Cyst Formation in Kidney via B-Raf Signaling in the PKD2 Transgenic Mice

• DOI: 10.1074/jbc.m805890200
• Publication Date: 2008-12-22T01:12:38Z
• Authors: Eun Young Park; Young Hoon Sung; Moon Hee Yang; Ji Yeun Noh; So Young Park; Tae Young Lee; Yeon Joo Yook; Kyung Hyun Yoo; Kyung Jin Roh; Ingyu Kim; Young-Hwan Hwang; Goo Taeg Oh; Je Kyung Seong; Curie Ahn; Han-Woong Lee; Jong Hoon Park
• Keywords: PKD1

The pathogenic mechanisms of human autosomal dominant polycystic kidney disease (ADPKD) have been well known to include the mutational inactivation PKD2. Although haploinsufficiency and loss heterozygosity at Pkd2 locus can cause cyst formation in mice, polycystin-2 is frequently expressed renal ADPKD, raising possibility that deregulated activation PKD2 may be associated with cystogenesis ADPKD. To determine whether increased expression physiologically pathogenic, we generated PKD2-overexpressing transgenic mice. These mice developed typical cysts an increase proliferation apoptosis, which are reflective ADPKD phenotype. manifestations were first observed six months, progressed age. In addition, found ERK was induced by overexpression via B-Raf signaling, providing a possible molecular mechanism cystogenesis. B-Raf/MEK/ERK sequential signaling up-regulated. Additionally, partially rescues lethality knock-out therefore demonstrates transgene functional product. 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Recently, caused tubular dysfunction centrosome overduplication (32Burtey Riera Ribe Pennekamp Passage Rance Dworniczak Fontes 2008; 23: 1157-1165Crossref (32) 33Burtey Pennenkamp Luciani Mattei M.G. 32: 1193-1198Crossref (37) investigate physiological effects enhanced vivo, overexpressing Interestingly, established reproducibly displayed phenotypes closely resembling Generation Overexpressing Mice-For construction cDNA 5′- 3′-untranslated regions full coding sequence (gift from Dr. Yale University), subcloned pCAGGS plasmid using XbaI XhoI sites. isolated digesting construct PvuI StuI standard techniques generate (34Nagao Yamaguchi Kusaka Maser R.L. Takahashi Cowley B.D. Grantham 63: 427-437Abstract Briefly, injected pronuclei fertilized eggs FVB/NJ female then transferred oviducts pseudo-pregnant ICR Founder routine genotyping PCR genomic DNAs extracted tails as templates primer pairs specific (5′-TGCTCAGTTGGCATACCTTG-3′ 5′-CTCCTCAGAGTCATCCAGGC-3′). For Southern blot analyses, digested XhoI, resolved on agarose gel, nylon membrane, hybridized 32P-labeled transgene-specific probe. probe created pair (forward, 5′-AGACCTGTTTGGCTTTGCTATT-3′ reverse, 5′-ATCTCAAAGGGAAGGGCCATACTG-3′). copy number measured real-time previously described (35Pennekamp Karcher Fischer Schweickert Skryabin Horst Blum 12: 938-943Abstract (366) pairs, exist single exon 6 both Pkd2, this analysis: 5′-ATTTCTTCCTGGCAGCCTGT-3′ 5′-TCACAACATCCAGCAATTCCA-3′. Reverse Transcriptase-PCR-Total RNAs kidneys wild RNeasy mini (Qiagen). total RNA (2 μg) Moloney murine leukemia virus reverse transcriptase according manufacturer's protocol (Promega). analysis were: 5′-AGACCTGTTTGGCTTTGCTATT 5′-ATCTCAAAGGGAAGGGCCATACTG-3′ (470 bp); 5′-GCAGCTTCGCAAACAGGTCAT-3′ 5′-GAAAGTATGCTGAGTTGCTGCCCT-3′ (419 5′-CGGTGCTGAGTATGTCGTGGAG-3′ 5′-TGTCATCATACTTGGCAGGTTTC-3′ Gapdh (internal control, 503 bp). separated 1% gel electrophoresis. Western Blot Analysis-Renal tissues sacrificed fibroblasts (MEFs) homogenized lysis buffer (Intron Biotechnology). Protein concentrations determined BCA assay (Sigma). Samples subjected SDS-PAGE, immunoblotted primary antibodies against following proteins: (a gift Wu, Vanderbilt phosphorylated B-Raf, Raf-1, (MEK), (ERK), Akt, p38, (JNK1/2), JNK1/2 (Cell Signaling), Raf-1 (Santa Cruz), β-actin Histological Immunohistochemical Analyses-Tissue samples fixed 4% buffered paraformaldehyde (Sigma), embedded paraffin. Sections stained hematoxylin eosin (H&E) histological examination. each documented scoring representative longitudinal section. images captured Nikon Eclipse E400 microscope, size Scion Image 1.62. calculate sizes tubules, analyzed Axiovision 3.1 software (Carl Zeiss). antigen (PCNA) polycystin-2, Tamm-Horsfall pERK signals detected horseradish peroxidase-conjugated secondary (Dako) Nova red (Vector Lab). Counterstaining carried out hematoxylin, coverslips mounted aqueous mounting medium (Zymed Laboratories Inc.). Lectin performed (36D'Agati Trudel 3: 975-983Crossref TUNEL Situ Death Detection Kit instructions (Chemicon). MEF Culture Transfection-MEFs derived line embryos (E13.5). Whole minced dispersed 0.05% trypsin, incubated 37 °C 15 min. MEFs plated Dulbecco's modified Eagle's (Invitrogen) supplemented 10% fetal bovine serum 5% CO2 70-80% confluence. transfected small interfering Cruz) Lipofectamine RNAi MAX (Invitrogen). concentration 10 nm transfection continued 48 h. treated 30 min MEK inhibitor PD98059 (Calbiochem). transfection, subcultured day before Effectene Statistical Analyses-Data presented mean ± S.D. one-way variance (ANOVA). An error probability less than (p < 0.05) considered significant. Cytogenesis Transgenic Mice-To analyze consequences overexpress under control chicken promoter cytomegalovirus immediate early enhancer (Fig. 1A). Four lines established, (data shown) orthologues 1B). results showed copies transgene. transcriptase-PCR orthologue significant transcript levels 1C). Consistently, also up-regulated 1D). characterize overexpression, analyses. Adult exhibited affected bilaterally (Table 1). When severely affected, numerous lesions, visible surface, 2A). Cysts tubules cortical medullar 2B), most region. glomeruli dilated morphology Bowman capsules altered 2C). commonly four lines. development classified distinct stages based characteristics during progressive stage, lined layer cuboidal cells proliferative hyperplastic 2D). intermediate cyst-lining still 2, E F), but several had transformed flat 2F, black arrowhead). late completely changed G H). Therefore, variable location stage.TABLE 1The numbers miceCyst diameter (mean S.D., μm)PKD2 lineAgeTotal sizeCyst size)<200 μm<200 μmMonthsC (n = 14)6-18130.04 58.2240 (128.37 57.83)4 (250.72 27.98)D 6)6-18161.87 97.5010 (104.00 31.30)5 (277.60 77.16)E 5)2-18170.33 84.7012 (134.76 37.61)4 (277.04 102.18)F 3)7-18257.59 182.594 (151.35 29.62)3 (399.24 214.54) Open table tab express transgene, immunohistochemically localized kidneys. Consistent previous reports, prominent distal wild-type 3, A B). As expected, strongly large D). our data indicated directly involved patients, arise tubule segment (37Wilson P.D. 350: 151-164Crossref (615) PKD2-mutated animal models originate segments. origins segments proximal rats 38Gallagher A.R. Hoffmann Brown Cedzich Meruvu Podlich Feng Vries Hammes H.P. Gretz 17: 2719-2730Crossref (56) origin cysts, immunohistochemical analyses nephron segment-specific lectins Lotus tetragonolobus (LTA, tubule), Dolichos biflorus (DBA, collecting (THP, tubule) 4). All originating ducts continuous lectin staining unlike Pkd2-deficient Large 4, Small originated F). range kidneys, clinical features Renal appeared months age examined 18 old. phenotype relatively correlated aging process. Cyst aged (6-month-old, 190.27 79.15 μm diameter; 18-month-old, 318.47 129.82 μm) 5). greater 200 (6-month old, n 4; 18-month 11). aging. Deregulation Apoptosis Proliferation Kidneys Mice-Because apoptosis prerequisites (39Zhou X.J. Kukes Diagn. Pathol. 65-68Crossref (22) 40Nadasdy Laszik Z. Lajoie Blick K.E. Wheeler D.E. Silva F.G. 1995; 5: 1462-1468Crossref evaluated processes 6). Most PCNA-positive around especially stage 6A). per significantly 6B). Compared cells, apoptotic mainly 6C). 6D). Coincidentally, patterns bromodeoxyuridine incorporation similar those PCNA shown). Downstream Effector Is Activated Signaling Formation-Progression delayed suppressing activity (41Omori Hida Fujita Tanimura Kohno Awazu 1604-1614Crossref (117) reported regulated calcium (42Yamaguchi Wallace D.P. Magenheimer B.S. Hempson S.J. Calvet J.P. 40419-40430Abstract (270) binds forming heterodimer, essential properties disrupting C-terminal fragment It plausible contribute modulating 43Yamaguchi Nagao Belibi Pelling J.C. 1983-1994Abstract (273) 44Yamaguchi Reif G.A. Hedge 178-187Crossref (210) test hypothesis, confirmed he level pathway analysis. phosphorylation activated 7A). Fig. 7B shows quantification 7A. Akt/total Akt higher NHK compared upstream down-regulated 7C). confirm checked change critical inhibition MEFs. reduced (15 μm, PD98058) 7D). Finally, MAPK pathways elevated p38 unaffected 7E). We investigated status next downstream effector kidney. 8A, relative Because causes 8A). Similar MEFs, suggest step induction because either deficiency evaluate precisely, cyst-surrounding 8, B-D). transiently exogenous 8E, consistent observations Product Functionally Active Mice-As nullizygosity 93