Translational research in ADPKD: lessons from animal models.

REVIEWS Translational research in ADPKD: lessons from animal models Hester Happé and Dorien J. M. Peters Abstract | Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2, which encode polycystin‑1 and polycystin‑2, respectively. Rodent models are available to study the pathogenesis of polycystic kidney disease (PKD) and for preclinical testing of potential therapies—either genetically engineered models carrying mutations in Pkd1 or Pkd2 or models of renal cystic disease that do not have mutations in these genes. The models are characterized by age at onset of disease, rate of disease progression, the affected nephron segment, the number of affected nephrons, synchronized or unsynchronized cyst formation and the extent of fibrosis and inflammation. Mouse models have provided valuable mechanistic insights into the pathogenesis of PKD; for example, mutated Pkd1 or Pkd2 cause renal cysts but additional factors are also required, and the rate of cyst formation is increased in the presence of renal injury. Animal studies have also revealed complex genetic and functional interactions among various genes and proteins associated with PKD. Here, we provide an update on the preclinical models commonly used to study the molecular pathogenesis of ADPKD and test potential therapeutic strategies. Progress made in understanding the pathophysiology of human ADPKD through these animal models is also discussed. Happé, H. and Peters, D. J. M. Nat. Rev. Nephrol. advance online publication 19 August 2014; doi:10.1038/nrneph.2014.137

Introduction

Department of Human Genetics, Leiden University Medical Center, S4‑P, PO Box 9600, Albinusdreef 2, Leiden, 2333 ZA Leiden, Netherlands (H.H., D.J.M.P.).

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common monogenetic diseases, with a prevalence ranging from 1 per 500 to 1 per 1,000 people worldwide.1 A large number of patients who require dialysis have ADPKD, but approved medication to prevent or postpone the need for renal replacement therapy in this disease is currently lacking. ADPKD is a systemic disease characterized by the progressive development of fluidfilled cysts in the kidneys, cardiovascular disease and cyst formation in the liver and pancreas. The formation of numerous cysts together with interstitial fibrosis causes chronic kidney disease in 50% of patients by the age of 55 years. Mutations in PKD1, which encodes polycystin‑1, are responsible for ADPKD in the majority of patients (~85–90%).2,3 Polycystin‑1 can interact with polycystin‑2, the protein encoded by PKD2, which is mutated in 10–15% of patients with ADPKD.2,3 The polycystins form multiprotein complexes, particularly at the cell membrane and at various subcellular locations. Correspondingly, involvement in cell–cell or cell–matrix interactions, signal transduction and mechanosensation are among their functions and a plethora of cellular changes have been observed in cystic epithelial cells and tissues. Several genetically engineered mutant Pkd1 or mutant Pkd2 mouse models have been generated to gain insight into the disease mechanisms and pathogenesis of ADPKD. These models, which enable step-by-step analysis of the pathogenesis of ADPKD, have provided

Correspondence to: D.J.M.P. [email protected]

Competing interests The authors declare no competing interests.

valuable insight into early and later progressive stages of the disease. Findings in tissues from models of early disease are complementary to those in renal tissues from patients, as kidneys are usually removed at a progressive stage of ADPKD or at end-stage renal disease (ESRD). Importantly, animal models of ADPKD also enable preclinical testing of potential therapies. A variety of rodent models with mutations in genes other than Pkd1 or Pkd2 have also been used to study various aspects of ADPKD. Some of these models have resulted from spontaneous mutations, whereas others are genetically engineered. They have enabled genetic confirmation that certain signalling pathways are critical and have led to new avenues of discovery and definition in this disease. Valuable lessons have been learned regarding the genetic mechanisms and molecular pathogenesis of polycystic kidney disease (PKD), disease initiation, progression, the role of renal epithelial injury, the involvement of various cellular structures (including primary cilia) and planar cell polarity. Moreover, studies in mouse models demonstrate the variety of signalling pathways implicated in cyst growth and potential targets for therapy. The stages of cystogenesis and disease progression in PKD are largely known; however, the detailed biochemical and cellular alterations are poorly understood. For example, what factors determine if and when cells devoid of polycystin‑1 or polycystin‑2 become cystic, and how the affected signalling pathways relate to this event, is unclear. To understand the fine, critical details in the molecular pathogenesis of ADPKD, a better understanding of the similarities and differences

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REVIEWS Key points ■■ Mice with mutations in Pkd1 or Pkd2 enable a step-by-step analysis of the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD) and valuable insights into early and progressive disease stages ■■ Mouse models have shown that mutations in genes encoding polycystin proteins cause renal cysts, but additional factors are also required for cyst formation ■■ Renal injury accelerates the rate of cyst formation, probably by increasing the chance that cysts are formed ■■ Animal studies have revealed a complex network of genetic and functional interactions between different renal cystic disease genes involved in polycystic kidney disease ■■ Differences in lifespan, metabolism, renal anatomy, involved nephron segment and genetic background mean that a mouse model cannot perfectly recapitulate human ADPKD ■■ The question of which mouse models of ADPKD are the most suitable to study disease mechanisms and for preclinical testing does not have a definitive answer

between various animal models is vital. Another challenge lies in fulfilling the need for models that closely resemble human disease. In this Review, we provide an update on the rodent models used to study the molecular pathogenesis of ADPKD and/or for preclinical testing of potential therapies. We also discuss and summarize the progress that has been made in understanding the pathophysiology of ADPKD in humans.

Phenotype and genotype of ADPKD

The hallmark of ADPKD is the formation of cysts in both kidneys. Gradual growth of existing cysts and new cyst formation over decades is coupled with a decline in renal function. By the age of 55 years, 50% of patients with ADPKD have developed ESRD and require dialysis or renal transplantation.1 In fact, ADPKD accounts for approximately 5–8% of patients with ESRD.4,5 Individual renal cysts can be detected in children with ADPKD during the first year of life, and massive cystic changes have been described in newborn children and in adolescents.6 Thus, age at onset of disease can vary greatly between individuals. In general, however, the first cysts can be diagnosed using ultrasound at around 20 years of age.7 Extrarenal manifestations of ADPKD include cysts in the liver, pancreas, seminal vesicles and arachnoid membrane.8–13 In addition, most patients have cardiovascular abnormalities, including hypertension, left ventricular hypertrophy, aortic root dilation, arterial aneurysms, heart valve abnormalities and sometimes intracranial aneurysms.14

Genetic mechanisms of disease The autosomal dominant pattern of inheritance means that patients with ADPKD carry one mutated copy and one normal copy of either PKD1 or PKD2 in all cells in their body. In renal epithelial cells, cysts can be formed when the level of the PKD1 or PKD2 gene product drops below a critical threshold. Two genetic mechanisms for cyst formation have been postulated. The first involves somatic inactivation of PKD1 or PKD2 by a ‘two-hit’ mechanism, suggesting that a germline mutation in PKD1 or PKD2 followed by a second somatic mutation in the normal copy of the gene later in life is responsible for

the phenotype. Complete loss of PKD1 or PKD2 makes cells more prone to cyst formation.15–17 Inactivating point mutations or deletions in the normal PKD1 or PKD2 allele have been demonstrated in epithelial cells isolated from the lining of single cysts.15,16 Furthermore, individuals with germline mutations in PKD1 or PKD2 and somatic mutations in the other PKD gene (transhetero zygous mutations) have been observed.17 Support for the somatic inactivation hypothesis also comes from the clonality of cysts; most cells in a cyst are derived from a single cell.15 However, the possibility that the observed somatic mutations are secondary events that give the cyst a survival or growth advantage rather than being required for initiation of cyst formation cannot be excluded. The second proposed genetic model of cyst formation suggests that haploinsufficiency (stochastic fluctuations in PKD1 or PKD2 gene dosage below a tissue-specific threshold) might be sufficient to cause cyst formation.18,19 Whether haploinsufficiency is sufficient for cyst initi ation in human ADPKD, or somatic mutations resulting in inactivation of the normal gene is always required, is unknown. Both mechanisms are likely to occur simultaneously in a PKD kidney, and probably involve inacti vating mutations and mutations that reduce protein function. Furthermore, data from murine models of PKD suggest that cyst formation requires the presence of additional factors, for example hormones or cytokines.20,21 After the first cysts are formed, cystic expansion leading to mechanical stress on the surrounding nephrons and nephron obstruction might contribute to additional cyst formation. Conceivably, ‘secondary’ cysts might form as a result of compression, obstruction and increased pressure on neighbouring nephrons rather than somatic gene mutations. Reduced levels of PKD1 or PKD2 gene expression might have a role in initiating the formation of these ‘secondary’ cysts.

Genotype–phenotype correlations Extreme phenotypic variability is seen in patients with ADPKD, ranging from renal disease and neonatal death owing to massive cystic expansion, hypertension and renal insufficiency, to normal-sized kidneys with just a few cysts and adequate renal function into old age.22 This variability, as well as the fact that most families carry a unique mutation, highlights the need for genotype–phenotype analyses.3,23 For many years the mutated gene has been known to be a major factor in determining phenotype; renal failure occurs approximately 20 years later in patients with mutations in PKD2 than in those with PKD1 mutations.23,24 Interestingly, genotyping of a large patient cohort showed that the type of mutation in PKD1, but not its position in the gene, correlates with renal survival.23 Patients carrying a missense mutation in PKD1 that results in reduced function of polycystin‑1 have a milder disease than those with a truncating mutation that results in a short, possibly unstable, inactive form of the protein.23 Thus, patients with mutations in PKD1 that result in a truncated protein are the most severely affected, followed by those with missense mutations that result in reduced functional protein. Patients who carry a PKD2 mutation

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REVIEWS Rodent models of PKD

Non-Pkd1/Pkd2 models ■ Mutations in genes other than Pkd1 or Pkd2 ■ Genes mutated in renal cystic diseases other than ADPKD

Pkd1/Pkd2 models ■ Deletion or mutations in Pkd1 or Pkd2

Reduced gene expression or missense mutations ■ Resemblance to human ADPKD phenotype, including extrarenal manifestations ■ All tissue and cell types affected ■ Some mimic human gene mutations

Germline knockout ■ Gene disruption is present in all cells ■ Embryonic lethal

Inducible ■ Timing of gene disruption can be controlled ■ Enables step-by-step analysis ■ Synchronized cyst formation ■ Rapid or slow progression dependent on age at gene disruption and percentage of cells with complete gene disruption

Gene overexpression ■ Resemblance to human ADPKD phenotype including extrarenal manifestations depending on the model Gene knockout models

Conditional knockout ■ Gene disruption is tissue or cell type specific

Noninducible ■ Gene disruption prenatal or neonatal ■ Usually early disease onset ■ In general rapidly progressive phenotype

Figure 1 | Rodent models of PKD. A schematic representation of the various rodent models of PKD and their general features. Abbreviations: ADPKD, autosomal dominant polycystic kidney disease; PKD, polycystic kidney disease.

have the mildest disease. A suggested correlation between cardiovascular abnormalities in ADPKD and the sequence position of the mutation in PKD1 could not be confirmed and is, therefore, unlikely.23,25 The clinical picture of ADPKD is sometimes complex; several families with a germline PKD1 mutation (truncating or missense) on one chromosome and an additional germline (missense) mutation in the other PKD1 allele or in PKD2, resulting in mild atypical or severe PKD with in utero presentation, have been reported.26,27 Similarly a homozygous inherited missense mutation in PKD2 caused severe early onset PKD.28 These data indicate that germline missense sequence variants in the other allele (PKD1 or PKD2) might modify disease progression. This observation is important given that one of the missense sequence variants identified in the families described has an incidence of 1%.26 The complexity of the situation increases when one considers that mutations in HNF1B, the gene mutated in patients with renal cysts and diabetes syndrome (RCAD), and PKHD1, the causative gene in autosomal recessive polycystic kidney disease (ARPKD), have been found in combination with mutations in PKD1 or PKD2.29 However, patients with mutations in multiple genes are rather unusual and comprise only a small fraction of cases, particularly of severe early onset disease. Although ADPKD is a monogenetic disorder, the disease phenotype is affected by additional factors, including genetic predisposition to hypertension,30 mutations in TCS231 and the environment. These factors might

contribute to intrafamilial and interfamilial variability in disease phenotype. The relative contributions of the various factors is not known, but given the large number of missense sequence variants in PKD1, these variants might turn out to be the strongest modifying factors.

Animal models of ADPKD Pkd1/Pkd2 models A variety of rodent models are available to study the pathogenesis of PKD and for preclinical testing of potential therapies (Figure 1). These can be categorized as Pkd1/Pkd2 and non-Pkd1/Pkd2 models (Table 1 and Table 2). The Pkd1/Pkd2 models have mutations in either Pkd1 or Pkd2 comparable to the human disease. They are genetically engineered and can be roughly subdivided into models with heritable Pkd1 or Pkd2 mutations, conditional knockout models with inducible or noninducible tissue-specific Pkd1 or Pkd2 disruption and hypomorphic models, which have reduced functional activity of either the Pkd1 or Pkd2 gene or their products. Finally, rodents with overexpression of Pkd1 or Pkd2 have been generated. Gene knockouts In Pkd1 or Pkd2 gene knockout models one or more exons are typically deleted32–37 or disrupted.38–40 These mutations are assumed to lead to the loss of expression of the protein. Although called knockouts, these models are not real genetic knockouts in which a gene is entirely deleted (that is ‘null’ mutants). Homozygous mutations in Pkd1 or Pkd2 are embryonic lethal in mice as they result in cardiovascular defects (oedema, vascular leaks and rupture of blood vessels). In addition, homozygous Pkd1 or Pkd2 knockout mice have massive cyst formation in the pancreas, kidneys and liver. By contrast, mice with heterozygous mutations are viable and have very mild cystic phenotypes in the kidneys, liver and sometimes in the pancreas. Models with tissue-specific gene disruption To bypass the embryonic lethality of homozygous Pkd1 or Pkd2 mutations and study tissue-specific effects, many conditional Pkd1 and Pkd2 knockout models have been generated using the Cre–loxP recombination system (Figure 2). In these models, the gene is disrupted only in those tissues or cell-types in which a tissue-specific promoter driving Cre recombinase is active. Various tissue and cell-type specific knockouts have been obtained by crossing mice with ‘floxed’ exons (that is, the target exon flanked by loxP sequences) in Pkd1 or Pkd2 with mice that have a transgene that expresses Cre under the control of a widespread or tissue-specific promoter.35–46 Importantly, specific disruption of Pkd1 or Pkd2 in the renal epithelium has been used to model the second-hit hypothesis and to study cystogenesis.35,41,42,45–47 Furthermore, Cre-expressing transgenic mouse lines are available that target specific nephron segments; that is, only the proximal tubules,46,47 the proximal and distal parts of the nephron41,48 or the principle or intercalating cells in the collecting ducts.49 In addition, SM22Cre, Tie2Cre and



REVIEWS Table 1 | Pkd1 and Pkd2 models of polycystic kidney disease* Model

Examples‡

Characteristics

Pkd1

Pkd1(del34)32,91 Pkd1(del43)33 Pkd1(del17)39 Pkd1(del2–6)34 Pkd1(del2–11)35 Pkd1(Bdgz)36 Pkd1(null)37 Pkd1(βgal–null)146 Pkd1(del17−21/geo)39

Homozygotes embryonic lethal Pkd1 deletion in all somatic cells

Pkd2

Pkd2(null)38,170 Pkd2(LacZ)40 Pkd2(d3)46

Homozygotes embryonic lethal Pkd2 deletion in all somatic cells

Knockouts

Conditional knockouts Pkd1

Pkd1flox/−:Ksp-Cre45 Pkd1flox:γGT.Cre47 Pkd1cond:MMTV.Cre43 Pkd1flox:NestinCre171 Pkd1flox:ATCB-Cre171 Pkd1flox:Pkhd1-Cre45 Pkd1lox:Tie2Cre50§ Pkd1con/–:Tie2-Cre51§ Pkd1lox:SM22Cre50§ Pkd1cond/–:Meox2-Cre51§ Pkd1KO/cond:Sm22α-Cre52§

Tissue, cell type and/or nephron-segment specific knockout of Pkd1 Timing of Pkd1 inactivation depends on the activity of the promoter that drives Cre expression Mutants with a floxed allele combined with a null allele can be produced Cystic phenotype is usually rapidly progressive

Pkd2

Pkd2F:Pkhd1-Cre131 Pkd2f3:γGT.Cre46 Pkd2fl:Wnt1Cre172§ Pkd2cond/cond:Meox2-Cre51§

Tissue, cell type and/or nephron-segment specific knockout of Pkd2 Timing of Pkd2 inactivation depends on the activity of the promoter that drives Cre expression Mutants with a floxed allele combined with a null allele can be produced Cystic phenotype is usually rapidly progressive

Inducible (conditional) knockouts Pkd1

Pkd1cond:R26CreER42 Pkd1lox:tam-KspCad-CreERT235,41 Pkd1flox:Mxi-Cre44 Pkd1cond:tam-Cre/Esr142 Pkd1flox:pCX-CreER173 Pkd1tm1Gztn:R26CreERT2174

Tissue, cell type and/or nephron-segment specific knockout of Pkd1 Pkd1 inactivation (Cre activity) can be induced by treatment with tamoxifen, interferon or doxycycline Severity and disease progression depends on age at Pkd1 inactivation, extent of Pkd1 inactivation and extent of renal injury (if applicable)

Pkd2

Pkd2fl:pCxCreER173§ Pkd2f3:Pdx1-Cre-ER™46 Pkd2f3:Mx1-Cre46

Tissue, cell type and/or nephron-segment specific knockout of Pkd2 Pkd2 inactivation (Cre activity) can be induced by treatment with tamoxifen, interferon or doxycycline Severity and disease progression depend on age at Pkd2 inactivation, extent of Pkd2 inactivation and extent of renal injury (if applicable)

Reduced expression

Pkd1(nl)19,88 Pkd1(L3)54 Pkd1miR TG61

Neonatal or perinatal onset Cyst formation most prominent in distal tubules and collecting ducts Progressive phase is characterized by extensive fibrosis and inflammation

Reduced or unstable expression

Pkd2(WS25)170 Pkd2(WS25/WS186)170,175 Pkd2(nf3)46

Cystic phenotype in kidneys, liver and pancreas Unstable Pkd2WS25 allele supports two-hit hypothesis, recapitulates human phenotype

Missense mutation

Pkd1(RC)55 Pkd1(V)53 Pkd1(mBei)56

Phenotypic severity depends on mutation Various nephron segments involved

Hypomorphic

Overexpression of human protein PKD1

PKD1(extra)60 PKD1TAG58 hPKD1 TPK57

Cystic kidneys and extrarenal manifestations, depending on the model

PKD2

hPKD2 TG59 hPKD295 hPKD2(1/703)176

Cystic kidneys and extrarenal manifestations, depending on the model

*An extensive, although not complete list of Pkd1 and Pkd2 mutant mice and a summary of their phenotypes is available online.177 ‡Transgenic mouse or rat nomenclature listed according to original source. §Cre activity in nonrenal epithelial cells. Abbreviations: TG, transgenic; TPK, transgenic polycystic kidney.



REVIEWS Table 2 | Non Pkd1/Pkd2 models of polycystic kidney disease. Model

Construct

Mutated gene

Protein

Human orthologue

Human disease

Pck rat178,179

NA

Pkhd1180

Fibrocystin*

PKHD1

ARPKD180

Pkhd1 mouse

Pkhd1 Pkhd1(del2/del2)65 Pkhd1(del4/del4)64,66 Pkhd1(e15GFP_16)67,68 Pkhd1(lacZ/lacZ)70 Pkhd1(del3–4/del3–4)71 Pkhd1(ex40)69

Pkhd1

Fibrocystin*

PKHD1

ARPKD180

Hnf1β mouse

KspCre Hnf1β(F/F)111 Alfp–Cre Hnf1β(F/F)181 (in liver) Pkhd1–CreHnf1β(F/F)111,182 HNF1β(lacz/+)183‡

Hnf1β

Hepatocyte nuclear factor 1β

HNF1β TCF2

Renal cysts and diabetes184

Bpk mouse185

NA

Bicc180

Bicaudal C

BICC1

Renal cystic dysplasia79

Jcpk mouse

NA

Bicc1

Bicaudal C

BICC1

Renal cystic dysplasia79

Cpk mouse187,188

NA

Cys1187

Cystin

NA

NA

Inv mouse

NA

Invs

Inversin

NPHP2

Nephronophthisis73

Pcy mouse

NA

Nphp3

Nephrocystin‑3

NPHP3

Nephronophthisis,76 Meckel–Gruber syndrome193

Kat mouse, kat2J/kat2J mouse194

NA

Nek1195

Nek1

NEK1

SRPS type II,196 SRPS type III197

Jck mouse198

NA

Nek875

Nek8

NEK8/NPHP9

Nephronophthisis74,75

Orpk mouse (also known as TgN737Rpw)199

NA

199

Tg737

Polaris

NA

NA

Tg737(δ2-3βGal) mouse200

NA

Tg737200

Polaris

NA

NA

Cy rat (also known as Han:SPRD-Cy)201

NA

Anks6 (Pkdr1)202

Ankyrin repeat and SAM domaincontaining protein 6

ANKS6

Nephronophthisis203

Wpk rat204

NA

Mks3205

Meckelin

MKS3205§

Meckel–Gruber syndrome205||

186

189–191 76,192

(null)63

80

190 76

*Also known as polyductin. ‡No renal cyst formation reported. §Also known as TMEM67. ||Mutations in the disease gene are also present in other ciliopathies. Abbreviations: ARPKD, autosomal recessive polycystic kidney disease; NA, not applicable; SRPS, short-rib polydactyly syndrome.

Meox2-Cre mice have been used to achieve Pkd1 or Pkd2 disruption in smooth muscle cells, endothelial cells and placental labyrinth respectively.50–52 Cre–loxP recombination systems can be non-inducible or inducible. In the non-inducible Pkd1 or Pkd2 conditional knockout models, renal cyst formation is generally early onset and progresses rapidly, as gene inactivation takes place as soon as the promoter driving Cre recombinase expression becomes active (usually during development). The inducible system has the advantage that gene disruption is achieved only after enabling nuclear entry of a modified Cre recombinase (for example by treatment with tamoxifen) at a chosen time point during postrenal development or in adult life. The inducible tissue-specific conditional knockout models enable a well-controlled step-by-step analysis of pathogenesis, including the early stages of cyst formation (Figure 3). Models with reduced Pkd1 or Pkd2 activity Hypomorphic mice have reduced Pkd1 or Pkd2 expression in all cells in which the gene is expressed, or carry a missense mutation that reduces protein expression and function.19,46,53–56 In contrast to homozygous Pkd1

or Pkd2 knockout models, hypomorphic mice survive embryonic development. These mice might also have extrarenal manifestations. Mice with genetic mutations that result in an amino acid substitution are particularly helpful to gain mechanistic insight into the function of protein domains. For example Pkd1V/V knockin mice, which have a missense mutation in the G protein-coupled receptor proteolysis site of polycystin‑1, express increased levels of uncleaved polycystin‑1 and have a postnatal renal cystic phenotype, indicating a critical but restricted role for cleavage of this protein in preventing cyst formation.53 Another knockin model, the Pkd1RC/RC mouse, carries a mutation that results in an amino acid substitution similar to a mutation identified in patients that is not located in a previously identified polycystin‑1 domain. These mice develop an ADPKD phenotype and functional studies suggest that the mutation results in improper protein folding and reduced cellular transport of polycystin‑1.55 Pkd1 or Pkd2 overexpression A variety of models do not fit within the above categories; mice that overexpress human or mouse PKD157,58



REVIEWS

Cre

Exon 1

Exons 2–11

Kidney

Cre

Exons 12–46 Ksp-Cad

Pkd1

lox

IoxP

IoxP

Cre

Cre Excision of exons 2–11 of Pkd1 exclusively in the kidneys

Exon 1 Exons 12–46 Pkd1del

Figure 2 | Tissue-specific Pkd1 knockout using the Cre–loxP recombination system. Using gene targeting, mice are generated in which loxP sites (arrowheads) are inserted on each side of an essential part of a gene, for example Pkd1 or Pkd2. These 34 base pair loxP sites are recognized by Cre recombinase. The loxP transgenic mouse is crossed with a transgenic mouse carrying the Cre gene under the control of a tissue-specific or cell-type-specific promoter. In the resulting offspring, Cre recognizes, aligns and brings together the loxP sites, resulting in excision of the loxP-flanked (floxed) DNA along with two half loxP sites. This leaves a loxP site in the genome. The system was further refined by modifying Cre so that it can only enter the nucleus and mediate recombination in the presence of a chemical agent such as tamoxifen. This refinement has enabled the generation of conditional inducible gene knockouts. Abbreviation: Ksp-Cad, kidney-specific cadherin.

or PKD2, 59 those that overexpress the extracellular domain of Pkd160 and those that express an artificial miRNA that manipulates Pkd1 transcript levels.61 Studies using models with overexpression or reduced expression of polycystin-1 or polycystin-2 have shown that proper polycystin function requires restrained expression levels within a critical window.57–59,61

Non-Pkd1/Pkd2 models The non-Pkd1/Pkd2 models have spontaneous or genetically engineered mutations in renal cystic disease genes other than Pkd1 or Pkd2. These include models with mutations in Pkhd1, the orthologue of the causative gene in ARPKD in humans. An example is the PCKrat, which carries a spontaneous mutation in Pkhd1. Identification of the mutated gene locus in this model contributed to identification of the mutated gene in patients with ARPKD.62 The PCK-rat has a renal and hepatic cystic disease with mild portal fibrosis. By contrast, most of the genetically engineered mouse models of ARPKD seem to be protected from renal cyst formation and have only mild tubular dilations with increasing age, although they have a strong hepatic cystic disease.63–69 Only two of these models clearly have renal cysts70,71 (Table 2). As Pkhd1 undergoes complex spli cing, the possibility that small protein products are generated that have functional effects cannot be excluded.

However, the literature is not consistent regarding the splice pattern of this gene.63,72 Several non-Pkd1/Pkd2 models have been investigated for many years. Mutations have been pinpointed to certain genes in animal models and patients have been identified with mutations in the orthologous human gene manifesting a disease that involves renal cyst formation. For example, the genes mutated in the human counterparts of Inv, Jck and Pcy mice are the NPHP2, NPHP9 and NPHP3, respectively, which are mutated in patients with nephronopthisis.73–76 Although these models have been used to study ADPKD, NPHP has a very different phenotype. In ADPKD, the kidneys are massively enlarged as a result of proliferating and expanding cysts, whereas in NPHP the kidneys are similar in size to a normal kidney with evidence of substantial apoptosis.68 Evidence exists, nevertheless, that some of the NPHPrelated mouse models have enlarged kidneys with proliferating cysts. The reason for these phenotypic differences is not entirely clear, but age of onset, genetic background and homozygosity are probably contributing factors.77,78 Another example is the human gene BICC1, which encodes bicaudal C. This gene was found to be mutated in two children diagnosed with cystic renal dysplasia.79 The human mutation is orthologous to the Bpk/Jcpk allelic mutation in mice (Table 2). However, Bpk and Jcpk mice carry a different type of mutation in Bicc1.80 Given the different genes involved in Pkd1/Pkd2 versus non-Pkd1/Pkd2 models, it is plausible that the first event in pathogenesis—the initiation of cyst formation—may not be identical but as the disease progresses, the mechanisms become increasingly similar and might involve the same cellular mechanisms that stimulate the growth of cysts, fluid accumulation and/or fibrosis (Figure 3). A potentially common pathway is indicated by the effectiveness of vasopressin receptor antagonists in ameliorating disease in Pkd1/Pkd2, Pcy mice and the PCK rat.81–84 Downstream signalling seems to be common to these models, however, treatment with drugs that target other signalling cascades might have different effects in these models, as seen with a epithelial growth factor receptor antagonist.85,86 Overall a comprehensive characterization of different models in parallel is required for more insight into common pathways.

Choice of appropriate model Each model of PKD has its own characteristics, for example age of onset and rate of disease progression, the affected nephron segment, the number of affected nephrons, synchronized or unsynchronized cyst formation and the extent of fibrosis and inflammation. These characteristics are strengths or weaknesses depending on the study questions to be answered. For example, to investigate alterations in gene or protein expression in epithelial cells during cystogenesis (early stages), the most appropriate model might have progressive and synchronized cyst formation without the involvement of extensive fibrosis and inflammation. Models that have extensive fibrosis and inflammation are more useful



REVIEWS

Normal allele and germline mutation allele in cells

Initiation Second hit or haploinsufficiency in a subset of cells

Growth and expansion Hyperproliferation—growth stimulating factors Altered apico–basal polarity and PCP Fluid transport ECM production Local tissue injury

Progression Fibrosis Apoptosis Collapse of cysts Infiltration of inflammatory cells

Injury

Common pathogenesis Proliferation of cells Presence of growth factors

Completed repair Grossly normal tubules Slightly altered cell integrity

Figure 3 | Disease progression in ADPKD. In patients with ADPKD, all tubular epithelial cells contain one mutated and one unaffected PKD1 or PKD2 allele. When expression of normal polycystin-1 and/or polycystin-2 drops below a critical threshold (as a result of a ‘second-hit’ or by chance; red cells) permissive conditions are created in which epithelial cells can become cystic. The time until a cyst forms varies (dashed arrows) and might be accelerated by renal injury. Tissue repair in the absence of polycystin‑1 or polycystin‑2 results in the creation of new cells with altered structural integrity that are sensitive to cyst formation. Once cysts are formed, they impose continuous stress onto surrounding tissue, resulting in local injury, which is probably accompanied by synthesis of growth factors and cytokines, triggering additional cyst formation. The next phase in cystogenesis is characterized by the growth and expansion of cysts owing to hyperproliferation and altered fluid transport as a result of further altered activation of various signalling pathways. During disease progression, the renal tissue becomes increasingly fibrotic and immune infiltrates accumulate. Although the initial stages in cyst formation might differ between animal models, the pathogenetic mechanisms become increasingly similar as the disease progresses and involves common pathways. Abbreviations: ECM, extracellular matrix; PCP, planar cell polarity.

to study the relative contribution of these processes to cystogenesis or disease progression. For preclinical testing, additional factors such as the pharmacological properties of the compound being tested, as well as target protein expression in different nephron segments and cystic epithelia should be taken into account. Clearly the choice of an appropriate model with which to study the pathogenesis of PKD and for preclinical studies is not straightforward.

Insights from rodent models Genetic mechanisms A variety of Pkd1-mutant and Pkd2-mutant mouse models that support proposed pathogenetic mechanisms in human disease have been created. The first supporting evidence for the two-hit hypothesis was provided by the Pkd2ws25/– model.38 In these mice, which have one unstable Pkd2 allele and an inactivating mutation in the other Pkd2 allele, somatic rearrangements accelerate renal cystic disease compared to mice that have only one inactivating mutation. Moreover, mice that carry one inactivating Pkd1 or Pkd2 mutation have only a very mild phenotype, whereas most patients who carry one inactivating PKD1 mutation develop PKD and ESRD.32–40 The lifespan of mice is probably too short for

heterozygotes to develop PKD; that is, the likelihood is lower that somatic mutations will occur and/or that the level of Pkd1 or Pkd2 will drop below a critical threshold for cyst formation (Figure 3). Further support for the two-hit hypothesis was obtained using genetically modified mice with somatic mutations that inactivate Pkd1 or Pkd2 in renal epithelial cells, resulting in renal cystic disease.35,42,45,46 Furthermore, inducible models in which Pkd1 or Pkd2 disruption can be activated at various time points indicate that gene disruption in adults can cause PKD.35,42,43

A critical window for Pkd1 and Pkd2 expression Mice with reduced levels of functional polycystin‑1 or polycystin‑2 are viable and have PKD.19,54,55,61,87,88 Although not formal proof of a gene-dosage effect (that is, the haploinsufficiency hypothesis), these mice indicate that reduced polycystin levels result in cyst formation. They also have extrarenal disease manifestations that are found in patients with ADPKD, such as cysts in the liver and pancreas and cardiovascular abnormalities, thereby corroborating the systemic nature of the disease.19,54,55,87,88 Whether extrarenal manifestations occur more frequently in patients who carry two somatic missense mutations or in the small subgroup of patients with an



REVIEWS inactivating germline mutation together with a missense somatic mutation requires further analysis.26–28 In the PKD phenotype in which there is blood vessel involvement, modifying genes in the genetic background seem to be very important; hypomorphic Pkd1nl/nl mice have severe dissecting aneurysms at 3 weeks of age depending on the genetic background of the mouse line.19,88,89 Furthermore, in patients with ADPKD the risk of cere bral aneurysms is higher in families with a history of aneurysms than in those without a similar family history, probably owing to the presence of m odifying genes.90 Homozygous Pkd1-deficient and Pkd2-deficient mice die during embryonic development or directly after birth because of severe cystic disease, vascular defects and/ or abnormalities of the placental labyrinth.38,91 Human embryos with homozygous inactivation of PKD1 or PKD2 are probably also not viable. Nonetheless, human or mouse embryos that are transheterozygous for a PKD1 mutant allele and a PKD2 mutant allele are viable but have more severe phenotypes than those carrying single gene mutations.92,93 In addition, mice that carry an inactivating germline mutation combined with a germ line missense mutation have a more severe phenotype than mice that carry only an inactivating mutation.55 These data, which strongly support the hypothesis that a somatic mutation in the previously normal allele or in the other PKD gene can modify disease progression, corroborate the observations made in humans.26 Both reduced and increased expression of Pkd1 or Pkd2 can lead to a renal cystic phenotype;57–59,94 however, overexpression of Pkd2 does not cause cystic disease in all models.95 These data indicate that for normal renal development, Pkd1 and Pkd2 expression levels need to be tightly maintained within a critical window. This observation is explained by the fact that the proteins form multiprotein complexes for which the stoichio metry will be altered when levels of the individual proteins are too high or too low. A variety of models have been proposed to explain how an imbalance between the levels of polycystin‑1 and polycystin‑2 might be pathogenetic. These include, for example, antagonistic effects of G protein-coupled receptor signalling, mechano transduction and functional consequences of altered polycystin‑2 expression or phosphorylation.60,96–99

Cyst formation in PKD Kinetics The rate of progression of cystogenesis in mice with an inducible Pkd1 deletion depends on their age at Pkd1 disruption; rapid progression occurs in young mice, whereas very slow progression of the disease occurs in adult mice.35,42 This difference correlates with the developmental stage and proliferative status of the kidneys at the moment of gene disruption.20,35,42 A similar phenomenon seems to occur in the human disease. In general, in adult patients with ADPKD the number and size of cysts increases over decades and cysts grow constantly but at different rates, from approximately 2–70% per year.6,100,101 The renal cysts detected in newborns with severe earlyonset ADPKD, however, must have grown in utero at

much higher rates than those observed in adults.6 Thus growth promoting conditions during renal development probably accelerate cystogenesis. In most animal models, deletions in the Pkd1 gene are relatively synchronized in much larger numbers of cells than in human ADPKD. Pkd1 disruption in a high percentage of cells in young adult mice resulted in tubular dilations after only 1–2 months.20 Interestingly, scattered Pkd1 disruption at the same age leads to a dormant period of roughly 6 months without tubular dilation, which is followed by a sudden massive and rapid onset of severe PKD. Cysts form in clusters and cyst-related signalling is altered in tubules near cysts. Conceivably the sudden shift in cystogenesis is caused by the initial cysts that trigger a ‘cystic snowball’ effect driving the formation of new cysts and progressing to severe PKD.102 Growing cysts probably consist of both mutant and ‘wild-type’ cells, at least in the early stages of disease. A germline chimeric mouse model that combines knockout mutant (Pkd1del2–6/del2–6) alleles and wild type (Pkd1+/+LacZ+) alleles elegantly supports this hypothesis.103 At early stages, the cysts in these mice consisted of many wild-type allele containing cells. In time these cells almost disappeared and the knockout cells overgrew. Interestingly, wild type but not knockout cells had increased apoptosis, which seems to be related to increased expression of proapoptotic pJNK and reduced expression of antiapoptotic Bcl–X l. 103 Although this model might not reflect the natural situation it highlights differences in signalling that might occur within early cysts. Role of renal injury Cellular proliferation and appropriate differentiation are required for tissue regeneration after injury; therefore, it has been hypothesized that renal injury could accelerate cyst formation in mouse models of ADPKD. Although heterozygous Pkd1+/-and Pkd2+/- mice have increased sensitivity to ischaemia–reperfusion injury, they do not have an overt PKD phenotype.104,105 By contrast, in conditional knockout models renal injury strongly accelerates the rate of cyst formation.20,48,106 Renal injury, however, does not result in unrestricted cell proliferation as observed in a Pkd1-deletion model treated with the nephrotoxin 1,2-dichlorovinyl-cysteine, in which cell proliferation returned to baseline levels after tissue repair. 20 Thus, accelerated cyst formation is not the result of uncontrolled cell proliferation, but is probably the result of altered integrity of the cells that are newly formed during the repair process in the absence of Pkd1, rendering them more susceptible to cyst formation.20 Increased sensitivity of cells that lack Pkd1 to cytokines or growth factors might be a crucial factor, as demonstrated in the double Brattleboro/Pkhd1–/– rat that lacks vasopressin and forms hardly any cysts unless the hormone is directly administered.21 Other studies reveal a progressive PKD phenotype after renal injury,48,106 leading to the proposal that renal injury is the ‘third-hit’ needed for cystogenesis.106 The Brattleboro/Pkhd1–/– rat study mentioned above21 as well



REVIEWS as more recent data strongly oppose this theory.20,102 The severity and type of injury, perhaps in combination with the age of the mice at the time of gene disruption and the nephron segment involved might, however, explain rapid cyst formation in at least one of these models.106 Renal injury is accompanied by a combination of processes, including repair-associated proliferation, secretion of growth factors and inflammation. To study the role of cell proliferation in cystogenesis, the transcription factor homeobox protein cut-like 1 (Cux‑1) was ectopically expressed in an intraflagellar transport protein 88 homologue (Ift88–/–) mutant. Cell proliferation was increased but overexpression of Cux‑1 did not result in accelerated cyst formation, suggesting a role for processes additional to renal injury and repair, other than cell proliferation.107 Although acute kidney injury is not the third-hit needed for cystogenesis, it clearly accelerates cyst formation, probably by increasing the chance that a cyst is formed.20,48,102 Regardless of the underlying mechanism the potential for renal injury to accelerate cystogenesis is relevant to the human disease. Patients with ADPKD sometimes have recurrent urinary tract infections that might be associated with rapidly progressive disease, although large scale studies have not been carried out.108,109 In addition, toxic waste products inherent to the function of the kidneys might cause local tissue injury. Over time these injuries might accumulate and contribute to impaired renal function and renal ageing. In most individuals renal function declines with age, resulting in a decrease in glomerular filtration rate of ≤50% by the age of 80–90 years.110 Renal ageing is a multifactorial process whereby several key mediators, such as chronic inflammation, oxidative stress and impaired capacity for kidney repair, have a substantial role.110 Conceivably superimposition of ‘ageing injury’ on top of genetic mutations in PKD could be an important factor in determining the clinical course of the disease. Mutations in either PKD1 or PKD2 prime the kidneys for cystogenesis. Somatic mutations and stochastic processes increase the chance that a cyst is formed. What determines if and when cells carrying the mutant alleles become cystic, and how the affected signalling pathways relate to this process, is so far unknown. Once a cyst is formed it imposes continuous stress on surrounding tissue, resulting in local tissue injury that is probably accompanied by increased synthesis of growth factors and/or cytokines, leading to additional cyst formation. Biological variation in the exposure of an individual to environmental factors, renal injury and renal ageing might accelerate the entire process, thereby contributing to variation in severity of the PKD phenotype between patients.

Cystic disease gene interactions Various studies have revealed a complex network of genetic and functional interactions between different cystic disease genes. For example, kidney-specific inactivation of Hnf1b leads to a PKD phenotype in mice and, interestingly, affects the expression of several genes

involved in cystic disease.111 Expression of these genes (Pkhd1, Pkd2 and Umod) is directly controlled by Hnf1β, indicating a transcriptional hierarchy between the protein and these genes.111,112 Hnf1β seems to be a critical regulator of a genetic cascade that controls proliferation and differentiation of renal tubular epithelial cells. This genetic interaction also explains the increased severity of the PKD phenotype in patients who carry a mutation in HNF1B and a mutation in an ADPKDassociated or ARPKD-associated gene.29 Another genetic interaction has been reported between Bicc1 (the gene mutated in Bpk and Jcpk mice) and Pkd2. Bicc1 encodes the RNA-binding protein bicaudal C homologue 1, which regulates the stability of Pkd2 mRNA and its translation efficiency.113 The protein–protein interaction between p olycystin‑1 and polycystin‑2 has been known for a long time.114,115 An interaction between polycystin‑2 and fibrocystin (also known as polyductin), which modulates p olycystin‑2 channel activity has also been found.64,67 Fibrocystin is encoded by the ARPKD gene Pkhd1. Crossbreeding of mouse lines revealed that reduced Pkd2 gene dosage can accelerate renal cystic disease in a Pkhd1 model, although this has not been observed in another model.64,67 However, reduced Pkd1 gene dosage led to a more severe phenotype in all Pkhd1 mutant mice studied so far.64,71 Functional interactions between the proteins encoded by PKD genes (PKD1, PKD2 and PKHD1) and polycystic liver disease (PLD) genes (PRKCSH and SEC63) add an extra level of complexity. PLD occurs frequently as an extrarenal manifestation of ADPKD, but also exists as a distinct, dominantly inherited genetic entity without kidney cysts. The autosomal dominant PLD genes, PRKCSH and SEC63, are involved in protein transport and quality control in the endoplasmic reticulum. Mouse studies indicate that these genes are required for adequate expression of polycystin‑1 and polycystin‑2.64,116–118 The formation of liver cysts and virtual absence of kidney cysts in patients with PLD could be related to differences in the expression levels of functional polycystin‑1. In summary, mouse models highlight the complex network of genetic and functional interactions between different cystic disease genes. In the future, new combinations of mutations might be identified in patients with renal cystic disease.

The role of primary cilia Polycystin‑1 and polycystin‑2, as well as many other proteins implicated in cystic diseases in humans or animal models, are expressed in the cilium and/or basal body. Primary cilia are evolutionarily conserved structures that protrude from the apical surface of cells and are involved in mechanosensing and chemosensing. Altered positioning of the basal body and signalling through cilia might be involved in increased cell proliferation, as proper docking and positioning of the basal body in the apical membrane is required for correct signalling by cilia.119 In inducible Pkd1-knockout mice, the position of the basal body is altered even in precystic stages and cell proliferation is slightly increased.20,35 Polycystin-regulated



REVIEWS cilia signalling has been postulated as a requirement to keep cells in their differentiated and quiescent state, and disturbed cilia function might lead to increased cell proliferation early in PKD.20,120 This hypothesis is supported by a reduced cystic phenotype observed in Pkd1 or Pkd2 mutant mice in the absence of cilia.121 A cilia-regulated signalling pathway that controls cell proliferation seems to be restrained by polycystin‑1 and/or polycystin‑2, but is activate in the absence of these proteins. In the absence of cilia, this signalling pathway is less effective, resulting in reduced cystogenesis.121 However, the components in this cascade are not yet known. Ca2+ signalling might be involved, owing to the fact that flow-induced cilia signalling is disrupted in cultured embryonic cells from Pkd1–/– mice, resulting in reduced release of Ca2+ from intracellular stores.122 Wnt and mTor signalling pathways might also be involved, but further research is needed.123,124 How do we reconcile reduced cystogenesis in the absence of cilia with delayed cyst formation in the kidneys of mice with inducible scattered Pkd1 disruption that become cystic after a long dormant phase?102 Perhaps the mechanism of cystogenesis first needs to be triggered before the polycystins can function as a ‘brake’, although polycystin‑1 and/or polycystin‑2 might not be the only ‘brake’ placed on the signalling pathway that controls cell proliferation. Alternatively, a higher proportion of cells with Pkd1 or Pkd2 disruption versus a lower proportion of cells with Pkd1 or Pkd2 disruption might increase the relative chances of cystogenesis, and also create a different cellular context with more Pkd1-negative cells in the local tissue environment. Overall, these data indicate that the requirement for polycystin‑1 and/or polycystin‑2 in the primary cilium to restrain proliferation-related signalling pathways, is context dependent.

Planar cell polarity In multicellular organisms, cells are polarized in the plane of the epithelial sheet. This property—planar cell polarity (PCP)—is recognized in some cell types by the orientation of hairs or cilia. In epithelial cells the apical– basal polarity is perpendicular to the planar polarity. The pathway that regulates planar polarization of cells is noncanonical Wnt signalling. PCP signalling is needed during renal development to ensure the correct morphology and diameter of the nephrons. Furthermore, the cilium or basal body—centrosome complex seems to have a role in determining PCP. Defects in the molecular pathways involved in PCP signalling might result in an increase in renal tubular diameter and it is hypothesized that defective PCP signalling might cause cyst formation in ADPKD.125,126 Indications for defects in PCP signalling in various models of renal cystic disease have been demonstrated using orientated cell division (OCD) or positioning of the centrosome.20,48,126–129 Furthermore, cystogenesis is seen in mice carrying a mutation in the PCP signalling pathway component protocadherin Fat4.130 However, not all studies confirm PCP signalling defects in undilated precystic tubules of Pkd1 or Pkd2 mutant mice,

suggesting that in these models misorientated cell division might not be involved in cystogenesis.131 In a model of ARPKD, misorientated cell division was observed but mice failed to develop dilated tubules or cystic kidneys.131 In this model misorientated cell division seems not to be sufficient to result in renal cysts; however, it should be noted that although PCP and OCD are closely related, they are not the same process. Additionally, establishment or maintenance of tubular architecture might not always be achieved using OCD, therefore, epithelial cell proliferation might not necessarily involve this process. Overall, the role of PCP in cystogenesis is unclear and complex.120

Fibrosis and inflammation The renal epithelium in ADPKD is characterised by epithelial cell proliferation, abnormal fluid secretion and increased extracellular matrix synthesis. Cyst growth and expansion is accompanied by interstitial fibrosis. Kidneys from patients with advanced ADPKD are characterized by cystic tissue and fibrotic areas with inflammation, but also regions with normal renal morphology.132–134 Although fibrosis and inflammation are common in patients, many PKD mouse models show these features to only a limited extend. Several models, however, do have inflammation and/or fibrosis, thereby resembling the more advanced stages of human ADPKD.46,53–55,88 These mice also have regression of renal volume owing to collapsing cysts that become fibrotic.55,88 Fibrosis results not only from molecular alterations in the cystic renal epithelium that directly affect interstitial cells, but also indirectly through secretion of proinflammatory cytokines that might attract inflammatory cells. These cells include macrophages that have been shown promote cyst formation. 135,136 In addition, cytokines activate the innate immune system,137,138 suggesting a ‘positive-feedback’ cycle, whereby interstitial inflammation is influenced by the pathological and molecular features of PKD and vice versa.139 Signalling in cystic epithelia In renal epithelial cells, deregulated PKD1 or PKD2 expression can result in cystogenesis. Both polycystin‑1 and polycystin‑2 are expressed in several cellular compartments and form various multimeric protein complexes, whereby they modulate several signalling pathways that in concert control essential cellular functions such as proliferation, apoptosis, cell adhesion and differentiation. Consequently, a large variety of cellular changes in cyst-lining cells have been observed. These changes include alterations in apical–basal polarity, PCP, OCD, cell proliferation, extracellular matrix production, fluid transport and cellular metabolism.120,140–142 Polycystin‑1 and/or polycystin‑2 transmit extracellular signals to the nucleus via multiple signalling pathways. A substantial body of evidence indicates that reduced Ca2+ influx, increased cAMP levels and aberrant Ras– Raf–MEK–ERK activation have roles in the growth of cysts.143,144 Other signalling mechanisms involve activation of G proteins, mTOR, PI3-kinase–Akt, Jak2–STAT1/



REVIEWS STAT3/STAT6, NFAT, NF‑κB or Wnt and Hippo signalling pathways.20,142–157 To a large extent these molecular pathways are involved in cell proliferation and thereby in cyst expansion. The possibility that some of these signalling pathways, or second messengers such as Ca2+, are involved in the early stages of cystogenesis cannot be excluded.

Preclinical testing

Therapeutic strategies in ADPKD focus on inhibiting cell proliferation and fluid secretion by targeting cellular signalling. Given the tight regulation of PKD1 and PKD2 expression, the complexity of the genes involved and the fact that most families are carriers of different mutations, strategies to directly target the genes are not currently an option. The complex network of signalling pathways that are deregulated in cystic kidneys provide many potential targets for therapeutic intervention. Consequently, an increasingly long list of compounds have been designed to target these pathways. Around 30 of these compounds have been tested in preclinical studies using a variety of Pkd1/Pkd2 and non-Pkd1/Pkd2 models.157–159 However, not all of these compounds have similar effects when tested in different models.159 Given the differences in life span, metabolism, renal anatomy, involved nephron segment and genetic background, a mouse model cannot perfectly mimic human ADPKD, a disease that develops and/or progresses over decades. Furthermore, long-term drug therapy might cause adverse effects that become difficult to tolerate. Although potential long-term adverse effects are not moni tored in mice, these models are indispensable to obtain mechanistic insights, screen drugs and test therapeutic effectiveness. A few drugs that target epithelial signalling pathways such as mTOR inhibitors, V2R-antagonists and somatostatin analogues, have been evaluated in clinical trials. At present, mTOR inhibitors are not proven to be effective in human ADPKD trials,160,161 but results for V2R-antagonists, and somatostatin analogues are certainly encouraging.162–166 Studies of the dual Src and Abl kinase inhibitor bosutinib167 and triptolide168 (used in traditional Chinese medicine), that restore cytosolic Ca2+ concentrations, are underway.169 The first clear mechanistic insights and direct indications of efficacy for V2R antagonists were 1.

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Torres, V. E., Harris, P. C. & Pirson, Y. Autosomal dominant polycystic kidney disease. Lancet 369, 1287–1301 (2007). Peters, D. J. M. & Sandkuijl, L. A. Genetic heterogeneity of polycystic kidney disease in Europe. Contrib. Nephrol. 128–139 (1992). Rossetti, S. et al. Comprehensive molecular diagnostics in autosomal dominant polycystic kidney disease. J. Am. Soc. Nephrol. 18, 2143–2160 (2007). Martinez, V. et al. Renal replacement therapy in ADPKD patients: a 25-year survey based on the Catalan registry. BMC Nephrol. 14, 186 (2013). Collins, A. J. et al. US Renal Data System 2010 Annual Data Report. Am. J. Kidney Dis. 57 (Suppl. 1), e1–e526 (2011). Grantham, J. J., Cook, L. T., Wetzel, L. H., Cadnapaphornchai, M. A. & Bae, K. T. Evidence

obtained in mouse models and the PCK rat.81–84 These studies led to a successful clinical trial, emphasizing the importance of animal models in ADPKD.164

Conclusions

In the past decade, rodent models have proven critical to study both the molecular basis of PKD and its natural history. These studies have provided us with many potential targets for therapeutic interventions and models have been used in preclinical testing. Nevertheless, the complexity of the signalling pathways and cellular alterations, as well as the slow progression of the disease in patients, makes the step from preclinical studies to treatment of human disease enormous. Despite the great number of animal models of PKD that are currently available, a need still exists for models that closely resemble the human disease. Ideally, this would be a Pkd1/Pkd2 model with a slow increase in the number and size of cysts, culminating in a massively cystic and fibrotic phenotype. Extended survival in addition to effects on kidney size and renal function would be of great value. On the other hand, novel interventions must be tested in a cost-effective and timely manner. There is no clear answer, therefore, to the question of which experimental models are most suitable to study PKD mechanisms or for preclinical testing of potential therapies. The use of at least two different models in parallel is the most desirable strategy. The best compromise seems to be to use a model with germline hypomorphic or missense mutations and an inducible conditional knockout. Review Criteria A search for original article was performed in PubMed using the search terms “ADPKD”, “ARPKD”, “PKD”, “polycystic kidney disease”, “autosomal dominant polycystic kidney disease”, “autosomal recessive polycystic kidney disease”, “polycystic”, “renal cyst”, “mouse models”, “conditional knock-out model”, “rat model”, “primary cilia”, “planar cell polarity”, “genetic interaction”, “functional interaction”, “signalling” and “fibrosis”, alone and in combination. Relevant English-language publications were identified and reviewed. Additional information was derived from the authors’ knowledge of the published literature and personal experience.

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