​GSK343 modulates macrophage M2 polarization through the EZH2/MST1/YAP1 signaling axis to mitigate neurological damage induced by hypercalcemia in CKD mice

Author links open overlay panelYaochen Cao【曹耀辰】 a b c e,Hongming Sun a d,Xitong Li b c,Wolfgang Pommer g,Yingquan Xiong b c,Xin Chen b c,Chang Chu c,Fabiao Yu【于法标】 a e,Berthold Hocher c e f h,Ziqiang Wang【王自强】 a e

• aDepartment of Medicine, Hainan Medical University, The First Affiliated Hospital of Hainan Medical University, No. 31 Longhua Road, Haikou 570102, Hannan, China

• bDepartment of Nephrology, Charité - Universitätsmedizin Berlin, Campus Mitte, Berlin 10117, Germany

• cFifth Department of Medicine (Nephrology/Endocrinology/Rheumatology), University Medical Centre Mannheim, University of Heidelberg, Heidelberg, Germany

• dDepartment of Neurology and Neuroscience, Okayama University School of Medicine, Okayama 700-8558, Japan

• eKey Laboratory of Emergency and Trauma, Ministry of Education, Key Laboratory of Hainan Trauma and Disaster Rescue, The First Affiliated Hospital of Hainan Medical University, College of Pharmacy, Institute of Functional Materials and Molecular Imaging, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China

• fReproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China

• gKuratorium für Dialyse und Nierentransplantation (KfH) - Bildungszentrum, Martin-Behaim-Str. 20, Neu-Isenburg 63263, Germany

• hInstitute of Medical Diagnostics, IMD Berlin, Berlin, Germany

Received 16 October 2023, Revised 11 January 2024, Accepted 16 January 2024, Available online 18 January 2024, Version of Record 29 January 2024.

Cellular Signalling    Volume 116, April 2024, 111063

https://doi.org/10.1016/j.cellsig.2024.111063Highlights

• •GSK343 promotes M2 macrophage polarization in CKD, reducing hypercalcemia-induced brain damage.

• •GSK343 decreases EZH2 and increases MST1 in CKD mice.

• •MST1 in CKD mice inhibits YAP1, aiding M2 polarization and neurological recovery.

• •GSK343 uses EZH2/MST1/YAP1 axis for M2 polarization, alleviating CKD-related brain harm.

• •The study offers new insights for treating hypercalcemia-induced neurological issues in CKD.

Abstract

Chronic kidney disease (CKD) often culminates in hypercalcemia, instigating severe neurological injuries that are not yet fully understood. This study unveils a mechanism, where GSK343 ameliorates CKD-induced neural damage in mice by modulating macrophage polarization through the EZH2/MST1/YAP1 signaling axis. Specifically, GSK343 downregulated the expression of histone methyltransferase EZH2 and upregulated MST1, which suppressed YAP1, promoting M2 macrophage polarization and thereby, alleviating neural injury in hypercalcemia arising from renal failure. This molecular pathway introduced herein not only sheds light on the cellular machinations behind CKD-induced neurological harm but also paves the way for potential therapeutic interventions targeting the identified axis, especially considering the M2 macrophage polarization as a potential strategy to mitigate hypercalcemia-induced neural injuries.

Keywords

GSK343

Chronic kidney disease

Hypercalcemia

Neurological injury

EZH2/MST1/YAP1 axis

Macrophage polarization

Introduction

Chronic Kidney Disease (CKD), particularly when it escalates to kidney failure, often precipitates a concurrent condition of hypercalcemia, with such instances evidenced in a plethora of prior research [1,2]. The accumulation of excessive calcium, or calcium overload, is a well-documented contributor to neuronal demise and substantive damage to the central nervous system [3]. In the intricate milieu of the central nervous system, macrophages, hailed for their pivotal roles in pathological events, can be stratified into two distinct phenotypes: the pro-inflammatory M1 and the anti-inflammatory M2, each harboring unique functional and therapeutic implications [4,5]. Several studies illuminate the potential of leveraging M2 macrophage polarization as a therapeutic avenue for nerve repair and regeneration [6].

Delving into the molecular mechanisms, GlaxoSmithKline 343 (GSK343) emerges as a potent inhibitor of EZH2, a catalytic component of PRC2 that is enmeshed in a myriad of biological cascades [7]. Interestingly, EZH2 has been spotlighted in recent narratives as a prospective target for kidney pathologies, given its capacity to mitigate acute kidney injury and impede renal fibrosis when inhibited [8]. Moreover, EZH2 has the ability to curtail the expression of STE20-like kinase-1 (MST1) by dampening the activity of the MST1 promoter [9], an enzyme intrinsically intertwined with the Hippo signaling pathway, a crucial regulator of cellular behaviors including proliferation and differentiation [10]. It has been illustrated that the diminution of MST1 expression can ameliorate the pathological alterations observed in diabetic kidneys and stymie the progression of diabetic nephropathy [11]. Concurrently, Yes-associated protein 1 (YAP1), a salient effector within the Hippo signaling cascade, is frequently implicated in oncogenic processes [12]. Notably, the activation of YAP instigated by MST1 inhibition can stimulate EMT and fibrosis within renal tubular epithelial cells [13], while elevated YAP expression has been demonstrated to inhibit macrophage M2 polarization [14].

In light of these insights, the present study endeavored to utilize a CKD mouse model to probe into whether GSK343, by modulating the EZH2/MST1/YAP1 axis, could facilitate M2 macrophage polarization, and subsequently ameliorate neural damage induced by hypercalcemia concomitant with kidney failure. This exploration aims to decipher the underlying mechanisms and offer viable therapeutic strategies for CKD management.

Fig. 1. Significance of critical genes related to resultant nerve damage from kidney failure-induced hypercalcemia. A, A heat map of the expression of significantly upregulated genes in the cerebral cortex with kidney failure-induced hypercalcemia from RNA sequencing. The expression value is shown in the color scale on the right. B, Venn diagram of the GeneCards RNA sequencing and DisGeNET prediction results. C, Expression of EZH2 in normal samples (blue box, n = 3) and CKD samples (red box, n = 3) following RNA-sequencing analysis. D, H3K27me3 modification at the promoter of MST1 analyzed by UCSC. E, Expression of MST1 in normal samples (blue box, n = 3) and CKD samples (red box, n = 3) following RNA-sequencing analysis. F, Expression of YAP1 in normal samples (blue box, n = 3) and CKD samples (red box, n = 3) following RNA-sequencing analysis. G, An interaction network of genes interacting with YAP1 constructed by GeneMANIA. H, Bubble chart of KEGG enrichment analysis of YAP1 and its interacting genes. The ordinate represents the enriched entry identifier, and the abscissa and bubble size represent the number of genes enriched in the identifier. The color of the bubble on the right indicates the -log p value. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. GSK343 polarizes macrophages toward the M2 phenotype and alleviates nerve damage induced by hypercalcemia in CKD mice. A, Body weight of CKD mice or those treated with GSK343 from day 0 to 7. B, The DAI of CKD mice or those treated with GSK343. C, Levels of Scr and BUN in the serum of CKD mice or those treated with GSK343. D, Blood calcium concentration in CKD mice or those treated with GSK343. E, HE and Masson's trichrome staining analysis of kidney tissue damage in CKD mice or those treated with GSK343. F, RT-qPCR and immunoblotting of albumin expression, Kim-1, PAI-1, FN, SMA, and Col. G, Nerve function changes in CKD mice or those treated with GSK343. H, Immunoblotting of iNOS and Arg1 proteins in the brain tissue of CKD mice or those treated with GSK343. I, Immunofluorescence staining of iNOS- and Arg1-positive macrophages in the brain tissues of CKD mice or those treated with GSK343. J, Flow cytometry used to observe CD38-positive (M1-type macrophages) and Egr2-positive (M2-type macrophages) macrophages in mouse brain tissue. K, TUNEL-positive cells in the hippocampal CA1 region of the CKD mice or those treated with GSK343. L, HE staining analysis of the hippocampal CA1 region and cerebral cortex of CKD mice or those treated with GSK343. n = 8 mice in each treatment. * p < 0.05, compared with normal mice. # p < 0.05, compared with CKD mice.

Fig. 3. GSK343 increases the expression of MST1 by reducing EZH2 expression. A, IHC staining and immunoblotting of EZH2 and MST1 proteins in the kidney tissue of CKD mice or those treated with GSK343 (n = 8, p < 0.05, compared with normal mice, # p < 0.05, compared with CKD mice). B, EZH2 and MST1 expression determined by RT-qPCR in TCMK-1 cells treated with GSK343. C, EZH2 and MST1 expression determined by immunoblotting in TCMK-1 cells treated with GSK343. D, EZH2 expression determined by RT-qPCR in TCMK-1 cells treated with oe-EZH2. E, EZH2 expression determined by immunoblotting in TCMK-1 cells treated with oe-EZH2. F, EZH2 and MST1 expression determined by immunoblotting in TCMK-1 cells treated with GSK343 or combined with oe-EZH2. * p < 0.05, compared with TCMK-1 cells treated with oe-NC or with control + oe-NC. # p < 0.05, compared with cells treated with GSK343 + oe-NC. All cell experiments were conducted three times independently.

Fig. 4. GSK343 polarizes macrophages toward the M2 phenotype and reduces nerve damage induced by hypercalcemia in CKD mice by regulating the EZH2/MST1 axis. CKD mice were treated with GSK343 + oe-NC, GSK343 + oe-EZH2 + oe-NC or GSK343 + oe-EZH2 + oe-MST1. A, the Transfection efficiency of MST1 overexpression determined by RT-qPCR in TCMK-1 cells. B, EZH2 and MST1 expression determined by RT-qPCR in kidney tissues of CKD mice. C, Body weight of CKD mice from day 0 to 7. D, DAI of CKD mice. E, Levels of Scr and BUN in the serum of CKD mice. F, Blood calcium concentration in CKD mice. G, HE and Masson's trichrome staining analysis of kidney tissue damage in CKD mice. H, RT-qPCR and immunoblotting of albumin expression, Kim-1, PAI-1, FN, SMA, and Col. I, Nerve function changes in CKD mice. J, immunoblotting of iNOS and Arg1 proteins in the brain tissue of CKD mice. K, Immunofluorescence staining of iNOS- and Arg1-positive macrophages in the brain tissues of CKD mice. L, Flow cytometry used to observe CD38-positive (M1-type macrophages) and Egr2-positive (M2-type macrophages) macrophages in the brain tissue of each group of mice. M, TUNEL-positive cells in the hippocampal CA1 region of the CKD mice. N, HE staining analysis of the hippocampal CA1 region and cerebral cortex of CKD mice. n = 8 mice in each treatment. *, p < 0.05, compared with CKD mice treated with GSK343 + oe-NC. #, p < 0.05, compared with CKD mice treated with GSK343 + oe-EZH2 + oe-NC. All cell experiments were conducted three times independently.

Fig. 5. MST1 polarizes macrophages toward the M2 phenotype and weakens nerve damage induced by hypercalcemia in CKD mice by repressing YAP1. CKD mice were treated with oe-MST1 or combined with oe-YAP1. A, IHC staining of YAP1 protein in the kidney tissue of CKD mice. B, Transfection efficiency of YAP1 overexpression determined by RT-qPCR in TCMK-1 cells. C, Transfection efficiency of YAP1 overexpression determined by immunoblotting in TCMK-1 cells. D, YAP1 and MST1 expression determined by immunoblotting in kidney tissues of CKD mice. E, Body weight of CKD mice from day 0 to 7. F, DAI of CKD mice. G, Levels of Scr and BUN in the serum of CKD mice. H, Blood calcium concentration in CKD mice. I, HE and Masson's trichrome staining analysis of kidney tissue damage in CKD mice. J, RT-qPCR and immunoblotting of expression of albumin, Kim-1, PAI-1, FN, SMA, and Col. K, Nerve function changes in CKD mice assessed by behavioral tests. L, Immunoblotting of iNOS and Arg1 proteins in the brain tissue of CKD mice. M, Flow cytometry was used to observe CD38-positive (M1-type macrophages) and Egr2-positive (M2-type macrophages) macrophages in the brain tissue of each group of mice. N, Immunofluorescence staining of iNOS- and Arg1-positive macrophages in the brain tissues of CKD mice. O, TUNEL-positive cells in the hippocampal CA1 region of the CKD mice. n = 8 for mice upon each treatment. P, HE staining for pathological changes in rat hippocampus CA1 region and cerebral cortex. n = 8 mice in each treatment. *, p < 0.05, compared with normal mice, TCMK-1 cells transfected with oe-NC or CKD mice treated with oe-NC. #, p < 0.05, compared with CKD mice or those treated with oe-MST1 + oe-NC. All cell experiments were conducted three times independently.

Fig. 6. GSK343 induces M2 polarization of macrophages and delays resultant nerve damage from kidney failure-induced hypercalcemia via the EZH2/MST1/YAP1 axis. CKD mice were treated with GSK343 or combined with oe-YAP1. A, EZH2, MST1 and YAP1 expression determined by RT-qPCR and immunoblotting in the kidney tissue of CKD mice. B, Body weight of CKD mice from day 0 to 7. C, DAI of CKD mice. D, Levels of Scr and BUN in the serum of CKD mice. E, Blood calcium concentration in CKD mice. F, HE staining of the kidney tissue damage of CKD mice. G, RT-qPCR and immunoblotting of expression of albumin, Kim-1, PAI-1, FN, SMA, and Col. H, Nerve function changes in CKD mice. I, Immunoblotting of iNOS and Arg1 proteins in the brain tissue of CKD mice. J, Immunofluorescence staining of iNOS- and Arg1-positive macrophages in the brain tissues of CKD mice. K, Flow cytometry used to observe CD38-positive (M1 macrophages) and Egr2-positive (M2 macrophages) macrophages in the brain tissues of each group of mice. L, TUNEL-positive cells in the hippocampal CA1 region of the CKD mice. M, HE staining analysis of the hippocampal CA1 region and cerebral cortex of CKD mice. n = 8 mice in each treatment. *, p < 0.05, compared with CKD mice treated with GSK343 + oe-NC. All cell experiments were conducted three times independently.

Fig. 7. Schematic diagram of GSK343 affecting the resultant nerve damage from kidney failure-induced hypercalcemia. GSK343 inhibits EZH2 to upregulate MST1 and downregulate YAP1, thereby promoting the M2 polarization of macrophages and reducing resultant nerve damage from kidney failure-induced hypercalcemia.

Supplementary material 1. Supplementary Fig. 1. Representative protein bands. A, Fig. 2F. B, Fig. 2H. C, Fig. 3A. D, Fig. 3C. E, Fig. 3F. F, Fig. 4H. G, Fig. 4J. H, Fig. 5C. I, Fig. 5D. J, Fig. 5J. K, Fig. 5L. L, Fig. 6A. M, Fig. 6G. N, Fig. 6I.

Supplementary material 2. Supplementary Fig. 2. Representative immunofluorescence images of iNOS and ARG1-positive macrophages in the CA1 region of the hippocampus and TUNEL staining images of apoptosis. A, C, E, and G indicate representative immunofluorescence images of quantitative results in Fig. 2I, 4K, 5M, and 6J, respectively. B, D, F, and H indicate TUNEL staining images of apoptotic cell statistics in Fig. 2K, 4M, 5O, and 6L, respectively.

Discussion

This study has brought forth intriguing findings, substantiating that GSK343, an inhibitor of EZH2, notably mitigates hypercalcemia-induced neurological damage in CKD-affected mice. This mitigation seemingly unfolds through the promotion of M2 macrophage polarization, orchestrated via the EZH2/MST1/YAP1 axis.

It has been previously delineated that the manifestation of M2 macrophage polarization can be indicated by the upregulation of arginase 1 (Arg1), while the concurrent release of inducible nitric oxide synthase (iNOS) heralds the polarization toward the M1 phenotype [23,24]. Present findings indicate that GSK343 notably suppresses iNOS expression, whilst simultaneously elevating Arg1 expression, thereby affirming its capability to propel M2 macrophage polarization. Such polarization has been demonstrated to not only attenuate neuronal damage, especially in the context of spinal cord ischemia-reperfusion injury [25], but also to proffer protective effects against kidney injury [26]. Consequently, GSK343 emerges as a potential candidate for mitigating neuronal damage ensuing from kidney failure-induced hypercalcemia.

Drilling down into the mechanistic depth of GSK343 action, the present investigations illustrate that the role played by this inhibitor in M2 macrophage polarization, and consequently, the amelioration of nerve damage induced by hypercalcemia secondary to kidney failure, is intertwined with the suppressed expression of EZH2 and an elevation in MST1 expression. Previously, GSK343 has been acknowledged as an inhibitor of EZH2 in various cancer subtypes [21]. Protection against acute kidney injury by EZH2 inhibition has been well-documented [27,28]. Moreover, EZH2's inhibitory action on MST1 expression, via reduction of H3K4me3 mark and RNA polymerase II occupancy on the MST1 promoter C-phosphate-G (CpG) region, has been substantiated [9]. Notably, MST1, whose deficiency is allied with CKD development [29], modulates M2 macrophage polarization by influencing phosphorylation processes [30]. The aforementioned evidence intimates that targeting the GSK343/EZH2/MST1 axis could emerge as a potentially viable strategy in ameliorating nerve damage cascading from kidney failure-induced hypercalcemia.

Moreover, this study uncovered that MST1, through the diminution of YAP1 expression, navigates macrophage polarization toward the M2 phenotype, thereby dampening hypercalcemia-triggered neuronal damage in the milieu of CKD. Aligning with these findings, prior research has documented that MST1 knockdown escalates YAP activity, culminating in renal failure [29]. Additionally, YAP activation amid renal injury can exacerbate CKD progression [31] and impede IL-4/IL-13-induced M2 macrophage polarization in the framework of inflammatory bowel disease [14]. Current experimental data reveal that GSK343 facilitates the polarization of macrophages toward the M2 phenotype, thereby attenuating hypercalcemia-induced nerve damage through the EZH2/MST1/YAP1 axis. While previous studies have illustrated interactions between EZH2 and the C-terminal of YAP [32], the exact mechanism governing their interplay is yet to be unveiled, necessitating further investigative endeavors for comprehensive elucidation.

Conclusions

Our study indicates that GSK343 can prevent nerve damage due to kidney failure-induced hypercalcemia. This prevention was associated with the potentiation of M2 polarization of macrophages, MST1 upregulation, and inhibition of the EZH2/YAP1 axis (Fig. 7). The current findings fill an essential gap in the knowledge of nerve damage due to kidney failure-induced hypercalcemia and provide a vital foundation for future GSK343-based targeted therapy for CKD treatment.