Renal ACE2 Expression Research Articles

Soluble ACE2 Is Filtered into the Urine.

ACE2 is a key enzyme in the renin-angiotensin system (RAS) capable of balancing the RAS by metabolizing angiotensin II (AngII). First described in cardiac tissue, abundance of ACE2 is highest in the kidney, and it is also expressed in several extrarenal tissues. Previously, we reported an association between enhanced susceptibility to hypertension and elevated renal AngII levels in global ACE2-knockout mice. To examine the effect of ACE2 expressed in the kidney, relative to extrarenal expression, on the development of hypertension, we used a kidney crosstransplantation strategy with ACE2-KO and WT mice. In this model, both native kidneys are removed and renal function is provided entirely by the transplanted kidney, such that four experimental groups with restricted ACE2 expression are generated: WT→WT (WT), KO→WT (KidneyKO), WT→KO (SystemicKO), and KO→KO (TotalKO). Additionally, we used nanoscale mass spectrometry-based proteomics to identify ACE2 fragments in early glomerular filtrate of mice. Although significant differences in BP were not detected, a major finding of our study is that shed or soluble ACE2 (sACE2) was present in urine of KidneyKO mice that lack renal ACE2 expression. Detection of sACE2 in the urine of KidneyKO mice during AngII-mediated hypertension suggests that sACE2 originating from extrarenal tissues can reach the kidney and be excreted in urine. To confirm glomerular filtration of ACE2, we used micropuncture and nanoscale proteomics to detect peptides derived from ACE2 in the Bowman's space. Our findings suggest that both systemic and renal tissues may contribute to sACE2 in urine, identifying the kidney as a major site for ACE2 actions. Moreover, filtration of sACE2 into the lumen of the nephron may contribute to the pathophysiology of kidney diseases characterized by disruption of the glomerular filtration barrier.

Regulation of SARS CoV-2 host factors in the kidney and heart in rats with 5/6 nephrectomy\u2014effects of salt, ARB, DPP4 inhibitor and SGLT2 blocker

BackgroundHost factors such as angiotensin-converting enzyme 2 (ACE2) and the transmembrane protease, serine-subtype-2 (TMPRSS2) are important factors for SARS-CoV-2 infection. Clinical and pre-clinical studies demonstrated that RAAS-blocking agents can be safely used during a SARS-CoV-2 infection but it is unknown if DPP-4 inhibitors or SGLT2-blockers may promote COVID-19 by increasing the host viral entry enzymes ACE2 and TMPRSS2.MethodsWe investigated telmisartan, linagliptin and empagliflozin induced effects on renal and cardiac expression of ACE2, TMPRSS2 and key enzymes involved in RAAS (REN, AGTR2, AGT) under high-salt conditions in a non-diabetic experimental 5/6 nephrectomy (5/6 Nx) model. In the present study, the gene expression of Ace2, Tmprss2, Ren, Agtr2 and Agt was assessed with qRT-PCR and the protein expression of ACE2 and TMPRSS2 with immunohistochemistry in the following experimental groups: Sham + normal diet (ND) + placebo (PBO); 5/6Nx + ND + PBO; 5/6Nx + high salt-diet (HSD) + PBO; 5/6Nx + HSD + telmisartan; 5/6Nx + HSD + linagliptin; 5/6Nx + HSD + empagliflozin.ResultsIn the kidney, the expression of Ace2 was not altered on mRNA level under disease and treatment conditions. The renal TMPRSS2 levels (mRNA and protein) were not affected, whereas the cardiac level was significantly increased in 5/6Nx rats. Intriguingly, the elevated TMPRSS2 protein expression in the heart was significantly normalized after treatment with telmisartan, linagliptin and empagliflozin.ConclusionsOur study indicated that there is no upregulation regarding host factors potentially promoting SARS-CoV-2 virus entry into host cells when the SGLT2-blocker empagliflozin, telmisartan and the DPP4-inhibitor blocker linagliptin are used. The results obtained in a preclinical, experimental non-diabetic kidney failure model need confirmation in ongoing interventional clinical trials.

Renin-Angiotensin System Induced Secondary Hypertension: The Alteration of Kidney Function and Structure

Long-term hypertension is known as a major risk factor for cardiovascular and chronic kidney disease (CKD). The Renin-angiotensin system (RAS) plays a key role in hypertension pathogenesis. Angiotensin II (Ang II) enhancement in Ang II-dependent hypertension leads to progressive CKD and kidney fibrosis. In the two-kidney one-clip model (2K1C), more renin is synthesized in the principal cells of the collecting duct than juxtaglomerular cells (JGCs). An increase of renal Ang I and Ang II levels and a decrease of renal cortical and medullary Ang 1–7 occur in both kidneys of the 2K1C hypertensive rat model. In addition, the activity of the angiotensin-converting enzyme (ACE) increases, while ACE2's activity decreases in the medullary region of both kidneys in the 2K1C hypertensive model. Also, the renal prolyl carboxypeptidase (PrCP) expression and its activity reduce in the clipped kidneys. The imbalance in the production of renal ACE, ACE2, and PrCP expression causes the progression of renal injury. Intrarenal angiotensinogen (AGT) expression and urine AGT (uAGT) excretion rates in the unclipped kidney are greater than the clipped kidney in the 2K1C hypertensive rat model. The enhancement of Ang II in the clipped kidney is related to renin secretion, while the elevation of intrarenal Ang II in the unclipped kidney is related to stimulation of AGT mRNA and protein in proximal tubule cells by a direct effect of systemic Ang II level. Ang II-dependent hypertension enhances macrophages and T-cell infiltration into the kidney which increases cytokines, and AGT synthesis in proximal tubules is stimulated via cytokines. Accumulation of inflammatory cells in the kidney aggravates hypertension and renal damage. Moreover, Ang II-dependent hypertension alters renal Ang II type 1 & 2 receptors (AT1R & AT2R) and Mas receptor (MasR) expression, and the renal interstitial fluid bradykinin, nitric oxide, and cGMP response to AT1R, AT2R, or BK B2-receptor antagonists. Based on a variety of sources including PubMed, Google Scholar, Scopus, and Science-Direct, in the current review, we will discuss the role of RAS-induced secondary hypertension on the alteration of renal function.

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.

Sex and kidney ACE2 expression in primary focal segmental glomerulosclerosis: A NEPTUNE study.

BackgroundAngiotensin-converting enzyme 2 (ACE2) has been implicated in the pathogenesis of experimental kidney disease. ACE2 is on the X chromosome, and in mice, deletion of ACE2 leads to the development of focal segmental glomerulosclerosis (FSGS). The relationship between sex and renal ACE2 expression in humans with kidney disease is a gap in current knowledge.MethodsWe studied renal tubulointerstitial microarray data and clinical variables from subjects with FSGS enrolled in the Nephrotic Syndrome Study Network (NEPTUNE) study. We compared relationships between ACE2 expression and age, estimated glomerular filtration rate (eGFR), urinary albumin to creatinine ratio (UACR), interstitial fibrosis, tubular atrophy, and genes implicated in inflammation and fibrosis in male and female subjects.ResultsACE2 mRNA expression was lower in the tubulointerstitium of males compared to females (P = 0.0026). Multiple linear regression analysis showed that ACE2 expression was related to sex and eGFR but not to age or treatment with renin angiotensin system blockade. ACE2 expression is also related to interstitial fibrosis, and tubular atrophy, in males but not in females. Genes involved in inflammation (CCL2 and TNF) correlated with ACE2 expression in males (TNF: r = -0.65, P < 0.0001; CCL2: r = -0.60, P < 0.0001) but not in females. TGFB1, a gene implicated in fibrosis correlated with ACE2 in both sexes.ConclusionsSex is an important determinant of ACE2 expression in the tubulointerstitium of the kidney in FSGS. Sex also influences the relationships between ACE2, kidney fibrosis, and expression of genes involved in kidney inflammation.

Combination of AT2R Agonist and Neprilysin Inhibitor is Superior Than the Combination of AT1R Antagonist and Neprilysin Inhibitor in Protecting Kidney from Injury in High‐Salt Fed Obese Rats

Angiotensin II type 1 receptor (AT1R) antagonists, angiotensin II type 2 receptor (AT2R) agonists, and atrial natriuretic peptide (ANP) are natriuretics that provide reno- and cardiovascular protection and reduce blood pressure and volume load. An approved combination therapy (Entresto) of the AT1R blocker valsartan (VAL), and inhibitor of the ANP degrading enzyme neprilysin (NEP) sacubitril (SAC), for patients with heart failure and reduced ejection fraction was found to be associated with increased albuminuria, an indicator of kidney injury. Based on studies showing that AT2R activation affects various renin angiotensin system (RAS) components by decreasing activity of the classical arm components, and increasing expression and activity of those of the protective arm such as angiotensin converting enzyme 2 (ACE2), we hypothesized that AT2R agonist treatment in combination with SAC will provide better reno-protection compared to VAL + SAC. We tested this hypothesis in obese Zucker rats (OZR), in which kidney injury was induced by feeding a high salt diet. Male OZR were fed a 0.4% normal sodium (NSD) or 4% high sodium diet (HSD) and administered vehicle, AT2R agonist C21 (1 mg/kg/day) + SAC (10 mg/kg/day), or VAL (10 mg/kg/day) + SAC (10 mg/kg/day) (16 days, daily gavage). While total food intake was significantly decreased and water intake significantly increased by HSD feeding, neither treatment had an effect compared to the control group. Yet, C21 + SAC treatment, not VAL + SAC, prevented increases in body weight gain and kidney weight, a sign of hypertrophy. Analysis of 24-hr urine samples revealed that HSD caused a marked increase in albuminuria and proteinuria, which was significantly prevented by only C21 + SAC treatment. ANP was similarly preserved and associated with comparable inhibition of NEP activity in both treatment groups compared to HSD control. Furthermore, C21 + SAC treatment caused an increase in the renal expression of ACE2, and a decrease in renal renin activity, unlike VAL + SAC treatment. Moreover, HSD caused a reduction in plasma renin levels, which were increased 3-times by VAL + SAC, but not by C21 + SAC. Renal AT2R expression was unchanged in all treatment groups compared with HSD control group. On the other hand, renal AT1R expression was modestly increased by only VAL + SAC treatment. Taken together, this data suggests the combination of C21 + SAC provides strong reno-protection in obese animals fed HSD and is superior to the protection afforded by the combination of VAL + SAC. Differential regulation of the opposing arms of the RAS may be the basis of the superiority of C21 + SAC over VAL + SAC.

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.

HYPERTENSION AND RENIN-ANGIOTENSIN SYSTEM BLOCKERS ARE NOT ASSOCIATED WITH EXPRESSION OF ANGIOTENSIN CONVERTING ENZYME 2 (ACE2) IN THE KIDNEY

Objective: Angiotensin converting enzyme 2 (ACE2) is the cellular entry point for severe acute respiratory syndrome coronavirus (SARS-CoV-2) – the cause of COVID-19 disease. However, the effect of RAS-inhibition on ACE2 expression in human tissues of key relevance to blood pressure regulation and COVID-19 infection has not previously been reported. Design and method: We examined how hypertension, its major metabolic co-phenotypes and antihypertensive medications relate to ACE2 renal expression using information from up to 436 patients whose kidney transcriptomes were characterised by RNA-sequencing. We further validated some of the key observations in other human tissues and/or a controlled experimental model. Results: Our data reveal increasing expression of ACE2 with age in both human lungs and the kidney. We show no association between renal expression of ACE2 and either hypertension or common types of RAS inhibiting drugs. We demonstrate that renal abundance of ACE2 is positively associated with a biochemical index of kidney function and show a strong enrichment for genes responsible for kidney health and disease in ACE2 co-expression analysis. Conclusions: Our results indicate that neither hypertension nor antihypertensive treatment are likely to alter the expression of the key entry receptor for SARS-CoV-2 in the human kidney. Our data further suggest that in the absence of SARS-CoV-2 infection, kidney ACE2 is most likely nephro-protective but the age-related increase in its expression within lungs and kidneys may be relevant to the risk of SARS-CoV-2 infection.

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.

Hypertension and renin-angiotensin system blockers are not associated with expression of angiotensin-converting enzyme 2 (ACE2) in the kidney.

AimsAngiotensin-converting enzyme 2 (ACE2) is the cellular entry point for severe acute respiratory syndrome coronavirus (SARS-CoV-2)—the cause of coronavirus disease 2019 (COVID-19). However, the effect of renin-angiotensin system (RAS)-inhibition on ACE2 expression in human tissues of key relevance to blood pressure regulation and COVID-19 infection has not previously been reported.Methods and resultsWe examined how hypertension, its major metabolic co-phenotypes, and antihypertensive medications relate to ACE2 renal expression using information from up to 436 patients whose kidney transcriptomes were characterized by RNA-sequencing. We further validated some of the key observations in other human tissues and/or a controlled experimental model. Our data reveal increasing expression of ACE2 with age in both human lungs and the kidney. We show no association between renal expression of ACE2 and either hypertension or common types of RAS inhibiting drugs. We demonstrate that renal abundance of ACE2 is positively associated with a biochemical index of kidney function and show a strong enrichment for genes responsible for kidney health and disease in ACE2 co-expression analysis.ConclusionOur results indicate that neither hypertension nor antihypertensive treatment is likely to alter the expression of the key entry receptor for SARS-CoV-2 in the human kidney. Our data further suggest that in the absence of SARS-CoV-2 infection, kidney ACE2 is most likely nephro-protective but the age-related increase in its expression within lungs and kidneys may be relevant to the risk of SARS-CoV-2 infection.

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.

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.

Abstract 136: Attenuation of Increased Blood Pressure by Chronic Estrogen Supplementation in Female IUGR Offspring at 12 months of Age is Associated With a Shift Towards ACE2

Low birth weight ( LBW ) women exhibit an earlier age at menopause and a two-fold greater prevalence of hypertension in later life. In a model of LBW induced via placental insufficiency in the rat, female LBW rats develop an increase in blood pressure ( BP ) by 12 months of age. This is associated with persistent estrous that occurs 6 months prior to controls, indicative of early reproductive senescence. Although the underlying mechanisms are unknown, a shift in the hormonal milieu in addition to dysregulation of the renin angiotensin system ( RAS ) are potential contributors to the increase in BP that occurs in women after menopause. The vasoconstrictor arm of the RAS is implicated in increased BP; ACE2 counterbalances this shift towards increased BP. We previously reported that renal ACE2 expression is elevated in female LBW rats that are normotensive in young adulthood; an increase that is not present in female LBW rats in later life. Thus, this study tested the hypothesis that chronic 17β-estradiol ( E2 ) supplementation would reduce BP and shift the balance of the RAS in female LBW rats. Placebo or E2 pellets (1.5mg 17β-estradiol, 60-day release) were implanted at 12 months of age for 6 weeks followed by measurement of BP in conscious, chronically catheterized animals. Kidneys were collected for molecular analysis. BP was significantly increased in placebo-treated LBW relative to placebo-treated Control (134 vs 122 mmHg, respectively, p &lt;0.05); an increase that was abolished by chronic E2 in LBW (116 mmHg, p &lt;0.05). Using Real-Time PCR, cortical ACE2 mRNA expression did not differ between Control or LBW rats. Medullary ACE2 mRNA expression was significantly increased in placebo-treated LBW compared to placebo-treated Controls (1-fold increase; p &lt;0.05). However, following chronic E2, medullary ACE2 mRNA expression was increased to a greater degree in chronic E2-treated LBW rats, almost two-fold higher, relative to chronic E2-treated Control. Therefore, these results suggest that E2 supplementation attenuates increased BP in female LBW rats in a manner that may involve a shift in the RAS towards ACE2 abundance suggesting that ACE2 via chronic E2 may be beneficial in the treatment of increased BP that occurs after reproductive senescence in LBW.

Overexpression of heterogeneous nuclear ribonucleoprotein F stimulates renal Ace-2 gene expression and prevents TGF-β1-induced kidney injury in a mouse model of diabetes.

Aims/hypothesisWe investigated whether heterogeneous nuclear ribonucleoprotein F (hnRNP F) stimulates renal ACE-2 expression and prevents TGF-β1 signalling, TGF-β1 inhibition of Ace-2 gene expression and induction of tubulo-fibrosis in an Akita mouse model of type 1 diabetes.MethodsAdult male Akita transgenic (Tg) mice overexpressing specifically hnRNP F in their renal proximal tubular cells (RPTCs) were studied. Non-Akita littermates and Akita mice served as controls. Immortalised rat RPTCs stably transfected with plasmid containing either rat Hnrnpf cDNA or rat Ace-2 gene promoter were also studied. ResultsOverexpression of hnRNP F attenuated systemic hypertension, glomerular filtration rate, albumin/creatinine ratio, urinary angiotensinogen (AGT) and angiotensin (Ang) II levels, renal fibrosis and profibrotic gene (Agt, Tgf-β1, TGF-β receptor II [Tgf-βrII]) expression, stimulated anti-profibrotic gene (Ace-2 and Ang 1–7 receptor [MasR]) expression, and normalised urinary Ang 1–7 level in Akita Hnrnpf-Tg mice as compared with Akita mice. In vitro, hnRNP F overexpression stimulated Ace-2 gene promoter activity, mRNA and protein expression, and attenuated Agt, Tgf-β1 and Tgf-βrII gene expression. Furthermore, hnRNP F overexpression prevented TGF-β1 signalling and TGF-β1 inhibition of Ace-2 gene expression.Conclusions/interpretationThese data demonstrate that hnRNP F stimulates Ace-2 gene transcription, prevents TGF-β1 inhibition of Ace-2 gene transcription and induction of kidney injury in diabetes. HnRNP F may be a potential target for treating hypertension and renal fibrosis in diabetes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-015-3700-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

The effects of Yishen-Pinggan decoction on the urine microalbumin and the expression of ACE2, AngIIin the spotnaneously hype

Objective To observe the effects of Yishen-Pinggan dcoction on the urine microalbumin and the expression of ACE2, AngⅡin the spontaneously hypertensive rat kidney（SHR）;to observe the kidney level hepatic side of the protective effect of early renal in SHR hypertension. Methods The same old male SHR and normal rats（Wistar-Kyoto rats, WKY） for the study, at 10 weeks of age, SHR rats were randomly divided into a model group, a Chinese medicine group, and a lotensin group;while WKY rats were served as a normal control group（n=8）. They were continuously treated for 12 weeks. Urine mAlb and β2-MG of rats were quantified at pre-experiment and after experiment, immuno-histochemical detection of renal ACE2 and AngⅡwere performed, the obtained results were statistically analyzed by computer image analysis. Result At the 12th week, compared with urinary mAlb（509.3±160.3）ng/ml,β2-MG（0.150±0.037）μg/ml, Ang kidneyⅡ（7 174.24±1 068.99）μm2,kidney ACE2（2 158.90±376.60）μm2 in the WKY group, the urinary mAlb（908.3 ±13.2）ng/ml, β2-MG（0.378±0.099）μg/ml, kidney AngⅡ（11 085.37±1 398.03）μm2 in the hypertension model group were significantly higher（P〈0.01）, and kidney ACE2（1 375.77±466.21）μm2 was decreased significantly（P〈0.01）; compared with hypertensive model group, rat urinary mAlb（574.9±197.8）ng/ml,β2-MG（0.198±0.040）μg/ml, kidney AngⅡ（8 462.91±781.23）μm2 in Chinese medicine group, rat urinary mAlb（644.4±147.5）ng/ml, β2-MG（0.206±0.044）μg/ml, kidney AngⅡ（8 696.88±679.70）μm2 in lotensin group were significantly decreased（P〈0.01）; while kidney ACE2（1 904.02 ± 454.08）μm2 in Chinese medicine group, kidney ACE2（1896.11±445.43）μm2 in lotensin group was significantly increased（P〈0.01）.＆amp;nbsp;Conclusion Yishen-Pinggan decoction can reduce the SHR urinary microalbumin excretion so as to reduce high blood pressure early kidney damage; its role may be associated with its increasing local renal ACE2 expression and reducing the kidney Ang Ⅱ expression. Key words: Yishen-Pinggan decoction; Hypertension; Early renal damage; Ang II

Insulin treatment attenuates renal ADAM17 and ACE2 shedding in diabetic Akita mice

Angiotensin-converting enzyme 2 (ACE2) is located in several tissues and is highly expressed in renal proximal tubules, where it degrades the vasoconstrictor angiotensin II (ANG II) to ANG-(1-7). Accumulating evidence supports protective roles of ACE2 in several disease states, including diabetic nephropathy. A disintegrin and metalloprotease (ADAM) 17 is involved in the shedding of several transmembrane proteins, including ACE2. Our previous studies showed increased renal ACE2, ADAM17 expression, and urinary ACE2 in type 2 diabetic mice (Chodavarapu H, Grobe N, Somineni HK, Salem ES, Madhu M, Elased KM. PLoS One 8: e62833, 2013). The aim of the present study was to determine the effect of insulin on ACE2 shedding and ADAM17 in type 1 diabetic Akita mice. Results demonstrate increased renal ACE2 and ADAM17 expression and increased urinary ACE2 fragments (≈70 kDa) and albumin excretion in diabetic Akita mice. Immunostaining revealed colocalization of ACE2 with ADAM17 in renal tubules. Renal proximal tubular cells treated with ADAM17 inhibitor showed reduced ACE2 shedding into the media, confirming ADAM17-mediated shedding of ACE2. Treatment of Akita mice with insulin implants for 20 wk normalized hyperglycemia and decreased urinary ACE2 and albumin excretion. Insulin also normalized renal ACE2 and ADAM17 but had no effect on tissue inhibitor of metalloproteinase 3 (TIMP3) protein expression. There was a positive linear correlation between urinary ACE2 and albuminuria, blood glucose, plasma creatinine, glucagon, and triglycerides. This is the first report showing an association between hyperglycemia, cardiovascular risk factors, and increased shedding of urinary ACE2 in diabetic Akita mice. Urinary ACE2 could be used as a biomarker for diabetic nephropathy and as an index of intrarenal ACE2 status.

Abstract 381: Upregulation of Tumor Necrosis Factor α-converting Enzyme (TACE) Protein Expression in db/db Mice is Reversed by Rosiglitazone

TACE, also known as ADAM17, is involved in the ectodomain shedding of several membrane bound proteins. Angiotensin converting enzyme 2 (ACE2), homologue of angiotensin converting enzyme (ACE), is known to be renoprotective by increasing the degradation of vasoactive peptide Angiotensin II (Ang II) to vasodilator peptide Ang - (1-7). It has been shown before that TACE mediates regulated ectodomain shedding of ACE2. PPARγ agonist, rosiglitazone, is known to impart renoprotection by attenuating albuminuria. However, the exact mechanism of renoprotection is not clear. Our previous results and others showed that renal ACE2 protein expression is increased during early stages of diabetes in db/db mice. We also demonstrated increased urinary ACE2 excretion in db/db mice. The goal of this study is to test the hypothesis that TACE is upregulated in db/db diabetic mice and treatment with rosiglitazone imparts renoprotection by attenuating the shedding of renal ACE2 via downregulating TACE protein expression. Male 6 week db/db mice were fed rosiglitazone (20mg/kg/day) for 10 weeks. Metabolic and urinary parameters were monitored weekly. Kidney lysate and urine was used to perform western blot and ACE2 activity (pmols/h/μg protein) respectively. db/db mice demonstrated glucose intolerance, hyperglycemia (348.2±29.9 mg/dL), and albuminuria (0.3±0.05 mg/day) at a very early age. Chronic rosiglitazone treatment normalized blood glucose levels (142.0±12.2 mg/dL), improved glucose tolerance and decreased albuminuria (0.17±0.03 mg/day) in treated db/db mice. Western blot showed increased renal TACE protein expression of db/db mice compared to controls (p&lt;0.05). Treatment with rosiglitazone significantly decreased renal TACE protein expression in treated db/db mice compared to untreated mice (p&lt;0.05). In addition, db/db mice demonstrated a 7 fold increase in urinary ACE2 activity (15.1±4.5) compared to controls (1.74±0.9). Treatment with rosiglitazone significantly attenuated and normalized ACE2 activity in treated db/db mice (0.1±0.06). In conclusion, TACE is upregulated in db/db mice and normalizing hyperglycemia with rosiglitazone could impart renoprotection by decreasing renal TACE protein expression and shedding of renal ACE2.

Role of angiotensin-converting enzyme (ACE and ACE2) imbalance on tourniquet-induced remote kidney injury in a mouse hindlimb ischemia–reperfusion model

In this study, the relationship between the local imbalance of angiotensin converting enzymes ACE and ACE2 as well as Ang II and Ang (1–7) and renal injury was observed in the different genotypes mice subjected to tourniquet-induced ischemia–reperfusion on hind limbs. In wild-type mice, renal ACE expression increased while renal ACE2 expression decreased significantly after reperfusion, accompanied by elevated serum angiotensin II (Ang II) level and lowered serum angiotensin (1–7) (Ang (1–7)) level. However, renal Ang (1–7) also increased markedly while renal Ang II was elevated. Renal injury became evident after limb reperfusion, with increased malondialdehyde (MDA), decreased super-oxide dismutase (SOD) activity and increased serum blood urea nitrogen (BUN) and creatinine (Cr), compared to control mice. These mice also developed severe renal pathology including infiltration of inflammatory cells in the renal interstitium and degeneration of tubule epithelial cells. In ACE2 knock-out mice with ACE up-regulation, tourniquet-induced renal injury was significantly aggravated as shown by increased levels of MDA, BUN and Cr, decreased SOD activity, more severe renal pathology, and decreased survival rate, compared with tourniquet-treated wild-type mice. Conversely, ACE2 transgenic mice with normal ACE expression were more resistant to tourniquet challenge as evidenced by decreased levels of MDA, BUN and Cr, increased SOD activity, attenuated renal pathological changes and increased survival rate. Our results suggest that the deregulation of ACE and ACE2 plays an important role in tourniquet-induced renal injury and that ACE2 up-regulation to restore the proper ACE/ACE2 balance is a potential therapeutic strategy for kidney injury.

Dual RAS blockade normalizes angiotensin-converting enzyme-2 expression and prevents hypertension and tubular apoptosis in Akita angiotensinogen-transgenic mice

We investigated the effects of dual renin-angiotensin system (RAS) blockade on angiotensin-converting enzyme-2 (Ace2) expression, hypertension, and renal proximal tubular cell (RPTC) apoptosis in type 1 diabetic Akita angiotensinogen (Agt)-transgenic (Tg) mice that specifically overexpress Agt in their RPTCs. Adult (11 wk old) male Akita and Akita Agt-Tg mice were treated with two RAS blockers (ANG II receptor type 1 blocker losartan, 30 mg·kg(-1)·day(-1)) and angiotensin-converting enzyme (ACE) inhibitor perindopril (4 mg·kg(-1)·day(-1)) in drinking water. Same-age non-Akita littermates and Agt-Tg mice served as controls. Blood pressure, blood glucose, and albuminuria were monitored weekly. The animals were euthanized at age 16 wk. The left kidneys were processed for immunohistochemistry and apoptosis studies. Renal proximal tubules were isolated from the right kidneys to assess gene and protein expression. Urinary ANG II and ANG 1-7 were quantified by ELISA. RAS blockade normalized renal Ace2 expression and urinary ANG 1-7 levels (both of which were low in untreated Akita and Akita Agt-Tg), prevented hypertension, albuminuria, tubulointerstitial fibrosis and tubular apoptosis, and inhibited profibrotic and proapoptotic gene expression in RPTCs of Akita and Akita Agt-Tg mice compared with non-Akita controls. Our results demonstrate the effectiveness of RAS blockade in preventing intrarenal RAS activation, hypertension, and nephropathy progression in diabetes and support the important role of intrarenal Ace2 expression in modulating hypertension and renal injury in diabetes.