Thiazide Diuretics Exacerbate Fructose-Induced Metabolic Syndrome

BASIC RESEARCH www.jasn.org

Sirirat Reungjui,* Carlos A. Roncal,* Wei Mu,* Titte R. Srinivas,* Dhavee Sirivongs,† Richard J. Johnson,* and Takahiko Nakagawa*

*Division of Nephrology, Hypertension and Transplantation, University of Florida, Gainesville, Florida; and †Division of Nephrology, Khon Kaen University, Khon Kaen, Thailand

ABSTRACT Fructose is a commonly used sweetener associated with diets that increase the prevalence of metabolic syndrome. Thiazide diuretics are frequently used in these patients for treatment of hypertension, but they also exacerbate metabolic syndrome. Rats on high-fructose diets that are given thiazides exhibit potassium depletion and hyperuricemia. Potassium supplementation improves their insulin resistance and hypertension, whereas allopurinol reduces serum levels of uric acid and ameliorates hypertension, hypertriglyceridemia, hyperglycemia, and insulin resistance. Both potassium supplementation and treatment with allopurinol also increase urinary nitric oxide excretion. We suggest that potassium depletion and hyperuricemia in rats exacerbates endothelial dysfunction and lowers the bioavailability of nitric oxide, which blocks insulin activity and causes insulin resistance during thiazide usage. Addition of potassium supplements and allopurinol with thiazides might be helpful in the management of metabolic syndrome.

J Am Soc Nephrol 18: 2624–2731, 2007. doi: 10.1681/ASN.2007040416

The metabolic syndrome (MS) is a constellation of adverse effects, they increase the incidence of new on- risk factors for cardiovascular disease and type 2 set of diabetes9 and can be associated with hypokale- diabetes and consists of abdominal obesity, hyper- mia, hyperuricemia, and hyperlipidemia.11 triglyceridemia, low HDL cholesterol, high BP, in- Understanding the precise mechanisms by which sulin resistance, and hyperglycemia.1,2 Endothelial HCTZ exacerbates MS is important. Some evidence dysfunction and hyperuricemia also are closely as- suggests that thiazide-induced hypokalemia may me- sociated with MS.3,4 diate insulin resistance.12,13 In addition, experimental Hydrochlorothiazide (HCTZ) is beneficial in hyperuricemia can cause endothelial dysfunction,3,14 patients with hypertension because it reduces mor- hypertension,15 and hyperinsulinemia.3,16 Further- bidity and mortality, especially the frequency of more, we recently reported that hyperuricemia causes stroke and congestive heart failure.5,6 As a result, the development of MS, and allopurinol, which lowers thiazides are recommended as the first-line therapy uric acid levels, improves these features of MS in fruc- for hypertension.6 However, many patients with tose (F)-fed rats.3 We therefore hypothesized that hy- hypertension have MS. In turn, HCTZ usage, although critical in the management of hypertension, can have several adverse effects, such as electrolyte Received April 6, 2007. Accepted June 20, 2007. disorders (hypokalemia, hyponatremia, and hypo- Published online ahead of print. Publication date available at magnesemia), hyperuricemia, hyperlipidemia, and www.jasn.org. impairment of glucose metabolism in addition to Correspondence: Dr. Sirirat Reungjui, current address is Division volume depletion.7–10 These adverse effects result in of Nephrology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand 40002. Phone: ϩ66-43-363746; Fax: ϩ66-43- the development or exacerbation of MS. Although 347542; E-mail: [email protected] low-dosage thiazides have led to a reduction of these Copyright © 2007 by the American Society of Nephrology

2724 ISSN : 1046-6673/1810-2624 J Am Soc Nephrol 18: 2624–2731, 2007 www.jasn.org BASIC RESEARCH pokalemia and hyperuricemia may be primary mechanisms by which the thiazides facilitate the MS and that acceleration of MS by thiazides may be prevented by potassium (K) supplementation and allopurinol. In this study, we administered thiazides to rats with MS, because this syndrome is common in patients with hypertension and it is important to determine whether thiazides can exacerbate its features. Although there are various models of MS in animals, we selected the F-induced model of MS. In- deed, the increasing incidence of MS in humans in the past two decades coincides with a marked increase in F intake.17,18 Im- portantly, that high F consumption causes features of the MS has been documented in clinical studies.19–21 Similarly, high-F diets cause MS in rodents, including hypertension, insulin re- Figure 1. SBP at weeks 4 and 16. The rats receiving F develop sistance, and hypertriglyceridemia.3,22,23 In this study, we hy- hypertension. HCTZ reduces the SBP. KCL further reduces BP at pothesized that HCTZ use exacerbates F-induced MS and ex- week 16. Similarly, allopurinol reduces BP reduction at both weeks 4 and 16. Data are means Ϯ SD. P at least Ͻ0.05 *versus normal diet, amined the role of hypokalemia and hyperuricemia in this &versus F, $versus FϩHCTZ, and #versus FϩHCTZϩKCL. process.

RESULTS HCTZ Reduced Systolic BP but Aggravated F-Induced MS Body Weights HCTZ use reduced systolic BP (SBP) in the FϩHCTZ group All rats were pair fed to eliminate effects of food intake on the compared with the F group at weeks 4 and 16 (Figure 1). The results, which can influence glucose, insulin, and triglyceride level of serum K was significantly lower in the FϩHCTZ and levels. Although this will not allow us to determine whether FϩHCTZϩallopurinol groups at week 20, whereas all thiazides can cause weight gain because access was not ad libi- HCTZ-treated rats also developed hypomagnesemia (Table tum, it did allow us to determine whether thiazides alter tri- Ϫ 1). An increase of serum HCO3 was also observed (F glyceride and glucose levels independent of effects on food 23.0 Ϯ 0.9; FϩHCTZ 24.7 Ϯ 1.1; FϩHCTZϩKCL 25.6 Ϯ intake. As such, the average body weights were similar in all 1.8; FϩHCTZϩallopurinol 25.7 Ϯ 1.5 mEq/L; all P Ͻ0.05 groups at week 20, as shown in Table 1. versus the F group). HCTZ use did not exacerbate MS early at week 4; the met- F-Induced Features of MS in Rats abolic profile of the F group and the FϩHCTZ group was sim- At week 4, the F-fed rats developed early features of MS, in- ilar at this time. However, HCTZ aggravated insulin resistance cluding hypertension (Figure 1), hypertriglyceridemia (nor- (Figure 3) and hyperuricemia at week 14 (F 2.2 Ϯ 0.5 versus Ϯ Ϯ Ͻ mal diet 125 55 versus F 325 104 mg/dl; P 0.001) and FϩHCTZ 2.7 Ϯ 0.5 mg/dl; P ϭ 0.04) and at week 20 (F 2.2 Ϯ Ϯ Ϯ hyperuricemia (normal diet 1.7 0.3 versus F 2.2 0.4 mg/dl; 0.6 versus FϩHCTZ 2.8 Ϯ 0.6 mg/dl; P ϭ 0.02). Serum glucose ϭ P 0.01). Serum cholesterol was significantly higher at week was also increased by HCTZ at week 14 (F 150 Ϯ 10 versus 14 (Figure 2). No significant difference in serum glucose was FϩHCTZ 176 Ϯ 13 mg/dl; P ϭ 0.006) and at week 20 (F 160 Ϯ observed between the F group and the normal diet group (Fig- 26 versus FϩHCTZ 186 Ϯ 22 mg/dl; P ϭ 0.02). Although se- ure 2). ϩ Table 1. Renal function, serum K, serum Mg2 , and urinary uric acid excretion evaluated at week 20 of the study F ؉ HCTZ ؉ F ؉ HCTZ ؉ Groups N F F ؉ HCTZ KCL Allopurinol Body weight (g) 543 Ϯ 45 565 Ϯ 66 560 Ϯ 88 558 Ϯ 58 557 Ϯ 89 BUN (mg/dL) 13.8 Ϯ 1.4 13 Ϯ 2.6 26.6 Ϯ 14a,b 25.3 Ϯ 6.6a,b 22.5 Ϯ 6.4a,b Cr (mg/dL) 0.44 Ϯ 0.05 0.43 Ϯ 0.05 0.46 Ϯ 0.11 0.44 Ϯ 0.07 0.44 Ϯ 0.05 Serum K (mEq/L) 4.4Ϯ 0.2 4.3 Ϯ 0.1 4.0 Ϯ 0.1a,b,d 4.5 Ϯ 0.3 4.1 Ϯ 0.1a,b,d ϩ Serum Mg2 (mg/dL) 1.6 Ϯ 0.28 1.5 Ϯ 0.23 1.15 Ϯ 0.1a,b 1.09 Ϯ 0.1a,b 1.1 Ϯ 0.1a,b Urine protein/Cr 0.24 Ϯ 0.4 2.29 Ϯ 2.5a 2.53 Ϯ 2.3a 3.18 Ϯ 2.8a 2.86 Ϯ 2.4a Urine uric acid (mg/d) 2.42 Ϯ 0.4 4.07 Ϯ 0.8a 2.93 Ϯ 1.0b 3.67 Ϯ 0.9a 3.38 Ϯ 1.1a Uric acid clearance (ml/min) 0.09 Ϯ 0.02 0.13 Ϯ 0.02a 0.08 Ϯ 0.03b 0.11 Ϯ 0.3 0.17 Ϯ 0.05a,c,d Data are expressed as meanϮ SD. N, normal diet group; F, fructose diet group; FϩHCTZ, fructose diet and hydrochlorothiazide group; FϩHCTZϩKCL, fructose diet and hydrochlorothiazide plus potassium chloride group; FϩHCTZϩAllopurinol, fructose diet and hydrochlorothiazide plus allopurinol group. All P values are at least Ͻ0.05. aP versus normal diet; bP versus fructose diet; cP versus FϩHCTZ; dP versus Fϩ HCTZϩKCL.

J Am Soc Nephrol 18: 2624–2731, 2007 Thiazides and Metabolic Syndrome 2725 BASIC RESEARCH www.jasn.org

ϩKCL rats at week 16 (FϩHCTZ 136 Ϯ 4 versus FϩHCTZ- ϩKCL 131 Ϯ 5 mmHg; P ϭ 0.04), whereas no effect was observed at week 4 (FϩHCTZ 129 Ϯ 5 versus FϩHCTZϩKCL 127 Ϯ 3 mmHg; P Ͼ 0.05). By insulin tolerance test, serum glucose was not decreased despite insulin administration in the FϩHCTZ group, whereas insulin-induced reduction of serum glucose was observed in the FϩHCTZϩKCL group (Figure 4). These data suggest that K supplementation improves insulin sensitivity, which was deteriorated by fructose with HCTZ. Compatibly, K supplementation tended to lower serum glucose at week 20 (FϩHCTZ 186 Ϯ 22 versus FϩHCTZϩKCL 166 Ϯ 20 mmHg; P ϭ 0.06). No significant change of serum uric acid and serum triglycerides was observed in the FϩHC- TZϩKCL group compared with the FϩHCTZ group.

Lowering Uric Acid with Allopurinol Improved Hypertension, Hypertriglyceridemia, Hyperglycemia, Figure 2. Time courses of serum uric acid (A), serum glucose (B), and Insulin Resistance serum cholesterol (C), and serum triglycerides (D). The rats receiving Allopurinol treatment significantly reduced the level of serum F developed hyperuricemia and hypertriglyceridemia throughout uric acid in the FϩHCTZ group. In addition, allopurinol sig- the study. Hypercholesterolemia was detected at week 14. The nificantly reduced SBP (Figure 1), and serum triglycerides combination of HCTZ with F causes more hyperuricemia and hyper- were also significantly lower than the FϩHCTZ group at weeks glycemia as shown at weeks 14 and 20. Serum cholesterol and triglyceride are numerically higher with HCTZ use but did not reach 4, 14, and 20 (Figure 2). statistical significance. KCL tends to reduce serum glucose at week Insulin resistance was improved by allopurinol (Figures 3 20 (P ϭ 0.06). Allopurinol treatment significantly reduces serum uric and 4). In addition, serum glucose was significantly lower at acid and triglycerides at all time points and serum glucose at week week 20 (FϩHCTZ 186 Ϯ 22 versus FϩHCTZϩallopurinol 20. Data are means Ϯ SD. P at least Ͻ0.05 *versus normal diet, 166 Ϯ 14 mg/dl; P ϭ 0.04). &versus F, $versus FϩHCTZ, and #versus FϩHCTZϩKCL. To investigate further the role of uric acid, we examined the correlation between uric acid and other factors. When individ- ual data on rats were examined at week 20, serum uric acid positively correlated with serum triglycerides, serum cholesterol, and serum glucose (Figure 5). Furthermore, there was a significant correlation between serum uric acid and SBP at week 4 (r ϭ 0.53, P ϭ 0.003) and serum insulin at week 14 (r ϭ 0.42, P ϭ 0.009).

Insulin and Other Factors At week 14, serum insulin concentrations in normal, F, FϩHCTZ,

Figure 3. QUICKI at week 14. The combination of FϩHCTZ significantly lowers insulin sensitivity than the normal diet or the F. Treatment with allopurinol improves insulin sensitivity. KCL tends to improve insulin sensitivity, but this was NS. Data are means Ϯ SD. P Ͻ 0.05 *versus normal diet, &versus F, and $versus FϩHCTZ.

Figure 4. The insulin tolerance test at week 18. Blood glucose rum triglycerides were higher in the FϩHCTZ group, they did levels were not lowered by insulin in rats receiving F with or not reach statistical significance (Figure 2). without HCTZ, consistent with insulin resistance. In contrast, KCL and allopurinol treatment improve the insulin response similar to K Supplementation Reduced SBP and Improved Insulin that observed with the normal diet group. Data are means Ϯ SD. Resistance P at least Ͻ0.05 *versus normal diet, &versus fructose diet, and K supplementation significantly reduced SBP in FϩHCTZ- $versus FϩHCTZ.

2726 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 2624–2731, 2007 www.jasn.org BASIC RESEARCH

FϩHCTZϩKCL, and FϩHCTZϩallopurinol groups were higher urinary uric acid excretion per day and uric acid clear- 1846 Ϯ452, 2561 Ϯ1286, 3449 Ϯ1200, 3058 Ϯ1514, and 2319 Ϯ ance compared with the normal group (Table 1), which sug- 971 pg/ml, respectively. Significant differences of serum insulin gested an increase of uric acid production. Similar to that ob- were detected between FϩHCTZ and normal (P ϭ 0.002) and served in humans, HCTZ enhanced hyperuricemia (Figure 2), borderline significance between FϩHCTZ and FϩHCTZϩallop- which could be attributed to a reduction of urinary uric acid urinol (P ϭ 0.05). Serum insulin levels positively correlated not excretion (Table 1). Allopurinol acutely reduced 32.3% of uri- only with serum glucose but also with serum triglycerides and nary uric acid excretion compared with the FϩHCTZ group serum cholesterol (Figure 5), suggesting an association between during the first week (P Ͻ 0.05), which could be due to a insulin resistance and dyslipidemia.24,25 reduced production of uric acid by allopurinol. However, it is of note that urinary uric acid excretion and uric acid clearance Renal Function and Urinary Excretion of Sodium, K, increased during the chronic phase despite allopurinol use and Uric Acid (Table 1). Although there was no significant difference in serum creatinine between groups, an increased urinary protein excretion K Supplementation or Allopurinol Increase Urinary by F suggests renal dysfunction. However, an elevation of Nitric Oxide Excretion blood urea nitrogen (BUN) in all HCTZ-treated groups Urine nitrate/nitrite excretion is a marker of nitric oxide (NO) (groups 3, 4, and 5) could be attributed to volume depletion bioavailability as well as endothelial function.26,27 F decreased induced by HCTZ, because F did not raise BUN. urine nitrate/nitrites, which were further lowered by HCTZ. With respect to urinary electrolytes, HCTZ significantly in- However, K supplementation or allopurinol treatment increased daily urinary sodium excretion at week 1 (normal creased urinary nitrate/nitrite excretion (Figure 6). 0.4 Ϯ 0.2; F 0.5 Ϯ 0.1; FϩHCTZ 0.8 Ϯ 0.4; FϩHCTZϩKCL 0.8 Ϯ 0.2; and FϩHCTZϩallopurinol 0.9 Ϯ 0.3 mEq/d; all P Ͻ 0.05 versus normal or F). Urinary K excretion was also signifi- DISCUSSION cantly higher in the FϩHCTZ group (1.5 Ϯ 0.4 mEq/d) and the FϩHCTZϩallopurinol group (1.4 Ϯ 0.3 mEq/d) as compared The principal new finding in this study is that we can show with the F group (1.2 Ϯ 0.1 mEq/d). Because of K supplemen- experimentally that correcting the serum K and uric acid ab- tation, the FϩHCTZϩKCL group had normal serum K level normalities in F-induced MS in rats can largely prevent the with high urine K (3.1 Ϯ 1.1). metabolic abnormalities that are associated with thiazides. The The F group developed hyperuricemia (Figure 2) with beneficial effects of these treatments were associated with an increase in urine nitrite/nitrates, suggesting the involvement of endothelial dysfunction on the development of MS with thiazide usage. Hypokalemia occurs in 6.5 to 50% of patients receiving diuretics,28–30 with the average reduction of serum K from thiazides reported at approximately 0.3 to 1.1 mEq/L.6,31,32 Previ- ous studies suggested that hypokalemia may be a factor causing hyperglycemia, hyperinsulinemia, and insulin resistance in pa-

Figure 6. Urine nitrate/nitrite level. The levels of urinary nitrate and nitrite in rats receiving F are lower than that observed in the normal diet group. HCTZ induces a further reduction of urinary nitrate and nitrite. KCL and allopurinol treatment increases the level of urinary nitrate and nitrite compared with the FϩHCTZ. Figure 5. Correlations of various metabolic factors in fructose/ Data are means Ϯ SD. P at least Ͻ0.05 *versus normal diet, HCTZ-induced MS in the rat. &versus F, and $versus FϩHCTZ.

J Am Soc Nephrol 18: 2624–2731, 2007 Thiazides and Metabolic Syndrome 2727 BASIC RESEARCH www.jasn.org tients receiving thiazides, because these features can be im- for this discrepancy is dosage. For example, when 1000 kcal proved with K supplements.13,33 It also has been shown that K is given, insulin resistance develops in 1 wk19; when 864 depletion, even without frank hypokalemia, can cause insulin kcal/d is consumed, insulin resistance is observed only in resistance.34 In our experiments, HCTZ produced a significant organs with high fructokinase activity (liver, fat cells) and reduction in serum K (0.3 mEq/L), although this did not reach takes 4 wk20; and when lower dosages are given, no insulin the criteria of hypokalemia (serum K Ͻ3.5 mEq/L). Our im- resistance is observed, even after 1 mo.21 Similar situations portant finding is that this mild K depletion was significantly occur in the rat. With high dosages (60% F), insulin resis- associated with exacerbation of hyperglycemia, insulin resistance occurs within 4 to 8 wk3; with dosages similar to that tance, and a reduction of urine NO excretion, all of which were in the current American diet (15% total energy intake), it corrected by K supplementation. The observation that K sup- takes slightly more than 1 yr.44 However, the rat is more plements prevented the reduction of urine NO in F-fed rats resistant because it has low uric acid levels; indeed, if uricase receiving HCTZ is consistent with an improvement in endo- is inhibited, then 20% F will induce hyperinsulinemia rap- thelial function. K supplementation has been shown to act as idly.45 Furthermore, F, by virtue of increasing the circula- an endothelium-derived hyperpolarizing factor35 and release tion of fatty acids, may lead to the ectopic deposition of fat NO to preserve endothelial function,36 whereas K depletion in liver and skeletal muscle that will cause insulin resistance was found to attenuate endothelial-dependent vasorelax- indirectly.46 This indirect induction of insulin resistance by ation.37 In turn, an inhibition of endothelial NO is known to F could also account for the discrepancy in some of these cause insulin resistance.38 studies. Because F and its metabolites fructose-3-phosphate Unlike glucose, F is the only sugar that rapidly increases and 3-deoxyglucosone cause oxidative stress and nonenzy- serum uric acid in humans as well as rodents.3 Recently, we matic glycation,47 the polyol pathway could also be involved found that F-induced hyperuricemia has a causal role in the in development of MS. pathogenesis of MS.3 This study demonstrated that HCTZ en- This study demonstrates that HCTZ aggravates the MS in hanced hyperuricemia in rats receiving a high-F diet by reduc- F-fed rats and that K supplementation and reducing uric acid ing urinary uric acid excretion, although F likely increases the levels may provide some protection. Nevertheless, it will be production of uric acid. Decreased urinary uric acid excretion important to determine whether similar protection can be pro- could be due to an increase in uric acid reabsorption in prox- vided by these maneuvers in humans. In this regard, we are imal tubule, which may be mediated by thiazide-induced vol- involved in a randomized, controlled trial to determine ume contraction.39 whether lowering of uric acid will improve features of MS as- Another important finding is that the lowering of serum sociated with thiazide use in black individuals with stage 1 uric acid by allopurinol was associated with an improvement of hypertension. the features of MS. We also found that a lowering of uric acid by allopurinol was associated with an increase in urinary NO excretion. Given that uric acid inhibits endothelial NO bio- CONCISE METHODS availability as well as endothelial function,3,14,40 a lowering of All animal studies were approved by the University of Florida Insti- uric acid could improve endothelial function in this model. tutional Animal Use and Care Committee. Collectively, these findings suggest that hyperuricemia, by virtue of reducing NO, plays a role in the pathogenesis of the MS Pilot Studies aggravated with HCTZ. Because variable dosages of HCTZ have been commonly used in The inhibition of uric acid production by allopurinol other studies (e.g., 3 to 80 mg/kg per d48,49), we performed pilot likely accounts for the reduction of urinary uric acid excre- studies to determine the minimum adequate dosage of HCTZ for tion during the first week. However, with long-term allo- this study. Because the aim of study was to examine the role of purinol treatment, the reduction in uric acid excretion was hypokalemia on adverse effects of HCTZ, we identified the lowest not observed. Although the precise mechanism remains un- dosage that can reduce BP along with a mild reduction of serum K. known, hyperuricemic rats are known to develop renal va- We therefore gave three different dosages of HCTZ (Sigma-Al- soconstriction with a reduction of renal blood flow, and this drich, St. Louis, MO) to normal rats. As a result, we found that 10 is reversed by allopurinol.41 In turn, an increase in renal mg/kg per d HCTZ is the lowest dosage that reduced BP and in- blood flow will result in increased uric acid excretion.42 In duced mild hypokalemia. this scenario, by improving endothelial dysfunction and renal blood flow, the lowering of uric acid could paradoxically Experimental Protocol enhance excretion. Male Sprague-Dawley rats (150 to 200 g; Charles River, Wil- The effect of high F intake on insulin sensitivity is still mington, MA) were placed on a standard diet (Harlan, Madi- debated. As recently reviewed,43 some groups have observed son, WI) for a 5-d run-in period, then the rats were divided that healthy individuals develop insulin resistance after high into five groups (n ϭ 8) with similar body weight and baseline F consumption, whereas others have reported no effect on blood chemistry: Group 1: Normal standard diet (Harlan); glucose metabolism as well as insulin sensitivity. One reason group 2 (F): 60% fructose diet (Harlan); group 3 (FϩHCTZ):

2728 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 2624–2731, 2007 www.jasn.org BASIC RESEARCH

In addition to 60% fructose diet, HCTZ 10 mg/kg per d was Statistical Analyses supplied in drinking water; group 4 (FϩHCTZϩKCL): 60% F All data are shown as means Ϯ SD. One-way ANOVA (SPSS 14.0; diet with HCTZ and 1% K chloride (KCL) in drinking water; SPSS, Chicago, IL) and post hoc multiple comparisons were used to the concentration of KCL in drinking water was increased at determine the significance between the mean of multiple groups with weeks 5 (1.5% KCL), 15 (1.75% KCL), and 17 (2% KCL) to the least-significant difference test for equal and Dunnett test for un- ϩ maintain normal serum K level through the whole study; and equal variances. The homogeneity of variance was clarified by Levene group 5 (FϩHCTZϩallopurinol): 60% F diet plus HCTZ and test. The paired and unpaired t tests were used to compare the con- allopurinol 150 mg/L (Sigma-Aldrich) in drinking water. tinuous variables of the specific two groups. Pearson correlation was We pair-fed rats to ensure equivalent caloric intake, thereby used to address potential associations between groups. Statistical sig- avoiding the influence of different food intake on the meta- nificance was defined as P Ͻ 0.05. bolic abnormalities. Body weight was measured weekly. At weeks 1, 4, 14, and 20, metabolic cages were used for urine collection overnight, where water but no food was freely ac- cessed. After 4 h of fasting, serum was also collected from the ACKNOWLEDGMENTS tail vein at weeks 4, 14, and 20. At the end of week 20, rats were killed. The work was supported by funding from the National Institutes of Health grants DK-52121, HL-68607, and HL-79352 and funds from Tail-Cuff BP Measurement Gatorade. S.R. is supported by a fellowship from the Anandamahidol SBP was measured in conscious rats with tail-cuff sphygmo- Foundation of Thailand. manometer (Visitech BP2000, Apex, NC) at weeks 4 and 16 as described previously.3 DISCLOSURES Biochemical Measurements T.N., S.R., and R.J.J. are listed as inventors on several patent applications Serum and urine K concentrations were determined with the atomic related to uric acid and cardiovascular disease from the University of Florida or absorption spectrophotometer (Perkin-Elmer 306, Downers Grove, University of Washington. R.J.J. is also on the Scientific Board of Nephromics, IL). By use of an autoanalyzer (VetAce; Alfa Wassermann, West Cald- Inc. well, NJ), the routine chemistries including glucose, cholesterol, triglycerides, uric acid, BUN, and creatinine were measured in serum. In addition, urine protein concentration and urine creatinine were de- REFERENCES termined.

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48. Webb RL, Navarrete AE, Davis S: Effects of valsartan and hydrochlo- 50. Straczkowski M, Stepien A, Kowalska I, Kinalska I: Comparison of rothiazide alone and in combination on blood pressure and heart rate simple indices of insulin sensitivity using the euglycemic hyperinsu- in conscious-telemetered spontaneously hypertensive rats (SHR). Am J linemic clamp technique. Med Sci Monit 10: CR480–CR484, 2004 Hypertens 11: 59–65, 1998 49. Kobayashi H, Sano T, Tarazi RC, Fouad-Tarazi FM: Effects of antihypertensive drugs on heart and resistance vessels. Cardiovasc Res 24: 137–143, 1990 See related editorial, ЉThe Fructose Nation,Љ on pages 2619–2621.

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