Early Nephrogenesis Research Articles

Transcriptional Control of Early Nephrogenesis in Xenopus tropicalis

Transcriptional Control of Early Nephrogenesis in Xenopus tropicalis

Dose-dependent responses to canonical Wnt transcriptional complexes in the regulation of mammalian nephron progenitors.

In vivo and in vitro studies argue that concentration-dependent Wnt signaling regulates mammalian nephron progenitor cell (NPC) programs. Canonical Wnt signaling is regulated through the stabilization of β-catenin, a transcriptional co-activator when complexed with Lef/Tcf DNA-binding partners. Using the GSK3β inhibitor CHIR99021 (CHIR) to block GSK3β-dependent destruction of β-catenin, we examined dose-dependent responses to β-catenin in mouse NPCs, using mRNA transduction to modify gene expression. Low CHIR-dependent proliferation of NPCs was blocked on β-catenin removal, with evidence of NPCs arresting at the G2-M transition. While NPC identity was maintained following β-catenin removal, mRNA-seq identified low CHIR and β-catenin dependent genes. High CHIR activated nephrogenesis. Nephrogenic programming was dependent on Lef/Tcf factors and β-catenin transcriptional activity. Molecular and cellular features of early nephrogenesis were driven in the absence of CHIR by a mutated stabilized form of β-catenin. Chromatin association studies indicate low and high CHIR response genes are likely direct targets of canonical Wnt transcriptional complexes. Together, these studies provide evidence for concentration-dependent Wnt signaling in the regulation of NPCs and provide new insight into Wnt targets initiating mammalian nephrogenesis.

Histone deacetylases 1 and 2 target gene regulatory networks of nephron progenitors to control nephrogenesis

Our studies demonstrated the critical role of Histone deacetylases (HDACs) in the regulation of nephrogenesis. To better understand the key pathways regulated by HDAC1/2 in early nephrogenesis, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) of HDAC1/2 on isolated nephron progenitor cells (NPCs) from mouse E16.5 kidneys. Our analysis revealed that 11,802 (40.4%) of HDAC1 peaks overlap with HDAC2 peaks, further demonstrates the redundant role of HDAC1 and HDAC2 during nephrogenesis. Common HDAC1/2 peaks are densely concentrated close to the transcriptional start site (TSS). GREAT Gene Ontology analysis of overlapping HDAC1/2 peaks reveals that HDAC1/2 are associated with metanephric nephron morphogenesis, chromatin assembly or disassembly, as well as other DNA checkpoints. Pathway analysis shows that negative regulation of Wnt signaling pathway is one of HDAC1/2′s most significant function in NPCs. Known motif analysis indicated that Hdac1 is enriched in motifs for Six2, Hox family, and Tcf family members, which are essential for self-renewal and differentiation of nephron progenitors. Interestingly, we found the enrichment of HDAC1/2 at the enhancer and promoter regions of actively transcribed genes, especially those concerned with NPC self-renewal. HDAC1/2 simultaneously activate or repress the expression of different genes to maintain the cellular state of nephron progenitors. We used the Integrative Genomics Viewer to visualize these target genes associated with each function and found that HDAC1/2 co-bound to the enhancers or/and promoters of genes associated with nephron morphogenesis, differentiation, and cell cycle control. Taken together, our ChIP-Seq analysis demonstrates that HDAC1/2 directly regulate the molecular cascades essential for nephrogenesis.

Renal Medullary Carcinomas Harbor a Distinct Methylation Phenotype and Display Aberrant Methylation of Genes Related to Early Nephrogenesis.

Simple SummaryRenal medullary carcinomas (RMC) are aggressive tumors of the kidneys, characterized by a loss of SMARCB1. As the tumors, arising predominantly in young males with sickle cell trait, are very rare and no standard method for detection or treatment has been described, prognosis for these patients is poor. We generated methylation profiles of seven RMC samples and compared the hitherto unexplored methylation landscape of these tumors to other renal tumors and malignant rhabdoid tumors as well as epithelioid sarcomas, constituting two prototypically SMARCB1 aberrant entities. Based on these valuable datasets, we found that—in accordance with the previous gene expression data—RMCs separate from other SMARCB1 deficient entities. In a focused analysis of genes that are important for nephrogenesis, we particularly detected genes that govern early nephrogenesis to be hypomethylated and expressed at high levels in RMCs.Renal medullary carcinomas (RMC) are rare aggressive tumors of the kidneys, characterized by a loss of SMARCB1. Characteristically, these tumors arise in patients with sickle cell trait or other hemoglobinopathies. Recent characterization efforts have unraveled oncogenic pathways that drive tumorigenesis. Among these, gene sets that characterize replicative stress and the innate immune response are upregulated in RMCs. Despite comprehensive genetic and transcriptomic characterizations, commonalities or differences to other SMARCB1 deficient entities so far have not been investigated. We analyzed the methylome of seven primary RMC and compared it to other SMARCB1 deficient entities such as rhabdoid tumors (RT) and epithelioid sarcomas using 850 K methylation arrays. Moreover, we evaluated the differential gene expression of RMC using RNA-sequencing in comparison to other rhabdoid tumors. In accordance with previous gene expression data, we found that RMCs separate from other SMARCB1 deficient entities, pointing to a potentially different cell of origin and a role of additional genetic aberrations that may drive tumorigenesis and thus alter the methylome when compared to rhabdoid tumors. In a focused analysis of genes that are important for nephrogenesis, we particularly detected genes that govern early nephrogenesis such as FOXI1 to be hypomethylated and expressed at high levels in RMC. Overall, our analyses underscore the fact that RMCs represent a separate entity with limited similarities to rhabdoid tumors, warranting specific treatment tailored to the aggressiveness of the disease.

Differences in Immunohistochemical and Ultrastructural Features between Podocytes and Parietal Epithelial Cells (PECs) Are Observed in Developing, Healthy Postnatal, and Pathologically Changed Human Kidneys.

During human kidney development, cells of the proximal nephron gradually differentiate into podocytes and parietal epithelial cells (PECs). Podocytes are terminally differentiated cells that play a key role in both normal and pathological kidney function. Therefore, the potential of podocytes to regenerate or be replaced by other cell populations (PECs) is of great interest for the possible treatment of kidney diseases. In the present study, we analyzed the proliferation and differentiation capabilities of podocytes and PECs, changes in the expression pattern of nestin, and several early proteins including WNT4, Notch2, and Snail, as well as Ki-67, in tissues of developing, postnatal, and pathologically changed human kidneys by using immunohistochemistry and electron microscopy. Developing PECs showed a higher proliferation rate than podocytes, whereas nestin expression characterized only podocytes and pathologically changed kidneys. In the developing kidneys, WNT4 and Notch2 expression increased moderately in podocytes and strongly in PECs, whereas Snail increased only in PECs in the later fetal period. During human kidney development, WNT4, Notch2, and Snail are involved in early nephrogenesis control. In kidneys affected by congenital nephrotic syndrome of the Finnish type (CNF) and focal segmental glomerulosclerosis (FSGS), WNT4 decreased in both cell populations, whereas Notch2 decreased in FSGS. In contrast, Snail increased both in CNF and FSGS, whereas Notch2 increased only in CNF. Electron microscopy revealed cytoplasmic processes spanning the urinary space between the podocytes and PECs in developing and healthy postnatal kidneys, whereas the CNF and FSGS kidneys were characterized by numerous cellular bridges containing cells with strong expression of nestin and all analyzed proteins. Our results indicate that the mechanisms of gene control in nephrogenesis are reactivated under pathological conditions. These mechanisms could have a role in restoring glomerular integrity by potentially inducing the regeneration of podocytes from PECs.

Hepatocyte nuclear factors play an important role in the pathogenesis of nephropathy

Hepatocyte nuclear factors play an important role in the pathogenesis of nephropathy

Computational modelling of nephron progenitor cell movement and aggregation during kidney organogenesis

During early kidney organogenesis, nephron progenitor (NP) cells move from the tip to the corner region of the ureteric bud (UB) branches in order to form the pretubular aggregate, the early structure giving rise to nephron formation. NP cells derive from metanephric mesenchymal cells and physically interact with them during the movement. Chemotaxis and cell–cell adhesion differences are believed to drive the cell patterning during this critical period of organogenesis. However, the effect of these forces to the cell patterns and their respective movements are known in limited details. We applied a Cellular Potts Model to explore how these forces and organizations contribute to directed cell movement and aggregation. Model parameters were estimated based on fitting to experimental data obtained in ex vivo kidney explant and dissociation–reaggregation organoid culture studies. Our simulations indicated that optimal enrichment and aggregation of NP cells in the UB corner niche requires chemoattractant secretion from both the UB epithelial cells and the NP cells themselves, as well as differences in cell–cell adhesion energies. Furthermore, NP cells were observed, both experimentally and by modelling, to move at higher speed in the UB corner as compared to the tip region where they originated. The existence of different cell speed domains along the UB was confirmed using self-organizing map analysis. In summary, we saw faster NP cell movements near aggregation. The applicability of Cellular Potts Model approach to simulate cell movement and patterning was found to be good during for this early nephrogenesis process. Further refinement of the model should allow us to recapitulate the effects of developmental changes of cell phenotypes and molecular crosstalk during further organ development.

Wilms tumour.

Wilms tumour (WT) is a childhood embryonal tumour that is paradigmatic of the intersection between disrupted organogenesis and tumorigenesis. Many WT genes play a critical (non-redundant) role in early nephrogenesis. Improving patient outcomes requires advances in understanding and targeting of the multiple genes and cellular control pathways now identified as active in WT development. Decades of clinical and basic research have helped to gradually optimize clinical care. Curative therapy is achievable in 90% of affected children, even those with disseminated disease, yet survival disparities within and between countries exist and deserve commitment to change. Updated epidemiological studies have also provided novel insights into global incidence variations. Introduction of biology-driven approaches to risk stratification and new drug development has been slower in WT than in other childhood tumours. Current prognostic classification for children with WT is grounded in clinical and pathological findings and in dedicated protocols on molecular alterations. Treatment includes conventional cytotoxic chemotherapy and surgery, and radiation therapy in some cases. Advanced imaging to capture tumour composition, optimizing irradiation techniques to reduce target volumes, and evaluation of newer surgical procedures are key areas for future research.

Exposure of human fetal kidneys to mild analgesics interferes with early nephrogenesis.

Acetaminophen, aspirin, and ibuprofen are mild analgesics commonly used by pregnant women, the sole current recommendation being to avoid ibuprofen from the fifth month of gestation. The nephrotoxicity of these three analgesics is well documented in adults, as is their interference with prostaglandins biosynthesis. Here we investigated the effect of these analgesics on human first trimester kidneys ex vivo. We first evaluated prostaglandins biosynthesis functionality by performing a wide screening of prostaglandin expression patterns in first trimester human kidneys. We demonstrated that prostaglandins biosynthesis machinery is functional during early nephrogenesis. Human fetal kidney explants aged 7-12 developmental weeks were exposed ex vivo to ibuprofen, aspirin or acetaminophen for 7 days, and analyzed by histology, immunohistochemistry, and flow cytometry. This study has revealed that these analgesics induced a spectrum of abnormalities within early developing structures, ranging from cell death to a decline in differentiating glomeruli density. These results warrant caution for the use of these medicines during the first trimester of pregnancy.

The low affinity p75 neurotrophin receptor is down-regulated in congenital anomalies of the kidney and the urinary tract: Possible involvement in early nephrogenesis

Congenital Anomalies of the Kidney and of the Urinary Tract (CAKUT) cover a broad range of disorders including abnormal kidney development caused by defective nephrogenesis. Here we explored the possible involvement of the low affinity p75 neurotrophin receptor (p75NTR) in CAKUT and nephrogenesis. In mouse, p75NTR was highly expressed in fetal kidney, located within cortical early nephrogenic bodies, and decreased rapidly after birth. In human control fetal kidney, p75NTR was also located within the early nephrogenic bodies as well as in the mature glomeruli, presumably in the mesangium. In CAKUT fetal kidneys, the kidney cortical structure and the localization of p75NTR were often disorganized, and quantification of p75NTR in amniotic fluid revealed a significant reduction in CAKUT compared to control. Finally, invalidation of p75NTR in zebrafish embryo with an antisense morpholino significantly altered pronephros development. Our results indicate that renal p75NTR is altered in CAKUT fetuses, and could participate to early nephrogenesis.

In vivo High-Content Screening in Zebrafish for Developmental Nephrotoxicity of Approved Drugs.

Despite widespread drug exposure, for example during gestation or in prematurely born children, organ-specific developmental toxicity of most drugs is poorly understood. Developmental and functional abnormalities are a major cause of kidney diseases during childhood; however, the potential causal relationship to exposure with nephrotoxic drugs during nephrogenesis is widely unknown. To identify developmental nephrotoxic drugs in a large scale, we established and performed an automated high-content screen to score for phenotypic renal alterations in the Tg(wt1b:EGFP) zebrafish line. During early nephrogenesis, embryos were exposed to a compound library of approved drugs. After treatment, embryos were aligned within microtiter plates using 3D-printed orientation tools enabling the robust acquisition of consistent dorsal views of pronephric kidneys by automated microscopy. To qualitatively and quantitatively score and visualize phenotypes, we developed software tools for the semi-automated analysis, processing and visualization of this large image-based dataset. Using this scoring scheme, we were able to categorize compounds based on their potential developmental nephrotoxic effects. About 10% of tested drugs induced pronephric phenotypes including glomerular and tubular malformations, or overall changes in kidney morphology. Major chemical compound groups identified to cause glomerular and tubular alterations included dihydropyridine derivatives, HMG CoA reductase inhibitors, fibrates, imidazole, benzimidazole and triazole derivatives, corticosteroids, glucocorticoids, acetic acid derivatives and propionic acid derivatives. In conclusion, the presented study demonstrates the large-scale screening of kidney-specific toxicity of approved drugs in a live vertebrate embryo. The associated technology and tool-sets can be easily adapted for other organ systems providing a unique platform for in vivo large-scale assessment of organ-specific developmental toxicity or other biomedical applications. Ultimately, the presented data and associated visualization and browsing tools provide a resource for potentially nephrotoxic drugs and for further investigations.

Birth Weight and Susceptibility to Chronic Kidney Disease.

The worldwide prevalence of noncommunicable diseases (NCDs) is projected to increase substantially over the next few decades. Chronic kidney disease (CKD) is a key determinant of poor health outcomes for major NCD. Genetic predisposition and environmental exposures are contributory factors, but increasingly, it is being recognized that fetal development is also an important modulator of the NCD risk. Low birth weight (LBW) and CKD affect more disadvantaged populations and ethnic minorities and, therefore, causes a disproportionate burden on the poor. Human nephron number is highly variable and may range from under half a million to almost over two million. Significant variability is already present at birth, highlighting the importance of early nephrogenesis. Nearly 60% of nephrons are developed in the third-trimester of pregnancy. Nephron numbers increase in proportion to birth weight and gestational age. This wide-variability probably contributes to individual susceptibility to develop CKD where individuals with nephron numbers on the lower side of the spectrum are those at higher risk of developing kidney dysfunction at higher rate and progress more toward end-stage CKD. This article aims at discussing LBW and the susceptibility to CKD. Furthermore, in postnatal environment, the weight gain or change at adult life increases the metabolic demand and determines the phenotypic expression of disease along with the spectrum of nephron number. Hence, a cycle of hyperfiltration mechanism of these nephrons leads to proteinuria, glomerulo- sclerosis, and progressive development of larger glomeruli, a greater risk of proteinuria and progressive CKD. Therefore, LBW offspring are at risk of developing CKD (defined as albuminuria, a reduced glomerular filtration rate, or renal failure) in later life. Furthermore, the impact of prenatal programming is expected to be compounded with age, and the association of LBW with the risk of CKD seen in younger adults may become greater with age. It would be prudent, to adopt policies of intensified life-long surveillance of LBW people, anticipating this risk.

The myriad possibility of kidney organoids.

Purpose of reviewHuman kidney development and the mechanisms of many kidney diseases are incompletely understood partly due to the lack of appropriate models. Kidney organoids derived from human pluripotent stem cells (hPSCs) are a new and rapidly developing in-vitro system covering the window of early nephrogenesis and having the capacity for disease modelling. The application of global analytic tools such as RNA sequencing and proteomics is providing new and unexpected insights into kidney organoids with relevance for development and disease. In this review, we focus on the most significant advances in the field over the last 2 years.Recent findingsThere have been several protocol modifications for the differentiation of hPSCs into kidney organoids, including the additional step of implantation into mice. These changes have improved the vascularization and maturity of the major cell types in the organoids, increased the production scale, and reduced the cost and labour intensity of culturing organoids. Single-cell RNA sequencing and global proteomics of kidney organoids have provided important insights into the multiple cell populations, origin of cells, and regulatory relationships between genes. There has been an increase in research using patient-derived induced pluripotent stem cells (iPSCs), or combining gene editing with iPSC-derived kidney organoids as a novel disease-modelling platform for improving our understanding of disease mechanisms, drug testing and discovery, and the potential for personalized therapy. Finally, there has been progress in culturing hPSCs-derived kidney cells in microfluidic kidney-on-a-chip devices and this may provide a means of further improving the maturity of kidney organoids.SummaryThe review summarizes the latest progress on kidney organoids including differentiation protocols, analysis tools, and applications. Despite some limitations, hPSC-derived kidney organoids are authentic and practical models for investigating kidney development and disease and progressing understanding about tissue regeneration, drug screening, and disease modelling.

Morphologic and morphometric study on microvasculature of developing mouse kidneys

A proper morphogenesis of the renal microvasculature is crucial not only for fulfilling the renal function but also to slow down the progression of chronic kidney disease in adulthood. However, the current description of the developing microvasculature is incomplete. The present study investigated the morphogenesis and volume densities of the renal microvasculature using computer-assisted tubular tracing, immunohistochemistry for CD34, and unbiased stereology. The earliest glomerular capillaries were observed at the lower cleft of the S-shaped nephrons, as simple loops connecting the afferent and efferent arterioles. In parallel with this, the peritubular capillaries were established. Noticeably, from early nephrogenesis on, the efferent arterioles of the early-formed glomeruli ran in close proximity to their own thick ascending limbs. In addition, the ascending vasa recta arising from the arcuate or interlobular veins also ran in close proximity to the thick descending limb. Thus, the tubules and vessels formed the typical countercurrent relation in the medulla. No loop bends were observed between descending and ascending vasa recta. The volume density of the cortical and medullary peritubular capillary increased 3.3- and 2.6-fold, respectively, from 2.34 (0.13) and 7.03 (0.09)% [means (SD)] at embryonic day 14.5 (E14.5) to 7.71 (0.44) and 18.27 (1.17)% at postnatal day 40 (P40). In contrast, the volume density of glomeruli changed only slightly during kidney development, from 4.61 (0.47)% at E14.5 to 6.07 (0.2)% at P7 to 4.19 (0.47)% at P40. These results reflect that the growth and formation of the renal microvasculature closely correspond to functional development of the tubules.

Wnt Signaling in Kidney Development and Disease.

Wnt signal cascade is an evolutionarily conserved, developmental pathway that regulates embryogenesis, injury repair, and pathogenesis of human diseases. It is well established that Wnt ligands transmit their signal via canonical, β-catenin-dependent and noncanonical, β-catenin-independent mechanisms. Mounting evidence has revealed that Wnt signaling plays a key role in controlling early nephrogenesis and is implicated in the development of various kidney disorders. Dysregulations of Wnt expression cause a variety of developmental abnormalities and human diseases, such as congenital anomalies of the kidney and urinary tract, cystic kidney, and renal carcinoma. Multiple Wnt ligands, their receptors, and transcriptional targets are upregulated during nephron formation, which is crucial for mediating the reciprocal interaction between primordial tissues of ureteric bud and metanephric mesenchyme. Renal cysts are also associated with disrupted Wnt signaling. In addition, Wnt components are important players in renal tumorigenesis. Activation of Wnt/β-catenin is instrumental for tubular repair and regeneration after acute kidney injury. However, sustained activation of this signal cascade is linked to chronic kidney diseases and renal fibrosis in patients and experimental animal models. Mechanistically, Wnt signaling controls a diverse array of biologic processes, such as cell cycle progression, cell polarity and migration, cilia biology, and activation of renin-angiotensin system. In this chapter, we have reviewed recent findings that implicate Wnt signaling in kidney development and diseases. Targeting this signaling may hold promise for future treatment of kidney disorders in patients.

Effects of bisphenol A treatment during pregnancy on kidney development in mice: a stereological and histopathological study.

Bisphenol A (BPA) is a chemical found in plastics that resembles oestrogen in organisms. Developmental exposure to endocrine-disrupting chemicals, such as BPA, increases the susceptibility to type 2 diabetes (T2DM) and cardiovascular diseases. Animal studies have reported a nephron deficit in offspring exposed to maternal diabetes. The aim of this study was to investigate the prenatal BPA exposure effects on nephrogenesis in a mouse model that was predisposed to T2DM. This study quantitatively evaluated the renal structural changes using stereology and histomorphometry methods. The OF1 pregnant mice were treated with a vehicle or BPA (10 or 100 μg/kg/day) during days 9-16 of gestation (early nephrogenesis). The 30-day-old offspring were sacrificed, and tissue samples were collected and prepared for histopathological and stereology studies. Glomerular abnormalities and reduced glomerular formation were observed in the BPA offspring. The kidneys of the BPA10 and BPA100 female offspring had a significantly lower glomerular number and density than those of the CONTROL female offspring. The glomerular histomorphometry revealed a significant difference between the female and male CONTROL offspring for the analysed glomerular parameters that disappeared in the BPA10 and BPA100 offspring. In addition, the kidney histopathological examination showed typical male cuboidal epithelial cells of the Bowman capsule in the female BPA offspring. Exposure to environmentally relevant doses of BPA during embryonic development altered nephrogenesis. These structural changes could be associated with an increased risk of developing cardiometabolic diseases later in life.

EpCAM controls morphogenetic programs during zebrafish pronephros development

Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein that is dynamically expressed in human and murine renal epithelia during development. The levels of EpCAM in the renal epithelium are upregulated both during regeneration after ischemia/reperfusion injury and in renal-derived carcinomas. The role of EpCAM in early kidney development, however, has remained unclear. The zebrafish pronephros shows a similar segmentation pattern to the mammalian metanephric nephron, and has recently emerged as a tractable model to study the regulatory programs governing early nephrogenesis. Since EpCAM shows persistent expression in the pronephros throughout early development, we developed a method to study the global changes in gene expression in specific pronephric segments of wild type and EpCAM-deficient zebrafish embryos. In epcam mutants, we found 379 differentially expressed genes. Gene ontology analysis revealed that EpCAM controls various developmental programs, including uretric bud development, morphogenesis of branching epithelium, regulation of cell differentiation and cilium morphogenesis.

Loss of Zeb2 in mesenchyme-derived nephrons causes primary glomerulocystic disease

Primary glomerulocystic kidney disease is a special form ofrenal cystic disorder characterized by Bowman's space dilatation in the absence of tubular cysts. ZEB2 is a SMAD-interacting transcription factor involved in Mowat-Wilson syndrome, a congenital disorder with an increased risk for kidney anomalies. Here we show that deletion of Zeb2 in mesenchyme-derived nephrons with either Pax2-cre or Six2-cre causes primary glomerulocystic kidney disease without tubular cysts in mice. Glomerulotubular junction analysis revealed many atubular glomeruli in the kidneys ofZeb2 knockout mice, which explains the presence of glomerular cysts in the absence of tubular dilatation. Gene expression analysis showed decreased expression of early proximal tubular markers in the kidneys of Zeb2 knockout mice preceding glomerular cyst formation, suggesting thatdefects in proximal tubule development during early nephrogenesis contribute to the formation of congenital atubular glomeruli. At the molecular level, Zeb2 deletion caused aberrant expression of Pkd1, Hnf1β, and Glis3, threegenes causing glomerular cysts. Thus, Zeb2 regulates the morphogenesis of mesenchyme-derived nephrons andis required for proximal tubule development and glomerulotubular junction formation. Our findings also suggest that ZEB2 might be a novel disease gene in patients with primary glomerular cystic disease.

Osr1 Interacts Synergistically with Wt1 to Regulate Kidney Organogenesis.

Renal hypoplasia is a common cause of pediatric renal failure and several adult-onset diseases. Recent studies have associated a variant of the OSR1 gene with reduction of newborn kidney size and function in heterozygotes and neonatal lethality with kidney defects in homozygotes. How OSR1 regulates kidney development and nephron endowment is not well understood, however. In this study, by using the recently developed CRISPR genome editing technology, we genetically labeled the endogenous Osr1 protein and show that Osr1 interacts with Wt1 in the developing kidney. Whereas mice heterozygous for either an Osr1 or Wt1 null allele have normal kidneys at birth, most mice heterozygous for both Osr1 and Wt1 exhibit defects in metanephric kidney development, including unilateral or bilateral kidney agenesis or hypoplasia. The developmental defects in the Osr1+/-Wt1+/- mouse embryos were detected as early as E10.5, during specification of the metanephric mesenchyme, with the Osr1+/-Wt1+/- mouse embryos exhibiting significantly reduced Pax2-positive and Six2-positive nephron progenitor cells. Moreover, expression of Gdnf, the major nephrogenic signal for inducing ureteric bud outgrowth, was significantly reduced in the metanephric mesenchyme in Osr1+/-Wt1+/- embryos in comparison with the Osr1+/- or Wt1+/- littermates. By E11.5, as the ureteric buds invade the metanephric mesenchyme and initiate branching morphogenesis, kidney morphogenesis was significantly impaired in the Osr1+/-Wt1+/- embryos in comparison with the Osr1+/- or Wt1+/- embryos. These results indicate that Osr1 and Wt1 act synergistically to regulate nephron endowment by controlling metanephric mesenchyme specification during early nephrogenesis.

LDL Receptor-Related Protein 6 Modulates Ret Proto-Oncogene Signaling in Renal Development and Cystic Dysplasia.

Hypoplastic and/or cystic kidneys have been found in both LDL receptor-related protein 6 (Lrp6)- and β-catenin-mutant mouse embryos, and these proteins are key molecules for Wnt signaling. However, the underlying mechanisms of Lrp6/β-catenin signaling in renal development and cystic formation remain poorly understood. In this study, we found evidence that diminished cell proliferation and increased apoptosis occur before cystic dysplasia in the renal primordia of Lrp6-deficient mouse embryos. The expression of Ret proto-oncogene (Ret), a critical receptor for the growth factor glial cell line-derived neurotrophic factor (GDNF), which is required for early nephrogenesis, was dramatically diminished in the mutant renal primordia. The activities of other representative nephrogenic genes, including Lim1, Pax2, Pax8, GDNF, and Wnt11, were subsequently diminished in the mutant renal primordia. Molecular biology experiments demonstrated that Ret is a novel transcriptional target of Wnt/β-catenin signaling. Wnt agonist lithium promoted Ret expression in vitro and in vivo. Furthermore, Lrp6-knockdown or lithium treatment in vitro led to downregulation or upregulation, respectively, of the phosphorylated mitogen-activated protein kinases 1 and 3, which act downstream of GDNF/Ret signaling. Mice with single and double mutations of Lrp6 and Ret were perinatal lethal and demonstrated gene dosage-dependent effects on the severity of renal hypoplasia during embryogenesis. Taken together, these results suggest that Lrp6-mediated Wnt/β-catenin signaling modulates or interacts with a signaling network consisting of Ret cascades and related nephrogenic factors for renal development, and the disruption of these genes or signaling activities may cause a spectrum of hypoplastic and cystic kidney disorders.