Direct reprogramming by forced expression of transcription factors can convert one cell type into another. Thus, desired cell types can be generated bypassing pluripotency. However, direct reprogramming towards renal cells remains an unmet challenge. Here, we identify renal cell fate-inducing factors on the basis of their tissue specificity and evolutionarily conserved expression, and demonstrate that combined expression of Emx2, Hnf1b, Hnf4a and Pax8 converts mouse and human fibroblasts into induced renal tubular epithelial cells (iRECs). iRECs exhibit epithelial features, a global gene expression profile resembling their native counterparts, functional properties of differentiated renal tubule cells and sensitivity to nephrotoxic substances. Furthermore, iRECs integrate into kidney organoids and form tubules in decellularized kidneys. Our approach demonstrates that reprogramming factors can be identified by targeted in silico analysis. Renal tubular epithelial cells generated ex vivo by forced expression of transcription factors may facilitate disease modelling, drug and nephrotoxicity testing, and regenerative approaches.

The increasing prevalence of end‐stage renal disease and persistent shortage of donor organs call for alternative therapies for kidney patients. Dialysis remains an inferior treatment as clearance of large and protein‐bound waste products depends on active tubular secretion. Biofabricated tissues could make a valuable contribution, but kidneys are highly intricate and multifunctional organs. Depending on the therapeutic objective, suitable cell sources and scaffolds must be selected. This study provides a proof‐of‐concept for stand‐alone kidney tubule grafts with suitable mechanical properties for future implantation purposes. Porous tubular nanofiber scaffolds are fabricated by electrospinning 12%, 16%, and 20% poly‐ε‐caprolactone (PCL) v/w (chloroform and dimethylformamide, 1:3) around 0.7 mm needle templates. The resulting scaffolds consist of 92%, 69%, and 54% nanofibers compared to microfibers, respectively. After biofunctionalization with L‐3,4‐dihydroxyphenylalanine and collagen IV, 10 × 106 proximal tubule cells per mL are injected and cultured until experimental readout. A human‐derived cell model can bridge all fiber‐to‐fiber distances to form a monolayer, whereas small‐sized murine cells form monolayers on dense nanofiber meshes only. Fabricated constructs remain viable for at least 3 weeks and maintain functionality as shown by inhibitor‐sensitive transport activity, which suggests clearance capacity for both negatively and positively charged solutes.

Fibroblasts can be directly reprogrammed to induced renal tubular epithelial cells (iRECs) using four transcription factors. These engineered cells may be used for disease modeling, cell replacement therapy or drug and toxicity testing. Direct reprogramming induces drastic changes in the transcriptional landscape, protein expression, morphological and functional properties of cells. However, how the metabolome is changed by reprogramming and to what degree it resembles the target cell type remains unknown. Using untargeted gas chromatography-mass spectrometry (GC-MS) and targeted liquid chromatography-MS, we characterized the metabolome of mouse embryonic fibroblasts (MEFs), iRECs, mIMCD-3 cells, and whole kidneys. Metabolic fingerprinting can distinguish each cell type reliably, revealing iRECs are most similar to mIMCD-3 cells and clearly separate from MEFs used for reprogramming. Treatment with the cytotoxic drug cisplatin induced typical changes in the metabolic profile of iRECs commonly occurring in acute renal injury. Interestingly, metabolites in the medium of iRECs, but not of mIMCD-3 cells or fibroblast could distinguish treated and non-treated cells by cluster analysis. In conclusion, direct reprogramming of fibroblasts into renal tubular epithelial cells strongly influences the metabolome of engineered cells, suggesting that metabolic profiling may aid in establishing iRECs as in vitro models for nephrotoxicity testing in the future.

Highlights d HNF4 controls lipid metabolism in Drosophila nephrocytes d The kidney disease mutation R85W shows dominantnegative effects in nephrocytes d Dephosphorylation at S87 prevents the dominant-negative effects d R85W mutation causes mitochondrial dysfunction in reprogrammed renal epithelial cells