Dynamic Fluid Flow and Endothelial Crosstalk Regulate Mesangial Homeostasis in a Simplified 3D Co-culture Model

Mesangial hypercellularity and excessive extracellular matrix (ECM) accumulation are defining lesions of mesangial proliferative glomerular diseases and drivers of progressive glomerulosclerosis and renal dysfunction. How mesangial cells (MCs) remain quiescent within the mechanically dynamic glomerular microenvironment, and why this control fails in disease, remain incompletely understood, in part because few in vitro systems combine three-dimensional architecture, heterotypic cell interactions, and defined mechanical loading. We therefore developed a simplified three-dimensional (3D) co-culture system using immortalized mouse cell lines: MCs embedded in a type I collagen gel, overlaid with a monolayer of mouse microvascular endothelial cells and subjected to orbital-shaking-induced fluid-flow-derived mechanical loading, intended to model indirect loading rather than pressure-driven interstitial perfusion. In MC monoculture, fluid flow markedly increased MC expansion and Ki-67-positive cell density, whereas an overlying endothelial monolayer substantially attenuated this proliferative response under flow and reduced cleaved caspase-3-positive (CC3) apoptotic MCs. Endothelial co-culture suppressed MC p38 MAPK and AKT phosphorylation under both static and fluid-flow conditions, while flow increased ERK phosphorylation in the endothelial layer despite reduced total ERK abundance. Fluid flow enhanced MC collagen accumulation (Picrosirius Red staining), whereas endothelial co-culture reduced total and type III collagen deposition under both conditions. These findings indicate that fluid flow and the endothelial monolayer are key determinants of the mesangial microenvironment, with flow prompting proliferation and the endothelium restraining proliferation and matrix deposition largely independently of flow. We propose that disruption of this endothelial-mesangial cross-talk may contribute to the mesangial expansion and fibrosis of mesangial proliferative glomerulonephritis.