Soluble FLT1 binds lipid microdomains in podocytes to control cell morphology and glomerular barrier function.

Vascular endothelial growth factor and its receptors, FLK1/KDR and FLT1, are key regulators of angiogenesis. Unlike FLK1/KDR, the role of FLT1 has remained elusive. FLT1 is produced as soluble (sFLT1) and full-length isoforms. Here, we show that pericytes from multiple tissues produce sFLT1. To define the biologic role of sFLT1, we chose the glomerular microvasculature as a model system. Deletion of Flt1 from specialized glomerular pericytes, known as podocytes, causes reorganization of their cytoskeleton with massive proteinuria and kidney failure, characteristic features of nephrotic syndrome in humans. The kinase-deficient allele of Flt1 rescues this phenotype, demonstrating dispensability of the full-length isoform. Using cell imaging, proteomics, and lipidomics, we show that sFLT1 binds to the glycosphingolipid GM3 in lipid rafts on the surface of podocytes, promoting adhesion and rapid actin reorganization. sFLT1 also regulates pericyte function in vessels outside of the kidney. Our findings demonstrate an autocrine function for sFLT1 to control pericyte behavior.

Bipolar disorder-iPSC derived neural progenitor cells exhibit dysregulation of store-operated Ca2+ entry and accelerated differentiation.

While most of the efforts to uncover mechanisms contributing to bipolar disorder (BD) focused on phenotypes at the mature neuron stage, little research has considered events that may occur during earlier timepoints of neurodevelopment. Further, although aberrant calcium (Ca2+) signaling has been implicated in the etiology of this condition, the possible contribution of store-operated Ca2+ entry (SOCE) is not well understood. Here, we report Ca2+ and developmental dysregulations related to SOCE in BD patient induced pluripotent stem cell (iPSC)-derived neural progenitor cells (BD-NPCs) and cortical-like glutamatergic neurons. First, using a Ca2+ re-addition assay we found that BD-NPCs and neurons had attenuated SOCE. Intrigued by this finding, we then performed RNA-sequencing and uncovered a unique transcriptome profile in BD-NPCs suggesting accelerated neurodifferentiation. Consistent with these results, we measured a slower rate of proliferation, increased neurite outgrowth, and decreased size in neurosphere formations with BD-NPCs. Also, we observed decreased subventricular areas in developing BD cerebral organoids. Finally, BD NPCs demonstrated high expression of the let-7 family while BD neurons had increased miR-34a, both being microRNAs previously implicated in neurodevelopmental deviations and BD etiology. In summary, we present evidence supporting an accelerated transition towards the neuronal stage in BD-NPCs that may be indicative of early pathophysiological features of the disorder.

Distinct Requirements for Adaptor Proteins NCK1 and NCK2 in Mammary Gland Development.

The adaptor proteins NCK1 and NCK2 are well-established signalling nodes that regulate diverse biological processes including cell proliferation and actin dynamics in many tissue types. Here we have investigated the distribution and function of Nck1 and Nck2 in the developing mouse mammary gland. Using publicly available single-cell RNA sequencing data, we uncovered distinct expression profiles between the two paralogs. Nck1 showed widespread expression in luminal, basal, stromal and endothelial cells, while Nck2 was restricted to luminal and basal cells, with prominent enrichment in hormone-sensing luminal subtypes. Next, using mice with global knockout of Nck1 or Nck2, we assessed mammary gland development during and after puberty (5, 8 and 12 weeks of age). Mice lacking Nck1 or Nck2 displayed significant defects in ductal outgrowth and branching at 5 weeks compared to controls, and the defects persisted in Nck2 knockout mice at 8 weeks before normalizing at 12 weeks. These defects were accompanied by an increase in epithelial cell proliferation at 5 weeks and a decrease at 8 weeks in both Nck1 and Nck2 knockout mice. We also profiled expression of several key genes associated with mammary gland development at these timepoints and detected temporal changes in transcript levels of hormone receptors as well as effectors of cell proliferation and migration in Nck1 and Nck2 knockout mice, in line with the distinct phenotypes observed at 5 and 8 weeks. Together these studies reveal a requirement for NCK proteins in mammary gland morphogenesis, and suggest that deregulation of Nck expression could drive breast cancer progression and metastasis.

Complementary Nck1/2 Signaling in Podocytes Controls α Actinin-4-Mediated Actin Organization, Adhesion, and Basement Membrane Composition.

BACKGROUND: Maintenance of the kidney filtration barrier requires coordinated interactions between podocytes and the underlying glomerular basement membrane (GBM). GBM ligands bind podocyte integrins, which triggers actin-based signaling events critical for adhesion. Nck1/2 adaptors have emerged as essential regulators of podocyte cytoskeletal dynamics. However, the precise signaling mechanisms mediated by Nck1/2 adaptors in podocytes remain to be fully elucidated. METHODS: We generated podocytes deficient in Nck1 and Nck2 and used transcriptomic approaches to profile expression differences. Proteomic techniques identified specific binding partners for Nck1 and Nck2 in podocytes. We used cultured podocytes and mice deficient in Nck1 and/or Nck2, along with podocyte injury models, to comprehensively verify our findings. RESULTS: Compound loss of Nck1/2 altered expression of genes involved in actin binding, cell adhesion, and extracellular matrix composition. Accordingly, Nck1/2-deficient podocytes showed defects in actin organization and cell adhesion in vitro, with podocyte detachment and altered GBM morphology present in vivo. We identified distinct interactomes for Nck1 and Nck2 and uncovered a mechanism by which Nck1 and Nck2 cooperate to regulate actin bundling at focal adhesions via α actinin-4. Furthermore, loss of Nck1 or Nck2 resulted in increased matrix deposition in vivo, with more prominent defects in Nck2-deficient mice, consistent with enhanced susceptibility to podocyte injury. CONCLUSION: These findings reveal distinct, yet complementary, roles for Nck proteins in regulating podocyte adhesion, controlling GBM composition, and sustaining filtration barrier integrity.

Multivalent nephrin-Nck interactions define a threshold for clustering and tyrosine-dependent nephrin endocytosis.

Assembly of signaling molecules into micrometer-sized clusters is driven by multivalent protein-protein interactions, such as those found within the nephrin-Nck (Nck1 or Nck2) complex. Phosphorylation on multiple tyrosine residues within the tail of the nephrin transmembrane receptor induces recruitment of the cytoplasmic adaptor protein Nck, which binds via its triple SH3 domains to various effectors, leading to actin assembly. The physiological consequences of nephrin clustering are not well understood. Here, we demonstrate that nephrin phosphorylation regulates the formation of membrane clusters in podocytes. We also reveal a connection between clustering and endocytosis, which appears to be driven by threshold levels of nephrin tyrosine phosphorylation and Nck SH3 domain signaling. Finally, we expose an in vivo correlation between transient changes in nephrin tyrosine phosphorylation, nephrin localization and integrity of the glomerular filtration barrier during podocyte injury. Altogether, our results suggest that nephrin phosphorylation determines the composition of effector proteins within clusters to dynamically regulate nephrin turnover and podocyte health.

Receptor Tyrosine Kinase Signaling and Targeting in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is amongst the deadliest of human cancers, with a median survival rate of just over one year following diagnosis. Characterized by rapid proliferation and diffuse infiltration into the brain, GBM is notoriously difficult to treat, with tumor cells showing limited response to existing therapies and eventually developing resistance to these interventions. As such, there is intense interest in better understanding the molecular alterations in GBM to guide the development of more efficient targeted therapies. GBM tumors can be classified into several molecular subtypes which have distinct genetic signatures, and they show aberrant activation of numerous signal transduction pathways, particularly those connected to receptor tyrosine kinases (RTKs) which control glioma cell growth, survival, migration, invasion, and angiogenesis. There are also non-canonical modes of RTK signaling found in GBM, which involve G-protein-coupled receptors and calcium channels. This review uses The Cancer Genome Atlas (TCGA) GBM dataset in combination with a data-mining approach to summarize disease characteristics, with a focus on select molecular pathways that drive GBM pathogenesis. We also present a unique genomic survey of RTKs that are frequently altered in GBM subtypes, as well as catalog the GBM disease association scores for all RTKs. Lastly, we discuss current RTK targeted therapies and highlight emerging directions in GBM research.

Protein Tyrosine Phosphatase Receptor Type G (PTPRG) Controls Fibroblast Growth Factor Receptor (FGFR) 1 Activity and Influences Sensitivity to FGFR Kinase Inhibitors.

Recently, FGFR1 was found to be overexpressed in osteosarcoma and represents an important target for precision medicine. However, because targeted cancer therapy based on FGFR inhibitors has so far been less efficient than expected, a detailed understanding of the target is important. We have here applied proximity-dependent biotin labeling combined with label-free quantitative mass spectrometry to identify determinants of FGFR1 activity in an osteosarcoma cell line. Many known FGFR interactors were identified (e.g. FRS2, PLCG1, RSK2, SRC), but the data also suggested novel determinants. A strong hit in our screen was the tyrosine phosphatase PTPRG. We show that PTPRG and FGFR1 interact and colocalize at the plasma membrane where PTPRG directly dephosphorylates activated FGFR1. We further show that osteosarcoma cell lines depleted for PTPRG display increased FGFR activity and are hypersensitive to stimulation by FGF1. In addition, PTPRG depletion elevated cell growth and negatively affected the efficacy of FGFR kinase inhibitors. Thus, PTPRG may have future clinical relevance by being a predictor of outcome after FGFR inhibitor treatment.

The ShcD phosphotyrosine adaptor subverts canonical EGF receptor trafficking.

Shc family signalling adaptors connect activated transmembrane receptors to proximal effectors, and most also contain a sequence involved in clathrin-mediated receptor endocytosis. Notably, this AP2 adaptin-binding motif (AD) is absent from the ShcD (also known as Shc4) homolog, which also uniquely promotes ligand-independent phosphorylation of the epidermal growth factor receptor (EGFR). We now report that cultured cells expressing ShcD exhibit reduced EGF uptake, commensurate with a decrease in EGFR surface presentation. Under basal conditions, ShcD colocalises with the EGFR and facilitates its phosphorylation, ubiquitylation and accumulation in juxtanuclear vesicles identified as Rab11-positive endocytic recycling compartments. Accordingly, ShcD also functions as a constitutive binding partner for the E3 ubiquitin ligase Cbl. EGFR phosphorylation and focal accumulation likewise occur upon ShcD co-expression in U87 glioma cells. Loss of ShcD phosphotyrosine-binding function or insertion of the ShcA AD sequence each restore ligand acquisition through distinct mechanisms. The AD region also contains a nuclear export signal, indicating its multifunctionality. Overall, ShcD appears to possess several molecular permutations that actively govern the EGFR, which may have implications in development and disease.

Localization of phosphotyrosine adaptor protein ShcD/SHC4 in the adult rat central nervous system.

BACKGROUND: Mammalian Shc (Src homology and collagen) proteins comprise a family of four phosphotyrosine adaptor molecules which exhibit varied spatiotemporal expression and signaling functions. ShcD is the most recently discovered homologue and it is highly expressed in the developing central nervous system (CNS) and adult brain. Presently however, its localization within specific cell types of mature neural structures has yet to be characterized. RESULTS: In the current study, we examine the expression profile of ShcD in the adult rat CNS using immunohistochemistry, and compare with those of the neuronally enriched ShcB and ShcC proteins. ShcD shows relatively widespread distribution in the adult brain and spinal cord, with prominent levels of staining throughout the olfactory bulb, as well as in sub-structures of the cerebellum and hippocampus, including the subgranular zone. Co-localization studies confirm the expression of ShcD in mature neurons and progenitor cells. ShcD immunoreactivity is primarily localized to axons and somata, consistent with the function of ShcD as a cytoplasmic adaptor. Regional differences in expression are observed among neural Shc proteins, with ShcC predominating in the hippocampus, cerebellum, and some fiber tracts. Interestingly, ShcD is uniquely expressed in the olfactory nerve layer and in glomeruli of the main olfactory bulb. CONCLUSIONS: Together our findings suggest that ShcD may provide a distinct signaling contribution within the olfactory system, and that overlapping expression of ShcD with other Shc proteins may allow compensatory functions in the brain.

ShcA Adaptor Protein Promotes Nephrin Endocytosis and Is Upregulated in Proteinuric Nephropathies.

Nephrin is a key structural component of the podocyte slit diaphragm, and proper expression of nephrin on the cell surface is critical to ensure integrity of the blood filtration barrier. Maintenance of nephrin within this unique cell junction has been proposed to require dynamic phosphorylation events and endocytic recycling, although the molecular mechanisms that control this interplay are poorly understood. Here, we investigated the possibility that the phosphotyrosine adaptor protein ShcA regulates nephrin turnover. Western blotting and immunostaining analysis confirmed that ShcA is expressed in podocytes. In immunoprecipitation and pulldown assays, ShcA, via its SH2 domain, was associated with several phosphorylated tyrosine residues on nephrin. Overexpression of ShcA promoted nephrin tyrosine phosphorylation and reduced nephrin signaling and cell surface expression in vitro In a rat model of reversible podocyte injury and proteinuria, phosphorylated nephrin temporally colocalized with endocytic structures coincident with upregulation of ShcA expression. In vivo biotinylation assays confirmed that nephrin expression decreased at the cell surface and correspondingly increased in the cytosol during the injury time course. Finally, immunostaining in kidney biopsy specimens demonstrated overexpression of ShcA in several human proteinuric kidney diseases compared with normal conditions. Our results suggest that increases in ShcA perturb nephrin phosphosignaling dynamics, leading to aberrant nephrin turnover and slit diaphragm disassembly.

Signaling adaptor ShcD suppresses extracellular signal-regulated kinase (Erk) phosphorylation distal to the Ret and Trk neurotrophic receptors.

Proteins of the Src homology and collagen (Shc) family are typically involved in signal transduction events involving Ras/MAPK and PI3K/Akt pathways. In the nervous system, they function proximal to the neurotrophic factors that regulate cell survival, differentiation, and neuron-specific characteristics. The least characterized homolog, ShcD, is robustly expressed in the developing and mature nervous system, but its contributions to neural cell circuitry are largely uncharted. We now report that ShcD binds to active Ret, TrkA, and TrkB neurotrophic factor receptors predominantly via its phosphotyrosine-binding (PTB) domain. However, in contrast to the conventional Shc adaptors, ShcD suppresses distal phosphorylation of the Erk MAPK. Accordingly, genetic knock-out of mouse ShcD enhances Erk phosphorylation in the brain. In cultured cells, this capacity is tightly aligned to phosphorylation of ShcD CH1 region tyrosine motifs, which serve as docking platforms for signal transducers, such as Grb2. Erk suppression is relieved through independent mutagenesis of the PTB domain and the CH1 tyrosine residues, and successive substitution of these tyrosines breaks the interaction between ShcD and Grb2, thereby promoting TrkB-Grb2 association. Erk phosphorylation can also be restored in the presence of wild type ShcD through Grb2 overexpression. Conversely, mutation of the ShcD SH2 domain results in enhanced repression of Erk. Although the SH2 domain is a less common binding interface in Shc proteins, we demonstrate that it associates with the Ptpn11 (Shp2) phosphatase, which in turn regulates ShcD tyrosine phosphorylation. We therefore propose a model whereby ShcD competes with neurotrophic receptors for Grb2 binding and opposes activation of the MAPK cascade.

A common haplotype containing functional CACNA1H variants is frequently coinherited with increased TPSAB1 copy number.

PurposeCaV3.2 signaling contributes to nociception, pruritus, gastrointestinal motility, anxiety, and blood pressure homeostasis. This calcium channel, encoded by CACNA1H, overlaps the human tryptase locus, wherein increased TPSAB1 copy number causes hereditary α-tryptasemia. Germ-line CACNA1H variants may contribute to the variable expressivity observed with this genetic trait.MethodsTryptase-encoding sequences at TPSAB1 and TPSB2, and TPSG1 and CACNA1H variants were genotyped in 46 families with hereditary α-tryptasemia syndrome. Electrophysiology was performed on tsA201 HEK cells transfected with wild-type or variant CACNA1H constructs. Effects on clinical phenotypes were interrogated in families with TPSAB1 duplications and in volunteers from the ClinSeq cohort.ResultsThree nonsynonymous variants in CACNA1H (rs3751664, rs58124832, and rs72552056) cosegregated with TPSAB1 duplications in 32/46 families and were confirmed to be in linkage disequilibrium (LD). In vitro, variant CaV3.2 had functional effects: reducing current densities, and altering inactivation and deactivation properties. No clinical differences were observed in association with the CACNA1H haplotype.ConclusionA previously unrecognized haplotype containing three functional CACNA1H variants is relatively common among Caucasians, and is frequently coinherited on the same allele as additional TPSAB1 copies. The variant CACNA1H haplotype, which in vitro imparts partial gain of function, does not result in detectable phenotypic differences in the heterozygous state.

Nephrin Suppresses Hippo Signaling through the Adaptor Proteins Nck and WTIP.

Podocytes are key components of the kidney blood filtration barrier, and their ability to withstand hemodynamic strain is proposed to be closely tied to their unique and flexible cytoarchitecture. However, the mechanisms that control such mechanotransduction are poorly understood. We have previously established that tyrosine phosphorylation of the transmembrane protein nephrin promotes recruitment of the Nck1/2 cytoskeletal adaptor proteins and downstream actin remodeling. We now reveal that Nck integrates nephrin with the Hippo kinase cascade through association with the adaptor protein WTIP. Using mutational analysis, we show that Nck sequesters WTIP and its binding partner Lats1 to phosphorylated nephrin, resulting in decreased phospho-activation of Lats1. We further demonstrate that, coincident with nephrin dephosphorylation in a transient model of podocyte injury in mice, Lats1 is rapidly activated, and this precedes significant down-regulation of the transcription regulator Yap. Moreover, we show reduced levels of Yap protein in mice with chronic disruption of nephrin phospho-signaling. Together, these findings support the existence of a dynamic molecular link between nephrin signaling and the canonical Hippo pathway in podocytes, which may facilitate the conversion of mechanical cues to biochemical signals promoting podocyte viability.

Nephrin Tyrosine Phosphorylation Is Required to Stabilize and Restore Podocyte Foot Process Architecture.

Podocytes are specialized epithelial cells of the kidney blood filtration barrier that contribute to permselectivity via a series of interdigitating actin-rich foot processes. Positioned between adjacent projections is a unique cell junction known as the slit diaphragm, which is physically connected to the actin cytoskeleton via the transmembrane protein nephrin. Evidence indicates that tyrosine phosphorylation of the intracellular tail of nephrin initiates signaling events, including recruitment of cytoplasmic adaptor proteins Nck1 and Nck2 that regulate actin cytoskeletal dynamics. Nephrin tyrosine phosphorylation is altered in human and experimental renal diseases characterized by pathologic foot process remodeling, prompting the hypothesis that phosphonephrin signaling directly influences podocyte morphology. To explore this possibility, we generated and analyzed knockin mice with mutations that disrupt nephrin tyrosine phosphorylation and Nck1/2 binding (nephrin(Y3F/Y3F) mice). Homozygous nephrin(Y3F/Y3F) mice developed progressive proteinuria accompanied by structural changes in the filtration barrier, including podocyte foot process effacement, irregular thickening of the glomerular basement membrane, and dilated capillary loops, with a similar but later onset phenotype in heterozygous animals. Furthermore, compared with wild-type mice, nephrin(Y3F/Y3F) mice displayed delayed recovery in podocyte injury models. Profiling of nephrin tyrosine phosphorylation dynamics in wild-type mice subjected to podocyte injury indicated site-specific differences in phosphorylation at baseline, injury, and recovery, which correlated with loss of nephrin-Nck1/2 association during foot process effacement. Our results define an essential requirement for nephrin tyrosine phosphorylation in stabilizing podocyte morphology and suggest a model in which dynamic changes in phosphotyrosine-based signaling confer plasticity to the podocyte actin cytoskeleton.

Diminution of signal transducer and activator of transcription 3 signaling inhibits vascular permeability and anaphylaxis.

BACKGROUND: During IgE-mediated immediate hypersensitivity reactions, vascular endothelial cells permeabilize in response to mast cell mediators. We have demonstrated previously that patients and mice with signal transducer and activator of transcription 3 (STAT3) mutations (autosomal dominant hyper-IgE syndrome [AD-HIES]) are partially protected from anaphylaxis. OBJECTIVES: We sought to study the mechanism by which STAT3 contributes to anaphylaxis and determine whether small-molecule inhibition of STAT3 can prevent anaphylaxis. METHODS: Using unaffected and STAT3-inhibited or genetic loss-of-function samples, we performed histamine skin prick tests, investigated the contribution of STAT3 to animal models of anaphylaxis, and measured endothelial cell permeability, gene and protein expression, and histamine receptor-mediated signaling. RESULTS: Although mouse mast cell degranulation was minimally affected by STAT3 blockade, mast cell mediator-induced anaphylaxis was blunted in Stat3 mutant mice with AD-HIES and in wild-type mice subjected to small-molecule STAT3 inhibition. Histamine skin prick test responses were diminished in patients with AD-HIES. Human umbilical vein endothelial cells derived from patients with AD-HIES or treated with a STAT3 inhibitor did not signal properly through Src or cause appropriate dissolution of the adherens junctions made up of the proteins vascular endothelial-cadherin and ß-catenin. Furthermore, we found that diminished STAT3 target microRNA17-92 expression in human umbilical vein endothelial cells from patients with AD-HIES is associated with increased phosphatase and tensin homolog (PTEN) expression, which inhibits Src, and increased E2F transcription factor 1 expression, which regulates ß-catenin cellular dynamics. CONCLUSIONS: These data demonstrate that STAT3-dependent transcriptional activity regulates critical components for the architecture and functional dynamics of endothelial junctions, thus permitting vascular permeability.

Increased SHP-1 protein expression by high glucose levels reduces nephrin phosphorylation in podocytes.

Nephrin, a critical podocyte membrane component that is reduced in diabetic nephropathy, has been shown to activate phosphotyrosine signaling pathways in human podocytes. Nephrin signaling is important to reduce cell death induced by apoptotic stimuli. We have shown previously that high glucose level exposure and diabetes increased the expression of SHP-1, causing podocyte apoptosis. SHP-1 possesses two Src homology 2 domains that serve as docking elements to dephosphorylate tyrosine residues of target proteins. However, it remains unknown whether SHP-1 interacts with nephrin and whether its elevated expression affects the nephrin phosphorylation state in diabetes. Here we show that human podocytes exposed to high glucose levels exhibited elevated expression of SHP-1, which was associated with nephrin. Coexpression of nephrin-CD16 and SHP-1 reduced nephrin tyrosine phosphorylation in transfected human embryonic kidney 293 cells. A single tyrosine-to-phenylalanine mutation revealed that rat nephrin Tyr(1127) and Tyr(1152) are required to allow SHP-1 interaction with nephrin. Overexpression of dominant negative SHP-1 in human podocytes prevented high glucose-induced reduction of nephrin phosphorylation. In vivo, immunoblot analysis demonstrated that nephrin expression and phosphorylation were decreased in glomeruli of type 1 diabetic Akita mice (Ins2(+/C96Y)) compared with control littermate mice (Ins2(+/+)), and this was associated with elevated SHP-1 and cleaved caspase-3 expression. Furthermore, immunofluorescence analysis indicated increased colocalization of SHP-1 with nephrin in diabetic mice compared with control littermates. In conclusion, our results demonstrate that high glucose exposure increases SHP-1 interaction with nephrin, causing decreased nephrin phosphorylation, which may, in turn, contribute to diabetic nephropathy.

Direct regulation of nephrin tyrosine phosphorylation by Nck adaptor proteins.

The transmembrane protein nephrin is a key component of the kidney slit diaphragm that contributes to the morphology of podocyte foot processes through signaling to the underlying actin cytoskeleton. We have recently reported that tyrosine phosphorylation of the cytoplasmic tail of nephrin facilitates recruitment of Nck SH2/SH3 adaptor proteins and subsequent actin remodeling and that phosphorylation of the Nck binding sites on nephrin is decreased during podocyte injury. We now demonstrate that Nck directly modulates nephrin phosphorylation through formation of a signaling complex with the Src family kinase Fyn. The ability of Nck to enhance nephrin phosphorylation is compromised in the presence of a Src family kinase inhibitor and when the SH3 domains of Nck are mutated. Furthermore, induced loss of Nck expression in podocytes in vivo is associated with a rapid reduction in nephrin tyrosine phosphorylation. Our results suggest that Nck may facilitate dynamic signaling events at the slit diaphragm by promoting Fyn-dependent phosphorylation of nephrin, which may be important in the regulation of foot process morphology and response to podocyte injury.

Advances in slit diaphragm signaling.

PURPOSE OF REVIEW: The podocyte slit diaphragm is a fundamental component of the glomerular filtration barrier and its function is highly dependent on the maintenance of specialized actin-based projections known as foot processes. In this review, we update the function of key slit diaphragm-associated proteins, and introduce some new players and emerging avenues of research within podocyte biology. RECENT FINDINGS: Studies using rodent models continue to support the long-held belief that precise regulation of actin dynamics at the slit diaphragm is essential for proper foot process organization. However, it is also becoming increasingly clear that alterations in actin remodeling can significantly contribute to damage in both animal models and human disease. In particular, the importance of signaling via the Rho family of GTPases has been recognized, as well as the requirement for proper localization and turnover of the slit diaphragm. SUMMARY: Regulation of the connection between the slit diaphragm and the podocyte actin network requires complex interplay between multiple signaling pathways. New discoveries contribute to an ever-expanding view of the slit diaphragm and serve to create a framework for the development of new therapeutic strategies targeting podocyte function in the future.

Vascular endothelial hyperpermeability induces the clinical symptoms of Clarkson disease (the systemic capillary leak syndrome).

The systemic capillary leak syndrome (SCLS) is a rare disorder characterized by transient episodes of hypotensive shock and anasarca thought to arise from reversible microvascular barrier dysfunction. Although the high prevalence of a monoclonal gammopathy of unknown significance in SCLS suggests a pathogenic contribution of endogenous immunoglobulins, the mechanisms of vascular hyperpermeability remain obscure. Herein, we report clinical and molecular findings on 23 patients, the largest SCLS case series to date. Application of episodic SCLS sera, but neither the purified immunoglobulin fraction nor sera obtained from patients during remission, to human microvascular endothelial cells caused vascular endothelial cadherin internalization, disruption of interendothelial junctions, actin stress fiber formation, and increased permeability in complementary functional assays without inducing endothelial apoptosis. Intravenous immunoglobulin, one promising therapy for SCLS, mitigated the permeability effects of episodic sera. Consistent with the presence of endogenous, nonimmunoglobulin, circulating permeability factor(s) constrained to SCLS episodes, we found that vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2), were elevated in episodic SCLS sera but not in remission sera. Ab-based inhibition of Ang2 counteracted permeability induced by episodic SCLS sera. Comparable experiments with anti-VEGF Ab (bevacizumab) yielded less interpretable results, probably because of endothelial toxicity of VEGF withdrawal. Our results support a model of SCLS pathogenesis in which nonimmunoglobulin humoral factors such as VEGF and Ang2 contribute to transient endothelial contraction, suggesting a molecular mechanism for this highly lethal disorder.