Inhibiting angiotensin-converting enzyme promotes renal repair by limiting progenitor cell proliferation and restoring the glomerular architecture

Abstract

We previously reported that angiotensin-converting enzyme inhibitor (ACEi) renoprotection in Munich Wistar Frömter (MWF) rats, which develop progressive glomerular injury, was associated with podocyte repopulation and preservation of glomerular architecture. Here, we studied the time course of the lesions, their cellular components, and the effect of ACEi. Early glomerular lesions were synechiae, followed by extracapillary crescents and glomerulosclerosis. The majority of cells forming crescents were claudin1(+) parietal epithelial cells and, to a lesser extent, WT1(+) podocytes, both in active proliferation. In crescents, cells expressing the metanephric mesenchyme marker NCAM were also found. Three distinct populations of parietal epithelial cells were identified in the rat Bowman's capsule: NCAM(+)WT1(-) cells, also expressing progenitor cell marker CD24, and NCAM(+)WT1(+) and NCAM(-)WT1(+) cells, the latter population representing parietal podocytes. After exposure to inductive medium, cultured parietal epithelial cells that were obtained by capsulated glomeruli generated podocytes, documenting their progenitor nature. Mitotic activity of cultured renal progenitors was induced by angiotensin II through the down-regulation of cell cycle inhibitor C/EBPδ expression. Treatment with ACEi reduced number and extension of crescents and glomerulosclerosis in MWF rats. Renoprotection was accomplished through the limitation of NCAM(+) progenitor proliferation via the modulation of C/EBPδ. Thus, chaotic migration and proliferation of the Bowman's capsule progenitor cells pave the way to crescent formation and subsequent sclerosis. ACEi, by moderating progenitor cell activation, restores glomerular architecture and prevents renal disease progression.

Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Populations of cells lining the Bowman's capsule of normal adult rat kidney. A: Representative picture of immunofluorescence staining of claudin1 (green) and WT1 (red) in the Bowman's capsule of Wistar rats used as controls. High-magnification insets show claudin1+WT1− cell (upper insets) and claudin1+WT1+ cell (lower insets). B: Representative pattern of immunogold labeling for nephrin in the Bowman's capsule of Wistar rats. Left panel shows the ultrastructure of parietal cells along the Bowman's capsule. High-magnification inset (right panel) shows that gold particles are localized along the foot processes of parietal cells. Original magnification: ×4400 (left panel); ×71,000 (right panel). C: Double immunolabeling for claudin1 (red) and NCAM (green) shows in most of the cells the colocalization of the two markers. D: Double immunofluorescence staining for NCAM (red) and CD24 (green) revealed that in the Bowman's capsule, the two antigens are expressed by the same cells (arrows). E: Representative photomicrograph showing three distinct cell populations in the Bowman's capsule of Wistar rats, identified by staining of NCAM (red) and WT1 (green): NCAM+WT1− progenitor cells (upper inset), NCAM+WT1+ transitional cells (middle inset), and NCAM−WT1+ parietal podocytes (lower inset). Original magnification, ×630. F: Schematic representation of the cell populations identified in the Bowman's capsule of Wistar rats. All of the cells were positive for PEC marker claudin1. A gradient of expression of NCAM and WT1 defined three populations of claudin1+ PECs with different degrees of differentiation. Seventy-six percent expressed only the progenitor marker NCAM, 12% expressed NCAM and the podocyte marker WT1, and 12% both progenitor and podocyte markers.

Figure 2

Time course of proteinuria and histological glomerular lesions in Wistar and MWF rats. A: Proteinuria levels progressively increased with age in MWF rats. *P < 0.01 versus Wistar rats and 10-week-old (10w) MWF; **P < 0.01 versus Wistar rats and 10w to 25w MWF. B: Age-related changes of the index of synechiae (striped bars) and crescents (dark gray bars) quantified in Wistar (n = 20) and MWF (n = 50) rats. ***P < 0.05 versus Wistar rats and 10w MWF; †P < 0.01 versus Wistar rats and 10w to 25w to 40w MWF. C: Renal section of 60-week-old Wistar rats was characterized by few synechiae (arrow). In MWF rats, synechiae were present from 10 weeks of age, and more severe crescentic lesions (arrowheads) developed from 25 weeks of age (D) and progressively increased with time (E and F). G: Synechiae in Wistar rats were not associated with accumulation of type III collagen (arrow). H: Also, early crescents, which were found in 25-week-old MWF rats, were negative for type III collagen staining (arrowhead). In 40-week-old MWF rats, crescents were characterized by scanty accumulation of type III collagen (I), which progressively increased at 60 weeks of age (arrowheads, J). Original magnification, ×400.

Figure 3

Phenotype of cell populations involved in crescentic lesions. A: Representative pictures of immunofluorescence staining for claudin1 (green), WT1 (white), Thy1.1 (red), and ED1 (red) in glomeruli of 60-week-old MWF rats. High-magnification insets show cells positive for the different markers. Dotted lines define the area of crescents in which positive cells were quantified. Nuclei are stained with DAPI (blue). Original magnification, ×630. B: Quantitative assessment of cells positive for each marker, revealed that the most represented cell population in crescents consisted of claudin1+ cells, and to a lesser extent of WT1+ cells. Thy1.1+ and ED1+ cells represented minor cell populations within the lesions.

Figure 4

ACE inhibitor reduces the number and extension of crescents and limits progenitor cell activation. A: A significant decrease in proteinuria levels and index of crescents was observed in 60-week-old (60w) MWF rats receiving ACEi for 10 weeks as compared with animals given saline. *P < 0.01 versus 60w MWF. B: The percentage of proliferating claudin1+WT1− cells (white bars), claudin1+WT1+ cells (gray bars), and claudin1−WT1+ cells (black bars) was lower in crescents of ACEi-treated MWF rats with respect to animals given saline. Cell proliferation was assessed by quantification of the number of BrdU+ cells in pulse-chased MWF rats. **P < 0.05 versus 60w MWF; *P < 0.01 versus 60w MWF. C: The expression of NCAM, which identifies renal progenitor cells, was evaluated in crescentic lesions of 60-week-old MWF rats. Note that NCAM+ cells (red) were localized in Bowman's capsule and capillary tuft, as well as in crescents. D: ACEi effectively reduced the presence of progenitor cells in crescents and restored the distribution of NCAM+ cells in the Bowman's capsule to the pattern observed in controls (shown in Figure 1). Nuclei are stained with DAPI (blue). Original magnification, ×630.

Figure 5

ACE inhibitor reduces proliferation of Bowman's capsule cells and regulates the expression of the cell cycle inhibitor C/EBPδ. A: In the Bowman's capsule of 60-week-old MWF rats given saline, the percentage of claudin1+WT1− cells (white bars) was markedly enhanced, and claudin1+WT1+ cells (striped bars) decreased with respect to Wistar rats. ACEi restored the percentage of both cell populations to control levels. B: Cell proliferation of claudin1+ cells along the Bowman's capsule was enhanced in 60-week-old MWF rats given saline as compared with controls. ACEi treatment significantly limited the number of proliferating cells within the Bowman's capsule (claudin1+BrdU+). *P < 0.05 versus Wistar; **P < 0.01 versus 60w MWF + saline. C: Representative picture of immunofluorescence staining for claudin1 (green) and BrdU (red) in the Bowman's capsule of 60-week-old MWF rats. Inset shows a cell expressing both markers. Nuclei are stained with DAPI (blue). Original magnification, ×630. D: The expression of C/EBPδ in the Bowman's capsule, evaluated by semiquantitative score, decreased at 60 weeks of age in MWF rats receiving saline as compared with Wistar rats, and was normalized by ACEi treatment. *P < 0.05 versus Wistar; ***P < 0.05 versus 60w MWF + saline. Representative photomicrographs of C/EBPδ expression in Wistar (E) and MWF rats given saline (F) or treated with ACEi (G) are shown. Note that in ACEi-treated rats, as in Wistar rats, most of the cells were positive for C/EBPδ (brown signal at high magnification); in MWF rats, the majority of the cells did not express the marker. Nuclei (blue) were counterstained with hematoxylin. Original magnification, ×400.

Figure 6

Characterization, differentiation, and proliferation of cultured PECs. A: Representative images of PECs, obtained from initial outgrowth of capsulated control rat glomeruli, that constitutively expressed claudin1 (red), NCAM (green), or CD24 (green). Cells were co-stained with DAPI (blue). B and C: Immunofluorescence micrographs of immunoisolated claudin1+ PECs co-stained with synaptopodin (SYN, green) and DAPI (blue). No signal for SYN (B) was observed in control cells. Cells treated with VRAD medium for 7 days expressed the podocyte marker SYN (C). D: Quantification of SYN+ cells: data are expressed as percentage of fluorescent cells per total DAPI+ cells in each HPF. *P < 0.001 versus control. E and F: PECs show a constitutive nuclear expression of the transcription factor C/EBPδ (green; E), that was markedly reduced after 6-hour incubation with angiotensin II (Ang II) (F). G and H: Representative images of H3p (green) and DAPI (blue) in immunoisolated claudin1+ PECs untreated (G) or treated (H) with Ang II for 24 hours. Nuclear H3p expression was markedly increased in cells exposed to Ang II (H) with respect to control cells (G). I: Quantification of mitotic cells: data are expressed as percentage of mitotic cells per total DAPI-positive cells in each HPF. *P < 0.001 versus control. All images were obtained at original magnification, ×630.