RAAS blockade, kidney disease, and expression of ACE2 , the entry receptor for SARS-CoV-2, in kidney epithelial and endothelial cells

  1. Ayshwarya Subramanian
  2. Katherine A Vernon
  3. Michal Slyper
  4. Julia Waldman
  5. Malte D Luecken
  6. Kirk Gosik
  7. Dan Dubinsky
  8. Michael S Cuoco
  9. Keith Keller
  10. Jason Purnell
  11. Lan Nguyen
  12. Danielle Dionne
  13. Orit Rozenblatt-Rosen
  14. Astrid Weins
  15. Human Cell Atlas Lung Biological Network
  16. Aviv Regev
  17. Anna Greka

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

SARS-CoV-2, the coronavirus that causes COVID-19, binds to angiotensin-converting enzyme 2 (ACE2) on human cells. Beyond the lung, COVID-19 impacts diverse tissues including the kidney. ACE2 is a key member of the Renin-Angiotensin-Aldosterone System (RAAS) which regulates blood pressure, largely through its effects on the kidney. RAAS blockers such as ACE inhibitors (ACEi) and Angiotensin Receptor Blockers (ARBs) are widely used therapies for hypertension, cardiovascular and chronic kidney diseases, and therefore, there is intense interest in their effect on ACE2 expression and its implications for SARS-CoV-2 pathogenicity. Here, we analyzed single-cell and single-nucleus RNA-seq of human kidney to interrogate the association of ACEi/ARB use with ACE2 expression in specific cell types. First, we performed an integrated analysis aggregating 176,421 cells across 49 donors, 8 studies and 8 centers, and adjusting for sex, age, donor and center effects, to assess the relationship of ACE2 with age and sex at baseline. We observed a statistically significant increase in ACE2 expression in tubular epithelial cells of the thin loop of Henle (tLoH) in males relative to females at younger ages, the trend reversing, and losing significance with older ages. ACE2 expression in tLoH increases with age in females, with an opposite, weak effect in males. In an independent cohort, we detected a statistically significant increase in ACE2 expression with ACEi/ARB use in epithelial cells of the proximal tubule and thick ascending limb, and endothelial cells, but the association was confounded in this small cohort by the underlying disease. Our study illuminates the dynamics of ACE2 expression in specific kidney cells, with implications for SARS-CoV-2 entry and pathogenicity.

Article activity feed

  1. Version published to 10.1101/2020.06.23.167098v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.06.23.167098: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementConsent: Single nucleus isolation from human kidney tissue: Frozen kidney biopsy specimens or frozen samples of macroscopically normal cortex from tumor nephrectomies (distant from the tumor site), were obtained after appropriate patient (discarded tissue) consent and in accordance with Partners Healthcare IRB and institutional guidelines.
    RandomizationWhenever feasible, we pooled 10x libraries on sequencing lanes to ensure that any individual sample was not confounded by batch (kidney section, day of sample collection, condition, timepoint) and were randomly distributed across lanes.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableIn addition to an internal unpublished single-cell cohort, we aggregated control kidney single-cell or single-nucleus RNA-seq data from 7 published studies for a total of 49 donors (29 males, 20 females) with a median age of 57 (min 2y, max 72y, IQR=14).

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Single nucleus isolation from human kidney tissue: Frozen kidney biopsy specimens or frozen samples of macroscopically normal cortex from tumor nephrectomies (distant from the tumor site), were obtained after appropriate patient (discarded tissue) consent and in accordance with Partners Healthcare IRB and institutional guidelines.
    Partners Healthcare
    suggested: (Partners HealthCare Biobank, RRID:SCR_001316)
    Preprocessing of 10x droplet-based sequencing outputs: We used the Cellranger toolkit (v2.1.1, v3, 10X Genomics https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger) to de-multiplex (cellranger mkfastq) the sequencing outputs, and for alignment (cellranger count) to the reference transcriptome (GRCh38 for human cells, GRCh38 pre-mRNA for human nuclei), and quantification of gene expression.
    https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger
    suggested: (Cell Ranger , RRID:SCR_017344)
    We used the R visualization packages ggplot2 v3.2.1 (34), cowplot v0.9.4 (35), ggpubr v0.2.5 (36) and patchwork v1.0.0.9 (36, 37) for generation of boxplots, violin plots, proportional bar plots, UMAP visualization and dotplots.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    cowplot
    suggested: (cowplot, RRID:SCR_018081)

    Results from OddPub: Thank you for sharing your code and data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    A caveat of the integrated analysis is that aggregating cells to broader cell categories across various batches may lead to loss of within-class cellular heterogeneity. Effect sizes may differ across the subclasses, thus impacting their estimation. In conclusion, at baseline, ACE2 expression had a statistically significant association with both age in females and sex at younger ages in the tLoH cells. Because of the higher median age in our integrated analysis dataset, larger cohorts with wider age ranges are necessary to validate the results. Association of RAAS blockade with ACE2 expression in the kidney epithelial and endothelial cells confounded by underlying disease: Next, to test the association of renal ACE2 expression with ACEi/ARB use, we surveyed ACE2 expression in 32,239 nuclei obtained by droplet-based snRNA-seq of frozen kidney samples from an independent cohort of 11 patients: 9 kidney biopsies with features of various kidney diseases including Lupus nephritis (LN) and IgA nephropathy (IgAN) (Table 1) and 2 cortical samples from a tumor nephrectomy and transplant nephrectomy with rejection and recurrent Focal Segmental Glomerulosclerosis (FSGS). 6 of the 9 biopsied patients were either on Lisinopril (ACEi) or Losartan (ARB). Nuclei were isolated using one of two protocols (Methods, Table 1). Graph based clustering and post hoc annotation (Methods) identified 14 broad cell classes (Figure S3A), including epithelial, immune and stromal cells, of which 8 classes ha...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2020.06.23.167098v1 on bioRxiv