Increased glomerular Vwf after kidney irradiation is not due to increased biosynthesis or endothelial cell proliferation.

Kuin, A., Citarella, F., Oussoren, Y. G., Van der Wal, A. F., Dewit, L. G. H. and Stewart, F. A. Increased Glomerular Vwf after Kidney Irradiation is not due to Increased Biosynthesis or Endothelial Cell Proliferation. Radiat. Res. 156, 20-27 (2001). Irradiation of the kidney induces dose-dependent, progressive renal functional impairment, which is partly mediated by vascular damage. It has previously been demonstrated that reduced renal function is preceded by an increased amount of von Willebrand factor (Vwf) in the glomerulus. The underlying mechanism and significance of this observation are unknown but, since it is an important mediator of platelet adhesion, Vwf in increased amounts could be implicated in glomerular thrombosis, resulting in impairment of renal function. Increased Vwf could be the result of increased biosynthesis by endothelial cells, or from increased numbers of endothelial cells after compensatory proliferation induced by irradiation, or it could be secondary to other events. In the present study, expression levels of mRNA for glomerular Vwf and glomerular cell proliferation rates were measured in control mouse kidneys and after irradiation with a single dose of 16 Gy. There were no significant changes in mRNA ratios for Vwf/beta-actin at 10 to 30 weeks after irradiation compared with unirradiated samples, whereas increased amounts of Vwf protein were seen in the glomeruli at these times. Labeling studies with IdU or staining for Ki67 demonstrated that glomerular proliferation was increased from 10 to 30 weeks after irradiation. Despite the increased proliferation rates, there was an absence of glomerular hyperplasia and no increase in the endothelial cell surface coverage in the glomeruli. Staining with antibodies against smooth muscle actin (SMAalpha) revealed that the observed proliferation mainly involved mesangial cells. These results indicate that the increased presence of glomerular Vwf after irradiation is not due to an increased number of endothelial cells per glomerulus, or to an increased production of Vwf. It is presumably secondary to other events, such as increased release of Vwf by damaged endothelial cells or entrapment of Vwf in the irradiated mesangial matrix.

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.

Mechanisms for optimising photodynamic therapy: second-generation photosensitisers in combination with mitomycin C.

Mechanisms for improving photodynamic therapy (PDT) were investigated in the murine RIF1 tumour using meso-tetrahydroxyphenylchlorin (m-THPC) or bacteriochlorin a (BCA) as photosensitisers and comparing these results with Photofrin-mediated PDT. The 86Rb extraction technique was used to measure changes in perfusion at various times after interstitial PDT. Non-curative combinations of light doses with m-THPC and BCA PDT markedly decreased vascular perfusion. This decrease was more pronounced for both new photosensitisers than for Photofrin. Comparison of tumour perfusion after PDT with tumour response revealed an inverse correlation for all three photosensitisers, but the relationship was less clear for m-THPC and BCA. In vivo/in vitro experiments were performed after Photofrin or m-THPC PDT in order to assess direct tumour kill (immediate plating) vs indirect vascular effects (delayed plating). For both photosensitisers, there was little direct cell killing but clonogenic survival decreased as the interval between treatment and excision increased. When m-THPC PDT was combined with mitomycin C (MMC), light doses could be decreased by a factor of 2 for equal tumour effects. Lower light and m-THPC doses could be used compared with Photofrin PDT in combination with MMC. BCA PDT with MMC did not result in a greater tumour response compared with BCA PDT alone. Reduction in both light and photosensitiser does for effective PDT regimes in combination with MMC offers substantial clinical advantages, since both treatment time and skin photosensitisation will be reduced.

Photosensitizing efficacy of MTHPC-PDT compared to photofrin-PDT in the RIF1 mouse tumour and normal skin.

The new photosensitizer, meso-tetrahydroxyphenylchlorin (mTPHC) was compared with Photofrin in the murine RIF1 tumour and in normal mouse skin. A range of mTHPC or Photofrin doses were given at intervals of 1 hr to 7 days before illumination. mTHPC-PDT resulted in much higher tumour phototoxicity with longer regrowth delays and more cures. The RIF1 tumour could be effectively treated with 30 J cm-1 (interstitial illumination) at 1 day after mTHPC, whereas 4 to 13 times higher light doses were required with Photofrin for an equivalent anti-tumour effect. High doses of mTHPC also caused more skin phototoxicity (superficial illumination) than Photofrin for the 1-day illumination interval. Evaluating both tumour and normal skin photosensitization, the largest therapeutic gain factor (TGF) for mTHPC-PDT was achieved with a low drug dose (0.15 mg kg-1) at 1 day before illumination (TGF = 5.6, relative to Photofrin PDT). The duration of cutaneous photosensitivity for mTHPC was shorter than for Photofrin. The light dose required to produce a desquamation response in 50% of the animals increased more than 20-fold over the period 1 to 7 days after high doses of mTHPC, whereas this light dose only increased by a factor of 2 from 1 to 7 days after Photofrin. The large therapeutic gains seen for mTHPC-mediated PDT compared to Photofrin, plus the rapid fading of skin photosensitization, suggest that mTHPC is a potent photosensitizer suitable for clinical testing.

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)

Changes in perfusion of mouse tumours after photodynamic therapy.

The influence of photodynamic therapy (PDT) on vascular perfusion was investigated in 2 s.c. mouse tumours, a radiation-induced fibrosarcoma (RIF I) and a squamous-cell carcinoma (SCCVII). The 86Rb extraction technique was used to measure changes in perfusion relative to cardiac output at various intervals after interstitial PDT. Control groups showed that vascular perfusion in the RIF I tumours decreased with increasing tumour size. For both tumours, of constant size, vascular perfusion decreased to less than 10% of control values within 5 min after high PDT doses. Significant decreases in vascular perfusion were also seen after lower, sub-curative doses. Thereafter there was slow recovery towards control levels. Photofrin given at shorter intervals before illumination generally resulted in even larger decreases in tumour perfusion, and slower recovery. Comparison of tumour perfusion measurements after PDT with tumour response revealed an inverse correlation with tumour growth delay both for the RIF I and for the SCCVII tumours. PDT with sub-curative light doses appears to decrease vascular perfusion in the RIF I and SCCVII for a period of at least 24 hr. The most severe reductions in tumour blood flow were associated with the longest regrowth delays, indicating a major role of vascular damage in tumour response to PDT.