Angiotensinogen Expression Research Articles

IUGR in rats is associated with changes in placental expression of MTAs

IUGR in rats is associated with changes in placental expression of MTAs

Mechanical stretch induces angiotensinogen expression through PARP1 activation in kidney proximal tubular cells.

Poly(ADP-ribose) polymerase 1 (PARP1) contributes to fibrosis in several disease models. Recent in vivo data indicate that loss of PARP1 attenuates renal fibrosis and inflammation independent on transforming growth factor-β (TGF-β); however, the role of PARP1 in kidney tubular cells in response to tubulointerstitial fibrosis remains to be defined. Here, we report that PARP1 activation after mechanical stretch of kidney proximal tubular cells enhances angiotensinogen expression via nuclear factor kappa B (NF-κB) activation. Mechanical stretch for 24 h increased PARP1 expression and activation in mouse cortical proximal tubular (MCT) cells. Treatment with 3-aminobenzamide, a PARP1 inhibitor, efficaciously reduced the PARP1 activation induced by mechanical stretch. PARP1 inhibition also reduced angiotensinogen expression and NF-κB p65 phosphorylation induced by mechanical stretch. TGF-β1 expression and secretion were enhanced by mechanical stretch, but PARP1 inhibition did not change the levels of TGF-β1. These data demonstrate that mechanical stretch-induced PARP1 activation contributes to angiotensinogen expression and NF-κB activation in kidney proximal tubular cells, resulting in the promotion of renal tubulointerstitial fibrosis and inflammation.

Activation of the renin-angiotensin system, specifically in the subfornical organ is sufficient to induce fluid intake.

Increased activity of the renin-angiotensin system within the brain elevates fluid intake, blood pressure, and resting metabolic rate. Renin and angiotensinogen are coexpressed within the same cells of the subfornical organ, and the production and action of ANG II through the ANG II type 1 receptor in the subfornical organ (SFO) are necessary for fluid intake due to increased activity of the brain renin-angiotensin system. We generated an inducible model of ANG II production by breeding transgenic mice expressing human renin in neurons controlled by the synapsin promoter with transgenic mice containing a Cre-recombinase-inducible human angiotensinogen construct. Adenoviral delivery of Cre-recombinase causes SFO-selective induction of human angiotensinogen expression. Selective production of ANG II in the SFO results in increased water intake but did not change blood pressure or resting metabolic rate. The increase in water intake was ANG II type 1 receptor-dependent. When given a choice between water and 0.15 M NaCl, these mice increased total fluid and sodium, but not water, because of an increased preference for NaCl. When provided a choice between water and 0.3 M NaCl, the mice exhibited increased fluid, water, and sodium intake, but no change in preference for NaCl. The increase in fluid intake was blocked by an inhibitor of PKC, but not ERK, and was correlated with increased phosphorylated cyclic AMP response element binding protein in the subfornical organ. Thus, increased production and action of ANG II specifically in the subfornical organ are sufficient on their own to mediate an increase in drinking through PKC.

Aliskiren in early postnatal life prevents hypertension and reduces asymmetric dimethylarginine in offspring exposed to maternal caloric restriction

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is involved in hypertension. We tested whether aliskiren treatment in early postnatal life can reduce ADMA and regulate the renin-angiotensin system to prevent hypertension in rat offspring exposed to maternal caloric restriction (CR). Four groups of 12-week-old male offspring were sacrificed: control, CR, CR+aliskiren, and CR+losartan group. The CR group included offspring from 50% food-restricted maternal rats. The CR+aliskiren and CR+losartan groups were produced by treating CR offspring with oral aliskiren 10 mg/kg/day or losartan 20 mg/kg/day between 2-4 weeks of age, respectively. Blood pressure increased in CR rats, which was prevented by aliskiren or losartan. CR increased plasma ADMA levels, which aliskiren prevented. Renal renin and prorenin receptor (PRR) expression increased in CR rats treated with aliskiren, whereas both were reduced by losartan. Both aliskiren and losartan decreased renal mRNA expression of angiotensinogen, angiotensin II type 2 receptor, and Mas in CR rats. However, aliskiren increased angiotensin II type 2 receptor and Mas protein levels in CR kidneys. Early aliskiren therapy prevents CR-induced hypertension via ADMA reduction, decreases angiotensinogen expression, and increases renal angiotensin II type 2 receptor and Mas protein.

P54 Hydrogen sulfide inhibits activation of the renin-angiotensin system in diabetic heart

Diabetic cardiomyopathy is a major contributing factor to morbidity in diabetics. A significant role of the cardiac renin-angiotensin system (RAS) in diabetic cardiomyopathy has been demonstrated clinically. Plasma levels of hydrogen sulfide (H 2 S), which have been demonstrated to inhibit plasma renin and endothelial ACE activity, are reduced in diabetic patients. The interaction between H 2 S and the RAS in the heart is unclear. We hypothesize that cardiac H 2 S levels are reduced in diabetes, resulting in cardiac RAS activation. Type 1 diabetes was induced in mice by streptozotocin. We observed that the expression of cystathionine gamma-lyase (CGL) in the heart was reduced significantly as diabetes progressed, whereas the cardiac RAS was activated. By using a fluorescent probe specific for H 2 S, we determined that H 2 S production was significantly impaired in neonatal rat ventricular myocytes (NRVM) cultured in high glucose medium (HG, 30 mM). In addition, DL-propargylglycine (PAG), a CGL inhibitor, increased the expression of angiotensinogen (AGT) in NRVM, suggesting that endogenous H 2 S is inhibitory to the cardiac RAS. HG significantly upregulated AGT expression in NRVM, which was further potentiated by PAG. GYY4137, a slow H 2 S releasing compound, completely abolished the effect of HG on RAS activation. Currently, we are using the transgenic mouse with cardiac specific over-expression of CGL to confirm these findings. In conclusion, downregulation of H 2 S synthesis may contribute to cardiac RAS activation in diabetes. Replenishing the H 2 S level in the diabetic heart, using an H 2 S donor, might provide a potential treatment for diabetic cardiomyopathy.

Effect of the renin inhibitor aliskiren against retinal ischemia-reperfusion injury

The purpose of this study was to investigate the effect of the renin inhibitor, aliskiren, on retinal ischemia-reperfusion injury. Retinal ischemia was induced by increasing intraocular pressure to 130 mmHg. At 7 days after ischemia, retinal damage was evaluated by measuring the retinal thickness and the number of retinal ganglion cells. Western blot was used to measure changes in the (pro)renin receptor expression. Retinal mRNA expressions of prorenin, angiotensinogen and angiotensin II type 1 receptor (AT1-R) were measured by real-time polymerase chain reaction. Rats were treated with the renin inhibitor, aliskiren. Although the number of retinal ganglion cells and the inner retinal thickness were significantly decreased at 7 days after ischemia, treatment with aliskiren significantly inhibited retinal ischemic injury. Administration of aliskiren increased mRNA expression of prorenin in the retina at 3 h after the reperfusion. The expression of the (pro)renin receptor was not changed after ischemia-reperfusion injury with or without aliskiren. Although there was an increase in the retinal expression of AT1-R at 3 h after the reperfusion, aliskiren administration suppressed this expression. A renin inhibitor attenuated subsequent ischemic damage in the rat retina via the inhibition of the prorenin-induced angiotensin generation.

Augmentation of kidney angiotensinogen expression and urinary angiotensinogen excretion by an intracellular angiotensin II fusion protein (1173.8)

Long‐term angiotensin II (ANG II) administration significantly increases ANG II levels in the kidney through two major mechanisms; AT1 receptor‐mediated augmentation of angiotensinogen (AGT) expression and uptake of circulating ANG II by the proximal tubule. However, it is not known if intracellular ANG II also stimulates AGT expression and secretion in the proximal tubule of the kidney. In the present study, we performed intrarenal adenoviral transfer of an intracellular cyan fluorescent fusion of ANG II (Ad‐sglt2‐ECFFP/ANG II) selectively in the proximal tubule using the sodium and glucose co‐transporter 2 (sglt2) promoter. AGT mRNA expression in the renal cortex and 24 h urinary AGT excretion were determined 2 and 4 weeks after ECFP/ANG II transfer. ECFP/ANG II was expressed in the proximal tubule in a time‐dependent manner, which was associated with significant increases in blood pressure 4 weeks after ECFP/ANG II transfer (p<0.01). AGT mRNA expression in the cortex was significantly increased at Week 4 by 61 ± 16% (p<0.05). Urinary AGT excretion rates were increased from 48.7 ± 5.7 (n=13) to 102 ± 13.5 (n=13) ng/24 h (p<0.05). However, plasma AGT levels were not altered. These results support the hypothesis that intracellular ANG II stimulates AGT expression in the proximal tubule leading to increased AGT formation and secretion into the tubular fluid.Grant Funding Source: Supported by P30GM10337 and RO1DK067299

The brain renin‐angiotensin system suppresses digestive efficiency (1126.13)

The renin‐angiotensin system (RAS) contributes to metabolic control through local actions within the brain and adipose. All components of the RAS are also expressed in the gastro‐intestinal (GI) tract, leading to the hypothesis that the RAS controls digestive efficiency. Mice with transgenic hyperactivity of the brain RAS (sRA) were generated through neuron‐specific expression of human renin, and expression of human angiotensinogen via its own promoter. sRA exhibit reduced body mass and elevated resting metabolic rate through elevated sympathetic nervous activity and a chronic suppression of the circulating RAS. Caloric densities of food/feces were assessed by bomb calorimetry. sRA had increased caloric intake (15.4±0.7 kcal/day, n=8) vs littermate control mice (13.0±0.7, n=7, P<0.01) as well as an increase in caloric loss to feces (6.3±0.8 vs 2.9±0.4 kcal/day, P<0.01). Thus, sRA had decreased digestive efficiency (59±5 vs 78±2 %, P<0.01) versus littermates on chow diet (Teklad 7013), but total daily caloric absorption (9.1±0.8 vs 10.0±0.5 kcal/day, P=0.36) was normal. Infusion of the angiotensin (Ang) II type 2 receptor agonist CGP‐42112a (50 ng/kg/min, s.c., 8 weeks) had no effect on efficiency or absorption (sRA 63±6 vs control 80±1 %; 9.3±0.8 vs 9.8±1.0 kcal/day). We conclude that the brain and circulating RAS help control digestive efficiency, and hypothesize a role for GI tract Ang II type 1 receptors.Grant Funding Source: Supported by NIH: HL098276, HL084207.

Eicosapentaenoic acid regulates brown adipose tissue gene expression and metabolism in high fat fed mice (1037.5)

Brown adipose tissue (BAT) is a thermogenic tissue, a key regulator of energy balance and a potential therapeutic target for obesity. We previously reported that eicosapentaenoic acid (EPA) reduced high fat (HF) diet‐induced obesity and insulin resistance in mice, independent of energy intake. We hypothesized that these effects are mediated in part by BAT thermogenesis. Using mice fed HF or HF‐EPA diets for 11 weeks, we demonstrated that BAT from HF‐EPA mice expressed higher mRNA levels of thermogenic genes such as fibronectin type lll domain containing 5 (FNDC5), peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1α) and uncoupling protein 3 (UCP3), compared to HF mice. EPA also induced expression of angiotensinogen (Agt) gene and other genes related to insulin sensitivity such as Glut 1 while downregulating arachidonate 5‐lipoxygenase (ALOX5), an inflammatory biomarker. At the protein level, EPA upregulated uncoupling protein (UCP) and downregulated fatty acid synthase (FAS). Thus, EPA exerts dual effects on adipose tissue depots, by reducing WAT inflammation and lipid accumulation, while activating BAT thermogenesis and reducing lipogenesis. In conclusion, EPA exerts differential tissue specific effects to reduce obesity‐associated metabolic disorders. Further molecular studies in cultured brown adipocytes are being conducted to dissect direct effects of EPA on brown fat.Grant Funding Source: Supported by AHA, USDA and TTU

Effect of Ang II on angiotensinogen expression levels in hypertensive rat astrocytes (1140.1)

Objective: To investigate the effects of Angiotensin II (Ang II) on Angiotensinogen (AGT) mRNA and protein levels in spontaneous hypertensive rats (SHR) astrocytes. Background: AGT is the precursor molecule for the synthesis of Ang II produced by the renin angiotensin system (RAS). Astrocytes are the major source of AGT in the brain, and regulation of brain AGT levels alters blood pressure and electrolyte homeostasis. In this study, SHR were used as a genetic hypertension model and the results were compared to normotensive Wistar rats. Methods: Astrocytes isolated from neonatal rat’s cerebellum (CB) and brainstem (BS) regions were treated with 100nM Ang II, and the time‐dependent effect of Ang II on AGT protein and mRNA levels were measured using Western blotting and quantitative polymerase chain reaction techniques, respectively. Results: Ang II significantly increased AGT protein levels but, decreased AGT mRNA levels over most time points examined in BS and CB Wistar and SHR astrocytes. There was no significant difference between Ang II’s ability to affect AGT protein and mRNA levels in SHR as compared to Wistar astrocytes. Conclusions: These findings showed that Ang II regulates it’s synthesis by inducing basal AGT protein and mRNA levels. Further, the ability of astrocytes to produce AGT may not be contributing to the hypertensive state in the SHR model. Grant Funding Source: Supported by Nova Southeastern University Chancellor's Faculty Research & Development Grant # 335872

Interleukin 6 derived from macrophages enhances angiotensinogen expression in renal proximal tubular cells (1173.3)

Chronic angiotensin II (Ang II) infusion leads to hypertension, intrarenal renin‐angiotensin system activation, stimulation of proximal tubular AGT and renal MΦ infiltration. Treatment with immunosuppressive drugs reduces MΦs infiltration, intrarenal Ang II levels and blood pressure, suggesting that MΦ contribute to intrarenal RAS activation and hypertension. However, the contribution of MΦ to AGT augmentation has not been delineated. To elucidate the role of MΦ in AGT augmentation, cultured rat MΦ were treated with 0.05‐5 μM Ang II up to 48 h. After removal of MΦ, the culture medium was incubated with rat PTC for 24 h. mRNA levels of interleukin 6 (IL‐6) in MΦ and AGT in PTC were evaluated by real‐time RT‐PCR. Treatment with 5 μM Ang II activated p38 MAP kinase and increased IL‐6 expression (2.65 ± 0.11, ratio to control) at 48 h; this response was attenuated by AT1 receptor blockade with olmesartan. Incubation of PTC with the culture medium of Ang II‐stimulated MΦ increased AGT expression in PTC (1.71 ± 0.49 by 5 μM Ang II). A neutralizing IL‐6 antibody added to culture medium attenuated the AGT augmentation in PTC (1.17 ± 0.27). These results demonstrate that Ang II mediated increases in IL‐6 production by MΦ increase AGT expression in PTC, suggesting that the Ang II mediated accumulation of MΦ in the kidney stimulates IL‐6 production in the MΦ, which augments intrarenal AGT expression contributing to hypertension.

Role for histone deacetylases in regulating angiotensinogen expression mediated by the JAK‐STAT pathway by suppressor of cytokine signaling in renal proximal tubular cells (1173.2)

Activated JAK‐STAT pathway stimulates SOCS expression, which inhibits JAK‐STAT pathway as a negative‐feedback system. Elevated SOCS limits AGT augmentation in PTC, suggesting that induction of SOCS elevation is an effective strategy to treat the renin‐angiotensin system (RAS) associated diseases. Thus, establishment of an approach to elevate SOCS without activation of the JAK‐STAT pathway is required. The aim of this study was to demonstrate that epigenetic modification by pharmacological treatments induces SOCS elevation leading to AGT suppression in PTC. Rat PTC were treated with sodium butyrate (NaB), a non‐selective HDAC inhibitor, for 6 hours. NaB induced SOCS1 (1.71 ± 0.04, ratio to control) and SOCS3 (1.67 ± 0.2) mRNA elevation in PTC. In the NaB‐treated cells, decreases in STAT3 phosphorylation levels (0.56 ± 0.02), AGT mRNA (0.19 ± 0.04) and protein (0.76 ± 0.04) levels were observed. PCI‐34051, an HDAC inhibitor exhibiting higher affinity to HDAC8 than other HDACs, also suppressed AGT expression. Inversely, Inhibition of class IIa HDACs by TMP269 increased AGT expression, indicating that class IIa HDACs include a repressor of AGT expression. These results suggest that an important role for HDACs, particularly HDAC8, in regulating AGT expression via the JAK‐STAT pathway in PTC. The findings will provide a more effective approach to treat RAS associated diseases by SOCS elevation.

Uric acid induces oxidative stress via an activation of the renin–angiotensin system in 3T3-L1 adipocytes

Hyperuricemia is recently reported involving in various obesity-related cardiovascular disorders, especially hypertension. However, the underlying mechanisms are not completely understood. In the present study, we investigated whether uric acid upregulates renin-angiotensin system (RAS) expression in adipocytes. We also examined whether RAS activation plays a role in uric acid-induced oxidative stress in adipocytes. The adipocytes of different phenotypes were incubated with uric acid for 48h, respectively. Losartan (10(-4)M) or captopril (10(-4)M) was used to block adipose tissue RAS activation. mRNA expressions of angiotensinogen (AGT), angiotensin-converting enzyme-1 (ACE-1), renin, angiotensin type 1 receptor (AT1R), and angiotensin type 2 receptor (AT2R) were evaluated with real-time PCR. Angiotensin II concentrations in supernatant were measured by ELISA. Intracellular reactive species (ROS) levels were measured by fluorescent probe DCFH-DA, DHR, or NBT assay. The uric acid upregulated both RAS (AGT, ACE1, renin, AT1R, and AT2R) mRNA expressions and angiotensin II protein secretion and caused a significant increase in ROS production in 3T3-L1 adipocytes. These effects could be prevented by RAS inhibitors, either losartan or captopril. RAS activation has been causally implicated in oxidative stress induced by uric acid in 3T3-L1 adipocytes, suggesting a plausible mechanism through which hyperuricemia contributes to the pathogenesis of obesity-related cardiovascular diseases.

Cushing's disease and hypertension: in vivo and in vitro study of the role of the renin-angiotensin-aldosterone system and effects of medical therapy

Cushing's disease (CD) is often accompanied by hypertension. CD can be treated surgically and, given the expression of somatostatin subtype 5 and dopamine 2 receptors by corticotroph pituitary adenomas, pharmacologically. Indeed, we recently observed that stepwise medical combination therapy with the somatostatin-analog pasireotide, the dopamine-agonist cabergoline, and ketoconazole (which directly suppresses steroidogenesis) biochemically controlled CD patients and lowered their blood pressure after 80 days. Glucocorticoids (GC) modulate the renin-angiotensin-aldosterone system (RAAS) among others by increasing hepatic angiotensinogen expression and stimulating mineralocorticoid receptors (MR). This study therefore evaluated plasma RAAS components in CD patients before and after drug therapy. In addition, we studied whether cabergoline/pasireotide have direct relaxant effects in angiotensin II (Ang II)-constricted iliac arteries of spontaneously hypertensive rats, with and without concomitant GR/MR stimulation with dexamethasone or hydrocortisone. Baseline concentrations of angiotensinogen were elevated, while renin and aldosterone were low and suppressed, respectively, even in patients treated with RAAS-blockers. This pattern did not change after 80 days of treatment, despite blood pressure normalization, nor after 4 years of remission. In the presence of dexamethasone, pasireotide inhibited Ang II-mediated vasoconstriction. The low plasma renin concentrations, even under RAAS blockade, in CD may be the consequence of increased GC-mediated MR stimulation and/or the elevated angiotensinogen levels in such patients. The lack of change in RAAS-parameters despite blood pressure and cortisol normalization suggests persisting consequences of long-term exposure to cortisol excess. Finally, pasireotide may have a direct vasodilating effect contributing to blood pressure lowering.

ROCK/NF-κB axis-dependent augmentation of angiotensinogen by angiotensin II in primary-cultured preglomerular vascular smooth muscle cells

In angiotensin II (ANG II)-dependent hypertension, the augmented intrarenal ANG II constricts the renal microvasculature and stimulates Rho kinase (ROCK), which modulates vascular contractile responses. Rho may also stimulate angiotensinogen (AGT) expression in preglomerular vascular smooth muscle cells (VSMCs), but this has not been established. Therefore, the aims of this study were to determine the direct interactions between Rho and ANG II in regulating AGT and other renin-angiotensin system (RAS) components and to elucidate the roles of the ROCK/NF-κB axis in the ANG II-induced AGT augmentation in primary cultures of preglomerular VSMCs. We first demonstrated that these preglomerular VSMCs express renin, AGT, angiotensin-converting enzyme, and ANG II type 1 (AT1) receptors. Furthermore, incubation with ANG II (100 pmol/l for 24 h) increased AGT mRNA (1.42 ± 0.03, ratio to control) and protein (1.68 ± 0.05, ratio to control) expression levels, intracellular ANG II levels, and NF-κB activity. In contrast, the ANG II treatment did not alter AT1a and AT1b mRNA levels in the cells. Treatment with H-1152 (ROCK inhibitor, 10 nmol/l) and ROCK1 small interfering (si) RNA suppressed the ANG II-induced AGT augmentation and the upregulation and translocalization of p65 into nuclei. Functional studies showed that ROCK exerted a greater influence on afferent arteriole responses to ANG II in rats subjected to chronic ANG II infusions. These results indicate that ROCK is involved in NF-κB activation and the ROCK/NF-κB axis contributes to ANG II-induced AGT upregulation, leading to intracellular ANG II augmentation.

Angiotensin II evokes angiogenic signals within skeletal muscle through co-ordinated effects on skeletal myocytes and endothelial cells.

Skeletal muscle overload induces the expression of angiogenic factors such as vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-2, leading to new capillary growth. We found that the overload-induced increase in angiogenesis, as well as increases in VEGF, MMP-2 and MT1-MMP transcripts were abrogated in muscle VEGF KO mice, highlighting the critical role of myocyte-derived VEGF in controlling this process. The upstream mediators that contribute to overload-induced expression of VEGF have yet to be ascertained. We found that muscle overload increased angiotensinogen expression, a precursor of angiotensin (Ang) II, and that Ang II signaling played an important role in basal VEGF production in C2C12 cells. Furthermore, matrix-bound VEGF released from myoblasts induced the activation of endothelial cells, as evidenced by elevated endothelial cell phospho-p38 levels. We also found that exogenous Ang II elevates VEGF expression, as well as MMP-2 transcript levels in C2C12 myotubes. Interestingly, these responses also were observed in skeletal muscle endothelial cells in response to Ang II treatment, indicating that these cells also can respond directly to the stimulus. The involvement of Ang II in muscle overload-induced angiogenesis was assessed. We found that blockade of AT1R-dependent Ang II signaling using losartan did not attenuate capillary growth. Surprisingly, increased levels of VEGF protein were detected in overloaded muscle from losartan-treated rats. Similarly, we observed elevated VEGF production in cultured endothelial cells treated with losartan alone or in combination with Ang II. These studies conclusively establish the requirement for muscle derived VEGF in overload-induced angiogenesis and highlight a role for Ang II in basal VEGF production in skeletal muscle. However, while Ang II signaling is activated following overload and plays a role in muscle VEGF production, inhibition of this pathway is not sufficient to halt overload-induced angiogenesis, indicating that AT1-independent signals maintain VEGF production in losartan-treated muscle.

Detailed localization of augmented angiotensinogen mRNA and protein in proximal tubule segments of diabetic kidneys in rats and humans.

In the intrarenal renin-angiotensin system, angiotensinogen levels are well known to be increased in diabetes, and these enhanced intrarenal angiotensinogen levels may initiate the development and accelerate the progression of diabetic nephropathy. However, the specific localization of the augmented angiotensinogen in proximal tubule segments in diabetes is still unknown. We investigated the detailed localization of angiotensinogen in 3 proximal tubule segments in the diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats and the control Long-Evans Tokushima Otsuka (LETO) rats. We also prepared OLETF rats treated with angiotensin II type 1 receptor blocker, olmesartan or with a combination of vasodilator agents. Moreover, biopsied samples of human kidney cortex were used to confirm the results of animal studies. We examined the co-localization of angiotensinogen with segment-specific markers by double staining using fluorescence in situ hybridization and/or immunofluorescence. Angiotensinogen mRNA expression was barely detectable in segment 1. In segment 3, the area of angiotensinogen mRNA expression was augmented in the OLETF rats compared with the LETO rats. Angiotensinogen protein expression areas in segments 1 and 3 were also increased in the OLETF rats compared with the LETO rats. Chronic treatment with olmesartan ameliorated these areas of augmented angiotensinogen expression. Biopsied human kidney samples showed similar results. These data suggest that the augmented angiotensinogen mRNA levels in segment 3 and angiotensinogen protein levels in segments 1 and 3 may contribute to the progression of diabetic nephropathy.

Oxidative Stress/Angiotensinogen/Renin-Angiotensin System Axis in Patients with Diabetic Nephropathy

Although recent studies have proven that renin-angiotensin system (RAS) blockades retard the progression of diabetic nephropathy, the detailed mechanisms of their reno-protective effects on the development of diabetic nephropathy remain uncertain. In rodent models, it has been reported that reactive oxygen species (ROS) are important for intrarenal angiotensinogen (AGT) augmentation in the progression of diabetic nephropathy. However, no direct evidence is available to demonstrate that AGT expression is enhanced in the kidneys of patients with diabetes. To examine whether the expression levels of ROS- and RAS-related factors in kidneys are increased with the progression of diabetic nephropathy, biopsied samples from 8 controls and 27 patients with type 2 diabetes were used. After the biopsy, these patients were diagnosed with minor glomerular abnormality or diabetes mellitus by clinical and pathological findings. The intensities of AGT, angiotensin II (Ang II), 4-hydroxy-2-nonenal (4-HNE), and heme oxygenase-1 (HO-1) were examined by fluorescence in situ hybridization and/or immunohistochemistry. Expression levels were greater in patients with diabetes than in control subjects. Moreover, the augmented intrarenal AGT mRNA expression paralleled renal dysfunction in patients with diabetes. These data suggest the importance of the activated oxidative stress/AGT/RAS axis in the pathogenesis of diabetic nephropathy.

Characterization of the Cardiac Renin Angiotensin System in Oophorectomized and Estrogen-Replete mRen2.Lewis Rats

The cardioprotective effects of estrogen are well recognized, but the mechanisms remain poorly understood. Accumulating evidence suggests that the local cardiac renin-angiotensin system (RAS) is involved in the development and progression of cardiac hypertrophy, remodeling, and heart failure. Estrogen attenuates the effects of an activated circulating RAS; however, its role in regulating the cardiac RAS is unclear. Bilateral oophorectomy (OVX; n = 17) or sham-operation (Sham; n = 13) was performed in 4-week-old, female mRen2.Lewis rats. At 11 weeks of age, the rats were randomized and received either 17 β-estradiol (E2, 36 µg/pellet, 60-day release, n = 8) or vehicle (OVX-V, n = 9) for 4 weeks. The rats were sacrificed, and blood and hearts were used to determine protein and/or gene expression of circulating and tissue RAS components. E2 treatment minimized the rise in circulating angiotensin (Ang) II and aldosterone produced by loss of ovarian estrogens. Chronic E2 also attenuated OVX-associated increases in cardiac Ang II, Ang-(1–7) content, chymase gene expression, and mast cell number. Neither OVX nor OVX+E2 altered cardiac expression or activity of renin, angiotensinogen, angiotensin-converting enzyme (ACE), and Ang II type 1 receptor (AT1R). E2 treatment in OVX rats significantly decreased gene expression of MMP-9, ACE2, and Ang-(1–7) mas receptor, in comparison to sham-operated and OVX littermates. E2 treatment appears to inhibit upsurges in cardiac Ang II expression in the OVX-mRen2 rat, possibly by reducing chymase-dependent Ang II formation. Further studies are warranted to determine whether an E2-mediated reduction in cardiac chymase directly contributes to this response in OVX rats.

Upregulation of a local renin–angiotensin system in the rat carotid body during chronic intermittent hypoxia

The carotid body (CB) plays an important role in the alteration of cardiorespiratory activity in chronic intermittent hypoxia (IH) associated with sleep-disordered breathing, which may be mediated by local expression of the renin-angiotensin system (RAS). We hypothesized a pathogenic role for IH-induced RAS expression in the CB. The CB expression of RAS components was examined in rats exposed to IH resembling a severe sleep-apnoeic condition for 7 days. In situ hybridization showed an elevated expression of angiotensinogen in the CB glomus cells in the hypoxic group when compared with the normoxic control group. Immunohistochemical studies and Western blot analysis revealed increases in the protein level of both angiotensinogen and angiotensin II type 1 (AT1) receptors in the hypoxic group, which were localized to the glomic clusters containing tyrosine hydroxylase. RT-PCR studies confirmed that levels of the mRNA expression of angiotensinogen, angiotensin-converting enzyme, AT1a and AT2 receptors were significantly increased in the CBs of the hypoxic rats. Functionally, the [Ca(2+)]i response to exogenous angiotensin II was enhanced in fura-2-loaded glomus cells dissociated from hypoxic rats when compared with those of the normoxic control animals. Pretreatment with losartan, but not PD123319, abolished the angiotensin II-induced [Ca(2+)]i response, suggesting an involvement of AT1 receptors. Moreover, daily treatment of the IH group of rats with losartan attenuated the levels of oxidative stress, gp91(phox) expression and macrophage infiltration in the CB. Collectively, the upregulated local RAS expression could play a pathogenic role in the augmented CB activity and local inflammation via AT1 receptor activation during IH conditions in patients with sleep-disordered breathing.