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Comment
. 2020 Sep;76(3):e26-e28.
doi: 10.1161/HYPERTENSIONAHA.120.15725. Epub 2020 Jul 9.

SARS-CoV-2 Receptor ACE2 (Angiotensin-Converting Enzyme 2) Is Upregulated in Colonic Organoids From Hypertensive Rats

Jing Li  1 , Bruce R Stevens  1 , Elaine M Richards  1 , Mohan K Raizada  1
Affiliations
Comment

SARS-CoV-2 Receptor ACE2 (Angiotensin-Converting Enzyme 2) Is Upregulated in Colonic Organoids From Hypertensive Rats

Jing Li et al. Hypertension. 2020 Sep.
No abstract available

Keywords: angiotensins; comorbidity; coronavirus disease; organoids; population.

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Figures

Figure.
Figure.
A, Representative images of colon (left), and the ratio of colon length/body weight of Wister Kyoto rat (WKY) and spontaneously hypertensive rat (SHR) rats (n=12 per group; right) from male 14-wk-old SHR and WKY rats (Charles River), mean systolic blood pressures 211±4 and 137±3 mm Hg, respectively, measured by Tail-Cuff Plethysmography.B, Phase microscopic images documenting growth of organoids from isolated colonic crypts of WKY and SHR rats stained with Trypan blue, dead cells are blue. Scale bar: 200 µmol/L. Primary colon organoids were grown from isolated crypts (gentle dissociation reagent [Stem Cell Technologies] in Matrigel [BD Biosciences] and organoid growth medium [Stem Cell Technologies] supplemented with recombinant mouse Noggin [PeproTech] and EGF [BioLegend], recombinant human IGF-1, FGF-basic [FGF-2;BioLegend] and R-spondin1 [R&D], Y-27632 [STEMCELL Technologies], and A83-01 [Tocris] described elsewhere [Cell Stem Cell, 2018, 23(6): 787–793. e6]). (n=12 rat colon cultures/group.)C, Organoid formation efficiency determined 3, 5, and 7 d after culture of isolated colonic crypts from WKY and SHR rats (n=12 per group; left) and 4% PFA-fixed organoids after 5 d in culture (right).D, Representative confocal immunofluorescence images of Ki67 and KRT20 in organoids from WKY and SHR (left, scale bar: 50 µmol/L); quantified and normalized fluorescent intensity (right, n=5 per group). Fixed, permeablilized organoids stained with Ki67 and KRT20 specific antibodies (Abcam) and DAPI.E, Comparison of mRNA for Ace2, Ace, Tmprss2, Ki67, and KRT20 in colonic organoids by real-time polymerase chain reaction (RT-PCR; left, shaded bars, n=9 per group) and in epithelium of proximal colon by RNA-seq (right, solid bars, n=4 per group) from WKY and SHR. For organoids, total RNA was purified using RNAeasy plus mini kit (Qiagen) and reverse-transcribed using iScript Reverse Transcription Supermix (Bio-Rad). Finally, RT-PCR (ABI Prism 7600) was performed with Taqman universal PCR master mix and specific probes. RNA-seq method.F, Analysis of Ace2 in organoids by confocal immunofluorescent imaging (left, Scale bar: 50 µmol/L) and by Western blot (right), quantified and normalized to actin fluorescence intensity (middle, n=5 per group). ACE-2 (Angiotensin-converting enzyme) and actin antibodies were from Abcam and Western blots were homogenates of organoids (2% SDS-Tris buffer, pH=7.5), run on 12%TGX precast gels, transferred to PVDF membranes (Biorad) and imaged on Odyssey imaging system with infrared light for Li-Cor Biosciences secondary antibodies. Fold change relative to WKY group. Values are means±SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, and nsP>0.05 vs WKY from SHR group, unpaired t test.
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