Glomerular parietal epithelial cells contribute to adult podocyte regeneration in experimental focal segmental glomerulosclerosis

Abstract

As adult podocytes cannot adequately proliferate following depletion in disease states, there has been interest in the potential role of progenitors in podocyte repair and regeneration. To determine whether parietal epithelial cells (PECs) can serve as adult podocyte progenitors following disease-induced podocyte depletion, PECs were permanently labeled in adult PEC-rtTA/LC1/R26 reporter mice. In normal mice, labeled PECs were confined to Bowman's capsule, whereas in disease (cytotoxic sheep anti-podocyte antibody) labeled PECs were found in the glomerular tuft in progressively higher numbers by days 7, 14, and 28. Early in disease, the majority of PECs in the tuft coexpressed CD44. By day 28, when podocyte numbers were significantly higher and disease severity was significantly lower, the majority of labeled PECs coexpressed podocyte proteins but not CD44. Neither labeled PECs on the tuft nor podocytes stained for the proliferation marker BrdU. The de novo expression of phospho-ERK colocalized to CD44 expressing PECs, but not to PECs expressing podocyte markers. Thus, in a mouse model of focal segmental glomerulosclerosis typified by abrupt podocyte depletion followed by regeneration, PECs undergo two phenotypic changes once they migrate to the glomerular tuft. Initially these cells are predominantly activated CD44 expressing cells coinciding with glomerulosclerosis, and later they predominantly exhibit a podocyte phenotype, which is likely reparative.

Figure 1. Podocyte depletion and glomerulosclerosis in PEC-Reverse Tetracycline-Transactivator (rtTA)/LC1/Rosa26 reporter mice

Podocytes were identified by p57 staining (brown, nuclear) and glomerulosclerosis by PAS staining. (A) Glomerulosclerosis increased at day 7, was maximal at day 14, and was lower by day 28. (B) Podocyte number was measured by quantitating the number of cells staining positive for p57 per glomerular tuft area. Podocyte number decreased abruptly at d7, with a nadir at d14, but was significantly higher at d28. (C-F) Representative p57 and PAS staining with scale bar. (C) Baseline mice (d0) showing typical p57 staining (examples represented by arrows), without sclerosis. (D) At FSGS d7, p57 staining was reduced, consistent with podocyte depletion. Proteinacious material was noted in some tubules (examples denoted by P). (E) FSGS d14 kidney section with 5 glomeruli marked by circles, named a-e. p57 staining was markedly reduced in glomeruli a-c, consistent with significant podocyte depletion, which was accompanied by glomerulosclerosis (arrow) and dilated capillary loops (arrows). Glomeruli d and e had a normal podocyte complement, with no sclerosis. Proteinacious material is present in some tubules (marked by P) (F) At day 28, the majority of glomeruli had a normal or near normal podocyte number; the glomerulus circled had continued podocyte depletion, accompanied by scarring.

Figure 2. Labeled PECs move onto the glomerular tuft in diseased PEC-Reverse Tetracycline-Transactivator (rtTA)/LC1/Rosa26 reporter mice

PECs were permanently labeled with the β-galactosidase reporter, only when mice are given doxycycline. The reporter was detected by X-gal (blue color, bright field) in panels C-J, and by β-gal staining (red color, confocal) in panels K-N. (A) Graph showing that compared to baseline, the percentage of glomeruli with one or more β-gal positive cells in the tuft was significantly increase at d7 (12.7±7.2%), d14 (16±3.2%) and d28 (26.75±5.4%) of FSGS. (B) Graph showing that within those glomeruli containing labeled PECs on the tuft, the absolute number of β-gal⁺cells increased progressively in number during disease. (C, E, G, I) show low power views of bright field micrographs of X-gal staining (blue, cytoplasmic) and PAS counterstain (pink), and the higher power views of the glomeruli marked by arrows are shown in panels D, F, H and J respectively. (D) Representative image at baseline of X-gal positive cells following doxycyline induction, restricted to Bowman's capsule within the glomerulus (dashed arrows). Following the onset of FSGS, there was a progressive increase in X-gal positive cells on the glomerular tuft at d7 (F), d14 (H) and d28 (I)(solid arrows indicate examples). (K-N) Confocal micrographs of β-gal (red, cytoplasmic) and DAPI (blue, nuclear) staining. (K) Representative image at baseline of β-gal positive cells, restricted to Bowman's capsule within the glomerulus (dashed arrows). Following the onset of FSGS, there was a progressive increase in β-gal positive cells on the glomerular tuft at d7 (L), d14 (M) and d28 (N)(solid arrows indicate examples).

Figure 3. Following the onset of FSGS, a subset of labeled PECs that move onto the glomerular tuft co-express the podocyte markers p57 and podocin

Confocal microscopy was used to identify permanently labeled PECs by β-gal staining (red color), and podocytes by p57 or podocin staining (green colors). (A-I) β-Gal and p57 double-staining and quantitation. (A) Graph showing the unbiased stereology data where a significantly higher number of β-gal⁺/p57⁺ double-positive cells on the glomerular tuft at day 28 of FSGS. (B-E) Baseline mouse. Staining for (B) p57 staining (green, nuclear) is limited to the glomerular tuft, (C) β-gal staining (labels PECs, red, cytoplasmic) is restricted to Bowman' capsule, and (D) DAPI (labels nuclei, blue) single channels and (E) merged images in a normal glomerulus at baseline. There is no overlap in staining, as PECs are restricted to Bowman's capsule. (F-I) FSGS day 28. (F) p57 staining. (G) β-gal positive cells are detected on the glomerular tuft. (H) DAPI staining. (I) The merged image shows a subset of PECs that migrated to the glomerular tuft co-express p57 (yellow color, solid arrows). An example of a PEC on the tuft that does not co-express p57 is shown (red, dashed arrow). (J-R) β-Gal and podocin double-staining and quantitation. (J) Graph showing that the number of β-gal⁺/podocin⁺ double-positive cells on the tuft was significantly higher on days 14 and 28 of FSGS. (K-N) Baseline mouse. (K) Podocin staining (green, cytoplasmic) is detected in podocytes, (L) β-gal staining is restricted to cells lining Bowman's capsule, (M) DAPI staining. (N) merged images showns no overlap, as PECs are restricted to Bowman's capsule. (O-R) FSGS day 28. (O) Podocin staining. (P) β-gal positive cells are detected on the glomerular tuft. (Q) DAPI staining. (R) The merged image shows a subset of PECs on the glomerular tuft co-express podocin (yellow color, arrows). Taken together, these results show that a subset of PECs that have migrated to the glomerular tuft in disease co-express the podocyte proteins p57 and podocin.

Figure 4. A subset of labeled PECs co-express the podocyte marker synaptopodin on the glomerular tuft in mice with FSGS

(A-D) Baseline mice. (A) Synaptopodin (green, membrane location in podocytes), (B) β-Gal (red, cytoplasmic, PEC label), (C) DAPI (blue, nuclear) and (D) Merge of all three stains, showing no colocalization, because PECs are restricted to Bowman's capsule (dashed arrows). (E-H) FSGS mice. (E) Synaptopodin staining, (F) β-Gal staining is detected in cells lining Bowman's capsule and inside the tuft, (G) DAPI. (H) Merge of all three stains, showing a subset of PECs on the tuft co-colocalize with synaptopodin (solid arrows). Because β-gal is restricted to the cell cytoplasm and synaptopodin is membrane bound, they do not merge to form a yellow color. Dashed arrows show no co-staining with synaptopodin in β-gal stained cells lining Bowman's capsule.

Figure 5. CD44, a marker of PEC activation, is detected in labeled cells along Bowman's capsule and on the tuft in PEC-Reverse Tetracycline-Transactivator (rtTA)/LC1/Rosa26 reporter mice with FSGS

Confocal images showing double-staining for CD44 (green color), β-gal (red color) and merge (yellow color). (A) Quantitation of CD44 staining in glomeruli. Graph showing the percentage of the glomerular area that was CD44⁺ was significantly higher on days 7 of FSGS, but was variable and did not reach significance on day 14 and 28. (B-E) Baseline mice. (B) Staining for CD44 (green, cytoplasmic) is not detectable in glomerular cross sections, but is present in occasional cells outside of glomeruli (dashed arrow). (C) Within glomeruli, β-gal staining (red) is limited to PECs lining Bowman's capsule. (D) DAPI staining (blue, nuclei). (E) When merged, there is no overlap with CD44 and β-gal staining. (F-I) FSGS day 7 (F) CD44 staining is detected in cells lining Bowman's capsule, and in cells in the glomerular tuft. (G) β-Gal staining shows PECs in the glomerular tuft. (H) DAPI staining. (I) The merge shows a subset of PECs that have migrated to the glomerular tuft co-express CD44 (β-gal⁺/CD44⁺) (solid arrows). (J-M) FSGS day 14 (J) The number of cells staining positive for CD44 on the glomerular tuft is substantially higher. CD44 stained cells are detected in the peri-glomerular distribution (dashed arrows). (K) β-Gal stained cells have migrated to the tuft. (L) DAPI staining. (M) The merge image shows a subset of PECs within the glomerular tuft co-express CD44 (β-gal⁺/CD44⁺, solid arrows). (N-Q) FSGS day 28. (N) The number of cells staining positive for CD44 on the glomerular tuft is substantially lower. CD44 stained cells are detected in the peri-glomerular distribution. (O) β-Gal stained cells continue to be detected on the glomerular tuft. (P) DAPI staining. (Q) The merge image shows labeled PECs on the tuft no longer co-express CD44, however, a subset of PECs along Bowman's capsule continue to co-express CD44 (β-gal⁺/CD44⁺, solid arrows).

Figure 6. In reporter mice with FSGS, phospho-ERK co-localizes with PECs co-expressing CD44, but not with PECs co-expressing podocin

Confocal images of triple stains, used to determine which PECs co-expressed the active form of ERK (phospho-ERK). (A-J) Triple staining for phosphor-ERK, podocin and β-gal. (A-E) Baseline mice: (A) Podocin (green) stains podocytes; (B) β-gal staining (red) is restricted to PECs along Bowman's capsule; (C) DAPI staining (blue, nuclear); (D) Phospho-ERK (purple) is not detected; (E) Merged image showing that PECs and podocyte markers do not overlap, and neither co-express phospho-ERK. (F-J) FSGS mice D14: (F) Podocin staining is reduced in the left lower quadrant of the glomerulus, consistent with reduced podocyte number; (G) β-gal stained PECs are detected on the tuft; (H) DAPI staining (blue, nuclear); (I) Phospho-ERK (purple) is detected in cells lining Bowman's capsule (dashed arrow) and in cells in the tuft (solid arrows); (J) Merged image showing that phospho-ERK staining is not co-expressed in PECs that stain for podocin. (K-N) Triple staining for phospho-ERK, CD44 and β-gal in FSGS mice D7. Three glomeruli are shown in a mouse with FSGS, each labeled (hatched circle) and demarcated (a-c) for ease of identification. (K) CD44 staining (green) is increased in cells lining Bowman's capsule (solid arrow) and in the glomerular tuft (dashed arrow) in glomeruli b and c. CD44 staining is not detected in glomerulus a. (L) β-gal staining (red) is restricted to PECs lining Bowman's capsule in glomerulus a (arrows), is detected in the tuft in glomerulus b, and is in both locations in glomerulus c. (M) Phospho-ERK staining (blue) is not detected in glomerulus a, is in the tuft in glomerulus b, and in both locations in glomerulus c. (N) Merge of triple stains: shows that β-gal staining is restricted to PECs lining Bowman's capsule in glomerulus a (dashed arrows), but CD44 staining, β-gal staining and Phospho-ERK staining co-localize in cells within the tuft in glomerulus b (solid arrows) and with cells lining Bowman's capsule (dashed arrows) as well as within the tuft (solid arrow) of glomerulus c.

Figure 7. Overall proposed schema based on results of the current studies

In experimental FSGS characterized by abrupt podocyte loss and subsequent repletion, a subset of adult PECs invade the glomerular tuft, and can participate in either a pro-fibrotic or regenerative pathway. We propose that activated PECs (CD44⁺) co-express phospho-ERK and are pro-fibrotic. However, PECs that have migrated to the glomerular tuft that do not express phospho-ERK are podocyte progenitors (express p57, podocin and synaptopodin) which are regenerative.