Cilia in PKD—Letting It All Hang Out
Authors: James P. Calvet
Journal: Journal of the American Society of Nephrology (2002)

Polycystic kidney disease (PKD) research has experienced a sort of celestial convergence, culminating with the article by Yoder, Hou, and Guay-Woodford (1) in this issue, which points to a role in PKD for the primary cilia of tubular epithelial cells. The question being asked is, "Why cilia?" This article by Yoder et al. (1) is the latest in a series of discoveries that have reported the involvement of PKD-related proteins in the assembly and function of cilia of mice and the nemotode, C. elegans, and of flagella of the unicellular green alga, Chlamydomonas. The events began with the discovery of the mouse Tg737 gene in an insertional mutagenesis screen (2). The original mutation was found to cause a recessive form of PKD in orpk mice; later when the gene was completely deleted, it was found to give rise to an embryonic lethal phenotype that is due to an absence of (or stunting of) cilia on ventral node epithelial cells (3,4), thus disrupting early morphologic left-right (L-R) axis determination (3). The protein product of this gene, polaris, was localized in the axoneme and basal body of primary cilia (4,5) and was found to be required for ciliary assembly (5,6). Another mouse mutant affecting L-R patterning and also causing PKD, inv (for inversion of embryonic turning) (7), also has a ciliary problem, which in this case appears to be a functional defect rather than a structural one (8). The recessive cpk mouse model of PKD also has a link with cilia. The cpk gene, which was just identified this year, encodes a protein, cystin, which was found to reside in the axonemal region of primary cilia, partially overlapping with polaris (9). This too may be a functional rather than structural defect, as no obvious structural abnormalities were noted by electron microscopy in the cilia of cpk mutant mice (10). At the same time that these studies were being carried out, work was also going on to examine mutations affecting male mating behavior in C. elegans, which identified homologs of the ADPKD genes PKD1 (lov-1 for location of vulva) and PKD2 (pkd-2), the protein products of which were found to localize in cilia of sensory neurons (11,12). These proteins also co-localize with another protein, OSM-5, which is a homolog of polaris (13,14). The lov-1 and pkd-2 mutations seem to affect ciliary function (12), whereas the osm-5 (osm for osmotic avoidance) mutations affect ciliary structure (13,14). To extend these studies to mammals, Pazour et al. (15) localized the PKD2 product, polycystin-2, to cilia of renal epithelial cells. And now Yoder et al. (1) report that the PKD1 product, polycystin-1, as well as polycystin-2, polaris, and cystin, all localize to primary cilia of cultured mouse cortical collecting duct (mCCD) cells. Thus, most of the major players in ARPKD and ADPKD have been linked to cilia (except for the product of the ARPKD PKHD1 gene, fibrocystin/polyductin, localization of which has not yet been reported but is most certainly being investigated). These discoveries are a testament to the power of genetics and the importance of animal models (16). Who would have thought to look at cilia at all as a basis for ADPKD if the direction had not been pointed out by these genetic studies? The observations in the animal models undoubtedly steered the polycystin work toward cilia. The cilia may otherwise have been overlooked or simply thought of as unlikely. There are several reasons. First, the polycystins have been localized to other more likely cellular compartments, such as membrane adhesion complexes, which can be easily reconciled with a role in abnormal cyst-forming behavior (17–21), or to the endoplasmic reticulum in the case of polycystin-2 (19). Second, cilia are not easy to see in cell culture, being near the resolution limit of conventional light microscopy (22). As pointed out by Yoder et al. (1), the mCCD cells they used had to be grown on cell culture inserts for at least 3 d in order to reach full polarization and cilia formation. Third, the function of cilia in renal tubules is not known — this being the next big challenge. One (or rarely two) primary (so-called 9+0) cilia can be found on most eukaryotic cells (23), and they are visible on cells in all kidney tubule segments (24). Being mostly non-motile (an exception being the embryonic ventral node cilia), primary cilia are thought to serve a sensory function, either chemo- or mechanosensory. These cilia often contain high concentrations of receptors; because they are "out there," they are ideally positioned to interact with their environment, as with the odorant receptors on olfactory cilia (25). Cilia also contain all the accoutrements for signal transduction, including heterotrimeric G proteins and ion channels. A mechanosensory function for renal tubular cilia seems quite possible. Primary cilia on cultured renal epithelial cells have been shown to reversibly bend in response to lateral fluid shear forces, as visualized by real-time video recording under conditions that resemble actual tubular flow rates (22,26), suggesting that the cilia may transduce flow-rate information to renal tubular cells. Such a possibility invites speculation about luminal flow rates and PKD. In thinking about a role for cilia in PKD, one should first consider what a cyst is. It is now well accepted that cell proliferation is an essential component of cyst formation; however, it is not clear what drives this proliferation. It seems likely that a renal tubule (to be a tubule) needs to undergo a highly regulated morphologic structuring that coordinates tubular elongation with tubular expansion. It is also likely that there is an ideal diameter for the tubule, and that this is a regulated process in the adult kidney, perhaps allowing the tubule to adapt to varying luminal flow rates and pressure gradients. If the cues for regulating tubular diameter are lost by mutation, the tubule may then begin to morph into a cyst. It is likely that cystic epithelial cells do not completely dedifferentiate; rather, they just lose their sense of being a tubule and revert to what is essentially an epithelial sheet (at least at first). Possibly the primary cilia function to measure luminal flow rates (or a lack of luminal flow) and thus regulate tubule diameter by generating signals that the cells interpret as morphologic cues. Although the cellular functions of the polycystins are still not known, there is a growing body of evidence suggesting that polycystin-1 is a novel G protein–coupled transmembrane receptor (27–29) that may regulate polycystin-2 calcium channel activity (28,30,31). There is also evidence that polycystin-1 and -2 together regulate the cyclin kinase inhibitor p21(waf1), thus directly inhibiting the cell cycle (32); in doing so, they may regulate tubulogenesis (33). It is also possible that polycystin-1 regulates the response of cells to cAMP by ensuring that cell proliferation is not stimulated by increased levels of intracellular cAMP (34–36). As such, polycystin-1, acting as a ciliary mechanoreceptor, may sense luminal flow rates and signal morphologically relevant information through a number of cellular signaling pathways to establish and maintain appropriate luminal dimensions. Polycystin-1 anchored into the ciliary plasma membrane may be configured to sense ciliary bending, perhaps using a mechanism that requires homophilic interactions between the PKD repeats in the long N-terminal ectodomain of the protein (37). Polycystin-2, acting as a tension-gated ion channel, would then respond to signals from polycystin-1 induced by this ciliary flexing to generate an influx of calcium. Indeed, fluid shear-force bending of the primary cilia of MDCK cells has been shown to increase intracellular calcium from both extracellular and intracellular sources and to transmit these signals to adjacent cells through gap junctions (38). The polycystin complex may act like a rheostat, where the degree of ciliary bending would govern the intensity of a variety of signals that would be integrated into morphologic information. If this process were to be nullified by mutation at the PKD loci or by mutations that affect ciliary assembly, the signals controlling cell growth would be lost altogether, and the result would be uninhibited cell proliferation and progressive cyst enlargement. The challenge is to propose a mechanism to explain how cilia-related functions of polycystin, polaris, and cystin may cause PKD. However, it should be appreciated that there are numerous other genes, such as those encoding c-Myc, β-catenin, Pax-2, H-ras, and many more (39,40), that can also cause PKD in animal models. Thus, cilia may only be part of the story when it comes to understanding what is able to generate cysts from tubules. Possibly the only connection between this triad—polycystin, polaris, and cystin—and PKD may be that their mutation can cause cellular dedifferentiation in various ways and that it is the cellular dedifferentiation per se that is the cause of cystic change (41). It is also possible that any or all of the numerous genes that cause (or modify the course of) PKD could regulate genes encoding ciliary components or downstream mechanosensory signaling targets, thus indirectly compromising ciliary form and function. If this is the case, it can be predicted that mutant alleles of other genes involved in making and operating cilia will be found to cause PKD — thus providing new depths to plumb for future research. Thanks to Jared Grantham and Robin Maser for helpful comments.