Part 1:Transplantation Of Chicken Egg White Extract induced Rabbit PBMCs As A Treatment For Renal Ischemia-reperfusion Injury in Rabbits
Mar 07, 2022
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Abstract
Ischemia-reperfusion injury is an important contributor to acute kidney injury and a major factor affecting early functional recovery after kidney transplantation. We conducted this experiment to investigate the protective effect of induced multipotent stem cell transplantation on renal ischemia-reperfusion injury. Forty rabbits were divided into four groups of 10 rabbits each. Thirty rabbits were used to establish the renal ischemia-reperfusion injury model, and ten rabbits served as the model group and were not treated. Among the 30 rabbits with renal ischemia-reperfusion injury, 10 rabbits were treated with induced peripheral blood mononuclear cells (PBMCs), and 10 other rabbits were treated with noninduced PBMCs. After three weekly treatments, the serum creatinine levels, urea nitrogen levels, and urine protein concentrations were quantified. The kidneys were stained with hematoxylin-eosin (HE), periodic acid-Schiff(PAS), and Masson's trichrome and then sent for commercial metabolomic testing. The kidneys of the rabbits in the model group showed different degrees of pathological changes, and the recovery of renal function was observed in the group treated with induced cells. The results indicate that PBMCs differentiate into multipotent stem cells after induction and exert a therapeutic effect on renal ischemia-reperfusion injury.
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Introduction
Clinical acute kidney injury, which has a very high incidence, might be caused by many factors, and ischemia-reperfusion injury is the most common of these factors [1]. The main pathological manifestation of acute kidney injury in renal tubular epithelial damage, and the recovery of renal function depends on renal tubular epithelial cell regeneration. In the event of renal tubular necrosis, blood purification is the only method that can maintain a stable internal bodily environment due to the impairment of kidney function. The development of drugs or interventions that accelerate renal tissue repair during renal tubular epithelial self-repair processes will undoubtedly shorten the course of acute kidney injury. Early recovery of a stable internal bodily environment can reduce the complications caused by renal failure and thereby reduce mortality [2].
Renal ischemia-reperfusion injury is clinically common in the context of emergency shock recovery, kidney surgery, and kidney transplantation and is thus an important factor affecting renal function [3]. The manifestations of renal ischemia-reperfusion injury include acute tubular epithelial cell injury, tubular peripheral microvascular damage, inflammation, and leukocyte infiltration. Mesenchymal stem cells are effective for the treatment of ischemia-related organ dysfunction, but the exact mechanism through which they improve renal function remains unclear [4]. In this study, we investigated the protective effect of induced multipotent stem cell transplantation on renal ischemia-reperfusion injury in rabbits, and our results provide experimental evidence and ideas for clinical treatment.
Induced multipotent stem cell regeneration of renal function represents a new method for the treatment of acute tubular necrosis. In our laboratory, induced multipotent stem cells are generated by the treatment of peripheral blood mononuclear cells(PBMCs) from rabbit peripheral blood with a homemade egg white extract to reverse their differentiation [5]. This study is the first to induce PBMCs with chicken egg white extract and use the induced PBMCs for the treatment of rabbit renal ischemia-reperfusion injury. Previous studies have shown that extracts of mammalian oocytes [6]and Xenopus oocytes |7|have the potential to reprogram cells. The identification of egg extracts with the ability to maintain and enhance the survival and differentiation of cells will be widely useful in cellular biology research. Many studies have shown that animal egg extracts are able to induce the reprogramming of somatic cells [6,8]. The chicken egg yolk is the largest egg cell, the yolk membrane comprises the cell membrane, and the egg white and eggshell, which have nutritional and protective roles, are formed by oviduct secretions. Therefore, chicken egg white extract has the capacity to induce stemness in PBMCs. In our previous research, we found that chicken egg white extract can induce the reverse differentiation of somatic cells into multipotent stem cells [5,9].
As determined by quantitative PCR, the induction of PBMCs with the chicken egg white extract significantly increased the relative expression of the multipotency-related genes OCT4, NANOG, and SOX2 and significantly decreased that of the somatic cell gene LMNA, which indicates that PBMCs differentiate into multipotent stem cells[9]. Further experiments are needed to identify the key molecules in the chicken egg white extract to further improve the induction efficiency and promote this method.
Mesenchymal stem cells with multidirectional differentiation potential have been differentiated into renal tubular epithelial cells and effectively promote the restoration of renal function in injured kidneys [4,10-13].In the present study, we assessed whether induced PBMCs function as mesenchymal stem cells. To this end, we divided 40 rabbits into four groups: a normal control group, a model control group, and induced cell treatment group, and a noninduced cell treatment group. After the successful establishment of the model, we compared the efficacies of the treatments and ultimately revealed that the induced PBMCs function as multipotent stem cells and participate in the repair of renal injury.
Materials and methods
1. Preparation of the rabbit model of renal ischemia-reperfusion injury
According to the inclusion and exclusion criteria, 40 wild-type Japanese white rabbits were numbered according to their body mass and randomly allocated to a group. The rabbits were purchased from Kunming Chushang Technology Co., Ltd., and the license number was SCXK (Dian)K2018-0001. The rabbits were anesthetized with intravenous injections of 3% sodium pentobarbital (Merck, Darmstadt, Germany) at a dose of 1 ml per kg of body weight. The abdominal skin of the rabbits was prepared, and an incision was made along the midline of the abdomen. The skin, subcutaneous tissue, and the peritoneum were separated layer by layer, and an incision was then made in the abdominal cavity. The renal pedicle was located, and the bilateral renal pedicle was rapidly clipped with a noninvasive arterial clip to restrict the blood flow for 1 hour. The artery clip was then released to restore the blood flow and allow reperfusion, which resulted in ischemia-reperfusion injury. The model was established in 30 rabbits, and the other 10 rabbits were not subjected to the operation and served as the normal control group. Among the rabbits with ischemia-reperfusion injury, 10 were used as the model control group, 10 other rabbits were injected with induced PBMCs, and the remaining 10 were injected with noninduced PBMCs. The cells were transfused on the first day after model establishment and then once a week for 3 consecutive weeks. All experimental protocols were approved by the Experimental Animal Ethics Committee of the 920th Hospital of the Joint Logistics Support Force of the People's Liberation Army(approved number: 2019-002-01). No human subjects were included in this study, and thus, informed consent is not applicable. The DOI link of experimental steps is dx doi.org/10.17504/protocols.io.bpyrmpv6.
2. Rabbit PBMC isolation, culture, induction, and labeling
Twenty milliliters of peripheral blood was harvested from each of the three rabbits, and the experiment was performed more than three times to ensure repeatability. PBMCs were isolated using a separation fluid (Haoyang Biological Products Technology Co., Ltd., Tianjin, China). After two washes, the cells were cultured in culture flasks. The cells in one flask were cultured in DMEM-F12 medium supplemented with 10% fetal bovine serum, and the cells in the other flask were cultured with 50% egg white extract in a culture medium. As shown in our previous study, cells grown in 50% chicken egg white extract in a culture medium will differentiate into multipotent stem cells and might be used for the treatment of diseases[9]. Thus, we used 50%chicken egg white extract-induced rabbit PBMCs as a treatment for kidney injury in the rabbit model. Three days after induction, CFSE-labeled cells were transfused into the rabbits belonging to the two treatment groups; CFSE fluoresces yellowish-green. The cells were centrifuged, and the supernatant was discarded. The cell pellet was suspended in 4 ml of PBS， and 10μl of 10 mM CFSE (from Abcam, ab145291, Cambridge, UK) was added. The mixture was incubated at 37°C for 10 minutes, and 3 ml of medium was added to terminate the labeling reaction. The cells were centrifuged, and the supernatant was removed. The cells were suspended in saline, and each rabbit received a transfusion of 2×10° cells. The cell survival rate was greater than 95%. The PBMCs were injected via the intravenous route. We observed that the fluorescence lasted for more than 1 month after the cells were labeled with CFSE, and because the experimental period was less than a month, the fluorescent cells could be traced.
3. Identification of noninduced and induced PBMCs
3.1.Quantitative PCR-based detection of the relative expression of pluripotency-related and somatic cell genes.
Three days after induction, RNA was extracted from the PBMCs, reverse transcribed, and subjected to quantitative PCR to determine the relative levels and fold changes in the expression of the pluripotency-related genes OCT4, NANOG, and SOX2 and the somatic cell gene LMNA. The primer sequences and product lengths are shown in Table 1.
https://doi.org/10.1371/journal.pone.0244160.t001
3.2.Flow cytometry.
Three days after induction, the PBMCs were labeled with SSEA-4-PE, OCT4-PE, and NANOG-PE, and the changes in the expression of pluripotency markers were detected.
SSEA-4 is a surface-expressed antigen. The PBMCs were centrifuged, and the supernatant was discarded [9]. The pellet was suspended in 50 ul of PBS, and 20 ul of an isotype control antibody or SSEA-4-PE was added to the wells. The mixtures were incubated for 1 hour at room temperature in the dark, and the cells were then washed once with PBS, fixed with 4%paraformaldehyde in PBS, and detected using a flow cytometer(Manufacturer: Becton Dickin-son; model: BD LSRFortessa; software version: BD FACSDiva Software v8.0.1, USA). All the antibodies were used in strict accordance with the manufacturer's instructions to avoid non-specific binding.
3.3. Immunohistochemical detection.
Noninduced and induced PBMCs were subjected to immunohistochemistry analysis. Specifically, noninduced and induced PBMCs were smeared on a slide, dried, and fixed with 4% paraformaldehyde for 10 minutes. The cells were stained with an Abcam immunohistochemistry kit using primary antibodies against OCT4 and NANOG at 1:50 dilution. The procedures were performed according to the manufacturer's instructions. DAB was used to develop the staining, and images were captured.
3.4. Western blot detection.
The western blot detection of noninduced and induced PBMCs was performed as follows. Proteins were extracted from noninduced and induced PBMCs, electrophoresed, and transferred to a membrane. The membrane was blocked for 1 hour and then incubated with a 1:500 dilution of an anti-OCT4 primary antibody for 1 hour at 37°C with shaking. The membrane was subjected to three 5-minute washes with TTBS and then incubated with a 1:500 dilution of the secondary antibody for 1 hour at 37°Cwith shaking. The membrane was subjected to three 5-minute washes with TTBS. Enhanced chemiluminescence (ECL)was used to detect the proteins, and images were captured using a chemiluminescence imaging camera (Tanon 5200, Shanghai).
3.5. Quantitative PCR detection of relative changes in the telomere length.
The relative changes in the telomere lengths in noninduced and induced PBMCs were detected as follows. Three days after induction, RNA was extracted from the PBMCs, reverse transcribed, and subjected to quantitative PCR to determine the relative changes in the telomere length. The telo-mere and internal control 36B4 primer sequences are shown in Table 2.
https://doi.org/10.1371/journal.pone.0244160.t002
4. Kidney function test
After three transplantations of the labeled cell, blood samples were collected from the ear veins of rabbits in the four groups. Serum was separated and sent to a laboratory to examine the changes in the creatinine and urea nitrogen contents.
5. Changes in the urinary protein content
After three transplantations of the labeled cells, urine samples were collected from the four groups of rabbits, and the urine protein contents were measured using the BCA method.
6. Observation of labeled cells in frozen sections
After three transplantations of the labeled cells, three rabbits from each group were euthanized through the introduction of an air embolism. The renal distributions of the labeled cells in frozen kidney sections were observed. The kidney tissue collected was partly frozen to observe fluorescent cells, and the histology was partially evaluated.
7. Histopathological assessment of the kidneys
Kidney tissue was used for HE staining and immunohistochemistry analysis of the fibrosis-related factor TGF-β, Masson's trichrome staining, and PAS staining. Changes in the renal tissues from each group were observed through HE staining. The degree of renal fibrosis was detected by immunohistochemistry. The extent of fibrosis in the kidneys from each group was detected by Masson's trichrome staining. The thickening of the renal basement membrane was detected by PAS staining. The kidney tissues of the treatment group were frozen, sectioned, incubated with the primary antibody aquaporin-1(from Abcam, ab9566, Cambridge, UK)specific for the proximal tubules, incubated with a red fluorescence-labeled secondary antibody(from Abcam,ab150115, Cambridge, UK), and counterstained with DAPI, and the distribution of red, green, and blue fluorescence was observed under a fluorescence microscope.
8. Metabolomics analysis of the kidney
After three transplantations of the labeled cells, five rabbits from each of the four groups were euthanized via the introduction of an air embolism. The kidney tissues were rapidly removed, placed in liquid nitrogen, refrozen at-80°C, and then sent to a company on dry ice for metabolomics analysis. Five biological replicates of rabbit kidney tissue samples from the four groups were analyzed, and the 20 samples were then analyzed by LC-QTOFMS. The ionization source of the LC-QTOFMS platform was electrospray ionization. Two ionization modes, positive ion mode(POS) and negative ion mode(NEG), were used for the detection of metabolites and increasing the coverage rate and thus the detection rate.
9. Statistical analysis
All statistical analyses were performed using SPSS 21.0 statistical software. The measurement results are expressed as the means±standard deviations. Four-group comparisons and the above results were analyzed by one-way analysis of variance (one-way ANOVA), and p<0.05 was considered statistically significant. Post hoc comparisons between individual groups after ANOVA were needed for the assessment of significant differences between specific groups. Pairwise comparisons between groups were performed using the LSD and SNK methods.
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