(Re)Building a Kidney

Abstract

(Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases-led consortium to optimize approaches for the isolation, expansion, and differentiation of appropriate kidney cell types and the integration of these cells into complex structures that replicate human kidney function. The ultimate goals of the consortium are two-fold: to develop and implement strategies for in vitro engineering of replacement kidney tissue, and to devise strategies to stimulate regeneration of nephrons in situ to restore failing kidney function. Projects within the consortium will answer fundamental questions regarding human gene expression in the developing kidney, essential signaling crosstalk between distinct cell types of the developing kidney, how to derive the many cell types of the kidney through directed differentiation of human pluripotent stem cells, which bioengineering or scaffolding strategies have the most potential for kidney tissue formation, and basic parameters of the regenerative response to injury. As these projects progress, the consortium will incorporate systematic investigations in physiologic function of in vitro and in vivo differentiated kidney tissue, strategies for engraftment in experimental animals, and development of therapeutic approaches to activate innate reparative responses.

Keywords: Directed differentiation; Organogenesis; Organoid; Regeneration.

Figure 1.

The RBK Consortium is taking multiple approaches to achieve its goals of engineering and engrafting new kidney tissue as well as enhancing kidney repair and regeneration to restore kidney function. (1) iPSC/ESC > NPCs: propagating human progenitor cells in their undifferentiated state and using them to generate kidney cells and organoids, populate tissue scaffolds, and recellularize kidney matrix are key goals of the RBK consortium. Starting with iPSCs or established ESC lines, NPCs will be generated using differentiation protocols for nephrogenic mesoderm. Propagation and self-renewal of these cells and mouse cap mesenchyme NPCs will be optimized using transgenic reporters of the progenitor cell type, quantitative RT-PCR of qualified human marker genes, and single-cell mRNA phenotyping as end points to detect expansion of the progenitor cell type. (2) Kidney organoids/kidney cell types: differentiation of self-organizing organoids or directed differentiation of individual kidney cell types will also be achieved by optimizing medium conditions and assaying outcomes using cell-type specific fluorescent protein transgenic reporter iPSC/ESC lines, quantitative RT-PCR of qualified human marker genes, and single-cell mRNA phenotyping. Fluorescent protein reporter human iPSC lines will also allow live imaging of ex vivo kidney differentiation as well as reisolation and transcriptional profiling of organoid-derived kidney cells, including nephron and stromal progenitors, podocytes, proximal tubules, distal tubules, and endothelium. Rigorously defined human kidney cell transcriptional signatures as well as cell injury markers derived from single-cell RNA sequencing and MARIS will be essential for organoid and cell type quality control and to establish baseline phenotypes for further functional characterization, disease modeling, and potential therapeutic use. (3) Scaffold tissue assembly: multiple approaches, including decellularized kidneys, silk, and synthetic polymers, and three-dimensional printed arrays of nephron tubules and blood vessels, will be tested to generate structures into which differentiated kidney cells or organoids can be seeded, instructed to differentiate, and interconnect nephron tubules to form functioning kidney tissue with the potential to replace failed kidney function. (4) Cell therapy/repair and regeneration factors: defined growth factor activities optimized for progenitor cell self-renewal and organoid differentiation as well as for directed differentiation of renal cell types will be integrated into new approaches to promote endogenous repair of nephrons. Adult kidney podocyte and interstitial progenitor cells will be profiled and characterized to develop new ways to promote endogenous repair. Contributions of nonepithelial cells, the endothelium, interstitium, and pericytes/vascular smooth muscle to kidney cell differentiation will also be assayed and modeled using fluorescent reporter lines and coculture with nephron cells in two-dimensional and three-dimensional cultures. ESC, embryonic stem cell lines.

Figure 2.

iPSC-derived kidney organoids contain all components of the fetal human kidney, including patterned and segmented nephrons, collecting ducts, renal interstitium, and endothelium. Comparison of mature and developing cell types between normal human fetal kidney (left) and iPSC-derived kidney organoids (right). Structures: CD, collecting duct; Endo, endothelium; Glom, glomerulus; PC, perivascular cell; PT, proximal tubule; SSB, s-shaped body nephron; STR, stroma. Markers: CALB1, collecting duct in early kidney and mature distal tubule in nephrons; CD31, endothelial cells; CUBN, proximal tubule; DAPI, nuclei; ECAD, distal epithelial cells; GATA3, collecting duct; KRT8, collecting duct in early kidney and mature distal tubule in nephrons; LTL, proximal tubule; MAFB, podocytes and glomeruli; MEIS1, interstitial cells; NPHS1, podocytes; PAX2, collecting duct and nephron; PDGFRA, pericytes; SOX17, endothelial cells. Images of human fetal kidney supplied by McMahon laboratory (Tracy Tran and Nils Lindström). Images of human iPSC-derived kidney organoid supplied by Little laboratory (Han Chiu, Minoru Takasato, Pei Er, Jessica Vanslambrouck, and Sara Howden).

Figure 3.

Multiple different bioengineering approaches to generating tissue scaffolds offer unique advantages for rebuilding kidney tissue. Polymer-based (A and B), decellularized tissue (C and D), and organ-on-a-chip (E and F) approaches are shown. (A) Scanning electron micrograph of 6% silk scaffold before cell seeding; a network of channels is formed into which NPCs can be seeded. Photo: Jeannine Coburn. (B) E-cadherin (epithelial cells) and DAPI (nuclei) staining reveals a network of tubules with lumens within the silk approximately 2 weeks after seeding NPCs. Photo: Jessica Davis-Knowlton. (C) Scanning electron micrograph of decellularized kidney tissue showing the extracellular matrix framework for tubules and glomeruli. Photo: Joseph Uzarski. (D) Immunostaining of a decellularized kidney that has been recellularized with MDCK cells 7 days after cell seeding. E-cadherin (green) and luminal cilia (acetylated tubulin, red) demonstrate epithelialization. Photo: Joseph Uzarski. (E) Schematic and top view picture of a vascular network-on-a-chip device that allows flow through the network via inlet (i) and outlet (o). Photo: Zheng laboratory. (F) An example view of a three dimensional microvessel network formed by mouse kidney endothelial cells. Red: CD31, blue: DAPI. The inset shows fluorescence immunostaining of a device in which podocytes (green) were cocultured with the vascular endothelial network (red). Photo: Zheng laboratory. EHT, extra high tension voltage setting; WD, working distance.

Figure 4.

Integration of vasculature, overcoming kidney fibrosis, and connecting bioengineered kidney tubules to the collecting system remain important challenges in advancing renal replacement therapy. The kidney vasculature represents a variety of unexplored endothelial cell subtypes that may affect nephron differentiation through reciprocal signaling events. (A) In the developing mouse kidney, vascular progenitor endothelial cells positive for Pecam (PE)/CD31(green) are intimately associated with the nephrogenic cortical fetal kidney domain populated by Six2-positive nephrogenic progenitor cells (red). E-cadherin (white) marks polarized epithelium of the branching ureter tips. Photo: Edward Daniel. (B) Kidney injury and fibrosis represents a major barrier to regeneration. Kidney injury recruits myofibroblasts, leading to fibrosis and CKD. Kim-1 immunostaining (green) identifies injured and dedifferentiated human proximal tubule whereas PDGFRb immunostaining reveals activated adjacent interstitial kidney fibroblasts (red). Photo: Monica Chang-Panesso and Farid Kadyrov. (C) Nephron interconnection is an essential process in generating a filtering kidney. New insights from the regenerating zebrafish kidney, where many new nephrons are added to the existing collecting system, can help define signaling pathways that stimulate cell rearrangements that lead to interconnected tubule lumens. The lhx1a:GFP transgene (green) marks new nephron cell projections, or invadopodia (arrowheads), that penetrate the collecting system as a prelude to interconnection. Photo: Caramai N. Kamei. CD, Collecting duct; nN, new nephron; blue fluorescence, DAPI nuclear stain.