Multi-omics and imaging mass cytometry characterization of human kidneys to identify pathways and phenotypes associated with impaired kidney function.

Despite the recent advances in our understanding of the role of lipids, metabolites, and related enzymes in mediating kidney injury, there is limited integrated multi-omics data identifying potential metabolic pathways driving impaired kidney function. The limited availability of kidney biopsies from living donors with acute kidney injury has remained a major constraint. Here, we validated the use of deceased transplant donor kidneys as a good model to study acute kidney injury in humans and characterized these kidneys using imaging and multi-omics approaches. We noted consistent changes in kidney injury and inflammatory markers in donors with reduced kidney function. Neighborhood and correlation analyses of imaging mass cytometry data showed that subsets of kidney cells (proximal tubular cells and fibroblasts) are associated with the expression profile of kidney immune cells, potentially linking these cells to kidney inflammation. Integrated transcriptomic and metabolomic analysis of human kidneys showed that kidney arachidonic acid metabolism and seven other metabolic pathways were upregulated following diminished kidney function. To validate the arachidonic acid pathway in impaired kidney function we demonstrated increased levels of cytosolic phospholipase A2 protein and related lipid mediators (prostaglandin E2) in the injured kidneys. Further, inhibition of cytosolic phospholipase A2 reduced injury and inflammation in human kidney proximal tubular epithelial cells in vitro. Thus, our study identified cell types and metabolic pathways that may be critical for controlling inflammation associated with impaired kidney function in humans.

Spatially resolved multiomics of human cardiac niches.

The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system1. The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug-target interactions to provide mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG+ and IgA+ plasma cells forming immune niches that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs.

Targeting succinate metabolism to decrease brain injury upon mechanical thrombectomy treatment of ischemic stroke.

Current treatments for acute ischemic stroke aim to reinstate a normal perfusion in the ischemic territory but can also cause significant ischemia-reperfusion (IR) injury. Previous data in experimental models of stroke show that ischemia leads to the accumulation of succinate, and, upon reperfusion, the accumulated succinate is rapidly oxidized by succinate dehydrogenase (SDH) to drive superoxide production at mitochondrial complex I. Despite this process initiating IR injury and causing further tissue damage, the potential of targeting succinate metabolism to minimize IR injury remains unexplored. Using both quantitative and untargeted high-resolution metabolomics, we show a time-dependent accumulation of succinate in both human and mouse brain exposed to ischemia ex vivo. In a mouse model of ischemic stroke/mechanical thrombectomy mass spectrometry imaging (MSI) shows that succinate accumulation is confined to the ischemic region, and that the accumulated succinate is rapidly oxidized upon reperfusion. Targeting succinate oxidation by systemic infusion of the SDH inhibitor malonate upon reperfusion leads to a dose-dependent decrease in acute brain injury. Together these findings support targeting succinate metabolism upon reperfusion to decrease IR injury as a valuable adjunct to mechanical thrombectomy in ischemic stroke.

Mechanism of succinate efflux upon reperfusion of the ischaemic heart.

AIMS: Succinate accumulates several-fold in the ischaemic heart and is then rapidly oxidized upon reperfusion, contributing to reactive oxygen species production by mitochondria. In addition, a significant amount of the accumulated succinate is released from the heart into the circulation at reperfusion, potentially activating the G-protein-coupled succinate receptor (SUCNR1). However, the factors that determine the proportion of succinate oxidation or release, and the mechanism of this release, are not known. METHODS AND RESULTS: To address these questions, we assessed the fate of accumulated succinate upon reperfusion of anoxic cardiomyocytes, and of the ischaemic heart both ex vivo and in vivo. The release of accumulated succinate was selective and was enhanced by acidification of the intracellular milieu. Furthermore, pharmacological inhibition, or haploinsufficiency of the monocarboxylate transporter 1 (MCT1) significantly decreased succinate efflux from the reperfused heart. CONCLUSION: Succinate release upon reperfusion of the ischaemic heart is mediated by MCT1 and is facilitated by the acidification of the myocardium during ischaemia. These findings will allow the signalling interaction between succinate released from reperfused ischaemic myocardium and SUCNR1 to be explored.

COVID-19 and pneumothorax: a multicentre retrospective case series.

INTRODUCTION: Pneumothorax and pneumomediastinum have both been noted to complicate cases of coronavirus disease 2019 (COVID-19) requiring hospital admission. We report the largest case series yet described of patients with both these pathologies (including nonventilated patients). METHODS: Cases were collected retrospectively from UK hospitals with inclusion criteria limited to a diagnosis of COVID-19 and the presence of either pneumothorax or pneumomediastinum. Patients included in the study presented between March and June 2020. Details obtained from the medical record included demographics, radiology, laboratory investigations, clinical management and survival. RESULTS: 71 patients from 16 centres were included in the study, of whom 60 had pneumothoraces (six with pneumomediastinum in addition) and 11 had pneumomediastinum alone. Two of these patients had two distinct episodes of pneumothorax, occurring bilaterally in sequential fashion, bringing the total number of pneumothoraces included to 62. Clinical scenarios included patients who had presented to hospital with pneumothorax, patients who had developed pneumothorax or pneumomediastinum during their inpatient admission with COVID-19 and patients who developed their complication while intubated and ventilated, either with or without concurrent extracorporeal membrane oxygenation. Survival at 28 days was not significantly different following pneumothorax (63.1±6.5%) or isolated pneumomediastinum (53.0±18.7%; p=0.854). The incidence of pneumothorax was higher in males. 28-day survival was not different between the sexes (males 62.5±7.7% versus females 68.4±10.7%; p=0.619). Patients aged ≥70 years had a significantly lower 28-day survival than younger individuals (≥70 years 41.7±13.5% survival versus <70 years 70.9±6.8% survival; p=0.018 log-rank). CONCLUSION: These cases suggest that pneumothorax is a complication of COVID-19. Pneumothorax does not seem to be an independent marker of poor prognosis and we encourage continuation of active treatment where clinically possible.

Targeting succinate dehydrogenase with malonate ester prodrugs decreases renal ischemia reperfusion injury.

Renal ischemia reperfusion (IR) injury leads to significant patient morbidity and mortality, and its amelioration is an urgent unmet clinical need. Succinate accumulates during ischemia and its oxidation by the mitochondrial enzyme succinate dehydrogenase (SDH) drives the ROS production that underlies IR injury. Consequently, compounds that inhibit SDH may have therapeutic potential against renal IR injury. Among these, the competitive SDH inhibitor malonate, administered as a cell-permeable malonate ester prodrug, has shown promise in models of cardiac IR injury, but the efficacy of malonate ester prodrugs against renal IR injury have not been investigated. Here we show that succinate accumulates during ischemia in mouse, pig and human models of renal IR injury, and that its rapid oxidation by SDH upon reperfusion drives IR injury. We then show that the malonate ester prodrug, dimethyl malonate (DMM), can ameliorate renal IR injury when administered at reperfusion but not prior to ischemia in the mouse. Finally, we show that another malonate ester prodrug, diacetoxymethyl malonate (MAM), is more potent than DMM because of its faster esterase hydrolysis. Our data show that the mitochondrial mechanisms of renal IR injury are conserved in the mouse, pig and human and that inhibition of SDH by 'tuned' malonate ester prodrugs, such as MAM, is a promising therapeutic strategy in the treatment of clinical renal IR injury.

Succinate accumulation drives ischaemia-reperfusion injury during organ transplantation.

During heart transplantation, storage in cold preservation solution is thought to protect the organ by slowing metabolism; by providing osmotic support; and by minimising ischaemia-reperfusion (IR) injury upon transplantation into the recipient1,2. Despite its widespread use our understanding of the metabolic changes prevented by cold storage and how warm ischaemia leads to damage is surprisingly poor. Here, we compare the metabolic changes during warm ischaemia (WI) and cold ischaemia (CI) in hearts from mouse, pig, and human. We identify common metabolic alterations during WI and those affected by CI, thereby elucidating mechanisms underlying the benefits of CI, and how WI causes damage. Succinate accumulation is a major feature within ischaemic hearts across species, and CI slows succinate generation, thereby reducing tissue damage upon reperfusion caused by the production of mitochondrial reactive oxygen species (ROS)3,4. Importantly, the inevitable periods of WI during organ procurement lead to the accumulation of damaging levels of succinate during transplantation, despite cooling organs as rapidly as possible. This damage is ameliorated by metabolic inhibitors that prevent succinate accumulation and oxidation. Our findings suggest how WI and CI contribute to transplant outcome and indicate new therapies for improving the quality of transplanted organs.

Efficient human cytomegalovirus reactivation is maturation dependent in the Langerhans dendritic cell lineage and can be studied using a CD14+ experimental latency model.

Studies from a number of laboratories have shown that the myeloid lineage is prominent in human cytomegalovirus (HCMV) latency, reactivation, dissemination, and pathogenesis. Existing as a latent infection in CD34(+) progenitors and circulating CD14(+) monocytes, reactivation is observed upon differentiation to mature macrophage or dendritic cell (DC) phenotypes. Langerhans' cells (LCs) are a subset of periphery resident DCs that represent a DC population likely to encounter HCMV early during primary infection. Furthermore, we have previously shown that CD34(+) derived LCs are a site of HCMV reactivation ex vivo. Accordingly, we have utilized healthy-donor CD34(+) cells to study latency and reactivation of HCMV in LCs. However, the increasing difficulty acquiring healthy-donor CD34(+) cells--particularly from seropositive donors due to the screening regimens used--led us to investigate the use of CD14(+) monocytes to generate LCs. We show here that CD14(+) monocytes cultured with transforming growth factor ß generate Langerin-positive DCs (MoLCs). Consistent with observations using CD34(+) derived LCs, only mature MoLCs were permissive for HCMV infection. The lytic infection of mature MoLCs is productive and results in a marked inhibition in the capacity of these cells to promote T cell proliferation. Pertinently, differentiation of experimentally latent monocytes to the MoLC phenotype promotes reactivation in a maturation and interleukin-6 (IL-6)-dependent manner. Intriguingly, however, IL-6-mediated effects were restricted to mature LCs, in contrast to observations with classical CD14(+) derived DCs. Consequently, elucidation of the molecular basis behind the differential response of the two DC subsets should further our understanding of the fundamental mechanisms important for reactivation.