Tissue Culture Models of Acute Kidney Injury: From Tubule Cells to Human Kidney Organoids

Acute kidney injury (AKI) affects approximately 13.3 million people around the world each year, causing chronic kidney disease and/or mortality. The mammalian kidney cannot generate new nephrons after postnatal renal damage and regenerative therapies for AKI are not available. Human kidney tissue culture systems can complement animal models of AKI and/or address some of their limitations. Donor-derived somatic cells, such as renal tubule epithelial cells or cell lines (RPTEC/hTERT, ciPTEC, HK-2, Nki-2, and CIHP-1), have been used for decades to permit drug toxicity screening and studies into potential AKI mechanisms. However, tubule cell lines do not fully recapitulate tubular epithelial cell properties in situ when grown under classic tissue culture conditions. Improving tissue culture models of AKI would increase our understanding of the mechanisms, leading to new therapeutics. Human pluripotent stem cells (hPSCs) can be differentiated into kidney organoids and various renal cell types. Injury to human kidney organoids results in renal cell type crosstalk and upregulation of kidney injury biomarkers that are difficult to induce in primary tubule cell cultures. However, current protocols produce kidney organoids that aren't mature and contain off-target cell types. Promising bioengineering techniques, such as bioprinting and "kidney-on-a17 chip" methods, as applied to kidney nephrotoxicity modeling advantages and limitations are discussed. This review explores the mechanisms and detection of AKI in tissue culture, with an emphasis on bioengineered approaches such as human kidney organoid models.

Recurrent Nephrotic Plasma Activates Pro-fibrotic Signalling Pathways Downstream of Protease-activated Receptor 1

Recurrence of steroid-resistant nephrotic syndrome (SRNS) is thought to be due to an unknown “circulating factor”, the identity of which has so far remained elusive. Our previous work suggests a signaling role for protease-activated receptor-1 (PAR-1), leading to impaired podocyte function. Here, we show that relapse nephrotic plasma (NP), but not paired remission plasma, induced a pro-fibrotic response. This change was inhibited by PAR-1 inhibitors, but not by TGF-β1 inhibition. Four PAR-1 inhibitors demonstrated distinct antagonistic properties. The phosphorylation of VASP and JNK in a 3D spheroid model (GlomSpheres) and kidney organoids corroborated the finding from a 2D ciPods model. Functionally, relapse NP induced podocyte motility, and podocyte loss from spheroids both of which were also selectively rescued by PAR-1 inhibitors. Also, it induced the loss of podocyte-specific markers in kidney organoids. We propose that the circulating factor acts as a pro-fibrotic effector by activating PAR-1, leading to increased podocyte injury.

Large-Scale Engineering and Cryopreservation of hiPSC-Derived Nephron Sheets

Background The generation of human induced pluripotent stem cells (hiPSCs) has opened a world of opportunities for stem cell-based therapies in regenerative medicine. Currently, several human kidney organoid protocols are available that generate organoids containing kidney structures. However, these kidney organoids are relatively small ranging up to 0.13 cm2 and therefore contain a small number of nephrons compared to an adult kidney, thus defying the exploration of future use for therapy. Method We have developed a scalable, easily accessible, and reproducible to increase the size of the organoid up to a nephron sheet of 2.5 cm2 up to a maximum of 12.6 cm2 containing a magnitude of nephrons. Results Confocal microscopy showed that the subunits of the nephrons remain evenly distributed throughout the entire sheet and that these tissue sheets can attain ~30,000-40,000 glomerular structures. Upon transplantation in immunodeficient mice, such nephron sheets became vascularized and matured. They also show reuptake of injected low-molecular mass dextran molecules in the tubular structures, indicative of glomerular filtration. Furthermore, we developed a protocol for the cryopreservation of intermediate mesoderm cells during the differentiation and demonstrate that these cells can be successfully thawed and recovered to create such tissue sheets. Conclusion The scalability of the procedures, and the ability to cryopreserve the cells during differentiation are important steps forward in the translation of these differentiation protocols to future clinical applications such as transplantable auxiliary kidney tissue.

Enhanced microvasculature formation and patterning in iPSC-derived kidney organoids cultured in physiological hypoxia

Functional kidney organoids have the potential to be used in implantable kidney grafts for patients with end-stage kidney disease, because they have been shown to self-organize from induced pluripotent stem cells into most important renal structures. To date, however, long-term kidney organoid culture has not succeeded, as nephrons lose their phenotype after approximately 25 days. Furthermore, the renal structures remain immature with diminishing endothelial networks with low connectivity and limited organoid invasion. We hypothesized that introducing long-term culture at physiological hypoxia, rather than the normally applied non-physiological, hyperoxic 21% O2, could initiate angiogenesis, lead to enhanced growth factor expression and improve the endothelial patterning. We therefore cultured the kidney organoids at 7% O2 instead of 21% O2 for up to 25 days and evaluated nephrogenesis, VEGF-A expression and vascularization. Whole mount imaging revealed a homogenous morphology of the endothelial network with enhanced sprouting and interconnectivity when the kidney organoids were cultured in hypoxia. Three-dimensional quantification confirmed that the hypoxic culture led to an increased average vessel length, likely due to the observed upregulation of proangiogenic VEGF-A189 mRNA and downregulation of the antiangiogenic protein VEGF-A165b measured in hypoxia. This research indicates the importance of optimization of oxygen availability in organoid systems and the potential of hypoxic culture conditions in improving the vascularization of organoids.

Revisiting The Role of Notch in Nephron Segmentation: Notch is required for proximal, not distal fate selection during mouse and human nephrogenesis

Notch signaling promotes maturation of nephron epithelia, but its proposed contribution to nephron segmentation into proximal and distal domains has been called into doubt. We leveraged single cell and bulk RNA-seq, quantitative immunofluorescent lineage/fate tracing, and genetically modified human iPSC to revisit this question in developing mouse kidneys and human kidney organoids. We confirmed that Notch signaling is needed for maturation of all nephron lineages, and thus mature lineage markers fail to detect a fate bias. By contrast, early markers identified a distal fate bias in cells lacking Notch2, and a concomitant increase in early proximal and podocyte fates in cells expressing hyperactive Notch1 was observed. Orthogonal support for a conserved role for Notch signaling in the distal/proximal axis segmentation is provided by the ability of Nicastrin-deficient hiPSCs-derived organoids to differentiate into TFA2B+ distal tubule and CDH1 connecting segment progenitors, but not into HNF4A+ or LTL+ proximal progenitors.