Dylan Burger

A Novel Mouse Model of Advanced Diabetic Kidney Disease

Currently available rodent models exhibit characteristics of early diabetic nephropathy (DN) such as hyperfiltration, mesangial expansion, and albuminuria yet features of late DN (hypertension, GFR decline, tubulointerstitial fibrosis) are absent or require a significant time investment for full phenotype development. Accordingly, the aim of the present study was to develop a mouse model of advanced DN with hypertension superimposed (HD mice). Mice transgenic for human renin cDNA under the control of the transthyretin promoter (TTRhRen) were employed as a model of angiotensin-dependent hypertension. Diabetes was induced in TTRhRen mice through low dose streptozotocin (HD-STZ mice) or by intercrossing with OVE26 diabetic mice (HD-OVE mice). Both HD-STZ and HD-OVE mice displayed more pronounced increases in urinary albumin levels as compared with their diabetic littermates. Additionally, HD mice displayed renal hypertrophy, advanced glomerular scarring and evidence of tubulointerstitial fibrosis. Both HD-OVE and HD-STZ mice showed evidence of GFR decline as FITC-inulin clearance was decreased compared to hyperfiltering STZ and OVE mice. Taken together our results suggest that HD mice represent a robust model of type I DN that recapitulates key features of human disease which may be significant in studying the pathogenesis of DN and in the assessment of putative therapeutics.

Assessment of urinary microparticles in normotensive patients with type 1 diabetes

Aims/hypothesisAssessment of urinary extracellular vesicles including exosomes and microparticles (MPs) is an emerging approach for non-invasive detection of renal injury. We have previously reported that podocyte-derived MPs are increased in diabetic mice in advance of albuminuria. Here, we hypothesised that type 1 diabetes and acute hyperglycaemia would increase urinary podocyte MP levels in uncomplicated diabetes.MethodsIn this post hoc exploratory analysis, we examined archived urine samples from normoalbuminuric patients with uncomplicated type 1 diabetes studied under clamped euglycaemia and hyperglycaemia and compared with healthy controls. Urinary vesicles were assessed by electron microscopy and nanoparticle tracking while podocyte MPs were assessed by flow cytometry.ResultsNeither vesicle size nor total number were significantly altered in type 1 diabetes or acute hyperglycaemia. By contrast, urinary podocyte MP levels were higher in type 1 diabetes (0.47 [0.00–3.42] MPs/μmol creatinine [Cr]) compared with healthy controls (0.00 [0.00–0.00] MPs/μmol Cr, p < 0.05) and increased under hyperglycaemic clamp (0.36 [0.00–4.15] MPs/μmol Cr during euglycaemia vs 2.70 [0.00–15.91] MPs/μmol Cr during hyperglycaemia, p < 0.05). Levels of urinary albumin to creatinine ratio and nephrin (surrogates of podocyte injury) were unchanged by type 1 diabetes or acute hyperglycaemia.Conclusion/interpretationTaken together, our data show that urinary podocyte MP levels are higher in patients with type 1 diabetes in advance of changes in other biomarkers (albuminuria, nephrin). Examination of podocyte MPs may serve as an early biomarker of glomerular injury in uncomplicated type 1 diabetes.

Thyroid-stimulating hormone acutely increases levels of circulating pro-coagulant microparticles.

Thyroid-stimulating hormone (TSH) acts on extra-thyroidal targets. When recombinant human (rh)TSH is administered to patients treated for thyroid cancer (thyroidectomy and radioablation) to screen for recurrence, metabolic and vascular stress occurs. This is indicated by elevations in levels of interleukin (IL)-6, C-reactive protein (CRP), oxidative stress and free fatty acids (FFA), as well as a decrease in endothelium-dependent relaxation. Microparticles (0 1–1 0 lm; or microvesicles) form by outward plasma membrane blebbing followed by shedding from cells. They display pro-coagulant activity by presenting surface phosphatidylserine. Platelet microparticles predominate; other sources include erythrocytes, leucocytes and endothelial cells. Elevations in total, and endothelial, microparticles correlate with indices of inflammation and vascular injury and may independently predict cardiovascular disease. Our objective was to determine whether rhTSH alters microparticle levels and procoagulant activity. Patients (12 women, six men) with a mean ( SD) age of 52 12 years and body mass index (BMI) of 29 5 kg/m, treated by total thyroidectomy and radioablation (14 papillary carcinoma, four follicular carcinoma), and on L-thyroxine therapy, were recruited (Ottawa Health Sciences Network Research Ethics Board, #2006558). Patients were disease free, that is no thyroglobulin response to rhTSH. They received two intramuscular doses of rhTSH (0 9 mg) on days 1 and 2 without discontinuation of L-thyroxine therapy. Blood was drawn on the mornings of days 1 (baseline; before rhTSH), 3 and 5 (serum separator tubes; BD Biosciences, Mississauga, ON, Canada). Samples were centrifuged at 1300 g for 10 min, and supernatants (sera) were used to measure TSH and free thyroxine (reference ranges 0 3–5 6 mU/l and 7–17 pmol/l, respectively) by AutoIA (Abbot DxI) in The Ottawa Hospital. Sera for microparticle and pro-coagulant measurements were frozen; they were thawed once for aliquoting and refrozen at 80 °C until thawed for analysis. For microparticle analysis, thawed samples (150 ll) were centrifuged at 2500 g (15 min, 4 °C). Supernatants were centrifuged at 2500 g (15 min, 4 °C). Supernatants were removed, then centrifuged at 20 000 g (20 min, 4 °C) to pellet the microparticles. Supernatants were aspirated, and microparticle-containing pellets were resuspended in Annexin V-binding buffer (10 mmol/l HEPES, pH 7 4, 140 mmol/l NaCl, 2 5 mmol/l CaCl2). Samples were labelled with 0 5 lg/ml Alexa-647 conjugated Annexin V (Biolegend, San Diego, CA, USA) and FITC-labelled CD144 antibody (1:50 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) for 30 min for optimal labelling. As negative controls, a subpopulation of microparticles was resuspended in Annexin Vbinding buffer lacking calcium, and a subpopulation of microparticles was labelled with FITC-conjugated IgG isotype controls. Microparticles were assessed using a MoFlo Fluorescence Activated Cell Sorter (Dako Canada Inc, Burlington, ON, Canada) as particles of <1 0 and >0 1 lm that exhibited more fluorescence than negative controls. Results were expressed as number of annexinV total microparticles/ll plasma (interassay CV 9 5%) or of annexinV/CD144 endothelial microparticles/ll (interassay CV 11 4%). Phosphatidylserine-positive microparticles were assessed using a Zymuphen MP-activity kit (Aniara). The assay utilizes immobilized annexin V to capture phosphatidylserine (PS)-expressing microparticles. Microparticles are detected by addition of coagulation factor Va, factor Xa, Ca2 + and prothrombin. The rate of thrombin production, as indicated by a chromogenic substrate (405 nm), is proportional to PS availability and thereby, microparticle concentration. Results are expressed in nmol/l of PS equivalents (interassay CV 10%). Differences between means were assessed by ANOVA with Student Newman Keul post hoc tests. Linear regression and Pearson’s correlation were used to compare rhTSH responses where indicated. P < 0 05 was considered significant. Following rhTSH injection, TSH levels (mean SD) increased from 0 5 0 9 to 98 3 7 1 mU/l (day 3) and then decreased to 17 9 4 3 mU/l (day 5). Free thyroxine values (mean SD) were 17 9 4 3 pmol/l. The level of total microparticles was 36 651 microparticles/ll at baseline, increased 1 6-fold to 57 890 microparticles/ll on day 3 and significantly increased 1 9-fold to 70 969 microparticles/ll on day 5 (Fig. 1a). Baseline pro-coagulant activity was 5 5 nmol/l PS equivalent and increased significantly by 1 5-fold to 8 5 nmol/l PS equivalent and by 1 9-fold to 10 1 nmol/l PS equivalent on days 3 and 5, respectively (Fig. 1b). Endothelial microparticles measured 7535 microparticles/ll at baseline (20 6% of total) and did not change in response to rhTSH (Fig. 1c). Increases in total microparticles and pro-coagulant activity (responses at day 5 for each person) were correlated (r = 0 70; P < 0 05). There was a negative correlation of total microparticle response at day 5 with age (r = 0 56; P < 0 05); a similar trend with pro-coagulant activity (r = 0 40, P = 0 10) did not reach significance. There was no correlation with BMI. There were too few men to compare responses by gender. As levels of total microparticles rose with rhTSH, but endothelial particles did not, the response may be due to platelet microparticles, the main subpopulation, although other microparticle populations (e.g. leucocyte) cannot be excluded. rhTSH increases platelet reactivity (soluble P-selectin and soluble CD40 ligand) and endothelial activation (soluble intercellular adhesion molecule-1, soluble E-selectin). These rhTSH-induced

Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry

Retroviruses and small EVs overlap in size, buoyant densities, refractive indices and share many cell-derived surface markers making them virtually indistinguishable by standard biochemical methods. This poses a significant challenge when purifying retroviruses for downstream analyses or for phenotypic characterization studies of markers on individual virions given that EVs are a major contaminant of retroviral preparations. Nanoscale flow cytometry (NFC), also called flow virometry, is an adaptation of flow cytometry technology for the analysis of individual nanoparticles such as extracellular vesicles (EVs) and retroviruses. In this study we systematically optimized NFC parameters for the detection of retroviral particles in the range of 115–130 nm, including viral production, sample labeling, laser power and voltage settings. By using the retroviral envelope glycoprotein as a selection marker, and evaluating a number of fluorescent dyes and labeling methods, we demonstrate that it is possible to confidently distinguish retroviruses from small EVs by NFC. Our findings make it now possible to individually phenotype genetically modified retroviral particles that express a fluorescent envelope glycoprotein without removing EV contaminants from the sample.

Isolation and Characterization of Circulating Microparticles by Flow Cytometry

Microparticles are small fragments (0.1-1.0 μm) of cellular membrane which are shed by cells under conditions of stress. As levels of circulating microparticles are elevated in disease, there has been significant interest in their assessment and quantification under pathological conditions. Here we describe a protocol for the isolation of microparticles from plasma samples and their characterization/quantification by flow cytometry. This assay has been employed for the assessment of microparticle levels in both human and animal plasma and may also be modified for the characterization of microparticles from culture media or from other biological samples (i.e., urine).

Thyroid-Stimulating Hormone-Stimulated Human Adipocytes Express Thymic Stromal Lymphopoietin

When recombinant human (rh) thyroid-stimulating hormone (TSH) is administered to thyroid cancer survivors, an acute extra-thyroidal effect raises pro-inflammatory cytokines and activates platelets. Thymic stromal lymphopoietin (TSLP) is a cytokine recently implicated in platelet activation. Our aim was to measure platelet microparticle levels after rhTSH stimulation in vivo, and to investigate TSLP expression in TSH-stimulated human adipocytes in culture. Blood samples for total and platelet microparticle analysis were obtained from thyroid cancer survivors before (day 1) and after rhTSH administration (day 5). Adipocytes, differentiated from stromal preadipocytes isolated from adipose tissue from surgical patients, were stimulated with TSH. TSLP mRNA expression, protein expression, and protein release into the adipocyte medium were measured. The level of platelet microparticles in thyroid cancer patients rose 5-fold after rhTSH stimulation. TSH upregulated TSLP mRNA expression in adipocytes in culture through a pathway that was inhibited by 66% by H89, a protein kinase A inhibitor. TSLP protein expression rose in response to TSH, and TSH-stimulated TSLP release into the medium was completely blocked by dexamethasone. In conclusion, TSLP is a novel TSH-responsive adipokine. Future studies will be needed to address the potential role of adipocyte-derived TSLP and whether it is linked to TSH-dependent platelet activation.

Survival Motor Neuron Protein is Released from Cells in Exosomes: A Potential Biomarker for Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is caused by homozygous mutation of the survival motor neuron 1 (SMN1) gene. Disease severity inversely correlates to the amount of SMN protein produced from the homologous SMN2 gene. We show that SMN protein is naturally released in exosomes from all cell types examined. Fibroblasts from patients or a mouse model of SMA released exosomes containing reduced levels of SMN protein relative to normal controls. Cells overexpressing SMN protein released exosomes with dramatically elevated levels of SMN protein. We observed enhanced quantities of exosomes in the medium from SMN-depleted cells, and in serum from a mouse model of SMA and a patient with Type 3 SMA, suggesting that SMN-depletion causes a deregulation of exosome release or uptake. The quantity of SMN protein contained in the serum-derived exosomes correlated with the genotype of the animal, with progressively less protein in carrier and affected animals compared to wildtype mice. SMN protein was easily detectable in exosomes isolated from human serum, with a reduction in the amount of SMN protein in exosomes from a patient with Type 3 SMA compared to a normal control. Our results suggest that exosome-derived SMN protein may serve as an effective biomarker for SMA.