Transition from acute kidney injury to chronic kidney disease in a long-term murine model of Shiga toxin-induced hemolytic-uremic syndrome.

Introduction: Up to 40% of patients with typical hemolytic-uremic syndrome (HUS), characterized by microangiopathic hemolytic anemia and acute kidney injury (AKI), develop long-term consequences, most prominently chronic kidney disease (CKD). The transition from AKI to CKD, particularly in the context of HUS, is not yet fully understood. The objective of this study was to establish and characterize a Shiga toxin (Stx)-induced long-term HUS model to facilitate the study of mechanisms underlying the AKI-to-CKD transition. Methods: C57BL/6J mice were subjected to 5, 10, 15, or 20 ng/kg Stx on days 0, 3, and 6 of the experiment and were sacrificed on day 14 or day 21 to identify the critical time of turnover from the acute to the chronic state of HUS disease. Results: Acute disease, indicated by weight loss, plasma neutrophil gelatinase-associated lipocalin (NGAL) and urea, and renal neutrophils, diminished after 14 days and returned to sham level after 21 days. HUS-associated hemolytic anemia transitioned to non-hemolytic microcytic anemia along with unchanged erythropoietin levels after 21 days. Renal cytokine levels indicated a shift towards pro-fibrotic signaling, and interstitial fibrosis developed concentration-dependently after 21 days. While Stx induced the intrarenal invasion of pro-inflammatory M1 and pro-fibrotic M2 macrophages after 14 days, pro-fibrotic M2 macrophages were the dominant phenotype after 21 days. Conclusion: In conclusion, we established and characterized the first Stx-induced long-term model of HUS. This tool facilitates the study of underlying mechanisms in the early AKI-to-CKD transition following HUS and allows the testing of compounds that may protect patients with AKI from developing subsequent CKD.

The Role of the N-Terminal Domain of Thrombomodulin and the Potential of Recombinant Human Thrombomodulin as a Therapeutic Intervention for Shiga Toxin-Induced Hemolytic-Uremic Syndrome.

Hemolytic-uremic syndrome (HUS) is a rare complication of an infection with Shiga toxin (Stx)-producing Escherichia coli (STEC-HUS), characterized by severe acute kidney injury, thrombocytopenia and microangiopathic hemolytic anemia, and specific therapy is still lacking. Thrombomodulin (TM) is a multi-domain transmembrane endothelial cell protein and its N-terminal domain has been implicated in the pathophysiology of some cases of HUS. Indeed, the administration of recombinant human TM (rhTM) may have efficacy in HUS. We used a Stx-based murine model of HUS to characterize the role of the N-terminal domain of TM. We show that mice lacking that domain (TMLed (-/-)) are more sensitive to Stx, with enhanced HUS progression seen at 4 days and increased mortality at 7 days post-HUS induction. In spite of these changes, renal function was less affected in surviving Stx-challenged TMLed (-/-) mice compared to their wild-type counterparts TMLed (+/+) at 7 days. Contrary to few clinical case reports from Japan, the administration of rhTM (0.06 mg/kg) to wild-type mice (C57BL/6J) with HUS did not protect against disease progression. This overall promising, but also contradictory body of evidence, requires further systematic preclinical and clinical investigations to clarify the role of TM in HUS as a potential therapeutic strategy.

Deletion of Sphingosine Kinase 2 Attenuates Acute Kidney Injury in Mice with Hemolytic-Uremic Syndrome.

Typical hemolytic uremic syndrome (HUS) can occur as a severe systemic complication of infections with Shiga toxin (Stx)-producing Escherichia coli. Its pathology can be induced by Stx types, resulting in toxin-mediated damage to renal barriers, inflammation, and the development of acute kidney injury (AKI). Two sphingosine kinase (SphK) isozymes, SphK1 and SphK2, have been shown to be involved in barrier maintenance and renal inflammatory diseases. Therefore, we sought to determine their role in the pathogenesis of HUS. Experimental HUS was induced by the repeated administration of Stx2 in wild-type (WT) and SphK1 (SphK1-/-) or SphK2 (SphK2-/-) null mutant mice. Disease severity was evaluated by assessing clinical symptoms, renal injury and dysfunction, inflammatory status and sphingolipid levels on day 5 of HUS development. Renal inflammation and injury were found to be attenuated in the SphK2-/- mice, but exacerbated in the SphK1-/- mice compared to the WT mice. The divergent outcome appeared to be associated with oppositely altered sphingolipid levels. This study represents the first description of the distinct roles of SphK1-/- and SphK2-/- in the pathogenesis of HUS. The identification of sphingolipid metabolism as a potential target for HUS therapy represents a significant advance in the field of HUS research.

Cell-free DNA in patients with sepsis: long term trajectory and association with 28-day mortality and sepsis-associated acute kidney injury.

Introduction: Outcome-prediction in patients with sepsis is challenging and currently relies on the serial measurement of many parameters. Standard diagnostic tools, such as serum creatinine (SCr), lack sensitivity and specificity for acute kidney injury (AKI). Circulating cell-free DNA (cfDNA), which can be obtained from liquid biopsies, can potentially contribute to the quantification of tissue damage and the prediction of sepsis mortality and sepsis-associated AKI (SA-AKI). Methods: We investigated the clinical significance of cfDNA levels as a predictor of 28-day mortality, the occurrence of SA-AKI and the initiation of renal replacement therapy (RRT) in patients with sepsis. Furthermore, we investigated the long-term course of cfDNA levels in sepsis survivors at 6 and 12 months after sepsis onset. Specifically, we measured mitochondrial DNA (mitochondrially encoded NADH-ubiquinone oxidoreductase chain 1, mt-ND1, and mitochondrially encoded cytochrome C oxidase subunit III, mt-CO3) and nuclear DNA (nuclear ribosomal protein S18, n-Rps18) in 81 healthy controls and all available samples of 150 intensive care unit patients with sepsis obtained at 3 ± 1 days, 7 ± 1 days, 6 ± 2 months and 12 ± 2 months after sepsis onset. Results: Our analysis revealed that, at day 3, patients with sepsis had elevated levels of cfDNA (mt-ND1, and n-Rps18, all p<0.001) which decreased after the acute phase of sepsis. 28-day non-survivors of sepsis (16%) had higher levels of cfDNA (all p<0.05) compared with 28-day survivors (84%). Patients with SA-AKI had higher levels of cfDNA compared to patients without AKI (all p<0.05). Cell-free DNA was also significantly increased in patients requiring RRT (all p<0.05). All parameters improved the AUC for SCr in predicting RRT (AUC=0.88) as well as APACHE II in predicting mortality (AUC=0.86). Conclusion: In summary, cfDNA could potentially improve risk prediction models for mortality, SA-AKI and RRT in patients with sepsis. The predictive value of cfDNA, even with a single measurement at the onset of sepsis, could offer a significant advantage over conventional diagnostic methods that require repeated measurements or a baseline value for risk assessment. Considering that our data show that cfDNA levels decrease after the first insult, future studies could investigate cfDNA as a "memoryless" marker and thus bring further innovation to the complex field of SA-AKI diagnostics.

Bruton's tyrosine kinase inhibition attenuates disease progression by reducing renal immune cell invasion in mice with hemolytic-uremic syndrome.

Hemolytic-uremic syndrome (HUS) can occur as a complication of an infection with Shiga-toxin (Stx)-producing Escherichia coli. Patients typically present with acute kidney injury, microangiopathic hemolytic anemia and thrombocytopenia. There is evidence that Stx-induced renal damage propagates a pro-inflammatory response. To date, therapy is limited to organ-supportive strategies. Bruton's tyrosine kinase (BTK) plays a pivotal role in recruitment and function of immune cells and its inhibition was recently shown to improve renal function in experimental sepsis and lupus nephritis. We hypothesized that attenuating the evoked immune response by BTK-inhibitors (BTKi) ameliorates outcome in HUS. We investigated the effect of daily oral administration of the BTKi ibrutinib (30 mg/kg) and acalabrutinib (3 mg/kg) in mice with Stx-induced HUS at day 7. After BTKi administration, we observed attenuated disease progression in mice with HUS. These findings were associated with less BTK and downstream phospholipase-C-gamma-2 activation in the spleen and, subsequently, a reduced renal invasion of BTK-positive cells including neutrophils. Only ibrutinib treatment diminished renal invasion of macrophages, improved acute kidney injury and dysfunction (plasma levels of NGAL and urea) and reduced hemolysis (plasma levels of bilirubin and LDH activity). In conclusion, we report here for the first time that BTK inhibition attenuates the course of disease in murine HUS. We suggest that the observed reduction of renal immune cell invasion contributes - at least in part - to this effect. Further translational studies are needed to evaluate BTK as a potential target for HUS therapy to overcome currently limited treatment options.

Targeting the innate repair receptor axis via erythropoietin or pyroglutamate helix B surface peptide attenuates hemolytic-uremic syndrome in mice.

Hemolytic-uremic syndrome (HUS) can occur as a systemic complication of infections with Shiga toxin (Stx)-producing Escherichia coli and is characterized by microangiopathic hemolytic anemia and acute kidney injury. Hitherto, therapy has been limited to organ-supportive strategies. Erythropoietin (EPO) stimulates erythropoiesis and is approved for the treatment of certain forms of anemia, but not for HUS-associated hemolytic anemia. EPO and its non-hematopoietic analog pyroglutamate helix B surface peptide (pHBSP) have been shown to mediate tissue protection via an innate repair receptor (IRR) that is pharmacologically distinct from the erythropoiesis-mediating receptor (EPO-R). Here, we investigated the changes in endogenous EPO levels in patients with HUS and in piglets and mice subjected to preclinical HUS models. We found that endogenous EPO was elevated in plasma of humans, piglets, and mice with HUS, regardless of species and degree of anemia, suggesting that EPO signaling plays a role in HUS pathology. Therefore, we aimed to examine the therapeutic potential of EPO and pHBSP in mice with Stx-induced HUS. Administration of EPO or pHBSP improved 7-day survival and attenuated renal oxidative stress but did not significantly reduce renal dysfunction and injury in the employed model. pHBSP, but not EPO, attenuated renal nitrosative stress and reduced tubular dedifferentiation. In conclusion, targeting the EPO-R/IRR axis reduced mortality and renal oxidative stress in murine HUS without occurrence of thromboembolic complications or other adverse side effects. We therefore suggest that repurposing EPO for the treatment of patients with hemolytic anemia in HUS should be systematically investigated in future clinical trials.

Involvement of NF-κB1 and the Non-canonical NF-κB Signaling Pathway in the Pathogenesis of Acute Kidney Injury in Shiga-toxin-2-induced Hemolytic-uremic Syndrome in Mice.

ABSTRACT: The hemolytic-uremic syndrome (HUS) is a thrombotic microangiopathy which can occur as a severe systemic complication after an infection with Shiga-toxin-(Stx)-producing Escherichia coli (STEC). Elevated levels of proinflammatory cytokines associated with the classical nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) signaling pathway were detected in the urine of HUS patients. Thus, we hypothesize that the immune response of the infected organism triggered by Stx can affect the kidneys and contributes to acute kidney injury. Hitherto, the role of the classical and non-canonical NF-κB signaling pathway in HUS has not been evaluated systematically in vivo. We aimed to investigate in a murine model of Shiga toxin-induced HUS-like disease, whether one or both pathways are involved in the renal pathology in HUS. In kidneys of mice subjected to Stx or sham-treated mice, protein or gene expression analyses were performed to assess the expression of receptors activating the classical and non-canonical pathway, such as Fn14 and CD40, levels of NF-κB1/RelA and NF-κB2/RelB including its upstream signaling proteins, and expression of cytokines as target molecules of both pathways. In line with a higher expression of Fn14 and CD40, we detected an enhanced translocation of NF-κB1 and RelA as well as NF-κB2 and RelB into the nucleus accompanied by an increased gene expression of the NF-κB1-target cytokines Ccl20, Cxcl2, Ccl2, Cxcl1, IL-6, TNF-α, Cxcl10, and Ccl5, indicating an activation of the classical and non-canonical NF-κB pathway. Thereby, we provide, for the first time, in vivo evidence for an involvement of both NF-κB signaling pathways in renal pathophysiology of STEC-HUS.

Sphingosine-1-phosphate promotes barrier-stabilizing effects in human microvascular endothelial cells via AMPK-dependent mechanisms.

Breakdown of the endothelial barrier is a critical step in the development of organ failure in severe inflammatory conditions such as sepsis. Endothelial cells from different tissues show phenotypic variations which are often neglected in endothelial research. Sphingosine-1-phosphate (S1P) and AMP-dependent kinase (AMPK) have been shown to protect the endothelium and phosphorylation of AMPK by S1P was shown in several cell types. However, the role of the S1P-AMPK interrelationship for endothelial barrier stabilization has not been investigated. To assess the role of the S1P-AMPK signalling axis in this context, we established an in vitro model allowing real-time monitoring of endothelial barrier function in human microvascular endothelial cells (HMEC-1) and murine glomerular endothelial cells (GENCs) with the electric cell-substrate impedance sensing (ECIS™) system. Following the disruption of the cell barrier by co-administration of LPS, TNF-α, IL-1ß, IFN-γ, and IL-6, we demonstrated self-recovery of the disrupted barrier in HMEC-1, while the barrier remained compromised in GENCs. Under physiological conditions we observed a rapid phosphorylation of AMPK in HMEC-1 stimulated with S1P, but not in GENCs. Consistently, S1P enhanced the basal endothelial barrier in HMEC-1 exclusively. siRNA-mediated knockdown of AMPK in HMEC-1 led to a less pronounced barrier enhancement. Thus we present evidence for a functional role of AMPK in S1P-mediated barrier stabilization in HMEC-1 and we provide insight into cell-type specific differences of the S1P-AMPK-interrelationship, which might influence the development of interventional strategies targeting endothelial barrier dysfunction.

Modeling Hemolytic-Uremic Syndrome: In-Depth Characterization of Distinct Murine Models Reflecting Different Features of Human Disease.

Diarrhea-positive hemolytic-uremic syndrome (HUS) is a renal disorder that results from infections with Shiga-toxin (Stx)-producing Escherichia coli. The aim of this study was to establish well-defined refined murine models of HUS that can serve as preclinical tools to elucidate molecular mechanisms of disease development. C57BL/6J mice were subjected to different doses of Stx2 purified from an E. coli O157:H7 patient isolate. Animals received 300 ng/kg Stx2 and were sacrificed on day 3 to establish an acute model with fast disease progression. Alternatively, mice received 25 ng/kg Stx2 on days 0, 3, and 6, and were sacrificed on day 7 to establish a subacute model with moderate disease progression. Indicated by a rise in hematocrit, we observed dehydration despite volume substitution in both models, which was less pronounced in mice that underwent the 7-day regime. Compared with sham-treated animals, mice subjected to Stx2 developed profound weight loss, kidney dysfunction (elevation of plasma urea, creatinine, and neutrophil gelatinase-associated lipocalin), kidney injury (tubular injury and loss of endothelial cells), thrombotic microangiopathy (arteriolar microthrombi), and hemolysis (elevation of plasma bilirubin, lactate dehydrogenase, and free hemoglobin). The degree of complement activation (C3c deposition), immune cell invasion (macrophages and T lymphocytes), apoptosis, and proliferation were significantly increased in kidneys of mice subjected to the 7-day but not in kidneys of mice subjected to the 3-day regime. However, glomerular and kidney volume remained mainly unchanged, as assessed by 3D analysis of whole mount kidneys using CD31 staining with light sheet fluorescence microscopy. Gene expression analysis of kidneys revealed a total of only 91 overlapping genes altered in both Stx2 models. In conclusion, we have developed two refined mouse models with different disease progression, both leading to hemolysis, thrombotic microangiopathy, and acute kidney dysfunction and damage as key clinical features of human HUS. While intrarenal changes (apoptosis, proliferation, complement deposition, and immune cell invasion) mainly contribute to the pathophysiology of the subacute model, prerenal pathomechanisms (hypovolemia) play a predominant role in the acute model. Both models allow the further study of the pathomechanisms of most aspects of human HUS and the testing of distinct novel treatment strategies.

5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation.

Leukotrienes (LTs) are proinflammatory lipid mediators formed from arachidonic acid in a 2-step reaction catalyzed by 5-lipoxygenase (5-LOX) requiring the formation of 5-HPETE [5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid] and its subsequent transformation to LTA4 5-LOX is thought to receive arachidonic acid from the nuclear membrane-embedded 5-LOX-activating protein (FLAP). The crystal structure of 5-LOX revealed an active site concealed by F177 and Y181 (FY cork). We examined the influence of the FY cork on 5-LOX activity and membrane binding in HEK293 cells in the absence and presence of FLAP. Uncapping the 5-LOX active site by mutation of F177 and/or Y181 to alanine (5-LOX-F177A, 5-LOX-Y181A, 5-LOX-F177/Y181A) resulted in delayed and diminished 5-LOX membrane association in A23187-stimulated cells. For 5-LOX-F177A and 5-LOX-F177/Y181A, formation of 5-LOX products was dramatically reduced relative to 5-LOX-wild type (wt). Strikingly, coexpression of FLAP in A23187-activated HEK293 cells effectively restored formation of 5-H(p)ETE (5-hydroxy- and 5-peroxy-6-trans-8,11,14-cis-eicosatetraenoic acid) by these same 5-LOX mutants (≈60-70% 5-LOX-wt levels) but not of LTA4 hydrolysis products. Yet 5-LOX-Y181A generated 5-H(p)ETE at levels comparable to 5-LOX-wt but reduced LTA4 hydrolysis products. Coexpression of FLAP partially restored LTA4 hydrolysis product formation by 5-LOX-Y181A. Together, the data suggest that the concealed FY cork impacts membrane association and that FLAP may help shield an uncapped active site.-Gerstmeier, J., Newcomer, M. E., Dennhardt, S., Romp, E., Fischer, J., Werz, O., Garscha, U. 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation.

Ca(2+)/calmodulin regulates Kvß1.1-mediated inactivation of voltage-gated K(+) channels.

A-type K(+) channels open on membrane depolarization and undergo subsequent rapid inactivation such that they are ideally suited for fine-tuning the electrical signaling in neurons and muscle cells. Channel inactivation mostly follows the so-called ball-and-chain mechanism, in which the N-terminal structures of either the K(+) channel's α or ß subunits occlude the channel pore entry facing the cytosol. Inactivation of Kv1.1 and Kv1.4 channels induced by Kvß1.1 subunits is profoundly decelerated in response to a rise in the intracellular Ca(2+) concentration, thus making the affected channel complexes negative feedback regulators to limit neuronal overexcitation. With electrophysiological and biochemical experiments we show that the Ca(2+) dependence is gained by binding of calmodulin to the "chain" segment of Kvß1.1 thereby compromising the mobility of the inactivation particle. Furthermore, inactivation regulation via Ca(2+)/calmodulin does not interfere with the ß subunit's enzymatic activity as an NADPH-dependent oxidoreductase, thus rendering the Kvß1.1 subunit a multifunctional receptor that integrates cytosolic signals to be transduced to altered electrical cellular activity.

Design, synthesis and evaluation of semi-synthetic triazole-containing caffeic acid analogues as 5-lipoxygenase inhibitors.

In this work the synthesis, structure-activity relationship (SAR) and biological evaluation of a novel series of triazole-containing 5-lipoxygenase (5-LO) inhibitors are described. The use of structure-guided drug design techniques provided compounds that demonstrated excellent 5-LO inhibition with IC50 of 0.2 and 3.2 µm in cell-based and cell-free assays, respectively. Optimization of binding and functional potencies resulted in the identification of compound 13d, which showed an enhanced activity compared to the parent bioactive compound caffeic acid 5 and the clinically approved zileuton 3. Compounds 15 and 16 were identified as lead compounds in inhibiting 5-LO products formation in neutrophils. Their interference with other targets on the arachidonic acid pathway was also assessed. Cytotoxicity tests were performed to exclude a relationship between cytotoxicity and the increased activity observed after structure optimization.