GRAPHICAL ABSTRACT

Highlights

• Chronic inflammation plays an important role in EPO production.

• RasGRP4 attenuates EPO in REPCs by modifying cytokine secretion from PBMCs.

• RasGRP4 reduces HIF2A through ubiquitin-proteasome degradation.

• RasGRP4 promotes myofibroblastic transformation in REPCs.

INTRODUCTION

Compared with patients with chronic kidney disease (CKD) from other causes, patients with diabetes develop renal anemia earlier and have more severe damage to the kidneys. Approximately 40% of patients with type 2 diabetes mellitus (T2DM) who are at high risk of progression to renal anemia as well as end-stage kidney disease (ESKD) will develop CKD [1]. Anemia in diabetic patients is often associated with impaired renal function secondary to diabetic nephropathy [2], and the vicious cycle between renal anemia and kidney dysfunction ultimately accelerates the progression of ESKD. The etiology of anemia in T2DM patients is multifactorial [3]. The pathogenesis of renal anemia includes chronic inflammation, iron deficiency, and a shortened half-life of red blood cells (RBCs), but the main cause is erythropoietin (EPO) [4] deficiency. Therefore, early prevention of decreased EPO production is important for preventing and delaying the progression of renal failure and CKD.

EPO, a human endogenous glycoprotein hormone, can stimulate RBC production and is mainly produced by renal EPO-producing cells (REPCs) [5]. The transcription of EPO is almost exclusively dominated by hypoxia-inducible factors (HIFs), predominantly HIF2A, which is usually associated with the response to chronic hypoxia [6]. In the HIF pathway, prolyl hydroxylase domain protein 2 (PHD2) constitutively prolyl-hydroxylates HIF2A, thereby inducing Von Hippel‒Lindau (VHL) tumor suppressor protein to degrade HIF2A, thereby reducing EPO production [7]. Previous studies have indicated that inflammation participates in regulating the HIF pathway and promoting the myofibroblastic transformation of REPCs to help further reduce EPO in a disease-specific manner [8-10]. Investigations into proinflammatory cytokines such as transforming growth factor β (TGFB), transforming growth factor α (TNFA), and interleukin-1β (IL1B) inhibiting EPO production have suggested that chronic inflammation plays a significant role in EPO production [11-14].

Ras guanine nucleotide-releasing protein-4 (RasGRP4), a guanine nucleotide exchange factor, is a molecule that activates Ras by promoting guanosine triphosphate binding. RasGRP4 has been shown to be an intracellular signaling protein that promotes inflammation by inducing proinflammatory cytokines and activating vital inflammatory signaling pathways [15-17]. Our previous research provided evidence that RasGRP4 can promote renal inflammatory injury in T2DM, probably by regulating the interaction between peripheral blood mononuclear cells (PBMCs) and glomerular vascular endothelial cells and further activating various inflammatory signaling pathways [18]. RasGRP4 deficiency in diabetic mice significantly decreases the infiltration of inflammatory cells and downregulates inflammatory signaling pathways in renal tissues [18], indicating that RasGRP4 might have value in preventing the reduction of EPO production. However, whether RasGRP4 can influence the renal production of EPO in T2DM remains unexplored.

Reznikoff et al. [19] have annunciated the founding of the C3H10T1/2 cell line deriving from the C3H mouse embryo. Studies have confirmed that C3H10T1/2 cell expressed makers for mesenchymal cells similar to renal EPO-producing (REP) pericytes, indicating C3H10T1/2 cells was a REP pericyte-like cell line [20-22]. Shih et al. [11] has used C3H10T1/2 cells as a REP pericyte-like cell line to investigate EPO production in vitro.

Herein, we demonstrated that RasGRP4 could decrease the production of EPO in T2DM. Since C3H10T1/2 cells have been shown to be a reliable cell line for studying EPO regulation and myofibroblast transformation, we explored the role of RasGRP4 in C3H10T1/2 cells stimulated with high glucose. Ultimately, we found that RasGRP4 could downregulate HIF2A and promote the inflammatory response in C3H10T1/2 cells cultured in high-glucose medium. These results suggested the involvement of RasGRP4 in renal anemia in T2DM and its potential as a therapeutic target.

METHODS

Cell culture

C3H10T1/2 cells (BeNa Culture Collection, Beijing, China) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin solution, and 0.1% mycoplasma inhibitor at 37°C in a humidified 5% CO₂ atmosphere.

PBMC processing

We used density gradient centrifugation to separate PBMCs [18] and then cultured them in medium supplemented with different glucose medium concentrations.

Mouse models

Male wild-type (WT) C57BL/6 mice (6 to 8 weeks old) were purchased from the Huafukang Animal Centre (Beijing, China). RasGRP4^−/− mice on a C57BL/6 background were generated by the Nanjing Biomedical Research Institute of Nanjing University. Genechem Technology (Shanghai, China) constructed nonspecific adeno-associated virus 9 (AAV9) carrying RasGRP4 or AAV9-RasGRP4 and control virus 9 carrying green fluorescent protein (GFP) or AAV9CON453. Mice were housed in specific pathogen-free conditions with freely available water and standard mouse chow. We randomly divided the mice into the following groups: WT C57BL/6J control (WT-normal control [NC]; n=6); diabetic C57BL/6J (WT-diabetes mellitus [DM]; n=6); RasGRP4^−/− (knockout [KO]-NC; n=6); diabetic RasGRP4^−/− (KO-DM; n=6); diabetic WT AAV9-GFP (WT-DM-GFP; n=6); diabetic WT AAV9-RasGRP4 (WT-DM-RasGRP4; n=6); diabetic KO AAV9-GFP (KO-DM-GFP; n=6); and diabetic KO AAV9-RasGRP4 (WT-DM-RasGRP4; n=6). Group assignments were random, and the experimenters were blinded to the histological assays and outcome assessments.

The establishment of diabetic mice has been previously described [18]. We induced RasGRP4 overexpression in WT and KO diabetic mice by tail vein injection of 1×10¹² vg/mL AAV9-RasGRP4 at 5 weeks after the final injection of streptozotocin; meanwhile, in the control group, WT and KO diabetic mice received injections of AAV9-GFP.

The animal experiments were approved by the Ethics Committee of Chu Hsien-I Memorial Hospital of Tianjin Medical University (approval No. DXBYY-IACUC-2021066). This study was carried out in accordance with Guidance on the operation of the Animals (Scientific Procedures) Act 1986 and associated guidelines, EU Directive 2010/63 for the protection of animals used for scientific purposes or the National Institutes of Health (National Research Council) Guide for the Care and Use of Laboratory Animals. All animal experiments were comply with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

Biochemical measurements

We measured blood glucose using a glucose analyzer (Sinocare, Hunan, China). We assessed aspartate aminotransferase, alanine aminotransferase, serum creatinine (SCr), blood urea nitrogen (BUN), total cholesterol, triglycerides, low-density lipoprotein cholesterol, urinary protein quantity, and urinary glucose using a biochemistry autoanalyzer with commercial kits.

Histology and immunohistochemical staining

Hematoxylin and eosin, periodic acid–Schiff, and Masson’s trichome staining were used to evaluate kidney injury, collagen deposition, and the degree of renal fibrosis, as described previously [18]. The distributions of EPO (ABclonal Technology, Wuhan, China) and alpha-smooth muscle actin (α-SMA)(Proteintech, Wuhan, China) were examined using immunohistochemical staining. We then imaged the sections under an optical microscope.

Quantitative real-time polymerase chain reaction

We extracted total ribonucleic acid (RNA) from mouse renal tissues, primary PBMCs, and C3H10T1/2 cells and purified it using TRIzol reagent (Invitrogen, Waltham, MA, USA). Reverse transcription was performed via a reverse transcription kit (Vazyme, Nanjing, China). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The primer sequences are shown in Supplementary Table 1.

Western blot

Protein expression in kidney and cell lysates were analyzed by Western blot analysis, as described previously [18]. The antibodies used were as follows: EPO (A4959, ABclonal), SMA (33187M, Bioss Antibodies, Woburn, MA, USA), HIF2A (DF2928, Affinity Biosciences, Cincinnati, OH, USA), VHL (sc-17780, Santa Cruz Biotechnology, Dallas, TX, USA), PHD2 (A14557, ABclonal), TGFB (AF0297, Beyotime Biotechnology, Shanghai, China), phosphorylated suppressor of mothers against decapentaplegic 3 (p-Smad3; AP1263, ABclonal), p-Smad2 (AP1007, ABclonal), Smad2/3 (A18674, ABclonal), p38 (AF6456, Affinity, Jiangsu, China), p-p38 (AF4001, Affinity), extracellular signal–related kinase 1/2 (ERK1/2; db758, Diablo, Shanghai, China), p-ERK1/2 (db269, Diablo), phosphorylated c-Jun N-terminal kinase (p-JNK; AP0631, ABclonal), JNK (A4867, ABclonal), TNFA (A11534, ABclonal), nuclear factor κB (NFKB) p65 (BF8005, Affinity), phosphorylated NFKB p65 (AF2006, Affinity), nucleotide-binding domain (NOD)–like receptor (NLR) family pyrin domain-containing 3 (NLRP3; ab214185, Abcam, Cambridge, UK), apoptosis-associated speck-like protein containing a cysteine–aspartic acid–specific protease/proteinase (Caspase) activation and recruitment domain (apoptosis-associated speck-like protein [ASC]; A1170, ABclonal), Caspase 1 (YT0652, ImmunoWay Biotechnology Company, Plano, TX, USA), IL1B (A1112, ABclonal), klotho (A12028, ABclonal), and glyceraldehyde 3-phosphate dehydrogenase (AC002, ABclonal). The membranes were then incubated with secondary antibodies, and signals were visualized using Immobilon Western Chemiluminescent HRP Substrate (Thermo Fisher, Waltham, MA, USA) and measured using Image J software (National Institutes of Health, Bethesda, MD, USA).

Enzyme-linked immunosorbent assay

Mice serum and cellular supernatant were collected, and the concentrations of EPO were measured with an EPO enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (F2194-A, FANKEWEI, Shanghai, China).

Immunofluorescence staining

We performed immunofluorescence (IF) staining to detect the expression of EPO and SMA in renal tissues and C3H10T1/2 cells. Frozen sections (4 μm) and C3H10T1/2 cells were fixed in 4% paraformaldehyde for 15 minutes, incubated in 0.05% Triton X-100 for 15 minutes, and then blocked in 1% bovine serum albumin (BSA) for 30 minutes. After BSA blocking, the sections were successively incubated overnight with primary antibodies, including EPO (ABclonal) and SMA (Proteintech). Then, the sections were stained with secondary antibodies conjugated with Alexa Fluor 488 or 594 (Servicebio, Wuhan, China) for 1 hour at 37°C. The nuclei were stained with 4´, 6-diamidino-2-phenylindole (DAPI; Solarbio) for 5 minutes. The images were observed under a fluorescence microscope (Olympus, Tokyo, Japan).

Statistical analyses

Comparisons between two groups were assessed using an independent-sample t-test. One-way analysis of variance (ANOVA) was used for comparisons among multiple groups. The data are expressed as the mean±standard deviation. P<0.05 was considered to indicate statistical significance. Statistical analysis was performed using SPSS statistical software version 25.0 (IBM Co., Armonk, NY, USA).

RESULTS

Effects of RasGRP4 modulation on renal inflammation in mice with T2DM

First, we generated RasGRP4-deficient (RasGRP4^−/−) mice and then achieved RasGRP4 overexpression in both WT and RasGRP4^−/− mice. The messenger RNA (mRNA) levels of RasGRP4 were verified by qRT-PCR (Supplementary Fig. 1A and D). We found that downregulation or upregulation of RasGRP4 in mice did not influence weight gain or hyperglycemia progression during the experimental period (Supplementary Fig. 1B, C, E, and F). However, pathological-histology analysis revealed that RasGRP4 aggravated kidney injury and fibrosis in mice with T2DM (Supplementary Fig. 1G and H). Moreover, we observed that the renal tissue infiltration of CD3⁺T lymphocytes and F4/80⁺ macrophages increased after the upregulation of RasGRP4 in T2DM mice, which indicated that RasGRP4 participates in the regulation of renal inflammatory reactions (Supplementary Fig. 1I and J). In addition, RasGRP4 did not affect the levels of liver enzymes, blood lipids, or blood glucose in mice, whereas it increased the levels of SCr, BUN, and total urinary protein (Supplementary Table 2).

RasGRP4 decreases serum EPO and promotes myofibroblastic transformation of REPCs in T2DM mice

We found that RBC counts and hemoglobin levels in diabetic mice were lower than those in normal mice and greater in RasGRP4^−/− diabetic mice than in WT diabetic mice, although the differences were not statistically significant (Fig. 1A and B). Serum EPO levels in T2DM mice decreased significantly compared with those in nondiabetic controls, and RasGRP4 significantly reduced the serum EPO concentration (Fig. 1C and D). REPCs synthesize approximately 90% of the total EPO in the kidney and are thought to be the main source of myofibroblasts in CKD [5,12]. Our IF experiment further demonstrated that RasGRP4 downregulated EPO expression and upregulated SMA expression in interstitial cells in mice with T2DM (Fig. 1E and F), which was further confirmed by the analysis of EPO and SMA protein levels (Fig. 1G and H). These results indicated that RasGRP4 promoted myofibroblastic transformation in REPCs.

RasGRP4 degrades HIF2A by regulating the ubiquitin–proteasome pathway in T2DM mice

EPO is one of the most important target genes of HIF2A. We discovered that RasGRP4 slightly upregulated the increase in HIF2A mRNA expression in T2DM mice, although the difference was not significant (Fig. 2A and B). However, the protein expression of HIF2A was significantly decreased by RasGRP4, probably via regulation of the ubiquitin–proteasome pathway. In contrast to those in RasGRP4^−/− diabetic mice, the protein levels of PHD2 and VHL were significantly increased, and the expression of HIF2A was decreased in WT diabetic mice (Fig. 2C). Moreover, the upregulation of RasGRP4 inhibited the activation of the HIF2A signaling pathway, probably by increasing the expression of PHD2 and VHL (Fig. 2D).

RasGRP4 regulates inflammatory signaling pathways associated with EPO production in vivo

Low-grade chronic systemic inflammation is a hallmark pathological feature of T2DM. Inflammatory factors can promote myofibroblastic transformation of REPCs, activate the ubiquitin–proteasome pathway and subsequently degrade HIF2A, resulting in a reduction in EPO production. We found that RasGRP4 deficiency inhibited the activation of both the Smad-dependent and non-Smad TGFB signaling pathways in T2DM mice. Compared with those in WT diabetic mice, the protein expression of TGFB and the phosphorylation of p38, ERK, JNK, Smad2, and Smad3 were significantly decreased in RasGRP4^−/− diabetic mice (Fig. 3A). In addition, overexpression of RasGRP4 in mice with T2DM led to increased activation of the relevant inflammatory signaling pathways (Fig. 3B). The TNFA/NFKB signaling pathway was also activated, as evidenced by increased TNFA, p-p65, and p-IκB levels (Fig. 3C and D). The NLRP3 inflammasomes, play an important role in the maturation and release of inflammatory cytokines, and have been proven to be stimulated by TGFB in a Smad3-dependent manner [23,24]. Moreover, we found that RasGRP4 could regulate the NLRP3 inflammasome signaling pathway in T2DM mice (Fig. 3E and F). In addition, compared with diabetic RasGRP4^−/− mice, diabetic WT mice showed decreased renal expression of klothoA, and the upregulation of RasGRP4 in T2DM mice further decreased the expression of klothoA (Fig. 3G and H). Overall, RasGRP4 plays an important role in EPO production in T2DM, probably by activating related inflammatory signaling pathways and reducing the levels of anti-inflammatory factors.

PBMCs from RasGRP4-deficient mice reduced inflammatory factor secretion by C3H10T1/2 cells under high-glucose conditions

Our prior research has shown that RasGRP4 is highly expressed in PBMCs and affects the function of PBMCs cultured in high-glucose medium, mainly by mediating the expression of inflammatory factors. After high-glucose intervention, PBMCs derived from KO mice (KO-HG) and those derived from WT mice (WT-HG) were screened for differences with a threshold of |log2-fold change|>1.0. We found a total of 674 DEGs in the KO-HG group compared with the WT-HG group, of which 442 genes were downregulated and 232 were upregulated based on the results of the volcano plot (Fig. 4A). The heatmap in Fig. 4B shows the main genes related to the regulation and control of TGFB, IL1B, and TNFA family members; upregulated and downregulated genes are represented in red and blue, respectively. The expression levels of TGFB, TNFA, and IL1B were increased in PBMCs isolated from WT mice compared with those from KO mice (Fig. 4C and D).

Conditioned media from RasGRP4-deficient PBMCs increased EPO production in C3H10T1/2 cells under high-glucose conditions

To determine how RasGRP4 influences the interaction between PBMCs and C3H10T1/2 cells under hyperglycemic conditions, we collected conditioned media from PBMCs isolated from WT or KO mice treated with low-glucose (WT-LG-CM and KO-LG-CM, respectively) or HG (WT-HG-CM and KO-HG-CM, respectively) and used it to stimulate C3H10T1/2 cells. Consistent with our in vivo observations, high glucose decreased the synthesis and secretion of EPO in C3H10T1/2 cells, and the production of EPO in C3H10T1/2 cells treated with WT-HG-CM was lower than that in those treated with KO-HG-CM. SMA, a hallmark of myofibroblasts, was expressed at a greater level in C3H10T1/2 cells treated with WT-HG-CM than in those treated with KO-HG-CM (Fig. 5).

Conditioned media from RasGRP4-deficient PBMCs could upregulate the HIF2A pathway and suppress inflammatory signaling pathways associated with EPO production in C3H10T1/2 cells

We further detected the expression of key proteins in the HIF2A pathway that regulate the expression of EPO. We found that HIF2A levels were lower and that PHD2 and VHL levels were significantly greater in C3H10T1/2 cells treated with WT-HG-CM than in those treated with KO-HG-CM (Fig. 6A). The TGFB and TNFA/NFKB signaling pathways and the NLRP3 inflammasome pathway were obviously activated in C3H10T1/2 cells treated with WT-HG-CM, while those treated with KO-HG-CM showed only slight effects (Fig. 6B-D). These results indicated that RasGRP4 could upregulate the expression of inflammatory factors in PBMCs under high-glucose conditions, thereby promoting myofibroblastic transformation and degrading HIF2A and further decreasing EPO production in C3H10T1/2 cells by activating critical inflammatory signaling pathways.

DISCUSSION

Our previous study indicated that RasGRP4 could promote the secretion of inflammatory cytokines by PBMCs and subsequently aggravate glomerular endothelial cell dysfunction, thereby promoting renal inflammatory injury in T2DM mice. In this study, we focused on whether RasGRP4 participates in the regulation of EPO synthesis and the development of renal anemia in patients with T2DM. We found that RBC counts and hemoglobin levels in diabetic mice were slightly decreased compared with those in normal mice, and the difference was not statistically significant, but the serum EPO concentration was significantly decreased in diabetic mice. This phenomenon suggested that these diabetic mice were in the early stage of diabetic nephropathy and had no clinical manifestations of renal anemia, and the decrease in the EPO level occurred earlier than that of clinical renal anemia in diabetic kidney disease patients. In this study, our results indicated that RasGRP4 plays a role in EPO production in T2DM patients. EPO is an indispensable erythropoietic hormone produced mainly by REPCs. Insufficient EPO can cause anemia, which is associated with aggravated kidney injury and increased CKD incidence and mortality. However, recombinant human EPO agents have been demonstrated to increase the risk of cardiovascular diseases [25]. Therefore, further elucidation of the mechanism underlying renal EPO synthesis defects and exploration of additional physiological therapies are necessary.

Several studies have indicated that HIF2A is important for regulating the transcription of EPO. Postnatal ablation of HIF2A can lead to anemia [26], and deletion of HIF2A can also impair hypoxia-induced renal EPO expression [6]. Although the HIF/PHD pathway is considered to be a chief regulator, the detailed mechanism of EPO generation has not been fully clarified. Under pathological conditions, REPCs become the major source of myofibroblasts, which express fibrogenic markers such as α-SMA and lose their EPO-producing ability. T2DM is a chronic inflammatory condition characterized by elevated levels of proinflammatory cytokines, which can be detected before the development of kidney damage [3]. Studies of proinflammatory cytokines, including TGFB, TNFA, and IL1B, have suggested that they play important roles in the occurrence and development of anemia in chronic diseases. For instance, injecting TGF into mice induced severe, progressive suppression of erythropoiesis, which was demonstrated by decreases in reticulocyte count and marrow erythroblasts [27]. Moreover, the accumulation of proinflammatory factors in CKD patients and model mice inhibited the expression of HIF2A and enhanced anemia progression. Therefore, a deeper understanding of how inflammation affects EPO production would likely enable the elucidation of anemic pathogenesis in chronic disease and the exploration of new intervention targets in the process of EPO synthesis.

In this study, IF staining revealed that RasGRP4 promoted the transformation of REPCs into myofibroblasts in mice with diabetes, which was further confirmed by the renal expression of EPO and SMA. We further examined the expression levels of the major regulatory genes involved in the production of EPO. Our results demonstrated that RasGRP4 could inhibit the HIF2A pathway, which is considered the major signaling pathway involved in EPO production. Upregulation of PHD2 and VHL expression promoted the degradation of HIF2A. Next, we detected a variety of critical proteins involved in signal transduction pathways associated with EPO production, and we demonstrated that RasGRP4 could decrease the expression of the anti-inflammatory factor klothoA and upregulate inflammatory pathways in T2DM, including the NLRP3 inflammasome, TGFB/Smad, TGFB/mitogen-activated protein kinases (MAPK), and IL1B, TNFA/NFKB signaling pathways. In addition, proinflammatory cytokine protein expression was upregulated in primary PBMC cultures under high glucose conditions, while TGFB, TNFA, and IL1B upregulation was significantly reduced in RasGRP4-deficient PBMCs. Through coculture experiments with conditioned medium, we found that RasGRP4 could increase the levels of inflammatory factors generated by PBMCs under high-glucose conditions and subsequently inhibit the production of EPO in C3H10T1/2 cells by regulating critical inflammatory signaling pathways.

Diabetic renal anemia has perpetually been a recalcitrant issue in the clinical realm. La Ferla et al. [28] found that both GATA binding protein 2 (GATA-2) and NFKB seem to be involved in the suppression of Epo gene expression by IL1B and TNFA in vitro and may be responsible for impaired Epo synthesis in inflammatory diseases in vivo. And several studies have confirmed the fact of IL1B and TNFA suppress Epo production [29,30]. Chronic inflammation plays a significant role in renal anemia, yet its precise mechanism remains indistinct. This research initially explored and confirmed that RasGRP4 exerts a crucial function in governing the incipient stage of compromised EPO production in diabetic kidneys by modulating the mechanism of cross-talk between PBMCs and REPCs, thereby offering a potential novel interventional target for the prophylaxis and therapeutics of early diabetic renal anemia. Nonetheless, there exist a number of limitations should also be borne in mind in this study. The first is the specific mechanism of how RasGRP4 influences the transformation of renal REPCs into myofibroblasts via inflammatory cells has not been further investigated. The second limitation concerns the supplementation of EPO in animal experiments was needed to further explore and verify the specific mechanisms. Meanwhile, the outcomes of this study require to be verified in diabetic nephropathy patients.

In conclusion, RasGRP4 deficiency in vivo reduced the myofibroblastic transformation of REPCs and increased HIF2A protein levels, further increasing EPO levels in mice with T2DM. Cell experiments confirmed that RasGRP4 regulated EPO production of REPCs by affecting the secretion of proinflammatory cytokines by primary PBMCs, degrading HIF2A, and activating inflammation in REPCs. Consequently, we provide new evidence that RasGRP4 participates in the production of renal EPO under diabetic conditions and could be a promising therapeutic target for improving renal anemia in patients with T2DM.

SUPPLEMENTARY MATERIALS

NOTES

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

AUTHOR CONTRIBUTIONS

Conception or design: J.W., P.Y., S.Z.

Acquisition, analysis, or interpretation of data: J.W., S.H., L.Z.

Drafting the work or revising: J.W., S.H., L.Z., S.Z.

Final approval of the manuscript: all authors.

FUNDING

This study was supported by National Natural Science Foundation of China (grant number 81600643), the China Endocrine Metabolism Talent Research Fund (grant number 2022-N-02-07 and 2023-N-03-15), the Scientific Research Funding of Tianjin Medical University Chu Hsien-I Memorial Hospital, Tianjin Key Laboratory of Metabolic Diseases (grant number ZXYZDSYSZD2022-1), Bethune Charitable Foundation (grant number Z04JKM2022E035), Hubei Chen Xiaoping Science and Technology Development Fund (grant number CXPJJH122012-002), Tianjin Science and Technology Plan Project Public Health Science and Technology Major Special Project (grant number 21ZXGWSY00100), the Scientific Research Program of Tianjin Education Commission (grant number 2022KJ248 and 2024ZD036), the Nature Science Foundation of Tianjin, China (grant number 21JCQNJC00460), and the Tianjin Key Medical Discipline (Specialty) Construction Project (grant number TJYXZDXK-032A).

Acknowledgements

We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

Fig. 1.

Ras guanine nucleotide-releasing protein-4 (RasGRP4) decreases serum erythropoietin (EPO) and promotes the myofibroblastic transformation of renal EPO-producing cells (REPCs) in type 2 diabetes mellitus (DM) mice. (A) The levels of red blood cell (RBC) in each group. (B) The levels of hemoglobin (Hb) in each group. (C) The serum EPO level in RasGRP4 deficient mice detected by enzyme-linked immunosorbent assay (ELISA). (D) The serum EPO level in RasGRP4 overexpressed mice detected by ELISA. (E, F) Immunofluorescence staining to determine the expression of EPO and smooth muscle actin (SMA) in renal tissues (×400). (G, H) Western blot to determine the expression of EPO and SMA in renal tissues. WT, wild-type; NC, normal control; KO, knockout; DAPI, 4´,6-diamidino-2-phenylindole; GFP, green fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ^aP<0.05, ^bP<0.01, ^cP<0.001, ^dP<0.0001.

Fig. 2.

Ras guanine nucleotide-releasing protein-4 (RasGRP4) promotes hypoxia-inducible factor 2-alpha (HIF2A) degradation by ubiquitin–proteasome pathway in type 2 diabetes mellitus (DM) mice. (A) The mRNA levels of HIF2A in renal tissues of RasGRP4 deficient mice detected by real-time polymerase chain reaction (PCR). (B) The mRNA levels of HIF2A in renal tissues of RasGRP4 overexpressed mice detected by real-time PCR. (C, D) Western blot to determine the protein levels of HIF2A, prolyl hydroxylase domain protein 2 (PHD2), and Von Hippel‒Lindau (VHL) in renal tissues. WT, wild-type; NC, normal control; KO, knockout; GFP, green fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ^aP<0.05, ^bP<0.01, ^cP<0.001, ^dP<0.0001.

Fig. 3.

Ras guanine nucleotide-releasing protein-4 (RasGRP4) up-regulates the inflammatory signal pathways associated with erythropoietin (EPO) production and inhibits the expression of klothoA. (A, B) Western blot to determine the protein levels of transforming growth factor β (TGFB), phosphorylated extracellular signal–related kinase (pho-ERK), ERK, pho-p38, p38, phosphorylated cJun N-terminal kinase (pho-JNK), JNK, pho-smad2, phosphorylated suppressor of mothers against decapentaplegic 3 (pho-Smad3), and Smad2/Smad3 in renal tissues. (C, D) Western blot to determine the protein levels of transforming growth factor α (TNFA), phop65, p65, phosphorylated IkappaB (pho-IκB), and IκB in renal tissues. (E, F) Western blot to determine the protein levels of nucleotide-binding domain (NOD)–like receptor (NLR) family pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein (ASC), cysteine–aspartic acid–specific protease/proteinase (Caspase) 1, and interleukin-1β (IL1B) in renal tissues. (G, H) Western blot to determine the protein levels of klothoA in renal tissues. WT, wild-type; NC, normal control; DM, diabetes mellitus; KO, knockout; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein. ^aP<0.05, ^bP<0.01, ^cP<0.001, ^dP<0.0001.

Fig. 4.

Reduced secretion of the inflammatory factors in conditioned media from Ras guanine nucleotide-releasing protein-4 (RasGRP4)-deficient peripheral blood mononuclear cells (PBMCs). (A) Differential genetic volcano plot. (B) Heatmap of the main genes related to the regulation and control of transforming growth factor β (TGFB), interleukin-1β (IL1B), and transforming growth factor α (TNFA) family members. (C) Quantitative real-time polymerase chain reaction to determine the mRNA levels of TGFB, TNFA, and IL1B in PBMCs isolated from wild-type (WT) and knockout (KO) mice. (D) Western blot to determine the protein levels of TGFB, TNFA, and IL1B in PBMCs isolated from WT and KO mice. HG, high-glucose; LG, low-glucose; DM, diabetes mellitus. ^aP<0.05, ^bP<0.01, ^cP<0.0001.

Fig. 5.

(A) The content of erythropoietin (EPO) in the supernatant of 10T1/2 cells cultured with conditioned media (CM) was detected by enzyme-linked immunosorbent assay (ELISA). (B) Western blot to determine the protein levels of EPO and smooth muscle actin (SMA) in 10T1/2 cells cultured with CM. (C) Immunofluorescence staining to determine the expression of EPO and SMA in 10T1/2 cells cultured with CM (×400). LG, low-glucose; WT, wild-type; KO, knockout; HG, high-glucose; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DAPI, 4´,6-diamidino-2-phenylindole. ^aP<0.05, ^bP<0.01, ^cP<0.001, ^dP<0.0001.

Fig. 6.

Conditioned media (CM) from Ras guanine nucleotide-releasing protein-4 (RasGRP4)-deficient peripheral blood mononuclear cells (PBMCs) showing the suppressed inflammatory signaling pathways associated with erythropoietin (EPO) production in C3H10T1/2 cells. (A) The mRNA levels of hypoxia-inducible factor 2-alpha (HIF2A) detected by quantitative real-time polymerase chain reaction and the protein levels of HIF2A, prolyl hydroxylase domain protein 2 (PHD2), and Von Hippel-Lindau (VHL) were determined by Western blot in 10T1/2 cells cultured with CM. (B) Western blot to determine the protein levels of transforming growth factor β (TGFB), phosphorylated extracellular signal–related kinase (pho-ERK), ERK, pho-p38, p38, phosphorylated c-Jun N-terminal kinase (pho-JNK), JNK, phosphorylated suppressor of mothers against decapentaplegic 2 (pho-Smad2), pho-Smad3, and Smad2/Smad3 in 10T1/2 cells cultured with CM. (C) Western blot to determine the protein levels of transforming growth factor α (TNFA), pho-p65, p65, phosphorylated IkappaB (pho-IκB), and IκB in 10T1/2 cells cultured with CM. (D) Western blot to determine the protein levels of nucleotide-binding domain (NOD)–like receptor (NLR) family pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein (ASC), cysteine–aspartic acid–specific protease/proteinase (Caspase) 1, and interleukin-1β (IL1B) in 10T1/2 cells cultured with CM. LG, low-glucose; WT, wild-type; KO, knockout; HG, high-glucose; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ^aP<0.05, ^bP<0.01, ^cP<0.001, ^dP<0.0001.

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Wang J, Huang S, Zhang L, He Y, Shao X, A-Ni-Wan ASJ, Kong Y, Meng X, Yu P, Zhou S. Ras Guanine Nucleotide-Releasing Protein-4 Inhibits Erythropoietin Production in Diabetic Mice with Kidney Disease by Degrading HIF2A. Diabetes Metab J. 2025 Jan 23. doi: 10.4093/dmj.2024.0398. Epub ahead of print.

Received: Jul 18, 2024; Accepted: Sep 07, 2024

DOI: https://doi.org/10.4093/dmj.2024.0398.

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