Human Renin Research Articles

Metformin modulates microbiota and improves blood pressure and cardiac remodeling in a rat model of hypertension.

Metformin has been attributed to cardiovascular protection even in the absence of diabetes. Recent observations suggest that metformin influences the gut microbiome. We aimed to investigate the influence of metformin on the gut microbiota and hypertensive target organ damage in hypertensive rats. Male double transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR), a model of angiotensin II-dependent hypertension, were treated with metformin (300 mg/kg/day) or vehicle from 4 to 7 weeks of age. We assessed gut microbiome composition and function using shotgun metagenomic sequencing and measured blood pressure via radiotelemetry. Cardiac and renal organ damage and inflammation were evaluated by echocardiography, histology, and flow cytometry. Metformin treatment increased the production of short-chain fatty acids (SCFA) acetate and propionate in feces without altering microbial composition and diversity. It significantly reduced systolic and diastolic blood pressure and improved cardiac function, as measured by end-diastolic volume, E/A, and stroke volume despite increased cardiac hypertrophy. Metformin reduced cardiac inflammation by lowering macrophage infiltration and shifting macrophage subpopulations towards a less inflammatory phenotype. The observed improvements in blood pressure, cardiac function, and inflammation correlated with fecal SCFA levels in dTGR. Invitro, acetate and propionate altered M1-like gene expression in macrophages, reinforcing anti-inflammatory effects. Metformin did not affect hypertensive renal damage or microvascular structure. Metformin modulated the gut microbiome, increased SCFA production, and ameliorated blood pressure and cardiac remodeling in dTGR. Our findings confirm the protective effects of metformin in the absence of diabetes, highlighting SCFA as a potential mediators.

Abstract P377: Short-Chain Fatty Acids Improve Cardiac Function in a Hypertensive Rat Model of Heart Failure

Hypertension-induced ventricular remodeling contributes significantly to the progression of heart failure (HF). The global increase in HF prevalence results in heightened cardiovascular morbidity and mortality, yet treatment options remain limited. The gut microbiota ferments indigestible dietary fibers, producing essential metabolites such as short-chain fatty acids (SCFAs), amino acids metabolites. We hypothesize that direct oral administration of SCFAs modulates mucosal immune cells, thereby ameliorating the heart failure with preserved ejection fraction (HFpEF) phenotype in the double-transgenic rat (dTGR) model. Four-week-old male rats, double-transgenic for human renin and angiotensinogen (dTGRs), were utilized. These dTGR rats exhibit classical symptoms of HFpEF, including hypertension, inflammation, and fibrosis in the heart and kidneys. The rats were randomized into five experimental groups: healthy non-transgenic vehicle-treated controls (wild type), vehicle-treated dTGR, and dTGR treated with either acetate, propionate, or butyrate. Following a 3-week treatment period, comprehensive phenotyping was conducted, including echocardiography, Millar-tip catheterization, gene expression analysis, and histological examination. SCFA administration improves diastolic function, as evidenced by alterations in the end-diastolic pressure-volume relationship measured via Millar-tip catheter. Notably, butyrate treatment had the most pronounced effect on alleviating diastolic dysfunction and reducing blood pressure. Additionally, gene expressions such as atrial natriuretic peptide, connective tissue growth factor, and myosin heavy chain alpha and beta showed improvement in SCFA-treated dTGR HFpEF rats compared to vehicle-treated counterparts. Furthermore, SCFA treatment ameliorated cardiac inflammation. Our data suggest that SCFAs, particularly butyric acid, enhance hypertensive diastolic dysfunction and cardiac gene expression in dTGR rats, underscoring their therapeutic potential in HF.

#2683 Targeted antibiotic modulation of the gut microbiome ameliorates hypertensive kidney damage

Abstract Background and Aims High blood pressure is associated with measurable inflammation, which precedes and promotes damage to the heart and kidneys. Microbiome-derived metabolites, particularly indole metabolites, modulate inflammation and host physiology, partially through aryl hydrocarbon receptor (AhR) signaling. To better understand the potential of a microbiome-targeting therapeutic approach to achieve organ protection in hypertension, we used narrow-spectrum antibiotics without enteral absorption to specifically deplete gram-negative or -positive bacteria in double transgenic rats (dTGR). Method Four-week-old dTGR (transgenic for human renin and angiotensinogen) were treated with oral Vancomycin (Vanco), Polymyxin B (Poly) or Vehicle (Veh) for 3 weeks. Seven-week-old SD rats were included as healthy controls. Microbiome, clinical and immune phenotype were analyzed by shotgun metagenomic sequencing, echocardiography, telemetric blood pressure (BP) measurement, clinical chemistry, bulk RNAseq and flow cytometry. Results Hypertensive kidney damage was ameliorated in Vanco treated dTGR, as assessed by renal Lcn2 expression, blood urea nitrogen and albuminuria. Vanco treated dTGR had significantly decreased cardiac hypertrophy. Poly treatment showed no effect. BP levels for both antibiotic treatments were not significantly different from Veh, despite a significantly improved endothelium-dependent and -independent vasorelaxation in isolated mesenteric arteries in both treated groups. Surprisingly, Vanco treatment led to a massive increase of gram-positive Lactobacilli and associated gene abundance for the production of indole lactic acid (ILA, via araT). We could previously show that ILA inhibits differentiation of pathological Th17 cells through AhR modulation. In line, Vanco treatment reduced the increased number of pathological Th17 in the kidney and intestine of dTGR. Poly treatment did not alter the inflammatory signature. Conclusion Modulation of the intestinal microbiome by narrow-spectrum antibiotics affects hypertensive organ damage. Our data underscores the importance of the gut microbiome in modulating hypertensive organ damage and helps to identify potential therapeutic strategies in the microbiome. Ongoing experiments investigate pro- and postbiotic therapeutic approaches and their AhR dependency.

Abstract 11373: Suppression of Plasma Angiotensin II by GLP-1 in Humans is Independent of Changes in Plasma Angiotensinogen

Introduction: In humans, the natriuretic effect of GLP-1 depends on GLP-1 receptor interaction, is independent of changes in renal blood flow, renin, ANP, and aldosterone but is accompanied by suppression of angiotensin II (ANGII). The mechanism by which GLP-1 reduces ANGII independently of changes in renin is not clarified. Hypothesis: It was hypothesized that GLP-1 suppresses renin substrate, angiotensinogen, in plasma. Methods: To test this proposal, we measured arterial angiotensinogen concentration in biobanked plasma samples from healthy males during a 3-hour GLP-1 infusion that induced natriuresis (1). Samples were from a study with fixed sodium intake where eight healthy men were examined twice in random order during a 3-hour infusion of either GLP-1(1.5 pmol kg -1 min -1 ) or vehicle together with an intravenous infusion of 0.9% NaCl (750 mL/hour). Arterial plasma angiotensinogen was measured in plasma by dilution (1:200) and addition of excess exogenous human renin (Inst. Of Biological Standards UK). After prolonged incubation, ANGI was quantified by radioimmunoassay. Intraassay variation was 9-12%. Results: Arterial plasma angiotensinogen and renin levels decreased similarly and modestly (~15 and ~30%, respectively) after NaCl infusion during both studies whereas ANGII levels decreased significantly (~25%) only during GLP-1 infusion. Conclusions: Suppression of plasma angiotensinogen is less likely to account for GLP-1-mediated ANGII suppression in healthy young males. In perspective, GLP-1 could enhance pathways of ANGII degradation rather than inhibit its formation. 1. Asmar A et al. Extracellular fluid volume expansion uncovers a natriuretic action of GLP-1: a functional GLP-1-renal axis in man. J Clin Endocrinol Metab. 2019 Jul 1;104(7):2509-2519.

Abstract 065: Reduction Of Endoplasmic Reticulum Stress By Treatment With Nifedipine In A Mouse Model Of Pre-eclampsia And In Human Placental Endothelial Cells

Introduction: Pre-eclampsia (PE) is characterized by high blood pressure (BP) and metabolic disorders during pregnancy, being the leading cause of maternal-neonatal mortality worldwide. The causes of PE are still unclear. We observed enhanced endoplasmic reticulum (ER) stress and increased cytoplasmic mobility of calreticulin, a Ca2+ ion binding chaperone protein, in placental endothelial cells (pECs) of mice with PE, indicating changes in Ca2+ mobilization. We aimed to describe the effect of nifedipine, a Ca2+ channel blocker, on ER stress in placentas of mice with PE and in human pECs exposed to hypoxia. Methods: We used heterozygous mice overexpressing human renin and angiotensinogen, developing high BP from gestational day (GD) 13.5, and controls (C57BL/6). Nifedipine (1 mg/kg in 0.5 ml saline, s.c.) or saline was administered at GD14.5 and placentas removed at GD18.5 (n=8-12/group). Protein expressions of IREα, ATF6 and PERK were evaluated by western blot. ECs were extracted from human placentas of healthy pregnancies by magnetic sorting and exposed to hypoxia (0.5% O2) or control conditions (8% O2) for 24h while treated with nifedipine (0.1 μM) or DMSO. Angiogenesis was assessed by tube formation assay on matrigel. Results were compared by 2-way ANOVA (mean±SD). Results: IREα expression was higher in placentas of PE mice treated with saline versus control (+306±64%, p=0.020) and significantly reduced with nifedipine versus saline in PE mice (-235±66%, p=0.009). Results were similar with PERK, which was increased in placentas of saline-treated PE mice versus saline controls (116±40%, p=0.037) and significantly reduced with nifedipine versus saline in PE mice (-141±38%, p=0.006). ATF6 was not different between groups, but was significantly reduced by nifedipine versus saline in PE mice (-76±24%, p=0.022). In pECs, nifedipine increased 17x the numbers of tubes formed on matrigel of cells exposed to hypoxia in comparison to DMSO control (p<0.001). Conclusion: The results describe an enhanced ER stress in placentas of PE mice and a positive effect of nifedipine on this stress. The pro-angiogenic effect of nifedipine was shown in pECs exposed to hypoxia. Further investigations are needed to confirm a potential angiogenic effect of nifedipine in PE.

Manipulation of components of the renin angiotensin system in renal proximal tubules fails to alter atherosclerosis in hypercholesterolemic mice.

Whole body manipulation of the renin-angiotensin system (RAS) consistently exerts profound effects on experimental atherosclerosis development. A deficit in the literature has been a lack of attention to the effects of sex. Also, based on data with gene-deleted mice, the site of RAS activity that influences lesion formation is at an unknown distant location. Since angiotensin (AngII) concentrations are high in kidney and the major components of the RAS are present in renal proximal tubule cells (PTCs), this study evaluated the role of the RAS in PTCs in atherosclerosis development. Mice with an LDL receptor -/- background were fed Western diet to induce hypercholesterolemia and atherosclerosis. We first demonstrated the role of AT1 receptor antagonism on atherosclerosis in both sexes. Losartan, an AngII type 1 (AT1) receptor blocker, had greater blood pressure-lowering effects in females than males, but equivalent effects between sexes in reducing atherosclerotic lesion size. To determine the roles of renal AT1a receptor and angiotensin-converting enzyme (ACE), either component was deleted in PTCs after weaning using a tamoxifen-inducible Cre expressed under the control of an Ndrg1 promoter. Despite profound deletion of AT1a receptor or ACE in PTCs, the absence of either protein did not influence development of atherosclerosis in either sex. Conversely, mice expressing human angiotensinogen and renin in PTCs or expressing human angiotensinogen in liver but human renin in PTCs did not change atherosclerotic lesion size in male mice. Whole-body AT1R inhibition reduced atherosclerosis equivalently in both male and female mice; however, PTC-specific manipulation of the RAS components had no effects on hypercholesterolemia-induced atherosclerosis.

1652-P: Suppression of Plasma Angiotensin II by GLP-1 in Humans Is Independent of Changes in Plasma Angiotensinogen

In humans, the natriuretic effect of GLP-1 depends on GLP-1 receptor interaction, is independent of changes in renal blood flow, renin, ANP, and aldosterone but is accompanied by suppression of angiotensin II (ANGII). The mechanism by which GLP-1 reduces ANGII independently of changes in renin is not clarified. It was hypothesized that GLP-1 suppresses renin substrate, angiotensinogen, in plasma. To test this proposal, we measured arterial angiotensinogen concentration in biobanked plasma samples from healthy males during a 3-hour GLP-1 infusion that induced natriuresis (1). Samples were from a study with fixed sodium intake where eight healthy men were examined twice in random order during a 3-hour infusion of either GLP-1(1.5 pmol kg-1 min-1) or vehicle together with an intravenous infusion of 0.9% NaCl (750 mL/hour). Arterial plasma angiotensinogen was measured in plasma by dilution (1:200) and addition of excess exogenous human renin (Inst. of Biological Standards UK). After prolonged incubation, ANGI was quantified by radioimmunoassay. Intraassay variation was 9-12%. Arterial plasma angiotensinogen and renin levels decreased similarly and modestly (~15 and ~30%, respectively) after NaCl infusion during both studies whereas ANGII levels decreased significantly (~25%) only during GLP-1 infusion. Suppression of plasma angiotensinogen is less likely to account for GLP-1-mediated ANGII suppression in healthy young males. In perspective, GLP-1 could enhance pathways of ANGII degradation rather than inhibit its formation. 1. Asmar A et al. Extracellular fluid volume expansion uncovers a natriuretic action of GLP-1: a functional GLP-1-renal axis in man. J Clin Endocrinol Metab. 2019 Jul 1;104(7):2509-2519. Disclosure B.L.Jensen: None. A.Asmar: Research Support; Novo Nordisk A/S.

Levistolide A ameliorates fibrosis in chronic kidney disease via modulating multitarget actions in vitro and in vivo.

The increasing incidence of chronic kidney disease (CKD) urgently calls for effective nephroprotective agents. Traditional Chinese Medicine Angelica sinensis and its formula are well known for CKD therapy, but the underlying mechanisms and effective substances of reno-protective effects remain unclear. To this end, we isolated eleven ligustilide dimers (1-11) from A. sinensis and examined the molecular mechanism of their nephroprotective effects. Because of internal RAS playing an important role in CKD, we used renin expression as a target and screened preliminarily for antifibrotic effects of ligustilide dimers (1-11) by constructing a dual luciferase reporter gene in vitro. Furthermore, the reno-protective effects of the ligustilides and their underlying mechanism were investigated in TGF-β1-stimulated HK-2 cells and 5/6 nephrectomy (Nx) mice. The ligustilide dimers exhibited anti-fibrotic effects by inhibiting human renin (hREN) promoter activity to decrease renin expression and down-regulate the expression of fibrosis-related factors, including α-SMA, collagen I, and fibronectin in vitro. Levistolide A (LA) and angeolide keto ester (AK) were screened out to identify their ability and underlying mechanism for treating CKD. Experimental validation further indicated that LA or AK treatment inhibited the expression of key molecules in RAS, TGF-β1/Smad, and MAPK pathways to downregulate ECM deposition. Furthermore, LA obviously meliorated renal injury in 5/6 Nx mice through ameliorating oxidant stress, inflammation, apoptosis and renal fibrosis. The experimental results demonstrated that ligustilide dimers were potential nephroprotective agents. LA might be an attractive drug candidate for renin-targeted CKD therapy.

The Proteome of Circulating Large Extracellular Vesicles in Diabetes and Hypertension.

Hypertension and diabetes induce vascular injury through processes that are not fully understood. Changes in extracellular vesicle (EV) composition could provide novel insights. Here, we examined the protein composition of circulating EVs from hypertensive, diabetic and healthy mice. EVs were isolated from transgenic mice overexpressing human renin in the liver (TtRhRen, hypertensive), OVE26 type 1 diabetic mice and wild-type (WT) mice. Protein content was analyzed using liquid chromatography-mass spectrometry. We identified 544 independent proteins, of which 408 were found in all groups, 34 were exclusive to WT, 16 were exclusive to OVE26 and 5 were exclusive to TTRhRen mice. Amongst the differentially expressed proteins, haptoglobin (HPT) was upregulated and ankyrin-1 (ANK1) was downregulated in OVE26 and TtRhRen mice compared with WT controls. Conversely, TSP4 and Co3A1 were upregulated and SAA4 was downregulated exclusively in diabetic mice; and PPN was upregulated and SPTB1 and SPTA1 were downregulated in hypertensive mice, compared to WT mice. Ingenuity pathway analysis identified enrichment in proteins associated with SNARE signaling, the complement system and NAD homeostasis in EVs from diabetic mice. Conversely, in EVs from hypertensive mice, there was enrichment in semaphroin and Rho signaling. Further analysis of these changes may improve understanding of vascular injury in hypertension and diabetes.

Increased angiotensin II coupled with decreased Adra1a expression enhances cardiac hypertrophy in pregnancy-associated hypertensive mice

Cardiac hypertrophy is a crucial risk factor for hypertensive disorders during pregnancy, but its progression during pregnancy remains unclear. We previously showed cardiac hypertrophy in a pregnancy-associated hypertensive (PAH) mouse model, in which an increase in angiotensin II (Ang II) levels was induced by human renin and human angiotensinogen, depending on pregnancy conditions. Here, to elucidate the factors involved in the progression of cardiac hypertrophy, we performed a comprehensive analysis of changes in gene expression in the hearts of PAH mice and compared them with those in control mice. We found that alpha-1A adrenergic receptor (Adra1a) mRNA levels in the heart were significantly reduced under PAH conditions, whereas the renin-angiotensin system was upregulated. Furthermore, we found that Adra1a-deficient PAH mice exhibited more severe cardiac hypertrophy than PAH mice. Our study suggests that Adra1a levels are regulated by renin-angiotensin system and that changes in Adra1a expression are involved in progressive cardiac hypertrophy in PAH mice.

PS-BPB04-7: PRO-ANGIOGENIC EFFECTS OF LSKL, A THROMBOSPONDIN-1 INHIBITOR, IN HUMAN AND EXPERIMENTAL MODELS OF PREECLAMPSIA

Introduction: Preeclampsia (PE) is a hypertensive disorder of pregnancy characterized by blood pressure above 140/90 mmHg. It accounts for 10% of all pregnancies and is responsible for 25% of preterm births and low birth weight. To date, no medications can treat or prevent PE. We identified an activation of the anti-angiogenic protein thrombospondin-1 (THBS1) in human and experimental preeclampsia. THBS1 is also a main activator of TGF-β. We postulate that THBS1-mediated TGF-β activation may contribute to impair placental angiogenesis during PE. Objective: We aimed to test the effects of LSKL, an inhibitor of THBS1-mediated TGF-β activation, on angiogenesis in placentas of a mouse model of PE and in human placental endothelial cells. Methods: Peptide LSKL or its control peptide SLLK (280 μM in 0.5 ml saline, s.c.) was administered at gestational day (GD) 14.5 in heterozygous transgenic mice overexpressing human renin and human angiotensinogen and controls (C57BL/6). Placentas (n = 12/group) were removed at GD 18.5. Protein expression of TGF-β1, TGF-β2, total and phosphorylated SMAD2 were assessed by western blot of placental lysates. Placental angiogenesis was assessed by CD31+ immunohistochemistry in placental sections. CD31+ endothelial cells were extracted by magnetic sorting from human placentas (n = 4) of healthy pregnancies. Cells (2–5 passages) were treated with LSKL or SLLK (30 μM) under hypoxia (0.5% vs. 8% O2) or thrombin (10 u/mL) exposure (4 hours). THBS1 expression in placental endothelial cells was assessed by immunofluorescence. Cell angiogenesis was quantified by the number of closed tubes formed on matrigel (per 15,000 cells, 4 hours). Results: Maternal LSKL treatment significantly reduced the expression of TGF-β2 (P < 0.01) and SMAD2 phosphorylation (P < 0.05) while improving angiogenesis (P < 0.05) in placentas of transgenic preeclamptic mouse compared to controls. In human placental endothelial cells, both thrombin (P < 0.05) and hypoxia (P < 0.05) significantly increased THBS1 expression. Thrombin (P < 0.01) and hypoxia (P < 0.05) significantly impaired cell angiogenesis on matrigel (P < 0.01), which was prevented by LSKL treatment (P < 0.01). In human cells, LSKL similarly reduced TGB-β2 (p < 0.05) and phospho-SMAD2 (P < 0.05) expressions under thrombin and hypoxia cell exposure. Conclusion: Our results describe a significant pro-angiogenic effect of LSKL treatment by regulating THBS1 and TGF-β2 mechanisms in human and experimental models of PE.

S-07-6: NEUTROPHIL EXTRACELLULAR TRAPS AS MEDIATORS OF ENDOTHELIAL DAMAGE IN HYPERTENSION AND DIABETES

Objective: Endothelial dysfunction (ED) plays a key role in the pathogenesis of hypertension and diabetes, yet its molecular determinants are poorly understood. Concomitant hypertension and diabetes can synergistically increase damage to the vasculature, with increasing evidence suggesting that resulting immune dysregulation may contribute to endothelial injury. Neutrophil extracellular traps (NETs), networks of chromatin and protein-containing extracellular fibers released to fight infection, are generated from neutrophils following activation of the citrullinating enzyme peptidylarginine deiminase 4 (PAD4). PAD4 has been shown to be upregulated in atherosclerosis, vasculitis, and various inflammatory autoimmune diseases, leading to NET-induced endothelial injury. Whether NETs could contribute to hypertension- and diabetes-induced ED has not been established. The objective of the present study is to investigate the impact of hypertension and diabetes on PAD4 activity and the contribution of NETs to resulting vascular injury. Design and Methods: Promyelocytic HL-60 cells were differentiated to neutrophil-like cells and treated with angiotensin II (10−7 M) and/or high glucose (25 mM) for 24 hours, with or without subsequent NET-inducing PMA stimulation (500 nM). NETs were isolated from the culture medium by differential centrifugation and assessed by Western blot analysis of citrullinated histone H3 (citH3, a primary NET component). Isolated NETs were used to treat Human Umbilical Vein Endothelial Cells (HUVECs) at 0.5–500 ng/ml for 24 hours, after which cell viability (XTT cell assay) and proliferation (BrdU assay) were measured. We also assessed NETosis in mice expressing human renin in the liver (TTRhRen) intercrossed with OVE26 diabetic mice [i.e. hypertensive-diabetic, HD mice], compared with wild-type (WT) FVBN/J mice. We examined kidney levels of citH3, neutrophil elastase (NE), and PAD4 expression by immunohistochemistry. Results: Treatment with high glucose, but not angiotensin II, increased citH3 levels in neutrophil-like cells. NETs isolated from PMA-stimulated cells decreased HUVEC viability by ∼2-fold at 5 ng/ml (P = < 0.0001), and dose dependently decreased proliferation from 0.5–500 ng/ml, with a ∼80% decrease at 500 ng/ml. In HD mice, we observed a significant increase in kidney levels of citH3 (P < 0.0001), NE (P < 0.0001), and PAD4 (P < 0.0001), which was associated with the presence of albuminuria and increased blood pressure levels. Conclusion: These results suggest that NETs are upregulated in hypertension and diabetes and may contribute to endothelial damage and microvascular injury.

Inhibitors of HIV-1 and Cathepsin L Proteases Identified from the Insect Gall of Hypericum kouytchense.

Hypericum kouytchense Lévl is a semi-evergreen plant of the Hypericaceae family. Its roots and seeds have been used in a number of traditional remedies for antipyretic, detoxification, anti-inflammatory, antimicrobial and antiviral functions. However, to date, no bioactivity compounds have been characterized from the insect gall of H. kouytchens. In this study, we evaluated the antiviral activities of different extracts from the insect gall of H. kouytchen against cathepsin L, HIV-1 and renin proteases and identified the active ingredients using UPLC-HRMS. Four different polar extracts (HW, H30, H60 and H85) of the H. kouytchense insect gall exhibited antiviral activities with IC50 values of 10.0, 4.0, 3.2 and 17.0 µg/mL against HIV-1 protease; 210.0, 34.0, 24.0 and 30.0 µg/mL against cathepsin L protease; and 180.0, 65.0, 44.0 and 39.0 µg/mL against human renin, respectively. Ten compounds were identified and quantified in the H. kouytchense insect gall extracts. Epicatechin, eriodictyol and naringenin chalcone were major ingredients in the extracts with contents ranging from 3.9 to 479.2 µg/mg. For HIV-1 protease, seven compounds showed more than 65% inhibition at a concentration of 1000.0 µg/mL, especially for hypericin and naringenin chalcone with IC50 values of 1.8 and 33.0 µg/mL, respectively. However, only hypericin was active against cathepsin L protease with an IC50 value of 17100.0 µg/mL, and its contents were from 0.99 to 11.65 µg/mg. Furthermore, we attempted to pinpoint the interactions between the active compounds and the proteases using molecular docking analysis. Our current results imply that the extracts and active ingredients could be further formulated and/or developed for potential prevention and treatment of HIV or SARS-CoV-2 infections.

Abstract 004: Pro-angiogenic Effects Of The Thrombospondin-1 Inhibitor Lskl In Preeclampsia Models

Introduction: Preeclampsia (PE) is characterized by high blood pressure, proteinuria and/or multi-organ damage, reaching 10% of all pregnancies globally. To date, no medication can treat PE. We recently identified an upregulation of the antiangiogenic protein Thrombospondin-1 (THBS1) in human and experimental PE. THBS1 is a main activator of latent TGF-β. We postulate that THBS1-mediated TGF-β activation can contribute to impair placental angiogenesis during PE. In this study, we aim to test the effects of LSKL, an inhibitor of TGF-β activation by THBS1, on angiogenesis in placentas of a mouse model of PE and in human placental endothelial cells exposed to PE-like conditions. Methods: Heterozygous female mice overexpressing human angiotensinogen and renin (PE) or C57BL/6 (control) were treated with LSKL or control peptide SLLK (280 μM, sc) at gestational day (GD) 14.5 (n=3 pregnant mouse/group). Placentas (n=4-9/pregnancy) were collected at GD 18.5. Placental expressions of TGF-β1, β2, total/phospho SMAD2 were assessed by western blot and angiogenesis by immunohistochemistry. Endothelial cells were extracted by magnetic sorting from human placentas (n=4) of healthy pregnancies. Cells were treated with LSKL or SLLK (30 μM) under hypoxia (0.5% vs. 8% O 2 ) or thrombin (10 u/mL). THBS1 expression in cells was assessed by immunofluorescence. Cell angiogenesis was quantified by the number of closed tubes formed on matrigel (per 15,000 cells, 4h). Results: LSKL treatment reduced the expression of TGF-β2 (P<0.01) and phospho-SMAD2 (P<0.05) and significantly improved angiogenesis (P<0.05) in placentas of PE mouse versus control. In cells, both thrombin (P<0.05) and hypoxia (P<0.05) significantly increased THBS1 expression. Thrombin (P<0.01) and hypoxia (P<0.05) significantly impaired cell angiogenesis on matrigel (P<0.01), which was prevented by LSKL treatment (P<0.01). LSKL similarly reduced TGB-β2 (p<0.05) and phospho-SMAD2 (P<0.05) expressions in endothelial cells under thrombin and hypoxia cell exposure. Conclusion: Our results describe a significant pro-angiogenic effect of LSKL treatment by regulating THBS1 and TGF-β2 mechanisms in an experimental model of PE and in human placental endothelial cells exposed to PE-like conditions.

Abstract P087: Metformin Treatment Decreases Blood Pressure But Does Not Ameliorate Hypertensive Cardio-Renal Damage In A Double Transgenic Rat Model

Introduction: Metformin (Met) is used as a first-line treatment in type II diabetes, reduces the cardiovascular (CV) risk in diabetes and may lead to decreased CV mortality independent of diabetes status. Met treatment has been shown to induce gut microbiome changes leading to enhanced production of protective short-chain fatty acids and potentially harmful metabolites (LPS). Our study aims to examine the effects of Met in a model of RAAS-mediated hypertension with cardio-renal damage. Methods: Four-week-old double transgenic rats (dTGR, transgenic for human renin and angiotensinogen) received oral Metformin (Met) or Vehicle (Veh) for 3 weeks. SD rats served as healthy controls. Flow cytometry (n=10 per group), echocardiography (n=14), radiotelemetric blood pressure (BP) measurements (n=5), clinical chemistry and gene expression analyses (n=14) were used to assess damage to kidney and heart. Results: Met treatment did not influence survival nor lead to lactate acidosis. Met treatment lowered BP significantly (systolic BP: Met: 218±5 mmHg, Veh: 237±3 mmHg). Interestingly, the decreased BP was accompanied by increased cardiac hypertrophy (heart weight to tibia length, SD: 34±1 g/m, Met: 40±1 g/m, Veh: 37±1 g/m). Echocardiographic systolic and diastolic function was deteriorated (EF: Met: 64±2 %, Veh: 68±4 %; E/A: Met: 0.75±0.1, Veh: 0.93±0.1). Plasma BNP and cardiac ß-to-α MHC ratio were higher in Met-treated dTGR. Intestines, spleens, kidneys and hearts of dTGR showed a strong pro-inflammatory phenotype with increased adaptive (e.g. cardiac T cells % of leucocytes: (SD: 10±0.003 %, Met: 15±0.01 %, Veh: 13±0.01 %) and innate (e.g. cardiac monocytes % of leucocytes: (SD: 2±0.001 %, Met: 3±0.004 %, Veh: 5±0.02 %) immune cell subsets in comparison to SD rats; with almost no differences between Met- and Veh-treated dTGR. Fecal metagenomic shotgun sequencing showed no large-scale taxonomic shifts between Veh- and Met-treated dTGR. Conclusion: dTGR display a pronounced pro-inflammatory immunophenotype across several organs. Met did not ameliorate hypertensive target organ damage in dTGR despite the BP lowering effect. These findings could help to understand the effects of Met on the microbiome, immunome and organ damage in the context of hypertension.

Hepatorenal Syndrome: A Way for Early and Accurate Diagnosis

BACKGROUND: Hepatorenal syndrome (HRS) is a devastating consequence of liver cirrhosis that is clinically categorized into two subtypes. Acute malfunction of renal role, as measured by an elevation in blood creatinine, significantly underestimates the loss in renal function in cirrhotic individuals; more accurate biomarkers are desperately required in cirrhotic patients. AIM: The present study set out to uncover new biomarkers for the early prediction of AKI in cirrhotic cases. A comprehensive panel of biomarkers was investigated to get a clear insight into the pathogenesis of HRS. PATIENTS AND METHODS: Participants in this study were 70 individuals from the hepatogastroenterology unit of the Theodor Bilharz Research Institute (TBRI). Detailed medical data and a physical examination were recorded. Three groups of patients have been identified; Group 1: 30 cases with compensated liver cirrhosis and normal kidney functions. Group 2: 20 cases with decompensated liver cirrhosis and normal kidney functions. Group 3: 20 cases with decompensated liver cirrhosis proved hepatorenal syndrome Type 2 h. The following biomarkers were detected in serum using the sandwich-ELISA method: Human L-arginine ELISA kit, human neutrophil gelatinase related lipocalin (NGAL), human noradrenaline (NA), human asymmetrical dimethylarginine (ADMA), human symmetric dimethylarginine (SDMA), human nitric oxide (NO), and human renin. RESULTS: There was a highly significant difference between Groups 1 and 2 in NITRIC and ADMA. Significant differences between Groups 2 and 3 in NGAL, noradrenalin, and SDMA were observed. There was a significant difference (Group 2 vs. Group 3) in renin, NITRIC, ADMA, and L-ARGININE. There was highly significant differentiation (Group 2 vs. Group 3) in NGAL, noradrenalin, and SDMA. There was highly significant variation as per odd ratio and confidence interval between (Group 3 vs. Group 2) in NGAL. CONCLUSION: Assessment of renal biomarkers in individuals with decompensated cirrhosis gives critical information on the etiology of AKI. Further, it may aid in the diagnosis and prognosis of AKI. Renin, NITRIC, ADMA, and L-ARGININE could be used as biomarkers to indicate HRS in individuals with advanced cirrhosis.

Discovery of Novel 2-Carbamoyl Morpholine Derivatives as Highly Potent and Orally Active Direct Renin Inhibitors.

The renin-angiotensin-aldosterone system (RAAS) plays a key role in the regulation of blood pressure. Renin, the first and rate-limiting enzyme of the RAAS, is an attractive target for the treatment of hypertension and cardiovascular/renal diseases. Therefore, various direct renin inhibitors (DRIs) have been researched over recent decades; however, most exhibited poor pharmacokinetics and oral bioavailability due to the peptidomimetic or nonpeptidomimetic structures with a molecular weight (MW) of >600, and only aliskiren is approved. This study introduces a novel class of DRIs comprised of a 2-carbamoyl morpholine scaffold. These compounds have a nonpeptidomimetic structure and a MW of <500. The representative compound 26 was highly potent despite not occupying S1'-S2' sites or the opened flap region used by other DRIs and exerted a significant antihypertensive efficacy via oral administration on double transgenic mice carrying both the human angiotensinogen and the human renin genes.

Pharmacophore modelling, docking and molecular dynamic simulation studies in the discovery of potential human renin inhibitors

Hypertension is the main cause of human death and the Renin- Angiotensin- Aldosterone System (RAAS) has a key role in the control of human blood pressure. In this research a multi-step virtual screening strategy was applied in order to find new potential renin inhibitors. The crystal structure of renin-aliskiren complex was explored and receptor-ligand pharmacophore model was developed and validated using pharmit. ZINC database was screened by pharmacophore model and Lipinski's rule of five. Thereafter, the retrieved hits were docked in the active site of renin by using Vina. ADME parameters and toxicity of the filtered compounds were approximated using in silico methods and the selected compounds were subjected to high performance docking in order to improve the accuracy of screening. The non-bond interactions of the hit molecules with renin were explored and the compounds with the highest affinity and appropriate interactions were selected. In the last step, molecular dynamic simulation studies were performed on the complex of renin with aliskiren and three top-ranked structures including ZINC6085004, ZINC426421106, and ZINC5481346. RMSD, RMSF, Rg and number of hydrogen bonds were calculated. The binding free energy was calculated using the MM/PBSA method. The result of MD simulation indicated the stable binding of ZINC426421106 and ZINC5481346 with the active site of renin. According to this in-silico study these two compounds are drug-like, nontoxic, and have a high potential for inhibiting renin and could serve as appropriate lead molecules for the development of renin inhibitors as antihypertensive agents.

Abstract 229: Effects Of Human Angiotensinogen And Human Renin In Proximal Tubule Cells On Development Of Atherosclerosis In Hypercholesterolemic Mice

Objective: This study determined whether angiotensinogen (AGT) interacted with renin in renal proximal tubule cells (PTCs) to promote atherosclerosis. Approach and Results: Since hepatocyte-produced AGT can be filtered by glomeruli and retained in renal PTCs, we first determined whether hepatocyte-derived AGT interacts with renin in PTCs to promote atherosclerosis. Transgenic mice expressing human renin in PTCs driven by a kidney androgen-related protein promoter (KAP-hREN) in an LDLR -/- background were used. To induce synthesis of human AGT in hepatocytes, an adeno-associated viral vector (AAV) containing human AGT with a liver-specific promoter was injected intraperitoneally. Three groups of male littermates were administered testosterone to activate human renin expression in PTCs: (1) wild type mice administered null AAV, (2) KAP-hREN transgenic mice administered null AAV, and (3) KAP-hREN transgenic mice administered AAV containing human AGT. Two weeks after administration of testosterone and AAVs, mice were fed a Western diet for 6 weeks. Induction of human AGT in liver and human renin in PTCs did not increase atherosclerosis, comparing to the other two groups. Next, to evaluate the direct interaction of AGT and renin in PTCs for promoting atherosclerosis, KAP-human AGT (KAP-hAGT) and KAP-hREN double transgenic mice were fed a Western diet for 12 weeks. Although immunostaining confirmed the presence of human AGT and human renin in PTCs, double transgenic mice did not have increased percent atherosclerotic lesion area, comparing to either wild type or single transgenic littermate groups. Surprisingly, retired male double transgenic breeders (46-67 weeks old) showed severe atherosclerosis spontaneously without Western diet feeding, while their single transgenic littermates had minimal to modest atherosclerotic lesions. The sample size was small so statistical analysis was not performed. Conclusions: The presence of human AGT in liver or PTCs with combination of human renin in PTCs did not augment Western diet-induced atherosclerosis in mice. Further studies with increased numbers are needed to determine whether retired male breeders containing human AGT and human renin in PTCs develop severe atherosclerosis.

Effects of Human Angiotensinogen and Human Renin in Proximal Tubule Cells on Development of Atherosclerosis in Hypercholesterolemic Mice

Effects of Human Angiotensinogen and Human Renin in Proximal Tubule Cells on Development of Atherosclerosis in Hypercholesterolemic Mice