Biomedical subjects

Lawrence B Holzman

Publications and source records attributed to Lawrence B Holzman.

18 recordsLinked to original sources

PubMed2006

Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible.

Nephrotic syndrome, a malfunction of the kidney glomerular filter, leads to proteinuria, edema and, in steroid-resistant nephrotic syndrome, end-stage kidney disease. Using positional cloning, we identified mutations in the phospholipase C epsilon gene (PLCE1) as causing early-onset nephrotic syndrome with end-stage kidney disease. Kidney histology of affected individuals showed diffuse mesangial sclerosis (DMS). Using immunofluorescence, we found PLCepsilon1 expression in developing and mature glomerular podocytes and showed that DMS represents an arrest of normal glomerular development. We identified IQ motif-containing GTPase-activating protein 1 as a new interaction partner of PLCepsilon1. Two siblings with a missense mutation in an exon encoding the PLCepsilon1 catalytic domain showed histology characteristic of focal segmental glomerulosclerosis. Notably, two other affected individuals responded to therapy, making this the first report of a molecular cause of nephrotic syndrome that may resolve after therapy. These findings, together with the zebrafish model of human nephrotic syndrome generated by plce1 knockdown, open new inroads into pathophysiology and treatment mechanisms of nephrotic syndrome.

Animals↗

PubMed2006

Podocyte-specific deletion of integrin-linked kinase results in severe glomerular basement membrane alterations and progressive glomerulosclerosis.

Alterations in glomerular podocyte cell-cell and cell-matrix contacts are key events in progressive glomerular failure. Integrin-linked kinase (ILK) has been implicated in podocyte cell-matrix interaction and is induced in proteinuria. For evaluation of ILK function in vivo, mice with a Cre-mediated podocyte-specific ILK inactivation were generated. These mice seemed normal at birth but developed progressive focal segmental glomerulosclerosis and died in terminal renal failure. The first ultrastructural lesions that are seen at onset of albuminuria are glomerular basement membrane (GBM) alterations with a significant increase in true harmonic mean GBM thickness. Podocyte foot process effacement and loss of slit diaphragm followed with progression to unselective proteinuria. No significant reduction of slit membrane molecules (podocin and nephrin), key GBM components (fibronectin, laminins, and collagen IV isoforms), or podocyte integrins could be observed at onset of proteinuria. However, alpha3-integrins were relocalized into a granular pattern along the GBM, consistent with altered integrin-mediated matrix assembly in ILK-deficient podocytes. As the increased GBM thickness precedes structural podocyte lesions and key components of the GBM were expressed at comparable levels to controls, these data suggest an essential role of ILK for the close interconnection of GBM structure and podocyte function.

Animals↗

PubMed2006

Nephrin ectodomain engagement results in Src kinase activation, nephrin phosphorylation, Nck recruitment, and actin polymerization.

A properly established and maintained podocyte intercellular junction, or slit diaphragm, is a necessary component of the selective permeability barrier of the kidney glomerulus. The observation that mutation or deletion of the slit diaphragm transmembrane protein nephrin results in failure of podocyte foot process morphogenesis and concomitant proteinuria first suggested the hypothesis that nephrin serves as a component of a signaling complex that directly integrates podocyte junctional integrity with cytoskeletal dynamics. The observations made herein provide the first direct evidence to our knowledge for a phosphorylation-mediated signaling mechanism by which this integrative function is derived. Our data support the model that during podocyte intercellular junction formation, engagement of the nephrin ectodomain induces transient Fyn catalytic activity that results in nephrin phosphorylation on specific nephrin cytoplasmic domain tyrosine residues. We found that this nephrin phosphorylation event resulted in recruitment of the SH2-SH3 domain-containing adapter protein Nck and assembly of actin filaments in an Nck-dependent fashion. Considered in the context of the role of nephrin family proteins in other organisms and the integral relationship of actin dynamics and junction formation, these observations establish a function for nephrin in regulating actin cytoskeletal dynamics.

Actins↗

PubMed2006

Imaging podocyte dynamics.

Cytoskeletal dynamics play important roles during normal podocyte development, in maintenance of the healthy glomerular filter, and in glomerular pathology. During the past few years, essential progress has been made in understanding the molecular mechanisms that govern podocyte cytoskeletal dynamics, nevertheless this knowledge remains incomplete. Examination of the podocyte cytoskeleton presents unique challenges to the experimentalist who is faced with attempting to model a morphologically complex cell that is situated in a complex microenvironment. This review will summarize recent progress in understanding cytoskeletal dynamics in the podocyte and will review modern imaging techniques relevant to studying this area of podocyte biology.

Actins↗

PubMed2006

Clinical impact of research on the podocyte slit diaphragm.

This Review summarizes recent research on the podocyte slit diaphragm. A growing number of molecules that function at the slit diaphragm have been identified in patients with inherited and sporadic nephrotic syndromes. Genetic deletion of nearly all of these molecules results in proteinuria and effacement of foot processes. Nephrin, Neph1 and podocin seem to form a multifunctional receptor complex at the slit diaphragm. Most of the other components of the slit diaphragm interact directly with this complex, in many cases coupling slit diaphragm components to the podocyte's actin cytoskeleton. These molecular findings are being applied to patients with glomerular disease. Over the next decade, these data might help to improve disease classification and prediction of which patients will respond to immunosuppressive treatment.

Gene Expression↗

PubMed2005

Glomerular disease workshop.

Recent observations regarding intrinsic glomerular cell biology, particularly in the podocyte, have provided exciting new insights into potential pathogenic mechanisms of human glomerular disease. Although both immune and nonimmune mechanisms of glomerular injury have been studied previously, experimental models of disease and recent techniques that provide tools for molecular profiling show great promise for identifying glomerular disease biomarkers. Despite these recent advances, additional work in both basic and clinical studies of glomerular disease is needed to advance the field. Standardization of animal models of distinct forms of glomerular disease would likely facilitate the search for biomarkers. Several factors limit current efforts to implement clinical trials of glomerular disease. Identification of disease biomarkers, development of disease-specific end points, and organization of collaborative clinical groups are critical for ultimately designing and implementing appropriately powered trials of glomerular disease.

Animals↗

PubMed2005

Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene.

Glomerular injury and proteinuria in diabetes (types 1 and 2) and IgA nephropathy is related to the degree of podocyte depletion in humans. For determining the causal relationship between podocyte depletion and glomerulosclerosis, a transgenic rat strain in which the human diphtheria toxin receptor is specifically expressed in podocytes was developed. The rodent homologue does not act as a diphtheria toxin (DT) receptor, thereby making rodents resistant to DT. Injection of DT into transgenic rats but not wild-type rats resulted in dose-dependent podocyte depletion from glomeruli. Three stages of glomerular injury caused by podocyte depletion were identified: Stage 1, 0 to 20% depletion showed mesangial expansion, transient proteinuria and normal renal function; stage 2, 21 to 40% depletion showed mesangial expansion, capsular adhesions (synechiae), focal segmental glomerulosclerosis, mild persistent proteinuria, and normal renal function; and stage 3, >40% podocyte depletion showed segmental to global glomerulosclerosis with sustained high-grade proteinuria and reduced renal function. These pathophysiologic consequences of podocyte depletion parallel similar degrees of podocyte depletion, glomerulosclerosis, and proteinuria seen in diabetic glomerulosclerosis. This model system provides strong support for the concept that podocyte depletion could be a major mechanism driving glomerulosclerosis and progressive loss of renal function in human glomerular diseases.

Animals↗

PubMed2005

An efficient system for tissue-specific overexpression of transgenes in podocytes in vivo.

The utility of promoter fragments isolated from the 5'-flanking region of endogenous mammalian genes to drive transgene expression in vivo is often limited by low expression levels. In this study, a bigenic system was established that allows constitutive overexpression of transgenes in a tissue-specific fashion in transgenic mice in a time- and cost-effective fashion. A modified floxed expression vector was constructed [CMVflox-enhanced green fluorescent protein (eGFP)], in which a lacZ cassette (beta-galactosidase) flanked by lox sites was placed between a CMV-promoter and the transgene of interest (eGFP). Before Cre recombination, expression of eGFP was effectively prevented by the interposed floxed lacZ cassette, whereas beta-galactosidase was strongly expressed in transiently transfected cells. Transcription of the gene of interest (eGFP) could be irreversibly activated by cotransfection with Cre recombinase. Mice transgenic for CMVflox-eGFP were generated by pronuclear injection. A rapid assay was developed to identify transgenic founders with active transgene expression by measuring transgene activity (beta-galactosidase) in tail biopsies. Transgene activity in tails correlated with transgene expression in most other tissues tested including podocytes within the kidney. To activate expression of the gene of interest in a tissue-specific fashion, founder mice were mated to the Cre mouse line 2.5P-Cre previously shown to mediate 100% Cre recombination exclusively in podocytes (Moeller MJ, Sanden SK, Soofi A, Wiggins RC, and Holzman LB. Genesis 35: 39-42, 2003). In doubly transgenic offspring, high-level eGFP expression resulting from Cre excision of the interposed lacZ cassette was detected in four of seven CMVflox-eGFP founder lines. This approach should also circumvent common limitations arising from lethality or transgene silencing as a consequence of transgene overexpression.

Animals↗

PubMed2004

Protocadherin FAT1 binds Ena/VASP proteins and is necessary for actin dynamics and cell polarization.

Cell migration requires integration of cellular processes resulting in cell polarization and actin dynamics. Previous work using tools of Drosophila genetics suggested that protocadherin fat serves in a pathway necessary for determining cell polarity in the plane of a tissue. Here we identify mammalian FAT1 as a proximal element of a signaling pathway that determines both cellular polarity in the plane of the monolayer and directed actin-dependent cell motility. FAT1 is localized to the leading edge of lamellipodia, filopodia, and microspike tips where FAT1 directly interacts with Ena/VASP proteins that regulate the actin polymerization complex. When targeted to mitochondrial outer leaflets, FAT1 cytoplasmic domain recruits components of the actin polymerization machinery sufficient to induce ectopic actin polymerization. In an epithelial cell wound model, FAT1 knockdown decreased recruitment of endogenous VASP to the leading edge and resulted in impairment of lamellipodial dynamics, failure of polarization, and an attenuation of cell migration. FAT1 may play an integrative role regulating cell migration by participating in Ena/VASP-dependent regulation of cytoskeletal dynamics at the leading edge and by transducing an Ena/VASP-independent polarity cue.

Actins↗

PubMed2004

Podocytes populate cellular crescents in a murine model of inflammatory glomerulonephritis.

Cellular crescents are a defining histologic finding in many forms of inflammatory glomerulonephritis. Despite numerous studies, the origin of glomerular crescents remains unresolved. A genetic cell lineage-mapping study with a novel transgenic mouse model was performed to investigate whether visceral glomerular epithelial cells, termed podocytes, are precursors of cells that populate cellular crescents. The podocyte-specific 2.5P-Cre mouse line was crossed with the ROSA26 reporter line, resulting in irreversible constitutive expression of beta-galactosidase in doubly transgenic 2.5P-Cre/ROSA26 mice. In these mice, crescentic glomerulonephritis was induced with a previously described rabbit anti-glomerular basement membrane antiserum nephritis approach. Interestingly, beta-galactosidase-positive cells derived from podocytes adhered to the parietal basement membrane and populated glomerular crescents during the early phases of cellular crescent formation, accounting for at least one-fourth of the total cell mass. In cellular crescents, the proliferation marker Ki-67 was expressed in beta-galactosidase-positive and beta-galactosidase-negative cells, indicating that both cell types contributed to the formation of cellular crescents through proliferation in situ. Podocyte-specific antigens, including WT-1, synaptopodin, nephrin, and podocin, were not expressed by any cells in glomerular crescents, suggesting that podocytes underwent profound phenotypic changes in this nephritis model.

Animals↗

PubMed2003

Recruitment of JNK to JIP1 and JNK-dependent JIP1 phosphorylation regulates JNK module dynamics and activation.

JIP1 is a scaffold protein that assembles and facilitates the activation of the mixed lineage kinase-dependent JNK module. Results of earlier work led us to propose a model for JIP1-JNK complex regulation that predicts that under basal conditions, JIP1 maintains DLK in a monomeric, unphosphorylated, and catalytically inactive state. Upon appropriate module stimulation, JNK-JIP1 binding affinity increases and DLK-JIP1 affinity decreases. Dissociation of DLK from JIP1 results in subsequent DLK oligomerization, autophosphorylation, and ultimately module activation. Our previous published results suggested the hypothesis that recruitment of JNK to JIP1 and phosphorylation of JIP1 by JNK is prerequisite for activation of the JNK module (Nihalani, D., Meyer, D., Pajni, S., and Holzman, L. B. (2001) EMBO J. 20, 3447-3458). The present study corroborated this hypothesis by demonstrating that JNK binding to JIP1 is necessary for stimulus-induced dissociation of DLK from JIP1, for DLK oligomerization, and for JNK activation. After mapping JNK-dependent JIP1 phosphorylation sites and testing their functional significance, it was observed that phosphorylation by JNK of JIP1 on Thr-103 and not other phosphorylated JIP1 residues is necessary for the regulation of DLK association with JIP1, DLK activation, and subsequent module activation. A refined model of JIP1-JNK module regulation is presented in which JNK phosphorylation of JIP1 is necessary prior to module activation.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Fyn binds to and phosphorylates the kidney slit diaphragm component Nephrin.

Recent investigations have focused on characterizing the molecular components of the podocyte intercellular junction, because several of these components, including Nephrin, are functionally necessary for development of normal podocyte structure and filter integrity. Accumulating evidence suggests that the Nephrin-associated protein complex is a signaling nexus. As such, Nephrin-dependent signaling might be mediated in part through Nephrin phosphorylation. Described are biochemical and mouse genetics experiments demonstrating that membrane-associated Nephrin is tyrosine-phosphorylated by the Src family kinase Fyn. Nephrin fractionated in detergent-resistant glomerular membrane fractions with Fyn and Yes. Fyn directly bound Nephrin via its SH3 domain, and Fyn directly phosphorylated Nephrin. Glomeruli in which Fyn, Yes, or Fyn and Yes were genetically deleted in mice were characterized to explore the relationship between these kinases and Nephrin. Fyn deletion resulted in coarsening of podocyte foot processes and marked attenuation of Nephrin phosphorylation in isolated glomerular detergent-resistant membrane fractions. Yes deletion had no identifiable effect on podocyte morphology but dramatically increased Nephrin phosphorylating activity. Similar to Fyn deletion, simultaneous deletion of Fyn and Yes reduced Nephrin phosphorylating activity. These results demonstrate that endogenous Fyn catalyzes Nephrin phosphorylation in podocyte detergent-resistant membrane fractions. Although Yes appears to effect the regulation of Nephrin phosphorylation, the mechanism by which this occurs requires investigation.

Animals↗

PubMed2003

Nephrin and Neph1 co-localize at the podocyte foot process intercellular junction and form cis hetero-oligomers.

Glomerular visceral epithelial cells (podocytes) appear to play a central role in maintaining the selective filtration barrier of the renal glomerulus. While the immunoglobulin superfamily member Nephrin was proposed to act as a cell adhesion molecule at the podocyte intercellular junction necessary for maintaining glomerular perm selectivity, the Nephrin ligand has not been identified. The existence of a new subfamily of Nephrin-like molecules including Neph1 was recently described. Genetic deletion of Nephrin or Neph1 resulted in similar phenotypes of podocyte foot process effacement and proteinuria. The subcellular localization of Neph1 and the possibility that Nephrin and Neph1 interact was investigated. Polyclonal antiserum for Neph1 was raised and characterized. Neph1 migrated as a 90-kDa protein on SDS-PAGE under reducing conditions. Neph1 was identified in a glomerular and podocyte-specific distribution in adult rat kidney. Like Nephrin and Podocin, Neph1 was enriched in Triton X-100 detergent-resistant membrane fractions. Consistent with this observation, immunogold electron microscopy demonstrated that Neph1 localized exclusively to lateral margins of podocyte foot processes at the insertion of the slit diaphragm. Neph1 and Nephrin participate in a direct cis-interaction involving their cytoplasmic domains. In addition, interactions between the extracellular domain of Nephrin and itself and between the extracellular domain of Nephrin and that of Neph1 were detected. Neph1 did not interact via a homophilic interaction. These observations suggest that Nephrin and Neph1 form a hetero-oligomeric receptor complex in the plane of the membrane that might interact across the foot process intercellular junction through interactions between Nephrin with itself and Neph1.

Animals↗

PubMed2003

Podocyte-specific expression of cre recombinase in transgenic mice.

We report a transgenic mouse line that expresses Cre recombinase exclusively in podocytes. Twenty- four transgenic founders were generated in which Cre recombinase was placed under the regulation of a 2.5-kb fragment of the human NPHS2 promoter. Previously, this fragment was shown to drive beta-galactosidase (beta-gal) expression exclusively in podocytes of transgenic mice. For analysis, founder mice were bred with ROSA26 mice, a reporter line that expresses beta-gal in cells that undergo Cre recombination. Eight of 24 founder lines were found to express beta-gal exclusively in the kidney. Histological analysis of the kidneys showed that beta-gal expression was confined to podocytes. Cre recombination occurred during the capillary loop stage in glomerular development. No evidence for Cre recombination was detected in any of 14 other tissues examined.

Animals↗

PubMed2003

Inducible podocyte-specific gene expression in transgenic mice.

The podocyte plays a key role in glomerular function and glomerular disease. To facilitate studies of podocyte function, we have developed a transgenic mouse model with inducible expression in the podocyte. The tetracycline-inducible transgenic system facilitates gene expression with restricted cellular distribution and tight temporal control. Recently, Bujard and colleagues have developed a functionally improved reverse tetracycline-controlled transcriptional activator (rtTA) with substantially lower background in the off state (the absence of tetracycline) and greater inducibility in the on state (the presence of tetracycline). We used the human podocin (NPHS2) gene promoter to control expression of the rtTA cassette and bred these mice with a reporter mouse line that contains the cytomegalovirus minimal promoter and tetO promoter elements together with LacZ, encoding beta-galactosidase. Dual transgenic mice, bearing both podocin-rtTA and tetO-LacZ transgenes, had no detectable expression in kidney or other organs in the absence of tetracycline. Administration of tetracycline in the drinking water was associated with podocyte expression of beta-galactosidase, in a fashion that was time dependent (maximal at 1 wk) and dose-dependent (maximal at 2 mg/ml). Podocyte expression was confirmed in two ways: histochemical staining for beta-galactosidase and double-immunostaining using the podocyte marker WT-1 and beta-galactosidase. This transgenic system should aid future investigations of podocyte function.

Animals↗

PubMed2002

Two gene fragments that direct podocyte-specific expression in transgenic mice.

Transgenic manipulation of the glomerular visceral epithelial cell offers a powerful approach for studying the biology of this morphologically complex cell type. It has been previously demonstrated that an 8.3-kb and a 5.4-kb fragment of the murine Nphs1 (nephrin) promoter-enhancer drives lacZ expression in podocytes, brain, and pancreas of transgenic mice, recapitulating the expression pattern of the endogenous nephrin gene. In this present study, two truly podocyte-specific promoters were identified that drive transgene expression in podocytes without expression in extrarenal tissues in adult or embryonic mice. A 1.25-kb fragment driving a lacZ reporter gene (p1.25N-nlacF) was derived from murine Nphs1 promoter similar to a human NPHS1 promoter fragment previously reported. Transgenic mice were generated and beta-galactosidase (beta-gal) expression was analyzed. Four of twelve founder mice were found to express beta-gal in podocytes (33% penetrance). Expression in brain and pancreas was absent in all animals, suggesting that nephrin expression in these organs might be driven by distinct cis-regulatory elements that can be removed to obtain podocyte-specific expression. A 2.5-kb fragment derived from the human NPHS2 (podocin) gene was designed in a similar fashion to drive lacZ expression in transgenic mice (p2.5P-nlacF). Twelve of twlve NPHS2 mouse founder lines expressed beta-gal exclusively in podocytes (100% penetrance). Beta-gal activity was not observed extrinsic to the kidney in p1.25N-nlacF or p2.5P-nlacF mouse embryos at gestational time points between 8.5 d post coitus and birth. In conclusion, the 2.5-kb NPHS2 promoter fragment may be useful for podocyte-specific transgenic expression when extrarenal expression of a transgene is problematic.

Animals↗

PubMed2001

Phosphorylation of Pax2 by the c-Jun N-terminal kinase and enhanced Pax2-dependent transcription activation.

The Pax gene family encodes DNA-binding proteins that can both activate and repress transcription of specific target genes during embryonic development. Pax proteins are required for pattern formation and cell differentiation in a broad spectrum of developing tissues. Consistent with its expression in the intermediate mesoderm, the optic cup and stalk, and the otic vesicle, Pax2, a member of the Pax2/5/8 subfamily, is essential for the development of the renal epithelia, the optic cup, and the inner ear. In addition to a DNA binding domain, the Pax2 protein contains a carboxyl-terminal transactivation domain rich in serine, threonine, and tyrosine. In this report, we demonstrate that the Pax2 transactivation domain is phosphorylated by the c-Jun N-terminal kinase, but not the ERK1/2 or p38 MAP kinases and that phosphorylation is coincident with increased transactivation of a Pax2-dependent reporter gene. Activation of JNK by either upstream kinase MEKK1 or DLK or by expression of Wnt signaling proteins significantly enhances Pax2 phosphorylation in cells. In vitro kinase assays using immunoprecipitated JNK or constitutively active, recombinant JNK show phosphorylation of GST-Pax2 fusion proteins. In transfected cells, phosphorylation of Pax2 correlates with increased transactivation of a Pax2-dependent reporter gene, suggesting that serine/threonine phosphorylation of the transactivation domain is important for Pax2 activity. Pax2 can form a complex with the JNK scaffolding protein JIP1, and this interaction is enhanced by activation of the JNK signaling module with the upstream kinase DLK. The data demonstrate that Pax2 is a new target for the JNK signaling module and point to a novel mechanism for mediating Pax-dependent transcription regulation.

Cell Line↗

PubMed2000

Evaluation of a new tool for exploring podocyte biology: mouse Nphs1 5' flanking region drives LacZ expression in podocytes.

Study of podocyte biology has been hampered by limitations in available experimental models that both recapitulate the in vivo phenotypes of this cell and can be readily and specifically manipulated at the molecular level. Transgenic manipulation of the podocyte represents one approach that might circumvent these limitations. The purpose of this study was to identify a promoter-enhancer that would direct the expression of transgenes in a podocyte-specific manner. The nephrin (Nphs1) promoter was considered a good candidate for this purpose, because nephrin was thought to be expressed exclusively in podocytes. Two independent BAC clones that contained the murine Nphs1 gene were identified. An 8.3-kb and a 5.4-kb fragment containing the 5' flanking promoter sequence were identified and characterized. Two constructs were generated by placing a bacterial lacZ reporter with a nuclear localization signal under the control of these two DNA fragments. Mice transgenic for both constructs were generated. Using a chemiluminescence assay, beta-galactosidase activity significantly above control was detected only in tissue homogenates of kidneys and brain of transgenic mice. In X-gal stained sections of transgenic adult kidneys, only podocyte nuclei expressed beta-galactosidase. In adult brain examined by tissue sectioning, beta-galactosidase activity was confined to a discrete area in the medulla. Identical patterns of beta-galactosidase expression were observed in multiple transgenic founders, suggesting that the expression pattern observed was independent of the site of transgene integration. The developmental expression of beta-galactosidase in transgenic embryos was also analyzed. Transgenes regulated by this promoter should be useful for studying the biology of gene products that regulate podocyte phenotype and function.

Animals↗