Migration of apical and basolateral polarity proteins during podocyte differentiation.

<p>Frozen kidney sections of newborn Wistar rat (P0) were stained using antibodies against the apical membrane protein Podocalyxin, the apical polarity protein Par3 and the basolateral polarity protein Scribble and were subjected to confocal laser microscopy. Since glomerular development is asynchronous, kidneys of newborn rats display various glomerular developmental stages. Each panel displays the expression pattern of the accordant proteins during glomerular development (from left to right): Developmental stages ranging from comma-shaped body (I), s-shaped body (II), capillary loop stage (III to IV), to a maturing glomerulus (V). (<b>A</b>) Whereas Par3 is expressed during comma-shaped body stage and localizes to the apical sited cell-cell junctions, expression of Podocalyxin starts during s-shaped body stage, when Par3 and the cell-cell contacts translocate along the lateral side of immature podocytes to basal. During this translocation the apical membrane area, marked by Podocalyxin, increases while the basolateral membrane area shrinks relatively. Arrows indicate translocation of Par3 from the apical cell-cell contacts in I to the developing foot processes in V. (<b>B</b>) Scribble localizes basal of Par3 at the cell-cell junctions and at the basolateral membrane during comma-shaped body stage (I) and translocates like Par3 during podocyte differentation to the developing foot processes in V. (<b>C</b>) While Podocalyxin and Par3 as well as Par3 and Scribble display an partial overlap of their localization (yellow in A and B), no overlap of Podocalyxin and Scribble can be detected indicating a localization to completely distinct membrane areas with Podocalyxin as an apical membrane marker and Scribble as a basolateral marker protein. Scale bars: 5 µm.</p

<i>Scribble^Δpodocyte</i> mice develop normal podocyte foot processes and show no increased susceptibility to glomerular stress.

<p>(<b>A, B</b>) No obvious histological abnormalities can be detected in PAS staining of <i>Scribble^Δpodocyte</i> kidney sections at P1 and at 6 weeks of age compared to control littermates. (<b>C, D</b>) Transmission electron micrographs display normal podocyte architecture with foot processes without any obvious ultrastructural defect. (<b>E</b>) No difference in the number of podocytes per sectioned glomerulus could be detected (ns, not significant, n = 3 each, 30 glomeruli for each mouse were analyzed). (<b>F</b>) During a one year follow up <i>Scribble^Δpodocyte</i> mice develop no significant albuminuria. (<b>G</b>) No difference in albuminuria can be detected between <i>Scribble^Δpodocyte</i> mice and control littermates in the BSA-overload model (n = 5 each) and (<b>H</b>) the ADR model (n = 6 each). Scale bars: 20 µm in (A) and (B), 1 µm in (C) and (D).</p

Scribble localizes to the cell-cell contacts and the basolateral membrane of podocytes.

<p>(<b>A</b>) Immunogold electron microscopy of P0 rat kidney sections displays localization of Scribble (arrows) at immature podocyte cell-cell contacts. (<b>B</b>) In adult rat kidney sections Scribble localizes predominantly at the basolateral side of podocyte foot processes and partially at the slit-diaphragm. Scale bars: 200 nm.</p

Generation of podocyte-specific Scribble knockout mice, <i>Scribble^Δpodocyte</i>.

<p><i>Scribble^flox/flox</i> mice were crossed with <i>NPHS2.Cre</i> mice to generate podocyte-specific <i>Scribble</i> knockout mice <i>Scribble^flox/flox; NPHS2.Cre</i> (<i>Scribble^Δpodocyte</i>). (<b>A</b>) Generation of tissue-specific <i>Scribble</i> knockout mice. (<b>B</b>) PCR analysis of genomic DNA from isolated glomeruli confirmed genomic deletion of exon 2–8 in <i>Scribble^Δpodocyte</i> mice. (<b>C, D</b>) Frozen kidney sections of control and <i>Scribble^Δpodocyte</i> mice were stained using antibodies against Scribble and the podocyte foot process marker Nephrin and were subjected to confocal laser microscopy. Arrows indicate the podocyte foot process compartment. While Scribble localizes to the podocyte foot process compartment of control mice, no Scribble expression can be detected in podocytes of <i>Scribble^Δpodocyte</i> mice. Scale bars: 5 µm.</p

Development of podocyte foot processes in cultured embryonic <i>Scribble</i> knockout kidneys and in <i>circletail</i> mutant mice.

<p>(<b>A</b>) Western blot of total embryo lysates shows complete loss of Scribble protein in <i>Scribble</i> knockout embryos (<i>Scribble^−/−</i>) (ns, non specific). (<b>B</b>) Kidneys of <i>Scribble</i> knockout and wildtype littermate embryos were harvested at P12.5 and grew in DMEM medium for 6 days. (<b>C</b>) Immunofluorescence staining against WT1, which is expressed in podocytes and embryonic kidney epithelial cells, and Scribble reveals complete loss of Scribble in <i>Scribble</i> knockout kidney culture (arrows indicate glomeruli), (<b>D</b>) while expression of the podocyte marker proteins Podocin and Nephrin as well as the apical polarity protein Par3 can be detected in <i>Scribble</i> knockout kidney culture glomeruli. (<b>E</b>) Electron micrographs show development of podocyte foot processes connected by slit diaphragms in <i>Scribble</i> knockout and wildtype kidney culture glomeruli (P, podocyte; GBM, glomerular basement membrane). Arrow heads indicate localization of foot processes, arrows indicate slit diaphragms. (<b>F</b>) <i>Circletail</i> mutant mice, bearing a mutation in the <i>Scribble</i> gene, which results in a shortened protein lacking third and fourth c-terminal PDZ domains, die during late embryonic development. Kidneys of <i>Crc/Crc</i> mutant mice and control littermates where harvested for electron microscopy at E18.5. Transmission electron micrographs display podocytes with normal foot process architecture and slit-diaphragms and without any obvious abnormalities. Scale bars: 400 µm in (B), 20 µm in (C) and (D), 2 µm in (E) left and middle panel, 500 nm in (E) right panel, 2 µm in (F) upper panel, and 500 nm in (F) lower panel.</p