HNF4A is a nuclear hormone receptor that binds DNA as an obligate homodimer. While all known human heterozygous mutations are associated with the autosomal-dominant diabetes form MODY1, one particular mutation (p.R85W) in the DNA-binding domain (DBD) causes additional renal Fanconi syndrome (FRTS). Here, we find that expression of the conserved fly ortholog dHNF4 harboring the FRTS mutation in Drosophila nephrocytes caused nuclear depletion and cytosolic aggregation of a wild-type dHNF4 reporter protein. While the nuclear depletion led to mitochondrial defects and lipid droplet accumulation, the cytosolic aggregates triggered the expansion of the endoplasmic reticulum (ER), autophagy, and eventually cell death. The latter effects could be fully rescued by preventing nuclear export through interfering with serine phosphorylation in the DBD. Our data describe a genomic and a non-genomic mechanism for FRTS in HNF4A-associated MODY1 with important implications for the renal proximal tubule and the regulation of other nuclear hormone receptors.

Scalable fabrication concepts of 3D kidney tissue models are required to enable their application in pharmaceutical high-throughput screenings. Yet the reconstruction of complex tissue structures remains technologically challenging. We present a novel concept reducing the fabrication demands, by using controlled cellular self-assembly to achieve higher tissue complexities from significantly simplified construct designs. We used drop-on-demand bioprinting to fabricate locally confined patterns of renal epithelial cells embedded in a hydrogel matrix. These patterns provide defined local cell densities (cell count variance <11%) with high viability (92 ± 2%). Based on these patterns, controlled self-assembly leads to the formation of renal spheroids and nephron-like tubules with a predefined size and spatial localization. With this, we fabricated scalable arrays of hollow epithelial spheroids. The spheroid sizes correlated with the initial cell count per unit and could be stepwise adjusted, ranging from Ø = 84, 104, 120–131 µm in diameter (size variance <9%). Furthermore, we fabricated scalable line-shaped patterns, which self-assembled to hollow cellular tubules (Ø = 105 ± 22 µm). These showed a continuous lumen with prescribed orientation, lined by an epithelial monolayer with tight junctions. Additionally, upregulated expression of kidney-specific functional genes compared to 2D cell monolayers indicated increased tissue functionality, as revealed by mRNA sequencing. Furthermore, our concept enabled the fabrication of hybrid tubules, which consisted of arranged subsections of different cell types, combining murine and human epithelial cells. Finally, we integrated the self-assembled fabrication into a microfluidic chip and achieved fluidic access to the lumen at the terminal sites of the tubules. With this, we realized flow conditions with a wall shear stress of 0.05 ± 0.02 dyne cm⁻² driven by hydrostatic pressure for scalable dynamic culture towards a nephron-on-chip model.

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.