Investigation of Several Compounds for the Treatment of Autosomal Dominant Polycystic Kidney Disease
Polycystic kidney disease (PKD) is a leading cause of kidney failure, and one of the most prominent rare hereditary diseases in New Zealand, as well as globally. The autosomal dominant form of this disease (ADPKD) has a country dependant prevalence rate of between one in 400 and one in 1000, and a 50% inheritance rate of the causal polycystin gene mutation in the offspring of an affected individual. The polycystin proteins act as a cell signalling complex in many cell types including the cilia of kidney tubule cells. Polycystin-1 makes contact with the extracellular matrix while polycystin-2 promotes the flow of calcium into the cell to regulate a cellular response during environmental change. A malfunction in the polycystin complex caused by genetic mutation results in the dysregulation of cellular processes due to a change in cyclic-AMP mediated activity and release of the cells from the highly moderated cell cycle, leading to uncontrolled proliferation and cyst formation. Once the kidney cells become cystic they lose the ability to exchange material with surrounding capillaries which ultimately reduces the kidney’s ability to filter the blood. Tolvaptan treatment is the only current means of slowing the process of cyst formation, by targeting the vasopressin pathway and inhibiting the conversion of ATP to cyclic AMP, thus reducing fluid uptake into the cells. Alongside Tolvaptan, a number of other drug targets have proven promising in PKD models, yet these therapies often fail to proceed past clinical trials due to drug toxicity and off target side effects which cause the continuation of treatment to be intolerable for patients. Therefore, investigation of a long-term solution for cyst reduction to treat PKD is imperative. This project investigated the efficacy of novel compounds to reduce cyst growth by targeting pathways modified in ADPKD, as well as the use of a kidney specific peptide as a compound chaperone. Ultimately this study aimed to identify the impact of novel compounds on cyst growth, compound associated pathway alterations, and kidney specific peptide uptake. The novel compounds (SAHA, EG1, Gymnodimine and Portimine) were optimised for treatment of the ADPKD cyst growth cell line models MDCK and LLC-PK1. These cell lines were used to measure the effect compound treatment had on cystic area and changes in the activity of the compound’s target. The actions of the compounds were determined via HDAC activity assay, western blotting or Annexin V flow cytometry assays. Uptake of the G3-C12 peptide into cells of the kidney tubules was achieved through flow cytometry detection of a fluorescently labelled peptide in vitro, as well as immunofluorescent staining of kidney derived cells and peptide injected mouse kidneys. Treating the cyst growth assays with EG1 and Portimine caused significant decreases in cystic growth. The EG1 treated cultures had 2 to 7-fold less area growth than the controls, and the Portimine treated cultures had 2 to 9-fold less area growth than the controls. Gymnodimine treatment produced a significant reduction in the LLC-PK1 spheroid growth area, with these cultures having 22-fold less area growth than the controls. Additionally, the therapeutic compound SAHA caused the MDCK spheroids to have reduced cystic area growth but was unable to reach statistical significance, yet when attached to the peptide there was a significant 2 to 3-fold decrease in the cyst growth area of the MDCK cultures. The cyst growth assays suggested that novel therapeutic compound treatment of EG1 and Portimine consistently had the greatest impact on cyst growth area change in vitro, however the attachment of the kidney specific peptide to SAHA also enabled this compound to slow cystic growth. Additionally, kidney targeted peptide uptake in vivo and in vitro identified peptide accumulation within the proximal tubular cells of mice kidneys, and in vitro human ADPKD cells.
Advisor: Eccles, Michael; Stayner, Cherie
Degree Name: Master of Science
Degree Discipline: Pathology
Publisher: University of Otago
Keywords: ADPKD; Compounds; Therapy
Research Type: Thesis