Background
Myofibroblasts secrete extracellular matrix and contribute to renal fibrosis [
1‐
4], which is a hallmark of poor repair in acute kidney injury and progression to chronic kidney disease. In recent years, despite divergent opinions on the source of renal myofibroblasts [
5‐
7], an increasing number of comprehensive genetic fate mapping studies have demonstrated that pericytes are the main progenitors of renal scar-forming myofibroblasts [
2,
8‐
10]. Moreover, pericytes have been increasingly exploited in cell therapy to inhibit renal fibrosis and promote tissue regeneration [
11‐
14].
Transforming growth factor β-1 (TGF-β1) is a pluripotent cytokine that drives organ fibrosis and regeneration [
15,
16]. Following unilateral ureteral obstruction, injured epithelial cells primarily secrete TGF-β1, which can then trigger pericyte-myofibroblast transition (PMT) [
16]. A previous study established an in vitro PMT model induced by TGF-β1 [
16,
17].
Differentiation is a complicated process that involves dynamic epigenetic, transcriptional, and metabolic remodeling [
18‐
20]. Metabolic remodeling, also known as metabolic reprogramming (MR), refers to the change in the primary production of ATP by cells from oxidative phosphorylation to aerobic glycolysis [
21,
22]. Increasing research has revealed that metabolic reprogramming is no longer limited to changes in the balance of glycolysis and oxidative phosphorylation but extends to changes in the metabolism of various nutrients, such as fatty acids, amino acids and glutamine [
23‐
25]. Studies have found a novel link between metabolism and epigenetic modulation in transdifferentiation [
11,
20,
26], in which metabolites can regulate differentiation [
18,
27]. PMT is an important pathological factor in the progression of renal fibrosis. However, changes in metabolic profiles and metabolic pathways in PMT remain poorly understood, and numerous questions regarding TGF-β1 biology and the pathophysiology of PMT remain unanswered.
In this study, we first report that glycolysis is increased during PMT and that the PI3K-Akt-mTOR pathway plays a role in this effect. Our data reveal that glycolysis and HKII expression levels were significantly elevated in PMT, and inhibiting glycolysis with an HKII competitive antagonist (2-DG) could reverse PMT. Moreover, when the PI3K-Akt-mTOR pathway was inhibited, PMT was blunted, accompanied by decreased HKII expression and activity. An HKII rescue experiment further clarified that the PI3K-Akt-mTOR pathway affects TGF-β1-induced PMT by regulating HKII.
Methods
To identify the main transcriptomic changes that accompany the process of PMT, we downloaded the gene expression profiles of GSE50439 [
28], whose raw data were extracted with the GPL1261 platform, (Mouse430_2) Affymetrix Mouse Genome 430 2.0 Array. In this database, Col1a1-eGFPL10a mice were subjected to sham or unilateral ureteral obstruction (UUO) surgery. From the mice subjected to the sham surgery, we extracted samples GSM1219324, GSM121932 and GSM1219326 as the control group, while from those subjected to UUO after 2 or 7 days, we extracted GSM1219330, GSM1219331, GSM1219332, GSM1219333, GSM1219334 and GSM1219335 as the PMT group. The preprocessing of the gene expression profile data, which included background correction, quantile normalization, median polish summarization, and log2 transformation, was performed by R and RStudio software. The limma R package was applied to identify differentially expressed genes (DEGs) by comparing expression values between pericyte samples and myofibroblast samples. The criteria for assessing DEGs were |log2-fold change (FC)|≥ 1 and
p < 0.05 in the gene expression between pericytes and myofibroblasts and an adjusted
p < 0.05. A volcano plot of DEGs was generated by the ggplot2 package in R. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analyses of the DEGs were performed with the Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool.
Samples were collected and immediately stored at −80 °C according to previous literature [
29]. Samples were extracted with 80% methanol aqueous solution and analyzed by ultraperformance liquid chromatography (UPLC) and tandem mass spectrometry (MS). Metabolite quantification was performed by using multiple reaction monitoring (MRM) mode in a triple-quadrupole mass spectrometer. Analyst 1.6.3 software was used to process the mass spectrometry data. Based on the local metabolic database, the metabolites of the samples were analyzed qualitatively and quantitatively by mass spectrometry.
Primary pericyte isolation, culture and cell treatment
Pericytes were isolated from C57/BL6 WT mice (male, 6–8 weeks old, purchased from the Animal Center of Chinese PLA General Hospital) by separating the kidneys aseptically, then dicing and incubating them with Liberase (0.5 mg/ml, Roche Applied Science, USA) and DNase (100 U/ml, Roche Applied Science, USA) at 37 °C for 30 min in DMEM (Corning, USA). Digestion was inactivated with DMEM containing 10% FBS (Corning, USA). The suspension was filtered with a 40 μm cell strainer to remove glomeruli and multicellular debris. Pericytes were purified by isolating PDGFRβ + cells using MACS (Miltenyi Biotech, Germany) and cultured in DMEM-F12 (Gibco Life Technologies, USA) with 10% FBS, 1% penicillin/streptomycin (Gibco Life Technologies, USA) and 1% ITS (Invitrogen, USA) in collagen-type I-coated (Sigma‒Aldrich, USA) T25 flasks. P1 primary pericytes were used for experiments. Additional details on the above procedure can be found in the referenced literature [
16,
30,
31]. Pericytes were pretreated with 5 mmol/L 2DG (Sigma‒Aldrich, USA), 50 μmol/L LY294002 (HY-10108, MCE, USA) or 100 nmol/L rapamycin (HY-10219, MCE, USA) to inhibit glycolysis [
32] or the PI3K-Akt-mTOR pathway [
33‐
35].
Model of PMT in vitro
When the cell confluence reached approximately 80%, the culture medium was replaced with 1640 medium without glucose and serum and cultured for 12 h. After the cells were synchronized, they were divided into two groups: (1) the control group, in which pericytes were cultured with complete culture medium, and (2) the TGF-β1-treated group, in which pericytes were cultured with complete culture medium containing 5 ng/mL TGF-β1 (1218209, PeproTech) for 24 or 48 h [
16]. The cell phenotype was verified by determining the expression levels of α-SMA, vimentin and desmin. According to our results, we considered that the in vitro PMT model was successfully established by 48 h of treatment with TGF-β1.
Quantitative real-time polymerase chain reaction (PCR) analysis
Briefly, the cells were homogenized using TRIzol (Invitrogen, USA), and the total RNA was reverse-transcribed to cDNA by a ProtoScript II FirstStrand cDNA Synthesis Kit (Applied Biosystems, USA). Real-time PCR was carried out (Bio-Rad, USA) with FastStart Universal SYBR Green Master Mix (Roche, Germany). The primers are shown in Table S1. The relative mRNA level was determined with the 2
−ΔΔCt method normalized by 18S as described previously [
36,
37].
Western blotting
Cells were lysed in RIPA buffer with 1% PMSF on ice for 20 min, and the protein concentrations were evaluated with a BCA protein assay kit (Thermo, Rockford, IL, USA). The same quality proteins were separated in 10–15% polyacrylamide gel and then transferred to NC membranes by Trans-Blot Turbo (Bio-Rad, Hercules, CA, USA). After the membranes were blocked in 20% casein, they were incubated overnight with the following primary antibodies: GAPDH (1/50000, 60004–1, Proteintech), α-SMA (1/3000, ab7817, Abcam), vimentin (1/10000, 60330, Proteintech), desmin (1/1000, sc-23879, Santa Cruz Biotechnology), hexokinase II (HKII) (1/1000, ab209847, Abcam), PKM (1/5000, ab150377, Abcam), and LDH (1/5000, ab52488, Abcam). Quantification was performed by measuring the intensity of the gels with ImageJ (Rawak Software, Inc. Germany).
Immunofluorescence staining
Immunofluorescence staining was described previously [
38]. Cells cultured on coverslips were washed twice with cold PBS and fixed with 4% paraformaldehyde for 3 min at room temperature and 15 min at 4℃. Following three extensive washes with PBS, the slides were permeabilized with 0.2% Triton X-100 for 10 min, blocked with 5% BSA for 30 min at room temperature, and then incubated with the specific primary antibodies previously described. The slides were probed with Cy3‐conjugated secondary antibody (red) and FITC‐conjugated secondary antibody (green) at room temperature for 1 h. The slides were imaged by confocal fluorescence microscopy. Each experiment was repeated three times, and immunofluorescence images were captured with identical exposure settings. The signal intensity of immunofluorescence images was quantified with the same parameter settings using ImageJ software, and scores were expressed as the percentage of total area.
P1 pericytes were seeded in an XF 24-well cell culture microplate (Seahorse Bioscience, Copenhagen, Denmark) at a density of 2 × 10
4 cells/well and washed in glucose-free XF base medium. A Seahorse XFe24 Extracellular Flux Analyzer was used to measure the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) in the medium [
39]. The ECAR was measured after serial injections with 100 mmol/L D-glucose, 10 μmol/L oligomycin, and 500 mmol/L 2-deoxyglucose. OCR was measured after serial injections with 15 μmol/L oligomycin, 20 μmol/L FCCP, and 5 μmol/L rotenone/antimycin A. Glycolysis was defined as the ECAR response to oligomycin after glucose injection, and the glycolytic reserve was calculated as ΔECAR
Oligomycin-Glucose. The maximum OCR value stimulated by FCCP was defined as the maximal respiration, and the difference between the maximum respiration and the basal respiration was the spare respiratory capacity. Data were analyzed by the XF Glycolysis Stress Test Report Generator macro provided by Seahorse Biosciences.
HK activity assays
We measured HK activity by using an HK activity assay kit (MAK091, Sigma, USA). In brief, glucose is converted to glucose-6-phosphate, which is oxidized by glucose-6-phosphate dehydrogenase to form NADH. The resulting NADH reduces a colorless probe, resulting in a colorimetric product (at λ = 450 nm). The HK activity was accurately measured by a microplate reader (Thermo Fisher, USA).
The pCMV6 plasmid (EX-Mm03044-M02, GeneCopoeia) containing the HKII cDNA sequence was transformed into TOP10 competent E. coli (CB104, Tiangen). After amplification, the plasmid was extracted with a Plasmid Mini Kit II according to the manufacturer’s protocol (D6945, Omega). When the cell confluence reached approximately 50%, the HKII plasmid was transfected into P1 pericytes with EndoFectin™ Max Transfection Reagent (EF003, GeneCopoeia) according to the instructions. The expression of HKII was measured by PCR.
Statistical analyses
All Western blot and immunofluorescence images are representative of at least three independent experiments. RT‒qPCR assays were performed in quadruplicate. Data shown are the mean ± SD for three or more independent experiments. Student’s t test was used for two-group comparisons. P < 0.05 indicated statistical significance.
Discussion
In this study, we compared the gene expression profiles of renal pericytes and pericyte-derived myofibroblasts through bioinformatics analysis. The results illustrated that cell metabolism changes significantly during PMT, involving carbon metabolism, HKII and the PI3K-Akt pathway. Then, we built an in vitro model of PMT to observe the specific changes in glycolysis. Moreover, we found that the PI3K-Akt-mTOR pathway may participate in the regulation of PMT by affecting HKII and glycolysis.
Renal fibrosis is the basic pathological feature of the progression of various renal diseases, and myofibroblasts are regarded as the predominant effector cells in fibrosis. However, the origins of myofibroblasts remain controversial, and possibilities include resident fibroblasts, pericytes, bone marrow-derived fibroblasts, tubular epithelial cells and endothelial cells [
5,
7,
10,
40,
41]. In 2021, Kuppe C et al. published an article in Nature, and they identified distinct subpopulations of pericytes and fibroblasts as the main cellular sources of scar-forming myofibroblasts during human kidney fibrosis by using single-cell RNA sequencing and spatial transcriptomics in human samples and genetic fate tracing, time-course single-cell RNA sequencing and ATAC-seq experiments in mice [
42]. Since an increasing number of in vitro and in vivo studies have confirmed that PMT is the main source of renal myofibroblasts in both humans and rodents [
10,
42,
43], the role of PMT in renal diseases is self-evident.
The relationship between glycometabolism and renal disease has attracted the attention of many researchers. The mRNA and protein levels of key enzymes in glycolysis, as well as glycolysis levels, have been shown to be elevated in fibroblasts in a UUO mouse model [
44], endothelial cells [
45] and macrophages [
46] in diabetic kidney disease (DKD). Inhibiting aerobic glycolysis can attenuate the progression of polycystic kidney disease, DKD, proliferative kidney disease, chronic kidney disease and kidney cancer [
45,
47‐
50]. Our study revealed the changes in carbon metabolism in PMT, especially the role of the PI3K-Akt-mTOR pathway. We believe that our study may provide new ideas for preventing renal fibrosis and renal disease.
Metabolic studies on pericytes vary between pericyte subpopulations. Placental pericytes in the proliferative state rely heavily on glycolysis, which is responsible for producing approximately 85% of their ATP [
51], while our results suggest that the level of ATP produced by mitochondria is 2–7 times that produced by glycolysis in primary renal pericytes at different cell densities (Additional file
1: Fig. S10D). The difference may be partly explained by the long-term hypoxia in malignant microenvironments and the abundant blood and oxygen supply in the kidney. Both GLUT1 and GLUT4 are detectable in renal pericytes, indicating that glucose uptake occurs through both insulin-dependent and insulin-independent pathways in pericytes [
26], consistent with studies in murine brain pericytes [
52]. Under TGF-β1 stimulation, the expression of GLUT1 and GLUT4 was increased, and the level of glucose in culture medium was lower in myofibroblasts (data not shown). These results illustrate that glucose uptake and consumption are higher in myofibroblasts than in pericytes. Cells in a quiescent state usually have low cellular metabolism, but when stimulated by growth factors, myofibroblasts increase ATP production to support cellular contractility [
53]. Researchers have observed increased glycolysis and oxidative phosphorylation during the differentiation of neurons, podocytes and lung fibroblasts [
53‐
55]. Moreover, metabolites such as the oxidized form of nicotinamide adenine dinucleotide (NAD
+) can orchestrate transcription and differentiation [
56]. Studies have found that glycolysis mainly provides most of the energy in the early stage of neuronal cell differentiation [
54]. However, the levels of most metabolites begin to increase in the late stage of TGF-β1–induced PMT. N-cadherin, Notch3 and FoxO3A signaling not only participate in preserving cell‒cell interactions but also maintain a quiescent state in pericytes by limiting cellular metabolism, mainly glycolysis [
11,
57,
58]. After blocking PFKFB3 with 3PO, glycolysis in pericytes is reduced, proliferation and migration are impaired, and attachment to epithelial cells by N-cadherin is enhanced [
51]. After inhibiting the expression of PDK4 in human pulmonary pericytes, a similar phenomenon is observed, and the interaction between endothelial cells and pericytes is enhanced, thereby preventing vascular loss in pulmonary hypertension [
59].
Renal pericytes, which have been neglected by researchers for years, are mesenchymal cells that surround endothelial cells in the capillary bed and postcapillary venules [
60]. In recent studies, PMT has become a tenable target for preventing renal fibrosis [
9,
61]. Pericytes activated by platelet-derived growth factor receptor signaling, TGF-β1 signaling or the Wnt pathway can detach from capillaries and transdifferentiate into myofibroblasts [
16,
62]. TGF-β1 is a primary cytokine that stimulates PMT. In our study, pericytes cultured with TGF-β1 for 48 h strongly expressed α-SMA (a myofibroblast marker), while primary pericytes weakly expressed α-SMA.
TGF-β1 is a key mediator in the development of renal fibrosis, and our study demonstrated that TGF-β1 activated the PI3K-Akt-mTOR pathway and participated in pericyte differentiation by increasing glycolysis. Indeed, TGF-β regulates renal fibrosis via both canonical and noncanonical pathways [
63]. TGF-β exerts biological effects by activating Smad2 and Smad3, which are negatively regulated by an inhibitory Smad7 [
64]. In our bioinformatics analysis, even though the gene expression of most Smads (including Smad1-6) did not differ between pericytes and myofibroblasts derived from pericytes, we found that Smad7 is downregulated during renal fibrosis (data not shown), which is consistent with previous reports [
65,
66]. The overexpression of Smad7 has been shown to be a therapeutic agent for renal fibrosis in various models of kidney diseases [
66]. Moreover, Smads also interact with other signaling pathways, such as the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways [
65], to positively or negatively regulate renal fibrosis. In our previous bioinformatics analysis, 27 DEGs were highly enriched in the MAPK signaling pathway, including MAPK9, MAPK11, MAPK13, MAPK14, MAPK8IP1 and MAPK8IP2 (data not shown). Some studies have demonstrated that the MAPK signaling pathway may participate in neural or cardiovascular pericyte differentiation [
67‐
69], but whether the TGF-β1/MAPK pathway participates in renal pericyte metabolism and differentiation needs further study.
The results of bioinformatics analysis indicated that the PI3K-Akt-mTOR pathway may be involved in PMT; we verified that the PI3K-Akt-mTOR pathway participated in the regulation of PMT by regulating HKII in glycolysis. Some studies have indicated that Akt stimulates the phosphorylation of HKII and results in the protection of mitochondria against oxidant- or Ca
2+-stimulated permeability transition pore opening [
70]. In addition to playing an important role in cell proliferation, apoptosis and metabolism in malignant cells and neural stem cells [
71‐
73], the PI3K-Akt-mTOR pathway is essential in cell pluripotency and differentiation [
74]. It has been reported that when activated by insulin and insulin-like growth factor 1 (IGF1), the PI3K-Akt pathways maintain cell pluripotency by inhibiting the MEK/ERK signaling pathway [
74].
In the present study, we first described metabolic reprogramming with enhanced glycolysis as a characteristic of PMT, including increased glucose consumption and an elevated level of glycolysis, and we demonstrated the vital role of the PI3K-Akt-mTOR pathway in PMT. Our study further suggests that the PI3K-Akt-mTOR pathway may affect glycolysis by mediating HKII. In addition, our work has implications not only for the fundamental understanding of metabolic reprogramming in PMT but also in prompting new ideas for inhibiting renal fibrosis. This research, however, is subject to several limitations. Although our research is based on previous transcriptome data analysis, it has not been verified in animal experiments, which will also be our future work. Meanwhile, the PMT model is induced by TGF-β1, and TGF-β1 is a multifunctional cytokine that is interconnected with multiple pathways. To explain how this complex network plays a role in PMT, more in-depth and systematic research is needed.
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