Differentiating Glomerular Inflammation from Fibrosis in a Bone Marrow Chimera for Rat Anti-Glomerular Basement Membrane Glomerulonephritis

Abstract

Background: Many types of glomerulonephritis (GN) undergo tandem connected phases: inflammation and fibrosis. Fibrosis in human GNs leads to irreversible end-stage disease. This study investigated how these 2 phases were controlled.

Methods: Using a rat anti-glomerular basement membrane GN model, we established bone marrow (BM) chimeras between GN-resistant Lewis (LEW) and GN-susceptible Wistar Kyoto (WKY) rats. Glomerular inflammation and fibrosis were compared between chimeras.

Results: LEW's BM to WKY chimeras with or without co-transfer of host WKY's T cells were GN-resistant. On the other hand, WKY's BM to LEW (LEW(WKY)) chimeras developed glomerular inflammation and albuminuria upon immunization. Quantitative analysis showed that the number and composition of inflammatory cells in glomeruli of immunized LEW(WKY) chimeras were similar to those in immunized WKY rats at their inflammatory peak. Thus, glomerular inflammation was controlled by BM-derived non-T cell populations. However, unlike WKY rats, LEW(WKY) rats did not develop fibrosis until the end of experiments (84 days) in spite of persistent inflammation and albuminuria.

Conclusion: Inflammation alone was not sufficient to trigger fibrosis, suggesting a critical role of glomerular cells in the fibrotic process. As LEW(WKY) chimera allows us to separate glomerular inflammation from fibrosis, this model provides a useful tool to study how fibrosis is initiated following inflammation.

Fig. 1

Development of anti-GBM glomerulonephritis (GN) in BM chimeras post immunization with nephritogenic T epitope pCol(28–40). (a and b) Time courses of albuminuria in WKY^LEW chimera and related auto-MB graft WKY^WKY controls (a), or in LEW^WKY and related control LEW^LEW (b). Albuminuria in each rat is shown separately by different symbols. Closed symbols connected with solid lines are for experimental rats WKY^LEW and LEW^WKY, and open symbols with dashed lines for controls WKY^WKY and LEW^LEW. Note that the data for most of WKY^LEW and LEW^LEW rats are overlapped due to none or insignificant albuminuria. (c and d) Summaries of GN in WKY^LEW chimera and its controls (c) or LEW^WKY and its controls (d). Shot horizontal lines represent means of each group. One-way ANOVA with post-hoc Tukey’s HSD test was performed. *p<0.05, ***, p<0.001. (e and f) Representative H-E staining for each group as indicated. Bar = 50 μm.

Fig. 2

WKY^LEW chimeras co-transferred with WKY’s T cells (WKY^LEW/T^WKY) do not develop anti-GBM GN after immunization with pCol(28–40). (a) Flow cytometry shows donor LEW’s BM cells before (left) and after (right) removal of T cells. Note a significant reduction in number of CD3⁺ cells (gate) post removal of pan-T cells. (b) PCR genotyping of PBL T cells or non-T cells in WKY^LEW or WKY^LEW/T^WKY chimeras. Note that T cells from a WKY^LEW/T^WKY chimera were of WKY’s origin, while those of a control WKY^LEW show both genotypes. D3R201 was used as a probe. (c) Genotyping of T cells of draining lymph node (LN) in WKY^LEW/T^WKY chimera at day 20 post immunization. (d) Time course of albuminuria in each WKY^LEW/T^WKY rat. Note overlapped lines due to low or none albuminuria in many rats. (e) Summary of GN scores in immunized WKY^LEW/T^WKY chimeras. Each symbol represents an individual rat. (f) H-E staining shows a representative glomerulus from a WKY^LEW/T^WKY chimera at day 40. Bar =50 μm. (g) In vitro T cell proliferation in responding to pCol(28–40) at day 20 in a representative WKY rat (dashed line) and a WKY^LEW/T^WKY (solid line). Horizontal lines are background cpm without pCol(28–40). (h) Summary of T cell proliferation in responding to pCol(28–40) in different groups. T cell proliferation is expressed as a stimulation index.

Fig. 3

Absence of fibrotic tissue in glomeruli of LEW^WKY chimera immunized with pCol(28–40). (a) PAS staining patterns in representative glomeruli from different groups and times as indicated at the top of each panel. (b) Summary of glomerular size in different groups as indicated. (c) Immunofluorescence shows collagen 1α1 (Col1α1) deposition in glomeruli of different chimeras to reveal fibrotic tissue. Arrows show initiating site of fibrosis. (d) Quantitation of collagen1α1 deposition in each glomerulus in different groups as indicated. Deposition is expressed as percentage of entire glomerular area. (e) Immunofluorescence shows α smooth muscle actin (αSMA) distribution in glomeruli of different groups. Glomerular αSMA⁺ cells (arrows) were present in WKY^WKY at day 35, but absent in LEW^WKY rats. BV, blood vessels, as internal positive control for αSMA. Dash lines outline glomeruli. (f) Summary of quantity of αSMA area in each glomerulus in different groups, expressed as percentage of entire glomerular area. One-way ANOVA with post-hoc Tukey’s HSD test was performed for b, d and f; all three with variance p<0.0001. *p<0.05, **p<0.01, ***, p<0.001. Red horizontal bars represent mean of each group. Bars in a, c and e =50 μm. (g) Electron microscopic (EM) micrographs show glomeruli in different groups of rats as indicated. Arrows indicate abnormal foot processes of podocytes. Note segmental effacement of the processes in WKY at day 45.

Fig. 4

Flow cytometry on glomeruli-infiltrating leukocytes (GILs) in chimera LEW^WKY and WKY rats post immunization. (a) Representative density plots for detection of T cells in each group as indicated. A dominant CD4⁺CD3⁺ population with a minor CD4⁻CD3⁺ one is presented in LEW^WKY day 35, WKY at day 35, but absent in WKY at day 45. (b) Representative density plots show CD11⁺CD8⁻, CD11⁺CD8⁺, and CD11⁻CD8⁺ populations among GILs in chimera LEW^WKY at day 35 and day 70, as well as in WKY at day 35. (d) Summary of relative sizes (%) of three GIL populations from different chimera or time as indicated. n=3 for each group, Error bars show SD; MΦ, macrophage. T-test was performed. ***p<0.001

Fig. 5

Glomerular macrophages and GBM-bound autoantibody in chimeras post immunization with pCol(28–40). (a) Upper panels show staining for ED1 and collagen 1α1. Lower panels are staining for RT1B and ED1 to show three macrophage subsets. Clustered macrophages are present in glomeruli of LEW^WKY chimera at day 70 and WKY^WKY rat at day 35, but absent in WKY^WKY rat at day 45. Arrows show macrophages in Bowman’s capsules. Nuclei were counter-stained by DAPI. Note a scarcity of cells in glomeruli of WKY^WKY at day 45. Bars =50μm. (b) Summary of glomeruli-infiltrating macrophage subsets in chimeras at various times as indicated. (c) Surface RT1B expression levels in RT1B⁺ macrophages, expressed as fluorescent intensity, in rats as indicated. Short horizontal bars show mean for each group. One-way ANOVA with post-hoc Tukey’s HSD test was performed for b and c. (d) PCR genotyping demonstrates that RT1B⁺ GILs were of WKY origin in LEW^WKY rats (right panel). Left panel shows two populations in purified RT1B⁺ cells. (e) Immunofluorescence shows GBM-bound IgG autoantibodies in LEW^WKY chimera and WKY^WKY control at indicated times. Arrowheads outline a glomerulus for WKY^WKY at day 45, revealing a large fibrotic tissue surrounding GBM.