Molecular biomarkers and regulators of susceptibility to drug induced kidney injury

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

Assessing the Effectiveness of a Far-Red Fluorescent Reporter for Tracking Stem Cells In Vivo

Far-red fluorescent reporter genes can be used for tracking cells non-invasively in vivo using fluorescence imaging. Here, we investigate the effectiveness of the far-red fluorescent protein, E2-Crimson (E2C), for tracking mouse embryonic cells (mESCs) in vivo following subcutaneous administration into mice. Using a knock-in strategy, we introduced E2C into the Rosa26 locus of an E14-Bra-GFP mESC line, and after confirming that the E2C had no obvious effect on the phenotype of the mESCs, we injected them into mice and imaged them over nine days. The results showed that fluorescence intensity was weak, and cells could only be detected when injected at high densities. Furthermore, intensity peaked on day 4 and then started to decrease, despite the fact that tumour volume continued to increase beyond day 4. Histopathological analysis showed that although E2C fluorescence could barely be detected in vivo at day 9, analysis of frozen sections indicated that all mESCs within the tumours continued to express E2C. We hypothesise that the decrease in fluorescence intensity in vivo was probably due to the fact that the mESC tumours became more vascular with time, thus leading to increased absorbance of E2C fluorescence by haemoglobin. We conclude that the E2C reporter has limited use for tracking cells in vivo, at least when introduced as a single copy into the Rosa26 locus

Multicolour In Vivo Bioluminescence Imaging Using a NanoLuc‐Based BRET Reporter in Combination with Firefly Luciferase

The ability to track the biodistribution and fate of multiple cell populations administered to rodents has the potential to facilitate the understanding of biological processes in a range of fields including regenerative medicine, oncology, and host/pathogen interactions. Bioluminescence imaging is an important tool for achieving this goal, but current protocols rely on systems that have poor sensitivity or require spectral decomposition. Here, we show that a bioluminescence resonance energy transfer reporter (BRET) based on NanoLuc and LSSmOrange in combination with firefly luciferase enables the unambiguous discrimination of two cell populations in vivo with high sensitivity. We insert each of these reporter genes into cells using lentiviral vectors and demonstrate the ability to monitor the cells’ biodistribution under a wide range of administration conditions, including the venous or arterial route, and in different tissues including the brain, liver, kidneys, and tumours. Our protocol allows for the imaging of two cell populations in the same imaging session, facilitating the overlay of the signals and the identification of anatomical positions where they colocalise. Finally, we provide a method for postmortem confirmation of the presence of each cell population in excised organs

Imaging technologies for monitoring the safety, efficacy and mechanisms of action of cell-based regenerative medicine therapies in models of kidney disease

AbstractThe incidence of end stage kidney disease is rising annually and it is now a global public health problem. Current treatment options are dialysis or renal transplantation, which apart from their significant drawbacks in terms of increased morbidity and mortality, are placing an increasing economic burden on society. Cell-based Regenerative Medicine Therapies (RMTs) have shown great promise in rodent models of kidney disease, but clinical translation is hampered due to the lack of adequate safety and efficacy data. Furthermore, the mechanisms whereby the cell-based RMTs ameliorate injury are ill-defined. For instance, it is not always clear if the cells directly replace damaged renal tissue, or whether paracrine effects are more important. Knowledge of the mechanisms responsible for the beneficial effects of cell therapies is crucial because it could lead to the development of safer and more effective RMTs in the future. To address these questions, novel in vivo imaging strategies are needed to monitor the biodistribution of cell-based RMTs and evaluate their beneficial effects on host tissues and organs, as well as any potential adverse effects. In this review we will discuss how state-of-the-art imaging modalities, including bioluminescence, magnetic resonance, nuclear imaging, ultrasound and an emerging imaging technology called multispectral optoacoustic tomography, can be used in combination with various imaging probes to track the fate and biodistribution of cell-based RMTs in rodent models of kidney disease, and evaluate their effect on renal function

Preventing Plasmon Coupling between Gold Nanorods Improves the Sensitivity of Photoacoustic Detection of Labeled Stem Cells in Vivo

© 2016 American Chemical Society.Gold nanorods are excellent contrast agents for imaging technologies which rely on near-infrared absorption such as photoacoustic imaging. For cell tracking applications, the cells of interest are labeled with the contrast agent prior to injection. However, after uptake into cells by endocytosis, the confinement and high concentration in endosomes leads to plasmon band broadening and reduced absorbance. This would limit the potential of multispectral optoacoustic tomography in terms of spectral processing and, consequently, sensitivity. Here, we show that steric hindrance provided by silica coating of the nanorods leads to the preservation of their spectral properties and improved photoacoustic sensitivity. This strategy allowed the detection and monitoring of as few as 2 × 104 mesenchymal stem cells in mice over a period of 15 days with a high spatial resolution. Importantly, the silica-coated nanorods did not affect the viability or differentiation potential of the transplanted mesenchymal stem cells

Early childhood respiratory morbidity and antibiotic use in ex-preterm infants: A primary care population-based cohort study

Background Globally, bronchopulmonary dysplasia (BPD) continues to increase in preterm infants. Recent studies exploring subsequent early childhood respiratory morbidity have been small or focused on hospital admissions.Primary aim Examine early childhood rates of primary care consultations for respiratory tract infections (RTI), lower respiratory tract infections (LRTI), wheeze and antibiotic prescriptions (Abx Px) in ex-preterm and term children. Secondary aim: examine differences between preterm infants discharged home with or without oxygen.Methods Retrospective cohort study using linked electronic primary care and hospital databases of children born between 1997 and 2014. We included 253 677 eligible children, with 1666 born preterm [less than] 32 weeks' gestation, followed up from primary care registration to age 5 years. Adjusted incidence rate ratios (aIRR) were calculated.Results Ex-preterm infants had higher rates of morbidity across all respiratory outcomes. After adjusting for confounders, aIRRs for RTI (1.37, 95% CI 1.33–1.42), LRTI (2.79, 95% CI 2.59–3.01), wheeze (3.05, 95% CI 2.64–3.52) and Abx Px (1.49, 95% CI 1.44–1.55) were higher for ex-preterm infants. Ex-preterm infants discharged home on oxygen had significantly greater morbidity across all respiratory diagnoses and Abx Px compared to those without home oxygen. The highest rates of respiratory morbidity were observed in children from the most deprived socioeconomic groups.Conclusion Ex-preterm infants, particularly those with BPD requiring home oxygen, have significant respiratory morbidity and antibiotic prescriptions in early childhood. With the increasing prevalence of BPD, further research should focus on strategies to reduce the burden of respiratory morbidity in these high-risk infants after hospital discharge

A Noninvasive Imaging Toolbox Indicates Limited Therapeutic Potential of Conditionally Activated Macrophages in a Mouse Model of Multiple Organ Dysfunction

Cell-based regenerative medicine therapies require robust preclinical safety, efficacy, biodistribution, and engraftment data prior to clinical testing. To address these challenges, we have developed an imaging toolbox comprising multispectral optoacoustic tomography and ultrasonography, which allows the degree of kidney, liver, and cardiac injury and the extent of functional recovery to be assessed noninvasively in a mouse model of multiorgan dysfunction. This toolbox allowed us to determine the therapeutic effects of adoptively transferred macrophages. Using bioluminescence imaging, we could then investigate the association between amelioration and biodistribution. Macrophage therapy provided limited improvement of kidney and liver function, although not significantly so, without amelioration of histological damage. No improvement in cardiac function was observed. Biodistribution analysis showed that macrophages homed and persisted in the injured kidneys and liver but did not populate the heart. Our data suggest that the limited improvement observed in kidney and liver function could be mediated by M2 macrophages. More importantly, we demonstrate here the utility of the imaging toolbox for assessing the efficacy of potential regenerative medicine therapies in multiple organs

Multimodal cell tracking from systemic administration to tumour growth by combining gold nanorods and reporter genes.

Understanding the fate of exogenous cells after implantation is important for clinical applications. Preclinical studies allow imaging of cell location and survival. Labelling with nanoparticles enables high sensitivity detection, but cell division and cell death cause signal dilution and false positives. By contrast, genetic reporter signals are amplified by cell division. Here, we characterise lentivirus-based bi-cistronic reporter gene vectors and silica-coated gold nanorods (GNRs) as synergistic tools for cell labelling and tracking. Co-expression of the bioluminescence reporter luciferase and the optoacoustic reporter near-infrared fluorescent protein iRFP720 enabled cell tracking over time in mice. Multispectral optoacoustic tomography (MSOT) showed immediate biodistribution of GNR-labelled cells after intracardiac injection and successive clearance of GNRs (day 1-15) with high resolution, while optoacoustic iRFP720 detection indicated tumour growth (day 10-40). This multimodal cell tracking approach could be applied widely for cancer and regenerative medicine research to monitor short- and long-term biodistribution, tumour formation and metastasis

Perylene Diimide Nanoprobes for In Vivo Tracking of Mesenchymal Stromal Cells Using Photoacoustic Imaging.

Noninvasive bioimaging techniques are critical for assessing the biodistribution of cellular therapies longitudinally. Among them, photoacoustic imaging (PAI) can generate high-resolution images with a tissue penetration depth of ∼4 cm. However, it is essential and still highly challenging to develop stable and efficient near-infrared (NIR) probes with low toxicity for PAI. We report here the preparation and use of perylene diimide derivative (PDI) with NIR absorbance (around 700 nm) as nanoprobes for tracking mesenchymal stromal cells (MSCs) in mice. Employing an in-house synthesized star hyperbranched polymer as a stabilizer is the key to the formation of stable PDI nanoparticles with low toxicity and high uptake by the MSCs. The PDI nanoparticles remain within the MSCs as demonstrated by in vitro and in vivo assessments. The PDI-labeled MSCs injected subcutaneously on the flanks of the mice are clearly visualized with PAI up to 11 days postadministration. Furthermore, bioluminescence imaging of PDI-labeled luciferase-expressing MSCs confirms that the administered cells remain viable for the duration of the experiment. These PDI nanoprobes thus have good potential for tracking administered cells in vivo using PAI

Measures of kidney function by minimally invasive techniques correlate with histological glomerular damage in SCID mice with adriamycin-induced nephropathy

Maximising the use of preclinical murine models of progressive kidney disease as test beds for therapies ideally requires kidney function to be measured repeatedly in a safe, minimally invasive manner. To date, most studies of murine nephropathy depend on unreliable markers of renal physiological function, exemplified by measuring blood levels of creatinine and urea, and on various end points necessitating sacrifice of experimental animals to assess histological damage, thus counteracting the principles of Replacement, Refinement and Reduction. Here, we applied two novel minimally invasive techniques to measure kidney function in SCID mice with adriamycin-induced nephropathy. We employed i) a transcutaneous device that measures the half-life of intravenously administered FITC-sinistrin, a molecule cleared by glomerular filtration; and ii) multispectral optoacoustic tomography, a photoacoustic imaging device that directly visualises the clearance of the near infrared dye, IRDye 800CW carboxylate. Measurements with either technique showed a significant impairment of renal function in experimental animals versus controls, with significant correlations with the proportion of scarred glomeruli five weeks after induction of injury. These technologies provide clinically relevant functional data and should be widely adopted for testing the efficacies of novel therapies. Moreover, their use will also lead to a reduction in experimental animal numbers