Mesenchymal stem cells (MSCs) are recognised as a promising tool to improve renal recovery in experimental models of cisplatin-induced acute kidney injury. However, these preclinical studies were performed on severely immunodeficient animals. Here, we investigated whether human umbilical cord derived MSC treatment could equally ameliorate acute kidney injury induced by cisplatin and prolong survival in mice with a normal immune system and those with a suppressed immune system by polyclonal antithymocyte globulin (ATG). We demonstrated that ATG pretreatment, when followed by MSC transplantation, significantly improved injured renal function parameters, as evidenced by decreased blood urea nitrogen and serum creatinine concentration, as well as improved renal morphology. This tissue restoration was also supported by increased survival of mice. The beneficial effects of ATG were associated with reduced level of inflammatory protein serum amyloid A3 and induced antioxidative expression of superoxide dismutase-1 (SOD-1), glutathione peroxidase (GPx), and hem oxygenase-1 (HO-1). Infused MSCs became localised predominantly in peritubular areas and acted to reduce renal cell death. In conclusion, these results show that ATG diminished in situ inflammation and oxidative stress associated with cisplatin-induced acute kidney injury, the effects that may provide more favourable microenvironment for MSC action, with consequential synergistic improvements in renal injury and animal survival as compared to MSC treatment alone.
Over the last decade, mesenchymal stem cells (MSCs) were shown to be one of the promising tools to treat acute kidney injury (AKI) in various animal models, including cisplatin-induced nephrotoxicity [
It is known that both innate and adaptive immune systems are important contributor to the pathogenesis of cisplatin-induced AKI and can significantly affect the extent of cisplatin nephrotoxicity [
Antithymocyte globulin (ATG) has been successfully used for decades in clinical transplantation due to its immunosuppressive role in GVHD and solid organ rejection. The immunosuppressive effects of ATG in clinical transplantation have been mostly attributed to its ability to reduce circulating T lymphocytes. However, recent data suggest that ATG has many pleiotropic immunomodulatory properties including inhibition of B lymphocytes, dendritic and natural killer cells as well as modulation of surface adhesion molecules and chemokine receptor expression [
To evaluate the interference of compromised immune system on MSC therapy in cisplatin-induced AKI, we analyzed morphological, functional, oxidative, and inflammatory alterations in the kidney of mice with a normal immune system and those with a suppressed immune system by ATG. Our aim was to evaluate both the separate and combined effects of ATG and MSCs on cisplatin-induced acute nephrotoxicity.
Umbilical cord (UC) derived MSCs were isolated from Wharton’s jelly according to standard protocol [
Detection and intrarenal localization of MSCs labeled with red fluorescence cell dye DiI (Invitrogen, Carlsbad, CA, USA) was analyzed by fluorescence microscope Nikon Eclipse TE 300 (Amstelveen, Netherlands). Firstly, MSCs in culture were labeled with lipophilic dye DiI (5
All procedures involving animals were approved by the National Ethical Committee and the Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection (Permit number 34401-54/2012/5). Animal care and treatment were conducted in accordance with the institutional guidelines and international laws and policies (Directive 2010/63/EU on the protection of animals used for scientific purposes).
Experiment was carried out on 8–12-week-old male BALB/cOlaHsd mice (Harlan, Italy;
Models of AKI were induced by intraperitoneal (ip) injection of a single dose of 17 mg/kg of cisplatin (Pliva-Teva, Croatia) dissolved in 0.9% saline solution (1 mg/2 mL). The dosage of cisplatin was based on literature analysis and results of our preliminary experiment, showing renal function impairment, as indicated by significantly increased levels of blood urea nitrogen (BUN) and creatinine as well as marked histologic changes in animals at day 4 after cisplatin application.
Antimouse antithymocyte globulin (ATG, Fresenius-Biotech-GMBH, Germany) was used as pretreatment to induce partial immunotolerance in mice. ATG is a polyclonal purified IgG fraction of sera from rabbits immunized with mouse thymocytes. ATG was provided as solution of 15.2 mg/mL and was injected at a dosage of 1.8 mg ip 3 consecutive days before cisplatin application (Figure
Shematic representation of the protocol and all control and experimental groups.
Half of the animals for each experimental group (
Mice in all groups were treated in the same manner and have undergone the same procedures. For instance, mice received saline instead of cisplatin or 0.2 mL of PBS instead of MSC. The animals were divided into 4 control groups (
On the day of euthanasia, blood was immediately collected by cardiac puncture and serum levels of blood urea nitrogen (BUN) and creatinine were measured using a blood chemistry analyzer.
For renal histology, 4
The severity of renal toxicity, including damage to the glomeruli, tubules, interstitium, and renal blood vessels, was assessed as described previously [
The criteria of a semiquantitative scoring scale of 0–5 were as follows: 0 is normal tubules, glomerulus, interstitium, and vessels. 1 is scant number of tubular epithelial cells showing minimal degeneration, mild tubular dilatation, small number of proteinaceous casts, no regeneration, and no definitely significant necrosis or apoptosis and no changes in the glomerulus, interstitium, and vessels. 2 is <25% of tubular epithelial cells showing mild degeneration (large cytoplasmic vacuoles, a few hyaline droplets in the cytoplasm), mild degree of tubular dilation and proteinaceous casts, slight change in tubular brush border loss, acute tubular necrosis in individual cell or small group of cells, a few apoptotic cells, and no regeneration and no changes in the glomerulus, interstitium, and vessels. 3 is 25%–50% of tubular epithelial cells showing moderate degeneration (multiple large-sized vacuoles, multiple foci of hyaline droplets), mild regeneration, moderate tubular brush border loss, moderate acute tubular necrosis in small group of tubules, and increased number of apoptotic cells; little involvement of mild glomerular vacuolization; and no changes in the interstitium and vessels. 4 is 51%–75% of tubular epithelial cells showing extensive moderate degeneration; moderate regeneration; severe tubular brush border loss; severe acute tubular necrosis; and a large number of apoptotic cells, with apoptotic bodies in clusters of tubules, and little involvement of mild glomerular vacuolization and interstitial lymphocytic infiltration. 5 is >75% of tubular epithelial cells showing severe degeneration, regeneration, severe tubular brush border loss, acute tubular necrosis, and a large number of apoptotic cells with numerous apoptotic bodies; mild involvement of glomerular injury (vacuolization, mesangial cell proliferation, and increase in mesangial matrix) and interstitial lymphocytic infiltration; and no significant changes in the vessels.
Tissue pieces were fixed in 4% formaldehyde overnight. After embedding in paraffin and deparaffinization through graded alcohols, 4
Examination of ultrastructural changes of renal proximal tubule cells was performed by transmission electron microscopy. Pieces of kidney tissue were fixed in a mixture of 4% paraformaldehyde and 2% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.3) for 3 hours at 4°C. After rinsing in 0.33 M sucrose in the same buffer and postfixation in 1% OsO4 for 1 hour, tissue samples were dehydrated through ascending grades of ethanol and embedded in Epon (Serva Electrophoresis, Heidelberg, Germany). Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Jeol 100 CX (Tokyo, Japan) electron microscope.
Equivalent parts of the mouse kidney were homogenized in lysis buffer with TissueLyser LT (Qiagen, Hilden, Germany) and total RNA was isolated from homogenate using RNeasy Micro Kit (Qiagen, Hilden, Germany) following the manufacturers’ instructions. The purity and amount of RNA were determined by measuring the OD at a ratio of 260 to 280 nm. cDNA was generated from 1
Mouse primers used for QPCR analysis of inflammatory cytokines and oxidative stress enzyme expression in renal tissue.
Parameter | Primer |
---|---|
IL-1 |
F: 5′-CAACCAACAAGTGATATTCTCCATG-3′ |
R: 5′-GATCCACACTCTCCAGCTGCA-3′ | |
|
|
ICAM-1 | F: 5′-CAATTTCTCATGCCGCACAG-3′ |
R: 5′-AGCTGGAAGATCGAAAGTCCG-3′ | |
|
|
HO-1 | F: 5′-GGTGATGGCTTCCTTGTACC-3′ |
R: 5′-AGTGAGGCCCATACCAGAAG-3′ | |
|
|
SOD1 | F: 5′-CCAGTGCAGGACCTCATTTT-3′ |
R: 5′-CACCTTTGCCCAAGTCATCT-3′ | |
|
|
SOD2 | F: 5′-GGCCAAGGGAGATGTTACAA-3′ |
R: 5′-GAACCTTGGACTCCCACA-3′ | |
|
|
CAT | F: 5′-CCGACCAGGGCATCAAAA-3′ |
R: 5′-GAGGCCATAATCCGGATCTTC-3′ | |
|
|
GPx | F: 5′-CCACCGTGTATGCCTTCTCC-3′ |
R: 5′-GATCGTGGTGCCTCAGAGAG-3′ | |
|
|
TGF |
F: 5′-GACCGCAACAACGCCATCTA-3′ |
R: 5′-GGCGTATCAGTGGGGGTCAG-3′ | |
|
|
SAA3 | F: 5′-TGC CAT CAT TCT TTG CAT CTT GA-3′ |
R: 5′-CCG TGA ACT TCT GAA CAG CCT-3′ |
SAA3: serum amyloid A3, HO-1: heme oxygenase-1, GPx: glutathione peroxidase, CAT: catalase, SOD-1: superoxide dismutase-1, SOD-2: superoxide dismutase 2, IL-1
The results are expressed as mean ± SEM. Statistical comparison of the data was performed using the
Results show that ATG caused selective immunosuppression with depletion of lymphocytes, eosinophils, and basophils without significant influence on other parameters of differential white cell spectrum or on platelet or erythrocyte count (Table
Effect of ATG treatment on haematologic parameters in adult BALB/cOlaHsd mice (
ATG | Control | |
---|---|---|
WBC (10/ |
705 ± 354 | 1434 ± 416 |
RBC (104/ |
1041 ± 121 | 883 ± 176 |
Hb (g/L) | 152 ± 21 | 137 ± 26 |
Hct (10−1%) | 496 ± 45 | 415 ± 59 |
MCV (10−1 fL) | 478 ± 33 | 473 ± 26 |
MCH (10−1 pg) | 146 ± 6 | 155 ± 2 |
MCHC (g/L) | 306 ± 22 | 328 ± 15 |
PLT (10−3/ |
1518 ± 238 | 1425 ± 289 |
NEUT (10/ |
282 ± 214 | 750 ± 384 |
LYM (10/ |
|
676 ± 245 |
MONO (10/ |
91 ± 50 | 201 ± 105 |
EOS (10/ |
|
25 ± 1 |
BAS (10/ |
|
1.3 ± 1.2 |
A
Body weight, relative kidney weight, and renal function in BALB/cOlaHsd mice 4 days after cisplatin administration.
Control | MSCs | ATG | ATG + MSCs | CIS | CIS + MSCs | ATG + CIS | ATG + CIS + MSCs | |
---|---|---|---|---|---|---|---|---|
Body weight (g), day 0 | 27.3 ± 0.6 | 25.6 ± 1.2 | 27.6 ± 2.2 | 27.4 ± 2.7 | 24.9 ± 0.7 | 24.6 ± 0.8 | 26.6 ± 0.4 | 26.9 ± 0.3 |
Body weight change (g) (day 0–day 4) | 0.9 ± 0.4 |
0.2 ± 0.2 |
0.3 ± 0.4 |
0.5 ± 0.4 |
(−) |
(−) |
(−) |
(−) |
Kidney relative weight ×100 | 1.68 ± 0.05 |
1.69 ± 0.06 |
1.67 ± 0.05 |
1.74 ± 0.05 |
1.71 ± 0.27 |
1.67 ± 0.17 |
1.79 ± 0.19 |
1.51 ± 0.06 |
BUN (mmol/L) | 8.05 ± 0.77 |
7.43 ± 0.2 |
8.4 ± 0.47 |
6.95 ± 0.37 |
43.87 ± 6.57 |
42.7 ± 4.63 |
40.26 ± 6.84 |
31.34 ± 3.99 |
Creatinine ( |
30.5 ± 0.77 |
18.0 ± 0.2 |
18.25 ± 0.47 |
31.5 ± 7.63 |
83.67 ± 19.43 |
48.0 ± 16.83 |
42.0 ± 3.96 |
27.5 ± 1.91 |
ANOVA followed by Duncun multicomparison test; results are expressed as mean ± SEM; values with different superscript letters in rows are statistically different (
Labeled MSCs were detected in different organs (lungs, liver, intestine, and kidney). In kidney, MSCs were found predominantly in peritubular regions; occasionally, they were localized within the proximal tubule epithelium and never in glomeruli (Figure
Representative images of kidney tissue in mice with cisplatin-induced AKI treated with DiI labeled MSCs (day 4). (a) DiI labeled MSCs (red fluorescence) in peritubular area. Original magnification: ×1000. (b) DiI labeled MSCs (red fluorescence) within proximal tubule epithelium. Original magnification: ×1000. Nuclei are stained with DAPI (blue fluorescence). Basal lamina of proximal tubule is marked with a gray line.
Administration of cisplatin resulted in reduction of body weights in all cisplatin treated groups. Mice with cisplatin-induced AKI had the lowest survival, while mice treated with single injection of MSCs after ATG immunosuppression had the highest survival among cisplatin treated groups (43.5% survival at day 30). Survival data and curves of cisplatin treated mice given MSCs or/and ATG are shown in Figure
Schematic representation of survival curves in BALB/cOlaHsd cisplatin treated mice. The survival was estimated by Kaplan-Meier statistical analysis. Mice pretreated with ATG had an increased survival after MSCs treatment during their follow-up (log rank
In contrast to all control groups, mice treated with cisplatin showed morphological and functional changes in kidneys 4 days after treatment as demonstrated by a significant increase in histology score, serum levels of BUN (
MSC treatment after ATG immunosuppression improves renal function and morphology as well as intestine morphology in BALB/cOlaHsd mice 4 days after cisplatin administration. (a) Representative micrographs of renal histology of healthy control mice and of mice treated with cisplatin and saline or MSCs and/or ATG (original magnification: ×200, periodic acid-Schiff (PAS) staining). (b) Serum concentration of blood urea nitrogen (BUN) and creatinine and caspase 3 score. Graphical results are expressed as mean ± SEM; ANOVA followed by Duncun multicomparison test:
Improvement of both renal morphology and function was found only in mice treated with both ATG and MSCs. Cisplatin mice treated with ATG and MSCs had significantly lower relative kidney weight (
At day 4 after cisplatin administration, kidneys of mice treated with ATG showed tubular lesions consisting of multiple large-sized vacuoles, multiple foci of luminal cell debris and hyaline casts, moderate tubular brush border loss, and moderate acute tubular necrosis in small group of tubules. Changes were seen in proximal as well as distal tubules, while glomerular changes were not detected. Mice treated with MSCs showed less damage in the kidney, but the difference was not significant. On the other hand, treatment with MSCs after ATG immunosuppression resulted in significant improvement. In particular, tubular epithelial cells showed minimal degeneration with rare necrosis and apoptosis, and hyaline casts were absent (Figure
Interestingly, although treatment with ATG or MSCs alone had no significant improvement on morphology of cisplatin-damaged kidney, serum creatinine concentrations (but not BUN) were reduced in both instances. It is known that serum concentration of creatinine is not always in strict correlation with glomerular filtration. Intestinal disturbances caused by some chemotherapeutics, including cisplatin, could raise serum creatinine concentration. Thus, processes other than a decline in renal function need to be excluded if BUN concentration remains unchanged.
Based on observation of this discrepancy and the presence of bloody diarrhea in cisplatin treated mice, the intestine was morphologically evaluated. Histological analyses revealed that cisplatin administration resulted in acute inflammation of the intestine with moderate to severe injury of crypts evidenced by crypt abscesses, cryptitis, and crypt dilatation. The villi shortened or in some cases completely disappeared (Figure
Results of transmission electron microscopy analysis confirmed histological results and revealed that cisplatin administration resulted in markedly changed ultrastructure of tubular epithelial cells, which showed typical characteristics of necrotic cell death such as ruptured apical plasma membrane, disrupted microvilli, swollen nucleus with disintegrating chromatin, dilatation of the endoplasmic reticulum, Golgi apparatus and perinuclear space, and vacuolized and disintegrated cytoplasm eventually (Figures
Kidney ultrastructure. (a) Ultrastructure of epithelial cell with large and rounded nucleus (N), numerous mitochondria (M), and pronounced microvilli (MV). (b) Necrotic epithelial cell (asterisk) with typical ultrastructural signs of necrosis such as electron pale and disintegrated cytoplasm, disrupted microvilli, and few mitochondria (M). L-lumen of proximal tubule. (c) Details of necrotic epithelial cell with strongly dilated perinuclear space (arrowhead), ruptured Golgi apparatus (asterisk), and highly demolished cytoplasmic integrity with hardly recognizable structures. (d) Epithelial cell with no brush border on apical surface, multilamellar bodies (arrowheads), and extremely large autophagic vacuoles (arrows) and fulfilling the entire cytoplasm. N-nucleus. (e) Apoptotic epithelial cell with condensed chromatin in nuclear fragments (N), lipid droplets (L), disappeared microvilli, and electron dense and condensed cytoplasm (asterisk). (f) Epithelial cells of proximal tubule with normal ultrastructure. Under basal lamina (arrow) of tubule, peritubular cell (asterisk) of connective tissue with typical signs of apoptosis such as cytoplasm condensation and chromatin fragmentation and condensation is present. Representative TEM images of kidney samples taken from control mice (a), mice receiving ATG and cisplatin (b–d), and mice receiving MSCs after ATG and cisplatin (e, f). Original magnifications: ×4400 (a), ×2600 (b), ×5800 (c), ×2600 (d), ×4400 (e), and ×2000 (f).
In all experimental groups, the number of caspase 3 positive tubular cells was low (on average
SAA3 is an important inflammatory marker and acute phase protein in mice. As expected, cisplatin treatment resulted in a significant increase in SAA3 expression (5-fold increase compared to control). Interestingly, MSC treatment had no beneficial effects on SAA3 expression (
Cisplatin treatment significantly reduced expression of the main oxidative enzymes in kidney, such as CAT, SOD1, and SOD2, but had no significant influence on GPx. The upregulation of ICAM-1 or downregulation of CAT, SOD1, and SOD2 was not restored by ATG or MSC treatment.
ATG pretreatment attenuated the mRNA expression of SAA3 and accelerated expression of HO-1 in kidneys of cisplatin treated mice. ATG pretreatment resulted also in significant increase in GPx expression in kidneys of both healthy and cisplatin treated mice. In contrast, MSC treatment had no significant beneficial effect on altered expression of measured proteins induced by cisplatin. Likewise, MSC or ATG treatment alone had no influence on the expression pattern of other proteins measured in kidneys of healthy mice. Gene expression of inflammatory and oxidative proteins in kidneys is shown in Figure
Gene expression of inflammatory and oxidative proteins in kidneys of 8–12-week-old male BALB/cOlaHsd mice after cisplatin-induced AKI treated with ATG and/or MSCs. Bars represent alterations in expression of cytokines, inflammatory, and oxidative proteins as measured by RT-PCR in kidney tissues of sacrificed mice. Results are expressed as mean ± SEM. ANOVA followed by Duncun multicomparison test:
The present study demonstrates for the first time that suppression of an immune system by ATG significantly improves beneficial effects of MSC transplantation in cisplatin-induced AKI in mice. Results demonstrate that treatment with MSCs alone resulted in tendency but not significant improvement of functional and morphological changes in cisplatin-induced AKI, while administration of MSCs after ATG immunosuppression resulted in amelioration of renal function and in increased survival of mice. Mice treated with MSCs after ATG immunosuppression had restored structure of kidney tissue on histologic and ultrastructural level. Only mild lesions, such as mild degeneration and slight change in tubular brush border with rare apoptotic or necrotic cells, were observed in renal tubular epithelial cells.
It is believed that one of MSCs’ beneficial effects is related to impaired apoptosis [
Various mechanisms are involved in cisplatin nephrotoxicity, including oxidative stress and inflammation [
To evaluate immunosuppressive efficacy of ATG and MSCs on cisplatin-induced AKI, one of the most known proteins produced in the acute phase of inflammation, serum amyloid A3 protein (SAA3), was assessed. Many acute phase proteins are upregulated early after renal failure as a consequence of inflammatory response in AKI [
Nevertheless, our results show that ATG significantly suppressed SAA3 in cisplatin-induced AKI. It was shown that ATG has ability to reduce infiltration of CD3+, CD4+, and CD8+ lymphocytes as well as neutrophils in the injured tissue [
Cisplatin-induced AKI is associated with the release of many soluble mediators and activation of adhesion molecules from activated or injured kidney cells (such as ICAM-1, TGF-
It was reported that ATG and MSCs have immunomodulatory effects, ATG by modulating surface adhesion molecules such as ICAM-1 [
To evaluate oxidative mechanisms of ATG alone and in combination with MSCs, we assessed expression of antioxidant enzymes in renal tissue. In agreement with others [
On the other hand, MSCs administration alone had no influence on white cells in the blood or the expression of SAA3 or other oxidative and inflammatory proteins (ICAM-1, HO-1, IL-1
Since we used lethal murine model of AKI with multiorgan failure, it is very likely that single administration of MSCs was insufficient to cope with extremely harmful environment. As demonstrated, cisplatin caused not only acute nephrotoxicity but also acute severe intestinal inflammation with bloody diarrhea, which affected creatinine levels and very likely also other molecules. Multiorgan failure results in altered hemodynamic state and renal vascular autoregulation, which further worsens renal function. Such models introduce much complexity in treatment strategy, especially in regard to systemic treatment and cell based therapies. It was suggested that one of the major problems influencing the efficacy of stem cell therapy is the poor MSCs survival following transplantation. This could at least partly be attributed to insufficient resistance of transplanted stem cells to oxidative and inflammatory stresses at the injured sites. Although MSCs may require activation by signals from a proinflammatory environment to modulate the activity of surrounding cells [
Taken together, our study shows that ATG pretreatment in MSC therapy, directed against cisplatin-induced AKI, significantly improved renal status as observed by morphological and functional parameters of injured kidney as well as prolonged lifespan of mice. The observed positive effects were due to reduced inflammation and cell death, caused by cisplatin, as measured also by immunological mediators and oxidative enzymes. ATG pretreatment created favourable immunological environment in immunocompetent mice, by both diminishing the level of inflammation and oxidative stress at the site of injury that may allowed further immunomodulatory activities of MSCs. As reviewed by Motaln and Lah [
The authors declare no conflict of interests regarding the publication of this paper.
The authors acknowledge Slovenian Research Agency (ARRS) Program Grants P3-0054, P3-0108, P3-0323, P1-0245, and P3-0314 in supporting part of this work. They then thank Dr. Helmut Schneeberger from Fresenius Biotech GmbH, Germany, for providing mice polyclonal ATG; Roche, Slovenia, for financial support in providing reagents for immunohistochemistry analysis; and Pliva-Teva, Croatia, for providing cisplatin. The authors acknowledge Mrs. Ksenija Kodra for dedicated animal care and Mr. Andrej Haler for valuable technical help in performing bioinformatics analysis.