Podocalyxin in Rat Platelets and Megakaryocytes

Miettinen, Aaro; Solin, Marja-Liisa; Reivinen, Jukka; Juvonen, Eeva; Väisänen, Riitta; Holthöfer, Harry

doi:10.1016/s0002-9440(10)65328-x

Cited by 63 publications

(51 citation statements)

References 18 publications

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“…PC on endothelia may also help to keep the vascular lumen open and repel circulating leukocytes. Because PC has recently been found to be present in thrombocytes (McNagny et al, 1997;Miettinen et al, 1999), it may also prevent thrombocytes from inappropriately adhering to endothelia.…”

Section: Discussionmentioning

confidence: 99%

Expression of Podocalyxin Inhibits Cell–Cell Adhesion and Modifies Junctional Properties in Madin-Darby Canine Kidney Cells

Takeda¹,

Go²,

Orlando

et al. 2000

MBoC

184

181

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Podocalyxin is a major membrane protein of the glomerular epithelium and is thought to be involved in maintenance of the architecture of the foot processes and filtration slits characteristic of this unique epithelium by virtue of its high negative charge. However, until now there has been no direct evidence for podocalyxin's function. Podocalyxin is a type 1 transmembrane sialoprotein with an N-terminal mucin-like domain. To assess its function, we cloned rat podocalyxin and examined the effects of its expression on the cell adhesion properties of stably transfected Chinese hamster ovary (CHO)-K1 and Madin-Darby canine kidney (MDCK) cells and inducible ecdysone receptor-expressing (EcR)-CHO cells. In a cell aggregation assay, CHO-K1 cells expressing high levels of podocalyxin showed complete inhibition of cell aggregation, and MDCK transfectants showed greatly reduced aggregation (ϳ60 -80%) compared with parental cells. In EcR-CHO cells, the expression level of podocalyxin induced by increasing levels of ecdysone analogue correlated closely with the antiadhesion effect. The inhibitory effect of podocalyxin was reversed by treatment of the cells with Arthrobacter ureafaciens sialidase, indicating that sialic acid is required for inhibition of cell adhesion. Overexpression of podocalyxin also affected transepithelial resistance and the distribution of junctional proteins in MDCK cells by an unknown mechanism that may involve interaction with the actin cytoskeleton. These results provide direct evidence that podocalyxin functions as an antiadhesin that maintains an open filtration pathway between neighboring foot processes in the glomerular epithelium by charge repulsion.

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Section: Discussionmentioning

confidence: 99%

Expression of Podocalyxin Inhibits Cell–Cell Adhesion and Modifies Junctional Properties in Madin-Darby Canine Kidney Cells

Takeda¹,

Go²,

Orlando

et al. 2000

MBoC

184

181

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“…The following primary antibodies were used: rabbit polyclonal anti-rat nephrin antiserum with or without Protein A purification as described previously, 19 rabbit polyclonal anti-human ZO-1 (Zymed Laboratories, San Francisco, CA), rabbit polyclonal anti-mouse CD2AP (courtesy of Dr. Andrey Shaw, Washington University, St. Louis, MO), rabbit polyclonal anti-human podocalyxin, 22 rabbit polyclonal anti-rat ␣3-integrin (Chemicon Int., Temecula, CA), rabbit polyclonal anti-human caveolin 1 (Santa Cruz Biotechnology, San Diego, CA), mouse monoclonal antihuman transferrin receptor (Zymed Laboratories), and mouse monoclonal anti-phosphotyrosine antibody PY20 (Transduction Laboratories, Lexington, KY). All antibodies have been shown to react with the respective rat proteins.…”

Section: Immunoblottingmentioning

confidence: 99%

Involvement of Lipid Rafts in Nephrin Phosphorylation and Organization of the Glomerular Slit Diaphragm

Simons

Schwarz

Kriz

et al. 2001

The American Journal of Pathology

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145

126

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NPHS1 has recently been identified as the gene whose mutations cause congenital nephrotic syndrome of the Finnish type. The respective gene product nephrin is a transmembrane protein expressed in glomerular podocytes and primarily localized to the glomerular slit diaphragm. This interpodocyte junction functions in the glomerular filtration by restricting the passage of plasma proteins into the urinary space in a size-selective manner. The functional role of nephrin in this filtration process is so far not very well understood. In this study, we show that nephrin associates in an oligomerized form with signaling microdomains, also known as lipid rafts, and that these localize to the slit diaphragm. We also show that the nephrin-containing rafts can be immunoisolated with the 27A antibody recognizing a podocyte-specific 9-Oacetylated GD3 ganglioside. In a previous study it has been shown that the in vivo injection of this antibody leads to morphological changes of the filtration slits resembling foot process effacement. Here, we report that, in this model of foot process effacement, nephrin dislocates to the apical pole of the narrowed filtration slits and also that it is tyrosine phosphorylated. We suggest that lipid rafts are important in the spatial organization of the glomerular slit diaphragm under physiological and pathological conditions. One of the main functions of the kidney is formation of the primary urine in the glomerulus by plasma ultrafiltration. The structural correlate of ultrafiltration is the glomerular capillary wall with its distinctly layered filtration barrier. It consists of the fenestrated vascular endothelium, the glomerular basement membrane, and a layer of morphologically highly specialized epithelial cells, podocytes.Podocytes form a tight network of multiple interdigitating cellular extensions, called foot processes, which are bridged by so-called "slit diaphragms." 1 The filtration barrier is freely permeable for water, small solutes, and ions but does not allow passage for proteins larger than albumin and other large molecules. The charge selectivity in this process has been attributed to the glomerular basement membrane whereas the slit diaphragm supposedly plays a major role in the size selectivity. 2,3 The importance of the filtration barrier is reflected by a number of human diseases in which the filtration barrier function is disrupted resulting in the loss of plasma proteins into the urine. Patients with persistent proteinuria ultimately develop a nephrotic syndrome with a variety of symptoms including edema, hypoalbuminemia, and hyperlipidemia. 4

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“…17 Briefly, the library was constructed using total RNA isolated from glomeruli of 1-month-old rats by the isothiocyanate/CsCl Ϫ method. 18 Furthermore, poly(Aϩ) mRNA was isolated using a PolyATrac mRNA isolation System III kit (Promega, Madison, WI).…”

Section: Cloning Of Rat Nephrin Cdnamentioning

confidence: 99%

“…The prehybridization and further hybridization were performed as described previously. 17 Briefly, the filter was prehybridized at 42°C and further hybridized in the hybridization solution containing the 32 P probe (10 7 cpm; see below) labeled with [␣-32 P]CTP (3000 Ci/mmol, 10 mCi/ml; Amersham), using a Random priming kit (Boehringer Mannheim, Germany). After an overnight hybridization the filter was washed twice with low-stringency buffer at 42°C, twice with highstringency buffer at 50°C, and exposed to the film (Fuji Photo Film, Japan) with intensifying screens at Ϫ70°C.…”

Section: Cloning Of Rat Nephrin Cdnamentioning

confidence: 99%

Cloning and Expression of the Rat Nephrin Homolog

Ahola¹,

Wang²,

Luimula³

et al. 1999

The American Journal of Pathology

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Despite of the increased availability of genetically modified mouse strains , the experimental models in the rat have provided the most widely employed and versatile models for the study of renal pathophysiology and functional genetics. The identification of the human gene mutated in the congenital nephrotic syndrome of the Finnish type (NPHS1) has recently been reported , and its protein product has been termed nephrin. Here we report the molecular cloning and characterization of rat nephrin cDNA. Rat nephrin cDNA has an open reading frame of 3705 bp , shows 82% sequence identity with human nephrin cDNA, and shows characteristic rat-specific splicing variants. The translated nucleotide sequence has 89% sequence identity at the amino acid level. The signal sequence , glycosylation , and cysteine localization patterns are nearly identical to those of human nephrin. As in the human , the rat nephrin transcript is expressed in a tissue-restricted pattern. Antipeptide antibodies raised to the intracellular nephrin-specific domain identified immunoreactivity exclusively within the rat kidney glomerulus by indirect immunofluorescence. Initial results with semiquantitative reverse transcriptase-polymerase chain reaction analysis showed a remarkable down-regulation of nephrin-specific mRNA in the puromycin nephrosis of the rat. (Am J Pathol 1999, 155:907-913)The molecular pathogenesis of diseases that result in abnormalities of glomerular filtration has remained poorly understood. Recent results indicate that podocytes play an important role in regulating the passage of macromolecules and have several structural properties suitable for allowing the rapid modulation of the permeability barrier.

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Podocalyxin in Rat Platelets and Megakaryocytes

Cited by 63 publications

References 18 publications

Expression of Podocalyxin Inhibits Cell–Cell Adhesion and Modifies Junctional Properties in Madin-Darby Canine Kidney Cells

Expression of Podocalyxin Inhibits Cell–Cell Adhesion and Modifies Junctional Properties in Madin-Darby Canine Kidney Cells

Involvement of Lipid Rafts in Nephrin Phosphorylation and Organization of the Glomerular Slit Diaphragm

Cloning and Expression of the Rat Nephrin Homolog

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