William H. Beierwaltes

Angiotensin dependence of endothelium-mediated renal hemodynamics.

Endothelium-derived relaxing factor has been shown to regulate renal blood flow, and inhibition of its synthesis increases blood pressure and renal vascular resistance and decreases renal blood flow. Using the substrate antagonist NW-nitro-L-arginine methyl ester (L-NAME), we tested whether renal vasoconstriction induced by endothelium-derived relaxing factor synthesis inhibition could be mediated in part by angiotensin II. In 14 control rats, 10 mg/kg body wt L-NAME increased blood pressure from 106 +/- 6 to 126 +/- 6 mm Hg (p < 0.001), increased renal vascular resistance by 74% (from 19.3 +/- 2.6 to 33.6 +/- 2.9 resistance units), and decreased renal blood flow by 34% (from 5.9 +/- 0.5 to 3.9 +/- 0.3 ml.min-1.g kidney wt-1, p < 0.005). When six rats were treated with 10 mg/kg body wt of the angiotensin receptor antagonist DuP 753, L-NAME increased blood pressure from 84 +/- 4 to 106 +/- 4 mm Hg (p < 0.001); however, renal vascular resistance increased by only 27% (from 13 +/- 2 to 17 +/- 3 resistance units, p < 0.01; p < 0.05 different from control value) and renal blood flow was unchanged. Likewise, after pretreatment of six rats with 32 micrograms/100 g body wt of the angiotensin converting enzyme inhibitor enalaprilat, L-NAME increased blood pressure from 88 +/- 5 to 124 +/- 6 mm Hg (p < 0.001) and renal vascular resistance by 54% (from 12 +/- 1 to 18 +/- 3 resistance units, p < 0.01; p < 0.05 different from control value) but renal blood flow was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal nitric oxide and angiotensin II interaction in renovascular hypertension.

In two-kidney, one clip (2K1C) renovascular hypertension, blood flow is reduced to the clipped but not to the nonclipped kidney, despite elevated angiotensin II. To determine possible interactions between endothelium-derived nitric oxide and angiotensin, we studied bilateral renal blood flow using radioactive microspheres in anesthetized 2K1C hypertensive rats 4 weeks after clipping. We studied the response to nitric oxide synthesis inhibition with 10 mg/kg body wt NG-nitro-L-arginine- methyl ester (L-NAME) in hypertensive rats untreated (n = 5) or treated (n = 5) with 10 mg/kg body wt of the angiotensin II antagonist losartan. 2K1C rats had a blood pressure of 159 +/- 9 mm Hg, and renal blood flow to the clipped kidney was reduced 87% compared with the nonclipped kidney. L-NAME increased blood pressure 36 +/- 5 mm Hg and decreased renal blood flow in the nonclipped kidney 61% (4.9 +/- 0.5 to 1.9 +/- 0.4 mL/min per gram kidney weight, P < .001). Renal vascular resistance increased 200% (33.4 +/- 2.2 to 100.7 +/- 15.0 resistance units [RU], P < .005). Renal blood flow and resistance in the clipped kidney were unchanged by L-NAME. Treatment of 2K1C rats with losartan reduced blood pressure (154 +/- 8 to 116 +/- 11 mm Hg, P < .01), did not change blood flow in the nonclipped, but normalized it in the clipped kidney (4.8 +/- 0.8 mL/min per gram kidney weight).(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased intracellular calcium stimulates renin release via calcium-inhibitable adenylyl cyclase

Intracellular calcium and cAMP are the 2 second messengers that regulate renin release; cAMP stimulates renin release from juxtaglomerular (JG) cells, whereas increased intracellular calcium inhibits it. We hypothesized that decreased intracellular calcium acts by activating calcium-inhibitable isoforms of adenylyl cyclase, increasing cAMP, and stimulating renin secretion. We used a primary culture of JG cells isolated from C-57/B6 mice. Cells were plated to a density of 70% in serum-free medium and incubated for 2 hours with or without 100 &mgr;mol/L of the cytosolic calcium chelator 5′5-dimethyl-1,2-bis-(2-aminophenoxy)-ethane-N,N,N′,N′-tetra-acetic acid (BAPTA-AM) to decrease intracellular calcium. JG cell cAMP content and renin release were determined by radioimmunoassay. Intracellular cAMP content was 4.04±0.92 pM/mL per milligram of protein, and it increased by125±33% (P<0.01) with BAPTA-AM. Basal renin was 1.28±0.40 &mgr;g of angiotensin I per milliliter per hour per milligram of protein, and BAPTA-AM increased it by 182±62% (P<0.025). Western blots using an antibody that recognizes adenylyl cyclase types V and VI yielded a characteristic band of ≈135 kDa. When primary cultures of isolated JG cells were tested for the calcium-inhibitable isoforms of adenylyl cyclase, they showed intense focal cytoplasmic staining. Cells stained for both renin and adenylyl cyclase V/VI showed colocalization in the cytoplasm, primarily on the granules. An adenylyl cyclase inhibitor (SQ 22,536) completely blocked BAPTA-AM–stimulated renin release and JG cell cAMP content. We conclude that calcium-inhibitable isoform(s) of adenylyl cyclase (types V and/or VI) exist within the JG cell. Thus, decreased intracellular calcium stimulates adenylyl cyclase, resulting in cAMP synthesis and, consequently, renin release.

Adenylyl Cyclase Isoform V Mediates Renin Release From Juxtaglomerular Cells

We have shown previously that decreasing intracellular calcium in the juxtaglomerular cells increases both cAMP formation and renin release. We hypothesized that this is because of an interaction between intracellular calcium and the calcium-inhibitable isoform of adenylyl cyclase, type-V. We used primary cultures of juxtaglomerular cells isolated from C-57/B6 mice at 70% to 80% confluence. Western blots were performed on isolated juxtaglomerular cells using antibodies against either of the 2 calcium inhibitable isoforms of adenylyl cyclase, types-V and -VI. Only the antibody against adenylyl cyclase-V gave us a strong band at 120 kDa as expected. Immunolabeling in juxtaglomerular cells with confocal microscopy found immunofluorescence for the adenylyl cyclase-V–specific antibody compared with either negative controls or cells stained with the adenylyl cyclase-VI antibody. Reducing isolated juxtaglomerular intracellular calcium with 100 &mgr;mol/L of the cytosolic calcium chelator BAPTA-AM stimulated both cAMP (3.49±0.70 to 10.09±0.81 pmol/mL per milligram of protein; P<0.002) and renin release (1001.8±81.5 to 1648.0±139.1 ng of angiotensin I per milliliter per hour per milligram of protein; P<0.01). The selective adenylyl cyclase-V inhibitor NKY80 completely blocked both BAPTA-AM–stimulated cAMP formation and renin release. We conclude that lowering intracellular calcium is permissive, allowing an increased activity of the calcium-inhibitable isoform adenylyl cyclase-V (but not adenylyl cyclase-VI) in the juxtaglomerular cell, producing cAMP, which stimulates renin secretion.

Expression and Function of the Calcium-Sensing Receptor in Juxtaglomerular Cells

Calcium-sensing receptors sense and translate micromolar changes of extracellular calcium into changes in intracellular calcium. Renin, a component of the renin-angiotensin system, is synthesized by, stored in, and released from the juxtaglomerular cells through a cAMP-dependent pathway. Increased intracellular calcium inhibits the adenylyl cyclase isoform type V, cAMP formation, and renin release from juxtaglomerular cells. We hypothesized that calcium-sensing receptors are expressed in juxtaglomerular cells and mediate changes in intracellular calcium and renin release. To test this we used primary cultures of isolated mouse juxtaglomerular cells in which we ran RT-PCR, Western blots, and immunofluorescence. RT-PCR showed a positive band at the expected 151 bp consistent with calcium-sensing receptor. Western blots showed a 130- to 150-kDa band confirming the calcium-sensing receptor in juxtaglomerular cells. Immunofluorescence and confocal microscopy using 2 different antibodies against the calcium-sensing receptor in juxtaglomerular cells showed positive fluorescence in the juxtaglomerular cells, which also had positive labeling for renin. To test whether calcium-sensing receptors regulate renin release, juxtaglomerular cells were incubated with a calcium-sensing receptor agonist, the calcimimetic cinacalcet-HCl, at concentrations of 50 and 1000 nmol/L in 0.25 mmol/L of calcium medium. Cinacalcet-HCl decreased juxtaglomerular cell cAMP formation to 47.3±6.8% and 44.2±9.7% of basal, respectively (P<0.001), and decreased renin release from 541.9±86.2 to 364.6±64.1 (P<0.05) and 279.6±56.9 (P<0.005) ng of angiotensin I per milliliter per hour per milligram of protein, respectively. We conclude that juxtaglomerular cells express the calcium-sensing receptor and that their activation leads to inhibition of adenylyl cyclase-V activity, decreasing cAMP formation and suppressing renin release.

Arginine vasopressin-induced renal vasodilation mediated by nitric oxide.

The vasoconstrictor vasopressin has been reported to induce paradoxical local vasodilation in the basilar vasculature through stimulation of the endothelium-derived relaxing factor nitric oxide (NO). We investigated the possibility that at subpressor doses, exogenous arginine vasopressin (AVP) might have a similar effect in the kidney. Ten Inactin-anesthetized rats were infused with sequential doses of AVP from 25 to 6,400 microU/min in 30-min increments. Subpressor infusion resulted in progressive renal vasodilation; renal blood flow (RBF) increased significantly going from 14 +/- 6% above basal at 200 microU/min (p < 0.02) to 27 +/- 5% (p < 0.01) at 1,600 microU/min accompanied by a 24 +/- 5% decrease in renal vascular resistance (RVR). At 6,400 microU/min, blood pressure (BP) increased 29 +/- 6 mm Hg and RVR increased. A second group of 8 rats were first given 10 mg/kg b.w. of the NO synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME) before infusion of AVP. L-NAME increased BP 22 +/- 3 mm Hg (p < 0.001), and decreased RBF 16 +/- 3% (p < 0.005). After L-NAME, no dose of AVP had any further effect on either BP, RBF, or RVR. Continuous infusion of a single subpressor dose of 100 microU AVP resulted in a 26% increase in RBF (from 7.52 +/- 0.68 to 9.49 +/- 0.54 ml/min/g kidney weight, p < 0.001). AVP doubled urinary cyclic guanosine monophosphate excretion, a marker for renal NO synthesis, from 8.51 +/- 1.01 to 17.48 +/- 4.26 pM/min (p < 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic cyclooxygenase-2 inhibition blunts low sodium-stimulated renin without changing renal haemodynamics.

Background Cyclooxygenase-2 (COX-2), the inducible isoform of cyclooxygenase, is found in the macula densa of the renal cortex and is upregulated by dietary sodium restriction. Because of this discrete cortical localization, we hypothesized that COX-2 plays a role in the chronic stimulation of renin via the macula densa pathway. Methods We examined the effect of the selective COX-2 inhibitor NS 398 in male Sprague–Dawley rats. Results A low sodium diet (0.02% NaCl) for 14 days elevated plasma–renin activity (PRA) nine-fold, from 6.1 ± 2.0 to 54.9 ± 6.5 ng angiotensin I (Ang I)/ml per h (P < 0.0001). Selective COX-2 inhibition with NS 398 had no effect on PRA in animals on normal sodium (5.1 ± 1.3 ng Ang I/ml per h), but decreased PRA by 41% in sodium-restricted rats, to 33.3 ± 3.6 ng Ang I/ml per h (P < 0.05). Chronic treatment with NS 398 did not decrease renal renin content (31.8 ± 1.8 versus 33.5 ± 2.6 ng Ang I/mg per h, with NS 398 versus controls), nor did it influence systemic blood pressure or renal haemodynamics. Neither urinary sodium excretion nor prostaglandin (PG)E2 excretion was altered in rats given NS 398. Chronic treatment with the non-selective COX inhibitor indomethacin during sodium restriction over 5 days reduced PRA by 35%, from 29.36 ± 4.81, to 19.13 ± 2.88 ng Ang I/ml per h (P < 0.05). Indomethacin had no effect on blood pressure or renal blood flow but reduced urinary PGE2 excretion by 70%. Conclusions One component of the chronic stimulation of PRA by dietary sodium restriction via the macula densa pathway appears to involve the induction of COX–2.

Influence of Nitric Oxide in the Chronic Phase of Two-Kidney, One Clip Renovascular Hypertension

Chronic two-kidney, one clip (2K1C) renovascular hypertension is characterized by a largely angiotensin-independent elevated blood pressure (BP). We hypothesized that the long-term effect of hypertension would compromise endothelium-derived nitric oxide (NO) and diminish its influence in controlling renal perfusion. We determined the influence of endothelium-derived NO on renal hemodynamics and the angiotensin-NO interaction regulation of renal perfusion in rats with chronic 2K1C hypertension. Renal blood flow (RBF) was measured by radioactive microspheres in rats with either early-phase (4 weeks after clipping, n=7) or chronic-phase (13 to 16 weeks after clipping, n=7) 2K1C hypertension. The systemic and renal response to NO synthesis inhibition was determined with 10 mg/kg body wt N omega-nitro-L-arginine methyl ester (L-NAME). In rats with early-phase 2K1C hypertension, BP was 149+/-3 mm Hg, which increased by 42+/-3 mm Hg with L-NAME (P<.001). L-NAME decreased RBF by 20% (P<.02) and 17% (P<.005) and increased renal vascular resistance (RVR) by 58% (P<.005) and 62% (P<.02) in the nonclipped and clipped kidneys, respectively. In rats with chronic 2K1C hypertension, BP was 166+/-3 mm Hg, and L-NAME increased this by 35+/-6 mm Hg (P<.001). In the nonclipped and clipped kidneys of chronic 2K1C hypertensive rats, L-NAME decreased RBF by 20% (P<.01) and 17% (P<.01) and increased RVR by 51% (P<.005) and 60% (P<.02), respectively. There were no differences in L-NAME-induced changes between early- and chronic-phase 2K1C hypertensive rats. Next, we treated seven chronic-phase 2K1C hypertensive rats with 10 mg/kg body wt losartan, which reduced BP by only 7.7% (P<.005). After losartan, L-NAME increased BP by 41+/-3 mm Hg (P<.001), decreased RBF to the nonclipped kidney by 44% (P<.05), and increased RVR by 110% (P<.005); the decrease in RBF was significantly greater compared with untreated chronic-phase controls (P<.05). In the clipped kidney, L-NAME decreased RBF by 26% (P<.05) and increased RVR by 76% (P <.05). Thus, angiotensin blockade did not attenuate the systemic or renal vasoconstriction to L-NAME. Our results suggest that in both early and chronic phases of 2K1C hypertension, NO contributes significant dilator tone to buffer the hypertension and maintains perfusion of both kidneys by counterbalancing angiotensin-independent vasoconstriction.

Extracellular ATP Stimulates NO Production in Rat Thick Ascending Limb

NO produced by NO synthase (NOS) 3 acts as an autacoid to regulate NaCl absorption in the thick ascending limb. ATP induces NO production by NOS 3 in endothelial cells. We hypothesized that extracellular ATP activates NOS in thick ascending limbs through P2 receptors. To test this, we measured intracellular NO production using the NO-selective fluorescent dye DAF-2 in suspensions of rat medullary thick ascending limbs. We found that ATP increased DAF-2 fluorescence in a concentration-dependent manner, reaching saturation at ≈200 &mgr;mol/L with an EC50 of 37 &mgr;mol/L. The increase was blunted by 74% by the nonselective NOS inhibitor l-&ohgr;-nitro-arginine-methyl-ester (2 mmol/L; 60±7 versus 16±6 arbitrary fluorescence units; P<0.02; n=5). In the presence of the P2 receptor antagonist suramin (300 &mgr;mol/L), ATP-induced NO production was reduced by 64% (101±11 versus 37±5 arbitrary fluorescence units; P<0.002; n=5). Blocking ATP hydrolysis with a 5′-ectonucleotidase inhibitor, ARL67156 (30 &mgr;mol/L) enhanced the response to ATP and shifted the EC50 to 0.8 &mgr;mol/L. In the presence of ARL67156, the EC50 of the P2X-selective agonist β,γ-methylene-adenosine 5′-triphosphate was 4.8 &mgr;mol/L and the EC50 for the P2Y–selective agonist UTP was 40.4 &mgr;mol/L. The maximal responses for both agonists were similar. Taken together, these data indicate that ATP stimulates NO production in the thick ascending limb primarily through P2X receptor activation and that ATP hydrolysis may regulate NO production.

Role of Nitric Oxide in the Renal Hemodynamic Response to Unilateral Nephrectomy

Reduction of renal mass by unilateral nephrectomy results in an immediate increase in renal blood flow (RBF) to the remnant kidney, followed by compensatory renal hypertrophy. Whether the increase in RBF after unilateral nephrectomy is mediated by nitric oxide (NO) was tested. It was found that immediately after nephrectomy, blood flow to the remaining kidney increased by 8% (P < 0.01), and inhibition of NO synthesis with Nomega-nitro-L-arginine methyl ester (L-NAME) blocked the increase in RBF. In addition, 2 d after nephrectomy, there was a 49% increase in RBF (corrected per gram of kidney weight), a 25% increase at 7 and 14 d, and a 16% increase after 28 d. Acute inhibition of NO synthesis with L-NAME in uninephrectomized rats caused a greater decrease in RBF on days 2 and 7 compared with controls, whereas by 14 and 28 d, the response to L-NAME was similar to controls. Urinary excretion of cyclic guanosine monophosphate, a marker for renal NO production, increased 2.5-fold by 2 d after uninephrectomy (P < 0.005) and remained at this level through 28 d. Pretreating rats chronically with a subpressor dose of L-NAME beginning 2 d before nephrectomy blocked the increase in RBF seen at 2 and 7 d and retarded the renal hypertrophy that should have developed by 7 d. It is concluded that after unilateral nephrectomy, immediate and sustained increases in RBF are mediated at least in part by NO. The hypertrophic response to unilateral nephrectomy may be partially initiated by the signal of hemodynamic changes.