Hypertension

Hypertension (BP>140/90 mmHg) is present in approximately 50% of 20-34 year old ADPKD patients with normal renal function and increases to nearly 100% of patients with ESRD. Its development is accompanied by a reduction in renal blood flow, increased filtration fraction, abnormal renal handling of sodium, and extensive remodeling of the renal vasculature.

The association between renal size and prevalence of hypertension has supported the hypothesis that stretching and compression of the vascular tree by cyst expansion causes ischemia and activation of the renin–angiotensin system. Whether the classic, circulating renin-angiotensin system is inappropriately activated is controversial. There is stronger evidence for the activation of a local intrarenal renin-angiotensin system. Evidence for the latter includes (1) partial amelioration of the reduced renal blood flow, increased renal vascular resistance and increased filtration fraction in the setting of acute or chronic administration of an ACEI; (2) shift of immunoreactive renin from the juxtaglomerular apparatus to the walls of the arterioles and small arteries; (3) ectopic synthesis of renin in the epithelium of dilated tubules and cysts; and (4) ACE-independent generation of angiotensin II by a chymase-like enzyme.

It has been suggested that a primary disruption of polycystin protein function in the vasculature may also play a role in the early development of hypertension and renal vascular remodeling. Supportive evidence includes expression of the respective PKD1 and PKD2 gene products in vascular smooth muscle and endothelium, and enhanced vascular smooth muscle contractility and impaired endothelial dependent vasorelaxation in heterozygous blood vessels. Recent studies have shown that nitric oxide endothelium-dependent vasorelaxation is impaired in small subcutaneous resistance vessels from patients with normal renal function before development of hypertension. Other factors proposed to contribute to hypertension in ADPKD include increased sympathetic nerve activity and plasma endothelin-1 levels and insulin resistance.

The diagnosis of hypertension in ADPKD is often made late. Twenty-four hours ambulatory blood pressure monitoring of children or young adults without hypertension may reveal elevated blood pressures and attenuated nocturnal blood pressure dipping and exaggerated blood pressure response during exercise, which may be accompanied by left ventricular hypertrophy and diastolic dysfunction. Early detection and treatment of hypertension is important because cardiovascular disease is the main cause of death and uncontrolled blood pressure increases the risk for proteinuria, hematuria, faster decline of renal function and morbidity and mortality from valvular heart disease and aneurysms.

Pain

Pain is the most frequent symptom (~60%) reported by adult patients. Acute pain may be associated with renal hemorrhage, passage of stones and urinary tract infections. Some patients develop chronic flank pain without identifiable etiology other than the cysts.

VEGF produced by the cystic epithelium may promote angiogenesis, hemorrhage into cysts and gross hematuria. Rarely, a hemorrhagic cyst can rupture into the subcasular or retroperitoneal space. Symptomatic episodes likely underestimate the frequency of cyst hemorrhage as over 90% of ADPKD patients have hyperdense (CT) or high signal (MRI) cysts reflecting blood or high protein content. Most hemorrhages resolve within two to seven days. If symptoms last longer than one week or if the initial episode occurs after the age of 50 years, investigation to exclude neoplasm should be undertaken.

Approximately 20% of ADPKD patients have kidney stones. Their composition is usually uric acid and/or calcium oxalate. Metabolic factors include decreased ammonia excretion, low urinary pH and low urinary citrate concentration. Urinary stasis secondary to the distorted renal anatomy may also play a role. Stones may be difficult to differentiate from cyst wall and parenchymal calcification, which also occur with increased frequency.

As in the general population urinary tract infections affect females more frequently than males. Most are caused by enterobacteriaciae. CT and MRI are sensitive to detect complicated cysts and provide anatomic definition, but the findings are not specific for infection. Nuclear imaging ( ⁶⁷ Ga or ¹¹¹ In-labeled leukocyte scans) is useful but false negative and positive results are possible. ¹⁸ fluorodeoxyglucose ( ¹⁸ FDG) positron-emission computed tomography (PET/CT) is currently the most helpful imaging modality to detect a cyst infection, but it is not widely available, is expensive, and currently not approved by the Centers for Medicare and Medicaid Services (CMS) for the diagnosis of infection. Cyst aspiration should be considered when the clinical setting and imaging are suggestive and blood and urine cultures are negative.

Renal cell carcinoma is a rare cause of pain in ADPKD. It does not occur more frequently than in the general population, but it may present at an earlier age with frequent constitutional symptoms and a higher proportion of sarcomatoid, bilateral, multicentric, and metastatic tumors. A solid mass on ultrasound, speckled calcifications on CT, and contrast enhancement, tumor thrombus and regional lymphadenopathies on CT or MRI should raise the suspicion for a carcinoma.

Renal Failure

The development of renal failure is highly variable. In most patients renal function is maintained within the normal range, despite relentless growth of cysts, until the 4 ^th to 6 ^th decade of life. By the time renal function starts declining, the kidneys usually are markedly enlarged and distorted with little recognizable parenchyma on imaging studies. At this stage, the average rate of GFR decline is approximately 4.4–5.9 mL/min/year. The mutated gene ( PKD1 or PKD2 ) is a significant determinant of the severity of disease and position of the mutation within PKD1 and possible modifier genes also contribute to the clinical course of ADPKD (see following sections). Other risk factors for a worse prognosis include male gender (particularly in PKD2 ), black race, first episode of hematuria before the age of 30, onset of hypertension before the age of 35, hyperlipidemia, low HDL, and sickle cell trait.

Several mechanisms account for renal function decline. The CRISP study has confirmed previous studies suggesting a strong relationship between renal enlargement and functional decline. CRISP has shown that kidney and cyst volumes are the strongest predictors of renal functional decline. CRISP also found that renal blood flow (or vascular resistance) is an independent predictor of functional decline. This points to the importance of vascular remodeling in the progression of the disease and may account for cases where the decline of renal function seems to be out of proportion to the severity of the cystic disease. Angiotensin II, transforming growth factor-β, and reactive oxygen species may contribute to the vascular lesions and interstitial fibrosis by stimulating the synthesis of chemokines, extracellular matrix, and metalloproteinase inhibitors. Other factors such as heavy use of analgesics may contribute to chronic kidney disease (CKD) progression in some patients.

Polycystic Liver Disease

PLD is the most common extrarenal manifestation. It is associated with both PKD1 and PKD2 genotypes. The cysts arise by excessive proliferation and dilatation of biliary ductules and peribiliary glands. Estrogen receptors are expressed in the epithelium lining the hepatic cysts and estrogens stimulate hepatic cyst derived cell proliferation. Bile duct cyst growth is also promoted by growth factors and cytokines secreted into the cyst fluid.

Hepatic cysts are rare in children. Their frequency increases with age and may have been underestimated by ultrasound and CT studies. Their prevalence by MRI in the CRISP study is 58%, 85 and 94% in 15 to 24, 25 to 34 and 35 to 46 year old participants. Hepatic cysts are more prevalent and hepatic cyst volume is larger in women than in men. Women who have multiple pregnancies or who have used oral contraceptive agents or estrogen replacement therapy have worse disease suggesting an estrogen effect on hepatic cyst growth.

Typically, PLD is asymptomatic, but symptoms have become more frequent as the lifespan of ADPKD patients has lengthened with dialysis and transplantation. Symptoms may result from mass effect or from complications related to the cysts. Symptoms typically caused by massive enlargement of the liver or by mass effect from a single or a limited number of dominant cysts include dyspnea, early satiety, gastroesophageal reflux, and mechanical low back pain. Other complications caused by mass effect include hepatic venous outflow obstruction, inferior vena cava compression, portal vein compression or bile duct compression presenting as obstructive jaundice.

Symptomatic liver cyst complications include cyst hemorrhage, infection and rarely torsion or rupture. The typical presentation of cyst infection is with localized pain, fever, leukocytosis, elevated sedimentation rate and often elevated alkaline phosphatase. It is usually monomicrobial and caused by enterobacteriaceae. MRI is very sensitive for identifying complicated hepatic cysts. On CT scanning, fluid-debris levels within cysts, cyst wall thickening, intracystic gas bubbles, and heterogeneous or increased density have been associated with infection. Radionuclide imaging and more recently ¹⁸ F-fluorodoxyglucose PET/CT scanning have been used for diagnosis.

Mild dilatation of the common bile duct has been observed in 40% of patients studied by CT and may rarely be associated with episodes of cholangitis. Rare associations of PLD include congenital hepatic fibrosis, adenomas of the ampulla of Vater, and cholangiocarcinoma.

Cysts in other Organs

Seminal vesicles, pancreas, and arachnoid membrane cysts are present in 40% (males), 5%, and 8% of patients, respectively. Seminal vesicle cysts rarely result in infertility. Defective sperm motility is another cause of male infertility in ADPKD. Pancreatic cysts are almost always asymptomatic, with very rare occurrences of recurrent pancreatitis and possibly chance associations of intraductal papillary mucinous tumor or carcinoma reported in ADPKD. Arachnoid membrane cysts are asymptomatic, but may increase the risk for subdural hematomas. Spinal meningeal diverticula may occur with increased frequency and rarely present with intracranial hypotension due to cerebrospinal fluid leak. Ovarian cysts are not associated with ADPKD.

Vascular Manifestations

These include intracranial aneurysms and dolichoectasias, thoracic aortic and cervicocephalic artery dissections, and coronary artery aneurysms. They are caused by alterations in the vasculature directly linked to mutations in PKD1 or PKD2 . The respective protein products, PC1 and PC2, are expressed in vascular smooth muscle cells (VSMC). Pkd2 ^+/− VSMCs from mice exhibit increased rates of proliferation and apoptosis and Pkd2 ^+/− mice have an increased susceptibility to vascular injury and premature death when induced to develop hypertension. Defective structural integrity of blood vessels occur in mice lacking PC1.

Intracranial aneurysms (ICA) occur in approximately 6% of patients with a negative, and 16% of those with a positive family history of aneurysms. They are most often asymptomatic. Focal findings such as cranial nerve palsy or seizure result from compression of local structures. The risk of rupture depends on many factors including the size of the aneurysm. Rupture carries a 35–55% risk of combined severe morbidity and mortality. The mean age at rupture is lower than in the general population (39 years versus 51 years). Most patients with ICA have normal renal function and up to 29% will have normal blood pressure at the time of rupture.

Cardiac Manifestations

Mitral valve prolapse occurs in up to 25% of ADPKD patients on echocardiography. Aortic insufficiency may occur in association with dilatation of the aortic root. Although these lesions may progress with time, they rarely require valve replacement. Screening echocardiography is not indicated unless a murmur is detected on examination. Clinically inconsequential pericardial effusions are a common incidental finding in ADPKD.

Diverticular Disease

Colonic diverticulosis and diverticulitis are more common in ESRD patients with ADPKD than in those with other renal diseases. Whether this increased risk extends to patients prior to ESRD is uncertain. There have been reports of extracolonic diverticular disease. It may become clinically significant in a minority of patients. Subtle alterations in polycystin function that may enhance the smooth muscle dysfunction during aging may underlie the development of diverticula.

Isolated Polycystic Liver Disease

Isolated autosomal dominant PLD (ADPLD; MIM 174050) also occurs as a genetically distinct disease in the absence of renal cysts. Like ADPKD, ADPLD is genetically heterogeneous, with two genes identified ( PRKCSH and SEC63 ) accounting for approximately one-third of isolated ADPLD cases. ADPLD often goes undetected even in first-degree relatives of patients with highly symptomatic polycystic liver disease. As in the case of polycystic liver disease associated with ADPKD, isolated ADPLD is more severe in women than in men. Liver function tests remain normal and when symptoms develop, these are related to mass effects or complications such as cyst hemorrhage or infection. Patients with isolated ADPLD may also be at increased risk for intracranial aneurysms and valvular heart disease.

Recessive Human Ciliopathies

Primary cilia are increasingly implicated in a wide variety of important morphogenic signaling pathways (e.g., ) which in turn account for the wide spectrum of clinical features associated with syndromes when cilia function is disturbed. Association with cilia, basal body or pericentriolar region has also been reported for the ever expanding groups of genes mutated in recessive “ciliopathy” syndromic disorders with pleiotropic manifestations that include varying degrees and penetrance of kidney cyst formation. These diseases include nephronophthisis (NPHP), Joubert syndrome (JBTS), Meckel-Gruber syndrome (MKS), Bardet-Biedl syndrome (BBS) and oro-facial-digital syndrome (OFD). In addition, fibrocystin, the ARPKD gene product is expressed in cilia as well as the apical membrane of distal nephron tubular cells.

Subsets of the recessive ciliopathies have coalesced into phenotypic and genetic continuums. NPHP, JBTS, and MKS comprise a phenotypic spectrum roughly proceeding from less severe to more severe, respectively. Manifestations at the NPHP end of the spectrum include kidney and liver fibrosis, kidney cysts and retinal defects. JBTS patients also have cerebellar vermal hypoplasia and cognitive impairment while at the most severe end of the spectrum, MKS patients manifest with occipital encephalocele and are commonly nonviable. BBS shares kidney and retinal defects and in addition is characterized by digital defects, obesity, anosmia and cognitive impairment. Recessive mutations in over thirty genes have been identified among the ciliopathy syndromes. Mutations in same genes (e.g., CEP290 , NPHP1 , BBS4 , MKS1 ) give rise to more than one of clinical syndromes indicating that the phenotypic continuum is mimicked by genotypic overlap. In the most extreme example, mutations in CEP290 has been associated with NPHP, JBTS MKS and BBS. This interrelationship of a spectrum of clinical syndromes with recessive mutations in the same gene likely results from a complex interplay of locus heterogeneity, multiple allelism, modifier gene effects and possibly more complex multigenic inheritance.

Recent work has established that the functional protein complexes composed of the recessive ciliopathy gene products are part of the transition zone and trafficking complexes that determine the molecular composition cilia ( Figure 80.2 ). Mutations in at least nine genes have been associated each with NPHP and JBTS, six genes with MKS, and an additional 14 genes associated with BBS. Extensive cell biological and proteomic analysis has shown that the NPHP, JBTS and MKS associated gene products comprise the transition zone fibers that are critical gatekeepers determining the entrance and exit of components into and out of cilia. The BBS gene products assemble into the BBSome which functions in the trafficking of integral membrane proteins to and from cilia. The other key elements of cilia structure and function are the IFT proteins that, in conjunction with the kinesin and dynein motor proteins, are required to form structurally intact cilia. While mice and fish exhibit polycystic phenotypes with mutations in IFT proteins that result in abnormal or absent cilia, no human diseases with recessive loss of function in IFT genes and absent cilia have been identified possibly because these are embryonically lethal. Taken together, these recent discoveries suggest that recessive ciliopathies result in complex perturbations in the molecular composition of cilia. These findings also define the molecular relationship between the syndromic kidney cystic diseases resulting from ciliopathies with the non-syndromic polycystic kidney diseases, ADPKD and ARPKD. The latter both result from mutations in individual integral membrane proteins that are among the much larger group of client proteins of the BBSome and transition zone complex as they function to determine molecular composition of cilia. Ciliopathy syndromes therefore result from complex perturbations of cilia composition whereas ADPKD and ARPKD result from the discrete absence of individual molecular components of cilia that are not know to otherwise affect delivery or retention of additional ciliary proteins.

Molecular Genetic Mechanisms of Cyst Formation in ADPKD

The molecular genetic mechanisms of cyst formation in ADPKD are required to explain two salient clinical features of the disease. The first is to explain why the disease occurs in such a focal manner given that the ADPKD gene mutations are autosomally inherited and all cells in the body carry a mutated allele. The second is to determine the basis of the marked intra-and interfamilial variability in disease severity.

The apparent paradox of the focal nature of ADPKD is highlighted by the observation in microdissection studies of early ADPKD kidneys that localized cystic dilatations occur in kidney tubules that appear otherwise normal despite the presence of the same germline mutation in all the cells. A molecular explanation for this observation has come with the discovery that cyst lining cells from human ADPKD cysts have loss of heterozygosity (LOH) in the chromosomal regions of the respective PKD genes in both kidney tubular and bile ductular cysts. The LOH indicates loss of the functional gene from the wild type allele through focal somatic second hit mutations that define cyst formation as a recessive phenotype at the cellular level. The absence of a remnant wild type allele in a subset of cells from a cyst with LOH suggests that the cysts are clonal in origin—i.e., arising from a single cell.

The conclusions from these studies were challenged on the basis of: (1) Immunolocalization studies using anti-PC1 antibodies showing evidence of residual expressed protein in human cysts, (2) the relatively low rate of detection of LOH in cysts, (3) the relatively high rate of somatic mutations required to account for the thousands of independent cysts, and (4) the fact that the finding of LOH in cysts established an association, but not a causal link. In retrospect, immunolocalization studies were focused on cellular staining patterns that did not examine cilia expression of the protein; the latter is now thought to be the primary site of PC1 function in ADPKD. Additionally, approximately 30% of germline mutations in PKD1 are predicted to be non-synonymous amino acid substitution mutations which are predicted to yield immunoreactive protein products. If a similar proportion of missense variants occurs among second hit mutations, resulting cysts be positive for PC1 by immunostaining. The relatively low detection rate of LOH in cysts may reflect limitations in comprehensive screening of PKD1 for pathogenic mutations. Even a decade later, the success rate of PKD1 mutation detection is still ~85%. When more effective sequence-based mutation detection was applied to cysts from patients with germline PKD2 mutations, the rate of detection of second hits was the same as the sensitivity of mutation detection suggesting that effectively all cysts had second hits. The rate of somatic mutations required to account for the thousands of cyst that develop in ADPKD are in keeping with the rate of measured HPRT mutation frequencies in kidney epithelial cells ranging from 5×10 ⁻⁵ in the first decade to 2.5×10 ⁻⁴ after the eighth decade of life.

A variant of the two hit hypothesis was suggested by studies examining kidney cysts from patients with defined PKD2 mutations. Cyst cells were initially screened for second hit mutations in PKD2 , followed by screening for PKD1 mutations in the subset of cysts in which second hit PKD2 mutations could not be found. Evidence of loss of a PKD1 allele in these latter cysts led to the proposal the trans-heterozygous somatic mutations involving an allele of the PKD gene other than the one affected by the germline mutation can give rise to cysts. However, trans-heterozygous mutations are unlikely to be sufficient for cyst formation. Compound heterozygous individuals with bilineal inheritance of a PKD1 mutation and a PKD2 mutation show more severe polycystic kidney disease as do trans-heterozygous mice, but the overall severity of phenotype in both man and mouse is within the range observed for individuals with mutations at just one of the gene loci.

Mouse models have established the causal link between second hits in a single Pkd gene and cyst formation ( Figure 80.3 ). The occurrence of kidney cysts after embryonic day 14.5 in conventional germline knockout animal models of Pkd1 and Pkd2 anticipated a link between homozygous loss of polycystin genes and cyst formation. A novel mouse model of Pkd2 was the first to established a causal relationship between somatic inactivation of the normal allele and cyst formation in adult mice. In this model, a serendipitous modified allele, Pkd2 ^WS25 , resulted from insertion of a disrupted exon 1 in tandem with the wild type exon1. Pkd2 ^WS25 expresses functional PC2 but is prone to genomic rearrangement converting it to either a null or true wild type allele. The dosage of this allele correlates with propensity toward cyst formation and the cyst lining cells in adult mouse kidneys did not express PC2. Subsequently, the conditional Cre-lox system has been used to bypass the embryonic lethality of null mice and achieve homozygous inactivation of Pkd genes in a tissue in a selective and temporally controlled manner. These studies confirmed that somatic second hit mutations in either Pkd gene are sufficient for cyst formation. Similar conditional inactivation approaches have been applied to cilia ablation models targeting genes whose products are required for intraflagellar transport ( Kif3a , Ift88 ). The latter showed that somatic loss of intact cilia also results in a polycystic kidney phenotype. The Cre-lox system offers spatial and temporal control of Pkd gene inactivation but has the limitation that inactivation events occur simultaneously in large tracts of cells in either selected nephron segments or along the entire nephron. This is not believed to be the prevalent pattern of inactivation in human disease; in this regard the Pkd2 ^WS25 model remains unique in that Pkd2 inactivation is thought to occur by stochastic mechanism involving individual cells. Interestingly, the molecular events underlying Pkd2 inactivation in this model are not believed to have a nephron segment specific bias, yet the majority of cysts arise from distal nephron segments. More recent mouse studies suggest that collecting duct cyst growth is particularly sensitive to PC1 dosage. These findings have been part of the rationale for the potential benefit of vassopress-2 receptor antagonists in the treatment of ADPKD.

Mouse models of polycystic kidney disease. a : Conditional mouse model of polycystic kidney disease due to homozygous inactivation of Pkd1 in some nephron segments. Cells in the kidney that have undergone inactivation of Pkd1 are marked by the β-galactosidase expression (blue) from a Cre-recombinase reporter expressing lacZ . Tubule segments (*) and glomeruli ( arrowheads ) in which Pkd1 is not inactivated appear normal but are confined to septa between expanding cysts lined by cells stained blue indicating Pkd1 has been inactivated. b : Chimeric mice produced from Pkd1 − / − ES cells aggregated with wild type morulae marked by constitutive lacZ expression show mosaic cyst formation. Cyst lining are comprised of cuboidal wild type cells ( blue staining ) and flat Pkd1 − / − cells that do not express lacZ and are therefore unstained (indicated by arrows). (Images courtesy of Stefan Somlo and Saori Nishio). c : Early inactivation of Pkd1 results in rapidly progressive cyst formation; cystic kidneys postnatal day 19 following inactivation of Pkd1 on postnatal day 2. d,e : Adult inactivation of Pkd1 results in slowly progressive polycystic kidney disease. Kidneys at 3 months ( d ) following inactivation at 1.5 months show no overt cysts; kidneys at 6 months ( e ) following inactivation at 1.5 months show progressive cyst formation. d–e : Scale bars, 2 mm (left panels); 100 μm (right panel).

(Modified and reprinted by permission from Macmillan Publishers Ltd: Piontek K et al., A critical developmental switch defines the kinetics of kidney cyst formation after loss of Pkd1, Nature Medicine , 13:1490–95, copyright 2007.).

While somatic second hits are sufficient to initiate cyst formation, an increasing number of additional molecular factors affecting progression of polycystic kidney disease have been discovered using in vivo model systems. These factors include: (1) The critical role of the timing of second hit mutations with respect to developmental stage, (2) the discovery that reduced dosage of PC1, not just complete absence, is sufficient for graded cystic dilation, (3) the potential role of kidney injury in fostering PKD progression, and (4) the finding that loss of polycystins can exert effects on surrounding cells to promote disease progression. Evidence that timing of polycystin inactivation has a profound impact on the rate of cyst growth came from inducible conditional inactivation mouse models permissive for inactivation of Pkd genes either during kidney development or during adult life. Using such a model, Piontek et al. showed that inactivating Pkd1 in the early postnatal period when the mouse kidney is still undergoing active nephrogenesis resulted in rapid progression of polycystic kidney disease whereas inactivation of Pkd1 in the post-developmental adult kidney resulted in very slow progression polycystic kidney disease. A similar finding of rapid cyst growth following early inactivation and slow cyst growth following late inactivation was observed in a polycystic model based on cilia ablation following inactivation of Kif3a . The discovery of the timing of gene inactivation as a contributing factor to disease progression has led to consideration of the possibility that the majority of somatic second hit mutations in human ADPKD as well as the critical rapid growth phase of cysts occur during embryogenesis. Such a mechanism, if confirmed in patient studies, would potentially have significant implications for the timing of the most effective therapy in ADPKD.

The impact of reduced PC1 dosage, as opposed to complete loss, in polycystic kidney disease has emerged over the past several years. Two animal models expressing hypomorphic (i.e., reduced function) alleles of Pkd1 showed progressive cyst formation despite the presence of some residual Pkd1 activity. Similarly, loss of the protein bicaudal C results in reduced PC2 expression and cyst formation through de-repression of inhibitory microRNAs. The significant interplay of gene dosage and polycystic kidney disease has, however, best been defined by studies centered on the genes responsible for isolated polycystic liver disease without kidney cysts (ADPLD)—a disease with indistinguishable clinical features from the polycystic liver disease that occurs in ADPKD patients. Heterozygous loss of function mutations in two genes have been identified so far for this dominantly inherited disease: PRKCSH and SEC63 . PRKCSH encodes the non-catalytic β-subunit of glucosidase II (GIIβ), an N -linked glycan processing (glucose trimming) enzyme in the ER. The SEC63p protein is highly conserved from yeast to man and functions as part of the ER protein translocation machinery to deliver proteins into and through the ER membrane. The functional unit for this process is the multicomponent translocon ( Figure 80.4 ). The activities of the translocon are tightly coordinated with downstream events of protein folding, modification and assembly, thereby providing a direct link between membrane targeting (including the cilial membrane) and protein maturation, and between Sec63p and GIIβ functional pathways. While neither protein is expressed in cilia, the connection of these proteins to polycystic kidney diseases is underscored by the finding that Prkcsh and Sec63 conditional mouse models develop kidney cysts in addition to liver cysts by a two hit mechanism when either gene is inactivated in a tissue selective manner. The severity of the polycystic phenotype in the ADPLD gene knockouts can be worsened by reducing the Pkd1 gene dosage. Increasing Pkd1 dosage by either genetic or pharmacologic means on the other hand eliminated cyst formation. The ability to modulate severity of polycystic kidney disease in these models by manipulating Pkd1 dosage establishes PC1 is the rate limiting component of the cystic pathway in both ADPKD and ADPLD. Interestingly, the same study showed that PC1 dosage also affects the severity of the kidney disease Due to mutations in Pkhd1 , the ARPKD disease gene.

The inter-relationship of isolated polycystic liver disease (ADPLD) with polycystic kidney disease (ADPKD). a : The two known polycystic liver disease gene products, the β subunit of glucosidase-II (GIIβ, indicated as Gluc II, red box) and SEC63 (red oval), are shown in relation to the multicomponent co-translational translocon. GII is an N-linked glycan processing (glucose trimming) enzyme in the ER that plays a major role in regulation of proper folding and maturation of glycoproteins. SEC63 acts as a docking site localizing the ER chaperone BiP to the luminal exit site of the translocon. BiP plays a direct role in gating of the translocation channel and in polypeptide transport via an ATP-dependent reaction that is activated by SEC63. There is a tight interconnection between protein translocation mediated by the multicomponent translocon and protein maturation involving the N-linked glycan processing enzymes, providing a direct link between the functions of the ADPLD gene products. Co-translational maturation events in this pathway include signal peptide cleavage, the transfer and trimming of N -linked glycans including the activity of GII, disulfide bond formation, transmembrane domain integration, chaperone binding and protein folding. SPC, signal peptidase complex; SS, signal sequence; PDI, protein disulfide isomerase; OST, oligosaccharyl transferase; Gluc I, glucosidase I; Gluc II, glucosidase II (α and β subunits); CNX, calnexin; CRT, calreticulin. (Modified and reprinted by permission from Elsevier: ref. [ ], copyright 2003.) b: The critical effect of polycystin-1 (PC1) dosage on polycystic disease severity. Inactivation of the APDLD gene Prkcsh encoding GIIβ in collecting ducts result in kidney cyst formation due to impaired PC1 biogenesis. The severity of cyst formation is markedly increased by further reducing PC1 with Pkd1 ^+/− background and moderately increased by reducing polycystin-2 (PC2) on the Pkd2 ^+/− background. Over-expression of PC1 using the Pkd1 ^F/H -BAC transgene rescues the Prkcsh ^flox/flox ; Ksp-Cre;Pkd2 +/− cystic phenotype; Pkd2 -BAC has no effect. Ages, P42; scale bar, 2 mm.

(Reprinted by permission from Macmillan Publishers Ltd: Fedeles S et al., A genetic interaction network of five genes for human polycystic kidney and liver diseases defines polycystin-1 as the central determinant of cyst formation, Nature Genetics , 43:1639–647, copyright 2011.)

Acute kidney injury following ischemia, nephrotoxin exposure or reduction of renal mass results in markedly augmented progression of cyst formation in animal models based on Pkd1 , Pkd2 , Kif3a or Ift88 . This is particularly true in adult onset models that otherwise show indolent progression in the absence of injury. These environmental “third hits” have been proposed as an essential part of disease progression in human ADPKD. While it is likely that kidney injury can exacerbate polycystic kidney disease, its contribution to disease progress in human ADPKD is uncertain given that cyst progression also occurs in animal models in the absence of injury. Finally, evidence is accumulating that there are effects promoting cyst formation that are active in the context of the whole kidney organ and may impact cells that have not lost polycystin expression. Perhaps the most intriguing of these was suggested by a unique chimeric animal model produced by aggregation of Pkd1 ^−/− embryonic stem cells with wild type morulae expressing LacZ as the wild type lineage marker ( Figure 80.3 ). These mice formed kidney cysts commensurate with the degree of Pkd1 ^−/− chimerism. The cyst lining epithelia were mosaic with both Pkd1 ^–/– and Pkd1 ^+/+ epithelial cells present in the early stages of cystogenesis. The Pkd1 ^–/– cyst cells appeared flat while the Pkd1 ^+/+ cells were cuboidal. Over time, Pkd1 ^−/− cells replaced the Pkd1 ^+/+ cells by inducing apoptosis via the c-Jun N-terminal kinase (JNK)-mediated pathway. These findings suggest the possibility that cells that lose PC1 expression induce cystic degeneration followed by programmed cell death in surrounding wild type tubular cells. Another study based on kidney specific inactivation of Pkd1 demonstrated the importance of the immune response mediated by macrophages in the progression of cyst formation in ADPKD. Orthologous gene models of ADPKD based on either Pkd1 or Pkd2 show infiltration of alternatively activated macrophages into the pericystic space early in the course of the disease. Pharmacologic depletion of the macrophages markedly slows the rate of growth of kidney cysts in these models. And aggregately, these animal models based on genes orthologous to human ADPKD are providing an increasingly complex and complete picture of the molecular genetic basis for disease progression. These studies also serve to emphasize the importance of using in vivo models to validate and refine data obtained from ex vivo tissue culture-based systems–ADPKD is after all a disease of three-dimensional organ structure and is most appropriately studied using in vivo models.

Molecular Determinants of Disease Progression

Studies have begun to provide clues as to the genetic determinants of disease progression. Interfamilial variation in ADPKD is strongly influenced by gene locus. PKD1 causes more severe disease than PKD2 , yet the gene products are thought to function in the same genetic and biophysical pathway. Either of two explanations can underlie this observed difference: mutation of PKD1 is inherently a more severe molecular lesion than mutation of PKD2 or the occurrence of the somatic second hit alterations that precipitate cyst formation more readily affect PKD1 than PKD2 . In general, the data support the latter mechanism. Homozygous inactivation of Pkd1 or Pkd2 in the mouse results in similar severity of kidney and pancreatic cystic disease, embryo edema, hemorrhage and embryonic lethality. While PC1 is rate limiting in models of dosage reduction, there is an absolute requirement for functional PC2 for PC1 to function normally. Indeed, the Pkd2 ^−/− mouse has the additional phenotypic features of randomization of left-right axis formation and defects in heart septum formation, neither of which are seen in Pkd1 ^−/− mice. Defects of the conotruncus, such as dual outlet right ventricle, have been a variable finding in Pkd1 ^−/− mice as have defects of bone ossification, and vascular integrity. These phenotypes have not been specifically investigated in Pkd2 ^−/− mice. In the genetic model organism Caenorhabditis elegans (nematode), mutation to either the Pkd1 ortholog, lov-1 or the Pkd2 ortholog, pkd-2 , results in identical defects in the stereotypical male mating behavior. Doubly mutant lov-1:pkd-2 worms do not show increased phenotypic severity.

The higher population frequency and increased clinical severity of PKD1 disease compared to PKD2 may be explained in large part by a higher frequency, respectively, of germline and somatic mutation affecting PKD1 . This in turn is certain to be influenced by the four-fold larger coding sequence of PKD1 compared to PKD2 . The recent recognition that PC1 is the rate limiting component of the polycystin complex coupled with the significantly higher rate of pathogenic non-synonymous amino acid substitution mutations in PC1 compared to PC2, further suggests that a broader range of mutations in PC1, than in PC2, will result in ADPKD. The strongest support for this theory that the frequency of mutation is primarily responsible for the locus specific differences in disease severity comes from the CRISP. At baseline, PKD1 kidneys had more cysts and were larger than PKD2 kidneys. However, while the absolute changes in kidney volume were greater for PKD1 (74.9 mL/year) than for PKD2 (32.2 mL/year), the relative rates of growth were not significantly different for the two genotypes (5.68% per year vs. 4.82% per year). This suggests that cyst initiation, but not cyst enlargement, is modulated by the disease gene; PKD1 is more prone to second hits than PKD2 , but there is no inherent difference in cyst growth potential once second hits occur in either gene.

Most of the several hundred PKD1 mutations described to date are unique to individual families. This suggests that each mutation essentially arose as a de novo event that was fixed in the population since it confers little or no significant reproductive disadvantage. The de novo mutation rate for PKD1 is estimated to range from 1.8×10 ⁻⁵ to 6.9×10 ⁻⁵ per generation. Studies in heterozygous mouse models support these theories. Pkd2 ^+/− mice uniformly show loss of PC2 immunoreactivity in cyst linings suggesting that homozygous somatic loss is the underlying cystogenic event; cyst linings in Pkd1 ^+/− mice uniformly showed presence of PC2 immunoreactivity allowing for the inference that it was PC1 that was lost in these cysts. In compound heterozygous Pkd1 ^+/− :Pkd2 ^+/− animals, 160 of 171 cysts (93%) showed uniformly positive staining for PC2, 8 (5%) were negative for PC2 and 3 (2%) could not be evaluated. This suggests that in compound heterozygous mice, Pkd1 was far more often the target of somatic loss in vivo .

Genotype-Phenotype Correlations

Genotype-phenotype correlations refer to the association between specific germline mutations (genotype) and the resulting spectrum of disease expression (phenotype). Genotype-phenotype correlations are largely absent in ADPKD with two notable exceptions. The first is the occurrence of contiguous gene deletion syndromes with loss of PKD1 in conjunction with loss of adjacent TSC2 , the gene mutated in tuberous sclerosis complex 2. This contiguous gene syndrome results in early onset severe APDKD and clinical features of tuberous sclerosis. The second example of genotype-phenotype correlation in ADPKD occurs with recessive inheritance of hypomorphic alleles (i.e., partial, as opposed to complete, loss of function) in either PKD1 or PKD2. The phenotype observed in individuals with hypomorphic alleles affecting both copies of the respective PKD gene can mimic ARPKD with early-onset polycystic disease manifest by diffuse and more homogeneous, albeit smaller cyst formation.

The marked intrafamilial variation otherwise seen in ADPKD suggest that factors other than the germline mutation determine the course of the disease. This intrafamilial variation was illustrated in a study of dizygotic twins, one of whom developed severe polycystic kidneys in utero while the sibling showed a more typical course of adult onset disease. Similarly, in an analysis of age of renal death in 74 parent-offspring pairs, the median difference within the pairs was 0 years, but with a range −26.3 to +27.2 years that followed a normal Gaussian distribution. A systematic analysis of genotype-renal function correlation in 461 affected individuals from 71 ADPKD families with known PKD2 mutations revealed that the location of PKD2 mutations did not influence the age of onset of ESRD. This study also found that marked intrafamilial variability in disease severity occurs in PKD2 as well, with several individuals showing presentation of ESRD before age 50 while affected family members had the typical late onset disease.

Over 90% of mutations in PKD2 are predicted to be chain terminating loss of function alleles. In PKD1 , about one third of putative pathogenic variants are predicted to be amino acid substitution or other in-frame variants. Most of the non-truncating mutant alleles are thought to be loss-of-function although missense variants behaving as hypomorphic alleles that result in mild disease expression in the heterozygous state have been identified. Two studies have correlated 5’ location of mutations in PKD1 with more severe phenotypes. In one study examining 324 patients from 80 families with known PKD1 mutations, location of the mutations 5’ to the median position correlated significantly with about a 3 year earlier onset of ESRD. In a second study, 51 patients with vascular complications and known PKD1 mutations were evaluated. The median position of the PKD1 mutation was significantly further 5’ in the vascular population than in PKD1 families without vascular complications. Within the vascular population, more severe disease as defined by aneurysmal rupture, early rupture, or more than one vascular case in the family had median mutation locations more 5’ than the PKD1 vascular population as a whole. These data correspond to a previous report of a smaller number of cases and raises the possibility that the large 5’ extracellular domain of PC1 may act as a dominant factor in aneurysmal complications in ADPKD.

Modifier Effects in ADPKD

Modifier effects, either genetic or environmental, are an alternative explanation to stochastic factors such as second hit timing and frequency for the phenotypic variation seen in ADPKD. The observed intrafamilial variability that argues against strong allelic effects may in fact argue for genetic background effects within families modulating disease expression. A study comparing intrafamilial variation in age to ESRD among siblings to that in monozygotic twins found significant excess variability among the siblings relative to the genetically identical twin pairs. The genetic contribution to intrafamilial disease variability, defined as the proportion of the variance explained by modifier genes (i.e., heritability), was assessed using variance component analysis in two studies. The first examined 406 patients from 66 PKD1 families and estimated the heritability for creatinine clearance (C _cr ) among those without ESRD to be 42% and the heritability for ESRD to be 78% among those with ESRD. Although conclusions from this data are limited by power, one possible interpretation that emerges is that genetic factors contribute to differing extents to progression in early stage disease and late stage disease. A second study examined variance of a number of phenotypic traits in 315 affected relatives in 83 PKD1 families. The heritability for these traits ranged from 18 to 59% of the variance. In this study, modifier genes accounted for 32% of the variance for C _cr and 43 to 50% of the variance for age of ESRD, again showing the trend toward more heritability for late stage disease. One putative modifier gene locus was identified in a severely affected PKD1 family in which affected individuals also carried a heterozygous mutation for HNF-1β, a transcription factor that has been implicated in regulating of PKD gene expression. A second study analyzing candidate genes as modifiers in a large cohort of ADPKD families identified the canonical Wnt inhibitor Dickkopf 3 as a candidate for modifying ADPKD expression. Much larger studies than these will be required to actually begin to identify modifier loci in genome-wide association studies.

Polycystin-1

Polycystin-1 (PC1) is an eleven membrane spanning 4302 amino acid protein with a ~3000 amino acid extracellular NH ₂ -terminus and a ~220 amino acid cytosolic COOH-terminus ( Fig. 80.5 ). The extracellular NH ₂ -terminal domain contains distinct combination protein motifs including leucine-rich repeats, a WSC homology domain, C-type lectin domain, an LDL-A related domain, and 16 immunoglobin-like domains PKD repeats, a receptor egg jelly (REJ) module containing four fibronectin type III FNIII) domains and a GPCR proteolytic site (GPS motif) within a newly defined GPC autoproteolysis inducing domain (GAIN domain) shift. Additional domains identified either by homology or computational prediction include a PLAT/lipoxygenase homology 2 (LH2) domain between the first and second transmembrane spans and a polycystin motif region in the extracellular loop between the sixth and seventh transmembrane spans. The cytoplasmic COOH-terminus of PC1 contains a coiled-coil domain. The region of the last five transmembrane spans of PC1 share sequence homology with PC2 (PC2) although there are critical differences in the sequence that suggest that PC1 does not function directly as a channel protein the way PC2 does (see Polycystin Channel Function subheading below).

The location and topology of the predicted 11 transmembrane spans of PC1 was demonstrated by experimentally using glycosylation reporter analysis. The NMR solution structure of one the PKD domains, so named because they were first described in PC1, showed the domain to have a β-sandwich fold that belongs to a distinct family uniquely conserved among vertebrate polycystin homologs. Atomic force spectroscopy has shown that PKD domains confer increased tensile strength to the PC1 molecule. However, another study found that the PC1 extracellular region is highly extensible and that this extensibility is mainly caused by the unfolding of its immunoglobulin-like PKD domains. Stretching the native PC1 extracellular region results in a sawtooth pattern with equally spaced force peaks resulting from the sequential unfolding of individual PKD domains and domains refold after mechanical force unloading. The tensile properties of the PKD domains is altered by naturally occurring missense mutations found in patients. The PKD domains, along with the WSC homology domain, C-type lectin domain, an LDL-A related domain are thought to be involved in protein-protein or protein-carbohydrate interactions which have led to speculation that PC1 could serve as a receptor for unknown ligands. A mechanosenory role in flow sensation has also been proposed for PC1. PC1 signaling events might be regulated through force driven unfolding/refolding events with or without associated ligand interactions.

PC1 has broad tissue expression, including kidney, brain, heart, bone, and muscle. Its expression is developmentally regulated with high levels of expression in developing mouse tissues that subsequently decline. The subcellular localization of PC1 in tissues has been difficult to establish likely due to a combination of very low abundance of the native protein and limitations of the antibody reagents. Several studies identified PC1 in the plasma membrane of kidney tubule cells, particularly in the distal nephron segments including collecting ducts. Within these segments, PC1 appeared at the lateral membrane at sites of intercellular adhesion and at desmosomes. Over-expressed, epitope-tagged full length PC1 has been localized in the lateral membrane. Similar lateral location of a PC1–PC2 complex was reported in kidney tissue over-expressing PC1 as a transgene, although a similar pattern was not reported in epitope tag knockin model. Following the paradigm shift resulting from the discovery of the role of primary cilia in ADPKD and related disorders, much of the emphasis on the subcellular localization of native PC1 has shifted to primary cilia of epithelial cells from the kidney and other tissues. Most recently, PC1 has been found to be a component in urinary exosome-like vesicles, whose potential role in the functioning of polycystin signaling and ADPKD is just beginning to be explored.

PC1 Cleavage

The complexity and potential diversity of PC1 function is further amplified by a series of proteolytic events yielding fragments in both the extracellular and intracellular compartments. PC1 undergoes autoproteolytic cleavage at the GPS motif within the GAIN domain to produce an NH ₂ -terminal fragment (NTF) comprised of the first extracellular domain and a COOH-terminal fragment (CTF) containing the eleven membrane spans and the cytosolic tail ( Figure 80.5 ). This cleavage process requires an intact REJ module and GAIN domain and likely occurs early in the biosynthetic pathway, probably at the level of the endoplasmic reticulum (ER). The NTF and CTF remain non-covalently associated with each other and functional GPS cleavage is required for PC1 to be able to stimulate spontaneous tubulogenesis of MDCK cells in three dimensional matrix culture. GPS cleavage is conserved in proteins evolutionarily related to PC1 and has been reported as an essential step for folding, trafficking, and bioactivity of non-polycystin proteins. The crystal structure of the GAIN domain, which includes the GPS motif at its COOH-terminal region, has recently been solved in two distantly related adhesion-GPCRs which share this domain with the PC1 related proteins in higher organisms. The GPS cleavage process is thought to be necessary for proper trafficking of PC1 and for autoinhibitory function in adhesion-GPCRs. GPS cleavage deficient knockin mice show a partial rescue of the Pkd1 null phenotype suggesting that uncleaved PC1 may behave as hypomorphic allele.

A second intra-membranous cleavage product of PC1 results in the P100 intramembranous fragment with six membrane spanning domains. P100 acts to reduce store operated calcium entry through a mechanism involving the ER calcium sensor protein STIM1. The cytoplasmic tail of PC1 is also cleaved. Two processes have been described that liberate different sized fragments, both of which traffic to the nucleus and affect gene transcription. These processes are discussed in detail in the section on C-terminal Cleavage below.

Interactions

The most significant biochemical interaction for PC1 remains its association with PC2. PC1 and PC2 interact through their respective COOH-termini ( Figure 80.5 ). The structural data suggest a stoichiometry of one PC1 molecule and three PC2 molecules in the complex. The interaction depends on the integrity of the coiled coil domains in the COOH-termini of PC1 and PC2 and has been demonstrated using full length PC1 and PC2 constructs. All known pathogenic predicted truncating mutations in either gene result in loss of this interaction. The recognition of this interaction soon after the discovery of the genes led to the hypothesis that PC1 may serve a receptor function that controls the channel activity of PC2 as part of the polycystin signaling complex.

Several other associated complexes for PC1 have been defined although their roles in the pathogenesis of ADPKD are less clear (reviewed in ). The extracellular domains of PC1 are capable of in vitro homotypic interactions via their PKD domain repeats that may mediate intercellular or intermolecular adhesion as part of polycystin function. In C. elegans , the β-subunit of ATP synthase (ATP-2) co-localizes in cilia and interacts with the PLAT/LH2 domain of the nematode PC1 homolog, LOV-1. Casein kinase II also interacts with LOV-1 and this interaction may regulate trafficking of the C. elegans PC2 homolog into cilia in a phosphorylation dependent manner. Interaction with the COOH-terminal domain of PC1 inhibits degradation and results in re-localization of regulators of G protein signaling (RGS7), perhaps fitting with a postulated role for PC1 as an atypical G-protein coupled receptor. The cytoplasmic tail of PC1 also interacts with tuberin, the tuberous sclerosis complex 2 ( TSC2 ) gene product (see mTOR). PC1 interacts with intermediate filament components including vimentin, cytokeratin 8 and 18 and desmin. PC1 has also been shown to co-localize and complex with the intercellular adhesion molecules E-cadherin and β-catenin suggesting a possible role in stabilization of adherens junctions and the maintenance of the differentiated polarized state of epithelia in kidney tubules. PC1 may mediate communication with the extracellular matrix as suggested by co-localization with α2, β1 integrin and interaction with αv,β3 integrin via the COOH-terminal region of the β3 integrin. This diversity of interacting partners highlights the likelihood that PC1 and PC2 (see below) serve a broad array of cellular and tissue functions, not all of which will be related to polycystic kidney disease.

Polycystin-2

From the time of its discovery, polycystin-2 (PC2) was hypothesized to be a cation channel most likely functioning in calcium-based signaling events. PC2 is a 968 amino acid integral membrane protein with six membrane spans and intracellular NH ₂ – and COOH-termini ( Figure 80.5 ). It belongs to the transient receptor potential (TRP) channel family. TRP channels constitute a family of channel proteins found throughout metazoan evolution that generally function as receptor gated sensory channels. The mammalian TRP channels encompass at least 28 different proteins that are grouped into six subfamilies based the complement of structural motifs in their respective cytosolic NH ₂ − and COOH-termini (reviewed in ). The polycystins belong to the TRPP subfamily and PC2 is also called TRPP2. The last five transmembrane spans bear a strong TRP channel signature and the region between S5 and S6 (transmembrane segments 5 and 6) contains the putative pore region. In addition, the large first extracellular loop between S1 and S2 contains a polycystin motif of unknown function that is highly conserved in all PC1- and PC2-related proteins. The cytoplasmic tail contains a putative calcium binding EF-hand. The EF hand binds calcium and may have a role in modulating channel activation and inhibition.

PC2 is widely expressed in tissues, particularly the kidney, heart, ovary, testis, vascular smooth muscle, and small intestine. In the kidney, PC2 is expressed in all nephron segments, with the possible exception of the thin limbs of the loops of Henle. PC2 is not expressed in the glomerulus. As with PC1, expression in the distal nephron segments may be more robust than in the proximal segments. A domain in the COOH-terminus functions to restrict the membrane location of PC2 to the ER and cilia. At a subcellular level, there is general agreement that both native and heterologously over-expressed PC2 is strongly expressed in the ER membrane of both cultured cells and tissues. The plasma membrane localization of PC2 has been the subject of debate (reviewed in ). Native PC2, but not heterologously over-expressed protein, has been reported on the plasma membrane both by anti-native protein antibodies and by channel physiology. It has also been proposed that PC2 requires co-assembly with PC1 to traffic to the cell membrane and co-expression of both polycystins results in appearance of a novel plasma membrane cation channel activity. Conversely, studies using the C-terminal fragment of PC1 suggested a possible reciprocal role for PC2 in the cellular distribution of PC1. There is consensus and strong evidence to show that PC2 is localized to the primary cilia in kidney tissues and cultured epithelial cells. Native and over-expressed PC2 can be detected in the primary cilium. The NH ₂ -terminus domain of PC2 containing an RVxP amino acid motif has been identified as both necessary and sufficient for cilia location of type 2 membrane proteins including PC2. Trafficking of PC2 to cilia is independent of PC1.

PC2 Homologous Proteins

Two homologs of PC2, PKD2L1 (or PCL) and PKD2L2, have been identified in the mammalian genome. PKD2L1 is expressed in striated muscle (heart and skeletal), brain, spleen, and testis, whereas PKD2L2 is expressed only in testis. PKD2L1 has been shown to function in conjunction with PKD1L3 in sensing sour taste and cerebrospinal fluid pH. PC2 is highly conserved in metazoan evolution and homologs have been identified in sea urchin, C. elegans , Drosophila melanogaster and zebrafish. In sea urchin, the PC2 homolog associates with suREJ3, the PC1 homolog, and both localize to acrosomal region of spermatozoa suggesting a possible role in the ionic channel events that accompany the acrosome reaction during fertilization. In C. elegans , mutations in the PC2 homolog result in defects in location-of-vulva and response behavior in the male worms that is indistinguishable from the phenotype seen in LOV-1 (PC1 homolog) mutants. As in mammalian systems, PC2 shows strong expression in the ER membrane and the cilia membrane in C. elegans . However, there is little sequence conservation of the cystolic NH ₂ − and COOH termini from mammals to invertebrates ( C. elegans and Drosophila ) and there appear to be differences in the phosphorylation pattern and trafficking signals with mammalian protein. The PC2 homolog in Drosophila is expressed in sperm and knockout in fly results in sperm that retain normal backward motility and ability to fertilize but lose hyperactivation and directed movement required for reaching the female storage organs. Flies with a loss of function PC2 allele showed reduced contractility of the visceral smooth muscle cells through a mechanism that requires cooperativity with the ryanodine receptor in regulating intracellular calcium homeostasis. In mammalian systems, PC2 directly interacts with and regulates the activity of the ryanodine receptor and alters smooth muscle cell calcium homeostasis. Finally, zebrafish have proven to be a valuable vertebrate genetic model of polycystic kidney disease. Zebrafish have a pronephric kidney as well as left-right body axis asymmetry and knockdown or mutation of the PC2 homolog in fish results in cystic dilation of the pronephric tubule as well as abnormalities of left-right body axis formation.

Channel Function

PC2 functions as non-selective cation channel permeable to calcium. The initial studies used a variety of biophysical systems to characterize the channel and this may account for qualitative differences in the channel properties described for PC2. The systems used include co-expressed PC1 and PC2 in CHO-K1 cells studied by patch clamping, the outer membrane of human placental syncytiotrophoblasts and reconstituted purified protein studied in lipid bilayers, ER vesicles prepared from cells over-expressing wild type or mutant PC2 fused to lipid bilayers or epithelial cells in culture treated with chemical chaperones and studied by patch clamp technique. While the channel is non-selective for cations, it does pass calcium and most investigators believe that in vivo PC2 function is calcium-based second messenger signaling. PC2 channel activity is stimulated by physiological concentrations of calcium and is inhibited by higher concentrations of calcium that may be achieved locally as a result of PC2 channel activity. The critical role of the channel activity in the in vivo functioning of PC2 was demonstrated by a pathogenic patient mutation altering a single amino acid (D511V) that resulted in isolated loss of channel activity. Aside from biophysical studies, the in vivo cellular effects of polycystins have been evaluated by monitoring changes in cellular calcium using fluorescent indicator. PC2 can function as an ER calcium release channel. Phosphorylation of PC2 at a casein kinase 2 site, serine 812, modulates channel activity and has a role in trafficking of PC2 between ER, Golgi and plasma membrane compartments. The function of PC2 as a putative mechanosensory channel in cilia is discussed in the section on cellular calcium signaling below.

Interactions

The functional association of PC2 with PC1 was discussed in previous sections. PC2 associates with a number of calcium channel proteins. PC2 homomultimerizes with itself via its COOH terminus as a predicted tetramer. PC2 forms functional heteromeric channels through interaction with at least two other TRP channels, TRPC1 and TRPV4. In keeping with its putative role in regulating cellular calcium homeostasis via intracellular calcium pools, PC2 interacts directly with the inositol 1,4,5-trisphosphate receptor (IP ₃ R) and also regulates the activity of the ryanodine receptor through direct interaction. PC2 interacts with the ER t-SNARE protein syntaxin-5 which acts to inactivate the channel. Additionally, the mitotic centrosomal kinase Aurora A binds and phosphorylates PC2 at serine 829 and reduces the endoplasmic reticulum calcium release activity of PC2.

Protein interaction screens have yielded several putative PC2 binding partners potentially involved in cytoskeletal interactions. PC2 binds Hax-1 which in turn associates with the F-actin-binding protein cortactin suggesting a link between PKD2 and the actin cytoskeleton. The COOH-terminus of PC2 interacts with CD2AP which also interacts directly with the actin cytoskeleton. PC2 interacts with tropomyosin-1, troponin-1 and alpha-actinin, components of the actin microfilament complex. Yeast two hybrid screening also identified mDia1/Drf1 (mammalian Diaphanous or Diaphanous-related formin 1 protein) as a PC2-interacting protein. PC2 and mDia1 co-localize prominently at the mitotic spindles of dividing cells suggesting an association with microtubule cytoskeleton components.

Clues as to regulation of PC2 trafficking have also begun to emerge from studies of associated proteins. In addition to the previously noted co-assembly of PC1 and PC2, a novel interacting protein PIGEA-14 (chibby homolog 1) has been identified that plays a role in PC2 trafficking. PIGEA-14 co-expression with PC2 resulted in translocation of both proteins to the trans-Golgi network, suggesting role for PIGAE-14 in regulating the intracellular location of PC2. PC2 has been proposed to bind to phosphofurin acidic cluster sorting protein (PACS)-1 and PACS-2 via an acidic cluster in the COOH-terminal domain of PC2 that also contains the serine 812 phosphorylation site. The phosphorylation of PC2 regulates the binding of these adapter proteins that in turn are required for the routing of PC2 between ER, Golgi and plasma membrane compartments. In an attempt to unify the mechanisms of ADPKD with those for autosomal recessive polycystic kidney disease (ARPKD), association between PC2 and the PKHD1 (ARPKD) gene product fibrocystin has been investigated. Fibrocystin and PC2 co-localize and co-immumoprecipitate suggesting that they may exist in the same complex and this complex may also include the kinesin-2 anterograde cilia transport motor which may mediate fibrocystin dependent regulation of PC2 channel function. Although direct association was not shown, the klp-6 gene product in C. elegans encoding a kinesin-3 family member functions in the same genetic pathway as the polycystins (i.e., male mating behavior) and is required for localization and function C. elegans homolog of PC2 in cilia.

Wnt Signaling

The canonical Wnt signaling pathway plays a central role in controlling cell proliferation and differentiation. Aberrant Wnt signaling during embryogenesis can lead to axis duplication or dysgenesis of critical body structures, and excessive Wnt signaling activity is associated with the malignant transformation and metastatic potential of neoplasms. At the heart of the Wnt pathway is the multifunctional β-catenin protein, which is both a key constituent of the epithelial intercellular adhesive apparatus and an arbiter of a transcriptional pathway that profoundly influences cell proliferation and differentiation. The adhesive junctions that alert epithelial cells to the proximity of their neighbors are mediated by the transmembrane calcium-dependent adhesion molecule E-cadherin. The cytoplasmic COOH-terminal tail of E-cadherin binds to several soluble cytosolic proteins, including α-catenin, β-catenin and p120, which together link E-cadherin clustered at sites of cell-cell contact to the subcortical cytoskeleton, which in turns allows cells to exploit their intercellular adhesive junctions in order to organize their membranes into polarized domains. The pool of β-catenin protein that is not bound to E-cadherin is able to enter the nucleus, where it binds to and activates the TCF transcription factor. Many of the genes whose expression is controlled by TCF stimulate cell division, including c-myc, c-jun and cyclin D1. Thus, sequestration of β-catenin at sites of cell-cell contacts prevents it from activating a transcriptional program that results in proliferation. In contrast, loss of cell-cell contact liberates E-cadherin-bound β-catenin, freeing it to pursue its pro-proliferative agenda.

Before β-catenin can enter the nucleus, however, it must negotiate a major hurdle in the form of the cytoplasmic Axin/GSK3/APC complex. The primary purpose of this protein assembly is to capture and phosphorylate free β-catenin and, by so doing, doom it to degradation by the proteasome. Activation of the canonical Wnt signaling cascade leads to inhibition of the GSK3 kinase and hence to an increase in the size of the pool of β-catenin that is free to enter the nucleus and activate TCF-mediated transcription. The Wnt ligands are a family of secreted polypeptides that bind to cell surface receptors composed of a transmembrane protein called Frizzled and a second transmembrane co-receptor that can be any one of several different members of the family of LDL receptor related (LRP) proteins. Ligation of Frizzled by a Wnt protein leads to activation of its cytosolic partner protein Disheveled, which interacts with the Axin/GSK3/APC complex to prevent it from targeting β-catenin for proteasomal destruction. Stimulation of the Frizzled receptor can also elicit several β-catenin-independent effects, including transient elevations in cytosolic calcium concentrations and activation of c-jun kinase (jnk). These additional consequences of Frizzled activation are collectively referred to as non-canonical Wnt signaling. Non-canonical Wnt signaling is intimately involved in the establishment of planar cell polarity (see following sections).

Several lines of evidence indicate that the polycystin proteins participate in modulating Wnt signaling and that perturbations of Wnt signaling can lead to renal cystic disease. Studies of genetically manipulated mouse models reveal that either kidney-specific inactivation of the APC gene or expression of constitutively active β-catenin both result in the development of renal cysts that closely resemble those found in ADPKD. PC1 can be co-immunoprecipitated in a complex that includes E-cadherin and β-catenin, indicating that the potential exists for PC1 to exert a direct influence over the stability or extent of membrane bound pool of β-catenin. Overexpression studies employing membrane tethered constructs incorporating the C-terminal of PC1 offer conflicting results. One such study suggests that the membrane tethered C-terminal tail of PC1 can activate Wnt signaling reporters, whereas another study employing similar construct did not detect this activity. The basis for this discrepancy remains to be elucidated. Recent results indicate that the expression of genes associated with activation of the Wnt pathway is elevated in cystic renal tissue derived from the excised kidneys of ADPKD patients, as compared to the expression levels detected in normal-appearing non-cystic tissue derived from the same kidneys.

A seminal study on inversin, the protein encoded by the gene mutated in nephronophthisis type II, illustrates a fascinating paradigm through which a polypeptide associated with a renal cystic disease can influence signal transduction through both the canonical and non-canonical Wnt pathways. Inversin marks Disheveled for degradation in the proteasome, thus turning off canonical Wnt signaling by preventing Disheveled from inhibiting GSK3. Inversin also enhances manifestations of planar cell polarity that are under the control of the non-canonical Wnt pathway. As discussed in a later section, cleavage of the PC1 COOH-terminal tail releases a fragment that is capable of activating jnk and therefore may be similarly capable of upregulating the non-canonical Wnt pathway. Finally, as discussed below in greater detail, a cleaved COOH-terminal fragment of PC1 can interact directly with the TCF transcription factor and, through this interaction, prevent the TCF transcription factor from activating the expression of genes under the control of the Wnt pathway. Thus, both inversin and PC1 may act through distinct but analogous mechanisms as molecular switches that control the relative strengths of the canonical and non-canonical Wnt signaling pathways. Taken together, these diverse observations suggest that a failure to suppress canonical Wnt signaling and to activate non-canonical Wnt signaling constitutes a plausible mechanism that could account for at least some of the pathogenesis of PKD. In this context, however, it is important to note two recent studies whose results are not consistent with this model. Analysis of mitotic spindle axis, a parameter thought to be related to planar cell polarity, finds that it is not perturbed in advance of cyst formation in mouse models of ADPKD. Furthermore, a study found that the cells lining the cysts that develop in mouse models of ADPKD that have been engineered to express a Wnt/TCF reporter in vivo did not demonstrate elevated levels of TCF transcription activity. It remains to be determined whether activation of TCF-mediated transcription is involved in early phases of cyst formation and is terminated by the time that cysts are evident.

Cellular Calcium

PC2 belongs to the TRP family of ion channel proteins. PC2 can function as a non-selective calcium-permeable cation channel at the endoplasmic reticulum and perhaps at the plasma membrane as well. The importance of the channle activity is suggested by the fact that the disease causing non-synonomous D511V mutation abrogates PC2 channel activity. Functional studies demonstrate that PC2 expression enhances the release of calcium from intracellular stores. PC2 can physically interact directly with the IP3 receptor and, perhaps by virtue of this association, can alter the kinetics of cytoplasmic calcium transients induced by IP3 and by agonists of receptors that signal through IP3.

Seminal studies of MDCK cells performed by Praetorius and Spring illuminated an entirely new perspective on the potential function of the primary cilia that grace the apical surfaces of renal epithelial cells. These investigators found that primary cilia can serve a mechanosensory function and that bending them induces transient elevations of cytosolic calcium concentrations. Subsequent research using cell lines that lack expression of the polycystin proteins as well as antibodies directed against these proteins’ extracellular domains suggests that the PC2 channel activity, as well as polcystin-1 receptor activity, is required for induction of calcium transients in response to ciliary bending. Taken together, these studies gave rise to the hypothesis that cilia on apical surface kidney epithelia sense flow via the PC1/PC2 complex and this flow signal is important in establishing and maintaining kidney tubule lumen morphology in vivo . More recently, PC2 has been shown to inhibit the activity of stretch-activated ion channels in smooth muscle cells, indicating a possible role of PC2 in pressure sensing.

The binding of EGF to its receptor initiates elevations in cytosolic calcium concentrations that appear to require participation of the channel activity of PC2. The mechanism responsible for this EGF effect on PC2 channel activity appears to involve a protein called mammalian diaphanous-related formin 1 (mDia1), a member of the formin family of actin and microtubule regulatory proteins whose activity is modulated by the RhoA small GTPase. The mDia protein binds to PC2 and exerts a membrane potential-dependent block on its channel activity. The binding of EGF to its receptor appears to activate RhoA, which in turn releases mDia from PC2 and relieves the associated block of the PC2 channel.

A role for PC2 in governing the size and properties of intracellular calcium stores is suggested by the observation that vascular smooth muscle cells isolated from mice heterozygous for inactivation of the PC2 gene exhibit reduced size of the releasable intracellular calcium pool as well as reduced capacitative calcium entry. PC1 may also modulate the properties of intracellular calcium stores. Over-expression of PC1 in MDCK cells may inhibit capacitative calcium entry and speed calcium re-uptake by the endoplasmic reticulum. While the relationship between these effects of the polycystins on intracellular calcium stores and the pathophysiology of cystic disease remains to be determined, it is clear that through their involvement in modulating intracellular calcium levels the polycystins impact a physiologically critical second messenger pathway that influences almost every aspect of cellular behavior.

Cyclic AMP

A large body of evidence indicates that fluid secretion into cyst lumens makes use of the same machinery and mechanisms that drive fluid secretion by the well-studied secretory epithelia of the airway, the small intestinal crypt and the shark rectal gland. Chloride accumulates in the cytosol above its electrochemical equilibrium through secondary active transport mediated by a basolateral Na,K,2Cl co-transporter. An apical conductive pathway allows chloride to travel passively down its electrochemical gradient into the cyst lumen and the resultant lumen-negative potential causes sodium to follow paracellularly. The osmotic gradient created by this NaCl transport draws water into the cyst lumen, presumably contributing to the expansion of cyst volume. Several studies demonstrate that cAMP levels are elevated in cyst epithelial cells and furthermore that cAMP stimulates cyst fluid and electrolyte secretion. The identification, through both functional and cell biological experiments, of CFTR as the apical chloride channel involved in cyst fluid secretion provides a molecular explanation for cAMP’s pro-secretory effects. The CFTR channel opens in response to protein kinase A-dependent phosphorylation and thus the channel is activated in response to elevations in cytoplasmic cAMP. In vivo support for the role of CFTR as a mediator of cyst expansion has come from preclinical studies that showed some efficacy for CFTR inhibitors in reducing cyst formation in an orthologous gene model of Pkd1 . Additionally, case reports of families in which ADPKD coexists with cystic fibrosis have suggested that individuals with both diseases manifest a milder form of ADPKD, although this anecdotal evidence has been challenged. It has also been shown that cAMP acts as a mitogen in ADPKD renal epithelial cells and that this effect may be mediated through stimulation of the B-Raf/MEK/ERK signaling cascade, which is discussed in greater detail later in the chapter.

While the involvement of CFTR in cyst fluid secretion provides a plausible connection between cAMP and the production of cyst fluid, it does not explain why cyst epithelial cells manifest high cytosolic cAMP concentrations. It is possible that the perturbations in cytosolic calcium levels discussed in the preceding section may in part account for cAMP dysregulation in cyst cells. Several isoforms of both adenylate cyclase and phosphodiesterase, which generate and degrade cAMP, respectively, are subject to positive or negative regulation by calcium. In fact, recent evidence indicates that the primary cilium contains a cAMP signaling complex that includes two calcium-sensitive isoforms of adenylyl cylclase, AC5 and AC6. This complex also includes protein kinase A as well as A kinase anchoring protein 150, which acts as a scaffold on which components of the cAMP signaling network assemble. This complex interacts with PC2. Loss of PC2 expression or of its channel activity leads to dis-inhibition of AC5 and AC6, resulting in elevated levels of cAMP.

Finally, the polycystin proteins may directly or indirectly alter the activities of G-protein coupled receptors that signal through cAMP. Expression and activity of the V2 vasopressin receptor, for example, is elevated in a number of animal models of PKD. As noted in a later section, this fact is being exploited through the development of V2 receptor antagonists as potential therapeutic agents that can potentially slow or prevent cyst fluid accumulation by reducing cytosolic cAMP levels.

G-Protein Coupled Receptor Signaling

While their effects on V2 receptor activity suggest that the polycystins may influence G-protein coupled receptors, other studies suggest that PC1 may itself function as a non-traditional G-protein coupled receptor. The COOH-terminal tail of PC1 contains a short sequence that is capable of activating purified trimeric G proteins. Furthermore, the ability of a membrane tethered construct of the PC1 COOH-terminal tail to activate c-jun kinase and the AP-1 pathway depends upon the involvement of trimeric G-proteins. Expression of PC1 is apparently sufficient to initiate activation of Gα subunits and dissociation of the Gβ and γ polypeptides, as detected through measurement of the activities of ion channels whose open probabilities are modulated by βγ interactions. The presence of PC2 prevented PC1 from exerting this effect on G-proteins. The expression of the membrane tethered C terminal tail of PC1 has been found to activate Gαq and thus to increase the activity of phospholipase C (PLC). Increased PLC activity leads to the generation of IP3 and consequently to elevations in cytosolic calcium levels which, in turn, can lead to the activation of the calcium-dependent calcineurin/NFAT-dependent transcription pathway. The NFAT transcription factor governs the expression of genes involved in cell growth and differentiation. Activation of the calcium-sensitive phosphatase calcineurin by sustained elevation of cytosolic calcium levels results in dephosphorylation of NFAT, leading to its nuclear accumulation. Recently, PC1 was shown to bind Gα ₁₂ and modulate the activity of Gα12/JNK apoptosis pathway. Mutations in Gα ₁₂ or PC1 that eliminate interaction between these proteins also abrogated the PC1 dependent inhibition of Gα ₁₂ stimulated apoptosis.

Thus, PC1 appears to possess the capacity to modulate the activities of trimeric G-proteins and their downstream effectors in much the same manner as traditional seven transmembrane domain G-protein coupled receptors. The nature of the stimuli or ligands that regulate this capacity of PC1 and the relationship of this capacity to the major physiological functions of PC1 remain to be determined.

Mitogen Activated Protein Kinase/Extracellular Regulated Kinase

The mitogen activated protein kinase/extracellular regulated kinase (MAPK/ERK) cascade couples extracellular signals received by a variety of surface receptors, through the activation of small G proteins and the involvement of a variety of adaptors, to the successive phosphorylation of Raf (MAP kinase kinase kinase), MEK (MAP kinase kinase) and MAP kinase/ERK. Activated MAPK/ERK can modulate protein translation and can enter the nucleus to regulate the activities of transcription factors and the cell cycle. The MAPK/ERK pathway receives input from and influences a variety of other signaling pathways, including those associated with G-protein coupled receptors, calcium, cAMP, PKA, PKC, EGF receptor (and other receptor tyrosine kinases) and integrins.

In renal epithelial cells the MAPK/ERK cascade influences a variety of morphogenetic phenomena, including cell spreading, branching and tubulogenesis. In wild type subconfluent cells, PC1 associates with focal adhesion contacts and promotes cell spreading through a mechanism that involves both the ERK cascade as well as the focal adhesion kinase, FAK. The absence of PC1 impedes spreading. Furthermore, the MAPK/ERK cascade is found to be activated in a number of animal models of PKD including orthologous gene models for Pkd1 and Pkd2 . Studies in cultured human ADPKD cells suggest that ERK activation may be attributable to the high levels of cAMP present in their cytosols, which acts through protein kinase A to activate the MAP kinase kinase kinase B-Raf; however, such B-RAf dependent activation was not observed in vivo models based on Pkd1 and Pkd2 . It is also worth noting that in the absence of PC2 there is a dramatic increase in the quantity of ERK detectable in the nucleus, suggesting that both of the polycystin proteins participate in providing tonic suppression of the activity of the MAPK/ERK cascade. Clearly, the MAPK/ERK pathway is intimately related to a variety of processes that control epithelial growth and morphogenesis. In light of the numerous observations that the MAPK/ERK cascade is excessively active in a variety of cell and animal models of PKD, efforts are underway to test the potential of inhibitors of the relevant MAPK/ERK cascade kinases to prevent the aberrant cell proliferation and differentiation associated with renal cystic disease.

mTOR

The mTOR (mammalian target of rapamycin) protein is a kinase whose activation leads to increased protein translation and cell growth. Proteins that inhibit the formation of the mRNA cap structure are phosphorylated and inactivated by mTOR. In addition, mTOR phosphorylates and activates ribosomal S6 kinases, which act to enhance the translation of mRNAs that encode proteins involved in translation. Thus, any signal that upregulates mTOR activity produces an enhancement of the cell’s capacity to synthesize protein and consequently to increase its size. The mTOR pathway is stimulated by cell surface receptors that signal through PI3 kinase to activate the AKT kinase. Activated AKT phosphorylates the tuberous sclerosis complex, composed of the TSC1 and TSC2 proteins (hamartin and tuberin). The TSC complex acts as a GTPase activating protein (GAP) for the small G protein Rheb. When in the GTP bound state, Rheb acts to enhance mTOR function. Phosphorylation of the TSC complex by AKT inhibits its GAP activity, resulting in elevated levels of GTP bound Rheb and thus in elevated levels of active mTOR kinase.

Since activation of mTOR prepares the cell for additional growth, it is perhaps not surprising that dysregulation of the mTOR pathway that leads to inappropriately high levels of mTOR activation are associated with a variety of neoplastic syndromes. Loss of the TSC complex and its associated Rheb GAP activity, for example, results in loss of suppression of mTOR. Tuberous sclerosis is the hereditary disease caused by inactivating mutations in the genes encoding tuberin and hamartin, and it is characterized by the development of multiple hamartomas as well as renal cysts. It is interesting to note that the TSC2/tuberin gene lies extremely close to the PC1 gene and the two genes have an associated contiguous gene deletion syndrome. Furthermore, PC1 interacts with tuberin through its C-terminus and it has been suggested that tuberin may play a role in controlling the trafficking of newly synthesized PC1 to the cell surface. Phosphorylation of TSC2 by ERK leads to dissociation of the TSC1/TSC2 complex and loss of its Rheb GAP activity. This phosphorylation appears to be prevented by PC1. The interaction between PC1 and tuberin also appears to protect tuberin from inactivating phosphorylation by AKT. Thus, the interaction between PC1 and tuberin maintains the Rheb GAP capacity of the TSC1/TSC2 complex and contributes to the suppression of mTOR activity.

Administration of rapamycin markedly slows cyst development in studies of the Han:SPRD rat model of PKD, suggesting the possibility that inappropriate activation of the mTOR pathway is associated with or in part responsible for the excessive proliferation of renal epithelial cells that characterizes PKD. Further support for this hypothesis is provided by studies demonstrating that downstream effectors of the mTOR pathway are inappropriately activated in cyst lining cells. These studies also extend the beneficial effects of rapamycin to two mouse models of polycystic disease. Taken together, these data suggest under normal circumstances the polycystin proteins exert a potentially significant inhibitory influence on the strength of mTOR signaling. It is possible that loss of this mTOR suppression could be responsible for at least some of the hyperplastic component of PKD pathophysiology and human clinical trials using inhibitors of mTOR in ADPKD have been completed (see Prospects for Therapy in ADPKD).

C-Terminal Cleavage

Until fairly recently, the prevailing view of signal transduction from the cell surface to the nucleus held that extracellular signals received by plasma membrane receptors must undergo an obligate translation into the language of the intracellular second messenger systems before they could exert any influence on events in the nucleus. Revelations from studies of the Drosophila Notch protein and a variety of other transmembrane receptors have led to the definiation of a new signaling paradigm known as regulated intramembranous proteolysis (RIP). During the course of its postsynthetic processing Notch undergoes proteolytic cleavages that release its extracellular domain, which remains attached to the transmembrane segment via non-covalent interactions. This preparatory cleavage permits Notch to become the substrate for a subsequent intramembraneous cleavage event mediated by γ-secretase that is initiated by the binding of Notch to its ligand Delta, a transmembrane constituent of the plasma membranes of neighboring cells. The fragment released through this final cleavage travels to the nucleus, where it influences transcriptional activities that play a key role in cell fate determination. Thus, the C-terminal tail of a membrane protein can itself act as a direct messenger that can carry a signal from the surface to the nucleus without any need for the intercession of traditional second messenger molecules. It has now become clear that such cleavage events are quite common, playing critical roles in a variety of physiological processes, including the unfolded protein response, cholesterol metabolism and the adaptation of pancreatic islet cells to changes in extracellular glucose concentrations.

Both PC1 and fibrocystin undergo cleavages that release COOH-terminal tail fragments capable of entering the nucleus and modulating activities therein. Two C-terminal tail cleavage fragments have been reported for PC1. The larger of these is approximately 200 amino acid residues in length, apparently comprising the entire extent of the protein’s predicted C-terminal tail. This fragment’s production is dependent upon the activity of the γ-secretase protease, and it includes the amino acid motif previously identified as a G-protein activation sequence, which is rich in basic residues and appears to be capable of serving as an autonomous nuclear localization motif. Expression of a protein corresponding to this fragment in transfected cells leads to activation of jun kinase and the AP-1 pathway. This fragment can also act as an inhibitor of canonical Wnt signaling by virtue of its ability to bind directly to the TCF transcription factor and impede TCF’s interaction with the p300 transcriptional co-activator complex. The cleaved C terminal tail also interacts with the CHOP-10/GADD153 transcription factor and similarly prevents its interaction with p300. CHOP is activated as a final step in the endoplasmic reticulum/unfolded protein response pathway and induces the expression of genes that activate apoptotic pathways. Apoptosis has been described in cyst lining epithelial cells and may contribute to cyst formation. Expression of the PC1 tail fragment reduces CHOP activity, as well as the level of apoptotis in Pkd1 ^–/– cultured epithelial cells.

The smaller fragment of the PC1 C terminal tail encompasses roughly 17 kDa and does not include the nuclear localization signal that has been identified in the larger fragment. This fragment can interact with STAT6 and the coactivator P100 and is apparently translocated into nuclei by virtue of these associations. The 17 kDa fragment directly activates STAT6, and it activates STAT3 through a mechanism that is dependent upon STAT phosphorylation activated by growth factors or cytokines. The activation of the STAT transcription factors is pro-proliferative and pro-apoptotic. It has been suggested that accumulation of the 17 kDa PC1 fragment in the nucleus may contribute to ADPKD pathogenesis, while inhibition of STAT activity may dramatically slow cyst development.

The relationship between these two fragments is currently unclear. It is possible that the smaller fragment is the product of a secondary cleavage of the larger fragment. It is also possible that these fragments arise through separate cleavage mechanisms that respond to and transduce distinct messages. Nuclear accumulation of the PC1 COOH-terminal tail is detected in the renal epithelial cells of transgenic mice that over-express PC1, of mice that manifest kidney specific agenesis of cilia, as well as of wild type mice that have been subjected to ureteral ligation. These data suggest that cleavage and nuclear translocation of the PC1 COOH-terminal tail is suppressed by intact cilia function. Absence of cilia or loss of flow appears to initiate both of the reported PC1 COOH-terminal tail cleavages. It is interesting to note that the 17 kDa fragment appears to increase proliferation and apoptosis, whereas the larger fragments appear to suppress these processes. This observation prompts the as yet untested speculation that the two cleavage fragments may participate in distinct phases of the renal epithelial response to injury. According to this model, the early phases of the injury response, which involve apoptosis of epithelial proliferation to replace lost or damaged cells may be mediated at least in part by activation of the cleavage that produces the 17 kDa fragment. Activation of the production of the larger cleavage fragment may accompany the later phases of repair and may serve as a break to prevent repairative proliferation and apoptosis from becoming excessive and potentially pathogenic.

The observations that polcystin-1 and fibrocystin are substrates for COOH-terminal tail cleavage raise a large number of interesting and potentially important questions. Where within the cell do the cleavage events occur? What proteolytic enzymes are responsible? Where are the cleavage sites within the primary structures of PC1 and fibrocystin, and what are the sequence parameters that determine their susceptibility to cleavage? Is the NH ₂ -terminal GPS cleavage a prerequisite for PC1 COOH-terminal tail cleavage? How does PC2 expression stimulate PC1 C-terminal tail cleavage? Finally, and perhaps most importantly, do mutations that prevent the cleavage produce a phenotype in cultured renal epithelial cells and in renal tubules in vivo ? Future research into these and other questions related to COOH-terminal tail cleavages are likely to shed important light on the normal physiological functions of the PC1 and fibrocystin proteins. These studies may also suggest new pathways that can be exploited in the continuing search for novel strategies applicable to the development of therapeutics for renal cystic diseases.