Modulation of radiation-induced damage of human glomerular endothelial cells by SMPDL3B.

The intracellular molecular pathways involved in radiation-induced nephropathy are still poorly understood. Glomerular endothelial cells are key components of the structure and function of the glomerular filtration barrier but little is known about the mechanisms implicated in their injury and repair. The current study establishes the response of immortalized human glomerular endothelial cells (GEnC) to ionizing radiation (IR). We investigated the role of sphingolipids and the lipid-modifying enzyme sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) in radiation-induced GEnC damage. After delivering a single dose of radiation, long and very-long-chain ceramide species, and the expression levels of SMPDL3b were elevated. In contrast, levels of ceramide-1-phosphate (C1P) dropped in a time-dependent manner although mRNA and protein levels of ceramide kinase (CERK) remained stable. Treatment with C1P or knocking down SMPDL3b partially restored cell survival and conferred radioprotection. We also report a novel role for the NADPH oxidase enzymes (NOXs), namely NOX1, and NOX-derived reactive oxygen species (ROS) in radiation-induced GEnC damage. Subjecting cultured endothelial cells to radiation was associated with increased NOX activity and superoxide anion generation. Silencing NOX1 using NOX1-specific siRNA mitigated radiation-induced oxidative stress and cellular injury. In addition, we report a novel connection between NOX and SMPDL3b. Treatment with the NOX inhibitor, GKT, decreased radiation-induced cellular injury and restored SMPDL3b basal levels of expression. Our findings indicate the importance of SMPDL3b as a potential therapeutic target in radiation-induced kidney damage.

Histochemical detection of ischemia-like alterations induced in kidney tissue in vitro--different sensitivity to oxidant stress of glomerular ENTPD1 versus E5NT.

The expression of ENTPD1 (ecto-nucleoside triphosphate diphosphohydrolase) along the glomerular microvasculature of the kidney is downregulated in ischemic conditions, in contrast to E5NT (ecto-5'-nucleotidase), which may explain the increased tendency for intraglomerular microthrombus formation in vivo. It has been suggested that in ischemia, reactive oxygen species (ROS) affect glomerular ENTPD1, whereas E5NT seems less sensitive to oxidant stress. To test this hypothesis, a soluble ATP and ADP hydrolyzing enzyme solution (apyrase) [0.4 U/ml] or 5'-nucleotidase solution [0.33 U/ml] as well renal tissue were exposed to ROS, generated by gamma-irradiation in vitro. The enzymes diluted in distilled water or cryostat rat kidney sections were exposed to gamma-irradiation (0.037 Gy/s) for 0, 2, 5, 10, or 15 min, with or without supplementation of the ROS scavenger dimethylsulfoxide (DMSO). The enzyme activity of the samples was biochemically tested using standard methods, before and after irradiation. The reaction product of irradiated versus nonirradiated kidney sections was semiquantitatively evaluated after histochemical staining for either glomerular ENTPD1 or glomerular E5NT expression. The results show that the enzyme activity in samples of soluble apyrase was significantly decreased after irradiation. This effect was inhibited by DMSO. In contrast, 5'-nucleotidase activity showed only a limited decline of the activity curve after irradiation, which could also be restored following supplementation of DMSO. Glomerular ENTPD1 expression showed significant decrease after irradiation of kidney sections; again, this was inhibitable by DMSO. Glomerular E5NT activity was not altered by irradiation and DMSO supplementation did not affect its activity. It is concluded that soluble apyrase as well as the glomerular ENTPD1 are sensitive to oxidant stress, which may explain their downregulation in the ischemic condition in vivo. However, soluble 5'-nucleotidase and E5NT seem much less sensitive to ROS. This relative insensitivity of E5NT to oxidant injury may counteract ischemic injury by promoting local generation of adenosine in the ischemic micro-environment.

Radiation Nephropathy in a Nonhuman Primate Model of Partial-Body Irradiation With Minimal Bone Marrow Sparing-Part 2: Histopathology, Mediators, and Mechanisms.

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 at approximately 180 d 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 d postirradiation 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.

Radiation-induced premature cellular senescence involved in glomerular diseases in rats.

Currently, cellular senescence has emerged as a fundamental contributor to chronic organ diseases. Radiation is one of the stress factors that induce cellular senescence. Although the kidney is known as a radiosensitive organ, whether and how radiation-induced cellular senescence is associated with kidney diseases remains unclear. In this study, we performed experiments on 7-8-week-old male rats that received a single dose of 18-Gy radiation in the unilateral kidney. The irradiated kidneys showed hallmarks of cellular senescence, including increased SA-ß-gal activity, upregulation of cyclin-dependent kinase inhibitor (p53, p21, and p16), and absence of DNA proliferation marker (Ki-67). Furthermore, combined with in-vitro experiments, we demonstrated that radiation-induced senescent glomerular endothelial cells acquired altered gene expression, namely, senescence-associated secretory phenotype (particularly, IL-6), which might be triggered by NF-kB signaling pathway. Pathological analysis suggested severe glomerular endothelial cell injury, as evidenced by thrombotic microangiopathy, collapsing glomeruli, and reduced endothelial cell numbers. We suggested that glomerular endothelial cells were more susceptible to radiation-induced cellular senescence. In conclusion, the current study is the first to identify the important role of radiation-induced cellular senescence, mainly derived from glomerular endothelial cells, for the development of glomerular injury.

Radioprotective effect of atorvastatin against ionizing radiation-induced nephrotoxicity in mice.

PURPOSE: Kidneys are exposed to ionizing radiation during radiotherapy in patients with abdominal malignancy. The aim of this study is to investigate the protective effect of atorvastatin (ATV) against ionizing radiation-induced nephrotoxicity in mice. MATERIALS AND METHODS: Sixty male BALB/c mice were randomly divided into six groups (10 mice per group); control, irradiation (IR), IR plus ATV (10, 20 and 50 mg/kg) and only ATV (50 mg/kg). ATV groups received ATV for seven days via oral gavage before exposure to IR. Animals were exposed to 2 Gy whole body of X-ray on day 8. After exposure to IR, biochemical, histological and immunohistological assays were performed. RESULTS: ATV significantly decreased the level of oxidative stress biomarkers in irradiated mice in comparison with IR alone. A significant reduction in the urea and creatinine levels was observed in ATV plus IR group compared to IR alone. Tubular degeneration, glomerular atrophy, interstitial expansion and fibrosis were observed in irradiated mice. Tubular degeneration and atrophy in the kidneys of IR plus ATV group were less than IR group. In addition, pre-treated animal with ATV significantly showed reduction in caspase-3 immunoreactivity. CONCLUSION: ATV has significant protective effect against radiation-induced nephrotoxicity in mice and is a promising medication for protection of patients during radiotherapy.

Outcome of the acute glomerular injury in proliferative lupus nephritis.

Treatment with total lymphoid irradiation (TLI) and corticosteroids markedly reduced activity of systemic lupus erythematosis in 10 patients with diffuse proliferative lupus nephritis (DPLN) complicated by a nephrotic syndrome. Physiologic and morphometric techniques were used serially before, and 12 and 36 mo post-TLI to characterize the course of glomerular injury. Judged by a progressive reduction in the density of glomerular cells and immune deposits, glomerular inflammation subsided. A sustained reduction in the fractional clearance of albumin, IgG and uncharged dextrans of radius greater than 50 A, pointed to a parallel improvement in glomerular barrier size-selectivity. Corresponding changes in GFR were modest, however. A trend towards higher GFR at 12 mo was associated with a marked increase in the fraction of glomerular tuft area occupied by patent capillary loops as inflammatory changes receded. A late trend toward declining GFR beyond 12 mo was associated with progressive glomerulosclerosis, which affected 57% of all glomeruli globally by 36 mo post-TLI. Judged by a parallel increase in volume by 59%, remaining, patent glomeruli had undergone a process of adaptive enlargement. We propose that an increasing fraction of glomeruli continues to undergo progressive sclerosis after DPLN has become quiescent, and that the prevailing GFR depends on the extent to which hypertrophied remnant glomeruli can compensate for the ensuing loss of filtration surface area.

Arachidonic acid metabolites mediate the radiation-induced increase in glomerular albumin permeability.

Radiation-induced renal injury is characterized by proteinuria, hypertension, and progressive decline in renal function. We have previously shown that in vivo or in vitro irradiation of glomeruli with a single dose of radiation (9.5 Gy) increases glomerular albumin permeability (P(alb)) within 1 hr. The current studies tested the hypothesis that this early radiation-induced increase in P(alb) is caused by the release of arachidonic acid and by the generation of specific arachidonic acid metabolites. Glomeruli obtained from WAG/Rij/MCW rats and cultured rat glomerular epithelial and mesangial cells were studied after irradiation (9.5 Gy, single dose). Arachidonic acid release and eicosanoid synthesis by glomeruli or cultured glomerular cells were measured after irradiation, and the effect of inhibitors of phospholipase A2 (PLA2) and cyclooxygenase (COX) on the irradiation-induced increase in P(alb) was assessed. Arachidonic acid release was demonstrated within 10 mins of irradiation of isolated glomeruli and monolayer cultures of glomerular epithelial and mesangial cells. Prostaglandin F(2alpha) (PGF(2alpha)) and PGE2 release was increased after irradiation of isolated glomeruli. Blocking arachidonic acid release or COX activity before irradiation completely prevented the increase in P(alb). COX inhibition immediately after irradiation also diminished the radiation-induced increase in P(alb). We conclude that arachidonic acid and its COX metabolites play an essential role in the early cellular changes that lead to the radiation-induced increase in P(alb). Understanding of the early epigenetic effects of irradiation may lead to new intervention strategies against radiation-induced injury of normal tissues.

Effects of prenatal 900 MHz electromagnetic field exposures on the histology of rat kidney.

PURPOSE: To research the harmful effects of prenatal exposure of 900 megahertz (MHz) electromagnetic field (EMF) on kidneys of four-week-old male rats and to determine protective effects of melatonin (MEL) and omega-3 (ω-3). MATERIALS AND METHODS: Twenty-one Wistar albino rats were randomly placed into seven groups as follows: Control (Cont), Sham, MEL, ω-3, EMF, EMF+ MEL and EMF+ω-3. After mating, three groups (EMF, EMF+ MEL, EMF+ ω-3) were exposed to an EMF. In the fourth week subsequent to parturition, six rats were randomly chosen from each group. Mean volume of kidneys and renal cortices, the total number of glomeruli and basic histological structure of kidney were evaluated by stereological and light microscopical methods, respectively. RESULTS: Stereological results determined the mean volume of the kidneys and cortices were significantly increased in EMF-exposed groups compared to the Cont group. However, EMF-unexposed groups were not significantly modified compared to the Cont group. Additionally, the total number of glomeruli was significantly higher in EMF-unexposed groups compared to the Cont group. Alternatively, the number of glomeruli in EMF-exposed groups was decreased compared to the Cont group. CONCLUSIONS: Prenatal exposure of rat kidneys to 900 MHz EMF resulted in increased total kidney volume and decreased the numbers of glomeruli. Moreover, MEL and ω-3 prevented adverse effects of EMF on the kidneys.

The distribution of colony-forming cells in the kidney.

A method is described for producing outgrowths of small nephron segments (average 24 cells) in culture. The method was used to estimate an overall colony-forming efficiency of 4.6% for cells constituting the segments. Efficiency was found to be lower for thick segments (1%) than for thin segments (6%) from Henle's loop. The latter higher level indicates that precursor cells are concentrated near the middle of the nephron. For comparison, a two-dose irradiation technique was used to calculate a mean number of 5 +/- 2 (SE) clonogens per segment producing outgrowths. This tended to be higher than the value of about 1 calculated from the 65% of segments producing outgrowths, as expected if the remaining segments contained no clonogens.

Radiation-induced renal disease. A clinicopathologic study.

Radiation injury to the renal parenchyma is an unusual cause of renal insufficiency. Light, immunofluorescence and electron microscopic studies were performed on the renal tissue from two patients in whom renal insufficiency developed within a year after they received abdominal irradiation. The glomerular lesion in both patients was similar. Mild endothelial cell swelling and basement membrane splitting were noted consistently on light microscopy. The electron microscopic examination revealed marked subendothelial expansion with electron-lucent material associated with deposition of basement membrane-like material adjacent to the endothelial cells. In some capillary loops, the endothelial cell lining appeared to be completely lost. The pathogenesis of radiation-induced renal injury is still uncertain. It is speculated that local activation of the coagulation system with consequent thrombosis of the renal microvasculature may be extremely important.

Radiation nephropathy in the rhesus monkey: morphometric analysis of glomerular and tubular alterations.

PURPOSE: The morphologic responses of the monkey kidney glomeruli and tubules to fractionated irradiation were assessed. METHODS AND MATERIALS: Both kidneys of adult female rhesus monkeys were irradiated with doses of gamma-rays ranging from 24 Gy in 12 fractions up to 36 Gy in 18 fractions. Serial renal biopsies were taken between 1 and 12 weeks after irradiation. The kidneys were removed at necropsy 16-23 weeks after irradiation. Glomeruli were assessed for the presence of pathologic features, including intercapillary eosinophilic material, ectatic capillaries, thrombi, hemorrhage, adhesions, and sclerosis. The relative proportion of renal cortex occupied by glomeruli, interstitium, or tubules was determined using a Chalkley point grid. Tubules were further scored as being either normal or abnormal in appearance. RESULTS: Examination of the renal biopsies revealed that progressive glomerular lesions were evident within 4-12 weeks after irradiation. Tubular changes were mild and focal. Morphometric analysis of whole kidneys removed at necropsy demonstrated that numbers of glomeruli with ectatic capillaries, thrombi, and hemorrhage were significantly different from controls at 16-23 weeks after irradiation by all of the doses in the range of 24 to 36 Gy. A significant (p < 0.05) increase in the relative proportion of renal cortex occupied by glomeruli and interstitium was indicative of tubule loss. Further analysis of these tubular changes revealed a highly significant (p < 0.001) dose-dependent increase in the proportion of abnormal to normal tubules. Thus following a dose of 24 Gy in 12 fractions, the ratio of abnormal: normal tubules was approximately 1:2; after 36 Gy in 18 fractions the ratio was 3:1. CONCLUSIONS: Glomeruli appeared to be very radiosensitive because after the clinically relevant dose of 24 Gy in 12 fractions essentially all glomeruli were altered in the irradiated kidneys as compared to controls. Thus, efforts aimed at increasing the threshold dose for development of radiation nephropathy should be directed primarily at preventing the glomerular lesions.

A morphometric analysis of glomerular and tubular alterations following fast-neutron irradiation of the pig and monkey kidney.

PURPOSE: The morphologic responses of the pig and monkey kidney to fractionated fast-neutron irradiation were assessed. METHODS AND MATERIALS: The right kidney of approximately 14-week-old female Large White pigs was irradiated with 6.6-12.2 Gy of fast neutrons (42 MeVd-->Be) given as 12 fractions over 18 days; the left kidney served as the contralateral unirradiated kidney. Both kidneys were removed at necropsy 2 years postirradiation. In addition, the remaining hypertrophied kidney of four unilaterally nephrectomized adult rhesus monkeys was irradiated with a total dose of 11.0 Gy fast neutrons (45 MeVp-->Be) given in an identical fractionation regimen to that used in the pig studies. These kidneys were removed when the animals exhibited renal failure, between 32-94 weeks postirradiation. Glomeruli were assessed for the presence of pathologic features, including intercapillary eosinophilic material (ICE), ectatic capillaries, thrombi, hemorrhage, and sclerosis. The relative proportion of renal cortex occupied by glomeruli, interstitium, normal, or abnormal tubules was determined using a Chalkley point grid. RESULTS: The incidence of normal glomeruli, ectatic capillaries, thrombosis, and periglomerular fibrosis were significantly different in the irradiated pig kidneys compared with the unirradiated contralateral kidneys (p < or = 0.02). Linear regression analysis demonstrated a significant dose relationship in terms of normal glomeruli, ectatic capillaries, and ICE (r > or = 0.64; p < or = 0.04). Irradiation was also associated with a significant (p < 0.0001) decrease and increase in the volume of renal cortex occupied by normal and abnormal tubules, respectively. Similar morphometric changes were noted in the irradiated monkey kidneys. CONCLUSIONS: The morphologic changes seen in the pig and monkey kidney after fractionated irradiation with fast neutrons are similar to those previously noted after single-dose or fractionated-photon irradiation. These findings support the hypothesis that the development of radiation nephropathy in these various models involves common pathophysiological mechanisms.

Radiation-induced changes in glomerular and tubular cell kinetics and morphology following irradiation of a single kidney in the pig.

PURPOSE: Radiation-induced changes in glomerular and tubular cell kinetics and morphology following irradiation of a single pig kidney were assessed. METHODS AND MATERIALS: The right kidney of 13 adult female Large White pigs was irradiated with a single dose of 9.8 Gy gamma rays. Animals were serially killed between 2 and 24 weeks postirradiation (PI); 1 h prior to postmortem each pig received 500 mg bromodeoxyuridine (BrdUrd). At postmortem, both kidneys were removed and tissue taken to prepare cell suspensions. The labeling index (LI) of these suspensions was measured using flow cytometry; in vivo BrdUrd incorporation in glomerular and tubular cells was determined immunohistochemically. The kidneys were also assessed histologically. RESULTS: Irradiation of the right kidney alone resulted in a significant increase in renal cell LI in both the irradiated and the contralateral unirradiated kidney within 2 weeks of irradiation; peak values of 1.57 +/- 0.32% and 1.04 +/- 0.13%, respectively, were seen 4 weeks PI, significantly greater (P < 0.001) than the preirradiation value of 0.18 +/- 0.01%. The LI values then declined with time, but remained greater than those seen prior to irradiation. A similar pattern of response was determined from counts of labeled glomerular and tubular cells identified immunohistochemically. The increase in labeled glomerular cells was seen 2 weeks PI, whereas that for the tubular cells did not occur until 4 weeks PI. The irradiated kidney exhibited diffuse, progressive glomerular alterations. In contrast, tubular damage was focal; the irradiated kidney also exhibited a prominent vasculopathy, involving arteriolar and peripheral interlobular artery thickening. The contralateral unirradiated kidney appeared unchanged. CONCLUSION: These findings confirm the hypothesis that the morphologic and kinetic responses observed after irradiation of a single kidney are similar to those observed after irradiation of both kidneys. Renal irradiation results in significant alterations in glomerular and tubular cell proliferation and morphology within 2-4 weeks of irradiation; glomerular changes appear predominant.

Quantitative morphology of late renal radiation injury.

Late radiation injury manifests itself in all morphologic compartments of the kidney, but loss of cell mass is most significant in the proximal convoluted tubules. Development of an end stage or nonfunctional kidney requires 12 or more months after single fraction X ray exposures of about 12 Gy (2, 5, 13) and is associated with marked morphologic alterations of renal tubules. Radiation induced changes were studied at 6 months after irradiation, a time interval when histological alterations appear minor, but in previous studies were shown to correlate with the later end stage alterations (5, 8, 9). Renal alterations were graded objectively based on renal weight, variation in size of tubule cell nuclei, and glomerular nuclear volume fraction. Irradiation was associated with loss of renal weight, increased variability of tubule nuclear size, and previously unappreciated changes in glomerular nuclear volume fraction. A classification index derived from a weighted combination of renal weight ratio and tubule cell nuclear variability correlates with radiation dose and with previously established subjective histologic grading of renal damage, and allows objective comparisons of various fractionation schedules.

Radiation-induced glomerular thrombus formation and nephropathy are not prevented by the ADP receptor antagonist clopidogrel.

PURPOSE: To assess the effects of kidney irradiation on glomerular adenosine diphosphatase (ADPase) activity and intraglomerular microthrombus formation, and their correlation to the development of renal functional impairment. METHODS AND MATERIALS: C3H/HenAf-nu(+) mice were given single-dose or fractionated kidney irradiations. Glomerular ADPase activity was measured using a cerium-based histochemical method. Microthrombus formation within the glomeruli was assessed by a semiquantitative immunohistochemical analysis of fibrinogen/fibrin deposits. Renal function was assessed by the [(51)Cr]EDTA retention assay. RESULTS: The ADPase activity was significantly reduced, to approximately 50% of pretreatment value, 4--40 weeks after 10--16 Gy single-dose irradiation and at 44 weeks after 20 x 2 Gy. No dose--effect relationship was found. An approximately fourfold increase in glomerular fibrinogen/fibrin staining was observed at 1 year after irradiation. This increase was not influenced by treating the mice with daily, oral clopidogrel, a platelet ADP receptor antagonist, which reduced platelet aggregation by more than 75%. Radiation-induced impairment of glomerular filtration was also not affected by the clopidogrel treatment. CONCLUSION: These data indicate that irradiation significantly reduced glomerular ADPase activity, which correlated with an increased glomerular fibrinogen/fibrin deposition. We were not able to reduce these prothrombotic changes, nor to protect against radiation nephropathy, by pharmacological intervention with an ADP-receptor antagonist.

Radiation nephritis following total-body irradiation and cyclophosphamide in preparation for bone marrow transplantation.

Two children prepared for bone marrow transplantation with total-body irradiation and cyclophosphamide developed hypertension, microscopic hematuria, proteinuria, diminished renal function, and anemia six months after transplantation. Light microscopy of the kidneys revealed mesangial expansion, glomerular capillary wall thickening, and lumenal thrombosis. Electron microscopy demonstrated widening of the subendothelial space due to the deposition of amorphous fluffy material. In one patient, immunofluorescence microscopy revealed glomerular capillary wall deposition of fibrin and immunoglobulins. The clinical and histologic findings support the diagnosis of radiation nephritis. Patients prepared for bone marrow transplantation with total-body irradiation and cyclophosphamide should be followed closely after transplantation for the development of hypertension, proteinuria, and renal insufficiency.

A modified cerium-based histochemical method for detection of experimentally-induced ATPase impairment in glomeruli of the rat kidney.

We studied glomerular ATPase activity, as detectable at the light microscopic (LM) level in cryostat sections of the rat kidney, after unilateral local X-irradiation. The biochemically detectable reduction in glomerular ATPase activity after X-irradiation could be demonstrated at the LM level by application of a modified cerium-based technique. Results show a clear reduction of reaction product in glomeruli in X-irradiated kidneys as compared with the contralateral control kidney. Technical parameters (i.e., tissue fixation, second thickness, cerium concentration of the incubation mixture, and percentage H2O2 added for the amplification step) were established for optimal reproducibility of the staining results. We show that this modified staining protocol allows detection of differences of ATPase activity in contrast to conventional histochemical methods. Inhibition studies with various phosphatase inhibitors and competitive substrate inhibition experiments revealed that the enzyme is specific for nucleoside di- and triphosphatases. Since reduced glomerular adenine nucleotidase activity has recently been recognized as an early event in (experimental) glomerulonephritis, we feel that the new staining protocol presented here may be highly relevant for routine tissue section screening in nephropathological research.

Perinatal radiation-induced renal damage in the beagle.

The developing perinatal kidney is particularly sensitive to radiation. The pathogenesis of the radiation-induced lesion is related to the destruction of outer cortical developing nephrons and direct radiation injury with secondary hemodynamic alterations in remnant nephrons. In this study, which is part of a life span investigation of the effects of whole-body gamma radiation during prenatal and early postnatal life, dogs were given 0, 0.16, 0.83, or 1.25 Gy irradiation at either 55 days postcoitus or 2 days postpartum and were examined morphometrically and histopathologically at 70 days of age. Although irradiated dogs showed no reduction in the total number of nephrons per kidney, there was a significant increase in the total number and relative percentage of immature, dysplastic glomeruli. In addition, deeper cortical glomeruli of irradiated kidneys exhibited mesangial sclerosis similar to that associated with progressive renal failure in our previous studies. These findings are in accord with those reported at doses of 2.24 to 3.57 Gy and demonstrate that the perinatal kidney is affected by radiation doses much lower than previously demonstrated.

A morphological study of radiation nephropathy in the pig.

Both kidneys of mature pigs received a single dose of 9.8 Gy 60Co gamma rays. Pigs were killed between 2 and 24 weeks after irradiation and the kidneys examined histologically. Glomerular and tubular changes were observed within 2 weeks of irradiation. Neutrophils and other leukocytes were seen within glomerular capillary loops; mesangial matrix and cell number increased. A progressive increase in thickening of the basement membrane and a decrease in capillary lumina were then noted. Basement membrane duplication occurred within 12 weeks. By 24 weeks these lesions had increased in severity, sclerotic endstage glomeruli, predominantly subcapsular or juxtamedullary, being evident. Tubular lesions initially consisted of focal areas of tubular atrophy in the juxtamedullary region. By 6 weeks subcapsular foci of tubular degeneration, regeneration, and necrosis were found; these appeared to resolve 12 weeks after irradiation. At later times the severity of the tubular lesions varied between pigs, with some exhibiting interstitial fibrosis involving a complete band of subcapsular tissue, while others showed relatively mild changes. There was no apparent change in the vasculature. These findings indicate that (a) there is no one target or dose-limiting cell, and (b) the vasculature does not play a primary role in the development of radiation nephropathy.

Early detection of radiation-induced glomerular injury by albumin permeability assay.

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