3D fractal topography attenuates inflammation and confers resilience to glomerular podocytes

In vivo, podocytes wrap around a looping capillary forming interdigitations required to establish a barrier function in kidney filtration. Cultured human podocytes can mimic key in vivo disease-associated changes—such as junctional and cytoskeletal remodeling, but their intricate, self-similar 3D branching structure remains poorly understood and technically difficult to replicate in vitro, especially in the context of inflammatory injury. Here, we established 3D fractal hemispheres capturing key features of glomerular architecture using nanoscale two-photon polymerization (2PP), enabling a detailed investigation into the effects of 3D topographies with various fractal indices on podocyte morphology and function. Podocytes cultured on intermediate fractal hemispheres, exhibited enhanced cytoskeletal branching, increased expression of slit diaphragm markers, and upregulation of nephrogenesis-related genes. To explore cellular responses to inflammatory stimuli by TNF-a and IL-6, we established a culture of fluorescently labelled podocytes within otherwise optically silent monolayer. The changes of single podocyte structure, fractal dimension and RNA sequencing revealed that podocytes grown on intermediate fractal hemispheres exhibited an enhanced immunosuppressive response, highlighting the potential of fractal topological structures in mitigating inflammation induced podocyte damage and kidney disease progression. A co-culture system with reporter THP-1-derived macrophages demonstrated a pronounced shift toward the M1 macrophage phenotype and heightened activation of inflammation pathways when podocytes were co-cultured on smooth hemispheres, compared to those on intermediate fractal hemispheres. These findings suggest that fractal microenvironments may play a crucial role in modulating podocyte-macrophage interactions and inflammatory responses in kidney disease. Overall design: To investigate the behavior of PODO/TERT256 human podocytes, cultured on 3D hemispheres with either smooth or fractal topography, in response to inflammation induced by cytokines (TNF-a and IL-6) or by THP-1-derived macrophages, we performed RNA sequencing of PODO/TERT256 human podocytes from the following groups: 1. Cells cultured on 3D hemispheres with smooth topography under the control condition 2. Cells cultured on 3D hemispheres with fractal topography under the control condition 3. Cells cultured on 3D hemispheres with smooth topography under the inflammation condition induced by cytokines (TNF-a and IL-6) 4. Cells cultured on 3D hemispheres with fractal topography under the inflammation condition induced by cytokines (TNF-a and IL-6) 5. Cells cultured on 3D hemispheres with smooth topography under the inflammation condition induced by THP-1-derived macrophages 6. Cells cultured on 3D hemispheres with fractal topography under the inflammation condition induced by THP-1-derived macrophages

在活体环境中，足细胞（podocytes）包裹于环状毛细血管表面，形成相互交错的指状突起，这是构建肾脏滤过屏障功能的必要结构。体外培养的人类足细胞能够模拟与疾病相关的关键活体变化——如连接复合体与细胞骨架重塑，但其复杂的自相似三维分支结构在体外仍鲜有研究，且技术上难以复刻，尤其是在炎症损伤的背景下。

本研究通过纳米级双光子聚合（two-photon polymerization, 2PP）技术，构建了能够模拟肾小球关键结构特征的三维分形半球模型，借此可深入探究不同分形指数的三维拓扑结构对足细胞形态与功能的影响。在中等分形指数的半球表面培养的足细胞，其细胞骨架分支更为发达，裂隙隔膜标志物的表达水平显著升高，同时与肾发生相关的基因表达也出现上调。

为探究足细胞对TNF-α与IL-6炎症刺激的应答反应，我们构建了荧光标记足细胞与光学静默单层细胞的共培养体系。通过对单足细胞结构、分形维度及转录组测序（RNA sequencing）的分析发现，在中等分形半球表面培养的足细胞展现出更强的免疫抑制应答能力，这凸显了分形拓扑结构在缓解炎症诱导的足细胞损伤及延缓肾脏病进展方面的应用潜力。

与报告基因标记的THP-1源性巨噬细胞（THP-1-derived macrophages）共培养实验显示，相较于中等分形半球表面的足细胞，在光滑半球表面培养的足细胞会显著促进巨噬细胞向M1巨噬细胞表型极化，并增强炎症通路的激活程度。上述研究结果表明，分形微环境在调控肾脏病中足细胞-巨噬细胞相互作用及炎症应答过程中发挥关键作用。

总体实验设计：为探究在光滑或分形拓扑结构的三维半球表面培养的PODO/TERT256人类足细胞，在受到细胞因子（TNF-α与IL-6）或THP-1源性巨噬细胞诱导的炎症刺激时的行为特征，我们对以下组别中的PODO/TERT256人类足细胞进行了转录组测序：
1. 对照组：于光滑拓扑结构三维半球表面培养的细胞
2. 分形拓扑组：于分形拓扑结构三维半球表面培养的细胞
3. 细胞因子炎症刺激组（光滑半球）：于光滑拓扑结构三维半球表面培养，并经TNF-α与IL-6诱导炎症的细胞
4. 细胞因子炎症刺激组（分形半球）：于分形拓扑结构三维半球表面培养，并经TNF-α与IL-6诱导炎症的细胞
5. THP-1源性巨噬细胞炎症刺激组（光滑半球）：于光滑拓扑结构三维半球表面培养，并经THP-1源性巨噬细胞诱导炎症的细胞
6. THP-1源性巨噬细胞炎症刺激组（分形半球）：于分形拓扑结构三维半球表面培养，并经THP-1源性巨噬细胞诱导炎症的细胞