Diabetic nephropathy model: animal model, two-dimensional cell simulation and three-dimensional organoid model

doi:10.12307/2025.634

Abstract

Abstract: BACKGROUND: In recent years, human pluripotent stem cell derived kidney organoids and rodent models of diabetic kidney disease have also made some progress. However, because the pathogenesis of diabetic kidney disease is affected by environmental factors and genetic factors, the pathogenesis is complex, and the clinical treatment for diabetic kidney disease patients needs to vary from person to person. Therefore, more flexible and integrated approaches need to be developed, leading to the discovery of strong preclinical evidence to support more targeted interventions in patients with diabetic kidney disease.
OBJECTIVE: To reviewthe research progress of diabetic kidney disease model from the aspects of diabetic kidney disease animal model, two-dimensional cell culture simulation diabetic kidney disease model, and three-dimensional organoid diabetic kidney disease model, to provide clues and ideas for further research.
METHODS: China National Knowledge Network and PubMed databases were searched with“diabetes, diabetic nephropathy, diabetic kidney diseases, diabetic nephropathy models, diabetic nephropathy animal models, organoids, diabetic and organoids, diabetic and kidney organoids, kidney organoids, diabetic nephropathy cell models, diabetic nephropathy syndrome combined animal models” as Chinese and English search terms. Totally 101 articles were finally included for review and analysis.
RESULTS AND CONCLUSION: Both in vivo and in vitro models of diabetic kidney disease are powerful tools to further study the pathogenesis of diabetic kidney disease. The interactions between multiple systems can be observed in animal models. Two-dimensional cell culture is easy to operate and low cost. The emerging kidney organoids fill the gap between two-dimensional and global levels, without species differences, and simulate the complexity of human kidney to a certain extent. With the continuous development of organoid technology, kidney organoids are expected to provide a new perspective for exploring the pathogenesis, pathophysiology, and drug screening of diabetic kidney disease.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Diabetic kidney disease, kidney organoids, diabetic nephropathy organoid model, cell models, animal models, TCM syndrome combined with diabetic nephropathy model, engineered tissue construction

CLC Number:

Qian Zuping, Chen Yong, Ran Yan, Da Jingjing, Zha Yan. Diabetic nephropathy model: animal model, two-dimensional cell simulation and three-dimensional organoid model [J]. Chinese Journal of Tissue Engineering Research, 2025, 29(17): 3632-3640.

Figures/Tables 7

2.1 肾类器官近年来，兴起的“类器官”为研究糖尿病疾病模型提供了一种新的、具有潜力的模型。类器官是由干细胞产生的、能够在体外自发形成组织的3D培养系统，具有稳定的遗传背景，可以再现器官结构和功能[17]。三维培养的肾类器官模拟了人体复杂的组织结构和一些简单的功能，它们的发展标志着二维细胞培养的巨大飞跃，二维细胞培养和动物模型对研究肾脏疾病均做出了巨大贡献，然而这些模型往往不能完全复制人体器官的生理和病理过程[18]；并且由于物种之间的差异影响，限制了将研究结果转化为临床应用[19]；此外，由于人体器官生理和病理过程的复杂性，依靠单纯的二维细胞培养不能满足细胞之间错综复杂的相互作用关系，一个结构化的三维类器官为解决这些问题提供了一种可能[20-21]。最早有关肾类器官的报道是通过不添加任何细胞因子的血清自分化而产生胚胎后肾以及少量的肾祖细胞[11]。随后，经过科研人员的不断探索[22]，将人多能干细胞诱导分化形成包含足细胞、近端小管上皮细胞、远端小管上皮细胞及血管内皮细胞等多种肾脏固有细胞三维肾脏类器官[23]。尽管不同的诱导方法在诱导时间、复杂性和可扩展性方面存在差异，但最终的类器官在组织复杂性和成熟度方面是相似的，能提供多种细胞间相互作用及协作发育并形成具有一定结构和功能的迷你肾，能更好地模拟人肾脏发育及生理病理状态，并且已经有许多研究利用肾类器官模拟肾脏疾病模型，如遗传性多囊肾[24-25]、急性肾损伤和肾癌等[26-28]。同时肾类器官也是未来建立糖尿病肾病模型的潜在工具，来自不同个体的多能干细胞衍生的肾脏类器官包含人类遗传异质性，可以分析糖尿病肾病患者个体间的差异性；还能从糖尿病肾病的动物模型在人类背景下进行验证，而不需要患者的肾组织样本；同时还能在类器官中进行高通量药物筛选和治疗评估[29]。肾类器官不仅在肾脏发育、疾病建模及药物筛选方面具有重要作用，而且，根据最新的研究报道，利用免疫逃避诱导的肾类器官有望用于临床移植为临床肾移植带来了一定的希望[30]。 2.2 糖尿病肾病类器官模型研究显示，由人多能干细胞诱导分化的含有内皮细胞及周细胞的血管类器官，暴露在高血糖或炎症细胞因子条件下能较好地模拟糖尿病对血管的损伤，出现与临床患者相似的病理改变即血管基底膜增厚[15](如图4)。由于糖尿病肾病是糖尿病晚期微血管并发症之一，因此该研究为后续建立糖尿病肾病类器官模型奠定基础。近期的一项研究表明，将人多能干细胞源肾类器官放在体外建立的糖尿病环境中(以24 h为一周期，在奇数天加入5 mmol/L葡萄糖，在偶数天加入25 mmol/L 葡萄糖)，与模拟健康个体的葡萄糖恒定为5 mmol/L的肾类器官相比，糖尿病环境下的肾类器官出现了纤维化等糖尿病肾病病理特征以及改变了肾类器官的代谢[29](如图5)。"

目前，一方面，虽然肾类器官仍处于胎肾阶段，如何促进其成熟并具备功能是未来利用类器官研究糖尿病肾病的关键因素[23]；另一方面，肾脏类器官的有限寿命将是糖尿病肾病研究需要克服的一个重要限制，尽管已经有研究报道可以将类器官培养至少60多天[14]，但是这些类器官在培养30多天后会发生纤维化和脱靶细胞的扩张[23]，这种自发性纤维化也会使发现糖尿病肾病的晚期纤维化变得更加困难[31]。肾类器官模拟糖尿病肾病的体外培养条件，如葡萄糖浓度、作用时间及是否要加入炎症因子等因素仍需探索。尽管以上提到了肾脏类器官的一些限制，然而，随着对类器官的研究，不断有研究人员提出一些解决办法，如有研究人员发现微环境的流变学和力学特性对类器官培养及成熟至关重要[32]。因此，有许多研究人员将生物材料与类器官相结合，通过改变培养类器官的微环境，从而促进了类器官的成熟，如水凝胶[33]、肾脱细胞细胞外基质水凝胶和流体动力学等[34-35]。肾脏微血管的损伤对于糖尿病肾病发生发展过程具有重要意义，最近有研究报道他们创建了一个可灌注的、血管化的肾类器官芯片模型，证明了右旋糖酐和红细胞可以通过大血管、微血管网络灌注到肾类器官中足细胞簇中，这增加了建立糖尿病肾病类器官模型的可行性[36]。在糖尿病肾病早期，系膜细胞的改变对于疾病的发生发展具有重要作用[37]，最新的研究报道通过CHIR99021、SAG、SB431542、IL-1b和RA选择性地诱导了人多能干细胞来源的肾间充质干细胞，从而诱导分化出系膜细胞和erythropoietin(EPO)细胞[38]，这为研究糖尿病肾病早期系膜细胞与足细胞及小管细胞之间的相互作用机制提供了一个新的可能。此外，肾类器官缺乏免疫细胞，而免疫细胞在糖尿病肾病中的作用不容忽视，尽管目前的手段无法在肾类器官中诱导出免疫细胞，但最近有研究人员将人诱导多能干细胞衍生的肾类器官与人外周血单个核细胞共培养，观察两者的相互作用，为在肾脏类器官中引入免疫细胞研究肾脏疾病及药物相互的作用创造了一个更现实的模型[39]。同时，有研究人员采用人单核细胞和人多能干细胞诱导分化的巨噬细胞利用transwell共培养的方法分化人多能干细胞，发现这种方法导致肾类器官数量更多、形态结构更好，为提高肾脏类器官模型的实用性提供了一种新的途径[40]。说明免疫细胞不仅在肾脏疾病中不可或缺，在肾脏发育过程中也具有重要作用。在利用肾类器官建立糖尿病肾病模型时，一方面不仅可以通过改变体外培养条件来建立，也可以尝试利用糖尿病肾病患者的细胞来建立肾类器官，以概括糖尿病的表观遗传易感性，还可以从基因修饰的人多能干细胞中诱导分化，用于测试特定基因的功能，如缺乏内皮型一氧化氮合酶从而建立糖尿病肾病小鼠模型，同样可以通过基因编辑的方式建立糖尿病肾病肾类器官模型；另一方面，可以将肾类器官移植到小鼠肾包膜下，用链脲佐菌素诱导携带肾类器官的小鼠成为糖尿病模型，使肾类器官暴露在糖尿病环境下，建立糖尿病肾病模型；前文提到的糖尿病血管类器官模型利用这种方法建立了与体外干预类似的病理变化[41]。总之，肾类器官的出现为糖尿病肾病模型提供了一个新的、具有巨大潜力的平台。 2.3 糖尿病肾病细胞模型糖尿病肾病体外模型包括二维肾源细胞系培养模型和新兴的三维肾类器官模型，大量基于二维肾源细胞系培养模型的研究揭示了糖尿病肾病损伤机制，同时也为药物筛选提供了便捷的平台。肾小球的损伤在糖尿病肾病的病理变化过程中起着重要作用，主要包括内皮细胞、足细胞和系膜细胞等固有细胞的损伤[37]。肾小管内皮细胞在肾小球滤过及尿液形成中具有重要作用，在高血糖条件下容易引发细胞氧化应激、炎症及细胞凋亡，最近的研究发现肾小球内皮细胞中内皮-间质转分化在糖尿病肾病进程中具有重要作用[42]。肾小球系膜细胞对调节肾小球血流动力学、维持细胞外基质稳态和吞噬凋亡细胞方面发挥着重要作用[43]。持续高血糖直接刺激系膜细胞导致系膜细胞功能障碍，如系膜细胞过度增殖、肥大、分泌促炎、促纤维化、促凋亡因子并激活诸多信号通路，分泌过量的细胞外基质，造成系膜基质增加和基底膜增厚，导致肾小球硬化[44]。基于系膜细胞系二维培养的研究表明：在高糖培养环境下系膜细胞会显著增殖，核因子κB、转化生长因子β、TRPC6和纤维连接蛋白的表达亦上调，高糖培养也会诱导系膜细胞凋亡[41]。足细胞的损伤和丢失是糖尿病肾病早期重要的病理标志，在糖尿病环境下培养的足细胞由于高糖、生长因子、脂肪酸、血管紧张素Ⅱ、转化生长因子β、激素和机械拉伸等因素，导致足细胞损伤、凋亡以及修复和再生能力受到限制，使足细胞数量减少，加速糖尿病肾病的发展[45-46]。虽然许多关于糖尿病肾病的研究都集中在肾小球上，但越来越多的证据表明肾小管在疾病进展中的重要性[47]。因此，将二维培养的小管细胞暴露于模拟糖尿病肾病的病理生理介质，包括高糖[48]、高脂和炎症因子及糖化蛋白和血管紧张素Ⅱ方式[49-50]，已被广泛用于糖尿病肾病的体外模型探索肾小管细胞损伤的机制(表1)。"

尽管有研究人员提出二维细胞系培养系统不能提供多种细胞之间的相互作用且不能形成肾脏样结构[51]、也不能准确地模拟肾脏病理生理过程[52-53]，且原代细胞长期培养或扩增较困难，它会在几天内去分化并衰老[54]。然而，最近许多研究人也开始关注在糖尿病肾病中肾脏各种细胞之间的相互作用[37，55-56]，以及由于二维的肾源细胞培养的操作简单和建模时间周期短依然成为了目前研究中建立糖尿病肾病体外模型的首选。 2.4 糖尿病肾病动物模型糖尿病并发症动物模型联盟提出了理想的糖尿病肾病小鼠模型的3个标准：高血糖情况下进行性肾功能不全、蛋白尿及特征性病理改变(包括电镜下基底膜增厚、系膜基质扩张或结节性系膜硬化、小管间质纤维化和小动脉透明质病变)[57]。一个理想的疾病模型对于探索疾病的发病机制具有重要作用，目前根据造模的原理不同，糖尿病肾病动物模型可以分为转基因型、自发型、诱导型及中医结合病证型。 2.4.1 转基因糖尿病肾病模型 OVE26小鼠是在胰腺β细胞中过表达钙调蛋白，从而导致胰岛素分泌不足进而导致系膜基质增加、肾小球硬化和足细胞数量减少[58]。有研究发现OVE26小鼠肾脏损伤程度与小鼠遗传背景及性别有关，FVB遗传背景下及雌性OVE26小鼠肾脏损伤更严重[58-59]。有研究人员为了更快速地产生糖尿病肾病模型，在OVE26的背景上对2 月龄小鼠进行单侧肾切除，从而加速了肾脏的损伤[60]。有研究报道内皮型一氧化氮合酶(eNOS)的缺乏会加速糖尿病小鼠肾脏的损伤，因此研究人员把内皮型一氧化氮合酶基因敲除(eNOS KO)发现小鼠出现明显的蛋白尿、高血压和肾小球系膜溶解[61]。作为适合研究2型糖尿病引起糖尿病肾病的Zucker糖尿病性脂肪(ZDF)大鼠在12周龄时出现明显的高血糖，肾脏无病理变化；在22周龄时尿白蛋白与肌酐比显著增加，肾小球出现微动脉瘤；40周龄时，肾脏出现不可逆损伤包括肾小球萎缩、微动脉瘤和硬化[62]。含有天然胰岛素抵抗基因的黑褐色、短尾(BTBR)小鼠与ob/ob瘦素突变小鼠杂交的子代小鼠在8周时出现明显的高血糖和蛋白尿及肾脏肾小球系膜基质积累，在22周龄时出现糖尿病肾病相对严重的病理改变(包括弥漫性、局灶性结节性肾小球硬化及广泛的系膜基质扩张)[63]。见表2。"

2.4.3 诱导型糖尿病肾病模型链脲佐菌素(STZ)是化学诱导法制备糖尿病肾病模型最常用的诱导剂，具有胰腺毒性，会破坏胰腺细胞[83]，链脲佐菌素诱导的糖尿病动物已成为研究1型糖尿病及其并发症的主要实验模型[84]。利用链脲佐菌素诱导1型糖尿病主要有两种方式，一种是单次大剂量，一种是多次小剂量，多次小剂量链脲佐菌素诱导小鼠引起高血糖的发病机制和形态学变化方面更类似于1型糖尿病，并且肾脏表现出更接近糖尿病肾病的病理特征且药物毒性更小，小鼠存活率高[85-86]，但建模时间更长[87]。由于2型糖尿病患者大多数属于肥胖型患者，多有高脂血症，高脂高糖共同促进了肾脏的损伤，因此，有研究利用高脂饲料喂养联合腹腔注射低剂量链脲佐菌素来制备糖尿病肾病大鼠模型，病理染色显示该模型除了早期的肾脏变化，还出现部分肾小球硬化、肾小管部分出现细胞肥大以及管腔变窄[88]，但该方法的建模时间周期较长，要在12周时肾脏才开始出现病理改变[87]。有研究人员应用“三联法”：高糖高脂喂养联合腹腔注射小剂量链脲佐菌素联合单侧肾脏切除，从而达到缩短造模时间的目的[89-90]。见表4。 2.4.4 中医病证结合建立糖尿病肾病模型中医认为糖尿病肾病可归属于“消渴”“水肿”“尿浊”“溺毒”等范畴，辨证施治对疾病有一定疗效[96]，因此建立病证结合动物模型对于中医治疗糖尿病肾病具有重要作用。病证结合动物模型是指在中医药理论与现代医学理论及实验动物学结合下构建疾病模型，使模型同时具备西医疾病与中医证候特征的优势，从而观察中医证候对疾病的影响[97]。中医认为，血瘀证始终贯穿糖尿病肾病病程，既是该病的"

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