LUIZ FERNANDO ONUCHIC
Projetos de Pesquisa
Unidades Organizacionais
Departamento de Clínica Médica, Faculdade de Medicina - Docente
LIM/29 - Laboratório de Nefrologia Celular, Genética e Molecular, Hospital das Clínicas, Faculdade de Medicina - Líder
LIM/29 - Laboratório de Nefrologia Celular, Genética e Molecular, Hospital das Clínicas, Faculdade de Medicina - Líder
3 resultados
Resultados de Busca
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- Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1(2014) BODDU, Ravindra; YANG, Chaozhe; O'CONNOR, Amber K.; HENDRICKSON, Robert Curtis; BOONE, Braden; CUI, Xiangqin; GARCIA-GONZALEZ, Miguel; IGARASHI, Peter; ONUCHIC, Luiz F.; GERMINO, Gregory G.; GUAY-WOODFORD, Lisa M.Autosomal recessive polycystic kidney disease (ARPKD) results from mutations in the human PKHD1 gene. Both this gene, and its mouse ortholog, Pkhd1, are primarily expressed in renal and biliary ductal structures. The mouse protein product, fibrocystin/polyductin complex (FPC), is a 445-kDa protein encoded by a 67-exon transcript that spans > 500 kb of genomic DNA. In the current study, we observed multiple alternatively spliced Pkhd1 transcripts that varied in size and exon composition in embryonic mouse kidney, liver, and placenta samples, as well as among adult mouse pancreas, brain, heart, lung, testes, liver, and kidney. Using reverse transcription PCR and RNASeq, we identified 22 novel Pkhd1 kidney transcripts with unique exon junctions. Various mechanisms of alternative splicing were observed, including exon skipping, use of alternate acceptor/donor splice sites, and inclusion of novel exons. Bioinformatic analyses identified, and exon-trapping minigene experiments validated, consensus binding sites for serine/arginine-rich proteins that modulate alternative splicing. Using site-directed mutagenesis, we examined the functional importance of selected splice enhancers. In addition, we demonstrated that many of the novel transcripts were polysome bound, thus likely translated. Finally, we determined that the human PKHD1 R760H missense variant alters a splice enhancer motif that disrupts exon splicing in vitro and is predicted to truncate the protein. Taken together, these data provide evidence of the complex transcriptional regulation of Pkhd1/PKHD1 and identified motifs that regulate its splicing. Our studies indicate that Pkhd1/PKHD1 transcription is modulated, in part by intragenic factors, suggesting that aberrant PKHD1 splicing represents an unappreciated pathogenic mechanism in ARPKD.
- International Multi-Specialty Delphi Survey: Identification of Diagnostic Criteria for Hepatic and Renal Cyst Infection(2016) LANTINGA, Marten A.; DARDING, Alexander J. M.; SEVAUX, Ruud G. L. de; ALAM, Ahsan; BLEEKER-ROVERS, Chantal P.; BOBOT, Mickael; GALL, Emilie Cornec-Le; GEVERS, Tom J. G.; HASSOUN, Ziad; MEIJER, Esther; MRUG, Michal; NEVENS, Frederik; ONUCHIC, Luiz F.; PEI, York; PICCOLI, Giorgina B.; PIRSON, Yves; RANGAN, Gopala K.; TORRA, Roser; VISSER, Folkert W.; JOURET, Francois; KANAAN, Nada; OYEN, Wim J. G.; SUWABE, Tatsuya; TORRES, Vicente E.; DRENTH, Joost P. H.Background: Cyst infection is one of the complications of autosomal dominant polycystic kidney disease and polycystic liver disease. The diagnosis is typically made on a mix of clinical, laboratory and imaging abnormalities but the importance of individual items is uncertain. We aimed to perform a Delphi survey amongst physicians to achieve consensus on diagnostic criteria. Methods: We retrieved diagnostic items from the literature and conducted physician and patient interviews. All items were combined to create the online questionnaire. Participants rated each item during 3 consecutive rounds. Items were rated for diagnostic helpfulness for hepatic and renal cyst infection on a 9-point scale with anchors, from extremely unimportant (n = 1) to extremely important (n = 9). We determined consensus with the disagreement index. The median rating of each item was calculated and categorized into inappropriate (<= 3.4), uncertain (3.5-6.4) or appropriate (>= 6.5). By combining all items that reached an appropriate consensus rating, we developed a diagnostic algorithm based on expert consensus. Results: We invited 58 physicians to participate in the survey. In total, 35 (60%) responded to round 1 of which 91% (n = 32) and 86% (n = 30) responded to round 2 and 3, respectively. The final panel included 23 nephrologists, 5 hepatologists, a nuclear medicine specialist and an infectious disease physician from 11 countries (male 67%, mean age 47 +/- 11 years, median clinical experience 21 years). The panel rated the diagnostic helpfulness of 59 potential items. Ultimately, 22 hepatic and 26 renal items were rated appropriate, including positive blood cultures and fluorodeoxyglucose positron-emission CT imaging. Ultrasonography and absence of intracystic bleeding were amongst those deemed uncertain or inappropriate. Subsequently, by combining items rated appropriate, we developed a clinical tool to diagnose hepatic and renal cyst infection. Conclusions: We identified diagnostic items for hepatic and renal cyst infection and developed an expert-based diagnostic algorithm, which may aid physicians in the diagnostic work-up. A prospective study is necessary to validate this algorithm. (C) 2016 S. Karger AG, Basel
- NEDD4-family E3 ligase dysfunction due to PKHD1/Pkhd1 defects suggests a mechanistic model for ARPKD pathobiology(2017) KAIMORI, Jun-ya; LIN, Cheng-Chao; OUTEDA, Patricia; GARCIA-GONZALEZ, Miguel A.; MENEZES, Luis F.; HARTUNG, Erum A.; LI, Ao; WU, Guanqing; FUJITA, Hideaki; SATO, Yasunori; NAKANUMA, Yasuni; YAMAMOTO, Satoko; ICHIMARU, Naotsugu; TAKAHARA, Shiro; ISAKA, Yoshitaka; WATNICK, Terry; ONUCHIC, Luiz F.; GUAY-WOODFORD, Lisa M.; GERMINO, Gregory G.Autosomal recessive polycystic kidney disease (ARPKD) is an important childhood nephropathy, occurring 1 in 20,000 live births. The major clinical phenotypes are expressed in the kidney with dilatation of the collecting ducts, systemic hypertension, and progressive renal insufficiency, and in the liver with biliary dysgenesis, portal tract fibrosis, and portal hypertension. The systemic hypertension has been attributed to enhanced distal sodium reabsorption in the kidney, the structural defects have been ascribed to altered cellular morphology, and fibrosis to increased TGF-beta signaling in the kidney and biliary tract, respectively. The pathogenic mechanisms underlying these abnormalities have not been determined. In the current report, we find that disrupting PKHD1 results in altered sub-cellular localization and function of the C2-WWW-HECT domain E3 family of ligases regulating these processes. We also demonstrate altered activity of RhoA and increased TGF-beta signaling and ENaC activity. Linking these phenomena, we found that vesicles containing the PKHD1/Pkhd1 gene product, FPC, also contain the NEDD4 ubiquitin ligase interacting protein, NDFIP2, which interacts with multiple members of the C2-WWW-HECT domain E3 family of ligases. Our results provide a mechanistic explanation for both the cellular effects and in vivo phenotypic abnormalities in mice and humans that result from Pkhd1/PKHD1 mutation.