Superoxide anion curbs nitric oxide modulation of afferent arteriolar ANG II responsiveness in diabetes mellitus

Gwynn C. Schoonmaker, Rachel W. Fallet, and Pamela K. Carmines

Department of Physiology and Biophysics, University of Nebraska College of Medicine, Omaha, Nebraska 68198-4575


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experiments were performed to test the hypothesis that the impact of endogenous nitric oxide (NO) on ANG II-induced renal arteriolar constriction is reduced in rats with insulin-dependent diabetes mellitus (65 mg/kg streptozotocin; STZ). Arteriolar diameter responses to exogenous ANG II were quantified before and during NO synthase inhibition (100 µM Nomega -nitro-L-arginine; L-NNA) by using the in vitro blood-perfused juxtamedullary nephron technique. Afferent arteriolar lumen diameter averaged 20.7 ± 2.0 µm in Sham kidneys and 25.9 ± 1.3 µm in STZ kidneys (P < 0.05). Efferent arteriolar diameter did not differ between Sham and STZ rats. In kidneys from Sham rats, afferent and efferent arteriolar responses to ANG II (0.1-10.0 nM) were exaggerated significantly by L-NNA. L-NNA also augmented efferent arteriolar ANG II responses in kidneys from STZ rats (high-glucose bath) but did not alter ANG II responses in afferent arterioles from STZ rats. L-NNA also accentuated efferent, but not afferent, arteriolar ANG II responses in STZ kidneys during acute restoration of bath glucose to normal levels. Superoxide dismutase (150 U/ml) restored the ability of L-NNA to allow exaggerated afferent arteriolar responses to ANG II in kidneys from STZ rats. These observations indicate that superoxide anion suppresses the modulatory influence of endogenous NO on ANG II-induced afferent arteriolar constriction in diabetes mellitus.

efferent arteriole; Nomega -nitro-L-arginine; rat; streptozotocin; superoxide dismutase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RENAL HEMODYNAMIC DYSFUNCTION plays a central role in the pathophysiological consequences of insulin-dependent diabetes mellitus (IDDM). Specifically, glomerular hyperfiltration (and/or consequent maladaptations to this process) is believed to ultimately engender reductions in glomerular filtration rate and the development of diabetic glomerulopathy. Although some investigators have advanced the postulate that increased nitric oxide (NO) production contributes to the renal hemodynamic maladaptations of IDDM (6, 23, 28, 39) other studies have failed to discern any effect of IDDM on the renal hemodynamic response to nitric oxide synthase (NOS) inhibition (17, 22, 26, 34). Data from our laboratory indicate that NOS inhibition exerts a diminished impact on renal arteriolar baseline diameter in streptozotocin (STZ)-treated rats and that superoxide dismutase (SOD) normalizes the diameter response to NOS inhibition (30). These observations suggest that superoxide anion accelerates NO degradation in IDDM, thus reducing the functional impact of endogenous NO on the renal microvasculature in IDDM.

In addition to its direct vasodilator effect, endogenously produced NO normally tempers renal vascular responsiveness to a variety of constrictor stimuli (7, 27, 33, 36). In particular, the counterbalancing influences of ANG II and NO on the renal microvasculature appear to represent primary determinants of renal perfusion and glomerular filtration. It is easy to envision that an imbalance in the vasoactive actions of these paracrine substances could contribute to the disruption of renal hemodynamic function in IDDM. Indeed, a recent analysis of glomerular filtration rate responses to NOS inhibition and/or ANG II receptor blockade suggested that combined alterations in ANG II and NO may contribute to the hyperfiltration in STZ rats (26). However, functional interactions between ANG II and NO have not yet been probed by direct means in renal arterioles from diabetic animals. The purpose of the present study was to test the postulate that IDDM is associated with an impaired modulatory impact of NO on ANG II-induced renal arteriolar vasoconstriction. Additional experiments assessed the ability of SOD to restore defective NO modulation of afferent arteriolar ANG II responsiveness in kidneys from diabetic rats.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Induction of Diabetes Mellitus

Male Sprague-Dawley rats were anesthetized with methohexital sodium (50 mg/kg Brevital ip; Eli Lilly, Indianapolis, IN) to facilitate intravenous injection of STZ (65 mg/kg; Sigma Chemical, St. Louis, MO). Sham rats received vehicle treatment. The rats recovered from anesthesia and were housed overnight with ad libitum access to food and water. The following morning, blood glucose levels were measured (Accu-Check III model 766, Boehringer Mannheim, Indianapolis, IN) and the rats were anesthetized again to facilitate subcutaneous insertion of a 2.3 × 2.0-mm sustained-release insulin implant (Linplant; Linshin Canada, Scarborough, Ontario, Canada) into STZ rats via a 16-G needle. Sham rats received vehicle implants (microrecrystallized palmitic acid). Blood glucose concentration and body weight were measured twice weekly thereafter. Some animals were housed individually in metabolic cages (Nalgene, Nalge, Rochester, NY) for 2 days immediately before the terminal experiment. The total volume of urine collected during the final 24-h period was centrifuged (to remove sediment) and stored at -70°C. These samples were assayed for creatinine concentration by a picric acid-based microplate assay (35).

In Vitro Blood-Perfused Juxtamedullary Nephron Technique

Two to three weeks after induction of diabetes, experiments were performed by using the in vitro blood-perfused juxtamedullary nephron technique (11). The rat was anesthetized with pentobarbital sodium (50 mg/kg Nembutal ip; Abbott Laboratories, North Chicago, IL), and an angiotensin-converting enzyme inhibitor (enalaprilat, 2 mg ia; Merck Research Laboratories, Rahway, NJ) was administered to suppress endogenous ANG II formation and its impact on vascular tone. Thirty minutes later, the right renal artery was cannulated via the superior mesenteric artery. This procedure initiated in situ perfusion of the kidney with Tyrode solution containing 52 g/l dialyzed BSA and D-glucose at a concentration of either 90 mg/dl (Sham rats) or 300 mg/dl (STZ rats). The rat was then exsanguinated via a carotid arterial cannula into a heparinized syringe, and the kidney was harvested for in vitro study. Renal perfusion was maintained throughout the dissection procedure needed to reveal the tubules, glomeruli, and vasculature of juxtamedullary nephrons. Ligatures were placed around the distal segments of the large arterial branches that supplied the exposed microvasculature.

When necessary to obtain an adequate volume of blood for renal perfusion, an additional blood-donor rat was anesthetized, treated with enalaprilat, subjected to acute bilateral nephrectomy, and exsanguinated via a carotid arterial cannula into a heparinized syringe. The collected blood was pooled with that harvested from the kidney donor and processed to remove leukocytes and platelets (19). The resulting perfusate was stirred continuously in a closed reservoir that was pressurized under 95% O2-5% CO2, thus providing both oxygenation and the driving force for perfusion of the dissected kidney at a constant renal arterial pressure of 110 mmHg. Kidneys from STZ rats were perfused with blood from STZ rats, and kidneys from Sham rats were perfused with blood from Sham rats.

The renal perfusion chamber was warmed, and the tissue surface was superfused with Tyrode solution containing 10 g/l BSA at 37°C. Except when otherwise noted, this bathing solution contained 300 mg/dl glucose for experiments utilizing STZ kidneys and 90 mg/dl glucose for experiments using Sham kidneys. The renal microvasculature was transilluminated on the stage of a compound microscope, and a single afferent or efferent arteriole was selected for study on the basis of visibility and blood flow. Video images of each microvessel were generated continuously and stored on videotape for later analysis. In one experiment in which two vessels could be visualized within the same field of view, responses of both vessels were recorded simultaneously and analyzed separately during videotape playback.

Experimental Protocols

Effect of NOS inhibition on ANG II-induced arteriolar vasoconstriction. Arteriolar diameter responses to increasing concentrations of ANG II (Sigma Chemical) were evaluated by exposing kidneys from Sham and STZ rats to the following superfusate bathing solutions: 1) Tyrode solution alone (5-10 min); 2) Tyrode solution containing 0.1, 1.0, and 10 nM ANG II (3 min at each concentration); and 3) Tyrode solution alone (10 min). After this recovery period, endogenous NOS inhibition was achieved by addition of 100 µM Nomega -nitro-L-arginine (L-NNA; Aldrich Chemical, Milwaukee, WI) to the Tyrode bathing solution, as described previously (31). Juxtamedullary afferent arteriolar diameter responses to 100 µM L-NNA are apparent within 5 min, maximal at 10 min, and stable for >30 min thereafter (19). Accordingly, a 15-min L-NNA treatment period preceded initiation of the second ANG II exposure sequence (0.1, 1.0, and 10 nM ANG II; 3 min each) in the continued presence of L-NNA. This was followed by a recovery period during which the tissue was exposed to Tyrode solution containing L-NNA alone. Imposition of two consecutive ANG II exposure sequences according to this protocol (but in the absence of L-NNA) evokes indistinguishable juxtamedullary arteriolar diameter responses in normal kidneys, even at peptide concentrations 10-fold higher than those employed in the present study (8).

Effect of reduced extracellular glucose concentration on ANG II-NO interactions in IDDM. Additional experiments addressed the possibility that an acute decrease in extracellular glucose toward normal levels might restore NO modulation of ANG II responses in STZ rats. Afferent or efferent arteriolar diameter responses to ANG II were documented in STZ kidneys before and during L-NNA treatment according to the protocol described above, except that all bath solutions contained 90 mg/dl glucose.

Effect of SOD on ANG II-NO interactions. The contribution of endogenous superoxide anion to the altered NO modulation of arteriolar ANG II responses in IDDM was explored through the use of acute SOD treatment. SOD from bovine erythrocytes (Sigma Chemical; 3,100-5,100 U/mg) was added to the perfusate blood to achieve a final concentration of 150 U/ml. This concentration of SOD has been reported to achieve complete scavenging of extracellular superoxide anion (16). Experiments were performed by using kidneys from both Sham rats and STZ rats perfused with SOD-supplemented blood (treatment groups designated Sham+SOD and STZ+SOD, respectively). Afferent arteriolar diameter responses to ANG II were obtained in these kidneys before and during L-NNA treatment, according to the protocol described above.

Data Analysis

Arteriolar lumen diameter was measured from videotaped images at 12-s intervals from a single point along the length of the vessel. The average diameter during the final minute of each treatment period was utilized for statistical analysis by repeated-measures ANOVA or Friedman repeated-measures ANOVA on ranks, as appropriate, followed by a Newman-Keuls test. Simple between-group comparisons were performed by using the unpaired t-test. All statistical computations were performed utilizing the SigmaStat 2.03 software package (SPSS, Chicago, IL). Statistical significance was defined as P values <0.05. All data are reported as means ± SE (n = number of arterioles, unless specified otherwise).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Characteristics

Table 1 summarizes the salient characteristics of Sham and STZ rats utilized in the present study. At the time of injection of either STZ or vehicle, the rats receiving STZ weighed somewhat more than those receiving vehicle (Sham rats). However, because Sham rats gained more weight (80 ± 7 g; n = 20 rats) than STZ rats (24 ± 5 g; n = 31 rats; P < 0.05) during the ensuing 2- to 3-wk period, the Sham rats weighed significantly more than STZ rats at the time of the microvascular function studies. The average blood glucose concentration for all rats before injection was 81 ± 2 mg/dl (n = 51 rats). Multiple measurements of blood glucose concentration during the 17 ± 1 days between initial injection (of STZ or vehicle) and the terminal experiment confirmed that STZ rats were significantly hyperglycemic relative to Sham rats. The STZ rats were polydipsic and polyuric, as evidenced by increases in water intake and urine flow during the 24-h before the microvascular function studies (during which time the animals were housed in metabolic cages), and had elevated creatinine excretion rates.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Summary of data characterizing Sham and STZ rats

Effect of NOS Inhibition on ANG II-Induced Arteriolar Vasoconstriction

Table 2 summarizes arteriolar diameters in the various treatment groups before (untreated) and during NOS inhibition (100 µM L-NNA). As reported previously (32), baseline afferent arteriolar diameter was significantly increased in kidneys from STZ rats, but efferent arteriolar diameter did not differ significantly between Sham and STZ rats. The afferent arteriolar dilation associated with IDDM remained evident in SOD-treated STZ kidneys but did not achieve statistical significance in STZ kidneys bathed in low-glucose (90 mg/dl) solution.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Baseline arteriolar diameters measured before (Untreated) and during NOS inhibition

Figure 1A illustrates the effect of L-NNA on ANG II-induced constriction of afferent arterioles in kidneys from Sham rats. Addition of 1 and 10 nM ANG II to the bathing solution reduced lumen diameter to 17.2 ± 1.6 and 9.9 ± 2.2 µm, respectively (n = 6), and removal of ANG II from the bathing solution restored diameter to 19.8 ± 1.4 µm [not significant (NS) vs. baseline]. Addition of L-NNA to the bathing solution decreased afferent arteriolar diameter by 6.0 ± 1.4 µm (Table 2). During continued L-NNA treatment, the same concentrations of ANG II reduced afferent arteriolar lumen diameter to 9.7 ± 1.5 µm (1 nM) and 4.1 ± 1.4 µm (10 nM). These responses significantly exceeded those observed in the same vessels before L-NNA treatment. Thus L-NNA treatment amplified afferent arteriolar vasoconstrictor responses to exogenous ANG II in kidneys from Sham rats. After removal of ANG II from the bathing solution, but in the continued presence of L-NNA, afferent diameter was restored to 14.0 ± 2.0 µm (NS vs. L-NNA baseline). This observation confirms that the responses to ANG II during L-NNA treatment were not merely a time-related decline in arteriolar diameter.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1.   Impact of nitric oxide synthase (NOS) inhibition on ANG II-induced contractile responses of juxtamedullary afferent arterioles from Sham (A) and streptozotocin (STZ; B) rats. Arteriolar diameter responses to ANG II were monitored before (Untreated; closed symbols) and during (open symbols) exposure to 100 µM Nomega -nitro-L-arginine (L-NNA). * P < 0.05 vs. baseline. dagger  P < 0.05 Untreated vs. L-NNA.

Figure 1B illustrates the impact of NOS inhibition on ANG II-induced constriction of afferent arterioles from STZ rats. Addition of ANG II (1 and 10 nM) to the solution bathing STZ kidneys reduced afferent diameter to 19.6 ± 1.8 and 10.7 ± 2.0 µm, respectively. These responses did not differ significantly (either as absolute change in diameter or as %change in diameter) from those observed in Sham kidneys. On removal of ANG II from the bathing solution, afferent diameter was restored to 24.7 ± 1.4 µm (NS vs. baseline). Addition of 100 µM L-NNA to the bathing solution reduced afferent arteriolar diameter in STZ kidneys by 6.4 ± 1.0 µm (Table 2), a response that did not differ from that observed in Sham kidneys. During continued exposure to L-NNA, ANG II reduced afferent arteriolar lumen diameter in STZ kidneys to 13.2 ± 1.4 µm (1 nM) and 6.7 ± 1.8 µm (10 nM). These responses to ANG II in the presence of L-NNA did not differ from those observed in the same arterioles before L-NNA treatment. On removal of ANG II from the L-NNA-containing bath, arteriolar diameter was restored to 16.7 ± 1.3 µm (NS vs. L-NNA baseline). Thus, in contrast to the behavior of afferent arterioles in kidneys from Sham rats, L-NNA failed to augment vasoconstrictor responses to exogenous ANG II in kidneys from STZ rats.

The effects of L-NNA on efferent arteriolar vasoconstrictor responses to ANG II in kidneys from Sham and STZ rats are summarized in Fig. 2. In Sham rats, efferent arteriolar diameter averaged 20.6 ± 2.8 µm during exposure to 1 nM ANG II (NS vs. baseline) and further declined to 14.3 ± 3.3 µm when exposed to 10 nM ANG II (P < 0.05 vs. baseline). Removal of ANG II from the bath restored efferent diameter to 23.1 ± 2.0 µm (NS vs. baseline). Subsequent L-NNA treatment reduced efferent arteriolar diameter by 1.6 ± 0.2 µm (Table 2). During L-NNA treatment, 1 and 10 nM ANG II significantly reduced efferent arteriolar diameter to 16.1 ± 3.5 and 10.4 ± 3.3 µm, respectively, and diameter was restored to 18.2 ± 3.0 µm during the recovery period (NS vs. L-NNA baseline). The efferent arteriolar response to 10 nM ANGII was significantly amplified by L-NNA treatment in kidneys from Sham rats (Fig. 2A).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2.   Influence of NOS inhibition on ANG II-induced contractile responses of juxtamedullary efferent arterioles from Sham (A) and STZ (B) rats. Arteriolar diameter responses to ANG II were monitored before (Untreated; closed symbols) and during (open symbols) exposure to 100 µM L-NNA. * P < 0.05 vs. baseline. dagger  P < 0.05 Untreated vs. L-NNA.

In kidneys from STZ rats, efferent arteriolar diameter was unaffected by 0.1 and 1 nM ANG II before L-NNA treatment but decreased significantly to 21.1 ± 2.7 µm on exposure to 10 nM ANG II (Fig. 2B). These responses did not differ significantly from those observed in efferent arterioles from Sham rats. Removal of ANG II from the bathing solution restored efferent arteriolar diameter to 27.0 ± 2.7 µm (100 ± 3% of baseline). The tendency for L-NNA to decrease efferent diameter failed to achieve statistical significance in kidneys from STZ rats (-2.4 ± 1.3 µm; Table 2). However, efferent arteriolar responses to 10 nM ANG II were exaggerated significantly by L-NNA treatment, with lumen diameter averaging 14.5 ± 3.7 µm under these conditions. Removal of ANG II from the L-NNA-containing bathing solution restored efferent diameter to 22.9 ± 2.2 µm (NS vs. L-NNA baseline). Thus the modulatory impact of L-NNA on efferent arteriolar ANG II responsiveness was sustained in kidneys from STZ rats.

Effect of Reduced Extracellular Glucose Concentration on ANG II-NO Interactions in IDDM

In experiments performed exclusively using kidneys from STZ rats, afferent and efferent arteriolar function was assessed by using bath solutions containing 90 mg/dl glucose. Under these conditions that impose on STZ kidneys an acute restoration of extracellular glucose concentration toward normal levels, arteriolar baseline diameters did not differ significantly from those observed in either Sham kidneys or STZ kidneys studied in the high-glucose environment (Table 2). Figure 3 illustrates the effects of L-NNA on arteriolar ANG II responsiveness in STZ kidneys studied using 90 mg/dl glucose bath solutions. Afferent arteriolar diameter averaged 18.4 ± 2.6 µm during exposure to 1 nM ANG II and further declined to 10.9 ± 2.3 µm when exposed to 10 nM ANG II (P < 0.05 vs. baseline). Removal of ANG II from the bath restored diameter to 22.3 ± 1.3 µm (NS vs baseline). Subsequent L-NNA treatment reduced afferent arteriolar diameter by 4.4 ± 1.4 µm (Table 2). During continued exposure to L-NNA in the 90 mg/dl glucose bath, ANG II reduced afferent arteriolar lumen diameter in STZ kidneys to 13.3 ± 1.7 µm (1 nM) and 7.2 ± 0.9 µm (10 nM). These responses to ANG II in the presence of L-NNA did not differ from those observed in the same arterioles before L-NNA treatment (Fig. 3A). Thus the inability of L-NNA treatment to allow exaggerated afferent arteriolar ANG II responsiveness remained evident in STZ kidneys bathed in solutions containing 90 mg/dl glucose.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of NOS inhibition on afferent (A) and efferent (B) arteriolar contractile responses to ANG II in kidneys from STZ rats during exposure to low-glucose bath (90 mg/dl). Arteriolar diameter responses to ANG II were monitored before (Untreated; closed symbols) and during (open symbols) exposure to 100 µM L-NNA. *P < 0.05 vs. baseline. dagger  P < 0.05 Untreated vs. L-NNA.

The effect of reduced bath glucose concentration on efferent arteriolar responses in STZ kidneys is shown in Fig. 3B. Efferent arteriolar diameter averaged 22.2 ± 2.2 µm during exposure to 1 nM ANG II (NS vs. baseline) and further declined to 16.8 ± 3.3 µm when exposed to 10 nM ANG II (P < 0.05 vs. baseline). Removal of ANG II from the bath restored efferent diameter to 23.3 ± 2.4 µm (NS vs. baseline). Subsequent L-NNA treatment only reduced efferent arteriolar diameter by 2.1 ± 0.7 µm (Table 2). During L-NNA treatment, 1 and 10 nM ANG II decreased efferent arteriolar diameter to 17.3 ± 3.0 and 11.2 ± 2.6 µm (both P < 0.05 vs. L-NNA baseline), respectively. Efferent arteriolar responses to 1 and 10 nM ANG II during L-NNA treatment significantly exceeded those observed in the same vessels under untreated conditions. After removal of ANG II from the bathing solution, but in the continued presence of L-NNA, efferent diameter was restored to 19.5 ± 2.7 µm (NS vs. L-NNA baseline). Thus the modulatory influence of L-NNA on efferent arteriolar ANG II responsiveness was sustained in STZ kidneys bathed in solutions containing low (normal) glucose concentrations.

Effect of SOD on NO-ANG II Interactions

Figure 4 depicts the effects of L-NNA on afferent arteriolar responses to ANG II in kidneys perfused with blood containing 150 U/ml SOD. In Sham+SOD kidneys, afferent arteriolar responsiveness to ANG II was similar to that observed in kidneys from Sham rats studied without SOD supplementation, with 10 nM ANG II decreasing arteriolar diameter to 10.0 ± 1.4 µm. After recovery of kidneys from the initial ANG II challenge (20.6 ± 0.8 µm; NS vs. baseline), L-NNA evoked a 4.2 ± 0.9-µm decline in afferent diameter (Table 2; NS vs. Sham without SOD). During continued L-NNA treatment, 1 and 10 nM ANG II decreased afferent diameter in Sham+SOD kidneys from 17.1 ± 1.0 to 11.3 ± 1.3 and 4.6 ± 1.0 µm, respectively. Removal of ANG II from the bathing solution restored afferent diameter to 15.3 ± 1.0 µm (NS vs. L-NNA baseline). Thus, as observed in Sham kidneys studied in the absence of SOD supplementation (Fig. 1A), the afferent arteriolar responses to 1 and 10 nM ANG II in Sham+SOD kidneys were exaggerated significantly by L-NNA treatment (Fig. 4A).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 4.   Impact of NOS inhibition on ANG II-induced contractile responses of juxtamedullary afferent arterioles during superoxide dismutase (SOD) supplementation (150 U/ml blood). Diameter responses to ANG II were monitored in Sham+SOD (A) and STZ+SOD (B) kidneys both before (Untreated; closed symbols) and during (open symbols) exposure to 100 µM L-NNA. *P < 0.05 vs. baseline. dagger  P < 0.05 Untreated vs. L-NNA.

Afferent arteriolar diameter tended to be greater in STZ+SOD kidneys than in STZ kidneys without SOD supplementation; however, this trend did not achieve statistical significance (Table 2). Addition of 1 and 10 nM ANG II to the solution bathing STZ+SOD kidneys reduced afferent diameter to 27.3 ± 1.9 and 16.8 ± 1.9 µm, respectively (Fig. 4B), responses that did not differ significantly from those in STZ kidneys without SOD supplementation. On removal of ANG II from the bath, afferent arteriolar diameter was restored to 29.6 ± 1.9 µm (NS vs. baseline). Addition of L-NNA to the bathing solution decreased afferent diameter in STZ+SOD kidneys by 4.2 ± 0.4 µm (Table 2; NS vs. STZ without SOD or Sham). During continued L-NNA treatment, the same concentrations of ANG II (1 and 10 nM) reduced afferent arteriolar lumen diameter to 20.5 ± 2.1 and 10.9 ± 1.2 µm, respectively. These responses to 1 and 10 nM ANG II significantly exceeded those observed in the same vessels before L-NNA treatment. After removal of ANG II from the bathing solution, but in the continued presence of L-NNA, afferent diameter was restored to 23.6 ± 2.0 µm (NS vs. L-NNA baseline). Therefore, SOD supplementation restored the ability of L-NNA to amplify afferent arteriolar vasoconstrictor responses to exogenous ANG II in kidneys from STZ rats.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Considerable attention has focused on elucidating the role of ANG II in the maladaptive renal hemodynamic events accompanying IDDM. Conventional wisdom asserts that the renin-angiotensin system is suppressed during the early stages of IDDM, as evidenced by the frequent observation of reduced plasma renin activity under these conditions. However, ACE inhibition retards development of glomerular injury in IDDM (25, 43), and most evidence indicates that this occurs through an ANG II-dependent mechanism (2, 37, 40). These observations suggest that the renal hemodynamic impact of endogenous ANG II may be exaggerated in IDDM (21, 42), despite normal or reduced renal tissue ANG II levels. Increased ANG II sensitivity may occur as a consequence of the hyperfiltration process (or may arise despite the development of hyperfiltration) and could contribute to the subsequent development of diabetic glomerulopathy and end-stage renal disease. The results of the present study support the contention that renal arteriolar ANG II responsiveness is retained during the early stage of a IDDM in a moderately hyperglycemic rat model characterized by polydipsia, polyuria, and increased creatinine excretion (consistent with hyperfiltration). Moreover, our data reveal that the normal modulatory influence of NO on afferent arteriolar ANG II responsiveness is absent under these conditions (but is restored by superoxide dismutase), whereas NO modulation of ANG II-induced efferent arteriolar constriction remains intact.

Numerous studies have revealed that the renal hemodynamic impact of ANG II is exaggerated by NOS inhibition, suggesting that endogenous NO tempers the renal vasoconstrictor impact of ANG II under normal conditions. In accord with this contention, juxtamedullary afferent and efferent arteriolar diameter responses to ANG II are exaggerated by treatment with L-NNA (18, 19). In the present study, the modulatory impact of 100 µM L-NNA on ANG II responsiveness was confirmed in both afferent and efferent arterioles from Sham rats. At this concentration, L-NNA is maximally effective in reducing afferent arteriolar baseline diameter and abolishing the afferent arteriolar vasodilator response to acetylcholine in our experimental setting (18, 31). The modulatory impact of L-NNA on ANG II responsiveness cannot be attributed to the L-NNA-induced decrease in diameter per se, because preconstriction with norepinephrine does not exaggerate juxtamedullary afferent arteriolar ANG II responsiveness (19). Thus the increased ANG II responsiveness observed during L-NNA treatment indicates that endogenous NO normally tempers ANG II-induced renal vasoconstrictor responsiveness.

IDDM is generally reported to impair renal vasodilator responses to acetylcholine and other endothelium-dependent agents (14, 15, 20, 41). However, the effect of IDDM on the increase in renal vascular resistance evoked by NOS inhibition remains controversial (6, 17, 22, 23, 26, 28, 34, 39). We previously observed markedly diminished afferent and efferent arteriolar vasoconstrictor responses to L-NNA in kidneys from STZ rats (30), suggesting that the tonic functional impact of NO on the renal microvasculature is decreased in IDDM. In the present study, however, the effect of NOS inhibition on baseline afferent arteriolar diameter remained intact in STZ kidneys, whereas efferent arteriolar responses were abated. At least two phenomena may contribute to this discrepancy with our previous data. First, the present study employed acute enalaprilat treatment to facilitate evaluation of arteriolar responsiveness to exogenous ANG II, whereas our previous study was performed in the setting of an intact renin-angiotensin system. In light of the complex interplay between tissue ANG II and NO levels under normal conditions, and our incomplete understanding of the status of the intrarenal renin-angiotensin system in IDDM (1), the impact of enalaprilat treatment on NO-dependent baseline tone in the present studies cannot be reasonably estimated. Second, our earlier study specifically targeted the tonic influence of endogenous NO on baseline diameter by generating L-NNA concentration-response relationships in kidneys from Sham and STZ rats, whereas the present study employed a complex experimental design that optimized our ability to detect NO modulation of ANG II responses through paired observations. This latter design is not a powerful means of detecting between-group differences in the effect of NOS inhibition on baseline diameter. Nevertheless, coupled with the use of enalaprilat-treated tissue to provide a functional baseline of reduced tissue ANG II formation, the design of the present studies provides a powerful assessment of the modulatory influence of NO on contractile responses to exogenous ANG II.

Short-term exposure to high-glucose media exaggerates ANG II-induced constriction of rabbit afferent arterioles by inhibiting the modulatory action of NO (4). The results of the present study extend this observation by demonstrating that the usual effect of L-NNA to allow enhanced afferent arteriolar ANG II responsiveness is absent in kidneys from diabetic rats. NO modulation of afferent arteriolar ANG II responsiveness was also absent in STZ kidneys studied during an acute reduction of extracellular glucose concentration toward normal levels, indicating that this phenomenon is not osmotic in origin. Pflueger and co-workers (34) have documented loss of the ability of L-NNA to promote renal vasoconstrictor responses to adenosine in IDDM. Thus it appears that neither basal nor agonist-stimulated NO levels are sufficient to provide the normal modulatory impact of NO on renal vasoconstrictor events in IDDM. Although this phenomenon can be expected to promote exaggerated renal vasoconstrictor responsiveness to ANG II in IDDM, afferent arteriolar ANG II responses observed in the absence of NOS inhibition did not differ between Sham and STZ kidneys in the present study. The multiple paracrine influences on the renal microvasculature, as well as alterations in endogenous ANG II synthesis, AT1 receptor density, and/or intracellular signaling events are all potential mechanisms through which the IDDM might influence the impact of ANG II on the renal microvasculature. We suspect that the loss of NO-dependent modulation of afferent arteriolar ANG II responsiveness in IDDM is offset by the diminished response of this arteriolar segment to membrane depolarization and opening of voltage-gated Ca2+ channels (9), processes that are critical in eliciting the constrictor response to ANG II. Baseline afferent arteriolar tone is also heavily dependent on membrane potential and Ca2+ influx through these channels (10). Hence, decreased functional expression of voltage-gated Ca2+ channels and/or a hyperpolarizing shift in membrane potential (e.g., increased K+ conductance) could engender the reduced preglomerular resistance characteristic of diabetic hyperfiltration, despite a reduced microvascular impact of endogenous NO.

Ichihara and co-workers (18) have provided evidence that both NOS-1 and NOS-3 provide NO that influences afferent arteriolar basal tone, whereas NOS-3 is the primary source of NO that modulates afferent arteriolar ANG II responsiveness. Hence, one could postulate that reduced NOS-3 expression might underlie the decreased impact of NO on afferent tone (30) and the absence of NO modulation of afferent arteriolar contractile responsiveness evident in IDDM. This scenario is improbable in light of the increase in afferent arteriolar NOS-3 immunostaining and NADPH-diaphorase staining that has been described at 1, 2, and 4 wk after induction of IDDM (38). It is more likely that increased degradation, rather than decreased synthesis, underlies the reduced bioavailability of NO in IDDM. Chronic exposure of cultured endothelial cells to a high-glucose environment promotes production of superoxide anion (12) which, in a virtually diffusion-limited reaction, reacts with NO to form peroxynitrite. Although not representing a significant pathway for NO degradation under normal conditions, peroxynitrite formation gains functional significance as a means of reducing the half-life of NO in states of oxidant stress such as hypertension or IDDM. Accordingly, although not altering responses in normal vessels, treatment with SOD or free radical scavengers ameliorates the impaired endothelium-dependent vasodilator function accompanying IDDM in a variety of nonrenal vascular beds (3, 14, 24, 29) and in renal interlobar arteries (13). We previously reported the ability of exogenous SOD to restore normal afferent and efferent arteriolar diameter responses to NOS blockade in IDDM (30). This observation suggests that superoxide anion accumulation promotes a reduction in ambient NO concentration and its impact on the renal microvasculature in IDDM, even in the face of normal or increased NO synthesis. A deleterious impact of superoxide anion on the renal microvascular actions of NO in IDDM is further implicated by our present observation that exogenous SOD unmasked a modulator effect of endogenous NO on ANG II-induced afferent arteriolar constriction in STZ kidneys, while not altering ANG II responses in kidneys from Sham rats. Thus superoxide anion acting in the vicinity of the afferent arteriole appears responsible for the impaired availability of NO to temper ANG II-induced constriction of that vascular segment.

Although the influence on NO on efferent arteriolar baseline diameter is suppressed in STZ rats (30), the results of the present study reveal that the modulatory impact of NO on efferent arteriolar ANG II responsiveness is preserved. This surprising observation may relate to the distinct sources of the NO that influences juxtamedullary arteriolar baseline diameter and constrictor responsiveness. Through the use of inhibitors that are selective for NOS-1 or nonselective (affecting all 3 NOS isoforms), Ichihara et al. (18) provided evidence that baseline diameters of juxtamedullary efferent arterioles are maintained under the tonic influence of NO derived from both NOS-3 (from endothelial sources) and NOS-1 [expressed in macula densa cells and the efferent arteriolar endothelium (5)]. Moreover, their work indicated that juxtamedullary efferent arteriolar ANG II responsiveness is modulated solely by NO produced through the enzymatic activity of NOS-1 (18). It is possible that the NO produced by NOS-1 is either anatomically or physiologically protected from reaction with superoxide anion in IDDM, or that superoxide anion production in IDDM is relatively compartmentalized at endothelial sites and thus reacts predominantly with NO derived from endothelial sources (mainly NOS-3). Further experiments are necessary to address these postulates.

In summary, the results of the present study confirm that NOS inhibition allows exaggerated juxtamedullary arteriolar diameter responses to ANG II, reflecting the modulatory impact of endogenous NO on agonist-induced vasoconstriction. NO modulation of afferent arteriolar ANG II responsiveness was absent in afferent arterioles from diabetic rats but was unmasked by acute treatment with superoxide dismutase. NO modulation of efferent arteriolar ANG II responsiveness was retained in kidneys from diabetic rats, perhaps due to compartmentalization of either NO or superoxide anion production. These observations support the contention that the renal microvascular impact of endogenous NO is suppressed in IDDM, largely as a result of NO degradation via reaction with superoxide anion.


    ACKNOWLEDGEMENTS

We thank David M. Pollock and Jennifer S. Pollock for performing the creatinine assays.


    FOOTNOTES

Merck Research Laboratories (Rahway, NJ) provided the enalaprilat used in these studies. This work was supported by a grant from the Juvenile Diabetes Foundation International (197008). G. C. Schoonmaker was the recipient of a Medical Student Research Scholarship from the Nebraska Medical Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. K. Carmines, Dept. of Physiology and Biophysics, Univ. of Nebraska College of Medicine, 984575 Nebraska Medical Center, Omaha, NE 68198-4575 (E-mail: pkcarmin{at}unmc.edu).

Received 20 May 1999; accepted in final form 13 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anderson, S. Physiologic actions and molecular expression of the renin-angiotensin system in the diabetic rat. Min. Electrolyte Metab. 24: 406-411, 1998[ISI][Medline].

2.   Anderson, S., F. F. Jung, and J. R. Ingelfinger. Renal renin-angiotensin system in diabetes: functional, immunohistochemical, and molecular biological correlations. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 265: F477-F486, 1993[Abstract/Free Full Text].

3.   Angulo, J., L. Rodríguez-Mañas, C. Peiró, M. Neira, J. Marín, and C. F. Sánchez-Ferrer. Impairment of nitric oxide-mediated relaxations in anaesthetized autoperfused streptozotocin-induced diabetic rats. Naunyn Schmiedebergs Arch. Pharmacol. 358: 529-537, 1998[ISI][Medline].

4.   Arima, S., S. Ito, K. Omata, K. Takeuchi, and K. Abe. High glucose augments angiotensin II action by inhibiting NO synthesis in in vitro microperfused rabbit afferent arterioles. Kidney Int. 48: 683-689, 1995[ISI][Medline].

5.   Bachmann, S., H. M. Bosse, and P. Mundel. Topography of nitric oxide synthesis by localizing constitutive NO synthases in mammalian kidney. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 268: F885-F898, 1995[Abstract/Free Full Text].

6.   Bank, N., and H. S. Aynedjian. Role of EDRF (nitric oxide) in diabetic renal hyperfiltration. Kidney Int. 43: 1306-1312, 1993[ISI][Medline].

7.   Barrett, R. J., and D. A. Droppleman. Interactions of adenosine A1 receptor-mediated renal vasoconstriction with endogenous nitric oxide and ANG II. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 265: F651-F659, 1993[Abstract/Free Full Text].

8.   Carmines, P. K. Segment-specific effect of chloride channel blockade on rat renal arteriolar contractile responses to angiotensin II. Am. J. Hypertens. 8: 90-94, 1995[ISI][Medline].

9.   Carmines, P. K., K. Ohishi, and H. Ikenaga. Functional impairment of renal afferent arteriolar voltage-gated calcium channels in rats with diabetes mellitus. J. Clin. Invest. 98: 2564-2571, 1996[Abstract/Free Full Text].

10.   Casellas, D., and P. K. Carmines. Control of the renal microvasculature: cellular and integrative perspectives. Curr. Opin. Nephrol. Hypertens. 5: 57-63, 1996[Medline].

11.   Casellas, D., and L. G. Navar. In vitro perfusion of juxtamedullary nephrons in rats. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 246: F349-F358, 1984[Abstract/Free Full Text].

12.   Cosentino, F., K. Hishikawa, Z. S. Katusic, and T. F. Luscher. High glucose increases nitric oxide synthase expression and superoxide anion generation in human aortic endothelial cells. Circulation 96: 25-28, 1997[Abstract/Free Full Text].

13.   Dai, F.-X., A. Diederich, J. Skopec, and D. Diederich. Diabetes-induced endothelial dysfunction in streptozotocin-treated rats: role of prostaglandin endoperoxides and free radicals. J. Am. Soc. Nephrol. 4: 1327-1336, 1993[Abstract].

14.   Diederich, D., J. Skopec, A. Diederich, and F.-X. Dai. Endothelial dysfunction in mesenteric resistance arteries of diabetic rats: role of free radicals. Am. J. Physiol. Heart Circ. Physiol. 266: H1153-H1161, 1994[Abstract/Free Full Text].

15.   Forti, A. C., and M. C. Fonteles. Decreased endothelium dependent relaxation (nitric oxide) in diabetic kidneys. Horm. Metab. Res. 30: 55-57, 1998[ISI][Medline].

16.   Fridovich, I. Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase. J. Biol. Chem. 245: 4053-4057, 1970[Abstract/Free Full Text].

17.   Goor, Y., G. Peer, A. Iaina, M. Blum, Y. Wollman, T. Chernihovsky, D. Silverberg, and S. Cabili. Nitric oxide in ischaemic acute renal failure of streptozotocin diabetic rats. Diabetologia 39: 1036-1040, 1996[ISI][Medline].

18.   Ichihara, A., E. W. Inscho, J. D. Imig, and L. G. Navar. Neuronal nitric oxide synthase modulates rat renal microvascular function. Am. J. Physiol. Renal Physiol. 274: F516-F524, 1998[Abstract/Free Full Text].

19.   Ikenaga, H., R. W. Fallet, and P. K. Carmines. Basal nitric oxide production curtails arteriolar vasoconstrictor responses to ANG II in rat kidney. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 271: F365-F373, 1996[Abstract/Free Full Text].

20.   Kamata, K., and M. Hosokawa. Endothelial dysfunction in the perfused kidney from the streptozotocin-induced diabetic rat. Res. Comm. Mol. Pathol. Pharmacol. 96: 57-70, 1997[ISI][Medline].

21.   Kennefick, T. M., T. T. Oyama, M. M. Thompson, J. P. Vora, and S. Anderson. Enhanced renal sensitivity to angiotensin actions in diabetes mellitus in the rat. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 271: F595-F602, 1996[Abstract/Free Full Text].

22.   Kiff, R. J., S. M. Gardiner, A. M. Compton, and T. Bennett. The effects of endothelin-1 and NG-nitro-L-arginine methyl ester on regional haemodynamics in conscious rats with streptozotocin-induced diabetes mellitus. Br. J. Pharmacol. 103: 1321-1326, 1991[Abstract].

23.   Komers, R., T. J. Allen, and M. E. Cooper. Role of endothelium-derived nitric oxide in the pathogenesis of the renal hemodynamic changes of experimental diabetes. Diabetes 43: 1190-1197, 1994[Abstract].

24.   Langenstroer, P., and G. M. Pieper. Regulation of spontaneous EDRF release in diabetic rat aorta by oxygen free radicals. Am. J. Physiol. Heart Circ. Physiol. 263: H257-H265, 1992[Abstract/Free Full Text].

25.   Lewis, E. J., L. G. Hunsicker, R. P. Bain, and R. D. Rohde. The effect of angiotensin-converting-enzyme inhibition on diabetic nephropathy. N. Engl. J. Med. 329: 1456-1462, 1993[Abstract/Free Full Text].

26.   Mathis, K. M., and R. O. Banks. Role of nitric oxide and angiotensin II in diabetes mellitus-induced glomerular hyperfiltration. J. Am. Soc. Nephrol. 7: 105-112, 1996[Abstract].

27.   Matsumura, Y., Y. Egi, H. Maekawa, A. Miura, S. Murata, and S. Morimoto. Enhancement of norepinephrine and angiotensin II-induced renal effects by NG-nitro-L-arginine, a nitric oxide synthase inhibitor. Biol. Pharm. Bull. 18: 496-500, 1995[ISI][Medline].

28.   Mattar, A. L., C. K. Fujihara, M. O. Ribeiro, G. De Nucci, and R. Zatz. Renal effects of acute and chronic nitric oxide inhibition in experimental diabetes. Nephron 74: 136-143, 1996[ISI][Medline].

29.   Mayhan, W. G. Superoxide dismutase partially restores impaired dilatation of the basilar artery during diabetes mellitus. Brain Res. 760: 204-209, 1997[ISI][Medline].

30.   Ohishi, K., and P. K. Carmines. Superoxide dismutase restores the influence of nitric oxide on renal arterioles in diabetes mellitus. J. Am. Soc. Nephrol. 5: 1559-1566, 1995[Abstract].

31.   Ohishi, K., P. K. Carmines, E. W. Inscho, and L. G. Navar. EDRF-angiotensin II interactions in rat juxtamedullary afferent and efferent arterioles. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 263: F900-F906, 1992[Abstract/Free Full Text].

32.   Ohishi, K., M. I. Okwueze, R. C. Vari, and P. K. Carmines. Juxtamedullary microvascular dysfunction during the hyperfiltration stage of diabetes mellitus. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 267: F99-F105, 1994[Abstract/Free Full Text].

33.   Parekh, N., L. Dobrowolski, A. P. Zou, and M. Steinhausen. Nitric oxide modulates angiotensin II- and norepinephrine-dependent vasoconstriction in rat kidney. Am. J. Physiol. Regulatory Integrative Comp. Physiol. 270: R630-R635, 1996[Abstract/Free Full Text].

34.   Pflueger, A. C., H. Osswald, and F. G. Knox. Adenosine-induced renal vasoconstriction in diabetes mellitus rats: role of nitric oxide. Am. J. Physiol. Renal Physiol. 276: F340-F346, 1999[Abstract/Free Full Text].

35.   Pollock, D. M., J. S. Polakowski, T. J. Opgenorth, and J. S. Pollock. Role of ETA receptors in the hypertension produced by 4-day L-NAME and cyclosporine treatment. Eur. J. Pharmacol. 346: 43-50, 1998[ISI][Medline].

36.   Reid, J. J., and M. J. Rand. Renal vasoconstriction is modulated by nitric oxide. Clin. Exp. Pharmacol. Physiol. 19: 376-379, 1992[ISI][Medline].

37.   Remuzzi, A., N. Perico, C. S. Amuchastegui, B. Malanchini, M. Mazerska, C. Battaglia, T. Bertani, and G. Remuzzi. Short- and long-term effect of angiotensin II receptor blockade in rats with experimental diabetes. J. Am. Soc. Nephrol. 4: 40-49, 1993[Abstract].

38.   Sugimoto, H., K. Shikata, M. Matsuda, M. Kushiro, Y. Hayashi, K. Hiragushi, J. Wada, and H. Makino. Increased expression of endothelial cell nitric oxide synthase (ecNOS) in afferent and glomerular endothelial cells is involved in glomerular hyperfiltration of diabetic nephropathy. Diabetologia 41: 1426-1434, 1998[ISI][Medline].

39.   Tolins, J. P., P. J. Shultz, L. Raij, D. Brown, and S. M. Mauer. Abnormal renal hemodynamic response to reduced renal perfusion pressure in diabetic rats: Role of nitric oxide. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 265: F886-F895, 1993[Abstract/Free Full Text].

40.   Vora, J. P., T. T. Oyama, M. M. Thompson, and S. Anderson. Interactions of the kallikrein-kinin and renin-angiotensin systems in experimental diabetes. Diabetes 46: 107-112, 1997[Abstract].

41.   Wang, Y. X., D. P. Brooks, and R. M. Edwards. Attenuated glomerular cGMP production and renal vasodilation in streptozotocin-induced diabetic rats. Am. J. Physiol. Regulatory Integrative Comp. Physiol. 264: R952-R956, 1993[Abstract/Free Full Text].

42.   Wilkes, B. M., P. F. Mento, and M. A. Vernace. Angiotensin responsiveness in hyperfiltering and nonhyperfiltering diabetic rats. J. Am. Soc. Nephrol. 4: 1346-1353, 1993[Abstract].

43.   Zatz, R., B. R. Dunn, T. W. Meyer, S. Anderson, H. G. Rennke, and B. M. Brenner. Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension. J. Clin. Invest. 77: 1925-1930, 1986[ISI][Medline].


Am J Physiol Renal Physiol 278(2):F302-F309
0363-6127/00 $5.00 Copyright © 2000 the American Physiological Society