Epirubicin for the Treatment of Sepsis and Septic Shock (EPOS-1): study protocol for a randomised, placebo-controlled phase IIa dose-escalation trial.

INTRODUCTION: Sepsis remains the major cause of death among hospitalised patients in intensive care. While targeting sepsis-causing pathogens with source control or antimicrobials has had a dramatic impact on morbidity and mortality of sepsis patients, this strategy remains insufficient for about one-third of the affected individuals who succumb. Pharmacological targeting of mechanisms that reduce sepsis-defining organ dysfunction may be beneficial. When given at low doses, the anthracycline epirubicin promotes tissue damage control and lessens the severity of sepsis independently of the host-pathogen load by conferring disease tolerance to infection. Since epirubicin at higher doses can be myelotoxic, a first dose-response trial is necessary to assess the potential harm of this drug in this new indication. METHODS AND ANALYSIS: Epirubicin for the Treatment of Sepsis and Septic Shock-1 is a randomised, double-blind, placebo-controlled phase 2 dose-escalation phase IIa clinical trial to assess the safety of epirubicin as an adjunctive in patients with sepsis. The primary endpoint is the 14-day myelotoxicity. Secondary and explorative outcomes include 30-day and 90-day mortality, organ dysfunction, pharmacokinetic/pharmacodynamic (PK/PD) and cytokine release. Patients will be randomised in three consecutive phases. For each study phase, patients are randomised to one of the two study arms (epirubicin or placebo) in a 4:1 ratio. Approximately 45 patients will be recruited. Patients in the epirubicin group will receive a single dose of epirubicin (3.75, 7.5 or 15 mg/m2 depending on the study phase. After each study phase, a data and safety monitoring board will recommend continuation or premature stopping of the trial. The primary analyses for each dose level will report the proportion of myelotoxicity together with a 95% CI. A potential dose-toxicity association will be analysed using a logistic regression model with dose as a covariate. All further analyses will be descriptive. ETHICS AND DISSEMINATION: The protocol is approved by the German Federal Institute for Drugs and Medical Devices. The results will be submitted for publication in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT05033808.

Empagliflozin prevents neointima formation by impairing smooth muscle cell proliferation and accelerating endothelial regeneration.

Background: Empagliflozin, an inhibitor of the sodium glucose co-transporter 2 (SGLT2) and developed as an anti-diabetic agent exerts additional beneficial effects on heart failure outcomes. However, the effect of empagliflozin on vascular cell function and vascular remodeling processes remains largely elusive. Methods/Results: Immunocytochemistry and immunoblotting revealed SGLT2 to be expressed in human smooth muscle (SMC) and endothelial cells (EC) as well as in murine femoral arteries. In vitro, empagliflozin reduced serum-induced proliferation and migration of human diabetic and non-diabetic SMCs in a dose-dependent manner. In contrast, empagliflozin significantly increased the cell count and migration capacity of human diabetic ECs, but not of human non-diabetic ECs. In vivo, application of empagliflozin resulted in a reduced number of proliferating neointimal cells in response to femoral artery wire-injury in C57BL/6J mice and prevented neointima formation. Comparable effects were observed in a streptozocin-induced diabetic model of apolipoprotein E-/- mice. Conclusive to the in vitro-results, re-endothelialization was not significantly affected in C57BL/6 mice, but improved in diabetic mice after treatment with empagliflozin assessed by Evan's Blue staining 3 days after electric denudation of the carotid artery. Ribonucleic acid (RNA) sequencing (RNA-seq) of human SMCs identified the vasoactive peptide apelin to be decisively regulated in response to empagliflozin treatment. Recombinant apelin mimicked the in vitro-effects of empagliflozin in ECs and SMCs. Conclusion: Empagliflozin significantly reduces serum-induced proliferation and migration of SMCs in vitro and prevents neointima formation in vivo, while augmenting EC proliferation in vitro and re-endothelialization in vivo after vascular injury. These data document the functional impact of empagliflozin on vascular human SMCs and ECs and vascular remodeling in mice for the first time.

Effects of therapeutic plasma exchange on the endothelial glycocalyx in septic shock.

BACKGROUND: Disruption of the endothelial glycocalyx (eGC) is observed in septic patients and its injury is associated with multiple-organ failure and inferior outcomes. Besides this biomarker function, increased blood concentrations of shedded eGC constituents might play a mechanistic role in septic organ failure. We hypothesized that therapeutic plasma exchange (TPE) using fresh frozen plasma might influence eGC-related pathology by removing injurious mediators of eGC breakdown while at the time replacing eGC protective factors. METHODS: We enrolled 20 norepinephrine-dependent (NE > 0.4 µg/kg/min) patients with early septic shock (onset < 12 h). Sublingual assessment of the eGC via sublingual sidestream darkfield (SDF) imaging was performed. Plasma eGC degradation products, such as heparan sulfate (HS) and the eGC-regulating enzymes, heparanase (Hpa)-1 and Hpa-2, were obtained before and after TPE. A 3D microfluidic flow assay was performed to examine the effect of TPE on eGC ex vivo. Results were compared to healthy controls. RESULTS: SDF demonstrated a decrease in eGC thickness in septic patients compared to healthy individuals (p = 0.001). Circulating HS levels were increased more than sixfold compared to controls and decreased significantly following TPE [controls: 16.9 (8-18.6) vs. septic patients before TPE: 105.8 (30.8-143.4) µg/ml, p < 0.001; vs. after TPE: 70.7 (36.9-109.5) µg/ml, p < 0.001]. The Hpa-2 /Hpa-1 ratio was reduced in septic patients before TPE but normalized after TPE [controls: 13.6 (6.2-21.2) vs. septic patients at inclusion: 2.9 (2.1-5.7), p = 0.001; vs. septic patients after TPE: 13.2 (11.2-31.8), p < 0.001]. Ex vivo stimulation of endothelial cells with serum from a septic patient induced eGC damage that could be attenuated with serum from the same patient following TPE. CONCLUSIONS: Septic shock results in profound degradation of the eGC and an acquired deficiency of the protective regulator Hpa-2. TPE removed potentially injurious eGC degradation products and partially attenuated Hpa-2 deficiency. Trial registration clinicaltrials.gov NCT04231994, retrospectively registered 18 January 2020.

Autonomous Calcium Signaling in Human and Zebrafish Podocytes Controls Kidney Filtration Barrier Morphogenesis.

BACKGROUND: Podocytes are critical to maintaining the glomerular filtration barrier, and mutations in nephrotic syndrome genes are known to affect podocyte calcium signaling. However, the role of calcium signaling during podocyte development remains unknown. METHODS: We undertook live imaging of calcium signaling in developing podocytes, using zebrafish larvae and human kidney organoids. To evaluate calcium signaling during development and in response to channel blockers and genetic defects, the calcium biosensor GCaMP6s was expressed in zebrafish podocytes. We used electron microscopy to evaluate filtration barrier formation in zebrafish, and Fluo-4 to detect calcium signals in differentiating podocytes in human kidney organoids. RESULTS: Immature zebrafish podocytes (2.5 days postfertilization) generated calcium transients that correlated with interactions with forming glomerular capillaries. Calcium transients persisted until 4 days postfertilization, and were absent after glomerular barrier formation was complete. We detected similar calcium transients in maturing human organoid glomeruli, suggesting a conserved mechanism. In both models, inhibitors of SERCA or IP3 receptor calcium-release channels blocked calcium transients in podocytes, whereas lanthanum was ineffective, indicating the calcium source is from intracellular podocyte endoplasmic-reticulum stores. Calcium transients were not affected by blocking heartbeat or by blocking development of endothelium or endoderm, and they persisted in isolated glomeruli, suggesting podocyte-autonomous calcium release. Inhibition of expression of phospholipase C-γ1, but not nephrin or phospholipase C-ε1, led to significantly decreased calcium activity. Finally, blocking calcium release affected glomerular shape and podocyte foot process formation, supporting the critical role of calcium signaling in glomerular morphogenesis. CONCLUSIONS: These findings establish podocyte cell-autonomous calcium signaling as a prominent and evolutionarily conserved feature of podocyte differentiation and demonstrate its requirement for podocyte foot process formation.

Role of endothelial microRNA 155 on capillary leakage in systemic inflammation.

BACKGROUND: Capillary leakage is a key contributor to the pathological host response to infections. The underlying mechanisms remain incompletely understood, and the role of microRNAs (MIR) has not been investigated in detail. We hypothesized that specific MIRs might be regulated directly in the endothelium thereby contributing to vascular leakage. METHODS: SmallRNA sequencing of endotoxemic murine pulmonary endothelial cells (ECs) was done to detect regulated vascular MIRs. In vivo models: transgenic zebrafish (flk1:mCherry/l-fabp:eGFP-DPB), knockout/wildtype mouse (B6.Cg-Mir155tm1.1Rsky/J); disease models: LPS 17.5 mg/kgBW and cecal ligation and puncture (CLP); in vitro models: stimulated human umbilical vein EC (HUVECs), transendothelial electrical resistance. RESULTS: Endothelial MIR155 was identified as a promising candidate in endotoxemic murine pulmonary ECs (25 × upregulation). Experimental overexpression in a transgenic zebrafish line and in HUVECs was sufficient to induce spontaneous vascular leakage. To the contrary, genetic MIR155 reduction protects against permeability both in vitro and in endotoxemia in vivo in MIR155 heterozygote knockout mice thereby improving survival by 40%. A tight junction protein, Claudin-1, was down-regulated both in endotoxemia and by experimental MIR155 overexpression. Translationally, MIR155 was detectable at high levels in bronchoalveolar fluid of patients with ARDS compared to healthy human subjects. CONCLUSIONS: We found that MIR155 is upregulated in the endothelium in mouse and men as part of a systemic inflammatory response and might contribute to the pathophysiology of vascular leakage in a Claudin-1-dependent manner. Future studies have to clarify whether MIR155 could be a potential therapeutic target.

Effect of Therapeutic Plasma Exchange on Immunoglobulin Deficiency in Early and Severe Septic Shock.

BACKGROUND: Deficiency of immunoglobulins of the classes IgG, IgG1, IgA and IgM is associated with severity of disease and mortality in sepsis and septic shock. Therapeutic plasma exchange (TPE) with fresh frozen plasma (FFP) has recently gained attention as an adjunctive therapeutic option in early septic shock. We hypothesized that TPE might modulate immunoglobulin deficiencies besides sole elimination of circulating injurious molecules. METHODS: We conducted a prospective single center study with TPE in 33 patients with early septic shock (onset < 12 h) requiring high doses of norepinephrine (NE > 0.4µg/kg/min). Clinical and biochemical data, including measurement of immunoglobulin subgroups IgG, IgG1, IgM and IgA were obtained before and after TPE. The following immunoglobulin cut-off values were used to analyze subgroups with low immunoglobulin concentrations at baseline (IgG ≤ 6.5, IgG1 ≤ 3, IgM ≤ 1.5 and IgA ≤ 0.35 g/L). RESULTS: At inclusion, median (IQR) SOFA score was 18 (15-20) and NE dose was 0.8 (0.6-1.2) µg/kg/min. The majority of patients demonstrated profound reductions in immunoglobulins levels of all classes. Globally, immunoglobulin levels were not significantly changed after a single TPE session. However, in patients with low baseline immunoglobulin levels a significant increase in all classes was observed (IgG 1.92 (0.96-3) g/L (+41%), IgG1 2.1 (1.46-2.32) g/L (+96%), IgA 0.44 (0.12-0.62) g/L (59%) and IgM 0.18 (0.14-0.34) g/L (+55%), p < 0.001 for comparison to patients above cut-off). CONCLUSIONS: The majority of early and severe septic shock patients had reduced immunoglobulin levels and a single TPE could attenuate immunoglobulin deficiencies of all classes. The clinical relevance of this observation has to be investigated in a proper designed trial.

Correction: Calcium dobesilate reduces VEGF signaling by interfering with heparan sulfate binding site and protects from vascular complications in diabetic mice.

[This corrects the article DOI: 10.1371/journal.pone.0218494.].

Calcium dobesilate reduces VEGF signaling by interfering with heparan sulfate binding site and protects from vascular complications in diabetic mice.

Inhibiting vascular endothelial growth factor (VEGF) is a therapeutic option in diabetic microangiopathy. However, VEGF is needed at physiological concentrations to maintain glomerular integrity; complete VEGF blockade has deleterious effects on glomerular structure and function. Anti-VEGF therapy in diabetes raises the challenge of reducing VEGF-induced pathology without accelerating endothelial cell injury. Heparan sulfate (HS) act as a co-receptor for VEGF. Calcium dobesilate (CaD) is a small molecule with vasoprotective properties that has been used for the treatment of diabetic microangiopathy. Preliminary evidence suggests that CaD interferes with HS binding sites of fibroblast growth factor. We therefore tested the hypotheses that (1) CaD inhibits VEGF signaling in endothelial cells, (2) that this effect is mediated via interference between CaD and HS, and (3) that CaD ameliorates diabetic nephropathy in a streptozotocin-induced diabetic mouse model by VEGF inhibition. We found that CaD significantly inhibited VEGF165-induced endothelial cell migration, proliferation, and permeability. CaD significantly inhibited VEGF165-induced phosphorylation of VEGFR-2 and suppressed the activity of VEGFR-2 mediated signaling cascades. The effects of CaD in vitro were abrogated by heparin, suggesting the involvement of heparin-like domain in the interaction with CaD. In addition, VEGF121, an isoform which does not bind to heparin, was not inhibited by CaD. Using the proximity ligation approach, we detected inhibition of interaction in situ between HS and VEGF and between VEGF and VEGFR-2. Moreover, CaD reduced VEGF signaling in mice diabetic kidneys and ameliorated diabetic nephropathy and neuropathy, suggesting CaD as a VEGF inhibitor without the negative effects of complete VEGF blockade and therefore could be useful as a strategy in treating diabetic nephropathy.

Sulfatases, in Particular Sulf1, Are Important for the Integrity of the Glomerular Filtration Barrier in Zebrafish.

The 6-O-endosulfatases (sulfs) are important enzymatic components involved in the regulation of heparan sulfate by altering the sulfatation pattern. Specifically in the kidney, sulfs have been implicated in the glomerular podocyte-endothelial cell crosstalk and in the preservation of the glomerular filtration barrier (GFB) in different mouse models. Since it has been shown that in zebrafish larvae, Sulf1, Sulf2a, and Sulf2b are expressed in the pronephric kidney we set out to establish if a reduction in sulf expression leads to GFB dysfunction. Here, we show that a reduced sulf expression following morpholino (MO) induced knockdown in zebrafish larvae promotes damage to the GFB leading to renal plasma protein loss from the circulation. Moreover, a combined knockdown of Sulf1, Sulf2a, and Sulf2b is associated with severe morphologic changes including narrowing of the fenestration between glomerular endothelial cells as well as thickening of the glomerular basement membrane and podocyte foot process effacement, suggesting that glomerular damage is an underlying cause of the circulatory protein loss observed after MO injection. Additionally, we show that a decrease in sulf expression reduces the bioavailability of VegfA in the glomerulus of the pronephros, which may contribute to the structural changes observed in the glomeruli of morphant fish. Furthermore, consistent with previous results, knockdown of the sulfs is associated with arteriovenous malformations in particular in the tail region of the larvae. Overall, taken together our results suggest that 6-O-endosulfatases are important in the preservation of GFB integrity and a reduction in their expression levels induces phenotypic changes that are indicative of renal protein loss.

Mutation of microphthalmia-associated transcription factor (mitf) in zebrafish sensitizes for glomerulopathy.

Different glomerular diseases that affect podocyte homeostasis can clinically present as nephrotic syndrome with massive proteinuria, hypoalbuminemia, hyperlipidemia and edema. Up to now, no drugs that specifically target the actin cytoskeleton of podocytes are on the market and model systems for library screenings to develop anti-proteinuric drugs are of high interest. We developed a standardized proteinuria model in zebrafish using puromycin aminonucleoside (PAN) via treatment in the fish water to allow for further drug testing to develop anti-proteinuric drugs for the treatment of glomerular diseases. We noticed that fish that carry the nacre-mutation show a significantly higher susceptibility for the disruption of the glomerular filtration barrier following PAN treatment, which results in a more pronounced proteinuria phenotype. Nacre zebrafish inherit a mutation yielding a truncated version of microphthalmia-associated transcription factor/melanogenesis associated transcription factor (mitf). We hypothesized that the nacre mutation may lead to reduced formin expression and defects in cytoskeletal rearrangement. Based on the observations in zebrafish, we carried out a PAN treatment on cultured human podocytes after knockdown with MITF siRNA causing a rearrangement of the actin cytoskeleton.

Removal of focal segmental glomerulosclerosis (FSGS) factor suPAR using CytoSorb.

Treatment of primary focal segmental glomerulosclerosis (FSGS) and its recurrence after kidney transplantation associated with rapid deterioration of kidney function remains to be challenging despite advances in immunosuppressive therapy. The presence of circulating factors has been postulated to be a pivotal player in the pathogenesis of FSGS, although suPAR and CLCF-1 have been identified as the most promising causative factors. The potential therapeutic effect of suPAR elimination in an FSGS patient using CytoSorb, a hemoadsorption device that gained attention in the cytokine elimination in septic patients, was studied. Efficiency of total plasma exchange to remove suPAR was determined. CytoSorb hemoadsorption caused a 27.33% reduction of the suPAR level in a single treatment, whereas total plasma exchange showed a suPAR level reduction of 25.12% (n = 3; 95% confidence interval, 0.2777-0.8090; P < 0.01), which may indicate therapeutic potential in the treatment of primary FSGS and its recurrence in a kidney transplant.

Disease modeling in genetic kidney diseases: zebrafish.

Growing numbers of translational genomics studies are based on the highly efficient and versatile zebrafish (Danio rerio) vertebrate model. The increasing types of zebrafish models have improved our understanding of inherited kidney diseases, since they not only display pathophysiological changes but also give us the opportunity to develop and test novel treatment options in a high-throughput manner. New paradigms in inherited kidney diseases have been developed on the basis of the distinct genome conservation of approximately 70 % between zebrafish and humans in terms of existing gene orthologs. Several options are available to determine the functional role of a specific gene or gene sets. Permanent genome editing can be induced via complete gene knockout by using the CRISPR/Cas-system, among others, or via transient modification by using various morpholino techniques. Cross-species rescues succeeding knockdown techniques are employed to determine the functional significance of a target gene or a specific mutation. This article summarizes the current techniques and discusses their perspectives.

Overexpression of TGF-ß Inducible microRNA-143 in Zebrafish Leads to Impairment of the Glomerular Filtration Barrier by Targeting Proteoglycans.

BACKGROUND: TGF-ß is known as an important stress factor of podocytes in glomerular diseases. Apart from activation of direct pro-apoptotic pathways we wanted to analyze micro-RNA (miRs) driven regulation of components involved in the integrity of the glomerular filtration barrier induced by TGF-ß. Since miR-143-3p (miR-143) is described as a TGF-ß inducible miR in other cell types, we examined this specific miR and its ability to induce glomerular pathology. METHODS: We analyzed miR-143 expression in cultured human podocytes after stimulation with TGF-ß. We also microinjected zebrafish eggs with a miR-143 mimic or with morpholinos specific for its targets syndecan and versican and compared phenotype and proteinuria development. RESULTS: We detected a time dependent, TGF-ß inducible expression of miR-143 in human podocytes. Targets of miR-143 relevant in glomerular biology are syndecans and versican, which are known components of the glycocalyx. We found that syndecan 1 and 4 were predominantly expressed in podocytes while syndecan 3 was largely expressed in glomerular endothelial cells. Versican could be detected in both cell types. After injection of a miR-143 mimic in zebrafish larvae, syndecan 3, 4 and versican were significantly downregulated. Moreover, miR-143 overexpression or versican knockdown by morpholino caused loss of plasma proteins, edema, podocyte effacement and endothelial damage. In contrast, knockdown of syndecan 3 and syndecan 4 had no effects on glomerular filtration barrier. CONCLUSION: Expression of versican and syndecan isoforms is indispensable for proper barrier function. Podocyte-derived miR-143 is a mediator for paracrine and autocrine cross talk between podocytes and glomerular endothelial cells and can alter expression of glomerular glycocalyx proteins.

Histamine regulates murine primary dendritic cell functions.

OBJECTIVE AND DESIGN: The modulation of antigen uptake and activation of dendritic cells (DCs) by histamine may function as a regulator of inflammation. Therefore, we sought to determine the impact of histamine on antigen uptake by and activation of murine DCs. MATERIAL AND METHODS: DCs from spleen and lung were either identified by flow cytometry or were immunomagnetically enriched. Cells were stimulated with histamine, and the regulation of MHC-II and co-stimulatory molecule expression (CD80, CD86, and ICOS-L) and antigen uptake were quantified by flow cytometry. Individual contributions of the histamine receptor subtypes were determined by using the antagonists mepyramine (histamine H1-receptor: H1R), famotidine (H2R), and JNJ 7777120 (H4R). RESULTS: Histamine accelerated the uptake of soluble antigen via the H1R, H2R, and H4R in splenic DCs. Co-stimulatory molecule expression was enhanced already by enrichment procedures, thus, the analyses were performed in unseparated cell populations. Histamine enhanced the expression of CD86 and ICOS-L while expression of CD80 was unaffected. Antagonism at H1R, H2R, and H4R and at H1R and H4R reduced the histamine-induced enhanced expression of CD86 and ICOS-L, respectively. CONCLUSIONS: Histamine contributes to the regulation of the immunological synapse by stimulation of antigen uptake and activation of DCs via H1R, H2R, and H4R.

CIN85 Deficiency Prevents Nephrin Endocytosis and Proteinuria in Diabetes.

Diabetic nephropathy (DN) is the major cause of end-stage renal disease worldwide. Podocytes are important for glomerular filtration barrier function and maintenance of size selectivity in protein filtration in the kidney. Podocyte damage is the basis of many glomerular diseases characterized by loss of interdigitating foot processes and decreased expression of components of the slit diaphragm. Nephrin, a podocyte-specific protein, is the main component of the slit diaphragm. Loss of nephrin is observed in human and rodent models of diabetic kidney disease. The long isoform of CIN85 (RukL) is a binding partner of nephrin that mediates nephrin endocytosis via ubiquitination in podocytes. Here we demonstrate that the loss of nephrin expression and the onset of proteinuria in diabetic mice correlate with an increased accumulation of ubiquitinated proteins and expression of CIN85/RukL in podocytes. CIN85/RukL deficiency preserved nephrin surface expression on the slit diaphragm and reduced proteinuria in diabetic mice, whereas overexpression of CIN85 in zebrafish induced severe edema and disruption of the filtration barrier. Thus, CIN85/RukL is involved in endocytosis of nephrin in podocytes under diabetic conditions, causing podocyte depletion and promoting proteinuria. CIN85/RukL expression therefore shows potential to be a novel target for antiproteinuric therapy in diabetes.