Lymphocytes are dispensable for glomerulonephritis but required for renal interstitial fibrosis in matrix defect-induced Alport renal disease

Abstract

One current theory for the emergence of glomerular nephritis implicates Th1-type cellular responses associated with delayed-type hypersensitivity, involving T cells and macrophages. Using a mouse model for progressive glomerulonephritis, we investigate the role of B and T cells in the pathogenesis of glomerular inflammation. Deletion of a3 chain of type IV collagen in mice (a3(IV) collagen null mice) results in GBM defects, glomerulonephritis and tubulointerstitial inflammation, fibrosis and significant immune infiltration including activated B-and T-lymphocytes. To evaluate the contribution of lymphocytes to the pathogenesis of glomerulonephritis and renal fibrosis, we generated mice that are deficient in both the a3(IV) collagen and Rag-1 (a3/Rag-1 DKO). Lymphocyte deficiency significantly reduces fibrosis in the renal interstitium, but ultrastructural GBM defects persist. Interestingly, glomerulonephritis in the double null mice persists at a similar level with comparable proteinuria. Here we demonstrate that despite the presence of B-cell and T-cells in the inflamed glomeruli, their deletion does not impede the emergence of glomerulonephritis but has a negative impact on the progression of renal interstitial fibrosis.

Feasibility of Repairing Glomerular Basement Membrane Defects in Alport Syndrome

“…11,12 Several approaches have been used in attempts to cure Alport syndrome in mice, or to at least slow progression of the disease and lengthen lifespan. These include efforts to prevent the development of GBM lesions and glomerulosclerosis [13][14][15] ; to attenuate the glomerular and tubulointerstitial inflammation that inevitably occurs [16][17][18] ; and to transplant or infuse various types of stem cells, with or without irradiation, with the goal of replacing mutant podocytes with exogenous cells capable of expressing the missing collagen a3a4a5(IV). [19][20][21][22] Despite claims of success at replacing podocytes and/or restoring GBM composition, [19][20][21] these results stirred controversy 23 and have not been convincingly replicated.…”

Stem Cell Therapies Benefit Alport Syndrome

“…In our BM transplant studies, mature lymphocytes and monocytes seemed to play a limited role in the rescue of the renal pathology in Col4A3 knockout mice. Despite the significant protection of the tubulointerstitial compartment after ablation of mature B and T lymphocytes, 16 this improvement did not result in an increase in the lifespan of Col4a3/Rag-1 DKO in comparison with Col4A3 knockout mice. Ablation of B and T cells and monocytes with the WT allele for COL4A3KO in the reconstituted BM did not preclude the expression of the missing chain of type IV collagen in Rag-1KO or CD11bKO BMtransplanted Col4A3 knockout mice.…”

“…Morphometric analyses for the histologic assessment of renal injury, here glomerular sclerosis, tubular atrophy, and interstitial volume were performed as described previously. 11,16 Immunocytochemistry Thin frozen sections (4 m) from kidneys embedded in OCT compound were denatured with 6 M urea/0.1M glycine (pH 3.5) and immunostained against rabbit anti-mouse ␣3 and rabbit anti-mouse ␣5 type IV collagen chains (a gift from Dr. Cosgrove, Boys Town National Research Center, Omaha, NE) as described previously. 11 FITC-conjugated secondary antibodies (Jackson Immunoresearch) were used at a 1:300 dilution.…”

“…11,16 Immunoprecipitation Immunoprecipitation was performed as described previously. 2 For each immunoprecipitation sample, 100 g of extracted native NC1 hexamers containing solution in 500 l of extraction buffer (50 mM TRIS [pH7.5], 150 mM NaCl, and 1% Triton X100) with "Complete" Protease Inhibitor Cocktail (Roche) was incubated with 10 l of type IV collagen ␣3 chain antibody or anti-FLAG M2 antibody as a control (Sigma) overnight at 4°C under constant gentle rotation.…”

“…13 Unlike the human disease, kidney disease progression in Col4A3 knockout mice involves significant immune infiltration; therefore, a case can be made that total body irradiation and subsequent BM transplantation in Col4A3 knockout mice could modulate disease progression by diminishing renal immune infiltration. Our previous report demonstrated that lymphocyte ablation improves renal interstitial fibrosis in Col4a3/Rag-1 double-KO mice on C57BL/6 background 16 as a result of diminished interstitial infiltrates. Recently, Katayama et al 13 provided evidence for an increase in the survival of Col4A3 knockout mice on 129Sv background, associated with improvement in renal function and histologic findings after total body irradiation; however, that study does not conclusively negate the specific therapeutic potential of BM-derived cells in the recovery of the renal phenotype.…”