Expression of alpha-actinin-4 in acquired human nephrotic syndrome: a quantitative immunoelectron microscopy study.

BACKGROUND: The molecular basis for the alteration of glomerular podocyte phenotype in nephrotic syndrome probably involves adaptive changes of the actin cytoskeleton. alpha-Actinin-4 is an actin cross-linking protein that also interacts with intra- and intercellular adhesion molecules and elements of the transmembrane signal transduction pathway and is implicated in nephrotic syndrome by animal models and human genetic studies. METHODS: We have performed the first quantitative immunoelectron microscopy study of alpha-actinin-4 expression in humans, analysing 12 cases of minimal change nephrosis (MCNS) and 16 cases of idiopathic membranous nephropathy (MGN), and comparing this with expression in normal tissue from seven nephrectomies (Nx). alpha-Actinin-4 was visualized by immunogold labelling of plastic-embedded whole glomerular cross-sections, and analysed using LUCIA software. RESULTS: Despite podocyte effacement, alpha-actinin-4 expression (group mean+/-1 SD) in MCNS was similar to that seen in normal Nx podocytes. In contrast, alpha-actinin-4 expression in MGN was significantly higher than in MCNS or Nx (P<0.001). Furthermore, in MGN cases showing a segmental deposition, expression of alpha-actinin-4 was significantly higher only in those capillary loop segments containing deposits, whereas in those segments without deposits expression was unchanged compared with that seen in Nx (P<0.001). alpha-Actinin-4 expression was higher in MGN cases where subepithelial deposits abutted podocyte cytoplasm, and lower in disease stages where deposits were contained within the glomerular basement membrane or were being reabsorbed. CONCLUSIONS: Elevated alpha-actinin-4 expression is observed only in MGN, and only in areas of subepithelial deposits. Further investigation into the cause of this may reveal insights into the pathogenesis of acquired nephrosis.

Post-embedding double-labeling of antigen-retrieved ultrathin sections using a silver enhancement-controlled sequential immunogold (SECSI) technique.

In electron microscopy, the post-embedding immunogold technique provides a high degree of resolution and the possibility of quantitation owing to the intrinsic characteristics of the colloidal gold marker. Application of this technique to the subcellular localization of multiple antigens by differential labeling using gold markers of different sizes, or to double labeling using the same primary antibody isotype with serial silver enhancement, has been reported. We have incorporated this double labeling technique into a modified procedure that produces excellent labeling and ultrastructural preservation, even after exposure of ultrathin sections large enough to cover a 300- micro m-diameter single-hole grid to hot antigen retrieval solutions and prolonged labeling protocols.