Review

Polycystin-1: a master regulator of intersecting cystic pathways

1 1 1,2

Sorin V. Fedeles , Anna-Rachel Gallagher , and Stefan Somlo

1

Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA

2

Department of Genetics, Yale University School of Medicine, New Haven, CT, USA

Autosomal dominant polycystic disease (ADPKD) belonging to the transient receptor potential (TRP) sensory

is the most common potentially lethal monogenic dis- channel family. Together, PC1 and PC2 are thought to

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order, with more than 12 million cases worldwide. The function as a Ca -permeable receptor channel complex [4].

two causative for ADPKD, PKD1 and PKD2, formation in ADPKD requires a germline mutation

encode products polycystin-1 (PC1) and polycys- in either PKD1 or PKD2. Although every cell in the body

tin-2 (PC2 or TRPP2), respectively. Recent data have shed

light on the role of PC1 in regulating the severity of the

Glossary

cystic phenotypes in ADPKD, autosomal recessive poly-

Autosomal dominant polycystic kidney disease (ADPKD): the most common

cystic kidney disease (ARPKD), and isolated autosomal

monogenic disorder in which kidneys develop fluid-filled derived from

dominant polycystic disease (ADPLD). These stu-

the tubule epithelial cells.

dies showed that the rate for cyst growth was a regu- Autosomal dominant polycystic (ADPLD): a monogenic condition

characterized by the growth of -derived cystic lesions.

lated trait, a process that can be either sped up or slowed

Autosomal recessive polycystic kidney disease (ARPKD): a rare pediatric

down by alterations in functional PC1. These findings

disease characterized by fusiform dilation of the collecting ducts in the kidneys

redefine the previous understanding that cyst formation and congenital hepatic fibrosis.

Bacterial artificial (BAC): a DNA construct used in transgenic

occurs as an ‘on–off’ process. Here, we review these and

mouse technology. BAC transgenic models offer the advantages that they are

other related studies with an emphasis on their transla-

low copy number and can faithfully reproduce the native splicing and

tional implications for polycystic diseases. expression pattern of genes. BACs can be modified by homologous

recombination in Escherichia coli and transgenic lines can be readily generated

by pronuclear injection.

Polycystic kidney disease and related cystic diseases

Cilia: apical cellular microtubular projection involved in mechanosensory

Autosomal dominant polycystic kidney disease (ADPKD) signaling. In the mammalian kidney, the ‘primary’ (which belongs to the

group of immotile ‘9+0’ cilia, i.e., nine peripheral microtubule doublets and no

(see Glossary) is part of a spectrum of inherited cystic

central pair) is located on the apical surface of epithelial cells and extends into

diseases that also includes autosomal dominant polycystic

the tubule lumen.

liver disease (ADPLD), autosomal recessive polycystic kid- Cystic fibrosis transmembrane receptor (CFTR): a transmembrane protein

involved in chloride conductance whose deletion results in cystic fibrosis.

ney disease (ARPKD), and an expanding group of reces-

Endoplasmic reticulum (ER): a cellular organelle that forms a network of

sively inherited syndromic (Table 1). ADPKD

membrane sacs called cisternae. It serves many fundamental functions

is the most common monogenic disorder that can lead to including protein folding, translocation, and transport.

ER-associated degradation (ERAD): a cellular pathway through which mis-

kidney failure with an incidence of 1 in 600–800 live births,

folded ER are retro-translocated, ubiquitinated, and targeted for

and affecting 600 000 people in the USA [1]. In most cases, degradation by the proteasome.

ADPKD manifests during adult life and is characterized by /polyductin (FPC): a large integral membrane protein encoded by

PKHD1, the responsible for the occurrence of ARPKD.

extensive cystic enlargement of both kidneys. The two

Immunoglobulin heavy chain binding protein (BiP): an ER Hsp70 chaperone

causative genes for ADPKD, PKD1 located on chromosome involved in protein translocation and folding. SEC63 (in addition to ERdj1)

16p13.3 and PKD2 located on chromosome 4q21, were recruits BiP to the translocation site via its DnaJ-like domain and provides the

necessary ATPase activity to cycle BiP to the ADP-bound high-affinity peptide

isolated by positional cloning and their respective protein

interaction state.

products, polycystin-1 (PC1) and polycystin-2 (PC2 or Kidney specific promoter Cre (Ksp-Cre): used to specifically inactivate proteins

in the thick ascending limb, distal, and collecting duct regions of the nephron.

TRPP2) [2,3], have been extensively studied. PC1 and

PRKCSH: the gene that encodes the C substrate heavy chain

PC2 are integral membrane proteins, with PC1 having

(which is now known to function as the b-subunit of glucosidase II). It is

structural and functional features suggestive of receptor involved in the deglycosylation of N-glycan moieties on nascent polypeptides

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as part of the calnexin–calreticulin cycle.

function, whereas PC2 is a Ca -permeable cation channel

Protein biogenesis: the process through which a protein is translated,

translocated into the endoplasmic reticulum, folded, and transported to its

physiological cellular location.

Corresponding authors: Fedeles, S.V. ([email protected]);

SEC63: the gene encoding the ER translocon-associated protein SEC63,

Somlo, S. ([email protected]).

involved in co-translational protein translocation.

Keywords: polycystic kidney disease; ; polycystin-1 dosage;

Unfolded protein response (UPR): the unfolded protein response, which

cyst progression; protein biogenesis; chaperone therapy.

involves the coordinate transcriptional activation of a set of genes that encode

1471-4914/$ – see front matter ER chaperones and certain cell death signals, occurs as a result of the

ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2014.01.004 accumulation of misfolded proteins in the ER. The UPR is activated by agents

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that affect ER homeostasis, such as tunicamycin, thapsigargin, Ca ionophores,

and reducing agents. In mammalian cells, UPR activation is mediated by three ER

transmembrane proteins that function as proximal sensors, designated IRE1,

ATF6, and PERK, of which IRE1 is the most conserved from yeast to .

Trends in Molecular Medicine, May 2014, Vol. 20, No. 5 251

Review Trends in Molecular Medicine May 2014, Vol. 20, No. 5

a

Table 1. Genes and protein products involved in dominant and recessive polycystic diseases

Disease (OMIM) Gene Key clinical features Protein products Pathogenetic mechanism

of cyst formation

ADPKD PKD1 Kidney and bile duct cysts Polycystin-1/ciliary receptor Two-hit model combined

MIM 173900 protein involved in with a PC1 dosage-

or dependent mechanism

chemosensation

ADPKD PKD2 Kidney and bile duct cysts Polycystin-2/ciliary and Two-hit model combined

MIM 173900 endoplasmic reticulum membrane with a PC1 dosage-

protein functioning as a non- dependent mechanism

selective cation channel

ARPKD PKHD1 Fusiform collecting duct Fibrocystin/polyductin/ciliary, Homozygous inactivation,

MIM 263200 dilatations, liver cysts, and plasma membrane protein oriented cell division

congenital hepatic fibrosis defects, PC1 dosage-

dependent mechanism

ADPLD PRKCSH Bile duct cystic dilatations GIIb/ER luminal protein/N-glycan Two-hit model combined

MIM 174050 trimming as part with a PC1 dosage-

of the calnexin–calreticulin cycle of dependent mechanism

protein folding

ADPLD SEC63 Bile duct cystic dilatations SEC63/ER transmembrane protein Two-hit model combined

MIM 174050 involved co-translational protein with a PC1-dosage

translocation together with SEC61 dependent mechanism

a

Abbreviations: OMIM, Online Mendelian Inheritance in Man; ADPKD, autosomal dominant polycystic kidney disease; MIM, Mendelian Inheritance in Man; PC1, polycystin-

1; PKD1/2, polycystic kidney disease gene 1/2; PKHD1, polycystic kidney and hepatic disease 1; ARPKD, autosomal recessive polycystic kidney disease; ADPLD, autosomal

dominant polycystic liver disease; PRKCSH, protein kinase C substrate heavy chain; GIIb, b subunit of glucosidase II; ER, endoplasmic reticulum.

carries this germline mutation, cyst formation is focal, 85% of cases ascertained by clinical presentation. The

arising only from a minority of kidney tubules and hepatic PKD1 gene product, PC1, is a very large protein consisting

bile ducts. This apparent paradox has been explained by the of 4302 amino acids with a 3074 extracellular

occurrence of somatic second hit mutations in the remaining amino terminus, 197 amino acid cytosolic carboxy termi-

normal copy of the affected gene, leading to recessive loss of nus, and 11 transmembrane domains [2,21]. PC1 is prob-

function in a subset of tubule epithelial cells that actually ably expressed in most nephron segments, although

give rise to cysts in adult tissues [5–10]. Although somatic expression at the tissue level has been difficult to deter-

second hit mutations are a generally accepted mechanism mine due to very low protein levels. At the subcellular

for ADPKD, additional factors have been shown to level, PC1 is localized to the primary cilium on the apical

influence the extent of cyst formation. Mouse and human surface of epithelial cells (Figure 1) [4,22], in addition to

studies have shown that these additional factors include other reported locations in the cell such as the lateral

non-cell autonomous effects on cells still expressing poly- membrane at sites of intercellular adhesion [23] and at

cystins [11], the developmental timing of PKD1 inactivation [24]. Cilia are linked to a wide range of cystic

[12], and milder effects of PC1 hypomorphic mutations diseases and the central role for cilia in many human

compared with complete loss of function [13–19]. Reduction diseases has been thoroughly reviewed [25,26]. Defects

in functional PC1 dosage has been shown to underlie expres- in proteins localized in the ciliary membrane,

sion of the ADPLD and ARPKD phenotypes in which the and complex are linked with a wide clinical

extent of tubule dilation and cyst formation is inversely spectrum of diseases, collectively termed ‘ciliopathies’.

correlated with the level of PC1 function [20]. These studies Clinical findings in ciliopathies include cyst formation in

also suggest that sensitivity to PC1 dosage differs between kidney tubules, bile and pancreatic ducts, retinal degen-

bile ducts and kidney tubules, and between the different eration and retinitis pigmentosa, situs inversus (incorrect

segments of the nephron. The following review focuses on left–right body axis), anosmia (loss of sense of smell), and

the common role of PC1 dosage in the cyst progression in hydrocephalus [27].

ADPKD, ARPKD, and ADPLD. PKD2, the gene mutated in 15% of clinically ascertained

polycystic kidney disease cases, encodes PC2 [28]. PC2 has

Genes and proteins been described as a non-selective permeable cation

Autosomal dominant polycystic kidney disease (ADPKD) channel belonging to the transient receptor potential poly-

ADPKD is characterized by the formation and growth of cystic (TRPP) subfamily of TRP cation channels [28]. PC2

multiple fluid-filled kidney cysts that progress over dec- is an integral membrane protein with six transmembrane

ades with attendant inflammation and fibrosis. Conse- segments, and both the amino and carboxy termini face the

quent loss of functional nephrons leads to end-stage cytoplasmic compartment [29,30]. PC2 contains several

renal disease (ESRD) in over 50% of affected individuals trafficking motifs: the RVxP motif at the amino terminus

by late adulthood. A major extrarenal manifestation of of PC2 is necessary and sufficient for the ciliary location of

ADPKD is polycystic liver disease, which does not affect the protein [31], whereas a region at the carboxy terminus

liver function but can lead to symptoms related to mass may restrict the membrane location of PC2 to the endo-

effects when significant liver enlargement occurs. plasmic reticulum (ER) and cilia. In the kidney, PC2 is

There are two known genes mutated in ADPKD: PKD1 found in all nephron segments, with the exception of the

and PKD2. PKD1 mutations account for approximately thin loops of Henle and the glomerulus.

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effects of concomitant inactivation of cilia and polycystins

would be. Cilia were ablated by mutation of intraflagellar

PC2

transport protein encoding genes that are required for the

PC1 ADPKD

formation and maintenance of cilia. Surprisingly, inactiva-

Cilium FPC ARPKD

tion of cilia in the setting of absent polycystins resulted in

much milder progression of polycystic disease than was

seen with inactivation of polycystin alone [37]. In the

Golgi ADPLD models used, polycystin protein levels in the kidney epithe-

(PRKCSH:SEC63)

lial cells were attenuated before the disappearance of cilia

and the severity of the polycystic disease was directly

related to the duration of the interval between the initial

ER

loss of polycystins and the subsequent involution of cilia.

Nucleus

These findings define the existence of a novel cilia-

dependent pathway promoting cyst growth that requires

the presence of intact cilia devoid of polycystins to produce

TRENDS in Molecular Medicine

kidney cysts [37]. The most straightforward interpretation

Figure 1. Schematic view of the relevant subcellular localization of the proteins of these findings is that polycystins provide baseline tonic

involved in autosomal dominant polycystic kidney disease (ADPKD), autosomal inhibition of this cilia-dependent signal. As part of normal

recessive polycystic kidney disease (ARPKD), and autosomal dominant polycystic

physiological function of polycystins, this inhibition is

liver disease (ADPLD). The two ADPLD proteins glucosidase IIb (GIIb) and Sec63p

are localized in the endoplasmic reticulum (ER) and are involved in protein reduced in a regulated manner based on mechanical flow

translocation (Sec63p) and folding (GIIb). The proteins associated with ADPKD,

or ligand-mediated regulation of PC1. The normal in vivo

polycystin-1 (PC-1, encoded by PKD1), and polycystin-2 (PC-2, encoded by PKD2),

function of these pathways is uncertain, but one hypothesis

in addition to the ARPKD protein fibrocystin/polyductin, (FPC, encoded by PKHD1)

are all ciliary transmembrane proteins which need to go through the co- may be that they regulate renal tubular lumen diameter by

translational translocation pathway (Figure 2) in the ER, which Sec63p and GIIb

subtle changes in tubule cell morphology through regula-

are integral members of, in order to be properly folded and trafficked to Golgi and

tion of the actin cytoskeleton (e.g., transitioning cells from

onto their final physiological destination, the primary cilia.

more columnar to more cuboidal shaped). Based on these

studies, we propose that polycystins act as suppressors of

The working model of polycystin function is that PC1 an as yet undefined cilia-dependent pathway, and that

functions as a mechanosensor or chemosensor at the pri- discovery of this latter pathway will provide insight into

mary cilium and regulates the activity of the PC2 calcium novel kidney tubule homeostatic mechanisms as well as

channel [32–34]. Beyond that, a myriad of cellular path- provide a universally applicable target for reducing cyst

ways have been implicated as effector pathways in ciliary growth in both the kidney and liver in ADPKD.

and cystic diseases, including planar cell polarity (PCP),

Wnt, mammalian target of rapamycin (mTOR), cyclic ade- Autosomal recessive polycystic kidney disease (ARPKD)

nosine monophosphate (cAMP), G-protein-coupled recep- ARPKD is a rare pediatric disease with an incidence of

tor (GPCR), cystic fibrosis transmembrane conductance approximately 1 in 20 000 births. ARPKD is characterized

regulator (CFTR), epidermal growth factor receptor by fusiform dilation of the collecting ducts in the kidneys,

(EGFR), mitogen-activated protein kinase (MAPK) cellular and biliary ductal ectasia leading to congenital hepatic

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Ca , and the cell cycle (reviewed in [35,36]). Extensive fibrosis of the portal tracts in the liver, and is associated

evidence that cAMP promotes cyst growth has led to the with a high level of morbidity and mortality in affected

hypothesis that loss-of-functional PC1 at the cilium results individuals. PKHD1 is the only known responsible for

in reduced cellular calcium signaling [4] that in turn ARPKD [38,39]. In ARPKD, there is a genotype–phenotype

results in increased adenylate cyclase and decreased phos- correlation, in that the presence of two completely inacti-

phodiesterase activity, leading to elevated intracellular vating PKHD1 mutations results in a more severe clinical

cAMP. Among the effects of increased cAMP in cystic outcome associated with perinatal mortality. Patients with

is increased activity, which at least one hypomorphic missense mutation have a more

can result in increased cell proliferation and deregulated juvenile presentation, suggesting that a subset of missense

fluid secretion, features associated with cyst growth [36]. changes result in reduced rather than absent function of

Lacking through all of these proposed functional pathway the PKHD1 gene product [40,41].

hypotheses is an understanding of the relationship of PKHD1 encodes the protein fibrocystin (FPC), which is a

polycystins with other ciliary component proteins and a large 4074 amino acid complex integral membrane protein.

clear definition of the direct role of polycystin signaling FPC contains a signal peptide, a predicted glycosylated

within cilia. extracellular amino terminus subsuming most of the pro-

A recent study based on genetic manipulation of poly- tein (3858 amino acids), a single transmembrane domain,

cystin and cilia in mutant mice has the potential to sig- and a 192 amino acid cytoplasmic tail [38,39,42]. FPC

nificantly recalibrate the concepts about the localizes in the cortical and medullary collecting ducts

interrelationship between polycystin and cilia function and thick ascending limbs of the loop of Henle in the kidney

[37]. Based on the observation that inactivation of the [43]. Elsewhere, FPC is expressed in the biliary and pan-

entire cilium structure or of a single cilium membrane creatic tracts, and in salivary ductal epithelia. FPC is

component protein, such as polycystin, both give rise to expressed in the cilia and apical membrane of renal tub-

cyst formation in the kidney, the study asked what the ular and bile duct cells [44–47].

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cis γ , β , Gluc Sec61 α Sec62 I CNX trans ERp57 ERj1 Sec63p Gluc II α β BiP Gluc II β α Key: CRT Ribosome Glucose ERp57

Precursor pepde Glycan moiety

TRENDS in Molecular Medicine

Figure 2. The co-translational translocation pathway highlighting (red contour) the two autosomal dominant polycystic liver disease (ADPLD) proteins, Sec63p and

glucosidase IIb (GIIb). At this stage, the precursor protein [e.g., polycystin-1 (PC1)] is threaded through the translocon, with its central subunit, the Sec61 complex, and the

auxiliary subunits that are present in the membrane (Sec63 and ERdj1/Erj1). Via their DnaJ domains, Sec63p and Erdj1 provide a link to the ATPase cycle of the luminal

chaperone binding immunoglobulin protein (BiP). Downstream of Sec63p, GIIb is involved (as the regulatory subunit of the glucose trimming enzyme, glucosidase II) in

glucose trimming, which takes place as part of the calnexin–calreticulin cycle. Cystic proteins such as PC1 have to go through this pathway to achieve their final, functional

cellular location (the primary cilia). Defects along this pathway may affect both the translocation (due to Sec63p inactivation) and glucose trimming/folding (via GIIb) of

cystic proteins as described in [20].

FPC is involved in regulation of PCP, a process in which an ER luminal N terminus, a cytoplasmic C terminus with

specialized structures are distributed in an oriented man- a coiled-coil region, and a luminal DnaJ domain between

ner within the plane of the epithelial sheet. Some examples the second and third transmembrane spans. SEC63p

of PCP include the scales of fish being oriented in the same associates with the SEC61/62 ER translocon, which forms

direction, and similarly the feathers of birds, the fur of the hydrophilic pore in the ER membrane through which

mammals, and the cuticular projections on the bodies and the nascent integral membrane or secreted polypeptides

appendages of flies and other insects [27]. Complete loss of are translocated. SEC63p is responsible for the tight bind-

FPC leads to loss of oriented cell division (OCD), a man- ing of the ribosome to the ER membrane during the co-

ifestation of PCP which allows oriented elongation of kid- translational transport process [58]. Two recent biochem-

ney tubules while maintaining a constant luminal ical studies have highlighted the role of SEC63p in the

diameter during postnatal growth of the organism initial phase of co-translational protein transport into the

[36,48–50]. Loss of OCD following inactivation of Pkhd1, ER in a precursor-specific manner: SEC63p functions in

however, is not sufficient to initiate kidney cyst formation determining substrate specificity in the initial insertion of

in mouse models of ARPKD, suggesting that additional certain precursors into the SEC61 translocon complex [59]

functional factors play a critical role in ARPKD [49]. and has a role in determining the steady-state quantity of

multi-spanning membrane proteins [60]. SEC63p recruits

Autosomal dominant polycystic liver disease (ADPLD) the Hsp70 homolog, binding immunoglobulin protein

Isolated ADPLD without kidney cysts manifests a clinical (BiP), to the translocation site via its DnaJ-like domain

phenotype of biliary cystic malformations that are indis- and provides the necessary adenosine triphosphatase

tinguishable from those occurring in ADPKD. The two (ATPase) activity to cycle BiP to the adenosine di-phos-

known disease causative genes for ADPLD are PRKCSH phate (ADP)-bound high-affinity peptide interaction state

and SEC63, which encode for non-cilial, ER-localized pro- [61,62]. PRKCSH and SEC63 are involved in the same

teins involved in protein biogenesis and post-translational functional pathway of co-translational protein transloca-

modification [51–54] (Figure 2). PRKCSH encodes the non- tion and maturation [54,63,64], and defects associated with

catalytic b-subunit of glucosidase II (GIIb) [55]. Glucosi- either of them have a significant impact on the biogenesis

dase II acts in the calnexin–calreticulin cycle of folding and of polycystic disease-associated proteins [20]. Along these

quality control for integral membrane and secreted pro- lines it would be interesting to examine the association

teins passing through the ER translocon [56]. Computa- between Prkcsh/Sec63 deletion and the upregulation of the

tional analysis predicts that GIIb contains a signal peptide unfolded protein response (UPR) in the context of murine

for translocation across the ER membrane, a low density ADPLD models and/or ADPLD patients.

lipoprotein domain class A (LDL-A), two EF-hand (named

after the E and F helices of parvalbumin where the cal- Mechanisms of cyst formation

cium-binding domain is located) domains, a glutamic acid- Following the discovery of the two ADPKD causative

rich region, a mannose-6-phosphate receptor domain, and genes, PKD1 and PKD2, the next significant step forward

a conserved C-terminal HDEL amino acid sequence for ER in understanding the underlying mechanisms came from

luminal retention. the discovery that cyst lining cells derived from human

Human SEC63p is an 83-kDa protein [57] that is pre- cystic kidney and liver tissues display somatic second hit

dicted to span the ER membrane three times. It contains mutations, which lead to homozygous inactivation of the

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respective genes in a cellular recessive mechanism [5,7–9]. By contrast, a recent study found that increased back-

Demonstration of a causal connection between somatic ground proliferation induced by a Cux-1 transgene did

inactivation of Pkd2 and cyst formation came from a not promote cyst growth in a model of polycystic disease

WS25

modified allele in the Pkd2 mouse line that is prone based on loss of cilia [73]. This suggests that the underlying

WS25

to genomic rearrangement converting Pkd2 to either a background proliferative potential is important but not

null or true wild type allele [6]. Somatic conversion to a null sufficient for cyst growth in adult models of polycystic

allele correlated with cyst formation in the kidney, liver, kidney disease. The severity of ADPKD is likely to be

and pancreas, showing that somatic loss of Pkd2 is suffi- modulated by factors including the timing and degree of

cient for cyst formation [6,14]. gene and protein inactivation, the occurrence of local injury

Similar to ADPKD, the mechanism of cyst formation in and the specific proliferative response that these changes

ADPLD involves somatic second hit mutations at either the engender.

PRKCSH or SEC63 loci [20,65,66]. In mice, conditional

tissue-specific inactivation of either gene triggered liver PC1 dosage in ADPKD

and kidney cyst formation, whereas age-matched hetero- PC1 dosage has sporadically been described to be a mod-

zygous mice maintained normal organ architecture [20]. ulator of cystic disease. Lantinga van-Leeuwen and collea-

Genetic and biochemical studies showed that homozygous gues [15] were the first to report that reduced levels of PC1

loss of either Prkcsh or Sec63 affects the dosage of the could lead to the clinical features of ADPKD without

functional PC1/PC2 complex, leading to cyst formation complete inactivation. The authors showed that the intro-

[20]. The implications for these findings are discussed later. nic insertion of a neomycin cassette into Pkd1 had a

Although the second hit hypothesis is the generally profound effect on splicing efficiency, resulting in a mixture

accepted mechanism for ADPKD, other mechanisms and of mutant and wild type sequences. Approximately 15–20%

factors that may influence the rate of cyst growth have of the Pkd1 transcripts were correctly spliced in the major-

nl/nl

been experimentally demonstrated in mouse models. Chi- ity of the homozygous Pkd1 mice. These hypomorphic

meric transgenic mice that were made by combining wild mice escape the embryonic lethality seen in complete

À/À

type morulae containing a lacZ lineage tracer with Pkd1 knockout mice where PC1 is totally absent. Although

nl/nl

null embryonic stem cells developed cysts whose size was the hypomorphic Pkd1 mice survive, they develop

À/À

dependent on the relative contribution of Pkd1 cells to severe cystic kidneys, with cysts originating from collecting

the chimeras [11]. The cysts displayed a mosaic pattern duct, thick ascending limb, and distal tubules. Another

with both null and wild type Pkd1 cells. Over time, null study by Jiang et al. [16] provided evidence that dimin-

cells dominated the entire cystic area by increased prolif- ished expression of native PC1 is sufficient to induce renal

eration while also inducing in the wild type cells cystic lesions of ADPKD. The authors showed that the

via a c-Jun N-terminal kinase (JNK)-mediated pathway intronic insertion of the neomycin cassette in reverse

[11]. orientation into Pkd1 interfered with the transcription

Cyst formation resulting from trans-heterozygous and/or splicing machinery, resulting in low (around 20%)

somatic mutations involving PKD1 and PKD2 has also but not completely absent Pkd1 expression. Only wild type

been described [67]. The effect of trans-heterozygosity on Pkd1 transcript and protein were detected in either het-

L3/+ L3/L3

the overall cyst formation appears to be small, and not erozygous Pkd1 or homozygous Pkd1 mice with no

enough to explain the cystic burden as it occurs in ADPKD. mutant transcript with the neomycin cassette present. The

L3/L3

For example, individuals with bilineal transmittance of Pkd1 mice developed a severe cystic phenotype around

PKD1 and PKD2 mutations [13] or trans-heterozygous P30, with cysts forming exclusively from the collecting

mice [14] display increased polycystic kidney disease, duct, thick ascending limb, and distal tubules. A similar

but the overall severity is within the range consistent with follow-up study used microRNA technology to knock-down

additive or small extra-additive effects of single gene the Pkd1 transcript [18]. Based on these studies, one can

mutations. hypothesize that although 50% reduction in PC1 levels, as

The timing of Pkd1 inactivation significantly impacts occurs in true heterozygote ADPKD patients, does not lead

the rate of cyst growth. In studies where temporally con- to frank cyst formation, 80% reduction may be sufficient to

trolled inducible Cre recombinase expression was used to trigger a polycystic phenotype. What remains unknown is

knock-out conditional Pkd1 alleles at different develop- whether the 50% reduction in PC1 in ADPKD patients

mental time points, early inactivation during ongoing affects tubular morphology in more subtle ways, perhaps

kidney development resulted in a fast rate of cyst growth. by increasing luminal diameter without unchecked expan-

In contrast to this, later inactivation in post-developmental sion leading to cysts.

or adult kidneys resulted in slower cystic development A recent study showed that a knockin mouse model,

RC/RC

[12,68]. The transition from rapid to slower cyst growth Pkd1 , mimicking a naturally occurring PKD1 variant

correlated with a switch in occurring p.R3277C [17], developed gradual cystic disease over 1

RC/null

around postnatal day 12 in the mouse, which may be linked year, whereas the Pkd1 mice had more rapidly

to a different proliferative potential of the kidney at early progressive disease [19]. These observations, coupled with

versus late stages of development [12]. These findings in biochemical data showing that the p.R3277C mutation

the developing kidney are complemented by further stu- leads to a decrease in the steady-state levels of PC1 that

dies showing that the ischemia–reperfusion-dependent can be partially rescued in a temperature-sensitive man-

injury response, which has a proliferative component, ner, point to the importance of PC1 dosage in the modula-

increases the rate of cyst growth in adult mice [69–72]. tion of cyst severity. Conversely, high copy number (>30)

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Pkd1 transgenic lines have reported the presence of kidney persistent tubule dilation and cyst growth, indicating that

cysts [74], suggesting that it is not only reduced dosage of these are not on–off phenomena.

PC1 that may lead to cysts. Very high levels of PC1 over- These in vivo differences in cyst severity reflect the fact

expression may generate cysts possibly by disrupting the that GIIb deletion is a ‘milder’ molecular defect, due to

stoichiometry of the PC1/PC2 complex, thereby impacting existence of alternative N-glycan trimming pathways,

its signaling function. Overall, these data imply that PC1 whereas Sec63p deletion is more ‘severe’, based on its

dosage is a bona fide mechanism of cyst formation in both proximal location in the ER translocation pathway. The

mouse and human ADPKD. critical point in understanding the implications of these

models is that the population of kidney tubule cells in

PC1 dosage in ADPLD which either GIIb or Sec63p are inactivated, as well as

New findings have defined the genetic interactions among the timing, are largely the same in all models because the

ADPKD, ADPLD, and ARPKD and have brought into the same Cre recombinase transgenic lines were used to knock-

spotlight the central importance of PC1 dosage across this out the respective genes. Therefore differences in the

spectrum of human polycystic diseases [20,75]. Pkd1 and severity of cyst progression translate to differences in

Pkd2 interact genetically with both Prkcsh and Sec63, the cell autonomous levels of residual PC1 function, as

respectively, encoding GIIb or Sec63p. The background opposed to differences in the number or types of cells

levels of the polycystin proteins in mouse models of ADPLD affected. In this formulation, the more severe molecular

were reduced by introducing a heterozygous null allele for lesion will have a more significant reduction in PC1 at the

the respective Pkd gene or were increased by introducing individual cell level, thereby giving rise to more severe

low copy number bacterial artificial chromosome (BAC) cystic disease based on differential intrinsic properties of

transgenes expressing either Pkd gene. The dosage of PC1 the cyst cells. These differences are determined by the

protein proved to be the ultimate arbiter in the severity amount of residual PC1 activity.

and timing of cyst formation following recessive tissue- Taken together, these data show that kidney tubule

specific loss of GIIb or Sec63p. In the absence of GIIb or diameter is a continuously distributed function of PC1

Sec63p, decreasing expression of Pkd1 using a heterozy- dosage and time (Figure 3). The greater the reduction in

+/–

gous Pkd1 background exacerbated the cystic pheno- PC1 dosage, the faster the cysts grow; the longer the time

type. Increasing expression of Pkd1, using a three-copy in the setting of reduced PC1 dosage, the larger the cysts

F/H

Pkd1 -BAC transgene, ameliorated the cystic pheno- become. The ‘asymptotic’ extreme is reached in the com-

type. Although reduction of Pkd2 dosage showed an inter- plete absence of PC1 when kidney tubule luminal dia-

mediate level of exacerbation in polycystic disease, the meters expand rapidly in full blown polycystic kidney

rescue of the cystic phenotype was not seen when Pkd2 disease. In defining the ADPLD mouse models as models

dosage was increased using a Pkd2-BAC transgenic. It has of hypomorphic PC1 function that give rise to a broad range

to be noted here that PC1 modulated the cystic phenotype from tubule dilation to frank cyst formation as a time-

when PC2 was present. In the complete absence of PC2, dependent function, this study [20] strongly supports the

overexpression of PC1 did not have any effect on cyst hypothesis that polycystins normally function to regulate

formation, suggesting that the functional PC1/PC2 com- tubule lumen diameter under physiological cues that

plex is strictly required for the rescue to take place. These remain to be identified. Coupled with a recent study sug-

findings define PC1 as the rate-limiting component of the gesting that PC1/PC2 tonically inhibits an unknown

polycystin complex in cyst formation in ADPLD.

These genetic studies were correlated with biochemical

experiments revealing that deletion of GIIb or Sec63p

leads to reduction in the steady-state expression, post-

mid Golgi and ciliary trafficking, of PC1, as well as other

integral membrane proteins. The ADPLD gene products,

via their role in the biogenesis of PC1, act as modifiers of

the PC1-dependent cystic phenotype.

When compared with GIIb, the Sec63 knockout mice

have earlier kidney cyst formation and faster progressing

cysts [20]. Biochemically, they display more severe defects

in the biogenesis of PC1 than PC2 and other transmem-

Polycysn-1

brane proteins [20,59]. Genetic changes in PC1 dosage

achieved by either reducing or increasing gene copy num- Time

ber further modify the rate of cyst progression in the

TRENDS in Molecular Medicine

ADPLD orthologous gene models. In addition, there is a

clear temporal component to cyst progression in the Figure 3. Schematic view showing that polycystin-1 (PC1) levels can dictate the

timing and severity of the phenotype in a time- (x-axis) and dose- (y-axis)

ADPLD models. At a common age, Sec63 knockouts are

dependent manner. For a certain PC1 threshold (depicted by the red line) no cyst

more severe than Prkcsh knockouts and the BAC trans-

formation occurs. When the dose of PC1 goes below this threshold, cyst initiation/

genic rescue kidneys are milder than non-transgenic progression ensues with lower levels leading to faster cyst growth. This is

evidenced by the autosomal dominant polycystic liver disease (ADPLD) mouse

littermates. Despite being milder at matched time points,

models where the lower dosage of PC1 on the Sec63-deficient background leads to

the Prkcsh knockouts and the BAC transgenic rescue

an earlier manifestation of the phenotype as compared with the Prkcsh null

kidneys continue to progress with slower but nonetheless background.

256

Review Trends in Molecular Medicine May 2014, Vol. 20, No. 5

cilia-dependent cyst promoting signaling pathway [37], it the dosage data from animal studies detailed above. A

can be hypothesized that a regulated signal, such as flow or subset of missense variants probably produce some PC1

ligands or both, modulate the PC1/PC2 complex to reduce protein with residual function resulting in slower growth of

the inhibitory activity. This allows the cilia-dependent cysts and therefore a delay in the median time to ESRD for

pathway to alter kidney tubular structure or increase the group of patients with these mutations. The genotype

luminal diameter, which can be reversed when the PC1/ of the normal PKD1 allele in ADPKD patients can also

PC2 is returned to the unstimulated, maximally inhibitory affect the course of disease, in this instance worsening

state. severity. Two recent studies have reported the existence

of partially penetrant PKD1 alleles in ADPKD patients

Organ and tissue specificity in ADPLD that occurred in trans with a fully penetrant truncating

Whereas conditional inactivation of GIIb or Sec63p in liver mutation or another missense variant [17,79]. In both

and kidney leads to cysts in mice, in humans ADPLD is cases, the individuals carrying two mutations of which

restricted to a bile duct-specific phenotype. The increased at least one was incompletely penetrant showed more

susceptibility of bile ducts to cyst formation compared with severe progression of ADPKD, findings again consistent

kidney tubules in human ADPLD may reflect that normal with the dosage correlated severity model. We are not yet

PC1 expression in the bile ducts is closer to a critical in a position to assess individual mutations for their

threshold below which cyst formation ensues. Mutations relative molecular severity, but this should be a goal for

in PRKCSH or SEC63 in bile duct cells results in reduced studies in the near future. Such information can inform

functional PC1 which falls below this threshold, whereas in patients of their family’s prognosis and may also serve as

kidney tubules the reduction in PC1 resulting from the the basis for therapeutic decisions once therapies become

same ADPLD gene mutations may not be sufficient to drop available.

below the critical threshold, and hence no cysts occur. Of Beyond allelic determinants of disease severity, two

course, the converse explanation is equally plausible, that recent clinical reports have described severe cases in

bile ducts require higher levels of PC1 to avert cystic patients with ADPKD who carry, in addition to their

dilation than kidney tubules. A corollary to the above expected familial germline defect in PKD1, additional

hypothesis is that as PC1 levels are reduced but farther mutations in either hepatocyte nuclear factor 1b (HNF-

above a critical threshold in the kidneys in ADPLD 1b) or Sec63 [80,81]. The effects from mutations in genes in

patients, kidney cysts grow more slowly and thus do not trans with PKD1 are similar to previous reports of bilineal

become clinically apparent in the normal lifespan of the inheritance of PKD1 and PKD2 mutations in the same

general population. Either way, the kidneys appear to be individual, also resulting in more severe ADPKD [13].

more ‘tolerant’ of changes in PC1 dosage resulting from These data suggest that patients with ADPKD who present

ADPLD gene mutations than bile ducts. with an early and severe phenotype clinically indistin-

In terms of intra-organ sensitivity to reduced PC1 guishable from ARPKD may carry mutations in other

dosage, collecting ducts are more sensitive to cyst forma- genes in trans with PKD1 that affect either the transcrip-

tion than the medullary thick ascending loops of Henle tional control of PKD1 expression such as HNF-1b [82] or

(mTAL) following similar levels of inactivation of either the biogenesis of PC1 as is the case with SEC63 and

Prkcsh or Sec63 (using Ksp-Cre). The exacerbation of the PRKCSH. Clinical testing application of next-generation

cystic kidney phenotype following reduction of PC1 dosage sequencing technologies such as whole-exome sequencing

+/–

on the Pkd1 background is accounted for almost exclu- may shed further light on the clinical phenotypic variation

sively by larger (i.e., faster growing) cyst formation in noted in ADPKD patients and better define the role of

collecting ducts. Applying the same reasoning as above, other PKD-related genes in the progression of ADPKD in

we postulate that the mTAL is more tolerant of changes in humans.

PC1 dosage than the collecting ducts, and thus the effect of

decreased PC1 dosage is more easily unmasked in the Relationship between PC1 expression and ARPKD

latter versus the former. This can be clinically significant Studies of the role of ADPLD gene mutations and PC1

as evidenced by the recent trial in ADPKD showing reduc- dosage also revealed an intriguing relationship between

tion of total kidney volume with a vasopressin-2 receptor PC1 dosage and ARPKD progression. Despite the absence

antagonist [76]. Vasopressin-2 is only expressed in the of any polycystic kidney phenotype in mice with loss of

collecting ducts and therefore this intervention was Pkhd1, compound Sec63/Pkhd1 mutant mice showed

thought to primarily target collecting ducts cysts. marked exacerbation of cyst formation compared with

the Sec63 background alone [20]. There is a precedent

Modifiers of human ADPKD severity for genetic interaction between the Pkhd1 and Pkd1 loci

Recently, genetic modifiers of the severity of polycystic in that loss of one copy of Pkd1 exacerbates cyst progres-

kidney disease in humans, beyond differences between sion in Pkhd1-deficient mice [20,83]. Inactivation of Sec63

PKD1 and PKD2 genotypes, have been identified. Allelic results in a more severe reduction in Pkd1 than the het-

variation in PKD1 has now been shown to affect disease erozygous state [20]. The most compelling evidence of the

progression [77,78]. The median age to ESRD in PKD1 PC1 dosage dependence of the Pkhd1 kidney phenotype,

patients with predicted truncating mutations was 55.6 however, came from the observation that overexpression of

years, whereas the median age to ESRD for PKD1 patients PC1 by a three-copy BAC transgenic allele rescued the

with non-synonymous potentially pathogenic mutations severe cystic disease in the compound Sec63/Pkhd1 double

was 67.9 years. These clinical findings are in keeping with mutant mice [20]. This showed that the worsening of

257

Review Trends in Molecular Medicine May 2014, Vol. 20, No. 5

Funconal interacon Box 1. Outstanding questions

Polycysn-1 Fibrocysn  How is PC1 modulating the ARPKD phenotype?

 Does overexpression of FPC have a genetic effect on the PC1-

dependent phenotype?

 Can proteasome inhibition/chemical chaperones improve the

biogenesis of PC1 missense mutants?

 Is FPC deficiency affecting the biogenesis of PC1?

Cell proliferaon Planar cell polarity  What are the organ differences between liver and kidney ARPKD

that make the former resistant to the beneficial role of PC1?

 Is proteasome therapy efficient in slowing cyst formation in the

absence of Sec63p?

2+

 What is the effect of losing GIIb/Sec63p on the ER Ca home-

ostasis?

 What is the effect of losing GIIb/Sec63p on the status of the

unfolded protein response?

Cyst progression

 What is the role of physiological polycystin signaling within cilia?

TRENDS in Molecular Medicine  How can one assess individual missense mutations in PKD1 in

terms of their molecular severity?

Figure 4. Diagram of the genetic interaction between Pkd1 and the autosomal

recessive polycystic kidney disease (ARPKD) locus. Fibrocystin plays a role in cyst

progression through its involvement in the planar cell polarity pathway (green

arrow). Polycystin-1 (PC1), through its link to cell proliferation (blue arrow), can

led to increased levels of PC1 and an amelioration of the

further contribute to the severity of the phenotype, as seen in the double Pkd1/

Pkhd1 mutants. Because defects in planar cell polarity in the absence of Pkhd1 do cystic phenotype in a model of ADPLD with inactivation of

not lead to polycystic kidney disease, the contribution of planar cell polarity to cyst

Prkcsh [20]. In that case, proteasome inhibition led to an

progression occurs in the context of increased proliferation (red arrow). Is it not

increase in the levels of PC1, which, coupled with the

clear what the functional interaction between PC1 and fibrocystin (dotted arrow) is,

for example, whether there is a direct biochemical connection between the two increase in apoptosis and decrease in proliferation of

proteins impacting their respective function/level.

cyst-lining epithelia, resulted in an amelioration of the

cystic phenotype. When compared with wild type cells,

polycystic kidney disease resulting from introduction of the the absence of GIIb presumably sensitized cells to apop-

recessive Pkhd1 mutation onto the Sec63 mutant back- tosis due to the accumulation of unfolded proteins by

ground was largely a function of PC1 dosage. Reduced PC1 proteasome inhibition. The reduced proliferation may have

dosage sensitizes mice with mutations in Pkhd1 to poly- resulted from the increased levels of PC1 function. It would

cystic kidney phenotypes that are not observed with a wild be interesting to test whether the beneficial effect of pro-

type complement of Pkd1 expression. teasome inhibition is also maintained in the case of Sec63

del4

The Pkhd1 mutation, although not resulting in cystic inactivation (Box 1).

dilation, nonetheless causes loss of oriented cell division in Based on these studies, PKD1 alleles with incomplete

elongating nephrons of the postnatal mouse kidney [49]. penetrance that produce a full length, although mutant,

Oriented cell division is a feature of planar cell polarity PC1 protein may be amenable to therapies akin to protea-

[48,50]. It is possible that coupling loss of planar cell some inhibition that can increase effective functional levels

polarity in Pkhd1 mutants with markedly reduced PC1 of PC1 and further slow cyst growth. Missense mutant PC1

dosage aggravates cyst growth due to reduced PC1 alone may maintain functionality but nevertheless become retro-

(Figure 4). It is worth noting that the rescue by the Pkd1 translocated from the ER and targeted for degradation via

BAC transgene in the Sec63/Pkhd1 double mutant mice is the ER-associate degradation (ERAD)-cytosolic ubiquitin

not as complete as in the Sec63 single mutants. One (Ub)–proteasome system [84] or via the recycling endo-

interpretation of these results may be that in the absence some/lysosomal pathway in a manner somewhat analogous

of the Pkhd1 gene product, FPC, the amount of PC1 to the cystic fibrosis transmembrane receptor (CFTR)

required to avert cysts is higher than on a wild type Pkhd1 DF508 mutation [85]. For these scenarios, proteasome

background, supporting the functional interdependence of and/or lysosome inhibition may increase PC1 dosage by

these two human PKD genes. The most intriguing finding blocking the PC1-dependent degradation pathways. This

of these studies is that PC1 dosage can ameliorate the approach could be coupled with chemical chaperone treat-

murine ARPKD phenotype in the kidney, which begs the ment as has been done for CFTR [86]. This concept sug-

question of what the relationship between PC1 and FPC is gests that sequencing of PKD1 mutations should be given

in human ARPKD. Perhaps there is a dose-dependent consideration for implications in personalized medicine.

relationship where FPC deficiency leads to PKD in humans

because the relative level of PC1 activity is insufficient to Acknowledgments

prevent kidney cysts. In this formulation, it is possible that The authors thank members of the Somlo laboratory for helpful

ARPKD may be amenable to therapies that increase the discussions. S.V.F. was supported by a National Institutes of Health

training grant (T32 DK007276). This work was supported by grants from

dosage or function of PC1.

the National Institutes of Health (DK51041, DK54053, DK100592) to S.S.

and the American Heart Association (10SDG4590028) to A-R.G.

Concluding remarks and future perspectives

Based on these insights, modulating levels of PC1 could

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