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Title: Metabolic biomarkers of aminoglycoside nephrotoxicity
Author: Rodrigues, Alison
ISNI:       0000 0004 5363 2087
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2014
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Drug-induced nephrotoxicity is a limiting factor to the efficacy and safety of various therapeutics including the aminoglycoside antibiotics. Aminoglycosides, such as gentamicin, cause proximal tubule injury in a significant proportion of individuals they are given to. The onset of this adverse drug reaction is currently managed by the monitoring of serum peak and trough levels and measurement of the classic renal functional markers serum creatinine and blood urea nitrogen. The limitations of these biomarkers are well established but novel, sensitive proximal tubule-specific biomarkers, such as kidney injury molecule-1, are gradually coming to the fore. Still, management of aminoglycoside nephrotoxicity is lacking a personalised strategy whereby the risk of a patient developing proximal tubule injury can be established at the individual level before exposure. Prior studies of gentamicin nephrotoxicity pin-pointed that HMG-CoA reductase inhibitors, also known as statins, could inhibit the accumulation of gentamicin in vitro and therefore reduce the cytotoxicity of the drug. In order to study HMG-CoA reductase and its relationship to aminoglycoside accumulation further, an LC-MS/MS based assay was developed and validated to measure the product of the enzyme, mevalonic acid, in the urine of rats and humans. Urinary mevalonic acid was converted to mevalonolactone at pH 2, extracted alongside a deuterated internal standard using ethyl acetate and quantified by reversed-phase LC-MS/MS. The assay had a broad dynamic range of 0.0156–10 μg/mL with precision <15% CV and accuracy 85–115% to suit the natural variation within species and between non-clinical and clinical samples. To demonstrate the utility of the assay and to ascertain the natural diurnal oscillations in HMG-CoA reductase activity, mevalonic acid was quantified in the urine of rats, mice and healthy children. In rats the excretion of mevalonic acid was significantly greater in urine collected during 22:00–10:00 h (mean 9.7 ± 2.3 μg/mg UCr) compared with 10:00–22:00 h (mean 3.4 ± 1.3 μg/mg UCr). In a human paired urine study, in 60% of individuals, morning collections had significantly greater concentrations of mevalonic acid than evening collections where the morning excretion was, on average, 105% greater. The diurnal rhythm of HMG-CoA reductase activity was investigated in relation to aminoglycoside nephrotoxicity in a repeat-dose gentamicin rat model. A strong positive relationship between pre-dose mevalonate excretion, gentamicin accumulation and kidney injury in the renal cortex was observed. Animals administered gentamicin at 10:00 h experienced greater gentamicin accumulation and kidney injury, compared to animals on the 22:00 h dosing schedule which corresponded to greater mevalonate excretion in the hours prior to 10:00 h compared to 22:00 h. These data support the idea that there is a contributory relationship between HMG-CoA reductase activity, the uptake of gentamicin and subsequent nephrotoxicity. Investigations with the aminoglycoside tobramycin did not reach the same conclusion as no clear relationship was observed. In contrast to the HMG-CoA reductase focussed research, a non-targeted metabonomic approach to understanding gentamicin nephrotoxicity was undertaken. Multivariate analyses of 1H-NMR spectra from gentamicin-exposed rats revealed multiple major perturbations in the urine and serum metabolome, prior to kidney injury molecule-1 elevation (urine OPLS-DA model 12 h post-dose Q2Y=0.93, p=0.007). Depletion of metabolites related to energy production and elevation of metabolites implicated in oxidative stress suggests gentamicin had a profound effect on the mitochondria of the proximal tubule epithelial cells. Quantification of metabolites such as the TCA cycle intermediates could be a non-invasive alternative to monitoring the toxicity of aminoglycosides prior to overt renal functional changes. Multivariate analyses of 1H-NMR urine spectra were also subjected to a pharmacometabonomic approach whereby the pre-dose or early post-dose metabolomewas integrated with post-dose kidney injury molecule-1 measurements in order to group individuals based on their differential response to gentamicin. Early-intervention metabolite signatures were identified to have an inverse relationship to kim-1 excretion, providing further evidence that the TCA cycle intermediates could be useful prognostic biomarkers of gentamicin nephrotoxicity. Analysis of pre-dose profiles identified gut- microbial metabolite 3-HPPA as correlated to the post-dose toxicity of gentamicin; follow up studies demonstrated that 3-HPPA excretion also had a positive relationship to urinary mevalonic acid. Hence, the pharmacometabonomic analyses implicated gut microbial and host HMG-CoA reductase activity as related to the extent of gentamicin nephrotoxicity, which certainly warrants additional investigations. The adoption of targeted and non-targeted biomarker identification techniques has proven successful in this research. Mevalonic acid and HMG-CoA reductase are promising mechanistic factors which may affect susceptibility to aminoglycoside nephrotoxicity in man and research will be facilitated by the development of the LC-MS/MS assay described herein. Certainly, the use of statins as a prophylactic measure against aminoglycoside nephrotoxicity will be explored. Comprehensive analysis of the metabolome has identified the importance of the perturbation of energy metabolism and oxidative stress in the onset and development of gentamicin nephrotoxicity and in addition, integration of these vast data sets with the novel biomarker kidney injury molecule-1 has revealed that the gut microbiome could also influence an individual’s susceptibility to this adverse drug reaction.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: RM Therapeutics. Pharmacology