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The Egyptian Journal of Histology:
doi: 10.1097/EHX.0000398849.13587.58
Original articles

Histological study on the effect of haematopoietic stem cells on the adriamycin model of nephropathy in albino rats

Metawe, Shaimaa M.a; Naim, Magda M.c; El Wazir, Yasser M.b; Saleh, Sohair A.a

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Author Information

aDepartment of Physiology, Faculty of Medicine, Menofiya University, Menofiya

bDepartment of Physiology, Faculty of Medicine

cDepartment of Histology, Suez Canal University, Egypt

Correspondence to Magda M. Naim, Department of Histology, Suez Canal University, Ismailia, Egypt Tel: +105185262; fax: +643208543; e-mail: magdanaim20032000@yahoo.com

Received March 10, 2010

Accepted April 12, 2011

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Abstract

Introduction and aim of the study: End-stage renal disease is a major health problem. Recent studies have reported the efficacy of stem cell therapy in nephropathy animal models. This study was carried out to investigate the effect of stem cells on renal structure and functions in nephropathy rat models.

Materials and methods: Thirty male albino rats were divided into three groups: control (I) and nephropathy (II, III) groups. Nephropathy was induced by an intravenous injection of adriamycin. Animals that were found to be nephropathic were divided into two groups: group II (animals were not subjected to treatment) and group III (animals were treated with an intravenous injection of stem cells). Five weeks after the start of the experiment, urine and blood samples were collected for biochemical investigation and kidney tissue was used for histological examination. The percentages of affected renal tubular cells were calculated, in addition to the area percentage of periodic acid-Schiff-positive material and collagen and their results were statistically analyzed.

Results: Adriamycin produced a significant increase in serum urea, creatinine and urinary proteins, with a significant decrease in creatinine clearance and serum albumin. Histological examination showed acute focal tubular necrosis, the renal corpuscles showed thickened membranes, changes in Bowman's capsule parietal cells and effacement of podocyte foot processes and the interstitium showed mononuclear cellular infiltration. A significant increase in damaged tubular cells, area percentage of periodic acid-Schiff-positive material and collagen was also detected. The use of stem cells produced a significant amelioration of all these results.

Conclusion: Stem cell transplantation is effective in improving both the structure and the function of kidneys in nephropathic rat models.

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Introduction

The incidence of end-stage renal disease (ESRD) is increasing worldwide and represents a significant burden to countries not only because of the financial costs of providing ESRD care but also because of loss of productivity and significant morbidity and mortality of the affected patients. There is clearly a pressing need for the identification and early treatment of patients with nephropathy to prevent progression to ESRD. Research in the last 25 years has made great advances in the understanding of the progression of chronic renal diseases. There are now effective treatment options such as kidney transplantation; however, the shortage of donor organs and maintaining patients on dialysis for long periods of time limit its use [1,2].

Stem or progenitor cells are defined by their capacity to undergo cell renewal and asymmetric cell division, which leads to their differentiation and formation of one or more mature tissues and preservation of the stem cell population [3]. Pluripotential cells exist in the inner mass of blastocysts, so-called embryonic stem cells, and in the developing gonadal ridge, germ stem cells [4]. In some tissues, like the bone marrow or intestinal epithelium, the presence of adult stem cells has been recognized for a long time and has been studied extensively [5]. Other types of stem cells include mesenchymal progenitor cells [6] and endothelial precursors [7]. Human umbilical cord blood represents a rich source of hematopoietic stem cells. It has the advantages of being highly abundant and easy to collect, with no serious ethical dilemmas [8].

This study has been carried out to determine the effect of transplanted hematopoietic stem cells on renal structure and function in experimentally induced nephropathy in rats.

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Materials and methods

This study was carried out at the Histology and Physiology Departments, Faculty of Medicine, Suez Canal University, and Physiology Department, Menofiya University. Thirty apparently healthy male albino rats, weighing 150–200 g, were used in this study. They were divided into two main groups: the control group (I; n=10) and the nephropathy group (n=20). In the control group, each rat received a single intravenous injection of saline 0.9% (vehicle of adriamycin) in the rat-tail vein after inducing venodilataion by rubbing the tail with alcohol; the animals were then allowed to live normally for 5 weeks. In the nephropathy group, animals received a single intravenous injection of adriamycin (Adriablastina, 10 mg, Pharmacia, Italy) at a dose of 5 mg/kg body weight in the rat-tail vein [9]. One week after the adriamycin injection (when animals were considered to be nephropathic), 24-h urine was collected using metabolic cages to assess proteinuria and rats with proteinuria levels higher than20 mg/24 h were considered nephropathic [10]. Half of these animals were not subjected to treatment for four weeks (group II) and the other half received a single intravenous injection of CD34+ stem cells in the rat-tail vein [11] (group III). The dose of stem cells was 2×106 umbilical cord blood (UCB) stem cells dissolved in 1 ml isolation buffer/rat [12]. Rats of this group were also allowed to live for 4 weeks after the stem cell injection.

All animals were provided with food and water, maintained at 25–28°C and were housed in large cages with free mobility. Five weeks after the start of the experiment, 24-h urine was collected from all groups using metabolic cages to assess proteinuria and creatinine concentration in urine (to calculate creatinine clearance). Each rat was placed in an individual metabolic cage, which was placed over a funnel. The top of the funnel was covered by wire mesh to avoid faecal contamination of urine. During urine collection, the animals received free access to water but no food was given to avoid contamination.

Blood samples were collected from the retroorbital venous plexus, under mild anaesthesia, using a fine heparinized capillary tube introduced into the medial epicanthus of the rat's eye [13]. Two millilitres of blood was collected in a clean graduated centrifuge tube, allowed to clot at room temperature for 10 min and then centrifuged at 3000 rpm for 20 min. The supernatant serum was collected in a dry clean tube to estimate serum urea, creatinine and albumin, and then creatinine clearance was calculated.

Rats were killed under general anaesthesia. One kidney from each animal was removed and fixed in a 10% neutral-buffered formalin solution and then processed to prepare 5 μm thick paraffin sections suitable for use in the following histological techniques: haematoxylene and eosin, periodic acid-Schiff (PAS) and Masson's trichrome.

For transmission electron microscopy (TEM), small pieces of the other kidneys were fixed in a 2.5% glutaraldehyde solution at 4°C for 24 h, and then washed overnight in several changes of 0.1 mol/l sodium phosphate buffer, pH 7.4, at 4°C. Postfixation was carried out in 2% osmium tetroxide in 0.1 mol/l sodium phosphate buffer, pH 7.4, at room temperature. Specimens were dehydrated in a graded series of cold ethanol and propylene oxide and then embedded in spurr. Semithin sections (1 μm) were stained with toluedine blue. Ultrathin sections (50 nm) were prepared on a Reichert–June ultracut microtome and subsequently stained for 10 min with 3% aqueous uranyl acetate and for 10 min in lead citrate. Sections were then examined and photographed using a Philips 400 T TEM. Electron microscopic processing and examination was carried out at the Electron microscopy Unit, Specialized Ain Shams University Hospital.

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Preparation and transplantation of stem cells

The UCB (cells' source) was collected from normal healthy volunteer donors undergoing full-term normal vaginal deliveries after obtaining informed consent. Women with a known history of hepatitis, infectious diseases, diabetes mellitus, severe hypertension, abortions or poor obstetric history were excluded.

The UCB was collected before the expulsion of the placenta. Using strict aseptic techniques, 50 ml of UCB was withdrawn from the umbilical vein and collected in sterile tubes containing 5 ml of a citrate phosphate dextrose adenine-l anticoagulant. Samples were collected separately, stored at 4°C and processed within 24 h [14].

Separation of UCB CD34+ stem cells was carried out according to the immunomagnetic separation technique [15]. By mixing and incubation, CD34+ cells were bound to Dynabeads M-450 CD34 (Invitrogen Corporation, Carlsbad, California). The rosettes formed were isolated from the suspension using a DYNAL Magnetic Particle Concentrator (DYNAL MPC). On subsequent incubation with DETACHaBEAD, CD34 gently induced detachment of isolated cells from the beads. A DYNAL MPC was then used to separate the purified, positively selected CD34+ cells from the released Dynabeads M-450 CD34.

The quantity of the isolated CD34+ cells was assessed by placing the sample on an automated cell counter. The quality of the isolated CD34+ cells was determined using the trypan blue dye exclusion test, in which the viable cells were not stained [16].

After the preparation of UCB stem cells, a dose of 2×106 UCB stem cells/rat was injected intravenously into the rat-tail vein.

The following quantifications were carried out:

(1) The percentages of degenerated cells [cloudy swelling (swollen cells) and hydropic degeneration (vacuolated cytoplasm) both without nuclear changes] and necrotic (pyknotic or karyolytic nuclei or nuclei with chromatin margination) cells, in each group, were calculated. All cells from each of the proximal convoluted tubule (PCT), distal convoluted tubule (DCT) and collecting tubules (CT) in five nonoverlapping fields from five different sections of five different rats, in each group, were counted at ×400.

(2) Measurements were carried out using the image analyzer (Leica Imaging System, Leica Microsystems GmbH, Wetzlar, Germany) at the Histology Department, Faculty of Medicine, Cairo University, to measure: (a) colour area percentage of PAS-positive material in PAS-stained sections and (b) colour area percentage of collagen fibres (green coloured) in Masson's trichrome-stained sections. Ten images were captured per animal.

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Statistical analysis

The chi-square test or Fisher's exact test and Student's t-test were used as appropriate to assess differences between the groups. All values were presented as mean±standard deviation. Significance was set at a P value of less than 0.05 for all comparisons. All statistical analyses were carried out using statistical package for social sciences (SPSS 9) (Chicago, Illinois, USA) statistical analysis software.

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Results

Biochemical results

As illustrated in Table 1, results of this study showed that adriamycin injection (in group II), compared with the control group, resulted in a significant increase in the mean values of serum urea, serum creatinine and urinary proteins; however, there was a significant decrease in the mean values of both creatinine clearance and serum albumin. All previous results were significantly reversed in the stem cell-treated group (group III).

Table 1
Table 1
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Histological results
Control group (group I)

Examination of haematoxylene and eosin-stained sections from the control kidney showed a normal architecture in the cortex and medulla, with the normal appearance of the renal corpuscles and tubules (Fig. 1). The majority of cells of the proximal and distal convoluted tubules and the collecting tubules were normal; however, a small percentage showed cloudy swelling, hydropic degeneration and necrosis (Table 2).

Table 2
Table 2
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Figure 1
Figure 1
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PAS-stained sections showed PAS-positive material in the basement membranes and in the brush border of the PCT (Fig. 2). Sections stained with Masson's trichrome technique showed scanty greenish-coloured collagen fibres in the interstitium and the mesangium (Fig. 3).

Figure 2
Figure 2
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Figure 3
Figure 3
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TEM examination showed that cells of the PCT showed a spherical euchromatic nucleus, a large number of mitochondria, small number of lysosomes, peroxisomes and abundant microvilli forming the brush border below which there were small vesicles and larger vacuoles (Fig. 4). The renal corpuscles were consisted of Bowman's capsules (which had an outer parietal layer and an inner visceral layer) and glomeruli separated by the capsular space. The outer parietal layer of Bowman's capsules was consisted of flat cells (simple squamous epithelium) lining the capsular space and resting on the capsular basement membrane. The glomerulus showed capillaries and podocytes in addition to the mesangial cells. The glomerular capillary wall was consisted of fenestrated endothelium and continuous, regular and thin basement membrane. Their lumina contained red blood cells. The podocytes closely enveloped the glomerular capillaries and developed cytoplasmic extensions (foot processes, both major and minor), which were planted on the glomerular capillary basement membrane and separated by filtration slits (Fig. 5).

Figure 4
Figure 4
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Figure 5
Figure 5
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Nephropathy group (group II)

Haematoxylene and eosin-stained sections showed acute focal tubular necrosis, whereas the renal corpuscles showed varying degrees of degeneration (Fig. 6). Some of the tubules showed necrosis, in which their cells had a vacuolated cytoplasm and pyknotic or karyolytic nuclei. Other cells showed hydropic degeneration with vacuolation of their cytoplasm. The lumen of some tubules showed hyaline casts. The majority of cells of the PCT (43.37±3.04%) showed hydropic degeneration, whereas 17.66±2.64% showed necrosis and 10.07±0.97% showed cloudy swelling. The percentage of each type of affection was significantly increased, compared with the control group (Table 2). Hydropic degeneration affected 60.54±5.09% of the DCT, whereas 9.45±1.59% showed necrosis and 0.52±0.19% showed cloudy swelling. The first two parameters were significantly increased, compared with the control group, whereas the third was insignificant (Table 2). The computed tomographic scanning also showed hydropic degeneration (29.27±3.25%), necrosis (38.11±4.64%) and cloudy swelling (0.86±0.38%). These were all significantly increased, compared with the control group (Table 2). Most of the renal corpuscles showed dilated glomerular capillaries and some showed parietal cells with large rounded nuclei. Mononuclear cellular infiltration and peritubular capillary dilatation were observed in the areas of focal necrosis (Fig. 6).

Figure 6
Figure 6
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PAS-stained sections, from this group, showed thickened basement membranes of Bowman's capsule, glomerular capillaries and renal tubules (Fig. 7). There was also a loss in the PAS-stained brush border in some of the PCT. Hyaline casts also appeared positively stained. The mean colour area percentage of the PAS-positive material was 5.59+0.77, which was significantly increased compared with the control group (Table 2). In Masson's trichrome-stained sections, an increase in the green-stained collagen fibres was observed in the interstitium, in the areas of focal tubular necrosis (Fig. 8). The mean colour area percentage of collagen was 6.87+0.96, which was significantly increased compared with the control group (Table 2).

Figure 7
Figure 7
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Figure 8
Figure 8
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TEM examination showed that both the PCT and the renal corpuscles were affected. The PCT showed small hyperchromatic nuclei, hydropic degeneration of the cytoplasm, degenerated mitochondria with disrupted cristae and thickened basement membranes (Figs 9 and 10). The renal corpuscles showed parietal cells with rounded nuclei and small vacuoles. They were resting on an irregular basement membrane below which collagen of the interstitium appeared. The podocytes showed hyperchromatic nuclei with effacement of their foot processes. The glomerular capillaries showed focal thickening and splitting of the basement membranes with small subendothelial electron-dense deposits (Figs 11 and 12).

Figure 9
Figure 9
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Figure 10
Figure 10
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Figure 11
Figure 11
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Figure 12
Figure 12
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Stem cell-treated group (groupIII)

Haematoxylene and eosin-stained sections, from this group, showed a picture nearly similar to the control group kidney (Fig. 13). The majority (91.05±1.29%) of cells of the PCT were normal; however, a small percentage showed hydropic degeneration, cloudy swelling or necrosis. The percentage of each type of effect was significantly decreased, compared with the nephropathy group (Table 2). Cells of the DCT were almost (84.48±2.83%) normal; however, a small percentage was affected and was significantly decreased compared with the nephropathy group (Table 2). Collecting tubules also showed improvement, and the percentages showed a significant decrease, compared with the nephropathy group (Table 2).

Figure 13
Figure 13
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PAS-stained sections, from this group, showed a more or less normal appearance of the basement membranes and the brush border of the PCT (Fig. 14). The mean colour area percentage of the PAS-positive material was 2.25±0.57, which was significantly decreased compared with the nephropathy group (Table 2). Sections stained with Masson's trichrome technique showed scanty greenish-coloured collagen fibres in the interstitium, similar to the control group (Fig. 15). The mean colour area percentage of collagen was 1.1±0.42, which was significantly decreased compared with the nephropathy group (Table 2).

Figure 14
Figure 14
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Figure 15
Figure 15
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Examination with TEM showed a nearly normal appearance of cells of the PCT and the renal corpuscles (Figs 16 and 17). The PCT showed rounded, euchromatic nuclei, several mitochondria, few lysosomes and basal infoldings (Fig. 16). The renal corpuscles also showed a more or less normal appearance of the glomerulus, especially the glomerular capillary basement membranes and the podocyte foot processes (Fig. 17).

Figure 16
Figure 16
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Figure 17
Figure 17
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Discussion

Chronic renal failure is a major medical, social and economic problem for patients and their families. The availability and quality of dialysis programmes largely depend on the prevailing economic conditions [17]. Alternate methods have led to rapid progression of new approaches such as therapeutic cloning and stem cell therapy [2].

Recent studies have demonstrated that stem cells have greater plasticity and can differentiate down multiple cell lineages in rodents [18]. Therefore, this study aimed to determine the effect of hematopoietic stem cell transplantation on renal functional and structural changes in nephropathic rats.

In this study, a single intravenous injection of adriamycin to rats at a dose of 5 mg/kg body weight caused nephropathy. Biochemical results showed a significant increase in serum urea, creatinine and 24-h urinary proteins. There was also a significant decrease in creatinine clearance and serum albumin, compared with the control group. These results were confirmed by the histological analysis, which revealed acute focal tubular necrosis and changes in the renal corpuscles in the form of thickened membranes, changes in the shape of parietal cells in addition to the interstitial mononuclear cellular infiltration and capillary dilation. Furthermore, TEM examination showed degenerative or necrotic changes in the PCT and focal thickening of the glomerular capillary basement membrane and effacement of the podocyte foot processes, which corresponds to membranous nephritis (which is characterized by massive proteinuria) [19] in addition to acute focal tubular necrosis. These results were in agreement with those of other studies [9,20] in which researchers reported that adriamycin caused a progressive reduction in the glomerular filtration rate as measured by increased serum urea and creatinine and decreased creatinine clearance in addition to massive proteinurea accompanied by hypoalbuminemia. These biochemical abnormalities appeared within 5–7 days after the injection of adriamycin and reached maximum levels by 4–5 weeks. They added that rats exhibited segmental glomerulosclerosis by week 4 and progressive global sclerosis and interstitial fibrosis by week 6.

Several mechanisms for adriamycin nephrotoxicity have been postulated: formation of free radicals, disturbed mitochondrial metabolism and a direct toxic effect on renal tissue; however, the mechanism of formation of free radicals was the most accepted one [9,21]. Adriamycin is a potential source of free radicals. The formation of these radicals is considered to be the rate-limiting step in lipid peroxidation [22]. Some researchers [23] reported that oxidation of lipids generates lipid radicals that can, in turn, initiate and self-sustain lipid oxidation. Thus, cell membranes and basement membranes that depend on the integration of nonoxidized lipids, to maintain their orderly architecture, may be deranged, a process that could be important in the induction of glomerular proteinuria because it affects the capillary basement membrane, the main factor in glomerular filtration barrier. Podocyte injury also contributes to this proteinuria [19]. The generation of reactive oxygen species also leads to mitochondrial DNA damage, with subsequent respiratory chain dysfunction [24]. Thus, the delicate balance between antioxidant defenses and the production of reactive oxygen species may be disrupted, leading to oxidative insult that causes tissue damage and eventually cell death [25]. Oxidative stress, produced by adriamycin, could also induce apoptosis, which may lead to a certain signalling pathway resulting in the development of nephropathy [26].

The direct acute cytotoxicity of adriamycin is thought to be secondary to DNA intercalation, cross-linking or binding, free radical generation with subsequent induction of DNA damage and cell death by means of necrosis or apoptosis [27]. This acute cytotoxicity affects the tubular epithelial cells, which are particularly vulnerable to toxic injuries [19]; therefore, they showed structural alterations, in response to adriamycin injection, in the form of degenerative or necrotic changes in this study. These structural alterations led to functional alterations with a subsequent significant increase in urinary proteins and hypoalbuminemia because of defective reabsorption of amino acids and protein molecules that pass through the filtration barrier (as a result of a change in glomerular basement membrane permeability) by the PCT. The direct effect of adriamycin also led to tubulointerstitial nephritis, which was manifested by inflammatory mononuclear cell infiltration, attributed by some researchers [28] to chemokine release by injured kidney cells, with subsequent interstitial fibrosis as was evident by a significant increase in area percentage of collagen, in the nephropathy group in this study, compared with the control group. The apparent change in the shape of parietal cells of Bowman's capsule could also be a sequel of nephritis. The appearance of hyaline casts, a characteristic finding in acute tubular necrosis, in this study, was reported by other researchers [29] to be a result of leakage and precipitation of proteins because the membranes were affected.

In this study, the administration of hematopoietic stem cells to nephropathic rats (in the stem cell-treated group) led to a significant biochemical and histological improvement of the kidneys. These results were in agreement with those of other researchers [30–32], who reported significantly better renal function, lower renal injury and apoptotic scores after stem cell transplantation in different models of experimental nephropathy such as ischaemic acute renal failure and experimental glomerulonephritis.

The different mechanisms by which stem cell transplantation results in tissue repair have been discussed. One of the studies [32] found that stem cells secrete high concentrations of growth factors such as the vascular endothelial growth factor and others which by a paracrine mechanism results in an improvement of renal functions mediated by their antiapoptotic, mitogenic and angiogenic [31] effects. A study by previous researchers [33] concluded that the paracrine action results in renal downregulation of proinflammatory cytokines and upregulation of anti-inflammatory ones, leading to tissue repair and preservation.

Another mechanism reported was the transdifferentiation of bone marrow stem cells and contribution toward the repair of ischaemically injured renal tubules [34]. Other researchers [12,35] disagreed with the transdifferentiation mechanism. The first group of researchers [12] failed to find any evidence of transdifferentiation of stem cells in glomerular, tubular or even renal interstitial cells. Therefore, the reduction of tubulointerstitial fibrosis that was observed in their study, and in this one, was a secondary effect related to better preservation of glomeruli. The other group of researchers [35] reported that the human CD45 antibody represents a universal marker for hematopoietic differentiation; therefore, it is useful in monitoring CD34+ cell engraftment. However, they only identified human CD45-expressing cells in the interstitium and perivascular spaces, in close proximity to tubules and glomeruli but clearly distinguishable from the parenchymal structures. Several explanations related to differences in the protocols for stem cell isolation, transplantation and detection might account for this discrepancy.

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Conclusion

In conclusion, haematopoietic stem cell transplantation improved both the histological structures and the renal functions in nephropathy rat models. Therefore, it is a promising therapy in future clinical applications.

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References

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Keywords:

adriamycin; kidney and rats; stem cells

© 2011 The Egyptian Journal of Histology

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