title: Kinetics of Label Retaining Cells in the Developing Rat Kidneys.
abstract: The kidney is a specialized low-regenerative organ with several different types of cellular lineages. The BrdU label-retaining cell (LRCs) approach has been used as part of a strategy to identify tissue-specific stem cells in the kidney; however, because the complementary base pairing in double-stranded DNA blocks the access of the anti-BrdU antibody to BrdU subunits, the stem cell marker expression in BrdU-labeled cells are often difficult to detect. In this study, we introduced a new cell labeling and detection method in which BrdU was replaced with 5-ethynyl-2-deoxyuridine (EdU) and examined the time-dependent dynamic changes of EdU-labeled cells and potential stem/progenitor markers in the development of kidney.
  
  
  
  Newborn rats were intraperitoneally injected with EdU, and their kidneys were harvested respectively at different time points at 1 day, 3 days, 1 week, 2 weeks, and 6 weeks post-injection. The kidney tissues were processed for EdU and cellular markers by immunofluorescence staining.
  
  
  
  At the early stage, LRCs labeled by EdU were 2176.0 ± 355.6 cells at day one in each renal tissue section, but dropped to 168 ± 48.4 cells by week 6. As time increased, the numbers of LRCs were differentially expressed in the renal cortex and papilla. At the postnatal day one, nearly twice as many cells in the cortex were EdU-labeled as compared to the papilla (28.6 ± 3.6% vs. 15.6 ± 3.4%, P<0.05), while there were more LRCs within the renal papilla since the postnatal week one, and at the postnatal week 6, one third as many cells in the cortex were EdU-labeled as compared to the papilla (2.5 ± 0.1% vs. 7.7 ± 2.7%, P<0.05). The long-term LRCs at 6-week time point were associated exclusively with the glomeruli in the cortex and the renal tubules in the papilla. At 6 weeks, the EdU-labeled LRCs combined with expression of CD34, RECA-1, Nestin, and Synaptopodin were discretely but widely distributed within the glomeruli; Stro-1 around the glomeruli; and α-smooth muscle actin (SMA) in arteries. Conversely, co-expression of CD34, RECA-1, and Nestin with the long term EdU-labeled LRCs was significantly lower in renal tubules (P<0.01), while Stro-1 and Synaptopodin were not detected.
  
  
  
  Our data found that at 6-week time point, EdU-labeled LRCs existing in the glomeruli expressed undifferentiated podocyte and endothelial markers at high rates, while those in the renal tubules expressed Nestin and vascular markers at low rates. To understand the characterization and localization of these EdU-LRCs, further studies will be needed to test cell lineage tracing, clonogenicity and differentiation potency, and the contributions to the regeneration of the kidney in response to renal injury/repair.
authors.affiliation: Department of Urology, Beijing ChaoYang Hospital, Capital Medical University, 8 Gongtinanlu, Beijing, 100020, China.|Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America.
authors.forename: Jianwen
authors.initials: J
authors.lastname: Wang
authors.affiliation: Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America.
authors.forename: Guiting
authors.initials: G
authors.lastname: Lin
authors.affiliation: Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America.
authors.forename: Amjad
authors.initials: A
authors.lastname: Alwaal
authors.affiliation: Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America.
authors.forename: Guifang
authors.initials: G
authors.lastname: Wang
authors.affiliation: Department of Urology, Beijing ChaoYang Hospital, Capital Medical University, 8 Gongtinanlu, Beijing, 100020, China.
authors.forename: Xingyuan
authors.initials: X
authors.lastname: Jia
authors.affiliation: Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America.
authors.forename: Lia
authors.initials: L
authors.lastname: Banie
authors.affiliation: Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Villalta
authors.affiliation: Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America.
authors.forename: Ching-Shwun
authors.initials: CS
authors.lastname: Lin
authors.affiliation: Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America.
authors.forename: Tom F
authors.initials: TF
authors.lastname: Lue
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism*; D001973:Bromodeoxyuridine / Q000378:metabolism*; D002454:Cell Differentiation; D019070:Cell Lineage; D049109:Cell Proliferation; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007091:Image Processing, Computer-Assisted; D007668:Kidney / Q000166:cytology* / Q000378:metabolism; D007700:Kinetics; D008297:Male; D038081:Organogenesis / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D015415:Biomarkers; D001973:Bromodeoxyuridine; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine