Calcineurin is required in urinary tract mesenchyme for the development of the pyeloureteral peristaltic machinery

Abstract

Congenital obstructive nephropathy is the principal cause of renal failure in infants and children. The underlying molecular and cellular mechanisms of this disease, however, remain largely undetermined. We generated a mouse model of congenital obstructive nephropathy that resembles ureteropelvic junction obstruction in humans. In these mice, calcineurin function is removed by the selective deletion of Cnb1 in the mesenchyme of the developing urinary tract using the Cre/lox system. This deletion results in reduced proliferation in the smooth muscle cells and other mesenchymal cells in the developing urinary tract. Compromised cell proliferation causes abnormal development of the renal pelvis and ureter, leading to defective pyeloureteral peristalsis, progressive renal obstruction, and, eventually, fatal renal failure. Our study demonstrates that calcineurin is an essential signaling molecule in urinary tract development and is required for normal proliferation of the urinary tract mesenchymal cells in a cell-autonomous manner. These studies also emphasize the importance of functional obstruction, resulting from developmental abnormality, in causing congenital obstructive nephropathy.

Figure 1

Deletion of calcineurin in cells expressing Pax3Cre. We generated mice carrying both the floxed-Cnb1 allele and the Pax3Cre transgene. In cells without Pax3Cre expression, the floxed-Cnb1 allele remained intact and functional. In cells with Pax3Cre expression, recombination occurred between the two loxP sites, leading to the deletion of exons 3–5 of Cnb1 and the inactivation of the gene.

Figure 2

Conditional deletion of Cnb1 resulted in congenital obstructive nephropathy. (A–F) Urinary systems from controls (A, C, and E) and their littermate mutants (B, D, and F). Pictures were taken under different magnifications according to their size. The unit on the ruler is 1 mm. (G–L) H&E-stained paraffin sections from the controls (G, I, and K) and mutants (H, J, and L). Arrow in J points to a mildly dilated collecting tubule.

Figure 3

Pax3Cre directs Cnb1 deletion in the metanephric and the ureteric mesenchyme. (A) RT-PCR shows the expression of Cnb1 in the kidney and ureter of the mutant (Pax3Cre^T/+;Cnb1^F/Δ) is reduced compared with that in the control (Pax3Cre^T/+;Cnb1^F/+). GAPDH indicates equal loading. One hundred nanograms of total RNA was used in each lane. (B) Extensive Cre expression (revealed by lacZ expression; blue) was found in the kidney (K), ureter, and bladder (B) of the Pax3Cre^T/+;ROSA^T/+ but not the Pax3Cre^+/+;ROSA^T/+ mice. (C–E and G) Samples from P1 Pax3Cre^T/+;ROSA^T/+ mice. LacZ expression is evident in the glomeruli and tubules that are derived from the metanephric mesenchyme but not in the collecting duct system originated from the UB (C, ×20). The filled arrow in D points to the developing glomeruli. The open arrow points to one of the UB branches. In the developing renal pelvis, the SM layers (SM) and the adventitia (AD) express LacZ, while the UB-derived urothelium (UT) remains LacZ negative (D, ×40, and E, ×60). U, ureter; PP, papilla. The SM layers in the developing renal pelvic wall are illustrated by αSMA staining on a wild-type newborn sample (F, ×60). LacZ is also selectively expressed in the SM layers in the ureter but not in the urothelium (G, ×60). (H–K) Cnb1 protein can be detected by immunostaining in every cell in the control (H and J) but is absent in metanephric mesenchyme– and ureteric mesenchyme–derived structures in the mutant littermates (I and K). Cnb1 proteins remain in the UB-derived collecting duct (CD) system (open arrow) and the urothelium (filled arrow).

Figure 4

Defective pyeloureteral peristalsis in the mutant mice. These are a series of images taken at 2-second intervals during a peristaltic cycle in samples from a control (A–E) and a mutant littermate (F–J) at P5. The black bars indicate the change of length in the most proximal segment of the ureter.

Figure 5

Samples from controls (Pax3Cre^T/+;Cnb1^F/+) (A, C, and E); and samples from their mutant littermates (Pax3Cre^T/+;Cnb1^F/Δ) (B, D, and F). Arrows in each panel indicate the locations of the UPJs. (A and B) Hemisected kidneys. (C–F) UPJ areas immunostained with an αSMA antibody at P1 (C and D) and P5 (E and F) showing the developing renal pelvic extension in the controls (C and E) and the underdevelopment of such an extension in the same area in the mutants (D and F).

Figure 6

A defective ureteric wall may contribute to the defective peristalsis. (A and B) Replica moldings of the urinary tracts (from the pelvis to the distal end of the ureter) of the control (A) and the mutant (B) littermates at P5. (C and D) The ureteric wall is straight and smooth in the control (C) but irregular in the mutant (D). (E–H) The SM layers are disorganized in the mutants (F and H) but not the controls (E and G) at P5. Arrow in H points to the epithelial cells in the lumen of the cyst-like structure. (I and J) The mutant shown in J has hydroureter marked by the white triangle. Hydroureter has never been observed in the controls (I). PC, pelvicaliceal space.

Figure 7

Calcineurin regulates cell proliferation in the developing renal pelvic wall. (A and B) Ki67 staining of the developing renal pelvic wall of control (A) and mutant (B) samples. (C and D) DAPI images of A and B, respectively. (E) The controls have significantly more proliferating mesenchymal cells along the developing renal pelvic wall. M, mesenchymal derivatives.

Figure 8

No developmental defects in the innervation of the urinary system in the mutants. (A and B) Immunostaining with an anti-neurofilament (NF) antibody revealed similar numbers of nerve fibers per transverse section between the control (A) and its littermate mutant at P1. (C and D) DAPI-stained sections from A and B, revealing the nuclei of the cells. (E) By counting only the nerve fibers occupying more than one-tenth the size of a nucleus of an average SMC, we found that the average numbers of nerve fibers are similar between the control samples and the mutant samples.

Figure 9

A model for the role of calcineurin (Cn) in the signaling events during urinary tract development. Deletion of Cnb1 in the mesenchyme along the urinary tract results in abnormal development of the renal pelvis and ureter by disrupting the proliferation of the mesenchymal cells and the development of the peristaltic machinery. Deletion of Shh in the urothelium (36) and deletion of Agtr1 and other genes in the renin-angiotensin axis (7, 37) also result in congenital obstructive phenotypes suggesting the possibility of interactions between these pathways. Ptch repression of Smo is relieved upon Shh binding, leading to the activation of Gli. p-NFAT, phosphorylated NFAT.