| dc.description.abstract | Chronic kidney disease (CKD) is the ninth leading cause of death in the United States and affects 15% of the adult population globally. Renal transplantation, the best and most economical treatment, is severely limited by the scarcity of donor organs. On average, over 3,000 patients are added to the kidney waitlist per month and 13 people pass away each day waiting for a transplant. One solution to eliminating this scarcity problem and treating kidney failure is to bioengineer a mass-produced universal donor kidney. The current barrier to developing a universal donor kidney is producing fully differentiated human renal tubule epithelial cells (HREC) in vitro. Native renal epithelia are characterized by an elaborate array of sodium transporters which enable the orchestrated reabsorption and secretion of a diverse array of ions whilst maintaining normal cell homeostasis. Transforming Growth Factor-β (TGF-β) is a key modulator of HREC functional phenotype in vitro that also contributes to the development of tubulointerstitial fibrosis in CKD. In this work, we seek to understand the molecular mechanisms underlying TGF-β induced cell dedifferentiation to establish culture conditions that restore differentiated phenotype to HREC in vitro.
First, we demonstrate that control of the TGF-β signal transduction pathway is sufficient to reverse the erosion of some functional phenotypes associated with renal tubule epithelial cell culture in conventional flat inelastic scaffolds. TGF-β inhibition leads to the appearance of functions characteristic of native tubule cells, including diuretic-inhibitable fluid transport, phlorizin-inhibitable glucose uptake, probenecid-inhibitable organic anion excretion, and apical acid extrusion. Second, we explore the roles of TGF-β and that AMP-activated protein kinase activator metformin in governing renal tubule epithelial cell glycolysis, oxidative phosphorylation, and substrate dependency. We find that metformin and SB431542 ameliorate the metabolic dysregulation brought on by cell culture stress by increasing the flux of glucose and fatty acids towards oxidative phosphorylation. Finally, we investigate the mechanism by which TGF-β governs apicobasal transport. We demonstrate that TGF-β suppresses Na-K-2Cl cotransporter 2 (NKCC2) transcription through the canonical Smad/Snail signaling axis which represses NKCC2 transcription factor Hepatic Nuclear Factor-1α (HNF-1α). Genetic knockout and rescue models are used to confirm the roles of Smad2, Smad3, Smad4, Snail1, and HNF-1α on renal NKCC2 expression.
These studies establish new genetic targets for the in vitro differentiation of renal tubule epithelial cells and provide greater insight to the mechanisms of tubulointerstitial fibrosis and metabolic dysregulation driving CKD. This provides a valuable resource for the wider scientific community to pursue the study of immunosuppression-free transplants that have not previously been possible due to the limitations of existing cell models. | |
