Abstract-We reported previously that urinary angiotensinogen (UAGT) levels provide a specific index of the intrarenal renin-angiotensin system (RAS) status in angiotensin II-dependent hypertensive rats. To study this system in humans, we recently developed a human angiotensinogen ELISA. To test the hypothesis that UAGT is increased in hypertensive patients, we recruited 110 adults. Four subjects with estimated glomerular filtration levels Ͻ30 mL/min per 1.73 m 2 were excluded because previous studies have already shown that UAGT is highly correlated with estimated glomerular filtration in this stage of chronic kidney disease. Consequently, 106 paired samples of urine and plasma were analyzed from 70 hypertensive patients (39 treated with RAS blockers [angiotensin-converting enzyme inhibitors or angiotensin II type 1 receptor blockers; systolic blood pressure: 139Ϯ3 mm Hg] and 31 not treated with RAS blockers [systolic blood pressure: 151Ϯ4 mm Hg]) and 36 normotensive subjects (systolic blood pressure: 122Ϯ2 mm Hg). UAGT, normalized by urinary concentrations of creatinine, were not correlated with race, gender, age, height, body weight, body mass index, fractional excretion of sodium, plasma angiotensinogen levels, or estimated glomerular filtration. However, UAGT/urinary concentration of creatinine was significantly positively correlated with systolic blood pressure, diastolic blood pressure, urinary albumin:creatinine ratio (rϭ0.5994), and urinary protein:creatinine ratio (rϭ0.4597). UAGT/urinary concentration of creatinine was significantly greater in hypertensive patients not treated with RAS blockers (25.00Ϯ4.96 g/g) compared with normotensive subjects (13.70Ϯ2.33 g/g). Importantly, patients treated with RAS blockers exhibited a marked attenuation of this augmentation (13.26Ϯ2.60 g/g). These data indicate that UAGT is increased in hypertensive patients, and treatment with RAS blockers suppresses UAGT, suggesting that the efficacy of RAS blockade to reduce the intrarenal RAS activity can be assessed by measurements of UAGT.

We previously reported that urinary excretion rates of angiotensinogen (AGT) provide a specific index of the activity of the intrarenal renin-angiotensin system in angiotensin II-dependent hypertensive rats. Meanwhile, we have recently developed direct enzyme-linked immunosorbent assays (ELISAs) to measure plasma and urinary AGT in humans. This study was performed to test a hypothesis that urinary AGT levels are enhanced in chronic kidney disease (CKD) patients and correlated with some clinical parameters. Eighty patients with CKD (37 women and 43 men, from 18 to 94 years old) and seven healthy volunteers (two women and five men, from 27 to 43 years old) were included. Plasma AGT levels showed a normal distribution; however, urinary AGT-creatinine ratios (UAGT/UCre) deviated from the normal distribution. When a logarithmic transformation was executed, Log(UAGT/UCre) levels showed a normal distribution. Therefore, Log(UAGT/UCre) levels were used for further analyses. Log(UAGT/UCre) levels were not correlated with age, gender, height, body weight, body mass index, systolic blood pressure, diastolic blood pressure, serum sodium levels, serum potassium levels, urinary sodium-creatinine ratios, plasma renin activity, or plasma AGT levels. However, Log(UAGT/UCre) levels were significantly correlated positively with urinary albumin-creatinine ratios, fractional excretion of sodium, urinary protein-creatinine ratios, and serum creatinine, and correlated negatively with estimated glomerular filtration rate. Log(UAGT/ UCre) levels were significantly increased in CKD patients compared with control subjects (1.8801 ± 0.0885 vs. 0.9417 ± 0.1048; P = .0024). These data confirmed our earlier report and showed that a new ELISA assay is a valid approach for measuring urinary AGT.

Reduced expression of bone morphogenetic protein receptor 2 subverts a stress granule response, heightens GM-CSF mRNA translation, and increases inflammatory cell recruitment to exacerbate pulmonary arterial hypertension.

Background We previously reported that kidney and urinary angiotensinogen levels were significantly increased before the development of diabetic nephropathy in diabetic rats. To address this system in humans, we have developed an enzyme-linked immunosorbent assay for human angiotensinogen and reported that urinary excretion of angiotensinogen levels is enhanced in patients with chronic kidney disease, including patients with type 2 diabetes. On the basis of these findings, this study was performed to demonstrate that urinary angiotensinogen levels increased before the onset of microalbuminuria and that urinary angiotensinogen can be an early biomarker of intra-renal renin-angiotensin system status in normoalbuminuric patients with type 1 diabetes compared with age- and sex-matched control subjects. Methods The study included 28 patients with type 1 diabetes and 21 control subjects. No subject received renin-angiotensin system blockades. Random spot urine samples as well as blood samples were obtained and analyzed. Results Urinary albumin:creatinine ratio or urinary protein:creatinine ratio did not increase in patients compared with control subjects, suggesting that these patients were in their premicroalbuminuric phase of diabetic nephropathy. However, the urinary angiotensinogen:creatinine ratio was significantly higher in patients than in control subjects (12.1 ± 3.2 µg/g versus 4.2 ± 0.7 µg/g). Importantly, an increase in plasma angiotensinogen levels was not observed (26.3 ± 1.3 µg/mL versus 29.5 ± 3.3 µg/mL). Conclusions Thus, in patients, an increase in urinary angiotensinogen levels is observed, and this increase is precedent to an increase in urinary albumin levels, suggesting that urinary angiotensinogen may function as an early marker of diabetic nephropathy.

In 2 animal models of PH based on Treg deficiency, females developed more severe PH than males. The data suggest that females are especially reliant on the normal Treg function to counteract the effects of pulmonary vascular injury leading to PH.

Rationale: Maintaining endothelial cells (EC) as a monolayer in the vessel wall depends on their metabolic state and gene expression profile, features influenced by contact with neighboring cells such as pericytes and smooth muscle cells (SMC). Failure to regenerate a normal EC monolayer in response to injury can result in occlusive neointima formation in diseases such as atherosclerosis and pulmonary arterial hypertension. Objective: We investigated the nature and functional importance of contact-dependent communication between SMC and EC to maintain EC integrity. Methods and Results: We found that in SMC and EC contact co-cultures, bone morphogenetic protein receptor 2 (BMPR2) is required by both cell types to produce collagen IV to activate integrin-linked kinase. This enzyme directs phospho c-Jun N-terminal kinase (p-JNK) to the EC membrane, where it stabilizes presenilin1 and releases Notch1 intracellular domain (N1ICD) to promote EC proliferation. This response is necessary for EC regeneration following carotid artery injury. It is deficient in EC-SMC Bmpr2 double heterozygous mice in association with reduced collagen IV production, decreased N1ICD and attenuated EC proliferation, but can be rescued by

Background Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue and elevated cytokines have been related to PAH pathogenesis but without clear understanding of how these abnormalities are initiated, perpetuated and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies. Methods Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry (LCMS), confirmed by ELISA, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next generation sequencing, functional studies in cultured monocytes and endothelial cells (EC) and hemodynamic and lung studies in a rat. Results SAM domain and HD1 domain-containing protein (SAMHD1), an innate immune factor that suppresses HIV replication was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH vs. 12 control lungs. Elevated SAMHD1 was localized to endothelial cells (EC), perivascular dendritic cells and macrophages and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH vs. control lungs (n=4 each). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) mRNAs were elevated in PAH vs. control lungs (n=10) and proteins were localized to macrophages. HERV-K dUTPase induced SAMHD1 and pro-inflammatory cytokines (e.g., IL6, IL1β and TNFα) in circulating monocytes and pulmonary arterial (PA) EC, and activated B cells. Vulnerability of PAEC to apoptosis was increased by HERV-K dUTPase in an IL6 independent manner. Furthermore, three weekly injections of HERV-K dUTPase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8), and elevated IL6. Conclusions Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH.

We recently reported that urinary excretion rates of angiotensinogen provide a specific index of the intrarenal renin-angiotensin system status in angiotensin II-dependent hypertensive rats. Angiotensinogen concentrations in mouse plasma are thought to be much lower than those in rat plasma; however, detailed information is deficient due to lack of direct quantitative measurements of rodent angiotensinogen. To elucidate this issue, we have developed a quantitative method for measurement of rodent angiotensinogen using a sandwich-type ELISA. The standard curve for mouse and rat angiotensinogen exhibited a high linearity at 0.16-10 and 0.08-5 ng/ml, respectively, with correlation coefficients >0.99. While plasma angiotensinogen concentrations of male high serum IgA (HIGA) mice (IgA nephritis model animals, 1,308 +/- 47 ng/ml; n = 10) were lower than those of control BALB/c mice (1,620 +/- 384; n = 12), urinary angiotensinogen concentrations of HIGA mice (14.6 +/- 1.5 ng/ml; n = 34) were higher than those of BALB/c mice (4.6 +/- 0.1; n = 2). In a similar manner, while plasma angiotensinogen concentrations of Zucker diabetic fatty (ZDF) obese rats (type 2 diabetic model animals, 1,789 +/- 50 ng/ml; n = 5) were lower than those of control ZDF lean rats (2,296 +/- 47; n = 5), urinary angiotensinogen concentrations of ZDF obese rats (88.2 +/- 11.4 ng/ml; n = 15) were higher than those of ZDF lean rats (31.3 +/- 1.9; n = 15). These data indicate that plasma and urinary angiotensinogen concentrations are less in mice than rats. However, these data suggest that urinary angiotensinogen levels are different from plasma angiotensinogen levels in rodents. The development of rodent angiotensinogen ELISA allows quantitative comparisons in mouse and rat angiotensinogen levels in models of hypertension and cardiovascular and kidney diseases.