Long-term treatment of rats with FGF-2 results in focal segmental glomerulosclerosis.

Long-term treatment (8 and 13 weeks) of rats with FGF-2 led to albuminuria and to increase in serum creatinine indicating the development of chronic renal failure. Histologically, the classic picture of focal segmental glomerulosclerosis (FSGS) was found; males were more severely affected than females. Among the early changes podocyte lesions were most prominent. Surprisingly, mitotic figures in podocytes and a considerable fraction of bi(multi)nucleated podocyte profiles were found in treated animals (roughly 16% in males, 8% in females). Since an increase of cell number of podocytes was not evident, we conclude that FGF-2 stimulates podocytes to re-enter the cell cycle and to undergo mitosis (nuclear division). However, podocytes-probably due to their highly differentiated cell shape in the adult-are unable to complete cell division (cytokinesis) resulting in bi- or multinucleated cells; in others cell division may fail totally leading to podocyte degeneration. Most podocytes in FGF-2-treated rats exhibited degenerative changes including cell body attenuation, extensive pseudocyst formation, widespread foot process effacement, as well as detachments from the glomerular basement membrane (GBM). The development of FSGS in this model is very uniform. In the case of podocyte detachments from peripheral capillaries, parietal cells become attached to naked GBM-areas, establishing the nidus for development of a tuft adhesion to Bowman's capsule. Tuft adhesions grow by encroaching of parietal cells onto adjacent capillary loops, resulting eventually in a solid synechia with collapsed capillaries, that is, what represents segmental sclerosis. The distribution of adhesions on the inner surface of Bowman's capsule appeared to be random, including all locations between the vascular and urinary pole. The two main aspects of this study (inability of podocytes to replicate and development of FSGS based on progressing podocyte degeneration) may be part of a vicious cycle. FGF-2 stimulates podocytes to enter cell division thereby conveying them into a hazardous situation. If a podocyte fails and degenerates it cannot be replaced, aggravating the situation for the remaining cells and possibly increasing their predisposition to respond to mitogenic stimuli. Similar mechanisms may constitute the development of FSGS in other experimental as well as human glomerulopathies.

The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3.

The ras-related rho A protein expressed in E. coli, was ADP-ribosylated by botulinum ADP-ribosyltransferase C3. C3 also modified the valine-14 mutant rho protein but not the products of H-ras, R-ras, ral, ypt, and rap 1 genes. A ras-rho chimaera consisting of 60 amino acids from the amino terminus of ras fused to 133 amino acids from the carboxy terminus of rho was not modified by C3. Antibodies raised against the porcine brain cytosolic substrate of C3 cross reacted with the rho, valine-14 rho and ras-rho proteins, but not with the gene products of H-ras, R-ras, ral or rap 1. Polyclonal anti-H-ras antibodies cross reacted with H-ras but not with ral, rho, or the C3 substrate purified from porcine brain.

Botulinum toxin-induced ADP-ribosylation and inhibition of exocytosis are unrelated events.

The hypothesis that inhibition of secretion by botulinum neurotoxin type D occurs by an intracellular process involving ADP-ribosylation has been directly tested by measuring both the extent of inhibition of secretion and of ADP-ribosylation in the same cells. Although the inhibitory effect of unpurified toxin closely parallels intracellular ribosylation, the two events are clearly unrelated, as using purified D and C3 toxins together with their antibodies, each of these events can be either stimulated or inhibited independently of each other.

Botulinum ADP-ribosyltransferase C3. Purification of the enzyme and characterization of the ADP-ribosylation reaction in platelet membranes.

A novel ADP-ribosyltransferase C3 was purified to homogeneity from filtrates of certain strains of Clostridium botulinum type C by ammonium sulfate precipitation, gel filtration, ion-exchange chromatography and heat treatment. The molecular mass of botulinum ADP-ribosyltransferase C3 was found to be 25 kDa. In the presence of [32P]NAD but not with [carbonyl-14C]NAD, C3 labelled 21-24-kDa protein(s) in membranes of human platelets and other tissues. The Km value of the ADP-ribosylation reaction for NAD was about 2 microM. Labelling of the 21-24-kDa protein(s) by C3 was largely reduced by addition of nicotinamide. Snake venom phosphodiesterase cleaved the ADP-ribose attached to the 21-24-kDa protein(s) by C3 and released 5'AMP. C3 catalyzed hydrolysis of [carbonyl-14C]NAD and released [carbonyl-14C]nicotinamide. ADP-ribosylation of 21-24-kDa platelet membrane protein(s) was biphasically regulated by Mg2+, Mn2+ and Ca2+. In the absence of free divalent cations GTP, GTP[gamma S] and GDP but not GDP[beta S], GMP, ATP or ATP[gamma S] increased labelling by C3. In the presence of Mg2+, GTP[gamma S] was inhibitory. Guanine nucleotides prevented heat inactivation of the substrate protein(s) with the rank order GTP[gamma S] = GTP = GDP greater than GDP[beta S] greater than GMP much greater than ATP = GMP = ATP[gamma S]. The data support the view that the novel ADP-ribosyltransferase C3 modifies eukaryotic 21-24-kDa GTP-binding protein(s).

Botulinum ADP-ribosyltransferase C3 but not botulinum neurotoxins C1 and D ADP-ribosylates low molecular mass GTP-binding proteins.

Botulinum ADP-ribosyltransferase C3 modified 21-24 kDa proteins in a guanine nucleotide-dependent manner similar to that described for botulinum neurotoxin C1 and D. Whereas GTP and GTP gamma S stimulated C3-catalyzed ADP-ribosylation in the absence of Mg2+, in the presence of added Mg2+ ADP-ribosylation was impaired by GTP gamma S. C3 was about 1000-fold more potent than botulinum C1 neurotoxin in ADP-ribosylation of the 21-24 kDa protein(s) in human platelet membranes. Antibodies raised against C3 blocked ADP-ribosylation of the 21-24 kDa protein by C3 and neurotoxin C1 but neither cross reacted with neurotoxin C1 immunoblots nor neutralized the toxicity of neurotoxin C1 in mice. The data indicate that the ADP-ribosylation of low molecular mass GTP-binding proteins in various eukaryotic cells is not caused by botulinum neurotoxins but is due to the action of botulinum ADP-ribosyltransferase C3. The weak enzymatic activities described for botulinum neurotoxins appear to be due to the contamination of C1 and D preparations with ADP-ribosyltransferase C3.