Biallelic Mutations in Nuclear Pore Complex Subunit NUP107 Cause Early-Childhood-Onset Steroid-Resistant Nephrotic Syndrome

Abstract

The nuclear pore complex (NPC) is a huge protein complex embedded in the nuclear envelope. It has central functions in nucleocytoplasmic transport, nuclear framework, and gene regulation. Nucleoporin 107 kDa (NUP107) is a component of the NPC central scaffold and is an essential protein in all eukaryotic cells. Here, we report on biallelic NUP107 mutations in nine affected individuals who are from five unrelated families and show early-onset steroid-resistant nephrotic syndrome (SRNS). These individuals have pathologically focal segmental glomerulosclerosis, a condition that leads to end-stage renal disease with high frequency. NUP107 is ubiquitously expressed, including in glomerular podocytes. Three of four NUP107 mutations detected in the affected individuals hamper NUP107 binding to NUP133 (nucleoporin 133 kDa) and NUP107 incorporation into NPCs in vitro. Zebrafish with nup107 knockdown generated by morpholino oligonucleotides displayed hypoplastic glomerulus structures and abnormal podocyte foot processes, thereby mimicking the pathological changes seen in the kidneys of the SRNS individuals with NUP107 mutations. Considering the unique properties of the podocyte (highly differentiated foot-process architecture and slit membrane and the inability to regenerate), we propose a "podocyte-injury model" as the pathomechanism for SRNS due to biallelic NUP107 mutations.

Figure 1

Genetic Analysis and Clinical Course of Early-Onset SRNS in Affected Individuals with NUP107 Mutations (A) Familial pedigrees and NUP107 mutations. Mutant alleles are colored in red. WT indicates the wild-type allele. Filled and unfilled symbols represent affected and unaffected members, respectively. (B) Clinical course of the affected individuals. The onset of renal symptoms and diagnosis of ESRD are represented by squares and crosses, respectively. Blue and red horizontal bars indicate the period leading to ESRD and the period before completed ESRD, respectively. SRNS-1 II-2 died from a viral infection before the advent of ESRD.

Figure 2

Kidney Histopathology of Affected Individuals with Biallelic NUP107 Mutations (A–C) Light micrographs of kidney biopsy specimens from SRNS-TWH II-1. (A) A low-power view (periodic acid-Schiff stain, 100× magnification) of two representative abnormal glomeruli (arrows). Half of the glomerulus is sclerosed (arrowheads). (B and C) Enlarged images (periodic acid methenamine silver stain, 400× magnification) show the collapse of glomerular tufts with hypertrophy and hyperplasia of the glomerular epithelial cells that fill the urinary space. Tubular injury accompanying atrophy of epithelia and interstitial fibrosis is noted. (D–F) Electron micrographs of biopsy specimens from SRNS-2 II-1 (D), SRNS-2 II-3 (E), and SRNS-2 II-4 (F). Effacement of podocyte foot processes and some mesangial expansion with sub-endothelial electron-dense deposits are apparent. The thickness of the glomerular basement membrane appears normal and shows no evidence of splitting, lamellation, or fragmentation, thereby excluding the possibility of a primary basement-membrane defect. Accumulation of storage materials and dysmorphic mitochondria were not found in the podocyte cytoplasm. Abbreviations are as follows: E, endothelial cell; M, mesangial cell; P, podocyte; Pa, papillary epithelia. Arrowheads indicate effacement of podocyte foot processes, yellow arrows represent electron dense deposits, black arrows show flattened podocyte foot processes, and yellow asterisks show paramesangial deposits. Scale bars represent100 μm (A), 40 μm (B and C), 2 μm (D and E), and 5 μm (F).

Figure 3

Decreased Intermolecular Interactions between NUP107 and NUP133 (A) In vitro protein-protein binding assay of altered NUP107 with NUP133. The FLAG-tagged NUP133 mixed with biotinylated altered NUP107 proteins was subjected to a pull-down assay with streptavidin magnetic beads. The bound proteins were separated by SDS-PAGE and then detected with an anti-FLAG antibody or with streptavidin-horseradish peroxidase. The corresponding protein inputs are shown in the middle and bottom panels. (B) Evaluation of the interaction between NUP107 and NUP133 with the use of wild-type NUP107 and its alterations. Wild-type GFP-NUP107 or its alterations were transiently produced in HeLa cells and precipitated with an anti-GFP antibody. The NUP107-NUP133 interaction was analyzed via immunoblotting using the antibodies indicated. (C) Subcellular localization of NUP107 or its alterations. For visualizing localization of altered or wild-type GFP-NUP107 in HeLa cells, the cells were fixed and stained with a MAb414 antibody recognizing the NPC on the nuclear envelopes. Scale bars represent 20 μm. The following abbreviation is used: WT, wild-type.