Urinary Bladder Dysfunction in Transgenic Sickle Cell Disease Mice

RESEARCH ARTICLE Urinary Bladder Dysfunction in Transgenic Sickle Cell Disease Mice Mário Angelo Claudino1,4, Luiz Osório Silveira Leiria1, Fábio Henrique da Silva2, Eduardo Costa Alexandre1, Andre Renno1, Fabiola Zakia Mónica1, Gilberto de Nucci1, Kleber Yotsumoto Fertrin3, Edson Antunes1, Fernando Ferreira Costa2, Carla Fernanda FrancoPenteado2* 1 Department of Pharmacology, State University of Campinas, Campinas, SP, Brazil, 2 Hematology and Hemotherapy Center, State University of Campinas, Campinas, SP, Brazil, 3 Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas, Campinas, SP, Brazil, 4 Laboratory of Multidisciplinary Research, São Francisco University Medical School, Bragança Paulista, SP, Brazil a11111 * Abstract OPEN ACCESS Citation: Claudino MA, Leiria LOS, da Silva FH, Alexandre EC, Renno A, Mónica FZ, et al. (2015) Urinary Bladder Dysfunction in Transgenic Sickle Cell Disease Mice. PLoS ONE 10(8): e0133996. doi:10.1371/journal.pone.0133996 Editor: Agustin Guerrero-Hernandez, Cinvestav-IPN, MEXICO Received: May 12, 2015 Background Urological complications associated with sickle cell disease (SCD), include nocturia, enuresis, urinary infections and urinary incontinence. However, scientific evidence to ascertain the underlying cause of the lower urinary tract symptoms in SCD is lacking. Objective Thus, the aim of this study was to evaluate urinary function, in vivo and ex vivo, in the Berkeley SCD murine model (SS). Accepted: July 4, 2015 Published: August 4, 2015 Copyright: © 2015 Claudino et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This study was supported by grants from the Brazilian funding bodies: Fundação de Amparo a Pesquisa do Estado de São Paulo (www.fapesp.br), Grant number: 2008/57441-0 FFC, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (www.cnpq.br), Grant number: 481761/ 2008-0 CFFP. Competing Interests: The authors have declared that no competing interests exist. Methods Urine output was measured in metabolic cage for both wild type and SS mice (25-30 g). Bladder strips and urethra rings were dissected free and mounted in organ baths. In isolated detrusor smooth muscle (DSM), relaxant response to mirabegron and isoproterenol (1nM10μM) and contractile response to (carbachol (CCh; 1 nM-100μM), KCl (1 mM-300mM), CaCl2 (1μM-100mM), α,β-methylene ATP (1, 3 and 10 μM) and electrical field stimulation (EFS; 1-32 Hz) were measured. Phenylephrine (Phe; 10nM-100μM) was used to evaluate the contraction mechanism in the urethra rings. Cystometry and histomorphometry were also performed in the urinary bladder. Results SS mice present a reduced urine output and incapacity to produce typical bladder contractions and bladder emptying (ex vivo), compared to control animals. In DSM, relaxation in response to a selective β3-adrenergic agonist (mirabegron) and to a non-selective β-adrenergic (isoproterenol) agonist were lower in SS mice. Additionally, carbachol, α, β-methylene ATP, KCl, extracellular Ca2+ and electrical-field stimulation promoted smaller bladder PLOS ONE | DOI:10.1371/journal.pone.0133996 August 4, 2015 1 / 15 Underactive Bladder in Sickle Mice contractions in SS group. Urethra contraction induced by phenylephrine was markedly reduced in SS mice. Histological analyses of SS mice bladder revealed severe structural abnormalities, such as reductions in detrusor thickness and bladder volume, and cell infiltration. Conclusions Taken together, our data demonstrate, for the first time, that SS mice display features of urinary bladder dysfunction, leading to impairment in urinary continence, which may have an important role in the pathogenesis of the enuresis and infections observed the SCD patients. Introduction Sickle cell disease (SCD), an inherited disorder of hemoglobin synthesis, is caused by a single nucleotide substitution (GTG for GAG) in the sixth codon of the β-globin gene. This mutation results in the substitution of valine for glutamic acid on the surface of the variant β-globin chain [1]. The multiple pleiotropic effects of the abnormal hemoglobin S production include vaso-occlusive crisis, stroke, pulmonary hypertension, osteonecrosis, leg ulcers and priapism [1–3]. SCD-associated urological complications have also been described, such as nocturia, enuresis, increased frequency of urinary infections and urinary incontinence [4]. Among the SCD patients presenting urinary tract infection, one to two thirds exhibit recurrent infection that may be accompanied by fever [5]. There is also an increased incidence of urinary tract infection during pregnancy in sickle cell trait and SCD [6–9]. As part of the renal complications of sickling, renal medullary infarcts lead to decreased ability to concentrate urine, yielding higher daily urinary volumes [10], compensatory polydipsia, and possibly the need for nocturnal bladder voiding [4]. Additionally, in SCD, there is a strong association between enuresis and overactive bladder symptoms such as daytime incontinence, urgency and frequency [11– 15]. However, scientific evidence to ascertain the underlying cause of the lower urinary tract symptoms (LUTS) in SCD patients is lacking. Some hypotheses to explain LUTS in these patients include inability of the kidneys to concentrate urine, social and genetic factors, delays in neurophysiological development, and urinary bladder dysfunction [16, 17] [4, 18]. The abilities of the lower urinary tract to store and to release urine are regulated by neural circuits located in the brain, spinal cord and peripheral ganglia. The sacral parasympathetic outflow provides the main excitatory input to the urinary bladder via the release of both cholinergic and non-adrenergic, non-cholinergic transmitters [19–21]. Detrusor smooth muscle (DSM) expresses muscarinic M2 and M3 receptors in a variety of animal species, but M3 receptors have been reported to be functionally more important for urinary bladder contractions and efficient emptying than M2 receptors [22, 23]. Non-cholinergic excitatory transmission mediated by ATP via purinergic P2X receptors in DSM may also contribute to bladder contractions [24]. Sympathetic innervation of the bladder arises in the thoracolumbar outflow of the spinal cord and releases noradrenaline, which activates inhibitory β-2 and β-3 adrenoceptors in DSM, causing bladder relaxation and contributing to the urine storage phase [19]. In addition, sympathetic stimulation will also stimulate α1-adrenoceptors in the urethra to provide bladder outlet resistance and prevent involuntary leakage of urine [25]. Changes to the contractile and relaxant mechanisms of the urethra and DSM may lead to either overacti (...truncated)