key: cord-0705124-2c7xwokb
authors: Garcia, Gabriela E.; Truong, Luan D.; Johnson, Richard J.
title: ACE2 Decreased Expression During Kidney Inflammatory Diseases: Implications to Predisposing to COVID 19 Kidney Complications
date: 2021-08-30
journal: Kidney Int
DOI: 10.1016/j.kint.2021.08.016
sha: 76448919eaf66ba5bffbecb74cb11d8c54f19e45
doc_id: 705124
cord_uid: 2c7xwokb

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another study, rats with anti-GBM GN received Angiotensin 1-7 (Ang-(1-7), 24 µg/kg/hour (Millipore Sigma, St. Louis, MO) or saline by osmotic minipumps (Alzet 2002, Alzet osmotic pumps, Cupertino, CA) as described S2 . The mini-osmotic pumps were incubated in sterile saline at 37°C overnight (to the pumps star working immediately) and implanted subcutaneously 4 hours after the injection of anti-GBM antibody (maximum glomerular deposition of IgG occurs one hour after injection of anti-GBM antibody S3 ). Rats were euthanized at day 7 after the injection of the antibody to collect kidney tissue since significant macrophage infiltration and kidney injury is observed at this time of the disease.

Tubulointerstitial nephritis model (TIN) was generated in Brown Norway rats (Envigo, Placentia, CA) by tail base injection of 100 µg of bovine tubular basement membrane in complete Freund's adjuvant with 4 mg of dried Mycobacterium tuberculosis, H37Ra strain (BD Difco, Sparks, MD). The rats also received a Bordetella pertussis vaccine (Massachusetts Public Health Biologics Laboratory, Boston, MA) containing around 22.2 x 10 8 cells in a separate intradermal injection into the flank as described S4,S5 . Control animals were immunized with ovalbumin (100 µg) in the same adjuvant. Rats were euthanized at different time points to collect kidney tissues.

RNA expression of ACE2: Total RNA was isolated from kidneys using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Five micrograms of total RNA from each sample was used for RNase protection assay (RPA). A rat ACE2 405 bp probe (nucleotide 1161 to 1566 as defined in GenBank sequence accession number AY881244) was generated by RT-PCR using kidney tissue. RNase protection assay was performed using the Torrey Pines Biolabs kit (East Orange, NJ) as described S6-S9 . Phosphoimage quantitation of blots was performed using the PhosphorImager SI scanning instrument and ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA) S6,S10,S11 . L-32 was used as a housekeeping gene. Final values were expressed as a ratio of the counts per minute (cpm) for the specific mRNA/L-32 mRNA to ensure a constant quantity of RNA in each sample. American Tissue Type Culture Collection, Manassas, VA) were used to have enough cells to perform this assay. Despite being a cell line, these cells are well recognized as having many characteristic of macrophages and have been extensively used to study macrophage function S12,S13 . The RAW cells were suspended in DMEM 3% FBS to a concentration of 4X10 6 cell/ml. Chemokine (100nM) alone, chemokine (100 nM) and Ang-(1-7) (10 nM), or medium were placed in lower wells of the chamber and separated from cell suspension in upper wells by 5-µm pore-size PVP-free polycarbonate filters. Results were expressed as mean ± SEM cell number per five high-power fields (x400) and were a representative of n=3 experiments performed in duplicated.

provided by Rolf A.K. Stah (Department of Medicine, Division of Nephrology and Osteology, Hamburg, Germany). Cells were cultured in DMEM medium supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM glutamine, and 10% heat inactivated FCS at 37C in 5% CO2. Cells were serum-deprived overnight and then, treated with 1 µM angiotensin II (Millipore Sigma) or angiotensin II plus angiotensin1-7 (Millipore Sigma,) 1µM, for 2, 4, 12, 16, and 24 hours and total RNA was extracted with TRIzol reagent. RNase protection assay was performed to detect IP10/CXCl10 mRNA expression.

Morphological analysis, immunohistochemical phenotyping, and quantitation of leukocytes: Kidney samples fixed in formalin or methanol-Carnoy fixative solution were embedded in paraffin. Two to three-µm sections were stained with periodic acid-Schiff reagent to assess kidney injury. Infiltrating macrophages were immunohistochemically stained for ED1 + (Bio-Rad/AbD Serotec, Hercules, CA), as described S8,S14,S15 . Positively stained cells per 100 glomeruli were counted and expressed per glomerular section. All quantitative morphological analyses were performed in a blinded fashion. 

Evaluation of phenotypic and functional stability of RAW 264.7 cell line through serial passages

In vivo inhibition of CC and CX3C chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in Wistar-Kyoto (WKY) rats by vMIP-II

IL-18 translational inhibition restricts IFN-gamma expression in crescentic glomerulonephritis