Positive clones in yeast two-hybrid assay using dendrin as a bait.

<p>Positive clones in yeast two-hybrid assay using dendrin as a bait.</p

Albuminuria in LPS-nephropathy and BSA-overload models.

<p>(a) LPS-injection induce significant albuminuria in both dendrin knockout and control mice. Urinary albumin levels were similar in both mice (8 mice in each group). (b) BSA overload caused massive albuminuria in both dendrin-deficient and control mice. However, no significant differences were observed between the two groups (6 knockout and 5 control mice were included). Units in the Y axes indicate albuminuria level as analyzed by Quantity One software.</p

Generation of dendrin knockout mouse line.

<p>(a) The targeting construct was designed to replace both coding exons of dendrin with a cassette encoding for eGFP and neomycin-resistance gene. (b) PCR-based genotyping generated 574 bp for a wild-type allele and 409 bp fragment for a knockout allele. The absence of dendrin mRNA was confirmed using RT-PCR in where cDNA generated from wildtype (wt) glomeruli and brain tissue showed dendrin expression (exon 1, exon 2 and the combination of exon 1–2), whereas cDNA from knockout (KO) animals remained negative for dendrin expression. Gapdh was used as a loading control. (c) Double immunofluorescence labeling for dendrin (two different antiboides, dendrin 1 and dendrin 2) and nephrin (green) showed that strong expression in wt glomeruli in where they colocalized (yellow). In KO glomeruli, no signal for dendrin was observed. DAPI staining (blue) showing nuclei. Magnifications: ×200.</p

The expression of podocyte proteins in dendrin-deficient glomeruli.

<p>Slit diaphragm proteins nephrin (a–b) and podocin (c–d) are observed as linear line around capillary loops in control (+/+) and knockout (−/−) mice. (e–f) The expressioin of foot process protein synaptopodin is unchanged in dendrin knockout glomeruli. DAPI staining (blue) showing nuclei. Magnifications: ×200. (g) Western blotting for nephrin, podocin and synaptopodin do not show any significant expressional difference between wildtype (wt) and knockout (ko) glomeruli. β-Actin, used as a loading control, shows similar expression levels in both fractions.</p

The kidney phenotype in dendrin knockout mouse.

<p>(a) Dendrin-deficient mice exhibit normal renal morphology by light microscopic examination (hematoxylin-eosin stained section from 2-month old dendrin knockout mouse). Inset: a dendrin-deficient glomerulus with normal morphological features. (b) In electron microscopy podocytes, the glomerular basement membrane and glomerular endothelial cells show normal morphological features. Fine slender podocyte foot processes interconnected by intact slit diaphragms are observed. Bar = 500 nm. (c) 2 ul of urine from 1-year-old wildtype (wt) and knockout (ko) mice was run on SDS-page gel. No albuminuria is detected Ladder showing 50 kD (blue) and 70 kD (orange) bands.</p

Schip1 Is a Novel Podocyte Foot Process Protein that Mediates Actin Cytoskeleton Rearrangements and Forms a Complex with Nherf2 and Ezrin

<div><p>Background</p><p>Podocyte foot process effacement accompanied by actin cytoskeleton rearrangements is a cardinal feature of many progressive human proteinuric diseases.</p><p>Results</p><p>By microarray profiling of mouse glomerulus, SCHIP1 emerged as one of the most highly enriched transcripts. We detected Schip1 protein in the kidney glomerulus, specifically in podocytes foot processes. Functionally, Schip1 inactivation in zebrafish by morpholino knock-down results in foot process disorganization and podocyte loss leading to proteinuria. In cultured podocytes Schip1 localizes to cortical actin-rich regions of lamellipodia, where it forms a complex with Nherf2 and ezrin, proteins known to participate in actin remodeling stimulated by PDGFβ signaling. Mechanistically, overexpression of Schip1 in vitro causes accumulation of cortical F-actin with dissolution of transversal stress fibers and promotes cell migration in response to PDGF-BB stimulation. Upon actin disassembly by latrunculin A treatment, Schip1 remains associated with the residual F-actin-containing structures, suggesting a functional connection with actin cytoskeleton possibly via its interaction partners. A similar assay with cytochalasin D points to stabilization of cortical actin cytoskeleton in Schip1 overexpressing cells by attenuation of actin depolymerisation.</p><p>Conclusions</p><p>Schip1 is a novel glomerular protein predominantly expressed in podocytes, necessary for the zebrafish pronephros development and function. Schip1 associates with the cortical actin cytoskeleton network and modulates its dynamics in response to PDGF signaling <i>via</i> interaction with the Nherf2/ezrin complex. Its implication in proteinuric diseases remains to be further investigated.</p></div

Schip1 localizes to cell lamellipodia and associates with the cortical actin cytoskeleton.

<p><b>(A)</b> Both endogenous (arrowheads, upper panel) and ectopic (arrowheads, lower panel) Schip1 localize to lamellipodia in cultured human podocytes. <b>(B)</b> To test Schip1 association to actin-rich lamellipodia regions, transiently transfected human podocytes were treated with the standard procedure (fixation and Triton X-100 permeabilization, upper panel), or incubated with saponin prior to fixation and staining for MycSchip1 (lower panel). Peripheral Schip1 expression is partially retained after saponin treatment, indicating association of the protein with detergent-insoluble cytoskeletal/plasma membrane structures. The same was not observed in control cells transfected with Stx8 (syntaxin 8). <b>(C)</b> Schip1 colocalizes with cortical F-actin in the podocyte lamellipodia. Both the Z- and XY-scanning indicate considerable signal overlap between Schip1 and F-actin along the plasma membrane in cells presenting well-developed lamellipodia. <b>(D)</b> Treatment with latrunculin A results in the dissolution of actin fibers in both control and Schip1-transfected podocytes. However, Schip1 signal remains associated with disturbed F-actin positive residues. In contrast, treatment with cytochalasin D results mostly in preservation of the cortical actin in Schip1 transfected podocytes.</p

Schip1, ezrin and Nherf2 colocalize in the podocyte foot processes of the human kidney glomerulus.

<p><b>(A)</b> Immunofluorescence on human kidney sections shows partial colocalization of Schip/ezrin and Schip/Nherf2 in the glomerulus (arrowheads). <b>(B)</b> By IEM, Schip1 is localized to the podocyte foot processes (FP), often to the apical but also to the basolateral side. Similar localization is seen for ezrin and Nherf2 (arrowheads). <b>(C)</b> Double IEM for Schip1 (10 nm gold particle) and ezrin or Nherf2 (5 nm gold particle) indicates that the proteins colocalize at the same subcellular area in the foot processes (arrowheads, zoom).</p

Schip1 overexpression promotes cortical F-actin accumulation, dissolution of stress fibers and motility in PDGF-BB-stimulated cells by attenuating actin depolymerisation.

<p><b>(A)</b> In control cells PDGF-BB treatment enhances development of lamellipodia. In Schip1-transfected cells PDGF-BB stimulation induces similar changes but also marked actin cytoskeleton rearrangement with cortical actin accumulation and dissolution of the actin stress fibers (box, zoom). Observe the neighboring non-Myc-Schip1 expressing cells, presenting with normal actin cytoskeleton and pronounced stress fibers. <b>(B)</b> Stable Schip1-expressing and control HEK293 cells were stimulated with 10% FCS or PDGF-BB, scratched, and left to migrate for 24 h (the wound healing assay). Schip1 transfected cells exhibit similar migration rate as controls in medium supplemented with 10% FCS, but migrate faster when induced with PDGF-BB (graph). Microscopic images of control and Schip1-expressing cell monolayers 18 and 24 h after wound scratching (left). <b>(C)</b> In vitro actin polymerization (right panel) and depolymerization (left panel) assays with lysates from GFP–Schip1-expressing HEK293 cells and controls show that Schip1-overexpression slows down actin depolymerization in presence of PDGFBB in comparison to cells treated with 10% FCS (p<0.0001). Results are representative of three separate experiments. RFU-relative fluorescence units.</p

Schip1 inactivation in zebrafish causes distortion of podocyte foot processes, podocytopenia and leakage of the filtration barrier.

<p><b>(A)</b> Brightfield images of PAS stained histological sections showing dilated Bowman’s space and distortion of proper podocyte structures (arrowheads) in Schip1 morphant zebrafish with milder (second panel) and more severe phenotype (third panel). Higher magnifications (size bars) shown in lower panel, including a cell nuclei quantification graph. <b>(B)</b> Electron microscopic analyses of zebrafish morphants show effaced podocyte foot processes with various degrees of damage (middle, arrowheads and right). However, slit diaphragms are consistently present (right, arrowheads). <b>(C)</b> Dye filtration assay in control and Schip1 zebrafish morphants. Rhodamine conjugated 10kDa is freely filtered into the tubuli of both control and morphant fish, whereas FITC labeled 500kDa dye remains in the pronephros in control embryos. In Schip1 morphants 500kDa dye accumulates in the Bowman’s space and also leaks into the tubuli. STD-standard control morpholino, F-fins, T-nephric tubuli, S-somites, B-brain, N-notochord, P-pronephros, G-gut, Y-yolk.</p