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Xue Z, Xuan H, Lau K, Su Y, Wegener M, Li K, Turner L, Adams M, Shi X, Wen H. Expression of ENL YEATS domain tumor mutations in nephrogenic or stromal lineage impairs kidney development. Nat Commun 2025; 16:2531. [PMID: 40087269 PMCID: PMC11909213 DOI: 10.1038/s41467-025-57926-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2024] [Accepted: 03/05/2025] [Indexed: 03/17/2025] Open
Abstract
Recurrent gain-of-function mutations in the histone reader protein ENL have been identified in Wilms tumor, the most prevalent pediatric kidney cancer. However, their pathological significance in kidney development and tumorigenesis in vivo remains elusive. Here, we generate mouse models mimicking ENL tumor (ENLT) mutations and show that heterozygous mutant expression in Six2+ nephrogenic or Foxd1+ stromal lineages leads to severe, lineage-specific kidney defects, both resulting in neonatal lethality. Six2-ENLT mutant kidneys display compromised cap mesenchyme, scant nephron tubules, and cystic glomeruli, indicative of premature progenitor commitment and blocked differentiation. Bulk and spatial transcriptomic analyses reveal aberrant activation of Hox and Wnt signaling genes in mutant nephrogenic cells. In contrast, Foxd1-ENLT mutant kidneys exhibit expansion in renal capsule and cap mesenchyme, with dysregulated stromal gene expression affecting stroma-epithelium crosstalk. Our findings uncover distinct pathways through which ENL mutations disrupt nephrogenesis, providing a foundation for further investigations into their role in tumorigenesis.
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Affiliation(s)
- Zhaoyu Xue
- Department of Epigenetics, Van Andel Institute, Grand Rapids, MI, 49503, USA
| | - Hongwen Xuan
- Department of Epigenetics, Van Andel Institute, Grand Rapids, MI, 49503, USA
| | - Kin Lau
- Bioinformatics and Biostatistics Core, Van Andel Institute, Grand Rapids, MI, 49503, USA
| | - Yangzhou Su
- Department of Epigenetics, Van Andel Institute, Grand Rapids, MI, 49503, USA
| | - Marc Wegener
- Genomics Core, Van Andel Institute, Grand Rapids, MI, 49503, USA
| | - Kuai Li
- Department of Epigenetics, Van Andel Institute, Grand Rapids, MI, 49503, USA
| | - Lisa Turner
- Pathology Core, Van Andel Institute, Grand Rapids, MI, 49503, USA
| | - Marie Adams
- Genomics Core, Van Andel Institute, Grand Rapids, MI, 49503, USA
| | - Xiaobing Shi
- Department of Epigenetics, Van Andel Institute, Grand Rapids, MI, 49503, USA
| | - Hong Wen
- Department of Epigenetics, Van Andel Institute, Grand Rapids, MI, 49503, USA.
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Kota SB, Kota SK. Lysine-specific demethylase 1a is obligatory for gene regulation during kidney development. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.02.25.640014. [PMID: 40060432 PMCID: PMC11888273 DOI: 10.1101/2025.02.25.640014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 03/14/2025]
Abstract
Histone methyltransferases and demethylases play crucial roles in gene regulation and are vital for proper functioning of multiple tissues. Lysine-specific histone demethylase 1A (Kdm1a), is responsible for the demethylation of specific lysines, namely K4 and K9, on histone H3. In this study, we investigated the functions of Kdm1a during mouse kidney development upon targeted deletion in renal progenitor cells. Loss of Kdm1a in Six2-positive nephron progenitors resulted in significant reduction in renal mass, tissue structural changes and impaired function. To further understand the molecular function of Kdm1a during kidney development, we conducted multi-omics analyses that included transcriptome profiling, Chromatin immunoprecipitation (ChIP) sequencing, and methylome assessments. These omic analyses identified Kdm1a as a critical gene regulator required for sustained expression of several nephron segment marker genes, as well as vast number of solute carrier (Slc) genes and a few imprinted genes. Absence of Kdm1a in kidneys led to an increase in global H3K9 methylation peaks, which correlated with the transcriptional downregulation of numerous genes. Among these were markers of nephron progenitors and presumptive tubular precursors. We also observed that specific gene bodies exhibited altered DNA methylation patterns at intragenic differentially methylated regions (DMRs) upon Kdm1a deletion, while the overall global levels of DNA methylation remained unchanged. Our data point to a key regulatory role for Kdm1a in the renal progenitor epigenome, influencing kidney specific gene expression in the developing nephrons. Together the study highlights an indispensable role for Kdm1a for proper development of mouse kidneys, and its absence leading to significant developmental and functional impairment.
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Affiliation(s)
- Savithri Balasubramanian Kota
- Nephrology Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA; Current affiliation: Bayer U.S. LLC
| | - Satya K. Kota
- Division of Bone and Mineral Research, Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Harvard University, Boston, USA
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Corkins ME, Romero-Mora A, Achieng MA, Lindström NO, Miller RK. Comparative analysis of Xenopus mesonephric transcriptomics: Conservation of the developmental lineage of nephron stages. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.01.13.632850. [PMID: 39868253 PMCID: PMC11760729 DOI: 10.1101/2025.01.13.632850] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 01/28/2025]
Abstract
The mammalian kidney develops in three sequential stages referred to as the pronephros, mesonephros, and metanephros, each developing from the preceding form. All three phases of kidney development utilize epithelized tubules called nephrons, which function to take in filtrate from the blood or coelom and selectively reabsorb solutes the organism needs, leaving waste products to be excreted as urine. The pronephros are heavily studied in aquatic organisms such as zebrafish and Xenopus, as they develop quickly and are functional. The metanephros is a preferred mammalian kidney model, as it best recapitulates human disease. However, very little is known about the mesonephric stage of kidney development in any organism. The pronephros extend to form the mesonephric duct, which ultimately develops into the Wolffian duct in male amniotes. Meanwhile, in organisms that lay their eggs in aquatic environments, the mesonephric kidney is the final form that is generated. Therefore, further understanding of the development and physiology of these kidneys will provide insight into the urogenital system as well as its evolutionary conservation. To gain a better understanding of its structure and cell types, we analyzed the developing mesonephros by in situ and single-cell mRNA sequencing of cells the that make up the developing mesonephros. By comparing these data to those published for the Xenopus pronephros and mammalian metanephros, we were able to evaluate nephron conservation between the three kidney stages.
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Affiliation(s)
- Mark E Corkins
- Department of Pediatrics, Pediatric Research Center, UTHealth McGovern Medical School, Houston, Texas 77030
- Department of Cell, Developmental & Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY. 10029
| | - Adrian Romero-Mora
- Department of Pediatrics, Pediatric Research Center, UTHealth McGovern Medical School, Houston, Texas 77030
| | - MaryAnne A Achieng
- Department of Stem Cell Biology & Regenerative Medicine, Keck School of Medicine of USC, Los Angeles CA. 90033
| | - Nils O Lindström
- Department of Stem Cell Biology & Regenerative Medicine, Keck School of Medicine of USC, Los Angeles CA. 90033
| | - Rachel K Miller
- Department of Pediatrics, Pediatric Research Center, UTHealth McGovern Medical School, Houston, Texas 77030
- Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, Texas 77030
- MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Program in Genetics and Epigenetics, Houston, Texas 77030
- MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Program in Biochemistry and Cell Biology, Houston, Texas 77030
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4
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Lan YZ, Wu Z, Chen WJ, Yu XN, Wu HT, Liu J. Sine oculis homeobox homolog family function in gastrointestinal cancer: Progression and comprehensive analysis. World J Clin Oncol 2025; 16:97163. [PMID: 39867730 PMCID: PMC11528897 DOI: 10.5306/wjco.v16.i1.97163] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/25/2024] [Revised: 09/20/2024] [Accepted: 10/20/2024] [Indexed: 10/30/2024] Open
Abstract
The sine oculis homeobox homolog (SIX) family, a group of transcription factors characterized by a conserved DNA-binding homology domain, plays a critical role in orchestrating embryonic development and organogenesis across various organisms, including humans. Comprising six distinct members, from SIX1 to SIX6, each member contributes uniquely to the development and differentiation of diverse tissues and organs, underscoring the versatility of the SIX family. Dysregulation or mutations in SIX genes have been implicated in a spectrum of developmental disorders, as well as in tumor initiation and progression, highlighting their pivotal role in maintaining normal developmental trajectories and cellular functions. Efforts to target the transcriptional complex of the SIX gene family have emerged as a promising strategy to inhibit tumor development. While the development of inhibitors targeting this gene family is still in its early stages, the significant potential of such interventions holds promise for future therapeutic advances. Therefore, this review aimed to comprehensively explore the advancements in understanding the SIX family within gastrointestinal cancers, focusing on its critical role in normal organ development and its implications in gastrointestinal cancers, including gastric, pancreatic, colorectal cancer, and hepatocellular carcinomas. In conclusion, this review deepened the understanding of the functional roles of the SIX family and explored the potential of utilizing this gene family for the diagnosis, prognosis, and treatment of gastrointestinal cancers.
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Affiliation(s)
- Yang-Zheng Lan
- Department of The Breast Center, Cancer Hospital of Shantou University Medical College, Shantou 515041, Guangdong Province, China
| | - Zheng Wu
- Department of The Breast Center, Cancer Hospital of Shantou University Medical College, Shantou 515041, Guangdong Province, China
| | - Wen-Jia Chen
- Department of The Breast Center, Cancer Hospital of Shantou University Medical College, Shantou 515041, Guangdong Province, China
| | - Xin-Ning Yu
- Department of General Surgery, First Affiliated Hospital of Shantou University Medical College, Shantou 515041, Guangdong Province, China
| | - Hua-Tao Wu
- Department of General Surgery, First Affiliated Hospital of Shantou University Medical College, Shantou 515041, Guangdong Province, China
| | - Jing Liu
- Department of The Breast Center, Cancer Hospital of Shantou University Medical College, Shantou 515041, Guangdong Province, China
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Chung E, Nosrati F, Adam M, Potter A, Sayed M, Humphreys BD, Lim HW, Hu YC, Potter SS, Park JS. The thin descending limb of the loop of Henle originates from proximal tubule cells during mouse kidney development. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.01.14.633065. [PMID: 39868227 PMCID: PMC11761803 DOI: 10.1101/2025.01.14.633065] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 01/28/2025]
Abstract
Background The thin descending limb (DTL) of the loop of Henle is crucial for urine concentration, as it facilitates passive water reabsorption. Despite its importance, little is known about how DTL cells form during kidney development. Single-cell RNA sequencing (scRNA-seq) studies have not definitively identified DTL cells in the developing mouse kidney. Methods We assembled a large scRNA-seq dataset by integrating multiple datasets of non-mutant developing mouse kidneys to identify developing DTL cells. To test whether DTL cells originate from proximal tubule (PT) cells, we generated a PT-specific Cre line, Slc34a1eGFPCre, and conducted lineage tracing of PT cells. Additionally, given that the transcription factor Hnf4a directly binds to the Aqp1 gene, we examined whether the loss of Hnf4a affects Aqp1 expression in DTL cells. Results From our scRNA-seq dataset, we identified a small cluster of cells distinct from both the proximal tubule and the thick ascending limb of the loop of Henle. Those cells exhibited high expression of DTL marker genes, including Aqp1 and Bst1. Notably, a subset of PT cells also expressed DTL marker genes, suggesting that PT cells may give rise to DTL cells. Using lineage tracing with the Slc34a1eGFPCre line, we found that DTL cells were positive for the Rosa26 reporter, confirming that DTL cells are descendants of PT cells. Furthermore, the loss of Hnf4a, a transcription factor essential for mature PT cell formation, disrupted proper Aqp1 expression in DTL cells, providing additional evidence of a developmental link between PT cells and DTL cells. Conclusion Our findings shed new light on the developmental origin of DTL cells and highlight the importance of Hnf4a in regulating their formation.
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Affiliation(s)
- Eunah Chung
- Division of Nephrology and Hypertension, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
- Feinberg Cardiovascular and Renal Research Institute, Chicago, IL, USA
| | - Fariba Nosrati
- Division of Nephrology and Hypertension, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
- Feinberg Cardiovascular and Renal Research Institute, Chicago, IL, USA
| | - Mike Adam
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
| | - Andrew Potter
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
| | - Mohammed Sayed
- Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
| | - Benjamin D. Humphreys
- Division of Nephrology, Department of Medicine, Washington University, St. Louis, MO, USA
- Department of Developmental Biology, Washington University, St. Louis, MO, USA
| | - Hee-Woong Lim
- Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
- Department of Pediatrics, University of Cincinnati, Cincinnati, OH, USA
| | - Yueh-Chiang Hu
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
- Department of Pediatrics, University of Cincinnati, Cincinnati, OH, USA
| | - S. Steve Potter
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
- Department of Pediatrics, University of Cincinnati, Cincinnati, OH, USA
| | - Joo-Seop Park
- Division of Nephrology and Hypertension, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
- Feinberg Cardiovascular and Renal Research Institute, Chicago, IL, USA
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6
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Zhao Z, Dai X, Jiang G, Lin F. Absent, Small, or Homeotic 2-Like-Mediated H3K4 Methylation and Nephrogenesis. J Am Soc Nephrol 2025:00001751-990000000-00522. [PMID: 39774048 DOI: 10.1681/asn.0000000600] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2024] [Accepted: 01/02/2025] [Indexed: 01/11/2025] Open
Abstract
Key Points
Deficits in nephron numbers are associated with higher risk of adult-onset kidney disease seen in congenital anomalies of the kidney and urinary tract.Mouse model experiments suggested that absent, small, or homeotic 2-like was vital for kidney development by activating cell cycle genes through histone methylation.Our findings identified absent, small, or homeotic 2-like–regulated genes as a potential target for treating congenital anomalies of the kidney and urinary tract.
Background
Many congenital anomalies of the kidney and urinary tract involve deficits in the number of nephrons, which are associated with a higher risk of hypertension and CKD later in life. Prior work has implicated histone modifications in regulating kidney lineage–specific gene transcription and nephron endowment. Our earlier study suggested that absent, small, or homeotic 2-like (ASH2L), a core subunit of the H3K4 methyltransferase complex, plays a role in ureteric bud morphogenesis during mammalian kidney development. However, the potential involvement of ASH2L in nephron formation remains an open question.
Methods
To investigate the role of ASH2L in nephron development, we inactivated Ash2l specifically in nephron progenitor cells by crossing Six2-e(Kozak-GFPCre-Wpre-polyA)1 mice with Ash2l
fl/fl mice. We used RNA sequencing combined with Cleavage Under Targets and Tagmentation sequencing to screen for gene and epigenomic changes, which were further verified by rescue experiments conducted on ex vivo culture explants.
Results
Inactivating ASH2L in nephron progenitor cells disrupted H3K4 trimethylation establishment at promoters of genes controlling nephron progenitor cell stemness, differentiation, and cell cycle, inhibiting their progression through the cell cycle and differentiation into epithelial cell types needed to form nephrons. Inhibition of the TGF-β/suppressor of mothers against decapentaplegic signaling pathway partially rescued the dysplastic phenotype of the mutants.
Conclusions
ASH2L-mediated H3K4 methylation was identified as a novel epigenetic regulator of kidney development. Downregulation of ASH2L expression or H3K4 trimethylation may be linked to congenital anomalies of the kidney and urinary tract.
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Affiliation(s)
- Ziyi Zhao
- Renal Division, Department of Internal Medicine, Xin Hua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Xuantong Dai
- Renal Division, Department of Internal Medicine, Xin Hua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Gengru Jiang
- Renal Division, Department of Internal Medicine, Xin Hua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Centre for Rare Disease, Shanghai, China
| | - Fujun Lin
- Renal Division, Department of Internal Medicine, Xin Hua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Centre for Rare Disease, Shanghai, China
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7
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Lund-Ricard Y, Calloch J, Glippa V, Vandenplas S, Huysseune A, Witten PE, Morales J, Boutet A. Postembryonic Maintenance of Nephron Progenitor Cells with Low Translational Activity in the Chondrichthyan Scyliorhinus canicula. J Am Soc Nephrol 2024:00001751-990000000-00488. [PMID: 39699552 DOI: 10.1681/asn.0000000558] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2024] [Accepted: 11/12/2024] [Indexed: 12/20/2024] Open
Abstract
Key Points
Unlike mammals, chondrichthyan species exhibit postembryonic nephrogenesis, where new nephrons are continuously added in the kidney.Nephron progenitor cells in catsharks display slow cycling property, akin to other somatic stem cells, indicating their potential for tissue renewal and regeneration.Molecular analysis suggests a potential link between protein synthesis rate and nephron progenitor cell maintenance.
Background
While adult mammals are unable to grow new nephrons, cartilaginous fish kidneys display nephrogenesis throughout life. In this study, we investigated the molecular properties of nephron progenitor cells (NPCs) within the kidney of the catshark (Scyliorhinus canicula).
Methods
We used branched DNA in situ hybridization to analyze markers expressed in catshark NPCs. Bromodesoxyuridine pulse-chase labeling was also performed to test whether NPCs are slow-cycling cells. To question the mechanisms allowing NPC maintenance in the catshark postembryonic kidney, we measured global protein synthesis rates using in vivo OP-puromycin incorporation. We also investigated the expression of two targets of the mammalian target of rapamycin pathway, an important signaling pathway for translation initiation.
Results
We found that NPCs express molecular markers previously identified in mice and teleost embryonic NPCs, such as the transcription factors Six2, Pax2, and Wt1. At postembryonic stages, these NPCs are integrated into a specific nephrogenic area of the kidney and contain slow-cycling cells. We also evidenced that NPCs have lower protein synthesis levels than the differentiated cells present in forming nephrons. Such transition from low to high translation rates has been previously observed in several populations of vertebrate stem cells as they undergo differentiation. Finally, we reported the phosphorylation of two targets of the mammalian target of rapamycin pathway, p4E-BP1 and pS6K1, in catshark differentiated epithelial cells but not in the NPCs.
Conclusions
This first molecular analysis of NPCs in a chondrichthyan species indicates that translation rate increases in NPCs as they differentiate into epithelial cells of the nephron.
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Affiliation(s)
- Yasmine Lund-Ricard
- Laboratory of Integrative Biology of Marine Models (LBI2M), Station Biologique, CNRS, Sorbonne Université, Roscoff, France
| | - Julien Calloch
- Laboratory of Integrative Biology of Marine Models (LBI2M), Station Biologique, CNRS, Sorbonne Université, Roscoff, France
| | - Virginie Glippa
- Laboratory of Integrative Biology of Marine Models (LBI2M), Station Biologique, CNRS, Sorbonne Université, Roscoff, France
| | - Sam Vandenplas
- Biology Department, Evolutionary Developmental Biology Group, Ghent University, Ghent, Belgium
| | - Ann Huysseune
- Biology Department, Evolutionary Developmental Biology Group, Ghent University, Ghent, Belgium
| | - P Eckhard Witten
- Biology Department, Evolutionary Developmental Biology Group, Ghent University, Ghent, Belgium
| | - Julia Morales
- Laboratory of Integrative Biology of Marine Models (LBI2M), Station Biologique, CNRS, Sorbonne Université, Roscoff, France
| | - Agnès Boutet
- Laboratory of Integrative Biology of Marine Models (LBI2M), Station Biologique, CNRS, Sorbonne Université, Roscoff, France
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Sekimoto A, Takaso Y, Saruyama H, Ookawa M, Yamamoto M, Toyohara T, Saigusa D, Fukuuchi T, Otsuka M, Fushiki Y, Yamakoshi S, Tanaka K, Ikeda T, Tanaka T, Takahashi N, Mishima E, Sato E. Impacts of low birthweight on kidney development and intergenerational growth of the offspring. iScience 2024; 27:111159. [PMID: 39524353 PMCID: PMC11546680 DOI: 10.1016/j.isci.2024.111159] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2024] [Revised: 07/10/2024] [Accepted: 10/09/2024] [Indexed: 11/16/2024] Open
Abstract
Low birthweight (LBW) increases the risk of adult-onset diseases, including kidney diseases, with intergenerational consequences; however, the underlying mechanisms and effective interventions are unclear. To examine the cross-generational effects of LBW, we established an LBW mouse model through reduced uterine perfusion pressure (RUPP) and investigated the therapeutic potential of tadalafil, a phosphodiesterase 5 inhibitor, on LBW-associated consequences. RUPP-pups (R1) had lower fetal and birth weights, delayed renal development, and fewer glomeruli than Sham-pups. In adulthood, R1 mice exhibited persistently fewer glomeruli and elevated blood pressure, while Tadalafil-R1 mice showed reduced hypertension in both sexes and improved renal pathological changes in males. Additionally, pregnant R1 mice displayed inadequate gestational liver hypertrophy, impaired hepatic purine metabolism, and diminished placental angiogenesis, resulting in fetal growth restriction in the subsequent generation. These findings underscore the lasting impact of LBW on adult health and future generations and suggest tadalafil's potential to mitigate LBW-associated risks.
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Affiliation(s)
- Akiyo Sekimoto
- Division of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578, Japan
- Tohoku Medical and Pharmaceutical University, Sendai, Japan
| | - Yoko Takaso
- Division of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578, Japan
| | - Haruka Saruyama
- Division of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578, Japan
| | - Masataka Ookawa
- Division of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578, Japan
| | - Mari Yamamoto
- Division of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578, Japan
| | - Takafumi Toyohara
- Department of Nephrology, Tohoku University Graduate School of Medicine, Sendai 983-8536, Japan
| | - Daisuke Saigusa
- Laboratory of Biomedical and Analytical Sciences, Faculty of Pharma-Science, Teikyo University, Tokyo 173-8605, Japan
| | - Tomoko Fukuuchi
- Laboratory of Biomedical and Analytical Sciences, Faculty of Pharma-Science, Teikyo University, Tokyo 173-8605, Japan
| | - Mayu Otsuka
- Laboratory of Biomedical and Analytical Sciences, Faculty of Pharma-Science, Teikyo University, Tokyo 173-8605, Japan
| | - Yui Fushiki
- Division of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578, Japan
| | - Seiko Yamakoshi
- Division of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578, Japan
| | - Kayo Tanaka
- Department of Obstetrics and Gynecology, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Tomoaki Ikeda
- Department of Obstetrics and Gynecology, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Tetsuhiro Tanaka
- Department of Nephrology, Tohoku University Graduate School of Medicine, Sendai 983-8536, Japan
| | - Nobuyuki Takahashi
- Division of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578, Japan
- Department of Nephrology, Tohoku University Graduate School of Medicine, Sendai 983-8536, Japan
| | - Eikan Mishima
- Department of Nephrology, Tohoku University Graduate School of Medicine, Sendai 983-8536, Japan
- Institute of Metabolism and Cell Death, Helmholtz Zentrum München, 85764 Neuherberg, Germany
| | - Emiko Sato
- Division of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578, Japan
- Department of Nephrology, Tohoku University Graduate School of Medicine, Sendai 983-8536, Japan
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9
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Nishimura Y, Hanada S. Origins and Molecular Mechanisms Underlying Renal Vascular Development. KIDNEY360 2024; 5:1718-1726. [PMID: 39115947 DOI: 10.34067/kid.0000000000000543] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/21/2024] [Accepted: 08/05/2024] [Indexed: 08/10/2024]
Abstract
Kidneys play a crucial role in maintaining homeostasis within the body, and this function is intricately linked to the vascular structures within them. For vascular cells in the kidney to mature and function effectively, a well-coordinated spatial alignment between the nephrons and complex network of blood vessels is essential. This arrangement ensures efficient blood filtration and regulation of the electrolyte balance, blood pressure, and fluid levels. Additionally, the kidneys are vital in regulating the acid-base balance and producing hormones involved in erythropoiesis and blood pressure control. This article focuses on the vascular development of the kidneys, summarizing the current understanding of the origin and formation of the renal vasculature, and the key molecules involved. A comprehensive review of existing studies has been conducted to elucidate the cellular and molecular mechanisms governing renal vascular development. Specific molecules play a critical role in the development of renal vasculature, contributing to the spatial alignment between nephrons and blood vessels. By elucidating the cellular and molecular mechanisms involved in renal vascular development, this study aims to advance renal regenerative medicine and offer potential avenues for therapeutic interventions in kidney disease.
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Affiliation(s)
- Yusuke Nishimura
- Department of Clinical Engineering, Faculty of Medical Science and Technology, Gunma Paz University, Takasaki, Japan
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10
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Bugacov H, Der B, Briantseva BM, Guo Q, Kim S, Lindström NO, McMahon AP. Dose-dependent responses to canonical Wnt transcriptional complexes in the regulation of mammalian nephron progenitors. Development 2024; 151:dev202279. [PMID: 39250420 PMCID: PMC11463962 DOI: 10.1242/dev.202279] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2023] [Accepted: 09/02/2024] [Indexed: 09/11/2024]
Abstract
In vivo and in vitro studies argue that concentration-dependent Wnt signaling regulates mammalian nephron progenitor cell (NPC) programs. Canonical Wnt signaling is regulated through the stabilization of β-catenin, a transcriptional co-activator when complexed with Lef/Tcf DNA-binding partners. Using the GSK3β inhibitor CHIR99021 (CHIR) to block GSK3β-dependent destruction of β-catenin, we examined dose-dependent responses to β-catenin in mouse NPCs, using mRNA transduction to modify gene expression. Low CHIR-dependent proliferation of NPCs was blocked on β-catenin removal, with evidence of NPCs arresting at the G2-M transition. While NPC identity was maintained following β-catenin removal, mRNA-seq identified low CHIR and β-catenin dependent genes. High CHIR activated nephrogenesis. Nephrogenic programming was dependent on Lef/Tcf factors and β-catenin transcriptional activity. Molecular and cellular features of early nephrogenesis were driven in the absence of CHIR by a mutated stabilized form of β-catenin. Chromatin association studies indicate low and high CHIR response genes are likely direct targets of canonical Wnt transcriptional complexes. Together, these studies provide evidence for concentration-dependent Wnt signaling in the regulation of NPCs and provide new insight into Wnt targets initiating mammalian nephrogenesis.
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Affiliation(s)
- Helena Bugacov
- Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90089, USA
- Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Balint Der
- Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90089, USA
- Department of Urology, Faculty of Medicine, Semmelweis University, Budapest 1082, Hungary
- Institute of Translational Medicine, Faculty of Medicine, Semmelweis University, Budapest 1094, Hungary
| | - Bohdana-Myroslava Briantseva
- Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90089, USA
| | - Qiuyu Guo
- Discovery Biomarkers, Amgen Research, 1 Amgen Center Drive, Thousand Oaks, CA 91320, USA
| | - Sunghyun Kim
- Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90089, USA
| | - Nils O. Lindström
- Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90089, USA
| | - Andrew P. McMahon
- Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90089, USA
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11
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Ibi Y, Nishinakamura R. Generating kidney organoids based on developmental nephrology. Eur J Cell Biol 2024; 103:151450. [PMID: 39137450 DOI: 10.1016/j.ejcb.2024.151450] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2024] [Revised: 08/05/2024] [Accepted: 08/08/2024] [Indexed: 08/15/2024] Open
Abstract
Over the past decade, the induction protocols for the two types of kidney organoids (nephron organoids and ureteric bud organoids) from pluripotent stem cells (PSCs) have been established based on the knowledge gained in developmental nephrology. Kidney organoids are now used for disease modeling and drug screening, but they also have potential as tools for clinical transplantation therapy. One of the options to achieve this goal would be to assemble multiple renal progenitor cells (nephron progenitor, ureteric bud, stromal progenitor) to reproduce the organotypic kidney structure from PSCs. At least from mouse PSCs, all the three progenitors have been induced and assembled into such "higher order" kidney organoids. We will provide an overview of the developmental nephrology required for the induction of renal progenitors and discuss recent advances and remaining challenges of kidney organoids for clinical transplantation therapy.
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Affiliation(s)
- Yutaro Ibi
- Department of Kidney Development, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan
| | - Ryuichi Nishinakamura
- Department of Kidney Development, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan.
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12
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Beamish JA, Watts JA, Dressler GR. Gene regulation in regeneration after acute kidney injury. J Biol Chem 2024; 300:107520. [PMID: 38950862 PMCID: PMC11325799 DOI: 10.1016/j.jbc.2024.107520] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2024] [Revised: 06/19/2024] [Accepted: 06/21/2024] [Indexed: 07/03/2024] Open
Abstract
Acute kidney injury (AKI) is a common condition associated with significant morbidity, mortality, and cost. Injured kidney tissue can regenerate after many forms of AKI. However, there are no treatments in routine clinical practice to encourage recovery. In part, this shortcoming is due to an incomplete understanding of the genetic mechanisms that orchestrate kidney recovery. The advent of high-throughput sequencing technologies and genetic mouse models has opened an unprecedented window into the transcriptional dynamics that accompany both successful and maladaptive repair. AKI recovery shares similar cell-state transformations with kidney development, which can suggest common mechanisms of gene regulation. Several powerful bioinformatic strategies have been developed to infer the activity of gene regulatory networks by combining multiple forms of sequencing data at single-cell resolution. These studies highlight not only shared stress responses but also key changes in gene regulatory networks controlling metabolism. Furthermore, chromatin immunoprecipitation studies in injured kidneys have revealed dynamic epigenetic modifications at enhancer elements near target genes. This review will highlight how these studies have enhanced our understanding of gene regulation in injury response and regeneration.
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Affiliation(s)
- Jeffrey A Beamish
- Division of Nephrology, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA
| | - Jason A Watts
- Epigenetics and Stem Cell Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA
| | - Gregory R Dressler
- Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA.
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13
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Rinta-Jaskari MM, Naillat F, Ruotsalainen HJ, Ronkainen VP, Heljasvaara R, Akram SU, Izzi V, Miinalainen I, Vainio SJ, Pihlajaniemi TA. Collagen XVIII regulates extracellular matrix integrity in the developing nephrons and impacts nephron progenitor cell behavior. Matrix Biol 2024; 131:30-45. [PMID: 38788809 DOI: 10.1016/j.matbio.2024.05.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2023] [Revised: 05/19/2024] [Accepted: 05/21/2024] [Indexed: 05/26/2024]
Abstract
Renal development is a complex process in which two major processes, tubular branching and nephron development, regulate each other reciprocally. Our previous findings have indicated that collagen XVIII (ColXVIII), an extracellular matrix protein, affects the renal branching morphogenesis. We investigate here the role of ColXVIII in nephron formation and the behavior of nephron progenitor cells (NPCs) using isoform-specific ColXVIII knockout mice. The results show that the short ColXVIII isoform predominates in the early epithelialized nephron structures whereas the two longer isoforms are expressed only in the later phases of glomerular formation. Meanwhile, electron microscopy showed that the ColXVIII mutant embryonic kidneys have ultrastructural defects at least from embryonic day 16.5 onwards. Similar structural defects had previously been observed in adult ColXVIII-deficient mice, indicating a congenital origin. The lack of ColXVIII led to a reduced NPC population in which changes in NPC proliferation and maintenance and in macrophage influx were perceived to play a role. The changes in NPC behavior in turn led to notably reduced overall nephron formation. In conclusion, the results show that ColXVIII has multiple roles in renal development, both in ureteric branching and in NPC behavior.
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Affiliation(s)
- Mia M Rinta-Jaskari
- Oulu Center for Cell-Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Aapistie 7, Oulu 90230, Finland
| | - Florence Naillat
- Oulu Center for Cell-Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Aapistie 7, Oulu 90230, Finland
| | - Heli J Ruotsalainen
- Oulu Center for Cell-Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Aapistie 7, Oulu 90230, Finland
| | | | - Ritva Heljasvaara
- Oulu Center for Cell-Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Aapistie 7, Oulu 90230, Finland
| | - Saad U Akram
- Center for Machine Vision and Signal Analysis (CMVS), University of Oulu, Helsinki, Finland
| | - Valerio Izzi
- Oulu Center for Cell-Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Aapistie 7, Oulu 90230, Finland; Research Unit of Biomedicine and Internal Medicine, Faculty of Medicine, University of Oulu, Finland
| | | | - Seppo J Vainio
- Oulu Center for Cell-Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Aapistie 7, Oulu 90230, Finland; InfoTech Oulu, Finland; Kvantum Institute, University of Oulu, Finland
| | - Taina A Pihlajaniemi
- Oulu Center for Cell-Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Aapistie 7, Oulu 90230, Finland.
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14
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Hinostroza F, Araya-Duran I, Piñeiro A, Lobos I, Pastenes L. Transcription factor roles in the local adaptation to temperature in the Andean Spiny Toad Rhinella spinulosa. Sci Rep 2024; 14:15158. [PMID: 38956427 PMCID: PMC11220030 DOI: 10.1038/s41598-024-66127-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2024] [Accepted: 06/27/2024] [Indexed: 07/04/2024] Open
Abstract
Environmental temperature strongly influences the adaptation dynamics of amphibians, whose limited regulation capabilities render them susceptible to thermal oscillations. A central element of the adaptive strategies is the transcription factors (TFs), which act as master regulators that orchestrate stress responses, enabling species to navigate the fluctuations of their environment skillfully. Our study delves into the intricate relationship between TF expression and thermal adaptation mechanisms in the Rhinella spinulosa populations. We sought to elucidate the dynamic modulations of TF expression in prometamorphic and metamorphic tadpoles that inhabit two thermally contrasting environments (Catarpe and El Tatio Geyser, Chile) and which were exposed to two thermal treatments (25 °C vs. 20 °C). Our findings unravel an intriguing dichotomy in response strategies between these populations. First, results evidence the expression of 1374 transcription factors. Regarding the temperature shift, the Catarpe tadpoles show a multifaceted approach by up-regulating crucial TFs, including fosB, atf7, and the androgen receptor. These dynamic regulatory responses likely underpin the population's ability to navigate thermal fluctuations effectively. In stark contrast, the El Tatio tadpoles exhibit a more targeted response, primarily up-regulating foxc1. This differential expression suggests a distinct focus on specific TFs to mitigate the effects of temperature variations. Our study contributes to understanding the molecular mechanisms governing thermal adaptation responses and highlights the resilience and adaptability of amphibians in the face of ever-changing environmental conditions.
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Affiliation(s)
- Fernando Hinostroza
- Centro de Investigación de Estudios Avanzados del Maule (CIEAM), Vicerrectoría de Investigación y Postgrado, Universidad Católica del Maule, Talca, Chile
- Centro de Investigación en Neuropsicología y Neurociencias Cognitivas, Facultad de Ciencias de la Salud, Universidad Católica del Maule, Talca, Chile
- Escuela de Química y Farmacia, Departamento de Medicina Traslacional, Facultad de Medicina, Universidad Católica del Maule, Talca, Chile
- Centro Para la Investigación Traslacional en Neurofarmacología, Universidad de Valparaíso, Valparaíso, Chile
| | - Ingrid Araya-Duran
- Center for Bioinformatics and Integrative Biology, Facultad de Ciencias de la Vida, Universidad Andrés Bello, Santiago, Chile
| | - Alejandro Piñeiro
- Laboratorio de Genética y Microevolución, Departamento de Biología y Química, Facultad de Ciencias Básicas, Universidad Católica del Maule, Talca, Chile
| | - Isabel Lobos
- Laboratorio de Genética y Microevolución, Departamento de Biología y Química, Facultad de Ciencias Básicas, Universidad Católica del Maule, Talca, Chile
| | - Luis Pastenes
- Laboratorio de Genética y Microevolución, Departamento de Biología y Química, Facultad de Ciencias Básicas, Universidad Católica del Maule, Talca, Chile.
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15
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Stansak KL, Baum LD, Ghosh S, Thapa P, Vanga V, Walters BJ. PCP auto count: a novel Fiji/ImageJ plug-in for automated quantification of planar cell polarity and cell counting. Front Cell Dev Biol 2024; 12:1394031. [PMID: 38827526 PMCID: PMC11140036 DOI: 10.3389/fcell.2024.1394031] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2024] [Accepted: 04/19/2024] [Indexed: 06/04/2024] Open
Abstract
Introdution: During development, planes of cells give rise to complex tissues and organs. The proper functioning of these tissues is critically dependent on proper inter- and intra-cellular spatial orientation, a feature known as planar cell polarity (PCP). To study the genetic and environmental factors affecting planar cell polarity, investigators must often manually measure cell orientations, which is a time-consuming endeavor. To automate cell counting and planar cell polarity data collection we developed a Fiji/ImageJ plug-in called PCP Auto Count (PCPA). Methods: PCPA analyzes binary images and identifies "chunks" of white pixels that contain "caves" of infiltrated black pixels. For validation, inner ear sensory epithelia including cochleae and utricles from mice were immunostained for βII-spectrin and imaged with a confocal microscope. Images were preprocessed using existing Fiji functionality to enhance contrast, make binary, and reduce noise. An investigator rated PCPA cochlear hair cell angle measurements for accuracy using a one to five agreement scale. For utricle samples, PCPA derived measurements were directly compared against manually derived angle measurements and the concordance correlation coefficient (CCC) and Bland-Altman limits of agreement were calculated. PCPA was also tested against previously published images examining PCP in various tissues and across various species suggesting fairly broad utility. Results: PCPA was able to recognize and count 99.81% of cochlear hair cells, and was able to obtain ideally accurate planar cell polarity measurements for at least 96% of hair cells. When allowing for a <10° deviation from "perfect" measurements, PCPA's accuracy increased to 98%-100% for all users and across all samples. When PCPA's measurements were compared with manual angle measurements for E17.5 utricles there was negligible bias (<0.5°), and a CCC of 0.999. Qualitative examination of example images of Drosophila ommatidia, mouse ependymal cells, and mouse radial progenitors revealed a high level of accuracy for PCPA across a variety of stains, tissue types, and species. Discussion: Altogether, the data suggest that the PCPA plug-in suite is a robust and accurate tool for the automated collection of cell counts and PCP angle measurements.
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Affiliation(s)
| | | | | | | | | | - Bradley J. Walters
- University of Mississippi Medical Center, Department of Otolaryngology—Head and Neck Surgery, Jackson, MS, United States
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16
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Neal SJ, Rajasekaran A, Jusić N, Taylor L, Read M, Alfandari D, Pignoni F, Moody SA. Using Xenopus to discover new candidate genes involved in BOR and other congenital hearing loss syndromes. JOURNAL OF EXPERIMENTAL ZOOLOGY. PART B, MOLECULAR AND DEVELOPMENTAL EVOLUTION 2024; 342:212-240. [PMID: 37830236 PMCID: PMC11014897 DOI: 10.1002/jez.b.23222] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/24/2023] [Revised: 08/15/2023] [Accepted: 09/14/2023] [Indexed: 10/14/2023]
Abstract
Hearing in infants is essential for brain development, acquisition of verbal language skills, and development of social interactions. Therefore, it is important to diagnose hearing loss soon after birth so that interventions can be provided as early as possible. Most newborns in the United States are screened for hearing deficits and commercially available next-generation sequencing hearing loss panels often can identify the causative gene, which may also identify congenital defects in other organs. One of the most prevalent autosomal dominant congenital hearing loss syndromes is branchio-oto-renal syndrome (BOR), which also presents with defects in craniofacial structures and the kidney. Currently, mutations in three genes, SIX1, SIX5, and EYA1, are known to be causative in about half of the BOR patients that have been tested. To uncover new candidate genes that could be added to congenital hearing loss genetic screens, we have combined the power of Drosophila mutants and protein biochemical assays with the embryological advantages of Xenopus, a key aquatic animal model with a high level of genomic similarity to human, to identify potential Six1 transcriptional targets and interacting proteins that play a role during otic development. We review our transcriptomic, yeast 2-hybrid, and proteomic approaches that have revealed a large number of new candidates. We also discuss how we have begun to identify how Six1 and co-factors interact to direct developmental events necessary for normal otic development.
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Affiliation(s)
- Scott J. Neal
- Department of Neuroscience and Physiology, Upstate Medical University, Syracuse, NY, USA
| | - Anindita Rajasekaran
- Department of Neuroscience and Physiology, Upstate Medical University, Syracuse, NY, USA
| | - Nisveta Jusić
- Department of Neuroscience and Physiology, Upstate Medical University, Syracuse, NY, USA
| | - Louis Taylor
- Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, Amherst, MA, USA
| | - Mai Read
- Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, Amherst, MA, USA
| | - Dominique Alfandari
- Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, Amherst, MA, USA
| | - Francesca Pignoni
- Department of Neuroscience and Physiology, Upstate Medical University, Syracuse, NY, USA
| | - Sally A. Moody
- Department of Anatomy and Cell Biology, George Washington University, School of Medicine and Health Sciences, Washington, DC, USA
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17
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Perl AJ, Liu H, Hass M, Adhikari N, Chaturvedi P, Hu YC, Jiang R, Liu Y, Kopan R. Reduced Nephron Endowment in Six2-TGCtg Mice Is Due to Six3 Misexpression by Aberrant Enhancer-Promoter Interactions in the Transgene. J Am Soc Nephrol 2024; 35:566-577. [PMID: 38447671 PMCID: PMC11149036 DOI: 10.1681/asn.0000000000000324] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2023] [Accepted: 02/27/2024] [Indexed: 03/08/2024] Open
Abstract
Key Points Aberrant enhancer–promoter interactions detected by Hi-C drive ectopic expression of Six3 in the Six2TGCtg line. Disruption of Six3 in the Six2TGCtg line restores nephron number, implicating SIX3 interference with SIX2 function in nephron progenitor cell renewal. Background Lifelong kidney function relies on the complement of nephrons generated during mammalian development from a mesenchymal nephron progenitor cell population. Low nephron endowment confers increased susceptibility to CKD. Reduced nephron numbers in the popular Six2TGC transgenic mouse line may be due to disruption of a regulatory gene at the integration site and/or ectopic expression of a gene(s) contained within the transgene. Methods Targeted locus amplification was performed to identify the integration site of the Six2TGC transgene. Genome-wide chromatin conformation capture (Hi-C) datasets were generated from nephron progenitor cells isolated from the Six2TGC +/tg mice, the Cited1 CreERT2/+ control mice, and the Six2TGC +/tg ; Tsc1 +/Flox mice that exhibited restored nephron number compared with Six2TGC +/tg mice. Modified transgenic mice lacking the C-terminal domain of Six3 were used to evaluate the mechanism of nephron number reduction in the Six2TGC +/tg mouse line. Results Targeted locus amplification revealed integration of the Six2TGC transgene within an intron of Cntnap5a on chr1, and Hi-C analysis mapped the precise integration of Six2TGC and Cited1 CreERT2 transgenes to chr1 and chr14, respectively. No changes in topology, accessibility, or expression were observed within the 50-megabase region centered on Cntnap5a in Six2TGC +/tg mice compared with control mice. By contrast, we identified an aberrant regulatory interaction between a Six2 distal enhancer and the Six3 promoter contained within the transgene. Increasing the Six2TGC tg to Six2 locus ratio or removing one Six2 allele in Six2TGC +/tg mice caused severe renal hypoplasia. Furthermore, clustered regularly interspaced short palindromic repeats disruption of Six3 within the transgene (Six2TGC ∆Six3CT ) restored nephron endowment to wild-type levels and abolished the stoichiometric effect. Conclusions These findings broadly demonstrate the utility of Hi-C data in mapping transgene integration sites and architecture. Data from genetic and biochemical studies together suggest that in Six2TGC kidneys, SIX3 interferes with SIX2 function in nephron progenitor cell renewal through its C-terminal domain.
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Affiliation(s)
- Alison J. Perl
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
| | - Han Liu
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
| | - Matthew Hass
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
- Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
| | - Nirpesh Adhikari
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
| | - Praneet Chaturvedi
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
| | - Yueh-Chiang Hu
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
| | - Rulang Jiang
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
| | - Yaping Liu
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio
- Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
- Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
| | - Raphael Kopan
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
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18
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Alfaifi J. miRNAs Role in Wilms tumor pathogenesis: Signaling pathways interplay. Pathol Res Pract 2024; 256:155254. [PMID: 38460245 DOI: 10.1016/j.prp.2024.155254] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/14/2024] [Revised: 03/01/2024] [Accepted: 03/04/2024] [Indexed: 03/11/2024]
Abstract
Wilms' tumors (WTs) are the most common type of kidney tumor in children, and a negative outlook is generally associated with widespread anaplastic. MicroRNAs (miRNAs) are crucial in the development of WT by regulating the expression of specific genes. There is an increasing amount of research that connects the dysregulation of miRNAs to the development of various renal illnesses. The conditions encompassed are renal fibrosis, renal cancers, and chronic and polycystic kidney disease. Dysregulation of several important miRNAs, either oncogenic or tumor-suppressing, has been found in WT. The present state of knowledge on the involvement of dysregulated miRNAs in the progression of WT is summarized in this review.
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Affiliation(s)
- Jaber Alfaifi
- Department of Child Health, College of Medicine, University of Bisha, Bisha 61922, Saudi Arabia.
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19
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Long HY, Qian ZP, Lan Q, Xu YJ, Da JJ, Yu FX, Zha Y. Human pluripotent stem cell-derived kidney organoids: Current progress and challenges. World J Stem Cells 2024; 16:114-125. [PMID: 38455108 PMCID: PMC10915962 DOI: 10.4252/wjsc.v16.i2.114] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/23/2023] [Revised: 12/18/2023] [Accepted: 01/29/2024] [Indexed: 02/26/2024] Open
Abstract
Human pluripotent stem cell (hPSC)-derived kidney organoids share similarities with the fetal kidney. However, the current hPSC-derived kidney organoids have some limitations, including the inability to perform nephrogenesis and lack of a corticomedullary definition, uniform vascular system, and coordinated exit pathway for urinary filtrate. Therefore, further studies are required to produce hPSC-derived kidney organoids that accurately mimic human kidneys to facilitate research on kidney development, regeneration, disease modeling, and drug screening. In this review, we discussed recent advances in the generation of hPSC-derived kidney organoids, how these organoids contribute to the understanding of human kidney development and research in disease modeling. Additionally, the limitations, future research focus, and applications of hPSC-derived kidney organoids were highlighted.
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Affiliation(s)
- Hong-Yan Long
- Graduate School, Zunyi Medical University, Zunyi 563000, Guizhou Province, China
| | - Zu-Ping Qian
- Graduate School, Zunyi Medical University, Zunyi 563000, Guizhou Province, China
| | - Qin Lan
- Graduate School, Zunyi Medical University, Zunyi 563000, Guizhou Province, China
| | - Yong-Jie Xu
- Department of Laboratory Medicine, Guizhou Provincial People's Hospital, Guiyang 550002, Guizhou Province, China
| | - Jing-Jing Da
- Department of Nephrology, Guizhou Provincial People's Hospital, Guiyang 550002, Guizhou Province, China
| | - Fu-Xun Yu
- Key Laboratory of Diagnosis and Treatment of Pulmonary Immune Diseases, National Health Commission, Guizhou Provincial People's Hospital, Guiyang 550002, Guizhou Province, China
| | - Yan Zha
- Graduate School, Zunyi Medical University, Zunyi 563000, Guizhou Province, China
- Department of Nephrology, Guizhou Provincial People's Hospital, Guiyang 550002, Guizhou Province, China.
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20
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Stansak KL, Baum LD, Ghosh S, Thapa P, Vanga V, Walters BJ. PCP Auto Count: A Novel Fiji/ImageJ plug-in for automated quantification of planar cell polarity and cell counting. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.01.30.578047. [PMID: 38352473 PMCID: PMC10862842 DOI: 10.1101/2024.01.30.578047] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/22/2024]
Abstract
Background During development, planes of cells give rise to complex tissues and organs. The proper functioning of these tissues is critically dependent on proper inter- and intra-cellular spatial orientation, a feature known as planar cell polarity (PCP). To study the genetic and environmental factors affecting planar cell polarity investigators must often manually measure cell orientations, which is a time-consuming endeavor. Methodology To automate cell counting and planar cell polarity data collection we developed a Fiji/ImageJ plug-in called PCP Auto Count (PCPA). PCPA analyzes binary images and identifies "chunks" of white pixels that contain "caves" of infiltrated black pixels. Inner ear sensory epithelia including cochleae (P4) and utricles (E17.5) from mice were immunostained for βII-spectrin and imaged on a confocal microscope. Images were preprocessed using existing Fiji functionality to enhance contrast, make binary, and reduce noise. An investigator rated PCPA cochlear angle measurements for accuracy using a 1-5 agreement scale. For utricle samples, we directly compared PCPA derived measurements against manually derived angle measurements using concordance correlation coefficients (CCC) and Bland-Altman limits of agreement. Finally, PCPA was tested against a variety of images copied from publications examining PCP in various tissues and across various species. Results PCPA was able to recognize and count 99.81% of cochlear hair cells (n = 1,1541 hair cells) in a sample set, and was able to obtain ideally accurate planar cell polarity measurements for over 96% of hair cells. When allowing for a <10° deviation from "perfect" measurements, PCPA's accuracy increased to >98%. When manual angle measurements for E17.5 utricles were compared, PCPA's measurements fell within -9 to +10 degrees of manually obtained mean angle measures with a CCC of 0.999. Qualitative examination of example images of Drosophila ommatidia, mouse ependymal cells, and mouse radial progenitors revealed a high level of accuracy for PCPA across a variety of stains, tissue types, and species. Altogether, the data suggest that the PCPA plug-in suite is a robust and accurate tool for the automated collection of cell counts and PCP angle measurements.
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Affiliation(s)
- Kendra L. Stansak
- Department of Otolaryngology - Head and Neck Surgery, University of Mississippi Medical Center, Jackson, Mississippi, United States of America
| | - Luke D. Baum
- Department of Otolaryngology - Head and Neck Surgery, University of Mississippi Medical Center, Jackson, Mississippi, United States of America
| | - Sumana Ghosh
- Department of Otolaryngology - Head and Neck Surgery, University of Mississippi Medical Center, Jackson, Mississippi, United States of America
| | - Punam Thapa
- Department of Otolaryngology - Head and Neck Surgery, University of Mississippi Medical Center, Jackson, Mississippi, United States of America
| | - Vineel Vanga
- Department of Otolaryngology - Head and Neck Surgery, University of Mississippi Medical Center, Jackson, Mississippi, United States of America
| | - Bradley J. Walters
- Department of Otolaryngology - Head and Neck Surgery, University of Mississippi Medical Center, Jackson, Mississippi, United States of America
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Wang SX, Streit A. Shared features in ear and kidney development - implications for oto-renal syndromes. Dis Model Mech 2024; 17:dmm050447. [PMID: 38353121 PMCID: PMC10886756 DOI: 10.1242/dmm.050447] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/16/2024] Open
Abstract
The association between ear and kidney anomalies has long been recognized. However, little is known about the underlying mechanisms. In the last two decades, embryonic development of the inner ear and kidney has been studied extensively. Here, we describe the developmental pathways shared between both organs with particular emphasis on the genes that regulate signalling cross talk and the specification of progenitor cells and specialised cell types. We relate this to the clinical features of oto-renal syndromes and explore links to developmental mechanisms.
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Affiliation(s)
- Scarlet Xiaoyan Wang
- Centre for Craniofacial and Regenerative Biology, King's College London, London SE1 9RT, UK
| | - Andrea Streit
- Centre for Craniofacial and Regenerative Biology, King's College London, London SE1 9RT, UK
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22
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Bahrami M, Darabi S, Roozbahany NA, Abbaszadeh HA, Moghadasali R. Great potential of renal progenitor cells in kidney: From the development to clinic. Exp Cell Res 2024; 434:113875. [PMID: 38092345 DOI: 10.1016/j.yexcr.2023.113875] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2023] [Revised: 12/02/2023] [Accepted: 12/03/2023] [Indexed: 12/23/2023]
Abstract
The mammalian renal organ represents a pinnacle of complexity, housing functional filtering units known as nephrons. During embryogenesis, the depletion of niches containing renal progenitor cells (RPCs) and the subsequent incapacity of adult kidneys to generate new nephrons have prompted the formulation of protocols aimed at isolating residual RPCs from mature kidneys and inducing their generation from diverse cell sources, notably pluripotent stem cells. Recent strides in the realm of regenerative medicine and the repair of tissues using stem cells have unveiled critical signaling pathways essential for the maintenance and generation of human RPCs in vitro. These findings have ushered in a new era for exploring novel strategies for renal protection. The present investigation delves into potential transcription factors and signaling cascades implicated in the realm of renal progenitor cells, focusing on their protection and differentiation. The discourse herein elucidates contemporary research endeavors dedicated to the acquisition of progenitor cells, offering crucial insights into the developmental mechanisms of these cells within the renal milieu and paving the way for the formulation of innovative treatment modalities.
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Affiliation(s)
- Maryam Bahrami
- Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Laser Applications in Medical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Shahram Darabi
- Cellular and Molecular Research Center, Research Institute for Non-Communicable Diseases, Qazvin University of Medical Sciences, Qazvin, Iran
| | | | - Hojjat Allah Abbaszadeh
- Laser Applications in Medical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
| | - Reza Moghadasali
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
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23
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Lindoso RS, Collino F, Kasai-Brunswick TH, Costa MR, Verdoorn KS, Einicker-Lamas M, Vieira-Beiral HJ, Wessely O, Vieyra A. Resident Stem Cells in Kidney Tissue. RESIDENT STEM CELLS AND REGENERATIVE THERAPY 2024:159-203. [DOI: 10.1016/b978-0-443-15289-4.00009-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2025]
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24
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Mederacke M, Conrad L, Doumpas N, Vetter R, Iber D. Geometric effects position renal vesicles during kidney development. Cell Rep 2023; 42:113526. [PMID: 38060445 DOI: 10.1016/j.celrep.2023.113526] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2022] [Revised: 07/25/2023] [Accepted: 11/15/2023] [Indexed: 12/30/2023] Open
Abstract
During kidney development, reciprocal signaling between the epithelium and the mesenchyme coordinates nephrogenesis with branching morphogenesis of the collecting ducts. The mechanism that positions the renal vesicles, and thus the nephrons, relative to the branching ureteric buds has remained elusive. By combining computational modeling and experiments, we show that geometric effects concentrate the key regulator, WNT9b, at the junctions between parent and daughter branches where renal vesicles emerge, even when uniformly expressed in the ureteric epithelium. This curvature effect might be a general paradigm to create non-uniform signaling in development.
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Affiliation(s)
- Malte Mederacke
- Department of Biosystems Science and Engineering, ETH Zürich, Schanzenstrasse 44, 4056 Basel, Switzerland; Swiss Institute of Bioinformatics, Mattenstrasse 26, 4058 Basel, Switzerland
| | - Lisa Conrad
- Department of Biosystems Science and Engineering, ETH Zürich, Schanzenstrasse 44, 4056 Basel, Switzerland; Swiss Institute of Bioinformatics, Mattenstrasse 26, 4058 Basel, Switzerland; Department for BioMedical Research (DBMR), University of Bern, Murtenstrasse 35, 3008 Bern, Switzerland
| | - Nikolaos Doumpas
- Department of Biosystems Science and Engineering, ETH Zürich, Schanzenstrasse 44, 4056 Basel, Switzerland; Swiss Institute of Bioinformatics, Mattenstrasse 26, 4058 Basel, Switzerland
| | - Roman Vetter
- Department of Biosystems Science and Engineering, ETH Zürich, Schanzenstrasse 44, 4056 Basel, Switzerland; Swiss Institute of Bioinformatics, Mattenstrasse 26, 4058 Basel, Switzerland
| | - Dagmar Iber
- Department of Biosystems Science and Engineering, ETH Zürich, Schanzenstrasse 44, 4056 Basel, Switzerland; Swiss Institute of Bioinformatics, Mattenstrasse 26, 4058 Basel, Switzerland.
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25
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Honeycutt SE, N'Guetta PEY, Hardesty DM, Xiong Y, Cooper SL, Stevenson MJ, O'Brien LL. Netrin 1 directs vascular patterning and maturity in the developing kidney. Development 2023; 150:dev201886. [PMID: 37818607 PMCID: PMC10690109 DOI: 10.1242/dev.201886] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2023] [Accepted: 10/02/2023] [Indexed: 10/12/2023]
Abstract
The intricate vascular system of the kidneys supports body fluid and organ homeostasis. However, little is known about how vascular architecture is established during kidney development. More specifically, how signals from the kidney influence vessel maturity and patterning remains poorly understood. Netrin 1 (Ntn1) is a secreted ligand that is crucial for vessel and neuronal guidance. Here, we demonstrate that Ntn1 is expressed by Foxd1+ stromal progenitors in the developing mouse kidney and conditional deletion (Foxd1GC/+;Ntn1fl/fl) results in hypoplastic kidneys with extended nephrogenesis. Wholemount 3D analyses additionally revealed the loss of a predictable vascular pattern in Foxd1GC/+;Ntn1fl/fl kidneys. As vascular patterning has been linked to vessel maturity, we investigated arterialization. Quantification of the CD31+ endothelium at E15.5 revealed no differences in metrics such as the number of branches or branch points, whereas the arterial vascular smooth muscle metrics were significantly reduced at both E15.5 and P0. In support of our observed phenotypes, whole kidney RNA-seq revealed disruptions to genes and programs associated with stromal cells, vasculature and differentiating nephrons. Together, our findings highlight the significance of Ntn1 to proper vascularization and kidney development.
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Affiliation(s)
- Samuel E. Honeycutt
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Pierre-Emmanuel Y. N'Guetta
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Deanna M. Hardesty
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Yubin Xiong
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Shamus L. Cooper
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Matthew J. Stevenson
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Lori L. O'Brien
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
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26
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Belcher B, Vestal J, Lane S, Kell M, Smith L, Camarata T. The zebrafish paralog six2b is required for early proximal pronephros morphogenesis. Sci Rep 2023; 13:19699. [PMID: 37952044 PMCID: PMC10640633 DOI: 10.1038/s41598-023-47046-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2023] [Accepted: 11/08/2023] [Indexed: 11/14/2023] Open
Abstract
The transcription factor Six2 plays a crucial role in maintaining self-renewing nephron progenitor cap mesenchyme (CM) during metanephric kidney development. In mouse and human, expression at single-cell resolution has detected Six2 in cells as they leave the CM pool and differentiate. The role Six2 may play in these cells as they differentiate remains unknown. Here, we took advantage of the zebrafish pronephric kidney which forms directly from intermediate mesoderm to test six2b function during pronephric tubule development and differentiation. Expression of six2b during early zebrafish development was consistent with a role in pronephros formation. Using morpholino knock-down and CRISPR/Cas9 mutagenesis, we show a functional role for six2b in the development of proximal elements of the pronephros. By 48 h post-fertilization, six2b morphants and mutants showed disrupted pronephric tubule morphogenesis. We observed a lower-than-expected frequency of phenotypes in six2b stable genetic mutants suggesting compensation. Supporting this, we detected increased expression of six2a in six2b stable mutant embryos. To further confirm six2b function, F0 crispant embryos were analyzed and displayed similar phenotypes as morphants and stable mutants. Together our data suggests a conserved role for Six2 during nephrogenesis and a role in the morphogenesis of the proximal tubule.
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Affiliation(s)
- Beau Belcher
- Biological Sciences, Arkansas State University, Jonesboro, USA
| | - Justin Vestal
- Biomedical Sciences, NYIT College of Osteopathic Medicine at Arkansas State University, Jonesboro, USA
| | - Samuel Lane
- Biological Sciences, Arkansas State University, Jonesboro, USA
| | - Margaret Kell
- Biomedical Sciences, NYIT College of Osteopathic Medicine at Arkansas State University, Jonesboro, USA
| | - Luke Smith
- Biological Sciences, Arkansas State University, Jonesboro, USA
| | - Troy Camarata
- Biomedical Sciences, NYIT College of Osteopathic Medicine at Arkansas State University, Jonesboro, USA.
- Biomedical Sciences, Baptist University College of Osteopathic Medicine, Baptist Health Sciences University, 1003 Monroe Ave, Memphis, TN, 38104, USA.
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27
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Stevenson MJ, Phanor SK, Patel U, Gisselbrecht SS, Bulyk ML, O'Brien LL. Altered binding affinity of SIX1-Q177R correlates with enhanced WNT5A and WNT pathway effector expression in Wilms tumor. Dis Model Mech 2023; 16:dmm050208. [PMID: 37815464 PMCID: PMC10668032 DOI: 10.1242/dmm.050208] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2023] [Accepted: 09/27/2023] [Indexed: 10/11/2023] Open
Abstract
Wilms tumors present as an amalgam of varying proportions of tissues located within the developing kidney, one being the nephrogenic blastema comprising multipotent nephron progenitor cells (NPCs). The recurring missense mutation Q177R in NPC transcription factors SIX1 and SIX2 is most correlated with tumors of blastemal histology and is significantly associated with relapse. Yet, the transcriptional regulatory consequences of SIX1/2-Q177R that might promote tumor progression and recurrence have not been investigated extensively. Utilizing multiple Wilms tumor transcriptomic datasets, we identified upregulation of the gene encoding non-canonical WNT ligand WNT5A in addition to other WNT pathway effectors in SIX1/2-Q177R mutant tumors. SIX1 ChIP-seq datasets from Wilms tumors revealed shared binding sites for SIX1/SIX1-Q177R within a promoter of WNT5A and at putative distal cis-regulatory elements (CREs). We demonstrate colocalization of SIX1 and WNT5A in Wilms tumor tissue and utilize in vitro assays that support SIX1 and SIX1-Q177R activation of expression from the WNT5A CREs, as well as enhanced binding affinity within the WNT5A promoter that may promote the differential expression of WNT5A and other WNT pathway effectors associated with SIX1-Q177R tumors.
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Affiliation(s)
- Matthew J. Stevenson
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Sabrina K. Phanor
- Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA
| | - Urvi Patel
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Stephen S. Gisselbrecht
- Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA
| | - Martha L. Bulyk
- Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA
- Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA
| | - Lori L. O'Brien
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
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28
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Habib R, Fahim S, Wahid M, Ainuddin J. Optimisation of a Method for the Differentiation of Human Umbilical Cord-derived Mesenchymal Stem Cells Toward Renal Epithelial-like Cells. Altern Lab Anim 2023; 51:363-375. [PMID: 37831588 DOI: 10.1177/02611929231207774] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/15/2023]
Abstract
Human umbilical cord-derived mesenchymal stem cells (hucMSCs) can differentiate into multiple cell lineages, but few methods have been developed to generate kidney lineage cells. Due to their human origin, pluripotent nature and immunomodulatory properties, these stem cells are attractive candidates for clinical applications such as the repair or regeneration of damaged organs. This study evaluated the renal differentiation potential of hucMSCs, when exposed for 10 days to optimised concentrations of retinoic acid, activin-A and bone morphogenetic protein-7 (BMP-7) in various combinations, with and without the priming of the cells with a Wnt signalling pathway activator (CHIR99021). The hucMSCs were isolated and characterised according to surface marker expression (CD73, CD90, CD44, CD146 and CD8) and tri-lineage differentiation potential. The expression of key marker genes (OSR1, TBXT, HOXA13, SIX2, PAX2, KRT18 and ZO1) was examined by qRT-PCR. Specific marker protein expression (E-cadherin, cytokeratin-8 and cytokeratin-19) was analysed by immunocytochemistry. CHIR99021-primed cells treated with the retinoic acid, activin-A and BMP-7 cocktail showed epithelial cell-like differentiation - i.e. distinct phenotypic changes, as well as upregulated gene and protein expression, were observed that were consistent with an epithelial cell phenotype. Thus, our results showed that hucMSCs can efficiently differentiate into renal epithelial-like cells. This work may help in the development of focused therapeutic strategies, in which lineage-defined human stem cells can be used for renal regeneration.
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Affiliation(s)
- Rakhshinda Habib
- Dow Research Institute of Biotechnology and Biomedical Sciences, Dow University of Health Sciences (Ojha campus), Karachi, Pakistan
| | - Shumaila Fahim
- Dow Research Institute of Biotechnology and Biomedical Sciences, Dow University of Health Sciences (Ojha campus), Karachi, Pakistan
| | - Mohsin Wahid
- Department of Pathology, Dow International Medical College, Dow University of Health Sciences (Ojha campus), Karachi, Pakistan
| | - Jahanara Ainuddin
- Department of Gynaecology and Obstetrics, Dow University Hospital, Karachi, Pakistan
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29
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Perl AJ, Liu H, Hass M, Adhikari N, Chaturvedi P, Hu YC, Jiang R, Liu Y, Kopan R. Reduced nephron endowment in the common Six2-TGC tg mouse line is due to Six3 misexpression by aberrant enhancer-promoter interactions in the transgene. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.10.06.561202. [PMID: 37873415 PMCID: PMC10592608 DOI: 10.1101/2023.10.06.561202] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/25/2023]
Abstract
Lifelong kidney function relies on the complement of nephrons generated during mammalian development from a mesenchymal nephron progenitor cell (NPC) population. Low nephron endowment confers increased susceptibility to chronic kidney disease. We asked whether reduced nephron numbers in the popular Six2TGC transgenic mouse line 1 was due to disruption of a regulatory gene at the integration site or to ectopic expression of a gene(s) contained within the transgene. Targeted locus amplification identified integration of the Six2TGC transgene within an intron of Cntnap5a on chr1. We generated Hi-C datasets from NPCs isolated from the Six2TGC tg/+ mice, the Cited1 CreERT2/+ control mice, and the Six2TGC tg/+ ; Tsc1 +/Flox,2 mice that exhibited restored nephron number compared with Six2TGC tg/+ mice, and mapped the precise integration of Six2TGC and Cited1 CreERT2 transgenes to chr1 and chr14, respectively. No changes in topology, accessibility, or expression were observed within the 50-megabase region centered on Cntnap5a in Six2TGC tg/+ mice compared with control mice. By contrast, we identified an aberrant regulatory interaction between a Six2 distal enhancer and the Six3 promoter contained within the transgene. Increasing the Six2TGC tg to Six2 locus ratio or removing one Six2 allele in Six2TGC tg/+ mice, caused severe renal hypoplasia. Furthermore, CRISPR disruption of Six3 within the transgene ( Six2TGC Δ Six3CT ) restored nephron endowment to wildtype levels and abolished the stoichiometric effect. Data from genetic and biochemical studies together suggest that in Six2TGC, SIX3 interferes with SIX2 function in NPC renewal through its C-terminal domain. Significance Using high-resolution chromatin conformation and accessibility datasets we mapped the integration site of two popular transgenes used in studies of nephron progenitor cells and kidney development. Aberrant enhancer-promoter interactions drive ectopic expression of Six3 in the Six2TGC tg line which was correlated with disruption of nephrogenesis. Disruption of Six3 within the transgene restored nephron numbers to control levels; further genetic and biochemical studies suggest that Six3 interferes with Six2 -mediated regulation of NPC renewal.
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30
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Bugacov H, Der B, Kim S, Lindström NO, McMahon AP. Canonical Wnt transcriptional complexes are essential for induction of nephrogenesis but not maintenance or proliferation of nephron progenitors. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.08.20.554044. [PMID: 37662369 PMCID: PMC10473675 DOI: 10.1101/2023.08.20.554044] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/05/2023]
Abstract
Wnt regulated transcriptional programs are associated with both the maintenance of mammalian nephron progenitor cells (NPC) and their induction, initiating the process of nephrogenesis. How opposing transcriptional roles are regulated remain unclear. Using an in vitro model replicating in vivo events, we examined the requirement for canonical Wnt transcriptional complexes in NPC regulation. In canonical transcription, Lef/Tcf DNA binding proteins associate the transcriptional co-activator β-catenin. Wnt signaling is readily substituted by CHIR99021, a small molecule antagonist of glycogen synthase kinase-3β (GSK3β). GSK3β inhibition blocks Gskβ-dependent turnover of β-catenin, enabling formation of Lef/Tcf/β-catenin transcriptional complexes, and enhancer-mediated transcriptional activation. Removal of β-catenin activity from NPCs under cell expansion conditions (low CHIR) demonstrated a non-transcriptional role for β-catenin in the CHIR-dependent proliferation of NPCs. In contrast, CHIR-mediated induction of nephrogenesis, on switching from low to high CHIR, was dependent on Lef/Tcf and β-catenin transcriptional activity. These studies point to a non-transcriptional mechanism for β-catenin in regulation of NPCs, and potentially other stem progenitor cell types. Further, analysis of the β-catenin-directed transcriptional response provides new insight into induction of nephrogenesis. Summary Statement The study provides a mechanistic understanding of Wnt/ β-catenin activity in self-renewal and differentiation of mammalian nephron progenitors.
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31
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Zheng H, Liu J, Pan X, Cui X. Biomarkers for patients with Wilms tumor: a review. Front Oncol 2023; 13:1137346. [PMID: 37554168 PMCID: PMC10405734 DOI: 10.3389/fonc.2023.1137346] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2023] [Accepted: 06/27/2023] [Indexed: 08/10/2023] Open
Abstract
Wilms tumor, originating from aberrant fetal nephrogenesis, is the most common renal malignancy in childhood. The overall survival of children is approximately 90%. Although existing risk-stratification systems are helpful in identifying patients with poor prognosis, the recurrence rate of Wilms tumors remains as high as 15%. To resolve this clinical problem, diverse studies on the occurrence and progression of the disease have been conducted, and the results are encouraging. A series of molecular biomarkers have been identified with further studies on the mechanism of tumorigenesis. Some of these show prognostic value and have been introduced into clinical practice. Identification of these biomarkers can supplement the existing risk-stratification systems. In the future, more biomarkers will be discovered, and more studies are required to validate their roles in improving the detection rate of occurrence or recurrence of Wilms tumor and to enhance clinical outcomes.
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Affiliation(s)
| | | | - Xiuwu Pan
- Department of Urology, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Xingang Cui
- Department of Urology, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
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32
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Wurmser M, Madani R, Chaverot N, Backer S, Borok M, Dos Santos M, Comai G, Tajbakhsh S, Relaix F, Santolini M, Sambasivan R, Jiang R, Maire P. Overlapping functions of SIX homeoproteins during embryonic myogenesis. PLoS Genet 2023; 19:e1010781. [PMID: 37267426 DOI: 10.1371/journal.pgen.1010781] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2022] [Accepted: 05/10/2023] [Indexed: 06/04/2023] Open
Abstract
Four SIX homeoproteins display a combinatorial expression throughout embryonic developmental myogenesis and they modulate the expression of the myogenic regulatory factors. Here, we provide a deep characterization of their role in distinct mouse developmental territories. We showed, at the hypaxial level, that the Six1:Six4 double knockout (dKO) somitic precursor cells adopt a smooth muscle fate and lose their myogenic identity. At the epaxial level, we demonstrated by the analysis of Six quadruple KO (qKO) embryos, that SIX are required for fetal myogenesis, and for the maintenance of PAX7+ progenitor cells, which differentiated prematurely and are lost by the end of fetal development in qKO embryos. Finally, we showed that Six1 and Six2 are required to establish craniofacial myogenesis by controlling the expression of Myf5. We have thus described an unknown role for SIX proteins in the control of myogenesis at different embryonic levels and refined their involvement in the genetic cascades operating at the head level and in the genesis of myogenic stem cells.
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Affiliation(s)
- Maud Wurmser
- Université de Paris Cité, Institut Cochin, INSERM, CNRS, Paris, France
| | - Rouba Madani
- Université de Paris Cité, Institut Cochin, INSERM, CNRS, Paris, France
| | - Nathalie Chaverot
- Université de Paris Cité, Institut Cochin, INSERM, CNRS, Paris, France
| | - Stéphanie Backer
- Université de Paris Cité, Institut Cochin, INSERM, CNRS, Paris, France
| | - Matthew Borok
- Univ Paris Est Creteil, INSERM, EnvA, EFS, AP-HP, IMRB, Creteil, France
| | | | - Glenda Comai
- Stem Cells & Development, Institut Pasteur, Paris, France
- CNRS UMR 3738, Institut Pasteur, Paris, France
| | - Shahragim Tajbakhsh
- Stem Cells & Development, Institut Pasteur, Paris, France
- CNRS UMR 3738, Institut Pasteur, Paris, France
| | - Frédéric Relaix
- Univ Paris Est Creteil, INSERM, EnvA, EFS, AP-HP, IMRB, Creteil, France
| | - Marc Santolini
- Université de Paris Cité, Interaction Data Lab, CRI Paris, INSERM. Paris, France
| | - Ramkumar Sambasivan
- Indian Institute of Science Education and Research (IISER) Tirupati, Tirupati, Andhra Pradesh, India
| | - Rulang Jiang
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America
| | - Pascal Maire
- Université de Paris Cité, Institut Cochin, INSERM, CNRS, Paris, France
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33
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Honeycutt SE, N’Guetta PEY, Hardesty DM, Xiong Y, Cooper SL, O’Brien LL. Netrin-1 directs vascular patterning and maturity in the developing kidney. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.04.14.536975. [PMID: 37131589 PMCID: PMC10153117 DOI: 10.1101/2023.04.14.536975] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/04/2023]
Abstract
Blood filtering by the kidney requires the establishment of an intricate vascular system that works to support body fluid and organ homeostasis. Despite these critical roles, little is known about how vascular architecture is established during kidney development. More specifically, how signals from the kidney influence vessel maturity and patterning remains poorly understood. Netrin-1 (Ntn1) is a secreted ligand critical for vessel and neuronal guidance. Here, we demonstrate that Ntn1 is expressed by stromal progenitors in the developing kidney, and conditional deletion of Ntn1 from Foxd1+ stromal progenitors (Foxd1GC/+;Ntn1fl/fl) results in hypoplastic kidneys that display extended nephrogenesis. Despite expression of the netrin-1 receptor Unc5c in the adjacent nephron progenitor niche, Unc5c knockout kidneys develop normally. The netrin-1 receptor Unc5b is expressed by embryonic kidney endothelium and therefore we interrogated the vascular networks of Foxd1GC/+;Ntn1fl/fl kidneys. Wholemount, 3D analyses revealed the loss of a predictable vascular pattern in mutant kidneys. As vascular patterning has been linked to vessel maturity, we investigated arterialization in these mutants. Quantification of the CD31+ endothelium at E15.5 revealed no differences in metrics such as the number of branches or branch points, whereas the arterial vascular smooth muscle metrics were significantly reduced at both E15.5 and P0. In support of these results, whole kidney RNA-seq showed upregulation of angiogenic programs and downregulation of muscle-related programs which included smooth muscle-associated genes. Together, our findings highlight the significance of netrin-1 to proper vascularization and kidney development.
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Affiliation(s)
- Samuel Emery Honeycutt
- Department of Cell Biology and Physiology University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | | | - Deanna Marie Hardesty
- Department of Cell Biology and Physiology University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Yubin Xiong
- Department of Cell Biology and Physiology University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Shamus Luke Cooper
- Department of Cell Biology and Physiology University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Lori Lynn O’Brien
- Department of Cell Biology and Physiology University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
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34
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Anderson MJ, Misaghian S, Sharma N, Perantoni AO, Lewandoski M. Fgf8 promotes survival of nephron progenitors by regulating BAX/BAK-mediated apoptosis. Differentiation 2023; 130:7-15. [PMID: 36527791 PMCID: PMC10718080 DOI: 10.1016/j.diff.2022.12.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2022] [Revised: 12/05/2022] [Accepted: 12/05/2022] [Indexed: 12/12/2022]
Abstract
Fibroblast growth factors (Fgfs) have long been implicated in processes critical to embryonic development, such as cell survival, migration, and differentiation. Several mouse models of organ development ascribe a prosurvival requirement specifically to FGF8. Here, we explore the potential role of prosurvival FGF8 signaling in kidney development. We have previously demonstrated that conditional deletion of Fgf8 in the mesodermal progenitors that give rise to the kidney leads to renal aplasia in the mutant neonate. Deleterious consequences caused by loss of FGF8 begin to manifest by E14.5 when massive aberrant cell death occurs in the cortical nephrogenic zone in the rudimentary kidney as well as in the renal vesicles that give rise to the nephrons. To rescue cell death in the Fgf8 mutant kidney, we inactivate the genes encoding the pro-apoptotic factors BAK and BAX. In a wild-type background, the loss of Bak and Bax abrogates normal cell death and has minimal effect on renal development. However, in Fgf8 mutants, the combined loss of Bak and Bax rescues aberrant cell death in the kidneys and restores some measure of kidney development: 1) the nephron progenitor population is greatly increased; 2) some glomeruli form, which are rarely observed in Fgf8 mutants; and 3) kidney size is rescued by about 50% at E18.5. The development of functional nephrons, however, is not rescued. Thus, FGF8 signaling is required for nephron progenitor survival by regulating BAK/BAX and for subsequent steps involving, as yet, undefined roles in kidney development.
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Affiliation(s)
- Matthew J Anderson
- Genetics of Vertebrate Development Section, Cancer and Developmental Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA
| | - Salvia Misaghian
- Genetics of Vertebrate Development Section, Cancer and Developmental Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA
| | - Nirmala Sharma
- Renal Differentiation and Neoplasia Section, Cancer and Developmental Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA
| | - Alan O Perantoni
- Renal Differentiation and Neoplasia Section, Cancer and Developmental Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA
| | - Mark Lewandoski
- Genetics of Vertebrate Development Section, Cancer and Developmental Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA.
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35
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Royer-Pokora B, Wruck W, Adjaye J, Beier M. Gene expression studies of WT1 mutant Wilms tumor cell lines in the frame work of published kidney development data reveals their early kidney stem cell origin. PLoS One 2023; 18:e0270380. [PMID: 36689432 PMCID: PMC9870146 DOI: 10.1371/journal.pone.0270380] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2022] [Accepted: 11/21/2022] [Indexed: 01/24/2023] Open
Abstract
In order to get a better insight into the timing of WT1 mutant Wilms tumor development, we compared the gene expression profiles of nine established WT1 mutant Wilms tumor cell lines with published data from different kidney cell types during development. Publications describing genes expressed in nephrogenic precursor cells, ureteric bud cells, more mature nephrogenic epithelial cells and interstitial cell types were used. These studies uncovered that the WT1 mutant Wilms tumor cells lines express genes from the earliest nephrogenic progenitor cells, as well as from more differentiated nephron cells with the highest expression from the stromal/interstitial compartment. The expression of genes from all cell compartments points to an early developmental origin of the tumor in a common stem cell. Although variability of the expression of specific genes was evident between the cell lines the overall expression pattern was very similar. This is likely dependent on their different genetic backgrounds with distinct WT1 mutations and the absence/presence of mutant CTNNB1.
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Affiliation(s)
- Brigitte Royer-Pokora
- Institute of Human Genetics, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
| | - Wasco Wruck
- Institute for Stem Cell Research and Regenerative Medicine, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
| | - James Adjaye
- Institute for Stem Cell Research and Regenerative Medicine, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
| | - Manfred Beier
- Institute of Human Genetics, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
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36
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Hamon M, Cheng HM, Johnson M, Yanagawa N, Hauser PV. Effect of Hypoxia on Branching Characteristics and Cell Subpopulations during Kidney Organ Culture. BIOENGINEERING (BASEL, SWITZERLAND) 2022; 9:bioengineering9120801. [PMID: 36551007 PMCID: PMC9774677 DOI: 10.3390/bioengineering9120801] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/17/2022] [Revised: 12/04/2022] [Accepted: 12/08/2022] [Indexed: 12/23/2022]
Abstract
During early developmental stages, embryonic kidneys are not fully vascularized and are potentially exposed to hypoxic conditions, which is known to influence cell proliferation and survival, ureteric bud branching, and vascularization of the developing kidney. To optimize the culture conditions of in vitro cultured kidneys and gain further insight into the effect of hypoxia on kidney development, we exposed mouse embryonic kidneys isolated at E11.5, E12.5, and E13.5 to hypoxic and normal culture conditions and compared ureteric bud branching patterns, the growth of the progenitor subpopulation hoxb7+, and the expression patterns of progenitor and differentiation markers. Branching patterns were quantified using whole organ confocal imaging and gradient-vector-based analysis. In our model, hypoxia causes an earlier expression of UB tip cell markers, and a delay in stalk cell marker gene expression. The metanephric mesenchyme (MM) exhibited a later expression of differentiation marker FGF8, marking a delay in nephron formation. Hypoxia further delayed the expression of stroma cell progenitor markers, a delay in cortical differentiation markers, as well as an earlier expression of medullary and ureteral differentiation markers. We conclude that standard conditions do not apply universally and that tissue engineering strategies need to optimize suitable culture conditions for each application. We also conclude that adapting culture conditions to specific aspects of organ development in tissue engineering can help to improve individual stages of tissue generation.
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Affiliation(s)
- Morgan Hamon
- Medical and Research Services, Greater Los Angeles Veterans Affairs Healthcare System at Sepulveda, North Hills, CA 91344, USA
- Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA
- Correspondence: (M.H.); (P.V.H.)
| | - Hsiao-Min Cheng
- Medical and Research Services, Greater Los Angeles Veterans Affairs Healthcare System at Sepulveda, North Hills, CA 91344, USA
- Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA
| | - Ming Johnson
- Medical and Research Services, Greater Los Angeles Veterans Affairs Healthcare System at Sepulveda, North Hills, CA 91344, USA
| | - Norimoto Yanagawa
- Medical and Research Services, Greater Los Angeles Veterans Affairs Healthcare System at Sepulveda, North Hills, CA 91344, USA
- Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA
| | - Peter V. Hauser
- Medical and Research Services, Greater Los Angeles Veterans Affairs Healthcare System at Sepulveda, North Hills, CA 91344, USA
- Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA
- Correspondence: (M.H.); (P.V.H.)
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37
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Liu H, Ngo NYN, Herzberger KF, Gummaraju M, Hilliard S, Chen CH. Histone deacetylases 1 and 2 target gene regulatory networks of nephron progenitors to control nephrogenesis. Biochem Pharmacol 2022; 206:115341. [PMID: 36356658 DOI: 10.1016/j.bcp.2022.115341] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2022] [Revised: 10/31/2022] [Accepted: 11/01/2022] [Indexed: 11/09/2022]
Abstract
Our studies demonstrated the critical role of Histone deacetylases (HDACs) in the regulation of nephrogenesis. To better understand the key pathways regulated by HDAC1/2 in early nephrogenesis, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) of HDAC1/2 on isolated nephron progenitor cells (NPCs) from mouse E16.5 kidneys. Our analysis revealed that 11,802 (40.4%) of HDAC1 peaks overlap with HDAC2 peaks, further demonstrates the redundant role of HDAC1 and HDAC2 during nephrogenesis. Common HDAC1/2 peaks are densely concentrated close to the transcriptional start site (TSS). GREAT Gene Ontology analysis of overlapping HDAC1/2 peaks reveals that HDAC1/2 are associated with metanephric nephron morphogenesis, chromatin assembly or disassembly, as well as other DNA checkpoints. Pathway analysis shows that negative regulation of Wnt signaling pathway is one of HDAC1/2's most significant function in NPCs. Known motif analysis indicated that Hdac1 is enriched in motifs for Six2, Hox family, and Tcf family members, which are essential for self-renewal and differentiation of nephron progenitors. Interestingly, we found the enrichment of HDAC1/2 at the enhancer and promoter regions of actively transcribed genes, especially those concerned with NPC self-renewal. HDAC1/2 simultaneously activate or repress the expression of different genes to maintain the cellular state of nephron progenitors. We used the Integrative Genomics Viewer to visualize these target genes associated with each function and found that HDAC1/2 co-bound to the enhancers or/and promoters of genes associated with nephron morphogenesis, differentiation, and cell cycle control. Taken together, our ChIP-Seq analysis demonstrates that HDAC1/2 directly regulate the molecular cascades essential for nephrogenesis.
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Affiliation(s)
- Hongbing Liu
- Department of Pediatrics, School of Medicine, Tulane University, United States.
| | - Nguyen Yen Nhi Ngo
- Department of Pediatrics, School of Medicine, Tulane University, United States
| | - Kyra F Herzberger
- Department of Pediatrics, School of Medicine, Tulane University, United States
| | - Manasi Gummaraju
- Department of Pediatrics, School of Medicine, Tulane University, United States; School of Arts and Science, Washington University in St. Louis, United States
| | - Sylvia Hilliard
- Department of Pediatrics, School of Medicine, Tulane University, United States
| | - Chao-Hui Chen
- Department of Pediatrics, School of Medicine, Tulane University, United States
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Perl AJ, Schuh MP, Kopan R. Regulation of nephron progenitor cell lifespan and nephron endowment. Nat Rev Nephrol 2022; 18:683-695. [PMID: 36104510 PMCID: PMC11078284 DOI: 10.1038/s41581-022-00620-w] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/27/2022] [Indexed: 11/08/2022]
Abstract
Low nephron number - resulting, for example, from prematurity or developmental anomalies - is a risk factor for the development of hypertension, chronic kidney disease and kidney failure. Considerable interest therefore exists in the mechanisms that regulate nephron endowment and contribute to the premature cessation of nephrogenesis following preterm birth. The cessation of nephrogenesis in utero or shortly after birth is synchronized across multiple niches in all mammals, and is coupled with the exhaustion of nephron progenitor cells. Consequently, no nephrons are formed after the cessation of developmental nephrogenesis, and lifelong renal function therefore depends on the complement of nephrons generated during gestation. In humans, a tenfold variation in nephron endowment between individuals contributes to differences in susceptibility to kidney disease; however, the mechanisms underlying this variation are not yet clear. Salient advances in our understanding of environmental inputs, and of intrinsic molecular mechanisms that contribute to the regulation of cessation timing or nephron progenitor cell exhaustion, have the potential to inform interventions to enhance nephron endowment and improve lifelong kidney health for susceptible individuals.
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Affiliation(s)
- Alison J Perl
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA
| | - Meredith P Schuh
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA
- Division of Nephrology and Hypertension, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA
| | - Raphael Kopan
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.
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Kwon HN, Kurtzeborn K, Iaroshenko V, Jin X, Loh A, Escande-Beillard N, Reversade B, Park S, Kuure S. Omics profiling identifies the regulatory functions of the MAPK/ERK pathway in nephron progenitor metabolism. Development 2022; 149:276992. [PMID: 36189831 PMCID: PMC9641663 DOI: 10.1242/dev.200986] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2022] [Accepted: 08/25/2022] [Indexed: 11/07/2022]
Abstract
Nephron endowment is defined by fetal kidney growth and crucially dictates renal health in adults. Defects in the molecular regulation of nephron progenitors contribute to only a fraction of reduced nephron mass cases, suggesting alternative causative mechanisms. The importance of MAPK/ERK activation in nephron progenitor maintenance has been previously demonstrated, and here, we characterized the metabolic consequences of MAPK/ERK deficiency. Liquid chromatography/mass spectrometry-based metabolomics profiling identified 42 reduced metabolites, of which 26 were supported by in vivo transcriptional changes in MAPK/ERK-deficient nephron progenitors. Among these, mitochondria, ribosome and amino acid metabolism, together with diminished pyruvate and proline metabolism, were the most affected pathways. In vitro cultures of mouse kidneys demonstrated a dosage-specific function for pyruvate in controlling the shape of the ureteric bud tip, a regulatory niche for nephron progenitors. In vivo disruption of proline metabolism caused premature nephron progenitor exhaustion through their accelerated differentiation in pyrroline-5-carboxylate reductases 1 (Pycr1) and 2 (Pycr2) double-knockout kidneys. Pycr1/Pycr2-deficient progenitors showed normal cell survival, indicating no changes in cellular stress. Our results suggest that MAPK/ERK-dependent metabolism functionally participates in nephron progenitor maintenance by monitoring pyruvate and proline biogenesis in developing kidneys.
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Affiliation(s)
- Hyuk Nam Kwon
- Helsinki Institute of Life Science, University of Helsinki, Helsinki, FIN-00014, Finland,Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, FIN-00014, Finland
| | - Kristen Kurtzeborn
- Helsinki Institute of Life Science, University of Helsinki, Helsinki, FIN-00014, Finland,Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, FIN-00014, Finland
| | - Vladislav Iaroshenko
- Helsinki Institute of Life Science, University of Helsinki, Helsinki, FIN-00014, Finland,Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, FIN-00014, Finland
| | - Xing Jin
- College of Pharmacy, Natural Product Research Institute, Seoul National University, Seoul 08826, Korea
| | - Abigail Loh
- Institute of Molecular and Cellular Biology (IMCB), A*STAR, Singapore 138648, Singapore
| | - Nathalie Escande-Beillard
- Institute of Molecular and Cellular Biology (IMCB), A*STAR, Singapore 138648, Singapore,Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, FIN-00014, Finland
| | - Bruno Reversade
- Institute of Molecular and Cellular Biology (IMCB), A*STAR, Singapore 138648, Singapore,Medical Genetics Department, School of Medicine, Koç University, Istanbul 34010, Turkey
| | - Sunghyouk Park
- College of Pharmacy, Natural Product Research Institute, Seoul National University, Seoul 08826, Korea
| | - Satu Kuure
- Helsinki Institute of Life Science, University of Helsinki, Helsinki, FIN-00014, Finland,Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, FIN-00014, Finland,GM-unit, Laboratory Animal Center, Helsinki Institute of Life Science, University of Helsinki, Helsinki, FIN-00014, Finland,Author for correspondence ()
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40
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Schnell J, Achieng M, Lindström NO. Principles of human and mouse nephron development. Nat Rev Nephrol 2022; 18:628-642. [PMID: 35869368 DOI: 10.1038/s41581-022-00598-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/10/2022] [Indexed: 12/17/2022]
Abstract
The mechanisms underlying kidney development in mice and humans is an area of intense study. Insights into kidney organogenesis have the potential to guide our understanding of the origin of congenital anomalies and enable the assembly of genetic diagnostic tools. A number of studies have delineated signalling nodes that regulate positional identities and cell fates of nephron progenitor and precursor cells, whereas cross-species comparisons have markedly enhanced our understanding of conserved and divergent features of mammalian kidney organogenesis. Greater insights into the complex cellular movements that occur as the proximal-distal axis is established have challenged our understanding of nephron patterning and provided important clues to the elaborate developmental context in which human kidney diseases can arise. Studies of kidney development in vivo have also facilitated efforts to recapitulate nephrogenesis in kidney organoids in vitro, by providing a detailed blueprint of signalling events, cell movements and patterning mechanisms that are required for the formation of correctly patterned nephrons and maturation of physiologically functional apparatus that are responsible for maintaining human health.
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Affiliation(s)
- Jack Schnell
- Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at University of Southern California, Los Angeles, CA, USA
| | - MaryAnne Achieng
- Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at University of Southern California, Los Angeles, CA, USA
| | - Nils Olof Lindström
- Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at University of Southern California, Los Angeles, CA, USA.
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41
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Li J, Cheng C, Xu J, Zhang T, Tokat B, Dolios G, Ramakrishnan A, Shen L, Wang R, Xu PX. The transcriptional coactivator Eya1 exerts transcriptional repressive activity by interacting with REST corepressors and REST-binding sequences to maintain nephron progenitor identity. Nucleic Acids Res 2022; 50:10343-10359. [PMID: 36130284 PMCID: PMC9561260 DOI: 10.1093/nar/gkac760] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2022] [Revised: 08/18/2022] [Accepted: 08/27/2022] [Indexed: 11/15/2022] Open
Abstract
Eya1 is critical for establishing and maintaining nephron progenitor cells (NPCs). It belongs to a family of proteins called phosphatase-transcriptional activators but without intrinsic DNA-binding activity. However, the spectrum of the Eya1-centered networks is underexplored. Here, we combined transcriptomic, genomic and proteomic approaches to characterize gene regulation by Eya1 in the NPCs. We identified Eya1 target genes, associated cis-regulatory elements and partner proteins. Eya1 preferentially occupies promoter sequences and interacts with general transcription factors (TFs), RNA polymerases, different types of TFs, chromatin-remodeling factors with ATPase or helicase activity, and DNA replication/repair proteins. Intriguingly, we identified REST-binding motifs in 76% of Eya1-occupied sites without H3K27ac-deposition, which were present in many Eya1 target genes upregulated in Eya1-deficient NPCs. Eya1 copurified REST-interacting chromatin-remodeling factors, histone deacetylase/lysine demethylase, and corepressors. Coimmunoprecipitation validated physical interaction between Eya1 and Rest/Hdac1/Cdyl/Hltf in the kidneys. Collectively, our results suggest that through interactions with chromatin-remodeling factors and specialized DNA-binding proteins, Eya1 may modify chromatin structure to facilitate the assembly of regulatory complexes that regulate transcription positively or negatively. These findings provide a mechanistic basis for how Eya1 exerts its activity by forming unique multiprotein complexes in various biological processes to maintain the cellular state of NPCs.
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Affiliation(s)
- Jun Li
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA
| | - Chunming Cheng
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA
| | - Jinshu Xu
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA
| | - Ting Zhang
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA
| | - Bengu Tokat
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA
| | - Georgia Dolios
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA
| | | | - Li Shen
- Department of Neurosciences, New York, NY 10029, USA
| | - Rong Wang
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA
| | - Pin-Xian Xu
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA.,Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
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Motojima M, Tanaka M, Kume T. Foxc1 and Foxc2 are indispensable for maintenance of progenitors of nephron and stroma in the developing kidney. J Cell Sci 2022; 135:276938. [PMID: 36073617 DOI: 10.1242/jcs.260356] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2022] [Accepted: 08/31/2022] [Indexed: 11/20/2022] Open
Abstract
Nephron development proceeds with reciprocal interactions among three layers: nephron progenitors (NPs), ureteric buds, and stromal progenitors (SPs). We found Foxc1 and Foxc2 (Foxc1/2) expression in NPs and SPs. Systemic deletion of Foxc1/2 two days after the onset of metanephros development (E13.5) resulted in epithelialization of NPs and ectopic formation of renal vesicles. NP-specific deletion did not cause these phenotypes, indicating that Foxc1/2 in other cells (likely in SPs) contributed to the maintenance of NPs. Single-cell RNA-seq analysis revealed NP and SP subpopulations, the border between committed NPs and renewing NPs, and similarity among cortical interstitium and vascular smooth muscle type cells. Integrated analysis of the control and knockout data indicated transformation of some NPs to strange cells expressing markers of vascular endothelium, reduced numbers of self-renewing NP and SP populations, downregulation of crucial genes for kidney development such as Fgf20 and Frem1 in NPs, and Foxd1 and Sall1 in SPs. It also revealed upregulation of genes that were not usually expressed in NPs and SPs. Thus, Foxc1/2 maintains NPs and SPs by regulating the expression of multiple genes.
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Affiliation(s)
- Masaru Motojima
- Department of Clinical Pharmacology, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan
| | - Masayuki Tanaka
- Medical Science College Office, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan
| | - Tsutomu Kume
- Feinberg Cardiovascular Research Institute, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA
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Urine-Derived Stem Cells for Epithelial Tissues Reconstruction and Wound Healing. Pharmaceutics 2022; 14:pharmaceutics14081669. [PMID: 36015295 PMCID: PMC9415563 DOI: 10.3390/pharmaceutics14081669] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2022] [Revised: 08/09/2022] [Accepted: 08/09/2022] [Indexed: 11/17/2022] Open
Abstract
Epithelial tissue injury can occur on any surface site of the body, particularly in the skin or urethral mucosa tissue, due to trauma, infection, inflammation, and toxic compounds. Both internal and external body epithelial tissue injuries can significantly affect patients’ quality of life, increase healthcare spending, and increase the global economic burden. Transplantation of epithelial tissue grafts is an effective treatment strategy in clinical settings. Autologous bio-engineered epithelia are common clinical skin substitutes that have the specific advantages of avoiding tissue rejection, obviating ethical concerns, reducing the risk of infection, and decreasing scarring compared to donor grafts. However, epithelial cells are often obtained from the individual’s skin and mucosa through invasive methods, which cause further injury or damage. Urine-derived stem cells (USC) of kidney origin, obtained via non-invasive acquisition, possess high stemness properties, self-renewal ability, trophic effects, multipotent differentiation potential, and immunomodulatory ability. These cells show versatile potential for tissue regeneration, with extensive evidence supporting their use in the repair of epidermal and urothelial injuries. We discuss the collection, isolation, culture, characterization, and differentiation of USC. We also discuss the use of USC for cellular therapies as well as the administration of USC-derived paracrine factors for epidermal and urothelial tissue repair. Specifically, we will discuss 3D constructions involving multiple types of USC-loaded hydrogels and USC-seeded scaffolds for use in cosmetic production testing, drug development, and disease modeling. In conclusion, urine-derived stem cells are a readily accessible autologous stem cell source well-suited for developing personalized medical treatments in epithelial tissue regeneration and drug testing.
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Huang B, Zeng Z, Zhang CC, Schreiber ME, Li Z. Approaches to kidney replacement therapies—opportunities and challenges. Front Cell Dev Biol 2022; 10:953408. [PMID: 35982852 PMCID: PMC9380013 DOI: 10.3389/fcell.2022.953408] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2022] [Accepted: 07/01/2022] [Indexed: 11/29/2022] Open
Abstract
One out of seven people develop chronic kidney disease (CKD). When kidney function continues to decline, CKD patients may develop end-stage renal disease (ESRD, or kidney failure). More than 2 out of 1,000 adults develop ESRD and these patients must live on dialysis or get a kidney transplant to survive. Each year, more than $51 billion is spent to treat patients with ESRD in the United States. In addition, ESRD greatly reduces longevity and quality of life for patients. Compared to dialysis, kidney transplant offers the best chance of survival, but few donor organs are available. Thus, there is an urgent need for innovative solutions that address the shortage of kidneys available for transplantation. Here we summarize the status of current approaches that are being developed to solve the shortage of donor kidneys. These include the bioartificial kidney approach which aims to make a portable dialysis device, the recellularization approach which utilizes native kidney scaffold to make an engineered kidney, the stem cell-based approach which aims to generate a kidney de novo by recapitulating normal kidney organogenesis, the xenotransplantation approach which has the goal to make immunocompatible pig kidneys for transplantation, and the interspecies chimera approach which has potential to generate a human kidney in a host animal. We also discuss the interconnections among the different approaches, and the remaining challenges of translating these approaches into novel therapies.
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Affiliation(s)
- Biao Huang
- USC/UKRO Kidney Research Center, Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
- Deptartment of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
| | - Zipeng Zeng
- USC/UKRO Kidney Research Center, Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
- Deptartment of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
| | - Chennan C. Zhang
- USC/UKRO Kidney Research Center, Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
- Deptartment of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
| | - Megan E. Schreiber
- USC/UKRO Kidney Research Center, Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
- Deptartment of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
| | - Zhongwei Li
- USC/UKRO Kidney Research Center, Division of Nephrology and Hypertension, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
- Deptartment of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States
- *Correspondence: Zhongwei Li,
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Tran T, Song CJ, Nguyen T, Cheng SY, McMahon JA, Yang R, Guo Q, Der B, Lindström NO, Lin DCH, McMahon AP. A scalable organoid model of human autosomal dominant polycystic kidney disease for disease mechanism and drug discovery. Cell Stem Cell 2022; 29:1083-1101.e7. [PMID: 35803227 PMCID: PMC11088748 DOI: 10.1016/j.stem.2022.06.005] [Citation(s) in RCA: 69] [Impact Index Per Article: 23.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2020] [Revised: 01/28/2022] [Accepted: 06/08/2022] [Indexed: 12/13/2022]
Abstract
Human pluripotent stem-cell-derived organoids are models for human development and disease. We report a modified human kidney organoid system that generates thousands of similar organoids, each consisting of 1-2 nephron-like structures. Single-cell transcriptomic profiling and immunofluorescence validation highlighted patterned nephron-like structures utilizing similar pathways, with distinct morphogenesis, to human nephrogenesis. To examine this platform for therapeutic screening, the polycystic kidney disease genes PKD1 and PKD2 were inactivated by gene editing. PKD1 and PKD2 mutant models exhibited efficient and reproducible cyst formation. Cystic outgrowths could be propagated for months to centimeter-sized cysts. To shed new light on cystogenesis, 247 protein kinase inhibitors (PKIs) were screened in a live imaging assay identifying compounds blocking cyst formation but not overall organoid growth. Scaling and further development of the organoid platform will enable a broader capability for kidney disease modeling and high-throughput drug screens.
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Affiliation(s)
- Tracy Tran
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Cheng Jack Song
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA; Amgen Research, Cardiometabolic Disorders, 1120 Veterans Blvd, South San Francisco, CA 94080, USA
| | - Trang Nguyen
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Shun-Yang Cheng
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Jill A McMahon
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Rui Yang
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Qiuyu Guo
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Balint Der
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Nils O Lindström
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Daniel C-H Lin
- Amgen Research, Cardiometabolic Disorders, 1120 Veterans Blvd, South San Francisco, CA 94080, USA
| | - Andrew P McMahon
- Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA.
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Hilliard S, Tortelote G, Liu H, Chen CH, El-Dahr SS. Single-Cell Chromatin and Gene-Regulatory Dynamics of Mouse Nephron Progenitors. J Am Soc Nephrol 2022; 33:1308-1322. [PMID: 35383123 PMCID: PMC9257825 DOI: 10.1681/asn.2021091213] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2021] [Accepted: 03/22/2022] [Indexed: 11/03/2022] Open
Abstract
BACKGROUND We reasoned that unraveling the dynamic changes in accessibility of genomic regulatory elements and gene expression at single-cell resolution will inform the basic mechanisms of nephrogenesis. METHODS We performed single-cell ATAC-seq and RNA-seq both individually (singleomes; Six2GFP cells) and jointly in the same cells (multiomes; kidneys) to generate integrated chromatin and transcriptional maps in mouse embryonic and neonatal nephron progenitor cells. RESULTS We demonstrate that singleomes and multiomes are comparable in assigning most cell states, identification of new cell type markers, and defining the transcription factors driving cell identity. However, multiomes are more precise in defining the progenitor population. Multiomes identified a "pioneer" bHLH/Fox motif signature in nephron progenitor cells. Moreover, we identified a subset of Fox factors exhibiting high chromatin activity in podocytes. One of these Fox factors, Foxp1, is important for nephrogenesis. Key nephrogenic factors are distinguished by strong correlation between linked gene regulatory elements and gene expression. CONCLUSION Mapping the regulatory landscape at single-cell resolution informs the regulatory hierarchy of nephrogenesis. Paired single-cell epigenomes and transcriptomes of nephron progenitors should provide a foundation to understand prenatal programming, regeneration after injury, and ex vivo nephrogenesis.
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Affiliation(s)
- Sylvia Hilliard
- Section of Pediatric Nephrology, Department of Pediatrics, Tulane University School of Medicine, New Orleans, Louisiana
| | - Giovane Tortelote
- Section of Pediatric Nephrology, Department of Pediatrics, Tulane University School of Medicine, New Orleans, Louisiana
| | - Hongbing Liu
- Section of Pediatric Nephrology, Department of Pediatrics, Tulane University School of Medicine, New Orleans, Louisiana
| | - Chao-Hui Chen
- Section of Pediatric Nephrology, Department of Pediatrics, Tulane University School of Medicine, New Orleans, Louisiana
| | - Samir S. El-Dahr
- Section of Pediatric Nephrology, Department of Pediatrics, Tulane University School of Medicine, New Orleans, Louisiana
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Röck R, Rizzo L, Lienkamp SS. Kidney Development: Recent Insights from Technological Advances. Physiology (Bethesda) 2022; 37:0. [PMID: 35253460 DOI: 10.1152/physiol.00041.2021] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
The kidney is a complex organ, and how it forms is a fascinating process. New technologies, such as single-cell transcriptomics, and enhanced imaging modalities are offering new approaches to understand the complex and intertwined processes during embryonic kidney development.
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Affiliation(s)
- Ruth Röck
- Institute of Anatomy, University of Zurich, Zurich, Switzerland.,Swiss National Centres of Competence in Research (NCCR) Kidney Control of Homeostasis (Kidney.CH), Zurich, Switzerland
| | - Ludovica Rizzo
- Institute of Anatomy, University of Zurich, Zurich, Switzerland.,Swiss National Centres of Competence in Research (NCCR) Kidney Control of Homeostasis (Kidney.CH), Zurich, Switzerland.,PhD program "Molecular and Translational Biomedicine," Life Science Zurich Graduate School, Zurich, Switzerland
| | - Soeren S Lienkamp
- Institute of Anatomy, University of Zurich, Zurich, Switzerland.,Swiss National Centres of Competence in Research (NCCR) Kidney Control of Homeostasis (Kidney.CH), Zurich, Switzerland
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Freedman BS. Physiology assays in human kidney organoids. Am J Physiol Renal Physiol 2022; 322:F625-F638. [PMID: 35379001 PMCID: PMC9076410 DOI: 10.1152/ajprenal.00400.2021] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2021] [Revised: 03/28/2022] [Accepted: 03/29/2022] [Indexed: 01/15/2023] Open
Abstract
Kidney organoids derived from human pluripotent stem cells constitute a novel model of disease, development, and regenerative therapy. Organoids are human, experimentally accessible, high throughput, and enable reconstitution of tissue-scale biology in a petri dish. Although gene expression patterns in organoid cells have been analyzed extensively, less is known about the functionality of these structures. Here, we review assays of physiological function in human kidney organoids, including best practices for quality control, and future applications. Tubular structures in organoids accumulate specific molecules through active transport, including dextran and organic anions, and swell with fluid in response to cAMP stimulation. When engrafted into animal models in vivo, organoids form vascularized glomerulus-like structures capable of size-selective filtration. Organoids exhibit metabolic, endocrine, injury, and infection phenotypes, although their specificity is not yet fully clear. To properly interpret organoid physiology assays, it is important to incorporate appropriate negative and positive controls, statistical methods, data presentation, molecular mechanisms, and clinical data sets. Improvements in organoid perfusion, patterning, and maturation are needed to enable branching morphogenesis, urine production, and renal replacement. Reconstituting renal physiology with kidney organoids is a new field with potential to provide fresh insights into classical phenomena.
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Affiliation(s)
- Benjamin S Freedman
- Division of Nephrology, Kidney Research Institute, and Institute for Stem Cell and Regenerative Medicine, Department of Medicine, Department of Laboratory Medicine and Physiology (adjunct), and Department of Bioengineering (adjunct), University of Washington School of Medicine, Seattle, Washington
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Comparative whole-genome transcriptome analysis in renal cell populations reveals high tissue specificity of MAPK/ERK targets in embryonic kidney. BMC Biol 2022; 20:112. [PMID: 35550069 PMCID: PMC9102746 DOI: 10.1186/s12915-022-01309-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2021] [Accepted: 04/25/2022] [Indexed: 12/19/2022] Open
Abstract
Background MAPK/ERK signaling is a well-known mediator of extracellular stimuli controlling intracellular responses to growth factors and mechanical cues. The critical requirement of MAPK/ERK signaling for embryonic stem cell maintenance is demonstrated, but specific functions in progenitor regulation during embryonic development, and in particular kidney development remain largely unexplored. We previously demonstrated MAPK/ERK signaling as a key regulator of kidney growth through branching morphogenesis and normal nephrogenesis where it also regulates progenitor expansion. Here, we performed RNA sequencing-based whole-genome expression analysis to identify transcriptional MAPK/ERK targets in two distinct renal populations: the ureteric bud epithelium and the nephron progenitors. Results Our analysis revealed a large number (5053) of differentially expressed genes (DEGs) in nephron progenitors and significantly less (1004) in ureteric bud epithelium, reflecting likely heterogenicity of cell types. The data analysis identified high tissue-specificity, as only a fraction (362) of MAPK/ERK targets are shared between the two tissues. Tissue-specific MAPK/ERK targets participate in the regulation of mitochondrial energy metabolism in nephron progenitors, which fail to maintain normal mitochondria numbers in the MAPK/ERK-deficient tissue. In the ureteric bud epithelium, a dramatic decline in progenitor-specific gene expression was detected with a simultaneous increase in differentiation-associated genes, which was not observed in nephron progenitors. Our experiments in the genetic model of MAPK/ERK deficiency provide evidence that MAPK/ERK signaling in the ureteric bud maintains epithelial cells in an undifferentiated state. Interestingly, the transcriptional targets shared between the two tissues studied are over-represented by histone genes, suggesting that MAPK/ERK signaling regulates cell cycle progression and stem cell maintenance through chromosome condensation and nucleosome assembly. Conclusions Using tissue-specific MAPK/ERK inactivation and RNA sequencing in combination with experimentation in embryonic kidneys, we demonstrate here that MAPK/ERK signaling maintains ureteric bud tip cells, suggesting a regulatory role in collecting duct progenitors. We additionally deliver new mechanistic information on how MAPK/ERK signaling regulates progenitor maintenance through its effects on chromatin accessibility and energy metabolism. Supplementary Information The online version contains supplementary material available at 10.1186/s12915-022-01309-z.
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COUP-TFII in Kidneys, from Embryos to Sick Adults. Diagnostics (Basel) 2022; 12:diagnostics12051181. [PMID: 35626336 PMCID: PMC9139597 DOI: 10.3390/diagnostics12051181] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2022] [Revised: 05/02/2022] [Accepted: 05/06/2022] [Indexed: 11/16/2022] Open
Abstract
Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is an orphan nuclear hormone receptor of unknown ligands. This molecule has two interesting features: (1) it is a developmental gene, and (2) it is a potential hormone receptor. Here, we describe the possible roles of COUP-TFII in the organogenesis of the kidneys and protection from adult renal diseases, primarily in mouse models. COUP-TFII is highly expressed in embryos, including primordial kidneys, and is essential for the formation of metanephric mesenchyme and the survival of renal precursor cells. Although the expression levels of COUP-TFII are low and its functions are unknown in healthy adults, it serves as a reno-protectant molecule against acute kidney injury. These are good examples of how developmental genes exhibit novel functions in the etiology of adult diseases. We also discuss the ongoing research on the roles of COUP-TFII in podocyte development and diabetic kidney disease. In addition, the identification of potential ligands suggests that COUP-TFII might be a novel therapeutic target for renal diseases in the future.
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