This characteristic of hOHT4 gave us a good hint to test whether one can provide an ideal tool without side effects to cure hyperuricemia. Since urine pH is determined by acid generation from food metabolism, we were able to generate urine having an intentional pH by eating designed diets. In this report, we will show that designed diets are effective to remove uric acid from the body, in such a way that alkaline urine is more favorable for removal than acid urine. Twenty-six healthy university female students years old, kg in body weight and cm in height , who had no health problem records in the physical check up conducted by the university, participated in this study.
The ethics committee at Hiroshima Jogakuin University approved the study protocol. All subjects signed informed consent documents. All participants lodged in the dormitory in the campus while the project was going. Values for protein, energy and purine contents were extracted from the available data in the 5 th ed nutritional table issued by the Japanese Health, Welfare and Labor Department for all diets ingested by 26 subjects.
Each diet period lasted 5 days. During each 5-day period, the diets made by different recipes but using the same compositions of natural food materials listed as an appendix were served.
All food materials were purchased from local supermarkets. Subjects had free access to mineral water. Twenty-four-hour urine specimens were collected in bottles and stored in a refrigerator during collection. Volume, pH, titration acid, organic acid and creatinine were measured in a sample from urine collected the day before measurement.
Four ml urine sample of each experimental day for every person was stored in a deep freezer for later ion analysis. According to Lennon, Lemann Jr. Data necessary for estimation of acid production were collected using HPLC. Tokyo, Japan. An aliquot of the filtered and properly diluted urine was injected into an HPLC apparatus with 2.
Titrable acid was estimated as the amount of 0. Bicarbonate concentration [HCO 3 - ] was calculated using the Henderson-Hasselbach equation for which solubility coefficient of carbon dioxide was taken as 0. Organic acid salts were measured by the Van Slyke and Palmer method with the modification by Lennon, Leman and Litzow [ 5 ]. Briefly, urine was mixed with Ca OH 2 and shaken for precipitating out phosphate.
Portions of the filtrate were brought to pH 2. The solution then titrated with 0. The organic acid salt measured was corrected for titration of creatinine which was determined with the Folin method. Uric acid was measured by conventional uricase-peroxidase method, using an autoanalyzer.
A series of studies on the same principle has been conducted for three years consecutively. Each year 10, 7 and 9 participants were assigned to either an acid or alkali diet at the beginning of a 5-day study and then switched to the other kind of diet. The first and second diet periods were separated by one month.
Because several participants were obliged to discontinue the project due to menstruation, two group populations were treated as independent samples instead of being treated as a paired set of data.
The student t test was used to test the significance of changes in measured parameters between the acid and alkali periods. It took 3 days to reach a steady level of urine pH of 6. Effect of acid square and alkali diamond diets on urine pH.
In order to confirm the achievement of proper loading of acid, we measured several factors relating with the endogenous fixed acid production and the urinary acid excretion and listed urinary ammonium, phosphate and sulfate together with urinary pH as a typical representative in Table 1.
Urinary ammonium, phosphate and sulfate were inversely related to the course of urinary pH. On the intake of the alkali diet, these values were significantly lower than those in the acid diet Table. Relationship between endogenous acid generation and renal acid excretion. Diamonds indicate data for the alkali diet and squares those for the acid diet.
The amount of excreted uric acid increased with increase in luminal pH. These calculated values are very close to those observed shown in Table 1. Relationship between excreted uric acid as expressed in mg uric acid in urine per day and urine pH. These data demonstrate that excretion of uric acid is suppressed in acidic medium. In the Framingham cohort, investigators concluded that hyperuricemia was a covariable of other known cardiovascular risk factors for cardiac deaths and coronary heart disease More recently, data appear to be swinging the interpretation the other way, indicating that hyperuricemia predisposes to plaque formation and endothelial dysfunction, as assessed by ultrasonography 19 — Hyperuricemia was also reported to be an independent risk factor for cardiovascular mortality 22 , In patients with established cardiovascular disease, elevated urate levels were an independent predictor of cardiovascular events 24 ; and finally, in a meta-analysis of the association between hyperuricemia and stroke, a small but increased risk was found even after adjustment for known cardiovascular risk factors In most studies, the increased risk of hyperuricemia for cardiovascular morbidity by odds ratio or hazard ratio was around 1.
If hyperuricemia is an etiological factor in cardiovascular morbidity, what are the mechanisms, and will its modification affect outcome? Data from a rodent model suggested that uric acid—mediated vasoconstriction leads to endothelial dysfunction, activation of the renin-angiotensin system, and hypertension Critics will, however, argue that it is impossible to disentangle hyperuricemia from hypertension and that hyperuricemia is a surrogate marker for early subclinical renal dysfunction, and the cardiovascular complications are secondary.
In the absence of more compelling experimental data, this debate can only be resolved by large-scale intervention studies in a hyperuricemic population analyzing the effects on blood pressure and cardiovascular events over time. The relative hyperuricemia in humans has raised questions about its evolutionary advantages, and its association with diseases requires understanding how it can become deleterious at high concentrations.
Initially, uric acid was considered an inert waste product that crystallizes at high concentrations to form renal stones and provoke gouty arthritis. Subsequently, uric acid was recognized to be a powerful antioxidant that scavenges singlet oxygen, oxygen radicals, and peroxynitrite and chelates transition metals, to reduce, for instance, iron ion—mediated ascorbic acid oxidation.
Urate thus accounts for approximately half of the antioxidant capacity of human plasma, and its antioxidant properties are as powerful as those of ascorbic acid 27 , As illustrated in Figure 2 A, uric acid can prevent peroxynitrite-induced protein nitrosation 29 , lipid and protein peroxidation 30 , and inactivation of tetrahydrobiopterin 31 , a cofactor necessary for NOS.
Together, these antioxidant actions underlie the protective effects of uric acid action in cardiovascular diseases, aging, and cancer Antioxidant and pro-oxidant effect of uric acid.
Antioxidant activities. Peroxynitrites can induce protein nitrosation and lipid and protein peroxidation and block tetrahydrobiopterin HB4 , a cofactor necessary for NOS activity. C By enhancing arginase activity, uric acid diverts l -arginine from NO production to urea production. Uric acid can also directly react with NO to generate nitrosated uric acid, and the nitroso group can then be transferred to glutathione GSH for transport to another recipient molecule.
In the presence of oxygen, uric acid reacts with NO to produce the stable species 6-aminouracil. Blue arrows, chemical reactions; green arrows, products from enzymatic or signaling pathways; red arrows, activation of enzymatic activities. In vitro and cellular studies have nevertheless demonstrated that depending on its chemical microenvironment, uric acid may also be pro-oxidant.
As illustrated in Figure 2 A, when uric acid is oxidized by peroxynitrites, urate radicals are produced that could propagate the pro-oxidant state 34 , but in the plasma they are rapidly inactivated by reaction with ascorbic acid NO, described initially as an endothelial cell—derived relaxing factor, is an important regulatory molecule in the cardiovascular system, and reduced NO levels are associated with hypertension and insulin resistance 35 — Urate can react directly with NO under aerobic conditions to generate an unstable nitrosated uric acid product that can transfer NO to other molecules such as glutathione ref.
Under anaerobic conditions, urate is converted in the presence of NO into stable 6-aminouracil The possibility that increased urate plasma levels can reduce NO bioavailability has been tested in rats treated with the uricase inhibitor oxonic acid. Similarly, direct exposure of endothelial cells to uric acid slightly reduces basal or VEGF-stimulated NO production Thus, uric acid can dose-dependently reduce NO bioavailability.
Although a direct chemical reaction of urate with NO could explain the decrease in plasma NOx, there is evidence that in vivo urate can decrease NO production by interfering with its biosynthesis. For instance, in pulmonary endothelial cells, uric acid reduces NO production by a mechanism that depends on uric acid increasing the activity of arginase, which diverts l-arginine to urea production instead of to NO production by eNOS Figure 2 C and ref.
Another pro-oxidant action of urate has been described during adipogenic differentiation of 3T3-L1 cells Figure 2.
When these cells are induced to differentiate into adipocytes, addition of uric acid at physiological concentrations further increases ROS production by a mechanism that involves activation of NADPH oxidase This effect in adipocytes may participate in the induction of inflammation and insulin resistance of adipose tissue observed in obesity Together, the available information indicates that uric acid has complex chemical and biological effects and that its pro-oxidant or NO-reducing properties may explain the association among hyperuricemia, hypertension, the metabolic syndrome, and cardiovascular disease In addition, when hyperuricemia leads to the formation of microcrystals, it leads to joint and renal inflammation.
Chronic inflammation as in tophaceous gout leads to bone and cartilage destruction, and chronic hyperuricemia and hyperuricosuria in gouty patients are also frequently associated with tubulointerstitial fibrosis and glomerulosclerosis, signs of local renal inflammation There is thus no simple explanation for the possible protective or pathogenic effect of hyperuricemia, and there is clearly a need for more animal models to study this link.
Urate homeostasis depends on the balance between production and complex processes of secretion and reabsorption in the kidney tubule and excretion in the intestine. In patients presenting with gout and primary hyperuricemia, the majority underexcrete urate when the fractional clearance of urate is measured Urate transport by the kidney has been investigated for many years, in part to search for uricosuric drugs to decrease plasma urate levels.
So far, several classes of uricosuric drugs have been identified that decrease plasma urate levels, such as benzbromarone, probenecid, sulfinpyrazone, or losartan, whereas other pharmacological agents such as pyrazinoate, the active metabolite of pyrazinamide, nicotinate, and lactate, are antiuricosuric.
In the human kidney, urate handling involves urate glomerular filtration followed by a complex array of reabsorptive and secretory mechanisms taking place in the proximal tubule. In the mouse, both the proximal and distal convoluted tubule appear to be involved in urate reabsorption and secretion, as determined by the localization of the various urate carriers that are discussed below and depicted in Figure 3.
It has to be noted that the relative importance of the reabsorption and secretion mechanisms differ among species. Humans, mice, and rats predominantly reabsorb uric acid, whereas pigs, rabbits, reptiles, and birds have more active secretory mechanisms. Urate transporters in kidney epithelial cells of humans and mice. Urate transport in the mouse kidney involves both the proximal and distal convoluted tubules middle and lower left panels. The same urate-transporting proteins present in humans are found in the mouse proximal tubules, except for Glut9, which is present at an extremely low levels.
In mice, in contrast to humans, Glut9 is present at very high levels in both the apical and basolateral poles of distal convoluted tubule cells. However, it is not known which isoform of Glut9 is present in the apical and basolateral membranes.
URAT1 is a transmembrane domain—containing protein found in the apical membrane of proximal tubule epithelial cells and transports urate in exchange for Cl — or organic anions. The antiuricosuric agents lactate, pyrazinoate, and nicotinate can serve as substrate for the antiporter activity of URAT1 to increase urate reabsorption. On the other hand, URAT1 is inhibited by the classical uricosuric agents benzbromarone, probenecid, and losartan. Inactivating mutations in URAT1 have been found in Japanese patients with idiopathic renal hypouricemia 51 , 52 , These patients are mostly asymptomatic but may develop exercise-induced acute renal failure Knockout of the Urat1 gene in the mouse leads to increased urate excretion but no significant hypouricemia, indicating that in the mouse, this transporter plays a less important role than in humans for the control of uricemia OAT4 encoded by the SLC22A11 gene is a multispecific anion transporter present in the apical membrane of epithelial cells from the proximal tubule 57 , It is involved in luminal urate reabsorption by a mechanism that is transactivated by intracellular dicarboxylates but not by the antiuricosuric agents; it is also affected by the diuretic hydrochlorothiazide OAT10 SLC22A13 is a urate and high-affinity nicotinate transporter expressed in brush border membrane vesicles from proximal tubules and, interestingly, also in cortical collecting ducts in rats Males had higher h Uur than females a.
The level of h Uur was lower in stage 1 and 3 hypertension group than that in normo-tension group c. Furthermore, participants in the macro-albuminuria group had significantly lower h Uur compared with those in the normo-albuminuria group d. Error bars represent the standard deviation values.
Levels of FEur were compared among different groups in gender a , CKD stages b , hypertension status c , and albuminuria d using a multivariate analysis. Females had higher levels in FEur than males a. And FEur was higher in stage 3 hypertension group than that in normo-tension group c. Furthermore, participants in the macro-albuminuria group had significantly higher FEur compared with those in the normo-albuminuria group d.
Error bars represent the interquartile range IQR. Levels of Cur according to CKD, hypertension, and albuminuria status. Levels of Cur were compared among different groups in CKD stages a , hypertension status b , and albuminuria status c using a multivariate analysis.
The level of Cur was lower in stage 1—3 hypertension than that in normo-tension group b. Furthermore, participants in the macro-albuminuria group had significantly lower Cur compared with those in the normo-albuminuria group c. CKD1, H0, A1 respectively. These factors were recognized as confounding factors for albuminuria on urinary uric acid excretion.
Our analyses showed that h Uur and Cur were lower and FEur was higher in the hypertension group, stage 3—5 CKD and macro-albuminuria group than those in the normotensive group, stage 1 CKD and the normo-albuminuria group. Moreover, males had higher h Uur and lower FEur than females. In addition, we found that albuminuria was negatively associated with h Uur and Cur, after adjusting for multiple confounding factors.
The prevalence of hyperuricemia has been studied in general populations. In the present study, we first analyzed the incidence of hyperuricemia in our patients with CKD, which was In addition, among the patients with hyperuricemia, low renal excretion of uric acid accounted for Therefore, it indicated that whether the general population or CKD patients, insufficient renal uric acid excretion is the main reason of hyperuricemia.
Additionally, the analysis showed that h Uur was lower in females, which is contradictory to the previous indication that estrogen promoted urinary uric acid excretion [ 28 ]. It can be explained by the fact that most women in our study were postmenopausal. Furthermore, we found that h Uur and Cur significantly reduced in patients with stage 3—5 CKD, which was consistent with the established knowledge that GFR is an important influencing factor.
A large body of evidence indicated that hypertension has reciprocally influenced on or been affected by hyperuricemia [ 29 , 30 , 31 ], and this might support our result that hypertensive patients had lower Cur. Indeed, several investigators have reported that sodium-sensitive hypertension is associated with hypercalciuria [ 32 ]. On the other hand, the study by Belge et al.
Accordingly, we speculated that renal uric acid transport was also altered in hypertensive diseases. According to previous studies, high glucose levels are associated with high serum urate levels, while frank glycosuria is associated with hypouricemia [ 3 ]. Additionally, SUA concentrations may also be different according to the type of diabetes [ 35 ].
Levels of SUA are often lower in patients with type 1 diabetes compared with their nondiabetic peers [ 36 ], which may be induced by glycosuria. But whether GLUT9 is involved in this phenomenon has not been confirmed. In contrast, type 2 diabetes is often correlated to higher SUA concentrations [ 37 , 38 ], although some studies have reported the presence of hypouricemia in type 2 diabetes [ 39 , 40 ].
It is likely that the presence of insulin resistance and hyperinsulinemia stimulate the renal tubular cells to reabsorb sodium coupling with uric acid [ 35 ]. The failure to detect a change in urate excretion in diabetes in our study may be explained by the fact that increase in FEur in all the studies was related to glycosuria, not diabetes per se, which may not be prominent in a well-treated population.
Currently, the association of albuminuria with urinary uric acid excretion has not been extensively studied. Many studies focused on the association between albuminuria and SUA, and documented that SUA is positively associated with albuminuria and predicts the development of albuminuria, including general population [ 15 ], diabetes mellitus [ 41 ], and hypertension [ 18 ]. Moreover, in another study conducted in general population, Scheven [ 43 ] found a positive association of albuminuria with tubular uric acid reabsorption, independent of potential confounders such as.
As to the negative association between h Uur, Cur and UACR, it may well be that albumin or concomitant non-albumine compounds, such as plasmin, found in urinary of albuminuric subjects, can specifically upregulate or downregulate the expression of genes encoding for tubular uric acid transporters. This phenomenon has been shown for other, non-uric acid membrane transporters in tubular proximal epithelial cells [ 20 , 21 , 22 , 23 ]. Since the patients with CKD are in steady state in these studies, this must reflect either a change in uric acid production or an increase in non-renal excretory pathways induced by metabolic changes, such as protein-energy wasting, accumulated uremic toxins, dyslipidemia and insulin resistance, in severe CKD [ 9 , 12 ].
In genetic analyses of CKD cohorts, Bhatnagar et al. Their data supported the notion that uric acid transporters in remote organs intestine versus kidney may regulate serum uric acid levels, especially after acute or chronic organ damage, as suggested in the Remote Sensing and Signaling Hypothesis [ 45 ]. As far as we know, it is possible that actually uric acid changes proteinuria or that proteinuria and uric acid are dependent on the same variable.
A well-designed study is needed to verify a causal relation between albuminuria and uric acid. As has been previously described, with increasing severity of CKD, FEur of residual nephrons increased in order to maintain homeostasis of uric acid.
This finding may actually reflect the compensation of residual nephrons or may arise from a presently undefined influence of uremia per se. Recently, researcher Andrew Rule and colleagues have developed a method to determine GFR at the level of the single nephron, and found that CKD risk factors were associated with increased single-nephron GFR, but they thought uric acid levels were associated with lower nephron number rather than single-nephron GFR, leading to a lower total GFR [ 46 ].
Indeed, our study demonstrated that UACR was negatively associated with h Uur and Cur, but not with FEur, after adjusting for confounders, suggesting that albuminuria may be associated with total uric acid excretion but was not strong enough to be correlated with the residual tubular excretion of uric acid in CKD patients. Accordingly, our findings indicate that in clinical practice, it is better to use h Uur and Cur to estimate the capacity of renal uric acid excretion in CKD patients, while FEur reflects more on the compensatory residual renal tubular function and should be valued with caution in patients with reduced kidney function.
Our study has some limitations that need to be mentioned. First, the cross-sectional nature of the present study makes it hard to determine any causal relationship. Second, the effect of specific etiology of CKD was not explored, since the extent of specific renal tubular damage was different. Finally, UACR was measured once, which is known to be subject to more variability. Influencing factors of renal uric acid excretion is complicated, and albuminuria, evaluated as UACR, was negatively correlated with h Uur and Cur after adjusting for multiple confounders in Chinese CKD patients.
This phenomenon may explain in part the association between albuminuria and serum uric acid. Further studies are required to determine the exact mechanism of the association of albuminuria with renal uric acid excretion. Renal transport of uric acid: evolving concepts and uncertainties.
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