Description
COVID-19 associated acute kidney injury (COVID-AKI) is a common complication of SARS-CoV-2 infection in hospitalized patients. It is unclear how susceptible human kidneys are to direct SARS-CoV-2 infection and whether pharmacologic manipulation of the renin-angiotensin II signaling (RAS) pathway modulates this susceptibility. Using induced pluripotent stem cell derived kidney organoids, SARS-CoV-1, SARS-CoV-2 and MERS-CoV tropism, defined by the paired expression of a host receptor (ACE2, NRP1 or DPP4) and protease (TMPRSS2, TMPRSS4, FURIN, CTSB or CTSL), was identified primarily amongst proximal tubule cells. Losartan, an angiotensin II receptor blocker being tested in COVID-19 patients, inhibited angiotensin II mediated internalization of ACE2, upregulated interferon stimulated genes (IFITM1 and BST2) known to restrict viral entry, and attenuated the infection of proximal tubule cells by SARS-CoV-2. Our work highlights the susceptibility of proximal tubule cells to SARS-CoV-2 and reveals a putative protective role for RAS inhibitors during SARS-CoV-2 infection.
Overall Design
Cell Lines:; Human iPSC lines were purchased from Gibco (human episomal iPSC line, Cat. #A18944-derived from neonate female umbilical cord blood) or were derived from cryopreserved human dermal fibroblast (Cell Applications, Inc. Cat. #106-05n, lot #1481) using CytoTune-iPS 2.0 Sendai reprogramming kit (Life Technologies Cat. #A16517) at the Harvard Stem Cell Institute iPSC Core Facility, as a gift from Dr. Martin Pollak at Harvard Medical School. iPSCs were confirmed to be of normal karyotype and maintained at 37 C with 5% CO2 with daily medium changes of mTeSR1 medium in 6 well plates coated with Matrigel (Corning, Cat. #354277). iPSC colonies were passaged using GCDR (Stem Cell Technologies, Cat. #05275) and transferred to T25 flasks coated with Matrigel prior to differentiation. iPSCs were routinely tested and confirmed negative for mycoplasma. Vero E6 cells (ATCC, Cat. #C1008) were maintained in a humidified 37C incubator with 5% CO2 and cultured in DMEM (Thermo Fisher) supplemented with 100 U/mL penicillin, 100 g/mL streptomycin, 2 mM L-glutamine (Thermo Fisher, Cat. #10378-016; herein referred to as 1% PSQ) and 10% heat-inactivated FBS (Thermo Fisher; herein referred to as complete DMEM). ; ; Generation of kidney organoids:; Kidney organoids were differentiated using the Takasato protocol with minor modifications51 that included APEL2 medium (Stem Cell Technologies, Cat. #05275) supplemented with 5% PFHM-II (Thermo Fisher, Cat. #12040077) (APEL2 + PFHM) in replacement of APEL. Induced PSCs were grown on a feeder-free system using Matrigel matrix coated plates. Cells were treated with 8 M CHIR99021 (R&D Systems, Cat. #4423) for 4 days followed by recombinant human FGF-9 (200 ng/mL) and heparin (1 g/mL) for an additional 3 days. At day 7, cells were dissociated into single cell suspension using trypsin-EDTA (0.05%) for 2 min and 250,000 or 500,000 cells were centrifuged to make a pellet at 400 x g for 2 min. The pellet was transferred onto a six-well transwell plate (Corning, Cat. # 07-200-170) with four or nine pellets per well, respectively. Pellets were incubated with a pulse of 5 M CHIR99021 in APEL2 + PFHM medium for 1 hr at 37 C. After 1 hr, the medium was changed to APEL2 + PFHM supplemented with FGF-9 (200 ng/mL, R&D Systems, Cat. #273-F9-025) and heparin (1 g/mL, Millipore Sigma, Cat. #4784) for an additional 5 days, and then were maintained in APEL2 + PFHM medium for 14 days with medium change every other day.; ; Treatment with RAAS modulators:; Kidney organoids on day 28 or hTEC at passage 5 were pretreated with or without losartan (100 M, Tocris, Cat. #3798) for 30 min prior to Ang II (1 M, Millipore Sigma, Cat. #A9525) treatment for 24 hr. DMSO was used as a vehicle control.; ; scRNA-Seq Library Construction:; Single (n=1) technical replicates of each treatment were kidney organoid group derived from the 1481 iPSC line were prepared for sequencing. Organoids were dissociated to single cells using TrypLE Select (Thermo Fisher Scientific) at 37C with mechanical trituration. Single cell suspensions were visually inspected under a microscope, counted using an automated hand-held cell counter (Millipore Scepter 2.0 Cell Counter), and resuspended at a concentration of 15,000 single-cells in 50 microliters of FACS buffer (0.1% BSAPBS). All four samples were processed according to 10X Genomics ChromiumTM Next GEM Single Cell 3’ Reagent Kits v3.1 as per manufacturers protocol. Briefly, single cells were partitioned together with barcoded Gel Bead-In-EMulsions (GEMs) using 10x GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating cDNA from poly-adenylated mRNA. DynaBeads MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using Agilent D1000 ScreenTape System. Libraries were pooled and first sequenced on NovaSeq 100 (plus 25 additional) Cycle SP dual lane flow cell to approximate the number of recovered cells in each sample. We recovered 4,235 cells in Control, 5,702 cells in losartan-alone, 5,800 cells in angiotensin II alone, and 5,855 cells in angiotensin II plus losartan treated groups with an estimated doublet rate of 4%. Based on these, we determined proportions for Illumina NovaSeq 100 (plus 25 additional) Cycle S2 dual lane flow cell with a targeted sequencing depth of ~30,000 reads per cell. Illumina sequencer’s BCL files were demultiplexed using cellranger mkfastq (a wrapper around Illumina’s bcl2fastq). Downstream barcode processing, alignment to pre-built GRCh38, and single-cell 3 gene counting were performed using CellRanger software version 3.0.1 (10X Genomics). All four libraries were aggregated using cellranger aggr (with mapped normalization). The resulting filtered feature-barcode matrix was imported into Seurat v3 for quality control, dimensionality reduction, cell clustering, and differential expression analysis using default or recommended parameters.
Curator
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