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. Author manuscript; available in PMC: 2020 Aug 1.
Published in final edited form as: Hypertension. 2019 Jun 24;74(2):313–322. doi: 10.1161/HYPERTENSIONAHA.119.12723

Fumarase Overexpression Abolishes Hypertension Attributable to eNOS Haploinsufficiency in Dahl Salt-Sensitive Rats

Hong Xue 1,2, Aron M Geurts 2,3, Kristie Usa 2, Feng Wang 2,4, Yingying Lin 2,4, Jenifer Phillips 2, Lisa Henderson 2, Maria Angeles Baker 2, Zhongmin Tian 5, Mingyu Liang 2,*
PMCID: PMC6620137  NIHMSID: NIHMS1530053  PMID: 31230549

Abstract

Human blood pressure salt sensitivity is associated with changes in urinary metabolites related to fumarase (Fh) and nitric oxide (NO) metabolism, and fumarase promotes NO production through an arginine regeneration pathway. We examined the role of the fumarase-NO pathway in the development of hypertension using genetically engineered rat models. Dahl salt-sensitive (SS) rats with heterozygous mutation of endothelial NO synthase (eNOS or Nos3) (SS-Nos3+/−) were bred with SS rats with a hemizygous Fh transgene. SS-Nos3+/− rats without the Fh transgene (SS-Nos3+/−/Fh0/0) developed substantial hypertension with a mean arterial pressure (MAP) of 134.2 ± 3.7 mmHg on a 0.4% NaCl diet, and 178.0 ± 3.5 mmHg after 14 days on a 4% NaCl diet. MAP decreased remarkably to 123.1 ± 1.4 mmHg on 0.4% NaCl, and 143.3 ± 1.5 mmHg on 4% NaCl in SS-Nos3+/− rats with a Fh transgene (SS-Nos3+/−/Fh0/1), and proteinuria, renal fibrosis, and tubular casts were attenuated in SS-Nos3+/−/Fh0/1 rats compared to SS-Nos3+/−/Fh0/0 rats. eNOS protein abundance decreased in rats with the Nos3 heterozygous mutation, which was not influenced by Fh overexpression in rats on the 0.4% NaCl diet. However, the decrease in NO metabolite in the renal outer medulla of SS-Nos3+/−/Fh0/0 rats on the 0.4% NaCl diet was reversed in SS-Nos3+/−/Fh0/1 rats, and levels of L-arginine, but not the other 12 amino acids analyzed, were significantly higher in SS-Nos3+/−/Fh0/1 rats than in SS-Nos3+/+/Fh0/0 rats. In conclusion, fumarase has potent effects in restoring NO production and blunting the development of hypertension attributable to eNOS haploinsufficiency.

Keywords: hypertension, metabolite, fumarase, kidney, nitric oxide

Introduction

Hypertension is associated with increased risk of cardiovascular disease (CVD), end-stage renal disease (ESRD), subclinical atherosclerosis, and all-cause mortality [1–4], and emerging evidence suggests that human hypertension is associated with abnormalities in cellular intermediary metabolism [5, 6]. Human blood pressure (BP) salt sensitivity is associated with changes in urinary levels of metabolic intermediaries [7]. Experimental alterations of several metabolic intermediaries influence BP, suggesting that metabolites could be causal contributors to the development of hypertension [7–9]. However, the mechanisms underlying the effect of metabolic intermediaries on BP regulation largely remain unclear [10].

Fumarase catalyzes the reversible conversion between fumarate and L-malate in the tricarboxylic acid (TCA) cycle. Human blood pressure salt sensitivity is associated with urinary levels of fumarate [7]. The Dahl salt-sensitive SS rat, a model of human salt-sensitive hypertension and renal injury used widely [11, 12], exhibits fumarase insufficiency compared to salt-insensitive consomic SS-Chr13BN rats or Sprague-Dawley (SD) rats [13–15]. Elevating fumarase activities by fumarase transgenic overexpression in the genetic background of the SS rat attenuates the development of hypertension [15], while elevating fumarate by intravenous infusion of a fumarate precursor in SS-Chr13BN rats [14] or silencing intra-renal fumarase with an siRNA in SD rats [15] exacerbates salt-induced hypertension.

A decreased level of nitric oxide (NO), particularly NO produced by the endothelial nitric oxide synthase (eNOS, encoded by Nos3 gene), can cause endothelial dysfunction and BP elevation [16, 17]. NOS converts L-arginine to NO and L-citrulline, and a defective L-arginine/NO pathway has been linked to NO deficiency in hypertension in both animal and human studies [18–23]. BP in humans on low- or high-sodium intake is positively associated with urinary levels of citrulline [7]. Long-term nitric oxide blockade by Nω-nitro-l-arginine methyl ester (L-NAME) leads to severe and progressive hypertension, which is aggravated by salt overload and prevented largely by salt restriction [24, 25]. L-arginine administration restores NO production and prevents salt-induced hypertension in SS rats [26]. Recent studies have identified a novel malate-aspartate-arginine pathway by which fumarase promotes NO production [27]. In this pathway, fumarase drives the generation of L-malate and oxaloacetate in the TCA cycle. Oxaloacetate is converted to aspartate, which is combined with citrulline to re-generate L-arginine, the substrate of NO production.

However, the in vivo pathophysiological relevance of this novel fumarase-NO pathway is unclear. In this study, we produced SS littermate rats with an eNOS heterozygous mutation, a fumarase transgene, or both, to examine the relevance of the fumarase-NO pathway in the development of salt-sensitive hypertension and renal injury. Remarkably, transgenic overexpression of fumarase increased L-arginine levels and reversed the decrease of NO in the renal outer medulla, and abolished the exacerbation of hypertension and renal injury induced by eNOS haploinsufficiency completely.

Methods

Detailed methods are available online. Data, analytical methods and study materials are available from the corresponding author upon reasonable request.

Production of SS-Nos3+/−/Fh0/1rats

SS rats with hemizygous transgenic overexpression of fumarase (SS-Fh0/1) or a targeted heterozygous mutation of the eNOS gene (Nos3) (SS-Nos3+/−) were produced as we described recently [15, 28]. SS-Fh0/1 rats were bred with SS-Nos3+/− rats. The offspring included four genotypes: SS-Nos3+/+/Fh0/0 (genetically equivalent to SS rats); SS-Nos3+/−/Fh0/0 (SS rats with a heterozygous mutation of Nos3); SS-Nos3+/+/Fh0/1 (SS rats with a hemizygous fumarase transgene), and SS-Nos3+/−/Fh0/1 (SS rats with a heterozygous mutation of Nos3 and a hemizygous fumarase transgene). Littermates of the four genotypes obtained from multiple litters were used for experimental analysis at eight to ten weeks of age. Equal numbers of males and females were included in each experimental group.

Animal experiments, tissue and urine collection, renal morphological analyses, Western blot and NO measurement

These experiments were performed as described previously [7, 15, 29] and in detailed methods in the online supplement.

High-performance liquid chromatographic (HPLC) analysis of amino acids

Amino acid levels in renal outer medulla tissues were measured according to the procedure for the analysis of o-phthaldialdehyde (OPA)/ethanethiol derivatives of amino acids [30].

Statistics

Data were analyzed using one- or two-way ANOVAs. Holm-Sidak method was the post-hoc test. p < 0.05 was considered significant. Data are shown as means ± SEMs.

Results

eNOS heterozygous mutation in the genetic background of SS rats exacerbated hypertension substantially

The mean arterial pressure (MAP) of SS-Nos3+/− rats was 129.6 ± 1.0 mmHg on the 0.4% NaCl diet and 171.5 ± 2.6 mmHg after two weeks on the 4% NaCl diet, which was significantly and substantially higher than that in their SS-Nos3+/+ littermates on both the 0.4% and 4% NaCl diets (122.3 ± 0.6 mmHg and 153.7 ± 2.9 mmHg, respectively: Figure 1A). The SS-Nos3+/+ littermates were genetically equivalent to the SS rat. The high salt-induced increase of MAP was significantly greater in SS-Nos3+/− rats (Figure 1B). Systolic and diastolic BP (SBP and DBP) followed the same trend as MAP (Figure 1C and 1D).

Figure 1. An eNOS heterozygous mutation exacerbates hypertension in Dahl salt-sensitive rats.

Figure 1.

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Rats were implanted with telemetry to record blood pressure continuously in vivo. A. Mean arterial blood pressure (MAP); B. Changes in MAP on the 4% NaCl diet compared to the 0.4% NaCl diet; C. Systolic blood pressure (SBP); D. Diastolic blood pressure (DBP). n = 5 SS-Nos3+/+ and 6 SS-Nos3+/−. * p < 0.05, ** p < 0.01 vs. SS-Nos3+/+, two-way repeated measures ANOVA. Δ p < 0.05 vs. SS-Nos3+/+ for individual days by Holm-Sidak test.

Fumarase transgenesis abolished the exacerbated hypertension induced by eNOS heterozygous mutation

We developed fumarase transgenic rats in the genetic background of SS rats with the eNOS heterozygous mutation by breeding SS-Fh0/1 with SS-Nos3+/−, and studied the offspring with the four combinations of genotypes.

The BP of SS-Nos3+/−/Fh0/0 rats was substantially higher than that of their SS-Nos3+/+/Fh0/0 wild-type littermates (Figure 2), which is consistent with the results from SS-Nos3+/− rats shown in Figure 1. On the 0.4% NaCl diet, SS-Nos3+/−/Fh0/1 rats’ MAP was 123.1 ± 1.4 mmHg, which was significantly lower than that of the SS-Nos3+/−/Fh0/0 rats (134.2 ± 3.7 mmHg) (Figure 2A). After the switch to the 4% NaCl diet, the increase in MAP was attenuated significantly in SS-Nos3+/−/Fh0/1 rats compared to SS-Nos3+/−/Fh0/0 rats (Figure 2A, 2B). The difference between SS-Nos3+/−/Fh0/1 and SS-Nos3+/−/Fh0/0 rats became statistically significant beginning on day 4 on the 4% salt diet (Figure 2B). On day 14 on the 4% salt diet, the MAP of SS-Nos3+/−/Fh0/1 and SS-Nos3+/−/Fh0/0 rats was 143.3 ± 1.5 mmHg and 178.0 ± 3.5 mmHg, respectively. SBP and DBP followed the same trend as MAP (Figure 2C, 2D). On the 14th day of the 4% NaCl diet, the SBP of SS-Nos3+/+/Fh0/0, SS-Nos3+/+/Fh0/1, SS-Nos3+/−/Fh0/0 and SS-Nos3+/−/Fh0/1 rats were 179.6 ± 3.5 mmHg, 166.2 ± 5.6 mmHg, 204.6 ± 4.0 mmHg and 166.1 ± 2.2 mmHg, respectively. The interaction of diet and genotypes was significant for MAP, SBP, and DBP (p < 0.001). No obvious difference was observed between male and female rats of each genotype.

Figure 2. Hemizygous fumarase transgenesis abolishes the exacerbated hypertension attributable to an eNOS heterozygous mutation in Dahl salt-sensitive rats.

Figure 2.

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Rats were implanted with telemetry to record blood pressure continuously in vivo. A. MAP; B. Changes of MAP on the 4% NaCl diet compared to the 0.4% NaCl diet; C. SBP; D. DBP. n = 8 for each group. * p < 0.05, ** p < 0.01 vs. SS-Nos3+/+/Fh0/0, ## p < 0.01 SS-Nos3+/−/Fh0/1 vs. SS-Nos3+/−/Fh0/0, two-way repeated measures ANOVA for comparison among genotypes. Δ and & next to individual days, Δ p < 0.05 vs. SS-Nos3+/+/Fh0/0, & p < 0.05 SS-Nos3+/−/Fh0/1 vs. SS-Nos3+/−/Fh0/0, Holm-Sidak test. Closed circle, SS-Nos3+/+/Fh0/0; closed diamond, SS-Nos3+/+/Fh0/1; open circle, SS-Nos3+/−/Fh0/0; and open diamond, SS-Nos3+/−/Fh0/1.

The MAP of SS-Nos3+/−/Fh0/1 rats on the 0.4% NaCl diet was similar to that of their SS-Nos3+/+/Fh0/0 wild-type littermates (Figure 2A). The MAP in SS-Nos3+/−/Fh0/1 rats in response to the 4% salt diet increased less than did that in the SS-Nos3+/+/Fh0/0 wild-type littermates beginning on day 9 on the 4% salt diet (Figure 2B). SBP and DBP followed the same trend as MAP (Figure 2C, 2D).

These data indicated that the fumarase transgenesis abolished the exacerbated hypertension induced by the eNOS heterozygous mutation in SS rats completely.

In the absence of the eNOS heterozygous mutation, the BP of SS-Nos3+/+/Fh0/1 rats on the 0.4% NaCl diet was similar to that of their SS-Nos3+/+/Fh0/0 littermates (Figure 2). After one week on the 4% NaCl diet, the increase in BP tended to be attenuated in SS-Nos3+/+/Fh0/1 rats. Differences in MAP, SBP, and DBP between SS-Nos3+/+/Fh0/1 rats and their SS-Nos3+/+/Fh0/0 wild-type littermates became statistically significant on the 14th day after the diet switch (Figure 2). The results indicated that the fumarase transgenesis alone attenuated hypertension in SS rats, which is consistent with our published data on SS-Fh0/1 rats (previously referred to as SS-TgFh1) [15]. The BP differences between fumarase transgenic rats and their SS littermates were greater in the previous study compared to this study, which might be because we used rats of different ages. SS rats’ BP increases gradually with age even when maintained on the 0.4% NaCl diet, and the rats used in the previous study were 3 to 5 wks older than were those used in this study.

Fumarase transgenesis abolished the exacerbation of renal injury attributable to the eNOS heterozygous mutation

SS rats on a high-salt diet develop significant renal injury, similar to some sub-groups of hypertensive patients, such as African American hypertensive patients. On the 0.4% NaCl diet, no statistical difference in albuminuria or proteinuria was observed between different genotypes. Within each genotype, levels of urine albumin and protein increased significantly after two weeks on the 4% NaCl diet (Figure 3). On the 4% NaCl diet, urine protein and albumin in SS-Nos3+/−/Fh0/0 rats were significantly higher compared to their SS-Nos3+/+/Fh0/0 wild-type littermates, indicating that the eNOS heterozygous mutation exacerbated kidney damage (Figure 3A and B). The fumarase transgenesis in the genetic background of SS rats with the eNOS heterozygous mutation abolished the exacerbated elevation of urine protein and albumin (Figure 3A and B). Urine protein in SS-Nos3+/−/Fh0/1 rats was even significantly lower compared to their SS-Nos3+/+/Fh0/0 wild-type littermates (Figure 3A). Urine protein/creatinine and albumin/creatinine ratios followed the same trend (Figure S1A, S1B).

Figure 3. Hemizygous fumarase transgenesis abolishes the exacerbated proteinuria, albuminuria and renal fibrosis attributable to an eNOS heterozygous mutation in Dahl salt-sensitive rats.

Figure 3.

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Urine was collected in metabolic cages from individual rats on 0.4% NaCl diet. After a switch to a 4% NaCl diet for two weeks, urine was collected again. Kidneys were collected after rats were fed with a 4% NaCl diet for two weeks. A. Urine protein; B. Urine albumin; C. Representative kidney sections illustrating fibrosis from SS-Nos3+/+/Fh0/0, SS-Nos3+/+/Fh0/1, SS-Nos3+/−/Fh0/0, and SS-Nos3+/−/Fh0/1 rats. Magnification, ×200; D and E. Quantification of area of fibrosis by Masson Trichrome staining in (D) renal outer medulla and (E) renal cortex. n=8; * p < 0.05 vs. SS-Nos3+/+/Fh0/0, # p < 0.05 and ## p < 0.01 SS-Nos3+/−/Fh0/1 vs. SS-Nos3+/−/Fh0/0, two-way ANOVA for urine protein and albumin and one-way ANOVA for fibrosis area followed by Holm-Sidak test.

In the renal outer medulla, the area of fibrosis increased significantly in SS-Nos3+/−/Fh0/0 rats compared to their SS-Nos3+/+/Fh0/0 wild-type littermates (Figure 3C and D). In SS-Nos3+/−/Fh0/1 rats, the area of fibrosis was significantly smaller compared to SS-Nos3+/−/Fh0/0 rats and did not differ significantly from that of their SS-Nos3+/+/Fh0/0 wild-type littermates. In the renal cortex, the area of fibrosis showed a trend similar to that in the renal outer medulla (Figure 3C and E). Tubular casts in the renal cortex and outer medulla, another index of renal injury, also followed a trend similar to that of the area of fibrosis (Figure S1C to E).

eNOS protein abundance decreased in SS-Nos3+/−/Fh0/0 rats and was not influenced by the transgenic overexpression of fumarase on the 0.4% NaCl diet

To examine the mechanism by which fumarase transgenesis abolished the exacerbation of hypertension and renal injury attributable to the eNOS heterozygous mutation, we asked first whether the fumarase transgenesis influenced eNOS protein abundance.

The fumarase transgenesis resulted in significant increases in the abundance of fumarase protein in the renal outer medulla, and the degree of increase was similar regardless of whether eNOS was mutated heterozygously (Figure 4A). The eNOS protein abundance in the renal outer medulla of rats with the eNOS heterozygous mutation decreased significantly by approximately 50%, consistent with the presence of an eNOS haploinsufficiency. The degree of decrease in eNOS protein abundance was similar in the presence or absence of the fumarase transgenic overexpression (Figure 4B). Protein abundance of fumarase and eNOS in the renal cortex followed a similar trend (Figure 4C and D). These results indicated that the eNOS heterozygous mutation leads to the loss of approximately 50% of eNOS protein, which is not influenced by the transgenic overexpression of fumarase when the rats were on a 0.4% NaCl diet. After 14 days of 4% NaCl diet, eNOS abundance in renal outer medulla of SS-Nos3+/−/Fh0/1 rats was higher than that in SS-Nos3+/−/Fh0/0 rats (Figure S2A).

Figure 4. eNOS protein abundance decreases in the kidneys of SS rats with an eNOS heterozygous mutation and fumarase overexpression does not affect the decrease on a 0.4% NaCl diet.

Figure 4.

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Kidney tissues were collected from rats maintained on a 0.4% NaCl diet. A and B. Representative Western blot images and quantification of (A) fumarase and (B) eNOS protein in the renal outer medulla. Coomassie blue stains shown under the Western blot were used for normalization; C and D. Representative Western blot images and quantification of (C) fumarase and (D) eNOS protein in the renal cortex. Lanes in Western blot images: 1 and 5, SS-Nos3+/+/Fh0/0; 2 and 6, SS-Nos3+/+/Fh0/1; 3 and 7, SS-Nos3+/−/Fh0/0; 4 and 8, SS-Nos3+/−/Fh0/1. n = 4 for each group. * p < 0.05, ** p < 0.01 vs. SS-Nos3+/+/Fh0/0, ## p < 0.01 SS-Nos3+/−/Fh0/0 vs. SS-Nos3+/−/Fh0/1, one-way ANOVA followed by Holm-Sidak test.

NO levels in the renal outer medulla were reduced by the eNOS heterozygous mutation and restored by fumarase overexpression on the 0.4% NaCl diet

We asked next whether the transgenic overexpression of fumarase influenced NO levels. The NO level was measured as nitrite levels in the kidney tissues collected when the rats were maintained on a 0.4% NaCl diet first. NO levels in the renal outer medulla of SS-Nos3+/−/Fh0/0 rats were significantly lower than in their SS-Nos3+/+/Fh0/0 wild-type littermates (Figure 5A). In SS-Nos3+/−/Fh0/1 rats, NO levels were significantly higher than in SS-Nos3+/−/Fh0/0 rats and not different than those of their SS-Nos3+/+/Fh0/0 wild-type littermates (Figure 5A). Among the genotypes, no significant difference in NO levels was observed in the renal cortex (Figure 5B). After 14 days of 4% NaCl diet, renal outer medullary nitrite levels in SS-Nos3+/−/Fh0/1 rats tended to be higher than that in SS-Nos3+/−/Fh0/0 strain, but the difference was not significant (Figure S2B).

Figure 5. Fumarase overexpression restored the decreased NO levels and promoted L-arginine generation in the renal outer medulla of SS rats with eNOS haploinsufficiency on a 0.4% NaCl diet.

Figure 5.

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Kidney tissues were collected from rats maintained on a 0.4% NaCl diet. A. Levels of nitrite in the renal outer medulla; B. Levels of nitrite in the renal cortex; C to E. (C) Levels of citrulline; (D) Levels of L-arginine and (E) L-arginine/citrulline ratio in the renal outer medulla. n = 8 for each group. * p < 0.05 vs. SS-Nos3+/+/Fh0/0, ## p < 0.01 SS-Nos3+/−/Fh0/0 vs. SS-Nos3+/−/Fh0/1, one-way ANOVA followed by Holm-Sidak test.

Fumarase overexpression increased the L-arginine level and L-arginine/citrulline ratio in the renal outer medulla of SS rats with the eNOS heterozygous mutation on the 0.4% NaCl diet

Fumarase could drive L-arginine regeneration from citrulline through a malate-aspartate-arginine pathway [27]. The level of citrulline tended to be lower in SS-Nos3+/−/Fh0/1 rats than that in the other genotypes, but the difference was not statistically significant (Figure 5C). The level of L-arginine in the renal outer medulla of SS-Nos3+/−/Fh0/1 rats was significantly higher compared to that of their SS-Nos3+/+/Fh0/0 wild-type littermates (Figure 5D). The level of L-arginine in SS-Nos3+/−/Fh0/0 rats showed a tendency to increase, but the difference was not statistically significant. The L-arginine/citrulline ratio in SS-Nos3+/−/Fh0/1 rats also was significantly higher compared to their SS-Nos3+/+/Fh0/0 wild-type littermates (Figure 5E). The levels of other amino acids that could be analyzed by the HPLC method, including serine, glutamine, glycine, threonine, alanine, tyrosine, valine, methionine, isoleucine, phenylalanine, and lysine, did not differ significantly between the genotypes (Figure S3A to S3K). After 14 days of 4% NaCl diet, the arginine/citrulline ratio still tended to be higher in SS-Nos3+/−/Fh0/1 rats, however, no significant difference was observed for levels of citrulline and L-arginine between genotypes (Figure S2C to S2E) or for the other amino acids analyzed (Figure S4A to S4K).

Discussion

We identified a remarkably potent effect of fumarase overexpression in abolishing eNOS haploinsufficiency-induced exacerbation of hypertension and renal injury. This occurs in the genomic and physiological context of the SS rat, a classic model that reproduces many characteristics of human salt-sensitive hypertension and renal injury [11, 12]. The findings support the pathophysiological significance of the recently discovered fumarase-NO pathway [27] and suggest that targeting fumarase-related metabolism might be a new therapeutic approach in treating hypertension and other diseases that may result from eNOS insufficiencies.

Fumarase can promote NO production through a malate-aspartate-arginine pathway [27] in which fumarase converts fumarate to L-malate, and malate dehydrogenase in the TCA cycle converts L-malate to oxaloacetate. Oxaloacetate can leave the TCA cycle and be converted to aspartate by aspartate transaminase. Aspartate can be combined with citrulline to generate argininosuccinate, catalyzed by argininosuccinate synthase, and argininosuccinate can be converted back to L-arginine by argininosuccinate lyase. Evidence from animal models and cultured cells supports the presence of this malate-aspartate-arginine pathway and its importance in NO production [27]. The kidney plays a central role in handling a variety of metabolites [31], including L-arginine synthesis and salvage [32, 33]. Approximately 60% of arginine is generated in the kidney by recycling citrulline, while only approximately 15% of arginine is synthesized de novo in the endothelium. Plasma citrulline was associated with sodium intake [34]. Our previous findings showed that renal levels of citrulline and L-arginine were significantly lower in SS rats than in SS-Chr13BN rats [27]. Arginine synthesis is critically dependent on the delivery of citrulline to the kidney where citrulline is converted to arginine [35].

In this study, we found that on the 0.4% salt diet, fumarase overexpression in eNOS haploinsufficient SS rats increased L-arginine levels and L-arginine/citrulline ratios in the renal outer medulla, and restored NO levels in this kidney region. These results are consistent with fumarase overexpression driving L-arginine regeneration from citrulline. In addition, we observed a tendency for increased arginine but significantly lower NO levels in the outer medulla of SS rats with the eNOS heterozygous mutation (SS-Nos3+/−/Fh0/0). Fumarase activity is decreased in SS rats [13]. It has been reported that in fumarase deficient cells, argininosuccinate is produced from arginine and fumarate instead of citrulline and aspartate [36]. It is possible that, in SS- Nos3+/−/Fh0/0 rats, some of the L-arginine is used to produce argininosuccinate instead of NO. In SS- Nos3+/−/Fh0/1 rats, fumarase activity is increased and more of the L-arginine may be directed toward NO generation.

In SS rats with fumarase overexpression, but not eNOS heterozygous mutation (SS-Nos3+/+/Fh0/1), L-arginine levels and L-arginine/citrulline ratios did not differ from those of their wild-type littermates. This suggests the fumarase mechanism might be capable of elevating L-arginine levels only in the presence of eNOS insufficiencies. The mechanism for that remains to be elucidated. Dimethly fumarate (DMF), a fumarate precursor, has been reported to reduce NOS or NOS activity [37, 38]. In the current study, overexpression of fumarase, which converts fumarate to L-malate, leads to increased NO levels. Our findings are consistent with these previous studies in that fumarate decreases NO in general, although the specific NOS isoform and mechanisms involved may be different depending on the tissue or disease process.

Fumarase overexpression restores NO in the renal outer medulla of SS rats with eNOS haploinsufficiency, but not in the renal cortex, when the rats were on the 0.4% salt diet. A myriad of pathways and mechanisms regulates NO levels, including NOS expression, posttranslational modifications and co-factors, presence of NOS isoforms, levels of the NOS substrate (L-arginine), calcium levels, and NO scavenging. The renal cortex and medulla have distinct structures and functions. Over 90% of renal blood flow occurs in the renal cortex, while approximately 5% takes place in the renal outer medulla, suggesting that circulating factors might have a greater influence on NO levels in the cortex than in the outer medulla. NOS isoform distribution also differs between the renal cortex and medulla. For example, nNOS is expressed highly in the macula densa [39, 40]. In addition, the robust production of arginine in the renal cortex might compensate for the lower eNOS protein abundance in SS rats with the eNOS heterozygous mutation and prevent a decrease in the NO level in the cortex. The fumarase/L-arginine regeneration pathway might be particularly important in NO production in the renal outer medulla, especially when eNOS protein abundance decreases. However, eNOS protein, NO, and amino acid levels in rats after 14 days of the 4% salt diet may be influenced by adaptational response to the 4% salt diet and secondary effects of hypertension, in addition to the effect of genotypes, the detailed mechanisms of which remains to be fully elucidated.

The renal outer medulla is known to play a particularly important physiological role in regulating BP and the development of hypertension [41], including hypertension associated with abnormalities in L-arginine, NO and fumarase. Direct administration of L-arginine in the renal medullary interstitium attenuates hypertension in SS rats [42]. Renal interstitial administration of anti-miR-214 attenuates hypertension in SS rats by increasing eNOS protein abundance in the renal outer medulla [28]. Renal medullary interstitial administration of fumarase siRNA in SD rats results in higher BP on the high salt diet [15]. It remains to be determined which cell types in the renal outer medulla are involved in fumarase’s effect on BP, although both endothelial cells and tubular epithelial cells might be important. Fumarase is an obligatory enzyme in the TCA cycle that is present in nearly all cells. eNOS is expressed widely in endothelial cells, as well as tubular segments, including the proximal tubule, medullary thick ascending limb of the loop of Henle, and collecting duct [43, 44]. In addition, it is possible that extra-renal sites also contribute to the observed phenotypes. It would be valuable to develop organ or cell specific transgenic animal models to further understand the role of fumarase metabolism pathway in salt sensitive hypertension.

Fumarate is the substrate of fumarase. DMF has been reported to ameliorate pulmonary hypertension by mitigating oxidative stress damage and inflammation via activating NRF2 [45]. In our study of salt sensitive hypertension models, we found that fumarase overexpression restored NO production by promoting L-arginine regeneration and resulted in lower blood pressure. The mechanisms by which fumarate and fumarase overexpression influence blood pressure may not be identical. Administration of exogenous fumarate and alteration of the endogenous fumarase gene expression level may influence shared biological mechanisms because both of them change fumarate levels. However, these experimental manipulations also may drive different changes in metabolic flux and signaling mechanisms because of differences in the magnitude, duration, and source (exogenous vs. endogenous) of the manipulations.

The BP phenotypes that we observed in SS rats with eNOS haploinsufficiency appeared different from mice with heterozygous knockout of eNOS. Homozygous knockout of eNOS in mice results in the development of hypertension and increased BP salt sensitivity [46–48]. As there is no evidence that homozygous eNOS deficiency occurs in the human population, heterozygous deficiency of eNOS might be more relevant [32]. However, mice with heterozygous mutations of eNOS did not develop significant hypertension or abnormal vascular phenotypes [46, 49, 50]. In contrast, SS rats with eNOS halploinsufficiency develop substantially exacerbated hypertension and a significantly greater BP response to a high-salt diet, as this study showed. Full expression of eNOS might be particularly important in SS rats because their level of NO, including that in the renal outer medulla, might be marginal from the outset [27]. Moreover, SS rats exhibit other pro-hypertensive factors, including increased oxidative stress [15]. These findings highlight the importance of the genomic and physiological context in our understanding of disease mechanisms [15].

Perspectives

Fumarase’s potent effect on NO and hypertension has significant clinical implications. In a study that analyzed 60 metabolites in urine samples obtained from 103 participants in the Dietary Approaches to Stop Hypertension (DASH)-Sodium trial, fumarate ranked the highest in the ability to classify the participants as salt-sensitive or -insensitive correctly, and citrulline and L-arginine were among the metabolites that were likely associated with SBP with low or high sodium intake [7]. Fumarase-related metabolism in the kidney also might be involved in other diseases. Chronic kidney diseases are associated with an impaired L-arginine/NO pathway [51, 52]. Fumarate is higher in urine samples from diabetic patients [53], and fumarase is lower in kidney biopsies from patients with diabetic nephropathy [54]. Among 622 metabolites analyzed, fumarate was one of six metabolites associated significantly with mortality over a median follow-up of 17 years in the Modification of Diet in Renal Disease (MDRD) cohort. The association was replicated in the African American Study of Kidney Disease and Hypertension (AASK) cohort over a median follow-up of ten years [55]. The correlational data on humans and the mechanistic data provided in this study support the potential therapeutic value of targeting fumarase-related metabolism in hypertension and other diseases.

Supplementary Material

COA
Graphical Abstract
Long In Vivo Checklist
Supplemental Material

Figure 6. Summary diagram of the pathway by which fumarase blunts hypertension induced by eNOS insufficiencies.

Figure 6.

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See reference 27 for additional evidence supporting the malate-aspartate-arginine pathway. FH. fumarase; NOS, nitric oxide synthases; NO, nitric oxide.

Novelty and Significance.

What is new?

We produced littermate rats with fumarase hemizygous transgene, eNOS heterozygous mutation, or both, in the genetic background of the Dahl SS rat, a classic model of human salt-sensitive hypertension and renal injury, to examine the pathophysiological relevance of a fumarase-NO pathway in vivo.

What is relevant?

Human hypertension is associated with abnormal metabolic intermediary metabolism. The mechanisms underlying the effect of metabolic intermediaries on BP regulation largely remain unclear. Fumarase contributes to driving NO generation through a malate-aspartate-arginine pathway. The current study demonstrates the pathophysiological significance of the fumarase-NO pathway in vivo.

Summary

Fumarase overexpression has a remarkably potent effect in restoring NO production and abolishing the exacerbation of hypertension and renal injury attributable to eNOS haploinsufficiency in SS rats.

Acknowledgements

This work was supported by US National Institutes of Health grant HL116264-5244 (M.L.).

Footnotes

Disclosures

None.

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Long In Vivo Checklist
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Supplemental Material
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