Ng ANG Research Articles

PDGF receptor antagonism prevents the increase in kidney ANG II levels in Cyp1a1‐Ren2 transgenic rats with ANG II‐dependent malignant hypertension (1136.1)

This study was performed to determine if chronic PDGF receptor blockade prevents the augmentation of intrarenal ANG II levels in Cyp1a1‐Ren2 transgenic rats with ANG II‐dependent malignant hypertension. Male Cyp1a1‐Ren2 rats (n=5/group) were induced to develop malignant hypertension by dietary administration of indole‐3‐carbinol (I3C, 0.3% wt/wt) for 10 days. Systolic blood pressures (SBP) were measured daily by tail‐cuff plethysmography. On day 10 of I3C administration, rats were subjected to conscious decapitation and trunk blood was collected for measurement of plasma renin activity (PRA) and plasma ANG II levels. The kidneys were harvested for determination of total kidney ANG II levels. Dietary I3C resulted in increases in SBP (130±5 to 171±9 mmHg, P<0.05), PRA (64±6 vs. 5±1 ng ANG I/ml/hr, P<0.001), plasma ANG II levels (186±26 vs. 139±10 fmol/ml, P<0.05), and total kidney ANG II content (550±31 vs. 253±24 fmol/g, P<0.001). Chronic imatinib administration (60 mg/kg/day, oral gavage) did not prevent the development of hypertension (187±10 vs. 141±2 mmHg, P<0.001) or the increase in PRA (72±6 vs. 5±1 ng ANG I/ml/hr, P<0.001), but prevented the I3C‐induced augmentation of intrarenal ANG II levels (285±128 vs. 253±24 fmol/g). The present findings indicate that PDGF receptor activation contributes to the increase in the elevations of intrarenal ANG II levels in ANG II‐dependent malignant hypertension.Grant Funding Source: 5P30GM103337

Loss of WNK3 is compensated for by the WNK1/SPAK axis in the kidney of the mouse

WNK3 kinase is expressed throughout the nephron and acts as a positive regulator of NKCC2 and NCC in vitro. Here we addressed the in vivo relevance of WNK3 using WNK3-deficient mice. WNK3-/- mice were viable and showed no gross abnormalities. The net tubular function was similar in wild-type (WT) and WNK3-/- mice as assessed by determination of 24-h urine output (1.63 ± .06 in WT and 1.55 ± .1 ml in WNK3-/-, n=16; P=0.42) and ambient urine osmolarity (1,804 ± 62 in WT vs. 1,819 ± 61 mosmol/kg in WNK3-/-, n=40; P=0.86). Water restriction (48 h) increased urine osmolarity similarly in both genotypes to 3,440 ± 220 and 3,200 ± 180 mosmol/kg in WT and WNK3-/- mice, respectively (n=11; P=0.41). The glomerular filtration rate (343 ± 22 vs. 315 ± 13 ml/min), renal blood flow (1.35 ± 0.1 vs. 1.42 ± 0.04 ml), and plasma renin concentration (94 ± 18 vs. 80 ± 13 ng ANG I·ml(-1)·h(-1)) were similar between WT and WNK3-/- mice (n=13; P=0.54). WNK1 was markedly upregulated in WNK3-deficient mice, whereas the expression of WNK4 was similar in both genotypes. When the mice were fed a salt-restricted diet [0.02% NaCl (wt/wt)] the levels of pSPAK/OSR1, pNKCC2, and pNCC were enhanced in both genotypes compared with the baseline conditions, with the levels in WNK3-/- exceeding those in WT mice. The upregulation of pSPAK/OSR1, pNKCC2, and pNCC in WNK3-/- mice relative to the levels in WT mice when fed a low-salt diet was paralleled by an increased diuresis in response to hydrochlorothiazide. In summary, the overall relevance of WNK3 for the renal reabsorption of NaCl appears to be limited and can be largely compensated for by the activation of WNK3-independent pathways. Consequently, our data suggest that WNK3 may serve as a member of a kinase network that facilitates the fine-tuning of renal transepithelial NaCl transport.

Parathyroid hormone stimulates juxtaglomerular cell cAMP accumulation without stimulating renin release

Parathyroid hormone (PTH) is positively coupled to the generation of cAMP via its actions on the PTH1R and PTH2R receptors. Renin secretion from juxtaglomerular (JG) cells is stimulated by elevated intracellular cAMP, and every stimulus that increases renin secretion is thought to do so via increasing cAMP. Thus we hypothesized that PTH increases renin release from primary cultures of mouse JG cells by elevating intracellular cAMP via the PTH1R receptor. We found PTH1R, but not PTH2R, mRNA expressed in JG cells. While PTH increased JG cell cAMP content from (log(10) means ± SE) 3.27 ± 0.06 to 3.92 ± 0.12 fmol/mg protein (P < 0.001), it did not affect renin release. The PTH1R-specific agonist, parathyroid hormone-related protein (PTHrP), also increased JG cell cAMP from 3.13 ± 0.09 to 3.93 ± 0.09 fmol/mg protein (P < 0.001), again without effect on renin release. PTH2R receptor agonists had no effect on cAMP or renin release. PTHrP increased cAMP in the presence of both low and high extracellular calcium from 3.31 ± 0.17 to 3.83 ± 0.20 fmol/mg protein (P < 0.01) and from 3.29 ± 0.18 to 3.63 ± 0.22 fmol/mg protein (P < 0.05), respectively, with no effect on renin release. PTHrP increased JG cell cAMP in the presence of adenylyl cyclase-V inhibition from 2.85 ± 0.17 to 3.44 ± 0.14 fmol/mg protein (P < 0.001) without affecting renin release. As a positive control, forskolin increased JG cell cAMP from 3.39 ± 0.13 to 4.48 ± 0.07 fmol/mg protein (P < 0.01) and renin release from 2.96 ± 0.10 to 3.29 ± 0.08 ng ANG I·mg prot(-1)·h(-1) (P < 0.01). Thus PTH increases JG cell cAMP via non-calcium-sensitive adenylate cyclases without affecting renin release. These data suggest compartmentalization of cAMP signaling in JG cells.

In Vivo Imaging of Macrophages during the Early-Stages of Abdominal Aortic Aneurysm Using High Resolution MRI in ApoE−/− Mice

BackgroundAngiotensin II (ANG II) promotes vascular inflammation and induces abdominal aortic aneurysm (AAA) in hyperlipidemic apolipoprotein E knock-out (apoE−/−) mice. The aim of the present study was to detect macrophage activities in an ANG II-induced early-stage AAA model using superparamagnetic iron oxide (SPIO) as a marker.Methodology/Principal FindingsTwenty-six male apoE−/− mice received saline or ANG II (1000 or 500 ng/kg/min) infusion for 14 days. All animals underwent MRI scanning following administration of SPIO with the exception of three mice in the 1000 ng ANG II group, which were scanned without SPIO administration. MR imaging was performed using black-blood T2 to proton density -weighted multi-spin multi-echo sequence. In vivo MRI measurement of SPIO uptake and abdominal aortic diameter were obtained. Prussian blue, CD68,α-SMC and MAC3 immunohistological stains were used for the detection of SPIO, macrophages and smooth muscle cells. ANG II infusion with 1000 ng/kg/min induced AAA in all of the apoE−/− mice. ANG II infusion exhibited significantly higher degrees of SPIO uptake, which was detected using MRI as a distinct loss of signal intensity. The contrast-to-noise ratio value decreased in proportion to an increase in the number of iron-laden macrophages in the aneurysm. The aneurysmal vessel wall in both groups of ANG II treated mice contained more iron-positive macrophages than saline-treated mice. However, the presence of cells capable of phagocytosing haemosiderin in mural thrombi also induced low-signal-intensities via MRI imaging.Conclusions/SignificanceSPIO is taken up by macrophages in the shoulder and the outer layer of AAA. This alters the MRI signaling properties and can be used in imaging inflammation associated with AAA. It is important to compare images of the aorta before and after SPIO injection.

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.

Acute activation of the calcium-sensing receptor inhibits plasma renin activity in vivo.

In vitro, the renin-secreting juxtaglomerular cells express the calcium-sensing receptor, and its activation with the calcimimetic cinacalcet inhibits renin release. To test whether the activation of calcium-sensing receptor similarly inhibits plasma renin activity (PRA) in vivo, we hypothesized that the calcium-sensing receptor is expressed in juxtaglomerular cells in vivo, and acutely administered cinacalcet would inhibit renin activity in anesthetized rats. Since cinacalcet inhibits parathyroid hormone, which may stimulate renin activity, we sought to determine whether cinacalcet inhibits renin activity by decreasing parathyroid hormone. Lastly, we hypothesized that chronically administered cinacalcet would inhibit basal and stimulated renin in conscious rats. Calcium-sensing receptors and renin were localized in the same juxtaglomerular cells using immunofluorescence in rat cortical slices fixed in vivo. Cinacalcet was administered acutely via intravenous bolus in anesthetized rats and chronically in conscious rats by oral gavage. Acute administration of cinacalcet decreased basal renin activity from 13.6 ± 2.4 to 6.1 ± 1.1 ng ANG I·ml(-1)·h(-1) (P < 0.001). Likewise, cinacalcet decreased furosemide-stimulated renin from 30.6 ± 2.3 to 21.3 ± 2.3 ng ANG I·ml(-1)·h(-1) (P < 0.001). In parathyroidectomized rats, cinacalcet decreased renin activity from 9.3 ± 1.3 to 5.2 ± 0.5 ng ANG I·ml(-1)·h(-1) (P < 0.05) similar to sham-operated controls (13.5 ± 2.2 to 6.6 ± 0.8 ng ANG I·ml(-1)·h(-1), P < 0.05). Chronic administration of cinacalcet over 7 days had no significant effect on PRA under basal or stimulated conditions. In conclusion, calcium-sensing receptors are expressed in juxtaglomerular cells in vivo, and acute activation of these receptors with cinacalcet inhibits PRA in anesthetized rats, independent of parathyroid hormone.

Greater orthostatic tolerance in young black compared to white women

We hypothesized that orthostatic tolerance (OT) is higher in young, healthy black (BW) compared to white women (WW). To determine OT, sixteen women (8 BW, 8 WW) underwent graded lower body negative pressure (LBNP) to presyncope. We measured blood pressure, heart rate, and R‐R interval (ECG) continuously at baseline and through all levels of LBNP. We took blood samples at baseline and presyncope for the measurement of plasma catecholamine and serum aldosterone concentrations and plasma renin activity. Cumulative stress index (CSI), the sum of the product of time and LBNP, was the indicator of OT. OT in the BW was ~ 50% greater than in the WW [CSI=−932.1 (402.1) vs. −496.9 (220.2) P &lt; 0.05]. While P[NE] increased in both groups at presyncope, the increase was greater in BW [Δ P[NE] 171.0 (112.3)] versus WW [73.9 (70.9), P &lt; 0.05], as were the increases in PRA [Δ PRA 2.6 (1.0) versus 0.6 (0.9) ng ANG II·ml−1 ·hr−1, P &lt; 0.05, for BW and WW, respectively). Although heart rate increased and R‐R interval decreased to a greater extent during LBNP in BW compared to WW [ANOVA, P &lt; 0.05)], baroreflex function (i.e. slope R‐R interval vs. LBNP) was unaffected by race. These data indicate that OT is greater in black compared to white women, which appears to be a function of greater sympathetic nervous system responses orthostatic stress. HL071159

Enhanced NMDA receptor function in the hypothalamic PVN contributes to support of sympathetic nerve discharge and blood pressure in Ang II + Salt hypertensive rats

Rats consuming a high salt diet and treated with angiotensin II (ANG II) develop neurogenic hypertension that involves activation of sympathetic nerve discharge (SND). Here, we investigated control of renal SND and mean arterial pressure (MAP) by NMDA receptors in the hypothalamic PVN. Control (0.4 % NaCl diet, no ANG II, n=6) and ANG II + Salt hypertensive rats (2.0% NaCl, 150 ng ANG II/kg/min, s.c., n=4) were anesthetized (urethane/chloralose) and MAP and renal SND responses to PVN microinjection of the NMDA receptor antagonist AP5 (3.0 nmol in 50 nl) were recorded. Baseline MAP was higher by ~12 mmHg in ANG II + Salt rats compared to controls. MAP was unchanged by AP5 in control rats, but decreased by 13 ± 2 mmHg (P&lt;0.05) in the ANG II+ Salt group. Whereas renal SND was unchanged by AP5 in control rats, it was reduced from baseline by 13 ± 4 % in ANG II + Salt rats. These data suggest that maintenance of renal SND and MAP in rats with ANG II + Salt hypertension involves an enhanced role for NMDA receptor activation in the PVN. Whether common adaptive mechanisms underlie this enhancement of NMDA receptor function and that reported to occur in other sympathoexcitatory states (e.g., water deprivation, heart failure, intermittent hypoxia) requires further investigation. Support: HL076312 (NCDC)

Salt sensitivity of blood pressure in NKCC1-deficient mice

NKCC1 is a widely expressed isoform of the Na-2Cl-K cotransporter that mediates several direct and indirect vascular effects and regulates expression and release of renin. In this study, we used NKCC1-deficient (NKCC1-/-) and wild-type (WT) mice to assess day/night differences of blood pressure (BP), locomotor activity, and renin release and to study the effects of high (8%) or low (0.03%) dietary NaCl intake on BP, activity, and the renin/aldosterone system. On a standard diet, 24-h mean arterial blood pressure (MAP) and heart rate determined by radiotelemetry, and their day/night differences, were not different in NKCC1-/- and WT mice. Spontaneous and wheel-running activities in the active night phase were lower in NKCC1-/- than WT mice. In NKCC1-/- mice on a high-NaCl diet, MAP increased by 10 mmHg in the night without changes in heart rate. In contrast, there was no salt-dependent blood pressure change in WT mice. MAP reductions by hydralazine (1 mg/kg) or isoproterenol (10 microg/mouse) were significantly greater in NKCC1-/- than WT mice. Plasma renin (PRC; ng ANG I.ml(-1).h(-1)) and aldosterone (aldo; pg/ml) concentrations were higher in NKCC1-/- than WT mice (PRC: 3,745+/-377 vs. 1,245+/-364; aldo: 763+/-136 vs. 327+/-98). Hyperreninism and hyperaldosteronism were found in NKCC1-/- mice during both day and night. High Na suppressed PRC and aldosterone in both NKCC1-/- and WT mice, whereas a low-Na diet increased PRC and aldosterone in WT but not NKCC1-/- mice. We conclude that 24-h MAP and MAP circadian rhythms do not differ between NKCC1-/- and WT mice on a standard diet, probably reflecting a balance between anti- and prohypertensive factors, but that blood pressure of NKCC1-/- mice is more sensitive to increases and decreases of Na intake.

Aldosterone receptor antagonism alleviates proteinuria, but not malignant hypertension, in Cyp1a1-Ren2 transgenic rats

The contribution of elevated aldosterone to the pathogenesis of malignant, ANG II-dependent hypertension remains uncertain. Therefore, we examined whether chronic mineralocorticoid receptor blockade attenuates the development of malignant hypertension in transgenic rats (TGRs) with inducible expression of the Ren2 gene [TGR(Cyp1a1Ren2)]. Systolic blood pressure (SBP) was measured by radiotelemetry in male TGRs in three groups: 1) control (n = 9), 2) hypertensives (HT; n = 8), and 3) hypertensives + spironolactone (11 mg.kg(-1).day(-1) sc; HTS; n = 8). Malignant hypertension was induced with dietary indole-3-carbinol (0.3%) for 10 days. Metabolic measurements were taken at the beginning of the study and at days 2 and 9. HT exhibited elevated SBP (125 +/- 3 vs. 187 +/- 5 mmHg), plasma renin activity (5 +/- 1 vs. 29 +/- 10 ng ANG I.ml(-1).h(-1)), plasma ANG II (175 +/- 39 vs. 611 +/- 74 fmol/ml), and plasma aldosterone (0.31 +/- 0.04 vs. 5.42 +/- 1.02 nmol/l). Urinary aldosterone excretion increased 5.5-fold by day 2 and an additional 90% by day 9. HT was associated with a 1.8-fold increase in proteinuria by day 9 that was alleviated by treatment with spironolactone (25 +/- 5 vs. 13 +/- 3 mg/day), suggesting that aldosterone contributes to the renal damage observed in malignant hypertension. Urinary Na+ excretion was decreased 76% on day 2, despite a sixfold increase in urinary aldosterone excretion. Decrease in urinary Na+ excretion on day 2 in HT suggests that Na+ reabsorption was increased in response to the increase in aldosterone; however, the lack of a change in SBP between HT and HTS suggests that mechanisms independent of aldosterone stimulation make a greater contribution to the maintenance of elevated arterial pressure in malignant hypertension in Cyp1a1-Ren2 transgenic rats.

Low plasma renin and reduced renin secretory responses to acute stimuli in conscious COX-2-deficient mice

In the current experiments, we determined the response of plasma renin concentration (PRC) to acute intraperitoneal administration of furosemide (40 mg/kg), hydralazine (2 mg/kg), isoproterenol (10 mg/kg), candesartan (50 microg), or quinaprilate (50 microg) in conscious wild-type (WT) and cyclooxygenase (COX)-2-/- mice on three different genetic backgrounds (mixed, C57BL/6, 129J). PRC was measured in plasma obtained by tail vein puncture. Basal PRC was significantly lower in COX-2-/- than WT mice independent of genetic background (51, 10, and 17% of WT in mixed, 129J, and C57BL/6). All five acute interventions caused significant increases of PRC in both COX-2+/+ and -/- mice, but the response was consistently less in COX-2-deficient mice (e.g., DeltaPRC in ng ANG I x ml(-1) x h(-1) caused by furosemide, isoproterenol, hydralazine, quinaprilate, or candesartan 4,699 +/- 544, 3,534 +/- 957, 2,522 +/- 369, 9,453 +/- 1,705, 66,455 +/- 21,938 in 129J WT, and 201 +/- 78, 869 +/- 275, 140 +/- 71, 902 +/- 304, 2,660 +/- 954 in 129J COX-2-/-). A low-NaCl diet and enalapril for 1 wk caused a 14-fold elevation of PRC in COX-2-/- mice and was associated with a greatly increased PRC response to acute furosemide (DeltaPRC 201 +/- 78 before and 15,984 +/- 2,397 after low Na/enalapril). As measured by radiotelemetry, blood pressure and heart rate responses to furosemide, hydralazine, isoproterenol, candesartan, or quinaprilate were not different between COX-2 genotypes. In conclusion, chronic absence of COX-2 reduces renin expression, release, and PRC and is associated with a reduced ability to alter PRC during acute stimulation regardless of the nature of the stimulus. COX-2 activity does not appear to be a mandatory and specific requirement for furosemide-stimulated renin secretion.

Increased renal vascular reactivity to ANG II after unilateral nephrectomy in the rat involves 20-HETE

This study examined the role of intrarenal ANG II in the renal vascular reactivity changes occurring in the remaining kidney undergoing adaptation following contralateral nephrectomy. Renal blood flow responses to intrarenal injections of ANG II (0.25 to 5 ng) were measured in anesthetized euvolemic male Wistar rats 1, 4, 12, and 24 wk after uninephrectomy (UNX) or sham procedure (SHAM). At week 4, renal vasoconstriction induced by 2 ng ANG II was greater in UNX (69 +/- 5%) than in SHAM rats (50 +/- 3%; P < 0.01). This response was inhibited, by 50 and 66%, and by 20 and 25%, in SHAM and UNX rats, after combined injections of ANG II and losartan, or PD-123319 (P < 0.05), respectively. Characteristics of ANG II receptor binding in isolated preglomerular resistance vessels were similar in the two groups. After prostanoid inhibition with indomethacin, renal vasoconstriction was enhanced by 42 +/- 8% (P < 0.05), only in SHAM rats, whereas after 20-HETE inhibition with HET0016, it was reduced by 53 +/- 16% (P < 0.05), only in UNX rats. These differences vanished after concomitant prostanoid and 20-HETE inhibition in the two groups. After UNX, renal cortical protein expression of cytochrome P-450 2c23 isoform (CYP2c23) and cyclooxygenase-1 (COX-1) was unaltered, but it was decreased for CYP4a and increased for COX-2. In conclusion, renal vascular reactivity to ANG II was significantly increased in the postuninephrectomy adapted kidney, independently of protein expression, but presumably involving interactions between 20-HETE and COX in the renal microvasculature and changes in the paracrine activity of ANG II and 20-HETE.

CGMP stimulates renin secretion in vivo by inhibiting phosphodiesterase-3

The interaction between renin, nitric oxide (NO), and its second messenger cGMP is controversial. cAMP is the stimulatory second messenger for renin but is degraded by phosphodiesterases (PDEs). We previously reported that increasing endogenous cGMP in rats by inhibiting its breakdown by PDE-5 stimulated renin secretion rate (RSR). This could be reversed by selective inhibition of neuronal nitric oxide synthase (nNOS). PDE-3 metabolizes cAMP, but this can be inhibited by cGMP, suggesting that renal cGMP could stimulate RSR by diminishing PDE-3 degradation of cAMP. Rats were anesthetized with Inactin before determination of blood pressure (BP), renal blood flow (RBF), and sampling of renal venous and arterial blood to determine RSR. In 13 rats, basal BP was 104 +/- 2 mmHg, RBF was 6.1 ml x min(-1) x g kidney wt(-1) and RSR was 2.9 +/- 1.4 ng ANG I x h(-1) x min(-1). Inhibiting PDE-5 with 20 mg/kg body wt i.p. Zaprinast did not change hemodynamic parameters but increased RSR fivefold to 12.2 +/- 4.9 ng ANG I x h(-1) x min(-1) (P < 0.05). Renal venous cAMP was increased by Zaprinast from 93.8 +/- 27.9 to 149.2 +/- 36.0 pM x min(-1) x g kidney wt(-1) (P < 0.05). When another 10 rats were treated with the PDE-3 inhibitor Milrinone (0.4 microg/min over 30 min, which did not affect hemodynamics), RSR was elevated to 10.4 +/- 4.4 ng ANG I x h(-1) x min(-1). Milrinone also increased renal venous cAMP from 212 +/- 29 to 304 +/- 29 pM x min(-1) x g kidney wt(-1) (P < 0.025). Administration of Zaprinast to rats pretreated with Milrinone (n = 10) did not further increase in RSR (7.5 +/- 3.3 ng ANG I x h(-1) x min(-1)). These results are consistent with endogenous renal cGMP inhibiting PDE-3, which diminishes renal metabolism of cAMP. The resulting increase in cAMP serves as an endogenous stimulus for renin secretion. This suggests a pathway by which NO can indirectly stimulate RSR through its second messenger cGMP.

Adenosine as a mediator of macula densa-dependent inhibition of renin secretion

Adenosine acting through A(1) adenosine receptors (A1AR) has been shown previously to be required for the vasoconstriction elicited by high luminal NaCl concentrations at the macula densa (MD). The present experiments were performed to investigate a possible role of A1AR in MD control of renin secretion in conscious wild-type (WT) and A1AR-deficient mice. The intravenous injection of NaCl (5% body wt) reduced plasma renin concentration (PRC; ng ANG I x ml(-1) x h(-1)) from 1,479 +/- 129 to 711 +/- 77 (P < 0.0001; n = 18) in WT mice but did not significantly change PRC in A1AR-/- mice (1,352 +/- 168 during control vs. 1,744 +/- 294 following NaCl; P = 0.19; n = 17). NaCl injections also caused a significant reduction in PRC in beta(1)/beta(2)-adrenergic receptor-/- mice (298 +/- 47 vs. 183 +/- 42; P = 0.03; n = 6). Injections of isotonic NaHCO(3) (5% body wt) elicited significant increases in PRC in both WT and A1AR-/- mice. NaCl as well as NaHCO(3) injections were accompanied by transient increases in blood pressure, heart rate, and activity that were similar in WT and A1AR-/- mice. The increase in PRC caused by an intraperitoneal injection of furosemide (40 mg/kg) was comparable in WT and A1AR-/- mice, and it was accompanied by similar transient increases in blood pressure, heart rate, and activity. Similarly, the stimulation of PRC caused by hydralazine was the same in WT and A1AR-/- mice. We conclude that the inhibition of renin secretion in response to an increase in NaCl at the MD requires A1AR and therefore appears to be adenosine dependent, whereas the stimulation of renin secretion during reductions in MD NaCl transport or arterial pressure does not require functional A1AR.

Influence of Prolonged Baroreflex Activation on Arterial Pressure in Angiotensin Hypertension

Despite recent evidence indicating sustained activation of the baroreflex during chronic infusion of angiotensin II (Ang II), sinoaortic denervation does not exacerbate the severity of the hypertension. Therefore, to determine whether Ang II hypertension is relatively resistant to the blood pressure-lowering effects of the baroreflex, the carotid baroreflex was electrically activated bilaterally for 7 days in 5 dogs both in the presence and absence of a continuous infusion of Ang II (5 ng/kg per minute) producing high physiological plasma levels of the peptide. Under control conditions, basal values for mean arterial pressure (MAP) and plasma norepinephrine concentration (NE) were 93+/-1 mm Hg and 99+/-25 pg/mL, respectively. By day 7 of baroreflex activation, MAP and NE were reduced to 72+/-4 mm Hg (-21+/-3 mm Hg) and 56+/-15 pg/mL, respectively, but PRA was unchanged (control=0.41+/-0.06 ng ANG I/mL per hour). All values returned to basal levels by the end of a 7-day recovery period. After 7 days of Ang II infusion, MAP increased from 93+/-3 to 129+/-3 mm Hg, whereas NE fell from 117+/-15 to 86+/-23 pg/mL. During the next 7 days of baroreflex activation/Ang II infusion, further reductions in NE were not statistically significant, and on the final day of baroreflex activation, the reduction in MAP was only 5+/-1 mm Hg, compared with 21+/-3 mm Hg in the control normotensive state. These findings indicate that long-term baroreflex-mediated reductions in arterial pressure are markedly diminished, but not totally eliminated, in the presence of hypertension produced by chronic infusion of Ang II.

Contribution of the basolateral isoform of the Na-K-2Cl−cotransporter (NKCC1/BSC2) to renin secretion

Acute administration of loop diuretics like furosemide leads to a stimulation of renin secretion, an effect thought to result from inhibition of Na-K-2Cl cotransporter (NKCC2)-mediated salt transport at the luminal surface of the macula densa (MD). However, loop diuretics also inhibit NKCC1, the second isoform of the Na-K-2Cl cotransporter, with similar potency. In the present study, we examined the influence of furosemide on renin secretion in NKCC1-deficient mice to distinguish between effects of the loop diuretic involving NKCC2 and, by implication, the MD pathway, and effects that might occur via inhibition of NKCC1. Baseline plasma renin concentration (PRC) was 1,212 +/- 211 in NKCC1+/+ (n = 13) and 3,851 +/- 579 ng ANG I.ml(-1).h(-1) in NKCC1-/- mice (n = 14; P = 0.00024). Acute administration of furosemide (50 mg/kg i.p.) increased PRC significantly to 9,324 +/- 1,018 ng ANG I.ml(-1).h(-1) in NKCC1+/+ (n = 13; P < 0.0001 compared with basal) and to 14,188 +/- 2,274 ng ANG I.ml(-1).h(-1) in NKCC1-/- mice [n = 14; P = 0.0002 compared with basal; P = 0.034 compared with wild-type (WT) plus furosemide]. Renin mRNA expression was about threefold higher in NKCC1-/- compared with WT mice. There was considerable recruitment of granular cells to upstream regions of afferent arterioles in NKCC1-/- mice. Patch-clamp studies in single juxtaglomerular granular (JG) cells from WT mice showed an approximately 10% increase in membrane capacitance during incubation with furosemide (10(-4) M), indicating a direct effect of the loop diuretic on renin secretion. No effect of furosemide on membrane capacitance was observed in JG cells from NKCC1-deficient mice. Furosemide (10(-3) M) significantly stimulated renin release from primary cultures of JG cells from WT mice, whereas no response was observed in NKCC1-/- mice. Our data suggest that a functional NKCC1 suppresses basal renin release, at least in part, through a direct effect on JG cells.

AT1receptor-mediated enhancement of collecting duct renin in angiotensin II-dependent hypertensive rats

Angiotensin II (ANG II)-infused rats exhibit increases in distal nephron renin expressed in principal cells of connecting tubules and collecting ducts. This study was performed to determine whether the augmentation of distal nephron renin involves ANG II type 1 (AT1) receptor activation. Male Sprague-Dawley rats (200-220 g) were divided into three groups: 1) sham operated (n = 8); 2) ANG II infused (80 ng/min, 13 days, n = 8); and 3) ANG II infused plus AT1 receptor blocker (ARB), olmesartan (5 mg/days, n = 8). ANG II infusion increased systolic blood pressure (BP; 178 +/- 4 vs. 122 +/- 1 mmHg; P < 0.001) and suppressed plasma renin activity (PRA; 0.08 +/- 0.1 vs. 5.3 +/- 0.8 ng ANG I x ml(-1) x h(-1)). ARB treatment prevented the increase in BP (113 +/- 6 mmHg) and led to increases in PRA (15.8 +/- 1.5 ng ANG I x ml(-1) x h(-1)). Renin protein levels measured in the kidney medulla, to avoid contribution from juxtaglomerular apparatus cells, were higher in ANG II-infused rats [1.64 +/- 0.3 vs. 1.00 +/- 0.1 densitometric units (DU) compared with sham-operated rats; P < 0.05], and ARB treatment prevented this increase (1.01 +/- 0.1). Similarly, renin immunoreactivity increased in medullary collecting ducts of ANG II-infused compared with sham-operated rats (2.5 +/- 0.3 vs. 1.0 +/- 0.2 DU; P < 0.001), which was also prevented by ARB (1.01 +/- 0.06). Renin qRTPCR in ANG II-infused rats showed higher mRNA levels in the kidney medulla compared with sham-operated rats (5.5 +/- 2.3 vs. 0.04 +/- 0.02 ratio to GAPDH mRNA levels; P < 0.001); however, renin transcript levels were normalized in the ARB-treated rats. These data demonstrate that the augmentation of distal nephron renin in ANG II-infused hypertensive rats is AT1 receptor mediated. The augmented distal tubular renin may contribute to increased intratubular ANG II levels and distal nephron sodium reabsorption in ANG II-dependent hypertension.

C-Jun NH2-terminal kinase mediation of angiotensin II-induced proliferation of human mesangial cells

Angiotensin II (ANG II) has been shown to activate c-Jun NH2-terminal kinase (JNK) in cultured mesangial cells, but the functional implication of this phenomenon remains to be determined, largely due to the lack of an effective approach to block JNK. Therefore, the present study was carried out to examine whether JNK is involved in ANG II-induced cell proliferation in cultured human mesangial cells (HMCs) with the use of a newly developed JNK-selective blocker, SP-600125. Within minutes, treatment with 100 nM ANG II activated all three members of MAP kinase family, including extracellular signal-regulated protein kinase (Erk) 1/2, JNK, and p38 in cultured HMCs, as assessed by immunoblotting detection of phosphorylation of MAP kinases. ANG II-dependent activation of JNK was further confirmed by detection of increased phosphorylation and transcription activity of c-Jun after the ANG II treatment. SP-600125 ranging from 5 to 10 microM almost completely abolished the activation of JNK by ANG II without affecting the activities of Erk1/2 and p38. After treatment with 100 ng ANG II, there was a steady increase in [3H]thymidine incorporation that was blocked by SP-60025 in a dose- and time-dependent manner. Similarly, SP-600125 dose dependently reduced the ANG II-induced increase in cell number. The antiproliferative effect of SP-60025 was further determined by cell-cycle analysis with flow cytometry. Twenty-four hours after ANG II treatment, 50% of the quiescent HMCs (G0/G1) progressed into the S phase, and the cell cycle progression was almost completely prevented in the presence of SP-60025. Our data suggest that JNK mediates the proliferative effect of ANG II in cultured HMCs and thus represents a novel therapeutic target for treatment of chronic renal diseases.

Permissive role of nitric oxide in macula densa control of renin secretion.

Experiments were performed in neuronal (nNOS)- and endothelial nitric oxide synthase (eNOS)-deficient mice to study the role of nitric oxide (NO) in macula densa control of renin secretion in vivo and in the isolated, perfused mouse kidney. Acute and chronic administration of loop diuretics was used as a method to stimulate macula densa-mediated renin secretion. Increases in plasma renin concentration (PRC) in response to a 3-day infusion of bumetanide (50 mg.kg(-1).day(-1)) or an acute injection of furosemide (50 mg/kg ip) were not markedly altered in nNOS-/- mice. Responses to furosemide were also maintained in eNOS-/- mice, but the administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) markedly attenuated the PRC response to furosemide in these mice. In the isolated kidney preparation, bumetanide caused similar relative increases in renin secretion in kidneys of wild-type, nNOS-/-, and eNOS-/- mice. Bumetanide only marginally increased renin secretion in L-NAME-treated kidneys, but the bumetanide effect was normalized by S-nitroso-N-acetyl-penicillamine. Basal PRC was significantly reduced in male nNOS-/- mice compared with nNOS+/+ (189 +/- 28 vs. 355 +/- 57 ng ANG I.ml(-1).h(-1); P = 0.017). There was no significant difference in PRC between eNOS+/+ and eNOS-/- mice. Basal renin secretion rates in perfused kidneys isolated from nNOS-/- or eNOS-/- mice were markedly reduced compared with wild-type controls. Our data suggest that NO generated by macula densa nNOS does not play a specific mediator role in macula densa-dependent renin secretion. However, NO independent of its exact source permits the macula densa pathway of renin secretion to function normally.

Extended acclimatization is required to eliminate stress effects of periodic blood-sampling procedures on vasoactive hormones and blood volume in beagle dogs.

Important in all experimental animal studies is the need to control stress stimuli associated with environmental change and experimental procedures. As the stress response involves alterations in levels of vasoactive hormones, ensuing changes in cardiovascular parameters may confound experimental outcomes. Accordingly, we evaluated the duration required for dogs (n = 4) to acclimatized to frequent blood sampling that involved different procedures. On each sampling occasion during a 6-week period, dogs were removed from their pen to a laboratory area and blood was collected either by venepuncture (days 2, 15, 34, 41) for plasma renin activity (PRA), epinephrine (EPI), norepinephrine, aldosterone, insulin, and atrial natriuretic peptide, or by cannulation (dogs restrained in slings; days 1, 8, 14, 22, 30, 33, 37, 40) for determination of haematocrit (HCT) alone (days 1 to 22) or HCT with plasma volume (PV; days 30 to 40). PRA was higher on days 2 and 15 compared with days 34 and 41 and had decreased by up to 48% by the end of the study (day 41 vs day 15; mean/SEM: 1.18/0.27 vs 2.88/0.79 ng ANG I/ml/h, respectively). EPI showed a time-related decrease from days 2 to 34, during which mean values had decreased by 51% (mean/SEM: 279/29 vs 134/20.9 pg/ml for days 2 and 34, respectively), but appeared stable from then on. None of the other hormones showed any significant variability throughout the course of the study. HCT was relatively variable between days 1 to 22 but stabilized from day 30, after which all mean values were approximately 6% lower than those between days 1 and 8. We conclude that an acclimatization period of at least 4 weeks is required to eliminate stress-related effects in dogs associated with periodic blood sampling.