Bowman's capsule provides a protective niche for podocytes from cytotoxic CD8+ T cells

Abstract

T cells play a key role in immune-mediated glomerulonephritis, but how cytotoxic T cells interact with podocytes remains unclear. To address this, we injected EGFP-specific CD8+ T cells from just EGFP death inducing (Jedi) mice into transgenic mice with podocyte-specific expression of EGFP. In healthy mice, Jedi T cells could not access EGFP+ podocytes. Conversely, when we induced nephrotoxic serum nephritis (NTSN) and injected Jedi T cells, EGFP+ podocyte transgenic mice showed enhanced proteinuria and higher blood urea levels. Morphometric analysis showed greater loss of EGFP+ podocytes, which was associated with severe crescentic and necrotizing glomerulonephritis. Notably, only glomeruli with disrupted Bowman's capsule displayed massive CD8+ T cell infiltrates that were in direct contact with EGFP+ podocytes, causing their apoptosis. Thus, under control conditions with intact Bowman's capsule, podocytes are not accessible to CD8+ T cells. However, breaches in Bowman's capsule, as also noted in human crescentic glomerulonephritis, allow access of CD8+ T cells to the glomerular tuft and podocytes, resulting in their destruction. Through these mechanisms, a potentially reversible glomerulonephritis undergoes an augmentation process to a rapidly progressive glomerulonephritis, leading to end-stage kidney disease. Translating these mechanistic insights to human crescentic nephritis should direct future therapeutic interventions at blocking CD8+ T cells, especially in progressive stages of rapidly progressive glomerulonephritis.

Keywords: Chronic kidney disease; Immunology; Nephrology; T cells.

Figure 1. Effects of Jedi T cell injections on normal pod-EGFP mice.

(A) Time course for the experimental protocol. Four days after intraperitoneal injection of PBS (0.1 ml), pod-EGFP mice were coinjected with LV.EGFP and either control T cells (n = 4) or Jedi T cells (n = 4). Mice with PBS injection only served as controls (n = 4). (B) Urinary albumin-to-creatinine ratios (UACR, μg/μg) tested for all time points were within the normal range (<0.15 μg/μg) for both PBS plus control T cell– and PBS plus Jedi T cell–injected mice. (C) Representative H&E-stained images of pod-EGFP kidneys. Original magnification, ×200 (upper panels); ×600 (lower panels). (D) Representative images of EGFP fluorescence (top row) and merged images of EGFP, DAPI, and differential interference contrast (DIC) to identify glomeruli and tubules (bottom row). Scale bar: 50 μm. (E and F) Quantification of the EGFP⁺ area in pod-EGFP mice, as EGFP⁺ area per glomerulus (glom) (E) and size distribution curves for EGFP⁺ area per glomerulus (F) (n = 4 mice, 381 glomeruli analyzed for PBS-only group; n = 4 mice, 442 glomeruli analyzed for PBS plus control T cell group; n = 4 mice, 420 glomeruli analyzed for PBS plus Jedi T cell groups).

Figure 2. Effects of Jedi T cell injections in the setting of NTSN.

(A) Time course for the experimental protocol. Four days after injection of NTS (0.1 ml), pod-EGFP mice were coinjected with LV.EGFP and either control T cells (n = 5) or Jedi T cells (n = 6). Mice with NTS injection only (n = 8) and EGFP^– WT injected with Jedi T cells (NTS plus Jedi T cell; EGFP^–) (n = 6) served as controls. (B) Following NTS injection, urinary albumin-to-creatinine ratios increased initially in all groups, but slowly declined over time in all groups, with the exception of the NTS plus Jedi T cell group. **P < 0.01 when compared with all other groups by ANOVA with Bonferroni’s modification for multiple comparison. (C) Representative EGFP fluorescence images of kidneys from mice. Original magnification, ×200 (top row); ×400 (bottom row). Scale bars: 50 μm (top row); bottom row: 20 μm (bottom row). Dotted lines indicate the BC, with arrows pointing to the sites of ruptures. (D and E) Quantification of the EGFP⁺ area in pod-EGFP mice, as EGFP⁺ area per glomerulus (D) and size distribution curves for EGFP⁺ area per glomerulus (E) (n = 4 mice, 381 glomeruli analyzed for PBS-only group; n = 4 mice, 471 glomeruli analyzed for NTS-only group; n = 5 mice, 539 glomeruli analyzed for NTS plus control T cell group; and n = 6, 649 glomeruli analyzed for NTS plus Jedi T cell group). **P < 0.01, compared with all other groups by ANOVA with Bonferroni’s correction for multiple comparisons.

Figure 3. Histopathological analysis of mouse kidneys.

(A) Representative images of H&E- and PAS-stained kidneys from mice injected with NTS. Destruction of glomeruli with inflammatory infiltrates and apoptosis and massive pathological worsening with defects in BC are apparent in kidneys of NTS plus Jedi T cell–injected mice. BC rupture is indicated by black arrows in PAS images. Bottom panel shows the immunofluorescence of MHC-I in all groups. Original magnification, ×200 (top row); ×600 (second and third rows); ×400 (bottom row). (B) Quantification of histopathology from 90 to 170 glomeruli per kidney section for each mouse of the 4 to 6 mice per group. ***P < 0.001, compared with all other groups by ANOVA with Bonferroni’s correction for multiple comparisons.

Figure 4. Effects of BC rupture on CD8/Jedi T cell infiltrations of glomerular space and EGFP⁺ podocytes.

(A) Frozen kidney sections were immunostained for all CD8⁺ (red) and Jedi CD45.1 (green) T cells. EGFP⁺ podocytes were pseudocolored in blue to allow separation from the CD8/CD45.1 T cells. Merged images show costaining of Jedi CD45.1⁺ and CD8⁺ T cells. Differential interference contrast allows identification of glomeruli and BC. Ruptures in the BC are indicated by white arrows. Original magnification, ×400. (B and C) Quantification of intraglomerular CD8⁺ T cells and EGFP⁺ podocyte area per glomerulus from NTS plus Jedi T cell group (n = 6 mice, ~150 glomeruli analyzed). Pearson’s correlation of the number of intraglomerular CD8⁺ T cells and the EGFP⁺ podocytes area/per glomerular tuft area (B) shows a significant inverse correlation between infiltrating CD8⁺ T cells and the remaining percentage of EGFP⁺ podocyte area. Quantification of CD8⁺ T cells in glomeruli with or without BC rupture (C). ****P < 0.0001, compared between indicated groups by ANOVA with Bonferroni’s correction for multiple comparisons.

Figure 5. Effects of BC rupture on glomerular localization of CD68 macrophages.

(A) Representative immunostained images of CD68⁺ macrophages in glomeruli with intact versus ruptured BC. Ruptured BC is associated with an increase in CD68⁺ macrophages infiltrating the glomerular space (indicated by white arrows). Original magnification, ×400. (B) Quantification of intraglomerular CD68⁺ area for intact versus ruptured BC (n = 53 glomeruli for intact, n = 29 glomeruli for ruptured BC in NTS plus control T cell group; n = 51 glomeruli for intact, n = 48 ruptured BC in NTS plus Jedi T cell group). ****P < 0.0001, compared between indicated groups by paired t test.

Figure 6. Effects of BC rupture on the distribution of proliferating CD8⁺ T cells.

(A) Proliferation of peri- and intraglomerular CD8⁺ T cells was evaluated by double immunostaining with Ki-67. Inner dotted line indicates the BC and outer line the limits of periglomerular infiltrates. Original magnification, ×400. (B and C) Quantification of periglomerular (B) and intraglomerular (C) numbers of CD8⁺ and percentages of CD8⁺Ki-67⁺ cells in glomeruli with intact or ruptured BC (n = 88 glomeruli with intact BC, n = 94 glomeruli with ruptured BC from 2 different NTS plus Jedi T cell–injected mice). ****P < 0.0001.

Figure 7. Immunohistologic analysis of CD8 in human biopsies with anti-GBM nephritis and ANCA-GN.

(A–F) Representative images of CD8 immunohistochemistry. (A) CD8⁺ cells originating from the periglomerular interstitium infiltrate a cellular crescent through discrete ruptures of BC (arrow). PAS counterstain. Original magnification, ×600. (B) CD8⁺ cells (arrows) penetrate a cellular crescent through multiple small breaks in BC, which appears fragmented and discontinuous. PAS counterstain. Original magnification, ×600. (C) A CD8⁺ cell located deep within a crescent overlies a GBM, suggesting direct podocyte contact (arrow). PAS counterstain. Original magnification, ×600. (D) BC has a broad rupture involving more than half the glomerular circumference, associated with abundant CD8⁺ cells infiltrating the crescent from the adjacent interstitium. PAS counterstain. Original magnification, ×400. (E) A glomerulus with complete destruction of BC shows circumferential infiltration of its cellular crescent by numerous CD8⁺ cells and merging of the crescent with the adjacent interstitium. PAS counterstain. Original magnification, ×400. (F) A glomerulus with intact BC has no infiltrating CD8⁺ cells within its cellular crescent despite many CD8⁺ cells in the periglomerular interstitium. A single circulating CD8⁺ cell is present in a glomerular capillary. PAS counterstain. Original magnification, ×400. (G) Quantification of percentage of glomeruli with crescents, with ruptured BC with CD8 cells in the crescents, or with intact BC with CD8 cells in the crescents. APIGN, acute postinfectious glomerulonephritis; MGN, membranous glomerulonephritis. ****P < 0.0001.