Potential and Uncertainties of RejectClass in Acute Kidney Graft Dysfunction: An Independent Validation Study.

BACKGROUND: Kidney graft rejections are classified based on the Banff classification. The RejectClass algorithm, initially derived from a cohort comprising mostly protocol biopsies, identifies data-driven phenotypes of acute rejection and chronic pathology using Banff lesion scores. It also provides composite scores for inflammation activity and chronicity. This study independently evaluates the performance of RejectClass in a cohort consisting entirely of indication biopsies. METHODS: We retrospectively applied RejectClass to 441 patients from the German TRABIO (TRAnsplant BIOpsies) cohort who had received indication biopsies. The primary endpoint was death-censored graft failure during 2 y of follow-up. RESULTS: The application of RejectClass to our cohort demonstrated moderately comparable phenotypic features with the derivation cohort, and most clusters indicated an elevated risk of graft loss. However, the reproduction of all phenotypes and the associated risks of graft failure, as depicted in the original studies, was not fully accomplished. In contrast, adjusted Cox proportional hazards analyses substantiated that both the inflammation score and the chronicity score are independently associated with graft loss, exhibiting hazard ratios of 1.7 (95% confidence interval, 1.2-2.3; P = 0.002) and 2.2 (95% confidence interval, 1.8-2.6; P < 0.001), respectively, per 0.25-point increment (scale: 0.0-1.0). CONCLUSIONS: The composite inflammation and chronicity scores may already have direct utility in quantitatively assessing the disease stage. Further refinement and validation of RejectClass clusters are necessary to achieve more reliable and accurate phenotyping of rejection.

The Tacrolimus Concentration/Dose Ratio Does Not Predict Early Complications After Kidney Transplantation.

Early-on post kidney transplantation, there is a high risk of graft rejection and opportunistic viral infections. A low tacrolimus concentration/dose (C/D) ratio as a surrogate marker of fast tacrolimus metabolism has been established for risk stratification 3 months post-transplantation (M3). However, many adverse events occurring earlier might be missed, and stratification at 1 month post-transplantation (M1) has not been investigated. We retrospectively analyzed case data from 589 patients who had undergone kidney transplantation between 2011 and 2021 at three German transplant centers. Tacrolimus metabolism was estimated by use of the C/D ratio at M1, M3, M6, and M12. C/D ratios increased substantially during the year, particularly between M1 and M3. Many viral infections and most graft rejections occurred before M3. Neither at M1 nor at M3 was a low C/D ratio associated with susceptibility to BKV viremia or BKV nephritis. A low C/D ratio at M1 could not predict acute graft rejections or impaired kidney function, whereas at M3 it was significantly associated with subsequent rejections and impairment of kidney function. In summary, most rejections occur before M3, but a low C/D ratio at M1 does not identify patients at risk, limiting the predictive utility of this stratification approach.

Patient-focused outcomes after initiation of dialysis for ESRD: mortality, hospitalization and functional impairment.

BACKGROUND: Outcome data regarding clinically relevant endpoints after starting dialysis for end-stage renal disease (ESRD) are sparse, and early events after starting dialysis are particularly underestimated. The aim of this study was to describe patient-focused outcomes in ESRD patients starting from first dialysis. METHODS: The data basis for this retrospective observational study were anonymized healthcare data from Germany's largest statutory health insurer. We identified ESRD patients who initiated dialysis in 2017. Deaths, hospitalizations and occurrence of functional impairment within 4 years after starting dialysis were recorded starting from first treatment. Hazard ratios in dialysis patients compared with an age- and sex-matched reference population without dialysis were generated, stratified by age. RESULTS: The dialysis cohort included 10 328 ESRD patients who started dialysis in 2017. First dialysis was performed in-hospital for 7324 patients (70.9%), and 865 of these died during the same hospitalization. One-year mortality for ESRD patients initiating dialysis was 33.8%. Functional impairment occurred in 27.1% of patients, while 82.8% of patients required hospitalization within 1 year. Hazard ratios of dialysis patients compared with the reference population for mortality, functional impairment and hospitalization at 1-year were 8.6, 4.3 and 6.2. Dialysis patients <50 years were disproportionately affected, with >40-fold increased risk of adverse events compared with their peers. CONCLUSIONS: The emergence of morbidity and mortality after starting dialysis for ESRD is significant, especially in younger patients. Patients have a right to be informed about the prognosis associated with their condition.

High Macrophage Densities in Native Kidney Biopsies Correlate With Renal Dysfunction and Promote ESRD.

Introduction: Macrophages and monocytes are main players in innate immunity. The relevance of mononuclear phagocyte infiltrates on clinical outcomes remains to be determined in native kidney diseases. Methods: Our cross-sectional study included 324 patients with diagnostic renal biopsies comprising 17 disease entities and normal renal tissues for comparison. All samples were stained for CD68+ macrophages. Selected groups were further subtyped for CD14+ monocytes and CD163+ alternatively activated macrophages. Using precise pixel-based digital measurements, we quantified cell densities as positively stained areas in renal cortex and medulla as well as whole renal tissue. Laboratory and clinical data of all cases at the time of biopsy and additional follow-up data in 158 cases were accessible. Results: Biopsies with renal disease consistently revealed higher CD68+-macrophage densities and CD163+-macrophage densities in cortex and medulla compared to controls. High macrophage densities correlated with impaired renal function at biopsy and at follow-up in all diseases and in diseases analyzed separately. High cortical CD68+-macrophage densities preceded shorter renal survival, defined as requirement of permanent dialysis. CD14+ monocyte densities showed no difference compared to controls and did not correlate with renal function. Conclusion: Precise quantification of macrophage densities in renal biopsies may contribute to risk stratification to identify patients with high risk for end-stage renal disease (ESRD) and might be a promising therapeutic target in renal disease.

Epidemiology of haemophagocytic lymphohistiocytosis at the population level in Germany.

We retrospectively analysed all German inpatient cases of haemophagocytic lymphohistiocytosis (HLH) from 2014 to 2020 to describe the epidemiology, clinical course, and underlying diseases of 4065 HLH patients. The age-standardized incidence rate of HLH in Germany was 0.52/100 000 people in 2014 and steadily increased by 10% per year to 0.97/100 000 in 2020 (mean 0.70/100 000). Inpatient deaths related to HLH increased from 0.84/1 000 000 people in 2014 to 2.32/1 000 000 people in 2020, caused by rising numbers of older HLH patients. Overall, HLH is more frequent than previously expected and incidence as well as HLH-related deaths increased significantly.

The role of RHIM in necroptosis.

The RIP homotypic interaction motif (RHIM) is a conserved protein domain that is approximately 18-22 amino acids in length. In humans, four proteins carrying RHIM domains have been identified: receptor-interacting serine/threonine protein kinase (RIPK) 1, RIPK3, Z-DNA-binding protein 1 (ZBP1), and TIR domain-containing adapter-inducing IFN-ß (TRIF), which are all major players in necroptosis, a distinct form of regulated cell death. Necroptosis is mostly presumed to be a fail-safe form of cell death, occurring in cells in which apoptosis is compromised. Upon activation, RIPK1, ZBP1, and TRIF each hetero-oligomerize with RIPK3 and induce the assembly of an amyloid-like structure of RIPK3 homo-oligomers. These act as docking stations for the recruitment of the pseudokinase mixed-lineage kinase domain like (MLKL), the pore-forming executioner of necroptosis. As RHIM domain interactions are a vital component of the signaling cascade and can also be involved in apoptosis and pyroptosis activation, it is unsurprising that viral and bacterial pathogens have developed means of disrupting RHIM-mediated signaling to ensure survival. Moreover, as these mechanisms play an essential part of regulated cell death signaling, they have received much attention in recent years. Herein, we present the latest insights into the supramolecular structure of interacting RHIM proteins and their distinct signaling cascades in inflammation and infection. Their uncovering will ultimately contribute to the development of new therapeutic strategies in the regulation of lytic cell death.

Progress and Setbacks in Translating a Decade of Ferroptosis Research into Clinical Practice.

Ten years after its initial description, ferroptosis has emerged as the most intensely studied entity among the non-apoptotic forms of regulated cell death. The molecular features of ferroptotic cell death and its functional role have been characterized in vitro and in an ever-growing number of animal studies, demonstrating that it exerts either highly detrimental or, depending on the context, occasionally beneficial effects on the organism. Consequently, two contrary therapeutic approaches are being explored to exploit our detailed understanding of this cell death pathway: the inhibition of ferroptosis to limit organ damage in disorders such as drug-induced toxicity or ischemia-reperfusion injury, and the induction of ferroptosis in cancer cells to ameliorate anti-tumor strategies. However, the path from basic science to clinical utility is rocky. Emphasizing ferroptosis inhibition, we review the success and failures thus far in the translational process from basic research in the laboratory to the treatment of patients.

Evolving epidemiology of pneumocystis pneumonia: Findings from a longitudinal population-based study and a retrospective multi-center study in Germany.

Background: Pneumocystis pneumonia (PCP) is a life-threatening opportunistic infectious disease of immunocompromised patients. Its incidence has decreased worldwide in the past, but data concerning its recent epidemiology are lacking. Methods: We retrospectively analyzed all German inpatient cases from January 1, 2014 to December 31, 2019, to describe the recent epidemiology, incidence, clinical course, mortality and underlying diseases of PCP. Simultaneously, we conducted a retrospective multi-center study at two German university hospitals, and analyzed PCP cases treated there to gain deeper insights on the basis of primary patient data. Findings: The incidence of PCP significantly increased from 2·3 to 2·6 per 100,000 population from 2014 to 2019 (1,857 to 2,172 cases, +17·0%, p < 0·0001), as well as PCP-related deaths (516 to 615 cases, +19·2%, p = 0·011). The spectrum of underlying diseases changed: Risk groups with established chemoprophylaxis for PCP based on international guidelines (HIV, hematologic malignancies, and transplantation) showed a significant decrease in PCP cases and deaths. Others, especially those with solid malignancies, and autoimmune and pulmonary diseases showed a significant increase in case numbers and deaths. Data from the retrospective multi-center study added information regarding prophylaxis and diagnostics of PCP. Interpretation: The incidence of PCP has reversed its trend, showing a significant increase in mortality on population level. Patients who were not previously considered in prophylactic measures are increasingly affected by PCP. This development deserves further investigation, and additional comprehensive guidelines for the use of chemoprophylaxis in new risk groups are needed. Funding: Department of Nephrology and Hypertension, University Hospital Schleswig-Holstein, Kiel.

Vitamin K1 inhibits ferroptosis and counteracts a detrimental effect of phenprocoumon in experimental acute kidney injury.

Ferroptosis, a type of iron-dependent programmed cell death distinct from apoptosis, necroptosis, and other types of cell death, is characterized by lipid peroxidation, reactive oxygen species production, and mitochondrial dysfunction. Accumulating evidence has highlighted vital roles for ferroptosis in multiple diseases, including acute kidney injury. Therefore, ferroptosis has become a major focus for translational research. However, despite its involvement in pathological conditions, there are no pharmacologic inhibitors of ferroptosis in clinical use. In the context of drug repurposing, a strategy for identifying new uses for approved drugs outside the original medical application, we discovered that vitamin K1 is an efficient inhibitor of ferroptosis. Our findings are strengthened by the fact that the vitamin K antagonist phenprocoumon significantly exacerbated ferroptotic cell death in vitro and also massively worsened the course of acute kidney injury in vivo, which is of utmost clinical importance. We therefore assign vitamin K1 a novel role in preventing ferroptotic cell death in acute tubular necrosis during acute kidney injury. Since the safety, tolerability, pharmacokinetics, and pharmacodynamics of vitamin K1 formulations are well documented, this drug is primed for clinical application, and provides a new strategy for pharmacological control of ferroptosis and diseases associated with this mode of cell death.

Study protocol: the TRAnsplant BIOpsies (TRABIO) study - a prospective, observational, multicentre cohort study to assess the treatment of kidney graft rejections.

INTRODUCTION: Despite continued efforts, long-term outcomes of kidney transplantation remain unsatisfactory. Kidney graft rejections are independent risk factors for graft failure. At the participating centres of the TRAnsplant BIOpsies study group, a common therapeutic standard has previously been defined for the treatment of graft rejections. The outcomes of this strategy will be assessed in a prospective, observational cohort study. METHODS AND ANALYSIS: A total of 800 kidney transplantation patients will be enrolled who undergo a graft biopsy because of deteriorating kidney function. Patients will be stratified according to the Banff classification, and the influence of the treatment strategy on end points will be assessed using regression analysis. Primary end points will be all-cause mortality and graft survival. Secondary end points will be worsening of kidney function (≥30% decline of estimated Glomerular Filtration Rate [eGFR] or new-onset large proteinuria), recurrence of graft rejection and treatment response. Baseline data and detailed histopathology data will be entered into an electronic database on enrolment. During a first follow-up period (within 14 days) and subsequent yearly follow-ups (for 5 years), treatment strategies and clinical course will be recorded. Recruitment at the four participating centres started in September 2016. As of August 2020, 495 patients have been included. ETHICS AND DISSEMINATION: Ethical approval for the study has been obtained from the ethics committee of Kiel (AZ B 278/16) and was confirmed by the committees of Munich, Mainz and Stuttgart. The results will be reported in a peer-reviewed journal, according to the Strengthening the Reporting of Observational Studies in Epidemiology criteria. TRIAL REGISTRATION NUMBER: ISRCTN78772632; Pre-results.

TAT-RHIM: a more complex issue than expected.

Murine cytomegalovirus protein M45 contains a RIP homotypic interaction motif (RHIM) that is sufficient to confer protection of infected cells against necroptotic cell death. Mechanistically, the N-terminal region of M45 drives rapid self-assembly into homo-oligomeric amyloid fibrils, and interacts with the endogenous RHIM domains of receptor-interacting serine/threonine protein kinases (RIPK) 1, RIPK3, Z-DNA-binding protein 1, and Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-ß. Remarkably, all four aforementioned mammalian proteins harbouring such a RHIM domain are key components of inflammatory signalling and regulated cell death (RCD) processes. Immunogenic cell death by regulated necrosis causes extensive tissue damage in a wide range of diseases, including ischaemia reperfusion injury, myocardial infarction, sepsis, stroke, and solid organ transplantation. To harness the cell death suppression properties of M45 protein in a therapeutically usable manner, we developed a synthetic peptide encompassing only the RHIM domain of M45. To trigger delivery of RHIM into target cells, we fused the transactivator protein transduction domain of human immunodeficiency virus 1 to the N-terminus of the peptide. The fused peptide could efficiently penetrate eukaryotic cells, but unexpectedly it eradicated or destroyed all tested cancer cell lines and primary cells irrespective of species without further stimulus through a necrosis-like cell death. Typical inhibitors of different forms of RCD cannot impede this process, which appears to involve a direct disruption of biomembranes. Nevertheless, our finding has potential clinical relevance; reliable induction of a necrotic form of cell death distinct from all known forms of RCD may offer a novel therapeutic approach to combat resistant tumour cells.

Primidone blocks RIPK1-driven cell death and inflammation.

The receptor-interacting serine/threonine protein kinase 1 (RIPK1) is a key mediator of regulated cell death and inflammation. Recent studies suggest that RIPK1 inhibition would fundamentally improve the therapy of RIPK1-dependent organ damage in stroke, myocardial infarction, kidney failure, and systemic inflammatory response syndrome. Additionally, it could ameliorate or prevent multi-organ failure induced by cytokine release in the context of hyperinflammation, as seen in COVID-19 patients. Therefore, we searched for a RIPK1 inhibitor and present the aromatic antiepileptic and FDA-approved drug primidone (Liskantin®) as a potent inhibitor of RIPK1 activation in vitro and in a murine model of TNFα-induced shock, which mimics the hyperinflammatory state of cytokine release syndrome. Furthermore, we detected for the first time RIPK1 activation in the respiratory tract epithelium of hospitalized patients who tested positive for SARS-CoV-2 infection. Our data provide a strong rationale for evaluating the drug primidone in conditions of hyperinflammation in humans.

Evaluation of underidentification of potential organ donors in German hospitals.

BACKGROUND: Since 2010, the number of organ donations in Germany has decreased by one third, mostly due to undetected organ donors. It is unclear, how the undetected potential donor pool is distributed among the different German hospital categories (A = university hospital, B = hospitals with neurosurgery, C = hospitals without neurosurgery) and region types. METHODS: We performed a nationwide secondary data analysis of all German inpatient cases of the year 2016 (n = 20,063,689). All fatalities were regarded as potential organ donors, in which primary or secondary brain damage was encoded and organ donation was not excluded by a contraindication or a lack of ventilation therapy. RESULTS: In 2016, 28,087 potential organ donors were identified. Thereof 21% were found in category A, 28% in category B and 42% in category C hospitals. The contact rate (= organ donation related contacts/ potential organ donors) and realization rate (= realized organ donations/ potential organ donors) of category A, B and C hospitals was 10.6% and 4.6%, 10.9% and 4.8% and 6.0% and 1.7%, respectively. 58.2% of the donor potential of category C hospitals was found in the largest quartile of category C hospitals. 51% (n = 14,436) of the potential organ donors were treated in hospitals in agglomeration areas, 28% (n = 7,909) in urban areas and 21% (n = 5,742) in rural areas. The contact- and realization rate did not significantly differ between these areas. CONCLUSIONS: The largest proportion of potential organ donors and the lowest realization rate are found in category C hospitals. Reporting and donation practice do not differ between urban and rural regions.

Plasma Membrane Pores Drive Inflammatory Cell Death.

Necroptosis and pyroptosis are two forms of regulated cell death. They are executed by the proteins mixed-lineage kinase domain-like (MLKL) and gasdermin D (GSDMD), respectively. Once activated by numerous pathways, these proteins form membrane pores that allow the influx and efflux of various ions, proteins, and water, ultimately resulting in the death of the cell. These modalities of cell death are considered highly inflammatory because of the release of inflammatory cytokines and damage-associated molecular patterns, and are thereby not only deleterious for the dying cell itself, but also its environment or the entire organism. The relevance for these processes has been observed in various physiological and pathophysiological conditions, ranging from viral and bacterial infections over autoimmune and chronic inflammatory diseases to ischemic organ damage. In recent years, initial in vitro experiments have shed light on a range of connections between necroptosis and pyroptosis. Initial in vivo studies also indicate that, in many disease models, these two forms of cell death cannot be considered individually, as they demonstrate a complex interaction. In this article, we provide an overview of the currently known structure, pathways of activation, and functions of MLKL and GSDMD. With emerging evidence for an interconnection between necroptosis and pyroptosis in not only in vitro, but also in vivo models of disease, we highlight in particular the clinical relevance of the crosslinks between these two forms of inflammatory cell death and their implications for novel therapeutic strategies in a variety of diseases.

Regulatory cell therapy in kidney transplantation (The ONE Study): a harmonised design and analysis of seven non-randomised, single-arm, phase 1/2A trials.

BACKGROUND: Use of cell-based medicinal products (CBMPs) represents a state-of-the-art approach for reducing general immunosuppression in organ transplantation. We tested multiple regulatory CBMPs in kidney transplant trials to establish the safety of regulatory CBMPs when combined with reduced immunosuppressive treatment. METHODS: The ONE Study consisted of seven investigator-led, single-arm trials done internationally at eight hospitals in France, Germany, Italy, the UK, and the USA (60 week follow-up). Included patients were living-donor kidney transplant recipients aged 18 years and older. The reference group trial (RGT) was a standard-of-care group given basiliximab, tapered steroids, mycophenolate mofetil, and tacrolimus. Six non-randomised phase 1/2A cell therapy group (CTG) trials were pooled and analysed, in which patients received one of six CBMPs containing regulatory T cells, dendritic cells, or macrophages; patient selection and immunosuppression mirrored the RGT, except basiliximab induction was substituted with CBMPs and mycophenolate mofetil tapering was allowed. None of the trials were randomised and none of the individuals involved were masked. The primary endpoint was biopsy-confirmed acute rejection (BCAR) within 60 weeks after transplantation; adverse event coding was centralised. The RTG and CTG trials are registered with ClinicalTrials.gov, NCT01656135, NCT02252055, NCT02085629, NCT02244801, NCT02371434, NCT02129881, and NCT02091232. FINDINGS: The seven trials took place between Dec 11, 2012, and Nov 14, 2018. Of 782 patients assessed for eligibility, 130 (17%) patients were enrolled and 104 were treated and included in the analysis. The 66 patients who were treated in the RGT were 73% male and had a median age of 47 years. The 38 patients who were treated across six CTG trials were 71% male and had a median age of 45 years. Standard-of-care immunosuppression in the recipients in the RGT resulted in a 12% BCAR rate (expected range 3·2-18·0). The overall BCAR rate for the six parallel CTG trials was 16%. 15 (40%) patients given CBMPs were successfully weaned from mycophenolate mofetil and maintained on tacrolimus monotherapy. Combined adverse event data and BCAR episodes from all six CTG trials revealed no safety concerns when compared with the RGT. Fewer episodes of infections were registered in CTG trials versus the RGT. INTERPRETATION: Regulatory cell therapy is achievable and safe in living-donor kidney transplant recipients, and is associated with fewer infectious complications, but similar rejection rates in the first year. Therefore, immune cell therapy is a potentially useful therapeutic approach in recipients of kidney transplant to minimise the burden of general immunosuppression. FUNDING: The 7th EU Framework Programme.

Strategies for Improving Influenza Vaccination Rates in Patients with Chronic Renal Disease.

BACKGROUND: The influenza vaccination rate among older and chronically ill patients in Germany has declined in the past decade in spite of vaccination campaigns. METHODS: The influenza vaccination rate among persons with chronic renal disease was studied with the aid of billing data from various Associations of Statutory Health Insurance Physicians (Kassenärztliche Vereinigungen, ASHIPs) in Germany. It was tested in a randomized controlled trial whether a written vaccination appeal, sent by physicians to patients, led to an increase in the vaccination rate. It was tested in a further such trial whether the vaccination rate among patients with renal disease could be improved by an appeal for vaccination that was sent by the ASHIPs to the treating nephrologists. Finally, it was also tested in a prospective interventional study whether the vaccination rate could be improved by an appeal for vaccination sent by a health- insurance carrier directly to the patients. RESULTS: In 2012-2017, the vaccination rate among persons with chronically impaired renal function ranged from 41.1% to 46.9%; it ranged from 31.7% to 33.7% in kidney transplant recipients and from 42.7% to 44.7% in dialysis patients. An appeal for vaccination that was sent from physicians to patients raised the vaccination rate by 8.3% in the intervention group compared to the control group (p = 0.03; number needed to treat [NNT]: 13). On the other hand, an appeal for vaccination that was sent to the nephrologists lowered the vaccination rate by 0.8% in the intervention group compared to the control group. Finally, an appeal for vaccination that was sent by the health-insurance fund to the patients raised the vaccination rate by 3.2% (p<0.001; NNT: 32). CONCLUSION: Fewer than half of all patients with chronic renal failure in Germany are vaccinated against influenza. The vaccination rate was found to be increased only after an appeal for vaccination that was sent directly to the patients. A letter sent to the treating physicians had no positive effect at all.

Effect of Sodium Bicarbonate in Kidney Transplant Recipients With Chronic Metabolic Acidosis.

BACKGROUND: Metabolic acidosis (MA) is a common complication after kidney transplantation and regarded to increase mortality, graft failure, and bone fractures. Here, we conducted a retrospective cohort study to analyze the effect of sodium bicarbonate on those events. METHODS: All kidney transplant recipients of the German health insurance Allgemeine Ortskrankenkasse (AOK) were selected, who received their transplantation between 2007 and 2015. Three groups were formed: (1) control group (no acidosis, n = 3602), (2) acidosis group (encoded acidosis, n = 370), and (3) treatment group (encoded therapy, n = 769). The study endpoints were mortality, death-censored graft failure, and bone fractures. RESULTS: The prevalence of MA in the first year after transplantation was 46.2%. The 5-year patient and graft survival were 89.8% and 89.3% in the control group, 90% and 90.8% in the acidosis group, and 87.5% and 81.6% in the treatment group, respectively. The rate of bone fractures did not differ between the groups. Neither log-rank tests nor multivariable Cox regression analyses could detect a negative impact of MA on mortality (hazard ratio [HR] 0.94; confidence interval [CI] 0.67-1.30), graft failure (HR1.18; CI 0.82-1.72), or the incidence of bone fractures (HR1.19; CI 0.92-1.55). Treatment with sodium bicarbonate was associated with an increased risk of graft failure (HR1.52; CI 1.03-2.25), whereas mortality (HR0.86; CI 0.59-1.26) and the incidence of bone fractures (HR1.16; CI 0.86-1.56) were not altered. CONCLUSIONS: MA is common after kidney transplantation but not associated with an increased frequency of death, graft failure, or bone fractures. Conversely, sodium bicarbonate therapy increased the incidence of graft failure.

Combined Knockout of RIPK3 and MLKL Reveals Unexpected Outcome in Tissue Injury and Inflammation.

Necroptosis, initially identified as a backup cell death program when apoptosis is hindered, is a prominent feature in the etiology and progression of many human diseases, such as ischemic injury and sepsis. Receptor-interacting protein kinase 3 (RIPK3) is the cardinal regulator of this cell death modality, recruiting and phosphorylating the executioner mixed lineage kinase domain-like protein (MLKL) to signal necroptosis, which is terminated by a cellular plasma membrane rupture and the leakage of intracellular contents from dying cells. Experimental data to date indicate that RIPK3 and MLKL is the core machinery essential for all necroptotic cell death responses. By using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) technology, we showed that Ripk3 and Mlkl knockout and Ripk3/Mlkl double-knockout in necroptosis-sensitive cell lines extensively block susceptibility to necroptosis, in each case to an indistinguishable degree. In vivo studies using Ripk3- or Mlkl-deficient mice validated kidney ischemia reperfusion injury and high-dose tumor necrosis factor (TNF) availability, as druggable targets in necroptotic-mediated pathologies. Here, we demonstrated that Ripk3 or Mlkl-deficient mice are protected to a similar extent from kidney ischemia reperfusion injury and TNF-induced toxicity. Remarkably, in contrast to each single knockout, Ripk3/Mlkl double-deficient mice did not have appreciable protection from either of the above necroptotic-mediated pathologies. Paradoxically, the double-knockout mice resembled, in each case, the vulnerable wild-type mice, revealing novel complexities in the mechanisms of inflammation-driven diseases, due to aberrant cell death.

The anticonvulsive Phenhydan® suppresses extrinsic cell death.

Different forms of regulated cell death-like apoptosis and necroptosis contribute to the pathophysiology of clinical conditions including ischemia-reperfusion injury, myocardial infarction, sepsis, and multiple sclerosis. In particular, the kinase activity of the receptor-interacting serine/threonine protein kinase 1 (RIPK1) is crucial for cell fate in inflammation and cell death. However, despite its involvement in pathological conditions, no pharmacologic inhibitor of RIPK1-mediated cell death is currently in clinical use. Herein, we screened a collection of clinical compounds to assess their ability to modulate RIPK1-mediated cell death. Our small-scale screen identified the anti-epilepsy drug Phenhydan® as a potent inhibitor of death receptor-induced necroptosis and apoptosis. Accordingly, Phenhydan® blocked activation of necrosome formation/activation as well as death receptor-induced NF-κB signaling by influencing the membrane function of cells, such as lipid raft formation, thus exerting an inhibitory effect on pathophysiologic cell death processes. By targeting death receptor signaling, the already FDA-approved Phenhydan® may provide new therapeutic strategies for inflammation-driven diseases caused by aberrant cell death.