scholarly journals Efficient Gene Transfer to Kidney Mesenchymal Cells Using a Synthetic Adeno-Associated Viral Vector

2018 ◽  
Vol 29 (9) ◽  
pp. 2287-2297 ◽  
Author(s):  
Yoichiro Ikeda ◽  
Zhao Sun ◽  
Xiao Ru ◽  
Luk H. Vandenberghe ◽  
Benjamin D. Humphreys
Keyword(s):  
High Efficiency ◽  
Viral Vector ◽  
Mesangial Cells ◽  
Critical Role ◽  
Genetic Material ◽  
Human Kidney ◽  
Mesenchymal Cells ◽  
Kidney Fibrosis ◽  
Kidney Organoids

BackgroundAfter injury, mesenchymal progenitors in the kidney interstitium differentiate into myofibroblasts, cells that have a critical role in kidney fibrogenesis. The ability to deliver genetic material to myofibroblast progenitors could allow new therapeutic approaches to treat kidney fibrosis. Preclinical and clinical studies show that adeno-associated viruses (AAVs) efficiently and safely transduce various tissue targets in vivo; however, protocols for transduction of kidney mesenchymal cells have not been established.MethodsWe evaluated the transduction profiles of various pseudotyped AAV vectors expressing either GFP or Cre recombinase reporters in mouse kidney and human kidney organoids.ResultsOf the six AAVs tested, a synthetic AAV called Anc80 showed specific and high-efficiency transduction of kidney stroma and mesangial cells. We characterized the cell specificity, dose dependence, and expression kinetics and showed the efficacy of this approach by knocking out Gli2 from kidney mesenchymal cells by injection of Anc80-Cre virus into either homozygous or heterozygous Gli2-floxed mice. After unilateral ureteral obstruction, the homozygous Gli2-floxed mice had less fibrosis than the Gli2 heterozygotes had. We observed the same antifibrotic effect in β-catenin–floxed mice injected with Anc80-Cre virus before obstructive injury, strongly supporting a central role for canonical Wnt signaling in kidney myofibroblast activation. Finally, we showed that the Anc80 synthetic virus can transduce the mesenchymal lineage in human kidney organoids.ConclusionsThese studies establish a novel method for inducible knockout of floxed genes in mouse mesangium, pericytes, and perivascular fibroblasts and are the foundation for future gene therapy approaches to treat kidney fibrosis.

scholarly journals Modeling oxidative injury response in human kidney organoids

2021 ◽  
Author(s):  
Aneta Przepiorski ◽  
Thitinee Vanichapol ◽  
Eugenel B. Espiritu ◽  
Amanda E. Crunk ◽  
Emily Parasky ◽  
...  
Keyword(s):  
Ischemia Reperfusion ◽  
Organic Anion ◽  
Kidney Injury ◽  
Induced Pluripotent Stem Cell ◽  
Human Kidney ◽  
Mitochondrial Morphology ◽  
Tubular Atrophy ◽  
Kidney Fibrosis ◽  
Kidney Organoids

Abstract BackgroundPersistent acute kidney injury (AKI) leads to tubular atrophy, kidney fibrosis, and, if severe enough, chronic kidney disease (CKD). A common feature of AKI is the generation of excessive reactive oxygen species (ROS) which damage cells and induce inflammation. MethodsHuman kidney organoids were treated with hemin, an iron-containing porphyrin derived from lysed red blood cells, that generates ROS in disease settings such as rhabdomyolysis, sepsis and ischemia reperfusion leading to AKI. In addition, we developed an induced pluripotent stem cell line expressing the biosensor, CytochromeC-GFP (CytoC-GFP), which provides a real-time readout of mitochondrial morphology, health, and early apoptotic events. ResultsWe found that hemin-treated kidney organoids show oxidative damage, increased expression of injury markers, impaired functionality of organic anion and cation transport and undergo fibrosis. Tubule injury could be detected in live CytoC-GFP organoids by cytoplasmic localization of fluorescence. Finally, we show that 4-(phenylthio)butanoic acid, an HDAC inhibitor with anti-fibrotic effects in vivo , reduces hemin-induced human kidney organoid fibrosis. ConclusionTogether this work establishes a hemin-induced model of kidney organoid injury and fibrosis as a new model to study renal repair and a human platform for developing AKI therapeutics.

scholarly journals 3D kidney organoids for bench-to-bedside translation

2020 ◽  
Author(s):  
Navin Gupta✉ ◽  
Emre Dilmen ◽  
Ryuji Morizane
Keyword(s):  
Collecting Duct ◽  
Developmental Toxicity ◽  
Human Kidney ◽  
Great Promise ◽  
Duct Systems ◽  
Recent Developments ◽  
Induced Pluripotent ◽  
Kidney Organoids

Abstract The kidneys are essential organs that filter the blood, removing urinary waste while maintaining fluid and electrolyte homeostasis. Current conventional research models such as static cell cultures and animal models are insufficient to grasp the complex human in vivo situation or lack translational value. To accelerate kidney research, novel research tools are required. Recent developments have allowed the directed differentiation of induced pluripotent stem cells to generate kidney organoids. Kidney organoids resemble the human kidney in vitro and can be applied in regenerative medicine and as developmental, toxicity, and disease models. Although current studies have shown great promise, challenges remain including the immaturity, limited reproducibility, and lack of perfusable vascular and collecting duct systems. This review gives an overview of our current understanding of nephrogenesis that enabled the generation of kidney organoids. Next, the potential applications of kidney organoids are discussed followed by future perspectives. This review proposes that advancement in kidney organoid research will be facilitated through our increasing knowledge on nephrogenesis and combining promising techniques such as organ-on-a-chip models.

scholarly journals Organoid single-cell profiling identifies a transcriptional signature of glomerular disease

2018 ◽  
Author(s):  
Jennifer L. Harder ◽  
Rajasree Menon ◽  
Edgar A. Otto ◽  
Jian Zhou ◽  
Sean Eddy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kidney Disease ◽  
Single Cells ◽  
Gene Expression Signature ◽  
Human Kidney ◽  
Glomerular Disease ◽  
Podocyte Injury ◽  
Compensation Mechanisms ◽  
Kidney Organoids

ABSTRACTPodocyte injury is central to many forms of kidney disease, but transcriptional signatures reflecting podocyte injury and compensation mechanisms are challenging to analyze in vivo. Human kidney organoids derived from pluripotent stem cells (PSCs), a new model for disease and regeneration, present an opportunity to explore the transcriptional plasticity of podocytes. Here, transcriptional profiling of over 12,000 single cells from human PSC-derived kidney organoid cultures was used to identify robust and reproducible cell-lineage gene expression signatures shared with developing human kidneys based on trajectory analysis. Surprisingly, the gene expression signature characteristic of developing glomerular epithelial cells was also observed in glomerular tissue from a kidney disease cohort. This signature correlated with proteinuria and inverse eGFR, and was confirmed in an independent podocytopathy cohort. Three genes in particular were further identified as critical components of the glomerular disease signature. We conclude that cells in human PSC-derived kidney organoids reliably recapitulate the developmental transcriptional program of podocytes and other cell lineages in the human kidney, and that the early transcriptional profile seen in developing podocytes is reactivated in glomerular disease. Our findings demonstrate an innovative approach to identifying novel molecular programs involved in the pathogenesis of glomerulopathies.

scholarly journals Maturation of Nephrons by Implanting hPSC-Derived Kidney Progenitors Under Kidney Capsules of Unilaterally Nephrectomized Mice

2021 ◽  
Author(s):  
Xin Yu ◽  
Shan Jiang ◽  
Kailin Li ◽  
Xianzhen Yang ◽  
Zhihe Xu ◽  
...  
Keyword(s):  
Disease Modeling ◽  
Vascular System ◽  
Human Kidney ◽  
Target Cells ◽  
Mice Model ◽  
Immunodeficient Mice ◽  
Filtration Barrier ◽  
Kidney Organoids

Abstract Background Human pluripotent stem cell (hPSCs)-derived kidney organoids may contribute to disease modeling and generation of kidney replacement tissues. However, realization of such applications requires the induction of hPSCs into functional mature organoids. One of the key questions for this process is whether a specific vascular system exists for nephrogenesis. Our previous study showed that implantation of hPSC-derived organoids below the kidney capsules of unilaterally nephrectomized immunodeficient mice for a short-term (2 weeks) resulted in the enlargement of organoids and production of vascular cells, although signs of maturation were lacking. Methods In this study, organoids are induced in vitro during 15 days and then sub-capsularly grafted into kidneys, we used the same unilaterally nephrectomized immunodeficient mice model to examine whether a medium -term (4 weeks) implantation could improve organoid maturation and vascularization, as evaluated by immunofluorescence and transmission electron microscopy(TEM). Results We demonstrate that after 2–4 weeks implantation, implanted renal organoids can form host-derived vascularization and mature in the absence of any exogenous vascular endothelial growth factor. Glomerular filtration barrier maturation was evidenced by glomerular basement membrane deposition, perforated glomerular endothelial cell development, as well as apical to basal podocyte polarization. A polarized monolayer epithelium and extensive brush border were also observed for tubular epithelial cells. Conclusions Our results indicate that the in vivo microenvironment is important for the maturation of human kidney organoids. Stromal expansion and a reduction of nephron structures were observed following longer-term (12 weeks) implantation,suggesting effects on off-target cells during the induction process. Accordingly, induction efficiency and transplantation models should be improved in the future.

scholarly journals Gene Therapy Approaches

2021 ◽  
Vol 1 (1) ◽  
pp. 52-56
Author(s):  
Hogir Saadi
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Nuclear Translocation ◽  
Viral Vector ◽  
Ex Vivo ◽  
Genetic Material ◽  
Target Tissue ◽  
Human Beings ◽  
Vector Systems

Gene therapy can be described broadly as the transfer of genetic material to control a disease or at least to enhance a patient's clinical status. The transformation of viruses into genetic shuttles is one of the core principles of gene therapy, which will introduce the gene of interest into the target tissue and cells. To do this, safe strategies have been invented, using many viral and non-viral vector delivery. Two major methods have emerged: modification in vivo and modification ex vivo. For gene therapeutic approaches which are focused on lifelong expression of the therapeutic gene, retrovirus, adenovirus, adeno-associated viruses are acceptable. Non-viral vectors are much less successful than viral vectors, but because of their low immune responses and their broad therapeutic DNA ability, they have advantages. The addition of viral functions such as receptor-mediated uptake and nuclear translocation of DNA may eventually lead to the development of an artificial virus in order to improve the role of non-viral vectors. For human use in genetic conditions, cancers and acquired illnesses, gene transfer techniques have been allowed. The ideal delivery vehicle has not been identified, although the accessible vector systems are capable of transporting genes in vivo into cells. Therefore, only with great caution can the present viral vectors be used in human beings and further progress in the production of vectors is required. Current progresses in our understanding of gene therapy approaches and their delivery technology, as well as the victors used to deliver therapeutic genes, are the primary goals of this review. For that reason, a literature search on PubMed and Google Scholar was carried out using different keywords.

scholarly journals Interleukin-1β Activates a MYC-Dependent Metabolic Switch in Kidney Stromal Cells Necessary for Progressive Tubulointerstitial Fibrosis

2018 ◽  
Vol 29 (6) ◽  
pp. 1690-1705 ◽  
Author(s):  
Dario R. Lemos ◽  
Michael McMurdo ◽  
Gamze Karaca ◽  
Julia Wilflingseder ◽  
Irina A. Leaf ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Target Genes ◽  
Kidney Injury ◽  
Human Kidney ◽  
Metabolic Switch ◽  
Metabolic Derangement ◽  
Sequestosome 1 ◽  
Kidney Organoids

Background Kidney injury is characterized by persisting inflammation and fibrosis, yet mechanisms by which inflammatory signals drive fibrogenesis remain poorly defined.Methods RNA sequencing of fibrotic kidneys from patients with CKD identified a metabolic gene signature comprising loss of mitochondrial and oxidative phosphorylation gene expression with a concomitant increase in regulators and enzymes of glycolysis under the control of PGC1α and MYC transcription factors, respectively. We modeled this metabolic switch in vivo, in experimental murine models of kidney injury, and in vitro in human kidney stromal cells (SCs) and human kidney organoids.Results In mice, MYC and the target genes thereof became activated in resident SCs early after kidney injury, suggesting that acute innate immune signals regulate this transcriptional switch. In vitro, stimulation of purified human kidney SCs and human kidney organoids with IL-1β recapitulated the molecular events observed in vivo, inducing functional metabolic derangement characterized by increased MYC-dependent glycolysis, the latter proving necessary to drive proliferation and matrix production. MYC interacted directly with sequestosome 1/p62, which is involved in proteasomal degradation, and modulation of p62 expression caused inverse effects on MYC expression. IL-1β stimulated autophagy flux, causing degradation of p62 and accumulation of MYC. Inhibition of the IL-1R signal transducer kinase IRAK4 in vivo or inhibition of MYC in vivo as well as in human kidney organoids in vitro abrogated fibrosis and reduced tubular injury.Conclusions Our findings define a connection between IL-1β and metabolic switch in fibrosis initiation and progression and highlight IL-1β and MYC as potential therapeutic targets in tubulointerstitial diseases.

The role of endoglin in kidney fibrosis

2014 ◽  
Vol 16 ◽  
Author(s):  
Jose M. Muñoz-Felix ◽  
Barbara Oujo ◽  
Jose M. Lopez-Novoa
Keyword(s):  
Transforming Growth Factor Beta ◽  
Renal Fibrosis ◽  
Transforming Growth Factor ◽  
Mesangial Cells ◽  
Cell Types ◽  
Experimental Models ◽  
Kidney Fibrosis ◽  
End Stage

Tubulointerstitial fibrosis and glomerulosclerosis, are a major feature of end stage chronic kidney disease (CKD), characterised by an excessive accumulation of extracellular matrix (ECM) proteins. Transforming growth factor beta-1 (TGF-β1) is a cytokine with an important role in many steps of renal fibrosis such as myofibroblast activation and proliferation, ECM protein synthesis and inflammatory cell infiltration. Endoglin is a TGF-β co-receptor that modulates TGF-β responses in different cell types. In numerous cells types, such as mesangial cells or myoblasts, endoglin regulates negatively TGF-β-induced ECM protein expression. However, recently it has been demonstrated that ‘in vivo’ endoglin promotes fibrotic responses. Furthermore, several studies have demonstrated an increase of endoglin expression in experimental models of renal fibrosis in the kidney and other tissues. Nevertheless, the role of endoglin in renal fibrosis development is unclear and a question arises: Does endoglin protect against renal fibrosis or promotes its development? The purpose of this review is to critically analyse the recent knowledge relating to endoglin and renal fibrosis. Knowledge of endoglin role in this pathology is necessary to consider endoglin as a possible therapeutic target against renal fibrosis.

scholarly journals A tissue-bioengineering strategy for modeling rare human kidney diseases in vivo

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
J. O. R. Hernandez ◽  
X. Wang ◽  
M. Vazquez-Segoviano ◽  
M. Lopez-Marfil ◽  
M. F. Sobral-Reyes ◽  
...  
Keyword(s):  
Kidney Diseases ◽  
Human Kidney ◽  
Cell Phenotype ◽  
Transcriptional Signature ◽  
Disease Mechanisms ◽  
Genetic Mechanisms ◽  
Experimental Approaches ◽  
Kidney Lesions ◽  
Kidney Organoids

AbstractThe lack of animal models for some human diseases precludes our understanding of disease mechanisms and our ability to test prospective therapies in vivo. Generation of kidney organoids from Tuberous Sclerosis Complex (TSC) patient-derived-hiPSCs allows us to recapitulate a rare kidney tumor called angiomyolipoma (AML). Organoids derived from TSC2−/− hiPSCs but not from isogenic TSC2+/− or TSC2+/+ hiPSCs share a common transcriptional signature and a myomelanocytic cell phenotype with kidney AMLs, and develop epithelial cysts, replicating two major TSC-associated kidney lesions driven by genetic mechanisms that cannot be consistently recapitulated with transgenic mice. Transplantation of multiple TSC2−/− renal organoids into the kidneys of immunodeficient rats allows us to model AML in vivo for the study of tumor mechanisms, and to test the efficacy of rapamycin-loaded nanoparticles as an approach to rapidly ablate AMLs. Collectively, our experimental approaches represent an innovative and scalable tissue-bioengineering strategy for modeling rare kidney disease in vivo.

scholarly journals Activation of MAIT cells plays a critical role in viral vector vaccine immunogenicity

2019 ◽  
Author(s):  
Nicholas M. Provine ◽  
Ali Amini ◽  
Lucy C. Garner ◽  
Christina Dold ◽  
Claire Hutchings ◽  
...  
Keyword(s):  
Cell Activation ◽  
Viral Vector ◽  
Critical Role ◽  
Vector Vaccine ◽  
Future Design ◽  
Mait Cells ◽  
Lethal Infection ◽  
Mait Cell

AbstractMucosal-associated invariant T (MAIT) cells can be activated by viruses through a cytokine-dependent mechanism, and thereby protect from lethal infection. Given this, we reasoned MAIT cells may have a critical role in the immunogenicity of replication-incompetent adenovirus vectors, which are novel and highly potent vaccine platforms. In vitro, ChAdOx1 (Chimpanzee Adenovirus Ox1) induced potent activation of MAIT cells. Activation required transduction of monocytes and plasmacytoid dendritic cells to produce IL-18 and IFN-α, respectively. IFN-α-induced monocyte-derived TNF-α was identified as a novel intermediate in this activation pathway, and activation required combinatorial signaling of all three cytokines. Furthermore, ChAdOx1-induced in vivo MAIT cell activation in both mice and human volunteers. Strikingly, MAIT cell activation was necessary in vivo for development of ChAdOx1-induced HCV-specific CD8 T cell responses. These findings define a novel role for MAIT cells in the immunogenicity of viral vector vaccines, with potential implications for future design.One sentence summaryRobust immunogenicity of candidate adenovirus vaccine vectors requires the activation of unconventional T cells.

MO010COLLAGEN TYPE VI PRODUCTION BY KIDNEY FIBROBLASTS IS DEPENDENT ON THE RIGHT STIMULI AND MAY SELF-PERPETUATE FIBROSIS

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Alexandra Louise Møller ◽  
Daniel G. K. Rasmussen ◽  
Morten A. Karsdal ◽  
Federica Genovese
Keyword(s):  
Cell Model ◽  
Human Kidney ◽  
Fold Increase ◽  
Type I ◽  
Kidney Fibrosis ◽  
Collagen Formation ◽  
Ecm Proteins ◽  
Time Point ◽  
Stimulation Of

Abstract Background and Aims Accumulation of extracellular matrix (ECM) proteins is a hallmark of kidney fibrosis, which can lead to altered tissue homeostasis, kidney failure, and ultimately death. Many different cell types are involved in this process, but fibroblasts are the main source of ECM proteins such as collagen type I (COL I), III (COL III), and VI (COL VI). Recently, it was suggested that a fragment of COL VI, released during collagen maturation, is a bioactive molecule (endotrophin; ETP) with signaling potential, indicating that collagens are not just passive structural proteins. In this study, we investigated the effect of different pro-fibrotic stimulants on COL VI production and the effect of ETP itself on human kidney fibroblasts in the scar-in-a-jar (SiaJ) cell model. Method Cells were seeded in 48-well plates at 30.000 cells/well and incubated for 24h in DMEM + 10% FBS for adherence. Cells were then starved by incubating them for further 24h in DMEM + 0.4% FBS. To induce fibrogenesis, fresh medium was added at day 0 with 225/150 mg/mL Ficoll 70/400 and 1% ascorbic acid, containing either 7-, 0.7-, or 0.07 nM PDGF-AA, 8-, 0.8-, or 0.08 nM PDGF-BB, 4-, 0.4-, or 0.04 nM PDGF-CC, 7-, 0.7-, or 0.07 nM PDGF-DD, 0.9-, or 0.09 nM CTGF, 0.02 nM TGF-β or 30 nM ETP. Medium was changed and collected on days 3, 6, 10, and 13. Biomarkers of COL I (PRO-C1), III (PRO-C3), and VI (PRO-C6) formation were assessed in the medium by enzyme-linked immunosorbent assays developed at Nordic Bioscience. Results The stimulation of kidney fibroblasts with PDGF-AA, -BB, -CC, and -DD caused an increase in PRO-C6 compared to the unstimulated cells at every time point (P<0.0001). The increase in formation peaked at day 10, and a dose-dependent increase in COL VI levels was observed with PDGF-DD treatment. Interestingly, CTGF treatment did not enhance the synthesis of COL VI at any time point, and TGF-β treatment suppressed PRO-C6 levels compared to the untreated cells (not significant). The stimulation with 30 nM ETP caused an increase in PRO-C1 (P<0.0001) and PRO-C3 (P<0.0001) compared to the unstimulated cells on days 6, 10, and 13. The increase in collagen formation peaked at day 10 for both markers, with a 7.28-fold increase for COL I and a 4.13-fold increase for COL III. Conclusion The production of COL VI, an important mediator of fibrosis and inflammation through its bioactive fragment endotrophin, shows a differential expression after stimulation of kidney fibroblasts with different pro-fibrotic growth factors. Interestingly, members of the PDGF family induced COL VI production, whereas CTGF and TGF-β did not. Moreover, we confirmed that ETP itself could stimulate kidney fibroblasts to produce more ECM proteins; hence COL VI may self-perpetuate fibrosis. This SiaJ model, combined with ECM formation biomarkers, could be used to elucidate the mechanisms behind acute and sustained matrix production profiles in vivo.

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