Kidney organoids recapitulate human basement membrane assembly in health and disease
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Evaluation Summary:
Kidney organoid cultures derived from human induced pluripotent stem cells represent a new tool with which to study renal morphogenesis in both normal and pathological states. In the current study, the authors have combined morphological evaluation with proteomics to elucidate aspects of the temporal sequence of basement membrane composition during normal renal development and in the setting of a pathogenic collagen type IV alpha 5 chain variant associated with Alport syndrome, an inherited kidney disease. This model system may help us to better understand the pathogenesis of inherited diseases that affect renal basement membrane composition.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Basement membranes (BMs) are complex macromolecular networks underlying all continuous layers of cells. Essential components include collagen IV and laminins, which are affected by human genetic variants leading to a range of debilitating conditions including kidney, muscle, and cerebrovascular phenotypes. We investigated the dynamics of BM assembly in human pluripotent stem cell-derived kidney organoids. We resolved their global BM composition and discovered a conserved temporal sequence in BM assembly that paralleled mammalian fetal kidneys. We identified the emergence of key BM isoforms, which were altered by a pathogenic variant in COL4A5 . Integrating organoid, fetal, and adult kidney proteomes, we found dynamic regulation of BM composition through development to adulthood, and with single-cell transcriptomic analysis we mapped the cellular origins of BM components. Overall, we define the complex and dynamic nature of kidney organoid BM assembly and provide a platform for understanding its wider relevance in human development and disease.
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Author Response
Reviewer #1 (Public Review):
This group has examined basement membrane composition using sophisticated technical methods previously. Here they have methodically examined kidney organoids for their resemblance to mammalian foetal kidneys in the temporal expression of membrane proteins. They continue this through to adulthood and use peripheral blood leucocytes to demonstrate the effect of a COL4A5 mutation on the expression of basement membrane components. The manuscript's strengths are its methodical nature and the number of proteins examined, as well as building on previous work. Its weaknesses are that we do not know how good a model the organoid is for Alport syndrome and whether it results in an intact glomerular basement membrane. So far, this manuscript has demonstrated that the organoids are consistent with …
Author Response
Reviewer #1 (Public Review):
This group has examined basement membrane composition using sophisticated technical methods previously. Here they have methodically examined kidney organoids for their resemblance to mammalian foetal kidneys in the temporal expression of membrane proteins. They continue this through to adulthood and use peripheral blood leucocytes to demonstrate the effect of a COL4A5 mutation on the expression of basement membrane components. The manuscript's strengths are its methodical nature and the number of proteins examined, as well as building on previous work. Its weaknesses are that we do not know how good a model the organoid is for Alport syndrome and whether it results in an intact glomerular basement membrane. So far, this manuscript has demonstrated that the organoids are consistent with what we know - but can it also tell us new things? In addition, it has only examined one pathogenic Alport COL4A5 variant and this person also had a COL4A4 variant and thus complicated disease.
Thank you for reviewing our paper and for highlighting the strengths of our work. Regarding the novelty, we consider the primary advance of our manuscript is the focus on the assembly and remodelling of extracellular matrix in kidney development. Through this focus we demonstrate that kidney organoids are a valuable human, multicellular system correlating with the matrix changes observed in mammalian kidney development at both gene expression and protein levels. Finally, we have shown that human kidney organoids can be used to study basement membrane assembly in health and disease, using Alport patient-derived organoids. As far as we are aware, this is the first time-course study using organoids to track the intrinsic changes in basement membranes during development. As such it will facilitate further studies into developmental transitions in basement membrane components during kidney development and permit detailed evaluation of the early changes that occur in genetic conditions that affect basement membrane assembly.
Reviewer #2 (Public Review):
Morais et al provide a convincing model for understanding basement membrane (BM) biology and interactions of BM components. The key findings of this paper are to establish a model that recapitulates the same biology and chemistry that is occuring during equivalent kidney development in humans, primarily. Utilizing kidney organoids, the authors characterize the spatiotemporal relationship of the proteins within kidney organioids as they form distinct basement membrane structures. They kidney is vital system in itself for understanding basement membranes among many different organs/tissues as they kidney has served as a genesis for discoveries over the last 60 years. Here the authors describe not only the timing of proteins in the development of kidney organoid BMs, but also the spatial relationships. Importantly, as a kidney BM model, the authors recapitulated the disease state of Alport syndrome (AS), a syndrome involving the disruption of the collagen IV α345 network in kidneys, an essential component of kidney BMs. Furthermore, they find that this model of kidney organoids derived from AS patients had the same hallmarks during development as AS in a human patient, including laminin overcompensation as a result of α345 network disruption.
This manuscript provides an invaluable model for understanding overall BM biology and disease progression, and especially so for kidney BM biology and kidney diseases. The potential for this model to study any number of missense variations within any number of proteins within a tractable and functionally identical BM is worth noting and exploring by other researchers.
We thank the reviewer for highlighting the strengths of our manuscript.
In general, the weaknesses of this article are insignificant as this manuscript aims to provide functional proof of concept of kidney organoids as a model for understanding human BM disease. Importantly, however, is the assumption that kidney BMs might represent all BMs. The diversity of BMs across tissues within humans alone is significant. Amongst different organisms from a broader evolutionary standpoint than just fly, C. elegans, mouse, and human, BMs are very likely exceptionally diverse from the earliest animal BMs to different human tissues BMs. While this model provides an important model for understanding BM biology, a caveat that a kidney BM will functionally differ from a lens BM should be apparent and noted. However, the open-ended question of how to create tractable models like kidney organoids in other tissues systems will be of use in stimulating the matrix, proteomic, and structural biology fields.
Thank you for this insightful comment. We agree that BMs are diverse and dynamic both in composition and structure throughout life. We have made alterations throughout the manuscript to highlight this point and to further emphasise the focus of this manuscript on kidney development.
Reviewer #3 (Public Review):
The emergence of methods to convert human induced pluripotent stem cells (iPSCs) into cultured kidney organoids that phenocopy the normal progression of embryonic and fetal differentiation represent a major advance in the study of normal and defective renal morphogenesis. This progress has been enriched by the addition of temporal/cell-type specific proteomics.
The current study largely focusses on the site-specific compositional changes that occur in basement membranes (BM) that form on different abluminal cell surfaces as differentiation advances. A general model of BM assembly from earlier studies provides a foundation upon which to interpret organoid kidney development. Laminins initiate BM assembly by binding to cognate cell-surface receptors, polymerizing, and binding to secreted nidogens, proteoglycans and collagen type IV, the last forming a second stabilizing polymer network. The iPSC differentiation system reveals the assembly and turnover of BM components consistent with the above, but now provides detailed information on the accumulation and turnover of different components in the key cell types through the different steps of differentiation with proteomic correlation. The approach also enables the analysis of the assembly defects and consequences arising from human congenital diseases as was shown with a type IV collagen alpha 5 subunit in organoids derived from Alport cells.
In combining organoid kidney culturing with laser microdissection and proteomic analysis, the authors have advanced use of the new tool compared to a 2018 study (Hale LJ et al., Nat. Communications), pushing the model from 18 to 25 days of differentiation and focusing more on BM formation during development. Evidence is presented to show that the major cell types, importantly including vascular endothelial cells, appear in the organoids in a temporal sequence. Relevant changes in BM-associated components are also shown. BM staining patterns are shown to change with emergence of laminin alpha5, laminin beta2 and collagen-IV alpha3 (replacing laminin beta1 and collagen-IV alpha1/2) at later stages. Organoids generated from iPSC cells derived from an X-linked missense variant of COL4A5 generated glomeruli containing alpha3/4/5, but with increases in laminin beta2, a known compensatory outcome.
The evaluation of later renal differentiation stages is particularly critical for the study of the glomerulus in which BM components undergo isoform switches that normally correlate with glomerular vascularization. A limitation of previous differentiation glomerular models has been the inability to show formation of the vascular tuft and the associated morphological changes as well as to show podocytes form inter-digitations. In that light, the current study could be strengthened by showing the ultrastructure of the day 25 glomeruli with identification of the BMs and different glomerular cell types (noting in particular if vascular endothelial cells are beginning to organize into the morphology of vascular tufts), and revealing the appearance of podocyte processes. It would also benefit the reader to enumerate the strengths as well as limitations with the culture model and how this work compares to previous studies.
The current submissions addresses temporal and tissue-specific BM changes during organoid kidney development. Day 25 kidney organoids contained tubules, stroma, and glomeruli with partial resemblance to (mouse) E19 kidney. Tubular and glomerular BMs are seen to form, the latter showing the expected switch from alpha-1/beta-1 laminins to alpha-5/beta-2 laminins, and alpha1/2 type IV collagens to alpha3-containing type IV collagens required for glomerular maturation.
Thank you for reviewing our manuscript and for your summary of how our findings relate to other seminal studies in the field of basement membrane assembly.
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Evaluation Summary:
Kidney organoid cultures derived from human induced pluripotent stem cells represent a new tool with which to study renal morphogenesis in both normal and pathological states. In the current study, the authors have combined morphological evaluation with proteomics to elucidate aspects of the temporal sequence of basement membrane composition during normal renal development and in the setting of a pathogenic collagen type IV alpha 5 chain variant associated with Alport syndrome, an inherited kidney disease. This model system may help us to better understand the pathogenesis of inherited diseases that affect renal basement membrane composition.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the …
Evaluation Summary:
Kidney organoid cultures derived from human induced pluripotent stem cells represent a new tool with which to study renal morphogenesis in both normal and pathological states. In the current study, the authors have combined morphological evaluation with proteomics to elucidate aspects of the temporal sequence of basement membrane composition during normal renal development and in the setting of a pathogenic collagen type IV alpha 5 chain variant associated with Alport syndrome, an inherited kidney disease. This model system may help us to better understand the pathogenesis of inherited diseases that affect renal basement membrane composition.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This group has examined basement membrane composition using sophisticated technical methods previously. Here they have methodically examined kidney organoids for their resemblance to mammalian foetal kidneys in the temporal expression of membrane proteins. They continue this through to adulthood and use peripheral blood leucocytes to demonstrate the effect of a COL4A5 mutation on the expression of basement membrane components.
The manuscript's strengths are its methodical nature and the number of proteins examined, as well as building on previous work.
Its weaknesses are that we do not know how good a model the organoid is for Alport syndrome and whether it results in an intact glomerular basement membrane. So far this manuscript has demonstrated that the organoids are consistent with what we know - but can …
Reviewer #1 (Public Review):
This group has examined basement membrane composition using sophisticated technical methods previously. Here they have methodically examined kidney organoids for their resemblance to mammalian foetal kidneys in the temporal expression of membrane proteins. They continue this through to adulthood and use peripheral blood leucocytes to demonstrate the effect of a COL4A5 mutation on the expression of basement membrane components.
The manuscript's strengths are its methodical nature and the number of proteins examined, as well as building on previous work.
Its weaknesses are that we do not know how good a model the organoid is for Alport syndrome and whether it results in an intact glomerular basement membrane. So far this manuscript has demonstrated that the organoids are consistent with what we know - but can it also tell us new things? In addition it has only examined one pathogenic Alport COL4A5 variant and this person also had a COL4A4 variant and thus complicated disease.
-
Reviewer #2 (Public Review):
Morais et al provide a convincing model for understanding basement membrane (BM) biology and interactions of BM components. The key findings of this paper are to establish a model that recapitulates the same biology and chemistry that is occuring during equivalent kidney development in humans, primarily. Utilizing kidney organoids, the authors characterize the spatiotemporal relationship of the proteins within kidney organioids as they form distinct basement membrane structures. They kidney is vital system in itself for understanding basement membranes among many different organs/tissues as they kidney has served as a genesis for discoveries over the last 60 years. Here the authors describe not only the timing of proteins in the development of kidney organoid BMs, but also the spatial relationships. …
Reviewer #2 (Public Review):
Morais et al provide a convincing model for understanding basement membrane (BM) biology and interactions of BM components. The key findings of this paper are to establish a model that recapitulates the same biology and chemistry that is occuring during equivalent kidney development in humans, primarily. Utilizing kidney organoids, the authors characterize the spatiotemporal relationship of the proteins within kidney organioids as they form distinct basement membrane structures. They kidney is vital system in itself for understanding basement membranes among many different organs/tissues as they kidney has served as a genesis for discoveries over the last 60 years. Here the authors describe not only the timing of proteins in the development of kidney organoid BMs, but also the spatial relationships. Importantly, as a kidney BM model, the authors recapitulated the disease state of Alport syndrome (AS), a syndrome involving the disruption of the collagen IV α345 network in kidneys, an essential component of kidney BMs. Furthermore, they find that this model of kidney organoids derived from AS patients had the same hallmarks during development as AS in a human patient, including laminin overcompensation as a result of α345 network disruption.
This manuscript provides an invaluable model for understanding overall BM biology and disease progression, and especially so for kidney BM biology and kidney diseases. The potential for this model to study any number of missense variations within any number of proteins within a tractable and functionally identical BM is worth noting and exploring by other researchers.
In general, the weaknesses of this article are insignificant as this manuscript aims to provide functional proof of concept of kidney organoids as a model for understanding human BM disease. Importantly, however, is the assumption that kidney BMs might represent all BMs. The diversity of BMs across tissues within humans alone is significant. Amongst different organisms from a broader evolutionary standpoint than just fly, C. elegans, mouse, and human, BMs are very likely exceptionally diverse from the earliest animal BMs to different human tissues BMs. While this model provides an important model for understanding BM biology, a caveat that a kidney BM will functionally differ from a lens BM should be apparent and noted. However, the open-ended question of how to create tractable models like kidney organoids in other tissues systems will be of use in stimulating the matrix, proteomic, and structural biology fields.
-
Reviewer #3 (Public Review):
The emergence of methods to convert human induced pluripotent stem cells (iPSCs) into cultured kidney organoids that phenocopy the normal progression of embryonic and fetal differentiation represent a major advance in the study of normal and defective renal morphogenesis. This progress has been enriched by the addition of temporal/cell-type specific proteomics.
The current study largely focusses on the site-specific compositional changes that occur in basement membranes (BM) that form on different abluminal cell surfaces as differentiation advances. A general model of BM assembly from earlier studies provides a foundation upon which to interpret organoid kidney development. Laminins initiate BM assembly by binding to cognate cell-surface receptors, polymerizing, and binding to secreted nidogens, …
Reviewer #3 (Public Review):
The emergence of methods to convert human induced pluripotent stem cells (iPSCs) into cultured kidney organoids that phenocopy the normal progression of embryonic and fetal differentiation represent a major advance in the study of normal and defective renal morphogenesis. This progress has been enriched by the addition of temporal/cell-type specific proteomics.
The current study largely focusses on the site-specific compositional changes that occur in basement membranes (BM) that form on different abluminal cell surfaces as differentiation advances. A general model of BM assembly from earlier studies provides a foundation upon which to interpret organoid kidney development. Laminins initiate BM assembly by binding to cognate cell-surface receptors, polymerizing, and binding to secreted nidogens, proteoglycans and collagen type IV, the last forming a second stabilizing polymer network. The iPSC differentiation system reveals the assembly and turnover of BM components consistent with the above, but now provides detailed information on the accumulation and turnover of different components in the key cell types through the different steps of differentiation with proteomic correlation. The approach also enables the analysis of the assembly defects and consequences arising from human congenital diseases as was shown with a type IV collagen alpha 5 subunit in organoids derived from Alport cells.
In combining organoid kidney culturing with laser microdissection and proteomic analysis, the authors have advanced use of the new tool compared to a 2018 study (Hale LJ et al., Nat. Communications), pushing the model from 18 to 25 days of differentiation and focusing more on BM formation during development. Evidence is presented to show that the major cell types, importantly including vascular endothelial cells, appear in the organoids in a temporal sequence. Relevant changes in BM-associated components are also shown. BM staining patterns are shown to change with emergence of laminin alpha5, laminin beta2 and collagen-IV alpha3 (replacing laminin beta1 and collagen-IV alpha1/2) at later stages. Organoids generated from iPSC cells derived from an X-linked missense variant of COL4A5 generated glomeruli containing alpha3/4/5, but with increases in laminin beta2, a known compensatory outcome.
The evaluation of later renal differentiation stages is particularly critical for the study of the glomerulus in which BM components undergo isoform switches that normally correlate with glomerular vascularization. A limitation of previous differentiation glomerular models has been the inability to show formation of the vascular tuft and the associated morphological changes as well as to show podocytes form inter-digitations. In that light, the current study could be strengthened by showing the ultrastructure of the day 25 glomeruli with identification of the BMs and different glomerular cell types (noting in particular if vascular endothelial cells are beginning to organize into the morphology of vascular tufts), and revealing the appearance of podocyte processes. It would also benefit the reader to enumerate the strengths as well as limitations with the culture model and how this work compares to previous studies.
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