Murine Models for Polycystic Kidney Disease
Towards Therapeutic Intervention Strategies for Polycystic Kidney Disease
6
Ngoc Hang Le, Nanna Claij, Martijn H. Breuning, Dorien J.M. Peters
Department of Human Genetics, Leiden University Medical Center;
Leiden, The Netherlands
Murine models for Polycystic Kidney Disease
Murine models for PKD have greatly contributed to further understanding of the defects associated with the disease and are crucial to test potential therapeutic compounds pre-clinically. In the following section several murine models for ADPKD and ARPKD are discussed. We also refer to the outstanding review by Guay-Woodford for a comprehensive description of murine models for PKD (1).
Models for ADPKD
Models obtained by targeted disruption of the genes mutated in the most common variant of polycystic kidney disease, ADPKD, the Pkd1 and Pkd2 gene, are outlined in the following section (Table 1). These models are the “Þ rst choice” strategy to obtain models for ADPKD since it is genetically most related to the human disease.
They have proven to be instrumental in further understanding of Pkd1 and Pkd2 function and in analysis of other organ systems besides the kidney where Pkd1 and Pkd2 have important functions, such as liver, pancreas, and the cardiovascular system. Following this, the HAN:SPRD-cy and kat and kat2J models are discussed in more detail. The HAN:SPRD-cy rat model has developed spontaneously and has been widely used for two reasons: it has been available for a long time and the disease progresses relatively slowly (2,3).
Targeted disruption of Pkd1
Several mouse models have been established to study ADPKD using targeted disruption of the Pkd1 gene, responsible for the majority of all PKD cases. In the Þ rst model that was developed, a Pkd1 truncation mutation was introduced (Pkd1del34; 4). Homozygous mice died peri-natally due to rapidly progressive polycystic kidney disease, pancreatic cysts, and pulmonary hypoplasia. Renal cyst formation was observed as early as embryonic day 15.5 (E15.5) in proximal
Table 1. Models for ADPKD.
tubules. However, heterozygous mice only exhibited polycystic kidney disease at extremely high age (5). This presented a clear discrepancy between mouse and human ADPKD pathology. Therefore, additional mouse models were called for.
Lu et al. later introduced a null mutation in Pkd1 by targeted disruption of exon 4 (Pkd1null; 6). Homozygous mice suff ered from renal and pancreatic cystic disease, polyhydramnios, hydrops fetalis, spina biÞ da occulta, and osteochondrodysplasia resulting in death at E13.5-16.5. Heterozygous mice developed adult-onset
pancreatic disease, although again no clear renal pathology comparable to human ADPKD was observed. This model does show that polycystin-1 is not only important in the kidney, but also essential for vascular and skeletal development and pancreatic function. Several other models further clearly demonstrated that polycystin-1 is not only essential for normal function of the kidneys, but also of other organs. Kim et al. developed a mouse model with renal cysts, pancreatic cysts, hydrops fetalis, vascular leaks, and hemorrhage (Pkd1L; 7). Pkd1L was developed via targeted disruption of exon 43-45 and illustrates that polycystin-1 is important for maintaining the integrity of blood vessels. Lethality occurred during embryonic development at E15.5.
Boulter et al. Þ rst reported a model where heterozygous adult mice developed renal cysts comparable to human ADPKD (8). Pkd1 was disrupted by insertion of a lacZ reporter gene in exon 17-21 (Pkd1del17-21βgeo). Heterozygous adult mice developed renal and liver cysts and skeletal defects. Homozygous embryos died from cardiovascular defects at E13.5-14.5. The model demonstrates that polycystin-1 is required for kidney function as well as cardiovascular and skeletal development.
Cardiovascular defects, a common denominator in Pkd1 mouse models, were reported also in homozygous embryos with targeted deletion of exons 2-6 (Pkd1-/-; 9). Also in this model, homozygous embryos developed severe polycystic kidney disease from E15.5-16.5. Lethality occurred during embryonic development due to the cardiovascular abnormalities. Total protein levels of polycystin-1 were decreased in heart and kidney of Pkd1-/- embryos. In the kidneys, β-catenin, c-myc and basolateral localization of E-cadherin were decreased also, whereas tyrosine phosphorylation of epidermal growth factor receptor (EGFR) was increased.
Interestingly, maternally administered pioglitazone, a thiazolidinedione compound that acts as a peroxisome proliferation activated receptor γ (PPARγ) agonist, improved survival of Pkd1-/- embryos and ameliorated the cardiac defects and cystogenesis. Recently, it was reported that JNK was increased in kidney tissue and primary mouse embryonic Þ broblasts derived from mice with targeted disruption of exon 2-6 (10). In addition, these cells showed increased growth and spontaneous immortalization. In summary, Pkd1-/- defects could be mediated by several signaling pathways. The implications of these data are excellently reviewed by Horie et al. (11).
In conclusion, several mouse models have been developed using targeting disruption of Pkd1. These models have proven to be crucial in further
understanding of Pkd1 function and in identifying several other organ systems besides the kidney where Pkd1 has important functions, such as liver, pancreas, and the cardiovascular system. However, some considerations should be taken
into account. So far, signiÞ cant diff erences in disease spectrum between mouse and human have been observed. Therefore, environmental factors, such as diet and micro-environment should be taken into account. In addition, intrinsic diff erences between mouse and human may also inß uence disease manifestation.
Hypomorphic Pkd1 mouse models
Two hypomorphic Pkd1 mouse mouse models are available that develop polycystic kidney disease shortly aft er birth, Pkd1nl/nl and Pkd1L3/L3 respectively (12,13). Pkd1nl/nl was developed by insertion of the neomycin gene ß anked by loxP sites in intron 1 of Pkd1. Pkd1L3/L3 was developed by insertion of a inverted neomycin gene ß anked by loxP sites in intron 34. Both models show early lethality within a month aft er birth. These models greatly beneÞ t research because the disease is relatively slowly progressive.
Targeted disruption of Pkd2
Two mouse models were developed using targeted disruption of Pkd2, the other gene known to be mutated in ADPKD. Wu et al. introduced a mutant exon 1 in tandem with the wild-type exon 1 at the mouse Pkd2 locus to produce an unstable allele, Pkd2WS25. Pkd2WS25 then undergoes somatic inactivation via intragenic homologous recombination resulting in a true null allele, Pkd2WS183 (14,15). Germ- line transmitt ed clones with a deletion of exon 1, Pkd2-/-, were also developed by Wu et al. Both heterozygous and homozygous mice developed polycystic kidney disease, similar to the human phenotype. Cyst-lining epithelial cells were all negative for polycystin-2. Pancreatic and liver cysts, defects in cardiac septation, and laterality defects were reported as well (14-16). Therefore, polycystin-2, similar to polycystin-1, seems to play an important role not only in the kidney but also in other organ systems. Pennekamp et al. also generated a Pkd2 knockout allele by insertion of a LacZ reporter gene in exon 1 (17). Homozygous mutant embryos did not only show polycystic kidney disease, but also right pulmonary isomerism, randomization of embryonic turning, heart looping, and abdominal situs inversus, indicating that polycystin-2 plays an important role in both kidney function and left -right axis determination.
The HAN:SPRD-cy and kat/kat2J models
The HAN:SPRD-cy rat model occurred spontaneously in a strain of Sprague- Dawley rats (2,3). Because of the slowly progressive nature of the disease, the HAN:SPRD-cy model has been extensively utilized for studies on therapeutic interventions for ADPKD. Proximal tubule cysts were evident at postnatal day 7 with death occurring at week 3-4 in cy/cy males. Heterozygous males died at 6 months and female mice showed even milder phenotypes. Recently, Brown et al.
identiÞ ed the disease causing mutation in the Polycystic Kidney Disease Rat 1, Pkdr1 gene, encoding the novel protein Samcystin (18).
The kat and kat2J mouse models showed proximal and glomerular cysts at 2 months
of age due to mutations in never in mitosis A (NIMA)-related kinase 1, Nek1 19,20).
Kat2J mice developed more progressive polycystic kidney disease compared to kat.
Where HAN:SPRD rats do not show extra-renal pathology, kat and kat2J mice show a wide spectrum of extra-renal manifestations including facial dysmorphisms, dwarÞ ng, male sterillity, anemia, and choroid plexus cysts.
Models for ARPKD
Murine models for the autosomal recessive variant of polycystic kidney disease, ARPKD, were available long before the models for the more frequently occurring variant, ADPKD, were developed using targeted disruption. Therefore, these models with a mutation in a variety of genes are generally more extensively characterized and more data are available on eff ects of potential therapeutic intervention strategies. In the following section, diff erent models for ARPKD are discussed in more detail (Table 2). These models all have recessive inheritance and severe early onset polycystic kidney disease in common. However, there are also diff erences. Some models show proximal cyst development during initial stages of the disease shift ing to pre-dominantly collecting duct cysts as the disease progresses (in bpk, cpk, Tg737orpk models). Other models develop cysts in more or even all nephron segments (jcpk, pck, wpk, and pcy). The models also diff er in disease severity. Lethality occurs post-natally between week 1-2 in bpk, jcpk, Tg737orpk, and inv models. Other models show less severe disease progression with lethality between week 3-4 (C57BL/6J-cpk/cpk, and wpk) or even later, between 4-9 months (jck and pcy). Extra-renal disease manifestations were detected in all models, except in the cpk model on C57BL/6J background (see sections below). On other genetic backgrounds, cpk mice did show extra-renal pathology, suggesting the presence of modiÞ er eff ects. Finally, most of the identiÞ ed disease causing mutations in the ARPKD models have been reported to encode proteins that localize in the cilium, an organelle that is thought to act as a mechanosensor in the renal epithelium (21). Since Pkd1 and Pkd2 also localize in this cellular compartment, there seems to be a direct correlation between ciliary proteins and polycystic kidney disease. This potential connection has been excellently reviewed previously (22-25).
The bpk and jcpk models
The BALB/c polycystic kidney, bpk, mouse model with a mutation in the Bicc1 gene encoding Bicaudal C, develops severe proximal tubule cysts (26-28). As the disease progresses, collecting duct cysts are predominantly detected. Bpk mice also develop liver cysts. Death usually occurred around postnatal day 8.
The chlorambucil induced juvenile congenital polycystic kidney, jcpk, model is also caused by a mutation in Bicc1 and develops cysts in all renal tubule segments with death occurring at postnatal day 10 (28). Liver and pancreatic dilatation were reported also.
The cpk mouse model
C57BL/6J-cpk/cpk mice with a mutation in the ciliary protein, cystin, suff ered from severe ARPKD with proximal tubule cysts evident at E16 (congenital polycystic kidney disease; 29-32). Similar to the bpk mouse model, collecting duct cysts are pre-dominantly detected as the disease progresses. Lethality usually occurs at the age of 3-4 weeks. Interestingly, cpk/cpk mice on C57BL/6J background did not show extra-renal pathology (33), whereas extra-renal pathology was observed on other genetic backgrounds (30). BALB/c-cpk/cpk mice also develop liver and pancreatic cysts (33). These data suggest that there are additional modiÞ er eff ects in this model for polycystic kidney disease.
The jck mouse model
Juvenile polycystic kidney, jck, mice develop a less severe ARPKD phenotype than the other models for ARPKD. Renal cyst development starts at postnatal day 3 resulting in lethality at approximately 4 months of age (34). Jck mice have defects in a protein implicated in cytoskeletal integrity, never in mitosis A (NIMA)-related kinase 8, Nek8 (35).
The Tg737orpk mouse model
Tg737orpk mice with a mutation in polaris die around postnatal day 7 due to cysts in kidney and liver, skeletal abnormalities, and pancreatic defects (oak ridge polycystic kidney; 36). Similar to the other models for ARPKD, proximal tubule cysts developed initially with later on pre-dominantly collecting duct cysts. Cilia length is decreased and mice have defects in left -right axis determination (37- 39). Interestingly, the Chlamydomonas orthologue of polaris is an intraß agellar transport protein involved in transport of ciliary proteins (IFT88; 40). Therefore, both polaris and polycystin-2 have essential functions in kidney and left -right axis determination.
Table 2. Models for ARPKD.
The pck and wpk rat models
There are two rat models for ARPKD. The polycystic kidney, pck, rat model for ARPKD has a mutation in the Polycystic Kidney and Hepatic Disease 1, Pkhd1 gene, the rat orthologue of PKDH1, the gene responsible for human ARPKD (41).
Pkhd1 encodes for a protein called Þ brocystin or polyductin. Pck rats suff er from distal tubule and collecting duct cysts and hepatic cysts. Where most models for polycystic kidney disease show overexpression and mislocalization of EGFR, this model does not.
Wister polycystic kidney, wpk, rats develop cysts in all renal tubule segments from E19 (43,44). At later stages, cysts mostly originate from the collecting duct. Wpk rats suff er from extensive extra-renal pathology with thymic and splenic hypoplasia and abnormalities of the central nervous system. Lethality usually occurs at week 4.
The inv and pcy mouse models for nephronophtisis
In addition to ARPKD, there are also relatively rare variants of autosomal recessive PKD, called nephronophtisis (NPH). Nephronophtisis is divided in diff erent subtypes depending on which gene is mutated. All types are characterized by severe and early onset polycystic kidney disease. Several mouse models for NPH are outlined below because they have provided signiÞ cant insight into PKD in general. The inversion of embryo turning, inv, mice are a model for
nephronophtisis type II (NPH type II). Inv mice develop renal and pancreatic cysts, situs inversus, liver, heart and lung defects with death occurring around postnatal day 7 (45,46). Inversin, the protein defect in inv, associates with tubulin (47) and may facilitate cross-talk between Wnt/β-catenin and Wnt/Ca2+ signaling (48).
Pcy mice are a model for nephronophtisis/medullary cystic kidney disease (NPH/
MCD), a late onset autosomal recessive polycystic kidney disease att ributed to a mutation in NPHP3 (49-50). Pcy mice develop cysts in all nephron segments with death occurring at week 30-36. Cerebral aneurysms are reported at end-stage of the disease. Pcy mice show increased proliferation and increased level of ERK activity (51), phosphatidyl-inositol kinase and phospholipase C, PLC (52), and vasopressin 2 receptor, V2R (53). ERK2, or p44 MAPK, was increased also in human ADPKD (54).
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Towards Therapeutic Intervention Strategies for Polycystic Kidney Disease
Abstract
Polycystic kidney diseases (PKD) are one of the major causes of chronic renal failure. The diseases are characterized by the formation of ß uid-Þ lled cysts in both kidneys. This review focuses on the defects reported in cystic cells and tissue, including Epidermal Growth Factor (EGF) and Arginine Vasopressin (AVP) signalling. In vitro and in vivo eff ects of compounds targeting these signalling pathways in cyst formation and progression are discussed.
Introduction
Polycystic kidney diseases (PKD) are characterized by the formation of ß uid-Þ lled cysts in both kidneys (Figure5A, Chapter 1, page 15). PKD is of the major causes of chronic renal failure. PKD patients require renal transplantation or, when donor kidneys are not available, renal dialysis (1). Generally, PKD is genetically inherited, although de novo mutations also occur. The most common inherited variant of PKD is Autosomal Dominant Polycystic Kidney Disease (ADPKD; Figure 5B, Chapter 1, page 15). Progressive development of cysts in both kidneys results in chronic renal failure in 50% of patients by the age of 50-60 years. ADPKD can be caused by a mutation in either the Polycystic Kidney Disease 1 (PKD1) or the Polycystic Kidney Disease 2 (PKD2) gene (2-6). Mutation analysis shows that the majority of all ADPKD cases results from a mutation in the PKD1 gene (~85%;7). Approximately 15% is caused by a mutation in the PKD2 gene. The autosomal recessive variant, called Autosomal Recessive Polycystic Kidney Disease (ARPKD;Figure 5B, Chapter 1, page 15) is much less frequent than ADPKD and is characterized by severe and early onset polycystic kidney disease which is frequently accompanied by congenital hepatic Þ brosis. This autosomal recessive variant is caused by a mutation in the Polycystic Kidney and Hepatic Disease 1 (PKDH1) gene (8,9).
Polycystin-1, polycystin-2, and Þ brocystin or polyductin, the protein products of the PKD1, PKD2, and PKDH1 gene respectively, are all glycoproteins. Although the genes responsible for ADPKD and ARPKD have been identiÞ ed and much work has been done to elucidate their precise function, the mechanism of cyst formation still remains unknown. Excellent reviews are available on polycystin-1 and polycystin-2 function (11-15). Murine models for PKD have greatly contributed to further understanding of the defects associated with the disease and are crucial to test potential therapeutic compounds pre-clinically. In the following section several murine models for ADPKD and ARPKD are brieß y discussed. We also refer to the outstanding review by Guay-Woodford for a comprehensive description of murine models for PKD (16). This review focuses on the defects reported in cystic cells and tissue, including Epidermal Growth Factor (EGF) and Arginine Vasopressin (AVP) signalling, and discusses in vitro and in vivo eff ects of compounds targeting these signalling pathways in cyst formation and progression.
Models for ADPKD
Targeted disruption of the genes mutated in the most common variant of polycystic kidney disease, ADPKD, the Pkd1 and Pkd2 gene, are the “Þ rst choice” strategy to obtain animal models for ADPKD since it is genetically most related to the human disease (Table 1, page 88). These models have proven to be instrumental in further understanding of Pkd1 and Pkd2 function and in analysis of other organ systems besides the kidney where Pkd1 and Pkd2 have important functions, such as liver,pancreas, and the cardiovascular system. Interestingly, pioglitazone, a thiazolidinedione compound that acts as a peroxisome proliferation activated receptor γ (PPARγ) agonist, improved survival of Pkd1-/- embryos and ameliorated the cardiac defects and cystogenesis (17). However, the molecular basis of this eff ect has yet to be determined. The HAN:SPRD-cy rat model was not obtained by targeted disruption of Pkd1 or Pkd2, but deserves special mentioning. It has developed spontaneously and has been widely used for two reasons: it has been available for a long time and the disease progresses relatively slowly (18,19).
Models for ARPKD
Murine models for the autosomal recessive variant of polycystic kidney disease, ARPKD, were available long before the models for ADPKD, were generated.
Therefore, these models that have a mutation in a variety of genes, are generally more extensively characterized and more data are available on eff ects of potential therapeutic intervention strategies. In the following section, diff erent models for ARPKD are discussed in more detail (Table 2, page 92). These models all have recessive inheritance and severe early onset polycystic kidney disease in common. However, there are also diff erences. Some models show proximal cyst development during initial stages of the disease shift ing to pre-dominantly collecting duct cysts as the disease progresses (in bpk, cpk, Tg737orpk models).
Other models develop cysts in more or even all nephron segments (jcpk, pck, wpk, and pcy). The models also diff er in disease severity. Lethality occurs post- natally between week 1-2 in bpk, jcpk, Tg737orpk, and inv models. Other models show less severe disease progression with lethality between week 3-4 (C57BL/6J- cpk/cpk, and wpk) or even later, between 4-9 months (jck and pcy). Extra-renal disease manifestations were detected in all models, except in the cpk model on C57BL/6J background. On other genetic backgrounds, cpk mice did show extra- renal pathology, suggesting the presence of modiÞ er eff ects. Finally, most of the identiÞ ed disease causing mutations in the ARPKD models have been reported to encode proteins that localize in the cilium, an organelle that is thought to act as a mechanosensor in the renal epithelium (20). Since Pkd1 and Pkd2 also localize in this cellular compartment, there seems to be a direct correlation between ciliary proteins and polycystic kidney disease. This potential connection has been excellently reviewed previously (21-24).
Abnormal expression of EGF and EGFR in polycystic kidney disease Several growth factors have been shown to be present at abnormal levels in polycystic kidneys. TGFα was increased in human ADPKD and in a mouse model of methylprednisone acetate, MPA, induced polycystic kidney disease (25-27).
TNFα and TGFβ mRNA was reported to be increased in cpk mice with ARPKD (28). Of the growth factors identiÞ ed to be abnormally expressed in polycystic kidney disease, EGF has been the most extensively studied. EGF is a growth factor that can bind to its receptor, EGFR. This results in activation of several intra- cellular signalling pathways. Depending on the cell type, diff erentiation status and availability of the EGFR, diff erent cellular responses can be generated.
Several reports have been published on EGF, some suggesting hyperactive EGF signaling activity in polycystic kidney disease, others indicating decreased levels of EGF. EGF was decreased in kidneys of C57BL/6J-cpk/cpk, DBA-2FG-pcy, and Han:SPRD-cy/+ (28-32). However, EGFR and also Erb-B2 were overexpressed and mislocalized at the apical membrane of human ADPKD and ARPKD, C57BL/6J-cpk/
cpk, BALB/c-bpk/bpk, HAN:SPRD-cy/+, Tg737orpk, and Pkd1-/- models (17,27,33-37).
Du and Wilson demonstrated that EGF binding to basolateral and apical localized EGFR in cystic cells was functional (37). Cyst ß uid of human and murine polycystic kidney disease was reported to contain increased levels of EGF-like peptides (38- 41). Regardless of the net eff ect, these data suggest that EGF/EGFR signalling is aff ected in polycystic kidney disease.
Eff ect of EGF treatment on (cystic) epithelial cells
Conß icting data are again reported on the biological eff ect of EGF on cultured renal cells. This may be explained by the biphasic eff ect of EGF on metanephric kidney culture Þ rst reported by Avner and Sweeney (42). Low dosage induced a mitogenic response, whereas a high dose inhibited proliferation. Yamaguchi et al. later on demonstrated that both control and cystic human kidney cortex cells (HKC) showed increased proliferation and ERK activity upon stimulation with EGF (serum-starved in 0.0002% FBS; 43). EGF induced proliferation could be blocked by both MEK or receptor tyrosine kinase inhibitors, PD98059 or genistein, respectively. Several reports further support this mitogenic eff ect of EGF. ADPKD and bpk cystic cells were hyper-responsive to EGF (>1ng/ml) induced mitogenesis, as assessed by [3H]-Thymidine incorporation (37,44,45). In contrast, Carone et al.
showed that EGF induced proliferation less in 3D collagen gel culture of human ADPKD cystic cells than normal kidney cells (46). EGF receptors were expressed at similar levels in normal and cystic cells.
EGF treatment for polycystic kidney disease in vivo
In the following section, in vivo eff ects of EGF treatment for polycystic kidney disease are outlined. Similar to the in vitro data, EGF treatment has dual eff ects on polycystic kidney disease in vivo. This may depend on dosage and time eff ects.
Treatment aft er postnatal day 10 accelerated disease progression, whereas early
treatment with EGF had beneÞ cial eff ects on polycystic kidney disease in bpk mice (47). In accordance, early EGF treatment of C57BL/J6- and BALB/c-cpk/cpk mice suff ering from severe ARPKD decreased renal cysts, increased renal function, and also decreased extra-renal pathology including liver and bile duct cysts, and eyelid opening (subcutaneous injection of 1 µg/g body weight during postnatal day 3-9; 48,49). Interestingly, prolonged treatment with EGF until postnatal day 14 increased mortality (100% lethal at postnatal day 21). These data suggest that if administered at the right dose and time range, EGF may prove a potential target for treatment of polycystic kidney disease.
EGFR inhibition based treatment for polycystic kidney disease in vivo Not only administration of EGF, but also the apparent opposite, inhibition of EGF signaling via the EGFR has been explored as a potential target for therapy.
EGFR inhibition leads to blockage of the signaling pathway, thereby preventing the cellular response to EGF stimulation. To inhibit EGFR several pharmaceutical compounds are available. EKI-785 is a compound inhibitor of EGFR that binds irreversibly to the ATP binding site of EGFR thereby inhibiting tyrosine kinase activity and auto-phosphorylation of EGFR and also Erb-B2 , an EGFR related receptor tyrosine kinase (50). Administration of EKI-785 from week 3 to 10 decreased phosphorylated EGFR levels in HAN:SPRD-cy/+ rats (90 mg/kg intra- peritoneally every 3 days; 51). The same eff ect was achieved with EKI-569, a derivative of EKI-785 (20 mg/kg intra-peritoneally every 3 days or enteral daily treatment with 5-20 mg/kg). Both compounds slowed polycystic kidney disease progression without growth retardation or liver function abnormalities, although liver enlargement was detected. EKI-785 and EKB-569 also slowed disease progression in bpk mice with proximal tubule and collecting duct cysts (30-90 mg/
kg intra-peritoneally every 3 days from day 7-19; 52,53).
Interestingly, administration of WTACE2 during postnatal day 7-21 slowed disease progression and increased expression of TGFα in proximal tubules and collecting ducts (54). WTACE2 is a competitive inhibitor of TNFα converting enzyme, TACE, a matrix metalloprotease (MMP) that cleaves membrane-bound TGFα to release secreted TGFα. This soluble TGFα can then activate EGFR. Therefore, WTACE2 indirectly also acts as an inhibitor of EGF signaling. Co-treatment with WTACE2 in between EKB-569 injections reduced eff ective EKB-569 dosage (to 30 mg/kg with 100 mg/kg WTACE2 intraperitoneally daily from day 8-20; 53).
EGFR blockade also slowed disease progression in Tg737orpk mice (53-55). Tg737orpk mice were reported to develop a milder cystic phenotype when a mutation was introduced that decreases EGFR tyrosine kinase activity, waved-2 (wa-2; 55).
Interestingly, transgenic overexpression of TGFα, Erb-b2, and T24ras, a component of EGFR signaling, all resulted in polycystic kidney disease (56-58). In addition, expression of a TGFα transgene in kidneys of pcy mice increased progression of polycystic kidney disease (59). Treatment of pck rats with ARPKD that do not show overexpression or mislocalization of EGFR, with EKI-785 or EKI-569 had no eff ect on disease progression (60).
In summary, these data indicate that the EGF/EGFR axis is relevant in at least several models for polycystic kidney disease and that therapeutic intervention in this pathway may have beneÞ cial eff ects.
AVP signalling in renal epithelial cells
Another signaling pathway that is crucial for renal epithelial cell function is Arginine Vasopressin, AVP, signaling. AVP, also called Anti-Diuretic hormone, ADH, regulates permeability of collecting ducts cells for water via binding to the receptor, arginine vasopressin 2 receptor, AVP-V2R or V2R. This subsequently results in activation of G-proteins and adenylyl cyclase. Adenylyl cyclase then increases cAMP levels, leading to transcription and apical translocation of AQP2 channels (61). Together with basolateral localization of AQP3, urine is thus concentrated in the collecting duct.
Abnormal cAMP levels in polycystic kidney disease
The signalling component cAMP, that is also an important partner in AVP signaling, has been shown to be elevated in kidney and urine of pcy mice with polycystic kidney disease (62). Furthermore, cAMP levels correlated with cyst enlargement and proliferation (32,63,64). cAMP is thought to stimulate cyst ß uid accumulation by activating the apical chloride transporter, CFTR (65,66).
Eff ect of cAMP treatment on cystic epithelial cells
Eff ects of cAMP treatment were studied in vitro in cultured renal cystic epithelial cells. Renal cystic cells showed increased proliferation with concomitant up-
regulation of ERK activity upon stimulation with 8-Br-cAMP and various activators of cAMP such as forskolin (activator of adenylyl cyclase, increases cAMP levels), isobutylmethylxanthine (IBMX, inhibitor of phospho-di-esterase (PDE), increases cAMP levels), AVP ( activates adenylyl cyclase via V2R), DDAVP (synthetic AVP), secretin, Vasoactive Intestinal Peptide (VIP; activates adenylyl cyclase via G-protein coupled receptors), and prostaglandin E2 (PGE2, activates adenylyl cyclase; 43,44).
Increased ERK activity correlated with increased proliferation and was observed in ADPKD cystic cells only, not in control human kidney cortex cells (HKC; 43,67).
Interestingly, overexpression of the C-terminal 193 amino acids of polycystin-1 in the SV40-Large T immortalized mouse cortical collecting duct cell line, M1, resulted in increased proliferation upon cAMP stimulation as well (68). cAMP induced proliferation could be blocked by co-treatment with inhibitors of Protein Kinase A (PKA, H89 and Rp-cAMP) or MEK (PD98059; 43,67). Genistein, a receptor tyrosine kinase inhibitor, had no eff ect. These data suggest that cAMP signals to PKA and MEK to induce proliferation. However, cAMP had similar eff ects on serum induced c-Fos transcriptional expression in control and cystic kidney cells of C57BL/6-cpk/cpk mice (69). In summary, cAMP seems to have promising potential on some aspects of polycystic kidney disease. Using a related strategy, polycystic
kidney disease mouse models were succesfully treated with inhibitors of V2R, a more upstream component of AVP signaling. These data are discussed in the following section.
AVP signaling based treatment for polycystic kidney disease
Treatment of C57BL/6-cpk/cpk and pcy mice, and pck rats with a V2R antagonist, OPC31260, slowed disease progression and decreased renal cAMP levels (61,70- 72). No eff ect was observed on liver cysts. Interestingly, treatment of a Pkd2 mouse model that develops renal cysts within 3 months aft er birth with the AVP-V2R antagonist, OPC31260, also slowed disease progression and decreased cAMP, AQP2, and AVP-V2R levels in the kidney (73). Wang et al. recently reported a new potent and more selective human V2R antagonist, OPC-41061, specially selected for use in clinical trials (72). OPC-41061 was conÞ rmed to be more eff ective than OPC-31260 in pck rats. In conclusion, intervention in the V2R/cAMP signalling pathway using V2R antagonists has high potential for therapeutic application.
Conclusions
Much eff ort has been taken to understand the molecular defects in polycystic kidney disease to aid the development of therapeutic intervention strategies. So far, two signalling routes prove to have serious potential in providing beneÞ cial therapies for PKD: EGF/EGFR signaling and the AVP/cAMP cascade. In the case of EGF, further evidence is required regarding the dosage- and time-dependent eff ects of treatment. Also, the contradicting reports on the eff ect of EGF treatment and of EGFR inhibition on PKD progression need to be addressed. Compounds targeting V2R/cAMP signaling have had more success in several pre-clinical animal models. OPC31260 mediated inhibition of V2R/cAMP has so far un-controversially presented as a beneÞ cial therapy for polycystic kidney disease. OPC31260 has even proven useful in a Pkd2 mouse model for ADPKD, a much more frequently occurring disease than ARPKD. It should be interesting to study biological eff ects of this compound in the recently developed Pkd1 mouse models, since the PKD1 gene is aff ected in the vast majority of ADPKD patients. In this respect, the PPARγ agonist, pioglitazone, reported by Muto et al. to have beniÞ cial eff ects in a Pkd1 mouse model, also warrants further investigation (17,74).
Thus, to provide a solid basis for targeted therapeutic strategies for PKD and to resolve some of the discrepancies, further molecular insight is required into the intracellular signalling components that are aff ected in PKD.
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