Animal Models for Human Polycystic Kidney Disease

Exp. Anim. 61(5), 477–488, 2012

—Review— Animal Models for Human Polycystic Kidney Disease

Shizuko Nagao, Masanori Kugita, Daisuke Yoshihara, and Tamio Yamaguchi

Education and Research Center of Animal Models for Human Diseases, Fujita Health University, 1–98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan

Abstract: Polycystic kidney disease (PKD) is a hereditary disorder with abnormal cellular proliferation, fluid accumulation in numerous cysts, remodeling of extracellular matrix, inflammation, and fibrosis in the kidney and liver. The two major types of PKD show autosomal dominant (ADPKD) or autosomal recessive inheritance (ARPKD). ADPKD is one of the most common genetic diseases, with an incidence of 1:500–1,000. Approximately 50% of patients with ADPKD develop end-stage renal disease (ESRD) by the age of 60. On the other hand, ARPKD is relatively rare, with an incidence of approximately 1:20,000–40,000. ARPKD is diagnosed early in life, often prenatally. The gene products responsible for ADPKD and ARPKD distribute in primary cilia and are thought to control intercellular Ca2+. Two types of animal model of PKD have been established: spontaneous hereditary models identified by the typical manifestations of PKD and gene-engineered models established by modification of human orthologous genes. Both types of animal models are used to study the mechanism of cystogenesis and efficacy of medical treatments. In PKD progression, critical roles of signaling pathways including MAPK, mTOR, and PPAR-γ have been discovered with these models. Therefore, experimental animal models are indispensable for investigating molecular mechanisms of PKD onset and progression as well as potential therapeutic treatments. Key words: Ca2+, cilia, gene engineering, PKD, PPAR-γ

Introduction (PC1), is a large receptor-like protein containing a long N-terminal extracellular domain that interacts with the Polycystic kidney disease (PKD) is characterized by PKD2 gene product, polycystin-2 (PC2), via a short the presence of numerous cysts that originate from the cytoplasmic C-terminus to form a heterodimeric complex renal tubules. In humans, autosomal dominant PKD [38]. PC1 and PC2 may form a heterotetramer [81]. PC2 (ADPKD) is one of the most common genetic diseases, is homologous to the transient receptor potential (TRP) with an incidence of 1:500–1,000, and is caused by mu- family of store-operated calcium (Ca2+) channels and is tations in either PKD1 or PKD2 [15]. About 85% of thought to function as a nonselective Ca2+ channel [14]. cases of ADPKD are caused by mutations in PKD1 on PC2 interacts with the PKHD1 gene product, FPC, as chromosome 16, and 15% are caused by mutations in well [67]. The PC complex is reported to localize in the PKD2 on chromosome 4. Autosomal recessive PKD primary cilia and act as a mechanosensor for maintaining (ARPKD) is also a monogenetic disorder, with a lower the differentiated state of cells in the renal tubular epi- incidence of 1:20,000–40,000, and is caused by a muta- thelium and hepatic biliary tract [36]. In order to make tion in the polycystic kidney and hepatic disease 1 use of basic research findings in the efforts to progress (PKHD1) gene, which encodes fibrocystin/polyductin towards new clinical therapeutic approaches, animal (FPC) [15, 64]. The gene product of PKD1, polycystin-1 models are imperative precursors for translational stud-

(Received 1 March 2012 / Accepted 28 April 2012) Address corresponding: S. Nagao, Education and Research Center of Animal Models for Human Diseases, Fujita Health University, 1–98 Den- gakugakubo, Kutsukake-cho Toyoake, Aichi 470-1192, Japan ©2012 Japanese Association for Laboratory Animal Science 478 S. NAGAO, ET AL. ies. In this review, widely-recognized animal models for certain types of kinases and GTPases in intracellular human PKD are introduced along with their possible signaling pathways is apparent in cystic kidney tissues uses in investigating the molecular mechanisms of PKD (Fig. 1F and 1I). The Pkdr1 (Cy) gene product, SamCys- onset and progression as well as their potential use in tin, is mainly distributed in the proximal tubules in which establishing therapeutic treatments. the initial cysts originate [6, 31]. In Cy/+ rats, SamCys- tin is overexpressed in the mature kidney after 21 days PKD Animal Models of age, whereas the protein is underexpressed in immature stages of kidney development compared with the There are two major types of PKD animal models: kidney from normal rats [31]. Thus, the abnormal expres- spontaneous hereditary models identified by the typical sion of SamCystin may affect the normal structure and manifestations of PKD and modified models established function of the proximal tubules. Neudecker et al. de- by mutation of human orthologous genes. These models veloped transgenic rats with Anks6(p.R823W), a missense are used for the elucidation of molecular-based disease mutation (C>T) of the responsible PKD gene in the Cy outcomes, determination of endogenous or exogenous strain [37]. In this model, the expression of Anks6 is factors related to disease progression, and establishment increased either in the immature or mature stages of of therapeutic interventions. kidney development, and the cysts are derived from the proximal tubules as in the Cy/+ rats of the spontaneous Spontaneous hereditary models of PKD model, suggesting that gain-of-function by the mutated This type of animal model is identified according to gene product may cause the disease. symptoms that are typical of human PKD. Therefore, PCK rats: Katusyama et al. discovered a PCK rat these animals have an obvious PKD phenotype, although strain from a Sprague Dawley (SD) outbreeding colony the responsible genes are not always orthologous to hu- [19, 23]. The responsible gene, Pkhd1, located on rat man genes. chromosome 9 is orthologous to the gene affected in Han:SPRD-Cy rats: The Han:SPRD-Cy (Cy) rat strain human ARPKD [69]. In this rat, polycystic kidney and was discovered by Kaspareit-Rittinghausen et al.; the liver disease results from a frameshift mutation (exon original colony was established in Hanover, Germany 36 is deleted by a change of A to T in intron 35) in the [18]. Numerous renal cysts are caused by a missense Pkhd1 gene. The gene product, FPC is located in the mutation (C to T, R823W) in exon 13 of the Pkdr1 (also cilia and expressed in the kidney, liver, and pancreas. called Cy, Samd6, or Anks6) gene on rat chromosome 5. The life span of PCK rats is approximately 1.5 years. At Three genotypes, wild-type (+/+), heterozygous (Cy/+) 1 year of age, numerous cysts are observed on the surface and homozygous (Cy/Cy), have been identified by PCR of the kidney and liver (Fig. 2A). In the kidney, the ini- analysis [6] relying on the inability of the MspI restric- tial cysts originate from the collecting duct, and the tion enzyme to recognize and cleave the region contain- growing cysts diffusely affect whole nephron segments ing the missense mutation [6]. Renal cysts are observed in the end-stage disease (Fig. 2B). In the liver, enlarged in both homozygous (Cy/Cy) and heterozygous (Cy/+) bile ducts with fibrosis are observed in the initial stage, animals. In Cy/Cy animals, expanded renal tubules and and cysts with fibrotic thickening are observed in the initial cysts are observed in the neonatal stage. Rapid later stage (Fig. 2C). In the pancreas, cysts derived from disease progression results in a short 3-week life span the pancreatic duct are evident (Fig. 2D). Extracellular (Fig. 1A), whereas in Cy/+ animals, disease progression signal-regulated kinase (ERK) and ribosomal S6 kinase is relatively slow (Fig. 1B), and the average life span is (S6) pathways are involved in stimulated cell prolifera- approximately 1.5 years in females and 1 year in males. tion in the kidney (Fig. 2E). In the liver, stimulation of In animals with the Cy/+ genotype, renal cysts are the ERK signaling pathway is involved in cell prolifera- mainly derived from the proximal tubules in the initial tion in hepatic cysts, and upregulated expression of stage [35] (Fig. 1C). Some of them are observed as chi- transforming growth factor-β (TGF-β) may cause fibro- meric cysts with normal and thickened extracellular sis [78]. matrix surrounding chimeric cysts and infiltrated mono- Pcy mice: The pcy mouse was originally discovered cytes in the interstitium [10, 35] (Fig. 1D and 1E). in the KK strain by Takahashi et al. [55]. It has nephro- Stimulated cellular proliferation with overexpression of nophthisis caused by a missense mutation (T1841G) in ANIMAL MODELS FOR POLYCYSTIC KIDNEY DISEASE 479

Fig. 1. characteristics of the kidney in Cy rats. (A) Hematoxylin and eosin (HE)-stained kidneys of 7-day-old +/+ (wild-type) (a, d), Cy/+ (b, e), and Cy/Cy (c, f) rats. The ratio of total kidney weight to body weight is approximately 2-fold higher in Cy/+ rats and 6-fold higher in Cy/Cy rats compared with +/+ (wild- type) rats. At 7 days of age, expanded renal tubules are observed microscopically in Cy/+ rats. In Cy/ Cy rats, enlarged kidneys with numerous cysts are observed at the same age. Bars=1 mm (a–c) and 100 µm (d–f). (B) Macroscopic photographs of kidneys from +/+ (wild-type) (a) and Cy/+ (b) rats at 8 months of age. Numerous renal cysts are obvious in the kidney. In contrast, no cysts are observed in the liver (photo not shown). (C–H) Histological findings of theCy/+ rat kidney. By Periodic acid-Schiff (PAS) staining, expansion of the renal tubules (C) and thickening of the extracellular matrix (D) are shown in the kidneys of 3-week-old Cy/+ rats. Chimeric renal tubules with normal and cystic transformed epithelia in the kidneys of 5-week-old Cy/+ rats (E). In the cystic transformed epithelium, thickening of the extracellular matrix (E: asterisks) and phosphorylated ERK (pERK) expression (F: arrow) are observed. After staining with Sirius Red, the red areas (#) indicate fibrosis in the kidney of a 6-week-old Cy/+ rat (G, kidney of a +/+ (wild-type) rat; H, polycystic kidney of a Cy/+ rat). Bars=50 µm (C and D) and 100 µm (E–H). (I) Overexpressed cell signaling proteins in the Cy rat kidney. In the kidneys of 12-week-old Cy/+ rats, the abundance, activity, or abundance and activity of Src, Rap-1, B-Raf, ERK, and AKT are upregulated compared with the +/+ (wild-type) rat kidney (n=2). These kinases and GTPases in cell signaling pathways are thought to be involved in accelerated cell proliferation. 480 S. NAGAO, ET AL.

Fig. 2. characteristics of the liver and kidney in PCK rats. (A) Macroscopic photographs of the liver (a), kidney (c), and HE-stained liver (b) and kidney (d) from a PCK rat at 12 months of age. Numerous diffuse cysts are obvious in both the kidney and liver. Bar=1 mm (b and d). (B) HE-stained kidney from PCK rats. The cysts are initially derived from collecting ducts and develop in whole nephron segments in later stages (HE-stained kidney; a, 5 weeks; b, 10 weeks, c, 15 weeks; and d, 25 weeks). Bar=1 mm. (C) Sirius Red-stained liver from PCK rats. Con- genital hepatic fibrosis (CHF) is observed in the liver. The cysts are derived from bile ducts, and fibrosis is marked from 6 days of age (Sirius Red-stained liver; a, 1 day; b, 6 days; c, 12 days; and d, 21 days). Bar=100 µm. (D) HE-stained pancreas from PCK rats. In the pancreas, the cysts are derived from the pancreatic duct (HE-stained pancreases at 1 year of age; a, trivial; b, mild; c, moderate; and d, severe). Bar=100 µm. (E) Overexpressed cell-signaling proteins in the PCK rat kidney. In the kidneys of 14-week-old PCK rats, the abundance, activity, or abundance and activity of Src, Rap-1, B-Raf, ERK, and AKT are upregulated compared with that in the normal SD rat kidney. These kinases and GTPases in cell-signaling pathways are thought to be involved in accelerated cell proliferation. ANIMAL MODELS FOR POLYCYSTIC KIDNEY DISEASE 481

Fig. 3. characteristics of the kidney in pcy and jck mice. (A) Computed tomographic (CT) images (a, whole body; b, kidneys) of a contrast medium-injected pcy mouse and macroscopic photograph (c) of a pcy mouse kidney at 30 weeks of age. (B) HE- and PAS-stained kidneys of a wild-type mouse (a and b) and a pcy mouse (c–d). Numerous renal cysts are obvious in the HE- (c, low magnification; e, high magnification) and PAS-stained (d, low magnification; f, high magnification) kidney of the pcy mouse. Bars=100 µm (a–f). (C) Upregulated ERK activity in 26-week-old pcy mice. Expression of the activated form of ERK (pERK) is upregulated in the pcy mouse kidney compared with that in the normal mouse kidney. (D) HE-stained kidneys of 4-, 10-, and 20-week-old wild-type (a, 4 weeks; b, 10 weeks; and c, 20 weeks) and jck (d, 4 weeks; e, 10 weeks; and f, 20 weeks) mice. The ratios of total kidney weight to body weight (K/B%) are approximately 5- to 10- fold higher in jck mice at 20 weeks of age compared with wild-type mice. Microscopically, a few cysts are observed at 4 weeks of age, and enlarged kidneys with numerous cysts are observed at 10 weeks of age in jck mice. Bar=1 mm. (E) Histological findings in the jck mouse kidney. Initial renal cysts (a: HE staining) are shown in the kidney of a 4-week-old jck mouse. At the same age, Ki67 (b) and pERK (c) are obviously expressed in the initial renal cysts and normal-shaped renal tubules. At 10 weeks of age, larger cysts are observed (d: HE staining). In epithelial cells with cysts, expression of cell proliferation markers Ki67 (e) and pERK (f) is obvious. Bar=100 µm. (F) Upregulated ERK activity in 10-week-old jck mice. ERK activity in the kidneys of jck mice is upregulated compared with that in a normal mouse kidney. 482 S. NAGAO, ET AL. the gene orthologous to human Nphp3 [40]. The gene Gene-modified models generated by mutation of human product, nephrocystin-3, is expressed in the primary orthologous genes cilia of epithelial cells in the kidney, pancreas, heart, and Transgenic mice: Transgenic mouse models with in- liver. The lifespan of pcy mice with either the DBA/2 or creased expression of the human orthologous PKD1 gene ICR strain background is approximately 40 weeks. Nu- have important roles for understanding the mechanism merous cysts are observed on the kidney surface at 30 of cystogenesis in human disease because expression of weeks of age (Fig. 3A). Enlarged renal tubules are ob- the PKD1 gene product, PC1, is thought to be increased served at embryonic day 15, and growing cysts are evi- in patients with ADPKD. After the identification of the dent in the corticomedullary junction at 3 weeks of age. entire sequence of the PKD1 gene in 1995 [59, 60], sev- Initially, cysts are derived from distal tubules, and whole eral groups tried to make transgenic animals. For ex- nephron segments become diffusely occupied by cysts ample, development of renal cysts was observed in the by 30 weeks of age, often with the occurrence of end- kidney and liver in transgenic mice in which a P1-derived stage renal disease (ESRD). In cystic epithelia, stimu- artificial chromosome (PAC) for PKD1 and TSC2 was lated cellular proliferation with upregulation of the ERK used [43]. Recently, Thivierge et al. reported a novel signaling pathway is observed (Fig. 3C) [39]. The longer transgenic mouse model produced with a bacterial arti- 2-year life span reported in pcy mice with the C57BL/6 ficial chromosome (BAC) [61]. In this model, the expres- strain background suggests that modifier genes may in- sion of the PKD1 gene is increased in tissues including fluence the rate of disease progression [29]. Although the kidney, liver, and heart, and overexpression of the retinal degeneration and hepatic fibrosis are observed in gene product, PC1, is observed in renal cysts. In addition, human patients with nephronophthisis, these phenom- hepatic cysts and fibrosis and cardiac and vascular ab- ena are not observed in the pcy mouse strain. normalities are shown as in human ADPKD. Jck mice: Jck (juvenile cystic kidney) mice were iso- Gene targeting mice: Pkd1 Gene targeting mice. Gene lated from a transgenic line by Atala et al. in 1993, but targeting models are produced by deletion of human the effects are unrelated to the transgene [2]. The jck orthologous PKD genes, PKD1 and PKD2 for ADPKD gene, also called Nek8 or Nphp9, is located on mouse and PKHD1 for ARPKD, respectively. Particularly, be- chromosome 11 [17]. The mutated gene product is found cause PKD1 is the responsible gene in 85% of ADPKD in the primary cilia and affects normal expression of the cases, quite a few Pkd1 knockout mice have been estab- PKD1 and PKD2 gene products, PC1 and PC2 [50]. The lished with null or deleted exons (2–6, 2–11, 33 or 43– lifespan of jck mice is approximately 20–25 weeks. The 45) [21, 24, 25, 28]. Slow or no disease progression is initial renal cysts are observed at 4 weeks of age, and observed in heterozygous Pkd1 targeting mice. Interest- the cysts are obvious at 10 weeks of age (Fig. 3D). Ex- ingly, in heterozygous Pkd1del2-6/+ gene targeting mice, pression of activated ERK is shown in proliferative cells Pkd1 haploinsufficiency is observed, and it causes a in the cystic epithelia (Fig. 3E and 3F) [49]. syndrome of inappropriate antidiuresis [1]. On the other Other models: The cpk (congenital polycystic kidney) hand, almost all types of homozygous Pkd1 targeting and bpk (BALB/c polycystic kidney) mice are other mice are lethal in the infant period [21, 24, 25, 28]. spontaneous models of PKD. Cpk mice have renal cysts Various devices are used for establishing appropriate derived from the proximal tubules and collecting ducts PKD models. For example, in a model obtained by con- by mutation of the Cys1 gene located on mouse chromo- ditional knockout using Pkd1-Cre/loxp and Aqp2-Cre some 12 [16]. The gene product, cystin, is located in the systems with the principal cells of the collecting duct, primary cilia of renal epithelial cells with polaris, which the progression of PKD is rapid, and the mean life span is known to be a gene product of TgN737Rpw, in orpk is relatively short (8.2 weeks) [44]. Furthermore, in the mice [76]. The bpk mice have cysts derived from the transgenic knockdown mouse developed by microRNA- collecting ducts in the kidney and from bile ducts in the based conditional RNAi against Pkd1, numerous renal liver caused by mutation of the Bicc1 gene located on cysts are observed with fibrosis and increased cell pro- mouse chromosome 10 [9]. The Bicc1 gene product is liferation [66]. also located in the primary cilia. It is intriguing that The time period of inactivation of the Pkd1 gene in various gene products in the spontaneous hereditary conditional knockout mice may influence the progression models are located in the primary cilia. of PKD. In fact, Piontek et al. reported that inactivation ANIMAL MODELS FOR POLYCYSTIC KIDNEY DISEASE 483 of Pkd1 before postnatal day 13 caused severe PKD Cy/+ rat model, a Raf kinase inhibitor reduced cell pro- within 3 weeks, whereas inactivation at day 14 or later liferation in the cystic epithelia [7]. An inhibitor of MEK resulted in the formation of renal cysts only after 5 ameliorated renal cystic disease in pcy mice and was months [42]. Takakura et al. have made similar observa- associated with decreased downstream ERK activity tions in Pkd1 conditional knockout mice as well [56]. [39]. In the kidney tissue of the Cy/+ rat and two mouse Therefore, the kidney seems to possess a critical check- PKD models, orpk and bpk, overexpression and activa- point for determining the severity of PKD progression. tion of EGFR are observed, and treatment with tyrosine Pkd2 gene targeting mice. Several gene targeting mice kinase inhibitors ameliorated PKD progression [46, 54, for Pkd2 gene have been reported. In particular, the 62]. On the other hand, activation of the cAMP signaling Pkd2WS25/− mouse with an unstable allele is one of the cascade through the adenylyl cyclase-activating receptor important models used for drug treatment studies be- is known to stimulate MAPK as well. In fact, decreased cause it has cysts in both the kidney and liver, as in intracellular cAMP achieved through reduction of plas- human ADPKD patients [73]. It was established by ma levels of vasopressin by increasing water intake or crossbreeding of Pkd2+/− and Pkd2WS25/+. In Pkd2WS25/− blocking the cAMP-mediated cellular effect of vasopres- mice, the weight (% of body weight) of the kidney and sin with an arginine vasopressin V2-receptor antagonist liver is 2-fold heavier compared with that in wild-type ameliorated PKD progression in PCK rats [13, 68], mice [52]. The rates of proliferation and apoptosis in Pkd2−/tm1Som mice [63], and pcy mice [13]. These find- epithelial cells of either renal cysts or cystic cholangio- ings suggest that blocking of the MAPK pathway acti- cytes are upregulated compared with those in normal vated by cAMP is also important for ameliorating PKD tissues [52]. progression. On the basis of the results from animal Pkhd1 gene targeting mice. In homozygous Pkhd1 models, a phase III clinical trial of a vasopressin V2- gene targeting mice produced with a BAC, renal cysts receptor antagonist is being performed in ADPKD pa- derived from the collecting ducts and dilatation of the tients. In addition, in order to avoid elevation of endog- intrahepatic bile ducts are seen in adulthood, which is enous vasopressin levels, physicians recommend that similar to the symptoms of the PCK rat, a spontaneous early-stage patients who do not have concerns about ARPKD model with a mutation in the rat gene ortholo- reduced renal function drink water continuously. gous to the human ARPKD gene [72]. Other pathways are also known to be related to MAPK signaling. For example, Src kinase is activated by EGFR Signaling Pathways Related to PKD and G protein-coupled receptors. The non-RTK activity Progression in Animal Models of Src is upregulated in the polycystic kidney and/or liver in BPK mice and PCK rats and in the polycystic In PKD, kidney enlargement is caused by stimulated kidney of Pkd+/− mice, and inhibition of Src ameliorates cellular proliferation of the tubule epithelia, leading to disease progression [12, 53]. Therefore, the inhibition the formation of numerous fluid-filled cysts with exten- of kinases in the accelerated MAPK pathway may have sive loss of functional nephrons, increased inflammation, therapeutic potential to ameliorate disease progression and interstitial fibrosis. It is thought that these phenom- in PKD patients. ena are related to the alteration of cell signaling pathways. Mammalian target of rapamycin (mTOR) signaling pathway Receptor tyrosine kinase (RTK) and cyclic AMP (cAMP) mTOR regulates cell proliferation by upstream signals signaling pathways from a serine-threonine kinase, AKT (also called Protein In the PKD kidney, the RTK pathway appears to have Kinase B). The mTOR signaling pathway is upregulated a major role in cell proliferation. Activation of RTK with in the kidneys of Cy rats [58], PCK rats [45], Pkd2WS25/− sequential stimulation of mitogen-activated protein ki- mice [79], and Pkd2flox/− mice [51]. Several inhibitors nase (MAPK) components, Raf, MEK, and ERK in- of mTOR are known to suppress disease progression in creases the number of cyst-lining cells in the kidneys of these models [51, 58, 79]. Cy rats (Fig. 1I) [33, 35], PCK rats (Fig. 2E) [32], pcy mTOR kinase forms at least two distinct complexes, mice (Fig. 3C) [39], and jck mice (Fig. 3F) [49]. In the mTOR complex 1 (mTORC1), which phosphorylates S6 484 S. NAGAO, ET AL. protein, and mTOR complex 2 (mTORC2), which acti- the RXR-mediated pathway is upregulated in the Cy rat vates AKT by the phosphorylation of Ser 473. Sirolimus, model [22]. This finding suggests that the regulation of a major mTOR inhibitor, suppressed the activity of either this pathway may control PKD progression through cell mTORC1 or mTORC2 in male Cy/+ rats; on the other proliferation mechanisms in this model. hand, there were no effects against mTORC2 in females Androgen receptor: Progression of PKD in male Cy/+ [3]. Although two clinical trials of mTOR inhibitor did rats and jck mice is more severe than that in females. not show a significant effect on disease progression in One of the factors that causes this increased severity is ADPKD patients [48, 65], the efficacy in relation to se- thought to be androgen, a male hormone [30, 49]. An- vere renal insufficiency in ADPKD is currently under drogen stimulates PKD progression via the androgen investigation. receptor with the activation of downstream MAPK pathways in males or testosterone-treated females after ovari- Intracellular Ca2+ ectomy [30, 49]. Careful hormone therapy may be re- It is thought that PC2 interacts with PC1 and FPC to quired in male PKD patients. regulate Ca2+ influx [14, 67]. Therefore, intracellular Signal transducers and activators of transcription 3 Ca2+ may have a key role in causing the characteristic (STAT3): STAT3 is a key signaling component in path- cellular phenotype of PKD. In fact, in primary culture ways activated by growth-factor receptors. Activated cells obtained from the kidneys of PKD1 knockout het- STAT3 makes dimers and stimulates cell proliferation erozygous mice, the concentration of intercellular Ca2+ and has anti-apoptotic activity. Recently, it was reported is decreased to approximately half of that in cells from that STAT3 activation is upregulated in the cystic epi- wild-type mice [1]. In Cy/+ rats, decrease of intercel- thelial cells of the PKD1 knockout mouse model, and lular Ca2+ by treatment with L-type calcium channel STAT3 inhibitors, pyrimethamine and S3I-201, amelio- blocker (CCB) accelerates PKD progression with in- rate PKD progression with downregulated expression of creased ERK activity in the cystic cells [33]. After these STAT3 and Bcl-x and decreased activity of cyclin D and preclinical findings were reported, a careful clinical c-Myc [57]. Because pyrimethamine is already used study was performed, and the results suggested that CCB clinically as an antimalarial drug, STAT3 inhibitors po- should be avoided in ADPKD patients unless CCB treat- tentially have therapeutic value in ADPKD patients in ment for resistant hypertension is necessary [27]. the near future. Cyclooxygenase-derived prostanoids: Cyclooxygen- Other signaling cascades ase-2 (COX-2) and its metabolites, including pros- Peroxisome proliferator-activated receptor (PPAR)-γ: tanoids, are thought to be involved in the inflammatory Activation of PPAR-γ has a crucial role in growth inhi- response. Activated COX-2 was observed in the kidney bition, cell cycle arrest, induction of apoptosis, fibrosis, of Cy/+ rats, and inhibition of this enzyme resulted in and inflammation in cancer cells [5, 20, 75]. PPAR-γ reduced fibrosis and cyst expansion as well as ameliora- agonists inhibit cell proliferation by downregulation of tion of inflammation, oxidative injury, and cell prolif- ERK and/or mTOR signaling pathways and decrease eration, possibly by decreased production of thrombox- inflammation and fibrosis by downregulation of TGF-β ane B2 and prostaglandin E2 [47]. Because [34, 77, 78]. Therefore, PPAR-γ may have therapeutic COX-2-selective inhibitors are widely used as nonste- value in ameliorating PKD progression. roidal anti-inflammatory drugs (NSAIDs), clinical ap- Monocyte chemoattractant protein-1 (MCP-1): MCP- plication in PKD patients may be considered. 1 is one of the factors that activate inflammation. Be- cause urinary MCP-1 is increased in ADPKD patients “Two-Hit” Theory and “Third-Hit” Theory and overexpression of MCP-1 is observed in the kidney of Cy/+ rats [10, 80], it seems to be a potential bio- Currently, the “two-hit” theory is widely advocated to marker for disease severity [26, 80]. Interestingly, explain the initial stage of disease progression in PKD. PPAR-γ agonists decrease the expression of MCP-1 in Namely, a germ-line mutation in one allele and a so- Cy/+ rats [11] with a concomitant reduction in disease matic mutation or insufficient expression in another al- progression. lele of either of the PKD genes causes cystogenesis [41, Retinoid X receptor (RXR): Recently, we reported that 70, 74]. It is thought that the variability in progression ANIMAL MODELS FOR POLYCYSTIC KIDNEY DISEASE 485 of PKD depends on the timing of the somatic mutation References or insufficient expression of the PKD gene in each renal cell. On the other hand, Weimbs et al. recently proposed A1. hrabi, A.K., Terryn, S., Valenti, G., Caron, N., Serradeil-Le Gal, C., Raufaste, D., Nielsen, S., Horie, S., Verbavatz, J.M., a “third-hit” theory of PKD progression [4, 56, 71], in and Devuyst, O. 2007. PKD1 haploinsufficiency causes a which unknown gene modification and/or environmental syndrome of inappropriate antidiuresis in mice. J. Am. 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