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      Tracking the stochastic fate of cells of the renin lineage after podocyte depletion using multicolor reporters and intravital imaging

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          Abstract

          Podocyte depletion plays a major role in focal segmental glomerular sclerosis (FSGS). Because cells of the renin lineage (CoRL) serve as adult podocyte and parietal epithelial cell (PEC) progenitor candidates, we generated Ren1cCre/R26R-ConfettiTG/WT and Ren1dCre/R26R-ConfettiTG/WT mice to determine CoRL clonality during podocyte replacement. Four CoRL reporters (GFP, YFP, RFP, CFP) were restricted to cells in the juxtaglomerular compartment (JGC) at baseline. Following abrupt podocyte depletion in experimental FSGS, all four CoRL reporters were detected in a subset of glomeruli at day 28, where they co-expressed de novo four podocyte proteins (podocin, nephrin, WT-1 and p57) and two glomerular parietal epithelial cell (PEC) proteins (claudin-1, PAX8). To monitor the precise migration of a subset of CoRL over a 2w period following podocyte depletion, intravital multiphoton microscopy was used. Our findings demonstrate direct visual support for the migration of single CoRL from the JGC to the parietal Bowman’s capsule, early proximal tubule, mesangium and glomerular tuft. In summary, these results suggest that following podocyte depletion, multi-clonal CoRL migrate to the glomerulus and replace podocyte and PECs in experimental FSGS.

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          Most cited references33

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          Surgical implantation of an abdominal imaging window for intravital microscopy.

          High-resolution intravital microscopy through imaging windows has become an indispensable technique for the long-term visualization of dynamic processes in living animals. Easily accessible sites such as the skin, the breast and the skull can be imaged using various different imaging windows; however, long-term imaging studies on cellular processes in abdominal organs are more challenging. These processes include colonization of the liver by metastatic tumor cells and the development of an immune response in the spleen. We have recently developed an abdominal imaging window (AIW) that allows long-term imaging of the liver, the pancreas, the intestine, the kidney and the spleen. Here we describe the detailed protocol for the optimal surgical implantation of the AIW, which takes ∼1 h, and subsequent multiphoton imaging, which takes up to 1 month.
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            Genetic engineering of glomerular sclerosis in the mouse via control of onset and severity of podocyte-specific injury.

            This study aimed to generate a mouse model of acquired glomerular sclerosis. A model system that allows induction of podocyte injury in a manner in which onset and severity can be controlled was designed. A transgenic mouse strain (NEP25) that expresses human CD25 selectively in podocytes was first generated. Injection of anti-Tac (Fv)-PE38 (LMB2), an immunotoxin with specific binding to human CD25, induced progressive nonselective proteinuria, ascites, and edema in NEP25 mice. Podocytes showed foot process effacement, vacuolar degeneration, detachment and downregulation of synaptopodin, WT-1, nephrin, and podocalyxin. Mesangial cells showed matrix expansion, increased collagen, mesangiolysis, and, later, sclerosis. Parietal epithelial cells showed vacuolar degeneration and proliferation, whereas endothelial cells were swollen. The severity of the glomerular injury was LMB2 dose dependent. With 1.25 ng/g body wt or more, NEP25 mice developed progressive glomerular damage and died within 2 wk. With 0.625 ng/g body wt of LMB2, NEP25 mice survived >4 wk and developed focal segmental glomerular sclerosis. Thus, the study has established a mouse model of acquired progressive glomerular sclerosis in which onset and severity can be preprogrammed by experimental maneuvers.
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              Tracing the fate of limbal epithelial progenitor cells in the murine cornea.

              Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreER(T2)-Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K-14(+) progenitor cell dynamics within the corneal epithelium in live animals. Multicolored columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 µm/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolor genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound-healing, disease, and following transplantation.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                22 March 2017
                2017
                : 12
                : 3
                : e0173891
                Affiliations
                [1 ]Department of Medicine, Division of Nephrology, University of Washington, Seattle, WA, United States of America
                [2 ]Department of Physiology & Biophysics, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States of America
                [3 ]Department of Nephrology and Hypertension, Kawasaki Medical School, Kurashiki, Japan
                [4 ]Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY, United States of America
                [5 ]Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, Virginia, United States of America
                University of Houston, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: NK JWP SJS HK JPP.

                • Data curation: NK JWP SJS HK DGE JPP.

                • Formal analysis: NK HK.

                • Funding acquisition: SJS JPP.

                • Investigation: NK DGE HK MR KWG.

                • Methodology: NK DGE HK MR KWG.

                • Project administration: RAG MLSSL.

                • Resources: NK DGE HK MR KWG SJS JPP.

                • Software: NK HK.

                • Supervision: SJS JWP JPP KWG.

                • Validation: SJS JPP JWP.

                • Visualization: NK HK DGE SJS JPP JWP.

                • Writing – original draft: NK HK DGE SJS JPP JWP.

                • Writing – review & editing: KWG MLSSL RAG SJS JPP.

                ‡ NK and HK are co-first authors on this work

                Article
                PONE-D-16-45488
                10.1371/journal.pone.0173891
                5362207
                28329012
                03953c90-5d6f-4e86-a74d-bdb4cb93930d
                © 2017 Kaverina et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 23 November 2016
                : 28 February 2017
                Page count
                Figures: 12, Tables: 0, Pages: 23
                Funding
                Funded by: 5 R01 DK
                Award ID: 056799-10
                Award Recipient :
                Funded by: 5 R01 DK
                Award ID: 056799-12
                Award Recipient :
                Funded by: 1 R01 DK
                Award ID: 097598-01A1
                Award Recipient :
                Funded by: 2 R01 DK
                Award ID: 064324-11A1
                Award Recipient :
                Funded by: 5 R01 DK
                Award ID: 100944-02
                Award Recipient :
                Funded by: RO1HL
                Award ID: 048459-17
                Award Recipient :
                Funded by: 5P30CA (CCSG Core Grant)
                Award ID: 016056-39
                Funded by: R37 HL
                Award ID: 066242
                Award Recipient :
                Funded by: RO1 DK
                Award ID: 091330
                Award Recipient :
                Funded by: P50 DK
                Award ID: 096373
                Funded by: American Diabetes Association grant
                Award ID: 4-15-CKD-56
                Award Recipient :
                Funded by: American Heart Association grant
                Award ID: 15GRNT23040039
                Award Recipient :
                US National Institutes of Health grants 5 R01 DK 056799-10, 5 R01 DK 056799-12, 1 R01 DK097598-01A1, DK64324 and DK100944, American Diabetes Association grant 4-15-CKD-56, and American Heart Association grant 15GRNT23040039. RO1HL048459-17; 5P30CA016056-39(CCSG Core Grant) R37 HL066242, RO1 DK091330, P50 DK096373.
                Categories
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