Mutations in MAPKBP1 Cause Juvenile or Late-Onset Cilia-Independent Nephronophthisis

Abstract

Nephronophthisis (NPH), an autosomal-recessive tubulointerstitial nephritis, is the most common cause of hereditary end-stage renal disease in the first three decades of life. Since most NPH gene products (NPHP) function at the primary cilium, NPH is classified as a ciliopathy. We identified mutations in a candidate gene in eight individuals from five families presenting late-onset NPH with massive renal fibrosis. This gene encodes MAPKBP1, a poorly characterized scaffolding protein for JNK signaling. Immunofluorescence analyses showed that MAPKBP1 is not present at the primary cilium and that fibroblasts from affected individuals did not display ciliogenesis defects, indicating that MAPKBP1 may represent a new family of NPHP not involved in cilia-associated functions. Instead, MAPKBP1 is recruited to mitotic spindle poles (MSPs) during the early phases of mitosis where it colocalizes with its paralog WDR62, which plays a key role at MSP. Detected mutations compromise recruitment of MAPKBP1 to the MSP and/or its interaction with JNK2 or WDR62. Additionally, we show increased DNA damage response signaling in fibroblasts from affected individuals and upon knockdown of Mapkbp1 in murine cell lines, a phenotype previously associated with NPH. In conclusion, we identified mutations in MAPKBP1 as a genetic cause of juvenile or late-onset and cilia-independent NPH.

Keywords: DNA damage; MAP kinase; MAPKBP1; WDR62; ciliopathy; digenism; kidney; mitotic spindle; nephronophthisis; retinitis pigmentosa.

Figure 1

Biallelic Mutations in MAPKBP1 Cause Nephronophthisis (A) Exon structure of MAPKBP1 cDNA. Positions of start codon (ATG) and stop codon (TGA) are indicated. (B) Domain structure of the protein. MAPKBP1 contains 12 WD repeats (green), located at the N terminus, a JNK-binding region located in the C-terminal part (red), and conserved coiled-coil domains at the C terminus (CC, purple). (C) Relation of four homozygous (H) and two compound heterozygous mutations (h) to exons and protein domains is indicated by black arrows. (D and E) Pedigrees (D) and chromatograms and segregation (E). Mutated nucleotides are shown above wild-type. In addition, individuals BN-21 and BN-23 also presented with retinitis pigmentosa (RP) linked to a homozygous mutation in PDE6A. See Figure S5. Segregation of the MAPKBP1 mutations was not examined in family NPH2139 due to the unavailability of the DNA of the affected brother and the parents.

Figure 2

Histological and Sonographic Kidney Lesions in Individuals with MAPKBP1 Mutations (A1) Periodic acid-Schiff staining of a kidney biopsy from individual BN-23 showing tubular atrophy (arrowheads), interstitial fibrosis (F), focal interstitial infiltration (Inf), and normal glomerulus (G). Scale bar represents 100 μm. (A2) Ultrastructure (transmission electron microscopy picture) from a tubule with abrupt transition from thin to thickened and disorganized basement membrane (arrow). Scale bar represents 5 μm. (A3) Example of a primary cilium found at the apical membrane of a tubular epithelial cell (arrows). Scale bar represents 250 μm. (B) Trichrome staining of a kidney biopsy from individual F1323-21 showing fibrosis, thickened basal membrane, as well as atrophic and dilated tubules. (C) PAS trichrome staining of a kidney biopsy from individual NPH2139-21 showing tubular atrophy (arrowheads in C1), interstitial fibrosis (F) and focal interstitial infiltration (Inf, C2), and sclerotic glomeruli (G, C2) surrounded by a thickened capsular basement membrane (arrows, C1 and C2). Scale bar represents 100 μm. (D1) Histological analysis of individual A3977-22 shows massive interstitial fibrosis and atrophic tubules. Scale bar represents 100 μm. (D2 and D3) Upon renal ultrasound, kidneys present small with narrowed parenchyma (white arrow), increased echogenicity, and single cysts on both sides (white asterisk). D2 right kidney, D3 left kidney.

Figure 3

MAPKBP1 Is Not Involved in Ciliogenesis (A and B) Serum-starved (24 hr) nontransfected RPE1 cells (A) or RPE1 cells transiently transfected (FuGENE [Promega]) with V5-tagged MAPKBP1 encoding plasmid (B) were fixed (paraformaldehyde; PFA) and stained for acetylated α-tubulin (AcTub, green, cilia; mouse monoclonal antibody [6-11-B-1, Sigma], dil 1/10,000) and for endogenous MAPKBP1 (A) (red; rabbit polyclonal [HPA030832, Sigma], dil 1/300) or overexpressed MAPKBP1 (B) (red, V5 rabbit polyclonal antibody [Sigma V8137], dil 1/10,000). Insets on the right show higher magnifications of representative cilia indicated by a white square in the corresponding images. Arrows indicate cilia, arrowheads cilium base region. (C–E) Serum-starved fibroblasts from controls (CT1 [unrelated healthy], CT2-BN-12 [mother of BN-22, heterozygous for the c.592C>T (p.Arg198^∗) mutation]) or from the affected individuals (BN-22, F1323-21, NPH309-21) were stained for acetylated-tubulin (AcTub, green) to identify cilia (C). Insets on the right show higher magnifications of representative cilia. Ciliogenesis (% of ciliated cells [D]) and length of cilia (E) were analyzed and quantified by ImageJ from three independent experiments (n > 50 cells for each experiment). ns,^∗p < 0.01, and ^∗∗∗p < 0.0001 were calculated via Dunn’s multiple comparison test after the analysis of variance ANOVA test. Note that cilia length in cells from individual BN-22 is not significantly different from that of cilia from cells of his mother (CT2-BN-12). Cilia in cells from individuals F1323-21 and NPH309-21 were not significantly different than in control cells (CT1) but longer than in cells from individuals of the BN family. Scale bars represent 5 μm.

Figure 4

MAPKBP1 Is Recruited to Mitotic Spindle Poles during Early Stages of Mitosis (A) Cycling RPE1 cells were fixed (PFA) and stained for MAPKBP1 (red) and for DNA with DAPI (blue). Arrows stress accumulation of MAPKBP1 staining at spindle poles in mitotic cells. The white arrowhead and star indicate representative ciliated and non-ciliated cells, respectively. Corresponding MAPKBP1 staining are shown in black and white in the lower panels. (B) Scheme indicating mitotic spindle poles (MSP, red) surrounding centrosomes (green) at each pole of the mitotic spindle (pink) during metaphase (chromosomes in blue). (C and D) Cycling RPE1 cells were fixed (PFA) and stained for MAPKBP1 (red) and either with p150 glued (MSP, green [C]; mouse monoclonal antibody [610473, BD Biosciences], dil 1/100) or acetylated α-tubulin (AcTub, spindle, green [D]) and γ-tubulin (γ-tub, centrioles, white [D]; goat polyclonal antibody [sc-7396, Santa Cruz], dil 1/2,000) together with DAPI (blue). Insets on the right show higher magnifications of representative MSP indicated by a white square in the corresponding images. Arrows and arrowheads point to MAPKBP1 staining at the MSP and to γ-tub, respectively. Scale bars represent 5 μm.

Figure 5

Localization of MAPKBP1 at Mitotic Spindle Poles Is Disturbed in Fibroblasts from Affected Individuals (A) Cycling RPE1 cells were fixed (PFA) and stained for endogenous MAPKBP1 (red) and WDR62 (green; mouse monoclonal [Clone 3G8, Sigma], dil 1/1,000) together with DAPI (blue). Representative pictures of cells in prometaphase and telophase are shown. Insets on the right show higher magnifications of representative MSP indicated by a white square in the corresponding images. (B–D) Cycling fibroblasts from control subjects (CT1 and CT2-BN-12) or from affected individuals (BN-22, F1323-21, NPH309-21) were stained for both MAPKBP1 (red) and WDR62 (green). Representative pictures of cells in metaphase are shown in (B). Arrows stress MSPs. Intensity of MAPKBP1 (C) and WDR62 (D) stainings at the mitotic spindle poles was quantified with ImageJ (3 independent experiments, n > 65 cells). ns, ^∗∗p < 0.001 and ^∗∗∗p < 0.0001 were calculated via Dunn’s multiple comparison test after the analysis of variance ANOVA test. Scale bars represent 5 μm.

Figure 6

Mutations in MAPKBP1 Affect JNK2 and/or WDR62 Binding HEK293 cells were transiently transfected (lipofectamine [ThermoFischer]) with plasmids encoding V5-tagged WT and mutant forms of MAPKBP1 and Myc-tagged WDR62 (A) or HA-tagged JNK2 (B), as indicated. Cells were lysed and immunoprecipitated with the anti-V5 antibody (rabbit polyclonal [Sigma, V8137]). Lysates and immunoprecipitates (IP) were analyzed by western blot as indicated (WB; V5: Mouse monoclonal clone SV5-Pk1 [AbD Serotec MCA-1360], dil 1/5,000; HA: mouse monoclonal, clone 16B12 [Biolegend, 901502], dil 1/5,000; MYC: mouse monoclonal, Ab-2 clone 9E10.3 [Fisher Scientific MS-139-P1], dil 1/5,000).

Figure 7

DNA Damage Response Is Increased in MAPKBP1 Mutant Cells (A) Fibroblasts from control subjects (CT1; CT2-BN-12) and affected individuals (BN-22, F1323-21, NPH309-21) were fixed and stained for phosphorylated γH2AX (red; mouse monoclonal, clone JBW301 [MILLIPORE 05-636], dil 1/200) together with DAPI (blue). Scale bar represents 5 μm. (B) The intensity of nuclear γH2AX staining was quantified from three independent experiments (n > 200). ns and ^∗∗∗p < 0.0001 were calculated via Dunn’s multiple comparison test after the analysis of variance ANOVA test. (C) Kidney biopsies from a control unrelated individual (CT3) and from the affected individual BN-23 were stained for γH2AX (green; rabbit polyclonal, dil 1/200). Scale bar represents 20 μm. (D and E) NIH 3T3 cells (fibroblasts) were stably transduced with lentiviral vectors expressing either control scrambled (shScr) or Mapkbp1 targeting shRNA (shMapkbp1#5; Figures S16 and S17). Immunoblots show the expression of different components of DNA damage response signaling including γH2AX (Cell Signaling, rabbit polyclonal, dil 1/1,000); (D) and phospho-53BP1 (Cell Signaling, rabbit polyclonal, dil 1/1,000); (E).