Flow induces common and specific transcriptional changes in renal tubular epithelial cells involving the PI3K pathway.

Flow-induced shear stress affects renal epithelial cells in the nephron tubule with potential implications for differential functionalities of the individual segments. Disruptions of cellular mechanosensation or flow conditions are associated with the development and progression of various renal diseases. This study investigates the effects of flow on the transcriptome of various renal tubular epithelial cell types. We analyzed the transcriptome of induced renal epithelial cells (iREC) cultured under physiological flow (0.57 ± 0.05 dyn/cm2 ) or in static conditions for 72 h. RNA sequencing showed 861 differentially expressed genes (DEGs), with 503 up- and 358 downregulated under flow. DEGs were linked to extracellular matrix (ECM) components (e.g. Col1a1, Col4a3, Col4a4, Fn1, Smoc2), junctions (Gja1, Tubb5), channel activities (Abcc4, Aqp1), and transcription factors (Foxq1, Lgr6). Next, we performed a meta-analysis comparing our data with three published datasets that subjected epithelial cell lines from distinct segments to flow, including proximal tubule and collecting duct cells. We found that TGF-ß, p53, MAPK, and PI3K are common flow-regulated pathways. Tfrc expression and thus the capability of iron uptake is commonly upregulated under flow. Many DEGs were related to kidney diseases, such as fibrosis (e.g. Tgfb1-3 and Serpine1). To obtain further mechanistic insights we investigated the role of the PI3K pathway in flow sensing. Applying flow and inhibition of PI3K showed significantly altered expression of transcripts related to ECM remodeling, angiogenesis, and ion transport. This suggests that the PI3K pathway is a critical mediator in flow-dependent cellular processes and gene expression, potentially influencing renal development and tissue remodeling. Finally, we derived a cross-cell-line summary of common as well as segment-specific transcriptomic effects, thus providing insights into the molecular mechanisms underlying flow sensing in the nephron tubule.

HNF1B Alters an Evolutionarily Conserved Nephrogenic Program of Target Genes.

SIGNIFICANCE STATEMENT: Mutations in hepatocyte nuclear factor-1 ß ( HNF1B ) are the most common monogenic causes of congenital renal malformations. HNF1B is necessary to directly reprogram fibroblasts to induced renal tubule epithelial cells (iRECs) and, as we demonstrate, can induce ectopic pronephric tissue in Xenopus ectodermal organoids. Using these two systems, we analyzed the effect of HNF1B mutations found in patients with cystic dysplastic kidney disease. We found cross-species conserved targets of HNF1B, identified transcripts that are differentially regulated by the patient-specific mutant protein, and functionally validated novel HNF1B targets in vivo . These results highlight evolutionarily conserved transcriptional mechanisms and provide insights into the genetic circuitry of nephrogenesis. BACKGROUND: Hepatocyte nuclear factor-1 ß (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 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 R295C mutant. CRISPR/Cas9 genome editing in Xenopus embryos evaluated transcriptional targets in vivo . RESULTS: HNF1B is essential for reprogramming mouse fibroblasts to induced renal tubular epithelial cells 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.

Tuning the 3D microenvironment of reprogrammed tubule cells enhances biomimetic modeling of polycystic kidney disease.

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.

Deep Learning-Assisted Nephrotoxicity Testing with Bioprinted Renal Spheroids.

We used arrays of bioprinted renal epithelial cell spheroids for toxicity testing with cisplatin. The concentration-dependent cell death rate was determined using a lactate dehydrogenase assay. Bioprinted spheroids showed enhanced sensitivity to the treatment in comparison to monolayers of the same cell type. The measured dose-response curves revealed an inhibitory concentration of the spheroids of IC 50 = 9 ± 3 µM in contrast to the monolayers with IC 50 = 17 ± 2 µM. Fluorescent labeling of a nephrotoxicity biomarker, kidney injury molecule 1 indicated an accumulation of the molecule in the central lumen of the spheroids. Finally, we tested an approach for an automatic readout of toxicity based on microscopic images with deep learning. Therefore, we created a dataset comprising images of single spheroids, with corresponding labels of the determined cell death rates for training. The algorithm was able to distinguish between three classes of no, mild, and severe treatment effects with a balanced accuracy of 78.7%.

Metabolic and Lipidomic Assessment of Kidney Cells Exposed to Nephrotoxic Vancomycin Dosages.

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 of renal spheroids and nephron-like tubules by bioprinting and controlled self-assembly of epithelial cells.

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, 120-131µm in diameter (size variance <9%). Furthermore, we fabricated scalable line-shaped patterns, which self-assembled to hollow cellular tubules (Ø = 105 ± 22µm). 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-2driven by hydrostatic pressure for scalable dynamic culture towards a nephron-on-chip model.

Molecular Basis for Autosomal-Dominant Renal Fanconi Syndrome Caused by HNF4A.

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.

Impact of Diabetic Stress Conditions on Renal Cell Metabolome.

Diabetic kidney disease is a major complication in diabetes mellitus, and the most common reason for end-stage renal disease. Patients suffering from diabetes mellitus encounter glomerular damage by basement membrane thickening, and develop albuminuria. Subsequently, albuminuria can deteriorate the tubular function and impair the renal outcome. The impact of diabetic stress conditions on the metabolome was investigated by untargeted gas chromatography-mass spectrometry (GC-MS) analyses. The results were validated by qPCR analyses. In total, four cell lines were tested, representing the glomerulus, proximal nephron tubule, and collecting duct. Both murine and human cell lines were used. In podocytes, proximal tubular and collecting duct cells, high glucose concentrations led to global metabolic alterations in amino acid metabolism and the polyol pathway. Albumin overload led to the further activation of the latter pathway in human proximal tubular cells. In the proximal tubular cells, aldo-keto reductase was concordantly increased by glucose, and partially increased by albumin overload. Here, the combinatorial impact of two stressful agents in diabetes on the metabolome of kidney cells was investigated, revealing effects of glucose and albumin on polyol metabolism in human proximal tubular cells. This study shows the importance of including highly concentrated albumin in in vitro studies for mimicking diabetic kidney disease.

Fabrication of Kidney Proximal Tubule Grafts Using Biofunctionalized Electrospun Polymer Scaffolds.

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.

Metabolic characterization of directly reprogrammed renal tubular epithelial cells (iRECs).

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.

Engineering kidney cells: reprogramming and directed differentiation to renal tissues.

Growing knowledge of how cell identity is determined at the molecular level has enabled the generation of diverse tissue types, including renal cells from pluripotent or somatic cells. Recently, several in vitro protocols involving either directed differentiation or transcription-factor-based reprogramming to kidney cells have been established. Embryonic stem cells or induced pluripotent stem cells can be guided towards a kidney fate by exposing them to combinations of growth factors or small molecules. Here, renal development is recapitulated in vitro resulting in kidney cells or organoids that show striking similarities to mammalian embryonic nephrons. In addition, culture conditions are also defined that allow the expansion of renal progenitor cells in vitro. Another route towards the generation of kidney cells is direct reprogramming. Key transcription factors are used to directly impose renal cell identity on somatic cells, thus circumventing the pluripotent stage. This complementary approach to stem-cell-based differentiation has been demonstrated to generate renal tubule cells and nephron progenitors. In-vitro-generated renal cells offer new opportunities for modelling inherited and acquired renal diseases on a patient-specific genetic background. These cells represent a potential source for developing novel models for kidney diseases, drug screening and nephrotoxicity testing and might represent the first steps towards kidney cell replacement therapies. In this review, we summarize current approaches for the generation of renal cells in vitro and discuss the advantages of each approach and their potential applications.

Direct reprogramming of fibroblasts into renal tubular epithelial cells by defined transcription factors.

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.