Despite the large number of variants identified in 23 NPHP genes to date, up to 40% of NPH patients remain genetically unsolved [24]. In some cases with convincing phenotype, only one heterogeneous gene defect is found even when applying state-of-the-art sequencing techniques. Many reported variants are classified as a “variant of unknown significance” and cannot be regarded as “disease-causing” with absolute certainty. Different approaches have been used in order to tackle this challenge with varying degrees of success [25, 26] risking that genetic results remain inconclusive.

Immunofluorescence (IF) staining of respiratory epithelial cells offers a technique to verify the pathogenic impact of inconclusive genetic results confirming the clinical diagnosis. Respiratory epithelial cells are obtained via non-invasive nasal brush biopsy [14], are not impacted in their structure by kidney transplantation, and can be analyzed directly after sampling. In motile ciliopathies such as primary ciliary dyskinesia (PCD), IF analysis has already been proven to be a convenient diagnostic tool with high comfort and minimal invasiveness for the patient [13].

In this study, we applied an IF-based approach to a large cohort of 111 individuals with either nephronophthisis (n = 58) or other renal ciliopathies (n = 53) using antibodies directed against NPHP1 and NPHP4. We included both NPHP1 and NPHP4 null mutants proving the specificity of used antibodies as recommended by standard good practice guidelines [14].

The pathogenicity of all NPHP1 variants was verified by showing a complete absence of the NPHP1 IF signal. This included one individual carrying a homozygous NPHP1 missense variant (c.1027G > A; p.Gly343Arg) formerly classified as a “variant of unknown significance” according to ACMG criteria (PP5, PP3, and PM2). Furthermore, the results revealed an absence of NPHP1 in individuals with an NPHP4 genotype. Vice versa, NPHP4 expression at the ciliary transition zone was severely reduced or completely absent in individuals with an NPHP1 genotype. A complete axonemal absence of NPHP4 was observed in the molecular scenario of a homozygous NPHP4 loss of function variant proving the specificity of the antibody’s NPHP4 recognition at the transition zone. However, in the same individual, we observed some weak cytoplasmic signals. These were most probably explained by unspecific staining effects that are frequently observed in polyclonal antibody use (also variably present in other individuals, Fig. 3). According to the current state of knowledge, nephrocystins—the gene products of NPHP genes—are organized in at least four distinct functional modules: the NPHP1-4–8 module, NPHP2-3–9-ANKS6 module, NPHP5-6 module, and the MKS module. These nephrocystin modules are related to different signaling pathways, including the Wnt-, Hedgehog-, DNA damage response (DDR)-, Hippo-, intracellular calcium signaling-, cAMP signaling-, and mTOR pathway [27]. However, so far, these observations were obtained by cell culture-based functional interaction studies only. Our results verify this molecular relationship between NPHP1 and NPHP4 through abnormal protein expression in vivo.

Previously, the expression of other proteins associated with a renal ciliopathy phenotype like CEP290/NPHP6 and polycystin1 and 2 has been reported at airway epithelia as well as at the olfactory bulb [28, 29]. We were able to reveal a significant olfactory deficit not only in BBS patients but also in individuals with a TMEM67/NPHP11-related phenotype [30]. Yet, despite the expression and an obvious interplay of nephrocystins at the transition zone, the molecular relevance of these proteins for motile cilia functional integrity largely remains unclear. Brndiarova et al. reported an altered motile cilia function in individuals affected by primary renal ciliopathies [31]. Furthermore, an increased prevalence of bronchiectasis has been observed in ADPKD patients, suggesting disturbed mucociliary clearance [12]. Further functional studies characterizing and quantifying ciliary motility and most of all standardized assessments of clinical airway-related symptoms will be needed to evaluate the functional impact of nephrocystins and polycystins on motile ciliary function.

BBS proteins and the transcription factor HNF1B also have a close functional relationship to cilia architecture and cilia-related transport processes. While BBS proteins—organized in a multimeric complex called BBSome—play a key role in cilia-related protein transport, HNF1B has been proven to directly regulate the expression of PKHD1, PKD2, and other genes causative for renal ciliopathies with a cystic phenotype [32]. In our study, we discovered that BBS and HNF1B genes do not have an impact on nephrocystin expression at the ciliary transition zone as IF results of all individuals with a genotype other than NPHP1/4 including BBS (n = 29) and HNF1B (n = 15) did not show any effect on NPHP1 or NPHP4 expression.

Non-specific staining of basal bodies by polyclonal antibodies has previously been reported as problematic in IF analyses [14]. To address potential issues, we performed western blot analyses using lysates obtained from healthy controls and NPHP1 individuals. Bands for NPHP1 and NPHP4 in lysates from healthy controls were specific and comparable in intensity while severely reduced or completely absent in NPHP1 individuals. We congruently verified the absence or severe reduction of NPHP1 and NPHP4 by both techniques—IF staining and western blot analysis. Unfortunately, there were no cilia lysates available for western blot analysis from the two individuals with an NPHP4 genotype as those samples were submitted as slides instead of native cell culture material, making cell culture impossible.

Our study suffers from several limitations—above all the small NPHP4 sample size comprising only two individuals with a corresponding genotype. However, one must take into account that NPHP4 variants make up only 3–7% of NPH cases overall [33, 34]. Another limitation was the relatively high number of poor-quality samples, particularly in the BBS cohort. There is no clear explanation for this observation. Bacterial contamination due to frequent respiratory infections seems to play a certain role. Yet, this assumption still must be objectively confirmed. Also, there might have been some kind of sampling error since the majority of BBS samples have been collected during BBS patient days which were held abroad. Despite these limitations, the results obtained in our study were congruent in both IF staining and western blots and underlined previous findings from functional studies regarding NPHP1 and NPHP4 interaction [8, 35, 36].

In our study, the IF-based approach was not only suitable to verify inconclusive genetic results but also could be applied as an efficient screening tool to streamline genetic analysis. This was exemplified by the two cases ON-143 and ON-243, which, at the beginning of the study, were registered as genetically unsolved despite a convincing phenotype for NPH and the Senior-Løken syndrome, respectively. IF results revealed a complete absence of NPHP1 and NPHP4 in one case (ON-243)—as observed in the NPHP1 cohort—and a combination of absent NPHP4 with severely reduced NPHP1 in the other one (ON-143)—comparable to the pattern found in NPHP4 individuals—prompting us to apply a comprehensive genetic analysis. Strikingly, in both individuals, we were able to ascertain a genetic diagnosis matching their IF pattern: a homozygous NPHP1 deletion in ON-243 and a homozygous deletion resulting in a frameshift of NPHP4 (c.811-2144del; deletion Ex8-16) in ON-143. Thus, in cases presenting a coherent phenotype, preceding IF screening might help to stratify and limit genetical efforts.

Certainly, in the scenario of fast and low-cost genetic analyses, the IF-based approach neither has the potential nor was intended to compete with or even replace genetic diagnostics in renal ciliopathies. However, this method has the potential to act as an ideal complement to the current standard of genetic diagnostics particularly in cases with inconclusive genotypes. Beyond that, it can serve as a model system to investigate the molecular interplay of nephrocystins in human tissue rather than animal models.