ACE2 internalization induced by a SARS-CoV-2 recombinant protein is modulated by angiotensin II type 1 and bradykinin 2 receptors.

AIMS: Angiotensin-converting enzyme 2 (ACE2) is a key regulator of the renin-angiotensin system (RAS) recently identified as the membrane receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we aim to study whether two receptors from RAS, the angiotensin receptor type 1 (AT1R) and the bradykinin 2 receptor (B2R) modulate ACE2 internalization induced by a recombinant receptor binding domain (RBD) of SARS-CoV-2 spike protein. Also, we investigated the impact of ACE2 coexpression on AT1R and B2R functionality. MATERIALS AND METHODS: To study ACE2 internalization, we assessed the distribution of green fluorescent protein (GFP) signal in HEK293T cells coexpressing GFP-tagged ACE2 and AT1R, or B2R, or AT1R plus B2R in presence of RBD alone or in combination with AT1R or B2R ligands. To estimate ACE2 internalization, we classified GFP signal distribution as plasma membrane uniform GFP (PMU-GFP), plasma membrane clustered GFP (PMC-GFP) or internalized GFP and calculated its relative frequency. Additionally, we investigated the effect of ACE2 coexpression on AT1R and B2R inhibitory action on voltage-gated calcium channels (CaV2.2) currents by patch-clamp technique. KEY FINDINGS: RBD induced ACE2-GFP internalization in a time-dependent manner. RBD-induced ACE2-GFP internalization was increased by angiotensin II and reduced by telmisartan in cells coexpressing AT1R. RBD-induced ACE2-GFP internalization was strongly inhibited by B2R co-expression. This effect was mildly modified by bradykinin and rescued by angiotensin II in presence of AT1R. ACE2 coexpression impacted on B2R- and AT1R-mediated inhibition of CaV2.2 currents. SIGNIFICANCE: Our work contributes to understand the role of RAS modulators in the susceptibility to SARS-CoV-2 infection and severity of COVID-19.

Angiotensin II receptor type 1 - An update on structure, expression and pathology.

The AT1 receptor, a major effector of the renin-angiotensin system, has been extensively studied in the context of cardiovascular and renal disease. Moreover, angiotensin receptor blockers, sartans, are among the most frequently prescribed drugs for the treatment of hypertension, chronic heart failure and chronic kidney disease. However, precise molecular insights into the structure of this important drug target have not been available until recently. In this context, seminal studies have now revealed exciting new insights into the structure and biased signaling of the receptor and may thus foster the development of novel therapeutic approaches to enhance the efficacy of pharmacological angiotensin receptor antagonism or to enable therapeutic induction of biased receptor activity. In this review, we will therefore highlight these and other seminal publications to summarize the current understanding of the tertiary structure, ligand binding properties and downstream signal transduction of the AT1 receptor.

Activation of the Central Renin-Angiotensin System Causes Local Cerebrovascular Dysfunction.

Background and Purpose: Hypertension is a leading risk factor for cerebrovascular disease and loss of brain health. While the brain renin-angiotensin system (RAS) contributes to hypertension, its potential impact on the local vasculature is unclear. We tested the hypothesis that activation of the brain RAS would alter the local vasculature using a modified deoxycorticosterone acetate (DOCA) model. Methods: C57BL/6 mice treated with DOCA (50 mg SQ; or shams) were given tap H2O and H2O with 0.9% NaCl for 1 to 3 weeks. Results: In isolated cerebral arteries and parenchymal arterioles from DOCA-treated male mice, endothelium- and nitric oxide-dependent dilation was progressively impaired, while mesenteric arteries were unaffected. In contrast, cerebral endothelial function was not significantly affected in female mice treated with DOCA. In males, mRNA expression of renal Ren1 was markedly reduced while RAS components (eg, Agt and Ace) were increased in both brain and cerebral arteries with central RAS activation. In NZ44 reporter mice expressing GFP (green fluorescent protein) driven by the angiotensin II type 1A receptor (Agtr1a) promoter, DOCA increased GFP expression ≈3-fold in cerebral arteries. Impaired endothelial responses were restored to normal by losartan, an AT1R (angiotensin II type 1 receptor) antagonist. Last, DOCA treatment produced inward remodeling of parenchymal arterioles. Conclusions: These findings suggest activation of the central and cerebrovascular RAS impairs endothelial (nitric oxide dependent) signaling in brain through expression and activation of AT1R and sex-dependent effects. The central RAS may be a key contributor to vascular dysfunction in brain in a preclinical (low renin) model of hypertension. Because the brain RAS is also activated during aging and other diseases, a common mechanism may promote loss of endothelial and brain health despite diverse cause.

Aldosterone-Regulating Receptors and Aldosterone-Driver Somatic Mutations.

Background: Somatic gene mutations that facilitate inappropriate intracellular calcium entrance have been identified in most aldosterone-producing adenomas (APAs). Studies suggest that angiotensin II and adrenocorticotropic hormone (ACTH) augment aldosterone production from APAs. Little is known, however, regarding possible variations in response to hormonal stimuli between APAs with different aldosterone-driver mutations. Objective: To analyze the transcript expression of type 1 angiotensin II receptors (AGTR1), ACTH receptors (MC2R), and melanocortin 2 receptor accessory protein (MRAP) in APAs with known aldosterone-driver somatic mutations. Methods: RNA was isolated from APAs with mutations in: KCNJ5 (n = 14), ATP1A1 (n = 14), CACNA1D (n = 14), and ATP2B3 (n = 5), and from normal adjacent adrenal tissue (n = 45). Transcript expression of MC2R, MRAP, AGTR1, aldosterone synthase (CYP11B2), 17α-hydroxylase/17,20-lyase (CYP17A1), and 11ß-hydroxylase (CYP11B1) were quantified using quantitative RT-PCR and normalized to ß-actin. Results: Compared to adjacent normal adrenal tissue, APAs had higher transcript levels of CYP11B2 (2,216.4 [1,112.0, 2,813.5]-fold, p < 0.001), MC2R (2.88 [2.00, 4.52]-fold, p < 0.001), and AGTR1 (1.80 [1.02, 2.80]-fold, p < 0.001]), and lower transcript levels of MRAP, CYP17A1, and CYP11B1 (0.28-0.36, p < 0.001 for all). MC2R and CYP11B2 transcripts were lower in APAs with KCNJ5 vs. other mutations (p < 0.01 for both). MC2R expression correlated positively with that of AGTR1 in APAs harboring KCNJ5 and CACNA1D mutations, and with MRAP expression in APAs harboring ATPase mutations. Conclusions: While MC2R and AGTR1 are expressed in all APAs, differences were observed based on the underlying aldosterone-driver somatic mutations. In tandem, our findings suggest that APAs with ATPase-mutations are more responsive to ACTH than KCNJ5-mutated APAs.

trans-Anethole attenuated renal injury and reduced expressions of angiotensin II receptor (AT1R) and TGF-ß in streptozotocin-induced diabetic rats.

Fibrosis is a pathological process in diabetic nephropathy that causes renal failure and dysfunction. Given the known anti-diabetic effects of trans-Anethole (TA), we aimed to investigate its renoprotective and anti-fibrotic effect alone and in combination with losartan in diabetic nephropathy. Male Wistar rats received a single intraperitoneal injection of 65 mg/kg streptozotocin (STZ) for diabetes induction. Diabetic rats were treated orally with saline, TA (80 mg/kg), losartan (Los; 10 mg/kg), or the combination of TA and losartan (TA-Los) daily for five weeks. Renal function was monitored during the study, and renal fibrosis, oxidative stress markers, apoptotic cells, and the expression and localization of AT1R, TGF-ß1, and Col-IV were detected in the kidney. Results showed that TA alone and in combination with losartan was able to decrease blood glucose, urea, and creatinine levels and improve kidney function parameters. TA, Los, and TA-Los significantly reduced tubule vascular degeneration, glomerular and tubulointerstitial sclerosis, oxidative stress, and apoptotic cells. Immunohistochemistry analyses showed that TA, losartan, and TA-losartan combination downregulated the AT1R, Col IV, and TGF-ß1 expression and distribution in diabetic rat kidneys. Results suggest that TA is able to suppress diabetic nephropathy in rats effectively, probably by decreasing blood glucose levels and downregulating AT1R and TGF-ß1 expression.

ACE (Angiotensin-Converting Enzyme) Inhibition Reverses Vasoconstriction and Impaired Dilation of Pial Collaterals in Chronic Hypertension.

Leptomeningeal anastomoses (LMAs) are pial collaterals that perfuse the penumbra and important for stroke outcome. We previously showed LMAs from SHRs (spontaneously hypertensive rats) were vasoconstricted compared with normotensive Wistar rats. Here, we investigated mechanisms by which hypertension causes LMA vasoconstriction. SHRs were treated with the ACE (angiotensin-converting enzyme) inhibitor captopril, an Ang II (angiotensin II)-independent antihypertensive agent hydralazine, or vehicle for 5 weeks in drinking water (n=8/group). A group of Wistar rats (n=8) had regular drinking water served as controls. Blood pressure was measured twice weekly by tail-cuff. LMAs were isolated and studied under pressurized conditions. Vasoreactivity of LMAs, including myogenic responses, reactivity to Rho-kinase inhibitor Y-27632, and nitric oxide were measured. Both captopril and hydralazine lowered blood pressure in SHRs similar to Wistar. However, only captopril normalized LMA increased tone compared with untreated SHRs (15±2% versus 50±3%; P<0.01) that was similar to Wistar (16±2%) but not hydralazine (38±6%; P>0.05). Vasodilatory response of LMAs to Y-27632 was impaired in SHRs compared with Wistar (28±3% versus 81±4%; P<0.01) that was restored by captopril (84±5%; P<0.01) and partially hydralazine (59±4%). LMAs from all groups constricted similarly to NOS (NO synthase) inhibition; however, the vasodilatory response of LMAs to the nitric oxide donor sodium nitroprusside was impaired in SHRs compared with Wistar rats (29±4% versus 80±2%; P<0.01) that was restored by captopril (84±4%; P<0.01), not hydralazine (38±8%; P>0.05). These results suggest that ACE inhibition during chronic hypertension reversed vascular dysfunction and hyperconstriction of LMAs that could improve stroke outcome by increasing collateral perfusion.

Angiotensin II type 1 receptor blockers favorably affect renal angiotensin II and MAS receptor expression in patients with diabetic nephropathy.

INTRODUCTION: The aims of this study were to assess the renal expression of angiotensin II type 1 receptor (AT1R), angiotensin II type 2 receptor (AT2R), and MAS receptor in human type 2 diabetic nephropathy (DN). MATERIALS AND METHODS: In total, 115 patients diagnosed with DN by renal biopsy were enrolled in this study. The protein expression levels of the AT1R, AT2R, and MAS receptors were assessed by immunohistochemistry. RESULTS: The protein expression levels of AT1R, AT2R, and MAS receptor in the renal biopsy tissue were correlated with the pathologic classification of DN. Tubulointerstitial AT1R expression in patients of class IIb was significantly stronger than control samples (p < 0.05). Expression of AT2R and MAS receptors were highest with class IIb DN patients. When DN patients were treated with AT1R blocker (ARB), the expression of AT1R was downregulated (p < 0.05), and the MAS receptor was upregulated in tubular interstitial (p < 0.05). CONCLUSIONS: Our results directly observed that renal expression levels of AT1R increase during the early stages of DN, ARB reducing AT1R while increasing MAS receptor. Therefore, ARB should be used as soon as possible in patients with DN.

The Role of Overexpressed Apolipoprotein AV in Insulin-Resistant Hepatocytes.

In this paper, we sought to explore the relationship between apolipoprotein AV (APOAV) overexpression and insulin resistance in hepatocytes. The insulin-resistant HepG2 cell model was constructed, and then, APOAV-overexpressed HepG2 cells (B-M) were induced by infecting with a recombinant adenovirus vector. Microarray data were developed from B-M samples compared with negative controls (A-con), and the microarray data were analyzed by bioinformatic methods. APOAV-overexpression induced 313 upregulated genes and 563 downregulated ones in B-M sample. The differentially expressed genes (DEGs) were significantly classified in fat digestion and absorption pathway. Protein-protein interaction network was constructed, and AGTR1 (angiotensin II receptor type 1) and P2RY2 (purinergic receptor P2Y, G-protein coupled 2) were found to be the significant nodes closely related with G-protein related signaling. Additionally, overexpression of APOAV could change the expression of Glut4 and release the insulin resistance of hepatic cells. Thus, APOAV overexpression may prevent the insulin resistance in liver cells by mediating the genes such as AGTR1 and P2RY2.

Differences in the expression of the renin angiotensin system and the kallikrein-kinin system during the course of myocardial infarction in male and female Wistar rats.

BACKGROUND: There is some evidence that components of the renin-angiotensin system and kallikrein-kinin system are not similarly regulated in both sexes. The aim of this work was to analyze the expression of angiotensin-converting enzyme, angiotensin-converting enzyme 2, angiotensin 1 receptor, angiotensin 2 receptor, beta-1 receptor, and beta-2 receptor during the evolution of myocardial infarction. METHODS: Thirty-six male and 36 female Wistar rats were used. Myocardial infarction was induced. Six groups of both sexes were formed, (n=6): (a) sham; (b) 48 h myocardial infarction; (c) one week myocardial infarction; (d) two weeks myocardial infarction; (e) three weeks myocardial infarction and (f) four weeks myocardial infarction. The expression was evaluated by real-time polymerase chain reaction on the penumbra of left ventricle. RESULTS: The mRNA expression of most biomarkers was lower in females than in males. During acute infarction, an increase of all protein expression was found in female and at two weeks while in the male only biomarker changes occurred at three weeks. In addition, in male biomarkers mRNA expression decreased during chronic infarction while in females it did not. CONCLUSIONS: The renin-angiotensin system and kallikrein-kinin system biomarkers expression occurs at earlier times in the female than in the male rat. In addition, during chronic myocardial infarction these biomarkers remained unchanged in females while in males they decreased.

AT1R/GSK-3ß/mTOR Signaling Pathway Involved in Angiotensin II-Induced Neuronal Apoptosis after HIE Both In Vitro and In Vivo.

OBJECTIVE: The focus of the present study is to evaluate the effects of Angiotensin II (Ang II) on neuronal apoptosis after HIE and the potential underlying mechanisms. METHODS: Primary neonatal rat cortical neurons were used to study the oxygen-glucose deprivation (OGD) cell model. The expressions of Ang II, AT1R, GSK-3ß, p-GSK-3ß, mTOR, p-mTOR, Bax, Bcl-2, and cleaved caspase-3 were detected via western blot. IF and flow cytometry were used to evaluate neuronal apoptosis. Hypoxic-ischemic encephalopathy (HIE) was established to evaluate the therapeutic effects of Ang II in vivo. Cerebral infarction areas were detected by 2,3,5-Triphenyltetrazolium chloride staining. The righting and geotaxis reflexes were also recorded. In addition, Fluoro-Jade C staining and TUNEL staining were performed to evaluate neuronal degeneration and apoptosis. RESULTS: Ang II significantly increased the rate of neuronal apoptosis, upregulated the expression of cleaved caspase-3, and downregulated Bcl-2/Bax ratio after OGD insult. For vivo assay, the expressions of endogenous Ang II and AT1R gradually increased and peaked at 24 h after HIE. Ang II increased NeuN-positive AT1R cell expression. In addition, Ang II increased the area of cerebral infarction, promoted neuronal degeneration and apoptosis, aggravated neurological deficits on righting and geotaxis reflexes, and was accompanied by increased expressions of phosphorylated GSK-3ß and mTOR. The application of valsartan (Ang II inhibitor) or SB216763 (GSK-3ß inhibitor) reversed these phenomena triggered by Ang II following HIE. CONCLUSION: Ang II increased neuronal apoptosis through the AT1R/GSK-3ß/mTOR signaling pathway after experimental HIE both in vitro and in vivo, and Ang II may serve as a novel therapeutic target to ameliorate brain injury after HIE.

Ethanol Withdrawal Alters the Oxidative State of the Heart Through AT1-Dependent Mechanisms.

AIMS: We investigated the cardiac effects of ethanol withdrawal and the possible role of AT1 receptors in such response. METHODS: Male Wistar rats were treated with increasing doses of ethanol (3 to 9%, vol./vol.) for 21 days. The cardiac effects of ethanol withdrawal were investigated 48 h after abrupt discontinuation of ethanol. Some animals were orally treated with losartan (10 mg/kg/day), a selective AT1 receptor antagonist. RESULTS: Ethanol withdrawal did not affect serum levels of creatine kinase (CK)-MB. Losartan prevented ethanol withdrawal-induced increase in superoxide anion (O2•-) production in the left ventricle (LV). However, ethanol withdrawal did no alter the levels of thiobarbituric acid reactive substances (TBARS) or the expression of Nox1, Nox2 or Nox4 were found in the LV. Ethanol withdrawal reduced the concentration of hydrogen peroxide (H2O2) in the LV and this response was prevented by losartan. Ethanol withdrawal increased catalase activity in the LV and losartan attenuated this response. No changes on superoxide dismutase (SOD) activity or expression were detected in the LV during ethanol withdrawal. The expression of AT1, AT2 or angiotensin converting enzyme (ACE) was not affected by ethanol withdrawal. Similarly, no changes on the expression of ERK1/2, SAPK/JNK, COX-1 or COX-2 were found in the LV during ethanol withdrawal. CONCLUSIONS: Ethanol withdrawal altered the cardiac oxidative state through AT1-dependent mechanisms. Our findings showed a role for angiotensin II/AT1 receptors in the initial steps of the cardiac effects induced by ethanol withdrawal.

Differential expression of angiotensin II type 1 receptor subtypes within the cerebral microvasculature.

Arteries and arterioles constrict in response to intraluminal pressure to generate myogenic tone, but the molecular nature of the vascular force-sensing mechanism is not fully characterized. Here, we investigated the role of angiotensin II type 1 receptors (AT1Rs) on vascular smooth muscle cells in the development of myogenic tone in cerebral parenchymal arterioles from mice. We found that pretreatment with the AT1R blocker losartan inhibited the development of myogenic tone in these vessels but did not alter the luminal diameter of arterioles with preestablished tone. Rodents express two AT1R isotypes: AT1Ra and AT1Rb. We previously demonstrated that AT1Rb is expressed at much higher levels compared with AT1Ra in cerebral pial arteries and is required for myogenic contractility in these vessels, whereas AT1Ra is unnecessary for this function. Here, we found that AT1Ra and AT1Rb are expressed at similar levels in parenchymal arterioles and that genetic knockout of AT1Ra blunted the ability of these vessels to generate myogenic tone. We also found that AT1Rb and total AT1R expression levels are much lower in parenchymal arterioles compared with pial arteries and that parenchymal arterioles are less sensitive to the vasoconstrictive effects of the endogenous AT1R ligand angiotensin II (ANG II). We conclude that 1) AT1Rs are critical for the initiation, but not the maintenance, of myogenic tone in parenchymal arterioles, and 2) lower levels of AT1Rb and total AT1R in parenchymal arterioles compared with pial arteries result in differences in myogenic and ANG II-induced vasoconstriction between these vascular segments.NEW & NOTEWORTHY Myogenic tone is critical for appropriate regulation of cerebral blood flow, but the mechanisms used by vascular smooth muscle cells to detect changes in intraluminal pressure are not fully characterized. Here, we demonstrate angiotensin II receptor type 1 (AT1R) is indispensable to initiation, but not maintenance, of myogenic tone in cerebral parenchymal arterioles. Furthermore, we demonstrate differences in AT1R expression levels lead to critical differences in contractile regulation between parenchymal arterioles and cerebral pial arteries.

Attenuation of Angiotensin II-Induced Hypertension in BubR1 Low-Expression Mice Via Repression of Angiotensin II Receptor 1 Overexpression.

Background Angiotensin II (Ang II) can cause hypertension and tissue impairment via AGTR1 (Ang II receptor type 1), particularly in renal proximal tubule cells, and can cause DNA damage in renal cells via nicotinamide adenine dinucleotide phosphate oxidase. BubR1 (budding uninhibited by benzimidazole-related 1) is a multifaceted kinase that functions as a mitotic checkpoint. BubR1 expression can be induced by Ang II in smooth muscle cells in vitro, but the relationship between systemic BubR1 expression and the Ang II response is unclear. Methods and Results Twenty 24-week-old male BubR1 low-expression mice (BubR1L/L mice) and age-matched BubR1+/+ mice were used in this study. We investigated how Ang II stimulation affects BubR1L/L mice. The elevated systolic blood pressure caused by Ang II stimulation in BubR1+/+ mice was significantly attenuated in BubR1L/L mice. Additionally, an attenuated level of Ang II-induced perivascular fibrosis was observed in the kidneys of BubR1L/L mice. Immunohistochemistry revealed that the overexpression of AGTR1 induced by Ang II stimulation was repressed in BubR1L/L mice. We evaluated AGTR1 and Nox-4 (nicotinamide adenine dinucleotide phosphate oxidase-4) levels to determine the role of BubR1 in the Ang II response. Results from in vitro assays of renal proximal tubule cells suggest that treatment with small interfering RNA targeting BubR1 suppressed Ang II-induced overexpression of AGTR1. Similarly, the upregulation in Nox4 and Jun N-terminal kinase induced by Ang II administration was repressed by treatment with small interfering RNA targeting BubR1. Conclusions Ang II-induced hypertension is caused by AGTR1 overexpression in the kidneys via the upregulation of BubR1 and Nox4.

Functional relevance of promoter CpG island of human Angiotensin II type 1 receptor (AT1R) gene.

Angiotensin II type 1 receptor can activate number of signalling pathways upon stimulation and consequently its involvement in cancer progression have also been revealed. But which epigenetic mechanisms are involved in its regulation, need to be further explored. In-silico analysis revealed a promoter CpG island (CGI) which was cloned and assayed for functional activity using reporter gene system. The effect of methylation on this CGI was analysed through varying degree of methyltransferase treatment of cloned fragment. Results thus obtained were validated by direct sequencing. To further establish the status of this effect, in-vivo analysis was done through screening of methylation pattern in the targeted region among hypertensives (HTN) and normotensives (NTN) using PCR-Bisulphite sequencing. Additionally, clinical details of all participants, biochemical parameters and lifestyle related information was also collected and statistically evaluated. Reporter gene assay assigned functional activity to the cloned promoter CpG island. Increased dose and durations of methyltransferase treatment decreased the expression of reporter gene indicating the role of promoter DNA methylation. Among all the human samples screened, only one of the HTN individual was found to have single hemi-methylated CpG site at a position which happens to be a part of Sp1 transcription factor binding site. To conclude, CpG island in the promoter region of AT1R (CpG.P.AT1R) gets regulated through epigenetic mechanism of DNA methylation.

Decreased H3K9ac level of AT2R mediates the developmental origin of glomerulosclerosis induced by prenatal dexamethasone exposure in male offspring rats.

This study aimed to demonstrate that prenatal dexamethasone exposure (PDE) can induce kidney dysplasia in utero and adult glomerulosclerosis in male offspring, and to explore the underlying intrauterine programming mechanisms. Pregnant rats were subcutaneously administered dexamethasone 0.2 mg/kg.d from gestational day (GD) 9 to GD20. The male fetus on GD20 and the adult offspring at age of postnatal week 28 were analyzed. The adult offspring kidneys in the PDE group displayed glomerulosclerosis, elevated levels of serum creatinine and urine protein, ultrastructural damage of podocytes, the reduced expression levels of podocyte marker genes, nephrin and podocin. The histone 3 lysine 9 acetylation (H3K9ac) level in the promoter of renal angiotensin II receptor type 2 (AT2R) and its expression were reduced, whereas the angiotensin II receptor type 1a (AT1aR)/AT2R expression ratio was increased. The fetal kidneys in the PDE group displayed an enlarged Bowman's space and a shrunken glomerular tuft, a reduced cortex width and an increase in the nephrogenic zone/cortical zone ratio, reduced the expression level of glial-cell-line derived neurotrophic factor/c-Ret tyrosine kinase receptor (GDNF/c-Ret) signal pathway and podocyte marker genes. Moreover, the H3K9ac and H3K27ac levels of AT2R as well as the gene and protein expression levels of AT2R in fetal kidneys were inhibited by PDE. In vitro, primary metanephric mesenchyme stem cells (MMSCs) were treated with dexamethasone. Overexpression of AT2R reversed the inhibited expression of GDNF/c-Ret and podocin/nephrin induced by dexamethasone, and glucocorticoids receptor antagonist abolished the decreased H3K9ac level and gene expression of AT2R. In conclusion, PDE induced the offspring's kidney dysplasia as well as adult glomerulosclerosis, which was mediated by a sustained decrease in renal AT2R expression via decreasing the H3 K9ac level.

Expression of the intrarenal angiotensin receptor and the role of renin-angiotensin system inhibitors in IgA nephropathy.

The critical role of the intrarenal renin-angiotensin system (RAS) in the development of kidney disease has been well demonstrated in animal and cell-culture experiments, but evidence from human kidney tissues is lacking. In this study, we screened 438 patients with IgA nephropathy (IgAN) and analyzed their clinical characteristics. Renal biopsy revealed the expression of angiotensin II type 1 receptor (AT1R), angiotensin II type 2 receptor (AT2R), and MAS receptor (MASR) in the tissues of 260 patients not treated with RAS inhibitors, 32 patients treated with angiotensin-converting enzyme inhibitors (ACEIs), and 89 patients treated with angiotensin receptor blockers (ARBs). The correlations in expression among these three receptors and the results of Oxford typing were analyzed, together with the ability of ACEIs and ARBs to reduce proteinuria and the effects of ARBs on AT1R and AT2R expression. The results showed significantly higher AT1R, AT2R, and MASR expression in the M1 group (mesangial score > 0.5) than in the M0 group (mesangial score < 0.5), significantly higher AT1R expression in the S1 group (presence of segmental glomerulosclerosis) than in the S0 group (absence of segmental glomerulosclerosis); AT1R expression in the C2 group (crescent formation > 25%) was significantly higher than in the C0 (crescent formation = 0) and C1 (crescent formation < 25%) groups. Patients treated with an ARB for < 6 months had significantly lower urinary protein levels than those taking these drugs for > 6 months. These findings imply that overexpression of AT1R on the mesangial cells of IgAN patients is associated with mesangial cell proliferation, glomerular segmental sclerosis, and crescent formation. In addition, long-term administration of ARB may decrease the efficacy of these medications in terms of reducing proteinuria.

Interaction between angiotensin II and glucose sensing at the subfornical organ.

The subfornical organ (SFO) lacks the normal blood-brain barrier and senses the concentrations of many different circulating signals, including glucose and angiotensin II (ANG II). ANG II has recently been implicated in the control of food intake and body weight gain. The present study assessed whether single SFO neurones sense changes in glucose and ANG II, and also whether changes in glucose concentration alter the responsiveness of these neurones to ANG II. SFO neurones dissociated from male Sprague-Dawley rats (100-175 g) were used. We first examined whether glucose concentration modulates AT1 receptor expression. Similar AT1a mRNA expression levels were found at glucose concentrations of 1, 5 and 10 mmol L-1 in dissociated SFO neurones. Glucose responsiveness of SFO neurones was assessed using perforated current-clamp recordings and switching between 5 and 10 mmol L-1 glucose artificial cerebrospinal fluid to classify single neurones as nonresponsive (nGS), glucose-excited (GE) or glucose-inhibited (GI). In total, 26.7% of the SFO neurones were GI (n = 24 of 90), 21.1% were GE (n = 19 of 90) and 52.2% were nGS (n = 47 of 90). Once classified, the effects of 10 nmol L-1 ANG II on the excitability of these neurones were tested, with 52% of GE (n = 10 of 19), 71% of GI (n = 17 of 24) and 43% of nGS (n = 20 of 47) neurones being ANG II sensitive. Finally, we tested whether acute changes in glucose concentration modified the response to ANG II and showed that some neurones (4/17) only respond to ANG II at 10 mmol L-1 glucose. Our data demonstrate that the same SFO neurone can sense glucose and ANG II and that acute changes in glucose concentration may change ANG II responsiveness.

Histopathological Relevance of Angiotensin II Type 1 Receptor in Renal Transplant Biopsy.

The occurrence of anti-angiotensin II type 1 receptor (AT1R) antibodies is thought to be a risk factor for transplant injury, but the relationship of AT1R to graft loss in renal transplantation has not been assessed. The aim of our study was to evaluate the expression of AT1R and its relationship with graft loss in patients who had a renal transplant biopsy for cause. METHODS: AT1R immunoreactivity was analyzed in 170 renal transplant biopsies. Immunohistochemical evaluation of AT1R expression was performed on 4 µm-thick paraffin sections mounted on silanized slides. AT1R expression was analyzed in 5 compartments: 1. glomeruli, 2. renal blood vessels (small and intermediate arteries), 3. peritubular capillaries, 4. tubular epithelium, and 5. interstitium based on a 3-step scale. RESULTS: Initially we checked 170 consecutive samples of biopsies for the immunoreactivity of the AT1R. The study finally included 118 renal transplant patients in 1-year observation after the biopsy. The renal allograft biopsy was performed between 6 days and 24 years after transplantation and the diagnosis was based on Banff criteria. We observed positive immunostaining of AT1R in tubular epithelium in 26.3% (42/118) of patients. A total of 7 patients had staining assessed as 2 and 35 as 1. One year post-biopsy graft loss in the AT1R (+) patients was 35.7 % (15/42) compared to 14.5% (11/76) in the AT1R (-) group (P = .008). CONCLUSIONS: The expression of AT1R in tubular epithelium of the biopsy for cause was associated with significantly higher graft loss. The relevance of AT1R should be considered for better transplant immunological risk assessment.

Pregnancy upregulates angiotensin type 2 receptor expression and increases blood flow in uterine arteries of rats.

Normal pregnancy is associated with decreased uterine vascular contraction and increased blood flow even though angiotensin II (AngII) levels are increased. AngII not only activates the angiotensin type 1 receptor (AT1R) to mediate vasoconstriction but also angiotensin type 2 receptor (AT2R) to cause vasodilation. We hypothesized that upregulation of AT2R expression and function accounts for increased uterine artery blood flow during pregnancy. Virgin, pregnant (at different days of gestation) and post-partum Sprague-Dawley rats were used to determine uterine artery hemodynamics using micro ultrasound and plasma angiotensin II levels by ELISA. Isolated uterine arteries were examined for AT1R and AT2R expression and isometric contraction/relaxation. Plasma AngII levels were steady up to mid-pregnancy, increased as pregnancy advanced, reaching a peak in late pregnancy, and then restored to pre-pregnant levels after delivery. The pattern of increase in AngII levels mirrored a parallel increase in uterine blood flow. AT1R expression did not change, but AT2R expression increased during pregnancy correlating with uterine blood flow increase. Treatment with the AT2R antagonist PD123319 reduced uterine arterial blood flow. Vasoconstriction to angiotensin II was blunted in pregnant rats. Treatment with PD123319 caused greater enhancement of AngII contraction in pregnant than virgin rats. Ex vivo exposure of estradiol to uterine arterial rings dose dependently upregulated AT2R expression, that was inhibited by estrogen receptor antagonist. These results demonstrate that elevated AngII levels during gestation induce an increase in uterine blood flow via heightened AT2R-mediated signaling. Estrogens appear to directly upregulate uterine vascular AT2R independent of any endogenous factors.

A novel rodent model of pregnancy complications associated with genetically determined angiotensin-converting enzyme (ACE) activity.

Brown Norway (BN) and Lewis (LW) inbred rat strains harbor different angiotensin-converting enzyme ( Ace) polymorphisms that result in higher ACE activity in BN than LW rats. Thus we hypothesized that pregnant BN rats would show pregnancy complications linked to angiotensin II (AII) activity. We performed longitudinal and cross-sectional studies in pregnant LW and BN rats. We found that BN rats have significantly higher ACE activity and AII levels at prepregnancy and throughout pregnancy compared with LW rats, except at midgestation. BN placentas and maternal kidneys had significantly higher expression of AII receptor 1 (AGTR1) and lower expression of AGTR2 than the respective LW placentas and maternal kidneys. Renin-angiotensin system activation in BN rats correlated with hypertension and proteinuria at gestational days 17-21, which were resolved after delivery. In addition, BN rat pregnancies were characterized by significant fetal loss, restricted growth in surviving fetuses, decreased uteroplacental blood flows, and decreased trophoblast remodeling of uterine arteries compared with LW pregnancies. Short-term losartan treatment significantly increased uteroplacental blood flow and fetal weight and decreased maternal blood pressure (BP) and proteinuria in BN pregnancies. In contrast, losartan treatment significantly decreased uteroplacental blood flow and fetal weight but had no significant effect on maternal BP in LW pregnancies. We conclude that Ace polymorphisms play an important role in the reproductive phenotype of BN and LW rats and that BN rats are a novel model of pregnancy complications in association with genetically controlled, increased ACE activity.