Janice G. Douglas

Determinants of Salt Sensitivity in Black and White Normotensive and Hypertensive Women

Abstract—Salt sensitivity (SS) has been linked to human hypertension. We examined ethnic differences in the relation between SS; erythrocyte sodium (Na+i), calcium (Ca2+i), potassium (K+i), and magnesium (Mg2+i); and sodium pump activity in African-American (AA) and white women. In a crossover protocol, similar numbers of normotensive, hypertensive, AA, and white women were randomized to 7 days of a 20 meq/d and a >200 meq/d salt diet (n=199). After an overnight inpatient stay, group differences in supine blood pressure (BP), heart rate, erythrocyte cations, and sodium pump activity were measured. The prevalence of SS (53.5% vs 51%) and salt resistance (26.3% vs 30.0%) was similar in both races. Greater mean BP increase with salt loading was seen in AA vs white hypertensives but not between the normotensive women. In hypertensives, increase in mean arterial pressure was 12.6 vs 8.2 mm Hg in AAs vs whites, respectively (P <0.01), and for systolic BP, it was 23 vs 14.8 mm Hg (P <0.01). Higher Na+i and Ca2+i were noted in SS and salt-intermediate AA than in the corresponding white subjects. Na+i, Ca2+i, and the ratios of Na+i to K+i and of Ca2+i to Mg2+i were positively correlated with salt responsiveness in AA but not in white women. Sodium pump activity was similar between groups, although the change in maximal activity trended to vary inversely with SS in AA. In closely matched AA and white women, the prevalence of SS is similarly high in both races, although the magnitude of BP increase is greater in AA hypertensives. In AA but not in whites, SS is positively associated with Na+i, Ca2+i, and the ratios of Na+i to K+i and of Ca2+i to Mg2+i.

The Rationale and Design of the AASK Cohort Study

Hypertensive kidney disease commonly progresses. The primary objective of the AASK (African American Study of Kidney Disease and Hypertension) Cohort Study is to determine prospectively the course of kidney function and risk factors for kidney disease progression in African Americans with hypertensive kidney disease who receive recommended anti-hypertensive therapy. The AASK Cohort Study is a prospective, observational study that is an extension of the AASK trial. The AASK trial tested the effects of three medications used as initial anti-hypertensive therapy (ramipril, metoprolol, and amlodipine) and two levels of BP control. Of the 1094 trial participants, approximately 650 to 700 individuals who have not reached ESRD will likely enroll in the Cohort Study. Risk factors to be studied include environmental, genetic, physiologic, and socioeconomic variables. The primary renal outcome is a composite clinical outcome defined by doubling of serum creatinine, ESRD, or death. Medication treatment for hypertension, beginning with the angiotensin converting enzyme inhibitor ramipril, is offered to all participants. In this fashion, the study directly controls two of the major determinants of kidney disease progression: treatment of hypertension and use of renoprotective, anti-hypertensive medication. The minimum duration of follow-up in the Cohort Study is 5 yr (total of 9 to 12 yr, including the period of the AASK trial). Ultimately, data from the AASK Cohort Study should enhance our understanding of the risk factors and processes that determine the progression of kidney disease. Such results might eventually lead to new strategies that delay or prevent ESRD.

PATHOPHYSIOLOGY AND TREATMENT IMPLICATIONS OF HYPERTENSION IN THE AFRICAN-AMERICAN POPULATION

Regardless of the etiology, hypertension remains a major public health problem in African-Americans and is associated with significant morbidity and mortality. Additional data on the pathophysiology of this disease in this population are needed, as are data on the best therapies to decrease the high complication rate. Because many of the large studies on hypertension have included few African-Americans, recruitment of this ethnic group into clinical trials should be promoted. Further studies into the genetic factors in the pathophysiology of racial differences in hypertension may shed more light on this complex issue.

A Comparison of Iothalamate-GFR and Serum Creatinine-Based Outcomes: Acceleration in the Rate of GFR Decline in the African American Study of Kidney Disease and Hypertension

In renal clinical trials, both slope-based and time-to-event renal outcomes have been used. These outcomes are typically based on estimates of GFR obtained using creatinine or iothalamate GFR (iGFR). The African American Study of Kidney Disease and Hypertension (AASK) was a trial in 1094 African Americans with hypertensive nephrosclerosis, which examined the effects of two levels of BP control and three antihypertensive regimens. This study compared the effects of the AASK interventions on outcomes based on serum creatinine with corresponding outcomes based on iGFR using 9742 matched pairs of iGFR and serum creatinine measurements. The iGFR-based outcomes included (1) a time-to-event composite outcome including a 50% GFR decline, ESRD, or death; (2) a composite outcome including a 50% GFR decline or ESRD; (3) mean decline in GFR in the first 3 mo after randomization (acute slope); (4) mean decline in GFR starting 3 mo after randomization (chronic slope); and (5) mean decline in GFR from baseline (total slope). The corresponding creatinine-based outcomes were (1) a composite of doubling of serum creatinine, ESRD, or death and (2) a composite of doubling of serum creatinine or ESRD and acute, chronic, and total slopes defined by the mean change in estimated GFR (eGFR), where eGFR was estimated from a regression equation for GFR depending primarily on serum creatinine and developed in AASK enrollees. Mean changes in iGFR and eGFR were also compared under extended models that allowed for the possibility that the rate of GFR decline may change over time during the chronic phase. an apparent acceleration in rate of decline of renal function over time was found. Subtle differences were observed between effects of the interventions on some of the creatinine and iGFR slope-based outcomes, but the main conclusions of the trial were similar for the serum creatinine and iothalamate-based measurements. This has important implications for the design of clinical trials with renal outcomes.

Angiotensin II Type 2 Receptor Subtype Mediates Phospholipase A2–Dependent Signaling in Rabbit Proximal Tubular Epithelial Cells

We investigated the ability of angiotensin II (Ang II) or the stable analogue [Sar1]-Ang II to increase intracellular and extracellular free arachidonic acid in primary cultures of rabbit proximal tubular epithelial cells to better characterize the receptor subtype and orientation of phospholipase A2 (PLA2)-mediated signaling. Proximal tubular cells were labeled with [3H]arachidonic acid for 4 hours and then treated with Ang II or [Sar1]-Ang II. Lipids were extracted from labeled cells, separated by thin-layer chromatography, and quantified by liquid scintillation counting. Ang II (10 mumol/L, 1 minute) stimulated an increase in intracellular free [3H]arachidonic acid from 21.0 +/- 2.0 to 32.2 +/- 2.8 disintegrations per minute/microgram protein, an effect that was potentiated by EGTA. [Sar1]-Ang II stimulated a time- and concentration-dependent increase in [3H]arachidonic acid release from labeled cells. Release of [3H]arachidonic acid was maximal at 10 mumol/L [Sar1]-Ang II, with an EC50 of approximately 3 mumol/L. Ang II receptor antagonists caused concentration-dependent inhibition of [Sar1]-Ang II-stimulated [3H]arachidonic acid release with the following order of potency: CGP 42112 = PD 123319 > losartan. Furthermore, in proximal tubular epithelial cells grown on polyester membrane filters, the Ang II receptor that mediated arachidonic acid release was predominantly apical rather than basolateral. These observations are consistent with activation of a Ca(2+)-independent, apical PLA2 isoform in epithelial cells through an Ang II type 2 receptor subtype.

Design and Statistical Aspects of the African American Study of Kidney Disease and Hypertension (AASK)

The African American Study of Kidney Disease and Hypertension (AASK) is a multicenter randomized clinical trial designed to test the effectiveness of three anti-hypertensive drug regimens and two levels of BP control on the progression of hypertensive kidney disease. Participants include African-American men and women aged 18 to 70 yr who have hypertensive kidney disease and GFR between 20 and 65 ml/min per 1.73 m(2). The three anti-hypertensive drug regimens include an angiotensin converting enzyme inhibitor (ramipril), a dihydropyridine calcium channel blocker (amlodipine) or a beta-blocker (metoprolol) as initial therapy. The BP control levels are a lower goal (mean arterial pressure, </=92 mmHg) and a usual goal (mean arterial pressure, 102 to 107 mmHg inclusive). The primary outcome is rate of change in renal function as measured by GFR, assessed by (125) I-iothalamate clearance. The main secondary patient outcome is a composite including the following events: (1) reduction in GFR by 50%, (2) end-stage renal disease, or (3) death.

Gβγ-independent constitutive association of Gαs with SHP-1 and angiotensin II receptor AT2 is essential in AT2-mediated ITIM-independent activation of SHP-1

Conventional mode of activation of SH2 domain-containing phosphatase 1 (SHP-1) by a single transmembrane (TM) inhibitory receptor such as killer cell inhibitory receptor, Fcγ receptor type IIb1, and paired Ig-like receptors of inhibitory types requires tyrosine phosphorylation of immunoreceptor tyrosine-based inhibitory (ITIM) motifs in the cytoplasmic domains of the inhibitory receptors. Contrary to this paradigm, AT2, a G protein-coupled 7TM receptor that does not undergo tyrosine phosphorylation in response to angiotensin II (Ang II) stimulation, also activates SHP-1. Here we show that SHP-1 constitutively and physically associates with AT2 receptor in transfected COS-7 cells. On stimulation by Ang II, SHP-1 becomes activated and dissociated from AT2 receptor, independent of pertussis toxin. Cotransfection of transducin Gβγ inhibits SHP-1/AT2 association and the SHP-1 activation, whereas cotransfection of C-terminal of β-adrenergic receptor kinase, which abrogates Gβγ signaling, facilitates SHP-1 activation. Surprisingly, SHP-1/AT2 association and the SHP-1 activation requires the presence of Gαs as shown by differential coimmunoprecipitation, dominant negative Gαs, constitutively active Gαs, and Gα peptides. A mutant AT2 receptor D141A–R142L that is inactive in Gα protein activation constitutively associates with SHP-1 and activates it. Together, these results indicate that Gαs alone, rather than exclusively in the form of Gαβγ heterotrimer may facilitate signal transduction for G protein-coupled receptors, suggesting a novel mechanism distinct from the classic paradigm of heterotrimeric G proteins. The AT2-mediated ITIM-independent activation of SHP-1 that is distinct from the conventional mode of activation, may represent a general paradigm for activation of SHP-1/2-class tyrosine phosphatases by G protein-coupled receptors.

Hypertension-related disease in African Americans. Factors underlying disparities in illness and its outcome.

PREVIEW African Americans have a higher prevalence of hypertension, diabetes, cardiovascular disease (CVD), stroke, and renal disease than white Americans. The high rates of diabetes and hypertension in children and of type 2 diabetes, stroke, and CVD in women are particularly striking. In this article, Drs Sowers, Ferdinand, Bakris, and Douglas examine the biologic, social, and genetic factors that contribute to these health disparities, the risk for which appears in early childhood.

A novel angiotensin receptor subtype in rat mesangium. Coupling to adenylyl cyclase.

The diversity of angiotensin II (Ang II) actions implies multiple receptor subtypes. To characterize these subtypes in rat mesangial cells, we used the angiotensin subtype 1A (AT1A) antagonist losartan (DuP 753), the subtype 2/1B (AT2/AT1B) antagonist PD 123319, and the AT2 antagonist CGP 42112A in radioreceptor and adenylyl cyclase assays. In radioligand binding competition experiments, approximately 25% of the specific binding sites labeled by 125I-[Sar1]Ang II were inhibited by low concentrations of PD 123319 (0.1 to 10 nM), whereas the AT2 antagonist CGP 42112A was inactive at concentrations less than 0.1 microM. Conversely, losartan inhibited 75% of the binding at low concentrations (0.1 nM to 0.1 microM), but higher concentrations (up to 10 microM) were required to inhibit the second component of 125I-[Sar1]Ang II binding. The effects of the different antagonists on the inhibition by Ang II of forskolin-stimulated cyclic AMP production were also analyzed. Ang II inhibited forskolin-stimulated adenylyl cyclase in a concentration-dependent fashion (IC50, 35 +/- 7 nM), and the maximal inhibition of adenylyl cyclase was 44 +/- 2%. In the radioligand binding experiments, both losartan and PD 123319 antagonized the inhibition of adenylyl cyclase elicited by 0.1 microM Ang II (IC50, 0.5 +/- 0.2 and 1.2 +/- 0.4 microM, respectively), whereas CGP 42112A was less potent (IC50, 5.7 +/- 1.6 microM). Comparison of binding affinities at AT1B receptor sites with antagonist potencies in the adenylyl cyclase assay show good agreement for losartan and CGP 42112A, whereas PD 123319 is less potent than expected from membrane binding assays, possibly because of partial agonist properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonate-Induced Tyrosine Phosphorylation of Epidermal Growth Factor Receptor and Shc-Grb2-Sos Association

-Protein tyrosine phosphorylation induced by arachidonic acid (AA), an important lipid second messenger, was investigated in rabbit renal proximal tubule epithelial cells. AA stimulated tyrosine phosphorylation of a number of proteins with estimated molecular weights of 42, 44, 52, 56, 85, and 170/180 kDa. The phosphoproteins pp44 and pp42 were identified as 2 isoforms of mitogen-activated protein kinase (MAPK). Phosphorylation of MAPK in response to AA was transient, dose-dependent, and accompanied by an increase in its activity. The mechanism of AA-induced MAPK activation in RTE cells was protein kinase C-independent and involved tyrosine phosphorylation of adaptor protein Shc and its association with Grb2-Sos complex. Moreover, stimulation of RTE cells with AA resulted in significant phosphorylation of epidermal growth factor (EGF) receptor and its association with Shc. The effect of AA on EGF receptor phosphorylation, its association with Shc, and MAPK activation was similar to the effect of 1 ng/mL EGF. Tyrphostin AG1478, a specific inhibitor of EGF receptor tyrosine kinase activity, completely blocked the effects of AA and EGF but not phorbol ester on MAPK phosphorylation. These data suggest that in renal tubular epithelial cells, the mechanism of AA-induced MAPK activation involves tyrosine phosphorylation of EGF receptor and its association with Shc and Grb2-Sos complex. Given the critical role of AA in signaling linked to G protein-coupled receptors (GPCRs), these observations provide a mechanism for cross talk between GPCRs linked to phospholipases and the tyrosine kinase receptor signaling cascades.