Radiation-induced glomerular thrombus formation and nephropathy are not prevented by the ADP receptor antagonist clopidogrel.

PURPOSE: To assess the effects of kidney irradiation on glomerular adenosine diphosphatase (ADPase) activity and intraglomerular microthrombus formation, and their correlation to the development of renal functional impairment. METHODS AND MATERIALS: C3H/HenAf-nu(+) mice were given single-dose or fractionated kidney irradiations. Glomerular ADPase activity was measured using a cerium-based histochemical method. Microthrombus formation within the glomeruli was assessed by a semiquantitative immunohistochemical analysis of fibrinogen/fibrin deposits. Renal function was assessed by the [(51)Cr]EDTA retention assay. RESULTS: The ADPase activity was significantly reduced, to approximately 50% of pretreatment value, 4--40 weeks after 10--16 Gy single-dose irradiation and at 44 weeks after 20 x 2 Gy. No dose--effect relationship was found. An approximately fourfold increase in glomerular fibrinogen/fibrin staining was observed at 1 year after irradiation. This increase was not influenced by treating the mice with daily, oral clopidogrel, a platelet ADP receptor antagonist, which reduced platelet aggregation by more than 75%. Radiation-induced impairment of glomerular filtration was also not affected by the clopidogrel treatment. CONCLUSION: These data indicate that irradiation significantly reduced glomerular ADPase activity, which correlated with an increased glomerular fibrinogen/fibrin deposition. We were not able to reduce these prothrombotic changes, nor to protect against radiation nephropathy, by pharmacological intervention with an ADP-receptor antagonist.

Radiation nephropathy--the link between functional damage and vascular mediated inflammatory and thrombotic changes.

The extent of radiation-induced nephropathy, which develops progressively over periods of months to years after treatment, is strongly influenced by both total dose and dose per fraction. In this study we examined the relationship between the early expression of various thrombotic and inflammatory markers of endothelial cell (EC) damage in irradiated mouse kidneys and the subsequent development of nephropathy. Decreased levels of glomerular ADPase and increased levels of glomerular Vwf were seen from 4 or 20 weeks after irradiation, respectively. These pro-thrombotic changes were associated with increased fibrin/fibrinogen deposits, indicative of microthrombus formation, at later times. These events were, however, not sensitive to changes in total dose or dose per fraction, therefore they cannot be quantitatively linked to the development of radiation nephropathy. Increased leucocyte invasion of the renal cortex was also seen after irradiation; this was quantitatively dependent on both total dose and dose per fraction. Linear quadratic analysis of the leucocyte dose-response curves yielded an alpha/beta ratio of 7.7 Gy, which is significantly greater than the alpha/beta ratio or 2.7 Gy determined for nephropathy, indicating less fractionation sensitivity for the inflammatory response. We conclude that inflammatory changes contribute to, but do not entirely explain, radiation nephropathy. The role of thrombotic changes is less clear.

Influence of acetylsalicylic acid on development of radiation-induced nephropathy.

PURPOSE: Previous studies have demonstrated that long-term treatment with acetylsalicylic acid (ASA) can significantly reduce the renal functional impairment that develops after high doses of irradiation. The effect is hypothesized to be mediated by selective inhibition of thromboxane A2 synthesis and inhibition of platelet aggregation. The present study was undertaken to investigate this phenomenon further using more clinically relevant fractionated and re-irradiation schedules. METHODS AND MATERIALS: Groups of mice were given bilateral renal irradiation with a series of four or 20 daily fractions of X-rays, or 10 daily fractions with a single dose of re-irradiation (0-10 Gy) after 27 weeks. Half the mice received ASA in drinking water (2.4 g x l(-1)) from 1 week before the start of irradiation and continuously throughout the follow-up period. Renal function was assessed by clearance of [51Cr]EDTA, about every 4 weeks for up to 80 weeks after the start of treatment. Histological damage in representative groups of mice was also assessed. RESULTS: Oral administration of ASA caused inhibition of thromboxane A2 synthesis (to < 36% of controls) and a strong inhibition of platelet aggregation in whole mouse blood (ex vivo). Prolonged treatment with ASA also resulted in a small, non-significant reduction of radiation-induced renal functional damage. No reduction in histological damage was seen in the ASA treated mice. CONCLUSION: Long-term oral administration of ASA gave only a modest, non-significant reduction of renal radiation injury after clinically relevant fractionated irradiation schedules.

Long-term effects of total-body irradiation on the kidney of Rhesus monkeys.

PURPOSE: To investigate the long-term effects of total-body irradiation (TBI) on kidneys in non-human primates. METHODS AND MATERIALS: The kidneys of Rhesus monkeys were histologically examined at 6-8 years after TBI with low single doses of 4.5-8.5Gy or two fractions of 5.4Gy. The kidneys of age-matched non-irradiated monkeys served as controls. Irradiation was performed on adult monkeys aged about 3 years; 6-8 years later animals were sacrificed and the kidneys removed and processed for histology. A semi-quantitative scoring system was used to evaluate overall histological damage. Glomerular changes were also morphometrically analysed according to previously published criteria. In selected dose groups (pro)thrombotic and inflammatory changes were investigated by immunostaining cryosections with antibodies against von Willebrand factor (vWF), leukocytes and macrophages. RESULTS: Histological changes were generally mild and only seen in kidneys irradiated with doses higher than 7 Gy. Glomerular changes were characterized by increased mesangial matrix and capillary dilatation. Tubulo-interstitial changes included hypercellularity, fibrosis and mild tubular atrophy. The mean glomerular area expressing vWF protein in the irradiated kidneys was not different from that in the age-matched controls. Numbers of infiltrating leukocytes were not significantly different between irradiated kidneys and controls. However, slightly increased numbers of macrophages were present in the renal cortex after irradiation. CONCLUSIONS: Renal damage after TBI of Rhesus monkeys with single doses of 4.5-8.5 Gy or two fractions of 5.4 Gy was mild, even after follow-up times of 6-8 years.

Increased expression of glomerular von Willebrand factor after irradiation of the mouse kidney.

Ionizing irradiation has been shown to induce an increased release of von Willebrand factor (vWF) in human endothelial cells in vitro. The present study was undertaken to investigate whether an increase in expression of vWF also occurs in glomerular endothelial cells in vivo after irradiation of the kidney. Increased expression of vWF may initiate prothrombotic changes, and the resultant vascular damage could cause renal failure. The amount of adherent leukocytes in the renal cortex after irradiation was also quantified, since this may contribute to the histological changes that occur after irradiation. Changes in expression of glomerular vWF and in the amount of leukocytes were related to the development of impairment of renal function, as assessed with the [51Cr]EDTA retention assay. Mice were given bilateral irradiation (single dose of 16 Gy) or were sham-irradiated and were sacrificed at intervals of 1 day to 40 weeks after irradiation. Immunohistochemical analysis of kidney cryosections was performed using a polyclonal vWF antibody or monoclonal CD45 antibody (leukocyte common antigen). The amount of glomerular vWF staining and CD45 staining in the renal cortex (percentage surface coverage) was quantified using a computerized image analyzer. The mean glomerular vWF staining in the nonirradiated kidneys was 34.4 +/- 6.2% (mean +/- SEM, 10 weeks after sham treatment). After irradiation, the expression of glomerular vWF increased gradually from 10 weeks to 53.4 +/- 3.6% at 40 weeks. The total number of leukocytes in the renal cortex of nonirradiated mice at 10 weeks after sham treatment was low, with a mean area of 1.0 +/- 0.09%, whereas in the irradiated kidneys the relative tissue area covered by leukocytes increased to 7.6 +/- 2.1% at 40 weeks. These alterations preceded impairment of renal function. The extent to which these changes are causally related to impairment of function will be the subject of future study using specific antithrombotic and anti-inflammatory agents.

A comparison of functional bladder damage after intravesical photodynamic therapy with three different photosensitizers.

The influence of type of photosensitizer, drug and light dose, and time interval between photosensitizer and illumination on the extent of photodynamic therapy (PDT)-induced bladder damage and recovery was investigated using a mouse model. The three photosensitizers studied were Photofrin, meso-tetrahydroxyphenylchlorin (m-THPC) and bacteriochlorin a (BCA). Functional bladder damage was quantitatively assessed from increases in urination frequency index (FI) at 1-35 weeks after illumination and histological damage was qualitatively assessed at 1 day, 1, 2 and 12 weeks. Photofrin-mediated PDT caused an acute increase in FI at 1 week, with recovery within 2-8 weeks after light doses of 2.7-8.2 J/cm2. After higher light doses there was only partial recovery. Previous results indicated that the acute response and rate of recovery was the same whether Photofrin was given at 1 day or up to 7 days before illumination. The m-THPC-mediated PDT at drug doses of > or = 0.3 mg/kg also resulted in a marked acute response with good recovery, even after 10.8 J/cm2. Lower drug doses in combination with 5.4 J/cm2 did not result in acute or late damage. There was no significant difference in acute response when m-THPC was given 1, 3 or 7 days before illumination, although recovery was faster for the longer illumination intervals (3 or 7 days). Illumination at 1 h after 20 mg/kg BCA induced an acute response within 2 days after illumination, with recovery within 4-8 weeks. Lower drug doses did not result in damage. The most prominent histological changes during the acute period with all three photosensitizers were submucosal edema and vessel dilation, with epithelial denudation (depending on drug/light dose). We conclude that BCA and m-THPC are both potent new photosensitizers. They can induce a moderate to severe acute bladder response with complete healing over a period of a few weeks. The photosensitizer m-THPC is very effective with low doses of photosensitizer and light, whereas relatively high doses of BCA and light are required to obtain equivalent functional bladder damage in our mouse model.

The influence of intravesical photodynamic therapy on subsequent bladder irradiation tolerance.

The aim of this project was to measure the irradiation tolerance of normal (non tumour bearing) mouse bladder after previous intravesical photodynamic therapy (PDT). Illumination with a range of light doses at 24 h after Photofrin was used as the initial PDT treatment and irradiation with a range of X-ray doses was given at 12 or 24 weeks after the initial therapy. Functional bladder damage was assessed from changes in micturition frequency (tested regularly for a follow-up period of 53 weeks after irradiation) and from cystometry measurements of the bladder at 53-56 weeks. PDT alone caused a marked increase in micturition frequency, with (partial) recovery by the time of irradiation. Irradiation alone caused a modest, transient acute response within 5 weeks and a progressive, permanent late response starting from about 25 weeks depending on X-ray dose. A reduced bladder capacity was also evident at 53-56 weeks after 20 Gy X-rays and after PDT alone. Irradiation after previous intravesical PDT caused an acute reaction similar to X-rays alone, but there was a much earlier expression of late functional bladder damage. The final level of damage prior to sacrifice at 53-56 weeks, was not significantly greater than after X-rays alone. These results suggest that irradiation after previous whole bladder PDT, for refractory bladder tumours, may lead to an increased risk of persistent increases in micturition frequency and reduced bladder capacity, beginning at very early times after irradiation.

Radiation tolerance of normal mouse bladders after intravesical chemotherapy.

The aim of this study was to compare functional damage in normal mouse bladder after various initial intravesical therapies and to investigate tolerance to subsequent irradiation. Six consecutive weekly intravesical instillations of Mitomycin C (MMC) or doxorubicin (DOX) were used as the initial therapy. Irradiation with single doses of 10-25 Gy (X-rays) was given at 4 or 12 weeks after intravesical treatment. Functional bladder damage was assessed from changes in the micturition frequency, expressed as frequency index (FI, number of urination events/ml urine in a 24-h test period) and from cystometry measurements of bladder volume at 52-56 weeks. Irradiation alone caused a temporary acute response (increased FI) within the first 4 weeks and a progressive late response starting from 15 to 37 weeks, depending on the radiation dose. A reduced bladder capacity was also evident at 52-56 weeks after 25 Gy. Intravesical MMC or DOX caused a 3-fold increase in FI during intravesical therapy with recovery to control levels within 2-3 weeks after cessation of treatment. Irradiation 4 weeks after MMC, or 4 or 12 weeks after DOX resulted in acute responses very similar to irradiation alone. There was no difference in time of onset or extent of late bladder damage when irradiation was given after DOX, compared with irradiation alone as assessed from repeated measurements of FI or cystometry at 52-56 weeks. In contrast, irradiation 12 weeks after MMC led to a decrease in acute radiation response compared with X-rays alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Bladder damage in mice after single and repeated intravesical instillations of mitomycin C or doxorubicin.

Functional and histological damage after intravesical administration of mitomycin C or doxorubicin were studied in mouse bladders. Dosing schedules were based on those commonly used in the clinic, and bladder function was assessed from changes in urination frequency. Repeated weekly instillations of 1 mg./ml. mitomycin C induced a transient three-fold increase in Frequency Index (FI) with recovery to control levels within 3 weeks. Weekly instillations of 6 mg./ml. doxorubicin also resulted in a three-fold increase in FI, but lower doses produced only minimal changes. There was, again, full recovery within 3 weeks. Our experiments indicate that repeated intravesical instillations of mitomycin C or doxorubicin are well tolerated, with rapid recovery from functional damage within a few weeks of cessation of therapy. There was significantly less damage after repeated instillations of clinically relevant doses of doxorubicin than mitomycin C.

Functional and histological damage in the mouse bladder after photodynamic therapy.

Bladders of anaesthetised mice were illuminated with laser light of 630 nm at 24 h after intraperitoneal administration of the photosensitiser Photofrin II (10 mg kg-1). A range of light doses, at a power setting of 100 mW, was delivered intravesically by a fibre optic inserted into the centre of the bladder via the urethra. Functional bladder damage was assessed from increases in urination frequency and the presence of urethra. Functional bladder damage was assessed from increases in urination frequency and the presence of haematuria at 1 to 26 weeks after treatment. Whole bladder illumination with incident light doses exceeding 18.75 J cm-2 caused extensive oedema, haemorrhage and necrosis of the bladder wall and mice had to be sacrificed within 24 h. PDT with incident light doses of 3.75 to 15.0 J cm-2 caused haematuria and increased urination frequency during the first week in nearly all mice, but there was complete functional recovery by 6 to 10 weeks after doses of up to 7.5 J cm-2. Recovery was slower after higher doses and up to 50% of mice still had some increased urination frequency at 10 weeks after greater than or equal to 11.25 J cm-2, although haematuria was rare at this time. Histologically, early damage (one day after PDT) consisted of epithelial sloughing, submucosal oedema, fibrin imbibition, vascular ectasia and, rarely, thrombosis. Doses exceeding 7.5 J cm-2 were often associated with foci of necrosis. In some instances, necrosis was complicated by bacterial infection, resulting in an acute transmural inflammation with a tendency to suppuration. After doses of up to 11.25 J cm-2 there was a gradual recovery and only a mild degree of fibrosis of the bladder wall (with some increase in vascularity) remained at 6 months.