Too Much of a Good Thing
Authors: Yiqiang Cai, Stefan Somlo
Journal: Journal of the American Society of Nephrology (2008)

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by age-dependent occurrence of bilateral, multiple renal cysts resulting in kidney enlargement in association with a variable spectrum of extrarenal manifestations, most commonly simple cysts arising from bile ducts in the liver. Nephronophthisis (NPHP) is an autosomal recessive kidney disease characterized by tubular basement membrane disruption, tubular atrophy, and tubulointerstitial nephritis, associated with corticomedullary cysts typically without kidney enlargement.1 Liver disease, when it occurs in NPHP, is characterized by portal tract fibrosis with little or no bile duct proliferation. ADPKD and NPHP, respectively, are the most common genetic causes of end-stage kidney disease in adults and children or adolescents. Cysts are considered the initiating pathogenetic event in ADPKD to the point where recent studies suggest that serial measurements of cyst and kidney volumes can be used as determinants of disease progression.2 Cysts in NPHP do not have a similar central role in pathogenesis of the disease. Nonetheless, a growing body of cellular and molecular evidence has come to suggest an interrelationship among these diseases and a number of others, such as autosomal recessive polycystic kidney disease (ARPKD), Bardet-Biedl syndrome (BBS), and Meckel-Gruber syndrome. These relationships are based on the association of many of the causative disease gene protein products with the cilia/basal body complex. There is little doubt that the primary cilium plays a central role in both establishing and maintaining the complex yet reproducible three-dimensional structure of the kidney. In addition to “guilt by association” arising from the finding that many gene products that are mutated in fibrocystic kidney diseases in humans and mice localize to cilia, there are several prospective studies confirming this association. For example, kidney cysts develop when Kif3a, a component of the anterograde transport machinery required for structural integrity of cilia but not otherwise known to be associated with cystic diseases, is inactivated in the kidney.3 Similarly, a forward genetic screen in zebrafish using pronephric kidney tubule dilation as the phenotype identified 10 mutant genes among which there was a marked overrepresentation of cilia-associated protein products.4 This convergence on the importance of the cilia/basal body complex in fibrocystic diseases has fostered a reductionist approach to mechanism based on the premise that at some level all of these proteins should be working together. In fact, disease protein interactions and complexes have been readily identified within disease groups. For example, the two ADPKD proteins, polycystin-1 and polycystin-2, have long been known to associate with each other.5 Several of the NPHP gene products interact among themselves,1 and several of the BBS gene products form a functional complex termed the BBSome.6 Functional association or complex formation among proteins across these disease entities have been more difficult to document. Protein and genetic data raise the possibility that ADPKD and ARPKD gene products affect a common pathway,7 but direct links between NPHP or BBS with either autosomal polycystic kidney disease has been less apparent. Two articles in this issue of JASN are among the first to suggest a direct link between ADPKD and NPHP, a link based on the NIMA (never-in-mitosis gene A)-related kinase family member NEK8. Otto et al.8 identified amino acid substitution mutations in NEK8 in families with NPHP and propose NEK8 as the NPHP9 gene. Sohara et al.9 report that Nek8 and polycystin-2 exist in a complex and describe increased expression of polycystin-1 and polycystin-2 along with abnormal phosphorylation of polycystin-2 in the Nek8 mutant jck mouse. The combined conclusions that NEK8 is an NPHP gene and its protein product may modulate ciliary targeting of polycystin-1 and -2 poses Nek8 as a linchpin between NPHP and ADPKD. Nek8 belongs to the family of NIMA-related serine-threonine kinases, which has 11 members in mammalian species.10,11 Among these, Nek2 is involved in G2-M regulation and centrosome separation; Nek6 and Nek7 are components of a mitotic kinase cascade; Nek9 plays a role in chromosome alignment and segregation during mitosis; and Nek1 and Nek8 play important roles in cilia, cell cycle, and polycystic kidney disease.10 The first evidence suggesting involvement of Neks in cystogenesis was the identification of mutations in Nek1 as causing polycystic kidney disease and pleiotropic extrarenal effects, including facial dysmorphism, dwarfism, male sterility, anemia, and cystic choroid plexus in kat and kat2J mice.12 Subsequently, a missense mutation in the RCC1 domain of Nek8 was identified in a mouse model of autosomal recessive juvenile cystic kidney disease, the jck mouse.13 Nek1 localizes to the centrosomes in interphase, whereas Nek8 localizes to the proximal region of the primary cilia of mouse renal epithelial cells.14 Mutant Nek8 is absent from cilia of cultured kidney tubule epithelial cells from jck mice, but expression of polycystin-1 and -2 is enhanced and the cilia are longer in these cells.15 In addition, Nek8 and polycystin-1 have recently been associated with reciprocal effects on each other's cilia location. Nek8 expression is increased in cilia of Pkd1−/− cultured kidney explants, and, conversely, polycystin-1 is increased in cilia of jck kidney explants.16 The study by Sohara et al.9 extends these findings to kidney tissue and more directly addresses the interrelationship of Nek8 with the polycystins. The authors show that polycystin-2 co-immunoprecipitates with Nek8 and that this interaction is not lost with the jck mutation. Expression of polycystin-1 and -2 at the level of both transcript and protein is increased in jck kidneys, although only polycystin-1 shows a marked increase. In keeping with the previously reported studies in cells,15 this increase in global cellular expression is associated with increased expression of polycystins in the primary cilia as well. Although these results are suggestive, quantitative conclusions based on native protein immunofluorescence is very difficult to interpret conclusively. This is highlighted by the discrepancy in Nek8 expression in cell lines from jck mice, where it is absent,15 and jck kidney tissue, where it expressed along the entire length of the cilium.9 In addition, in the jck mouse kidney, polycystin-2 has an altered electrophoretic migration, which the authors propose is an aberrantly phosphorylated form of the protein on the basis of its disappearance after treatment with protein phosphatase-1. In aggregate, the study by Sohara et al.9 as well as previous studies15,16 raise the possibility that polycystin-1 and -2 may be downstream targets of Nek8. Some genetic evidence to support this comes from the observation that doubly heterozygous Pkd1 and Nek8 mutant mice have discernible kidney cysts at 16 weeks, a finding not observed in singly heterozygous mice for either gene.16 Although this finding is suggestive of a genetic interaction, it does not permit determination of whether either gene functions upstream or downstream of the other in a pathway. Studies examining the effects of Pkd1+/− genotype on disease progression in jck/jck homozygous mice and the reciprocal studies with kidney-specific Pkd1 knockouts and jck heterozygous mice could address this hypothesis further. In the second article, Otto et al.8 used a candidate gene approach to screen NEK8 in 188 patients with NPHP using direct sequencing and an additional 400 patients with NPHP using polymorphisms near NEK8 followed by sequencing of those showing homozygosity. They identified three amino acid substitution variants in highly conserved residues, but only one occurred in a homozygous state as would be expected for a causative NPHP gene. One of the two heterozygous changes occurred in an individual with homozygous NPHP5 mutations. The three variants were not observed among a relatively small number of control subjects. A directed yeast two-hybrid screen for interaction of NEK8 with NPHP, BBS, intraflagellar transport, and murine polycystic kidney disease gene products yielded no interacting partners despite the high sensitivity and limited specificity of this approach. The authors go on to show that NEK8 variants observed in patients with NPHP markedly reduce or abolish centrosomal and ciliary location of NEK8, suggesting that these may be pathogenic mutations. In aggregate, the data support the conclusion the mutations in NEK8 may be a rare cause of NPHP in humans. The work of Otto et al.8 and Sohara et al.9 together pose the question of whether the mechanisms of NPHP and ADPKD are interrelated, but they do not answer that question. It is not clear that a disease in which nonmutant polycystins are overexpressed in lengthened cilia as a result of Nek8 mutations (NPHP9) speaks to the same mechanism as a disease in which polycystins are nonfunctional or absent from cilia (ADPKD). It remains possible that the observed alterations in polycystin protein in cilia are the result of a more generalized alteration in cilia composition, and if one looked at a spectrum of cilia proteins, then a generalized abnormality not restricted to polycystins may be observed. It is important in studying cilia-related structural diseases of the kidney that we continue to seek unbiased approaches toward understanding mechanism and remain cautious in focusing only on what we know. This was a lesson well learned once before in this field when cilia were unknown in cystic disease and may yet be the lesson for the future as well. DISCLOSURES None. The author's are members of the Yale Center for Polycystic Kidney Disease Research (NIH P50 DK57328).