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 kidney 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
2+
order, with more than 12 million cases worldwide. The function as a Ca -permeable receptor channel complex [4].
two causative genes for ADPKD, PKD1 and PKD2, Cyst formation in ADPKD requires a germline mutation
encode protein 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 cysts derived from
dominant polycystic liver disease (ADPLD). These stu-
the tubule epithelial cells.
dies showed that the rate for cyst growth was a regu- Autosomal dominant polycystic liver disease (ADPLD): a monogenic condition
characterized by the growth of bile duct-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 chromosome (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’ cilium (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 ciliopathies (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 proteins 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 Fibrocystin/polyductin (FPC): a large integral membrane protein encoded by
PKHD1, the gene 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 protein kinase 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
2+
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; polycystic liver 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 humans.
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-
mechanosensation 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 glucose trimming enzyme 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 amino acid 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 human 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 desmosomes [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, axoneme
sion of the ADPLD and ARPKD phenotypes in which the and basal body 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 calcium 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
2+
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 locus 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
epithelium is increased protein kinase A 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 pep de 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 apoptosis 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 gene expression 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-
Polycys n-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
Func onal interac on Box 1. Outstanding questions
Polycys n-1 Fibrocys n 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 prolifera on 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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