Michael Kavran

Autoimmunity to Uroplakin II Causes Cystitis in Mice: A Novel Model of Interstitial Cystitis

BACKGROUND The pathophysiology of interstitial cystitis (IC) is unknown. Deficits in urothelial cell layers and autoimmune mechanisms may play a role. OBJECTIVE To examine whether immunization of mice with recombinant mouse uroplakin II (rmUPK2), a bladder-specific protein, would provoke an autoimmune response sufficient to create an IC phenotype. DESIGN, SETTING, AND PARTICIPANTS RmUPK2 complementary DNA was generated, transferred into a bacterial expression vector, and the generated protein was purified. Eight-week-old SWXJ female mice were immunized with rmUPK2 protein via subcutaneous injection of 200μg of rmUPK2 protein in 200μl of an emulsion. MEASUREMENTS Mice were euthanized 5 wk after immunization. Axillary and inguinal lymph node cells were tested for antigen-specific responsiveness and cytokine production, serum isotype antibody titers against rmUPK2 were determined, and gene expression of inflammatory mediators was measured in the bladder and other organs. For functional analysis, mice were placed in urodynamic chambers for 24-h micturition frequency and total voided urine measurements. RESULTS AND LIMITATIONS Immunization with rmUPK2 resulted in T-cell infiltration of the bladder urothelium and increased rmUPK2-specific serum antibody responses in the experimental autoimmune cystitis (EAC) mice models compared with controls. The ratio of bladder to body weight was increased in EAC mice. Quantitative reverse transcriptase polymerase chain reaction analysis showed elevated gene expression of tumor necrosis factor α, interferon γ, interleukin (IL)-17A, and IL-1β in bladder urothelium but not in other organs. Evaluation of 24-h micturition habits of EAC mice showed significantly increased urinary frequency (p<0.02) and significantly decreased urine output per void (p<0.021) when compared with control mice. CONCLUSIONS Our study showed that a bladder-specific autoimmune response sufficient to induce inflammation and EAC occurs in mice following immunization with rmUPK2. EAC mice displayed significant evidence of urinary frequency and decreased urine output per void. Further phenotype characterization of EAC mice should include evidence for pain and/or afferent hypersensitivity, and evidence of urothelial cell layer damage.

Chronic Pelvic Allodynia is Mediated by CCL2 through Mast Cells in an Experimental Autoimmune Cystitis Model

The cause of chronic pelvic pain in interstitial cystitis/painful bladder syndrome (IC/PBS) remains unclear; autoimmunity is a possible etiology. We have recently shown that injection of a single immunogenic peptide of uroplakin 3A (UPK3A 65-84) induces experimental autoimmune cystitis (EAC) in female BALB/cJ mice that is unique among experimental models in accurately reflecting both the urinary symptoms and pelvic pain of IC/PBS. The aim of this project was to identify the roles of mast cells and mast cell chemoattractant/activator monocyte chemoattractant protein-1 [chemokine (C-C motif) ligand 2 (CCL2)] in the allodynia in this model. We immunized 6- to 8-wk-old female BALB/cJ mice with UPK3A 65-84 peptide and, 5-40 days later, observed increased responses to stimulation of the suprapubic abdominal and hindpaw surfaces with von Frey monofilaments compared with mice injected with adjuvant alone. Suprapubic and hindpaw tactile allodynia responses by EAC mice were blocked by instillation of lidocaine into the bladder but not by lidocaine in the uterus, confirming the bladder as the source of the hypersensitivity. Markedly increased numbers of activated mast cells and expression of CCL2 were found in the bladder after immunization with UPK3A 65-84. Hypersensitive responses were inhibited by mast cell stabilizer cromolyn sodium and antagonists of histamine receptors 1 and 2. Furthermore, BALB/cJ mice with deletion of the Ccl2 or chemokine (C-C motif) receptor 2 gene exhibited markedly reduced allodynia and accumulation of mast cells after UPK3A 65-84 immunization. These results show that UPK3A 65-84 immunization causes chronic visceral allodynia and suggest that it is mediated by CCL2-driven mast cell accumulation in the bladder.

Expression of Monocyte Chemotactic Protein 3 Following Simulated Birth Trauma in a Murine Model of Obesity

OBJECTIVE To determine the effect of obesity on simulated birth trauma in leptin-deficient obese mice as measured by relative monocyte chemotactic protein 3 (MCP-3) expression. MATERIALS AND METHODS A total of 25 wild-type and 25 obese C57BL/6 virgin female mice underwent 1 hour of vaginal distension (VD), sham VD, or anesthesia without VD. Pelvic organ tissues were then harvested either immediately or 24-hours post VD and subsequent real-time polymerase chain reaction analysis was performed. RESULTS Urethral MCP-3 levels in wild-type mice were elevated from baseline at 0 hours with a return to baseline at 24 hours in both VD and sham VD groups. In obese mice, there was a 6-fold elevation in MCP-3 levels at 0 hours after sham VD vs control (P <.05), which then returned to baseline levels at 24 hours. After undergoing VD, MCP-3 levels increased to 6-fold baseline values (P = .002) at 0 hours, with continued elevation in MCP-3 levels to 15 times control levels (P = .0003) at 24 hours. CONCLUSIONS MCP-3 is significantly over-expressed in the urethral tissues of both wild-type and obese mice immediately after any urethral manipulation. At 24 hours, the MCP-3 expression patterns become divergent between VD and sham VD in obese mice. With a greater degree of trauma, MCP-3 continued to rise at 24 hours, suggesting that the underlying obesity resulted in alterations in response to tissue injury, paralleling the degree of injury. Such associations warrant further investigation into the role of MCP-3 as a chemokine for stem cell migration, with implications for subsequent tissue repair mechanisms after birth trauma.

Setting a new standard: Updating the vaginal distention translational model for stress urinary incontinence

The vaginal distention (VD) translational model for postpartum stress urinary incontinence (SUI) is potentially biased for use in evaluating animals with increasing phenotypic size (obesity) due to a fixed VD volume. Our study had three principle and two secondary aims. First, to examine both ex vivo and in vivo catheter pressure changes during volume distention. Secondly, to determine mean pressure at current volume standard for use as target pressure (TP) for VD under isobaric (IB) conditions. Thirdly, to demonstrate feasibility and equivalence of VD at TP versus isovolumetric (IV) standard. Secondary aims were to demonstrate decreased variability (IB vs. IV) and to review the effect of weight.

Short-term diabetes- and diuresis-induced alterations of the bladder are mostly reversible in rats

To determine whether diabetes mellitus‐ and diuresis‐induced alterations in the bladder can be reversed in rats.

Stem cell homing factor, CCL7, expression in mouse models of stress urinary incontinence.

Objectives Animal models of vaginal distention (VD) have demonstrated increased expression of chemokine (C-C motif) ligand 7 (CCL7) In this study, we investigated the expression of CCL7 in mice models of simulated birth trauma–induced urinary incontinence using VD and pudendal nerve transection (PNT). Methods Forty-nine mice were divided into 6 groups: VD, sham VD, PNT, sham PNT, anesthesia, and age-matched controls. The urethra, vagina, and rectum were harvested for the expression of CCL7 immediately or 24 hours after assigned procedure. Venous sampling for quantification of serum CCL7 was also performed. An analysis of variance model was used to compare the relative expression of CCL7 in each group. Results Urethral CCL7 expression in the VD group was significantly higher than control group after 24 hours (P < 0.01). There was no difference in the urethral CCL7 expression in PNT, sham PNT, sham VD, or anesthesia groups compared with the controls. No statistically significant difference was noted in the vaginal and rectal expression of CCL7 between any of the groups except for sham PNT. Statistically significant differences were noted in the serum CCL7 expression in the VD, PNT, and sham PNT (P < 0.01 in all) groups after 24 hours compared with the control group. Conclusions This study demonstrates overexpression of urethral CCL7 after VD but not PNT. This suggests that nerve injury does not contribute to the CCL7 overexpression. The overexpression of CCL7 in the serum of mice after VD suggests a translational potential where CCL7 measurement could be used as a surrogate for injury after delivery.

Functional and morphological alterations of the urinary bladder in type 2 diabetic FVBdb/db mice

AIMS Diabetic bladder dysfunction (DBD) has been extensively studied in animal models of type 1 diabetes. We aimed to examine the functional and morphological alterations of the urinary bladder in a type 2 diabetes model, FVB(db/db) mice. METHODS FVB(db/db) mice and age-matched FVB/NJ control mice were tested at either 12, 24 or 52weeks of age. Body weight, blood glucose and glycated hemoglobin (HbA1c) levels were measured. Bladder function was assessed by measurement of 24-h urination behavior and conscious cystometry. Bladder was harvested for Massons Trichrome staining and morphometric analysis. RESULTS The body weights of FVB(db/db) mice were twice as those of FVB/NJ control mice. The blood glucose and HbA1c levels were higher in FVB(db/db) mice at 12 and 24weeks, but not at 52weeks. A significant increase in the mean volume per void, but decrease in the voiding frequency, in FVB(db/db) mice was observed. Cystometry evaluation showed increased bladder capacity, voided volume, and peak micturition pressure in FVB(db/db) mice compared with FVB/NJ mice. Morphometric analysis revealed a significant increase in the areas of detrusor muscle and urothelium in FVB(db/db) mice. In addition, some FVB(db/db) mice, especially males at 12 and 24weeks, showed small-volume voiding during 24-h urination behavior measurement, and detrusor overactivity in the cystometry measurement. CONCLUSIONS The FVB(db/db) mouse, displaying DBD characterized by not only increased bladder capacity, void volume, and micturition pressure, but also bladder overactivity, is a useful model to further investigate the mechanisms of type 2 diabetes-related bladder dysfunction.

Bladder function in mice with inducible smooth muscle-specific deletion of the manganese superoxide dismutase gene

Manganese superoxide dismutase (MnSOD) is considered a critical component of the antioxidant systems that protect against oxidative damage. We are interested in the role of oxidative stress in bladder detrusor smooth muscle (SM) in different disease states. In this study, we generated an inducible, SM-specific Sod2(-/-) mouse model to investigate the effects of MnSOD depletion on the function of the bladder. We crossbred floxed Sod2 (Sod2(lox/lox)) mice with mice containing heterozygous knock-in of a gene encoding a tamoxifen-activated Cre recombinase in the SM22α promoter locus [SM-CreER(T2)(ki)(Cre/+)]. We obtained Sod2(lox/lox),SM-CreER(T2)(ki)(Cre/+) mice and injected 8-wk-old males with 4-hydroxytamoxifen to induce Cre-mediated excision of the floxed Sod2 allele. Twelve weeks later, SM-specific deletion of Sod2 and depletion of MnSOD were confirmed by polymerase chain reaction, immunoblotting, and immunohistochemistry. SM-specific Sod2(-/-) mice exhibited normal growth with no gross abnormalities. A significant increase in nitrotyrosine concentration was found in bladder SM tissue of SM-specific Sod2(-/-) mice compared with both wild-type mice and Sod2(+/+), SM-CreER(T2)(ki)(Cre/+) mice treated with 4-hydroxytamoxifen. Assessment of 24-h micturition in SM-specific Sod2(-/-) mice revealed significantly higher voiding frequency compared with both wild-type and SM-specific Cre controls. Conscious cystometry revealed significantly shorter intercontraction intervals and lower functional bladder capacity in SM-specific Sod2(-/-) mice compared with wild-type mice. This novel model can be used for exploring the mechanistic role of oxidative stress in organs rich in SM in different pathological conditions.

Local release from affinity-based polymers increases urethral concentration of the stem cell chemokine CCL7 in rats

The protein chemokine (C-C motif) ligand 7 (CCL7) is significantly over-expressed in urethral and vaginal tissues immediately following vaginal distention in a rat model of stress urinary incontinence. Further evidence, in this scenario and other clinical scenarios, indicates CCL7 stimulates stem cell homing for regenerative repair. This CCL7 gradient is likely absent or compromised in the natural repair process of women who continue to suffer from SUI into advanced age. We evaluated the feasibility of locally providing this missing CCL7 gradient by means of an affinity-based implantable polymer. To engineer these polymers we screened the affinity of different proteoglycans, to use them as CCL7-binding hosts. We found heparin to be the strongest binding host for CCL7 with a 0.323 nM dissociation constant. Our experimental approach indicates conjugation of heparin to a polymer backbone (using either bovine serum albumin or poly (ethylene glycol) as the base polymer) can be used as a delivery system capable of providing sustained concentrations of CCL7 in a therapeutically useful range up to a month in vitro. With this approach we are able to detect, after polymer implantation, significant increase in CCL7 in the urethral tissue directly surrounding the polymer implants with only trace amounts of human CCL7 present in the blood of the animals. Whole animal serial sectioning shows evidence of retention of locally injected human mesenchymal stem cells (hMSCs) only in animals with sustained CCL7 delivery, 2 weeks after affinity-polymers were implanted.

MP82-01 A GENETIC FEMALE MOUSE MODEL WITH CONGENITAL GENITOURINARY ANOMALIES AND URINARY INCONTINENCE

INTRODUCTION AND OBJECTIVES: Hedgehog signaling pathway is known to have important role in the urogenital development. Transcription mediators of the pathway, Gli2 and Gli3, have been shown to be heavily involved in proper urogenital sinus formation. Both Gli2 and Gli3 null mice are non-viable, and display severe urogenital ad hindgut malformations. Here, we have generated a compound genetic mutant, Gli2+/-;Gli3D699/+, that is viable well into adulthood, and displaying variable urogenital malformations including urinary incontinence in its females. We aim to characterize the urinary incontinence observed in Gli2+/-; Gli3D699/+ female mice and assess its functional, anatomical, and histological characteristics. METHODS: Gli2+/and Gli3D699/+ mice were crossed to generate the double mutant (Gli2+/-; Gli3D699/+) female mice and wild type female mice were used as comparison controls; which all were verified via Polymerase Chain Reactions. Void measurements, Cystometrogram (CMG) and leak point pressure (LPP) were performed in all genotypes to assess bladder functions. The mice were then sacrificed to harvest the bladders and urethras for gross characterization via ink injection and histological assays. Differences were reported as mean and standard errors of mean (SEM) and analyzed using univariate analysis. Statistical significance set at 0.05. RESULTS: No significant differences between the mutant and wild type mice were detected for 24 hour urinary output [(n1⁄4 13) mean 26.5cc 5 vs ( n1⁄47) mean 22.15cc 6, p1⁄40.13]. CMG studies revealed a decrease in peak micturition pressure values and significantly reduced LPP in Gli2+/-; Gli3D699/+ mice compared to wild type mice [(n1⁄45) 4.28 cmH2O 2.4 vs (n1⁄44) 20.24 cmH2O 6.45, p<0.0001; (n1⁄45) 6.66 cmH2O 1.6 vs (n1⁄45) 26.5cmH2O 5, p<0.05; respectively]. Gross characterization revealed that the ano-genital distance was severely reduced in double mutant mice; however, the urethra, vagina, and anus all remain separate and distinctly identifiable in these mice. Histological analyses revealed Gli2+/-; Gli3D699/+ mice exhibited a widened urethra and a decrease in smooth muscle layer thickness in the bladder outlet and urethra, with increased mucosal folding. CONCLUSIONS: Gli2+/-; Gli3D699/+ female mice display persistent urinary incontinence with evident malformation of the bladder outlet and urethra. This presents a genetic mouse model for female urinary incontinence and alludes to potential genetic factors involved in the human condition.