Tomás P. Griffin

Mesenchymal stem cell secretion of chemokines during differentiation into osteoblasts, and their potential role in mediating interactions with breast cancer cells

Over 70% of patients with advanced breast cancer will develop bone metastases for which there is no cure. Mesenchymal Stem Cells (MSCs) and their derivative osteoblasts are subpopulations of cells within the bone marrow environment, postulated as potential interacting targets for disseminating cancer cells because of their ability to secrete a range of chemokines. This study aimed to investigate chemokine secretion throughout MSC differentiation into osteoblasts and their effect on the breast cancer cells. Primary MSCs and osteoblast progenitors were cultured in appropriate conditions to induce differentiation into mature osteoblasts. Chemokines secreted throughout differentiation were detected using ChemiArray and ELISA. Migration of breast cancer cells in response to the bone‐derived cells was quantified using Transwell inserts. Breast cancer cells were cocultured with MSCs, retrieved using magnetic beads, and changes in CCL2 expression were analyzed. MSCs secreted a range of factors including IL‐6, TIMP‐1 and CCL2, the range and level of which changed throughout differentiation. CCL2 secretion by MSCs increased significantly above control cells as they differentiated into mature osteoblasts (p < 0.05). The bone‐derived cells stimulated migration of breast cancer cells, and this was inhibited (21–50%) in the presence of a CCL2 antibody. CCL2 gene expression in breast cancer cells was upregulated following direct coculture with MSCs. The varying levels of chemokines secreted throughout MSC differentiation may play an important role in supporting tumor cell homing and progression. These results further highlight the distinct effect MSCs have on breast cancer cells and their potential importance in supporting development of metastases.

The Promise of Mesenchymal Stem Cell Therapy for Diabetic Kidney Disease

Diabetes mellitus (DM) commonly leads to progressive chronic kidney disease despite current best medical practice. The pathogenesis of diabetic kidney disease (DKD) involves a complex network of primary and secondary mechanisms with both intra-renal and systemic components. Apart from inhibition of the renin angiotensin aldosterone system, targeting individual pathogenic mediators with drug therapy has not, thus far, been proven to have high clinical value. Stem or progenitor cell therapies offer an alternative strategy for modulating complex disease processes through suppressing multiple pathogenic pathways and promoting pro-regenerative mechanisms. Mesenchymal stem cells (MSCs) have shown particular promise based on their accessibility from adult tissues and their diverse mechanisms of action including secretion of paracrine anti-inflammatory and cyto-protective factors. In this review, the progress toward clinical translation of MSC therapy for DKD is critically evaluated. Results from animal models suggest distinct potential for systemic MSC infusion to favourably modulate DKD progression. However, only a few early phase clinical trials have been initiated and efficacy in humans remains to be proven. Key knowledge gaps and research opportunities exist in this field. These include the need to gain greater understanding of in vivo mechanism of action, to identify quantifiable biomarkers of response to therapy and to define the optimal source, dose and timing of MSC administration. Given the rising prevalence of DM and DKD worldwide, continued progress toward harnessing the inherent regenerative functions of MSCs and other progenitor cells for even a subset of those affected has potential for profound societal benefits.

β‐Blocker withdrawal is preferable for accurate interpretation of the aldosterone–renin ratio in chronically treated hypertension

To evaluate the effects of β‐adrenoreceptor antagonists (β‐blockers) on the aldosterone–renin ratio (ARR) in the context of antihypertensive polypharmacy in chronic hypertension. To determine the optimal duration of β‐blocker withdrawal required to normalize the ARR.

Screening for phaeochromocytoma and paraganglioma: impact of using supine reference intervals for plasma metanephrines with samples collected from fasted/seated patients.

Background The Endocrine Society Clinical Practice Guideline on Phaeochomocytoma and Paraganglioma recommends phlebotomy for plasma-free metanephrines with patients fasted and supine using appropriately defined reference intervals. Studies have shown higher diagnostic sensitivities using these criteria. Further, with seated-sampling protocols, for result interpretation, reference intervals that do not compromise diagnostic sensitivity should be employed. Objective To determine the impact on diagnostic performance and financial cost of using supine reference intervals for result interpretation with our current plasma-free metanephrines fasted/seated-sampling protocol. Methods We conducted a retrospective cohort study of patients who underwent screening for PPGL using plasma-free metanephrines from 2009 to 2014 at Galway University Hospitals. Plasma-free metanephrines were measured using liquid chromatography-tandem mass spectrometry. Supine thresholds for plasma normetanephrine and metanephrine set at 610 pmol/L and 310 pmol/L, respectively, were used. Results A total of 183 patients were evaluated. Mean age of participants was 53.4 (±16.3) years. Five of 183 (2.7%) patients had histologically confirmed PPGL (males, n=4). Using seated reference intervals for plasma-free metanephrines, diagnostic sensitivity and specificity were 100% and 98.9%, respectively, with two false-positive cases. Application of reference intervals established in subjects supine and fasted to this cohort gave diagnostic sensitivity of 100% with specificity of 74.7%. Financial analysis of each pretesting strategy demonstrated cost-equivalence (€147.27/patient). Conclusion Our cost analysis, together with the evidence that fasted/supine-sampling for plasma-free metanephrines, offers more reliable exclusion of PPGL mandates changing our current practice. This study highlights the important advantages of standardized diagnostic protocols for plasma-free metanephrines to ensure the highest diagnostic accuracy for investigation of PPGL.

A cross-sectional study of the effects of β-blocker therapy on the interpretation of the aldosterone/renin ratio: can dosing regimen predict effect?

Context and aim: Aldosterone/renin ratio (ARR) is used as the primary screening tool for primary aldosteronism. Its interpretation is often challenging because of the interference of antihypertensive medication. &bgr;-blocker therapy suppresses renin production by inhibiting &bgr;-adrenergic receptors in the juxtaglomerular apparatus of the kidney and consequently aldosterone secretion (to a lesser extent). Therefore, &bgr;-blocker therapy has the potential to elevate the ARR. The aim of this study was to investigate whether or not the effect of &bgr;-blocker therapy on the ARR could be predicted from the dosing regimen. Methods: A prospective cross-sectional study was conducted. Participants were stratified into one of four groups (control/low/medium/high) based on the quantity of &bgr;-blocker prescribed. ARR was calculated from renin/aldosterone, measured using two assay systems. Results: Eighty-nine volunteers were recruited to our study. In the control group, zero patients had a positive ARR using plasma renin activity (PRA)/direct renin concentration (DRC). In the low, medium, and high-dose &bgr;-blocker groups between 8–25% of patients demonstrated screen positive ARR results for primary aldosteronism using DRC and PRA. DRC was significantly lower in patients in the medium/high-dose groups and PRA significantly lower in the low/medium/high-dose groups compared with controls. ARR using DRC was significantly higher in the medium/high-dose groups and ARR using PRA was significantly higher in the low/medium/high-dose groups compared with controls. Conclusion: Our study suggests that &bgr;-blocker therapy is associated with an increased risk of positive ARR screens for primary aldosteronism irrespective of the dose of &bgr;-blocker prescribed, in patients in whom it is clinically reasonable to expect that primary aldosteronism may be present.

ANCA-associated vasculitis: a comparison of cases presenting to nephrology and rheumatology services

BACKGROUND Anti-neutrophil cytoplasmic antibody (ANCA) -associated vasculitis (AAV) is a disease characterized by inflammation of small vessels and detectable ANCA in the circulation. Patients may develop a broad spectrum of clinical features ranging from indolent sino-nasal disease and rashes to fulminant renal failure or acute life-threatening pulmonary haemorrhage. Consequently, patients with AAV present to a variety of specialties including nephrology and rheumatology, whose training and approaches to management of such patients may differ. There is little literature comparing patients presenting to different specialties and their outcomes. METHODS We compared two cohorts of patients with ANCA-positive AAV presenting to either the rheumatology or nephrology department at Galway University Hospitals from June 2002 to July 2011. A standardized data collection form was used to collect information regarding baseline demographics, manifestations of AAV, initial management, relapses and complications. RESULTS Forty-five patients were included in this study (15 rheumatology/30 nephrology). The nephrology cohort was older, had a higher C-reactive protein, Birmingham Vascular Activity Score and ANCA titer at presentation compared to the rheumatology group. Induction treatment varied between the cohorts with rheumatology patients most commonly receiving a combination of oral corticosteroids (73%) and methotrexate (60%) and nephrology patients receiving a combination of intravenous corticosteroids (93%) and cyclophosphamide (90%). Fifty-three percent of the rheumatology patients who completed induction therapy relapsed compared to 30% of the nephrology patients. CONCLUSION This study presents two different cohorts of patients with the same disease that were managed by two different disciplines. It highlights the heterogeneity of AAV and the importance of interdisciplinary communication and cooperation when managing these patients.

Screening for primary aldosteronism using the newly developed IDS-iSYS® automated assay system

Background The recommended approach to screening for primary aldosteronism (PA) in at-risk populations is to determine the ratio of aldosterone concentration (serum (SAC)/plasma (PAC)) to renin measured in plasma as activity (PRA) or concentration (DRC). However, lack of assay standardisation mandates the need for method-specific decision thresholds and clinical validation in the local population. Aim The study objective was to establish method-specific aldosterone: renin ratio (ARR) cut-offs for PA in men and women using the IDS-iSYS® assay system (IDS plc). Methods A prospective cohort study design was used. PAC and DRC were measured immunochemically in ethylenediamine-tetraacetic acid (EDTA) plasma on the IDS-iSYS® instrument. Results A total of 437 subjects (218 men, 219 women) were recruited including: healthy normotensive volunteers (n=266) and women taking the oral contraceptive pill (OCP; n=15); patients with essential hypertension (EH; n=128); confirmed PA (n=16); adrenal cortical carcinoma (ACC; n=3); Addisons disease (AD; n=4) and phaeochromocytoma/paraganglioma (PPGL; n=5). In this population, an ARR cut-off at >37.4 pmol/mIU provided 100% diagnostic sensitivity, 96% specificity and positive likelihood ratio for PA of 23:1. When the ARR decision threshold was stratified according to gender, a cut-off of >26.1 pmol/mIU in men and >113.6 pmol/mIU in women resulted in diagnostic sensitivity and specificity of 100%. Conclusion This study demonstrates that decision thresholds for PA should not only be method-specific but also gender-specific. However, given the small number of PA patients (n=16), particularly women (n=4), further validation through a prospective study with a larger PA cohort is required before the thresholds presented here could be recommended for routine clinical use.

Evaluating the optimum rest period prior to blood collection for fractionated plasma free metanephrines analysis

Introduction The high diagnostic accuracy of plasma metanephrines (PMets) in the diagnosis of Phaeochromocytoma/Paraganglioma (PPGL) is well established. Considerable controversy exists regarding optimum sampling conditions for PMets. The use of reference intervals that do not compromise diagnostic sensitivity is recommended. However, the optimum rest period prior to sampling has yet to be clearly established. The aim of this study was to evaluate PMets concentrations in paired blood samples collected following 30 and 40 min seated-rest prior to sampling, in patients in whom it was clinically reasonable to suspect that PPGL may be present. Design and Methods A retrospective cross-sectional study design was used. PMets results from paired blood samples collected after 30 and 40 min seated-rest between January 2009 and June 2015 were recorded. Results were interpreted using reference intervals established in subjects seated and supine. Results A total of 410 patient results were eligible for analysis. There was no statistical difference between plasma normetanephrine (NMN) or metanephrine (MN) concentrations in samples collected following 30 and 40 min seated-rest in subjects with PPGL (n=11), post-resection of PPGL (n=20) or in whom PPGL was excluded (n=379). Using reference intervals established in the seated position, diagnostic sensitivity was 100% at 30 min and 90.9% at 40 min. Diagnostic specificity was approximately 95% at both time points. When supine reference intervals were used, diagnostic sensitivity was 100% and diagnostic specificity was reduced by ≈22% at both time points. Conclusion Based on these data, we recommend at most 30 min continuous rest prior to sampling for PMets measurement.

Associations between glycaemic control and activation of the renin-angiotensin-aldosterone system in participants with type 2 diabetes mellitus and hypertension

Introduction Hyperglycaemia increases succinate concentrations and succinate receptor activation in the kidney resulting in renin release. The aim of our study was to determine if there is an association between glycaemic control as evidenced by glycated haemoglobin values and activation of the renin-angiotensin-aldosterone system in patients with type 2 diabetes mellitus and hypertension. Methods A cross-sectional study was conducted at Galway University Hospitals between December 2014 and March 2015. Participants (n = 66) were identified following interrogation of the electronic database for patients with type 2 diabetes mellitus. Baseline clinical demographics, aldosterone, plasma renin activity, direct renin concentration, urea and electrolytes, glycated haemoglobin, cholesterol, urine sodium and albumin creatinine ratio were recorded. Results There was a significant positive linear correlation between glycated haemoglobin and renin (both plasma renin activity [P = 0.002] and direct renin concentration [P = 0.008]) and between serum creatinine and aldosterone measured using both radioimmunoassay (P = 0.008) and immunochemiluminometric assay (P = 0.008). A significant negative linear correlation was demonstrated between serum sodium and plasma renin activity (P = 0.005) and direct renin concentration (P = 0.015) and between estimated glomerular filtration rate and aldosterone measured using radioimmunoassay (P = 0.02) and immunochemiluminometric assay (P = 0.016). A significant negative linear correlation existed between urine sodium and plasma renin activity (P = 0.04) and aldosterone measured using radioimmunoassay (P = 0.045). Conclusions There is a direct positive association between glycaemic control and renin. We advocate for renin measurement to be part of the diabetologists armamentarium to assess, guide and optimize therapeutic strategies in patients with diabetes.

Influence of Referral to a Combined Diabetology and Nephrology Clinic on Renal Functional Trends and Metabolic Parameters in Adults With Diabetic Kidney Disease

Objective To examine the impact of a diabetes renal clinic (DRC) on renal functional and metabolic indices in adults who have diabetes mellitus (DM) and chronic kidney disease (CKD). Patients and Methods All patients evaluated at a DRC in a single tertiary referral center from January 1, 2008, to December 31, 2012, were identified. Serial renal and metabolic indices from January 1, 2004, to December 31, 2014, were recorded, and trends over time were analyzed by linear mixed-effects models. Results A total of 200 patients who had DM and CKD were identified and subdivided into 3 categories based on presumptive CKD etiology: 43 (21.5%) with type 1 DM (T1D) only, 127 (63.5%) with type 2 DM (T2D) only, and 30 (15.0%) with DM and an additional CKD etiology. Average annual absolute (mL/min per body surface area per year) and percentage (%/year) changes, respectively, in Chronic Kidney Disease Epidemiology Collaboration estimated glomerular filtration rate before vs after first DRC attendance were: −1.59 vs −3.10 (P=.31) and −1.22 vs −9.39 (P=.06) for T1D; −5.64 vs −3.07 (P=.004) and −10.88 vs −9.94 (P=.70) for T2D; and −6.50 vs +0.91 (P<.001) and −13.28 vs −2.29 (P=.001) for DM with an additional CKD etiology. Glycemic control worsened in those who had T2D, whereas trends in total cholesterol levels improved in those who had T1D. Conclusion After first DRC attendance, the absolute rate of estimated glomerular filtration rate decline remained similar for those who had T1D, but it slowed for those who had T2D or DM with additional CKD etiology. Thus, benefits of combined diabetology and nephrology consultation may vary for different diabetic subpopulations.