Podocyte repopulation by renal progenitor cells following glucocorticoids treatment in experimental FSGS

Abstract

Prednisone is a mainstay of treatment for patients with focal segmental glomerulosclerosis (FSGS), a disease characterized by reduced podocyte number and glomerulosclerosis. Although the systemic immune-modulatory effects of prednisone are well-known, direct tissue effects on glomerular cells are poorly understood. Experimental FSGS was induced in mice with a cytotoxic anti-podocyte antibody, resulting in an abrupt decrease in podocyte number by day 3, proteinuria, and the development of glomerulosclerosis. Administering daily prednisone to mice with FSGS, beginning at day 3, significantly increased podocyte number at weeks 2 and 4. Podocyte number did not increase in control mice with FSGS given DMSO. The increase in podocyte number in prednisone-treated mice correlated significantly with reduced glomerulosclerosis. Prednisone reduced podocyte apoptosis measured by synaptopodin⁺/caspase-3⁺ double staining. Additionally, the number of podocyte progenitors, defined as cells expressing both a parietal epithelial cell protein and a podocyte protein, was significantly increased in prednisone-treated mice with FSGS at weeks 2 and 4. This was associated with increased phospho-ERK staining in both parietal epithelial cells (PAX2⁺/p-ERK⁺) and in podocyte progenitors (WT-1⁺/p-ERK⁺ lining Bowman's capsule). These data show that in this model of experimental FSGS, prednisone augments glomerular repair by increasing podocyte number through direct effects on both glomerular epithelial cells. Prednisone limits podocyte loss by reducing apoptosis, and it increases regeneration by augmenting the number of podocyte progenitors. The data support a direct glomerular cell action for prednisone in improving outcomes in FSGS.

Keywords: CD44; apoptosis; focal segmental glomerulosclerosis; glomerulosclerosis; parietal epithelial cell; prednisone; proteinuria; regeneration; repair.

Fig. 1.

Renal function in experimental focal segmental glomerulosclerosis (FSGS). Renal function, as measured by urine protein:creatinine ratio, was not different in all normal age-matched animals compared with baseline before experiment. Compared with normal mice (open bars), there was an increase in the urine protein:creatinine ratio in mice with FSGS given DMSO at 2 and 4 wk (gray bars). Prednisone treatment decreased the urine protein:creatinine ratio in mice with FSGS at 2 and 4 wk (filled bars).

Fig. 2.

Prednisone improves glomerulosclerosis in experimental FSGS. A–C: representative images of Sirius red staining at ×200 original magnification (arrowheads indicate examples of glomerulosclerosis, * shows an example of interstitial fibrosis in untreated mice with FSGS at 4 wk). D: glomerulosclerosis index increased in mice with FSGS given DMSO at 2 and 4 wk (hatched bars) compared with age-matched normal mice (open bars). Prednisone treatment (filled bars) was associated with a decrease in glomerulosclerosis index in mice with FSGS. E: percentage of glomeruli with global sclerosis, defined as over 75% tuft scarring (score 4), increased in mice with FSGS given DMSO (hatched bars). Prednisone treatment significantly decreased global sclerosis at 2 and 4 wk (filled bars).

Fig. 3.

Prednisone increases podocyte number in experimental FSGS. A–C: representative images of p57 staining (brown, nuclear) at 2 and 4 wk in normal mice (A-1, A-2), mice with FSGS given DMSO (B-1, B-2), and prednisone-treated mice with FSGS (C-1, C-2; ×630 original magnification). Arrows show examples of positive staining. D: staining is not detected in the negative control where the p57 primary antibody was omitted (×200 original magnification). E: podocyte number, measured as the number of p57-positive cells/glomerular cross section, was significantly depleted by day 3 of FSGS. Podocyte number remained low in mice with FSGS mice given DMSO at 2 and 4 wk (dotted line). Podocyte number increased significantly in mice with FSGS given prednisone (solid line) at 2 and 4 wk. These data show that when prednisone is administered following abrupt podocyte depletion, podocyte number increases compared with controls.

Fig. 4.

Prednisone decreases podocyte apoptosis in experimental FSGS. A–C: podocyte apoptosis was measured by double staining for caspase-3 (red color) and synaptopodin (green color), with DAPI staining nuclei (blue color; ×630 original magnification). A1–3: normal mice: synaptopodin staining is present in a typical podocyte distribution (arrows); caspase-3 staining is absent. B1–3: mice with FSGS given DMSO. B-1: caspase-3-stained cell (arrowhead) is shown. B-2: areas of reduced synaptopodin staining are noted (*). B-3: caspase-3 and synaptopodin double positive cell (yellow color, arrowhead) in the tuft. C1–3: mice with FSGS given prednisone. C-1: caspase-3-positive cells in the tubule (dashed arrow) adjacent to the glomerulus. C-2: intensity of synaptopodin staining is more marked in mice with FSGS given DMSO. C-3: no double staining is detected in the glomerulus. D: quantitation for caspase-3⁺/synaptopodin⁺ double staining: apoptosis is increased in mice with FSGS given DMSO at 2 and 4 wk (hatched bars) compared with normal mice (open bars). Prednisone treatment decreased apoptosis at 2 and 4 wk (filled bars).

Fig. 5.

Podocyte proliferation measured by p57/Ki-67 double staining. A–C: representative images of p57/Ki-67 staining at 2 and 4 wk at ×630 original magnification. A-1, A-2: normal mice: p57-positive nuclei (blue/gray) are detected in a typical podocyte distribution (arrowheads). No Ki-67 staining is detected in normal glomeruli. A tubular cell stains positive (red, dashed arrow) at 4 wk (A-2). B-1, B-2: mice with FSGS given DMSO: overall fewer p57 staining cells are detected; p57 staining is not detected in areas of sclerosis (*). At 2 wk, Ki-67 staining is detected in a cell lining Bowman's capsule, and at 4 wk positive staining is detected in a tubular cell. C-1, C-2: mice with FSGS given prednisone: at 2 wk, Ki-67 staining is detected in a cell lining Bowman's capsule, and at 4 wk positive staining is detected in a tubular cell. D1–3: negative controls (×200 magnification). D-1: staining for Ki-67 was not detected when the primary antibody was omitted (dashed circles represent glomeruli with p57 staining). D-2: staining for p57 was not detected when the primary antibody was omitted (dashed circles represent glomeruli; arrow represents a positive Ki-67 cell). D-3: staining for p57 or Ki-67 was not detected when both primary antibodies were omitted (dashed circles represent glomeruli). These data show that podocytes do not proliferate in mice with FSGS given DMSO or prednisone. Ki-67 staining is detected in cells lining Bowman's capsule.

Fig. 6.

Glomerular progenitor cells, defined as cells double staining for PAX2 and synaptopodin, increase in experimental FSGS. A–C: representative images at ×630 original magnification for PAX2 (blue/gray nuclear, dashed arrow) and synaptopodin (brown, cytoplasmic, solid arrow) double staining. Arrowheads indicate double positive PAX2⁺/synaptopodin⁺ cells. A-1, A-2: normal mice: PAX2-stained cells are confined to Bowman's capsule, and synaptopodin stains in a typical podocyte distribution at 2 and 4 wk. B-1, B-2: mice with FSGS given DMSO: PAX2⁺/synaptopodin⁺ double-stained cells are detected at 2 and 4 wk (arrowheads). Segmental decreases in synaptopodin staining are represented by the *. C-1, C-2: mice with FSGS given prednisone: PAX2⁺/synaptopodin⁺ double-stained cells are detected at 2 and 4 wk. D1–3: negative controls (×200 magnification). D-1: staining for synaptopodin was not detected when the primary antibody was omitted (dashed circles represent glomeruli with PAX2 staining). D-2: staining for PAX2 was not detected when the primary antibody was omitted (dashed circles represent glomeruli). D-3: staining for PAX2 and synaptopodin was not detected when both primary antibodies were omitted (dashed circles represent glomeruli). E: progenitor cell number, measured as the number of PAX2⁺/synaptopodin⁺ double positive cells/glomerular cross section, was augmented by prednisone treatment (filled bar) at 2 and 4 wk compared with control mice with FSGS given DMSO.

Fig. 7.

Glomerular progenitor cells, defined as cells double staining for PAX2 (blue/gray) and Wilms' Tumor-1 (WT-1; red), increase in experimental FSGS. A: representative images of PAX2⁺/WT-1⁺ double staining at ×630 original magnification in normal mice. A-1: arrows indicate PAX2 (blue/gray)/WT-1 (red) double staining at 2 wk. Arrows indicate examples of a PAX2-positive (blue/gray only, WT-1-negative) cell and a WT-1-positive (red only, PAX2-negative) cell. A-2: fluorescent microscopic view of the A-1 bright-field view, where only WT-1 staining is seen because only the warp-red substrate is visible by fluorescent microscopy. * Indicates the site where PAX2 stains but WT-1 (red fluorescence) is negative in the nucleus. A-3 and A-4: represent PAX2/⁺WT-1⁺ staining in normal mice at 4 wk. Arrows indicate PAX2 or WT-1 single positive cells, and * indicates the site where PAX2 stains but WT-1 is negative in the nucleus (A-4). B: representative images of PAX2⁺/WT-1⁺ double staining at ×630 original magnification in untreated mice with FSGS at 2 wk. B-1: arrows indicate PAX2 (blue/gray) or WT-1 (red) single positive cells. Arrowhead indicates a PAX2⁺/WT-1⁺ double positive cell lining along Bowman's capsule. B-2: fluorescent microscopic view of B-1 bright-field view, where only WT-1 staining is seen. Arrowhead indicates the same cell from B-1, termed glomerular progenitor cell, which is visible. B-3 and B-4: same staining in untreated mice with FSGS at 4 wk. Arrows indicate PAX2 or WT-1 single positive cells, arrowheads indicate a glomerular progenitor cell (PAX2⁺/WT-1⁺ double positive). C: representative images of PAX2⁺/WT-1⁺ double staining at ×630 original magnification in prednisone-treated mice with FSGS at 2 wk. C-1: arrows indicate PAX2 (blue/gray) or WT-1 (red) single positive cells. Arrowheads indicate PAX2⁺/WT-1⁺ double positive cells lining Bowman's capsule and in the glomerular tuft. C-2: fluorescent microscopic view of the C-1 bright-field view, only WT-1 staining is seen. Arrowheads indicate the same cells from C-1, which are visible by fluorescence. C-3 and C-4: same staining in prednisone-treated mice with FSGS at 4 wk. Arrows indicate PAX2 or WT-1 single positive cells; arrowheads indicate glomerular progenitor cells (PAX2⁺/WT-1⁺ double positive). D: number of cells staining positive for PAX2 and WT-1 per glomerular cross section was significantly higher at 2 and 4 wk in untreated mice with FSGS (hatched bar) compared with normal mice (open bar). Prednisone treatment increases the number of PAX2/WT-1-positive cells/glomerular cross section at 2 and 4 wk (filled bars), compared with untreated mice with FSGS.

Fig. 8.

Prednisone augments p-ERK1/2 in parietal epithelial cells (PECs) in experimental FSGS. A–C: representative images of PAX2/p-ERK double staining at ×630 original magnification. The arrowheads show double positive cells along Bowman's capsule, arrows show PAX2 single positive cells. D-1 to D-3: staining was not detected when the primary antibodies were omitted as negative controls (×200). PAX2 only (D-1), p-ERK only (D-2), no primary antibody (D-3). Glomeruli are circled for easier identification. E: number of PECs with phosphorylated ERK, measured as the number of PAX2/p-ERK double positive cells/glomerulus along Bowman's capsule, increased at 2 and 4 wk in mice with FSGS mice (hatched bar). Prednisone treatment was associated with an increase in this number at 2 wk (filled bar).

Fig. 9.

Prednisone augments glomerular progenitor cells' p-ERK1/2 along Bowman's capsule in experimental FSGS. A–C: representative images of WT-1/p-ERK double staining at ×630 original magnification. The arrowheads show double positive cells along Bowman's capsule; arrows show WT-1 or p-ERK single positive cells. D-1 to D-3: staining was not detected when the primary antibodies were omitted as negative controls (×200). WT-1 only (D-1), p-ERK only (D-2), and no primary antibody (D-3). Glomeruli are circled for easier identification. E: number of progenitor cells with phosphorylated ERK, measured as the number of WT-1/p-ERK double positive cells/glomerulus along Bowman's capsule, increased at 2 and 4 wk in mice with FSGS (hatched bar). Prednisone treatment was associated with an increase in this number at 2 wk (filled bar).