Experiments in wild-type (WT; C57BL/6J) mice, endothelial nitric oxide synthase null mutant [eNOS(-/-)] mice, and neuronal NOS null mutant [nNOS(-/-)] mice were performed to determine which NOS isoform regulates renal cortical and medullary blood flow under basal conditions and during the infusion of ANG II. Inhibition of NOS with Nω-nitro-l-arginine methyl ester (l-NAME; 50 mg/kg iv) in Inactin-anesthetized WT and nNOS(-/-) mice increased arterial blood pressure by 28–31 mmHg and significantly decreased blood flow in the renal cortex (18–24%) and the renal medulla (13–18%). In contrast, blood pressure and renal cortical and medullary blood flow were unaltered after l-NAME administration to eNOS(-/-) mice, indicating that NO derived from eNOS regulates baseline vascular resistance in mice. In subsequent experiments, intravenous ANG II (20 ng·kg−1·min−1) significantly decreased renal cortical blood flow (by 15–25%) in WT, eNOS(-/-), nNOS(-/-), and WT mice treated with l-NAME. The infusion of ANG II, however, led to a significant increase in medullary blood flow (12–15%) in WT and eNOS(-/-) mice. The increase in medullary blood flow following ANG II infusion was not observed in nNOS(-/-) mice, in WT or eNOS(-/-) mice pretreated with l-NAME, or in WT mice administered the nNOS inhibitor 5-(1-imino-3-butenyl)-l-ornithine (1 mg·kg−1·h−1). These data demonstrate that NO from eNOS regulates baseline blood flow in the mouse renal cortex and medulla, while NO produced by nNOS mediates an increase in medullary blood flow in response to ANG II.
- nitric oxide synthase
- laser-Doppler flowmetry
- angiotensin II
- endothelial and neuronal nitric oxide synthase
nitric oxide (no) plays an important role in the regulation of renal tubular and vascular function. One of the primary influences of NO on the kidney is to decrease renal vascular resistance by altering the diameter of large preglomerular vessels (12), the afferent and efferent arterioles (8, 9, 29), and vasa recta (21). Despite these observations, it is still unclear which NO synthase (NOS) isoforms are important in the regulation of renal vascular resistance. Experimental evidence indicates that neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3) are all present in the normal kidney (2, 19, 27, 28). Moreover, mRNA and/or immunoreactive protein of each NOS isoform have been identified in different segments of the renal vasculature, although nNOS and eNOS appear to be the isoforms most prominent in the vasculature (2, 19, 28).
It is generally accepted that eNOS, found in the vascular endothelium, is important in blood flow control. The localization of nNOS and iNOS mRNA or protein in the vasculature and/or macula densa indicates that these isoforms may also play a role in blood flow regulation during physiological or pathophysiological conditions. The functional importance of the different NOS isoforms in the regulation of renal blood flow is not clear. Experiments have indicated that systemic administration of a pharmacological inhibitor of iNOS does not alter blood flow in the normal rat kidney (16, 17, 25). The results with inhibitors of nNOS, however, have been inconclusive. In several studies, administration of selective inhibitors of nNOS had no effect on renal vascular resistance in normal rats (5, 6, 14), although other experiments indicated that renal blood flow is decreased by pharmacological inhibition of nNOS in rats on a low-salt diet (5) or in rats fed either a low- or high-salt diet (26).
One of the more likely reasons for the differences in experimental results has been the dependence of these studies upon pharmacological agents. A variety of inhibitors, with differing potency, selectivity, and potentially different cellular distribution has been used to examine the importance of the individual NOS isoforms in the regulation of renal blood flow (5, 6, 14, 16, 25). Because it is difficult to document selective in vivo inhibition of individual NOS isoforms in the kidney, the results of these experiments are difficult to interpret. With the availability of NOS null mutant mice, we now have the opportunity to determine the importance of the different NOS isoforms in mice that are administered pharmacological agents, as well as those in which one of the NOS isoforms has been genetically deleted. This combined genetic and pharmacological approach should permit a more accurate understanding of the role of these isoforms in the regulation of renal vascular function. Preliminary Western blotting experiments indicated that iNOS was not detectable in kidney homogenates obtained from normal, wild-type mice, eNOS null mutant (-/-) mice, or nNOS(-/-) mice. Moreover, previous reports indicate that systemic administration of iNOS inhibitors does not alter blood flow in the rat kidney (16, 17, 25). The present studies therefore focused upon the role of eNOS and nNOS in the control of renal cortical and medullary blood flow in anesthetized mice under baseline conditions and during the intravenous infusion of ANG II.
MATERIALS AND METHODS
Experiments were performed on male C57BL/6J mice, nNOS(-/-) mice, 129S/SvImj mice, and F2 hybrid B6129SF2/J mice obtained from the Jackson Laboratory (Bar Harbor, ME) and male eNOS(-/-) mice obtained from a breeding colony at the Medical College of Wisconsin. The nNOS(-/-) were originally developed by Huang and colleagues (13); the founders of the eNOS(-/-) mouse colony were obtained from the Jackson Laboratory and were originally derived by Shesely and colleagues (23). Mice were provided with normal rodent chow and tap water ad libitum. All animal procedures were approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee.
Protein Isolation and Western Blotting
Western blotting protocol.
Initial immunoblotting protocols were performed to identify the NOS isoforms in the kidneys of the different mouse strains. The C57BL/6J, eNOS(-/-), and nNOS(-/-) mice were euthanized with an overdose of sodium pentobarbital (100 mg/kg ip), and the kidneys were rapidly removed and frozen on dry ice. Isolated tissue was stored at −80°C until protein extraction. In the extraction procedure, pieces of whole tissue were homogenized using a Potter-Elvehjem tissue grinder in a solution containing 250 mmol/l sucrose, 1 mmol/l EDTA, and 5 mmol/l potassium phosphate, pH 7.7. A protease inhibitor cocktail was added to give final concentrations of 4-(2-aminoethyl)benzenesulfonyl fluoride (780 μmol/l), aprotinin (0.6 μmol/l), leupeptin (15 μmol/l), bestatin (30 μmol/l), pepstatin A (11 μmol/l), and E64 (10 μmol/l). Large tissue debris and nuclear fragments were removed by a low-speed centrifuge spin (14,000 g, 4°C, 20′). The protein concentration of the tissue homogenate was determined by a Coomassie protein assay (Pierce, Rockford, IL) with albumin as a standard. Protein samples were electrophoretically size-separated using a discontinuous system consisting of a 12% polyacrylamide resolving gel and a 5% polyacrylamide stacking gel. Broad-range molecular weight markers (∼10–250 kDa) were loaded into one lane as a size standard. Equivalent amounts of total protein from the same tissue of different mice were added to adjacent lanes, and the samples were run at 200 V for 45–60 min on an 8 × 10 cm electrophoresis cell (Bio-Rad, Hercules, CA). After separation, the proteins were electrophoretically transferred to a nitrocellulose membrane at 100 V for 1 h. Membranes were washed in Tris-buffered saline (TBS), blocked with 10% nonfat dried milk in TBS (NFM/TBS) overnight for 2 h, and incubated with a 1:1,000 dilution of anti-nNOS, anti-eNOS, anti-iNOS, or anti-β-actin antibodies (1:1,000) for 2 h at room temperature. The membranes were then rinsed and incubated with an appropriate horseradish peroxidase-labeled anti-rabbit or anti-mouse secondary antibody in 4% NFM/TBS for 2 h. Bound antibody was detected by chemiluminescence (ECL, Amersham, Arlington Heights, IL) on X-ray film. A monoclonal mouse antibody raised against the structural protein β-actin was used as a loading control. Membranes were stripped between incubations with the different antibodies in a Tris-buffered solution containing 2% SDS and 100 mmol/l β-mercaptoethanol at 50°C.
Anesthetized Mouse Studies
Mice were deeply anesthetized with ketamine (30 mg/kg im) and acepromazine (3 mg/kg im) plus Inactin (100 mg/kg ip) for all in vivo procedures. Supplemental Inactin anesthesia was administered as needed through a cannula in the left femoral vein The mice were maintained on a thermostatically controlled warming table, and body temperature was constantly monitored. A tracheotomy was performed to facilitate breathing. The influence of anesthesia to depress the respiratory rate and decrease the baseline arterial blood pressure was dramatic. To maintain the stability of the preparation for the time necessary to perform the present experiments, the mice were supplemented with 100% O2, as described previously (15). Although it is possible that the supplementation of O2 led to oxidative stress in the mice, the supplementation was found to be absolutely necessary to maintain the hemodynamic stability and ensure the survival of the anesthetized mice during this study.
The right femoral vein and artery were cannulated to infuse 2% BSA in saline (10 ml·kg−1·h−1) and to measure arterial blood pressure, respectively. The left kidney was exposed via a midline incision and placed in a plastic holder with the dorsal surface facing upward. The ureter was carefully removed to expose the papilla. Changes in the red blood cell flow in the superficial renal cortex and in the papilla were measured using a laser-Doppler flowmeter (Model BLF21D, Transonic Systems, Ithaca, NY), as described previously in rats (18). The laser-Doppler flow probe (1.2-mm diameter, Type N, Transonic Systems) was used to obtain a flow reading from six different spots on the surface of the renal cortex and from the surface of the exposed renal papilla. The average laser-Doppler flow signal from the renal cortex and the signal from the renal papilla are expressed as a percentage of the respective laser-Doppler flow signal measured from that region during the control period. As such, this technique permits the comparison of changes in blood flow but does not permit comparison of absolute flow values.
Influence of l-NAME on Renal Cortical and Medullary Blood Flow in Anesthetized Mice
Mice [C57BL/6J, nNOS(-/-), eNOS(-/-), 129S/SvImj, and B6129SF2/J] were prepared as described above; a 30-min equilibration period followed the surgical preparation. Baseline measurements of renal cortical and medullary blood flow, as well as mean arterial blood pressure, were obtained after the equilibration period. Each mouse was then administered a 50 mg/kg iv bolus of the nitric oxide inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) and permitted to equilibrate for an additional 30 min before additional measurements of cortical and medullary blood flow were obtained. Two additional groups of mice [C57BL/6J and eNOS(-/-)] were prepared as described above but were infused with the nNOS-selective inhibitor 5-(1-imino-3-butenyl)-l-ornithine (v-NIO; 1 mg·kg−1·h−1) (1) for 30 min before the experimental measurements. An adjustable aortic occluder was placed on the suprarenal aorta to ensure that renal perfusion pressure was not increased compared with the control arterial pressure during each experimental manipulation.
Assessment of NOS Blockade with l-NAME and v-NIO
Three separate groups of C57BL/6J mice were prepared for assessment of NOS blockade after administration of the different NOS enzyme inhibitors. After the equilibration period from surgery, the mice were administered a vehicle, a bolus dose of l-NAME (50 mg/kg iv), or were continuously infused with v-NIO (1 mg·kg−1·h−1). One-half hour later, the kidneys were rapidly removed and frozen on dry ice. Total tissue protein was prepared, as described above, and the conversion of radiolabeled l-arginine to l-citrulline, as an index of NOS enzymatic activity, was quantified as we have previously described (17, 19, 28). The total tissue homogenate (100 μg) was incubated with 2 mmol/l CaCl2, 1 mmol/l NADPH, 25 μmol/l FAD, 1.25 μg/ml calmodulin, 10 μmol/l BH4, and [3H]arginine (∼1, 500,000 dpm, specific activity 68 Ci/mmol) in 20 mmol/l HEPES buffer, pH 7.2, at 37°C for 60 min. The arginine and converted citrulline were separated by isocratic reverse-phase HPLC with a Supelco (Bellefonte, PA) LC-18-DB column (mobile phase 11.5% methanol, 11.5% acetonitrile, 1% tetrahydrofuran, 0.1 mol/L KH2PO4 pH 5.9). The amount of [3H]arginine converted to [3H]citrulline was quantified by radiochemical detection (Packard, Tampa, FL).
Influence of ANG II on Renal Cortical and Medullary Blood Flow in Anesthetized Mice
Mice [C57BL/6J, nNOS(-/-), and eNOS(-/-)] were surgically prepared, as described above. After the equilibration period after surgery, baseline levels of renal cortical and medullary blood flow and arterial pressure were measured. An intravenous infusion of ANG II (20 ng·kg−1·min−1) was then begun; 30 min later, blood flow and arterial blood pressure were again measured. The ANG II infusion was then stopped, and postcontrol data were collected. An adjustable aortic occluder was placed on the suprarenal aorta to ensure that renal perfusion pressure was not increased compared with the control arterial pressure during each experimental manipulation.
Additional experiments were performed in a separate group of C57BL/6J and eNOS(-/-) mice. Mice were prepared as described above; arterial blood pressure, renal cortical blood flow, and renal medullary blood flow were measured after a 30-min equilibration period. The mice were then administered a bolus dose of the nonselective NOS inhibitor l-NAME (50 mg/kg iv) or the nNOS-selective inhibitor v-NIO (1 mg·kg−1·h−1). An intravenous infusion of ANG II (20 ng·kg−1·min−1) was then administered to both groups of mice and renal cortical and medullary blood flow and arterial blood pressure were measured during an experimental period. As described for the above experiments, an adjustable aortic occluder was used to prevent increases in renal perfusion pressure during each experimental manipulation.
Data are expressed as means ± SE; P ≤ 0.05 were considered significant. Within-group changes were evaluated with a t-test or a one way repeated-measures ANOVA followed by a Student-Newman-Keuls post hoc test. Differences in NOS activity between groups were evaluated with a one-way ANOVA and a Student-Newman-Keuls post hoc test. The statistical analysis was conducted using commercially available software (SigmaStat 2.03; Systat Software; Richmond, CA). Laser-Doppler blood flow data are normalized to the control value for each individual group; all statistical analyses were performed on the raw blood flow data from these groups of mice.
Identification of NOS Isoforms in the Mouse Kidney
Western blotting protocols were performed to identify the immunoreactive protein of the different NOS isoforms in the kidneys of C57BL/6J, eNOS(-/-), and nNOS(-/-) mice (n = 4/group). As shown in Fig. 1, both eNOS and nNOS immunoreactive protein were readily detectable in whole kidney homogenates obtained from C57BL/6J mice. As expected, eNOS protein was not detectable in the kidney of eNOS(-/-) mice, and nNOS was not detected in the kidneys of nNOS(-/-) mice. A densitometric comparison of the blots indicated that nNOS was significantly upregulated by three-fold in the eNOS(-/-) compared with the C57BL/6J (P = 0.012). Approximately 50% more eNOS protein was observed in the nNOS(-/-) compared with the C57BL/6J kidney, though this difference was not significant. Immunoreactive iNOS protein was not detectable in the kidney tissue homogenates of any of the mice (data not shown). The density of the β-actin band was not different between samples, indicating equal loading in all lanes.
Influence of l-NAME and v-NIO on Renal Cortical and Medullary Blood Flow in Anesthetized Mice
The influence of l-NAME administration on systemic arterial blood pressure and renal blood flow is illustrated in Fig. 2. Mean arterial pressure (MAP) significantly increased by 20–30 mmHg after administration of the nonselective NOS inhibitor l-NAME (50 mg/kg iv; n = 6–7/group) to the anesthetized C57BL/6J (P ≤ 0.001), nNOS(-/-) (P = 0.001), 129S/SvImj (P ≤ 0.001), and B6129SF2/J (P ≤ 0.001) mice but was unaltered from control values in the eNOS(-/-) mice (n = 6–7/group). Renal cortical and medullary blood flow significantly decreased after l-NAME (50 mg/kg ip) in the anesthetized C57BL/6J (P = 0.003, 0.020, respectively), nNOS(-/-) (P = 0.002, 0.003, respectively), 129S/SvImj (P = 0.001, 0.029, respectively), and B6129SF2/J (P = 0.001, 0.044, respectively) mice. Neither cortical nor medullary flow were altered in the eNOS(-/-) mice. Renal perfusion pressure was maintained at the same level as observed in the control period by the use of a clamp on the suprarenal aorta.
The effect of administration of the nNOS-selective inhibitor v-NIO in C57BL/6J and eNOS(-/-) mice is illustrated in Fig. 3. MAP was not significantly altered in the C57BL/6J or the eNOS(-/-) mice after a 30-min infusion of v-NIO (n = 6/group). Blood flow in the renal cortex and medulla was also not altered during the v-NIO administration to eNOS(-/-). Renal cortical blood flow increased (P = 0.033), while medullary flow was unaltered in the C57BL/6J after v-NIO administration.
The influence of acute administration of the nonspecific NOS inhibitor l-NAME and the nNOS-selective inhibitor v-NIO on NOS activity is illustrated in Fig. 4 (n = 6–8/group). Compared with the vehicle-treated control mice, the NOS enzymatic activity in the kidney, as assessed by the conversion of radiolabeled arginine to citrulline, was significantly decreased by treatment with l-NAME (P < 0.001) or v-NIO (P = 0.030). The NOS enzymatic activity in the kidneys of the l-NAME-treated mice was decreased to a greater extent than observed in the kidneys of the v-NIO-treated mice (P < 0.001).
Influence of ANG II on Renal Cortical and Medullary Blood Flow in Anesthetized Mice
The influence of intravenous ANG II on systemic arterial blood pressure, renal cortical blood flow, and renal medullary blood flow in C57BL/6J, eNOS(-/-), and nNOS(-/-) mice is illustrated in Fig. 5 (n = 6/group). Compared with control levels, intravenous ANG II (20 ng·kg−1·min−1) significantly increased systemic arterial blood pressure in eNOS(-/-) mice by 13 ± 2 mmHg (P < 0.001) and in C57BL/6J mice by 7 ± 3 mmHg (P = 0.012). MAP was not significantly increased in the nNOS(-/-) mice during the infusion of ANG II. The ANG II infusion led to a significant decrease in renal cortical blood flow in the C57BL/6J (P < 0.001), eNOS(-/-) (P < 0.001), and nNOS(-/-) mice (P = 0.010), which ranged from 12 to 22%. In contrast, renal medullary blood flow was unaltered during ANG II infusion in the nNOS(-/-) mice, while medullary blood flow was significantly increased by 11 ± 4% in the C57BL/6J mice (P = 0.030) and by 27 ± 4% in the eNOS(-/-) mice (P < 0.001) during intravenous infusion of ANG II. Renal perfusion pressure was prevented from increasing from control levels during the experiments in this study. In the nNOS(-/-) mice in this protocol, however, systemic arterial blood pressure and renal perfusion pressure significantly decreased in the postcontrol period compared with the control period (P = 0.029).
The influence of ANG II infusion on systemic arterial blood pressure and renal cortical and medullary blood flow in C57BL/6J mice pretreated with either the nonspecific NOS inhibitor l-NAME or the nNOS-selective inhibitor v-NIO and in a group of eNOS(-/-) mice pretreated with l-NAME are presented in Fig. 6. Intravenous ANG II infusion led to an equivalent reduction in cortical blood flow in the C57BL/6J treated with l-NAME (P = 0.027) or v-NIO (P = 0.002), or in the eNOS(-/-) treated with l-NAME (P ≤ 0.001). In contrast, medullary blood flow was unaltered during infusion of ANG II to the C57BL/6J or eNOS(-/-) mice pretreated with l-NAME or the C57BL/6J pretreated with v-NIO. Together with the results illustrated in Fig. 5, these experiments indicate that NO produced by nNOS mediates an increase in renal medullary blood flow during the infusion of ANG II.
The present experiments assessed the importance of the different NOS isoforms in the regulation of renal cortical and medullary blood flow under baseline conditions and during the administration of ANG II. Initial experiments demonstrated that eNOS is the likely source of NO, which influences renal cortical and medullary blood flow under baseline conditions. This conclusion is supported by the observations that the nonspecific NOS inhibitor l-NAME significantly decreased renal cortical and medullary perfusion in normal mice and nNOS(-/-) mice but did not alter renal perfusion in eNOS(-/-) mice. These experiments reveal that the presence of eNOS is necessary for NOS inhibitors to alter baseline blood flow. In support of the conclusions reached from the l-NAME studies, the nNOS-selective inhibitor v-NIO did not alter renal perfusion in C57BL/6J or eNOS(-/-) mice. In addition to the effects on renal blood flow, the present experiments demonstrated that l-NAME administration increased mean arterial blood pressure in normal and nNOS (-/-) mice; MAP was unaffected in eNOS(-/-) mice administered l-NAME, and v-NIO had no effect on arterial blood pressure in C57BL/6J or eNOS(-/-) mice. These observations indicate that NO derived from eNOS is of primary importance in the normal regulation of systemic arterial vascular resistance, as well as renal vascular resistance.
The present studies, which show that the NO, which influences renal blood flow under baseline conditions is derived from eNOS, are in agreement with previous data from our laboratory. The administration of two different nNOS-selective inhibitors did not alter baseline renal hemodynamics in anesthetized rats despite significantly decreasing NO in the interstitial space (14). The similarity of the results obtained between rats and mice in the different studies and the consistency of the results obtained with the different treatments in mice in the present study all support the conclusion that NO derived from nNOS has a minimal influence on baseline renal hemodynamics. Despite this evidence from our laboratory, other groups have reported that administration of nNOS-selective inhibitors leads to a reduction in renal blood flow in anesthetized rats under different conditions (5, 26). The explanation for the differences in results between these previous studies is unclear, but the present approach, in which both genetically manipulated animals and pharmacological inhibitors were used in combination, provides confirmation of our conclusions using mechanistically different approaches. It is important to note, however, that the presence of nNOS in blood vessels (2, 19, 28), the macula densa (27), collecting ducts (28), and perivascular and pelvic nerves (2) indicate that this isoform is likely important for both vascular and extravascular function in the kidney.
Further studies were performed to determine the importance of nNOS and eNOS in the maintenance of renal blood flow during the administration of exogenous ANG II. Infusion of ANG II led to a significant decrease in renal cortical blood flow in C57BL/6J mice, eNOS(-/-) mice, and nNOS(-/-) mice, but no differences were detected in the magnitude of the decrease between the groups. In contrast to the results observed in the renal cortical circulation, renal medullary blood flow significantly increased during ANG II infusion to C57BL/6J and eNOS(-/-) mice, while medullary blood flow was unaltered in the renal medulla of nNOS(-/-) mice during ANG II infusion. Further studies demonstrated that the increase in medullary blood flow, which occurs during ANG II infusion, was not present in C57BL/6J or eNOS(-/-) mice pretreated with l-NAME or C57BL/6J mice pretreated with v-NIO. These studies indicate that NO derived from nNOS has a vasodilatory influence on the renal medullary circulation when ANG II levels are increased by intravenous administration.
It is well recognized that intravenous or renal arterial infusion of ANG II leads to a marked reduction in whole kidney blood flow and in renal cortical blood flow, but the effects of this peptide in the circulation of the renal medulla are not clear. In different studies, ANG II has been shown to have a vasoconstrictor effect on the renal medullary circulation (11, 21), to have no influence on the medullary circulation (4, 10, 18), or even to lead to an increase in medullary blood flow after bolus or intravenous administration (3, 20, 22). The actions of ANG II in the medullary circulation have been reported to be attenuated by NO (24, 30), prostaglandins (4, 7, 18, 21), and/or kinins (22), which may lead to variable responses, depending on the experimental preparation. The present experiments demonstrate that NO produced by nNOS acts as a vasodilator in the renal medulla when ANG II levels are increased, but it is likely that other vasodilator systems are also activated in the renal medulla to blunt the effects of ANG II. Renal cortical blood flow significantly decreased with ANG II infusion, but medullary blood flow was either increased or unchanged when nNOS was genetically deleted or blocked with enzyme inhibitors. It is therefore likely that another vasodilator system blunts the constrictor effects of ANG II in the renal medulla. One possible mediator is the prostaglandin system that has been demonstrated to blunt the constrictor effects of ANG II in the rat renal medullary circulation (7, 18), though other systems remain to be explored.
Western blotting protocols verified the absence of immunoreactive protein in the null mutant animals, and NOS enzymatic activity assays documented the inhibitory effect of l-NAME in the kidney. The in vitro conversion of l-arginine to l-citrulline was significantly reduced in the kidneys of mice administered l-NAME or v-NIO by intravenous infusion, indicating that NOS enzymatic activity was reduced in these kidneys. The reduction in NOS enzymatic activity was greater in the mice administered l-NAME compared with mice treated with v-NIO. This difference presumably reflects the inhibition of both eNOS and nNOS by l-NAME, which would be expected to exceed the inhibitory effect of an nNOS-selective agent.
An interesting finding that arose from the Western blotting studies is the observation that nNOS protein is increased in the kidneys of eNOS(-/-) mice, while eNOS protein is increased in the kidneys of nNOS(-/-) mice. Immunoreactive iNOS was not detectable in the kidneys of any of the mice. These data indicate that significant upregulation of the other constitutive NOS isoforms occurs in the kidney of the NOS knockout mice. This apparent upregulation, however, did not appear to affect the functional responses during experimental maneuvers in these studies. Experiments in the WT and nNOS(-/-) mice demonstrated equivalent elevations of MAP and reductions in renal cortical and medullary blood flow after administration of l-NAME, while the systemic and renal vasoconstrictor response to l-NAME was completely eliminated in the eNOS(-/-) mice. These results suggest that the upregulation of nNOS in the eNOS(-/-) mice and upregulation of eNOS in the nNOS (-/-) mice did not lead to qualitative differences in the functional responses. The cellular localization of the upregulated NOS mRNA and/or protein remains to be further explored in the control strains, as well as the nNOS(-/-) and eNOS(-/-) mice.
Three different strains were used in the initial experiments as control animals for the eNOS(-/-) and nNOS(-/-) mice. The C57BL/6J is a control for the eNOS (-/-) mouse, which has been backcrossed to the C57BL/6J background for over eight generations. The nNOS(-/-) mouse has a mixed genetic background; we therefore used the two parental strains, the C57BL/6J and the 129S/SvImj, as well as an F2 hybrid B6129SF2/J strain as controls for this strain. The functional responses in the C57BL/6J, the 129S/SvImj, and the B6129SF2/J mice were not different in the l-NAME protocol, indicating that the effects observed were likely due to the genetic deletion rather than the background genetics of the parental strains. After the l-NAME protocol, we used the C57BL/6J as the sole wild-type control strain in the remaining experiments.
In conclusion, data from the present study indicate that both nNOS and eNOS produce NO, which influences renal cortical and/or medullary perfusion. The results of these experiments demonstrate that eNOS is the predominant isoform involved in the regulation of renal blood flow under baseline conditions, although NO produced by nNOS appears to be an important buffer of the constrictor actions of ANG II in the renal medulla.
This work was partially supported by National Institutes of Health Grants HL-29587 and DK-50739.
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