Antagonism of Bmp4 Signaling Disrupts Smooth Muscle Investment of the Ureter and Ureteropelvic Junction

Abstract

Purpose-Congenital ureteropelvic junction obstruction has been associated with aberrant ureteral smooth muscle organization. Recent evidence has shown that BMP4 may be involved in ureteral morphogenesis. We determined whether the disruption of BMP4 signaling results in abnormal smooth muscle investment of the ureter and ureteropelvic junction. Materials and Methods-We used a Cre mediated Bmp4 knockout system to conditionally excise the Bmp4 gene in developing mouse embryos. Kidney rudiments were isolated from embryos at varying gestational ages from WT and conditional knockout mice. Metanephric kidney explants were cultured in the presence or absence of the BMP antagonist Noggin. Agarose beads pre-incubated with Gremlin, another BMP antagonist, were used for localized disruption of BMP signaling. Frozen sections and whole metanephric explants were then analyzed by immunofluorescence. Results-Bmp4 gene excision resulted in a dose dependent loss of ureteral smooth muscle. Antagonism of BMP signaling inhibited ureteral smooth muscle investment in a dose dependent manner and was paralleled by a dose dependent decrease in the immediate downstream targets of BMP signaling, phosphorylated Smad1, 5 and 8. Localized antagonism of BMP resulted in the focal disruption of ureteral smooth muscle investment. Conclusions-We report that decreased BMP signaling, whether by the loss of BMP4 in vivo or direct antagonism in vitro, results in a gradual reduction of the normal, well organized coat of smooth muscle surrounding the ureter. Our results also suggest that this occurs via a direct Smad dependent pathway. This raises the possibility that abnormalities in BMP4 signaling may have a role in the development of congenital ureteropelvic junction obstruction. Keywords ureter; ureteral obstruction; abnormalities; bone morphogenetic protein 4; gene expression Congenital UPJ obstruction is the most common cause of urinary tract dilatation in children, occurring in 1/1,000 to 2,000 newborns. 1 Recent studies indicate that congenital obstructions may in part be due to aberrant ureteral smooth muscle organization, resulting in impaired peristalsis. 2-4 Additionally, surgical specimens of obstructed human UPJ segments

Absence of Canonical Smad Signaling in Ureteral and Bladder Mesenchyme Causes Ureteropelvic Junction Obstruction

“…To our surprise, unlike the previous studies on Bmp4 disruption, 16,17 Smad4 mutants have SM differentiation and formation of SM layers around the epithelium ( Figure 3, A-D). We investigated the SM layer thickness in mutant and control ureters at E18.5, when hydronephrosis has occurred, and E16.5, just before the onset of hydronephrosis.…”

“…7,10,19,22,23 Previous studies have shown that inactivation of Bmp4 disrupts the structural and functional organization of the kidney and lower urinary tract. 16 To specifically investigate the role of canonical Smaddependent TGF-b superfamily signaling in the development of the lower urinary tract, we have used the Tbx18-Cre transgene to mediate the inactivation of Smad4, the common Smad indispensible for the transcriptional responses of TGF-b superfamily signaling, in the ureteral mesenchyme. Loss of canonical Smad signaling in the mesenchyme led to UPJ obstruction and severe hydronephrosis.…”

“…Although the ureteral SMs showed a number of alterations, SM development in Smad4 mutants seems to be much more advanced than in mutants with loss of Bmp4. 16,17 The TGF-b superfamily seems to also have an Smad-independent noncanonical pathway by r-GTPase, phosphatidylinositol 39 kinase, and MAPK. 32 Because less-severe SM defects are observed in Smad4 mutants (this study) compared with the Bmp4 mutants previously reported, Bmp4 effects in ureteral SM development, such as the differentiation of the mesenchymal cells into SMCs, may be partially Smad-independent.…”

“…Pathogenesis of UPJ obstruction in ureters of the Smad4 mutants. Because the SM layer development seems less affected in Smad4 mutants than Bmp4 mutants 16 and TGF-b superfamily signaling may have Smad4-independent downstream pathways mediated by r-GTPase, phosphatidylinositol 39 kinase, and MAPK, it is possible that the part of Bmp4 signaling in ureteral SM development may be Smad4-independent. (A) At the same time, other phenotypes in Smad4 mutants could be attributed to the lack of TGF-b/Bmp4 signaling by Smad4.…”

Cell Biology of Ureter Development

“…53 Peri-urothelial mesenchymal cells are also stimulated to express BMP4, which itself effects their own differentiation into SM. 51,53 Here, BMP4 enhances intracellular levels of phospho-SMADs 43,54 and upregulates TSHZ3, a transcription factor-like protein. TSHZ3 is needed for MYOCD expression within nascent ureteric SM cells.…”