A computational analysis of the role of integrins and Rho-GTPases in the emergence and disruption of apical-basal polarization in renal epithelial cells.

Apical-basal polarization in renal epithelial cells is crucial to renal function and an important trigger for tubule formation in kidney development. Loss of polarity can induce epithelial-to-mesenchymal transition (EMT), which can lead to kidney pathologies. Understanding the relative and combined roles of the involved proteins and their interactions that govern epithelial polarity may provide insights for controlling the process of polarization via chemical or mechanical manipulations in an in vitro or in vivo setting. Here, we developed a computational framework that integrates several known interactions between integrins, Rho-GTPases Rho, Rac and Cdc42, and polarity complexes Par and Scribble, to study their mutual roles in the emergence of polarization. The modeled protein interactions were shown to induce the emergence of polarized distributions of Rho-GTPases, which in turn led to the accumulation of apical and basal polarity complexes Par and Scribble at their respective poles, effectively recapitulating polarization. Our multiparametric sensitivity analysis suggested that polarization depends foremost on the mutual inhibition between Rac and Rho. Next, we used the computational framework to investigate the role of integrins and GTPases in the generation and disruption of polarization. We found that a minimum concentration of integrins is required to catalyze the process of polarization. Furthermore, loss of polarization was found to be only inducible via complete degradation of the Rho-GTPases Rho and Cdc42, suggesting that polarization is fairly stable once it is established. Comparison of our computational predictions against data from in vitro experiments in which we induced EMT in renal epithelial cells while quantifying the relative Rho-GTPase levels, displayed that EMT coincides with a large reduction in the Rho-GTPase Rho. Collectively, these results demonstrate the essential roles of integrins and Rho-GTPases in the establishment and disruption of apical-basal polarity and thereby provide handles for the in vitro or in vivo regulation of polarity.

The Importance of Effective Ligand Concentration to Direct Epithelial Cell Polarity in Dynamic Hydrogels.

Epithelial cysts and organoids are multicellular hollow structures formed by correctly polarized epithelial cells. Important in steering these cysts from single cells is the dynamic regulation of extracellular matrix presented ligands, and matrix dynamics. Here, control over the effective ligand concentration is introduced, decoupled from bulk and local mechanical properties, in synthetic dynamic supramolecular hydrogels formed through noncovalent crosslinking of supramolecular fibers. Control over the effective ligand concentration is realized by 1) keeping the ligand concentration constant, but changing the concentration of nonfunctionalized molecules or by 2) varying the ligand concentration, while keeping the concentration of non-functionalized molecules constant. The results show that in 2D, the effective ligand concentration within the supramolecular fibers rather than gel stiffness (from 0.1 to 8 kPa) regulates epithelial polarity. In 3D, increasing the effective ligand concentration from 0.5 × 10-3 to 2 × 10-3 m strengthens the effect of increased gel stiffness from 0.1 to 2 kPa, to synergistically yield more correctly polarized cysts. Through integrin manipulation, it is shown that epithelial polarity is regulated by tension-based homeostasis between cells and matrix. The results reveal the effective ligand concentration as influential factor in regulating epithelial polarity and provide insights on engineering of synthetic biomaterials for cell and organoid culture.

Engineering Strategies to Move from Understanding to Steering Renal Tubulogenesis.

Rebuilding the kidney in the context of tissue engineering offers a major challenge as the organ is structurally complex and has a high variety of specific functions. Recreation of kidney function is inherently connected to the formation of tubules since the functional subunit of the kidney, the nephron, is based on tubular structures. In vivo, tubulogenesis culminates in a perfectly shaped, patterned, and functional renal tubule via different morphogenic processes that depend on delicately orchestrated chemical, physical, and mechanical interactions between cells and between cells and their microenvironment. This review summarizes the current understanding of the role of the microenvironment in the morphogenic processes involved in in vivo renal tubulogenesis. We highlight the current state-of-the-art of renal tubular engineering and provide a view on the design elements that can be extracted from these studies. Next, we discuss how computational modeling can aid in specifying and identifying design parameters and provide directions on how these design parameters can be incorporated in biomaterials for the purpose of engineering renal tubulogenesis. Finally, we propose that a step-by-step reciprocal interaction between understanding and engineering is necessary to effectively guide renal tubulogenesis. Impact statement Tubular tissue engineering lies at the foundation of regenerating kidney tissue function, as the functional subunit of the kidney, the nephron, is based on tubular structures. Guiding renal tubulogenesis toward functional renal tubules requires in-depth knowledge of the developmental processes that lead to the formation of native tubules as well as engineering approaches to steer these processes. In this study, we review the role of the microenvironment in the developmental processes that lead to functional renal tubules and give directions how this knowledge can be harnessed for biomaterial-based tubular engineering using computational models.

Substrate Stiffness Determines the Establishment of Apical-Basal Polarization in Renal Epithelial Cells but Not in Tubuloid-Derived Cells.

Mechanical guidance of tissue morphogenesis is an emerging method of regenerative medicine that can be employed to steer functional kidney architecture for the purpose of bioartificial kidney design or renal tissue engineering strategies. In kidney morphogenesis, apical-basal polarization of renal epithelial cells is paramount for tubule formation and subsequent tissue functions like excretion and resorption. In kidney epithelium, polarization is initiated by integrin-mediated cell-matrix adhesion at the cell membrane. Cellular mechanobiology research has indicated that this integrin-mediated adhesion is responsive to matrix stiffness, raising the possibility to use matrix stiffness as a handle to steer cell polarization. Herein, we evaluate apical-basal polarization in response to 2D substates of different stiffness (1, 10, 50 kPa and glass) in Madin Darby Canine Kidney cells (MDCKs), a classic canine-derived cell model of epithelial polarization, and in tubuloid-derived cells, established from human primary cells derived from adult kidney tissue. Our results show that sub-physiological (1 kPa) substrate stiffness with low integrin-based adhesion induces polarization in MDCKs, while MDCKs on supraphysiological (>10 kPa) stiffness remain unpolarized. Inhibition of integrin, indeed, allows for polarization on the supraphysiological substrates, suggesting that increased cellular adhesion on stiff substrates opposes polarization. In contrast, tubuloid-derived cells do not establish apical-basal polarization on 2D substrates, irrespective of substrate stiffness, despite their ability to polarize in 3D environments. Further analysis implies that the 2D cultured tubuloid-derived cells have a diminished mechanosensitive capacity when presented with different substrate stiffnesses due to immature focal adhesions and the absence of a connection between focal adhesions and the cytoskeleton. Overall, this study demonstrates that apical-basal polarization is a complex process, where cell type, the extracellular environment, and both the mechanical and chemical aspects in cell-matrix interactions performed by integrins play a role.