Objectives: To study the effect of ischemic preconditioning (Ipre) on the protective effect of adipose-derived mesenchymal stem cells (ADMSCs) against renal ischemia/reperfusion (I/R) injury in a rat model. Methods: 90 male Sprague Dawely rats divided into 5 equal groups; sham (right nephrectomy without ischemia), control (right nephrectomy with 45 min left renal ischemia), Ipre group as control group with 3 cycles of Ipre just before renal ischemia, ADMSCs-treated group (as control with 0.1 ml of ADMSCs containing 106 cells via penile vein at the time of reperfusion) and Ipre + ADMSCs group as ADMCs group with 3 cycles of Ipre just before renal ischemia. Serum creatinine and BUN were measured at basal and at the end of experiment. The harvested kidneys were examined for the expression of ki67, caspase-3, HIF-??1 and stromal derived growth factor (SDF-1)-?? by immunohistochemistry and for CD31 and CD45 by immunoflourescence at 24 h, 48 hr and 72 hrs after ischemia. Results: treatment Ipre with ADMSCs caused more significant improvement in all the studied parameters than each modality did alone. Conclusion: Ipre potentiates the renoprotective effect of ADMSCs against renal I/R injury probably by up regulation of SDF-1?? which increases homing of stem cells to the injured kidney tissues as well as by enhancing angiogenesis and tubular cell regeneration and suppression of apoptosis and inflammatory reaction.
Renal ischemia/reperfusion (I/R) injury is a common clinical problem that encountered in renal transplantation, nephron-sparing surgery, renal vascular surgery and aortic cross-clamping for abdominal aortic aneurisms1. It is a major cause of acute renal failure (ARF). In kidney transplantation, I/R injury is a common cause of renal cell death, ARF, delayed graft function, and renal graft rejection2. Ischemia-reperfusion injury is a complex process that involves ATP depletion, accumulation of intracellular Ca2 and reactive oxygen species (ROS), proinflammatory cytokine production and apoptotic pathway activation. ROS are involved in tissue damage that occurs following I/R3, and their role in the pathophysiology of I/R injury is supported by the increased formation of lipid peroxidation and other toxic products that occur following such an injury4.
The efficacy and therapeutic potentials of using mesenchymal stem cell (MSC) in treatment of ischemia/reperfusion injury have been extensively investigated in different organs such as the kidney and heart5-7. Several experimental studies demonstrated the renoprotective actions of MSC therapy against acute ischemic kidney injury through homing and recruitment of MSCs in both glomerular and tubular structures, regeneration of tubular epithelium and enhancement of peri-tubular capillary regeneration5, 6, 8. Compared with other types of MSCs such as bone marrow derived-MSCs (ADMSCs), adipose-derived MSCs (ADMSCs) have the advantages of being abundant, minimal invasiveness in harvesting and unlimited supply from in vitro culturing9. Banas et al.10 demonstrated a therapeutic superiority of ADMSCs over bone marrow-derived mesenchymal stem cells in an animal model of liver injury. In addition, Banas et al.10 reported that ADMSCs have more potent anti-inflammatory and immuno-modulating effects than MSCs derived from bone marrow. Moreover, Chen et al11 demonstrated that, ADMSC therapy minimized kidney damage after IR injury through suppressing oxidative stress and inflammatory response.
Ischemic preconditioning (Ipre), a well-established phenomenon that describes tissue adaptation to stress by taking profit of intrinsic defense mechanisms, was initially described in the heart by Murry et al12 in 1986. We demonstrated in previous studies by our groups the renoprotective effect Ipre against renal I/R injury13,14. Previous studies demonstrated improvement of mobilization of BMSCs into peripheral blood by ischemic stimulation and many IPC-induced factors, such as VEGF, erythropoietin, and SDF-1?? 15-17. Also, several studies have also demonstrated an increase of circulating BMSCs after Ipre18,19. Patschan et al.,19 reported that renal ischemia rapidly (within 3- 6 hrs) mobilizes of transplanted endothelial progenitor cells (EPC) which transiently home to the spleen and acting as temporary reservoir of mobilized EPCS and accumulation of EPCS in renal medulla in papillary region at late phase of Ipre. Moreover, in clinical studies, a technique of repetitive balloon dilations (brief exposure to ischemia) that is used for intracoronary cell delivery has been found to facilitate the transfer of BMSCs20,21. These data suggested a potential relationship between Ipre and MSCs. So, this study was designed to investigate the possible enhancing effect for Ipre on mobilization and the recruitment of ADMSCs on renal I/R in a rat model as well as to study its underlying mechanism.
Material and Methods
The study included ninety male Sprague Dawely rats weighing 220-250 g (aged 3 -4 months) that were bred and housed in the animal house of the Urology and Nephrology center, Mansoura, Egypt. The animals were housed in separate cages with free access to the tape water. The experiment was performed according to the international guidelines for the care of the experimental animals and approved by local ethical committee of Mansoura Faculty of Medicine, Egypt.
Rats were randomly divided into five groups (18 rats each); a) sham group; left renal pedicle was surgically explored without ischemia and right nephrectomy was done, b) control (I/R) group; in which clamping of left renal pedicle for 45 min with right nephrectomy were done with iv injection of 0.1 ml of culture media, c) Ipre group as control group with three cycles of 2-min ischemia followed by 5-min reperfusion period before the definitive 45-min ischemia14, d) ADMSC-treated group; as control group with iv administration of 0.1 ml ADMSC containing (106 cells) 60 min before ischemia and e) ADMSC + IPre- treated group; as ADMSC group with 3 cycles of Ipre. Each group is subdivided into 3 subgroups (6 each) according to the time of sacrifice 24 hrs, 48 hrs and 72 hrs after ischemia.
In all rat, anaesthesia was induced by intraperitoneal (i.p) injection of a mixture of ketamine (75 mg/kg) and diazepam (5 mg/kg). After anesthesia; the rat was fixed supine on a thermoregulated heating board to maintain a body temperature of 37?? C. In sham group, a midline laparotomy was performed and the left kidney and its pedicle were dissected off the surrounding perinephric fat and exposed without clamping of the renal pedicle. Then, the pedicle of the right kidney was exposed and ligated using 3-0 silk sutures twice and the kidney was removed. The abdomen was irrigated with isotonic saline and the abdominal incision was closed by continuous stitches using 3/0 vicryl sutures. In control (I/R) group, the same was done as sham group with clamping of the left renal artery for 45 min and right kidney was removed 5 min before removal of the vascular clamp.
Isolation and characterization of ADMSC
To obtain ADMSCs the scrotum was opened under anesthesia, then the paragonadal fat was obtained and cutted into small pieces which were added to 3 volumes of PBS in a 50-ml conical tube, vortexed at full speed for 1 min, and centrifuged at 220 g for 10 min. The adipose tissue is transferred from the upper phase to a fresh tube, and digested in 0.075% collagenase I for 1 hr at 37oC with shaking. Then the digest was centrifuged at 220 g for 10 min and the supernatant was removed and the pellet was re-suspended in 40 ml PBS and re-centrifuged at 220 g for 10 min. The supernatant was removed and the pellet was re-suspended in 10 ml 160 nM NH4Cl for 10 min, followed by the addition of 10 ml of PBS and centrifuged at 220 g for 10 min. 5 ml of DMEM (supplemented with 10% FBS) was added to re-suspend the pellet, followed by filtration through a 100-mm cell strainer into a 10-cm culture dish. The culture dish was placed in a 5% CO2 incubator for 3’5 days to allow the formation of ADSC colonies, which was then trypsinized and propagated. FACS analysis revealed that ADMSCs were positive (98%) for the expression of CD44 (phycoerythrin, PE) and CD105 (phycoerythrin, PE) and negative for the expression of the CD34 (phycoerythrin, PE) and CD31 markers (phycoerythrin, PE) (fig.1a).
Adipogenic and osteogenic differentiation of ADMSCs
For induction of adipocyte differentiation, 5’104 MSCs were cultured in DMEM with 4.5 g/L glucose supplemented with 10% FBS, 0.5 mM isobutyl-methylxanthine, 200 ‘M indomethacin, 10-6 M dexamethasone, and 10 ‘/ml insulin in T-25 flasks, with changing the medium twice per week. After 3 weeks, the cells were fixed in 10% neutral buffered formalin for 10 minutes and stained with Sudan black stain for visualizing fat droplets into the cell (fig. 1b)
For induction of osteogenic differentiation, 5’104 MSCs were plated in six-well microplates in DMEM supplemented with 10% FBS, 10 mM ‘-glycerolphosphate, 0.2 mM ascorbic acid, and 10-8 M dexamethasone. The medium was changed twice per week. To demonstrate osteogenic differentiation, the cultures were washed with PBS, fixed with ice-cold ethanol 70% for 1 hour and stained with alizarin red S for 10 minutes in order to assess calcium accumulation (fig. 1c).
Identification and homing of Mesenchymal stem cells
Before cell transplantation, the cultured cells were impregnated with ferumoxides injectable solution (feridex) (Bayer Health Care Pharmaceuticals Inc.). The impregnated stem cells could be stained by Prussian blue stain to detect homing into the injured kidney; they were assessed using several kidney specimens taken within different intervals but within 24 hours after mesenchymal stem cell injection into penile vein. Iron labeled ADMSCs were detected in many compartments of the kidney such as glomerular capillaries (fig.1d) and peritubular capillary in the cortex and outer strip outer medulla (OSOM) (Fig1e).
Collection of blood and urine samples and harvesting the kidney
Blood samples were collected before the procedure (basal) and before sacrifice of the animal at 24 h, 48 h and 72 hrs (endpoint). Blood samples were obtained under inhalational general anesthesia from the ophthalmic venous plexus. The blood sample was centrifuged and the serum was isolated and stored at 20 oC for measurement of serum creatinine and blood urea nitrogen (BUN). Then, the rats were anesthetized by i.p. thiopental sodium (12 mg/100 g body weight). A midline laparotomy was done and the left kidney is removed and bisected longitudinally into two halves by a scalpel. Both halves were placed in a container filled with formalin 10% and sent for histopathological and immunohistochemical examination.
Assessment of renal function
Serum creatinine and blood urea nitrogen (BUN) levels were estimated from blood samples by using an auto-analyser (CX 7; Beckman, Foster City, CA, USA).
The kidney specimen was processed for embedding in paraffin and sectioned in 3??m thick slices and stained with hematoxylin and eosin and examined by light microscopy in a blinded fashion. Tubulointerstitial injury was examined for tubular atrophy, tubular dilatation, distal tubular cast, loss of proximal tubular brush border, patchy loss of tubular cells, peritubular vascular congestion and endothelial damage and leukocyte accumulation. The degree of tubulointerstitial damage was assessed using a well-defined grading system4, where 0= no abnormality, 1= minimal damage involve ?? 25% of the cortex and outer medulla, 2= mild damage involve 25 to 50% of the cortex and outer medulla, 3= moderate damage involve 50 to75% of the cortex and outer medulla and 4 = sever damage involve ?? 75% of the cortex and outer medulla22. Each kidney is given a score determined by two observers.
Paraformaldehyde-fixed paraffin-embedded 3 ??m thick sections were deparafinized, and sodium citrate antigen retrieval (10 mM, pH = 6.0, boiled for 20 minutes then cooled in buffer at RT for 15 min) was performed. Following antigen retrieval, sections were washed and blocked with 5% normal goat serum (Sigma-Aldrich) for 30 minutes. Next, 3% hydrogen peroxide (Fisher Scientific) was used for 15 minutes to block endogenous peroxidase activity, then sections were washed and incubated with the primary antibody (caspase-3 (RB-1197-P1), ki67 (RB-9043-P1), HIF-1?? (MS-1164-P1) and SDF-1?? (MAB350)) overnight at 4’C, followed by biotinylated secondary antibody (Power – Stain’ 1.0 Poly HRP DAB Kit for Mouse + Rabbit, cat# 52-0017) for 30 minutes. Sections were washed and treated with R.T.U. Vectastain Elite ABC reagent (Vector) for 30 minutes followed by incubation with substrate reagent diaminobenzidine (DAB) for 2 minutes. Sections were then rinsed with water, counterstained using Mayer’s hematoxylin solution for 1 minute, and mounted. The sections were observed on an Olympus BX51 light microscope. Pictures were obtained by a PC-driven digital camera (Olympus E-620).
Semiquantitative scoring for immunohistochemical staining
Scoring was done in a blinded manner. For apoptotic index, the number of caspase-3 positive cells was counted in ten non-overlapping randomly selected x400 fields of each slide and the apoptotic index was expressed as the average scores of the 10 ‘elds 23. The proliferation index was defined as the percentage of the counted immunoreactive nuclei for ki67 per at least 1000 tubular cells24. For HIF-1alpha, HIF-1?? positive cells were counted on 10 to 15 randomly selected 200 fields or entire specific area25. The immunohistochemical examination for SDF-1?? showed cytoplasmic, membranous and sometimes nuclear distribution of the stain in renal tubular cells and scored as follows: 0 = no staining; 1= staining of <25% of cells, 2= staining of 25-50% of cells, 3 = >50% of cells26.
Sections (3 ??m) were prepared on a cryostat and mounted on superfrost slides, fixed in ice-cold acetone for 1’2 min, and allowed to air dry. These sections were then blocked with 1:100 normal rabbit serum in phosphate buffered saline (PBS). Primary antibodies (CD31 (BBA7) and CD45 (BAM1430)) were then added to the sections and incubated at room temperature for 1 h. Sections were then rinsed in PBS and incubated anti-rat IgG secondary antibody (Anti-Mouse IgG (H+L), F(ab,)2 Fragment ( Alexa Fluor ?? 488 Conjugate) cat # 4408) for 35 min at room temperature. Sections were washed and developed with 3,3-diaminobenzidine (Vector Laboratories Inc, Burlingame, CA). Tissue sections were examined for positive staining, and representative photos were taken. Assessment of the expression of these markers were done as follow, the number of CD45 (LCA) and CD31 (PECAM) positive cells were counted in ten randomly selected 200x fields and the average number of positive cells was calculated.
Real time PCR for HIF-1??, SDF-1?? and caspase-3 genes
The expression of mRNA of HIF-1??, HIF-1?? and caspase-3 genes were done according to previously mentioned technique14.The primer sequences for tested genes were , SDF-1?? (forward SDF-1′ F59-GAGCCATGTCGCCAGAGCCAAC-39, R59 CACACCTCTCACATCTTGAGCCTCT- 395′- HIF-1?? forward 5′-TGCTTGGTGCTGATTTGTGA-3′ (nt 681’700) and reverse primers 5′-GGTCAGATGATCAGAGTCCA-3′ (nt 871’890), caspase-3, F: 5#-GGACCTGTGGACCTGAAAAA-3# 159 R: 5#-GCATGCCATATCATCGTCAG-3, housekeeping internal control gene (GAPDH) (140 bp) forward: 5′ TATCGGACGCCTGGTTAC-3′, reverse: 5′-CTGTGCCGTTGAACTTGC-3’.
Statistical analysis was performed using SPSS version 16 (SPSS, Chicago, IL, USA). Qualitative data were presented as numbers and percents, while quantitative data were expressed as means ?? SD. One way ANOVA test with Scheffe’s posthoc test were used to study the statistical significance of other parameters among all groups. P-value < 0.05 was considered significant.
Serum creatinine (mg/dl) and BUN (mg/dl) in different groups
The results of serum creatinine (mg/dl) and BUN in all studied groups at basal and test conditions are shown in table 1. Means of basal serum creatinine and BUN were comparable between the all studied groups. Test values of serum creatinine and BUN were significantly higher in control (I/R) group compared to sham group at 24h, 48h and 7 days (p< 0.05). Pretreatment with either ADMSCs or Ipre alone caused significant attenuation of serum creatinine and BUN levels compared to control (I/R) group at different end points (p< 0.05). Pretreatment with combination caused more significant improvement of serum creatinine and BUN than each one did alone at different end points (p< 0.05).
Routine histopathological examination of kidney tissues
Histopathological examination revealed significant increase in the tubulointerstitial damage score in control group compared to sham group (p <0.001). This effect was significantly attenuated in both Ipre and ADMSCs groups and the maximum significant attenuation was noticed in combination group compared to control group (p <0.001) (fig.3a). Kidneys specimens obtained from the sham group showed normal kidney architecture (fig.3b), while those obtained from control group showed the severe and pronounced injury in the cortex and the outer stripe of the outer medulla (OSOM) in the form of marked tubular necrosis, detachment of epithelial cells from the basement membrane, tubular dilatation, intratubular cast and luminal congestion with extensive loss of brush border and neutrophilic infiltrate (fig. 3c). Kidney sections obtained from studied groups showed dilated irregular tubules with prominent nucleoli with few scattered apoptotic cells (ADMSCs group), few apoptotic cells – regeneration in the form of irregular dilated tubules and solid tubules (Ipre group) and cortex-prominent nucleoli, few apoptotic cells & mitotic figures (combination group (Fig3d-e respectively).
Assessment of the expression of HIF-1?? and SDF-1?? in kidney tissues
Examination of expression of HIF-1?? by real time PCR and immunostaining showed significant increase in control group compared to sham group (p <0.001), an effect significantly attenuated in both Ipre and ADMSCs groups and the maximum significant attenuation was noticed in combination group compared to control group (p <0.001) (table 2 and fig.4a) at all time intervals of the study. Immunopositivity for HIF-1?? appeared as nuclear staining in renal tubules of inner strip and outer strip of outer medulla. Representative samples from different groups include fig4b (for sham group), fig4c (control group), fig4d (Ipre group), fig4e (ADMSCs group) and fig4f (combined group at 48 hrs).
While the expression of SDF-1?? by real time PCR and immunostaining showed significant increase in the in control group compared to sham group (p <0.001), with significant enhanced in both Ipre and ADMSCs groups and the maximum significant enhancement in its expression was noticed in combination group compared to control group (p <0.001) (table 2 and fig.4g). Immunopositivity for HIF-1?? appeared as cytoplasmic, membranous and sometimes nuclear distribution of the stain in renal tubular cells. Figures (4h-l) are representative samples for HIF-1?? immunostaining in different groups at 48 hrs.
Assessment of the expression of proliferation (ki67) and apoptotic (caspase-3) markers in kidney tissues
Immunostaining examination for ki67 showed significant increase in control group compared to sham group (p <0.001), an effect significantly enhanced in both Ipre and ADMSCs groups and the maximum significant enhancement was noticed in combined group compared to control group (p <0.001) (fig.4a). Immunopositivity for ki67 appeared as nuclear staining in renal tubules. Figures (4b-f) are representative samples for ki67 expression in different groups at 48 hrs.
Also, examination of caspase-3 by real time PCR and immunostaining showed significant increase in the expression of caspase-3 in control group compared to sham group (p <0.001), which was significantly attenuated in both Ipre and ADMSCs groups and the maximum significant attenuation was noticed in combination group compared to control group (p <0.001) (table 2 and fig.4g). Immunopositivity for caspase-3 appeared as cytoplasmic staining in renal tubules. Figures (4h-l) are representative samples for caspase-3 expression in different groups at 48 hrs.
Immunoflourescent examination for the expression of CD45 (lymphocyte marker) and CD31 (vascular endothelial marker) in kidney tissues
Immunostaining showed significant increase in the expression of CD45 (cytoplasmic staining associated with the membrane staining for the recruited lymphocytes at site of injury) in control group compared to sham group (p <0.001), an effect significantly attenuated in both Ipre and ADMSCs groups and the maximum significant attenuation was noticed in combination group compared to control group (p <0.001) (fig.5a). Figures (8b-f) are representative samples for CD45 expression in different groups at 48 hrs.
Also, immunostaining showed non-significant change in the expression of CD31 (appeared as cytoplasmic and membranous staining for endothelial cells in peritubular capillaries) between control and sham groups (p <0.001). Treatment with either Ipre and ADMSCs groups caused significant increase in the expression of CD31 compared to control group with the maximum significant attenuation was noticed in combination group compared to control group (p <0.001) (fig.5g). Figures (5h-l) are representative samples for caspase-3 expression in different groups at 48 hrs.
The present study, which investigated the possible potentiating effect for Ipre on the renoprotective of ADMSCs against renal I/R injury and its underlying mechanisms in a rat model has several important implications. First, treatment with either ADMSCs or Ipre alone improved the kidney functions and morphology after renal I/R injury and Ipre enhanced significantly the effect of ADMSCs on kidney functions and morphology. Second, therapy with ADMSCs or Ipre alone caused significant increase in the expression of markers of cell proliferation (ki67), stem cell migration and homing (SDF-1??) and angiogenic markers (CD31) with significant decrease in the expression of markers of hypoxia (HIF-1??) and apoptosis (caspase-3). The effects of ADMSCs were potentiated by Ipre which enhanced migration and homing of ADMSCs to the site of injury leading to more improvement in kidney functions.
We selected ADMSCs in the present study because a) adipose tissue contains 100’1,000 times more pluripotent cells per-cubic centimeter than bone marrow27, so adipose tissues are abundant source for stem cells, b) ADMSCs can be easily procured and can differentiate into other mature cell types including endothelial vascular cells28 c) ADMSCs are immunoprivileged both in vitro and in vivo29. The present study demonstrated that ADMSCs caused significant improvement of renal injury caused by I/R at the level of functions and morphology. These findings are in agreement with previous experimental studies which demonstrated the effectiveness of using stem cell therapy for acute kidney injury5, 6,8, 30,31. Also, in consistence with previous studies by our group13,14, the present study demonstrated significant improvement in kidney functions and morphology by Ipre.
This renoprotective actions for ADMSCs or Ipre alone against renal I/R injury were associated significant increase in ki67, SDF-1?? and CD31 expression and significant decrease in expression of caspase-3, CD45, HIF-1?? suggesting that the possible underlying mechanisms for the renoprotective effect of ADMSCs against renal ischemia include improvement of renal tubular cell proliferation, upregulation of SDF-1?? which enhance the migration and homing of stem cells to the site of injury, improvement of medullary blood flow (evidenced by significant reduction in HIF-1?? and increase in CD31 expression), and attenuation of apoptosis (caspase-3) and lymphocyte recruitment in kidney tissues (as evidenced by significant reduction of CD45). Previous studies reported that the renoprotective effect for stem cell therapy might include angiogenesis, stem cell homing, anti-inflammatory reaction, anti-oxidative stress, and immunomodulation5, 6,8, 30-32. Moreover, we recently demonstrated activation of nrf2, HO-1, NQO-1 antioxidant genes and attenuation of inflammatory cytokines and apoptotic genes in the protective action by Ipre14. Mohammadzadeh-Verdin et al.,33 found that induction of Nrf2 system in MSCs by adenoviruses caused protection against renal I/R injury. Also, previous studies reported the renoprotective effect for remote limb Ipre against renal I/R injury and explained this effect via an intra-renal mechanism acting within cortical cells might underpin the reno-protective function of Ipre34, reductions in lipid peroxidation, intensification of anti-oxidant systems and downregulation of COX-2 expression35.
Non-hypoxic cells do not contain a detectable level of HIF-1?? protein, whereas cells exposed to hypoxia show HIF-1?? expression within 30 min36.The renal tubules at the corticomedullary and outer medullary region of the kidney are more prone to ischemic injury because they are sensitive to decreases in oxygen concentration37,38. The present study demonstrated nuclear localization of HIF-1?? suggesting detection of an active from of HIF-1??. Sham operated rats showed low expression of HIF-1?? in inner strip of medulla without expression in the cortex suggesting low PO2 in medulla relative to the cortex. These findings are in agreement with Manotham et al.,25, Stroka et al 39 and Yuan et al40 who reported expression of HIF-1?? in the medulla of normal kidney. Yuan et al 40 also suggested that HIF-1?? may exert its role on peritubular capillary of the kidney. However, Rosenberger et al41 failed to detect expression of HIF-1?? in the medulla of normal rat kidney. Also, in the present study, the expression of HIF-1?? was significantly increased in renal tubules and extends to S3 in the outer strip of the medulla and S2 of proximal tubules in the cortex of control group suggesting extension of hypoxia to the outer strip of medulla and inner cortex in ischemic kidney caused by renal vasoconstriction. Treatment with either Ipre or ADMSCs separately significantly attenuated HIF-1?? expression. Moreover, Ipre before ADMSCs caused marked attenuation of HIF-1?? in the medulla suggesting improved blood flow and oxygenation to the renal cortex and medulla in combined group. These findings could be explained by enhanced angiogenesis by a combination of both modalities as evidenced by marked enhancement of CD31 (marker of endothelial cell lining peritubular capillaries) in combined group.
Several studies have suggested a significant role for stromal cell-derived factor-1 (SDF-1) in mediating tissue repair by promoting stem cell migration to sites of ischemic injury to facilitate tissue repair42-45. Unlike, other chemokines which need selective induction by certain stimuli, SDF-1?? is up-regulated in a variety of damaged tissues and organs as part of the injury or DNA damage response. In addition, SDF-1?? is highly inducible in several pathologic conditions such as ischemia and/or hypoxia46-48. Ceradini et al.,44 reported that SDF-1?? expression is directly proportional to reduced Oxygen tension. In agreement with the results of previous studies, the present study demonstrated significant elevation of SDF-1?? expression in I/R injury group compared to sham operated group. Recently, Yu et al., 49 demonstrated involvement of SDF-1??-CXCR4 axis in migration of MSCs to hypoxic-ischemic brain lesion in rat model. Wan et al., 48 demonstrated significant increase in SDF-1?? production in kidney tissues (started in the cortex and then diffused to the outer medulla) in response to renal I/R injury and macrophage depletion significantly enhanced this response. Also, the present study demonstrated that, Ipre and ADMSCs increased significantly the expression of SDF-1?? compared to control group. Previous studies reported similar results for ADMSCs on SDF-1?? expression in kidney tissues49 and heart50. Also, previous studies demonstrated that, SDF-1?? has been shown to enhance the recruitment of intravenously infused extragenous stem cells into heart and brain ischemic tissues51,52. Also, Gallagher et al.,53 demonstrated in diabetic mice, that local administration of SDF-1?? at the wound site caused more epithelial progenitor cells chemo-attracted to the wound area and wound healing was improved.
A combination of ADMSCs and Ipre caused more significant up-regulation of SDF-1?? compared to Ipre and ADMSCs groups. These findings suggested up regulation of SDF-1?? could be a possible mechanism for the enhancing effect of Ipre on ADMSCs which increases the homing and migration of ADMSCs to the site of injury to improve the kidney function. Bo et al., 54 found that Ipre protected kidney from I/R in late phase through enhanced migration and recruitment of EPCs and VEGF and SDF-1?? might play an important role in this protective effect. In a mice model of myocardial infarction, Kamoto et al.,50 found significant increase in SDF-1?? and VEGF after Ipre (1 and 3 hrs), which was associated with increase in number of stem cells in the myocardium. Also, Liu et al.,55 investigated the role of EPCS in modulation of IR in partial nephrectomy rat model using early phase of Ipre. They found significant increase in number of EPCs in kidneys at 12 hrs following reperfusion in Ipre group. Also, the demonstrated enhanced markers of endothelial and tubular cell proliferation, angiogenesis, VEGF-A and SDF-1?? expression in Ipre group. Although the present study, demonstrated for the first time, up to the best of our knowledge, upregulation of the chemo attractant factor SDF1?? by Ipre which might help migration and homing of ADMSCs to the ischemic kidney, this needs further studies to explain in details the underlying mechanism for up regulation of SDF1??.This is considered as one of the limitations of the present study.
Several animal studies have demonstrated angiogenesis as one of the essential mechanisms underlying the improvement of ischemic organ dysfunction after stem cell therapy 7,11,56. The present study we examined the expression of endothelial marker (CD31) in interstitial and peritubular areas of kidney specimens. The expression of CD31 was increased significantly in Ipre group as well as in ADMSCs group compared to control group as well addition of Ipre to ADMSCs enhanced its expression markedly at all different time-intervals of the study. In agreement with these findings, Chen et al.,11 reported significant increase in CM-Dil-positive cells and cells positive for endothelial markers (i.e. CD31 and vWF) in kidney tissues obtained from animals receiving ADMSC. Moreover, Chen et al.,11 reported significant increase in eNOS mRNA expression, an indicator of angiogenesis, in animals after ADMSC administration. All of these studies suggest taken together, our findings, in addition to corroborating those of previous studies7, 9, 31, 56 suggest that ADMSC treatment may improve renal function after IR injury through enhanced angiogenesis56. Moreover, the findings of the present study suggest that improvement of angiogenesis might be a possible mechanism for the enhancing effect for Ipre on ADMSCs renoprotective effect against renal IR injury.
We concluded that, Ipre enhanced the renoprotective effects of ADMSC against acute IR injury. The key mechanisms underlying the positive impact of Ipre on ADMSC treatment on renal function could be due to enhancement of SDF-1?? expression, angiogenesis and renal tubular cell proliferation and suppression of inflammatory response and apoptotic cell death.