Pulmonary, cardiac and renal distribution of ACE2, furin, TMPRSS2 and ADAM17 in rats with heart failure: Potential implication for COVID-19 disease

Abstract

Congestive heart failure (CHF) is often associated with kidney and pulmonary dysfunction. Activation of the renin-angiotensin-aldosterone system (RAAS) contributes to avid sodium retention, cardiac hypertrophy and oedema formation, including lung congestion. While the status of the classic components of RAAS such as renin, angiotensin converting enzyme (ACE), angiotensin II (Ang II) and angiotensin II receptor AT-1 is well studied in CHF, the expression of angiotensin converting enzyme-2 (ACE2), a key enzyme of angiotensin 1-7 (Ang 1-7) generation in the pulmonary, cardiac and renal systems has not been studied thoroughly in this clinical setting. This issue is of a special interest as Ang 1-7 counterbalance the vasoconstrictory, pro-inflammatory and pro-proliferative actions of Ang II. Furthermore, CHF predisposes to COVID-19 disease severity, while ACE2 also serves as the binding domain of SARS-CoV-2 in human host-cells, and acts in concert with furin, an important enzyme in the synthesis of BNP in CHF, in permeating viral functionality along TMPRSST2. ADAM17 governs ACE2 shedding from cell membranes. Therefore, the present study was designed to investigate the expression of ACE2, furin, TMPRSS2 and ADAM17 in the lung, heart and kidneys of rats with CHF to understand the exaggerated susceptibility of clinical CHF to COVID-19 disease. Heart failure was induced in male Sprague Dawley rats by the creation of a surgical aorto-caval fistula. Sham-operated rats served as controls. One week after surgery, the animals were subdivided into compensated and decompensated CHF according to urinary sodium excretion. Both groups and their controls were sacrificed, and their hearts, lungs and kidneys were harvested for assessment of tissue remodelling and ACE2, furin, TMPRSS2 and ADAM17 immunoreactivity, expression and immunohistochemical staining. ACE2 immunoreactivity and mRNA levels increased in pulmonary, cardiac and renal tissues of compensated, but not in decompensated CHF. Furin immunoreactivity was increased in both compensated and decompensated CHF in the pulmonary, cardiac tissues and renal cortex but not in the medulla. Interestingly, both the expression and abundance of pulmonary, cardiac and renal TMPRSS2 decreased in CHF in correlation with the severity of the disease. Pulmonary, cardiac and renal ADAM17 mRNA levels were also downregulated in decompensated CHF. Circulating furin levels increased in proportion to CHF severity, whereas plasma ACE2 remained unchanged. In summary, ACE2 and furin are overexpressed in the pulmonary, cardiac and renal tissues of compensated and to a lesser extent of decompensated CHF as compared with their sham controls. The increased expression of the ACE2 in heart failure may serve as a compensatory mechanism, counterbalancing the over-activity of the deleterious isoform, ACE. Downregulated ADAM17 might enhance membranal ACE2 in COVID-19 disease, whereas the suppression of TMPRSS2 in CHF argues against its involvement in the exaggerated susceptibility of CHF patients to SARS-CoV2.

Keywords: ADAM17; TMPRSS2; angiotensin converting enzyme 2; furin; heart; heart failure; kidney; lung.

Figure 1

Impact of aorto‐caval placement on heart, lung and kidney weights as compared with sham‐operated controls. Cardiac, lung, and kidney weights expressed as absolute values (A–C, respectively) of rats with compensated and decompensated CHF and their sham controls (n = 9‐11). Values are means ± SEM

Figure 2

Immunoreactive levels and expression of ACE2 in the lungs, LV, RV and kidneys of compensated, decompensated CHF and sham controls as were determined with western blots analysis and RT‐qPCR. A, Representative western‐blot analysis of tissue lysates with antibody for ACE2. Western‐blot analysis quantification of ACE2 in the lungs, LV, RV and kidneys (B, D, F and H, respectively) of sham, compensated CHF and decompensated CHF (n = 3‐10), where GAPDH was used as loading control. Quantification of RT‐qPCR analysis for ACE2 mRNA normalized to GAPDH are depicted in C, E, G and I. Values are means ± SEM

Figure 3

Immunoreactive levels and expression of furin in the lungs, LV, RV and kidneys of compensated, decompensated CHF and sham controls as were determined with western blots analysis and RT‐qPCR. A, Representative western blot analysis of tissue lysates with antibody for furin. Western blot analysis quantification of furin in the lungs, LV, RV and kidneys (B, D, F and H, respectively) of sham, compensated CHF and decompensated CHF (n = 3‐11), where GAPDH was used as loading control. Quantification of RT‐qPCR analysis for furin mRNA normalized to GAPDH are depicted in C, E, G and I. Values are means ± SEM

Figure 4

Immunoreactive levels and expression of TMPRSS2 in the lungs, LV, RV and kidneys of compensated, decompensated CHF and sham controls as were determined with western blots analysis and RT‐qPCR. A, Representative western‐blot analysis of tissue lysates with antibody for TMPRSS2. Western‐blot analysis quantification of TMPRSS2 in the lungs, LV, RV and kidneys (B, D, F and H, respectively) of sham, compensated CHF and decompensated CHF (n = 3‐10), where GAPDH was used as loading control. Quantification of RT‐qPCR analysis for TMPRSS2 mRNA normalized to GAPDH are depicted in C, E, G and I. Values are means ± SEM

Figure 5

Immunoreactive levels and expression of ADAM17 in the lungs, LV, RV and kidneys of compensated, decompensated CHF and sham controls as were determined with western blots analysis and RT‐qPCR. A, Representative western blot analysis of tissue lysates with antibody for ADAM17. Western‐blot analysis quantification of ADAM17 in the lungs, LV, RV and kidneys (B, D, F and H, respectively) of sham, compensated CHF and decompensated CHF (n = 3‐10), where GAPDH was used as loading control. Quantification of RT‐qPCR analysis for ADAM17 mRNA normalized to GAPDH are depicted in C, E, G and I. Values are means ± SEM

Figure 6

Histological changes in lung, heart and kidney tissues from rats with compensated and decompensated CHF as compared to their sham controls. Slices from lung, heart and kidneys of the various experimental groups were stained with primary antibodies of ACE2 (A), Furin (B) and TMPRSS2 (C). Cy™3 Donkey Anti‐Rabbit IgG, Cy™3 Donkey Anti‐Goat IgG and Cy™3 Donkey Anti‐Mouse IgG were used as secondary antibodies together with DAPI Fluoromount‐G^® for nuclear staining. Images were captured using a Widefield Zeiss Upright microscope and analysed with Zen software. Representative images of the cardiac, pulmonary and renal tissues were obtained at 40×, 20× and 20× magnification, respectively

Figure 7

Circulatory levels of soluble ACE2 (sACE2) and furin in rats with compensated and decompensated CHF and their sham controls. Plasma samples from the various experimental groups (n = 4‐9) were collected and analysed for ACE2 (A) and furin (B) levels by utilizing commercial ELISA assays. Values are means ± SEM

Figure 8

SARS‐CoV‐2 binding, activation, invasion and replication in target cells. The initial step after the invasion of SARS‐CoV‐2 is binding to membranal ACE2 widely expressed in vital organs including lung, heart and kidney. ACE2 is responsible for the conversion of Ang II to Ang 1‐7 which exerts beneficial effects on the cardiac tissue such as vasodilation, anti‐fibrosis and anti‐inflammation via Mas receptor (MasR). The binding of SARS‐CoV‐2 to ACE2 is preceded by TMPRSS2/furin‐mediated exposure of the viral receptor binding protein (RBP) localized to S‐glycoprotein (S1 domain of the viral spike) and revealing the viral effusion site on S2 domain. Furin is expressed in these tissues both intracellularly and in the circulation as a free enzyme, making it a key factor along TMPRSS2 in the uncovering of RBP and eventually in SARS‐CoV‐2 transmission. In addition, furin enhances the affinity of the virus to ACE2, not only by exposing the viral binding site on S1 domain but also by revealing the effusion site on the S2 domain in the viral spike. Consequently, the virus undergoes endocytosis and massive replication accompanied by profound activation by the abundant intracellular furin and Cathepsin L (Cat‐L). The activated intracellular SARS‐CoV‐2 undergoes exocytosis where it binds again to ACE2 elsewhere, thus creating a vicious feed‐forward devastating cycle. According to the current study, compensated, but not decompensated congestive heart failure (CHF) is characterized by enhanced expression of myocardial ACE2 and downregulation of TMPRSS2. ADAM17 is responsible for shedding of ACE2, a process stimulated by AT1 receptor (AT1‐R) and may explain why renin angiotensin aldosterone system (RAAS) inhibitors augment ACE2 expression.