Agonists of prostaglandin E2 receptors as potential first in class treatment for nephronophthisis and related ciliopathies.

Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy belonging to the ciliopathy disorders and known as the most common cause of hereditary end-stage renal disease in children. Yet, no curative treatment is available. The major gene, NPHP1, encodes a protein playing key functions at the primary cilium and cellular junctions. Using a medium-throughput drug-screen in NPHP1 knockdown cells, we identified 51 Food and Drug Administration-approved compounds by their ability to alleviate the cellular phenotypes associated with the loss of NPHP1; 11 compounds were further selected for their physicochemical properties. Among those compounds, prostaglandin E1 (PGE1) rescued ciliogenesis defects in immortalized patient NPHP1 urine-derived renal tubular cells, and improved ciliary and kidney phenotypes in our NPH zebrafish and Nphp1 knockout mouse models. Furthermore, Taprenepag, a nonprostanoid prostaglandin E2 receptor agonist, alleviated the severe retinopathy observed in Nphp1−/− mice. Finally, comparative transcriptomics allowed identification of key signaling pathways downstream PGE1, including cell cycle progression, extracellular matrix, adhesion, or actin cytoskeleton organization. In conclusion, using in vitro and in vivo models, we showed that prostaglandin E2 receptor agonists can ameliorate several of the pleotropic phenotypes caused by the absence of NPHP1; this opens their potential as a first therapeutic option for juvenile NPH-associated ciliopathies.

The renal inflammatory network of nephronophthisis.

Renal ciliopathies are the leading cause of inherited kidney failure. In autosomal dominant polycystic kidney disease (ADPKD), mutations in the ciliary gene PKD1 lead to the induction of CCL2, which promotes macrophage infiltration in the kidney. Whether or not mutations in genes involved in other renal ciliopathies also lead to immune cells recruitment is controversial. Through the parallel analysis of patients' derived material and murine models, we investigated the inflammatory components of nephronophthisis (NPH), a rare renal ciliopathy affecting children and adults. Our results show that NPH mutations lead to kidney infiltration by neutrophils, macrophages and T cells. Contrary to ADPKD, this immune cell recruitment does not rely on the induction of CCL2 in mutated cells, which is dispensable for disease progression. Through an unbiased approach, we identified a set of inflammatory cytokines that are upregulated precociously and independently of CCL2 in murine models of NPH. The majority of these transcripts is also upregulated in NPH patient renal cells at a level exceeding those found in common non-immune chronic kidney diseases. This study reveals that inflammation is a central aspect in NPH and delineates a specific set of inflammatory mediators that likely regulates immune cell recruitment in response to NPH genes mutations.

Mouse genetics reveals Barttin as a genetic modifier of Joubert syndrome.

Genetic and phenotypic heterogeneity and the lack of sufficiently large patient cohorts pose a significant challenge to understanding genetic associations in rare disease. Here we identify Bsnd (alias Barttin) as a genetic modifier of cystic kidney disease in Joubert syndrome, using a Cep290-deficient mouse model to recapitulate the phenotypic variability observed in patients by mixing genetic backgrounds in a controlled manner and performing genome-wide analysis of these mice. Experimental down-regulation of Bsnd in the parental mouse strain phenocopied the severe cystic kidney phenotype. A common polymorphism within human BSND significantly associates with kidney disease severity in a patient cohort with CEP290 mutations. The striking phenotypic modifications we describe are a timely reminder of the value of mouse models and highlight the significant contribution of genetic background. Furthermore, if appropriately managed, this can be exploited as a powerful tool to elucidate mechanisms underlying human disease heterogeneity.

Mutations in GREB1L Cause Bilateral Kidney Agenesis in Humans and Mice.

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute a major cause of chronic kidney disease in children and 20% of prenatally detected anomalies. CAKUT encompass a spectrum of developmental kidney defects, including renal agenesis, hypoplasia, and cystic and non-cystic dysplasia. More than 50 genes have been reported as mutated in CAKUT-affected case subjects. However, the pathophysiological mechanisms leading to bilateral kidney agenesis (BKA) remain largely elusive. Whole-exome or targeted exome sequencing of 183 unrelated familial and/or severe CAKUT-affected case subjects, including 54 fetuses with BKA, led to the identification of 16 heterozygous variants in GREB1L (growth regulation by estrogen in breast cancer 1-like), a gene reported as a target of retinoic acid signaling. Four loss-of-function and 12 damaging missense variants, 14 being absent from GnomAD, were identified. Twelve of them were present in familial or simplex BKA-affected case subjects. Female BKA-affected fetuses also displayed uterus agenesis. We demonstrated a significant association between GREB1L variants and BKA. By in situ hybridization, we showed expression of Greb1l in the nephrogenic zone in developing mouse kidney. We generated a Greb1l knock-out mouse model by CRISPR-Cas9. Analysis at E13.5 revealed lack of kidneys and genital tract anomalies in male and female Greb1l-/- embryos and a slight decrease in ureteric bud branching in Greb1l+/- embryos. We showed that Greb1l invalidation in mIMCD3 cells affected tubulomorphogenesis in 3D-collagen culture, a phenotype rescued by expression of the wild-type human protein. This demonstrates that GREB1L plays a major role in early metanephros and genital development in mice and humans.

Mutations in MAPKBP1 Cause Juvenile or Late-Onset Cilia-Independent Nephronophthisis.

Nephronophthisis (NPH), an autosomal-recessive tubulointerstitial nephritis, is the most common cause of hereditary end-stage renal disease in the first three decades of life. Since most NPH gene products (NPHP) function at the primary cilium, NPH is classified as a ciliopathy. We identified mutations in a candidate gene in eight individuals from five families presenting late-onset NPH with massive renal fibrosis. This gene encodes MAPKBP1, a poorly characterized scaffolding protein for JNK signaling. Immunofluorescence analyses showed that MAPKBP1 is not present at the primary cilium and that fibroblasts from affected individuals did not display ciliogenesis defects, indicating that MAPKBP1 may represent a new family of NPHP not involved in cilia-associated functions. Instead, MAPKBP1 is recruited to mitotic spindle poles (MSPs) during the early phases of mitosis where it colocalizes with its paralog WDR62, which plays a key role at MSP. Detected mutations compromise recruitment of MAPKBP1 to the MSP and/or its interaction with JNK2 or WDR62. Additionally, we show increased DNA damage response signaling in fibroblasts from affected individuals and upon knockdown of Mapkbp1 in murine cell lines, a phenotype previously associated with NPH. In conclusion, we identified mutations in MAPKBP1 as a genetic cause of juvenile or late-onset and cilia-independent NPH.

Novel NEK8 Mutations Cause Severe Syndromic Renal Cystic Dysplasia through YAP Dysregulation.

Ciliopathies are a group of genetic multi-systemic disorders related to dysfunction of the primary cilium, a sensory organelle present at the cell surface that regulates key signaling pathways during development and tissue homeostasis. In order to identify novel genes whose mutations would cause severe developmental ciliopathies, >500 patients/fetuses were analyzed by a targeted high throughput sequencing approach allowing exome sequencing of >1200 ciliary genes. NEK8/NPHP9 mutations were identified in five cases with severe overlapping phenotypes including renal cystic dysplasia/hypodysplasia, situs inversus, cardiopathy with hypertrophic septum and bile duct paucity. These cases highlight a genotype-phenotype correlation, with missense and nonsense mutations associated with hypodysplasia and enlarged cystic organs, respectively. Functional analyses of NEK8 mutations in patient fibroblasts and mIMCD3 cells showed that these mutations differentially affect ciliogenesis, proliferation/apoptosis/DNA damage response, as well as epithelial morphogenesis. Notably, missense mutations exacerbated some of the defects due to NEK8 loss of function, highlighting their likely gain-of-function effect. We also showed that NEK8 missense and loss-of-function mutations differentially affect the regulation of the main Hippo signaling effector, YAP, as well as the expression of its target genes in patient fibroblasts and renal cells. YAP imbalance was also observed in enlarged spheroids of Nek8-invalidated renal epithelial cells grown in 3D culture, as well as in cystic kidneys of Jck mice. Moreover, co-injection of nek8 MO with WT or mutated NEK8-GFP RNA in zebrafish embryos led to shortened dorsally curved body axis, similar to embryos injected with human YAP RNA. Finally, treatment with Verteporfin, an inhibitor of YAP transcriptional activity, partially rescued the 3D spheroid defects of Nek8-invalidated cells and the abnormalities of NEK8-overexpressing zebrafish embryos. Altogether, our study demonstrates that NEK8 human mutations cause major organ developmental defects due to altered ciliogenesis and cell differentiation/proliferation through deregulation of the Hippo pathway.

A homozygous missense mutation in the ciliary gene TTC21B causes familial FSGS.

Several genes, mainly involved in podocyte cytoskeleton regulation, have been implicated in familial forms of primary FSGS. We identified a homozygous missense mutation (p.P209L) in the TTC21B gene in seven families with FSGS. Mutations in this ciliary gene were previously reported to cause nephronophthisis, a chronic tubulointerstitial nephropathy. Notably, tubular basement membrane thickening reminiscent of that observed in nephronophthisis was present in patients with FSGS and the p.P209L mutation. We demonstrated that the TTC21B gene product IFT139, an intraflagellar transport-A component, mainly localizes at the base of the primary cilium in developing podocytes from human fetal tissue and in undifferentiated cultured podocytes. In contrast, in nonciliated adult podocytes and differentiated cultured cells, IFT139 relocalized along the extended microtubule network. We further showed that knockdown of IFT139 in podocytes leads to primary cilia defects, abnormal cell migration, and cytoskeleton alterations, which can be partially rescued by p.P209L overexpression, indicating its hypomorphic effect. Our results demonstrate the involvement of a ciliary gene in a glomerular disorder and point to a critical function of IFT139 in podocytes. Altogether, these data suggest that this homozygous TTC21B p.P209L mutation leads to a novel hereditary kidney disorder with both glomerular and tubulointerstitial damages.

Integrin alpha 8 recessive mutations are responsible for bilateral renal agenesis in humans.

Renal hypodysplasia (RHD) is a heterogeneous condition encompassing a spectrum of kidney development defects including renal agenesis, hypoplasia, and (cystic) dysplasia. Heterozygous mutations of several genes have been identified as genetic causes of RHD with various severity. However, these genes and mutations are not associated with bilateral renal agenesis, except for RET mutations, which could be involved in a few cases. The pathophysiological mechanisms leading to total absence of kidney development thus remain largely elusive. By using a whole-exome sequencing approach in families with several fetuses with bilateral renal agenesis, we identified recessive mutations in the integrin α8-encoding gene ITGA8 in two families. Itga8 homozygous knockout in mice is known to result in absence of kidney development. We provide evidence of a damaging effect of the human ITGA8 mutations. These results demonstrate that mutations of ITGA8 are a genetic cause of bilateral renal agenesis and that, at least in some cases, bilateral renal agenesis is an autosomal-recessive disease.

Dishevelled stabilization by the ciliopathy protein Rpgrip1l is essential for planar cell polarity.

Cilia are at the core of planar polarity cellular events in many systems. However, the molecular mechanisms by which they influence the polarization process are unclear. Here, we identify the function of the ciliopathy protein Rpgrip1l in planar polarity. In the mouse cochlea and in the zebrafish floor plate, Rpgrip1l was required for positioning the basal body along the planar polarity axis. Rpgrip1l was also essential for stabilizing dishevelled at the cilium base in the zebrafish floor plate and in mammalian renal cells. In rescue experiments, we showed that in the zebrafish floor plate the function of Rpgrip1l in planar polarity was mediated by dishevelled stabilization. In cultured cells, Rpgrip1l participated in a complex with inversin and nephrocystin-4, two ciliopathy proteins known to target dishevelled to the proteasome, and, in this complex, Rpgrip1l prevented dishevelled degradation. We thus uncover a ciliopathy protein complex that finely tunes dishevelled levels, thereby modulating planar cell polarity processes.

Control of the Wnt pathways by nephrocystin-4 is required for morphogenesis of the zebrafish pronephros.

Nephronophthisis is a hereditary nephropathy characterized by interstitial fibrosis and cyst formation. It is caused by mutations in NPHP genes encoding the ciliary proteins, nephrocystins. In this paper, we investigate the function of nephrocystin-4, the product of the nphp4 gene, in vivo by morpholino-mediated knockdown in zebrafish and in vitro in mammalian kidney cells. Depletion of nephrocystin-4 results in convergence and extension defects, impaired laterality, retinal anomalies and pronephric cysts associated with alterations in early cloacal morphogenesis. These defects are accompanied by abnormal ciliogenesis in the cloaca and in the laterality organ. We show that nephrocystin-4 is required for the elongation of the caudal pronephric primordium and for the regulation of cell rearrangements during cloaca morphogenesis. Moreover, depletion of either inversin, the product of the nphp2 gene, or of the Wnt-planar cell polarity (PCP) pathway component prickle2 increases the proportion of cyst formation in nphp4-depleted embryos. Nephrocystin-4 represses the Wnt-ß-catenin pathway in the zebrafish cloaca and in mammalian kidney cells in culture. In these cells, nephrocystin-4 interacts with inversin and dishevelled, and regulates dishevelled stability and subcellular localization. Our data point to a function of nephrocystin-4 in a tight regulation of the Wnt-ß-catenin and Wnt-PCP pathways, in particular during morphogenesis of the zebrafish pronephros. Moreover, they highlight common signalling functions for inversin and nephrocystin-4, suggesting that these two nephrocystins are involved in common physiopathological mechanisms.

The ciliary pocket: an endocytic membrane domain at the base of primary and motile cilia.

Cilia and flagella are eukaryotic organelles involved in multiple cellular functions. The primary cilium is generally non motile and found in numerous vertebrate cell types where it controls key signalling pathways. Despite a common architecture, ultrastructural data suggest some differences in their organisation. Here, we report the first detailed characterisation of the ciliary pocket, a depression of the plasma membrane in which the primary cilium is rooted. This structure is found at low frequency in kidney epithelial cells (IMCD3) but is associated with virtually all primary cilia in retinal pigment epithelial cells (RPE1). Transmission and scanning electron microscopy, immunofluorescence analysis and videomicroscopy revealed that the ciliary pocket establishes closed links with the actin-based cytoskeleton and that it is enriched in active and dynamic clathrin-coated pits. The existence of the ciliary pocket was confirmed in mouse tissues bearing primary cilia (cumulus), as well as motile cilia and flagella (ependymal cells and spermatids). The ciliary pocket shares striking morphological and functional similarities with the flagellar pocket of Trypanosomatids, a trafficking-specialised membrane domain at the base of the flagellum. Our data therefore highlight the conserved role of membrane trafficking in the vicinity of cilia.

Nephrocystin-1 and nephrocystin-4 are required for epithelial morphogenesis and associate with PALS1/PATJ and Par6.

Nephronophthisis (NPH) is an autosomal recessive disorder characterized by renal fibrosis, tubular basement membrane disruption and corticomedullary cyst formation leading to end-stage renal failure. The disease is caused by mutations in NPHP1-9 genes, which encode the nephrocystins, proteins localized to cell-cell junctions and centrosome/primary cilia. Here, we show that nephrocystin mRNA expression is dramatically increased during cell polarization, and shRNA-mediated knockdown of either NPHP1 or NPHP4 in MDCK cells resulted in delayed tight junction (TJ) formation, abnormal cilia formation and disorganized multi-lumen structures when grown in a three-dimensional collagen matrix. Some of these phenotypes are similar to those reported for cells depleted of the TJ proteins PALS1 or Par3, and interestingly, we demonstrate a physical interaction between these nephrocystins and PALS1 as well as their partners PATJ and Par6 and show their partial co-localization in human renal tubules. Taken together, these results demonstrate that the nephrocystins play an essential role in epithelial cell organization, suggesting a plausible mechanism by which the in vivo histopathologic features of NPH might develop.

The ciliary gene RPGRIP1L is mutated in cerebello-oculo-renal syndrome (Joubert syndrome type B) and Meckel syndrome.

Cerebello-oculo-renal syndrome (CORS), also called Joubert syndrome type B, and Meckel (MKS) syndrome belong to the group of developmental autosomal recessive disorders that are associated with primary cilium dysfunction. Using SNP mapping, we identified missense and truncating mutations in RPGRIP1L (KIAA1005) in both CORS and MKS, and we show that inactivation of the mouse ortholog Rpgrip1l (Ftm) recapitulates the cerebral, renal and hepatic defects of CORS and MKS. In addition, we show that RPGRIP1L colocalizes at the basal body and centrosomes with the protein products of both NPHP6 and NPHP4, known genes associated with MKS, CORS and nephronophthisis (a related renal disorder and ciliopathy). In addition, the RPGRIP1L missense mutations found in CORS individuals diminishes the interaction between RPGRIP1L and nephrocystin-4. Our findings show that mutations in RPGRIP1L can cause the multiorgan phenotypic abnormalities found in CORS or MKS, which therefore represent a continuum of the same underlying disorder.

Characterization of the nephrocystin/nephrocystin-4 complex and subcellular localization of nephrocystin-4 to primary cilia and centrosomes.

Nephrocystin and nephrocystin-4 are newly identified proteins involved in familial juvenile nephronophthisis, an autosomal recessive nephropathy characterized by cyst formation and renal fibrosis. Nephrocystin is an adaptor protein that is able to associate with signaling molecules involved in cell adhesion and actin cytoskeleton organization, such as p130Cas, Pyk2, tensin and filamins. Nephrocystin was recently shown to interact and to co-localize with the microtubule component beta-tubulin to the primary cilia in renal epithelial cells, an organelle known to play a key role in the pathogenesis of cystic kidney diseases. In this study, we demonstrated that nephrocystin-4 also localizes to the primary cilia in polarized epithelial tubular cells, particularly at the basal bodies, and associates with microtubule component alpha-tubulin, suggesting a common role for the nephrocystin proteins in ciliary function. However, the co-localization of nephrocystin-4 with the microtubules is not restricted to the primary cilia, as nephrocystin-4 was also detected at the centrosomes of dividing cells and close to the cortical actin cytoskeleton in polarized cells. We also detected p130Cas and Pyk2 in the nephrocystin-4-containing complex, confirming the role of the nephrocystin proteins in cell-cell and cell-matrix adhesion signaling events. Finally, we refined the structural and functional regions involved in the interaction between nephrocystin and nephrocystin-4. These data suggest that nephrocystin and nephrocystin-4 belong to a multifunctional complex localized in actin- and microtubule-based structures involved in cell-cell and cell-matrix adhesion signaling as well as in cell division.

The gene mutated in juvenile nephronophthisis type 4 encodes a novel protein that interacts with nephrocystin.

Nephronophthisis, the most common genetic cause of chronic renal failure in children, is a progressive tubulo-interstitial kidney disorder that is inherited as an autosomal recessive trait. The disease is characterized by polyuria, growth retardation and deterioration of renal function during childhood or adolescence. The most prominent histological features are modifications of the tubules with thickening of the basement membrane, interstitial fibrosis and, in the advanced stages, medullary cysts. Nephronophthisis can also be associated with conditions affecting extrarenal organs, such as retinitis pigmentosa (Senior-Løken syndrome) and ocular motor apraxia (Cogan syndrome). Three loci are associated with the juvenile, infantile and adolescent forms, on chromosomes 2q13 (NPHP1; refs 5,6), 9q22 (NPHP2; ref. 7) and 3q21 (NPHP3; ref. 8), respectively. NPHP1, the only gene identified so far, encodes nephrocystin, which contains a Src homology 3 (SH3) domain and interacts with intracytoplasmic proteins involved in cell adhesion. Recently, a second locus associated with the juvenile form of the disease, NPHP4, was mapped to chromosome 1p36 (ref. 14). We carried out haplotype analysis of families affected with nephronophthisis that were not linked to the NPHP1, NPHP2 or NPHP3 loci, using markers covering this region. This allowed us to reduce the NPHP4 interval to a one centimorgan interval between D1S2795 and D1S2870, which contains six genes. We identified five different mutations in one of these genes, designated NPHP4, in unrelated individuals with nephronophthisis. The NPHP4 gene encodes a 1,250-amino acid protein of unknown function that we named nephrocystin-4. We demonstrated the interaction of nephrocystin-4 with nephrocystin suggesting that these two proteins participate in a common signaling pathway.