Einosuke Mizuta

Uricosuric Action of Losartan via the Inhibition of Urate Transporter 1 (URAT 1) in Hypertensive Patients

BACKGROUND The angiotensin receptor blocker losartan inhibited urate transporter 1 (URAT1) according to in vitro experiments. However, it is still unknown whether the inhibitory effect of losartan on URAT1 contributes to its uricosuric action in humans. METHODS Thirty-two patients with hypertension and nine patients with idiopathic renal hypouricemia (five with and four without hypertension) were enrolled for this study. Hypertensive patients were prescribed oral losartan (50 mg/day, n = 16) or candesartan (8 mg/day, n = 16). Before and after 1-month treatment, the serum concentration of urate (Sur) and creatinine (Scr), and the clearance value of urate (Cur) and creatinine (Ccr) were determined. Clearance studies using the URAT1 inhibitor benzbromarone (100 mg/day) or losartan (50 mg/day) loading test were also performed in these patients. RESULTS Blood pressure (BP) significantly decreased in the patients treated with either losartan or candesartan. Losartan significantly reduced Sur, which was associated with a concomitant increase in the Cur/Ccr ratio, whereas candesartan did not alter these parameters. In hypertensive patients with loss-of-function mutation of URAT1, losartan did not alter either Sur or Cur/Ccr, nor did benzbromarone. The lack of effect of URAT1 inhibitors on renal excretion of urate was independent of the renal function of hypouricemic patients. On the other hand, both losartan and benzbromarone increased Cur/Ccr ratio in hypertensive patients harboring the wild URAT1 gene, regardless of the presence of hypouricemia. CONCLUSIONS These findings suggested that losartan inhibited URAT1 and thereby it lowered Sur levels in hypertensive patients.

Ubiquitin-Proteasome System Impairment Caused by a Missense Cardiac Myosin-binding Protein C Mutation and Associated with Cardiac Dysfunction in Hypertrophic Cardiomyopathy

The ubiquitin-proteasome system is responsible for the disappearance of truncated cardiac myosin-binding protein C, and the suppression of its activity contributes to cardiac dysfunction. This study investigated whether missense cardiac myosin-binding protein C gene (MYBPC3) mutation in hypertrophic cardiomyopathy (HCM) leads to destabilization of its protein, causes UPS impairment, and is associated with cardiac dysfunction. Mutations were identified in Japanese HCM patients using denaturing HPLC and sequencing. Heterologous expression was investigated in COS-7 cells as well as neonatal rat cardiac myocytes to examine protein stability and proteasome activity. The cardiac function was measured using echocardiography. Five novel MYBPC3 mutations -- E344K, DeltaK814, Delta2864-2865GC, Q998E, and T1046M -- were identified in this study. Compared with the wild type and other mutations, the E334K protein level was significantly lower, it was degraded faster, it had a higher level of polyubiquination, and increased in cells pretreated with the proteasome inhibitor MG132 (50 microM, 6 h). The electrical charge of its amino acid at position 334 influenced its stability, but E334K did not affect its phosphorylation. The E334K protein reduced cellular 20 S proteasome activity, increased the proapoptotic/antiapoptotic protein ratio, and enhanced apoptosis in transfected Cos-7 cells and neonatal rat cardiac myocytes. Patients carrying the E334K mutation presented significant left ventricular dysfunction and dilation. The conclusion is the missense MYBPC3 mutation E334K destabilizes its protein through UPS and may contribute to cardiac dysfunction in HCM through impairment of the ubiquitin-proteasome system.

Leptin Gene and Leptin Receptor Gene Polymorphisms Are Associated with Sweet Preference and Obesity

Leptin is an adipocyte-secreted hormone that regulates food intake and body weight, and that was recently reported to suppress sweet sensitivity in an animal model. We investigated the associations among sweet preference, obesity, and polymorphisms of the leptin gene (LEP) or leptin receptor gene (LEPR). A total of 3,653 residents randomly selected from among the citizens of Suita City, Osaka, Japan were enlisted as subjects, in whom we investigated sweet preference, clinical characteristics, including obesity and serum leptin level, and the polymorphisms of LEP and LEPR (G-2548A and A19G for LEP; R109K, R223Q, and rs3790439 for LEPR). We determined the associations among the parameters using logistic regression analysis, in order to consider potential confounding factors for sweet preference and/or obesity. The LEP A19G and LEPR R109K polymorphisms were associated with sweet preference, whereas the serum leptin level was not. Further, the LEPR 109KK genotype was found to be associated with obesity along with sweet preference. In conclusion, our results are the first to show associations of LEP and LEPR polymorphisms with sweet preference, and may provide useful information for diagnosis and treatment of lifestyle-related diseases.

Co-induction of heme oxygenase-1 and peroxiredoxin I in astrocytes and microglia around hemorrhagic region in the rat brain

Heme[none1] oxygenase-1 (HO-1) and peroxiredoxin I (PrxI) are known to be oxidative stress- and heme-related proteins. The antioxidant activity of PrxI is inhibited by heme, therefore co-expression of HO-1 and PrxI is considered to be a reasonable mechanism to maintain its antioxidative function. Immunoblotting demonstrated that HO-1 and PrxI were induced around the hemorrhagic region. Immunohistochemical studies revealed that, in acute phase, HO-1 and PrxI were induced primarily in microglia. In the subacute and chronic phase, the immunoreactivity of HO-1 and PrxI in astrocytes was the most intense. These data are the first to demonstrate co-induction of HO-1 and PrxI in the brain. Our results suggest that HO-1 and PrxI are localized in a similar manner to assure the antioxidant activity of PrxI under stress conditions associated with intracerebral hemorrhage.

Impairment of Ubiquitin–Proteasome System by E334K cMyBPC Modifies Channel Proteins, Leading to Electrophysiological Dysfunction

Cardiac arrhythmogenesis is regulated by channel proteins whose protein levels are in turn regulated by the ubiquitin-proteasome system (UPS). We have previously reported on UPS impairment induced by E334K cardiac myosin-binding protein C (cMyBPC), which causes hypertrophic cardiomyopathy (HCM) accompanied by arrhythmia. We hypothesized that UPS impairment induced by E334K cMyBPC causes accumulation of cardiac channel proteins, leading to electrophysiological dysfunction. Wild-type or E334K cMyBPC was overexpressed in HL-1 cells and primary cultured neonatal rat cardiac myocytes. The expression of E334K cMyBPC suppressed cellular proteasome activities. The protein levels of K(v)1.5, Na(v)1.5, Hcn4, Ca(v)3.2, Ca(v)1.2, Serca, RyR2, and Ncx1 were significantly higher in cells expressing E334K cMyBPC than in wild type. They further increased in cells pretreated with MG132 and had longer protein decays. The channel proteins retained the correct localization. Cells expressing E334K cMyBPC exhibited higher Ca(2+) transients and longer action potential durations (APDs), accompanied by afterdepolarizations and higher apoptosis. Those augments of APD and Ca(2+) transients were recapitulated by a simulation model. Although a Ca(2+) antagonist, azelnidipine, neither protected E334K cMyBPC from degradation nor affected E334K cMyBPC incorporation into the sarcomere, it normalized the APD and Ca(2+) transients and partially reversed the levels of those proteins regulating apoptosis, thereby attenuating apoptosis. In conclusion, UPS impairment caused by E334K cMyBPC may modify the levels of channel proteins, leading to electrophysiological dysfunction. Therefore, UPS impairment due to a mutant cMyBPC may partly contribute to the observed clinical arrhythmias in HCM patients.

Transcriptional activation of the anchoring protein SAP97 by heat shock factor (HSF)-1 stabilizes Kv1.5 channels in HL-1 cells

BACKGROUND AND PURPOSE The expression of voltage‐dependent K+ channels (Kv) 1.5 is regulated by members of the heat shock protein (Hsp) family. We examined whether the heat shock transcription factor 1 (HSF‐1) and its inducer geranylgeranylacetone (GGA) could affect the expression of Kv1.5 channels and its anchoring protein, synapse associated protein 97 (SAP97).

Effects of a low-dose antihypertensive diuretic in combination with losartan, telmisartan, or candesartan on serum urate levels in hypertensive patients

BACKGROUND A combination therapy of a low-dose antihypertensive diuretic with an angiotensin II receptor blocker (ARB) may have unfavorable effects on serum urate levels. METHODS Forty-two hypertensive patients without hyperuricemia (18 men and 24 women, mean age 65 years) were randomly divided into three groups. Each of the group was allocated to a combination therapy with losartan (LOS; CAS 124750-99-8; 50 mg/day)/hydrochlorothiazide (HCTZ; CAS 58-93-5; 12.5 mg/day) (LOS/HCTZ group), telmisartan (TEL; CAS 144701-48-4; 40 mg/day)/HCTZ (12.5 mg/day) (TEL/HCTZ group), or candesartan (CND; CAS 145040-37-5; 8 mg/day)/HCTZ (12.5 mg/day) (CND/HCTZ group), respectively. Before and after the treatment, blood pressure and biochemical parameters of blood and urine were evaluated. RESULTS Both systolic and diastolic blood pressures significantly decreased in all groups (p < 0.01) without any statistical differences. The LOS/HCTZ group showed no changes in serum urate levels (5.8 +/- 1.0 mg/dl to 5.8 +/- 1.4 mg/dl) and in % fractional excretion of urate (FEUA). In the TEL/HCTZ group, the serum urate level was significantly increased, from 5.5 +/- 0.9 mg/dl to 6.5 +/- 1.2 mg/dl (p < 0.01), whereas FEUA significantly decreased (p < 0.01). Similarly, the CND/HCTZ group showed a significant increase in the serum urate level from 5.4 +/- 0.9 mg/dl to 6.0 +/- 1.2 mg/dl (p < 0.01) and a significant decrease in FEUA (p < 0.01). No significant differences were found in fasting plasma glucose and electrolytes levels in any of the groups. CONCLUSIONS A combination therapy with a low-dose HCTZ and ARBs resulted in reduced urate excretion and elevated serum urate levels. A combination therapy with the ARB losartan was not accompanied with these effects, likely because of its inhibitory action on urate transporter 1. The study limitations deserve mention in consideration of ethic restrictions, small size, short term examination and uncontrolled design.

Effect of losartan and benzbromarone on the level of human urate transporter 1 mRNA

Both an angiotensin II receptor blocker, losartan (CAS 124750-99-8) and a serum urate lowering agent, benzbromarone (CAS 3562-84-3) exert a uricosuric action by inhibiting urate transporter 1 (URAT1). A recent clinical trial indicated that losartan could reduce the level of serum urate in hypertensive patients treated with urate lowering agents, suggesting the different mode of action of losartan from benzbromarone. In the present study, the effect of losartan and benzbromarone on the level of URAT1 mRNA was determined in transfected HEK293 cells. Losartan caused a significant reduction of its mRNA level, whereas it was not affected by benzbromarone. These results indicate that losartan decreases the level of human URAT1 mRNA, which may underlie the uricosuric action of losartan in hypertensive patients treated with serum urate lowering agents.

Effects of azelnidipine on uric acid metabolism in patients with essential hypertension

Abstract Purpose: To examine effects of a long-acting calcium channel blocker (CCB) azelnidipine on uric acid metabolism in hypertensive patients. Methods: Azelnidipine was administered to 72 patients at a daily dose of 8 mg or 16 mg. In 22 cases out of the 72 patients, a different CCB was switched to azelnidipine. Blood pressure was measured and biochemical parameters of blood and urine were evaluated before and 2–3 months after the administration. Results: Azelnidipine significantly decreased both systolic and diastolic blood pressure and the heart rate. It decreased both serum urate levels and the urinary uric acid to creatinine ratio (Uur/Ucr), but did not affect the uric acid clearance to creatinine clearance ratio (Cur/Ccr). Azelnidipine decreased both Uur/Ucr and Cur/Ccr in patients with Uur/Ucr ≥0.5 or ≥0.34, although it did not change these clearance parameters in patients with Uur/Ucr <0.5 or <0.34. Azelnidipine decreased the serum urate levels and Uur/Ucr in hyperuricemic patients with uric acid levels ≥7.0 mg/dL in males and ≥6.0 mg/dL in females. It did not change these parameters in normouricemic patients with serum urate levels <7.0 mg/dL in males and <6.0 mg/dL in females. Azelnidipine decreased Uur/Ucr and Cur/Ccr in hyperuricemic patients with normal or overexcretion of uric acid, although it did not change these clearance parameters in hyperuricemic patients with uric acid hypoexcretion. Conclusions: Azelnidipine decreased the serum urate acid levels and Uur/Ucr, and this response was most prominent in hyperuricemic patients or patients with normal and overexcretion of uric acid.

Effects of Uric Acid on the NO Production of HUVECs and its Restoration by Urate Lowering Agents

BACKGROUND Although urate impaired the endothelial function, its underlying mechanism remains unknown. We hypothesized that urate impaired nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) via activation of uric acid transporters (UATs). PURPOSE AND METHOD In the present study, we studied effects of urate on NO production and eNOS protein expression in HUVEC cells in the presence and absence of urate lowering agents using molecular biological and biochemical assays. RESULTS HUVECs expressed the 4 kinds of UATs, URATv1, ABCG2, MRP4 and MCT9. Exposure to urate at 7 mg/dl for 24 h significantly reduced production of NO. Pretreatment with benzbromarone, losartan or irbesartan normalized NO production. The same exposure resulted in dephosphorylation of endothelial NO synthase (eNOS) in HUVECs. Again pretreatment with benzbromarone, losartan or irbesartan abolished this effect. CONCLUSION Urate reduced NO production by impaired phosphorylation of eNOS in HUVEC via activation of UATs, which could be normalized by urate lowering agents.