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Title: Molecular biomarkers and regulators of susceptibility to drug induced kidney injury
Author: Sharkey, Jack William
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2013
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Adverse drug reactions (ADRs) are undesirable effects of any therapeutic compound beyond its desired pharmacological effect. They are not only a serious issue for the sufferer but also bear a large societal cost and are one of the main reasons for the withdrawal of drugs from the market. Nephrotoxicity is the toxic affect of any exogenous compound on the kidney. The kidney is particularly susceptible to adverse effects of drugs due to its adaptations which allow it to carry out its physiological role efficiently. The kidney receives approximately 25 % of the cardiac output so is exposed to any blood borne toxin at high levels. The kidney also concentrates the filtrate as it passes through the nephron which exposes the epithelial cells of the nephron to much greater concentrations of any toxin present. The fact that the cells of the nephron are metabolically active and have active transport mechanisms also contribute towards the susceptibility of the kidney to ADRs. Adverse events which affect the kidney are often initially very subtle but can rapidly progress into more serious events if not detected early. A biomarker is any quantifiable change in an endogenous protein or molecule which can be indicative of a disease process or state. Current gold standard biomarkers of kidney injury include Serum Creatinine and blood urea nitrogen. Both of these are suboptimal biomarkers of kidney injury in terms of sensitivity and specificity so there is a real need for the development of more specific, translational biomarkers of kidney injury. Ideally these next generation biomarkers would also provide information on the location of the injury or the extent of the injury. MicroRNAs (miRNAs) are short ribonucleic acid sequences which are the smallest functional non-coding RNA units in plants and animals. Recent research has implicated miRNAs in many disease states, particularly cancers where many miRNA species have been shown to be aberrantly expressed. MiRNAs have also been shown to have potential as biomarkers of drug induced liver injury (DILI). This thesis focuses on the potential of miRNAs to serve as translational biomarkers of kidney injury. Techniques used to isolate, purify and quantify miRNA species were validated to determine the suitability of them for routine quantification in any laboratory. The qPCR technique was shown to be highly precise and sensitive for miRNA quantification. Intra-assay and inter-assay variation was low (<12 %), recovery was variable (54 – 89 %). Variable recovery was controlled for by the development of an internal synthetic c.elegans standard. Significant urinary elevations in four kidney enriched miRNAs were observed at all time points (3hr) preceding SCr elevations (9hr IR; 0.2±0.01 mg/dL. 9hr Control; 0.1±0.01 mg/dL) following 10 minute ischemic reperfusion (IR). MiRNA elevations (3hr IR; miR-26b 7.01. 3hr control; 0.38 (ΔΔCt normalised to U6snRNA)) were also comparable to Kim-1 (3hr IR; 443±71pg/mg.UCr. 3hr Control; 96±3171pg/mg.UCr) and histological changes (3hr). No significant elevations in miR-26b, 30b or 30c were observed in the serum of a murine model of paracetamol induced liver injury whereas miR-192 was found to be significantly elevated. NAG (Control 122 mU/ml mg UCr, Cisplatin 313 mU/ml mg UCr) and Kim-1 (Control 1003 pg/mg UCr, Cisplatin 10617pg/mg UCr) were significantly elevated in the urine of a Cisplatin murine model of kidney injury 72 hours post-administration whereas a total of 18 miRNA species were significantly elevated by greater than twofold 48 hours post-administration. Five miRNAs were found to be significantly elevated and two decreased in the serum and four were elevated in the kidney tissue of Cisplatin administered mice. In the urine of an Adriamycin induced model of kidney injury one miRNA was significantly elevated and three reduced. 28 were significantly elevated in the serum and three were reduced while 14 were significantly elevated in the kidney tissue. MiR-34a was found to be significantly elevated in the kidney tissue of both the Adriamycin and Cisplatin models. Transfection of HEK293 cells with a miR-34a mimic was confirmed through the quantification of the level of Bcl-2 and CDK6 protein (mean 0.56 and 0.83 respectively). Cisplatin (50 µM) caused a significant reduction in cell viability in transfected cells (MTS fold change 0.12) whereas it did not in control cells. Cisplatin (50 µM) caused significantly greater expression of cleaved caspase 3 in miR-34a mimic transfected cells than in control cells. This work shows that the assays and techniques used to isolate, purify and quantify miRNA species are precise and sensitive. It shows that kidney enriched miRNA species can be quantified in the urine of animals with IR induced kidney injury and are significantly elevated when compared to control animals with at least the same sensitivity as urinary Kim-1 and greater sensitivity than SCr. It also identifies a number of miRNA species which are significantly altered in a Cisplatin and Adriamycin murine model of drug induced kidney injury (DIKI) which may have utility as biomarkers. They may also present locational biomarkers of kidney injury and warrant further investigation. Finally it shows that the modulation of miR-34a can alter susceptibility to toxicity in vitro which suggests that miRNAs may represent therapeutic interventions in the future.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available