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A number of inherited disorders result in renal cyst development. The most common form, autosomal dominant polycystic kidney disease (ADPKD), is a disorder most often diagnosed in adults and caused by mutation in PKD1 or PKD2. The PKD1 protein, polycystin-1, is a large receptor-like protein, whereas polycystin-2 is a transient receptor potential channel. The polycystin complex localizes to primary cilia and may act as a mechanosensor essential for maintaining the differentiated state of epithelia lining tubules in the kidney and biliary tract. Elucidation of defective cellular processes has highlighted potential therapies, some of which are now being tested in clinical trials. ARPKD is the neonatal form of PKD and is associated with enlarged kidneys and biliary dysgenesis. The disease phenotype is highly variable, ranging from neonatal death to later presentation with minimal kidney disease. ARPKD is caused by mutation in PKHD1, and two truncating mutations are associated with neonatal lethality. The ARPKD protein, fibrocystin, is localized to cilia/basal body and complexes with polycystin-2. Rare, syndromic forms of PKD also include defects of the eye, central nervous system, digits, and/or neural tube and highlight the role of cilia and pathways such as Wnt and Hh in their pathogenesis.
Polycystic kidney disease (PKD) is a group of monogenic disorders that result in renal cyst development. The morbidity associated with the most common forms, autosomal dominant PKD (ADPKD) and autosomal recessive PKD (ARPKD), is largely limited to the kidney and liver (and, in ADPKD, vasculature) and extends from neonates to old age. In a rarer group of mainly recessively inherited pleiotropic disorders, cystic kidneys or cystic dysplasia are part of a set of developmental phenotypes. Over the past 15 years, positional cloning approaches have identified more than 20 genes causing these disorders. The common forms of PKD are associated with a low level of genic heterogeneity but extreme allelic heterogeneity, whereas marked genic heterogeneity is found in syndromic forms of PKD. Novel protein families have been identified, the functions of which are only now coming to light. A factor that unites all these disorders is the involvement of the primary cilium and the base of the cilium, the basal body, in their pathogenesis. PKD proteins, and orthologs in other species, have been localized to cilia/basal body, and loss or abnormalities of cilia in the kidney are associated with cyst development. Rapid progress toward understanding the etiology and pathogenesis of these diseases is revealing possible therapeutic targets that are now being assessed.
ADPKD is typically diagnosed in adults and, with an incidence of 1:400–1000, is one of the most common monogenic disorders. The disease is characterized by the progressive, bilateral development and enlargement of focal cysts that in many cases ultimately result in end-stage renal disease (ESRD) (1). In the United States, 4.4% of patients requiring renal replacement therapy (dialysis or transplant) have ADPKD. There is considerable phenotypic variability, however, from rare infantile cases with large cystic kidneys to patients in their tenth decade with adequate renal function (2). ADPKD is a systemic disease, with cyst development also occurring in the liver, pancreas, seminal vesicles, and arachnoid. In the liver, it can (rarely; more often in women) result in severe polycystic liver disease (PLD) requiring surgical intervention. Other notable phenotypes in ADPKD involve the vasculature, with intracranial aneurysms approximately five times more common than in the general population and significant morbidity/mortality associated with aneurysmal rupture (1). Typical presenting symptoms include flank pain, hematuria, renal colic, urinary tract infections, and hypertension (3). ADPKD can generally be diagnosed in at-risk adults by imaging of the kidney by ultrasound, CT, or MRI with multiple cysts generally visible that increase in size and number with age. Recent MRI data from an observational trial of ADPKD showed that kidneys increase on average 5.27% per year, but with a wide range of variability, and that renal volume can be used to monitor disease progression before a measurable decline in function (4).
ADPKD is genetically heterogeneous with two genes identified, PKD1 (16p13.3) and PKD2 (4q21), plus a few unlinked families described (5-8). PKD1 accounts for ~85% of cases in clinically identified populations (9) and is a significantly more severe disease; the average age at onset of ESRD is 20 years younger in PKD1 than in PKD2 [54.3 years versus 74.0 years (10)]. The greater severity of PKD1 appears to be due to the development of more cysts at an early age, not to faster growth (11). Both PKD1 and PKD2 can be associated with severe PLD and vascular abnormalities. A separate, genetically heterogeneous disease, autosomal dominant polycystic liver disease (ADPLD), causes severe PLD but with no or minimal renal cysts (12, 13).
The PKD1 gene (46 exons; genomic extent, 50 kb) encodes a large transcript with an open reading frame (ORF) of 12,909 bp (6, 7). The 5′ 33 exons of PKD1 lie in a region that is duplicated six times elsewhere on chromosome 16, complicating molecular diagnostics. PKD2 (15 exons; 68 kb) has an ORF of 2904 bp (8). Screening of ADPKD patients indicates that a wide range of mutations cause this disease; 314 different truncating mutations (in 400 families) to PKD1 and 91 truncating mutations (in 166 families) to PKD2 have been described. An additional one quarter of mutations are missense. Likely mutations are detected in ~90% of patients, making molecular diagnostics an option when diagnostics by renal imaging is equivocal (9). This is particularly helpful for obtaining a definite diagnosis in a young person who is a potential living related donor. The type of mutation (truncating or missense) does not seem to be strongly associated with the phenotype (14, 15), but mutation position in PKD1 has been weakly associated with severity of renal disease and to the development of intracranial aneurysms (14, 16). The majority of patients have an affected parent, but >10% of families can be traced to a new mutation; two cases of mosaicism have recently been described (17, 18).
Mice homozygous for Pkd1 or Pkd2 mutations typically die at E12.5–16.5 with grossly cystic kidneys and pancreas, indicating that loss of all gene product results in cyst development (19). Heterozygous animals develop only a few cysts, mainly in the liver, at older ages (20). Recent studies of conditional knockouts of Pkd1 show that inactivation before postnatal day 13 results in rapidly progressive cystic disease whereas later inactivation results in a much milder course, indicating a developmental switch that may be associated with normal downregulation of the protein at that time (21, 22). The focal cyst development seen in human ADPKD may be due to somatic second hits; screening of PKD1 and PKD2 in cyst lining cells has identified loss of heterozygosity and other somatic mutations (23, 24). A PKD2 model (WS25) with a hypermutable allele also develops progressive cystic disease in combination with a null allele (PKD2WS25/−) (25). However, dosage reduction of the protein in two Pkd1 animal models with hypomorphic alleles that generate <20% of the normally spliced product indicates that cysts can develop even if the protein is not completely lost (26).
The PKD1 protein, polycystin-1, is a large (4303 amino acids) integral membrane protein with 11 transmembrane domains and an extracellular region consisting of a variety of domains, including 12 PKD domains (an immunoglobulin-like fold), which in other proteins are associated with protein-protein and protein-carbohydrate interactions (6, 27, 28) (Figure 1). Overall, polycystin-1 has the structure of a receptor or adhesion molecule. Polycystin-2 (the PKD2 protein) is a nonselective cation channel that transports calcium (29, 30). The polycystins form a distinct sub-family (TRPP) of transient receptor potential (TRP) channels. Polycystin-2 shows homology with the final six-transmembrane region of polycystin-1. Polycystin-1 is cleaved at the G protein–coupled receptor proteolytic site (GPS) domain, which may be important to activate the protein (31). Interestingly, mice with a mutation that disrupts cleavage are viable (to P28) with enlarged, cystic kidneys, suggesting a role for the uncleaved product during development (32). Cleavage of the C-terminal tail and migration to the nucleus (similar to notch signaling) has also been suggested in two studies (33, 34). Polycystin-2 may also regulate proliferation and differentiation by controlling progression through the cell cycle (35).
There is good evidence that the polycystins form a complex, and a major role of this complex may be to regulate levels of intracellular Ca2+. The polycystins have been localized to various sites. These include involvement in cell-cell and cell-matrix interactions, as well as in the endoplasmic reticulum in the case of polycystin-2, but the most compelling evidence associating them with PKD supports a location on primary cilia (36, 37). Most cells have a single primary cilium. In the case of tubular epithelial cells, this extends into the lumen of the tubule. This cilium has a 9+0 microtubule arrangement (nonmotile) and+has a sensory function. The base of the cilium, the basal body, is derived from the mother centriole; the external part of the cilium consists of a microtubule core (the axoneme) covered by a ciliary membrane. Evidence that cilia are important in PKD comes from animal models in which intraflagellar transport (IFT), which is required for movement of proteins in the cilium and hence ciliary formation, was disrupted by kidney-specific inactivation of the anterograde IFT motor component KIF3A (38). These animals develop renal cysts. Data indicate that the polycystin complex acts as a flow sensor on the cilium (39). Mechanical or flow-induced bending of cilia is associated with a Ca2+ influx into the cell, which occurs through the polycystin-2 channel (40). This mechanosensory response is lost in polycystin-1-deficient cells (39). Urinary vesicles that contain polycystin-1 may help mediate this flow response (41, 42), similar to the role that nodal vesicular parcels play in transmitting signals in the node (43). The node is a ciliated structure formed during development that is involved in establishing left-right body asymmetry. Asymmetric calcium signaling through the polycystin-2 channel appears to be important in this process, which is consistent with laterality defects found in Pkd2−/− mice (44, 45).
A family of polycystin-like proteins have now been identified, four with structures similar to polycystin-1 and three like polycystin-2 (46), although no others seem to be linked to PKD. PKDREJ (PKD receptor for egg jelly) is only expressed in the male germline, and Pkdrej−/− mice are fertile but with reduced reproductive success. PKDREJ appears to have a role in the zona pellucida–evoked acrosome reaction (47). The polycystins PKD2L1 and PKD1L3 are expressed in taste cells and form a complex that is the sour taste receptor (48). These proteins are also important for pH sensing in cerebrospinal fluid (49).
Consistent with the polycystin complex having a role in Ca2+ regulation, PKD cells display alterations in intracellular Ca2+ homeostasis and possibly have a reduced level of intracellular Ca2+ (50). In addition, increased levels of cAMP have been found in several PKD animal models and this may be directly related to the reduced Ca2+ (Figure 2) (51-53). In PKD cells, cAMP stimulates MAPK/ERK signaling, which depends on Src, Ras and B-Raf, compared to an inhibitory response in normal cells (54, 55). This altered response can be reversed by treatment with a Ca2+ channel activator or a Ca2+ ionophore to increase intracellular Ca2+ levels in PKD cells (56). As well as activating the MAPK/ERK pathway and cell proliferation in PKD cells, cAMP also stimulates chloride-driven fluid secretion.
Patients with contiguous deletions of PKD1 and the adjacent tuberous sclerosis gene (TSC2) have unusually severe PKD, suggesting synergy between these intersecting pathways (Figure 2) (57). Activation of mTOR in PKD and possible interaction of the TSC2 protein (tuberin) and polycystin-1 also show crosstalk between these pathways (58).
Until now, therapy for ADPKD has been directed toward limiting its complications. Cardiovascular complications, to a large extent related to hypertension, are a major cause of morbidity and the leading cause of mortality. Despite this, optimal antihypertensive regimens and blood pressure targets remain uncertain. HALT-PKD is a clinical trial designed to determine whether combined therapy with an angiotensin I–converting enzyme inhibitor (ACEI) and an angiotensin II–receptor blocker (ARB) is superior to an ACEI alone in delaying the progression of cystic kidney disease (CKD) in patients with CKD stage 1 or 2 or in slowing down the decline of renal function in patients with CKD stage 3. HALT-PKD will also determine whether a low blood pressure target (<110/75) is superior to a standard blood pressure target (120–130/70–80) in the group of patients with preserved function. A smaller clinical trial seeks to determine whether treatment of pediatric or young adult ADPKD patients with a statin can slow down the progression of kidney and heart disease (Table 1).
Better understanding of signaling pathways and cellular changes associated with ADPKD has suggested possible therapies to directly inhibit the development or growth of cysts, some of which are now being tested in clinical trials (Table 1). It is known that vasopressin, via V2 receptors (VPV2R), has a role in regulating cAMP levels in the distal nephron and collecting duct, a major site of ADPKD cysts. This suggested that VPV2R antagonists may be beneficial. Animal studies, including in a Pkd2 model, showed a dramatic reduction in cyst development (51, 52). Phase II clinical trials of tolvaptan, a VPV2R inhibitor, have now been completed and a phase III double-blind placebo-controlled trial has begun. Another means to regulate cellular cAMP levels is through somatostatin's action on SST2 receptors that are present in the liver and kidney. A stable somatostatin analogue, octreotide, has proved effective at limiting progression in liver and kidney cystic disease in a rat model of PKD (59). Preliminary studies in human ADPKD indicate that octreotide may inhibit cyst growth, and further clinical trials of long-acting somatostatin analogs are under way (Table 1) (60). The suggested activation of the mTOR pathway in PKD has prompted testing of mTOR inhibitors. Administration in three rodent models has resulted in a reduction in cyst growth and preservation of renal function (58). In addition, analysis of transplanted ADPKD patients who received mTOR inhibitors as the immunosuppressive agent showed a reduction in size of the native kidney and the liver compared to other immunosuppressive regimens, thus justifying further clinical trials (58, 61).
A number of other compounds have shown efficacy in animal models of PKD but have not yet been tested in clinical trials (Figure 2). Administration of small-molecule CFTR inhibitors of the thiazolidinone and glycine hydrazide classes in a Pkd1flox/−;Ksp-Cre mouse model of rapidly progressive ADPKD slowed cyst expansion and protected renal function (62). Chloride-driven fluid secretion into cysts requires the coordinated activity of apical CFTR and basolateral Na/K/2Cl cotransporter NKCC1, plus basolateral recycling of Na+ and K+ and maintenance of a hyperpolarized membrane potential by the basolateral Na+,K+-ATPase and by K+ channels such as the Ca2+-dependent K+ channel KCa3.1. Direct inhibitors of KCa3.1 that are well tolerated in vivo and are in phase III clinical trials for sickle cell disease inhibited cystogenesis in vitro (63, 64). Triptolide, a product from a traditional Chinese herb that induces Ca2+ release in a polycystin-dependent manner, inhibited cyst development and protected renal function in Pkd1flox/−;Ksp-Cre mice when administered very early through lactation, but not when the treatment was started later at P4 (65, 66). ErbB tyrosine kinase and c-Src inhibitors inhibited both renal and hepatic cyst development and protected renal function in various rodent models of polycystic kidney disease, while MAPK/ERK kinase (MEK) and cyclin-dependent kinase inhibitors inhibited the development of PKD in the pcy model and in the jck and cpk models, respectively (67-69, 74).
In addition, some compounds have shown efficacy in animal models of PKD by mechanisms not illustrated in Figure 2. Ribozymes targeting either vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1) or 2 (VEGFR2) mRNA slowed the progression of cystic disease in Han:SPRD Cy/ rats (70). A tumor necrosis factor-alpha (TNF-α) inhibitor prevented cyst development in Pkd2+/− animals, presumably because TNF-α in cystic tissues and cyst fluids disrupts the localization of PC2 to the plasma membrane and primary cilia (71). An inhibitor of 20-hydroxyeicosatetraenoic acid (20-HETE), an endogenous cytochrome P450 metabolite of arachidonic acid with mitogenic properties, reduced kidney size and improved survival in bpk mice (72). Diets enriched in long-chain n-3 polyunsaturated fatty acids have been shown to exert anti-inflammatory effects and to slow renal cystic enlargement and functional deterioration in animal models of PKD. However, a small prospective clinical trial of eicosapentaenoic acid (EPA) failed to demonstrate a beneficial effect in ADPKD patients with a mean total kidney volume of 1563 ml and a mean serum creatinine of 1.56 mg/dl (73).
ARPKD has been estimated to have an incidence of 1:20,000 and typically presents in utero or during the neonatal period with greatly enlarged, echogenic kidneys. The most extreme cases are associated with the Potter's phenotype, consisting of pulmonary hypoplasia, characteristic facies and spine and limb abnormalities. A significant proportion of cases (up to 30%) die by the neonatal period, primarily of respiratory insufficiency. In the survivors, hypertension and renal insufficiency, including ESRD (up to a third of children require renal replacement therapy), are the major signs of renal disease (75). The other pathognomonic sign of disease is biliary dysgenesis resulting in congenital hepatic fibrosis, plus intrahepatic bile duct dilatation (Caroli disease). The disease presentation is not limited to the neonatal period; it can be diagnosed in childhood, or even adolescence or adulthood (76). These later-presenting cases typically have less severe kidney disease, and complications of the liver disease are more common. These complications include those associated with portal hypertension, such as bleeding esophageal or gastric varices, and cholangitis related to Caroli disease.
ARPKD is caused by mutation in one gene, PKHD1 (6q21). PKHD1 (polycystic kidney and hepatic disease 1) has 67 exons and covers a genomic region of 470 kb. The longest ORF has 12,222 bp, but there may be other alternatively spliced products (77, 78). PKHD1 exhibits a high level of allelic heterogeneity, with 303 different mutations described on >650 alleles. Approximately 40% of mutations are predicted to be truncating and 60% missense. About one third of mutations are unique to a single family, and a small number are enriched in specific geographic areas. One missense change, T36M, is relatively frequent (17% of mutant alleles) and is of European origin. A clear genotype/phenotype correlation has been described in ARPKD, with two truncating mutations always associated with the most severe phenotype, neonatal death (79). This indicates that many missense changes are not fully penetrant alleles; they are associated with production of some functional protein. Molecular diagnostics in ARPKD is helpful to make a firm diagnosis, especially in cases with less severe disease, and to facilitate preimplementation genetic diagnostics and prenatal diagnostics.
Six different knockout mouse models of ARPKD have been described with a variety of different Pkhd1 mutations, plus a spontaneous rat model, PCK (78, 80, 81). All of the mouse models develop biliary dysgenesis and fibrosis, similar to human ARPKD, but kidney disease is generally mild and late-onset compared to severe human ARPKD. Pancreatic cysts, including grossly enlarged pancreatic cystic disease, is common. The PCK rat has progressive cystic disease in the liver and kidney and has been a favored model to test potential therapies (51). Shortened and abnormal ciliary structures, described in several rodent models, are associated with reduced Pkhd1 expression in cells (81-83). This is in contrast to ADPKD, where cilia are thought to be normal. Whereas most PKD genes are widely expressed, PKHD1 has more limited expression in kidney, liver, pancreas, and lung, perhaps explaining the limited range of phenotypes compared to syndromic forms of PKD that are associated with ciliary defects (78).
The PKHD1 protein, fibrocystin (also called polyductin), has 4074 amino acids and is attached to the membrane via a single transmembrane domain (Figure 1) (78). The large extracellular region contains 12 TIG/IPT domains (immunoglobulin-like fold shared by plexins and transcription factors). Fibrocystin has been found in the same complex as polycystin-2 and has been localized to primary cilia, as well as to the basal body (83-87). Fibrocystin is thought to be cleaved at a proprotein convertase site in the extracellular region (42, 88). In addition, similar to polycystin-1, cleavage and transport of the C-tail to the nucleus has been described (88, 89). The precise function of fibrocystin is at present unknown, but it may mediate its activity through polycystin-2. One fibrocystin homolog, fibrocystin-L, has been described in mammals; it has a similar overall structure but does not seem to be associated with PKD (90).
A number of rare syndromic disorders have renal cysts or cystic dysplasia as part of their phenotype (91). These disorders have provided important insights into mechanisms that may be associated with cystogenesis and, in particular, the role of cilia and/or the basal body in that process. Most of the implicated proteins have been localized to the centrosomal/basal body and/or the cilium, and in many disorders, ciliary defects have been described (91). These ciliopathies have considerable phenotypic overlap, and recently it has become clear that often the same gene is associated with more than one disease. The syndromic PKD disorders include nephronophthisis (NPHP), which in turn includes Senior-Loken syndrome (SLS) (92); Joubert syndrome and related disorders ( JSRD) (93); Meckel syndrome (MKS); Bardet-Biedel syndrome (BBS) (94); and orofacial digital syndrome (OFDS) (95). These diseases and the genes involved are summarized in Figure 3.
As well as renal cystic disease/cystic dysplasia, a number of other phenotypes are found in two or more of the diseases. These include biliary dysgenesis/hepatic fibrosis; eye defects; central nervous system/neural tube abnormalities; distal limb/digit anomalies; and developmental delay/mental retardation. A high level of genetic heterogeneity is also evident: BBS has been linked to 14 different genes, MKS to nine, JSRD to five, and NPHP to nine. Many genes are implicated in multiple disorders, which both reveals the deficiencies of a phenotype-based classification system and illustrates the clinical variability associated with mutation at the same locus (93). Some of this variability may be related to allelic effects; the most severe phenotype (lethal MKS) is associated with two inactivating mutations in MKS3 or RPGRIP1L, whereas cases with at least one missense change or other hypomorphic allele at these loci are more often associated with JSRD. However, such a clear relationship is not evident with CEP290, and genetic background factors are likely to be most important. Indeed, in BBS and NPHP, oligogenic inheritance has been demonstrated: mutation at more than one locus is required for disease development (94, 96, 97). Similarly, hypomorphic MKS1 alleles have been found to modify the phenotype in BBS (98).
The pleiotropic phenotypes in these syndromic disorders can likely be linked to defects in ciliary function and provide an insight into the signaling pathways that require functional cilia to operate. These disorders also shed light on other possible mechanisms by which ciliary abnormalities may be linked to cyst development.
Vision defects due to retinal degeneration are a feature of BBS, JSRD, and SLS that is found in ~20% of patients with NPHP. In the rods and cones of the eye, a connecting cilium transports components to the outer segment, which contains discs of the light-absorbing pigment. Retinal degeneration has been demonstrated in several forms of BBS, and the implicated proteins are thought to be involved in ciliary formation and function, including involvement with IFT (94). A protein recently associated with JSRD and MKS, RPGRIPL1, is a homolog of the retinitis pigmentosa GTPase regulator-interacting protein (RPGRIP1) that causes Leber congenital amaurosis and has been localized to the connecting cilium of photoreceptors (99). Other sensory defects, such as loss of smell in BBS, are also likely due to defects in sensory cilia.
A number of developmental defects are associated with these ciliopathies, including polydactyly and other limb abnormalities (MKS, OFDS, BBS), defects of the neural tube (exencephaly), and other defects of the central nervous system (MKS, JSRD). Defects in Hedgehog (Hh) signaling have been associated with polydactyly and craniofacial abnormalities, and two murine IFT mutants were found to have similar defects (100). GLI1-3, the transcription factors activated by Hh signaling, have been localized to the distal tip of cilia, where cleavage of GLI3 into the repressed form, GLI3R, occurs (101). This processing is suppressed when Hh binds patched-1, releasing active GLI3A to be transported to the nucleus to activate target genes. Hence, functioning IFT (cilia) is required for activation of the Hh pathway. Recently, GLIS2 mutations have been associated with NPHP in one family (102).
Certain phenotypes found in mouse models of BBS, particularly neural tube defects and disrupted stereociliary bundles in the cochlea, resemble phenotypes associated with disruption of the planar cell polarity (PCP) pathway, also known as the noncanonical Wnt pathway (103). PCP was discovered in Drosophila as a process determining organization, such as wing hairs and sensory bristles, that requires coordinated organization (polarity) of cells within the phase of a single cellular layer. In vertebrates, PCP controls developmental processes such as convergent extension and mitotic spindle orientation. A protein defective in NPHP2, inversin, has been suggested to be a switch between the β-catenin-dependent canonical Wnt pathway and the noncanonical PCP pathway, a change that may be dependent on fluid flow (104). Inversin also appears to be required for convergent extension during Xenopus gastrulation. Orientation of the mitotic spindle during division is thought to be important for the development of tubules so that epithelial cells divide along the longitudinal axis of the tubule. Defects in the orientation of the spindle have been detected in PKD animal models, including the PCK rat. This finding suggests that disoriented cell division may result in tubule enlargement in the transverse phase, similar to the fusiform dilation characteristic of some forms of PKD, such ase ARPKD (105).
Gene identification in PKD has revealed a wide range of proteins associated with cyst development and has led to the discovery of two new protein families, the polycystins and the fibrocystins. An unanticipated connection has been found between PKD and the primary cilium. Polycystin-1 and -2 may act as a sensor on the cilium, essential for maintaining the differentiated state of the epithelia lining tubules in the kidney and biliary tract. Functioning cilia also seem important in other forms of PKD, and the diverse phenotypes associated with syndromic forms of PKD are likely explained by a lack of fully functional cilia and possible disruption of the Hh and Wnt pathways. Better understanding of the downstream consequences of PKD mutations has identified a number of therapeutic targets that are now being tested in preclinical and clinical trials.
Dr. Torres receives research funding from Otsuka Pharmaceuticals for clinical trials.