Nephronophthisis

1Nephronophthisis (NPH), an autosomal recessive disorder initially described in 1945 by Smith and Graham and in 1951 by Fanconi, is a chronic tubulointerstitial nephritis that uniformly progresses to end-stage renal disease (ESRD). With regard to the age of onset for ESRD, three clinical variants have been described: infantile, juvenile, and adolescent forms. Of these, juvenile NPH is the most common, which accounts for 5–10% of cases of ESRD in children. In the past, NPH and medullary cystic kidney disease (MCKD) were considered in the same complex. Whereas these disorders share a number of clinical as well as histological features (tubular basement membrane disintegration, tubular cyst formation, and tubulointerstitial inflammation and fibrosis), MCKD is distinct from NPH by its autosomal dominant inheritance and by the late onset of renal failure after the fourth decade of life. In this review, we only consider NPH.

2Juvenile NPH is an uncommon condition that affects girls and boys equally. The incidence is approximately 0.13 for 10,000 live births in Finland, whereas in Canada, it is 1 per 50,000 live births and in United States 9 per 8.3 million. The disorder has been reported worldwide. The first symptoms generally develop around 4–6 years of age. Polyuria and polydipsia related to a reduced urinary concentrating ability and loss of sodium conservation occurs early in the course of the disease, whereas glomerular filtration rate (GFR) remains normal. Decreased urinary concentrating defect is demonstrated by a low urinary osmolarity (<400 mosm/kg in the first urine sample in the morning), which does not increase after desmopressin acetate administration. Urinary sodium wasting may be responsible for hyponatremia and hypovolemia in cases of decreased sodium intake. Decreased growth velocity related to chronic dehydration and later to renal insufficiency results in growth retardation. Hematuria and proteinuria are absent or minimal. Blood pressure is normal before the onset of renal failure.

3Renal insufficiency is often present when the diagnosis is made. Late symptoms are related to the progressive renal insufficiency and include anemia, metabolic acidosis, nausea, anorexia, and weakness. ESRD develops at a mean age of about 13 years but can also occurs in some rare cases much later during adulthood.

4Renal ultrasound may be normal, with normal-sized kidneys, but renal parenchymal hyperechogenicity and loss of corticomedullary differentiation are often observed. At later stages, small cysts are present in the medulla. Renal biopsy shows severe tubular damage on light microscopy. Groups of atrophic tubules with thickened basement membranes alternate with groups of dilated or collapsed tubules. Homogeneous or multilayered thickening of tubular basement membranes is prominent, but disintegration of the basement membrane can also occur (Fig. 1). Abrupt transition from one abnormality to another is highly suggestive of juvenile NPH. These various changes in the tubular basement membrane, although nonspecific, occur in NPH more extensively than in any kidney disorders with abnormal tubules. There is moderate to massive interstitial fibrosis with few inflammatory cells. The glomeruli are often normal, although secondary sclerosis is observed in advanced disease.

5This form of autosomal recessive NPH has been called the adolescent form following the identification of the NPHP3 gene in a large Venezuelan family in which ESRD occurred at a mean age of 19 years. However, there is no clear correlation between the age at ESRD and the genotype, as some patients with an NPHP3 mutation progress to ESRD before 10 years of age, whereas ESRD occurs in adulthood in some patients with NPHP1 deletion. Histological lesions are similar to those observed in juvenile NPH.

6A chronic autosomal recessive tubulointerstitial nephritis with cortical microcyts progressing to ESRD before 2 years of age was initially described by Gagnadoux et al.. The disease differs from juvenile NPH not only by its early onset but also by the histopathologic features. Whereas cystic dilatations of the collecting ducts are seen in these patients, the typical changes in the tubular basement membranes seen in juvenile NPH are usually absent. Ultrasonography usually shows moderately enlarged kidneys. Severe hypertension is common.

7In 10–20% of cases, extrarenal symptoms are present, in particular, retinitis pigmentosa (RP) [Senior-Løken syndrome (SLS)], cerebellar ataxia [Joubert syndrome (JS)], oculomotor apraxia type Cogan, mental retardation, bone anomalies and hepatic fibrosis. Situs inversus and ventricular cardiac septal defect are associated in some patients with the infantile form (Table 1).

8SLS, in which tapetoretinal degeneration (also known as RP) accompanies juvenile NPH is seen in approximately 10–15% percent of cases. Initially, Senior and Løken described patients with early and severe visual impairment resembling Leber congenital amaurosis, but the syndrome has thereafter been extended to all patients with NPH and degenerative retinopathy. In the late-onset form, children may develop night blindness followed by complete visual impairment in the following years. Electroretinogram (ERG) shows complete extinction before RP may be observed by funduscopic examination (Fig. 2). Some patients have only an attenuated ERG, but visual acuity is normal. RP has been observed in association with mutations in most NPHP genes (except NPHP7), but whereas RP is always present and severe in patients with NPHP5 and NPHP6 mutations, the symptoms are in general mild in patients with mutations in the other NPHP genes. Georges et al. reported on four patients, from three different families, with RP responsible for severe visual impairment during childhood who developed chronic interstitial nephritis with histological lesions characteristic of NPH and renal failure only between 42 and 56 years of age. No NPHP1 deletion was found in these patients, but the other genes were not analyzed.

9Nephrocystin proteins encoded by NPHP4, 5, 6, and 8 genes have been shown to localize to the connecting cilia between the inner and outer segments of the photoreceptors. All components necessary for assembly, maintenance, and turnover of the outer segment where the phototransduction takes place are synthesized in the cell body and are transported through the connecting cilia. Proteins implicated in other syndromes with retinal degeneration, such as Bardet-Biedl, Alstrom or Usher syndromes, have been shown to be localized to the connecting cilia where they are probably involved in the transport of phototransduction proteins, i.e. rhodopsin. Perturbation of these transporters leads to degeneration of the photoreceptor.

10JS is an autosomal recessive neurological disorder that associates congenital hypotonia evolving into cerebellar ataxia, developmental delay, oculomotor apraxia, and abnormal breathing pattern during the first month of life. JS is characterized by a complex cerebellar and brainstem malformation, the so-called “molar tooth sign” (MTS) observed by magnetic resonance imaging (MRI) (Fig. 3). JS can be associated with juvenile NPH and/or retinal involvement (JS type B). To date, three gene loci have been mapped to 9q34.3 (JBTS1), 11p11.2-q12.3 (JBTS2), and 6q23 (JBTS3), and mutations in the AHI1 (Abelson helper integration site) gene have been identified in JBTS3-linked families with a pure cerebellar phenotype.

11NPHP1 homozygous deletions (identical to the deletions observed in patients with isolated NPH) have been identified in a small percentage of patients with a mild neurological form of JS and NPH (and RP in one case). A recent survey of 56 families with NPHP1 deletion revealed that 5 (8.9%) had JS-related disorders with a variable phenotype. Most of them had no mental retardation, the characteristic MTS aspect was present in only one family, and thickened superior peduncles was the only malformation on brain MRI in two other families, whereas it was unremarkable in one family. Recently, mutations in the NPHP6/CEP290 and NPHP8/RPGRIP1L genes have been found in patients with JS. They were associated with severe RP in the first case, whereas there was no or only mild retinopathy in patients with RPGRIP1L mutations. In a series of 28 patients with NPH and at least one JS-related neurological symptom, Tory et al. found NPHP1 and NPHP6 homozygous or compound heterozygous mutations in 13 (46%).

12Meckel-Gruber syndrome (MKS) is an autosomal recessive lethal disorder characterized by central nervous system malformation (typically occipital encephalocele), postaxial polydactyly, cystic kidney dysplasia, and ductal proliferation in the portal area of the liver. Interestingly, mutations in the NPHP6, NPHP8, and MKS3 genes have been found in patients with MKS as well as in patients with JS, suggesting that these two conditions represent a broad spectrum of the same underlying disorder. As with other genes implicated in cystic kidney diseases, most mutated proteins responsible for MKS and JS have been shown to be localized to kidney primary cilia, further suggesting a connection between these syndromes.

13Bone anomalies can lead to phalangeal cone-shaped epiphyses, which are usually associated with other extrarenal manifestations (Saldino-Mainzer syndrome). Other skeletal dysplasia are associated in different syndromes. Hepatic involvement may be characterized by hepatosplenomegaly and portal fibrosis, with no or only mild bile duct proliferation. Mutations of the NPHP3 gene were reported in affected members with hepatic fibrosis and NPH from one family.

14Situs inversus has been reported in a patient with infantile NPH and mutation of the NPHP2 gene. This patient also had a cardiac ventricular septal defect (Table 2).

15Several other syndromes that feature NPH have been described, such as Jeune, Ellis van Creveld (chondroectodermal dysplasia), RHYNS (retinitis pigmentosa, hypopituitarism, and skeletal dysplasia), Alstrom, Sensenbrenner (cranioectodermal dysplasia), and Arima-Dekaban syndromes (Table 3). The description of these rare syndromes is beyond the scope of this paper.

16Positional cloning and candidate-gene approaches led to the identification of causative genes. To date, mutations in eight different genes (NPHP1, 3, 4, 5, 6, 7, 8, and 9) have been identified in juvenile NPH, whereas in the infantile form, mutations have been found in the NPHP2 gene (Fig. 4 and Table 2).

17In 1993, the first gene responsible for juvenile NPH was localized on chromosome 2q13 by positional cloning in consanguineous families. Homozygous deletions of about 250 kb in the 2q13 region were initially detected in 70% of patients and allowed identification of the responsible gene, NPHP1, in 1997. NPHP1 contains 20 exons and encodes for a protein, named nephrocystin or nephrocystin-1, that has an src homology 3 (SH3) and coil-coiled domains that interact with proteins (including products of other NPHP genes). Nephrocystin and its partners are localized at the cell–cell junction (adherens junction) and at the cell–matrix interface (focal adhesion), suggesting important functions in maintaining tubular epithelium (Fig. 5). Moreover, nephrocystin-1 is also localized at the primary cilia-like proteins associated with other cystic kidney diseases, such as polycystins. The detection of homozygous mutations by polymerase chain reaction (PCR) amplification permits fast and accurate diagnosis of the disease without the need for renal biopsy. In large series of patients with a presumptive diagnosis of NPH based mainly on clinical and radiological data, NPHP1 homozygous deletion is present in 20–40% of the cases ( and personal data). Heterozygous deletions are found in 6% of patients, with concomitant point mutation of the NPHP1 gene on the second allele (personal data). Whereas most patients with NPHP1 deletions or mutations have no extrarenal symptoms, a moderate form of retinal degeneration or JS has been reported in some cases.

18Mutations in the NPHP2 (INVS) gene are responsible for the infantile form of NPH. This gene, located on chromosome 9q31, encodes inversin, a protein that is critical for normal left–right patterning in the vertebrate embryo. In proximal tubular cells, inversin is associated with nephrocystin-1 and with components of microtubule cytoskeleton. It is localized to different subcellular compartments, including nuclei, cytoplasm, and cell–cell junction. Its interaction with APC2 suggests that inversin might play a role during the cell cycle. Knockout mice for the INVS gene show large renal cysts, altered left–right laterality (situs inversus), and hepatobiliary-duct malformations.

19NPHP3 located on chromosome 3q22 was initially mapped in a large family of NPH patients from Venezuela. Mutations in NPHP3 were described in families with renal disease alone, as well as in families with renal disease associated with hepatic fibrosis or retinal degeneration. Age at onset of ESRD was highly variable in patients with NPHP3 mutations from 4 to 37 years of age. Recently, two NPH patients with ESRD at 4 years of age were found to carry NPHP3 mutations. In another recently published by Bergmann et al., NPHP3 mutations were found in patients with a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas and multicystic kidneys with perinatal death in some cases. NPHP3 encodes a 1,330 amino acid protein with a tubulin-tyrosine ligase domain that interacts with nephrocystin. Interestingly, the pcy mouse, a spontaneously occurring renal cystic disease model that closely resembles NPH, harbors a homozygous missense mutation in the mouse NPHP3 ortholog (Nphp3) that most likely causes the kidney phenotype. Recent observations that the pcy-associated renal cystic disease is amenable to treatment with a vasopressin-2 receptor antagonist opens new perspectives for potential therapeutic strategies in NPH, for which no effective treatment is available.

20The NPHP4 gene located on chromosome 1p36 encodes a 1,426 amino acid protein called nephrocystin-4/nephroretinin. Nephrocystin-4 interacts with nephrocystin-1 and is probably involved in the same intracellular signaling pathway. Interestingly, nephrocystin-4 is conserved in the nematode Caenorhabditis elegans, which exhibits male mating phenotype defect upon Nphp4 knockdown, a phenotype also observed with orthologs of polycystic kidney disease genes. Direct sequencing of the NPHP4 gene has been performed in 250 individuals, 190 with isolated NPH, 50 with RP and ten with oculomotor apraxia. Twenty-three different NPHP4 mutations were found in 26 (10%) unrelated patients (13 with isolated NPH, eight with RP, and two with oculomotor apraxia). Of note, nephrocystin-4 localizes to the connecting cilium of photoreceptor cells and interacts with RPGRIP1 [RP guanosine riphosphatase (GTPase) regulator interacting protein 1], a component of cone and rod photoreceptors that is mutated in patients with Leber amaurosis.

21In contrast to the previous NPHP genes, mutations in the IQCB1 gene, now referred to as NPHP5, were reported only in patients with NPH in combination with severe retinal degeneration and early blindness—SLS. NPHP5 mutations, involving both alleles in all cases, were found in 16 of 92 patients with early onset RP. NPHP5 encodes an IQ-domain protein called IQCB1 or nephrocystin-5 that is expressed in connecting cilia of photoreceptors, where it is associated with calmodulin and retinitis pigmentosa GTPase regulator (RPGR). Nephrocystin-5 is also present in the primary cilia of renal epithelial cells.

22The NPHP6 gene, also known as CEP290, encodes a centrosomal protein that activates ATF4, a transcription factor involved in the control of the cell cycle. Thirteen different mutations in the NPHP6 gene were initially reported in 12 families with JS. Most of the patients had congenital blindness or severe visual defect in the first years of life. In two families (patients aged 3, 9.5, 10, and 15 years), there was no renal disease. Interestingly, the NPHP6 gene was thereafter reported to be mutated in more than 20% of patients with severe congenital blindness but no renal involvement (Leber congenital amaurosis). A hypomorphic mutation (c.2991+1655A>G) that creates a strong splice-donor site and inserts a cryptic exon in the CEP290 ribonucleic acid (RNA) was detected in 16 (21%) of 76 unrelated patients with blindness but without clinical signs of renal disease. Moreover, these patients had no neurological symptoms typical of JS and had normal cognitive function. Another group has confirmed that mutations in the NPHP6 gene is the most common cause of Leber amaurosis. Given the broad spectrum of the phenotype associated with NPHP6 mutations, Helou et al. screened this gene in 99 patients with cerebellar ataxia (JS), 75 patients with RP, and 21 patients with isolated NPH and found mutations in seven, two, and one case, respectively. In four patients, only single heterozygous mutations were found, and in one of them, an additional heterozygous NPHP4 missense mutation was present, arguing for a digenic inheritance. A genome-wide linkage scan in families with MKS led to the identification of NPHP6 mutations in some patients.

23The NPHP7 gene, also known as the GLIS2 gene, contains six coding exons and encodes a Kruppel-like zinc-finger transcription factor, which has been found mutated in one consanguineous Oji-Cree Canadian family with isolated NPH in three children who developed ESRD by 8 years of age. This gene seems to be very rarely involved, as no other mutation was found in a cohort of 470 individuals with NPH-like phenotypes. The kidneys of mice with a targeted disruption of the Glis2 gene are atrophic, with fibrosis starting at 8 weeks of age. Apoptosis is increased in renal tubular cells, whereas cell proliferation is not. Interestingly, the genes promoting epithelial–mesenchymal transition and fibrosis are up-regulated in the absence of Glis2.

24Mutations in a novel gene on chromosome 16, RPGRIP1L, have been found in patients with JS and in fetuses with MKS. Interestingly, Delous et al. reported that MKS fetuses carried two truncating mutations, whereas JS patients carried missense mutations and/or one truncating mutation, suggesting a genotype–phenotype correlation. RPGRIP1L is a cytosolic protein that colocalizes at the basal bodies, centrosomes, and primary cilia in renal tubular cells with nephrocystin-4 and nephrocystin-6. Interestingly, missense mutations found in JS patients decrease the interaction of RPGRIP1L with nephrocystin-4 without affecting its localization, suggesting that a defect in this association may contribute to the phenotype. In the same lines, NPHP4 missense mutations known to cause NPH with RP also disrupt this interaction. Recently, it was shown that Rpgrip1l participates in sonic hedgehog (Shh) signaling and by this means plays a critical role in patterning of the developing neural tube and limb through the cilium. The RPGRIP1L gene was thereafter analyzed in a cohort of 56 patients with JS. RPGRIP1L mutations were identified in five kindreds, including six individuals (8%). Of note, patients with RPGRIP1L mutations had normal retina, except two patients out of 12 with moderate visual impairment. Additional clinical symptoms were present in some patients with RPGRIP1L mutations, such as liver fibrosis, postaxial polydactyly, pituitary agenesis, and partial growth hormone deficiency. These last findings indicate a possible overlap with RHYNS syndrome.

25The jck cystic kidney mouse model is associated with a mutation in the Nek8 gene. Analysis of the NEK8 gene in a cohort of 588 patients with NPH led to the identification of three missense mutations in patients with isolated NPH. In one patient, an additional mutation in the NPHP5 gene was also present. Interestingly, mutant forms of NEK8 showed defects in ciliary localization.

26Overall, NPHP1 to NPHP9 mutations have been reported in cases of juvenile NPH with or without extrarenal symptoms, except for mutations in NPHP2 that have been found only in patients with infantile NPH. NPHP1 mutations were found in ~20% to 40% of cases, whereas mutations in the other genes seem to account for a very low percentage of cases (Table 1). Analysis of the NPHP1, NPHP3, and NPHP4 genes in a cohort of 94 different families have shown that a mutation or a deletion in one of these three genes was identified in 44 (47%) patients. Interestingly, in six families, three mutations in two NPHP genes were found, whereas two mutations in two NPHP genes (NPHP3 and NPHP4) were found in another kindred. Finally, a single mutation in one of these three genes was discovered in nine other patients. In the same lines, Tory et al. found that some patients with NPH and at least one JS-related neurological symptom had both an NPHP1 deletion and either a heterozygous NPHP6 or AHI1 mutation. These findings demonstrate that similar to the inheritance patterns described in Bardet-Biedl syndrome (BBS), NPH, at least in some patients, follows a digenic or oligogenic inheritance with heterozygous mutations in different genes in the same patients. Sequencing of all the known NPH genes in a large cohort of patients will be necessary to validate this model and appreciate the mutation load that could account for the severity of the nephropathy as well as the extrarenal symptoms.

27Cilia are present in almost all cells in the organism and act as sensory organelles that connect visual, mechanosensory, odorant or other stimuli to cell-cycle, control of epithelial architecture, or other yet unknown processes. The finding that polycystin-1 and polycystin-2—the proteins responsible for autosomal dominant polycystic kidney disease and proteins involved in other cystic kidney diseases (BBS, oro-facio-digital syndrome)—were present in cilia in renal tubular cells indicates a possible connection between this organelle and cyst formation. It has been suggested that the primary cilium senses fluid movement in the renal tubules. The presence of cystoproteins (such as nephrocystins) in cilia of various organs (photoreceptor, ependymal cells, cholangiocytes, chondrocytes) explains the multiple organ involvement in some patients with NPH. To illustrate this unifying theory, the term ciliopathies has been coined to design all the syndromes related to dysfunction of ciliary proteins. For more information on this subject, the readers are referred to an excellent recent review.

28In conclusion, and from a practical point of view, the diagnosis of NPH should be considered if a child presents with polyuria, urinary sodium loss, growth failure, renal insufficiency without hematuria or proteinuria, normal blood pressure, and normal-sized kidneys without dilatation of the urinary tract. These patients should be screened for homozygous or heterozygous NPHP1 deletion, which is found in 20–40% of cases. In the absence of such deletion, renal biopsy may be proposed to confirm the diagnosis. At present, screening for mutation in all the other NPHP genes is not routinely performed due to the low frequency of detected mutations and the high cost of the procedure. Patients with associated disorders should be considered separately. In case of severe RP, the NPHP5 gene may be screened for mutations, whereas in patients with neurological symptoms such as cerebellar ataxia, the NPHP6 and NPHP8 genes should be analyzed first. In case of early onset tubulointerstitial nephritis with cortical microcysts, the NPHP2 gene should be screened for mutation (Fig. 6).