Experiments were performed to determine whether l-arginine transport regulates nitric oxide (NO) production and hemodynamics in the renal medulla. The effects of renal medullary interstitial infusion of cationic amino acids, which compete with l-arginine for cellular uptake, on NO levels and blood flow in the medulla were examined in anesthetized rats. NO concentration in the renal inner medulla, measured with a microdialysis-oxyhemoglobin trapping technique, was significantly decreased by 26–44% and renal medullary blood flow, measured by laser Doppler flowmetry, was significantly reduced by 20–24% during the acute renal medullary interstitial infusion of l-ornithine, l-lysine, and l-homoarginine (1 μmol·kg−1·min−1 each; n = 6–8/group). In contrast, intramedullary infusion of l-arginine increased NO concentration and medullary blood flow. Flow cytometry experiments with 4-amino-5-methylamino-2′,7′-difluorescein diacetate, a fluorophore reactive to intracellular NO, demonstrated that l-ornithine, l-lysine, and l-homoarginine decreased NO by 54–57% of control, whereas l-arginine increased NO by 21% in freshly isolated inner medullary cells (1 mmol/l each, n > 1,000 cells/experiment). The mRNA for the cationic amino acid transporter-1 was predominantly expressed in the inner medulla, and cationic amino acid transporter-1 protein was localized by immunohistochemistry to the collecting ducts and vasa recta in the inner medulla. These results suggest that l-arginine transport by cationic amino acid transport mechanisms is important in the production of NO and maintenance of blood flow in the renal medulla.
- laser Doppler flowmetry
several functional studies provide evidence supporting the concept that endogenous production of nitric oxide (NO) by NO synthase (NOS) in the kidney may be partially dependent on extracellular l-arginine concentration and cellular l-arginine uptake mechanisms. Both renal perfusion rate and glomerular filtration rate were decreased when l-arginine was eliminated from the perfusate of the isolated perfused kidney preparation (32). Studies in anesthetized rats have demonstrated that intravenous administration of l-arginine leads to renal vasodilation (8), whereas cellular l-arginine uptake by the y+ transporter appears necessary for the NO-mediated effects on tubuloglomerular feedback (36). Moreover, the addition of either NO donors or increased extracellular l-arginine concentration inhibited chloride absorption in the isolated perfused thick ascending loop of Henle (31). The results of these functional studies support the hypothesis that cellular uptake of l-arginine is important in the regulation of NO formation in the kidney.
Supplementation of extracellular l-arginine has also proven beneficial in animal models of hypertension. Chronic oral or intravenous l-arginine administration prevents sodium-dependent hypertension in Dahl salt-sensitive (Dahl S) rats (1, 2, 13, 29, 28). Consistent with an antihypertensive effect, l-arginine treatment led to a normalization of the pressure natriuresis relationship and increased transmission of pressure into the renal interstitium in anesthetized Dahl S rats (28, 29). Experiments performed in our laboratory have since demonstrated that sodium-induced hypertension in the Dahl S rats can be prevented by selective infusion of l-arginine into the renal medullary interstitial space (26), indicating that the antihypertensive effect of l-arginine administration in the Dahl S may be due to alterations in l-arginine availability in the renal medulla.
To further investigate the mechanisms of cellular l-arginine uptake in the renal medulla, we recently reported that l-arginine uptake in the inner medullary collecting duct is mediated by a y+ transporter mechanism encoded as cationic amino acid transporter-1 (CAT1). Experiments with freshly isolated cells from the inner medulla subsequently demonstrated that both l-arginine uptake and NO production could be blocked by the cationic amino acids l-ornithine and l-lysine, which compete with l-arginine for cationic amino acid transport (38), and Zou and Cowley demonstrated that intravenous infusion of l-arginine increases NO concentration in both the renal cortex and medulla of anesthetized rats (41). Moreover, we recently reported (14) that chronic blockade of l-arginine transport in the renal medulla with an antisense oligonucleotide against CAT1 or the cationic amino acids l-ornithine and l-lysine leads to a sustained decrease in NO in the renal medulla and the development of systemic hypertension (14). These experimental observations indicate that cellular l-arginine transport may be a critical factor in NO formation in this portion of the kidney. Furthermore, the results of these studies raise the possibility that the in vivo ratio of l-arginine to other cationic amino acids may regulate NO production and consequently NO-dependent function in this portion of the kidney. To date, the influence of manipulation of arginine-transport mechanisms on the regulation of renal medullary blood flow has not been experimentally investigated.
In the present study, we tested the hypotheses that l-arginine transport by a y+ mechanism influences NO formation in the renal medulla in vivo, and that an alteration in the balance of l-arginine and other cationic amino acids in the renal medulla affects NO production and NO-dependent function. Initial studies examined the concentration of l-arginine and other cationic amino acids in the renal medulla. Studies were then performed to determine the influence of renal medullary interstitial infusion of the cationic amino acids l-ornithine, l-lysine, l-homoarginine, and l-arginine on renal medullary interstitial NO levels and renal medullary blood flow in anesthetized Sprague-Dawley rats. The effects of l-arginine and other cationic amino acids on intracellular NO levels in a serum-free, freshly isolated suspension of renal medullary cells were also studied. Finally, to determine which cationic amino acid transporter is likely affected, quantitative RT-PCR was performed to compare the abundance of CAT1, CAT2, and CAT3 mRNA in the renal cortex and medulla and an immunohistochemistry protocol was performed to examine the distribution of CAT1 in the inner medulla.
Male Sprague-Dawley rats weighing between 280 and 350 g were used in this study. The animals were obtained from Harlan Sprague Dawley (Madison, WI), and housed in the Animal Resource Center at the Medical College of Wisconsin. Food and water were available ad libitum. All experiments were conducted in accordance with the Medical College of Wisconsin Institutional Animal Care and Use Committee's guidelines.
Concentration of l-arginine, l-ornithine, and l-lysine in plasma and renal tissue.
Rats were euthanized with an overdose of pentobarbital sodium (100 mg/kg ip). A sample of arterial blood was obtained and spun at 5,000 g for 15′ to separate the plasma. The kidneys were also removed from the rat and grossly divided into renal cortical, outer medullary, and inner medullary tissue before snap freezing on dry ice. The samples were kept at −80°C until analysis. The renal tissue was homogenized using a Potter-Elvehjem tissue grinder at 3,000 rpm in a solution containing (in mmol/l) 250 sucrose, 1 EDTA, 0.1 PMSF, and 5 potassium phosphate, pH 7.7. All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise noted. The homogenate was centrifuged at low speed (15,000 g, 4°C, 20′), and the protein concentration of the supernatant was determined using a Coomassie blue protein assay (Pierce; Rockford, IL) with bovine serum albumin as a standard.
Amino acids were separated by reverse phase (RP)-HPLC and quantitated by fluorometric detection as we previously described (21, 26). Before RP-HPLC separation, the plasma or tissue samples were deproteinized with 0.14 M sulfosalicylic acid containing known concentrations of l-β-alanine (internal standard). The samples were mixed, centrifuged at 10,000 g for 15 min, derivatized with o-phthaldialdehyde (1 mg/ml), and separated by RP-HPLC with a system consisting of a Bio-Rad AS-100 Auto Sampler, Hitachi L-7100 Gradient Pump, Waters (15 cm × 3.9 mm, 5 μm) column, Waters 474 Fluorescent Detector (Excitation: 338λ Emission: 425λ) and a Hitachi D-2500 Integrator. Validation experiments demonstrated a clean separation of a standard cocktail consisting of 17 amino acids. With six repeated injections, the coefficient of variation for the arginine peak was 0.3% for retention time, 3.3% for peak area, and 1.65% for peak height. The calculated concentration of l-arginine in a plasma pool injected 23 times on 6 different days yielded a coefficient of variation of 5%.
Surgical preparation for microdialysis and laser Doppler flowmetry.
Rats were anesthetized with an injection of thiobutabarbital (Inactin; 100 mg/kg body wt ip) and a tracheotomy was performed. The right jugular vein and the right femoral artery were cannulated with polyethylene (PE)-50 to infuse saline (154 mmol/l NaCl) intravenously (5 ml·kg−1·h−1) and to measure arterial pressure, respectively. The left kidney was exposed via a flank incision for the implantation of optical fibers or microdialysis probes. Interstitial infusion catheters were constructed as previously described (18), inserted into a depth of 5.5 mm, and infused with saline vehicle or saline with amino acids or drugs at a rate of 0.5 ml/h.
Measurement of NO by oxyhemoglobin-trapping technique.
In vivo microdialysis studies in the renal medulla of rats were performed as previously described (15, 14, 41). Microdialysis probes which permit both infusion and collection of microdialysis fluid (IBR-2; Bioanalytical Systems, Indianapolis, IN) were implanted to a depth of 5.5 mm beneath the renal cortical surface for NO measurements in the renal medulla. The microdialysis port of these probes were perfused at a rate of 2 μl/min with a solution (pH 7.4) containing (in mmol/l): 40.5 Na2HPO4, 9.5 NaH2PO4, 500 NaCl, and 0.003 oxyhemoglobin (Human Ao hemoglobin [ferrous]; Sigma). A spectrophotometric assay of NO-induced methemoglobin formation in the dialysate was then performed (DU-640; Beckmann Instruments, Fullerton, CA). Because oxyhemoglobin is stoichiometrically converted to methemoglobin on oxidation by NO, methemoglobin concentration was used as an index of NO concentration in the renal interstitial space. Methemoglobin (or NO) concentration was calculated from the equation c = A/εb, where c is methemoglobin or NO concentration, A is the absorbance difference between 401 and 411 nm, ε is the extinction coefficient of methemoglobin (112,000 M−1·cm−1), and b is the light path (in cm).
In the experimental protocols, saline was infused as a vehicle at a rate of 0.5 ml/h via the infusion port of the microdialysis probes. After a 120-min equilibration period, dialysate fluid was collected during a 60-min control period. Thereafter, l-ornithine (N = 8, 1 μmol·kg−1·min−1), l-lysine (N = 8, 1 μmol·g−1·min−1), l-homoarginine (N = 8, 1 μmol·kg−1·min−1), or l-arginine (N = 8, 1 μmol·kg−1·min−1) were infused into the interstitial space of the renal medulla. All experimental compounds were infused in saline. Because the cationic amino acids l-lysine (17), l-homoarginine (27), and l-ornithine (Wu and Mattson, unpublished observations) do not directly inhibit NOS enzymatic activity, the effects of these amino acids should be mediated by effects independent of direct NOS enzyme inhibition. After a 60-min equilibration period, dialysate fluid was collected for 60 min. Finally, the NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 100 μg·kg−1·min−1) was administered to each group for a final collection period. This large dose of l-NAME was administered to demonstrate the functional effects of a maximum dose of a NOS enzyme inhibitor; l-NAME does not compete with l-arginine for cellular uptake in inner meduallry collecting ducts (IMCD) (38) or other cell types (34). To test for the reversibility of the effect of the cationic amino acids, excess l-arginine (N = 8; 10 μmol·kg−1·min−1) was administered in the final period to a separate group of rats infused in the initial experimental period with l-ornithine (1 μmol·kg−1·min−1). As a final set of control experiments, mannitol (N = 8; 1 μmol·kg−1·min−1) was infused into the interstitial space for both experimental periods to examine the influence of nonspecific osmotic effects of the amino acid solutions on NO in the renal medullary interstitial space.
Laser Doppler flowmetry.
Single-mode optical fibers (Edmund Scientific; Barrington, NJ) were inserted into the renal medulla (5.5 mm). The fibers were sheathed with PE-50 and anchored in place on the kidney surface with cyanoacrylate adhesive. Interstitial infusion catheters were constructed as previously described (18) and inserted into the renal inner medulla so the tip of the catheters were located within 1 mm of the optical fibers. The flow signals from the renal medulla were measured with a laser-Doppler flowmeter (model BLF21D, Transonic Systems; Ithaca, NY) via the optical fiber as previously described (15, 20).
Laser-Doppler flowmetry experiments were performed on six groups of rats. After a 120-min equilibration period after surgery, the control level of renal medullary blood flow was obtained during a 30-min control period, in which saline (0.5 ml/h) was infused into the renal medullary interstitial catheter. The following amino acids in saline were then infused into the renal medulla in separate groups for a 30-min experimental period: l-ornithine (n = 6, 1 μmol·kg−1·min−1), l-lysine (n = 6, 1 μmol·kg−1·min−1), l-homoarginine (n = 6, 1 μmol·kg−1·min−1), l-ornithine (1 μmol·kg−1·min−1) with excess l-arginine (n = 6, 10 μmol·kg−1·min−1), l-arginine (n = 6, 1 μmol·kg−1·min−1), and l-NAME (n = 7, 1 μmol·kg−1·min−1). Mean arterial pressure and medullary blood flow were averaged during the final 5 min of each period.
Flow cytometry for 4-amino-5-methylamino-2′,7′-difluorescein diacetate fluorescence.
Isolated renal inner medullary cells were prepared as described (14, 38), resuspended in Locke's solution composed of (in mmol/l) 154 NaCl, 5.6 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 3.6 NaHCO3, and 5.6 glucose, and preincubated for 60 min in either Locke's solution, Locke's solution containing a 1,000-fold dilution of rabbit antiserum raised against CAT1 (14), or Locke's solution containing a 1,000-fold dilution of nonimmune rabbit serum. After this preincubation, the cells were subsequently incubated with Locke's solution containing 1 mmol/l of l-ornithine, l-lysine, l-homoarginine, l-arginine, l-NAME, or d-mannitol for 60 min at 37°C. Additional experiments were performed, in which l-arginine (1 mmol/l) was added in addition to 1 mmol/l solution of l-lysine, l-ornithine, and l-homoarginine. Thereafter, the cells were loaded with 10 μmol/l of 4-amino-5-methylamino-2′,7′-difluorescein diacetate (DAF-FM diacetate; Molecular Probes, Eugene, OR), a fluorophore reactive to intracellular NO, for 60 min at 37°C. Flow cytometry for the triazolo-fluorescein analog (DAF-FM-T) was performed using a FACScan cytometer (Becton-Dickinson, Franklin Lakes, NJ) equipped with an argon laser for excitation at 488 nm. A minimum of 1,000 events was collected for each analysis. The DAF-FM-T fluorescence was detected with a 530/30-nm band-pass filter. Data acquisition and analysis were performed with Summit version 3.0 software (DakoCytomation, Fort Collins, CO).
Tissue was homogenized in the lysis buffer and RNA was extracted using ABI6700 workstation. Expression of the mRNA for rat CAT1–3 was measured by real-time quantitative reverse transcription-PCR with the Perkin-Elmer ABI 7700 Sequence Detection System as previously described with 100 ng total RNA (17). Primers for CAT-1 (Genbank gi6981555) amplification were 5′-TGAACATTTCAGCCGGCCT-3′ (Fwd) and 5′-TCTTCCAAGCACGGCCACA-3′ (Rev). The probe for CAT1 detection was 5′-FAM-AGCCGTTCTTATCATCACCGTGTGCA-Tamra-3′. Primers for CAT2 (for both CAT2a and CAT2b; Genbank gi5114429) amplification were 5′-ATGGCGCTTGGCTTTCTGAT-3′ (Fwd) and 5′-AAGGCATCCTCATCGTCTTC-3′ (Rev). The probe for CAT2 detection was 5′-FAM-TCGTCCCTGGGGTTACCCTCCAAG-Tamra-3′. Primers for CAT3 (Genbank gi8392943) amplification were 5′-GTGAATGTTTACCTGATGATGC-3′ (Fwd) and 5′-GTAGATAGCAAATCCAATCAGC-3′ (Rev). The probe for CAT3 detection was 5′-FAM-ATGACAGCTGACACTTGGGCCCGA-Tamra-3′. Serially diluted sense RNAs transcribed with T3 RNA polymerase (Ambion) from a cDNA fragment subcloned into pBluescript II (Stratagene) for each of the genes of interest were used as external controls. All amplifications were duplicated. The PCR products were sequenced to confirm the specificity of the reaction.
Immunohistochemistry of CAT1 in the renal medulla.
The excised kidneys were fixed by immersion in 4% paraformaldehyde for 48 h and embedded in paraffin. Four micrometer sections prepared on Silane-coated slide glass were deparaffinized with xylene and washed with PBS. The sections were incubated in 3% H2O2 in methanol for 10 min to eliminate endogenous peroxidase activity. After being rinsed with PBS, the sections were treated with 10% goat serum for 15 min. After being washed with PBS, the sections were exposed to rabbit antiserum against rat CAT1 (14) in a dilution of 1:100 for 60 min. After being rinsed with PBS, the sections were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories) in a dilution of 1:200 for 30 min. The sections were then rinsed and incubated for 60 min with peroxidase-labeled streptavidin. After being treated with diaminobenzidine for 10 min, the sections were counterstained with Gill's hematoxylin. The slides were examined with an inverted microscope (model IX70, Olympus). Digitized images were produced with a Spot RT Slider cooled digital camera and Spot version 3.5.4 software (Diagnostic Instruments).
Data are expressed as means ± SE. Within-group changes were evaluated with a one-way ANOVA for repeated measurements, followed by a Student-Newman-Keuls post hoc test. Between-group changes were evaluated with a two-way ANOVA, followed by a Student-Newman-Keuls post hoc test. A confidence level of P < 0.05 was considered significant.
Concentration of l-arginine, l-ornithine, and l-lysine in plasma and renal tissue.
Initial experiments were performed to determine the concentration of l-arginine and the other cationic amino acids, l-ornithine and l-lysine, in the plasma and renal tissue of SD rats. As illustrated in Fig. 1, the plasma concentration of l-lysine was significantly greater than the concentrations of l-arginine and l-ornithine. In the homogenized kidney tissue, normalized to quantity of tissue protein, the concentration of each amino acid was significantly greater in the renal cortical tissue than in the outer medulla or inner medulla. In the renal cortex, the concentration of l-arginine and l-lysine was six- to sevenfold greater than the concentration of l-ornithine. The l-arginine and l-lysine levels were not significantly different from each other in any of the kidney regions examined; l-ornithine was not detectable in the homogenates of the renal outer and inner medulla.
Effect of infusion of cationic amino acids on NO concentration in the renal medulla.
The concentration of NO in situ was evaluated in the rat renal medulla by an in vivo microdialysis-oxyhemoglobin trapping technique. The summarized data are illustrated in Fig. 2. The baseline NO levels (∼120 nmol/l) are in agreement with levels previously reported for the renal medulla (14, 15, 41). NO concentration in the renal medulla was significantly decreased by the 60-min intramedullary infusion of the cationic amino acids l-ornithine, l-lysine, and l-homoarginine. Subsequent infusion of the NOS inhibitor l-NAME after infusion of these amino acids further reduced NO in the renal medulla. Moreover, infusion of an excessive dose of l-arginine after l-ornithine restored the decrease in NO by l-ornithine in the renal medulla. The 60-min infusion of l-arginine alone into the renal medulla significantly increased NO concentration. As a control for osmotic effects of the amino acid solutions, the concentration of NO in the renal medulla was not significantly affected by the intramedullary infusion of an equimolar amount of d-mannitol.
Influence of CAT infusion on renal medullary blood flow.
The influence of infusion of the different amino acids and l-NAME on renal medullary blood flow is illustrated in Fig. 3. Renal medullary blood flow was significantly reduced during the acute intramedullary infusion of the cationic amino acids l-ornithine, l-lysine, and l-homoarginine. Infusion of a 10-fold excess of l-arginine during the infusion of l-ornithine prevented the change in medullary blood flow. In contrast, infusion of l-arginine alone significantly increased blood flow in the medulla. The average mean arterial pressure ranged from 96–126 mmHg during the control period in all groups and was unaltered during interstitial infusion of the cationic amino acids. The mean arterial pressure significantly increased, however, from 112 ± 4 to 144 ± 2 mmHg during the infusion of this large dose of l-NAME. Nevertheless, medullary interstitial infusion of the NOS inhibitor l-NAME significantly decreased medullary blood flow by 38 ± 4%.
Influence of cationic amino acids on NO in isolated renal inner medullary cells.
The influence of incubation of the cells with the different amino acids, l-NAME, and d-mannitol on the DAF-FM-T fluorescence is illustrated in Fig. 4. The intensity of DAF-FM-T fluorescence was significantly increased during the incubation of the cells with l-arginine. In contrast, incubation of the cells with the cationic amino acids l-ornithine, l-lysine, and l-homoarginine attenuated the development of NO fluorescence in the cells. The effects of l-ornithine, l-lysine, and l-homoarginine to attenuate the development of DAF-FM fluorescence were partially reversed when l-arginine was added to the cells. In an effect similar to that observed in the microdialysis study, incubation with l-NAME greatly decreased NO production in the cells while mannitol did not alter NO production in the isolated cells. To begin to identify a mechanism for these observed effects, this same experiment was run on two other groups of cells which were incubated with either nonimmune rabbit serum or antisera raised against CAT1. Whereas the nonimmune serum (1:1,000) had no effect on the response to any of the amino acids, the addition of a 1:1,000 dilution of the anti-CAT1 antisera blunted the increase in NO when additional l-arginine was added to the cells and also attenuated the decrease in NO observed when the cells were incubated with l-ornithine, l-lysine, or l-homoarginine. The addition of the nonimmune sera and the anti-CAT1 sera did not influence DAF-FM fluorescence in the mannitol group or the decrease in NO observed when the NOS inhibitor l-NAME was added to the cells.
Quantification of rat CAT mRNA.
The expression of mRNA for rat CAT1–3 measured by real-time quantitative reverse transcription-PCR is shown in shown in Fig. 5. The predominant CAT mRNA measured in the brain was CAT1; CAT1 was also the most prevalent mRNA measured in each portion of the kidney. A gradient of CAT1 expression was observed in the kidney; the expression was highest in the inner medulla and least in the renal cortical tissue. In the liver, CAT2 was the most prevalent species measured. Significant levels of CAT3 mRNA were measured only in the brain.
Immunohistochemistry of CAT1 in the renal medulla.
An immunohistochemical localization of CAT1 in the renal medulla is shown in Fig. 6. The immunoreactivity for CAT1 in the renal inner medulla was detected in collecting duct cells (black arrow), the wall of vasa recta (red arrow), and in epithelial cells of the pelvis (green arrow). These observations are consistent with previous studies, which have demonstrated that CAT1 is present in IMCD (38) and in endothelial cells (5).
The present experiments demonstrate that the concentration of l-arginine and l-lysine, another cationic amino acid which competes with l-arginine for cellular uptake, are in approximately equivalent concentrations in the renal inner and outer medulla. To determine whether the ratio of l-arginine to other cationic amino acids is important in NO production in vivo, selective infusion of l-ornithine, l-lysine, and l-homoarginine, which not only compete with l-arginine for cellular uptake but also facilitate the efflux of intracellular l-arginine by y+-mediated mechanisms (3, 4, 5, 9, 33, 37) led to a selective decrease in NO concentration in the renal medullary interstitial space. Accompanying the decrease in NO was a reduction in renal medullary blood flow as assessed by implanted optical fibers for laser-Doppler flowmetry. In contrast to the suppressive effects of the competing cationic amino acids, medullary interstitial infusion of l-arginine increased medullary NO and blood flow. Consistent with the observations made in vivo, subsequent in vitro experiments with freshly isolated inner medullary cells demonstrated that intracellular NO was increased by l-arginine and was suppressed by l-ornithine, l-lysine, and l-homoarginine. Further studies then demonstrated that CAT1 mRNA is predominantly expressed in the renal medulla and CAT1 immunoreactive protein is localized to the IMCD and vasa recta. Combined, these data indicate that l-arginine uptake is important in NO production and blood flow regulation in the rat renal medulla.
Several experiments were performed to test for the specificity of the effects of l-arginine and the other cationic amino acids. First, administration of l-arginine reversed the effects of l-ornithine, l-lysine, and l-homoarginine to decrease NO in both the in vivo and in vitro experiments. These experiments indicate that l-arginine and the other cationic amino acids are competing for the same transporter. To control for the osmotic effects associated with the infusion of the amino acids, the interstitial infusion of equimolar amounts of mannitol did not alter NO levels in the renal medullary interstitial space of anesthetized rats. Moreover, because l-arginine increased NO whereas equimolar amounts of the other cationic amino acids decreased NO, nonspecific osmotic effects are unlikely. Finally, as a positive control, renal medullary interstitial infusion of the nonspecific NOS inhibitor l-NAME decreased NO concentration and blood flow in the medulla as has been previously reported (15, 22, 41). Administration of l-NAME also decreased NO in the freshly isolated medullary cells. These control experiments lend support to the conclusion that l-arginine competes with other cationic amino acids for cellular uptake.
The in vitro experiments support the in vivo observations and provide further insight into the mechanisms leading to the observed effects in the intact kidney. First, these experiments indicate that the addition of l-arginine to the media stimulates NO production, and the addition of the other cationic amino acids along with l-arginine decreases NO production. These findings are consistent with a mechanism of competitive inhibition of l-arginine uptake. Second, the in vitro findings demonstrated that the other cationic amino acids decrease NO in the absence of extracellular l-arginine. This finding suggests that the NO-depleting effect of l-ornithine, l-lysine, and l-homoarginine are due not only to competitive inhibition. Instead, this observation is consistent with trans-stimulation of system y+, which leads to transmembrane exchange of extracellular and intracellular cationic amino acids, as has been previously described in several different cell types (3–5, 9, 33, 37). In this case, stimulation of the y+ transporter by cationic acids on the extracellular surface results in the efflux of l-arginine from the inside to the outside of the cell. The NO-depleting effect of l-ornithine, l-lysine, and l-homoarginine observed in vivo may therefore be because of competition with l-arginine for cellular uptake and/or due to the facilitation of the efflux of intracellular l-arginine out of the cells by a y+-mediated mechanism (3–5, 9, 33, 37).
The present study also addressed the potential mechanism at work in the present study. In the in vitro studies, the addition of the anti-CAT1 serum decreased the NO response to l-arginine, indicating that the effects of l-arginine to increase NO in the cell suspension are at least partially mediated by CAT1. Moreover, there was a greater DAF signal in cells incubated with the other cationic amino acids plus the anti-CAT1 antisera compared with cells incubated with the l-lysine, l-ornithine, or l-homoarginine alone. The nonimmune sera did not affect the response in any group and the immune antisera did not alter the response in the vehicle-treated group, the mannitol group, or the l-NAME group. Together, the results of these experiments indicate that the influence of l-arginine, the influence of the other cationic amino acids, or the influence of l-arginine in combination with the other amino acids are mediated, at least in part, by the CAT-1 transporter. Consistent with this conclusion is the observation that there is greater expression of mRNA for CAT-1 than the other CAT transporters in the inner medulla of the rat. In addition, previous studies from our laboratory indicated that chronic blockade of CAT-1 with an antisense oligonucleotide decreased NO in the renal medullary interstitial space and led to the development of hypertension in normal rats, suggesting the importance of CAT-1 in the renal medulla.
Though NO production in the renal medulla appears to be dependent on cellular l-arginine transport, it is unclear what cell types or what NOS isoforms in the renal medulla are responsible for this effect. Previous work from our laboratory demonstrated that the IMCD is enriched in NOS (39), and NOS enzymatic activity, as quantified by the conversion of l-arginine to l-citrulline, in freshly isolated IMCD is dependent on l-arginine transport by a y+-mediated mechanism (38). The influence of the other cationic amino acids to decrease NO production in freshly isolated cells obtained from the renal medulla was confirmed in the present study by measuring DAF fluorescence. It is therefore possible that renal medullary interstitial infusion of cationic amino acids blocked NO production by the IMCD. Alternatively, other structures in the renal medulla, in particular, the vasa recta, also contain relatively large amounts of NOS (23, 39). Experiments in the present study indicate that CAT1 mRNA is predominantly expressed in the renal medulla and CAT1 immunoreactive protein is localized in the collecting ducts and vasa recta. The release of NO from any of these structures may therefore have been altered in these experiments.
The specific transporter affected by the cationic amino acids was not directly addressed in the in vivo experiments. In previous studies from our laboratory, we demonstrated that l-arginine uptake in the IMCD was mediated by a y+ transporter encoded as CAT1 (38). Further studies then demonstrated that chronic infusion of l-ornithine, l-lysine, or an antisense oligonucleotide for CAT1 lead to a sustained decrease in NO in the rat renal medulla, which is accompanied by the development of hypertension (14). Although we did not use agents that specifically targeted individual transporters in these in vivo experiments, we observed that antisera raised against CAT1 partially blunted the effects of the cationic amino acids on NO in renal medullary cells. This result suggests that CAT1, which has been shown to be the predominant CAT in the rat renal inner medulla (38), mediates the change in NO caused by cationic amino acids.
The partial dependence of NO production and medullary blood flow on cellular l-arginine transport in the renal medulla is surprising because it has been considered that l-arginine is abundantly present in the intracellular space (100–3,800 μM) when compared with the Km of NOS for l-arginine assessed in vitro (5 μM) (5). Furthermore, the enzymatic mechanisms necessary to recycle l-citrulline to l-arginine are present in endothelial cells (12, 40) and endothelial cells can sustain intracellular l-arginine levels despite continuous NO release (25). Nevertheless, several studies have indicated that the supplementation of exogenous l-arginine ameliorates the decrease in endothelial function observed in hypertension (11), diabetes mellitus (30), and hypercholesterolemia (6, 7). This discrepancy that NO generation is dependent on the uptake of extracellular l-arginine despite the sufficient intracellular l-arginine has been referred as the “arginine paradox” (10). Furthermore, a previous study has demonstrated that extracellular l-lysine (2 mmol/l) decreases intracellular l-arginine from 800 to 100 μmol/l in cytokine-stimulated macrophages, which should still be sufficient for NO formation, but NO generated from these cells is almost totally inhibited by l-lysine (5). Several explanations have been proposed to address this phenomenon. One possibility is that endogenous NOS inhibitors such as NG-monomethyl-l-arginine and NG,NG-dimethyl-l-arginine in the plasma (35) are present in the intracellular compartment in such concentrations to increase the functional Km value of the NOS enzymes. Another explanation is that intracellular l-arginine may be spatially sequestered, and therefore NOS cannot easily access the intracellular pool. Interestingly, the CAT1 transporter has been shown to colocalize with endothelial NOS in the caveolae of endothelial cells (24), suggesting that extracellular l-arginine is easily available to endothelial NOS in these cells.
An additional unanswered question raised by these experiments is whether the physiological concentration of l-ornithine or l-lysine in the renal medulla can suppress l-arginine availability in vivo. Although the minimal concentration of these cationic amino acids that can affect NO formation is not known, it is possible that changes in the endogenous concentration of these other cationic amino acids can affect l-arginine availability and NO production in the renal medulla under different physiological and/or pathophysiological conditions. In support of this idea is the observation from this and previous studies (41) that administration of l-arginine increased NO in the kidney of the anesthetized rat. Moreover, the present experiments demonstrate that the concentration of l-arginine and l-lysine are equivalent in the renal medulla, indicating that competition between l-arginine and other cationic amino acids may occur under physiological conditions. These findings suggest that NO production is indeed dependent on extracellular l-arginine in vivo; this observation may be due to competition with l-arginine for cellular uptake or the facilitation of the efflux of intracellular l-arginine by y+-mediated mechanisms (37).
In conclusion, the present experiments were performed to determine if endogenous renal NO production might be regulated by l-arginine transport mechanisms. Renal medullary interstitial infusion of the cationic amino acids l-ornithine, l-lysine, or l-homoarginine, which compete with l-arginine for cellular transport by CAT1, led to a significant decrease in NO in the renal medullary interstitial space. Consistent with the decrease in NO observed with infusion of these cationic amino acids, blood flow in the renal medulla was decreased when the cationic amino acids l-ornithine, l-lysine, or l-homoarginine were administered. These same amino acids were then demonstrated to decrease intracellular NO in freshly isolated cells obtained from the rat renal inner medulla. The present experiments therefore provide in vivo data indicating that NO production and blood flow regulation in the renal medulla of the rat kidney is partially dependent upon cellular transport of l-arginine by a y+ mechanism.
This work was partially supported by National Institutes of Health Grants HL-29587, DK-62803 (to D. L. Mattson), and HL-02334 (to W. J. Arendshorst), a fellowship program of the Japanese Heart Foundation, and of Sankyo Life Science Foundation (to M. Kakoki). This work was performed while D. L. Mattson was an Established Investigator of the American Heart Association.
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