1

Effects of febuxostat on metabolic and renal alterations in rats with

fructose-induced metabolic syndrome

Laura G. Sánchez-Lozada1, Edilia Tapia1, Pablo Bautista-García1, Virgilia Soto2,

Carmen Ávila-Casado2, Iliana P. Vega-Campos1, Takahiko Nakagawa3, Lin Zhao4,

Martha Franco1, Richard J. Johnson3

Departments of 1Nephrology and 2Pathology, Instituto Nacional de Cardiología

Ignacio Chávez, Mexico City, Mexico. 3Nephrology, Hypertension and

Transplantation, University of Florida, Gainesville, FL, USA. 4TAP Pharmaceutical

Products Inc., Lake Forest, IL, USA .

Correspondence to:

L. Gabriela Sánchez-Lozada, PhD

Department of Nephrology

Instituto Nacional de Cardiología Ignacio Chavez

Juan Badiano 1. 14080-Mexico City

Mexico

Tel: 52 55 55 73 69 02

Fax: 52 55 55 73 77 16

e-mail: lgsanchezlozada@hotmail.com

Running Title: Febuxostat ameliorates features of metabolic syndrome and renal

damage in fructose-fed rats

Page 1 of 42Articles in PresS. Am J Physiol Renal Physiol (January 23, 2008). doi:10.1152/ajprenal.00454.2007

Copyright © 2008 by the American Physiological Society.

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ABSTRACT

Increased fructose consumption is associated with hyperuricemia, metabolic

syndrome and renal damage. This study evaluated whether febuxostat (Fx), an

investigational non-purine and selective xanthine oxidase inhibitor, could alleviate

the features of metabolic syndrome as well as the renal hemodynamic alterations

and afferent arteriolopathy induced by a high-fructose diet in rats. Two groups of

rats were fed a high-fructose diet (60% fructose) for 8 weeks, and two groups

received a normal diet. For each diet, one group was treated with Fx (5-6

mg/kg/day in drinking water) during the last 4 weeks (ie after the onset of metabolic

syndrome) and the other received no treatment (placebo, P). Body weight was

measured daily. Systolic blood pressure and fasting plasma uric acid (UA), insulin

and triglycerides were measured at baseline and at 4 and 8 weeks. Renal

hemodynamics and histomorphology were evaluated at the end of the study. A

high-fructose diet was associated with hyperuricemia, hypertension as well as

increased plasma triglycerides and insulin. Compared to Fructose+P, Fructose+Fx

rats showed significantly lowered blood pressure, UA, triglycerides and insulin

(p<0.05 for all comparisons). Moreover, Fructose+Fx rats had significantly reduced

glomerular pressure, renal vasoconstriction and afferent arteriolar area relative to

Fructose+P rats. Fx treatment in rats on a normal diet had no significant effects. In

conclusion, normalization of plasma UA with Fx in rats with metabolic syndrome

alleviated both metabolic and glomerular hemodynamic and morphologic

alterations. These results provide further evidence for a pathogenic role of

hyperuricemia in fructose-mediated metabolic syndrome.

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Key Words: febuxostat, metabolic syndrome, hyperuricemia, glomerular

hypertension, arteriolopathy.

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INTRODUCTION

Increasing evidence associates fructose consumption with the increased

incidence of metabolic syndrome and obesity seen in Western societies (7; 14). In

contrast to the metabolic effects of other sugars, fructose induces hyperuricemia

through the stimulation of nucleotide catabolism via the conversion of hepatic ATP

to ADP by fructokinase; fructose also decreases the renal excretion of uric acid

(UA) through an increase of lactate synthesis (19).

Interestingly, epidemiologic studies have demonstrated that the prevalence

of metabolic syndrome shows a graded increase with increasing serum UA levels

(6; 22; 26; 29; 56), suggesting that hyperuricemia could be included in the

definition of metabolic syndrome (10; 44; 51). Moreover, serum UA concentration

increases in parallel to the number of metabolic syndrome traits (11; 30; 56); thus,

serum UA concentration is positively correlated with blood pressure, waist-to-hip

ratio, homeostasis model assessment index (an index of insulin resistance), body

mass index, and levels of fasting plasma glucose, insulin, triglycerides, C-reactive

protein, and is inversely correlated with HDL-cholesterol levels (reviewed in

reference (51)). In addition, it has been found that serum UA levels may be a

reliable predictor of pre-metabolic syndrome in obese youth (13).

There is evidence that the increased serum UA occurring with metabolic

syndrome may not only be a consequence of hyperinsulinemia (33; 34), but it may

actually promote or exacerbate features of metabolic syndrome. For example, UA

may play an important role in the pathogenesis of metabolic syndrome due to its

ability to inhibit nitric oxide (NO) bioavailability and thereby induce endothelial

dysfunction, which is the hallmark of insulin resistance (25; 39). Supporting this

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contention is the observation that increasing amounts of fructose in the diet results

in escalating levels of hyperuricemia which correlates with increasing features of

metabolic syndrome in rats (38). Additional experimental studies have shown that

chronic administration of allopurinol [an inhibitor of xanthine oxidase (XO)] or

benzbromarone (a uricosuric agent) can reduce fructose-induced hyperinsulinemia

and hypertriglyceridemia (33). Another potential mechanism activated by UA that

may contribute to the pathogenesis of metabolic syndrome is oxidative stress. In

this regard, it was shown that soluble UA stimulated an increase in NADPH

oxidase activity and the production of reactive oxygen species (ROS) in mature

adipocytes which resulted in activation of MAP kinases p38 and Erk1/2, reduction

in NO bioavailability and increased protein nitrosylation and lipid oxidation (43).

Collectively, these studies suggest that increased levels of UA play a fundamental

role in the pathogenesis of the metabolic syndrome.

Clinical and experimental observations suggest that both metabolic

syndrome and hyperuricemia confer a higher susceptibility to develop chronic

kidney disease (9; 21). Clinical studies have shown that the greater the number of

metabolic syndrome traits exhibited, the greater the risk of developing

microalbuminuria (9). Moreover, a recent study suggests that increased metabolic

risk in young healthy males is associated with a 6.9-fold increase in the likelihood

of glomerular hyperfiltration occurring prior to the onset of overt cardiovascular

disease (49). Hyperuricemia has also been associated with chronic renal damage.

In a study of 6,400 subjects with normal renal function, a value of serum UA >8.0

mg/dL was associated with a 2.9- and 10.0-fold increased risk for developing renal

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insufficiency within 2 years in men and women, respectively (21). Increased serum

UA has also been found to be a predictor of poor outcome in patients with IgA

nephropathy (47). Recently, it was shown that allopurinol treatment for 12 months

in hyperuricemic patients with mild-to-moderate chronic renal disease significantly

decreased serum UA and helped to preserve kidney function when compared to

patients receiving no allopurinol (46). However, even more compelling evidence of

the contribution of hyperuricemia to the development and progression of renal

damage comes from experimental studies. Rats made hyperuricemic by the

administration of oxonic acid (inhibitor of uricase) developed systemic and

glomerular hypertension, mild tubulointerstitial damage, arteriolopathy of the

afferent arteriole and cortical vasoconstriction (32; 40; 41); these effects were

prevented by reducing serum UA levels through the simultaneous administration of

oxonic acid and allopurinol or benziodarone (32; 40; 41). Interestingly, the

hyperuricemia induced by high fructose intake in rats produced renal effects similar

to those seen in experimental hyperuricemia; a 60% fructose diet raised plasma

UA and induced glomerular hypertension, cortical vasoconstriction and afferent

arteriole thickening (38).

Febuxostat, an investigational potent, non-purine molecule is being

developed as an inhibitor of XO for the treatment of hyperuricemia in gout patients

(3; 4). Febuxostat is different from allopurinol in that it does not inhibit other

enzymes in purine and pyrimidine metabolism pathways (48). Moreover, studies

have shown that febuxostat inhibits XO activity simply by obstructing substrate

binding and that this inhibition is not influenced by changes in the redox status of

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the cofactor (36). Because it has been shown previously that administration of

allopurinol, as a prophylactic agent or as treatment, prevented or reversed features

of metabolic syndrome (33), the present study was undertaken to determine if

chronic treatment with febuxostat can alleviate the major traits of fructose-induced

metabolic syndrome as well as the accompanying renal hemodynamic alterations

and afferent arteriolopathy in rats.

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METHODS

Experimental Design: Four groups of male Sprague-Dawley rats

(n=10/group; 290-350 g) were studied over a period of 8 weeks. Two groups

(Normal) received a regular diet and the other two groups (Fructose) were fed a

60% fructose diet to induce development of the metabolic syndrome. Details on the

composition of the diets are presented in Table 1. After 4 weeks, febuxostat (Fx)

was administered in drinking water (50 mg/L; approximately 5-6 mg/kg/day) for an

additional 4 weeks in one normal-diet group (Normal+Fx) and one fructose-diet

group (Fructose+Fx). Respective placebo (P) control groups (Normal+P and

Fructose+P) received no treatment, except for an additional amount of NaCl in the

drinking water (5.84 mg/L, to maintain a salt concentration equivalent to that of the

Fx-containing water) for 4 additional weeks.

Two additional groups of fructose-fed rats (n=5/group) were studied in order

to assess the effects of a longer period (14 weeks) of fructose feeding and Fx

treatment on systemic blood pressure. Both groups received a 60% fructose diet

for the entire 14 weeks; one group received Fx (50 mg/L in drinking water) for 10

weeks (from Weeks 5 to 14) and the other group served as the placebo control . All

experiments were approved by the Ethics Committee of Instituto Nacional de

Cardiologia Ignacio Chavez.

Measurements: Body weight and food intake were measured daily. Systolic

blood pressure (SBP) was measured in conscious rats by a tail-cuff

sphygmomanometer (XBP-1000 Kent Scientific Corp., Torrington, CT, USA). All

animals were preconditioned for blood pressure measurements one week before

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each experiment. Fasting plasma UA (Diagnostic Chem Ltd., Charlottetown, PEI,

Canada), insulin (Crystal Chem Inc., Downers Grove, IL, USA) and triglycerides

(Spinreact, Girona, Spain) were measured using commercial kits. SBP and all

biochemical parameters were determined prior to the start of fructose feeding (ie

baseline) and at the end of 4 and 8 weeks.

For the two groups administered fructose for 14 weeks, body weight was

measured at baseline and weekly thereafter. SBP was measured at baseline and

at the end of 4, 8 and 12 weeks. No other measurements were conducted in these

groups.

Micropuncture: Animals were anesthetized with pentobarbital sodium (30

mg/kg, ip) and placed on a thermoregulated table to maintain body temperature at

37°C. Trachea, jugular veins, femoral arteries and the left ureter were catheterized

with polyethylene tubing (PE-240, PE-50, and PE-10). The left kidney was

exposed, placed in a Lucite holder, sealed with agar, and covered with Ringer’s

solution. Mean arterial pressure (MAP) was monitored with a pressure transducer

(Model p23 db; Gould, San Juan, PR) connected to the catheter in the femoral

artery and recorded on a polygraph (Grass Instruments; Quincy, MA, USA). Blood

samples were taken periodically and replaced with blood from a donor rat. Rats

were maintained under euvolemic conditions by infusion of 10 ml/kg of body weight

of isotonic rat plasma during surgery, followed by an infusion of 25% polyfructosan,

at 2.2 ml/h (Inutest, Fresenius Kabi, Linz, Austria). After 60 min, five to seven

samples of proximal tubular fluid were obtained to determine flow rate and

polyfructosan concentrations. Intratubular pressure under free-flow (FF) and stop-

flow (SFP) conditions and peritubular capillary pressure (Pc) were measured in

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other proximal tubules with a servo-null device (Servo Nulling Pressure System;

Instrumentation for Physiology and Medicine, San Diego, CA, USA). Glomerular

colloid osmotic pressure was estimated from protein concentrations obtained from

blood of the femoral artery and surface efferent arterioles Polyfructosan was

measured in plasma and urine samples by the anthrone-based technique of

Davidson and Sackner (12). Total GFR was calculated using the following formula:

GFR = (U × V) / P, where U is the polyfructosan concentration in urine, V is urine

flow rate, and P is the polyfructosan concentration in plasma.

The volume of fluid collected from individual proximal tubules was estimated

from the length of the fluid column in a constant bore capillary tube of known

internal diameter. The concentration of tubular polyfructosan was measured by the

microfluorometric method of Vurek and Pegram (54). Single nephron glomerular

filtration rate (SNGFR) was calculated using the formula: SNGFR = (TF/P)PF x V,

where PF is the concentration of polyfructosan in tubular fluid (TF) and plasma (P),

and V is the tubular flow rate which is obtained by timing the collection of tubular

fluid (2). Protein concentration in afferent and efferent samples was determined

according to the method of Viets et al (53). MAP, GFR, glomerular capillary

hydrostatic pressure (PGC), single-nephron plasma flow (QA), afferent artieriole

(AR), efferent arteriole (ER) and total (TR) resistances and ultrafiltration coefficient

(Kf) were calculated according to equations previously reported (2).

Renal Histology and Quantification of Morphology: After the micropuncture

study, kidneys were washed by perfusion with phosphate-buffered saline (PBS)

and fixed with 4% paraformaldehyde. Renal biopsies were embedded in paraffin.

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Four-µm sections of fixed tissue were stained with periodic acid Schiff (PAS)

reagent. Arteriolar morphology was assessed by indirect peroxidase

immunostaining for alpha smooth-muscle actin (DAKO Corp.; Carpinteria, CA,

USA) (40; 41). Sections of kidney tissue incubated with normal rabbit serum were

used as negative controls for immunostaining against alpha smooth-muscle actin.

For each arteriole, and in 10 arterioles per biopsy, the outline of the vessel

and its internal lumen (excluding the endothelium) were generated using computer-

based analysis to calculate the total medial area (outline – inline). The

media/lumen (M/L) ratio was calculated by the outline/inline relationship.

Quantifications were performed blinded.

Statistical Analysis: Values are expressed as mean ± standard error (SE).

Body weight, food intake, SBP and the biochemical parameters measured over the

course of the study were analyzed for treatment and diet effects, time effect and

treatment-by-time interaction using a two-way ANOVA for repeated-measures test.

Single-time measurements (MAP, whole-kidney GFR, glomerular hemodynamics

and histological parameters) were analyzed using standard two-way ANOVA..

When the ANOVA p value was <0.05, post-test comparisons were made using a

Bonferroni multiple-comparison test. The relationship between variables was

assessed by correlation analysis. In the 14-week fructose feeding study,

comparisons were made using an unpaired Student’s t-test.

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RESULTS

Metabolic parameters, blood pressure and body weight: Plasma UA

increased in fructose-fed rats and was reversed with febuxostat treatment to

normal levels (Figure 1), while fructose-fed rats treated with placebo continued to

show increased levels of plasma UA. Fasting triglyceride levels were <100 mg/dL

in all groups at baseline (Figure 1). By Week 4, rats fed a high-fructose diet had

triglyceride levels that were twice those of rats fed a normal diet. Treatment with

febuxostat during Weeks 5-8 resulted in a significant decrease in triglyceride

levels, while the levels in the untreated fructose-fed animals continued to rise. A

positive correlation between UA concentrations and triglycerides (TG) at Week 8 in

all groups of rats was observed (UA vs TG: r= 0.69, p<0.0001).

Fasting plasma insulin levels rose with a high-fructose diet, becoming

significantly elevated at Week 8 (Figure 1). Febuxostat treatment resulted in

normalization of insulin levels, while continued administration of fructose alone

caused a progressive increase. A positive correlation between UA and insulin

concentrations in all groups of rats was present at Week 8 (UA vs insulin: r= 0.73,

p<0.0001).

A high-fructose diet resulted in a significant rise in SBP at Week 4 which

persisted until the end of the study. Febuxostat tended to reduce blood pressure in

rats fed a high-fructose diet, although the reduction was not statistically significant

relative to the Fructose+P group (Figure 1). MAP measured by intra-arterial

measurement at Week 8 was also increased in fructose-fed rats and was

normalized by febuxostat treatment (p<0.05). A positive correlation between UA

and MAP in all groups of rats was observed (r=0.54, p=0.001). Febuxostat

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treatment in rats fed a normal diet had no significant effects on metabolic

parameters and blood pressure.

Due to the discrepancy between the SBP and MAP data in the 8-week

study, an additional set of rats were fed a high-fructose diet for 14 weeks (Figure

2). Baseline values of SBP were similar between groups (Fructose Diet+P, 120±4

mmHg; Fructose Diet+Fx, 117±4 mmHg; p=ns). After four weeks of fructose diet

and before the start of placebo or febuxostat treatment, both groups developed

hypertension (Fructose Diet+P 153±4 mmHg; Fructose Diet+Fx 152±4 mmHg;

p=ns). SBP increased progressively with continued fructose feeding and reached

a value of 163±6 mmHg at 12 weeks. Febuxostat treatment completely abrogated

this increase in SBP (121±4 mmHg at Week 12, p=0.0003). Thus, treatment with

febuxostat for longer than 8 weeks may be needed to enable detection of systemic

blood pressure changes measured by the tail-cuff method in conscious rats.

Daily food intake and body weight measured during the course of the 8-

week study are presented in Figure 3. The interaction between time and treatment

was found to be significant for both parameters. However, the results of the

Bonferroni post-hoc test between treatments rendered statistical significance only

at isolated time points, mostly at the beginning of the study when the rats’ diet was

switched from normal to high-fructose. Nonetheless, the overall tendency in food

intake and body weight was for no significant difference among the four groups,

indicating that treatment with febuxostat did not significantly affect food intake nor

body-weight gain in these rats, regardless of the type of diet. In support of this

contention are the results for body weight from the additional two groups of rats fed

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a high-fructose diet for 14 weeks; febuxostat treatment during Weeks 5-14 did not

affect body-weight gain (data not shown).

Glomerular hemodynamics: Glomerular hemodynamics results are depicted

in Figure 4 and in Table 2. Two rats from each group of Normal+P, Normal+Fx and

Fructose+P were discarded due to complications during preparation of the

micropuncture experiment. Fructose-fed rats developed renal cortical

vasoconstriction, manifested as significantly lower SNGFR and elevated

glomerular pressure compared to the Normal+P group (Figure 4). The reduction of

SNGFR was caused by a significant decrement of ultrafiltration coefficient and

glomerular plasma flow, despite the presence of increased glomerular pressure.

Relative to rats on a normal diet, a lower renal cortical perfusion in fructose-fed rats

(Fructose+P) was the result of numerically higher afferent and efferent arteriole

resistances (Table 2). The glomerular hemodynamic changes accompanying the

fructose-induced metabolic syndrome tended to improve with febuxostat treatment;

glomerular hypertension was prevented and the ultrafiltration coefficient was

normalized in the Fructose+Fx group. Additionally, the values of SNGFR,

glomerular plasma flow, AR and ER in the Fructose+Fx group were not different

compared to the Normal+P group. A positive correlation was shown to exist

between plasma UA and glomerular pressure in all groups of rats (r=0.54,

p=0.001).

Renal arteriolar morphology: Table 3 and Figure 5 show results for the renal

arteriolar morphology. Fructose-fed rats (Fructose+P) developed a thickening of

the afferent arteriole as indicated by a significantly higher arteriolar area; however,

in the Fructose+Fx group, this alteration was reversed to a value similar to that in

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the Normal+P group. In addition, UA level in all groups of rats correlated with

arteriolar area (r= 0.58, p=0.0003). There were no significant differences in M/L

ratios among the various groups (Table 3). Febuxostat treatment in rats fed a

normal diet had no significant effects on glomerular hemodynamics and renal

arteriolar morphology.

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DISCUSSION

In the present study we examined whether the effect of reducing

hyperuricemia with febuxostat during fructose-induced metabolic syndrome was

associated with preservation of glomerular hemodynamic function and

preglomerular vessel morphology. Administration of a 60% fructose diet to rats for

4 weeks induced features of metabolic syndrome such as hypertension,

hypertriglyceridemia and hyperuricemia as has been previously shown (33; 38).

Two groups of rats were maintained on a high-fructose diet for an additional 4

weeks, but only one group was then treated with the XO inhibitor febuxostat.

Similar to what has been shown with allopurinol (33), febuxostat treatment

reversed the hyperuricemia, the increases of both blood pressure and plasma

triglycerides, and also prevented the increase of fasting plasma insulin. While the

effect of febuxostat on SBP (measured by the tail-cuff method) was marginal at 8

weeks, when a separate group of rats was fed a high-fructose diet for a longer time

period (12 weeks), a more pronounced effect of lowering UA on reducing blood

pressure was observed. Moreover, glomerular hypertension, decreased

ultrafiltration coefficient and afferent arteriolopathy induced by a high-fructose diet

were also mitigated by febuxostat treatment. Therefore, the present results support

a causal role for UA in the pathogenesis of fructose-induced metabolic syndrome

and the accompanying renal damage.

Accumulating evidence dating back to the mid-1800s suggests that

metabolic syndrome and obesity epidemics parallel sugar and fructose intake (23).

Fructose is present in table sugar (sucrose), honey, high-fructose corn syrup

(HFCS), and fruit. The widespread use of HFCS, coupled with the continued intake

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of table sugar, has led to a 30% increase in fructose intake over the last 20 years

(14). Recent epidemiological analysis has found that greater intake of added

sugars or sugar-sweetened drinks is associated with higher plasma UA

concentration (18). Moreover, a graded positive association between

concentrations of serum UA and the prevalence of metabolic syndrome traits has

been described (11; 30); this relationship appears to apply to children and

adolescents as well (17).

Recent evidence indicates that metabolic syndrome is related to the

development of renal disease and is also a predictor of poor outcome in patients

with chronic renal failure (9). However, because of the considerable overlap

between clinical features of the metabolic syndrome and diabetes, a cause-effect

relationship cannot be clearly established from clinical and epidemiological studies.

One approach to identifying a distinction would be to study a model of primary

metabolic syndrome as exemplified by the fructose -fed rat. The results of the

present study support previous findings regarding the deleterious effect of

metabolic syndrome induced by a high-fructose diet on renal structure and function

(38). In addition, recent studies in humans have shown that metabolic syndrome is

associated with an increased risk for a reduced glomerular filtration rate (GFR) and

microalbuminuria (9; 20). Interestingly, it has been reported that patients with

essential hypertension and metabolic syndrome have glomerular hypertension and

increased albumin excretion (52).

Our group recently demonstrated that treatment of fructose-fed rats with

allopurinol, as well as with a uricosuric agent (benzodiarone), normalized plasma

UA and triglycerides and significantly decreased SBP, suggesting that UA plays a

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causal role in the pathogenesis of fructose-induced metabolic syndrome (33). The

primary importance of the current study is that we have now extended the studies

to investigate the effects of XO inhibition on renal hemodynamics and structure

associated with fructose ingestion. In addition we also demonstrated the ability of a

second XO inhibitor to improve features of the metabolic syndrome in this model;

thus treatment with febuxostat also normalized plasma UA and reduced

triglycerides, and the SBP measured in conscious animals showed a trend toward

reduction, whereas MAP measured in anesthetized animals at Week 8 was

significantly lower in febuxostat-treated animals on a high-fructose diet as

compared to the untreated rats.

In regard to the discrepancy between SBP and MAP in the 8-week study,

this phenomenon may derive from an inherent limitation of the tail-cuff method to

detect small-to-moderate changes in blood pressure (28); by extending the time to

12 weeks, when SBP differences could become greater, the tail-cuff method was

able to detect significant differences (Figure 2). Direct intra-arterial MAP, on the

other hand, is more sensitive and is likely the reason we could detect the

differences in blood pressure at 8 weeks.

There is evidence that reactive oxygen species (ROS) contribute to the

pathophysiology of metabolic syndrome. In this regard, fructose intake has been

associated with oxidative stress mediated by several different pathways: 1)

reduction of copper intestinal reabsorption and the resulting decrease in the activity

of Cu-Zn superoxide-dismutase (SOD) (8; 37); 2) diminished mRNA expression of

catalase, Cu-ZN SOD as well as Mn-SOD in several tissues (8); and 3) increased

activity of NADPH oxidase via angiotensin II type 1 receptor activation (35; 45).

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Fructose also increases xanthine oxidase activity (which is why it increases uric

acid) and hence increases xanthine-oxidase associated oxidants (5). However, in

aortic vessels of fructose-fed rats only NADPH oxidase inhibitors such as apocynin

and diphenylene iodinium were able to abrogate the increased synthesis of

superoxide; in contrast, the xanthine oxidase blocker oxypurinol did not show such

inhibitory effect (45). These findings suggest a primary role of NADPH oxidase

over-activation in the pathogenesis of fructose-induced metabolic syndrome.

Interestingly, uric acid may have a role in NADPH oxidase activation.

Recently our group reported that soluble uric acid stimulated an increase in

NADPH oxidase activity and ROS production in mature adipocytes but not in

preadipocytes (43). The stimulation of NADPH oxidase-dependent ROS by uric

acid resulted in activation of MAP kinases p38 and ERK1/2, a decrease in nitric

oxide bioavailability, and an increase in protein nitrosylation and lipid oxidation. In

fructose fed rats the primary sites metabolizing fructose (liver, intestine, kidney and

adipocytes) might therefore increase uric acid levels that could affect other

(vascular and endothelial) sites.

Previously, we demonstrated that elevated UA levels induced by a 60%

fructose diet in rats may be partially responsible for the glomerular hypertension

and cortical vasoconstriction induced by metabolic syndrome (38). In that study we

did not find evidence of glomerular or tubulointerstitial structural damage, nor was

any fructose-induced albuminuria detected. These observations suggest that, at

least at 8 weeks, the primary renal abnormalities induced by fructose are

hemodynamic in nature, with renal structural changes limited to the afferent

arteriole. This generalization is supported by the fact that normalization of plasma

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UA with febuxostat in the present study was associated with normal values of

glomerular pressure and conserved ultrafiltration coefficient and afferent arteriolar

morphology.

The mechanisms that contribute to renal damage during metabolic

syndrome are not completely understood. However, there exists clinical and/or

experimental evidence suggesting that endothelial dysfunction, oxidative stress,

serum lipids abnormalities and inflammatory cytokines synthesized by adipose

tissue may play a role (1; 27; 42; 50; 55). In this respect, there are studies

suggesting that UA participates in the development of these alterations. Thus,

increased UA decreases NO levels and induces endothelial dysfunction (24; 25);

UA stimulates NADPH oxidase with oxidant generation, reduction in NO levels, and

the formation of peroxynitrite in cultured adipocytes (43); and finally, hyperuricemia

closely correlates with hypertriglyceridemia (15; 16; 33) and predicts the

development of obesity (31).

In summary, hyperuricemia induced by a high-fructose diet was associated

with hypertension, hypertriglyceridemia and hyperinsulinemia as well as glomerular

hypertension, renal cortical arteriole vasoconstriction and preglomerular

arteriolopathy. Normalization of plasma UA with febuxostat in rats with fructose-

induced metabolic syndrome alleviated both metabolic and glomerular

hemodynamic alterations; these results support a pathogenic role of hyperuricemia

in the fructose-mediated metabolic syndrome and renal damage. Although more

studies are needed, the results provide a possible explanation by which metabolic

syndrome is associated with chronic renal disease.

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ACKNOWLEDGEMENTS

We thank Jose Santamaría, Magdalena Cristóbal and Benito Chávez-

Rentería for their technical assistance. We also wish to acknowledge our sincere

gratitude to the late Jaime Herrera-Acosta, M.D., for his contributions and

stimulating discussions during the conception of this project.

DISCLOSURES

This study was supported by a grant provided by TAP Pharmaceutical

Products Inc., Lake Forest, IL, USA. Lin Zhao is an employee of TAP

Pharmaceutical Products Inc. Dr. Johnson is listed as an inventor on several patent

applications by the University of Florida and the University of Washington related to

the lowering of uric acid or the inhibition of fructose as a means to prevent or treat

cardiorenal and obesity related diseases. Dr. Johnson will also have a book on

fructose and obesity that will be published in April 2008 (Rodale press). Dr.

Nakagawa is listed as inventor on patent applications by the University of Florida

related to the role of fructose in hypertension and metabolic syndrome.

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TABLE 1. Relevant nutrient content of the rodent experimental diets.

Constituent Normal diet Fructose diet

Energy (kcal/g) 3.1 3.3

Starch (%) 41.2 trace

Sucrose (%) 4.9 trace

Fructose (%) trace 60.0

Protein (%) 18.8 20.0

Fat (%) 6.0 5.0

Vitamins (%) 1.0 1.0

Minerals (%) 4.8 5.0

Normal diet: Cat No. 2018, Teklad Global, 18% protein rodent diet.

Fructose diet: Cat No. TD 78463. Harlan Teklad, Madison, WI, USA.

Nutrient composition presented as % of weight.

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TABLE 2. Effect of febuxostat on glomerular hemodynamics in normal and fructose-fed rats

Parameter Normal Diet

+ Placebo

(n=8)

Normal Diet

+ Febuxostat

(n=8)

Fructose Diet

+ Placebo

(n=8)

Fructose Diet

+ Febuxostat

(n=10)

Hct (%) 0.47±0.01 0.48±0.01 0.50±0.01 0.51±0.01

GFR (ml/min) 0.7±0.1 0.9±0.1 1.0±0.1 0.8±0.1

AR (dyn.s.cm-5) 2.6±0.9 2.5±0.2 3.5±0.4 2.7±0.2

ER (dyn.s.cm-5) 1.2±0.3 1.3±0.1 1.7±0.2 1.2±0.1

TR (dyn.s.cm-5) 3.8±1.1 3.8±0.3 5.2±0.6 3.9±0.3

Hct: hematocrit; GFR: glomerular filtration rate; AR: afferent arteriole resistance; ER: efferent arteriole resistance; TR: total resistance.

Two-way ANOVA: diet was a significant factor for Hct; diet, treatment and the interaction between diet and treatment were not

significant in the other parameters.

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TABLE 3. Effect of febuxostat on arteriolar morphology in normal and fructose-fed rats

Parameter Normal Diet

+ Placebo

(n=8)

Normal Diet

+ Febuxostat

(n=8)

Fructose Diet

+ Placebo

(n=8)

Fructose Diet

+ Febuxostat

(n=10)

Arteriolar Area (µm2) 263.6±12.6 259.4±11.2 332.3±12.8a 248.5±10.3b

Media/Lumen ratio 2.98±0.20 2.46±0.19 2.43±0.15 2.67±0.32

Two-way ANOVA: Diet, treatment and the interaction diet and treatment were significant in arteriolar area, but not in media/lumen

ratio.

Bonferroni post-test comparisons: a p<0.001 vs Normal Diet + Placebo group; b= p<0.001 vs Fructose Diet + Placebo group.

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FIGURE LEGENDS

FIGURE 1. Effects of a high-fructose diet and febuxostat on plasma uric acid (UA),

triglycerides (TG) and insulin (Ins), and on systolic blood pressure (SBP) in

conscious rats (two-way ANOVA for repeated measurements, n=10/group). Mean

arterial pressure (MAP) in anesthetized rats at Week 8 (standard two-way ANOVA,

n=8/group, except for Fructose Diet + Fx group, n=10). Two-way ANOVA for

repeated measurement: p<0.0001 for treatment and time interaction for UA, TG,

Ins and SBP. Two-way ANOVA: p=0.003 for diet and treatment interaction in MAP.

Bonferroni post-test comparisons: a, b p<0.05.

FIGURE 2. Effects of a high-fructose diet and febuxostat on systolic blood pressure

in two additional groups of rats fed a high-fructose diet for 14 weeks and treated

with placebo (P) or febuxostat (Fx) beginning on Week 5; *p<0.05 vs Fructose +

Placebo group by unpaired Student’s t-test.

FIGURE 3. Effects of a high-fructose diet and febuxostat on body weight and food

intake in rats (two-way ANOVA for repeated measurements, n=10/group). Two-way

ANOVA for repeated measurement: p<0.0001 for time and treatment interaction in

both parameters. Bonferroni post-test comparisons: a p<0.05.

FIGURE 4. Glomerular hemodynamics in rats administered high-fructose or normal

diets and treated with placebo or febuxostat. P = Placebo; Fx = Febuxostat.

n=8/group, except for Fructose Diet + Fx group (two-way ANOVA, n=10).

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Single nephron GFR ANOVA: Diet p=0.002, Treatment p=ns, Interaction p=ns.

Glomerular pressure ANOVA: Diet p=0.04, Treatment p=0.04, Interaction p=0.02.

Glomerular plasma flow ANOVA: Diet p=0.02, Treatment p=ns, Interaction p=ns.

Ultrafiltration coefficient ANOVA: Diet p=ns, Treatment p=ns, Interaction p=0.01.

Bonferroni post-test comparisons: a, b p<0.05.

FIGURE 5. Afferent arteriolar morphology in normal and fructose-fed rats treated

with placebo or febuxostat. Antibody anti-smooth muscle alpha-actin

counterstained with PAS (1000X). P = Placebo; Fx = Febuxostat.

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