Albumin-bound kynurenic acid is an appropriate endogenous biomarker for assessment of the renal tubular OATs-MRP4 channel

Renal tubular secretion mediated by organic anion transporters(OATs)and the multidrug resistanceassociated protein 4(MRP4)is an important means of drug and toxin excretion.Unfortunately,there are no biomarkers to evaluate their function.The aim of this study was to identify and characterize an endogenous biomarker of the renal tubular OATs-MRP4 channel.Twenty-six uremic toxins were selected as candidate compounds,of which kynurenic acid was identified as a potential biomarker by assessing the protein-binding ratio and the uptake in OAT1-,OAT3-,and MRP4-overexpressing cell lines.OAT1/3 and MRP4 mediated the transcellular vectorial transport of kynurenic acid in vitro.Serum kynurenic acid concentration was dramatically increased in rats treated with a rat OAT1/3(rOAT1/3)inhibitor and in rOAT1/3 double knockout(rOAT1/3^(-/-))rats,and the renal concentrations were markedly elevated by the rat MRP4(rMRP4)inhibitor.Kynurenic acid was not filtered at the glomerulus(99%of albumin binding),and was specifically secreted in renal tubules through the OAT1/3-MRP4 channel with an appropriate affinity(Km)(496.7 mM and 382.2 mM for OAT1 and OAT3,respectively)and renal clearance half-life(t1/2)in vivo(3.7±0.7 h).There is a strong correlation in area under the plasma drug concentration-time curve(AUC0et)between cefmetazole and kynurenic acid,but not with creatinine,after inhibition of rOATs.In addition,the phase of increased kynurenic acid level is earlier than that of creatinine in acute kidney injury process.These results suggest that albumin-bound kynurenic acid is an appropriate endogenous biomarker for adjusting the dosage of drugs secreted by this channel or predicting kidney injury.

外源性kynurenic acid对大鼠黑质多巴胺能神经元损伤的保护作用研究

目的 观察评价预先应用谷氨酸 (Glu)受体拮抗剂kynurenicacid(KYNA)对黑质多巴胺(DA)能神经元及神经传导纤维损伤的保护性作用。方法 雌性SD大鼠 4 0只 ,随机分为 4组 ,每组10只 ,应用江湾I型C立体定向仪 ,在单侧黑质致密部及中脑被盖腹侧部 ,A组注射生理盐水 ,B组注射KYNA ,C组注射KYNA和 6 羟基多巴胺 (6 OHDA) ,KYNA先于 6 OHDA 30min ,D组注射 6 OHDA。注射药物 3d后 ,进行症状观察 ,4周后处死大鼠。切片HE染色观察黑质细胞的形态特点 ,冰冻切片免疫组化特殊染色观察酪氨酸羟化酶 (TH)阳性细胞及TH阳性纤维着色情况。结果 正常黑质细胞体形较大 ,富含黑色素颗粒 ,可见尼氏体。TH着色结果提示B组与A组之间无显著差异 ,P >0 .0 5。实验组C与A、B、D组比较均有显著性差异 ,P <0 .0 1。结论 外源性Glu受体拮抗剂KYNA通过阻滞Glu受体一定时间阶段内能减轻 6 OHDA诱导的黑质DA能神经元毒性损害。

腹外侧眶皮层内给予谷氨酸抑制福尔马林诱发的大鼠脊髓背角Fos表达

目的:观察腹外侧眶皮层(ventrolateral orbital cortex,VLO)内微量注射谷氨酸对大鼠后爪注射福尔马林诱发的腰段脊髓背角Fos表达的抑制效应以及该效应是否通过中脑导水管周围灰质(PAG)下行抑制系统介导。方法:免疫组织化学染色技术。结果:(1)VLO内微量注射谷氨酸(100nmol/0.5μl)明显抑制大鼠后爪注射福尔马林诱发的脊髓背角Fos表达,与注射生理盐水相比,差异显著(P<0.001),并且这种抑制效应可被VLO内预先注射非选择性谷氨酸受体拮抗剂kynurenic acid(2nmol/0.5μl)所翻转,与单独注射谷氨酸相比差异显著(P<0.001),但与注射生理盐水相比无显著差异(P>0.05);(2)双侧腹外侧PAG内微量注射局麻剂利多卡因(0.2nmol/0.5μl)可以明显阻断VLO内微量注射谷氨酸(100nmol/0.5μl)对大鼠脊髓背角Fos表达的抑制效应,与单独注射谷氨酸相比差异显著(P<0.001),而双侧腹外侧PAG内微量注射生理盐水不影响VLO内微量注射谷氨酸对大鼠脊髓背角Fos表达的抑制效应(P>0.05)。结论:VLO内谷氨酸可能通过其受体激活PAG脑干下行抑制系统在脊髓水平抗炎性持续性伤害感受效应。

Measurement of Tryptophan Metabolites in Healthy Old Men and Patients of Type 2 Diabetes Mellitus (T2DM)

Background: Plasma levels of tryptophan (TRP) metabolites have not been measured extensively in patients of type 2 diabetes mellitus (T2DM). Methods: Metabolites analysis was performed by a liquid chromatograph tandem mass spectrometer, the LCMS-8060 quadrupole mass spectrometer combined with Nexera X2 liquid chromatograph system (Shimadzu Corporation, Kyoto, Japan). Body mass index (BMI) and TRP metabolites have been measured in healthy old men (n = 20) and patients of T2DM (n = 20). TRP metabolites were measured by using the ultrahigh speed liquid chromatography-mass spectros-copy (Shimadzu Corporation). Results: The plasma levels of 5-hydroxytryptophan (5-HTRP), 5-hydroxyindoleacetic acid (5-HIAA), kynurenic acid (KNA), 3-hydroxykynurenine (3-HKN), and 3-hydroxyanthranilic acid (3-HAA) were higher in patients of T2DM than healthy old men. Since 5HTRP and 5-HIAA belong to the serotonin pathway, and KNA, 3-HKN, and 3-HAA belong to the KN pathway of TRP metabolism, these pathways were activated more in the patients of T2DM. Since plasma levels of Indole-3-acetic acid were not elevated in T2DM, that pathway was not activated more in T2DM. Serotonin levels were not increased but 5-HIAA levels were increased in the plasma of T2DM patients, which may mean that serotonin was quickly metabolized to 5-HIAA in the patients of T2DM. Conclusion: Plasma levels of tryptophan metabolites in serotonin and kynurenine pathways increased in T2DM patients. Obesity expressed by BMI may not influence tryptophan metabolism in healthy old men and T2DM patients. These results indicate that our new method of the simultaneous measurements of all the tryptophan metabolites is the powerful measure to identify factor related to endogenous stresses seen in DM.

Differences of Plasma Levels of Tryptophan, Serotonin, 5-Hydroxyindole Acetic Acid, and Kynurenine between Healthy People and Patients of Major Monopolar Depression at Various Age and Gender

Background: It is not well analyzed whether there are differences in plasma levels of tryptophan (TRP) metabolites between healthy control people (HC) and patients of major monopolar depression (MMD). Methods: Ultra high-speed liquid chromatography/mass spectrometry has been used for the simultaneous determination of plasma levels of tryptophan metabolites in depressive patients. Results: There are no significant differences between plasma levels of TRP between HC and MMD. Plasma levels of TRP of HC are higher in young men, young women, old men, and old women in this order. Serotonin (5-HT) levels are higher in MMD than HC. Plasma levels of 5-HIAA of HC are also higher than those of patients of MMD. Plasma levels of kynurenine (KYN) of healthy old men and old women are higher than those of young men and old women. Plasma levels of KYN are higher in old women and young men of MMD than those of HC. Conclusion: Plasma levels of 5-HT are higher in patients of MMD than those of HC, which may suggest that use of drugs inhibiting the 5-HT transportation may increase plasma levels of 5-HT in MMD.

Effects of Gender, Age, and Clot Formation on the Measurements of Tryptophan Metabolites in Blood

Background: Tryptophan metabolism plays important roles in health and diseases. Although simultaneous measurements of tryptophan metabolites are successfully measured, influences of age, gender, and clot formation on the measurements have not been reported. Methods: We took blood from young and old Japanese men and women and compared plasma levels of tryptophan metabolites. We also took plasma and serum from the blood of middle-aged men (n = 10). Metabolites analysis was performed by a liquid chromatograph tandem mass spectrometer, the LCMS-8060 quadrupole mass spectrometer combined with Nexera X2 liquid chromatograph system (Shimadzu Corporation, Kyoto, Japan). Body mass index (BMI) and TRP metabolites have been measured in healthy young men (n = 48), young women (n = 47), old men (n = 44), and old women (n = 39). TRP metabolites were measured by using the ultrahigh speed liquid chromatography-mass spectroscopy (Shimadzu Corporation). Results: Tryptophan and its metabolites such as serotonin, 5-hydroxyindole acetic acid, indole-3-acetic acid, kynurenine, anthranilic acid, were higher in young women and old men than young men and old women. Plasma levels of 3-hydroxykynurenine and xanthurenic acid were lower in young women and old men. Comparison of plasma and serum indicates that most of metabolites were higher in serum than plasma except for 3-hydroxy-kynurenine and quinolinic acid. Conclusion: Metabolites of the upper stream of degradation of tryptophan were higher in young women and old men than young men and old women, which suggests that the degradation of tryptophan was accelerated in young men and old women than young women and old men. Serum preparation may activate tryptophan degradation resulting in higher levels of metabolites in serum than in plasma.

Comparison of Age and Gender Differences of Tryptophan Metabolites in Patients of Major Monopolar and Bipolar Depression

Background: No research has been done for the determination of plasma levels of tryptophan metabolites in patients of monopolar and bipolar depression. Methods: Ultra high-speed liquid chromatography/mass spectrometry has been used for the simultaneous determination of plasma levels of tryptophan metabolites in depressive patients. Results: No significant age and gender differences were shown in monopolar depressive patients and some differences were shown in bipolar patients. The administration of drugs such as antidepressants, antipsychotics, mood stabilizers do not seem to have affected the results. Conclusion: In patients of major monopolar depression age and gender differences of plasma levels of tryptophan metabolites disappear although significant differences are observed in healthy volunteers. Some differences of age and gender differences were shown between monopolar and bipolar depressive patients.

G protein-coupled receptor 35 attenuates nonalcoholic steatohepatitis by reprogramming cholesterol homeostasis in hepatocytes

Nonalcoholic fatty liver disease(NAFLD)is the most common chronic liver disease worldwide.Fat accumulation“sensitizes”the liver to insult and leads to nonalcoholic steatohepatitis(NASH).G protein-coupled receptor 35(GPR35)is involved in metabolic stresses,but its role in NAFLD is unknown.We report that hepatocyte GPR35 mitigates NASH by regulating hepatic cholesterol homeostasis.Specifically,we found that GPR35 overexpression in hepatocytes protected against high-fat/cholesterol/fructose(HFCF)diet-induced steatohepatitis,whereas loss of GPR35 had the opposite effect.Administration of the GPR35 agonist kynurenic acid(Kyna)suppressed HFCF diet-induced steatohepatitis in mice.Kyna/GPR35 induced expression of StAR-related lipid transfer protein 4(STARD4)through the ERK1/2 signaling pathway,ultimately resulting in hepatic cholesterol esterification and bile acid synthesis(BAS).The overexpression of STARD4 increased the expression of the BAS rate-limiting enzymes cytochrome P450 family 7 subfamily A member 1(CYP7A1)and CYP8B1,promoting the conversion of cholesterol to bile acid.The protective effect induced by GPR35 overexpression in hepatocytes disappeared in hepatocyte STARD4-knockdown mice.STARD4 overexpression in hepatocytes reversed the aggravation of HFCF diet-induced steatohepatitis caused by the loss of GPR35 expression in hepatocytes in mice.Our findings indicate that the GPR35–STARD4 axis is a promising therapeutic target for NAFLD.