Genetic and environmental factors driving congenital solitary functioning kidney.

BACKGROUND: Congenital solitary functioning kidney (CSFK) is an anomaly predisposing to hypertension, albuminuria and chronic kidney disease. Its aetiology is complex and includes genetic and environmental factors. The role of gene-environment interactions (G×E), although relevant for other congenital anomalies, has not yet been investigated. Therefore, we performed a genome-wide G×E analysis with six preselected environmental factors to explore the role of these interactions in the aetiology of CSFK. METHODS: In the AGORA (Aetiologic research into Genetic and Occupational/environmental Risk factors for Anomalies in children) data- and biobank, genome-wide single-nucleotide variant (SNV) data and questionnaire data on prenatal exposure to environmental risk factors were available for 381 CSFK patients and 598 healthy controls. Using a two-step strategy, we first selected independent significant SNVs associated with one of the six environmental risk factors. These SNVs were subsequently tested in G×E analyses using logistic regression models, with Bonferroni-corrected P-value thresholds based on the number of SNVs selected in step one. RESULTS: In step one, 7-40 SNVs were selected per environmental factor, of which only rs3098698 reached statistical significance (P = .0016, Bonferroni-corrected threshold 0.0045) for interaction in step two. The interaction between maternal overweight and this SNV, which results in lower expression of the Arylsulfatase B (ARSB) gene, could be explained by lower insulin receptor activity in children heterozygous for rs3098698. Eight other G×E interactions had a P-value <.05, of which two were biologically plausible and warrant further study. CONCLUSIONS: Interactions between genetic and environmental factors may contribute to the aetiology of CSFK. To better determine their role, large studies combining data on genetic and environmental risk factors are warranted.

Uncovering risk factors for kidney injury in children with a solitary functioning kidney.

Children with a solitary functioning kidney (SFK) have an increased risk of kidney injury. The exact risk of and risk factors for kidney injury remain unknown, which impedes personalized care. Here, we recruited a nationwide multicenter cohort of 944 patients with SFK to get more insight into this by consenting patients born in 1993-2020 and diagnosed with congenital or acquired SFK before adulthood. The median follow-up was 12.8 years and four indications of kidney injury were studied: urine protein-creatinine ratios, blood pressure, estimated glomerular filtration rate and use of anti-hypertensive/proteinuric medication. For each indicator except medication use, separate cut-off values for any injury and severe injury were used. Survival analyses indicated that at 18 years of age, any or severe kidney injury were present in 75% and 39% of patients with congenital SFK, respectively. Risk factors for kidney injury included kidney agenesis as cause of the SFK, anomalies in the SFK, and high body mass index at last follow-up. Kidney agenesis and being overweight were specifically associated with proteinuria and high blood pressure, whereas anomalies in the SFK were associated with reduced estimated glomerular filtration rates. The high prevalence of kidney injury in patients with SFK emphasizes the need for long-term follow-up, in which lifestyle is an important topic to address. More research into the etiological role of risk factors will help to translate our findings into individualized care strategies. Thus, our study shows that a significant proportion of children with SFK will develop kidney injury over time.

Environmental and parental risk factors for congenital solitary functioning kidney - a case-control study.

BACKGROUND: The etiology of congenital solitary functioning kidney (CSFK) is largely unknown but likely includes various risk factors. We performed a case-control study to compare exposure to environmental and parental risk factors during embryonic kidney development between children with CSFK and healthy controls. METHODS: We included 434 children with CSFK and 1302 healthy controls from the AGORA data- and biobank matched on year of birth. Exposure to potential risk factors was investigated using parental questionnaire data. Crude and adjusted odds ratios (aORs) with 95% confidence intervals (CIs) were estimated for each potential risk factor. Multiple imputation was used to deal with missing values. Confounders for each potential risk factor were selected using directed acyclic graphs. RESULTS: Maternal stress was newly identified as a risk factor for CSFK (aOR 2.1, 95% CI 1.2-3.5). Known associations with conception using in vitro fertilization/intracytoplasmic sperm injection (aOR 1.8, 95% CI 1.0-3.2), maternal infections during pregnancy (aOR 2.5, 95% CI 1.4-4.7), smoking during pregnancy (aOR 1.4, 95% CI 1.0-2.0), and parental CAKUT (aOR 6.6, 95% CI 2.9-15.1) were confirmed, but previous associations with diabetes and obesity could not be replicated. Folic acid supplement use and younger maternal age seemed to reduce the risk of CSFK (aORs 0.7, 95% CI 0.5-1.0, and 0.8, 95% CI 0.6-1.0, respectively). CONCLUSIONS: Environmental and parental risk factors are likely to be involved in the development of CSFK and future studies should combine genetic, environmental, and gene-environment interaction analyses. Women wanting to become pregnant should consider optimizing their health and lifestyle. A higher-resolution version of the Graphical abstract is available as Supplementary information.

Kidney injury rates after unilateral nephrectomy in childhood-a systematic review and meta-analysis.

BACKGROUND: Unilateral nephrectomy is a relatively common procedure in children which results in a solitary functioning kidney (SFK). Living with an SFK predisposes to kidney injury, but it remains unknown which children are most at risk. We aimed to investigate kidney injury rates in patients who underwent unilateral nephrectomy in childhood and to investigate differences among nephrectomies performed for a congenital anomaly, malignancy or other condition. METHODS: MEDLINE and EMBASE were searched for studies reporting kidney injury rates [i.e. proteinuria, hypertension and/or a decreased glomerular filtration rate (GFR)] of patients who underwent unilateral nephrectomy during childhood. Studies including five or more patients with at least 12 months of follow-up were eligible. Analyses were performed using random effects models and stratified by indication for nephrectomy. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Meta-analysis Of Observational Studies in Epidemiology (MOOSE) guidelines were used for reporting. RESULTS: Over 5000 unique articles were screened, of which 53 studies reporting on >4000 patients were included in the analyses. Proteinuria, hypertension and a decreased GFR were present in 15.3, 14.5 and 11.9% of patients, respectively. Heterogeneity among the studies was large in several subgroups, impairing quantitative meta-analyses. However, none of our analyses indicated differences in injury rates between a congenital anomaly or malignancy as an indication for nephrectomy. CONCLUSIONS: Unilateral nephrectomy during childhood results in signs of kidney injury in >10% of patients, with no clear difference between the indications for nephrectomy. Therefore, structured follow-up is necessary in all children who underwent nephrectomy, regardless of the indication.

The functioning and the complication rate of extreme long existing urinary diversions.

PURPOSE OF REVIEW: This review is timely and relevant because many patients live many years with urinary diversions. Knowledge about the long term outcome with respect to function and complications are important for patient counseling and for the manner to follow-up patients. This study was performed to investigate the functioning of urinary diversions constructed > 25 years earlier. RECENT FINDINGS: Most studies have a relatively shorter follow-up, mainly focussing on short term postoperative complications. Focussing on the long term, urinary tract infections (UTI) including pyelonephritis are common. Mild kidney function deterioration is described. SUMMARY: Retrospective study (2018-2019); 43 patients with regular follow-up at the Radboud University Medical Centre Nijmegen the Netherlands. Ileal conduit (n = 19) and ureterosigmoidostomy (n = 11) are the most common diversion types for reasons such as: bladder exstrophy (n = 15), urinary incontinence (n = 9) and malignancy (n = 8). This series with a median follow-up of 40 years, shows it is possible to live and cope with a urinary diversion for a very long time. Ureterosigmoidostomies give relatively good results. Ileal conduits are functioning properly with acceptable complication rates. 95% suffers from chronic UTI's. Kidney function deterioration was mild. Diversions for benign reasons have more complex complications compared to diversion constructed for malignant reasons. VIDEO ABSTRACT: http://links.lww.com/COU/A32.

Outcome of a thorough screening of lower urinary tract function in all pediatric kidney recipients.

LUTD is one of the possible factors influencing pediatric kidney graft outcomes. This study evaluates the results of a thorough assessment of voiding behavior in pediatric transplants. Data of patients with kidney disease of nephrological origin are compared to those with urological origin. A single-center analysis of pediatric kidney transplants performed from 2005 to the present was executed. Donor and recipient characteristics as well as voiding and drinking habits were documented using FVCs and uroflowmetry with PVR measurements. LUTD was defined by a mean MVV >150% or <65% of the EBC for age, abnormal uroflowmetry, PVR repeatedly >15% of EBC or >20 mL, abnormal voiding patterns or behavior, and presence of LUT symptoms. LUTD was diagnosed in 71% of the 56 screened children and more present in urological origin of kidney disease (100%) compared to nephrological origin (61%, P = .005). Individual presence of LUT symptoms, abnormal voiding behavior, FVC parameters, UTIs, and uroflowmetry/PVR parameters were not different between the two groups. Polyuria after transplantation was seen in 63% of patients, mainly in the first post-transplant years and recipients aged <10 years. Time after transplantation was a significant independent predictive factor for the presence of LUTD. LUTD is common in all pediatric kidney recipients and underestimated in those with a nephrological origin of disease. Active screening, monitoring and a care attention plan prior to transplantation and during follow-up, is advocated to optimize outcomes for all patients.

Sox11 gene disruption causes congenital anomalies of the kidney and urinary tract (CAKUT).

Congenital abnormalities of the kidney and the urinary tract (CAKUT) belong to the most common birth defects in human, but the molecular basis for the majority of CAKUT patients remains unknown. Here we show that the transcription factor SOX11 is a crucial regulator of kidney development. SOX11 is expressed in both mesenchymal and epithelial components of the early kidney anlagen. Deletion of Sox11 in mice causes an extension of the domain expressing Gdnf within rostral regions of the nephrogenic cord and results in duplex kidney formation. On the molecular level SOX11 directly binds and regulates a locus control region of the protocadherin B cluster. At later stages of kidney development, SOX11 becomes restricted to the intermediate segment of the developing nephron where it is required for the elongation of Henle's loop. Finally, mutation analysis in a cohort of patients suffering from CAKUT identified a series of rare SOX11 variants, one of which interferes with the transactivation capacity of the SOX11 protein. Taken together these data demonstrate a key role for SOX11 in normal kidney development and may suggest that variants in this gene predispose to CAKUT in humans.

The effect of a cyclic uniaxial strain on urinary bladder cells.

PURPOSE: Pre-conditioning of a cell seeded construct may improve the functional outcome of a tissue engineered construct for augmentation cystoplasty. The precise effects of mechanical stimulation on urinary bladder cells in vitro are not clear. In this study we investigate the effect of a cyclic uniaxial strain culture on urinary bladder cells which were seeded on a type I collagen scaffold. METHODS: Isolated porcine smooth muscle cells or urothelial cells were seeded on a type I collagen scaffolds and cultured under static and dynamic conditions. A uniform cyclic uniaxial strain was applied to the seeded scaffold using a Bose Electroforce Bio-Dynamic bioreactor. Cell proliferation rate and phenotype were investigated, including SEM analysis, RT-PCR and immunohistochemistry for α-Smooth muscle actin, calponin-1, desmin and RCK103 expression to determine the effects of mechanical stimulation on both cell types. RESULTS: Dynamic stimulation of smooth muscle cell seeded constructs resulted in cell alignment and enhanced proliferation rate. Additionally, expression of α-Smooth muscle actin and calponin-1 was increased suggesting differentiation of smooth muscle cells to a more mature phenotype. CONCLUSIONS: Mechanical stimuli did not enhance the proliferation and differentiation of urothelial cells. Mechanical stimulation, i.e., preconditioning may improve the functional in vivo outcome of smooth muscle cell seeded constructs for flexible organs such as the bladder.

Moderate agreement between bladder capacity assessed by frequency volume charts and uroflowmetry, in adolescent and adult enuresis patients.

AIMS: Frequency Volume Charts (FVC) are recommended for the evaluation of enuretic patients. Although this is a good instrument for the assessment of functional bladder capacity, it is known that patient compliance could introduce problems. Therefore, we assessed whether uroflowmetry and post-void residual volume could replace FVC recordings in specific cases, by comparing the bladder capacity as measured by FVC or uroflowmetry. METHODS: We performed post-hoc analyses using data from a retrospective cohort study, in secondary/tertiary care. This included 907 patients between 2003 and 2013, aged ≥11 years, suffering from enuresis (≥1 wet night/fortnight). Data were collected from the medical files. Bland Altman plots were made to compare the two methods. RESULTS: Agreement between uroflowmetry and FVC was reasonable only when uroflowmetry was between 200 and 450 ml. CONCLUSIONS: For individual clinical purposes, uroflowmetry can be used if values are in this range. For future research, we recommend to keep measuring bladder capacity with an FVC. Neurourol. Urodynam. 36:745-747, 2017. © 2016 Wiley Periodicals, Inc.

Reference values for frequency volume chart and uroflowmetry parameters in adolescent and adult enuresis patients.

AIMS: Reference values of Frequency Volume Chart (FVC) and uroflowmetry parameters for adolescent and adult enuresis patients are lacking. In this study, we aim to describe those parameters, in order to interpret findings from FVCs and uroflowmetries in those patients. METHODS: Retrospective, descriptive cohort study, concerning 907 patients aged 11 years and older, suffering from enuresis of at least one wet night per fortnight, treated in a secondary/tertiary centre, between 2003 and 2013. The main FVC parameters of interest were: maximum voided volume (MVV), 24 hr urine production and nocturnal urine volume (NUV) including first morning void (FMV). Nocturnal polyuria (NP) was defined based on both International Children's Continence Society (ICCS, 2014) and International Continence Society (ICS, 2002) definitions. Data of all patients were collected from the medical files. RESULTS: Age had an impact on diurnal and nocturnal FVC parameters. Median MVV excluding FMV was 250 ml in the youngest, 11-year-old males and 363 ml in the eldest, ≥18-yr-old males. For females, these values were 230 ml and 310 ml. Median 24 hr urine production increased from 1,025 ml to 1,502 ml (males) and from 1,007 ml to 1,557 ml (females). Median NUV showed an increase from 387 ml to 519 ml (males) and from 393 ml to 525 (females). Forty-two percent of men and 30% of women had a small MVV (for age). Prevalence of NP differed when assessed by the ICS or the ICCS definition: following ICS guidelines, NP was present in 96% of our male and 93% of our female population, compared to 27% and 41%, respectively, following ICCS guidelines. CONCLUSIONS: Both small MVV and NP were found frequently in our adolescent and adult enuresis patients, which is in line with the current thoughts on causal factors. NP prevalence is quite different when using ICS or ICCS definitions, respectively. We would like to encourage the development of an unambiguous definition of NP to use both in pediatric and adult urology. Neurourol. Urodynam. 36:463-468, 2017. © 2016 Wiley Periodicals, Inc.

Prioritization and burden analysis of rare variants in 208 candidate genes suggest they do not play a major role in CAKUT.

The leading cause of end-stage renal disease in children is attributed to congenital anomalies of the kidney and urinary tract (CAKUT). Familial clustering and mouse models support the presence of monogenic causes. Genetic testing is insufficient as it mainly focuses on HNF1B and PAX2 mutations that are thought to explain CAKUT in 5­15% of patients. To identify novel, potentially pathogenic variants in additional genes, we designed a panel of genes identified from studies on familial forms of isolated or syndromic CAKUT and genes suggested by in vitro and in vivo CAKUT models. The coding exons of 208 genes were analyzed in 453 patients with CAKUT using next-generation sequencing. Rare truncating, splice-site variants, and non-synonymous variants, predicted to be deleterious and conserved, were prioritized as the most promising variants to have an effect on CAKUT. Previously reported disease-causing mutations were detected, but only five were fully penetrant causal mutations that improved diagnosis. We prioritized 148 candidate variants in 151 patients, found in 82 genes, for follow-up studies. Using a burden test, no significant excess of rare variants in any of the genes in our cohort compared with controls was found. Thus, in a study representing the largest set of genes analyzed in CAKUT patients to date, the contribution of previously implicated genes to CAKUT risk was significantly smaller than expected, and the disease may be more complex than previously assumed.

Tubular Constructs as Artificial Urinary Conduits.

PURPOSE: A readily available artificial urinary conduit might be substituted for autologous bowel in standard urinary diversions and minimize bowel associated complications. However, the use of large constructs remains challenging as host cellular ingrowth and/or vascularization is limited. We investigated large, reinforced, collagen based tubular constructs in a urinary diversion porcine model and compared subcutaneously pre-implanted constructs to cell seeded and basic constructs. MATERIALS AND METHODS: Reinforced tubular constructs were prepared from type I collagen and biodegradable Vicryl® meshes through standard freezing, lyophilization and cross-linking techniques. Artificial urinary conduits were created in 17 female Landrace pigs, including 7 with a basic untreated construct, 5 with a construct seeded with autologous urothelial and smooth muscle cells, and 5 with a free graft formed by subcutaneous pre-implantation of a basic construct. All pigs were evaluated after 1 month. RESULTS: The survival rate was 94%. At evaluation 1 basic and 1 cell seeded conduit were occluded. Urinary flow was maintained in all conduits created with pre-implanted constructs. Pre-implantation of the basic construct resulted in a vascularized tissue tube, which could be used as a free graft to create an artificial conduit. The outcome was favorable compared to that of the other conduits. Urinary drainage was better, hydroureteronephrosis was limited and tissue regeneration was improved. CONCLUSIONS: Subcutaneous pre-implantation of a basic reinforced tubular construct resulted in a vascularized autologous tube, which may potentially replace bowel in standard urinary diversions. To our knowledge we introduce a straightforward 2-step procedure to create artificial urinary conduits in a large animal model.

Interaction between MTHFR 677C>T and periconceptional folic acid supplementation in the risk of Hypospadias.

BACKGROUND: Hypospadias is a congenital malformation with both environmental factors and genetic predisposition involved in the pathogenesis. The role of maternal periconceptional folic acid supplement use in the development of hypospadias is unclear. As folate levels may also be influenced by the C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene, we hypothesize that a gene-environment interaction between this polymorphism and folic acid use is involved in the etiology of hypospadias. METHODS: We conducted a case-control study among 855 hypospadias cases and 713 population-based controls from the AGORA data- and biobank. Folic acid supplement use was derived from maternal questionnaires and infant and maternal DNA was used to determine the MTHFR C677T polymorphism using Taqman assays. We performed separate analyses for different hypospadias phenotypes (anterior/middle/posterior). RESULTS: Hypospadias was neither associated with folic acid use or the MTHFR C677T polymorphism, nor with their interaction. However, we did find an association with middle hypospadias when no supplements were used (odds ratio = 1.6; 95% confidence interval, 1.1-2.4), especially in infants carrying the CT/TT genotype (odds ratio = 2.5; 95% confidence interval, 1.4-4.7). In addition, more infants with these genotypes seemed to have posterior hypospadias, regardless of folic acid use. CONCLUSION: Our study does not suggest a major role for folic acid supplements or the MTHFR C677T polymorphism in the etiology of hypospadias in general, but not using folic acid and/or carrying the MTHFR C677T polymorphism may be associated with middle and posterior hypospadias. Therefore, we stress the importance of studying gene-environment interactions preferably in stratified analyses for different hypospadias phenotypes.

Maternal risk factors involved in specific congenital anomalies of the kidney and urinary tract: A case-control study.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) comprise a heterogeneous group of birth defects with a variety of genetic and nongenetic factors suspected of involvement in the etiology. However, little is known about risk factors in specific CAKUT phenotypes. Therefore, we studied potential maternal risk factors in individual phenotypes within the CAKUT spectrum. METHODS: Questionnaire data were collected from parents of 562 children with CAKUT and 2139 healthy controls within the AGORA data- and biobank. Potential maternal risk factors investigated included folic acid use, overweight and obesity, smoking, alcohol consumption, subfertility, and diabetes mellitus. We performed logistic regression analyses to assess associations between these potential risk factors and CAKUT phenotypes. RESULTS: Increased risks of CAKUT were observed for folic acid use and maternal obesity, while fertility treatment by in vitro fertilization or intrauterine insemination and diabetes diagnosed during pregnancy also seem to be associated with CAKUT. Use of multivitamins reduced the risk (odds ratio [OR], 0.5; 95% confidence interval [CI], 0.2-1.0) as opposed to use of folic acid supplements only (OR, 1.3; 95% CI, 1.0-1.8). Folic acid use was associated with duplex collecting systems (OR, 1.8; 95% CI, 1.0-3.4) and vesicoureteral reflux (OR, 1.8; 95% CI, 1.1-2.9) in particular. A relatively strong association was observed between diabetes during pregnancy and posterior urethral valves (OR, 2.6; 95% CI, 1.1-5.9). CONCLUSION: Use of folic acid only seems to be counterproductive for prevention of CAKUT, in contrast to multivitamin use. Furthermore, we observed differences in risk factor patterns among CAKUT phenotypes, which stress the importance of separate analyses for each phenotype. Birth Defects Research (Part A) 106:596-603, 2016. © 2016 Wiley Periodicals, Inc.

AGORA, a data- and biobank for birth defects and childhood cancer.

BACKGROUND: Research regarding the etiology of birth defects and childhood cancer is essential to develop preventive measures, but often requires large study populations. Therefore, we established the AGORA data- and biobank in the Netherlands. In this study, we describe its rationale, design, and ongoing data collection. METHODS: Children diagnosed with and/or treated for a structural birth defect or childhood cancer and their parents are invited to participate in the AGORA data- and biobank. Controls are recruited through random sampling from municipal registries. The parents receive questionnaires about demographics, family and pregnancy history, health status, prescribed medication, lifestyle, and occupational exposures before and during the index pregnancy. In addition, blood or saliva is collected from children and parents, while medical records are reviewed for diagnostic information. RESULTS: So far, we have collected data from over 6,860 families (3,747 birth defects, 905 childhood cancers, and 2,208 controls). The types of birth defects vary widely and comprise malformations of the digestive, respiratory, and urogenital tracts as well as facial, cardiovascular, kidney, skeletal, and central nervous system anomalies. The most frequently occurring childhood cancer types are acute lymphatic leukemia, Hodgkin and non-Hodgkin lymphoma, Wilms' tumor, and brain and spinal cord tumors. Our genetic and/or epidemiologic studies have been focused on hypospadias, anorectal malformations, congenital anomalies of the kidney and urinary tract (CAKUT), and orofacial clefts. CONCLUSION: The large AGORA data- and biobank offers great opportunities for investigating genetic and nongenetic risk factors for disorders in children and is open to collaborative initiatives. Birth Defects Research (Part A) 106:675-684, 2016. © 2016 Wiley Periodicals, Inc.

Surgical treatment of neurogenic stress urinary incontinence: A systematic review of quality assessment and surgical outcomes.

BACKGROUND: There are many opinions but little firm knowledge about the optimal treatment of neurogenic stress urinary incontinence (NSUI). OBJECTIVE: To scrutinize the quality and surgical outcomes of the available treatment modalities in the published literature. EVIDENCE ACQUISITION: A systematic review of the published literature from Pub Med and Web of Science was undertaken for studies describing surgical treatment of NSUI between 1990 and 2013. A checklist of criteria of methodological and reporting quality of interventions in urological publications was applied to assess quality of the retrieved publications. Surgical outcomes of success, failure, and reoperation were calculated. Statistical analyses included one-way ANOVA and post-hoc tests to determine significant differences between groups. EVIDENCE SYNTHESIS: Thirty studies were identified with Level 3 evidence. The quality of reporting was 43-81%, with significantly higher quality noted in studies published after 2002 (64% vs. 45%, P < 0.0001). None of the studies followed a randomized controlled trial (RCT) design. Three primary surgical procedures were used in 29 of 30 studies: artificial urinary sphincter (AUS), urethral slings, and urethral bulking agents. One study used a ProACT device. AUS was considered more successful than urethral bulking agents (77 ± 15% vs. 27 ± 20%, P = 0.002). Urethral bulking agents reported higher failures than urethral sling procedures (49 ± 16% vs. 21 ± 19%, P = 0.016) and AUS (21 ± 19% vs. 10 ± 11%, P < 0.002). CONCLUSIONS: The quality of evidence obtained from non-RCTs is modest. Surgeries for NSUI have relatively high success rates but also high complication rates in this highly heterogeneous population. More studies using modern techniques are required to update our knowledge.

New technique for needle-less implantation of eukaryotic cells.

BACKGROUND AIMS: On review of the use of stem cells in the literature, promissory outcomes for functional organ recovery in many subspecialties in medicine underscore its therapeutic potential. The application of stem cells through the use of a needle can result in additional scar formation, which is undesired for delicate organs. The present work describes the use of a needle-less stem cell injector with the Immediate Drop on Demand Technology (I-DOT) for cell injection in vitro. METHODS: Mesenchymal stromal cells from human bone marrow were labeled with ethynyl-deoxyuridine (EdU) for 2 days and then were re-suspended. With the use of I-DOT, the cells were applied to type 1 collagen matrices or pig bladder tissue specimens with or without mucosa at different levels of energy. The collagen matrices were analyzed after 4 h and 5 days; bladder tissue specimens were analyzed 4 h after cell implantation. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT) assay was performed immediately after cell application to the collagen matrices. Histological analysis with the use of frozen sections and immunofluorescence was used to localize EdU-labeled cells. RESULTS: A considerable number of cells were detected by use of the MTT assay for collagen matrices. In the collagen matrix, the mean measured depth immediately after application ranged between 210 µm and 489 µm, 220 µm and 270 µm for entire bladder specimens, and 230 µm and 370 µm for bladder without mucosa. Cells survived for up to 5 days in the collagen matrix in both bladder specimens. CONCLUSIONS: Cells can survive during I-DOT application, which suggests that the I-DOT device may be a potentially suitable technology for needle-less cell application onto tissues.

Sequencing of the DKK1 gene in patients with anorectal malformations and hypospadias.

UNLABELLED: Anorectal malformations (ARM) are rare congenital malformations of the gastrointestinal tract. Approximately 60% of the patients have additional congenital malformations, such as hypospadias. A recently published article showed that deletion of one single gene, dickkopf WNT signaling pathway inhibitor-1 (Dkk1), resulted in an imperforate anus with rectourinary fistula and preputial hypospadias in mice. To determine whether DKK1 also plays a role in the etiology of ARM and hypospadias in humans, we sequenced the four exons of the DKK1 gene in 17 patients affected with both ARM and hypospadias. No new potential disease-causing variant was identified. However, we detected a known non-synonymous variant in one patient, which was predicted in silico to be damaging, and the corresponding unaffected amino acid is highly conserved. CONCLUSION: In this human study, a potential interesting non-synonymous variant was found in the DKK1 gene. Whether this variant plays a contributory role in the genesis of ARM or hypospadias would require a much larger study.

Recent advances in ureteral tissue engineering.

Reconstruction of long ureteral defects often warrants the use of graft tissue and extensive surgical procedures to maintain the safe transport of urine from the kidneys to the urinary bladder. Complication risks, graft failure-related morbidity, and the lack of suitable tissue are major concerns. Tissue engineering might offer an alternative treatment approach in these cases, but ureteral tissue engineering is still an underreported topic in current literature. In this review, the most recent published data regarding ureteral tissue engineering are presented and evaluated, with a focus on cell sources, implantation strategies, and (bio)materials.