A biomimetic gelatin-based platform elicits a pro-differentiation effect on podocytes through mechanotransduction

Abstract

Using a gelatin microbial transglutaminase (gelatin-mTG) cell culture platform tuned to exhibit stiffness spanning that of healthy and diseased glomeruli, we demonstrate that kidney podocytes show marked stiffness sensitivity. Podocyte-specific markers that are critical in the formation of the renal filtration barrier are found to be regulated in association with stiffness-mediated cellular behaviors. While podocytes typically de-differentiate in culture and show diminished physiological function in nephropathies characterized by altered tissue stiffness, we show that gelatin-mTG substrates with Young’s modulus near that of healthy glomeruli elicit a pro-differentiation and maturation response in podocytes better than substrates either softer or stiffer. The pro-differentiation phenotype is characterized by upregulation of gene and protein expression associated with podocyte function, which is observed for podocytes cultured on gelatin-mTG gels of physiological stiffness independent of extracellular matrix coating type and density. Signaling pathways involved in stiffness-mediated podocyte behaviors are identified, revealing the interdependence of podocyte mechanotransduction and maintenance of their physiological function. This study also highlights the utility of the gelatin-mTG platform as an in vitro system with tunable stiffness over a range relevant for recapitulating mechanical properties of soft tissues, suggesting its potential impact on a wide range of research in cellular biophysics.

Engineering and Functionalization of Gelatin Biomaterials: From Cell Culture to Medical Applications

“…When podocytes were cultured in substrate with stiffness similar to that of in vivo glomeruli tissues (2-5 kPa), they exhibited phenotypes indicative of cellular differentiation. 86 This supports the theory that in general, the physical and mechanical properties of gelatin substrate whether it is because of the increased ECM concentration or the increase in the degree of crosslinking affects cell adhesion, proliferation, migration, and differentiation.…”

Similar Biophysical Abnormalities in Glomeruli and Podocytes from Two Distinct Models

“…adhesion, and potentially, altered gene expression patterns (dedifferentiation). 5,6 We show that increasing numbers of biophysically defective podocytes (Tg26) causes progressive loss of three-dimensional collagen gel contraction, a simple model of the mechanical role of podocytes in a glomerulus (Figure 7). Defective structure and reduced adhesion of these cells would increase their susceptibility to detachment and loss in response to shear force.…”

“…culturing podocytes on soft matrix (,2 kPa) causes a reduction in mRNAs coding for filamin A, nonmuscle myosin IIa, WT-1 protein expression, and expression of other differentiation markers. 5,6,26,29 Inhibition of integrin and cadherin function reduced glomerular E, indicating that cell matrix and cell-cell adhesion both contribute to a normal glomerular mechanical environment. 5 The reduced E value of an individual injured podocyte would not only provide inadequate structural support to a capillary at that location but could also reduce the ability of normal neighboring podocytes to Figure 8.…”

“…The cytoskeletal structure of podocytes supports glomerular capillary structure in response to normal and pathologic hemodynamic forces, although the relative contributions of podocytes and the glomerular basement membrane (GBM) are not fully resolved. 4 Additionally, mechanical environment can affect the differentiated state of numerous cell types, including podocytes, [5][6][7] which are determinants of the mechanical properties of glomerular capillaries and glomeruli. 8,9 To understand the biophysical characteristics of glomeruli and podocytes in disease, we studied the mechanical properties of glomeruli and podocytes in two mechanistically distinct, established models of extreme and mild glomerular disease: Tg26 mice (podocytes expressing HIV, severe) and protamine sulfateinduced minimal change disease (acute, reversible, mild).…”

Substrate Stiffness Modulates Renal Progenitor Cell Properties via a ROCK-Mediated Mechanotransduction Mechanism

“…Hu et al [49] and, more recently, Abdallah et al [31] studied the effects of substrate mechanical characteristics on podocytes. Hu et al reported a pro-differentiation and maturation effect of gelatin substrates with Young's modulus near that of healthy glomeruli (in the 2-5 kPa range) and suggested that Src-mediated activation of RacGAPs may be responsible for the mechanoresponse of podocytes [31].…”