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ABSTRACT: The renal vasopressin V(2) receptor (V(2)R) plays a critical role in physiological and pathophysiological processes associated with arginine vasopressin (AVP)-induced antidiuresis. Because clinical data suggests that females may be more prone to hyponatremia from AVP-mediated antidiuresis, we investigated whether there are sex differences in the expression and function of the renal V(2)R. In normal Sprague-Dawley rat kidneys, V(2)R mRNA and protein expression was 2.6- and 1.7-fold higher, respectively, in females compared with males. To investigate the potential physiological implications of this sex difference, we studied changes in urine osmolality induced by the AVP V(2)R agonist desmopressin. In response to different doses of desmopressin, there was a graded increase in urine osmolality and decrease in urine volume during a 24-h infusion. Females showed greater mean increases in urine osmolality and greater mean decreases in urine volume at 0.5 and 5.0 ng/h infusion rates. We also studied renal escape from antidiuresis produced by water loading in rats infused with desmopressin (5.0 ng/h). After 5 days of water loading, urine osmolality of both female and male rats escaped to the same degree physiologically, but V(2)R mRNA and protein in female kidneys was reduced to a greater degree (-63% and -73%, respectively) than in males (-32% and -48%, respectively). By the end of the 5-day escape period, renal V(2)R mRNA and protein expression were reduced to the same relative levels in males and females, thereby abolishing the sex differences in V(2)R expression seen in the basal state. Our results demonstrate that female rats express significantly more V(2)R mRNA and protein in kidneys than males, and that this results physiologically in a greater sensitivity to V(2)R agonist administration. The potential pathophysiological implications of these results are that females may be more susceptible to the development of dilutional hyponatremia because of a greater sensitivity to endogenously secreted AVP.
AJP Renal Physiology 02/2011; 300(2):F433-40. · 4.42 Impact Factor
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ABSTRACT: Sex differences in mean arterial pressure (MAP) are reported in many experimental models of hypertension and are ascribed to gonadal sex based on studies showing that gonadectomy and gonadal hormone replacement affect MAP. The interpretation of these studies, however, has been confounded by differences in the sex chromosome complement (XX versus XY). To investigate the sex chromosome complement independent of gonadal sex, we used the 4 core genotype mouse model in which gonadal sex is separated from the sex chromosome complement enabling comparisons among XX and XY females and XX and XY males. We found that, in the gonadectomized (GDX) 4 core genotype, MAP after 2 weeks of angiotensin II infusion (200 ng/kg per minute) was greater in XX than XY (MAP [in millimeters of mercury]: GDX-XX-female, 148+/-4.5; GDX-XY-female, 133+/-4.4; GDX-XX-male, 149+/-9.4; GDX-XY-male, 138+/-5.5; P<0.03, XX versus XY; n=8 to 9 per group). In contrast, no sex chromosome effects were found on heart rate, body weight, or plasma angiotensin II 2 weeks after angiotensin II infusion. This study suggests that, in addition to effects of gonadal hormones on blood pressure, X- or Y-linked genes, parental imprinting, or X mosaicism contributes to sex differences in hypertension. Furthermore, the finding that MAP was greater in XX mice compared with XY mice in the GDX state suggests that adverse sex chromosome effects encoded within the XX sex chromosome complement could contribute to hypertension in women with ovarian hormone deficiency, such as postmenopausal women and women with premature ovarian failure.
Hypertension 03/2010; 55(5):1275-82. · 6.21 Impact Factor
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ABSTRACT: Angotensin converting enzyme 2 (ACE2) is a newly discovered monocarboxypeptidase that counteracts the vasoconstrictor effects of angiotensin II (Ang II) by converting Ang II to Ang-(1-7) in the kidney and other tissues.
ACE2 activity from renal homogenates was investigated by using the fluorogenic peptide substrate Mca-YVADAPK(Dnp)-OH, where Mca is (7-methoxycoumarin-4-yl)-acetyl and Dnp is 2,4-dinitrophenyl.
We found that ACE2 activity expressed in relative fluorescence units (RFU) in the MF1 mouse is higher in the male (M) compared to the female (F) kidney [ACE2 (RFU/min/μg protein): M 18.1 ± 1.0 versus F 11.1 ± 0.39; P < 0.0001; n = 6]. Substrate concentration curves revealed that the higher ACE2 activity in the male was due to increased ACE2 enzyme velocity (Vmax) rather than increased substrate affinity (Km). We used the four core genotypes mouse model in which gonadal sex (ovaries versus testes) is separated from the sex chromosome complement enabling comparisons among XX and XY gonadal females and XX and XY gonadal males. Renal ACE2 activity was greater in the male than the female kidney, regardless of the sex chromosome complement [ACE2 (RFU/min/μg protein): intact-XX-F, 7.59 ± 0.37; intact-XY-F, 7.43 ± 0.53; intact-XX-M, 12.1 ± 0.62; intact-XY-M, 12.7 ± 1.5; n = 4-6/group; P < 0.0001, F versus M, by two-way ANOVA]. Enzyme activity was increased in gonadectomized (GDX) female mice regardless of the sex chromosome complement whereas no effect of gonadectomy was observed in the males [ACE2 (RFU/min/μg protein): GDX-XX-F, 12.4 ± 1.2; GDX-XY-F, 11.1 ± 0.76; GDX-XX-M, 13.2 ± 0.97; GDX-XY-M, 11.6 ± 0.81; n = 6/group]. 17β-oestradiol (E2) treatment of GDX mice resulted in ACE2 activity that was only 40% of the activity found in the GDX mice, regardless of their being male or female, and was independent of the sex chromosome complement [ACE2 (RFU/min/μg protein): GDX+E2-XX-F, 5.56 ± 1.0; GDX+E2-XY-F, 4.60 ± 0.52; GDX+E2-XX-M, 5.35 ± 0.70; GDX+E2-XY-M, 5.12 ± 0.47; n = 6/group].
Our findings suggest sex differences in renal ACE2 activity in intact mice are due, at least in part, to the presence of E2 in the ovarian hormone milieu and not to the testicular milieu or to differences in sex chromosome dosage (2X versus 1X; 0Y versus 1Y). E2 regulation of renal ACE2 has particular implications for women across their life span since this hormone changes radically during puberty, pregnancy and menopause.
Biology of sex differences. 01/2010; 1(1):6.
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ABSTRACT: 17beta-Oestradiol (E2)-mediated inhibition of angiotensin-converting enzyme (ACE) protects the E2-replete kidney from the progression of hypertensive renal disease. Angiotensin-converting enzyme 2 (ACE2), a homologue of ACE, counters the actions of ACE by catalysing the conversion of angiotensin II (Ang II) to angiotensin(1-7) [Ang(1-7)]. We investigated E2 regulation of ACE2 in the renal wrap (RW) model of hypertension in rats. After 6 weeks on a high-sodium diet (4% NaCl), the activity of ACE2 was reduced in the renal cortex by 31%, which was mirrored by similar decreases in ACE2 protein (30%) and mRNA expression (36%) in the ovariectomized RW rat (RW-OVX); E2 replacement prevented these effects. The RW-OVX rats exhibited greater renal injury, including 1.7-fold more tubulointerstitial fibrosis and 1.6-fold more glomerulosclerosis than E2-replete females (RW-Intact and RW-OVX+E2). Angiotensin(1-7) infusion prevented these exacerbating effects of ovariectomy on renal pathology; no differences in indicators of renal injury were observed between RW-OVX-Ang(1-7) and RW-Intact rats. These renal protective effects of Ang(1-7) infusion were not attributable to increased ACE2 activity or to changes in heart rate or body weight, since these parameters were unchanged by Ang(1-7) infusion. Furthermore, Ang(1-7) infusion did not attenuate renal injury by reducing mean arterial pressure (MAP), since infusion of the peptide did not lower MAP but rather caused a slight increase during a 6 week chronic treatment for Ang(1-7). These results suggest that E2-mediated upregulation of renal ACE2 and the consequent increased Ang(1-7) production contribute to E2-mediated protection from hypertensive renal disease. These findings have implications for E2-deficient women with hypertensive renal disease and suggest that therapeutics targeted towards increasing ACE2 activity and Ang(1-7) levels will be renal protective.
Experimental Physiology 06/2008; 93(5):648-57. · 3.21 Impact Factor
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ABSTRACT: PET imaging has been recently introduced for investigating the type 1 angiotensin II receptor (AT(1)R) in vivo. The goal of the present study was to investigate the effects of acute and chronic exposure to angiotensin converting enzyme inhibitors (ACEI) on the AT(1)R in the dog kidney.
Animals were imaged at baseline, after acute intravenous ACEI treatment and after a chronic 2-week exposure to an oral ACEI. Control animals were imaged at identical time points in the absence of ACEI treatment.
In vivo AT(1)R binding expressed by K (i) was increased in the renal cortex by chronic ACEI treatment (p < 0.05). In vitro measurements of AT(1)R density (B (max)) also revealed significant increases in AT(1)R in isolated glomeruli (p < 0.05). Plasma renin activity was increased, but angiotensin II (Ang II) and the Ang II/Ang I ratio showed a weak correlation with chronic ACEI treatment, consistent with an Ang II escape phenomenon.
This study reveals, for the first time, that chronic ACEI treatment increases AT(1)R binding in vivo in the dog renal cortex.
European journal of nuclear medicine and molecular imaging 06/2008; 35(6):1109-16. · 4.99 Impact Factor
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ABSTRACT: The effects of high-sodium (HS) and normal-sodium (NS) diets on ovarian hormone modulation of mean arterial pressure (MAP) were examined in Dahl salt-resistant (DR) and salt-sensitive (DS) rats. Ovariectomy increased MAP (OVX-Sham) to a greater extent in DS rats maintained for 2 wk on a HS (22 mmHg) compared with a NS (6 mmHg) diet. Ovariectomy had no effect on MAP in DR rats on NS but did increase MAP in rats on HS (10 mmHg) diets. On HS diets, glomerular filtration rate (GFR) was 36% less in the DS-Sham than DR-Sham animals; ovariectomy increased GFR in both strains by 1.4-1.5-fold; glomerular angiotensin II type 1 receptor (AT(1)R) densities were 1.6-fold higher in the DS-Sham than in the DR-Sham group; ovariectomy increased glomerular AT(1)R densities by 1.3-fold in DR rats but had no effect in DS rats; 17beta-estradiol (E(2)) downregulated adrenal AT(1)R densities in both strains on either diet; ovariectomy reduced estrogen receptor-alpha (ER-alpha) protein expression in the renal cortex by 40-50% although renal ER-alpha expression was 34% lower in DS than in DR rats. These observed effects of gonadectomy were prevented by E(2) treatment, suggesting that E(2) deficiency mediates the effects of ovariectomy on MAP, GFR, AT(1)R densities, and renal ER-alpha protein expression. In conclusion, ovariectomy-induced increases in MAP are augmented by HS diet in both strains, and this effect is not mediated by a reduction in GFR. Aberrant renal AT(1)R regulation and reduced renal ER-alpha expression are potential contributors to the hypertensive effects of E(2) deficiency in DS rats. These findings have implications for women with salt-sensitive hypertension and women who are E(2) deficient, such as postmenopausal women.
AJP Heart and Circulatory Physiology 05/2008; 294(4):H1508-13. · 3.71 Impact Factor
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ABSTRACT: This study examined the effects of ovariectomy (OVX) and 17beta-estradiol (E(2)) replacement (OVX + E(2)) on renal function in Sprague-Dawley rats. OVX caused a 40% decrease in the fractional excretion of potassium (FE(K(+))) that was prevented by E(2) replacement [Sham, 24.2 +/- 2.9%; OVX, 14.5 +/- 2.1% (P < 0.05 vs. OVX + E(2)); and OVX + E(2), 26.2 +/- 2.7%; n = 7-11] and that corresponded to significant increases in plasma potassium [(in mmol/l): Sham, 3.15 +/- 0.087; OVX, 3.42 +/- 0.048 (P < 0.05 vs. OVX + E(2)); and OVX + E(2), 3.19 +/- 0.11; n = 7-11]. No effects of OVX were detected on plasma levels of sodium and aldosterone. Angiotensin II type 1 receptor (AT(1)R) densities in ovariectomized rats were 1.4-fold and 1.3-fold higher in glomerular [maximum binding capacity (B(max); in fmol/mg protein): Sham, 482 +/- 21; OVX, 666 +/- 20 (P < 0.05 vs. OVX + E(2)); and OVX + E(2), 504 +/- 26; n = 7-11] and proximal tubular [B(max) (in fmol/mg protein): Sham, 721 +/- 16; OVX, 741 +/- 24 (P < 0.05 vs. OVX + E(2)); and OVX + E(2), 569 +/- 23; n = 7-11] membranes compared with E(2) replete animals, respectively. Both the angiotensin-converting enzyme inhibitor captopril and the AT(1)R antagonist losartan prevented the OVX-induced decrease in the FE(K(+)) and the increase in renal AT(1)R densities, suggesting that E(2) deficiency reduces potassium excretion in an ANG II/AT(1)R-dependent manner. These findings may have implications for renal function in postmenopausal women as well as contribute to the reasons underlying the age-induced increase in susceptibility to hypertension-associated disease in women.
AJP Heart and Circulatory Physiology 07/2007; 293(1):H17-22. · 3.71 Impact Factor
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ABSTRACT: Several types of renal disease progress at a faster rate in men compared with women, but the reasons for this sex difference are not well understood. Chronic renal disease is associated with elevated levels of toxic reactive oxygen species (ROS). Superoxide, the major ROS in the kidney, is generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.
To determine if female protection from renal disease progression is consistent with 17beta-estradiol (E2) attenuation of superoxide production, this study was conducted to assess superoxide production in the renal cortex of male and female control and renal wrap (RW) rats, as well as in ovariectomized rats treated with vehicle or E2.
Sprague-Dawley rats were divided into 2 sham operation male (Sham-M) and female (Sham-F) control groups, and 4 RW hypertensive groups: RW-M; RW-F; RW ovariectomized females treated with vehicle (RW-OVX); and RW ovariectomized females treated with E2, supplied as a 0.24 mg/60-day release pellet (RW-OVX+E2). All groups were maintained on a high-sodium (4% NaCl) diet for 6 weeks.
Mean (SEM) markers of renal injury and oxidative stress, including urinary protein (mg/24 h: RW-M, 298 [31] vs RW-F, 169 [22]; P < 0.001), microalbuminuria (RW/Sham arbitrary units [AU]/24 h: M, 8.78 [0.58] vs F, 4.31 [1.0]; P < 0.005), and malondialdehyde (nmol/24 h: RW-M, 167 [23] vs RW-F, 117 [8.5]; P < 0.05) levels, as well as mean glomerular volume (microm3 x 10(6): RW-M, 2.25 [0.16] vs RW-F, 1.25 [0.04]; P < 0.001) and the glomerulosclerotic index (AU: RW-M, 2.64 [0.19] vs RW-F, 1.10 [0.09]; P < 0.001) were greater in both control and RW males compared with females in the same treatment groups. Though RW surgery increased mean arterial pressure in both male and female rats, no sex difference was observed. Under these conditions, mean (SEM) renal cortical NADPH oxidase activity was 1.3-fold higher in RW males compared with RW females (relative light units [RLU]/180 sec: RW-M, 4080 [240] vs RW-F, 3200 [260]; P < 0.05). Ovariectomy increased NADPH oxidase activity by 1.4-fold (RLU/180 sec: RW-OVX, 4520 [184]; P < 0.01) under conditions in which the mean glomerular volume and glomerulosclerotic index were both increased by 1.5-fold, whereas E2 replacement (RLU/180 sec: RW-OVX+E2, 2745 [440]) prevented these effects. Furthermore, the effects on NADPH oxidase activity were mirrored by changes in the protein abundance of NADPH oxidase subunit p22P(phox).
These results suggest that E2 protects the female kidney in part by attenuating injury-induced increases in renal superoxide production.
Gender Medicine 04/2007; 4(1):56-71. · 2.10 Impact Factor
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ABSTRACT: The adrenal mineralocorticoid aldosterone promotes sodium (Na(+)) reabsorption and potassium (K(+)) loss from the kidney. Female sex steroids such as estrogen and progesterone are known modulators of the renin-angiotensin-aldosterone system.
We conducted studies to determine if there is a sex difference in plasma Na(+) concentration ([Na(+)]) and plasma K(+) concentration ([K(+)]), and if interactions between female sex steroids and aldosterone contribute to a sex difference in these electrolytes.
Plasma [Na(+)] and [K(-)] were determined in weight-matched male and female Sprague-Dawley rats using an ion-selective electrode system. To assess the sensitivity of males and females to aldosterone, the mineralocorticoid was infused chronically by osmotic minipump. The role of female sex steroids in the regulation of plasma electrolyte concentrations was determined in bilaterally ovariectomized (OVX) female rats treated daily with SC injections of progesterone, 17beta-estradiol (E(2)), or selective estrogen receptor (ER) modulators. The role of plasma [K(+)] in the regulation of adrenal angiotensin II type 1 receptor (AT(1)R) expression was determined by manipulating plasma [K(+)] by varying dietary K(-). Adrenal AT(1)R expression was assessed using a radioligand binding assay.
Plasma [Na(-)] was not different between male and female rats, but plasma [K(-)] was reduced in females compared with males (P = 0.003). In aldosterone-infused female rats, plasma [Na(+)] was increased and plasma [K(+)] was reduced further than in male rats infused with aldosterone (both, P = 0.001). In OVX female rats, progesterone reduced plasma [Na(+)] (P = 0.04) but had no effect on plasma [K(+)]. In contrast, E(2) increased plasma [Na(+)] (P = 0.01) and reduced plasma [K(+)] (P = 0.001). Dietary K supplementation in E(2)-treated rats returned plasma [K(+)] and adrenal AT(1)R binding to levels observed in control rats. Both an ERa and ERP agonist decreased plasma [K(+)] and decreased adrenal AT(1)R binding (both, P < 0.01).
In these studies, plasma [K(+)] was reduced in female Sprague-Dawley rats compared with males. The effects of aldosterone on plasma electrolytes were enhanced in females compared with males. E(2) treatment reduced plasma [K(+)] and adrenal AT(1)R binding in OVX rats, and the decrease in plasma [K(+)] contributed to the decrease in adrenal AT(1)R binding. Both ERalpha and ERbeta contributed to the estrogen-induced decrease in plasma [K(+)] and adrenal AT(1)R binding.
Gender Medicine 04/2006; 3(1):43-53. · 2.10 Impact Factor
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ABSTRACT: Neutral endopeptidase (NEP) inhibition attenuates renal damage in the diabetic kidney, but little is known about the mechanisms of this renoprotective effect.
We examined the interaction between angiotensin II (Ang II) and atrial natriuretic peptide (ANP) under low (5 mM) and high (30 mM) glucose conditions, on cell proliferation and extracellular matrix (ECM) synthesis in renomedullary interstitial cells (RMICs) derived from wild-type (WT) and NEP-deficient (NEP-) mice.
Under high glucose conditions, Ang II (10(-6)M) increased cell proliferation (control, 174.3 +/- 16.9; Ang II, 846.3 +/- 91.0 cpm/well) and ECM synthesis (control, 22.3 +/- 3.1; Ang II, 79.0 +/- 9.6 cpm/cell) in RMICs derived from WT and NEP- mice to a similar extent. ANP (10(-7)M) reduced Ang II-induced cell proliferation and ECM synthesis in RMICs derived from both strains, but more efficiently in RMICs derived from NEP- mice. The Ang II-induced cell proliferation and ECM synthesis was attenuated with AT1 receptor blockade, but more efficiently in RMICs-derived NEP- mice.
This data shows that ANP and AT1 receptor blockade attenuate Ang II-induced RMIC proliferation and ECM synthesis more efficiently in the absence of NEP. These results support the concept that NEP inhibition is beneficial in attenuating abnormal cell growth and ECM metabolism associated with diabetic nephropathy.
Nephron Physiology 02/2006; 103(3):p149-56. · 2.55 Impact Factor
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ABSTRACT: The current study examined angiotensin receptor (ATR) regulation in proliferating rat aortic vascular smooth muscle cells (VSMCs) in culture. Radioligand competition analysis coupled with RNase protection assays (RPAs) revealed that angiotensin type 1a receptor (AT(1a)R) densities (B(max)) increased by 30% between 5 and 7 days in culture [B(max) (fmol/mg protein): day 5, 379 +/- 8.4 vs. day 7, 481 +/- 12, n = 3, P < 0.05] under conditions in which no significant changes in AT(1a)R mRNA expression occurred [in RPA arbitrary units (AU): day 5, 0.23 +/- 0.01 vs. day 7, 0.24 +/- 0.04, n = 4] or in mRNA synthesis determined by nuclear run-on assays [AU: day 5, 0.35 +/- 0.14 vs. day 7, 0.33 +/- 0.11, n = 5]. In contrast, polysome distribution analysis indicated that AT(1a)R mRNA was more efficiently translated in day 7 cells compared with day 5 [% of AT(1a)R mRNA in fraction 2 out of total AT1R mRNA recovered from the sucrose gradient: day 5, 20.9 +/- 9.9 vs. day 7, 56.8 +/- 5.6, n = 3, P < 0.001]. Accompanying the polysome shift was 50% less RNA-protein complex (RPC) formation between VSMC cytosolic RNA binding proteins in day 7 cells compared with 5-day cultures and the 5' leader sequence (5'LS) of the AT(1a)R [5'LS RPC (AU): day 5, 0.62 +/- 0.15 vs. day 7, 0.23 +/- 0.03; n = 4, P < 0.05] and also with exon 2 [Exon 2 RPC (AU): day 5, 35.0 +/- 5.7 vs. day 7, 17.2 +/- 3.6; n = 4, P < 0.05]. Taken together, these results suggest that AT(1a)R expression is regulated by translation during VSMC proliferation in part by RNA binding proteins that interact within exon 2 in the 5'LS of the AT(1a)R mRNA.
AJP Regulatory Integrative and Comparative Physiology 02/2006; 290(1):R50-6. · 3.34 Impact Factor
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ABSTRACT: The antidiuretic effects of arginine vasopressin (AVP) on the kidney are mediated by V2 subtype AVP receptors (V2R). To investigate the role of regulation of V2R in water and sodium homeostasis, we have developed a method for small interfering RNA (siRNA)-mediated inhibition of V2R expression in vivo. Three 21-nt siRNA sequences were chosen that specifically targeted the mouse V2R but shared no appreciable sequence homology to any other known mouse genes, including the vasopressin V1a and V1b receptors. Additionally, an siRNA sequence that shared no significant matches to any known mammalian gene sequences was chosen for use as a control. Chemically synthesized siRNA was complexed with the liposomal transfection reagent DOTAP. Each mouse (male C57BL/6) received 3.6 nmol (approximately 50 microg) of either the control (nonsilencing) or one of the V2R-targeting siRNAs via intravenous injection. Forty-eight hours after injection membranes were prepared from the inner medulla of the kidneys, and V2R expression was measured by a radioligand binding assay and Western immunoblotting. Treatment with one of the V2R-targeting siRNAs (R2) caused a 39.7 +/- 8.7% reduction in V2R-specific binding compared with the control (n = 11, P < 0.05) and a 37.0 +/- 2.3% reduction in V2R protein expression as measured by Western immunoblotting (n = 4, P < 0.001). Additionally, real-time PCR revealed that R2 siRNA treatment induced a 68.8 +/- 2.2% reduction in V2R mRNA. However, this siRNA treatment did not alter the animals' basal urine concentrating capacity under unstimulated conditions. In subsequent experiments, treatment with R2 siRNA was found to significantly attenuate the antidiuretic effects the V2R-specific AVP agonist 1-desamino-[8-D-arginine]vasopressin (dDAVP). Mice were infused with dDAVP (0.25 ng/h) for 3 days to produce maximal antidiuresis and then were injected with either the R2 siRNA or the nonsilencing control. On day 2 after treatment, urine osmolality was significantly decreased from 3,455 +/- 72 in control animals (n = 12) to 3,155 +/- 129 mosmol/kgH2O in R2 siRNA-treated animals (n = 12) (P < 0.05); similarly, on day 2 24-h urine volume was significantly increased from 0.86 +/- 0.07 ml/day to 1.11 +/- 0.06 ml/day in R2 siRNA-treated animals (P < 0.05). In summary we have demonstrated that RNA interference methodology can be used successfully in vivo to significantly reduce functional expression of the V2R in the mouse kidney.
Physiological Genomics 05/2005; 21(3):382-8. · 2.73 Impact Factor
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ABSTRACT: Renal injury is greater in male compared with female rats after renal wrap (RW) hypertension. We investigated the role of gonadal steroids in the sex differences in RW disease severity in male (M) and female (F), castrated (Cast), and ovariectomized (OVX) rats and after dihydrotestosterone (DHT) and 17beta-estradiol (E2) treatment. Male castration attenuated the severity of RW-induced glomerulosclerosis (GS) [GS index (GSI): RW-M, 2.1 +/- 0.2; RW-Cast, 1.3 +/- 0.2; RW-Cast+DHT, 2.4 +/- 0.4], mean glomerular volume (MGV; microm3 x 10(6): RW-M, 1.9 +/- 0.1; RW-Cast, 1.45 +/- 0.15; RW-Cast+DHT, 1.91 +/- 0.15), tubular damage, and proteinuria (mg/day: RW-M, 130 +/- 8; RW-Cast, 105 +/- 5; RW-Cast+DHT, 142 +/- 9), whereas DHT treatment abrogated these effects. Ovariectomy increased the GSI (RW-F, 0.69 +/- 0.05; RW-OVX, 1.2 +/- 0.1; RW-OVX+E2, 0.65 +/- 0.05), tubular damage, and MGV (microm3 x 10(6): RW-F, 1.0 +/- 0.06; RW-OVX, 1.5 +/- 0.05; RW-OVX+E2, 0.96 +/- 0.06), whereas E2 treatment prevented these effects. Furthermore, DHT treatment of RW-OVX animals exacerbated the GSI (1.9 +/- 0.19), MGV (1.7 +/- 0.2 x 10(6) microm3), and proteinuria (171 +/- 21 mg/day) even further. Our data show that the lack of E2 and presence of androgens contribute to progressive renal disease induced by RW hypertension, suggesting that gonadal steroid status is an independent factor in the greater susceptibility men exhibit toward hypertension-associated renal disease compared with women.
American journal of physiology. Renal physiology 04/2005; 288(3):F513-20. · 3.68 Impact Factor
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ABSTRACT: To investigate the faster rate of renal disease progression in men compared with women, we addressed the following questions in the renal wrap (RW) model of hypertension: 1) Do sex differences exist in RW-induced renal injury, which are independent of sex differences in blood pressure? 2) Do sex differences in nitric oxide (NO) production exist in RW hypertension? Male (M) and female (F) rats underwent sham-operated (M-Sham, n = 7; F-Sham, n = 10) or RW (M-RW, n = 13; F-RW, n = 14) surgery for 9 wk. Markers of renal injury, including the glomerulosclerosis index (F-RW, 0.70 +/- 0.1 vs. M-RW, 2.2 +/- 0.6; P < 0.05), mean glomerular volume (F-RW, 1.05 +/- 0.050 x 10(6) vs. M-RW, 1.78 +/- 0.15 x 10(6) microm(3); P < 0.001), and proteinuria (F-RW, 68.7 +/- 15 vs. M-RW, 124 +/- 7.7 mg/day; P < 0.001) were greater in RW males compared with RW females. Endothelial NO synthase protein expression was elevated in the renal cortex (3.2-fold) and medulla (2.2-fold) 9 wk after RW in males, whereas no differences were observed in females. Neuronal NO synthase protein expression was unchanged in the renal cortex in males and in both the renal cortex and medulla in females, whereas in the male medulla, neuronal NOS was decreased by 57%. These data suggest the degree of renal injury is greater in male compared with female rats in RW hypertension despite similar degrees of hypertension and renal function and may involve sex differences in renal NO metabolism.
AJP Heart and Circulatory Physiology 01/2005; 288(1):H43-7. · 3.71 Impact Factor
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ABSTRACT: The rat angiotensin type 1a receptor (AT1aR) is comprised of three exons. Two transcripts are possible due to alternative splicing of exon 2 (E1,3 and E1,2,3). Both transcripts code for identical AT1aR proteins since they differ only in the length of their 5' leader sequence (5'LS). We investigated the functional differences of these two transcripts in stably transfected Chinese hamster ovary (CHO) cells and also determined the splice variant composition in rat tissues. E1,3 expressing cells exhibited 1.8-fold higher AT1R densities and five-fold higher levels of Ang II-stimulated inositol phosphate production compared to E1,2,3 expressing cells. No differences in E1,3 and E1,2,3 mRNA levels or mRNA stability were seen. In vitro translation assays revealed 1.8-fold higher AT1aR protein levels from E1,3 compared to E1,2,3 transcripts, suggesting exon 2 reduces functional AT1R expression by inhibiting translation. Deletion of 10 nucleotides in exon 2 increased translation of the mutated E1,2,3 transcript to levels which were indistinguishable from E1,3, suggesting that this loop region of a predicted hairpin contributes to the inhibitory RNA cis element within exon 2. Comparison of AT1aR exonic composition and AT1R densities in rat tissues suggests alternative splicing is regulated in a tissue-specific manner and contributes to tissue-specific differences in AT1R density.
Gene 11/2004; 341:93-100. · 2.34 Impact Factor
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ABSTRACT: The ovariectomized (OVX) Dahl salt-sensitive (DS) rat fed a low-salt diet is a model of postmenopausal hypertension. In addition to estrogen loss, aging can also contribute to postmenopausal hypertension. We hypothesized that: (1) female DS rats on a low-salt diet become hypertensive with age; (2) ovariectomy accelerates age-dependent hypertension in the DS rat caused by estrogen depletion; and (3) this hypertension correlates with increased type 1 angiotensin receptor (AT1R) number (Bmax). Blood pressure was monitored by telemetry from 3 to 12 months and AT1R Bmax was determined by Scatchard analysis in glomeruli and adrenal cortex. Three groups of DS rats were studied: intact, OVX, and 17beta-estradiol-replaced OVX (OVX+E). In intact rats, aging to 12 months resulted in hypertension (159+/-6 mm Hg) and an 82% decrease in estrogen. Blood pressure in OVX was significantly higher than OVX+E through 12 months of age (173+/-4 versus 150+/-8 mm Hg). At 4 months, OVX increased AT1R Bmax compared with intact and OVX+E in both glomeruli and adrenal cortex. Aging also increased AT1R Bmax in these tissues in intact rats. In summary, female DS rats fed a low-salt diet have hypertension develop with age, that is accelerated by OVX and attenuated by estrogen replacement. Concurrently, AT1Rs are upregulated by age and OVX, which is prevented by estrogen replacement. This study suggests that an increased activity of the renin angiotensin system contributes to the development of hypertension, and estrogen protects against this process.
Hypertension 11/2004; 44(4):405-9. · 6.21 Impact Factor
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ABSTRACT: Rat angiotensin type 1a receptor (AT(1a)R) is regulated by four upstream AUGs present in the 5' leader sequence (5'-LS). Disruption of all four upstream AUGs (QM) results in 2-3-fold higher levels of angiotensin type 1 receptor (AT(1)R) densities in transiently transfected rat aortic smooth muscle cells (A10 cells) and stably transfected Chinese hamster ovary cells. Cells expressing QM have 5-fold higher levels of angiotensin II-induced inositol phosphate production than wild type (WT). Polysome analysis showed that QM mRNA is present in heavier fractions than the WT transcript, and 5.7-fold more AT(1)R protein is produced by in vitro translation from QM transcripts compared with WT transcripts. The AT(1a)R comprises 3 exons. Exon 3 (E3) encodes the entire open reading frame and 3'-untranslated region. Exons 1 and 2 (E1 and E2) and 52 nucleotides of E3 encode the 5'-LS. The AUGs in both exons contribute to the inhibitory effect on AT(1)R expression but not to the same degree. Disruption of the AUGs in exon 2 (DM2) relieves half of the inhibition, whereas disruption of the AUGs in exon 1 (DM1) is without effect. Disruption of the AUGs in exon 2 results in levels of receptor expression and translation that are indistinguishable from the alternative splice variant E1,3, which we previously showed was more efficiently translated than the E1,2,3 transcript. Individual mutations revealed that only the fourth AUG increased AT(1)R translation. In conclusion, all four AUGs present in the 5'-LS function cumulatively to suppress AT(1a)R expression and signaling by inhibiting translation. These data also show that both AUGs in E2 contribute to the inhibitory cis element present in this alternatively spliced exon.
Journal of Biological Chemistry 11/2004; 279(44):45322-8. · 4.77 Impact Factor
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ABSTRACT: Sex differences exist in the mechanisms initiating early compensatory renal growth after unilateral nephrectomy (UNX); remnant kidney growth is growth hormone (GH) independent in adult female rats and GH dependent in adult male rats. The present study determined whether sex differences also exist in angiotensin type 1 receptor (AT1R) regulation during early remnant kidney (REM) growth after UNX, and if so, whether GH modulates AT1R expression after UNX in the male rat. Scatchard analysis of radioligand binding in glomeruli demonstrated that 48 h post-UNX, AT1R density (Bmax) was significantly decreased by 20% in female REM compared with control kidneys. In contrast, male REM glomerular Bmax was significantly increased by 28% compared with control kidneys. Furthermore, GH-suppressed male rats displayed attenuated REM growth, which was associated with a 35% decrease in AT1R Bmax. Losartan treatment also decreased REM AT1R Bmax by 55%. The activity of mRNA binding proteins that bind to the 5' leader sequence of the AT1R was regulated by UNX and GH treatment in an inverse manner to AT1R expression. These findings suggest that in rats 1) there are sex differences in the regulation of glomerular AT1R expression after UNX; 2) the increase in AT1R binding sites in the male REM is regulated by GH and mediates early remnant kidney growth; and 3) AT1R 5' leader sequence mRNA binding proteins play a role in UNX and GH regulation of glomerular AT1Rs in both males and females.
American journal of physiology. Renal physiology 01/2004; 285(6):F1085-91. · 3.68 Impact Factor
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ABSTRACT: Hypertension and associated cardiovascular disease increase after menopause. Angiotensin AT(1) receptor (AT(1)R) antagonists are effective treatments, in part, by inhibiting angiotensin II (Ang II)-induced aldosterone release from the adrenal zona glomerulosa (ZG). Estrogen decreases the number of AT(1)Rs in the adrenal gland and attenuates acute Ang II-induced aldosterone release. Here, we examined the effects of 17beta-estradiol (E(2)) on AT(1)R gene regulation in the rat adrenal cortex (AC). Female rats were ovariectomized and injected with vehicle or E(2). Immunohistochemistry revealed the presence of both estrogen receptor (ER)alpha and ERbeta in the ZG, and E(2) treatment increased the intensity of their nuclear staining. Under conditions in which AT(1)R maximal binding capacity was decreased by 46%, chronic miniosmotic pump Ang II-induced aldosterone secretion was reduced by 43%. E(2) treatment had no effect on AT(1a)R and AT(1b)R mRNA levels in the AC, whereas the AT(1)R mRNA polysome distribution in sucrose gradients was shifted to lighter fractions, indicating that E(2) treatment reduces AT(1)R translation. RNA binding proteins (RBPs) in AC extracts formed complexes with the 5' leader sequence (5'LS), coding region, and the 3'-untranslated region (3'UTR); however, only the activity of 5'LS RBPs was regulated by E(2) treatment. These data suggest that E(2), acting through its receptors in the ZG, reduces AT(1)R density and Ang II-induced aldosterone release, primarily by inhibiting AT(1)R translation, possibly by blocking ribosomal scanning caused by increased steric hindrance from 5'LS RBPs. Dysregulation of this posttranscriptional mechanism may contribute to the increased incidence of cardiovascular disease associated with menopause.
Endocrinology 08/2003; 144(7):3251-61. · 4.46 Impact Factor
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ABSTRACT: Estrogen inhibits adrenal angiotensin type 1 receptor (AT(1)R) binding sites and attenuates the adrenal responsivity to angiotensin II (Ang II). Ang II modulates AT(1)R expression. Here, we determined if estrogen-induced down-regulation of adrenal AT(1)Rs involves modulation of adrenal Ang II. Female rats were ovariectomized (OVX) and injected with 17beta-estradiol benzoate (E(2); 40 micro g/kg) or vehicle for 7 d. Adrenal Ang II was separated from other angiotensin peptides by HPLC and measured by RIA. Scatchard analysis of radioligand binding curves showed that E(2) or captopril (Cap; 0.5 g/liter water) significantly reduced adrenal AT(1)R binding (maximum binding capacity) by 22% and 19%, respectively, compared with OVX (276 +/- 2.09 fmol/mg protein). E(2) and Cap lowered adrenal Ang II levels by 39% and 21%, respectively, compared with OVX (4.10 +/- 0.44 pmol/g). E(2) caused no further reductions in adrenal AT(1)R binding or in Ang II levels in Cap-treated OVX rats. High-dose Ang II infusion (1000 ng/kg.min) increased adrenal Ang II levels by 71% and lowered AT(1)R binding by 18%. Under these infusion conditions, E(2) did not reduce adrenal Ang II or AT(1)R binding. No differences in AT(1)R affinity (dissociation constant) were observed among groups. These data suggest that E(2) regulates the number of adrenal AT(1)R binding sites indirectly by modulating adrenal Ang II.
Endocrinology 05/2003; 144(4):1350-6. · 4.46 Impact Factor
