Biallelic Expression of Mucin-1 in Autosomal Dominant Tubulointerstitial Kidney Disease: Implications for Nongenetic Disease Recognition

Abstract

Background: Providing the correct diagnosis for patients with tubulointerstitial kidney disease and secondary degenerative disorders, such as hypertension, remains a challenge. The autosomal dominant tubulointerstitial kidney disease (ADTKD) subtype caused by MUC1 mutations (ADTKD-MUC1) is particularly difficult to diagnose, because the mutational hotspot is a complex repeat domain, inaccessible with routine sequencing techniques. Here, we further evaluated SNaPshot minisequencing as a technique for diagnosing ADTKD-MUC1 and assessed immunodetection of the disease-associated mucin 1 frameshift protein (MUC1-fs) as a nongenetic technique.

Methods: We re-evaluated detection of MUC1 mutations by targeted repeat enrichment and SNaPshot minisequencing by haplotype reconstruction via microsatellite analysis in three independent ADTKD-MUC1 families. Additionally, we generated rabbit polyclonal antibodies against MUC1-fs and evaluated immunodetection of wild-type and mutated allele products in human kidney biopsy specimens.

Results: The detection of MUC1 mutations by SNaPshot minisequencing was robust. Immunostaining with our MUC1-fs antibodies and an MUC1 antibody showed that both proteins are readily detectable in human ADTKD-MUC1 kidneys, with mucin 1 localized to the apical membrane and MUC1-fs abundantly distributed throughout the cytoplasm. Notably, immunohistochemical analysis of MUC1-fs expression in clinical kidney samples facilitated reliable prediction of the disease status of individual patients.

Conclusions: Diagnosing ADTKD-MUC1 by molecular genetics is possible, but it is technically demanding and labor intensive. However, immunohistochemistry on kidney biopsy specimens is feasible for nongenetic diagnosis of ADTKD-MUC1 and therefore, a valid method to select families for further diagnostics. Our data are compatible with the hypothesis that specific molecular effects of MUC1-fs underlie the pathogenesis of this disease.

Keywords: CKD; IF/TA; MCKD; TIN; interstitial nephritis.

Graphical abstract

Figure 1.

Expression and localization of mucin 1 in human kidney. (A) Immunohistochemical detection of mucin 1 in kidney tissues of healthy controls and a patient with ADTKD-MUC1 (ADTKD-0034) with two independent antibodies ([C-term]: ab80952 and [VNTR]: VU4H5). (B) Immunofluorescent staining of healthy control kidney shows partial colocalization of mucin 1 (red), stained with the VNTR mucin 1 antibody and Tamm Horsfall protein (THP; green), an established kidney tubule marker of the thick ascending limb. Arrows indicate two tubules with coexpression of both proteins. Asterisks mark two tubular segments that solely express mucin 1.

Figure 2.

Families with ADTKD selected for MUC1 mutation confirmation on the basis of haplotype analysis. Black symbols indicate affected individuals, white symbols indicate unaffected status, and slashed symbols indicate deceased individuals. Generations are numbered in roman, and individuals are numbered in Arabic numerals. DNA for in vitro analysis was available from all haplotype-equipped individuals, and they were subjected to microsatellite-based haplotype reconstruction (six individuals in family A-11, 34 individuals in family A-29, and ten individuals in family A-30). Bars in magenta represents the intrafamilial mutation-carrying haplotypes. The C duplication in mutated MUC1 VNTRs is indicated with a C in haplotype bars. For visual clearness, only the mutation-carrying haplotypes are colored in family A-29.

Figure 3.

Specific detection of overexpressed and endogenous MUC1-fs with pAb3-fs via immunoblot. (A) Scheme of the wild-type and mutated allele products as they occur in patients with ADTKD-MUC1 (dark blue boxes represent the wild-type VNTR motif, and red boxes represent the frameshift motif of the mutated MUC1). The arrow indicates the distinct VNTR repeat where the cytosine insertion (+C) has occurred, leading to the novel MUC1-fs from that point onward that terminates in a premature STOP. Also, the recognition sites of the MUC1 antibodies used (VU4H5, pAbx-fs, and ab80952) are indicated in the color corresponding to their binding sites. The light blue boxes indicate the N-terminal end of protein, the gold box indicates the C-terminal end of the MUC1 wild-type protein, and the green box indicates the novel C-terminal end of MUC1-fs. C, C-terminal; N, N-terminal. (B) Immunoblots showing detection of MUC1-fs protein after transient transfection of HeLa cells with the empty backbone vector pcDNA3 (Ø), an MUC1 wild-type cDNA harboring 22 tandem repeats in its VNTR (WT), and an MUC1 frameshift cDNA harboring 22 frameshifted tandem repeats in its VNTR (FS). Actin was used as a loading control. In vitro transcribed and translated (IVTT) reticulocyte lysates generated from linearized plasmids (WT and FS) were blotted and incubated with either MUC1-fs (pAb3-fs) or VU4H5 (WT, VNTR) antibody. (C) pAb3-fs and VU4H5 detect identical signals in immunoblotting lysates from human urinary primary tubular cells (hUPTs) generated from the urine of three patients with ADTKD-MUC1 (ADTKD-0061, ADTKD-0014, and ADTKD-0071) corresponding to families A-14, A-11, and A-43, respectively. Single-molecule real time sequencing provided knowledge of the number of VNTRs as well as the position of the duplication mutation (dupC), which are depicted schematically below (blue represents the wild type, and red represents mutated repeats of the VNTR from the affected family members). Asterisks indicate the migrating species that we regard as MUC1-fs. (D) The individual lanes on the membranes of the three indicated ADTKD-MUC1 families were cut in half and incubated with either pAb3-fs or VU4H5 antibody. The bands regarded as specific for MUC1-fs are indeed recognized by both antibodies. (E) Four separate siRNAs for MUC1 (siMUC1_5, siMUC1_7, siMUC1_10, and siMUC1_11) successfully reduced levels of MUC1-fs protein in lysates of hUPTs after being subjected to siRNA knockdown; “oligofectamine only” and “siGFP” were implied as controls. α-Tubulin was blotted as a loading control.

Figure 4.

Immunodetection of MUC1-fs in human kidneys. (A) Immunohistochemical staining of MUC1-fs in human kidneys compared with the serum of the corresponding rabbit before immunization (preimmune) and secondary antibody only (ADTKD-0034). (B) Immunohistochemical staining of mucin 1 and MUC1-fs in human kidney biopsies (VU4H5 and pAb3-fs, respectively) from living donors before transplantation (healthy kidney) or fibrotic kidneys, where no specific MUC1-fs expression could be detected. Biopsy from a patient with ADTKD-MUC1 (ADTKD-0042) serves as positive control for staining. (C) Immunohistochemical staining of serial sections of an ADTKD-MUC1 kidney biopsy (ADTKD-0038) for MUC1-fs with preadsorption of pAb3-fs with “no peptide,” nonspecific “control peptide” (hSPAG4 peptide used for generation of hSPAG4 antibody), or frameshift peptide used for immunization against MUC1-fs (“MUC1-fs peptide”).

Figure 5.

Mucin 1 and MUC1-fs expression in patients with ADTKD-MUC1. Immunohistochemical detection of MUC1-fs and mucin 1 expression in affected members of three ADTKD-MUC1 families (A-29 [ADTKD-0032], A-30 [ADTKD-0043] and A-33 [ADTKD-0048]). Family A-32 (ADTKD-UMOD) serves as a negative control; it shows no MUC1-fs expression, but it does show mucin 1 expression. *The nomenclature of renamed families as cited in ref. .

Figure 6.

Distinct biallelic expression of mucin 1 and MUC1-fs in patients with ADTKD-MUC1. (A) Immunohistochemical detection of mucin 1 (C-term) and MUC1-fs (pAb3-fs) in a patient with ADTKD-MUC1 (ADTKD-0075). Preimmune serum serves as a control. (B) Immunofluorescent detection of mucin 1 (C-term) and MUC1-fs in tubules of a patient with ADTKD-MUC1 (ADTKD-0034; green, MUC1-fs; red, wild-type mucin 1).

Figure 7.

Identification of patients with ADTKD-MUC1 by unbiased tissue array screening. (A) Immunohistochemical identification of two individual patients with ADTKD-MUC1 (ADTKD-0024 and ADTKD-0026) on a randomly spotted kidney array, applying pAb3-fs for detection. Array was spotted on two slides containing 85 individually spotted kidney samples from 30 kidney nephrectomies, usually in triplicates, dating from 2005 to 2010. “Slide 1” with the two positive individuals is depicted. “Slide 2” with 29 negative samples is not shown. The 5× and 40× magnifications show tissue spots of the two identified ADTKD-MUC1 individuals from families A-21 and A-23 and one random individual negative for staining with pAb3-fs (negative sample). (B) Pedigrees of the two identified families (A-21 and A-23). Arrows indicate the immunohistochemically identified individuals. Asterisks indicate individuals with performed and confirmed SNaPshot minisequencing for ADTKD-MUC1. The striped individual in family A-21 has low-degree renal insufficiency (serum creatinine, 1.3 mg/dl) but has not yet been tested by SNaPshot minisequencing. The striped individual in family A-23 died in early adulthood of unknown cause.