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GSK343 modulates macrophage M2 polarization through the EZH2MST1YAP1 signaling axis to mitigate neurological damage induced by hypercalcemia in CKD mice
发布时间:2024-01-30 发布者: 浏览次数:

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

Fig. 4

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

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

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

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 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 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.




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