Loss of Roundabout Guidance Receptor 2 (Robo2) in Podocytes Protects Adult Mice from Glomerular Injury by Maintaining Podocyte Foot Process Structure.

Roundabout guidance receptor 2 (ROBO2) plays an important role during early kidney development. ROBO2 is expressed in podocytes, inhibits nephrin-induced actin polymerization, down-regulates nonmuscle myosin IIA activity, and destabilizes kidney podocyte adhesion. However, the role of ROBO2 during kidney injury, particularly in mature podocytes, is not known. Herein, we report that loss of ROBO2 in podocytes [Robo2 conditional knockout (cKO) mouse] is protective from glomerular injuries. Ultrastructural analysis reveals that Robo2 cKO mice display less foot process effacement and better-preserved slit-diaphragm density compared with wild-type littermates injured by either protamine sulfate or nephrotoxic serum (NTS). The Robo2 cKO mice also develop less proteinuria after NTS injury. Further studies reveal that ROBO2 expression in podocytes is up-regulated after glomerular injury because its expression levels are higher in the glomeruli of NTS injured mice and passive Heymann membranous nephropathy rats. Moreover, the amount of ROBO2 in the glomeruli is also elevated in patients with membranous nephropathy. Finally, overexpression of ROBO2 in cultured mouse podocytes compromises cell adhesion. Taken together, these findings suggest that kidney injury increases glomerular ROBO2 expression that might compromise podocyte adhesion and, thus, loss of Robo2 in podocytes could protect from glomerular injury by enhancing podocyte adhesion that helps maintain foot process structure. Our findings also suggest that ROBO2 is a therapeutic target for podocyte injury and podocytopathy.

Integrin-Linked Kinase Deficiency in Collecting Duct Principal Cell Promotes Necroptosis of Principal Cell and Contributes to Kidney Inflammation and Fibrosis.

BACKGROUND: Necroptosis is a newly discovered cell death pathway that plays a critical role in AKI. The involvement of integrin-linked kinase (ILK) in necroptosis has not been studied. METHODS: We performed experiments in mice with an Ilk deletion in collecting duct (CD) principal cells (PCs), and cultured tubular epithelial cells treated with an ILK inhibitor or ILK siRNA knockdown. RESULTS: Ilk deletion in CD PCs resulted in acute tubular injury and early mortality in mice. Progressive interstitial fibrosis and inflammation associated with the activation of the canonical TGF-ß signaling cascade were detected in the kidneys of the mice lacking ILK in the CD PCs. In contrast to the minimal apoptosis detected in the animals' injured CDs, widespread necroptosis was present in ILK-deficient PCs, characterized by cell swelling, deformed mitochondria, and rupture of plasma membrane. In addition, ILK deficiency resulted in increased expression and activation of necroptotic proteins MLKL and RIPK3, and membrane translocation of MLKL in CD PCs. ILK inhibition and siRNA knockdown reduced cell survival in cultured tubular cells, concomitant with increased membrane accumulation of MLKL and/or phospho-MLKL. Administration of a necroptosis inhibitor, necrostatin-1, blocked cell death in vitro and significantly attenuated inflammation, interstitial fibrosis, and renal failure in ILK-deficient mice. CONCLUSIONS: The study demonstrates the critical involvement of ILK in necroptosis through modulation of the RIPK3 and MLKL pathway and highlights the contribution of CD PC injury to the development of inflammation and interstitial fibrosis of the kidney.

Identification of direct negative cross-talk between the SLIT2 and bone morphogenetic protein-Gremlin signaling pathways.

Slit guidance ligand 2 (SLIT2) is a large, secreted protein that binds roundabout (ROBO) receptors on multiple cell types, including neurons and kidney podocytes. SLIT2-ROBO-mediated signaling regulates neuronal migration and ureteric bud (UB) outgrowth during kidney development as well as glomerular filtration in adult kidneys. Additionally, SLIT2 binds Gremlin, an antagonist of bone morphogenetic proteins (BMPs), and BMP-Gremlin signaling also regulates UB formation. However, direct cross-talk between the ROBO2-SLIT2 and BMP-Gremlin signaling pathways has not been established. Here, we report the discovery of negative feedback between the SLIT2 and BMP-Gremlin signaling pathways. We found that the SLIT2-Gremlin interaction inhibited both SLIT2-ROBO2 signaling in neurons and Gremlin antagonism of BMP activity in myoblasts and fibroblasts. Furthermore, BMP2 down-regulated SLIT2 expression and promoter activity through canonical BMP signaling. Gremlin treatment, BMP receptor inhibition, and SMAD family member 4 (SMAD4) knockdown rescued BMP-mediated repression of SLIT2. BMP2 treatment of nephron progenitor cells derived from human embryonic stem cells decreased SLIT2 expression, further suggesting an interaction between the BMP2-Gremlin and SLIT2 pathways in human kidney cells. In conclusion, our study has revealed direct negative cross-talk between two pathways, previously thought to be unassociated, that may regulate both kidney development and adult tissue maintenance.

Crim1 regulates integrin signaling in murine lens development.

The developing lens is a powerful system for investigating the molecular basis of inductive tissue interactions and for studying cataract, the leading cause of blindness. The formation of tightly controlled cell-cell adhesions and cell-matrix junctions between lens epithelial (LE) cells, between lens fiber (LF) cells, and between these two cell populations enables the vertebrate lens to adopt a highly ordered structure and acquire optical transparency. Adhesion molecules are thought to maintain this ordered structure, but little is known about their identity or interactions. Cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is strongly expressed in the developing lens and its mutation causes ocular disease in both mice and humans. How Crim1 regulates lens morphogenesis is not understood. We identified a novel ENU-induced hypomorphic allele of Crim1, Crim1(glcr11), which in the homozygous state causes cataract and microphthalmia. Using this and two other mutant alleles, Crim1(null) and Crim1(cko), we show that the lens defects in Crim1 mouse mutants originate from defective LE cell polarity, proliferation and cell adhesion. Crim1 adhesive function is likely to be required for interactions both between LE cells and between LE and LF cells. We show that Crim1 acts in LE cells, where it colocalizes with and regulates the levels of active ß1 integrin and of phosphorylated FAK and ERK. The RGD and transmembrane motifs of Crim1 are required for regulating FAK phosphorylation. These results identify an important function for Crim1 in the regulation of integrin- and FAK-mediated LE cell adhesion during lens development.

Mutations in TBX18 Cause Dominant Urinary Tract Malformations via Transcriptional Dysregulation of Ureter Development.

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life. Identification of single-gene mutations that cause CAKUT permits the first insights into related disease mechanisms. However, for most cases the underlying defect remains elusive. We identified a kindred with an autosomal-dominant form of CAKUT with predominant ureteropelvic junction obstruction. By whole exome sequencing, we identified a heterozygous truncating mutation (c.1010delG) of T-Box transcription factor 18 (TBX18) in seven affected members of the large kindred. A screen of additional families with CAKUT identified three families harboring two heterozygous TBX18 mutations (c.1570C>T and c.487A>G). TBX18 is essential for developmental specification of the ureteric mesenchyme and ureteric smooth muscle cells. We found that all three TBX18 altered proteins still dimerized with the wild-type protein but had prolonged protein half life and exhibited reduced transcriptional repression activity compared to wild-type TBX18. The p.Lys163Glu substitution altered an amino acid residue critical for TBX18-DNA interaction, resulting in impaired TBX18-DNA binding. These data indicate that dominant-negative TBX18 mutations cause human CAKUT by interference with TBX18 transcriptional repression, thus implicating ureter smooth muscle cell development in the pathogenesis of human CAKUT.

A Dominant Mutation in Nuclear Receptor Interacting Protein 1 Causes Urinary Tract Malformations via Dysregulation of Retinoic Acid Signaling.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of CKD in the first three decades of life. However, for most patients with CAKUT, the causative mutation remains unknown. We identified a kindred with an autosomal dominant form of CAKUT. By whole-exome sequencing, we identified a heterozygous truncating mutation (c.279delG, p.Trp93fs*) of the nuclear receptor interacting protein 1 gene (NRIP1) in all seven affected members. NRIP1 encodes a nuclear receptor transcriptional cofactor that directly interacts with the retinoic acid receptors (RARs) to modulate retinoic acid transcriptional activity. Unlike wild-type NRIP1, the altered NRIP1 protein did not translocate to the nucleus, did not interact with RARα, and failed to inhibit retinoic acid-dependent transcriptional activity upon expression in HEK293 cells. Notably, we also showed that treatment with retinoic acid enhanced NRIP1 binding to RARα RNA in situ hybridization confirmed Nrip1 expression in the developing urogenital system of the mouse. In explant cultures of embryonic kidney rudiments, retinoic acid stimulated Nrip1 expression, whereas a pan-RAR antagonist strongly reduced it. Furthermore, mice heterozygous for a null allele of Nrip1 showed a CAKUT-spectrum phenotype. Finally, expression and knockdown experiments in Xenopus laevis confirmed an evolutionarily conserved role for NRIP1 in renal development. These data indicate that dominant NRIP1 mutations can cause CAKUT by interference with retinoic acid transcriptional signaling, shedding light on the well documented association between abnormal vitamin A levels and renal malformations in humans, and suggest a possible gene-environment pathomechanism in this disease.

Blocking peptides and molecular mimicry as treatment for kidney disease.

Protein mimotopes, or blocking peptides, are small therapeutic peptides that prevent protein-protein interactions by selectively mimicking a native binding domain. Inexpensive technology facilitates straightforward design and production of blocking peptides in sufficient quantities to allow preventive and therapeutic trials in both in vitro and in vivo experimental disease models. The kidney is an ideal peptide target, since small molecules undergo rapid filtration and efficient bulk absorption by tubular epithelial cells. Because the half-life of peptides is markedly prolonged in the kidneys compared with the bloodstream, blocking peptides are an attractive tool for treating diverse renal diseases, including ischemia, proteinuric states, such as membranous nephropathy and focal and segmental glomerulosclerosis, and renal cell carcinoma. Therapeutic peptides represent one of the fastest-growing reagent classes for novel drug development in human disease, partly because of their ease of administration, high binding affinity, and minimal off-target effects. This review introduces the concepts of blocking peptide design, production, and administration and highlights the potential use of therapeutic peptides to prevent or treat specific renal diseases.

MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus.

Cardiac left ventricular outflow tract (LVOT) defects represent a common but heterogeneous subset of congenital heart disease for which gene identification has been difficult. We describe a 46,XY,t(1;5)(p36.11;q31.2)dn translocation carrier with pervasive developmental delay who also exhibited LVOT defects, including bicuspid aortic valve (BAV), coarctation of the aorta (CoA) and patent ductus arteriosus (PDA). The 1p breakpoint disrupts the 5' UTR of AHDC1, which encodes AT-hook DNA-binding motif containing-1 protein, and AHDC1-truncating mutations have recently been described in a syndrome that includes developmental delay, but not congenital heart disease [Xia, F., Bainbridge, M.N., Tan, T.Y., Wangler, M.F., Scheuerle, A.E., Zackai, E.H., Harr, M.H., Sutton, V.R., Nalam, R.L., Zhu, W. et al. (2014) De Novo truncating mutations in AHDC1 in individuals with syndromic expressive language delay, hypotonia, and sleep apnea. Am. J. Hum. Genet., 94, 784-789]. On the other hand, the 5q translocation breakpoint disrupts the 3' UTR of MATR3, which encodes the nuclear matrix protein Matrin 3, and mouse Matr3 is strongly expressed in neural crest, developing heart and great vessels, whereas Ahdc1 is not. To further establish MATR3 3' UTR disruption as the cause of the proband's LVOT defects, we prepared a mouse Matr3(Gt-ex13) gene trap allele that disrupted the 3' portion of the gene. Matr3(Gt-ex13) homozygotes are early embryo lethal, but Matr3(Gt-ex13) heterozygotes exhibit incompletely penetrant BAV, CoA and PDA phenotypes similar to those in the human proband, as well as ventricular septal defect (VSD) and double-outlet right ventricle (DORV). Both the human MATR3 translocation breakpoint and the mouse Matr3(Gt-ex13) gene trap insertion disturb the polyadenylation of MATR3 transcripts and alter Matrin 3 protein expression, quantitatively or qualitatively. Thus, subtle perturbations in Matrin 3 expression appear to cause similar LVOT defects in human and mouse.

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

Primary glomerulocystic kidney disease is a special form of renal 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 of Zeb2 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 that defects 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, Hnf1ß, and Glis3, three genes causing glomerular cysts. Thus, Zeb2 regulates the morphogenesis of mesenchyme-derived nephrons and is 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.

Mutations of the SLIT2-ROBO2 pathway genes SLIT2 and SRGAP1 confer risk for congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) account for 40-50% of chronic kidney disease that manifests in the first two decades of life. Thus far, 31 monogenic causes of isolated CAKUT have been described, explaining ~12% of cases. To identify additional CAKUT-causing genes, we performed whole-exome sequencing followed by a genetic burden analysis in 26 genetically unsolved families with CAKUT. We identified two heterozygous mutations in SRGAP1 in 2 unrelated families. SRGAP1 is a small GTPase-activating protein in the SLIT2-ROBO2 signaling pathway, which is essential for development of the metanephric kidney. We then examined the pathway-derived candidate gene SLIT2 for mutations in cohort of 749 individuals with CAKUT and we identified 3 unrelated individuals with heterozygous mutations. The clinical phenotypes of individuals with mutations in SLIT2 or SRGAP1 were cystic dysplastic kidneys, unilateral renal agenesis, and duplicated collecting system. We show that SRGAP1 is expressed in early mouse nephrogenic mesenchyme and that it is coexpressed with ROBO2 in SIX2-positive nephron progenitor cells of the cap mesenchyme in developing rat kidney. We demonstrate that the newly identified mutations in SRGAP1 lead to an augmented inhibition of RAC1 in cultured human embryonic kidney cells and that the SLIT2 mutations compromise the ability of the SLIT2 ligand to inhibit cell migration. Thus, we report on two novel candidate genes for causing monogenic isolated CAKUT in humans.

Digital pathology assessment of kidney glomerular filtration barrier ultrastructure in an animal model of podocytopathy.

Background: Transmission electron microscopy (TEM) images can visualize kidney glomerular filtration barrier ultrastructure, including the glomerular basement membrane (GBM) and podocyte foot processes (PFP). Podocytopathy is associated with glomerular filtration barrier morphological changes observed experimentally and clinically by measuring GBM or PFP width. However, these measurements are currently performed manually. This limits research on podocytopathy disease mechanisms and therapeutics due to labor intensiveness and inter-operator variability. Methods: We developed a deep learning-based digital pathology computational method to measure GBM and PFP width in TEM images from the kidneys of Integrin-Linked Kinase (ILK) podocyte-specific conditional knockout (cKO) mouse, an animal model of podocytopathy, compared to wild-type (WT) control mouse. We obtained TEM images from WT and ILK cKO littermate mice at 4 weeks old. Our automated method was composed of two stages: a U-Net model for GBM segmentation, followed by an image processing algorithm for GBM and PFP width measurement. We evaluated its performance with a 4-fold cross-validation study on WT and ILK cKO mouse kidney pairs. Results: Mean (95% confidence interval) GBM segmentation accuracy, calculated as Jaccard index, was 0.54 (0.52-0.56) for WT and 0.61 (0.56-0.66) for ILK cKO TEM images. Automated and corresponding manual measured PFP widths differed significantly for both WT (p<0.05) and ILK cKO (p<0.05), while automated and manual GBM widths differed only for ILK cKO (p<0.05) but not WT (p=0.49) specimens. WT and ILK cKO specimens were morphologically distinguishable by manual GBM (p<0.05) and PFP (p<0.05) width measurements. This phenotypic difference was reflected in the automated GBM (p=0.06) more than PFP (p=0.20) widths. Conclusions: These results suggest that certain automated measurements enabled via deep learning-based digital pathology tools could distinguish healthy kidneys from those with podocytopathy. Our proposed method provides high-throughput, objective morphological analysis and could facilitate podocytopathy research and translate into clinical diagnosis.

Efficient generation and mapping of recessive developmental mutations using ENU mutagenesis.

Treatment with N-ethyl-N-nitrosourea (ENU) efficiently generates single-nucleotide mutations in mice. Along with the renewed interest in this approach, much attention has been given recently to large screens with broad aims; however, more finely focused studies have proven very productive as well. Here we show how mutagenesis together with genetic mapping can facilitate the rapid characterization of recessive loci required for normal embryonic development. We screened third-generation progeny of mutagenized mice at embryonic day (E) 18.5 for abnormalities of organogenesis. We ascertained 15 monogenic mutations in the 54 families that were comprehensively analyzed. We carried out the experiment as an outcross, which facilitated the genetic mapping of the mutations by haplotype analysis. We mapped seven of the mutations and identified the affected locus in two lines. Using a hierarchical approach, it is possible to maximize the efficiency of this analysis so that it can be carried out easily with modest infrastructure and resources.

Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells.

Several proteins implicated in the pathogenesis of polycystic kidney disease (PKD) localize to cilia. Furthermore, cilia are malformed in mice with PKD with mutations in TgN737Rpw (encoding polaris). It is not known, however, whether ciliary dysfunction occurs or is relevant to cyst formation in PKD. Here, we show that polycystin-1 (PC1) and polycystin-2 (PC2), proteins respectively encoded by Pkd1 and Pkd2, mouse orthologs of genes mutated in human autosomal dominant PKD, co-distribute in the primary cilia of kidney epithelium. Cells isolated from transgenic mice that lack functional PC1 formed cilia but did not increase Ca(2+) influx in response to physiological fluid flow. Blocking antibodies directed against PC2 similarly abolished the flow response in wild-type cells as did inhibitors of the ryanodine receptor, whereas inhibitors of G-proteins, phospholipase C and InsP(3) receptors had no effect. These data suggest that PC1 and PC2 contribute to fluid-flow sensation by the primary cilium in renal epithelium and that they both function in the same mechanotransduction pathway. Loss or dysfunction of PC1 or PC2 may therefore lead to PKD owing to the inability of cells to sense mechanical cues that normally regulate tissue morphogenesis.

ZEB2 controls kidney stromal progenitor differentiation and inhibits abnormal myofibroblast expansion and kidney fibrosis.

FOXD1+ cell-derived stromal cells give rise to pericytes and fibroblasts that support the kidney vasculature and interstitium but are also major precursors of myofibroblasts. ZEB2 is a SMAD-interacting transcription factor that is expressed in developing kidney stromal progenitors. Here we show that Zeb2 is essential for normal FOXD1+ stromal progenitor development. Specific conditional knockout of mouse Zeb2 in FOXD1+ stromal progenitors (Zeb2 cKO) leads to abnormal interstitial stromal cell development, differentiation, and kidney fibrosis. Immunofluorescent staining analyses revealed abnormal expression of interstitial stromal cell markers MEIS1/2/3, CDKN1C, and CSPG4 (NG2) in newborn and 3-week-old Zeb2-cKO mouse kidneys. Zeb2-deficient FOXD1+ stromal progenitors also took on a myofibroblast fate that led to kidney fibrosis and kidney failure. Cell marker studies further confirmed that these myofibroblasts expressed pericyte and resident fibroblast markers, including PDGFRß, CSPG4, desmin, GLI1, and NT5E. Notably, increased interstitial collagen deposition associated with loss of Zeb2 in FOXD1+ stromal progenitors was accompanied by increased expression of activated SMAD1/5/8, SMAD2/3, SMAD4, and AXIN2. Thus, our study identifies a key role of ZEB2 in maintaining the cell fate of FOXD1+ stromal progenitors during kidney development, whereas loss of ZEB2 leads to differentiation of FOXD1+ stromal progenitors into myofibroblasts and kidney fibrosis.

Network Pharmacology-Based Study on the Active Ingredients and Mechanism of Pan Ji Sheng Traditional Chinese Medicine Formula in the Treatment of Inflammation.

Background: Pan Ji Sheng Formula is a Chinese medicine formula that enables heat-free detoxification as well as anti-inflammatory and immune-boosting properties. This formula contains eight herbs. Its underlying mechanism is unknown. The bioactive ingredients were screened in our work, and the mechanism of this formula was investigated. Methods: Using traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP), ingredients in Pan Ji Sheng Chinese medicine formula were screened, and we selected the main bioactive ingredients for web-based research. The targets of bioactive ingredients are primarily obtained from the SwissTargetPrediction and TCMSP databases, and the text mining method is used. STRING and Cytoscape were then used to examine the protein-protein interaction (PPI) networks. To explore the biological function and related pathways, functional annotation and pathway analysis were performed. Results: This research discovered 96 bioactive ingredients. Then, 215 potential targets of bioactive ingredients were screened. Through the analysis of the PPI network, we discovered 25 key target genes, which can be described as hub target genes regulated by bioactive ingredients. Bioactive ingredients primarily regulate CASP3, AKT1, JUN, and other proteins. The formula works synergistically to enhance immune response and antiinfection by regulating immune-related pathways, TNF signaling pathways, and apoptosis. Conclusions: A variety of bioactive ingredients in the formula could play roles in regulating CASP3, AKT1, and other genes in immune, infection, apoptosis, and tumor-related signaling pathways. Our data point the way forward for future studies on the mechanism of action of this formula.

Copy Number Variation Analysis Facilitates Identification of Genetic Causation in Patients with Congenital Anomalies of the Kidney and Urinary Tract.

Background: Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease among children and adults younger than 30 yr. In our previous study, whole-exome sequencing (WES) identified a known monogenic cause of isolated or syndromic CAKUT in 13% of families with CAKUT. However, WES has limitations and detection of copy number variations (CNV) is technically challenging, and CNVs causative of CAKUT have previously been detected in up to 16% of cases. Objective: To detect CNVs causing CAKUT in this WES cohort and increase the diagnostic yield. Design setting and participants: We performed a genome-wide single nucleotide polymorphism (SNP)-based CNV analysis on the same CAKUT cohort for whom WES was previously conducted. Outcome measurements and statistical analysis: We evaluated and classified the CNVs using previously published predefined criteria. Results and limitations: In a cohort of 170 CAKUT families, we detected a pathogenic CNV known to cause CAKUT in nine families (5.29%, 9/170). There were no competing variants on genome-wide CNV analysis or WES analysis. In addition, we identified novel likely pathogenic CNVs that may cause a CAKUT phenotype in three of the 170 families (1.76%). Conclusions: CNV analysis in this cohort of 170 CAKUT families previously examined via WES increased the rate of diagnosis of genetic causes of CAKUT from 13% on WES to 18% on WES + CNV analysis combined. We also identified three candidate loci that may potentially cause CAKUT. Patient summary: We conducted a genetics study on families with congenital anomalies of the kidney and urinary tract (CAKUT). We identified gene mutations that can explain CAKUT symptoms in 5.29% of the families, which increased the percentage of genetic causes of CAKUT to 18% from a previous study, so roughly one in five of our patients with CAKUT had a genetic cause. These analyses can help patients with CAKUT and their families in identifying a possible genetic cause.

Assessing vesicoureteral reflux in live inbred mice via ultrasound with a microbubble contrast agent.

Vesicoureteral reflux (VUR) is a common pediatric anomaly linked to renal scarring and hypertension. Although there are many mouse VUR models, cystograms have previously only been performed in euthanized animals, thus preventing serial assessments for VUR in the same animal and not delineating "live" physiology. Our purpose was to develop a live murine cystogram assay that could be used serially to track reflux. We injected microbubbles via transurethral catheters into bladders of C57BL6/J and C3H/HeJ inbred mouse strains that are known to have low and high VUR rates, respectively. We performed ultrasound to visualize microbubbles in the renal pelvis to determine feasibility of the procedure. We then repeated the microbubble ultrasound using a transducer allowing for visualization of both kidneys and ureters simultaneously and for 3 dimensional (3D) reconstruction. We then performed "euthanized" cystograms on all mice for comparison. C3H/HeJ mice had a strong and persistent microbubble signal in the renal pelvis and ureters bilaterally with low-contrast infusion volumes (<100 µl) and similarly showed bilateral reflux by euthanized cystograms. With larger infused volumes (≥150 µl), C57BL6/J mice had small volumes of microbubbles in the renal pelvis that cleared quickly and did not show reflux on euthanized cystograms. Thus, using animal models of known VUR, we demonstrate the utility of contrast-enhanced ultrasound to visualize reflux in live mice.

The fate of Notch-deficient nephrogenic progenitor cells during metanephric kidney development.

To determine which nephron segments require Notch signals for development, we conditionally deleted Rbpj, a transcription factor required for canonical Notch signaling, in nephrogenic progenitors (NPs) of the metanephric mesenchyme. The retinoic acid receptor-ß2 (Rarb2) promoter efficiently directed Cre-recombinase (Cre) activity to these progenitors. Conditional knockout of Rbpj in mice (Rarb2Cre(+)/Rbpj (f/-)) caused severe renal hypoplasia, as indicated by a 70-95% reduction in nephron number and the development of tubular cysts. To track the fate of NPs following Rarb2Cre expression, we labeled them with membrane-associated enhanced green fluorescent protein (GFP). In TomatoGFP(+)/Rarb2Cre(+) control mice, NPs differentiated into epithelia of all nephron segments, except into collecting ducts. In TomatoGFP(+)/Rarb2Cre(+)/Rbpj (f/-) conditional knockout mice, NPs developed into podocytes or distal tubular epithelia, indicating that canonical Notch signals were not required for mesenchymal-to-epithelial transition or for the specification of these nephron segments. Conversely, the few proximal tubules and associated cysts that developed in these mice were derived from the 5-10% of NPs that had failed to express Cre and, therefore, had intact Notch signaling. Thus, our fate mapping studies establish that the profound effect of Notch signaling on nephrogenesis is due to the specification of proximal but not distal tubules or podocytes.

Characterization of apparently balanced chromosomal rearrangements from the developmental genome anatomy project.

Apparently balanced chromosomal rearrangements in individuals with major congenital anomalies represent natural experiments of gene disruption and dysregulation. These individuals can be studied to identify novel genes critical in human development and to annotate further the function of known genes. Identification and characterization of these genes is the goal of the Developmental Genome Anatomy Project (DGAP). DGAP is a multidisciplinary effort that leverages the recent advances resulting from the Human Genome Project to increase our understanding of birth defects and the process of human development. Clinically significant phenotypes of individuals enrolled in DGAP are varied and, in most cases, involve multiple organ systems. Study of these individuals' chromosomal rearrangements has resulted in the mapping of 77 breakpoints from 40 chromosomal rearrangements by FISH with BACs and fosmids, array CGH, Southern-blot hybridization, MLPA, RT-PCR, and suppression PCR. Eighteen chromosomal breakpoints have been cloned and sequenced. Unsuspected genomic imbalances and cryptic rearrangements were detected, but less frequently than has been reported previously. Chromosomal rearrangements, both balanced and unbalanced, in individuals with multiple congenital anomalies continue to be a valuable resource for gene discovery and annotation.

PODO: Trial Design: Phase 2 Study of PF-06730512 in Focal Segmental Glomerulosclerosis.

INTRODUCTION: Focal segmental glomerulosclerosis (FSGS) is characterized by proteinuria and a histologic pattern of glomerular lesions of diverse etiology that share features including glomerular scarring and podocyte foot process effacement. Roundabout guidance receptor 2 (ROBO2)/slit guidance ligand 2 (SLIT2) signaling destabilizes the slit diaphragm and reduces podocyte adhesion to the glomerular basement membrane (GBM). Preclinical studies suggest that inhibition of glomerular ROBO2/SLIT2 signaling can stabilize podocyte adhesion and reduce proteinuria. This clinical trial evaluates the preliminary efficacy and safety of ROBO2/SLIT2 inhibition with the ROBO2 fusion protein PF-06730512 in patients with FSGS. METHODS: The Study to Evaluate PF-06730512 in Adults With FSGS (PODO; ClinicalTrials.gov identifier NCT03448692), an open-label, phase 2a, multicenter trial in adults with FSGS, will enroll patients into 2 cohorts (n = 22 per cohort) to receive either high- or low-dose PF-06730512 (intravenous) every 2 weeks for 12 weeks. Key inclusion criteria include a confirmed biopsy diagnosis of FSGS, an estimated glomerular filtration rate (eGFR) ≥45 ml/min/1.73 m2 based on the Chronic Kidney Disease Epidemiology Collaboration formula (30-45 with a recent biopsy), and urinary protein-to-creatinine ratio (UPCR) >1.5 g/g. Key exclusion criteria include collapsing FSGS, serious/active infection, ≥50% tubulointerstitial fibrosis on biopsy, and organ transplantation. The primary endpoint is change from baseline to week 13 in UPCR; secondary endpoints include safety, changes in eGFR, and PF-06730512 serum concentration. RESULTS: This ongoing trial will report the efficacy, safety, pharmacokinetics, and biomarker results of PF-06730512 for patients with FSGS. CONCLUSION: Findings from this proof-of-concept study may support further development and evaluation of PF-06730512 to treat FSGS and warrant assessment in phase 3 clinical trials.