Jacqueline Zanen

Tubular Injury and Regeneration in the Rat Kidney Following Acute Exposure to Gentamicin: A Time-Course Study

Aminoglycoside antibiotics act as nephrotoxic drugs, inducing a lysosomal phospholipidosis and necrotic lesions essentially in convoluted proximal tubules. Previous studies have demonstrated that tubular injury caused by these compounds elicits a process of renal tissue repair (tubular regeneration) involving an increase of cell turnover in tubular epithelium. The present study was performed in order to: (i) achieve further insight into the temporal relationship between aminoglycoside-induced phospholipidosis, tubular necrosis, and tubular regeneration; and (ii) approach the control of tubular regeneration after nephrotoxin-induced insult. To investigate the latter point, we examined by immunocytochemistry the intrarenal distribution of epidermal growth factor (EGF) during tubular regeneration. Five groups of female Sprague-Dawley rats (n = 5) were treated for 4 days with gentamicin i.p. at a daily dose of 50 mg/kg delivered in 2 injections per day. Sham-treated animals (n = 5) received an equivalent amount of vehicle (0.9% NaCl) according to the same protocol. Groups of treated rats, and controls, were terminated 16 h (day 1), 4 days, 7 days, 14 days, and 21 days after the end of gentamicin administration. One hour prior to necropsy, each animal was given an i.p. injection of 40 mg 5-bromo-2-deoxyuridine (BrdU) for the immunocytochemical demonstration of S-phase cells, using an anti-BrdU monoclonal antibody. Renal tissue was processed for light microscopy analysis, namely: a computer-aided morphometry of lysosomes in proximal tubular cells, a single-blind evaluation of gentamicin-induced tubular injury, the measurement of cell proliferation by immunocytochemical detection of BrdU-labeled nuclei, the demonstration of EGF-like immunoreactive material in renal tissue by using anti-rat EGF antiserum and immunogold-silver staining. As revealed by the morphometry of lysosomes in proximal tubular epithelium, the degree of gentamicin-induced phospholipidosis was maximum at day 1 (relative area occupied by lysosomes was increased 25-fold over mean control value) and declined thereafter. In contrast, tubular necrosis reached a peak 4 days after the end of drug administration. In proximal tubular epithelium, the stimulation of cell turnover associated with tubular regeneration showed a peak at day 7 (15-fold the mean control value). Tubular regeneration was also accompanied by mild interstitial hyperplasia. Three weeks after treatment with gentamicin, morphological evidence of drug-induced injury had disappeared due to the tissue repair process, except for the occasional presence of small hyperplastic foci in renal cortex interstitium. In both treated animals and controls, EGF immunoreactivity as revealed by immunocytochemical staining was associated with distal tubules (renal cortex and outer medulla).(ABSTRACT TRUNCATED AT 400 WORDS)

A CNBR peptide located in the middle region of diphtheria toxin fragment B induces conductance change in lipid bilayers: Possible role of an amphipathic helical segment

Abstract Conductance measurements on planar lipid bilayers demonstrate that CB1, a CNBr peptide of diphtheria toxin fragment B located in its middle region, possesses the unique property to destabilize the lipid bilayer organization. It is suggested that a segment of 25 amino acids in the N-terminal sequence of CB1 could be responsible for this effect. Its very low polarity, its predicted amphipathic helical structure and a helix length corresponding to the thickness of the hydrocarbon region of the lipid bilayer should specifically favor its insertion in the membrane. The existence of such a transverse lipid-associating domain could confer upon the molecule the properties leading to the anchoring of diphtheria toxin in the cytoplasmic membrane.

Modification of immunoreactive EGF and EGF receptor after acute tubular necrosis induced by tobramycin or cisplatin.

Acute tubular necrosis induced by aminoglycoside antibiotics and various other nephrotoxins is followed by a regenerative process which leads to the restoration of damaged tubules. Several lines of evidence indicate that tubular regeneration is mediated by polypeptide growth factors such as epidermal growth factor (EGF). Previous studies devoted to cisplatin nephrotoxicity have shown that this agent causes tubular cystic degeneration possibly related to an impairment of renal tissue repair. Thus, we examined on a comparative basis the time course of the regenerative response subsequent to tubular damage induced by tobramycin or cisplatin, particular attention being paid to renal EGF and its receptor. Female Sprague-Dawley rats (160-180 g body weight) were treated during 4 consecutive days with daily doses of 200 mg/kg tobramycin i.p. (BID) or 2 mg/kg cisplatin (once a day). Sham-treated rats were given 0.9% NaCl i.p. following the same protocol. Groups of experimental animals (n = 5-10) were terminated at increasing time intervals (1, 4, 7, 14, 21, 60 days) after cessation of treatment. One hour prior to sacrifice, each individual received i.p. 200 mg/kg 5-bromo-2-deoxyuridine (BrdU) for the immunohistochemical demonstration of cell proliferation. Blood was collected at the time of sacrifice in order to assess glomerular filtration rate by measuring serum creatinine and BUN levels. Kidneys were analyzed with respect to total EGF determined by RIA in renal tissue homogenates, and soluble EGF was assayed in extracts prepared by centrifugation. Renal tissue was processed for the immunohistochemical detection of S-phase cells, of EGF, of EGF receptors, and of the intermediate filament vimentin, the latter being used as a marker of epithelium dedifferentiation. In absence of nephrotoxic alterations, EGF was immunolocalized in distal tubules, whereas EGF receptor immunostaining was seen in proximal tubules cells. Vimentin immunostaining was confined to glomeruli and blood vessels. Tobramycin and cisplatin caused acute tubular necrosis in proximal convoluted tubules and proximal straight tubules, respectively. Tissue damage was accompanied by renal dysfunction reflected by an elevation of serum creatinine and BUN levels. Tubular necrosis was followed by a proliferative response indicative of tubular regeneration. Regenerative hyperplasia was associated with a reduction of total immunoreactive EGF due to a decrease of tissue-bound proEGF. Tubules undergoing regenerative repair were characterized by a disappearance of EGF receptors and the presence of immunoreactive vimentin. In tobramycin-treated rats, renal dysfunction lasted for 4-7 days and was fully reversible, as indicated by the return of serum markers to normal values.

Structure-activity relationships of the B fragment of diphtheria toxin: The lipid-binding domains

Two different lipid-associating domains have previously been identified in diphtheria toxin fragment B: one of the surface type in the N-terminus of B and one of the transverse type in its middle region. We have now determined about 85% of the primary structure of fragment B and show, here, that the middle part of fragment B contains a highly hydrophobic region of 72 amino acid residues (polarity index: 295) which includes the transverse lipid-associating domain. That this domain is actually involved in a process of membrane penetration is suggested by lipid bilayer conductance measurements of the CNBr peptides of fragment B and trypsin treatment of fragment B-multilamellar liposome complexes.

Phenotypic variations and dynamic topography of transformed cells in an experimental model of diethylstilbestrol-induced renal tumour in male Syrian hamster.

This work explores the phenotypic changes affecting transformed cells in an experimental model of diethylstilbestrol (DES)-induced renal tumours in male Syrian hamster. This estrogen-induced neoplasm presents an important cytological pleomorphism and its origin remains largely controversial. In order to characterize phenotypic variations during tumour progression, the occurrence of seven lineage markers was analysed by a morphometric approach in kidney sections of DES-exposed hamsters (6–;11 months). S100 protein, neuron-specific enolase (NSE) and vimentin are expressed by a large percentage of malignant cells during tumour development. Glial fibrillary acidic protein (GFAP), protein gene product 9.5 (PGP 9.5) and desmin are mostly evidenced in advanced neoplasm whereas Leu 7 always presents a focal expression. As evidenced by double-label immunofluorescence, the coexpression of three important neuroectodermal lineage markers (S100, NSE and PGP 9.5) in earliest tumour buds points to a peripheral nerve sheath origin for this neoplasm thus confirming previously published data. For each marker, the fluctuations of expression levels during tumour progression as well as the spatial heterogeneity of distribution suggest variable phenotypic differentiation of transformed cell populations. This observation is largely corroborated by double-label immunofluorescence showing coexpression modification of several markers during tumour progression. This points to a complex dynamic and spatial self-organization of different phenotypes within neoplasms. Altogether, these results support the concept that DES-induced kidney tumours are not made of unstructured cell populations but represent adaptive complex dynamic biosystems.

Immunohistochemical analysis of diethylstilbestrol-induced renal tumors in adult male Syrian hamsters: evidence for relationship to peripheral nerve sheath tumors.

Abstract. Estrogen-induced Syrian hamster kidney tumors (SHKT) are widely used as experimental models for the study of hormonal and renal carcinogenesis. In order to characterize the direction of differentiation of SHKT, kidney sections of diethylstilbestrol (DES)-treated hamsters (1–11xa0months) were analyzed by immunohistochemistry using a panel of lineage-specific markers. The first tumorous buds found in animals exposed to DES for 4–6xa0months exhibited prominent S100, Leu-7, and vimentin immunoreactivities. Immunopositivities for neuron-specific enolase, PGPxa09.5, desmin, and glial fibrillary acidic protein were mostly detected in medium-sized and large tumors after prolonged exposure to DES (>6xa0months). All neoplasms, irrespective of the size and the duration of treatment, appeared negative for cytokeratin, neurofilaments, synaptophysin, and CD99 antibodies. Western blotting confirmed to a large extent the immunohistochemical observations. The systematic analysis of serial kidney sections by confocal microscopy after double immunostaining for S100 and neurofilaments revealed that early neoplastic buds could stem from S100-positive cells associated with nerves bundles. Altogether, these observations suggest that DES-induced SHKT could be related to malignant peripheral nerve sheath tumor and originate from a yet unidentified precursor cell present in the sheath of peripheral nerves.

Dynamic of pituitary cell populations in male syrian hamster exposed to diethylstilbestrol

TCFP MEDIATES THE ACTION OF RETINOIDS AND VITAMIN D3 ON U937 CELLS DIFFERENTIATION. COMMES T&&e, PIQUEMAL David, DEFACQIJE H&ne , SEVILLA Claude et MARTI Jacques. INSERM u431. Univ-A4ontpII 34 095 Montpellier FRANCE Retinoids and vitamin D (VD) cooperate to inhibit the proliferation and induce the differentiation of human myelomonocytic leukemia cells. in order to investigate the role of TGFP as a possible mediator in this process, we used antibodies neutralizing the cytokine activity (aTGFb-Ab) r,nd studied their effects on the differentiation of U937 cells induced by various combinations of VD and synthetic retinoids. Our data demonstrate that aTGFP-Ab partially inhibit the expression of the differentiated phenotype, as assessed by measurement of phagocytic activity, response to chemobctic peptides, secretion of lysozyme and CD1 1 b expression. We also used a& sense oligonucleotides (TGF-AS) to inhibit the expression of TGFP in U937 cells, as monitored by immunofluorescence labelling. We found hat combinations of TGF-AS and aTGF-Ab had cumulative effects. Cell growth inhibition induced by VD and retinoids was nearly completely revened ad cell differentiation was largely reduced. Tie-course experiments, based on the delayed additions of aTGFb-Ab and differentiation inducers, showed that neutralizing antibodies have an effect only if added tit& he first 24h of treatment, suggesting that TGFP is involved in an early step of the differentiation process. Studies on the expression of TGFP receptor n&NAs by RT-PCR revealed that, while typ-e II receptor r&NA level remained stable, the amount of Type I TGFP receptor mRNA decreased within the first 12 h of treatment by differentiation inducers, suggesting further desensitization of cells to TGFP. Northern blot analyses showed detectable levels of TGFP mRNA in proliferating U937 cells. No variations were observed at this level upon differentiation, but the secretion of the latent TGFP protein precursor, as measured by ELBA, was increased from <SO0 pg/ml up to 2.5 &ml after 96 h in cultures supematants (normalized for 106 cells/ml) indicating a positive regulation at the post-transcriptional level. The amount of TGFP protein precursor remained low during the first 24 hours and the biologically active form of thi cytokine was not d&e&d in cell supematants. However, independent experiments showed that active recombinant TGFP actually potentiates the action of VD on cell growth inhibition and differentiation, at concenlra;ltions as low as 10 pg/ml. All these results support the notion that an autocrine TGFP pathway, activated in U937 cells treated by VD and retinoids, is involved in the early steps of the process leading to cell growth arrest and differentiation.