Cells of renin lineage are progenitors of podocytes and parietal epithelial cells in experimental glomerular disease

Abstract

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.

Figure 1

Characterization of adult Ren1cCreER×Rs-tdTomato-R mice. Double-staining was performed for renin protein (red) and for the reporter for cells of renin lineage using a fluorescein-conjugated antibody to RFP (green). A merge of the two appears yellow. A: In baseline (day 0) mice not given tamoxifen, renin staining was restricted to the JGC (arrows). The boxed region is shown at higher magnification in the inset. Staining for the reporter was not detected. B: In baseline mice given tamoxifen, the majority of renin-staining cells costained for RFP (white; dashed arrows), and these costaining cells were confined to the JGC. Occasional JGC cells only stained for renin (arrow). The boxed region is shown at higher magnification in the inset, where costaining JGC are more clearly seen. C: In diseased Ren1cCreER×Rs-tdTomato-R mice not given tamoxifen (day 14), renin staining was restricted to the JGC (arrows). The boxed region is shown at higher magnification in the inset, where renin staining is seen to be restricted to the JGC. Staining for RFP was not detected. D: In diseased mice given tamoxifen (day 14), renin staining was restricted to the JGC and costained with RFP (white; dashed arrow). The boxed region is shown at higher magnification in the inset, where costaining JGC are more clearly seen. No renin was detected in the diseased glomerulus. A labeled cell of renin lineage in the glomerulus stains green (thick arrow). Original magnification: ×100 (main images); ×630 (insets).

Figure 2

Cells of renin lineage are present in glomeruli of Ren1cCreER×Rs-tdTomato-R mice with experimental FSGS. Ren1cCreER×Rs-tdTomato-R mice were given tamoxifen for three alternate days to temporally and permanently label only cells of renin lineage. A survival biopsy was then performed (baseline, day 0), after which experimental FSGS was induced in each mouse. A: At day 0, labeled cells of renin lineage reporter (red) were restricted to the JGC (arrows). No labeled cells were detected in individual glomeruli (circles). B: A higher magnification view of the boxed region in panel A shows reporter-labeled cells of renin lineage in the JGC (arrow) but not within the glomerulus (circle). C: At day 14 of disease, reporter-labeled cells were detected in the JGC (thick arrows) and in a glomerulus (thin arrows). Labeled cells within glomeruli were focal in nature; fluorescence was absent in two glomeruli (circles). D: A higher magnification view of the boxed region in panel C shows reporter-labeled cells in the JGC (thick arrow) and within the glomerulus (thin arrows). E: At day 14 of disease, reporter-labeled cells were detected in the JGC (thick arrows) and in the glomerulus (thin arrows). F and G: Higher magnification views of the two boxed regions in panel E show reporter-labeled cells in the JGC (thick arrow) and within the glomerulus (thin arrows). Original magnification: ×100 (A, C, and E); ×630 (B, D, F, and G).

Figure 3

Cells of renin lineage express proteins considered to be podocyte-specific in glomeruli of Ren1cCreER×Rs-tdTomato-R mice with experimental FSGS. Double-staining was performed for WT-1, nephrin, or podocin (red), and for the cells of renin lineage reporter with a fluorescein-conjugated antibody to RFP (green). A: Staining for WT-1 (red; thin arrows) was confined to the glomerular tuft at baseline (day 0). Staining for RFP was confined to the JGC (green; thick arrows). Red blood cell autofluorescence (yellow-orange) was noted in the glomerular tuft. B: Costaining for WT-1 and RFP (yellow-orange; thick dashed arrows) in three cells within the glomerulus at day 14 of disease. One reporter-labeled cell in the glomerulus did not costain for WT-1 (thin dashed arrow). Most glomerular cells stained for WT-1 alone (red; thin arrows). C: A higher magnification view of the boxed region in panel B. D: Staining for nephrin (red; thin arrows) was confined to the glomerular tuft at baseline (day 0). Staining for RFP was confined to the JGC (green; thick arrow). E: Costaining for nephrin and RFP (yellow-orange; dashed arrow) within the glomerulus at day 14 of disease. An RFP-labeled cell outside the glomerulus did not costain with nephrin (thick arrow). Ribbon-like Nephrin staining alone (red; thin arrow). F: A higher magnification view of the boxed region in panel E. G: Staining for podocin (red; thin arrows) was confined to the glomerular tuft at baseline (day 0). Staining for RFP was confined to the JGC (green; thick arrow). H: Costaining for podocin and RFP (yellow-orange; dashed arrow) within the glomerulus at day 14 of disease. An RFP-labeled cell outside the glomerulus did not costain with podocin (thick arrow). Cytoplasmic podocin staining alone (red; thin arrow). I: A higher magnification view of the boxed region in panel H. These representative examples show that a subset of cells of renin lineage that have entered the glomeruli of diseased Ren1cCreER×Rs-tdTomato-R mice begin to express WT-1, nephrin, and podocin, all of which are considered to be podocyte proteins. Original magnification: ×400 (A, B, D, E, G, and H); ×1000 (C, F, and I).

Figure 4

Cells of renin lineage express glomerular PEC proteins in glomeruli of Ren1cCreER×Rs-tdTomato-R mice with experimental FSGS. Double-staining was performed for PEC proteins with antibodies to PAX2 or claudin-1 (red) and for the cells of renin lineage reporter with a fluorescein-conjugated antibody to RFP (green). A: At baseline (day 0) staining for PAX2 (thin arrows) was confined to Bowman's capsule, and did not overlap with staining for the reporter (thick arrow) restricted to the JGC. B: At day 14 of disease, a subset of cells lining Bowman's capsule within the glomerulus costained for PAX2 and the reporter (yellow; thick arrow). Other PAX2-staining cells did not costain with the reporter (thin arrows). C: A higher magnification view of the boxed region in panel B. D: In baseline mice, staining for claudin-1 (red; thin arrows) did not overlap with staining for the reporter (green; thick arrow), which was confined to the JGC. E: In diseased mice, RFP-labeled cells were detected lining Bowman's capsule; these cells costained for claudin-1 (yellow-orange; thick arrow). Staining for claudin-1 alone (red; thin arrows). Red blood cell autofluorescence (light green) was noted in the glomerular tuft. F: A higher magnification view of the boxed region in panel E. These representative examples show that, in diseased Ren1cCreER×Rs-tdTomato-R mice, a subset of cells of renin lineage that line Bowman's capsule express the PEC proteins PAX2 and claudin-1.

Figure 5

Cells of renin lineage increase in glomeruli of Ren1cCre×Rs-ZsGreen-R mice with experimental FSGS. Experimental disease was induced in Ren1cCre×Rs-ZsGreen-R mice to fate-map cells of renin lineage with ZsGreen reporter. No antibody was required to visualize ZsGreen, which stains green in both cytoplasm and nucleus; DAPI was used to counterstain nuclei blue. A and B: Representative ZsGreen labeling at baseline day 0. A: Low-power view of five glomeruli (circles). ZsGreen fluorescence is detected in cells within the JGC (arrows) alongside three of the glomeruli; only one cell is detected within a glomerulus (dashed arrow). B: A higher magnification view of the boxed region in panel A shows that the ZsGreen fluorescence (arrow) was confined to cells within the JGC. C–F: Representative ZsGreen labeling at day 14 of disease. C: Low-power view of four glomeruli (circles). The two glomeruli on the upper left are devoid of ZsGreen-labeled cells. The glomerulus on the upper right has several ZsGreen-labeled cells, which were predominantly located within the glomerular tuft (dashed arrows). In the circled glomerulus within the boxed region, ZsGreen-labeled cells are predominantly lining Bowman's capsule (thin arrows), but also appear in the JGC (thick arrow). D: A higher magnification view of the boxed region in panel C more clearly shows the ZsGreen-labeled cells lining Bowman's capsule (arrows). E: At day 14, one glomerulus (left circle) had no ZsGreen-labeled cells, but was accompanied by ZsGreen-labeled cells in the JGC (thick arrow) and in blood vessels (dotted arrows). Another glomerulus (right circle) had several ZsGreen-labeled cells (dashed arrows). F: A higher magnification view of the boxed region in panel E shows the ZsGreen-labeled cells as within the glomerular tuft (dashed arrows). G: Quantification of the number of ZsGreen-labeled cells. ZsGreen-positive cells lining Bowman's capsule (circles) were rarely detected at baseline (day 0). In disease, however, the numbers of these cells were significantly increased at all time points, relative to baseline. The number of ZsGreen-positive cells within the glomerular tuft (squares) had more than doubled at day 3, and continued to increase significantly at days 7 and 14. These data show an increase in the number of cells of renin lineage in glomeruli of mice with experimental FSGS. Labeled cells were detected either along Bowman's capsule, within the glomerular tuft, or at both locations. Data are expressed as means ± SEM. *P = 0.03, ^†P = 0.001, ^‡P = 0.04 versus d0 Bowman's capsule; ^§P = 0.03, ^¶P = 0.003, ^⋮P = 0.002 versus d0 glomerular tuft.

Figure 6

In Ren1cCre×Rs-ZsGreen-R mice with experimental FSGS, cells of renin in glomeruli costain for podocyte proteins. To determine whether the cells of renin lineage express proteins considered to be podocyte specific, experimental FSGS was induced in Ren1cCre×Rs-ZsGreen-R mice; confocal microscopy was performed after staining for WT-1 or synaptopodin (used as podocyte markers). ZsGreen fluorescence, used to fate-map cells of renin lineage, does not require antibody detection. A–C: Double-staining for ZsGreen and WT-1. A: In a representative glomerulus at baseline (day 0), ZsGreen-labeled cells were detected outside the glomerulus, in the JGC (green; thick arrow); ZsGreen staining did not overlap with WT-1 staining (red; thin arrows) within the glomerulus. B: At day 14 of disease, ZsGreen fluorescence was detected in the JGC (thick arrows) and also in a cell within the glomerular tuft (dashed arrow), where it overlapped with WT-1, appearing yellow. Nuclear staining for WT-1 alone (red; thin arrows). C: A higher magnification view of the boxed region in panel B more clearly shows the coexpression overlap of ZsGreen and WT-1 (yellow; dashed arrow). D–F: Double-staining for ZsGreen and synaptopodin. D: At day 0, ZsGreen fluorescence was seen in the afferent arteriole (thick arrow); ZsGreen staining did not overlap with synaptopodin staining (red; thin arrows) in the adjoining glomerulus. E: At day 14, ZsGreen fluorescence was still seen in the afferent arteriole (thick arrow), and an overlap of ZsGreen and synaptopodin staining (yellow; dashed arrows) was seen in a cell within the glomerular tuft. F: A higher magnification view of the boxed region in panel B more clearly shows the coexpression overlap of ZsGreen and synaptopodin (dashed arrows). These representative examples show that, in Ren1cCre×Rs-ZsGreen-R mice with experimental FSGS, a subset of cells of renin lineage labeled by ZsGreen within the glomerulus express the podocyte proteins WT-1 and synaptopodin.

Figure 7

In Ren1cCre×Rs-ZsGreen-R mice with experimental FSGS, cells of renin lineage that have entered the glomerulus coexpress PEC proteins. Experimental FSGS was induced in Ren1cCre×Rs-ZsGreen-R mice and confocal microscopy was performed after staining with antibodies to PAX2 and claudin-1 (used as PEC markers). ZsGreen fluorescence, used to fate-map cells of renin lineage, does not require antibody detection. A–C: Double-staining for ZsGreen and PAX2. A: In a representative glomerulus at baseline (day 0), ZsGreen-labeled cells were detected in cells outside the glomerulus in the JGC (green; thick arrow); within the glomerulus, ZsGreen staining did not overlap with PAX2 staining (red; thin arrows). Red blood cell autofluorescence (light green) was noted in the glomerular tuft. B: At day 14, in a subset of cells along Bowman's capsule, ZsGreen staining overlapped with PAX2 staining (yellow; thick arrows). In another subset, ZsGreen-labeled cells lining Bowman's capsule did not express PAX2 (dashed arrows), and for some PAX2-positive cells the staining did not overlap with ZsGreen (thin arrow). C: A higher magnification view of the boxed region in panel B more clearly shows the coexpression overlap of ZsGreen and PAX2 staining (thick arrow), along with single PAX2 stain (thin arrow). D–F: Double-staining for ZsGreen and claudin-1. D: At baseline, ZsGreen fluorescence in the afferent arteriole (thick arrow) did not overlap with claudin-1 staining (red; thin arrows) in the adjoining glomerulus. E: At day 14, in two cells lining Bowman's capsule ZsGreen staining overlapped with claudin-1 staining (yellow; thick arrows). One ZsGreen-labeled cell lining Bowman's capsule did not stain for claudin-1 (dashed arrow). F: A higher magnification view of the boxed region in panel E more clearly shows the coexpression overlap of ZsGreen and claudin-1 (arrows). These representative examples show that, in Ren1cCre×Rs-ZsGreen-R mice with experimental FSGS, a subset of cells of renin lineage labeled by ZsGreen within the glomerulus express the PEC proteins PAX2 and claudin-1 when these cells line Bowman's capsule.