TEPP-46

Gene Regulatory Effect of Pyruvate Kinase M2 is Involved in Renal Inflammation in Type 2 Diabetic Nephropathy

Authors
Le Li1, 2, Lei Tang2, 3, Xiaoping Yang2, 4, Ruifang Chen2, Zhen Zhang2, Yiping Leng2, Alex F. Chen1, 2, 5

Affiliations
1 Department of Cardiology, The Third Xiangya Hospital, Central South University, Changsha, China
2 The Center for Vascular Disease and Translational Medicine, The Third Xiangya Hospital, Central South University, Changsha, China
3 Department of Pharmacy, The Third Xiangya Hospital, Central South University, Changsha, China
4 Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Hunan Normal University, Changsha, China
5 Department of Cardiology, and Institute for Cardiovascu- lar Development and Regenerative Medicine, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China

Key words : PKM2, gene regulatory effects, type 2 diabetic nephropathy, renal inflammation, ICAM-1

ABSTRACT

Background and Aims The inflammation of glomerular en- dothelial cells induces and promotes the activation of mac- rophages and contributes to the development of diabetic ne- phropathy. Thus, this study aimed to investigate the gene regulatory effect and potential role of pyruvate kinase M2 (PKM2) in inflammatory response in diabetic nephropathy.

Methods The plasma PKM2 levels of patients with diabetes were evaluated. Eight-week-old mice were divided into three groups (WT, db/db mice, and db/db mice treated with TEPP-46) and raised for 12 weeks. Blood and kidney samples were col- lected at the end of the experiment. Endothelial cells were stimulated with high glucose with or without TEPP-46. The expression of intercellular adhesion molecule 1 (ICAM-1), in- terleukin 6 (IL-6), interleukin 1 beta (IL-1β), phospho-PKM2, PKM2, phospho-STAT3, STAT3, nuclear factor kappa B (NF-kB), and phospho-NF-kB in vivo and in vitro were determined using Western blot. The activation of macrophages (CD68 + CD86 + ) in the glomeruli was assessed via fluorescent double staining. Moreover, immune endothelial adhesion experiments were performed.

Results The plasma PKM2 levels of patients with type 2 diabetes increased. P-PKM2 was up-regulated in vivo and in vitro. TEPP-46 decreased inflammatory cell infiltration and ICAM-1 expression in vivo and in vitro and inhibited the differentiation of macrophages to M1 cells in db/db mice with diabetic ne- phropathy. PKM2 regulated the phosphorylation of STAT3 and NF-kB. Furthermore, high glucose levels induced the transition from tetramer to dimer and the nuclear translocation of PKM2. Conclusion The gene regulatory effect of PKM2 is involved in renal inflammation in type 2 diabetic nephropathy by promot- ing the phosphorylation of STAT3 and NF-kB and the expression of intercellular adhesion molecule 1. Thus, the down-regulation of phosphorylated PKM2 may have protective effects against diabetic nephropathy by inhibiting renal inflammation.

Introduction

Diabetic nephropathy is the primary complication of vascular inju- ry in patients with diabetes mellitus and is the leading cause of end- stage renal failure. Inflammation has an essential role in the patho- genesis and progression of diabetic nephropathy [1]. Both gene regulatory and metabolic abnormalities induced by high glucose (HG) levels are involved in the inflammatory network in diabetic nephropathy [2]. Therefore, we assessed the critical nodes in this network for the control of renal inflammation.

Pyruvate kinase M2 (PKM2) is a key rate-limiting enzyme in gly- colysis and exists in two interconvertible, active conformations, which have different functions in both metabolic and gene regula- tion. Conformational isomerization between tetramer and dimer can be observed under multiple pathological events [3, 4]. Tetra- meric PKM2 is located in the cytoplasm and regulates glycolysis, acting as a canonical pyruvate kinase with high catalytic activity. Dimeric PKM2 translocates to the nucleus and participates in gene regulation during inflammation with its noncanonical protein ki- nase activity [4]. A previous study has shown that PKM2 tetramer is down-regulated in diabetic nephropathy, thereby leading to de- creased glucose metabolic flux and accumulation of toxic glucose metabolites in podocytes [5]. Previously, dimeric PKM2 was be- lieved to be present in tumor cells [6] and exhibit gene regulatory effects similar to a protein kinase. However, whether the gene reg- ulatory effect of PKM2 is involved in pathological changes and in- flammatory response in diabetic nephropathy must be validated. Thus, the role of PKM2 dimers in renal inflammation in diabetic ne- phropathy and its underlying mechanisms were investigated.

Long-term exposure to HG levels induces glomerular endothelial cell inflammation and injury [7]. Different inflammatory molecules, including chemokines, adhesion molecules, and proinflammatory cytokines, are critical factors that may be involved in diabetic ne- phropathy [8]. The up-regulation of intercellular adhesion molecule 1 (ICAM-1) in endothelial cells is the initial step in inflammatory cell infiltration; however, the mechanism underlying ICAM-1 regu- lation in diabetic nephropathy has not been fully elucidated. Nu- clear factor kappa B (NF-kB) and STAT3 are transcriptional factors that are important in the development and progression of diabet- ic nephropathy [9, 10]. In addition, these factors can up-regulate ICAM-1 expression in multiple pathological models [11]. Thus, the regulation of dimeric PKM2 to STAT3 and NF-kB via its protein ki- nase ability was further evaluated.

Neither wonder drugs nor specific treatment can achieve the goal of radical cure in diabetic nephropathy [12]. TEPP-46 can switch dimeric PKM2 to tetramers, thereby inhibiting PKM2 phos- phorylation in addition to its gene regulatory effect. TEPP-46 at- tenuates the up-regulation of inflammatory factors in LPS-stimu- lated macrophages [4]. Thus, whether TEPP-46 can inhibit inflam- mation in vivo and the potential mechanisms underlying diabetic nephropathy were investigated. Our results can provide a new di- rection for research on the therapeutic target of renal inflamma- tion in diabetic nephropathy.

Materials and Methods

Clinical characteristics and biochemical analyses

A total of 23 patients with diabetes and 14 aged-matched partici- pants without diabetes were recruited from May 2016 to August 2016. However, patients with autoimmune diseases, malignancies, infectious diseases, and severe renal or hepatic dysfunction were excluded. All procedures in this study were in accordance with the ethical standards of the responsible committee on human experi- mentation and with the Declaration of Helsinki, which was revised in 2000. Informed consent was obtained from all patients included in the study. Patients’ data, including current conditions and med- ical histories, were recorded. The plasma PKM2 levels of the par- ticipants were assessed with an enzyme-linked immunosorbent assay (ELISA) kit.

Animals

Laboratory animals were purchased from the Department of Lab- oratory Animals of Central South University. In this study, male db/ db and C57 mice matched by age were selected. After the mice were placed in the barrier environment, they were fed adaptively 1 week before the start of the experiment.

The mice were divided into three groups: C57 mice that served as non-diabetic control (WT), db/db mice treated with TEPP-46 (db/ db + TEPP-46, 30 mg/kg per day with sodium carboxymethyl cellu- lose as the solvent, administered as gavage), and untreated db/db mice (db/db). Both the WT and db/db mice were treated with 0.5 % sodium carboxymethyl cellulose once a day. Five mice were includ- ed in each group, and the experiment lasted for 12 weeks. This study was approved by the Animal Use Committee of Central South University (no. 2018sydw0143). All institutional and national guidelines for the care and use of laboratory animals were followed. During the experiment, the mice were weighed every day, and the fasting blood glucose (FBG) levels of the mice were assessed every week using a glucometer with blood obtained from the tail vein (Accu-Check, Roche Diagnostics). At the end of the experiment, the urinary protein-to-creatinine ratio (mg/mg) was assessed. Then, all mice were euthanized, and the renal cortex was collected for further experiments.

Glucose tolerance test

Oral glucose tolerance test (OGTT) was performed at the end of the experiment. The plasma glucose concentrations were meas- ured using blood samples collected from the tail vein using a glu- cometer (Accu-Chek, Roche Diagnostics) at 0, 30, 60, 90, and 120 min after the administration of glucose (3 g/kg).

Cell culture

Human umbilical vein endothelial cells (HUVECs) were cultured in an incubator at a constant temperature of 37 °C with a concen- tration of 5 % CO2. DMEM medium was used for cell culture and was mixed with 10 % FBS, 100 U/mL of penicillin, and 100 ug/mL of streptomycin. In total, 6–10 generations of endothelial cells were obtained for further analysis. The glucose concentrations of the control and HG groups (5.5 and 25 mmol/L, respectively) were used in the cell culture medium. The siRNA sequence of PKM2 was 5′-CCAUAAUCGUCCUCACCAAUU-3′ (Ribobio, China). The control sequence was also purchased from Ribobio.

Adhesion assay

The THP-1 cells were labeled with PKH26 and were cultured in the incubator for 30 min. Then, they were added into the confluent monolayers of HUVECs, which had been pretreated with either HG alone or HG combined with TEPP-46. The adherent THP-1 cells were counted with a fluorescence microscope, compared after incuba- tion for 1 h, and rinsed with phosphate-buffered saline.

Western blot analysis

Western blot analysis was performed to measure the relative pro- tein expression in the HUVECs and renal cortex. The whole proteins in HUVECs were collected with a whole-cell lysis kit, and the sub- sequent steps were followed. The following antibodies were used in this experiment: PKM2 (CST, the USA), p-PKM2 (Y105) (CST, the USA), p-STAT3 (CST, the USA), p-NF-kB p65 (Ser536) (CST, the USA), NF-kB p65 (CST, the USA), ICAM-1 (CST, the USA), STAT3 (Santa Cruz, the USA), IL-6 (CST, the USA), and interleukin 1 beta (IL-1β) (CST, the USA).

Staining

In this experiment, 5-μm-thick paraffin-embedded sections were used for hematoxylin and eosin, periodic acid–Schiff (PAS), and Masson’s trichrome staining and immunofluorescence analyses. The tissue sections were double-stained. Macrophages were de- tected via red fluorescence staining with an anti-CD68 antibody (macrophage marker). Meanwhile, M1 macrophages were detect- ed via green fluorescence staining with an anti-CD86 antibody.

Quantitative real-time polymerase chain reaction (PCR) analysis

Peripheral blood mononuclear cells (PBMCs) were prepared from buffy coats via Ficoll-Hypaque density gradient centrifugation. Total RNA was extracted from PBMC with TRizol (Invitrogen) Rea- gent, and purity and concentration were analyzed using the Nan- oDrop 2000 spectrophotometer (Thermo Fisher Scientific). The primer sequences used for quantitative real-time PCR analysis were as follows: human PKM2 (F5′-ATGTCGAAGCCCCATAGTGAA-3′ and R5′-TGGGTGGTGAATCAATGTCCA-3′) and human GAPDH (F5′- TGTGGGCATCAATGGATTTGG-3′ and R5′-ACACCATGTATTCCGGGT- CAAT-3′).

ELISA

PKM2 in the plasma of patients and controls were detected with an ELISA kit (Cusabio, China) according to the manufacturer’s instructions.

Statistical analysis

GraphPad Prism 7.0 (GraphPad Software, San Diego, CA) was used for statistical analysis. Data were expressed as mean ± SEM. One- way analysis of variance was used for data analysis among groups, and Duncan’s multiple analysis was conducted for multiple com- parisons. A p value < 0.05 was considered statistically significant. Results Phosphorylated PKM2 is up-regulated in vivo and in vitro To determine the role of PKM2 in diabetes, the PKM2 expression in the plasma of diabetic patients was assessed since it can be used as a putative biomarker for inflammatory diseases. Result showed that the plasma PKM2 levels were significantly higher in the type 2 diabetes mellitus (T2DM) group than in the control group (3.666 ± 0.4889 vs. 5.026 ± 0.3768 pg/mL, p = 0.0337; ▶ Fig. 1a), whereas no significant difference was observed in the PKM2 mRNA levels in PBMCs between the control and T2DM groups (▶ Fig. 1b). The FBG and blood urea nitrogen levels of the T2DM group were significant- ly higher than those of the control group (▶ Fig. 1S). The phospho- rylation of PKM2 significantly increased in the kidneys of db/db mice (▶ Fig. 1c) and was up-regulated in HUVECs incubated with HG in a time-dependent manner (▶ Fig. 2S) . Osmotic pressure did not influence p-PKM2 levels(▶ Fig. 1d). These results indicated that p-PKM2 is up-regulated under HG environment. TEPP-46 inhibits inflammatory cell infiltration and ICAM-1 expression in vivo and in vitro The effects of TEPP-46 on diabetic nephropathy was assessed, and results showed that the renal function of and the morphology of glomeruli in db/db mice improved (▶ Fig. 2a and ▶ Fig. 3S) after treatment with TEPP-46. The treatment down-regulated the ex- pressions of ICAM-1, IL-6, and IL-1β and the phosphorylation of PKM2 in the renal cortex of db/db mice (▶ Fig. 2b). To evaluate the effects of TEPP-46 on macrophage infiltration and activation, we assessed M1 macrophages via double immunofluorescence stain- ing. The number of M1 macrophages significantly decreased in the db/db + TEPP-46 mice compared with the db/db mice (▶ Fig. 2c). These results indicated that TEPP-46 inhibited macrophage infil- tration and the differentiation of macrophages to M1 cells in the db/db mice with diabetic nephropathy. In addition, TEPP-46 did not affect FBG levels and the OGTT results (▶ Fig. 3S and 4S). As shown in ▶ Fig. 2d and in reference to non-treated cells, hy- perglycemia induced the adhesion of THP-1 cells to HUVECs by ap- proximately 2.8-fold higher. However, the adhesion of THP-1 cells to endothelial cells was only 1.7-fold higher after pretreatment with TEPP-46. Thus, TEPP-46 significantly attenuated the adhesion of immune cells to endothelial cells induced by hyperglycemia. The regulation of ICAM-1, IL-6, and IL-1β and the phosphorylation of PKM2 in vitro (▶ Fig. 2e) were consistent with those in vivo. These results indicated that phosphorylated PKM2 plays a crucial role in renal inflammation in type 2 diabetic nephropathy. PKM2 regulates the phosphorylation of STAT3 and NF-kB STAT3 and NF-kB can up-regulate ICAM-1 expression. To investi- gate the underlying mechanism of PKM2 in ICAM-1 regulation, we assessed the activation of STAT3 and NF-kB in vivo and in vitro. Results showed that the inhibition of phosphorylated PKM2 re- duced the activation of STAT3 and NF-kB both in the renal cortex of db/db mice and HUVECs incubated with HG (▶ Figs. 3a and b). PKM2 silencing inhibited the phosphorylation of STAT3 and NF-kB (▶ Fig. 3c and ▶ Fig. 5S). These results showed that phosphorylated PKM2 induced the activation of STAT3 and NF-kB. PKM2 can be dimerized and translocated into the nucleus in HUVECs under HG condition The isomerization of PKM2 and nuclear translocation under HG condition was further validated. The PKM2 dimer levels significant- ly increased along with the decrease in tetramer levels (▶ Fig. 4a). Dimeric PKM2 translocated to the nucleus in endothelial cells incu- bated with HG (▶ Fig. 4b). These results indicated that PKM2 can be dimerized and translocated into the nucleus in endothelial cells incubated with HG. Discussion and Conclusion This study revealed the noncanonical function of PKM2. That is, it regulates gene expression in renal inflammation in diabetic ne- phropathy. PKM2 could be phosphorylated, dimerized under HG condition, and then translocated into the nucleus. Phosphorylated PKM2 activated the STAT3 and NF-kB pathways, thereby inducing ICAM-1 expression, which eventually led to renal inflammation. The inhibition of phosphorylated PKM2 with pharmacological in- terventions may have protective effects against renal inflammation in diabetic nephropathy. Both metabolic disorders and gene regulation partially consti- tute the regulatory network of renal inflammation. Qi et al. have shown the crucial role of metabolic pathways regulated by PKM2 in diabetic nephropathy [5]. Reducing glycolysis and enforcing PKM2 tetramerization correct the proinflammatory phenotype of CAD macrophages [13]. Our results showed that the gene regula- tory effect of dimeric PKM2 had a vital role in renal inflammation via alleviating immune endothelial adhesion and improving infil- tration and activation of macrophages. In non-alcoholic liver dis- ease, the nucleation of PKM2 causes macrophages to transform into the M1 type, which can promote inflammation and steatohep- atitis progression [14]. The prevention of PKM2 nuclear accumula- tion had anti-inflammatory benefits in fatal lung inflammation [15]. Based on previous studies and the current study, PKM2 is a central node in the regulatory network of inflammation. Moreover, switching PKM2 from dimers to tetramers with TEPP- 46 down-regulated phosphorylated PKM2, alleviated ICAM-1 ex- pression in endothelial cells, and decreased infiltration and activa- tion of macrophages, which showed that inhibiting phosphorylat- ed PKM2 with TEPP-46 could relieve inflammatory response in diabetic nephropathy. ICAM-1 is an adhesive molecule that plays a vital role in the progression of diabetic nephropathy [16, 17], and it can be up-regulated to recruit activated macrophages and to se- crete multiple cytokines. Inhibiting or knocking out ICAM-1 can improve renal function in animal models with type 1 diabetes mel- litus or T2DM [18]. In addition, PKM2 also enhances the release of other inflammatory factors. PKM2 promotes the transcription and releasing of IL-1β in coronary artery disease [3, 19]. The inhibition of PKM2 expression reduces the release of interferon gamma in CD4 + T induced by Hcy [20]. PKM2 is a critical regulatory factor of multiple inflammatory molecules, including chemokines, adhesion molecules, and proinflammatory cytokines. The NF-kB and STAT3 signaling pathways are classic inflamma- tory pathways, and their activation can up-regulate ICAM-1 expression in multiple pathological conditions [21]. This study showed that NF-kB and STAT3 were phosphorylated in db/db mice and in vitro. The tetramerization of PKM2 with TEPP-46 down-regulated the phosphorylation of STAT3 and NF-kB. Activated NF-kB promotes the expression of various inflammation-related genes and up-reg- ulates inflammation-related cytokines and adhesion proteins; thus, it plays a role in the pathogenesis of diabetic nephropathy [22, 23]. The inhibition of the STAT3 pathway, which was found to have a role in intracellular processes in a diabetic nephropathy model, has broad protective effects [24–26]. ▶ Fig. 2 TEPP-46 alleviated endothelial cell inflammatory response and inflammatory cell infiltration in vivo and in vitro. a Urinary protein-to-creati- nine ratio of the WT, db/db, and db/db + TEPP-46 mice (n = 5 for each group). * p < 0.05. b Western blot analysis of p-PKM2, PKM2, intercellular adhesion molecule 1 (ICAM-1), interleukin (IL)-6, and IL-1 beta (IL-1β) in the renal cortex of WT, db/db, and db/db + TEPP-46 mice (n = 5 for each group). c Representative images of double immunofluorescence staining and quantitative analysis of the kidneys. M1 macrophages tested positive for CD68 (red) and CD86 (green). DAPI was used to stain the cell nucleus. Scale bars, 100 μm (n = 5). d Representative fluorescence microscopic images of THP-1 cells attached to human umbilical vein endothelial cells and quantitative analysis results. Scale bar, 100 μm (n = 3). e Western blot analysis of p-PKM2, PKM2, ICAM-1, IL-6, and IL-1β in endothelial cells treated with high glucose with or without TEPP-46 (n = 3). * p < 0.05. Endothelial cell dysfunction is the initial step in inflammatory cell infiltration [27, 28]. Endothelial cells in the glomeruli induces inflammatory responses and loses their function when treated with HG, thereby leading to proteinuria [29, 30]. HUVECs under diabet- ic environment are used as an in vitro model of diabetes and exhib- its inflammation with modified gene and protein expression [24]. We found that the plasma PKM2 levels increased in patients with diabetes, which was consistent with the findings of previous stud- ies showing elevated serum PKM2 level in some inflammatory dis- eases [31–33]. Plasma PKM2 level is associated with type 1 and type 2 diabetic nephropathy; thus, the assessment of serum PKM2 level is important [34]. Th17 differentiation is involved in the patholog- ical progression of DN [35]. Our results indicated that the IL-6 level decreased with the down-regulation of p-PKM2 induced by TEPP- 46 in endothelial cells. Previous studies have shown that IL-6 pro- motes Th17 cell differentiation, leading to the activation of inflammatory cascade [36]. Since the pharmacological inhibition of PKM2 reduced glycolysis and in vitro differentiation to Th1 and Th17 cells in experimental autoimmune encephalomyelitis [37], the mecha- nism of Th17 cell differentiation induced by PKM2 in diabetic ne- phropathy must be validated. TEPP-46 did not influence the FBG levels and OGTT results of db/db mice, indicating that its beneficial effect on diabetes may be associated with the improvement of in- flammation rather than its direct glucose-related effect. However, further studies must be conducted to validate glucose utilization in db/db mice treated with TEPP-46. PKM2 is acetylated on lysine 305 in tumor cells under HG condition; thus, PK activity is down- regulated [38]. Moreover, PKM2 in endothelial cells might be phos- phorylated, isomerized, and translocated to the nucleus to regu- late inflammatory response in diabetic nephropathy. However, the role of other reactions, particularly acetylation, in the pathogen- esis of renal inflammation was not evaluated.

This study first showed that PKM2 has a gene regulatory effect on renal inflammation in type 2 diabetic nephropathy. Tetrameri- zation, which decreases phosphorylated PKM2, can down-regulate ICAM-1 expression and decrease macrophage adhesion and the activation of the NF-kB and STAT3 pathways. PKM2 is a critical node in the regulatory network of renal inflammation. Moreover, this study presented novel therapeutic targets for the inhibition of in- flammation in diabetic nephropathy.

Conflict of Interest

The authors declare that they have no conflict of interest.

Funding

This work was supported, in part, by the National Science Founda- tion of China (NSFC) Projects 81930012 and 81730013, and the Chinese Postdoctoral Science Foundation 2018M643010.

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