Predictors of the Late Renal Outcome after Posterior Urethral Valves Ablation in a Developing Country: Long Term Study

Introduction: Posterior urethral valves are the commonest form of obstructive uropathy in pediatrics and a common cause of chronic kidney disease (CKD) during childhood with estimated renal failure rate of 25-40%. This study aims at evaluating long term changes in kidney and bladder functions of children with posterior urethral valves after ablation, and at assessing predictors of late renal outcome, considering challenges in Egypt as a developing country.   Materials and Methods: A retrospective study of 30 surgically managed  PUVs patients who attended at Alexandria University Children’s Hospital for follow up. Patients underwent surgery between 2005 and 2016. Mean postoperative follow up period was 6.7±3.8 years (range 3.1 to 14.6 years). Data collected included age at presentation, clinical presentation, serum creatinine (initial, nadir, and last follow up), eGFR at last follow up, renal ultrasound (initial, and last follow up), voiding cystourethrogram (initial, and last follow up), and urodynamic studies at last follow up.   Results: Thirty patients underwent PUVs ablation at a median age of 9 months. Ten (33.3%) patients were diagnosed antenatally. At the last follow up visit, 14 (46.7%) patients had moderate-severe CKD. Twenty-five (83.3%) patients had abnormalities in their urodynamic studies. Univariate analysis showed the need for re-ablation, use of anti-cholinergics, high initial serum creatinine, high nadir creatinine, presence of VUR, history of febrile UTIs and presence of proteinuria were significantly associated with low eGFR. Multivariate analysis showed that high nadir creatinine and presence of VUR were independent factors associated with lower e-GFR at last follow-up. Antenatal diagnosis was significantly associated with better e-GFR. Conculsion: Nadir creatinine and vesicoureteral reflux have high prognostic value for late renal functions, and antenatal diagnosis is associated with better renal functions in patients with posterior urethral valves. Increasing family awareness, antenatal care facilities,and referal to tertiary care centers are priorities for promoting the antenatal diagnosis and management in developing countries.Facilities and training for prenatal intervention should be encouraged

Tissue Engineering of Rat Bladder Using Marrow-Derived Mesenchymal Stem Cells and Bladder Acellular Matrix

Bladder replacement or augmentation is required in congenital malformations or following trauma or cancer. The current surgical solution involves enterocystoplasty but is associated with high complication rates. Strategies for bladder tissue engineering are thus actively sought to address this unmet clinical need. Because of the poor efficacy of synthetic polymers, the use of bladder acellular matrix (BAM) has been proposed. Indeed when cellular components are removed from xenogenic or allogeneic bladders, the extracellular matrix scaffold thus obtained can be used alone or in combination with stem cells. In this study, we propose the use of BAM seeded with marrow-derived mesenchymal stem cells (MSCs) for bladder tissue engineering. We optimized a protocol for decellularization of bladder tissue from different species including rat, rabbit and swine. We demonstrate the use of non-ionic detergents followed by nuclease digestion results in efficient decellularization while preserving the extracellular matrix. When MSCs were seeded on acellular matrix scaffold, they remained viable and proliferative while adopting a cellular phenotype consistent with their microenvironment. Upon transplantation in rats after partial cystectomy, MSC-seeded BAM proved superior to unseeded BAM with animals recovering nearly 100% normal bladder capacity for up to six months. Histological analyses also demonstrated increased muscle regeneration.ISSN:1932-620

Linguistic translation and validation of the Arabic version of International Female Coital Incontinence Questionnaire (IFCI-Q)

ABSTRACTObjectives Aim of the study was to translate the International Female Coital Incontinence Questionnaire (IFCI-Q) into Arabic (Egyptian) and validate it into among Egyptian population complaining of coital urinary incontinence (CI).Methods Original questionnaire has been translated and back-translated by an expert panel, to produce the Arabic version. A pilot study was performed to make sure the questionnaire was understandable. Sixty patients included in the study were divided into two groups: Group A comprised patients with CI, and Group B comprised females who attended the urology clinic for other complaints, without CI. Reliability of the Arabic IFCI-Q was evaluated for internal consistency using Cronbach alpha coefficient. Test–retest reliability was determined using the Weighted Cohen’s k-test. Discrimination validity was evaluated by comparing scores of patients with those of healthy females not complaining of CI using Mann–Whitney test.Results 83.3% of women of both groups (mean age: 43.1 ± 10.6 yrs [Group A], 38.9 ± 8 [Group B] yrs) reported OAB symptoms, 73.3% had stress urinary incontinence and 46.7% reported mixed urinary incontinence. Regarding Group A, 10 patients had CI during penetration, 12 during orgasm and 8 had both forms of CI. The comparison of the responses between Group A and Group B demonstrated a statistically difference (p < 0.00). The content validity was assessed by a panel of expert functional urologists. The Cronbach’s alpha coefficients for the total score were high (0.9–1), indicating high internal consistency. The difference between the two groups revealed an internal consistency of IFCI-Q of 0.563–0.851. The test–retest procedure revealed that the k-values of each item are very good.Conclusions The Arabic version will allow utilizing this tool in a large population of Arabic-speaking countries, with different ethnic and demographic backgrounds

Derivation and characterization of BAMs from multiple species.

<p>A) Swine, rabbit and rat bladders were decellularized as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111966#s2" target="_blank">methods</a> section. Detergent treatment alone was not sufficient to remove all cellular debris, in particular nuclei, indicated with black arrows (ii). DNase type I was used to remove residual DNA, followed by extensive washes in PBS (iii). Paraffin sections were stained with hematoxylin-eosin (H&E). In all species tested including thick porcine bladders, this protocol resulted in complete removal of cellular components with no obvious damage to extracellular matrix architecture. Scale bars  = 500 µm (swine), 100 µm (rat). B) Confocal microscopy analysis of decellularized rat bladder. In the smooth muscle region of the bladder, the extracellular matrix protein collagen 1 could be detected as long fibrillar proteins that were preserved after decellularizarion. The smooth muscle cell-specific proteins calponin and α-smooth muscle actin (α-SMA, middle and right panels, respectively) were highly expressed in normal bladder tissue but only residual staining could be observed after treatment. DAPI counterstain (white arrows) was also used to confirm complete removal of DNA after treatment. The extracellular matrix protein collagen 4 was observed surrounding and between CD31+ blood vessels in normal bladders and was preserved in BAM. The basement membrane laminin was mainly observed around CD31+ blood vessels in normal bladder but also faintly between them. Although CD31+ endothelial cells were removed in BAM, laminin was preserved sourronding decellularized blood vessels. Asterisks indicate bladder cavity.</p

Histological analysis of transplanted engineered bladders.

<p>A) Engineered bladders from BAM alone and MSCs-seeded BAMs were retrieved at 1 (not shown) and 6 months (shown) and compared to normal bladders. H&E staining reveals that all bladders possess a tri-layered organization including urothelium, lamina propria and smooth muscle. Masson's trichrome staining shows that unseeded BAMs had more fibrosis (**) and myofibroblastic proliferation (*) compared to normal controls and MSCs-seeded BAMs. IHC for pancytokeratins AE1/3 reveals multilayered urothelium in both unseeded and MSCs-seeded BAMs. IHC for α-SMA shows that smooth muscle fibers in MSCs-seeded BAMs were thicker, more organized and more fascicular than in unseeded BAMs. Scale bars  = 150 µm. B) Histomorphometry of SMA+ area over total tissue volume confirms higher smooth muscle regeneration in MSC-seeded BAM group over BAM alone group at six months post-transplant. C) Urothelium layer thickness was comparable in all groups at six months post-transplant.</p

Performance of engineered bladders <i>in vivo</i>.

<p>A) Gross appearance of MSCs-seeded BAMs immediately after transplantation (0 month) and before retrieval (1 month and 6 months). The anastomosis between native (*) and engineered (**) bladder is indicated by a dashed line. Note that the anastomosis is inconspicuous at 1 and 6 months and that the graft appears normal and well vascularized. B) Representative CMG graphs of animals from normal control group, partial cystectomy (PC) group and MSCs-seeded BAM group. Arrowheads indicate micturition. C) Urodynamic data from all animal tested was plotted and compared using Kruskal-Wallis tests. MSCs-seeded BAMs performed better than unseeded BAMs at 6 months. At 6 months, MSCs-seeded BAMs had restored full bladder capacity when compared to normal controls. NS: not significant. D) Bladder compliance of all animals at 6 months shows that MSC-seeded BAM group outperforms BAM alone group.</p

In vitro characterization of rat MSCs alone and on BAM.

<p>A) Rat mesenchymal stem cells (MSCs) isolated from bone marrow flushes by plastic adherence. These cells where shown to possess typical MSC plasticity <i>in vitro</i>, including the capacity to differentiate into Nile Red/PPARγ positive adipocytes, collagen 1/osteocalcin (OC)/Runx2 positive osteoblasts, and collagen 2/Sox-9/FGFR3 positive chondrocytes. Cells at passage three were used. Scale bars  = 100 µm for all panels. B) Rat MSCs at passage three also possessed the capacity to differentiate into smooth muscle cells in vitro when stimulated with TGF-β, as shown by their upregulation of smooth muscle cell markers calponin and α-SMA. C) Flow cytometry performed on passage four rat MSCs confirmed the cells used in subsequent experiments possessed a typical MSC immunophenotype including expression of SMC markers CD44, CD73 and CD105 and were devoid of endothelial, hematopoietic, and monocytic cells (tested using CD31, CD45 and MAC-1, respectively). D) Bladder tissue was decellularized and seeded with MSCs. After seven days in culture, tissues were formalin-fixed and paraffin-embedded for histological analysis (H&E shown). MSCs were found to adhere to and colonize bladder tissue efficiently, as shown by their broad distribution even deep within tissue. BC: bladder cavity. Scale bar  = 250 µm. E) Rat MSCs found in the middle circular (MC) layer of smooth muscle matrix where found to adopt a smooth muscle cell phenotype, including the expression of the smooth muscle cell-specific protein calponin. Scale bar  = 50 µm. F) Rat MSCs adopt a phenotype specific to their localization within acellular bladder matrix. Cells found within the MC layer of smooth muscle express α-SMA whereas cell attached to the mucosal surface or submucosa remain undifferentiated (not shown). Scale bar  = 100 µm. G) Rat MSCs seeded on BAMs for seven days remain alive and proliferative, as suggested by their expression of the proliferation marker Ki67. Scale bar  = 100 µm.</p