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.

No abstract

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 The data support the view that the novel ADP-ribosyltransferase C3 modifies eukaryotic 21 -24-kDa GTP-binding protein(s).Eight different botulinum toxins (A -G) produced by certain strains of Clostridium botulinum have been described. Among these botulinum toxins A, B, C1, D, E, F, and G are typical neurotoxins, and are the most potent of the known biological or chemical toxins [I, 21. These toxins inhibit the neurotransmitter release at cholinergic and non-cholinergic synapses by an as yet unknown mechanism [l]. In contrast, botulinum C2 toxin is not neurotoxic but cytotoxic [2,4]. This toxin belongs to the family of microbial ADP-ribosyltransferases such as cholera, pertussis and diphtheria toxins [5-71. It has been shown that component I of the binary botulinum C2 toxin ADP-ribosylates specifically non-muscle actin, thereby severely impairing the ability of actin to polymerize [3, 5, 8, 91. Recently it has been reported that besides botulinum C2 toxin another botulinum ADP-ribosyltransferase is produced by C. botulinum type C [lo, 111. In order to distinguish this novel microbial enzyme from botulinum toxins C1 and C2, we termed the enzyme botulinum ADP-ribosyltransferase C3. The novel ADP-ribosyltransferase modified 21 -24-kDa eukaryotic protein(s) in several tissues including human platelets, fibroblasts, neuroblastoma x glioma hybrid cells, S49 lymphoma cells and brain tissue [lo, 111. The well-studied bacterial ADP-ribosyltransferases, cholera and pertussis toxin, are known to act on eukaryotic cells by modifying the GTP-binding proteins G, and Gi, respectively [6]. GTP-binding proteins play key roles in hormonal and sensory signal transduction of eukaryotic cells by regulating the functional coupling between receptors and effector systems such as adenylate cyclase, phospholipase C and ion channels [12]. Interestingly it has been suggested that also botulinum ADP-ribosyltransferase C3 modifies GTP-binding proteins, since hydrolysis-resistent GTP analogues reportedly inhibit ADP-ribosylation by C3 [ll].As so far basic information on the novel botulinum ADPribosyltransferase is lacking we report in this communication on the purification and characterization of botulinum ADPribosyltransferase C3 in detail. In order to gain more insight into the C3-catalyzed reaction and to substantiate the hypothesis that the eukaryotic substrate is a GTP-binding protein, we studied the influence of divalent cations and guanine nucleotides on ADP-ribosylation by C3.

Podocytes are lost by detachment from the GBM as viable cells; details are largely unknown. We studied this process in the rat after growth stimulation with FGF-2. Endothelial and mesangial cells responded by hyperplasia, podocytes underwent hypertrophy, but, in the long run, developed various changes that could either be interpreted showing progressing stages in detachment from the GBM or stages leading to a tighter attachment by foot process effacement (FPE). This occurred in microdomains within the same podocyte; thus, features of detachment and of reinforced attachment may simultaneously be found in the same podocyte. (1) Initially, hypertrophied podocytes underwent cell body attenuation and formed large pseudocysts, i.e., expansions of the subpodocyte space. (2) Podocytes entered the process of FPE starting with the retraction of foot processes (FPs) and the replacement of the slit diaphragm by occluding junctions, thereby sealing the filtration slits. Successful completion of this process led to broad attachments of podocyte cell bodies to the GBM. (3) Failure of sealing the slits led to gaps of varying width between retracting FPs facilitating the outflow of the filtrate from the GBM. (4) Since those gaps are frequently overarched by broadened primary processes, the drainage of the filtrate into the Bowman’s space may be hindered leading to the formation of small pseudocysts associated with bare areas of GBM. (5) The merging of pseudocysts created a system of communicating chambers through which the filtrate has to pass to reach Bowman’s space. Multiple flow resistances in series likely generated an expansile force on podocytes contributing to detachment. (6) Such a situation appears to proceed to complete disconnection generally of a group of podocytes owing to the junctional connections between them. (7) Since such groups of detaching podocytes generally make contact to parietal cells, they start the formation of tuft adhesions to Bowman’s capsule.

Botulinum ADP-ribosyltransferase C3 modified 21-24 kDa proteins in a guanine nucleotide-dependent manner similar to that described for botulinum neurotoxin CI and D. Whereas GTP and GTP~S stimulated C3-catalyzed ADP-ribosylation in the absence of Mg a +, in the presence of added Mg 2 + ADP-ribosylation was impaired by GTPyS. 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 CI 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 CI and D preparations with ADP-ribosyltransferase C3.ADP-ribosylation; Botulinum ADP-ribosyltransferase C3; Botulinum-neurotoxin C1; Botulinum neurotoxin D; GTP-binding protein