Investigation of microRNAs as markers of human vascular dysfunction in kidney disease

Vascular dysfunction commonly co-exists with kidney disease, which results in a substantially increased risk of cardiovascular disease (CVD). A severe, acute form of this vascular-renal phenotype is seen in those presenting with systemic vasculitis associated with autoantibodies to neutrophil cytoplasmic antigens (ANCA) - a rare inflammatory disorder. The most frequent severe manifestation of ANCA vasculitis involves endothelial injury giving rise to rapidly progressive glomerulonephritis in the kidney. Despite current treatments, overall survival remains poor with many patients suffering chronic inflammation, a major contributor to the development and progression of both CVD and chronic kidney disease (CKD). Indeed, those with CKD have a substantially higher chance of dying from CVD than of progressing to end-stage renal disease (ESRD). Furthermore, those who respond to treatment remain at risk of further disease relapses. microRNAs (miRs) are selectively expressed in certain tissues and cell states, which makes them potential biomarker candidates. In the circulation, the miRs are bound to Argonaute 2 proteins or encapsulated in extracellular vesicles (ECVs). This makes them resistant to RNase degradation providing further support for their potential use as novel biomarkers. miR-126 is enriched in endothelial cells and is a regulator of vascular integrity and angiogenesis. miR-126 was measured in patients with active vasculitis and once in treatment-induced remission. At disease presentation, miR-126 was at a low concentration and increased with successful treatment. Patients with vasculitis - both active disease and in remission states - had lower circulating miR-126 than in healthy volunteers. miR-126 was also measured in patients with ESRD to establish whether it was also a marker of chronic vascular dysfunction. Circulating levels were measured in patients receiving haemodialysis before and after the treatment. Before haemodialysis, miR-126 was at a low level in circulating blood and increased after the treatment. The levels of miR-126 in patients with CKD were also measured and were at a higher concentration than patients with ESRD. These data suggest that miR-126 has a potential role as a biomarker of vascular health and could track the progression of vascular disease. Using small RNA sequencing, the miR profiles of patients with active vasculitis and once in remission were established. These results were analysed in order to determine appropriate miRs for biomarker discovery. Using the data obtained, the miR hits were measured in larger patient cohorts for validation. This did not result in the discovery of a specific miR biomarker for the identification of acute vascular dysfunction in humans. ECVs were isolated from healthy volunteers and patients with active vasculitis. Uptake of ECVs was established in human and mouse macrophages, as well as renal proximal tubules in vitro. This did not result in increased cytokine production in the cultured macrophages or renal cells. This suggests that uptake is possible, however, the ECV cargo does not activate immune and renal cells in vitro. These findings, therefore, show that circulating miRs are potential markers of acute and chronic human vascular dysfunction. miR-126 was a successful marker for differentiating between patients with active disease and treatment-induced remission. The miR profile of these patients did not differ as determined by RNA sequencing. In addition, ECVs can be isolated from patient blood and taken up by recipient macrophage and renal proximal tubule cells, however, this does not result in a phenotypic change. These data suggest that miR-126 has a potential role as a biomarker of vascular health and could track the progression of vascular disease and response to treatment in acute and chronic vascular dysfunction

Molecular identification of different trypanosome species and subspecies in tsetse flies of northern Nigeria

Background: Animal African Trypanosomiasis (AAT) is caused by several species of trypanosomes including Trypanosoma congolense, T. vivax, T. godfreyi, T. simiae and T. brucei. Two of the subspecies of T. brucei also cause Human African Trypanosomiasis. Although some of them can be mechanically transmitted by biting flies; these trypanosomes are all transmitted by tsetse flies which are the cyclical vectors of Trypanosoma congolense, T. godfreyi, T. simiae and T. brucei. We present here the first report assessing the prevalence of trypanosomes in tsetse flies in Nigeria using molecular tools. Methods: 488 tsetse flies of three species, Glossina palpalis palpalis, G. tachinoides and G. morsitans submorsitans were collected from Wuya, Niger State and Yankari National Park, Bauchi State in 2012. Trypanosomes were detected and identified using an ITS1 PCR assay on DNA purified from the ‘head plus proboscis’ (H + P) and abdomen (ABD) parts of each fly. Results: T. vivax and T. congolense Savannah were the major parasites detected. Trypanosomes prevalence was 7.1 % in G. p. palpalis, 11.9 % in G. tachinoides and 13.5 % in G. m. submorsitans. Prevalences of T. congolense Savannah ranged from 2.5 to 6.7 % and of T. vivax were approximately 4.5 %. Trypanosoma congolense Forest, T. godfreyi and T. simiae were also detected in the site of Yankari. The main biological and ecological determinants of trypanosome prevalence were the fly sex, with more trypanosomes found in females than males, and the site, with T. congolense subspp. being more abundant in Yankari than in Wuya. As expected, the trypanosome species diversity was higher in Yankari National Park than in the more agricultural site of Wuya where vertebrate host species diversity is lower. Conclusions: Our results show that T. congolense Savannah and T. vivax are the main species of parasite potentially causing AAT in the two study sites and that Yankari National Park is a potential reservoir of trypanosomes both in terms of parasite abundance and species diversity

Glucocorticoid receptor alters isovolumetric contraction and restrains cardiac fibrosis

Corticosteroids directly affect the heart and vasculature and are implicated in the pathogenesis of heart failure. Attention is focussed upon the role of the mineralocorticoid receptor (MR) in mediating pro-fibrotic and other adverse effects of corticosteroids upon the heart. In contrast, the role of the glucocorticoid receptor (GR) in the heart and vasculature is less well understood. We addressed this in mice with cardiomyocyte and vascular smooth muscle deletion of GR (SMGRKO mice). Survival of SMGRKO mice to weaning was reduced compared with that of littermate controls. Doppler measurements of blood flow across the mitral valve showed an elongated isovolumetric contraction time in surviving adult SMGRKO mice, indicating impairment of the initial left ventricular contractile phase. Although heart weight was elevated in both genders, only male SMGRKO mice showed evidence of pathological cardiomyocyte hypertrophy, associated with increased myosin heavy chain-β expression. Left ventricular fibrosis, evident in both genders, was associated with elevated levels of mRNA encoding MR as well as proteins involved in cardiac remodelling and fibrosis. However, MR antagonism with spironolactone from birth only modestly attenuated the increase in pro-fibrotic gene expression in SMGRKO mice, suggesting that elevated MR signalling is not the primary driver of cardiac fibrosis in SMGRKO mice, and cardiac fibrosis can be dissociated from MR activation. Thus, GR contributes to systolic function and restrains normal cardiac growth, the latter through gender-specific mechanisms. Our findings suggest the GR:MR balance is critical in corticosteroid signalling in specific cardiac cell types

Cytokeratin-18 is a sensitive biomarker of alanine transaminase increase in a placebo-controlled, randomized, crossover trial of therapeutic paracetamol dosing (PATH-BP biomarker substudy)

Drug-induced liver injury (DILI) is a challenge in clinical medicine and drug development. Highly sensitive novel biomarkers have been identified for detecting DILI following a paracetamol overdose. The study objective was to evaluate biomarker performance in a 14-day trial of therapeutic dose paracetamol. The PATH-BP trial was a double-blind, placebo-controlled, crossover study. Individuals (n = 110) were randomized to receive 1 g paracetamol 4× daily or matched placebo for 2 weeks followed by a 2-week washout before crossing over to the alternate treatment. Blood was collected on days 0 (baseline), 4, 7, and 14 in both arms. Alanine transaminase (ALT) activity was measured in all patients. MicroRNA-122 (miR-122), cytokeratin-18 (K18), and glutamate dehydrogenase (GLDH) were measured in patients who had an elevated ALT on paracetamol treatment (≥50% from baseline). ALT increased in 49 individuals (45%). All 3 biomarkers were increased at the time of peak ALT (K18 paracetamol arm: 18.9 ± 9.7 ng/ml, placebo arm: 11.1 ± 5.4 ng/ml, ROC-AUC = 0.80, 95% CI 0.71–0.89; miR-122: 15.1 ± 12.9fM V 4.9 ± 4.7fM, ROC-AUC = 0.83, 0.75–0.91; and GLDH: 24.6 ± 31.1U/l V 12.0 ± 11.8U/l, ROC-AUC = 0.66, 0.49–0.83). All biomarkers were correlated with ALT (K18 r = 0.68, miR-122 r = 0.67, GLDH r = 0.60). To assess sensitivity, biomarker performance was analyzed on the visit preceding peak ALT (mean 3 days earlier). K18 identified the subsequent ALT increase (K18 ROC-AUC = 0.70, 0.59–0.80; miR-122 ROC-AUC = 0.60, 0.49–0.72, ALT ROC-AUC = 0.59, 0.48–0.70; GLDH ROC-AUC = 0.70, 0.50–0.90). Variability was lowest for ALT and K18. In conclusion, K18 was more sensitive than ALT, miR-122, or GLDH and has potential significant utility in the early identification of DILI in trials and clinical practice

Exosome signalling in the kidney

Urine contains exosomes originating from the circulation and all cells lining the urinary tract. Exosomes are a route of inter-cellular communication along the nephron potentially able to transfer of protein and/or RNA. It is not known whether this is a regulated process analogous to other cell-to-cell signalling systems. The aims of this study were to develop nanoparticle tracking analysis (NTA) as a technique to quantify exosomes in urine. Secondly, the hormonal regulation of exosome uptake in vitro and in vivo was investigated. Thirdly, exosome excretion in a central diabetes insipidus (DI) patient and a patient group after radiocontrast exposure was measured to investigate exosome excretion along the kidney in injury. Using the fluorescent capabilities of NTA, urinary exosomes were quantified in urine samples. NTA was able to detect changes in aquaporin 2 levels in vitro and in vivo. Storage conditions for human urinary exosomes were also optimised using NTA. A kidney cortical collecting duct cell line (CCDs) was used to model regulation of exosome uptake in vitro. CCDs were stimulated with desmopressin, a vasopressin analogue, and uptake of fluorescently-loaded or microRNA-loaded exosomes was measured. Desmopressin stimulated exosome uptake into collecting duct cells via V2 receptor stimulation. Intra-cellular uptake of exosomes was confirmed by microRNA specific mRNA down-regulation. Mechanistically, exosome uptake in response to desmopressin required cyclic AMP production, was mediated by clathrin-dependent endocytosis and was selective for exosomes from kidney tubular cells. In mice, fluorescently-loaded exosomes were systemically injected before and after administration of the V2 antagonist, tolvaptan, and urinary exosome excretion was measured. Basally, 2.5% of injected exosomes were recovered in urine; tolvaptan treatment resulted in a 5-fold increase. By combining antibodies to nephron segment-specific proteins with NTA we measured human urinary exosome excretion in central diabetes insipidus (DI) and after radiocontrast exposure (n=37). In DI, desmopressin reduced the excretion of exosomes derived from upstream glomerular and proximal tubule cells. In patients exposed to radiocontrast, urinary exosomes from the glomerulus were positively correlated with the tubular injury markers KIM- 1 and NGAL. These findings therefore show that tubular exosome uptake is a specific, hormonally regulated process that is reduced with injury. Physiologically, exosomes are a mechanism of inter-cellular communication; therapeutically, exosomes represent a novel vehicle by which RNA therapy could be targeted for the treatment of kidney disease

Effect of Cannabinoids and Stress on Motor Maps and Behaviour

Motor maps exhibit experience dependent changes and are therefore sensitive to the type and amount of neurotransmitters present in the motor cortex and changes in motor map expression often results in impairments of skilled behaviour. Endocannabinoids have been shown to affect spinal locomotion and stress has been shown to affect spontaneous locomotion therefore, the effect of cannabinoid signaling and stress on motor map expression and behaviour was examined. The first study determined that cannabinoid 1 receptor (CB1R) signaling dampened motor map expression and that both anandamide (AEA) and 2-arachidonoylglycerol (2-AG) contributed to this effect. Activation of CB1R signaling did not affect skilled reaching task performance. However, a lack of cannabinoid signaling resulted in impairment on performing the reaching motion. The second study characterized acute and repeated stress effects on motor map expression. Acute stress did not alter map expression while repeated stress decreased forelimb map expression and resulted in skilled motor behaviour deficits. It was then established that 2-AG mediates the stress effects seen on motor map expression. The last study observed the effects of delta-9-tetrahydrocannabinol (THC) on motor map expression as well as skilled and unskilled behaviour. It was discovered that THC dose dependently affected motor map expression in that a low dose of THC increased map expression while higher doses decreased forelimb map expression. THC decreased the number of reach attempts made in one 15 minute reach training session but did not impair the execution of reaching behaviour. Unskilled behaviour was assessed using the bar task, rotorod, horizontal ladder rung walking task and the open field test. No differences were found between rats that received THC and rats that received DMSO on the rotorod, rung walking task and the open field test. The highest dose of THC used (2.5 mg/kg) resulted in a longer latency for removal of the forepaws from the bar in the bar task. This research is important and necessary due to increasing usage of cannabinoids for recreation as well as to treat neurological illness. The present findings suggest that endocannabinoids and repeated stress via 2-AG have a dampening effect on motor map expression

The role of serotonin on the expression of rat motor maps

Bibliography: p. 83-105Some pages are in colour

Additional file 2: of Molecular identification of different trypanosome species and subspecies in tsetse flies of northern Nigeria

R code used for the multiple logistic regression analyses: file “Isaac_et_al_additional_file_2_R_code_for_multiple_logistic_regressions.txt”. (TXT 7 kb