Single Nucleus RNA Sequencing of Remnant Kidney Biopsies and Urine Cell RNA Sequencing Reveal Cell Specific Markers of Covid-19 Acute Kidney Injury (preprint)

Acute kidney injury (AKI) in COVID-19 patients is associated with high mortality and morbidity. Critically ill COVID-19 patients are at twice the risk of in-hospital mortality compared to non-COVID AKI patients. We know little about the cell-specific mechanism in the kidney that contributes to worse clinical outcomes in these patients. New generation single cell technologies have the potential to provide insights into physiological states and molecular mechanisms in COVID-AKI. One of the key limitations is that these patients are severely ill posing significant risks in procuring additional biopsy tissue. We recently generated single nucleus RNA-sequencing data using COVID-AKI patient biopsy tissue as part of the human kidney atlas. Here we describe this approach in detail and report deeper comparative analysis of snRNAseq of 4 COVID-AKI, 4 reference, and 6 non-COVID-AKI biopsies. We also generated and analyzed urine transcriptomics data to find overlapping COVID-AKI-enriched genes and their corresponding cell types in the kidney from snRNA-seq data. We identified all major and minor cell types and states by using by using less than a few cubic millimeters of leftover tissue after pathological workup in our approach. Differential expression analysis of COVID-AKI biopsies showed pathways enriched in viral response, WNT signaling, kidney development, and cytokines in several nephron epithelial cells. COVID-AKI profiles showed a much higher proportion of altered TAL cells than non-COVID AKI and the reference samples. In addition to kidney injury and fibrosis markers indicating robust remodeling we found that, 17 genes overlap between urine cell COVID-AKI transcriptome and the snRNA-seq data from COVID-AKI biopsies. A key feature was that several of the distal nephron and collecting system cell types express these markers. Some of these markers have been previously observed in COVID-19 studies suggesting a common mechanism of injury and potentially the kidney as one of the sources of soluble factors with a potential role in disease progression. Translational StatementThe manuscript describes innovation, application and discovery that impact clinical care in kidney disease. First, the approach to maximize use of remnant frozen clinical biopsies to inform on clinically relevant molecular features can augment existing pathological workflow for any frozen tissue without much change in the protocol. Second, this approach is transformational in medical crises such as pandemics where mechanistic insights are needed to evaluate organ injury, targets for drug therapy and diagnostic and prognostic markers. Third, the cell type specific and soluble markers identified and validated can be used for diagnoses or prognoses in AKI due to different etiologies and in multiorgan injury.

Exome-wide association study to identify rare variants influencing COVID-19 outcomes: Results from the Host Genetics Initiative (preprint)

Host genetics is a key determinant of COVID-19 outcomes. Previously, the COVID-19 Host Genetics Initiative genome-wide association study used common variants to identify multiple loci associated with COVID-19 outcomes. However, variants with the largest impact on COVID-19 outcomes are expected to be rare in the population. Hence, studying rare variants may provide additional insights into disease susceptibility and pathogenesis, thereby informing therapeutics development. Here, we combined whole-exome and whole-genome sequencing from 21 cohorts across 12 countries and performed rare variant exome-wide burden analyses for COVID-19 outcomes. In an analysis of 5,048 severe disease cases and 571,009 controls, we observed that carrying a rare deleterious variant in the SARS-CoV-2 sensor toll-like receptor TLR7 (on chromosome X) was associated with a 5.3-fold increase in severe disease (95% CI: 2.75-10.05, p=5.41x10-7). These results further support TLR7 as a genetic determinant of severe disease and suggest that larger studies on rare variants influencing COVID-19 outcomes could provide additional insights.

Urine test predicts kidney injury and death in COVID-19 (preprint)

Background: Kidney injury is common in COVID-19 infection, but serum creatinine (SCr) is not a sensitive or specific marker of kidney injury. We hypothesized that molecular markers of tubular injury could diagnose COVID-19 associated kidney damage and predict its clinical course. Methods: This is a prospective cohort study of 444 consecutive COVID-19 patients (43.9% females, 20.5% African American, 54.1% Latinx) in Columbia University's Emergency Department at the peak of the New York pandemic (March-April 2020). Urine and blood were collected simultaneously at admission (median time of day 0, IQR 0-2 days) and within 1 day of a positive SARS-CoV-2 test in 70% of patients. Biomarker assays were blinded to clinical data. Results: Urinary NGAL (uNGAL) was strongly associated with AKI diagnosis (267{+/-}301 vs. 96{+/-}139 ng/mL, P=1.6x10-10). uNGAL >150ng/mL had 80% specificity and 75% sensitivity to diagnose AKIN stage 2 or higher. uNGAL quantitatively predicted the duration of AKI and outcomes, including death, dialysis, shock, and longer hospital stay. The risk of death increased 73% per standard deviation of uNGAL [OR (95%CI): 1.73 (1.29-2.33), P=2.8x10-4] and was independent of baseline SCr, co-morbidities, and proteinuria [adjusted OR (95%CI): 1.51 (1.10-2.11), P=1.2x10-2]. Proteinuria and uKIM-1 also indicated tubular injury, but were not diagnostic of AKI. Typically, distal nephron segments transcribe NGAL, but in COVID-19 biopsies with widespread acute tubular injury (ATI), NGAL expression overlapped KIM-1 in proximal tubules. Conclusion: uNGAL predicted the diagnosis, duration, and severity of AKI and ATI, as well as hospital stay, dialysis, shock, and death in patients with acute COVID-19.

Alternative splicing of OAS1 alters the risk for severe COVID-19 (preprint)

ABSTRACT A locus containing OAS1/2/3 has been identified as a risk locus for severe COVID-19 among Europeans ancestry individuals, with a protective haplotype of ∼75 kilobases derived from Neanderthals. Here, we show that among several potentially causal variants at this locus, a splice variant of OAS1 occurs in people of African ancestry independently of the Neanderthal haplotype and confers protection against COVID-19 of a magnitude similar to that seen in individuals without African ancestry.

Age-dependent impact of the major common genetic risk factor for COVID-19 on severity and mortality (preprint)

Background: There is considerable variability in COVID-19 outcomes amongst younger adults and some of this variation may be due to genetic predisposition. We characterized the clinical implications of the major genetic risk factor for COVID-19 severity, and its age-dependent effect, using individual-level data in a large international multi-centre consortium. Method: The major common COVID-19 genetic risk factor is a chromosome 3 locus, tagged by the marker rs10490770. We combined individual level data for 13,424 COVID-19 positive patients (N=6,689 hospitalized) from 17 cohorts in nine countries to assess the association of this genetic marker with mortality, COVID-19-related complications and laboratory values. We next examined if the magnitude of these associations varied by age and were independent from known clinical COVID-19 risk factors. Findings: We found that rs10490770 risk allele carriers experienced an increased risk of all-cause mortality (hazard ratio [HR] 1.4, 95% confidence interval [CI] 1.2-1.6) and COVID-19 related mortality (HR 1.5, 95%CI 1.3-1.8). Risk allele carriers had increased odds of several COVID-19 complications: severe respiratory failure (odds ratio [OR] 2.0, 95%CI 1.6-2.6), venous thromboembolism (OR 1.7, 95%CI 1.2-2.4), and hepatic injury (OR 1.6, 95%CI 1.2-2.0). Risk allele carriers [≤] 60 years had higher odds of death or severe respiratory failure (OR 2.6, 95%CI 1.8-3.9) compared to those > 60 years OR 1.5 (95%CI 1.3-1.9, interaction p-value=0.04). Amongst individuals [≤] 60 years who died or experienced severe respiratory COVID-19 outcome, we found that 31.8% (95%CI 27.6-36.2) were risk variant carriers, compared to 13.9% (95%CI 12.6-15.2%) of those not experiencing these outcomes. Prediction of death or severe respiratory failure among those [≤] 60 years improved when including the risk allele (AUC 0.82 vs 0.84, p=0.016) and the prediction ability of rs10490770 risk allele was similar to, or better than, most established clinical risk factors. Interpretation: The major common COVID-19 risk locus on chromosome 3 is associated with increased risks of morbidity and mortality and these are more pronounced amongst individuals [≤] 60 years. The effect on COVID-19 severity was similar to, or larger than most established risk factors, suggesting potential implications for clinical risk management. Funding: Funding was obtained by each of the participating cohorts individually.

High rate of renal recovery in survivors of COVID-19 associated acute renal failure requiring renal replacement therapy.

INTRODUCTION: A large proportion of patients with COVID-19 develop acute kidney injury (AKI). While the most severe of these cases require renal replacement therapy (RRT), little is known about their clinical course. METHODS: We describe the clinical characteristics of COVID-19 patients in the ICU with AKI requiring RRT at an academic medical center in New York City and followed patients for outcomes of death and renal recovery using time-to-event analyses. RESULTS: Our cohort of 115 patients represented 23% of all ICU admissions at our center, with a peak prevalence of 29%. Patients were followed for a median of 29 days (2542 total patient-RRT-days; median 54 days for survivors). Mechanical ventilation and vasopressor use were common (99% and 84%, respectively), and the median Sequential Organ Function Assessment (SOFA) score was 14. By the end of follow-up 51% died, 41% recovered kidney function (84% of survivors), and 8% still needed RRT (survival probability at 60 days: 0.46 [95% CI: 0.36-0.56])). In an adjusted Cox model, coronary artery disease and chronic obstructive pulmonary disease were associated with increased mortality (HRs: 3.99 [95% CI 1.46-10.90] and 3.10 [95% CI 1.25-7.66]) as were angiotensin-converting-enzyme inhibitors (HR 2.33 [95% CI 1.21-4.47]) and a SOFA score >15 (HR 3.46 [95% CI 1.65-7.25). CONCLUSIONS AND RELEVANCE: Our analysis demonstrates the high prevalence of AKI requiring RRT among critically ill patients with COVID-19 and is associated with a high mortality, however, the rate of renal recovery is high among survivors and should inform shared-decision making.

Failure to replicate the association of rare loss-of-function variants in type I IFN immunity genes with severe COVID-19 (preprint)

A recent report found that rare predicted loss-of-function (pLOF) variants across 13 candidate genes in TLR3- and IRF7-dependent type I IFN pathways explain up to 3.5% of severe COVID-19 cases. We performed whole-exome or whole-genome sequencing of 1,934 COVID-19 cases (713 with severe and 1,221 with mild disease) and 15,251 ancestry-matched population controls across four independent COVID-19 biobanks. We then tested if rare pLOF variants in these 13 genes were associated with severe COVID-19. We identified only one rare pLOF mutation across these genes amongst 713 cases with severe COVID-19 and observed no enrichment of pLOFs in severe cases compared to population controls or mild COVID-19 cases. We find no evidence of association of rare loss-of-function variants in the proposed 13 candidate genes with severe COVID-19 outcomes.

Furin cleaves SARS-CoV-2 spike-glycoprotein at S1/S2 and S2'for viral fusion/entry: indirect role for TMPRSS2 (preprint)

The Spike (S)-protein of SARS-CoV-2 binds host-cell receptor ACE2 and requires proteolytic 'priming' (S1/S2) and 'fusion-activation' (S2') for viral entry. The S-protein furin-like motifs PRRAR685{downarrow} and KPSKR815{downarrow} indicated that proprotein convertases promote virus entry. We demonstrate that furin and PC5A induce cleavage at both sites, ACE2 enhances S2' processing, and their pharmacological inhibition (BOS-inhibitors) block endogenous cleavages. S1/S2-mutations (S1/S2) limit S-protein-mediated cell-to-cell fusion, similarly to BOS-inhibitors. Unexpectedly, TMPRSS2 does not cleave at S1/S2 or S2', but it can: (i) cleave/inactivate S-protein into S2a/S2b; (ii) shed ACE2; (iii) cleave S1-subunit into secreted S1', activities inhibited by Camostat. In lung-derived Calu-3 cells, BOS-inhibitors and S1/S2 severely curtail 'pH-independent' viral entry, and BOS-inhibitors alone/with Camostat potently reduce infectious viral titer and cytopathic effects. Overall, our results show that: furin plays a critical role in generating fusion-competent S-protein, and indirectly, TMPRSS2 promotes viral entry, supporting furin and TMPRSS2 inhibitors as potential antivirals against SARS-CoV-2

SARS-CoV-2 protein ORF3a is pathogenic in Drosophila and causes phenotypes associated with COVID-19 post-viral syndrome (preprint)

The Coronavirus Disease 2019 (COVID-19) pandemic has caused millions of deaths and will continue to exact incalculable tolls worldwide. While great strides have been made toward understanding and combating the mechanisms of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection, relatively little is known about the individual SARS-CoV-2 proteins that contribute to pathogenicity during infection and that cause neurological sequela after viral clearance. We used Drosophila to develop an in vivo model that characterizes mechanisms of SARS-CoV-2 pathogenicity, and found ORF3a adversely affects longevity and motor function by inducing apoptosis and inflammation in the nervous system. Chloroquine alleviated ORF3a induced phenotypes in the CNS, arguing our Drosophila model is amenable to high throughput drug screening. Our work provides novel insights into the pathogenic nature of SARS-CoV-2 in the nervous system that can be used to develop new treatment strategies for post-viral syndrome.