Evidence that renal arginine transport is impaired in spontaneously hypertensive rats.

Low renal nitric oxide (NO) bioavailability contributes to the development and maintenance of chronic hypertension. We investigated whether impaired l-arginine transport contributes to low renal NO bioavailability in hypertension. Responses of renal medullary perfusion and NO concentration to renal arterial infusions of the l-arginine transport inhibitor l-lysine (10 µmol·kg(-1)·min(-1); 30 min) and subsequent superimposition of l-arginine (100 µmol·kg(-1)·min(-1); 30 min), the NO synthase inhibitor N(G)-nitro-l-arginine (2.4 mg/kg; iv bolus), and the NO donor sodium nitroprusside (0.24 µg·kg(-1)·min(-1)) were examined in Sprague-Dawley rats (SD) and spontaneously hypertensive rats (SHR). Renal medullary perfusion and NO concentration were measured by laser-Doppler flowmetry and polarographically, respectively, 5.5 mm below the kidney surface. Renal medullary NO concentration was less in SHR (53 ± 3 nM) compared with SD rats (108 ± 12 nM; P = 0.004). l-Lysine tended to reduce medullary perfusion (-15 ± 7%; P = 0.07) and reduced medullary NO concentration (-9 ± 3%; P = 0.03) while subsequent superimposition of l-arginine reversed these effects of l-lysine in SD rats. In SHR, l-lysine and subsequent superimposition of l-arginine did not significantly alter medullary perfusion or NO concentration. Collectively, these data suggest that renal l-arginine transport is impaired in SHR. Renal l-[(3)H]arginine transport was less in SHR compared with SD rats (P = 0.01). Accordingly, we conclude that impaired arginine transport contributes to low renal NO bioavailability observed in the SHR kidney.

Role of L-arginine uptake mechanisms in renal blood flow responses to angiotensin II in rats.

AIM: To examine whether reduced renal arginine transport increases the responsiveness of the renal circulation to angiotensin II in salt sensitivity, renal perfusion responses to angiotensin II were examined in the presence of L-arginine transport inhibitor, L-lysine and subsequent L-arginine in Sprague Dawley (SD) and Dahl salt-sensitive (Dahl S) rats. METHODS: Laser Doppler probes and a transonic flow probe were used to measure regional renal perfusion and total renal perfusion respectively. Renal perfusion responses to intravenous (i.v.) angiotensin II were sequentially examined under control conditions and during i.v. infusion of L-lysine, L-arginine or nitric oxide synthase inhibitor, N(G)-nitro-L-arginine. RESULTS: Angiotensin II (10 and 100 ng kg(-1) min(-1) , i.v.) reduced total renal (-10 ± 3 and -36 ± 5%) and cortical (-10 ± 2 and -28 ± 4%) but not medullary perfusion in SD rats. In these rats L-lysine enhanced the renal perfusion response (P = 0.003), whereas subsequent L-arginine reversed this effect (P = 0.04). Angiotensin II reduced total renal, cortical and medullary perfusion in Dahl S rats. In Dahl S rats fed high salt, L-lysine did not affect renal perfusion responses to angiotensin II, but subsequent L-arginine blunted the renal blood flow response (P = 0.01) and increased the medullary perfusion during angiotensin II infusion (P = 0.006). CONCLUSION: Intact renal L-arginine transport attenuates the vasoconstrictor effects of circulating angiotensin II in the renal cortex in SD rats. L-arginine also plays an important role in protecting the renal medullary circulation from the ischemic effects of angiotensin II in Dahl S rats.

Importance of the renin-angiotensin system in the regulation of arterial blood pressure in conscious mice and rats.

AIM: The present experiments were designed to determine the mechanism(s) for increased sensitivity to blockade of the renin-angiotensin system in mice in comparison with rats. METHODS: Mice and rats, with indwelling femoral arterial and venous catheters, were chronically administered angiotensin II or pharmacological inhibitors of the renin-angiotensin system as sodium intake was altered. RESULTS: Increasing sodium intake led to suppression of circulating renin, angiotensin II, and aldosterone in rats and mice in the absence of alterations in arterial blood pressure. Additional experiments demonstrated that continuous intravenous infusion of angiotensin II (20 ng kg(-1) min(-1)) significantly increased arterial blood pressure by approximately 35 mmHg in conscious rats at all levels of sodium intake (n = 6). In contrast, arterial pressure was unaffected by angiotensin II infusion in conscious mice under conditions of low sodium intake, although arterial pressure was increased by 16 mmHg when mice were administered a high sodium intake while infused with angiotensin II (n = 6). In comparison, blockade of the endogenous renin-angiotensin system led to significantly greater effects on arterial pressure in mice than rats. Continuous infusion of captopril (30 microg kg(-1) min(-1)) or losartan (100 microg kg(-1) min(-1)) resulted in a 55-90% greater fall in blood pressure in conscious mice in comparison with conscious rats. CONCLUSION: The present studies indicate that arterial pressure in mice is more dependent upon the endogenous renin-angiotensin system than it is in rats, but mice are more resistant to the hypertensive effects of exogenous angiotensin II.

Role of the renin-angiotensin system during alterations of sodium intake in conscious mice.

The present studies were performed to quantify circulating components of the renin-angiotensin-aldosterone axis and to determine the functional importance of this system during alterations in sodium intake in conscious mice. Increasing sodium intake from approximately 200 to 1,000 microeq/day significantly decreased plasma renin concentration from 472 +/- 96 to 304 +/- 83 ng ANG I. ml(-1). h(-1) (n = 5) but did not alter plasma renin activity from the low-sodium level of 7.7 +/- 1.1 ng ANG I. ml(-1). h(-1). Despite the elevated plasma renin concentration, plasma ANG II in mice on low-sodium level averaged 14 +/- 3 pg/ml and was significantly suppressed to 6 +/- 1 pg/ml by high-sodium intake (n = 7). Consistent with the modulation of ANG II, plasma aldosterone significantly decreased from 41 +/- 8 to 8 +/- 3 ng/dl when sodium intake was elevated (n = 6). In a final set of experiments, the continuous infusion of ANG II (20 ng. kg(-1). min(-1)) led to a mild salt-sensitive increase in mean arterial pressure from 108 +/- 2 to 131 +/- 2 mmHg as sodium intake was varied from low to high (n = 7). In vehicle-infused mice, mean arterial pressure was unaltered from 109 +/- 2 mmHg when sodium intake was increased (n = 6). These studies indicate that the physiological suppression of circulating ANG II may be required to maintain a constancy of arterial pressure during alterations in sodium intake in normal mice.

The influence of nitric oxide synthase 1 on blood flow and interstitial nitric oxide in the kidney.

The role of nitric oxide (NO) produced by NO synthase 1 (NOS1) in the renal vasculature remains undetermined. In the present study, we investigated the influence of systemic inhibition of NOS1 by intravenous administration of N(omega)-propyl-L-arginine (L-NPA; 1 mg. kg(-1). h(-1)) and N(5)-(1-imino-3-butenyl)-L-ornithine (v-NIO; 1 mg. kg(-1). h(-1)), highly selective NOS1 inhibitors, on renal cortical and medullary blood flow and interstitial NO concentration in Sprague-Dawley rats. Arterial blood pressure was significantly decreased by administration of both NOS1-selective inhibitors (-11 +/- 1 mmHg with L-NPA and -7 +/- 1 mmHg with v-NIO; n = 9/group). Laser-Doppler flowmetry experiments demonstrated that blood flow in the renal cortex and medulla was not significantly altered following administration of either NOS1-selective inhibitor. In contrast, the renal interstitial level of NO assessed by an in vivo microdialysis oxyhemoglobin-trapping technique was significantly decreased in both the renal cortex (by 36-42%) and medulla (by 32-40%) following administration of L-NPA (n = 8) or v-NIO (n = 8). Subsequent infusion of the nonspecific NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 50 mg. kg(-1). h(-1)) to rats pretreated with either of the NOS1-selective inhibitors significantly increased mean arterial pressure by 38-45 mmHg and significantly decreased cortical (25-29%) and medullary (37-43%) blood flow. In addition, L-NAME further decreased NO in the renal cortex (73-77%) and medulla (62-71%). To determine if a 40% decrease in NO could alter renal blood flow, a lower dose of L-NAME (5 mg. kg(-1). h(-1); n = 8) was administered to a separate group of rats. The low dose of L-NAME reduced interstitial NO (cortex 39%, medulla 38%) and significantly decreased blood flow (cortex 23-24%, medulla 31-33%). These results suggest that NOS1 does not regulate basal blood flow in the renal cortex or medulla, despite the observation that a considerable portion of NO in the renal interstitial space appears to be produced by NOS1.

Comparison of arterial blood pressure in different strains of mice.

The present study was performed to compare the resting level of arterial blood pressure when monitored for 24 h/day in outbred Swiss Webster (SW) and inbred C57BL/ 6J, A/J, C3HeB/FeJ, and SWR/J mice. Mean arterial pressure (MAP) and heart rate (HR) varied throughout the day, with maximal values observed in the hours of darkness. Systolic (SAP), MAP, and diastolic (DAP) arterial blood pressure averaged 122 +/- 2, 112 +/- 2, and 102 +/- 2 mm Hg, respectively, in conscious SW mice (N = 6). No differences were detected in the 24-h averages of MAP between SW, C57BL/6J (N = 7), A/J (N = 5), C3HeB/FeJ (N = 5), or SWR/J (N = 7) mice maintained on a normal sodium diet. Average daily heart rate (HR) was highest in the C3HeB/FeJ (665 +/- 15 beats/min) and lowest in the C57BL/6J (594 +/- 9 beats/min). The MAP was significantly increased in SW mice administered L-NAME (133 +/- 2 mm Hg, N = 5) and significantly decreased in SW mice administered captopril (99 +/- 2 mm Hg, N = 5). These studies demonstrate similar levels of resting arterial pressure in different mouse strains under baseline conditions.

Measurement of regional blood flow in the kidney using laser-Doppler flowmetry.

The study of regional blood flow in the kidney has been fraught with difficulties. Quantitation of perfusion of the inner cortex and medulla of the kidney has been especially challenging to measure because there is no way to directly visualize the large or small blood vessels in these areas. Furthermore, there has been a lack of reliable techniques to measure tissue blood flow in the kidney. In order to appreciate fully the technical demands of measuring changes in regional blood flow within the kidney, it is necessary to first understand the complex structure of the renal microcirculation.

Characterization of L-arginine transporters in rat renal inner medullary collecting duct.

Previous work from our laboratory has demonstrated that the inner medullary collecting duct (IMCD) expresses a large amount of nitric oxide synthase (NOS) activity. The present study was designed to characterize the transport of NOS substrate, L-arginine, in a suspension of bulk-isolated IMCD cells from the Sprague-Dawley rat kidney. Biochemical transport studies demonstrated an L-arginine transport system in IMCD cells that was saturable and Na(+) independent (n = 6). L-Arginine uptake by IMCD cells was inhibited by the cationic amino acids L-lysine, L-homoarginine, and L-ornithine (10 mmol/l each) and unaffected by the neutral amino acids L-leucine, L-serine, and L-glutamine. Both L-ornithine (n = 6) and L-lysine (n = 6) inhibited NOS enzymatic activity in a dose-dependent manner in IMCD cells, supporting the important role of L-arginine transport for NO production by this tubular segment. Furthermore, RT-PCR of microdissected IMCD confirmed the presence of cationic amino acid transporter CAT1 mRNA, whereas CAT2A, CAT2B, and CAT3 were not detected. These results indicate that L-arginine uptake by IMCD cells occurs via system y(+), is encoded by CAT1, and may participate in the regulation of NO production in this renal segment.

Control of arterial blood pressure and renal sodium excretion by nitric oxide synthase in the renal medulla.

Work in our laboratory has focused on the role of nitric oxide synthase (NOS) in the regulation of renal medullary function. Biochemical studies demonstrated that the renal medulla is enriched in immunoreactive NOS protein and NOS enzymatic activity when compared with the renal cortex. Further experiments showed large amounts of NOS activity in the inner medullary collecting ducts, while moderate NOS activity was found in glomeruli and vasa recta and minimal NOS activity was detected in other nephron segments examined. In subsequent functional studies, selective renal medullary infusion of NOS stimulators (bradykinin or acetylcholine) or inhibitors (L-NAME) preferentially altered medullary blood flow. The alterations in medullary flow were associated with parallel changes in sodium and water excretion. Similar to the effects observed in anaesthetized rats, chronic infusion of L-NAME directly into the renal medullary interstitial space of conscious, uninephrectomized SD rats selectively decreased renal medullary blood flow throughout a 5-day L-NAME infusion period. The decrease in medullary blood flow was associated with retention of sodium and the development of hypertension, and the effects were reversible. In contrast to the effects of chronic NOS inhibition, renal medullary infusion of NOS substrate L-arginine prevented the development of sodium-sensitive hypertension in the Dahl salt-sensitive rat placed on a high sodium diet. The data reviewed in this paper indicate that NOS isoforms expressed in the renal medulla have a potent influence on renal medullary tubular and vascular function with consequential effects on fluid and electrolyte homeostasis and arterial blood pressure.

Nitric oxide synthase activity and isoforms in rat renal vasculature.

Experiments were performed to quantify nitric oxide synthase (NOS) activity and identify the NOS isoforms present in the Sprague-Dawley rat renal vasculature. NOS enzymatic activity was measured by adding [(3)H]arginine to microdissected renal blood vessels and quantifying the conversion to [(3)H]citrulline by reverse-phase high-performance liquid chromatography. Total NOS activity was greatest in microdissected vasa recta (123+/-41 pmol. mg(-1). h(-1), n=5) and significantly less in glomeruli (46+/-9 pmol. mg(-1). h(-1), n=6) and afferent arterioles (42+/-10 pmol. mg(-1). h(-1), n=6) and averaged <5 pmol. mg(-1). h(-1) in arcuate (n=8) and interlobular (n=9) arteries. Addition of 1.0 mmol/L EDTA to the reaction decreased NOS activity to <5 pmol. mg(-1). h(-1) in afferent arterioles, glomeruli, and vasa recta (n=5 each), indicating that the NOS enzymatic activity in these segments is primarily a result of constitutive NOS. Both neuronal and endothelial NOS mRNA were identified in each vascular segment by reverse transcription-polymerase chain reaction, but inducible NOS mRNA was detected only in microdissected arcuate arteries. The present experiments indicate that the vasa recta, glomeruli, and afferent arterioles contain large amounts of calcium-dependent NOS enzymatic activity and that neuronal NOS and endothelial NOS mRNA are present in these segments.

Increase in renal medullary nitric oxide synthase activity protects from norepinephrine-induced hypertension.

Studies were performed in conscious Sprague-Dawley rats to determine the role of the alpha(2)-adrenergic receptor-mediated increase in the renal medullary nitric oxide synthase (NOS) activity as a counterregulatory mechanism of blood pressure control in response to increased renal adrenergic stimulation. A subpressor dose of norepinephrine (NE, 8 microg. kg(-1). h(-1)) was infused intravenously, and NOS activity was determined with arginine-citrulline conversion by high-performance liquid chromatography in renal cortical and outer and inner medullary tissues. It was found that after 7 days of intravenous NE infusion, NOS activity was significantly higher in both the outer and inner medullary tissues (158+/-45 versus 30+/-24 pmol. mg(-1). h(-1) [outer medulla] and 5.1+/-0.7 versus 2.0+/-0.5 nmol. mg(-1). h(-1) [inner medulla] for NE-treated versus control rats, respectively). To determine whether the increase of NOS activity was mediated through renal medullary alpha(2)-receptors, the receptor antagonist rauwolscine (RAU, 1 microg. kg(-1). min(-1)) was infused via an implanted renal medullary interstitial catheter, and the consequences of intravenous NE administration were evaluated. NOS activity was significantly lower in the RAU-infused animals and did not increase with infusion of NE. To determine the systemic effects of the renal medullary alpha(2)-receptors, studies were performed to determine the consequences of chronic intravenous infusion of subpressor amounts of NE in the presence and absence of renal medullary alpha(2)-receptor inhibition. Under conditions in which RAU was continuously infused into the renal medulla, the same subpressor dose of NE caused sustained and reversible hypertension (mean arterial pressure increased from 120+/-3 to 131+/-3 mm Hg). Chronic blunting of the renal medullary NOS activity with N(G)-nitro-L-arginine methyl ester (75 microg. kg(-1). h(-1)) also enabled NE to produce a significant rise in mean arterial pressure (from 117+/-2 to 134+/-4 mm Hg). We conclude that the hypertensive effects of moderate elevations of renal adrenergic activity were chronically buffered by the alpha(2)-receptor-mediated increase in NOS activity within the renal medulla.

Use of antisense techniques in rat renal medulla.

The experiments outlined in this chapter utilized a novel infusion technique to deliver an antisense oligonucleotide (and an enzyme inhibitor) directly into the renal medullary interstitial space of conscious rats. Antisense treatment led to a selective decrease in nNOS protein and reduced total NOS enzymatic activity in the renal medulla of the infused rats while three other gene products found in the renal medulla (iNOS, eNOS, and beta-actin) were unaltered. Physiological studies in rats demonstrated that infusion of the antisense oligonucleotide into the renal medullary interstitial space increased mean arterial pressure. The increase in blood pressure was dependent on the sodium intake of the rats, was not mimicked when a scrambled oligonucleotide was infused, and was reversible when the antisense infusion was stopped. To confirm the functional data obtained with the antisense oligonucleotide, renal medullary interstitial infusion of the nNOS enzyme inhibitor 7-NI was also shown to lead to a similar increase in arterial pressure and decrease in total NOS activity in the renal medulla. Together, the antisense oligonucleotide, the enzyme inhibitor, and the interstitial infusion technique were used to demonstrate that nNOS found in the renal medulla is important in the chronic regulation of arterial pressure. The experiments summarized in this chapter outline a strategy that can potentially be used to examine the functional effects of many different proteins in this region of the body. Through the use of antisense oligonucleotides and other pharmacological agents, we can hope to gain a more comprehensive understanding of the factors that control renal medullary tubular and vascular function and consequently fluid and electrolyte homeostasis and blood pressure.

Quantification of nitric oxide synthase activity in microdissected segments of the rat kidney.

This study was designed to quantify nitric oxide synthase (NOS) activity in microdissected glomeruli (Glm), pars convoluta, pars recta, cortical collecting duct, cortical thick ascending limb, outer medullary collecting duct, medullary thick ascending limb and thin limb, inner medullary collecting duct (IMCD) and thin limb, and vasa recta (VR). Total protein from microdissected segments was incubated with L-[3H]arginine and appropriate cofactors, and the L-arginine and converted L-citrulline were separated by reverse-phase HPLC and radiochemically quantitated. NOS activity was found to be greatest in IMCD (11.5 +/- 1.0 fmol citrulline. mm-1. h-1) and moderate in Glm (1.9 +/- 0.3 fmol. glomerulus-1. h-1) and VR (3.2 +/- 0.8 fmol. mm-1. h-1). All other renal structures studied exhibited significantly less NOS activity. The mRNA for NOS isoforms in the NOS activity-positive segments was then identified by RT-PCR. The IMCD contained mRNA for neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS), but Glm and VR only expressed the mRNA for nNOS and eNOS. These experiments demonstrate that the greatest enzymatic activity for NO production in the kidney is in the IMCD, three- to sixfold less activity is present in the Glm and VR, and minimal NOS activity is found in other segments studied.

Chronic sodium balance and blood pressure response to captopril in conscious mice.

The influence of chronic administration of the converting enzyme inhibitor captopril on blood pressure and sodium balance was evaluated in conscious Swiss Webster mice. Arterial pressure was measured with chronic indwelling catheters, and sodium balance was determined by infusing sodium intravenously in isotonic saline and collecting urine 24 h/d. Experiments to validate sodium balance measurements in mice demonstrated recovery of 100+/-3% of sodium intake under steady-state conditions (n=20 mice on 70 individual days, sodium intake range 160 to 1000 micromol/d). It was further demonstrated that mean arterial pressure, heart rate, and body weight were unaltered from 115+/-7 mm Hg, 646+/-12 bpm, and 34+/-0.6 g, respectively, as sodium intake was increased stepwise from 150 to 900 micromol NaCl per day. An additional validation group (n=7) demonstrated that daily and cumulative sodium balance can be accurately determined during and after the intravenous administration of an agent known to alter renal sodium handling (furosemide 50 mg. kg-1. d-1). Experiments were then performed to examine the influence of intravenous captopril infusion (40 mg. kg-1. d-1, n=7) in mice in which the daily sodium intake was fixed at approximately 200 micromol/d. This dose of captopril was determined to significantly decrease the pressor response to a 10-ng bolus of angiotensin I (Ang I) from 24+/-5 in the control state to 6+/-2 mm Hg (n=5). After 5 days of infusion of the converting enzyme inhibitor, mean arterial pressure significantly fell from 114+/-3 to 58+/-2 mm Hg, body weight significantly decreased from 36+/-1 to 33+/-1 g, and cumulative sodium balance significantly decreased to -270+/-55 micromol. These parameters returned toward control during 5 postcontrol days. Results of this study demonstrate that accurate sodium balance measurements can be obtained from individual conscious mice over a 5-fold range of sodium intake. The experiments also indicate that converting enzyme inhibition has a potent influence to lower blood pressure in normal mice; the hypotensive response appears to be due in part to increased urinary sodium excretion.

Renal medullary interstitial infusion of L-arginine prevents hypertension in Dahl salt-sensitive rats.

Studies were designed to examine the effects of renal medullary interstitial infusion of L-arginine (L-Arg) on the development of high-salt-induced hypertension in Dahl salt-sensitive/Rapp (DS) rats. The threshold dose of L-Arg (300 micrograms . kg-1 . min-1) that increased the renal medullary blood flow without altering the cortical blood flow was first determined in anesthetized DS rats. Studies were then carried out to determine the effects of this dose of L-Arg on salt-induced hypertension in DS rats. In the absence of chronic medullary L-Arg infusion, mean arterial pressure (MAP) increased in DS rats from 125 +/- 2 to 167 +/- 5 mmHg by day 5 of a high-salt diet (4.0%), with no change observed in Wistar-Kyoto (WKY) or Dahl salt-resistant/Rapp (DR) rats. MAP did not change significantly with medullary infusion of L-Arg alone in DR rats (control = 104 +/- 1 mmHg) or in WKY rats (control = 120 +/- 3 mmHg) and was not significantly changed from these levels during the 7 days of L-Arg infusion combined with high-NaCl diet. The same amount of L-Arg that prevented salt-induced hypertension in DS rats when infused into the renal medulla (300 micrograms . kg-1 . min-1) failed to blunt salt-induced hypertension when administered intravenously to DS rats. DS rats receiving L-Arg (300 micrograms . kg-1 . min-1 iv) exhibited an increase in plasma L-Arg from control concentrations of 138 +/- 11 to 218 +/- 4 micromol/l, while MAP, which averaged 124 +/- 3 mmHg during the 3-day control period, rose to 165 +/- 5 mmHg by day 5 of high salt (4%) intake. These results indicate that the prevention of salt sensitivity in DS rats was due specifically to the action of L-Arg on renal medullary function and that DS rats may have a deficit of medullary substrate availability and NO production.

Effects of daily sodium intake and ANG II on cortical and medullary renal blood flow in conscious rats.

Implanted optical fibers and laser-Doppler flow measurement techniques were used for the sequential measurement of regional renal blood flow in conscious rats to determine the effects of an increase of daily NaCl intake on the renal cortical blood flow and blood flow to the outer and inner medulla. Cortical blood flow was increased significantly (32%) by the second day when NaCl intake was increased from 1 to 7 meq/day and was increased further (50%) on the second day after a further elevation of NaCl intake to 13 meq/day. Blood flow to the outer and inner medulla was not changed as NaCl intake was elevated. The increase in renal cortical flow was closely associated with significant reductions in circulating concentrations of ANG II from 31 to 16 pg/ml. Rats given a continuous infusion of nonpressor does of ANG II (5.0 ng.kg(-1).min-1) to maintain constant plasma concentrations of ANG II as sodium intake was increased exhibited no increase of cortical flow. We conclude that reductions of plasma ANG II associated with incremental increases of daily sodium intake result in a rise of renal cortical flow. The elevated blood flow to the renal cortex may enhance sodium excretion and contribute to long-term sodium homeostasis.

Iron attenuates nitric oxide level and iNOS expression in endotoxin-treated mice.

The effect of exogenous Fe-citrate complex (Fe doses of 120 and 240 micromol/kg) on nitric oxide (NO) production in vivo has been studied in blood and liver tissue of endotoxin-treated mice. Fe-citrate complex was administered to mice subcutaneously at the same time with intravenous injection of Escherichia coli lipopolysaccharide (LPS). Iron-dependent decrease in NO2-/NO3- and nitrosyl hemoglobin levels in blood of animals was detected at 6 h after LPS administration, suggesting systemic attenuation of NO generation. NO production in the liver tissue of LPS-treated mice was decreased after Fe administration judging from the amount of mononitrosyl-iron complexes formed in the tissue by diethyldithiocarbamate. The iNOS protein determination in the liver tissue of LPS-treated mice demonstrated iron-dependent inhibition of iNOS expression. We have found previously that exogenous iron does not affect systemic NO level when it is given at 6 h after LPS injection, i.e. after iNOS expression. This is a first report demonstrating iron-dependent iNOS down-regulation in endotoxin-treated mice.

Long-term measurement of arterial blood pressure in conscious mice.

This study describes a technique for the direct daily measurement of arterial blood pressure, sampling of arterial blood, and continuous intravenous infusion in free-moving, conscious, Swiss-Webster mice. Catheters were chronically implanted in the femoral artery and vein, tunneled subcutaneously, exteriorized at the back of the neck in a lightweight tethering spring, and attached to a swivel device at the top of the cage. Time-control experiments (n = 8) demonstrated stable values of mean arterial pressure (MAP, 116 +/- 1 mmHg) and heart rate (HR, 627 +/- 21 beats/min) for up to 35 days after catheter implantation. It was further observed that restraining mice (n = 7) increased MAP by 10 +/- 3 mmHg and HR by 78 +/- 8 beats/min from the values observed under free-moving conditions. To demonstrate the chronic use of the venous catheter, intravenous infusion of NG-nitro-L-arginine methyl ester (L-NAME, 8.6 mg.kg-1.day-1, n = 6) for 5 days significantly increased MAP from 117 +/- 4 to 131 +/- 4 mmHg without altering HR. In a final group of mice (n = 5), oral L-arginine (2% in drinking water) increased plasma arginine concentration from 90 +/- 7 to 131 +/- 17 microM and prevented L-NAME hypertension. These experiments illustrate the feasibility of long-term intravenous infusion, direct arterial blood pressure measurements, and arterial blood sampling in conscious mice.

Inducible nitric oxide synthase and blood pressure.

In the present studies, the influence of inducible nitric oxide synthase (NOS) inhibition with aminoguanidine on renal function and blood pressure was examined in rats. Intravenous aminoguanidine infusion (60 mg x kg-1 x hr-1) for 40 minutes to anesthetized Sprague-Dawley rats (n=7) resulted in no significant changes in mean arterial pressure or renal cortical blood flow, while medullary blood flow was slightly increased. Despite minimal effects on renal blood flow, urine flow was significantly decreased from 14.2+/-2.7 to 10.4+/-2.3 microL x min-1 x g kidney wt-1 during aminoguanidine infusion. To examine the possible effects of inducible NOS on blood pressure, aminoguanidine (10 mg x kg-1 x h-1 IV) was infused chronically into uninephrectomized rats maintained on a high salt (4.0% NaCl) diet. Mean arterial pressure significantly increased from 104+/-2 to 118+/-3 mm Hg after 6 days of aminoguanidine infusion (n=7) and returned to levels not different from those in the control group after 2 days of postcontrol infusion. Calcium-independent NOS activity in the renal medulla, a tissue that expresses inducible NOS in normal rats, was significantly decreased by 49% in the aminoguanidine-infused group (n=6) compared with that activity in the vehicle-infused control animals (n=6). In contrast, calcium-dependent NOS activity in the renal medulla was not significantly altered by aminoguanidine infusion, indicating specificity of aminoguanidine for inducible NOS in these experiments. In a final group of rats (n=5), oral L-arginine administration in drinking water (2% wt/vol) increased plasma arginine levels from 118+/-5 to 232+/-16 micromol/L and blocked the increase in arterial pressure after 6 days of aminoguanidine infusion. The present experiments provide evidence supporting a role for inducible NOS in the control of arterial pressure, possibly by renal tubular effects.

Evidence for the presence of smooth muscle alpha-actin within pericytes of the renal medulla.

This study was designed to determine whether smooth muscle alpha-actin mRNA and smooth muscle alpha-actin contractile protein elements were present within the renal medullary pericytes. Extraction of total RNA from microdissected outer medullary descending vasa recta allowed for the detection of smooth muscle alpha-actin mRNA expression using reverse transcription-polymerase chain reaction (RT-PCR). Expression of smooth muscle alpha-actin was specific to the descending vasa recta and not a result of tubular contamination because RT-PCR amplification of the vasopressin V2 receptor, which is a specific tubular marker, did not occur. To determine the exact cell type(s) that translate the mRNA into protein, we performed immunohistochemistry on the renal outer and inner medulla using a monoclonal smooth muscle alpha-actin antibody, whose specificity was determined by immunoblot analysis. Smooth muscle alpha-actin protein was found selectively within the pericytes surrounding the descending vasa recta from the outer and inner medullary tissue sections. This study demonstrates that the pericytes alone that surround the descending vasa recta within the outer and inner medulla contain smooth muscle alpha-actin mRNA and protein and are therefore the site of the contractile elements that could play a vasomodulatory role in the control of renal medullary blood flow and its distribution within the renal medulla.