validated in human biosamples. We report that diabetic mice have suppressed IR expression and are more susceptible to UTIs caused by uropathogenic Escherichia coli (UPEC). Systemic IR inhibition increases UPEC susceptibility, while IR activation reduces UTIs. Localized IR deletion in bladder urothelium promotes UTI by increasing barrier permeability and suppressing antimicrobial peptides. Mechanistically, IR deletion reduces nuclear factor κB (NF-κB)-dependent programming that co-regulates urothelial tight junction integrity and antimicrobial peptides. Exfoliated urothelial cells or urine samples from diabetic youths show suppressed expression of IR, barrier genes, and antimicrobial peptides. These observations demonstrate that urothelial insulin signaling has a role in UTI prevention and link IR to urothelial barrier maintenance and antimicrobial peptide expression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Abstract: A 55-year-old woman with multiple medical problems, including anuric, dialysis-dependent, end-stage renal disease, presented with persistent fever of unknown origin. Despite extensive workup with cross-sectional imaging and panculture, the etiology was not found. Eventually, an 111In-labeled WBC scan was performed to evaluate for occult infection, which revealed intense heterogeneous uptake in the urinary bladder. Subsequent bladder catheterization showed pus and blood, which grew Klebsiella pneumoniae. The fevers resolved with adjustment of the therapy. Although urinary analysis and culture are standard practice in the workup of fever of unknown origin, anuria may obscure this common source of infection., Competing Interests: Conflicts of interest and sources of funding: none declared., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

The bladder epithelial cells elicit robust innate immune responses against urinary tract infections (UTIs) for preventing the bacterial colonization. Physiological fluctuations in circulating estrogen levels in women increase the susceptibility to UTI pathogenesis, often resulting in adverse health outcomes. Dr adhesin bearing Escherichia coli (Dr E. coli) cause recurrent UTIs in menopausal women and acute pyelonephritis in pregnant women. Dr E. coli bind to epithelial cells via host innate immune receptor CD55, under hormonal influence. The role of estrogens or estrogen receptors (ERs) in regulating the innate immune responses in the bladder are poorly understood. In the current study, we investigated the role of ERα, ERβ and GPR30 in modulating the innate immune responses against Dr E. coli induced UTI using human bladder epithelial carcinoma 5637 cells (HBEC). Both ERα and ERβ agonist treatment in bladder cells induced a protection against Dr E. coli invasion via upregulation of TNFα and downregulation of CD55 and IL10, and these effects were reversed by action of ERα and ERβ antagoinsts. In contrast, the agonist-mediated activation of GPR30 led to an increased bacterial colonization due to suppression of innate immune factors in the bladder cells, and these effects were reversed by the antagonist-mediated suppression of GPR30. Further, siRNA-mediated ERα knockdown in the bladder cells reversed the protection against bacterial invasion observed in the ERα positive bladder cells, by modulating the gene expression of TNFα, CD55 and IL10, thus confirming the protective role of ERα. We demonstrate for the first time a protective role of nuclear ERs, ERα and ERβ but not of membrane ER, GPR30 against Dr E. coli invasion in HBEC 5637 cells. These findings have many clinical implications and suggest that ERs may serve as potential drug targets towards developing novel therapeutics for regulating local innate immunity and treating UTIs., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments 1,2 . Here we create multilayer bladder 'assembloids' by reconstituting tissue stem cells with stromal components to represent an organized architecture with an epithelium surrounding stroma and an outer muscle layer. These assembloids exhibit characteristics of mature adult bladders in cell composition and gene expression at the single-cell transcriptome level, and recapitulate in vivo tissue dynamics of regenerative responses to injury. We also develop malignant counterpart tumour assembloids to recapitulate the in vivo pathophysiological features of urothelial carcinoma. Using the genetically manipulated tumour-assembloid platform, we identify tumoural FOXA1, induced by stromal bone morphogenetic protein (BMP), as a master pioneer factor that drives enhancer reprogramming for the determination of tumour phenotype, suggesting the importance of the FOXA1-BMP-hedgehog signalling feedback axis between tumour and stroma in the control of tumour plasticity.

Resident macrophages are abundant in the bladder, playing key roles in immunity to uropathogens. Yet, whether they are heterogeneous, where they come from, and how they respond to infection remain largely unknown. We identified two macrophage subsets in mouse bladders, MacM in muscle and MacL in the lamina propria, each with distinct protein expression and transcriptomes. Using a urinary tract infection model, we validated our transcriptomic analyses, finding that MacM macrophages phagocytosed more bacteria and polarized to an anti-inflammatory profile, whereas MacL macrophages died rapidly during infection. During resolution, monocyte-derived cells contributed to tissue-resident macrophage pools and both subsets acquired transcriptional profiles distinct from naïve macrophages. Macrophage depletion resulted in the induction of a type 1-biased immune response to a second urinary tract infection, improving bacterial clearance. Our study uncovers the biology of resident macrophages and their responses to an exceedingly common infection in a largely overlooked organ, the bladder., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Uropathogenic Escherichia coli (UPEC), a leading cause of urinary tract infections, is associated with prostate and bladder cancers. Cytotoxic necrotizing factor 1 (CNF1) is a key UPEC toxin; however, its role in bladder cancer is unknown. In the present study, we found CNF1 induced bladder cancer cells to secrete vascular endothelial growth factor (VEGF) through activating Ras homolog family member C (RhoC), leading to subsequent angiogenesis in the bladder cancer microenvironment. We then investigated that CNF1-mediated RhoC activation modulated the stabilization of hypoxia-inducible factor 1α (HIF1α) to upregulate the VEGF. We demonstrated in vitro that active RhoC increased heat shock factor 1 (HSF1) phosphorylation, which induced the heat shock protein 90α (HSP90α) expression, leading to stabilization of HIF1α. Active RhoC elevated HSP90α, HIF1α, VEGF expression, and angiogenesis in the human bladder cancer xenografts. In addition, HSP90α, HIF1α, and VEGF expression were also found positively correlated with the human bladder cancer development. These results provide a potential mechanism through which UPEC contributes to bladder cancer progression, and may provide potential therapeutic targets for bladder cancer., (© 2020 Federation of American Societies for Experimental Biology.)

Urinary tract infection is one of the most common bacterial infections which is mainly caused by Escherichia coli (UPEC). Autophagy plays a key role in immune response to eliminate invading pathogens. Exploring the effect of autophagy on UPEC infection and the molecular mechanisms will be benefit for the treatment of urinary tract infection. High-mobility group protein N2 (HMGN2), a highly conserved nuclear protein and an antibacterial peptide, has been associated with bacterial infection induced immune response; however, whether this function is due to the regulation of autophagy remains unclear. In this study, we demonstrate for the first time that HMGN2 is upregulated in UPEC infection of bladder epithelial cell line 5637 (BEC 5637). Furthermore, HMGN2 enhances autophagy in BEC 5637 via activation of AMPK and ULK1, whereas UPEC suppresses autophagy. In addition, the enhanced autophagy activity by HMGN2 overexpression or rapamycin boosts the proliferation of UPEC J96 in BEC 5637. In summary, our data indicate that HMGN2 activates autophagy via AMPK/ULK1 pathway which can be utilized by UPEC J96 for their proliferation within bladder epithelial cells., (Copyright © 2019. Published by Elsevier Inc.)

Iron is a critical nutrient required by hosts and pathogens. Uropathogenic Escherichia coli (UPEC), the principal causative agent of urinary tract infections (UTIs), chelate iron for their survival and persistence. Here, we demonstrate that dietary modulation of iron availability limits UPEC burden in a mouse model of UTI. Mice on a low-iron diet exhibit reduced systemic and bladder mucosal iron availability and harbor significantly lower bacterial burden, concomitant with dampened inflammation. Hepcidin is a master regulator of iron that controls iron-dependent UPEC intracellular growth. Hepcidin-deficient mice ( Hamp1 -/- ) exhibit accumulation of iron deposits, persistent bacterial burden in the bladder, and a heightened inflammatory response to UTI. However, a low-iron dietary regimen reversed the iron overload and increased bacterial burden phenotypes in Hamp1 -/- mice. Thus modulation of iron levels via diet can reduce UPEC infection and persistence, which may have significant implications for clinical management of UTI.

The open probability of calcium-activated voltage-gated potassium channel (BK channel) on bladder umbrella urothelial cells is increased by lipopolysaccharide (LPS). It is hypothesized that this channel's activity is important in the urothelial innate immune response during urinary tract infection (UTI). We performed in vivo studies using female C57BL/6 mice whose bladders were inoculated with LPS (150 μl of 1 mg/ml) or uropathogenic Escherichia coli (UPEC, UTI89), without and with intravesical BK inhibitor iberiotoxin (IBTX, 1 μM). Inflammatory biomarkers (chemokines and cytokines) were measured in urine specimens collected 2 h after inoculation using a 32-multiplex ELISA. Of these 32 biomarkers, 19 and 15 were significantly elevated 2 h after LPS and UPEC exposure, respectively. IBTX significantly abrogated the elevations of 15 out of 19 biomarkers after LPS inoculation and 12 out of 15 biomarkers after UPEC inoculation. In a separate experiment, qPCR for IL-6, interferon-γ-induced protein 10 (CXCL10), and macrophage inflammatory protein 2 (CXCL2) in urothelium paralleled the changes measured in urine of these same biomarkers, supporting that urinary changes in biomarker levels reflected urothelial expression changes. These in vivo data demonstrated that BK channel activity is crucial in the urothelial host innate immune response, as measured by changes in urinary biomarkers, in UTI pathogenesis.

The signaling networks regulating antimicrobial activity during urinary tract infection (UTI) are incompletely understood. Interleukin-6 (IL-6) levels increase with UTI severity, but the specific contributions of IL-6 to host immunity against bacterial uropathogens are unknown. To clarify this we tested whether IL-6 activates the Stat3 transcription factor, to drive a program of antimicrobial peptide gene expression in infected urothelium during UTI. Transurethral inoculation of uropathogenic Escherichia coli led to IL-6 secretion, urothelial Stat3 phosphorylation, and activation of antimicrobial peptide transcription, in a Toll-like receptor 4-dependent manner in a murine model of cystitis. Recombinant IL-6 elicited Stat3 phosphorylation in primary urothelial cells in vitro, and systemic IL-6 administration promoted urothelial Stat3 phosphorylation and antimicrobial peptide expression in vivo. IL-6 deficiency led to decreased urothelial Stat3 phosphorylation and antimicrobial peptide mRNA expression following UTI, a finding mirrored by conditional Stat3 deletion. Deficiency in IL-6 or Stat3 was associated with increased formation of intracellular bacterial communities, and exogenous IL-6 reversed this phenotype in IL-6 knockout mice. Moreover, chronic IL-6 depletion led to increased renal bacterial burden and severe pyelonephritis in C3H/HeOuJ mice. Thus, IL-6/Stat3 signaling drives a transcriptional program of antimicrobial gene expression in infected urothelium, with key roles in limiting epithelial invasion and ascending infection., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Bladder urothelium plays an active role in response to bacterial infection. There is little known about the electrophysiological activity in urothelial cells in this process. We used a nonenzymatic method to isolate bladder urothelial tissue and to patch clamp umbrella cells in situ. A 200 pS conductance potassium (K + ) channel was detected from female C57BL6 mice. Of 58 total patches, 17.2% patches displayed the 200 pS K + conductance channel. This K + conductance channel showed Ca 2+ sensitivity and voltage dependence. Specific big-conductance potassium channel (BK) inhibitors (paxilline, iberiotoxin) blocked the 200 pS K + conductance channel activity. RT-PCR and immunoblot confirmed BK channel pore-forming α-subunit (BK-α) mRNA and protein in urothelium. Immunohistochemistry also showed the BK-α located in urothelium. The above data provided evidence that the 200 pS K + conductance channel was a BK channel. Lipopolysaccharide (LPS), a component of uropathogenic Escherichia coli, was used to investigate the role of BK channel in the pathogenesis of urinary tract infection. BK channel activity as NP o increased threefold within 30 min of exposure to LPS. mRNAs for LPS receptors (TLR4, CD14, MD-2) were expressed in the urothelium but not in lamina propria or detrusor. Blockade of the receptors by an antagonist (polymyxin B) abrogated LPS's effect on BK channel. The involvement of protein kinase A (PKA) on BK channel activity was demonstrated by applying PKA blockers (H89 and PKI). Both PKA inhibitors abolished the BK channel activity induced by LPS. In conclusion, BK channel was identified in bladder umbrella cells, and its activity was significantly increased by LPS.

Urinary tract infection (UTI) is a broad term referring to an infection of the kidneys, ureters, bladder, and/or urethra. Because of its prevalence, frequent recurrence, and rising resistance to antibiotics, UTI has become a challenge in clinical practice. Autosomal-dominant polycystic kidney disease (ADPKD) is the most common monogenic disorder of the kidney and is characterized by the growth of fluid-filled cysts in both kidneys. Progressive cystic enlargement, inflammation, and interstitial fibrosis result in nephron loss with subsequent decline in kidney function. ADPKD patients frequently develop UTI; however, the cellular and molecular mechanisms responsible for the high UTI incidence in ADPKD patients remain virtually unaddressed. Emerging evidence suggests that α-intercalated cells (α-ICs) of the collecting ducts function in the innate immune defense against UTI. α-ICs inhibit bacterial growth by acidifying urine and secreting neutrophil gelatinase-associated lipocalin (NGAL) that chelates siderophore-containing iron. It is necessary to determine, therefore, if ADPKD patients with recurrent UTI have a reduced number and/or impaired function of α-ICs. Identification of the underlying cellular and molecular mechanisms may lead to the development of novel strategies to reduce UTI in ADPKD., (Copyright © 2017 the American Physiological Society.)

Methicillin-resistant Staphylococcus aureus (MRSA) is an emerging cause of catheter-associated urinary tract infection (CAUTI), which frequently progresses to more serious invasive infections. We adapted a mouse model of CAUTI to investigate how catheterization increases an individual's susceptibility to MRSA UTI. This analysis revealed that catheterization was required for MRSA to achieve high-level, persistent infection in the bladder. As shown previously, catheter placement induced an inflammatory response resulting in the release of the host protein fibrinogen (Fg), which coated the bladder and implant. Following infection, we showed that MRSA attached to the urothelium and implant in patterns that colocalized with deposited Fg. Furthermore, MRSA exacerbated the host inflammatory response to stimulate the additional release and accumulation of Fg in the urinary tract, which facilitated MRSA colonization. Consistent with this model, analysis of catheters from patients with S. aureus -positive cultures revealed colocalization of Fg, which was deposited on the catheter, with S. aureus Clumping Factors A and B (ClfA and ClfB) have been shown to contribute to MRSA-Fg interactions in other models of disease. We found that mutants in clfA had significantly greater Fg-binding defects than mutants in clfB in several in vitro assays. Paradoxically, only the ClfB - strain was significantly attenuated in the CAUTI model. Together, these data suggest that catheterization alters the urinary tract environment to promote MRSA CAUTI pathogenesis by inducing the release of Fg, which the pathogen enhances to persist in the urinary tract despite the host's robust immune response., Competing Interests: The authors declare no conflict of interest.

One of the most common urologic problems afflicting millions of people worldwide is urinary tract infection (UTI). The severity of UTIs ranges from asymptomatic bacteriuria to acute cystitis, and in severe cases, pyelonephritis and urosepsis. The primary cause of UTIs is uropathogenic Escherichia coli (UPEC), for which current antibiotic therapies often fail. UPEC forms multicellular communities known as biofilms on urinary catheters, as well as on and within bladder epithelial cells. Biofilm formation protects UPEC from environmental conditions, antimicrobial therapy, and the host immune system. Previous studies have investigated UPEC biofilm formation in aerobic conditions (21% oxygen); however, urine oxygen tension is reduced (4-6%), and urine contains molecules that can be used by UPEC as alternative terminal electron acceptors (ATEAs) for respiration. This study was designed to determine whether these different terminal electron acceptors utilized by E. coli influence biofilm formation. A panel of 50 urine-associated E. coli isolates was tested for the ability to form biofilm under anaerobic conditions and in the presence of ATEAs. Biofilm production was reduced under all tested sub-atmospheric levels of oxygen, with the notable exception of 4% oxygen, the reported concentration of oxygen within the bladder., Competing Interests: The authors declare no conflict of interest.

Positive selection in the two-domain type 1 pilus adhesin FimH enhances Escherichia coli fitness in urinary tract infection (UTI). We report a comprehensive atomic-level view of FimH in two-state conformational ensembles in solution, composed of one low-affinity tense (T) and multiple high-affinity relaxed (R) conformations. Positively selected residues allosterically modulate the equilibrium between these two conformational states, each of which engages mannose through distinct binding orientations. A FimH variant that only adopts the R state is severely attenuated early in a mouse model of uncomplicated UTI but is proficient at colonizing catheterized bladders in vivo or bladder transitional-like epithelial cells in vitro. Thus, the bladder habitat has barrier(s) to R state-mediated colonization possibly conferred by the terminally differentiated bladder epithelium and/or decoy receptors in urine. Together, our studies reveal the conformational landscape in solution, binding mechanisms, and adhesive strength of an allosteric two-domain adhesin that evolved "moderate" affinity to optimize persistence in the bladder during UTI.

Autophagy is a cellular recycling pathway, which in many cases, protects host cells from infections by degrading pathogens. However, uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections (UTIs), persist within the urinary tract epithelium (urothelium) by forming reservoirs within autophagosomes. Iron is a critical nutrient for both host and pathogen, and regulation of iron availability is a key host defense against pathogens. Iron homeostasis depends on the shuttling of iron-bound ferritin to the lysosome for recycling, a process termed ferritinophagy (a form of selective autophagy). Here, we demonstrate for the first time that UPEC shuttles with ferritin-bound iron into the autophagosomal and lysosomal compartments within the urothelium. Iron overload in urothelial cells induces ferritinophagy in an NCOA4-dependent manner causing increased iron availability for UPEC, triggering bacterial overproliferation and host cell death. Addition of even moderate levels of iron is sufficient to increase and prolong bacterial burden. Furthermore, we show that lysosomal damage due to iron overload is the specific mechanism causing host cell death. Significantly, we demonstrate that host cell death and bacterial burden can be reversed by inhibition of autophagy or inhibition of iron-regulatory proteins, or chelation of iron. Together, our findings suggest that UPEC persist in host cells by taking advantage of ferritinophagy. Thus, modulation of iron levels in the bladder may provide a therapeutic avenue to controlling UPEC persistence, epithelial cell death, and recurrent UTIs.

Settling of (18)F-FDG in the bladder is often noted on whole-body PET/CT images, but this phenomenon has never received any careful attention and the mechanism has been unclear. The 2 patients described in this report, one with a T1 pathologic fracture and another with widespread bone and lymph node metastases from an unknown primary tumor, underwent PET/CT. Both had urinary tract infection and a distended bladder during scanning. The interesting layering of (18)F-FDG in the urinary bladder was observed in both patients. The presence of this phenomenon demands careful evaluation of the urine by the clinician, and the mechanism is hypothesized to be slow (18)F-FDG excretion in patients with a distended urinary bladder, resulting in delayed mixing with urine. In addition, urinary tract infection may be a potential cause. Images showing this interesting layering should be interpreted with care., (© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

S5D-SRCRB is a novel mouse secretory glycoprotein belonging to the ancient and highly conserved scavenger receptor cysteine-rich superfamily of protein receptors. Available evidence indicates that S5D-SRCRB interacts with conserved microbial cell wall components, as well as with some endogenous proteins, and presents a restricted tissue expression pattern. This study further analyzes the expression of S5D-SRCRB along the mouse urogenital tract. Immunohistochemical staining for S5D-SRCRB was observed in spermatocytes from seminiferous tubules and in the epithelial surface from urethra and bladder, as well as in kidney tubules, mainly from medulla and papilla. Double stainings showed that S5D-SRCRB is expressed in both principal (P) and intercalated (IC) cells from renal collecting ducts (CD). By using an in vitro cell model of IC cell differentiation, preferential expression of S5D-SRCRB was observed in the apical border of terminally differentiated IC. Colocalization of S5D-SRCRB with galectin-3 (Gal-3) was also observed in kidney and bladder, but not in testis, supporting concurrent biochemical studies demonstrating the carbohydrate-dependent interaction of Gal-3 and S5D-SRCRB. Furthermore, upregulation of S5D-SRCRB expression was observed in in vitro and in vivo models of bacterial aggression, reinforcing the emerging view that CD, and specially IC, are important players in innate defense of the urinary tract against infection. Taken together, the results indicate that S5D-SRCRB is an integral component of the urogenital tract involved in innate immune functions., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The adherence of uropathogenic Escherichia coli (UPEC) to the host urothelial surface is the first step for establishing UPEC infection. Uroplakin Ia (UPIa), a glycoprotein expressed on bladder urothelium, serves as a receptor for FimH, a lectin located at bacterial pili, and their interaction initiates UPEC infection. Surfactant protein D (SP-D) is known to be expressed on mucosal surfaces in various tissues besides the lung. However, the functions of SP-D in the non-pulmonary tissues are poorly understood. The purposes of this study were to investigate the possible function of SP-D expressed in the bladder urothelium and the mechanisms by which SP-D functions. SP-D was expressed in human bladder mucosa, and its mRNA was increased in the bladder of the UPEC infection model in mice. SP-D directly bound to UPEC and strongly agglutinated them in a Ca(2+)-dependent manner. Co-incubation of SP-D with UPEC decreased the bacterial adherence to 5637 cells, the human bladder cell line, and the UPEC-induced cytotoxicity. In addition, preincubation of SP-D with 5637 cells resulted in the decreased adherence of UPEC to the cells and in a reduced number of cells injured by UPEC. SP-D directly bound to UPIa and competed with FimH for UPIa binding. Consistent with the in vitro data, the exogenous administration of SP-D inhibited UPEC adherence to the bladder and dampened UPEC-induced inflammation in mice. These results support the conclusion that SP-D can protect the bladder urothelium against UPEC infection and suggest a possible function of SP-D in urinary tract.

Objective: To establish an in vivo mouse model of the urinary bladder microcirculation, and characterize the molecular mechanisms of endotoxin-induced leukocyte recruitment., Methods: The murine model was adapted from a technique previously reported for the rat. Mouse bladder microcirculation was observed using intravital microscopy, four hours after intravesical challenge with lipopolysaccharide (LPS) and leukocyte-endothelial interactions were examined. Molecular mechanisms of leukocyte recruitment were identified using antibodies to adhesion molecules and chemokines., Results: LPS from Escherichia coli administered intravesically resulted in a significant increase in leukocyte adhesion and rolling at four hours post stimulation. LPS from Pseudomonas aeruginosa administered at similar doses resulted in a significant, but lower increase in leukocyte adhesion after four hours compared with E. coli LPS. Leukocyte adhesion within the bladder microcirculation was dependent on α(4) -integrins and ICAM-1, whereas leukocyte rolling was P-selectin dependent, but α(4) -integrin independent. Blockade of MIP-2 and KC did not alter leukocyte-endothelial interactions. The bladder endothelium expressed P-selectin, ICAM-1, VCAM-1, MIP-2, and MCP-1. Only VCAM-1 endothelial expression was significantly increased after LPS stimulation., Conclusion: The mouse model of the urinary bladder microcirculation is suitable for the study of inflammatory responses during urinary tract infection (UTI) in vivo., (© 2011 John Wiley & Sons Ltd.)

Ethnopharmacological Relevance: Labisia pumila var. alata (LPva) is a traditional medicinal herb used by Malaysian women to treat many ailments of the genitourinary tract. Its phytoestrogenic properties suggest potential to prevent recurrent urinary tract infection (UTI) in women post menopause. The aim of this study was therefore to investigate the mechanisms of action of LPva in an in vitro model of UTI., Materials and Methods: Bladder epithelial cell lines T24 and 5637 and uropathogenic Escherichia coli (UPEC) strain CFT073 were used to model uroepithelial infection. The ability of LPva to induce programmed cell death was tested using the Annexin-V-FLUOS and TUNEL assays. Expression of caveolin-1, β1 integrin and antimicrobial peptides HBD-2 and LL-37 in response to LPva treatment and/or infection, was assessed using RT real-time PCR. Effects on protein expression were confirmed by Western blot analysis. Sensitivity and yeast agglutination assays were employed to determine if LPva had antimicrobial activities and/or interacted with type 1 fimbriae, respectively. Finally, bacterial adherence and invasion to cells treated with LPva was examined., Results: LPva induced uroepithelial apoptosis which was coupled with upregulated expression of caveolin-1 and downregulation of β1 integrin. LPva did not exhibit direct antimicrobial properties and did not influence antimicrobial peptide levels in cells. Additionally, LPva did not interact with type 1 fimbriae and did not affect adherence in comparison to non-treated control cells. However, LPva significantly reduced the number of intracellular UPEC in bladder epithelial cells., Conclusions: Our findings suggest that LPva has beneficial applications against UPEC infection due to its ability to induce programmed cell death and reduce bacterial invasion of the uroepithelium., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The urinary tract is frequently being exposed to potential pathogens and rapid defence mechanisms are therefore needed. Cathelicidin, a human antimicrobial peptide is expressed and secreted by bladder epithelial cells and protects the urinary tract from infection. Here we show that vitamin D can induce cathelicidin in the urinary bladder. We analyzed bladder tissue from postmenopausal women for expression of cathelicidin, before and after a three-month period of supplementation with 25-hydroxyvitamin D3 (25D3). Cell culture experiments were performed to elucidate the mechanisms for cathelicidin induction. We observed that, vitamin D per se did not up-regulate cathelicidin in serum or in bladder tissue of the women in this study. However, when the bladder biopsies were infected with uropathogenic E. coli (UPEC), a significant increase in cathelicidin expression was observed after 25D3 supplementation. This observation was confirmed in human bladder cell lines, even though here, cathelicidin induction occurred irrespectively of infection. Vitamin D treated bladder cells exerted an increased antibacterial effect against UPEC and colocalization to cathelicidin indicated the relevance of this peptide. In the light of the rapidly growing problem of resistance to common urinary tract antibiotics, we suggest that vitamin D may be a potential complement in the prevention of UTI.

As new and intriguing details of how uropathogens initiate infections and persist within the urinary tract have emerged, so has important information regarding how the immune system functions within the urinary tract. Recent studies have revealed the existence of a multifaceted innate immune response triggered by Toll-like receptor (TLR) 4 on superficial bladder epithelial cells directed at clearing infection by Gram negative pathogens. Other studies have reported that the adaptive immune response in the urinary tract is effective and that vaccines comprised of bacterial virulence factors or whole dead bacteria can evoke protective immunity against urinary tract infections (UTIs) in animals and humans. As antibiotic therapy becomes increasingly ineffective, modulating the innate and adaptive immune system in the urinary tract using TLR4 ligands and other immunomodulators may become viable options to combat UTIs.

Background: Uropathogenic Escherichia coli (UPEC) is a common cause of urinary-tract infection. The mechanisms by which bacteria cause the symptoms of cystitis remain unclear., Methods: The contractions of isolated rat detrusor strips evoked by electrical field stimulations (EFS) or by exogenous agonists and immunoblotting for the detection of protein expression in the bladder were measured in the short (1 h) and long (24 h) term after the intravesical instillation of J96 (O4:K6) strain or UPEC isolated from patients with acute E. coli pyelonephritis or E. coli lipopolysaccharide (LPS)., Results: One hour after the instillation of UPEC, the level of endothelial nitric oxide synthase (eNOS) and the contractile response, but not protein kinase C (PKC) and extracellular signal-regulated kinase (ERK)-1/2 activation, were higher. Twenty-four hours after UPEC treatment, detrusor contractions were decreased, and inducible (i) NOS protein expression and ERK1/2 phosphorylation, but not PKC activation, were observed. Both aminoguanidine and PD98059 treatment markedly reversed the decrease of EFS- and acetylcholine-evoked detrusor contractions induced by UPEC. The instillation of LPS triggered PKC activation but not ERK1/2 phosphorylation., Conclusions: Short-term intravesical instillation of UPEC enhances detrusor contractions through an eNOS-related pathway, but iNOS-regulated ERK1/2 signaling may be involved in long-term UPEC treatment-induced responses. There are different mechanisms involved in the responses induced by UPEC and LPS.

Aims: To determine the relative density of nerve fibres immunoreactive to growth associated protein-43 (GAP-43, an indicator of neuronal sprouting) in the subepithelium and detrusor of patients with idiopathic detrusor overactivity (IDO). To investigate the effect, if any, of age and previous recurrent bacterial cystitis on neuronal sprouting in such patients., Materials and Methods: A series of 18 women with urodynamically proven IDO (median age 62 years, range 39-85), who were refractory to treatment, underwent cystoscopy and cold cup biopsy. Controls (n=26, median age 65, range 32-79) were females without urgency/urge incontinence, undergoing cystoscopy for other indications. Recurrent proven bacterial cystitis (rUTI) was documented. Frozen sections were stained with specific antibodies to GAP-43 and protein gene product 9.5 (PGP, a general neuronal marker). The area represented by immunoreactive (ir) subepithelial or muscle nerve fibres was measured., Results: The density of GAP-43ir and PGPir nerves did not differ significantly between IDO patients and controls, in either subepithelium or detrusor. The GAP-43ir nerve density (as percent of PGPir) increased significantly with advancing age amongst patients with IDO in the detrusor muscle but not in the subepithelium; density in controls was unaltered. In IDO patients with rUTI, a significant increase in GAP-43 (as percent of PGPir) was observed in the subepithelium., Conclusions: Although we found no evidence of increased neuronal proliferation in patients with IDO generally, the increase in GAP-43 with age and with previous cystitis history suggests that neuronal sprouting is important in some subsets of patients with IDO., (Copyright (c) 2005 Wiley-Liss, Inc.)

Purpose: Bladder wall fibrosis is a sequela of recurrent urinary tract infection (UTI). Inducible nitric oxide synthase (iNOS) has been shown to mediate the fibrotic response to inflammation in other tissues. We determined if iNOS could be involved in the fibrotic response to recurrent UTI., Materials and Methods: Human bladder smooth muscle cells (SMC) were treated with bacterial lipopolysaccharides (LPS) and a mixture of inflammatory cytokines. The level of collagen type III, and the levels of iNOS mRNA, protein and activity were determined. The effect of the iNOS inhibitor aminoguanidine on collagen type III expression was then assessed., Results: Expression of collagen type III, iNOS mRNA and iNOS protein as well as iNOS activity were increased in bladder SMC treated with the combination of LPS and cytokines. The increase in collagen type III expression was inhibited by pretreatment of cells with aminoguanidine., Conclusions: LPS and inflammatory cytokines induce collagen type III expression in an iNOS dependent manner in human bladder SMC. This finding suggests that iNOS may be a critical mediator of the bladder wall fibrotic response to chronic UTI and iNOS inhibitors may be of therapeutic value in patients with chronic UTI.

In rats with chronic urinary tract infections, urine and blood were examined for two classes of compounds (nitrosamines and interferon) which may lead to the development of urothelial hyperplasia and neoplasia. In vitro, Escherichia coli, a Proteus species or a mixture of both were able to induce high levels of interferon which theoretically could reduce the host's cellular immune surveillance. These high levels were not detected in vivo in either short-term (5 hr. to 2 wk.) or long-term (2 wk. to 24 wk.) infected rats. In contrast, N, N dimethylnitrosamine was detected in the majority (greater than or equal to 50 per cent) of long term infected rats after 12 wk. although individual rats showed detectable levels as early as 2 wk. post infection. Sterile human or rat urine supported bacterial growth and subsequent production of N, N dimethylnitrosamine, but only after 16 wk. of subculturing in vitro. Gas chromatography was able to detect small amounts of nitrosamines extracted from urine. The mass spectrometer yielded quantitatively and qualitatively better detection. With long term infections, the appearance of a potential carcinogen, N, N dimethylnitrosamine, occurs in vivo and in vitro and correlates with previous findings that describe the development of hyperplastic and early neoplastic changes in the rat urothelium.

To study the relative concentrations and appearance of immunoglobulin in the bladder and kidneys of rats with acute and chronic urinary tract infections, three groups of female Sprague-Dawley rats were studied. Group I underwent weekly intraurethral inoculation with Escherichia coli 07. Group II received inoculation of 0.2 ml of PBS alone. Group III were unmanipulated controls. Bladder and kidney washings were obtained for bacterial colony counts and organism identification. Bladder and kidney tissue was prepared for immunofluorescent examination using goat or rabbit antirat IgA, IgM, or IgG layered with fluorescent-labeled rabbit antigoat or goat antirabbit serum. Negative immunofluorescent controls substituted PBS for antirat serum. No significant difference was noted between bladder and kidney cultures in groups I and II. Comparison of immunofluorescent intensity between groups I and III demonstrated that IgM intensity was greatest between weeks one and three in bladders and kidneys, then decreased to non-significant levels. Bladder IgG intensity peaked at week four and remained elevated throughout the remainder of the study while renal IgG intensity was significant throughout the study period. Renal and bladder IgA was noted only sporadically. Comparison of culture positive animals to culture negative animals (groups II and III) demonstrated a lower immunofluorescent intensity score for culture negative animals. Discrete cellular immunofluorescence was evident in 19.1% of bladders and compared to 0.6% of kidneys. This study suggests that the immunologic response in the rat to intraurethral inoculation and infection is biphasic (IgM followed by IgG) and that a local cellular immunologic response may exist in the bladder.

The mucopolysaccharide layer produced by the transitional cells coating the bladder plays an important role in the defense mechanism of the lower urinary tract. Carbenoxolone, a drug derived from licorice, has been shown to be beneficial to patients with peptic ulcer disease by stimulating increased mucous production in the stomach. Since the bladder epithelium has an endodermal derivation, the question of whether carbenoxolone has an effect on the mucopolysaccharide layer in the bladder was put forth. Carbenoxolone was shown to provide a protective effect to laboratory-induced lower urinary tract infections in the rabbit model.