Jeanine-Anne Heuson-Stiennon

Redistribution of Epidermal Growth Factor Immunoreactivity in Renal Tissue after Nephrotoxin-Induced Tubular Injury

Tubular necrosis elicits a process of renal tissue repair characterized by an increase of cell turnover in tubular epithelium. The present study was undertaken to examine the distribution of epidermal growth factor (EGF) and/or of its larger precursor proEGF in the kidney undergoing tubular regeneration. Sprague-Dawley rats were exposed to various drugs (aminoglycosides or platinum-based anticancer agents) known to induce tubular necrosis. The proliferative response resulting from renal tissue damage was measured by the incorporation of [3H]thymidine into DNA of renal cells. EGF immunoreactivity was evidenced by immunocytochemical staining, using anti-EGF antibody and immunogold-silver staining. Concomitantly with the increase of cell proliferation resulting from tubular injury, a redistribution of EGF immunoreactivity was observed in renal tissue (from the inner stripe of outer medulla towards renal cortex). Amazingly, EGF was detected in proximal tubules of nephrotoxin-treated rats whereas, in the kidneys of control animals, it was almost exclusively found in distal tubules and collecting ducts. Insofar as the administration of exogenous EGF has recently been shown to enhance renal tubular regeneration after ischaemic injury [Humes et al: J Clin Invest 1989; 84:1757-1761], our observations lend further support to the concept that EGF might be involved in renal tissue repair.

Demonstration of estrogen receptors and of estrogen responsiveness in the HKT-1097 cell line derived from diethylstilbestrol-induced kidney tumors

SummaryThis study was undertaken in order to examine the estrogen sensitivity of HKT-1097, an established cell line recently derived from diethylstilbestrol (DES)-induced kidney tumors in Syrian hamsters. Estrogen receptor (ER) level in HKT-1097, determined by enzyme-linked immunoassay, was 67 fmol/mg protein, i.e., a value approx. 30% lower than that found in Syrian hamster kidney tumors. ER immunostaining in cells fixed with Carnoys mixture, as well as ER demonstration by Western blotting, suggested DES-induced nuclear translocation or stabilization of the receptor within the nucleus. Kinetic parameters of estrogen binding to ER in HKT-1097 cells were 8.4×10−11M and 60.8 fmol/mg protein for Kd and Bmax, respectively. The Kd of estrogen binding to ER in HKT-1097 was close to that evaluated for the receptor in breast cancer-derived MCF-7 cell line, whereas the Bmax value was approx. seven times lower in HKT-1097 as compared to MCF-7. In HKT-1097 cells, antiestrogens ICI 182,780 and RU 58,668 induced ER downregulation and competed with estrogen binding to the receptor. As demonstrated by Western blot analysis, DES exposure led to an increased expression of progesterone receptor (PgR) in HKT-1097 cells. Addition of DES to estrogen-free medium produced a stimulation of growth in both HKT-1097 and MCF-7 cells, but the mitogenic effect was less marked for HKT-1097. Despite the fact that ICI 182,780 and RU 58,668 clearly interact with HKT-1097 cell ER, they appeared unable to suppress DES-induced stimulation of growth and increase of PgR expression.

Renal epidermal growth factor precursor: proteolytic processing in an in vitro cell-free system

The enzymatic processing of the membrane-bound renal epidermal growth factor precursor (proEGF) could be an important step in the control of nephrogenic repair consecutive to kidney insult. The enzyme machinery responsible for that processing was examined in a cell-free system consisting of renal membranes isolated from kidney homogenates by differential centrifugation, and incubated in vitro. After a 24-h incubation at 37 degrees C, 6-14% of membrane-bound proEGF was processed and soluble products with EGF immunoreactivity were released. As revealed by HPLC and Western blotting analysis, the products of proEGF proteolysis consisted of 6 kDa EGF (the molecular weight of mature EGF) and two polypeptides with molecular weights around 45 kDa. Interestingly the 45 kDa EGF forms, like the 6 kDa EGF, exhibited mitogenic activity toward growth-arrested NRK-52E renal cell line. The kinetic study of proEGF degradation gave data consistent with the 45 kDa product(s) being processing intermediate(s) between proEGF and 6 kDa EGF. The enzymatic activity responsible for proEGF nicking was inhibited by divalent heavy metal ions (Cu2+ or Zn2+) and several protease inhibitors (aprotinin, PMSF, leupeptin, soybean trypsin inhibitor), suggesting that proEGF is processed by kallikrein-like serine proteases present in the membrane preparations. Along with previous studies, the current observations suggest that renal kallikreins might play a role in renal tubular regeneration by promoting the release of soluble EGF in renal tissue.

Characterization of a cell line established from diethylstilbestrol-induced renal tumors in Syrian hamsters.

SummaryThis article describes HKT-1097, a new cell line established from renal tumors induced by the protracted administration of diethylstilbestrol (DES) to male Syrian golden hamsters. Cell culture was initiated from tumor samples obtained from two 14-mo.-old animals which had undergone exposure to DES for a period of 11 mo. The HKT-1097 cell line was characterized between Passages 16 and 22 with respect to cell morphology, growth properties, karyology, and the presence of estrogen receptors. Moreover, immunostaining with a panel of antisera was performed to identify the cytological profile of the cell line and establish a parallel with tumor tissue in vivo. HKT-1097 cells are fibroblastoid; their most distinctive feature is that they exhibit strikingly long processes. The HKT-1097 cell line grows as a monolayer with a tendency toward a less stringent density-dependent inhibition of growth. The modal chromosome number is 44, but more than 50% of the cells are aneuploid, suggesting a substantial degree of karyotype instability. HKT-1097 cells express estrogen receptors. They contain immunoreactive vimentin and desmin, but appear negative upon cytokeratin immunostaining. In addition, these cells express glial fibrillary acidic protein and other markers of the neuroectodermal lineage, but lack neurofilament protein. Insofar as the same lineage markers have been demonstrated in DES-induced Syrian hamster kidney tumors (SHKT), we conclude that HKT-1097 cells retain some of the original tumor cell phenotype. The current observations suggest that estrogen-induced SHKT derive from the renal interstitium and point to an involvement of neuroectodermal cells in the development of these neoplasms.

Light and electron microscopic characterization of the proliferative response induced by tobramycin in rat kidney cortex.

Administration of aminoglycoside antibiotics is frequently associated with tubular necrosis which can eventually lead to renal dysfunction. Previously, we have shown that renal tissue injury due to aminoglycoside nephrotoxicity elicits a process of tissue repair characterized by stimulation of cell proliferation. The present study was undertaken to examine both quantitatively and qualitatively the cell proliferation associated with renal tissue repair. Female Sprague-Dawley rats (180-200 g body weight) were treated ip for 10 days with various doses of tobramycin (10, 20, or 50 mg/kg twice daily). Each animal received 200 microCi [3H]thymidine 1 hr before sacrifice to evaluate the extent of cell proliferation in renal cortex. The rate of DNA synthesis in renal cortex was estimated by measuring the specific radioactivity of the nucleic acid. The frequency and localization of S-phase cells in cortex tissue were determined on paraffin and plastic tissue sections processed for histoautoradiography. In addition, the ultrastructure of proliferating cells was characterized by electron microscopic examination of consecutive ultrathin sections. An excellent correlation (r = 0.993) was found between the rate of DNA synthesis and the frequency of S-phase cells evaluated in rats receiving various doses of tobramycin. The stimulation of cell proliferation involved mostly proximal tubular cells and interstitial cells. The latter cells had the ultrastructural appearance of fibroblasts at various stages of differentiation. Similarly, S-phase cells in proximal tubules were either fully differentiated epithelial cells or immature elements. Taken together, the present experimental data illustrate the capacity of the kidney to trigger complex tissue reactions in response to nephrotoxic injury.

In vitro demonstration of a mitogenic activity in renal tissue extracts during regenerative hyperplasia

Normal rat kidney (NRK-52E) cells, an established cell line of renal origin, were used as a bioassay system to reveal a possible mitogenic activity in tissue extracts prepared from kidneys undergoing tubular regeneration. Acute tubular injury was induced in female Wistar rats by a 4-day treatment with gentamicin at daily doses of 50 or 100 mg/kg twice daily. Animals were killed either 1 or 4 days after cessation of gentamicin administration. Proximal tubule regeneration in treated animals was confirmed by morphological examination after proliferating cell nuclear antigen staining. Tissue extracts from regenerating kidneys stimulated DNA synthesis in growth-arrested cells to a higher extent than extracts from intact kidneys. Sera from treated and control animals showed no difference with respect to mitogenic activity. The mitogenic effect of tissue extracts was sensitive to the tyrosine kinase inhibitor tyrphostin A46. The cell proliferative response to regenerating kidney extracts, but not that to intact kidney extracts, was partly suppressed by the addition of anti-insulin-like growth factor I (anti-IGF-I) antiserum. These data indicate that nephrogenic repair entails an elevation of biologically active IGF-I in kidney tissue.Normal rat kidney (NRK-52E) cells, an established cell line of renal origin, were used as a bioassay system to reveal a possible mitogenic activity in tissue extracts prepared from kidneys undergoing tubular regeneration. Acute tubular injury was induced in female Wistar rats by a 4-day treatment with gentamicin at daily doses of 50 or 100 mg/kg twice daily. Animals were killed either 1 or 4 days after cessation of gentamicin administration. Proximal tubule regeneration in treated animals was confirmed by morphological examination after proliferating cell nuclear antigen staining. Tissue extracts from regenerating kidneys stimulated DNA synthesis in growth-arrested cells to a higher extent than extracts from intact kidneys. Sera from treated and control animals showed no difference with respect to mitogenic activity. The mitogenic effect of tissue extracts was sensitive to the tyrosine kinase inhibitor tyrphostin A46. The cell proliferative response to regenerating kidney extracts, but not that to intact kidney extracts, was partly suppressed by the addition of anti-insulin-like growth factor I (anti-IGF-I) antiserum. These data indicate that nephrogenic repair entails an elevation of biologically active IGF-I in kidney tissue.

Tubular Regeneration in Rat Kidney Cortex During Treatment with Gentamicin at a Low Dose

Tissue injury induced in rat kidney cortex by gentamicin at a low dose (10 mg/kg) has been studied by measuring subsequent regeneration after treatment for 4, 7, and 14 days. Cell proliferation was demonstrated by the incorporation of [3H]-thymidine into kidney cortex DNA. In treated animals, the specific radioactivity of DNA increased up to 4.6 times the mean value found in the controls. Autoradiography showed labelling of nuclei in proximal tubular cells. Electron microscopy showed that a subpopulation of tubular cells, devoid of myeloid bodies, which are characteristic of gentamicin intoxication, is also poorer in peroxisomes, and therefore may belong to less differentiated, regenerating cells. It is concluded that a low dose of gentamicin induces measurable tissue regeneration.

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.

APOPTOSIS AND CELL PROLIFERATION IN A MODEL OF RENAL TUMOR IN THE MALE SYRIAN HAMSTER EXPOSED TO DIETHYLSTILBESTROL

Mutation in the dlg gene cause neoplastic imaginal discs overgrowth. It encodes a protein required for septate junctions structure. Several vertebrate homologs of dlg have been identified. One of them., the dog 20-2 IS a protein component of epithelial tight junctions. Tight junchons form a belt around the cell, maintaining apical-basal cell polarity. In the course of a chromosomal walking in tbe Friderich ataxia chromosomic region, the Xl04 gene was isolated by exon trapping and further identified as the human 20-2 gene. A mZO-2 cDNA was cloned from a 10.5 day mouse embryo library using a human 20-2 cDNA probe. DNA sequence anal member of the MAGUK familv with PDZ. SH 3 sis indicates that mZO-2 is a and GUK domains. We have examined the RNA expression pattern of the 20-2 gene during mouse embryogenesis by means of in siru RNA hybridization. The transcript mZG2 is specifically expressed in the colon, and tightly regulated during lung and kidney organogenesis. Detailed pattern of expression for mZG-2 will be presented. These data suggest a specific role for mZO-2 during mouse embryogenesis. The cnzrtion of a null mutation for 20-2 in mouse will be of importance since no protein component of the tight junction have been knock out in a whole organism. In other hand, we can test ZG-2 as a tumor suppressor gene by bansfecting its cDNA into tumor cells which do not express the protein: preliminary results will be presented.

Transforming growth factor-α and epidermal growth factor in hamster tissues: Biochemical and immunohistochemical studies

Abstract In Syrian golden hamster kidneys and submaxillary glands, the levels of EGF, determined by radioimmunoassay, were much lower than in the same organs of two other rodent species, mouse and rat. In submaxillary glands, the EGF/TGF-α receptor-binding activities were also much lower in hamster than in mouse and rat. In contrast, the TGF-α content of hamster kidneys, determined by radioimmunoassay, was higher than in the kidneys of the other animals, as was the EGF/TGF-α receptor-binding activity. Using immunohistochemistry, the TGF-α immunoreactivity in hamster kidneys was localized both in proximal and distal tubules with the exception of the macula densa area. The levels of TGF-α in the submaxillary glands were very low in all the animals tested. Hamster kidney extracts contained a specific immunoreactive protein with the M r and the TV-terminal amino acid sequence (VVSHFNECPD) expected for mature hamster TGF-α. Western blot analysis of hamster renal solubilized membrane proteins using anti-EGF receptor antibodies revealed three immunoreactive protein bands of which one had the M r expected for the EGF/TGF-α receptor. The immunohistochemical pattern of this receptor in hamster kidneys proximal tubular cells was very similar to the other tested rodent species.