Deletion of Fibroblast Growth Factor Receptor 2 from the Peri-Wolffian Duct Stroma Leads to Ureteric Induction Abnormalities and Vesicoureteral Reflux

Purpose: Pax3cre-mediated deletion of fibroblast growth factor receptor 2 (Fgfr2) broadly in renal and urinary tract mesenchyme led to ureteric bud (UB) induction defects and vesicoureteral reflux (VUR), although the mechanisms were unclear. Here, we investigated whether Fgfr2 acts specifically in peri-Wolffian duct stroma (ST) to regulate UB induction and development of VUR and the mechanisms of Fgfr2 activity. Methods: We conditionally deleted Fgfr2 in ST (Fgfr2 ST-/- ) using Tbx18cre mice. To look for ureteric bud induction defects in young embryos, we assessed length and apoptosis of common nephric ducts (CNDs). We performed 3D reconstructions and histological analyses of urinary tracts of embryos and postnatal mice and cystograms in postnatal mice to test for VUR. We performed in situ hybridization and real-time PCR in young embryos to determine mechanisms underlying UB induction defects. Results: We confirmed that Fgfr2 is expressed in ST and that Fgfr2 was efficiently deleted in this tissue in Fgfr2 ST-/- mice at embryonic day (E) 10.5. E11.5 Fgfr2 ST-/- mice had randomized UB induction sites with approximately 1/3 arising too high and 1/3 too low from the Wolffian duct; however, apoptosis was unaltered in E12.5 mutant CNDs. While ureters were histologically normal, E15.5 Fgfr2 ST-/- mice exhibit improper ureteral insertion sites into the bladder, consistent with the ureteric induction defects. While ureter and bladder histology appeared normal, postnatal day (P) 1 mutants had high rates of VUR versus controls (75% versus 3%, p = 0.001) and occasionally other defects including renal hypoplasia and duplex systems. P1 mutant mice also had improper ureteral bladder insertion sites and shortened intravesicular tunnel lengths that correlated with VUR. E10.5 Fgfr2 ST-/- mice had decreases in Bmp4 mRNA in stromal tissues, suggesting a mechanism underlying the ureteric induction and VUR phenotypes. Conclusion: Mutations in FGFR2 could possibly cause VUR in humans. © 2013 Walker et al

Comparison of the Gene Expression Profiles from Normal and Fgfrl1 Deficient Mouse Kidneys Reveals Downstream Targets of Fgfrl1 Signaling

Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron

Genome-wide linkage and association study implicates the 10q26 region as a major genetic contributor to primary nonsyndromic vesicoureteric reflux

Abstract Vesicoureteric reflux (VUR) is the commonest urological anomaly in children. Despite treatment improvements, associated renal lesions – congenital dysplasia, acquired scarring or both – are a common cause of childhood hypertension and renal failure. Primary VUR is familial, with transmission rate and sibling risk both approaching 50%, and appears highly genetically heterogeneous. It is often associated with other developmental anomalies of the urinary tract, emphasising its etiology as a disorder of urogenital tract development. We conducted a genome-wide linkage and association study in three European populations to search for loci predisposing to VUR. Family-based association analysis of 1098 parent-affected-child trios and case/control association analysis of 1147 cases and 3789 controls did not reveal any compelling associations, but parametric linkage analysis of 460 families (1062 affected individuals) under a dominant model identified a single region, on 10q26, that showed strong linkage (HLOD = 4.90; ZLRLOD = 4.39) to VUR. The ~9Mb region contains 69 genes, including some good biological candidates. Resequencing this region in selected individuals did not clearly implicate any gene but FOXI2, FANK1 and GLRX3 remain candidates for further investigation. This, the largest genetic study of VUR to date, highlights the 10q26 region as a major genetic contributor to VUR in European populations

Genetic approaches to human renal agenesis/hypoplasia and dysplasia

Congenital abnormalities of the kidney and urinary tract are frequently observed in children and represent a significant cause of morbidity and mortality. These conditions are phenotypically variable, often affecting several segments of the urinary tract simultaneously, making clinical classification and diagnosis difficult. Renal agenesis/hypoplasia and dysplasia account for a significant portion of these anomalies, and a genetic contribution to its cause is being increasingly recognized. Nevertheless, overlap between diseases and challenges in clinical diagnosis complicate studies attempting to discover new genes underlying this anomaly. Most of the insights in kidney development derive from studies in mouse models or from rare, syndromic forms of human developmental disorders of the kidney and urinary tract. The genes implicated have been shown to regulate the reciprocal induction between the ureteric bud and the metanephric mesenchyme. Strategies to find genes causing renal agenesis/hypoplasia and dysplasia vary depending on the characteristics of the study population available. The approaches range from candidate gene association or resequencing studies to traditional linkage studies, using outbred pedigrees or genetic isolates, to search for structural variation in the genome. Each of these strategies has advantages and pitfalls and some have led to significant discoveries in human disease. However, renal agenesis/hypoplasia and dysplasia still represents a challenge, both for the clinicians who attempt a precise diagnosis and for the geneticist who tries to unravel the genetic basis, and a better classification requires molecular definition to be retrospectively improved. The goal appears to be feasible with the large multicentric collaborative groups that share the same objectives and resources

Connexin45 expression in the human obstructed detrusor muscle

PURPOSE: Patients with benign prostatic hyperplasia (BPH) and bladder outlet obstruction (BOO) frequently develop lower urinary tract symptoms (LUTS). To elucidate the underlying pathomechanisms we focused on altered cellular communication between detrusor cells. METHODS: Bladder biopsies were collected from eight BPH patients with compensated BOO and from eight non-obstructed patients. Detrusor areas were separated by laser capture microdissection microscopy, and extracted RNA was subjected to quantitative RT-PCR for connexin43 and connexin45. Furthermore, localization of connexin45 and lysosome membrane associated protein 1 was studied by immunohistochemistry. RESULTS: We found the human detrusor to express connexin45 rather than connexin43. Compared to controls, connexin45 expression was not significantly changed in detrusors of obstructed patients. However, connexin45 protein patterns were focally altered in obstruction. CONCLUSIONS: Our study is the first to provide evidence that connexin45-coupling of detrusor cells may be regionally impaired in patients with BOO due to BPH. The altered connexin45 coupling may contribute to LUTS

GDNF/Ret signaling and renal branching morphogenesis: From mesenchymal signals to epithelial cell behaviors

Signaling by GDNF through the Ret receptor tyrosine kinase is required for the normal growth and morphogenesis of the ureteric bud (UB) during kidney development. Recent studies have sought to understand the precise role of Ret signaling in this process, and the specific responses of UB cells to GDNF. Surprisingly, the requirement for Gdnf and Ret was largely relieved by removing the negative regulator Spry1, revealing unexpected functional overlap between GDNF and FGF10. However, the kidneys that developed without Gdnf/Ret and Spry1 displayed significant branching abnormalities, suggesting a unique role for GDNF in fine-tuning UB branching. GDNF/Ret signaling alters patterns of gene expression in UB tip cells, and one critical event is upregulation of the ETS transcription factors Etv4 and Etv5. Mice lacking Etv4 and Etv5 fail to develop kidneys. Thus, these genes represent key components of a regulatory network downstream of Ret. Studies of chimeric embryos in which a subset of cells lack either Ret, Etv4/5 or Spry1 have revealed an important role for this pathway in cell movement. Ret signaling, via Etv4 and Etv5, promotes competitive cell rearrangements in the nephric duct, in which the cells with the highest level of Ret signaling preferentially migrate to form the first ureteric bud tip