Nephrin and Podocin Expression Around the Onset of Puromycin Aminonucleoside Nephrosis

Hosoyamada, Makoto; Kou, Yan; Nishibori, Yukino; Takiue, Yuichi; Kudo, Akihiko; Kawakami, Hayato; Shibasaki, Toshiaki; Endou, Hitoshi

doi:10.1254/jphs.fp0040802

Cited by 28 publications

(24 citation statements)

References 21 publications

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“…This was characterized by a change from a linear to a discontinuous and granular pattern of staining on immunofluorescence, similar to our IL-13-transfection model. It is interesting that in the puromycin nephropathy model, onset of proteinuria and effacement of podocyte foot processes were found to parallel decreased protein expression rather than changes in mRNA expression of podocin (40,41).…”

Section: Discussionmentioning

confidence: 98%

Overexpression of Interleukin-13 Induces Minimal-Change–Like Nephropathy in Rats

Lai¹,

Wei²,

Tan³

et al. 2007

Journal of the American Society of Nephrology

194

130

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IL-13 has been implicated in the pathogenesis of minimal-change nephrotic syndrome. This study aimed to investigate the role of IL-13 on the development of proteinuria and expression of podocyte-related genes that are associated with nephrotic syndrome. IL-13 was overexpressed in Wistar rats through transfection of a mammalian expression vector cloned with the rat IL-13 gene, into the quadriceps by in vivo electroporation. Serum IL-13, albumin, cholesterol, and creatinine and urine albumin were measured serially. Kidneys were harvested after day 70 for histology and electron microscopy. Glomerular gene expression of nephrin, podocin, dystroglycan, B7-1, and IL-13 receptor subunits were examined using real-time PCR with hybridization probes and expressed as an index against ␤-actin. Protein expression of these molecules was determined by immunofluorescence staining. The IL-13-transfected rats (n ‫؍‬ 41) showed significant albuminuria, hypoalbuminemia, and hypercholesterolemia when compared with control rats (n ‫؍‬ 17). No significant histologic changes were seen in glomeruli of IL-13-transfected rats. However, electron microscopy showed up to 80% of podocyte foot process fusion. Glomerular gene expression was significantly upregulated for B7-1, IL-4R␣, and IL-13R␣2 but downregulated for nephrin, podocin, and dystroglycan. Immunofluorescence staining intensity was reduced for nephrin, podocin, and dystroglycan but increased for B7-1 and IL-4R␣ in IL-13-transfected rats compared with controls. In conclusion, these results suggest that IL-13 overexpression in the rat could lead to podocyte injury with downregulation of nephrin, podocin, and dystroglycan and a concurrent upregulation of B7-1 in the glomeruli, inducing a minimal change-like nephropathy that is characterized by increased proteinuria, hypoalbuminemia, hypercholesterolemia, and fusion of podocyte foot processes.

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Section: Discussionmentioning

confidence: 98%

Overexpression of Interleukin-13 Induces Minimal-Change–Like Nephropathy in Rats

Lai¹,

Wei²,

Tan³

et al. 2007

Journal of the American Society of Nephrology

194

130

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“…A plasmid encoding GST-Neph1CD fusion protein that includes the entire cytoplasmic domain of mouse Neph1 was prepared in pGEX4T-1 (Amersham Biosciences) using standard techniques (16). GST-Grb2 was a gift from B. Margolis, University of Michigan, and GST-Grb2-SH2 was a gift from A. Pandey, Johns Hopkins University.…”

Section: Methodsmentioning

confidence: 99%

Neph1 Cooperates with Nephrin To Transduce a Signal That Induces Actin Polymerization

Garg

Verma

Nihalani

et al. 2007

Molecular and Cellular Biology

128

183

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While the mechanisms that regulate actin dynamics in cellular motility are intensively studied, relatively little is known about signaling events that transmit outside-in signals and direct assembly and regulation of actin polymerization complexes at the cell membrane. The kidney podocyte provides a unique model for investigating these mechanisms since deletion of Nephrin or Neph1, two interacting components of the specialized podocyte intercellular junction, results in abnormal podocyte morphogenesis and junction formation. We provide evidence that extends the existing model by which the Nephrin-Neph1 complex transduces phosphorylation-mediated signals that assemble an actin polymerization complex at the podocyte intercellular junction. Upon engagement, Neph1 is phosphorylated on specific tyrosine residues by Fyn, which results in the recruitment of Grb2, an event that is necessary for Neph1-induced actin polymerization at the plasma membrane. Importantly, Neph1 and Nephrin directly interact and, by juxtaposing Grb2 and Nck1/2 at the membrane following complex activation, cooperate to augment the efficiency of actin polymerization. These data provide evidence for a mechanism reminiscent of that employed by vaccinia virus and other pathogens, by which a signaling complex transduces an outside-in signal that results in actin filament polymerization at the plasma membrane.Precisely regulated actin polymerization provides the motive force necessary for intercellular junction formation and contributes to defining the shape and polarity of the cell. Similarly, directed actin polymerization proximate to the leading edge of the plasma membrane drives cell motility and is required in the complicated dynamics of lamellipodia, filopodia, and other specialized membrane structures including invadopodia and podosomes (6). While substantial progress has been made in understanding the cellular and molecular mechanisms that determine actin dynamics in these model systems (32), less is known about membrane-based proximal signaling events that transmit outside-in signals and direct assembly and regulation of actin polymerization complexes at these sites.Kidney glomerular visceral epithelial cells or podocytes are necessary for maintaining the glomerular filtration barrier (reviewed in reference 20). When mature, these cells have a unique octopus-like structure comprised of a central cell body that gives off branching primary, secondary, and tertiary processes. The tertiary processes or "foot processes" attach the podocyte to the glomerular capillary basement membrane, where they surround the glomerular capillary and where they interdigitate to form a specialized intercellular junction called the "slit diaphragm." Here foot processes provide a necessary element of the permeability-selective glomerular filter, allowing passage of water, solutes, and other small macromolecules from the capillary lumen to the urinary space while restricting the flux of cells and larger macromolecules.The podocyte provides a unique model for investiga...

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“…28 Kidney cortex was obtained from rats at day 0-5 rat after the injection of PAN and examined immunohistochemically as described below. Protein lysates was obtained from isolated glomeruli and analyzed for protein expression of Gal-1, nephrin and b-actin.…”

Section: Puromycin Aminonucleoside Nephrosismentioning

confidence: 99%

Expression of galectin-1, a new component of slit diaphragm, is altered in minimal change nephrotic syndrome

Shimizu

Khoshnoodi

Akimoto

et al. 2009

Laboratory Investigation

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Nephrin is an essential structural component of the glomerular slit diaphragm (SD), a highly organized intercellular junction that constitutes the ultrafiltration barrier of the kidney. Recent studies have identified two additional nephrininteracting SD proteins (NEPH1 and NEPH2), suggesting that the zipper-like pattern of the SD is formed through complex intra-and intermolecular interactions of these proteins. However, the complexity of the SD structure suggests that additional SD components remain to be discovered. In this study, we identified galectin-1 (Gal-1) as a new component of the SD, binding to the ectodomain of nephrin. Using dual-immunofluorescence and confocal microscopy and dual-immunoelectron microscopy, we found Gal-1 colocalizing with the ectodomain of nephrin at the SD in normal human kidney. By immunoprecipitation and surface plasmon resonance, we confirmed a direct molecular interaction between Gal-1 and nephrin. Moreover, recombinant Gal-1 induced tyrosine phosphorylation of the cytoplasmic domain of nephrin and activation of the extracellular signal-regulated kinase 1/2 in podocytes. We also showed that podocytes are a major site of biosynthesis of Gal-1 in the glomerulus and that the normal expression patterns and levels of Gal-1 are altered in patients with minimal change nephrotic syndrome. Finally, in puromycin aminonucleoside-induced rat nephrosis, an apparent reduction in the levels of Gal-1 and nephrin around the onset of heavy proteinuria was also revealed. Our data present Gal-1 as a new extracellular ligand of nephrin localized at the glomerular SD, and provide further insight into the complex molecular organization, interaction, and structure of the SD, which is an active site of intracellular signaling necessary for podocyte function. Nephrin is a principal component of the glomerular slit diaphragm (SD), a highly specialized intercellular junction that constitutes the ultrafiltration barrier of the kidney.1 Structurally, nephrin is a type-1 transmembrane glycoprotein with 10 predicted asparagine-(N)-linked glycosylation sites located on its ectodomain.2 Recently, the sites of these N-linked glycans on the ectodomain of nephrin were mapped by mass spectrometry; and their terminal sugar residues were identified by the use of carbohydrate-specific lectins, which study showed that human nephrin is a sialoprotein with terminal sugars of high mannose glycans and galactose. 3 We have also shown that N-linked glycosylation is absolutely critical for the biosynthesis of the newly synthesized nephrin molecules in the endoplasmic reticulum (ER) and for their proper transport to the plasma membrane. 4 Moreover, we recently demonstrated that N-linked glycosylation of nephrin in the ER is highly dependent on the orchestration by calreticulin as a lectin chaperone and the levels of intracellular ATP as an energy source. 5,6

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Nephrin and Podocin Expression Around the Onset of Puromycin Aminonucleoside Nephrosis

Cited by 28 publications

References 21 publications

Overexpression of Interleukin-13 Induces Minimal-Change–Like Nephropathy in Rats

Overexpression of Interleukin-13 Induces Minimal-Change–Like Nephropathy in Rats

Neph1 Cooperates with Nephrin To Transduce a Signal That Induces Actin Polymerization

Expression of galectin-1, a new component of slit diaphragm, is altered in minimal change nephrotic syndrome

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