Redox modulation of the pro-fibrogenic mediator plasminogen activator inhibitor-1 following ionizing radiation.

Fibrosis is a common form of normal tissue damage after exposure to a wide variety of insults believed to involve oxidative stress. Plasminogen activator inhibitor-1 (PAI-1) is thought to play a major role in the development of progressive fibrosis via the inhibition of extracellular matrix degradation. Because radiation causes oxidative injury, which has been shown to trigger fibrogenic responses, the present study was designed to test the hypothesis that PAI-1 expression is redox-regulated after irradiation. Irradiating rat kidney tubule epithelial cells (NRK52E) with 1-20 Gy gamma-rays led to dose-dependent increases in steady-state levels of PAI-1 mRNA and immunoreactive protein within 24 and 48 h, respectively. Enhancement of intracellular soluble thiol pools after incubation with N-acetylcysteine (2.5 mM), from 3.27 +/- 0.27 nM/mg protein to 5.34 +/- 0.52 nM/mg protein in cells incubated with N-acetylcysteine 30 min before and assessed 4 h after irradiation, abolished the radiation-induced up-regulation of PAI-1. In addition, overexpression of catalase inhibited radiation-induced increases in PAI-1 expression, suggesting a mechanistic role for hydrogen peroxide (H2O2) in regulating PAI-1 expression after oxidative insult. In support of this notion, incubating NRK52E cells with H2O2 (100 microM) also led to a nearly 3-fold increase in PAI-1 gene expression. These results demonstrate that PAI-1 is redox-regulated after exposure to ionizing radiation or H2O2 and suggest that H2O2 scavenging might represent a fundamental mechanism for modulating fibrogenic disease via inhibition of the induction of profibrogenic mediators after acute or chronic oxidative stress.

A novel immunological model for the study of prostate cancer.

The Dunning R-3327 rat prostatic adenocarcinoma is a widely accepted model for in vivo experimental studies of prostate cancer. We have previously derived phenotypically distinct cell lines from a s.c. tumor resulting from the inoculation of the R-3327-5 subclone into Copenhagen rats. In this study, we report studies using a gelatin sponge model for the delivery of tumor cells and the retrieval of tumor-specific leukocytes responsive to different prostatic cell lines. S.c. preimplanted sponges were inoculated with tumor cells previously selected for differential properties of tumor formation and metastasis and examined for leukocyte content at time points of 1, 3, and 5 weeks after tumor cell inoculation. Cytospin and flow cytometric analyses revealed fewer tumor-associated leukocytes present in sponges inoculated with tumorigenic R-3327-5' and R-3327-5'B lines, with lesser sponge degradation, than in experiments with the nontumorigenic R-3327-5'A line, suggestive of a tumor cell-induced immunomodulatory mechanism. Morphological studies indicate an intermittent tumor growth pattern that gradually disappears in sponges inoculated with the nontumorigenic R-3327-5'A cells but a robust growth pattern in sponges inoculated with the tumorigenic cell lines. Cytokine analyses show the secretion of higher levels of active transforming growth factor-beta by the more invasive and metastatic lines. Total transforming growth factor-beta levels are higher in the epithelial, tumorigenic R-3327-5'B line. Additionally, the more tumorigenic lines secrete interleukin 10, a potent immunosuppressive molecule. In this report, we demonstrate the ability to retrieve viable leukocyte populations from a prostate tumor line bearing sponges, which offers an important model for further in vitro and in vivo manipulations and holds promise for testing adoptive immunotherapeutic strategies.

Role of antioxidant enzyme expression in the selective cytotoxic response of glioma cells to gamma-linolenic acid supplementation.

We hypothesized that the cytotoxic effect of GLA observed in glioma but not normal glial cells reflects differences in GLA metabolism and/or antioxidant enzyme levels between these cells. The PUFA content of unsupplemented glioma cells was approximately 50% of that seen in unsupplemented astrocytes. Supplementation with 20 microM GLA for 24 h led to a 230 and 22% increase in glioma and astrocyte PUFA content, respectively, such that both supplemented cell types contained similar levels of PUFA. No major differences were seen in terms of GLA metabolites retained in the cells or secreted into the media following incubation with [(3)H]-GLA. No significant differences were observed in activity of MnSOD or CuZn-SOD between the cells. However, CAT and GPx activity in the glioma cells was significantly higher and lower, respectively, than observed in normal astrocytes. GLA supplementation resulted in a significant increase in CAT activity in normal astrocytes; glioma CAT activity was unchanged. No significant change was seen in the other antioxidant enzymes following GLA supplementation. These results suggest that the cytotoxic effect of GLA on glioma cells reflects both increased PUFA content and an inability to upregulate CAT.

v-Ha-RaS oncogene upregulates the 92-kDa type IV collagenase (MMP-9) gene by increasing cellular superoxide production and activating NF-kappaB.

Matrix metalloproteinase 9 (MMP-9) degrades basement membrane type IV collagen and is expressed during cellular migration and invasion. Here we show that v-Ha-Ras overexpression in rat kidney epithelial cells (REC) caused upregulation of MMP-9 gene expression in part by increasing cellular oxidant levels. v-Ha-Ras mediated the production of superoxide in Ras-transfected cells, which was associated with upregulated MMP-9 gene expression. Conversely, v-Ha-Ras expression decreased steady-state levels of mRNAs from tissue inhibitor of metalloproteinase 1 (TIMP-1), an inhibitor of MMP-9; plasminogen activator inhibitor 1 (PAI-1), which indirectly activates MMP-9 by increasing plasmin levels; and collagen IV, a substrate of MMP-9 and a major component of basement membrane. Gel mobility shift assays demonstrated that Ras overexpression enhanced NF-kappaB, but not AP-1 DNA binding to motifs in the MMP-9 gene promoter. The Ras-induced increase in NF-kappaB DNA binding could be inhibited by treatment with the antioxidants N-acetyl-L-cysteine and glutathione monoester, suggesting that intracellular oxidant levels can mediate MMP-9 transcription. Our findings identify an important role for Ras in the regulation of MMP-9 expression, and suggest that increased superoxide production can upregulate MMP-9 expression and thus contribute to malignant conversion.

Nephropathy in the mature pig after the irradiation of a single kidney: a comparison with the immature pig.

The right kidney of 11 mature 10-month-old Large White female pigs was irradiated with single doses of 9.8-14.0 Gy of 60Co gamma rays. Individual kidney glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured using 99mTc-DTPA and 131I-hippuran renography for periods up to 24 weeks after irradiation. Renal function was assessed either as a functional index, FI (FI = irradiated/unirradiated kidney function), or as the individual kidney GFR and ERPF. The radiation-induced changes after the irradiation of a single kidney (unilaterally irradiated--UI) of mature pigs were compared with those previously observed in 14-week-old immature pigs. Irradiation resulted in a dose-dependent reduction in the FI for both GFR and ERPF. However, these reductions were significantly less than those previously seen in immature pigs. Within 2 weeks of irradiation GFR increased in both the irradiated and the unirradiated kidneys in each animal, compared with unirradiated age-matched control kidneys. No marked changes in renal hemodynamics were seen in mature animals after a single dose of 9.8 Gy. This was in marked contrast to the pronounced reduction in the GFR and ERPF in the irradiated kidney previously observed in immature animals irradiated with an equivalent single dose of X rays. After higher doses, the irradiated kidney in mature pigs showed a dose-dependent reduction in GFR and ERPF. However, the extent of this reduction was significantly less than that seen in immature animals. There was no apparent difference in the response of the unirradiated kidneys in mature or immature pigs. The ED50 values, based on a probit fit to the data for the proportion of functional tests in which the irradiated kidney showed a greater than or equal to 50% reduction in GFR or ERPF, were higher in the mature animals; for example for ERPF the ED50 values were 11.76 +/- 0.28 Gy and 7.67 +/- 0.34 Gy for mature and immature animals, respectively. Thus, the UI kidney in mature pigs appears to be less radiosensitive than the UI kidney in immature animals.

A morphometric analysis of glomerular and tubular alterations following fast-neutron irradiation of the pig and monkey kidney.

PURPOSE: The morphologic responses of the pig and monkey kidney to fractionated fast-neutron irradiation were assessed. METHODS AND MATERIALS: The right kidney of approximately 14-week-old female Large White pigs was irradiated with 6.6-12.2 Gy of fast neutrons (42 MeVd-->Be) given as 12 fractions over 18 days; the left kidney served as the contralateral unirradiated kidney. Both kidneys were removed at necropsy 2 years postirradiation. In addition, the remaining hypertrophied kidney of four unilaterally nephrectomized adult rhesus monkeys was irradiated with a total dose of 11.0 Gy fast neutrons (45 MeVp-->Be) given in an identical fractionation regimen to that used in the pig studies. These kidneys were removed when the animals exhibited renal failure, between 32-94 weeks postirradiation. Glomeruli were assessed for the presence of pathologic features, including intercapillary eosinophilic material (ICE), ectatic capillaries, thrombi, hemorrhage, and sclerosis. The relative proportion of renal cortex occupied by glomeruli, interstitium, normal, or abnormal tubules was determined using a Chalkley point grid. RESULTS: The incidence of normal glomeruli, ectatic capillaries, thrombosis, and periglomerular fibrosis were significantly different in the irradiated pig kidneys compared with the unirradiated contralateral kidneys (p < or = 0.02). Linear regression analysis demonstrated a significant dose relationship in terms of normal glomeruli, ectatic capillaries, and ICE (r > or = 0.64; p < or = 0.04). Irradiation was also associated with a significant (p < 0.0001) decrease and increase in the volume of renal cortex occupied by normal and abnormal tubules, respectively. Similar morphometric changes were noted in the irradiated monkey kidneys. CONCLUSIONS: The morphologic changes seen in the pig and monkey kidney after fractionated irradiation with fast neutrons are similar to those previously noted after single-dose or fractionated-photon irradiation. These findings support the hypothesis that the development of radiation nephropathy in these various models involves common pathophysiological mechanisms.

Radiation-induced changes in glomerular and tubular cell kinetics and morphology following irradiation of a single kidney in the pig.

PURPOSE: Radiation-induced changes in glomerular and tubular cell kinetics and morphology following irradiation of a single pig kidney were assessed. METHODS AND MATERIALS: The right kidney of 13 adult female Large White pigs was irradiated with a single dose of 9.8 Gy gamma rays. Animals were serially killed between 2 and 24 weeks postirradiation (PI); 1 h prior to postmortem each pig received 500 mg bromodeoxyuridine (BrdUrd). At postmortem, both kidneys were removed and tissue taken to prepare cell suspensions. The labeling index (LI) of these suspensions was measured using flow cytometry; in vivo BrdUrd incorporation in glomerular and tubular cells was determined immunohistochemically. The kidneys were also assessed histologically. RESULTS: Irradiation of the right kidney alone resulted in a significant increase in renal cell LI in both the irradiated and the contralateral unirradiated kidney within 2 weeks of irradiation; peak values of 1.57 +/- 0.32% and 1.04 +/- 0.13%, respectively, were seen 4 weeks PI, significantly greater (P < 0.001) than the preirradiation value of 0.18 +/- 0.01%. The LI values then declined with time, but remained greater than those seen prior to irradiation. A similar pattern of response was determined from counts of labeled glomerular and tubular cells identified immunohistochemically. The increase in labeled glomerular cells was seen 2 weeks PI, whereas that for the tubular cells did not occur until 4 weeks PI. The irradiated kidney exhibited diffuse, progressive glomerular alterations. In contrast, tubular damage was focal; the irradiated kidney also exhibited a prominent vasculopathy, involving arteriolar and peripheral interlobular artery thickening. The contralateral unirradiated kidney appeared unchanged. CONCLUSION: These findings confirm the hypothesis that the morphologic and kinetic responses observed after irradiation of a single kidney are similar to those observed after irradiation of both kidneys. Renal irradiation results in significant alterations in glomerular and tubular cell proliferation and morphology within 2-4 weeks of irradiation; glomerular changes appear predominant.

Radiation nephropathy in the rhesus monkey: morphometric analysis of glomerular and tubular alterations.

PURPOSE: The morphologic responses of the monkey kidney glomeruli and tubules to fractionated irradiation were assessed. METHODS AND MATERIALS: Both kidneys of adult female rhesus monkeys were irradiated with doses of gamma-rays ranging from 24 Gy in 12 fractions up to 36 Gy in 18 fractions. Serial renal biopsies were taken between 1 and 12 weeks after irradiation. The kidneys were removed at necropsy 16-23 weeks after irradiation. Glomeruli were assessed for the presence of pathologic features, including intercapillary eosinophilic material, ectatic capillaries, thrombi, hemorrhage, adhesions, and sclerosis. The relative proportion of renal cortex occupied by glomeruli, interstitium, or tubules was determined using a Chalkley point grid. Tubules were further scored as being either normal or abnormal in appearance. RESULTS: Examination of the renal biopsies revealed that progressive glomerular lesions were evident within 4-12 weeks after irradiation. Tubular changes were mild and focal. Morphometric analysis of whole kidneys removed at necropsy demonstrated that numbers of glomeruli with ectatic capillaries, thrombi, and hemorrhage were significantly different from controls at 16-23 weeks after irradiation by all of the doses in the range of 24 to 36 Gy. A significant (p < 0.05) increase in the relative proportion of renal cortex occupied by glomeruli and interstitium was indicative of tubule loss. Further analysis of these tubular changes revealed a highly significant (p < 0.001) dose-dependent increase in the proportion of abnormal to normal tubules. Thus following a dose of 24 Gy in 12 fractions, the ratio of abnormal: normal tubules was approximately 1:2; after 36 Gy in 18 fractions the ratio was 3:1. CONCLUSIONS: Glomeruli appeared to be very radiosensitive because after the clinically relevant dose of 24 Gy in 12 fractions essentially all glomeruli were altered in the irradiated kidneys as compared to controls. Thus, efforts aimed at increasing the threshold dose for development of radiation nephropathy should be directed primarily at preventing the glomerular lesions.

Radiation response of the monkey kidney following contralateral nephrectomy.

PURPOSE: The long-term functional and morphologic responses of the hypertrophied monkey kidney after unilateral nephrectomy to fractionated irradiation were assessed. METHODS AND MATERIALS: The right kidney of 13 adult female rhesus monkeys was removed. Twelve weeks after unilateral nephrectomy (UN) the remaining kidney received fractionated doses of gamma-rays ranging from 35.2 Gy/16 fractions (F) up to 44 Gy/20 F. Glomerular filtration rate, effective renal plasma flow, blood urea nitrogen, serum creatinine, and hematocrit values were measured up to 107 weeks postirradiation (PI). The monkeys were killed and the remaining kidneys were removed 107 weeks PI or earlier when end-stage renal failure was exhibited. Glomeruli were scored for the presence/absence of several pathologic features including increased intercapillary eosinophilic material (ICE), ectatic capillaries, and thrombi. The relative proportion of renal cortex occupied by glomeruli, interstitium, normal tubules or abnormal tubules was determined using a Chalkley point grid. These quantal dose response data were analyzed using a logistic regression model. RESULTS: Irradiation of the remaining kidney in UN monkeys resulted in a dose-dependent reduction in renal function and anemia. Glomerular dysfunction preceded tubular dysfunction. Animals receiving 44 Gy all manifested progressive clinical renal failure. Conversely, those receiving < or = 39.6 Gy showed stable, albeit impaired, renal function for the duration of the observation period of 107 weeks. Morphologically, the incidence of ICE, ectatic glomerular capillaries, thrombi, and periglomerular fibrosis was significantly dose-related (p < 0.005). A significant (p < 0.001) dose-related increase in the relative proportion of renal cortex occupied by abnormal tubules was indicative of tubular injury. A highly significant (p < 0.001) dose-dependent increase in the proportion of abnormal to normal tubules was also seen. CONCLUSION: The pathogenesis of radiation nephropathy is difficult to fully understand because of the complex and dynamic interactions among all components of the nephron that make discrimination between primary radiation effects and secondary pathophysiological consequences very difficult. Notwithstanding, the current experiment shows that the functional and morphological expressions of radiation injury in the kidney are dose dependent. Renal failure occurs when both the glomeruli and tubules are dysfunctional. In monkeys following UN, a total dose of 44 Gy to the remaining kidney damages all components of the nephron and causes renal failure in less than 45 weeks. With lower doses, changes to the glomeruli predominate and the animals survive. Kidney doses of up to 39.6 Gy/18 fractions of 2.2 Gy are compatible with survival for at least 2 years in primates.

Amelioration of both early and late radiation-induced damage to pig skin by essential fatty acids.

PURPOSE: To evaluate the possible role of essential fatty acids, specifically gamma-linolenic and eicosapentaenoic acid, in the amelioration of early and late radiation damage to the skin. METHODS AND MATERIALS: Skin sites on the flank of 22-25 kg female large white pigs were irradiated with either single or fractionated doses (20 F/28 days) of beta-rays from 22.5 mm diameter 90Sr/90Y plaques at a dose rate of approximately 3 Gy/min. Essential fatty acids were administered orally in the form of two 'active' oils, So-1100 and So-5407, which contained gamma-linolenic acid and a mixture of that oil with eicosapentaenoic acid, respectively. Oils (1.5-6.0 ml) were given daily for 4 weeks prior, both 4 weeks prior and 10-16 weeks after, or in the case of one single dose study, just for 10 weeks after irradiation. Control animals received a 'placebo' oil, So-1129, containing no gamma linolenic acid or eicosapentaenoic acid over similar time scales before and after irradiation. Acute and late skin reactions were assessed visually and the dose-related incidence of a specific reaction used to compare the effects of different treatment schedules. RESULTS: A reduction in the severity of both the early and late radiation reactions in the skin was only observed when 'active' oils were given over the time course of the expression of radiation damage. Prior treatment with oils did not modify the radiation reaction. A 3.0 ml daily dose of either So-1100 or So-5407 given prior to, but also after irradiation with single and fractionated doses of beta-rays produced the most significant modification to the radiation reactions, effects consistent with dose modification factors between 1.06-1.24 for the acute reactions of bright red erythema and/or moist desquamation, and of 1.14-1.35 for the late reactions of dusky/mauve erythema and dermal necrosis. There was the strong suggestion of an effect produced by the 'placebo' oil, So-1129, after higher daily doses of oil. CONCLUSIONS: Essential fatty acids can modulate normal tissue reactions when given over the time when radiation damage is normally expressed. Dose modification factors suggest that a > or = 10% higher dose is required to produce the same level of normal tissue injury. Clinical application of selected essential fatty acids at appropriate doses may lead to a significant increase in the therapeutic gain in patients treated for cancer by radiotherapy.

Effects of single doses of X-rays on renal function in the pig after the irradiation of both kidneys.

Irradiation of a single kidney in the pig with relatively low doses of X-rays, in the order of 8 Gy, produces a pronounced reduction in both glomerular filtration rate (GFR) and effective renal plasma flow (ERPF). This apparent high radiosensitivity may be due, in part, to the compensatory hypertrophy displayed by the contralateral unirradiated kidney. This could suppress any potential for recovery by the irradiated kidney. To test this hypothesis, both kidneys of 14-week-old Large White pigs were sequentially irradiated with single doses of 250 kV X-rays, in the range 8.8 to 12.6 Gy. Sequential measurements of individual kidney GFR and ERPF were made for periods up to 24 weeks after irradiation. Time-related changes in haematocrit (Hct) were also studied. Two weeks after irradiation, GFR and ERPF increased markedly in all irradiated kidneys; levels then declined in a dose-dependent manner. Following a dose of 8.8 Gy renal haemodynamics returned to control values within 4 weeks of irradiation and remained essentially constant throughout the study. After higher doses, GFR and ERPF decreased markedly and remained below control values up to 24 weeks after irradiation. Associated with these changes in renal haemodynamics was a fall in Hct within 3 weeks of irradiation, with minimal levels being found approximately 8 weeks after irradiation. Although there was some recovery between weeks 12 and 24, Hct values remained below those of age-matched controls. At all doses the mean functional status of irradiated kidneys in animals in which both kidneys were irradiated (BI) was significantly greater than that previously observed in the irradiated kidney of pigs in which only one kidney was irradiated (UI). Moreover, in BI pigs there appeared to be a marked imbalance between the contribution each kidney makes to the total renal function. In terms of ERPF, the functional status of the right kidney, relative to that of the left kidney, showed a dose-related decline. These findings support the hypothesis that the compensatory response exhibited by the contralateral unirradiated kidney in UI pigs suppresses the potential for functional recovery by the irradiated kidney. The findings also indicate that individual kidneys in the same animal may differ in their response to a similar nephrotoxic insult.

Effects of single doses of X-rays on renal function in unilaterally irradiated pigs.

The right kidney of 13 Large White female pigs was irradiated with single doses of 250 kV X-rays in the range 7-12.6 Gy. Sequential measurements of individual kidney glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were carried out by means of 99mTc-DTPA and 131I-hippuran renography for periods up to 24 weeks after irradiation. GFR levels increased in irradiated and unirradiated contralateral kidneys 2 weeks after treatment compared with age-matched controls. ERPF values exhibited a small increase in a proportion of animals. Renal function then declined in irradiated kidneys in a dose-dependent manner. A dose of 7 Gy resulted in a decline followed by subsequent recovery. After doses of greater than or equal to 8.8 Gy GFR and ERPF declined rapidly, reaching minimal levels by 6-12 weeks, the time depending on the dose. The reduction in ERPF was quantitatively greater than that for GFR. In animals receiving greater than 8.8 Gy the irradiated kidney contributed in the order of only 10% of the total ERPF. The reduction in GFR resulted in a prompt functional compensatory response in GFR in the unirradiated contralateral kidney. In terms of ERPF, a compensatory response was not evident until weeks 20-24. The results indicated that the radiation tolerance dose of the pig kidney following unilateral irradiation with single doses of X-rays was approximately 8 Gy.

Functional recovery in the irradiated kidney following removal of the contralateral unirradiated kidney.

The right kidneys of seven Large White female pigs, approximately 14 weeks of age, were irradiated with single doses of 7-12.6 Gy of 250 kV X-rays. Sequential measurements of individual kidney glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were carried out using 99mTc-DTPA and [131I]hippuran renography for time periods up to 24 weeks after irradiation. From this data, kidneys receiving a dose of 7 Gy were found to be functioning (F), while kidneys which received greater than or equal to 8.8 Gy were assessed as having no significant function (NF). When the pigs were approximately 10 months of age the contralateral unirradiated kidney was removed; the left kidney of three age-matched unirradiated pigs was also removed. The response of the right kidney to unilateral nephrectomy (UN) in these animals was assessed in terms of changes in haemodynamics (i.e. GFR and ERPF) for periods up to 24 weeks after UN. At post-mortem, the length and weight of the remaining kidney was measured. A marked increase in renal length was observed in irradiated kidneys following UN. In addition, the weights of irradiated kidneys following UN were greater than those of irradiated kidneys in age-matched pigs where the unirradiated kidney had not been removed. Four weeks after UN there was a pronounced increase in GFR and, in particular, ERPF in previously NF irradiated kidneys. The mean increase in these parameters, measured at the end of the follow-up period, when compared with the pre-surgery values, was 350.1 +/- 84.3 and 781.8 +/- 151.0% respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The response of the pig kidney to the combined effects of cisplatin and unilateral renal irradiation.

Seven mature Large White female pigs, approximately 10 months of age received a single dose of cis-dichlorodiammineplatinum(II), c-DDP (2.5 mg/kg body weight). Prior to, and 4 weeks after c-DDP administration, individual kidney glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured by [99mTc]DTPA and [131I]hippuran renography. Of the 5 pigs surviving the c-DDP treatment most exhibited a reduction in both GFR and ERPF; the mean reduction in GFR (36.2 +/- 18.9%) was more pronounced than that for ERPF (12.6 +/- 19.4%). However, the difference in the severity of the impairment in these two parameters was not significant (p greater than 0.55). Haematocrit, haemoglobin and red blood cell counts were markedly reduced 14 days after c-DDP infusion, and despite some recovery evident 21 days after treatment, all three haematological parameters were still reduced 28 days after c-DDP administration. The right kidneys of these 5 animals, plus 5 pigs which did not receive c-DDP, were irradiated with a single dose of 11.9 Gy of 60Co gamma-rays. Individual kidney GFR and ERPF was routinely measured up to 24 weeks after irradiation. Pigs in which only the right kidney was irradiated showed a marked increase in both GFR and ERPF values 2 weeks after irradiation. This was followed by a decline in function with a reduction of 50% in terms of ERPF 16 weeks after irradiation. Values then showed some evidence of a recovery in function. There was a concomitant compensatory response by the contralateral unirradiated kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Repair, repopulation and cell cycle redistribution in rat foot skin.

The influence of the phenomena of the repair of sublethal damage, repopulation and the role of the reassortment of surviving clonogenic target cells within the cell cycle have been examined in the foot skin of rats using a series of split dose experiments. The dose-related incidence of moist desquamation was used as an end-point. Initially the iso-effect dose for moist desquamation (ED50) increased with an increasing time interval (1-22 h) between two equal fractions. This effect was attributed to the well established phenomenon of the repair of sublethal damage. This appeared to be maximal with a 22 h gap between fractions. A further increase in the time interval, from 2-7 days, between two equal fractions resulted in a decrease in the ED50 value for moist desquamation. The phenomenon is most likely to be explained by a shortening of the cell cycle time in surviving epithelial target cells as repopulation first initiated. With intervals between two fractions of greater than 10 days the ED50 for moist desquamation again increased. This is likely to represent an increase in the number of epidermal target cells (repopulation). Further evidence for the effect of a reassortment of cells in the cell cycle has come from another study in which a half-tolerance priming dose of 16.8 Gy was followed by three daily fractions starting 48 or 125 h after the priming dose. The ED50 for moist desquamation based on the total fractionated dose (three fractions) was significantly lower (P < 0.05) after the longer time interval, i.e. fractions given on days 5, 6 and 7 after the primary dose. These findings were supported by the results of a cell proliferation kinetic study and jointly question the validity of a frequently made assumption of equal biological effect per fraction in a prolonged fractionated irradiation schedule.

Radiation-induced alteration of rat mesangial cell transforming growth factor-beta and expression of the genes associated with the extracellular matrix.

We hypothesized that the altered mesangial cell phenotype observed in radiation nephropathy reflects, at least partly, radiation-induced changes in expression of the genes associated with transforming growth factor-beta (TGF-beta) and extracellular matrix (ECM). To test this hypothesis, rat mesangial cells were used between passages 7 and 11 after primary isolation from glomeruli. Cells were placed in serum-free medium 24 h prior to irradiation and irradiated with single doses of 5-20 Gy 137Cs gamma rays; control cells received sham irradiation. After irradiation, the cells were maintained in serum-free medium for up to 48 h postirradiation. Total RNA was isolated, and Northern analysis was performed using cDNA probes for TGF-beta 1, beta 2 and beta 3 and several ECM genes. Irradiation resulted in isoform-specific alterations in TGF-beta mRNA; TGF-beta 1 levels showed a dose-independent increase 24-48 h postirradiation; TGF-beta 3 mRNA levels showed a progressive dose-independent decrease over the same period, decreasing to levels approximately 25% of those seen in controls. These changes were associated with a concomitant increase in levels of mRNA expressed by genes for the components of the ECM; no changes were observed in TGF-beta 2, collagen I, collagen III or decorin. Thus radiation can alter mesangial cell TGF-beta and the expression of the genes involved in ECM, although the nature of this alteration varies for the TGF-beta isoforms and specific ECM genes.

Angiotensin II-induced modulation of rat mesangial cell phenotype.

Inhibition of angiotensin II (AII) can ameliorate the severity of experimental radiation nephropathy. To determine the ability of AII to modulate mesangial cell phenotype, primary cultures of rat mesangial cells (passage number 6-11) were placed in serum-free medium 24 h prior to addition of AII (10(-9)-10(-5) M); control cells received serum-free medium alone. Cells were maintained in serum-free medium for a further 48 h. Addition of AII to quiescent mesangial cells resulted in significant (P < 0.05) time- and/or dose-dependent increases in Fn and Pail mRNA and/or immunoreactive protein. No significant change was observed in terms of Tgfb1 mRNA. A significant increase in total Tgfb1 protein (P < 0.01) secreted by AII-treated mesangial cells was noted; however, this increase was primarily in terms of latent TGF-beta; the relative proportion of active TGF-beta secreted decreased after AII incubation. AII had no effect on the activity of Mmp2 or Mmp9. However, AII-treated mesangial cells did show an increase in the amount of tissue inhibitor of metalloproteinase-2 (Timp2) immunoreactive protein secreted into the medium. The AII-mediated increase in Pail mRNA levels appeared due in part to activation of the AT1 receptor and was independent of TGF-beta; co-incubation with TGF-beta-neutralizing antibody failed to inhibit the AII-mediated increase in Pail mRNA. Thus mesangial cells treated with AII exhibit a pro-fibrosis phenotype.

Irradiation of rat mesangial cells alters the expression of gene products associated with the development of renal fibrosis.

To determine the ability of radiation to modulate mesangial cell expression of various molecules involved in promoting extracellular matrix (ECM) accumulation [fibronectin, plasminogen activator-inhibitor 1 (Pai1), and tissue inhibitor of metalloproteinase-2 (Timp2)] and degradation (Tgfb, plasminogen activators u-PA or t-PA, matrix metalloproteinases Mmp2 and Mmp9), primary cultures of rat mesangial cells (passage number 6-11) were placed in serum-free medium 24 h prior to irradiation with single doses of 0.5-20 Gy (137)Cs gamma rays. After irradiation, cells were maintained in serum-free medium for a further 48 h. Irradiation of quiescent mesangial cells resulted in significant (P < 0.05) time- and dose-dependent increases in Fn and Pai1 mRNA and/or immunoreactive protein. Despite an increase in Tgfb1 mRNA, there was little evidence for an increase in total Tgfb protein. Indeed, active levels remained unaltered after irradiation. Irradiation led to differential changes in MMP expression; active Mmp2 levels increased, while Mmp9 levels appeared unaltered. In addition, secretion of plasminogen activators into the medium was unchanged after irradiation, while secretion of Timp2 increased. We conclude that irradiating mesangial cells leads to altered production of various molecules involved in accumulation and degradation of extracellular matrix.

The role of the tubulointerstitium in radiation-induced renal fibrosis.

The functional and morphological response of the remaining hypertrophied kidney in unilaterally nephrectomized rats to single doses of 0-20 Gy X rays was investigated. Functional and histological end points were assessed serially 4-24 weeks postirradiation. Renal irradiation led to time- and dose-dependent reductions in renal function, seen in terms of a decreased glomerular filtration rate, increased blood urea nitrogen, and reduced hematocrit. These changes were accompanied by morphological changes in the glomerular, tubular and interstitial portions of the kidney. However, dose-dependent changes were observed only in terms of tubulointerstitial lesions. Significant increases in the degree of interstitial staining for collagen type III and fibronectin were observed 24 weeks postirradiation. These increases in extracellular matrix components were accompanied by a significant increase in interstitial alpha smooth muscle actin, suggesting activation of interstitial fibroblasts into myofibroblasts. There was no evidence of glomerular Tgfb after renal irradiation. A significant increase in tubular Tgfb staining was only seen 8 weeks postirradiation. In contrast, there was a shift of staining to the interstitium such that by 24 weeks postirradiation interstitial Tgfb staining was significantly greater than that seen in controls. These findings suggest that the tubule epithelial cell and the interstitial fibroblast are both active participants in the development and/or progression of radiation-induced renal fibrosis.

Radiation-induced alterations in rat mesangial cell Tgfb1 and Tgfb3 gene expression are not associated with altered secretion of active Tgfb isoforms.

Despite evidence of selective radiation-induced modulation of expression of rat mesangial cell Tgfb gene isoforms, it is unclear whether these changes in gene expression are accompanied by changes in protein secretion. To address this issue, primary cultures of rat mesangial cells (passage number 6- 11) were placed in serum-free medium 24 h prior to irradiation with single doses of 0.5-20 Gy of (137)Cs gamma rays. After irradiation, cells were maintained in serum-free medium for a further 24 h. Irradiation of quiescent mesangial cells resulted in a significant (P