We have previously shown significant pathology in the heart and kidney of murine hematopoietic-acute radiation syndrome (H-ARS) survivors of 8.7–9.0 Gy total-body irradiation (TBI). The goal of this study was to determine temporal relationships in the development of vasculopathy and the progression of renal and cardiovascular delayed effects of acute radiation exposure (DEARE) at TBI doses less than 9 Gy and to elucidate the potential roles of senescence, inflammation and oxidative stress. Our results show significant loss of endothelial cells in coronary arteries by 4 months post-TBI (8.53 or 8.72 Gy of gamma radiation). This loss precedes renal dysfunction and interstitial fibrosis and progresses to abnormalities in the arterial media and adventitia and loss of coronary arterioles. Major differences in radiation-induced pathobiology exist between the heart and kidney in terms of vasculopathy progression and also in indices of inflammation, senescence and oxidative imbalance. The results of this work suggest a need for different medical countermeasures for multiple targets in different organs and at various times after acute radiation injury to prevent the progression of DEARE.

The development of medical countermeasures against acute and delayed multi-organ injury requires animal models predictive of the human response to radiation and its treatment. Late chronic injury is a well-known feature of radiation nephropathy, but acute kidney injury has not been reported in an appropriate animal model. We have established a single-fraction partial-body irradiation model with minimal marrow sparing in non-human primates. Subject-based medical management was used including parenteral fluids according to prospective morbidity criteria. We show herein that 10 or 11 Gy exposures caused both acute and chronic kidney injury. Acute and chronic kidney injury appear to be dose-independent between 10 and 11 Gy. Acute kidney injury was identified during the first 50 days postirradiation and appeared to resolve before the occurrence of chronic kidney injury, which was progressively more severe up to 180 days postirradiation, which was the end of the study. These findings show that mitigation of the acute radiation syndrome by medical management will unmask delayed late effects that occur months after partial-body irradiation. They further emphasize that both acute and chronic changes in kidney function must be taken into account in the use and timing of mitigators and medical management for acute radiation syndrome and delayed effects of acute radiation exposure (DEARE).

A workshop entitled “Radiation-Induced Fibrosis: Mechanisms and Opportunities to Mitigate” (held in Rockville, MD, September 19, 2016) was organized by the Radiation Research Program and Radiation Oncology Branch of the Center for Cancer Research (CCR) of the National Cancer Institute (NCI), to identify critical research areas and directions that will advance the understanding of radiation-induced fibrosis (RIF) and accelerate the development of strategies to mitigate or treat it. Experts in radiation biology, radiation oncology and related fields met to identify and prioritize the key areas for future research and clinical translation. The consensus was that several known and newly identified targets can prevent or mitigate RIF in pre-clinical models. Further, basic and translational research and focused clinical trials are needed to identify optimal agents and strategies for therapeutic use. It was felt that optimally designed preclinical models are needed to better study biomarkers that predict for development of RIF, as well as to understand when effective therapies need to be initiated in relationship to manifestation of injury. Integrating appropriate endpoints and defining efficacy in clinical trials testing treatment of RIF were felt to be critical to demonstrating efficacy. The objective of this meeting report is to (a) highlight the significance of RIF in a global context, (b) summarize recent advances in our understanding of mechanisms of RIF, (c) discuss opportunities for pharmacological mitigation, intervention and modulation of specific molecular pathways, (d) consider the design of optimal clinical trials for mitigation and treatment and (e) outline key regulatory nonprescriptive frameworks for approval.

Angiotensin-converting enzyme inhibitors (ACEi) are effective mitigators of radiation nephropathy. To date, their experimental use has been in fixed-dose regimens. In clinical use, doses of ACEi and other medication may be escalated to achieve greater benefit. We therefore used a rodent model to test the ACEi enalapril as a mitigator of radiation injury in an escalating-dose regimen. Single-fraction partial-body irradiation (PBI) with one hind limb out of the radiation field was used to model accidental or belligerent radiation exposures. PBI doses of 12.5, 12.75 and 13 Gy were used to establish multi-organ injury. One third of the rats underwent PBI alone, and two thirds of the rats had enalapril started five days after PBI at a dose of 30 mg/l in the drinking water. When there was established azotemic renal injury enalapril was escalated to a 60 mg/l dose in half of the animals and then later to a 120 mg/l dose. Irradiated rats on enalapril had significant mitigation of combined pulmonary and renal morbidity and had significantly less azotemia. Dose escalation of enalapril did not significantly improve outcomes compared to fixed-dose enalapril. The current data support use of the ACEi enalapril at a fixed and clinically usable dose to mitigate radiation injury after partial-body radiation exposure.

We recently reported that daily dietary supplementation with 100 µg selenium (a dose exceeding a rat's nutritional requirement by about 33-fold) initiated immediately after total-body irradiation (TBI) and maintained for 21 weeks mitigates radiation nephropathy in a rat model as indicated by blood urea nitrogen (BUN) levels and histopathological criteria (Radiat Res. 2009; 17:368–73). In this follow-up study, we explored the risks and benefits of delaying the onset of supplementation, shortening periods of supplementation, and escalating selenium supplementation beyond 100 µg/day. Supplementation with 200 µg selenium/day (as selenite or seleno- l -methionine) substantially improved the mitigation of radiation nephropathy by lowering BUN levels at 4 months after TBI from 115 to as low as 34 mg/dl and by proportionally lowering the incidence of histopathological abnormalities. Shortening the period of supplementation to 3 or 2 months did not compromise efficacy. Delaying the onset of supplementation for 1 week reduced but did not abrogate the mitigation of radiation nephropathy. Supplementation with 300 µg/day mitigated radiation nephropathy less effectively than 200 µg and was poorly tolerated. Rats that had been given 10 Gy of TBI were less tolerant of high-dose selenium than nonirradiated rats. This reduced tolerance of high-dose selenium would need to be taken into consideration when selenium is used for the mitigation of radiation injury in victims of nuclear accidents or acts of radiological terrorism. The high dose requirements, the pronounced threshold effect, and the superior performance of selenite suggest that the mitigation of radiation nephropathy involves mechanisms that go beyond the induction of selenoproteins.

It is known that angiotensin converting enzyme inhibitors (ACEIs) and angiotensin II type-1 receptor blockers (ARBs) can be used to mitigate radiation-induced renal injury. However, for a variety of reasons, these previous results are not directly applicable to the development of agents for the mitigation of injuries caused by terrorism-related radiation exposure. As part of an effort to develop an animal model that would fit the requirements of the U.S. Food and Drug Administration (FDA) “Animal Efficacy Rule”, we designed new studies which used an FDA-approved ACEI (captopril) or an FDA-approved ARB (losartan, Cozaar®) started 10 days after a single total-body irradiation (TBI) at drug doses that are equivalent (on a g/m 2 /day basis) to the doses prescribed to humans. Captopril and losartan were equally effective as mitigators, with DMFs of 1.23 and 1.21, respectively, for delaying renal failure. These studies show that radiation nephropathy in a realistic rodent model can be mitigated with relevant doses of FDA-approved agents. This lays the necessary groundwork for pivotal rodent studies under the FDA Animal Efficacy Rule and provides an outline of how the FDA-required large-animal studies could be designed.

Radiation exposures from accidents, nuclear detonations or terrorist incidents are unlikely to be homogeneous; however, current biodosimetric approaches are developed and validated primarily in whole-body irradiation models. A workshop was held at the Armed Forces Radiobiology Research Institute in May 2008 to draw attention to the need for partial-body biodosimetry, to discuss current knowledge, and to identify the gaps to be filled. A panel of international experts and the workshop attendees discussed the requirements and concepts for a path forward. This report addresses eight key areas identified by the Workshop Program Committee for future focus: (1) improved cytogenetics, (2) clinical signs and symptoms, (3) cutaneous bioindicators, (4) organ-specific biomarkers, (5) biophysical markers of dose, (6) integrated diagnostic approaches, (7) confounding factors, and (8) requirements for post-event medical follow-up. For each area, the status, advantages and limitations of existing approaches and suggestions for new directions are presented.

Persistent, chronic oxidative injury may play a mechanistic role in late radiation injury. Thus antioxidants may be useful as mitigators of radiation injury. The antioxidants deferiprone, genistein and apocynin were tested in a rat radiation nephropathy model that uses single-fraction total-body irradiation (TBI) followed by syngeneic bone marrow transplant. Deferiprone was added to the drinking water at 1.0 or 2.5 g/liter, starting 3 days after the TBI. Urinary bleomycin-detectable iron, which could enhance production of oxygen radicals, was reduced in the rats on deferiprone compared to untreated rats, but deferiprone did not mitigate radiation nephropathy. Genistein added to the chow at 750 mg/kg starting immediately after TBI did not mitigate radiation nephropathy. Apocynin added to the drinking water at 250 mg/liter immediately after TBI did not mitigate radiation nephropathy. Thus three different types of antioxidants, when used at doses consistent with an antioxidant effect, had no mitigation efficacy against radiation nephropathy.

Sieber, F., Muir, S. A., Cohen, E. P., North, P. E., Fish, B. L., Irving, A. A., Mäder, M. and Moulder, J. E. High-Dose Selenium for the Mitigation of Radiation Injury: A Pilot Study in a Rat Model. Radiat. Res. 171, 368–373 (2009). The purpose of this study was to evaluate in an animal model the safety and efficacy of dietary supplementation with high doses of selenium for the mitigation of the type of radiation injury that might be sustained during a nuclear accident or an act of radiological terrorism. Age-matched male rats were exposed to 10 Gy (single dose) of total-body irradiation (TBI) followed by a syngeneic bone marrow transplant, then randomized to standard drinking water or drinking water supplemented with sodium selenite or seleno- l -methionine. At 21 weeks after TBI, most rats on standard drinking water had severe renal failure with a mean blood urea nitrogen (BUN) level of 124 ± 29 mg/dl (geometric mean ± SE) whereas rats on selenium-supplemented drinking water (100 μg/day) had a mean BUN level of 67 ± 12 mg/dl. The mitigating effect of selenium was confirmed by histopathological analyses. None of the animals on high-dose selenium showed signs of selenium toxicity. Our results suggest that dietary supplementation with high-dose selenium may provide a safe, effective and practical way to mitigate radiation injury to kidneys.

Lenarczyk, M., Cohen, E. P., Fish, B. L., Irving, A. A., Sharma, M., Driscoll, C. D. and Moulder, J. E. Chronic Oxidative Stress as a Mechanism for Radiation Nephropathy. Radiat. Res. 171, 164–172 (2009). Suppression of the renin-angiotensin system has proven efficacy for mitigation and treatment of radiation nephropathy, and it has been hypothesized that this efficacy is due to suppression of radiation-induced chronic oxidative stress. It is known that radiation exposure leads to acute oxidative stress, but direct evidence for radiation-induced chronic renal oxidative stress is sparse. We looked for evidence of oxidative stress after total-body irradiation in a rat model, focusing on the period before there is physiologically significant renal damage. No statistically significant increase in urinary 8-isoprostane (a marker of lipid peroxidation) or carbonylated proteins (a marker of protein oxidation) was found over the first 42 days after irradiation, while a small but statistically significant increase in urinary 8-hydroxydeoxy-guanosine (a marker of DNA oxidation) was detected at 35–55 days. When we examined renal tissue from these animals, we found no significant increase in either DNA or protein oxidation products over the first 89 days after irradiation. Using five different standard methods for detecting oxidative stress in vivo , we found no definitive evidence for radiation-induced renal chronic oxidative stress. If chronic oxidative stress is part of the pathogenesis of radiation nephropathy, it does not leave widespread or easily detectable evidence behind.

Moulder, J. E., Fish, B. L. and Cohen, E. P. Impact of Angiotensin II Type 2 Receptor Blockade on Experimental Radiation Nephropathy. Radiat. Res. 161, 312–317 (2004). In the rat, blockade of angiotensin II type 1 receptors diminishes the functional changes that occur after kidney irradiation. It has been hypothesized that some of the beneficial effects of angiotensin II type 1 blockers in renal disease are caused by a rise in angiotensin II that stimulates the angiotensin II type 2 receptor. If this hypothesis applied in this model, blockade of the type 2 receptor should exacerbate radiation nephropathy and/or counteract the beneficial effects of type 1 receptor blockade. To assess this hypothesis, rats were given total-body irradiation plus bone marrow transplantation and then treated for 12 weeks with a type 1 receptor blocker (L158,809), a type 2 blocker (PD123319), both blockers, or no blockers. Rats were assessed for renal function (proteinuria, hypertension, azotemia) and renal failure for up to 62 weeks. Contrary to the hypothesis, the type 2 blocker alone produced a temporary delay in the development of radiation nephropathy, and it substantially enhanced the efficacy of the type 1 blocker. This implies that both type 1 and type 2 angiotensin receptors need to be blocked to achieve the maximum level of prophylaxis of radiation nephropathy. We speculate that the beneficial effect of the angiotensin II type 2 receptor blocker is due to a reduction in radiation-induced renal cell proliferation or fibrosis.

Moulder, J. E., Fish, B. L., Regner, K. R. and Cohen, E. P. Angiotensin II Blockade Reduces Radiation-Induced Proliferation in Experimental Radiation Nephropathy. Radiat. Res. 157, 393–401 (2002). Total-body irradiation or renal irradiation is followed by a well-defined sequence of changes in renal function leading eventually to renal failure. Previous studies in a rat model have shown that inhibition of angiotensin-converting enzyme or blockade of angiotensin II receptors can prevent the structural and functional changes that occur after renal irradiation, and that these interventions are particularly important between 3 and 10 weeks after irradiation. We have now shown that in the same rat model, total-body irradiation induces proliferation of renal tubular cells (i.e., an increase in the number of cells staining positive for proliferating cell nuclear antigen) within 5 weeks after irradiation. Treatment with an angiotensin II receptor blocker delays this radiation-induced tubular proliferation and decreases its magnitude. Renal radiation also induces proliferation of glomerular cells, but the relative increase in glomerular proliferation is not as great as that seen in renal tubular cells, and the increase is not delayed or decreased by treatment with an angiotensin II receptor blocker. We hypothesize that angiotensin II receptor blockers exert their beneficial effect in radiation nephropathy by delaying the proliferation (and hence the eventual mitotic death) of renal tubular cells that have been genetically crippled by radiation.

Moulder, J. E., Fish, B. L., Regner, K. R., Cohen, E. P. and Raife, T. J. Retinoic Acid Exacerbates Experimental Radiation Nephropathy. Radiat. Res. 157, 199–203 (2002). Studies have shown that angiotensin-converting enzyme inhibitors and an angiotensin II receptor blocker can delay, but cannot reverse, the progression of experimentally induced radiation nephropathy. In an effort to find a method for reversing injury, three agents were tested in a rat model of radiation nephropathy. Pirfenidone (a phenyl-pyridone antifibrotic) and thiaproline (an inhibitor of collagen deposition) were not capable of retarding the development of radiation nephropathy. However, all-trans retinoic acid (an anti-inflammatory agent) exacerbated radiation nephropathy. We speculated that the detrimental effects of retinoic acid might be the result of stimulation of renal cell proliferation. However, retinoic acid had no effect on tubular or glomerular cell proliferation in normal animals and did not enhance radiation-induced proliferation. A recent report that retinoic acids inhibit nitric oxide production suggested an alternative mechanism, since inhibition of production of nitric oxide is known to exacerbate radiation nephropathy. Experiments demonstrated that retinoic acid exacerbated the radiation-induced drop in renal production of nitric oxide, suggesting that the detrimental effect of all-trans retinoic acid might be explained by inhibition of renal nitric oxide activity. Particularly in view of the recent clinical report of enhancement of radiation nephropathy by retinoic acid in patients receiving bone marrow transplantation, the combination of retinoic acid and renal irradiation should be carried out with great caution.

Datta, P. K., Moulder, J. E., Fish, B. L., Cohen, E. P. and Lianos, E. A. Induction of Heme Oxygenase 1 in Radiation Nephropathy: Role of Angiotensin II. Radiat. Res. 155, 734–739 (2001). In a rat model of radiation-induced nephropathy, we investigated changes in expression of heme oxygenase 1 (Hmox1, also known as HO-1), an enzyme that catalyzes conversion of heme into biliverdin, carbon monoxide and iron. The study explored whether radiation induces Hmox1 expression in the irradiated kidney and whether angiotensin II (AII) mediates Hmox1 expression in glomeruli isolated from irradiated kidneys. To assess the effects of radiation on Hmox1 expression, rats received 20 Gy bilateral renal irradiation and were randomized to groups receiving an AII type 1 (AT 1 ) receptor antagonist (L-158,809) or no treatment. Drug treatment began 9 days prior to bilateral renal irradiation and continued for the duration of the study. Estimation of Hmox1 levels in glomerular protein lysates assessed by Western blot analysis revealed a significant increase in Hmox1 protein at 50 and 65 days postirradiation. In animals treated with the AT 1 receptor antagonist, there was no induction of Hmox1, suggesting that AII may be a mediator of Hmox1 induction. To confirm that AII stimulates Hmox1 expression, animals were infused with 200, 400 or 800 ng/kg min –1 of AII for 18–19 days, and Hmox1 protein levels in glomeruli were assessed. There was a significant induction of Hmox1 in glomeruli of animals infused with 800 ng/kg min –1 of AII. These studies demonstrate that glomerular Hmox1 expression is elevated in the middle phase of radiation nephropathy and that AII can increase glomerular Hmox1 levels.

Sharma, M., Sharma, R., McCarthy, E. T., Ge, X. L., Fish, B. L., Savin, V. J., Cohen, E. P. and Moulder, J. E. Early Detection of Radiation-Induced Glomerular Injury by Albumin Permeability Assay. Renal irradiation leads predictably to glomerular vascular injury, cell lysis, matrix accumulation, sclerosis and loss of renal function. The immediate effects of renal irradiation that may be associated with glomerular pathology and proteinuria are not clear in the human disease or its rat model. We hypothesized that radiation-induced injury causes immediate and subtle alterations in glomerular physiology independent of the neurohumoral and hemodynamic regulatory mechanisms. We employed a sensitive in vitro functional assay of glomerular albumin permeability (P alb ) to demonstrate radiation-induced damage to the glomerular filtration barrier immediately after total-body irradiation of rats. In blinded experiments, control rats were sham-treated, and experimental rats received 9.5 Gy X rays. Rats were killed humanely at 1 h to 9 weeks after irradiation and glomeruli were isolated. In parallel experiments, glomeruli were isolated from normal rats and irradiated in vitro. The change in glomerular capillary permeability due to an experimental oncotic gradient was determined using videomicroscopy and P alb was calculated. Results show that in vivo or in vitro irradiation of glomeruli caused an increased P alb at 1 h. Increased P alb was observed up to 3 weeks after irradiation. Glomeruli from mice irradiated with 9.5 or 19.0 Gy X rays did not show increased P alb at 1 h postirradiation. We conclude that glomerular protein permeability of irradiated rats increases in a dose-dependent manner immediately after irradiation and that it appears to be independent of hemodynamic or systemic influences.

Angiotensin-converting enzyme (ACE) inhibitors can be used to prevent the development of radiation nephropathy. Current studies were designed to determine whether blocking the angiotensin II (AII) receptor, rather than preventing the creation of AII, would be effective in the prophylaxis of radiation nephropathy. Rats received 17 Gy total-body irradiation (TBI) in six fractions followed by syngeneic bone marrow transplantation (BMT). Prior to TBI the rats were randomized to groups receiving an ACE inhibitor (captopril), an AII type 1 ( AT 1 ) receptor antagonist (L-158,809) or no treatment. Renal function was assessed over 40 weeks, and a subset of animals were sacrificed for histopathology at 33 weeks. Renal function deteriorated progressively in animals receiving TBI alone, leading to renal failure by 34 weeks. Renal function was significantly better in animals receiving either captopril or L-158,809, and none of these animals had progressed to renal failure by 43 weeks. Analysis of both renal function and histopathology shows that the AII receptor antagonist is more effective than the ACE inhibitor in the prophylaxis of BMT nephropathy. This indicates that a reduction of activation of the AT 1 receptor by AII, by itself, is sufficient for the prophylaxis of radiation nephropathy.

Chronic renal failure occurs in about 20% of long-term survivors treated with bone marrow transplant (BMT) regimens that include total-body irradiation (TBI); this syndrome is called BMT nephropathy. In a previous study in a syngeneic rat BMT model it was shown that captopril (an inhibitor of angiotensin-converting enzyme) could be used to treat experimental BMT nephropathy. Current studies were designed to determine whether captopril could also be used to prevent BMT nephropathy. Rats received 14 to 18.5 Gy TBI in six fractions over 3 days followed by syngeneic BMT. Seven days before TBI half the rats were started on captopril (500 mg/liter in the drinking water). Blood urea nitrogen, ratios of urine protein to creatinine, serum creatinine, and blood pressure were used to assess renal function. In animals receiving TBI alone, BMT nephropathy developed 3 to 6 months after transplant. At 6 months after TBI, captopril-treated animals had lower systolic blood pressure and better-preserved renal function than animals receiving TBI alone, with dose-modifying factors of about 1.3. The captopril treatment had no effect on bone marrow ablation by TBI. Captopril appears to be safe and effective in the prophylaxis of BMT nephropathy.

Both experimental and clinical radiation nephropathy are typically progressive, evolving to kidney failure over weeks to months. Other late radiation injuries (spinal cord, lung) are also progressive and have no known specific antidote. Recent reports of the efficacy of captopril in modifying radiation injury of the lung prompted this trial of captopril in treating established radiation nephropathy. Six months after 15-27 Gy in 12 fractions bilateral renal irradiation, 72 rats with blood urea nitrogen > 4.1 mmol/liter were started on captopril (500 mg/liter) or no drug in the drinking water. Subsequent survival was significantly enhanced in rats receiving captopril as opposed to no drug (P = 0.0013), and the rate of rise of blood urea nitrogen was significantly diminished (P < 0.0001) in the animals on captopril. Urine protein excretion was also reduced from initially elevated levels in the rats on captopril compared to levels in rats given no drug. We conclude that captopril therapy preserves kidney function, reduces proteinuria, and enhances survival in experimental radiation nephropathy.