Metformin and Inhibition of Transforming Growth Factor-Beta Stimulate In Vitro Transport in Primary Renal Tubule Cells.

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. We previously identified transforming growth factor-beta as a signaling pathway necessary for in vitro differentiation of renal tubule cells. Inhibition of TGF-ß receptor-1 led to active inhabitable 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. The signals that undermine in vitro differentiation are complex, but susceptible to pharmacologic intervention. This achievement overcomes a major hurdle limiting the development of a bioreactor of cultured cells for renal replacement therapy that encompasses not only endocrine and metabolic functions but also transport and excretion. Impact statement Clinical tissue engineering requires functional fidelity of the cultured cell to its in vivo counterpart, but this has been elusive in renal tissue engineering. Typically, renal tubule cells in culture have a flattened morphology and do not express key transporters essential to their function. In this study, we build on our prior work by using small molecules to modulate pathways affected by substrate elasticity. In doing so, we are able to enhance differentiation of these cells on conventional noncompliant substrates and show transport. These results are fundamentally enabling a new generation of cell-based renal therapies.

Middle-molecule clearance in CRRT: in vitro convection, diffusion and dialyzer area.

Several studies have attempted to compare different doses and modes of therapy in continuous renal replacement therapies in critically ill patients. It is commonly asserted in the literature that convective therapies can achieve higher clearance of middle molecules than achieved by dialysis alone. However, regardless of the actual prescription, most therapies will actually contain a mixture of both diffusive and convective clearance. Molecular transport in purely convective prescriptions may be hindered by clotting and protein interactions with the dialyser. We measured middle molecule clearance using a tracer molecule, Ficoll, in citrated bovine blood. Using a 2 x 2 factorial design, we examined the impact of prescription [postdilution continuous venovenous hemofiltration (CVVH) vs. continuous venovenous hemodialysis (CVVHD)] and membrane area (0.4 m2 vs. 2.0 m2) on blood-side and dialysate-side middle-molecule clearance. In large dialysers, convective and diffusive prescriptions resulted in nearly identical middle molecule clearance from 10 to 100 kDa molecular weight. In the smaller dialyser, middle molecule clearance was higher when a diffusive therapy (CVVHD) was prescribed versus a convective therapy (postdilution CVVH). We hypothesized that high ultrafiltration rates in the smaller dialyzer resulted in a concentration polarization at the membrane that formed a prefilter, limiting middle-molecule clearance. This effect has implications for design and analysis of clinical trials of continuous renal replacement therapy (CRRT).