Dynamic fluid flow and endothelial cross talk regulate mesangial homeostasis in a simplified 3-D coculture 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 (3-D) architecture, heterotypic cell interactions, and defined mechanical loading. We therefore developed a simplified three-dimensional (3-D) coculture 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 coculture suppressed MC p38 MAPK and AKT phosphorylation under both static and fluid flow conditions, whereas flow increased ERK phosphorylation in the endothelial layer despite reduced total ERK abundance. Fluid flow enhanced MC collagen accumulation (Picrosirius Red staining), whereas endothelial coculture 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.NEW & NOTEWORTHY We established a three-dimensional (3-D) coculture model integrating fluid flow-derived mechanical loading, extracellular matrix architecture, and mesangial-endothelial interactions. Fluid flow intrinsically promoted MC expansion, whereas endothelial coculture robustly suppressed it. Thus, the endothelium restrains mesangial expansion through biological signaling rather than by physically shielding the matrix, whereas fluid flow reaches mesangial cells by enhancing convective solute transport rather than by direct mechanical loading, identifying endothelial signaling and flow-driven solute transport as regulators of the dynamic glomerular microenvironment.