兰州大学机构库 >第一临床医学院
创伤性颅脑损伤致肾功能亢进的临床及机制研究
Alternative TitleClinical and Mechanism Research of Augmented Renal Clearance Induced by Traumatic Brain Injury
党子龙
Subtype博士
Thesis Advisor武新安
2023-05-30
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name医学博士
Degree Discipline内科学
Keyword创伤性颅脑损伤 Traumatic Brain Injury, Augmented Renal Clearance 肾功能亢进 Transporters 转运体 Brain-kidney axis 脑-肾轴 5-methoxyindole-3-acetic acid (5M) 5-甲氧基吲哚-3-乙酸(5M)
Abstract

目的

肾功能亢进(Augmented Renal Clearance,ARC)严重影响药物疗效。国外研究显示创伤性颅脑损伤(Traumatic Brain Injury,TBI)后ARC发生率高达85%,但国内尚未见相关报道。中国TBI患者数量多,因此探讨中国人TBI致ARC的流行病学、识别手段以及危险因素等显得极为迫切。TBI患者的药物治疗特别是抗感染等治疗对其预后至关重要,头孢曲松是TBI致ARC患者首选的抗感染药物之一。TBI致ARC可能会对其药动学和药效学产生不利影响,但影响程度到底如何,目前常用剂量是否足够有效尚无相关研究。肾清除过程包括肾小球滤过、肾小管分泌和肾小管的重吸收,TBI致ARC患者肾清除率显著增加,到底是肾清除的哪个或哪些过程发生变化,有待探讨。同时,TBI致ARC机制尚待阐明。基于此,本论文拟(1)初步调查TBI致ARC患者的发病率,明确此类患者危险因素,并构建识别ARC的模型;(2)探索TBI致ARC对头孢曲松药动学和药效学的影响及机制;(3)考察TBI致ARC后肾功能的变化及其机制;(4)基于转运体远端遥感理论探究TBI致ARC的发生机制。最终为TBI致ARC患者的精准用药提供理论依据,并阐明TBI致ARC的分子机制,为该类患者的防治及精准用药提供理论依据。

方法

(1)TBI致ARC的现状、危险因素和预测模型构建

设计前瞻性单中心横断面研究,根据纳排标准连续纳入2018年10月1日至2019年9月30日在兰州大学第一医院重症监护病房收治的所有TBI患者。统计TBI致ARC患者发病率,分析人口统计学特征;评价常用的4个肾小球滤过率估算公式和创伤患者肾功能亢进评分系统(ARCTIC)诊断ARC 的准确性;利用ARC患者和非ARC患者差异特征进多元逻辑回归,找出患者ARC发生的独立危险因素;纳入危险因素和4种肾小球滤过率估算公式的诊断界值构建预测ARC的模型。

(2)TBI致ARC对头孢曲松药动学和药动学/药效学靶值达标概率的影响及其机制

设计前瞻性观察性队列研究,纳入使用头孢曲松进行抗感染的TBI致ARC和非ARC患者各15名。于首次用药后于设计的时间点采集患者静脉血样,同时从给药起始收集患者12h尿液;用高效液相色谱测定患者头孢曲松血浆总浓度、游离浓度以及尿中浓度,计算头孢曲松药动学参数和原型药物尿中排泄率;测定两组患者头孢曲松的蛋白结合率;用蒙特卡洛模拟头孢曲松在不同给药方案下的药动学/药效学靶值达标概率。为了进一步探讨TBI致ARC对头孢曲松药动学的影响机制,将SD实验大鼠分为四组:对照组(N组)、TBI致ARC组(A组)、中剂量抑制剂组(C组)以及高剂量抑制剂组(D组);于造模24h后分别腹腔注射给予C组和D组大鼠中剂量(MK571 30mg/kg)、高剂量(MK571 60mg/kg)多药耐药相关转运蛋白(MRP2/4)的抑制剂;抑制剂给药后2h,分别经静脉给予四组大鼠头孢曲松(175.14 mg/kg),然后采集血样和尿样考察四组大鼠药动学的变化。

(3)TBI致ARC后肾功能的变化及其机制

使用Marmarou法构建TBI致ARC大鼠模型,测定对照组大鼠和实验组大鼠24h内生肌酐清除率(24h CrCl);考察TBI致ARC大鼠ARC的持续时间; HE染色后观察TBI致ARC大鼠肾小球和肾小管形态的变化;通过在体/离体泵注菊粉、对氨基马尿酸和二甲双胍的方法考察TBI致ARC大鼠在体/离体肾小球滤过率和肾小管中分泌有机阴离子和阳离子的转运体功能的变化;使用实时荧光定量PCR(qPCR)、Western Blot以及免疫组化考察TBI致ARC对大鼠肾皮质中有机阴离子和有机阳离子转运体mRNA和蛋白表达变化的影响。

(4)基于转运体远端遥感理论探究TBI致ARC的发生机制

通过测定脑组织和脑脊液中伊文思蓝的水平,考察TBI致ARC大鼠的血脑屏障(BBB)和血脑脊液屏障(BCSFB)完整性;提取分离TBI致ARC大鼠BBB和BCSFB后,用qPCR和Western Blot分别分析这两个屏障中Oat3、Mrp4 的mRNA和蛋白表达水平变化;使用靶向代谢组学考察TBI致ARC大鼠脑组织、脑脊液、血液及尿液中OATs-MRPs(Organic anion transporters-Multidrug resistance-associated proteins)内源性底物的变化;考察大鼠松果体中乙酰复合胺-O-甲基转移酶 (ASMT)的mRNA和蛋白表达水平变化;细胞实验考察5-甲氧基吲哚-3-乙酸(5-Methoxyindole-3-acetic acid,5M)对hCMECD3(永生化的BBB细胞)、Z310(永生化的BCSFB细胞)以及RPTEC/TERT1(永生化的肾小管上皮细胞)中MRP2/4和Nrf2的mRNA和蛋白表达水平的影响;利用分子对接和双荧光素酶报告实验考察5M对LXRβ的激动作用;细胞实验考察5M对hCMECD3、Z310以及RPTEC/TRET1中LXRβ的诱导作用;在细胞水平通过给予Nrf2和LXRβ抑制剂考察5M经Nrf2和LXRβ上调hCMECD3、Z310以及RPTEC/TRET1中MRP2/4的表达;动物水平考察TBI致ARC大鼠对BBB、BCSFB以及肾脏中Nrf2和LXRβ的mRNA和蛋白表达水平的影响。

结果

(1)我院TBI致ARC患者发生率为50%;TBI致ARC患者其平均24h CrCl显著高于非ARC患者、既往高血压患病率和血肌酐水平显著低于非ARC患者;由4个肾小球滤过率估算公式计算所得估算肾小球滤过率(Estimated Glomerular Filtration Rate,eGFR)与24h CrCl中等相关,其会高估eGFR较低患者的24h CrCl,而低估eGFR正常和较高患者的24h CrCl,且在ARC组观察到更明显的偏差和更低的精确度;ARCTIC与24h CrCl弱相关;4个eGFR公式和ARCTIC用于诊断ARC时敏感性较高,但特异性均较差;纳入患者的BMI、既往高血压患病率以及eGFR的诊断界值构建的ARC预测模型其特异性、敏感性、阳性预测值和阴性预测值均在74.1%以上。

(2)TBI致ARC不会改变患者的头孢曲松蛋白结合率,但ARC后头孢曲松肾清除率显著增加,尿中12h 累积排泄量显著增加,同时t1/2β和头孢曲松的AUC显著降低。相关性分析发现头孢曲松游离药物清除率、尿液累积排泄量与24h CrCl呈极强相关性。ARC会导致患者的头孢曲松PK/PD靶值达标概率显著降低,常规给药方案用于治疗敏感和耐药的大肠杆菌科细菌感染时均达不到目标治疗效果,敏感细菌感染时可增加用药频次和剂量以提高药效,耐药细菌感染时需考虑更换药物。TBI致ARC大鼠使头孢曲松肾清除显著增加,半衰期明显缩短,AUC显著减低,使用MRP2/4抑制剂MK571后可明显逆转这一趋势。

(3)使用Marmarou法成功构建了TBI致大鼠模型;TBI致ARC大鼠在造模后一周内均会发生ARC。但其肾小管和肾小球的形态与对照组相比没有明显改变。通过在体和离体实验证实TBI致ARC大鼠其肾小球滤过率和肾小管分泌有机阴离子和有机阳离子的功能均显著增加。通过qPCR,Western Blot以及免疫组化从基因水平和蛋白水平均证实肾小管中有机阴离子转运体Oat1、Oat3、Mrp2以及Mrp4,有机阳离子转运体Oct2和Mate1的表达均显著上调。

(4)TBI模型不会破坏BBB和BCSFB的完整性,且这两个屏障中Oat3和Mrp4的mRNA和蛋白表达水平均明显升高;靶向代谢组学分析了24个OATs-MRPs底物,发现只有有机阴离子5M在TBI致ARC大鼠的脑组织、脑脊液以及血液中浓度显著升高,且尿液中12h累积排泄量明显增加,当给予大鼠MPRs的抑制剂后,其在脑组织和脑脊液中浓度进一步显著升高,同时伴有血液中浓度和尿液中累积排泄量的显著减少;TBI致ARC大鼠其松果体中ASMT(代谢产生5M的酶)的mRNA和蛋白表达水平均显著上调;5M对hCMECD3、Z310以及RPTEC/TRET1细胞中MRP2/4中mRNA和蛋白表达水平呈剂量依赖性诱导作用;5M能分别诱导hCMECD3、Z310以及RPTEC/TRET1细胞中Nrf2 和LXRβ mRNA和蛋白表达水平的显著上调;当分别给予三种细胞Nrf2和LXRβ的抑制剂后5M诱导三种细胞中MRP2/4蛋白表达水平的现象被显著逆转;TBI致ARC大鼠其BBB、BCSFB以及肾皮质中Nrf2和LXRβ的mRNA和蛋白表达水平均显著上调。

结论

(1)TBI患者ARC发生率约为50%。无论是eGFR的估算方法还是ARCTIC评分,直接作为识别ARC的工具时都不够准确。将eGFR的诊断界值作为其中一个风险因素,并与患者其他特征,如BMI、是否有高血压病史进行建模,可作为预测TBI患者是否发生ARC的一个非常有用的工具。

(2)TBI 致ARC 患者的头孢曲松肾排泄明显加快,其半衰期明显缩短, AUC和PK/PD 靶值达标概率均明显降低,严重影响其药效。发生这一现象的机制可能与肾小管转运体MRP2/4的转运功能显著上调有关。

(3)TBI 致ARC后肾小球和肾小管形态不会发生改变,但肾小球滤滤过率和肾小管分泌功能均显著增强,且以后者更为显著。肾小管分泌功能的增强与肾小管上皮细胞中有机阴离子OATs-MRPs和OCT2-MATE1的表达及功能上调有关。

(4)TBI 后脑中5M 增多,其通过Nrf2 和LXRβ上调脑屏障中MRP4、肾小管中MRP2/4 表达和功能形成脑-肾远端遥感信号通路,也即脑-肾轴的形成,从而导致ARC;且我们首次发现5M是LXRβ激动剂。

Other Abstract

Objective

Augmented Renal Clearance (ARC) severely reduces the efficacy of the drugs. Foreign studies have shown that the incidence of ARC after traumatic brain injury (TBI) is as high as 85%, but no relevant report exists in China. There are many TBI patients in China, so it is highly urgent to explore the epidemiology, identification methods, and risk factors of ARC induced by TBI in the Chinese population. Drug treatment, especially anti-infection, is crucial to the prognosis of TBI patients. Ceftriaxone is one of the preferred anti-infective drugs for TBI-induced ARC patients. TBI-induced ARC may have adverse effects on its pharmacokinetics and pharmacodynamics. Still, there is no relevant research on the extent of the impact and whether the current commonly used doses are sufficient. The renal clearance process includes glomerular filtration, renal tubular secretion, and renal tubular reabsorption. The renal clearance rate in ARC patients significantly increased after TBI, but which processes of renal clearance are changed remains to be explored. Meanwhile, the mechanismTBI-induced ARC remains to be elucidated. Based on this, the paper intends to (1) Preliminarily investigate the incidence of ARC patients induced by TBI in China, clarify the risk factors of such patients, and construct a model for identifying ARC; (3) To investigate the changes in renal function and its mechanism after TBI-induced ARC; (4) To explore the mechanism of TBI-induced ARC based on the remote sensing theory of drug transporters. Ultimately, it provides a theoretical basis for the precise medication of TBI-induced ARC patients; and clarifies the molecular mechanism of TBI-induced ARC, providing a theoretical basis for such patients' prevention and precision medication.

Methods

(1) The status, risk factors, and prediction model construction of TBI-induced ARC

A prospective single-center cross-sectional study was designed. All patients with TBI admitted to the intensive care unit of Lanzhou University First Hospital from October 1, 2018, to September 30, 2019, were continuously included according to inclusion and exclusion criteria. The incidence rate of ARC patients caused by TBI was counted, and the demographic characteristics were analyzed; The four commonly used glomerular filtration rate estimation formulas and the accuracy of the Augmented Renal Clearance in Trauma Intensive Care Scoring System(ARCTIC) in diagnosing ARC were evaluated; Used the different characteristics of ARC patients and non-ARC patients to conduct multiple logistic regression to find out the independent risk factors of ARC patients; The risk factors and the diagnostic cut-off values of the four glomerular filtration rate estimation formulas were included to construct a model for predicting ARC.

(2) Effects of TBI-induced ARC on the PK and PK/PD target achievement probability of ceftriaxone and its mechanism

A prospective observational cohort study was designed, including each 15 patients in ARC and non-ARC groups caused by TBI who were treated with ceftriaxone for anti-infection. Collect venous blood samples from patients at 0, 0.5, 1, 1.5, 3.5, 5.5, 7.5, and 11.5 hours after the first administration, and collect 12h urine of patients from the beginning of administration; The plasma total concentration, free concentration and urine concentration of ceftriaxone were measured by high-performance liquid chromatography, and the pharmacokinetic parameters of ceftriaxone total drug and free drug were calculated by DAS2.0 software; Calculate the urinary excretion rate of unchanged ceftriaxone; Determine the protein binding rate of ceftriaxone in two groups of patients; Use Monte Carlo to simulate the probability of target attainment of pharmacokinetics/pharmacodynamics of ceftriaxone under different dosage regimens.

The SD experimental rats were divided into four groups: control group (N group), TBI-induced ARC group (A group), middle-dose inhibitor group (C group), and high-dose inhibitor group (D group); Group C and D were given medium-dose (MK571 30mg/kg) and high-dose (MK571 60mg/kg) inhibitors of multidrug resistance-related transporter (MRP2/4) by intraperitoneal injection, respectively. Two hours after administering the inhibitor, ceftriaxone (175.14mg/kg) was administered intravenously to the four groups of rats. Then blood and urine samples were collected to investigate the pharmacokinetic changes of the four groups of rats.

(3) Changes and mechanisms of renal function after TBI-induced ARC

Use the Marmarou method to construct a TBI-induced ARC rat model, measure the 24h creatinine clearance rate (24h CrCl) of the rats in the control and the experimental group. Investigated the duration of ARC in TBI-induced ARC rats; Used HE staining to examine TBI-induced ARC Morphological changes of rat glomeruli and renal tubules; In vivo injection of inulin, p-aminohippuric acid, and metformin to investigate changes in glomerular filtration rate and functions of organic anions and cations transporter in renal tubules of TBI-induced ARC rats. Real-time PCR (qPCR), Western Blot, and immunohistochemistry were used to investigate the effect of TBI-induced ARC on the mRNA and protein expression changes of organic anion and organic cation transporter in rat renal cortex.

(4) Explore the mechanism of TBI-induced ARC based on the remote sensing theory of transporters

The integrity of the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) of ARC rats induced by TBI was investigated by measuring Evans blue in brain tissue and cerebrospinal fluid; After extracting and separating the BBB and BCSFB of ARC rats caused by TBI, the mRNA and protein expression levels of Oat3 and Mrp4 in these two barriers were analyzed by qPCR and Western Blot respectively; Using targeted metabolomics to investigate the changes of endogenous substrates of OATs-MRPs in the brain tissue, cerebrospinal fluid, blood and urine of TBI-induced ARC rats; Investigate the changes of mRNA and protein expression levels of acetylserosamine-O-methyltransferase (ASMT) in the pineal gland of TBI-induced ARC rats; The cytotoxicity effects of 5-methoxyindole-3-acetic acid (5M) on hCMECD3 (immortalized BBB cells), Z310 (immortalized BCSFB cells) and RPTEC/ TERT1 (immortalized renal tubular epithelial cells) was investigated by CCK-8 method; Cell experiments were conducted to examine the effect of 5M on the mRNA and protein expression levels of MRP2/4 and Nrf2 in hCMECD3, Z310 and RPTEC/TRET1; Molecular docking of 5M with nuclear receptor proteins was carried out using AutoDock to investigate whether 5M is a ligand for nuclear receptors; Use dual luciferase reporter assay to investigate the agonistic effect of 5M on LXRβ; Cell experiment was used to investigate the induction effect of 5M on LXRβ in hCMECD3, Z310 and RPTEC/TRET1; By administering Nrf2 and LXRβ inhibitors, to explore 5M can up-regulates the expression of MRP2/4 in RPTEC/TRET1, hCMECD3 and Z310 via Nrf2 and LXRβ at the cellular level; The Nrf2 and LXRβ mRNA and protein expression levels were investigated in BBB, BCSFB, and kidney of TBI-induced ARC rats.

Results

(1) The incidence rate of TBI-induced ARC patients in our hospital was 50%; The average 24h CrCl of TBI-induced ARC patients was significantly higher than that of non-ARC patients, and the prevalence of medical history of hypertension and serum creatinine level were substantially lower than those of non-ARC patients; The estimated glomerular filtration rate (eGFR) calculated by the 4 glomerular filtration rate estimation formulas was moderately correlated with 24h CrCl, which would overestimate 24h CrCl in patients with low eGFR, and underestimate 24h CrCl in patients with normal and higher eGFR, and more pronounced bias and lower precision were observed in the ARC group; ARCTIC was weakly correlated with 24h CrCl; 4 eGFR formulas and ARCTIC had high sensitivity but poor specificity when used in the diagnosis of ARC; The ARC prediction model’s specificity, sensitivity, positive predictive value and negative predictive value of the predictive model were all above 74.1%, which constructed by including patients' BMI, prevalence of medical history of hypertension and eGFR diagnostic cut-off values.

(2) TBI-induced ARC did not change the protein binding rate of ceftriaxone. Still, after ARC, the renal clearance and the 12h cumulative excretion of ceftriaxone in urine increased significantly, while the AUC and t1/2β of ceftriaxone decreased significantly. Correlation analysis found that the free drug clearance rate and cumulative urine excretion of ceftriaxone highly correlated with 24h CrCl. ARC would dramatically reduce the probability of patients' target attainment of ceftriaxone PK/PD. The target therapeutic effect cannot be achieved when the conventional dosing regimen is used to treat sensitive and drug-resistant Escherichia coli bacterial infections. In the case of sensitive bacterial infection, the frequency and dosage of medication can be increased to improve the drug's efficacy. In the case of drug-resistant bacterial infection, drug replacement should be considered. In TBI-induced ARC rats, the renal clearance of ceftriaxone was significantly increased, the half-life was shortened considerably, and the AUC was significantly decreased. This trend can be reversed after the MRP2/4 inhibitor MK571 was administered.

 (3) The TBI-induced rat model was successfully established using the Marmarou method; ARC rats with TBI-induced ARC will develop ARC within one week after the model is established. However, the renal tubules and glomeruli morphology did not change significantly compared with the control group. In vivo and in vitro experiments confirmed that TBI-induced ARC rats had increased glomerular filtration rate and renal tubular secretion of organic anions and organic cations, and the increase in renal tubular secretion was more prominent, that is, after TBI, renal tubules secretion functionality of both organic anion and organic cation transporters is increased. The expression of organic anion transporters(Oat1, Oat3, Mrp2, and Mrp4) and organic cation transporters(Oct2 and Mate1) in renal tubules were up-regulated at the gene level and protein level.

(4) The TBI model didn’t destroy the integrity of BBB and BCSFB, and the mRNA and protein expression levels of Oat3 and Mrp4 in these two barriers are increased; Targeted metabolomics analysis of 24 OATs and MRPs substrates found that only the organic anion 5M was significantly increased in the brain tissue, cerebrospinal fluid and blood of TBI-induced ARC rats, and its’ 12-h cumulative excretion in the urine was also considerably increased. When the inhibitor of MPRs was administered to rats, its concentration in brain tissue and cerebrospinal fluid was further significantly increased, accompanied by a significant decrease in blood concentration and cumulative excretion in urine; The mRNA and protein expression levels of ASMT (an enzyme that metabolizes 5M) in the pineal gland of TBI-induced ARC rats were significantly up-regulated; 5M has a dose-dependent induction effect on the mRNA and protein expression levels of MRP2/4 in hCMECD3, Z310 and RPTEC/TRET1 cells; 5M induced significant up-regulation of Nrf2 and LXRβ mRNA and protein expression levels in hCMECD3, Z310 and RPTEC/TRET1 cells, respectively; When the inhibitors of Nrf2 and LXRβ were given to the three kinds of cells respectively, the phenomenon that 5M induced the up-regulated protein expression levels of MRP2/4 was significantly reversed; The mRNA and protein expression levels of Nrf2 and LXRβ in BBB, BCSFB, and the renal cortex of TBI-induced ARC rats were significantly up-regulated.

Conclusion

(1) The incidence of ARC in TBI patients is about 50%. Neither the estimation method of eGFR nor the ARCTIC score was accurate enough when directly used to identify ARC. Using the diagnostic cutoff value of eGFR as one of the risk factors and modeling it with other patient characteristics, such as BMI and history of hypertension, can be a handy tool for predicting ARC in TBI patients.

(2) The renal excretion of ceftriaxone in patients with ARC caused by TBI was significantly accelerated, its half-life was shortened considerably, and the ceftriaxone AUC and the probability of target attainment of PK/PD was significantly reduced, which seriously affected the efficacy of ceftriaxone. The mechanism of this phenomenon may be related to the up-regulation of the transport function of MRP2/4.

(3) After TBI-induced ARC, the morphology of glomeruli and renal tubules wasn’t change. Still, the glomerular filtration rate and renal tubular secretion function increased significantly, especially the latter, associated with the expression and functional upregulation of OATs-MRPs and OCT2-MATE1 in renal tubular epithelial cells.

(4) 5M increased in the brain after TBI, which can up-regulate the expression and function of MRP4 in the brain barrier and MRP2/4 in the renal tubules through Nrf2 and LXRβ, forms the brain-kidney remote sensing signal pathway that is the formation of the brain-kidney axis, which leads to ARC. And for the first time, we found that 5M is an LXRβ agonist.

 

MOST Discipline Catalogue医学 - 临床医学 - 内科学(含:心血管病、血液病、呼吸系病、消化系病、内分泌与代谢病、肾病、风湿病、传染病)
URL查看原文
Language中文
Other Code262010_120180901451
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/537614
Collection第一临床医学院
Affiliation
兰州大学第一临床医学院
Recommended Citation
GB/T 7714
党子龙. 创伤性颅脑损伤致肾功能亢进的临床及机制研究[D]. 兰州. 兰州大学,2023.
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