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.

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.

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.

Endoglin haploinsufficiency reduces radiation-induced fibrosis and telangiectasia formation in mouse kidneys.

BACKGROUND AND PURPOSE: Endoglin is a transforming growth factor beta (TGF-beta) co-receptor mainly expressed in dividing endothelial cells. It regulates cell proliferation and survival and is upregulated at sites of vessel repair. Mutations in endoglin have been linked to the vascular disease hereditary hemorrhagic telangiectasia (HHT). HHT patients display dilated capillaries (telangiectasia) that are prone to rupture. Cancer patients receiving radiotherapy develop similar vascular damage in normal tissues lying in the irradiation field. If located in the mucosa, irradiation-induced telangiectasia can lead to severe bleeding. Therefore, this study was aimed at investigating the role of endoglin in radiation-induced telangiectasia formation. MATERIALS AND METHODS: Kidneys of endoglin heterozygous (Eng(+/-)) or wild type mice were irradiated with 16 Gy. Mice were sacrificed after 20 weeks and changes in gene expression and protein levels were analysed. RESULTS: Expression of TGF-beta target genes involved in radiation-induced fibrosis and fibrosis development in the kidney decreased in Eng(+/-) compared to wild type mice. Unexpectedly, Eng(+/-) mice also displayed reduced telangiectasia formation in the irradiated kidney. CONCLUSIONS: Endoglin plays an important role in the development of irradiation-induced normal tissue damage. Future studies will show whether interfering with endoglin functions protects tissues from late radiation toxicity.

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.

Fractalkine: an important candidate for directing periglomerular leukocyte accumulation in irradiated mouse kidneys.

Radiation-induced impairment of renal function is preceded by capillary endothelial cell damage, which initiates a cascade of inflammatory and thrombotic events. Accumulation of leukocytes in the irradiated kidney, especially in areas surrounding the glomeruli, has been clearly demonstrated. The chemokine fractalkine has recently been identified as a key mediator of leukocyte adhesion that functions without the requirement of integrins or selectin-mediated rolling. In this study we investigate the possible involvement of fractalkine in the inflammatory response of the irradiated kidney. Mouse kidneys were irradiated with single doses of 16 or 0 Gy, and protein and mRNA levels of fractalkine and PECAM-1 were examined after 10 to 40 weeks. These changes were correlated with the progressive increase and distribution of leukocytes in the irradiated kidneys. Increased fractalkine immunoreactivity was seen at glomerular sites 30 to 40 weeks after irradiation. This fractalkine expression was strongly associated with the presence of leukocytes surrounding the Bowman's capsule of the same glomeruli. No significant changes in mRNA levels of fractalkine were seen in whole kidney extracts after irradiation, but expression levels were not determined for isolated glomeruli. PECAM-1 protein levels did not change with time after irradiation, although a significant decrease in mRNA expression was seen at 10 weeks. This study is the first demonstration of increased fractalkine after irradiation and the results suggest that fractalkine may be an important mechanism of leukocyte trafficking in the development of a radiation induced inflammatory response.

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.

Decrease in laminin content and protein excretion rate after five sixths nephrectomy and low-dose irradiation in the rat.

The effect of low-dose irradiation on laminin distribution and urine protein excretion in the remnant rat kidney has been studied. The rat remnant kidney formed after 5/6 nephrectomy is an experimental model of chronic renal failure. In the remnant kidney, focal segmental glomerulosclerosis is developed characterized by focal or segmental sclerosis in glomeruli, alterations in the tubules and thickening of the glomerular basement membrane. Low dose irradiation has been presumed to suppress sclerotic processes. In this study 24 male Wistar rats were subdivided into the nephrectomized group, nephrectomized and irradiated groups (1 or 3 Grey), and healthy control group. Animals were sacrificed at 2, 4 and 8 weeks after beginning the experiment. Laminin immunohistochemical staining was found along the tubular and glomerular basement membranes in all experimental groups, but with varying intensity. Laminin content in the basement membranes was decreased in early stages (week 2), especially after irradiation followed by increase during the later stages with relatively high levels at the end of the experiment (week 8). Irradiation at a dose of 3 Grey decreased protein excretion compared to the nephrectomized rats at all stages, while 1 Grey dose was ineffective. Based on decreased proteinuria we conclude that moderate low-dose irradiation has beneficial effects on the rat remnant kidney and that laminin in basement membranes is probably not the most crucial component in regulating membrane permeability.

[Morphological changes in the kidney after 5/6 nephrectomy and low-dose radiation]. / Morfologicheskie izmeneniia v pochke posle 5/6 nefréktomii i vozdeistviia malykh doz radiatsii.

In the present study, we investigated the effect of low-dose irradiation of experimental nephrectomized rats. We hypothized that the low-dose irradiation may slow down the development of focal-segmented glomerulosclerosis (FSGS) after 5/6 nephrectomy. Experiments were performed with 32 male Wistar rats, divided into four groups. The first group contained only operated animals. Animals in the second and third groups were irradiated on the next day after operation with 1 and 3 Gy, respectively. The healthy animals made the forth, control group. Attention was focused on physiological and morphological changes after low-dose (1 and 3 Gy) irradiation. We measured blood pressure, proteinuria, serum creatinin and cysC. Morphological changes of glomerulus and tubules were studied. Animals of the first group had significantly thicker glomerular basement membrane, compared to animals of other groups. The morphological study demonstrated degeneration of the tubular epithelium, tubular atrophy and FSGS. Besides, it was shown that changes in the third group (3 Gy) were less than in nephrectomized (first group) and 1 Gy (second group). The animals of the third group (3 Gy) had significantly lower proteinuria and FSGS. We conclude that our hypothesis, suggesting that low-dose irradiation slows down the development of FSGS, was confirmed.

Bone-marrow-derived cells contribute to glomerular endothelial repair in experimental glomerulonephritis.

Glomerular endothelial injury plays an important role in the pathogenesis of renal diseases and is centrally involved in renal disease progression. Glomerular endothelial repair may help maintain renal function. We examined whether bone-marrow (BM)-derived cells contribute to glomerular repair. A rat allogenic BM transplant model was used to allow tracing of BM-derived cells using a donor major histocompatibility complex class-I specific mAb. In glomeruli of chimeric rats we identified a small number of donor-BM-derived endothelial and mesangial cells, which increased in a time-dependent manner. Induction of anti-Thy-1.1-glomerulonephritis (transient mesangial and secondary glomerular endothelial injury) caused a significant, more than fourfold increase in the number of BM-derived glomerular endothelial cells at day 7 after anti-Thy-1.1 injection compared to chimeric rats without glomerular injury. The level of BM-derived endothelial cells remained high at day 28. We also observed a more than sevenfold increase in the number of BM-derived mesangial cells at day 28. BM-derived endothelial and mesangial cells were fully integrated in the glomerular structure. Our data show that BM-derived cells participate in glomerular endothelial and mesangial cell turnover and contribute to microvascular repair. These findings provide novel insights into the pathogenesis of renal disease and suggest a potential role for stem cell therapy.

Tissue trace element change after total body irradiation.

PURPOSE: In this study, we examined the changes of tissue contents of trace elements and iron after total body irradiation (TBI) and their possible impact on late toxicities. MATERIAL AND METHODS: 20 female Wistar rats were randomly assigned to two groups - either radiation (n = 10) or control (n = 10). Rats in the radiation group received TBI of 5 Gy in a single fraction. Rats were sacrificed and tissue samples of heart, lung and kidney were taken 8 weeks after radiation. Tissue levels of zinc, copper, magnesium, manganese and iron analysis were performed with an atomic absorption spectrophotometer and suprapure grade standard solutions. One kidney of each animal was taken for electron microscopic analysis. Blood samples were collected from all animals and the blood chemistry related to kidney function was studied. RESULTS: The kidney levels of Fe and Cu significantly increased 8 weeks after irradiation (p < 0.05). The Cu/Zn ratio did not reach statistical significance in any tissue, however in kidney, there was a tendency to rise (p = 0.08). Myocardium and lung content of trace elements and iron did not show any significant change 8 weeks after irradiation. Electron microscopic analysis showed significant injury in glomerular endothelial cells, renal tubules and thickening of basement membrane. Blood chemistry showed a significant rise in serum creatinine (p = 0.008) and calcium (p = 0.01) in the TBI group. Serum creatinine levels were 0.73 and 0.84 mg/dl, and serum calcium levels were 10.1 and 11.3 mg/dl in control and TBI groups, respectively. CONCLUSION: A sublethal dose of TBI causes deposition of Cu and Fe within the kidney after TBI. Deposition of these elements may have some additional role on the toxicity caused by direct radiation on the kidney.

Role of heat shock protein 47 on tubulointerstitium in experimental radiation nephropathy.

The molecular mechanisms of fibrosis in radiation nephropathy have received scant attention. Heat shock protein 47 (HSP47), a collagen-binding stress protein, helps in the intracellular processing of procollagen molecules during collagen synthesis. We investigated the role of HSP47 in the progression of radiation nephropathy using experimental radiation nephropathy. Experimental rat groups were as follows: (i) group I, sham operated (n = 12); (ii) group II, single doses of irradiation, either 7, 15 or 25 Gy to left kidney (n = 60); and (iii) group III, a similar irradiation procedure as group II after right nephrectomy (n = 60). The rats were followed up until 9 months after renal exposure to radiation. Renal dysfunction (as determined by serum creatinine and blood urea nitrogen) and hypertension were noted in group III rats, along with inflammatory cell infiltration and interstitial fibrosis (as determined by increased deposition of collagens). Compared to control rat kidneys, an increased expression of HSP47 was noted in kidneys obtained from irradiated rats. By double immunostaining, HSP47-expressing cells were identified as alpha-smooth muscle actin-positive myofibroblasts and vimentin-positive tubular epithelial cells. Increased expression of HSP47 was closely associated with increased deposition of collagens in the widened interstitium of irradiated rats. Overexpression of HSP47 by phenotypically altered tubulointerstitial cells might play a role in excessive assembly/synthesis of collagens and could contribute to tubulointerstitial fibrosis in radiation nephropathy.

Angiotensin II blockade reduces radiation-induced proliferation in experimental radiation nephropathy.

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

Radiation-induced kidney injury: a role for chronic oxidative stress?

Kidney irradiation clearly leads to a progressive reduction in function associated with concomitant glomerulosclerosis and/or tubulointerstitial fibrosis. However, the particular cell types, mediators and/or mechanisms involved in the development and progression of radiation nephropathy remain ill defined. Angiotensin II (Ang II) plays a major pathogenic role; administration of Ang II blockers markedly abrogates the severity of radiation nephropathy in experimental models. Both ionizing radiation and Ang II signal via generation of reactive oxygen species (ROS). Thus, we hypothesized that localized kidney irradiation might lead to a chronic oxidative stress. In view of the difficulty in measuring ROS in vivo we adopted an indirect immunohistochemical approach in which we used a monoclonal antibody specific for 8-hydroxy-2'-deoxyguanosine (8-OHdG), one of the most commonly used markers of DNA oxidation. The right kidney of 7-8 week-old male Sprague-Dawley rats was removed. Five to 6 weeks later the remaining hypertrophied kidney was irradiated with single doses of 0-20.0 Gy X-rays. Groups of rats, three per dose, were killed at 4, 8, 16 and 24 weeks post-irradiation, their kidneys fixed, and sections stained with the 8-OHdG-specific antibody N45.1. For quantitation of glomerular DNA oxidation with the N45.1 antibody stained sections, 50 glomeruli/animal were counted. The presence of any intensely stained nuclei within the glomerular tuft was scored as positive. Quantitation of tubular DNA oxidation employed a 10 x 10 point ocular grid. Sections were examined at 400 magnification; 250 tubular profiles were counted. All tubules with any nuclear staining were scored as positive.Sham-irradiated kidneys showed little evidence of DNA oxidation over the experimental period. In contrast, localized kidney irradiation led to a marked, dose-independent increase in glomerular and tubular cell nuclear DNA oxidation. This increase was evident at the first time point studied, i.e. 4 weeks after irradiation, and persisted for up to 24 weeks postirradiation. DNA oxidation in the irradiated kidney was only seen in apparently viable glomerular and tubular cells. Thus, while from 16 to 24 weeks post-irradiation structural alterations had progressed to glomerular sclerosis and tubular atrophy, positive staining for 8-OHdG was not observed in severely atrophic tubules. Similarly, fewer positive staining cells were noted in glomeruli undergoing sclerosis, while none were seen in totally sclerotic glomeruli. These data support the hypothesis that renal irradiation is associated with a chronic and persistent oxidative stress.

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.

Increased glomerular Vwf after kidney irradiation is not due to increased biosynthesis or endothelial cell proliferation.

Kuin, A., Citarella, F., Oussoren, Y. G., Van der Wal, A. F., Dewit, L. G. H. and Stewart, F. A. Increased Glomerular Vwf after Kidney Irradiation is not due to Increased Biosynthesis or Endothelial Cell Proliferation. Radiat. Res. 156, 20-27 (2001). Irradiation of the kidney induces dose-dependent, progressive renal functional impairment, which is partly mediated by vascular damage. It has previously been demonstrated that reduced renal function is preceded by an increased amount of von Willebrand factor (Vwf) in the glomerulus. The underlying mechanism and significance of this observation are unknown but, since it is an important mediator of platelet adhesion, Vwf in increased amounts could be implicated in glomerular thrombosis, resulting in impairment of renal function. Increased Vwf could be the result of increased biosynthesis by endothelial cells, or from increased numbers of endothelial cells after compensatory proliferation induced by irradiation, or it could be secondary to other events. In the present study, expression levels of mRNA for glomerular Vwf and glomerular cell proliferation rates were measured in control mouse kidneys and after irradiation with a single dose of 16 Gy. There were no significant changes in mRNA ratios for Vwf/beta-actin at 10 to 30 weeks after irradiation compared with unirradiated samples, whereas increased amounts of Vwf protein were seen in the glomeruli at these times. Labeling studies with IdU or staining for Ki67 demonstrated that glomerular proliferation was increased from 10 to 30 weeks after irradiation. Despite the increased proliferation rates, there was an absence of glomerular hyperplasia and no increase in the endothelial cell surface coverage in the glomeruli. Staining with antibodies against smooth muscle actin (SMAalpha) revealed that the observed proliferation mainly involved mesangial cells. These results indicate that the increased presence of glomerular Vwf after irradiation is not due to an increased number of endothelial cells per glomerulus, or to an increased production of Vwf. It is presumably secondary to other events, such as increased release of Vwf by damaged endothelial cells or entrapment of Vwf in the irradiated mesangial matrix.

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.

Effects of ionizing radiation on progressive experimental renal disease: a hemodynamic approach.

In order to evaluate the progression of renal disease, Munich-Wistar rats were submitted to 5/6 nephrectomy and given whole-body x- or gamma-irradiation with or without remnant kidney protection or were submitted only to remnant kidney irradiation. All groups received a single 6-Gy dose immediately after surgery. Whole-kidney function, glomerular hemodynamics, 24-hour proteinuria and histopathology were assessed 60 days after surgery and irradiation. The irradiated nephrectomized animals presented whole-kidney function parameters comparable to those of normal rats. In addition, they were less hypertensive and had higher hematocrit. They showed glomerular hyperfiltration and hypertension even greater than their respective nephrectomized controls. However, the interrelations among the glomerular filtration determinants were somewhat different in irradiated animals. Their 24-hour proteinuria was significantly lower and the sclerosis index and tubulointerstitial injury score were markedly smaller. Among irradiated animals, the worst sclerosis index was observed in those with a shielded remnant kidney and the best in those without protection of the remnant kidney. This led us to speculate about a possible influence of resident mesangial cells on the early events following renal mass ablation and on the maintenance of subsequent physiopathologic changes. Therefore, radiation undoubtedly provoked a beneficial change in the course of renal disease when the renal mass ablation model was employed. Many factors could have contributed to this favorable feature including lower levels of systemic arterial pressure, less increment in DeltaP, diminished proteinuria, and maintenance of tubulointerstitial space integrity. Our data also suggest that development of glomerulosclerosis seems to be determined by events occurring immediately after injury.