Basic And Clinical Study About The High Insulin Suppressed Renal Excretion Of Uric Acid And The Intervention Mechanics Of Losartan

Backgrounds: The metabolic syndrome(Met S), which is closely associated with incidence and mortality of diabetes and cardiovascular or kidney diseases, has become more prevalent following rapid economic growth in C hina in recent decades. So it has been becoming both a public health and clinical problems. Elevated serum uric acid(SUA) levels or hyperuricemia related to insulin resistance are commonly seen in patients with the Met S which characterized by hyperinsulinemia and/or insulin resistance. It was considered that the impairment of renal excretion of uric acid was the most frequent mechanism for the development of increasing in serum uric acid concentration or hyperuricemia. Urate transporter 1(URAT1 encoded by SLC22A12), located at the apical membrane in the proximal tubules, is the most significant renal tubular urate transporter for reabsorption and fluctuations of serum urate levels. Elevated serum uric acid(SUA) levels or hyperuricemia is also considered to be the high-risk factors for cardiovascular and kidney diseases or even as a component of metabolic syndrome because it was associated with an increased prevalence of metabolic disorders such as obesity, dyslipidemia and hypertension. So it is necessary to treat asymptomatic hyperuricemia with urate- lowering drugs. Many evidence showed that decreasing serum uric acid levels may be of great benefit in reducing CV events, protecting kidney in patients with asymptomatic hyperuricemia. But the clinical applications of present antihyperuricemic or uricosuric drugs are limited because of their adverse drug reactions(ADR). It has been reported that the angiotensin II–receptor blocker(ARB) losartan also has the effect of decreasing serum uric level in patients with hypertension and other cardiovascular risk factors except for controlling BP, reducing cardiovascular events, reducing urine protein and protecting renal function. While the precise mechanism of the metabolism of uric acid in the state of insulin resistance or hyperinsulinaemia remains still need to be further elaborated. Whether Losartan has the effects on insulin- induced expression of URAT1 via the PI3K/Akt1 pathway in human renal tubular epithelial cells or not remains need to be clarified.Objectives: To provide basis for both the precise mechanisms and prevention of hyperuricemia related to metabolic syndrome in the state of insulin resistance or hyperinsulinaemia, the correlation between serum insulin and the renal excretion of uric acid was investigated in patients with Met S in present cross-sectional study, and the effects of losartan on insulin- induced expression of urate transporter-1(URAT1) via the PI3K/Akt1 pathway are tested in cultured human renal tubular epithelial cells in vitro.Methods: 1. A cross-sectional study was conducted for one year in clinic and inpatient department of endocrinology from March 2014. In present study, a 108 subjects(74 males, 34 females) with Met S were enrolled, who were in the age of 54.58(55.31±10.68) years. Basic informations, history of diseases and recent medication history were collected. Fasting blood insulin(FIns), postprandial blood insulin(2h PIns) and related serum biochemical indexes were measured. 24 hours urine sample of patients were collected to calculate the renal excretion indexes of uric acid such as fractional urate excretion(FEUA, %), uric acid clearance(CUA, ml/min/1.73 m2), excretion of uric acid per volume of glomerular filtration(Eur GF, mg/dl/1.73 m2) and urinary uric acid to creatinine ratio(UUA/Ucr). Estimated glomerular filtration rate(e GFR) were calculated according to the modified MDRD equations of C hina. The clinical characteristics of the study population between male and female were compared. Subjects were divided into 4 groups b ased on Fins, 2h PIns and HOMA-IR quartiles. The various group values of renal excretion index of uric acid were compared. To analyze the correlations between renal excretion of uric acid and the components of metabolic syndrome, the study population was divided into different groups according to the components and the renal excretion of uric acid values were compared. Relative risks were analyzed by using multiple logistic regression analysis and P-values of < 0.05 were considered statistically significant. 2. HK-2 cells were treated with high insulin(1, 10 and 100 n M) for 6, 24, 48 or 72 hrs. Cells were then harvested for RNA or protein isolation at 6, 24, 48 or 72 hrs, respectively. Expression of URAT1 gene was assessed by RT-PCR and Western Blot. To determinate the activity of PI3K/Akt1, the insulin(100 n M) induced cells were treated either with 10 μM LY294002(PI3K/Akt inhibitor) or not for 48 hrs.LY294002 was added 1 hour prior to insulin applied to inhibit PI3K/Akt signaling pathway. The protein levels of URAT1, Akt1 and phosphor(p)-Akt1 were assessed by Western Blot. 3. Human proximal tubule epithelial cell line HK-2 were treated with various concentrations of Losartan(1, 10 and 100 n M) for 48 hrs, and the expression of URAT1 protein were asse ssed by Western Blot and compared with that of control group. Then the cells were divided into 4 groups, that is, control group, high concentration of insulin group(insulin 100 n M), high concentration of Losartan group(insulin 100 n M plus Losartan 100 μM) and LY294002 group(LY294002 10 μM + insulin 100 n M), the expression of URAT1, Akt1 and p-Akt1 protein were assessed by Western Blot in 48 hrs.Results: 1.(1) The levels of serum uric acid and creatinine in male were higher than that in female(P<0.001).(2) FEUA showed a distinct decreasing trend along with elevating of the FIns level(F=8.920,P=0.004). 24 h UUA(2h PIns ≥ 22.2 m U/L)、FEUA and CUA showed a distinct decreasing trend along with elevating of the 2h PIns level(F=7.548,P=0.007;F=9.620,P=0.002;F=15.736,P<0.001, respectively). There were no statistical significance of variation in renal excretion index of uric acid along with the fluctuate of HOMA-IR(P>0.05).(3) Univariate analysis of influencing factors about renal excretion of uric acid in patients with Met S. The level of UUA/Ucr in male was lower than that in female(P=0.002). The level of 24 h UUA、CUA in non-senile group(<60 y) were higher than that in senile group(≥60 y) significantly(P < 0.01). The level of CUA in patients who had a duration of diabetes was higher than that in patients who had no the duration of diabetes(P<0.05). The levels of 24 h UUA, FEUA, CUA and UUA/Ucr in patients who had a history of hypertension were lower than that in patients who had no history of hypertension(P<0.05). The level of 24 h UUA in elevated systolic blood pressure group(≥130 mm Hg) was lower than that in decreased systolic blood pressure group(<130 mm Hg)(P<0.05). There was no statistical significance of 24 h UUA, FEUA, CUA, Eur GF and UUA/Ucr among different groups when patients were grouped according to BMI and waist circumference(P>0.05). The levels of 24 h UUA and CUA in high PBG(≥7.8 mmol/L) group were higher than that in normal PBG group(< 7.8 mmol/L) significantly(P < 0.05) when patients were grouped according to PBG but not FBG and Hb Alc. 24 h UUA in high TG group(TG ≥ 1.7 mmol/L) was higher than that in normal TG group(TG< 1.7 mmol/L)(P<0.05). 24 h UUA and Eur GF in low HDL-C group(male < 1.0 mmol/L, female < 1.3 mmol/L) were lower than that in normal HDL-C group(male ≥1.0 mmol/L, female ≥1.3 mmol/L)(P<0.05). CUA and UUA/Ucr in normal e GFR group(e GFR≥90 ml/min/1.73 m2) were higher than that in low e GFR gro up(e GFR<90 ml/min/1.73 m2) significantly(P<0.01) but Eur GF just was the opposite(P<0.01). 24 h UUA and CUA in high 24 h UTP group(24h UTP≥0.15 g/24h) were higher than that in normal 24 h UTP group(24h UTP <0.15 g/24h)(P<0.05).(4) FEUA, CUA and UUA/Ucr were negatively correlated with SUA levels(P<0.05).(5) Multivariate regression analysis of the influence factors about renal uric acid excretion. Age and 2h PIns was negatively correlated with 24 h UUA but PBG just was the opposite. Age, HDL-C and 2h PIns in men whereas age, 24 h UTP and TG in women were risk factors for 24 h UUA. PBG was positively correlated with FEUA but FIns was the opposite. The risk factors for FEUA were FIns in men but PBG in women. e GFR and PBG were positively correlated with CUA but 2h PIns was the opposite. The risk factors for CUA were e GFR and 2h PIns in men but PBG and age in women. e GFR and BMI were negatively correlated with Eur GF. e GFR was positively correlated with UUA/Ucr whereas patients whether or not had history of hypertension was the opposite. The risk factors for UUA/Ucr were e GFR in men but history of hypertension and PBG in women. 2. RT-PCR and Western Blot results revealed that URAT1 m RN A and protein expression in different concentrations of insulin groups were higher than that in control group. Insulin induced URAT1 m RN A and protein up-expression in a time- and dose-dependent manner(P<0.05). URAT1 m RN A and protein expression peaked at 100 n M insulin and 48 hrs(1.57- fold and 1.80-fold vs control respectively, P<0.05); therefore, this concentration and time was used for all subsequent experiments. The levels of phosphorylated Akt1 increased in HK-2 cells when exposured to insulin(100 n M) for 48 hrs(P<0.05, compared with control), indicating that the activation of the PI3K/Akt1 pathway increased. Compared with high insulin group, LY294002 significantly diminished the expression of p-Akt1 protein induced by insulin in HK-2 cells(P < 0.01). The PI3K/Akt1 inhibitor LY294002 eliminated the effect of insulin-triggered increase of URAT1 expression when compared with high insulin group(P<0.05). 3. Compared with high insulin group, Losartan down-regulated the insulin- induced expression of URAT1 protein in a dose-dependent manner(P<0.01). Compared with control group, the expression of URAT1 and p-Akt1 but not Akt1 protein induced by high insulin increased significantly in HK-2 cells(P<0.05). Compared with high insulin group, the expression of URAT1 and p-Akt1 protein induced by high insulin decreased significantly in high concentration of Losartan group(P<0.05). Compared with high insulin plus high concentration of Losartan group, the expression of URAT1 and p-Akt1 protein induced by high insulin decreased significantly in LY294002 group(P<0.05). The effect of Losartan was weaker than that of LY294002 between two groups.Conclusions: 1. Elevated serum insulin levels was a primary influence factor for renal underexcretion of uric acid which might play an important role in the development of hyperuricemia in patients with Met S. The level of SUA was higher in men than that in women which might mainly be attributed to the renal underexcretion of uric acid induced by elevated serum insulin levels. So it has a positive significance in improving hyperinsulinemia in patients with the Met S to prevent elevated serum uric acid levels or hyperuricemia. 2. Insulin could induce URAT1 expression in a time- and dose-dependent manner in cultured HK-2 cells. This indicated that high insulin may increase SUA levels by up-regulating URAT1 expression and then increasing reabsorption of UA at renal tubular epithelial cells in the status of hyperinsulinemia. High insulin may upregulated URAT1 expression via the activation of PI3K/Akt1 pathway in HK-2 cells. 3. Losartan can significantly inhibit the high insulin- induced up-expression of URAT1 in a dose-dependent manner in HK-2 cells. Losartan might down-regulate the high insulin- induced URAT1 expression by partly inhibiting the active of PI3K/Akt1 pathway in HK-2 cells. Losartan play its uricosuric action by reducing proximal tubular reabsorption of uric acid which leading to decreased serum uric acid concentrations in patients with the Met S related hyperuricemia.