PubMed Health⌕ Search

Biomedical subjects

John R Sedor

Publications and source records attributed to John R Sedor.

At least 19 recordsLinked to original sources
PubMed2006

Beta8 integrin binds Rho GDP dissociation inhibitor-1 and activates Rac1 to inhibit mesangial cell myofibroblast differentiation.

Alpha(v)beta8 integrin expression is restricted primarily to kidney, brain, and placenta. Targeted alpha(v) or beta8 deletion is embryonic lethal due to defective placenta and brain angiogenesis, precluding investigation of kidney alpha(v)beta8 function. We find that kidney beta8 is localized to glomerular mesangial cells, and expression is decreased in mouse models of glomerulosclerosis, suggesting that beta8 regulates normal mesangial cell differentiation. To interrogate beta8 signaling pathways, yeast two-hybrid and co-precipitation studies demonstrated beta8 interaction with Rho guanine nucleotide dissociation inhibitor-1 (GDI). Selective beta8 stimulation enhanced beta8-GDI interaction as well as Rac1 (but not RhoA) activation and lamellipodia formation. Mesangial cells from itgb8-/- mice backcrossed to a genetic background that permitted survival, or gdi-/- mice, which develop glomerulosclerosis, demonstrated RhoA (but not Rac1) activity and alpha-smooth muscle actin assembly, which characterizes mesangial cell myofibroblast transformation in renal disease. To determine whether Rac1 directly modulates RhoA-associated myofibroblast differentiation, mesangial cells were transduced with inhibitory Rac peptide fused to human immunodeficiency virus-Tat, resulting in enhanced alpha-smooth muscle actin organization. We conclude that the beta8 cytosolic tail in mesangial cells organizes a signaling complex that culminates in Rac1 activation to mediate wild-type differentiation, whereas decreased beta8 activation shifts mesangial cells toward a RhoA-dependent myofibroblast phenotype.

Animals↗
PubMed2006

Math6 expression during kidney development and altered expression in a mouse model of glomerulosclerosis.

Math6 is a tissue-restricted member of the Atonal family of basic helix-loop-helix (bHLH) transcription factors and has been implicated in specification and differentiation of cell lineages in the brain. We identify here Math6 as a podocyte-expressed bHLH protein that was down-regulated in human immunodeficiency virus-associated nephropathy (HIVAN); a collapsing glomerulopathy characterized by podocyte dedifferentiation. Early in metanephric development, Math6 was expressed in metanephric mesenchyme but not ureteric bud-derived cells, with overall Math6 expression most abundant in the nephrogenic zone, including developing glomeruli. In adult kidney, Math6 expression was restricted to podocytes. In adult podocyte cell lines and kidneys from the transgenic mouse model of HIVAN, Math6 podocyte expression was reduced concurrent with previously reported reductions in Nephrin and Synaptopodin expression, suggesting a correlation between the loss of Math6 expression and typical podocyte terminal differentiation markers. These studies suggest that Math6 may participate in kidney development and may be a permissive factor for podocyte differentiation.

Amino Acid Sequence↗
PubMed2005

Mechanical force-activated phospholipase D is mediated by Galpha12/13-Rho and calmodulin-dependent kinase in renal epithelial cells.

The renal glomerulus, the site of plasma ultrafiltration, is exposed to mechanical force in vivo arising from capillary blood pressure and fluid flow. Studies of cultured podocytes demonstrate that they respond to stretch by altering the structure of the actin cytoskeleton, but the mechanisms by which physical force triggers this architectural change and the signaling pathways that lead to generation of second messengers are not defined. In the present study, we found that in renal epithelial cells [podocytes and Madin-Darby canine kidney (MDCK) cells], application of mechanical force to the cell surface through fibronectin-coated ferric beads and exposure of the cells to magnetic force lead to Rho translocation and actin cytoskeleton reorganization. This application of force recruited Rho and filamentous actin (F-actin) to bead loci and subsequently stimulated phospholipase D (PLD), a downstream effector of Rho. Using MDCK cells that stably express regulators of G protein-signaling (RGS) proteins [RGS4 attenuates Galpha(i) and Galpha(q), and the p115RhoGEF-RGS domain (p115-RGS) attenuates Galpha(12/13)] to define the signaling pathway, we found that mechanical force induced Galpha(12/13)-Rho activation and increased F-actin to stimulate PLD activity. The activation can be partially prevented by the C(3) exoenzyme. Pretreatment of the cells with chemical inhibitors of several kinases showed that calmodulin-dependent kinase is also involved in stretch-induced PLD activation by a separate pathway. Taken together, our data demonstrate that in cultured podocytes and MDCK cells, mechanical force leads to actin cytoskeleton reorganization and PLD activation. The signaling pathways for PLD activation involve Galpha(12/13)/Rho/F-actin and calmodulin-dependent kinase.

Actins↗
PubMed2005

WT1-interacting protein and ZO-1 translocate into podocyte nuclei after puromycin aminonucleoside treatment.

Podocyte differentiation is required for normal glomerular filtration barrier function and is regulated by the transcription factor WT1. We identified WT1-interacting protein (WTIP) and hypothesized that it functions as both a scaffold for slit diaphragm proteins and a corepressor of WT1 transcriptional activity by shuttling from cell-cell junctions to the nucleus after injury. Endogenous WTIP colocalizes with zonula occludens-1 (ZO-1) in cultured mouse podocyte adherens junctions. To model podocyte injury in vitro, we incubated differentiated podocytes with puromycin aminonucleoside (PAN; 100 microg/ml) for 24 h, which disassembled cell-cell contacts, rearranged actin cytoskeleton, and caused process retraction. Podocyte synaptopodin expression diminished after PAN treatment, consistent with podocyte dedifferentiation in some human glomerular diseases. To assess podocyte function, we measured albumin flux across differentiated podocytes cultured on collagen-coated Transwell filters. Albumin transit across PAN-treated cells increased to levels observed with undifferentiated podocytes. Consistent with our hypothesis, WTIP, as well as ZO-1, translocated from podocyte adherens junctions to nuclei in PAN-treated cells. Because WTIP is a transcriptional corepressor for WT1, we examined the effect of PAN on expression of retinoblastoma binding protein Rbbp7 (also known as RbAp46), a WT1 target gene expressed in S-shaped bodies during nephrogenesis. Rbbp7 expression in PAN-treated podocytes was reduced compared with untreated cells. In conclusion, WTIP translocates from cell-cell junctions to the nucleus in PAN-treated podocytes. We suggest that WTIP monitors slit diaphragm protein assembly and shuttles into the nucleus after podocyte injury, translating changes in slit diaphragm structure into altered gene expression and a less differentiated phenotype.

Albumins↗
PubMed2005

Variants in the Wilms' tumor gene are associated with focal segmental glomerulosclerosis in the African American population.

Wilms' tumor gene (WT1) is important for nephrogenesis and gonadal growth. WT1 mutations cause Denys-Drash and Frasier syndromes, which are characterized by glomerular scarring. To test whether genetic variations in WT1 and WIT1 (gene immediately 5' to WT1) associate with focal segmental glomerulosclerosis (FSGS), patients with biopsy-proven idiopathic and HIV-1-associated FSGS were enrolled in a multicenter study. We genotyped SNP rs6508 located in WIT1 exon 1, three SNPs (rs2301250, rs2301252, rs2301254) in the promoter shared by WT1 and WIT1, rs2234590 in exon 6, rs2234591 in intron 6, rs16754 in exon 7, and rs1799937 in intron 9 of WT1. Cases (n = 218) and controls (n = 281) were compared in the African American population. Stratification by HIV-1 infection status showed that SNPs rs6508, rs2301254, and rs1799937 were significantly associated with FSGS [rs6508 odds ratio (OR) 1.82, P = 0.006; rs2301254 OR 1.65, P = 0.049; rs1799937 OR 1.91, P = 0.005] in the non-HIV-1 group and rs2234591 (OR 0.234, P = 0.011) in the HIV-1 group. Haplotype analyses in the population revealed that seven SNPs were associated with FSGS; five SNPs had the highest contingency score [-log10(P value) = 13.57] in the HIV-1 group. This association could not be explained by population substructure. We conclude that SNPs in WT1 and WIT1 genes are significantly associated with FSGS, suggesting that variants in these genes may mediate pathogenesis by altering WT1 function. Furthermore, HIV-1 infection status interacts with genetic variations in both genes to influence this phenotype. We speculate that nephropathy liability alleles in WT1 pathway genes cause podocyte dysfunction and glomerular scarring.

Black or African American↗
PubMed2005

A dipstick protein and specific gravity algorithm accurately predicts pathological proteinuria.

BACKGROUND: The proper strategy to screen for early chronic kidney disease is debatable, but protein-creatinine ratio from a random urine sample (UPC) commonly is used. The purpose is to determine whether dipstick data effectively identify patients with increased UPC ratios. Because urine concentration affects proteinuria interpretation, we hypothesized that dipstick protein (DSP) and specific gravity (SG) are sufficient for screening. METHODS: A hospital laboratory database was searched for urine samples simultaneously assayed for UPC ratio, DSP, and SG (n = 2,098). A random 70% of the cohort was used to generate a development model, which was validated in the remaining 30%. Samples were stratified according to DSP and SG values. A DSP versus SG matrix was created, and each sample was allocated to a discrete DSP-SG category. Proportions of samples with overt (UPC ratio > or = 500 mg/g) and nephrotic-range proteinuria (UPC ratio > or = 3,000 mg/g) were calculated for all 40 cells. RESULTS: Optimum correlations between DSP-SG cells and UPC ratios were determined for the development model, yielding 97.0% negative predictive value (NPV) for a UPC ratio of 500 mg/g or less and 97.5% positive predictive value (PPV) for a UPC ratio of 500 mg/g or greater. NPV for a UPC ratio of 3,000 mg/g or less was 99.7%. Application of the model to the validation sample showed a 96.5% NPV for a UPC ratio of 500 mg/g or less, 99.4% PPV for a UPC ratio of 500 mg/g or greater, and 99.0% NPV for a UPC ratio of 3,000 mg/g or less. CONCLUSION: DSP and SG values effectively identify patients requiring proteinuria quantification by means of UPC ratio. A Web-based tool was developed that allows DSP and SG value entry and provides a recommendation regarding the need for proteinuria quantification.

Biomarkers↗
PubMed2004

An activating mutant of Cdc42 that fails to interact with Rho GDP-dissociation inhibitor localizes to the plasma membrane and mediates actin reorganization.

Cdc42 is a member of the Rho family of GTPases and plays an important role in the regulation of actin cytoskeletal organization. Activation of Cdc42 and associated signal transduction cascades are dependent upon proper localization of this GTPase. The studies described herein address the hypothesis that Rho GDP-dissociation inhibitor, RhoGDI, plays an essential role in the translocation of Cdc42 to signaling complexes at the plasma membrane and is essential for Cdc42-mediated actin cytoskeletal rearrangements. An activating mutant of Cdc42 that is RhoGDI-binding defective (Cdc42(G12V/R66E)) is characterized and used as a tool to study the functional importance of the Cdc42-RhoGDI interaction. Overexpression of mycCdc42(G12V/R66E) in COS-7 cells results in actin cytoskeletal rearrangements that are indistinguishable from those stimulated by overexpression of mycCdc42(G12V). In addition, the G12V activating mutant of Cdc42 was overexpressed in mesangial cells that are null for RhoGDI expression. MycCdc42(G12V) stimulation of filopodia formation in these cells was indistinguishable from that observed in wild-type mesangial cells. Taken together, the results presented herein indicate that although RhoGDI is a critical regulator of guanine nucleotide binding, cycling of Cdc42 between membranes and the cytosol and cellular transformation, it is not essential for Cdc42-mediated organization of the actin cytoskeleton.

Actins↗
PubMed2004

The NHE1 Na+/H+ exchanger recruits ezrin/radixin/moesin proteins to regulate Akt-dependent cell survival.

Apoptosis results in cell shrinkage and intracellular acidification, processes opposed by the ubiquitously expressed NHE1 Na(+)/H(+) exchanger. In addition to mediating Na(+)/H(+) transport, NHE1 interacts with ezrin/radixin/moesin (ERM), which tethers NHE1 to cortical actin cytoskeleton to regulate cell shape, adhesion, motility, and resistance to apoptosis. We hypothesize that apoptotic stress activates NHE1-dependent Na(+)/H(+) exchange, and NHE1-ERM interaction is required for cell survival signaling. Apoptotic stimuli induced NHE1-regulated Na(+)/H(+) transport, as demonstrated by ethyl-N-isopropyl-amiloride-inhibitable, intracellular alkalinization. Ectopic NHE1, but not NHE3, expression rescued NHE1-null cells from apoptosis induced by staurosporine or N-ethylmaleimide-stimulated KCl efflux. When cells were subjected to apoptotic stress, NHE1 and phosphorylated ERM physically associated within the cytoskeleton-enriched fraction, resulting in activation of the pro-survival kinase, Akt. NHE1-associated Akt activity and cell survival were inhibited in cells expressing ERM binding-deficient NHE1, dominant negative ezrin constructs, or ezrin mutants with defective binding to phosphoinositide 3-kinase, an upstream regulator of Akt. We conclude that NHE1 promotes cell survival by dual mechanisms: by defending cell volume and pH(i) through Na(+)/H(+) exchange and by functioning as a scaffold for recruitment of a signalplex that includes ERM, phosphoinositide 3-kinase, and Akt.

Apoptosis↗
PubMed2004

An activating mutant of Rac1 that fails to interact with Rho GDP-dissociation inhibitor stimulates membrane ruffling in mammalian cells.

Rac1, a member of the Rho family of small GTP-binding proteins, is involved in the regulation of the actin cytoskeleton via activation of lamellipodia and membrane ruffle formation. RhoGDI (Rho-family-specific GDP-dissociation inhibitor) forms a complex with Rho proteins in the cytosol of mammalian cells. It not only regulates guanine nucleotide binding to Rho proteins, but may also function as a molecular shuttle to carry Rho proteins from an inactive cytosolic pool to the membrane for activation. These studies tested if RhoGDI is necessary for the translocation of Rac1 from the cytosol to the plasma membrane for the formation of membrane ruffles. We describe a novel mutant of Rac1, R66E (Arg66-->Glu), that fails to bind RhoGDI. This RhoGDI-binding-defective mutation is combined with a Rac1-activating mutation G12V, resulting in a double-mutant [Rac1(G12V/R66E)] that fails to interact with RhoGDI in COS-7 cells, but remains constitutively activated. This double mutant stimulates membrane ruffling to a similar extent as that observed after epidermal growth factor treatment of non-transfected cells. To confirm that Rac1 can signal ruffle formation in the absence of interaction with RhoGDI, Rac1(G12V) was overexpressed in cultured mesangial cells derived from a RhoGDI knockout mouse. Rac1-mediated membrane ruffling was indistinguishable between the RhoGDI(-/-) and RhoGDI(+/+) cell lines. In both the COS-7 and cultured mesangial cells, Rac1(G12V) and Rac1(G12V/R66E) co-localize with membrane ruffles. These findings suggest that interaction with RhoGDI is not essential in the mechanism by which Rac1 translocates to the plasma membrane to stimulate ruffle formation.

Animals↗
PubMed2004

A WT1 co-regulator controls podocyte phenotype by shuttling between adhesion structures and nucleus.

Glomerular podocyte differentiation state is critical for filtration barrier function and is regulated by WT1, a zinc finger transcription factor. A yeast two-hybrid assay identified a novel, WT1-interacting protein (WTIP) that maps to human chromosome 19q13.1, a region with genes linked to familial focal segmental glomerulosclerosis. The domain structure of WTIP is similar to the zyxin subfamily of cytosolic LIM domain-containing proteins, which contain three carboxyl-terminal LIM protein-protein interaction domains and a proline-rich, pre-LIM region with a nuclear export signal. Other LIM domain-containing proteins (zyxin and mouse muscle LIM protein) did not interact with WT1 in two-hybrid assays, and WTIP did not interact with an unrelated transcription factor, LMX1B. WTIP mRNA was detected in cultured podocytes and was developmentally regulated, with expression peaking in mouse kidney at embryonic day 15-16 (E15-E16) in kidney but persisting into adulthood. In situ hybridization demonstrated WTIP expression in developing E15 glomeruli and in cultured podocytes. The partial WTIP clone, which interacted with WTIP in the two-hybrid assay, co-localized with WT1 in nuclei, co-precipitated with WT1, and inhibited WT1-dependent transcriptional activation of the amphiregulin promoter. In contrast, full-length WTIP was excluded from cell nuclei, but after the addition of leptomycin B, an inhibitor of Crm1-mediated nuclear export, it accumulated in the nucleus and co-precipitated with WT1 in whole cell lysates. Epitope-tagged WTIP co-localized with the adaptor protein CD2AP (CMS) in podocyte actin spots and with Mena at cell-cell junctions. We propose that WTIP monitors slit diaphragm protein assembly as part of a multiple protein complex, linking this specialized adhesion junction to the actin cytoskeleton, and shuttles into the nucleus after podocyte injury, providing a mechanism whereby changes in slit diaphragm structure modulate gene expression.

Actins↗
PubMed2004

Renal phenotype is exacerbated in Os and lpr double mutant mice.

BACKGROUND: ROP-Os/+ mice are born with oligosyndactyly and oligonephronia and develop renal dysfunction, which includes renal tubular epithelial cell (RTC) Fas-dependent apoptosis and tubular atrophy. MRL/lpr mice harbor a Fas-inactivating mutation and develop glomerulonephritis, whereas mice expressing lpr on a C3H background demonstrate no renal phenotype. We hypothesized that crossing ROP-Os/+ with CH3-lpr/lpr mice would rescue the Os/+ renal phenotype by reducing Fas-dependent RTC apoptosis. METHODS: ROP-Os/+ mice were intercrossed with C3H-lpr/lpr mice and F(2) generation animals were phenotyped by kidney weight, serum creatinine, and albuminuria. Kidney sections were scored for histopathology and apoptosis. Univariate and multivariate analyses were used to examine additive effects of Os and lpr on renal phenotype. RESULTS: By 16 weeks, F(2)Os/+ lpr/lpr mice developed significantly more albuminuria, glomerulosclerosis, and interstitial inflammation compared to Os/++/+ mice. Glomerular cell apoptosis was increased in Os/+ lpr/lpr compared to Os/++/+ mice, with no significant difference in RTC apoptosis. A statistically significant Os-lpr effect on renal phenotype was demonstrated by multivariate analysis, which exceeded the combined independent effects if Os and lpr, indicating a biologic interaction exists between Os and lpr. CONCLUSION: Os/+ mice with a superimposed lpr mutation displayed a more severe renal phenotype, rather than phenotype rescue, suggesting that Fas pathway activation is necessary to delete cells resulting from Os-dependent injury. We further propose that an Os-lpr gene interaction and/or mixed ROP-C3H genetic background regulated the renal phenotype, consistent with the concept that chronic renal disease pathogenesis reflects effects of multiple nephropathy susceptibility alleles.

Animals↗
PubMed2003

Congenital diaphragmatic hernia, kidney agenesis and cardiac defects associated with Slit3-deficiency in mice.

Slit3 along with Slit1 and Slit2 comprise the Slit family of proteins. The latter two proteins are known to be involved in axon guidance and cell migration during animal development. However, little is know about the functions of Slit3. We created a Slit3-deficient mouse model from an OmniBank ES cell line with a Slit3 allele trapped by insertional mutagenesis to analyze the in vivo functions of this protein. In this model, congenital diaphragmatic hernia is the most obvious phenotype. Herniation was found to be caused by a defective central tendon (CT) of the diaphragm that remained fused with the liver. Electron microscopic analyses of the defective CT revealed disorganized collagen fibrils that failed to form tight collagen bundles. The hearts of Slit3-deficient mice have an enlarged right ventricle. In addition, 20% of homozygous mice also showed a range of kidney defects that include unilateral or bilateral agenesis of the kidney and ureter, or varying degrees of renal hypoplasia. Thus, we concluded that Slit3 is involved in the development of multiple organ systems that include the diaphragm and the kidney. Slit3-deficient mice represent a genetic animal model for physiological and pathological studies of congenital diaphragmatic hernia.

Animals↗
PubMed2003

Kidney disease, genotype and the pathogenesis of vasculopathy.

PURPOSE OF REVIEW: The two leading causes of end-stage renal disease in the United States are diabetes mellitus and hypertensive nephrosclerosis, accounting for over two-thirds of all cases. In many patients both diseases are associated with small- and large-vessel disease, commonly attributed to hypertension or accelerated atherosclerosis. Recent investigations, however, have suggested that renal large-vessel and microvascular disease may be independent contributors to progressive kidney failure. RECENT FINDINGS: Although genes have not been definitely linked to renal vascular disease, population- and family-based epidemiology of kidney disease, segregation analysis of Pima and Caucasian families in which diabetic nephropathy is clustered, and the positional cloning of genes responsible for rare, familial glomerulosclerosis syndromes support the hypothesis that genes regulate the pathogenesis of renal disease. This review highlights developments in our current understanding of vasculopathy and its role in renal disease, and it summarizes evidence suggesting that genetic determinants for the vascular phenotype are associated with common causes of chronic renal failure. SUMMARY: With the application of genomics and proteomics methodologies to drug discovery, the identification of renal susceptibility genes should identify new mechanisms of progressive renal disease pathogenesis and generate novel target molecules for the treatment of kidney disease.

Animals↗
PubMed2003

DNA expression analysis: serial analysis of gene expression, microarrays and kidney disease.

PURPOSE OF REVIEW: Expression profiling using serial analysis of gene expression and microarray technologies allows global description of expressed genes present in biological systems. Although relatively new technologies, each having been developed in the mid-1990s, both have become established and widely used tools for identification of gene networks and gene function. RECENT FINDINGS: This review highlights DNA expression analyses published in 2002, emphasizing primarily serial analysis of gene expression and microarray technologies. We focus on the applicability of DNA expression analysis to renal disease, especially as some investigators have developed custom serial analysis of gene expression kidney libraries and kidney disease-specific 'designer chip' microarrays. Data analysis techniques and statistics are also discussed, since the challenge is generation of accurate messenger RNA profiles and interpretation of data in a manner that is both coherent and reproducible. SUMMARY: Because kidney disease pathophysiology is complex, expression analysis can identify candidate nephropathy pathogenesis genes and gene networks, which eventually could become targets for therapeutic intervention.

Base Sequence↗
PubMed2003

Nucleic acid-based techniques for post-transcriptional regulation of molecular targets.

PURPOSE OF REVIEW: Messenger RNA, transfer RNA and ribosomal RNA were defined long ago as essential components for transmission of genetic code from DNA. However, there are many other, less commonly recognized RNAs, such as ribozymes and small interfering RNAs, which are distinguished by their ability to inhibit RNA function. This review describes the basic molecular concepts and potential therapeutic applications of RNA inhibition by a variety of molecules, including ribozymes, antisense oligonucleotides, aptamers and small interfering RNAs. RECENT FINDINGS: A tremendous amount of data has recently emerged about double-stranded small interfering RNAs, which bind and degrade corresponding messenger RNAs by a process called RNA interference. Though native small interfering RNAs have been shown to be biologically relevant in animals and plants, synthetic types have rapidly become powerful tools for post-transcriptional inhibition of specific gene products to determine functional consequences in simple organisms and in-vitro model systems. More established means of RNA inhibition, such as with ribozyme and antisense strategies, continue to be viable options for in-vitro experiments, and form the basis for many ongoing clinical trials. SUMMARY: Ribozymes, antisense oligonucleotides, aptamers and small interfering RNAs are potentially useful reagents for in-vitro investigation and for treatment of kidney and hypertension diseases.

Animals↗