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Abstract-- Although the kidneys are the main target organs for uranium (U) toxicity, the aim of the present study is to examine the histological changes in kidney’s rats administrated with uranyl nitrate compared with controls by light microscope. The present study was carried out on 60 albino rats. 20 animals control and 40 animals were administrated with 75mg/Kg body weight of uranyl nitrate (UN ) per day orally for one month Half the animals sacrificed on 30nd day(group1). Small pieces of kidney were procured, fixed in 10% neutral buffered formalin and embedded in paraffin. Sections of 5 µ thickness were cut and stained with Haematoxylin and Eosin for general morphology. The remain animals (group2) were left for another month and used the same treatment of (group1) after two months of the experiment this treatment added as long term study. Significant histological changes were observed. Significant increment changes were observed in kidney’s rats treated with uranyl nitrate for two months more than kidney’s rats treated with uranyl nitrate for one month, degenerative changes were seen more in cortex as compared to medulla .Hemorrhage and inflammatory cell reaction was also observed in cortex, tubular necrosis were seen. Melanodyldehyd (MDA) as Lipid Peroxidation was significantly higher in homogenized kidney’s tissue of rats than in the controls, while (GSH) was significantly lower in homogenized kidney’s tissue of rats than in the control subjects. Index Term-- Uranyl nitrate, Uranium toxicity, Histopathological changes in kidney, kidney toxicity.

I. INTRODUCTION The chemical toxicity of depleted uranium is about a million times greater in vitro than its radiological hazard1 three main pathways exist by which internalization of uranium may occur: inhalation, ingestion, and embedded fragments contamination1. Depleted uranium is a by-product of the enrichment process for that metal, during which the more radioactive isotopes U235 and U234 are removed2. This depleted form of uranium has about 60% of the radioactivity of the natural mineral, and its density, availability, and relatively low cost make

Widad Jumaa Hamid is serving as Lecturer, Al-Mustanseriya University, College of Pharmacy/Baghdad/Iraq

wedad_jouma@yahoo.com

it attractive for military purposes, specifically in armor and projectiles2 Uranium (uranyl salts), has been used for more than 40 years to stain DNA for electron microscopy. Other stains were favored by electron microscope, which may be due to the more recent findings (1990s) that uranyl ion can catalyze hydrolysis of DNA (strand breaks) in the presence of light. The electronic structure of uranyl nitrate UO2 (NO3)2·2H2O. Binding of DNA to nucleic acids and nucleotides and its catalysis of chemical modification of these molecules would clearly implicate this heavy metal as a cytotoxic3, The behavioral and chemical toxicity of U in the kidney: a reassessment. This gives a thorough comprehensive review of uranium nephrotoxicity, with detailed descriptions of what is known about how the kidney handles uranium and the specific sites of damage to kidney cells by uranium. Although published in 1989, there is relatively little to add in terms of our current understanding of uranium toxicity to the kidney. This paper is strongly recommended for anyone wishing to understand the biomedical aspects of uranium nephrotoxicity4

A histological kidney study of uranium and non-uranium workers. Kidney tissue sections were inspected by pathologists for damage from uranium exposure in 7 uranium industry workers on autopsy and compared with six workers who would have had no known uranium exposure5.

Morphologic changes in uranyl nitrate-induced acute renal failure in saline- and water-drinking rats. Describes the sequential changes in renal morphology within hours the proximal tubules showed loss of brush border and increased vacuolization and necrosis6,

Uranyl nitrate induced glomerular basement membrane alterations in rabbits: a quantitative analysis. Uranyl nitrate caused a dose dependent increase in glomerular basement membrane thickness with little or no sign of recovery with clean drinking water7

Glomerular endothelial cells in uranyl nitrate-induced acute renal failure in rats. It is suggested that U causes alterations in endothelial cells resulting in reduced glomerular ultrafiltration7

Uranyl Nitrate Induce Histological Changes in The Kidney

Widad Jumaa Hamid

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II. MATERIALS AND METHODS

60 male rats (Rattus norvegicus) with a starting weight of 250 to 300 grams were used in this study. 40 animals administrated orally with two ml of 75 mg/kg body weight UN. After one month we divided the animals into two groups (group 1 & group 2). 20 rats (group 1 ) were sacrificed on days 30 , the remained animals(group 2 ) were sacrificed on days 60, 20 animals administrated orally with ,two ml of, physiological saline PBS (Phosphate Buffer Saline ) and used as control( group 3 ). Histopathology, formalin fixed kidney tissue was embedded in paraffin, sectioned at 5 μm thickness and stained with hematoxylin and eosin (H&E). The byproduct of lipid peroxidation malondialdehyde (MDA) level was measured in kidney’s tissues homogenates depends on the formation of pink chromosphere because of the reaction between (MDA) and thiobar bituuric acid TBA, which can be measured spectrophotometric ally according to the method of (Buege and Aust,) 10 In this method 2 ml of TBA reagent (10.375) g (TBA), (15g) trichloroacetic acid (TCA) dissolved in 100 ml of 0.25 N hydrochloric acid at 45C0, was added to 1 ml of the homogenate. The mixture was incubated in a boiling water bath for 10 minutes, cooled and then centrifuged at 3000 r pm for 10 minutes. Light absorbance of the clear supernatant was determined spectrophotometric ally at 535 nm against Blank. MDA concentration was calculated using a molar absorptivity coefficient 1.5 x 105 m-1 cm-1 (Sedlak and Lindsay) 11. The results were expressed as µmol MDA/g tissue. Glutathione (GSH) levels in tissue homogenized were determined according to the method of Ellman 12. In which 0.5 ml of 4% sulphosalcylic acid was added to equal volume of tissue homogenate for precipitation of protein. After centrifugation 0.5 ml of cleer supernatant was mixed with 4.5 ml (DTNB) reagent [0.1Mm DTNB IN 0.1 m phosphate buffer pH8]. The light absorbance of the mixture was measured spectrophotometric ally at 412 nm after 2 minutes.

III. STATISTICAL ANALYSIS

All data are expressed as the means ± S.E.M. Statistical analysis was performed using analysis of variance for multiple comparisons and linear regression between groups.. The data of this study used statistical analysis of variance

(ANOVA) test and least significantly difference (LSD) test by probability of less than 0.05 (P<0.05) according to 13

IV. RESULT AND DISCUSSIONS

Kidney’s rats were studied after uranyl nitrate administration to observe histological changes in kidney for one month and for two months.30 fields were examined per slide and the histological changes were observed (Table 1) .

TABLE I showed histological changes in kidney’s rats administrated with 75mgUN/kg

body weight for one month or two and f months that systematically compared with control.

TABLE II

Oxidative and antioxidant parameters in tissue homogenized compared to control

The present study showed histological changes in kidney’s rats after administration with Uranyl Nitrate for one month and for two months (Fingers,2,3,4,5,6,7) showed these changes. Varicose grades of cloudy swelling of tubules obliterated bowman’s space, increased cellularity of glomeruli, and inflammatory cell infiltration. In contrast 30% slides of control group showed only congestion (Table 1).

Parameters After one month of administration with UN

(mean±SD)

After two months of administration with UN

(mean±SD)

Control (mean±SD)

MDA µmol/L 3.47±0.33 3.98±0.78 1.51±0.72 GSH mmol/L 1.56±0.21 1.76±0.43 4.29±0.58

Finding

After one month of

administration with UN (30)fields

After two months of

administration with

UN(30)fields

Control (

30)fields

Cloudy swelling

25% 40% Non

Necrosis 10% 20% Non Inflammatory cells Infiltration

25% 45% Non

Obliterated bowman’s space

35% 50% Non

Congestion 45% 75% 30%

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Fig. 1. Normal section of control kidney (H&E×200) (group 3). ). Showing

congestion CG

Fig. 2. section of kidney rat administrated with 75mgUN/kg body weight for one month.(H&E×200) ( group 1 ). Showing renal tubules desquamated from the Underlying basement membrane congestion CG & decreased Bowman’s

space DBS.

Fig. 3. section of kidney rat administrated with 75mgUN/kg body weight for

one month. H&E×200) (group 2). Showing renal tubules desquamated from the underlying basement membrane congestion CG, decreased Bowman’s space

DBS cell infiltration ICI hemorrhages & cloudy swelling CLS

Fig. 4. section of kidney rat administrated with 75mgUN/kg body weight for 2 months. H&E×200) (group 2). Showing congestion CG, increased cellularity

IC, decreased Bowman’s space DBS cell infiltration ICI hemorrhages & cloudy swelling CLS

Fig. 5. section of kidney rat administrated with 75mgUN/kg body weight. For 2

months (H&E×200) (group 2). Showing congestion CG, increased cellularity IC, decreased Bowman’s space DBS cell infiltration ICI hemorrhages & cloudy

swelling CLS

Fig. 6. section of kidney rat administrated with 75mgUN/kg body weight for 2 months. (H&E×200) (Group 2). Showing congestion CG, increased cellularity IC, decreased Bowman’s space DBS cell infiltration ICI hemorrhages & cloudy

swelling CLS

CG

CG

DBS

CG

ICI

CLS

DBS

GS IC

CLS

DBS

CR

CLS

CG

NI

ICI

DBS

DBS

IC

NI

CG

CR

CG

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Fig. 7. section of kidney rat administrated with 75mgUN/kg body weight for 2 months. (H&E×200) (Group 2). Showing congestion CG, increased cellularity IC, decreased Bowman’s space DBS cell infiltration ICI hemorrhages & cloudy

swelling CLS Although the nephrotoxicity of uranium has been established through numerous animal studies, relatively little is known about the effects of long-term environmental uranium exposure. Examination of the H&E stained sections revealed a sequence of change, as Compared to controls (Figure1), there was cytoplasmic swelling and vacuolization, and the presence of necrotic cells ( Figure 4). This was followed by expansion of necrosis to involve large numbers of tubular epithelial cells, with sloughing of cellular debris into the lumen (Figure 3). Subsequently, focal regions of flattened basophilic regenerating cells appeared in the walls of the tubules, displacing necrotic cells (Figure 4), These regenerating cells became more prominent, occupying large regions of the affected tubular surface ( Figure 5,6,7)., although there was some residual focal scarring and interstitial inflammation after two months of exposure to UN . Experimental work showed that administration of uranium causes extreme toxicity and some authors referred to it as the most toxic metal 14. It was also observed that uranium-damaged tubular epithelium demonstrates a remarkable rate of atypical tubular regeneration15. Early observations from the beginning of the 19th century reported nephrotoxicity caused by uranium poisoning, with predominant changes described by necrosis in the proximal convoluted tubule and a moderate degree of inflammatory and fibrotic changes, resulting in scarred kidney16. There have been other experimental animal studies of soluble depleted uranium nephrotoxicity. Depleted uranium salts have been administered using single or multiple exposures. The injury in these appeared to be due to heavy metal toxicity rather than radiation effects17 so , Luessentrop,etal ,observed well marked dose dependent morphological changes in kidney rats, using dosages of 5, 15

or 30 mg/kg administered as intravenous uranyl nitrate. Despite these considerably higher doses, the sequences of clinical and renal pathology findings were agreed with the present study 18 The chemical toxicity of depleted uranium is about a million times greater in vitro than its radiological hazard19.

It has been reported that Uranium, as other heavy metals, is a well-known nephrotoxic, accumulating in the kidney and injuring proximal tubular epithelial cells, It subsequently binds to anionic sites in the proximal tubular epithelial brush Injury to the cell is related to alterations in cell and lysosome membranes, injured mitochondria leading to impaired energy production and altered calcium ion homeostasis. These may be based upon uranium-related induction of cellular oxidative stress1819 The chemical toxicity of depleted uranium is about a million times greater in vitro than its radiological hazard. It has been reported recently that UN exposure would severely damage kidney tissues that agreed with recent and following studies. In an early series of experiments, very high doses (up to 20% in the diet) of a variety of uranium compounds were fed to rats, dogs and rabbits for periods ranging from 30 days to 2 years. On the basis of very limited histopathological investigations, renal damage was reported in each kidney tissues 20. The present study found that exposure rats with U induced enhancement of lipid peroxidation, which is a major mechanism for some toxic effects of uranium in different organ and tissues have certainly been suggested earlier by (Prasad et al)21.

The activity of lipid peroxidation is accelerated in animals treated with UN. Higher level of MDA is associated with depletion of antioxidants and several forms of scavengers (Dublineau et al.,: Stevens et al.,)22,23.

Exposure to Uranium results in significant accumulation in most of vital organs like kidney and free radical damage have been proposed as cause of uranium induced tissue damage where oxidative stress is a likely molecular mechanism.24 The decrease in GSH in animals treated with UN when compared with control group can be explained by the role of the toxic effects of UN.

V. CONCLUSIONS Degenerative changes like cloudy swelling inflammatory cell infiltration, decreased bowman’s space and increased of glomeruli have been observed in kidney of albino rats in this work after administration of UN orally for one month ,so the changes were more prominent in kidney after administration of UN orally for two months.

ICI DBS

I CLS

NI

CG

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VI. REFERENCES

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Nitrate and Syzygium aramaticum (L.) Merr. PhD Thesis, University of Baghdad/ College of sciences. Chapter 5;87-88.