A diffusion immunogold procedure for accurate ultrastructural investigation of renal cell membrane epitopes.

A role for renal antigenic targets has been supposed and sometimes convincingly demonstrated in the development of various types of experimental glomerulonephritides. In this report we describe a reliable protocol for accurate ultrastructural investigation of antigens on the renal cell surface by means of a pre-embedding technique associated with colloidal gold staining. Sprague-Dawley rats were injected with a monoclonal antibody specific for a 90-kD cell membrane glycoprotein and killed 12 or 48 h later; after prefixation, renal fragments were cryoprotected and snap-frozen. Cryostat sections were incubated with a 5-nm colloidal gold-goat antimouse antibody, postfixed in osmium tetroxide reduced with potassium ferrocyanide and embedded in Durcupan ACM. At the glomerular level, gold granules were localized on the endothelial cell surface. In the proximal tubules uniform labelling was noticed on the brush border microvilli, followed by later marking of the basolateral membranes. By this pre-embedding immunogold method we obtained suitable histological preservation and fine resolution of the cell membrane immunoreactive sites. This procedure represents a useful tool for ultrastructural studies on the interaction of circulating antibodies with renal cell surface antigens.

Role of a 90-kD glycoprotein in Heymann's nephritis.

Heymann's nephritis can be induced in rats by injection of antibodies (Ab) against the major nephritogenic antigen (Ag) (gp330). We previously reported on a 90-kD tubular-glomerular glycoprotein (gp90) having enzymatic activity (dipeptidyl-peptidase IV) which, after antibody interaction, induces a transient Heymann-like immunofluorescence pattern. In the present study we have analysed the ultrastructural features of the renal immune reaction occurring in rats after injection of a monoclonal antibody to gp90, revealed by a pre-embedding immunogold staining (5-nm particles). In the early stage (10-60 min) gold granules were found along the glomerular capillary walls mainly in the endothelial region. During the intermediate stage (24-48 h) the brush border microvilli were diffusely labelled. In the late stage (1-2 weeks) gold particles were still noticed at the tubular level. This work suggests that the immune reaction in Heymann's nephritis also occurs on the endothelial side of the glomerular capillary walls, thus facilitating the passage of epithelial-specific antibody. The tubular kinetics confirms that gp90 may play a role in the metabolic activity of tubular proximal cells. Because this antigen has been demonstrated in human kidney as well, it may be relevant to some membranous or other human nephropathies.

[Etiopathogenesis of membranous nephropathy: is there a correlation between experimental and human pathology?]. / Eziopatogenesi della nefropatia membranosa: quale correlazione tra la patologia sperimentale ed umana?

The Authors discuss the etiologic, pathogenetic and immunopathologic aspects of Heymann nephritis, in order to compare the numerous acquisitions concerning this nephropathy with the scanty knowledge of human membranous nephropathy, of which it represents the experimental counterpart. This rat disease can be obtained by inoculation of tubular brush border preparations (active form) or of the relevant antibodies (passive form); after an initial hypothesis of glomerular deposition of circulating immune complexes, studies on its pathogenetic mechanisms, instead demonstrated that in situ immunoaggregates, caused by an interaction between circulating antibodies and fixed glomerular antigens, are formed. Recent investigations have led to the identification of a major nephritogenic antigen (gp330), which is a tubular brush border glycoprotein expressed by coated pits located at the glomerular epithelial cell surface. Studies on antigen-antibody interactions at this level have demonstrated that there is a quick redistribution and accumulation of the so-formed immune complexes, and when polyclonal antibodies were utilized, growth of subepithelial electron dense deposits was observed. Although other tubulo-glomerular antigens, which can also be expressed by endothelial cells, play an uncertain role, they seem to favour transmembrane passing of anti-gp330 antibodies. Immune complex formation gives rise to the onset of proteinuria through complement system activation, without leukocyte involvement: in particular a MAC and C9 fraction lytic effect was demonstrated on cultured epithelial cells. In conclusion, studies on Heymann nephritis contribute to our understanding of the etiopathogenetic mechanisms regarding human membranous nephropathy, and emphasize a possible role played by tubular antigens and in situ formed immune complexes.

[The physiopathologic bases of the neurotoxicity of phosphorus chelating agents containing soluble aluminum salts in patients with renal insufficiency]. / Le basi fisiopatologiche della neurotossicità dei chelanti del fosforo contenenti sali solubili di alluminio nel paziente con insufficienza renale.

To what extent can damage to the central and peripheral nervous systems be ascribed to chronic aluminum (Al) intoxication taken as a chelating agent for phosphorus, to limit hyperphosphatemia in uremic patients? Since Al is normally eliminated by the renal route, its accumulation in uremia has to be ascribed to a reduced or abolished renal clearance of the metal, which results in preferential toxicity for certain tissues, especially nervous tissue, which shows difficulty in eliminating Al, even after intake has been stopped. This review discusses, on the basis of toxicologic, experimental and clinical data, the possible pathogenic steps of Al neurotoxicity in uremia, considering: the damage to axonal transport in which Al intoxication tends to affect the components of the cytoskeleton, the polymerization phase of the alpha and beta tubulin constituents of neurotubules, and the normal translocation of neurofilaments from the perikaryon to more distal positions of the axon; the abnormalities in the brain pool of adrenergic, cholinergic and GABA neurotransmitters; the increase in permeability and changes in perm-selectivity of the blood-brain-barrier, with further loss of neurotransmitters and with acquisition, from the systemic circulation, of neurotransmitter-like substances such as hormones, monoamines and peptides, which may adversely modulate synaptic and membrane functions; the cerebral energy metabolism and particularly the hexokinase reaction, by Al replacement of the Mg-ion in the Mg-ATP complex, so that phosphorylation of glucose to G6P is blocked; the interaction of Al with calmodulin by displacement of the Ca-ion and subsequent formation of a stable Al-calmodulin complex with a cytotoxic effect due to the increase in the intracellular calcium concentration.(ABSTRACT TRUNCATED AT 250 WORDS)