Darren Bridgewater

Canonical WNT/β-catenin signaling is required for ureteric branching

WNT/beta-catenin signaling has an established role in nephron formation during kidney development. Yet, the role of beta-catenin during ureteric morphogenesis in vivo is undefined. We generated a murine genetic model of beta-catenin deficiency targeted to the ureteric bud cell lineage. Newborn mutant mice demonstrated bilateral renal aplasia or renal dysplasia. Analysis of the embryologic events leading to this phenotype revealed that abnormal ureteric branching at E12.5 precedes histologic abnormalities at E13.5. Microarray analysis of E12.5 kidney tissue identified decreased Emx2 and Lim1 expression among a small subset of renal patterning genes disrupted at the stage of abnormal branching. These alterations are followed by decreased expression of genes downstream of Emx2, including Lim1, Pax2, and the ureteric tip markers, c-ret and Wnt 11. Together, these data demonstrate that beta-catenin performs essential functions during renal branching morphogenesis via control of a hierarchy of genes that control ureteric branching.

GLI3 repressor controls nephron number via regulation of Wnt11 and Ret in ureteric tip cells.

Truncating GLI3 mutations in Pallister-Hall Syndrome with renal malformation suggests a requirement for Hedgehog signaling during renal development. HH-dependent signaling increases levels of GLI transcriptional activators and decreases processing of GLI3 to a shorter transcriptional repressor. Previously, we showed that Shh-deficiency interrupts early inductive events during renal development in a manner dependent on GLI3 repressor. Here we identify a novel function for GLI3 repressor in controlling nephron number. During renal morphogenesis, HH signaling activity, assayed by expression of Ptc1-lacZ, is localized to ureteric cells of the medulla, but is undetectable in the cortex. Targeted inactivation of Smo, the HH effector, in the ureteric cell lineage causes no detectable abnormality in renal morphogenesis. The functional significance of absent HH signaling activity in cortical ureteric cells was determined by targeted deletion of Ptc1, the SMO inhibitor, in the ureteric cell lineage. Ptc1−/−UB mice demonstrate ectopic Ptc1-lacZ expression in ureteric branch tips and renal hypoplasia characterized by reduced kidney size and a paucity of mature and intermediate nephrogenic structures. Ureteric tip cells are remarkable for abnormal morphology and impaired expression of Ret and Wnt11, markers of tip cell differentiation. A finding of renal hypoplasia in Gli3 −/− mice suggests a pathogenic role for reduced GLI3 repressor in the Ptc1−/−UB mice. Indeed, constitutive expression of GLI3 repressor via the Gli3Δ699 allele in Ptc1−/−UB mice restores the normal pattern of HH signaling, and expression of Ret and Wnt11 and rescued the renal phenotype. Thus, GLI3 repressor controls nephron number by regulating ureteric tip cell expression of Wnt11 and Ret.

Stimulatory and inhibitory signaling molecules that regulate renal branching morphogenesis

Branching morphogenesis, defined as the growth and branching of epithelial tubules, is a fundamental developmental process involved in the formation of a variety of mammalian tissues, including the kidney. Defective renal branching may result in a number of clinically relevant abnormalities, including renal agenesis, renal dysplasia, multiplex kidneys, and hypertension. In this review we describe the morphological events that generate the characteristic tree-like structure of the mammalian collecting system. We also highlight new knowledge related to both established and novel signaling systems that are important for stimulating and inhibiting branching morphogenesis.

Penetrance of biallelic SMARCAL1 mutations is associated with environmental and genetic disturbances of gene expression

Biallelic mutations of the DNA annealing helicase SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1) cause Schimke immuno-osseous dysplasia (SIOD, MIM 242900), an incompletely penetrant autosomal recessive disorder. Using human, Drosophila and mouse models, we show that the proteins encoded by SMARCAL1 orthologs localize to transcriptionally active chromatin and modulate gene expression. We also show that, as found in SIOD patients, deficiency of the SMARCAL1 orthologs alone is insufficient to cause disease in fruit flies and mice, although such deficiency causes modest diffuse alterations in gene expression. Rather, disease manifests when SMARCAL1 deficiency interacts with genetic and environmental factors that further alter gene expression. We conclude that the SMARCAL1 annealing helicase buffers fluctuations in gene expression and that alterations in gene expression contribute to the penetrance of SIOD.

Stromally expressed β-catenin modulates Wnt9b signaling in the ureteric epithelium.

The mammalian kidney undergoes cell interactions between the epithelium and mesenchyme to form the essential filtration unit of the kidney, termed the nephron. A third cell type, the kidney stroma, is a population of fibroblasts located in the kidney capsule, cortex and medulla and is ideally located to affect kidney formation. We found β-catenin, a transcriptional co-activator, is strongly expressed in distinctive intracellular patterns in the capsular, cortical, and medullary renal stroma. We investigated β-catenin function in the renal stroma using a conditional knockout strategy that genetically deleted β-catenin specifically in the renal stroma cell lineage (β-cats-/-). β-cats-/- mutant mice demonstrate marked kidney abnormalities, and surprisingly we show β-catenin in the renal stroma is essential for regulating the condensing mesenchyme cell population. We show that the population of induced mesenchyme cells is significantly reduced in β-cats-/- mutants and exhibited decreased cell proliferation and a specific loss of Cited 1, while maintaining the expression of other essential nephron progenitor proteins. Wnt9b, the key signal for the induction of nephron progenitors, was markedly reduced in adjacent ureteric epithelial cells in β-cats-/-. Analysis of Wnt9b-dependent genes in the neighboring nephron progenitors was significantly reduced while Wnt9b-independent genes remained unchanged. In contrast mice overexpressing β-catenin exclusively in the renal stroma demonstrated massive increases in the condensing mesenchyme population and Wnt9b was markedly elevated. We propose that β-catenin in the renal stroma modulates a genetic program in ureteric epithelium that is required for the induction of nephron progenitors.

GRP78 and CHOP modulate macrophage apoptosis and the development of bleomycin‐induced pulmonary fibrosis

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been associated with fibrotic lung disease, although exactly how they modulate this process remains unclear. Here we investigated the role of GRP78, the main UPR regulator, in an experimental model of lung injury and fibrosis. Grp78+/−, Chop−/− and wild type C57BL6/J mice were exposed to bleomycin by oropharyngeal intubation and lungs were examined at days 7 and 21. We demonstrate here that Grp78+/− mice were strongly protected from bleomycin‐induced fibrosis, as shown by immunohistochemical analysis, collagen content and lung function measurements. In the inflammatory phase of this model, a reduced number of lung macrophages associated with an increased number of TUNEL‐positive cells were observed in Grp78+/− mice. Dual immunohistochemical and in situ hybridization experiments showed that the macrophage population from the protected Grp78+/− mice was also strongly positive for cleaved caspase‐3 and Chop mRNA, respectively. In contrast, the administration of bleomycin to Chop−/− mice resulted in increased quasi‐static elastance and extracellular matrix deposition associated with an increased number of parenchymal arginase‐1‐positive macrophages that were negative for cleaved caspase‐3. The data presented indicate that the UPR is activated in fibrotic lung tissue and strongly localized to macrophages. GRP78‐ and CHOP‐mediated macrophage apoptosis was found to protect against bleomycin‐induced fibrosis. Overall, we demonstrate here that the fibrotic response to bleomycin is dependent on GRP78‐mediated events and provides evidence that macrophage polarization and apoptosis may play a role in this process. Copyright

The GDNF Target Vsnl1 Marks the Ureteric Tip

Glial cell line-derived neurotrophic factor (GDNF) is indispensable for ureteric budding and branching. If applied exogenously, GDNF promotes ectopic ureteric buds from the Wolffian duct. Although several downstream effectors of GDNF are known, the identification of early response genes is incomplete. Here, microarray screening detected several GDNF-regulated genes in the Wolffian duct, including Visinin like 1 (Vsnl1), which encodes a neuronal calcium-sensor protein. We observed renal Vsnl1 expression exclusively in the ureteric epithelium, but not in Gdnf-null kidneys. In the tissue culture of Gdnf-deficient kidney primordium, exogenous GDNF and alternative bud inducers (FGF7 and follistatin) restored Vsnl1 expression. Hence, Vsnl1 characterizes the tip of the ureteric bud epithelium regardless of the inducer. In the tips, Vsnl1 showed a mosaic expression pattern that was mutually exclusive with β-catenin transcriptional activation. Vsnl1 was downregulated in both β-catenin-stabilized and β-catenin-deficient kidneys. Moreover, in a mouse collecting duct cell line, Vsnl1 compromised β-catenin stability, suggesting a counteracting relationship between Vsnl1 and β-catenin. In summary, Vsnl1 marks ureteric bud tips in embryonic kidneys, and its mosaic pattern demonstrates a heterogeneity of cell types that may be critical for normal ureteric branching.

Developmental Origins for Kidney Disease Due to Shroom3 Deficiency

CKD is a significant health concern with an underlying genetic component. Multiple genome-wide association studies (GWASs) strongly associated CKD with the shroom family member 3 (SHROOM3) gene, which encodes an actin-associated protein important in epithelial morphogenesis. However, the role of SHROOM3 in kidney development and function is virtually unknown. Studies in zebrafish and rat showed that alterations in Shroom3 can result in glomerular dysfunction. Furthermore, human SHROOM3 variants can induce impaired kidney function in animal models. Here, we examined the temporal and spatial expression of Shroom3 in the mammalian kidney. We detected Shroom3 expression in the condensing mesenchyme, Bowmans capsule, and developing and mature podocytes in mice. Shroom3 null (Shroom3Gt/Gt) mice showed marked glomerular abnormalities, including cystic and collapsing/degenerating glomeruli, and marked disruptions in podocyte arrangement and morphology. These podocyte-specific abnormalities are associated with altered Rho-kinase/myosin II signaling and loss of apically distributed actin. Additionally, Shroom3 heterozygous (Shroom3Gt/+) mice showed developmental irregularities that manifested as adult-onset glomerulosclerosis and proteinuria. Taken together, our results establish the significance of Shroom3 in mammalian kidney development and progression of kidney disease. Specifically, Shroom3 maintains normal podocyte architecture in mice via modulation of the actomyosin network, which is essential for podocyte function. Furthermore, our findings strongly support the GWASs that suggest a role for SHROOM3 in human kidney disease.

β-Catenin Overexpression in the Metanephric Mesenchyme Leads to Renal Dysplasia Genesis via Cell-Autonomous and Non–Cell-Autonomous Mechanisms

Renal dysplasia, a developmental disorder characterized by defective ureteric branching morphogenesis and nephrogenesis, ranks as one of the major causes of renal failure among the pediatric population. Herein, we demonstrate that the levels of activated β-catenin are elevated in the nuclei of ureteric, stromal, and mesenchymal cells within dysplastic human kidney tissue. By using a conditional mouse model of mesenchymal β-catenin overexpression, we identify two novel signaling pathways mediated by β-catenin in the development of renal dysplasia. First, the overexpression of β-catenin within the metanephric mesenchyme leads to ectopic and disorganized branching morphogenesis caused by β-catenin directly binding Tcf/lef consensus binding sites in the Gdnf promoter and up-regulating Gdnf transcription. Second, β-catenin overexpression in the metanephric mesenchyme leads to elevated levels of transcriptionally active β-catenin in the ureteric epithelium. Interestingly, this increase of β-catenin-mediated transcription results from a novel Ret/β-catenin signaling pathway. Consistent with these findings, analysis of human dysplastic renal tissue demonstrates that undifferentiated mesenchymal cells expressing high levels of β-catenin also express increased GDNF. Furthermore, dysplastic ureteric tubules that were surrounded by high levels of GDNF also exhibited increased levels of activated β-catenin. Together, these data support a model in which the elevation of β-catenin in the metanephric mesenchyme results in cell-autonomous and non-cell-autonomous events that lead to the genesis of renal dysplasia.

Matrix metalloproteinase 9 is associated with peritoneal membrane solute transport and induces angiogenesis through β-catenin signaling

Background. For patients using peritoneal dialysis (PD), the peritoneal membrane can develop fibrosis and angiogenesis, leading to ultrafiltration failure, chronic hypervolemia and increased risk of technique failure and mortality. Matrix metalloproteinases (MMPs), and specifically the gelatinases (MMP2 and MMP9), may be involved in peritoneal membrane injury. Methods. From stable PD patients, mesothelial cells were assayed for MMP gene expression. MMP9 was overexpressed in mouse peritoneum by adenovirus, and MMP9−/− mice were subjected to transforming growth factor &bgr; (TGF-&bgr;)–induced peritoneal fibrosis. Results. MMP9 mRNA expression correlated with peritoneal membrane solute transport properties. Overexpression of MMP9 in the mouse peritoneum induced submesothelial thickening and angiogenesis. MMP9 induced mesothelial cell transition to a myofibroblast phenotype measured by increased alpha smooth muscle actin and decreased E-cadherin expression. Angiogenesis was markedly reduced in MMP9−/− mice treated with an adenovirus expressing active TGF-&bgr; compared with wild-type mice. TGF-&bgr;-mediated E-cadherin cleavage was MMP9 dependent, and E-cadherin cleavage led to &bgr;-catenin-mediated signaling. A &bgr;-catenin inhibitor blocked the angiogenic response induced by AdMMP9. Conclusions. Our data suggest that MMP9 is involved in peritoneal membrane injury possibly through cleavage of E-cadherin and induction of &bgr;-catenin signaling. MMP9 is a potential biomarker for peritoneal membrane injury and is a therapeutic target to protect the peritoneal membrane in PD patients.