Increased angiotensinogen expression, urinary angiotensinogen excretion, and tissue injury in nonclipped kidneys of two-kidney, one-clip hypertensive rats.

In angiotensin II (ANG II)-dependent hypertension, there is an angiotensin type 1 receptor-dependent amplification mechanism enhancing intrarenal angiotensinogen (AGT) formation and secretion in the tubular fluid. To evaluate the role of increased arterial pressure, AGT mRNA, protein expression, and urinary AGT (uAGT) excretion and tissue injury were assessed in both kidneys of two-kidney, one-clip Sprague-Dawley hypertensive rats subjected to left renal arterial clipping (0.25-mm gap). By 18-21 days, systolic arterial pressure increased to 180 ± 3 mmHg, and uAGT increased. Water intake, body weights, 24-h urine volumes, and sodium excretion were similar. In separate measurements of renal function in anesthetized rats, renal plasma flow and glomerular filtration rate were similar in clipped and nonclipped kidneys and not different from those in sham rats, indicating that the perfusion pressure to the clipped kidneys remained within the autoregulatory range. The nonclipped kidneys exhibited increased urine flow and sodium excretion. The uAGT excretion was significantly greater in nonclipped kidneys compared with clipped and sham kidneys. AGT mRNA was 2.15-fold greater in the nonclipped kidneys compared with sham (1.0 ± 0.1) or clipped (0.98 ± 0.15) kidneys. AGT protein levels were also greater in the nonclipped kidneys. The nonclipped kidneys exhibited greater glomerular expansion and immune cell infiltration, medullary fibrosis, and cellular proliferation than the clipped kidneys. Because both kidneys have elevated ANG II levels, the greater tissue injury in the nonclipped kidneys indicates that an increased arterial pressure synergizes with increased intrarenal ANG II to stimulate AGT production and exert greater renal injury.

PKC-α-dependent augmentation of cAMP and CREB phosphorylation mediates the angiotensin II stimulation of renin in the collecting duct.

In contrast to the negative feedback of angiotensin II (ANG II) on juxtaglomerular renin, ANG II stimulates renin in the principal cells of the collecting duct (CD) in rats and mice via ANG II type 1 (AT1R) receptor, independently of blood pressure. In vitro data indicate that CD renin is augmented by AT1R activation through protein kinase C (PKC), but the exact mechanisms are unknown. We hypothesize that ANG II stimulates CD renin synthesis through AT1R via PKC and the subsequent activation of cAMP/PKA/CREB pathway. In M-1 cells, ANG II increased cAMP, renin mRNA (3.5-fold), prorenin, and renin proteins, as well as renin activity in culture media (2-fold). These effects were prevented by PKC inhibition with calphostin C, PKC-α dominant negative, and by PKA inhibition. Forskolin-induced increases in cAMP and renin expression were prevented by calphostin C. PKC inhibition and Ca2+ depletion impaired ANG II-mediated CREB phosphorylation and upregulation of renin. Adenylate cyclase 6 (AC) siRNA remarkably attenuated the ANG II-dependent upregulation of renin mRNA. Physiological activation of AC with vasopressin increased renin expression in M-1 cells. The results suggest that the ANG II-dependent upregulation of renin in the CD depends on PKC-α, which allows the augmentation of cAMP production and activation of PKA/CREB pathway via AC6. This study defines the intracellular signaling pathway involved in the ANG II-mediated stimulation of renin in the CD. This is a novel mechanism responsible for the regulation of local renin-angiotensin system in the distal nephron.

Renal angiotensin-converting enzyme is essential for the hypertension induced by nitric oxide synthesis inhibition.

The kidney is an important source of angiotensin-converting enzyme (ACE) in many species, including humans. However, the specific effects of local ACE on renal function and, by extension, BP control are not completely understood. We previously showed that mice lacking renal ACE, are resistant to the hypertension induced by angiotensin II infusion. Here, we examined the responses of these mice to the low-systemic angiotensin II hypertensive model of nitric oxide synthesis inhibition with L-NAME. In contrast to wild-type mice, mice without renal ACE did not develop hypertension, had lower renal angiotensin II levels, and enhanced natriuresis in response to L-NAME. During L-NAME treatment, the absence of renal ACE was associated with blunted GFR responses; greater reductions in abundance of proximal tubule Na(+)/H(+) exchanger 3, Na(+)/Pi co-transporter 2, phosphorylated Na(+)/K(+)/Cl(-) cotransporter, and phosphorylated Na(+)/Cl(-) cotransporter; and greater reductions in abundance and processing of the γ isoform of the epithelial Na(+) channel. In summary, the presence of ACE in renal tissue facilitates angiotensin II accumulation, GFR reductions, and changes in the expression levels and post-translational modification of sodium transporters that are obligatory for sodium retention and hypertension in response to nitric oxide synthesis inhibition.

Activation of the renin-angiotensin system by a low-salt diet does not augment intratubular angiotensinogen and angiotensin II in rats.

In angiotensin II (ANG II) infusion hypertension, there is an augmentation of intratubular angiotensinogen (AGT) and ANG II leading to increased urinary AGT and ANG II excretion rates associated with tissue injury. However, the changes in urinary AGT and ANG II excretion rates and markers of renal injury during physiologically induced stimulation of the renin-angiotensin system (RAS) by a low-salt diet remain unclear. Male Sprague-Dawley rats received a low-salt diet (0.03% NaCl; n = 6) and normal-salt diet (0.3% NaCl, n = 6) for 13 days. Low-salt diet rats had markedly higher plasma renin activity and plasma ANG II levels. Kidney cortex renin mRNA, kidney AGT mRNA, and AGT immunoreactivity were not different; however, medullary renin mRNA, kidney renin content, and kidney ANG II levels were significantly elevated by the low-salt diet. Kidney renin immunoreactivity was also markedly increased in juxtaglomerular apparati and in cortical and medullary collecting ducts. Urinary AGT excretion rates and urinary ANG II excretion rates were not augmented by the low-salt diet. The low-salt diet caused mild renal fibrosis in glomeruli and the tubulointerstitium, but no other signs of kidney injury were evident. These results indicate that, in contrast to the response in ANG II infusion hypertension, the elevated plasma and intrarenal ANG II levels caused by physiological stimulation of RAS are not reflected by increased urinary AGT or ANG II excretion rates or the development of renal injury.

Involvement of the brain (pro)renin receptor in cardiovascular homeostasis.

RATIONALE: Despite overwhelming evidence of the importance of brain renin-angiotensin system (RAS), the very existence of intrinsic brain RAS remains controversial. OBJECTIVE: To investigate the hypothesis that the brain (pro)renin receptor (PRR) is physiologically important in the brain RAS regulation and cardiovascular functions. METHODS AND RESULTS: PRR is broadly distributed within neurons of cardiovascular-relevant brain regions. The physiological functions of PRR were studied in the supraoptic nucleus (SON) because this brain region showed greater levels of PRR mRNA in the spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto (WKY) rats. Adeno-associated virus (AAV)-mediated overexpression of human PRR in the SON of normal rats resulted in increases in plasma and urine vasopressin, and decreases in H(2)O intake and urine output without any effects on mean arterial pressure and heart rate. Knockdown of endogenous PRR by AAV-short hairpin RNA in the SON of SHRs attenuated age-dependent increases in mean arterial pressure and caused a decrease in heart rate and plasma vasopressin. Incubation of neuronal cells in culture with human prorenin and angiotensinogen resulted in increased generation of angiotensin I and II. Furthermore, renin treatment increased phosphorylation of extracellular signal-regulated kinase ½ in neurons from both WKY rats and SHRs; however, the stimulation was 50% greater in the SHR. CONCLUSIONS: The study demonstrates that brain PRR is functional and plays a role in the neural control of cardiovascular functions. This may help resolve a long-held controversy concerning the existence of intrinsic and functional brain RAS.

Increased renin excretion is associated with augmented urinary angiotensin II levels in chronic angiotensin II-infused hypertensive rats.

Renin expression in principal cells of collecting ducts (CD) is upregulated in angiotensin II (ANG II)-dependent hypertensive rats; however, it remains unclear whether increased CD-derived renin undergoes tubular secretion. Accordingly, urinary levels of renin (uRen), angiotensinogen (uAGT), and ANG II (uANG II) were measured in chronic ANG II-infused Sprague-Dawley rats (80 ng/min for 14 days, n = 10) and sham-operated rats (n = 10). Systolic blood pressure increased in the ANG II rats by day 5 and continued to increase throughout the study (day 13; ANG II: 175 ± 10 vs. sham: 116 ± 2 mmHg; P < 0.05). ANG II infusion increased renal cortical and medullary ANG II levels (cortical ANG II: 606 ± 72 vs. 247 ± 43 fmol/g; P < 0.05; medullary ANG II: 2,066 ± 116 vs. 646 ± 36 fmol/g; P < 0.05). Although plasma renin activity (PRA) was suppressed in the ANG II-infused rats (0.3 ± 0.2 vs. 5.5 ± 1.8 ng ANG I·ml(-1)·h(-1); P < 0.05), renin content in renal medulla was increased (12,605 ± 1,343 vs. 7,956 ± 765 ng ANG I·h(-1)·mg(-1); P < 0.05). Excretion of uAGT and uANG II increased in the ANG II rats [uAGT: 1,107 ± 106 vs. 60 ± 26 ng/day; P < 0.0001; uANG II: 3,813 ± 431 vs. 2,080 ± 361 fmol/day; P < 0.05]. By day 13, despite suppression of PRA, urinary prorenin content increased in ANG II rats [15.7 ± 3 vs. 2.6 ± 1 × 10(-3) enzyme units excreted (EUE)/day, P < 0.01] as was the excretion rate of renin (8.6 ± 2 × 10(-6) EUE/day) compared with sham (2.8 ± 1 × 10(-6) EUE/day; P < 0.05). Urinary renin and prorenin protein levels examined by Western blot were augmented ∼10-fold in the ANG II-infused rats. Concomitant AT(1) receptor blockade with candesartan prevented the increase. Thus, in ANG II-dependent hypertensive rats with marked PRA suppression, increased urinary levels of renin and prorenin reflect their augmented secretion by CD cells into the luminal fluid. The greater availability of renin and AGT in the urine reflects the capability for intratubular ANG II formation which stimulates sodium reabsorption in distal nephron segments.

Major role for ACE-independent intrarenal ANG II formation in type II diabetes.

Combination therapy of angiotensin-converting enzyme (ACE) inhibition and AT(1) receptor blockade has been shown to provide greater renoprotection than ACE inhibitor alone in human diabetic nephropathy, suggesting that ACE-independent pathways for ANG II formation are of major significance in disease progression. Studies were performed to determine the magnitude of intrarenal ACE-independent formation of ANG II in type II diabetes. Although renal cortical ACE protein activity [2.1 +/- 0.8 vs. 9.2 +/- 2.1 arbitrary fluorescence units (AFU) x mg(-1) x min(-1)] and intensity of immunohistochemical staining were significantly reduced and ACE2 protein activity (16.7 +/- 3.2 vs. 7.2 +/- 2.4 AFU x mg(-1) x min(-1)) and intensity elevated, kidney ANG I (113 +/- 24 vs. 110 +/- 45 fmol/g) and ANG II (1,017 +/- 165 vs. 788 +/- 99 fmol/g) levels were not different between diabetic and control mice. Afferent arteriole vasoconstriction due to conversion of ANG I to ANG II was similar in magnitude in kidneys of diabetic (-28 +/- 3% at 1 microM) and control (-23 +/- 3% at 1 microM) mice; a response completely inhibited by AT(1) receptor blockade. In control kidneys, afferent arteriole vasoconstriction produced by ANG I was significantly attenuated by ACE inhibition, but not by serine protease inhibition. In contrast, afferent arteriole vasoconstriction produced by intrarenal conversion of ANG I to ANG II was significantly attenuated by serine protease inhibition, but not by ACE inhibition in diabetic kidneys. In conclusion, there is a switch from ACE-dependent to serine protease-dependent ANG II formation in the type II diabetic kidney. Pharmacological targeting of these serine protease-dependent pathways may provide further protection from diabetic renal vascular disease.

Chymase inhibition retards albuminuria in type 2 diabetes.

Chymase released from mast cells produces pro-fibrotic, inflammatory, and vasoconstrictor agents. Studies were performed to test the hypothesis that chronic chymase inhibition provides a renal protective effect in type 2 diabetes. Diabetic (db/db) and control mice (db/m) were chronically infused with a chymase-specific inhibitor or vehicle for 8 weeks. Baseline urinary albumin excretion (UalbV) averaged 42 ± 3 and 442 ± 32 microg/d in control (n = 22) and diabetic mice (n = 27), respectively (p < .05). After administration of chymase inhibitor to diabetic mice, the change in UalbV was significantly lower (459 ± 57 microg/d) than in vehicle-treated diabetic mice (645 ± 108 microg/d). UNGAL V was not different at baseline between diabetic mice that would receive the chymase inhibitor (349 ± 56 ng/d, n = 6) and vehicle (373 ± 99 ng/d, n = 6) infusions, but increased significantly only in the vehicle-treated diabetic mice (p < .05). Glomeruli of diabetic kidneys treated chronically with chymase inhibition demonstrated reduced mesangial matrix expansion compared to glomeruli from untreated diabetic mice. Plasma angiotensin II levels were not altered by chymase inhibitor treatment. In summary, chronic chymase inhibition slowed the progression of urinary albumin excretion in diabetic mice. In conclusion, renal chymase may contribute to the progression of albuminuria in type 2 diabetes renal disease.

Genetic clamping of renin gene expression induces hypertension and elevation of intrarenal Ang II levels of graded severity in Cyp1a1-Ren2 transgenic rats.

INTRODUCTION: Transgenic rats with inducible angiotensin II (Ang II)-dependent hypertension (strain name: TGR[Cyp1a1-Ren2]) were generated by inserting the mouse Ren2 renin gene, fused to the cytochrome P450 1a1 (Cyp1a1) promoter, into the genome of the rat. The present study was performed to characterise the changes in plasma and kidney tissue Ang II levels and in renal haemodynamic function in Cyp1a1-Ren2 rats following induction of either slowly developing or malignant hypertension in these transgenic rats. MATERIALS AND METHODS: Arterial blood pressure (BP) and renal haemodynamics and excretory function were measured in pentobarbital sodium-anaesthetised Cyp1a1- Ren2 rats fed a normal diet containing either a low dose (0.15%, w/w for 1415 days) or high dose (0.3%, w/w for 1112 days) of the aryl hydrocarbon indole-3-carbinol (I3C) to induce slowly developing and malignant hypertension, respectively. In parallel experiments, arterial blood samples and kidneys were harvested for measurement of Ang II levels by radioimmunoassay. RESULTS: Dietary I3C increased plasma renin activity (PRA), plasma Ang II levels, and arterial BP in a dose-dependent manner. Induction of different fixed levels of renin gene expression and PRA produced hypertensive phenotypes of varying severity with rats developing either mild or malignant forms of hypertensive disease. Administration of I3C, at a dose of 0.15% (w/w), induced a slowly developing form of hypertension whereas administration of a higher dose (0.3%) induced a more rapidly developing hypertension and the clinical manifestations of malignant hypertension including severe weight loss. Both hypertensive phenotypes were characterised by reduced renal plasma flow, increased filtration fraction, elevated PRA, and increased plasma and intrarenal Ang II levels. These I3C-induced changes in renal haemodynamics, PRA and kidney Ang II levels were more pronounced in Cyp1a1-Ren2 rats with malignant hypertension. Chronic administration of the AT1-receptor antagonist, hypertension, the associated changes in renal haemodynamics, and the augmentation of intrarenal Ang II levels. CONCLUSIONS: Activation of AT1-receptors by Ang II generated as a consequence of induction of the Cyp1a1-Ren2 transgene mediates the increased arterial pressure and the associated reduction of renal haemodynamics and enhancement of intrarenal Ang II levels in hypertensive Cyp1a1-Ren2 transgenic rats.

The role of IL-6 in the physiologic versus hypertensive blood pressure actions of angiotensin II.

Angiotensin II (AngII) is a critical physiologic regulator of volume homeostasis and mean arterial pressure (MAP), yet it also is known to induce immune mechanisms that contribute to hypertension. This study determined the role of interleukin-6 (IL-6) in the physiologic effect of AngII to maintain normal MAP during low-salt (LS) intake, and whether hypertension induced by plasma AngII concentrations measured during LS diet required IL-6. IL-6 knockout (KO) and wild-type (WT) mice were placed on LS diet for 7 days, and MAP was measured 19 h/day with telemetry. MAP was not affected by LS in either group, averaging 101 ± 4 and 100 ± 4 mmHg in WT and KO mice, respectively, over the last 3 days. Seven days of ACEI decreased MAP ~25 mmHg in both groups. In other KO and WT mice, AngII was infused at 200 ng/kg per minute to approximate plasma AngII levels during LS. Surgical reduction of kidney mass and high-salt diet were used to amplify the blood pressure effect. The increase in MAP after 7 days was not different, averaging 20 ± 5 and 22 ± 6 mmHg in WT and KO mice, respectively. Janus Kinase 2 (JAK2)/signal transducer of activated transcription (STAT3) phosphorylation were not affected by LS, but were increased by AngII infusion at 200 and 800 ng/kg per minute. These data suggest that physiologic levels of AngII do not activate or require IL-6 to affect blood pressure significantly, whether AngII is maintaining blood pressure on LS diet or causing blood pressure to increase. JAK2/STAT3 activation, however, is tightly associated with AngII hypertension, even when caused by physiologic levels of AngII.

Transcription factor avian erythroblastosis virus E26 oncogen homolog-1 is a novel mediator of renal injury in salt-sensitive hypertension.

Transcription factor E26 transformation-specific sequence-1 (ETS-1) is a transcription factor that regulates the expression of a variety of genes, including growth factors, chemokines, and adhesion molecules. We recently demonstrated that angiotensin II increases the glomerular expression of ETS-1 and that blockade of ETS-1 ameliorates the profibrotic and proinflammatory effects of angiotensin II. The Dahl salt-sensitive rat is a paradigm of salt-sensitive hypertension associated with local activation of the renin-angiotensin system. In these studies, we determined whether: (1) salt-sensitive hypertension is associated with renal expression of ETS-1 and (2) ETS-1 participates in the development of end-organ injury in salt-sensitive hypertension. Dahl salt-sensitive rats were fed a normal-salt diet (0.5% NaCl diet) or a high-salt diet (4% NaCl) for 4 weeks. Separate groups on high-salt diet received an ETS-1 dominant-negative peptide (10 mg/kg/d), an inactive ETS-1 mutant peptide (10 mg/kg/d), the angiotensin II type 1 receptor blocker candesartan (10 mg/kg/d), or the combination high-salt diet/dominant-negative peptide/angiotensin II type 1 receptor blocker for 4 weeks. High-salt diet rats had a significant increase in the glomerular expression of the phosphorylated ETS-1 that was prevented by angiotensin II type 1 receptor blocker. ETS-1 blockade reduced proteinuria, glomerular injury score, fibronectin expression, urinary transforming growth factor-ß excretion, and macrophage infiltration. Angiotensin II type 1 receptor blocker reduced proteinuria, glomerular injury score, and macrophage infiltration, whereas concomitant ETS-1 blockade and angiotensin II type 1 receptor blocker had additive effects and reduced interstitial fibrosis. Our studies demonstrated that salt-sensitive hypertension results in increased glomerular expression of phosphorylated ETS-1 and suggested that ETS-1 plays an important role in the pathogenesis of end-organ injury in salt-sensitive hypertension.

Angiotensin II type 1 receptor-mediated augmentation of renal interstitial fluid angiotensin II in angiotensin II-induced hypertension.

BACKGROUND: Angiotensin II (Ang II)-dependent hypertension is associated with augmented intrarenal concentrations of Ang II; however, the distribution of the increased intrarenal Ang II has not been fully established. OBJECTIVE: To determine the changes in renal interstitial fluid Ang II concentrations in Ang II-induced hypertension and the consequences of treatment with an angiotensin II type 1 (AT1) receptor blocker. DESIGN AND METHODS: Rats were selected to receive vehicle (5% acetic acid subcutaneously; n = 6), Ang II (80 ng/min subcutaneously, via osmotic minipump; n = 7) or Ang II plus an AT1 receptor antagonist, candesartan cilexetil (10 mg/kg per day, in drinking water; n = 6) for 13-14 days, at which time, experiments were performed on anesthetized rats. Microdialysis probes were implanted in the renal cortex and were perfused at 2 microl/min. The effluent dialysate concentrations of Ang I and Ang II were measured by radioimmunoassay and reported values were corrected for the equilibrium rates at this perfusion rate. RESULTS: Ang II-infused rats developed greater mean arterial pressures (155 +/- 7 mmHg) than vehicle-infused rats (108 +/- 3 mmHg). Ang II-infused rats showed greater plasma (181 +/- 30 fmol/ml) and kidney (330 +/- 38 fmol/g) Ang II concentrations than vehicle-infused rats (98 +/- 14 fmol/ml and 157 +/- 22 fmol/g, respectively). Renal interstitial fluid Ang II concentrations were much greater than plasma concentrations, averaging 5.74 +/- 0.26 pmol/ml in Ang II-infused rats - significantly greater than those in vehicle-infused rats (2.86 +/- 0.23 pmol/ml). Candesartan treatment prevented the hypertension (87 +/- 3 mmHg) and led to increased plasma Ang II concentrations (441 +/- 27 fmol/ml), but prevented increases in kidney (120 +/- 15 fmol/g) and renal interstitial fluid (2.15 +/- 0.12 pmol/ml) Ang II concentrations. CONCLUSIONS: These data indicate that Ang II-infused rats develop increased renal interstitial fluid concentrations of Ang II, which may contribute to the increased vascular resistance and reduced sodium excretion. Furthermore, the augmentation of renal interstitial fluid Ang II is the result of an AT1 receptor-mediated process and can be dissociated from the plasma concentrations.

Olmesartan improves endothelin-induced hypertension and oxidative stress in rats.

Recent studies have indicated that both endothelin (ET) and angiotensin (Ang) II stimulate oxidative stress, which contributes to the development of hypertension. Here, we examined the effects of Ang II type 1 (AT1) receptor blockade on reactive oxygen species (ROS) formation in ET-dependent hypertension. Chronic ET-1 infusion (2.5 pmol/kg/min, i.v., n=7) into rats for 14 days increased systolic blood pressure from 113+/-1 to 141+/-2 mmHg. ET-1-infused rats showed greater plasma renin activity (8.1+/-0.8 Ang I/ml/h), and greater Ang I (122+/-28 fmol/ml) and Ang II levels (94+/-13 fmol/ml) than vehicle (0.9% NaCl)-infused rats (3.1+/-0.6 Ang I/ml/h, 45+/-8 and 47+/-7 fmol/ml, respectively, n=6). Angiotensin converting enzyme and AT1 receptor expression in aortic tissues were similar between the vehicle- and ET-1-infused rats. Vascular superoxide anion (O2-) production and plasma thiobarbituric acid-reactive substance (TBARS) levels were greater in ET-1-infused rats (27+/-1 counts per minutes [CPM]/mg dry tissue weight and 8.9+/-0.8 micromol/l, respectively) than vehicle-infused rats (16+/-1 CPM/mg and 5.1+/-0.1 micromol/l, respectively). The ET-1-induced hypertension was prevented by simultaneous treatment with a new AT1 receptor antagonist, olmesartan (0.01% in chow, 117+/-5 mmHg, n =7), or hydralazine (15 mg/kg/day in drinking water, 118+/-4 mmHg, n=6). Olmesartan prevented ET-1-induced increases in vascular O2- production (15+/-1 CPM/mg) and plasma TBARS (5.0+/-0.1 micromol/l). Vascular O2- production and plasma TBARS were also decreased by hydralazine (21+/-1 CPM/mg and 7.0+/-0.3 micromol/l, respectively), but these levels were significantly higher than in vehicle-infused rats. These data suggest that ET-dependent hypertension is associated with augmentation of Ang II levels and ROS formation. The combined effects of the elevations in circulating ET-1 and Ang II, as well as the associated ROS production, may contribute to the development of hypertension induced by chronic ET-1 infusion.

Angiotensin II increases the expression of (pro)renin receptor during low-salt conditions.

BACKGROUND: Evidence indicates that chronic angiotensin II (AngII) infusion increases (pro)renin receptor ((P)RR) expression in renal inner medullary collecting duct (IMCD) cells. Recently, it has been shown that renal (P)RR expression is augmented during a low-salt (LS) diet. However, the role of AngII in mediating the stimulation of (P)RR during LS conditions is unknown. We hypothesized that AngII mediates the increased expression of (P)RR during low-salt conditions in IMCDs. METHODS: (P)RR expression and AngII levels were evaluated in Sprague-Dawley rats fed a LS diet (0.03% NaCl) and normal salt (NS; 0.4% NaCl) for 7 days. We examined the effects of sodium reduction (130 mM NaCl) and AngII on (P)RR expression in IMCDs isolated in hypertonic conditions (640 mOsmol/L with 280 mM NaCl). RESULTS: Plasma renin activity in LS rats was significantly higher than rats fed with NS (28.1 ± 2.2 versus 6.7 ± 1.1 ng AngI·mL⁻¹·hr⁻¹; P < 0.05), as well as renin content in renal cortex and medulla. The (P)RR mRNA and protein levels were higher in medullary tissues from LS rats but did not change in the cortex. Intrarenal AngII was augmented in LS compared with NS rats (cortex: 710 ± 113 versus 277 ± 86 fmol/g, P < 0.05; medulla: 2093 ± 125 versus 1426 ± 126 fmol/g, P < 0.05). In cultured IMCDs, (P)RR expression was increased in response to LS or AngII treatment and potentiated by both treatments (both at 640 mOsmol/L). CONCLUSIONS: These data indicate that (P)RR is augmented in medullary collecting ducts in response to LS and that this effect is further enhanced by the increased intrarenal AngII content.

(Pro)renin receptor mediates both angiotensin II-dependent and -independent oxidative stress in neuronal cells.

The binding of renin or prorenin to the (pro)renin receptor (PRR) promotes angiotensin (Ang) II formation and mediates Ang II-independent signaling pathways. In the central nervous system (CNS), Ang II regulates blood pressure via inducing oxidative stress; however, the role of PRR-mediated Ang II-independent signaling pathways in oxidative stress in the CNS remains undefined. To address this question, Neuro-2A cells were infected with control virus or an adeno-associated virus encoding the human PRR. Human PRR over-expression alone increased ROS levels, NADPH oxidase activity, as well as NADPH oxidase (NOX) isoforms 2 and 4 mRNA expression levels and these effects were not blocked by losartan. Moreover, the increase in NOX 2 and NOX 4 mRNA levels, NADPH oxidase activity, and ROS levels induced by PRR over-expression was prevented by mitogen activated protein kinase/extracellular signal-regulated kinase 1 and 2 (MAPK/ERK1/2) inhibition, and phosphoinositide 3 kinase/Akt (IP3/Akt) inhibition, indicating that PRR regulates NOX activity and ROS formation in neuro-2A cells through Ang II-independent ERK1/2 and IP3/Akt activation. Interestingly, at a concentration of 2 nM or higher, prorenin promoted Ang II formation, and thus further increased the ROS levels in cultured Neuro-2A cells via PRR. In conclusion, human PRR over-expression induced ROS production through both angiotensin II-dependent and -independent mechanisms. We showed that PRR-mediated angiotensin II-independent ROS formation is associated with activation of the MAPK/ERK1/2 and PI3/Akt signaling pathways and up-regulation of mRNA level of NOX 2 and NOX4 isoforms in neuronal cells.

The absence of intrarenal ACE protects against hypertension.

Activation of the intrarenal renin-angiotensin system (RAS) can elicit hypertension independently from the systemic RAS. However, the precise mechanisms by which intrarenal Ang II increases blood pressure have never been identified. To this end, we studied the responses of mice specifically lacking kidney angiotensin-converting enzyme (ACE) to experimental hypertension. Here, we show that the absence of kidney ACE substantially blunts the hypertension induced by Ang II infusion (a model of high serum Ang II) or by nitric oxide synthesis inhibition (a model of low serum Ang II). Moreover, the renal responses to high serum Ang II observed in wild-type mice, including intrarenal Ang II accumulation, sodium and water retention, and activation of ion transporters in the loop of Henle (NKCC2) and distal nephron (NCC, ENaC, and pendrin) as well as the transporter activating kinases SPAK and OSR1, were effectively prevented in mice that lack kidney ACE. These findings demonstrate that ACE metabolism plays a fundamental role in the responses of the kidney to hypertensive stimuli. In particular, renal ACE activity is required to increase local Ang II, to stimulate sodium transport in loop of Henle and the distal nephron, and to induce hypertension.

The Prorenin and (Pro)renin Receptor: New Players in the Brain Renin-Angiotensin System?

It is well known that the brain renin-angiotensin (RAS) system plays an essential role in the development of hypertension, mainly through the modulation of autonomic activities and vasopressin release. However, how the brain synthesizes angiotensin (Ang) II has been a debate for decades, largely due to the low renin activity. This paper first describes the expression of the vasoconstrictive arm of RAS components in the brain as well as their physiological and pathophysiological significance. It then focus on the (pro)renin receptor (PRR), a newly discovered component of the RAS which has a high level in the brain. We review the role of prorenin and PRR in peripheral organs and emphasize the involvement of brain PRR in the pathogenesis of hypertension. Some future perspectives in PRR research are heighted with respect to novel therapeutic target for the treatment of hypertension and other cardiovascular diseases.

Sexual dimorphism in urinary angiotensinogen excretion during chronic angiotensin II-salt hypertension.

BACKGROUND: The intrarenal renin-angiotensin system contributes to hypertension by regulating sodium and water reabsorption throughout the nephron. Sex differences in the intrarenal components of the renin-angiotensin system have been involved in the greater incidence of high blood pressure and progression to kidney damage in males than females. OBJECTIVE: This study investigated whether there is a sex difference in the intrarenal gene expression and urinary excretion of angiotensinogen (AGT) during angiotensin II (Ang II)-dependent hypertension and high-salt (HS) diet. METHODS: Male and female Sprague-Dawley rats were divided into 5 groups for each sex: Normal-salt control, HS diet (8% NaCl), Ang II-infused (80 ng/min), Ang II-infused plus HS diet, and Ang II-infused plus HS diet and treatment with the Ang II receptor blocker, candesartan (25 mg/L in the drinking water). Rats were evaluated for systolic blood pressure (SBP), kidney AGT mRNA expression, urinary AGT excretion, and proteinuria at different time points during a 14-day protocol. RESULTS: Both male and female rats exhibited similar increases in urinary AGT, with increases in SBP during chronic Ang II infusion. HS diet greatly exacerbated the urinary AGT excretion in Ang II-infused rats; males had a 9-fold increase over Ang II alone and females had a 2.5-fold increase. Male rats displayed salt-sensitive SBP increases during Ang II infusion and HS diet, and female rats did not. In the kidney cortex, males displayed greater AGT gene expression than females during all treatments. During Ang II infusion, both sexes exhibited increases in AGT gene message compared with same-sex controls. In addition, HS diet combined with Ang II infusion exacerbated the proteinuria in both sexes. Concomitant Ang II receptor blocker treatment during Ang II infusion and HS diet decreased SBP and urinary AGT similarly in both sexes; however, the decrease in proteinuria was greater in the females. CONCLUSION: During Ang II-dependent hypertension and HS diet, higher intrarenal renin-angiotensin system activation in males, as reflected by higher AGT gene expression and urinary excretion, indicates a mechanism for greater progression of high blood pressure and might explain the sex disparity in development of salt-sensitive hypertension.

Soluble form of the (pro)renin receptor is augmented in the collecting duct and urine of chronic angiotensin II-dependent hypertensive rats.

Renin synthesis and secretion by principal cells of the collecting duct are enhanced in angiotensin (Ang) II-dependent hypertension. The presence of renin/(pro)renin and its receptor, the (pro)renin receptor ([P]RR), in the collecting duct may provide a pathway for Ang I generation with further conversion to Ang II. To assess whether (P)RR activation occurs during Ang II-dependent hypertension, we examined renal (P)RR levels and soluble (P)RR excretion in the urine of chronic Ang II-infused rats (80 ng/min; for 2 weeks; n=10) and sham-operated rats (n=10). Systolic blood pressure and Ang II levels in the plasma and kidney were increased whereas plasma renin activity was suppressed in Ang II-infused rats. Renal (P)RR transcripts were upregulated in the cortex and medulla of Ang II-infused rats. (P)RR immunoreactivity in collecting duct cells and the protein levels of the full-length form (37-kDa band) were significantly decreased in the medulla of Ang II-infused rats. The soluble (P)RR (28-kDa band) was detected in the renal medulla and urine samples of Ang II-infused rats, which also showed increases in urinary renin content. To determine whether the soluble (P)RR could stimulate Ang I formation, urine samples were incubated with recombinant human (pro)renin. Urine samples of Ang II-infused rats exhibited increased Ang I formation compared with sham-operated rats. Thus, in chronic Ang II-infused rats, the catalytic activity of the augmented renin produced in the collecting duct may be enhanced by the intraluminal soluble (P)RR and cell-surface located (P)RR, thus contributing to enhanced intratubular Ang II formation.

Angiotensin II stimulates renin in inner medullary collecting duct cells via protein kinase C and independent of epithelial sodium channel and mineralocorticoid receptor activity.

Collecting duct (CD) renin is stimulated by angiotensin (Ang) II, providing a pathway for Ang I generation and further conversion to Ang II. Ang II stimulates the epithelial sodium channel via the Ang II type 1 receptor and increases mineralocorticoid receptor activity attributed to increased aldosterone release. Our objective was to determine whether CD renin augmentation is mediated directly by Ang II type 1 receptor or via the epithelial sodium channel and mineralocorticoid receptor. In vivo studies examined the effects of epithelial sodium channel blockade (amiloride; 5 mg/kg per day) on CD renin expression and urinary renin content in Ang II-infused rats (80 ng/min, 2 weeks). Ang II infusion increased systolic blood pressure, medullary renin mRNA, urinary renin content, and intrarenal Ang II levels. Amiloride cotreatment did not alter these responses despite a reduction in the rate of progression of systolic blood pressure. In primary cultures of inner medullary CD cells, renin mRNA and (pro)renin protein levels increased with Ang II (100 nmol/L), and candesartan (Ang II type 1 receptor antagonist) prevented this effect. Aldosterone (10(-10) to 10(-7) mol/L) with or without amiloride did not modify the upregulation of renin mRNA in Ang II-treated cells. However, inhibition of protein kinase C with calphostin C prevented the Ang II-mediated increases in renin mRNA and (pro)renin protein levels. Furthermore, protein kinase C activation with phorbol 12-myristate 13-acetate increased renin expression to the same extent as Ang II. These data indicate that an Ang II type 1 receptor-mediated increase in CD renin is induced directly by Ang II via the protein kinase C pathway and that this regulation is independent of mineralocorticoid receptor activation or epithelial sodium channel activity.