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.
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.
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 β-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 α-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.
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.
