CITED4 gene therapy protects against maladaptive cardiac remodeling after ischemia/reperfusion injury in mice.

Cardiac signaling pathways functionally important in the heart's response to exercise often protect the heart against pathological stress, potentially providing novel therapeutic targets. However, it is important to determine which of these pathways can be feasibly targeted in vivo. Transgenic overexpression of exercise-induced CITED4 has been shown to protect against adverse remodeling after ischemia/reperfusion injury (IRI). Here we investigated whether somatic gene transfer of CITED4 in a clinically relevant time frame could promote recovery after IRI. Cardiac CITED4 gene delivery via intravenous AAV9 injections in wild type mice led to an approximately 3-fold increase in cardiac CITED4 expression. After 4 weeks, CITED4-treated animals developed physiological cardiac hypertrophy without adverse remodeling. In IRI, delivery of AAV9-CITED4 after reperfusion resulted in a 6-fold increase in CITED4 expression 1 week after surgery, as well as decreased apoptosis, fibrosis, and inflammatory markers, culminating in a smaller scar and improved cardiac function 8 weeks after IRI, compared with control mice receiving AAV9-GFP. Somatic gene transfer of CITED4 induced a phenotype suggestive of physiological cardiac growth and mitigated adverse remodeling after ischemic injury. These studies support the feasibility of CITED4 gene therapy delivered in a clinically relevant time frame to mitigate adverse ventricular remodeling after ischemic injury.

Long-Term Outcomes of Rituximab-Treated Adult Patients with Podocytopathies.

BACKGROUND: Long-term outcomes of rituximab-treated adult patients with podocytopathies (either minimal change disease or focal segmental glomerulosclerosis) are largely unknown. METHODS: A retrospective study at 30 nephrology departments from 15 countries worldwide included rituximab-treated adults with primary podocytopathies and a minimum clinical follow-up of 36 months. The primary outcome was relapse-free survival at 36 months. RESULTS: 183 adult patients (n=64 with focal segmental glomerulosclerosis and n=119 with minimal change disease) with difficult-to-treat nephrotic syndrome (68% steroid-dependent/frequently relapsing, 22% steroid-resistant, 85% previously treated with two or more lines of immunosuppressive therapy) were treated with rituximab as part of a remission induction regimen. Complete or partial remission at 6 months after rituximab treatment was achieved in 82%. Eighty-three of 151 (55%) initial responders achieved long-term relapse-free survival over three years. Maintenance therapy with rituximab was associated with a better relapse-free survival (HR 2.05, 95% CI: 1.07-3.91), irrespective of the dosing regimen. At 36 months, 61% of initial responders receiving maintenance therapy with rituximab achieved long-term relapse-free survival and withdrawal of all concomitant immunosuppressive medication compared to 36% of patients without maintenance treatment (OR 2.69, 95% CI: 1.27-5.73). Relapses per year were reduced from an annual relapse rate of 1.0 (95% CI: 1.0-1.7) before to 0.17 (95% CI: 0.00-0.24) relapses/year after rituximab initiation. Over the 36 months of follow-up, a stable course of estimated glomerular filtration rate (eGFR) was observed in those who initially responded with either complete or partial remission, whereas non-responders experienced a reduction in eGFR reaching -11 (95% CI: -18 to -8) mL/min/1.73m2 . CONCLUSIONS: Rituximab facilitated achievement of initial and long-term response in a majority of adult patients with difficult-to-treat podocytopathies. Maintenance treatment with rituximab further associated with long-term relapse-free survival over three years. Non-response to initial rituximab treatment was associated with poor kidney prognosis.

Deceased donor urinary Dickkopf-3 associates with future allograft function following kidney transplantation.

Predicting future kidney allograft function is challenging. Novel biomarkers, such as urinary Dickkopf-3 (uDKK3), may help guide donor selection and improve allograft outcomes. In this prospective multicenter pilot trial, we investigated whether donor uDKK3 reflects organ quality and is associated with future allograft function. We measured uDKK3/crea ratios (uDKK3/crea) from 95 deceased and 46 living kidney donors. Prenephrectomy uDKK3/crea levels were 100× higher in deceased than in living donors (9888 pg/mg vs 113 pg/mg; P < .001). Among deceased donor transplantations, recipients were stratified by their corresponding uDKK3/crea donor levels ranging below (group A, n = 68) or above (group B, n = 65) median. The primary end point of best estimated glomerular filtration rate (eGFR) within the first 3 months after kidney transplantation was superior in group A (56.3 mL/min/1.73 m2) than that in group B (44.2 mL/min/1.73 m2; P = .0139). Second, the composite clinical end point consisting of death, allograft failure or eGFR decline >50% occurred less frequent in group A. By mixed linear regression modeling, donor uDKK3/crea remained an independent predictor of eGFR after transplantation, with a slope of -4.282 mL/min/1.73 m2 per logarithmic increase in donor uDKK3/crea. In summary, uDKK3 may serve as a noninvasive, donor-dependent biomarker for assessing organ quality and future allograft function.

S100A1's single cysteine is an indispensable redox switch for the protection against diastolic calcium waves in cardiomyocytes.

The EF-hand calcium (Ca2+) sensor protein S100A1 combines inotropic with antiarrhythmic potency in cardiomyocytes (CMs). Oxidative posttranslational modification (ox-PTM) of S100A1's conserved, single-cysteine residue (C85) via reactive nitrogen species (i.e., S-nitrosylation or S-glutathionylation) has been proposed to modulate conformational flexibility of intrinsically disordered sequence fragments and to increase the molecule's affinity toward Ca2+. Considering the unknown biological functional consequence, we aimed to determine the impact of the C85 moiety of S100A1 as a potential redox switch. We first uncovered that S100A1 is endogenously glutathionylated in the adult heart in vivo. To prevent glutathionylation of S100A1, we generated S100A1 variants that were unresponsive to ox-PTMs. Overexpression of wild-type (WT) and C85-deficient S100A1 protein variants in isolated CM demonstrated equal inotropic potency, as shown by equally augmented Ca2+ transient amplitudes under basal conditions and ß-adrenergic receptor (ßAR) stimulation. However, in contrast, ox-PTM defective S100A1 variants failed to protect against arrhythmogenic diastolic sarcoplasmic reticulum (SR) Ca2+ waves and ryanodine receptor 2 (RyR2) hypernitrosylation during ßAR stimulation. Despite diastolic performance failure, C85-deficient S100A1 protein variants exerted similar Ca2+-dependent interaction with the RyR2 than WT-S100A1. Dissecting S100A1's molecular structure-function relationship, our data indicate for the first time that the conserved C85 residue potentially acts as a redox switch that is indispensable for S100A1's antiarrhythmic but not its inotropic potency in CMs. We, therefore, propose a model where C85's ox-PTM determines S100A1's ability to beneficially control diastolic but not systolic RyR2 activity.NEW & NOTEWORTHY S100A1 is an emerging candidate for future gene-therapy treatment of human chronic heart failure. We aimed to study the significance of the conserved single-cysteine 85 (C85) residue in cardiomyocytes. We show that S100A1 is endogenously glutathionylated in the heart and demonstrate that this is dispensable to increase systolic Ca2+ transients, but indispensable for mediating S100A1's protection against sarcoplasmic reticulum (SR) Ca2+ waves, which was dependent on the ryanodine receptor 2 (RyR2) nitrosylation status.

More than fear? Brain activation patterns of dental phobic patients before and after an exposure-based treatment.

Hyperactivation of brain networks conferring defensive mobilization is assumed to underlie inappropriate defensive-preparation in patients with Specific Phobia. However, studies targeting Dental Phobia (DP) yielded quite heterogeneous results and research concerning the effects of exposure treatments on phobic brain activation so far is missing. This functional Magnetic Resonance Imaging (fMRI) study aimed to investigate activation patterns in DP patients during exposure to phobia-related stimuli and the effects of an exposure-based fear treatment on phobia-related activation. Seventeen patients with DP and seventeen non-phobic, healthy controls participated in this fMRI experiment presenting dental-related and neutral auditory and visual stimuli. After completing a short exposure-based CBT program, patients were scanned a second time to illustrate treatment-related changes in brain activation patterns. Pre-treatment fMRI results demonstrate enhanced activation in DP-patients mainly in the precuneus and lateral parietal cortex. Moreover, a small activation focus was observed in the amygdala and anterior cingulate cortex (ACC) as parts of classically fear-related structures. Activation in all these clusters decreased significantly from pre- to post-treatment assessment and in the case of the ACC was correlated with dental fear reduction. Activation changes in the precuneus and lateral parietal cortex suggest a pronounced first-person perspective memory processing including a vivid recall of contextual information from an egocentric perspective triggered by exposure to phobia-related stimuli. Besides a treatment-sensitive hyperactivity of fear-sensitive structures, DP may also be characterized by a disturbed memory retrieval that can be reorganized by successful exposure treatment.

Interactive exploration of adverse events and multimorbidity in CKD.

BACKGROUND AND HYPOTHESIS: Persons with chronic kidney disease (CKD) are at increased risk of adverse events, early mortality, and multimorbidity. A detailed overview of adverse event types and rates from a large CKD cohort under regular nephrological care is missing. We generated an interactive tool to enable exploration of adverse events and their combinations in the prospective, observational German CKD (GCKD) study. METHODS: The GCKD study enrolled 5217 participants under regular nephrological care with an estimated glomerular filtration rate of 30-60 or >60 mL/min/1.73m2 and an overt proteinuria. Cardio-, cerebro- and peripheral vascular, kidney, infection, and cancer events, as well as deaths were adjudicated following a standard operation procedure. We summarized these time-to-event data points for exploration in interactive graphs within an R shiny app. Multivariable adjusted Cox models for time to first event were fitted. Cumulative incidence functions, Kaplan-Meier curves and intersection plots were used to display main adverse events and their combinations by sex and CKD etiology. RESULTS: Over a median of 6.5 years, 10 271 events occurred in total and 680 participants (13.0%) died while 2947 participants (56.5%) experienced any event. The new publicly available interactive platform enables readers to scrutinize adverse events and their combinations as well as mortality trends as a gateway to better understand multimorbidity in CKD: incident rates per 1000 patient-years varied by event type, CKD etiology, and baseline characteristics. Incidence rates for the most frequent events and their recurrence were 113.6 (cardiovascular), 75.0 (kidney), and 66.0 (infection). Participants with diabetic kidney disease and men were more prone to experiencing events. CONCLUSION: This comprehensive explorative tool to visualize adverse events (https://gckd.diz.uk-erlangen.de/), their combination, mortality, and multimorbidity among persons with CKD may manifest as a valuable resource for patient care, identification of high-risk groups, health services, and public health policy planning.

Amdoparvovirus-associated disease in red pandas (Ailurus fulgens).

The roster of amdoparvoviruses (APVs) in small carnivores is growing rapidly, but in most cases, the consequences of infection are poorly understood. Red panda amdoparvovirus (RPAV) is highly prevalent in zoo-housed red pandas and has been detected in both healthy and sick animals. Clarifying the clinical impact of RPAV in this endangered species is critical, and zoological collections offer a unique opportunity to examine viral disease association in carefully managed populations. We evaluated the potential impact of RPAV in captive red pandas with a combination of prospective and retrospective analyses. First, we collected feces from 2 healthy animals from one collection over a 6-year period and detected virus in 72/75 total samples, suggesting that RPAV can be a long-term subclinical infection. We next investigated the infections using a retrospective study of infection status and tissue distribution in a cohort of necropsied animals. We performed polymerase chain reaction and in situ hybridization on 43 necropsy cases from 4 zoo collections (3 from the United States, 1 from Europe, 1997-2022). RPAV was present in these populations for at least 2 decades before its discovery and is detectable in common and significant lesions of zoo-housed red pandas, including myocarditis (3/3 cases), nephritis (9/10), and interstitial pneumonia (2/4). RPAV is also detectable in sporadic lesions, including multisystemic pyogranulomatous inflammation, oral/pharyngeal mucosal inflammation, and dermatitis. The colocalization of virus with lesions supports a role in causation, suggesting that despite the apparently persistent and subclinical carriage of most infections, RPAV may have a significant impact in zoo collections.

Effects of Early Empagliflozin Initiation on Diuresis and Kidney Function in Patients With Acute Decompensated Heart Failure (EMPAG-HF).

BACKGROUND: Effective diuretic regimens using loop diuretics in patients with acute decompensated heart failure are often limited by the development of worsening kidney function. Sodium-glucose cotransporter-2 inhibitors induce glucosuria and sodium excretion with nephroprotective effects in patients with stable heart failure but their role in acute decompensated heart failure is unclear. METHODS: In this single-center, prospective, double-blind, placebo-controlled, randomized study, we randomly assigned patients with acute decompensated heart failure to empagliflozin 25 mg daily or placebo in addition to standard decongestive treatments that included loop diuretics. The primary end point was cumulative urine output over 5 days. Secondary end points included diuretic efficiency, dynamics in markers of kidney function and injury, and NT-proBNP (N-terminal pro-B-type natriuretic peptide). RESULTS: Sixty patients were randomized within 12 hours of hospitalization for acute decompensated heart failure. Addition of empagliflozin daily to standard medical treatment of acute decompensated heart failure resulted in a 25% increase in cumulative urine output over 5 days (median 10.8 versus 8.7 L mL in placebo, group difference estimation 2.2 L [95% CI, 8.4 to 3.6]; P=0.003). Empagliflozin increased diuretic efficiency compared with placebo (14.1 mL urine per milligram furosemide equivalent [95% CI, 0.6-27.7]; P=0.041) without affecting markers of renal function (estimated glomerular filtration rate, 51±19 versus 54±17 mL/min per 1.73 m²; P=0.599) or injury (total urinary protein, 492±845 versus 503±847 mg/g creatinine; P=0.975; and urinary α1-microglobulin, 55.4±38.6 versus 31.3±33.6 mg/g creatinine; P=0.066) with more pronounced decrease in NT-proBNP in the empagliflozin group compared with placebo (-1861 versus -727.2 pg/mL after 5 days; quotient in slope, 0.89 [95% CI, 0.83-0.95]; P<0.001). There were no differences in the incidence of safety events between groups. CONCLUSIONS: Early addition of empagliflozin to standard diuretic therapy increases urine output without affecting renal function in patients with acute decompensated heart failure. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT04049045.

Different roles of protein biomarkers predicting eGFR trajectories in people with chronic kidney disease and diabetes mellitus: a nationwide retrospective cohort study.

BACKGROUND: Chronic kidney disease (CKD) is a common comorbidity in people with diabetes mellitus, and a key risk factor for further life-threatening conditions such as cardiovascular disease. The early prediction of progression of CKD therefore is an important clinical goal, but remains difficult due to the multifaceted nature of the condition. We validated a set of established protein biomarkers for the prediction of trajectories of estimated glomerular filtration rate (eGFR) in people with moderately advanced chronic kidney disease and diabetes mellitus. Our aim was to discern which biomarkers associate with baseline eGFR or are important for the prediction of the future eGFR trajectory. METHODS: We used Bayesian linear mixed models with weakly informative and shrinkage priors for clinical predictors (n = 12) and protein biomarkers (n = 19) to model eGFR trajectories in a retrospective cohort study of people with diabetes mellitus (n = 838) from the nationwide German Chronic Kidney Disease study. We used baseline eGFR to update the models' predictions, thereby assessing the importance of the predictors and improving predictive accuracy computed using repeated cross-validation. RESULTS: The model combining clinical and protein predictors had higher predictive performance than a clinical only model, with an [Formula: see text] of 0.44 (95% credible interval 0.37-0.50) before, and 0.59 (95% credible interval 0.51-0.65) after updating by baseline eGFR, respectively. Only few predictors were sufficient to obtain comparable performance to the main model, with markers such as Tumor Necrosis Factor Receptor 1 and Receptor for Advanced Glycation Endproducts being associated with baseline eGFR, while Kidney Injury Molecule 1 and urine albumin-creatinine-ratio were predictive for future eGFR decline. CONCLUSIONS: Protein biomarkers only modestly improve predictive accuracy compared to clinical predictors alone. The different protein markers serve different roles for the prediction of longitudinal eGFR trajectories potentially reflecting their role in the disease pathway.

Circulating MicroRNAs as Biomarker for Vessel-Associated Retinal Diseases.

INTRODUCTION: Vessel-associated retinal diseases are a major cause of blindness and severe visual impairment. The identification of appropriate biomarkers is of great importance to better anticipate disease progression and establish more targeted treatment options. MicroRNAs (miRNAs) are short, single-stranded, noncoding ribonucleic acids that are involved in the posttranscriptional regulation of gene expression through hybridization with messenger RNA. The expression of certain miRNAs can be different in patients with pathological processes and can be used for the detection and differentiation of various diseases. In this study, we investigate to what extent previously in vitro identified miRNAs are present as cell-free circulating miRNAs in the serum and vitreous of human patients with and without vessel-associated retinal diseases. METHODS: Relative quantification by quantitative real-time polymerase chain reaction was used to analyze miRNA expression in patients with vessel-associated retinal diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), and retinal vein occlusion compared with control patients. RESULTS: In serum samples, miR-29a-3p and miR-192-5p showed increased expression in patients with neovascular AMD relative to control patients. Similarly, miR-335-5p, miR-192-5p, and miR-194-5p showed increased expression in serum from patients with proliferative DR. In vitreous samples, miR-100-5p was decreased in patients with proliferative DR. Differentially expressed miRNAs showed good diagnostic accuracy in receiver operating characteristic (ROC) and area under the ROC curve analysis. CONCLUSION: The miRNAs investigated in this study may have the potential to serve as biomarkers for vessel-associated retinal diseases. Combining multiple miRNAs may enhance the predictive power of the analysis.

Differential Prognostic Utility of Adiposity Measures in Chronic Kidney Disease.

OBJECTIVE: Adipose tissue contributes to adverse outcomes in chronic kidney disease (CKD), but there is uncertainty regarding the prognostic relevance of different adiposity measures. We analyzed the associations of neck circumference (NC), waist circumference (WC), and body mass index (BMI) with clinical outcomes in patients with mild to severe CKD. METHODS: The German Chronic Kidney Disease study is a prospective cohort study, which enrolled Caucasian adults with mild to severe CKD, defined as estimated glomerular filtration rate : 30-60 mL/min/1.73 m2, or >60 mL/min/1.73 m2 in the presence of overt proteinuria. Associations of NC, WC, and BMI with all-cause death, major adverse cardiovascular events (MACE: a composite of nonfatal stroke, nonfatal myocardial infarction, peripheral artery disease intervention, and cardiovascular death), and kidney failure (a composite of dialysis or transplantation) were analyzed using multivariable Cox proportional hazards regression models adjusted for confounders and the Akaike information criteria were calculated. Models included sex interactions with adiposity measures. RESULTS: A total of 4537 participants (59% male) were included in the analysis. During a 6.5-year follow-up, 339 participants died, 510 experienced MACE, and 341 developed kidney failure. In fully adjusted models, NC was associated with all-cause death in women (hazard ratio 1.080 per cm; 95% CI 1.009-1.155) but not in men. Irrespective of sex, WC was associated with all-cause death (hazard ratio 1.014 per cm; 95% CI 1.005-1.038). NC and WC showed no association with MACE or kidney failure. BMI was not associated with any of the analyzed outcomes. Models of all-cause death, including WC offered the best (lowest) Akaike information criteria. CONCLUSION: In Caucasian patients with mild to severe CKD, higher NC (in women) and WC were significantly associated with increased risk of death from any cause but BMI was not.

Heart-Type Fatty Acid Binding Protein, Cardiovascular Outcomes, and Death: Findings From the German CKD Cohort Study.

RATIONALE & OBJECTIVE: Heart-type fatty acid binding protein (H-FABP) is a biomarker that has been shown to provide long-term prognostic information in patients with coronary artery disease independently of high-sensitivity troponin T (hs-TNT). We examined the independent associations of H-FABP with cardiovascular outcomes in patients with chronic kidney disease (CKD). STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: 4,951 patients enrolled in the German Chronic Kidney Disease (GCKD) study with an estimated glomerular filtration rate of 30-60 mL/min/1.73 m2 or overt proteinuria (urinary albumin-creatinine ratio > 300 mg/g or equivalent). EXPOSURE: Serum levels of H-FABP and hs-TNT were measured at study entry. OUTCOME: Noncardiovascular (non-CV) death, CV death, combined major adverse CV events (MACE), and hospitalization for congestive heart failure (CHF). ANALYTICAL APPROACH: Hazard ratios (HRs) for associations of H-FABP and hs-TNT with outcomes were estimated using Cox regression analyses adjusted for established risk factors. RESULTS: During a maximum follow-up of 6.5 years, 579 non-CV deaths, 190 CV deaths, 522 MACE, and 381 CHF hospitalizations were observed. In Cox regression analyses adjusted for established risk factors, H-FABP was associated with all 4 outcomes, albeit with lower HRs than those found for hs-TNT. After further adjustment for hs-TNT levels, H-FABP was found to be associated with non-CV death (HR, 1.57 [95% CI, 1.14-2.18]) and MACE (HR, 1.40 [95% CI, 1.02-1.92]) but with neither CV death (HR, 1.64 [95% CI, 0.90-2.99]) nor CHF hospitalizations (HR, 1.02 [95% CI, 0.70-1.49]). LIMITATIONS: Single-point measurements of H-FABP and hs-TNT. Uncertain generalizability to non-European populations. CONCLUSIONS: In this large cohort of patients with CKD, H-FABP was associated with non-CV death and MACE, even after adjustment for hs-TNT. Whether measurement of H-FABP improves cardiovascular disease risk prediction in these patients warrants further studies.

Assessment of angiogenesis-related parameters in juvenile idiopathic arthritis-associated uveitis.

PURPOSE: Juvenile idiopathic arthritis-associated uveitis (JIAU) may run a chronic and treatment-resistant course, and occasionally, alterations of the iris vasculature may be observed clinically. METHODS: Iris tissue (IT), aqueous humor (AH) and serum samples from patients with clinically inactive JIAU (n = 30), acute anterior uveitis (AAU; n = 18), and primary open angle glaucoma (POAG; n = 20) were obtained during trabeculectomy or cataract surgery. Samples were analyzed by RNA-Seq, qRT-PCR, LC-IMS, Western-Blot, and LEGENDplex™ analysis. Pattern of iris vasculature in JIAU patients was assessed qualitatively via fluorescein and indocyanine green angiography (FLA/ICGA). RESULTS: RNA-Seq of IT showed significantly differential expression (DE) of 136 genes between JIAU and POAG, of which 15 were associated with angiogenesis. qRT-PCR, performed to validate RNA-Seq results, showed upregulation of the angiogenesis-related genes Kdr, Angpt-1, Tie-1, Tie-2 and Mmrn2 in IT (JIAU vs POAG, p > 0.05). LC-IMS of IT revealed a total number of 56 DE proteins (JIAU vs POAG), of which Angiopoetin, Lumican and Decorin were associated with angiogenesis and showed increased (p > 0.05) expression on Western-Blot analysis. LEGENDplex™ analysis showed upregulation of ANGPT-2 in AH from JIAU compared to AAU and POAG, whereas VEGF was upregulated in AAU. Iris vascular leakage, hypoperfusion and neovascularization were observed by FLA/ICGA in JIA patients with treatment-refractory complicated course of uveitis. CONCLUSION: Angiogenesis-related factors could play a role in long-standing complicated JIAU, leading to clinically visible alterations in selected cases.

Updated and enhanced pig cardiac transcriptome based on long-read RNA sequencing and proteomics.

Clinically translatable large animal models have become indispensable for cardiovascular research, clinically relevant proof of concept studies and for novel therapeutic interventions. In particular, the pig has emerged as an essential cardiovascular disease model, because its heart, circulatory system, and blood supply are anatomically and functionally similar to that of humans. Currently, molecular and omics-based studies in the pig are hampered by the incompleteness of the genome and the lack of diversity of the corresponding transcriptome annotation. Here, we employed Nanopore long-read sequencing and in-depth proteomics on top of Illumina RNA-seq to enhance the pig cardiac transcriptome annotation. We assembled 15,926 transcripts, stratified into coding and non-coding, and validated our results by complementary mass spectrometry. A manual review of several gene loci, which are associated with cardiac function, corroborated the utility of our enhanced annotation. All our data are available for download and are provided as tracks for integration in genome browsers. We deem this resource as highly valuable for molecular research in an increasingly relevant large animal model.

Development of an AAV9-RNAi-mediated silencing strategy to abrogate TRPM4 expression in the adult heart.

The cation channel transient receptor potential melastatin 4 (TRPM4) is a calcium-activated non-selective cation channel and acts in cardiomyocytes as a negative modulator of the L-type Ca2+ influx. Global deletion of TRPM4 in the mouse led to increased cardiac contractility under ß-adrenergic stimulation. Consequently, cardiomyocyte-specific inactivation of the TRPM4 function appears to be a promising strategy to improve cardiac contractility in heart failure patients. The aim of this study was to develop a gene therapy approach in mice that specifically silences the expression of TRPM4 in cardiomyocytes. First, short hairpin RNAmiR30 (shRNAmiR30) sequences against the TRPM4 mRNA were screened in vitro using lentiviral transduction for a stable expression of the shRNA cassettes. Western blot analysis identified three efficient shRNAmiR30 sequences out of six, which reduced the endogenous TRPM4 protein level by up to 90 ± 6%. Subsequently, the most efficient shRNAmiR30 sequences were delivered into cardiomyocytes of adult mice using adeno-associated virus serotype 9 (AAV9)-mediated gene transfer. Initially, the AAV9 vector particles were administered via the lateral tail vein, which resulted in a downregulation of TRPM4 by 46 ± 2%. Next, various optimization steps were carried out to improve knockdown efficiency in vivo. First, the design of the expression cassette was streamlined for integration in a self-complementary AAV vector backbone for a faster expression. Compared to the application via the lateral tail vein, intravenous application via the retro-orbital sinus has the advantage that the vector solution reaches the heart directly and in a high concentration, and eventually a TRPM4 knockdown efficiency of 90 ± 7% in the heart was accomplished by this approach. By optimization of the shRNAmiR30 constructs and expression cassette as well as the route of AAV9 vector application, a 90% reduction of TRPM4 expression was achieved in the adult mouse heart. In the future, AAV9-RNAi-mediated inactivation of TRPM4 could be a promising strategy to increase cardiac contractility in preclinical animal models of acute and chronic forms of cardiac contractile failure.

Peritoneal Level of CD206 Associates With Mortality and an Inflammatory Macrophage Phenotype in Patients With Decompensated Cirrhosis and Spontaneous Bacterial Peritonitis.

BACKGROUND & AIMS: Peritoneal macrophages (PMs) regulate inflammation and control bacterial infections in patients with decompensated cirrhosis. We aimed to characterize PMs and associate their activation with outcomes of patients with spontaneous bacterial peritonitis (SBP). METHODS: We isolated PMs from ascites samples of 66 patients with decompensated cirrhosis (19 with SBP) and analyzed them by flow cytometry, quantitative real-time polymerase chain reaction, functional analysis, and RNA microarrays. We used ascites samples of a separate cohort of 111 patients with decompensated cirrhosis (67 with SBP) and quantified the soluble form of the mannose receptor (CD206) and tumor necrosis factor by enzyme-linked immunosorbent assay (test cohort). We performed logistic regression analysis to identify factors associated with 90-day mortality. We validated our findings using data from 71 patients with cirrhosis and SBP. Data from 14 patients undergoing peritoneal dialysis for end-stage renal disease but without cirrhosis were included as controls. RESULTS: We used surface levels of CD206 to identify subsets of large PMs (LPM) and small PMs (SPM), which differed in granularity and maturation markers, in ascites samples from patients with cirrhosis. LPMs vs SPMs from patients with cirrhosis had different transcriptomes; we identified more than 4000 genes that were differentially regulated in LPMs vs SPMs, including those that regulate the cycle, metabolism, self-renewal, and immune cell signaling. LPMs had an inflammatory phenotype, were less susceptible to tolerance induction, and released more tumor necrosis factor than SPMs. LPMs from patients with cirrhosis produced more inflammatory cytokines than LPMs from controls. Activation of PMs by Toll-like receptor agonists and live bacteria altered levels of CD206 on the surface of LPMs and release of soluble CD206. Analysis of serial ascites fluid from patients with SBP revealed loss of LPMs in the early phase of SBP, but levels increased after treatment. In the test and validation cohorts, patients with SBP and higher concentrations of soluble CD206 in ascites fluid (>0.53 mg/L) were less likely to survive for 90 days than those with lower levels. CONCLUSIONS: Surface level of CD206 can be used to identify mature, resident, inflammatory PMs in patients with cirrhosis. Soluble CD206 is released from activated LPMs and increased concentrations in patients with cirrhosis and SBP indicate reduced odds of surviving for 90 days.

Association of the metabolic syndrome with mortality and major adverse cardiac events: A large chronic kidney disease cohort.

BACKGROUND: Metabolic syndrome with its key components insulin resistance, central obesity, dyslipidaemia, and hypertension is associated with a high risk for cardiovascular events and all-cause mortality in the general population. However, evidence that these findings apply to patients with chronic kidney disease (CKD) with moderately reduced estimated glomerular filtration rate and/or albuminuria is limited. OBJECTIVES: We aimed to investigate the association between metabolic syndrome and its components with all-cause mortality and cardiovascular outcomes in CKD patients. METHODS: Prospective observation of a cohort of 5110 CKD patients from the German Chronic Kidney Disease study with 3284 (64.3%) of them having a metabolic syndrome at baseline. RESULTS: During the follow-up of 6.5 years, 605 patients died and 650 patients experienced major cardiovascular events. After extended data adjustment, patients with a metabolic syndrome had a higher risk for all-cause mortality (hazard ratio [HR] = 1.26, 95% confidence interval [CI]: 1.04-1.54) and cardiovascular events (HR = 1.48, 95% CI: 1.22-1.79). The risk increased steadily with a growing number of metabolic syndrome components (increased waist circumference, glucose, triglycerides, hypertension and decreased HDL cholesterol): HR per component = 1.09 (95% CI: 1.02-1.17) for all-cause mortality and 1.23 (95% CI: 1.15-1.32) for cardiovascular events. This resulted in hazard ratios between 1.50 and 2.50 in the case when four or five components are present. An analysis of individual components of metabolic syndrome showed that the glucose component led to the highest increase in risk for all-cause mortality (HR = 1.68, 95% CI: 1.38-2.03) and cardiovascular events (HR = 1.81, 95% CI: 1.51-2.18), followed by the HDL cholesterol and triglyceride components. CONCLUSIONS: We observed a high prevalence of metabolic syndrome among patients with moderate CKD. Metabolic syndrome increases the risk for all-cause mortality and cardiovascular events. The glucose and lipid components seem to be the main drivers for the association with outcomes.

Retention in the Austrian opioid agonist treatment system: a national prospective cohort study.

BACKGROUND: Retention in care is a prerequisite for successful recovery, especially for a chronic condition like opioid dependence. Though retention varies greatly depending on the different substitution medication and treatment model, treatment retention is used as an indicator of treatment quality and effectiveness of care on a system and individual level. To monitor the overall quality of the Austrian opioid agonist treatment (OAT) system and to monitor patient satisfaction within the system, a new online-based registry called "eSuchmittel" was introduced in Austria at the beginning of 2011. The objective of this study is to analyze retention rates within the Austrian treatment system and to identify patient characteristics associated with retention, using data collected by the substitution registry. METHODS: The complete Austrian sample of 4778 registered patients starting treatment between 1.1.2011 to 31.12.2012 were included in the prospective cohort study using data from the Austrian substitution registry. For the statistical analysis, multivariate Cox Regression and Kaplan-Meier survival analysis were used to evaluate retention in treatment. RESULTS: The retention rate of the total cohort after two years was around 61%. Retention rates were significantly lower for men (exp(B) = .806, 95% CI 0.714-0.908) and significantly higher for patients aged 30 and older (exp(B) = 1.155, 95% CI 1.044-1.279), among patients located in Vienna (exp(B) = 1.439, 95% CI 1.273-1.626) and among patients prescribed oral slow-release morphine (SROM) (exp(B) = 2.141, 95% CI 1.885-2.430). CONCLUSIONS: Average retention in the Austrian system is high in comparison to international retention rates. Nationally, SROM demonstrates higher treatment retention when compared to other available substitution medications. Sociodemographic and regional indicators also contribute to higher retention in care. A systematic monitoring of retention rates within a national registry is an important tool helping to evaluate the quality of care. In this study, the Austrian OAT system proves very high retention in care, an important success criterion.

Transcriptomic and proteomic analysis of iris tissue and aqueous humor in juvenile idiopathic arthritis-associated uveitis.

Gene and protein expression profiles of iris biopsies, aqueous humor (AqH), and sera in patients with juvenile idiopathic arthritis-associated uveitis (JIAU) in comparison to control patients with primary open-angle glaucoma (POAG) and HLA-B27-positive acute anterior uveitis (AAU) were investigated. Via RNA Sequencing (RNA-Seq) and mass spectrometry-based protein expression analyses 136 genes and 56 proteins could be identified as being significantly differentially expressed (DE) between the JIAU and POAG group. Gene expression of different immunoglobulin (Ig) components as well as of the B cell-associated factors ID3, ID1, and EBF1 was significantly upregulated in the JIAU group as compared to POAG patients. qRT-PCR analysis showed a significantly higher gene expression of the B cell-related genes CD19, CD20, CD27, CD138, and MZB1 in the JIAU group. At the protein level, a significantly higher expression of Ig components in JIAU than in POAG was confirmed. The B cell-associated protein MZB1 showed a higher expression in JIAU patients than in POAG which was confirmed by western blot analysis. Using bead-based immunoassay analysis we were able to detect a significantly higher concentration of the B cell-activating and survival factors BAFF, APRIL, and IL-6 in the AqH of JIAU and AAU patients than in POAG patients. The intraocularly upregulated B cell-specific genes and proteins in iris tissue suggest that B cells participate in the immunopathology of JIAU. The intracameral environment in JIAU may facilitate local effector and survival functions of B cells, leading to disease course typical for anterior uveitis.