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dc.contributor.advisorEccles, Michael
dc.contributor.advisorStayner, Cherie
dc.contributor.authorBates, Michael
dc.date.available2016-08-18T21:47:04Z
dc.date.copyright2016
dc.identifier.citationBates, M. (2016). DNA Methylation in Polycystic Kidney Disease and Bio-distribution of the C3-G12 peptide (Thesis, Master of Science). University of Otago. Retrieved from http://hdl.handle.net/10523/6740en
dc.identifier.urihttp://hdl.handle.net/10523/6740
dc.description.abstractAutosomal dominant polycystic kidney disease (ADPKD) is the most common inherited form of renal disease, and is characterised by the formation of fluid-filled cysts within organs, particularly in the kidneys. The two genes that are mutated in ADPKD, PKD1 and PKD2, encode proteins in a plasma membrane complex that facilitates calcium ion release in the cell. Patients typically have an inherited mutation in one of these genes from birth and it is postulated that a second hit in the same gene is required for pathogenesis. Changes in downstream signalling pathways then give rise to the cysts, physically limiting the function of the kidney, and without intervention can lead to renal failure. Mutations in the second allele have been found to be responsible for the second hit in some but not all cysts. We postulate that this “second hit” could also be caused by epigenetic mechanisms, particularly involving changes in DNA methylation. Epigenetic modifying drugs have been shown to reduce cyst formation and limit the pathology of the disease. The use of a targeted delivery system conjugated to an epigenetic modifying drug could ensure the drug concentration is sufficient within the renal tissue, with minimal exposure elsewhere in the body, reducing side effects. Conjugates such as this could initially be most effectively be tested in a small animal model, such as in mice. To facilitate these types of studies, we have investigated DNA methylation in two regions of the mouse Pkd1 gene. Bisulfite sequencing of kidney DNA samples from wildtype mice, and the del34 Pkd1 mouse model heterozygous for a Pkd1 mutation, were performed. We found that the promotor CpG island was unmethylated whereas the gene body region examined was hypermethylated in both Pkd1 heterozygous and wildtype mouse kidneys. However, quantitative PCR expression analysis revealed consistently higher expression of the Pkd1 ii gene in Pkd1 heterozygous samples when compared to wildtype. Hematoxylin and eosin staining of mouse kidneys sections revealed at least one confirmed cyst in a Pkd1 heterozygous sample, but cysts are considerably fewer in number when compared with the human disease. To extend the limited data available on the methylation landscape in human PKD1, we have performed reduced representation bisulfite sequencing (RRBS) on two ADPKD human kidney samples, and three wildtype kidney samples. In contrast to the mouse data, we found methylation differences in the PKD1 gene in ADPKD samples, when compared to wildtype kidney tissue, and changes in other key genes involved in cystogenesis. These data further support a potential role for DNA methylation in ADPKD pathogenesis. To be able to target a potential methylation difference specifically in the kidney, as a therapeutic option for ADPKD, we have investigated the ability of the galectin-3-avid (C3 G12) binding peptide to specifically localise to the kidney. We injected the peptide bound to a Cy3.5 fluorescent probe into wildtype mice. We observed that the C3-G12-Cy3.5 conjugate accumulated in the mouse kidneys and was retained there for at least 30 minutes. In contrast, little to no fluorescence was observed in the heart, liver, lungs and spleen. These data suggest that the C3-G12 peptide could be used in a selective delivery mechanism for future therapies. This research provides new data on the mouse and human methylation landscapes in ADPKD and confirms the kidney targeting nature of the C3-G12 peptide in mice.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectADPKD
dc.subjectPKD
dc.subjectPolycystic
dc.subjectKidney
dc.subjectRenal
dc.subjectMethylation
dc.subjectBisulfite
dc.subjectSequencing
dc.subjectNext-gen
dc.subjectPKD1
dc.subjectdel34
dc.subjectCpG
dc.subjectRRBS
dc.subjectC3-G12
dc.titleDNA Methylation in Polycystic Kidney Disease and Bio-distribution of the C3-G12 peptide
dc.typeThesis
dc.date.updated2016-08-18T08:53:46Z
dc.language.rfc3066en
thesis.degree.disciplineGenetics
thesis.degree.nameMaster of Science
thesis.degree.grantorUniversity of Otago
thesis.degree.levelMasters
otago.openaccessOpen
otago.evidence.presentYes
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