Ablation of developing podocytes disrupts cellular interactions and nephrogenesis both inside and outside the glomerulus.

Podocyte loss in adults leads to glomerulosclerosis. However, the impact of podocyte loss on glomerulogenesis and the development of the kidney as a whole has not been directly studied. Here, we used a podocyte-specific Cre transgene to direct the production of diphtheria toxin (DTA) inside podocytes during nephrogenesis. Affected podocytes underwent translational arrest and apoptosis, leading to oliguria, proteinuria, hematuria, interstitial hemorrhage, and perinatal death. Glomerular cell-cell interactions were disrupted, even before overt podocyte apoptosis. VEGF production by podocytes was greatly decreased, and this was associated with reduced endothelial fenestration and altered glomerular vascular architecture. In addition to these glomerular anomalies, embryonic podocyte ablation also led to structural changes and increased apoptosis in proximal tubules. The collecting ducts, however, only showed molecular changes that are likely an indirect effect of the greatly reduced urine flow. Although podocyte loss significantly impacted the development and maintenance of the vasculature both inside and outside the glomerulus, our results suggest that there is a lack of long-range signaling from deep-seated, mature glomeruli to the differentiating cells in the outer nephrogenic zone. This study illustrates the tight integration of various cell types in the developing kidney and shows that the impact of podocyte loss during development is much greater than that in adults. This study also shows the specificity and effectiveness of a genetically controlled podocyte ablation system in mice where the additional readily available tools can further expand its applications.

Vasopressin-stimulated increase in phosphorylation at Ser269 potentiates plasma membrane retention of aquaporin-2.

Vasopressin controls water excretion through regulation of aquaporin-2 (AQP2) trafficking in renal collecting duct cells. Using mass spectrometry, we previously demonstrated four phosphorylated serines (Ser256, Ser261, Ser264, and Ser269) in the carboxyl-terminal tail of rat AQP2. Here, we used phospho-specific antibodies and protein mass spectrometry to investigate the roles of vasopressin and cyclic AMP in the regulation of phosphorylation at Ser269 and addressed the role of this site in AQP2 trafficking. The V2 receptor-specific vasopressin analog dDAVP increased Ser(P)269-AQP2 abundance more than 10-fold, but at a rate much slower than the corresponding increase in Ser256 phosphorylation. Vasopressin-mediated changes in phosphorylation at both sites were mimicked by cAMP addition and inhibited by protein kinase A (PKA) antagonists. In vitro kinase assays, however, demonstrated that PKA phosphorylates Ser256, but not Ser269. Phosphorylation of AQP2 at Ser269 did not occur when Ser256 was replaced by an unphosphorylatable amino acid, as seen in both S256L-AQP2 mutant mice and in Madin-Darby canine kidney cells expressing an S256A mutant, suggesting that Ser269 phosphorylation depends upon prior phosphorylation at Ser256. Immunogold electron microscopy localized Ser(P)269-AQP2 solely in the apical plasma membrane of rat collecting duct cells, in contrast to the other three phospho-forms (found in both apical plasma membrane and intracellular vesicles). Madin-Darby canine kidney cells expressing an S269D "phosphomimic" AQP2 mutant showed constitutive localization at the plasma membrane. The data support a model in which vasopressin-mediated phosphorylation of AQP2 at Ser269:(a) depends on prior PKA-mediated phosphorylation of Ser256 and (b) enhances apical plasma membrane retention of AQP2.

Smad signaling in the neural crest regulates cardiac outflow tract remodeling through cell autonomous and non-cell autonomous effects.

Neural crest cells (NCCs) are indispensable for the development of the cardiac outflow tract (OFT). Here, we show that mice lacking Smad4 in NCCs have persistent truncus arteriosus (PTA), severe OFT cushion hypoplasia, defective OFT elongation, and mispositioning of the OFT. Cardiac NCCs lacking Smad4 have increased apoptosis, apparently due to decreased Msx1/2 expression. This contributes to the reduction of NCCs in the OFT. Unexpectedly, mutants have MF20-expressing cardiomyocytes in the splanchnic mesoderm within the second heart field (SHF). This may result from abnormal differentiation or defective recruitment of differentiating SHF cells into OFT. Alterations in Bmp4, Sema3C, and PlexinA2 signals in the mutant OFT, SHF, and NCCs, disrupt the communications among different cell populations. Such disruptions can further affect the recruitment of NCCs into the OFT mesenchyme, causing severe OFT cushion hypoplasia and OFT septation failure. Furthermore, these NCCs have drastically reduced levels of Ids and MT1-MMP, affecting the positioning and remodeling of the OFT. Thus, Smad-signaling in cardiac NCCs has cell autonomous effects on their survival and non-cell autonomous effects on coordinating the movement of multiple cell lineages in the positioning and the remodeling of the OFT.

Calcineurin-NFATc signaling pathway regulates AQP2 expression in response to calcium signals and osmotic stress.

The aquaporin (AQP)2 channel mediates the reabsorption of water in renal collecting ducts in response to arginine vasopressin (AVP) and hypertonicity. Here we show that AQP2 expression is induced not only by the tonicity-responsive enhancer binding protein (TonEBP)/nuclear factor of activated T cells (NFAT)5-mediated hypertonic stress response but also by the calcium-dependent calcineurin-NFATc pathway. The induction of AQP2 expression by the calcineurin-NFATc pathway can occur in the absence of TonEBP/NFAT5. Mutational and chromatin immunoprecipitation analyses revealed the existence of functional NFAT binding sites within the proximal AQP2 promoter responsible for regulation of AQP2 by NFATc proteins and TonEBP/NFAT5. Contrary to the notion that TonEBP/NFAT5 is the only Rel/NFAT family member regulated by tonicity, we found that hypertonicity promotes the nuclear translocation of NFATc proteins for the subsequent induction of AQP2 expression. Calcineurin activity was also found to be involved in the induction of TonEBP/NFAT5 expression by hypertonicity, thus further defining the signaling mechanisms that underlie the TonEBP/NFAT5 osmotic stress response pathway. The coordinate regulation of AQP2 expression by both osmotic stress and calcium signaling appears to provide a means to integrate diverse extracellular signals into optimal cellular responses.

Congenital progressive hydronephrosis (cph) is caused by an S256L mutation in aquaporin-2 that affects its phosphorylation and apical membrane accumulation.

Congenital progressive hydronephrosis (cph) is a spontaneous recessive mutation that causes severe hydronephrosis and obstructive nephropathy in affected mice. The mutation has been mapped to the distal end of mouse chromosome 15, but the mutated gene has not been found. Here, we describe the identification of a single base pair change in aquaporin-2 (Aqp2) in cph mutants through genetic linkage mapping. The C-T change led to the substitution of a Ser (S256) by a Leu in the cytoplasmic tail of the Aqp2 protein, preventing its phosphorylation at S256 and the subsequent accumulation of Aqp2 on the apical membrane of the collecting duct principal cells. The interference with normal trafficking of Aqp2 by this mutation resulted in a severe urine concentration defect. cph homozygotes demonstrated polydipsia and produced a copious amount of hypotonic urine. The urine concentration defect could not be corrected by [deamino-Cys1,D-Arg8]-vasopressin (DDAVP, a vasopressin analog), characteristic of nephrogenic diabetes insipidus. The nephrogenic diabetes insipidus symptoms and the absence of developmental defects in the pyeloureteral peristaltic machinery in the mutants before the onset of hydronephrosis suggest that the congenital obstructive nephropathy is most likely a result of the polyuria. This study has revealed the genetic basis for the classical cph mutation and has provided direct genetic evidence that S256 in Aqp2 is indispensable for the apical accumulation, but not the general glycosylation or membrane association, of Aqp2.

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

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.

Expression of a truncated Sall1 transcriptional repressor is responsible for Townes-Brocks syndrome birth defects.

Townes-Brocks syndrome (TBS, OMIM #107480) is an autosomal dominant disorder that causes multiple birth defects including renal, ear, anal and limb malformations. Mutations in SALL1 have been postulated to cause TBS by haploinsufficiency; however, a mouse model carrying a sall1-null allele does not mimic the human syndrome. Since the mutations that cause TBS could express a truncated SALL1 protein containing the domain necessary for transcriptional repression but lacking the complete DNA binding domain, we hypothesized that TBS is due to dominant-negative or gain-of-function activity of a mutant protein. To test this hypothesis, we have created a mutant allele, sall1-DeltaZn2-10, that produces a truncated protein and recapitulates the abnormalities found in human TBS. Heterozygous mice mimic TBS patients by displaying high-frequency sensorineural hearing loss, renal cystic hypoplasia and wrist bone abnormalities. Homozygous sall1-DeltaZn2-10 mutant mice exhibit more severe defects than sall1-null mice including complete renal agenesis, exencephaly, limb and anal deformities. We demonstrate that truncated Sall1 mediates interaction with all Sall family members and could interfere with the normal function of all Sall proteins. These data support a model for the pathogenesis of TBS in which expression of a truncated SALL1 protein causes abnormal development of multiple organs.

Murine Sall1 represses transcription by recruiting a histone deacetylase complex.

The multi-zinc finger proteins of the Sal family regulate organogenesis. Genetic evidence from Drosophila has shown that spalt (sal) can alter gene expression in a cell autonomous fashion, but Sal proteins have never been directly analyzed for their ability to activate or repress transcription. In this report, we show that a member of the Sal family, mouse Sall1, is a potent transcriptional repressor. When fused to a heterologous DNA-binding domain, Sall1 represses transcription of a luciferase reporter by over 100-fold. Expression of the N terminus alone is sufficient for dose-responsive repression that, as shown by deletion analysis, requires the extreme N-terminal amino acids of the protein. The N terminus of Sall1 can repress at both short and long range relative to the promoter, and treatment with the histone deacetylase (HDAC) inhibitor, trichostatin A, alleviates repression by 3-fold. The same regions of the protein that are required for repression physically interact with components of chromatin remodeling complexes, HDAC1, HDAC2, RbAp46/48, MTA-1, and MTA-2. Finally, we demonstrate that Sall1 is localized to discrete nuclear foci and this localization depends on the N-terminal repression domain. Together, these results suggest that the N terminus of mouse Sall1 can recruit HDAC complexes to mediate transcriptional repression.