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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 7  |  Issue : 4  |  Page : 153-164

Effect of suramin on renal proximal tubular cells damage induced by cisplatin in rats (histological and immunohistochemical study)


Department of Histology, Faculty of Medicine, Tanta University, Tanta, Egypt; Department of Anatomy, College of Medicine, Imam Mohammad Ibn Saud Islamic University, Riyadh, Saudi Arabia

Date of Submission20-Apr-2019
Date of Decision16-May-2019
Date of Acceptance22-May-2019
Date of Web Publication18-Nov-2019

Correspondence Address:
Dr. Eman Ali El-Kordy
Department of Histology, Faculty of Medicine, Tanta University, Tanta, Egypt

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JMAU.JMAU_21_19

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  Abstract 

Background: Renal toxicity is the most common complication of cispaltin therapy that has broad-spectrum antitumor activity against a variety of human solid tumor. Suramin, a Food and Drug Administration-approved old drug is a polysulfonated compound of napthylurea originally designed to treat trypanosomiasis. Aim: The current work aimed to investigate the possible protective effect of different doses of suramin against cisplatin-induced renal proximal tubular cells (RPTCs) damage. Material and Methods: Fifty adult male rats were used and divided into five equal groups. Group I served as a control, group II received suramin alone (10 mg/kg). Groups III, IV and V were administered cisplatin once (5 mg/kg, intraperitoneally) alone or combined with low dosage suramin (5 mg/kg) or high dosage suramin (10 mg/kg) once intravenously respectively. Results: Compared with control rats, cisplatin administration caused proximal tubules damage, RPTCs vacuolation with pyknotic nuclei, loss of brush border and widespread caspase-3 immunostaining. Cisplatin-induced RPTCs toxicity was further confirmed morphometrically (a significantly decreased proximal tubular epithelium height and increased mean number of caspase-3-immunopositive cells). These changes were accompanied by biochemical alteration manifested as a significant increase of blood urea nitrogen and serum creatinine. Simultaneous administration of high-dose but not low-dose suramin to the cisplatin-treated rats improved the deleterious morphological and morphometrical effects on RPTCs and restored the aforementioned biochemical parameters to control values. Conclusion: In conclusion suramin in a dose dependant manner protects RPTCs from damage induced by cisplatin.

Keywords: Cisplatin renal toxicity, histopathology, proximal tubules, suramin


How to cite this article:
El-Kordy EA. Effect of suramin on renal proximal tubular cells damage induced by cisplatin in rats (histological and immunohistochemical study). J Microsc Ultrastruct 2019;7:153-64

How to cite this URL:
El-Kordy EA. Effect of suramin on renal proximal tubular cells damage induced by cisplatin in rats (histological and immunohistochemical study). J Microsc Ultrastruct [serial online] 2019 [cited 2019 Dec 6];7:153-64. Available from: http://www.jmau.org/text.asp?2019/7/4/153/267747


  Introduction Top


Cancer continues to be the world's largest cause of death. In 2018, the World Health Organization reported that cancer was the second leading cause of death with an estimated 9.6 million deaths worldwide.[1] Cisplatin (cis-diammine-dichloro-platinum) is one of the most effective chemotherapy agents that has broad-spectrum antitumor activity against a variety of human solid tumors.[2],[3],[4] Renal toxicity is the most common complication of cispaltin therapy that limits its clinical uses accounting for about 30% of hospitalized patients.[5],[6],[7] Cisplatin is metabolized in renal proximal tubular cells (RPTCs) to its nephrotoxic metabolite which develops mainly in the S3 segment 48–72 h after its administration.[8],[9],[10]

The exact mechanism of cisplatin-induced RPTCs damage has not been fully elucidated. However, several biological responses, including mitochondrial dysfunction, activation of cell death pathways, generation of reactive oxygen metabolites (ROM) and a robust inflammatory response resulting in RPTCs death by apoptosis and necrosis have been involved in playing an important roles in cisplatin-induced renal toxicity.[10],[11]

Suramin, a Food and Drug Administration-approved drug is a polysulfonated compound of napthylurea originally designed in 1920 to treat trypanosomiasis as well as infections of Onchocerca volvulus.[12] Research interest in this old drug has turned in recent years to a new use. Several studies have recently shown that suramin protects animal models against muscle and liver fibrosis as well as reduces brain and kidney injury caused by ischemia reperfusion in rodents.[13],[14] In addition, delayed suramin administration was also effective in mitigating renal fibrosis progression in obstructive nephropathy and in attenuating liver injury following exposure to D-galactosamine and lipopolysaccharide in mice.[12],[13],[14],[15] The mechanisms by which suramin protects these organs from injury are linked to apoptosis inhibition, inactivation of nuclear factor-kB and reduction of toxic proinflammatory cytokine formation as tumor necrotic factor α (TNF-α).[14],[16]

However, the studies of suramin effect as renoprotective agent on cisplatin treated animals induced RPTCs damage are very limited. Based on these, we examined in the current study the possible protective effect of different doses of suramin on histological, immunohistochemical and morphometrical RPTCs changes of cisplatin treated rats and correlated these effects with blood urea nitrogen and serum creatinine levels.


  Materials and Methods Top


Animals

The experiment was conducted on 50 adult male albino rats, 8 weeks of age (180–200 g) that were housed in temperature and humidity controlled conditions under a light/dark photocycle with food and water supplied ad libitum. Ethical animal treatment protocols were followed, Tanta Faculty of Medicine.

Chemicals

Cisplatin was obtained as a vial of 10 mg/10 ml saline (Unistin; EIMC United Pharmaceuticals, Badr city, Cairo, ARE). Suramin was purchased from Sigma Aldrich (St. Louis, MO), each 100 mg was dissolved in 10 ml saline (10 mg/ml). All other chemicals used in the experiment were of analytical grade. Suramin was administered by tail vein injection while cisplatin intraperitoneally.

Experiment design

After 2 weeks of acclimatization, rats were classified into 5 groups, 10 rats each.

  • Group I (control group): Was subdivided into two subgroups, five animals were left untreated, whereas the others were given a single intravenous injection (tail vein) of 1 ml saline
  • Group II (suramin group): Were administered a single dose of suramin (10 mg/kg)[17]
  • Group III (cisplatin treated group): Were injected with a single dose of cisplatin (5 mg/kg)[18],[19]
  • Group IV (low dosage suramin treated group) and Group V (high dosage suramin treated group): Rats were injected with cisplatin as in group III and were concomitantly given a single suramin dosage (5 mg/kg) or (10 mg/kg) respectively 2 h after cisplatin injection.


All rats were sacrificed under ether inhalation after 3 days from the beginning of the experiment, Histological, immunohistochemical, morphometrical and biochemical studies were conducted.

Histological and immunohistochemical study

Fresh small pieces from the right renal cortex were taken, fixed in 10% buffered formalin and processed for examination by light microscopy to gain (5–7 im) sections of paraffin to be stained with:[20],[21],[22]

  1. Hematoxylin and eosin (H and E)
  2. Periodic acid Schiff method (PAS)
  3. Immunohistochemical staining using avidinngstoche peroxidase system to detect caspase-3. For detection of apoptosis, paraffin sections of 5 μm thickness had been stained with modified avidineds, par peroxidase for caspase-3. The primary antibodies used were monoclonal anticaspase-3 mouse (Thermo Fisher Scientific Inc., Rockford, Illinois, USA) at 1:100 dilution, which incubated at room temperature with slides for 1 h. Then, the sections were counterstained with Meyerer hematoxylin. Caspase-3 marker shows brown cellular cytoplasmic localization in early apoptosis and brown nuclear localization in late apoptosis.


For transmission electron microscope (TEM), thin slices of the left renal cortex were immediately fixed in 2.5% phosphate-buffered glutaraldehyde (pH 7.4), post fixed in 1% cold osmium tetroxide for 2 h in the same buffer at 4°C, dehydrated and embedded in epoxy resin. Sections of ultrathin (50–80 nm) were stained with uranyl acetate and lead citrate[23] for examination by (JEM-1010) transmission electron microscopy (TEM; Jeol, Tokyo, Japan) in Tanta University Electron Microscopy Unit.

Morphometrical study

The image analyzer computer system Leica Qwin 500 (England) in the Central laboratory, Faculty of Medicine, Tanta University was used to evaluate:

  1. Proximal tubular epithelial height and proximal tubular diameter in hematoxylin and eosin-stained sections (×400)
  2. Number of caspase-3 immunopositive cells per low power field.


The data were then collected in an Excel sheet and submitted to statistical analysis.

Biochemical study

At time of sacrifice, venous blood samples from all animals were obtained from the retro-orbital sinus, collected in heparinized tubes and centrifuged at 3000 rpm for 15 min. The serum was stored at-20°C until it was assayed for following biochemical parameters:[24]

  1. Blood urea nitrogen (BUN)
  2. Serum creatinine (SCr)


All performed procedures were strictly in accordance with a commercially available spectrophotometric enzymatic kit (Thermo Trace BECGMAN, Germany).

Statistical analysis

Statistical analysis were performed using SPSS program, version 17 (IBM Corporation, Somers, New York, USA) in the form of mean (X) ± standard deviation. Differences between the groupsn means were assessed using a one-way analysis of variance. P <0.05 was considered to be statistically significant.


  Results Top


Histological and immunohistochemical results

Control and suramin groups (Groups I and II)

Renal cortex sections of both control subgroups and suramin group showed the same histological structure. Sections stained with H and E showed renal corpuscles, proximal convoluted tubules (PCTs) and distal convoluted tubules. The PCTs constituted the main bulk of the cortex, their cells (RPTCs) had narrow lumina and cuboidal acidophilic cells containing rounded vesicular nuclei with prominent nucleoli [Figure 1]a and b].
Figure 1: (a and b) Proximal convoluted tubules (p) have narrow lumina, acidophilic cuboidal cells containing round vesicular nuclei with prominent nucleoli (arrows). G, a renal corpuscle containing glomerulus and D, a distal convoluted tubule. Renal cortex of control rat H and E, (a) scale bar 100 μm and (b) scale bar 50 μm

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Regarding PAS reaction, renal cortex sections revealed a PAS-positive reaction along the prominent apical brush borders of the RPTCs as well as at their basement membrane [Figure 2].
Figure 2: Proximal convoluted tubules (p) with PAS-positive reaction along apical brush border (bb) of RPTCs and continuous clear basement membrane (arrow). Renal cortex of control rat, PAS, scale bar 50 μm

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Caspase-3 immunostained sections, demonstrated negative staining of the RPTCs [Figure 3].
Figure 3: Proximal convoluted tubules (p) with negative immunoreactivity for caspase-3 of RPTCs. Renal cortex of control rat Caspase-3 immunostaining ×400

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Ultrastructurally, RPTCs showed profuse long closely packed apical microvilli with multiple vesicles immediately beneath it. Their cytoplasm had numerous elongated moderately dense mitochondria with lamellar cristae arranged perpendicular on the trilamellar basal lamina and the nuclei were large, basal and euchromatic. The RPTCs were observed to be joined by junctional complexes along the apical part of the lateral cell boundaries together with narrow intercellular space [Figure 4]a, [Figure 4]b, [Figure 4]c.
Figure 4: (a-c) Renal proximal tubular cells show profuse long apical microvilli (MV), elongated abundant mitochondria (m) arranged perpendicular on trilamillar basal lamina (arrow), multiple vesicles (v) and euchromatic nuclei (n). Note intercellular space (dotted arrow) and junctional complexes (j). Renal cortex of control rat TEM, (a) ×1000, (b) ×1500 and (c) ×2500

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Cisplatin treated group (group III)

H and E-stained renal cortex sections of cisplatin treated rats showed obvious histological changes affecting most of the PCTs that appeared destroyed with areas of disorganization and loss of cellular architecture. RPTCs exhibited vacuolated cytoplasm with pyknotic densely stained nuclei and others appeared separated from their basement membrane. Some tubules appeared with widened irregular lumina comprising dense acidophilic hyaline casts as well as shedding and desquamation of lining epithelium. Interstitial mononuclear cellular infiltration as well as interstitial hemorrhage were also observed [Figure 5]a, [Figure 5]b and [Figure 6].
Figure 5: (a and b) Marked destruction and disorganization of most PCTs with loss of normal architecture (stars), proximal tubular cells with vacuolated cytoplasm (v) and small pyknotic nuclei (dotted arrow). Note RPTCs separation from basement membrane (arrow) and interstitial hemorrhage (h). Renal cortex of cisplatin treated rat H and E, (a) scale bar 100 μm and (b) scale bar 50 μm

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Figure 6: Marked degenerative changes of PCTs, some exhibit irregular dilated lumina (stars) comprising dense acidophilic hyaline casts (hy), RPTCs with vacuolated cytoplasm (v) and small pyknotic nuclei (dotted arrows), shaded epithelium (arrow head) and interstitial mononuclear cellular infiltration (IM). Renal cortex of cisplatin treated rat H and E, scale bar 50 μm

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PAS-stained sections revealed RPTCs with an apparent weak disrupted reaction at their brush border and prominent positive reaction of the basement membrane that appeared thickened in many area and interrupted at other sites. In addition, some PCTs appeared with evident complete loss PAS reaction at the brush border and others with positively stained hyaline casts in the lumina surrounded by strong positive PAS membrane [Figure 7]a and [Figure 7]b.
Figure 7: (a and b) RPTCs with an apparent weak disrupted PAS- positive reaction brush border (bb), strong positive reaction of thickening basement membrane (arrows) with interrupted one in many areas (double arrows). Note some PCTs with evident complete loss PAS reaction at the brush border (stars) and positively stained hyaline casts in tubular lumina (hy). Renal cortex of cisplatin treated rat PAS, (a and b) scale bar 50 μm

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Regarding caspase-3 immunostained sections, there was widespread brown staining of most RPTCs with intensive expression in exfoliated cells [Figure 8].
Figure 8: Widespread brown immunoreactivity for caspase-3 of most proximal tubular cells (arrows) with intensive expression in exfoliated cells (dotted arrow). Renal cortex of cisplatin treated rat Caspase-3 immunostaining ×400

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The electron micrographs of renal cortex of cisplatin treated rats showed remarkable changes in the proximal tubular cells, most of the cells exhibited marked vacuolated cytoplasm contained vacuolated mitochondria with destroyed cristae and multiple heterogeneous vacuoles studded with electron dense particles [Figure 9]a, [Figure 9]b, [Figure 9]c and [Figure 10]. Other RPTCs exhibited marked loss of their mitochondria in rarefied cytoplasm leaving empty spaces and the remaining mitochondria were small in size with electron dense matrix [Figure 11]. Their nuclei appeared dark shrunken heterochromatic with irregular nuclear envelope and dilated perinuclear space, Moreover apical displacement of the nuclei was observed [Figure 10] and [Figure 11]. Some RPTCs appeared with interrupted basal lamina, meanwhile others showed focal thickening and splitting of these lamina [Figure 9]a, [Figure 9]b, [Figure 9]c and [Figure 10]. In addition, widening of intercellular spaces as well as distortion and focal loss of microvilli were observed [Figures 10 and [Figure 11].
Figure 9: (a-c) Proximal tubular cells show vacuolated cytoplasm (v) contains vacuolated mitochondria with destructed cristae (m) and interrupted basal lamina (double arrows) meanwhile its focal thickening in other sites (arrow). Stars, rarified cytoplasm with remnants of cytoplasmic components and N, nucleus. Renal cortex of cisplatin treated rat TEM, (a) ×1000, (b) ×1500 and (c) ×2500

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Figure 10: Proximal tubular cells show heterogenous irregular multiple cytoplasmic vacuoles (V), apical displacement of small heterchromatic nucleus (N), focal splitting of basal lamina (double arrows) and dilated intercellular space (arrow). MV, microvilli. Renal cortex of cisplatin treated rat TEM, ×2500

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Figure 11: Proximal tubular cells show rarefied cytoplasm (CY) contains small sized disorganized electron dense mitochondria (M) and small irregular heterchromatic nucleus (N) with dilated perinuclear space (arrow). Note a few distorted microvilli (MV). Renal cortex of cisplatin treated rat TEM, ×2500

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Low dosage suramin treated group (group IV)

By H and E stain, renal cortex sections of low dosage suramin treated rats showed the similar changes as those in group III. The PCTs were still affected with dilated lumina. Most RPTCs appeared with marked cytoplasmic vacuolation and small pyknoic nuclei. Shedding epithelium and separated cells from their basement membrane were also observed [Figure 12]a and [Figure 12]b.
Figure 12: (a and b) Degenerated PCTs with dilated lumina (stars), RPTCs with vacuolated cytoplasm (v), small pyknoic nuclei (dotted arrows), shedding epithelium (arrow head) and others separated from their basement membrane (arrow). Renal cortex of low dosage suramin treated rat H and E, (a) scale bar 100 μm and (b) scale bar 50 μm

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Regardless, few RPTCs displayed normal PAS-positive reaction along the apical brush border, most cells had still an apparent weak interrupted PAS-positive brush border with strong positive reaction of the basement membrane [Figure 13]a and [Figure 13]b.
Figure 13: (a and b) Most RPTCs have an apparent weak interrupted PAS- positive brush border (bb) and strong positive reaction of the basement membrane (arrows). Note a few PCTs with more or less normal intact brush border of their cells (stars). Dotted arrows, RPTCs with detached basement membrane. Renal cortex of low dosage suramin treated rat PAS, (a and b) scale bar 50 μm

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Immunohistochemical reactions revealed marked brown immunoreaction for caspase-3 of most RPTCs and only a few one displayed weak reaction [Figure 14].
Figure 14: Most RPTCs have brown nuclear immunoreaction for caspase-3 (arrows) and a few one displayed weak reaction (dotted arrows). Renal cortex of low dosage suramin treated rat Caspase-3 immunostaining ×400

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The electron micrographs confirmed the results observed by light microscope as no obvious improvement in the RPTCs. Most cells were still affected and appeared with electron dense irregular arrangement different shaped mitochondria in disrupted cytoplasm, small dark heterochromatic nuclei and widening intercellular spaces as well as focal loss of microvilli [Figure 15]. Moreover, multiple lysosomes and vacuoles in the cytoplasm of some cells were seen [Figure 16]. Few RPTCs appeared with normal euchromatic nuclei [Figure 17].
Figure 15: Proximal tubular cells show electron dense different sized and shaped disoriented mitochondria (M), a shrunken heterchromatic nucleus (N) and disrupted focal loss microvilli (MV). Note dilated intercellular spaces (arrow). Renal cortex of low dosage suramin treated rat TEM, ×2500

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Figure 16: Proximal tubular cells with multiple lysosomes (Ly) and vacuoles (V). Note elongated mitochondria (M) and microvilli (MV). Renal cortex of low dosage suramin treated rat TEM, ×2000

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Figure 17: Proximal tubular cells with normal euchromatic nuclei (N) and electron dense mitochondria (M). MV, microvilli. Renal cortex of low dosage suramin treated rat TEM, ×2500

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High dosage suramin treated group (group V)

H and E stained renal cortex sections of high dosage suramin treated rats, showed partial improvement in the histological structure of the PCTs which appeared more or less normal. Most RPTCs appeared with acidophilic cytoplasm and rounded vesicular nuclei, while some one showed cytoplasmic vacuolation with pyknotic nuclei [Figure 18]a and [Figure 18]b.
Figure 18: (a and b) An apparent normal PCTs (P), have more or less normal cells with vesicular nuclei (arrows). Note a few RPTCs with vacuolated cytoplasm (v) and pyknotic nuclei (dotted arrow). H, interstitial hemorrhage. Renal cortex of high dosage suramin treated rat H and E, (a) scale bar 100 μm and (b) scale bar 50 μm

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PAS-stained sections revealed preserved intact brush border and continuous clear basement membrane of most RPTCs, while a few one appeared with weak interrupted brush border [Figure 19]a and [Figure 19]b.
Figure 19: (a and b) Most RPTCs appear with preserved intact brush border (bb) and continuous clear basement membrane (arrows). Note a few PCTs with weak interrupted brush border (stars). Renal cortex of high dosage suramin treated rat PAS (a and b) scale bar 50 μm

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Regarding the immunohistochemistry, most RPTCs were observed with weak immunoreactivity for caspase-3 in comparison to cisplatin treated rats. Otherwise, a few one appeared with brown reactions [Figure 20].
Figure 20: PCTs with an appearing weak immunoreactivity for caspase-3 of their cells (stars). Note a few tubules with brown immunoreactions (P). Renal cortex of high dosage suramin treated rat Caspase-3 immunostaining ×400

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Ultrastructurally, RPTCs showed large euchromatic nuclei and numerous intact elongated mitochondria perpendicular to trilamellar basal lamina with minimal vacuolation of the cytoplasm. Restoration of the long apical microvilli was observed as well as preserved junctional complexes with narrow intercellular space [Figure 21]a and [Figure 21]b. However, some cells were still affected and had irregular shaped electron dense mitochondria and dark shrunken heterochromatic nuclei [Figure 22].
Figure 21: (a and b) Proximal tubular cells show euchromatic nuclei (N) and numerous intact mitochondria (M) with minimal cytoplasmic vacuolation (star). Note microvilli (MV), trilamillar basal lamina (arrow) and preserved junctions (J) with narrow intercellular space (dotted arrow). Renal cortex of high dosage suramin treated rat TEM, (a) ×1500, (b) ×2500

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Figure 22: Proximal tubular cells with irregular shaped electron dense mitochondria and a small heterochromatic nucleus (N). MV, microvilli. Renal cortex of high dosage suramin treated rat TEM, ×2500

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Morphometric results

As shown in [Table 1] and [Graph 1], [Graph 2], cisplatin treated rats (group III) had a significantly decreased proximal tubular epithelium height and a significantly increased proximal tubular diameter as compared to control animals (P < 0.05). Treating rats with low dosage suramin (group IV) did not show significant changes in both parameters while high dosage suramin treated rats (group V) had a significantly increased proximal tubular epithelium height and a significantly decreased proximal tubular diameter (P < 0.05) as compared to cisplatin group. However, no significant change (P > 0.05) was observed in both aforementioned parameters in the suramin alone treatment group (group II) as compared to control one.

Table 1: Proximal tubular epithelium height and proximal tubular diameter (μm) in control and experimental groups

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The mean number of caspase-3-immunopositive cells for all groups is presented in [Table 2] and [Graph 3]. There was a significant increase (P < 0.05) in cisplatin group (group III) as compared to the control one that remained significantly high in low dosage treated rats (group IV). A statistically significant decrease (P < 0.05) in the number of caspase-3-immunopositive cells was observed in rats treated with high dosage suramin (group V) compared with group III. This parameter was nonsignificant different (P > 0.05) in suramin group (group II) in comparison to the control.

Table 2: Number of caspase-3-immunopositive cells (N/1000 μm2) in control and experimental groups

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Biochemical results

As shown in [Table 3] and [Graph 4] and [Graph 5], cisplatin treated rats (group III) had a high significantly increased levels of BUN and SCr as compared to control animals (P < 0.05). Treating rats with low dosage suramin (group IV) did not show a significant decrease in both measured parameters while high dosage suramin treated rats (group V) had a significantly decreased levels of BUN and SCr (P < 0.05) as compared to cisplatin group. However, no significant change (P > 0.05) was observed in both aforementioned parameters in the suramin alone treatment group (group II) as compared to control one.

Table 3: Blood urea nitrogen and serum creatinine levels (mg/dL) in control and experimental groups

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  Discussion Top


The kidneys are the major target for the toxic effects of various chemical agents owing to their high blood supply as they receive up to 25% of the cardiac output.[25] Drug nephrotoxicity was found to be responsible for 19% of acute kidney injury as reported in the epidemiological survey of critically ill patients.[26] Cisplatin nephrotoxicity developed primarily in the proximal tubules and its concentration in the RPTCs is 5 times higher than that of the serum, thus contributing to its toxicity.[27]

Researches on whether suramin has an effect on RPTCs after their damage by cisplatin are very limited and to our knowledge, no morphological and morphometrical study up till now has examined that effect. So, our goal was to determine whether suramin at different doses enhances morphological and morphometrical improvement of RPTCs after their damage by cisplatin.

In the present study, all results of suramin treated rats (group II) indicated its safety and nontoxicity as they were similar to the control one. Other researchers reported that the suramin at a dose of 0.1esent study, all results of suramin treated rats (group in mice after ischemia perfusion and that 10 mg/kg was not toxic.[14] Given the fact that concentration of suramin in the kidney is much higher than that of serum as it has a long half-life (21 days), so a one dose may achieve a therapeutic effect.[28]

Our study revealed PCTs of cisplatin treated rats (group III) had various degenerative changes including loss of the cellular architecture, tubular lumen dilatation, brush border damage and marked vacuolated cytoplasm with pyknotic nuclei of the RPTCs. Furthermore, TEM showed vacuolated destructed and small dense mitochondria as well as shrunken heterochromatic nuclei and interrupted basal lamina with its focal thickening. These results were confirmed morphometriclly that revealed a significant decrease of proximal tubular epithelium height associated with significantly increased tubular diameter compared with the control group. These results were consistent with those reported previously.[29],[30],[31],[32],[33],[34] Moreover other investigators stated that cisplatin causes renal tubular toxicity without evident morphologic changes in glomeruli.[9],[23]

Within the RPTCs, cisplatin is transformed into a highly reactive form that reacts rapidly with antioxidant molecules results in increased oxidative stress and ROM generation as well as lipid peroxidation production.[35] Consequently, the observed RPTCs desquamation into tubular lumen and tubular epithelium detachment may be related to damaged cell membranes as a result of production of ROM and lipid peroxidation. The intratubular hyaline casts might represent cellular debris combined with leakage precipitated proteins present in the tubular lumen.[33],[36]

Preservation of brush border is very important for the function and survival of RPTCs that have been lost partially or completely after cisplatin injection as was shown by PAS reaction and confirmed utrastructurally. Previous researchers contributed this finding and the observable interrupted basal laminae to ROM generation which led to rapid loss of cytoskeletal integrity.[37]

The observed mononuclealar cellular infiltration after cisplatin injection was in consistence with other investigators[10],[38],[39] who reported that cisplatin evoked an inflammatory response with cell infiltration of renal tissue mainly mast cells, T cells, macrophages and neutrophils. It is well established both RPTCs and leukocytes have multiple P2 receptors that is playing an important role in increasing an inflammatory response through the realization of danger signals.[40],[41] Furthermore, these receptors regulate the release of proinflammatory cytokines and chemokines molecules which attract and activate neutrophils to sites of damage, exacerbating the renal damage by potentiating an inflammatory response leading to the generation of ROM.[14],[42]

In cisplatin treated rats ROM production leads to respiratory chain dysfunction as a result of mitochondrial DNA damage. Consequently the imbalance between oxidation and antioxidation leading to RPTCs damage and death.[43],[44],[45] Our data strongly supported that as the cisplatin induced sever alterations in mitochondrial shape and structure and this was consistence with other investigators.[32],[46] Maintaining the orderly architecture of RPTCs and their basement membrane depends on the integration of nonoxidized lipids that have been lost after cisplatin treatment as a result of ROM formation with lipid peroxidation leading to lipid containing membranes dilation, suggesting cytoplasmic vacuolation and multiple heterogeneous vacuoles observed in this current study.

Furthermore, the results of previous study indicated that cytochrome P450 (CYP) 2E1 has an important role in cisplatin-induced RPTCs damage. This enzyme is localized to the RPTCs but not the glomeruli and is predominantly present in the endoplasmic reticulum. The interaction between cisplatin and CYP2E1 results in ROM generation and initiation of both RPTCs necrosis and apoptosis.[47]

There is growing evidence indicating RPTCs apoptosis increased the pathogenesis of cispaltin induced renal damage.[39],[48],[49] In the current study, the observed widespread positive caspase 3 immunoreactivity of cisplatin treated rats pointing to abundant apoptotic cells that was confirmed morphometrically as shown a significant increase in the number of caspase-3-positive cells. These results were in agreement with other study that reported involvement of cisplatin-induced tubular epithelial cells is in caspase-3 activation.[50]

Several apoptotic pathways are activated during cisplatin treatment including intrinsic and extrinsic one. The intrinsic pathway is associated with upregulation of mitochondrial permeability and caspase-3 activation by BAX as a result of activation of a tumor suppressor P53,[50],[51],[52] while in the extrinsic one TNF-αNor other death receptor ligands may activate death receptors on the proximal tubular cell surface resulting in caspase-8 activation followed by downstream effector caspases such as caspase-3.[53],[54]

In the current work the biochemical data of rats injected with cisplatin (group III) indicated impairment of renal function as there was a significance increase of both BUN and SCr. These data came hand by hand with histological and morphomertical results that could explain this disturbance. Impairment of the renal function is considered as a result of RPTCs damage as a consequence of ROM formation and it has been already reported by other studies.[34],[47],[55],[56] In addition decreased glomerular filtration rate even if there is no glomerular damage can cause BUN and SCr elevations with subsequently aggravation of tubular damage.[57]

Comparing with cisplatin group, not low dosage treated suramin rats (group IV), but high dosage treated suramin one (group V) showed amelioration of toxic effects of cisplatin on RPTCs. Low dosage suramin failed to improve the morphological changes of the RPTCs as well as the widespread positive caspase immunoreactivity induced by cispaltin while, high dosage suramin resulted in partial morphological RPTCs improvement associated with weak caspase immunoreactivity. Electron microscopic results supported these findings that revealed appearance of RPTCs with large euchromatic nuclei, numerous intact elongated mitochondria and restoration of most cells to their normal structures than those observed in low dosage suramin group.

Suramin acts on oxidative formation downstream and its effect as a pan P2 receptor antagonist attenuates expression of the proinflammatory factors as cytokines and consequently protects RPTCs from the inflammatory response and oxidative stress associated with cisplatin nephrotoxicity.[58],[59],[60] In addition, previous study found that inhibition of mast cell degranulation in mice after cisplatin treatment with subsequently decreasing the release of cytokine TNF-αNfrom RPTCs dampened the proinflammatory response and further attenuated its toxicity.[61]

Moreover, the observed morphometric results confirmed the histological one as Low dosage suramin showed no significant changes in both the proximal tubular epithelial height and tubular diameter as well as the caspase-3-immunopositive cells that remained significantly high compared with cisplatin group. In contrast high dosage suramin was able to significantly decrease the caspase-3-immunopositive cells and showed significant proximal tubular epithelial height increasing and tubular diameter decreasing.

Consistent with our results, previous researchers[14] found that suramin attenuated renal tubular damage and decreased the number of apoptotic cells of proximal tubules in ischemic reperfusion injured kidneys. The possibility of antiapotptotic effect of suramin may be through blocking death receptor activation on RPTCs as apoptosis is mediated by TNF-αNleading to reducing caspases activation, decreasing endoplasmic reticulum stress induced apoptosis and preventing mitochondrial membrane potential loss.[16] The observed intact mitochondria in group V with subsequently reducing caspases activation and cytochrome C release are important factors for RPTCs survival and function.

In the current work, the improvement of renal function by suramin occurred in a dose-dependent manner in concomitant with histological and morphometrical results. The biochemical data showed the rats treated with high dosage suramin but not a lower one improved the cisplatin-induced decreased in kidney function. There was a significance increased in both parameters (BUN and SCr) that indicated the morphological RPTCs improvement. It was found that suramin accelerates renal function recovery after acute kidney injury as it promotes RPTCs proliferationin vitro andin vivo and through interference of late-onset apoptosis.[14]

Interestingly, previous data revealed suramin induced renal growth in rats, predominantly in the RPTCs with no glomeruli alterations through Src-dependent activation of the PI3K/Akt pathway. These two important signaling molecules (Src and Akt) are associated with cell survival and proliferation, may mediate recovery of renal function through this mechanism.[59] Moreover, an increased number of intact mitochondria of RPTCs that were noted in group V may reflect compensatory adaptation to produce much energy to improve the renal function.


  Conclusion Top


The present study showed that simultaneous administration of suramin to the cisplatin-treated rats abrogates the toxic effect of cisplatin on RPTCs in a dosage-dependent manner. High dosage suramin has a potential renoprotective effect at both morphological and morphometrical levels as well as restores renal function to normal one.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3]



 

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