高葡萄糖所誘導脂肪細胞外泌體在腎臟病變之作用機制的探討

Abstract

糖尿病是現代人群當中，最常見的慢性疾病之一，長期的高血糖會導致各種併發症，包括像是心臟、腦血管、周邊血管疾病和腎臟病變。我們的研究重點是糖尿病高血糖導致的腎臟併發症，因為大約 40% 的第 2 型糖尿病患者，具有糖尿病的腎臟病變，這種疾病的發生和死亡率很高。和肥胖有關的脂肪細胞功能障礙，是第 2 型糖尿病重要的危險因子。除了在儲存能量方面的作用之外，脂肪細胞還發揮內分泌細胞的作用，分泌一系列激素和細胞激素，它們是代謝和平衡的重要調節劑。最近的研究顯示，功能失調的脂肪組織會分泌病理訊號，直接損害其他組織，可能也會導致腎臟疾病的發展。近年來，外泌體已經成為，細胞之間通訊的重要介質，這些細胞外囊泡可以將許多的生物活性分子（如RNA和蛋白質）運送到遠處的目標細胞，從而影響遠端細胞和組織的功能。本研究是在探討，高糖條件下所誘導的脂肪細胞，分泌的外泌體，是否帶有影響遠端腎臟細胞的異常分子訊號，進而導致糖尿病腎臟病變的發病機制。為了去驗證我們的假設，利用模擬第 2 型糖尿病的環境，培養脂肪細胞，分別將脂肪細胞培養在低糖環境（5.5 mM）模擬正常人血糖環境和高糖環境（33 mM），並且收集培養脂肪細胞的培養液。隨後，透過差速離心從這些脂肪細胞中分離外泌體，確保外泌體的純度和質量，並使用奈米粒子追蹤分析（NTA）、電子顯微鏡(TEM)和蛋白質印跡進行表徵，以確認其大小和蛋白質的量。外泌體PKH26染色結果證實脂肪細胞來源的外泌體可被CIHP-1和HK-2細胞內化。此外，高糖條件下的外泌體處理的CIHP-1、HK-2細胞活力顯著降低。以高糖條件下來源的外泌體處理CIHP-1和HK-2細胞的形態學結果顯示，細胞形態明顯轉變為長條狀，表明HK-2已成為上皮間質轉化（EMT）類型。CIHP-1細胞經過高糖來源的外泌體處理後，鬼筆環肽F-肌動蛋白染色結果顯示細胞骨架紊亂，足突消失，白蛋白內流實驗結果顯示狹縫隔膜的完整性受到一定破壞，損害了狹縫隔膜的濾過屏障功能。為了探究外泌體糖尿病併發症的分子機制，採用蛋白質體學對外泌體蛋白進行蛋白質體學分析。研究結果表明，外泌體蛋白與 ECM 積聚、纖維化途徑密切相關，可能導致糖尿病腎臟併發症。未來的實驗將對透過蛋白質體學分析確定的目標路徑進行，以確認這些假設的準確性。

Diabetes is among the most prevalent chronic diseases in modern populations. Prolonged hyperglycemia can lead to various complications, including Cardiovascular diseases, cerebrovascular diseases, peripheral vascular diseases and nephropathy. Our research focuses on diabetic kidney complications, as approximately 40% of patients with type 2 diabetes (T2D) develop diabetic nephropathy, a condition associated with high morbidity and mortality rates. Obesity-related adipocyte dysfunction is a significant risk factor for T2D. Beyond their role in energy storage, adipocytes function as endocrine cells, secreting a range of hormones and cytokines collectively referred to as adipokines, which are critical regulators of metabolic homeostasis. Recent studies have demonstrated that dysfunctional adipose tissue can secrete pathological signals that directly damage other tissues, contributing to the development of kidney disease. In recent years, exosomes have emerged as key mediators of intercellular communication. These extracellular vesicles can transport bioactive molecules, such as proteins, lipids, and RNAs, to distant target cells, thereby influencing the function of remote cells and tissues. This study aims to investigate whether exosomes secreted by adipocytes under high-glucose conditions carry abnormal molecular signals that affect distal renal cells, contributing to the pathogenesis of diabetic kidney complications. To test this hypothesis, we cultured adipocytes by simulating the environment of type 2 diabetes, isolating the exosomes they secreted during the adipogenesis process of cells under both low (5.5 mM) and high (30 mM) glucose concentrations. Following this, exosomes isolated from these adipocytes through differential centrifugation, ensuring the purity and quality of the exosomes and characterized using Nanoparticle Tracking Analysis (NTA), transmission electron microscopy and western blot, which confirmed their size and protein content. The result of exosomes PKH26 staining confirmed that adipocyte-derived exosomes could be taken up by CIHP-1 and HK-2 cell lines. In addition, CIHP-1, HK-2 cells treated with exosomes derived from high glucose conditions exhibited a significant reduction in viability. Morphology of CIHP-1 and HK-2 cells treated with exosomes derived from high glucose conditions, the results showed that the morphology obviously changed into a long strip shape, indicating that HK-2 has become an Epithelial–mesenchymal transition (EMT) type and CIHP-1 also showed an EMT type and gradually develops into renal fibrosis. After CIHP-1 cells were treated with high glucose-derived exosomes, phalloidin F-actin staining results showed cytoskeleton disorder , the loss of foot processes and the results of the albumin influx assay clearly indicate compromised slit diaphragm integrity, leading to impaired filtration barrier function in the podocyte monolayer. This disruption ultimately contributes to proteinuria and glomerulosclerosis. To find out the molecular mechanism of exosome-diabetic complication, exosome protein was performed by LC MS/MS for proteomics analysis. The result show that exosome proteins were high relative with excessive extracellular matrix (ECM) accumulation, fibrosis pathway which could lead to diabetic kidney complications. Future experiments will be conducted on the target pathways identified through proteomic analysis to confirm the accuracy of these hypotheses.