Kidney organoids derived from human pluripotent stem cells exhibit glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here, we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which greatly expands their endogenous pool of endothelial progenitor cells (EPCs) and generates vascular networks with perfusable lumens surrounded by mural cells. Vascularized kidney organoids cultured under flow exhibit more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression, compared to static controls. However, the association of vessels with these compartments is reduced upon disrupting the endogenous VEGF gradient. Glomerular vascular development progresses through intermediate stages akin to the embryonic mammalian kidney’s formation of capillary loops abutting foot processes. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studying kidney development, disease, and regeneration.

Kidney injury is characterized by persisting inflammation and fibrosis, yet mechanisms by which inflammatory signals drive fibrogenesis remain poorly defined. RNA sequencing of fibrotic kidneys from patients with CKD identified a metabolic gene signature comprising loss of mitochondrial and oxidative phosphorylation gene expression with a concomitant increase in regulators and enzymes of glycolysis under the control of PGC1 and MYC transcription factors, respectively. We modeled this metabolic switch , in experimental murine models of kidney injury, and in human kidney stromal cells (SCs) and human kidney organoids. In mice, MYC and the target genes thereof became activated in resident SCs early after kidney injury, suggesting that acute innate immune signals regulate this transcriptional switch. , stimulation of purified human kidney SCs and human kidney organoids with IL-1 recapitulated the molecular events observed , inducing functional metabolic derangement characterized by increased MYC-dependent glycolysis, the latter proving necessary to drive proliferation and matrix production. MYC interacted directly with sequestosome 1/p62, which is involved in proteasomal degradation, and modulation of p62 expression caused inverse effects on MYC expression. IL-1 stimulated autophagy flux, causing degradation of p62 and accumulation of MYC. Inhibition of the IL-1R signal transducer kinase IRAK4 or inhibition of MYC as well as in human kidney organoids abrogated fibrosis and reduced tubular injury. Our findings define a connection between IL-1 and metabolic switch in fibrosis initiation and progression and highlight IL-1 and MYC as potential therapeutic targets in tubulointerstitial diseases.

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In mouse embryos, the Zfhx1 transcription factor genes, Sip1 and ␦EF1, are expressed in complementary domains in many tissues. Their possible synergism in embryogenesis was investigated by comparing the phenotype of Sip1؊/؊;␦EF1؊/؊ double homozygotes with single homozygous embryos. Unexpectedly, in Sip1؊/؊ embryos ␦EF1 was ectopically activated, suggesting a negative regulation of ␦EF1 expression by Sip1. Sip1؊/؊;␦EF1؊/؊ embryos were similar to Sip1؊/؊ embryos in short somite production and developmental arrest around E8.5, but showed more severe defects in dorsal neural tube morphogenesis accompanied by a larger reduction of Sox2 expression, ascribable to the loss of the ectopic ␦EF1 expression. Sip1؉/؊;␦EF1؊/؊ embryos develop various morphological defects after E10 that were absent in ␦EF1؊/؊ embryos even in tissues without significant overlap of Sip1 and ␦EF1 expression, and arrested during mid gestation earlier than ␦EF1؊/؊ embryos. These findings indicate that complex synergistic interactions occur between Zfhx1 transcription factor genes during mouse embryogenesis. Developmental Dynamics 235: 1941-1952, 2006.

Kidneys have the capacity for intrinsic repair, preserving kidney architecture with return to a basal state after tubular injury. When injury is overwhelming or repetitive, however, that capacity is exceeded and incomplete repair results in fibrotic tissue replacing normal kidney parenchyma. Loss of nephrons correlates with reduced kidney function, which defines chronic kidney disease (CKD) and confers substantial morbidity and mortality to the worldwide population. Despite the identification of pathways involved in intrinsic repair, limited treatments for CKD exist, partly because of the limited throughput and predictivity of animal studies. Here, we showed that kidney organoids can model the transition from intrinsic to incomplete repair. Single-nuclear RNA sequencing of kidney organoids after cisplatin exposure identified 159 differentially expressed genes and 29 signal pathways in tubular cells undergoing intrinsic repair. Homology-directed repair (HDR) genes including Fanconi anemia complementation group D2 ( FANCD2 ) and RAD51 recombinase ( RAD51 ) were transiently up-regulated during intrinsic repair but were down-regulated in incomplete repair. Single cellular transcriptomics in mouse models of obstructive and hemodynamic kidney injury and human kidney samples of immune-mediated injury validated HDR gene up-regulation during tubular repair. Kidney biopsy samples with tubular injury and varying degrees of fibrosis confirmed loss of FANCD2 during incomplete repair. Last, we performed targeted drug screening that identified the DNA ligase IV inhibitor, SCR7, as a therapeutic candidate that rescued FANCD2/RAD51-mediated repair to prevent the progression of CKD in the cisplatin-induced organoid injury model. Our findings demonstrate the translational utility of kidney organoids to identify pathologic pathways and potential therapies.

The kidney is one of the most complex organs composed of multiple cell types, functioning to maintain homeostasis by means of the filtering of metabolic wastes, balancing of blood electrolytes, and adjustment of blood pressure. Recent advances in 3D culture technologies in vitro enabled the generation of "organoids" which mimic the structure and function of in vivo organs. Organoid technology has allowed for new insights into human organ development and human pathophysiology, with great potential for translational research. Increasing evidence shows that kidney organoids are a useful platform for disease modeling of genetic kidney diseases when derived from genetic patient iPSCs and/or CRISPR-mutated stem cells.Although single cell RNA-seq studies highlight the technical difficulties underlying kidney organoid generation reproducibility and variation in differentiation protocols, kidney organoids still hold great potential to understand kidney pathophysiology as applied to kidney injury and fibrosis. In this review, we summarize various studies of kidney organoids, disease modeling, genome-editing, and bioengineering, and additionally discuss the potential of and current challenges to kidney organoid research.

Organoids serve as a novel tool for disease modeling in three-dimensional multicellular contexts. Static organoids, however, lack the requisite biophysical microenvironment such as fluid flow, limiting their ability to faithfully recapitulate disease pathology. Here, we unite organoids with organ-on-a-chip technology to unravel disease pathology and develop therapies for autosomal recessive polycystic kidney disease. PKHD1 -mutant organoids-on-a-chip are subjected to flow that induces clinically relevant phenotypes of distal nephron dilatation. Transcriptomics discover 229 signal pathways that are not identified by static models. Mechanosensing molecules, RAC1 and FOS, are identified as potential therapeutic targets and validated by patient kidney samples. On the basis of this insight, we tested two U.S. Food and Drug Administration–approved and one investigational new drugs that target RAC1 and FOS in our organoid-on-a-chip model, which suppressed cyst formation. Our observations highlight the vast potential of organoid-on-a-chip models to elucidate complex disease mechanisms for therapeutic testing and discovery.

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