Posttranslationally modified self-peptides promote hypertension in mouse models.

Posttranslational modifications can enhance immunogenicity of self-proteins. In several conditions, including hypertension, systemic lupus erythematosus, and heart failure, isolevuglandins (IsoLGs) are formed by lipid peroxidation and covalently bond with protein lysine residues. Here, we show that the murine class I major histocompatibility complex (MHC-I) variant H-2Db uniquely presents isoLG-modified peptides and developed a computational pipeline that identifies structural features for MHC-I accommodation of such peptides. We identified isoLG-adducted peptides from renal proteins, including sodium glucose transporter 2, cadherin 16, Kelch domain-containing protein 7A, and solute carrier family 23, that are recognized by CD8+ T cells in tissues of hypertensive mice, induce T cell proliferation in vitro, and prime hypertension after adoptive transfer. Finally, we find patterns of isoLG-adducted antigen restriction in class I human leukocyte antigens that are similar to those in murine analogs. Thus, we have used a combined computational and experimental approach to define likely antigenic peptides in hypertension.

Inhibition of Transforming Growth Factor-ß Improves Primary Renal Tubule Cell Differentiation in Long-Term Culture.

Patient-oriented applications of cell culture include cell therapy of organ failure like chronic renal failure. Clinical deployment of a cell-based device for artificial renal replacement requires qualitative and quantitative fidelity of a cultured cell to its in vivo counterpart. Active specific apicobasal ion transport reabsorbs 90-99% of the filtered load of salt and water in the kidney. In a bioengineered kidney, tubular transport concentrates wastes and eliminates the need for hemodialysis, but renal tubule cells in culture transport little or no salt and water due to dedifferentiation that mammalian cells undergo in vitro thereby losing important cell-type specific functions. We previously identified transforming growth factor-ß (TGF-ß) as a signaling pathway necessary for in vitro differentiation of renal tubule cells. Inhibition of TGF-ß receptor-1 led to active and inhibitable electrolyte and water transport by primary human renal tubule epithelial cells in vitro. Addition of metformin increased transport, in the context of a transient effect on 5'-AMP-activated kinase phosphorylation. These data motivated us to examine whether increased transport was an idiosyncratic effect of SB431542, probe pathways downstream of TGF-ß receptors possibly responsible for the improved differentiation, evaluate whether TGF-ß inhibition induced a range of differentiated tubule functions, and to explore crosstalk between the effects of SB431542 and metformin. In this study, we use multiple small-molecule inhibitors of canonical and noncanonical pathways to confirm that inhibition of canonical TGF-ß signaling caused the increased apicobasal transport. Hallmarks of proximal tubule cell function, including sodium reabsorption, para-amino hippurate excretion, and glucose uptake increased with TGF-ß inhibition, and the specificity of the response was shown using inhibitors of each transport protein. We did not find any evidence of crosstalk between metformin and SB431542. These data suggest that the TGF-ß signaling pathway governs multiple features of differentiation in renal proximal tubule cells in vitro. Inhibition of TGF-ß by pharmacologic or genome engineering approaches may be a viable approach to enhancing differentiated function of tubule cells in vitro. Impact statement Cell therapy of renal failure requires qualitative and quantitative fidelity between in vitro and in vivo phenotypes, which has been elusive. We show that control of transforming growth factor-ß signaling can promote differentiation of renal tubule cells grown in artificial environments. This is a key enabling step for cell therapy of renal failure.

Functionalizing Polyacrylamide Hydrogels for Renal Cell Culture Under Fluid Shear Stress.

A functional renal tubule bioreactor needs to reproduce the reabsorption and barrier functions of the renal tubule. Our prior work has demonstrated that primary human renal tubule cells respond favorably when cultured on substrates with elasticity similar to healthy tissue and when subjected to fluid shear stress. Polyacrylamide (PA) is widely used in industrial processes such as water purification because it is electrically neutral and chemically inert. PA is a versatile tool as the concentration and mechanical properties of the gel are easily adjusted by varying the proportions of monomer and crosslinker. Control of mechanical properties is attractive for preparing cell culture substrates with tunable stiffness, but PA's inert chemical properties require additional steps to prepare PA for cell attachment, such as chemical reactions to bind extracellular matrix proteins. Methods based on protein functionalization for cell attachment work well in the short term but fail to provide sufficient attachment to withstand the mechanical traction of fluid shear stress. In our present work, we tested the effects of subjecting primary renal tubule cells to fluid shear stress on an elastic substrate by developing a simple method of incorporating N-(3-Aminopropyl) methacrylamide hydrochloride (APMA) into PA hydrogels. Integration of APMA into the PA hydrogel formed a nondegradable elastic substrate promoting excellent long-term cell attachment despite the forces of fluid shear stress. Impact statement Cell culture on artificial materials requires the presence of ligands on the surface to which extracellular matrix receptors on the cell can bind. Simple nonspecific adsorption or covalent linkage of plasma or extracellular matrix proteins only suffices for short-term static culture. Prolonged culture may result in degradation of the original protein such that linkage is severed but new proteins secreted by the cell are blocked from adsorbing to the artificial scaffold. This results in detachment and loss of cell mass, as well as defects in monolayers. We present a simple technique to integrate amine moeities into a polyacrylamide hydrogel that resist degradation and support long-term culture.