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Am J Physiol Regul Integr Comp Physiol 294: R1744-R1751, 2008. First published March 19, 2008; doi:10.1152/ajpregu.00871.2007
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WATER AND ELECTROLYTE HOMEOSTASIS

L-arginine-induced glomerular hyperfiltration response: the roles of insulin and ANG II

Mario Ruiz, Prabhleen Singh, Scott C. Thomson, Karen Munger, Roland C. Blantz, and Francis B. Gabbai

Division of Nephrology-Hypertension, VA San Diego Healthcare System and the University of California, San Diego School of Medicine, La Jolla, California

Submitted 5 December 2007 ; accepted in final form 12 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Infusion of L-arginine produces an increase in glomerular filtration via kidney vasodilation, correlating with increased kidney excretion of nitric oxide (NO) metabolites, but the specific underlying mechanisms are unknown. We utilized clearance and micropuncture techniques to examine the whole kidney glomerular filtration rate (GFR) and single nephron GFR (SNGFR) responses to 1) L-arginine (ARG), 2) ARG+octreotide (OCT) to block insulin release, 3) ARG+OCT+insulin (INS) infusion to duplicate ARG-induced insulin levels, and 4) losartan (LOS), an angiotensin AT-1 receptor blocker, +ARG+OCT. ARG infusion increased GFR, while increasing insulin levels. OCT coinfusion prevented this increase in GFR, but with insulin infusion to duplicate ARG induced rise in insulin, the GFR response was restored. Identical insulin levels in the absence of ARG had no effect on GFR. In contrast to ARG infusion alone, coinfusion of OCT with ARG reduced proximal tubular fractional and absolute reabsorption potentially activating tubuloglomerular feedback. Losartan infusion, in addition to ARG and OCT (LOS+ARG+OCT), restored the increase in both SNGFR and proximal tubular reabsorption, without increasing insulin levels. In conclusion, 1) hyperfiltration responses to ARG require the concurrent, modest, permissive increase in insulin; 2) inhibition of insulin release after ARG reduces proximal reabsorption and prevents the hyperfiltration response; and 3) inhibition of ANG II activity restores the hyperfiltration response, maintains parallel increases in proximal reabsorption, and overrides the arginine/octreotide actions.

nitric oxide; glomerular filtration rate; octreotide

L-ARGININE INFUSION PRODUCES systemic and kidney vasodilation, leading to an increase in glomerular filtration rate (GFR) (11, 26). While this effect of L-arginine on renal function is well established, the mechanism by which L-arginine increases GFR has not been defined. Increases in nitric oxide (NO) generation have been proposed as a mediator of the effects of L-arginine on renal hemodynamics since L-arginine is the substrate for NO. Nitric oxide metabolites increase in the urine during infusion of L-arginine, and blockers of nitric oxide synthase blunt the renal vasodilatory response to solutions containing mixed amino acids, including L-arginine (2, 19, 25).

An important implication of the above hypothesis is that changes in plasma concentration of L-arginine modulate the activity of the endothelial nitric oxide synthase. This enzyme is located in the cellular cytoplasm and plasma membrane, and increases in the activity of this enzyme due to changes in plasma levels of L-arginine should enhance NO generation leading to renal vasodilation and increases in GFR (34). Support for an additional mechanism of L-arginine-mediated vasodilation was provided in studies performed by Guigliano et al. (14), when the response of the peripheral vascular bed (lower extremities) to L-arginine infusion was examined in the presence and absence of octreotide, an inhibitor of insulin secretion. While infusion of L-arginine in normal individuals reduced peripheral vascular resistance, administration of L-arginine with octreotide did not modify vascular resistance. Simultaneous administration of octreotide and insulin restored the vasodilatory response of the peripheral vascular bed to L-arginine. Overall, the elegant study by Guigliano et al. does not refute an important role for NO production but clearly support an important, and potentially permissive, role for insulin as a mediator between L-arginine infusion and peripheral vascular responses.

The kidney is a substantial vascular bed, which shares several similarities with the peripheral vascular bed, but also important differences. One of the most important differences is that the tone of the afferent and efferent arteriolar resistances is determined not only by endothelial factors but also by specific intrarenal mechanisms such as the tubuloglomerular feedback system (TGF) (38, 39). TGF couples changes in tubular reabsorption to changes in glomerular resistances, renal blood flow, and GFR to maintain glomerulotubular balance. Such unique characteristics of the renal vascular bed could have important implications with respect to the response of renal resistances to agonists or antagonists acting within the systemic vasculature. Specifically, while the renal vascular bed responds to L-arginine infusion in a similar manner as peripheral vascular resistance, the mechanism involved in the renal response might be quite different.

This study was designed to examine the mechanism(s) involved in L-arginine-induced increases in GFR by focusing on the potential role of insulin as a mediator of L-arginine response in the kidney (14, 20, 36, 37, 43). To this end, clearance and micropuncture experiments were performed to evaluate the effect of L-arginine infusion on GFR and plasma insulin levels in normal rats, as well as the role of insulin during L-arginine infusion. We have shown in prior studies that in many pathophysiological models of disease, the renal vasodilatory and hyperfiltration response to amino acid infusions is lacking, and blockade of ANG II restores the response (3–7, 10, 12). Others have also studied the role of ANG II in the renal responses to arginine infusion (17, 18, 42). To evaluate the interactions of such hormonal systems in the renal response to L-arginine infusion, micropuncture experiments were also performed to clarify the effect of ANG II blockade on proximal tubular reabsorption during L-arginine infusion. Experimental protocols were designed to address five specific questions: 1) what is the effect of L-arginine infusion on GFR and plasma insulin levels? 2) does blockade of insulin secretion with octreotide modify the GFR response to L-arginine? 3) can exogenous replacement of insulin restore the GFR response in rats treated with L-arginine and octreotide? 4) what is the role of insulin in modifying proximal tubular reabsorption during L-arginine infusion? and 5) what is the role of ANG II in the tubular and glomerular response during ARG+OCT?


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All experimentation was conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All our experimental studies were conducted under approvals of the Institutional Animal Care and Use Committee. The studies were conducted in 200- to 300-g, male Wistar-Fromter rats bred and housed in the Veterinary Medical Unit at the Veterans Affairs Medical Center (San Diego, CA). All rats received free access to tap water and standard rat chow.

Experimental Protocols

The experimental protocols are presented in Fig. 1. Studies were designed as two-period studies with control and experimental periods. Separate animals were used in each of the experimental protocols A, B, and C. Comparisons between animal groups and protocols are described in Table 1, along with the number of animals in each group. At the end of surgery and completion of the equilibration period, two 15-min urine collections were obtained to determine baseline GFR values, and a plasma sample was collected for insulin measurements. After completion of the control measurements, rats received either NaCl-NaHCO3 solution alone, L-arginine (Sigma Chemical, St. Louis, MO) at a dose of 7.5 mg·kg–1·min–1 (ARG), octreotide (OCT) (Sandostatin, Novartis Pharmaceuticals, East Hanover, NJ) at a dose of 50 µg·kg–1·h–1 or insulin (INS) (Novolin-Regular, human insulin, recombinant DNS origin, Novo Nordisk Pharmaceuticals, Princeton, NJ) 3 mU·kg–1·h–1 or a combination of ARG, OCT and INS (ARG+OCT, ARG+OCT+INS, or OCT+INS) at the doses previously mentioned. The dose of OCT was selected using a Medline literature search to establish midrange intravenous doses capable of inhibiting insulin secretion in rats. Losartan was administered as a 10 mg/kg iv bolus at the beginning of the equilibration period to evaluate the specific effects of AT-1 blockade on kidney function, and then arginine and octreotide were given at the end of the equilibration period. After a 50-min period of equilibration, two 15-min clearance periods were obtained to determine GFR. A plasma sample for insulin measurement was collected again at the end of this period. Urine samples were also collected for urinary nitric oxide metabolite measurements.


Figure 1
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  		Fig. 1. Experimental protocols A, B, and C.

 

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  		Table 1. Clearance studies for two-kidney GFR, insulin measurements and urinary NOx measurements and micropuncture studies for single-kidney GFR, SNGFRl, FR, APR measurements

 
Clearance Experiments

Surgical preparation. Under Inactin anesthesia (100 mg/kg body wt ip; Andrew Lockwood Associates, Ann Arbor, MI) a tracheostomy tube [polyethylene (PE)-250 tubing] was placed and left internal jugular (PE-50), left femoral artery (PE-50), and urinary bladder (PE-50) were cannulated. Body temperature was regulated by placing the rat on a water-heated blanket at 98°F (Baxter K-MOD 100 Heat Therapy Pump, Baxter Healthcare, Deerfield, IL). At the end of the surgical preparation, rats underwent a 60-min equilibration period. During the equilibration period, they received two infusions, one containing 3H-inulin in NaCl-NaHCO3 at a rate of 0.8 ml/h and the other containing NaCl-NaHCO3 solution at 1.4 ml/h. The latter infusion served as a vehicle for L-arginine (ARG), octreotide (OCT), insulin (INS), or various combinations, including ARG+OCT, ARG+OCT+INS, or OCT+INS administered during the second or experimental period.

Mean arterial blood pressure (MAP) was monitored by connecting the femoral artery catheter to a transducer and to a desktop computer loaded with the WinDaq software (DATAQ Instruments, Akron OH). Blood samples for glucose, hematocrit, and 3H-inulin were obtained from the femoral artery catheter at the beginning and the end of each urine collection. Glucose was measured via a Glucometer Elite XL (Bayer, Elkhart, IN). Urine was collected in preweighed containers under oil for two 15-min periods. GFR was calculated as the clearance of inulin using the formula: GFR = UV/P inulin, where U represents the concentration of inulin in the urine, V is the volume of urine per minute, and P is the concentration of inulin in the plasma. These clearance experiments provided GFR values from two kidneys and are expressed as ml·min–1·100 g body wt–1.

Micropuncture Experiments

Measurements of single-nephron glomerular filtration rate, absolute proximal reabsorption, and fractional proximal reabsorption. Anesthesia and preparatory surgery were similar to clearance experiments, including placement of tracheostomy, right jugular, left femoral artery, and bladder. The left kidney was exposed and placed in a Lucite cup. The cup surrounding the kidney was packed with cotton and 2% agar, and the surface was covered with warm (37°C) NaCl-NaHCO3 solution. All studies were performed using a euvolemic protocol, with infusion of 1% body wt donor plasma over 1-h period followed by 0.15% body wt donor plasma per hour. All rats received two additional infusions of NaCl-NaHCO3 solutions, one containing [3H]inulin 100 µCi/h in a volume of 0.8 ml/h and the other infusion (1.4 ml/h) served as a control for the solution containing L-arginine, and L-arginine+octreotide in doses previously mentioned. Both solutions were initiated at the end of the surgical preparation and maintained throughout the study.

After 1 h of equilibration, late surface segments of proximal tubules were identified by intratubular injection of diluted food dye and color (FD&C) contained in a micropipette of 3- to 5-µm outer diameter. Three timed tubular fluid collections (2.5 min) were randomly obtained from late proximal segment with the use of an injected oil block. Plasma samples were obtained for [3H]-labeled inulin concentration to compute single nephron glomerular filtration rate (SNGFR), and the tubular fluid to plasma (TF/P) inulin ratio. Two period studies were performed in all rats. After completing baseline measurements, rats received 1) L-arginine (ARG) or 2) L-arginine+octreotide (ARG+OCT). In the third group (LOS+ARG+OCT), losartan was administered at the beginning of the equilibration period, as described previously, and then L-arginine+octreotide were administered at the end of equilibration period after baseline measurements. A new set of proximal tubular collections was obtained 50 min after initiating L-arginine, or L-arginine+octeotride. Whole kidney GFR values represent left micropuncture kidney GFR only, while clearance experiments described above use two-kidney GFR, and are expressed as ml·min–1·100 g body wt–1.

Measurement of Plasma Insulin Levels

Blood (~0.8 ml) was collected in an Eppendorf tube and centrifuged for 3 to 4 min. Approximately 250 µl of plasma was micropipetted and immediately frozen at –70°C for later analysis. The removed plasma was replaced with NaCl-NaHCO3 solution, and the packed red blood cells were resuspended and infused into the rat via the left femoral artery to avoid excessive blood loss and volume depletion. Insulin levels were measured using a commercial kit (Rat Insulin Radioimmunoassay kit; Linco Research, St. Charles, MO).

Measurements of Nitrate/Nitrite Products

Urine samples were kept frozen at –70°C after completion of the experiments. These samples were then thawed out and analyzed using a commercial NO kit (Biomol Quantizyme Assay System Nitric Oxide Colorimetric Assay Kit, Biomol Research Laboratories, Plymouth Meeting, PA). This NO kit allows for the total determination of NO2 and NO3 in the specimen by conversion of the specimen nitrate by the enzyme nitrate reductase. This is followed by measurement of nitrite in the specimen as a colored azo dye product of the Griess reaction that absorbs visible light at 540 nm. The final urinary excretion of nitrate/nitrite (NOx) is expressed as picomoles per milliliter of GFR adjusted per 100 g body wt.

Other Analytic Methods

[3H]inulin activity in plasma, urine, and tubular fluid was monitored on a model B4530 Tri/Carb Packard liquid scintillation counter (Packard Instruments, Downers Grove, IL). SNGFR, absolute proximal reabsorption (APR), and fractional proximal reabsorption (FR) were determined as described in previous studies from this laboratory (3, 27, 28).

Statistical Analysis

Systat 6.0.1 for Windows (SPSS, 1996) was used to perform ANOVA with post hoc testing and repeated-measures ANOVA. Statistical significance was defined as P < 0.05 or lower when Bonferroni correction was used for multiple comparisons. Results are presented as means ± SE. GFR values are normalized to 100 g body wt. SNGFR and FR were compared using paired t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Clearance Studies

Thirty-two rats divided into four groups [Control (CON), ARG, ARG+OCT, and OCT] were used to address questions 1 and 2 using protocol A. Table 2 summarizes the characteristics of the four groups of rats and the results that were obtained during baseline and experimental periods. No significant differences were observed in body weight, mean arterial pressure, and hematocrit among the four groups when both periods were compared. Glucose levels were similar in CON, ARG, and ARG+OCT with mild but significant reductions observed during the experimental period in OCT. Changes in insulin, GFR, and urinary NOx products between experimental and baseline values are represented as {Delta} values in Fig. 2. GFR, insulin levels, and NOx remained unchanged in CON rats. Administration of L-arginine leads to significant increases in GFR, insulin levels, and NOx products in ARG group. Simultaneous administration of L-arginine and octreotide (ARG+OCT) prevented the changes in GFR, insulin levels, and NOx products. Infusion of octreotide alone produced no significant effect on GFR, insulin, or NOx products.


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  		Table 2. General characteristics protocol A

 

Figure 2
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  		Fig. 2. Changes in values of insulin (A); two-kidney GFR (B) and urinary NOx (C) between the control and experimental period in the four groups of rats for protocol A (n = 8 in each group). A–C: P < 0.05 between control and ARG group and between ARG and ARG+OCT.

 
To answer our third question about whether increases in insulin levels during L-arginine infusion are critical for the increase in GFR, we performed a second group of studies in which exogenous insulin was administered to duplicate the changes in plasma insulin levels during L-arginine infusion (protocol B). Two groups of rats were studied (n = 4 per group). A control group constituted by rats receiving octreotide and insulin (OCT+INS) was compared with rats receiving L-arginine, octreotide, and insulin (ARG+OCT+INS). Baseline characteristics including weight, MAP, and HCT were similar in the two groups (Table 3). Blood glucose was slightly higher during baseline conditions in the OCT+INS group and fell as a consequence of simultaneous infusion of octreotide and insulin. Changes in insulin levels, GFR, and NOx products between experimental and baseline values are represented as {Delta} values in Figs. 3A–C. Exogenous insulin administration increased insulin levels by ~1 pg/ml in both OCT+INS and OCT+INS+ARG (Fig. 3A). This increase in insulin was similar to the increase observed in ARG-treated rats (Fig. 2A). The increase in plasma insulin levels was associated with increases in GFR and NOx products in ARG+OCT+INS, while no changes in either one of these two parameters was observed in OCT+INS-treated rats (Fig. 3C).


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  		Table 3. General characteristics protocol B

 

Figure 3
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  		Fig. 3. Changes in values of insulin (A); two-kidney GFR (B) and urinary NOx (C) between the control and experimental period in the two groups of rats for protocol B (n = 4 in each group). B and C: P < 0.05 between OCT+INS and ARG+OCT+INS.

 
To answer question five, in a separate group of animals (n = 11), losartan was added prior to ARG+OCT (protocol C). This group was compared with the groups ARG and ARG+OCT from protocol A. LOS administration alone in the control period did not increase GFR. In the experimental period, there was a change in GFR similar to that with ARG alone (0.60 ± 0.06 vs. 0.49 ± 0.08 ml·min–1·100 g body wt–1) as shown in Table 4. Changes in the values of urinary NOx products were also similar in the LOS+ARG+OCT group to ARG alone group (3,942 ± 417 pmol/ml GFR in LOS+ARG+OCT vs. 3,333 ± 335 pmol/ml GFR in ARG). This was in spite of OCT administration blocking the insulin effects in the LOS+ARG+OCT group, as no change in insulin levels were seen in this group similar to controls [{Delta} insulin 0.16 ± 0.28 pg/ml in LOS+ARG+OCT vs. 0.21 ± 0.64 pg/ml in controls, P = not significant (NS)].


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  		Table 4. Changes in insulin, GFR, and UNOX levels in ARG, ARG+OCT, and LOS+ARG+OCT in clearance studies

 
Micropuncture Studies

Micropuncture techniques were used to investigate the changes in SNGFR, APR, and FR in rats treated with ARG, ARG+OCT, and LOS+ARG+OCT. Thirty-eight nephrons were evaluated in both control and experimental periods in 12 rats. SNGFR, left kidney GFR, APR, and FR results are presented in Fig. 4. Infusion of L-arginine increased SNGFR from 37.4 ± 2 to 42.4 ± 2.5 nl/min (P < 0.05). Associated with the increase in SNGFR, absolute proximal reabsorption increased such that FR remained unchanged (0.45 ± 0.02 vs. 0.41 ± 0.01, NS).


Figure 4
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  		Fig. 4. Changes in single nephron filtration rate (SNGFR), whole kidney GFR (single left kidney values via left ureteral catheter collection), absolute proximal reabsorption (APR), and fractional reabsorption (FR) in control condition and after arginine infusion for three groups-arginine (ARG), arginine+octreotide (ARG+OCT) and losartan+arginine+octreotide (LOS+ARG+OCT) (n = 4 in each group), 14 nephron collections each for the respective control and experimental periods in the ARG and ARG+OCT groups, 10 nephron collections each for the control and experimental periods in the LOS+ARG+OCT group.

 
The response in ARG+OCT group was quite different from the ARG-treated rats. There was no significant change in SNGFR (43.4 ± 1.7 vs. 39.1 ± 2.7 nl/min, NS) but a highly significant reduction in APR and FR (17.84 ± 1.28 vs. 10.43 ± 1.52 nl/min, P = 0.002 and 0.42 ± 0.03 vs. 0.28 ± 0.04, P = 0.01 respectively), suggesting that preventing the insulin increase during L-arginine infusion leads to significant changes in proximal reabsorption, thus answering our question on the role of insulin in modifying proximal tubular reabsorption during L-arginine infusion. Administration of LOS prior to ARG and OCT resulted in a dramatic increase in SNGFR from 39.9 ± 2.5 vs. 54.4 ± 1.6 nl/min (P < 0.05) while FR remained essentially constant (0.40 ± 0.02 vs. 0.41 ± 0.01), with a significant increase in APR (16.22 ± 1.82 vs. 21.97 ± 0.92 nl/min, P = 0.017) in parallel with SNGFR. These changes in SNGFR and APR were significant whether analyzed by ANOVA with individual nephron collections or when analyzed using the mean values for SNGFR and APR in control and experimental periods. There was no change in MAP in the control or experimental periods between ARG alone and LOS+ARG+OCT group [110 ± 7 in ARG group vs. 114 ± 2 in LOS+OCT+ARG group in the control period (P = 0.58) and 115 ± 4 in ARG group vs. 115 ± 4 in LOS+OCT+ARG group in the experimental period]. These data answer question five and demonstrate a critical role for ANG II in the glomerular and tubular responses in ARG+OCT-treated rats. These findings also raise the question of whether the large increase in APR is merely in response to the major increase in filtered load and perfect glomerulotubular balance or that LOS+OCT+ARG actually produces a primary increase in APR. The latter alternative mechanism might require an inhibitory effect of ANG II during ARG+OCT and a reversal of these effects by LOS.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Intravenous infusion of L-arginine, like several other amino acids, increases GFR as a result of kidney vasodilation (11, 26). The mechanism(s) involved in amino acid or L-arginine-induced increases in GFR has been a topic of great interest to many investigators (14, 17, 18, 42). Research has focused on increases in NO generation as the mechanism directly responsible for the increase in GFR since L-arginine is the substrate for nitric oxide production (2, 19, 36, 37). A relationship between L-arginine infusion and generation and activity of NO is perfectly logical and presumably of functional relevance. Since L-arginine is the substrate for nitric oxide synthase, an increase in extracellular substrate levels could increase NO levels in parallel via any of the three NOS isoforms (2, 19, 25). The results from the current study support this general conclusion in that 1) increases in GFR during L-arginine infusion correlate with increased NO metabolites in the urine and 2) absence of changes in GFR correlate with absence of changes in NO metabolites during systemic L-arginine administration. However, this simple hypothesis is difficult to reconcile with 1) the findings of the current study that insulin is required for L-arginine-induced changes in GFR and increase in NO generation, and 2) the finding that other hormonal systems, including ANG II play a role in this response and restore the SNGFR/GFR changes and NO generation as indexed by increased urinary NOx products in response to ARG (4–7).

Our results clearly demonstrate the importance of insulin in L-arginine-mediated changes in GFR. L-arginine administration 1) increases extracellular insulin levels, 2) blockade of insulin secretion with octreotide prevents the increase in GFR, and 3) exogenous administration of insulin to octreotide-treated rats restores the increase in GFR. Insulin not only promotes glucose uptake but also upregulates cellular transport of amino acids (23). The fact that insulin replacement alone restored the effects of L-arginine in the kidney demonstrates that other effects of octreotide on glucagon release were not involved. It seems likely that permissive insulin levels are required for L-arginine uptake by kidney cells to exert the functional kidney responses. In addition, the proximal tubular reabsorptive response to L-arginine infusion also plays a critical role in the response. Hence, the aggregate examination of mechanisms provided by these results suggests that the overall L-arginine-induced kidney functional responses are far more complex than merely the supply of kidney NO leading to vasodilation.

Previous studies by Tucker et al. (40) demonstrated that larger doses of insulin (5 units) increase GFR, suggesting that insulin alone augments GFR. Interestingly, administration of insulin alone in much lower doses (milliunits), sufficient to restore GFR responses in L-arginine+octreotide rats, did not produce changes in GFR, demonstrating that insulin alone at the normal post L-arginine levels is not responsible for the increase in GFR. Rather, it is the interaction between changes in L-arginine plasma levels and insulin that is critical to the increase in GFR and the generation of NO, as reflected by levels of metabolites in the urine. Our findings are in agreement with previous studies by Guigliano et al. (14), who examined the vasodilatory effect of L-arginine in the limb vasculature of normal human subjects. These investigators demonstrated that systemic administration of L-arginine reduces peripheral vascular resistance. The reduction in peripheral resistance was prevented by simultaneous infusion of L-arginine and octreotide and restored by exogenous insulin to reproduce the changes in insulin levels produced by infusion of L-arginine alone. Whether one examines either the peripheral vasculature or the kidney, changes in resistances during L-arginine administration require modest, simultaneous changes in insulin levels (8, 13, 21). These results therefore suggest that neither insulin nor L-arginine alone is the unique mediator of the vasodilator response, but they are both critical elements.

Our laboratory has a longstanding interest in examining the changes in proximal reabsorption during the infusion of various amino acids, including glycine in normal rats and in various experimental models of disease, including Goldblatt hypertension, diabetes mellitus, cyclosporine administration, and chronic glomerulonephritis (3–7). Administration of L-arginine in this study increased GFR and SNGFR, similar to those obtained in normal rats with glycine and other vasodilatory amino acids. The increase in SNGFR in normal rats is coupled to parallel increases in absolute proximal tubular reabsorption such that glomerulotubular balance is well maintained (29). Interestingly, absence of a GFR or SNGFR increase in arginine+octreotide rats was associated with a reduction in APR and FR during L-arginine administration. This reduction in proximal reabsorption is similar to the response we have previously observed during glycine infusion in experimental models of disease that do not elicit an increase in GFR/SNGFR (3–7).

We propose that the reduction in APR and FR during arginine+octreotide increases delivery of sodium chloride to the macula densa, which, in turn, activates the tubuloglomerular feedback system and limits any potential hyperfiltration response. The reduction in APR and FR in arginine+octreotide rats implies that insulin plays an important role in modulating or sustaining proximal tubular reabsorption. The results of our study also provide evidence for a regulatory role of ANG II in modifying the hemodynamic and reabsorptive responses to L-arginine infusion. Blockade of the AT-1 ANG II receptors in rats receiving arginine and octreotide (to block insulin) not only prevented the reduction in proximal reabsorption but also led to a restoration of the increase in SNGFR.

This could perhaps be predominantly a hemodynamic consequence due to the effect of losartan on the vasculature or it could be due to a direct tubular effect to increase proximal reabsorption. Although insulin levels were not affected, suggesting that there was no direct action of ANG II blockade on insulin release, there could be improved insulin sensitivity due to losartan despite unchanged insulin levels. There are data in the literature supporting the role of ANG II in the etiology of whole body and skeletal muscle insulin resistance, and inhibition of ANG II by AT1 receptor blockade in improving whole body insulin action in several insulin-resistant rodent models (9, 16, 22, 30, 35). In addition, prior studies from our group in rats did not demonstrate any changes in plasma and kidney ANG II concentrations after amino acid and specifically arginine administration (11).

Previous studies from our laboratory and others have shown that the interaction and balance between NO and ANG II responses in the kidney dictates the net hemodynamic and reabsorptive functional responses in a variety of physiological and pathophysiological conditions (3–7, 10, 12). We have observed that the hemodynamic and tubular reabsorptive kidney responses to nonselective inhibition of NOS with L-NMMA can be largely prevented by coadministration of losartan, an angiotensin AT-1 receptor blocker (3). The blockade of AT-1 receptor permitted a large increase in proximal tubular reabsorption in spite of the normal, proreabsorptive effects of ANG II via the AT-1 receptor (15, 33). This antiabsorptive effect of ANG II could be based upon a previously recognized biphasic effect on proximal tubular reabsorption (15). Losartan may have also dampened the strength of TGF helping to restore the normal GFR response (24, 28, 32). However, prevention of a reduction in proximal tubular reabsorption and suppression of TGF in concert may not be a sufficient explanation for restoration of the SNGFR response observed in rats receiving octreotide, losartan, and the L-arginine infusion. Losartan was infused prior to L-arginine; therefore the SNGFR response was not just the consequence of ANG II blockade since the control SNGFR was not significantly elevated above normal prior to L-arginine infusion. However, the overall response to L-arginine during octreotide and the restoration of a normal response by losartan was qualitatively quite similar to that observed previously in a variety of pathophysiological models that lack a normal GFR and vasodilatory response to amino acid infusions (3–7, 10, 12). Recent studies from our laboratory in a remnant kidney model have demonstrated that blockade of ANG II activity can restore protein expression of NOS-1 levels and functional responses to NOS blockade (unpublished observations). This is significant, as this model has been characterized by NO deficiency (1, 31, 44). Reductions in ANG II activity may compensate by some mechanism and restore NO responses to ARG in the presence of OCT (17, 40, 42) and perhaps improve insulin sensitivity without increasing insulin levels.

Perspectives and Significance

In conclusion, our findings demonstrate that while the hyperfiltration response after arginine infusion correlates with nitric oxide generation, an increase in insulin levels appears to be important to this response, and in the absence of insulin increase, blockade of ANG II activity restores this response. This suggests a complex interplay among several hormonal systems requiring coordinated responses of insulin release and ANG II activity for the GFR response to occur. The underlying mechanisms of this interaction and exact intrarenal site of action need to be elucidated in future studies.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by funds supplied from the National Institutes of Health (DK-28602, DK-56248, and DK-62831) and the Research Service of the Department of Veterans Affairs. Dr. Ruiz was supported by a Minority Supplement to DK-28602. Dr. Singh was supported by funds supplied by the National Kidney Foundation of Southern California.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. C. Blantz, Nephrology-Hypertension (9111-H), Univ. of California, San Diego School of Medicine, 3350 La Jolla Village Dr., San Diego, CA 92161 (e-mail: rblantz{at}ucsd.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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