Mutations in multiple components of the nuclear pore complex cause nephrotic syndrome
Daniela A. Braun, … , Mustafa K. Khokha, Friedhelm Hildebrandt
Daniela A. Braun, … , Mustafa K. Khokha, Friedhelm Hildebrandt
Published September 4, 2018
Citation Information: J Clin Invest. 2018;128(10):4313-4328. https://doi.org/10.1172/JCI98688.
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Research Article Genetics Nephrology

Mutations in multiple components of the nuclear pore complex cause nephrotic syndrome

Abstract

Steroid-resistant nephrotic syndrome (SRNS) almost invariably progresses to end-stage renal disease. Although more than 50 monogenic causes of SRNS have been described, a large proportion of SRNS remains unexplained. Recently, it was discovered that mutations of NUP93 and NUP205, encoding 2 proteins of the inner ring subunit of the nuclear pore complex (NPC), cause SRNS. Here, we describe mutations in genes encoding 4 components of the outer rings of the NPC, namely NUP107, NUP85, NUP133, and NUP160, in 13 families with SRNS. Using coimmunoprecipitation experiments, we showed that certain pathogenic alleles weakened the interaction between neighboring NPC subunits. We demonstrated that morpholino knockdown of nup107, nup85, or nup133 in Xenopus disrupted glomerulogenesis. Re-expression of WT mRNA, but not of mRNA reflecting mutations from SRNS patients, mitigated this phenotype. We furthermore found that CRISPR/Cas9 knockout of NUP107, NUP85, or NUP133 in podocytes activated Cdc42, an important effector of SRNS pathogenesis. CRISPR/Cas9 knockout of nup107 or nup85 in zebrafish caused developmental anomalies and early lethality. In contrast, an in-frame mutation of nup107 did not affect survival, thus mimicking the allelic effects seen in humans. In conclusion, we discovered here that mutations in 4 genes encoding components of the outer ring subunits of the NPC cause SRNS and thereby provide further evidence that specific hypomorphic mutations in these essential genes cause a distinct, organ-specific phenotype.

Authors

Daniela A. Braun, Svjetlana Lovric, David Schapiro, Ronen Schneider, Jonathan Marquez, Maria Asif, Muhammad Sajid Hussain, Ankana Daga, Eugen Widmeier, Jia Rao, Shazia Ashraf, Weizhen Tan, C. Patrick Lusk, Amy Kolb, Tilman Jobst-Schwan, Johanna Magdalena Schmidt, Charlotte A. Hoogstraten, Kaitlyn Eddy, Thomas M. Kitzler, Shirlee Shril, Abubakar Moawia, Kathrin Schrage, Arwa Ishaq A. Khayyat, Jennifer A. Lawson, Heon Yung Gee, Jillian K. Warejko, Tobias Hermle, Amar J. Majmundar, Hannah Hugo, Birgit Budde, Susanne Motameny, Janine Altmüller, Angelika Anna Noegel, Hanan M. Fathy, Daniel P. Gale, Syeda Seema Waseem, Ayaz Khan, Larissa Kerecuk, Seema Hashmi, Nilufar Mohebbi, Robert Ettenger, Erkin Serdaroğlu, Khalid A. Alhasan, Mais Hashem, Sara Goncalves, Gema Ariceta, Mercedes Ubetagoyena, Wolfram Antonin, Shahid Mahmood Baig, Fowzan S. Alkuraya, Qian Shen, Hong Xu, Corinne Antignac, Richard P. Lifton, Shrikant Mane, Peter Nürnberg, Mustafa K. Khokha, Friedhelm Hildebrandt

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Figure 4

CRISPR/Cas9–mediated knockout of NUP107, NUP85, or NUP133 induces filopodia formation and increases active Cdc42 in human podocytes.

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CRISPR/Cas9–mediated knockout of NUP107, NUP85, or NUP133 induces filopo...
Immortalized human podocytes underwent lentiviral transduction with a plasmid expressing a Cas9-GFP fusion construct under the control of a doxycycline-inducible promoter and a single gRNA. For each gene, 2 different cell lines were generated expressing gRNAs against NUP107 (targeting exon 4 or 11), NUP85 (targeting exon 1 or 15), or NUP133 (targeting exon 1 or 5), respectively. Experiments were performed 72 hours after induction of Cas9 expression with doxycycline (1 μg/ml). (A) We stained immortalized human podocytes expressing empty vector (MOCK) or individual gRNAs targeting NUP107, NUP85, or NUP133 with phalloidin to detect F-actin fibers. Podocytes that had either 3 actin-based protrusions or 1 filamentous protrusion of more than one-quarter of the cell body were quantified as “filopodia positive.” Representative images showing “filopodia-negative” control cells and NUP107-, NUP85-, or NUP133-knockout podocytes that exhibited filopodia (arrowheads). Scale bars: 25 μm. The result was confirmed in 3 independent experiments. (B) Quantification of approximately 50 cells for each condition resulted in 22% of MOCK-expressing cells with filopodia (11/50), in contrast to 58% (29/50) for NUP107 gRNA exon 4 (ex4) and 46% (23/50) for NUP107 gRNA ex11; 50% (25/50) for NUP85 gRNA ex1 and 54% (30/56) for NUP85 gRNA ex15; and 44% (22/50) for NUP133 gRNA ex1 and 38% (19/50) for NUP133 gRNA ex5. Note that knockout podocytes show increased filopodia formation. (C) Using the colorimetric G-Lisa Cdc42 Activation Assay Biochem Kit (Cytoskeleton), we demonstrate an increase in the active state of Cdc42 following CRISPR/Cas9–mediated knockout of NUP107, NUP85, or NUP133 in human podocytes. Data points represent 3 independent experiments (highlighted in different colors) and are displayed with mean and SD. P values calculated by 1-way ANOVA are indicated in the figure as *P < 0.05; **P < 0.01.