2016
DOI: 10.1172/jci.insight.86177
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A flexible, multilayered protein scaffold maintains the slit in between glomerular podocytes

Abstract: Vertebrate life critically depends on renal filtration and excretion of low molecular weight waste products. This process is controlled by a specialized cell-cell contact between podocyte foot processes: the slit diaphragm (SD). Using a comprehensive set of targeted KO mice of key SD molecules, we provided genetic, functional, and high-resolution ultrastructural data highlighting a concept of a flexible, dynamic, and multilayered architecture of the SD. Our data indicate that the mammalian SD is composed of NE… Show more

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Cited by 75 publications
(90 citation statements)
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“…A conditional knockin mouse line of the Neph2 locus was generated by homologous recombination in 129 Sv/J (129S7/SvEvBrd) embryonic stem cells. Briefly, exon 2 of the endogenous Neph2 locus was replaced by a cDNA consisting of exon 2‐17 flanked by loxP sites that was followed by an internal ribosomal entry site to enable expression of beta‐galactosidase from the bacterial lacZ gene . Resulting chimaeras were tested for germline transmission, interbred with C57BL/6 mice and the positive selection neomycin/kanamycin cassette removed by mating with a flip‐recombinase expressing mouse line.…”
Section: Methodsmentioning
confidence: 99%
See 1 more Smart Citation
“…A conditional knockin mouse line of the Neph2 locus was generated by homologous recombination in 129 Sv/J (129S7/SvEvBrd) embryonic stem cells. Briefly, exon 2 of the endogenous Neph2 locus was replaced by a cDNA consisting of exon 2‐17 flanked by loxP sites that was followed by an internal ribosomal entry site to enable expression of beta‐galactosidase from the bacterial lacZ gene . Resulting chimaeras were tested for germline transmission, interbred with C57BL/6 mice and the positive selection neomycin/kanamycin cassette removed by mating with a flip‐recombinase expressing mouse line.…”
Section: Methodsmentioning
confidence: 99%
“…replaced by a cDNA consisting of exon 2-17 flanked by loxP sites that was followed by an internal ribosomal entry site to enable expression of beta-galactosidase from the bacterial lacZ gene. 18 Resulting chimaeras were tested for germline transmission, interbred with C57BL/6 mice and the positive selection neomycin/kanamycin cassette removed by mating with a flip-recombinase expressing mouse line. Resulting mice (Neph2 flox/+ ) were then bred to a Cytomegalovirus (CMV)-promoter driven cre-transgenic mouse line in C57BL/6 background to generate a constitutive knockout (Neph2 Δ/Δ , ie, Neph2 flox/ flox ; CMV.cre tg/+ ).…”
mentioning
confidence: 99%
“…Several proteins are known to participate in maintaining the structural integrity and composition of slit diaphragm including NEPHRIN and NEPH1, whose genetic defects in humans and mice lead to FSGS, which is characterized by podocyte dysfunction and nephrotic range proteinuria [19,27,[34][35][36][37][38]. The prevailing hypotheses suggest that the extracellular domains of NEPHRIN and NEPH1 constitute the structural framework of slit diaphragm, thus, maintaining its structural integrity [17,37,39,40].…”
Section: Discussionmentioning
confidence: 99%
“…Neph1 mice were developed by Tobias Huber (33) and genotyped using the following primer pairs: 5′-GAAAAGAGAGATACGGAGAACCGAGGG-3′ (forward, common), 5′-AGAACACTAAGGCAGCAAAAGAGAAGACGA-3′ (reverse, wild type), and 5′-CACAAAGCTGAGAAGAAAGGAAACCGT-3′ (reverse, knock-out). Expected product lengths are 750 bp for wild type allele and 600 bp for knock-out allele.…”
Section: Methodsmentioning
confidence: 99%