Disease of the Month

Nephronophthisis-Associated Ciliopathies

Friedhelm Hildebrandt and Weibin Zhou Departments of Pediatrics and of Human Genetics, University of Michigan, Ann Arbor, Michigan

Nephronophthisis (NPHP), an autosomal recessive cystic kidney disease, represents the most frequent genetic cause of end-stage kidney disease in the first three decades of life. Contrary to polycystic kidney disease, NPHP shows normal or diminished kidney size, cysts are concentrated at the corticomedullary junction, and tubulointerstitial fibrosis is dominant. NPHP can be associated with retinitis pigmentosa (Senior-Løken syndrome), liver fibrosis, and cerebellar vermis aplasia (Joubert syndrome) in approximately 10% of patients. Positional cloning of six novel genes (NPHP1 through 6) as mutated in NPHP and functional characterization of their encoded proteins have contributed to the concept of “ciliopathies.” It has helped advance a new unifying theory of cystic kidney diseases. This theory states that the products of all genes that are mutated in cystic kidney diseases in humans, mice, or zebrafish are expressed in primary cilia or centrosomes of renal epithelial cells. Primary cilia are sensory organelles that connect mechanosensory, visual, osmotic, and other stimuli to mechanisms of cell-cycle control and epithelial cell polarity. The ciliary theory explains the multiple organ involvement in NPHP regarding retinitis pigmentosa, liver fibrosis, ataxia, situs inversus, and mental retardation. Mutations in NPHP genes cause defects in signaling mechanisms, including the noncanonical Wnt signaling pathway. The “ciliopathy” NPHP thereby is caused by defects in tissue differentiation and maintenance as a result of impaired processing of extracellular cues. Nephrocystins, the proteins that are encoded by NPHP genes, are highly conserved in evolution. Positional cloning of additional causative genes of NPHP will elucidate further signaling mechanisms that are involved, thereby establishing therapeutic approaches using animal models in mouse, zebrafish, and Caenorhabditis elegans. J Am Soc Nephrol 18: 1855–1871, 2007. doi: 10.1681/ASN.2006121344

Nephronophthisis: A Frequent Genetic which there is moderate renal enlargement), cysts seem to develop Cause of Kidney Failure in Children e vacuo by loss of normal tissue. This is in contrast to polycystic Renal Involvement kidney disease (PKD), in which cysts are evenly spread out over Nephronophthisis (NPHP) is an autosomal recessive cystic kid- the entire organ and lead to gross enlargement of the kidneys (4). ney disease that constitutes the most frequent genetic cause for NPHP is inherited in an autosomal recessive mode. This end-stage kidney disease (ESKD) in the first three decades of life includes NPHP variants with extrarenal manifestations (4). (1–4). Three clinical forms of NPHP have been distinguished by NPHP has previously been grouped together with the clinical onset of ESKD: Infantile (5), juvenile (6), and adolescent NPHP (7), entity of medullary cystic kidney disease (MCKD) (6,10) be- which manifest with ESKD at the median ages of 1, 13, and 19 yr, cause of similarities of clinical and pathologic features (12). respectively. Initial symptoms are relatively mild (except in infan- Both NPHP and MCKD feature corticomedullary cysts in kid- tile NPHP type 2) and consist of polyuria, polydipsia with regular neys of normal or slightly reduced size. However, MCKD is fluid intake at nighttime, secondary enuresis, and anemia (8). At clearly distinct from NPHP regarding multiple aspects: (1) an average age of 9 yr, a slightly raised serum creatinine is noted, MCKD follows autosomal dominant inheritance, (2) ESKD oc- before ESKD invariably develops within a few years. Renal ultra- curs in the fourth decade and later, and (3) in MCKD there is no sound reveals increased echogenicity. Later, cysts appear at the extrarenal involvement other than hyperuricemia and gout. corticomedullary junction within kidneys of normal or slightly NPHP was first described by Smith and Graham in 1945 (2) reduced size (Figure 1, A and B) (9). Renal histology reveals a and by Fanconi et al. (3), who introduced the term “familial characteristic triad of tubular basement membrane disruption, juvenile nephronophthisis.” Since then, Ͼ300 cases have been tubulointerstitial nephropathy, and corticomedullary cysts (Figure published in the literature (10). In NPHP, the earliest presenting 1C) (10,11). In NPHP, cysts arise from the corticomedullary junc- symptoms are polyuria, polydipsia, decreased urinary concen- tion of the kidneys (Figure 1, A and B). Because kidneys size is trating ability, and secondary enuresis. They occur in Ͼ80% of normal or slightly reduced (except in infantile NPHP type 2, in cases (13) and start at approximately 6 yr of age. Anemia and growth retardation develop later in the course of the disease (8). Regular fluid intake at nighttime is a characteristic feature of Published online ahead of print. Publication date available at www.jasn.org. the patient’s history and starts at approximately age 6 yr. Address correspondence to: Dr. Friedhelm Hildebrandt, Departments of Pediat- Because of the mild nature of symptoms and the lack of edema, rics and of Human Genetics, University of Michigan Health System, 8220C MSRB hypertension, or urinary tract infections, there is often a delay III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0646. Phone: 734-615- 7285 (office), 734-615-7895 or 7896 (laboratories); Fax: 734-615-1386 or 7770; in the diagnosis of NPHP. This causes a risk for sudden death E-mail: [email protected] from fluid and electrolyte imbalance. Disease recurrence has

which follows autosomal dominant inheritance, ESKD occurs later in life. MCKD types 1 and 2 show a median onset of ESKD at 62 (25) and 32 yr (26), respectively. MCKD type 2 can be positively diagnosed by detection of mutations in the UMOD gene that encodes uromodulin/Tamm-Horsfall protein.

Epidemiology NPHP has been reported from virtually all regions of the world (13). The incidence of the disease has been given as nine patients per 8.3 million (27) in the United States or one in 50,000 live births in Canada (10,28). Although a rare disorder, it represents the most frequent genetic cause of ESKD in the first three decades of life and is a major cause of ESKD in children, accounting for 10 to 25% of these patients in Europe (13,29–31). In the North American pediatric ESKD population, pooled data indicate a prevalence of approximately 5% of all children with ESKD (32,33).

Extrarenal Manifestations of Eye, Brain, and Figure 1. Morphology of nephronophthisis (NPHP). (A) Macro- Liver scopic pathology reveals cysts that arise from the corticomed- NPHP may be associated with tapetoretinal degeneration (Se- ullary junction of normal-sized kidneys. (B) Renal ultrasound nior-Løken syndrome [SLSN] [34,35]), cerebellar vermis aplasia demonstrates increased echogenicity, loss of corticomedullary (Joubert syndrome [JBTS] [36,37]), ocular motor apraxia type differentiation, and the presence of cysts. In contrast to poly- Cogan (38), mental retardation (21,39), liver fibrosis (40), or cone- cystic kidney disease (PKD), in NPHP, cysts are concentrated at shaped epiphyses of the phalanges (Mainzer-Saldino syndrome the corticomedullary border and kidneys are not enlarged. (C) [41]). Infantile NPHP type 2 (17) can be associated with situs Renal histology in NPHP shows the characteristic triad of renal inversus (17), retinitis pigmentosa (42), or cardiac ventricular septal tubular (and glomerular) cysts, tubular membrane disruption, and tubulointerstitial cell infiltrates with interstitial fibrosis and defect (17). The ciliary theory of NPHP elucidates the pathogenic periglomerular fibrosis. Adapted from Hildebrandt et al. (9a); basis of extrarenal organ involvement regarding retinitis pigmentosa, and courtesy of D. Bockenhauer. liver fibrosis, ataxia, situs inversus, and mental retardation.

Retinal Involvement (SLSN) never been reported in kidneys that were transplanted to pa- SLSN, represented by the concomitant occurrence of NPHP tients with NPHP (14). By positional cloning, we and others with retinitis pigmentosa, was first described by Contreras (43), have identified recessive mutations in six different novel genes Senior (34), and Løken (35). Three different terms have been used as causing NPHP: NPHP1 (15,16), NPHP2/inversin (17), NPHP3 in the literature to describe the retinal findings of SLSN: Retinitis (18), NPHP4 (19), NPHP5 (20), and NPHP6 (21), defining NPHP pigmentosa, tapetoretinal degeneration, and retinal-renal dyspla- types 1, 2, 3, 4, 5, and 6, respectively. This has made definite sia. This most likely reflects a spectrum within the pathogenesis molecular genetic diagnostics possible (www.renalgenes.org). that includes developmental defects (dysplasia) as well as defects Homozygous deletions in the NPHP1 gene account for approx- of tissue maintenance (degeneration) (44). In children with reces- imately 25% of all NPHP cases, whereas the other genes con- sive mutations in the NPHP1, 2, 3, and 4 genes, retinitis pigmen- tribute Ͻ2% each. As expected in a recessive disease, pene- tosa occurs in approximately 10% of all affected families, without trance of the renal phenotype seems to be 100%. any obvious genotype/phenotype correlation. Whether patients In NPHP, chronic renal failure develops within the first three with retinal involvement carry an additional mutation in an un- decades of life (7,22,23). Infantile NPHP, which is characterized known modifier gene is an open question. Early-onset and late- by mutations in NPHP2/inversin, leads to ESKD between birth onset types of SLSN have been distinguished. The early-onset type and age 3 yr (17,23). In a study conducted in 46 children who seems to represent a form of Leber’s congenital amaurosis, be- had juvenile NPHP type 1 caused by mutations in the NPHP1 cause children exhibit coarse nystagmus and/or blindness at birth gene, a serum creatinine of 6 mg/dl was reached at a median or develop these symptoms within the first 2 yr of life (45). It is age of 13 yr (range 4 to 20 yr) (6,22). Similarly, the median age interesting that patients with mutations in the NPHP5 or NPHP6 of ESKD in patients with mutations in the NPHP5 gene was 13 genes exhibit early-onset retinitis pigmentosa in all known cases yr (20). The median time lapse between a serum creatinine of 2 (20,21). Fundoscopic alterations are present in all patients with and 4 mg/dl was 32 mo and between 4 and 6 mg/dl was 10 mo late-onset SLSN by the age of 10 yr. The late-onset form manifests (24). In patients with adolescent NPHP as a result of mutations first with night blindness, followed by development of blindness in the NPHP3 gene, ESKD develops by 19 yr of age (18). If renal during school age. Retinal degeneration is characterized by a failure has not developed by the age of 25 yr, then the diagnosis constant and complete extinction of the electroretinogram, which of recessive NPHP should be questioned and autosomal dom- precedes the development of visual and fundoscopic signs of inant MCKD considered as a differential diagnosis. In MCKD, retinitis pigmentosa (46). The kidney involvement in SLSN is J Am Soc Nephrol 18: 1855–1871, 2007 Nephronophthisis-Associated Ciliopathies 1857 identical clinically to what is known from patients with NPHP patient with situs inversus also had a cardiac ventricular septal without ocular involvement regarding age of onset, symptoms, defect as a heterotaxy phenotype. This finding was analogous to and histology of renal disease. the randomization of heart looping that was seen in nphp2/inversin knockdown experiments in zebrafish. Recently, it has become Cerebellar Vermis Aplasia (JBTS) apparent that products of other genes that are associated with In JBTS, a developmental disorder with multiple organ involve- renal cystic disease (in addition to inversin) are important for ment, NPHP or cystic dysplasia occurs in association with left–right axis determination of the body plan (66,67). The gene coloboma of the eye (or retinal degeneration); with aplasia/hyp- PKD2, mutations in which cause autosomal dominant PKD and oplasia of the cerebellar vermis causing ataxia; and with the fac- that encodes the calcium release channel polycystin-2, had been Ϫ Ϫ ultative symptoms of psychomotor retardation, polydactyly, oc- shown in a Pkd2 / mouse model to represent a gene that regu- cipital encephalocele, and episodic neonatal tachypnea/dyspnea lates left–right axis determination, acting upstream of Nodal, Ebaf, (36,37,47–49). A pathognomonic diagnostic feature of JBTS on Leftb, and Pitx2 (68,69). axial magnetic resonance imaging of the brain is the presence of prominent superior cerebellar peduncles, termed the “molar tooth Other Syndromes with NPHP sign” of the midbrain-hindbrain junction (49,50). In patients with Bardet-Biedl syndrome (BBS) exhibits renal histology that is the association of JBTS and NPHP, mutations have been described similar to NPHP (70,71). Positional cloning of recessive genes in three different genes, NPHP1 (37,49,50), AHI (51,52) (JBTS type that are mutated in BBS has revealed that the molecular relation 3), and NPHP6 (21,53). Patients with JBTS have abnormal axonal between NPHP and BBS may lie in coexpression of the respec- decussation (crossing in the brain) that affects the corticospinal tive gene products in primary cilia, basal bodies, and centro- tract and superior cerebellar peduncles, thereby explaining the somes of renal epithelial cells (72). For BBS, an oligogenic motor and behavioral abnormalities (54). Some patients also have inheritance pattern has been described. This refers to the find- abnormal cerebral structure with cortical polymicrogyria (55). Oc- ing that mutations in more than one BBS gene may be required ular motor apraxia type Cogan, defined as the transient inability of for full penetrance of some aspects of organ involvement (73). horizontal eye movements in the first few years of life, may be Further disease variants have been described in association with associated with JBTS. This symptom has been described in pa- NPHP, including Jeune syndrome (asphyxiating thoracic dyspla- tients with mutations in the NPHP1 (38,56) (“JBTS4”) and NPHP4 sia) (74–77), Ellis van Creveld syndrome (78), RHYNS syndrome (57) genes. It may be due to defects in the nuclei of the abducens (retinitis pigmentosa, hypopituitarism, NPHP, and skeletal dys- nerve, which contain both ipsilaterally projecting motor neurons plasia) (79), Alstrom syndrome (retinitis pigmentosa, deafness, and contralaterally projecting interneurons, or supranuclear con- obesity, and diabetes without mental defect, polydactyly, or hy- trol regions such as the pontine paramedian reticular formation pogonadism) (80), and Meckel-Gruber syndrome (81,82), which in that projects to the abducens and oculomotor nuclei, which has the case of MKS3 mutations can be allelic with JBTS (83). In been postulated for other forms of horizontal gaze palsy (58). Alstrom syndrome, the single underlying gene, ALMS1, encodes a Defects in axon guidance may be related to defects in renal tubule novel protein that contains coiled-coil domains and a putative development by shared signaling pathways (see Signal Mecha- nuclear localization signal, as well as serine-rich and histidine-rich nisms Relevant for NPHP). Two additional loci for JBTS have been regions (84,85). ALMS1 forms a part of the centrosome (86,87). identified: JBTS1 on chromosome 9q34.3 (59) and JBTS2/CORS2 on This, together with the finding that BBS proteins localize to cen- chromosome 11p12-q13.3 (60). trosomes, confirms the role of centrosomal proteins in cystic kidney diseases that are associated with diabetes, obesity, and retini- Liver Fibrosis and Skeletal Changes tis pigmentosa (88,89). Additional NPHP-associated disorders are NPHP may be associated with liver fibrosis (40,61–63). Pa- Sensenbrenner syndrome (cranioectodermal dysplasia) (90,91) tients develop hepatomegaly and moderate portal fibrosis with and Arima syndrome (cerebro-oculo-hepato-renal syndrome) (92– mild bile duct proliferation. This pattern differs from that of 94). NPHP has also been described in association with ulcerative classical congenital hepatic fibrosis, whereby biliary dysgenesis colitis (95). is prominent. A recessive mutation in the NPHP3 gene was recently described in a patient with NPHP and liver fibrosis (18). Hepatic involvement in NPHP type 2 (infantile NPHP) Positional Cloning Reveals Seven Causative seems to involve only transient elevation of transaminases (23). Genes for NPHP The association of NPHP with cone-shaped epiphyses of the Since its first description in 1945 (2,3), the pathogenesis of phalanges (type 28 and 28A), known as Mainzer-Saldino syn- NPHP had been elusive. Positional cloning revealed novel drome, was first published by Mainzer et al. (41) in patients genes that cause NPHP when mutated. These are monogenic who also had retinal degeneration and cerebellar ataxia. recessive genes, suggesting that mutations in each single one of these genes is sufficient to cause NPHP in a patient who bears Situs Inversus these mutations, indicating that their products are necessary for The presence of situs inversus was shown in a patient with normal kidney function. Positional cloning thereby generated infantile NPHP and mutations in the NPHP2/inversin gene (17). new insights into disease mechanisms of NPHP and demon- Therefore, the role of inversin in left–right axis specification that strated that they are related to signaling mechanisms of sensory had been described in mice was confirmed in humans (64,65). The cilia, centrosomes, and planar cell polarity (1,72,96). Seven 1858 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1855–1871, 2007

NPHP-associated genes have been identified so far: NPHP1 fluid movement in the kidney tubule, where polycystin-1 trans- through 6 and AHI1 (15–21,54,55,57) (Table 1). mits the signal to polycystin-2, which is a TRP type calcium ϩ channel. This would produce sufficient Ca2 influx to induce ϩ NPHP1 Ca2 release from intracellular storage, which then regulates We identified mutations in NPHP1 as causing juvenile NPHP numerous intracellular signaling activities that are linked to the type 1 (15,16). NPHP1 encodes nephrocystin-1, a protein that regulation of cell cycle and planar cell polarity (103). In partic- interacts with components of cell–cell and cell–matrix signaling, ular, inversin/NPHP2 function has been implicated in signal- including p130Cas (97), focal adhesion kinase 2 (98), tensin, and ing mechanisms of planar cell polarity (see The Wnt Pathway) filamin A and B (99,100). It also interacts with the products of (104). Okada et al. (105) previously demonstrated that inversin other NPHP genes, such as nephrocystin-2/inversin (17), nephro- is needed to position the cilia in cells of the ventral node. cystin-3 (18), and nephrocystin-4 (57,101). Nephrocystin-1 localizes to adherens junctions and focal adhesions of renal epithelial NPHP3 cells (99,100), which are involved in cell–cell and cell–basement By positional cloning in a large Venezuelan kindred (7), we membrane communications, respectively (101,102) (Figure 2). identified mutations in NPHP3 as responsible for adolescent NPHP (18). We demonstrated that mutations in the murine NPHP2 ortholog Nphp3 cause the renal cystic mouse mutant pcy (18), The renal cystic changes of infantile NPHP (NPHP type 2) which was recently shown to be responsive to treatment with a combine clinical features of NPHP and of PKD (5). Guided by vasopressin receptor antagonist (106). mapping of a locus for infantile NPHP to chromosome 9q21- q22 (23) and by the observation that a deletion in the inversin NPHP4 (Invs) gene causes renal cystic phenotype in the inv/inv mouse Mutations in NPHP4 were identified by homozygosity map- model (64,65), we identified mutations in human inversin ping and total genome search for linkage (19,57,107). The en- (INVS) as the cause of infantile NPHP (type 2) with and with- coded protein, nephrocystin-4/nephroretinin, is in a complex out situs inversus (17). Inversin interacts with nephrocystin-1 with other proteins that are involved in cell adhesion and actin and with ␤-tubulin, which constitutes the microtubule axoneme cytoskeleton organization, such as nephrocystin-1, p130Cas, of primary cilia. We demonstrated that nephrocystin-1 and Pyk2, tensin, filamin, and ␣-tubulin. In polarized epithelial inversin localize to primary cilia of renal tubular cells (17)—the cells, nephrocystin-4 localizes to primary cilia, basal bodies, same subcellular compartment that was identified as central to and the cortical actin cytoskeleton, whereas in dividing cells, it the pathogenesis of PKD (1,66,103). This was one of the first localizes to centrosomes (101). Nephrocystin-4 is conserved in findings to support a unifying theory of renal cystogenesis Caenorhabditis elegans and expressed in ciliated head and tail (1,72), which states that proteins that, when mutated, cause neurons of the nematode (108). Upon knockdown, it exhibits a renal cystic disease in humans, mice, or zebrafish (“cystopro- male mating phenotype, similar to orthologs of other genes that teins”) are expressed in primary cilia, basal bodies, or centro- are mutated in cystic kidney disease (108). somes (66,72). The interaction and co-localization to cilia of nephrocystin-1, inversin, and ␤-tubulin provided a functional NPHP5 link between the pathogenesis of NPHP, the pathogenesis of Recently, we identified another novel gene (NPHP5) as being PKD, primary cilia function, and left–right axis determination mutated in NPHP type 5 (20). It is interesting that all mutations (17). The functional relationship between ciliary expression of found were truncations of the encoded protein nephrocystin-5, these so-called “cystoproteins” (proteins mutated in cystic kid- and all patients had early-onset retinitis pigmentosa (SLSN). ney disease) and the renal cystic phenotype, however, is still Nephrocystin-5 contains two IQ domains, which directly inter- somewhat unclear. One of the first concepts for this relation- act with calmodulin (20) and is in a complex with the retinitis ship proposes that cilia may act as mechanosensors to sense pigmentosa GTPase regulator, which when defective causes

Table 1. Genetics and frequency of extrarenal associations in NPHP

NPHP Type Parameter 123456

Mutated gene NPHP1 NPHP2 NPHP3 NPHP4 NPHP5 NPHP6 AHI1 Encoded protein Nephrocystin-1 Nephrocystin-2 Nephrocystin-3 Nephrocystin-4 Nephrocystin-5 Nephrocystin-6 Jouberin Frequency of gene mutation (%)b 20 1 1 2 2 1 Ͻ2 Kidney cysts (%) 100 100 100 100 100 100 ? Retinal degeneration (%) 7 10 Ϸ40 15 100 100 ? Cerebellar vermis aplasia (%) Ϸ1— ———Ϸ90 100 Oculomotor apraxia (Cogan) (%) 1 — — Ͻ1— —?

aNPHP, nephronophthisis. bPercentage of 976 patients from different families with NPHP-associated disorders as evaluated in the authors’ worldwide cohort. Different frequencies have been reported by Saunier et al. (157). J Am Soc Nephrol 18: 1855–1871, 2007 Nephronophthisis-Associated Ciliopathies 1859

Figure 2. Cystoproteins are proteins of genes that are mutated in cystic kidney diseases of humans, mice, or zebrafish. They share the common feature of expression in primary cilia, basal bodies, or centrosomes. Depending on cell-cycle stage, some cystoproteins localize to adherens junctions or focal adhesions. Many cystoproteins have been localized to more than one intracellular domain. The speckled arrow in the primary cilium indicates the direction of anterograde transport along the microtubule system mediated by kinesin 2, a heterotrimeric protein that is composed of two motor units (Kif3a and Kif3b) and one nonmotor unit (KAP3). AJ, adherens junction; BB, basal body; Cen, centriole; ER, endoplasmic reticulum; FAP, focal adhesion plaque; TJ, tight junction; PC-1, polycystin-1; PC-2, polycystin-2. Modified from Watnick and Germino (72), with permission.

X-linked retinitis pigmentosa. Both nephrocystin-5 and retinitis function and tissue architecture in the pathogenesis of cystic kid- pigmentosa GTPase regulator localize to connecting cilia of pho- ney disease, retinitis pigmentosa, and central nervous system de- toreceptors and in primary cilia of renal epithelial cells (20). The velopment. Mutations in NPHP6/CEP290 have been confirmed as fact that connecting cilia of photoreceptors are the structural causing JBTS with and without renal involvement (21,53). It is equivalents of primary cilia of renal epithelial cells may explain interesting that a 300–amino acid in-frame deletion of NPHP6/ retinal involvement in the retinal-renal syndrome SLSN. CEP290 caused retinal degeneration only, without renal or cerebellar involvement in the rds16 mouse model (109). This is in NPHP6 accordance with the recent finding that a hypomorphic mutation Very recently, we identified by positional cloning of recessive of NPHP6/CEP290 represents the most frequent cause of Leber’s truncating mutations in a novel gene NPHP6/CEP290, which en- congenital amaurosis (110). codes a centrosomal protein, as the cause of NPHP type 6 and JBTS type 5 (21). We demonstrated that abrogation of NPHP6 AHI1 function in zebrafish causes planar cell polarity (convergent ex- Finally, mutations in AHI1 have been detected in patients tension) defects and recapitulates the human phenotype of NPHP with JBTS with and without renal involvement (51,52,54,55). type 6, including renal cysts, retinitis pigmentosa, and cerebellar Taken together, these findings indicate that the nephrocystin defects (21). In addition, a defect in cell size control and morpho- proteins are involved in functions of sensory cilia, cell polarity, genesis was found upon nphp6 knockdown in the nonvertebrate and cell division (101). Ciona intestinalis. Nephrocystin-6 modulates the activity of ATF4/ CREB2, a transcription factor that is implicated in cAMP-depen- Cilia: A Unifying Theory for Cystic dent renal cyst formation (106). Nephrocystin-6 is expressed in Kidney Disease centrosomes and the mitotic spindle in a cell cycle–dependent The demonstration that nephrocystin-1 and inversin/ manner. Its identification establishes a link between centrosome NPHP-2 localize to primary cilia of renal tubular cells (17) was 1860 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1855–1871, 2007 among the first findings to support a new unifying theory of renal cystogenesis (72). This theory states that proteins that, when mutated, cause renal cystic disease in humans, mice, or zebrafish (“cystoproteins”) are expressed in primary cilia, basal bodies, or centrosomes (66,72) (Figure 3).

Cilia Structure and Function The cilium is a hair-like structure that extends from the cell surface into the extracellular space. Virtually all vertebrate cell types have cilia in developing or mature tissue. Cilia consist of a microtubule-based axoneme covered by a specialized plasma membrane. The axoneme has nine peripheral microtubule doublets arranged around a central core. There may be two central microtubules (9 ϩ 2or9ϩ 0 axoneme; Figure 3). 9 ϩ 2 cilia usually have dynein arms that link the microtubule doublets and are motile, whereas most 9 ϩ 0 cilia lack dynein arms and are nonmotile (“primary cilia”). The ciliary axoneme is an- chored in the basal body, a microtubule-organizing center de- rived from the older of the two centrioles. The transition zone at the junction of the basal body acts as a filter for the molecules that can pass into or out of the cilium. Nephrocystin-1 is localized at the transition zone of epithelial cells (111). During generation of cilia (ciliogenesis), cilia elongate from the basal body by the addition of new axonemal subunits to the distal tip, the plus end of the microtubules. Axonemal and membrane components are transported in raft macromolecular particles (complex A and B) by so-called intraflagellar transport (IFT) along the axonemal doublet microtubules (Figure 3) (112). An- terograde transport toward the tip is driven by heterotrimeric kinesin 2, which contains motor subunits Kif3a and Kif3b and a nonmotor subunit. Mutations of Kif3a cause renal cysts in mice (113). Kinesins also help form signaling complexes within the ciliary membrane. Retrograde transport back to the cell body occurs via the motor protein cytoplasmic dynein 1B (114). The unexpected convergence in primary cilia of proteins that underlie cystic kidney diseases is supported by the following findings: (1) there is high evolutionary conservation of the proteins involved (Figure 4, A through D), (2) most of these proteins interact with each other (Figure 4C), and (3) clinical phenotypes are related to tissue-specific expression in the sensory cilia (Figure 4, E through G).

Cilia: Conserved Modules to Sense Cell External Signals Figure 3. Cilia structure and intraflagellar transport (IFT). (A) A typ- It is becoming apparent that primary cilia are highly con- ical cilium consists of an axoneme of nine doublet microtubules. Each served structures for sensing of a wide variety of extracellular doublet arises from the inner two microtubules of the basal body cues by a broad variety of specialized tissues. A common microtubule triplets. The axoneme is surrounded by a specialized ciliary membrane that is separated from the cell membrane by a zone “theme” conserved through evolution seems to suggest that of transition fibers. (B) A cross-section of 9 ϩ 2 and 9 ϩ 0 cilium. Cilia many times when cells are to receive cues from the outside of are broadly divided into two types on the basis of the presence or the cell, they use a primary cilium. A broad range of cues can be absence of a central pair of microtubule singlets in the axoneme (9 ϩ received by specific ciliary receptors. These include photosen- 2or9ϩ 0 structure, respectively). Inner and outer dynein arms, sation (rhodopsin), mechanosensation (polycystin-1 and -2), which are usually associated with 9 ϩ 2 cilia, can be present in either osmosensation, and olfactory sensation (seven-membrane type of cilium and are important for ciliary motility. Ciliary assembly spanning olfactory receptors). The decision rules by which cells and maintenance is accomplished by IFT, which relies on the micro- place a specific receptor molecule into the cilium are unknown. tubule motor proteins kinesin 2 and cytoplasmic dynein to transport In general, it seems that the pathogenesis of ciliopathies is IFT protein complexes and their associated cargo up and down the based on an inability of epithelial cells to sense or process length of the cilium (depicted in A). Eb1, end-binding protein 1. extracellular cues (115). Adapted from Bisgrove and Yost (110a). J Am Soc Nephrol 18: 1855–1871, 2007 Nephronophthisis-Associated Ciliopathies 1861

Figure 4. The cilia/basal body hypothesis of renal cystic disease and related disorders. This illustration summarizes the new unifying pathogenic theory of cystic kidney disease, which states that virtually all proteins that are mutated in cystic kidney disease of humans, mice, or zebrafish (“cystoproteins”) show expression of their encoded proteins in primary cilia, basal bodies, or centrosomes. The horizontal axis symbolizes the flow of genetic information from genes over proteins to disease phenotypes. The vertical axis represents evolutionary time. (A) On the evolutionary scale, genes that are responsible for renal cystic disease in vertebrates are shown, including the unicellular organism Chlamydomonas reinhardtii. (B) Many of the orthologs of human cystic kidney disease genes are expressed in ciliated neurons of head and tail of the nematode Caenorhabditis elegans, where they lead to a male mating phenotype when knocked down (e.g., lov-1 or nph-4) (see E). Most proteins that are mutated in Bardet-Biedl syndrome (BBS) are conserved as basal body components of motile cilia (flagella) C. reinhardtii, where mutations in these genes lead to a phenotype of defective IFT or propulsion (see E). (C) Many cystoproteins directly interact with each other (e.g., NPHP1 and NPHP2/inversin). (D) Recently, it was discovered that the products of all genes that are mutated in cystic kidney diseases of humans, mice, or zebrafish show expression of their encoded proteins in primary cilia, basal bodies, or centrosomes. These findings placed primary cilia and centrosomes at the center of these disease processes (D has no vertical dimension). (E through G) The convergence of the pathogenesis at sensory cilia that serve distinct functions in different tissues may explain the broad organ involvement (pleiotropy) of NPHP and BBS that includes many different organ systems through defects of sensory cilia. Pleiotropic phenotypes in NPHP or BBS include cystic kidney disease, retinitis pigmentosa, infertility, diabetes, and other diseases of premature aging of organs.

Evolutionary Conservation of Cystoproteins in the lifespan of the worm has been demonstrated (121). In C. elegans, For many “cystoproteins,” the renal cystic phenotype is con- bbs-7 and bbs-8 are required for the correct localization/motility of the served among vertebrates. For example, mutations in inversin IFT proteins osm-5/polaris and che-11 (122). A bioinformatics ap- lead to NPHP type 2 in humans, mice, and zebrafish (Figure 4) proach based on the ciliary/basal body hypothesis of BBS pathogen- (17). At least two nephrocystins are conserved even in the nema- esis also helped to identify the cystoprotein BBS5 (123). Using bioin- tode C. elegans by amino acid sequence, functional features, and formatic screens for ciliary genes in combination with data from their expression patterns and knockdown phenotypes: Nphp-1 positional cloning, mutations in ARL6 were identified as being re- and nphp-4 are expressed in head (amphid) and tail (phasmid) sponsible for BBS type 3 (124,125). ARL6, a small GTPase, is specifi- neurons, which are ciliated osmosensor neurons of C. elegans (Figure 4A) (108,116). Localization of nephrocystin-1 and -4 to cally expressed in ciliated cells and undergoes IFT. some of these ciliated neurons overlaps with localization of the Evolutionary conservation of cystoproteins goes even fur- Ͼ cystoprotein orthologs polycystin-1 (lov-1) and polycystin-2 ther: Some cystoproteins have been conserved over 1.5 billion (pkd-2) and with many orthologs of BBS proteins (117). Knock- years of evolution from the unicellular organism Chlamydomo- down of nphp-1 and nphp-4 leads to impaired male mating behav- nas reinhardtii to vertebrates (Figure 4, A and B). C. reinhardtii ior (108), similar to what has been described for lov-1 and pkd-2 uses two motor cilia (flagella) for locomotion. Strikingly, mutants (118). These data have been confirmed and refined for nephrocystin-4 and at least six proteins that are mutated in BBS specific neuronal cell type (119,120). In addition, a role for nphp-4 are conserved in C. reinhardtii where they are part of the basal 1862 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1855–1871, 2007 body proteome (117,126). Defects of cystoprotein orthologs in Retinal Degeneration. The renal-retinal involvement in C. reinhardtii have deficient IFT and flagellar propulsion (127). SLSN can be explained by the fact that the primary cilium of renal epithelial cells is a structural equivalent of the connecting Nephrocystin Complexes cilium of photoreceptor cells in the retina. We have shown that Many cystoproteins participate in protein–protein interaction nephrocystin-5 and nephrocystin-6 are expressed in the con- complexes (Figure 4C). These interactions may partially explain necting cilia of photoreceptors (20,21). In analogy to motor why mutations in different NPHP genes lead to similar pheno- transport along the axoneme of primary cilia in kidney epithe- types. Some of the domains that occur in cystoproteins are shared lial cells, in the connecting cilia of photoreceptors, cargo is between different proteins. Coiled-coil domains, for example, oc- trafficked along microtubule tracks from the photoreceptor in- cur in six of eight proteins that are defective in NPHP-like diseases ner segment to the outer segment via a motor protein complex (nephrocystin-1, -2, -3, -5, and -6 and ALMS1). Nephrocystin-1 is that contains kinesin 2 and back to the cell body via a cytoplas- targeted to the transition zone of motile and primary cilia (111), mic dynein (Figure 5) (129). In this way, 10 billion molecules of and casein kinase 2–mediated phosphorylation of three critical the visual pigment rhodopsin are trafficked up and down the serine residues within a cluster of acidic amino acids in nephro- connecting cilia per human retina per day. cystin mediates phosphofurin acidic cluster sorting protein Liver Fibrosis. Both NPHP and BBS can be associated with (PACS)-1 binding and is essential for co-localization of nephrocys- liver fibrosis (40), whereas autosomal recessive PKD is associ- tin with PACS-1 at the base of cilia (128). The basal body/centro- ated with bile duct ectasia. Bile duct involvement in these cystic somal expression of proteins that are involved in NPHP has led to kidney diseases may be explained by the ciliary theory, because identification of mutations in NPHP6/CEP290 as the cause of the epithelial cells lining bile ducts (cholangiocytes) possess NPHP type 6 (JBTS type 5). Its gene product nephrocystin-6/ primary cilia (Figure 4, F and G). Cep290 (21) is part of the centrosomal proteome (86). Central Nervous System. Recent findings suggest that oc- Extrarenal Organ Involvement by Ciliary Dysfunction ulomotor apraxia type Cogan (associated with NPHP1 and A prominent feature of NPHP is that in a certain percentage NPHP4 mutations), cerebellar vermis hypoplasia (in JBTS), and of cases, there can be involvement of multiple organs (pleiot- mental retardation (in NPHP type 6) may be due to defects in ropy) other than the kidney. In some instances, there seems to microtubule-associated functions during neurite outgrowth be a genotype/phenotype correlation regarding pleiotropy. For and axonal guidance. Mechanotransport along microtubules instance, there is involvement of the retina in all known cases plays a role not only in intraciliary but also in axonal transport with mutations of NPHP5 or NPHP6. In other instances, such as (130). An example is the motor protein KIF3A, which is mu- NPHP1 mutations, the molecular basis of eye involvement is tated in a renal cystic mouse model and also plays a role in unknown. The pleiotropy of NPHP has now found a potential axonal transport (113). Because the malformations of the cere- explanation in the ciliary hypothesis of cystic kidney diseases bellum that occur in JBTS (37) consist of abnormal “wiring” of (Figure 4, E through G). The extrarenal organ involvement in decussating (crossing) neurons, impaired axonal outgrowth NPHP by organ system is discussed as follows. and axon guidance may be central to the neurologic defects in

Figure 5. Primary (nonmotile) cilia of renal epithelial cells and connecting cilia of retinal photoreceptors are analogous structures. In the primary cilium (A) of renal epithelial cells (B), “cargo” proteins are trafficked along the microtubule tracks from the region of the Golgi stack to the tip of the cilia via the motorprotein kinesin 2 and back down via cytoplasmic dynein 1b. (C) In an analogous manner, approximately 109 molecules of the visual pigment rhodopsin are transferred up and down the connecting cilia per human retina per day. Adapted from references (66,129). J Am Soc Nephrol 18: 1855–1871, 2007 Nephronophthisis-Associated Ciliopathies 1863

JBTS, in analogy to the lissencephaly phenotype that is caused caused by mutations in uromodulin, which has so far not been by the centrosomal proteins LIS1 and doublecortin (131). detected in cilia, basal bodies, or centrosomes. Congenital Cardiac Malformations. In a patient with infantile NPHP, we observed a ventricular septal defect as a congenital cardiac malformation (17). This developmental defect may be Focal Adhesion Hypothesis viewed as a “heterotaxy” (left–right orientation) phenotype that is When NPHP1 was first identified (15), we proposed a patho- caused by the same mechanism (68) that leads to situs inversus in genic hypothesis that tied in nephrocystin-1 with defects of this patient. The phenotypic combination of NPHP, situs inversus, cell–cell and cell–matrix signaling (102,138). This was based on and cardiac septal defect on the basis of inversin mutations is the finding that nephrocystin-1 contains an SH3 domain, local- observed in humans, mice, and zebrafish (17). izes to adherens junctions and focal adhesions of renal epithe- Obesity. Obesity is part of the clinical spectrum of the lial cells, and interacts with integral components of these struc- ciliopathy BBS, and excessive obesity has been described in tures, such as p130CAS (99,100). This “adherens junction/focal children with NPHP6 mutations after renal transplantation (B. adhesion hypothesis” of NPHP pathogenesis (102,138) recently Hoppe, University of Cologne, Germany, personal communi- was partially reconciled with the “cilia/centrosome” hypothe- cation, 2006). It is interesting that in the Bbs6 knockout mouse sis in an integrative hypothesis by showing that nephrocystin- model, obesity was associated with hyperphagia and decreased 4/nephroretinin in polarized epithelial cells co-localizes with ␤ activity of the mice (132). -catenin at cell–cell contact sites and to primary cilia, whereas in dividing cells, it localizes to centrosomes (101) (Figure 6, A Signaling Mechanisms Relevant for NPHP and B). Nephrocystins and other cystoproteins are expressed in different subcellular compartments in a cell cycle–dependent The Wnt Pathway manner (21,133). These subcellular compartments include focal Recent results on inversin/NPHP2 shed light on the mech- adhesions, adherens junctions, cilia, basal bodies, centrosomes, anosensory hypothesis of bending of primary cilia by tubular and the mitotic spindle (Figure 6). It is still mostly unclear flow. They have provided data on downstream signaling events which of these subcellular localizations are most proximal to that are necessary to maintain normal tubular development and the pathogenesis of NPHP or other cystic kidney diseases. In morphology (104): In this model (Figure 6,C, F, and H) the canon- relation to these subcellular localizations, many hypotheses on ical Wnt signaling occurs primarily through ␤-catenin–dependent mechanisms of cystogenesis have been put forward. Among pathways in the absence of tubular flow. Stimulation of the pri- them are (1) the mechanosensory hypothesis of renal cilia func- mary cilium by flow, however, increases expression of inversin, tion, (2) participation in signaling at focal adhesions and adhe- which then reduces levels of cytoplasmic disheveled through pro- rens junctions, (3) a role in the maintenance of planar cell teasomal degradation and subsequently switches off the canonical polarity within the noncanonical Wnt signaling pathway, and pathway by allowing activation of the ␤-catenin destruction com- (4) a role in centrosome-related functions of cell-cycle regula- plex (96). When inversin is defective (as in NPHP type 2), the tion. These hypotheses are discussed next (Figure 6). canonical Wnt pathway will prevail and disrupt apical-basolateral polarity of the renal epithelium (96). Because planar cell polarity The Mechanosensory Hypothesis signaling is important for oriented cell division, it seems logical On the basis of the initial finding that bending the primary that Fisher et al. (139) recently were able to demonstrate abnormal 2ϩ cilium elicits Ca influx (134–136), it was shown that cilia can orientation of the mitotic spindle in two different rodent models of act as mechanosensors to sense fluid movement in the kidney cystic kidney disease. tubule, in cooperation with polycystins. In this model, polycystin-1 transmits the signal to polycystin-2, which is a TRP cal- ϩ cium channel (103). This produces sufficient Ca2 influx to Centrosomes ϩ induce Ca2 release from intracellular stores, which then reg- Nephrocystin-6, which is mutated in JBTS, is a component of ulates numerous signaling activities inside the cell that are the centrosomal proteome (86). In addition, BBS4 is instru- linked to cell-cycle regulation. It is thought that defects in mental in recruiting proteins to the pericentrosomal matrix, cell-cycle regulation may be ultimately responsible for the de- implicating the centrosome and its relation to cell-cycle control velopment of kidney cysts (103). In support of the ciliary hy- in the pathogenesis of BBS (117,140). The products of the genes pothesis of cystic kidney diseases, a motor protein of the kine- mutated in the ciliopathy Meckel-Gruber syndrome MKS1 (81) sin II family, KIF3A, which is involved in intraciliary transport, and MKS3 (82) recently were shown to localize to basal bodies was shown to cause a murine renal cystic disease when mu- and centrosomes (141) (Figure 6D). tated (113). Kramer-Zucker et al. (137) recently showed that cilia of larval zebrafish kidney tubules have a 9 ϩ 2 configuration Cell Cycle and are motile. Disruption of cilia structure or motility resulted A balance between hyperproliferation and apoptosis may in pronephric cyst formations, with left–right asymmetry de- play an important role in the pathogenesis of cystic kidney fects. Despite many data in support of the ciliary hypothesis, diseases (Figure 6, F and G). For example, whereas in PKD, some data are still hard to reconcile with this model; for exam- kidneys are grossly enlarged, in NPHP and BBS, kidney size ple, the autosomal dominant variant of NPHP, MCKD2, is remains normal and cysts grow at the expense of normal tissue 1864 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1855–1871, 2007

Figure 6. Nephrocystins localize to different subcellular compartments in a cell cycle–dependent manner and participate in multiple signaling pathways together with other “cystoproteins” (proteins that are mutated in cystic kidney disease). Functional complexes that play a role in the planar cell polarity (PCP) pathway are highlighted for focal adhesion (A), adherens junction (B), cilium (C), basal body (D), centrosome (E), nucleus (F), mitotic spindle (G), and the Wnt pathways (H). Cystoproteins are shown on colored background and in bold type using blue for nephrocystins (NPHP), orange for BBS proteins, green for polycystins (PKD) and fibrocystin (PKHD1), and yellow for cystoproteins of renal cystic mouse models. Proteins that are not bold have been described in the context of the pathogenesis of cystic kidney disease (e.g., as a binding partner to a bona fide cystoprotein). Associated proteins with no known role in cystic kidney diseases are shown on gray background. Black dots connect proteins that directly interact. (A) Focal adhesions. (1) Nephrocystin-1 directly interacts with the focal adhesion adapter protein p130Cas (“crk-associated substrate”) (99,100,102), which is a major mediator of focal adhesion assembly, binds to focal adhesion kinase, and mediates stress fiber formation (149). Nephrocystin-1 competes for binding to p130Cas with the proto-oncogene products Src and Fyn (99). (2) Nephrocystin-1 is in a protein complex with the focal adhesion proteins Pyk2/Fak2 (focal adhesion kinase 2), tensin (98), and filamin A and B. Its overexpression leads to activation of extracellular signal–regulated kinases 1 and 2 (ERK1 and ERK2) (98). (3) In children with NPHP, overexpression of ␣5␤1 integrin was described in proximal tubules, which most likely results from defective ␣6-integrin expression (150). (4) The knockout mouse models for tensin (151) and for the Rho GDI␣ gene (152) both exhibit an NPHP-like phenotype, thereby implicating further proteins of the focal adhesion signaling cascade in the pathogenesis of NPHP-like diseases. (B) Adherens junctions. (1) Nephrocystin-1 co-localizes with E-cadherin and p130Cas to adherens junctions. (2) The C-terminal half of nephrocystin, the “nephrocystin homology domain,” is able to promote nephrocystin self-association and epithelial adherens junctional targeting (100). (3) Disruption of this targeting leads to reduced transepithelial resistance (100). (4) Nephrocystin-4 is in a protein complex with nephrocystin-1, NPHP2/inversin, p130Cas, and Pyk2/Fak2 and has been J Am Soc Nephrol 18: 1855–1871, 2007 Nephronophthisis-Associated Ciliopathies 1865

(e vacuo). It seems that hyperproliferation may be the predom- NPHP: Defects of Tissue Differentiation inant mechanism in PKD-like diseases (142), whereas apoptosis and Maintenance is predominant in diseases of the NPHP and BBS group. A role It is striking that in renal cystic diseases (and in the associ- Ϫ/Ϫ Ϫ/Ϫ of apoptosis was in fact confirmed in the Bbs2 and Bbs4 ated extrarenal organ involvement), mutations of monogenic mouse models (143,144). Polycystin-1 and -2 signaling and the disease genes may lead to developmental defects (dysplasia), in renal cystic phenotype may be linked by a function of these which a structural organ defect is present at birth, but also to proteins in cell growth regulation. Polycystin-1 expression ac- degenerative defects (degeneration), in which organ structure tivates the JAK-STAT pathway, thereby upregulating and function are normal at birth but deteriorate over time. p21(waf1) and inducing cell-cycle arrest in G0/G1 (145). The Examples of this phenomenon from the side of cystic kidney cell-cycle arrest requires polycystin-2. Involvement of polycys- disease are autosomal recessive PKD, in which there is a struc- tin-1/2 signaling in the JAK/STAT pathway might explain how tural defect of the kidney present at birth, as opposed to NPHP, mutations of either gene can result in dysregulated growth in which kidneys are normal at birth (with the exception of (145). Very recently, this hypothesis was confirmed by demon- NPHP2) but degeneration leads to loss of renal function over stration that two mouse models of PKD (jck and cpk) can be the course of years. Regarding retinal involvement, NPHP can efficiently treated with the cyclin-dependent kinase inhibitor be associated with the developmental defect of retinal (R) roscovitine (146). coloboma (lack of retinal tissue) in JBTS but also with the

localized to adherens junctions in confluent MDCK cells (101). (C) Primary cilia. Recently, the development of a unifying hypothesis of renal cystogenesis was established (72). This hypothesis states that proteins that, when mutated, cause renal cystic disease in humans, mice, or zebrafish are part of a functional module, as defined by their subcellular localization to primary cilia, basal bodies, or centrosomes. This applies to polycystin-1 and -2; fibrocystin/polyductin; nephrocystin-1, -2 (inversin), -3, -4, and -5; BBS-associated proteins; cystin; polaris; ALMS1; and many others. Because nephrocystins interact, they are represented here as the “nephrocystin complex.” On the basis of positional cloning, mutations in the inversin gene were identified as causing infantile NPHP (type 2) (17). This established a link between the pathogenesis of NPHP and disease mechanisms of PKD (17), in which nephrocystin-1 interacts with both inversin and ␤-tubulin. Because ␤-tubulin is a major component of primary cilia, this led to demonstration of co-localization of all three proteins in the primary renal cilia of epithelial cells (17). The complex also contains NPHP3, which is mutated in adolescent NPHP (type 3) and in the renal cystic mouse model pcy. The ciliary hypothesis of cystic kidney disease was confirmed by revealing that Nphp3 mRNA was expressed in kidney, retinal connecting cilia, ciliated bile ducts, and the node, which regulates left–right body axis in mice (18), and by identifying mutations in nephrocystin-5 (IQCB1), which co-localizes with calmodulin to primary cilia of renal epithelial cells and retinal connecting cilia (20). (D) Basal bodies. Basal bodies are short cylindrical arrays of microtubules and other proteins that are found at the base of cilia and that organize the assembly of the ciliary axoneme. They are analogous to centrosomes. Nephrocystins localize to the transition zone of basal bodies (111). Proteins that are mutated in BBS are components of the basal body transitional zone and are highly conserved in evolution. (E) Centrosomes. For a protein that is mutated in the related disease BBS4, it was shown that BBS4 is instrumental in recruiting proteins (e.g., PCM1) to the pericentrosomal matrix, confirming the role of centrosomal function in the pathogenesis of BBS (116). (F) Transcriptional programs. The transcription factor HNF1␤ regulates transcription of multiple genes that are mutated in cystic kidney disease–related genes (153). (G) Cell-cycle regulation. The hypotheses of functional involvement of the nephrocystin complex at focal adhesions and adherens junction on the one hand and the ciliary/centrosomal hypotheses on the other hand may be integrated by demonstration that different locations of the complex may predominate in a cell cycle–dependent manner. This is evidenced by the findings that inversin/nephrocystin-2 (17), nephrocystin-4 (101), and nephrocystin-6 (21) expression occurs in a cell cycle–dependent manner. Inversin exhibits a dynamic expression pattern in MDCK cells that show expression at centrosomes in early prophase, at spindle poles in metaphase and anaphase, and at the midbody in cells that undergo cytokinesis (133,154). Nephrocystin-4 was detected in MDCK cells at centrosomes of dividing cells and in polarized cells close at the cytoskeleton and in the vicinity of the cortical actin cytoskeleton, with co-localization of p130Cas, Pyk2, and ␤-catenin (101). (H) Wnt pathways. Recent data demonstrated that in renal tubules, inversin/NPHP2 may induce switching from the canonical to the noncanonical Wnt signaling pathway in response to flow sensing by primary cilia of renal tubular cells (104). It is thought that this function of inversin is important to maintain renal epithelial cell polarity (96). The hypothesis that the renal cystic disease phenotype is due to defects in the maintenance of planar cell polarity seems plausible for multiple reasons: (1) It would reconcile previous functional hypotheses, because focal adhesions, adherens junctions, cilia, centrosomes, basal bodies, and regulation of the cell cycle all play a pivotal role in the regulation and maintenance of planar cell polarity (155); (2) because planar cell polarity plays an important role in developmental morphogenesis and also in the regeneration of differentiated tissue, a defect in planar cell polarity may explain both the occurrence of cysts during organogenesis and degenerative cystogenesis as it occurs in NPHP; and (3) the mechanism of convergent extension, which may be central to renal tubular morphology, was shown to be disturbed in many renal cystic diseases (156). APC, adenomatous polyposis coli protein; APC2, anaphase promoting complex subunit 2; CBP, CREB-binding protein; CREB, cAMP response element–binding protein; CK1␧, casein kinase ␧; DKK, Dickkopf-related protein; CK2, casein kinase II; GAP, RhoGTPase activating protein; GEF, guanine nucleotide exchange factor; GSK3, glycogen synthase kinase 3; JUNK, JUN kinase; PCM1, pericentriolar material 1; p150, p150glued/dynactin-1; PKC, protein kinase C; ROCK, Rho-associated protein kinase. 1866 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1855–1871, 2007 degenerative processes of tapetoretinal degeneration/retinitis dorf F, Reis A, Hildebrandt F: Identification of a new gene pigmentosa (21,44,109). The joint occurrence of developmental locus for adolescent nephronophthisis, on chromosome and degenerative defect may be explained by the fact that many 3q22 in a large Venezuelan pedigree. Am J Hum Genet 66: developmental transcriptional programs are reinitiated for 118–127, 2000 mechanisms of tissue maintenance and repair. Because planar 8. Ala-Mello S, Kivivuori SM, Ronnholm KA, Koskimies O, Siimes MA: Mechanism underlying early anaemia in chil- cell polarity plays an important role in developmental morpho- dren with familial juvenile nephronophthisis. Pediatr Neph- genesis and also in the regeneration of differentiated tissue, a rol 10: 578–581, 1996 defect in planar cell polarity may explain both occurrence of 9. Blowey DL, Querfeld U, Geary D, Warady BA, Alon U: cysts during organogenesis and degenerative cystogenesis as it Ultrasound findings in juvenile nephronophthisis. Pediatr occurs in NPHP. Nephrol 10: 22–24, 1996 9a. Hildebrandt F, Sayer JA, Jungers P, Gru¨nfield J-P: Therapeutic Approaches to NPHP Nephronophthisis—medullary cystic and medullary No effective prophylaxis or treatment is available for NPHP. sponge kidney disease. In: Diseases of the Kidney and Urinary The only therapeutic options are supportive treatment once Tract, edited by Schrier WB, Philadelphia, Lippincott, Wil- chronic renal failure has developed and dialysis and transplan- liams & Wilkins, 2007, pp 478–501 tation for terminal renal failure. An important future challenge 10. Waldherr R, Lennert T, Weber HP, Fodisch HJ, Scharer K: The nephronophthisis complex. A clinicopathologic study will be the development of therapies that capitalize on what we in children. Virchows Arch A Pathol Anat Histol 394: 235– have learned about the biology of NPHP and other cystic 254, 1982 diseases of the kidney. Gattone et al. (106) recently showed that 11. Zollinger HU, Gaboardi F, Imbasciati E, Lennert T: the renal cystic phenotype of pcy mice, which is the equivalent Nephronophthisis (medullary cystic disease of the kidney). of human NPHP type 3, can be strongly mitigated or even A study using electron microscopy, immunofluorescence, reversed by treatment with the vasopressin V2 receptor antag- and a review of the morphological findings. Helv Paediatr onist OPC31260. Similar results were obtained using a pkd2 Acta 35: 509–530, 1980 mouse model (147). This effect is thought to be mediated by a 12. Gardner KD Jr: Juvenile nephronophthisis and renal med- reduction in intracellular cAMP levels, a finding that awaits ullary cystic disease. Perspect Nephrol Hypertens 4: 173–185, reconciliation with the ciliary/centrosome hypothesis of 1976 NPHP. In this context, it is interesting that nephrocystin-6 13. Kleinknecht C: The inheritance of nephronophthisis. 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