Empagliflozin reduces kidney fibrosis and improves kidney function by alternative macrophage activation in rats with 5/6-nephrectomy

Background Sodium glucose cotransporter 2 (SGLT2) inhibitors originally developed for the treatment of type 2 diabetes are clinically very effective drugs halting chronic kidney disease progression. The underlying mechanisms are, however, not fully understood. Methods We generated single-cell transcriptomes of kidneys from rats with 5/6 nephrectomy before and after SGLT2 inhibitors treatment by single-cell RNA sequencing. Findings Empagliflozin treatment decreased BUN, creatinine and urinary albumin excretion compared to placebo by 39.8%, 34.1%, and 55%, respectively (p < 0.01 in all cases). Renal interstitial fibrosis and glomerulosclerosis was likewise decreased by 51% and 66.8%; respectively (p < 0.05 in all cases). 14 distinct kidney cell clusters could be identified by scRNA-seq. The polarization of M2 macrophages from state 1 (CD206-CD68- M2 macrophages) to state 5 (CD206+CD68+ M2 macrophages) was the main pro-fibrotic process, as CD206+CD68+ M2 macrophages highly expressed fibrosis-promoting genes and can convert into fibrocytes. Empagliflozin remarkably inhibited the expression of fibrosis-promoting (IFG1 and TREM2) and polarization-associated genes (GPNMB, LGALS3, PRDX5, and CTSB) in CD206+CD68+ M2 macrophages and attenuated inflammatory signals from CD8+ effector T cells. The inhibitory effect of empagliflozin on CD206+CD68+ M2 macrophages polarization was mainly achieved by affecting mitophagy and mTOR pathways. Interpretation We propose that the beneficial effects of empagliflozin on kidney function and morphology in 5/6 nephrectomyiced rats with established CKD are at least partially due to an inhibition of CD206+CD68+ M2 macrophage polarization by targeting mTOR and mitophagy pathways and attenuating inflammatory signals from CD8+ effector T cells. Fundings A full list of funding bodies that contributed to this study can be found in the Acknowledgements section

The effect of Eucalyptus camaldulensis leaf extracts from different environmental harvesting locations on Plasmodium chabaudi-induced malaria outcome

Abstract In every country in the globe, malaria is still regarded as a health risk. In terms of therapeutic medicines against parasites, medicinal plants are a promising source, although the types and concentrations of their active compounds can vary depending on the environment. In this study, the effect of Eucalyptus camaldulensis leaf extracts (ECE) collected from two different habitats (sandy and muddy) on the antioxidant and antimalarial activity was examined. Phytochemical analysis of ECE from muddy (ECEM) and sandy (ECES) habitats were compared. ECEM contained 13 expected compounds while ECES contained only 10 compounds. This was evidenced through infrared spectroscopy. Also, ECEM contained more phenolics and flavonoids than ECES as well as processes higher antioxidant activity that reached 55%. Plasmodium chabaudi-infected mice were treated with ECEM and ECES and the parasitemia was compared. ECEM could significantly suppress the parasitemia by approximately 84% and also was able to decrease the spleen index better than EXCS. Moreover, ECEM was better than ECES in ameliorating the induced decrease in erythrocyte number and hemoglobin content in mice infected with P. chabaudi. It is possible to use E. camaldulensis, which grows in muddy environments, as an antimalarial drug with the largest efficacy gradients

An in vitro approach to understand contribution of kidney cells to human urinary extracellular vesicles

Extracellular vesicles (EV) are membranous particles secreted by all cells and found in body fluids. Established EV contents include a variety of RNA species, proteins, lipids and metabolites that are considered to reflect the physiological status of their parental cells. However, to date, little is known about cell-type enriched EV cargo in complex EV mixtures, especially in urine. To test whether EV secretion from distinct human kidney cells in culture differ and can recapitulate findings in normal urine, we comprehensively analysed EV components, (particularly miRNAs, long RNAs and protein) from conditionally immortalised human kidney cell lines (podocyte, glomerular endothelial, mesangial and proximal tubular cells) and compared to EV secreted in human urine. EV from cell culture media derived from immortalised kidney cells were isolated by hydrostatic filtration dialysis (HFD) and characterised by electron microscopy (EM), nanoparticle tracking analysis (NTA) and Western blotting (WB). RNA was isolated from EV and subjected to miRNA and RNA sequencing and proteins were profiled by tandem mass tag proteomics. Representative sets of EV miRNAs, RNAs and proteins were detected in each cell type and compared to human urinary EV isolates (uEV), EV cargo database, kidney biopsy bulk RNA sequencing and proteomics, and single-cell transcriptomics. This revealed that a high proportion of the in vitro EV signatures were also found in in vivo datasets. Thus, highlighting the robustness of our in vitro model and showing that this approach enables the dissection of cell type specific EV cargo in biofluids and the potential identification of cell-type specific EV biomarkers of kidney disease.Peer reviewe

The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase&nbsp;1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation&nbsp;disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age&nbsp; 6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score&nbsp; 652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc&nbsp;= 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N&nbsp;= 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in&nbsp;Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in&nbsp;Asia&nbsp;and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701

Characterization of Micro-RNA Changes during the Progression of Type 2 Diabetes in Zucker Diabetic Fatty Rats

The aim of the present pilot study was the identification of micro-RNA changes over time during the development and progression of type 2 diabetes (T2D) in Zucker diabetic fatty rats (ZDF rats). T2D is a complex metabolic disorder that is characterized, inter alia, by progressive failure of pancreatic β cells to produce insulin, but also by functional or morphological modifications of others organ, such as liver, adipose tissue and the cardiovascular system. Micro-RNAs are a novel class of biomarkers that have the potential to represent biomarkers of disease progression. In this study, the onset and progression of diabetes was followed in ZDF rats from six weeks until 17 weeks of age. After an initial phase of hyperinsulinemia, the animals developed T2D and lost the capacity to produce sufficient insulin. Circulating miRNAs were measured from plasma samples at four time points: pre-diabetes (six weeks of age), hyperinsulinemia (eight weeks), β cell failure (11 weeks) and late-stage diabetes (17 weeks) using TaqMan miRNA arrays. Bioinformatic analysis revealed distinct changes of circulating miRNAs over time. Several miRNAs were found to be increased over the course of the disease progression, such as miR-122, miR-133, miR-210 and miR-375. The most significantly decreased miRNAs were miR-140, miR-151-3p, miR-185, miR-203, miR-434-3p and miR-450a. Some of the miRNAs have also been identified in type 2 diabetic patients recently and, therefore, may have the potential to be useful biomarkers for the disease progression of T2D and/or the treatment response for anti-diabetic medications

Comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease

Urinary Extracellular Vesicles (uEV) have emerged as a source for biomarkers of kidney damage, holding potential to replace the conventional invasive techniques including kidney biopsy. However, comprehensive studies characterizing uEV isolation methods with patient samples are rare. Here we compared performance of three established uEV isolation workflows for their subsequent use in transcriptomics analysis for biomarker discovery in diabetic kidney disease. We collected urine samples from individuals with type 1 diabetes with macroalbuminuria and healthy controls. We isolated uEV by Hydrostatic Filtration Dialysis (HFD), ultracentrifugation (UC), and a commercial kit- based isolation method (NG), each with different established urine clearing steps. Purified EVs were analysed by electron microscopy, nanoparticle tracking analysis, and Western blotting. Isolated RNAs were subjected to miRNA and RNA sequencing. HFD and UC samples showed close similarities based on mRNA sequencing data. NG samples had a lower number of reads and different mRNA content compared to HFD or UC. For miRNA sequencing data, satisfactory miRNA counts were obtained by all methods, but miRNA contents differed slightly. This suggests that the isolation workflows enrich specific subpopulations of miRNA-rich uEV preparation components. Our data shows that HFD,UC and the kit-based method are suitable methods to isolate uEV for miRNA-seq. However, only HFD and UC were suitable for mRNA-seq in our settings.Peer reviewe

A balanced pyrimidine pool is required for optimal Chk1 activation to prevent ultrafine anaphase bridge formation

International audienceCytidine deaminase (CDA) deficiency induces an excess of cellular dCTP, which reduces basal PARP-1 activity, thereby compromising complete DNA replication, leading to ultrafine anaphase bridge (UFB) formation. CDA dysfunction has pathological implications, notably in cancer and in Bloom syndrome. It remains unknown how reduced levels of PARP-1 activity and pyrimidine pool imbalance lead to the accumulation of unreplicated DNA during mitosis. We report that a decrease in PARP-1 activity in CDA-deficient cells impairs DNAdamage-induced Chk1 activation, and, thus, the downstream checkpoints. Chemical inhibition of the ATR-Chk1 pathway leads to UFB accumulation, and we found that this pathway was compromised in CDA-deficient cells. Our data demonstrate that ATR-Chk1 acts downstream from PARP-1, preventing the accumulation of unreplicated DNA in mitosis, and, thus, UFB formation. Finally, delaying entry into mitosis is sufficient to prevent UFB formation in both CDA-deficient and CDA-proficient cells, suggesting that both physiological and pathological UFBs are derived from unreplicated DNA. Our findings demonstrate an unsuspected requirement for a balanced nucleotide pool for optimal Chk1 activation both in unchallenged cells and in response to genotoxic stress

Protective vaccination against blood-stage malaria of Plasmodium chabaudi: differential gene expression in the liver of Balb/c mice towards the end of crisis phase

Protective vaccination induces self-healing of otherwise fatal blood-stage malaria of Plasmodium chabaudi in female Balb/c mice. To trace processes critically involved in self-healing, the liver, an effector against blood-stage malaria, is analyzed for possible changes of its transcriptome in vaccination-protected in comparison to non-protected mice towards the end of the crisis phase. Gene expression microarray analyses reveal that vaccination does not affect constitutive expression of mRNA and lincRNA. However, malaria induces significant (p3-fold as compared to the corresponding constitutive expressions. Massive up-regulations, partly by >100-fold, are found for genes as RhD, Add2, Ank1, Ermap, and Slc4a, which encode proteins of erythrocytic surface membranes, and as Gata1 and Gfi1b, which encode transcription factors involved in erythrocytic development. Also, Cldn13 previously predicted to be expressed on erythroblast surfaces is up-regulated by >200-fold, though claudins are known as main constituents of tight junctions acting as paracellular barriers between epithelial cells. Other genes are up-regulated by 10-fold, which can be subgrouped in genes encoding proteins known to be involved in mitosis, in cell cycle regulation, and in DNA repair. Our data suggest that protective vaccination enables the liver to respond to P. chabaudi infections with accelerated regeneration and extramedullary erythropoiesis during crisis, which contributes to survival of otherwise lethal blood-stage malaria