Darius Bagli

Mammalian Target of Rapamycin (mTOR) Induces Proliferation and De-Differentiation Responses to Three Coordinate Pathophysiologic Stimuli (Mechanical Strain, Hypoxia, and Extracellular Matrix Remodeling) in Rat Bladder Smooth Muscle

Maladaptive bladder muscle overgrowth and de-differentiation in human bladder obstructive conditions is instigated by coordinate responses to three stimuli: mechanical strain, tissue hypoxia, and extracellular matrix remodeling.( 1,2) Pathway analysis of genes induced by obstructive models of injury in bladder smooth muscle cells (BSMCs) identified a mammalian target of rapamycin (mTOR)-specific inhibitor as a potential pharmacological inhibitor. Strain-induced mTOR-specific S6K activation segregated differently from ERK1/2 activation in intact bladder ex vivo. Though rapamycins antiproliferative effects in vascular smooth muscle cells are well known, its effects on BSMCs were previously unknown. Rapamycin significantly inhibited proliferation of BSMCs in response to mechanical strain, hypoxia, and denatured collagen. Rapamycin inhibited S6K at mTOR-sensitive phosphorylation sites in response to strain and hypoxia. Rapamycin also supported smooth muscle actin expression in response to strain or hypoxia-induced de-differentiation. Importantly, strain plus hypoxia synergistically augmented mTOR-dependent S6K activation, Mmp7 expression and proliferation. Forced expression of wild-type and constitutively active S6K resulted in loss of smooth muscle actin expression. Decreased smooth muscle actin, increased Mmp7 levels and mTOR pathway activation during in vivo partial bladder obstruction paralleled our in vitro studies. These results point to a coordinate role for mTOR in BSMCs responses to the three stimuli and a potential new therapeutic target for myopathic bladder disease.

Phenotypic switching induced by damaged matrix is associated with DNA methyltransferase 3A (DNMT3A) activity and nuclear localization in smooth muscle cells (SMC).

Extracellular matrix changes are often crucial inciting events for fibroproliferative disease. Epigenetic changes, specifically DNA methylation, are critical factors underlying differentiated phenotypes. We examined the dependency of matrix-induced fibroproliferation and SMC phenotype on DNA methyltransferases. The cooperativity of matrix with growth factors, cell density and hypoxia was also examined. Primary rat visceral SMC of early passage (0–2) were plated on native collagen or damaged/heat-denatured collagen. Hypoxia was induced with 3% O2 (balanced 5% CO2 and 95% N2) over 48 hours. Inhibitors were applied 2–3 hours after cells were plated on matrix, or immediately before hypoxia. Cells were fixed and stained for DNMT3A and smooth muscle actin (SMA) or smooth muscle myosin heavy chain. Illumina 450 K array of CpG sites was performed on bisulfite-converted DNA from smooth muscle cells on damaged matrix vs native collagen. Matrix exquisitely regulates DNMT3A localization and expression, and influences differentiation in SMCs exposed to denatured matrix +/− hypoxia. Analysis of DNA methylation signatures showed that Matrix caused significant DNA methylation alterations in a discrete number of CpG sites proximal to genes related to SMC differentiation. Matrix has a profound effect on the regulation of SMC phenotype, which is associated with altered expression, localization of DNMTs and discrete changes DNA methylation.

Uropathogenic Escherichia coli infection: potential importance of epigenetics

Patients suffering from recurrent urinary tract infections (UTIs) may be maintained on antibiotic prophylaxis, or even treated by surgery. However, there are no biological data on which to base such treatment selection for the individual patient. This highlights the need for a biological marker that might predict UTI recurrence risk. Infection of mammalian tissues with bacteria, viruses and other pathogens results in the modification of the host cell epigenome, particularly DNA methylation. We recently demonstrated that in vitro infection of bladder uroepithelial cells with uropathogenic Escherichia coli results in hypermethylation of the tumor suppressor gene CDKN2A, providing proof-of-concept that uropathogenic E. coli infection modulates the host cell epigenome. If postinfection persistence of UTI-induced uroepithelial DNA hypermethylation were to be associated with subsequent UTI propensity, these epigenetic marks could act as a potential biomarker for UTI recurrence risk and could be used to rationalize and improve treatment of patients with infection-associated uropathies.

Physiological Changes in Transperitoneal Versus Retroperitoneal Laparoscopy in Children: A Prospective Analysis

PURPOSEnThe choice of minimally invasive surgical approaches in pediatric urology is largely influenced by surgeon preference and experience. Little is known about the differences in physiological variables that might objectively influence the choice of surgical approach. We compared the cerebral and systemic hemodynamic effects of transperitoneal vs retroperitoneal CO(2) insufflation in children.nnnMATERIALS AND METHODSnAfter receiving ethical review board approval and written parental consent 36 pediatric patients undergoing transperitoneal (18) or retroperitoneal (18) laparoscopic surgery were enrolled in this study. A standardized anesthetic technique of isoflurane 1 MAC and remifentanil 0.2 mcg/kg per minute was used. Measured parameters included end tidal CO(2), middle cerebral artery blood flow velocity, heart rate and noninvasive mean arterial blood pressure. Transcranial Doppler ultrasound was used to measure middle cerebral artery blood flow velocity. Data were collected before, during and after CO(2) insufflation to 12 mm Hg pneumoperitoneum at regular intervals, including every minute for 10 minutes and every 2 minutes thereafter. Within group analysis was done using repeated measures ANOVA. Nonlinear regression analysis was used to determine the best fit and the relationship of each variable with time with p <0.05 considered significant.nnnRESULTSnPatient age and weight were comparable in the 2 groups. Transperitoneal CO(2) insufflation resulted in a rapid parallel increase in middle cerebral artery blood flow velocity, mean arterial pressure and end tidal CO(2) during the first 8 minutes of pneumoperitoneum (p <0.05). Despite a continued increase in end tidal CO(2) thereafter middle cerebral artery blood flow velocity and mean arterial pressure attained a plateau within the first 8 minutes (p <0.05). In contrast, middle cerebral artery blood flow velocity and end tidal CO(2) increased progressively throughout the retroperitoneal CO(2) insufflation period (p <0.01).nnnCONCLUSIONSnCerebral blood flow velocity and end tidal CO(2) seem to increase progressively and gradually during retroperitoneal laparoscopy, in contrast to the more rapid increase and plateau effect during transperitoneal laparoscopy. Presumably the smaller absorptive surface in the retroperitoneal space explains this physiological difference.

Pathologic Bladder Microenvironment Attenuates Smooth Muscle Differentiation of Skin Derived Precursor Cells: Implications for Tissue Regeneration

Smooth muscle cell containing organs (bladder, heart, blood vessels) are damaged by a variety of pathological conditions necessitating surgery or organ replacement. Currently, regeneration of contractile tissues is hampered by lack of functional smooth muscle cells. Multipotent skin derived progenitor cells (SKPs) can easily be isolated from adult skin and can be differentiated in vitro into contractile smooth muscle cells by exposure to FBS. Here we demonstrate an inhibitory effect of a pathologic contractile organ microenvironment on smooth muscle cell differentiation of SKPs. In vivo, urinary bladder strain induces microenvironmental changes leading to de-differentiation of fully differentiated bladder smooth muscle cells. Co-culture of SKPs with organoids isolated from ex vivo stretched bladders or exposure of SKPs to diffusible factors released by stretched bladders (e.g. bFGF) suppresses expression of smooth muscle markers (alpha SMactin, calponin, myocardin, myosin heavy chain) as demonstrated by qPCR and immunofluorescent staining. Rapamycin, an inhibitor of mTOR signalling, previously observed to prevent bladder strain induced de-differentiation of fully differentiated smooth muscle cells in vitro, inhibits FBS-induced smooth muscle cell differentiation of undifferentiated SKPs. These results suggest that intended precursor cell differentiation may be paradoxically suppressed by the disease context for which regeneration may be required. Organ-specific microenvironment contexts, particularly prevailing disease, may play a significant role in modulating or attenuating an intended stem cell phenotypic fate, possibly explaining the variable and inefficient differentiation of stem cell constructs in in vivo settings. These observations must be considered in drafting any regeneration strategies.

Finding NeMO—Nerve-sparing Mid-urethral Obstruction: A Pathophysiologically Accurate Model of Rodent Partial Bladder Outlet Obstruction

OBJECTIVEnTo develop and evaluate a novel technique modeling partial bladder outlet obstruction (pBOO) using a nerve-sparing mid-urethral obstruction (NeMO) approach.nnnMATERIALS AND METHODSnFemale unoperated rats were compared to rats after NeMO, NeMO sham, proximal urethral (PU) obstruction, or PU sham. Residual volume, bladder capacity, voiding volume, and bladder mass were recorded; the contractile characteristics of isolated bladder strips were also analyzed. Additionally, we quantitated nerve fibers at the bladder neck as well as the extracellular matrix in the bladder wall.nnnRESULTSnNeMO yields a more predictable degree of obstruction vs PU, causes no animal mortality, and is easy to release. NeMO also results in a more moderate increase in bladder mass commensurate with human disease vs the exaggerated response to PU, and does not lead to the excessive bladder dilation observed after PU while showing increased residual urine and fibrosis over time, thus closely modeling human pBOO pathophysiology. Importantly, PU shams significantly incite both an undesirable mass increase as well as bladder dysfunction, correlating with a denervation injury making them unsuitable as controls when modeling a non-neurogenic pBOO. The bladder physiology and structure of NeMO-sham animals were indistinguishable from those of unoperated controls. The low complication rate and low variability of NeMO also can be applied to mice, opening the pBOO field to the full spectrum of transgenic manipulation.nnnCONCLUSIONnNeMO is a pathophysiologically accurate modeling approach, with low variability and mortality, and newly paves the way for realistic and robust interpretation of omics and sequencing analytical methodologies. We therefore suggest NeMO as a new standard model when investigating pBOO.

Do patients with classic bladder exstrophy have fecal incontinence? A web-based study.

OBJECTIVESnTo explore the occurrence of fecal incontinence in patients with classic bladder exstrophy (CBE) by administering a web-based pilot study.nnnMETHODSnA questionnaire assessing fecal continence status was devised. Questions included demographics, age to achieve toilet training for bowels, and the patient perception of the degree of fecal soiling during day and night, if present. The CBE contact list of our institutions social worker was addressed (324 patients) and directed to fill the survey posted at the website http://www.SurveyMonkey.com.nnnRESULTSnThere were 94 responders (29%) to the survey. They were analyzed as 2 groups: pediatric (age up to 18 years, n = 69, 9 excluded for not achieving toilet training) and adult (age >18 years, n = 25). In the pediatric group, fecal incontinence was reported in 57% of patients during the day and 32% during night. In the adult group, fecal incontinence was reported in 44% of patients during the day and 40% during night. Seven patients reported having undergone ureterosigmoidostomy (US) diversion. Stratifying patients based on US diversion showed fecal incontinence of 100% vs 22% during the day (P <.001), and 86% vs 22% during the night (P <.01), for the US vs non-US subgroups, respectively.nnnCONCLUSIONSnOur preliminary survey suggests that fecal incontinence in CBE may be a significant overlooked issue that may persist into adulthood of CBE patients. With the potential functional and psychological burden, clinical awareness and management of this issue is crucial. Further exploration of this issue, with detailed attention to surgical procedure(s) involved and degree of quality of life impairment, needs to be initiated.

Hypertension in children with poorly functioning unilateral kidneys: predictors of resolution after nephrectomy

Study Type – Prognosis (case series)u2028Level of Evidenceu20034

Uropathogenic E. coli (UPEC) Infection Induces Proliferation through Enhancer of Zeste Homologue 2 (EZH2).

Host-pathogen interactions can induce epigenetic changes in the host directly, as well as indirectly through secreted factors. Previously, uropathogenic Escherichia coli (UPEC) was shown to increase DNA methyltransferase activity and expression, which was associated with methylation-dependent alterations in the urothelial expression of CDKN2A. Here, we showed that paracrine factors from infected cells alter expression of another epigenetic writer, EZH2, coordinate with proliferation. Urothelial cells were inoculated with UPEC, UPEC derivatives, or vehicle (mock infection) at low moi, washed, then maintained in media with Gentamycin. Urothelial conditioned media (CM) and extracellular vesicles (EV) were isolated after the inoculations and used to treat naïve urothelial cells. EZH2 increased with UPEC infection, inoculation-induced CM, and inoculation-induced EV vs. parallel stimulation derived from mock-inoculated urothelial cells. We found that infection also increased proliferation at one day post-infection, which was blocked by the EZH2 inhibitor UNC1999. Inhibition of demethylation at H3K27me3 had the opposite effect and augmented proliferation. CONCLUSION: Uropathogen-induced paracrine factors act epigenetically by altering expression of EZH2, which plays a key role in early host cell proliferative responses to infection.

A Review of the Effect of Injected Dextranomer/Hyaluronic Acid Copolymer Volume on Reflux Correction Following Endoscopic Injection

The current literature suggests that multiple variables affect vesicoureteric reflux (VUR) resolution rates following dextranomer/hyaluronic acid copolymer (Dx/HA) injection. This article reviews the evidence pertaining to the effect of injected Dx/HA volume on success rates following endoscopic correction. Lack of prospective studies which use injected volume as a continuous variable coupled with a nonstandardized injection technique and endpoint hinders the ability to reach a definite conclusion.