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Kidney Week

Abstract: FR-PO1020

Precise Modeling of NUP93 Highlights Downstream Pathways

Session Information

Category: Genetic Diseases of the Kidney

  • 1002 Genetic Diseases of the Kidney: Non-Cystic

Authors

  • Bull, Katherine R., University of Oxford, Oxford, United Kingdom
  • Saleem, Moin, University of Bristol, Bristol, United Kingdom
  • Cornall, Richard John, University of Oxford, Oxford, United Kingdom
Background

Variants in nuclear pore complex components, including NUP93, cause steroid resistant nephrotic syndrome (SRNS), but underlying mechanisms are not understood. NUP93 null mutations are likely to be lethal.
Genetic validation and investigation requires tools to link genetics to cellular function. Conventional methods in immortalized podocytes fail to model non-null variants under physiological promotors, and are limited by transfection resistance. To better mimic human disease we applied CRISPR/Cas9 to model a NUP93 variant.

Methods

Lentivirus was used to generate human podocytes constitutively expressing Cas9 and introduce guide sequences. Motility was assessed by scratch assay.
150 bp PE polyA RNA-Seq (HiSeq4000) was analysed in R, fitting a quasi likelihood generalised linear model with edgeR.

Results

Lentiviral podocyte CRISPR is efficient, disrupting 43% of alleles, 98% after single cell selection.
We modeled a homozygous mutation in exon 13 of NUP93, identified in a child with SRNS. Edited podocytes had NUP93 protein but reduced motility.
We performed RNA-Seq on NUP93, wildtype and two Wilms Tumor 1 (WT1) podocyte lines. WT1 requires cofactor translocation; the WT1 transcriptomes clustered, but were distant from NUP93, suggesting NUP93 is not essential for WT1 function.
Comparing NUP93 to wildtype, 3011 genes were significantly differentially expressed. Gene ontology analysis highlighted cell cycle, proliferation and interferon signalling. Highly overexpressed genes included ID2, which regulates proliferation and is repressed by SMAD4. Since NUP93 facilitates SMAD4 nuclear transport, this suggests a pathway.

Conclusion

Lentiviral CRISPR efficiently induces mutation in immortalized podocytes, providing a new technique for genetic manipulation of this cell line. Using this approach we modeled human SRNS disease due to aberrant NUP93 and highlight potential downstream targets.

Funding

  • Private Foundation Support