Nerimiah Emmett

Alterations in Aldosterone and Angiotensin II Levels in Salt-Induced Hypertension

Several studies have demonstrated that plasma renin-angiotensin activity is reduced in rats administered a high salt diet. We evaluated changes in plasma and tissue levels of aldosterone (ALDO) and angiotensin II (A-II), as well as the reduced-to-oxidized glutathione ratio. Male Dahl salt-sensitive (SS) rats were placed on either a high-salt (8% NaCl; HS) or a normal-salt (0.3% NaCl; NS) diet for 3 weeks. Prior to and weekly on the diets, systolic blood pressure was measured by tail cuff plethysmography. Levels of A-II and ALDO in plasma, heart, and kidney were analyzed by enzyme immunoassay. Reduced and oxidized gluthatione were simultaneously measured by HPLC fluorescence detection. Heart and kidney tissues were prepared for histological analysis. Systolic blood pressure in animals on a HS diet was significantly elevated above that of those on a NS diet. High salt caused a reduction in both plasma A-II and ALDO levels; while their levels in the heart and kidney were increased. Exposure to a high-salt diet led to the enlargement of both heart and kidney. The reduced-to-oxidized glutathione ratio in plasma, heart and kidney was lowered by exposure to a HS diet. Kidneys from animals on a high-salt diet showed fibroid necrosis associated with wrinkling and thickening of the glomerular capillary wall, while hearts were hypertrophic. Taken together, high dietary salt induces inappropriate activation of the local renin-angiotensin-aldosterone systems. Tissue levels of angiotensin II and aldosterone may be more reflective of the severity of vascular maladaptations than are plasma levels, and may play a greater role in the maintenance of hypertension.

Fenofibrate attenuates tubulointerstitial fibrosis and inflammation through suppression of nuclear factor-κB and transforming growth factor-β1/Smad3 in diabetic nephropathy.

Fibrates, the ligands of peroxisome proliferator-activated receptor-α, have been shown to have a renal protective action in diabetic models of renal disease, but the mechanisms underlying this effect are unknown. In the present study, we sought to investigate in greater detail the effect of fenofibrate and its mechanism of action on renal inflammation and tubulointerstitial fibrosis in an animal model of type 2 diabetes mellitus. Twelve-week-old non-diabetic Zucker lean (ZL) and Zucker diabetic fatty (ZD) rats were treated with vehicle or fenofibrate for 10 weeks. mRNA and protein analyses were performed by real-time polymerase chain reaction, Western blot and immunostaining. The diabetic condition of ZD rats was associated with an increase in collagen and α-smooth muscle actin accumulation in the kidney, which was significantly reduced by fenofibrate. Chronic treatment of ZD rats with fenofibrate attenuated renal inflammation and tubular injury as evidenced by a decrease in mRNA and protein expression of secreted phosphoprotein-1, monocyte chemotactic protein-1 and kidney injury molecule-1 in the kidneys. Renal interstitial macrophage infiltration was also significantly reduced in the kidneys of fenofibrate-treated diabetic animals. Moreover, renal nuclear factor (NF)-κB DNA-binding activity, transforming growth factor (TGF)-β1 and phospho-Smad3 proteins were significantly higher in ZD animals compared with ZL ones. This increase in NF-κB activity, TGF-β1 expression and Smad3 phosphorylation was greatly attenuated by fenofibrate in the diabetic kidneys. Taken together, fenofibrate suppressed NF-κB and TGF-β1/Smad3 signaling pathways and reduced renal inflammation and tubulointerstitial fibrosis in diabetic ZD animals.

1A-779 attenuates angiotensin-(1-7) depressor response in salt-induced hypertensive rats.

Chronic infusion of angiotensin-(1-7) [Ang-(1-7)] lowers blood pressure in salt-induced and spontaneously hypertensive (SHR) rats. In the present study, we have examined the acute effect of Ang-(1-7) in salt-induced hypertension using Dahl salt-sensitive rats placed on low (0.3%) or high (8.0% NaCl) salt diets for 2 weeks. Rats fed a high salt diet showed a greater rise in BP than those fed a low salt diet. Ang-(1-7) (24 microg/kg) reduced mean arterial pressure (MAP), enhanced the release of prostacyclin and nitric oxide, and suppressed thromboxane A(2) levels. A-779 (48 microg/kg, i.v), a selective Ang-(1-7) antagonist, partially blocked these effects of Ang-(1-7). The Ang-(1-7)-induced depressor response observed in these animals was related to an increase in vasodilatory prostanoids, a decrease in the constrictor prostanoid thromboxane A(2), and an increase in nitric oxide levels in both plasma and isolated aortic rings.

Gender differences in the attenuation of salt-induced hypertension by angiotensin (1-7).

Chronic infusion of angiotensin (1-7) [Ang-(1-7)] lowers blood pressure in spontaneously hypertensive rats (SHR). To assess the role of Ang-(1-7) in salt-induced hypertension, Ang-(1-7) (24 microg/kg/hr) or saline was administered chronically via osmotic minipump into the jugular vein of 5-6 wk-old male (M) and female (F) Dahl salt-sensitive rats placed on a high-salt (8% NaCl) diet for 2 weeks. Blood pressure (BP) and heart rate were measured prior to the start of the diet and weekly thereafter. Ang-(1-7) significantly attenuated the BP increase after 1 wk on the diet in both M and F rats, but after 2 weeks only in F rats. Enhanced release of prostacyclin, (6-keto PGF1 alpha), following Ang-(1-7) treatment was observed in both M and F rats. In addition, significant increases in aortic blood flow and plasma levels of nitric oxide were observed in the F rats following Ang-(1-7) treatment. These findings demonstrate that the reduction in BP is due to both prostacyclin and NO and that there is a gender difference in the attenuation of salt-induced hypertension by Ang-(1-7).

Plasma 25-hydroxyvitamin D concentrations are inversely associated with blood pressure of Dahl salt-sensitive rats.

Dietary salt is a contributing factor to the development of hypertension in individuals who are salt-sensitive. The vitamin D endocrine system has been reported to modulate vascular structure and function. Since elderly hypertensive females with low plasma renin activity, typical of salt-sensitivity, had significantly lower 25-hydroxyvitamin D concentrations compared with normotensive elderly and young females, we have used Dahl salt-sensitive and salt-resistant rats fed high (80 g/kg diet) and low (3 g/kg diet) salt diets as models to examine the relationship between salt-sensitivity and 25-hydroxyvitamin D, the precursor of the hormonal form of vitamin D, 1,25-dihydroxyvitamin D. Plasma 25-hydroxyvitamin D concentrations of salt-resistant rats were unaffected by a high salt diet, but plasma 25-hydroxyvitamin D concentrations of salt-sensitive rats were significantly reduced within three weeks to lower than 25%. There was a negative association between plasma 25-hydroxyvitamin D concentrations of salt-sensitive rats and the number of days that the rats were fed a high salt diet (r = -0.98, P < 0.02) and a positive association between blood pressure and the number of days that the rats were fed a high salt diet (r = 0.97, P < 0.05). An inverse relationship was found between plasma 25-hydroxyvitamin D concentrations and blood pressure (r = -0.99, P < 0.01). Spontaneously hypertensive rats did not have low plasma 25-hydroxyvitamin D concentrations, suggesting that reduction of plasma 25-hydroxyvitamin D concentration might be specific to salt-induced hypertension.

Glomerular Expression of Kidney Injury Molecule-1 and Podocytopenia in Diabetic Glomerulopathy

Background/Aims: Studies have shown that kidney injury molecule-1 (KIM-1) is upregulated in damaged renal proximal tubules. In this study, we examined KIM-1 expression in glomerular epithelial cells in diabetic glomerulopathy. Methods: Renal histology, immunostaining and Western blot for protein level, and real-time PCR for mRNA expression of KIM-1 and podocyte markers were evaluated in untreated or losartan-treated Zucker lean (Fa/+) and Zucker diabetic fatty (Fa/Fa) rats. Results: The diabetic rats showed an increased glomerular expression of KIM-1. KIM-1 staining was localized primarily in the hyperplastic parietal epithelium of Bowman’s capsule in the early stages of diabetes with subsequent increase in KIM-1-positive cells in the glomerular tuft in the more advanced stages. The increase in glomerular KIM-1 was associated with a decrease in podocytes in Fa/Fa rats. Antiproteinuric treatment with losartan attenuated podocytopenia and decreased renal expression of KIM-1 in treated diabetic rats. In an in vitro study, albumin overload increased KIM-1 protein in the primary cultures of rat glomerular epithelial cells. Conclusion: These results show that glomerular KIM-1 expression was increased, in proportion to the extent of proteinuria and podocytopenia in the diabetic animals, supporting that KIM-1 could be used as a potential biomarker for glomerular injury in proteinuric kidney disease.

Possible Mechanisms of Salt-Induced Hypertension in Dahl Salt-Sensitive Rats

Genetic factors, diet, and salt sensitivity have all been implicated in hypertension. To further understand the mechanisms involved in salt-induced hypertension, cardiovascular, hemodynamics, and biochemical parameters in Dahl salt-sensitive rats were evaluated in animals on high- and low-sodium diets. During a 4-week treatment period, blood pressure was significantly elevated in the high (8.0%) salt group compared to the low (0.3%) salt group (p< or =0.05 for weeks 2 and 4, respectively). No significant changes were observed in heart rate. The increase in blood pressure was associated with significant increases in lower abdominal aortic and renal vascular resistance, along with a reduction in blood flow. A fourfold increase in arginine vasopressin was observed in animals on the high-salt diet. In contrast, there was no effect on plasma sodium, potassium, or aldosterone levels during the treatment period. As measured in isolated aortic rings, the high-salt diet also caused a significant elevation in stimulated norepinephrine release and a reduction in cyclic GMP levels. These data suggest that salt-induced elevation in blood pressure is due to activation of both the sympathetic and arginine vasopressin systems via mechanisms involving decreased cyclic GMP generation in vascular smooth muscle.

Influence of low density lipoproteins on vascular smooth muscle cell growth and motility: Modulation by extracellular matrix

Low density lipoproteins (LDL) are thought to play a major role in cardiovascular diseases such as atherosclerosis. Much remains to be done to understand the cellular effects of LDL and how the extracellular matrix (ECM) influences these effects. We found that LDL produced a dose dependent increase in vascular smooth muscle cell (SMC) proliferation. The ECM altered the proliferative response of SMC to LDL: on collagen I there was a 66% inhibition, endothelial cell derived-ECM a 2-fold increase, and collagen IV no difference in proliferation compared to paired controls. LDL affected SMC motility (cell area and shape factor) but the extent and direction of the effect depended on whether the cells were cultured on uncoated or coated dishes. LDL treated cultures had a 5-fold lower migration rate but net movement was not different, suggesting that LDL decreased SMC random movement. There was a dose-dependent accumulation of lipid by SMC incubated with LDL and, subsequently, cytoplasmic lipid droplets were observed. Cells cultured on uncoated plates showed an increased cholesterol content as a function of LDL concentration. In contrast, cells cultured on a collagen IV matrix showed no net change in cholesterol content over the range of LDL concentrations studied. Hence, the uptake of LDL cholesterol appears to be completely inhibited by this matrix. These studies indicate that the influence of LDL on several SMC parameters is modulated by ECM components.

Plasma 24,25-dihydroxyvitamin D concentration of Dahl salt-sensitive rats decreases during high salt intake

Dahl salt-sensitive rats, but not salt-resistant rats, develop hypertension in response to high salt intake. We have previously shown an inverse relationship between plasma 25-hydroxyvitamin D (25-OHD) concentration and blood pressure of Dahl salt-sensitive rats during high salt intake. In this study, we report on the relationship between high salt intake and plasma 24,25-dihydroxyvitamin D (24,25-(OH)(2)D) concentration of Dahl salt-sensitive and salt-resistant rats. Rats were fed a high salt diet (8%) and sacrificed at day 2, 7, 14, 21, and 28. Plasma 24,25-(OH)(2)D concentrations of salt-sensitive rats were reduced to 50% of that at baseline at day 2-when blood pressure and plasma 25-OHD concentration were unchanged, but 25-OHD content in the kidney was 81% of that at baseline. Plasma 24,25-(OH)(2)D concentration was reduced further to 10% of that at baseline from day 7 to 14 of high salt intake, a reduction that was prevented in rats switched to a low salt (0.3%) diet at day 7. Exogenous 24,25-dihydroxycholecalciferol (24,25-(OH)(2)D(3)), administered at a level that increased plasma 24,25-(OH)(2)D concentration to five times normal, did not attenuate the salt-induced hypertension of salt-sensitive rats. Plasma 24,25-(OH)(2)D concentration of salt-resistant rats was gradually reduced to 50% of that at baseline at day 14 and returned to baseline value at day 28 of high salt intake. We conclude that the decrease in plasma 24,25-(OH)(2)D concentration in salt-sensitive rats during high salt intake is caused by decreased 25-OHD content in the kidney and also by another unidentified mechanism.

Spatial Control of Proton Pump H,K-ATPase Docking at the Apical Membrane by Phosphorylation-coupled Ezrin-Syntaxin 3 Interaction

Background: Polarized acid secretion in gastric parietal cells requires ezrin and its phosphorylation at Ser-66. Results: Phosphorylation of Ser-66 induces ezrin conformational change, which enables ezrin to interact with syntaxin 3. Conclusion: Conformational change of ezrin provides a spatial cue for apical trafficking of H,K-ATPase. Significance: Ezrin conformation orchestrates the polarized vesicle trafficking in epithelial cells. The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of a cAMP-dependent protein kinase (PKA) cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. A coupling protein is ezrin whose phosphorylation at Ser-66 by PKA is required for parietal cell activation. However, little is known regarding the molecular mechanism(s) by which ezrin operates in gastric acid secretion. Here we show that phosphorylation of Ser-66 induces a conformational change of ezrin that enables its association with syntaxin 3 (Stx3) and provides a spatial cue for H,K-ATPase trafficking. This conformation-dependent association is specific for Stx3, and the binding interface is mapped to the N-terminal region. Biochemical analyses show that inhibition of ezrin phosphorylation at Ser-66 prevents ezrin-Stx3 association and insertion of H,K-ATPase into the apical plasma membrane of parietal cells. Using atomic force microscopic analyses, our study revealed that phosphorylation of Ser-66 induces unfolding of ezrin molecule to allow Stx3 binding to its N terminus. Given the essential role of Stx3 in polarized secretion, our study presents the first evidence in which phosphorylation-induced conformational rearrangement of the ezrin molecule provides a spatial cue for polarized membrane trafficking in epithelial cells.