Angiotensin-converting Enzyme Shedding Research Articles

Effects of Angiotensin II Type 1A Receptor on ACE2, Neprilysin and KIM-1 in Two Kidney One Clip (2K1C) Model of Renovascular Hypertension.

Activation of the renin angiotensin system plays a pivotal role in the regulation of blood pressure, which is mainly attributed to the formation of angiotensin-II (Ang II). The actions of Ang II are mediated through binding to the Ang-II type 1 receptor (AT1R) which leads to increased blood pressure, fluid retention, and aldosterone secretion. In addition, Ang II is also involved in cell injury, vascular remodeling, and inflammation. The actions of Ang II could be antagonized by its conversion to the vasodilator peptide Ang (1–7), partly generated by the action of angiotensin converting enzyme 2 (ACE2) and/or neprilysin (NEP). Previous studies demonstrated increased urinary ACE2 shedding in the db/db mouse model of diabetic kidney disease. The aim of the study was to investigate whether renal and urinary ACE2 and NEP are altered in the 2K1C Goldblatt hypertensive mice. Since AT1R is highly expressed in the kidney, we also researched the effect of global deletion of AT1R on renal and urinary ACE2, NEP, and kidney injury marker (KIM-1). Hypertension and albuminuria were induced in AT1R knock out (AT1RKO) and WT mice by unilateral constriction of the renal artery of one kidney. The 24 h mean arterial blood pressure (MAP) was measured using radio-telemetry. Two weeks after 2K1C surgery, MAP and albuminuria were significantly increased in WT mice compared to AT1RKO mice. Results demonstrated a correlation between MAP and albuminuria. Unlike db/db diabetic mice, ACE2 and NEP expression and activities were significantly decreased in the clipped kidney of WT and AT1RKO compared with the contralateral kidney and sham control (p < 0.05). There was no detectable urinary ACE2 and NEP expression and activity in 2K1C mice. KIM-1 was significantly increased in the clipped kidney of WT and AT1KO (p < 0.05). Deletion of AT1R has no effect on the increased urinary KIM-1 excretion detected in 2K1C mice. In conclusion, renal injury in 2K1C Goldblatt mouse model is associated with loss of renal ACE2 and NEP expression and activity. Urinary KIM-1 could serve as an early indicator of acute kidney injury. Deletion of AT1R attenuates albuminuria and hypertension without affecting renal ACE2, NEP, and KIM-1 expression.

Angiotensin-converting enzyme 2 and COVID-19 in cardiorenal diseases.

The rapid spread of the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has brought into focus the key role of angiotensin-converting enzyme 2 (ACE2), which serves as a cell surface receptor required for the virus to enter cells. SARS-CoV-2 can decrease cell surface ACE2 directly by internalization of ACE2 bound to the virus and indirectly by increased ADAM17 (a disintegrin and metalloproteinase 17)-mediated shedding of ACE2. ACE2 is widely expressed in the heart, lungs, vasculature, kidney and the gastrointestinal (GI) tract, where it counteracts the deleterious effects of angiotensin II (AngII) by catalyzing the conversion of AngII into the vasodilator peptide angiotensin-(1-7) (Ang-(1-7)). The down-regulation of ACE2 by SARS-CoV-2 can be detrimental to the cardiovascular system and kidneys. Further, decreased ACE2 can cause gut dysbiosis, inflammation and potentially worsen the systemic inflammatory response and coagulopathy associated with SARS-CoV-2. This review aims to elucidate the crucial role of ACE2 both as a regulator of the renin–angiotensin system and a receptor for SARS-CoV-2 as well as the implications for Coronavirus disease 19 and its associated cardiovascular and renal complications.

Activation of Kinin B1R Upregulates ADAM17 and Results in ACE2 Shedding in Neurons.

Angiotensin converting enzyme 2 (ACE2) is a critical component of the compensatory axis of the renin angiotensin system. Alterations in ACE2 gene and protein expression, and activity mediated by A Disintegrin And Metalloprotease 17 (ADAM17), a member of the “A Disintegrin And Metalloprotease” (ADAM) family are implicated in several cardiovascular and neurodegenerative diseases. We previously reported that activation of kinin B1 receptor (B1R) in the brain increases neuroinflammation, oxidative stress and sympathoexcitation, leading to the development of neurogenic hypertension. We also showed evidence for ADAM17-mediated ACE2 shedding in neurons. However, whether kinin B1 receptor (B1R) activation has any role in altering ADAM17 activity and its effect on ACE2 shedding in neurons is not known. In this study, we tested the hypothesis that activation of B1R upregulates ADAM17 and results in ACE2 shedding in neurons. To test this hypothesis, we stimulated wild-type and B1R gene-deleted mouse neonatal primary hypothalamic neuronal cultures with a B1R-specific agonist and measured the activities of ADAM17 and ACE2 in neurons. B1R stimulation significantly increased ADAM17 activity and decreased ACE2 activity in wild-type neurons, while pretreatment with a B1R-specific antagonist, R715, reversed these changes. Stimulation with specific B1R agonist Lys-Des-Arg9-Bradykinin (LDABK) did not show any effect on ADAM17 or ACE2 activities in neurons with B1R gene deletion. These data suggest that B1R activation results in ADAM17-mediated ACE2 shedding in primary hypothalamic neurons. In addition, stimulation with high concentration of glutamate significantly increased B1R gene and protein expression, along with increased ADAM17 and decreased ACE2 activities in wild-type neurons. Pretreatment with B1R-specific antagonist R715 reversed these glutamate-induced effects suggesting that indeed B1R is involved in glutamate-mediated upregulation of ADAM17 activity and ACE2 shedding.

Cancer progression in COVID-19: integrating the roles of renin angiotensin aldosterone system, angiopoietin-2, heat shock protein-27 and epithelial mesenchymal transition.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has affected millions worldwide and has been found to cause severe disease in patients with underlying comorbidities. In patients with known malignancies, in addition to constraints in routine healthcare, the risk of being susceptible to developing severe forms of the disease is of grave concern.While follow-up studies on survivors of the severe acute respiratory syndrome (SARS) 2003 outbreak revealed increased susceptibility to infections, tumours and cardiovascular abnormalities, recent studies implicating angiopoietin 2 in induction of inflammatory intussusceptive angiogenesis and diffuse alveolar damage in COVID-19 patients raises the possibility of progression of carcinogenetic processes in patients with known malignancies. Angiotensin converting enzyme-2 (ACE-2) mediated cellular entry of SARS-Cov2 leads to receptor shedding of ACE-2 and disrupts the renin angiotensin aldosterone axis (RAAS). This augments the pro-inflammatory and proliferative effects of RAAS, while attenuating the anti-inflammatory and anti-proliferative angiotensin 1-7 /Mas pathway. Angiopoietin-2, a molecule responsible for angiogenesis and cancer progression which corelates with tumour load in certain cancers, is upregulated by angiotensin 2-AT1 Receptor axis. Tumour microenvironment—comprising of various cells, blood vessels and extra cellular matrix which express the RAAS peptides—plays a key role in cancer initiation, progression and metastasis. Angiotensin 2 induces the formation of a desmoplastic environment, favouring cancer cell growth. ACE-2 downregulation causes bradykinin accumulation which may exert its proliferative action via mitogen activated protein kinase pathways which has established roles in cancers of breast and kidney.In addition to cytokine storm causing organ damage, acute inflammation in COVID-19 may also cause epithelial mesenchymal transition and heat shock protein 27 phosphorylation, both of which are key mediators in cancer signalling pathways.We hypothesise that SARS-Cov2, by impacting the RAAS and immune system, has the potential to cause tumour cell proliferation, apoptosis evasion and metastasis, thereby increasing the possibility of cancer progression in patients with known malignancies.

ACE2 (Angiotensin-Converting Enzyme 2) in Cardiopulmonary Diseases

Discovery of ACE2 (angiotensin-converting enzyme 2) revealed that the renin-angiotensin system has 2 counterbalancing arms. ACE2 is a major player in the protective arm, highly expressed in lungs and gut with the ability to mitigate cardiopulmonary diseases such as inflammatory lung disease. ACE2 also exhibits activities involving gut microbiome, nutrition, and as a chaperone stabilizing the neutral amino acid transporter, B0AT1, in gut. But the current interest in ACE2 arises because it is the cell surface receptor for the novel coronavirus, severe acute respiratory syndrome coronavirus-2, to infect host cells, similar to severe acute respiratory syndrome coronavirus-2. This suggests that ACE2 be considered harmful, however, because of its important other roles, it is paradoxically a potential therapeutic target for cardiopulmonary diseases, including coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2. This review describes the discovery of ACE2, its physiological functions, and its place in the renin-angiotensin system. It illustrates new analyses of the structure of ACE2 that provides better understanding of its actions particularly in lung and gut, shedding of ACE2 by ADAM17 (a disintegrin and metallopeptidase domain 17 protein), and role of TMPRSS2 (transmembrane serine proteases 2) in severe acute respiratory syndrome coronavirus-2 entry into host cells. Cardiopulmonary diseases are associated with decreased ACE2 activity and the mitigation by increasing ACE2 activity along with its therapeutic relevance are addressed. Finally, the potential use of ACE2 as a treatment target in COVID-19, despite its role to allow viral entry into host cells, is suggested.

Blood flow restriction exercise stimulates mobilization of hematopoietic stem/progenitor cells and increases the circulating ACE2 levels in healthy adults.

Adult CD34+ hematopoietic stem/progenitor cells (HSPC) in the systemic circulation are bone marrow-derived and have the propensity of maintaining cardiovascular health. Activation of angiotensin-converting enzyme-2 (ACE2)-angiotensin-(1-7)-Mas receptor pathway, the vascular protective axis of the renin-angiotensin system (RAS), stimulates vasculogenic functions of HSPCs. In a previous study, exposure to hypoxia increased the expressions of ACE2 and Mas, and stimulated ACE2 shedding. The current study tested if blood flow restriction exercise (BFR)-induced regional hypoxia recapitulates the in vitro observations in healthy adults. Hypoxia was induced by 80% limb occlusion pressure (LOP) via inflation cuff. Muscle oxygen saturation was determined using near-infrared spectroscopy. Peripheral blood was collected 30 min after quiet sitting (control) or after BFR. Lin-CD45lowCD34+ HSPCs were enumerated by flow cytometry, and ACE and ACE2 activities were determined in plasma and cell lysates and supernatants. Regional hypoxia resulted in muscle oxygen saturation of 17.5% compared with 49.7% in the control condition (P < 0.0001, n = 9). Circulating HSPCs were increased following BFR (834.8 ± 62.1/mL) compared with control (365 ± 59, P < 0.001, n = 7), which was associated with increased stromal-derived factor 1α and vascular endothelial growth factor receptor levels by four- and threefold, respectively (P < 0.001). ACE2 activity was increased in the whole cell lysates of HSPCs, resulting in an ACE2-to-ACE ratio of 11.7 ± 0.5 in BFR vs 9.1 ± 0.9 in control (P < 0.05). Cell supernatants have threefold increase in the ACE2-to-ACE ratio following BFR compared with control (P < 0.001). Collectively, these findings provide strong evidence for the upregulation of ACE2 by acute regional hypoxia in vivo. Hypoxic exercise regimens appear to be promising means of enhancing vascular regenerative capacity.NEW & NOTEWORTHY Although many studies have explored the mechanisms of skeletal muscle growth and adaptation with hypoxia exercise interventions, less attention has been given to the potential for vascular adaptation and regenerative capacity. This study shows for the first time an acute upregulation of the angiotensin-converting enzyme 2 and increase in CD34+ vasculogenic cells following an acute bout of blood flow restriction with low-intensity exercise. These rapid changes collectively promote skeletal muscle angiogenesis. Therefore, this study supports the potential of hypoxic exercise interventions with low intensity for vascular and muscle health.

Coronavirus SARS-CoV-2: filtering fact from fiction in the infodemic: Q&A with virologist Professor Urs Greber.

Coronavirus SARS-CoV-2: filtering fact from fiction in the infodemic: Q&A with virologist Professor Urs Greber.

Sound Science before Quick Judgement Regarding RAS Blockade in COVID-19.

There has been much speculation in journals as well as social and traditional media about a link between popularly used classes of drugs that inhibit the renin-angiotensin system (RAS) and novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) infection or coronavirus

Abstract P2036: Effects of Angiotensin II Type 1 A Receptor (AT1aR) on Renal and Urinary Biomarkers of Acute Kidney Injury in Two Kidney One Clip (2K1C) Model of Renovascular Hypertension

Renin angiotensin system (RAS) plays a key role in the pathogenesis and progression of hypertension and renal injury. Angiotensin-II (Ang-II) type 1 receptor (AT1R) mediates the actions of Ang II to increase blood pressure, fluid retention, and aldosterone secretion. Ang II is involved in cell injury, vascular remodeling, and inflammation by releasing inflammatory and pro-inflammatory mediators. The effects of Ang II could be counteracted through its catabolism into the vasodilator peptide Ang (1-7) by angiotensin converting enzyme 2 (ACE2) and/or neprilysin (NEP). Diabetes and high blood pressure are the major causes of chronic kidney disease which could lead to end stage renal disease. Our previous studies demonstrated that shedding of renal ACE2 and NEP in the urine of db / db diabetic mice could act as biomarkers for diabetic kidney disease. The aim of the study is to test the hypothesis that there is increased renal ACE2 and NEP shedding in the urine of two-kidney one clip (2K1C) model of renovascular hypertension. In addition, we investigated the effects of deleting AT1aR on these RAS components and on kidney injury molecule-1 (KIM-1). Renovascular hypertension was induced in AT1aR knock out (KO) and WT mice by 2K1C surgery. Blood pressure (BP) measurement by radio-telemetry revealed significant increase of 46.1± 3.6 mmHg in 2K1C mice compared to control mice (p&lt;0.001). Deleting AT1aR significantly decreased the BP in 2K1C mice compared to 2K1C WT mice (69.86 ± 0.19 vs. 151.69 ± 12.22 mmHg, p&lt;0.001). Renal expression and activity of ACE2 and NEP were significantly decreased in the clipped kidney of WT and AT1KO group (p&lt;0.05). In contrast to diabetes, there was no detectable urinary ACE2 and NEP expression and activity in 2KIC mice. In addition, urinary expression of KIM-1 was increased in the clipped kidney. Although deleting AT1R attenuated albuminuria and hypertension, it did not altered urinary expression of KIM-1. These results suggest that the decreased renal NEP and ACE2 in the clipped kidney of 2K1C model may thus worsen kidney injury via impairment of Ang (1-7) formation. In conclusion, urinary KIM-1 could serve as an early indicator of non-diabetic acute kidney injury. Urinary ACE2 and NEP could be used as biomarker of diabetic kidney disease.

Blood Flow Restriction Exercise Increases the ACE2/ACE Ratio and ACE2 Shedding in CD34+ Cells in Healthy Individuals

Adult CD34+ hematopoietic stem/progenitor cells in the systemic circulation are bone marrow‐derived and have the propensity of maintaining cardiovascular health. We have previously shown that activation of the vascular protective axis of renin angiotensin system (RAS), Angiotensin Converting enzyme‐2 (ACE2)/Angiotensin‐(1–7)/Mas receptor axis stimulates vasoreparative functions of CD34+ cells in health and disease. Importantly, in vitro exposure of CD34+ cells to hypoxia enhanced the expressions of ACE2 and Mas, and stimulated ACE2 shedding, but ACE and AT1 receptors, members of the vascular detrimental axis of RAS were not affected. This study tested if acute regional hypoxia recapitulates the in vitro observations in healthy individuals. Blood flow restriction exercise (BFR) was performed in the lower body by applying 80% limb occlusion pressure (LOP). Delphi personalized tourniquet system was used to accomplish the target LOP. Exercise protocol consisted of total four sets (30/15/15/15 repetitions) of movements at 10% of maximal strength. Muscle oxygen saturation was determined by using MOXY muscle oxygen monitor. Peripheral blood was collected two weeks before (control) and 30 min after BFR. CD34+ cells were enumerated by flow cytometry, and ACE and ACE2 activities in plasma, and in cell lysates and cell‐free supernatants were determined. Plasma concentrations of SDF and VEGF were analyzed by ELISA. BFR prescribed at 80% LOP resulted in muscle oxygen levels of 14.1% compared to 50.9% in the resting condition. As predicted, circulating CD34+ cells were increased following BFR (before 356±59.1 and after 835±62.1/mL blood, P&lt;0.001, n=5), which was associated with increased plasma levels of SDF and VEGF (4‐ and 3‐fold, respectively, compared to control (P&lt;0.001, n=5)). ACE2 activity was increased in the whole cell lysates of Lineage‐negative cells resulting in the ACE2/ACE ratio of 11.7±0.5 compared to the baseline ratio of 9.1±0.9 (P&lt;0.05, n=5). Cell supernatants have 3‐fold increase in ACE2/ACE ratio following BFR compared to the baseline levels (P&lt;0.001, n=5). Collectively, these findings provide compelling evidence for the upregulation of ACE2, the vasoprotective enzyme of RAS, by acute regional hypoxia in HSPCs. BFR‐exercise regimen could be a promising approach for enhancing or restoring cardiovascular health in individuals that are poor mobilizers or those at risk for the development of cardiovascular complications.Support or Funding InformationNational Institute of General Medical Sciences (NIGMS) and National Institute of Aging (NIA) AG056881.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The use of unlicensed bone marrow–derived platelet lysate–expanded mesenchymal stromal cells in colitis: a pre-clinical study

BackgroundMesenchymal stromal cells (MSCs) are a promising candidate for treatment of inflammatory disorders, but their efficacy in human inflammatory bowel diseases (IBDs) has been inconsistent. Comparing the results from various pre-clinical and clinical IBD studies is also challenging due to a large variation in study designs. MethodsIn this comparative pre-clinical study, we compared two administration routes and investigated the safety and feasibility of both fresh and cryopreserved platelet-lysate–expanded human bone marrow–derived MSCs without additional licensing in a dextran sodium sulfate (DSS) colitis mouse model both in the acute and regenerative phases of colitis. Body weight, macroscopic score for inflammation and colonic interleukin (IL)-1β and tumor necrosis factor (TNF)α concentrations were determined in both phases of colitis. Additionally, histopathology was assessed and Il-1β and Agtr1a messenger RNA (mRNA) levels and angiotensin-converting enzyme (ACE) protein levels were measured in the colon in the regenerative phase of colitis. ResultsIntravenously administered MSCs exhibited modest anti-inflammatory capacity in the acute phase of colitis by reducing IL-1β protein levels in the inflamed colon. There were no clear improvements in mice treated with fresh or cryopreserved unlicensed MSCs according to weight monitoring results, histopathology and macroscopic score results. Pro-inflammatory ACE protein expression and shedding were reduced by cryopreserved MSCs in the colon. ConclusionsIn conclusion, we observed a good safety profile for bone marrow–derived platelet lysate–expanded MSCs in a mouse pre-clinical colitis model, but the therapeutic effect of MSCs prepared without additional licensing (i.e. such as MSCs are administered in graft-versus-host disease) was modest in the chosen in vivo model system and limited to biochemical improvements in cytokines without a clear benefit in histopathology or body weight development.

Angiotensin-converting enzyme 2 and renal disease.

The renin-angiotensin system (RAS) is a pivotal player in the physiology and pathophysiology of cardiovascular and renal systems. Discovery of angiotensin-converting enzyme 2 (ACE2), capable of cleaving RAS effector peptide angiotensin (Ang) II into biologically active Ang-(1-7), has increased the complexity of our knowledge of the RAS. ACE2 expression is abundant in the kidney and is thought to provide protection against injury. This review emphasizes current experimental and clinical findings that examine ACE2 in the context of kidney injury and its potential therapeutic impact for treatment of kidney disease. Clinical studies have reported upregulation of ACE2 in urine from diabetic patients, which may be reflective of pathological shedding of renal ACE2 as suggested by mechanistic experiments. Studies in experimental models have investigated the feasibility of pharmacological induction of ACE2 for improvement of renal function, inflammation, and fibrosis. Emerging concepts about the RAS indicate that ACE2 is a critical regulator of angiotensin peptide metabolism and the pathogenesis of renal disease. Human recombinant ACE2 is available and may be a practical clinical approach to enzyme replacement. Elucidating precise roles of ACE2 throughout disease progression will enrich our view of the RAS and help identify novel targets and appropriate strategies for intervention.

Abstract 071: Adam17-mediated Neurogenic Hypertension Requires Neuronal Activation of At1a Receptors

We previously reported that neurogenic hypertension is associated with a reduction of Angiotensin Converting Enzyme 2 (ACE2) activity and an increase in A Disintegrin And Metalloprotease 17 (ADAM17) activity in the hypothalamus. Since ADAM17 is known to be expressed in multiple cell types and can be activated by various receptors, we tested the hypothesis that neuronal AT1a receptors (AT1aR) are necessary for ADAM17-mediated ACE2 shedding during neurogenic hypertension. DOCA-salt treatment (DOCA 1mg/g body weight sc + 1% saline p.o./3 weeks) was given to male neuronal AT1aR knockdown mice (AT1aR floxed crossed with Nefh-cre mice, 14-16 week-old) and their non-transgenic (NT) littermates (n=8/group). Following DOCA-salt treatment, enzyme activity assays were performed in hypothalamus of both DOCA-salt-treated and sham mice. Unlike in NT mice, ADAM17 activity was not increased by DOCA-salt treatment in the hypothalamus of neuronal AT1aR knockdown mice (153.7 ±23.8 vs. 128.5 ±11.6 FU/min, P =0.22). To assess whether impaired ADAM17 activation resulted from down-regulation of neuronal AT1aR, or was due to an attenuated BP rise, wild-type mice were infused with norepinephrine (NE; 4 ug/kg/min/14 d, n=6/group). Contrary to DOCA-salt hypertension, ACE2 activity in the hypothalamus remained intact during NE-induced hypertension and there was no significant increase in ADAM17 activity (59.1 ±4.3 vs. 52.3 ±11.7 FU/min, P =0.29). Using FACS, neurons, astrocytes and non-neuronal/non-astrocyte cells were sorted from the hypothalamus. A significant increase in ADAM17 mRNA resulting from DOCA-salt treatment (+1.72 ±0.19 fold vs. sham, P &lt;0.05) was observed only in the neuronal population of NT mice but not in neuronal AT1aR knockdown mice. ERK phosphorylation, NOX4 expression and ROS production, all of which are involved in both intra- and extracellular mechanisms of ADAM17 activation were then assessed using Western blotting and DHE-staining. All were significantly impaired in the hypothalamus of neuronal AT1aR knock-down mice, compared to NT, after DOCA-salt treatment. Taken together, our data provide strong evidence that activation of neuronal AT1aR is responsible for ADAM17-mediated ACE2 shedding and the maintenance of neurogenic hypertension.

Protein Kinase C-δ Mediates Shedding of Angiotensin-Converting Enzyme 2 from Proximal Tubular Cells.

Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin (Ang) II to Ang-(1–7), and protects against diabetic renal injury. Soluble ACE2 fragments are shed from the proximal tubule, and appear at high levels in the urine with diabetes. High glucose-induced shedding of ACE2 from proximal tubular cells is mediated by the enzyme “a disintegrin and metalloproteinase-17″ (ADAM17). Here, we investigated the mechanism for constitutive shedding of ACE2. Mouse proximal tubular cells were cultured and ACE2 shedding into the media was assessed by enzyme activity assay and immunoblot analysis. Cells were incubated with pharmacologic inhibitors, or transfected with silencing (si) RNA. Incubation of proximal tubular cells with increasing concentrations of D-glucose stimulated ACE2 shedding, which peaked at 16 mM, while L-glucose (osmotic control) had no effect on shedding. In cells maintained in 7.8 mM D-glucose, ACE2 shedding was significantly inhibited by the pan-protein kinase C (PKC) competitive inhibitor sotrastaurin, but not by an inhibitor of ADAM17. Incubation of cells with the PKC-α and -β1-specific inhibitor Go6976, the PKC β1 and β2-specific inhibitor ruboxistaurin, inhibitors of matrix metalloproteinases-2,-8, and -9, or an inhibitor of ADAM10 (GI250423X) had no effect on basal ACE2 shedding. By contrast, the PKC-δ inhibitor rottlerin significantly inhibited both constitutive and high glucose-induced ACE2 shedding. Transfection of cells with siRNA directed against PKC-δ reduced ACE2 shedding by 20%, while knockdown of PKC-ε was without effect. These results indicate that constitutive shedding of ACE2 from proximal tubular cells is mediated by PKC-δ, which is also linked to high glucose-induced shedding. Targeting PKC-δ may preserve membrane-bound ACE2 in proximal tubule in disease states and diminish Ang II-stimulated adverse signaling.

Paricalcitol modulates ACE2 shedding and renal ADAM17 in NOD mice beyond proteinuria

Circulating and renal activity of angiotensin-converting enzyme 2 (ACE2) is increased in non-obese diabetic (NOD) mice. Because paricalcitol has been reported to protect against diabetic nephropathy, we investigated the role of paricalcitol in modulating ACE2 in these mice. In addition, renal ADAM17, a metalloprotease implied in ACE2 shedding, was assessed. NOD female and non-diabetic control mice were studied for 21 days after diabetes onset and divided into various treatment groups. Diabetic animals received either vehicle; 0.4 or 0.8 μg/kg paricalcitol, aliskiren, or a combination of paricalcitol and aliskiren. We then studied the effect of paricalcitol on ACE2 expression in proximal tubular epithelial cells. Paricalcitol alone or in combination with aliskiren resulted in significantly reduced circulating ACE2 activity in NOD mice but there were no changes in urinary albumin excretion. Serum renin activity was significantly decreased in mice that received aliskiren but no effect was found when paricalcitol was used alone. Renal content of ADAM17 was significantly decreased in animals that received a high dose of paricalcitol. Renal and circulating oxidative stress (quantified by plasma H2O2 levels and immunolocalization of nitrotyrosine) were reduced in high-dose paricalcitol-treated mice compared with non-treated diabetic mice. In culture, paricalcitol incubation resulted in a significant increase in ACE2 expression compared with nontreated cells. In NOD mice with type 1 diabetes, paricalcitol modulates ACE2 activity, ADAM17, and oxidative stress renal content independently from the glycemic profile and urinary albumin excretion. In tubular cells, paricalcitol may modulate ACE2 by blocking its shedding. In the early stage of diabetic nephropathy, paricalcitol treatment counterbalances the effect of diabetes on circulating ACE2 activity. Our results suggest that additional use of paricalcitol may be beneficial in treating patients with diabetes under standard therapeutic strategies.

Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice.

Angiotensin-converting enzyme 2 (ACE2) gene therapy aimed at counteracting pancreatic ACE2 depletion improves glucose regulation in two diabetic mouse models: db/db mice and angiotensin II-infused mice. A disintegrin and metalloproteinase 17 (ADAM17) can cause shedding of ACE2 from the cell membrane. The aim of our studies was to determine whether ADAM17 depletes ACE2 levels in pancreatic islets and β-cells. Dynamics of ADAM17-mediated ACE2 shedding were investigated in 832/13 insulinoma cells. Within a wide range of ACE2 expression levels, including the level observed in mouse pancreatic islets, overexpression of ADAM17 increases shed ACE2 and decreases cellular ACE2 levels. We provide a mathematical description of shed and cellular ACE2 activities as a function of the ADAM17 activity. The effect of ADAM17 on the cellular ACE2 content was relatively modest with an absolute control strength value less than 0.25 and approaching 0 at low ADAM17 activities. Although we found that ADAM17 and ACE2 are both expressed in pancreatic islets, the β-cell is not the major cell type expressing ACE2 in islets. During diabetes progression in 8-, 12-, and 15-week-old db/db mice, ACE2 mRNA and ACE2 activity levels in pancreatic islets were not decreased over time nor significantly decreased compared with nondiabetic db/m mice. Levels of ADAM17 mRNA and ADAM17 activity were also not significantly changed. Inhibiting basal ADAM17 activity in mouse islets failed to affect ACE2 levels. We conclude that whereas ADAM17 has the ability to shed ACE2, ADAM17 does not deplete ACE2 from pancreatic islets in diabetic db/db mice.

Abstract P208: Effect of Rosiglitazone on Renal Neprilysin Activity and Protein Expression in Db/db Diabetic Mice

Alteration in renin-angiotensin system has been implicated in the pathophysiology of diabetic kidney disease. The deleterious actions of angiotensin II (Ang II) could be antagonized by the formation of Ang (1-7) partly generated by the actions of angiotensin converting enzyme 2 (ACE2) and neprilysin (NEP). NEP is a member of the zinc-containing metallopeptidase family, and has a main role in the degradation of several peptides, including natriuretic peptides, bradykinin, amyloid beta, and Ang I. Our previous studies demonstrated increased shedding of renal ACE2 in db/db mouse model of type 2 diabetes. We also showed that treatment with the PPAR_ agonist, rosiglitazone normalized hyperglycemia, decreased urinary ACE2 and attenuated albuminuria in db/db mice. The aim of the study was to test the hypothesis that hyperglycemia down-regulates renal NEP in db/db diabetic mice and that treatment with rosiglitazone will normalize renal NEP expression and activity. Seven-week-old db/db male mice were subjected to rosiglitazone treatment (20 mg/kg/day) for 10 weeks. Treatment with rosiglitazone significantly lowered blood glucose levels in db/db mice (p&lt;0.001). Western blot analysis and immunohistochemistry demonstrated a significant decrease in renal and urinary NEP protein expression in 17wk db/db mice compared to lean control mice (p&lt;0.0001). Treatment of db/db mice with rosiglitazone attenuated albuminuria, increased renal NEP protein expression and activity. In conclusion, the renoprotective effects of rosiglitazone could be mediated by up-regulation of renal NEP expression and activity in db/db mice. Alteration in the balance between Ang II and Ang (1-7) forming enzymes could contribute to the development of albuminuria in db/db diabetic mice.

Abstract 033: Neuronal Knock-out of AT1 Receptor Attenuates the Development of Doca-salt-induced Hypertension

We previously reported that neurogenic hypertension is associated with an increase in A Disintegrin And Metalloprotease 17 (ADAM17) activity and a reduction of Angiotensin Converting Enzyme 2 (ACE2) activity in the hypothalamus. In addition, we showed that silencing ADAM17 or blocking Angiotensin (Ang)-II type 1 receptors (AT1R) in the central nervous system (CNS) prevented DOCA-salt hypertension, confirming the pivotal role of AT1R and ADAM17 in neurogenic hypertension. However, the interaction between AT1R, ADAM17, and ACE2 is still unclear. Since ADAM17 is known to be expressed in multiple cell types and can be activated by various receptors, we tested the hypothesis that neuronal AT1R are necessary for ADAM17-mediated ACE2 shedding in neurogenic hypertension. Male neuronal AT1R knockout (AT1R floxed crossed with Nefh-cre recombinase mice, 12-16 week-old, n=5) and littermate mice (n=8) were implanted with telemetry probes for continuous recording of blood pressure (BP) and heart rate (HR). Following DOCA-salt treatment, both strains developed hypertension, however mean arterial pressure (MAP; 123.9 ±6.4 vs. 138.4 ±4.3 mmHg) and HR (483.9 ±24.1 vs. 520.3 ±22.5 bmp) were significantly lower in neuronal AT1R knockout mice after 2 weeks of treatment, compared to controls. Western blot and enzyme activity assays from the hypothalamus of these DOCA-salt-treated mice revealed that the expression of pro-ADAM17 (inactive form) was significantly increased (+17.0 ±5.3%, P&lt;0.05), while the activity of ADAM17 was decreased (-37.4 ±10.5%) in neuronal AT1R knockout animals. Concomitant to the down-regulation of ADAM17, both the expression and activity of ACE2 were found to be significantly (P&lt;0.05) up-regulated in the hypothalamus of neuronal AT1R knockout mice, by +20.7 ±8.5% and +32.3 ±10.1%, respectively. These results suggest that activation of neuronal AT1R is responsible for ADAM17-mediated ACE2 shedding and the maintenance of neurogenic hypertension.

ADAM17‐Mediated Shedding of ACE2 in Pancreatic Beta‐Cells

Angiotensin‐converting enzyme 2 (ACE2) hydrolyzes angiotensin II. Our laboratory has previously shown that pancreatic ACE2 levels decrease as diabetes progresses and that pancreatic ACE2 gene therapy counteracts development of hyperglycemia in diabetic animal models. As a disintegrin and metalloprotease 17 (ADAM17) is an enzyme that can cleave enzymatically active ACE2 from the cell to the extracellular environment by so‐called shedding, we hypothesize that increased levels of ADAM17 in pancreatic beta‐cells lead to reduced cellular ACE2 levels. To this end, 832/13 cells (rat insulinoma cells derived from pancreatic beta‐cells) were transfected with expression plasmids for ACE2, ADAM17, and a hydrolytically inactive E406A ADAM17 mutant. Using fluorogenic enzyme substrates, we measured shed ACE2 activity after a 24 h incubation as well as cell‐associated ACE2 and ADAM17 activities. For a wide range of ACE2 expression levels, including the ACE2 expression level observed in pancreatic islets, a 4.6‐fold increase in ADAM17 activity had the following significant effects: shed ACE2 activity was increased 2 – 2.7‐fold, cellular ACE2 activity was decreased 33‐47%, and the ratio of shed ACE2/cellular ACE2 was increased 3.4 – 4.1‐fold. Adjusting for transfection efficiency, we further determined that the cellular ACE2 activity can be described as a power function of the ADAM17 activity with exponent ‐0.29 ±0.04. We conclude that ADAM17‐mediated shedding of ACE2 has the potential to decrease cellular ACE2 levels in pancreatic beta‐cells. More studies are underway to assess the role of this mechanism in diabetes. Support: NIH: GM103424, AHA: 12EIA8030004.

Functional and molecular evidence for expression of the renin angiotensin system and ADAM17-mediated ACE2 shedding in COS7 cells.

The renin angiotensin system (RAS) plays a vital role in the regulation of the cardiovascular and renal functions. COS7 is a robust and easily transfectable cell line derived from the kidney of the African green monkey, Cercopithecus aethiops. The aims of this study were to 1) demonstrate the presence of an endogenous and functional RAS in COS7, and 2) investigate the role of a disintegrin and metalloproteinase-17 (ADAM17) in the ectodomain shedding of angiotensin converting enzyme-2 (ACE2). Reverse transcription coupled to gene-specific polymerase chain reaction demonstrated expression of ACE, ACE2, angiotensin II type 1 receptor (AT1R), and renin at the transcript levels in total RNA cell extracts. Western blot and immunohistochemistry identified ACE (60 kDa), ACE2 (75 kDa), AT1R (43 kDa), renin (41 kDa), and ADAM17 (130 kDa) in COS7. At the functional level, a sensitive and selective mass spectrometric approach detected endogenous renin, ACE, and ACE2 activities. ANG-(1-7) formation (m/z 899) from the natural substrate ANG II (m/z 1,046) was detected in lysates and media. COS7 cells stably expressing shRNA constructs directed against endogenous ADAM17 showed reduced ACE2 shedding into the media. This is the first study demonstrating endogenous expression of the RAS and ADAM17 in the widely used COS7 cell line and its utility to study ectodomain shedding of ACE2 mediated by ADAM17 in vitro. The transfectable nature of this cell line makes it an attractive cell model for studying the molecular, functional, and pharmacological properties of the renal RAS.