Renal Angiotensin-converting Enzyme 2 Research Articles

Candesartan upregulates angiotensin-converting enzyme 2 in kidneys of male animals by decreased ubiquitination.

Candesartan is a common angiotensin-II receptor-1 blocker used for patients with cardiovascular and renal diseases. Angiotensin-converting enzyme 2 (ACE2) is a negative regulator of blood pressure (BP), and also a major receptor for coronaviruses. To determine whether and how candesartan upregulates ACE2, we examined BP and ACE2 in multi-organs from male and female C57BL/6J mice treated with candesartan (1 mg/kg, i.p.) for 7 days. Relative to the vehicle, candesartan lowered BP more in males than females; ACE2 protein abundances were increased in kidneys, not lungs, hearts, aorta, liver, spleen, brain, or serum, only from males. Ace2-mRNA was similar in kidneys. Candesartan also decreased BP in normal, hypertensive, and nephrotic male rats. The renal ACE2 was increased by the drug in normal and nephrotic male rats but not spontaneously hypertensive ones. In male mouse kidneys, ACE2 was distributed at sodium-hydrogen-exchanger-3 positive proximal-convoluted-tubules; ACE2-ubiquitination was decreased by candesartan, accompanied with increased ubiquitin-specific-protease-48 (USP48). In candesartan-treated mouse renal proximal-convoluted-tubule cells, ACE2 abundances and activities were increased while ACE2-ubiquitination and colocalization with lysosomal and proteosomal markers were decreased. The silence of USP48 by siRNA caused a reduction of ACE2 in the cells. Thus, the sex-differential ACE2 upregulation by candesartan in kidney from males may be due to the decreased ACE2-ubiquitination, associated with USP48, and consequent degradation in lysosomes and proteosomes. This is a novel mechanism and may shed light on candesartan-like-drug choice in men and women prone to coronavirus infections.

Sirt1 activation prevents high glucose-induced angiotensin converting enzyme 2 downregulation in renal tubular cells by regulating the TIMP3/ADAM17 pathway.

Angiotensin converting enzyme 2 (ACE2) exerts renoprotective effects in diabetic kidney disease (DKD) by converting angiotensin (Ang) II into Ang(1-7). Previous studies have demonstrated that ACE2 expression in renal tubules is downregulated in DKD, but the mechanism is not fully understood. Sirtuin-1 (Sirt1) is a protein deacetylase that may regulate the activity of the renin-angiotensin system. The present study investigated the effects of Sirt1 on ACE2 expression under high glucose (HG) conditions and the underlying signaling pathway. Rats with DKD and NRK-52E cells cultured with HG were employed in this study. Western blotting, immunohistochemistry detection and qRT-PCR were performed for protein and mRNA expression analyses. Rats subjected to DKD displayed downregulated expression of Sirt1 and ACE2 in kidneys. Resveratrol, an activator of Sirt1, restored ACE2 expression and ameliorated renal injuries. Similarly, pharmacological activation of Sirt1 with SRT1720 markedly upregulated ACE2 in NRK-52E cells cultured with HG, while Sirt1 small interfering RNA (siRNA) further suppressed ACE2 expression. In addition, A disintegrin and metalloproteinase (ADAM) 17 was observed to be upregulated, and its inhibitor, tissue inhibitor of metalloproteinase 3 (TIMP3), was downregulated in the kidneys of diabetic rats and NRK-52E cells incubated with HG. The TIMP3/ADAM17 pathway was involved in the regulation of ACE2 expression, as evidenced by decreased ACE2 expression levels after TIMP3-siRNA pretreatment. SRT1720 ameliorated the imbalance of TIMP3/ADAM17 induced by HG and consequently enhanced the expression of ACE2. Notably, the above effect of SRT1720 on ACE2 was interrupted by TIMP3-siRNA. Our findings suggest that Sirt1 activation may prevent HG-induced downregulation of renal tubular ACE2 by modulating the TIMP3/ADAM17 pathway. Sirt1 stimulation might be a potential strategy for the treatment of DKD.

S-39-2: EFFECTS OF RAS INHIBITORS ON TISSUE ACE2 EXPRESSION IN ANIMAL MODELS

Angiotensin-converting enzyme 2 (ACE2) protects against organ damages by overactivated renin-angiotensin system (RAS). Also, ACE2 exerts a role as the receptors for SARS-CoV-2 to enter host target cells. Based on limited number of articles reporting that RAS inhibitors induced upregulation of cardiac or renal ACE2 in animal models, concern have arisen that RAS inhibitors may aggravate SARS-CoV-2 infection and COVID-19 severity. Therefore, we performed a comprehensive review by applying the standard searching strategy for systematic reviews to investigate the effects of RAS inhibitors on cardiac, vascular, renal and pulmonary mRNA/protein ACE2 expression in healthy animals and disease animal models. We identified 88 eligible articles including 167 experiments. In RAS inhibitors-treated healthy animals, only 3 (heart 2, kidneys 1) of 37 experiments showed ACE2 expression greater than twice of the control (overexpression) whereas others showed no/marginal change in the heart, arteries, kidneys, lungs, and other organs. Among 102 disease models (130 experiments), baseline ACE2 was overexpressed in 16 models (18 experiments), less than half the control level (repression) in 28 models (40 experiments) and remained no/marginal change in 51 models (74 experiments). RAS inhibitors induced ACE2 overexpression from no/marginal change levels in only 7 experiments, 4 (myocardial infarction 2, heart failure 1, diabetic nephropathy 1) of which were not supported by other experiments under similar conditions. In 36 experiments, RAS inhibitors reversed or prevented disease-induced repression, yielding no/marginal change. Thus, ACE2 overexpression is a rare consequence of RAS inhibitors treatment in healthy animals and disease models. Future studies should clarify the pathophysiological significance of reversal or prevention of ACE2 repression induced by RAS inhibitors in disease models.

Activation of angiotensin-converting enzyme 2 ameliorates metabolic syndrome-induced renal damage in rats by renal TLR4 and nuclear transcription factor κB downregulation.

BackgroundMetabolic syndrome (MetS) is an independent risk factor for chronic kidney disease (CKD) through many mechanisms, including activation of the renin–angiotensin system. The deleterious effects of angiotensin II (Ang II) can be counterbalanced by angiotensin-converting enzyme 2 (ACE2). Diminazene aceturate (DIZE), an anti-trypanosomal drug, can activate ACE2.ObjectiveThis study aimed to investigate the possible reno-protective effects of DIZE in MetS rats with elucidation of related mechanisms.Materials and methodsThirty adult male Wistar albino rats were divided equally into control, MetS, and MetS + DIZE groups. Body weight, systolic blood pressure (SBP), and urinary albumin levels were measured. Serum levels of fasting blood glucose (FBG), insulin, uric acid, lipid profile, urea, and creatinine were measured. Homeostasis Model Assessment Index (HOMA-IR) was estimated. Subsequently, renal levels of ACE2, Ang II, malondialdehyde (MDA), reduced glutathione (GSH), and tumor necrosis factor-α (TNF-α) were measured with histopathological and immunohistochemical assessment of TLR4 and NF-κB in renal tissues.ResultsMetS caused dyslipidemia with significant increases in body weight, SBP, FBG, serum insulin, HOMA-IR, uric acid, urea, creatinine, urinary albumin, and renal levels of Ang II, MDA, and TNF-α, whereas renal ACE2 and GSH were significantly decreased. Renal TLR4 and NF-κB immunoreactivity in MetS rats was upregulated. DIZE supplementation of MetS rats induced significant improvements in renal function parameters; this could be explained by the ability of DIZE to activate renal ACE2 and decrease renal Ang II levels with downregulation of renal TLR4 and NF-κB expression.ConclusionDIZE exerts a reno-protective effect in MetS, mainly by downregulating renal TLR4 and NF-κB levels.

Tissue-specific expression of the SARS-CoV-2 receptor, angiotensin-converting enzyme 2, in mouse models of chronic kidney disease

Elevated angiotensin-converting enzyme 2 (ACE2) expression in organs that are potential targets of severe acute respiratory syndrome coronavirus 2 may increase the risk of coronavirus disease 2019 (COVID-19) infection. Previous reports show that ACE2 alter its tissue-specific expression patterns under various pathological conditions, including renal diseases. Here, we examined changes in pulmonary ACE2 expression in two mouse chronic kidney disease (CKD) models: adenine-induced (adenine mice) and aristolochic acid-induced (AA mice). We also investigated changes in pulmonary ACE2 expression due to renin–angiotensin system (RAS) blocker (olmesartan) treatment in these mice. Adenine mice showed significant renal functional decline and elevated blood pressure, compared with controls. AA mice also showed significant renal functional decline, compared with vehicles; blood pressure did not differ between groups. Renal ACE2 expression was significantly reduced in adenine mice and AA mice; pulmonary expression was unaffected. Olmesartan attenuated urinary albumin excretion in adenine mice, but did not affect renal or pulmonary ACE2 expression levels. The results suggest that the risk of COVID-19 infection may not be elevated in patients with CKD because of their stable pulmonary ACE2 expression. Moreover, RAS blockers can be used safely in treatment of COVID-19 patients with CKD.

Expression of ACE2 in the Intact and Acutely Injured Kidney.

The actions of angiotensin-converting enzyme 2 (ACE2) oppose those of the renin-angiotensin-aldosterone system. ACE2 may be a cytoprotectant in some tissues. This study examined ACE2 expression in models of AKI. ACE2 mRNA and protein expression and ACE2 activity were assessed in murine ischemic AKI. Renal ACE2 mRNA expression was evaluated in LPS-induced AKI in wild-type (C57BL/6J) mice, in heme oxygenase-1+/+ and heme oxygenase-1-/- mice, and after unilateral ureteral obstruction (UUO) in wild-type mice. The effect of sex and age on renal ACE2 protein expression was also assessed. In ischemic AKI, ACE2 mRNA and protein expression and ACE2 activity were reduced as compared with such indices in the intact kidney. In ischemic AKI, ACE2, which, in health, is prominently expressed in the tubular epithelium, especially proximal tubules, is decreased in expression in these segments. Decreased ACE2 expression in AKI did not reflect reduced GFR, because ACE2 mRNA expression was unaltered after UUO. LPS induced renal ACE2 mRNA expression in wild-type mice, but this effect did not occur in heme oxygenase-1-deficient mice. In ischemic and LPS-induced AKI, renal expression of the Mas receptor was increased. In the intact kidney, renal ACE2 protein expression decreased in female mice as compared with male mice, but was unaltered with age. We conclude that renal ACE2 expression is decreased in ischemic AKI, characterized by decreased GFR and abundant cell death, but is upregulated in LPS-induced AKI, an effect requiring heme oxygenase-1. Determining the significance of ACE2 expression in experimental AKI merits further study. We suggest that understanding the mechanism underlying ACE2 downregulation in AKI may offer insights relevant to COVID-19: ACE2 expression is downregulated after ACE2 mediates SARS-CoV-2 cellular entry; such downregulation is proinflammatory; and AKI commonly occurs and determines outcomes in COVID-19.

Angiotensin‐II type 2 receptor activation modulates renal neprilysin and coronavirus receptor angiotensin converting enzyme‐2 expression and activity in obese rats fed high salt diet

Neprilysin and coronavirus receptor angiotensin‐converting enzyme‐2 (ACE2) are peptidases critically involved in the breakdown of deleterious angiotensin‐II and the formation of protective angiotensin‐(1‐7). A disintegrin and metalloproteinase‐17 (ADAM17) is involved in the shedding of neprilysin and ACE2. We have reported the increase in renal angiotensin‐(1‐7) formation upon activation of angiotensin‐II type 2 receptor (AT2R). However, the modulation of expression and activity of these peptidases upon stimulation of AT2R in the kidney under the normal, obese, and high‐salt diet (HSD)‐fed condition is not known. Hence, we have treated, male, lean and obese Zucker rats (OZR) for 14 days without or with AT2R agonist C21 (1 mg/kg/day, via osmotic pumps) while fed HSD or normal diet. We have observed that the activity of neprilysin and ACE2 is decreased in the kidney of OZR fed either diet, however, only neprilysin activity has been found increased in the plasma (p<0.05). The C21‐treatment increased renal neprilysin expression and activity in the HSD‐fed OZR reflecting an increase in the urinary but decreased plasma activity (p<0.05). Likewise, The renal ACE2 expression is also increased upon C21‐treatment in HSD‐fed OZR and lean rats (p<0.05), but not the activity. These changes may be ascribed to ADAM17, as the renal expression of ADAM17 is paradoxically decreased upon HSD feeding in OZR (p<0.05) which is further reduced by C21‐treatment. Results indicate that AT2R activation differentially affects renal neprilysin and ACE2 which is critical in reference to current coronavirus pandemic.

Male bias in ACE2 basic science research: missed opportunity for discovery in the time of COVID-19.

Throughout the world, including the United States, men have worse outcomes from COVID-19 than women. SARS-CoV-2, the causative virus of the COVID-19 pandemic, uses angiotensin-converting enzyme 2 (ACE2) to gain cellular entry. ACE2 is a member of the renin-angiotensin system (RAS) and plays an important role in counteracting the harmful effects mediated by the angiotensin type 1 receptor. Therefore, we conducted Ovid MEDLINE and Embase database searches of basic science studies investigating the impact of the biological variable of sex on ACE2 expression and regulation from 2000, the year ACE2 was discovered, through December 31, 2020. Out of 2,131 publications, we identified 853 original research articles on ACE2 conducted in primary cells, tissues, and/or whole mammals excluding humans. The majority (68.7%) of these studies that cited the sex of the animal were conducted in males, while 11.2% were conducted solely in females; 9.26% compared ACE2 between the sexes, while 10.8% did not report the sex of the animals used. General findings are that sex differences are tissue-specific and when present, are dependent upon gonadal state. Renal, cardiac, and adipose ACE2 is increased in both sexes under experimental conditions that model co-morbidities associated with worse COVID-19 outcomes including hypertension, obesity, and renal and cardiovascular diseases; however, ACE2 protein was generally higher in the males. Studies in Ace2 knockout mice indicate ACE2 plays a greater role in protecting the female from developing hypertension than the male. Studying the biological variable of sex in ACE2 research provides an opportunity for discovery in conditions involving RAS dysfunction and will shed light on sex differences in COVID-19 severity.

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 improves diabetic nephropathy by targeting Smad7 for ubiquitin degradation.

Angiotensin‑converting enzyme 2(ACE2), an important component of the renin‑angiotensin system, protects against renal tubulointerstitial fibrosis, but its level of involvement in the mechanism of diabetic nephropathy(DN) currently remains unclear. Herein, the effects of ACE2 in DN and the associated mechanisms were investigated using serum and renal biopsy specimens from patients with DN and control participants, and human renal proximal tubular epithelial cells (HRPTEpiCs). The present study determined that the circulating concentration of ACE2 was high, but renal ACE2 expression was markedly lower, and there was abundant expression of Arkadia, an E3 ubiquitin ligase, in patients with DN. Invitro, ACE2 attenuated high‑glucose‑induced tubular epithelial to mesenchymal cell transition(EMT), which was demonstrated by increased expression of α‑SMA and loss of E‑cadherin expression, as demonstrated by western blot analysis and reverse transcription‑quantitative PCR. Adenovirus‑mediated ACE2 overexpression was also revealed to significantly inhibit Arkadia expression and alleviated high‑glucose‑induced EMT, while ACE2 inhibition had the opposite effects. Furthermore, western blot analysis demonstrated that ACE2‑alleviated EMT was associated with downregulated Arkadia and increased SMAD family member 7(Smad7) protein, followed by TGF‑β/Smad pathway inhibition in HRPTEpiCs. In conclusion, ACE2 is protective in DN, which may be due to the inhibition of Arkadia‑mediated Smad7 degradation, whereby TGF‑β/Smad‑mediated EMT is ameliorated in high‑glucose‑stimulated HRPTEpiCs.

Involvement of sex hormones, oxidative stress, ACE and ACE2 activity in the impairment of renal function and remodelling in SHR

AimsHypertension is a relevant sex and sex hormones-dependent risk factor where the cardiovascular and renal health of the population are concerned. Men experience greater losses of renal function (RF) than women, but the mechanisms remain somewhat unclear. Our goal was to evaluate the relationship between oxidative stress (OS), angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) activities and RF in male and female SHR. Main methodsTwelve-week-old spontaneously hypertensive rats (SHR) were submitted to either castration or SHAM surgery and divided into 4 groups, SHAM or Castrated (CAST) males or females. After 51 days we evaluated RF (inulin and sodium para-aminohippurate), ACE and ACE2 activities (fluorimetry), OS (flow cytometry), collagen deposition (picrosirius red) and protein expression (western blot). Key findingsMales presented lower RF than females and castration impaired this parameter in both groups. Sexual dimorphism was not observed regarding OS and inflammation; however, castration increased this parameter more severely in males than in females. SHAM males exhibited higher collagen deposition than females, though castration increased it in both sexes, eliminating the difference. We found sexual dimorphism regarding renal ACE and ACE2 activities, which were lower in males than in females. Although castration did not alter ACE activity, it reduced ACE2 activity in females and increased it in males. SignificanceThese results indicate that sex hormones affect RF in SHR. As alterations in the oxidative system were capable of promoting podocyte injury, inflammation, and collagen deposition, we put forward that these effects are differently modulated by ACE and ACE2.

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.

Concurrent neprilysin inhibition and renin-angiotensin system modulations prevented diabetic nephropathy

AimsRenin-angiotensin system (RAS) and natriuretic peptides system (NPS) perturbations govern the development of diabetic nephropathy (DN). Hence, in search of a novel therapy against DN, present study targeted both, NPS and RAS simultaneously using a neprilysin inhibitor (NEPi) in combination with either angiotensin receptor blocker (ARB) or angiotensin-converting enzyme 2 (ACE2) activator. MethodsWe induced diabetes in male Wistar rats by a single dose of streptozotocin (55 mg/kg, i.p.). After four weeks, we treated diabetic rats with thiorphan, telmisartan or diminazene aceturate (Dize) 0.1, 10, 5 mg/kg/day, p.o. alone as monotherapy, or both thiorphan/telmisartan or thiorphan/Dize as combination therapy, for four weeks. Then, plasma and urine biochemistry were performed, and kidneys from all the groups were collected and processed separately for histopathology, ELISA and Western blotting. Key findingsProposed combination therapies attenuated metabolic perturbations, prevented renal functional decline, and normalised adverse alterations in renal ACE, ACE2, Ang-II, Ang-(1–7), neprilysin and cGMP levels in diabetic rats. Histopathological evaluation revealed a significant reduction in glomerular and tubulointerstitial fibrosis by combination therapies. Importantly, combination therapies inhibited inflammatory, profibrotic and apoptotic signalling, way better than respective monotherapies, in preventing DN. ConclusionRenoprotective potential of thiorphan (NEPi)/telmisartan (ARB) and thiorphan/Dize (ACE2 activator) combination therapies against the development of DN is primarily attributed to normalisation of RAS and NPS components and inhibition of pathological signalling related to inflammation, fibrosis, and apoptosis. Hence, we can conclude that NEPi/ARB and NEPi/ACE2 activator combination therapies might be new therapeutic strategies in preventing DN.

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.

Murine recombinant angiotensin-converting enzyme 2 attenuates kidney injury in experimental Alport syndrome

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase in the renin-angiotensin system that catalyzes the breakdown of angiotensin II to angiotensin 1-7. We have reported that ACE2 expression in the kidney is reduced in experimental Alport syndrome but the impact of this finding on disease progression has not been studied. Accordingly, we evaluated effects of murine recombinant ACE2 treatment in Col4a3 knockout mice, a model of Alport syndrome characterized by proteinuria and progressive renal injury. Murine recombinant ACE2 (0.5 mg/kg/day) was administered from four to seven weeks of age via osmotic mini-pump. Pathological changes were attenuated by murine recombinant ACE2 treatment which ameliorated kidney fibrosis as shown by decreased expression of COL1α1 mRNA, less accumulation of extracellular matrix proteins, and inhibition of transforming growth factor-β signaling. Further, increases in proinflammatory cytokine expression, macrophage infiltration, inflammatory signaling pathway activation, and heme oxygenase-1 levels in Col4a3 knockout mice were also reduced by murine recombinant ACE2 treatment. Lastly, murine recombinant ACE2 influenced the turnover of renal ACE2, as it suppressed the expression of tumor necrosis factor-α converting enzyme, a negative regulator of ACE2. Thus, treatment with exogenous ACE2 alters angiotensin peptide metabolism in the kidneys of Col4a3 knockout mice and attenuates the progression of Alport syndrome nephropathy.

Effects and Mechanisms of Radiofrequency Ablation of Renal Sympathetic Nerve on Anti-Hypertension in Canine.

BackgroundRadiofrequency ablation of renal sympathetic nerve (RDN) shows effective BP reduction in hypertensive patients while the specific mechanisms remain unclear.ObjectiveWe hypothesized that abnormal levels of norepinephrine (NE) and changes in NE-related enzymes and angiotensinconverting enzyme 2 (ACE2), angiotensin (Ang)-(1-7) and Mas receptor mediate the anti-hypertensive effects of RDN.MethodsMean values of systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) were assessed at baseline and follow-up. Plasma and renal norepinephrine (NE) concentrations were determined using highperformance liquid chromatography with electrochemical detection, and levels of NE-related enzyme and ACE2-Ang(1-7)- Mas were measured using real time PCR, Western blot and immunohistochemistry or Elisa in a hypertensive canine model fed with high-fat diet and treated with RDN. The parameters were also determined in a sham group treated with renal arteriography and a control group fed with normal diet.ResultsRDN decreased SBP, DBP, MAP, plasma and renal NE. Compared with the sham group, renal tyrosine hydroxylase (TH) expression was lower and renalase expression was higher in the RDN group. Compared with the control group, renal TH and catechol-o-methyl transferase (COMT) were higher and renalase was lower in the sham group. Moreover, renal ACE2, Ang-(1-7) and Mas levels of the RDN group were higher than those of the sham group, which were lower than those of the control group.ConclusionRDN shows anti-hypertensive effect with reduced NE and activation of ACE2-Ang(1-7)-Mas, indicating that it may contribute to the anti-hypertensive effect of RDN.

Sex-Specific Changes in Renal Angiotensin-Converting Enzyme and Angiotensin-Converting Enzyme 2 Gene Expression and Enzyme Activity at Birth and Over the First Year of Life.

Angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) are key enzymes of the renin-angiotensin system. We investigated developmental changes in renal ACE and ACE2 gene expression and activity in both male and female sheep. Three groups of sheep (fetus, newborn, and adult) were used. Renal ACE and ACE2 activities, messenger RNA (mRNA), and protein expression were studied. Renal ACE and ACE2 activities increased at 1 year in males, while there were no changes throughout development in females. Renal ACE and ACE2 mRNA and protein showed no sex differences but increased by 1 year of age. There are sex-related differences in the development of renal-converting enzyme activities that may have functional implications in terms of the regulation of blood pressure and renal function in men and women. The difference in the patterns of gene expression and enzyme activity indicates that changes in gene expression may not accurately reflect changes in activity.

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.

Deletion of angiotensin-converting enzyme 2 exacerbates renal inflammation and injury in apolipoprotein E-deficient mice through modulation of the nephrin and TNF-alpha-TNFRSF1A signaling.

BackgroundThe renin-angiotensin system (RAS) has been implicated in atherosclerotic lesions and progression to chronic kidney diseases. We examined regulatory roles of angiotensin-converting enzyme 2 (ACE2) in the apolipoprotein E (ApoE) knockout (KO) kidneys.MethodsThe 3-month-old wild-type, ApoEKO, ACE2KO and ApoE/ACE2 double-KO (DKO) mice in a C57BL/6 background were used. The ApoEKO mice were randomized to daily deliver either Ang II (1.5 mg/kg) and/or human recombinant ACE2 (rhACE2; 2 mg/kg) for 2 weeks. We examined changes in pro-inflammatory cytokines, renal ultrastructure, and pathological signaling in mouse kidneys.ResultsDownregulation of ACE2 and nephrin levels was observed in ApoEKO kidneys. Genetic ACE2 deletion resulted in modest elevations in systolic blood pressure levels and Ang II type 1 receptor expression and reduced nephrin expression in kidneys of the ApoE/ACE2 DKO mice with a decrease in renal Ang-(1-7) levels. These changes were linked with marked increases in renal superoxide generation, NADPH oxidase (NOX) 4 and proinflammatory factors levels, including interleukin (IL)-1beta, IL-6, IL-17A, RANTES, ICAM-1, Tumor necrosis factor-alpha (TNF-alpha) and TNFRSF1A. Renal dysfunction and ultrastructure injury were aggravated in the ApoE/ACE2 DKO mice and Ang II-infused ApoEKO mice with increased plasma levels of creatinine, blood urea nitrogen and enhanced levels of Ang II in plasma and kidneys. The Ang II-mediated reductions of renal ACE2 and nephrin levels in ApoEKO mice were remarkably rescued by rhACE2 supplementation, along with augmentation of renal Ang-(1-7) levels. More importantly, rhACE2 treatment significantly reversed Ang II-induced renal inflammation, superoxide generation, kidney dysfunction and adverse renal injury in ApoEKO mice with suppression of the NOX4 and TNF-alpha-TNFRSF1A signaling. However, rhACE2 had no effect on renal NOX2 and TNFRSF1B expression and circulating lipid levels.ConclusionsACE2 deficiency exacerbates kidney inflammation, oxidative stress and adverse renal injury in the ApoE-mutant mice through modulation of the nephrin, NOX4 and TNF-alpha-TNFRSF1A signaling. While rhACE2 supplementation alleviates inflammation, renal dysfunction and glomerulus injury in the ApoE-mutant mice associated with upregulations of Ang-(1-7) levels and nephrin expression and suppression of the TNF-alpha-TNFRSF1A signaling. Strategies aimed at enhancing the ACE2/Ang-(1-7) actions may have important therapeutic potential for atherosclerotic renal injury and kidney diseases.

Daily exercise training protects against albuminuria and angiotensin converting enzyme 2 shedding in db/db diabetic mice.

Angiotensin II (Ang II) is involved in induction and progression of renal damage in diabetes. Angiotensin converting enzyme 2 (ACE2) is highly expressed in the kidney and has been shown to be renoprotective by degrading Ang II to Ang-(1-7). A disintegrin and metalloproteinase 17 (ADAM17)-mediated shedding of renal ACE2 contribute to diabetic nephropathy pathogenesis. Lifestyle modification and metformin are recommended as initial therapies for most patients with type 2 diabetes. The aim of this study was to investigate whether exercise training and/or metformin improve glucose homeostasis and albuminuria and downregulate renal ADAM17 and ACE2 shedding in db/db mice. Seven-week-old normal and db/db mice were subjected either to a sedentary existence or exercise training with and without metformin (150 mg/kg per day) for 10 weeks. Exercise training significantly lowered blood glucose, urinary albumin and ACE2 excretion in db/db mice. ADAM17 and ACE2 proteins were co-localized in cortical tubules of the kidney, indicating a possible interaction. Metformin treatment was effective in lowering hyperglycemia only during the first 2 weeks of treatment. Increased renal ADAM17 in 17-week-old db/db mice was corrected by physical exercise but not metformin. In addition, exercise training reduced plasma triglycerides and enhanced insulin levels of db/db mice. In conclusion, exercise training alone and in combination with metformin prevented shedding of renal ACE2 by decreasing ADAM17 protein. Urinary ACE2 could serve as a prognostic tool for the progression of kidney damage and its attenuation by exercise may partially contribute to its renal protection.