ABSTRACT
Senior-Løken syndrome is a rare genetic disorder which presents with
nephronophthisis
and retinal degeneration, leading to end-stage renal disease and
progressive blindness.
The
most frequent cause of juvenile nephronophthisis is mutation in the
nephronophthisis
type 1 (NPHP1) gene. NPHP1 encodes the protein nephrocystin-1,
which functions at the transition zone (TZ) of primary cilia. Here we
report a 9-year-old Senior-Løken syndrome patient with NPHP1 deletion,
who presents with a decreased electroretinogram consistent with early
retinal degeneration. The patient had undergone bilateral nephrectomy
and a renal transplant. Immunohistochemistry and electron microscopy of
the resected kidney showed disorganized cystic structures with loss of
cilia in renal tubules. Phosphoinositides have been recently recognized
as critical components of the ciliary membrane and immunostaining of
kidney sections for phosphoinositide 5-phosphatases (INPP5E) showed loss
of staining compared to a healthy control.
The
decreased expression of INPP5E specifically in the primary cilium,
coupled with disorganized cilia morphology, suggests a novel role of
NPHP1 that it is involved in regulating ciliary phosphoinositide in the
ciliary membrane of renal tubular cells.
Key Words: Primary cilia, NPHP1, Senior-Løken syndrome,
nephronophthisis, transition zone, INPP5E
The primary cilium is a solitary, immotile microtubule-based structure
extending from the surface of almost all mammalian cell types (Anderson
et al., 2008; Berbari, O’Connor, Haycraft, & Yoder, 2009; Satir &
Christensen, 2007; Veland, Awan, Pedersen, Yoder, & Christensen, 2009).
Defects in cilia result in a class of multi-organ diseases that usually
include retinal degeneration, renal disease and cerebral abnormalities
collectively called ciliopathies (Halbritter et al., 2013; Hildebrandt,
Attanasio, & Otto, 2009). Nephronophthisis (NPHP) is an autosomal
recessive cystic renal ciliopathy characterized in the clinic by
polyuria and anemia as core manifestations. NPHP constitutes the most
frequent genetic cause of end-stage kidney disease before age 30 (Ahmed
& Ali, 2011; Srivastava & Sayer, 2014; Stokman, Lilien, & Knoers,
1993). Deletion of NPHP1, an important ciliary protein residing at the
ciliary transition zone (TZ), is associated with development of
Senior-Løken
syndrome and NPHP, which mainly affects the kidneys and eyes (Ronquillo,
Bernstein, & Baehr, 2012).
In addition to retinal degeneration and kidney defects, clinical
manifestations of Senior-Løken syndrome may also include skeletal,
dermatological and cerebellar anomalies, small hand (short metacarpals)
and
madarosis
(Aggarwal, Jain, Yadav, Kaverappa, & Gupta, 2013; Ronquillo et al.,
2012). Currently, no treatment is available to prevent disease
pathogenesis. Deletion in the NPHP1 gene represents the leading
genetic abnormality reported in nephrophthisis patients to date
(Hildebrandt et al., 2009; Wolf, 2015). Similar to other NPHP gene
product, the NPHP1 protein has been found to localize at the basal body
and the TZ of primary cilia in renal tubular cells and retinal
photoreceptors (Fliegauf et al., 2006; Garcia-Gonzalo & Reiter, 2017;
Gogendeau et al., 2020; Goncalves & Pelletier, 2017; Omran, 2010).
NPHP1 has also been found in areas of cell to cell contact, including
tight junctions, adherens junctions and focal adhesions (Hildebrandt et
al., 2009; Zhou et al., 2012).
The ocular involvement in Senior-Løken syndrome
is
variable and includes different degrees of retinal dystrophy and
cataracts (Aggarwal et al., 2013). The most frequently reported
ophthalmologic manifestations of NPHP have been retinal degeneration and
ocular-motor apraxia (OMA) (Aggarwal et al., 2013; R, 2014). In this
study, we report the clinical phenotype of a 9-year-old male patient.
The child initially presented with progressive renal failure at age 2.
Subsequent ultrasound findings were consistent with cystic kidney
disease. He went on to develope renal failure and early signs of retinal
degeneration. The patient presented with polyuria and polydipsia, then
progressed to end-stage renal disease, for which he underwent bilateral
nephrectomy and a renal transplant. Ophthalmic exam showed his visual
acuity was 20/20 in both eyes. On slit lamp examination, the anterior
segment of the both eyes was also normal. Color fundus photographs of
both eyes were unremarkable, without pigmentary changes, lesions or
scars in the macula or periphery (Fig 1A ). Visual field
examination showed no focal defects in both eyes (Fig 1B ).
Color
fundus photographs in NPHP1 patients are typically normal despite
bilateral visual field loss. Full field ERG, which provides an
assessment of overall retinal function, was recorded after 30 minutes of
dark adaptation.
The
scotopic and photopic flash ERG showed a decreased rod and cone response
in both eyes (Fig 1C ). The amplitude of the b-wave was reduced
for the rod response (0.01, 3.0 and 10.0 scotopic) without implicit time
delay. The b value of the dark-adapted ERG (0.01) was
36
µV OD compared with 140 µV in the control patient. ERG (3.0) was 120 µV
OD compared with 212 µV in the control patient. ERG (10.0) was 152 µV OD
compared with 217 µV in the control patient. The amplitude of both a and
b-wave of the cone response was bilaterally reduced compared to the
age-matched control. The b value of the light-adapted ERG was 50 µV OD
in the patient and 143 µV in the control patient. No remarkable changes
were found in 30-Hz flicker in either eye (data not shown). In summary,
the patient with NPHP1 deletion demonstrated diminished rod responses
and overall impaired cone function.
Kidneys of NPHP1 patients display a characteristic triad of
corticomedullary cysts, tubular basement membrane disruption and
tubulointerstitial nephropathy (Srivastava & Sayer, 2014; Stokman,
Lilien, & Knoers, 1993; Wolf, 2015). To determine the renal phenotype
of this patient, histopathological analysis of his kidney obtained by
nephrectomy was performed alongside an identical analysis of a healthy
human kidney (Fig. 1D ). H&E staining of the patient’s kidney
sections showed a diffuse sclerosing tubulointerstitial process with a
predominance of tortuous and atrophic tubules at the corticomedullary
junction. Enlarged cortical cyst formation was present in the
corticomedullary region. Higher magnification highlighted a thickened
and multilayered tubular basement membrane (TBM).
In
order to determine the impact of mutation of NPHP1 on human primary
cilia and TBM of renal epithelia cells, and TBM profiles by transmission
electron microscopy. Electron microscopy revealed an irregular TBM
pattern, consisting frequently of two or three membrane layers which
were folded excessively while the lining of the tubular cysts was
flattened. As shown in Figure 1D, fewer primary cilia were seen on the
apical side of the tubular epithelial cells, indicating renal cilia
dysfunction.
It has been previously demonstrated that nephrocystin proteins localize
to the ciliary TZ of renal tubular epithelia in mice and cultured cells.
The TZ, a cilia sub-compartment just distal of the basal body, has a
unique membrane content and has also been implicated in regulating what
proteins enter the cilia signaling compartment in diverse model systems
(Awata et al., 2014; Fliegauf et al., 2006; Sang et al., 2011; Shi et
al., 2017; Szymanska & Johnson, 2012; Williams et al., 2011). To our
knowledge (Fliegauf et al., 2006) cilia frequency, morphology and
content of renal primary cilia in a human patient with NPHP1 deletion
has not been done. Here, we assess primary cilia in a kidney biopsy of a
patient with nephronophthisis. We examined the renal biopsy with
confocal immunofluorescence microscopy and compared the findings to
those in relevant control samples from a healthy kidney. Typical ciliary
markers, acetylated α-tubulin an axoneme marker, Arl13b a cilia membrane
marker and Intraflagellar Transport 88 a cilia specific IFT marker, were
employed to assess primary cilia. Compared with control kidney primary
cilia, NPHP1 patient renal cilia were disorganized and shortened
(Fig. 2A ). Quantification of cilia which are marked by Arl13b
antibody frequency showed loss of NPHP1 significantly reduced the
proportion of ciliated cells from 78.5
±12.3%
in the normal patient to 60 ±7.3% in the NPHP1 deletion patient,
indicating that NPHP1 influences ciliogenesis in human renal tubular
cells
(Fig.
2E ). In addition, quantification of immunofluorescently labeled primary
cilia revealed that ciliary length was reduced from 5.6 ±1.2 µm in the
healthy patient to 3.2 ±1.0
µm
in the NPHP1 patient (Fig. 2F ). Previous studies have shown
that mice with NPHP1 deletion do not develop pathological
characteristics of nephronophthisis (Jiang et al., 2008). However,
NPHP1
knockout significantly decreases ciliation and ciliary length in MDCK
cells, which is consistent with our data for patient-derived cilia
(Delous et al., 2009). This difference in phenotype might be due to
species differences in gene function.(Williams, Masyukova, & Yoder,
2010). Recent data have also begun to demonstrate that TZ function may
be cell type and tissue specific, with mutations in some TZ members
having specific phenotypes (Lewis et al., 2019). Our findings suggest
that NPHP1 may be required for normal cilia maintenance and function in
human kidney.
The TZ is a compartment of the proximal region of the cilium, between
the basal body and axoneme. Numerous studies in diverse model systems
have shown that TZ functions as a selective membrane diffusion barrier,
regulating ciliary protein entry and exit (Goncalves & Pelletier, 2017;
Omran, 2010). Two TZ protein complexes have been identified: the NPHP
complex and MKS complex. In mammalian cells,
AC3
and PKD2 (adenylyl cyclase III and PKD2 are both ciliary proteins) are
aberrantly located in MKS-complex-deficient cilia (Garcia-Gonzalo et
al., 2011). InC.elegans , TRAM protein and membrane-associated RP2 homologues are
abnormal in TZ-deficient cilia (Williams et al., 2011). Previous studies
have demonstrated by LAP-tagging and in vitro binding, that
NPHP1, NPHP4 and NPHP8 have a strong mutual interaction (Sang et al.,
2011): NPHP4 acts as a bridge that directly binds both NPHP1 and NPHP8in vitro , whereas NPHP1 and NPHP8 cannot directly bind to each
other. Furthermore,
NPHP4
and NPHP8 have been shown to localize to the base of the cilia of renal
tubular cells in vivo and in vitro (Delous et al., 2009;
Sang et al., 2011), evidence that they play a clear role within
cilia. Based on these studies, we hypothesized that loss of NPHP1 would
directly affect NPHP4 localization in vivo , but produce no
alteration of NPHP8. To test this hypothesis, we co-stained renal
sections from the patient with NPHP1 deletion and healthy controls for
either NPHP4 or NPHP8 and the ciliary marker Arl13b. Analysis of
confocal images revealed no alteration in NPHP8 localization, indicating
that NPHP1 exerts its function independently of NPHP8 (Fig 2B ).
In healthy control, NPHP4 localized only at the base of the cilia. In
contrast, in renal sections from the patient with NPHP1 deletion, NPHP4
was significantly increased not only at the base of the cilia but also
in the ciliary axoneme (Fig. 2C ). Quantitative analysis showed
that 97.78 ±3.85% renal epithelial cells in the NPHP1 deletion group
demonstrated an abnormal enrichment of NPHP4,
compared
to only 4.2 ±7.2% in the control group (Fig. 2G ). These
results suggest that the NPHP1-NPHP4 interaction is conserved and that
it plays a role in controlling NPHP4 localization in primary cilia of
human renal epithelial cells.
NPHP proteins are important regulators of ciliary entrance and exit for
a diverse array of components (Omran, 2010). Therefore, the regulation
of phosphoinositides, which recently have been recognized as a critical
component of the ciliary membrane, could also be influenced by these
complexes residing in the ciliary transition zone. Several groups have
shown that a mutation of inositol 5-phosphatase (INPP5E), which is found
to localize in the axoneme of the primary cilium, is responsible for
ciliary instability in Joubert syndrome (Chavez et al., 2015; Dyson et
al., 2017; Jacoby et al., 2009). Based on the previously described
distribution of NPHP1 in cilia, we hypothesized that NPHP1 may be
critical for controlling phosphoinositide entry into the cilia of human
renal epithelial cells. Using a monoclonal antibody against INPP5E, we
assessed the distribution of INPP5E within the primary cilia of renal
tissue from both a patient with NPHP1 deletion and a healthy control.
While INPP5E was expressed along the axoneme of primary cilia in normal
tissue, it was absent from the cilia of the NPHP1 deletion patient
(Fig. 2D ). Quantitative analysis showed that up to 42.40
±13.83% of renal epithelial cells in the NPHP1 deletion patient had
abnormal INPP5E expression, compared to healthy tissue (Fig.
2H ). This data indicates a critical role for NPHP1 in phosphoinositide
regulation.
Interestingly, nephronophthisis and
Joubert
syndrome are two types of ciliopathies with overlapping clinical
manifestations, including cystic kidney disease and retinal
degeneration. Previous publications showed that NPHP1 and INPP5E
mutations are responsible for Joubert syndrome(Constable, Long, Floyd,
Schurmans, & Caspary, 2020; Jacoby et al., 2009). Our study shows that
NPHP1 deletion results in abnormal expression of INPP5E, which supports
the hypothesis that Joubert syndrome and nephronophthisis represent the
mild and the severe versions of the clinical manifestation of the same
biological disorder. Revealing the mechanism by which NPHP1 regulates
the amount of INPP5E at the vertebrate TZ and elucidating the relevant
signaling pathways represent important steps toward developing novel
therapeutic targets.
Because its defects are associated with many severe ciliopathies, the
function of TZ is an active field of biomedical research. Numerous
publications have demonstrated that TZ proteins serve as the ciliary
gatekeeper, but the exact mechanisms by which they regulate ciliary
gating are still not clear. Although previous studies have shown that
NPHP1
acts as a ciliary gate in C. elegans (Williams et al., 2011),
NPHP1
deficiency did not alter ciliary levels of Arl13b or Sstr3 in vertebrate
cells, suggesting that NPHP1 is not involved in governing the transition
of these proteins in
vertebrate
cilia (Lin, Guo, & Dutcher, 2018). More recent studies have proposed
that the TZ could act as a specific ciliary gate for lipids (Goncalves
& Pelletier, 2017). Supporting this notion, the location of INPP5E
within vertebrate cilia has been shown to depend on TZ proteins, such as
TCTN1. RPGRIP1L/MKS5 has been reported to play a role in regulating the
amount
of the phosphoinositide PIP2 within cilia of C. elegans , but
little is known about the possible involvement of NPHP1 as a ciliary
gate for lipids. In the present study, we report a diagnosed case of
Senior-Løken syndrome, which derives from a deletion of a key TZ
protein. The patient had renal failure and retinal degeneration. We show
that primary cilia in the patient’s renal biopsy exhibit morphological
abnormalities which likely impaired normal function. Importantly, we
also show that the ciliary localization of INPP5E is altered in the
sample from the patient with NPHP1 deletion, confirming our hypothesis
that NPHP1 may act as a ciliary gate for lipids. Furthermore, we
detected an abnormal expression of NPHP4, which suggests that NPHP1 acts
as a structural scaffold for the NPHP4 present in the TZ. We bring into
focus the input of NPHP1 in forming a TZ in which NPHP4 functions
normally, which in turn regulates INPP5E transition and its ability to
influence cilia stability.