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

ABSTRACT: 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-α 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE300321 | GEO | 2025/12/21

REPOSITORIES: GEO

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Project description:Glomerular podocytes are highly differentiated cells that are key components of the kidney filtration units. The podocyte cytoskeleton builds the basis for the dynamic podocyte cytoarchitecture and plays a central role for proper podocyte function. Recent studies implicate that immunosuppressive agents including the mTOR-inhibitor everolimus have a protective role directly on the stability of the podocyte cytoskeleton. To elucidate mechanisms underlying mTOR-inhibitor mediated cytoskeletal rearrangements, we carried out microarray gene expression studies to identify target genes and corresponding pathways in response to everolimus. We analyzed the effect of everolimus in a puromycin aminonucleoside experimental in vitro model of podocyte injury. Upon treatment with puromycin aminonucleoside, microarray analysis revealed gene clusters involving cytoskeletal-associated pathways, adhesion, migration and extracellular matrix composition to be affected. Everolimus is capable of protecting podocytes from injury, both on the transcriptome and protein level. Rescued genes included TUBB2B and DCDC2, both involved in microtubule structure formation in neuronal cells but not identified in podocytes so far. Confirming gene expression data, Western-blot analysis in cultured podocytes showed an increase of TUBB2B and DCDC2 protein after everolimus treatment, and immunohistochemistry in healthy control kidneys confirmed a podocyte-specific expression. Microtubule-inhibitor experiments led to a maldistribution of TUBB2B and DCDC2 as well as an aberrant reorganization of the actin cytoskeleton. Tubb2bbrdp/brdp mice showed a delay in glomerular podocyte and capillary development. Taken together, our study suggests that off-target, non-immune mediated effects of the mTOR-inhibitor everolimus on the podocyte cytoskeleton might involve regulation of microtubules, revealing a potential novel role of TUBB2B and DCDC2 in glomerular podocyte development We analyzed the effect of everolimus on gene expression in a puromycin aminonucleoside experimental in vitro model of podocyte injury using microarrays

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

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