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Abstract
NO deficiency is associated with development of hypertension. Defects in the renal citrulline-arginine pathway or arginine reabsorption potentially reduce renal NO in prehypertensive spontaneously hypertensive rats (SHRs). Hence, we investigated genes related to the citrulline-arginine pathway or arginine reabsorption, amino acid pools, and renal NO in 2-week-old prehypertensive SHRs. In addition, because perinatally supporting NO availability reduces blood pressure in SHRs, we supplemented SHR dams during pregnancy and lactation with citrulline, the rate-limiting amino acid for arginine synthesis. In female offspring, gene expression of argininosuccinate synthase (involved in renal arginine synthesis) and renal cationic amino acid Y-transporter (involved in arginine reabsorption) were both decreased in 2-day and 2-week SHRs compared with normotensive WKY, although no abnormalities in amino acid pools were observed. In addition, 2-week-old female SHRs had much less NO in their kidneys (0.46±0.01 versus 0.68±0.05 nmol/g of kidney weight, respectively; P<0.001) but not in their heart. Furthermore, perinatal supplementation with citrulline increased renal NO to 0.59±0.02 nmol/g of kidney weight (P<0.001) at 2 weeks and persistently ameliorated the development of hypertension in females and until 20 weeks in male SHR offspring. Defects in both the renal citrulline-arginine pathway and in arginine reabsorption precede hypertension in SHRs. We propose that the reduced cationic amino acid transporter disables the developing SHR kidney to use arginine reabsorption to compensate for reduced arginine synthesis, resulting in organ-specific NO deficiency. This early renal deficiency and its adverse sequels can be corrected by perinatal citrulline supplementation persistently in female and transiently in male SHRs.
Hypertension is associated with oxidative stress, often caused by a self-perpetuating cycle of NO deficiency, increased reactive oxygen species, and a disturbed constrictor-dilator balance in the kidney.1 Inhibition of NO synthase (NOS) causes marked and persistent hypertension.2–4 Therefore, an impairment of the NO-generating pathway could be involved in the onset of hereditary hypertension. It is even conceivable that a reduction in renal NO precedes hypertension.
There are multiple causes of a reduced or deficient NO generation in the prehypertensive kidney. Among others, these are reduced availability of NOS or the NOS substrate l-arginine. In this regard, Vaziri et al5 reported an abundance of NOS isoforms in kidneys of juvenile spontaneously hypertensive rats (SHRs). Using microarray methodology, we identified a reduction in the expression of argininosuccinate synthase (ASS) and the arginine transporter slc7a7 in kidneys of young SHRs. This led us to investigate the challenging issue of whether NO deficiency precedes hypertension because of reduced arginine availability in SHRs. Arginine availability can be impaired because of a reduced dietary uptake, reduced endogenous synthesis, impaired transport, and/or shunting of arginine into other metabolic pathways.
We speculate that sufficient arginine availability to produce NO in the kidney mainly depends on both endogenous synthesis and reabsorption. NOS produces NO by converting arginine to citrulline. In kidney proximal tubular cells, arginine is synthesized by ASS and argininosuccinate lyase (ASL) that convert citrulline to argininosuccinate and arginine, respectively. Citrulline delivery to proximal tubule cells is rate limiting for renal arginine synthesis.6,7 A defect in this citrulline-arginine pathway could reduce arginine availability and, hence, cause an NO deficiency in the prehypertensive kidney. Indeed, in 3.5 and 6.0-week–old SHRs compared with normotensive Wistar-Kyoto (WKY) rats, Jones8 observed a smaller arginine pool in plasma and in skeletal muscle. A second source of arginine in the kidney is tubular uptake. Transepithelial arginine reabsorption is brought about by a transporter, system b0,+, which transports arginine from the tubular fluid, across the apical membrane, and by the so-called renal cationic amino acid Y-transporter that is responsible for arginine transport across the basolateral membrane.9 Although a citrulline carrier in the kidney has not yet been identified, citrulline transport does not depend on the y+ system.10
Taken together, we hypothesize that decreased arginine availability in the developing SHR kidney, which leads to NO deficiency preceding hypertension, is caused by a defect in the citrulline-arginine pathway, arginine reabsorption, or both. Hence, we investigated whether gene expression of enzymes related to the renal citrulline-arginine pathway or gene expression of transporters involved with arginine reabsorption was decreased in the developing SHR kidney. Furthermore, we measured renal and cardiac amino acid pools and NO in 2-week-old prehypertensive SHRs. Previously we observed that transient perinatal supplementation of SHRs with arginine, taurine, and vitamins C and E had a permanent antihypertensive effect.11 Accordingly, it is plausible that factors that support NO availability directly or indirectly by increasing arginine availability in the perinatal phase could decrease blood pressure later in life (reprogramming12). Therefore, our secondary hypothesis is that brief perinatal citrulline supplementation in SHRs corrects renal NO in the developing kidney and, hence, has long-term antihypertensive effects in conjunction with a shift of the renal vascular constrictor-dilator balance toward relaxation.
Methods
Animals
From day 7 of gestation, SHR dams and their offspring received either tap water (SHR) or citrulline in drinking water (2.5 g/L) until 6 weeks of age (SHRcitr). Pups were weaned at 4 weeks. The number of rats and litters in each group is presented in the tables and figure legends.
Renal NO Content at 2 Weeks
To determine tissue NO content, we modified a previously described protocol.13 Figure 1 represents a schematic cartoon of the protocol. We injected 2-week-old female SHRs, SHRcitrs, and WKY rats with a mixture of 37.5 mg/kg of FeSO4·7H2O and 190 mg/kg of sodium citrate SC and with 500 mg/kg of Na-DETC IP. The hydrophobic FE2+-(DETC)2 complex associates with cell membranes and traps NO trafficking into or out of cells resulting in the formation of paramagnetic mononitrosyl-iron complexes with DETC. After 30 minutes, rats were anesthetized with sodium pentobarbital (60 mg/kg) and whole-body perfused with saline for 3 minutes. Kidney and heart samples were halved, placed in a plastic syringe with HEPES buffer (150 mmol/L; pH 7.4), snap-frozen in liquid nitrogen, and subjected to electron paramagnetic resonance (EPR) spectroscopy.
Figure 1. Cartoon of the protocol used to measure tissue NO content. Rats are injected with FeSO4+Na-Citrate SC and Na-DETC IP. The hydrophobic FE2+-(DETC)2 complex traps NO by forming paramagnetic NO-Fe-DETC complexes (MNIC). After 30 minutes, rats are anesthetized and whole-body saline perfused. Kidney and heart samples are snap frozen and subjected to EPR spectroscopy. After reduction with dithionite, the NO-Fe-DETC complexes are clearly visible.
EPR spectra were recorded at 77 K on a modified X-band ESP 300 radiospectrometer (Bruker BioSpin) operating near 9.54 GHz with 20 mW of power. Frozen samples were placed in a quartz liquid finger dewar at the center of a Burker ER4103TM cavity. Field modulation was 0.5 mT, gain 2.105, time constant, and apparent diffusion coefficient conversion time 82 ms. Nine consecutive scans were accumulated to reduce instrumental noise. Spin densities were calibrated with frozen reference solutions of NO-Fe2+-(MGD)2 in PBS.13 The signals from Cu2+-DETC complexes, formed when DETC ligands chelate endogenous Cu2+, completely dominate the NO-Fe-DETC complexes and were removed by a reduction with dithionite (100 mmol/L for 15 minutes). After this reduction, NO-Fe-DETC complexes (mononitrosyl-iron complexes) were clearly visible and could be quantified. NO assessed by this approach is equivalent to the mononitrosyl-iron complex yield in tissue after 30 minutes of in vivo trapping. Because some NO will be shunted into other metabolic pathways before being trapped, this method will underestimate the actual tissue NO content. Renal perfusion, essential for iron removal, is not possible in 2-day-old rats.
Supplemental Methods
Diet, gene expression in renal cortex of 2-day and 2-week-old rats, amino acid concentrations at 2 weeks, long-term effects of perinatal citrulline in SHRs, terminal arterial pressure and renal function at 28 to 30 and 48 to 50 weeks, calculations, and statistics are described in the supplemental Methods section (available at http://hyper.ahajournals.org).
Results
Gene Expression in Renal Cortex of 2-Day and 2-Week-Old Rats
Microarray analysis identified persistent reduction in ASS and Slc7a7 expression in SHRs (data not shown). To confirm this, quantitative PCR was performed on specific components of these pathways. Gene expression of rate-limiting enzymes of arginine synthesis, the cationic anion transporter, and the isoforms of NOS were measured in SHRs and WKY rats at 2 days and 2 weeks. Expression of ASS was reduced in the SHR kidney at 2 days (P<0.01), and both ASS and ASL were decreased in the kidney of 2-week-old SHRs (P<0.01 and P<0.05, respectively; Table 1). Thus, both genes coding for enzymes that serve in synthesizing arginine from citrulline displayed lower expression in the developing SHR kidney. Moreover, expression of the cationic amino acid transporter Slc7a7, the so-called renal cationic amino acid Y-transporter transporter, was decreased in 2-day and 2-week SHRs (both P<0.01). Although renal gene expression of neuronal NOS was considerably higher in 2-week SHRs (P<0.001), there were no differences in endothelial NOS at either 2 days or 2 weeks. Inducible NOS was below the threshold of reliable detection.
Table 1. Renal Gene Expression Change (Log2) in Female SHRs as Compared With Female WKY Rats at 2 Days and 2 Weeks of Age
Amino Acid Concentrations at 2 Weeks
Amino acids in 2-week SHRs, SHRcitrs, and WKY rats in the kidney and heart tissue in 2-week SHR and WKY rats were quite similar (Table S1). Only ornithine was significantly lower in both male and female SHR heart tissue, and citrulline was higher in the female SHR kidney as compared with the WKY. Perinatal supplementation with citrulline in SHRs significantly increased arginine levels in the heart of both male and female offspring but not in their kidneys.
Renal NO Content at 2 Weeks
In both the left and right kidneys, NO content was markedly higher in 2-week WKY rats than in age-matched SHRs (P<0.01 and P<0.001, respectively; Figure 2a). This renal NO deficiency in very young female SHRs seems to be organ specific, because in the heart, NO yields were similar (Figure 2b). Perinatal supplementation with citrulline in female SHRs resulted in significantly higher renal NO (P<0.05; Figure 2a) without an effect on NO yields in the heart (Figure 2b). Kidney weight was similar in SHRs and WKY rats (153±11 mg versus 150±8 mg). Perinatal citrulline had no effect on organ weights.
Figure 2. a, NO yields determined by EPR in the left and right kidney in 2-week-old female SHRs (open circles and bars; n/litters= 10/5), SHRcitrs (closed circles and diagonally striped bars; n/litters=9/4), and WKY rats (triangles and horizontally striped bars; n/litters=10/5). b, NO yields in the heart determined by EPR in female SHRs (n/litters=7/2), SHRcitrs (n/litters=9/4), and WKY rats (n/litters=8/3). Both raw and average values are displayed. *P<0.05, †P<0.01, and ‡P<0.001 vs SHRs.
Long-Term Effects of Perinatal Citrulline in SHRs
In SHRcitrs, 24-hour citrulline intake was 416±11 and 281±21 mg/kg of body weight per day at 4 and 6 weeks of age, respectively. (Micro)nutrient intake during lactation could not be measured. Perinatal supplementation of citrulline increased body weight in both females and males up to 24 to 28 weeks of age (Figure S1). Systolic blood pressure (SBP) was reduced in female SHRcitrs at 8 weeks of age, ie, 2 weeks after cessation of supplementation, and this reduction in SBP persisted for a further 8 months (P<0.001; Figure 3). In male SHRcitrs, SBP was already reduced at 4 weeks, ie, during supplementation. Thereafter, this difference gradually diminished and was similar to control SHRs from 24 weeks onward.
Figure 3. SBP in control female (top left panel) and male (bottom left panel) SHRs (open circles; n=24 and 16, respectively) and SHRcitrs (closed circles; n=16 and 10, respectively) and WKY rats (triangles; n=9 and 10, respectively). WKY rat was lower than SHR from 8 weeks onward (P<0.001). MAP at 50 weeks of age in female (top right panel) and male (bottom right panel) SHRs (open bars), SHRcitrs (diagonally striped bars), and WKY rats (striped bars). *P<0.05 and ‡P<0.001 vs SHRs.
Terminal Arterial Pressure and Renal Function at 28 to 30 and 48 to 50 Weeks
Perinatal citrulline significantly reduced mean arterial pressure (MAP) at 28 to 30 weeks in both female and, unexpectedly, considering SBP, male SHRs (P<0.001 and P<0.05, respectively; Table 2). Renal hemodynamics was not affected by perinatal citrulline in female SHRs. Glomular filtration rate (GFR), effective renal plasma flow (ERPF), and renal blood flow (RBF) measured by flow probe were all significantly reduced in male SHRcitrs (P<0.05). Perinatal citrulline had no effect on filtration fraction (FF), para-aminohippurate (PAH) extraction, or fractional electrolyte excretion in male or female SHRs. Unexpectedly, irrespective of perinatal treatment, female SHRs displayed a much lower PAH extraction (0.60±0.03; n=24) than male SHRs (0.87±0.06; n=13; P<0.0001).
Table 2. Renal Function in Male and Female SHRs and SHRcitrs at 28 to 30 Weeks
During infusion of the superoxide dismutase mimetic Tempol, MAP decreased significantly in all of the groups. However, in female SHRcitrs, this decrease was significantly less than in SHR controls (Figure 4). Although in male SHRs and SHRcitrs RBF increased during Tempol, time-control experiments in control SHRs indicated that this was an aspecific effect. Tempol significantly decreased renal vascular resistance in all of the groups except female SHRcitrs. During Tempol, GFR, ERPF, PAH extraction, FF, and fractional electrolyte excretions were not different between SHR, and SHRcitr (Table S2).
Figure 4. MAP (left), RBF (middle), and renal vascular resistance (RVR) (right) before (baseline) and during Tempol in 28- to 30-week–old male and female SHRs (n=7 and 7, respectively) and SHRcitrs (n=4 and 5, respectively). *P<0.05, †P<0.01, and ‡P<0.001 vs SHRs; §P<0.05, ∥P<0.01, and ¶P<0.001 vs baseline.
At 48 to 50 weeks, female SHRcitrs had significantly lower MAP than SHR controls (P<0.05), but MAP was no different in males (Figure 3). As expected, in aged male SHRs, ERPF was lower and FF higher than in aged-matched WKY rats,14 and here we document that this is also the case in aged females (Table S3). These strain differences were not affected in either males or females by supplementing SHRs perinatally with citrulline.
Discussion
The aim of our study was to investigate whether a defect in the renal citrulline-arginine production pathway or a decrease in transporters involved in arginine reabsorption reduces renal NO preceding the development of hypertension in SHRs. Gene expression of ASS and a transporter responsible for arginine reabsorption were both decreased in young SHRs. This did not coincide with clear abnormalities in renal amino acid pools. Young SHRs displayed organ-specific renal NO deficiency, because the cardiac NO pool was normal. Furthermore, supporting renal NO by perinatal supplementation of citrulline persistently ameliorated hypertension in female SHRs. Administration of Tempol, a superoxide dismutase mimetic, did not reduce renal vascular resistance in adult female SHRs that had been perinatally exposed to citrulline, suggesting a persistent shift in the renal constrictor-dilator balance toward relaxation as compared with control female SHRs.
Although Jones8 observed a reduced arginine pool in plasma and skeletal muscle in SHRs, we did not find a difference in the SHR kidney compared with WKY rats. In addition, brief perinatal supplementation with citrulline, which increased renal NO, did not affect amino acid pools in the kidney. However, arginine concentrations were consistently increased in the heart tissue of SHRs perinatally supplemented with citrulline, suggesting that citrulline can indeed increase arginine levels and that there is clearly an organ-specific defect in the renal citrulline-arginine pathway. Therefore, it is likely that a defect in the tubular citrulline-arginine synthesis pathway and/or arginine reabsorption has a compartmentalized effect on arginine pools in the kidneys of 2-week-old SHRs.15 A defect in the citrulline-arginine synthesis pathway could impair the synthesis of NO, despite unrestricted or even excess amounts of NOS, as observed previously in 3-week–old SHRs.5 Indeed, at 2 days and 2 weeks of age, we observed decreased renal gene expression of ASS, and ASL was decreased at 2 weeks. Although not directly assessed, this suggests an impaired ability to convert citrulline via argininosuccinate to arginine, thereby decreasing arginine synthesis. How citrulline increases renal NO in young SHRs requires further investigation. Both ASS and ASL gene expression were decreased but not absent. Citrulline delivery to the proximal tubule cells is rate limiting for renal arginine synthesis.6,7 Therefore, we speculate that increasing delivery of citrulline to the juvenile SHR kidney could result in some increase in arginine synthesis in specific renal compartments resulting in a net increase of renal NO.
In the absence of enhanced citrulline delivery, the kidney may compensate for such arginine shortage by increasing arginine reabsorption. Thus, most likely, renal NO deficiency because of substrate shortage would only occur when both renal arginine synthesis and reabsorption are impaired. Indeed, expression of the cationic amino acid Y-transporter was also reduced at both 2 days and 2 weeks in SHRs. This transporter is basolateral in renal and intestinal epithelial cells and exchanges neutral for cationic amino acids.9 Because arginine is a cationic amino acid, Y-transporters may regulate the rate of renal NO synthesis by controlling the uptake of arginine. Welch et al16 observed an increase in renal neuronal NOS expression in adult SHRs versus WKY rats, and we found that this increase was already present at 2 weeks, thus clearly preceding the onset of hypertension. However, we did not observe any difference in the renal expression of endothelial NOS. Taken together, substrate deficiency because of reduced synthesis and impaired delivery to NOS could very well explain the reduction in renal NO content.
To determine renal NO content in renal tissue, we trapped NO in paramagnetic NO-Fe-DETC complexes, which were quantified by EPR spectroscopy. This new and direct determination of organ NO content in young rats revealed a markedly reduced NO yield in the kidney, but not in the heart, in SHRs compared with WKY rats. Cosentino et al17 could increase aortic NO production in vitro with tetrahydrobiopterin in 4-week–old prehypertensive SHRs, and they ascribed the enhanced vascular superoxide production, measured in the aorta, to uncoupled endothelial NOS. The present study clearly shows that, already in 2-week-old prehypertensive SHRs, renal NO production is markedly decreased. Interestingly, this deficiency was not found in the heart, suggesting that uncoupling of endothelial NOS in the vascular bed in prehypertensive SHRs17 is already a secondary effect. Although administration of tetrahydrobiopterin in young SHRs has antihypertensive effects,18 perinatal tetrahydrobiopterin or inducible NOS inhibition in SHRs had no persistent reproducible effects on blood pressure (M.P. Koeners, unpublished data, 2006). Outspoken defects in gene expression relating to renal arginine availability were found at 2 days and 2 weeks, and decreased renal NO was documented at 2 weeks. Technical limitations preclude the measurement of renal amino acids and NO in 2d old SHR.
Following up on our observations on defects in the citrulline-arginine pathway and arginine reabsorption in 2-day and 2-week-old SHRs, we postulated that perinatal supplementation of citrulline would correct renal NO deficiency, because intestinal citrulline supply determines renal arginine production in a master-slave relation, and citrulline is a neutral amino acid the reabsorption of which does not involve the y+L transporter.7 Indeed, this is exactly what we found, suggesting that dietary citrulline may be a novel way of supporting renal arginine availability, especially when arginine transport is disturbed. In 1991, Chen and Sanders19 showed that in 3-week-old Dahl/Rapp rats, daily IP l-citrulline injections over 5 days could prevent salt-sensitive hypertension as effectively as daily IP l-arginine injections. The present study shows that, in SHRs, this concept is also applicable to the development of hypertension in a much earlier phase, namely, during nephrogenesis. Supporting NO availability in the developing SHR kidney appears to be able to permanently reprogram blood pressure regulation in adult life, leading to a lower blood pressure. Intra-arterial MAP measured at the end of the protocol generally reproduced differences between groups found by external SBP measurements. These findings suggest that maternal amino acid supplements during pregnancy could be a feasible approach to prevent or ameliorate the development of hypertension in genetically or environmentally predisposed children.
Previously, we treated SHRs perinatally up to 4 or 8 weeks with a mixture of l-arginine, taurine, vitamin C, and vitamin E and observed a similar effect on blood pressure regulation in the offspring.11 Perinatal vitamin C and vitamin E alone had no persistent antihypertensive effects.20 Our new data suggest that, because reduced expression of the renal cationic amino acid transporter could have limited the effect of l-arginine supplements in our previous study, antioxidative effects of taurine could have contributed substantially to the persistent effects.11 Offspring of rat dams exposed to food restriction during pregnancy develop mild hypertension, and supplementation of l-arginine after weaning can reduce this blood pressure increase21 and eventually even protect the kidneys.22 Supplementing a low-protein diet during pregnancy with glycine can also reverse the development of hypertension in the offspring,23 and this has been postulated to be because of correction of reduced vascular endothelial NOS activity.24 However, perinatal supplementation of l-arginine, let alone l-citrulline, with the aim of protecting the offspring in experimental models of perturbed pregnancy, has to our knowledge not yet been tested.
Hypertension is characterized by a self-perpetuating cycle of NO deficiency and increased reactive oxygen species and a disturbed renal constrictor-dilator balance.1 To investigate whether the perinatal citrulline supplement had a persistent effect on renal vasoreactivity, we scavenged superoxide with a superoxide dismutase mimetic (Tempol) during acute experiments. Tempol had similar effects on all of the groups with the notable exception of female SHRcitrs. Female SHRcitrs displayed a smaller decrease in MAP and practically no decrease in renal vascular resistance during Tempol, suggesting a persistent decrease in renal superoxide-dependent vasoconstriction. This is compatible with the idea that, in SHRcitrs, a persistent shift in the renal vasoconstrictor-vasodilator balance toward relaxation underlies the antihypertensive effect of perinatal citrulline. Note that a response to Tempol does not prove that there is a specific defect in superoxide dismutase activity. Tempol and other superoxide dismutase mimetics have been shown to correct enhanced peripheral vasoconstriction in models of perinatal malnutrition,25,26 and we have observed that perinatal Tempol can also persistently reduce SBP in SHRs.12
Judging by the higher body weight, it is possible that perinatal citrulline has long-term stimulatory effects on muscle protein synthesis. Such an effect has been observed previously during refeeding in old malnourished rats.27 However, because monitoring growth was not a primary objective, we did not measure terminal organ weights, with the exception of the kidney and heart (ventricles). Thus, we cannot provide an explanation for this effect on body growth. Note that the increase was no longer present in the aged SHRcitrs.
In multiple studies of developmental plasticity in different species, including humans, gender differences have been observed.28 Although not a primary focus of our study, we also observed a marked gender difference in the response to perinatal citrulline supplementation in SHRs, with a more consistent and pronounced effect in females than in males. Because we did not measure renal gene expression and NO content in young male SHRs, we cannot speculate on the basis for this difference.
In conclusion, there is clearly reduced expression of both the renal citrulline-arginine synthesis pathway, as well as the arginine reabsorption pathway, during nephrogenesis in the female SHRs. We propose that reduced cationic amino acid transporter disables the kidney to use arginine reabsorption to compensate for reduced arginine synthesis. It seems plausible that this results in renal NO deficiency that precedes the development of hypertension in SHRs. This NO deficiency and its adverse sequels can be persistently corrected with perinatal citrulline supplementation.
Perspectives
Low levels of NO in the kidney precede hypertension in SHRs, an accepted model of essential hypertension. This organ-specific NO deficiency was measured in vivo with EPR. Previous studies indicated that supporting renal NO availability during renal development in SHRs, by maternal dietary supplements with antioxidants or an NO donor, had persistent antihypertensive effects. In the current study, this concept was taken a step further, and the citrulline-arginine pathway was used to generate more NO in the kidney. This also proved to be an effective antihypertensive strategy. A persistent shift in the renal vasoconstrictor-vasodilator balance toward relaxation appears to underlie the antihypertensive effect of perinatal citrulline in SHRs. Although more research needs to be done, it is promising that brief dietary supplementation of an amino acid during development can cause long-lasting improvements of blood pressure regulation in hereditary hypertension. This raises the possibility that simple dietary interventions in the mother might spare the next generation.
Acknowledgments
We thank Paula Martens, Ria de Winter, Adèle Dijk, Nel Willekes, and Dionne van der Giezen for expert laboratory assistance.
Sources of Funding
The Dutch Kidney Foundation (grants NS6013 and C03.2039) and the European Union Sixth Framework Programme for Research and Technical Development of the European Community (The Early Nutrition Programming Project: FOOD-CT-2005-007036) supported this study.
Disclosures
None.
- Received June 4, 2007.
- Revision received July 5, 2007.
- Accepted September 25, 2007.
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