Recent Advances in the Cell Biology of Polycystic Kidney Disease

Recent Advances in the Cell Biology of Polycystic Kidney Disease Brendan J. Smyth,* Richard W. Snyder,* Daniel F. Balkovetz,{ and Joshua H. Lipschutz{ *Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Departments of Medicine and Cell Biology, University of Alabama at Birmingham and Veterans Administration Medical Center, Birmingham, Alabama 35294 { Department of Medicine and Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, Pennsylvania 19104 {

Autosomal dominant polycy´stic kidney disease (ADPKD) is a significant familial disorder, crossing multiple ethnicities as well as organ systems. The goal of understanding and, ultimately, curing ADPKD has fostered collaborative eVorts among many laboratories, mustered on by the opportunity to probe fundamental cellular biology. Here we review what is known about ADPKD including wellaccepted data such as the identification of the causative genes and the fact that PKD1 and PKD2 act in the same pathway, fairly well-accepted concepts such as the ‘‘two-hit hypothesis,’’ and somewhat confusing information regarding polycystin-1 and -2 localization and protein interactions. Special attention is paid to the recently discovered role of the cilium in polycystic kidney disease and the model it suggests. Studying ADPKD is important, not only as an evaluation of a multisystem disorder that spans a lifetime, but as a testament to the achievements of modern biology and medicine. KEY WORDS: Polycystin, Polycystic kidney disease, ADPKD. ß 2003 Elsevier Inc.

‘‘Medicine is the tutor of Biology.’’ Dictum of medicine as noted in the comments at the presentation of the Albert Lasker Award for Clinical Medical Research, 1997. Joseph L. Goldstein, M. D., Nobel Laureate.

International Review of Cytology, Vol. 231 0074-7696/03 $35.00

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Copyright 2003, Elsevier Inc. All rights reserved.

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I. Historical Background Though this is a review of the cell biology of autosomal dominant polycystic kidney disease (ADPKD), medical aspects of this disease have largely driven research in the field. Therefore we thought it appropriate to spend some time reviewing the history and clinical features of ADPKD. Cystic diseases have been recognized for several millennia [for in-depth reviews of the history of ADPKD see Torres and Watson (1998) and Grantham (2001)]. For instance, Babylonian cuneiform texts contained detailed descriptions of hydatid cysts, which are composed of watery fluid and the larvae of certain tapeworms, especially of the genus Echinococcus (Nelson, 1988). Hippocrates (460 B.C.E.) recognized many types of kidney disease and Galen (600 A.C.E.) determined the etiologies of some types of hematuria (Torres and Watson, 1998). However, it was not until the Middle Ages, which heralded the link between disease and organ dysfunction, as opposed to the ancient Greek concept that had dominated medical science for 18 centuries and explained disease largely through the ‘‘humors,’’ that the presence of polycystic kidney disease (PKD) was recognized in western medicine. This was due, in large part, to the autopsy observations of artists, after permission for dissection was given by papal edict in 1480 (Benevieni, 1954). The misdiagnosis of what was actually PKD and uremia in 1586 by the court physicians of King Stefan Barthory, a Polish monarch, demonstrated the Renaissance’s continuing conflict between Greek humoral and scientific observation theories (Walter, 1934; Szpilczynski, 1977; Torres and Watson, 1998). It should be noted that Valsava (1666–1723) has been given credit for first studying the chemical composition of cyst fluid by tasting it (Torres and Watson, 1998). Advancements in anesthetics, antiseptics, and surgical technique made nephrectomy feasible by the late 1800s. Nevertheless, before the advent of modern imaging and surgical techniques, nephrectomy was a risky endeavor; something that was well appreciated by Lejars (1888). Lejars also recognized the bilaterality of PKD, which explained the disastrous results of nephrectomy in this disease. It was, in fact, Lejars who coined the term ‘‘polycystic kidney disease’’ (Torres and Watson, 1998). The picture became somewhat more complete when Steiner (1899) suggested that PKD was a genetic disorder. Osler (1902) described PKD in detail, including the multiorgan involvement, the characteristics of the urine, and a listing of the complete diagnostic criteria. Thus, by the end of the nineteenth century, PKD was recognized as a genetic disorder and its clinical manifestations were understood. ADPKD researchers made major discoveries over the past half-century (Grantham, 2001). In his 1957 doctoral thesis, Dalgaard established the autosomal dominant pattern of inheritance of this disease. Gardener (1969)

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established that renal cysts behave in a similar manner to renal tubules and Carone et al. (1974) described basement membrane weakness in PKD cysts. Evan et al. (1979) recognized the extensive degree of epithelial cell proliferation within cysts. Therefore, by 1980 it was determined that the formation and enlargement of cysts were due to three factors: the rates of epithelial cell proliferation, fluid collection, and tubular membrane dysfunction (Grantham, 2001). In 1982, the Polycystic Kidney Disease Foundation, dedicated to PKD research and treatment, was established by Joseph Bruening, whose wife had PKD, and Jared Grantham, a researcher at the Kansas University Medical Center (Foundation, 2002). PKD research benefited greatly from the general scientific revolution in genetics that was occurring during the 1980s. For example, Huntington disease (HD) is an autosomal dominant disorder with similarities to ADPKD, in that symptoms appear in adulthood and vary both in the rate of progression and in the age of onset. The HD gene was mapped to chromosome 4 by restriction fragment length polymorphisms in 1983 and ultimately cloned 10 years later (Gusella et al., 1983; MacDonald et al., 1992). Advances in PKD research, using many of the same techniques, soon followed. Reeders, a postdoctoral fellow in the Weatherall laboratory in Oxford, gathered together families with ADPKD and, in collaboration with Martin Breuning, located the region of the first ADPKD gene (PKD1) on the short arm of chromosome 16 (Reeders et al., 1985; Grantham, 2001). Although the exact PKD1 locus itself was not identified at this time, PKD1 was determined to be the causative gene in
85% of families, with 15% of families not demonstrating linkage to chromosome 16 markers (Kimberling et al., 1988). Kimberling and colleagues in 1993 located the region of the second gene for ADPKD (PKD2). The next step, cloning the genes, proved diYcult, especially for PKD1. By studying a family aZicted with combined PKD1 and tuberous sclerosis (TSC2) gene deletions (termed contiguous gene syndrome or PKDTS), the Oxford Group cloned PKD1 (Consortium, 1994). By 1995, researchers fully sequenced the PKD1 gene (Consortium, 1994, 1995; Hughes et al., 1995). In 1996, Somlo used a positional cloning approach to identify and sequence the PKD2 gene and described the protein product, polycystin 2 (Mochizuki et al., 1996). In 1996, the PKD Foundation established the first International PKD Gene Mutation Registry (Foundation, 2002). Relatively little is known about the protein product of the gene responsible for autosomal recessive polycystic kidney disease (ARPKD), fibrocystin (also called polyductin or tigmin), which was recently identified on chromosome 6, other than that it is predicted to have a single transmembrane (TM)-spanning domain and is a member of a novel class of proteins that shares structural features with c-met (the hepatocyte growth-factor receptor) and plexins (Onuchic et al., 2002; Ward et al., 2002; Xiong et al., 2002). Renal cysts are a common end point in many diseases such as von Hippel–Lindau disease

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(VHL), juvenile nephronophthisis, and chronic renal disease (Reichard et al., 1998; McKusick et al., 2002); however, this review will focus on the cell biology and pathogenesis of ADPKD, for which much is now known.

II. Clinical Features of Autosomal Dominant Polycystic Kidney Disease A. Organ Systems 1. Kidney The chief clinical manifestations of ADPKD are the triad of bilateral hyperechoic cysts, renal insuYciency, and hypertension (HTN) (Sessa et al., 1997). As a rule, the clinical diagnosis of ADPKD relies mainly on symptoms, family history, diagnostic imaging, and physical findings. On initial presentation, the most common symptoms are kidney related: flank pain, abdominal pain, symptoms of urinary tract infection, and episodic gross hematuria. As symptoms usually do not become apparent until age 30–50 years, ADPKD is often not discovered until adulthood (Stiasny et al., 2002). Acute and chronic flank pain are common complaints for ADPKD patients secondary to kidney enlargement, cyst infection, and nephrolithiasis (Sklar et al., 1987). As ADPKD patients are susceptible to the usual variety of insults that could aVect anyone, a systematic approach is essential to diVerentiate the etiology of abdominal pain in an ADPKD patient and to define a medical plan. There are also several conditions that are more specific for ADPKD. Referred pain is common in ADPKD, due to the kidney’s complex network of neuronal innervation. In addition, ADPKD patients are reported to have an increased incidence of uric acid and calcium oxalate stones. ADPKD patients with nephrolithiasis may also have distal acidification defects, abnormal transport of ammonium, low urine pH, and hypocitruria (Torres et al., 1993). Interestingly, dietary citrate treatment has been determined to slow the progression of ADPKD in Han:SPRD rats (Tanner and Tanner, 2000, 2003). Urinalysis in patients with ADPKD can show hematuria, pyuria, and mild proteinuria (Chapman et al., 1994). Approaches for the management of ADPKD pain and cyst infection have been extensively reviewed (Bajwa et al., 2001; Hemal, 2001). Physical examination may reveal easily palpable kidneys or be grossly normal depending on the size of the renal cysts (see Fig. 1). ADPKD is distinguished by bilateral symmetrical renal involvement. The kidneys are often significantly enlarged, and may weigh up to 8 kg each (Gabow, 1993). Microscopically, cysts originate from any portion of the renal tubule. Cysts of varying

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FIG. 1 The phenotypic eVect of ADPKD on the kidney. Gross pathological specimen of a kidney from a patient with ADPKD. The kidney is enormously enlarged and is filled with cysts. From Gabow (1993). Reprinted with permission from the New England Journal of Medicine.

size are typically seen in both the cortical and medullary areas. The cysts are lined with epithelium, thinned from the compression eVects of tubuloepithelial hypertrophy. Glomerulosclerosis, tubular atrophy, and interstitial fibrosis are typically present in kidneys with significant dysfunction (Zeier et al., 1992). Radiologic criteria exist for the diagnosis of ADPKD by renal computerized tomography (CT) scan or ultrasound (US). The presence of at least two renal cysts (unilateral or bilateral) in individuals at risk and younger than 30 years may be regarded as suYcient to establish a diagnosis; among those aged

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30–59 years, the presence of at least two cysts in each kidney is required; and among those aged 60 years and older, at least four cysts in each kidney is required for a positive diagnosis (Ravine et al., 1994). A CT scan is capable of measuring total kidney, renal cyst, and renal parenchymal volumes reproducibly. Recent studies have established that an increase in cyst volume correlated best with the decline in renal function and renal volume appears to be a good surrogate marker for disease progression in ADPKD (King et al., 2000). However, DNA linkage analysis is potentially more accurate than renal US, especially for prospective kidney donors less than 30 years of age (Hannig et al., 1992). Although ADPKD is typically a late-onset disorder, US has permitted the detection of the disorder in the newborn or infant in some instances and occasionally even prenatally as early as 20 weeks gestation (Pretorius et al., 1987; Ceccherini et al., 1989; Turco et al., 1993). Nonetheless, ADPKD cysts are rarely seen with antenatal US (Sedman et al., 1987). When cysts are discovered, neonatal ADPKD generally involves both kidneys. Prenatal genetic screening for ADPKD is rarely done, as very few people would consider terminating a pregnancy were the diagnosis of ADPKD to be confirmed. If prenatal diagnosis is considered, Breuning and colleagues (1990) recommend that chorionic villus sampling be attempted only after the linkage phase of the DNA markers has been established by haplotyping the index family. Furthermore, families should be of suYcient size to rule out mutations of the less common PKD2 gene. A high mutation rate with increased intrafamily variability is characteristic of neonatal ADPKD (Fick et al., 1994). Possible mechanisms of pathogenesis include DNA instability (Fick et al., 1994), inheritance of a gene modifier (Peters and Breuning, 2001), or a large deletion as seen in PKDTS (Dauwerse et al., 2002). Though it is somewhat controversial, there may be an increased risk for renal cell carcinoma in patients with ADPKD (Kumar et al., 1980; Bernstein et al., 1987; Gabow, 1993). A recently described model of ‘‘polycystic kidneys’’ and renal cell carcinoma in Japanese and Chinese toad hybrids may shed further light on the link between renal cystic disease and malignancy (Masahito et al., 2003). 2. Intracranial and Aortic Aneurysms Intracranial saccular aneurysms are one of the most devastating extrarenal manifestations of this disease. ADPKD was first associated with cerebral aneurysms in 1904 (Greenburg, 1997). The reported prevalence of intracranial ‘‘berry’’ aneurysm in ADPKD can range from 10 to 30% (Levey et al., 1983; Butler et al., 1996). Patients with ADPKD carry up to a 20fold increased risk of subarachnoid hemorrhage compared to the general population (Schievink et al., 1992). The average rate of rupture of incidental

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aneurysms is 2%/year (Greenburg, 1997). However, serious aneurysms tend to be familial and, given the small but real risk of angiography (
1%), general screening for cerebral aneurysms is not recommended unless the patient is symptomatic or has a strong family history. A patient with an unruptured aneurysm greater than 1 cm in diameter should undergo surgical repair. See Pirson et al. (2002) for a detailed review of the management of cerebral aneurysm. 3. Noncystic Cardiovascular System Although ADPKD primarily disrupts the function of ductal organs, such as the kidney, liver, and pancreas (Milutinovic et al., 1980; Grunfeld and Bennett, 1995), abnormalities of the cardiovascular system are of paramount importance (Iglesias et al., 1983; Ritz et al., 1994; Leier et al., 1984). HTNrelated cardiovascular complications in ADPKD, as in other patients with end-stage renal disease (ESRD), are the leading cause of death (Perrone et al., 2001; Schrier et al., 2002). ADPKD patients are prone to develop aortic aneurysms secondary to HTN and, possibly, ADPKD-associated connective tissue disorders, and renal size may obscure visualization of an aortic aneurysm. A clear pathogenic link between this lesion and ADPKD remains to be determined (Lacombe, 2000). The deleterious role of HTN in the ADPKD patient has been welldescribed (Geberth et al., 1995; Schrier et al., 2002). The pathogenesis of hypertension in ADPKD is complex and dependent on the interaction of hemodynamic, endocrine, and neurogenic factors. The final pathway may involve activation of the renin–angiotensin II–aldosterone axis with cyst expansion resulting in stretch and attenuation of the intrarenal vessels. In addition, the renal prognosis of ADPKD is found to be worse in individuals born to an unaVected parent with essential hypertension versus a normotensive unaVected parent (Geberth et al., 1995). Mitral valve prolapse aVects 25% of PKD1 patients while mitral regurgitation and left ventricular hypertrophy (LVH) are more likely to be a result of HTN (Lumiaho et al., 2001). Rigorous control of HTN is essential to decrease the incidence of LVH in all patients (Schrier et al., 2002). 4. Intrahepatic Biliary and Gastrointestinal System Involvement of the liver is more frequent, more severe, and has an earlier onset in females than in males (Sherstha et al., 1997). For both sexes, the number of hepatic cysts generally increases with age. Hepatic cysts occur in 75% of patients over the age of 60 years (Iglesias et al., 1983; Gabow, 1993). Nonobstructive diVuse dilatation of intrahepatic bile ducts may occur (Terada and Nakanuma, 1988). Liver cysts not communicating with the biliary tract

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lumen have also been seen (Jordon et al., 1989). A high incidence of diverticulosis and diverticulitis is seen in patients with chronic renal failure secondary to ADPKD. Colonic diverticula aVect about 80% of these patients and colonic perforation is a relatively frequent complication (ScheV et al., 1980). 5. Other Organ Systems Cysts secondary to ADPKD may also occur in the pancreas (5%), spleen (10%), and, very rarely, the lungs (Gabow, 1990). The literature is also replete with reports of many coexisting abnormalities, such as ADPKD and Marfan syndrome (Somlo et al., 1993; Kaplan et al., 1997; Hateboer et al., 2000). ADPKD has also been reported to have a higher incidence of seminal vesicle cyst-induced infertility (Belet et al., 2002; Vecchi et al., 2003). Given that ADPKD is a common disease, some of these reports likely represent chance associations. Despite the similarity in nomenclature, there is no association between ADPKD and polycystic ovaries (Heinonen et al., 2002).

B. Demographics and Factors Affecting Progression ADPKD is a progressive, multisystemic disorder of unregulated cyst formation, aVecting 1 in 500 to 1 in 1000 people and is the fourth leading cause of renal failure worldwide (Iglesias et al., 1983; Igarashi and Somlo, 2002). It is the most frequent genetic cause of renal failure in adults, aVecting 500,000 people in the United States alone and accounting for up to 5% of the total ESRD population in the United States (Gabow, 1993; USRDS, 2002). The rate of ADPKD progression varies due to multiple etiologies, which include the presence of a germline mutation in the PKD1 versus PKD2 gene, the nature of the mutation, and probably environmental factors (Peters and Breuning, 2001). The phenotype of patients with mutations in PKD2 is generally milder than PKD1, as PKD2 patients tend to live longer and reach ESRD later (Hateboer et al., 1999). The prevalence of ADPKD, over time, has remained unchanged due to the late onset of clinical manifestations. Many factors have been described to aVect the onset and rate of progression of ADPKD including gender, race, and age of diagnosis (FickBrosnahan et al., 2001; USRDS, 2002). Approximately 45% of ADPKD patients develop ESRD by the age of 60, and adult males generally reach ESRD 5–6 years earlier than females (Parfrey et al., 1990; Gabow et al., 1992; Pirson, 1996). Both human and animal studies suggest a possible role for testosterone (Cowley et al., 1997). Males represent 54% of the total ADPKD patients on hemodialysis (USRDS, 2002). On the other hand, hepatic cyst growth may be stimulated by estrogen (Sherstha et al., 1997). Comparing ethnicities, the course of ADPKD is accelerated in African-Americans, with

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the mean age of onset being 43 years versus 54 years in whites (Peters and Breuning, 2001). The etiology of these diVerences between ethnicities remains to be elucidated. ADPKD patients tend to fare better on dialysis compared to other causes of ESRD (Perrone et al., 2001), which is probably secondary to a better degree of general health, in comparison to, say diabetics, who make up
43% of the ESRD population (USRDS, 2002), and possibly to increased renal erythropoietin production prior to renal failure (Milutinovic et al., 1984; Fourtounas et al., 2002). To conclude this section, ADPKD is a common multisystem autosomal dominant genetic disorder with many challenging clinical problems. As noted, the past decade has witnessed impressive discoveries such as the cloning of the ADPKD causative genes. Evidence supporting the interaction of polycystin-1 and -2 in a common pathway is also quite compelling and is discussed in the next section.

C. A Common Pathway For in-depth reviews of the molecular genetics and other aspects of ADPKD refer to the OMIM website of the NCBI (http://www.ncbi.nlm.nih.gov/, specifically sections *601313, #173900, *173910) and Igarashi and Somolo (2002). The best evidence linking polycystin-1 and -2 to a common pathway comes from the medical literature, namely the near identity of the cystic phenotype in patients irrespective of the causative gene (Grantham, 2001). Strongly supporting the idea of a common pathway is the similarity of phenotypes caused by targeted disruptions in the homologous genes in mice. Himmelbauer and co-workers (1992) identified a conserved linkage group by mapping conserved sequences and cDNAs from the region surrounding the PKD1 gene in the mouse genome. The homologous region was located on mouse chromosome 17 (Himmelbauer et al., 1992). The mouse Pkd1 gene was located within a previously defined conserved region that included the mouse homologue of tuberous sclerosis 2 (TSC2) and, like their human counterparts, the mouse Tsc2 and Pkd1 genes were arranged in a tail-totail orientation (Olsson et al., 1996). Lu and colleagues introduced into mice, by homologous recombination, a Pkd1 truncation mutation, Pkd1, that mimicked a mutation found in ADPKD. Pkd1 heterozygotes had no discernible phenotype, whereas homozygotes died during the perinatal period with massively enlarged cystic kidneys, pancreatic ductal cysts, and pulmonary hypoplasia. Renal cyst formation began at embryonic Day 15.5 (E15.5) in proximal tubules and progressed rapidly to replace the entire renal parenchyma. The timing of cyst formation indicated that full-length polycystin is required for normal morphogenesis during elongation and maturation of

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tubular structures in the kidney and pancreas (Lu and Zhou, 1997). See Table I for a list of the various Pkd1 ‘‘knockout mice’’ that have been generated. Pritchard and colleagues (2000) generated transgenic mice that overexpressed a 108-kb human genomic fragment containing the entire ADPKD gene, PKD1, plus the tuberous sclerosis gene, TSC2. Two transgenic lines produced full-length PKD1 mRNA and polycystin-1 protein that was developmentally regulated, similar to the endogenous pattern, with expression during renal embryogenesis and neonatal life, and expression. Tuberin expression was limited to the brain. Transgenic animals from both lines (and the TPK2 founder animal) often displayed a renal cystic phenotype, typically consisting of multiple microcysts, mainly of glomerular origin. Hepatic cysts and bile duct proliferation, characteristic of ADPKD, were also seen. To test the functionality of the transgene, animals were bred with the Pkd1 (del34) knockout mouse. Both transgenic lines rescued the embryonically lethal Pkd1 (del34/del34) phenotype, demonstrating that human polycystin-1 can complement for loss of the endogenous protein (Pritchard et al., 2000). TABLE I Examples of Published Target Disruptions in Pkd1 and Pkd2a

Mouse locus

Line name

Mutation: deletion or disruption

Reference

PKD1 (Chr. 17)

Del 34

Exon 34 deletion

PKD1

Null

Exon 4 disruption

Lu et al. (2001)

PKD1

Del 17–21

Exons 17–21 deletion; IRES lacZ-neo fusion

Boulter et al. (2001)

PKD1

—

Exon 2–4 deletion with in-frame lacZ

Bhunia et al. (2002)

PKD1

—

Exon 2–6 deletion

Muto et al. (2002)

PKD1

—

Exon 1 disruption

Wu et al. (2002)

PKD1

—

Exon 43–45

Kim et al. (2000)

ENU mutagenesis

Herron et al. (2002)

PKD1 PKD2 (Chr. 5)

—

Lu and Zhou (1997)

Exon 1 disruption

Wu et al. (1998a)

PKD2

WS25

Exon 1 duplication with disruption

Wu et al. (2000)

PKD2

—lacZ

Exon 1 deletion LacZ ‘‘promotor trap’’

Pennekamp et al. (2002)

a Human ADPKD is a monogenetic disorder due to mutations in PKD1 (chromosome 16) or PKD2 (chromosome 4). The homologous mouse genes on chromosome 17 and 5 have been disrupted at various sites. ENU, ethylnitrosourea; IRES, internal ribosome entry site. Adapted from a presentation by Somlo (2002).

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Wu and colleagues (1997) cloned the murine homologue of PKD2. The protein product of Pkd2 was highly conserved with 91% identity and 98% similarity to human polycystin-2 at the amino acid level. Pkd2 mRNA was widely expressed in mouse tissues and mapped to mouse chromosome 5 (Wu et al., 1997). Two mutations were then induced in the mouse homologue Pkd2: an unstable allele (Pkd2WS25) that can undergo homologous-recombination-based somatic rearrangement to form a null allele and a true null mutation (Pkd2). Pkd2/ mice died in utero between embryonic Day (E) 13.5 and parturition. They had structural defects in cardiac septation and cyst formation in maturing nephrons and pancreatic ducts. As in human ADPKD, formation of kidney cysts in adult Pkd2WS25/ mice was associated with renal failure and early death (median survival, 65 weeks versus 94 weeks for controls). Adult Pkd2þ/ mice had intermediate survival in the absence of cystic disease or renal failure, providing the first indication of a deleterious eVect of haploinsuYciency at Pkd2 on long-term survival (Wu et al., 2000). In another study, six of the Pkd2WS25/ mice were sacrificed at 11 weeks and all had bilateral renal cysts involving 20–75% (mean, 45%) of the cut surface of the kidney (Wu et al., 1998a). Arguably, the Pkd2WS25/ mouse is the best animal model of human ADPKD currently available. See Table I for a list of Pkd2 ‘‘knockout mice’’ that have been generated. Given that the phenotype in patients with mutations in polycystin-1 and -2, as well as in mice with targeted disruptions in the homologous mouse genes, were quite similar (see above), it was suggested that the polycystins interact in a common pathway. Qian and colleagues (1997) described a coiled-coil domain within the C-terminus of polycystin-1 and showed that it bound specifically to the C-terminus of PKD2. Naturally occurring mutations of PKD1 and PKD2 disrupted this association (Qian et al., 1997). It was then shown that the interaction through the C-terminal cytoplasmic tails resulted in an up-regulation of polycystin-1 but not polycystin-2. Furthermore, the cytoplasmic tail of polycystin-2 but not polycystin-1 formed homodimers through a coiled-coil domain distinct from the region required for interaction with polycystin-1, suggesting that polycystin-1 may require the presence of polycystin-2 for stable expression (Tsiokas et al., 1997). Hanaoka and co-workers (2000) showed that polycystin-1 and -2 interacted to produce new calcium-permeable nonselective cation currents. Neither polycystin alone was capable of producing currents. Moreover, diseaseassociated mutant forms of either polycystin that were incapable of heterodimerization did not result in new channel activity. They also showed that polycystin-2 is localized inside the cell in the absence of polycystin-1, but is translocated to the plasma membrane in its presence (Hanaoka et al., 2000). A final bit of evidence for a common pathway comes from the worm Caenorhabditis elegans in which the PKD1 and PKD2 homologs, lov-1 and pkd-2, act in the same pathway in vivo. Mutations in either lov-1 or pkd-2

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resulted in identical male sensory behavioral defects. Also, pkd-2;lov-1 double mutants were no more severe than either of the single mutants, indicating that lov-1 and pkd-2 act together. LOV-1::GFP and PKD2::GFP were expressed in the same male-specific sensory neurons and were concentrated in cilia and cell bodies (Barr et al., 2001). This will be discussed later in detail (see Section IV.B).

D. Inheritance Pattern, the ‘‘Two-Hit Hypothesis’’ Evidence supporting the ‘‘two-hit hypothesis’’ is quite compelling though not yet definitive. The concept is discussed in this section. Classically three mechanisms account for disease with a dominant pattern of inheritance: haploinsuYciency (a situation in which the protein produced by a single copy of an otherwise normal gene is not suYcient to ensure normal function), gain-of-function mutations (including dominant-negative eVects), and a ‘‘second-hit’’ in the normal allele leading to a complete loss of function (Wunderle et al., 1994). HaploinsuYciency is a relatively rare cause of autosomal disease, though there are examples in the renal literature. For instance, the Pax family of paired box transcription factors has been demonstrated in both mice and humans to have crucial functions during organ development. The remarkable feature of some members of the Pax family is this semidominant character of loss of function mutations, haploinsuYciency, which in practical terms means that heterozygotes display abnormal phenotypes (Strachen and Read, 1994; Lipschutz, 1998). Kidney hypoplasia in humans has been reported for a heterozygous point mutation in the Pax-2 gene (Sanyanusin et al., 1995). In mice, targeted disruption of the Pax-2 gene led to renal hypoplasia in heterozygous knockout mice (Torres et al., 1995). Gain-offunction mutations and second-hit mutations are generally more common mechanisms of autosomal dominant disease. The pathogenesis of an unrelated disease, retinoblastoma (RB), provided a clue with respect to the genetic mechanism of ADPKD. ADPKD and RB are both autosomal dominant in inheritance (Reeders, 1992). Retinoblastoma is the most common intraocular tumor in children. Based on the epidemiology of retinoblastoma, Alfred Knudson (1971) developed the ‘‘two-hit hypothesis,’’ which is now a fundamental concept in many inherited diseases including HD, retinitis pigmentosa, achondroplasia, breast/ovarian cancer due to BRCA1 and BRCA2 mutations, myotonic dystrophy, neurofibromatosis, Marfan’s syndrome, and familial hypercholesterolemia. Knudson hypothesized that the gene responsible for RB was a tumor suppressor gene that, while autosomal dominant in character, was recessive at the cellular level (i.e., inactivation or loss of both alleles of the gene appears to be necessary for the

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development of the disease). The first ‘‘hit’’ occurs in the germline of the cancer susceptibility gene resulting in the constitutional mutation. The second ‘‘hit’’ occurs somatically in the other allele of the same gene, resulting in loss of heterozygosity (LOH). The population of somatic cells carrying the constitutional mutation is so large that there is a high probability of at least one cell undergoing LOH, i.e., acquiring a second hit (Knudson, 1971). Phenotypic variability across family members may be at least partially explained by the unpredictability of the second hit. Reeders (1992) later expanded Knudson’s hypothesis and devised the twohit mutational hypothesis for ADPKD. His two-hit hypothesis explained several features of ADPKD including the absence of detectable abnormalities in most nephrons (even in the end-stage disease, less than 10% of the nephrons in each kidney contain cysts), and the fact that any segment of the nephron, from the glomerulus to the collecting duct, can harbor a cyst. The hypothesis suggests that at the sites of cyst formation, a somatic mutation occurs in the normal copy of the PKD1 or PKD2 genes. The idea of a second hit is strongly supported by data showing that cysts are monoclonal in origin (Qian et al., 1996). Factors that would influence the rate of somatic mutation and, therefore, the variability in phenotypic presentation include genetic factors, toxic exposures, and the specific haplotype. Though the twohit hypothesis for ADPKD is widely accepted, the fact that several laboratories have shown that cystic epithelial cells express polycystin-1 and -2 and, in some of them, polycystin-1 is even overexpressed, prevents us from ruling out the possibility of haploinsuYciency or a dominant negative eVect (Ward et al., 1996; Ibraghimov-Beskrovnaya et al., 1997; Ong et al., 1999a,b). It should be noted, however, that these results can be reconciled with the ‘‘twohit hypothesis’’ by the probable frequent occurrence of somatic missense mutations not aVecting the epitope of the antibodies used, but nevertheless resulting in a nonfunctional ‘‘mutant protein’’ (Ong et al., 1999a; Koptides and Deltas, 2000).

III. Molecular Biology of Autosomal Dominant Polycystic Kidney Disease A. Structure of Polycystin-1 and -2 The genes containing mutations responsible for ADPKD are PKD1 and PKD2, which encode for polycystin-1 and polycystin-2 (Consortium, 1994; Mochizuki et al., 1996). The polycystins are a unique group of transmembrane glycoproteins now known to consist of at least six homologues in various species ranging from sea urchins, to C. elegans, to mammals. These

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homologues include polycystin-1, -2, -L, -1L1, -2L2, and REJ (receptor for egg jelly-like protein) (Nomura et al., 1998; Hughes et al., 1999; Veldhuisen et al., 1999; Guo et al., 2000; Yuasa et al., 2002). ADPKD is caused by mutations in PKD1 in 85% of cases (Parfrey et al., 1990) and PKD2 in 15% (Peters and Sandkuijl, 1992). Rare cases of ADPKD not linked to either PKD1 or PKD2 (Daoust et al., 1995) have been reported. However, recent confirmation of these finding is lacking and one such family actually had bilineal inheritance of both PKD1 and PKD2 (Pei, 2001; Igarashi and Somlo, 2002). Polycystins PKDL, PLD1L1, and PKD2L are unlikely to be involved in ADPKD; their involvement in other disease states is currently unknown (Nomura et al., 1998; Wu et al., 1998b; Yuasa et al., 2002). Nevertheless, these proteins may shed light on the function of polycystin-1 and -2. For instance, polycystin-L was the third polycystin to be isolated, but the first whose function was demonstrated as a Ca2þ-regulated, Ca2þ-permeable cation channel (Calvet and Grantham, 2001; Yuasa et al., 2002). Polycystin-2 has also been shown to be a Ca2þ-regulated, Ca2þ-permeable cation channel (Koulen et al., 2002). 1. PKD1 Gene and Protein It was previously mentioned that the PKD1 locus was discovered through the genetic analysis of a Portuguese child with PKDTS, a severe infantile polycystic kidney disease in which the kidneys contain a multitude of variably sized cysts closely resembling those more commonly seen in later life in the advanced stages of ADPKD (Longa et al., 1997). PKD1 and TSC2 were determined to be contiguous genes but located on opposite strands, abutted in a ‘‘tail-to-tail’’ (30 to 30 ) divergent orientation. This bidirectional gene organization could suggest that their expression is coordinately regulated (Calvet, 1998; Harris, 1999). Well-recognized in prokaryotes, bidirectional gene organization may be a more frequent occurrence in the human genome than previously thought (Adachi and Lieber, 2002). Supportive evidence of the existence of a coregulatory mechanism includes the role of the TSC2 gene product (tuberin) in intracellular polycystin 1 protein traYcking (Kleymenova et al., 2001; Kugoh et al., 2002). The exact coregulatory interaction, however, remains to be elucidated. Determining the precise structural organization of the PKD1 gene should be helpful in deciphering these mechanisms and ultimately understanding the eVects of the more than 60 mutations already identified for the PKD1 gene (Perrichot et al., 2000). The PKD1 gene maps to chromosome 16p13.3–p13.2 and spans 52 kb of genomic DNA, only 12.9 kb of which are coding sequence (Consortium, 1995). The PKD1 gene is comprised of 46 exons that encode an mRNA transcript of 14,148 bp, which, in turn, translates into a very large (>4000

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amino acid) protein. The PKD1 50 region encompasses about 60% of this large gene. The 50 sequence from exon 1 to intron 33 is repeated at six other sites on the same 16p chromosome, referred to as 50 repeats or homologous genes. This implies that only 3.9 kb of PKD1 mRNA is unique, which can be a major hindrance to cloning and mutation analysis as it can be diYcult to distinguish mutations in PKD1 from mutations in the homologous genes. The 50 PKD1 repeats are possible mutagenic pseudogenes that are transcribed as nonfunctional poly(A)-containing RNAs. Our understanding of the role of homologous genes as reservoirs of mutations will have to await the accumulation of more single nucleotide polymorphism (SNP) frequency data (McCluskey et al., 2002). ADPKD mutations include nonsense mutations, insertions or deletions, or splicing changes. Mutations occur throughout the gene and no ‘‘hot-spots’’ have been identified, though mutations most frequently occur at the 30 region. No correlation has been discovered between specific PKD1 mutations and clinical manifestations (Rossetti et al., 2001). Polycystin-1 is a 4302 amino acid integral membrane glycoprotein of molecular weight 462 kDa and is composed of an extracellular NH2 domain that contains a unique array of distinct protein motifs, 11 transmembrane domains, and an
200 amino acid intracellular signal transducing coiled-coil COOH terminus (Hughes et al., 1995) (see Fig. 2). It is widely expressed in many mammalian tissues, including the kidney, brain, heart, and muscle (Geng et al., 1997). 2. PKD2 Gene and Protein By the early 1990s, another form of ADPKD was recognized that was phenotypically similar to PKD1, yet was not linked to the PKD1 chromosome 16 locus (Kimberling et al., 1993; Peters et al., 1993). The identification of PKD2 in 1993 as the second gene mutation responsible for ADPKD was a major breakthrough in elucidating the pathogenesis of this common disorder. The PKD2 gene was later positionally cloned in 1996 (Kimberling et al., 1993; Peters et al., 1993; Mochizuki et al., 1996; Schneider et al., 1996). It was located on the long arm of chromosome 4 at 4q21–q23, in a region that contains a 2904-bp open reading frame and a 2086-bp untranslated region. But unlike PKD1, there are no DNA segments that repeat (Mochizuki et al., 1996). The PKD2 gene spans 68 kb of genomic DNA and consists of 15 exons (Hayashi et al., 1997). The PKD2 mutations reported are mainly nucleotide substitutions (missense/nonsense), small deletions, small insertions, and splicing changes (Mochizuki et al., 1996). Mutations occur over the entire sequence of the PKD2, without significant clustering (Deltas, 2001). Polycystin-2 is a highly conserved 968 amino acid protein with a molecular weight of 110 kDa, composed of six transmembranous domains

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and intracellular C- and N-termini (Fig. 2) (Mochizuki et al., 1996). It is strongly expressed in fetal and adult kidney, ovary, testis, and small intestine (Markowitz et al., 1999; Obermuller et al., 1999). Although their membrane-spanning segments share about 50% homology, polycystin-1 and -2 are very diVerent in terms of size and probable function. Polycystin-2 is considerably smaller than polycystin-1, and it lacks a large extracellular domain. Polycystin-2 shares significant structural similarity to the transient receptor potential (TRP) channels and voltage-activated calcium and sodium channels (Tsiokas et al., 1999; Igarashi and Somlo, 2002).

B. Cellular Localization and Interacting Proteins To begin to understand the function of polycystin-1 and -2 it is important to localize these proteins within the cell and identify interacting and downstream proteins. Unfortunately, the literature is somewhat confusing with the polycystins being localized to multiple sites within the cell, along with multiple interacting proteins being described. This could reflect the fact that the polycystins indeed have multiple sites of action and there is precedent for

FIG. 2

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FIG. 2

(continued)

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FIG. 2 Structure diagram of polycystin-1 and -2. Polycystin-1 and -2 are the protein products of PKD1 and PKD2, respectively. The predicted structures (not drawn to scale), along with the known domains, are shown. Three-dimensional representations of many of the domains are available online from SWISS-3DIMAGE (http://us.expasy.org/sw3d). Adapted from Igarashi and Somlo (2002).

this in the literature. For example, b-catenin is a protein with which we are familiar (Balkovetz et al., 1997; Lipschutz and Kissil 1998) and that has been associated with ADPKD (Huan and van Adelsberg, 1999; Rodova et al., 2002), which is present in cell–cell junctions and binds to the cytoplasmic domain of a family of Ca2þ-dependent cell adhesion molecules, the cadherins (Knudsen and Wheelock, 1992). b-Catenin also acts in the nucleus by binding to transcription factors of the T cell factor-lymphoid enhancer factor (Tcf-Lef) family (PfeiVer et al., 1985; Korinek et al., 1997; Morin et al., 1997; Rubinfeld et al., 1997). However, an alternate explanation for the multiple sites of localization and multiple binding partners is that the variable, and sometimes even contradictory, results are due to the many diVerent antibodies that have been used for localization (Harris, 1999). As noted by Ibraghimov-Beskrovnaya et al. (2000) ‘‘some of the reported antibodies were raised against rather short synthetic peptides derived from human sequences, and it is possible that they might not crossreact with diVerent cell types and/or between species.’’ Until the recent localization of the polycystins to the cilia, there has also been a lack of a global theory to explain how mutations in polycystin-1 and -2 lead to the cystic phenotype seen in ADPKD. Below is a summary of some of the localizations and interactions that have been described.

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To study the subcellular localization of polycystin-1, ScheVers et al. (2000) raised antibodies against various domains of polycystin-1. Using confocal laser scanning and immunoelectron microscopy, in Madin–Darby canine kidney (MDCK) cells, polycystin-1 was detected in the cytoplasm as well as colocalizing with desmosomes, but not with tight or adherens junctions. In another study, to identify proteins that interacted with the polycystin-1 Cterminal tail, this segment was used as bait in a yeast two-hybrid screening of a kidney epithelial cell library. The intermediate filament protein vimentin was identified as a strong polycystin-1-interacting partner. Cytokeratins K8 and K18 and desmin were also found to interact with polycystin-1. These interactions were mediated by coiled-coil motifs in polycystin-1 and intermediate filament proteins. In vivo a cell membrane-anchored form of recombinant polycystin-1 decorated the intermediate filament network and endogenous polycystin-1 distributed with intermediate filaments at desmosomal junctions (Xu et al., 2001). Another recent report utilized a three-dimensional MDCK in vitro model of tubulogenesis and cystogenesis to demonstrate that polycystin-1 is a component of desmosomal junctions in epithelial cells. A striking down-regulation of polycystin-1 mRNA was detected in cysts as compared to tubules, leading to altered protein expression and localization. Although polycystin-1 was localized to basolateral membranes of MDCK tubules, it was detected only in cytoplasmic pools in cystic cells. Furthermore, the expression of polycystin-1 was modulated during distinct stages of HGF-induced tubulogenesis from MDCK cysts (O Bukanov et al., 2002). On the other hand, Huan showed that polycystin-1 colocalized with the adherins junction molecules E-cadherin and a-, b-, and g-catenin. Polycystin-1 coprecipitated with those proteins and comigrated with them on sucrose density gradients, but did not colocalize, coprecipitate, or comigrate with focal adhesion kinase, a component of the focal adhesion complex (Huan and van Adelsberg, 1999). Supporting these results was the finding that a portion of the human PKD1 gene contained consensus sequences for numerous transactivating factors, including four T cell factor (TCF)binding elements (TBEs) that responded to b-catenin (Rodova et al., 2002). ScheVers et al. (2002) also showed, using two polyclonal antisera raised against polycystin-2, that there was distinct expression of the endogenous protein in the Golgi apparatus and the plasma membrane of MDCK cells. Most of the heterologously expressed polycystin-2 (PC2-EGFP) remained in the endoplasmic reticulum (ER). In contrast, glycosylation analysis of native and recombinant polycystin-2 has shown that it is completely endoglycosidase, (Endo) H sensitive, suggesting that most, if not all, polycystin-2 protein is normally located in the ER and/or cis-Golgi (Cai et al., 1999). In several studies, polycystin-2 was indeed localized to the ER in cells and tissue by immunohistochemistry, though some signal was detected at the basolateral

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cell membrane (Cai et al., 1999; Markowitz et al., 1999; Obermuller et al., 1999), which led to the suggestion that polycystin-1 and -2 could be members of the same signaling pathway but not directly interact with one another. Newby and colleagues (2002) recently demonstrated the existence of an intracellular native polycystin complex. Polycystin-1 is heavily N-glycosylated, and several glycosylated forms of polycystin-1 diVering in their sensitivity to Endo H were found. Using antibodies to both proteins, they showed that polycystin-2 associated selectively with two species of full-length polycystin-1, one Endo H sensitive and the other Endo H resistant; importantly, the latter could be further enriched in plasma membrane fractions and coimmunoprecipitated with polycystin-2. Finally, a subpopulation of this complex colocalized to the lateral cell borders of PKD1 transgenic kidney cells (Newby et al., 2002). Polycystin-1 has also been localized to epithelial cell–cell contacts in culture. It was proposed that polycystin-1 was involved in cell–cell adhesion via a cluster of Ig-like repeats and antibodies raised against Ig-like domains of polycystin-1 disrupted cell–cell interactions in MDCK cell monolayers (Ibraghimov-Beskrovnaya et al., 2000). Very recently, polycystin-1 and -2, have been localized to the cilia (Yoder et al., 2002a) and a plausible model for the pathogenesis of ADPKD has been generated. The background and rationale for this model will be extensively discussed in Section IV.B.

C. Molecular Pathways Given the lack of definitive cellular localization and identification of polycystin-binding partners, it has been diYcult to hypothesize and test molecular pathways. Nevertheless, several putative pathways have been identified. Boletta and co-workers (2000) described a cell culture system in which they overexpressed PKD1. They showed that expression of human PKD1 in MDCK cells slowed their growth and protected them from programmed cell death. MDCK cells expressing PKD1 also spontaneously formed branching tubules while control cells formed simple cysts. Increased cell proliferation and apoptosis have been implicated in the pathogenesis of cystic diseases and this study suggested that PKD1 may function to regulate both cellular proliferation and apoptosis, allowing cells to enter a diVerentiation pathway that results in tubule formation (Boletta et al., 2000). Another study that focused on pathway activation was by Bhunia and colleagues (2002) who recently showed that expression of polycystin-1 activated the JAK-STAT pathway, thereby up-regulating p21 (waf1) and inducing cell cycle arrest in G0/G1. This process required polycystin-2 as

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an essential cofactor. Mutations that disrupted polycystin-1/2 binding prevented activation of the pathway. Mouse embryos lacking Pkd1 had defective STAT1 phosphorylation and p21 (waf1) induction, suggesting that one function of the polycystin-1/2 complex was to regulate the JAK/ STAT pathway, which could explain why mutations in either PKD1 or PKD2 result in dysregulated growth (Bhunia et al., 2002).

IV. Disease Models A. ADPKD as a Disease of Stages Given the problems identifying binding partners and cellular sites of action of polycystin-1 and -2, it has also been diYcult to generate a mechanistic model. What has been done in lieu of a global model is to view ADPKD as a disease of stages and describe phenomenologically what happens during each stage. The three stages can be divided into (1) cyst initiation, (2) cyst enlargement, and (3) progression (Germino, 2002). Much of what is important for cyst initiation has been discussed earlier. For example, in cyst formation the timing and position of the second hit are crucial. There are also factors that influence the rate of somatic mutation such as genetics, toxic exposures, and the specific haplotype. During the stages of cyst enlargement and progression, abnormalities involving polarized proteins have been described. One of these proteins is the epidermal growth factor receptor (EGFR), which is the receptor for epidermal growth factor (EGF) and transforming growth factor-a (TGF-a) (Sweeney and Avner, 1998). EGFR is normally found on the basolateral membrane on the epithelia of the collecting tubule; however, in both human and murine models of ADPKD and ARPKD, EGFR was overexpressed and mislocalized to the apical membrane of the cystic tubular epithelium (Sweeney and Avner, 1998). In the aVected kidneys, there was also augmented protein synthesis and amplified EGFR mRNA, as well as an increase in the activity of tyrosine kinase (Richards et al., 1998; Sweeney and Avner, 1998). A physiologically active, autocrine/paracrine EGF–TGF-a–EGFR pathway has been postulated in cystic tubular epithelia to be responsible for cellular proliferation (Orellana and Avner, 1995). EGF and TGF-a experimentally are found to promote cyst formation, and cyst fluid from both human and murine models have extremely high levels of EGF and TGFa (Avner and Sweeney, 1990; Sweeney and Avner, 1998). This seems to be a local phenomenon, as Horikoshi and co-workers demonstrated a greatly increased concentration of EGF in the cystic fluid from aVected animals compared to urinary levels (Horikoshi et al., 1991).

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Using a murine model for ARPKD with a point mutation ameliorating tyrosine kinase activity, Richards et al., (1998) demonstrated that lessening tyrosine kinase activity decreased cystic formation. Furthermore, they demonstrated that using tyrosine kinase or EGFR inhibitors blunted any eVect when given stimulatory TGF-a. This points to a potentially regulatory role of tyrosine kinase in cyst formation. The mislocalization of the EGFR to the apical surface of collecting tubule epithelium appears to be a relatively selective defect of cell polarity, as most other receptor subtypes seem to maintain their positions (Sweeney and Avner, 1998). This abnormal localization to the apical surface has also been described for the Naþ/Kþ-ATPase pump. Wilson and co-workers (1991), in demonstrating altered polarity of the sodium/potassium pump, were unable to detect any change in the polarity of several other membrane proteins. Increased protein expression on the apical membrane may be related to increased secretion from the apical surface into the cyst lumen leading to cyst enlargement. In addition to EGFR, cyclic AMP (cAMP) has been postulated to have a role in cellular proliferation in collecting tubule epithelia. It has been documented in vitro that activation of cAMP regulates cyst production (Hanaoka and Guggino, 2000). One potential mechanism of cyst enlargement is increased epithelial secretion of chloride through a cAMP stimulatory mechanism (Wallace et al., 1996). Davidow and colleagues (1996) demonstrated that this secretion is mediated through the cystic fibrosis transmembrane regulator (CFTR). There is some question, however, as to whether this mechanism is similar for both ADPKD and ARPKD (Nakanishi et al., 2001). There may also be a role for cAMP in the stimulation of protein kinases. Marfella-Scivittaro et al. (2002), in evaluating cAMP-stimulated protein kinase A, found that the altered localization of PKA subtype 1 and 2 may, in turn, alter EGF activity. The exocyst is a conserved eight-protein complex involved in the biogenesis of polarity in species as diverse as yeast and mammals, and has been postulated to be the docking and targeting complex for vesicular traYc to the plasma membrane (GrindstaV et al., 1998; Guo et al., 1999). We have shown, using an in vitro model system in which MDCK cells, of tubular origin (Simons and Fuller, 1985), are grown in a collagen matrix, that the exocyst complex is centrally involved in cystogenesis and tubulogenesis (Lipschutz et al., 2000). The exocyst complex in polarized kidney epithelial cells is normally localized mainly to the tight junction; however, in primary cultures of cells from the kidneys of patients with ADPKD, the exocyst complex was mislocalized to the cytoplasm (Charron et al., 2000). In addition to its involvement in polarized membrane traYcking and cystogenesis/tubulogenesis, we have recently shown that the exocyst complex is also involved in protein synthesis, by interacting with the translational machinery of the ER (Lipschutz et al., 2003). Protein

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synthesis and epithelial cell proliferation have long been associated with ADPKD (Evan et al., 1979), with some investigators terming ADPKD a ‘‘neoplasia in disguise’’ (Qian et al., 2001). During the progression stage, there have been increases in chemokine expression (MCP-1, osteopontin) (Cowley et al., 2001), metalloproteinases (Obermuller et al., 2001; Norman et al., 1995), and matrix deposition/ composition (Germino, 2002, Wilson et al., 1996). B. Role of the Cilium in ADPKD: An Emerging Model 1. Relationship between Primary Renal Cilia and Polycystic Kidney Disease Cilia are thin rod-like organelles found on the surface of many eukaryotic cell types in nature. Cilia extend outward from the basal body, a cellular organelle related to the centriole. Cilia are classified as primary (nonmotile) and motile. In epithelium containing numerous cilia, the cilia have been observed to have a propulsive function (Fawcett and Porter, 1954). Structurally, cilia are covered by a membrane that is continuous with the plasma membrane and contain a central axoneme composed of microtubules. It has been known for over a hundred years that epithelial cells along the length of the nephron possess primary cilium (Zimmerman, 1898). In the mammalian kidney, cilia have been observed on cells in the parietal layer of Bowman’s capsule, the proximal tubule, the distal tubule, and in the principal, but not intercalated cells of the collecting duct (Webber and Lee, 1975). In contrast to epithelia with numerous motile cilia, the cells of these nephron segments exhibit a single cilium, or, less commonly, two cilia. In the kidney the primary cilium cells projects from the centriole of the cell, is nonmotile, and exhibits an axoneme microtubular pattern of 9 þ 0. This is in contrast to motile cilia that exhibit a typical 9 þ 2 axoneme microtubular pattern of organization. Despite the established anatomical presence of cilia in the kidney, little has been ascribed regarding specific function. It seems unlikely that they play a major propulsive role and some have considered the renal cilium as a vestigial organelle (Webber and Lee, 1975). Recently, the primary cilium of the kidney has been receiving increasing consideration from investigators in the field of polycystic kidney disease. Notably, several gene products, which when mutated result in the development of polycystic kidney disease, have been localized to and/or are important for the function of the primary cilium of the kidney epithelial cells. 2. orpk Mouse and Polaris The relationship between primary renal cilium and the pathogenesis of polycystic kidney disease was suggested by the orpk mouse, which is a model of autosomal recessive polycystic kidney disease due to a hypomorphic

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insertional mutation of a gene referred to as Tg737 (Moyer et al., 1994). In these animals, homozygotic mutation of the Tg737 gene product results in a complex phenotype including polycystic kidneys, portal fibrosis, pancreatic acinar cells atrophy, extra molar, midline cleft palate, and preaxial polydactyly. Curiously, Tg737 knockout mice are embryonic lethal, and are noted to have both situs inversis and neural tube defects at embryonic Day 9.5–10.5 (Murcia et al., 2000). In mice homozygous for the Tg737 gene knockout, the ventral node cells lack cilia on their apical surface. It is believed that the loss of cilia on these cells leads to defects in right–left patterning caused by an abnormal nodal flow of morphogens (nodal flow hypothesis). These investigators named the product of the Tg737 gene polaris, which is based on the various polarity related defects associated with the diVerent alleles of the Tg737 gene (Murcia et al., 2000). A similar phenotype of situs inversus has been observed in the Kif3B knockout animals, which also have structural defects in the cilium on the ventral node cells of the early embryo (Nonaka et al., 1998). Kif3B is a microtubuledependent motor that localizes to wild-type nodal cilia of early mouse embryos (Nonaka et al., 1998). Pazour and colleagues (2000) also demonstrated that Tg737 mutant mice have smaller cilia in renal tubular epithelia, suggesting a role for the Tg737 gene product in cilia formation. Localization of polaris to the cilia structure of renal epithelial cells was first demonstrated using the MDCK epithelial cell line (Balkovetz et al., 2000). The presence of polaris in renal epithelium was confirmed and localization of polaris was demonstrated in both motile and immotile cilium in the epithelial cells of the lung, testes, and brain as well as the flagella of sperm (Taulman et al., 2001). Western blot analysis showed that polaris exists as 95- and 75-kDa isoforms. Northern blot analysis demonstrates multiple Tg737 transcripts (Moyer et al., 1994). The smaller 75-kDa isoform is predicted to be the result of alternative splicing of the mRNA (Taulman et al., 2001). Using a cell biological approach, it was shown that polaris is required for the assembly of renal cilia. In MDCK cells, polaris localization and nonionic detergent solubility changed dramatically during cilia formation. These changes correlated with the formation of basal bodies. Moreover, a cortical collecting duct cell line derived from mice with a mutation in the Tg737 gene did not develop normal cilia and this defect was corrected by reexpression of the wild-type Tg737 gene (Yoder et al., 2002b). 3. cpk Mouse and Cystin The congenital polycystic kidney (cpk) mouse is another mouse model of polycystic kidney disease that mimics human disease and may be related to renal cilia function. The cpk mouse is a congenic stain that exhibits disease

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processes that closely resemble autosomal recessive human infantile polycystic kidney disease with cysts forming in the proximal tubules, reductions of both exocrine and endocrine pancreatic function, and minimal cyst formation in the liver (Fry et al., 1985). Homozygous cpk mutant mice die from renal failure within 4–5 weeks after birth. Recently, the gene disrupted in cpk mice has been identified by positional cloning (Hou et al., 2002). This cpk gene encodes a hydrophilic, 145-amino acid protein called cystin. Northern blot analysis in adult mouse tissues demonstrates cystin expression primarily in the kidney and liver, and, to a lesser extent, in the lung, brain, and heart. In fetal tissues, the cystin transcript is seen in kidney, but not brain, lung, or liver. Immunofluorescent analysis of mouse cortical collecting duct (mCCD) cells transfected with epitope-tagged cystin cDNA demonstrates cilia localization. In contrast to polaris, which is expressed both in the ciliary basal bodies and axonemes, epitope-tagged cystin expression was predominantly in the axonemes (Hou et al., 2002). This diVerence in distribution of cystin, as compared to polaris, suggested that cystin is not involved in a cilia formation pathway, like polaris, but rather plays a role in cilia function. Left–right patterning defects have not been reported, and no defects in renal cilia structure of cpk mutant mice have been demonstrated by electron microscopy (Ricker et al., 2000). Localization of endogenous cystin is in progress. 4. inv Mouse and Inversin The relationship between renal primary cilia and polycystic kidney disease is further supported by the inv mouse. The inv (inversion of embryonic turning) mutation in mice was created by random insertional mutagenesis, resulting in both a constant reversal of left–right axis polarity (situs inversus) and cyst formation in the kidneys and pancreas (Yokoyama et al., 1993; Mochizuki et al., 1998). Expression of inv mRNA is highest in adult liver and kidney, and is seen in early stage embryos. The cDNA of the inv gene encodes a 1062amino acid protein termed inversin. Inversin is without obvious signal peptide or transmembrane domains, indicating that the protein is intracellular (Mochizuki et al., 1998). The development of situs inversus in inv mice is preceded by abnormal nodal flow in the early embryo. In inv mice, the nodal cilia were motile but could produce only very weak leftward nodal flow. These data are consistent with the hypothesis that nodal flow produces a gradient of morphogens and initiates left–right axis determination (Okada et al., 1999), and strongly suggest a relationship between inversin and nodal cilia function. Morgan and colleagues (2002) performed an expression analysis, which suggests a role for inversin in primary cilia and involvement in the cell cycle. In this study the investigators demonstrated that inversin binds to the Apc2

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subunit of the anaphase-promoting complex. Inversin–Apc2 interaction is dependent on a conserved destruction box (D-box) motif. This same group used a rabbit polyclonal antiinversin antibody to show that inversin displays a dynamic expression throughtout the cell cycle and is strongly expressed in primary cilia, but is not essential for ciliogenesis (Morgan et al., 2002). In MDCK cells, immunofluorescence analysis revealed nuclear staining prior to nuclear envelope breakdown. Cells in early prophase showed strong staining in the centrosomes. In metaphase and anaphase inversin localized to the spindle poles (Schwartz et al., 1997). Inversin staining in MDCK cell cilia was not reported. The investigators did demonstrate inversin staining in the primary cilia of the mouse inner medullary collecting duct cell line (mIMCD3). Because renal cilia from homozygous inv mice appear normal, it seems that inversin is likely to be important for cilia function but not cilia formation (Morgan et al., 2002). Another group of investigators investigated the localization of inversin using an independently generated rabbit polyclonal antibody in an immortalized cell line from the early segment of the proximal tubule (S1 cells) (Nurnberger et al., 2002). Three distinct isoforms of inversin were identified using this antibody. A 125-kDa inversin protein isoform was found at cell–cell junction complexes containing N-cadherin and catenins. Two inversin isoforms, 140 and 90 kDa, were identified in the nuclear and perinuclear regions. The 90-kDa nuclear inversin complexes with b-catenin. These investigators did not observe inversin staining in cilia. These findings suggest that inversin, like b-catenin (Korinek et al., 1997; Morin et al., 1997; Rubinfeld et al., 1997), functions in both the nucleus and cell–cell adhesion sites. 5. PKD1/PKD2 and Polycystin-1/Polycystin-2 Work with C. elegans provided early clues that the gene products for PKD1 and PKD2 might also be involved with cilia structure and/or function. C. elegans is a small nematode, growing to about 1 mm in length, and lives in the soil (especially rotting vegetation) in many parts of the world, where it survives by feeding on microbes such as bacteria. During the examination of mutations that aVect the mating behavior in C. elegans, Barr and associates identified worm homologs of PKD1 as lov-1 (for location of vulva) and PKD2 as pkd2. The protein products for lov-1 and pkd2 were found to localize to the cilia of sensory neurons in C. elegans (Barr and Sternberg, 1999; Barr et al., 2001). Both lov-1 and pkd2 protein products colocalize with the C. elegans homolog of polaris (OSM-5) (Haycraft et al., 2001; Qin et al., 2001). The lov-1 and pkd2 mutations altered cilia function (Barr et al., 2001), while the OSM-5 mutations altered cilia structure in C. elegans (Haycraft et al., 2001). Another clue suggesting cilia localization of polycystin-2 was provided by Peenekamp et al. (2002). In this study, PKD2 allele knockout in

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mice resulted in a phenotype that included situs inversus (similar to the phenotype in orpk and inv knockout mice) (Pennekamp et al., 2002). These observations led investigators to hypothesize that the protein products for PKD1 and PKD2 (polycystin-1 and polycystin-2, respectively) might also be localized to the primary cilia of renal epithelial cells. Pazour et al. (2002) used an established polycystin-2 rabbit polyclonal antibody to study polycystin-2 expression in human renal proximal tubule epithelial cells (human RPTEC). The antibody detected a protein of
110 kDa on Western blot analysis and decorated the cilia of the cells on immunofluorescent analysis. Curiously, levels of polycystin-2 were shown to be more heavily concentrated in the runted cilia of renal tubular epithelial cells isolated from orpk mutant mice (Pazour et al., 2002). Previous reports regarding the localization of polycystin-1 and -2 in renal tissue did not identify the protein in cilia structures though it was found, as noted, in many other locations. It is possible, in earlier studies, using cultured cells, that the cells had not formed ciliary structures. Yoder and colleagues (2002a) recently used an immunofluorescence-based approach to demonstrate that polycystin-1, polycystin-2, polaris, and cystin are all found in the cilia of mCCD cells in culture. In this study the investigator used previously characterized antibodies to localize these proteins. To optimize cell polarity and cilia formation, the cells were grown on permeable supports. In this study, the investigators were able to localize endogenous polycystin-1, polycystin-2, and polaris. However, the analysis of cystin localization was done using epithelial cells overexpressing an epitope-tagged isoform of cystin (Yoder et al., 2002a). 6. Functions of Cilia Despite the emerging evidence of the relationship between primary cilia and polycystic kidney disease, little is known regarding the physiological function of renal cilia. Schwartz and colleagues (1997) proposed that renal primary cilia function as flow sensors. This hypothesis was based on the observation that flow rates that occur in renal tubules result in a deflection in the cilia shaft (Schwartz et al., 1997). Teleologically, this model is analogous to the bending of a flag stick on a golf green during a windy day—whereby the velocity of the wind could be approximated by the degree of flag stick bending. It now appears that the bending of the cilia is linked to the regulation of intracellular calcium concentrations. Artificial bending of the cilium of MDCK cells grown on coverslips causes intracellular calcium to substantially increase (Praetorius and Spring, 2001). Initially extracellular calcium enters through mechanically sensitive channels, followed by calcium-induced calcium release from intracellular stores. The increase of intracellular calcium then spreads to adjacent cells, probably by a process that is dependent on gap junctions.

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It now appears that polycystins-1 and -2 are important functional elements for mechanosensation by the primary cilium of kidney epithelial cells (Nauli et al., 2003). In this study Nauli et al. confirm localization of polycystin-1 and polycystin-2 to the primary cilia of kidney epithelium. They go on to demonstrate that renal epithelial cells from transgenic mice that lack functional polycystin-1 form cilia but do not demonstrate increases of intracellular calcium levels with mechanical stimulation of the cilia. Moreover, functionperturbing antibodies directed against polycystin-2 also abrogate the increases in intracellular calcium following mechanical stimulation of the cilia. Collectively, this work demonstrates the importance of cilia function in the regulation of renal tubule maintenance and development.

V. Conclusions The pace of discovery in ADPKD research has been truly astonishing. Building on a century of investigation, the last decade has witnessed the discovery of the causative genes, an elucidation of the ‘‘two-hit hypothesis,’’ and, with the recent cilia studies, a framework for establishing a model whereby mutations in PKD1 and PKD2 lead to the cystic phenotype seen in ADPKD. With continuing research, what was once an incurable and devastating disease may be rendered more manageable and, even, curable.

Acknowledgments We wish to thank Katherine Rogers for assistance with preparation of the manuscript. This work was supported in part by grants from the Polycystic Kidney Disease Foundation (J.H.L.), the N.I.H. (J.H.L., DK58090, DK02509), and the V.A. (D.F.B, V.A. Merit Award).

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