Radiation nephropathy in a nonhuman primate model of partial-body irradiation with minimal bone marrow sparing. Part 2: Histopathology, mediators, and mechanisms

Abstract

Male rhesus macaques were subjected to partial-body irradiation at 10, 11, or 12 Gy with 5% bone marrow protection. Animals were euthanized when dictated by prospectively determined clinical parameters, or approximately 180 days following irradiation. Histological sections of kidney were stained with hematoxylin and eosin as well as a battery of histochemical and immunohistochemical stains. Histopathological alterations were centered on glomerular changes and fibrosis of glomeruli and the interstitial compartment. These changes were first noted in animals necropsied approximately 100 days post-irradiation and continued in animals necropsied through the observation period. Glomerular changes included congestion, thrombosis, erythrocyte degeneration, capillary tuft dilation, fibrin deposition, altered quantity and dispersion pattern of von Willebrand factor, increased mesangial matrix, and mesangial deposits of material that stained positively with periodic acid-Schiff staining. Areas of interstitial and glomerular fibrosis, as demonstrated by Masson’s trichrome staining, were topographically associated with increased immunohistochemical staining for connective tissue growth factor, alpha smooth muscle actin and collagen 1, but there was little staining for transforming growth factor beta. Fibrotic glomeruli had reduced microvascularity as demonstrated by reduced CD31 immunohistochemical staining. Vascular congestion was commonly noted in the region of the corticomedullary junction, and proteinaceous casts were commonly noted in cortical and medullary tubules. Longitudinal analysis of histopathological alterations provided evidence defining the latency, severity and progression of delayed radiation-induced kidney injury.

INTRODUCTION

Delayed effects of acute radiation exposure (DEARE) are a known complication of radiation therapy, and are anticipated to be a contributor to overall morbidity and mortality associated with accidental or deliberate exposure to ionizing radiation. The goal of the current study was to determine the clinical and histopathological progress of radiation-associated alterations in the kidney of male rhesus macaques for approximately 185 days following radiation exposure. Herein, the time course of histopathological alterations in the kidney consequent to 10 or 11 Gy of partial-body irradiation with approximately 5% bone marrow sparing (PBI/BM5) provided a link and supportive database for the clinical evidence defining the latency, severity and progression of chronic kidney injury (CKI) in the nonhuman primate (NHP) (Cohen et al. 2017). In-life observations and clinical pathology alterations related to acute and delayed kidney injury, including response to Neupogen® therapy, are addressed in a companion article in this issue (Cohen et al. 2018).

METHODS

Details of study design, radiation exposure, post-irradiation clinical care, and clinical parameters that dictated euthanasia are described in the companion publication in this issue (Cohen et al. 2018). Briefly, male rhesus macaques, ranging in age from 3.5 to 9.8 years, were exposed to 6 MV Linear Accelerator (LINAC) photon radiation at a dose rate of 0.80 Gy min⁻¹ for a total dose of 10, 11, or 12 Gy, with 5% bone marrow sparing. Animals received supportive clinical care, including dexamethasone and antibiotic administration, in compliance with prospectively delineated clinical parameters. All studies were conducted under an IACUC-approved protocol. Animals were euthanized when necessitated via IACUC-approved clinical condition and humane considerations.

Gentamicin sulfate was administered at a dosage level of 3 ± 1.5 mg/kg via intramuscular injection when the absolute neutrophil count was ≤ 500/μL and body temperature was ≥ 103.0⁰F (39.4⁰C). Gentamicin therapy most commonly was instituted for two days, but in three animals extended for three, four or six days.

Specimens of kidney were fixed in neutral-buffered formalin at the time of necropsy. Tissues were processed to paraffin blocks via routine histology procedures, and hematoxylin and eosin (H&E)-stained sections of kidney were examined by routine light microscopy. Histological sections subjected to additional histochemical and immunohistochemical staining procedures were prepared and examined (Table 1). Treatment groups were known to the pathologist at the time of histopathological examination.

Table 1.

Histological and immunohistochemical stains on kidney specimens

Histochemical stains
Hematoxylin & eosin	X
Masson’s trichrome	X
Toluidine blue	X
Perls’ iron stain	X
Periodic acid-Schiff/hematoxylin	X
Phosphotungstic acid/hematoxylin	X
Immunohistochemical stains
Alpha smooth muscle actin	X
Tryptase	X
Collagen 1	X
Transforming growth factor-beta	X
Connective tissue growth factor	X
CD31	X
IL22	X
IL22R	X
Von Willebrand factor	X
Pro-renin receptor	X

The staining method for toluidine blue histochemical stain was from Carson (Carson 1997). The staining method for the Masson’s trichrome histochemical stain was from Bancroft (Bancroft and Stevens 1996). The staining methods for the Perls’ iron stain and periodic acid-Schiff/hematoxylin stains were from Sheehan (Sheehan and Hrapchak 1980).

Immunohistochemical staining was performed on 5μm thick formalin-fixed paraffin embedded sections using the Discovery XT and Discovery Ultra research instruments (Ventana Medical Systems). Sections were deparaffinized with Discovery EZ prep (Ventana Medical Systems), and then either heat retrieved with proprietary solutions Discovery RiboCC and Discovery CC1, or pretreated with Protease 2 (Ventana Medical Systems) at 37˚C. Whenever blocking was needed, blocking Sniper (Biocare Medical) was applied for four to eight minutes as pre-determined empirically during assay optimization. Sections were incubated with primary antibodies diluted in Antibody Diluent (Life Technologies) at varying concentrations, temperature and time based on protocols optimized with positive control tissues. Primary antibodies included anti-alpha smooth muscle actin (αSMA) (abcam, AB119952), anti-tryptase (abcam, AB81703, anti-IL-22 (abcam, AB 18499), anti-IL22RA1 (Sigma Aldrich, HPA042399), anti-von Willebrand factor (vWF) (abcam, AB6994), anti-CD31 (abcam, AB9498), anti-collagen I (abcam, AB34710), anti-pro-renin R/ATP6AP2 (Sigma, HPA003156), anti-transforming growth factor-beta (TGFβ) (abcam, AB92486), and anti-connective tissue growth factor (CTGF) (abcam, AB6992). Sections were labelled with HRP- conjugated secondary antibodies OmniMap anti-rabbit HRP or OmniMap anti-mouse HRP (Ventana Medical Systems), and then detected with the ChromoMap DAB kit (Ventana Medical Systems) prior to counterstaining with hematoxylin and bluing reagent (Ventana Medical Systems).

The stained histological sections were examined by light microscopy and observations were entered in Provantis histopathology data system. Histopathology data entries for selected histological changes were transferred into Microsoft Excel® worksheets for further analysis and generation of illustrative graphs.

Histopathological alterations were recorded using currently accepted diagnostic terminology to the extent possible. In order to convey the extent of histological alterations, each observation was given a subjectively determined severity grade of 1 to 5, with Grade 1 being the least pronounced alteration. A severity grade of zero indicates the histological alteration was not discernible in the sections. Tissue alterations in the irradiated animals were compared to equivalent sections taken from naïve control animals. The severity grades should be interpreted as subjective division of continuous data into ordinal classes, for the purpose of comparing the apparent degree of changes, rather than an attempt at morphometric analysis of the histological changes.

Severity scoring for most observations was empirical, but histological grading of alpha smooth muscle actin (αSMA) staining in the kidney was performed in compliance with a published scoring procedure (Cohen et al. 1996). In this scoring regimen, a grade of 1 indicated normal vascular staining by αSMA, with grades of 2 or more indicating pathological αSMA staining.

RESULTS

There was a clear radiation dose-associated reduction in survival time, which was most pronounced in animals irradiated at the 12 Gy level (Table 2). As previously shown (MacVittie et al. 2012), there was a clear radiation dose-dependent reduction in overall survival time for the 10, 11 or 12 Gy exposures throughout the 180-day study duration, particularly in animals irradiated at the 12 Gy level. Debilitation in animals euthanized within the first 30 days post-irradiation was largely related to gastrointestinal injury, as detected by hydration status and overall clinical status (MacVittie et al. 2012, MacVittie et al. 2012). Debilitation in animals in the later stages of the study were largely related to pulmonary injury, as detected by increased respiratory rate and decreased pO₂ levels.

Table 2.

Necropsy Date Related to Radiation Dose

Radiation dose (Gy)	10	11	12
Number of animals	30	42	17
Necropsy datesa
1–10	1	2	8
11–20	3	4	5
21–30	1	4	2
31–40	1	3
41–50	1	2
51–60		1
61–70
71–80		1
81–90		3	1
91–100	1	4
101–110	1	3	1
111–120	2
121–130		1
131–140	1	4
141–150		1
151–160
161–170	1	3
171–180	4
181–191	13	6

^a=animals died or were euthanized during the 10-day period ending on this day post-irradiation

The development of histological changes was based on necropsy dates, which were determined by clinical presentation of the animals rather than a predetermined termination schedule. For that reason, the results presented herein should not be interpreted as a time course study of lesion development, but rather an approximation of the lesion development schedule as afforded by specimen availability. The incidences of histological alterations presented below are based on availability of suitably preserved and stained sections rather than the number of animals in each radiation dose cohort, thus there are variations based on technical issues with tissue fixation and staining procedures. Sections of kidney from a naïve control male rhesus macaque are presented for comparison (Fig. 1a, b, c)

Figure 1a.

The renal glomerulus of a naïve control male rhesus macaque shows the vascular pole (v) and multiple glomerular tufts composed of thin-walled capillaries supported by scant mesangial matrix. A small amount of fibrous connective tissue (arrow) is present on the external aspect of the glomerular (Bowman’s) capsule. Darkly stained nuclei in the spaces surrounding tubules (t) denote the presence of endothelial cells in the intertubular interstitium, though the endothelial cell cytoplasm is too thin to be visualized in routine H&E-stained sections. A minimal amount of pink-stained fibrous connective tissue is visible in the intertubular interstitium. H&E stain, 40x objective magnification.

Figure 1b.

A Masson’s trichrome stain performed on a kidney section from a naïve control male rhesus macaque accentuates the fibrous connective tissue on the external aspect of Bowman’s capsule and reveals a minimal amount of blue-stained fibrous connective tissue in the intertubular interstitium. Low-level staining is also noted in the mesangial matrix of glomerular tufts. Masson’s trichrome stain, 40x objective magnification.

Figure 1c.

The magenta color produced by a PASH stain performed on a kidney section from a naïve control male rhesus macaque reveals the carbohydrate content of basement membranes in glomerular capillaries and tubules. The mesangial matrix is faintly stained. Periodic acid-Schiff/hematoxylin counterstain, 40x objective magnification.

The H&E-stained kidney sections had a spectrum of histologic changes, the preponderance of which first appeared, or increased in incidence/severity, at approximately 85–100 days post-irradiation. As presented below, various special staining procedures revealed histological alterations in animals euthanized at earlier time points. For example, Masson’s trichrome staining revealed grade 1 interstitial fibrosis in two irradiated animals that were euthanized on days 42 and 53.

Glomerular histopathology

Glomerular congestion (Fig 1d, e) of grade 1–4 was noted at day 93 and thereafter throughout the 180d time course. The glomerular congestion consisted of glomerular capillaries that were engorged with erythrocytes, while capillaries in the surrounding renal parenchyma were microscopically normal. Limitation of the capillary congestion to glomerular capillaries suggested local vascular alteration in the glomeruli, as opposed to generalized congestion of the kidney or corticomedullary congestion, as described below. It should be noted that Grade 2 glomerular congestion was observed in one animal at day 8 following 12 Gy irradiation and another animal at day 20 following 10 Gy irradiation, but the wide chronological separation of these observations from the bulk of the glomerular changes in the study suggested the early glomerular changes in two animals were outliers.

Glomerular thrombosis (Fig. 2a, b) ranging from grade 1 to grade 3 was commonly noted after day 85. Glomerular thrombosis consisted of accumulations of eosinophilic material consistent with partially degenerated erythrocytes, thus was considered to be a result of persistent glomerular congestion and coagulation alterations that were most likely secondary to endothelial injury and/or platelet activation. Glomerular thrombosis in a single animal at day 9 following 11 Gy irradiation had no additional glomerular changes, or tubular changes secondary to glomerular injury, thus the glomerular thrombosis was suspected to be an outlier observation.

Figure 2a.

Glomerular thrombosis was noted in the same general time period as glomerular congestion, and was considered to be a facet of the radiation-associated glomerular injury.

Figure 2b.

Glomerular thrombosis was present in the renal cortex of a male rhesus macaque collected at day 105 following irradiation at 11 Gy. Note accumulations of brightly eosinophilic erythrocyte debris. One glomerulus (*) is atrophic. H&E stain, 20x objective magnification.

Glomeruli of a few animals contained brightly eosinophilic globules of various sizes which were suspected to contain fragments of degenerated erythrocytes as well as other proteinic materials.These changes were randomly scattered among the irradiated animals, with no apparent relationship to radiation dose level or post-irradiation interval.

Marked dilation of one or more glomerular capillary loops was recorded as capillary tuft dilation. This histologic alteration (Fig. 2c, d) was not present until post-irradiation day 85, after which grade 1 or grade 2 capillary tuft dilation was noted in 26 of 44 animals. Capillary tuft dilation commonly occurred in conjunction with glomerular congestion and/or thrombosis, suggesting these morphologic alterations had shared pathogenesis. However, there were examples of capillary tuft dilation in glomeruli that had no evidence of glomerular congestion or thrombosis, possibly indicating capillary tuft dilation represented an unresolved or unresolvable component in the spectrum of glomerular capillary injury.

Figure 2c.

Glomerular capillary tuft dilation was noted in the same time frame as glomerular capillary congestion and thrombosis, and was considered to be a related manifestation of radiation-associated glomerular injury.

Figure 2d.

Glomerular capillary tuft dilatation (arrow) was present in the renal cortex of a male rhesus macaque collected at day 177 following irradiation at 11 Gy. Note the interstitial fibrosis (*) and atrophic glomerulus (**). H&E stain, 20x objective magnification.

Glomeruli of irradiated animals commonly had an increase in amorphous mesangial material that was recorded as increased mesangial matrix (Fig. 3a, b). Grade 1 increased mesangial matrix was first noted at day 77 in an animal irradiated at 11 Gy, and thereafter was noted in glomeruli of 33 of 48 animals necropsied through the remainder of the 180-day observation period. Increased mesangial matrix as noted in H&E-stained sections was commonly, but not invariably, associated with glomerular IHC staining for αSMA. The increased mesangial matrix often was coincident with increased PASH staining, and/or increased trichrome-positive staining in glomeruli.

Figure 3a.

An increase in the mesangial matrix of glomeruli was noted in the same time frame as other indicators of glomerular injury.

Figure 3b.

Increased glomerular mesangial matrix was present in the renal cortex of a male rhesus macaque collected at day 141 following irradiation at 11 Gy. Note the homogeneous, lightly eosinophilic material in the glomerulus. Interstitial fibrosis is apparent at the right side and above the glomerulus. H&E stain, 20x objective magnification.

Periodic acid/Schiff-hematoxylin (PASH) staining revealed Grade 1 deposits of PAS-positive material (Fig. 3c, d) in the glomerular mesangium of one animal at day 85 following irradiation at 11 Gy. Thereafter, PAS-positive mesangial deposits were noted in 23 of 44 animals necropsied through the remainder of the 180-day observation period. These observations in the PASH-stained sections were related to the increased mesangial matrix in the H&E-stained sections, glomerular fibrosis in the trichrome-stained sections, and positive αSMA immunohistochemical staining.

Figure 3c.

Mesangial deposits of PAS⁺ material were noted in a subset of animals starting at 85 days following irradiation. The increased PAS⁺ material was closely related to the increased mesangial matrix that was observed in H&E-stained sections, and was considered to be part of the spectrum of histologic changes associated with radiation-associated glomerular injury.

Figure 3d.

PAS⁺ mesangial deposits were present in renal glomeruli of a male rhesus macaque collected at day 141 following irradiation at 11 Gy. Note the marked increase in PAS⁺ material in the enlarged glomerulus on the right side of the image. PASH stain, 20x objective magnification.

The phosphotungstic acid-hematoxylin (PTAH) histochemical stain was initially performed in an effort to accentuate fibrin deposits in glomeruli, though it is known that PTAH staining reveals the presence of multiple proteins. The initial histopathological evaluation revealed glomeruli containing PTAH-positive droplets that did not appear to be fibrin. Positive PTAH staining was also noted in renal tubular casts. Each of these manifestations of PTAH staining was recorded separately.

Grade 1 glomerular fibrin deposition, as revealed by PTAH staining (Fig. 3e, f)) was noted in an animal that was necropsied on day 85 post-irradiation. Thereafter, grade 1 or grade 2 glomerular fibrin deposition was observed in 37 of 43 animals necropsied through the remainder of the observation period. Glomerular fibrin deposition in the 12 Gy exposure group was present only in the animal that survived to day 102 post-irradiation. The time course indicated glomerular fibrin deposition was not an acute effect of irradiation, and was more related to post-irradiation interval than the radiation dose levels used in the present study. The fibrin deposition, though commonly observed, involved only scattered glomeruli and was not of a degree that would be considered a major component of the overall pathological process in the kidney.

Figure 3e.

PTAH staining revealed fibrin deposition in thrombotic glomeruli of irradiated animals, commencing at the time many other glomerular changes were noted and continuing throughout the period of observation.

Figure 3f.

PTAH staining on the kidney of a male rhesus macaque collected at day 107 following 11 Gy irradiation reveals flocculent and fibrillar PTAH⁺ material in the glomerulus. The positively stained material was consistent with a mixture of fibrin and degenerated erythrocytes, which presented as glomerular capillary congestion and/or thrombosis in the H&E-stained sections. PTAH stain, 30x objective magnification.

The PTAH stain revealed positively stained droplets of various sizes in the glomerular mesangium of irradiated animals (Fig 4a, b). The droplets did not have the physical outline of fibrin deposits, and were not as intensely PTAH⁺ as fibrin deposits. The intensity of PTAH staining and general configuration of the droplets suggest they originated from degenerated erythrocytes. These droplets were also visible in the H&E-stained sections as brightly eosinophilic droplets, also consistent with erythrocyte fragments.

Figure 4a.

PTAH staining revealed variably sized PTAH⁺ droplets within the glomerular mesangium. The PTAH+ mesangial droplets were first noted at approximately 90 days post-irradiation, and were commonly noted in animals necropsied through the remainder of the observation period.

Figure 4b.

PTAH staining on the kidney of a male rhesus macaque collected at day 180 following irradiation at 10 Gy revealed homogeneous and punctate accumulations of PTAH⁺ material within capillaries. The smaller, punctate structures were consistent with degenerated, fragmented erythrocytes in congested or thrombotic capillaries. The larger, homogeneous accumulations of PTAH⁺ material (e.g., upper aspect of glomerulus on right) corresponded to the capillary tuft dilation that was noted in the H&E-stained sections. PTAH stain, 40x objective magnification.

Interstitial and glomerular fibrosis

Grade 1 interstitial fibrosis, as revealed by Masson’s trichrome staining, was first noted in the kidney of one animal necropsied on day 42 following 11 Gy irradiation and another animal irradiated at 11 Gy and necropsied on Day 53. From post-irradiation day 93 onward, interstitial fibrosis was present in the kidney of 38 of 43 animals necropsied through the remainder of the 180-day observation period (Fig. 4c, d). It is noteworthy that one animal irradiated at 12 Gy survived to Day 81, yet had no histological evidence of renal fibrosis, while the other long-term survivor irradiated at 12 Gy had grade 2 interstitial renal fibrosis when necropsied on day 102. These observations suggested that the onset of interstitial renal fibrosis was related primarily to post-irradiation interval, i.e., a latent period, rather than the radiation levels employed in this study. The severity of the renal interstitial fibrosis in animals after day 93 ranged from grade 1 to grade 3, with no progression to grades 4 or 5, but there was a time-related increase in severity within the range of grades 1 through 3. Grade 3 interstitial fibrosis started to appear at day 141, in an animal irradiated at 11 Gy. Thereafter, 16 of 28 animals necropsied had grade 3 interstitial fibrosis and only one animal, which was irradiated at 10 Gy, did not manifest some degree of renal interstitial fibrosis.

Figure 4c.

Interstitial fibrosis, as identified in sections stained with Masson’s trichrome stain, was frequently noted in the kidneys of irradiated animals starting approximately 90 days following irradiation. There was little difference between animals irradiated at 10 versus 11 Gy. Few animals irradiated at 12 Gy survived a sufficient post-irradiation interval for renal interstitial fibrosis to develop. The interstitial fibrosis became progressively more severe with increased post-irradiation time.

Figure 4d.

Interstitial fibrosis was present in the renal cortex of a male rhesus macaque collected at day 183 following irradiation at 10 Gy. Note the blue-stained collagenous tissue that tends to form linear bands in the renal cortex. Masson’s trichrome stain, 5x objective magnification.

Areas of fibrosis in kidneys occasionally had tubules lined by basophilic epithelial cells, which is suggestive but not confirmatory of tubular epithelial regeneration. Tubular basophilia was not a prominent histologic feature of the overall renal lesion complex.

Renal glomerular fibrosis was recorded separately from interstitial fibrosis. Glomerular fibrosis typically occurred in conjunction with interstitial fibrosis on an animal-by-animal basis, though the interstitial and glomerular fibrosis were not necessarily co-located within specific areas of the kidney of an individual animal. In general, glomerular fibrosis was not as pronounced as interstitial fibrosis. Grade 2 glomerular fibrosis (as revealed by Masson’s trichrome staining) was first noted in the kidney of an animal irradiated at 10 Gy and necropsied on day 105. Thereafter, glomerular fibrosis was commonly, but not invariably, present in animals that had renal interstitial fibrosis (Fig. 4e, f). A few animals had interstitial fibrosis with no discernible glomerular fibrosis, but the converse situation did not occur. The glomerular fibrosis was assigned grades 1 or 2 in all affected animals except for grade 3 glomerular fibrosis in one animal irradiated at 11 Gy and necropsied at the end of the observation period.

Figure 4e.

Glomerular fibrosis, as revealed by Masson’s trichrome staining, was noted in the same time frame and typically was contemporaneous with interstitial fibrosis.

Figure 4f.

Both interstitial and glomerular fibrosis were present in the renal cortex of a male rhesus macaque collected at day 141 following irradiation at 11 Gy. Note the increase in blue-stained collagenous tissue in the interstitium surrounding cortical tubules and the slight increase in blue-stained collagenous tissue in the glomerulus, as compared to the normal kidney shown in Figure 1b. The arrow indicates a constriction in the neck of a proximal cortical tubule. Masson’s trichrome stain, 30x objective magnification.

Alpha smooth muscle actin (αSMA) immunohistochemical staining

Immunohistochemical staining for αSMA in the kidney was classified by published criteria (Cohen et al. 1996), in which grade 1 indicated the normal background staining of blood vessels, and a grade of 2 or greater indicated an abnormal staining pattern. Grade 2 αSMA interstitial staining was noted in one animal irradiated at 11 Gy and necropsied on post-irradiation day 21, but there was no additional grade 2 αSMA interstitial staining until day 93. After day 93, αSMA interstitial staining was frequently noted, typically in animals that also had interstitial fibrosis as revealed by Masson’s trichrome staining (Fig. 5a, c).

Figure 5a.

Immunohistochemical staining for α-smooth muscle actin (αSMA) in the kidney was subjectively graded by a published scoring system (see text), whereby a grade of 1 represented the normal staining of renal interstitial vasculature. An increased level of αSMA staining was noted in the renal interstitium starting at day 93 following irradiation, and continuing through the observation period. The increased interstitial αSMA staining tended to be topographically associated with areas of interstitial fibrosis.

Figure 5c.

Glomerular and interstitial staining for αSMA was present in the renal cortex of a male rhesus macaque collected at day 77 following irradiation at 11 Gy. Note the αSMA⁺ staining in glomeruli (arrow) and interstitium (arrowhead). The increased αSMA staining correlated with fibrosis seen with Masson’s trichrome staining and increased collagen 1 IHC staining. Alpha smooth muscle actin immunohistochemical stain, 20x objective magnification. </igure_Caption>

Grade 2 αSMA glomerular staining was first noted at day 85 in an animal irradiated at 11 Gy, and was present in 39 of the 44 animals necropsied after day 85 (Fig 5b, c). It was interesting to note the affected animal at day 85 had no discernible interstitial or glomerular fibrosis on Masson’s trichrome staining, and no evidence of increased interstitial αSMA staining, but had grade 3 increased mesangial matrix in the glomeruli (see below). The observations in this animal suggest an association between increased mesangial matrix and glomerular αSMA staining that may be an early manifestation of glomerular injury. In contrast to interstitial αSMA staining, which was commonly associated with trichrome-positive interstitial fibrosis, glomerular αSMA staining was frequently observed in animals that had no evidence of trichrome-positive glomerular fibrosis. As presented below, the occurrence of glomerular αSMA staining appeared to be more closely associated with increased mesangial matrix than with glomerular fibrosis as demonstrated by Masson’s trichrome staining.

Figure 5b.

Immunohistochemical staining revealed an increased level of αSMA staining in renal glomeruli starting at day 93 following irradiation, and continuing through the observation period.

Collagen 1 immunohistochemical staining

A low level of positive staining for collagen was noted in the interstitium throughout the renal cortex and outer medulla. The level of collagen 1 staining in naive control animals was established as the baseline, and the degree of collagen 1 staining in irradiated animals was subjectively compared to that baseline, and subcategorized as glomerular or interstitial. Increased interstitial collagen 1 staining was commonly noted in animals irradiated at 10 or 11 Gy, but was present in only the longest surviving (day 102) animal exposed to 12 Gy irradiation. As with other indicators of renal injury, increased interstitial and glomerular collagen 1 deposition was noted more commonly from day 85 through the remainder of the observation period (Fig. 5d-f). Increased collagen 1 staining paralleled observations of interstitial fibrosis made on the Masson’s trichrome-stained sections. The collagen 1 IHC stains tended to reveal more collagen deposition than was discernible in the trichrome-stained sections. In some instances the interstitial collagen deposition was visible in the collagen 1-stained sections but not visible in the trichrome-stained sections. Increased collagen 1 staining in glomeruli was of lower intensity than the collagen 1 staining the interstitium.

Figure 5d.

Immunohistochemical staining for collagen 1 in the kidney revealed a generally mild (grade 2) increase in the amount of interstitial collagen starting at approximately day 85 post-irradiation, with a smaller number of affected animals in the day 20 to day 50 interval. The amount of interstitial collagen increased substantially in a number of animals necropsied at the end of the observation period.

Figure 5f.

An immunohistochemical stain for collagen 1 on the kidney of a male rhesus macaque collected at day 105 following irradiation at 11 Gy shows brown collagen staining in the interstitium surrounding tubules as well as within one glomerulus (*). Collagen 1 IHC stain, 20x objective magnification.

CD31 and von Willebrand factor (vWF) immunohistochemical staining

Positive staining for CD31 was present in vascular endothelial cells throughout the kidney, and was prominent in glomerular capillaries. The level of CD31 staining in naive control animals was established as the baseline, and the degree of CD31 staining in glomeruli of irradiated animals was subjectively compared to that baseline. Decreased glomerular CD31 staining (Fig. 6a, b) occurred in glomeruli that had other histologic indications of radiation-associated injury, particularly increased mesangial matrix and increased αSMA staining.

Figure 6a.

Immunohistochemical staining for the CD31 endothelial cell marker revealed reduced microvascular density in fibrotic glomeruli.

Figure 6b.

The renal cortex of a male rhesus macaque collected at day 180 following irradiation at 10 Gy has a normal pattern of CD31 staining in capillaries of the upper glomerulus, but reduced CD31 staining is present in the damaged lower glomerulus. This indicates a reduction in the population of normal capillary endothelial cells is a facet of the radiation-associated glomerular injury. CD31 IHC stain, 20x objective magnification.

Positive staining for von Willebrand factor (vWF) was noted in blood vessels throughout the kidney (Fig. 6c-e). The level of VWF staining in naive control animals was established as the baseline, and the degree of vWF staining in irradiated animals was subjectively compared to that baseline. The expected low level of vWF staining was present in the naive control animals, where the vWF staining was assigned a severity grade of 0. An increased level of vWF IHC staining was present in glomerular capillaries of irradiated animals, with temporal distribution similar to that of other radiation-associated glomerular changes. The vWF staining in naïve control animals typically presented as a crisp delineation of endothelial cells of glomerular capillaries. The vWF staining in glomeruli of irradiated animals occasionally had a smudged, indistinct presentation, suggesting breakdown of the glomerular capillary endothelium.

Figure 6c.

Increased vWF IHC staining in glomeruli was noted in a few animals, primarily those irradiated at 12 Gy, within the first 20 days following irradiation. A larger number of animals exhibited increased and morphologically altered glomerular vWF staining starting at approximately 100 days following irradiation, and continuing through the observation period.

Figure 6e.

Immunohistochemical staining for von Willebrand factor in the renal cortex of a naive control male rhesus macaque shows the well-delineated, moderate intensity vWF staining of glomerular capillaries and the more intense vWF staining in the centrally located arteriole. von Willebrand factor IHC stain, 20x objective magnification.

Growth factor immunohistochemical staining

Positive immunohistochemical staining for connective tissue growth factor (CTGF) was subcategorized by location in glomeruli, cortical tubules or collecting ducts (Fig. 7a-e). Positive CTGF staining was present in cortical tubules, collecting ducts, and glomeruli of irradiated animals, most commonly in animals necropsied from day 100 through day 180. Positive CTGF glomerular staining was topographically related to glomerular αSMA staining, and positive staining in tubules and collecting ducts was topographically related to interstitial fibrosis and interstitial αSMA staining.

Figure 7a.

Immunohistochemical staining for connective tissue growth factor (CTGF) in glomeruli revealed a pattern of increased CTGF staining that correlated with the glomerular fibrosis noted with Masson’s trichrome staining, increased mesangial matrix, and increased immunohistochemical staining for glomerular collagen and α-smooth muscle actin (αSMA).

Figure 7e.

Immunohistochemical staining for connective tissue growth factor (CTGF) in the kidney of a male rhesus macaque collected at day 105 following irradiation at 11 Gy shows positive staining of cells on the surface of glomerular loops, presumably podocytes. CTGF IHC stain, 20x objective magnification.

Positive staining for transforming growth factor beta (TGFβ) was subcategorized as interstitial, tubular, collecting duct or glomerular. There was little evidence of TGFβ staining in any subcategory location of naïve control or irradiated animals. Neutrophils within blood vessels were TGFβ⁺, thus served as an internal control for the staining procedure.

Immunohistochemical staining for IL22 and IL22R was subcategorized based on location in collecting ducts or cortical tubules. Positive staining for IL22 and IL22R was noted in all animals, including naïve controls, with no discernible alteration related to level of irradiation or post-irradiation interval.

Staining for renal pro-renin receptor was subcategorized as glomerular, interstitial or cortical collecting duct. Positive staining for pro-renin receptor was not present in the glomeruli or interstitium of the naive control animals, but pro-renin receptor staining of mild (grade 2) intensity was present in the cortical collecting ducts of those animals. Positive staining for pro-renin receptor was commonly present in the cortical collecting ducts of irradiated animals, as was seen in the naïve control animals, but was rarely noted in the renal interstitium or glomeruli of irradiated animals.

Iron staining

The Perls’ iron stain revealed iron-positive deposits in glomeruli of only one animal that was irradiated at 11 Gy and necropsied on day 140. The deposition of iron-positive material was presumed to be associated with the glomerular congestion noted in a larger number of animals. It may be noteworthy that the glomerular iron deposition was present in an animal that had the most pronounced (grade 3) glomerular capillary congestion, PAS⁺ mesangial deposits, and glomerular αSMA and vWF staining.

Mast cell populations

There was no definitive radiation-associated change in mast cell populations in the kidney, as determined by toluidine blue staining and tryptase IHC staining, but there was a tendency toward reduced mast cell staining in the period immediately following irradiation and a tendency toward increased mast cell populations in the later stages of the observation period (Fig. 8a). The staining procedures for mast cell identification were based on demonstration of the contents of mast cell granules, therefore, the apparent reduction in mast cell populations could be due to mast cell degranulation. Mast cell populations in the later stages of the observation period co-existed with increased interstitial fibrous connective tissue, but it is uncertain whether the increased mast cell population is a cause or an effect of the fibrosis.

Figure 8a.

Tryptase staining revealed a slight increase in mast cell populations in irradiated animals, particularly in animals necropsied after approximately 85 days post-irradiation.

The tryptase IHC stain revealed a greater number of mast cells than the toluidine blue stain, thus was considered to be a more reliable indicator of mast cell populations. Mast cells were absent or very sparse (grade 1) until approximately day 100, when mast cells were more commonly observed in the renal cortex. Prior to day 115, only 10 of 59 animals had grade 1 or grade 2 mast cell populations in the kidney, and only 2 of the 10 animals had grade 2 mast cell populations. From day 115 through the end of the observation period, 27 of 35 animals had grade 1 or grade 2 mast cell populations, with 17 of 27 animals having grade 2 mast cell populations. The increase in mast cell population closely paralleled the time course of interstitial fibrosis. Given the known proclivity of mast cells to locate near collagenous tissue, it was uncertain whether the increased mast cell population was a cause or an effect of the interstitial fibrosis.

Generalized vascular changes

Congestion of small blood vessels in the corticomedullary region was commonly observed in irradiated animals (Fig. 8b, c). This histological change is commonly encountered in nonhuman primates, which brings into question the relevance of the observation. However, the corticomedullary congestion was pronounced and commonly observed in irradiated animals in the present study, but was not present in the naïve control animals. There was no apparent relationship to radiation exposure level or post-irradiation survival time.

Figure 8b.

Vascular congestion in the region of the corticomedullary junction was commonly noted in irradiated animals throughout the observation period.

Figure 8c.

Corticomedullary congestion was present in the kidney of a male rhesus macaque collected at day 21 following irradiation at 11 Gy. The renal cortex is at the left side of the image, and the medulla is at the upper right. Note the brightly eosinophilic erythrocytes that are largely concentrated in blood vessels in the medulla near the corticomedullary junction. H&E stain, 3.08x objective magnification.

Renal tubular changes

Intratubular accumulation of homogeneous eosinophilic material, recorded as proteinaceous casts, was first noted at Day 98, after which it was noted in 28 of 42 animals (Fig. 8d, e). This manifestation of radiation-associated renal injury is well-known (Geraci et al. 1993), and is presumed to reflect damage to the glomerular filtration mechanism, with passage of proteins into the glomerular filtrate and subsequently into the tubules. The PTAH stains revealed the casts commonly had low-level PTAH positivity (Fig. 9a, b), similar to the degree of PTAH staining exhibited by erythrocytes.

Figure 8d.

Formation of intratubular proteinaceous casts was frequently noted in the kidneys of irradiated animals from approximately 100 days following irradiation throughout the remainder of the observation period.

Figure 8e.

Proteinaceous casts were present in the renal cortex of a male rhesus macaque collected at day 105 following irradiation at 11 Gy. Note the homogeneous eosinophilic cast material in dilated tubules (*) and increased mesangial matrix (arrow) in a glomerulus. H&E stain, 20x objective magnification.

Figure 9a.

Positive PTAH staining indicated the intratubular casts contained fibrin and erythrocyte fragments in addition to other proteinic materials that traversed the glomerular permeability barrier.

Figure 9b.

A PTAH stain on the kidney of a male rhesus macaque collected at day 141 following irradiation at 11 Gy reveals a dilated tubule in the center of the image containing intact erythrocytes (arrow) that were identical to those seen within blood vessels in the section, as well as additional PTAH⁺ globules and punctate bodies that were consistent with erythrocyte fragments. The cast in a nearby (perhaps the same) tubule (*) that is uniformly PTAH⁺ may represent an obstruction in the outflow from the adjacent dilated tubule segment. PTAH stain, 30x objective magnification.

Grade 1 or grade 2 tubular degeneration and/or hyaline droplet accumulation were first noted at day 85 in an animal irradiated at 11 Gy. Thereafter, tubular degeneration and/or hyaline droplet formation were noted with some frequency (Fig. 9c, d), but typically involved only widely scattered proximal convoluted tubules. These changes were consistent with early stages of degenerative changes in tubular epithelium, perhaps coupled with an excessive load of proteinic material received due to damaged glomerular structures. Punctate PAS⁺ cytoplasmic inclusions were present in renal tubules of a number of irradiated animals, but the exact nature of the cytoplasmic inclusions was not apparent. In some affected animals the punctate cytoplasmic inclusions coexisted with overt hyaline cytoplasmic droplets, suggesting both structures were of the same genesis.

Figure 9c.

Minimal to mild renal tubular degeneration was noted in animals necropsied at post-irradiation day 100 through the remainder of the observation period.

Figure 9d.

The renal cortex of a male rhesus macaque collected at day 102 following irradiation at 12 Gy has a degenerative tubule (*) lined by swollen tubular epithelial cells, some of which contain brightly eosinophilic hyaline droplets. Two partially thrombotic glomeruli (arrows) contain degenerated erythrocytes and erythrocyte fragments. H&E stain, 16.1x objective magnification.

Dilatation of renal tubular lumina was present in a number of animals, mostly commonly in animals that were irradiated at 12 Gy. This change was suspected to reflect hemodynamic and urinary alterations associated with administration of therapeutic fluids.

DISCUSSION

Animals were euthanized based on clinical indications of debilitation, presumably resulting from advanced radiation-associated injury to internal organs, primarily the lungs. The histopathological observations represent snapshots of organ injury at various time intervals during the observation period, but do not represent an organized time-course study of lesion development in each organ. Interrelationships between lesion pathogenesis in various organs may have influenced the histopathological status of tissue changes. E.g., advanced pulmonary fibrosis may have resulted in hypoxic effects on the kidney, thus accentuating the renal fibrosis in animals that had a particular predilection for pulmonary injury or inadequate response to clinical management directed at amelioration of pulmonary effects.

Multiple pathological processes in the kidney progress to a similar final morphological and functional status, known as end-stage kidney. Pathological processes initiated in one renal compartment, whether glomerular, tubular or interstitial, have a tendency to expand into the other compartments. Renal fibrosis commonly progresses to result in renal failure (Cohen 1995). Regardless of the initiating cause, renal fibrosis involves four overlapping phases: priming, activation, execution and progression (Liu 2011). Priming by an event such as irradiation triggers the activation and expansion of multiple fibrogenic cell populations of diverse origins, including interstitial fibroblasts, pericytes, circulating progenitor cells, and phenotypic transition of preexisting cell populations. Upon activation, the fibrogenic cells orchestrate the production and assembly of extracellular matrix proteins such as collagen. Presence of the increased extracellular matrix has an adverse effect on tubules, which become atrophic, and reduces the vascular supply to the degree that local tissue hypoxia results. At this point a vicious cycle is established, resulting in end-stage renal failure. The main risk in renal injury, whether acute or chronic, is initiation of a self-perpetuating renal fibrogenesis feedback loop that will increase in severity in the absence of the initiating factor. Injury-related changes in tubular epithelial cells contribute substantially to the development of interstitial fibrosis by producing growth factors that activate myofibroblasts. Renal tubular epithelial cells have a high metabolic rate that is heavily dependent on adequate oxygen supply, and rapidly respond to local tissue hypoxia. The high oxygen demand contributes to the vicious cycle in renal fibrosis, whereby interstitial fibrosis leads to local tissue hypoxia which stimulates tubular epithelial cells to elaborate growth factors, thus inducing myofibroblasts to produce increasing amounts of collagen. The post-irradiation observation period in the present study appeared to encompass the phase of initiation and early expansion of the delayed phase of radiation nephropathy. Histological changes noted in the present study were generally similar but more pronounced than those reported in rhesus macaques at lower radiation doses (4.5 −8.5 Gy single doses or two fractions of 5.4 Gy separated by 24 hours) with a longer post-irradiation interval (6–8 years) (van Kleef et al. 2000). Glomerular changes in the reported study included increased mesangial matrix and capillary dilatation, similar to the changes seen in the present study. Tubulointerstitial changes in the reported study included hypercellularity, fibrosis and mild tubular atrophy noted 6–8 years following low-dose radiation exposure. Hypercellularity was not specifically recorded as a histological observation in the present study, though there was an unequivocal increase in interstitial cellularity associated with interstitial fibrosis. Tubular atrophy was not a prominent feature in the present study, perhaps reflecting the relatively short post-irradiation observation period in the present study (185 days) versus the reported study (6–8 years). Leukocyte infiltration was negligible in both the reported study and the present study, but the reported study indicated a slight increase in macrophage populations of the renal cortex. Altered expression of von Willebrand factor was not noted in the reported study, possibly a reflection of the lower radiation dose and substantially longer post-irradiation interval in the reported study.

Connective tissue growth factor (CTGF), which is induced by transforming growth factor beta (TGFβ), has been shown to have a central role in the fibrosis associated with a number of human disease processes, including renal fibrosis (Ito et al. 1998), and has been shown to be an important factor in the development of renal fibrosis in the rat remnant kidney model (Frazier et al. 2000) as well as other models of renal fibrosis. Increased expression of CTGF was observed in intestinal biopsy specimens of patients experiencing post-irradiation enteritis (Vozenin-Brotons et al. 2003) as well as lung (Kalash et al. 2014), kidney (Liu and Wang 2008, Kruse et al. 2009), and liver (Cheng et al. 2015) that were target organs of radiation injury in rodents. Previous studies by our group have shown elevated tissue levels of CTGF in the kidney, lung, spleen, thymus and liver of rhesus macaques following partial-body irradiation with 5% bone marrow sparing (Zhang et al. 2015).

The CTGF promoter region has a TGFβ-specific response element that directly induces transcription of the CTGF gene (Grotendorst et al. 1996, Grotendorst 1997), thus CTGF serves as the direct mediator of many of the downstream connective tissue effects of TGFβ. CTGF has a mitogenic effect on fibroblasts (Frazier et al. 1996, Kothapalli et al. 1997), and induces extracellular matrix-associated genes such as collagen 1, fibronectin and α−5 integrin (Frazier et al. 1996, Frazier and Grotendorst 1997, Duncan et al. 1999). TGFβ can induce the transformation of fibroblasts into myofibroblasts (Desmouliere et al. 1993), contraction of which enhances renal interstitial scarring and tubular atrophy (Ng et al. 1998, Yang et al. 1998), leading to local tissue hypoxia that promotes additional CTGF expression. In addition to transformation from fibroblasts, myofibroblasts are also thought to form by epithelial-mesenchymal transdifferentiation from tubular epithelial cells (Ng et al. 1998).

The above background information raised expectations with regard to immunohistochemical staining for TGFβ, CTGF, aSMA as a marker for myofibroblast differentiation, and trichrome staining/collagen 1 immunostaining as indicators of the final collagen production. Most of these components were present as expected, however, TGFβ immunostaining was notably sparse in the kidney specimens of the present study. The sparse TGFβ staining was unexpected, given the evidence of renal fibrosis that was abundantly apparent in the trichrome-stained sections and evidence of CTGF activity in the kidney of irradiated animals. This incongruity raised suspicion that the TGFβ primary antibody used in the staining procedure was not optimal for this animal species, or the specimens were not optimally preserved for demonstration of TGFβ. The specimens originated from animals that were euthanized for cause, rather than through a planned sequential sacrifice, thus it is also possible that TGFβ activity was expressed at some time prior to necropsy. It is well established that CTGF, TGFB (which exclusively stimulates CTGF production in a paracrine manner), and aSMA are upregulated and expressed in renal tubule epithelium when the epithelium, and particularly the basement membrane, is damaged and the tubular epithelial cells are undergoing epithelial-mesenchymal transition (Frazier et al. 2000). However, expression of TGFB in tubules and glomeruli occurs only in the first several days following injury, after which the long-term expression of injury involves CTGF expression in a positive loop paracrine effect on myofibroblasts. Continued TGFB expression would be unexpected at the post-injury time point at which the majority of these kidney specimens were collected. Studies in cultured kidney cells have shown that continued expression of CTGF is maintained by hypoxia-inducible-factor-1 (HIF-1) in the absence of continued signaling by TGFβ.

In the present study, localization of aSMA, CTGF, and collagen 1 immunostaining as well as trichrome and PAS stains suggested intertubular as well as mesangial and endocapillary glomerular origin of cellular transitions to myofibroblasts, with resultant fibrosis and mesangial expansion that occurred globally throughout the interstitium and glomeruli, including visceral glomerular tufts.

Multiple glomerular and interstitial changes in the kidney started to appear at approximately 100 days following irradiation. Intratubular proteinaceous casts, indicating altered glomerular filtration function, were first noted in an animal that was necropsied on day 98 post-irradiation. The appearance of interrelated histological changes in different compartments of the kidney at approximately the same time post-irradiation suggests may reflect radiation-associated injury to a specific cell population (e.g., capillary endothelial cells or pericytes) that has multicentric distribution in glomeruli and the renal interstitium. Similar delays were seen in the lung and intestinal tract, as described by Parker et. al. in this issue (Parker et al. 2018, Parker et al. 2018).

In the present study the detection of altered macrophage or leukocyte populations was based solely on microscopic examination of routinely stained histologic sections. Inflammatory cell infiltration or increased macrophage populations were not prominent histological features of the radiation nephropathy, but that does not preclude the possibility of functional alterations in cellular populations. Functional alterations in the tissue-based population of macrophages have been implicated in fibrogenesis in multiple organs, therefore, more definitive studies of macrophage function and signaling processes would be necessary before dismissing the possible role of macrophages in post-irradiation renal fibrosis.

Gentamicin, which was administered to 10 of the irradiated animals as part of the medical management program, is known to be nephrotoxic under some circumstances. Review of the in-life records revealed that gentamicin therapy was initiated between study days 6 and 28, and animals that received gentamicin therapy were necropsied between study days 30 and 183. A minimum of 20 days elapsed between cessation of gentamicin therapy and necropsy of any animal. BUN and creatinine levels were monitored prior to and following gentamicin therapy to assess potential acute renal injury attributable to the antibiotic therapy. The clinical pathology parameters revealed no evidence of an adverse effect of the antibiotic therapy, and review of the histopathological observations revealed no gentamicin therapy-related alterations in the spectrum of radiation-related changes in the kidney. These observations indicated the gentamicin therapy had no influence on the renal lesions seen at termination.

The histomorphological features and time course of radiation-associated renal changes in the present study were similar to those encountered in other studies conducted in nonhuman primates and other species, with variations related to species differences, relative radiation dose, rate of radiation exposure, and post-irradiation interval before necropsy.

CONCLUSIONS

Histopathological alterations in the kidney were centered on fibrosis and a spectrum of glomerular changes that were noted in animals necropsied from approximately day 100 post-irradiation and extending through the observation period. Interstitial and glomerular fibrosis were apparent with Masson’s trichrome staining and collagen 1 immunohistochemical (IHC) staining. The fibrosis was associated with increased immunohistochemical staining for α-smooth muscle actin (αSMA) and connective tissue growth factor (CTGF), the latter expressed in collecting ducts, glomeruli and cortical tubules. In addition to the fibrosis noted on trichrome staining, glomeruli had increased αSMA IHC staining and increased mesangial matrix as noted on routinely stained sections. Fibrotic glomeruli had reduced microvascular density as seen in CD31 IHC staining. Glomeruli had multiple manifestations of microvascular injury, including capillary congestion, thrombosis, and capillary tuft dilation. Formation of proteinaceous casts in the lumen of cortical and medullary tubules indicated loss of proteins into the glomerular filtrate, further indicating a deficit in glomerular function. Less common glomerular changes included accumulation of PTAH⁺ particles that were consistent with fragmented erythrocytes, mesangial deposits of PAS⁺ material that was presumed to be of basement membrane origin, and increased quantity and atypical dispersion of von Willebrand factor (vWF) on IHC staining. In addition to glomerular congestion, the kidney commonly had regional vascular congestion that was most pronounced in the corticomedullary junction.

Figure 1d.

Congestion of glomerular capillaries, with sparing of capillaries in the surrounding renal cortex, was commonly noted starting at approximately 100 days following irradiation.

Figure 1e.

Glomerular congestion was present in the renal cortex of a male rhesus macaque collected at day 105 following irradiation at 11 Gy. Note multiple proteinaceous casts (*) and dilated and/or thrombotic glomerular capillaries (arrow) throughout the section. . H&E stain, 5x objective magnification.

Figure 5e.

Immunohistochemical staining for collagen 1 in the kidney revealed a grade 2 increase in the amount of glomerular collagen starting at approximately day 90 post-irradiation, and continuing through the observation period. Increased glomerular collagen was uncommonly noted in the kidneys of animals necropsied during the day 20 to day 50 interval.

Figure 6d.

Increased vWF IHC staining in interstitial blood vessels was less common than the increased vWF staining observed in glomerular capillaries, but the temporal sequence was similar to the glomerular change.

Figure 6f.

Immunohistochemical staining for von Willebrand factor in the renal cortex of a male rhesus macaque collected at day 182 following irradiation at 10 Gy shows an increased amount of somewhat dispersed vWF⁺ material in two glomeruli (*), as well as the reduced amount of vWF staining in the atrophic glomerulus (**). A more nearly normal glomerulus (arrow) is present. Von Willebrand Factor immunohistochemical stain, 20x objective magnification.

Figure 7b.

Increased immunohistochemical staining for connective tissue growth factor (CTGF) was noted in cortical tubules. As with other many other manifestations of radiation-associated renal injury, the increased expression of CTGF in cortical tubules was most commonly observed in animals necropsied approximately 100 days post-irradiation and continuing through the observation period to day 180.

Figure 7c.

Increased immunohistochemical staining for connective tissue growth factor (CTGF) was noted in renal collecting ducts of irradiated animals. As with other manifestations of radiation-associated renal injury, the increased expression of CTGF in collecting ducts was observed most commonly in animals necropsied at 85 to 180 days post-irradiation.

Figure 7d.

Immunohistochemical staining for connective tissue growth factor (CTGF) on the kidney of a male rhesus macaque collected at day 105 following irradiation at 10 Gy shows positive staining of collecting ducts and tubules in a somewhat linear array that included medullary rays in the renal cortex. CTGF IHC stain, 10x objective magnification.