NEK8 mutations affect ciliary and centrosomal localization and may cause nephronophthisis

Abstract

Nephronophthisis, an autosomal recessive kidney disease, is the most frequent genetic cause of chronic renal failure in the first 3 decades of life. Causative mutations in 8 genes (NPHP1-8) have been identified, and homologous mouse models for NPHP2/INVS and NPHP3 have been described. The jck mouse is another model of recessive cystic kidney disease, and this mouse harbors a missense mutation, G448V, in the highly conserved RCC1 domain of Nek8. We hypothesized that mutations in NEK8 might cause nephronophthisis in humans, so we performed mutational analysis in a worldwide cohort of 588 patients. We identified 3 different amino acid changes that were conserved through evolution (L330F, H425Y, and A497P) and that were absent from at least 80 ethnically matched controls. All 3 mutations were within RCC1 domains, and the mutation H425Y was positioned within the same RCC1 repeat as the mouse jck mutation. To test the functional significance of these mutations, we introduced them into full-length mouse Nek8 GFP-tagged cDNA constructs. We transiently overexpressed the constructs in inner medullary collecting duct cells (IMCD-3 cell line) and compared the subcellular localization of mutant Nek8 to wild-type Nek8. All mutant forms of Nek8 showed defects in ciliary localization to varying degrees; the H431Y mutant (human H425Y) was completely absent from cilia and the amount localized to centrosomes was decreased. Overexpression of these mutants did not affect overall ciliogenesis, mitosis, or centriole number. Our genetic and functional data support the assumption that mutations in NEK8 cause nephronophthisis (NPHP9), adding another link between proteins mutated in cystic kidney disease and their localization to cilia and centrosomes.

Figure 1.

Human mutations in NEK8 and evolutionary conservation. (A) Chromatograms of 3 different NEK8 mutations detected in 3 individuals with nephronophthisis. Family number, amino acid sequence change, and mutated nucleotide are given above sequence traces. Wild-type sequences are shown below mutated sequences. Reading frame is indicated by underlining codon triplets in the upper panel, and mutated nucleotides are indicated by an arrowhead. The two mutations L330F and A497P occurred in the heterozygous and H425Y in the homozygous state. All mutations were absent from at least 80 healthy control individuals. (B) Alignment of the Nek8 protein sequence of regions mutated in patients with homologues from various species. Amino acid residues that are within the same chemical group are coded in the same color. Mutated amino acids are indicated with arrowheads. The amino acid sequences are aligned with those of Homo sapiens (H.s.), Mus musculus (M.m), Xenopus tropicalis (X.t.), Danio rerio (D.r.), and Ciona intestinalis (C.i.).

Figure 2.

(A) Quantification of differential subcellular localization of GFP-tagged Nek8 constructs. Transiently transfected ciliated, mononucleate IMCD-3 cells with low to medium levels of expression were quantified for ciliary, centrosomal, perinuclear, and cell peripheral localization of GFP-mutant Nek8. The total number of cells counted from 3 independent experiments was n = 45, 50, 55, 55, and 45 for GFP alone, wild-type (WT), L336F, H431Y, and A503P, respectively. Error bars = SEM. Note that ciliary localization was significantly reduced for mutant constructs L336F and H431Y compared with the wild-type. Centrosomal localization was significantly reduced for mutant H431Y. Statistical significance was assessed using T-test assuming 2-sample unequal variances with 2-tailed probability. *P = 0.01, **P < 0.001. (B) Overexpression of Nek8 has no effect on ciliogenesis. Transfected and untransfected cells were categorized as ciliated with a pair of centrioles, lacking cilia with a pair of centrioles, undergoing mitosis, multinucleate, or having an aberrant number of centrioles, including none at all. n = total number of cells counted from three independent experiments. The total number of cells counted from three independent experiments was n = 1095, 150, 300, 250, 300, and 258 for untransfected, GFP alone, WT, L336F, H431Y, and A503P, respectively. Error bars = SEM.

Figure 3.

Mutant forms of Nek8 have defects in subcellular localization. Transient overexpression of N-terminal GFP-tagged mouse Nek8 cDNA in inner medullary collecting duct (IMCD-3) cells. (A) Cells were stained for anti-acetylated tubulin (red) to visualize cilia (arrows) and anti-γ-tubulin (red) to mark centrosomes (arrowheads) and DAPI to indicate the nucleus (blue). Wild-type GFP-Nek8 (green) localizes to the cytoplasm, centrosomes (arrowheads), and cilia (arrows). (B through E) Expression of GFP alone and mutant GFP-Nek8 constructs. Note that amino acid numbering differs in murine mNek8 and human NEK8 (Table 1). Cells were fixed and stained for anti-acetylated tubulin for cilia (red) and for anti-γ-tubulin for centrosomes (blue) and DAPI (blue). (B) GFP alone localizes to the cytoplasm, but not to cilia or centrosomes. (C) The GFP-L336F Nek8 mutant was detected in the cytoplasm and centrosomes, but not in cilia. (D) The GFP-H431Y Nek8 mutant is not associated with either centrosomes or cilia, in this example. (E) GFP-A503P localizes to the cytoplasm and centrosomes, but not to cilia in this example. Insets are ×2 original magnification in panels B and D and ×1.5 magnification in panels C and E.