RESUMEN
There are diverse pathophysiological mechanisms involved in acute kidney injury (AKI). Among them, overactivity of the renin angiotensin system (RAS) has been described. Angiotensin converting enzyme 2 (ACE2) is a tissue RAS enzyme expressed in the apical border of proximal tubules. Given the important role of ACE2 in the metabolism of Angiotensin II this study was aimed to characterize kidney and urinary ACE2 in amouse model of AKI. Ischemia reperfusion injury (IRI) was induced in C57BL/6 mice by clamping of the left renal artery followed by removal of the right kidney. In kidneys harvested 48 hours after IRI, immunostaining revealed a striking maldistribution of ACE2 including spillage into the tubular lumen and presence of ACE2 positive luminal casts in the medulla. In cortical membranes ACE2 protein and enzymatic activity were both markedly reduced (37±4 vs. 100±6 ACE2/ß-Actin, P=0.0004 and 96±14 vs. 152±6 RFU/µg protein/h P=0.006). In urine, the full-length membrane bound ACE2 protein (100kD) was markedly increased (1120±405 vs. 100±46 ACE2/µg Crea, P=0.04) and casts stained for ACE2 were recovered in the urine sediment. In AKI caused by IRI there is a marked loss of ACE2 from the apical tubular border with deposition of ACE2 positive material in the medulla and increased urinary excretion of the full length-membrane bound ACE2 protein. The deficiency of tubular ACE2 in AKI suggests that provision of this enzyme could have therapeutic applications and that its excretion in the urine may also serve as a diagnostic marker of severe proximal tubular injury.
RESUMEN
Urinary [Formula: see text] excretion is decreased in chronic kidney disease (CKD), but very little is known about fecal [Formula: see text] excretion. Sodium zirconium cyclosilicate (SZC) is a cation exchanger that selectively captures K+ in the gastrointestinal tract. We investigated if SZC can sequester [Formula: see text] in vivo and evaluated the effect of SZC on fecal [Formula: see text] in a mouse model of CKD. Mice with CKD induced by 5/6 kidney ablation were fed either a regular diet or a diet containing SZC (4 g/kg) and followed for 7 days. Fecal [Formula: see text] was measured before and after the addition of 50 meq KCl/L to release [Formula: see text] from SZC. [Formula: see text] sequestered in SZC in the gastrointestinal (GI) tract was estimated from the change in fecal [Formula: see text] observed when KCl was added to liberate the sequestered [Formula: see text]. In mice with CKD, fecal [Formula: see text] excretion was higher than in normal mice and also higher than urine [Formula: see text] excretion measured concurrently. Using data pooled from the SZC diet, the change in [Formula: see text] was 6.5 ± 0.6 compared with 0.6 ± 0.6 µmol/g on the normal diet (P < 0.0001). In conclusion, fecal [Formula: see text] excretion in CKD is increased and about sixfold higher than urine [Formula: see text] excretion, revealing an important route of elimination of [Formula: see text] present in the GI tract. SZC administration sequesters a substantial portion of [Formula: see text] in the GI tract, suggesting that the binding of [Formula: see text] offers therapeutic potential beyond its known primary action as a specific K+ binder.NEW & NOTEWORTHY Fecal [Formula: see text] excretion in chronic kidney disease is increased and about sixfold higher than urine [Formula: see text] excretion, revealing an important route of elimination of [Formula: see text] that is present in the gastrointestinal tract. Sodium zirconium cyclosilicate (SZC) administration sequesters a substantial portion of [Formula: see text], suggesting that binding of [Formula: see text] by SZC in the gastrointestinal tract offers therapeutic potential in chronic kidney disease and other clinical conditions beyond its known primary action of SZC as a specific K+ binder.
Asunto(s)
Hiperpotasemia , Insuficiencia Renal Crónica , Animales , Ratones , Potasio , Tracto GastrointestinalRESUMEN
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.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Enzima Convertidora de Angiotensina 2/uso terapéutico , Animales , COVID-19/terapia , Riñón/virología , Pulmón/virología , Ratones , SARS-CoV-2RESUMEN
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.
RESUMEN
Aminopeptidase A is one of the most potent enzymes within the renin-angiotensin system in terms of angiotensin II degradation. Here, we examined whether there is a kidney phenotype and any compensatory changes in other renin angiotensin system enzymes involved in the metabolism of angiotensin II associated with aminopeptidase A deficiency. Kidneys harvested from aminopeptidase A knockout mice were examined by light and electron microscopy, immunohistochemistry and immunofluorescence. Kidney angiotensin II levels and the ability of renin angiotensin system enzymes in the glomerulus to degrade angiotensin II ex vivo, their activities, protein and mRNA levels in kidney lysates were evaluated. Knockout mice had increased blood pressure and mild glomerular mesangial expansion without significant albuminuria. By electron microscopy, knockout mice exhibited a mild increase of the mesangial matrix, moderate thickening of the glomerular basement membrane but a striking appearance of knob-like structures. These knobs were seen in both male and female mice and persisted after the treatment of hypertension. In isolated glomeruli from knockout mice, the level of angiotensin II was more than three-fold higher as compared to wild type control mice. In kidney lysates from knockout mice angiotensin converting enzyme activity, protein and mRNA levels were markedly decreased possibly as a compensatory mechanism to reduce angiotensin II formation. Thus, our findings support a role for aminopeptidase A in the maintenance of glomerular structure and intra-kidney homeostasis of angiotensin peptides.
Asunto(s)
Membrana Basal Glomerular , Glutamil Aminopeptidasa , Riñón , Angiotensina II/metabolismo , Animales , Femenino , Membrana Basal Glomerular/metabolismo , Glutamil Aminopeptidasa/genética , Glutamil Aminopeptidasa/metabolismo , Riñón/metabolismo , Masculino , Ratones , Ratones Noqueados , Sistema Renina-Angiotensina/genéticaRESUMEN
Angiotensin converting enzyme 2 (ACE2) plays an important role in inflammation, which is attributable at least, in part, to the conversion of the pro-inflammatory angiotensin (Ang) II peptide into angiotensin 1-7 (Ang 1-7), a peptide which opposes the actions of AngII. ACE2 and AngII are present in many tissues but information on the cornea is lacking. We observed that mice deficient in the Ace2 gene (Ace2-/- ), developed a cloudy cornea phenotype as they aged. Haze occupied the central cornea, accompanied by corneal edema and neovascularization. In severe cases with marked chronic inflammation, a cell-fate switch from a transparent corneal epithelium to a keratinized, stratified squamous, psoriasiform-like epidermis was observed. The stroma contained a large number of CD11c, CD68, and CD3 positive cells. Corneal epithelial debridement experiments in young ACE2-deficient mice showed normal appearing corneas, devoid of haze. We hypothesized, however, that these mice are "primed" for a corneal inflammatory response, which once initiated, would persist. In vitro studies reveal that interleukins (IL-1a, IL-1b), chemokines (CCL2, CXCL8), and TNF-α, are all significantly elevated, resulting in a cytokine storm-like phenotype. This phenotype could be partially rescued by treatment with the AngII type 1 receptor (AT1R) antagonist, losartan, suggesting that the observed effect was mediated by AngII acting on its main receptor. Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes human ACE2 as the receptor for entry with subsequent downregulation of ACE2, corneal inflammation in Ace2-/- mice may have a similar mechanism with that in COVID-19 patients. Thus the Ace2-/- cornea, because of easy accessibility, may provide an attractive model to explore the molecular mechanisms, immunological changes, and treatment modalities in patients with COVID-19.
Asunto(s)
Enzima Convertidora de Angiotensina 2/genética , Córnea/patología , Síndrome de Liberación de Citoquinas/fisiopatología , Modelos Animales de Enfermedad , Angiotensina II/metabolismo , Animales , COVID-19 , Células Cultivadas , Quimiocinas/metabolismo , Células Epiteliales/metabolismo , Humanos , Interleucinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , SARS-CoV-2 , Células THP-1 , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Angiotensin-converting enzyme II (ACE2) is a homologue of angiotensin-converting enzyme discovered in 2000. From the initial discovery, it was recognized that the kidneys were organs very rich on ACE2. Subsequent studies demonstrated the precise localization of ACE2 within the kidney and the importance of this enzyme in the metabolism of Angiotensin II and the formation of Angiotensin 1-7. With the recognition early in 2020 of ACE2 being the main receptor of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), the interest in this protein has dramatically increased. In this review, we will focus on kidney ACE2; its localization, its alterations in hypertension, diabetes, the effect of ACE inhibitors and angiotensin type 1 receptor blockers (ARBs) on ACE2 and the potential use of ACE2 recombinant proteins therapeutically for kidney disease. We also describe the emerging kidney manifestations of COVID-19, namely the frequent development of acute kidney injury. The possibility that binding of SARS-CoV-2 to kidney ACE2 plays a role in the kidney manifestations is also briefly discussed.
Asunto(s)
Betacoronavirus/patogenicidad , Infecciones por Coronavirus/enzimología , Enfermedades Renales/enzimología , Riñón/enzimología , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/enzimología , Receptores Virales/metabolismo , Lesión Renal Aguda/enzimología , Lesión Renal Aguda/virología , Enzima Convertidora de Angiotensina 2 , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Antivirales/uso terapéutico , Betacoronavirus/efectos de los fármacos , COVID-19 , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Diabetes Mellitus/enzimología , Diabetes Mellitus/fisiopatología , Historia del Siglo XXI , Interacciones Huésped-Patógeno , Humanos , Hipertensión/enzimología , Hipertensión/fisiopatología , Riñón/fisiopatología , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/fisiopatología , Pandemias , Peptidil-Dipeptidasa A/historia , Peptidil-Dipeptidasa A/uso terapéutico , Neumonía Viral/virología , Receptores Virales/historia , SARS-CoV-2 , Tratamiento Farmacológico de COVID-19RESUMEN
A new coronavirus, referred to as SARS-CoV-2, is responsible for the recent outbreak of severe respiratory disease. This outbreak first detected in Wuhan, China in December 2019, has spread to other regions of China and to 25 other countries as of January, 2020. It has been known since the 2003 SARS epidemic that the receptor critical for SARS-CoV entry into host cells is the angiotensin-converting enzyme 2 (ACE2). The S1 domain of the spike protein of SARS-CoV attaches the virus to its cellular receptor ACE2 on the host cells. We thought that it is timely to explain the connection between the SARS-CoV, SARS-CoV-2, ACE2 and the rationale for soluble ACE2 as a potential therapy.
Asunto(s)
Betacoronavirus/patogenicidad , Infecciones por Coronavirus/tratamiento farmacológico , Peptidil-Dipeptidasa A , Neumonía Viral/tratamiento farmacológico , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Acoplamiento Viral , Enzima Convertidora de Angiotensina 2 , Animales , COVID-19 , Línea Celular , Haplorrinos , Humanos , Peptidil-Dipeptidasa A/fisiología , Proteínas Recombinantes/uso terapéutico , SARS-CoV-2 , Solubilidad , Replicación Viral , Tratamiento Farmacológico de COVID-19RESUMEN
Angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase that potently degrades angiotensin II to angiotensin 1-7. Previous studies showed that injection of the enzymatic ectodomain of recombinant ACE2 (rACE2) markedly increases circulatory levels of ACE2 activity, and effectively lowered blood pressure in angiotensin II-induced hypertension. However, due to the short plasma half-life of rACE2, its therapeutic potential for chronic use is limited. To circumvent this, we generated a chimeric fusion of rACE2 and the immunoglobulin fragment Fc segment to increase its plasma stability. This rACE2-Fc fusion protein retained full peptidase activity and exhibited greatly extended plasma half-life in mice, from less than two hours of the original rACE2, to over a week. A single 2.5 mg/kg injection of rACE2-Fc increased the overall angiotensin II-conversion activities in blood by up to 100-fold and enhanced blood pressure recovery from acute angiotensin II induced hypertension seven days after administration. To assess rACE2-Fc given weekly on cardiac protection, we performed studies in mice continuously infused with angiotensin II for 28 days and in a Renin transgenic mouse model of hypertension. The angiotensin II infused mice achieved sustained blood pressure control and reduced cardiac hypertrophy and fibrosis. In chronic hypertensive transgenic mice, weekly injections of rACE2-Fc effectively lowered plasma angiotensin II and blood pressure. Additionally, rACE2-Fc ameliorated albuminuria, and reduced kidney and cardiac fibrosis. Thus, our chimeric fusion strategy for rACE2-Fc is suitable for future development of new renin angiotensin system-based inhibition therapies.
Asunto(s)
Hipertensión/tratamiento farmacológico , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Peptidil-Dipeptidasa A/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Angiotensina II/administración & dosificación , Angiotensina II/sangre , Enzima Convertidora de Angiotensina 2 , Animales , Línea Celular , Modelos Animales de Enfermedad , Femenino , Semivida , Humanos , Hipertensión/etiología , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/aislamiento & purificación , Fragmentos Fc de Inmunoglobulinas/farmacología , Ratones , Ratones Endogámicos BALB C , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/aislamiento & purificación , Peptidil-Dipeptidasa A/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Renina/genética , Sistema Renina-Angiotensina/efectos de los fármacos , Resultado del TratamientoRESUMEN
The pathways implicated in diabetic kidney disease (DKD) are largely derived from animal models. To examine if alterations in renin-angiotensin system (RAS) in humans are concordant with those in rodent models, we measured concentration of angiotensinogen (AOG), cathepsin D (CTSD), angiotensin-converting enzyme (ACE), and ACE2 and enzymatic activities of ACE, ACE2, and aminopeptidase-A in FVB mice 13-20 wk after treatment with streptozotocin (n = 9) or vehicle (n = 15) and people with long-standing type 1 diabetes, with (n = 37) or without (n = 81) DKD. In streptozotocin-treated mice, urine AOG and CTSD were 10.4- and 3.0-fold higher than in controls, respectively (P < 0.001). Enzymatic activities of ACE, ACE2, and APA were 6.2-, 3.2-, and 18.8-fold higher, respectively, in diabetic animals (P < 0.001). Angiotensin II was 2.4-fold higher in diabetic animals (P = 0.017). Compared with people without DKD, those with DKD had higher urine AOG (170 vs. 15 µg/g) and CTSD (147 vs. 31 µg/g). In people with DKD, urine ACE concentration was 1.8-fold higher (1.4 vs. 0.8 µg/g in those without DKD), while its enzymatic activity was 0.6-fold lower (1.0 vs. 1.6 × 109 RFU/g in those without DKD). Lower ACE activity, but not ACE protein concentration, was associated with ACE inhibitor (ACEI) treatment. After adjustment for clinical covariates, AOG, CTSD, ACE concentration, and ACE activity remained associated with DKD. In conclusion, in mice with streptozotocin-induced diabetes and in humans with DKD, urine concentrations and enzymatic activities of several RAS components are concordantly increased, consistent with enhanced RAS activity and greater angiotensin II formation. ACEI use was associated with a specific reduction in urine ACE activity, not ACE protein concentration, suggesting that it may be a marker of exposure to this widely-used therapy.
Asunto(s)
Diabetes Mellitus Experimental/orina , Diabetes Mellitus Tipo 1/orina , Nefropatías Diabéticas/orina , Enzimas/orina , Insuficiencia Renal Crónica/orina , Sistema Renina-Angiotensina , Adulto , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Biomarcadores/orina , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/diagnóstico , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/diagnóstico , Nefropatías Diabéticas/diagnóstico , Nefropatías Diabéticas/etiología , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Insuficiencia Renal Crónica/diagnóstico , Insuficiencia Renal Crónica/etiología , Sistema Renina-Angiotensina/efectos de los fármacos , Regulación hacia Arriba , UrinálisisRESUMEN
Blockers of the renin-angiotensin system are effective in the treatment of experimental and clinical diabetic nephropathy. An approach different from blocking the formation or action of angiotensin II (1-8) that could also be effective involves fostering its degradation. Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase that cleaves angiotensin II (1-8) to form angiotensin (1-7). Therefore, we examined the renal effects of murine recombinant ACE2 in mice with streptozotocin-induced diabetic nephropathy as well as that of amplification of circulating ACE2 using minicircle DNA delivery prior to induction of experimental diabetes. This delivery resulted in a long-term sustained and profound increase in serum ACE2 activity and enhanced ability to metabolize an acute angiotensin II (1-8) load. In mice with streptozotocin-induced diabetes pretreated with minicircle ACE2, ACE2 protein in plasma increased markedly and this was associated with a more than 100-fold increase in serum ACE2 activity. However, minicircle ACE2 did not result in changes in urinary ACE2 activity as compared to untreated diabetic mice. In both diabetic groups, glomerular filtration rate increased significantly and to the same extent as compared to non-diabetic controls. Albuminuria, glomerular mesangial expansion, glomerular cellularity, and glomerular size were all increased to a similar extent in minicircle ACE2-treated and untreated diabetic mice, as compared to non-diabetic controls. Recombinant mouse ACE2 given for 4 weeks by intraperitoneal daily injections in mice with streptozotocin-induced diabetic nephropathy also failed to improve albuminuria or kidney pathology. Thus, a profound augmentation of ACE2 confined to the circulation failed to ameliorate the glomerular lesions and hyperfiltration characteristic of early diabetic nephropathy. These findings emphasize the importance of targeting the kidney rather than the circulatory renin angiotensin system to combat diabetic nephropathy.
Asunto(s)
Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/etiología , Riñón/enzimología , Peptidil-Dipeptidasa A/sangre , Albuminuria/enzimología , Albuminuria/etiología , Albuminuria/genética , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Enzima Convertidora de Angiotensina 2 , Animales , Autoantígenos/genética , Colágeno Tipo IV/deficiencia , Colágeno Tipo IV/genética , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/enzimología , Nefropatías Diabéticas/sangre , Nefropatías Diabéticas/enzimología , Nefropatías Diabéticas/fisiopatología , Femenino , Tasa de Filtración Glomerular , Riñón/patología , Riñón/fisiopatología , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos/metabolismo , Peptidil-Dipeptidasa A/administración & dosificación , Peptidil-Dipeptidasa A/genética , Proteínas Recombinantes/administración & dosificación , Factores de TiempoAsunto(s)
Antagonistas de Receptores de Angiotensina/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Betacoronavirus , Infecciones por Coronavirus/etiología , Riñón/enzimología , Pulmón/enzimología , Peptidil-Dipeptidasa A/genética , Neumonía Viral/etiología , Enzima Convertidora de Angiotensina 2 , Animales , COVID-19 , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Ratones , Pandemias , Peptidil-Dipeptidasa A/fisiología , SARS-CoV-2RESUMEN
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.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Melfalán , gammaglobulinas , Humanos , Ratones , Animales , Peptidil-Dipeptidasa A/metabolismo , SARS-CoV-2/metabolismoRESUMEN
Severe cases of COVID-19 are characterized by development of acute respiratory distress syndrome (ARDS). Water accumulation in the lungs is thought to occur as consequence of an exaggerated inflammatory response. A possible mechanism could involve decreased activity of the epithelial Na+ channel, ENaC, expressed in type II pneumocytes. Reduced transepithelial Na+ reabsorption could contribute to lung edema due to reduced alveolar fluid clearance. This hypothesis is based on the observation of the presence of a novel furin cleavage site in the S protein of SARS-CoV-2 that is identical to the furin cleavage site present in the alpha subunit of ENaC. Proteolytic processing of αENaC by furin-like proteases is essential for channel activity. Thus, competition between S protein and αENaC for furin-mediated cleavage in SARS-CoV-2-infected cells may negatively affect channel activity. Here we present experimental evidence showing that coexpression of the S protein with ENaC in a cellular model reduces channel activity. In addition, we show that bidirectional competition for cleavage by furin-like proteases occurs between ãENaC and S protein. In transgenic mice sensitive to lethal SARS-CoV-2, however, a significant decrease in gamma ENaC expression was not observed by immunostaining of lungs infected as shown by SARS-CoV2 nucleoprotein staining.
Asunto(s)
COVID-19 , Canales Epiteliales de Sodio , Furina , Ratones Transgénicos , Proteolisis , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Canales Epiteliales de Sodio/metabolismo , Animales , Humanos , Ratones , Furina/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/metabolismo , COVID-19/metabolismo , COVID-19/virología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/virología , Pulmón/metabolismo , Pulmón/virología , Pulmón/patología , Células HEK293RESUMEN
Angiotensin-converting enzyme-2 (ACE2) enhances the degradation of ANG II and its expression is altered in diabetic kidneys, but the regulation of this enzyme in the urine is unknown. Urinary ACE2 was studied in the db/db model of type 2 diabetes and stretozotocin (STZ)-induced type 1 diabetes during several physiological and pharmacological interventions. ACE2 activity in db/db mice was increased in the serum and to a much greater extent in the urine compared with db/m controls. Neither a specific ANG II blocker, telmisartan, nor an ACE inhibitor, captopril, altered the levels of urinary ACE2 in db/db or db/m control mice. High-salt diet (8%) increased whereas low-salt diet (0.1%) decreased urinary ACE2 activity in the urine of db/db mice. In STZ mice, urinary ACE2 was also increased, and insulin decreased it partly but significantly after several weeks of administration. The increase in urinary ACE2 activity in db/db mice reflected an increase in enzymatically active protein with two bands identified of molecular size at 110 and 75 kDa and was associated with an increase in kidney cortex ACE2 protein at 110 kDa but not at 75 kDa. ACE2 activity was increased in isolated tubular preparations but not in glomeruli from db/db mice. Administration of soluble recombinant ACE2 to db/m and db/db mice resulted in a marked increase in serum ACE2 activity, but no gain in ACE2 activity was detectable in the urine, further demonstrating that urinary ACE2 is of kidney origin. Increased urinary ACE2 was associated with more efficient degradation of exogenous ANG II (10(-9) M) in urine from db/db compared with that from db/m mice. Urinary ACE2 could be a potential biomarker of increased metabolism of ANG II in diabetic kidney disease.
Asunto(s)
Angiotensina II/metabolismo , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/orina , Peptidil-Dipeptidasa A/metabolismo , Peptidil-Dipeptidasa A/orina , Enzima Convertidora de Angiotensina 2 , Animales , Biomarcadores/metabolismo , Biomarcadores/orina , Western Blotting , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase that degrades angiotensin (Ang) II to Ang-(1-7). ACE2 is highly expressed within the kidneys, it is largely localized in tubular epithelial cells and less prominently in glomerular epithelial cells and in the renal vasculature. ACE2 activity has been shown to be altered in diabetic kidney disease, hypertensive renal disease and in different models of kidney injury. There is often a dissociation between tubular and glomerular ACE2 expression, particularly in diabetic kidney disease where ACE2 expression is increased at the tubular level but decreased at the glomerular level. In this review, we will discuss alterations in circulating and renal ACE2 recently described in different renal pathologies and disease models as well as their possible significance.
Asunto(s)
Enfermedades Renales/enzimología , Peptidil-Dipeptidasa A/metabolismo , Enzima Convertidora de Angiotensina 2 , Animales , Humanos , Enfermedades Renales/patología , Glomérulos Renales/enzimología , Túbulos Renales/enzimología , Sistema Renina-Angiotensina/fisiologíaRESUMEN
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.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Animales , Ratones , SARS-CoV-2 , EncéfaloRESUMEN
Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase that degrades angiotensin II with high efficiency leading to the formation of angiotensin-(1-7). ACE2 within the kidneys is largely localized in tubular epithelial cells and in glomerular epithelial cells. Decreased glomerular expression of this enzyme coupled with increased expression of ACE has been described in diabetic kidney disease, both in mice and humans with type 2 diabetes. Moreover, both ACE2 genetic ablation and pharmacological ACE2 inhibition have been shown to increase albuminuria and promote glomerular injury. Studies using recombinant ACE2 have shown the ability of ACE2 to rapidly metabolize Ang II in vivo and form the basis for future studies to examine the potential of ACE2 amplification in the therapy of diabetic kidney disease and cardiovascular disease.
Asunto(s)
Angiotensina II/metabolismo , Nefropatías Diabéticas/tratamiento farmacológico , Riñón/efectos de los fármacos , Peptidil-Dipeptidasa A/uso terapéutico , Angiotensina I/metabolismo , Enzima Convertidora de Angiotensina 2 , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Nefropatías Diabéticas/enzimología , Nefropatías Diabéticas/genética , Humanos , Riñón/enzimología , Fragmentos de Péptidos/metabolismo , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Proteínas Recombinantes/uso terapéuticoRESUMEN
Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin II to angiotensin-(1-7) and is expressed in podocytes. Here we overexpressed ACE2 in podocytes in experimental diabetic nephropathy using transgenic methods where a nephrin promoter drove the expression of human ACE2. Glomeruli from these mice had significantly increased mRNA, protein, and activity of ACE2 compared to wild-type mice. Male mice were treated with streptozotocin to induce diabetes. After 16 weeks, there was no significant difference in plasma glucose levels between wild-type and transgenic diabetic mice. Urinary albumin was significantly increased in wild-type diabetic mice at 4 weeks, whereas albuminuria in transgenic diabetic mice did not differ from wild-type nondiabetic mice. However, this effect was transient and by 16 weeks both transgenic and nontransgenic diabetic mice had similar rates of proteinuria. Compared to wild-type diabetic mice, transgenic diabetic mice had an attenuated increase in mesangial area, decreased glomerular area, and a blunted decrease in nephrin expression. Podocyte numbers decreased in wild-type diabetic mice at 16 weeks, but were unaffected in transgenic diabetic mice. At 8 weeks, kidney cortical expression of transforming growth factor-ß1 was significantly inhibited in transgenic diabetic mice as compared to wild-type diabetic mice. Thus, the podocyte-specific overexpression of human ACE2 transiently attenuates the development of diabetic nephropathy.