key: cord-0748034-9s9ctu9i authors: Alexander, Mariam P.; Mangalaparthi, Kiran K.; Madugundu, Anil K.; Moyer, Ann M.; Adam, Benjamin A.; Mengel, Michael; Singh, Smrita; Herrmann, Sandra M.; Rule, Andrew D.; Cheek, E. Heidi; Herrera Hernandez, Loren P.; Graham, Rondell P.; Denic, Aleksander; Aubry, Marie-Christine; Roden, Anja C.; Hagen, Catherine E.; Quinton, Reade A.; Bois, Melanie C.; Lin, Peter T.; Maleszewski, Joseph J.; Cornell, Lynn D.; Sethi, Sanjeev; Pavelko, Kevin D.; Charlesworth, Jon; Narasimhan, Ramya; Larsen, Christopher P.; Rizza, Stacey A.; Nasr, Samih H.; Grande, Joseph P.; McKee, Trevor D.; Badley, Andrew D.; Pandey, Akhilesh; Taner, Timucin title: Acute kidney injury in severe COVID-19 has similarities to sepsis-associated kidney injury- a multi-Omics study date: 2021-07-15 journal: Mayo Clin Proc DOI: 10.1016/j.mayocp.2021.07.001 sha: d157e3e4b5f614f272fac181cd76a7c395041736 doc_id: 748034 cord_uid: 9s9ctu9i Objective To compare COVID-19 acute kidney injury (AKI) to sepsis-AKI (S-AKI) the morphology, transcriptomic and proteomic characteristics of autopsy kidneys were analyzed. Patients and methods Individuals 18 years and older who died from COVID-19 and had an autopsy performed at Mayo Clinic between April 2020 to October 2020 were included. Morphological evaluation of the kidneys of 17 individuals with COVID-19 was performed. In a subset of 7 COVID-19 cases with post-mortem interval of ≤20 hours, ultrastructural and molecular characteristics (targeted transcriptome & proteomics analyses of tubulointerstitium) were evaluated. Molecular characteristics were compared to archived cases of S-AKI and non-sepsis causes of AKI (NS-AKI). Results The spectrum of COVID-19 renal pathology included macrophage dominant microvascular inflammation (glomerulitis and peritubular capillaritis), vascular dysfunction (peritubular capillary congestion & endothelial injury), tubular injury with ultrastructural evidence of mitochondrial damage. Investigation of the spatial architecture using a novel imaging mass cytometry revealed enrichment of CD3+CD4+ T cells in close proximity to antigen-presenting cells, and macrophage-enriched glomerular and interstitial infiltrates, suggesting an innate and adaptive immune tissue response. COVID-19 AKI and S-AKI, as compared to NS-AKI, had an enrichment of transcriptional pathways involved in inflammation (apoptosis, autophagy, MHC class I and II, and Th1 differentiation). Proteomic pathway analysis demonstrated that COVID-19 AKI & to a lesser extent S-AKI was enriched in necroptosis and sirtuin signaling pathways, both involved in regulatory response to inflammation. Upregulation of ceramide signaling pathway and downregulation of oxidative phosphorylation in COVID-19 AKI was noted. Conclusions This data highlights the similarities between S-AKI and COVID-19 AKI and suggests that mitochondrial dysfunction may play a pivotal role in COVID-19 AKI. This data may allow the development of novel diagnostic and therapeutic targets. Infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease-2019 (COVID- 19) , and is associated with acute kidney injury (AKI) in more than a third of hospitalized patients. 1 The AKI is often moderate to severe, and is an independent risk factor for in-hospital mortality. 2, 3 Acute tubular injury is considered the pathological hallmark of COVID-19 kidney disease as demonstrated in both native and autopsy renal studies. 4, [5] [6] [7] Microthrombi, described in heart and lungs 8, [9] [10] [11] [12] is infrequently reported in kidneys. 5, 13 Similarly, although severe COVID-19 is associated with a systemic inflammatory response and inflammation in both lungs and heart, little is known about the immune response in the kidneys. Direct viral toxicity, hemodynamic instability, cytokine storm and immune mediated injury have all been proposed as likely causes of renal injury in COVID- 19. 7,14,15 Recent studies have drawn similarities between sepsis-associated AKI (S-AKI) and COVID-19 related kidney injury. 16, 17 To date, reports evaluating the renal pathology in COVID-19 have largely focused on light microscopy and ultrastructural studies; molecular studies have been limited. Herein, we present a series of COVID-19 patients that underwent post-mortem examination, with emphasis on renal pathology findings. Our study sought to; (a) describe the pathological spectrum of COVID-19-associated renal injury; (b) characterize the molecular profile of COVID-19 associated AKI as compared to S-AKI and NS-AKI to elucidate similarities and differences in underlying pathophysiology. J o u r n a l P r e -p r o o f All cases of COVID-19, as confirmed by antemortem SARS-CoV-2 nasopharyngeal or oropharyngeal polymerase chain reaction (PCR), undergoing research-consented postmortem examination at Mayo Clinic (Rochester, MN) between April 2 and September 9, 2020, were included in the study. Salient information for all cases was abstracted from the electronic medical record, including demographic information, disease history (if available), and the presence or absence of chronic underlying conditions. Two autopsy-derived control cohorts with AKI were selected for comparative purposes; those who died of bronchopneumonia-associated sepsis (S-AKI, n=7) and those who died with AKI not secondary to sepsis or COVID-19 (NS-AKI, n=7). The study was approved by and conducted according to the requirements of the Institutional Review Board at Mayo Clinic. A detailed light microscopic evaluation of all 17 autopsies was performed by two renal pathologists (MPA and LPH). Tissue sections were stained with hematoxylin and eosin, PAS, Trichrome, and Jones Methenamine Silver stains. In addition, immunohistochemical evaluation with CD68, CD3, and CD20 to evaluate macrophages, T cells, and B cells was performed in all 17 autopsies. Detailed methods are provided in the Supplementary Appendix. Light microscopy analysis was also performed in the two control cohorts. In a subset of 7 COVID-19 cases that had postmortem interval (< 20hours) and only mild autolysis, ultrastructural studies, immunohistochemical evaluation for SARS-CoV-2 antigens and in-situ hybridization for viral RNA were also performed. A high-parameter image mass cytometry using 23 different molecular markers was performed on the COVID-19 cases. The detailed methods, including the markers, are provided in the Supplementary Appendix. Tissue from the well preserved 7 renal autopsy blocks from COVID-19 cases, as well as the two control groups (n=7 each) were analyzed by NanoString platform using the nCounter Human Organ Transplant Panel that profiles expression of 770 genes associated with different pathways related to immune response and tissue injury. Proteomics of the 7 cases each in three groups was performed following laser capture microdissection of the tubulointerstitial compartment excluding any glomeruli and or regions of interstitial fibrosis or tubular atrophy. Apoptosis in each group was assessed by TUNEL assay. Post-normalization gene expression analysis was performed using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria). Functional annotation gene sets were derived using NanoString-provided, literature-based annotations (available here: https://www.nanostring.com/products/ncounter-assays-panels/immunology/human-organtransplant/). Functional annotation gene set expression was determined using the z-scores of the mean normalized counts of the constituent genes. Gene set z-scores were compared between sample groups using Student's t-test. The top relatively upregulated and downregulated individual genes within each sample group (versus all other sample groups) were determined using fold change and Student's t-test. In case of quantitative proteomics data, TMT channel intensities of identified proteins were used for statistical analysis using Python 3.6.5. Channel intensities were log2 transformed and median normalized prior to applying J o u r n a l P r e -p r o o f independent two-tailed T-test. P-values were corrected for multiple hypotheses testing using Benjamini-Hochberg method. Differentially expressed proteins (p <0.05) were analyzed using QIAGEN's Ingenuity Pathway Analysis software (IPA, QIAGEN Redwood City, CA, USA) to identify altered signaling pathways. The significance values (p-value of overlap) for the canonical pathways were calculated by the right-tailed Fisher's exact test. We used nonparametric rank-sum test to compare the groups, because of small number of cases per group. All statistical analyses were performed using JMP (SAS Institute, Cary, NC; version 14.1). Pvalues <0.05 were considered to be statistically significant. The demographics of the 17 COVID-19 autopsy cases are provided in Table 1 . The cohort had a median age of 78 years (range, 29-94 years), and 14 (82%) were older than 70 years. There were 15 men, and 11 self-identified as White (64%). Three (18%) additional patients selfidentified as Asian, none as Black. The time spent in the hospital varied widely, with 12 (71%) having been hospitalized for 5 or more days prior to death. Frequent and notable comorbidities The kidney autopsy findings are characterized in Table 2 . Renal pathology was characterized by moderate-to-severe acute tubular injury in all ( Figure 1A) . Most showed marked simplification of the lining epithelium and luminal ectasia. Myoglobin casts were observed in two cases, and osmotic tubulopathy in one. Tubulointerstitial inflammation was mild with macrophage and CD3 positive T-cell dominance. Peritubular capillaritis, with a mixed T-cell and macrophage immunophenotype was seen in 13 (76%) (Figure 1B-F) . Similarly, glomerulitis, with a mixed T-cell and macrophage immunophenotype was seen in the same13 (76%). The four patients who did not show microvascular inflammation had been treated with a combination of remdesivir and either dexamethasone (2) or convalescent plasma (2) . Patients treated with dexamethasone alone or remdesivir alone had microvascular inflammation. Neutrophilic microvascular inflammation and interstitial infiltration was not a conspicuous feature in any of the cases. Non-immune glomerular pathology included nodular diabetic glomerulosclerosis in 1(6%); whereas collapsing FSGS or acute thrombotic microangiopathy was not identified. Marked peritubular capillary congestion was noted in all cases (Figure 1 B and C) . Table 3 . The mean age of the groups was 78, 71 and 74 (p=0.6). There were more women in the sepsis-associated AKI group than in either of the other two groups. The mean terminal serum creatinine was 3.0 mg/dl, 1.6 and 1.3 mg/dL respectively (p=0.32). All three groups had similar numbers of co-morbidities and were comparable with respect to chronic microstructural changes in the renal parenchyma. Microvascular inflammation (glomerulitis and peritubular capillaritis) was more prominent in patients with COVID-19 (5/7) and S-AKI (7/7), as compared to NS-AKI controls (0/7). Mononuclear inflammatory cells were the predominant inflammatory cell type in both, while neutrophils were noted only infrequently, and only in S-AKI. Parenchymal tubulointerstitial inflammation was patchy, mild (3/7) or moderate (4/7) and largely mononuclear in COVID-19 and S-AKI, as compared to minimal inflammation in the NS-AKI controls (7/7). On ultrastructural evaluation of Covid-19 AKI, there was evidence of tubular injury with cytoplasmic vacuolization, loss of brush border and mitochondrial injury. The latter was characterized by swelling, vacuolization and distortion of cristae. Condensed mitochondria and distorted circular mitochondriae were identified. Flocculent densities, and large vesicles with J o u r n a l P r e -p r o o f abundant clathrin-coated vesicles resembling viral particles were also identified (Figure 2A-D) Tubulo-reticular inclusions were not seen in the endothelial cytoplasm of COVID-19 cases. Peritubular capillary endothelial injury was noted ( Figure 2E) . Multi-layering of the glomerular and peritubular capillary basement membranes, features of more chronic endothelial injury, was not appreciated. Apoptosis of tubular epithelial cells was more common in COVID-19 (3.1/hpf) and S-AKI (4.1/hpf) as compared to NS-AKI controls (0.7/hpf) (p<0.001) (Figure 2F-G) . SARS-CoV-2 immunohistochemistry and in situ-hybridization studies on kidney sections were negative in all 7 COVID-19 cases. Definitive viral inclusions were not identified on ultrastructural studies of podocytes, endothelium, or tubules. To define the phenotype of cellular infiltrates and the spatial architecture of AKI in COVID-19 kidneys, high-parameter imaging mass cytometry to detect 23 cell surface markers was performed on a Hyperion imaging system coupled to a Helios time-of-flight mass cytometer (CyTOF) ( Table S1) conducted for all the proteins found in the three groups and a heat map was generated ( Figure 5A ). Ingenuity Pathway Analysis demonstrated that COVID-19 AKI kidneys were enriched in proteins involved in the sirtuin and necroptosis signaling pathways, both involved in regulatory response to inflammation Table S2 . Ceramide signaling pathway upregulation was unique in COVID-19 kidneys when compared to NS-AKI, whereas oxidative phosphorylation was downregulated in COVID-19 AKI (Figure 5B ). Our study examined the morphologic and molecular characteristics of kidneys obtained during autopsies of patients who died of COVID-19. The morphological and molecular profile of severe COVID-19 renal injury resembles S-AKI. 19 This includes microvascular dysfunction with inflammation, i.e. a macrophage-dominant cellular phenotype and a prominent T cell response in the tubulointerstitium in close proximity to antigen-presenting cells noted on spatial J o u r n a l P r e -p r o o f architectural analysis. The decreased oxidative phosphorylation and upregulation of ceramide signaling pathways with ultrastructural evidence of mitochondrial injury noted in COVID-19 AKI points to metabolic reprogramming of tubular epithelial cells that has been described in S-AKI. Overall, the COVID-19 kidneys had significantly more inflammation compared to NS-AKI controls. The molecular signals of inflammation and immune activation observed in COVID-19 kidneys suggests a global anti-viral response characterized by a macrophage and T cell-rich inflammation, and type-II interferon production, as widely suggested by blood-based assays. 20, 21 The CD4 + dominant T cell response, as we observed in the COVID-19 kidneys, has been associated with worse clinical outcomes. 22 SARS-CoV-2-specific CD4 + T cells directed to the spike (S) surface glycoprotein have been detected in patients with severe COVID-19, and may account for the CD4 + T cells found in the tubulointerstitial compartment of the kidneys. 23 The molecular pathways, investigated both at the genomic and proteomic level, confirmed that inflammation is the predominant driver of the renal injury seen in COVID-19, similar to S-AKI. In both groups, we found increased expression of gene sets associated with counter regulatory mechanisms normally seen in immune activation, including Treg differentiation and sirtuin signaling. 24 In severe COVID-19 cases, increase in circulating Treg frequency is associated with worse outcomes, presumed secondary to suppression of anti-viral T cell responses. 25 In COVID-19 kidneys, as well as in S-AKI, we found evidence of increased apoptosis. While apoptosis is potentially virally mediated via the extrinsic pathways 26 , the upregulated ceramide pathway noted in our COVID-19 AKI cohort, coupled with the reduced oxidative phosphorylation signals, suggests that ceramide-induced apoptosis via mitochondrial disruption might play a role in tubular cell death. 27 Indeed, mitochondria of the proximal tubular epithelial cells of the COVID-19 kidneys demonstrated marked vacuolization and distortion of the cristae as has been J o u r n a l P r e -p r o o f described previously 28 . The upregulated ceramide pathway in COVID-19 AKI underscores the role of lipid dysregulation in severe COVID-19 as suggested by others. 29 Necroptosis, an immunogenic cell death pathway that can eliminate virus-infected cells and mobilize both innate and adaptive immune responses to restrict virus replication, was also more profound in COVID-19 kidneys, compared to NS-AKI. Notably, necroptosis plays a major role in lung injury following SARS-COV2 infection. 26, 30, 31 Kellum et al. and others have suggested that COVID-19 AKI is very similar to sepsis-associated AKI. 17 ' 32 Indeed, our observations of microvascular dysfunction, inflammation, and the metabolic reprogramming ( decreased oxidative phosphorylation and increased ceramide signaling) in COVID-19 AKI are similar to the fundamental pathophysiologic mechanisms described in sepsis-induced AKI I 33 ' 34 ' 19 We summarize the key findings in our study and draw comparisons to published literature in COVID-AKI and S-AKI in Table 4 . These pathology demonstrate potential therapeutic targets for COVID-19. The main limitation of the current study is that while we had the ability to compare molecular characteristics of COVID-19-related pathology against sepsis-associated AKI autopsies, we were not able to compare to autopsies of patients who died of other viral-mediated acute kidney injury. This requires planning and prospective studies to obtain optimal preservation of autopsy tissue for omics studies. The preservation artifacts noted on ultrastructural studies of archived formalin-fixed non-COVID-19 autopsy tissue from prior patients did not allow for comparison. Additionally statistical analysis did not adjust for multiple testing and, a larger sample size could have provided a more robust gene expression analysis. The strength of our study is that we J o u r n a l P r e -p r o o f Acute kidney injury in patients hospitalized with COVID-19 Renal Involvement and Early Prognosis in Patients with COVID-19 Pneumonia Kidney disease is associated with in-hospital death of patients with COVID-19 Kidney Biopsy Findings in Patients with COVID-19 Renal histopathological analysis of 26 postmortem findings of patients with COVID-19 in China Postmortem Kidney Pathology Findings in Patients with COVID-19 Renal histopathological analysis of 26 postmortem findings of patients with COVID-19 in China COVID-19-Associated Nonocclusive Fibrin Microthrombi in the Heart Pathological features of COVID-19-associated myocardial injury: a multicentre cardiovascular pathology study The Spectrum of Histopathologic Findings in Lungs of Patients With Fatal Coronavirus Disease 2019 (COVID-19) Infection Pulmonary post-mortem findings in a series of COVID-19 cases from northern Italy: a two-centre descriptive study Histopathological findings and viral tropism in UK patients with severe fatal COVID-19: a post-mortem study Postmortem examination of COVID-19 patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings in lungs and other organs suggesting vascular dysfunction Multiorgan and Renal Tropism of SARS-CoV-2 SARS-CoV-2 and Viral Sepsis: Immune Dysfunction and Implications in Kidney Failure Histopathologic and Ultrastructural Findings in Postmortem Kidney Biopsy Material in 12 Patients with AKI and COVID-19 Sepsis-associated acute kidney injury: is COVID-19 different? Multiplexed color-coded probe-based gene expression assessment for clinical molecular diagnostics in formalin-fixed paraffinembedded human renal allograft tissue Sepsis-induced acute kidney injury Warriors of SARS-CoV-2 Infection SARS-CoV-2-specific T cell responses and correlations with COVID-19 patient predisposition Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications Phenotype and kinetics of SARS-CoV-2-specific T cells in COVID-19 patients with acute respiratory distress syndrome Nuclear sirtuins and inflammatory signaling pathways Profound Treg perturbations correlate with COVID-19 severity SARS-CoV-2 triggers inflammatory responses and cell death through caspase-8 activation Ceramide-induced apoptosis in renal tubular cells: a role of mitochondria and sphingosine-1-phoshate Tubular Epithelial and Peritubular Capillary Endothelial Injury in COVID-19 AKI Dysregulation of lipid metabolism and pathological inflammation in patients with COVID-19 Necroptosis in anti-viral inflammation Necroptosis: Mechanisms and Relevance to Disease Parallels in Sepsis and COVID-19 Conditions: Implications for Managing Severe COVID-19 Histopathology of septic shock induced acute kidney injury: apoptosis and leukocytic infiltration Sepsis and acute kidney injury Histopathology of septic shock induced acute kidney injury: apoptosis and leukocytic infiltration Sepsis and acute kidney injury Sepsis-induced acute kidney injury SARS-CoV-2 triggers inflammatory responses and cell death through caspase-8 activation COVID-19: NAD(+) deficiency may predispose the aged, obese and type2 diabetics to mortality through its effect on SIRT1 activity Emerging Evidence concerning the Role of Sirtuins in Sepsis Role of the mTOR Signalling Pathway in Human Sepsis-Induced Myocardial Dysfunction Necroptosis in anti-viral inflammation Necroptosis: Mechanisms and Relevance to Disease Covid-19) sepsis: revisiting mitochondrial dysfunction in pathogenesis, aging, inflammation, and mortality Dysregulation of lipid metabolism and pathological inflammation in patients with COVID-19 No of patients (%) <50 1 (6) J o u r n a l P r e -p r o o f Oxidative phosphorylation -- 10 Ceramide signaling pathway ++ 11