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.

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.

Deletion of Frs2alpha from the ureteric epithelium causes renal hypoplasia.

Fibroblast growth factor receptor 2 (Fgfr2) signaling is critical in maintaining ureteric branching architecture and mesenchymal stromal morphogenesis in the kidney. Fibroblast growth factor receptor substrate 2alpha (Frs2alpha) is a major docking protein for Fgfr2 with downstream targets including Ets variant (Etv) 4 and Etv5 in other systems. Furthermore, global deletion of Frs2alpha causes early embryonic lethality. The purpose of the study was to determine the role of Frs2alpha in mediating Fgfr2 signaling in the ureteric epithelium. To that end, we generated mice with conditional deletion of Frs2alpha in the ureteric epithelium (Frs2alpha(UB-/-)) and mice with point mutations in the Frs2alpha binding site of Fgfr2 (Fgfr2(LR/LR)). Frs2alpha(UB-/-) mice developed mild renal hypoplasia characterized by decreased ureteric branching morphogenesis but maintained normal overall branching architecture and had normal mesenchymal stromal development. Reduced nephron endowment in postnatal mutant mice was observed, corresponding with the reduction in branching morphogenesis. Furthermore, there were no apparent renal abnormalities in Fgfr2(LR/LR) mice. Interestingly, Etv4 and Etv5 expression was unaltered in Frs2alpha(UB-/-) mice, as was Sprouty1, an antagonist of Frs2alpha signaling. However, Ret and Wnt11 (molecules critical for ureteric branching morphogenesis) mRNA levels were lower in mutants vs. controls. Taken together, these findings suggest that Fgfr2 signals through adapter molecules other than Frs2alpha in the ureteric epithelium. Furthermore, Frs2alpha may transmit signals through other receptor kinases present in ureteric epithelium. Finally, the renal hypoplasia observed in Frs2alpha(UB-/-) mice is likely secondary to decreased Ret and Wnt11 expression.

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.

Link between reduced nephron number and hypertension: studies in a mutant mouse model.

Low birth weight (LBW) infants with reduced nephron numbers have significantly increased risk for hypertension later in life, which is a devastating health problem. The risk from a reduction in nephron number alone is not clear. Recently, using conditional knock-out approach, we have developed a mutant mouse with reduced nephron number in utero and no change in birth weight, by deleting fibroblast growth factor receptor 2 (fgfr2) in the ureteric bud. Our purpose was to investigate the role of in utero reduced nephron number alone in absence of LBW as a risk for developing hypertension in adulthood. Using tail cuff blood pressure measurements we observed significant increases in systolic blood pressure in one year old mutant mice versus controls. We also detected cardiac end-organ injury from hypertension as shown by significant increases in normalized heart weights, left ventricular (LV) wall thickness, and LV tissue area. Two-dimensional echocardiography revealed no changes in cardiac output and therefore significant increases in systemic vascular resistance in mutants versus controls. We also observed increases in serum blood urea nitrogen (BUN) levels and histologic evidence of glomerular and renal tubular injury in mutant mice versus controls. Thus, these studies suggest that our mutant mice may serve as a relevant model to study the link between reduction of nephron number in utero and the risk of hypertension and chronic renal failure in adulthood.

Role of fibroblast growth factor receptors 1 and 2 in the metanephric mesenchyme.

To determine the importance of fibroblast growth factor receptors (fgfrs) 1 and 2 in the metanephric mesenchyme, we generated conditional knockout mice (fgfr(Mes-/-)). Fgfr1(Mes-/-) and fgfr2(Mes-/-) mice develop normal-appearing kidneys. Deletion of both receptors (fgfr1/2(Mes-/-)) results in renal aplasia. Fgfr1/2(Mes-/-) mice develop a ureteric bud (and occasionally an ectopic bud) that does not elongate or branch, and the mice do not develop an obvious metanephric mesenchyme. By in situ hybridization, regions of mutant mesenchyme near the ureteric bud(s) express Eya1 and Six1, but not Six2, Sall1, or Pax2, while the ureteric bud expresses Ret and Pax2 normally. Abnormally high rates of apoptosis and relatively low rates of proliferation are present in mutant mesenchyme dorsal to the mutant ureteric bud at embryonic day (E) 10.5, while mutant ureteric bud tissues undergo high rates of apoptosis by E11.5. Thus, fgfr1 and fgfr2 together are critical for normal formation of metanephric mesenchyme. While the ureteric bud(s) initiates, it does not elongate or branch infgfr1/2(Mes-/-) mice. In metanephric mesenchymal rudiments, fgfr1 and fgfr2 appear to function downstream of Eya1 and Six1, but upstream of Six2, Sall1, and Pax2. Finally, this is the first example of renal aplasia in a conditional knockout model.