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Abstract Background Induced pluripotent stem cells (iPSCs)-derived kidney organoids are a promising model for studying disease mechanisms and renal development. Despite several protocols having been developed, further improvements are needed to overcome existing limitations and enable a wider application of this model. One of the approaches to improve the differentiation of renal organoids in vitro is to include in the system cell types important for kidney organogenesis in vivo, such as macrophages. Another approach could be to improve cell survival. Mesodermal lineage differentiation is the common initial step of the reported protocols. The glycogen synthase kinase-3 (GSK-3) activity inhibitor, CHIR99021 (CHIR), is applied to induce mesodermal differentiation. It has been reported that CHIR simultaneously induces iPSCs apoptosis that can compromise cell differentiation. We thought to interfere with CHIR-induced apoptosis of iPSCs using rapamycin. Methods Differentiation of kidney organoids from human iPSCs was performed. Cell survival and autophagy were analyzed using Cell counting kit 8 (CCK8) kit and Autophagy detection kit. Cells were treated with rapamycin or co-cultured with human monocytes isolated from peripheral blood or iPSCs-macrophages using a transwell co-culture system. Monocyte-derived extracellular vesicles (EVs) were isolated using polyethylene glycol precipitation. Expression of apoptotic markers cleaved Caspase 3, Poly [ADP-ribose] polymerase 1 (PARP-1) and markers of differentiation T-Box Transcription Factor 6 (TBX6), odd-skipped related 1 (OSR1), Nephrin, E-Cadherin, Paired box gene 2 (Pax2) and GATA Binding Protein 3 (Gata3) was assessed by RT-PCR and western blotting. Organoids were imaged by 3D-confocal microscopy. Results We observed that CHIR induced apoptosis of iPSCs during the initial stage of renal organoid differentiation. Underlying mechanisms implied the accumulation of reactive oxygen species and decreased autophagy. Activation of autophagy by rapamacin and by an indirect co-culture of differentiating iPSCs with iPSCs-macrophages and human peripheral blood monocytes prevented apoptosis induced by CHIR. Furthermore, monocytes (but not rapamycin) strongly promoted expression of renal differentiation markers and organoids development via released extracellular vesicles. Conclusion Our data suggest that co-culturing of iPSCs with human monocytes strongly improves differentiation of kidney organoids. An underlying mechanism of monocytic action implies, but not limited to, an increased autophagy in CHIR-treated iPSCs. Our findings enhance the utility of kidney organoid models.

Summary: Background: The after-care treatment project KTx360° aimed to reduce graft failure and mortality after kidney transplantation (KTx). Methods: The study was conducted in the study centers Hannover, Erlangen and Hannoversch Muenden from May 2017 to October 2020 under the trial registration ISRCTN29416382. The program provided a multimodal aftercare program including specialized case management, telemedicine support, psychological and exercise assessments, and interventions. For the analysis of graft failure, which was defined as death, re-transplantation or start of long-term dialysis, we used longitudinal claims data from participating statutory health insurances (SHI) which enabled us to compare participants with controls. To balance covariate distributions between these nonrandomized groups we used propensity score methodology, in particular the inverse probability of treatment weighting (IPTW) approach. Findings: In total, 930 adult participants were recruited at three different transplant centres in Germany, of whom 320 were incident (enrolled within the first year after KTx) and 610 prevalent (enrolled >1 year after KTx) patients. Due to differences in the availability of the claims data, the claims data of 411 participants and 418 controls could be used for the analyses. In the prevalent group we detected a significantly lower risk for graft failure in the study participants compared to the matched controls (HR = 0.13, 95% CI = 0.04–0.39, p = 0.005, n = 389 observations), whereas this difference could not be detected in the incident group (HR = 0.92, 95% CI = 0.54–1.56, p = 0.837, n = 440 observations). Interpretation: Our findings suggest that a multimodal and multidisciplinary aftercare intervention can significantly improve outcome after KTx, specifically in patients later after KTx. For evaluation of effects on these outcome parameters in patients enrolled within the first year after transplantation longer observation times are necessary. Funding: The study was funded by the Global Innovation fund of the Joint Federal Committee of the Federal Republic of Germany, grant number 01NVF16009.

Abstract Free heme is released from hemoproteins during hemolysis or ischemia reperfusion injury and can be pro‐inflammatory. Most studies on nephrotoxicity of hemolysis‐derived proteins focus on free hemoglobin (fHb) with heme as a prosthetic group. Measurement of heme in its free, non‐protein bound, form is challenging and not commonly used in clinical routine diagnostics. In contrast to fHb, the role of free heme in acute kidney injury (AKI) after cardiopulmonary bypass (CPB) surgery is unknown. Using an apo‐horseradish peroxidase‐based assay, we identified free heme during CPB surgery as predictor of AKI in patients undergoing cardiac valve replacement (n = 37). Free heme levels during CPB surgery correlated with depletion of hemopexin (Hx), a heme scavenger‐protein. In mice, the impact of high levels of circulating free heme on the development of AKI following transient renal ischemia and the therapeutic potential of Hx were investigated. C57BL/6 mice were subjected to bilateral renal ischemia/reperfusion injury for 15 min which did not cause AKI. However, additional administration of free heme in this model promoted overt AKI with reduced renal function, increased renal inflammation, and reduced renal perfusion on functional magnetic resonance imaging. Hx treatment attenuated AKI. Free heme administration to sham operated control mice did not cause AKI. In conclusion, free heme is a predictor of AKI in CPB surgery patients and promotes AKI in transient renal ischemia. Depletion of Hx in CPB surgery patients and attenuation of AKI by Hx in the in vivo model encourage further research on Hx therapy in patients with increased free heme levels during CPB surgery.

BackgroundExcessive inflammation, hemolysis, and accumulation of labile heme play an essential role in the pathophysiology of multi-organ dysfunction syndrome (MODS) in sepsis. Alpha1-antitrypsin (AAT), an acute phase protein with heme binding capacity, is one of the essential modulators of host responses to inflammation. In this study, we evaluate the putative protective effect of AAT against MODS and mortality in a mouse model of polymicrobial abdominal sepsis.MethodsPolymicrobial abdominal sepsis was induced in C57BL/6N mice by cecal ligation and puncture (CLP). Immediately after CLP surgery, mice were treated intraperitoneally with three different forms of human AAT—plasma-derived native (nAAT), oxidized nAAT (oxAAT), or recombinant AAT (recAAT)—or were injected with vehicle. Sham-operated mice served as controls. Mouse survival, bacterial load, kidney and liver function, immune cell profiles, cytokines/chemokines, and free (labile) heme levels were assessed. In parallel, in vitro experiments were carried out with resident peritoneal macrophages (MPMΦ) and mouse peritoneal mesothelial cells (MPMC).ResultsAll AAT preparations used reduced mortality in septic mice. Treatment with AAT significantly reduced plasma lactate dehydrogenase and s-creatinine levels, vascular leakage, and systemic inflammation. Specifically, AAT reduced intraperitoneal accumulation of free heme, production of cytokines/chemokines, and neutrophil infiltration into the peritoneal cavity compared to septic mice not treated with AAT. In vitro experiments performed using MPMC and primary MPMΦ confirmed that AAT not only significantly decreases lipopolysaccharide (LPS)-induced pro-inflammatory cell activation but also prevents the enhancement of cellular responses to LPS by free heme. In addition, AAT inhibits cell death caused by free heme in vitro.ConclusionData from the septic CLP mouse model suggest that intraperitoneal AAT treatment alone is sufficient to improve sepsis-associated organ dysfunctions, preserve endothelial barrier function, and reduce mortality, likely by preventing hyper-inflammatory responses and by neutralizing free heme.

Purpose Primary aldosteronism (PA), characterised by low-renin hypertension, confers a high cardiovascular risk and is the most common cause of secondary hypertension, with an increased prevalence in patients with treatment-resistant hypertension. However, it is estimated that only a small percentage of affected patients are identified in routine clinical practice. Inhibitors of the renin-angiotensin system cause an increase in renin levels in patients with intact aldosterone regulation, and inadequate low renin with concurrent RAS inhibition (RASi) may therefore indicate PA, which could serve as a first look screening test for selection for formal work-up. Methods We analysed patients between 2016–2018 with treatment-resistant hypertension who had inadequate low renin in the presence of RASi (i. e. at risk for PA) and who were offered systematic work-up with adrenal vein sampling (AVS). Results A total of 26 pts were included in the study (age 54.8 ± 11, male 65%). Mean office blood pressure (BP) was 154/95 mmHg on 4.5 antihypertensive drug classes. AVS had a high technical success rate (96%) and demonstrated unilateral disease in the majority of patients (57%), most of which (77%) were undetected by cross-sectional imaging. Conclusion In patients with resistant hypertension, low renin in the presence of RASi is a strong indicator for autonomous aldosterone secretion. It may serve as an on-medication screening test for PA to select for formal PA work up.

Rationale & Objective: Copeptin and Midrange pro-atrial natriuretic peptide (MR-pro-ANP) are associated with outcomes independently of N-terminal pro-brain natriuretic peptide (NT-pro-BNP) in patients with heart failure (HF). The value of these markers in patients with chronic kidney disease (CKD) has not been studied. Study Design: Prospective cohort study. Setting & Participants: A total of 4,417 patients enrolled in the German Chronic Kidney Disease (GCKD) study with an estimated glomerular filtration rate of 30-60 mL/min/1.73m2 or overt proteinuria (urinary albumin-creatinine ratio >300mg/g or equivalent). Exposures: Copeptin, MR-pro-ANP, and NT-pro-BNP levels were measured in baseline samples. Outcomes: Noncardiovascular death, cardiovascular (CV) death, major adverse CV event (MACE), and hospitalization for HF. Analytical Approach: HRs for associations of Copeptin, MR-pro-ANP, and NT-pro-BNP with outcomes were estimated using Cox regression analyses adjusted for established risk factors. Results: During a maximum follow-up of 6.5 years, 413 non-CV deaths, 179 CV deaths, 519 MACE, and 388 hospitalizations for HF were observed. In Cox regression analyses adjusted for established risk factors, each one of the 3 markers were associated with all the 4 outcomes, albeit the highest HRs were found for NT-pro-BNP. When models were extended to include all the 3 markers, NT-pro-BNP remained associated with all 4 outcomes. Conversely, from the 2 novel markers, associations remained only for Copeptin with non-CV death (HR, 1.62; 95% CI, 1.04-2.54 for highest vs lowest quintile) and with hospitalizations for HF (HR, 1.73; 95% CI, 1.08-2.75). Limitations: Single-point measurements of Copeptin, MR-pro-ANP, and NT-pro-BNP. Conclusions: In patients with moderately severe CKD, we confirm NT-pro-BNP to be strongly associated with all outcomes examined. As the main finding, the novel marker Copeptin demonstrated independent associations with non-CV death and hospitalizations for HF, and should therefore be evaluated further for risk assessment in CKD. Plain-Language Summary: A blood sample–based biomarker that indicates high cardiovascular risk in a patient with kidney disease would help to guide interventions and has the potential to improve outcomes. In 4,417 patients of the German Chronic Kidney Disease study, we assessed the relationship of Copeptin, pro-atrial natriuretic peptide, and N-terminal pro-brain natriuretic peptide (NT-pro-BNP) with important outcomes over a follow-up period of 6.5 years. NT-pro-BNP was strongly associated with all of the 4 outcomes, including death unrelated to cardiovascular disease, death because of cardiovascular disease, a major cardiovascular event, and hospitalization for heart failure. Copeptin was associated with death unrelated to cardiovascular disease and hospitalization for heart failure. NT-pro-BNP and Copeptin are, therefore, promising candidates for a blood sample–based strategy to identify patients with kidney disease at high cardiovascular risk.

Ischemia causes an inflammatory response featuring monocyte-derived macrophages (MF) involved in angiogenesis and tissue repair. Angiogenesis and ischemic macrophage differentiation are regulated by Notch signaling via Notch ligand Delta-like 1 (Dll1). Colony stimulating factor 1 (CSF-1) is an essential MF lineage factor, but its role in ischemic macrophage development and the interaction with Notch signaling is so far unclear. Using a mouse model of hind limb ischemia with CSF-1 inhibitor studies and Dll1 heterozygous mice we show that CSF-1 is induced in the ischemic niche by a subpopulation of stromal cells expressing podoplanin, which was paralleled by the development of ischemic macrophages. Inhibition of CSF-1 signaling with small molecules or blocking antibodies impaired macrophage differentiation but prolonged the inflammatory response, resulting in impaired perfusion recovery and tissue regeneration. Yet, despite high levels of CSF-1, macrophage maturation and perfusion recovery were impaired in mice with Dll1 haploinsufficiency, while inflammation was exaggerated. In vitro, CSF-1 was not sufficient to induce full MF differentiation from donor monocytes in the absence of recombinant DLL1, while the presence of DLL1 in a dose-dependent manner stimulated MF differentiation in combination with CSF-1. Thus, CSF-1 is an ischemic niche factor that cooperates with Notch signaling in a non-redundant fashion to instruct macrophage cell fate and maturation, which is required for ischemic perfusion recovery and tissue repair.

IntroductionEven during physiologic aging, the kidney experiences a loss of mass and a progressive functional decline. This is clinically relevant as it leads to an increased risk of acute and chronic kidney disease. The kidney tubular system plays an important role in the underlying aging process, but the involved cellular mechanisms remain largely elusive.MethodsKidneys of 3-, 12- and 24-month-old male C57BL/6J mice were used for RNA sequencing, histological examination, immunostaining and RNA-in-situ-hybridization. Single cell RNA sequencing data of differentially aged murine and human kidneys was analyzed to identify age-dependent expression patterns in tubular epithelial cells. Senescent and non-senescent primary tubular epithelial cells from mouse kidney were used for in vitro experiments.ResultsDuring normal kidney aging, tubular cells adopt an inflammatory phenotype, characterized by the expression of MHC class II related genes. In our analysis of bulk and single cell transcriptional data we found that subsets of tubular cells show an age-related expression of Cd74, H2-Eb1 and H2-Ab1 in mice and CD74, HLA-DQB1 and HLADRB1 in humans. Expression of MHC class II related genes was associated with a phenotype of tubular cell senescence, and the selective elimination of senescent cells reversed the phenotype. Exposure to the Cd74 ligand MIF promoted a prosenescent phenotype in tubular cell cultures.DiscussionTogether, these data suggest that during normal renal aging tubular cells activate a program of ‘tubuloinflammaging’, which might contribute to age-related phenotypical changes and to increased disease susceptibility.

Abstract Recent reports demonstrate that SARS-CoV-2 utilizes cell surface heparan sulfate as an attachment factor to facilitate the initial interaction with host cells. Heparan sulfate interacts with the receptor binding domain of SARS-CoV-2 spike glycoprotein, and blocking this interaction can decrease cell infection. We and others reported recently that the family of compounds of 2,5-dihydroxyphenylic acid interferes with the binding of the positively charged groove in growth factor molecules to negatively charged cell surface heparan sulfate. We hypothesized that Calcium Dobesilate (CaD)—calcium salt of 2,5-dihydroxyphenylic acid—may also interfere with the binding of SARS-CoV-2 spike protein to heparan sulfate. Using lentiviral SARS-CoV-2 spike protein pseudotyped particles we show that CaD could significantly reduce pseudovirus uptake into endothelial cells. On the contrary, CaD did not affect cell infection with VSVG-expressing lentivirus. CaD could also prevent retention of SARS-CoV-2 spike protein in ex vivo perfused mouse kidney. Using microfluidic culture of endothelial cells under flow, we show that CaD prevents spike protein interaction with heparan sulfate glycocalyx. Since CaD has no adverse side effects and is approved in humans for other medical indications, our findings can rapidly translate into clinical studies.

Abstract Background Recently, a randomized controlled trial (RCT) demonstrated rapid but individually variable hemodynamic improvement with therapeutic plasma exchange (TPE) in patients with septic shock. Prediction of clinical efficacy in specific sepsis treatments is fundamental for individualized sepsis therapy. Methods In the original RCT, patients with septic shock of

Loss of canonical Notch signaling in vascular endothelial cells induces spontaneous formation of proto-typical tertiary lymphoid structures in mouse kidney, liver and lungs, which form around central arteries that acquire a high endothelial cell signature

Abstract Background After kidney transplantation, pregnancy and graft function may have a reciprocal interaction. We evaluated the influence of graft function on the course of pregnancy and vice versa. Methods We performed a retrospective observational study of 92 pregnancies beyond the first trimester in 67 women after renal transplantation from 1972 to 2019. Pre-pregnancy eGFR was correlated with outcome parameters; graft function was evaluated by Kaplan Meier analysis. The course of graft function in 28 women who became pregnant after kidney transplantation with an eGFR of 25 to

Abstract Background Chronic kidney disease (CKD) is a common condition that increases mortality and the risk of cardiovascular and other morbidities regardless of underlying renal condition. Chronic inflammation promotes renal fibrosis. Recently, renal B cell infiltrates were described in chronic kidney disease of various etiologies beyond autoimmunity. Methods We here investigated B cells and indicators of tertiary lymphoid structure formation in human renal biopsies. Renal function was studied during long‐term B cell depletion in human patients with membranous nephropathy and with CKD of unknown origin. Results Cytokine profiles of tertiary lymphoid structure formation were detected in human renal interstitium in a range of kidney diseases. Complex B cell structures consistent with tertiary lymphoid organ formation were evident in human membranous nephropathy. Here, B cell density did not significantly associate with proteinuria severity, but with worse excretory renal function. Proteinuria responses mostly occurred within the first 6 months of B cell depletion. In contrast, recovery of excretory kidney function was observed only after 18 months of continuous therapy, consistent with a structural process. Renal tertiary lymphatic structures were also detected in the absence of autoimmune kidney disease. To start to address whether B cell depletion may affect CKD in a broader population, we assessed kidney function in neurologic patients with CKD of unknown origin. In this cohort, eGFR significantly increased within 24 months of B cell depletion. Conclusion Long‐term B cell depletion associated with significant improvement of excretory kidney function in human CKD. Kinetics and mechanisms of renal B cell aggregation should be investigated further to stratify the impact of B cells and their aggregates as therapeutic targets.

Background: Macrophages play an important role in renal ischemia reperfusion injury, but the functional changes of macrophages under hypoxia/reoxygenation and the related mechanism are unclear and need to be further clarified. Methods: The effects of hypoxia/reoxygenation on functional characteristics of RAW264.7 macrophages were analyzed through the protein expression detection of pro-inflammatory factors TNF-α and CD80, anti-inflammatory factors ARG-1 and CD206. The functional implications of C-X3-C motif chemokine receptor 1(CX3CR1) down-regulation in hypoxic macrophages were explored using small interfering RNA technology. Significance was assessed by the parametric t-test or nonparametric Mann–Whitney test for two group comparisons, and a one-way ANOVA or the Kruskal–Wallis test for multiple group comparisons. Results: Hypoxia/reoxygenation significantly increased the protein expression of M1-related pro-inflammatory factors TNF-α, CD80 and chemokine C-X3-C motif chemokine ligand 1 (CX3CL1)/CX3CR1 and inhibited the protein expression of M2-related anti-inflammatory factors ARG-1 and CD206 in a time-dependent manner in RAW264.7 cells. However, the silencing of CX3CR1 in RAW264.7 cells using specific CX3CR1-siRNA, significantly attenuated the increase in protein expression of TNF-α (P

Lipoprotein(a) (Lp(a)) is becoming increasingly important as an independent risk factor for cardiovascular disease. Since no effective therapy currently exists other than lipid apheresis, the recommendation remains to optimally adjust all other cardiovascular risk factors (CVRF). In a Northwest German population study, the frequency of elevated Lp(a) levels and all other CVRF was investigated. The aim was to investigate whether individuals with elevated Lp(a) levels were also more likely to have other CVRFs. To date, 4602 individuals have been enrolled in the study, and blood pressure, weight, lipids, diabetes, medications, and pre-existing conditions were recorded in addition to Lp(a). In addition, questionnaires assessed physical activity, psychological stress, depression, and brain dysfunction. All participants received detailed individual recommendation about their CVRF and its treatment. In the further follow-up of 5 years, it will be examined how persons with elevated Lp(a) implemented these recommendations in comparison with participants without elevated Lp(a). The first group Lp(a) 120 nmol/L of 538 (11.7%). 81.6% of all participants had one or more CVRF. Age, sex, and prevalence of hypertension, diabetes, smoking, obesity, and exercise did not differ among the 3 groups. As expected, LDL-Cholesterol was significantly elevated in the Lp(a) >120 nmol/L group despite significantly more frequent use of statins. Significantly more often hypertensive patients were found in the Lp(a) >120 nmol/L group who were inadequately controlled by medication and significantly less often persons without further CVRF. No differences existed in the frequency of psychological stress, depression, and mild cognitive impairment. CVRF occur with comparable frequency in individuals with elevated Lp(a) levels. However, individuals with Lp(a) above 120 nmol/L were more likely to have poorly controlled blood pressure, elevated LDL-C, and less likely to have no other risk factors. This underlines that in case of Lp(a) elevation all further CVRF should be intensively adjusted, especially in case of strongly elevated values >120 nmol/L. However, these recommendations have not been adequately implemented in clinical care in this population to date.

Abstract 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

Abstract Background Reduced endothelial Tie2 expression occurs in diverse experimental models of critical illness, and experimental Tie2 suppression is sufficient to increase spontaneous vascular permeability. Looking for a common denominator among different critical illnesses that could drive the same Tie2 suppressive (thereby leak inducing) phenotype, we identified “circulatory shock” as a shared feature and postulated a flow-dependency of Tie2 gene expression in a GATA3 dependent manner. Here, we analyzed if this mechanism of flow-regulation of gene expression exists in vivo in the absence of inflammation. Results To experimentally mimic a shock-like situation, we developed a murine model of clonidine-induced hypotension by targeting a reduced mean arterial pressure (MAP) of approximately 50% over 4 h. We found that hypotension-induced reduction of flow in the absence of confounding disease factors (i.e., inflammation, injury, among others) is sufficient to suppress GATA3 and Tie2 transcription. Conditional endothelial-specific GATA3 knockdown (B6-Gata3tm1-Jfz VE-Cadherin(PAC)-cerERT2) led to baseline Tie2 suppression inducing spontaneous vascular leak. On the contrary, the transient overexpression of GATA3 in the pulmonary endothelium (jet-PEI plasmid delivery platform) was sufficient to increase Tie2 at baseline and completely block its hypotension-induced acute drop. On the functional level, the Tie2 protection by GATA3 overexpression abrogated the development of pulmonary capillary leakage. Conclusions The data suggest that the GATA3–Tie2 signaling pathway might play a pivotal role in controlling vascular barrier function and that it is affected in diverse critical illnesses with shock as a consequence of a flow-regulated gene response. Targeting this novel mechanism might offer therapeutic opportunities to treat vascular leakage of diverse etiologies.

Introduction: Atypical hemolytic uremic syndrome (aHUS) is a rare, complex, multisystem disease of dysregulated complement activity, characterized by progressive thrombotic microangiopathy (TMA), acute kidney injury, and multiorgan dysfunction, which often progresses to chronic kidney disease. Results from the prospective clinical trial of ravulizumab (NCT02949128) reveal rapid resolution of TMA in patients with aHUS, with sustained efficacy and safety in a 26-week initial evaluation period. Methods: The aim of this analysis was to characterize the long-term efficacy and the safety profile of ravulizumab in adults with aHUS who had completed the initial evaluation period of the trial. Complete TMA response, hematologic and kidney functions, and safety were evaluated for all patients available for follow-up in the extension period (median follow-up: 76.7 weeks; range: 0.6–118.3). This trial included a total of 58 patients, 49 of whom entered the extension period. Results: A total of 4 additional patients achieved complete TMA response during the follow-up period. Normalization of platelet count, serum lactate dehydrogenase (LDH), and hemoglobin observed in the 26-week initial evaluation period was sustained until the last available follow-up, as were the improvements in the estimated glomerular filtration rate (eGFR) and patient quality of life. All efficacy endpoints were correlated with the sustained inhibition of complement C5. Most adverse events (AEs) occurred early during the initial evaluation period and decreased substantially during the extension period. No patient developed a meningococcal infection or died during the extension period. Conclusion: This analysis reveals that ravulizumab administered every 8 weeks is efficacious with an acceptable safety profile for the long-term treatment of adults with aHUS and provides additional clinical benefit beyond 6 months of treatment.

A kidney transplant is often the best treatment for end-stage renal disease. Although immunosuppressive therapy sharply reduces the occurrence of acute allograft rejection (AR), it remains the main cause of allograft dysfunction. We aimed to identify effective biomarkers for AR instead of invasive kidney transplant biopsy. We integrated the results of several proteomics studies related to AR and utilized public data sources. Gene ontology (GO) and pathway analyses were used to identify important biological processes and pathways. The performance of the identified proteins was validated using several public gene expression omnibus (GEO) datasets. Samples that performed well were selected for further validation through RNA sequencing of peripheral blood mononuclear cells of patients with AR (n = 16) and non-rejection (n = 19) from our medical center. A total of 25 differentially expressed proteins (DEPs) overlapped in proteomic studies of urine and blood samples. GO analysis showed that the DEPs were mainly involved in the immune system and blood coagulation. Pathway analysis showed that the complement and coagulation cascade pathways were well enriched. We found that immunoglobulin heavy constant alpha 1 (IGHA1) and immunoglobulin κ constant (IGKC) showed good performance in distinguishing AR from non-rejection groups validated with several GEO datasets. Through RNA sequencing, the combination of IGHA1, IGKC, glomerular filtration rate, and donor age showed good performance in the diagnosis of AR with ROC AUC 91.4% (95% CI: 82–100%). Our findings may contribute to the discovery of potential biomarkers for AR monitoring.

BackgroundAcute rejection (AR) in kidney transplantation is an established risk factor that reduces the survival rate of allografts. Despite standard immunosuppression, molecules with regulatory control in the immune pathway of AR can be used as important targets for therapeutic operations to prevent rejection.MethodsWe downloaded the microarray data of 15 AR patients and 37 non-acute rejection (NAR) patients from Gene Expression Omnibus (GEO). Gene network was constructed, and genes were classified into different modules using weighted gene co-expression network analysis (WGCNA). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Cytoscape were applied for the hub genes in the most related module to AR. Different cell types were explored by xCell online database and single-cell RNA sequencing. We also validated the SLAMF8 and TLR4 levels in Raw264.7 and human kidney tissues of TCMR.ResultsA total of 1,561 differentially expressed genes were filtered. WGCNA was constructed, and genes were classified into 12 modules. Among them, the green module was most closely associated with AR. These genes were significantly enriched in 20 pathway terms, such as cytokine–cytokine receptor interaction, chemokine signaling pathway, and other important regulatory processes. Intersection with GS > 0.4, MM > 0.9, the top 10 MCC values and DEGs in the green module, and six hub genes (DOCK2, NCKAP1L, IL2RG, SLAMF8, CD180, and PTPRE) were identified. Their expression levels were all confirmed to be significantly elevated in AR patients in GEO, Nephroseq, and quantitative real-time PCR (qRT-PCR). Single-cell RNA sequencing showed that AR patient had a higher percentage of native T, CD1C+_B DC, NKT, NK, and monocytes in peripheral blood mononuclear cells (PBMCs). Xcell enrichment scores of 20 cell types were significantly different (p

Abstract 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.

Purpose Hypertension is the most important risk factor for disease and premature death. Treatment strategies adjusted for cardiovascular risk have been proposed in guidelines, but real-life treatment strategies for patients with newly diagnosed hypertension in Germany are largely unknown. The aim of the study was to analyse initial drug treatment strategies and associated risk status in patients with newly diagnosed hypertension. Material and methods In the representative research database of the public health insurance system in Germany (2077899 individuals) we identified patients with newly diagnosed hypertension in 2012 and analysed co-existing cardiovascular co-morbidities and hypertension-mediated organ damage by ICD-codes as qualifiers for high risk. Health insurance billing datasets for redeemed prescriptions were analysed at several time points using ATC-codes. Results The incidence of hypertension was 2.6%, 33.6% of the patients were at high risk at diagnosis, mainly due to cardiovascular co-morbidities. Most patients initially received monotherapy (55.4%), of which ACE inhibitors (43.8%) or beta-blockers (32.4%) were the leading drug classes, while 21.7% of patients received no drug therapy during the first year. The treatment strategies of low and high-risk patients resembled each other – high-risk patients also received mostly monotherapy during the first year after diagnosis (53.4%), while 13.7% remained without drug therapy. Combination therapy was the most frequent treatment strategy one year after hypertension diagnosis (40.6%) and in the long term (68.4%). Conclusion Initial treatment strategies may not always be stratified according to cardiovascular risk. The majority of patients with hypertension receives initial monotherapy independent of their individual risk. However, combination therapy represents the major form of therapy in the long-term.

Acute rejection (AR) is closely associated with renal allograft dysfunction. Here, we utilised RNA sequencing (RNA-Seq) and bioinformatic methods to characterise the peripheral blood mononuclear cells (PBMCs) of patients with acute renal allograft rejection. Pretransplant blood samples were collected from 32 kidney allograft donors and 42 corresponding recipients with biopsies classified as T cell-mediated rejection (TCMR, n = 18), antibody-mediated rejection (ABMR, n = 5), and normal/non-specific changes (non-AR, n = 19). The patients with TCMR and ABMR were assigned to the AR group, and the patients with normal/non-specific changes (n = 19) were assigned to the non-AR group. We analysed RNA-Seq data for identifying differentially expressed genes (DEGs), and then gene ontology (GO) analysis, Reactome, and ingenuity pathway analysis (IPA), protein—protein interaction (PPI) network, and cell-type enrichment analysis were utilised for bioinformatics analysis. We identified DEGs in the PBMCs of the non-AR group when compared with the AR, ABMR, and TCMR groups. Pathway and GO analysis showed significant inflammatory responses, complement activation, interleukin-10 (IL-10) signalling pathways, classical antibody-mediated complement activation pathways, etc., which were significantly enriched in the DEGs. PPI analysis showed that IL-10, VEGFA, CXCL8, MMP9, and several histone-related genes were the hub genes with the highest degree scores. Moreover, IPA analysis showed that several proinflammatory pathways were upregulated, whereas antiinflammatory pathways were downregulated. The combination of NFSF14+TANK+ANKRD 33 B +HSPA1B was able to discriminate between AR and non-AR with an AUC of 92.3% (95% CI 82.8–100). Characterisation of PBMCs by RNA-Seq and bioinformatics analysis demonstrated gene signatures and biological pathways associated with AR. Our study may provide the foundation for the discovery of biomarkers and an in-depth understanding of acute renal allograft rejection.

BackgroundAcute kidney injury (AKI) after lung transplantation (LuTx) is associated with increased long-term mortality. In this prospective observational study, commonly used AKI-definitions were examined regarding prediction of long-term mortality and compared to simple use of the serum creatinine value at day 7 for patients who did not receive hemodialysis, and serum creatinine value immediately before initiation of hemodialysis (d7/preHD-sCr).Methods185 patients with LuTx were prospectively enrolled from 2013-2014 at our center. Kidney injury was assessed within 7 days by: (1) the Kidney Disease Improving Global Outcomes criteria (KDIGO-AKI), (2) the Acute Disease Quality Initiative 16 Workgroup classification (ADQI-AKI) and (3) d7/preHD-sCr. Prediction of all-cause mortality was examined by Cox regression analysis, and clinical as well as laboratory factors for impaired kidney function post-LuTx were analyzed.ResultsAKI according to KDIGO and ADQI-AKI occurred in 115 patients (62.2%) within 7 days after LuTx. Persistent ADQI-AKI, KDIGO-AKI stage 3 and higher d7/preHD-sCr were associated with higher mortality in the univariable analysis. In the multivariable analysis, d7/preHD-sCr in combination with body weight and intra- and postoperative platelet transfusions predicted mortality after LuTx with similar performance as models using KDIGO-AKI and ADQI-AKI (concordance index of 0.75 for d7/preHD-sCr vs., 0.74 and 0.73, respectively). Pre-transplant reduced renal function, diabetes, higher BMI, and intraoperative ECMO predicted higher d7/preHD-sCr (r2 = 0.354, p < 0.001).ConclusionOur results confirm the importance of AKI in lung transplant patients; however, a simple and pragmatic indicator of renal function, d7/preHD-sCr, predicts long-term mortality equally reliable as more complex AKI-definitions like KDIGO and ADQI.

Purpose: Most guidelines for treatment of hypertension in the setting of diabetes recommend a blood pressure (BP) target of 160/100 from 9 to 2%[[mean BP change −31 (15.7)/ −14 (9.8) mmHg]] and of 140–159/90–99 mmHg from 32 to 11% [[mean BP change −16(12.7)/ −8.9 (8.7) mmHg]], with corresponding increases in the prevalence of patients with baseline BP levels of 120–139/80–99 from 48 to 65% [[mean BP change −4.1 (10.6)/ −3.1 (7.8) mmHg]]and of

Abstract Background A dysbalanced coagulation system is part of the pathological host response to infection in sepsis. Activation of pro-coagulant pathways and attenuation of anti-coagulant activity ultimately lead to microvascular stasis and consequent organ failure. No treatment approaches specifically targeting this axis are available. We explored the effects of therapeutic plasma exchange (TPE) on microvascular coagulation dysbalance in septic shock. Methods We conducted a prospective single-center study enrolling 31 patients with early septic shock (onset 0.4 μg/kg/min). Clinical and biochemical data, including measurement of protein C; a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13 (ADAMTS13); and von Willebrand factor antigen (vWF:Ag), were obtained before and after TPE against fresh frozen plasma. Results Antithrombotic acting proteins such as antithrombin-III (ATIII) and protein C were markedly reduced in septic patients, but their activity increased after TPE (ATIII, 51% (41–61) vs. 63% (48–70), p = 0.029; protein C, 47% (38–60) vs. 62% (54–69), p = 0.029). Median ADAMTS13 activity was increased by TPE from 27 (21–42) % before to 47 (38–62) % after TPE (p

Abstract Background Surgical correction of hyperparathyroidism after kidney transplantation has been associated with significant graft function decline. We examined the effects of parathyroidectomy on short- and long-term graft function and its potential predictors. Methods For this retrospective, monocentric study we identified 48 (5.5%) out of 892 patients from our protocol biopsy program who received renal transplantation between 2000 and 2007, with parathyroidectomy after transplantation. Data from up to three years after parathyroidectomy was collected and analyzed with multivariable linear regression analyses. Results Main indications for parathyroidectomy were hypercalcemia and graft calcifications. Parathyroidectomy was successful in 47 patients, with a median drop in serum intact parathormone (iPTH) from 394 to 21 pg/ml. Mean estimated glomerular fitration rate (eGFR) before parathyroidectomy was 60 ± 26 ml/min. At three months after parathyroidectomy, the eGFR was 46 ± 18 ml/min (p

Aim: The aim of the case-control study was to investigate if serum biomarkers indicative of vascular inflammation and endothelial dysfunction can predict the development of microalbuminuria in patients with diabetes mellitus type 2. Methods: Among participants enrolled in the ROADMAP (Randomized Olmesartan And Diabetes MicroAlbuminuria Prevention) and observational follow-up (OFU) studies, a panel of 15 serum biomarkers was quantified from samples obtained at initiation of the study and tested for associations with the development of new-onset microalbuminuria during follow-up. A case-control study was conducted with inclusion of 172 patients with microalbuminuria and 188 matched controls. Nonparametric inferential, nonlinear regression, mediation, and bootstrapping statistical methods were used for the analysis. Results: The median follow-up time was 37 months. At baseline, mean concentrations of C-X-C motif chemokine ligand 16 (CXCL-16), transforming growth factor (TGF)–β1 and angiopoietin-2 were higher in patients with subsequent microalbuminuria. In the multivariate analysis, after adjustment for age, sex, body mass index, glycated hemoglobin, duration of diabetes, low-density lipoprotein (LDL), smoking status, blood pressure, baseline urine albumin-to-creatinine ratio (UACR), estimated glomerular filtration rate (eGFR), time of follow-up and cardiovascular disease, CXCL-16 (odds ratio [OR] 2.60, 95% confidence interval [CI] 1.71–3.96), angiopoietin-2 (OR 1.50, 95% CI 1.14–1.98) and TGF-β1 (OR 1.03, 95% CI 1.01–1.04) remained significant predictors of new-onset microalbuminuria (P < 0.001). Inclusion of these biomarkers in conventional clinical risk models for prediction of microalbuminuria increased the area under the curve (AUC) from 0.638 to 0.760 (P < 0.001). Conclusion: In patients with type 2 diabetes, elevated plasma levels of CXCL-16, angiopoietin-2, and TGF-β1 are independently predictive of microalbuminuria. Thus, these serum markers improve renal risk models beyond established clinical risk factors. Keywords: albuminuria, atheromatosis, cardiovascular disease, diabetic kidney disease, inflammation

Sepsis is a life-threatening syndrome caused by a pathological host response to an infection that eventually, if uncontrolled, leads to septic shock and ultimately, death. In sepsis, a massive aggregation of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) cause a cytokine storm. The endothelial glycocalyx (eGC) is a gel like layer on the luminal side of the endothelium that consists of proteoglycans, glycosaminoglycans (GAG) and plasma proteins. It is synthesized by endothelial cells and plays an active role in the regulation of inflammation, permeability, and coagulation. In sepsis, early and profound injury of the eGC is observed and circulating eGC components correlate directly with clinical severity and outcome. The activity of the heparan sulfate (HS) specific glucuronidase Heparanase-1 (Hpa-1) is elevated in sepsis, resulting in shedding of heparan sulfate (HS), a main GAG of the eGC. HS induces endothelial barrier breakdown and accelerates systemic inflammation. Lipopolysaccharide (LPS), a PAMP mainly found on the surface of gram-negative bacteria, activates TLR-4, which results in cytokine production and further activation of Hpa-1. Hpa-1 shed HS fragments act as DAMPs themselves, leading to a vicious cycle of inflammation and end-organ dysfunction such as septic cardiomyopathy and encephalopathy. Recently, Hpa-1′s natural antagonist, Heparanase-2 (Hpa-2) has been identified. It has no intrinsic enzymatic activity but instead acts by reducing inflammation. Hpa-2 levels are reduced in septic mice and patients, leading to an acquired imbalance of Hpa-1 and Hpa-2 paving the road towards a therapeutic intervention. Recently, the synthetic antimicrobial peptide 19–2.5 was described as a promising therapy protecting the eGC by inhibition of Hpa-1 activity and HS shed fragments in animal studies. However, a recombinant Hpa-2 therapy does not exist to the present time. Therapeutic plasma exchange (TPE), a modality already tested in clinical practice, effectively removes injurious mediators, e.g., Hpa-1, while replacing depleted protective molecules, e.g., Hpa-2. In critically ill patients with septic shock, TPE restores the physiological Hpa-1/Hpa-2 ratio and attenuates eGC breakdown. TPE results in a significant improvement in hemodynamic instability including reduced vasopressor requirement. Although promising, further studies are needed to determine the therapeutic impact of TPE in septic shock.

Cell-free hemoglobin (CFH), a pro-oxidant and cytotoxic compound that is released in hemolysis, has been associated with nephrotoxicity. Lung transplantation (LuTx) is a clinical condition with a high incidence of acute kidney injury (AKI). In this study, we investigated the plasma levels of CFH and haptoglobin, a CFH-binding serum protein, in prospectively enrolled LuTx patients (n = 20) with and without AKI. LuTx patients with postoperative AKI had higher CFH plasma levels at the end of surgery compared with no-AKI patients, and CFH correlated with serum creatinine at 48 h. Moreover, CFH levels inversely correlated with haptoglobin levels, which were significantly reduced at the end of surgery in LuTx patients with AKI. Because multiple other factors can contribute to AKI development in the complex clinical setting of LuTx, we next investigated the role of exogenous CFH administration in a mouse model of mild bilateral renal ischemia reperfusion injury (IRI). Exogenous administration of CFH after reperfusion caused overt AKI with creatinine increase, tubular injury, and enhanced markers of renal inflammation compared with vehicle-treated animals. In conclusion, CFH is a possible factor contributing to postoperative AKI after LuTx and promotes AKI in an experimental model of mild transient renal ischemia. Targeting CFH might be a therapeutic option to prevent AKI after LuTx.

Background: Zinc-alpha 2-glycoprotein (AZGP1), a secreted protein with ubiquitous tissue expression, has been controversially linked to the risk of cardiovascular disease. In a cohort of kidney transplant recipients, we measured serum AZGP1 levels after transplantation over a 2 year period and tested for an association with pulse wave velocity as an important parameter indicating future cardiovascular events.Methods: Annual blood sampling and pulse wave velocity measurements were longitudinally performed in 113 kidney transplant recipients. AZGP1 was measured in serum samples using standard ELISA. Association of AZGP1 with pulse wave velocity was longitudinally assessed during follow up of 2 years by mixed longitudinal modeling.Results: AZGP1 serum levels declined significantly after kidney transplantation. This decline was dependent on allograft function as indicated by inverse correlation with eGFR. When corrected for eGFR multivariable analysis revealed an inverse correlation between AZGP1 and pulse wave velocity. This analysis further showed independent associations of older age, higher blood pressure, and higher calcium phosphate product with higher pulse wave velocity.Conclusions: Improved kidney function after transplantation leads to a decline in AZGP1 serum levels. Independent of kidney function and other cardiovascular risk factors lower AZGP1 levels are associated with higher pulse wave velocity in the 2 years after kidney transplantation. These data suggest that AZGP1 might be a potential biomarker for cardiovascular health and a target for improving cardiovascular outcome.

Myeloid plasticity is a hallmark of the innate immune response to Toll-like receptor (TLR) activation. Here, we provide a protocol for monocyte cell fate tracking by adoptive transfer in the context of systemic inflammation induced by TLR7 activation, the principal innate immune receptor sensing viral RNA in mice. Defined monocyte subsets are isolated from the bone marrow of donor mice by cell sorting and adoptively transferred into the systemic circulation of congenic hosts, with or without concurrent activation of TLR7 via the topical application of the small molecule agonist, imiquimod, in a cream formulation that induces a systemic inflammatory response. Advantages are the precise definition of donor cell populations and resulting cell fate without the need for host conditioning in a model that recapitulates key aspects of the systemic inflammatory response to TLR7 stimulation.

Nephrotoxic drugs can cause acute kidney injury (AKI) and analgesic nephropathy. Diclofenac is potentially nephrotoxic and frequently prescribed for pain control. In this study, we investigated the effects of single and repetitive oral doses of diclofenac in the setting of pre-existing subclinical AKI on the further course of AKI and on long-term renal consequences. Unilateral renal ischemia–reperfusion injury (IRI) for 15 min was performed in male CD1 mice to induce subclinical AKI. Immediately after surgery, single oral doses (100 mg or 200 mg) of diclofenac were administered. In a separate experimental series, repetitive treatment with 100 mg diclofenac over three days was performed after IRI and sham surgery. Renal morphology and pro-fibrotic markers were investigated 24 h and two weeks after the single dose and three days after the repetitive dose of diclofenac treatment using histology, immunofluorescence, and qPCR. Renal function was studied in a bilateral renal IRI model. A single oral dose of 200 mg, but not 100 mg, of diclofenac after IRI aggravated acute tubular injury after 24 h and caused interstitial fibrosis and tubular atrophy two weeks later. Repetitive treatment with 100 mg diclofenac over three days aggravated renal injury and caused upregulation of the pro-fibrotic marker fibronectin in the setting of subclinical AKI, but not in sham control kidneys. In conclusion, diclofenac aggravated renal injury in pre-existing subclinical AKI in a dose and time-dependent manner and already a single dose can cause progression to chronic kidney disease (CKD) in this model.

Abstract Background Despite modern intensive care with standardized strategies against acute respiratory distress syndrome (ARDS), Pneumocystis pneumonia (PcP) remains a life-threatening disease with a high mortality rate. Here, we analyzed a large mixed cohort of immunocompromised patients with PcP, with regard to clinical course and treatment, and aimed at identifying predictors of outcome. Methods This was a single-center retrospective analysis in a tertiary care institution across 17 years. Diagnosis of PcP required typical clinical features and microbiological confirmation of Pneumocystis jirovecii. Epidemiological, clinical, laboratory and outcome data were collected from patient records. Results A total of 52,364 specimens from 7504 patients were sent for microbiological assessment (3653 with clinical suspicion of Pneumocystis pneumonia). PcP was confirmed in 240 patients, about half of them HIV positive (52%). The remaining subjects were either solid organ transplant recipients (16.3%) or suffered from malignancy (15.8%) or autoimmune diseases (11.7%). Of note, 95% of patients with PcP were not receiving chemoprophylaxis. Overall in-hospital mortality was 25.4%, increasing to 58% if ICU admission was required. Multivariable regression identified lactate dehydrogenase (LDH) as predictor of in-hospital mortality (adjusted OR 1.17 (95% CI 1.09–1.27), p

Abstract Background Given the pathophysiological key role of the host response to an infection rather than the infection per se, an ideal therapeutic strategy would also target this response. This study was designed to demonstrate safety and feasibility of early therapeutic plasma exchange (TPE) in severely ill individuals with septic shock. Methods This was a prospective single center, open-label, nonrandomized pilot study enrolling 20 patients with early septic shock (onset 0.4 μg/kg/min) out of 231 screened septic patients. Clinical and biochemical data were obtained before and after TPE. Plasma samples were taken for ex-vivo stimulation of human umbilical vein endothelial cells (HUVECs) to analyze barrier function (immunocytochemistry and transendothelial electrical resistance (TER)). Cytokines were measured by cytometric bead array (CBA) and enzyme-linked immunosorbent assays (ELISAs). An immediate response was defined as > 20% NE reduction from baseline to the end of TPE. Results TPE was well tolerated without the occurrence of any adverse events and was associated with a rapid reduction in NE (0.82 (0.61–1.17) vs. 0.56 (0.41–0.78) μg/kg/min, p = 0.002) to maintain mean arterial pressure (MAP) above 65 mmHg. The observed 28-day mortality was 65%. Key proinflammatory cytokines and permeability factors (e.g., interleukin (IL)-6, IL-1b, and angiopoietin-2) were significantly reduced after TPE, while the protective antipermeability factor angiopoietin-1 was not changed. Ex-vivo stimulation of HUVECs with plasma obtained before TPE induced substantial cellular hyperpermeability, which was completely abolished with plasma obtained after TPE. Conclusions Inclusion of early septic shock patients with high doses of vasopressors was feasible and TPE was safe. Rapid hemodynamic improvement and favorable changes in the cytokine profile in patients with septic shock were observed. It has yet to be determined whether early TPE also improves outcomes in this patient cohort. An appropriately powered multicenter randomized controlled trial is desirable. Trial registration Clinicaltrials.gov, NCT03065751. Retrospectively registered on 28 February 2017.

Background: Many patients have disabilities; it is therefore essential that medical education includes comprehensive teaching on disability and rehabilitation. In 2006 Hannover Medical School implemented an introductory course in the curriculum for medical students, on how to communicate with persons with disability and the need for rehabilitation. The course, entitled “Introduction to medicine”, has the main goals of teaching the strategy and systematic approach of medicine to solving patients’ problems. Methods: This paper describes the content, methods and outcomes of 1 of the 4 main themes of the “Introduction to medicine” course; the theme “Pain and disability”, which is covered in the second week of the course. Results: Evaluation of the “Pain and disability” module found that students’ ratings for the category “patient involvement” were very high (93%), whereas their ratings for the category “examination of student knowledge” were low. The overall rating of the module was “good” (10.8 out of 15 points), but not “very good”. Conclusion: The concept of the “Pain and disability” module is feasible and successful, even though it is scheduled early in the first year of the curriculum and approximately 350 students participate. Factors related to this success are: a mixture of teaching knowledge, supporting students’ understanding, and applying communication and physical examination skills.

GPI-anchored uPAR is the receptor for the extracellular serine protease urokinase-type plasminogen activator (uPA). Though uPAR role in inflammatory processes is documented, underlying mechanisms are not fully understood. In this study we demonstrate that uPAR is a part of Toll-like receptor 4 (TLR4) interactome. Downregulation of uPAR expression resulted in diminished LPS-induced TLR4 signaling, less activation of NFκB, and decreased secretion of inflammatory mediators in myeloid and non-myeloid cells in vitro. In vivo uPAR−/− mice demonstrated better survival, strongly diminished inflammatory response and better organ functions in cecal ligation and puncture mouse polymicrobial sepsis model. Mechanistically, GPI-uPAR and soluble uPAR colocalized with TLR4 on the cell membrane and interacted with scavenger receptor CD36. Our data show that uPAR can interfere with innate immunity response via TLR4 and this mechanism represents a potentially important target in inflammation and sepsis therapy.

Chronic kidney disease (CKD) is characterized by a long-term loss of kidney function and, in most cases, by progressive fibrosis. Zinc-alpha2-glycoprotein (AZGP1) is a secreted protein, which is expressed in many different tissues and has been associated with a variety of functions. In a previous study, we have shown in cell culture and in AZGP1 deficient mice that AZGP1 has protective anti-fibrotic effects. In the present study, we tested the therapeutic potential of an experimental increase in AZGP1 using two different strategies. (1) C57Bl/6J mice were treated systemically with recombinant AZGP1, and (2) a transgenic mouse strain was generated to overexpress AZGP1 conditionally in proximal tubular cells. Mice underwent unilateral uretic obstruction as a pro-fibrotic kidney stress model, and kidneys were examined after 14 days. Recombinant AZGP1 treatment was accompanied by better preservation of tubular integrity, reduced collagen deposition, and lower expression of injury and fibrosis markers. Weaker but similar tendencies were observed in transgenic AZGP1 overexpressing mice. Higher AZGP1 levels led to a significant reduction in stress-induced accumulation of tubular lipid droplets, which was paralleled by improved expression of key players in lipid metabolism and fatty acid oxidation. Together these data show beneficial effects of elevated AZGP1 levels in fibrotic kidney disease and highlight a novel link to tubular cell lipid metabolism, which might open up new opportunities for CKD treatment.

Chronic antibody-mediated rejection (AMR) is a key limiting factor for the clinical outcome of a kidney transplantation (Ktx), where early diagnosis and therapeutic intervention is needed. This study describes the identification of the biomarker CXC-motif chemokine ligand (CXCL) 9 as an indicator for AMR and presents a new aptamer-antibody-hybrid lateral flow assay (hybrid-LFA) for detection in urine. Biomarker evaluation included two independent cohorts of kidney transplant recipients (KTRs) from a protocol biopsy program and used subgroup comparisons according to BANFF-classifications. Plasma, urine and biopsy lysate samples were analyzed with a Luminex-based multiplex assay. The CXCL9-specific hybrid-LFA was developed based upon a specific rat antibody immobilized on a nitrocellulose-membrane and the coupling of a CXCL9-binding aptamer to gold nanoparticles. LFA performance was assessed according to receiver operating characteristic (ROC) analysis. Among 15 high-scored biomarkers according to a neural network analysis, significantly higher levels of CXCL9 were found in plasma and urine and biopsy lysates of KTRs with biopsy-proven AMR. The newly developed hybrid-LFA reached a sensitivity and specificity of 71% and an AUC of 0.79 for CXCL9. This point-of-care-test (POCT) improves early diagnosis-making in AMR after Ktx, especially in KTRs with undetermined status of donor-specific HLA-antibodies.

Ischemia reperfusion injury (IRI) is linked with inflammation in kidney transplantation (ktx). The chemokine CXCL13, also known as B lymphocyte chemoattractant, mediates recruitment of B cells within follicles of lymphoid tissues and has recently been identified as a biomarker for acute kidney allograft rejection. The goal of this study was to explore whether IRI contributes to the up-regulation of CXCL13 levels in ktx. It is demonstrated that systemic levels of CXCL13 were increased in mouse models of uni- and bilateral renal IRI, which correlated with the duration of IRI. Moreover, in unilateral renal IRI CXCL13 expression in ischemic kidneys was up-regulated. Immunohistochemical studies revealed infiltration of CD22+ B-cells and, single-cell RNA sequencing analysis a higher number of cells expressing the CXCL13 receptor CXCR5, in ischemic kidneys 7 days post IRI, respectively. The potential relevance of these findings was also evaluated in a mouse model of ktx. Increased levels of serum CXCL13 correlated with the lengths of cold ischemia times and were further enhanced in allogenic compared to isogenic kidney transplants. Taken together, these findings indicate that IRI is associated with increased systemic levels of CXCL13 in renal IRI and ktx.

Endothelial Tie2 signaling plays a pivotal role in vascular barrier maintenance at baseline and after injury. We previously demonstrated that a sharp drop in Tie2 expression observed across various murine models of critical illnesses is associated with increased vascular permeability and mortality. Matrix metalloprotease (MMP)−14-mediated Tie2 ectodomain shedding has recently been recognized as a possible mechanism for Tie2 downregulation in sepsis. Here, we identified the exact MMP14-mediated Tie2 ectodomain cleavage sites and could show that pharmacological MMP14 blockade in experimental murine sepsis exerts barrier protective and anti-inflammatory effects predominantly through the attenuation of Tie2 cleavage to improve survival both in a pre-treatment and rescue approach. Overall, we show that protecting Tie2 shedding might offer a new therapeutic opportunity for the treatment of septic vascular leakage.

Conventional Ly6Chi monocytes have developmental plasticity for a spectrum of differentiated phagocytes. Here we show, using conditional deletion strategies in a mouse model of Toll-like receptor (TLR) 7-induced inflammation, that the spectrum of developmental cell fates of Ly6Chi monocytes, and the resultant inflammation, is coordinately regulated by TLR and Notch signaling. Cell-intrinsic Notch2 and TLR7-Myd88 pathways independently and synergistically promote Ly6Clo patrolling monocyte development from Ly6Chi monocytes under inflammatory conditions, while impairment in either signaling axis impairs Ly6Clo monocyte development. At the same time, TLR7 stimulation in the absence of functional Notch2 signaling promotes resident tissue macrophage gene expression signatures in monocytes in the blood and ectopic differentiation of Ly6Chi monocytes into macrophages and dendritic cells, which infiltrate the spleen and major blood vessels and are accompanied by aberrant systemic inflammation. Thus, Notch2 is a master regulator of Ly6Chi monocyte cell fate and inflammation in response to TLR signaling.

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.

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

BACKGROUND:Sodium-glucose cotransporter-2 inhibitors (SGLT2is) represent a new class of oral hypoglycemic agents used in the treatment of type 2 diabetes mellitus. They have a positive effect on the progression of chronic kidney disease, but there is a concern that they might cause acute kidney injury (AKI). METHODS AND FINDINGS:We conducted a systematic review and meta-analysis of the effect of SGLT2is on renal adverse events (AEs) in randomized controlled trials and controlled observational studies. PubMed, EMBASE, Cochrane library, and ClinicalTrials.gov were searched without date restriction until 27 September 2019. Data extraction was performed using a standardized data form, and any discrepancies were resolved by consensus. One hundred and twelve randomized trials (n = 96,722) and 4 observational studies with 5 cohorts (n = 83,934) with a minimum follow-up of 12 weeks that provided information on at least 1 adverse renal outcome (AKI, combined renal AE, or hypovolemia-related events) were included. In 30 trials, 410 serious AEs due to AKI were reported. SGLT2is reduced the odds of suffering AKI by 36% (odds ratio [OR] 0.64 [95% confidence interval (CI) 0.53-0.78], p < 0.001). A total of 1,089 AKI events of any severity (AEs and serious AEs [SAEs]) were published in 41 trials (OR 0.75 [95% CI 0.66-0.84], p < 0.001). Empagliflozin, dapagliflozin, and canagliflozin had a comparable benefit on the SAE and AE rate. AEs related to hypovolemia were more commonly reported in SGLT2i-treated patients (OR 1.20 [95% CI 1.10-1.31], p < 0.001). In the observational studies, 777 AKI events were reported. The odds of suffering AKI were reduced in patients receiving SGLT2is (OR 0.40 [95% CI 0.33-0.48], p < 0.001). Limitations of this study are the reliance on nonadjudicated safety endpoints, discrepant inclusion criteria and baseline hypoglycemic therapy between studies, inconsistent definitions of renal AEs and hypovolemia, varying follow-up times in different studies, and a lack of information on the severity of AKI (stages I-III). CONCLUSIONS:SGLT2is reduced the odds of suffering AKI with and without hospitalization in randomized trials and the real-world setting, despite the fact that more AEs related to hypovolemia are reported.

Background: Ischemia reperfusion injury (IRI) plays a major role in solid organ transplantation. The length of warm ischemia time is critical for the extent of tissue damage in renal IRI. In this experimental study we hypothesized that local release of labile heme in renal tissue is triggered by the duration of warm ischemia (15 vs. 45 min IRI) and mediates complement activation, cytokine release, and inflammation.Methods: To induce IRI, renal pedicle clamping was performed in male C57BL/6 mice for short (15 min) or prolonged (45 min) time periods. Two and 24 h after experimental ischemia tissue injury labile heme levels in the kidney were determined with an apo-horseradish peroxidase assay. Moreover, renal injury, cytokines, and C5a and C3a receptor (C5aR, C3aR) expression were determined by histology, immunohistochemistry and qPCR, respectively. In addition, in vitro studies stimulating bone marrow-derived macrophages with LPS and the combination of LPS and heme were performed and cytokine expression was measured.Results: Inflammation and local tissue injury correlated with the duration of warm ischemia time. Labile heme concentrations in renal tissue were significantly higher after prolonged (45 min) as compared to short (15 min) IRI. Notably, expression of the inducible heme-degrading enzyme heme oxygenase-1 (HO-1) was up-regulated in kidneys after prolonged, but not after short IRI. C5aR, the pro-inflammatory cytokines IL-6 and TNF-α as well as pERK were up-regulated after prolonged, but not after short ischemia times. Consecutively, neutrophil infiltration and up-regulation of pro-fibrotic cytokines such as CTGF and PAI were more pronounced in prolonged IRI in comparison to short IRI. In vitro stimulation of macrophages with LPS revealed that IL-6 expression was enhanced in the presence of heme. Finally, administration of the heme scavenger human serum albumin (HSA) reduced the expression of pro-inflammatory cytokines, C3a receptor and improved tubular function indicated by enhanced alpha 1 microglobulin (A1M) absorption after IRI.Conclusions: Our data show that prolonged duration of warm ischemia time increased labile heme levels in the kidney, which correlates with IRI-dependent inflammation and up-regulation of anaphylatoxin receptor expression.

Abstract Resorptive activity of osteoclasts is important for maintaining bone homeostasis. Endogenous compounds such as oxidized low density lipoprotein (oxLDL) have been shown to disturb this activity. While some studies have investigated the effects of oxLDL on the process of osteoclastogenesis, the underlying mechanism are not fully understood. We show here that oxLDL concentrations of ~10–25 µg protein (0.43–1.0 µM MDA/mg protein) completely blocked the formation of functional osteoclasts. The underlying mechanism implies an inhibition of autophagy that in turn leads to a decreased fusion of cathepsin K (CatK)-loaded lysosomal vesicles with the ruffled border membrane. As result, a lower secretion of CatK and impaired protonation of the resorption lacunae by vacuolar-ATPase (v-ATPase) is observed in the presence of oxLDL. We demonstrate that scavenger receptor A (SR-A) mediates oxLDL effects on osteoclastogenesis and repressing this receptor partially rescued oxLDL effects. Collectively, our data provides an insight into the possible mechanism of oxLDL on osteoclastogenesis suggesting that it does not perturb the packaging of CatK and v-ATPase (V-a3) in the secretory lysosome, but inhibits the fusion of these lysosomes to the ruffled border. The relevance of our findings suggests a distinct link between oxLDL, autophagy and osteoclastogenesis.

Abstract Regeneration of arterial endothelium after injury is critical for the maintenance of normal blood flow, cell trafficking, and vascular function. Using mouse models of carotid injury, we show that the transition from a static to a dynamic phase of endothelial regeneration is marked by a strong increase in endothelial proliferation, which is accompanied by induction of the chemokine CX3CL1 in endothelial cells near the wound edge, leading to progressive recruitment of Ly6Clo monocytes expressing high levels of the cognate CX3CR1 chemokine receptor. In Cx3cr1‐deficient mice recruitment of Ly6Clo monocytes, endothelial proliferation and regeneration of the endothelial monolayer after carotid injury are impaired, which is rescued by acute transfer of normal Ly6Clo monocytes. Furthermore, human non‐classical monocytes induce proliferation of endothelial cells in co‐culture experiments in a VEGFA‐dependent manner, and monocyte transfer following carotid injury promotes endothelial wound closure in a hybrid mouse model in vivo. Thus, CX3CR1 coordinates recruitment of specific monocyte subsets to sites of endothelial regeneration, which promote endothelial proliferation and arterial regeneration.

Monocyte-to-macrophage differentiation results in the secretion of various inflammatory mediators and oxidative stress molecules necessary for atherosclerosis pathogenesis. Consequently, this differentiation represents a potential clinical target in atherosclerosis. Calcium dobesilate (CaD), an established vasoactive and angioprotective drug in experimental models of diabetic microvascular complications reduces oxidative stress and inhibits inflammation via diverse molecular targets; however, its effect on monocytes/macrophages is poorly understood. In this study, we investigated the anti-inflammatory mechanism of CaD during phorbol 12-myristate 13-acetate (PMA)-induced monocyte-to-macrophage differentiation in in vitro models of sepsis (LPS) and hyperglycemia, using THP-1 monocytic cell line. CaD significantly suppressed CD14, TLR4, and MMP9 expression and activity, lowering pro-inflammatory mediators, such as IL1β, TNFα, and MCP-1. The effects of CaD translated through to studies on primary human macrophages. CaD inhibited reactive oxygen species (ROS) generation, PKCδ, MAPK (ERK1/2 and p38) phosphorylation, NOX2/p47phox expression, and membrane translocation. We used hydrogen peroxide (H2O2) to mimic oxidative stress, demonstrating that CaD suppressed PKCδ activation via its ROS-scavenging properties. Taken together, we demonstrate for the first time that CaD suppresses CD14, TLR4, MMP9, and signature pro-inflammatory cytokines, in human macrophages, via the downregulation of PKCδ/NADPH oxidase/ROS/MAPK/NF-κB-dependent signaling pathways. Our data present novel mechanisms of how CaD alleviates metabolic and infectious inflammation.