No development of hypertension in the hyperuricemic liver-Glut9 knockout mouse
2015; Elsevier BV; Volume: 87; Issue: 5 Linguagem: Inglês
10.1038/ki.2014.385
ISSN1523-1755
AutoresFrédéric Preitner, Anabela Pimentel, Salima Metref, Corinne Berthonneche, Alexandre Sarre, Catherine Moret, Samuel Rotman, Gabriel Centeno, Dmitri Firsov, Bernard Thorens,
Tópico(s)Case Reports on Hematomas
ResumoUrate is the metabolic end point of purines in humans. Although supra-physiological plasma urate levels are associated with obesity, insulin resistance, dyslipidemia, and hypertension, a causative role is debated. We previously established a mouse model of hyperuricemia by liver-specific deletion of Glut9, a urate transporter that provides urate to the hepatocyte enzyme uricase. These LG9 knockout mice show mild hyperuricemia (120 μmol/l), which can be further increased by the urate precursor inosine. Here, we explored the role of progressive hyperuricemia on the cardiovascular function. Arterial blood pressure and heart rate were periodically measured by telemetry over 6 months in LG9 knockout mice supplemented with incremental amounts of inosine in a normal chow diet. This long-term inosine treatment elicited a progressive increase in uricemia up to 300 μmol/l; however, it did not modify heart rate or mean arterial blood pressure in LG9 knockout compared with control mice. Inosine treatment did not alter cardiac morphology or function measured by ultrasound echocardiography. However, it did induce mild renal dysfunction as revealed by higher plasma creatinine levels, lower glomerular filtration rate, and histological signs of chronic inflammation and fibrosis. Thus, in LG9 knockout mice, inosine-induced hyperuricemia was not associated with hypertension despite partial renal deficiency. This does not support a direct role of urate in the control of blood pressure. Urate is the metabolic end point of purines in humans. Although supra-physiological plasma urate levels are associated with obesity, insulin resistance, dyslipidemia, and hypertension, a causative role is debated. We previously established a mouse model of hyperuricemia by liver-specific deletion of Glut9, a urate transporter that provides urate to the hepatocyte enzyme uricase. These LG9 knockout mice show mild hyperuricemia (120 μmol/l), which can be further increased by the urate precursor inosine. Here, we explored the role of progressive hyperuricemia on the cardiovascular function. Arterial blood pressure and heart rate were periodically measured by telemetry over 6 months in LG9 knockout mice supplemented with incremental amounts of inosine in a normal chow diet. This long-term inosine treatment elicited a progressive increase in uricemia up to 300 μmol/l; however, it did not modify heart rate or mean arterial blood pressure in LG9 knockout compared with control mice. Inosine treatment did not alter cardiac morphology or function measured by ultrasound echocardiography. However, it did induce mild renal dysfunction as revealed by higher plasma creatinine levels, lower glomerular filtration rate, and histological signs of chronic inflammation and fibrosis. Thus, in LG9 knockout mice, inosine-induced hyperuricemia was not associated with hypertension despite partial renal deficiency. This does not support a direct role of urate in the control of blood pressure. Urate is a product of purine metabolism.1Hediger M.A. Johnson R.J. Miyazaki H. et al.Molecular physiology of urate transport.Physiology (Bethesda). 2005; 20: 125-133Crossref PubMed Scopus (274) Google Scholar,2So A. Thorens B. Uric acid transport and disease.J Clin Invest. 2010; 120: 1791-1799Crossref PubMed Scopus (553) Google Scholar In most mammals, urate is further metabolized to allantoin by hepatic enzyme uricase, leading to low plasma urate levels (30–60 μmol/l in mice). By contrast, humans and great apes lost uricase expression during evolution and therefore have higher plasma urate levels. In humans, plasma urate levels gradually increased from ∼200 μmol/l in the 1920s to 350 μmol/l in the 1970s, in parallel with the increased intake of foods known to increase urate levels, including fructose and purine-rich foods (meat, seafood, and beer).3Choi H.K. Atkinson K. Karlson E.W. et al.Purine-rich foods, dairy and protein intake, and the risk of gout in men.N Engl J Med. 2004; 350: 1093-1103Crossref PubMed Scopus (800) Google Scholar,4Choi J.W. Ford E.S. Gao X. et al.Sugar-sweetened soft drinks, diet soft drinks, and serum uric acid level: the Third National Health and Nutrition Examination Survey.Arthritis Rheum. 2008; 59: 109-116Crossref PubMed Scopus (315) Google Scholar The evolutionary advantage of high urate levels remains highly debated. Urate is an antioxidant in cell-free systems. Thus, uricase mutations and higher uricemia were postulated to provide a survival advantage by reducing oxidative stress.5Ames B.N. Cathcart R. Schwiers E. et al.Uric acid provides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer: a hypothesis.Proc Natl Acad Sci USA. 1981; 78: 6858-6862Crossref PubMed Scopus (2312) Google Scholar In line with this, longevity among primates highly correlates with serum and brain urate levels.6Cutler R.G. Urate and ascorbate: their possible roles as antioxidants in determining longevity of mammalian species.Arch Gerontol Geriatr. 1984; 3: 321-348Abstract Full Text PDF PubMed Scopus (108) Google Scholar Urate and/or its precursors are neuroprotective in vitro7Weisskopf M.G. O'Reilly E. Chen H. et al.Plasma urate and risk of Parkinson's disease.Am J Epidemiol. 2007; 166: 561-567Crossref PubMed Scopus (313) Google Scholar and in vivo in mice,8Hooper D.C. Spitsin S. Kean R.B. et al.Uric acid, a natural scavenger of peroxynitrite, in experimental allergic encephalomyelitis and multiple sclerosis.Proc Natl Acad Sci USA. 1998; 95: 675-680Crossref PubMed Scopus (493) Google Scholar,9Scott G.S. Spitsin S.V. Kean R.B. et al.Therapeutic intervention in experimental allergic encephalomyelitis by administration of uric acid precursors.Proc Natl Acad Sci USA. 2002; 99: 16303-16308Crossref PubMed Scopus (119) Google Scholar and the presence of high plasma urate levels in humans is predictive of a lower risk for Parkinson's disease.7Weisskopf M.G. O'Reilly E. Chen H. et al.Plasma urate and risk of Parkinson's disease.Am J Epidemiol. 2007; 166: 561-567Crossref PubMed Scopus (313) Google Scholar Also, urate infusion in humans acutely improves endothelial function.10Waring W.S. Convery A. Mishra V. et al.Uric acid reduces exercise-induced oxidative stress in healthy adults.Clin Sci (Lond). 2003; 105: 425-430Crossref PubMed Scopus (212) Google Scholar,11Waring W.S. McKnight J.A. Webb D.J. et al.Uric acid restores endothelial function in patients with type 1 diabetes and regular smokers.Diabetes. 2006; 55: 3127-3132Crossref PubMed Scopus (165) Google Scholar However, soluble urate causes intracellular oxidative stress in vitro in adipocytes and in vascular smooth cells.12Corry D.B. Eslami P. Yamamoto K. et al.Uric acid stimulates vascular smooth muscle cell proliferation and oxidative stress via the vascular renin-angiotensin system.J Hypertens. 2008; 26: 269-275Crossref PubMed Scopus (551) Google Scholar,13Baldwin W. McRae S. Marek G. et al.Hyperuricemia as a mediator of the proinflammatory endocrine imbalance in the adipose tissue in a murine model of the metabolic syndrome.Diabetes. 2011; 60: 1258-1269Crossref PubMed Scopus (313) Google Scholar Moreover, highly proinflammatory uric acid crystals can form in persistent clinical hyperuricemia that is often associated with gout, kidney stones, and nephropathies.1Hediger M.A. Johnson R.J. Miyazaki H. et al.Molecular physiology of urate transport.Physiology (Bethesda). 2005; 20: 125-133Crossref PubMed Scopus (274) Google Scholar,2So A. Thorens B. Uric acid transport and disease.J Clin Invest. 2010; 120: 1791-1799Crossref PubMed Scopus (553) Google Scholar Hyperuricemia is a risk marker for renal and cardiovascular diseases in patients with diabetes, hypertension, and heart failure.14Feig D.I. Kang D.H. Johnson R.J. Uric acid and cardiovascular risk.N Engl J Med. 2008; 359: 1811-1821Crossref PubMed Scopus (1754) Google Scholar,15Hayden M.R. Tyagi S.C. Uric acid: a new look at an old risk marker for cardiovascular disease, metabolic syndrome, and type 2 diabetes mellitus: the urate redox shuttle.Nutr Metab (Lond). 2004; 1: 10Crossref PubMed Scopus (319) Google Scholar,16Mene P. Punzo G. Uric acid: bystander or culprit in hypertension and progressive renal disease?.J Hypertens. 2008; 26: 2085-2092Crossref PubMed Scopus (102) Google Scholar However, in vivo evidence for causation remains scarce and highly controversial.17Bobulescu I.A. Moe O.W. Renal transport of uric acid: evolving concepts and uncertainties.Adv Chronic Kidney Dis. 2012; 19: 358-371Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar,18Johnson R.J. Sanchez-Lozada L.G. Mazzali M. et al.What are the key arguments against uric acid as a true risk factor for hypertension?.Hypertension. 2013; 61: 948-951Crossref PubMed Scopus (52) Google Scholar In rats, urate elevation (by oxonate-induced uricase inhibition) induced over weeks an elevation of blood pressure, a form of salt-sensitive hypertension, and renal injuries.19Mazzali M. Hughes J. Kim Y.G. et al.Elevated uric acid increases blood pressure in the rat by a novel crystal-independent mechanism.Hypertension. 2001; 38: 1101-1106Crossref PubMed Scopus (1030) Google Scholar,20Mazzali M. Kanellis J. Han L. et al.Hyperuricemia induces a primary renal arteriolopathy in rats by a blood pressure-independent mechanism.Am J Physiol Renal Physiol. 2002; 282: F991-F997Crossref PubMed Scopus (689) Google Scholar In a pilot clinical trial of 30 adolescents with primary hypertension and borderline hyperuricemia, blood pressure was decreased by the hypouricemiant drug allopurinol (that blocks the production of urate by xanthine oxidase (XO)).21Feig D.I. Soletsky B. Johnson R.J. Effect of allopurinol on blood pressure of adolescents with newly diagnosed essential hypertension: a randomized trial.JAMA. 2008; 300: 924-932Crossref PubMed Scopus (742) Google Scholar However, these results may not be generalized to adult hypertensive patients.17Bobulescu I.A. Moe O.W. Renal transport of uric acid: evolving concepts and uncertainties.Adv Chronic Kidney Dis. 2012; 19: 358-371Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar,22Feig D.I. Madero M. Jalal D.I. et al.Uric acid and the origins of hypertension.J Pediatr. 2013; 162: 896-902Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Genome-wide association studies recently identified polymorphisms in SLC2A9 encoding the urate transporter Glut9 as strong determinants of urate levels in humans.23Dehghan A. Kottgen A. Yang Q. et al.Association of three genetic loci with uric acid concentration and risk of gout: a genome-wide association study.Lancet. 2008; 372: 1953-1961Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar,24Doring A. Gieger C. Mehta D. et al.SLC2A9 influences uric acid concentrations with pronounced sex-specific effects.Nat Genet. 2008; 40: 430-436Crossref PubMed Scopus (339) Google Scholar,25Li S. Sanna S. Maschio A. et al.The GLUT9 gene is associated with serum uric acid levels in Sardinia and Chianti cohorts.PLoS Genet. 2007; 3: e194Crossref PubMed Scopus (234) Google Scholar,26Vitart V. Rudan I. Hayward C. et al.SLC2A9 is a newly identified urate transporter influencing serum urate concentration, urate excretion and gout.Nat Genet. 2008; 40: 437-442Crossref PubMed Scopus (596) Google Scholar Gene polymorphisms in SLC2A9 could explain up to 5–6% of the variance in serum urate, and were associated with an increased risk for gout, but not for hypertension or other features of the metabolic syndrome, reviewed in Hediger and in So.1Hediger M.A. Johnson R.J. Miyazaki H. et al.Molecular physiology of urate transport.Physiology (Bethesda). 2005; 20: 125-133Crossref PubMed Scopus (274) Google Scholar,2So A. Thorens B. Uric acid transport and disease.J Clin Invest. 2010; 120: 1791-1799Crossref PubMed Scopus (553) Google Scholar Glut9 is highly expressed in liver and kidney.1Hediger M.A. Johnson R.J. Miyazaki H. et al.Molecular physiology of urate transport.Physiology (Bethesda). 2005; 20: 125-133Crossref PubMed Scopus (274) Google Scholar,2So A. Thorens B. Uric acid transport and disease.J Clin Invest. 2010; 120: 1791-1799Crossref PubMed Scopus (553) Google Scholar Patients with inactivating mutations in GLUT9 show idiopathic renal hypouricemia with high renal urate fractional excretion.27Anzai N. Ichida K. Jutabha P. et al.Plasma urate level is directly regulated by a voltage-driven urate efflux transporter URATv1 (SLC2A9) in humans.J Biol Chem. 2008; 283: 26834-26838Crossref PubMed Scopus (292) Google Scholar They are usually asymptomatic except for the occasional development of uric acid nephrolithiasis, chronic renal dysfunction, or strenuous exercise-induced acute renal failure. We recently generated mice with systemic or liver-specific deletion of Glut9.28Preitner F. Bonny O. Laverriere A. et al.Glut9 is a major regulator of urate homeostasis and its genetic inactivation induces hyperuricosuria and urate nephropathy.Proc Natl Acad Sci USA. 2009; 106: 15501-15506Crossref PubMed Scopus (191) Google Scholar Systemic Glut9 knockout mice showed a very high urate fractional excretion reflecting a suppression of renal urate reabsorption. Moreover, both systemic and liver-specific Glut9 knockout mice were hyperuricemic and hyperuricosuric with a 20- to 30-fold higher urate excretion rate, reflecting the inability of plasma urate to enter hepatocytes and undergo degradation by uricase in the absence of the transporter.28Preitner F. Bonny O. Laverriere A. et al.Glut9 is a major regulator of urate homeostasis and its genetic inactivation induces hyperuricosuria and urate nephropathy.Proc Natl Acad Sci USA. 2009; 106: 15501-15506Crossref PubMed Scopus (191) Google Scholar Although elevated, plasma urate levels in liver-specific Glut9 knockout mice are still low compared with human values. Previously, we increased plasma urate levels acutely in liver-specific Glut9 knockout mice up to human levels by gavaging them with inosine, a metabolic precursor of urate.29Preitner F. Laverriere-Loss A. Metref S. et al.Urate-induced acute renal failure and chronic inflammation in liver-specific Glut9 knockout mice.Am J Physiol Renal Physiol. 2013; 305: F786-F795Crossref PubMed Scopus (28) Google Scholar We showed that hyperuricemia and high-fat diet feeding combine to induce extensive urate crystal deposition in the kidneys, acute renal failure, and long-term renal sterile inflammation. The consequence of the inosine treatment was much milder in mice fed a chow diet, with no acute renal failure and rare signs of inflammation.29Preitner F. Laverriere-Loss A. Metref S. et al.Urate-induced acute renal failure and chronic inflammation in liver-specific Glut9 knockout mice.Am J Physiol Renal Physiol. 2013; 305: F786-F795Crossref PubMed Scopus (28) Google Scholar To study the development of urate-induced pathologies in mice, we supplemented inosine in a chow diet at incremental doses. Here we measured arterial blood pressure and heart rate in LG9 knockout (KO) mice with controlled hyperuricemia. Alb-CreERT2; Glut9lox/lox and Glut9lox/lox (control) mice (N=8) fed a normal chow diet were implanted with indwelling carotid artery pressure telemetric devices at 8 weeks of age. One month post surgery, before induction of the hepatic deletion of Glut9, plasma urate was similar in both groups (Figure 1a). Tamoxifen-induced hepatic deletion of Glut9 in Alb-CreERT2; Glut9lox/lox (LG9KO) mice raised plasma urate levels to 170 μmol/l––i.e., 70% higher as compared with Glut9lox/lox (control) mice (Figure 1a and c). To further increase uricemia, we supplemented chow diet with incremental amounts of the urate precursor inosine (0, 7.5, 10, and 15 g inosine/kg chow diet, INO7.5, INO10, and INO15, respectively). The treatment progressively increased plasma urate up to 300 μmol/l (Figure 1a). Interestingly, in control mice inosine decreased plasma urate instead. The inosine treatment did not significantly alter plasma creatinine in LG9KO mice despite a consistent trend for elevated levels compared with controls (Figure 1b, P=0.11, repeated measure analysis of variance). In order to assess the potential effect of inosine-induced hyperuricemia on the cardiovascular function, we measured heart rate and arterial blood pressure by telemetry during 8 sessions spread over the course of the study, either during a treatment transition or further into a treatment. Figure 2a shows a representative telemetric measurement of blood pressure, during the transition from chow diet to chow diet supplemented with 7.5 g/kg inosine (INO7.5). The data show that arterial blood pressure (systolic, diastolic, and mean) was not altered in LG9KO mice as compared with controls. Longitudinal analysis of the evolution of mean arterial blood pressure over the course of the study was performed for each session on 24 h averages of data acquired during Saturday night and Sunday morning, which was observed to be an especially quiet period of the week (Figure 2b). The data show that mean arterial blood pressure was not increased by either the tamoxifen or the inosine supplementation treatments in LG9KO mice during the course of the study (P=0.53 vs. controls, repeated measure analysis of variance). Similar data were obtained for systolic and diastolic blood pressures (not shown). Thus, inosine supplementation does not increase blood pressure in hyperuricemic LG9KO mice as compared with controls. Next, we assessed the relationship between mean arterial blood pressure and plasma urate levels in individual mice, one day after the transition from chow to INO7.5 (Figure 2c), or six weeks later, under INO15 (Figure 2d). In each case, plasma urate was measured the Monday following the telemetric session. Strikingly, mean blood pressure did not vary with plasma urate levels ranging from 25 to 330 μmol/l under INO7.5 (Figure 2c) and 25 to 430 μmol/l under INO15 (Figure 2d). Thus, hyperuricemia in inosine-supplemented LG9KO mice is not correlated with a higher blood pressure. Similar data and conclusions were obtained for systolic and diastolic blood pressures (not shown). Overall, similar data and conclusions were also obtained for the evolution of heart rate during the course of the study (Figure 3). To address a possible effect of long-term inosine supplementation in LG9KO mice on heart function and morphology, we performed heart ultrasound echography 2 weeks after the last telemetry session, in mice maintained on the INO15 diet (Table 1). Inosine-treated LG9KO and control mice showed normal and similar values for parameters related to heart morphology (in particular left ventricle mass, volume, inner diameter, and wall thickness) and heart function (cardiac output, stroke volume, fractional shortening, ejection fraction). No sign of cardiac hypertrophy was observed in either group. Histological analysis at the end of the study confirmed the absence of morphological defect, inflammation, or fibrosis in LG9KO hearts (data not shown).Table 1Inosine treatment in LG9 knockout (KO) mice does not affect heart function or morphologyControlLG9KOn=8n=6Means.e.m.Means.e.m.T-testBody weight (g)31.71.531.80.90.939Plasma urate (μmol/l)55.66.0255.814.90.000Plasma creatinine (μmol/l)12.20.316.00.80.000Heart rate (b.p.m.)46921459160.718Heart wet weight (ventricles; mg)122311920.462Interventricular septum thickness, end diastole (mm)0.740.010.750.020.538Interventricular septum thickness, end systole (mm)1.010.040.960.030.353Left ventricular inner diameter, end diastole (mm)3.970.103.990.150.905Left ventricular inner diameter, end systole (mm)2.920.173.070.200.583Left ventricular posterior wall thickness, end diastole (mm)0.770.020.760.020.636Left ventricular posterior wall thickness, end systole (mm)1.020.050.980.040.556Left ventricle volume, end diastole (μl)69.33.870.56.50.864Left ventricle volume, end systole (μl)34.04.238.36.10.569Left ventricle mass (mg)107.82.7105.34.50.627Left ventricle mass per body weight (mg/g)3.460.203.310.100.555Cardiac output (ml/min)16.40.915.50.40.444Stroke volume (μl)34.91.033.91.60.594Fractional shortening (%)26.92.723.52.10.363Ejection fraction (%)52.44.047.33.70.386At the end of the telemetric experiment (inosine 15 g/kg for 3 months), mice were isoflurane anesthetized and heart rate was maintained around 400 b.p.m. during ultrasound echography. N=6–8. Open table in a new tab At the end of the telemetric experiment (inosine 15 g/kg for 3 months), mice were isoflurane anesthetized and heart rate was maintained around 400 b.p.m. during ultrasound echography. N=6–8. To quantify the renal dysfunction induced by long-term inosine supplementation in LG9KO mice, we assessed the glomerular filtration rate (GFR) by the inulin clearance test. As shown in Figure 4a, the GFR was 44% lower in inosine-treated LG9KO mice as compared with controls, revealing a significant renal dysfunction. In LG9KO mice histological analysis of the kidneys (Figure 4b–e) revealed birefringent crystals under polarized light (Figure 4b) with a severe nonspecific chronic cortico-medullar inflammation, although no granuloma (characteristic of a chronic specific inflammatory reaction to foreign bodies) was observed. These inflammatory infiltrates were composed of mononuclear cells (lymphocytes and macrophages as revealed by immunohistochemical detection of CD3 and F4/80, respectively) and plasma cells (Figure 4c and d). This chronic inflammation was associated with moderate focal tubulointerstitial fibrosis (as revealed by fuchsin acid orange staining, Figure 4e), tubular dilation, and tubular atrophy. In controls, kidney morphology was normal, with only very weak signs of chronic tubulointerstital inflammation, interstitial fibrosis, and no crystal deposit in the renal parenchyma (Figure 4b–e). Thus, inosine supplementation in LG9KO mice induced the deposition of urate crystals and severe chronic nonspecific inflammation, likely starting within the interstitial compartment (cortex) and progressing to the medullar region and leading to fibrotic lesions. On the contrary, in control mice, the inosine supplementation had only marginal effects. Here we describe that controlled hyperuricemia in LG9KO mice, achieved by dietary inosine supplementation, elicited mild chronic renal dysfunction and damage, but no sign of either heart dysfunction, altered heart morphology, or change in blood pressure at any hyperuricemic levels. These data therefore do not support a direct causal role of urate on blood pressure in mice. We previously reported that genetic inactivation of liver-Glut9 expression in LG9KO mice leads to moderate increase in plasma urate levels.28Preitner F. Bonny O. Laverriere A. et al.Glut9 is a major regulator of urate homeostasis and its genetic inactivation induces hyperuricosuria and urate nephropathy.Proc Natl Acad Sci USA. 2009; 106: 15501-15506Crossref PubMed Scopus (191) Google Scholar Our present experiments show that diet supplementation with inosine allows a gradual increase in plasma urate concentration over several months, reaching uricemic values comparable to the normal-to-high range in humans. Here, we successfully monitored arterial blood pressure and heart rate by cardiac telemetry, over a 20-week period, before and after tamoxifen-induced gene deletion, and following periods of supplementation with increasing amounts of inosine. LG9KO mice were on the C57BL/6J genetic background that we and others previously found suitable for hypertension studies.30Stauss H.M. Godecke A. Mrowka R. et al.Enhanced blood pressure variability in eNOS knockout mice.Hypertension. 1999; 33: 1359-1363Crossref PubMed Scopus (109) Google Scholar,31Cook S. Hugli O. Egli M. et al.Clustering of cardiovascular risk factors mimicking the human metabolic syndrome X in eNOS null mice.Swiss Med Wkly. 2003; 133: 360-363PubMed Google Scholar,32Van Vliet B.N. Chafe L.L. Montani J.P. Characteristics of 24 h telemetered blood pressure in eNOS-knockout and C57Bl/6J control mice.J Physiol. 2003; 549: 313-325Crossref PubMed Scopus (92) Google Scholar Throughout the present experiment, LG9KO mice did not show any difference in arterial blood pressure or heart rate as compared with controls despite much higher hyperuricemic levels. Thus, there was no correlation at all between uricemia and blood pressure. However, the inosine supplementation in LG9KO mice induced a mild, chronic renal dysfunction with a trend for increased plasma creatinine, a decreased GFR, and chronic renal inflammation and fibrosis. Thus, in our model, renal dysfunction is not associated with alterations in blood pressure or heart rate. In this study, inosine-induced renal alterations are consistent with observations in rats, in which a 7-week treatment with the uricase inhibitor oxonate induced a mild hyperuricemia (100–125 μmol/l vs. 60–80 μmol/l in controls), low-grade renal inflammation, fibrosis, subtle tissue injuries, and renal dysfunction.14Feig D.I. Kang D.H. Johnson R.J. Uric acid and cardiovascular risk.N Engl J Med. 2008; 359: 1811-1821Crossref PubMed Scopus (1754) Google Scholar,19Mazzali M. Hughes J. Kim Y.G. et al.Elevated uric acid increases blood pressure in the rat by a novel crystal-independent mechanism.Hypertension. 2001; 38: 1101-1106Crossref PubMed Scopus (1030) Google Scholar,20Mazzali M. Kanellis J. Han L. et al.Hyperuricemia induces a primary renal arteriolopathy in rats by a blood pressure-independent mechanism.Am J Physiol Renal Physiol. 2002; 282: F991-F997Crossref PubMed Scopus (689) Google Scholar However, the absence of hypertension development in hyperuricemic LG9KO mice is not consistent with the same rat studies. Indeed, the oxonate treatment induced systemic hypertension (143–150 vs. 125–110 mm Hg, as measured by tail-cuff sphygmomanometer), which was prevented by co-treatment with either the XO inhibitor allopurinol to suppress urate production or with the uricosuric agent benziodarone to increase urate excretion.14Feig D.I. Kang D.H. Johnson R.J. Uric acid and cardiovascular risk.N Engl J Med. 2008; 359: 1811-1821Crossref PubMed Scopus (1754) Google Scholar,19Mazzali M. Hughes J. Kim Y.G. et al.Elevated uric acid increases blood pressure in the rat by a novel crystal-independent mechanism.Hypertension. 2001; 38: 1101-1106Crossref PubMed Scopus (1030) Google Scholar,20Mazzali M. Kanellis J. Han L. et al.Hyperuricemia induces a primary renal arteriolopathy in rats by a blood pressure-independent mechanism.Am J Physiol Renal Physiol. 2002; 282: F991-F997Crossref PubMed Scopus (689) Google Scholar Differences in dietary salt content between these rat studies (0.1 or 0.05% sodium19Mazzali M. Hughes J. Kim Y.G. et al.Elevated uric acid increases blood pressure in the rat by a novel crystal-independent mechanism.Hypertension. 2001; 38: 1101-1106Crossref PubMed Scopus (1030) Google Scholar,20Mazzali M. Kanellis J. Han L. et al.Hyperuricemia induces a primary renal arteriolopathy in rats by a blood pressure-independent mechanism.Am J Physiol Renal Physiol. 2002; 282: F991-F997Crossref PubMed Scopus (689) Google Scholar) and the present study (0.2% sodium) are unlikely to explain the differences in blood pressure. For instance, a sodium restriction as low as 0.02% (low sodium diet) does not alter blood pressure in mice.33Oliverio M.I. Best C.F. Smithies O. et al.Regulation of sodium balance and blood pressure by the AT(1A) receptor for angiotensin II.Hypertension. 2000; 35: 550-554Crossref PubMed Scopus (93) Google Scholar Major experimental differences between aforementioned rat studies14Feig D.I. Kang D.H. Johnson R.J. Uric acid and cardiovascular risk.N Engl J Med. 2008; 359: 1811-1821Crossref PubMed Scopus (1754) Google Scholar,19Mazzali M. Hughes J. Kim Y.G. et al.Elevated uric acid increases blood pressure in the rat by a novel crystal-independent mechanism.Hypertension. 2001; 38: 1101-1106Crossref PubMed Scopus (1030) Google Scholar,20Mazzali M. Kanellis J. Han L. et al.Hyperuricemia induces a primary renal arteriolopathy in rats by a blood pressure-independent mechanism.Am J Physiol Renal Physiol. 2002; 282: F991-F997Crossref PubMed Scopus (689) Google Scholar and our mouse study include the species, the achieved hyperuricemic levels (125 vs. 300 μmol/l), and the experimental paradigm used to induce hyperuricemia (uricase pharmacological blockade vs. inosine supplementation as a model of excessive purine consumption). Rodents display much lower uricemic levels compared with humans and are not evolutionarily prepared to handle high levels of urate.17Bobulescu I.A. Moe O.W. Renal transport of uric acid: evolving concepts and uncertainties.Adv Chronic Kidney Dis. 2012; 19: 358-371Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar,22Feig D.I. Madero M. Jalal D.I. et al.Uric acid and the origins of hypertension.J Pediatr. 2013; 162: 896-902Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar It is however noteworthy that, in this study, we did not observe any correlation between urate levels and blood pressure over a 17-fold urate concentration range (25–430 μmol/l), reaching levels comparable to human higher range.14Feig D.I. Kang D.H. Johnson R.J. Uric acid and cardiovascular risk.N Engl J Med. 2008; 359: 1811-1821Crossref PubMed Scopus (1754) Google Scholar,19Mazzali M. Hughes J. Kim Y.G. et al.Elevated uric acid increases blood pressure in the rat by a novel crystal-independent mechanism.Hypertension. 2001; 38: 1101-1106Crossref PubMed Scopus (1030) Google Scholar,20Mazzali M. Kanellis J. Han L. et al.Hyperuricemia induces a primary renal arteriolopathy in rats by a blood pressure-independent mechanism.Am J Physiol Renal Physiol. 2002; 282: F991-F997Crossref PubMed Scopus (689) Google Scholar In humans, the association between uricemia and cardiovascular disease is found not only with frank hyperuricemia (defined as >350–400 μmol/l) but also in the normal-to-high uricemic range (310–330 μmol/l).14Feig D.I. Kang D.H. Johnson R.J. Uric acid and cardiovascular risk.N Engl J Med. 2008; 359: 1811-1821Crossref PubMed Scopus (1754) Google Scholar Urate is produced as a purine metabolite by the enzyme XO, which also generates oxidants as byproducts. Thus, urate may be a marker for XO–associated oxidants, and the benefit of XO inhibitors such as allopurinol may rely on blocking the production of oxidants rather than on lowering urate.18Johnson R.J. Sanchez-Lozada L.G. Mazzali M. et al.What are the key arguments against uric acid as a true risk factor for hypertension?.Hypertension. 2013; 61: 948-951Crossref PubMed Scopus (52) Google Scholar Therefore, although our data exclude a direct causal role of urate per se on blood pressure in LG9KO mice, further studies to address a potential role of high XO activity on the cardiovascular function are warranted. In epidemiological studies, hyperuricemia is associated with hypertension; however, evidence for causation is very limited and inconclusive.17Bobulescu I.A. Moe O.W. Renal transport of uric acid: evolving concepts and uncertainties.Adv Chronic Kidney Dis. 2012; 19: 358-371Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar,21Feig D.I. Soletsky B. Johnson R.J. Effect of allopurinol on blood pressure of adolescents with newly diagnosed essential hypertension: a randomized trial.JAMA. 2008; 300: 924-932Crossref PubMed Scopus (742) Google Scholar,22Feig D.I. Madero M. Jalal D.I. et al.Uric acid and the origins of hypertension.J Pediatr. 2013; 162: 896-902Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar As urate is not considered as a true cardiovascular risk factor, it is not routinely measured as a marker in at-risk patients, and treatment of asymptomatic hyperuricemia is not recommended.17Bobulescu I.A. Moe O.W. Renal transport of uric acid: evolving concepts and uncertainties.Adv Chronic Kidney Dis. 2012; 19: 358-371Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar,22Feig D.I. Madero M. Jalal D.I. et al.Uric acid and the origins of hypertension.J Pediatr. 2013; 162: 896-902Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar In conclusion, results of the present study in LG9KO mice are consistent with the current body of epidemiological studies, suggesting that urate per se does not have a direct causal role in the development of hypertension. Alb-CreERT2; Glut9lox/lox (LG9KO) and Glut9lox/lox (control) mice were generated28Preitner F. Bonny O. Laverriere A. et al.Glut9 is a major regulator of urate homeostasis and its genetic inactivation induces hyperuricosuria and urate nephropathy.Proc Natl Acad Sci USA. 2009; 106: 15501-15506Crossref PubMed Scopus (191) Google Scholar by sequentially crossing Glut9lox/lox mice with Alb-CreERT234Schuler M. Dierich A. Chambon P. et al.Efficient temporally controlled targeted somatic mutagenesis in hepatocytes of the mouse.Genesis. 2004; 39: 167-172Crossref PubMed Scopus (114) Google Scholar mice, then with resulting Alb-CreERT2; Glut9lox/+ and finally Alb-CreERT2; Glut9lox/lox offspring to produce 50% of both Alb-CreERT2; Glut9lox/lox and Glut9lox/lox mice. Mice were backcrossed to >99.9% C57BL/6J genetic background by five rounds of Speed Congenics (Genomouse, Polygene, Rümlang, Switzerland). At age 5 weeks, all mice received three daily intraperitoneal injections of tamoxifen at 1 mg/mouse (Sigma-Aldrich, Buchs, Switzerland, 100 μl of 10 mg/ml (1:10 EtOH:sunflower oil)). Tamoxifen-induced hepatic deletion of Glut9 in Alb-CreERT2; Glut9lox/lox (LG9KO) was systematically confirmed by PCR and western blot28Preitner F. Bonny O. Laverriere A. et al.Glut9 is a major regulator of urate homeostasis and its genetic inactivation induces hyperuricosuria and urate nephropathy.Proc Natl Acad Sci USA. 2009; 106: 15501-15506Crossref PubMed Scopus (191) Google Scholar and measurement of plasma urate levels after tamoxifen induction. Mice were housed individually at 23 °C, with 12:12 h light cycle and free access to water and plain or supplemented chow diet (Provimi Kliba Ag, Kaiseraugst, Switzerland, cat. no. 3436): protein 18.5%, fat 4.5%, carbohydrates 54%; energy density 3.85 kcal/g (proteins 28%, fat 11%, carbohydrates 57%); sodium content 0.20%, and potassium 0.78%. Experiments were approved by the Service Vétérinaire Cantonal Vaudois under licence no. VD2156.3. Pressure transmitters (DSI PhysioTel PA-C10) were implanted in mice under isoflurane anesthesia. The catheter tip was introduced into the carotid artery and positioned in the aortic arch and the implant secured subcutaneously on the flank. Mice were allowed to recover for 3 weeks. The DSI acquisition system (DSI, Saint Paul, MN, USA) with 16 receiver plates enabled the simultaneous telemetric measurement of all mice in the study. Heart rate and mean arterial blood pressure were measured during eight sessions of 3.5 days each (from Thursday afternoon to Monday morning), spread over a 20-week period and four different inosine-supplemented diets. Of the 16 mice with implanted pressure transmitters, 15 showed proper signal transmission for 15 weeks post surgery (N=7–8 up to INO7.5). Thereafter, signal was progressively lost (INO15, first run: N=6–7; INO15, second run: N=6; INO15, last run: N=4–6). Plasma was collected from the tail at 1300–1400 h on the Mondays immediately following the telemetric sessions. Bleed frequency was limited to prevent hypovolemic stress and mice had a recovery time of at least 10 days before the next telemetric measurement. Plasma urate and creatinine were measured using the Roche/Hitachi 902 robot system (Roche Diagnostics, Rotkreutz, Switzerland). The Creatinine Plus Enzymatic assay was used. Transthoracic echocardiography was performed in isoflurane anesthetized mice using the Vevo 2100 ultrasound machine with MS400 probe (VisualSonics, Toronto, ON, Canada). Heart rate was maintained at 400–550 b.p.m. and the heart was imaged in the 2-D mode in the parasternal long-axis view. An M-mode cursor was positioned perpendicular to the interventricular septum and the posterior wall of the left ventricle at the level of the papillary muscles. Left ventricular fractional shortening was calculated as [(LVID;d-LVID;s)/LVID;d] × 100 and ejection fraction as [(LV Vol;d-LV Vol;s)/LV Vol;d] × 100. The GFR was assessed by the inulin clearance test as previously described, in anesthetized mice.35Vallon V. Eraly S.A. Rao S.R. et al.A role for the organic anion transporter OAT3 in renal creatinine secretion in mice.Am J Physiol Renal Physiol. 2012; 302: F1293-F1299Crossref PubMed Scopus (89) Google Scholar Kidneys were embedded in paraffin and 4-μm sections were prepared for hematoxylin and eosin staining. Immunohistochemical detection of the macrophage marker F4/80 and lymphocyte marker CD3 was performed using respectively: rat anti-F4/80 primary antibody (1:800) and a horseradish peroxidase-conjugated goat anti-rat Ig secondary antibody (1:100) (Invitrogen, Basel, Switzerland); rabbit anti-human CD3 primary antibody (1:100, DAKO, Baar, Switzerland) and the DAKO EnVision+Rabbit HRP ready-to-use kit (cat. K4002). Cryosections of kidney were obtained from absolute ethanol-fixed kidneys to assess anisotropism of uric acid crystals under polarized light. Statistical analyses were performed using R (version 2.11.1) using two-tailed t-tests to compare single data points between LG9KO and control groups, or paired t-tests to compare different time points, or repeated measure analysis of variance to compare groups over time, as indicated. The authors thank Marianne Carrard and Armelle Bauduret for expert technical help, Frédéric Schütz for statistics, Annette Yves for the anti-CD3 antibody, Olivier Bonny for the anti-Glut9 antibody and helpful discussions, and OB and Nadia Preitner for careful manuscript reading. This work was supported by grants from the Swiss National Science Foundation No. 3100A0-113525 (BT), EU 7th FP Integrated project EDICT No. 201924 (BT), and the Swiss National Competence Center in Research Transcure (BT).
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