A prospective cohort study in patients with type 2 diabetes mellitus for validation of biomarkers (PROVALID) –study design and baseline characteristics

Background/Aims: The prevalence of diabetes mellitus type 2 and kidney disease in these patients varies widely between European countries. Methods: In addition to store biosamples the “Prospective cohort study in patients with type 2 diabetes mellitus for validation of biomarkers” collects information on history, physical status, laboratory measurements and medication in 4000 patients with diabetes mellitus type 2, being taken care of at the primary level of healthcare in 5 European countries (Austria, Hungary, Netherlands, Poland and Scotland). Next to comparing the rate of loss of eGFR between the countries, a further objective of the PROVALID study is to determine the 5-year cumulative incidence of renal and cardiovascular outcomes. Results: The mean age of the population recruited is 62.9±10 years, 54.6% are male and the mean BMI is 30.9±5.4 kg/m2 . Metabolic control (median HBA1c 6.8 % (6.2;7.5)) is achieved via administration of metformin in 67.4% of the patients and insulin in 30.3%. Median systolic and diastolic blood pressure at recruitment is 135 (125;146) and 80 (72;85) mmHg, 65.4% of subjects received RAAS blocking agents. Mean eGFR is 80.7±29.2 ml/min/1.73m2 and median baseline albumin/creatinine ratio 8.3 mg (IQR: 3.8 and 25.1). Conclusion: PROVALID will provide information on incidence and progression of renal and cardiovascular disease and therapy in patients with type 2 diabetes mellitus in different European countries. Thus, in contrast to many other cohort studies we will be able to associate national clinical practise pattern with outcome in this highly vulnerable patient population

Urinary peptidomics analysis reveals proteases involved in diabetic nephropathy

Mechanisms underlying the onset and progression of nephropathy in diabetic patients are not fully elucidated. Deregulation of proteolytic systems is a known path leading to disease manifestation, therefore we hypothesized that proteases aberrantly expressed in diabetic nephropathy (DN) may be involved in the generation of DN-associated peptides in urine. We compared urinary peptide profiles of DN patients (macroalbuminuric, n = 121) to diabetic patients with no evidence of DN (normoalbuminuric, n = 118). 302 sequenced, differentially expressed peptides (adjusted p-value < 0.05) were analysed with the Proteasix tool predicting proteases potentially involved in their generation. Activity change was estimated based on the change in abundance of the investigated peptides. Predictions were correlated with transcriptomics (Nephroseq) and relevant protein expression data from the literature. This analysis yielded seventeen proteases, including multiple forms of MMPs, cathepsin D and K, kallikrein 4 and proprotein convertases. The activity of MMP-2 and MMP-9, predicted to be decreased in DN, was investigated using zymography in a DN mouse model confirming the predictions. Collectively, this proof-of-concept study links urine peptidomics to molecular changes at the tissue level, building hypotheses for further investigation in DN and providing a workflow with potential applications to other diseases

Identification of molecular markers of delayed graft function based on the regulation of biological ageing

Introduction: Delayed graft function is a prevalent clinical problem in renal transplantation for which there is no objective system to predict occurrence in advance. It can result in a significant increase in the necessity for hospitalisation post-transplant and is a significant risk factor for other post-transplant complications. Methodology: The importance of microRNAs (miRNAs), a specific subclass of small RNA, have been clearly demonstrated to influence many pathways in health and disease. To investigate the influence of miRNAs on renal allograft performance post-transplant, the expression of a panel of miRNAs in pre-transplant renal biopsies was measured using qPCR. Expression was then related to clinical parameters and outcomes in two independent renal transplant cohorts. Results: Here we demonstrate, in two independent cohorts of pre-implantation human renal allograft biopsies, that a novel pre-transplant renal performance scoring system (GRPSS), can determine the occurrence of DGF with a high sensitivity (>90%) and specificity (>60%) for donor allografts pre-transplant, using just three senescence associated microRNAs combined with donor age and type of organ donation. Conclusion: These results demonstrate a relationship between pre-transplant microRNA expression levels, cellular biological ageing pathways and clinical outcomes for renal transplantation. They provide for a simple, rapid quantitative molecular pre-transplant assay to determine post-transplant allograft function and scope for future intervention. Furthermore, these results demonstrate the involvement of senescence pathways in ischaemic injury during the organ transplantation process and an indication of accelerated bio-ageing as a consequence of both warm and cold ischaemia

The composition and functional protein subsystems of the human nasal microbiome in granulomatosis with polyangiitis: a pilot study

Abstract: Background: Ear, nose and throat involvement in granulomatosis with polyangiitis (GPA) is frequently the initial disease manifestation. Previous investigations have observed a higher prevalence of Staphylococcus aureus in patients with GPA, and chronic nasal carriage has been linked with an increased risk of disease relapse. In this cross-sectional study, we investigated changes in the nasal microbiota including a detailed analysis of Staphylococcus spp. by shotgun metagenomics in patients with active and inactive granulomatosis with polyangiitis (GPA). Shotgun metagenomic sequence data were also used to identify protein-encoding genes within the SEED database, and the abundance of proteins then correlated with the presence of bacterial species on an annotated heatmap. Results: The presence of S. aureus in the nose as assessed by culture was more frequently detected in patients with active GPA (66.7%) compared with inactive GPA (34.1%). Beta diversity analysis of nasal microbiota by bacterial 16S rRNA profiling revealed a different composition between GPA patients and healthy controls (P = 0.039). Beta diversity analysis of shotgun metagenomic sequence data for Staphylococcus spp. revealed a different composition between active GPA patients and healthy controls and disease controls (P = 0.0007 and P = 0.0023, respectively), and between healthy controls and inactive GPA patients and household controls (P = 0.0168 and P = 0.0168, respectively). Patients with active GPA had a higher abundance of S. aureus, mirroring the culture data, while healthy controls had a higher abundance of S. epidermidis. Staphylococcus pseudintermedius, generally assumed to be a pathogen of cats and dogs, showed an abundance of 13% among the Staphylococcus spp. in our cohort. During long-term follow-up of patients with inactive GPA at baseline, a higher S. aureus abundance was not associated with an increased relapse risk. Functional analyses identified ten SEED protein subsystems that differed between the groups. Most significant associations were related to chorismate synthesis and involved in the vitamin B12 pathway. Conclusion: Our data revealed a distinct dysbiosis of the nasal microbiota in GPA patients compared with disease and healthy controls. Metagenomic sequencing demonstrated that this dysbiosis in active GPA patients is manifested by increased abundance of S. aureus and a depletion of S. epidermidis, further demonstrating the antagonist relationships between these species. SEED functional protein subsystem analysis identified an association between the unique bacterial nasal microbiota clusters seen mainly in GPA patients and an elevated abundance of genes associated with chorismate synthesis and vitamin B12 pathways. Further studies are required to further elucidate the relationship between the biosynthesis genes and the associated bacterial species

Association of Pulmonary Hemorrhage, Positive Proteinase 3, and Urinary Red Blood Cell Casts With Venous Thromboembolism in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

Objective To assess the frequency of venous thromboembolism (VTE) events in the Rituximab in Antineutrophil Cytoplasmic Antibody (ANCA)-Associated Vasculitis (RAVE) trial and identify novel potential risk factors. Methods VTE events in 197 patients enrolled in the RAVE trial were analyzed. Baseline demographic and clinical characteristics were recorded, and univariate and multivariate analyses were performed to identify factors associated with VTE in ANCA-associated vasculitis (AAV). Results VTE occurred in 16 patients (8.1%) with an overall average time to event of 1.5 months (range 1.0-2.75). In univariate analyses with calculation of hazard ratios (HRs) and 95% confidence intervals (95% CIs), heart involvement (HR 17.408 [95% CI 2.247-134.842]; P = 0.006), positive proteinase 3 (PR3)-ANCA (HR 7.731 [95% CI 1.021-58.545]; P = 0.048), pulmonary hemorrhage (HR 3.889 [95% CI 1.448-10.448]; P = 0.008), and the presence of red blood cell casts (HR 15.617 [95% CI 3.491-69.854]; P <0.001) were associated with the onset of VTE. In multivariate models adjusted for age and sex, the significant associations between VTE events and heart involvement (HR 21.836 [95% CI 2.566-185.805]; P = 0.005), PR3-ANCA (HR 9.12 [95% CI 1.158-71.839]; P = 0.036), pulmonary hemorrhage (HR 3.91 [95% CI 1.453-10.522]; P = 0.007), and urinary red blood cell casts (HR 16.455 [95% CI 3.607-75.075]; P <0.001) remained. Conclusion Patients diagnosed as having AAV with pulmonary hemorrhage, positive PR3-ANCA, heart involvement, and the presence of red blood cell casts are at an increased risk to develop VTE. Further studies are needed to confirm and expand these findings and to explore the mechanisms of hypercoagulability in these patients with the aim of informing potential targets for therapeutic intervention

Non-adherence to cardiometabolic medication as assessed by LC-MS/MS in urine and its association with kidney and cardiovascular outcomes in type 2 diabetes mellitus

Aims/hypothesis Non-adherence to medication is a frequent barrier in the treatment of patients with type 2 diabetes mellitus, potentially limiting the effectiveness of evidence-based treatments. Previous studies have mostly relied on indirect adherence measures to analyse outcomes based on adherence. The aim of this study was to use LC-MS/MS in urine—a non-invasive, direct and objective measure—to assess non-adherence to cardiometabolic drugs and analyse its association with kidney and cardiovascular outcomes. Methods This cohort study includes 1125 participants from the PROVALID study, which follows patients with type 2 diabetes mellitus at the primary care level. Baseline urine samples were tested for 79 cardiometabolic drugs and metabolites thereof via LC-MS/MS. An individual was classified as totally adherent if markers for all drugs were detected, partially non-adherent when at least one marker for one drug was detected, and totally non-adherent if no markers for any drugs were detected. Non-adherence was then analysed in the context of cardiovascular (composite of myocardial infarction, stroke and cardiovascular death) and kidney (composite of sustained 40% decline in eGFR, sustained progression of albuminuria, kidney replacement therapy and death from kidney failure) outcomes. Results Of the participants, 56.3% were totally adherent, 42.0% were partially non-adherent, and 1.7% were totally non-adherent to screened cardiometabolic drugs. Adherence was highest to antiplatelet and glucose-lowering agents and lowest to lipid-lowering agents. Over a median (IQR) follow-up time of 5.10 (4.12–6.12) years, worse cardiovascular outcomes were observed with non-adherence to antiplatelet drugs (HR 10.13 [95% CI 3.06, 33.56]) and worse kidney outcomes were observed with non-adherence to antihypertensive drugs (HR 1.98 [95% CI 1.37, 2.86]). Conclusions/interpretation This analysis shows that non-adherence to cardiometabolic drug regimens is common in type 2 diabetes mellitus and negatively affects kidney and cardiovascular outcomes

Immunologic Changes Implicated in the Pathogenesis of Focal Segmental Glomerulosclerosis

Focal segmental glomerulosclerosis is a histological pattern on renal biopsy caused by diverse mechanisms. In its primary form, a circulatory factor is implicated in disease onset and recurrence. The natural history of primary FSGS is unpredictable, since some patients are unresponsive towards immunosuppressive measures. Immunologic changes, leading to a proinflammatory or profibrotic milieu, have been implicated in disease progression, namely, glomerular scarring, eventually leading to end-stage renal disease. Among these, interleukin-1ß, tumor-necrosis factor-α (TNF-α), and transforming growth factor-ß1 (TGF-ß1) have emerged as important factors. Translating these findings into clinical practice dampened the enthusiasm, since both TNF-α and TGF-ß1 blockade failed to achieve significant control of the disease. More recently, a role of the complement system has been demonstrated which in fact may be another attractive target in clinical practice. Rituximab, blocking CD20-bearing cells, demonstrated conflicting data regarding efficacy in FSGS. Finally, the T-cell costimulating molecule B7-1 (CD80) is implicated in development of proteinuria in general. Blockade of this target demonstrated significant benefits in a small cohort of resistant patients. Taken together, this review focuses on immunology of FSGS, attributable to either the disease or progression, and discusses novel therapeutic approaches aiming at targeting immunologic factors.status: publishe

Immunologic Changes Implicated in the Pathogenesis of Focal Segmental Glomerulosclerosis

Focal segmental glomerulosclerosis is a histological pattern on renal biopsy caused by diverse mechanisms. In its primary form, a circulatory factor is implicated in disease onset and recurrence. The natural history of primary FSGS is unpredictable, since some patients are unresponsive towards immunosuppressive measures. Immunologic changes, leading to a proinflammatory or profibrotic milieu, have been implicated in disease progression, namely, glomerular scarring, eventually leading to end-stage renal disease. Among these, interleukin-1ß, tumor-necrosis factor-α (TNF-α), and transforming growth factor-ß1 (TGF-ß1) have emerged as important factors. Translating these findings into clinical practice dampened the enthusiasm, since both TNF-α and TGF-ß1 blockade failed to achieve significant control of the disease. More recently, a role of the complement system has been demonstrated which in fact may be another attractive target in clinical practice. Rituximab, blocking CD20-bearing cells, demonstrated conflicting data regarding efficacy in FSGS. Finally, the T-cell costimulating molecule B7-1 (CD80) is implicated in development of proteinuria in general. Blockade of this target demonstrated significant benefits in a small cohort of resistant patients. Taken together, this review focuses on immunology of FSGS, attributable to either the disease or progression, and discusses novel therapeutic approaches aiming at targeting immunologic factors