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Abstract
Though angiogenesis has been investigated in depth, vascular regression and rarefaction remain poorly understood. Regression of renal vasculature accompanies many pathological states such as diabetes, hypertension, atherosclerosis, and radiotherapy. Radiation decreases microvessel density in multiple organs, though the mechanism is not known. By using a whole animal (rat) model with a single dose of partial body irradiation to the kidney, changes in the volume of renal vasculature were recorded at two time points, 60 and 90 days after exposure. Next, a novel vascular and metabolic imaging (VMI) technique was used to computationally assess 3D vessel diameter, volume, branch depth, and density over multiple levels of branching down to 70 µm. Four groups of rats were studied, of which two groups received a single dose of 12.5 Gy X-rays. The kidneys were harvested after 60 or 90 days from one irradiated and one non-irradiated group at each time point. Measurements of the 3D vasculature showed that by day-90 post-radiation, when renal function is known to deteriorate, total vessel volume, vessel density, maximum branch depth, and the number of terminal points in the kidneys decreased by 55%, 57%, 28%, and 53%, respectively. Decreases in the same parameters were not statistically significant at 60 days post-irradiation. Smaller vessels with internal diameters of 70-450 µm as well as large vessels of diameter 451-850 µm, both decreased by 90 days post-radiation. Vascular regression in the lungs of the same strain of irradiated rats has been reported to occur before 60 days supporting the hypothesis that this process is regulated in an organ-specific manner and occurs by a concurrent decrease in luminal diameters of small as well as large blood vessels.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Vascular regression is not well understood, especially in mammals. This process occurs naturally during organ development and also in pathological states, but is difficult to replicate and investigate in the laboratory, using common rodent models. Reports from radiation accident survivors and preclinical animal experimentations implicate that radiation-induced injury or dysfunction of the vasculature is a major cause of organ injury [1]. The effects of radiation are well known to occur acutely, which are then followed by organ-specific delayed effects. Acute vascular effects include endothelial swelling and apoptosis, loss of vessel permeability, and infiltration of blood [2]. Delayed effects include capillary collapse, basement membrane thickening, fibrosis, telangiectasia, and loss of regenerative capacity [3]. Since all organs are perfused by blood vessels, vascular injury can be central to multiple organs after radiation.
With the rising threats of nuclear accidents, radiological attacks by terrorist groups, and increasing use of radiotherapy, the concern of injury to noncancerous tissue (normal tissue) is increasing. Yet, the consequences of radiation to the blood vessels of whole organs are still not well defined. The kidney is a radio-sensitive organ and can be dose-limiting for radiotherapy for pelvic cancers, gastrointestinal cancers, gynecologic cancers, lymphomas, and sarcomas of the upper abdomen. As a result, radiation-induced kidney injuries and diseases have been studied by many groups [4]. In our previous study, we published the results of different doses of radiation (0, 7.5, 10, and 12.5 Gray (Gy)) effects on the metabolic state of kidneys and livers. As mentioned in the paper, we measure two autofluorescence coenzymes called nicotinamide adenine dinucleotide (NAD) + hydrogen (H)(NADH) and Flavin Adenine Dinucleotide (FAD) and used the ratio of NADH/FAD as a biomarker of redox state oxidization of the organ. We found that with increases in the doses of radiation, the redox ratio decreases due to increased reactive oxygen species production [5] . In another study, we investigated the result of 13 Gy radiation on the metabolic state of kidney and then the mitigation effect of lisinopril on it. We showed that the redox ratio significantly decreased in the whole kidney and regular injection of lisinopril significantly mitigated the effects of radiation and reversed the decrease in redox ratio [6]. Kruse et al. showed that after 30 weeks (210 days) of a single dose of 16 Gy exposure, capillary networks of the renal cortex and glomeruli were replaced with tortuous and telangiectatic vessels [7]. Kuin et al. showed injuries caused by 16 Gy irradiation in mice renal capillaries after 20-40 weeks [8]. However, earlier effects in the irradiated kidney, as well as sequential changes over time, remain to be reported.
It is likely that if humans are exposed to > 5 Gy today, they will survive acute radiation injury that occurs within the first 60 days but will then suffer from the delayed effects to the kidney, which takes years to develop [9]. Cancer radiotherapy and accidental radiation to one or part of one kidney can cause radiation-induced renal fibrosis. This is characterized by the excessive accumulation of collagen and other extracellular matrix (ECM) components [10–13]. Fibrosis is a hallmark of many chronic kidney diseases and can be a consequence or cause of impairment of vascular function and morphology [14]. Animal models of radiation nephropathy are well described [15–21], with the best-studied being rat models [16,22–25]. Interestingly, clinical interventions, such as angiotensin converting enzyme inhibitors have mitigated or treated radiation nephropathy in rats [16,17,22,24–26], as well as in humans [27].
A major challenge in investigating vascular injury in rodent kidneys is the lack of imaging tools to easily examine 3D changes in the blood vessels over time. This is an important experimental endpoint, without which it is difficult to determine cause and effect using pharmacological or genetic interventions that regulate vascular regression. Imaging modalities such as micro-computed tomography (micro-CT) [28,29], ultra-microscopy [29], near-infrared fluorescence imaging [30], magnetic resonance imaging [31], and ultrasound imaging [32] are existing tools for 3D vascular imaging but, they are slow, complex, and costly. Labeling with a contrast agent antibody or filler is required for most of these technologies [32], each involving its own set of limitations. Recently, we uncovered a new and innovative technique, vascular and metabolic imaging (VMI), that does not use stains and other exogenous agents to assess the vasculature of different organs [33]. VMI generates 3D-structures of rodent renal vessels that are easily amenable to mathematical analyses. For the first time, the current study describes an innovative computational assessment of the vascular changes in the kidney over two time points at 60 and 90 days after radiation. This study analyzes regression of renal vessels of ≥70 µm diameter in a well-characterized rat model after 12.5 Gy irradiation, using VMI. Changes in vessel diameter and volume, the number of terminal points, and the maximum branch depth of the renal vascular tree are measured to reveal new insights into vascular remodeling and rarefaction in the kidney. Determining these endpoints will propel more studies in vascular regression.
2. Experiments and methods
2.1 Radiation exposure and animal care
The animal protocols were approved by the Institutional Animal Care and Use Committees (IACUC) at the Medical College of Wisconsin, Milwaukee. WAG/RijCmcr female rats were irradiated at 12-13 weeks of age (∼155 grams). Rats were randomized into two groups: I) non-irradiated that received no irradiation (i.e., 0 Gy irradiation) (n = 12); II) irradiated that received 12.5 Gy partial body irradiation shielding only part of one hind limb (leg-out partial body irradiation (PBI) [23]) (n = 14). The 12.5 Gy dose of radiation was selected since it is the highest dose that can be delivered without requiring supportive care with a daily injection of saline to treat lethal diarrhea between days 3-7 post-irradiation. To achieve proper radiation exposure, briefly, rats were restrained and irradiated. One hind limb of each rat was carefully externalized and shielded with a 0.25-inch lead block. An XRAD 320KV orthovoltage x-ray system (Precision X-Ray, North Branford, Connecticut) was operated at 320 kVp and 13 mAs with a half-value layer of 1.4 mm Copper (Cu) with a dose-rate of 1.75 Gy min-1 for a total dose of 12.5 Gy. Radiation was delivered from posterior to anterior to each rat. The dosimetry is as described by Medhora et al. [26]. Rats from each group were euthanized either at 60 or 90 days after radiation. The kidneys were harvested and flash-frozen in prechilled isopentane. In total kidneys from four groups of rats were studied: I) non-irradiated rats at day 60 post-irradiation (n = 6), II) irradiated rats receiving 12.5 Gy at 60 days post-irradiation (n = 7), III) non-irradiated rats at day 90 post-irradiation (n = 6), IV) irradiated rats receiving 12.5 Gy at day 90 after radiation (n = 7).
2.2 3D optical imaging
Cryo-imager is a custom-designed device built at the Biophotonics lab, Florida Atlantic University. This instrument enables the measurement of autofluorescence metabolic indices, NADH and FAD, in frozen tissues as described previously [33–35]. In brief, the snap-frozen kidneys are embedded in a dark medium, kept in an ultra-low freezer for more than 24 hours, and then mounted on the Cryo-imager. This device first acquires and saves NADH and FAD images of the sample surface, then cuts and again acquires similar images sequentially from thin cut sections (30-42 µm). Imaging a rat kidney with this instrument takes around 3 hours. A mercury arc lamp (200 W lamp, Oriel, Irvine, CA, with a bulb from Ushio Inc., Japan) is used as the excitation source. The excitation light passes through optical excitation filters for NADH (350 nm with 80 nm bandwidth) and FAD (437 nm with 20nm bandwidth). The emitted light from the sample is first filtered by emission optical filters of NADH (460 nm with 50nm bandwidth) and FAD (537 nm with 50nm bandwidth), then is captured by a CCD camera (QImaging Retiga R6, 16 bit). Acquired images are used to obtain the 3D images of the samples. Vasculatures are extracted from these images using FIJI [36] and IMARIS 9.5 software (Bitplane Inc, Zurich, Switzerland) as described in [33].
2.3 Vascular segmentation
This procedure was described in our recent publication [33]. In brief, the label-free vascular segmentation method uses the stack of images acquired from the NADH channel of the Cryo-imager. Metabolically active tissues appear bright due to the abundance of mitochondria. However, blood within vessels quenches the NADH fluorescence, leaving pixels from functional vessels to appear dark. Each slice of the stack of NADH fluorescent images acquired from the kidney is processed in a stepwise manner. First, the brightness and contrast of each slice are adjusted to make the dark perfused vessels against the remaining bright tissue darker. Second, the images are inverted so vessels are now bright, and tissue is dark (negative image). Third, the background is subtracted so other components of kidney tissues are removed from the vessels. Then by stacking the slices of each sample in the Z-direction the re-constructed 3D-vasculature is obtained.
For quantification, the 3D-vasculature is fed to Imaris (9.5.1 software, Bitplane Inc), and using a manual tracing algorithm based on local intensity contrast, the vessel network is extracted. The outline of each vessel using both automatic and manual methods is set. Based on the extracted vessel network, different parameters are calculated such as vessel mean diameter, vessel volume, maximum vessel branch depth, the number of terminal points, etc. To measure the vessel density, the total volume of each kidney was measured by delineating the surface area of the whole organ using Imaris. Then the total vessel volume is divided by the whole sample volume to obtain the vessel density.
2.4 Statistical analysis
All statistical analyses are conducted using MATLAB (MATLAB, Inc., Boston MA, USA). Dose-response and time-response effects of irradiation on kidneys are investigated. Dose-response is the changes that are observed in irradiated kidneys compared to non-irradiated kidneys. Time-response is the difference between the changes in the irradiated kidney 60 vs. 90 days post-radiation. The mean vessel diameter, total vessel volume, vessel density, maximum vessel branch depth, and the number of terminal points are assessed to compare the dose-response of irradiated kidneys using the non-parametric Mann–Whitney U test (Wilcoxon rank-sum test) with the significant criteria P < 0.05. To analyze the time-response, fold change is used for total vessel volume, vessel density, maximum vessel branch depth, and the number of terminal points, 60 and 90 days after radiation. The equations that are used for fold change are as follows:
3. Results
The representative 3D-extracted vasculature, vessel start points (blue spheres), and terminal points (green spheres) from four groups of rats are shown in Fig. 1. The sizes of spheres marking the start of the vessel and the terminal points vary based on the vessel diameter at that point. Figure 1(a), 1(b), 1(c), and 1d represent the day-60 non-irradiated, day-60 irradiated, day-90 non-irradiated, and day-90 irradiated kidneys, respectively. The green spheres in Fig. 1(b) (irradiated day-60) and 1d (irradiated day-90) appear both smaller and fewer in number compared to Fig. 1(a) (non-irradiated day-60) and 1c (non-irradiated day-90), respectively, suggesting that the diameter and the number of perfused vessels after radiation may have decreased on day-60 and day-90 post-radiation. Also, the effect of radiation is more evident in Fig. 1(d) (irradiated day-90) compared to Fig. 1(b) (irradiated day-60), suggesting that the changes in vascular architecture due to radiation were more pronounced at 90 days as compared to 60 days post-radiation. Similarly, sizes of blue and green spheres in Fig. 1 also shows that radiation reduced the diameter of the vessels, especially after 90 days. Quantitative analyses are given in Tables 1 and 2.
Fig. 1. The representative extracted 3D vasculature in which the terminal point of each vessel is marked with green spheres and the vessel start points are marked with blue spheres. (a) Non-irradiated kidney harvested at 60 days, (b) 12.5 Gy irradiated kidney harvested 60 days after irradiation, (c) non-irradiated kidney harvested at 90 days, (d) 12.5 Gy irradiated kidney harvested 90 days after irradiation. Results are quantitated in Tables 1 and 2. Videos of 3D images can be found in the supplemental document (see Visualization 1, Visualization 2, Visualization 3, and Visualization 4).
Table 1. Fold change of vascular parameters induced by radiation after day-60 and day-90 post-radiation
Table 2. Measurements of vascular structural parameters (mean ± standard deviation)
Figure 2 represents the 3D vessel structure of the same samples in Fig. 1 but color-coded based on the mean diameter of vessels ranging from 70 µm to 850 µm. The radius of each vessel is measured from the center to the surface of vessels. Violet is assigned to vessels with a diameter of ∼ 70 µm and red is assigned to vessels of ∼850 µm diameter with a color-coded range in between (see Fig. 2). Figures 2(a) (non-irradiated day-60) and 2c (non-irradiated day-90) show that in non-irradiated kidneys, the largest vessel branches are mostly colored red or yellow. However, Figs. 2(b) (irradiated day-60) and 2d (irradiated day-90) show that the largest vessel branches are mostly colored green or blue, suggesting that radiation reduced the lumen of the blood vessels. Comparison of Figs. 2(c) (non-irradiated day-90) and 2d (irradiated day-90) show that by 90 days post-radiation, vessel diameter in irradiated kidneys is markedly reduced from that of non-irradiated kidneys. Quantitative analyses are given in Table 3.
Fig. 2. The representative extracted 3D vasculature colored-coded based on diameter. The vessel diameter measured ranged from 70 µm to 850 µm. The largest vessels are colored red, and the smallest vessels are colored violet. (a) Non-irradiated kidney harvested at 60 days, b) 12.5 Gy irradiated kidney harvested 60 days after irradiation, (c) non-irradiated kidney harvested at 90 days, (d) 12.5 Gy irradiated kidney harvested 90 days after irradiation. Results are quantitated in Table 3. Videos of 3D images can be found in the supplemental document (see Visualization 5, Visualization 6, Visualization 7, and Visualization 8).
Table 3. The average number of vessels that were measured mostly in increments of 100 µm. Stars (*) mark the values in the irradiated group that are significantly different from the corresponding non-irradiated group i.e. P < 0.05
Figure 3 shows the same kidneys in Figs. 1 and 2 but color-coded to reflect the branch depth. Branch depth is the number of bifurcations or branch points in the shortest path, from the start to a given point in the vessel network. The maximum branch depth in a kidney sample is an indicator of the maximum number of times a vessel has been bifurcated to get to the smallest detected vessels (70 µm in this study). Therefore, maximum branch depth is also a marker of the highest branch order that could be observed in a sample.
Fig. 3. The representative extracted 3D vasculature colored-coded based on branch depth. The branch depth ranges from 0 to 19. Vessels with a branch depth of 0 are colored violet and vessels with branch depth of 19 are colored red. (a) non-irradiated kidney harvested at 60 days, b) 12.5 Gy irradiated kidney harvested 60 days after irradiation, (c) non-irradiated kidney harvested at 90 days, (d) 12.5 Gy irradiated kidney harvested 90 days after irradiation. Results are quantitated in Tables 1 and 2. Videos of 3D images can be found in the supplemental document (see Visualization 9, Visualization 10, Visualization 11, and Visualization 12).
The highest maximum vessel branch depth (branch order) that was observed, was branch depth (order) of 19. Therefore, in Fig. 3 vessels are colored from the branch depth of 0, colored by violet, to the branch depth of 19, colored by red. Figures 3(a) and 3(b) are representatives of the non-irradiated and irradiated kidneys harvested day-60 post-radiation, respectively. The smaller vessels (the vessels closer to the terminal points) in these figures are colored mainly blue and green. This suggests that first, the maximum branch depth by this method of analysis for both irradiated and non-irradiated kidneys harvested 60-days post-radiation was less than 19, and second, the branch depth had not been considerably affected by radiation after 60 days. In contrast, in Fig. 3(c) (non-irradiated day-90) the color of smaller vessels is yellow and sometimes orange and red, but in Fig. 3(d) (irradiated day-90) there is no orange or red. This indicates a conspicuous reduction in branch depth by day-90 post-radiation.
The value of fold change for 3D vessel structural parameters is calculated using Eqs. (1) and (2) (see Methods) to investigate the time response of irradiated kidneys. As described in the methods, the fold change for each day is the ratio of average value in the irradiated group over the average value in the corresponding non-irradiated group. The fold change provides a quantitative index for the severity of changes caused by radiation on day-60 and day-90. The smaller the value of fold change, the more severe the damage that is caused by radiation.
Table 1 presents the fold change value for total vessel volume, vessel density, maximum branch depth, and the number of terminal points at days-60 and 90 post-radiation. The results in Table 1 show that the fold change value for all parameters is lower on day-90 compared to day-60.
Table 2 shows average values ± standard deviation of measurements done on all 3D vessel structural parameters. As Table 2 shows, for non-irradiated kidneys, the average of total vessel volume, vessel density, maximum vessel branch depth, and the number of terminal points increased on day-90 compared to day-60. It also shows that for 12.5 Gy irradiated kidneys, the changes in the average of total vessel volume, vessel density, maximum vessel branch depth, and the number of terminal points by day 90 were greater compared to day 60. This observation shows the late effects of radiation on kidney vessels.
The results in Table 2 are graphically represented in Fig. 4 to easily visualize changes in total vessel volume, vessel density, maximum branch depth (branch order), and the number of terminal points in all samples of the four groups.
Fig. 4. Graphs representing mean ± standard deviation of the following parameters evaluated in Fig. 1–3: (a) total vessel volume, (b) vessel density, (c) maximum branch depth, and (d) number of terminal points. Values are shown for non-irradiated (0 Gy) and irradiated (12.5 Gy) groups at 60 (green bars) and 90 (blue bars) days post-irradiation. Decreases in values after 12.5 Gy are calculated with * P < 0.05.
Figure 4(a) demonstrates that a single dose of 12.5 Gy reduced the total vessel volume by 26% and 55% after 60 and 90 days, respectively. Figure 4(b) illustrates that vessel density decreased by 11% and 57% after 60- and 90-days post-radiation, respectively. Figure 4(c) displays that the maximum branch depth for 12.5 Gy decreased by 10% compared to non-irradiated kidneys (0 Gy) at day-60; however, this decrease was 28% on day 90 post-radiation. The number of terminal points is a good estimate of the number of vessels that were detected in a sample. The bar graphs in Fig. 4(d) show the number of vessels in all samples. While 12.5 Gy diminished the number of terminal points on day 60 only by 8%, this value decreased to 53% by day-90. None of the changes at day-60 post-irradiation reached statistical significance, whereas all values decreased at day-90 compared to the non-irradiated controls (0 Gy).
Table 3 shows the number of vessels in different ranges mostly spanning 100 µm. The data show that the number of vessels in all ranges on day-90 post-radiation decreased. With a closer look at day-60 data in Table 3, the number of vessels in smaller diameter ranges (70-150 µm, 251-450 µm) increased and the number of vessels in larger diameter ranges (451-850 µm) decreased in the irradiated group. This increase could be due to the decrease in lumens of the large vessels, moving them into the smaller diameter ranges.
The number of vessels in irradiated kidneys on day-90 was significantly lower in ranges of 151-250 µm, 251-350 µm, and 351-450 µm [37]. We also observed the number of vessels in irradiated kidneys of day-60 was significantly lower in vessels with a diameter of 551-650 µm.
4. Discussion
By implementing the VMI method [33], we measured changes in functional vessels of rat kidneys at two time points. For VMI images of the NADH channel are obtained with an excitation wavelength of 350 ±80 nm. Hemoglobin and oxy-hemoglobin light absorbance coefficients are maximum around that wavelength, so that vessels that carry hemoglobin (Hb) or oxygenated hemoglobin (HbO2) more readily absorb light in the range of 350 ± 80 nm [38]. As a result, NADH signals appear dark in vessels that carry blood, while the rest of the tissue is bright and functional vessels become distinct from the kidney parenchyma. The comparison of the color of larger vessels in Fig. 2(a) with Fig. 2(b) and Fig. 2(c) with Fig. 2(d) suggested that in macro-vessels (>100 µm), the lumen was reduced, decreasing total vessel volume and subsequently vessel density. The comparison of irradiated kidneys with non-irradiated kidneys in Fig. 1 and Fig. 3 suggests that the number of micro-vessels decreased by radiation since the irradiated kidney at 90 days has notably fewer terminal points (Fig. 1). Figure 3 shows that the maximum branch depth (branch order) detected in irradiated kidneys is also lower by 90 days post-radiation. These results can occur in the following ways: I) loss of microvessels post-radiation, a result confirmed by the lower number of termini; II) a decrease in vessel lumen diameter, so that the smallest branches fall below the 70 µm diameter cut-off and are eliminated from the count, reducing branch depth.
To illustrate the effect of radiation on the vessel diameter, we counted the number of vessels spanning different diameters because though the mean vessel diameter after radiation decreased, the number of vessels detected decreased as well. Consequently, the average vessel diameter in each sample did not change. Data presented in Table 3 show that in the irradiated group at 60 days, the number of vessels in the smaller diameter ranges (70-150 µm, 251-450 µm) tended to increase while the number of vessels of larger diameter (451-850 µm) appeared to decrease. However, at 60 days the total number of vessels did not change (328.7 for 0 Gy vs 343.7 for 12.5 Gy). As mentioned in the results, this suggests that the larger vessels that were irradiated decreased in diameter to move into lower ranges, implying that there was a constriction in the lumen of the larger vessels. However, this decrease did not appear to be as pronounced in the vessels with smaller diameters since their numbers seemed to increase, probably by the addition of vessels from the larger diameter groups. In contrast, at 90 days the number of vessels in the larger diameter range (451-850 µm) decreased and so did the numbers in the smaller diameter range (70-450 µm). But the total number of vessels counted also decreased (412.1 for 0 Gy vs 158.2 for 12.5 Gy). Taken together, these results support a new finding that by 90 days, there was a considerable constriction in the lumen of the irradiated vessels, accounting for the decrease in perfused volume (see Table 1 and Fig. 4(a)) as well as the marked decrease in vessel density observed in Fig. 4(b).
Though not statistically significant, the non-irradiated renal vessels increased in lumen diameter from 60 days to 90 days, especially in the 750-850 µm range (p = 0.018; n = 6/group). This could be by the growth of the kidneys (the bodyweight of non-irradiated rats increased by 5% during this time) and/or by vasodilation that may occur as the demand for blood increases when young adult rats mature. There is a significant decrease in body weight of ∼ 25% at 60 days in irradiated rats as compared to their age-matched non-irradiated siblings (results not shown). However, the reduction in vessel volume was not statistically significant between the same two groups at 60 days (Table 2). The vessel volume was considerably lower by 90 days. This again suggests that vascular remodeling, as well as vasoconstriction, may account for the decline in vessel volume measured in irradiated rats at 90 days. Since radiation is known to cause considerable vascular endothelial damage [39,40], the results point to endothelial injury after radiation, that is known to cause vasoconstriction, and lead to vascular rarefaction and decreased volume and branch depth.
Vascular regression in the rat lung has been monitored at 6 time points up to one year after 10 Gy radiation [41,42]. There was a loss of vasoreactivity by 56 days in the same strain of irradiated rats as the current study, while vascular density was lower by 28 days and further decreased by 56 days. In contrast, vessel regression was not statistically lower at 60 days post-radiation in the kidneys. The novel results indicate for the first time that the timing of vascular regression after radiation is organ specific. Interestingly the correlation between fall in vessel numbers corresponds to the time of lethal organ damage recorded following radiation. Lethal radiation nephropathy is reported only after 90 days whereas lethal radiation pneumonitis occurs between 42-80 days after exposure. Not only is the timing different, but the dose is also different. Lethal radiation pneumonitis does not occur at or below 10 Gy [23,41–44] whereas radiation nephropathy is lethal after doses as low as 10 Gy in the same strain of rats [23,27].
Partial body irradiation (PBI) shielding only part of one hind leg of a rat is the most advanced model designed to study organ damage in the context of crosstalk between multiple organs exposed simultaneously to a single dose of ionizing radiation. This would optimally mimic injury from a nuclear accident or radiological attack, which are rising threats to public health [45]. As reported [24], 12.5 Gy leg-out PBI is a well-selected dose of exposure, because it shows multiple phases of injuries without requiring supportive care consisting of repeated saline injections. Also, sufficient number of rats will survive to enable evaluation of the delayed effect of radiation exposure (up to 160 days) [23].To study lethal injuries to organs such as the lungs and kidneys that are observed within 100 days at doses above 12.5 Gy [24], enough bone marrow must be spared to survive hematopoietic death induced by lower doses such as 8 Gy [9,23,46]. Hematopoietic death occurs by 30 days in rats [23], whereas radiation pneumonitis and nephropathy are delayed effects of acute radiation exposure (DEARE) that occur after 30 days. Non-lethal renal dysfunction, as measured by an increase in the blood urea nitrogen (BUN) has been reported after 90 days following 12.5 Gy, which is why the 90-day time point was selected for the current study. Lethal radiation nephropathy occurred only after 110 days in the same model [24]. An earlier time point at 60 days was also chosen to determine if severe vascular injury preceded renal dysfunction at 90 days.
Abnormalities in the circulation are believed to play an important role in the initiation of several diseases [47,48]. Decreases in renal microvessel numbers have been described in pathological states such as diabetes, hypertension, and atherosclerosis [14]. In these examples, the effects are reported to be most likely initiated by sustained vasoconstriction in the small vessels (functional rarefaction) that promote vascular regression and vessel dropout leading to subsequent tissue injury [14]. Smaller arteries are described as the first to be affected by acute or chronic insults that would eventually impact the loss of renal hemodynamics and organ function. Such vascular regression are ascribed to dysfunction of the vascular endothelium [49,50], a target and a source of key vasoactive agents and growth factors e.g., nitric oxide, angiotensin II, endothelin-1, prostacyclin, vascular endothelial growth factor (VEGF) etc. [14]. Endothelial injury would tilt the balance to favor the loss of vascular tone as well as vascular permeability, enhancing inflammation and thrombosis via the regulation of adhesion molecules and chemokines [39]. Interventions with statins [51,52] angiotensin converting enzyme inhibitors or receptor blockers [53,54], or endothelin receptor blockers [55,56] have been shown to improve renal microvascular function [14]. Microvascular rarefaction was often accompanied by glomerulosclerosis and tubulointerstitial fibrosis in these conditions [57–61]. Also, in general, insults that induced apoptosis impaired regenerative capacity of the kidney [56,62] and exacerbated the probability of a reversal of microvessel regression, by interrupting repair that occurs through resident and circulating cell progenitors which facilitate angiogenesis [63,64]. Such pathological effects also occur after radiation.
Endothelial cell death is a primary lesion initiated by intestinal radiation damage in mice [2]. Apoptosis in endothelial cells occurs within hours of exposure in the intestine, lung [65] and nervous system [66], though results from the kidney remain unknown [67]. Early loss of endothelial cells in multiple organs coupled with the depletion of resident and bone marrow progenitors by radiation, could well account for the vascular injury in the gut, lung, central nervous system and possibly, as reported here, in the kidney. Radiation is known to induce glomerulosclerosis and tubulointerstitial fibrosis [17,27,68], which are well known to be mitigated by angiotensin converting enzymes [17,27]. In fact, the pathogenesis of radiation nephropathy are common to those of other renal debilitating vascular injuries that are unrelated to radiation [56–60]. Taken together it is very possible that vascular injury to the kidney that is described in this study is a major cause of lethal radiation nephropathy.
VMI is a novel, in-expensive, label-free, and rapid method that enables us to extract the 3D vessel structure of whole organ with good resolution, however, there are some limitations in the current study. Microvessels below 70 µm in diameter were not amenable to VMI. Increasing sample size may achieve statistical significance for the vascular parameters that were measured here at 60-day after radiation, since a decline was observed for all of these. And finally, only the lumen of the vessels was investigated without information regarding vascular wall thickness, to determine if remodeling of vessel walls also contributed to the decrease in vessel volume. Future studies must address these issues, which are beyond the scope of the current investigation.
5. Conclusion
In summary, this study is innovative and unique in several ways. A whole animal injury model was used in conjunction with a novel technique, VMI, to measure multiple levels of branching of the vascular tree in the kidney. The results indicated that besides vascular regression, which is still largely believed to be initiated by damage to the microvessels, simultaneous changes in lumen size in large vessels may play an important role in the process. The study supports reports that constriction of vessel diameter is an important step in vascular regression. And lastly, vascular remodeling is organ-specific, so that the same insult can cause injury at different times and with different severity in organs such as the kidney versus the lung. This supports a hypothesis that endothelial cells have distinct properties in each organ and vascular structure may largely be regulated by the surrounding parenchyma since these are different between organs such as the kidney and the lung.
Funding
National Institutes of Health (AI101898, AI107305, AI133594, EY031533).
Acknowledgements
We would like to thank Tracy Gasperetti and Dana Scholler for the excellent animal care and help with irradiations. We appreciate Buse Nur Ceyhan and Shalaka Konjalwar for editorial contributions.
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
Supplemental document
See Supplement 1 for supporting content.
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