IJMCM Spring 2021, Vol 10, No 2 Original Article DOI: 10.22088/IJMCM.BUMS.10.2.133 The Role of Bone Marrow-Derived Mesenchymal Stromal Cells and Hesperidin in Ameliorating Nephrotoxicity Induced by Cisplatin in Male Wistar Rats Khalid Mohamed Mazher1, Osama Mohamed Ahmed2, Hadeer Abdallah Sayed3, Taghreed Mohamed Nabil1 1. Department of Cytology and Histology, Faculty of Veterinary Medicine, Beni-Suef University, 62511, BeniSuef, Egypt. 2. Physiology Division, Zoology Department, Faculty of Science, Beni-Suef University, 62521, Beni-Suef, Egypt. 3. Faculty of Veterinary Medicine, Beni-Suef University, 62511, Beni-Suef, Egypt. Submmited 24 March 2021; Accepted 11 July 2021; Published 1 September 2021 Bone marrow-derived mesenchymal stromal cells (BM-MSCs) and antioxidants opened the way for many effective therapeutic experiments against damaged organs like kidneys. Nephrotoxicity is the main complication of chemotherapeutic drugs. Therefore, the present study aimed to investigate the efficacy of BM-MSCs and hesperidin to treat cisplatin-induced nephrotoxicity in rats. Fifty rats were divided into five equal groups of 10 each. Group-I served as a control group, group-II received a single dose of cisplatin (7.5 mg/kg) intraperitoneally to induce nephrotoxicity, group-III received a daily dose of hesperidin (40 mg/kg) orally for four weeks, and on the 5th day cisplatin was administered an hour before hesperidin administration. Group-IV consisted of cisplatintreated rats that were intravenously injected with 1х106 BM-MSCs cells/rat once per week. Group V contained cisplatin-treated rats that received a combination of hesperidin and BM-MSCs with the same dosage regimes. After four weeks, serum and kidney samples were collected for biochemical, histological, and immunohistochemical examinations were performed. Cisplatin administered rats showed deteriorated biochemical parameters and severe degenerative changes in renal tissue. Both single and combined hesperidin and BM-MSCs treatments restored the renal biochemical parameters. Histologically, the renal tissues significantly improved in the BM-MSCs treated group in comparison with the hesperidin treated group. Moreover, combined treatment (i.e., group V) showed complete restoration of the normal architecture in the renal tissue. Our data suggest that the combined treatment of BM-MSCs and hesperidin has a potent renoprotective efficacy against cisplatin-induced nephrotoxicity rather than the single treatment. Key words: Bone marrow-derived mesenchymal stromal cells, cisplatin, hesperidin, immunohistochemistry, nephrotoxicity, p53, apoptosis  Corresponding author: Taghreed Mohamed Nabil: Department of Cytology and Histology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt. E-mail: tagh_mhm24@yahoo.com This work is published as an open access article distributed under the terms of the Creative Commons Attribution 4.0 License (http://creativecommons.org/licenses/by-nc/4). Non-commercial uses of the work are permitted, provided the original work is properly cited. Mazher KM et al. T he kidney is the primary organ of the body They can also regulate the immune response and performing maintain secrete potent paracrine factors that promote hemostasis, regulate blood pressure, participate to structural and functional renal recovery. BM-MSCs detoxification and waste excretion. Therefore, the are considered as free-radical scavenging cells that kidney is the main organ affected by toxic can reduce oxidative stress by releasing exosomes metabolites and excreted drugs, especially the to prevent ROS accumulation (9). vital chemotherapeutic functions agents to resulting in kidney Hesperidin, a natural flavonoid antioxidant, is found in citrus and has antibacterial, anticancer, damage or nephrotoxicity (1). Cisplatin or cis-diamminedichloroplatinum (II) anti-inflammatory, and anti-apoptotic properties is one the most effective chemotherapeutic drugs (10). Due to its antioxidant activity it can attenuate used to treat broad types of malignant tumors. oxidative stress by inhibiting free radical formation However, cisplatin clinical use is greatly limited (11). The hesperidin antioxidant activity was due to nephrotoxic complication (2). Cisplatin attributed to its hydrogen-donating properties, nephrotoxicity results from excessive reactive which neutralize hydroxyl and superoxide free oxygen species (ROS) production, which induces radicals. As a result, much interest has been drawn oxidative stress leading to renal cytotoxicity, to hesperidin as a renoprotective compound against inflammation, cisplatin-induced renal damage (12). and congestion (3). Toxic metabolites produced by cisplatin metabolism The current study aimed to assess the effects primarily damage renal tubular cells, resulting in of BM-MSCs and hesperidin treatment alone or as a DNA damage, mitochondrial dysfunction, and combination treatment to ameliorate cisplatin- apoptosis. induced (4). The cisplatin-induced renal dysfunction is manifested by a significant increase nephrotoxicity and to restore renal structure and function in Wistar rats. of renal biochemical parameters such as urea and creatinine, and disturbance of electrolyte levels (5). Under certain conditions, bone marrow- Materials and methods BM-MSCs isolation, preparation, and culturing derived mesenchymal stromal cells (BM-MSCs) The BM-MSCs were isolated as previously can self-renew and differentiate into various described (13, 14). Briefly, male albino Wistar rats functional cells. (6). The BM-MSCs' advantages of (4 weeks old, 60-80 g body weight) were used to plasticity, ease of isolation from bone marrow, and isolate BM-MSCs. Rats were euthanized by an multiplication into cell-derived colonies have overdose of inhaled isoflurane anesthetic (15). After opened a promising way of a novel therapeutic sterilizing the whole-body surface with 70% ethyl option of incurable and degenerative diseases (7). alcohol, the hind limbs (femur and tibia) were The therapeutic potential of BM-MSCs in acute dissected and cleaned thoroughly from the adherent renal failure is dependent on their ability to homing, tissues by using sterile instruments. The bones were engrafting, and differentiating the sites of damaged stored on iced Petri dishes containing Dulbecco's tissue. The homing occurs due to the interaction modified Eagle's medium (DMEM) (Life Science between the molecular signals released from the Production, UK) supplemented with 1% penicillin- injured receptors streptomycin mixture (Lonza, Belgium). BM-MSCs expressed on BM-MSCs (8). Thus, BM-MSCs isolation and culturing were done under proper accelerate the regeneration of the damaged tissue sterile conditions in a vertical laminar airflow by promoting angiogenesis, stimulating mitosis, cabinet (Biobase, China). After cutting the bones decreasing apoptosis, and reducing inflammation. just below the bone marrow cavity, the bone tissue as chemokines and Int J Mol Cell Med Spring 2021; Vol 10 No 2 134 Anti-nephrotoxic effect of mesenchymal stromal cells and hesperidin marrow cells were isolated by flushing with pellet was re-suspended in 1 ml DMEM, and the DMEM cell viability was tested again. supplemented with 1% penicillin- streptomycin mixture. The bone marrow plug was Morphological examination of BM-MSCs then collected in a sterile 15 ml Falcon tube and BM-MSCs were examined under the inverted centrifuged at 2000-3000 rpm for 3-5 min, and the microscope to identify their characteristic fusiform step was repeated twice till obtaining a complete shape and well-developed cytoplasmic processes clear supernatant. The sediments were suspended in (18). a complete DMEM medium supplemented with Gene expression analysis by real-time quanti- 15% fetal bovine serum (FBS) (Life Science tative PCR (RT-qPCR) Production, UK) (16). The viable cell counts were RNA was extracted from the cultured BM- determined by staining them with 0.4% trypan blue MSCs using NucleoSpin RNA nucleic acids solution and using a hemocytometer (17). The extraction kit (Macherey-Nagel, Germany). The 2 cDNAs were synthesized from the mRNA by cell culture flasks and incubated in a 5% CO2 reverse transcription according to the manufa- humidified incubator (Biobase, China) at 37 ˚C. cturer's instructions. Quantitative real-time PCR After 3-4 days incubation, the non-adherent cells and data analysis were conducted using Step One were removed by replacing the old complete media real-time PCR Systromal (Applied Biosystromals, with a sterile fresh one. USA) and ViPrimePLUS One Step Taq RT-qPCR cellular suspensions were cultured in sterile 25 cm th On the 8 day of incubation, the adherent cells Green Master Mix I with ROX (SYBR Green (80-90% confluence) were washed twice by a Dye) (no. QLMM14-R) (Vivantis Technologies, sterile phosphate buffer saline (PBS) (Lonza, Malaysia). Belgium) to remove the excess FBS and trypsinized Ten μL of the master mix was prepared in 1x with 1.5 mL 0.25% trypsin/ 1 mM EDTA (Lonza, solution, and was added to 20 μL final volume Belgium) at 37 ˚C for 2 min. The trypsin action was containing 1 μL primers, 5 μL template cDNA, and neutralized by adding 3-5 mL complete DMEM 4 μL nuclease free water. Primers sequences media to the suspension, followed by centrifugation specific for CD105, CD73, CD34, CD45, and the β- and the clear supernatant was discarded. The cell actin housekeeping gene were used (Table 1). Table 1. Primers sequence for RT-qPCR. Primer 5’ to 3’ Gene bank Forward GGCAGCTTCAACAACCATCA XM_032900290.1 Reverse GGATGGACTAGATCGGAGCC Forward TGTTGGGACCAGCAACTCAA Reverse TTTGAGGCTCAGTGGTAGCC Forward GCGAGGCTTTCAACACAACC Reverse ACTCCACTGTCTTGATTCCC Forward TGTGAACATACGGATTGTGA Reverse ACTTTAACTTAACAAACTGC Forward TGACAGGATGCAGAAGGAGA Reverse TAGAGCCACCAATCCACACA Gene CD105 CD73 CD34 CD45 β-actin 135 Int J Mol Cell Med Spring 2021; Vol 10 No 2 NM_021576.2 XM_032903804.1 AF251010.1 NM_031144.3 Mazher KM et al. The thermal cycling profile of RT-qPCR was group and received carboxymethyl cellulose (CMC) set as follows: an initial cycle at 55 °C for 10 min, (Sigma-Aldrich, USA) 1%,once daily via oral followed by a cycle for 8 min at 95 °C, then 40 gavage for 4 weeks, and a single 1 ml distilled cycles at 95 °C for 10 s and 60 °C for 60 s. The data water intraperitoneally (IP) on the 5th day of were expressed as cycle threshold (Ct). The relative treatment (19), group-II received a single dose of quantification of each target gene was quantified cisplatin (7.5 mg/kg) IP to induce nephrotoxicity, according to delta-delta Ct calculation (2 -ΔΔCt ) as group-III received a daily dose of hesperidin in following: CMC 1% (40 mg/kg) orally (20) for 4 weeks and on ΔΔCt = [(Ct target, sample)- (Ct ref, sample)]- the 5th day of administering hesperidin, cisplatin [(Ct target, control)- (Ct ref, control)] where: (7.5 mg/kg) was administered IP an hour before Ct target, control = Ct value of gene of interest in hesperidin administration. Group-IV (cisplatin- control DNA. treated and BM-MSCs treated group) received Ct ref, control = Ct value of reference gene in distilled water orally once a day for 4 weeks, a control DNA. single cisplatin dose (7.5 mg/kg) intravenously on Ct target, sample = Ct value of gene of interest in the 5th day, and then was injected with 1х106 BM- the tested sample. MSCs cells/rat (BM-MSCs with ≥ 95% cell Ct ref, sample = Ct value of reference gene in the viability were used) once per week starting from the tested sample. 6th day till 4 weeks (13). Group V (cisplatin treated Animals and Adult male albino Wistar rats hesperidin/BM-MSCs group) received (Rattus hesperidin and cisplatin as group III and were norvegicus) weighing 150-200 g were used. The concomitantly administered BM-MSCs as group IV rats were obtained from the Egyptian Company for the day after cisplatin injection. the Production of sera and vaccines (Vacsera, At the end of the experiment (i.e. 4 weeks), Egypt). Animals were raised at the Faculty of venous blood samples were collected from rats Veterinary Medicine, Beni-Suef University, Egypt, under isoflurane anesthesia for measuring the in well-ventilated standard plastic cages, and biochemical parameters. Kidney tissues were maintained under standard laboratory conditions of obtained from euthanized rats for histological and controlled immunohistochemical examination. temperature (24±1 °C), humidity (50±5%), and 12:12 h light: dark cycle throughout Determination of serum creatinine and urea the experiment. Food and water were provided ad levels libitum. Rats were kept for one week as an The serum creatinine and urea levels were adaptation period before starting the experiment. measured by the colorimetric method utilizing Every effort was made to minimize the pain and Diamond reagents Kits (Diamond Diagnostic animal suffering. All experimental procedures were Chemical Company, Egypt) (21). conducted under the guidelines of the Institutional Determination of serum sodium and potassium Animal Care and Use of Ethics Committee of levels Faculty of Veterinary Medicine, Beni-Suef Serum sodium and potassium levels were University, Egypt (Ethical Approval Number: determined BSU/IACUC /2020/108) reagents (Spectrum Diagnostics, Egypt) using the Experimental design colorimetric method (22). Fifty healthy rats were divided into five equal groups (10 rats/group). Group-I served as control by Spectrum-Diagnostics Sodium Histological and histochemical examination Collected kidneys were dissected, washed with Int J Mol Cell Med Spring 2021; Vol 10 No 2 136 Anti-nephrotoxic effect of mesenchymal stromal cells and hesperidin physiological saline solution, cut into small pieces, retrieve antigens. The tissues were then immersed and fixed in 10% neutral buffered formalin. The in 3% hydrogen peroxide for 15 min at room fixed tissues were dehydrated in ascending alcohol temperature grades, and activity, followed by washing in double-distilled embedded in Paraplast®. Paraplast blocks were water and phosphate-buffered saline with 0.05% sectioned at 4‒ 5 µm thin layers, then stained with Tween® 20 (PBST). After that, the sections were hematoxylin and eosin (H&E) to examine the incubated with a 1:20 dilution of p53 mouse general histological structure, periodic acid - schiff monoclonal antibody (Invitrogen, Thermo Fisher (PAS) neutral Scientific, USA) for 1 h at room temperature. After mucopolysaccharides, and bromophenol blue to intense washing by PBST, the slides were incubated demonstrate the total protein content. Staining with HRP-conjugated secondary antibody (Thermo procedures were performed as outlined by Suvrna Scientific, USA) and rinsed again with PBST. et al. (23). Kidney sections were examined and Finally, the 3.3-diaminobenzidine (DAB) solution photographed research (Dako, Denmark) was added, then the slides were microscope (Leica, Hannover, Germany) attached rinsed with tap water and counterstained with with Canon digital camera (Canon, Japan). Mayer's hematoxylin, dehydrated, and mounted. cleared for in xylol, demonstration using Leica impregnated, of the binuclear to block endogenous peroxidase The morphometric analysis was performed The negative control sections were processed under light microscopy. For semiquantitative similarly but without adding the primary antibody. analysis of the severity of histopathological changes The p53 protein expression in the immunostained of the kidney, ten high-magnification (×400) fields sections was examined under a light microscope. of the cortex and medulla were randomly selected. The brown color immunostaining determined the Kidney damage was defined as glomerular atrophy, positive reaction in the cytoplasm and nucleus. tubular Quantification of histochemical and immun- epithelial vacuolar degeneration, desquamation, loss of brush border, hyaline cast ohistochemical staining formation, infiltration of inflammatory cells, and Ten different areas of each PAS, bromophenol vascular congestion. Kidney histological changes blue, and p53 stained images (X400)/ each group were scored from (0-4) with the following was analyzed by the Image J software (National semiquantitative scale: 0: no histological changes Institute of Health, Bethesda, Maryland, USA), to and the tissues appeared normal; 1: histological estimate PAS, bromophenol blue color intensity, changes in less than 25% studied microscopic and p53 immunopositivity of the renal tissue in domains; 2: histological changes in 26-50% studied different experimental groups. microscopic domains; 3: histological changes in 51- Statistical analysis 75% studied 4: The obtained data were expressed as mean histological changes in more than 75% studied ±SEM (n=10), and all values were analyzed by microscopic domains (24). SPSS Version 25 software package (SPSS, Inc., Immunohistochemistry USA) using one-way ANOVA followed by The p53 microscopic protein domains; and immunohistochemical staining was performed using the avidin-biotin- Duncan's test post hoc analysis. P values <0.05 were considered as significant. peroxidase method (25). Briefly, the tissue sections were deparaffinized and rehydrated using xylene Results and alcohol, respectively, and were then boiled in RT-qPCR was performed to characterize the 10 mM sodium citrate (pH 6) for 8-15 min to isolated BM-MSCs. BM-MSCs showed a high 137 Int J Mol Cell Med Spring 2021; Vol 10 No 2 Mazher KM et al. expression of specific markers of bone marrow Histological and histochemical investigations derived stromal cells (CD73 and CD105) and weak The microscopic examination of the control rat expression of CD45 and CD34 (hematopoietic kidney stained with H&E revealed normal renal markers) (Figure 1F). corpuscles and tubules. The renal corpuscles had Figure 1. Real-time quantitative PCR (RT-qPCR) amplification plot curves for in vitro expression profile of BM-MSCs markers. A) CD73 (red plot); B) CD105 (pink plot); C) CD45 (purple plot); D) CD34 (yellow plot); E) β-actin;F: gGene expression data analysis of BMMSCs markers (CD73, CD105, CD45, and CD34) in relation to β-actin as a housekeeping gene. Data are expressed as mean ± standard error. Int J Mol Cell Med Spring 2021; Vol 10 No 2 138 Anti-nephrotoxic effect of mesenchymal stromal cells and hesperidin well definitive glomeruli (glomerular tufts) In the hesperidin-treated rats (group III), most surrounded by a well-organized double layer of renal corpuscles appeared with intact glomeruli and Bowman's capsule. The outer parietal layer was Bowman's capsules. Nearly all tubular epithelial lined by simple squamous epithelium while cells had vesicular nuclei and few cytoplasmic podocytes and mesangial cells lined the inner vacuolations. visceral layer; the space between both layers is interstitial infiltration of inflammatory cells and called the urinary space (Bowman's space). The congestion in the glomerular tuft and renal blood renal tubules appeared with the typical proximal vessels were present (Figure 2E). Interestingly, the convoluted tubules pattern, distal convoluted BM-MSCs treated rats (group IV) showed a tubules, and collecting ducts. The proximal progressive renal tissue improvement in renal convoluted tubules were small-sized in diameter, corpuscles and renal tubules. A marked decrease in with narrow lumina, and lined by pyramidal cells the infiltrated inflammatory cells was also observed with apical brush borders. The cytoplasm was compared to that in the hesperidin-treated rats deeply acidophilic, containing rounded vesicular (group III); however, mild vascular congestion was nuclei. The distal convoluted tubules were large- still noticed (Figure 2F). sized in diameter, with wide lumina and lined by On the other hand, moderate The combined treatment with hesperidin and simple cuboidal epithelium with rounded central BM-MSCs vesicular nuclei surrounded by lightly stained improvement of the renal tissue. It appeared similar acidophilic cytoplasm. The loop of Henle's and to the control group with a pronounced absence of collecting ducts of the renal medulla lined with inflammatory cells and congestion. The renal simple squamous to cuboidal epithelium with tubules and corpuscles recovered their normal flattened to rounded vesicular nuclei, respectively architecture with preserved brush borders of the (Figure 2 A, B). tubular cells and the parietal layer of Bowman's However, the examined renal sections of the (group V) showed a marked capsule (Figure 2G). cisplatin-treated rats (group II) showed severe Figure 2H demonstrates the scoring results of degenerative changes in the renal corpuscles and the histopathological changes of the kidney in tubules. Most renal corpuscles appeared with different experimental atrophied glomeruli with prominent wide urinary significant increase (P <0.05) in pathological space, nuclear pyknosis in podocytes and mesangial changes in the cisplatin intoxicated group in cells, irregularity, and partial loss of the parietal comparison with the control and other treated layer of Bowman's capsule. Moreover, pronounced groups. Renal pathological changes significantly loss of the renal tubule's cellular architecture was decreased (P <0.05) in the BM-MSCs-administered observed. The affected tubular epithelial cells group in comparison with the hesperidin-treated exhibited swelling, cytoplasmic vacuolations, and group. Moreover, substantial recovery of the renal hydropic degeneration with pyknotic nuclei in architecture was observed in the group that received addition to partial and complete loss of the apical the combined treatment in comparison with those brush that were administered with hesperidin or BM- borders. Some renal tubules showed and was There was a dilatation with acidophilic hyaline materials in their MSCs lumina. Also, there were intense pericorpuscular different from the control group. The PAS and peritubular lymphocytic infiltrations in addition technique demonstrated mucopolysaccharides in the to marked congestion in the glomerular tufts and all renal sections of the control group with a strong cortical blood vessels (Figure 2 C, D). reaction to the PAS at the brush border, basement 139 Int J Mol Cell Med Spring 2021; Vol 10 No 2 separately, groups. non-significantly Mazher KM et al. Figure 2. Representative hematoxylin and eosin staining, and scores for renal injury. A and B: Kidney sections of control rats; A) The renal cortex appeared with normal renal architecture including renal corpuscles with normal glomerulus (G), normal Bowman’s capsule having well-developed outer parietal layer lined with simple squamous epithelium (arrowheads), and inner visceral layer lined by podocytes and mesangial cells (thick arrows) separated by a urinary space (U). The renal tubules had the typical characteristics features of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) with prominent apical brush borders (thin arrows); B) The renal medulla, which was lined with simple cuboidal cells and had normal collecting tubules (CT), had rounded vesicular nuclei and acidophilic cytoplasm; C and D: kidneys of cisplatin-treated rats; C) The renal cortex showed severe degenerations, renal corpuscles with atrophied glomeruli (G) and a wide urinary space (U), cytoplasmic vacuolations and hydropic degenerations (arrows), nuclear pyknosis (arrowheads) and loss of the apical brush borders of the renal tubular epithelium, glomerulus and cortical blood vessel congestion (C), and diffuse lymphocytic infiltrations; : the renal medulla shows dilatations and cellular degenerations (arrows) of the renal tubules and their lumina filled with acidophilic hyaline materials (H); E: the kidneys of hesperidin administered rats show that most renal corpuscles have intact glomeruli and Bowman’s capsules and the epithelial cells of proximal tubules (PCT) and distal tubules (DCT) exhibit vesicular nuclei and few cytoplasmic vacuolations despite the presence of vascular congestion (C) and lymphocytic infiltration (F); F) The kidneys of BM-MSC-treated rats had improved renal tissue with a marked decrease in lymphocytic infiltrations. However, some congestion in glomerular tufts and blood vessels (C) was present; G: the kidneys of hesperidin and BM-MSCs-treated rats (combined treatment) showed complete regeneration of renal tissue that looked similar to the control group with normal renal corpuscles with normal glomerulus (G), proximal tubules (PCT), and distal tubules (DCT) with absence of inflammatory cells and congestion; H: the bar graph shows the scores of the pathological changes. Results are expressed as means ± SD with dissimilar superscript letters (significantly differing at P < 0.05); a: significantly different from the control group; b: significantly different from the cisplatin administered group; c: significantly different from hesperidin -treated group; d: significantly differ from BM-MSCs -treated group. Scale bar: 50 µm. membrane of the renal tubules, and the basal lamina tubule's brush borders moderately reacted with PAS of renal corpuscle that appeared with dense (Figure 3C). In the BM-MSCs treated rats (group magenta coloration (Figure 3A). In the cisplatin- IV), treated rats (group II), the degenerated renal tubules significantly improved in comparison with the appeared with partial or complete loss of the brush hesperidin-treated rats (group III). The renal borders, which were negatively reacting with PAS. basement membranes and brush borders showed a However, tubule's marked increase in PAS reaction intensity from basement membrane showed weak to moderate moderate to strong reaction (Figure 3D). The reaction (Figure 3B). combined hesperidin and BM-MSCs treatment the renal corpuscles and the renal tissue's mucopolysaccharides Moderate improvement of the mucopoly- (group V) renal tissues reattained to a great saccharides was observed in the renal sections of extent the intense PAS-positive reaction, which hesperidin-treated rats (group III). Some renal was similar to the control group (Figure 3E) with Int J Mol Cell Med Spring 2021; Vol 10 No 2 140 Anti-nephrotoxic effect of mesenchymal stromal cells and hesperidin Figure 3. Representative photomicrographs of renal tissues submitted to PAS staining for mucopolysaccharides evaluation. A) Control rat kidney showed an intense PAS positive reaction (intense magenta) in the brush border (arrowheads) and the basement membrane of the renal tubules, as well as the basal lamina of the renal corpuscle in the control rat kidney (arrows); B) Cisplatin-treated rats showed a negative PAS reaction with loss of the brush border of the proximal tubules (PCT), while the basement membrane of the renal corpuscle and renal tubules were moderately reacting (arrows); C) Hesperidin-treated rats kidneys exhibited a mild to moderate PAS reaction of the brush border (arrowheads) and basement membrane of the renal tubules and the basal lamina of the renal corpuscle (arrows); D) BM-MSCs-treated rat's kidneys showed a moderate to strong PAS positive reaction in the brush border (arrowheads) and basement membrane of the renal tubules and renal corpuscles (arrows); E) Hesperidin and BM-MSCs treated rats kidneys having an intense PAS reaction similar to the control group in the brush border (arrowheads) and basement membrane of the renal tubules and the renal corpuscle (arrows); F) The bar graph shows the mean color intensity of PAS reaction in different experimental groups. Results were expressed as means ± SD with dissimilar superscript letters (significantly differing at P < 0.05); a: significantly different from the control group; b: significantly different from the cisplatin administered group; c: significantly different from hesperidin -treated group, d: significantly different from BM-MSCs treated group. Scale bar: 50 µm.. preserved tubular brush borders and basement with no significant difference in comparison with membranes of the renal corpuscles and tubules. the control group. The mean color intensity of the PAS reaction The total protein content was screened using in different experimental groups is represented in the bromophenol blue method. Kidney sections of Figure 3F. A significant decrease (P <0.05) in the control rats (group I) revealed normal total protein color intensity of the PAS reaction was noticed in content in the cellular cytoplasm of renal tubules, the cisplatin-administered group in comparison mesangial cells and podocytes, which were stained with the control group and other treated ones. There deep blue (Figure 4A). The cisplatin-treated rats was the (group II) renal tissues showed a significant groups. reduction of the total protein content in the Moreover, the PAS intensity reaction significantly cytoplasm of affected cells indicated by faint blue increased (P <0.05) in the combined-treatment staining (Figure 4B). In contrast, kidneys in both group in comparison with the groups that received single treatments with either hesperidin (group III) hesperidin or BM-MSCs separately. A complete or BM-MSCs (group IV) showed moderate staining restoring affinity no significant hesperidin and of difference BM-MSCs renal between treated cytoplasmic carbohydrates occurred after applying the combined treatment 141 Int J Mol Cell Med Spring 2021; Vol 10 No 2 of the cell lining renal tubules and corpuscles (Figure 4C, D). In the combined Mazher KM et al. Figure 4. Representative photomicrographs of renal tissues stained with bromophenol blue for total protein evaluation. A) Control rat's kidneys have normal protein content, as evidenced by dense blue staining of renal corpuscles and renal tubules; B) Cisplatin-treated rat's kidneys have a marked decrease in protein content, as indicated by a faint blue reaction of the tubular cells and glomeruli; C) Hesperidintreated rats kidneys have a moderate improvement in protein content, as evidenced by a moderate staining affinity of tubular cells; D) BMMSCs-treated rats kidneys have a marked increase in protein content, as evidenced by moderate to strong blue staining of the renal tissue; E: hesperidin and BM-MSCs treated rats kidneys showed pronounced improvement in protein content with intense blue staining; F) The bar graph shows the mean color intensity of bromophenol blue in different experimental groups. Values are expressed as mean ± SD with dissimilar superscript letters (significantly differing at P < 0.05); a: significantly different from the control group; b: significantly different from the cisplatin administered group; c: significantly different from hesperidin-treated group; d: significantly different from BM-MSCs treated group. Scale bar: 100 µm. treatment with hesperidin and BM-MSCs hesperidin or BM-MSCs separately. A complete (group V), the total protein content significantly restoring of the total proteins was occurred after the improved in the renal corpuscles and tubular cells, combined treatment. as indicated by a strong staining affinity similar to Immunohistochemistry of p53 the control group (Figure 4E). The p53 protein immunohistochemistry of Figure 4F demonstrates the morphometric renal tissues showed that most renal corpuscular analysis of the mean color intensity of the and tubular epithelial cells of the control group bromophenol (group I) exhibited a weak reaction for the p53 blue reaction in different experimental groups. There was a significant (Figure decrease (P <0.05) in the color intensity of the nephrotoxicity induced by cisplatin treatment bromophenol blue reaction in the cisplatin- (group II), a progressive and robust reaction to the administered group in comparison with the control p53 was noticed, with a widespread brown color in group and other treated groups. No significant the difference was present between the hesperidin, and immunostained sections of the hesperidin-treated BM-MSCs the rats (group III) showed moderate reaction (Figure bromophnol blue intensity reaction significantly 5C), while the BM-MSCs treated rats (group IV) increased (P <0.05) in the combined-treated group renal in comparison with the groups that received with the cisplatin group (Figure 5D). The combined treated groups. Moreover, 5A). affected tissues However, renal reacted cells in response (Figure weakly in 5B). to The comparison Int J Mol Cell Med Spring 2021; Vol 10 No 2 142 Anti-nephrotoxic effect of mesenchymal stromal cells and hesperidin Figure 5. p53 immunostaining of kidney sections. A) Very few apoptotic (p53 positive) cells were present in the renal corpuscle cells and tubules in the control rat group, there are; B) cisplatin-treated rats exhibited very strong immunoreactivity with p53, which is manifested by a widespread dark brown color; C) hesperidin-treated rats exhibit moderate p53 immunoreactivity in the tubular cells; D) the BM-MSCtreated rat group exhibited weak immunoreactivity to p53; E: p53 immunostaining is barely detectable in the kidneys of hesperidin and BMMSCs treated rat group; F: the bar graph shows the percentage of p53 immunopositivity in different experimental groups. Data are expressed as mean ± SD with dissimilar superscript letters (significantly differing at P < 0.05); a: significantly different from the control group; b: significantly different from the cisplatin administered group; c: significantly different from hesperidin-treated group; d: significantly different from BM-MSCs-treated group. Scale bar: 50 µm. treatment with hesperidin and BM-MSCs (group V) renal functions showed a marked decrease in the renal apoptotic Figure 6 demonstrates the effect of BM-MSCs cells. The renal corpuscles and tubular cells reacted and hesperidin treatments on the renal function weakly to the p53 antibody (Figure 5E). Figure 5F parameters in cisplatin-treated rats. There was a shows the morphometric analysis of the percentage significant increase in serum urea, creatinine, and of p53 immunopositive apoptotic cells in different potassium levels in the cisplatin-treated group (P experimental groups. The rate of immunopositive <0.05) in comparison with the control group. In p53 apoptotic cells is significantly higher (P <0.05) contrast, treatment of cisplatin-induced nephroto- in the cisplatin-administered group in comparison xicity with hesperidin, BM-MSCs, and their with the control and other treated groups. A combination for four weeks significantly reduced significant decrease in immunopositive apoptotic the serum urea and creatinine levels (P < 0.05) in cells (P <0.05) in the BM-MSCs-treated group was comparison with the cisplatin-treated group (Figure observed in comparison with the hesperidin- 6A, B, and D). The serum sodium level exhibited a administered group. Moreover, the immunopositive significant decrease in the cisplatin-treated group (P apoptotic cells significantly decreased (P <0.05) in < 0.05) in comparison with the control group. the combined treated group in comparison with the However, a significant elevation of the sodium single treated ones, while no significant difference levels was noticed (P < 0.05) in the hesperidin, with the control group was observed. Evaluation of biochemical parameters related to 143 Int J Mol Cell Med Spring 2021; Vol 10 No 2 BM-MSCs, and their combination treatment (Figure 6C) Mazher KM et al. Figure 6. Figure 6. The effect of BM-MSCs and/or hesperidin on serum biochemical parameters after cisplatin-induced nephrotoxicity. A) Urea; B) Creatinine levels; C) sodium levels; D) Potassium levels. The data are presented as mean ± standard error (N=6). The mean values with different superscript symbols differ significantly (P <0.05). Discussion faint bromophenol blue staining (27). Our study The current study revealed that hesperidin, confirmed the apparent apoptotic effect caused by BM-MSCs, or hesperidin and BM- MSCs admini- cisplatin administration in kidney as detected by the stration significantly reduced the nephrotoxic effect strong immunopositivity reaction for p53 (4). Some of of studies reported that the tubular cell apoptosis and hesperidin and BM-MSCs had the most potent renal toxicity caused by cisplatin are due to the ameliorative impact on restoring the damaged renal massive production of proinflammatory cytokines, tissue. including TNF-α (28). The apoptotic effect was cisplatin. Moreover, co-administration The present study showed that cisplatin more intense in the renal tubules, mainly proximal intoxication induced renal dysfunction as indicated convoluted by significant altered renal biochemical parameters, concentration of mitochondria and their functional including blood urea, creatinine, potassium, and role in concentration and reabsorption processes sodium levels (5). The biochemical findings were (27). Therefore, the renal tubules are the primary supported by the severe alteration of the renal target for cisplatin-induced oxidative stress and architecture induced by cisplatin injection. Severely activate apoptosis pathways (29). The massive degenerated tubular epithelial cells, dilated renal production of ROS, free radicals, and inflammatory tubules with intraluminal cast formation, atrophied cytokine increase of oxidative stress act directly on glomeruli with wide urinary spaces, interstitial the renal tubular cell components leading to lymphocytic of cytotoxicity, tubular mitochondria damage, tubular glomerular renal blood vessels were observed (26). transport system disturbances, protein synthesis Additionally, inhibition, and DNA injury (28). cytoplasmic infiltrations, significant and congestion depletion mucopolysaccharides and of the tubules, because of the high protein The imbalance between oxidant-antioxidant content was confirmed by a weak PAS reaction and statuses caused by cisplatin has led several studies Int J Mol Cell Med Spring 2021; Vol 10 No 2 144 Anti-nephrotoxic effect of mesenchymal stromal cells and hesperidin to recommend natural described (14). Their marker expression profiles antioxidants to enhance the renal antioxidant were confirmed by quantitative real-time PCR defense from analysis of CD105, CD73, CD34, and CD45. The nephrotoxicity (30,31). Several studies investigated cultured BM-MSCs highly expressed CD73 and the administration of exogenous antioxidants, CD105 (mesenchymal stromal cells) and lowly especially the natural type, to enforce the renal expressed CD34 and CD45 (hematopoietic) marker antioxidant defense and avoid nephrotoxicity genes (35), confirming their biological chara- induced by nephrotoxic drugs (32). It is generally cteristics. system, providing and exogenous protect kidneys accepted that natural antioxidants significantly The BM-MSCs treatment alone after cisplatin improve or prevent nephrotoxicity by trapping administration (group IV) improved the renal destructive biochemical parameters but with no significant free radicals and inhibiting inflammation (31). difference was observed in comparison with the In the current study, the treatment of cisplatinintoxicated rats with antioxidant renal architecture was significantly retrieved in substantially improved the renal biochemical comparison with rats that received hesperidin only. parameters in comparison with the cisplatin- treated BM-MSCs group similar to the findings of Kumar et al. (30). disappearance of inflammatory cells, decreased Regarding the histopathological findings, the vascular congestion, reduced cellular apoptosis (i.e., degenerative changes in renal corpuscles and less expression of p53). Our data supported the tubules cytoplasmic previous studies that showed a pivotal role of the mucopolysaccharides and protein content were BM-MSCs in kidney repair due to their ability to moderately vascular home the injured kidney, secrete anti-inflammatory congestion and lymphocytic infiltrations persisted. cytokines, and their easy differentiation into Our results coincide with previous studies that functional tubular epithelial cells (36). Furthermore, demonstrated that the antioxidant therapy using the BM-MSCs showed the ability to prevent the flavonoids, including hesperidin, Vitamin C, or pathological selenium had a partial inhibitory effect on the nephrotoxicity early, and reduce lipid peroxidation, cisplatin-induced oxidative stress in the kidney which enhances the kidneys’ histological and (33). The antioxidant effect of hesperidin is functional regeneration (37). decreased restored. hesperidin group that received hesperidin only. In contrast, the while Additionally, treated process rats of showed marked cisplatin-induced achieved by decreasing the ROS reaction by New strategies are directed to enhance the scavenging free radicals and chelating metals and treatment efficacy of BM-MSCs (38), and to converting them into non-toxic end products (12). overcome the low survival ratio of the transplanted Our study suggested that hesperidin showed stromal cells induced by their insufficient resistance nephroprotective against the oxidative and inflammatory stresses at capacities because of its antioxidant properties. the injured sites (31). Due to their insufficient The use of BM-MSCs is a promising resistance to oxidative and inflammatory stresses at therapeutic strategy for repairing renal damage and the damaged sites, the transplanted stromal cells restoring kidney function and structure (34). The have low survival ratios, which is the crucial BM-MSCs used in this work were characterized problem affecting stromal cell therapy (31). Hence, before being used in the experimental study. The our results further confirm that the antioxidant pre- prepared stem cells were spindle shaped with few treatments is able to significantly increase the and short cytoplasmic processes as previously stromal cell longevity, viability, and repair efficacy 145 Int J Mol Cell Med Spring 2021; Vol 10 No 2 Mazher KM et al. (6). Studies showed that the treatment of diabetic rats with hesperidin and BM-MSCs together was the most potent deteriorated lipid approach profile, in heart The authors declare that they have no conflict of interest. ameliorating and kidney References functions (13). Also, antioxidant preconditioning 1. Malyszko J, Kozlowska K, Kozlowski L, et al. Nephrotoxicity could effectively improve the therapeutic effect of of anticancer treatment. Nephrol Dial Transplant 2017;32: adipose-derived mesenchymal stem cell therapy for 924-36. liver fibrosis (39). 2. Aldossary SA. Review on Pharmacology of Cisplatin: Clinical Interestingly, the current study showed that the pre-treatment of cisplatin-treated rats with Use, Toxicity and Mechanism of Resistance of Cisplatin. Biomed Pharmacol J 2019;12:7-12. hesperidin antioxidant followed by BM-MSCs 3. Manohar S, Leung N. Cisplatin nephrotoxicity: a review of the injection remarkably improved the kidney function literature. J Nephrol 2018;31:15-25. and renal architecture. All treatments, either 4. Mi XJ, Hou JG, Wang Z, et al. The protective effects of separately or in combination, improved the renal maltol on cisplatin-induced nephrotoxicity through the AMPK- function parameters after cisplatin administration mediated PI3K/Akt and p53 signaling pathways. Sci Rep with no significant differences. However, according 2018;8:15922. to our histological assessment, we noticed a 5. Zhao Y, Dai W. Effect of phloretin treatment ameliorated the substantial restoration of the renal architecture cisplatin-induced compared to the single treatment, which became experimental rats. Pharmacogn Mag 2020;16:207-13. similar to the control group (40). 6. Shaban S, El-Husseny MWA, Abushouk AI, et al. Effects of Therefore, the current study revealed that nephrotoxicity and oxidative stress in Antioxidant Supplements on the Survival and Differentiation of combined treatment of hesperidin and BM-MSCs Stem Cells. Oxid Med Cell Longev 2017;2017:5032102. has the most significant restoring effect against the 7. Aly RM. Current state of stem cell-based therapies: an cisplatin nephrotoxicity than using them separately. overview. Stem Cell Investig 2020;7:8. In conclusion, cisplatin-induced 8. Wang G, Zhang Q, Zhuo Z, et al. Enhanced Homing of nephrotoxicity and biochemical renal parameters CXCR-4 Modified Bone Marrow-Derived Mesenchymal Stem imbalance was significantly improved by the Cells to Acute Kidney Injury Tissues by Micro-Bubble-Mediated treatment with BM-MSCs, hesperidin, and their Ultrasound Exposure. Ultrasound Med Biol 2016;42:539-48. combination. 9. Pan B, Fan G. Stem cell-based treatment of kidney diseases. Based the on the histological, histochemical and immunohistochemical results, Exp Biol Med (Maywood) 2020;245:902-10. the combination of BM-MSCs and hesperidin was 10. Abotaleb M, Samuel SM, Varghese E, et al. Flavonoids in the most effective approach in curing cisplatin- Cancer and Apoptosis. Cancers (Basel) 2018;11. induced nephrotoxicity in rats. The pre-treatment of 11. Mesallam DIA, Atef M. Hesperidin: a New Approach to hesperidin antioxidant significantly improved the Ameliorate Diazinon Induced Hepatotoxicity in Adult Male BM-MSCs Albino Rats. Zagazig Journal of Forensic Medicine 2020; therapy for cisplatin- induced nephrotoxicity, suggesting this approach to enhance 18:24-38. the stromal cell therapeutic efficacy. 12. Kaltalioglu K, Coskun-Cevher S. Potential of morin and Acknowledgments hesperidin in the prevention of cisplatin-induced nephrotoxicity. This research did not receive any specific grant Ren Fail 2016;38:1291-9. from funding agencies in the public, commercial, or 13. Ahmed OM, Hassan MA, Saleh AS. Combinatory effect of non-profit sectors. hesperetin and mesenchymal stem cells on the deteriorated lipid Conflict of Interest profile, heart and kidney functions and antioxidant activity in Int J Mol Cell Med Spring 2021; Vol 10 No 2 146 Anti-nephrotoxic effect of mesenchymal stromal cells and hesperidin STZ-induced diabetic rats. Biocell 2020;44:27. Protective Role of Vitamin C. Med J Cairo Univ 2019;87: 14. Chaudhary JK, Rath PC. A simple method for isolation, 547-55. propagation, characterization, and differentiation of adult mouse 27. El-Kordy EA. Effect of Suramin on Renal Proximal Tubular bone marrow-derived multipotent mesenchymal stem cells. J Cells Damage Induced by Cisplatin in Rats (Histological and Cell Sci Ther 2017;8:261. Immunohistochemical Study). J Microsc Ultrastruct 2019;7: 15. Leary S, Underwood W, Anthony R, et al. AVMA guidelines 153-64. for the euthanasia of animals: 2020 edition. American Veterinary 28. Mohamed ME, Abduldaium YS, Younis NS. Ameliorative Medical Association. 2020. effect of linalool in cisplatin-induced nephrotoxicity: the role of 16. Soleimani M, Nadri S. A protocol for isolation and culture of HMGB1/TLR4/NF-κB and Nrf2/HO1 pathways. Biomolecules mesenchymal stem cells from mouse bone marrow. Nat Protoc 2020;10:1488. 2009;4:102-6. 29. Ning Y, Shi Y, Chen J, et al. Necrostatin-1 Attenuates 17. Katsares V, Petsa A, Felesakis A. A Rapid and Accurate Cisplatin-Induced Nephrotoxicity Through Suppression of Method for the Stem Cell Viability Evaluation: The Case of the Apoptosis and Oxidative Stress and Retains Klotho Expression. Thawed Umbilical Cord Blood Lab Med 2009;40:557–60. Front Pharmacol 2018;9:384. 18. El–Amir Ahmad Ghanayem Y, Ahmad Ashour F, Ahmad 30. Kumar M, Dahiya V, Kasala ER, et al. The renoprotective Rashed L, et al. The role of bone marrow derived mesenchymal activity of hesperetin in cisplatin induced nephrotoxicity in rats: stem cells in attenuation of renal failure in adult male albino rats. Molecular and biochemical evidence. Biomed Pharmacother Al-Azhar Medical Journal 2016;45:135-48. 2017;89:1207-15. 19. Sahu BD, Kuncha M, Sindhura GJ, et al. Hesperidin 31. Liu D, Cheng F, Pan S, et al. Stem cells: a potential attenuates cisplatin-induced acute renal injury by decreasing treatment option for kidney diseases. Stem Cell Res Ther oxidative stress, inflammation and DNA damage. Phytomedicine 2020;11:249. 2013;20:453-60. 32. Elshama S, Abdalla ME, Mohamed AM. Role of Natural 20. Khan MH, Parvez S. Hesperidin ameliorates heavy metal antioxidants in treatment of toxicity. Journal of Toxicololgy induced toxicity mediated by oxidative stress in brain of Wistar Analysis 2018;1:3-7. rats. J Trace Elem Med Biol 2015;31:53-60. 33. Dennis JM, Witting PK. Protective Role for Antioxidants in 21. Young DS, Friedman RB, Washington DC. Effects of Acute Kidney Disease. Nutrients 2017;9. disease on clinical laboratory Tests. Clinical Chemistry2002. p. 34. Zhao L, Han F, Wang J, et al. Current understanding of the 682-3. administration of mesenchymal stem cells in acute kidney injury 22. Rifai N. Tietz Fundamentals of Clinical Chemistry and to chronic kidney disease transition: a review with a focus on Molecular Diagnostics. 8th ed2018. preclinical models. Stem Cell Res Ther 2019;10:385. 23. Suvarna SK, Layton C, Bancroft JD. Bancroft’s theory and 35. practice of histological techniques. 8th ed. China: Elsevier Ltd; Characterization and Classification of Mesenchymal Stem Cells 2019. in Several Species Using Surface Markers for Cell Therapy 24. Gibson-Corley KN, Olivier AK, Meyerholz DK. Principles Purposes. Indian J Clin Biochem 2018;33:46-52. for valid histopathologic scoring in research. Vet Pathol 36. Wu L, Rong C, Zhou Q, et al. Bone Marrow Mesenchymal 2013;50:1007-15. Stem Cells Ameliorate Cisplatin-Induced Renal Fibrosis via 25. Frost AR, Sparks D, Grizzle WE. Methods of antigen miR-146a-5p/Tfdp2 Axis in Renal Tubular Epithelial Cells. recovery vary in their usefulness in unmasking specific antigens Front Immunol 2020;11:623693. in 37. Mata-Miranda MM, Bernal-Barquero CE, Martinez-Cuazitl immunohistochemistry. Appl Immunohistochem Mol Ghaneialvar H, Soltani L, Rahmani HR, et al. Morphol 2000;8:236-43. A, et al. Nephroprotective Effect of Embryonic Stem Cells 26. Mohamed RH, Youssef YM, EL-Kafrawy MH. Effect of Reducing Lipid Peroxidation in Kidney Injury Induced by Cisplatin on the Kidney of the Albino Rat and Possible Cisplatin. Oxid Med Cell Longev 2019;2019:5420624. 147 Int J Mol Cell Med Spring 2021; Vol 10 No 2 Mazher KM et al. 38. Stavely R, Nurgali K. The emerging antioxidant paradigm of engraftment efficiency in a mouse liver fibrosis model. Stem mesenchymal stem cell therapy. Stem Cells Transl Med Cell Res Ther 2020;11:237. 2020;9:985-1006. 40. Zahran F, Nabil A, El Karef A. Effect of antioxidants and 39. Liao N, Shi Y, Wang Y, et al. Antioxidant preconditioning mesenchymal stem cells on cisplatin induced renal fibrosis in improves therapeutic outcomes of adipose tissue-derived rats. J Stem Cell Res Ther 2016;1:150-8. mesenchymal stem cells through enhancing intrahepatic Int J Mol Cell Med Spring 2021; Vol 10 No 2 148