Uric Acid, Hominoid Evolution, and the Pathogenesis of Salt-Sensitivity

Parallel Mutations in the Uricase Gene in Early Hominoids: Evolutionary Implications

Uric acid is generated during the metabolism of purines and in most mammals is further degraded to allantoin by the hepatic enzyme, uricase (urate oxidase), resulting in serum uric acid levels in the range of 0.5 to 1.5 mg/dL. In contrast, serum uric acid is higher in hominoids (apes and humans) as well as in certain New World monkeys. The increase in uric acid is due to distinct mutations in the uricase gene that made it nonfunctional. In humans, the chimpanzee (Pan), and the gorilla (Gorilla), 3 mutations have been identified, including a nonsense mutation of codon 33, a nonsense mutation of codon 187, and a splice mutation in exon 3.¹⁶ The mutation at codon 33 is also present in the orangutan (Pongo). In contrast, none of these mutations are present in the gibbon (Hylobates), whereas a separate 13 bp deletion was identified between codons 72 and 76 of exon 2.¹⁶ Based on the phylogeny of hominoid evolution as assessed by DNA-DNA hybridization,^17,18⇓ Wu et al¹⁶ proposed that the nonsense mutation at codon 33 occurred between 24 and 13 to 16 million years ago (affecting the Pongo/Gorilla/Pan/Homo lineage) and the 13 bp deletion in exon 2 occurred sometime after the split (ie, divergence node) of the Hylobates from the other hominoids, which occurred around 22 to 24 million years ago (Figure 1).

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Figure 1. Cladogram of hominoid evolution showing the proposed times of the various uricase mutations. The divergence nodes for the Cercopithecoidea are 27 to 33 million years ago (MYA), gibbons 18 to 22 MYA, orangutans 13 to 16 MYA, gorilla 8 to 10 MYA, chimpanzees/man 4.9 to 7.7 MYA. (Adapted with permission from Wu et al,¹⁶ Journal of Molecular Evolution, 1992, 34:78–84, © Springer-Verlag GmbH.)

The uricase gene is also altered within the New World (Ceboidea) and Old World (Cercopithecoidea) monkeys.¹⁹ Serum uric acid was found to be elevated in several species of New World monkeys, including the tamarin (Saguinus), the capuchin (Cebus), and the wooly monkey (Lagothrix), whereas uric acid was low in the squirrel monkey (Saimiri) and owl monkey (Aotus).¹⁹ The levels of uric acid correlated with changes in hepatic urate oxidase activity in these species. Although studies determining the site of the mutations responsible for the loss of uricase in the New World monkeys have not been performed, the mutations were likely independent from those observed during hominoid evolution, given evidence that the divergence of the Platyrrhini infraorder (the New World monkeys) from the Catarrhini (Old World monkeys and hominoids) occurred approximately 40 million years ago.²⁰ Interestingly, although urate oxidase was retained in the Old World monkeys (Cercopithecoidea), the enzyme activity is relatively unstable compared with other mammalian (nonprimate) species, suggesting that evolutionary mechanisms that impair urate oxidase activity may also have been operative in this superfamily.^19,21⇓

The observation that several independent mutations involving uricase occurred during hominoid evolution and in parallel during the evolution of the Old World and New World monkeys has been interpreted as evidence that there must have been an evolutionary advantage for early primates in having an elevated uric acid level.^16,19,21⇓⇓ The time frame of the mutations for the uricase gene indicates that they occurred during the Miocene (24 to 8 million years ago). It is thus important to understand the environmental pressures that were occurring during this period of hominoid history.

Fossil evidence suggests that the earliest hominoids, such as Proconsul, originated in East Africa approximately 22 to 17 million years ago, where they lived in lush subtropical forests and wetlands.^22–25⇓⇓⇓ These early hominoids were arboreal quadrupeds with a diet that was frugivorous (ingesting primarily soft fruits).^22–25⇓⇓⇓ By the early Miocene, there were numerous hominoid species,^22,23⇓ particularly in Eurasia.²⁵ However, by the middle to late Miocene (14 to 8 million years ago), there was an extinction of many Miocene apes, especially in Europe.^24,25⇓ This appears to be associated with an environmental change to a drier and more seasonal climate and with a change in the habitat to more open areas of savannas interspersed with tropical and subtropical forests.²⁵ In certain areas of East Africa, wet and wooded habitats may have been maintained.²⁶ Molecular DNA studies suggest that there was a rapid period of positive selection for various genes during this period, particularly for a gene family (morpheus) in the short arm of chromosome 16, ²⁷ and there were significant changes in dentition and axial skeleton that may have facilitated adaptation to a more arid environment.^22,23,28⇓⇓

As discussed above, the sodium intake of the early hunter-gatherers in the middle to late Pleistocene was in the range of 690 mg (1.9 g NaCl) per day. The sodium intake of early hominoids during the Miocene epoch (from 24 to 5 million years ago) was likely even lower, because the diets consisted primarily of fruits (frugivorous) or leaves (foliverous). Eaton and Konner estimated that the sodium content of a strictly vegetarian Paleolithic diet may have amounted to only 225 mg sodium (10 mEq Na⁺ or 0.6 g NaCl).¹² Thus, the climatic shift to more arid conditions in the middle to late Miocene may have placed selection pressure on the early primates toward a genotype that would maximally conserve sodium with the maintenance of blood pressure.

Uric Acid Maintains Blood Pressure Under Low-Sodium Conditions

Most authorities^16,19,21⇓⇓ have proposed that the mutations in the uricase gene provided an evolutionary advantage because uric acid may function as an antioxidant.²⁹ Although an antioxidant role for uric acid may be one mechanism to explain the beneficial effects of the uricase mutation, we examined the hypothesis that uric acid might regulate blood pressure. This hypothesis was based on previous studies demonstrating that serum uric acid correlates with blood pressure and predicts the development of hypertension in population-based studies.^30–32⇓⇓

To test this hypothesis, mild hyperuricemia was induced in rats by administering the uricase inhibitor, oxonic acid, and then, to recreate the prehistoric conditions, we placed the rats on a low-salt (0.125% NaCl) diet.^14,15⇓ Normal rats on a low-salt diet have either no change or a gradual fall in blood pressure; in contrast, hyperuricemic rats increase their blood pressure (Figure 2).¹⁴ The increase in blood pressure correlated with the uric acid levels and could be prevented by lowering the uric acid levels with allopurinol (a xanthine oxidase inhibitor) or with benziodarone (a uricosuric agent). The increase in blood pressure in hyperuricemic rats was shown to be mediated in part by stimulation of the renin angiotensin system.¹⁴ This hormonal system has a key role in maintaining blood pressure, glomerular filtration rate, and sodium balance under low-salt dietary conditions. We also found that hyperuricemic rats develop vascular disease, particularly of the afferent arteriole of the renal microvasculature, as well as mild renal interstitial inflammation and tubular injury.^14,15⇓ These renal lesions, as well as the renin activation, could be prevented by lowering the uric acid with either allopurinol or benziodarone. The arteriolar lesion resembled the arteriolosclerosis observed in patients with essential hypertension, but the lesion occurred independently of blood pressure as a consequence of a direct stimulation of the vascular smooth muscle cells by uric acid.¹⁵

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Figure 2. Hyperuricemia maintains blood pressure under low-salt dietary conditions in the rat. Sprague-Dawley rats (n=6) placed on a low Na⁺ diet (0.125% NaCl) have a gradual fall in blood pressure, whereas blood pressure is increased in rats (n=6) on a low-salt diet (LSD), which are administered the uricase inhibitor, oxonic acid (OA, 2%). The administration of allopurinol (150 mg/L in the drinking water, n=6) prevents the development of the hyperuricemia and the increase in blood pressure. Uric acid levels at 7 weeks: 2.1±0.2 mg/dL, oxonic acid; 1.3±0.2 mg/dL, low-salt control; 1.2±0.3 mg/dL, oxonic acid+allopurinol.

Goldblatt originally postulated that primary renal microvascular disease might be the major pathogenic mechanism for essential hypertension.³³ Recent studies support this hypothesis.³⁴ The induction of preglomerular arteriolar disease in experimental models leads to tubular ischemia, the interstitial infiltration of lymphocytes and macrophages, local oxidant generation, and alterations in the expression of vasoconstrictors and vasodilators that favor local vasoconstriction.³⁴ These changes result in both a decrease in sodium filtration (caused by a decrease in the ultrafiltration coefficient, Kf) and increased sodium reabsorption (via direct tubular effects) and result in an enhanced blood pressure response to sodium (salt sensitivity).³⁴

Hyperuricemia Induces Salt Sensitivity in Rats

Given that hyperuricemia induces primary renal arteriolar lesions, we hypothesized that chronic hyperuricemia might also induce salt sensitivity. The uricase inhibitor, oxonic acid (2%), was administered with a low-salt diet to rats for 7 weeks. The diet resulted in mild hyperuricemia (uric acid level at 1 week of 2.4±0.7 versus 1.2±0.1 mg/dL in low-salt diet controls, P<0.005, 6 rats tested per group). The oxonic acid diet was then stopped and rats continued on a low-salt diet for 2 weeks, allowing the serum uric acid levels to decrease to levels no different from those observed in low-salt diet controls. Renal tissue obtained from a random subset of rats sacrificed at 9 weeks showed that the previously hyperuricemic rats had a persistent afferent arteriolopathy, with decreased arteriolar lumen diameter and an increased media:lumen ratio compared with low-salt diet controls (Table 1). The rats also showed mild interstitial inflammation and low-grade interstitial collagen deposition (Table 1). Rats were then randomized to a low- or high-salt (2% NaCl) diet. An increase in blood pressure from the high-salt diet (ie, salt sensitivity) was observed only in the rats that had been previously hyperuricemic (Figure 3). This experiment therefore demonstrates that hyperuricemia in the rat can induce salt sensitivity and is consistent with other models in which salt sensitivity can be induced as a consequence of the development of preglomerular arteriolar disease.³⁴

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Figure 3. Hyperuricemia induces salt sensitivity. Sprague-Dawley rats were administered oxonic acid (2%) on a low-salt (0.125% NaCl) diet for 7 weeks (OA-LS group). During this time blood pressure increased compared with low-salt diet (LS) controls. The oxonic acid diet was then stopped, allowing uric acid levels to return to levels equivalent to the low-salt controls. At 9 weeks rats were randomized to continue their low-salt diet or switched to a high-salt (2% NaCl) diet (HS). Previously, hyperuricemic rats developed an increase in blood pressure documenting the development of salt sensitivity; no change in blood pressure was observed in the control group on switching to a high-salt diet. n=6 per group; #P<0.05 versus LS by Student t test; *P<0.05 versus other groups by ANOVA with Fisher exact test.

Uric Acid Stimulates Smooth Muscle Cell Proliferation

We further explored the mechanism by which uric acid causes vascular disease. Soluble, endotoxin-free uric acid (3 to 5 mg/dL) stimulates vascular smooth muscle cell proliferation with activation of the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (Erk1/2) and stimulation of platelet-derived growth factor (PDGF) and its receptor (Figure 4). Rao et al³⁵ have also showed that uric acid induces PDGFA-chain expression and that the smooth muscle cell proliferation can be prevented with neutralizing antibodies to PDGF. We also found that the smooth muscle cell proliferation is mediated by uric acid-induced cyclooxygenase-2 (COX-2) expression with the generation of thromboxane (Duk-Hee Kang, Takahiko Nakagawa, Lili Feng, Marilda Mazzali, Susumu Watanabe, Lin Han, Luan Truong, Raymond Harris, Richard J. Johnson. Unpublished manuscript, 2002), and partially by activation of the renin angiotensin system.¹⁵

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Figure 4. Uric acid induces smooth muscle cell proliferation, MAP kinase activation, and PDGF expression. Rat aortic vascular smooth muscle cells were serum starved and exposed to uric acid (3 mg/dL) for up to 96 hours. Uric acid (UA, 3 mg/dL) induces ERK1/2 MAP kinase phosphorylation. A, Uric acid (3 mg/dL) also induces PDGF-A-chain (B), C-chain (C), and α-receptor (D) mRNA expression, but not PDGF-B-chain mRNA expression. A, Western blot showing the presence of phosphorylated p44 and p 42 (upper panel) and unphosphorylated p44 and p42 MAP kinase (lower panel); B through D, RNAase protection assay.

The increase in uric acid that occurred with the mutation of uricase during the Miocene may have therefore provided a survival advantage (Figure 5). The stimulation of the renin angiotensin system would be expected to acutely increase blood pressure and sodium reabsorption, whereas the preglomerular vascular disease induced by activation of MAP kinase, PDGF, and COX-2 systems would lead to chronic salt sensitivity. The net effect would be to maintain blood pressure and sodium balance.

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Figure 5. Proposed mechanism by which the uricase mutation provided a survival benefit to Miocene hominoids. Environmental pressures during the middle to late Miocene resulted in a survival advantage for hominoids that could better conserve sodium and maintain blood pressure. In animals that had a mutation in the uricase gene, the increase in serum uric acid acutely increases blood pressure and maintains sodium conservation because of the action of uric acid to enhance activation of the renin angiotensin system in response to a low-salt diet. Uric acid also induces renal microvascular disease by stimulating smooth muscle cell proliferation with activation of MAP kinase and stimulation of PDGF, COX-2, and the renin angiotensin system. The development of renal microvascular disease and interstitial inflammation causes salt sensitivity and a chronic increase in blood pressure. The activation of these 2 main pathways (renin angiotensin and COX-2 and PDGF-induced microvascular disease) results in a persistent elevation in blood pressure with maintenance of sodium balance.

Uric Acid: Potential Role in Hypertension and Cardiovascular Disease in Industrialized Societies

Whereas an elevated serum uric acid might have been advantageous for maintaining blood pressure under low-salt dietary conditions, the induction of chronic salt sensitivity would be expected to result in hypertension in modern society with its a high-salt diet. It is of interest that hyperuricemia predicts the development of hypertension and is strongly linked to cardiovascular disease.^{30–32,36,37}⇓⇓⇓⇓ Serum uric acid is also higher in almost all high-risk groups, including males and postmenopausal women (because estrogen is uricosuric), in the obesity insulin-resistance syndrome (because insulin may stimulate uric acid reabsorption), in blacks, and in patients with renal disease (secondary to decreased excretion).^38,39⇓ One might speculate that the higher uric acid levels in blacks may reflect a longer period of environmentally induced selection pressure after the deletion of the uricase gene.

Controversy has existed over the role of uric acid in cardiovascular disease for several reasons. First, some authorities have viewed hyperuricemia as a “marker” for patients at increased cardiovascular risk, as opposed to being truly pathogenic.⁴⁰ This conclusion is based on several epidemiological studies that could not show uric acid to be independent of other factors such as hypertension for predicting cardiovascular events.⁴⁰ However, a factor does not need to be independent to have a causal role in a disease process. For example, if hyperuricemia is a cause of hypertension, then it would not be expected to be independent of it as a risk factor for cardiovascular events. Evidence that uric acid may have a causal role in hypertension is suggested by the experimental studies of mild hyperuricemia in rats.^14,15⇓ Furthermore, although hyperuricemia is not always an independent risk factor for cardiovascular events, it has always been found to be an independent risk factor for the development of hypertension.^30,31⇓ This suggests that a causal relationship between uric acid and hypertension may in part explain the variance in epidemiological studies about the role of uric acid in cardiovascular disease.

Second, some authorities have suggested that hyperuricemia may be a secondary response to the reduced renal blood flow that is a characteristic hemodynamic finding in hypertension.⁴¹ There is strong evidence that renal vasoconstriction results in increased proximal urate reabsorption and an increase in serum uric acid.⁴² Hyperuricemia may also occur in patients with congestive heart failure or peripheral vascular disease because of tissue ischemia that increases uric acid generation (from ATP breakdown) and reduces urate excretion (caused by the effects of lactate on the organic anion exchanger). Although there is no doubt that these conditions do result in an increase in serum uric acid levels, this should not necessarily imply that the increase in serum uric acid is without biological effect. Indeed, our studies would suggest that the increase in uric acid in these conditions may well represent a feedback mechanism to augment the renin angiotensin system to maximally stimulate sodium reabsorption and maintain blood pressure, because a reduced renal blood flow and/or tissue ischemia may be signaling the organism that its overall blood volume is low.

Finally, if uric acid truly causes hypertension, one might expect evidence showing that allopurinol treatment can lower blood pressure. We have not been able to document any controlled studies that have examined this possibility. The studies to examine whether allopurinol can lower blood pressure will need to be performed carefully. Thus, as we recently reported,³⁴ once animals have significant preglomerular disease, they will manifest salt-sensitive hypertension regardless of the mechanism that causes the arteriolopathy. This was further shown in this manuscript, because we demonstrated that salt sensitivity is induced once the vascular disease occurs and that this is independent of the uric acid level. Because modern man is on a high salt diet, it may be difficult to show that lowering uric acid will lower blood pressure in subjects with hypertension. It may still be possible, however, to show this in 2 situations. First, it may be possible to show this in early hypertension associated with a marked increase in uric acid levels and before the development of significant vascular disease, such as in newly transplanted patients placed on cyclosporine or patients with preeclampsia. Second, it may be possible to show a hypotensive effect with allopurinol in patients with established hypertension who are first sodium depleted, because this will remove the salt-sensitivity mechanism mediated by the vascular disease. The sodium restriction will be necessary to remove the role of the preglomerular vascular disease in mediating the blood pressure response.

Perspectives

We present the hypothesis that the uricase mutation that occurred during early hominoid evolution was originally advantageous because it helped to maintain blood pressure both acutely (via stimulation of the renin angiotensin system) and chronically (by inducing salt-sensitivity via the development of renal microvascular and interstitial disease). In modern society the switch to a high salt diet, coupled with this mutation, may have an important role in the current epidemic of hypertension and cardiovascular disease. Other dietary factors (such as dietary changes in potassium and magnesium) and the development of obesity are also likely to be contributory factors for the development of hypertension. Although one must be cautious in the interpretation of experimental models, we suggest that studies be performed to determine the effect of lowering uric acid in man on the development of hypertension, particularly before the development of significant microvascular and renal injury.