Adenovirus-Mediated Over-Expression of Nrf 2 Within Mesenchymal Stem Cells ( MSCs ) Protected Rats Against Acute Kidney Injury

© 2015 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2015, 5(2), 201-208 doi: 10.15171/apb.2015.028 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
Acute kidney injury (AKI) is described as a sudden and prolonged decrease in glomerular filtration rate.AKI is induced by various reasons including low blood volume from any cause, exposure to substances harmful to the kidney, and urinary tract obstruction.2][3] AKI has been reported in 5 to 7% of hospitalized patients and over 30% of ICU hospitalized patients.The mortality rate of patients with AKI is about 50% and in cases in which dialysis is needed the mortality rate could reach 88%. 4 Therefore, AKI has attracted much research interests in recent years because of its significant mortality rate.A novel strategy for treatment of AKI is MSC transplantation.Promising results of preclinical studies, ease of handling and enormous expansion potential of MSCs, and encouraging preliminary clinical trials of MSC transplantation in other diseases, have made the MSC based approaches very interesting for the treatment of human kidney injury. 5,6][8] The pathogenesis of AKI is complex, however, it has been shown that ischemia/reperfusion triggers AKI, mainly via aggravating stresses, inflammation and activation of renin-angiotensin system (RAS). 91][12] As mentioned before, MSC transplantation could treat AKI; however, the most important problem with the application of MSCs for the treatment is their low survival rate in such a stressful microenvironment.Therefore, protection of MSCs against various stresses is very important for improving their therapeutic potential.The nuclear factor erythroid-2 related factor 2 (Nrf2) is a critical transcription factor for protection of cells against numerous stresses. 13,146][17][18] Previously, in an in vitro study we showed that Nrf2 potentiates MSCs for combating with various stresses. 19Moreover, recent studies have shown that Nrf2 over-expression increases the secretion rate of some growth factors such as VEGF, which might enhance the AKI improvement. 20,21We hypothesized that Nrf2 overexpression in MSCs might exert beneficial therapeutic effects on the cells which could result in their higher survival rate against cisplatin induced stresses, and repairing much tissue than the nonrecombinant MSCs by their differentiation to kidney cells and/or secretion of growth factors.Hence, we transiently over-expressed Nrf2 in rat MSCs using an adenoviral transduction system, and then transplanted the cells to cisplatin-induced AKI rats.Then, we assayed that whether improvement rate of the AKI is higher following transplantation of the Nrf2 over-expressing MSCs comparing to non-manipulated control MSCs.

MSC isolation and identification
For isolation of MSCs, rats were anesthetized with ketamine (40mg/kg) and xylazine (10mg/kg).Then, femurs and tibias of both legs were dissected out and cleaned under sterile operating conditions.Afterward, the marrow cavity was exposed by cutting the spongious end of each bone.We followed the process as described above.MSCs were characterized by adhesion capacity and differentiation assays.All experiments were performed on cells after the third passage.

Nrf2 isolation and construction of recombinant adenovirus
Gateway pAd/CMV/V5-DEST vector and ViraPower Adenoviral Expression System (Invitrogen) were used to construct recombinant adenoviruses, as described elsewhere. 19Briefly, the human MSCs were UV irradiated for one hour, and then subjected to RNA extraction using TriPure isolation reagent (Roche).Afterwards, full-length Nrf2 cDNA was obtained through RT-PCR using specific primers.Then, the amplified cDNA fragment was cloned into pENTER/TEV/DTOPO vector using pENTER Directional TOPO Cloning kit (Invitrogen).Following confirmation of the cloned sequence, the Nrf2 expression cassette was transferred from pENTR gateway vector to the adenovirus expression plasmid pAd/CMV/V5-DEST (Invitrogen) and designated as pAd/CMV-Nrf2.To obtain virus particles, the recombinant pAd/CMV-Nrf2 plasmid was transfected to 293A cells.As a control, pAd/CMV/V5-GW/lacZ vector (Invitrogen) was transfected into 293A cells to produce lacZ-bearing adenoviruses.After infection of rat MSCs with the recombinant adenovirus, the expression of Nrf2 was evaluated with RT-PCR analyses.

In vitro cytotoxic effect of cisplatin on surveillance of Nrf2-MSCs
The cytotoxic effect of cisplatin on the rat MSCs was determined by MTT assay. 19,22Briefly, 2×10 4 rat MSCs/well were seeded in a 96-well plate and appropriate MOIs of the recombinant or control adenoviruses were added to the cells after 12 h.Six days post transfection, cisplatin was added to the wells at various concentrations of 0-250µg/ml, (Sigma, Germany) and after 48h, the cells were subjected to cytotoxicity assay.In this regard, 3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, Dusseldorf, Germany) was added to the cells at final concentration of 0.5 mg/ml and incubated at 37°C in a 5% CO2 atmosphere for 4 h.Finally, the reaction was stopped by addition of 10% SDS and 0.01 M HCl and the absorbance was read at 570 nm.

Animal model of AKI
Male Wistar rats (2 months old, 200-250 g) were obtained from Pasteur Institute of Iran.Animal care and experiments were according to the National Institute of Health's Guide for the Care and Use of Laboratory Animals.For induction of acute kidney injury, rats were given an intraperitoneal injection of cisplatin (5 mg/kg body wt).One day after receiving the cisplatin injection (day 1), the rats were divided into three groups of 10 animals, each received an intravenous injection as follows: (1) control group, which received 0.5ml normal saline; (2) Ad-MSCs group, which received 2×10 6 cells infected with control adenoviruses; (3) Nrf2-MSCs group, which received 2 ×10 6 cells infected with Nrf2 bearing adenoviruses.Blood samples for assessment of blood urea nitrogen (BUN) and determination of creatinine concentration were collected before the cisplatin dose (baseline) and in surviving rats on days 3, 6, 8 and 11 of the experiment.For determination of renal function, and histology and morphometric analysis, the rats were sacrificed on day eleven after the cisplatin injection.For morphologic analysis with light microscopy, kidney specimens were fixed in formaldehyde and paraffin sections of 5µm thickness were stained with hematoxylin and eosin.Then, the luminal hyaline casts and tubular necrosis (cell debris in tubular lumen, denudation of tubular basement membrane and nuclear fragmentation) were assessed in non-overlapping fields (up to 30 for each section) using 40x objective (high-power field).Sections were analyzed by a same pathologist, in a single-blind manner.

Statistical Analysis
The statistical significance of the results was evaluated using analysis of variance, ANOVA, and a Student's ttest.In all tests, p < 0.05 was considered significant.

Adenoviral-mediated gene delivery to MSCs
Recombinant adenovirus production results have been presented in our recent article. 19In a few words, Human MSCs were characterized by their adhesion to tissue culture surfaces, immunophenotyping and differentiation capacities.Nrf2 was isolated from human MSCs and cloned into pENTR/D-TOPO.Subsequently, the fidelity of the cloned sequence was confirmed by sequencing (GenBank accession number HM446346).Finally, recombinant adenoviruses were constructed in 293A Nrf2-MSCs protected rats against AKI Advanced Pharmaceutical Bulletin, 2015, 5(2), 201-208 cells.Our results showed that Nrf2 over-expression by adenovirus system is transient and its expression level increased to the highest at day 6.

Nrf2 protects MSCs against cisplatin induced-toxicities in vitro
To determine whether human Nrf2 can protect bone marrow derived MSCs against cisplatin induced cytotoxicity; the Nrf2 expressing cells were exposed to various concentrations of cisplatin and then subjected to cytotoxicity assay.As it is represented by Figure 1, the rat MSCs infected with Nrf2-adenoviruses were more resistant to 150µg/ml cisplatin comparing to the controls.This confirmed the protective effect of Nrf2 on MSCs against cisplatin induced toxicities.

Nrf2 improves efficacy of MSCs transplantation in animal model of cisplatin induced toxicity
To evaluate whether Nrf2-MSCs exert in vivo protective effects against cisplatin induced toxicity, acute kidney injury was induced in rats by cisplatin administration, an antitumor drug whose clinical use is accompanied by a high incidence of nephrotoxicity, mainly in the form of renal tubular damage.Under light microscope, the kidneys with induced cisplatin toxicity showed AKIassociated tubular lesions including loss of brush border, flattening and loss of the epithelial cells, luminal cell debris and hyaline casts.In addition, renal function tests (BUN and creatinine) showed significant increase (Figure 2).However, in the Ad-MSCs group, intravenous injection of Ad-MSCs strongly protected renal function as reflected by significantly lower amount of BUN and creatinine concentration comparing to the control group which only received normal saline.Furthermore, tubular necrosis and cast formation decreased significantly.Moreover, in the last group, administration of Nrf2-MSCs promoted much more functional and histological improvement.This confirmed the successful function, surveillance and resistance of Nrf2-MSCs in the reactive oxygen species (ROS)-rich microenvironment induced by cisplatin (Figure 3).

Discussion
The prevalence of acute kidney injury ranges from 5% of all hospitalized patients to 30 to 50% in critical care units. 23Application of some growth factors including insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF) and epidermal growth factor (EGF) for enhancement of tubular regeneration in experimental AKI has been one of the successful strategies. 24,25These growth factors rescue surviving tubular epithelial cells which consequently their regeneration repairs the injured kidneys.However, this approach requires plenty of surviving cells. 24,25Another critical strategy that involves a lot of studies in the world is the administration of MSCs.7][28] The mechanism of this treatment is based on the current theory about MSCs, stating that tissue stem cells replace dead tissue cells under physiologic conditions.Following tissue injury, mobilized stem cells replenish stem cell repertoire lost.When cell lost prevail stem cell repair, tissue injury occurs.Cytokines and chemokines secreted from injured tissue attract mobilized stem cells to repair site.Therefore, mesenchymal stem cells could not contribute in tissue repair and not enter organ in absence of injury. 29ifferent studies have shown that MSCs exert their therapeutic effects via two distinct mechanisms including differentiation to target tissue cells and secretion of growth factors.Morigi et al. and Herrera et al. reported that MSC mediated protection is a result of the capability of MSCs to engraft into a damaged kidney. 24,30In addition, differentiation of MSCs into renal cell types has been observed after AKI.Moreover, Poulsom et al. showed that when Y chromosome containing MSCs are injected into female mouse with AKI, the transplanted MSCs are recruited into peritubular sites. 31In contrast, other studies have shown that the valuable effects of MSCs are mediated via paracrine activities.Some growth factors and chemokines, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor, monocyte-chemoattractant protein-1, HGF, and insulin-like growth factor-1 (IGF-1), are secreted by MSCs which enhance epithelial proliferation, modulate inflammation, or promote angiogenesis, and therefore are good candidates for therapy of AKI. 6 Many stresses are involved in AKI pathogenesis.Over 50% of hospitalized cases of AKI are caused by renal ischemia. 32Ischemia is thought to create an inhospitable environment by inducing local expression of inflammatory cytokines that promote cell apoptosis. 33schemia has two important components including hypoxia and serum deprivation. 34Also, it has been demonstrated that ischemia can cause oxidative stress. 35n addition to tubular cells, these stresses cause apoptosis in transplanted MSCs. 19,34MSCs are more susceptible to death than other tissue cells. 36,37Therefore, not only MSCs alleviate the stresses, but they are died by stresses.Various factors such as strength and length of stresses, number of infused MSCs and the level of growth factors determine the outcome of this battle.Previously, we showed that overexpression of Nrf2 in mesenchymal stem cells reduces oxidative stress-induced apoptosis and cytotoxicity. 19Also, the protective role of Nrf2 has been previously shown by numerous studies. 14,16,38Higher surveillance rate of MSCs in stressful microenvironments could result in successful regeneration of the damaged tissue by the MSCs.Nrf2 is a transcription factor which activates expression of multiple antioxidants and detoxifying enzymes. 39,40herefore, Nrf2 overexpression in MSCs might decrease cytotoxic effect of stresses.2][43][44] As mentioned previously, administration of growth factors is one of the current strategies in AKI treatment.Consequently, Nrf2 overexpression in MSCs has a dual and synergistic protective effect on surveillance of MSCs which can result in successful treatment of AKI.Because of homology between human and rat Nrf2 proteins, 45 here we used the recombinant adenovirus vector harboring human Nrf2 coding sequence constructed in our previous study. 19Previously, we showed that the adenovirus mediated expression of Nrf2 protein is transient.In addition, Nrf2 overexpression has no deleterious effect on differentiation capacity of MSCs into adipocytic, osteoblastic and chonderocytic lineages.In this study we showed that human Nrf2 could protect rat MSCs against cisplatin in vitro.This capability makes Nrf2 an excellent protein for improving transplantation process, especially for treatment of acute kidney injury.In this study, we hypothesized that Nrf2, as a potent cytoprotective transcription factor, could protect MSCs against stresses during transplantation.To verify this hypothesis, Nrf2-MSCs were transplanted into cisplatininduced AKI rats and their therapeutic efficacy was assessed by measuring blood urea nitrogen and creatinine concentration and morphologic analysis.AKI was diagnosed according to RIFLE criteria.These criteria include three categories of injury (Risk, Injury, and Failure with increasing severity) and two classes of kidney outcome (loss of function, and end-stage renal disease).Serum creatinine increases to 1.5-fold from baseline in the risk class.][52][53][54][55] These morphologic changes have been also seen in cisplatin treated rats.Because of the beneficial therapeutic effect of MSCs, morphological abnormalities decreased in Ad-MSCs rats.However, the Nrf2-MSCs were more effective in decreasing the morphological abnormalities comparing to the Ad-MSCs.Therefore, it could be deduced that Nrf2 overexpression protects MSCs from stress-related conditions in vivo.Nevertheless, a possible disadvantage of Nrf2 overexpression is the risk of tumor progression. 56Recent studies have shown that an Nrf2 suppressor, Keap1, is often mutated in cancer cells resulting in a constitutive activation of Nrf2. 57,58In addition, it has been demonestrated that a hypoxic tumor microenvironment leads to increased transactivation of Nrf2. 591][62][63] However, in the present study, no sign of tumorogenic transformational changes have been observed.

Conclusion
The obtained results showed that Nrf2 could inhibit apoptosis induction in MSCs transplanted into the cisplatin-induced AKI rats.5][66][67] Therefore, MSCs probably exert their protective effect on cisplatininduced AKI in a paracrine manner, i.e. via production of growth factors and anti-inflammatory cytokines.Localization of MSCs in peritubular interstitial areas indicates that the MSCs acted by exerting paracrine activity. 23The Nrf2-MSCs produced higher amount of growth factors and therefore, can be more effective than the non-Nrf2 overexpressing Ad-MSCs in improvement of kidney functions.

Figure 2 .
Figure 2. Protective effects of Nrf2 on MSCs following their transplantation in rats suffering from cisplatin-induced acute kidney injury.A; Assessment of renal function by measuring BUN values.B; Evaluation of serum creatinine concentrations as an indicator of renal function (Mean±SE, **: P<0.01 and ***: P<0.001).

Figure 3 .
Figure 3. Renal histology following transplantation of Nrf2-MSCs and Ad-MSCs.A, B and C; Light microscopy images of renal tissue from rats (H&E stained kidney sections, 400×).A; Cisplatin group: tubules showed extensive and marked changes consisting of flattening, loss of epithelial cells and brush border.B; Ad-MSC group: less severe tubular damage and very mild cell swelling were observed.C; Nrf2-MSC group: higher recovery and similarity to normal morphology of kidney.D; Renal histology (casts and tubular necrosis, quantified as number per high power field (HPF)) of cisplatin treated rats.Tubular necrosis and lesions included loss of brush border and epithelial cells, flattening, , and luminal cell debris.(Mean±SE, *: P<0.05, **: P<0.01 and ***: P<0.001) Nrf2-MSCs developed injury and risk classes of AKI, respectively.It showed that MSC transplantation can reduce kidney damage and overexpression of Nrf2 in MSCs potentiates them against AKI.In the morphologic aspect, necrosis and apoptosis of tubular cells lead to tubular obstruction by cast formation.Casts are composed of shed epithelial cells and necrotic debris.Cytoskeletal disruption, ATP | 205 Nrf2-MSCs protected rats against AKI Advanced Pharmaceutical Bulletin, 2015, 5(2), 201-208