Immunofluorescence analyses of respiratory epithelial cells aid the diagnosis of nephronophthisis

Abstract

Background: Nephronophthisis (NPH) comprises a heterogeneous group of inherited renal ciliopathies clinically characterized by progressive kidney failure. So far, definite diagnosis is based on molecular testing only. Here, we studied the feasibility of NPHP1 and NPHP4 immunostaining of nasal epithelial cells to secure and accelerate the diagnosis of NPH.

Methods: Samples of 86 individuals with genetically determined renal ciliopathies were analyzed for NPHP1 localization using immunofluorescence microscopy (IF). A sub-cohort of 35 individuals was also analyzed for NPHP4 localization. Western blotting was performed to confirm IF results.

Results: NPHP1 and NPHP4 were both absent in all individuals with disease-causing NPHP1 variants including one with a homozygous missense variant (c.1027G > A; p.Gly343Arg) formerly classified as a "variant of unknown significance." In individuals with an NPHP4 genotype, we observed a complete absence of NPHP4 while NPHP1 was severely reduced. IF results were confirmed by immunoblotting. Variants in other genes related to renal ciliopathies did not show any impact on NPHP1/NPHP4 expression. Aberrant immunostaining in two genetically unsolved individuals gave rise for a further genetic workup resulting in a genetic diagnosis for both with disease-causing variants in NPHP1 and NPHP4, respectively.

Conclusions: IF of patient-derived respiratory epithelial cells may help to secure and accelerate the diagnosis of nephronophthisis-both by verifying inconclusive genetic results and by stratifying genetic diagnostic approaches. Furthermore, we provide in vivo evidence for the interaction of NPHP1 and NPHP4 in a functional module.

Keywords: Immunofluorescence; NPHP1; NPHP4; Nephronophthisis; Renal ciliopathies; Transition zone.

A higher-resolution version of the Graphical abstract is available as Supplementary information

Fig. 1

NPHP1 and NPHP4 co-localize at the ciliary transition zone in respiratory epithelial cells. a Schematic illustration of respiratory epithelial cells before and after deciliation. NPHP1 and NPHP4 (green) specifically localize to the transition zone, distal to the microtubule organizing center (MTOC, red) containing DNAH5. Upon deciliation, the cilia break off in the middle of the transition zone. Figure created with BioRender.com. b–e High-resolution immunofluorescence analysis of deciliated respiratory epithelial cells from healthy control individuals cultured under air–liquid interface (ALI) conditions. b Cells were stained with antibodies directed against DNAH5 (red) and NPHP1 (green). c Profiles of immunofluorescence intensity show a proximal staining of DNAH5 and a distal staining of NPHP1 with no overlap. d Cells were stained with antibodies directed against NPHP1 (red) and NPHP4 (green). NPHP1 and NPHP4 specifically co-localize at the transition zone. e Profiles of immunofluorescence intensity show an overlap of NPHP1 and NPHP4. Nuclei were stained with Hoechst33342 (blue). Scale bars represent 10 µm

Fig. 2

NPHP1 is absent from the ciliary transition zone of respiratory cells in individuals with pathogenic variants in NPHP1 and NPHP4. High-resolution immunofluorescence analysis of respiratory epithelial cells from healthy control individuals and individuals carrying pathogenic variants in NPHP1 and NPHP4. Cells were co-stained with antibodies against RSPH4a (ciliary marker, red) and NPHP1 (green). In control cells, NPHP1 localizes to the transition zone (a), whereas NPHP1 was absent in cells of individuals ON-111 (homozygous deletion of NPHP1), ON-243 (homozygous deletion of NPHP1), ON-114 (homozygous variant in NPHP1: c.1027G > A; p.Gly343Arg), ON-201 II1 (homozygous variant in NPHP4: c.3272del; p.Val1091fs), and ON-143 (homozygous variant in NPHP4: c.811-2144del; deletion Ex8-16). Nuclei were stained with Hoechst33342 (blue). Scale bars represent 10 µm

Fig. 3

NPHP4 is severely reduced or absent from the transition zone of respiratory cells in individuals with pathogenic variants in NPHP1 and NPHP4. High-resolution immunofluorescence analysis of respiratory epithelial cells from healthy control individuals and individuals carrying variants in NPHP1 and NPHP4. Cells were co-stained with antibodies against acetylated-α-tubulin (ciliary marker, red) and NPHP4 (green). In control cells, NPHP4 localizes to the transition zone (a), whereas NPHP4 was severely reduced or absent in cells of individuals ON-111 (homozygous deletion of NPHP1), ON-243 (homozygous deletion of NPHP1), ON-201 II1 (homozygous variant in NPHP4: c.3272del; p.Val1091fs), and ON-143 (homozygous variant in NPHP4: c.811-2144del; deletion Ex8-16). Nuclei were stained with Hoechst33342 (blue). Scale bars represent 10 µm

Fig. 4

Flow chart displaying an overview of the results of subsequent NPHP1/NPHP4 immunofluorescence and genetic analyses of 111 individuals with renal ciliopathies

Fig. 5

Western blot analysis of respiratory epithelial cells from individuals with pathogenic variants in NPHP1 demonstrate the absence of NPHP1 and the reduction of NPHP4. Immunoblotting of whole-cell lysates from respiratory epithelial cells after air–liquid interface (ALI) culture. a Immunoblotting with anti-NPHP1 demonstrates the detection of the 83 kDa large isoform of NPHP1 (O15259-1) in the control. In individuals ON-111, ON-116, and ON-243, carrying variants in NPHP1, this band was absent. The subtle smaller bands detectable in all lanes represent an unspecific binding of the antibody. b Immunoblotting with anti-NPHP4 demonstrates the detection of the 157 kDa large isoform of NPHP4 (O75161-1) in the control. In individuals ON-111 (homozygous deletion of NPHP1), ON-116 (homozygous deletion of NPHP1), and ON-243 (homozygous deletion of NPHP1), this band was either absent or severely reduced. The subtle higher bands detectable in all lanes represent an unspecific binding of the antibody. c Immunoblotting with anti-acetylated-α-tubulin confirmed equal amounts of ciliary proteins within the different lysates. d Immunoblotting with anti-GAPDH confirmed equal protein concentrations of the whole-cell lysates. e Silver staining demonstrates the integrity of protein recovery from the whole cell preparation