Part2:Transplantation Of Chicken Egg White Extract Induced Rabbit PBMCs As A Treatment For Renal Ischemia-reperfusion Injury in Rabbits

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Results

1. Successful labeling of PBMCs with CFSE

Carboxyfluorescein diacetate and succinimidyl ester(CFSE)-labeled PBMCs exhibited yellow-green fluorescence under a fluorescence microscope, as shown in Eig 1B. After counterstaining with DAPI, all the cells showed blue fluorescence, as shown in Fig 1A, and an analysis of the overlap in the fluorescence staining showed that the cells were successfully labeled, as shown in Eig 1C.

2. Identification of PBMCs after induction

2.1.Relative expression levels of multipotency-related and somatic cell genes in noninduced and induced PBMCs. The expression of NANOG, OCT4, and SOX2 was significantly increased in PBMCs stimulated with chicken egg white extract, and the expression of the somatic cell gene LMNA was decreased (Fig 1D), which indicated that the cells differentiated into multipotent cells. Statistical analysis showed that the two groups were significantly different (n=3,p=0.003). The cells were induced three times, and three biological replicates were included in the study. The difference in the somatic cell gene LMNA between the two groups was not statistically significant.

2.2.The pluripotency factor of induced PBMCs was significantly higher than that of noninduced PBMCs. Among the noninduced cells,0.081% were positive for OCT4-PE, whereas 99.3% of the induced cells were positive for OCT4-PE. The percentages of SSEA-4-PE-positive noninduced and induced cells were 1.08% and 16.5%, respectively. Moreover, 0.495% of the noninduced cells were positive for NANOG-PE, whereas 95.8% of the induced cells were found to be NANOG-PE-positive(Fig 1E-1M). The percentages of isotype control cells positive for OCT4-PE, SSEA-4-PE, and NANOG-PE were 0.546%,0.401%, and 0.249%, respectively.

2.3.Immunohistochemical analyses yielded positive results for the induced PBMCs. Immunohistochemical analyses of OCT4 and NANOG showed that induced PBMCs, but not noninduced PBMCs, expressed these markers (Fig 2A-2D). Fig2A and 2Cshow uninduced PBMCs, and Fig 2B and 2D show induced PBMCs. In addition, OCT4expression is shown in Fig 2A and2B, and Fig 2Cand 2D show the expression of NANOG.

2.4.Western blot analyses yielded positive results for induced PBMCs. According to the Western blot results, OCT4 was expressed in induced PBMCs but not in noninduced PBMCs (EFig 2E).

2.5. The relative telomere lengths were significantly longer in induced PBMCs. Based on the quantitative PCR results, the telomeres of induced PBMCs(1.83807±0.84756) were significantly longer than those of noninduced PBMCs(1±0.08307)(Eig 2E,means±standard deviations, n=5,p=0.013), which indicated that the cells differentiated into young stem cells. The relative telomere lengths actually increased after dedifferentiation.

3. The serum urea nitrogen and creatinine levels were decreased in the induced cell treatment group

The model control group had a urea nitrogen content of 22.1 mmol/l and a creatinine content of 452 umol/l. After three rounds of transplantation with induced cells, the urea nitrogen con-tent was 7 mmol/l， and the creatinine content was 74 μmol/l; these values were similar to the normal levels. The levels in the noninduced cell treatment group remained elevated (Fig 2G). Statistical analysis showed statistically significant differences among the results of the four groups (p= 0.031).

4. The urinary protein concentrations were decreased in the induced group 

The urinary protein concentration in the model control group was 8.17 mg/ml. After the three treatments, the urinary protein concentration in the induced group was 4.35 mg/ml, which was similar to the normal level. However, the urinary protein content of the noninduced group remained high at 7.96mg/ml(Fig 2H). Statistical analysis showed statistically significant differences among the results obtained from the four groups (p=0.001).

5. Labeled cells were detected in the induced group

As shown in Fig3A-3D, many fluorescent cells were distributed in the kidneys of the induced cell treatment group, whereas the kidney tissues from the other three groups did not display any fluorescent cells. A possible explanation is that induced cells are transported to the injured kidney to repair the damage. The distribution of fluorescently labeled induced PBMCs in frozen kidney sections suggested that these cells were involved in repairing the injured kidney.

6. The structure of the kidney of the induced group exhibited a normal phenotype

Based on the hematoxylin-eosin(HE) staining results, the structure of the kidneys of the rabbits in the model control group was damaged. After treatment with the induced cells, the renal tissue structure exhibited a normal phenotype. In contrast, the damage to the kidney tissue persisted in the noninduced cell treatment group (Fig 3E-3H). The acute tubular necrosis (ATN) score is shown in Fig 3I.

7. The IOD of the induced cell groups was significantly reduced

Image-Pro Plus 6.0(Media Cybernetics, Inc., Rockville, MD, USA) software was used to perform an immunohistochemical analysis of the cumulative optical density (IOD). For each group, at least six 200× magnification fields were randomly selected from each section, and images were captured. We attempted to view the entire field of vision to ensure that every photograph had the same background. Image-Pro Plus 6.0software was used to select the same brown color as a uniform standard for judging the positive staining in all the images. Each image was analyzed to determine the IOD of positive staining. The IOD was significantly increased in the model control group, whereas the IODs of the normal control and induced cell groups were reduced and significantly reduced, respectively. The IOD of the noninduced cell treatment group was not markedly reduced compared with that of the model control group (Fig3). Statistical analysis showed significant differences among the results of the four groups(p =0.001).

8. Fibrosis was improved in the induced group

Substantial collagen fiber deposition was observed in the model control group, and serious fibrosis occurred. This fibrosis was improved or eliminated in the induced cell treatment group, whereas severe fibrosis was still observed in the noninduced cell treated group (Eig 4A-4D).

9. Thickening of the basement membrane was not observed in the induced group

The thicknesses of the glomerular basement membrane, the basilar membrane of the renal capsule, and the tubulointerstitial membrane were significantly increased in the model control group. In the induced cell treatment group, the basement membrane did not display any significant thickening, whereas thicker basement membranes were still observed in the noninduced cell treatment group (Fig 4E-4H). The basement membrane thickness scores are shown in Fig 4I.

10. Transplanted cells differentiated into tubular epithelial cells

The immunofluorescence results showed that the transplanted cells exhibited green and red fluorescence at the same time, which indicated that the transplanted cells were differentiated into tubular epithelial cells(TECs) (Fig 4J).

11. The identified substances were increased in the model group and decreased after treatment

In the negative ion mode, the two substances that displayed significantly increased levels in the model control group were 2'-deoxy-D-ribose and N-acetylglucosamine 1-phosphate (Fig 5A). The levels of both of these compounds were significantly reduced after induced cell therapy compared with the levels found in the model control group (Fig5A).In the positive ion mode, the levels of three substances (D-pinitol, lysyl-glycine, and glutamyl-asparagine) were significantly increased in the model control group (Fig 5B), and the levels of these three substances were significantly reduced after induced cell therapy compared with those found in the model control group (Fig5B)

https://doi.org/10.1371/journal.pone.0244160.g005

In the negative ion mode, the levels of the two identified substances were increased in the model group and decreased after treatment, which indicated a meaningful correlation between the levels of the two substances.

In the positive ion mode, the levels of the three identified substances were increased in the model control group and decreased after treatment, which indicated meaningful correlations among the levels of these three substances.

12. Significant changes in the pyrimidine metabolism and phenylalanine, tyrosine, and tryptophan biosynthesis pathways were observed

In the negative ion mode, a significant change in the pyrimidine metabolism pathway was detected between the model control group and the normal control group (Eig6A). In addition,

significant changes in this pathway were observed in the induced cell treatment group compared with the model control group (Fig 6B), which indicated that the pyrimidine metabolism pathway is a significantly altered pathway. In the positive ion mode analysis, the phenylalanine, tyrosine, and tryptophan biosynthesis pathways were significantly altered in the model control group compared with the normal control group (Fig 6C), and these pathways were significantly altered in the induced cell treatment group compared with the model control group (Eig 6D), which indicated that the phenylalanine, tyrosine, and tryptophan biosynthesis path-ways are meaningful pathways. In Fig6, a darker bubble color and a larger volume indicate a more significant difference. As shown by the arrows in Fig 6A and6B, the difference in the pyrimidine metabolism pathway is significant, and as indicated by the arrows in Fig 6C and 6D, the differences in phenylalanine, tyrosine and tryptophan biosynthesis pathways are significant.

https://doi.org/10.1371/journal.pone.0244160.g006

Discussion

Acute kidney injury caused by ischemia-reperfusion is a common clinical disease, and the mortality rate of patients with acute kidney injury is approximately 30% to 50% due to limited treatment measures[14]. During ischemia, the activation of enzymes induces cytoskeletal destruction, cell membrane damage, and DNA degradation, which eventually leads to cell necrosis and apoptosis [15]. Ischemia-reperfusion also activates complement proteins, cytokines, and chemokines, among other molecules, and the mechanisms underlying ischemia-reperfusion injury are of substantial importance [16]. Additionally, transplanted stem cells promote the migration and recruitment of residual renal stem cells to the injury site and induce their differentiation into TECs, but the exact mechanism is unclear [17].

The mortality of patients with ischemia-reperfusion injury is high due to the lack of effective clinical treatment [18]. The condition requires long-term or lifelong renal replacement therapy or renal transplantation[19]. The pathophysiological mechanism of ischemia-reperfusion injury is very complicated and results from numerous interactions among inflammatory cells, vascular endothelial cells, and cytokines[20]. In recent years, mesenchymal stem cells have become the focus of research on ischemia-reperfusion injury [21] and have been shown to improve ischemia-reperfusion-induced renal injury. As shown in studies using ischemia-reperfusion models, mesenchymal stem cells play an important role in regulating immunity, participating in vascular reconstruction[22], and repairing the renal microenvironment by secreting paracrine antiapoptotic factors, mitogenic factors, and angiogenic factors. A recent study using stem cells of different origins revealed that induced pluripotent stem cells other types of stem cells [22]. We believe that the induced cells are more likely to be mesenchymal stem cells because they are more mature than induced pluripotent stem cells.

After induced pluripotent stem cell transplantation, molecules expressed on the surface of the induced pluripotent stem cells interact with T cells to regulate their biological activity. T cells can evade the immune system following damage and reduce the intensity of the immune response in the surrounding tissue, and these cells thus play a role in protecting the function of damaged tissue to some extent [23]. We successfully established a rabbit model of renal interstitial fibrosis and demonstrated that the transplantation of induced autologous stem cells can repair kidney damage within 8 weeks [24]. Stem cells provide an advantageous microenvironment for the repair of renal tubular epithelial damage [24]. In this experiment, after the administration of three consecutive weekly treatments, the serum creatinine and urea nitrogen levels in the induced cell treatment group were restored to the same level observed in the normal control group, whereas the levels in the noninduced cell treatment and model control groups were significantly elevated.

As shown by Haynesworth et al. [25], mesenchymal stem cells secrete a variety of growth factors, colony-stimulating factors, adhesion molecules, and interleukins(IL-6, IL-7, IL-8, I-11, IL-14, and IL-15), which promote the mitosis of renal TECs and tissue repair and thereby inhibit the elevation of creatinine and urea nitrogen concentrations.

In the present study, the expression levels of the pluripotency factors SSEA-4, NANOG, and OCT4 in induced PBMCs were detected by flow cytometry, and the results revealed that PBMCs dedifferentiated into multipotent cells. The quantitative PCR analysis demonstrated that the telomeres in induced PBMCs were significantly longer than those in noninduced PBMCs.Based on our evidence, PBMCs obtained from rabbit peripheral blood dedifferentiate into multipotent stem cells following treatment with chicken egg white extracts and exhibit the characteristics of mesenchymal stem cells [9], which allows their eventual participation in the repair of renal injury. Studies have shown that mammalian egg cells and Xenopus egg cell extracts can reprogram somatic cells [6,7]. Our previous research showed that chicken egg extract can also reprogram somatic cells [5,9]. The chicken egg is the largest egg cell, and its ability to reprogram somatic cells will thus advance cell biological research. The mammalian egg cell and Xenopus egg cell extract-induced reprogramming steps are cumbersome, and the extracts are difficult to obtain; however, chicken eggs are the largest egg cells, and a large amount of chicken egg white extract can thus be obtained. If the procedure is performed in a sterile manner, the obtained extract does not need to be filtered and sterilized. The activity of the extract can be maintained well. We repeated the induction experiment using final concentrations of chicken egg white extract of 10%,20%,30%,40%, and 50% to induce the PBMCs (see Sl to S3 Figs). As the concentration of the chicken egg white extract increased, the pluripotency factor positive rate gradually increased, and the highest positive rate was obtained with a final concentration of 50%. However, a chicken egg white extract concentration higher than 50% will affect cell growth. We used protease, DNase, and RNase to lyse the protein, DNA, and RNA in egg white extract, respectively, and then conducted our induction experiment. We found that the egg white extract obtained after protein lysis no longer has the ability to reprogram cells, and the chicken protein extract obtained after DNA and RNA lysis still has the ability to reprogram cells. Therefore, these findings confirmed that the main role of the extract is played by the protein component (results not shown).

We also analyzed animal blood samples after one week of treatment. However, the difference among the four groups was not significant; thus, we only show the results obtained for the four groups after three treatments. The distribution of fluorescently labeled induced PBMCs in frozen kidney sections suggested that these cells were involved in repairing the injured kidney. HE staining showed damage to the kidney structure in the model control group. After treatment with induced cells, the kidney exhibited a normal structure, whereas damage to the kidney structure was observed in the noninduced cell treatment group. After three treatments, Masson's trichrome staining of kidney sections from the four groups revealed substantial collagen fiber deposition and serious fibrosis in the model control group. This fibrosis was improved or eliminated by treatment with the induced cells, whereas severe fibrosis was still observed in the noninduced cell treatment group.A renal metabonomics analysis of the four groups conducted in the negative ion mode revealed significantly increased lev-els of 2'-deoxy-D-ribose and N-acetylglucosamine 1-phosphate in the model control group compared with the normal control group, and the levels of these substances were significantly reduced in the induced cell treatment group compared with the model control group. In the positive ion mode, the levels of five substances were significantly increased in the model control group compared with the normal control group, and the levels of three of these five substances were significantly reduced in the induced cell treatment group compared with the model control group.

The postulated main mechanisms underlying the observed repair of the kidney and the accompanying reduction in the inflammatory response are listed below. ① Induced PBMCs differentiate into TECs or fuse with surviving cells to directly promote the repair of renal tissue. ②The synergistic effects of various cytokines provide a good microenvironment for the repair of renal tissues [26]. After three transplantations of induced PBMCs, the renal function and pathological indexes returned to the normal levels, and these normal levels persisted until the end of the experiment, which suggested that the transplanted multipotent stem cells effectively promoted the repair of renal tissue structure and function.

Although the specific mechanism through which induced PBMCs promote structural and functional repair of the kidney has not yet been fully understood, the results of this study suggest that the intravenous transplantation of induced PBMCs promotes repair after acute kidney injury. These results provide a valuable reference for researchers investigating the function of multipotent stem cells in regenerating damaged kidneys and for the clinical treatment of acute and chronic kidney diseases.

In summary, induced multipotent stem cell transplantation has substantial significance for parenchymal cell repair in animal models of renal failure and other kidney diseases.

Conclusions

The treatment of PBMCs with chicken egg white extract significantly increased the expression of pluripotency-related genes and proteins, which indicated that the cells had dedifferentiated into multipotent stem cells. Thus, induced PBMCs dedifferentiate into multipotent stem cells and can potentially be used to treat kidney injury. Future research is needed to identify the key molecules in chicken egg white extract and thus further improve the induction efficiency.

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