scholarly journals Revisiting The Role of Notch in Nephron Segmentation: Notch is required for proximal, not distal fate selection during mouse and human nephrogenesis

2021 ◽  
Author(s):  
Kathryn Duvall ◽  
Lauren Bice ◽  
Alison J Perl ◽  
Naomi Pode Shakked ◽  
Praneet Chaturvedi ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Human Kidney ◽  
Distal Tubule ◽  
Rna Seq ◽  
Early Markers ◽  
Nephron Segmentation ◽  
Human Ipsc ◽  
Kidney Organoids ◽  
In Cells

Notch signaling promotes maturation of nephron epithelia, but its proposed contribution to nephron segmentation into proximal and distal domains has been called into doubt. We leveraged single cell and bulk RNA-seq, quantitative immunofluorescent lineage/fate tracing, and genetically modified human iPSC to revisit this question in developing mouse kidneys and human kidney organoids. We confirmed that Notch signaling is needed for maturation of all nephron lineages, and thus mature lineage markers fail to detect a fate bias. By contrast, early markers identified a distal fate bias in cells lacking Notch2, and a concomitant increase in early proximal and podocyte fates in cells expressing hyperactive Notch1 was observed. Orthogonal support for a conserved role for Notch signaling in the distal/proximal axis segmentation is provided by the ability of Nicastrin-deficient hiPSCs-derived organoids to differentiate into TFA2B+ distal tubule and CDH1 connecting segment progenitors, but not into HNF4A+ or LTL+ proximal progenitors.

scholarly journals Single cell census of human kidney organoids shows reproducibility and diminished off-target cells after transplantation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ayshwarya Subramanian ◽  
Eriene-Heidi Sidhom ◽  
Maheswarareddy Emani ◽  
Katherine Vernon ◽  
Nareh Sahakian ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Human Kidney ◽  
Mouse Kidney ◽  
Target Cells ◽  
Rna Seq ◽  
Kidney Capsule ◽  
Time Points ◽  
Human Ipsc ◽  
Kidney Organoids

AbstractHuman iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we profile four human iPSC lines for a total of 450,118 single cells to show how organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes are largely reproducible across time points, protocols, and replicates, we detect variability in cell proportions between different iPSC lines, largely due to off-target cells. To address this, we analyze organoids transplanted under the mouse kidney capsule and find diminished off-target cells. Our work shows how single cell RNA-seq (scRNA-seq) can score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells.

scholarly journals Kidney organoid reproducibility across multiple human iPSC lines and diminished off target cells after transplantation revealed by single cell transcriptomics

2019 ◽  
Author(s):  
Ayshwarya Subramanian ◽  
Eriene-Heidi Sidhom ◽  
Maheswarareddy Emani ◽  
Nareh Sahakian ◽  
Katherine Vernon ◽  
...  
Keyword(s):  
Single Cell ◽  
Human Kidney ◽  
Cell Types ◽  
Mouse Kidney ◽  
Target Cells ◽  
Rna Seq ◽  
Kidney Capsule ◽  
Human Ipsc ◽  
Kidney Organoids

AbstractHuman iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we used single cell RNA-Seq (scRNA-Seq) to profile 415,775 cells to show that organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes were largely reproducible across iPSC lines, time points, protocols, and replicates, cell proportions were variable between different iPSC lines. Off-target cell proportions were the most variable. Prolonged in vitro culture did not alter cell types, but organoid transplantation under the mouse kidney capsule diminished off-target cells. Our work shows how scRNA-seq can help score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells.

scholarly journals High throughput single cell RNA-seq of developing mouse kidney and human kidney organoids reveals a roadmap for recreating the kidney

2017 ◽  
Author(s):  
Alexander N. Combes ◽  
Belinda Phipson ◽  
Luke Zappia ◽  
Kynan T. Lawlor ◽  
Pei Xuan Er ◽  
...  
Keyword(s):  
Single Cell ◽  
Kidney Tissue ◽  
Human Kidney ◽  
Population Based ◽  
Mouse Kidney ◽  
Rna Seq ◽  
Kidney Morphogenesis ◽  
Human Ipsc ◽  
Kidney Organoids

AbstractRecent advances in our capacity to differentiate human pluripotent stem cells to human kidney tissue are moving the field closer to novel approaches for renal replacement. Such protocols have relied upon our current understanding of the molecular basis of mammalian kidney morphogenesis. To date this has depended upon population based-profiling of non-homogenous cellular compartments. In order to improve our resolution of individual cell transcriptional profiles during kidney morphogenesis, we have performed 10x Chromium single cell RNA-seq on over 6000 cells from the E18.5 developing mouse kidney, as well as more than 7000 cells from human iPSC-derived kidney organoids. We identified 16 clusters of cells representing all major cell lineages in the E18.5 mouse kidney. The differentially expressed genes from individual murine clusters were then used to guide the classification of 16 cell clusters within human kidney organoids, revealing the presence of distinguishable stromal, endothelial, nephron, podocyte and nephron progenitor populations. Despite the congruence between developing mouse and human organoid, our analysis suggested limited nephron maturation and the presence of ‘off target’ populations in human kidney organoids, including unidentified stromal populations and evidence of neural clusters. This may reflect unique human kidney populations, mixed cultures or aberrant differentiation in vitro. Analysis of clusters within the mouse data revealed novel insights into progenitor maintenance and cellular maturation in the major renal lineages and will serve as a roadmap to refine directed differentiation approaches in human iPSC-derived kidney organoids.

Immunolocalization of calcitriol receptor, 24-hydroxylase cytochrome P-450, and calbindin D28k in human kidney

1994 ◽  
Vol 266 (3) ◽  
pp. F477-F485 ◽  
Author(s):  
R. Kumar ◽  
J. Schaefer ◽  
J. P. Grande ◽  
P. C. Roche
Keyword(s):  
Vitamin D ◽  
Proximal Tubule ◽  
Polyclonal Antibodies ◽  
Collecting Duct ◽  
Human Kidney ◽  
Distal Tubule ◽  
Calbindin D28k ◽  
Parietal Epithelial Cells ◽  
Cytochrome P 450 ◽  
In Cells

The precise localization of the calcitriol (1 alpha,25-dihydroxyvitamin D3) receptor (VDR) and the 25-hydroxyvitamin D3 [25(OH)D3] 24-hydroxylase cytochrome P-450 in the human kidney is unknown. Using newly developed polyclonal antibodies against the human VDR, we demonstrate that the receptor is present in cells of the distal tubule, the collecting duct, the proximal tubule, and in the parietal epithelial cells of the glomerulus. In the distal tubule and collecting duct not all cells contain epitopes for the receptor. The protein is not detected in glomerular capillaries, in the glomerular mesangium, in the interstitium, or in blood vessels. Specific polyclonal antibodies directed against the 25(OH)D3 24-hydroxylase cytochrome P-450 demonstrate epitopes for the cytochrome in cells of the proximal tubule, the distal tubule, glomerular parietal epithelial cells, and mesangial cells. The protein is absent from interstitial cells. Calbindin D28k is present exclusively in principal cells of the distal tubule and collecting duct. In the human kidney, the VDR is present in cells where vitamin D-inducible proteins are found; conversely it is absent from cells where vitamin D-dependent proteins are not present.

scholarly journals Novel role of NF-κB-p65 in antioxidant homeostasis in human kidney-2 cells

2012 ◽  
Vol 302 (11) ◽  
pp. F1440-F1446 ◽  
Author(s):  
Liza E. George ◽  
Mustafa F. Lokhandwala ◽  
Mohammad Asghar
Keyword(s):  
Antioxidant Enzymes ◽  
Human Kidney ◽  
Redox Balance ◽  
Nuclear Factor Κb ◽  
D1 Receptor ◽  
Luciferase Reporter ◽  
Renal Cells ◽  
Direct Role ◽  
In Cells

Nuclear factor-κB (NF-κB) plays a role in inflammation. However, we recently reported an association between NF-κB and antioxidant enzymes in renal proximal tubules of exercise-trained rats, suggesting its role in antioxidant homeostasis (George L, Lokhandwala MF, Asghar M. Am J Physiol Renal Physiol 297: F1174–F1180, 2009). A direct role of NF-κB in antioxidant homeostasis in renal cells has not been elucidated and warrants investigation. Therefore, we examined whether NF-κB has a direct role in antioxidant homeostasis and redox balance in human kidney-2 cells overexpressing NF-κB-p65 and compared them with the cells overexpressing Nrf-2, a well-known transcription factor involved in antioxidant homeostasis. The ability of NF-κB-p65 to increase antioxidant enzymes, to reduce reactive oxygen species (ROS), and to rescue ROS-induced renal dopamine D1 receptor dysfunction, was studied. The transcription activity of NF-κB-p65 and Nrf-2, measured as luciferase reporter activity, increased in cells overexpressing these nuclear factors. The levels of mRNA and activity of glutathione peroxidase as well as the protein levels of superoxide dismutase-1 and glutamylcystein transferase were increased in cells overexpressing NF-κB-p65 and Nrf-2. Furthermore, the levels of ROS decreased and D1 receptor agonist SKF38393-mediated [35S]GTPγS binding (index of D1 receptor function) increased in the presence of hydrogen peroxide in cells overexpressing NF-κB-p65 and Nrf-2. These results suggest a direct role of NF-κB-p65 in antioxidant homeostasis, contributing to redox balance in renal cells.

scholarly journals Modeling acute kidney injury in kidney organoids with cisplatin

2019 ◽  
Author(s):  
Jenny L. M. Digby ◽  
Aneta Przepiorski ◽  
Alan J. Davidson ◽  
Veronika Sander
Keyword(s):  
Dna Damage ◽  
Acute Kidney Injury ◽  
Proximal Tubule ◽  
Kidney Injury ◽  
Induced Pluripotent Stem Cell ◽  
Human Kidney ◽  
Distal Tubule ◽  
Dependent Manner ◽  
Kidney Organoids

ABSTRACTAcute kidney injury (AKI) remains a major global healthcare problem and there is a need to develop human-based models to study AKI in vitro. Towards this goal, we have characterized induced pluripotent stem cell-derived human kidney organoids and their response to cisplatin, a chemotherapeutic drug that induces AKI and preferentially damages the proximal tubule. We found that a single treatment with 50 µM cisplatin induces HAVCR1 and CXCL8 expression, DNA damage (γH2AX) and cell death in the organoids in a dose-dependent manner but greatly impairs organoid viability. DNA damage was not specific to the proximal tubule but also affected the distal tubule and interstitial populations. This lack of specificity correlated with low expression of the proximal tubule-specific SLC22A2/OCT2 transporter for cisplatin. To improve viability, we developed a repeated low-dose regimen of 4x 5 µM cisplatin over 7 days and found this causing less toxicity while still inducing a robust AKI response that included secretion of known AKI biomarkers and inflammatory cytokines. This work validates the use of human kidney organoids to model aspects of AKI in vitro, with the potential to identify new AKI biomarkers and develop better therapies.

scholarly journals miR-449a Repression Leads to Enhanced NOTCH Signaling in TMPRSS2:ERG Fusion Positive Prostate Cancer Cells

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 964
Author(s):  
Simone Bauer ◽  
Leonie Ratz ◽  
Doreen Heckmann-Nötzel ◽  
Adam Kaczorowski ◽  
Markus Hohenfellner ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Notch Signaling ◽  
Target Genes ◽  
Negative Regulator ◽  
Rna Seq ◽  
Hes1 Expression ◽  
Enhanced Expression ◽  
Primary Effector

About 50% of prostate cancer (PCa) tumors are TMPRSS2:ERG (T2E) fusion-positive (T2E+), but the role of T2E in PCa progression is not fully understood. We were interested in investigating epigenomic alterations associated with T2E+ PCa. Using different sequencing cohorts, we found several transcripts of the miR-449 cluster to be repressed in T2E+ PCa. This repression correlated strongly with enhanced expression of NOTCH and several of its target genes in TCGA and ICGC PCa RNA-seq data. We corroborated these findings using a cellular model with inducible T2E expression. Overexpression of miR-449a in vitro led to silencing of genes associated with NOTCH signaling (NOTCH1, HES1) and HDAC1. Interestingly, HDAC1 overexpression led to the repression of HES6, a negative regulator of the transcription factor HES1, the primary effector of NOTCH signaling, and promoted cell proliferation by repressing the cell cycle inhibitor p21. Inhibition of NOTCH as well as knockdown of HES1 reduced the oncogenic properties of PCa cell lines. Using tissue microarray analysis encompassing 533 human PCa cores, ERG-positive areas exhibited significantly increased HES1 expression. Taken together, our data suggest that an epigenomic regulatory network enhances NOTCH signaling and thereby contributes to the oncogenic properties of T2E+ PCa.

scholarly journals A translational kidney organoid system bolsters human relevance of clinical development candidate

2019 ◽  
Author(s):  
Amy Westerling-Bui ◽  
Thomas W. Soare ◽  
Srinivasan Venkatachalan ◽  
Michael DeRan ◽  
Eva Maria Fast ◽  
...  
Keyword(s):  
Quality Control ◽  
Drug Discovery ◽  
Animal Studies ◽  
Channel Blocker ◽  
Clinical Development ◽  
Rna Seq ◽  
Human System ◽  
Functional Studies ◽  
Human Ipsc ◽  
Kidney Organoids

AbstractA major challenge in drug discovery is gaining confidence in the human relevance of pre-clinical animal studies. While human iPSC-derived organoids offer exciting opportunities to address this, concerns about applicability and scalability remain. Here, we report a high-throughput organoid platform for assessment of kidney disease targeting compounds in a human system. We confirmed platform reproducibility by single cell RNA-Seq (scRNA-Seq) and derived a NanoString panel for efficient quality control (QC). Organoid transplantation in rats for 2 to 4 weeks promoted organoid maturation and vascularization. In functional studies, cyclosporine A (CsA) and GFB-887, a novel TRPC5 channel blocker, protected kidney organoids from injury. Pharmacodynamic studies with GFB-887 delivered orally to rats were also successfully performed in human transplanted organoids. These data show how human organoids can deliver confidence in taking development candidate compounds to the clinic, fulfilling their promise to revolutionize drug discovery.

scholarly journals Human kidney organoids model the tacrolimus nephrotoxicity and elucidate the role of autophagy

2020 ◽  
Author(s):  
Jin Won Kim ◽  
Sun Ah Nam ◽  
Eunjeong Seo ◽  
Jong Young Lee ◽  
Dohui Kim ◽  
...  
Keyword(s):  
Human Kidney ◽  
Kidney Organoids

Angiotensin II biphasically regulates cell differentiation in human iPSC-derived kidney organoids

2021 ◽  
Vol 321 (5) ◽  
pp. F559-F571
Author(s):  
Stacy M. Yanofsky ◽  
Courtney M. Dugas ◽  
Akemi Katsurada ◽  
Jiao Liu ◽  
Zubaida Saifudeen ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Angiotensin Ii ◽  
Induced Pluripotent Stem Cell ◽  
Human Kidney ◽  
Developmental Potential ◽  
Novel Strategy ◽  
Induced Pluripotent ◽  
Human Ipsc ◽  
Kidney Organoids

This study demonstrates angiotensin II exertion of biphasic effects on cell differentiation through distinct mediatory roles of angiotensin II type 1 receptor and type 2 receptor in human induced pluripotent stem cell-derived kidney organoids, providing a novel strategy to establish and further characterize the developmental potential of the human kidney organoids.

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