Cells of Renin Lineage Are Progenitors of Podocytes and Parietal Epithelial Cells in Experimental Glomerular Disease
2013; Elsevier BV; Volume: 183; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2013.04.024
ISSN1525-2191
AutoresJeffrey W. Pippin, Matthew A. Sparks, Sean T. Glenn, Sandra Buitrago, Thomas M. Coffman, Jeremy S. Duffield, Kenneth W. Gross, Stuart J. Shankland,
Tópico(s)Chronic Kidney Disease and Diabetes
ResumoGlomerular injury leads to podocyte loss, a process directly underlying progressive glomerular scarring and decline of kidney function. The inherent repair process is limited by the inability of podocytes to regenerate. Cells of renin lineage residing alongside glomerular capillaries are reported to have progenitor capacity. We investigated whether cells of renin lineage can repopulate the glomerulus after podocyte injury and serve as glomerular epithelial cell progenitors. Kidney cells expressing renin were genetically fate-mapped in adult Ren1cCreER×Rs-tdTomato-R, Ren1cCre×Rs-ZsGreen-R, and Ren1dCre×Z/EG reporter mice. Podocyte depletion was induced in all three cell-specific reporter mice by cytotoxic anti-podocyte antibodies. After a decrease in podocyte number, a significant increase in the number of labeled cells of renin lineage was observed in glomeruli in a focal distribution along Bowman's capsule, within the glomerular tuft, or in both locations. A subset of cells lining Bowman's capsule activated expression of the glomerular parietal epithelial cell markers paired box protein PAX2 and claudin-1. A subset of labeled cells within the glomerular tuft expressed the podocyte markers Wilms tumor protein 1, nephrin, podocin, and synaptopodin. Neither renin mRNA nor renin protein was detected de novo in diseased glomeruli. These findings provide initial evidence that cells of renin lineage may enhance glomerular regeneration by serving as progenitors for glomerular epithelial cells in glomerular disease characterized by podocyte depletion. Glomerular injury leads to podocyte loss, a process directly underlying progressive glomerular scarring and decline of kidney function. The inherent repair process is limited by the inability of podocytes to regenerate. Cells of renin lineage residing alongside glomerular capillaries are reported to have progenitor capacity. We investigated whether cells of renin lineage can repopulate the glomerulus after podocyte injury and serve as glomerular epithelial cell progenitors. Kidney cells expressing renin were genetically fate-mapped in adult Ren1cCreER×Rs-tdTomato-R, Ren1cCre×Rs-ZsGreen-R, and Ren1dCre×Z/EG reporter mice. Podocyte depletion was induced in all three cell-specific reporter mice by cytotoxic anti-podocyte antibodies. After a decrease in podocyte number, a significant increase in the number of labeled cells of renin lineage was observed in glomeruli in a focal distribution along Bowman's capsule, within the glomerular tuft, or in both locations. A subset of cells lining Bowman's capsule activated expression of the glomerular parietal epithelial cell markers paired box protein PAX2 and claudin-1. A subset of labeled cells within the glomerular tuft expressed the podocyte markers Wilms tumor protein 1, nephrin, podocin, and synaptopodin. Neither renin mRNA nor renin protein was detected de novo in diseased glomeruli. These findings provide initial evidence that cells of renin lineage may enhance glomerular regeneration by serving as progenitors for glomerular epithelial cells in glomerular disease characterized by podocyte depletion. Glomerular diseases are the leading cause of progressive chronic and end-stage kidney disease.1Foley R.N. Collins A.J. End-stage renal disease in the United States: an update from the United States Renal Data System.J Am Soc Nephrol. 2007; 18: 2644-2648Crossref PubMed Scopus (369) Google Scholar Recent studies have demonstrated important roles for both podocytes and parietal epithelial cells (PECs) in these diseases. Diabetic nephropathy,2Pagtalunan M.E. Miller P.L. Jumping-Eagle S. Nelson R.G. Myers B.D. Rennke H.G. Coplon N.S. Sun L. Meyer T.W. 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Several seminal studies have shown that the neighboring glomerular PECs might serve this role.24Appel D. Kershaw D.B. Smeets B. Yuan G. Fuss A. Frye B. Elger M. Kriz W. Floege J. Moeller M.J. Recruitment of podocytes from glomerular parietal epithelial cells.J Am Soc Nephrol. 2009; 20: 333-343Crossref PubMed Scopus (394) Google Scholar, 25Swetha G. Chandra V. Phadnis S. Bhonde R. Glomerular parietal epithelial cells of adult murine kidney undergo EMT to generate cells with traits of renal progenitors.J Cell Mol Med. 2011; 15: 396-413Crossref PubMed Scopus (46) Google Scholar, 26Poulsom R. Little M.H. Parietal epithelial cells regenerate podocytes.J Am Soc Nephrol. 2009; 20: 231-233Crossref PubMed Scopus (15) Google Scholar, 27Sagrinati C. Netti G.S. Mazzinghi B. Lazzeri E. Liotta F. Frosali F. Ronconi E. Meini C. Gacci M. Squecco R. Carini M. Gesualdo L. Francini F. Maggi E. Annunziato F. Lasagni L. Serio M. Romagnani S. Romagnani P. 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A new role of the juxtaglomerular apparatus.J Clin Invest. 1997; 100: 786-794Crossref PubMed Scopus (122) Google Scholar The purpose of these studies was to apply genetic cell fate-mapping strategies in four transgenic gene–targeted mice that report for cells of renin lineage to test the hypothesis that these cells serve as progenitor cells for podocytes and PECs during experimental glomerular disease characterized by a decrease in podocyte number. Three newly generated renin-reporter mouse strains and one existing reporter mouse strain were used. Four different reporter mouse strains were used to genetically fate-map cells of renin lineage, three of which were newly generated. The newly generated Ren1cCreER×Rs-tdTomato-R mouse labels the Ren1c gene with tomato red protein only after the administration of tamoxifen (Sigma-Aldrich, St. Louis, MO). Because renin expression might be switched on later in life, thus confounding the data from the constitutive reporter mice, we introduced a Cre recombinase fused to the human estrogen receptor (ER) ligand-binding domain40Feil R. Brocard J. Mascrez B. LeMeur M. Metzger D. Chambon P. Ligand-activated site-specific recombination in mice.Proc Natl Acad Sci USA. 1996; 93: 10887-10890Crossref PubMed Scopus (694) Google Scholar into exon 1 of the Ren1c gene residing within a 240-kb bacterial artificial chromosome (BAC), here represented as RenCreER, using previously described methods41Sparwasser T. Gong S. Li J.Y. Eberl G. General method for the modification of different BAC types and the rapid generation of BAC transgenic mice.Genesis. 2004; 38: 39-50Crossref PubMed Scopus (57) Google Scholar, 42Glenn S.T. Jones C.A. Pan L. Gross K.W. In vivo analysis of key elements within the renin regulatory region.Physiol Genomics. 2008; 35: 243-253Crossref PubMed Scopus (22) Google Scholar (Supplemental Figure S1). The Ren1cCreER transgenic line, when crossed to the reporter Gt(ROSA)Sortm9(CAG-tdTomato)Hze/J,43Madisen L. Zwingman T.A. Sunkin S.M. Oh S.W. Zariwala H.A. Gu H. Ng L.L. Palmiter R.D. Hawrylycz M.J. Jones A.R. Lein E.S. Zeng H. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.Nat Neurosci. 2010; 13: 133-140Crossref PubMed Scopus (4169) Google Scholar allows for the permanent tagging of cells of renin lineage with tomato red protein, which is detected by red fluorescent protein (RFP), within temporal windows defined by treatment with tamoxifen (Supplemental Figure S2A). Accordingly, 5-week-old bigenic mice, weighing 16 to 20 g, were given either 100 mg/kg tamoxifen or vehicle by intraperitoneal injection on alternate days for 6 days. The newly generated Ren1cCre×Rs-ZsGreen-R reporter mouse labels the Ren1c gene with ZsGreen. To tag cells of renin lineage indefinitely, even after renin is no longer being transcribed, a Cre-recombinase cassette40Feil R. Brocard J. Mascrez B. LeMeur M. Metzger D. Chambon P. Ligand-activated site-specific recombination in mice.Proc Natl Acad Sci USA. 1996; 93: 10887-10890Crossref PubMed Scopus (694) Google Scholar was cloned into the Ren1c BAC using homologous recombination, as described previously42Glenn S.T. Jones C.A. Pan L. Gross K.W. In vivo analysis of key elements within the renin regulatory region.Physiol Genomics. 2008; 35: 243-253Crossref PubMed Scopus (22) Google Scholar, 43Madisen L. Zwingman T.A. Sunkin S.M. Oh S.W. Zariwala H.A. Gu H. Ng L.L. Palmiter R.D. Hawrylycz M.J. Jones A.R. Lein E.S. Zeng H. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.Nat Neurosci. 2010; 13: 133-140Crossref PubMed Scopus (4169) Google Scholar (Supplemental Figure S1). The renin regulatory region can then control Cre recombinase, which recognizes and excises loxP sites in DNA. When the Ren1cCre transgenic line is crossed with the commercially available B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J reporter mouse,43Madisen L. Zwingman T.A. Sunkin S.M. Oh S.W. Zariwala H.A. Gu H. Ng L.L. Palmiter R.D. Hawrylycz M.J. Jones A.R. Lein E.S. Zeng H. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.Nat Neurosci. 2010; 13: 133-140Crossref PubMed Scopus (4169) Google Scholar here represented as ZsGreen, a floxed stop cassette is excised, allowing for constitutive ZsGreen expression driven by a CAG promoter. Validation of the fidelity of this newly generated Ren1cCre×Rs-ZsGreen-R transgenic mouse in reporting cells of renin lineage was confirmed by visualizing adult kidney tissue (Supplemental Figure S2B). Ren1cCre×Rs-ZsGreen-R mice devoid of Cre were used as negative controls for labeling. The existing Ren1dCre×Z/EG reporter mouse labels the Ren1d gene with GFP, as previously reported.34Sequeira López M.L.S. Pentz E.S. Nomasa T. Smithies O. Gomez R.A. Renin cells are precursors for multiple cell types that switch to the renin phenotype when homeostasis is threatened.Dev Cell. 2004; 6: 719-728Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar The utility of this knock-in mouse is that all cells of renin lineage and their descendants permanently express GFP, even if renin expression is subsequently decreased or absent.44Novak A. Guo C. Yang W. Nagy A. Lobe C.G. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision.Genesis. 2000; 28: 147-155Crossref PubMed Scopus (730) Google Scholar Mice with Cre recombinase under control of the renin locus34Sequeira López M.L.S. Pentz E.S. Nomasa T. Smithies O. Gomez R.A. Renin cells are precursors for multiple cell types that switch to the renin phenotype when homeostasis is threatened.Dev Cell. 2004; 6: 719-728Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar were crossed with Z/EG reporter mice.44Novak A. Guo C. Yang W. Nagy A. Lobe C.G. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision.Genesis. 2000; 28: 147-155Crossref PubMed Scopus (730) Google Scholar Z/EG reporter mice constitutively express lacZ under the control of the CMV enhancer/chicken actin promoter. When crossed with a Cre recombinase-expressing strain, lacZ expression is replaced with enhanced GFP expression in tissues expressing Cre. The newly generated RenGFP reporter mouse serves to report renin promoter activity by the presence of a GFP reporter; that is, when renin is transcribed, GFP is expressed. Thus, to more comprehensively ensure control of reporter expression, homologous recombination was used to introduce a GFP cassette into exon one of the Ren1c gene residing within a 240-kb BAC to generate a construct that has GFP expression controlled from within the entire natural genomic sequence context for renin (Supplemental Figure S1). The insertion of GFP into the renin gene, which is centrally located within the BAC, allows large amounts of 5′- and 3′-flanking sequence to act on reporter expression. This is done in an effort to gain a more faithful representation of endogenous renin gene expression, as well as to insulate transgene expression from influences of surrounding sequence at the insertional site. The integration of GFP into the renin containing BAC to generate the RenGFP transgenic line has been described previously.42Glenn S.T. Jones C.A. Pan L. Gross K.W. In vivo analysis of key elements within the renin regulatory region.Physiol Genomics. 2008; 35: 243-253Crossref PubMed Scopus (22) Google Scholar The goal of these studies was to test the hypothesis that cells of renin lineage are progenitors for glomerular epithelial cells in glomerular disease characterized by an abrupt decline in podocyte number. Accordingly, an inducible model of experimental glomerular disease resembling classic clinical FSGS was induced with a cytotoxic antibody in all four strains of mice. We have previously reported that this experimental FSGS model induces an abrupt decline in podocyte number, which is associated clinically with the onset of proteinuria and histologically with focal and segmental glomerulosclerosis.30Ohse T. Vaughan M.R. Kopp J.B. Krofft R.D. Marshall C.B. Chang A.M. Hudkins K.L. Alpers C.E. Pippin J.W. Shankland S.J. De novo expression of podocyte proteins in parietal epithelial cells during experimental glomerular disease.Am J Physiol Renal Physiol. 2010; 298: F702-F711Crossref PubMed Scopus (91) Google Scholar, 45Zhang J. Pippin J.W. Vaughan M.R. Krofft R.D. Taniguchi Y. Romagnani P. Nelson P.J. Liu Z.H. Shankland S.J. Retinoids augment the expression of podocyte proteins by glomerular parietal epithelial cells in experimental glomerular disease.Nephron Exp Nephrol. 2012; 121: e23-e37Crossref PubMed Scopus (76) Google Scholar, 46Taniguchi Y. Pippin J.W. Hagmann H. Krofft R.D. Chang A.M. Zhang J. Terada Y. Brinkkoetter P. Shankland S.J. 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