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1.  Pathogenesis of Renal Injury in the Megabladder Mouse: A Genetic Model of Congenital Obstructive Nephropathy 
Pediatric research  2010;68(6):500-507.
Congenital obstructive nephropathy (CON) is the most common cause of chronic renal failure in children, often leading to end stage renal disease. The megabladder (mgb) mouse exhibits signs of urinary tract obstruction in utero resulting in the development of hydroureteronephrosis and progressive renal failure following birth. This study examined the development of progressive renal injury in homozygous mgb mice (mgb−/−). Renal ultrasound was utilized to stratify the disease state of mgb−/− mice, while surgical rescue was performed using vesicostomy. The progression of renal injury was characterized using a series of pathogenic markers including α-smooth muscle isoactin (α-SMA), TGF-β1, connective tissue growth factor (CTGF), E-cadherin, F4/80, Wilm’s Tumor 1 (WT-1), and paired box gene 2 (Pax2). This analysis indicated that mgb−/− mice are born with pathologic changes in kidney development that progressively worsen in direct correlation with the severity of hydronephrosis. The initiation and pattern of fibrotic development observed in mgb−/− kidneys appeared distinctive from prior animal models of obstruction. These observations suggest that the mgb mouse represents a unique small animal model for the study of CON.
doi:10.1203/PDR.0b013e3181f82f15
PMCID: PMC3121911  PMID: 20736884
2.  Struvite Urolithiasis and Chronic Urinary Tract Infection in a Murine Model of Urinary Diversion 
Urology  2013;81(5):943-948.
OBJECTIVES
To characterize the clinical course following cutaneous vesicostomy (CV) in megabladder (mgb−/−) mice with functional urinary bladder obstruction.
MATERIALS AND METHODS
Forty-five mgb−/− males underwent CV at a median age of 25 days. The 34 animals that survived longer than 3 days after CV were evaluated by serial observation and renal ultrasound. Moribund animals were euthanized. Urinary bladders and kidneys were analyzed by histopathology, and urine biochemical studies were performed.
RESULTS
At a median of 11 weeks after CV, 35% (12/34) of mgb−/− males became moribund with pelvic masses, which were identified as bladder stones at necropsy. Urine pH was alkaline and microscopy demonstrated struvite crystals. Urine contained Gram-positive cocci, while urine cultures were polymicrobial. Stone composition was chiefly struvite (88–94%) admixed with calcium phosphate. In 40% (2/5) of cases, retained intravesical polypropylene suture was identified as the presumed nidus. No stones were detected in over 100 males prior to CV or in 25 cases when CV was performed using polydioxanone suture. Kidneys from 33% (4/12) of animals with bladder stones contained staghorn calculi. Histopathology from animals with struvite stones demonstrated active cystitis, pyelitis, and chronic pyelonephritis.
CONCLUSIONS
These findings attest to the importance of the nidus in lithogenesis and provide a novel murine model for struvite urolithiasis and chronic infection of the diverted urinary tract.
doi:10.1016/j.urology.2013.02.003
PMCID: PMC3916187  PMID: 23523293
urinary tract infection; pyelonephritis; urinary diversion; struvite; urolithiasis; mouse model
3.  Megabladder mouse model of congenital obstructive nephropathy: genetic etiology and renal adaptation 
Congenital obstructive nephropathy remains one of the leading causes of chronic renal failure in children. The direct link between obstructed urine flow and abnormal renal development and subsequent dysfunction represents a central paradigm of urogenital pathogenesis that has far-reaching clinical implications. Even so, a number of diagnostic, prognostic, and therapeutic quandaries still exist in the management of congenital obstructive nephropathy. Studies in our laboratory have characterized a unique mutant mouse line that develops in utero megabladder, variable hydronephrosis, and progressive renal failure. Megabladder mice represent a valuable functional model for the study of congenital obstructive nephropathy. Recent studies have begun to shed light on the genetic etiology of mgb−/− mice as well as the molecular pathways controlling disease progression in these animals.
doi:10.1007/s00467-013-2658-6
PMCID: PMC3928515  PMID: 24276861
Congenital obstructive nephropathy; Kidney pathogenesis; Animal models of disease; Long-range enhancer
4.  Expression and Antimicrobial Function of Beta-Defensin 1 in the Lower Urinary Tract  
PLoS ONE  2013;8(10):e77714.
Beta defensins (BDs) are cationic peptides with antimicrobial activity that defend epithelial surfaces including the skin, gastrointestinal, and respiratory tracts. However, BD expression and function in the urinary tract are incompletely characterized. The purpose of this study was to describe Beta Defensin-1 (BD-1) expression in the lower urinary tract, regulation by cystitis, and antimicrobial activity toward uropathogenic Escherichia coli (UPEC) in vivo. Human DEFB1 and orthologous mouse Defb1 mRNA are detectable in bladder and ureter homogenates, and human BD-1 protein localizes to the urothelium. To determine the relevance of BD-1 to lower urinary tract defense in vivo, we evaluated clearance of UPEC by Defb1 knockout (Defb1-/-) mice. At 6, 18, and 48 hours following transurethral UPEC inoculation, no significant differences were observed in bacterial burden in bladders or kidneys of Defb1-/- and wild type C57BL/6 mice. In wild type mice, bladder Defb1 mRNA levels decreased as early as two hours post-infection and reached a nadir by six hours. RT-PCR profiling of BDs identified expression of Defb3 and Defb14 mRNA in murine bladder and ureter, which encode for mBD-3 and mBD-14 protein, respectively. MBD-14 protein expression was observed in bladder urothelium following UPEC infection, and both mBD-3 and mBD-14 displayed dose-dependent bactericidal activity toward UPEC in vitro. Thus, whereas mBD-1 deficiency does not alter bladder UPEC burden in vivo, we have identified mBD-3 and mBD-14 as potential mediators of mucosal immunity in the lower urinary tract.
doi:10.1371/journal.pone.0077714
PMCID: PMC3804605  PMID: 24204930
5.  Molecular Basis of Renal Adaptation in a Murine Model of Congenital Obstructive Nephropathy 
PLoS ONE  2013;8(9):e72762.
Congenital obstructive nephropathy is a common cause of chronic kidney disease and a leading indication for renal transplant in children. The cellular and molecular responses of the kidney to congenital obstruction are incompletely characterized. In this study, we evaluated global transcription in kidneys with graded hydronephrosis in the megabladder (mgb−/−) mouse to better understand the pathophysiology of congenital obstructive nephropathy. Three primary pathways associated with kidney remodeling/repair were induced in mgb−/− kidneys independent of the degree of hydronephrosis. These pathways included retinoid signaling, steroid hormone metabolism, and renal response to injury. Urothelial proliferation and the expression of genes with roles in the integrity and maintenance of the renal urothelium were selectively increased in mgb−/− kidneys. Ngal/Lcn2, a marker of acute kidney injury, was elevated in 36% of kidneys with higher grades of hydronephrosis. Evaluation of Ngalhigh versus Ngallow kidneys identified the expression of several novel candidate markers of renal injury. This study indicates that the development of progressive hydronephrosis in mgb−/− mice results in renal adaptation that includes significant changes in the morphology and potential functionality of the renal urothelium. These observations will permit the development of novel biomarkers and therapeutic approaches to progressive renal injury in the context of congenital obstruction.
doi:10.1371/journal.pone.0072762
PMCID: PMC3762787  PMID: 24023768
6.  3-Dimensional Morphometric Analysis of Murine Bladder Development and Dysmorphogenesis 
Developmental Dynamics  2012;241(3):522-533.
Disorders of the urinary tract represent a major cause of morbidity and impaired quality of life. To better understand the morphological events responsible for normal urinary tract development, we performed 3-D reconstructive analysis of developing mouse bladders in control, mgb−/−, and Fgfr2Mes−/− mice. Detrusor smooth muscle differentiation initiated in the bladder dome and progressed caudally with the leading edge extending down the right posterior surface of the bladder. Gender-specific differences in detrusor smooth muscle development were observed during early embryonic development. Bladder trigone morphology transitioned from an isosceles to equilateral triangle during development due to the preferential lengthening of the urethra to ureter distance. The primary defect observed in mgb−/− bladders was a significant reduction in detrusor smooth muscle differentiation throughout development. Deviations from normal trigone morphology correlated best with VUR development in Fgfr2Mes−/− mice, while alterations in intravesicular tunnel length did not. In conclusion, multivariate morphometric analysis provides a powerful tool to quantify and assess urinary tract development.
doi:10.1002/dvdy.23744
PMCID: PMC3288201  PMID: 22275180
bladder; development; detrusor smooth muscle; vesicoureteral reflux
7.  Analysis of the Sonic Hedgehog Signaling Pathway in Normal and Abnormal Bladder Development 
PLoS ONE  2013;8(1):e53675.
In this study, we examined the expression of Sonic Hedgehog, Patched, Gli1, Gli2, Gli3 and Myocardin in the developing bladders of male and female normal and megabladder (mgb−/−) mutant mice at embryonic days 12 through 16 by in situ hybridization. This analysis indicated that each member of the Sonic Hedgehog signaling pathway as well as Myocardin displayed distinct temporal and spatial patterns of expression during normal bladder development. In contrast, mgb−/− bladders showed both temporal and spatial changes in the expression of Patched, Gli1 and Gli3 as well as a complete lack of Myocardin expression. These changes occurred primarily in the outer mesenchyme of developing mgb−/− bladders consistent with the development of an amuscular bladder phenotype in these animals. These results provide the first comprehensive analysis of the Sonic Hedgehog signaling pathway during normal bladder development and provide strong evidence that this key signaling cascade is critical in establishing radial patterning in the developing bladder. In addition, the lack of detrusor smooth muscle development observed in mgb−/− mice is associated with bladder-specific temporospatial changes in Sonic Hedgehog signaling coupled with a lack of Myocardin expression that appears to result in altered patterning of the outer mesenchyme and poor initiation and differentiation of smooth muscle cells within this region of the developing bladder.
doi:10.1371/journal.pone.0053675
PMCID: PMC3538723  PMID: 23308271
8.  Gene-resolution analysis of DNA copy number variation using oligonucleotide expression microarrays 
BMC Genomics  2007;8:111.
Background
Array-based comparative genomic hybridization (aCGH) is a high-throughput method for measuring genome-wide DNA copy number changes. Current aCGH methods have limited resolution, sensitivity and reproducibility. Microarrays for aCGH are available only for a few organisms and combination of aCGH data with expression data is cumbersome.
Results
We present a novel method of using commercial oligonucleotide expression microarrays for aCGH, enabling DNA copy number measurements and expression profiles to be combined using the same platform. This method yields aCGH data from genomic DNA without complexity reduction at a median resolution of approximately 17,500 base pairs. Due to the well-defined nature of oligonucleotide probes, DNA amplification and deletion can be defined at the level of individual genes and can easily be combined with gene expression data.
Conclusion
A novel method of gene resolution analysis of copy number variation (graCNV) yields high-resolution maps of DNA copy number changes and is applicable to a broad range of organisms for which commercial oligonucleotide expression microarrays are available. Due to the standardization of oligonucleotide microarrays, graCNV results can reliably be compared between laboratories and can easily be combined with gene expression data using the same platform.
doi:10.1186/1471-2164-8-111
PMCID: PMC1868757  PMID: 17470268

Results 1-8 (8)