Abstract
Introduction
Acute kidney injury (AKI) is a clinical emergency caused by the rapid decline of renal function caused by various etiologies. Growth differentiation factor 11 (GDF11) can promote renal tubular regeneration and improve kidney function in AKI, but the specific mechanism remains unclear. Herein, we investigated the effect and mechanisms of GDF11 in ameliorating AKI induced by ischemia-reperfusion (I/R).
Methods
An animal model of AKI was established by I/R method, and the changes of serum urea nitrogen and creatinine were measured to evaluate the AKI. Enzyme-linked immunosorbent assay (ELISA) was used to measure cytokines, malondialdehyde, superoxide dismutase, nitric oxide synthase, and arginase 1 levels. Flow cytometry was used to count the M1/M2 macrophages. IHC, WB, and q-PCR experiments were used to evaluate the expression of GDF11.
Results
The changes in serum levels of urea nitrogen and creatinine after I/R suggest that an animal model of AKI induced by I/R was successfully established. AKI caused by I/R significantly changed the M1/M2 macrophage polarization balance, with an increase in M2 being significantly higher than M1 as well as increased oxidative stress. Treatment with GDF11 after I/R significantly increased the differentiation of M2 cells and inhibited the differentiation of M1 macrophages, as well as decreased oxidative stress.
Conclusion
GDF11 can promote the repair of AKI caused by I/R by regulating the balance of M1/M2 polarization in macrophages and oxidative stress.
Keywords: Ischemia-reperfusion, Acute kidney injury, Macrophage polarization, GDF11, Cytokines
Introduction
Acute kidney injury (AKI) is a common clinical emergency. A variety of clinical risk factors such as severe trauma, shock, sepsis, renal transplantation, and the use of nephrotoxic drugs can lead to the occurrence of AKI [1–3]. The incidence of AKI in developed countries accounts for 3.2–9.6% of hospitalized patients. In developing countries, the mortality rate of AKI is still as high as 50–60%. Nearly 2 million patients die of AKI every year worldwide [4, 5]. Therefore, in-depth analysis of the molecular mechanisms of AKI pathogenesis will facilitate the discovery of new therapeutic targets and markers for accurate diagnosis of AKI; this is significant for reducing the incidence and mortality of AKI.
The pathogenesis of AKI is complex with multiple etiology. Ischemia-reperfusion (I/R) injury is one of the main causes of AKI [6, 7]. Studies have shown that I/R leads to an increase in the permeability of renal blood vessels and the infiltration of inflammatory cells. Inflammatory cells on the surface of renal endothelial cells trigger the immune inflammatory response and subsequently induce AKI. Macrophages are highly plastic inflammatory cells both in vivo and in vitro [8]. During kidney injury, macrophages polarize into different phenotypes and play multiple roles in the local microenvironment [9]. Macrophages have been shown to contribute to renal repair after I/R by switching their phenotype from pro-inflammatory M1 to anti-inflammatory M2 type [10]. However, the specific mechanisms of macrophage polarization into M1 and M2 during renal I/R still require further study.
Growth differentiation factor 11 (GDF11) is an important member of the transforming growth factor beta (TGF-β) superfamily, which is widely present in muscle, brain, kidney, spleen, nerve tissue, etc. [11, 12]. Recent studies have found that GDF11 can promote M2 macrophage polarization and reduce M1 macrophage polarization through the TGFβ-R1/smad2 pathway in severe acute pancreatitis, thereby inhibiting the acute inflammatory injury in the pancreas [13]. However, the application of GDF11 in the treatment of AKI disease caused by I/R is rarely reported. GDF11 has been reported to promote the regeneration of renal tubules in aged mice, thereby improving the renal function of I/R in AKI [14]. Also, GDF11 has been revealed to stimulate the proliferation of renal fibroblasts and induce EMT in tubular epithelial cell, indicating that GDF11 may contribute to the renal fibrosis [15]. The relationship between GDF11 and pulmonary fibrosis is proven by in vitro intervention of GDF11 on lung proper cells and lung fibroblasts exposed to cigarette smoke extract that GDF11 can significantly inhibit cell aging and inflammation and improve tissue repair mediated by fibroblasts [16]; however, its specific mechanism for the treatment of AKI is still unclear. In addition, it still remains unknown whether GDF11 can regulate M1/M2 macrophage polarization to promote the repair of AKI.
In this study, the I/R-induced AKI model was established, and the M1/M2 polarization, inflammatory factor release, and GDF11 expression of macrophages at different time periods after surgery were detected. The effects of GDF11 intervention in the repair of AKI damage was explored through animal experiments and cell experiments.
Materials and Methods
Animals
A total of 40 eight-week-old female C57 mice were purchased from the Laboratory Animal Center of Guangdong Provincial and housed in the animal facility of Shenzhen University School of Medicine. This study was approved by the Laboratory Animal Ethics Association of Shenzhen University (No. 202100311).
Reagents
The enzyme-linked immunosorbent assay (ELISA) kits of mouse interleukin (IL)-1β, -4, -6, -10, tumor necrosis factor α (TNF-α), and transforming growth factor β1 (TGF-β1) were purchased from eBioscience (San Diego, CA, USA). The malondialdehyde (MDA) and superoxide dismutase (SOD) detection kits were purchased from Nanjing JianChen Inc. (Nanjing, China). The inducible nitric oxide synthase (iNOS) and arginase (Arg-1) assay kits were purchased from Sigma (St. Louis, MO, USA). Anti-CD11b, F4/80, and CD206 antibodies were purchased from BioLegend (San Diego, CA, USA). Recombinant GDF11 was purchased from Abnova (Taiwan, China). Anti-GDF11 polyclonal antibody was purchased from Solarbio Inc. (Beijing, China).
Methods
Establishment of I/R Mouse Model and GDF11 Treatment
A mouse model of renal I/R injury was established by bilateral renal pedicle clamping in 8-week-old C57BL/6 mice [7]. Briefly, the mice were anesthetized by injection of 1% sodium pentobarbital (80 mg/kg), and the bilateral kidneys were surgically separated and exposed in the abdominal renal region. After blocking the arterial blood flow for 45 min, the arterial clip was released and the blood perfusion was confirmed to be restored. The kidneys were repositioned, and the incision was sutured. At the same time, a sham-operation control group (sham group) was established without clamping the renal pedicle, but other conditions were kept the same as the I/R group. In addition, the death of experimental mice was observed for 7 days after operation and counted every day. In I/R mice, GDF11 (0.1 mg/kg/day) was injected into the tail vein of mice half an hour after the surgery and continuously injected daily for 7 days. After the experiment, tissue samples (such as from the kidney) and blood of mice were collected for relevant detection and analysis.
Specimen Collection
Mice were sacrificed 1 day, 3 days, and 7 days after the establishment of the I/R model. Blood was collected from the heart. Each kidney was cut into two pieces along the longitudinal section, and four kidney tissues were collected for subsequent experiments.
Renal Function Assessment
The serum was collected by centrifuging the blood samples at 5,000 r/min for 10 min and then stored in a refrigerator at −20°C. The serum levels of creatinine (Cr) and blood urea nitrogen were measured using ELISA kits in accordance with the instructions provided with the kits.
Cytokine Measurement
The serum levels of IL-1β, IL-6, TNF-α, IL-4, IL-10, and TGF-β were measured by ELISA in accordance with the user manual.
MDA and SOD Measurement
Mouse kidney tissues were washed with sterile PBS, and then 5 mg of tissue was weighed and placed in a 2 mL glass homogenizer containing 300 μL pre-cooled PBS on ice. After centrifuging at 10,000 rpm for 10 min at 4°C, the supernatant was collected and the protein concentration was measured using a BCA protein concentration assay kit. Samples were diluted with PBS solution to a protein concentration of 1.0 mg/mL. MDA and SOD were measured using commercial kits by following the user manual.
Measurement of iNOS and Arg-1 Level in Mouse Kidney Tissue
The sample was prepared from 5 mg of kidney tissue as described above. The iNOS and Arg-1 levels were measured according to the instructions of the iNOS and Arg-1 ELISA detection kit.
Flow Cytometry Assay of Macrophage Polarization
Kidney tissues were cut into pieces in saline aseptically, homogenized with a 2.5 mL syringe, and passed through a 400-mesh wire mesh to obtain a single cell suspension. The cell concentration was adjusted to 1 × 106/mL. 5 μL of anti-CD11b, F4/80, and CD206 antibodies were added to 200 μL of the cell suspension and incubated at room temperature for 25 min. M1/M2 macrophages were counted using flow cytometry.
HE Staining of Renal Tissues
The kidney tissues were fixed with 4% paraformaldehyde for 24 h before section. Antigen was retrieved by incubating the kidney tissue slices at 60°C for 30 min. The slices were deparaffinized with different concentrations of ethanol (100%, 90%, 80%, 70%). For HE staining, slices were incubated with hematoxylin for 15 min at room temperature, following by washing with clean water and dipping in eosin solution for 2–5 min. After gradient dehydration with different concentrations of ethanol (70%, 80%, 90%, 100%), the slices were sealed and observed under a microscope.
Immunofluorescence Staining of iNOS and CD206
The kidney tissue sections were prepared as described above. Slices were incubated with Alexa Fluor 488-labeled CD206 antibody and TRITC-labeled iNOS antibody, at 4°C overnight. After being washed with PBST, slices were incubated with 500 μL of DAPI staining solution at 37°C for 10 min. After being washed with 500 μL PBST and rinsed with DAPI, 50 μL anti-fluorescence quencher was then added and observed under a fluorescence microscope.
Western Blot of GDF11 Expression in Mouse Kidney Tissues
Ten milligrams of kidney tissues was homogenized in 90 μL of lysis buffer on ice. The protein concentration in homogenate was measured using a BCA protein concentration assay kit. 10 µg of renal tissue protein was applied to polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane, and then the membranes were incubated with 5% BSA at room temperature for 30 min, followed by incubating with the primary CDF11 antibody overnight at 4°C. After being washed with Tris-buffered saline with 0.1% Tween 20 detergent (TBST) for 3 × 10 min, the membranes were incubated with the secondary antibody (diluted 1:10,000 in TBST) for 60 min at room temperature, followed by washing with TBST for 3 × 10 min, and then incubating with ECL developer solution and exposing on X-film.
Quantitative Reverse Transcription PCR of GDF11 mRNA Expression in Mouse Kidney Tissues
The total RNA was extracted from the fresh kidney tissues using an RNA extraction kit under RNase-free conditions. GDF11 primer was applied to amplify the mRNA of GDF11 mRNA according to the quantitative reverse transcription PCR kit.
Immunohistochemistry Detection of Gdf11 Expression in Mouse Kidney Tissues
The kidney tissue sections were prepared as described above. The slices were incubated with 20 μg/mL proteinase K for 10–20 min. After being washed with PBS, slices were incubated with anti-GDF11 antibody at 4°C overnight, followed by being incubated with enzyme-labeled secondary antibodies at room temperature for 30 min, stained with 50 μL of freshly prepared DAB chromogenic solution for 5 min, rinsed with DAB staining solution, and then incubated with hematoxylin staining solution for 3 min. After being mounted, the slides were observed under a microscope.
In vitro Cell Co-Culture Assay of the Effect of GDF11 on M1/M2 Macrophage Polarization
The experiment contained five groups: normal control group (1 × 106 TECs cells were co-cultured with 1 × 106 bone marrow cells for 5 days); GDF11 (25 ng)-P group (1 × 106 TECs cells were stimulated with 25 ng/mL GDF11 for 3 days and then co-incubated with 1 × 106 bone marrow cells for 2 days); GDF11 (100 ng)-P group (1 × 106 TECs cells were treated with 100 ng/mL GDF11 for 3 days and then co-cultured with 1 × 106 bone marrow cells for 2 days); GDF11 (25 ng)-T (1 × 106 TECs cells was treated with 25 ng/mL GDF11 and co-incubated with 1 × 106 bone marrow cells for 5 days); GDF11 (100 ng)-T (1 × 106 TECs cells were co-cultured with 1 × 106 bone marrow cells and 100 ng/mL GDF11 for 5 days).
Statistical Analysis
The SPSS statistical software was used to analyze the association between the GDF11 treatment group and the control group. Some statistics were analyzed using the stat package (v 2.5.2) in R (v 3.5.2). Data are expressed as mean ± standard error (**p < 0.01; *p < 0.05; ns, no significant difference).
Results
I/R Establishment of Animal Model of AKI
As shown in Figure 1a, the survival rate of mice in the I/R model group was 75% with no death in the sham-operation group (Sham). Figure 1b shows the longitudinal sections of the mouse kidneys in each group. The I/R group showed obvious kidney damage, but the damage was slightly improved after the 7th day. Serum urea nitrogen (blood urine nitrogen) and creatinine (Cr) levels were significantly increased on the 1st day after I/R when compared to the sham-operation group, indicating that the animal model of AKI was successfully established. The creatinine and urea nitrogen levels were decreased significantly on the 3rd and 7th day compared to the 1st day after I/R (Fig. 1c).
Fig. 1.
Establishment of a mouse model of AKI induced by I/R. a Survival rate after I/R surgery and sham surgery. b Appearance of longitudinal section of kidney after I/R surgery and sham surgery. c Serum level of urea nitrogen and creatinine. Normal, normal group; Sham, sham-operated group; I/R 1D, 1 day after I/R; I/R 3D, 3 days after I/R; I/R 7D, 7 days after I/R. *p < 0.05, **p < 0.01; NS, p > 0.05 compared with the sham group.
I/R-Induced AKI Affects Serum Cytokines and Oxidative Stress
Among the M1 cytokines (IL-6, IL-1β, TNF-a), serum IL-6 was increased significantly on the 7th day after surgery, while IL-1β was decreased on the 3rd day and then increased on the 7th day, and TNF-a was decreased on the1st and 3rd day after surgery (Fig. 2a–c). Among the M2 cytokines (IL-4, IL-10, TGF-β1), serum IL-4 was decreased on the 3rd day, IL-10 was increased on the 1st and 7th day, and TGF-β1 was significantly decreased on the 1st and 3rd day after surgery (Fig. 2d–f). MDA level in the kidney tissues of the sham-operation group was slightly higher than that in the normal control group with no significant difference (p > 0.05); the MDA level was significantly higher on the 1st and 3rd day after I/R, with no significant difference on the 7th day compared to the sham-operation group (Fig. 2g). SOD was significantly decreased on the 1st and 3rd day after surgery compared to the sham group, with gradual increase after post-operation time (Fig. 2h). These results suggest that I/R can aggravate renal tissue inflammation by increasing renal oxidative stress.
Fig. 2.
Effects of I/R-induced AKI on serum cytokines levels. Serum levels of oxidative stress M1-type cytokines IL-6 (a), IL-1β (b), TNF-α (c) as well as M2-type cytokines IL-4 (d), IL-10 (e), and TGF-β (f) were measured. MDA (g) and SOD (h) levels were measured in mouse kidney tissue homogenates. Normal, normal group; Sham, sham-operated group; I/R 1D, 1 day after I/R; I/R 3D, 3 days after I/R; I/R 7D, 7 days after I/R. *p < 0.05, **p < 0.01; NS, p > 0.05 compared with the sham group.
Effects of I/R-Induced AKI on Macrophage Polarization
The polarization of macrophages was assessed by detecting the expression of iNOS and Arg-1 in kidney tissue. The iNOS expression in the I/R group was increased on the 1st and 3rd day after surgery compared to the sham group, while the expression of Arg-1 in I/R group was increased after surgery compared with that in the sham group (Fig. 3a). The effects of I/R-induced AKI on kidney tissue and tissue macrophages were analyzed by HE staining and immunofluorescence experiments. HE staining showed that the renal tissue cells were most affected on the 1st day after I/R. There was granular material deposition around the renal tubules, and atrophic glomeruli were occasionally seen. The histiocyte morphology was gradually recovered on the 3rd day, indicating that the injury began to repair. Immunofluorescence staining showed that the markers of M1 and M2 macrophages were significantly increased on the 1st day after I/R, and gradually decreased with the prolongation of the post-operation time, indicating that the polarization of M1/M2 macrophages was not stable (Fig. 3b). I/R-induced AKI repair may play an important role. In order to further clarify the effect of I/R-induced AKI on macrophage polarization, flow cytometry was used to count the macrophages. M1 (CD11b+F4/80+) macrophages in the I/R group reached a high peak on the 1st day (proportion: 5.46%) and gradually decreased with the prolongation of the post-operation time (Fig. 3c); Meanwhile, M2 (CD11b+CD206+) in the I/R group was higher than in the sham group throughout the entire post-operation time. The results showed that M1 macrophages may be highly expressed in the early stage of renal injury, while M2 macrophages are the main cells in the later stage involving the repair process.
Fig. 3.
Effects of AKI induced by I/R on macrophage polarization. a The content of iNOS and Arg-1 in mouse kidney tissue was detected by ELISA. b The pathological changes of kidney tissue were observed by HE staining and macrophage immunofluorescence. c M1/M2 polarization was analyzed by flow cytometry. Normal, normal group; Sham, sham group; I/R 1D, 1 day after I/R; I/R 3D, 3 days after I/R; I/R 7D, 7 days after I/R. *p < 0.05, **p < 0.01; NS, p > 0.05 compared to the sham group.
I/R-Induced AKI Affects the Expression of GDF11
CDF11 protein expression was significantly increased in the I/R group after surgery compared to the sham control group (Fig. 4a). Western blot showed that I/R-induced AKI increased GDF11 protein expression in kidney tissues on the 1st and 3rd day after surgery compared to the sham group (Fig. 4b). Quantitative reverse transcription PCR showed that CDF11 mRNA expression was significantly increased in the I/R group after surgery compared to the sham group (Fig. 4c). These results suggest that GDF11 may contribute to the repair of renal injury after I/R surgery through inhibiting the inflammation process of renal tissue.
Fig. 4.
Effect of AKI induced by I/R on GDF11 expression. a The protein expression of GDF11 in mouse kidney tissue was detected by IHC. The brown color in IHC represented the positive staining of GDF11. b The protein expression of GDF11 in mouse kidney tissue was detected by WB. c The mRNA level of GDF11 in kidney tissues was detected by q-PCR. Normal, normal group; Sham, sham group; I/R 1D, 1 day after I/R; I/R 3D, 3 days after I/R; I/R7D, 7 days after I/R. *p < 0.05, **p < 0.01; NS, p > 0.05 compared with the sham group.
GDF11 Promoted M1/M2 Cell Polarization in vitro
In order to clarify the role of GDF11 in promoting the polarization of M1/M2 cells, primary mouse TECs cells and bone marrow cells were co-cultured. The flow cytometry assay showed that GDF11 inhibited the differentiation of M1 macrophages with increasing concentration of GDF11, whereas GDF11 promoted the differentiation of M2 macrophages. When GDF11 was simultaneously treated with TECs cells and bone marrow cells in co-culture, GDP11 inhibited the differentiation of M1 macrophages and promoted the differentiation of M2 macrophages in a concentration-dependent manner. The results indicated that GDF11 may act on both TECs cells and myeloid cells at the same time (Fig. 5).
Fig. 5.
Flow cytometry assay of the effect of GDF11 on M1/M2 polarization of macrophages in vitro.
Effects of GDF11 on Serum Cytokines after I/R-Induced AKI
In order to verify the treatment effect of GDF11 on I/R-induced AKI, GDF11 was used to block AKI after I/R in mice. The results showed that GDF11 significantly inhibited the expression of IL-6 (Fig. 6a), but it had little effect on the expression of IL-1β (Fig. 6b) and TNF-α (Fig. 6c); this indicates that GDF11 may have an inhibitory effect on M1 macrophages. CDF11 promoted the expression of IL-4 (Fig. 6d), IL-10 (Fig. 6e), and TGF-β (Fig. 6f), indicating that GDF11 has a role in promoting M2 macrophage polarization.
Fig. 6.
Effect of GDF11 on the serum levels of cytokines after I/R treatment. The serum levels of IL-6 (a), IL-1β (b), TNF-α (c), IL-4 (d), IL-10 (e), and TGF-β (f) were measured. Normal, normal group; Sham, sham group; I/R 1D, 1 day after I/R; I/R 3D, 3 days after I/R; I/R 7D, 7 days after I/R. *p < 0.05, **p < 0.01; NS, p > 0.05 compared with the sham group.
GDF11 Affected Macrophage M1/M2 Polarization in I/R-Induced AKI
In order to explore the effect of CDF11 on M1/M2 polarization of macrophages during AKI, the M1/M2 macrophages were counted by flow cytometry. As shown in Figure 7, the M1 (CD11b+F4/80+) macrophages accounted for 1.61% in the normal group, 3.15% in the sham group, 5.42% in the I/R 7-day (D) group, and 5.42% in the I/R 7D group, and 4.54% in the I/R 7D + GDF11 group; this indicates that the number of M1 macrophages increased after I/R, but GDF11 treatment significantly decreased M1 macrophages, which also indicates that GDF11 has the effect of inhibiting the differentiation of M1 macrophages. The M2 (CD11b+CD206+) macrophages were 1.9% in the normal group, 2.82% in the sham model group, and 3.48% in the I/R model group. After GDF11 treatment, M2 macrophages were increased to 5%, indicating that GDF11 promoted M2 differentiation.
Fig. 7.
In vivo study of the effect of GDF11 treatment on M1/M2 polarization of macrophages in I/R mouse model.
Discussion
This study established an I/R-induced AKI mouse model to investigate the roles of CDF11 in the inflammatory factor expression, oxidative stress, and M1/M2 macrophages polarization in AKI. Our study suggests that macrophage polarization is involved in the process of kidney injury and repair, among which M1 macrophages play a key role in the early stage of kidney injury through a pro-inflammatory role, while M2 macrophages are the main cells involved in the later repair process. GDF11 can protect mice from I/R-induced AKI through regulating the polarization of M1/M2 macrophages by exerting an inhibitory effect on M1 macrophage polarization and a promoting effect on M2 macrophage polarization.
AKI is a clinical syndrome with increasing incidence year by year. During AKI, the renal function of patients is damaged in a short time, which is reflected in the increase of serum creatinine and serum urea nitrogen [17]. The pathophysiological mechanisms of AKI are complex, and I/R is one of the main causes that lead to AKI. I/R injury refers to the metabolic disorder of cells and the destruction of structure and function of kidney [18, 19]. In this study, we found that serum urea nitrogen and creatinine were significantly increased after I/R when compared with the sham-operation group, indicating that the I/R-induced AKI model was successfully established. The inflammatory response to I/R-induced AKI is caused by oxidative stress, inflammatory cytokine production, and infiltration of neutrophils and macrophages [20]. The M1/M2 polarization of macrophages is involved in the release and dynamic changes of many cytokines, including pro-inflammatory factors such as IL-6, IL-1β, and TNF-α from M1 macrophages and IL-4, IL-10, and TGF-β from M2 macrophages [21]. A previous study found that GDF11 treatment can antagonize TNF-a-induced inflammation in macrophages and protected against the development of inflammatory arthritis in mice [22]. Our results showed that the M1 and M2 macrophage cytokines were mainly decreased on the first day after surgery and then increased; this indicates that macrophages are differentiated into M1 and M2 during the repair process of AKI, and then they participate in the repair process of AKI. M1-type cytokines are associated with inflammatory damage; for example, IL-6 can recruit macrophages to infiltrate the renal interstitium, promoting further renal tissue damage [23]. M2-type cytokines mainly play a role in the repair process; for example, IL-4 is a multifunctional cytokine, which can reduce the production of pro-inflammatory cytokines, and the increase of IL-4 production is related to the reduction of renal injury in rats [24]. In I/R-induced AKI, oxidative stress increases macrophage infiltration and TECs damage, which in turn aggravates renal injury [25]. Our study showed that MDA was significantly increased, and SOD was significantly decreased on the first day after I/R; the corresponding oxidative stress-related MDA decreased with the prolongation of post-operation time, while the antioxidant SOD gradually increased, indicating a process of AKI repair. The level of oxidative stress was also reversed accordingly.
It has been demonstrated that in the different stages of AKI, there are different macrophage types in kidney tissues. For example, iNOS+ M1 macrophages are recruited to the kidney within 48 h after I/R injury, whereas Arg-1+ and CD206+ M2 macrophages predominate at subsequent time points [26, 27]. During the renal repair period, M1 macrophages can be transformed into the M2 type, and the corresponding function also changes from promoting damage to repairing renal tubules [28]. Our study showed that the number of M2-type macrophages was increased in the late stage of repair, while the M1 type of macrophages was decreased in the late stage of repair, suggesting that M2 macrophages play a dominant role in the late stage of AKI repair. GDF11 has been revealed to regulate the local inflammatory microenvironment and exert an anti-inflammatory effect through upregulating the transcription level of IL-10 anti-inflammatory factor and downregulating the expression of inflammatory factors such as IL-1β, IL-6, and TNF-α [29]; however, it has not yet been reported whether GDF11 regulates macrophage differentiation and promotes repair in AKI induced by I/R. In this study, both the CDF11 mRNA and protein expression were gradually increased during the repair of AKI. Although it was down-regulated on the seventh day, it was still significantly higher than that in the control and sham-operated groups, indicating that GDF11 may play a role in the repair of AKI. In addition, our in vitro cell experiments also found that GDF11 has an inhibitory effect on M1-type macrophages, whereas has a promoting effect on M2-type macrophages. These in vitro findings indicated that GDF11 could repair I/R-induced renal injury in AKI by regulating M1/M2 polarization. This was also confirmed in our animal experiments using GDF11 to treat I/R-induced AKI in mice.
In conclusion, our study proves that GDF11 is involved in the repair process of AKI caused by I/R. GDF11 can promote the differentiation of macrophages to the M2 type and inhibit the differentiation of macrophages to the M1 type. Therefore, GDF11 therapy may delay the damage process of AKI or promote the repair of AKI. Although the cause of GDF11 production in I/R-induced AKI is still unknown and needs further study, GDF11 regulates M1/M2 macrophage polarization and subsequently promotes repair which may provide help for clinical AKI treatment.
Statement of Ethics
This study was approved by the Laboratory Animal Ethics Association of Shenzhen University (No. 202100311), and all animal experiments are conducted in accordance with the regulations on the administration of experimental animals and relevant national laws and regulations.
Conflict of Interest Statement
The authors have no conflicts of interest to declare. Wei-hua Liu, Xuan Wang, Lixin Wei, and He-qun Zou declare that they have no conflict of interest. Ling Feng declares that she has no conflict of interest.
Funding Sources
This study is supported by the Natural Science Foundation of Fujian Province (2022J01998) and Natural Science Foundation of Guangdong Province (2021A1515010972) and National Natural Science Foundation of China (81873620), University Stability Support Project of Shenzhen Science and Technology Innovation Committee (20200822123122001), and Key Technology Research Project of Shenzhen Science and Technology Innovation Committee (JSGG20200225152709802).
Author Contributions
Wei-hua Liu wrote the manuscript and contributed to the final revision of the manuscript. Ling Feng participated in the design of this study. Xuan Wang participated in formal analysis and data curation of this study. He-qun Zou and Lixin Wei involved in the critical revision and approved the final version of the manuscript.
Funding Statement
This study is supported by the Natural Science Foundation of Fujian Province (2022J01998) and Natural Science Foundation of Guangdong Province (2021A1515010972) and National Natural Science Foundation of China (81873620), University Stability Support Project of Shenzhen Science and Technology Innovation Committee (20200822123122001), and Key Technology Research Project of Shenzhen Science and Technology Innovation Committee (JSGG20200225152709802).
Data Availability Statement
All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.
References
- 1. Bagshaw SM, Darmon M, Ostermann M, Finkelstein FO, Wald R, Tolwani AJ, et al. Current state of the art for renal replacement therapy in critically ill patients with acute kidney injury. Intensive Care Med. 2017;43(6):841–54. 10.1007/s00134-017-4762-8. [DOI] [PubMed] [Google Scholar]
- 2. Menting TP, Wever KE, Ozdemir-van Brunschot DM, Van der Vliet DJ, Rovers MM, Warle MC. Ischaemic preconditioning for the reduction of renal ischaemia reperfusion injury. Cochrane Database Syst Rev. 2017;3:CD010777. 10.1002/14651858.CD010777.pub2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Makris K, Spanou L. Acute kidney injury: definition, pathophysiology and clinical phenotypes. Clin Biochem Rev. 2016;37(2):85–98. [PMC free article] [PubMed] [Google Scholar]
- 4. Heung M, Yessayan L. Renal replacement therapy in acute kidney injury: controversies and consensus. Crit Care Clin. 2017;33(2):365–78. 10.1016/j.ccc.2016.12.003. [DOI] [PubMed] [Google Scholar]
- 5. Bouchard J, Mehta RL. Acute kidney injury in Western countries. Kidney Dis. 2016;2(3):103–10. 10.1159/000445091. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Zhou L, Zhuo H, Ouyang H, Liu Y, Yuan F, Sun L, et al. Glycoprotein non-metastatic melanoma protein b (Gpnmb) is highly expressed in macrophages of acute injured kidney and promotes M2 macrophages polarization. Cell Immunol. 2017;316:53–60. 10.1016/j.cellimm.2017.03.006. [DOI] [PubMed] [Google Scholar]
- 7. Malek M, Nematbakhsh M. Renal ischemia/reperfusion injury; from pathophysiology to treatment. J Ren Inj Prev. 2015;4(2):20–7. 10.12861/jrip.2015.06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Rodriguez F, Bonacasa B, Fenoy FJ, Salom MG. Reactive oxygen and nitrogen species in the renal ischemia/reperfusion injury. Curr Pharm Des. 2013;19(15):2776–94. 10.2174/1381612811319150014. [DOI] [PubMed] [Google Scholar]
- 9. Meng XM, Tang PMK, Li J, Lan HY. Macrophage phenotype in kidney injury and repair. Kidney Dis. 2015;1(2):138–46. 10.1159/000431214. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Wen Y, Crowley SD. The varying roles of macrophages in kidney injury and repair. Curr Opin Nephrol Hypertens. 2020;29(3):286–92. 10.1097/MNH.0000000000000595. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Walker RG, Poggioli T, Katsimpardi L, Buchanan SM, Oh J, Wattrus S, et al. Biochemistry and biology of GDF11 and myostatin: similarities, differences, and questions for future investigation. Circ Res. 2016;118(7):1125–41; discussion 1142. 10.1161/CIRCRESAHA.116.308391. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Egerman MA, Glass DJ. The role of GDF11 in aging and skeletal muscle, cardiac and bone homeostasis. Crit Rev Biochem Mol Biol. 2019;54(2):174–83. 10.1080/10409238.2019.1610722. [DOI] [PubMed] [Google Scholar]
- 13. Duan F, Wang X, Wang H, Wang Y, Zhang Y, Chen J, et al. GDF11 ameliorates severe acute pancreatitis through modulating macrophage M1 and M2 polarization by targeting the TGFβR1/SMAD-2 pathway. Int Immunopharmacol. 2022;108:108777. 10.1016/j.intimp.2022.108777. [DOI] [PubMed] [Google Scholar]
- 14. Zhang Y, Li Q, Liu D, Huang Q, Cai G, Cui S, et al. GDF11 improves tubular regeneration after acute kidney injury in elderly mice. Sci Rep. 2016;6:34624. 10.1038/srep34624. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Pons M, Koniaris LG, Moe SM, Gutierrez JC, Esquela-Kerscher A, Zimmers TA, et al. GDF11 induces kidney fibrosis, renal cell epithelial-to-mesenchymal transition and kidney dysfunction and failure. Surgery. 2018;164(2):262–73. 10.1016/j.surg.2018.03.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Frohlich J, Vinciguerra M. Candidate rejuvenating factor GDF11 and tissue fibrosis: friend or foe? Geroscience. 2020;42(6):1475–98. 10.1007/s11357-020-00279-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Zou YF, Zhang W. Role of microRNA in the detection, progression, and intervention of acute kidney injury. Exp Biol Med. 2018;243(2):129–36. 10.1177/1535370217749472. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Xu X, Kriegel AJ, Liu Y, Usa K, Mladinov D, Liu H, et al. Delayed ischemic preconditioning contributes to renal protection by upregulation of miR-21. Kidney Int. 2012;82(11):1167–75. 10.1038/ki.2012.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Salvadori M, Rosso G, Bertoni E. Update on ischemia-reperfusion injury in kidney transplantation: pathogenesis and treatment. World J Transplant. 2015;5(2):52–67. 10.5500/wjt.v5.i2.52. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Furuichi K, Kaneko S, Wada T. Chemokine/chemokine receptor-mediated inflammation regulates pathologic changes from acute kidney injury to chronic kidney disease. Clin Exp Nephrol. 2009;13(1):9–14. 10.1007/s10157-008-0119-5. [DOI] [PubMed] [Google Scholar]
- 21. Goerdt S, Politz O, Schledzewski K, Birk R, Gratchev A, Guillot P, et al. Alternative versus classical activation of macrophages. Pathobiology. 1999;67(5–6):222–6. 10.1159/000028096. [DOI] [PubMed] [Google Scholar]
- 22. Li W, Wang W, Liu L, Qu R, Chen X, Qiu C, et al. GDF11 antagonizes TNF-α-induced inflammation and protects against the development of inflammatory arthritis in mice. Faseb J. 2019;33(3):3317–29. 10.1096/fj.201801375RR. [DOI] [PubMed] [Google Scholar]
- 23. Lin M, Yiu WH, Wu HJ, Chan LYY, Leung JCK, Au WS, et al. Toll-like receptor 4 promotes tubular inflammation in diabetic nephropathy. J Am Soc Nephrol. 2012;23(1):86–102. 10.1681/ASN.2010111210. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24. Souza MK, Neves RVP, Rosa TS, Cenedeze MA, Arias SCA, Fujihara CK, et al. Resistance training attenuates inflammation and the progression of renal fibrosis in chronic renal disease. Life Sci. 2018;206:93–7. 10.1016/j.lfs.2018.05.034. [DOI] [PubMed] [Google Scholar]
- 25. Bolisetty S, Zarjou A, Hull TD, Traylor AM, Perianayagam A, Joseph R, et al. Macrophage and epithelial cell H-ferritin expression regulates renal inflammation. Kidney Int. 2015;88(1):95–108. 10.1038/ki.2015.102. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Saito H, Tanaka T, Tanaka S, Higashijima Y, Yamaguchi J, Sugahara M, et al. Persistent expression of neutrophil gelatinase-associated lipocalin and M2 macrophage markers and chronic fibrosis after acute kidney injury. Physiol Rep. 2018;6(10):e13707. 10.14814/phy2.13707. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. Lee S, Huen S, Nishio H, Nishio S, Lee HK, Choi BS, et al. Distinct macrophage phenotypes contribute to kidney injury and repair. J Am Soc Nephrol. 2011;22(2):317–26. 10.1681/ASN.2009060615. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28. Lin SL, Li B, Rao S, Yeo EJ, Hudson TE, Nowlin BT, et al. Macrophage Wnt7b is critical for kidney repair and regeneration. Proc Natl Acad Sci U S A. 2010;107(9):4194–9. 10.1073/pnas.0912228107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29. Loffredo FS, Steinhauser ML, Jay SM, Gannon J, Pancoast JR, Yalamanchi P, et al. Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy. Cell. 2013;153(4):828–39. 10.1016/j.cell.2013.04.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.