Kirk M. McHugh

Ribonuclease 7 is a potent antimicrobial peptide within the human urinary tract

Although the urinary tract is constantly challenged by microbial invasion, it remains free from colonization. Although little is known about how the urinary tract maintains sterility, the presence of antimicrobial peptides (AMPs) in the urine suggests that they may play a role in its protection from infection. Ribonuclease 7 (RNase 7) is a potent AMP that was first identified in the skin. Here, we characterize the expression and relevance of RNase 7 in the human kidney and urinary tract. Using RNA isolated from healthy human tissue, we performed quantitative real-time PCR and found basal RNASE7 expression in kidney and bladder tissue. Immunohistochemical and immunofluorescent analysis localized RNase 7 to the urothelium of the bladder, ureter, and the intercalated cells of the collecting tubules. In control urine samples from healthy individuals, the concentration of RNase 7 was found to be in the low micromolar range; very abundant for an AMP. Antibacterial neutralization assays showed that urinary RNase 7 has potent antimicrobial properties against Gram-negative and Gram-positive uropathogenic bacteria. Thus, RNase 7 is expressed in the human kidney and urinary tract and it may have an important antimicrobial role in maintaining tract sterility.

Role of fibroblast growth factor receptor 2 in kidney mesenchyme.

Conditional deletion of murine fibroblast growth factor receptors (Fgfrs) 1 and 2 in metanephric mesenchyme leads to renal agenesis with unbranched ureteric buds; however, there are occasionally two buds per nephric duct. Our goal was to determine whether conditional deletion of Fgfr1 or Fgfr2 alone resulted in multiple ureteric bud induction sites. Although deletion of Fgfr1 alone results in no abnormalities, loss of Fgfr2 often leads to multiple ureteric buds and anomalies including renal aplasia, misshaped kidneys, partially duplicated kidneys, duplicated ureters, and obstructed hydroureter. Deletion of Fgfr2 did not change expression domains of glial cell line-derived neurotrophic factor (GDNF), Robo2, bone morphogenetic protein 4, or Sprouty1, all of which regulate ureteric bud induction. Cultured Fgfr2 mutant nephric ducts were also not more sensitive to exogenous GDNF than controls. Whole mount in situ hybridization revealed that in mutant embryos, Fgfr2 was deleted from stromal cells around the nephric duct and ureteric bud base, which correlates well with the ureteric bud induction abnormalities. Thus, Fgfr2 is critical in ensuring that there is a single ureteric bud from the nephric duct. The plethora of later stage defects in Fgfr2 conditional knockouts is reminiscent of many human cases of genetic urogenital anomalies.

An illustrated anatomical ontology of the developing mouse lower urogenital tract

Malformation of the urogenital tract represents a considerable paediatric burden, with many defects affecting the lower urinary tract (LUT), genital tubercle and associated structures. Understanding the molecular basis of such defects frequently draws on murine models. However, human anatomical terms do not always superimpose on the mouse, and the lack of accurate and standardised nomenclature is hampering the utility of such animal models. We previously developed an anatomical ontology for the murine urogenital system. Here, we present a comprehensive update of this ontology pertaining to mouse LUT, genital tubercle and associated reproductive structures (E10.5 to adult). Ontology changes were based on recently published insights into the cellular and gross anatomy of these structures, and on new analyses of epithelial cell types present in the pelvic urethra and regions of the bladder. Ontology changes include new structures, tissue layers and cell types within the LUT, external genitalia and lower reproductive structures. Representative illustrations, detailed text descriptions and molecular markers that selectively label muscle, nerves/ganglia and epithelia of the lower urogenital system are also presented. The revised ontology will be an important tool for researchers studying urogenital development/malformation in mouse models and will improve our capacity to appropriately interpret these with respect to the human situation. SUMMARY: The developmental anatomy of the lower urinary and reproductive systems of developing and postnatal mice is described, providing a revised ontology to aid the understanding of human urogenital tract abnormalities.

High Incidence of Vesicoureteral Reflux in Mice With Fgfr2 Deletion in Kidney Mesenchyma

PURPOSE Mice with Fgfr2 conditional deletion in metanephric mesenchyma (Fgfr2(Mes-/-)) have ureteral bud induction abnormalities. We determined whether Fgfr2(Mes-/-) mutants developed abnormally positioned ureters predisposing to vesicoureteral reflux. MATERIALS AND METHODS We measured common nephric duct length and assayed for apoptosis in embryonic day 11.5 mice. We performed 3-dimensional reconstruction of, and real-time polymerase chain reaction and whole mount in situ hybridization for Fgfr2 in urinary tracts in embryonic day 15.5 embryos. We also performed cystograms followed by 3-dimensional reconstruction in postnatal animals. RESULTS Compared with controls Fgfr2(Mes-/-) embryos had increased common nephric duct length with no difference in apoptosis, indicating cranially displaced ureteral buds. Three-dimensional reconstruction at embryonic day 15.5 showed low ureteral insertion into the bladder near the bladder neck in Fgfr2(Mes-/-) mice. Postnatal Fgfr2(Mes-/-) mutants had a high rate of vesicoureteral reflux compared with controls (47.4% vs 4.0%, p = 0.00006). In postnatal mutants with unilateral reflux the refluxing ureters inserted closer to the bladder neck than nonrefluxing ureters. External ureteral insertional angles at the outer bladder wall formed by the ureteral insertion points and the bladder neck were greater in mutant refluxing ureters than in contralateral nonrefluxing ureters or control ureters. At embryonic day 15.5 Fgfr2 was decreased in Fgfr2(Mes-/-) kidneys compared with that in controls but not statistically different in ureters or bladders. CONCLUSIONS Fgfr2(Mes-/-) mice have ureteral induction abnormalities associated with abnormal ureteral insertion in the bladder and subsequent vesicoureteral reflux, consistent with the Mackie and Stephens hypothesis.

A Molecular Marker for the Development of Interstitial Cystitis in a Rat Model: Isoactin Gene Expression

PURPOSE To determine whether the differential expression of bladder smooth muscle isoactin can be used as a molecular marker for the development of interstitial cystitis (IC). METHODS Three groups of five female Sprague-Dawley rats each underwent urethral catheterization and intravesical instillation of 0.5 ml. of 0.4N HCl. One group was sacrificed one, two and four weeks after the application of HCl, and their bladders harvested for histologic examination and evaluation using Northern blot analysis of bladder smooth muscle isoactins. Five control animals were sacrificed and their bladders harvested to establish isoactin gene expression of bladder smooth muscle in the normal state. The bladders of the rats in each group were excised, immediately frozen in liquid nitrogen, pooled, then stored -70 degrees C until needed for RNA isolation. Isoactin cDNA probes have been developed, therefore isoactin specific cDNA insert fragments were isolated and insert DNA was purified by gel electrophoresis. Total cellular RNA was isolated from 1.0 gm. of bladder smooth muscle from each group. After spectrophotometric quantification, Northern Blot analysis was performed using 2% agarose-formaldehyde gels and Biotrans nylon membranes. Two complete Northern Blot series were run on a single gel and blotted to a single membrane to eliminate gel and blotting discrepancies. RESULTS Microscopic histologic analysis revealed detrusor mastocystosis and eosinophilia as has been noted in humans with chronic interstitial cystitis. Two weeks after the intravesical application of hydrochloric acid, the relative expression of gamma-smooth muscle isoactin was noted to increase by 1.7-fold, while alpha-smooth muscle isoactin expression increased by a factor of 9. These effects were seen to stabilize four weeks after acid application. CONCLUSIONS The intravesical application of dilute HCl in rats results in a histologic appearance which mimics that seen in humans with interstitial cystitis. The appearance of detrusor mastocytosis and eosinophilia was accompanied by a relative decrease in the expression of gamma- and a relative increase in alpha-smooth muscle isoactin gene expression. This pattern of smooth muscle isoactin expression is consistent with a more immature and possibly synthetic smooth muscle phenotype, which may be responsible for the clinical presentation of those with IC. Northern blot analysis of bladder smooth muscle cells may serve as an effective marker for the development of interstitial cystitis in humans.

Three-Dimensional Imaging Reveals Ureteric and Mesenchymal Defects in Fgfr2-Mutant Kidneys

Current techniques to morphologically characterize the processes of nephrogenesis and ureteric branching during kidney development have many limitations. Here, we used in vivo three-dimensional analysis to study renal development in mice lacking fibroblast growth factor receptor 2 in the ureteric bud (Fgfr2(UB-/-)) and in littermate controls. We found that Fgfr2(UB-/-) mice have more severe defects in ureteric branching morphogenesis than previously reported, including significantly fewer branches and tips than control mice. Furthermore, these mice had decreased ureteric volume and surface area and longer ureteric segments than control mice. We also observed previously unrecognized abnormalities in nephrogenesis, including a gradual increase in volume and surface area during maturation from renal vesicles to mature nephrons, in the mutant mice. Finally, we quantified many events of normal renal development that are either difficult or impossible to measure without this three-dimensional technique. In summary, the three-dimensional approach is a powerful and quantitative means to characterize branching morphogenesis and nephrogenesis.

Identification of a Unique Transgenic Mouse Line That Develops Megabladder, Obstructive Uropathy, and Renal Dysfunction

Urinary tract malformations, obstructive uropathy, and hypoplasia/dysplasia are extremely important in terms of pediatric health care costs, with end-stage renal failure in children estimated to cost >

Ribonucleases 6 and 7 have antimicrobial function in the human and murine urinary tract

15 billion annually in the United States alone. Even so, little is known regarding the mechanisms that control these processes. Identified was a unique mutant mouse model that develops in utero megabladder, resulting in variable hydroureteronephrosis and chronic renal failure secondary to obstructive uropathy. These animals, designated mgb for megabladder, possess a primary defect in bladder smooth muscle development that is apparent by embryonic day 15. The mgb mouse represents an excellent model for the study of normal and pathogenic bladder development, including the postnatal progression of chronic renal failure that results from the development of in utero obstructive uropathy.

Expression and antimicrobial function of beta-defensin 1 in the lower urinary tract.

Recent evidence suggests antimicrobial peptides protect the urinary tract from infection. Ribonuclease 7 (RNase 7), a member of the RNase A superfamily, is a potent epithelial-derived protein that maintains human urinary tract sterility. RNase 7 expression is restricted to primates, limiting evaluation of its antimicrobial activity in vivo. Here we identified Ribonuclease 6 (RNase 6) as the RNase A Superfamily member present in humans and mice that is most conserved at the amino acid level relative to RNase 7. Like RNase 7, recombinant human and murine RNase 6 has potent antimicrobial activity against uropathogens. Quantitative real-time PCR and immunoblot analysis indicate that RNase 6 mRNA and protein are up-regulated in the human and murine urinary tract during infection. Immunostaining located RNase 6 to resident and infiltrating monocytes, macrophages, and neutrophils. Uropathogenic E. coli induces RNase 6 peptide expression in human CD14+ monocytes and murine bone marrow derived macrophages. Thus, RNase 6 is an inducible, myeloid-derived protein with markedly different expression from the epithelial-derived RNase 7 but with equally potent antimicrobial activity. Our studies suggest RNase 6 serves as an evolutionarily conserved antimicrobial peptide that participates in the maintenance of urinary tract sterility.

Pathogenesis of Renal Injury in the Megabladder Mouse: A Genetic Model of Congenital Obstructive Nephropathy

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.