DPP-4抑制劑對腎臟疾病之藥理作用與分子機制研究

Study of pharmacological effect and molecular mechanism of DPP-4 inhibitor in kidney diseases

王偉傑(Wei-Jie Wang)

指導教授 ： 翁清松

中原大學/工學院/生物醫學工程研究所/博士(2014年)

https://doi.org/10.6840/cycu201400872

摘要

摘要
糖尿病腎病變是造成腎臟衰竭最主要的原發疾病；即使血糖獲得良好的控制，往往糖尿病仍會造成慢性腎臟病、甚至腎臟衰竭。 過去多類的口服降血糖用藥及胰島素並不能降低慢性腎臟病、 甚至腎臟衰竭的發生。過去幾年隨著新型降血糖用藥胰高血糖素樣肽-1 (GLP-!)及基肽酶-4抑制劑 (DPP-4 inhibitors)的上市，越來越多的研究發現這些藥物除了降低血糖之外，也可以保護微血管內皮。因此我們想要研究慢性腎病的致病機轉及基肽酶-4抑制劑是否對這樣的尿毒傷害有保護的作用。
為了研究尿毒素硫酸吲哚酚（indoxyl sulfate）對腎小球系膜細胞的作用。大鼠腎小球系膜細胞暴露於一系列濃度尿毒素中。尿毒素對腎小球系膜細胞的細胞毒性作用採用MTT法測定 裂解的半胱天冬-3(caspase-3)的蛋白水平，血管緊張素，血管緊張素轉換酶（ACE）和腎素是利用免疫印跡法檢測。反轉錄聚合酶鏈反應(PCR)技術來確定基因(mRNA)的表達。
接著我們觀察尿毒素對腎小管上皮細胞影響和二肽基肽酶-4抑制劑的保護作用。人類腎臟細胞2（HK-2）細胞暴露於二肽基肽酶-4抑制劑存在或不存在的硫酸吲哚酚所致的尿毒環境中。二肽基肽酶-4抑制劑對HK-2細胞存活率的影響採用MTT法測定。使用螢光顯微鏡來對活性氧簇（ROS）的產生進行研究。通過免疫印跡檢測裂解的半胱天冬-3，轉化生長因子-β（TGF-β）、α-平滑肌肌動蛋白（α-SMA）、NF-κB p65和AKT的磷酸化和細胞外信號調節激酶（ERK）的水平。
再接著我們探討基肽酶-4抑制劑對大鼠缺血再灌注急性腎損傷(AKI）的影響。小鼠經由手術誘發急性腎損傷，同時實驗組也給予基肽酶-4抑制劑，我們也採取血液，以測定血漿尿素氮，肌酐和電解質濃度。腎臟組織我們也在染色後用顯微鏡觀察。
最後我們進入人體實驗我們假設基肽酶-4抑制劑可以延緩糖尿病腎病患者腎小球濾過率的減少。我們招募 30位在醫院門診其罹患糖尿病及腎功能下降的患者作為研究。
在尿毒及腎臟系膜細胞的實驗中: 尿毒濃度越高則裂解的半胱天冬-3的水平越高，細胞的存活越低。經尿毒素處理之腎小球系膜細胞，其親腎素和血管緊張素轉換酶的基因(mRNA)的表達上升。腎素和血管緊張素轉換酶的水平與尿毒素處理時間有正相關性。
在尿毒及腎臟小管細胞的實驗中: 活性氧簇的產生和裂解的半胱天冬-3的水平與尿毒素濃度呈正相關。 NF-κB的激活伴隨著磷酸化AKT和ERK、 p65蛋白的降低。 在尿毒素處理過的HK-2細胞其轉化生長因子-β和α-平滑肌肌動蛋白的表達被上調。在尿毒素處理人類腎臟細胞2，基肽酶-4抑制劑顯著增加細胞存活和減少活性氧簇的產生。基肽酶-4抑制劑恢復AKT和ERK的磷酸化和抑制NF-κB路徑的活化。此外，基肽酶-4抑制劑也能抑制轉化生長因子-β和α-平滑肌肌動蛋白的表達。
在探討基肽酶-4抑制劑對大鼠缺血再灌注急性腎損傷（AKI）的影響的實驗中: 在實驗組，有用基肽酶-4抑制劑治療者其增加的血漿肌酸酐和尿素氮濃度與對照組相同。基肽酶-4抑制劑的治療能恢復的上升的NGAL和HO-1，而SDF-1和HMGB-1的水平不變。
在人體實驗中: 在第12週，飯前平均血糖下降到131.0±25.3毫克/升，有9.3％的減少。糖化血色素降低至6.6±0.5％，比基值減少了23％。觀察12週後， 24小時的尿蛋白流失量(UPCR)下降6.0％。然而，所有的數據在開始研究和12週後之間並沒有顯著差異。
根據上述的一系列研究，我們得到以下結論:
尿毒素對細胞活性的影響是通過調節凋亡途徑中半胱天冬-3活性。 尿毒素誘發腎小球系膜細胞中腎素-血管緊張素(RAS)系統的表達。
基肽酶-4抑制劑具有抗細胞凋亡活性，改善了尿毒素誘導的腎損害，其部分歸因於調節 ROS/p38MAPK/ERK和PI3K-AKT途徑以及下游NF-κB信號傳導途徑。
基肽酶-4抑制劑也可以可降低灌注損傷所造成的腎功能障礙和細胞損害。
基肽酶-4抑制劑對於有罹患糖尿病及腎功能下降的患者儘管經過三個月的研究，其血糖下降、腎功能的維持、蛋白尿的下降及各項生化指標皆沒有達到有意義的保護作用；本實驗的限制包括樣本量小，沒有對照組，開始研究和12週後抽血時病人是否空腹的狀態並不相同。未來修正之前的實驗缺失後再度實驗 其結果仍是值得期待的。

關鍵字

慢性腎臟病 ； 糖尿病 ； 尿毒素Indoxyl sulfate ； DPP-4 抑制劑 ； 急性腎損傷

並列摘要

Abstract
Background
Diabetes is the most common cause of kidney failure. Even when diabetes is controlled, the disease can lead to CKD and kidney failure. Conventional treatments such as insulin treatment fail to prevent diabetes patient from developing CKD that eventually progresses to kidney failure. Several classes of drug have been developed and prescribed in clinical practices, including GLP-1 agonist and DPP-4 inhibitor. Increasing evidence has demonstrated the beneficial effects of DPP-4 inhibitor on microvascular endothelia. It is of interest to bring the missing piece of pathogenesis of indoxyl sulfate-mediated CKD and to get better understanding of pharmacology of DPP-4 inhibitor in CKD setting.

Material and Methods
To investigate the deleterious effects of indoxyl sulfate on kidney mesangial cells, rat renal mesangial cells were exposed to indoxyl sulfate at a serial concentrations. Cytotoxicity of indoxyl sulfate on renal mesangial cells were determined using MTT assay. Protein levels of cleaved caspase-3, angiotensin, angiotensin converting enzyme (ACE) and renin were detected by immunoblotting. Reverse transcriptional PCR was performed to determine the mRNA expression. To investigate the deleterious effects of IS on proximal tubular cells and the protective role of DPP-4 inhibitor. Human kidney 2 (HK-2) cells were exposed to IS in the presence or absence of DPP-4 inhibitor. Effects of DPP-4 inhibitor on viability of HK-2 cells were determined by MTT assay. Reactive oxygen species (ROS) production was examined using fluorescent microscopy. Levels of cleaved caspase-3, transforming growth factor-beta (TGF-), α-smooth muscle actin (α-SMA) and NF-kappaB p65 and phosphorylation of AKT and extracellular signal-regulated kinase (ERK) were detected by immunoblotting. To study beneficial effects of DPP-4 inhibitor on acute kidney injury (AKI) induced by ischemia and reperfusion, mice were surgically developed AKI and given DPP-4 inhibitor At the end of this period, blood samples were also taken to determine plasma urea nitrogen, creatinine, and electrolyte concentrations. The kidney tissues were collected for histological studies. To study the effects of DPP-4 inhibitor on kidney function and renal microvasculature of diabetes patients, 30 outpatients with decreased GFR and diabetes were recruited and followed up in a nephrology clinic at a general hospital.

Results
Levels of cleaved caspase-3 were augmented while the cell viability was inhibited by indoxyl sulfate in a dose-dependent manner. The mRNA expressions of pro-renin and ACE were upregulated in mesangial cells exposed to indoxyl sulfate. Level of renin and ACE were increased in response to indoxyl sulfate exposure in time-dependent fashion. Indoxyl sulfate induces proliferation and cell death of renal mesangial cell, which is time-dependent.
Production of ROS and level of cleaved caspase-3 in HK-2 cells were increased by IS in a dose-dependent manner. The phosphorylation of AKT and ERK p65 were decreased alongside activation of NF-κB. Expression of TGF-β and α-SMA, were upregulated in IS-treated HK-2 cells. Treatment with DPP-4 inhibitor resulted in a significant increase in cell viability and a decrease of ROS production in IS-treated HK-2 cells. DPP-4 inhibitor restored IS-induced deactivations of AKT and ERK and inhibited activation of NF-κB in IS-treated HK-2 cells. Moreover, DPP-4 inhibitor could also attenuate IS-induced up-regulation of TGF-β and α-SMA expression.
In the DPP-4 treament group, the increase in plasma creatinine and urea nitrogen concentrations was significantly less following reperfusion, and their values reached the same level as that in the sham group. Creatinine clearance and urine osmolarity in the treatment group was higher in comparison with the AKI group. DPP-4 inhibitor treatment restored increased levels of NGAL and HO-1, whereas levels of SDF-1 and HMGB-1 were unchanged.
At 12 weeks, the mean AC declined to 131.0± 25.3 mg/dl, a 9.3% reduction at the end of the12-week study period. HbA1c was reduced to 6.6±0.5 %, a 23% reduction from baseline. A 6.0 % reduction in UPCR at 12 weeks was observed. However, there is no significant difference between baseline and 12-weeks in the population enrolled. Limitations included small sample size, differences before study entry in the physical conditions and absolute status of kidney function.

Conclusion
The loss of cell viability is attributed to caspase-3 activity through apoptosis pathway. RAS in renal mesangial cell is activated in response to indoxyl sulfate treatment. Our findings suggest that DPP-4 inhibitor possesses anti-apoptotic activity to ameliorate the IS-induced renal damage, which may be partly attributed to regulating ROS/p38MAPK/ERK and PI3K-AKT pathways as well as downstream NF-κB signaling pathway. DPP-4 inhibitor may decrease kidney functional disturbance and cellular damages following reperfusion injury in rats. DPP-4 inhibitor may slow the estimated GFR decrease in diabetes patients.

並列關鍵字

Acute kidney injury ； Diabetes mellitus ； Chronic kidney disease ； Indoxyl sulfate ； DPP-4 inhibitor

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