Ontology-based modeling, integration, and analysis of heterogeneous clinical, pathological, and molecular kidney data for precision medicine.

Many data resources generate, process, store, or provide kidney related molecular, pathological, and clinical data. Reference ontologies offer an opportunity to support knowledge and data integration. The Kidney Precision Medicine Project (KPMP) team contributed to the representation and addition of 329 kidney phenotype terms to the Human Phenotype Ontology (HPO), and identified many subcategories of acute kidney injury (AKI) or chronic kidney disease (CKD). The Kidney Tissue Atlas Ontology (KTAO) imports and integrates kidney-related terms from existing ontologies (e.g., HPO, CL, and Uberon) and represents 259 kidney-related biomarkers. We also developed a precision medicine metadata ontology (PMMO) to integrate 50 variables from KPMP and CZ CellxGene data resources and applied PMMO for integrative kidney data analysis. The gene expression profiles of kidney gene biomarkers were specifically analyzed under healthy control or AKI/CKD disease statuses. This work demonstrates how ontology-based approaches support multi-domain data and knowledge integration in precision medicine.

Energy depletion by cell proliferation sensitizes the kidney epithelial cells to injury.

Acute kidney injury activates both proliferative and antiproliferative pathways, the consequences of which are not fully elucidated. If an initial proliferation of the renal epithelium is necessary for the successful repair, the persistence of proliferation markers is associated with the occurrence of chronic kidney disease. We hypothesized that proliferation in stress conditions impacts cell viability and renal outcomes. We found that proliferation is associated with cell death after various stresses in kidney cells. In vitro, the ATP/ADP ratio oscillates reproducibly throughout the cell cycle, and cell proliferation is associated with a decreased intracellular ATP/ADP ratio. In vivo, transcriptomic data from transplanted kidneys revealed that proliferation was strongly associated with a decrease in the expression of the mitochondria-encoded genes of the oxidative phosphorylation pathway, but not of the nucleus-encoded ones. These observations suggest that mitochondrial function is a limiting factor for energy production in proliferative kidney cells after injury. The association of increased proliferation and decreased mitochondrial function was indeed associated with poor renal outcomes. In summary, proliferation is an energy-demanding process impairing the cellular ability to cope with an injury, highlighting proliferative repair and metabolic recovery as indispensable and interdependent features for successful kidney repair.NEW & NOTEWORTHY ATP depletion is a hallmark of acute kidney injury. Proliferation is instrumental to kidney repair. We show that ATP levels vary during the cell cycle and that proliferation sensitizes renal epithelial cells to superimposed injuries in vitro. More proliferation and less energy production by the mitochondria are associated with adverse outcomes in injured kidney allografts. This suggests that controlling the timing of kidney repair might be beneficial to mitigate the extent of acute kidney injury.

Human ureteric bud organoids recapitulate branching morphogenesis and differentiate into functional collecting duct cell types.

Directed differentiation of human pluripotent stem cells (hPSCs) into functional ureteric and collecting duct (CD) epithelia is essential to kidney regenerative medicine. Here we describe highly efficient, serum-free differentiation of hPSCs into ureteric bud (UB) organoids and functional CD cells. The hPSCs are first induced into pronephric progenitor cells at 90% efficiency and then aggregated into spheres with a molecular signature similar to the nephric duct. In a three-dimensional matrix, the spheres form UB organoids that exhibit branching morphogenesis similar to the fetal UB and correct distal tip localization of RET expression. Organoid-derived cells incorporate into the UB tips of the progenitor niche in chimeric fetal kidney explant culture. At later stages, the UB organoids differentiate into CD organoids, which contain >95% CD cell types as estimated by single-cell RNA sequencing. The CD epithelia demonstrate renal electrophysiologic functions, with ENaC-mediated vectorial sodium transport by principal cells and V-type ATPase proton pump activity by FOXI1-induced intercalated cells.

Organoid-on-a-chip model of human ARPKD reveals mechanosensing pathomechanisms for drug discovery.

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.

A reference tissue atlas for the human kidney.

Kidney Precision Medicine Project (KPMP) is building a spatially specified human kidney tissue atlas in health and disease with single-cell resolution. Here, we describe the construction of an integrated reference map of cells, pathways, and genes using unaffected regions of nephrectomy tissues and undiseased human biopsies from 56 adult subjects. We use single-cell/nucleus transcriptomics, subsegmental laser microdissection transcriptomics and proteomics, near-single-cell proteomics, 3D and CODEX imaging, and spatial metabolomics to hierarchically identify genes, pathways, and cells. Integrated data from these different technologies coherently identify cell types/subtypes within different nephron segments and the interstitium. These profiles describe cell-level functional organization of the kidney following its physiological functions and link cell subtypes to genes, proteins, metabolites, and pathways. They further show that messenger RNA levels along the nephron are congruent with the subsegmental physiological activity. This reference atlas provides a framework for the classification of kidney disease when multiple molecular mechanisms underlie convergent clinical phenotypes.

Kidney repair and regeneration: perspectives of the NIDDK (Re)Building a Kidney consortium.

Acute kidney injury impacts âˆ¼13.3 million individuals and causes âˆ¼1.7 million deaths per year globally. Numerous injury pathways contribute to acute kidney injury, including cell cycle arrest, senescence, inflammation, mitochondrial dysfunction, and endothelial injury and dysfunction, and can lead to chronic inflammation and fibrosis. However, factors enabling productive repair versus nonproductive, persistent injury states remain less understood. The (Re)Building a Kidney (RBK) consortium is a National Institute of Diabetes and Digestive and Kidney Diseases consortium focused on both endogenous kidney repair mechanisms and the generation of new kidney tissue. This short review provides an update on RBK studies of endogenous nephron repair, addressing the following questions: (i) What is productive nephron repair? (ii) What are the cellular sources and drivers of repair? and (iii) How do RBK studies promote development of therapeutics? Also, we provide a guide to RBK's open access data hub for accessing, downloading, and further analyzing data sets.

Anatomical structures, cell types and biomarkers of the Human Reference Atlas.

The Human Reference Atlas (HRA) aims to map all of the cells of the human body to advance biomedical research and clinical practice. This Perspective presents collaborative work by members of 16 international consortia on two essential and interlinked parts of the HRA: (1) three-dimensional representations of anatomy that are linked to (2) tables that name and interlink major anatomical structures, cell types, plus biomarkers (ASCT+B). We discuss four examples that demonstrate the practical utility of the HRA.

Orphan nuclear receptor COUP-TFII enhances myofibroblast glycolysis leading to kidney fibrosis.

Recent studies demonstrate that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts also induced production of alpha-smooth muscle actin (αSMA) and collagen 1. Knockout of COUP-TFII decreased glycolysis and collagen 1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs.

Modelling kidney disease using ontology: insights from the Kidney Precision Medicine Project.

An important need exists to better understand and stratify kidney disease according to its underlying pathophysiology in order to develop more precise and effective therapeutic agents. National collaborative efforts such as the Kidney Precision Medicine Project are working towards this goal through the collection and integration of large, disparate clinical, biological and imaging data from patients with kidney disease. Ontologies are powerful tools that facilitate these efforts by enabling researchers to organize and make sense of different data elements and the relationships between them. Ontologies are critical to support the types of big data analysis necessary for kidney precision medicine, where heterogeneous clinical, imaging and biopsy data from diverse sources must be combined to define a patient's phenotype. The development of two new ontologies - the Kidney Tissue Atlas Ontology and the Ontology of Precision Medicine and Investigation - will support the creation of the Kidney Tissue Atlas, which aims to provide a comprehensive molecular, cellular and anatomical map of the kidney. These ontologies will improve the annotation of kidney-relevant data, and eventually lead to new definitions of kidney disease in support of precision medicine.

Enhancer and super-enhancer dynamics in repair after ischemic acute kidney injury.

The endogenous repair process can result in recovery after acute kidney injury (AKI) with adaptive proliferation of tubular epithelial cells, but repair can also lead to fibrosis and progressive kidney disease. There is currently limited knowledge about transcriptional regulators regulating these repair programs. Herein we establish the enhancer and super-enhancer landscape after AKI by ChIP-seq in uninjured and repairing kidneys on day two after ischemia reperfusion injury (IRI). We identify key transcription factors including HNF4A, GR, STAT3 and STAT5, which show specific binding at enhancer and super-enhancer sites, revealing enhancer dynamics and transcriptional changes during kidney repair. Loss of bromodomain-containing protein 4 function before IRI leads to impaired recovery after AKI and increased mortality. Our comprehensive analysis of epigenetic changes after kidney injury in vivo has the potential to identify targets for therapeutic intervention. Importantly, our data also call attention to potential caveats involved in use of BET inhibitors in patients at risk for AKI.

Flow-enhanced vascularization and maturation of kidney organoids in vitro.

Kidney organoids derived from human pluripotent stem cells have 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 expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls. Glomerular vascular development progressed through intermediate stages akin to those involved in the embryonic mammalian kidney's formation of capillary loops abutting foot processes. The association of vessels with these compartments was reduced after disruption of the endogenous VEGF gradient. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studies of kidney development, disease, and regeneration.

CRISPR/Cas9-based Targeted Genome Editing for the Development of Monogenic Diseases Models with Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) represent a formidable tool for disease modeling, drug discovery, and regenerative medicine using human cells and tissues in vitro. Evolving techniques of targeted genome editing, specifically the CRISPR/Cas9 system, allow for the generation of cell lines bearing gene-specific knock-outs, knock-in reporters, and precise mutations. However, there are increasing concerns related to the transfection efficiency, cell viability, and maintenance of pluripotency provided by genome-editing techniques. The procedure presented here employs transient antibiotic selection that overcomes reduced transfection efficiency, avoids cytotoxic flow sorting for increased viability, and generates multiple genome-edited pluripotent hPSC lines expanded from a single parent cell. Avoidance of xenogeneic contamination from feeder cells and reduced operator workload, owing to single-cell passaging rather than clump passaging, are additional benefits. The outlined methods may enable researchers with limited means and technical experience to create human stem cell lines containing desired gene-specific mutations. © 2018 by John Wiley & Sons, Inc.

Disparate levels of beta-catenin activity determine nephron progenitor cell fate.

Formation of a functional kidney depends on the balance between renewal and differentiation of nephron progenitors. Failure to sustain this balance can lead to kidney failure or stem cell tumors. For nearly 60 years, we have known that signals from an epithelial structure known as the ureteric bud were essential for maintaining this balance. More recently it was discovered that one molecule, Wnt9b, was necessary for both renewal and differentiation of the nephron progenitor cells. How one ligand signaling through one transcription factor promoted two seemingly contradictory cellular processes was unclear. In this study, we show that Wnt9b/beta-catenin signaling alone is sufficient to promote both renewal and differentiation. Moreover, we show that discrete levels of beta-catenin can promote these two disparate fates, with low levels fostering progenitor renewal and high levels driving differentiation. These results provide insight into how Wnt9b regulates distinct target genes that balance nephron progenitor renewal and differentiation.

Repression of Interstitial Identity in Nephron Progenitor Cells by Pax2 Establishes the Nephron-Interstitium Boundary during Kidney Development.

The kidney contains the functional units, the nephrons, surrounded by the renal interstitium. Previously we discovered that, once Six2-expressing nephron progenitor cells and Foxd1-expressing renal interstitial progenitor cells form at the onset of kidney development, descendant cells from these populations contribute exclusively to the main body of nephrons and renal interstitial tissues, respectively, indicating a lineage boundary between the nephron and renal interstitial compartments. Currently it is unclear how lineages are regulated during kidney organogenesis. We demonstrate that nephron progenitor cells lacking Pax2 fail to differentiate into nephron cells but can switch fates into renal interstitium-like cell types. These data suggest that Pax2 function maintains nephron progenitor cells by repressing a renal interstitial cell program. Thus, the lineage boundary between the nephron and renal interstitial compartments is maintained by the Pax2 activity in nephron progenitor cells during kidney organogenesis.

Repair after nephron ablation reveals limitations of neonatal neonephrogenesis.

The neonatal mouse kidney retains nephron progenitor cells in a nephrogenic zone for 3 days after birth. We evaluated whether de novo nephrogenesis can be induced postnatally beyond 3 days. Given the long-term implications of nephron number for kidney health, it would be useful to enhance nephrogenesis in the neonate. We induced nephron reduction by cryoinjury with or without contralateral nephrectomy during the neonatal period or after 1 week of age. There was no detectable compensatory de novo nephrogenesis, as determined by glomerular counting and lineage tracing. Contralateral nephrectomy resulted in additional adaptive healing, with little or no fibrosis, but did not also stimulate de novo nephrogenesis. In contrast, injury initiated at 1 week of age led to healing with fibrosis. Thus, despite the presence of progenitor cells and ongoing nephron maturation in the newborn mouse kidney, de novo nephrogenesis is not inducible by acute nephron reduction. This indicates that additional nephron progenitors cannot be recruited after birth despite partial renal ablation providing a reparative stimulus and suggests that nephron number in the mouse is predetermined at birth.

(Re)Building a Kidney.

(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.

SOCS2 Balances Metabolic and Restorative Requirements during Liver Regeneration.

After significant injury, the liver must maintain homeostasis during the regenerative process. We hypothesized the existence of mechanisms to limit hepatocyte proliferation after injury to maintain metabolic and synthetic function. A screen for candidates revealed suppressor of cytokine signaling 2 (SOCS2), an inhibitor of growth hormone (GH) signaling, was strongly induced after partial hepatectomy. Using genetic deletion and administration of various factors we investigated the role of SOCS2 during liver regeneration. SOCS2 preserves liver function by restraining the first round of hepatocyte proliferation after partial hepatectomy by preventing increases in growth hormone receptor (GHR) via ubiquitination, suppressing GH pathway activity. At later times, SOCS2 enhances hepatocyte proliferation by modulating a decrease in serum insulin-like growth factor 1 (IGF-1) that allows GH release from the pituitary. SOCS2, therefore, plays a dual role in modulating the rate of hepatocyte proliferation. In particular, this is the first demonstration of an endogenous mechanism to limit hepatocyte proliferation after injury.

Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids.

Human-pluripotent-stem-cell-derived kidney cells (hPSC-KCs) have important potential for disease modelling and regeneration. Whether the hPSC-KCs can reconstitute tissue-specific phenotypes is currently unknown. Here we show that hPSC-KCs self-organize into kidney organoids that functionally recapitulate tissue-specific epithelial physiology, including disease phenotypes after genome editing. In three-dimensional cultures, epiblast-stage hPSCs form spheroids surrounding hollow, amniotic-like cavities. GSK3ß inhibition differentiates spheroids into segmented, nephron-like kidney organoids containing cell populations with characteristics of proximal tubules, podocytes and endothelium. Tubules accumulate dextran and methotrexate transport cargoes, and express kidney injury molecule-1 after nephrotoxic chemical injury. CRISPR/Cas9 knockout of podocalyxin causes junctional organization defects in podocyte-like cells. Knockout of the polycystic kidney disease genes PKD1 or PKD2 induces cyst formation from kidney tubules. All of these functional phenotypes are distinct from effects in epiblast spheroids, indicating that they are tissue specific. Our findings establish a reproducible, versatile three-dimensional framework for human epithelial disease modelling and regenerative medicine applications.

Nephron organoids derived from human pluripotent stem cells model kidney development and injury.

Kidney cells and tissues derived from human pluripotent stem cells (hPSCs) may enable organ regeneration, disease modeling and drug screening. We report an efficient, chemically defined protocol for differentiating hPSCs into multipotent nephron progenitor cells (NPCs) that can form nephron-like structures. By recapitulating metanephric kidney development in vitro, we generate SIX2+ SALL1+ WT1+ PAX2+ NPCs with 90% efficiency within 9 days of differentiation. The NPCs possess the developmental potential of their in vivo counterparts and form PAX8+ LHX1+ renal vesicles that self-organize into nephron structures. In both two- and three-dimensional culture, NPCs form kidney organoids containing epithelial nephron-like structures expressing markers of podocytes, proximal tubules, loops of Henle and distal tubules in an organized, continuous arrangement that resembles the nephron in vivo. We also show that this organoid culture system can be used to study mechanisms of human kidney development and toxicity.