Defects in ciliary localization of Nek8 is associated with cystogenesis

Abstract

Mutations in the human NIMA (Never in Mitosis gene A)-related kinase 8 (Nek8) are associated with a rare form of the juvenile renal cystic disease, nephronophthisis type 9, and mutations in murine Nek8 cause renal cysts in jck mice. Cystogenesis involves dysfunctional ciliary signaling, and we have previously reported that Nek8 localizes to the primary cilium in mouse kidney epithelial cells. We now report that in developing mouse kidney, Nek8 is detected in the cilia of a subset of ureteric-bud-derived tubules at embryonic day (E)15.5. An increasing proportion of ureteric-bud-derived tubules express ciliary Nek8 until E18.5. Postnatal day 1 and 7 Nek8 is observed with equal frequency in both ureteric-bud and non-ureteric-bud-derived tubules. To investigate the cell biological consequences of kinase-deficient and jck mutant forms of Nek8, we transiently expressed green fluorescent protein (GFP)-tagged constructs in vitro. Mutations in the kinase and C-terminal domains of Nek8 adversely affected ciliary targeting but did not affect ciliogenesis or ciliary length. Consistent with these in vitro observations, kidneys from homozygous jck mice revealed reduced ciliary expression of Nek8 compared with kidneys from heterozygous (unaffected) mice. These data indicate that the ciliary localization of Nek8 in a subset of ureteric-bud-derived kidney tubules is essential for maintaining the integrity of those tubules in the mammalian kidney.

Fig. 1

Variable spatial expression of ciliary NIMA (Never in Mitosis gene A)-related kinase 8 (Nek8) in developing mouse kidney. Paraffin-embedded sections of CD1/129 mouse embryonic kidney were stained with 4′,6-diamidino-2-phenylin-dole (DAPI) for nuclei (blue), anti-acetylated tubulin for cilia (red), and anti-Nek8 for endogenous protein (green). a A medullary tubule from embryonic day (E)18.5 with Nek8 colocalizing with the majority of cilia (arrows). b A cortical tubule from E16.5 with Nek8 absent in cilia (arrowheads). c Tubules from E16.5. Nek8 colocalizes with the majority of cilia in a medullary tubule (arrows and bottom inset) but exhibits no colocalization with cilia in an adjacent tubule (arrowhead and top inset). Insets are x3 magnification

Fig. 2

Tubules of ureteric-bud origin express NIMA (Never in Mitosis gene A)-related kinase 8 (Nek8). Paraffin-embedded sections of CD1/129 mouse kidneys stained with anti-Nek8 antibody, markers denoting kidney cell types, and 4′,6-diamidino-2-phenylindole (DAPI) (blue). a An example of an embryonic day (E)18.5 tubule in the medulla labeled with fluorescein isothiocyanate (FITC)-co6njugated Dolichos biflorus agglutinin (DBA) (green) expressing luminal Nek8 (red). b An example of an E18.5 tubule in the medulla labeled with anti-calbindin (red) expressing luminal Nek8 (green). c Kidney sections at different developmental stages were stained with anti-Nek8 and anti-calbindin antibodies. Shown are percentages of Nek8-positive tubules of ureteric-bud (UB) origin (grey diamonds) and tubules of ureteric-bud origin that have ciliary Nek8 (black squares). Error bars = standard error of mean

Fig. 3

Overexpression of mutant forms of NIMA (Never in Mitosis gene A)-related kinase 8 (Nek8) shows differential localization. Transient overexpression of N-terminal green fluorescent protein (GFP)-tagged mouse Nek8 cDNA in mouse inner medullary collecting duct (IMCD-3) cells. a Western blot of transfected cell extracts using anti-GFP antibody shows expected band size of 102kDa for the GFP-Nek8 fusion proteins. b–d Cells were fixed and stained for anti-acetylated tubulin for cilia and anti-gamma tubulin for centrosomes (both red) and 4′,6-diamidino-2-phenylindole (DAPI) to indicate the nucleus (blue). b Wild-type GFP-Nek8 localizes to the cytoplasm, centrosomes and cilia (arrow). c GFP-kinase-deficient (K33M) Nek8 localizes to the cytoplasm but not centrosomes and cilia (arrowhead) in this example. d GFP-jck Nek8 localizes to the cytoplasm and centrosomes but not cilia (arrowhead) in this example. Single cells are shown as representatives. b’, c’, and d’ are higher magnification images of the cells shown in b, c, and d

Fig. 4

In vitro expression of green fluorescent protein (GFP)-NIMA (Never in Mitosis gene A)-related kinase 8 (Nek8) mutations. a Quantification of differential subcellular localization of GFP-tagged Nek8 constructs. Transiently transfected ciliated, mononucleate inner medullary collecting duct (IMCD)-3 cells with low to medium levels of expression were quantified for localization of various forms of GFP-Nek8 to cilia, centrosomes, a perinuclear region, and/or cell periphery. The total number of cells counted from three independent experiments was n=45, 50, 65, and 40, respectively, for GFP alone, wt, K33M, and jck. Error bars = standard error of mean (SEM). b Overexpression of Nek8 has no effect on overall ciliogenesis. Transfected and untransfected IMCD-3 cells expressing various forms of GFP-Nek8 were categorized as ciliated with a pair of centrioles, lacking cilia with a pair of centrioles, undergoing mitosis, multinucleate, or having an abnormal number of centrioles, including none at all. The total number of cells counted from three independent experiments was n=950, 250, 300, 300, and 300, respectively for untransfected, GFP alone, wt, K33M, and jck. Error bars = SEM. * p< 0.01, ** p<0.001

Fig. 5

Differential NIMA (Never in Mitosis gene A)-related kinase 8 (Nek8) ciliary localization in diseased jck kidney. Paraffin-embedded sections of postnatal day 7 mouse kidneys from a wild type (+/+), b heterozygous (+/jck), and c, d homozygous jck (jck/jck) mice were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue), anti-acetylated tubulin for cilia (red), and anti-Nek8 for endogenous protein (green). The heterozygous and homozygous mice were litter mates. Shown are ciliated tubules within the medulla. Arrows indicate Nek8 localization, and arrowheads indicate lack of Nek8 in cilia

Fig. 6

NIMA (Never in Mitosis gene A)-related kinase 8 (Nek8) expression in ureteric-bud-derived tubules and ciliary lengthening in jck kidneys. a, b Paraffin-embedded sections of postnatal day (P)7 mouse kidney from heterozygous (+/jck) and homozygous (jck/jck) jck mouse litter mates were costained with anti-calbindin D-28K and anti-Nek8 antibodies. a Percentages indicate calbindin D-28K-positive tubules that express Nek8 in luminal cilia. b Percentages indicate Nek8-positive tubules that are of ureteric-bud origin. c P7 heterozygous (+/jck) and homozygous (jck/jck) jck mouse litter mates were costained with anti-acetylated tubulin and anti-Nek8 antibodies. The two bars on the left show the average length of Nek8-expressing cilia of non-cystic +/jck and jck/jck tubules. The two bars on the right are the average lengths of cilia of jck/jck cysts, where the cilia either express or do not express Nek8. *p<0.05