Fig. 3

Double in situ hybridization and immunohistochemistry detecting macrophage migration inhibitory factor (MIF) expression and macrophage accumulation. (A) Constitutive MIF mRNA expression (purple) by some glomerular and tubular epithelial cells in the kidney of oxonic acid (OA) group. Few ED1⁺ macrophages (brown, arrows) are found within the glomerulus and interstitium. (B) Constitutive weak MIF protein expression (blue) by most tubules with some ED1⁺ macrophages within glomeruli and occasional damaged tubules in the kidney of OA group. (C, D) Serial sections demonstrating differential up-regulation of MIF mRNA and protein expression in the early tubulointerstitial lesion in oxonic acid plus uric acid (UA) groups (G = glomerulus). A marked up-regulation of MIF mRNA (C, purple) by tubular epithelial cells is limited to severe tubulointerstitial lesions with many ED1⁺ macrophages (brown-purple) accumulation (*). Note that many ED1 + macrophages also expressed MIF mRNA. In contrast, immunostaining of the serial sections demonstrated that there is very weak or undetectable MIF protein expression by the damaged tubules and macrophages (brown) within the lesions (*); whereas, weak constitutive expression of MIF protein is unaltered in the areas of the kidney that are normal or minimally injured. (E) Strong MIF mRNA (purple) expression and macrophage accumulation (brown-purple) in a damaged tubules of UA groups, where some evidence for deposition of uric acid crystals was found within the lumen with marked tubular epithelial cell proliferation and MIF expression. Note that all macrophages (*) within the damaged tubules also strongly express MIF mRNA (brown-purple). But, there is no MIF up-regulation and macrophage accumulation found in those non-inflamed tubules. (F) Macrophage-rich (brown) granuloma formation in UA group. Note that a multinucleated giant cells with strong MIF protein expression (blue) is found within the granuloma (*). All sections are counterstained with periodic acid/Schiff reagent (PAS). Magnifications: 250X (A, B), 100X (C, D), 400X (E, F).