Impaired nutrient signaling and body weight control in a Na⁺ neutral amino acid cotransporter (Slc6a19)-deficient mouse

Amino acid uptake in the intestine and kidney is mediated by a variety of amino acid transporters. To understand the role of epithelial neutral amino acid uptake in whole body homeostasis, we analyzed mice lacking the apical broad-spectrum neutral (0) amino acid transporter BᴼAT1 (Slc6a19). A general neutral aminoaciduria was observed similar to human Hartnup disorder which is caused by mutations in SLC6A19. Na⁺ -dependent uptake of neutral amino acids into the intestine and renal brush-border membrane vesicles was abolished. No compensatory increase of peptide transport or other neutral amino acid transporters was detected. Mice lacking BᴼAT1 showed a reduced body weight. When adapted to a standard 20% protein diet, BᴼAT1-deficient mice lost body weight rapidly on diets containing 6 or 40% protein. Secretion of insulin in response to food ingestion after fasting was blunted. In the intestine, amino acid signaling to the mammalian target of rapamycin (mTOR) pathway was reduced, whereas the GCN2/ATF4 stress response pathway was activated, indicating amino acid deprivation in epithelial cells. The results demonstrate that epithelial amino acid uptake is essential for optimal growth and body weight regulation.This work was supported by National Health and Medical Research Council Grant 525415, Australian Research Council Grant DP0877897, University of Sydney Bridging Grant RIMS2009-02579), and by an anonymous foundatio

Self-organisation after embryonic kidney dissociation is driven via selective adhesion of ureteric epithelial cells

Human pluripotent stem cells, after directed differentiation in vitro, can spontaneously generate complex tissues via self-organisation of the component cells. Self-organisation can also reform embryonic organ structure after tissue disruption. It has previously been demonstrated that dissociated embryonic kidneys can recreate component epithelial and mesenchymal relationships sufficient to allow continued kidney morphogenesis. Here, we investigate the timing and underlying mechanisms driving self-organisation after dissociation of the embryonic kidney using time-lapse imaging, high-resolution confocal analyses and mathematical modelling. Organotypic self-organisation sufficient for nephron initiation was observed within a 24 h period. This involved cell movement, with structure emerging after the clustering of ureteric epithelial cells, a process consistent with models of random cell movement with preferential cell adhesion. Ureteric epithelialisation rapidly followed the formation of ureteric cell clusters with the reformation of nephron-forming niches representing a later event. Disruption of P-cadherin interactions was seen to impair this ureteric epithelial cell clustering without affecting epithelial maturation. This understanding could facilitate improved regulation of patterning within organoids and facilitate kidney engineering approaches guided by cell-cell self-organisation

Reprogramming somatic cells to a kidney fate

Recent years have challenged the view that adult somatic cells reach a state of terminal differentiation. Although the ultimate example of this, somatic cell nuclear transfer, has not proven feasible in human beings, dedifferentiation of mature cell types to a more primitive state, direct reprogramming from one mature state to another, and the reprogramming of any adult cell type to a pluripotent state via enforced expression of key transcription factors now all have been shown. The implications of these findings for kidney disease include the re-creation of key renal cell types from more readily available and expandable somatic cell sources. The feasibility of such an approach recently was shown with the dedifferentiation of proximal tubule cells to nephrogenic mesenchyme. In this review, we examine the technical and clinical challenges that remain to such an approach and how new reprogramming approaches also may be useful for kidney disease. (C) 2014 Elsevier Inc. All rights reserved

Direct transcriptional reprogramming of adult cells to embryonic nephron progenitors

Direct reprogramming involves the enforced re-expression of key transcription factors to redefine a cellular state. The nephron progenitor population of the embryonic kidney gives rise to all cells within the nephron other than the collecting duct through amesenchyme-to-epithelial transition, but this population is exhausted around the time of birth. Here, we sought to identify the conditions under which adult proximal tubule cells could be directly transcriptionally reprogrammed to nephron progenitors. Using a combinatorial screen for lineage-instructive transcription factors, we identified a pool of six genes (SIX1, SIX2, OSR1, EYA1, HOXA11, and SNAI2) that activated a network of genes consistent with a cap mesenchyme/ nephron progenitor phenotype in the adult proximal tubule (HK2) cell line. Consistent with these reprogrammed cells being nephron progenitors,we observed differential contribution of the reprogrammed population into the Six2+ nephron progenitor fields of an embryonic kidney explant. Dereplication of the pool suggested that SNAI2 can suppress E-CADHERIN, presumably assisting in the epithelial-to- mesenchymal transition (EMT) required to form nephron progenitors. However, neither TGFb-induced EMT nor SNAI2 overexpression alone was sufficient to create this phenotype, suggesting that additional factors are required. In conclusion, these results suggest that reinitiation of kidney development from a population of adult cells by generating embryonic progenitors may be feasible, opening the way for additional cellular and bioengineering approaches to renal repair and regeneration.</p

Renal imino acid and glycine transport system ontogeny and involvement in developmental iminoglycinuria

Renal maturation occurs post-natally in many species and reabsorption capacity at birth can vary substantially from the mature kidney. However, little is known regarding the maturation of amino acid transport mechanisms, despite the well-known physiological state of developmental iminoglycinuria. Commonly seen during early infancy, developmental iminoglycinuria is a transient version of the persistent inherited form of the disorder, referred to as iminoglycinuria, and manifests as a urinary hyperexcretion of proline, hydroxyproline and glycine. The transporters involved in developmental iminoglycinuria and their involvement in the improvement of renal reabsorption capacity remain unknown. qPCR (quantitative real-time PCR) and Western blot analysis in developing mouse kidney revealed that the expression of Slc6a18, Slc6a19, Slc6a20a and Slc36a2 was lower at birth (approx. 3.4-, 5.0-, 2.4- and 3.0-fold less than adult kidney by qPCR respectively) and increased during development. Furthermore, immunofluorescence confocal microscopy demonstrated the absence of apical expression of Slc6a18, Slc6a19, Slc6a20a and the auxiliary protein collectrin in kidneys of mice at birth. This correlated with the detection of iminoglycinuria during the first week of life. Iminoglycinuria subsided (proline reduction preceded glycine) in the second week of life, which correlated with an increase in the expression of Slc6a19 and Slc6a20a. Mice achieved an adult imino acid and glycine excretion profile by the fourth week, at which time the expression level of all transporters was comparable with adult mice. In conclusion, these results demonstrate the delayed expression and maturation of Slc6a18, Slc6a19, Slc6a20a and Slc36a2 in neonatal mice and thus the molecular mechanism of developmental iminoglycinuria. © The Authors

DevKidCC allows for robust classification and direct comparisons of kidney organoid datasets

BACKGROUND: While single-cell transcriptional profiling has greatly increased our capacity to interrogate biology, accurate cell classification within and between datasets is a key challenge. This is particularly so in pluripotent stem cell-derived organoids which represent a model of a developmental system. Here, clustering algorithms and selected marker genes can fail to accurately classify cellular identity while variation in analyses makes it difficult to meaningfully compare datasets. Kidney organoids provide a valuable resource to understand kidney development and disease. However, direct comparison of relative cellular composition between protocols has proved challenging. Hence, an unbiased approach for classifying cell identity is required. METHODS: The R package, scPred, was trained on multiple single cell RNA-seq datasets of human fetal kidney. A hierarchical model classified cellular subtypes into nephron, stroma and ureteric epithelial elements. This model, provided in the R package DevKidCC (github.com/KidneyRegeneration/DevKidCC), was then used to predict relative cell identity within published kidney organoid datasets generated using distinct cell lines and differentiation protocols, interrogating the impact of such variations. The package contains custom functions for the display of differential gene expression within cellular subtypes. RESULTS: DevKidCC was used to directly compare between distinct kidney organoid protocols, identifying differences in relative proportions of cell types at all hierarchical levels of the model and highlighting variations in stromal and unassigned cell types, nephron progenitor prevalence and relative maturation of individual epithelial segments. Of note, DevKidCC was able to distinguish distal nephron from ureteric epithelium, cell types with overlapping profiles that have previously confounded analyses. When applied to a variation in protocol via the addition of retinoic acid, DevKidCC identified a consequential depletion of nephron progenitors. CONCLUSIONS: The application of DevKidCC to kidney organoids reproducibly classifies component cellular identity within distinct single-cell datasets. The application of the tool is summarised in an interactive Shiny application, as are examples of the utility of in-built functions for data presentation. This tool will enable the consistent and rapid comparison of kidney organoid protocols, driving improvements in patterning to kidney endpoints and validating new approaches. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13073-022-01023-z

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

Abstract 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

Iminoglycinuria and hyperglycinuria are discrete human phenotypes resulting from complex mutations in proline and glycine transporters

Iminoglycinuria (IG) is an autosomal recessive abnormality of renal transport of glycine and the imino acids proline and hydroxyproline, but the specific genetic defect(s) have not been determined. Similarly, although the related disorder hyperglycinuria (HG) without iminoaciduria has been attributed to heterozygosity of a putative defective glycine, proline, and hydroxyproline transporter, confirming the underlying genetic defect(s) has been difficult. Here we applied a candidate gene sequencing approach in 7 families first identified through newborn IG screening programs. Both inheritance and functional studies identified the gene encoding the proton amino acid transporter SLC36A2 (PAT2) as the major gene responsible for IG in these families, and its inheritance was consistent with a classical semidominant pattern in which 2 inherited nonfunctional alleles conferred the IG phenotype, while 1 nonfunctional allele was sufficient to confer the HG phenotype. Mutations in SLC36A2 that retained residual transport activity resulted in the IG phenotype when combined with mutations in the gene encoding the imino acid transporter SLC6A20 (IMINO). Additional mutations were identified in the genes encoding the putative glycine transporter SLC6A18 (XT2) and the neutral amino acid transporter SLC6A19 (B0AT1) in families with either IG or HG, suggesting that mutations in the genes encoding these transporters may also contribute to these phenotypes. In summary, although recognized as apparently simple Mendelian disorders, IG and HG exhibit complex molecular explanations depending on a major gene and accompanying modifier genes

Direct reprogramming to human nephron progenitor-like cells using inducible piggyBac transposon expression of SNAI2-EYA1-SIX1

All nephrons in the mammalian kidney arise from a transient nephron progenitor population that is lost close to the time of birth. The generation of new nephron progenitors and their maintenance in culture are central to the success of kidney regenerative strategies. Using a lentiviral screening approach, we previously generated a human induced nephron progenitor-like state in vitro using a pool of six transcription factors. Here, we sought to develop a more efficient approach for direct reprogramming of human cells that could be applied in vivo. PiggyBac transposons are a non-viral integrating gene delivery system that is suitable for in vivo use and allows for simultaneous delivery of multiple genes. Using an inducible piggyBac transposon system, we optimized a protocol for the direct reprogramming of HK2 cells to induced nephron progenitor-like cells with expression of only 3 transcription factors (SNAI2, EYA1, and SIX1). Culture in conditions supportive of the nephron progenitor state further increased the expression of nephron progenitor genes. The refined protocol was then applied to primary human renal epithelial cells, which integrated into developing nephron structures in vitro and in vivo. Such inducible reprogramming to nephron progenitor-like cells could facilitate direct cellular reprogramming for kidney regeneration

Renal subcapsular transplantation of PSC-derived kidney organoids induces neo-vasculogenesis and significant glomerular and tubular maturation in vivo

Human pluripotent stem cell (hPSC)-derived kidney organoids may facilitate disease modeling and the generation of tissue for renal replacement. Long-term application, however, will require transferability between hPSC lines and significant improvements in organ maturation. A key question is whether time or a patent vasculature is required for ongoing morphogenesis. Here, we show that hPSC-derived kidney organoids, derived in fully defined medium conditions and in the absence of any exogenous vascular endothelial growth factor, develop host-derived vascularization. In vivo imaging of organoids under the kidney capsule confirms functional glomerular perfusion as well as connection to pre-existing vascular networks in the organoids. Wide-field electron microscopy demonstrates that transplantation results in formation of a glomerular basement membrane, fenestrated endothelial cells, and podocyte foot processes. Furthermore, compared with non-transplanted organoids, polarization and segmental specialization of tubular epithelium are observed. These data demonstrate that functional vascularization is required for progressive morphogenesis of human kidney organoids