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Background Risk of kidney function decline in immunoglobulin A (IgA) nephropathy (IgAN) is significant and may not be predicted by available clinical and histological tools. To serve this unmet need, we aimed at developing a urinary biomarker-based algorithm that predicts rapid disease progression in IgAN, thus enabling a personalized risk stratification. Methods In this multicentre study, urine samples were collected in 209 patients with biopsy-proven IgAN. Progression was defined by tertiles of the annual change of estimated glomerular filtration rate (eGFR) during follow-up. Urine samples were analysed using capillary electrophoresis coupled mass spectrometry. The area under the receiver operating characteristic curve (AUC) was used to evaluate the risk prediction models. Results Of the 209 patients, 64% were male. Mean age was 42 years, mean eGFR was 63 mL/min/1.73 m2 and median proteinuria was 1.2 g/day. We identified 237 urine peptides showing significant difference in abundance according to the tertile of eGFR change. These included fragments of apolipoprotein C-III, alpha-1 antitrypsin, different collagens, fibrinogen alpha and beta, titin, haemoglobin subunits, sodium/potassium-transporting ATPase subunit gamma, uromodulin, mucin-2, fractalkine, polymeric Ig receptor and insulin. An algorithm based on these protein fragments (IgAN237) showed a significant added value for the prediction of IgAN progression [AUC 0.89; 95% confidence interval (CI) 0.83–0.95], as compared with the clinical parameters (age, gender, proteinuria, eGFR and mean arterial pressure) alone (0.72; 95% CI 0.64–0.81). Conclusions A urinary peptide classifier predicts progressive loss of kidney function in patients with IgAN significantly better than clinical parameters alone., Graphical Abstract Graphical Abstract

(1) Background: Arterial hypertension (HTN) is one of the most relevant cardiovascular risk factors. Nowadays multiple pharmaceutical treatment options exist with novel interventional methods (e.g., baroreflex activation therapy (BAT)) as a last resort to treat patients with resistant HTN. Although pathophysiology behind resistant HTN is still not fully understood. There is evidence that selected biomarkers may be involved in the pathophysiology of HTN. (2) Methods: We investigated serum SDC4-levels in patients suffering from resistant HTN before and 6 months after BAT implantation. We collected 19 blood samples from patients with resistant HTN and blood pressure above target and measured serum SDC4-levels. (3) Results: Our results showed high serum SDC4-levels in patients with resistant HTN as compared to a healthy population. Patients with both, resistant HTN and diabetes mellitus type II, demonstrated higher serum SDC4-levels. &beta, blockers had lowering effects on serum SDC4-levels, whereas calcium channel blockers were associated with higher levels of serum SDC4. BAT implantation did not lead to a significant difference in serum SDC4-levels after 6 months of therapy. (4) Conclusion: Based on our results we propose SDC4 is elevated in patients suffering from resistant HTN. Thus, SDC4 might be a potential marker for endothelial dysfunction in patients with resistant hypertension.

Background and Aims Although IgA nephropathy (IgAN) is the most common primary glomerulonephritis in many parts of the world, risk of progressive kidney function decline is significant. Furthermore, the effect of immunosuppressive treatment in IgAN currently remains uncertain. There is no validated tool to predict disease progression or response to treatment. To serve this unmet need, we aimed at developing a urinary biomarker-based algorithm that predicts fast disease progression in patients with IgAN, thus enabling a personalized risk stratification and potentially improving patient management. Method In this multicenter study in 7 centers in Europe and in Canada urine samples were collected as part of clinical routine at time of biopsy in n= 300 patients (63% male, age 42±14 years) with biopsy proven IgAN. The follow-up data were collected for at least one year. Progressive disease was defined as an annual loss of kidney function (estimated glomerular filtration rate, eGFR) of more than -5ml/min/1.73m2 per year or when end-stage kidney disease (ESKD) was reached. The institutional review boards of each of the participating centers approved this study. Urine samples were analyzed using capillary electrophoresis coupled mass spectrometry (CE-MS). Whole proteome/peptidome profile was obtained for each sample. This study received funding from the European Union´s ERA PerMed program. Results Urine proteome/peptidome profiles were obtained from n=294 patients. On average, 2383 peptides were detected per sample. The data were subsequently divided into a discovery (n=154) and validation cohort (n=140). The comparison of the progressors (n=35) and non-progressors (n=119) in the discovery cohort resulted in the definition of more than 100 significant peptides. These included mainly fragments from collagen, mostly type 1 (decreased in progressors) and from different blood derived proteins like alpha-1-antitrypsin, alpha-2-HS-glycoprotein and apolipoproteins (increased in progressors). The distribution of the 100 most significant peptides in the progressor and non-progressor group is shown in the figure. The peptides were combined into a classifier using support vector machine. After optimizing the classifier employing a take-one-out procedure combined with n-1 cross-validation, the urine-peptide based algorithm enabled separation of progressors versus no progressors with an accuracy of 90% in take-one-out cross-validation. This classifier was subsequently applied to the validation cohort and resulted in highly significant separation of progressive from non-progressive IgAN patients. Furthermore, this classifier will be further applied blinded in an independent well characterized multicenter cohort of 267 IgAN patients. Conclusion We identified a urinary proteome profile which was associated with progressive loss of GFR in patients with IgAN. Further validation of this profile in an independent cohort is ongoing. The data indicate that CE-MS-based urinary proteomics enables identifying IgAN patients at high risk of disease progression. These patients may benefit from aggressive immunosuppressive treatment. Upon validation of the classifier in an independent cohort, its value in predicting response to immunosuppression will be assessed, aiming at establishing an innovative strategy that could improve patient management and personalize treatment of IgAN patients.

Multiple sclerosis (MS) is an inflammatory disease mainly affecting the central nervous system. In MS, abnormal immune mechanisms induce acute inflammation, demyelination, axonal loss, and the formation of central nervous system plaques. The long-term treatment involves options to modify the disease progression, whereas the treatment for the acute relapse has its focus in the administration of high-dose intravenous methylprednisolone (up to 1000 mg daily) over a period of three to five days as a first step. If symptoms of the acute relapse persist, it is defined as glucocorticosteroid-unresponsive, and immunomodulation by apheresis is recommended. However, several national and international guidelines have no uniform recommendations on using plasma exchange (PE) nor immunoadsorption (IA) in this case. A systematic review and meta-analysis was conducted, including observational studies or randomized controlled trials that investigated the effect of PE or IA on different courses of MS and neuromyelitis optica (NMO). One thousand, three hundred and eighty-three patients were included in the evaluation. Therapy response in relapsing-remitting MS and clinically isolated syndrome was 76.6% (95%CI 63.7&ndash, 89.8%) in PE- and 80.6% (95%CI 69.3&ndash, 91.8%) in IA-treated patients. Based on the recent literature, PE and IA may be considered as equal treatment possibilities in patients suffering from acute, glucocorticosteroid-unresponsive MS relapses.

During sepsis, the alarmin HMGB1 is released from tissues and promotes systemic inflammation that results in multi-organ damage, with the kidney particularly susceptible to injury. The severity of inflammation and pro-damage signaling mediated by HMGB1 appears to be dependent on the alarmin's redox state. Therefore, we examined HMGB1 redox in kidney cells during sepsis. Using intravital microscopy, CellROX labeling of kidneys in live mice indicated increased ROS generation in the kidney perivascular endothelium and tubules during lipopolysaccharide (LPS)-induced sepsis. Subsequent CellROX and MitoSOX labeling of LPS-stressed endothelial and kidney proximal tubule cells demonstrated increased ROS generation in these cells as sepsis worsens. Consequently, HMGB1 oxidation increased in the cytoplasm of kidney cells during its translocation from the nucleus to the circulation, with the degree of oxidation dependent on the severity of sepsis, as measured in in vivo mouse samples using a thiol assay and mass spectrometry (LC-MS/MS). The greater the oxidation of HMGB1, the greater the ability of the alarmin to stimulate pro-inflammatory cyto-/chemokine release (measured by Luminex Multiplex) and alter mitochondrial ATP generation (Luminescent ATP Detection Assay). Administration of glutathione and thioredoxin inhibitors to cell cultures enhanced HMGB1 oxidation during sepsis in endothelial and proximal tubule cells, respectively. In conclusion, as sepsis worsens, ROS generation and HMGB1 oxidation increases in kidney cells, which enhances HMGB1's pro-inflammatory signaling. Conversely, the glutathione and thioredoxin systems work to maintain the protein in its reduced state., Graphical abstract fx1, Highlights • Endotoxins (LPS) increase cellular oxidative stress during sepsis. • During its translocation, HMGB1 gets oxidized in the cytoplasm. • Thioredoxin and glutathione keep HMGB1 in a reduced redox state during sepsis. • HMGB1 oxidation enhances its stimulation of inflammatory cyto-/chemokine release.

The secretome, defined as a portion of proteins secreted by specific cells to the extracellular space, secures a proper microenvironmental niche not only for the donor cells, but also for the neighboring cells, thus maintaining tissue homeostasis. Communication via secretory products exists between endothelial cells and fibroblasts, and this local mechanism maintains the viability and density of each compartment. Endothelial dysfunction, apart from obvious cell-autonomous defects, leads to the aberrant secretome, which predisposes fibroblasts to acquire a myofibroblastic fibrogenic phenotype. In our recent profiling of the secretome of such dysfunctional profibrogenic renal microvascular endothelial cells, we identified unique profibrogenic signatures, among which we detected ligands of Notch and Wnt-β-catenin pathways. Here, we stress the role of reprogramming cues in the immediate microenvironment of (myo)fibroblasts and the contribution of the endothelial secretome to the panoply of instructive signals in the vicinity of fibroblasts. We hope that this brief overview of endothelial-fibroblast communication in health and disease will lead to eventual unbiased proteomic mapping of individual secretomes of glomerular and tubular epithelial cells, pericytes, and podocytes through reductionist approaches to allow for the synthetic creation of a complex network of secretomic signals acting as reprogramming factors on individual cell types in the kidney. Knowledge of profibrogenic and antifibrogenic signatures in the secretome may garner future therapeutic efforts.

BackgroundLow birth weight (LBW) neonates have impaired kidney development that leaves them susceptible to kidney disease and hypertension during adulthood. The study here identifies events that blunt nephrogenesis and kidney development in the murine LBW neonate.MethodsWe examined survival, kidney development, GFR, gene expression, and cyto-/chemokines in the LBW offspring of malnourished (caloric and protein-restricted) pregnant mice.ResultsMalnourished pregnant mothers gave birth to LBW neonates that had 40% reduced body weight and 54% decreased survival. Renal blood perfusion was reduced by 37%, whereas kidney volume and GFR were diminished in the LBW neonate. During gestation, the LBW neonatal kidney had 2.2-fold increased apoptosis, 76% decreased SIX2+ progenitor cells, downregulation of mesenchymal-to-epithelial signaling factors Wnt9b and Fgf8, 64% less renal vesicle formation, and 32% fewer nephrons than controls. At birth, increased plasma levels of IL-1β, IL-6, IL-12(p70), and granulocyte-macrophage colony-stimulating factor in the LBW neonate reduced SIX2+ progenitor cells.ConclusionIncreased pro-inflammatory cytokines in the LBW neonate decrease SIX2+ stem cells in the developing kidney. Reduced renal stem cells (along with the decreased mesenchymal-to-epithelial signaling) blunt renal vesicle generation, nephron formation, and kidney development. Subsequently, the mouse LBW neonate has reduced glomeruli volume, renal perfusion, and GFR.

Maternal undernutrition during pregnancy (MUN) often leads to low birth weight (LBW) neonates that have a reduced total nephron endowment, leaving these neonates susceptible to kidney disease throughout their lives. For reasons unknown, these LBW neonates have impaired kidney development due to a severe reduction in renal SIX2+ stem cells during nephrogenesis. Using a mouse model of MUN, we investigated SIX2+ stem cell reduction in the LBW neonate. Significant upregulation of the protein fetuin-B (measured by PCR and immunoblotting) in the MUN mother's placenta, organs and circulation yielded a 3-fold increase of this protein in the embryonic kidney. Recombinant fetuin-B, administered to healthy pregnant mothers at the concentration equivalent to that in the MUN mother, crossed the placenta and reduced both SIX2+ stem cells by 50% and nephron formation by 66% in embryonic kidneys (measured by immunofluorescence and the physical dissector/fractionator stereological method). Administration of fetuin-B to kidney explants yielded similar reductions in renal SIX2+ stem cells and nephron formation. Fetuin-B treatment of isolated embryonic renal SIX2+ stem cell primary cultures 1) increased NF-kB activity and apoptosis, 2) reduced cell proliferation due to upregulated p21 nuclear activity and subsequent cell cycle arrest, and 3) enhanced generation of reactive oxygen species (measured by fluorescence microscopy). In conclusion, MUN increases fetuin-B in the developing embryonic kidney. The increase in fetuin-B blunts nephrogenesis by reducing SIX2+ stem cells by promoting their apoptosis (via NF-kB upregulation), blunting their proliferative renewal (via p21 upregulation) and enhancing oxidative stress.

Background Our laboratory has previously demonstrated that Sirt1endo-/- mice show endothelial dysfunction and exaggerated renal fibrosis, whereas mice with silenced endothelial transforming growth factor beta (TGF-β) signaling are resistant to fibrogenic signals. Considering the fact that the only difference between these mutant mice is confined to the vascular endothelium, this indicates that secreted substances contribute to these contrasting responses. Methods We performed an unbiased proteomic analysis of the secretome of renal microvascular endothelial cells (RMVECs) isolated from these two mutants. We cultured renal fibroblasts and RMVECs and used microfluidic devices for coculturing. Results Dickkopf-3 (DKK3), a putative ligand of the Wnt/β-catenin pathway, was present exclusively in the fibrogenic secretome. In cultured fibroblasts, DKK3 potently induced myofibroblast activation. In addition, DKK3 antagonized effects of DKK1, a known inhibitor of the Wnt pathway, in conversion of fibroblasts to myofibroblasts. In RMVECs, DKK3 induced endothelial-mesenchymal transition and impaired their angiogenic competence. The inhibition of endothelial outgrowth, enhanced myofibroblast formation and endothelial-mesenchymal transition were confirmed in coculture. In reporter DKK3-eGFP × Col3.6-GFPcyan mice, DKK3 was marginally expressed under basal conditions. Adriamycin-induced nephropathy resulted in upregulation of DKK3 expression in tubular and, to a lesser degree, endothelial compartments. Sulindac sulfide was found to exhibit superior Wnt pathway-suppressive action and decreased DKK3 signals and the extent of renal fibrosis. Conclusions In conclusion, this unbiased proteomic screen of the profibrogenic endothelial secretome revealed DKK3 acting as an agonist of the Wnt pathway, enhancing formation of myofibroblasts and endothelial-mesenchymal transition and impairing angiogenesis. A potent inhibitor of the Wnt pathway, sulindac sulfide, suppressed nephropathy-induced DKK3 expression and renal fibrosis.

After briefly discussing endothelial glycocalyx and its role in vascular physiology and renal disease, this overview focuses on its degradation very early in the course of microbial sepsis. We describe our recently proposed mechanism for glycocalyx degradation induced by exocytosis of lysosome-related organelles and release of their cargo. Notably, an intermediate in nitric oxide synthesis, NG-hydroxy-l-arginine, shows efficacy in curtailing exocytosis of these organelles and improvement in animal survival. These data not only depict a novel mechanism responsible for very early glycocalyx degradation, but may also outline a potential preventive therapy. The second issue discussed in this article is related to the therapeutic acceleration of restoration of already degraded endothelial glycocalyx. Here, using as an example our recent findings obtained with sulodexide, we illustrate the importance of the expedited repair of degraded endothelial glycocalyx for the survival of animals with severe sepsis. These two focal points of the review on glycocalyx may not only have broader disease applicability, but they may also provide additional evidence to buttress the idea of the importance of endothelial glycocalyx and its maintenance and repair in the prevention and treatment of an array of renal and nonrenal diseases.