Building a Community Partnership for the Development of Health Ministries Within the African American Community: The Triad Pastors Network.

African Americans continue to have worse health outcomes despite attempts to reduce health disparities. This is due, in part, to inadequate access to healthcare, but also to the health care and medical mistrust experienced by communities of color. Churches and worship centers have historically served as cultural centers of trusted resources for educational, financial, and health information within African American communities and a growing number of collaborations have developed between academic institutions and community/faith entities. Herein, we describe the infrastructure of a true and sustainable partnership developed with > 100 prominent faith leaders within the Piedmont Triad region of North Carolina for the purpose of developing or expanding existing health ministries within houses of worship, to improve health literacy and overall health long-term. The Triad Pastors Network is an asset-based partnership between the Maya Angelou Center for Health Equity at Wake Forest University School of Medicine and faith leaders in the Piedmont Triad region of North Carolina that was created under the guiding principles of community engagement to improve health equity and decrease health disparities experienced by African American communities. A partnership in which co-equality and shared governance are the core of the framework provides an effective means of achieving health-related goals in a productive and efficient manner. Faith-based partnerships are reliable approaches for improving the health literacy needed to address health disparities and inequities in communities of color.

Enhancing undergraduate education and research in aging to eliminate health disparities (ENGAGED) - A pipeline program to advance diversity in aging research.

The Enhancing Undergraduate Education and Research in Aging to Eliminate Health Disparities (ENGAGED) program takes advantage of the broad, multidisciplinary research established in the area of aging at Wake Forest University School of Medicine and its partner institutions, Wake Forest University and Winston-Salem State University. The ENGAGED program is designed to provide undergraduate students who are underrepresented in the biomedical sciences an opportunity to participate in educational and research training in aging and health disparities. Funded since August 2019, ENGAGED has provided 73 academic year internships and 46 summer internships, with another 8 internships starting in Fall 2023. A total of 61 students (67% female, 64% African American, 21% Hispanic) have participated in the ENGAGED program, reflecting the fact that 49% participate in more than one session or program offering. To date, 38 of the ENGAGED trainees have graduated (100% with a science, technology, engineering, or math degree), 84% of whom are working or pursuing advanced degrees in the biomedical sciences. Given the unique strengths, existing infrastructure, and long-standing collaborations among the partner institutions, the ENGAGED program is helping advance diversity in aging and health disparities research by creating a pipeline for well-trained underrepresented students interested in biomedical research careers.

A pilot study to assess the circulating renin-angiotensin system in COVID-19 acute respiratory failure.

The renin-angiotensin system (RAS) is fundamental to COVID-19 pathobiology, due to the interaction between the SARS-CoV-2 virus and the angiotensin-converting enzyme 2 (ACE2) coreceptor for cellular entry. The prevailing hypothesis is that SARS-CoV-2-ACE2 interactions lead to an imbalance of the RAS, favoring proinflammatory angiotensin II (ANG II)-related signaling at the expense of the anti-inflammatory ANG-(1-7)-mediated alternative pathway. Indeed, multiple clinical trials targeting this pathway in COVID-19 are underway. Therefore, precise measurement of circulating RAS components is critical to understand the interplay of the RAS on COVID-19 outcomes. Multiple challenges exist in measuring the RAS in COVID-19, including improper patient controls, ex vivo degradation and low concentrations of angiotensins, and unvalidated laboratory assays. Here, we conducted a prospective pilot study to enroll 33 patients with moderate and severe COVID-19 and physiologically matched COVID-19-negative controls to quantify the circulating RAS. Our enrollment strategy led to physiological matching of COVID-19-negative and COVID-19-positive moderate hypoxic respiratory failure cohorts, in contrast to the severe COVID-19 cohort, which had increased severity of illness, prolonged intensive care unit (ICU) stay, and increased mortality. Circulating ANG II and ANG-(1-7) levels were measured in the low picomolar (pM) range. We found no significant differences in circulating RAS peptides or peptidases between these three cohorts. The combined moderate and severe COVID-19-positive cohorts demonstrated a mild reduction in ACE activity compared with COVID-19-negative controls (2.2 ± 0.9 × 105 vs. 2.9 ± 0.8 × 105 RFU/mL, P = 0.03). These methods may be useful in designing larger studies to physiologically match patients and quantify the RAS in COVID-19 RAS augmenting clinical trials.

Angiotensinogen import in isolated proximal tubules: evidence for mitochondrial trafficking and uptake.

The renal proximal tubules are a key functional component of the kidney and express the angiotensin precursor angiotensinogen; however, it is unclear the extent that tubular angiotensinogen reflects local synthesis or internalization. Therefore, the current study established the extent to which angiotensinogen is internalized by proximal tubules and the intracellular distribution. Proximal tubules were isolated from the kidney cortex of male sheep by enzymatic digestion and a discontinuous Percoll gradient. Tubules were incubated with radiolabeled 125I-angiotensinogen for 2 h at 37°C in serum/phenol-free DMEM/F12 media. Approximately 10% of exogenous 125I-angiotensinogen was internalized by sheep tubules. Subcellular fractionation revealed that 21 ± 4% of the internalized 125I-angiotensinogen associated with the mitochondrial fraction with additional labeling evident in the nucleus (60 ± 7%), endoplasmic reticulum (4 ± 0.5%), and cytosol (15 ± 4%; n = 4). Subsequent studies determined whether mitochondria directly internalized 125I-angiotensinogen using isolated mitochondria from renal cortex and human HK-2 proximal tubule cells. Sheep cortical and HK-2 mitochondria internalized 125I-angiotensinogen at a comparable rate of (33 ± 9 vs. 21 ± 10 pmol·min-1·mg protein-1; n = 3). Lastly, unlabeled angiotensinogen (100 nM) competed for 125I-angiotensinogen uptake to a greater extent than human albumin in HK-2 mitochondria (60 ± 2 vs. 16 ± 13%; P < 0.05, n = 3). Collectively, our data demonstrate angiotensinogen import and subsequent trafficking to the mitochondria in proximal tubules. We conclude that this pathway may constitute a source of the angiotensinogen precursor for the mitochondrial expression of angiotensin peptides.

Evidence for a mitochondrial angiotensin-(1-7) system in the kidney.

Evidence for an intracellular renin-angiotensin system (RAS) in various cell organelles now includes the endoplasmic reticulum, nucleus, and mitochondria (Mito). Indeed, angiotensin (ANG) AT1 and AT2 receptor subtypes were functionally linked to Mito respiration and nitric oxide production, respectively, in previous studies. We undertook a biochemical analysis of the Mito RAS from male and female sheep kidney cortex. Mito were isolated by differential centrifugation followed by a discontinuous Percoll gradient and were coenriched in Mito membrane markers VDAC and ATP synthase, but not ß-actin or cathepsin B. Two distinct renin antibodies identified a 37-kDa protein band in Mito; angiotensinogen (Aogen) conversion was abolished by the inhibitor aliskiren. Mito Aogen was detected by an Aogen antibody to an internal sequence of the protein, but not with an antibody directed against the ANG I N terminus. ANG peptides were quantified by three direct RIAs; mitochondrial ANG II and ANG-(1-7) contents were higher compared with ANG I (23 ± 8 and 58 ± 17 vs. 2 ± 1 fmol/mg protein; P < 0.01, n = 3). 125I-ANG I metabolism primarily revealed the formation of 125I-ANG-(1-7) in Mito that reflects the endopeptidases neprilysin and thimet oligopeptidase. Last, immunoblot studies utilizing the ANG-(1-7)/Mas receptor antibody revealed the protein in isolated Mito from sheep renal cortex. Collectively, the current data demonstrate that Mito actively metabolize the RAS precursor protein Aogen, suggesting that ANG-(1-7) may be generated within Mito to establish an intramitochondrial RAS tone and contribute to renal mitochondrial function.

Perceived Discrimination and Nocturnal Blood Pressure Dipping Among Hispanics: The Influence of Social Support and Race.

OBJECTIVE: Little is known about the relationship of perceived racism to ambulatory blood pressure (ABP) in Hispanics. We explored possible associations between ABP nocturnal dipping and perceived racism in a Hispanic cohort. METHODS: Participants included 180 community-dwelling Hispanics from the Northern Manhattan Study. Measures included perceived racism, socioeconomic status, social support, and ABP monitoring. Nocturnal ABP nondipping was defined as a less than 10% decline in the average asleep systolic blood pressure relative to the awake systolic blood pressure. RESULTS: Overall, 77.8% of participants reported some form of perceived racism (Perceived Ethnic Discrimination Questionnaire scores >1.0). Greater social support was associated with less perceived discrimination (Spearman r = -0.54, p < .001). Those with higher perceived discrimination scores reported more depressive symptoms (r = 0.25, p < .001). Those with higher Perceived Ethnic Discrimination Questionnaire scores were less likely to show nocturnal ABP nondipping in multivariate models (odds ratio = 0.40, confidence interval = 0.17-0.98, p = .045). Among those with low perceived racism, black Hispanic participants were more likely to have nocturnal ABP nondipping (82.6%) compared with white Hispanics (53.9%; p = .02). Among those with high perceived racism, no associations between race and the prevalence of ABP nondipping was found (black Hispanic = 61.5% versus white Hispanic = 51.4%, p = .39; p interaction = .89). CONCLUSIONS: Perceived racism is relatively common among US Hispanics and is associated with ABP. Nondipping of ABP, a potential cardiovascular risk factor, was more common in black Hispanic participants with low perceived racism. This finding may reflect different coping mechanisms between black versus white Hispanics and related blood pressure levels during daytime exposures to discrimination.

An angiotensin-(1-7) peptidase in the kidney cortex, proximal tubules, and human HK-2 epithelial cells that is distinct from insulin-degrading enzyme.

Angiotensin 1-7 [ANG-(1-7)] is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic, and pro-oxidant effects of ANG II. We previously identified an peptidase that preferentially metabolized ANG-(1-7) to ANG-(1-4) in the brain medulla and cerebrospinal fluid (CSF) of sheep (Marshall AC, Pirro NT, Rose JC, Diz DI, Chappell MC. J Neurochem 130: 313-323, 2014); thus the present study established the expression of the peptidase in the kidney. Utilizing a sensitive HPLC-based approach, we demonstrate a peptidase activity that hydrolyzed ANG-(1-7) to ANG-(1-4) in the sheep cortex, isolated tubules, and human HK-2 renal epithelial cells. The peptidase was markedly sensitive to the metallopeptidase inhibitor JMV-390; human HK-2 cells expressed subnanomolar sensitivity (IC50 = 0.5 nM) and the highest specific activity (123 ± 5 fmol·min(-1)·mg(-1)) compared with the tubules (96 ± 12 fmol·min(-1)·mg(-1)) and cortex (107 ± 9 fmol·min(-1)·mg(-1)). The peptidase was purified 41-fold from HK-2 cells; the activity was sensitive to JMV-390, the chelator o-phenanthroline, and the mercury-containing compound p-chloromercuribenzoic acid (PCMB), but not to selective inhibitors against neprilysin, neurolysin and thimet oligopeptidase. Both ANG-(1-7) and its endogenous analog [Ala(1)]-ANG-(1-7) (alamandine) were preferentially hydrolyzed by the peptidase compared with ANG II, [Asp(1)]-ANG II, ANG I, and ANG-(1-12). Although the ANG-(1-7) peptidase and insulin-degrading enzyme (IDE) share similar inhibitor characteristics of a metallothiolendopeptidase, we demonstrate marked differences in substrate specificity, which suggest these peptidases are distinct. We conclude that an ANG-(1-7) peptidase is expressed within the renal proximal tubule and may play a potential role in the renal renin-angiotensin system to regulate ANG-(1-7) tone.

Gender, Age and COVID-19 Vaccination Status in African American Adult Faith-Based Congregants in the Southeastern United States.

OBJECTIVES: The COVID-19 pandemic has revealed significant differences in COVID-19 vaccination rates, with African Americans reporting lower rates compared to other racial and ethnic groups. The purpose of these analyses was to assess whether COVID-19 vaccination status differed according to age in a sample of 1,240 African American adult congregants of faith-based organizations ages 18 years or older, and to examine whether this association was moderated by gender. DESIGN: We developed and administered a 75-item cross-sectional survey, the Triad Pastor's Network COVID-19 and COVID-19 Vaccination survey, to assess experiences and perceptions regarding the COVID-19 virus and vaccines. We assessed the association between age and having received > 1 dose of a COVID-19 vaccine using unadjusted and multivariable binary logistic regression models, and the interaction of age and gender with COVID-19 vaccination status in a multivariable model. RESULTS: Approximately 86% of participants reported having received ≥ 1 dose of a COVID-19 vaccine. The mean age (standard deviation) of the sample was 51.33 (16.62) years, and 70.9% of the sample was comprised of women. The age by gender interaction term in the multivariable model was significant (p = 0.005), prompting additional analyses stratified by gender. In women, increased age was significantly associated with higher odds of COVID-19 vaccination (odds ratio = 1.09; 95% Confidence Interval 1.06, 1.11; p < 0.001). In men, the association was not significant (p = 0.44). CONCLUSIONS: Older age was positively associated with COVID-19 vaccination in African American women, but not African American men, which may inform strategies to increase vaccination rates.

Assessing the Role of Trust in Public Health Agencies and COVID-19 Vaccination Status Among a Community Sample of African Americans in North Carolina.

BACKGROUND: Mistrust of the government and medical establishments are prominent reasons for vaccine hesitancy among African Americans (AAs). As COVID-19 research evolves in real time with some uncertainties remaining, AA communities may be less trusting of public health agencies. The purpose of these analyses was to assess the association between trust in public health agencies that recommend the COVID-19 vaccination and COVID-19 vaccination status among AAs in North Carolina. METHODS: A 75-item cross-sectional survey, the Triad Pastors Network COVID-19 and COVID-19 Vaccination survey, was developed and administered to African Americans in North Carolina. Multivariable logistic regression was used to examine the association between levels of trust in public health agencies who recommend the COVID-19 vaccine and COVID-19 vaccination status among AAs. RESULTS: Of the 1157 AAs included in these analyses, approximately 14% of AAs had not received the COVID-19 vaccine. These findings indicated that lower levels of trust in public health agencies significantly decreased the odds of getting the COVID-19 vaccination compared to those with higher levels of trust among AAs. The most trusted source for information on COVID-19 included federal agencies among all respondents. Among the vaccinated, primary care physicians were another trusted source of information. Pastors were another trusted source for those willing to be vaccinated. CONCLUSIONS: Despite the majority of the respondents in this sample receiving the COVID-19 vaccine, subgroups of AAs remain unvaccinated. Federal agencies have a high level of trust among AA adults; however, innovative approaches are needed to reach AAs who remain unvaccinated.

Novel roles of nuclear angiotensin receptors and signaling mechanisms.

The renin-angiotensin system (RAS) constitutes an important hormonal system in the physiological regulation of blood pressure. The dysregulation of the RAS is considered a major influence in the development and progression of cardiovascular disease and other pathologies. Indeed, experimental and clinical evidence indicates that blockade of this system with angiotensin-converting enzyme (ACE) inhibitors or angiotensin type 1 receptor (AT1R) antagonists is an effective therapy to attenuate hypertension and diabetic renal injury, and to improve heart failure. Originally defined as a circulating system, multiple tissues express a complete RAS, and compelling evidence now favors an intracellular system involved in cell signaling and function. Within the kidney, intracellular expression of the three predominant ANG receptor subtypes is evident in the nuclear compartment. The ANG type 1 receptor (AT1R) is coupled to the generation of reactive oxygen species (ROS) through the activation of phosphoinositol-3 kinase (PI3K) and PKC. In contrast, both ANG type 2 (AT2R) and ANG-(1-7) (AT7R) receptors stimulate nitric oxide (NO) formation, which may involve nuclear endothelial NO synthase (eNOS). Moreover, blockade of either ACE2-the enzyme that converts ANG II to ANG-(1-7)-or the AT7 receptor exacerbates the ANG II-ROS response on renal nuclei. Finally, in a model of fetal programmed hypertension, the nuclear ROS response to ANG II is enhanced, while both AT2 and AT7 stimulation of NO is attenuated, suggesting that an imbalance in the intracellular RAS may contribute to the development of programming events. We conclude that a functional intracellular or nuclear RAS may have important implications in the therapeutic approaches to cardiovascular disease.

Nuclear angiotensin-(1-7) receptor is functionally coupled to the formation of nitric oxide.

The kidney is an important target for the actions of the renin-angiotensin system (RAS) and this tissue contains a complete local RAS that expresses the bioactive peptides angiotensin II (ANG II) and Ang-(1-7). We find both angiotensin type 1 (AT(1)R) and type 2 (AT(2)R) receptors expressed on renal nuclei that stimulate reactive oxygen species and nitric oxide (NO), respectively. Since Ang-(1-7) also exhibits actions within the kidney and the Ang-(1-7)/Mas receptor protein contains a nuclear localization sequence, we determined the expression of Ang-(1-7) receptors in nuclei isolated from the kidneys of young adult sheep. Binding studies with (125)I-[Sar(1)Thr(8)]-ANG II revealed sites sensitive to the Ang-(1-7) antagonist [d-Ala(7)]-Ang-(1-7) (DALA, A779), as well as to AT(2) and AT(1) antagonists. Incubation of Ang-(1-7) [10(-15) to 10(-9) M] with isolated cortical nuclei elicited a dose-dependent increase in the fluorescence of the NO indicator [4-amino-5-methylamino-2',7']-difluorofluorescein diacetate. The NO response to Ang-(1-7) was abolished by the NO inhibitor N-nitro-l-arginine methyl ester and DALA, but not the AT(1) antagonist losartan or the AT(2) blocker PD123319. Immunofluorescent studies utilizing the Ang-(1-7)/Mas receptor antibody revealed immunolabeling of the proximal tubules but not staining within the glomerulus in cortical sections of the sheep kidney. In the nuclear fraction of isolated proximal tubules, immunoblots revealed the precursor angiotensinogen and renin, as well as functional activity for ACE, ACE2, and neprilysin. We conclude that renal nuclei express Ang-(1-7)/Mas receptors that are functionally linked to NO formation. The marked sensitivity of the intracellular NO response to Ang-(1-7) implicates a functional role of the Ang-(1-7) axis within the nucleus. Moreover, evidence for the precursor and enzymatic components of the RAS within the nuclear compartment of the proximal tubules provides a potential pathway for the intracellular generation of Ang-(1-7).

Lower urinary α-Klotho is associated with lower angiotensin-(1-7) and higher blood pressure in young adults born preterm with very low birthweight.

Early-life factors including preterm birth and VLBW increase the risk of hypertension, but the mechanisms remain poorly understood. Reductions in the anti-aging protein α-klotho are associated with hypertension, possibly due to angiotensin (Ang) II activation, but the mechanisms are incompletely understood and clinical evidence is lacking. The association of α-klotho with the alternative Ang-(1-7) pathway, which counteracts Ang II to lower BP, is undescribed. We hypothesized that lower urinary α-klotho is associated with higher BP and lower urinary Ang-(1-7) in preterm-born VLBW young adults. In a cross-sectional analysis of data from a prospective cohort of 141 preterm-born VLBW young adults, we assessed the associations among urinary α-klotho/creatinine, Ang II/creatinine, Ang-(1-7)/creatinine, Ang II/Ang-(1-7), and BP using generalized linear models adjusted for age and hypertensive pregnancy and conducted a sensitivity analysis in 32 term-born young adults. Among those born preterm, lower α-klotho/creatinine was associated with higher systolic BP (adjusted ß (aß): -2.58 mm Hg, 95% CI -4.99 to -0.17), lower Ang-(1-7)/creatinine (ln aß: 0.1, 0.04-0.16), and higher Ang II/Ang-(1-7) (ln aß: -0.14, -0.21 to -0.07). In term-born participants, α-klotho/creatinine was inversely associated with Ang II/creatinine (ln aß: -0.15, -0.27 to -0.03) and Ang II/Ang-(1-7) (ln aß: -0.15, -0.27 to -0.03). In preterm-born young adults with VLBW, lower urinary α-klotho/creatinine was associated with higher SBP, lower urinary Ang-(1-7)/creatinine, and higher urinary Ang II/Ang-(1-7). Reduced renal α-klotho expression could lead to renal Ang-(1-7) suppression as a novel mechanism for the development of hypertension among individuals born preterm with VLBW.

The angiotensin II-AT1 receptor stimulates reactive oxygen species within the cell nucleus.

We and others have reported significant expression of the Ang II Type 1 receptor (AT1R) on renal nuclei; thus, the present study assessed the functional pathways and distribution of the intracellular AT1R on isolated nuclei. Ang II (1nM) stimulated DCF fluorescence, an intranuclear indicator of reactive oxygen species (ROS), while the AT1R antagonist losartan or the NADPH oxidase (NOX) inhibitor DPI abolished the increase in ROS. Dual labeling of nuclei with antibodies against nucleoporin 62 (Nup62) and AT1R or the NADPH oxidase isoform NOX4 revealed complete overlap of the Nup62 and AT1R (99%) by flow cytometry, while NOX4 was present on 65% of nuclei. Treatment of nuclei with a PKC agonist increased ROS while the PKC inhibitor GF109203X or PI3 kinase inhibitor LY294002 abolished Ang II stimulation of ROS. We conclude that the Ang II-AT1R-PKC axis may directly influence nuclear function within the kidney through a redox sensitive pathway.

Programs to Recruit and Retain a More Diverse Workforce in Biomedical Sciences Research.

To improve overall healthcare and to reduce health disparities, efforts must focus on increasing the diversity of personnel trained in the biomedical sciences. Here, we describe the development, implementation, and relative outcomes of three pipeline training programs in biomedical sciences research designed to increase workforce diversity institutionally, regionally, and nationally. We report on their effectiveness in improving the recruitment and retention of underrepresented minorities with the long-term goal of remedying health inequities and disparities.

Clinical Relevance of Trace Bands on Serum Electrophoresis in Patients Without a History of Gammopathy.

Serum protein electrophoresis (SPE) and immunofixation is commonly used to screen for plasma cell dyscrasias. Interpretation of these tests is qualitative by nature and can yield trace, faint, or scarcely visible immunoglobulin bands (TFS), which can be difficult to classify. Whether these bands should be reported at all is challenging given their unknown clinical significance. In the present study, we retrospectively analyzed 14,036 physician-ordered protein SPE and immunofixation electrophoresis (IFE) tests on serum and urine specimens (from 4,091 patients) during the period of 2000-2010. We found that 17% of all IFE results evaluated for the presence of monoclonal gammopathies (2,389 out of 14,036) contained TFS bands, representing 4.2% (173 out of 4091) of all patients evaluated. Sixty of these patients (42%) had no previous history of gammopathy, and were clinically evaluated over a mean period of up to five years from the original diagnosis of plasma cell pathology. None of these patients had progressed to multiple myeloma, lymphoplasmacytic lymphoma, plasmacytoma, or leukemia. The remaining 82 patients (58%) had a previous history of gammopathy, but had not progressed to any symptomatic plasma cell dyscrasia. Evaluation of these patients was followed for a median period of 4.3 years, with a mean of 21.5 IFE tests per individual. These data suggest that for patients without a previous history of gammopathy, the presence of TFS bands on serum protein electrophoresis does not warrant frequent follow up investigation as commonly practiced. Routine follow up of patients with a prior history of gammopathy, conversely, are warranted and may contribute to overall survival with multiple treatment options now available. For those interpreting IFE results, it may be worth considering these data when composing comments regarding suggested repeat testing frequency by SPE/IFE or alternate test methods.

The renin-angiotensin-aldosterone system in adolescent offspring born prematurely to mothers with preeclampsia.

HYPOTHESIS/INTRODUCTION: Preeclampsia is associated with alterations in the maternal renin-angiotensin-aldosterone system (RAAS), increased blood pressure (BP), and cardiovascular risk in the offspring. We hypothesized that preeclampsia is associated with alterations in the RAAS in the offspring that persist into adolescence. MATERIALS AND METHODS: We compared components of the circulating (n = 111) and renal (n = 160) RAAS in adolescents born prematurely with very low birth weight (VLBW) of preeclamptic (PreE) and normotensive (NoHTN) pregnancies. Multivariable linear regression was used to evaluate potential confounding and intermediate variables. Analyses were stratified by sex. RESULTS: Adjusting for race and antenatal steroid exposure, male offspring of PreE mothers had higher circulating aldosterone than those of NoHTN mothers (adjusted mean difference = 109; 95% confidence limits: -9, 227 pmol/L). Further adjustment for current BMI attenuated this difference (adjusted mean difference: 93; 95% confidence limits: -30, 215 pmol/L). CONCLUSION: Among male preterm VLBW infants, maternal preeclampsia is associated with increased circulating aldosterone level in adolescence, which appears to be mediated in part by higher BMI.

Glucocorticoid-induced fetal programming alters the functional complement of angiotensin receptor subtypes within the kidney.

We examined the impact of fetal programming on the functional responses of renal angiotensin receptors. Fetal sheep were exposed in utero to betamethasone (BMX; 0.17 mg/kg) or control (CON) at 80 to 81 days gestation with full-term delivery. Renal nuclear and plasma membrane fractions were isolated from sheep age 1.0 to 1.5 years for receptor binding and fluorescence detection of reactive oxygen species (ROS) or nitric oxide (NO). Mean arterial blood pressure and blood pressure variability were significantly higher in the BMX-exposed adult offspring versus CON sheep. The proportion of nuclear AT(1) receptors sensitive to losartan was 2-fold higher (67 ± 6% vs 27 ± 9%; P<0.01) in BMX compared with CON. In contrast, the proportion of AT(2) sites was only one third that of controls (BMX, 25 ± 11% vs CON, 78 ± 4%; P<0.01), with a similar reduction in sites sensitive to the Ang-(1-7) antagonist D-Ala7-Ang-(1-7) with BMX exposure. Functional studies revealed that Ang II stimulated ROS to a greater extent in BMX than in CON sheep (16 ± 3% vs 6 ± 4%; P<0.05); however, NO production to Ang II was attenuated in BMX (26 ± 7% vs 82 ± 14%; P<0.05). BMX exposure was also associated with a reduction in the Ang-(1-7) NO response (75 ± 8% vs 131 ± 26%; P<0.05). We conclude that altered expression of angiotensin receptor subtypes may be one mechanism whereby functional changes in NO- and ROS-dependent signaling pathways may favor the sustained increase in blood pressure evident in fetal programming.

Angiotensin-(1-7)-angiotensin-converting enzyme 2 attenuates reactive oxygen species formation to angiotensin II within the cell nucleus.

The angiotensin (Ang) type 1 receptor (AT(1)R) is highly expressed on renal nuclei and stimulates reactive oxygen species (ROS). It is not known whether other functional components of the Ang system regulate the nuclear Ang II-AT(1)R ROS pathway. Therefore, we examined the expression of Ang receptors in nuclei isolated from the kidneys of young adult (1.5 years) and older adult (3.0 to 5.0 years) sheep. Binding studies in renal nuclei revealed the AT(2)R as the predominant receptor subtype ( approximately 80%) in young sheep, with the Ang-(1-7) (AT(7)R; Mas protein) and AT(1)R antagonists competing for the remaining sites. Conversely, in older sheep, the AT(1)R accounted for approximately 85% of nuclear sites, whereas the Ang type 2 receptor and AT(7)R subtypes comprise approximately 20% of remaining sites. Ang II increased nuclear ROS to a greater extent in older (97+/-22%; n=6) versus young animals (7+/-2%; P=0.01; n=4), and this was abolished by an AT(1)R antagonist. The AT(7)R antagonist D-Ala(7)-Ang-(1-7) increased ROS formation to Ang II by approximately 2-fold (174+/-5% versus 97+/-22%; P<0.05) in older adults. Immunoblots of renal nuclei revealed protein bands for the AT(7)R and Ang-converting enzyme 2 (ACE2), which metabolizes Ang II to Ang-(1-7). The ACE2 inhibitor MLN4760 also exacerbated the Ang II-dependent formation of ROS (156+/-15%) and abolished the generation of Ang-(1-7) from Ang II. We conclude that an ACE2-Ang-(1-7)-AT(7)R pathway modulates Ang II-dependent ROS formation within the nucleus, providing a unique protective mechanism against oxidative stress and cell damage.

Nuclear angiotensin II type 2 (AT2) receptors are functionally linked to nitric oxide production.

Expression of nuclear angiotensin II type 1 (AT(1)) receptors in rat kidney provides further support for the concept of an intracellular renin-angiotensin system. Thus we examined the cellular distribution of renal ANG II receptors in sheep to determine the existence and functional roles of intracellular ANG receptors in higher order species. Receptor binding was performed using the nonselective ANG II antagonist (125)I-[Sar(1),Thr(8)]-ANG II ((125)I-sarthran) with the AT(1) antagonist losartan (LOS) or the AT(2) antagonist PD123319 (PD) in isolated nuclei (NUC) and plasma membrane (PM) fractions obtained by differential centrifugation or density gradient separation. In both fetal and adult sheep kidney, PD competed for the majority of cortical NUC (> or =70%) and PM (> or =80%) sites while LOS competition predominated in medullary NUC (> or =75%) and PM (> or =70%). Immunodetection with an AT(2) antibody revealed a single approximately 42-kDa band in both NUC and PM extracts, suggesting a mature molecular form of the NUC receptor. Autoradiography for receptor subtypes localized AT(2) in the tubulointerstitium, AT(1) in the medulla and vasa recta, and both AT(1) and AT(2) in glomeruli. Loading of NUC with the fluorescent nitric oxide (NO) detector DAF showed increased NO production with ANG II (1 nM), which was abolished by PD and N-nitro-l-arginine methyl ester, but not LOS. Our studies demonstrate ANG II receptor subtypes are differentially expressed in ovine kidney, while nuclear AT(2) receptors are functionally linked to NO production. These findings provide further evidence of a functional intracellular renin-angiotensin system within the kidney, which may represent a therapeutic target for the regulation of blood pressure.