Kidney Organoids Generated Using an Allelic Series of NPHS2 Point Variants Reveal Distinct Intracellular Podocin Mistrafficking.

BACKGROUND: NPHS2 variants are the most common cause of steroid-resistant nephrotic syndrome in children >1 month old. Missense NPHS2 variants were reported to cause mistrafficking of the encoded protein, PODOCIN, but this conclusion was on the basis of overexpression in some nonpodocyte cell lines. METHODS: We generated a series of human induced pluripotent stem cell (iPSC) lines bearing pathogenic missense variants of NPHS2 , encoding the protein changes p.G92C, p.P118L, p.R138Q, p.R168H, and p.R291W, and control lines. iPSC lines were also generated from a patient with steroid-resistant nephrotic syndrome (p.R168H homozygote) and a healthy heterozygous parent. All lines were differentiated into kidney organoids. Immunofluorescence assessed PODOCIN expression and subcellular localization. Podocytes were transcriptionally profiled and PODOCIN-NEPHRIN interaction interrogated. RESULTS: All variant lines revealed reduced levels of PODOCIN protein in the absence of reduced transcription. Although wild-type PODOCIN localized to the membrane, distinct variant proteins displayed unique patterns of subcellular protein trafficking, some unreported. P118L and R138Q were preferentially retained in the endoplasmic reticulum (ER); R168H and R291W accumulated in the Golgi. Podocyte profiling demonstrated minimal disease-associated transcriptional change. All variants displayed podocyte-specific apoptosis, which was not linked to ER stress. NEPHRIN-PODOCIN colocalization elucidated the variant-specific effect on NEPHRIN association and hence NEPHRIN trafficking. CONCLUSIONS: Specific variants of endogenous NPHS2 result in distinct subcellular PODOCIN localization within organoid podocytes. Understanding the effect of each variant on protein levels and localization and the effect on NEPHRIN provides additional insight into the pathobiology of NPHS2 variants. PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_01_05_JASN2022060707.mp3.

Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids.

While pluripotent stem cell-derived kidney organoids are now being used to model renal disease, the proximal nephron remains immature with limited evidence for key functional solute channels. This may reflect early mispatterning of the nephrogenic mesenchyme and/or insufficient maturation. Here we show that enhanced specification to metanephric nephron progenitors results in elongated and radially aligned proximalised nephrons with distinct S1 - S3 proximal tubule cell types. Such PT-enhanced organoids possess improved albumin and organic cation uptake, appropriate KIM-1 upregulation in response to cisplatin, and improved expression of SARS-CoV-2 entry factors resulting in increased viral replication. The striking proximo-distal orientation of nephrons resulted from localized WNT antagonism originating from the organoid stromal core. PT-enhanced organoids represent an improved model to study inherited and acquired proximal tubular disease as well as drug and viral responses.

Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids.

While pluripotent stem cell-derived kidney organoids are now being used to model renal disease, the proximal nephron remains immature with limited evidence for key functional solute channels. This may reflect early mispatterning of the nephrogenic mesenchyme and/or insufficient maturation. Here we show that enhanced specification to metanephric nephron progenitors results in elongated and radially aligned proximalised nephrons with distinct S1 - S3 proximal tubule cell types. Such PT-enhanced organoids possess improved albumin and organic cation uptake, appropriate KIM-1 upregulation in response to cisplatin, and improved expression of SARS-CoV-2 entry factors resulting in increased viral replication. The striking proximo-distal orientation of nephrons resulted from localized WNT antagonism originating from the organoid stromal core. PT-enhanced organoids represent an improved model to study inherited and acquired proximal tubular disease as well as drug and viral responses.

Determining lineage relationships in kidney development and disease.

The lineage relationships of cells provide information about the origins of component cell types during development and repair as well as the source of aberrant cells during disease. Genetic approaches to lineage tracing applied in the mouse have revealed much about how the mammalian kidney forms, including the identification of key progenitors for the nephrons and stromal compartments. Inducible Cre systems have also facilitated lineage tracing studies in the postnatal animal that illustrate the changes in cellular fate that can occur during kidney injury. With the advent of single-cell transcriptional profiling and trajectory analyses, predictions of cellular relationships across development are now being made in model systems, such as the mouse, as well as in human fetal kidney. Importantly, these approaches provide predictions of lineage relationships rather than definitive evidence. Although genetic approaches to the study of lineage have not previously been possible in a human setting, the application of CRISPR-Cas9 gene editing of pluripotent stem cells is beginning to teach us about human lineage relationships.

Cellular extrusion bioprinting improves kidney organoid reproducibility and conformation.

Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here, we apply extrusion-based three-dimensional cellular bioprinting to deliver rapid and high-throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate the relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, three-dimensional bioprinting enables precise manipulation of biophysical properties, including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitates the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production delivers improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.

Single cell analysis of the developing mouse kidney provides deeper insight into marker gene expression and ligand-receptor crosstalk.

Recent advances in the generation of kidney organoids and the culture of primary nephron progenitors from mouse and human have been based on knowledge of the molecular basis of kidney development in mice. Although gene expression during kidney development has been intensely investigated, single cell profiling provides new opportunities to further subsect component cell types and the signalling networks at play. Here, we describe the generation and analysis of 6732 single cell transcriptomes from the fetal mouse kidney [embryonic day (E)18.5] and 7853 sorted nephron progenitor cells (E14.5). These datasets provide improved resolution of cell types and specific markers, including subdivision of the renal stroma and heterogeneity within the nephron progenitor population. Ligand-receptor interaction and pathway analysis reveals novel crosstalk between cellular compartments and associates new pathways with differentiation of nephron and ureteric epithelium cell types. We identify transcriptional congruence between the distal nephron and ureteric epithelium, showing that most markers previously used to identify ureteric epithelium are not specific. Together, this work improves our understanding of metanephric kidney development and provides a template to guide the regeneration of renal tissue.

Kidney micro-organoids in suspension culture as a scalable source of human pluripotent stem cell-derived kidney cells.

Kidney organoids have potential uses in disease modelling, drug screening and regenerative medicine. However, novel cost-effective techniques are needed to enable scaled-up production of kidney cell types in vitro We describe here a modified suspension culture method for the generation of kidney micro-organoids from human pluripotent stem cells. Optimisation of differentiation conditions allowed the formation of micro-organoids, each containing six to ten nephrons that were surrounded by endothelial and stromal populations. Single cell transcriptional profiling confirmed the presence and transcriptional equivalence of all anticipated renal cell types consistent with a previous organoid culture method. This suspension culture micro-organoid methodology resulted in a three- to fourfold increase in final cell yield compared with static culture, thereby representing an economical approach to the production of kidney cells for various biological applications.

Nephron progenitor commitment is a stochastic process influenced by cell migration.

Progenitor self-renewal and differentiation is often regulated by spatially restricted cues within a tissue microenvironment. Here, we examine how progenitor cell migration impacts regionally induced commitment within the nephrogenic niche in mice. We identify a subset of cells that express Wnt4, an early marker of nephron commitment, but migrate back into the progenitor population where they accumulate over time. Single cell RNA-seq and computational modelling of returning cells reveals that nephron progenitors can traverse the transcriptional hierarchy between self-renewal and commitment in either direction. This plasticity may enable robust regulation of nephrogenesis as niches remodel and grow during organogenesis.

Evaluation of variability in human kidney organoids.

The utility of human pluripotent stem cell-derived kidney organoids relies implicitly on the robustness and transferability of the protocol. Here we analyze the sources of transcriptional variation in a specific kidney organoid protocol. Although individual organoids within a differentiation batch showed strong transcriptional correlation, we noted significant variation between experimental batches, particularly in genes associated with temporal maturation. Single-cell profiling revealed shifts in nephron patterning and proportions of component cells. Distinct induced pluripotent stem cell clones showed congruent transcriptional programs, with interexperimental and interclonal variation also strongly associated with nephron patterning. Epithelial cells isolated from organoids aligned with total organoids at the same day of differentiation, again implicating relative maturation as a confounder. This understanding of experimental variation facilitated an optimized analysis of organoid-based disease modeling, thereby increasing the utility of kidney organoids for personalized medicine and functional genomics.

3D organoid-derived human glomeruli for personalised podocyte disease modelling and drug screening.

The podocytes within the glomeruli of the kidney maintain the filtration barrier by forming interdigitating foot processes with intervening slit diaphragms, disruption in which results in proteinuria. Studies into human podocytopathies to date have employed primary or immortalised podocyte cell lines cultured in 2D. Here we compare 3D human glomeruli sieved from induced pluripotent stem cell-derived kidney organoids with conditionally immortalised human podocyte cell lines, revealing improved podocyte-specific gene expression, maintenance in vitro of polarised protein localisation and an improved glomerular basement membrane matrisome compared to 2D cultures. Organoid-derived glomeruli retain marker expression in culture for 96 h, proving amenable to toxicity screening. In addition, 3D organoid glomeruli from a congenital nephrotic syndrome patient with compound heterozygous NPHS1 mutations reveal reduced protein levels of both NEPHRIN and PODOCIN. Hence, human iPSC-derived organoid glomeruli represent an accessible approach to the in vitro modelling of human podocytopathies and screening for podocyte toxicity.

Pericytes promote skin regeneration by inducing epidermal cell polarity and planar cell divisions.

The cellular and molecular microenvironment of epithelial stem/progenitor cells is critical for their long-term self-renewal. We demonstrate that mesenchymal stem cell-like dermal microvascular pericytes are a critical element of the skin's microenvironment influencing human skin regeneration using organotypic models. Specifically, pericytes were capable of promoting homeostatic skin tissue renewal by conferring more planar cell divisions generating two basal cells within the proliferative compartment of the human epidermis, while ensuring complete maturation of the tissue both spatially and temporally. Moreover, we provide evidence supporting the notion that BMP-2, a secreted protein preferentially expressed by pericytes in human skin, confers cell polarity and planar divisions on epidermal cells in organotypic cultures. Our data suggest that human skin regeneration is regulated by highly conserved mechanisms at play in other rapidly renewing tissues such as the bone marrow and in lower organisms such as Drosophila. Our work also provides the means to significantly improve ex vivo skin tissue regeneration for autologous transplantation.

Patient-iPSC-Derived Kidney Organoids Show Functional Validation of a Ciliopathic Renal Phenotype and Reveal Underlying Pathogenetic Mechanisms.

Despite the increasing diagnostic rate of genomic sequencing, the genetic basis of more than 50% of heritable kidney disease remains unresolved. Kidney organoids differentiated from induced pluripotent stem cells (iPSCs) of individuals affected by inherited renal disease represent a potential, but unvalidated, platform for the functional validation of novel gene variants and investigation of underlying pathogenetic mechanisms. In this study, trio whole-exome sequencing of a prospectively identified nephronophthisis (NPHP) proband and her parents identified compound-heterozygous variants in IFT140, a gene previously associated with NPHP-related ciliopathies. IFT140 plays a key role in retrograde intraflagellar transport, but the precise downstream cellular mechanisms responsible for disease presentation remain unknown. A one-step reprogramming and gene-editing protocol was used to derive both uncorrected proband iPSCs and isogenic gene-corrected iPSCs, which were differentiated to kidney organoids. Proband organoid tubules demonstrated shortened, club-shaped primary cilia, whereas gene correction rescued this phenotype. Differential expression analysis of epithelial cells isolated from organoids suggested downregulation of genes associated with apicobasal polarity, cell-cell junctions, and dynein motor assembly in proband epithelial cells. Matrigel cyst cultures confirmed a polarization defect in proband versus gene-corrected renal epithelium. As such, this study represents a "proof of concept" for using proband-derived iPSCs to model renal disease and illustrates dysfunctional cellular pathways beyond the primary cilium in the setting of IFT140 mutations, which are established for other NPHP genotypes.

Haploinsufficiency for the Six2 gene increases nephron progenitor proliferation promoting branching and nephron number.

The regulation of final nephron number in the kidney is poorly understood. Cessation of nephron formation occurs when the self-renewing nephron progenitor population commits to differentiation. Transcription factors within this progenitor population, such as SIX2, are assumed to control expression of genes promoting self-renewal such that homozygous Six2 deletion results in premature commitment and an early halt to kidney development. In contrast, Six2 heterozygotes were assumed to be unaffected. Using quantitative morphometry, we found a paradoxical 18% increase in ureteric branching and final nephron number in Six2 heterozygotes, despite evidence for reduced levels of SIX2 protein and transcript. This was accompanied by a clear shift in nephron progenitor identity with a distinct subset of downregulated progenitor genes such as Cited1 and Meox1 while other genes were unaffected. The net result was an increase in nephron progenitor proliferation, as assessed by elevated EdU (5-ethynyl-2'-deoxyuridine) labeling, an increase in MYC protein, and transcriptional upregulation of MYC target genes. Heterozygosity for Six2 on an Fgf20-/- background resulted in premature differentiation of the progenitor population, confirming that progenitor regulation is compromised in Six2 heterozygotes. Overall, our studies reveal a unique dose response of nephron progenitors to the level of SIX2 protein in which the role of SIX2 in progenitor proliferation versus self-renewal is separable.

Dermal Contributions to Human Interfollicular Epidermal Architecture and Self-Renewal.

The human interfollicular epidermis is renewed throughout life by populations of proliferating basal keratinocytes. Though interfollicular keratinocyte stem cells have been identified, it is not known how self-renewal in this compartment is spatially organized. At the epidermal-dermal junction, keratinocytes sit atop a heterogeneous mix of dermal cells that may regulate keratinocyte self-renewal by influencing local tissue architecture and signalling microenvironments. Focusing on the rete ridges and complementary dermal papillae in human skin, we review the identity and organisation of abundant dermal cells types and present evidence for interactions between the dermal microenvironment and the interfollicular keratinocytes.

Drosophila Dachsous and Fat polarize actin-based protrusions over a restricted domain of the embryonic denticle field.

Atypical cadherins Dachsous (Ds) and Fat coordinate the establishment of planar polarity, essential for the patterning of complex tissues and organs. The precise mechanisms by which this system acts, particularly in cases where Ds and Fat act independently of the 'core' frizzled system, are still the subject of investigation. Examining the deployment of the Ds-Fat system in different tissues of the model organism Drosophila, has provided insights into the general mechanisms by which polarity is established and propagated to coordinate outcomes across a field of cells. The Drosophila embryonic epidermis provides a simple model epithelia where the establishment of polarity can be observed from start to finish, and in the absence of proliferation, over a fixed number of cells. Using the asymmetric placement of f-actin during denticle assembly as a read-out of polarity, we examine the requirement for Ds and Fat in establishing polarity across the denticle field. Comparing detailed phenotypic analysis with steady state protein enrichment revealed a spatially restricted requirement for the Ds-Fat system within the posterior denticle field. Ectopic Ds signaling provides evidence for a model whereby Ds acts to asymmetrically enrich Fat in a neighboring cell, in turn polarizing the cell to specify the position of the actin-based protrusions at the cell cortex.

Ubiquitous expression of CUG or CAG trinucleotide repeat RNA causes common morphological defects in a Drosophila model of RNA-mediated pathology.

Expanded DNA repeat sequences are known to cause over 20 diseases, including Huntington's disease, several types of spinocerebellar ataxia and myotonic dystrophy type 1 and 2. A shared genetic basis, and overlapping clinical features for some of these diseases, indicate that common pathways may contribute to pathology. Multiple mechanisms, mediated by both expanded homopolymeric proteins and expanded repeat RNA, have been identified by the use of model systems, that may account for shared pathology. The use of such animal models enables identification of distinct pathways and their 'molecular hallmarks' that can be used to determine the contribution of each pathway in human pathology. Here we characterise a tergite disruption phenotype in adult flies, caused by ubiquitous expression of either untranslated CUG or CAG expanded repeat RNA. Using the tergite phenotype as a quantitative trait we define a new genetic system in which to examine 'hairpin' repeat RNA-mediated cellular perturbation. Further experiments use this system to examine whether pathways involving Muscleblind sequestration or Dicer processing, which have been shown to mediate repeat RNA-mediated pathology in other model systems, contribute to cellular perturbation in this model.

Double-stranded RNA is pathogenic in Drosophila models of expanded repeat neurodegenerative diseases.

The pathogenic agent responsible for the expanded repeat diseases, a group of neurodegenerative diseases that includes Huntington's disease is not yet fully understood. Expanded polyglutamine (polyQ) is thought to be the toxic agent in certain cases, however, not all expanded repeat disease genes can encode a polyQ sequence. Since a repeat-containing RNA intermediary is common to all of these diseases, hairpin-forming single-stranded RNA has been investigated as a potential common pathogenic agent. More recently, it has become apparent that most of the expanded repeat disease loci have transcription occurring from both strands, raising the possibility that the complementary repeat RNAs could form a double-stranded structure. In our investigation using Drosophila models of these diseases, we identified a fortuitous integration event that models bidirectional repeat RNA transcription with the resultant flies exhibiting inducible pathology. We therefore established further lines of Drosophila expressing independent complementary repeat RNAs and found that these are toxic. The Dicer pathway is essential for this toxicity and in neuronal cells accounts for metabolism of the high copy number (CAG.CUG)(100) double-stranded RNAs down to (CAG)(7) single-stranded small RNAs. We also observe significant changes to the microRNA profile in neurons. These data identify a novel pathway through which double-stranded repeat RNA is toxic and capable of eliciting symptoms common to neurodegenerative human diseases resulting from dominantly inherited expanded repeats.

Perturbation of the Akt/Gsk3-ß signalling pathway is common to Drosophila expressing expanded untranslated CAG, CUG and AUUCU repeat RNAs.

Recent evidence supports a role for RNA as a common pathogenic agent in both the 'polyglutamine' and 'untranslated' dominant expanded repeat disorders. One feature of all repeat sequences currently associated with disease is their predicted ability to form a hairpin secondary structure at the RNA level. In order to investigate mechanisms by which hairpin-forming repeat RNAs could induce neurodegeneration, we have looked for alterations in gene transcript levels as hallmarks of the cellular response to toxic hairpin repeat RNAs. Three disease-associated repeat sequences--CAG, CUG and AUUCU--were specifically expressed in the neurons of Drosophila and resultant common transcriptional changes assessed by microarray analyses. Transcripts that encode several components of the Akt/Gsk3-ß signalling pathway were altered as a consequence of expression of these repeat RNAs, indicating that this pathway is a component of the neuronal response to these pathogenic RNAs and may represent an important common therapeutic target in this class of diseases.