Immunohistochemistry of the golden hamster pituitary during chronic administration of diethylstilbestrol: a quantitative analysis using confocal laser scanning microscopy.

The aim of this study was to examine by immunohistochemistry the morphologic changes affecting pituitary cell populations in male Syrian hamsters undergoing chronic exposure (3 days to 9 months) to diethylstilbestrol (DES). Cell proliferation in the hypophysis was monitored by the immunohistochemical demonstration of S-phase cells after pulse labeling with 5-bromo-2'-deoxyuridine. Cell proliferation analysis was combined with the identification of different cell populations by immunostaining with antisera raised against hypophyseal hormones. Sections processed for double-label immunofluorescence were examined by confocal microscopy. In the adenohypophysis, the relative surface occupied by gonadotrophs and thyrotrophs decreased rapidly during the first months of treatment while corticotroph and somatotroph populations remained unaffected. Accordingly, the incidence of S-phase cells in these four cell populations was lower than or similar to control values. In contrast, lactotrophs increased gradually during the first month of exposure to DES to reach a maximum value at 2-4 months. At the beginning, this increase was primarily due to hyperplasia but later on it also involved cellular hypertrophy. Somatomammotrophs did not seem to be involved in this model. In the pars intermedia, the labeling index of melanotrophs rose rapidly to reach values 5-6 times higher than controls. After 4 months, neoplasms originating from the pars intermedia were seen invading both the neuro- and the adenohypophysis. At the end of treatment, the pituitary was markedly enlarged resulting from the development of an adenoma of the pars intermedia.

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

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 (1-11 months) were analyzed by immunohistochemistry using a panel of lineage-specific markers. The first tumorous buds found in animals exposed to DES for 4-6 months exhibited prominent S100, Leu-7, and vimentin immunoreactivities. Immunopositivities for neuron-specific enolase, PGP 9.5, desmin, and glial fibrillary acidic protein were mostly detected in medium-sized and large tumors after prolonged exposure to DES (> 6 months). 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.

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

This 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 Carnoy's 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 x 10(-11) M 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.

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

This 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.

Apoptosis and cell proliferation in the seminal vesicles and coagulating glands of male Syrian hamsters exposed to diethylstilboestrol (DES).

Regression of the accessory sex glands was induced in male Syrian hamsters by chronic exposure to diethylstilboestrol (DES), an agonist of 17beta-oestradiol. Experimental groups (n = 4-5) were killed at increasing time intervals up to 6 months after initiation of treatment. Organ atrophy was evaluated by morphological examination. Apoptosis in the seminal vesicles and coagulating glands was visualized by in situ analysis of DNA fragmentation. Cell proliferation was monitored by immunostaining nuclei of S-phase cells after pulse labelling with BrdU. The volume of the seminal vesicles decreased drastically after 2 weeks of DES administration due to a marked reduction of secretions in the lumen of the glands. Cell proliferation in the seminal vesicles was stimulated by chronic administration of DES. Mitotic activity mostly increased during the first month of treatment, leading to epithelial hyperplasia associated with progressive hyperplasia of the fibromuscular stroma. Evidence of epithelial dysplasia and metaplasia, often associated with an infiltration of polymorphonuclear leukocytes, was observed in animals exposed to DES for 4 months or more. Regression of the seminal vesicles was also associated with apoptosis in the gland epithelium. Apoptosis appeared 3 days after starting DES administration and culminated at 1 month. Thereafter the number of apoptotic cells decreased progressively, but apoptosis remained present until the end of treatment. In contrast, coagulating glands were less sensitive to DES. No major morphological changes were observed in these glands except for a moderate reduction of protein secretion. The levels of the apoptotic and proliferating cells indices were very low in the coagulating glands during DES treatment. In conclusion, these data point to different sensitivities of the accessory sex glands to DES exposure because DES induces extensive alterations of the normal morphology of the seminal vesicles, but shows only a moderate impact on the coagulating glands.

Sublethal alterations and sustained cell proliferation associated with the diethylstilbestrol-induced renal carcinogenesis in male Syrian golden hamsters.

The current study was initiated to explore the sublethal alterations and the tissue damage occurring in the hamster kidney during diethylstilbestrol-induced renal carcinogenesis. A total of 49 male Syrian golden hamsters (35 treated and 13 control animals) was utilized in the experimental procedure. Chronic exposure to diethylstilbestrol was achieved by s.c. insertion of implants containing 25 mg diethylstilbestrol. For long-term observation, adequate blood level of diethylstilbestrol was insured by renewing the implant every 2 months. Experimental groups (n = 4 to 9) were terminated 1, 2, 4, 6, 9 and 11 months after initial implantation for morphological examination of the kidney. Diethylstilbestrol carcinogenicity in this experimental model was confirmed by the observation that most animals undergoing drug exposure for 6 months or more exhibited renal neoplasms. The most striking nonneoplastic morphological abnormality disclosed by histological and cytological examination consisted in the accumulation of granular inclusions in proximal tubule cells. In renal tissue, the extent of cell proliferation determined by PCNA labeling progressively increased along with the duration of diethylstilbestrol exposure and suggested a sustained proliferative response in altered proximal tubules. The present data suggest that an impairment of functional tubular regeneration could promote, as well as the estrogen genotoxic effect, the tumorigenicity of diethylstilbestrol in the kidney of male hamsters.

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.

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.

Attenuation of gentamicin-induced nephrotoxicity in rats by fleroxacin.

The effect of fleroxacin on gentamicin-induced nephrotoxicity was evaluated with female Sprague-Dawley rats. Animals were injected during 4 or 10 days with saline (NaCl; 0.9%), gentamicin alone at doses of 10 and 40 mg/kg of body weight/12 h (subcutaneously), fleroxacin alone at a dose of 25 mg/kg/12 h (intraperitoneally), or the combination gentamicin-fleroxacin in the same regimen. Gentamicin induced a dose- and time-dependent renal toxicity as evaluated by gentamicin cortical levels, sphingomyelinase activity in the renal cortex, histopathologic and morphometric analysis, blood urea nitrogen and serum creatinine levels, and cellular regeneration ([3H]thymidine incorporation into DNA of cortical cells). The extent of these changes was significantly reduced when gentamicin was given in combination with fleroxacin. Although the mechanisms by which fleroxacin reduces the nephrotoxic potential of gentamicin are unknown, we propose that the fleroxacin-gentamicin combination enhances exocytosis activity in proximal tubular cells, as suggested by the higher excretion of urinary enzymes and lower cortical levels of gentamicin observed in animals treated with the combination fleroxacin-gentamicin compared with those treated with gentamicin alone. The protective effect of fleroxacin on gentamicin nephrotoxicity should be investigated further.

In situ demonstration of germinal cell apoptosis during diethylstilbestrol-induced testis regression in adult male Syrian hamsters.

Testis regression was induced in male Syrian hamsters by chronic exposure to diethylstilbestrol (DES), and estradiol-17 beta agonist. Experimental groups (n = 4-5) were killed at increasing time intervals over a period of 6 mo after initiation of treatment. Apoptosis in testes was demonstrated by in situ analysis of DNA fragmentation. Cell proliferation was monitored by immunostaining nuclei of S-phase cells after pulse labeling with 5-bromo-2'-deoxyuridine. Levels of FSH and testosterone, measured by RIA fell rapidly in DES-treated hamsters. In parallel, testis weight and seminiferous tubule area underwent an 80% decrease during the first 2 wk of DES administration. The composition of seminiferous epithelium was also drastically affected by DES, since it became progressively confined to Sertoli cells, spermatogonia, and spermatocytes. Testis regression was associated with an important increase of apoptosis, which started 3 days after the beginning of DES administration. Apoptosis was still 10- to 50-fold higher than in control testes by the end of treatment; it affected primarily spermatocytes and, to a much lesser extent, spermatogonia. Cell proliferation was not inhibited by chronic DES administration. In conclusion, these data indicate that apoptosis can by itself account for estrogen-induced testis regression.

Involvement of transforming growth factor-alpha and its receptor in a model of DES-induced renal carcinogenesis in the Syrian hamster.

This study explores the role played by TGF alpha in estrogen-induced renal tumors. Tumors were induced in male Syrian hamster by chronic administration of diethylstilbestrol (DES). Six experimental groups (n = 5-9) were chronically exposed to DES and sacrificed after 1, 2, 4, 6, 9 and 11 months, respectively. In the course of treatment, the nephrons were the site of an important increase of cell turnover, which was characterized by a process of hyperplasia/dysplasia in proximal tubules preceding the neoplastic transformation. In treated animals and in controls, the analysis of renal tissue by Western blot revealed the presence of a 6 kDa polypeptide crossreacting with anti-TGF alpha antibody. In controls, TGF alpha immunoreactivity was localized in proximal and in distal tubules. Before tumor development (1-4 months), TGF alpha RIA showed an increase of TGF alpha concentration in renal tissue, in parallel with the increased cell proliferation observed in proximal tubules. In addition, Western blot analysis also demonstrated in kidney tissue the presence of a 165 kDa protein displaying the immunoreactivity of EGF/TGF alpha receptor. The receptor immunoreactivity was localized in proximal tubular cells suggesting an involvement of TGF alpha in tubular epithelial growth through autocrine or paracrine pathways. In large neoplasms, immunocytochemistry revealed only clusters of transformed cells intensely stained by the anti-TGF alpha antibody. These cells displayed the appearance of stellate or polyhedric cells infiltrating adjacent neoplastic tissues. Antisera raised against intra- or extracytoplasmic domains of the EGF/TGF alpha receptor were assayed to localize this receptor in the tumors. In contrast with tubular structures, immunoreactivity to EGF/TGF alpha receptor was never detected in tumoral tissue. The apparent absence of EGF/ TGF alpha receptor immunoreactivity in malignant cells seems to exclude an involvement of this growth factor in the tumorigenic process, although it could be involved in tumor neovascularization.

Transforming growth factor-alpha and epidermal growth factor in hamster tissues: biochemical and immunohistochemical studies.

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-alpha receptor-binding activities were also much lower in hamster than in mouse and rat. In contrast, the TGF-alpha content of hamster kidneys, determined by radioimmunoassay, was higher than in the kidneys of the other animals, as was the EGF/TGF-alpha receptor-binding activity. Using immunohistochemistry, the TGF-alpha immunoreactivity in hamster kidneys was localized both in proximal and distal tubules with the exception of the macula densa area. The levels of TGF-alpha 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 N-terminal amino acid sequence (VVSHFNECPD) expected for mature hamster TGF-alpha. 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-alpha receptor. The immunohistochemical pattern of this receptor in hamster kidneys proximal tubular cells was very similar to the other tested rodent species.

Renal tissue expression of EGF and EGF receptor after ischaemic tubular injury: an immunohistochemical study.

Rat kidneys undergoing tubular regeneration after ischaemic injury have been examined with regard to EGF, EGF receptor and vimentin, using immunohistochemical techniques. Renal ischaemia was induced in male Sprague-Dawley rats by 35-min clamping of renal arteries. Groups (n = 4-6) of experimental animals were killed at different time intervals (12, 24, 48, 72 h, 7 and 14 days) after reperfusion. One hour before sacrifice, each rat received i.p. 200 mg/kg 5-bromo-2'-deoxyuridine (BrdU) for the immunocytological demonstration of DNA synthesis. Renal necropsies were processed to reveal by immunohistochemistry EGF, EGF receptor, vimentin, and BrdU incorporated into DNA of S-phase cells. Tubular necrosis particularly involved proximal straight tubules in the outer stripe of the outer medulla and was followed by tubular regeneration, with a peak of cell proliferation at 48-72 h and an apparent dedifferentiation of tubular epithelium. As soon as 12 h after ischaemia, there was a substantial reduction of EGF immunostaining and the incidence of distal tubules showing EGF immunoreactivity reached a nadir at 48 h. In control kidneys, EGF receptor was mostly immunolocalized in proximal tubules although juxtaglomerular cells also exhibited immunolabelling. EGF receptor immunostaining in tubular epithelium showed no major change during the episode of tubular necrosis (12-24 h) but disappeared in tubular profiles undergoing regenerative hyperplasia (48-72 h). No vimentin immunostaining was found in tubules of control kidneys. Tubular epithelium remained mostly vimentin negative during the early phase of tubular necrosis/regeneration (12-72 h). By contrast, 7 days after ischaemia numerous dedifferentiated tubules expressed vimentin. In conclusion, tubular regeneration after ischaemia is associated with a typical sequence of transient events: (1) reduction of EGF immunostaining; (2) disappearance of EGF receptors during the mitogenic response; (3) expression of vimentin in tubular epithelium, and (4) return to a normal appearance.

Potentiation of gentamicin nephrotoxicity in the rat by infusion of aprotinin.

The present study was undertaken to examine a possible effect of aprotinin, a 6.5-kDa polypeptide with an inhibitory effect on proteolysis, on aminoglycoside nephrotoxicity. Experimental animals (female Sprague-Dawley rats, 175-200 g body wt) were treated for 4 days with 40 mg/kg gentamicin given ip at 12-hr intervals. Aprotinin (40,000 kIU per animal) was infused i.v. over a period of 8 days, using subcutaneously implanted miniosmotic pumps. In protocol A, infusion pumps were placed 4 days before starting gentamicin treatment. In protocol B, pumps were implanted 15-18 hr prior to first gentamicin administration. In addition to rats exposed to both gentamicin and aprotinin (GAP), animals were treated with gentamicin ip+saline i.v. (G), saline ip+aprotinin i.v. (AP), or received only saline by both routes of administration (C). All rats were terminated 4 days after the end of gentamicin dosing. One hour before sacrifice, 200 microCi of [3H]thymidine was given ip to each animal in order to monitor cell turnover in renal tissue. The kidneys were analyzed with respect to (i) histopathological alterations and renal dysfunction, (ii) aminoglycoside tissue accumulation, and (iii) tubular regeneration (measurement of cell proliferation). In animals receiving aprotinin alone, histological examination of renal cortex on paraffin sections disclosed mild tubular injury with focal cell necrosis. In plastic-embedded tissue, proximal tubule epithelium was characterized by the presence of numerous inclusions densely stained with toluidine blue. At the ultrastructural level, these inclusions appeared filled with amorphous electron-dense material. In gentamicin-treated animals, cortical drug accumulation reached values higher than 0.3 mg/g renal tissue, but a significant 30-40% decrease of gentamicin accumulation was noted in GAP groups, compared to G groups. Histological examination of renal cortex (paraffin sections) revealed the development of acute tubular necrosis in both G and GAP groups. Tubular injury was accompanied by mild renal dysfunction, as shown by the level of serum creatinine which was increased almost 3-fold in the G group, compared to C and AP groups. Aprotinin infusion produced a further increase of serum creatinine, particularly in protocol A where it was 72% higher for the GAP group than for the G group. In both G and GAP groups, postnecrotic tubular regeneration was evidenced by determining the rate of DNA synthesis and the frequency of S-phase cells in renal cortex. Both methods gave consistent results and showed a 8- to 13-fold increase of cell proliferation in groups receiving gentamicin alone, compared to C groups.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Morphometric and tridimensional studies of tubular cystic degeneration in rat kidney following exposure to cisplatin.

Cisplatin, a widely used chemotherapeutic agent, is characterized by a dose-limiting renal toxicity. Cystic tubular dilatation is the most typical histopathological alteration encountered in cisplatin-treated rats. The purpose of the present study was to explore by a morphometric approach the development of cystic degeneration and, in particular, to analyse, by computer-assisted tridimensional reconstructions, the spatial structure and the tubular origin of cisplatin-induced renal cysts. This study was performed on rats given 8 mg/kg cisplatin i.p. for four days and sacrificed 4, 7, 14, 21, 50 and 60 days after last drug administration. The relative area occupied by cystic tubules increased rapidly in the outer stripe of outer medulla (OSOM) and reached a maximum 21 days after the end of treatment. Cystic dilatations appeared later in the kidney cortex and the inner stripe of outer medulla (ISOM). The tridimensional study of cystic tubules located in OSOM confirmed previous reports indicating that they arise from proximal straight tubules and showed that cystic degeneration was not associated with atrophy or degeneration in more proximal parts of the nephron. Moreover, cystic tubules located in ISOM were found to originate from distal straight tubules and/or the loop of Henle, an observation which, to our knowledge, has not been reported so far in cisplatin-treated rats.

Endogenous EGF as a potential renotrophic factor in ischemia-induced acute renal failure.

The time course for the increases in soluble renal epidermal growth factor (EGF) after ischemia has been established. These elevated levels of EGF have been compared with the degree of tissue injury as well as the extent of cell proliferation in the recovering tissue. Levels of soluble immunoreactive EGF (irEGF) in control animals were 9.74 +/- 1.1 ng/g wet wt (n = 4-8 for all values) and rose to 83.9 +/- 30 ng/g within 12 h after injury. Soluble irEGF content peaked at 88.8 +/- 15 ng/g at 24 h postinjury and returned to control values by 72 h. We previously reported that trypsin digestion of crude renal membranes (CRM) generates rat EGF that is indistinguishable from that isolated from the submandibular gland. Initial levels of trypsin-releasable membrane-associated irEGF were 439 +/- 26 ng/g. These levels fell to 46.6 +/- 9.6 ng/g at 48 h after injury. The total renal EGF demonstrated an 80% decline 48 h after injury but returned to 50% of the initial values after 72 h representing significant new synthesis of EGF-containing proteins between 48 and 72 h postinjury. Immunohistochemical staining of kidney paraffin sections for EGF immunoreactivity demonstrated staining intensities that paralleled the amount of irEGF in the trypsin-digested CRM fraction, suggesting that the membrane-associated irEGF is the predominant form detected by this technique. Regenerative hyperplasia subsequent to tubular insult was monitored by immunostaining nuclei of S phase cells after pulse labeling with the thymidine analogue 5-bromo-2'-deoxyuridine. Cell proliferation was particularly prominent in the outer stripe of outer medulla of kidneys exposed to ischemia and reached a maximum (19-fold higher than the baseline value) 48 h after reperfusion. Renal cell turnover returned to control values by day 7. The observation that the peak in soluble EGF levels (24 h) precedes the peak in tubular regeneration (48 h) by 24 h is consistent with the hypothesis that EGF is one of the mitogenic signals triggering regenerative hyperplasia after renal injury.

Immunocytological localization of epidermal growth factor in the rat kidney after drug-induced tubular injury.

The present study was undertaken to analyze, at the cytological level, the intrarenal distribution of epidermal growth factor (EGF) in normal conditions and after drug-induced tubular injury. Female Sprague-Dawley rats were injected with gentamicin (50 mg/kg.day) for 4 days to induce tubular necrosis and were terminated 4 days after the last drug administration. For light microscopy, EGF immunoreactivity was demonstrated by immunogold-silver staining. In the kidneys of control rats EGF was found associated with distal tubules (cortex and outer medulla). Kidney exposure to gentamicin resulted in a drastic decrease of EGF immunoreactivity. For the electron microscopy study, immunoreactive EGF was detected on ultrathin sections by immunogold labeling. Within distal tubules epithelium of control animals, immunoreactive material was predominantly seen on the basolateral membrane, and to a much lesser extent in the cytoplasm or on the apical membrane. After treatment with gentamicin, there was a reduction of the density of gold particles observed in distal tubule cells. Interestingly, gold particles also appeared, but at a lower density, in proximal tubule cells. In these cells, EGF immunoreactivity also seemed membrane-bound and was mostly observed on (or next to) the basolateral membrane.

Epidermal growth factor accelerates renal tissue repair in a model of gentamicin nephrotoxicity in rats.

Epidermal growth factor (EGF) is a potent mitogen for renal tubular cells that possess specific high-affinity binding sites for this polypeptide. However, actual function of EGF within the kidney remains to be elucidated. We evaluated the effect of exogenous EGF administration on the rate of tubular regeneration in an experimental model of gentamicin (GT) nephrotoxicity. Female Sprague-Dawley rats were anesthetized, and a miniosmotic pump filled with mouse EGF or saline was implanted subcutaneously. Twenty-four hours later, GT (40 mg.kg-1 x 12 h-1 ip) was given for 4 and 8 days. Groups of treated animals and controls were killed either the day after cessation of treatment (days 5 and 9) or 4 and 8 days after the end of 8-day GT administration (days 12 and 16). Cortical GT levels of groups killed at days 5, 9, 12, and 16 were similar in animals infused with saline or EGF. Serum creatinine levels were significantly higher in GT-treated animals infused with EGF or saline and killed at days 9 and 12 compared with saline-treated animals infused with EGF or saline alone (P < 0.01). Blood urea nitrogen (BUN) also increased as a result of GT administration. However, in animals receiving GT and EGF and killed at day 16, mean BUN level was significantly lower (P < 0.01) compared with rats dosed with GT alone. In treated rats, the extent of tubular regeneration, evaluated by the rate of [3H]thymidine incorporation into renal cortical DNA or by the frequency of S-phase cells (histoautoradiography), was increased in a dose- and time-dependent fashion.(ABSTRACT TRUNCATED AT 250 WORDS)