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Radiation nephropathy is treatable with an angiotensin converting enzyme inhibitor or an angiotensin II type-1 (AT1) receptor antagonist
Radiotherapy and Oncology 46 (1998) 307–315
Radiation nephropathy is treatable with an angiotensin converting enzyme inhibitor or an angiotensin II type-1 (AT1) receptor antagonist John E. Moulder a ,*, Brian L. Fish a, Eric P. Cohen b a
Department of Radiation Oncology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA b Department of Medicine, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA Received 4 July 1997; revised version received 2 September 1997; accepted 10 September 1997
Abstract Background and purpose: Previous studies showed that progression of established radiation nephropathy could be delayed by continuous treatment with high doses of captopril, an angiotensin-converting-enzyme (ACE) inhibitor. The current studies were designed to determine whether a lower dose or a shorter treatment with captopril would be effective and whether an angiotensin II type-1 (AT1) receptor antagonist (AII blocker) would be effective. Materials and methods: In the captopril studies, rats were given renal irradiation at doses sufficient to produce radiation nephropathy. Six months after irradiation, animals were stratified by azotemia and assigned to no treatment, continuous high- or low-dose captopril, or 6 weeks of high-dose captopril. Captopril was given in drinking water at 62.5 mg/l (low dose) or 500 mg/l (high dose). The AII blocker study had a similar design, except that the nephropathy was the result of total body irradiation and bone marrow transplantation and the treatments were no treatment or continuous treatment with an AII blocker, L-158,809 (20 mg/l in drinking water). Animals were followed for 1 year with periodic studies of renal function. Results: Survival and renal function were significantly enhanced by all treatments. Continuous captopril treatment was more effective than the 6-week course of treatment, but there was no difference in effectiveness between the high and low doses of captopril. In continuous therapy, captopril and the AII blocker had roughly equivalent efficacy. Conclusions: Both the ACE inhibitor and the AII blocker were effective treatments for established radiation nephropathy. The best results with the ACE inhibitor required continuous therapy, but could be achieved with a low dose of the drug. 1998 Elsevier Science Ireland Ltd. Keywords: Nephropathy; Radiation; ACE inhibitors; Angiotensin II type-1 receptor antagonist; Captopril; Radiation injuries
1. Introduction Late radiation-induced normal tissue morbidity has classically been viewed as due solely to a delayed reduction in the number of surviving clonogens and these late injuries have been held to be inevitable, progressive and untreatable [27]. Recently, a paradigm shift has begun in the characterization of late radiation-induced normal tissue morbidity, as data from a variety of tissues indicate that these injuries involve dynamic interactions among parenchymal and vascular cells within an organ [22]. This paradigm offers a fundamentally new approach to radiation-induced normal tissue injuries, as it allows for the possibility that normal * Corresponding author.
tissue injuries can be treated. These treatments would be directed at modulating steps in the cascade of events leading to the clinical expression of damage [22]. Since this sequence of events does not appear to occur in tumors (where direct clonogenic cell kill still appears to be dominant), such treatments would not be expected to reduce antitumor efficacy. The possibility that radiation nephropathy could be treated arose from work done in the late 1980s. In 1986, Robbins and Hopewell [26] showed that the early hemodynamic changes observed after renal irradiation were decreased by treatment with an angiotensin-converting-enzyme (ACE) inhibitor (Fig. 1), captopril and in 1988 Ward et al. [30] showed that captopril could be used to reduce radiationinduced pulmonary injury. Because of these results and
0167-8140/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0167-8140 (97 )0 0175-8
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Fig. 1. The renin-angiotensin system is a regulated network of enzymes and substrates with a primary role in cardiovascular homeostasis (enzymes are shown in bold-face, inhibitors are shown in italics). Renin, produced in the kidney, is secreted in response to decreased renal perfusion and adrenergic nerve stimulation and the level of renin activity is believed to be the major control point in the system. Its specific substrate is angiotensinogen. The product of that reaction, angiotensin I, is converted to angiotensin II (AII) by angiotensin converting enzyme (ACE). ACE is present in lung and other organ endothelia and on kidney proximal tubule brush borders. AII is the active angiotensin; it is a potent vasoconstrictor, elevates blood pressure, stimulates collagen synthesis and is a growth promoter for kidney epithelial and vascular smooth muscle cells. AII has two known receptors. The type-1 (AT1) receptors serve the main hemodynamic functions of AII. The type-2 (AT2) receptors are active in utero and may play a role in apoptosis and development. AII may directly suppress renin activation and the volume retentive effect of AII will also suppress renin activity. ACE is also known as kininase II and in that role degrades the vasodilator bradykinin into inactive peptides [14]. Thus, ACE inhibition results in accumulation of a vasodilator, providing an AII-independent route for the antihypertensive activity of ACE inhibitors.
the known efficacy of ACE inhibitors in the treatment of progressive renal failure [1,2,25], we investigated the role of captopril in the treatment and prophylaxis of radiation nephropathy [4,5,7,16,19]. Initial studies [4] were directed towards treatment of established radiation nephropathy. In this model, the rats had undergone bilateral renal irradiation 6 months earlier. Animals with elevated blood urea nitrogen (BUN) were stratified by BUN and placed on captopril (500 mg/l in drinking water) or no drug. Azotemia and proteinuria were attenuated and survival was enhanced by the captopril therapy [4]. Further studies showed that enalapril (a non-thiol ACE inhibitor) was also effective, that the kidneys of captopril-treated animals developed less severe histopathologic lesions and that captopril also preserved renal function in a rat bone marrow transplant (BMT) nephropathy model [19]. In other models of chronic renal failure, ACE inhibitors are not always effective once renal injury is established [8]. Thus, the use of an ACE inhibitor from the time of irradiation should be more effective than treatment of established injury. In experiments with 1 year follow-up, both captopril and enalapril were found to be effective when used prophylactically [7], but other types of antihypertensives (verapamil, hydralazine, hydrochlorothiazide and alpha-methyldopa) were not effective [7,22]. A reduction in the dose of captopril was associated with a reduction in the prophylactic
benefit, but doses as low as 62.5 mg/l had some efficacy [7]. Unlike the situation in lung, where cessation of captopril therapy was followed by rapid deterioration [22], preservation of renal function was sustained in animals taken off captopril 26 weeks after irradiation [20]. Since angiotensin II (AII) is a potent vasoconstrictor and hypertension plays a major role in radiation nephritis, inhibition of AII production by ACE inhibition (Fig. 1) provides an obvious explanation for the efficacy of ACE inhibitors in radiation nephropathy [21,22]. However, ACE has other substrates that have hemodynamic activity (e.g. bradykinin [9,14]) and captopril has activity that may not be the result of ACE inhibition (e.g. antimitotic activity [22]). In order to determine whether ACE inhibitors were functioning via inhibition of AII production, we tested an angiotensin II type-1 (AT1) receptor antagonist [9,14] (Fig. 1). Analysis of both renal function and histopathology showed that the AT1 receptor antagonist (AII blocker) was even more effective than the ACE inhibitor in the prophylaxis of radiation nephropathy [21]. The effectiveness of AII blockers in the prophylaxis of radiation nephropathy was subsequently confirmed by Oikawa et al. [24]. While our previous studies [4,19] clearly showed that radiation nephritis could be treated with ACE inhibitors, several questions remained. The dose of captopril used was well above that used in humans (on a g/m2/day basis). Does captopril need to be used at such a high dose? Does the therapy need to be continued indefinitely, or will shorter courses be effective in treatment as they are in prophylaxis [5]? Finally, will AII blockers be as effective in treatment as they are in prophylaxis? The studies reported here were designed to answer these questions.
2. Materials and methods 2.1. Animals These studies were performed with syngeneic WAG/Rij/ MCW rats bred and housed in a moderate security barrier. The animals were free of Mycoplasma Pulmonis, pseudomonas and common rat viruses. No antibiotics, immunosuppressive drugs, or known nephrotoxic agents were used. Animals were maintained in the Animal Care Facilities of the Medical College of Wisconsin, which are fully accredited by the American Association for Accreditation of Laboratory Animal Care. The experiments described here were conducted under protocols approved by the Medical College of Wisconsin Animal Care and Use Committee. 2.2. Captopril study In the captopril study, 6–7-week-old female rats received bilateral renal irradiation in a study of the kinetics of repair of renal radiation injury. This experiment used a wide range of treatment schedules and radiation doses (one or two frac-
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tions at total doses of 8–15 Gy and 20 or 40 fractions at total doses of 27–35 Gy) and generated a large pool of animals with varying degrees of renal injury. At 26 weeks after irradiation, blood urea nitrogen (BUN) was assessed in all animals and animals (n = 95) with BUN of ≥30 mg/dl and ≤60 mg/dl (19–21 mg/dl is normal) were stratified by BUN and placed into four treatment groups, i.e. continuous highdose captopril, continuous low-dose captopril, 6 weeks of high-dose captopril, or no drug. Captopril was given in drinking water and drug treatments started 28 weeks after irradiation (i.e. 2 weeks after the assessment of BUN). The high-dose captopril regimen was 500 mg/l (the same dose used in the previous treatment studies [4,19]) and the lowdose regimen was 62.5 mg/l (the lowest dose that had been used in the captopril prophylaxis studies [7]). All animals remained in the study until morbid from uremia or until sacrifice 80 weeks after irradiation. Three animals were lost to follow-up because of the development of tumors at 55 weeks (low-dose captopril arm), 60 weeks (high-dose captopril arm) and 73 weeks (6-week treatment arm). The study design was identical to that previously used [4,19], except for the additional treatment arms and the selection of a narrower range of BUN in the current study (30–60 versus 25–120 mg/dl). 2.3. AII blocker study In the AII blocker study, 6–7-week-old female rats were given total body irradiation (TBI) followed by syngeneic BMT [16,18] in a study of pulsed low dose rate radiotherapy. This experiment used a range of dose rates (0.016–0.19 Gy/min) and radiation doses (18.9–20.1 Gy) and generated a large pool of animals with varying degrees of renal injury. At 26 weeks after irradiation, BUN was assessed in all animals and animals (n = 66) with BUN of ≥35 mg/dl and ≤70 mg/dl were stratified by BUN and placed into two treatment groups, i.e. continuous treatment with an AII blocker, or no treatment. Drug treatment began at 28 weeks after irradiation and continued for the duration of the study. Losartan is the first AII blocker to reach clinical use [9, 14]. For our experiments, we used a closely-related compound, L-158,809, which is a potent, competitive and specific antagonist of the AT1 receptor [3,29]. This AII blocker has good absorption when taken orally, is stable in water and has antihypertensive efficacy in rats equal to ACE inhibitors [3,29]. The L-158,809 was given in drinking water at 20 mg/l, the same dose used in our prophylactic study [21], and a dose whose antihypertensive activity in our rats was confirmed in the subtotal nephrectomy model [21]. All animals remained in the study until morbid from uremia or until sacrifice 75 weeks after irradiation. Five animals were lost to follow-up because of the development of tumors at 31–55 weeks (four in the AII blocker arm, one in the control arm) and one animal was lost to follow-up in the control arm when it developed a non-healing wound at 41 weeks. The AII blocker study differed from the captopril
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study in the use of TBI rather than renal irradiation to cause nephropathy and in the severity of azotemia that was used to select animals for the trial (35–70 versus 30–60 mg/dl). 2.4. Renal function assessment Animals were monitored daily in all experiments and those with symptomatic uremia or other morbid conditions (e.g. tumors) were sacrificed. Severe nephritis (uremia) is the major cause of morbidity in this model [16]. BUN, urine protein, urine creatinine and systolic blood pressure (BP) were assessed at 26, 41, 50 and 59 weeks after irradiation (2 weeks prior to and 13, 22 and 31 weeks into drug therapy) in all surviving animals. Urine was collected in metabolic cages and blood was collected by orbital bleeding. BUN was assayed with a urease-nitroprusside colorimetric assay, urine protein was assayed with a Coomassie Blue colorimetric assay and urine creatinine was assayed with a modified Jaffe´ reaction colorimetric assay [7]. As a general rule, BUN is an imprecise indicator of glomerular filtration rate (GFR) [6]. However, in this model, the correlation of BUN and serum creatinine is very strong [20] and data from other studies [6] indicate that serum creatinine is an adequate marker of GFR. Because BUN is strongly correlated with serum creatinine and hence with GFR, BUN rather than serum creatinine or creatinine clearance was used to assess renal function. It should also be noted that the degree of elevation of BUN at 26 weeks after renal irradiation is strongly correlated with survival time in this model [5]. Urine protein excretion was expressed as the ratio of urine protein to urine creatinine (UP/UC) in the same urine sample. This was done to account for the known urinary concentration defect that occurs in renal radiation injury and to normalize for animal size differences [7]. Tail-cuff BP was measured in unanesthetized animals using a semiautomatic machine. The mean BP reading from a minimum of three readings on each of 3 consecutive days was used for each time point; animals were preconditioned to the BP apparatus [7]. 2.5. Statistical methods All statistical comparisons were done using non-parametric techniques [28]. Physiologic data are shown as medians with 25–75% ranges and were compared by Mann– Whitney tests. Medians, ranges and non-parametric tests were used because physiological values in the groups showing abnormal renal function were often not normally distributed and because some animals were sacrificed with uremia prior to some of the assay intervals. For the purpose of calculating median values in groups where some animals (but less than half) had developed renal failure (uremia) prior to the assay interval, the values of parameters in animals that had developed renal failure were considered to have been above those of all survivors. If half or more of
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Fig. 2. Actuarial risk of renal failure (uremia) during treatment of established radiation nephropathy with different captopril regimens. Animals received bilateral renal irradiation and those with BUN between 30 and 60 mg/dl at 26 weeks after irradiation were stratified by BUN and placed on treatments that began 28 weeks after irradiation. Data are shown for 24 animals that received no captopril treatment, 23 animals that were treated continuously with high-dose captopril, 22 animals that were treated continuously with low-dose captopril and 26 animals that were treated with high-dose captopril from 28 to 34 weeks after irradiation.
animals, leading to renal failure in 62% of the animals by 50 weeks (Fig. 3). In the captopril-treated animals, azotemia was stable for 13 weeks before the increase in BUN resumed (Fig. 3). When the progression of azotemia resumed, the progression was much faster in the animals on the 6-week treatment course (Fig. 3). The captopril treatment stabilized blood pressure and proteinuria in the animals getting continuous captopril, but only reduced the rate of progression in the animals on the 6-week treatment course (Fig. 3). As with the survival data (Fig. 2), the physiology data (Fig. 3) showed essentially no difference in efficacy between the high- and low-dose captopril treatments, except that the high-dose treatment was somewhat better at controlling blood pressure. The differences among the groups were not a result of any initial differences between the groups. While the irradiated animals were only stratified by BUN, the resulting groups had similar values of BP and UP/UC (Fig. 3). For all three parameters (BUN, BP and UP/UC), all groups had 26-week values that were significantly (all P , 0.01) higher than those of normal (unirradiated) animals and not significantly (all P . 0.10) different from each other.
the animals in a dose group had developed renal failure prior to the assay, the point was plotted as ‘renal failure’. Survival curves were done by the Kaplan–Meier technique and compared using the method of Lee and Desu [12]. Survival curves were not plotted beyond the time where the number of animals at risk dropped below four.
3. Results 3.1. Captopril study Survival was enhanced when animals with established radiation nephropathy were placed on any of the three captopril regimens (Fig. 2). For untreated animals, the median survival time was 49 weeks (21 weeks after therapy started in the treatment arms). For animals on the continuous captopril regimens, the median survival time was 72 weeks on the high-dose regimen (23 weeks longer than in the untreated animals) and 74 weeks on the low-dose regimen (25 weeks longer than in the untreated animals). For animals on the 6-week course of captopril therapy, the median survival time was 65 weeks (16 weeks longer than in the untreated animals). The increase in survival times observed was highly significant (P = 0.013 for the 6-week treatment and P , 0.0001 for both continuous therapies versus the untreated animals). The continuous therapies were clearly superior to the 6-week course (both P , 0.01), but the highand low-dose continuous therapies were equally effective (P . 0.20). The increased survival for the animals treated with captopril was reflected in the renal function assays (Fig. 3). There was a progressive increase in azotemia (as BUN), hypertension and proteinuria (as UP/UC) in the untreated
Fig. 3. Evolution of azotemia (as BUN), hypertension (as systolic BP) and proteinuria (as UP/UC) during treatment of established radiation nephropathy with different captopril regimens. These are the same animals as shown in Fig. 2. Data are shown for animals that received no treatment (X), animals that were treated continuously with high-dose captopril (W), animals that were treated continuously with low-dose captopril (S) and animals that were treated with high-dose captopril from 28 to 34 weeks after irradiation (A). Data are shown as medians with 25–75% ranges. For the purpose of calculating median values in groups where some animals (but less than half) had developed renal failure (uremia) prior to the assay interval, the values of animals that had developed renal failure were considered to have been above those of all survivors. If half or more of the animals in a dose group had developed renal failure prior to the assay, the point was plotted as ‘renal failure’.
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Fig. 4. Progression of azotemia during treatment of established radiation nephropathy with different captopril regimens. BUN levels are shown 13 weeks after the start of captopril therapy as a function of BUN at 26 weeks (2 weeks prior to the start of captopril therapy). Data are shown for 24 animals that received no treatment (X), 23 animals that were treated continuously with high-dose captopril (W), 22 animals that were treated continuously with low-dose captopril (S) and 26 animals that were treated with high-dose captopril from 28 to 34 weeks after irradiation (A). The dashed lines show what the BUN values would be if there were no progression of azotemia. These are the same animals as shown in Figs. 2 and 3.
After 13 weeks of therapy, continuous captopril treatment was equally effective across the range of azotemia used in the study (Fig. 4). However, for longer durations of therapy, continuous captopril was most effective in the animals with initial BUN levels of less than about 40 mg/dl (data not shown). In contrast, even 13 weeks after the start of the 6week course of captopril, there was progression of azotemia in the animals whose initial BUN had been greater than ~45 mg/dl (Fig. 4).
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AII blocker was reflected in the renal function assays (Fig. 6). In this study, as in the captopril study (Fig. 3), there was a progressive increase in azotemia, hypertension and proteinuria in the untreated animals, leading to renal failure in 58% of the animals by 50 weeks. In the animals treated with the AII blocker, the BUN rose slowly during treatment and had risen significantly by 50 weeks (P = 0.002 compared to values at 26 weeks). Although azotemia increased during treatment with the AII blocker, hypertension and proteinuria actually decreased. The decrease in blood pressure was statistically significant at all three assay times (all P , 0.005 compared to values at 26 weeks), while the decrease in proteinuria was only significant at 41 and 50 weeks (both P , 0.001 compared to values at 26 weeks). The differences among the treated and untreated animals were not a result of any initial differences between the groups. While the irradiated animals were only stratified by BUN, the resulting groups had similar values of BP and UP/UC (Fig. 6). For all three parameters (BUN, BP and UP/UC), all groups had 26-week values that were significantly (all P , 0.005) higher than those of normal (unirradiated) animals and not significantly (all P . 0.10) different from each other. Treatment with the AII blocker was most effective when the degree of azotemia at the start of treatment was less than about 45 mg/dl (Fig. 7). However, after 31 weeks of treatment, BUN had progressed in all but the animal with the lowest initial BUN (Fig. 7). 3.3. AII blocker versus ACE inhibitor A rigorous comparison of the captopril (Section 3.1) and AII blocker (Section 3.2) studies is not possible. While each study was internally randomized, the two studies were not done at the same time, they used different irradiation procedures (local irradiation versus TBI) and they used some-
3.2. AII blocker study Survival was also enhanced when animals with established BMT nephropathy were placed on the AII blocker regimen (Fig. 5). For untreated animals, the median survival time was 50 weeks (22 weeks after therapy started in the treatment arm). For animals on the AII blocker regimen, the median survival time was in excess of 75 weeks (25+ weeks longer than in the untreated animals). The increase in survival times observed was highly significant (P , 0.0001 versus the untreated animals). The increased survival for the animals treated with the
Fig. 5. Actuarial risk of renal failure (uremia) during treatment of established radiation nephropathy with an AII blocker. Animals received TBI plus BMT and those with BUN between 35 and 70 mg/dl at 26 weeks after irradiation were stratified by BUN and placed on treatments that began 28 weeks after irradiation. Data are shown for 33 animals that received no treatment and 33 animals that were treated continuously with the AII blocker, L-158,809.
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Fig. 6. Evolution of azotemia (as BUN), hypertension (as systolic BP) and proteinuria (as UP/UC) during treatment of established BMT nephropathy with an AII blocker. These are the same animals as shown in Fig. 5. Data are shown for animals that received no treatment (X) and animals that were treated continuously with the AII blocker, L-158,809 (W). Data are shown as medians with 25–75% ranges. For the purpose of calculating median values in groups where some animals (but less than half) had developed renal failure (uremia) prior to the assay interval, the values of animals that had developed renal failure were considered to have been above those of all survivors. If half or more of the animals in a dose group had developed renal failure prior to the assay, the point was plotted as ‘renal failure’.
what different ranges of BUN as entry criteria. As a result, the pre-treatment functional parameters were different. In the AII blocker study, the BUN values were slightly higher, BP was significantly higher and the UP/UC ratios were lower (Fig. 8). However, despite these differences the resulting renal failure incidence curves (Fig. 9) and the progression of physiological injury (Fig. 8) for the two control groups were remarkably similar. The renal failure incidence (Fig. 9) and azotemia progression (Fig. 8) curves for the two treated groups were also remarkably similar, so we can conclude that there is no great difference between the efficacy of the two agents for the treatment of radiation nephropathy. However, Fig. 8 does show that the AII blocker was much more effective in controlling proteinuria and somewhat more effective in controlling hypertension.
4. Discussion Chronic radiation injuries such as radiation nephropathy are classically held to be inexorably progressive, often
resulting in fibrosis and organ failure [27]. This has necessitated shielding of susceptible organs [11] or limiting the dose of radiation [13]. To date, treatment of renal radiation injuries in humans has been symptomatic at best. In keeping with results in other organ systems [22], our studies suggest that radiation nephropathy may be pharmacologically treatable. The effect of ACE inhibitors and AII blockers is especially impressive when treatment starts before azotemia is severe (i.e. before the BUN becomes more than twice normal) (Fig. 7). It is also important to note that these effects are obtained with drugs that are well-tolerated in animals [1,2,29], and in the case of captopril, with a drug approved for use in the treatment of hypertension in humans [25,32]. In combination with our previously published studies [4,19], these experiments show that radiation nephropathy is not inevitably fatal. The previous studies did not, however, establish that it was inhibition of AII production that accounted for the efficacy of captopril in the treatment of radiation nephropathy (Fig. 1). First, ACE is not a very specific enzyme. In particular, ACE is also kininase II which degrades the active vasodilator bradykinin. Thus, ACE inhibition results in accumulation of a vasodilator [9,14]. Second, captopril has actions, including antimitotic activity [23] and non-specific thiol effects [31], that may not
Fig. 7. Progression of azotemia during treatment of established BMT nephropathy with an AII blocker (L-158,809). BUN levels are shown after 13, 22 and 31 weeks of therapy as a function of BUN at 26 weeks (2 weeks prior to the start of captopril therapy). Data are shown for 33 animals that received no treatment (X) and 33 animals that were treated continuously from 28 weeks with the AII blocker (W). The dashed lines show what the BUN values would be if there were no progression of azotemia. These are the same animals shown in Figs. 5 and 6.
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be the result of ACE inhibition. Thus, the efficacy of captopril in the treatment of radiation nephropathy does not prove that the renin-angiotensin pathway is involved. However, the efficacy of the AII blocker (Figs. 5–7) means that blocking the AT1 receptor (Fig. 1) per se is sufficient for retarding the progression of established radiation nephropathy and thus provides direct evidence for the involvement of the renin-angiotensin system. The current results confirm previous studies [5,7,10] that suggested that the antihypertensive action of the ACE inhibitors was not sufficient to account for their efficacy in the treatment of radiation nephropathy. This is clearly seen in the AII blocker study, where L-158,809 lowered the blood pressure in animals with established radiation nephropathy (Fig. 6), but did not prevent slow progression of azotemia (Figs. 6 and 7) or the eventual development of renal failure in some animals (Fig. 5). Similarly, these results confirm previous studies [5] that suggested that these beneficial effects are not explained merely by a reduction of proteinuria. This is seen in the ability of both continuous captopril (Fig. 3) and continuous L-158,809 (Fig. 6) to control or reduce proteinuria without preventing the slow progression of azotemia or the eventual development of renal failure in some animals. The striking effectiveness of the continuous low-dose (62.5 mg/l) captopril regimen (Figs. 2–4) was a surprise. When captopril was used prophylactically [7], a dose of 62.5 mg/l was effective in reducing azotemia, but it had little effect on hypertension and proteinuria and was only modestly effective in delaying renal failure (compared to the 500 mg/l regimen). In contrast, in these studies of treatment (as opposed to prophylaxis), doses of 62.5 and 500 mg/l are equally effective for control of azotemia (Figs. 3 and 4), for control of proteinuria (Fig. 3) and for prevention of renal failure (Fig. 2). The only difference seen between the two regimens was that the 500 mg/l dose was more effective in controlling blood pressure (Fig. 4). The latter observation reinforces the observation that control of hypertension is not critical for the efficacy of captopril in the treatment of radiation nephropathy. The equal effectiveness of the high- and low-dose regimens for treatment, in the face of their inequality for prophylaxis, implies that the mechanisms of action of ACE inhibitors in treatment and prophylaxis are not entirely the same. It should be emphasized that the 62.5 mg/l captopril regimen is a low dose by human standards. Based on data collected as part of other studies, the actual captopril dose in the low dose regimen is 50–75 mg/m2/day compared to the 25–250 mg/m2/day used in humans [15,17]. While a continuous low-dose captopril regimen was effective in the treatment of radiation nephropathy, the 6week treatment with high-dose captopril was relatively ineffective, delaying the rise in azotemia by only 8–12 weeks (Fig. 3) and delaying progression to renal failure by only 10–16 weeks (Fig. 2). The short treatment was particularly ineffective in animals with high initial azotemia, as seen
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from the azotemia progression data (Fig. 4) and from the early portion of the renal failure time course (Fig. 2). The short course of treatment was assessed because of other studies which showed that properly timed short interventions could be effective in prophylaxis [5] and that prophylactic treatment could be stopped if azotemia was kept under control for a sufficient length of time after irradiation [20]. These results imply that while established radiation nephropathy may be treatable with relatively low doses of captopril, treatment may have to continue indefinitely. Again this is in contrast with the studies of prophylactic use of ACE inhibitors in radiation nephropathy when higher doses (≥250 mg/l) are required for maximum effect, but where continuous treatment is not required [5,15,20]. Although captopril and L-158,809 were not tested against each other in a randomized fashion and the study designs were not identical, the two agents were tested on animals with similar initial degrees of injury (Fig. 8) and with similar prognoses if left untreated (Figs. 8 and 9). The similar success of the two agents on these two groups of animals suggest that ACE inhibitors and AII blockers have roughly equal efficacy for the treatment of radiation nephropathy. This is yet another area in which prophylaxis and treatment appear different, as in our published [21,22] and still-inprogress studies that indicate that L-158,809 is superior to captopril when used prophylactically.
Fig. 8. Evolution of azotemia (as BUN), hypertension (as systolic BP) and proteinuria (as UP/UC) during treatment of established BMT nephropathy with an ACE inhibitor (captopril) versus an AII blocker (L-158,809). Data are shown for animals receiving continuous treatment with L-158,809 (A) (from Fig. 6) or captopril (W) (high- and low-dose data combined from Fig. 3). Because there are differences between the two experiments, the controls for the AII blocker (B) and the captopril (X) treatments are shown separately. Data are shown as medians with 25–75% ranges.
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References
Fig. 9. Actuarial risk of renal failure (uremia) during treatment of established radiation nephropathy with an ACE inhibitor (captopril) versus an AII blocker (L-158,809). Data are shown for animals receiving continuous treatment with L-158 809 (from Fig. 5) or captopril (high- and low-dose data combined from Fig. 2). Because there are differences between the two experiments, the controls are shown separately. These are the same animals as shown in Fig. 8.
In addition to providing further evidence that late radiation injuries can be treatable, these studies suggest that the mechanisms involved in the treatment of radiation nephropathy are not entirely the same as those involved in prophylaxis. However, the efficacy of the AII blocker in both treatment and prophylaxis clearly indicates that angiotensin II and/or the angiotensin type-1 receptor are intimately involved, both early and late, in the development and progression of radiation nephropathy. These and other studies [4,5,19,20,22,30,31] clearly show that established radiation-induced normal tissue injuries can be treated. The dogma of inexorable progression towards organ failure is no longer tenable and a clear-cut rationale for clinical studies now exists. In radiation nephropathy, where inhibition of the renin-angiotensin system has been so successful experimentally, ACE inhibitors and AII antagonists appear to be the treatments of choice. In lung, experimental work has also suggested that these agents may be particularly effective [22,30,31]. It remains possible that inhibition of different peptide regulatory systems will be effective in other radiation injuries, such as in liver, gut and spinal cord. In these latter tissues, agents other than ACE inhibitors or AII blockers may be required for the treatment of radiation-induced normal tissue injuries.
Acknowledgements This research was supported by grant CA24652 from the US National Cancer Institute. Captopril was supplied as a gift by the Bristol-Myers-Squibb Research Institute, Princeton, NJ. L-158,809 was supplied as a gift by Merck, Sharpe and Dohme, Rahway, NJ. Marylou Stott provided expert technical assistance. Yvonne Morauski assisted with the preparation of the manuscript.
[1] Anderson, S., Rennke, H.G. and Brenner, B.M. Therapeutic advantage of converting enzyme inhibitors in arresting progressive renal disease associated with systemic hypertension in the rat. J. Clin. Invest. 77: 1993–2000, 1986. [2] Beukers, J.J.B., Van der Wal, A., Hoedemaeker, P.J. and Weening, J.J. Converting enzyme inhibition and progressive glomerulosclerosis in the rat. Kidney Int. 32: 794–800, 1987. [3] Chang, R.S.L., Siegl, P.K.S. and Clineschmidt, B.V. et al. In vitro pharmacology of L-158,809, a new and highly potent and selective angiotensin II receptor antagonist. J. Pharmacol. Exp. Ther. 262: 133–138, 1992. [4] Cohen, E.P., Fish, B.L. and Moulder, J.E. Treatment of radiation nephropathy with captopril. Radiat. Res. 132: 346–350, 1992. [5] Cohen, E.P., Fish, B.L. and Moulder, J.E. Successful brief captopril treatment in radiation nephropathy. J. Lab. Clin. Med. 129: 536–547, 1997. [6] Cohen, E.P. and Lemann, J. The role of the laboratory in evaluation of kidney function. Clin. Chem. 37: 785–796, 1991. [7] Cohen, E.P., Moulder, J.E., Fish, B.L. and Hill, P. Prophylaxis of experimental bone marrow transplant nephropathy. J. Lab. Clin. Med. 124: 371–380, 1994. [8] Ikoma, M., Kawamura, T., Kakinuma, Y., Fogo, A. and Ichikawa, I. Cause of variable therapeutic efficiency of angiotensin converting enzyme inhibitor on glomerular lesions. Kidney Int. 40: 195–202, 1991. [9] Johnston, C.I. Angiotensin receptor antagonists: focus on losartan. Lancet 346: 1403–1407, 1995. [10] Juncos, L.I., Carrasco Duen˜as, S., Cornejo, J.C., Broglia, C.A. and Cejas, H. Long-term enalapril and hydrochlorothiazide in radiation nephritis. Nephron 64: 249–255, 1993. [11] Lawton, C.A., Barber-Derus, S.W., Murray, K.J., Cohen, E.P., Ash, R.C. and Moulder, J.E. Influence of renal shielding on the incidence of late renal dysfunction associated with T-lymphocyte deplete bone marrow transplantation in adult patients. Int. J. Radiat. Oncol. Biol. Phys. 23: 681–686, 1992. [12] Lee, E.T. and Desu, M.M. A computer program for comparing K samples with right-censored data. Comput. Programs Biomed. 2: 315–321, 1972. [13] Miralbell, R., Bieri, S. and Mermillod, B. et al. Renal toxicity after allogeneic bone marrow transplantation: the combined effects of total-body irradiation and graft-versus-host disease. J. Clin. Oncol. 14: 579–585, 1996. [14] Moser, M. Angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists and calcium channel blocking agents: a review of potential benefits and possible adverse reactions. J. Am. Coll. Cardiol. 29: 1414–1421, 1997. [15] Moulder, J.E., Cohen, E.P., Fish, B.L. and Hill, P. Prophylaxis of bone marrow transplant nephropathy with captopril, an inhibitor of angiotensin-converting enzyme. Radiat. Res. 136: 404–407, 1993. [16] Moulder, J.E. and Fish, B.L. Late toxicity of total body irradiation with bone marrow transplantation in a rat model. Int. J. Radiat. Oncol. Biol. Phys. 16: 1501–1509, 1989. [17] Moulder, J.E. and Fish, B.L. Angiotensin converting enzyme inhibitor captopril does not prevent acute gastrointestinal radiation damage in the rat. Radiat. Oncol. Invest. 5: 50–53, 1997. [18] Moulder, J.E., Fish, B.L. and Abrams, R.A. Renal toxicity following total body irradiation and syngeneic bone marrow transplantation. Transplantation 43: 589–592, 1987. [19] Moulder, J.E., Fish, B.L. and Cohen, E.P. Treatment of radiation nephropathy with ACE inhibitors. Int. J. Radiat. Oncol. Biol. Phys. 27: 93–99, 1993. [20] Moulder, J.E., Fish, B.L. and Cohen, E.P. Noncontinuous use of angiotensin converting enzyme inhibitors in the treatment of experimental bone marrow transplant nephropathy. Bone Marrow Transplant. 19: 729–736, 1997.
J.E. Moulder et al. / Radiotherapy and Oncology 46 (1998) 307–315 [21] Moulder, J.E., Fish, B.L., Cohen, E.P. and Bonsib, S.M. Angiotensin II receptor antagonists in the prevention of radiation nephropathy. Radiat. Res. 146: 106–110, 1996. [22] Moulder, J.E., Robbins, M.E.C., Cohen, E.P., Hopewell, J.W. and Ward, W.F. Pharmacologic modification of radiation-induced late normal tissue injury. In: Radiation Therapy, Ch. 6. Editors: B.B. Mittal, J.A. Purdy and K.K. Ang. Kluwer Academic, Norwell, MA, pp. 129–151, 1998. [23] Nguyen, L., Ward, W.F., Ts’ao, C.H. and Molteni, A. Captopril inhibits proliferation of human lung fibroblasts in culture: a potential antifibrotic mechanism. Proc. Soc. Exp. Biol. Med. 205: 80–84, 1994. [24] Oikawa, T., Freeman, M., Lo, W., Vaughan, D.E. and Fogo, A. Modulation of plasminogen activator inhibitor-1 in vivo: a new mechanism for the anti-fibrotic effect of renin-angiotensin inhibition. Kidney Int. 51: 164–172, 1997. [25] Parving, H.H., Hommel, E. and Smidt, U.M. Protection of kidney function and decrease in albuminuria by captopril in insulin dependent diabetics with nephropathy. Br. Med. J. 197: 1086–1091, 1988. [26] Robbins, M.E.C. and Hopewell, J.W. Physiological factors effecting
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Radiation nephropathy is treatable with an angiotensin converting enzyme inhibitor or an angiotensin II type-1 (AT1) receptor antagonist John E. Moulder a ,*, Brian L. Fish a, Eric P. Cohen b a
Department of Radiation Oncology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA b Department of Medicine, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA Received 4 July 1997; revised version received 2 September 1997; accepted 10 September 1997
Abstract Background and purpose: Previous studies showed that progression of established radiation nephropathy could be delayed by continuous treatment with high doses of captopril, an angiotensin-converting-enzyme (ACE) inhibitor. The current studies were designed to determine whether a lower dose or a shorter treatment with captopril would be effective and whether an angiotensin II type-1 (AT1) receptor antagonist (AII blocker) would be effective. Materials and methods: In the captopril studies, rats were given renal irradiation at doses sufficient to produce radiation nephropathy. Six months after irradiation, animals were stratified by azotemia and assigned to no treatment, continuous high- or low-dose captopril, or 6 weeks of high-dose captopril. Captopril was given in drinking water at 62.5 mg/l (low dose) or 500 mg/l (high dose). The AII blocker study had a similar design, except that the nephropathy was the result of total body irradiation and bone marrow transplantation and the treatments were no treatment or continuous treatment with an AII blocker, L-158,809 (20 mg/l in drinking water). Animals were followed for 1 year with periodic studies of renal function. Results: Survival and renal function were significantly enhanced by all treatments. Continuous captopril treatment was more effective than the 6-week course of treatment, but there was no difference in effectiveness between the high and low doses of captopril. In continuous therapy, captopril and the AII blocker had roughly equivalent efficacy. Conclusions: Both the ACE inhibitor and the AII blocker were effective treatments for established radiation nephropathy. The best results with the ACE inhibitor required continuous therapy, but could be achieved with a low dose of the drug. 1998 Elsevier Science Ireland Ltd. Keywords: Nephropathy; Radiation; ACE inhibitors; Angiotensin II type-1 receptor antagonist; Captopril; Radiation injuries
1. Introduction Late radiation-induced normal tissue morbidity has classically been viewed as due solely to a delayed reduction in the number of surviving clonogens and these late injuries have been held to be inevitable, progressive and untreatable [27]. Recently, a paradigm shift has begun in the characterization of late radiation-induced normal tissue morbidity, as data from a variety of tissues indicate that these injuries involve dynamic interactions among parenchymal and vascular cells within an organ [22]. This paradigm offers a fundamentally new approach to radiation-induced normal tissue injuries, as it allows for the possibility that normal * Corresponding author.
tissue injuries can be treated. These treatments would be directed at modulating steps in the cascade of events leading to the clinical expression of damage [22]. Since this sequence of events does not appear to occur in tumors (where direct clonogenic cell kill still appears to be dominant), such treatments would not be expected to reduce antitumor efficacy. The possibility that radiation nephropathy could be treated arose from work done in the late 1980s. In 1986, Robbins and Hopewell [26] showed that the early hemodynamic changes observed after renal irradiation were decreased by treatment with an angiotensin-converting-enzyme (ACE) inhibitor (Fig. 1), captopril and in 1988 Ward et al. [30] showed that captopril could be used to reduce radiationinduced pulmonary injury. Because of these results and
0167-8140/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0167-8140 (97 )0 0175-8
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Fig. 1. The renin-angiotensin system is a regulated network of enzymes and substrates with a primary role in cardiovascular homeostasis (enzymes are shown in bold-face, inhibitors are shown in italics). Renin, produced in the kidney, is secreted in response to decreased renal perfusion and adrenergic nerve stimulation and the level of renin activity is believed to be the major control point in the system. Its specific substrate is angiotensinogen. The product of that reaction, angiotensin I, is converted to angiotensin II (AII) by angiotensin converting enzyme (ACE). ACE is present in lung and other organ endothelia and on kidney proximal tubule brush borders. AII is the active angiotensin; it is a potent vasoconstrictor, elevates blood pressure, stimulates collagen synthesis and is a growth promoter for kidney epithelial and vascular smooth muscle cells. AII has two known receptors. The type-1 (AT1) receptors serve the main hemodynamic functions of AII. The type-2 (AT2) receptors are active in utero and may play a role in apoptosis and development. AII may directly suppress renin activation and the volume retentive effect of AII will also suppress renin activity. ACE is also known as kininase II and in that role degrades the vasodilator bradykinin into inactive peptides [14]. Thus, ACE inhibition results in accumulation of a vasodilator, providing an AII-independent route for the antihypertensive activity of ACE inhibitors.
the known efficacy of ACE inhibitors in the treatment of progressive renal failure [1,2,25], we investigated the role of captopril in the treatment and prophylaxis of radiation nephropathy [4,5,7,16,19]. Initial studies [4] were directed towards treatment of established radiation nephropathy. In this model, the rats had undergone bilateral renal irradiation 6 months earlier. Animals with elevated blood urea nitrogen (BUN) were stratified by BUN and placed on captopril (500 mg/l in drinking water) or no drug. Azotemia and proteinuria were attenuated and survival was enhanced by the captopril therapy [4]. Further studies showed that enalapril (a non-thiol ACE inhibitor) was also effective, that the kidneys of captopril-treated animals developed less severe histopathologic lesions and that captopril also preserved renal function in a rat bone marrow transplant (BMT) nephropathy model [19]. In other models of chronic renal failure, ACE inhibitors are not always effective once renal injury is established [8]. Thus, the use of an ACE inhibitor from the time of irradiation should be more effective than treatment of established injury. In experiments with 1 year follow-up, both captopril and enalapril were found to be effective when used prophylactically [7], but other types of antihypertensives (verapamil, hydralazine, hydrochlorothiazide and alpha-methyldopa) were not effective [7,22]. A reduction in the dose of captopril was associated with a reduction in the prophylactic
benefit, but doses as low as 62.5 mg/l had some efficacy [7]. Unlike the situation in lung, where cessation of captopril therapy was followed by rapid deterioration [22], preservation of renal function was sustained in animals taken off captopril 26 weeks after irradiation [20]. Since angiotensin II (AII) is a potent vasoconstrictor and hypertension plays a major role in radiation nephritis, inhibition of AII production by ACE inhibition (Fig. 1) provides an obvious explanation for the efficacy of ACE inhibitors in radiation nephropathy [21,22]. However, ACE has other substrates that have hemodynamic activity (e.g. bradykinin [9,14]) and captopril has activity that may not be the result of ACE inhibition (e.g. antimitotic activity [22]). In order to determine whether ACE inhibitors were functioning via inhibition of AII production, we tested an angiotensin II type-1 (AT1) receptor antagonist [9,14] (Fig. 1). Analysis of both renal function and histopathology showed that the AT1 receptor antagonist (AII blocker) was even more effective than the ACE inhibitor in the prophylaxis of radiation nephropathy [21]. The effectiveness of AII blockers in the prophylaxis of radiation nephropathy was subsequently confirmed by Oikawa et al. [24]. While our previous studies [4,19] clearly showed that radiation nephritis could be treated with ACE inhibitors, several questions remained. The dose of captopril used was well above that used in humans (on a g/m2/day basis). Does captopril need to be used at such a high dose? Does the therapy need to be continued indefinitely, or will shorter courses be effective in treatment as they are in prophylaxis [5]? Finally, will AII blockers be as effective in treatment as they are in prophylaxis? The studies reported here were designed to answer these questions.
2. Materials and methods 2.1. Animals These studies were performed with syngeneic WAG/Rij/ MCW rats bred and housed in a moderate security barrier. The animals were free of Mycoplasma Pulmonis, pseudomonas and common rat viruses. No antibiotics, immunosuppressive drugs, or known nephrotoxic agents were used. Animals were maintained in the Animal Care Facilities of the Medical College of Wisconsin, which are fully accredited by the American Association for Accreditation of Laboratory Animal Care. The experiments described here were conducted under protocols approved by the Medical College of Wisconsin Animal Care and Use Committee. 2.2. Captopril study In the captopril study, 6–7-week-old female rats received bilateral renal irradiation in a study of the kinetics of repair of renal radiation injury. This experiment used a wide range of treatment schedules and radiation doses (one or two frac-
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tions at total doses of 8–15 Gy and 20 or 40 fractions at total doses of 27–35 Gy) and generated a large pool of animals with varying degrees of renal injury. At 26 weeks after irradiation, blood urea nitrogen (BUN) was assessed in all animals and animals (n = 95) with BUN of ≥30 mg/dl and ≤60 mg/dl (19–21 mg/dl is normal) were stratified by BUN and placed into four treatment groups, i.e. continuous highdose captopril, continuous low-dose captopril, 6 weeks of high-dose captopril, or no drug. Captopril was given in drinking water and drug treatments started 28 weeks after irradiation (i.e. 2 weeks after the assessment of BUN). The high-dose captopril regimen was 500 mg/l (the same dose used in the previous treatment studies [4,19]) and the lowdose regimen was 62.5 mg/l (the lowest dose that had been used in the captopril prophylaxis studies [7]). All animals remained in the study until morbid from uremia or until sacrifice 80 weeks after irradiation. Three animals were lost to follow-up because of the development of tumors at 55 weeks (low-dose captopril arm), 60 weeks (high-dose captopril arm) and 73 weeks (6-week treatment arm). The study design was identical to that previously used [4,19], except for the additional treatment arms and the selection of a narrower range of BUN in the current study (30–60 versus 25–120 mg/dl). 2.3. AII blocker study In the AII blocker study, 6–7-week-old female rats were given total body irradiation (TBI) followed by syngeneic BMT [16,18] in a study of pulsed low dose rate radiotherapy. This experiment used a range of dose rates (0.016–0.19 Gy/min) and radiation doses (18.9–20.1 Gy) and generated a large pool of animals with varying degrees of renal injury. At 26 weeks after irradiation, BUN was assessed in all animals and animals (n = 66) with BUN of ≥35 mg/dl and ≤70 mg/dl were stratified by BUN and placed into two treatment groups, i.e. continuous treatment with an AII blocker, or no treatment. Drug treatment began at 28 weeks after irradiation and continued for the duration of the study. Losartan is the first AII blocker to reach clinical use [9, 14]. For our experiments, we used a closely-related compound, L-158,809, which is a potent, competitive and specific antagonist of the AT1 receptor [3,29]. This AII blocker has good absorption when taken orally, is stable in water and has antihypertensive efficacy in rats equal to ACE inhibitors [3,29]. The L-158,809 was given in drinking water at 20 mg/l, the same dose used in our prophylactic study [21], and a dose whose antihypertensive activity in our rats was confirmed in the subtotal nephrectomy model [21]. All animals remained in the study until morbid from uremia or until sacrifice 75 weeks after irradiation. Five animals were lost to follow-up because of the development of tumors at 31–55 weeks (four in the AII blocker arm, one in the control arm) and one animal was lost to follow-up in the control arm when it developed a non-healing wound at 41 weeks. The AII blocker study differed from the captopril
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study in the use of TBI rather than renal irradiation to cause nephropathy and in the severity of azotemia that was used to select animals for the trial (35–70 versus 30–60 mg/dl). 2.4. Renal function assessment Animals were monitored daily in all experiments and those with symptomatic uremia or other morbid conditions (e.g. tumors) were sacrificed. Severe nephritis (uremia) is the major cause of morbidity in this model [16]. BUN, urine protein, urine creatinine and systolic blood pressure (BP) were assessed at 26, 41, 50 and 59 weeks after irradiation (2 weeks prior to and 13, 22 and 31 weeks into drug therapy) in all surviving animals. Urine was collected in metabolic cages and blood was collected by orbital bleeding. BUN was assayed with a urease-nitroprusside colorimetric assay, urine protein was assayed with a Coomassie Blue colorimetric assay and urine creatinine was assayed with a modified Jaffe´ reaction colorimetric assay [7]. As a general rule, BUN is an imprecise indicator of glomerular filtration rate (GFR) [6]. However, in this model, the correlation of BUN and serum creatinine is very strong [20] and data from other studies [6] indicate that serum creatinine is an adequate marker of GFR. Because BUN is strongly correlated with serum creatinine and hence with GFR, BUN rather than serum creatinine or creatinine clearance was used to assess renal function. It should also be noted that the degree of elevation of BUN at 26 weeks after renal irradiation is strongly correlated with survival time in this model [5]. Urine protein excretion was expressed as the ratio of urine protein to urine creatinine (UP/UC) in the same urine sample. This was done to account for the known urinary concentration defect that occurs in renal radiation injury and to normalize for animal size differences [7]. Tail-cuff BP was measured in unanesthetized animals using a semiautomatic machine. The mean BP reading from a minimum of three readings on each of 3 consecutive days was used for each time point; animals were preconditioned to the BP apparatus [7]. 2.5. Statistical methods All statistical comparisons were done using non-parametric techniques [28]. Physiologic data are shown as medians with 25–75% ranges and were compared by Mann– Whitney tests. Medians, ranges and non-parametric tests were used because physiological values in the groups showing abnormal renal function were often not normally distributed and because some animals were sacrificed with uremia prior to some of the assay intervals. For the purpose of calculating median values in groups where some animals (but less than half) had developed renal failure (uremia) prior to the assay interval, the values of parameters in animals that had developed renal failure were considered to have been above those of all survivors. If half or more of
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Fig. 2. Actuarial risk of renal failure (uremia) during treatment of established radiation nephropathy with different captopril regimens. Animals received bilateral renal irradiation and those with BUN between 30 and 60 mg/dl at 26 weeks after irradiation were stratified by BUN and placed on treatments that began 28 weeks after irradiation. Data are shown for 24 animals that received no captopril treatment, 23 animals that were treated continuously with high-dose captopril, 22 animals that were treated continuously with low-dose captopril and 26 animals that were treated with high-dose captopril from 28 to 34 weeks after irradiation.
animals, leading to renal failure in 62% of the animals by 50 weeks (Fig. 3). In the captopril-treated animals, azotemia was stable for 13 weeks before the increase in BUN resumed (Fig. 3). When the progression of azotemia resumed, the progression was much faster in the animals on the 6-week treatment course (Fig. 3). The captopril treatment stabilized blood pressure and proteinuria in the animals getting continuous captopril, but only reduced the rate of progression in the animals on the 6-week treatment course (Fig. 3). As with the survival data (Fig. 2), the physiology data (Fig. 3) showed essentially no difference in efficacy between the high- and low-dose captopril treatments, except that the high-dose treatment was somewhat better at controlling blood pressure. The differences among the groups were not a result of any initial differences between the groups. While the irradiated animals were only stratified by BUN, the resulting groups had similar values of BP and UP/UC (Fig. 3). For all three parameters (BUN, BP and UP/UC), all groups had 26-week values that were significantly (all P , 0.01) higher than those of normal (unirradiated) animals and not significantly (all P . 0.10) different from each other.
the animals in a dose group had developed renal failure prior to the assay, the point was plotted as ‘renal failure’. Survival curves were done by the Kaplan–Meier technique and compared using the method of Lee and Desu [12]. Survival curves were not plotted beyond the time where the number of animals at risk dropped below four.
3. Results 3.1. Captopril study Survival was enhanced when animals with established radiation nephropathy were placed on any of the three captopril regimens (Fig. 2). For untreated animals, the median survival time was 49 weeks (21 weeks after therapy started in the treatment arms). For animals on the continuous captopril regimens, the median survival time was 72 weeks on the high-dose regimen (23 weeks longer than in the untreated animals) and 74 weeks on the low-dose regimen (25 weeks longer than in the untreated animals). For animals on the 6-week course of captopril therapy, the median survival time was 65 weeks (16 weeks longer than in the untreated animals). The increase in survival times observed was highly significant (P = 0.013 for the 6-week treatment and P , 0.0001 for both continuous therapies versus the untreated animals). The continuous therapies were clearly superior to the 6-week course (both P , 0.01), but the highand low-dose continuous therapies were equally effective (P . 0.20). The increased survival for the animals treated with captopril was reflected in the renal function assays (Fig. 3). There was a progressive increase in azotemia (as BUN), hypertension and proteinuria (as UP/UC) in the untreated
Fig. 3. Evolution of azotemia (as BUN), hypertension (as systolic BP) and proteinuria (as UP/UC) during treatment of established radiation nephropathy with different captopril regimens. These are the same animals as shown in Fig. 2. Data are shown for animals that received no treatment (X), animals that were treated continuously with high-dose captopril (W), animals that were treated continuously with low-dose captopril (S) and animals that were treated with high-dose captopril from 28 to 34 weeks after irradiation (A). Data are shown as medians with 25–75% ranges. For the purpose of calculating median values in groups where some animals (but less than half) had developed renal failure (uremia) prior to the assay interval, the values of animals that had developed renal failure were considered to have been above those of all survivors. If half or more of the animals in a dose group had developed renal failure prior to the assay, the point was plotted as ‘renal failure’.
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Fig. 4. Progression of azotemia during treatment of established radiation nephropathy with different captopril regimens. BUN levels are shown 13 weeks after the start of captopril therapy as a function of BUN at 26 weeks (2 weeks prior to the start of captopril therapy). Data are shown for 24 animals that received no treatment (X), 23 animals that were treated continuously with high-dose captopril (W), 22 animals that were treated continuously with low-dose captopril (S) and 26 animals that were treated with high-dose captopril from 28 to 34 weeks after irradiation (A). The dashed lines show what the BUN values would be if there were no progression of azotemia. These are the same animals as shown in Figs. 2 and 3.
After 13 weeks of therapy, continuous captopril treatment was equally effective across the range of azotemia used in the study (Fig. 4). However, for longer durations of therapy, continuous captopril was most effective in the animals with initial BUN levels of less than about 40 mg/dl (data not shown). In contrast, even 13 weeks after the start of the 6week course of captopril, there was progression of azotemia in the animals whose initial BUN had been greater than ~45 mg/dl (Fig. 4).
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AII blocker was reflected in the renal function assays (Fig. 6). In this study, as in the captopril study (Fig. 3), there was a progressive increase in azotemia, hypertension and proteinuria in the untreated animals, leading to renal failure in 58% of the animals by 50 weeks. In the animals treated with the AII blocker, the BUN rose slowly during treatment and had risen significantly by 50 weeks (P = 0.002 compared to values at 26 weeks). Although azotemia increased during treatment with the AII blocker, hypertension and proteinuria actually decreased. The decrease in blood pressure was statistically significant at all three assay times (all P , 0.005 compared to values at 26 weeks), while the decrease in proteinuria was only significant at 41 and 50 weeks (both P , 0.001 compared to values at 26 weeks). The differences among the treated and untreated animals were not a result of any initial differences between the groups. While the irradiated animals were only stratified by BUN, the resulting groups had similar values of BP and UP/UC (Fig. 6). For all three parameters (BUN, BP and UP/UC), all groups had 26-week values that were significantly (all P , 0.005) higher than those of normal (unirradiated) animals and not significantly (all P . 0.10) different from each other. Treatment with the AII blocker was most effective when the degree of azotemia at the start of treatment was less than about 45 mg/dl (Fig. 7). However, after 31 weeks of treatment, BUN had progressed in all but the animal with the lowest initial BUN (Fig. 7). 3.3. AII blocker versus ACE inhibitor A rigorous comparison of the captopril (Section 3.1) and AII blocker (Section 3.2) studies is not possible. While each study was internally randomized, the two studies were not done at the same time, they used different irradiation procedures (local irradiation versus TBI) and they used some-
3.2. AII blocker study Survival was also enhanced when animals with established BMT nephropathy were placed on the AII blocker regimen (Fig. 5). For untreated animals, the median survival time was 50 weeks (22 weeks after therapy started in the treatment arm). For animals on the AII blocker regimen, the median survival time was in excess of 75 weeks (25+ weeks longer than in the untreated animals). The increase in survival times observed was highly significant (P , 0.0001 versus the untreated animals). The increased survival for the animals treated with the
Fig. 5. Actuarial risk of renal failure (uremia) during treatment of established radiation nephropathy with an AII blocker. Animals received TBI plus BMT and those with BUN between 35 and 70 mg/dl at 26 weeks after irradiation were stratified by BUN and placed on treatments that began 28 weeks after irradiation. Data are shown for 33 animals that received no treatment and 33 animals that were treated continuously with the AII blocker, L-158,809.
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Fig. 6. Evolution of azotemia (as BUN), hypertension (as systolic BP) and proteinuria (as UP/UC) during treatment of established BMT nephropathy with an AII blocker. These are the same animals as shown in Fig. 5. Data are shown for animals that received no treatment (X) and animals that were treated continuously with the AII blocker, L-158,809 (W). Data are shown as medians with 25–75% ranges. For the purpose of calculating median values in groups where some animals (but less than half) had developed renal failure (uremia) prior to the assay interval, the values of animals that had developed renal failure were considered to have been above those of all survivors. If half or more of the animals in a dose group had developed renal failure prior to the assay, the point was plotted as ‘renal failure’.
what different ranges of BUN as entry criteria. As a result, the pre-treatment functional parameters were different. In the AII blocker study, the BUN values were slightly higher, BP was significantly higher and the UP/UC ratios were lower (Fig. 8). However, despite these differences the resulting renal failure incidence curves (Fig. 9) and the progression of physiological injury (Fig. 8) for the two control groups were remarkably similar. The renal failure incidence (Fig. 9) and azotemia progression (Fig. 8) curves for the two treated groups were also remarkably similar, so we can conclude that there is no great difference between the efficacy of the two agents for the treatment of radiation nephropathy. However, Fig. 8 does show that the AII blocker was much more effective in controlling proteinuria and somewhat more effective in controlling hypertension.
4. Discussion Chronic radiation injuries such as radiation nephropathy are classically held to be inexorably progressive, often
resulting in fibrosis and organ failure [27]. This has necessitated shielding of susceptible organs [11] or limiting the dose of radiation [13]. To date, treatment of renal radiation injuries in humans has been symptomatic at best. In keeping with results in other organ systems [22], our studies suggest that radiation nephropathy may be pharmacologically treatable. The effect of ACE inhibitors and AII blockers is especially impressive when treatment starts before azotemia is severe (i.e. before the BUN becomes more than twice normal) (Fig. 7). It is also important to note that these effects are obtained with drugs that are well-tolerated in animals [1,2,29], and in the case of captopril, with a drug approved for use in the treatment of hypertension in humans [25,32]. In combination with our previously published studies [4,19], these experiments show that radiation nephropathy is not inevitably fatal. The previous studies did not, however, establish that it was inhibition of AII production that accounted for the efficacy of captopril in the treatment of radiation nephropathy (Fig. 1). First, ACE is not a very specific enzyme. In particular, ACE is also kininase II which degrades the active vasodilator bradykinin. Thus, ACE inhibition results in accumulation of a vasodilator [9,14]. Second, captopril has actions, including antimitotic activity [23] and non-specific thiol effects [31], that may not
Fig. 7. Progression of azotemia during treatment of established BMT nephropathy with an AII blocker (L-158,809). BUN levels are shown after 13, 22 and 31 weeks of therapy as a function of BUN at 26 weeks (2 weeks prior to the start of captopril therapy). Data are shown for 33 animals that received no treatment (X) and 33 animals that were treated continuously from 28 weeks with the AII blocker (W). The dashed lines show what the BUN values would be if there were no progression of azotemia. These are the same animals shown in Figs. 5 and 6.
J.E. Moulder et al. / Radiotherapy and Oncology 46 (1998) 307–315
be the result of ACE inhibition. Thus, the efficacy of captopril in the treatment of radiation nephropathy does not prove that the renin-angiotensin pathway is involved. However, the efficacy of the AII blocker (Figs. 5–7) means that blocking the AT1 receptor (Fig. 1) per se is sufficient for retarding the progression of established radiation nephropathy and thus provides direct evidence for the involvement of the renin-angiotensin system. The current results confirm previous studies [5,7,10] that suggested that the antihypertensive action of the ACE inhibitors was not sufficient to account for their efficacy in the treatment of radiation nephropathy. This is clearly seen in the AII blocker study, where L-158,809 lowered the blood pressure in animals with established radiation nephropathy (Fig. 6), but did not prevent slow progression of azotemia (Figs. 6 and 7) or the eventual development of renal failure in some animals (Fig. 5). Similarly, these results confirm previous studies [5] that suggested that these beneficial effects are not explained merely by a reduction of proteinuria. This is seen in the ability of both continuous captopril (Fig. 3) and continuous L-158,809 (Fig. 6) to control or reduce proteinuria without preventing the slow progression of azotemia or the eventual development of renal failure in some animals. The striking effectiveness of the continuous low-dose (62.5 mg/l) captopril regimen (Figs. 2–4) was a surprise. When captopril was used prophylactically [7], a dose of 62.5 mg/l was effective in reducing azotemia, but it had little effect on hypertension and proteinuria and was only modestly effective in delaying renal failure (compared to the 500 mg/l regimen). In contrast, in these studies of treatment (as opposed to prophylaxis), doses of 62.5 and 500 mg/l are equally effective for control of azotemia (Figs. 3 and 4), for control of proteinuria (Fig. 3) and for prevention of renal failure (Fig. 2). The only difference seen between the two regimens was that the 500 mg/l dose was more effective in controlling blood pressure (Fig. 4). The latter observation reinforces the observation that control of hypertension is not critical for the efficacy of captopril in the treatment of radiation nephropathy. The equal effectiveness of the high- and low-dose regimens for treatment, in the face of their inequality for prophylaxis, implies that the mechanisms of action of ACE inhibitors in treatment and prophylaxis are not entirely the same. It should be emphasized that the 62.5 mg/l captopril regimen is a low dose by human standards. Based on data collected as part of other studies, the actual captopril dose in the low dose regimen is 50–75 mg/m2/day compared to the 25–250 mg/m2/day used in humans [15,17]. While a continuous low-dose captopril regimen was effective in the treatment of radiation nephropathy, the 6week treatment with high-dose captopril was relatively ineffective, delaying the rise in azotemia by only 8–12 weeks (Fig. 3) and delaying progression to renal failure by only 10–16 weeks (Fig. 2). The short treatment was particularly ineffective in animals with high initial azotemia, as seen
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from the azotemia progression data (Fig. 4) and from the early portion of the renal failure time course (Fig. 2). The short course of treatment was assessed because of other studies which showed that properly timed short interventions could be effective in prophylaxis [5] and that prophylactic treatment could be stopped if azotemia was kept under control for a sufficient length of time after irradiation [20]. These results imply that while established radiation nephropathy may be treatable with relatively low doses of captopril, treatment may have to continue indefinitely. Again this is in contrast with the studies of prophylactic use of ACE inhibitors in radiation nephropathy when higher doses (≥250 mg/l) are required for maximum effect, but where continuous treatment is not required [5,15,20]. Although captopril and L-158,809 were not tested against each other in a randomized fashion and the study designs were not identical, the two agents were tested on animals with similar initial degrees of injury (Fig. 8) and with similar prognoses if left untreated (Figs. 8 and 9). The similar success of the two agents on these two groups of animals suggest that ACE inhibitors and AII blockers have roughly equal efficacy for the treatment of radiation nephropathy. This is yet another area in which prophylaxis and treatment appear different, as in our published [21,22] and still-inprogress studies that indicate that L-158,809 is superior to captopril when used prophylactically.
Fig. 8. Evolution of azotemia (as BUN), hypertension (as systolic BP) and proteinuria (as UP/UC) during treatment of established BMT nephropathy with an ACE inhibitor (captopril) versus an AII blocker (L-158,809). Data are shown for animals receiving continuous treatment with L-158,809 (A) (from Fig. 6) or captopril (W) (high- and low-dose data combined from Fig. 3). Because there are differences between the two experiments, the controls for the AII blocker (B) and the captopril (X) treatments are shown separately. Data are shown as medians with 25–75% ranges.
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References
Fig. 9. Actuarial risk of renal failure (uremia) during treatment of established radiation nephropathy with an ACE inhibitor (captopril) versus an AII blocker (L-158,809). Data are shown for animals receiving continuous treatment with L-158 809 (from Fig. 5) or captopril (high- and low-dose data combined from Fig. 2). Because there are differences between the two experiments, the controls are shown separately. These are the same animals as shown in Fig. 8.
In addition to providing further evidence that late radiation injuries can be treatable, these studies suggest that the mechanisms involved in the treatment of radiation nephropathy are not entirely the same as those involved in prophylaxis. However, the efficacy of the AII blocker in both treatment and prophylaxis clearly indicates that angiotensin II and/or the angiotensin type-1 receptor are intimately involved, both early and late, in the development and progression of radiation nephropathy. These and other studies [4,5,19,20,22,30,31] clearly show that established radiation-induced normal tissue injuries can be treated. The dogma of inexorable progression towards organ failure is no longer tenable and a clear-cut rationale for clinical studies now exists. In radiation nephropathy, where inhibition of the renin-angiotensin system has been so successful experimentally, ACE inhibitors and AII antagonists appear to be the treatments of choice. In lung, experimental work has also suggested that these agents may be particularly effective [22,30,31]. It remains possible that inhibition of different peptide regulatory systems will be effective in other radiation injuries, such as in liver, gut and spinal cord. In these latter tissues, agents other than ACE inhibitors or AII blockers may be required for the treatment of radiation-induced normal tissue injuries.
Acknowledgements This research was supported by grant CA24652 from the US National Cancer Institute. Captopril was supplied as a gift by the Bristol-Myers-Squibb Research Institute, Princeton, NJ. L-158,809 was supplied as a gift by Merck, Sharpe and Dohme, Rahway, NJ. Marylou Stott provided expert technical assistance. Yvonne Morauski assisted with the preparation of the manuscript.
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