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Background: Hepatocyte nuclear factor 1-beta (HNF1B) is an essential transcription factor during embryogenesis. Mutations in HNF1B are the most common monogenic causes of congenital cystic dysplastic renal malformations. The direct functional consequences of mutations in HNF1B on its transcriptional activity are unknown. Methods: Direct reprogramming of mouse fibroblasts to induced renal tubular epithelial cells (iRECs) was conducted both with wild type HNF1B and with patient mutations. HNF1B was expressed in Xenopus ectodermal explants. Transcriptomic analysis by bulk RNA-Seq identified conserved targets with differentially regulated expression by the wild type or theR295C mutant. CRISPR genome editing in Xenopus embryos evaluated transcriptional targets in vivo Results: HNF1B is essential for reprogramming mouse fibroblasts to iRECs and induces development of ectopic renal organoids from pluripotent Xenopus cells. The mutation R295C retains reprogramming and inductive capacity but alters the expression of specific sets of downstream target genes, instead of diminishing overall transcriptional activity of HNF1B. Surprisingly, targets associated with polycystic kidney disease were less affected than genes affected in congenital renal anomalies. Cross-species conserved transcriptional targets were dysregulated in hnf1b CRISPR-depleted Xenopus embryos, confirming their dependence on hnf1b Conclusions: HNF1B activates an evolutionarily conserved program of target genes that disease-causing mutations selectively disrupt. These findings provide insights into the renal transcriptional network that controls nephrogenesis.

Renal tubular cells frequently lose differentiation markers and physiological properties when propagated in conventional cell culture conditions. Embedding cells in 3D microenvironments or controlling their 3D assembly by bioprinting can enhance their physiological properties, which is beneficial for modeling diseases in vitro. A potential cellular source for modeling renal tubular physiology and kidney diseases in vitro are directly reprogrammed induced renal tubular epithelial cells (iRECs). iRECs were cultured in various biomaterials and as bioprinted tubular structures. They showed high compatibility with the embedding substrates and dispensing methods. The morphology of multicellular aggregates was substantially influenced by the 3D microenvironment. Transcriptomic analyses revealed signatures of differentially expressed genes specific to each of the selected biomaterials. Using a new cellular model for autosomal-dominant polycystic kidney disease, Pkd1$^{-/-}$ iRECs showed disrupted morphology in bioprinted tubules and a marked upregulation of the Aldehyde dehydrogenase 1a1 (Aldh1a1). In conclusion, 3D microenvironments strongly influence the morphology and expression profiles of iRECs, help to unmask disease phenotypes, and can be adapted to experimental demands. Combining a direct reprogramming approach with appropriate biomaterials will facilitate construction of biomimetic kidney tubules and disease models at the microscale.

Vancomycin is a glycopeptide antibiotic used against multi-drug resistant gram-positive bacteria such as Staphylococcus aureus (MRSA). Although invaluable against resistant bacteria, vancomycin harbors adverse drug reactions including cytopenia, ototoxicity, as well as nephrotoxicity. Since nephrotoxicity is a rarely occurring side effect, its mechanism is incompletely understood. Only recently, the actual clinically relevant concentration the in kidneys of patients receiving vancomycin was investigated and were found to exceed plasma concentrations by far. We applied these clinically relevant vancomycin concentrations to murine and canine renal epithelial cell lines and assessed metabolic and lipidomic alterations by untargeted and targeted gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analyses. Despite marked differences in the lipidome, both cell lines increased anabolic glucose reactions, resulting in higher sorbitol and lactate levels. To the best of our knowledge, this is the first endometabolic profiling of kidney cells exposed to clinically relevant vancomycin concentrations. The presented study will provide a valuable dataset to nephrotoxicity researchers and might add to unveiling the nephrotoxic mechanism of vancomycin.

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 to 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/cm² driven by hydrostatic pressure for scalable dynamic culture towards a nephro

HNF4A is a nuclear hormone receptor that bindsDNA as an obligate homodimer. While all known hu-man heterozygous mutations are associated with theautosomal-dominant diabetes form MODY1, oneparticular mutation (p.R85W) in the DNA-bindingdomain (DBD) causes additional renal Fanconi syn-drome (FRTS). Here, we find that expression of theconserved fly ortholog dHNF4 harboring the FRTSmutation inDrosophilanephrocytes caused nucleardepletion and cytosolic aggregation of a wild-typedHNF4 reporter protein. While the nuclear depletionled to mitochondrial defects and lipid droplet accu-mulation, the cytosolic aggregates triggered theexpansion of the endoplasmic reticulum (ER), auto-phagy, and eventually cell death. The latter effectscould be fully rescued by preventing nuclear exportthrough interfering with serine phosphorylation inthe DBD. Our data describe a genomic and a non-genomic mechanism for FRTS in HNF4A-associatedMODY1 with important implications for the renalproximal tubule and the regulation of other nuclearhormone receptors.