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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Funding
  8. References

Ischemia and subsequent reperfusion (I/R) damage kidney tubular cells and consequently impair renal function. Rabbit bone marrow mesenchymal stem cells (BM-MSCs) expressing human bone morphogenic protein-7 (hBMP-7) regenerated tubular cells and improved renal function in a kidney I/R model. Rabbits were injected immediately after I/R with one of the following: (i) hBMP-7-transduced BM-MSCs (BM-MSCshBMP-7); (ii) enhanced green fluorescent protein–transduced BM-MSCs (BM-MSCsEGFP); or (iii) PBS. The activity of superoxide dismutase (SOD) was higher, and the amount of malondialdehyde (MDA) was lower in the BM-MSCshBMP-7 group than in the BM-MSCsEGFP group. Both the BM-MSCshBMP-7 group and the BM-MSCsEGFP group had higher SOD activity and lower amounts of MDA than the PBS group. Bcl-2- and Bcl-2-associated X protein levels, and other variables, indicated the regeneration of the kidney in both experimental groups. However, the BM-MSCs hBMP-7 group showed higher activity than the BM-MSCsEGFP group, indicating that the combined strategy of BM-MSC transplantation with hBMP-7 gene therapy could be a useful approach for the treatment of renal IRI.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Funding
  8. References

Renal ischemia–reperfusion injury (IRI) causes acute renal failure (ARF). Ischemia followed by reperfusion is a common cause of ARF in clinical practice and usually results in a high mortality (30%–50%) because of the absence of efficient therapeutic strategies (Rabb et al. 1997). Oxygen free radicals (OFR) play a critical role in the pathophysiology of IRI (Kadkhodaee et al. 1995; Nath & Norby 2000); their presence consumes a large amount of antioxida\nts and antioxidases (such as SOD, catalase [CAT], peroxidase [POD] and glutathione peroxidase [GSH-PX]). Thus, the balance between OFR production and clearance is interrupted, and the antioxidative defense is compromised during IRI. OFR damages the cell membrane, resulting in the entry of extracellular calcium and subsequent cell death (Geeraerts et al. 1991). Specifically, mitochondrial function of renal cortex cells decreases, which in turn reduces the synthesis of ATP and therefore inactivates ATP-dependent ion transport in the cell membrane (Tanaka et al. 2004). Intracellular calcium accumulation followed by mitochondrial uptake leads to cytoskeletal damage, cell membrane injury and DNA degradation, resulting in massive necrosis and apoptosis (Lien et al. 2003).

The necrosis and apoptosis of renal tubular cells is related not only to the degree of ischemia but also to the tolerance of these cells to ischemia. Specifically, the cells of the proximal renal tubule and thick ascending limb of loop of Henle are susceptible to IRI (Gobe et al. 1999). IRI alters tubular cell polarity and causes necrotic and apoptotic death (Sheridan & Bonventre 2000). The resulting loss in renal function may be recovered by repairing the damaged tubular cells to a certain extent following the removal of pathogenic factors (Esson & Schrier 2002); however, some renal damage is irreversible, thereby reducing the kidney function (Zeisberg et al. 2005).

Bone marrow mesenchymal stem cells isolated from bone marrow mononuclear cells are able to differentiate terminally to multiple types of cells, including renal tubular cells. They can be expanded ex vivo. These cells may also contribute to renal parenchymal turnover and regeneration (Jiang et al. 2002; Kale et al. 2003). They migrate to the injured kidney and regenerate and repair damaged tubules (Mollura et al. 2003) possibly through reduced inflammation by inducing anti-inflammatory-related cytokines, including interleukin-4 and interleukin-10 (Semedo et al. 2007, 2009). The significance of a potential BM-MSCs therapy for IRI-induced ARF is evident from the current poor prognosis of the disease (Mollura et al. 2003).

Bone morphogenic protein-7 (BMP-7) regulates the cell proliferation and is important for renal development and physiology (Li et al. 2004). The amount of renal BMP-7 decreases after IRI (Simon et al. 1999), a phenomenon that can be attributed to decreased histone acetyltransferase activity (Marumo et al. 2008). Thus, applying exogenous BMP-7 to an IRI-damaged kidney may regenerate the tubules and restore renal function (Vukicevic et al. 1998).

We therefore sought to find whether expressing BMP-7 in BM-MSCs would enhance the cellular regenerative effect of BM-MSCs in a renal I/R model. The results presented in this study show that BM-MSCshBMP-7 enhanced the cellular regenerative effect of BM-MSCs and improved renal function.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Funding
  8. References

The recombinant adenovirus–transduced BM-MSCs

To determine the optimal MOI for BM-MSC transduction, we determined a balance between transduction efficiency and cell viability. BM-MSCs were cultured for three passages, transduced with Ad-hBMP-7 or EGFP recombinant adenoviruses (Ad-EGFP) and analyzed using a flow cytometer. We experimented with four MOIs: 75, 100, 125 and 150 (results not shown). The MOI of 100 was optimal with a transduction efficiency of 90.52%.

hBMP-7 gene expression in BM-MSCs and their detection in renal tissue after transplantation

The expression of hBMP-7 in transduced BM-MSCs was detected by RT-PCR. A specific amplified band corresponding to an expected 162-bp PCR product was observed in hBMP-7-transduced BM-MSCs but not in non-hBMP-7-transduced BM-MSCs (Fig. 1A). Similarly, Western blot analysis detected a 55-kDa band, corresponding to hBMP-7 only in hBMP-7-transduced BM-MSCs (Fig. 1B).

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Figure 1.  Analysis of hBMP-7 expression in adenovirus-infected bone marrow mesenchymal stem cells (BM-MSCs). (A) RT-PCR detection of hBMP-7 in BM-MSCs. Three days after viral transduction, hBMP-7 mRNA was detected in Ad-hBMP-7-transduced BM-MSCs (lanes 3 and 4), but not in Ad-EGFP-transduced BM-MSCs (lane 2) or untransduced BM-MSCs (results not shown). (B) Western blot shows 55-kDa-specific bands in hBMP-7-transduced BM-MSCs lysate, indicating the expression of hBMP7 (lanes 3 and 4) compared with EGFP-transduced BM-MSCs (lane 1) or untransduced BM-MSCs (lane 2). (C) ELISA. Culture media collected at different time points were examined for hBMP-7 secretion. There was <25 pg/mL in Ad-EGFP-transduced BM-MSCs culture medium, but 800 pg/mL in Ad-hBMP-7-transduced BM-MSCs culture medium at 3 days and remained at a high level over a 1-week period (P < 0.001). (D) RT-PCR detection of hBMP-7 mRNA in renal tissue. RNA samples were prepared from renal tissues 3 days after cell transplantation. At 3 days, no hBMP-7 mRNA was detected in the samples extracted from the renal tissues of the BM-MSCsEGFP group (lanes 2–4), but it was highly expressed in the BM-MSCshBMP-7 group (lanes 5–7).

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We also measured hBMP-7 levels in the culture medium; it was markedly higher in Ad-hBMP-7-transduced BM-MSCs as compared with Ad-EGFP-transduced BM-MSCs in the same time point and reached a peak level at 7 day after transduction (< 0.001, Fig. 1C). At day 3 after transplantation, we isolated renal RNA and confirmed human BMP-7-specific mRNA expression by RT-PCR. Human BMP-7 mRNA was detected in the RNA sample extracted from the renal tissue of the BM-MSCshBMP-7 group animal but was not detected in the renal RNA extracted from the rabbits of the BM-MSCsEGFP group, PBS group or Sham group (Fig. 1D).

Detection of BM-MSCs in the renal tissue of rabbits received transplantation

To verify whether the transplanted BM-MSCs can be incorporated into the damaged renal tissue, BM-MSCs were treated with Hoechst33342 stain and injected into IRI rabbits. We determined that the treated BM-MSCs were localized in renal tissue by detecting cells with nuclear blue fluorescence in the kidney (Fig. 2A, arrow head). Immunostaining of the kidney showed the expression of cytokeratin 18 (CK18) in the cells of the renal tubule (Fig. 2B). Juxtaposition of Hoechst33342 and CK18 immunostaining showed the localization of BM-MSCs in the renal tubules (Fig. 2C).

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Figure 2.  Detection of BM-MSCs in the renal tissue of transduced rabbits. (A) The bone marrow mesenchymal stem (BM-MS) cells are stained with Hoechst33342 and then infused into IRI-treated rabbit. The blue fluorescence is from Hoechst33342. (B) CK18 (cytokeratin 18) staining for the renal tubular cell. (C) The merged image of 2A and 2B represents the presence of BM-MSC cell in the renal tissue. Scale bar = 50 μm.

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Detection of Bcl-2 and Bax protein in renal tissue

In the sham group, the expression of Bcl-2 and Bcl-2-associated X (Bax) protein was relatively low. At day 3 and day 7 after transplantation, the expression of Bcl-2 and Bax protein was significantly higher in IRI rabbits. The BM-MSCshBMP-7 group expressed high levels of Bcl-2 and the low levels of Bax protein, relative to the PBS group (P < 0.05). BM-MSCshBMP-7 group expressed significantly higher Bcl-2(Fig. 3A) and lower Bax protein (Fig. 3B) than the BM-MSCsEGFP group.

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Figure 3.  Quantitative analysis of apoptosis-related proteins using immunohistochemistry. (A) Bcl-2 and (B) Bax protein expression in kidney in all groups at 3 and 7 days after transplantation (IOD [sum]). Pairwise multiple comparisons between groups were made using Bonferroni’s method. Representative images of immunohistochemical characterization of renal Bcl-2 expression in PBS (C) and BM-MSCshBMP-7 groups (D) or Bax expression in PBS (E) and BM-MSCshBMP-7 (F) groups 3 days after cell transplantation (DAB, ×400). Scale bar = 100 μm. *P < 0.05 versus Sham; P < 0.05 versus. PBS; P < 0.05 versus BM-MSCsEGFP and §P < 0.05 versus BM-MSCshBMP-7.

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Immunohistochemistry showed Bcl-2-positive (Fig. 3C,D) and Bax-positive (Fig. 3E,F) cells with yellow and brown cytoplasm, respectively. Figure 3C,E shows representative staining images from PBS group. Figure 3D,F shows representative staining images from the BM-MSCshBMP-7 group. The comparative images of the various groups are not shown. After IRI, the expression of Bcl-2 and Bax protein increased significantly. The BM-MSCshBMP-7 group showed significantly higher levels of Bcl-2 and significantly lower levels of Bax protein compared with the PBS, Ad-hBMP-7 and the BM-MSCsEGFP groups.

Effects of hBMP-7-transduced BM-MSC transplantation on renal cell proliferation and apoptosis

To examine the effect of hBMP-7-transduced BM-MSC transplantation on the repair of renal tubular cell after IRI, the cell proliferation marker protein, proliferating cell nuclear antigen (PCNA), expression level and the cell apoptosis rate (TUNEL) were measured. Figure 4A,B shows that renal IRI significantly increased the absolute count of PCNA- and TUNEL-positive cells at days 3 and 7 in the PBS group as compared to the sham group. This suggests renal tissue self-repair by cell proliferation (PCNA) and removal of damaged cells (TUNEL). Cell proliferation was highest in the BM-MSCsBMP7 group, although BMP7 expression virus transduction alone or BM-MSCsEGFP transplantation also stimulated higher cell proliferation. Similarly, the transplantation of BM-MSCsBMP7 greatly reduced the level of renal cell apoptosis when compared with BMP7 expression virus transduction alone or BM-MSCsEGFP transplantation.

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Figure 4.  Evaluation of renal cell proliferation and apoptosis. Amount of (A) proliferating cell nuclear antigen (PCNA)- or (B) TUNEL-positive renal cells at 3 and 7 days after cell transplantation or viral transduction. Pairwise multiple comparisons between groups were made using Bonferroni’s method. Representative images of immunohistochemical characterization of renal PCNA expression in PBS (C) and BM-MSCshBMP-7 (D) or TUNEL in PBS (E) and BM-MSCshBMP-7 (F) groups 3 days after cell transplantation (DAB, ×400). Scale bar = 100 μm. *P < 0.05 versus Sham; P < 0.05 versus PBS; P < 0.05 versus BM-MSCsEGFP and §P < 0.05 versus BM-MSCshBMP-7.

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Immunohistochemistry showed PCNA- and TUNEL-positive cells with yellow and brown nuclei, respectively. Figure 4C,E shows representative staining images from the PBS group. Figure 4D,F shows representative staining images from the BM-MSCshBMP-7 group. The comparative results of the various groups are not included. In the sham group, the expression of PCNA and TUNEL was significantly lower compared with IRI groups. After IRI, the expression of PCNA and TUNEL increased significantly. The BM-MSCshBMP-7 group showed significantly higher levels of PCNA and significantly lower levels of TUNEL compared with the PBS, Ad-hBMP virus and the BM-MSCsEGFP groups.

Effects of hBMP-7-transduced BM-MSC transplantation on SOD activity and MDA content in kidney

The therapeutic effect of BM-MSCsBMP7 can also be measured by the reduction of oxidative stress that induced by renal IRI. The level of oxidative stress was measured by assessing superoxide dismutase (SOD) activity (Fig. 5A) and MDA level (Fig. 5B). SOD is the enzyme that reduces oxygen free radical that causes oxidative damage in IRI, and MDA is a metabolite of lipid oxidation. As compared with the PBS group, renal SOD activity was significantly higher in the hBMP-7 and BM-MSCshBMP-7 groups, and MDA content was significantly higher in the BM-MSCshBMP-7 group (P < 0.05). Moreover, compared with the BM-MSCsEGFP group, SOD activity was significantly higher and the content of MDA significantly was lower in the BM-MSCshBMP-7 group (P < 0.05). No significant differences in SOD activity and MDA content between BM-MSCsEGFP and hBMP-7 groups at days 3 and 7 after transplantation were observed (P > 0.05).

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Figure 5.  Effects of BM-MSCsBMP7 on oxidative stress induced by renal ischemia–reperfusion injury. (A) Activity of superoxide dismutase and (B) renal malondialdehyde content in all groups at day 3 and day 7 after transplantation or viral transduction was assessed. Pairwise multiple comparisons between groups were made using Bonferroni’s method. *P < 0.05 versus Sham; P < 0.05 versus PBS; P < 0.05 versus BM-MSCsEGFP and §P < 0.05 versus BM-MSCshBMP-7.

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Effects of hBMP-7-transduced BM-MSCs transplantation on renal function

Renal function was evaluated by analyzing blood serum creatinine and urea. Blood serum creatinine and urea levels were significantly lower in the experimental groups relative to the levels after renal IRI (Fig. 6A,B). Both the BM-MSCshBMP-7 and BM-MSCsEGFP groups showed significantly lower levels of serum creatinine and urea than the PBS group. The BM-MSCshBMP-7 group showed significantly lower levels of serum creatinine and urea than the BM-MSCsEGFP group.

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Figure 6.  Effects of BM-MSCsBMP7 on renal cell function and morphology. (A) Serum creatinine and (B) urea were measured in all groups at days 3 and 7 after transplantation or viral transduction. Pairwise multiple comparisons between groups were made using Bonferroni’s method. (C) The kidney section from the rabbit that received sham surgery was stained with hematoxylin and eosin. (D) The kidney section from the rabbit that received ischemia and reperfusion but treated with PBS injection was stained with hematoxylin and eosin. (E) The kidney section from the rabbit that received ischemia and reperfusion but treated with BM-MSCshBMP-7 infusion was stained with hematoxylin and eosin. Scale bar = 50 μm. *P < 0.05 versus Sham; P < 0.05 versus PBS; P < 0.05 versus BM-MSCsEGFP and §P < 0.05 versus BM-MSCshBMP-7.

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To verify the histological integrity of the kidney, tissue sections stained with hematoxylin and eosin (H&E) were observed under a light microscope. As shown in Fig. 6C, normal renal tubular cell morphology was observed in the kidneys of rabbits from the sham group that received the same treatment with the exception of ischemia and reperfusion. In rabbits that have renal IRI and receive PBS transfusion, IRI-induced renal tubule damage, including cell necrosis because of edema, was observed (Fig. 6D). Reduced renal tubular cell necrosis was detected upon transplantation of BM-MSCshBMP-7 after IRI (Fig. 6E).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Funding
  8. References

We used a model of severe kidney IRI, which is characterized by ARF with subsequent recovery. ARF is a common disease with high morbidity and mortality. Recovery from ARF is dependent on the replacement of necrotic tubular cells with functional tubular epithelium (Esson & Schrier 2002). In this IRI model, many tubular cells of the outer medulla die by necrosis and apoptosis (Sheridan & Bonventre 2000), and surviving cells suffer from widespread cytoskeletal disruption (Lien et al. 2003). The recovery phase following injury is marked by cytoskeletal reorganization and intense tubular cell proliferation (Kale et al. 2003). The discovery of immature BM-MSCs and their potential in repairing injured renal tissue have been described in recent studies (Kale et al. 2003). Bone marrow ablation has been shown to worsen the initial rise in BUN following bilateral renal ischemia and to slow the recovery of tubules in the outer medulla at the histological level (Kale et al. 2003). The fact that stem cell infusion prevented both of these effects suggests that stem cells play an early role in limiting the initial injury as well as a later role in enhancing tubular repair.

It seems unlikely that the effect of infused stem cells is dependent on their differentiation into functional tubular epithelia. Rather, it seems likely that the initial role of these cells is to limit tubular backleak of the glomerular filtrate, thus maintaining the glomerular filtration rate (GFR) (Moran & Myers 1985; Myers & Moran 1986). Repair of the renal tubule may occur as a cooperative effort involving both the local actions of surviving tubular cells within the injured tubular segments and the mobilization and homing of BM-MSCs to the areas of injury. These BM-MSCs appear to differentiate into the proper epithelial cells of the tubular segment that they populate, and to provide a functional role in limiting the degree of ARF (Kale et al. 2003).

The present results provide a conceptual basis for the development of therapeutic strategies aimed at treating ARF with stem cells. There is some evidence to suggest that infused BM-MSCs distribute with varying levels of efficiency to various organs suffering from ischemic injury, including the kidneys (Nakatomi et al. 2002; Orlic 2002; Otani et al. 2002). Importantly, BM-MSCs seem to migrate preferentially to sites of injury, suggesting that they can sense the local microenvironment, where they promote functional recovery (Chen et al. 2001). Their capacity to cross the basement membrane is regulated by metalloproteases secreted by BM-MSCs under the influence of inflammatory cytokines, such as TNF-α, TGF-β1 and interleukin-1β (Ries et al. 2007). Moreover, BM-MSCs express on their surface a limited array of functional chemokine receptors, such as chemokine receptor 4, which have a pivotal role in their recruitment to the site of injury (Sordi et al. 2005).

Therefore, in vivo, the functional responses of transplanted BM-MSCs, recruited to the local microenvironment, are probably dictated by danger signals such as inflammation and hypoxia. Despite a large body of evidence suggesting that BM-MSCs can differentiate into virtually any cell type in vitro (Pittenger et al. 1999), the therapeutic effect observed following in vivo administration seems to be mostly related to their immune modulatory capacity and their capacity to stimulate the survival and functional recovery of resident cells in injured organs by paracrine mechanisms (Togel et al. 2005; Lee et al. 2006) and by recruitment of local precursors (Munoz et al. 2005).

The differences between the BM-MSCsEGFP group and PBS group indicate that BM-MSCs have the ability to promote the proliferation and inhibit the apoptosis of tubular cells through different mechanisms. Also, the BM-MSCs have the ability to reduce the level of lipid peroxidation by decreasing the levels of MDA. They also have the ability to enhance the cleaning up of free radicals. Consequently, as our results show, there was significant structural and functional renal recovery.

BM-MSCs increase the risk of embolism; therefore, lower amounts were transplanted to minimize this risk. And the amount of BM-MSCs migrated to injured kidney is less in the bad environment after IRI. So, hBMP-7-modified BM-MSCs were used to stimulate the migration, proliferation and differentiation of BM-MSCs in order to enhance their therapeutic action on the kidney.

For several reasons, BM-MSCs are an attractive cellular vehicle for gene delivery. They can be obtained in relatively large numbers through a standard clinical procedure and are easily expanded in culture and capable of long-term transgene expression (Munoz et al. 2005). Their administration can be autologous or via banked stores, suggesting that they may be immunoprivileged (Uccelli et al. 2006). In the present study, we showed that BM-MSCs could be transduced with recombinant adenovirus carrying hBMP-7 with high efficiency and without any effect on cell viability. BM-MSCs hBMP-7 expressed hBMP-7 at both the mRNA and protein levels, and the secreted hBMP-7 could be detected as long as 14 days post-transduction. After transplantation of these cells, we also found hBMP-7 expression in renal tissue. These results indicate that BM-MSCs can be an effective vehicle for gene delivery.

Meanwhile, compared with the BM-MSCsEGFP group, the activity of SOD was higher and the content of MDA was lower in the BM-MSCshBMP-7 group. The measurement of expressed Bcl-2 and Bax protein and the amount of tubular cells at the proliferative stage or apoptosis stage indicated that the regeneration of the kidney was markedly promoted in BM-MSCshBMP-7. Structural and functional repair of the kidneys was significantly promoted in the BM-MSCshBMP-7 group (P < 0.05).

The present study shows that transplantation of hBMP-7-modified BM-MSCs through abdominal aorta lessened the renal injury and enhanced the repair of kidney and recovery of renal function. We have also shown the expression of hBMP-7 in the injured kidney treated with hBMP-7 gene-transduced BM-MSCs 1 week after cell transplantation. Therefore, grafted cells could supply a transient high level of hBMP-7 to the ischemic kidney in the acute phase. BMP-7 is a member of transforming growth factor-β (TGF-β) superfamily and is also known to have important functions in renal development and maintenance of normal renal physiological function (Li et al. 2004). BMP-7 has the important action on modulating the development, differentiation, proliferation and apoptosis of cells. BMP-7 activates the post-IRI regeneration partly by inhibiting TGF-β, a major effector in the fibrotic pathway that induces renal tissue damage, through its downstream targets, SMA- and MAD-related proteins 1/5/8 (Smad1/5/8) (Manson et al. 2011). The application of exogenous BMP-7 can promote the recovery of renal function (Vukicevic et al. 1998; Simon et al. 1999). Our results are consistent with others that have shown the facilitation from recovery by exogenous BMP-7.

Gene therapy and stem cell therapy hold promise for the treatment of ischemic diseases of many organs (Kurozumi et al. 2004; Yang et al. 2007). However, the combined therapy with stem cell and BMP-7 gene has not been reported up to now. In the present study, we observed that hBMP-7-modified BM-MSCs could increase SOD activity and proliferation of tubular cells and reduce MDA levels following IRI-induced ARF. We therefore believe that this combined strategy of BM-MSCs transplantation with hBMP-7 gene therapy could be a useful approach for the treatment of renal IRI.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Funding
  8. References

Cell isolation and culture

Bone marrow was extruded from rabbit femurs. BM-MSCs were isolated from the marrow using density gradient centrifugation and cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Hyclone) as in the study by Xu et al. (2010). Briefly, bone marrow aspirates (2–4 mL) were isolated from the sternum of iliac crest of rabbits; it was placed in a tube containing 4 mL of phosphate-buffered saline (PBS) and 1250 U heparin. The marrow sample was washed twice with PBS after centrifugation at 800 × g for 5 min. The residual cells were loaded onto the equal volume of 1.073 g/mL Percoll solution and centrifuged at 2000 × g for 20 min at 20 °C. The mononuclear cells were collected from the upper layer and interface, diluted with 2 volumes of PBS and then collected by centrifugation at 800 × g for 5 min. The cells were resuspended in the medium as described above and incubated at 37 °C in 5% CO2 and 95% humidity. After 18–24 h, the culture medium was replaced and subsequently changed twice/week. Nonadherent hematopoietic cells were removed. After 10–14 days of cultivation, the cells were dissociated with 0.25% trypsin and 1 mm EDTA and replaced at 104 cells/cm2 and grown to near confluence. At third passage, the BM-MSCs were used in the in vitro and in vivo experiments.

Adenovirus and gene transfer

We used AdMax™ system (Microbix Biosystems, Inc. Canada) to construct a recombinant adenovirus (Ad-hBMP-7) that contains and expresses hBMP-7.

Fluorescence microscopy and flow cytometry were used to determine the efficiency of Ad-hBMP-7 transduction into BM-MSCs. The best multiplicity of infection (MOI) for adenovirus-mediated gene transfer was determined by exposing BM-MSCs to concentrated adenovirus vectors at MOIs of 50, 75, 100, 125 and 150 for 48 h. Fluorescence microscopy was used to observe the transduction efficiency and cell viability based on the EGFP expression and cell morphology. The optimal MOI was chosen for both highest EGFP expression and cell viability. Flow cytometry was used to quantify the transduction efficiency of the adopted MOI based on the expression of EGFP. The BM-MSCs were harvested by standard trypsinization and then subjected to flow cytometric analysis on an EPICS XL flow cytometer (Beckman Coulter, USA) using CellQuest™ software with 10 000–20 000 events recorded for each sample.

Human BMP-7 expression

Total RNA was extracted from BM-MSCs 72 h after transduction or from renal tissue at an appropriate time point after cell transplantation with animal RNAout (Mianyang Tianze Gene Engineering Co.). First-strand cDNA synthesis was carried out using primer Oligo(dT)and reverse transcription with Rever Tra Ace (TOYOBO CO., Ltd, Japan) using the manufacturer’s recommended protocols. The hBMP-7-specific PCR primer sequences were forward 5′-GCTACGCCGCCTACTACTGTGA-3′ and reverse 5′-GAGGACGGAGATGGCATTGAG-3′. The rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer sequences were forward 5′-TGGTGAAGGTCGGAGTGAA-3′ and reverse 5′-GTGAAGACGCCAGTGGATT-3′. PCR conditions included denaturation at 94 °C for 10 s, annealing at 55 °C for 30 s and extension at 68 °C for 60 s. The reaction ran for 30 cycles. The PCR products were analyzed using a 1.0% agarose gel electrophoresis.

We used whole-cell lysates of BM-MSCs to perform Western blot analyses. After SDS gel electrophoresis using 10% polyacrylamide gels, proteins were probed with mouse anti-human BMP-7 antibody (sc-73748, Santa Cruz Biotechnology) and detected using immunohistochemistry (DAB colorimetric assay).

Immunofluorescent staining was performed with mouse anti-human BMP-7 as the primary antibody and polyclonal sheep anti-mouse CY3-conjugated antibody (Sigma) as a secondary antibody. BM-MSCs were observed for hBMP-7 expression after incubation with the antibodies under a fluorescent microscope.

The levels of secreted hBMP-7 in culture medium were quantified using a human BMP-7 immunoassay kit (R&D System Inc.). The culture medium was collected from BM-MSC cultures in 6-well plates at 0, 1, 3, 5, 7, 10 and 14 days after gene transduction (n = 6 at each time point).

Rabbit model of renal cold IRI and cell transplantation

To establish renal cold IRI, rabbits were anesthetized with 30 mg/mL sodium pentobarbital (30 mg/kg body weight) through an ear vein injection. Venous puncture was performed in the ear vein after which heparin was injected at 100 U/100 g, and normal saline was supplemented at 0.8 mL/100 g during the surgery at 0.8 mL/h. A transverse incision was made in the abdomen; the right kidney and right renal artery and vein were isolated. The renal pedicle was ligated with a 1-0 suture. The left kidney and left renal artery and vein were isolated using the same procedures, and the abdominal aorta and vena cava were exposed. A noninvasive clamp was applied to the right renal artery origin, and the renal pedicle was cut at the site close to the renal hilum after which the right kidney was removed. A catheter was then inserted through the stump of renal artery and fixed. Noninvasive clamps were applied to the abdominal aorta and vena cava above and below the bilateral renal arteries and veins. After the clamp at the stump of right renal vein was released, the left kidney was irrigated with 10–15 mL of normal saline at 0–4 °C using an epidural catheter. During the irrigation, the kidney was kneaded at the direction from peripheral kidney to the renal hilum until the kidney became white, and the fluid from the stump of right renal vein was clear. The left renal artery and vein were clamped at the origins along with the right renal artery. A 1-0 suture was used to ligate right renal vein, and the clamps at the abdominal aorta and vena cava were released. The left kidney was kept under icy-water bag. After 1 h, the clamps at the left renal vein and artery were released for reperfusion. The adenovirus solution and/or BM-MSCs suspension or normal saline (1.2 mL) was injected through the epidural catheter in the right renal artery followed by the removal of epidural catheter and ligation of right renal artery with a 1-0 suture. The incision was then closed. After surgery, the body temperature of the rabbits was maintained and the rabbits were housed. Venous blood was collected through the ear vein at day 0, day 3 and day 7. There were five animals/treatment and time point.

Labeling BM-MSCs with Hoechst33342 before transplantation

Before transplantation of BM-MSCs, cells were labeled with Hoechst33342. Hoechst33342 is a membrane-permeable active fluorescent dye and can enter the normal cells and bind to the DNA in the nucleus. Thus, these cells are labeled and can be detected at 460 nm. Under a fluorescent microscope, Hoechst33342-positive cells have blue fluorescence. At 2 h before transplantation, Hoechst33342 was added to the cultured cells at a final concentration of 10 μg/mL followed by incubation at 37 °C for 30 min. Immediately before transplantation, a fraction of cells were collected and observed under a fluorescence microscope to confirm whether cells were successfully labeled and representative photographs were captured [62]. Then, 0.5 mL of 1% paraformaldehyde was added to the cells that were then subjected to flow cytometry. A total of 10 000–20 000 cells were counted, and the proportion of Hoechst33342-positive cells was calculated.

Immunohistochemical staining, renal histology, cell proliferation and apoptosis

Five-micrometer paraffin slides were prepared from the kidneys of each experimental rabbit at 3 days after transplantation. Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 30 min. The slides were washed with PBS and incubated for 30 min in blocking solution (10% normal goat serum diluted in PBS) and then incubated overnight at room temperature with mouse anti-rabbit Bcl-2 or BAX or proliferating cell nuclear antigen (PCNA) (BOSTER, Wuhan) at 1 : 100 dilution. Next, they were incubated in biotinylated goat anti-mouse antibody at 1 : 50 dilution in blocking solution (37 °C, 20 min). The slides were then incubated in streptavidin–biotin–horseradish peroxidase complex (Beyotime Institute of Biotechnology, China) for 30 min in 37 °C, and the color was developed with 0.3 mg/mL of 3.3-diaminobenzidine (DAB). We performed normal hematoxylin and eosin (H&E) staining on some of the kidney slides. We used TUNEL In Situ Apoptosis Detection kit (Nanjing Keygen Biotech, China), following the manufacturer’s protocol to detect apoptosis in kidney at 3 days after transplantation. Photographs of the slides at 400× magnification were captured using an LCD camera (Olympus, Japan) connected to the microscope. PCNA-positive cells and TUNEL-positive cells in each of the photographs were counted. We determined the level of Bcl-2 and Bax expression from the integrated optical density (IOD) of the photographs using IPP6.0 image analysis software.

Testing of renal function

We collected 4 mL of whole blood by bleeding from rabbit ear vein in the presence of heparin. We used an automated biochemistry analyzer to perform ELISA to detect the blood serum levels of creatinine and urea. The ELISA kits for detecting creatinine and urea were purchased from Whitman Biotech (Nanjing, China).

Detection of SOD activity and MDA content in kidney

Renal tissue homogenates of 10% and 1% were used to determine the SOD activity and MDA content by chromometry according to the manufacturer’s recommended conditions. These kits were purchased from Nanjing Jiancheng Bioengineering Institute, China.

Statistical analysis

Continuous variables were compared by one-way analysis of variance (anova). When a significance between groups was apparent, multiple comparisons of means were made using the Bonferroni procedure with type-I error adjustment. Data are presented as means ± SD. All statistical assessments were two-sided and evaluated at the 0.05 level of confidence. Statistical analyses were performed using SPSS 15.0 statistics software (SPSS Inc, Chicago, IL).

Funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Funding
  8. References

This study is supported by Natural Science Foundation Project of CQ-CSTC (No. CSTC, 2009BB5154).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Funding
  8. References
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