Kidney Angiotensin in Cardiovascular Disease: Formation and Drug Targeting.

The concept of local formation of angiotensin II in the kidney has changed over the last 10-15 years. Local synthesis of angiotensinogen in the proximal tubule has been proposed, combined with prorenin synthesis in the collecting duct. Binding of prorenin via the so-called (pro)renin receptor has been introduced, as well as megalin-mediated uptake of filtered plasma-derived renin-angiotensin system (RAS) components. Moreover, angiotensin metabolites other than angiotensin II [notably angiotensin-(1-7)] exist, and angiotensins exert their effects via three different receptors, of which angiotensin II type 2 and Mas receptors are considered renoprotective, possibly in a sex-specific manner, whereas angiotensin II type 1 (AT1) receptors are believed to be deleterious. Additionally, internalized angiotensin II may stimulate intracellular receptors. Angiotensin-converting enzyme 2 (ACE2) not only generates angiotensin-(1-7) but also acts as coronavirus receptor. Multiple, if not all, cardiovascular diseases involve the kidney RAS, with renal AT1 receptors often being claimed to exert a crucial role. Urinary RAS component levels, depending on filtration, reabsorption, and local release, are believed to reflect renal RAS activity. Finally, both existing drugs (RAS inhibitors, cyclooxygenase inhibitors) and novel drugs (angiotensin receptor/neprilysin inhibitors, sodium-glucose cotransporter-2 inhibitors, soluble ACE2) affect renal angiotensin formation, thereby displaying cardiovascular efficacy. Particular in the case of the latter three, an important question is to what degree they induce renoprotection (e.g., in a renal RAS-dependent manner). This review provides a unifying view, explaining not only how kidney angiotensin formation occurs and how it is affected by drugs but also why drugs are renoprotective when altering the renal RAS. SIGNIFICANCE STATEMENT: Angiotensin formation in the kidney is widely accepted but little understood, and multiple, often contrasting concepts have been put forward over the last two decades. This paper offers a unifying view, simultaneously explaining how existing and novel drugs exert renoprotection by interfering with kidney angiotensin formation.

A Novel Soluble ACE2 Protein Provides Lung and Kidney Protection in Mice Susceptible to Lethal SARS-CoV-2 Infection.

BACKGROUND: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) uses full-length angiotensin converting enzyme 2 (ACE2) as a main receptor to enter target cells. The goal of this study was to demonstrate the preclinical efficacy of a novel soluble ACE2 protein with increased duration of action and binding capacity in a lethal mouse model of COVID-19. METHODS: A human soluble ACE2 variant fused with an albumin binding domain (ABD) was linked via a dimerization motif hinge-like 4-cysteine dodecapeptide (DDC) to improve binding capacity to SARS-CoV-2. This novel soluble ACE2 protein (ACE2-1-618-DDC-ABD) was then administered intranasally and intraperitoneally to mice before intranasal inoculation of SARS-CoV-2 and then for two additional days post viral inoculation. RESULTS: Untreated animals became severely ill, and all had to be humanely euthanized by day 6 or 7 and had pulmonary alveolar hemorrhage with mononuclear infiltrates. In contrast, all but one mouse infected with a lethal dose of SARS-CoV-2 that received ACE2-1-618-DDC-ABD survived. In the animals inoculated with SARS-CoV-2 that were untreated, viral titers were high in the lungs and brain, but viral titers were absent in the kidneys. Some untreated animals, however, had variable degrees of kidney proximal tubular injury as shown by attenuation of the proximal tubular brush border and increased NGAL and TUNEL staining. Viral titers in the lung and brain were reduced or nondetectable in mice that received ACE2-1-618-DDC-ABD, and the animals developed only moderate disease as assessed by a near-normal clinical score, minimal weight loss, and improved lung and kidney injury. CONCLUSIONS: This study demonstrates the preclinical efficacy of a novel soluble ACE2 protein, termed ACE2-1-618-DDC-ABD, in a lethal mouse model of SARS-CoV-2 infection that develops severe lung injury and variable degrees of moderate kidney proximal tubular injury.

A Novel Soluble ACE2 Variant with Prolonged Duration of Action Neutralizes SARS-CoV-2 Infection in Human Kidney Organoids.

BACKGROUND: There is an urgent need for approaches to prevent and treat SARS-CoV-2 infection. Administration of soluble ACE2 protein acting as a decoy to bind to SARS-CoV-2 should limit viral uptake mediated by binding to membrane-bound full-length ACE2, and further therapeutic benefit should result from ensuring enzymatic ACE2 activity to affected organs in patients with COVID-19. METHODS: A short variant of human soluble ACE2 protein consisting of 618 amino acids (hACE2 1-618) was generated and fused with an albumin binding domain (ABD) using an artificial gene encoding ABDCon, with improved albumin binding affinity. Human kidney organoids were used for infectivity studies of SARS-CoV-2 in a BSL-3 facility to examine the neutralizing effect of these novel ACE2 variants. RESULTS: Whereas plasma ACE2 activity of the naked ACE2 1-618 and ACE2 1-740 lasted about 8 hours, the ACE2 1-618-ABD resulted in substantial activity at 96 hours, and it was still biologically active 3 days after injection. Human kidney organoids express ACE2 and TMPRSS2, and when infected with SARS-CoV-2, our modified long-acting ACE2 variant neutralized infection. CONCLUSIONS: This novel ACE2 1-618-ABD can neutralize SARS-CoV-2 infectivity in human kidney organoids, and its prolonged duration of action should ensure improved efficacy to prevent viral escape and dosing convenience.

Soluble Angiotensin-Converting Enzyme 2 Protein Improves Survival and Lowers Viral Titers in Lethal Mouse Model of Severe Acute Respiratory Syndrome Coronavirus Type 2 Infection with the Delta Variant.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) utilizes angiotensin-converting enzyme 2 (ACE2) as its main receptor for cell entry. We bioengineered a soluble ACE2 protein termed ACE2 618-DDC-ABD that has increased binding to SARS-CoV-2 and prolonged duration of action. Here, we investigated the protective effect of this protein when administered intranasally to k18-hACE2 mice infected with the aggressive SARS-CoV-2 Delta variant. k18-hACE2 mice were infected with the SARS-CoV-2 Delta variant by inoculation of a lethal dose (2 × 104 PFU). ACE2 618-DDC-ABD (10 mg/kg) or PBS was administered intranasally six hours prior and 24 and 48 h post-viral inoculation. All animals in the PBS control group succumbed to the disease on day seven post-infection (0% survival), whereas, in contrast, there was only one casualty in the group that received ACE2 618-DDC-ABD (90% survival). Mice in the ACE2 618-DDC-ABD group had minimal disease as assessed using a clinical score and stable weight, and both brain and lung viral titers were markedly reduced. These findings demonstrate the efficacy of a bioengineered soluble ACE2 decoy with an extended duration of action in protecting against the aggressive Delta SARS-CoV-2 variant. Together with previous work, these findings underline the universal protective potential against current and future emerging SARS-CoV-2 variants.

Intranasal soluble ACE2 improves survival and prevents brain SARS-CoV-2 infection.

A soluble ACE2 protein bioengineered for long duration of action and high affinity to SARS-CoV-2 was administered either intranasally (IN) or intraperitoneally (IP) to SARS-CoV-2-inoculated k18hACE2 mice. This decoy protein (ACE2 618-DDC-ABD) was given either IN or IP, pre- and post-inoculation, or IN, IP, or IN + IP but only post-inoculation. Survival by day 5 was 0% in untreated mice, 40% in the IP-pre, and 90% in the IN-pre group. In the IN-pre group, brain histopathology was essentially normal and lung histopathology significantly improved. Consistent with this, brain SARS-CoV-2 titers were undetectable and lung titers reduced in the IN-pre group. When ACE2 618-DDC-ABD was administered only post-inoculation, survival was 30% in the IN + IP, 20% in the IN, and 20% in the IP group. We conclude that ACE2 618-DDC-ABD results in markedly improved survival and provides organ protection when given intranasally as compared with when given either systemically or after viral inoculation, and that lowering brain titers is a critical determinant of survival and organ protection.

Superiority of intranasal over systemic administration of bioengineered soluble ACE2 for survival and brain protection against SARS-CoV-2 infection.

The present study was designed to investigate the effects of a soluble ACE2 protein termed ACE2 618-DDC-ABD, bioengineered to have long duration of action and high binding affinity to SARS-CoV-2, when administered either intranasally (IN) or intraperitoneally (IP) and before or after SARS-CoV-2 inoculation. K18hACE2 mice permissive for SARS-CoV-2 infection were inoculated with 2Ã-10 4 PFU wildtype SARS-CoV-2. In one protocol, ACE2 618-DDC-ABD was given either IN or IP, pre- and post-viral inoculation. In a second protocol, ACE2 618-DDC-ABD was given either IN, IP or IN+IP but only post-viral inoculation. In addition, A549 and Vero E6 cells were used to test neutralization of SARS-CoV-2 variants by ACE2 618-DDC-ABD at different concentrations. Survival by day 5 was 0% in infected untreated mice, and 40% in mice from the ACE2 618-DDC-ABD IP-pre treated group. By contrast, in the IN-pre group survival was 90%, histopathology of brain and kidney was essentially normal and markedly improved in the lungs. When ACE2 618-DDC-ABD was administered only post viral inoculation, survival was 30% in the IN+IP group, 20% in the IN and 0% in the IP group. Brain SARS-CoV-2 titers were high in all groups except for the IN-pre group where titers were undetectable in all mice. In cells permissive for SARS-CoV-2 infection, ACE2 618-DDC-ABD neutralized wildtype SARS-CoV-2 at high concentrations, whereas much lower concentrations neutralized omicron BA. 1. We conclude that ACE2 618-DDC-ABD provides much better survival and organ protection when administered intranasally than when given systemically or after viral inoculation and that lowering brain titers is a critical determinant of survival and organ protection.

Evidence For and Against Direct Kidney Infection by SARS-CoV-2 in Patients with COVID-19.

Despite evidence of multiorgan tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients with coronavirus disease 2019 (COVID-19), direct viral kidney invasion has been difficult to demonstrate. The question of whether SARS-CoV2 can directly infect the kidney is relevant to the understanding of pathogenesis of AKI and collapsing glomerulopathy in patients with COVID-19. Methodologies to document SARS-CoV-2 infection that have been used include immunohistochemistry, immunofluorescence, RT-PCR, in situ hybridization, and electron microscopy. In our review of studies to date, we found that SARS-CoV-2 in the kidneys of patients with COVID-19 was detected in 18 of 94 (19%) by immunohistochemistry, 71 of 144 (49%) by RT-PCR, and 11 of 84 (13%) by in situ hybridization. In a smaller number of patients with COVID-19 examined by immunofluorescence, SARS-CoV-2 was detected in 10 of 13 (77%). In total, in kidneys from 102 of 235 patients (43%), the presence of SARS-CoV-2 was suggested by at least one of the methods used. Despite these positive findings, caution is needed because many other studies have been negative for SARS-CoV-2 and it should be noted that when detected, it was only in kidneys obtained at autopsy. There is a clear need for studies from kidney biopsies, including those performed at early stages of the COVID-19-associated kidney disease. Development of tests to detect kidney viral infection in urine samples would be more practical as a noninvasive way to evaluate SARS-CoV-2 infection during the evolution of COVID-19-associated kidney disease.

A novel soluble ACE2 protein totally protects from lethal disease caused by SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) uses full-length angiotensin converting enzyme 2 (ACE2), which is membrane bound, as its initial cell contact receptor preceding viral entry. Here we report a human soluble ACE2 variant fused with a 5kD albumin binding domain (ABD) and bridged via a dimerization motif hinge-like 4-cysteine dodecapeptide, which we term ACE2 1-618-DDC-ABD. This protein is enzymatically active, has increased duration of action in vivo conferred by the ABD-tag, and displays 20-30-fold higher binding affinity to the SARS-CoV-2 receptor binding domain than its des-DDC monomeric form (ACE2 1-618-ABD) due to DDC-linked dimerization. ACE2 1-618-DDC-ABD was administered for 3 consecutive days to transgenic k18-hACE2 mice, a model that develops lethal SARS-CoV-2 infection, to evaluate the preclinical preventative/ therapeutic value for COVID-19. Mice treated with ACE2 1-618-DDC-ABD developed a mild to moderate disease for the first few days assessed by a clinical score and modest weight loss. The untreated control animals, by contrast, became severely ill and had to be sacrificed by day 6/7 and lung histology revealed extensive pulmonary alveolar hemorrhage and mononuclear infiltrates. At 6 days, mortality was totally prevented in the treated group, lung histopathology was improved and viral titers markedly reduced. This demonstrates for the first time in vivo the preventative/ therapeutic potential of a novel soluble ACE2 protein in a preclinical animal model.