Cells of renin lineage are adult pluripotent progenitors in experimental glomerular disease

Abstract

Modified vascular smooth muscle cells of the kidney afferent arterioles have recently been shown to serve as progenitors for glomerular epithelial cells in response to glomerular injury. To determine whether such cells of renin lineage (CoRL) serve as progenitors for other cells in kidney disease characterized by both glomerular and tubulointerstitial injury, permanent genetic cell fate mapping of adult CoRL using Ren1cCreER × Rs-tdTomato-R reporter mice was performed. TdTomato-labeled CoRL were almost completely restricted to the juxtaglomerular compartment in healthy kidneys. Following 2 wk of antibody-mediated focal segmental glomerulosclerosis (FSGS) or 16 wk of ⅚ nephrectomy-induced chronic kidney diseases, tdTomato-mapped CoRL were identified in both interstitial and glomerular compartments. In the interstitium, PDGFβ receptor (R)-expressing cells significantly increased, and a portion of these expressed tdTomato. This was accompanied by a decrease in native pericyte number, but an increase in the number of tdTomato cells that coexpressed the pericyte markers PDGFβ-R and NG2. These cells surrounded vessels and coexpressed the pericyte markers CD73 and CD146, but not the endothelial marker ERG. Within glomeruli of reporter mice with the ⅚ nephrectomy model, a subset of labeled CoRL migrated to the glomerular tuft and coexpressed podocin and synaptopodin. By contrast, labeled CoRL were not detected in glomerular or interstitial compartments following uninephrectomy. These observations indicate that in addition to supplying new adult podocytes to glomeruli, CoRL have the capacity to become new adult pericytes in the setting of interstitial disease. We conclude that CoRL have the potential to function as progenitors for multiple adult cell types in kidney disease.

Keywords: FSGS; PDGFβ-R; interstitium; pericyte; podocyte; regeneration; remnant kidney; tubulointerstitium.

Fig. 1.

Increase in glomerular and interstitial scarring and peritubular microvascular damage in Ren1cCreER × Rs-tdTomato-R mice with focal segmental glomerulosclerosis (FSGS). Representative images show periodic acid-Schiff staining at baseline (A) and in a FSGS kidney (B). Arrows indicate dilated tubules, and proteinacious material within dilated tubules is indicated by p. Quantification of the percentage of area staining for PDGFβ receptor (R) significantly increased in FSGS (C). Representative images show PDGFβ-R staining (green) at baseline (D), which is increased in FSGS (E). Quantification of the percentage of area staining for CD31 significantly decreased in FSGS (F). Representative images show CD31 staining (green) at baseline (G) and in FSGS (H).

Fig. 2.

Interstitial pericyte number decreased in Ren1cCreER × Rs-tdTomato-R mice with FSGS. Triple staining identified pericytes as negative for α-smooth muscle actin (SMA) and positive for PDGFβ-R and NG2 (αSMA⁻ PDGFβ-R⁺ NG2⁺). A–C: single-channel images at baseline for α-SMA (A, green), PDGFβ-R (B, blue) and NG2 (C, red). When merged (D), αSMA⁻ PDGFβ-R⁺ NG2⁺ cells have a purple/pink color and are indicated by the numbers shown. αSMA⁺ PDGFβ-R⁺ NG2⁺ contained white/green color and were not considered pericytes. E–G: single-channel images at day 14 FSGS for α-SMA (E), PDGFβ-R (F), and NG2 (G). When merged (H), αSMA⁻ PDGFβ-R⁺ NG2⁺ cells have a purple/pink color and are indicated by the numbers shown. αSMA⁺ PDGFβ-R⁺ NG2⁺ contained white/green color and were not considered pericytes. Quantification showed that the number of pericytes (αSMA⁻ PDGFβ-R⁺ NG2⁺) was significantly lower in FSGS (I).

Fig. 3.

Cells of renin lineage (CoRL) were detected in the interstitium in FSGS mice, and a subset coexpress the pericyte marker PDGFβ-R. A: Ren1cCreER × Rs-tdTomato-R mice given tamoxifen had tdTomato-labeled cells (arrows, red color) in the juxtaglomerulus surrounding an arteriole (a) but not in the glomerulus (g). B: quantification showed the number of red fluorescent protein (RFP) reporter-positive and PDGFβ-R- positive cells significantly increased in FSGS. Representative images of FSGS show PDGFβ-R (C, green), Tdtomato reporter (D, red), and merge (E, yellow). F–H: higher magnification images (×1,000) of the areas shown in the dashed insets in C–E (arrows indicate the cells of particular interest).

Fig. 4.

CoRL in the interstitium in FSGS were pericytes defined as cells coexpressing the markers PDGF-R, NG2, CD73, and CD146. A: the number of pericytes derived from CoRL, identified by triple staining as RFP⁺NG2⁺PDGFβ-R⁺, were significantly higher in FSGS. B: quantification of the percentage of total pericytes that were derived from CoRL (RFP⁺) was significantly higher in FSGS. C: representative image in FSGS of a pericyte derived from CoRL detected by triple staining showing staining for the reporter (RFP, red), PDGFβ-R (blue), and NG2 (blue) colocalizing to create a purple and white color (arrows). D: higher magnification of the area shown in the dashed inset in C. E: representative images of triple staining showing reporter (RFP, red), PDGFβ-R (green), and CD73 (blue) colocalizing (purple and white) in FSGS (arrow). F: higher magnification of the area shown in the dashed inset in E. G: representative images of triple staining showing reporter (RFP, red), PDGFβ-R (blue), and CD146 (green) colocalizing (purple and white) in FSGS (arrow). H: higher magnification of the area shown in the dashed inset in G.

Fig. 5.

CoRL in the interstitium do not express endothelial cell markers. ERG and CD31 were used as endothelial-specific markers in the triple stains. A: representative image of triple staining showing reporter (RFP, red), PDGFβ-R (green), and the nuclear endothelial marker ERG (blue) did not colocalize in the glomerulus (g) at baseline (purple and white, arrow). PDGFβ-R (green) localized to interstitial cells and mesangial cells in the glomerular tuft (g) and did not colocalize with ERG. Reporter localized to cells in the juxtaglomerulus surrounding an arteriole (a) but did not colocalize with ERG. B: representative image of triple staining showing reporter (RFP, red), PDGFβ-R (green), and ERG (blue, nucleus) did not colocalize in CoRL in the interstitium (purple and white, arrow). The reporter and PDGFβ-R did colocalize. C: higher magnification (×1,000) of the area shown in the dashed inset in B. D: representative image of double staining showing reporter (RFP, red) and the endothelial surface marker CD31 (green) did not colocalize at baseline (arrow). CD31 (green) localized to endothelial cells in the glomerular tuft (g) and arteriole (a). Reporter localized to cells in the juxtaglomerulus surrounding an arteriole (a) but did not colocalize with CD31. E: representative image of double staining showing reporter (RFP, red) and CD31 (green) did not colocalize in CoRL in the interstitium (yellow) and that an RFP-positive cell surrounded the capillary endothelium (arrow) in FSGS. F: higher magnification of the area shown in the dashed inset in E.

Fig. 6.

CoRL in the interstitium exhibit ultrastructural features consistent with pericytes and costained for PDGFβ-R, but not for renin. A: representative image of triple staining at baseline showing reporter (RFP, red), PDGFβ-R (green), and renin (blue). As expected, reporter and renin staining colocalized in cells in the juxtaglomerulus surrounding the arteriole (a) but neither were detected in the glomerulus (g). B: representative image of triple staining in FSGS. The reporter (red) and PDGFβ-R (green) colocalized in cells in the interstitium (yellow color, arrow). Renin staining (blue) was not detected in the interstitium. A glomerulus is indicated (g) C: a higher magnification (×1,000) of the area shown in the dashed inset in B. These data show that a subset of interstitial pericytes derive from CoRL.

Fig. 7.

Decrease in glomerular podocyte number and increase in glomerular and interstitial scarring in Ren1cCreER × Rs-tdTomato-R mice with remnant kidneys. A: quantification of podocyte number, measured by p57 staining, was significantly lower in the remnant kidney compared with baseline. Representative images of p57 staining are shown at baseline (B) and in the remnant kidney (C). D: quantification of glomerular scarring, measured by Jones' stain, was significantly higher in the remnant kidney compared with baseline. Representative images of Jones' staining are shown at baseline (E) and in the remnant kidney (F). G: quantification of tubulointerstitial scarring by Jones' stain was significantly higher in the remnant kidney compared with baseline. Representative images showing Jones' staining at baseline (H) and in the remnant kidney (I).

Fig. 8.

CoRL in a subset of glomeruli in remnant kidneys costain for podocin and synaptopodin and have ultrastructural features consistent with podocytes in mice. A–C: representative images of double staining showing reporter (RFP, red, the permanent label for CoRL), podocin (green, cytoplasmic), and 4,6-diamidino-2-phenylindole (DAPI; blue, nuclear). A: at baseline, the reporter localized to cells in the juxtaglomerulus surrounding the arteriole (a) and was not detected in the glomerulus (g). Podocin localized to cells in the glomerular tuft. The 2 therefore did not colocalize. B: in the remnant glomerulus (g), CoRL labeled by the reporter colocalized with podocin creating a yellow-orange color (arrows). C: higher magnification of the area shown in the dashed inset in B. D–F: representative images of double staining for CoRL reporter (RFP, red), synaptopodin (green), and DAPI (blue). D: at baseline, the reporter localized to the arteriole (a), synaptopodin localized to the glomerular tuft (g), and therefore the 2 did not colocalize as expected. E: in remnant kidneys, the reporter colocalized with synaptopodin in a cell on the glomerular tuft (g) creating a yellow-orange color (arrows). F: higher magnification of the area shown in the dashed inset in E. G and H: brightfield images of immunoperoxidase staining for the tomato red reporter (black) in the juxtaglomerulus (indicated by arrow, positive control). I: brightfield image of immunoperoxidase staining for reporter (black) in the glomerular tuft appears in a podocyte distribution (indicated by arrows). J: TEM image of immunoperoxidase staining for tomato red reporter (black) in CoRL observed in the juxtaglomerulus served as a positive control (arrows); rg, renin granules observed in the cells. K: TEM image of podocytes that lacked reporter staining in the cell body or within foot processes (arrows indicate the lack of staining in foot processes; podo, podocytes; cap, capillary). Note fenestration of endothelium. L: TEM image of podocytes with staining for tomato red reporter (black) within foot processes (arrows indicate staining in foot processes). Definitions and note are as in K.

Fig. 9.

CoRL were detected in the interstitium in reporter mice with the remnant kidney model of chronic kidney disease, and a subset coexpress the pericyte marker PDGFβ-R, but not renin. Pericytes derived from CoRL were identified by double staining for the reporter (RFP, red) and PDGFβ-R (green). A: quantification of the number of RFP⁺ PDGFβ-R⁺ cells per ×200 interstitial field showed their number was significantly higher in remnant kidneys compared with baseline. B: representative image at baseline of triple staining showing the reporter (RFP, red) and renin (blue) colocalized in cells in the juxtaglomerulus surrounding an arteriole (a) but did not colocalize with renin either in the glomerulus (g) or in the interstitium. C: reporter (RFP, red) and PDGFβ-R (green) colocalized in the interstitium, but renin (blue) did not colocalize with CoRL (purple, arrow) in the remnant kidney. D: higher magnification of the area shown in the dashed inset in C.

Fig. 10.

CoRL in the interstitium in remnant kidneys are pericytes and not endothelial cells. A: representative image of triple staining showing the CoRL reporter (RFP, red), PDGFβ-R (blue), and the additional pericyte marker CD146 (green) colocalizating (purple and white) in the interstitium of remnant kidneys (arrows). B: higher magnification of image from A showing colocalization. C–E: to ensure that CoRL in the interstitium were not differentiating into endothelial cells, triple staining was performed for the CoRL reporter (RFP, red), PDGFβ-R (green), and the endothelial cell marker ERG (blue). C: there was no staining overlap at baseline (no purple and white color). PDGFβ-R (green) localized to interstitial cells and mesangial cells in the glomerular tuft (g) and did not colocalize with ERG. Reporter localized to PDGF-βR staining cells in the juxtaglomerulus surrounding (yellow color, arrow) an arteriole (a), but did not colocalize with ERG staining. D: in the remnant kidney, triple staining showed that the reporter (RFP, red) and PDGFβ-R (green) colocalized (yellow, arrow), but neither colocalized with ERG (blue; no purple and white colors detected). E: higher magnification of the area shown in the dashed inset in D.

Fig. 11.

CoRL differentially express mesenchymal stem cell (MSC) markers in FSGS. A: representative image of double staining showing reporter (RFP, red) and CD29 (green) colocalize in cells in the juxtaglomerulus surrounding an arteriole (a) at baseline. Double staining showing reporter (RFP, red) and CD29 (green) colocalize (yellow) in cells in the glomerular tuft (g; B) and perivascular cells in the interstitium (C) in FSGS. D: representative image of triple staining showing reporter (RFP, red), CD105 (green), and PDGFβ-R (blue) do not colocalize in cells in the juxtaglomerulus surrounding an arteriole (a) at baseline. E: triple staining showing reporter (RFP, red), CD105 (green), and PDGFβ-R (blue) colocalize (purple) in interstitial cells in FSGS. F: higher magnification of the area shown in the dashed inset in D.