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- Borestrom, C., et al.
(författare)
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A CRISP(e)R view on kidney organoids allows generation of an induced pluripotent stem cell-derived kidney model for drug discovery
- 2018
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Ingår i: Kidney International. - : Elsevier BV. - 0085-2538 .- 1523-1755. ; 94:6, s. 1099-1110
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Tidskriftsartikel (refereegranskat)abstract
- Development of physiologically relevant cellular models with strong translatability to human pathophysiology is critical for identification and validation of novel therapeutic targets. Herein we describe a detailed protocol for generation of an advanced 3-dimensional kidney cellular model using induced pluripotent stem cells, where differentiation and maturation of kidney progenitors and podocytes can be monitored in live cells due to CRISPR/Cas9-mediated fluorescent tagging of kidney lineage markers (SIX2 and NPHS1). Utilizing these cell lines, we have refined the previously published procedures to generate a new, higher throughput protocol suitable for drug discovery. Using paraffin-embedded sectioning and whole-mount immunostaining, we demonstrated that organoids grown in suspension culture express key markers of kidney biology (WT1, ECAD, LTL, nephrin) and vasculature (CD31) within renal cortical structures with microvilli, tight junctions and podocyte foot processes visualized by electron microscopy. Additionally, the organoids resemble the adult kidney transcriptomics profile, thereby strengthening the translatability of our in vitro model. Thus, development of human nephron-like structures in vitro fills a major gap in our ability to assess the effect of potential treatment on key kidney structures, opening up a wide range of possibilities to improve clinical translation.
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- Zambrano, S, et al.
(författare)
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FYVE domain-containing protein ZFYVE28 regulates EGFR-signaling in podocytes but is not critical for the function of filtration barrier in mice
- 2018
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8:1, s. 4712-
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Tidskriftsartikel (refereegranskat)abstract
- The kidney ultrafiltration barrier is formed of endothelial cells, the glomerular basement membrane and podocytes. Podocytes have a central role in normal physiology and disease pathogenesis of the glomerulus. Signaling through epidermal growth factor receptor (EGFR) in podocytes mediates development of many glomerular disease processes. In this work, we have identified zinc finger FYVE-type containing 28 (ZFYVE28) as a novel highly podocyte-enriched gene. We localize ZFYVE28 in podocyte foot processes in adult kidney. During glomerulogenesis, Zfyve28 is first expressed at the early capillary loop glomerulus. In cultured podocytes, we show that overexpression of ZFYVE28 promotes EGF-signaling, possibly by up-regulating EGFR expression and by modulating its localization. To study the role of ZFYVE28 in vivo, we generated both conventional and podocyte-specific knockout mouse lines. Kidneys developed normally in ZFYVE28-deficient mice. In adult mice, the absence of ZFYVE28 did not affect the maintenance of the filtration barrier. Moreover, ZFYVE28-deficiency did not affect the outcome of glomerular damage induced by injection of nephrotoxic serum. Taken together, we have identified Zfyve28 as a new molecular component of podocyte foot processes and show that it mediates EGF-signaling in podocytes. However, ZFYVE28 is not essential for the development or maintenance of the glomerulus filtration barrier.
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- Zambrano, S., et al.
(författare)
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GPRC5b Modulates Inflammatory Response in Glomerular Diseases via NF-kappa B Pathway
- 2019
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Ingår i: Journal of the American Society of Nephrology. - : Ovid Technologies (Wolters Kluwer Health). - 1046-6673 .- 1533-3450. ; 30:9, s. 1573-1586
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Tidskriftsartikel (refereegranskat)abstract
- Background Inflammatory processes play an important role in the pathogenesis of glomerulopathies. Finding novel ways to suppress glomerular inflammation may offer a new way to stop disease progression. However, the molecular mechanisms that initiate and drive inflammation in the glomerulus are still poorly understood. Methods We performed large-scale gene expression profiling of glomerulus-associated G protein-coupled receptors (GPCRs) to identify new potential therapeutic targets for glomerulopathies. The expression of Gprc5b in disease was analyzed using quantitative PCR and immunofluorescence, and by analyzing published microarray data sets. In vivo studies were carried out in a podocyte-specific Gprc5b knockout mouse line. Mechanistic studies were performed in cultured human podocytes. Results We identified an orphan GPCR, Gprc5b, as a novel gene highly enriched in podocytes that was significantly upregulated in common human glomerulopathies, including diabetic nephropathy, IgA nephropathy, and lupus nephritis. Similar upregulation of Gprc5b was detected in LPS-induced nephropathy in mice. Studies in podocyte-specific Gprc5b knockout mice showed that Gprc5b was not essential for normal development of the glomerular filtration barrier. However, knockout mice were partially protected from LPS-induced proteinuria and recruitment of inflammatory cells. Mechanistically, RNA sequencing in Gprc5b knockouts mice and experiments in cultured human podocytes showed that Gpr5cb regulated inflammatory response in podocytes via NF-kappa B signaling. Conclusions GPRC5b is a novel podocyte-specific receptor that regulates inflammatory response in the glomerulus by modulating the NF-kappa B signaling pathway. Upregulation of Gprc5b in human glomerulopathies suggests that it may play a role in their pathogenesis.
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