DNA hypomethylation of Syk induces oxidative stress and apoptosis via the PKCβ/P66shc signaling pathway in diabetic kidney disease

Epigenetic alterations, especially DNA methylation, have been shown to play a role in the pathogenesis of diabetes mellitus (DM) and its complications, including diabetic kidney disease (DKD). Spleen tyrosine kinase (Syk) is known to be involved in immune and inflammatory disorders. We, therefore, investigated the possible involvement of Syk promoter methylation in DKD, and the mechanisms underlying this process. Kidney tissues were obtained from renal biopsies of patients with early and advanced DKD. A diabetic mouse model (ApoE−/− DM) was generated from ApoE knockout (ApoE−/−) mice using a high‐fat and high‐glucose diet combined with low‐dose streptozocin intraperitoneal injection. We also established an in vitro model using HK2 cells. A marked elevation in the expression levels of Syk, PKCβ, and P66shc in renal tubules was observed in patients with DKD. In ApoE−/− DM mice, Syk expression and the binding of Sp1 to the Syk gene promoter were both increased in the kidney. In addition, the promoter region of the Syk gene exhibited hypomethylation. Syk inhibitor (R788) intervention improved renal function and alleviated pathologic changes in ApoE−/− DM mice. Moreover, R788 intervention alleviated oxidative stress and apoptosis and downregulated the expression of PKCβ/P66shc signaling pathway proteins. In HK2 cells, oxLDL combined with high‐glucose stimulation upregulated Sp1 expression in the nucleus (compared with control and oxLDL groups), and this was accompanied by an increase in the binding of Sp1 to the Syk gene promoter. SP1 silencing downregulated the expression of Syk and inhibited the production of reactive oxygen species and cell apoptosis. Finally, PKC agonist intervention reversed the oxidative stress and apoptosis induced by Syk inhibitor (R406). In DKD, hypomethylation at the Syk gene promoter was accompanied by an increase in Sp1 binding at the promoter. As a consequence of this enhanced Sp1 binding, Syk gene expression was upregulated. Syk inhibitors could attenuate DKD‐associated oxidative stress and apoptosis via downregulation of PKCβ/P66shc signaling pathway proteins. Together, our results identify Syk as a promising target for intervention in DKD.

and the mechanisms underlying this process.Kidney tissues were obtained from renal biopsies of patients with early and advanced DKD.A diabetic mouse model (ApoE −/− DM) was generated from ApoE knockout (ApoE −/− ) mice using a high-fat and high-glucose diet combined with low-dose streptozocin intraperitoneal injection.We also established an in vitro model using HK2 cells.A marked elevation in the expression levels of Syk, PKCβ, and P66shc in renal tubules was observed in patients with DKD.In ApoE −/− DM mice, Syk expression and the binding of Sp1 to the Syk gene promoter were both increased in the kidney.In addition, the promoter region of the Syk gene exhibited hypomethylation.Syk inhibitor (R788) intervention improved renal function and alleviated pathologic changes in ApoE −/− DM mice.Moreover, R788 intervention alleviated oxidative stress and apoptosis and downregulated the expression of PKCβ/P66shc signaling pathway proteins.In HK2 cells, oxLDL combined with high-glucose stimulation upregulated Sp1 expression in the nucleus (compared with control and oxLDL groups), and this was accompanied by an increase in the binding of Sp1 to the Syk gene promoter.SP1 silencing downregulated the expression of Syk and inhibited the production of reactive oxygen species and cell apoptosis.Finally, PKC agonist intervention reversed the oxidative stress and apoptosis induced by Syk inhibitor (R406).In DKD,

| INTRODUCTION
The prevalence of Type 2 diabetes mellitus (DM) has increased rapidly worldwide to the point where DM can be considered a global epidemic.2][3] Given the considerable health care burden of DKD, the search for treatments targeting the occurrence, progression, and outcome of DKD is a major priority.
DKD is morphologically characterized by a thickening of the glomerular basement membrane (GBM) underlying the podocytes, expansion of the extracellular matrix surrounding mesangial cells, and interstitial fibrosis and tubular atrophy (IFTA). 4][7][8][9][10][11] Evidence is accumulating that renal proximal tubule cells play an important role in the development of DKD. 12 Renal tubular epithelial cells contain abundant mitochondria, and these are the main site of ATP production, the location for the tricarboxylic acid cycle, and the energy source for maintaining transport function.Under highglucose stimulation, mitochondrial-dependent respiration in tubular epithelial cells is diminished, and reactive oxygen species (ROS) production is high, ultimately resulting in renal tubular epithelial cell injury. 13hus, mitochondrial dysfunction contributes to the development of DKD through the production of excessive ROS and consequent apoptosis.
Epigenetics plays an important role in the pathogenesis of DM and DKD.These epigenetic alterations can include DNA methylation and histone modifications.DNA methylation in the gene body or transcribed regions is known to regulate transcription elongation and splicing. 14,15Spleen tyrosine kinase (Syk) is an intracellular protein tyrosine kinase that binds immunoreceptors to transduce intracellular signals to initiate inflammatory responses to extracellular antigen IgG immune complexes.In particular, Syk is known to play an important role in the classical Fcγ receptor intracellular signaling response.Interestingly, Syk has already been demonstrated to play a role in mediating immune and inflammatory responses during the pathogenesis of autoimmune glomerulonephritis and chronic glomerulonephritis. 16n the present study, our aims were to explore the expression of Syk in kidney tissues of patients with DKD, to analyze changes in Syk promoter methylation in the DKD mouse model and their effect on oxidative stress and cell apoptosis, and to explore whether Syk is a potential therapeutic target for the alleviation of diabetic renal damage.

| Human kidney sample preparation
This study was approved by the Institutional Ethics Committee of the West China Hospital of Sichuan University.Patients with early and advanced DKD were included in this study, and kidney tissues were obtained from renal biopsies.Patients with renal carcinoma and eGFR >60 mL/min/1.73m 2 but without hypertension, diabetes, or other comorbidities served as a control.Control samples were obtained from healthy kidney poles.The clinical data and laboratory test results of these patients are shown in Supplementary Table S1.Using the pathological classification of the Renal Pathology Society in 2010, 17 DKD patients were divided into a combined grade I and grade II diabetic nephropathy (DN I/II) group or a combined grade III and grade IV (DN III/IV) group.The pathological features of these DKD patients are described in Supplementary Table S2.Frozen sections and paraffin hypomethylation at the Syk gene promoter was accompanied by an increase in Sp1 binding at the promoter.As a consequence of this enhanced Sp1 binding, Syk gene expression was upregulated.Syk inhibitors could attenuate DKDassociated oxidative stress and apoptosis via downregulation of PKCβ/P66shc signaling pathway proteins.Together, our results identify Syk as a promising target for intervention in DKD.

K E Y W O R D S
apoptosis, diabetic kidney disease, DNA methylation, oxidative stress, renal tubular epithelial cells, Syk sections of kidney tissue samples were processed for immunofluorescence and immunohistochemistry.

| Biochemical measurements
Prior to euthanasia, blood glucose was measured using a glucose analyzer after a 6-h daytime fast.Urine samples were collected while the mice were housed individually in metabolic cages for 24 h.Blood samples were obtained by cardiac puncture.Blood urea nitrogen, plasma creatinine, plasma total cholesterol, and plasma triglycerides were measured on a biochemistry autoanalyzer (BS-240VET, mindray) using commercial kits.Urinary albumin creatinine ratio values were measured using test kits (ZC-38536, ZC-38529, ZCIBIO Technology Co., Ltd).

| Transmission electron microscopy
Kidney sections were fixed in 3.7% glutaraldehyde in PBS buffer.After rinsing and postfixing in 1% osmium tetroxide, samples were embedded in 10% gelatin, fixed, and then cut into several blocks (<1 mm 3 ).After dehydration in increasing concentrations of alcohol and infiltration with increasing concentrations of Quetol-812 epoxy resin mixed with propylene oxide, samples were embedded in pure, fresh Quetol-812 epoxy resin and polymerized.Ultrathin sections (50 nm) were cut and poststained with uranyl acetate for 10 min and lead citrate for 2 min at room temperature before observation under a transmission electron microscope (JEM-1400PLUS).

| Bisulfite sequencing PCR
DNA extraction and bisulfite conversion of genomic DNA (150-500 ng) was performed using the EZ DNA methylation gold™ Kit (D5006, ZYMO RESEARCH) according to the manufacturer's instructions.PCR was performed using PCR MasterMix (DRR071A, Takara, Japan).For bisulfite sequencing of the Syk promoter, the PCR products were isolated by electrophoresis and sequenced. 142.10 | Transfection of siRNA HK2 cells were seeded in six-well plates and flasks and maintained in DMEM/F12 with 10% FBS.Silencernegative control siRNA and silencer validated siRNA against SP1 were purchased from Shanghai Genechem Co., Ltd.(Shanghai, China).The transfected cells were incubated with validated SP1 siRNA puromycin to screen cells with stable silencing gene expression.Transfection with the SP1 siRNAs diluted in Opti-MEM I (100 nmol/L) was performed using Lipofectamine 2000 Reagent (Invitrogen, USA) according to the manufacturer's protocol.The culture medium was changed 24 h after transfection.
2.12 | RNA isolation and real-time

quantitative PCR analysis
Total RNA from kidney tissue or cultured cells was extracted using TRIzol reagent (15596018, Thermo Fisher Scientific Inc., Waltham, MA).Total RNA concentration was then measured using a microspectrophotometer (Thermo Fisher Scientific Inc.).After confirming the RNA quality by agar gel electrophoresis, cDNA was synthesized from the total RNA samples.Real-time quantitative PCR was performed on the CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA) using SYBR Premix Ex Taq™ (RR42LR, Tli RNaseH Plus, Takara) as previously described. 17GAPDH was used as an internal standard.Relative mRNA levels were calculated as the ratio of target mRNA to GAPDH mRNA and expressed as mean ± standard error of the mean (SEM).reversed, and the DNA was purified.Purified DNA samples were then amplified by real-time quantitative PCR. 13 2.14 | Flow cytometry HK2 cells were seeded at a density of 2 × 10 5 cells/well in six-well plates. Afer treatment, cells were processed and assayed using an Annexin V-FITC/ PI apoptosis detection kit (556547, BD Biosciences, San Diego, CA, USA) according to the manufacturer's instructions.The samples were all analyzed within 1 h using a FACS Calibur Becton Dickinson flow cytometer. Finally, the results were analyzed using FlowJo software.

| Syk was increased in DKD renal biopsies
Using immunofluorescence and immunohistochemistry staining, we observed that the expression levels of Syk, PKCβ, and P66shc were increased in renal biopsies from DKD subjects in comparison with their corresponding levels in renal biopsies from control subjects (CON, n = 10) (Figure 1A,B).Importantly, the expression levels of Syk, PKCβ, and P66shc were not statistically different between DN I/II (n = 10) and DN III/IV (n = 10).In addition, the expression levels of Syk and P66shc were positively correlated with blood glucose and urinary albumin/ creatinine ratio (ACR) in all subjects, while the expression level of PKCβ was positively correlated with ACR (Table 1).

| Syk was increased in DKD mice, and Syk expression was modulated by DNA methylation
We first assessed the phosphorylation status of Syk in the kidney from ApoE −/− DM mice by immunofluorescence (Figure 2A).Phosphorylation of Syk (p-Syk) was markedly increased in the renal tubules of streptozocin (STZ)-induced diabetic mice, weakly expressed in nondiabetic ApoE −/− mice, and not detected in normal control mice.Western blot analysis revealed that the expression of p-Syk in ApoE −/− DM mice was significantly higher than that in normal control or ApoE −/− mice (Figure 2B).Next, we analyzed the promoter region of Syk using NCBI tools (Supplementary Figure S1).Meth Primer 2.0 that the promoter region contained abundant CpG sites (Supplementary Figure S2).The ApoE −/− DM group exhibited hypomethylation in the Syk promoter region compared with normal control and ApoE −/− groups (Figure 2C).Real-time quantitative PCR analysis revealed that the ApoE −/− DM group demonstrated higher Syk mRNA expression levels compared to the Con and ApoE −/− groups (Figure 2D).
Next, we explored Syk expression and Syk promoter methylation in HK2 cells.First, HK2 cells were stimulated with HG and oxLDL.In comparison with the NG and oxLDL groups, p-Syk protein expression was increased in the HG and oxLDL + HG groups (Figure 3A).Furthermore, the HG and oxLDL + HG groups exhibited Syk promoter hypomethylation (Figure 3B).Real-time quantitative PCR analysis revealed that Syk expression was also increased in the HG and oxLDL + HG groups (Figure 3C).This result was consistent with our in vivo experiments, confirming Syk promoter hypomethylation and a corresponding increase in Syk expression under HG and oxLDL + HG situations.

| DNA methylation modulated Syk expression through Sp1
In a search of the PROMO database, Sp1 was predicted to be a Syk transcription factor.Using JASPER, we predicted the Sp1 transcription factor binding site in Syk.We employed chip-qPCR to verify the enrichment of Sp1 in the Syk promoter region of mice (Figure 4A).mice compared with normal and ApoE −/− mice.Because a binding site for Sp1 was identified in the promoter region, we next examined Sp1 expression in all three groups of mice.Immunohistochemistry (Figure 4B), western blot (Figure 4C), and real-time quantitative PCR (Figure 4D) analyses revealed that ApoE −/− DM mice expressed Sp1 at a significantly higher level compared to control and ApoE −/− mice.In addition, we also found that enrichment of Sp1 was significantly increased in HK2 cells treated with HG + oxLDL (Figure 5A), and that Sp1 expression levels in HG and oxLDL + HG groups were higher than in NG and oxLDL groups (Figure 5B,C).Together, our in vivo and in vitro studies provide evidence that Sp1 was enriched at the syk promoter region.
To provide further validation that Sp1 regulates Syk, we silenced SP1 in HK2 cells using lentivirus.Western blot analysis revealed that Sp1 and p-Syk expression levels were inhibited in the SP1 silencing group (Figure 5D).This result demonstrates that silencing SP1 downregulated Syk expression.Next, the effects of Sp1 on oxidative stress and apoptosis were explored.We observed that GSH expression and COX-1 expression were downregulated and NOX expression was upregulated in the HG and oxLDL + HG groups, while GSH expression and COX-1 expression were upregulated and NOX was downregulated after SP1 silencing (Figure 6A,B).In addition, Bax and cleaved caspase-3 levels were increased, and the Bcl-2 level was decreased in the HG and oxLDL + HG groups compared to the NG and oxLDL + HG groups.Moreover, SP1 silencing inhibited apoptosis (Figure 6C).Together, these results demonstrate that silencing SP1 reduced ROS production and inhibited apoptosis in HK2 cells.| inhibitor improved renal function pathology in ApoE −/− DM mice Histological analysis revealed that Syk inhibitor (R788) treatment alleviated mesangial expansion, basement membrane thickening, and renal tubule injury (Figure 7A), as well as the ACR level (Supplementary Table S3), in comparison with untreated ApoE −/− DM mice.In scanning electron microscopy and transmission electron microscopy (TEM) images, R788 was observed to alleviate foot process fusion, basement membrane thickening, and mitochondrial dilation and swelling, and to increase the number of mitochondrial crest (Figure 7B,C).Moreover, R788 decreased the expression of Ngal (a marker of renal tubular epithelial cell injury) (Figure 7D).

| Syk inhibitors attenuated oxidative stress and apoptosis by downregulating the PKCβ/ P66shc pathway
Next, the effects of Syk inhibitor on oxidative stress and apoptosis were examined.We analyzed the expression levels of GSH, NOX, NDUFS1, GPx, and COX-1 in kidney tissues (Figure 8A,B), and found that GSH, NDUFS1, GPx, and COX-1 levels were upregulated in ApoE −/− DM mice treated with R788, while the level of NOX was downregulated.To investigate apoptosis TUNEL used to evaluate the number of apoptotic cells in different tissue sections (Figure 8C), and levels of the apoptosis-related proteins Bcl-2, Bax, and cleaved caspase-3 were detected by western blot (Figure 8D).While TUNEL staining was increased in the nuclei of renal tubules and glomeruli in ApoE −/− DM mice, TUNEL staining was decreased in ApoE −/− DM + R788 mice.
To further investigate the effects of Syk inhibitor on oxidative stress and apoptosis in renal tubular epithelial cells, HK2 cells were treated with R406.Compared with the NG and oxLDL groups, the HG and oxLDL + HG groups exhibited increased levels of NOX, Bax, and cleaved caspase-3, and decreased levels of NDUFS1, GPX, and Bcl-2.These changes were reversed by the administration of R406 (Figure 9A,B).Next, the mitochondrial membrane potential was evaluated using JC-1 (Figure 9C), and the results reveal that abnormal changes to the HK2 membrane potential were relieved after R406 administration.In addition, while reactive oxygen species (ROS) levels were increased after HG stimulation (an enhanced fluorescence signal from the reporter was observed), ROS levels were decreased following R406 treatment (Figure 9D).Finally, flow cytometry revealed that R406 treatment inhibited HG-induced apoptosis (Supplementary Figure S3).
Based on the observed expression changes in Syk and PKCβ/P66shc pathway proteins in DKD patients, we next examined the effect of Syk inhibitor on the PKCβ/P66shc pathway in ApoE −/− DM mice using western blot analysis (Figure 10A).Our results reveal that the expression levels of PKCβ and P66shc in ApoE −/− DM mice were decreased after treatment with R788.To further explore the involvement of the PKCβ/P66shc signaling pathway in oxidative stress and apoptosis, HK2 cells were treated with the PKC inhibitor LY333531 and the PKC agonist PMA.While the expression levels of NOX4, Bax, and cleaved 3 upregulated after PMA stimulation, levels of NDUFS1, GPX, and bcl-were downregulated (Figure 10B).Furthermore, the above protein expression level changes were reversed after administration of LY333531 (Figure 10B,C).Together, these results provide evidence that Syk inhibition attenuated oxidative stress and apoptosis in DKD by downregulating the PKCβ/ P66shc signaling pathway.Although evidence is emerging of the importance of epigenetic modification in the regulation of renal function, 21,22 our current understanding of the biological functions of Syk in the kidney is limited, and its role in epigeneticmediated renal regulation remains to be elucidated.In this study, we first revealed that DNA methylation regulates Syk expression through Sp1, and that Syk inhibitors protect against oxidative stress and apoptosis, at least in part through epigenetic regulation of PKCβ/P66shc signaling.
Dyslipidemia as a risk factor for accelerating the progression of DKD 23,24 has attracted widespread attention.ApoE is an important protein in lipid transport, and it is known to regulate lipid consumption and degradation.Indeed, ApoE deficiency can result in abnormal lipid deposition.In this study, we employed ApoE knockout mice to model lipid metabolism disorder in type 2 DM, and we injected these mice with STZ to construct a DKD model.Syk expression was significantly increased in ApoE −/− DM mice, which was consistent with the results of our clinical study.In contrast, no significant difference in expression was observed in −/− with the normal group).Moreover, no significant change in Syk expression was observed in HK2 cells after oxLDL treatment, suggesting that disturbances in lipid metabolism cannot increase Syk expression alone.Likewise, our clinical study did not reveal a correlation between Syk expression and high-fat levels.Thus, upregulation of Syk expression in renal tubules during DKD requires a contribution from other factors in addition to abnormal lipid metabolism.

| DNA methylation and Syk in DKD
Syk is known to mediate disease initiation in SLE, vasculitis, arthritis, and other diseases. 25In these previous studies, Syk was mainly expressed in tubular epithelial cells and glomeruli, and activated by upstream immune complexes.In our clinical study, Syk expression in the renal tubules of patients with DKD was strongly positive.We first investigated whether the observed upregulation of Syk expression in DKD was related to DNA methylation.DNA methylation is a marker of transcriptional inhibition, and a reduction of DNA methylation in CpG-rich promoters can induce an increase in gene expression. 26,27In previous studies, inhibition of DNA methylation was observed to activate Syk expression and subsequently inhibit breast cancer cell invasion. 28In addition, DNA methyltransferase 1 expression is known to be increased in diabetic mice. 29In our present study, we demonstrate that the Syk promoter region in ApoE −/− DM mice exhibited hypomethylation.Moreover, expression of the Syk gene was correspondingly increased, indicating that DNA methylation affects Syk expression in DKD.
Sp1 is a sequence-specific DNA-binding protein that directly binds to DNA to enhance gene transcription.This transcription factor is mainly expressed in the nucleus of glomeruli and proximal tubules, and there are clinical studies showing elevated Sp1 expression in the glomeruli and proximal tubules of patients with chronic glomerulonephritis (compared with normal human kidneys). 30In diabetic retinopathy, Sp1 is involved in highglucose-induced retinal endothelial cell migration. 31In a previous study on DKD, it was found that the miox promoter is hypomethylated in renal tubules, and that silencing SP1 inhibits the expression of miox. 32In our we found that Sp1 the Syk promoter region was DKD.Moreover, Syk expression was induced by high glucose and oxLDL, and downregulated by SP1 silencing, suggesting that Syk promoter methylation may regulate the expression of Syk through Sp1 in DKD.

| Syk inhibitors and PKCβ/P66shc pathway in DKD
ROS play an important role in cellular immune defense, cell growth, and necrosis.][35] Under the stimulation of high glucose, mitochondrialdependent respiration in HK2 cells is weakened, and ROS are produced in large quantities, leading to cell damage. 36Oxidative stress induced by glucose and lipid metabolism disorders can activate caspase-1, and ultimately the mitochondrial-dependent apoptosis pathway. 37Because changes in Syk expression were observed at both the epigenetic level and the protein level in DKD, we administered Syk inhibitor (R788) as an intervention.Renal pathological changes, oxidative stress, and apoptosis were all ameliorated in ApoE −/− DM + R788 mice (compared with ApoE −/− DM mice), providing evidence that Syk is a potential therapeutic target for DKD.In addition, while PKCβ and P66shc expression levels were elevated in ApoE −/− DM mice, R788 downregulated both PKCβ and P66shc expression levels.
PKCβ is known to promote DKD progression in several ways, including via regulation of endothelial permeability, vasoconstriction, extracellular matrix synthesis, cell growth, and cytokine activation. 38PKCβ-mediated phosphorylation of serine 36 in P66shc is considered to be a key regulatory component of mitochondrial translocation, ROS production, and apoptosis. 39,40P66shc, a member of the adaptor protein family, is the longest form of adaptor protein and uses electrons from the respiratory chain to oxidize cytochrome to form ROS, exerting its biological function in mitochondria. 41Studies have shown that hyperglycemia can induce PKC activation of P66shc in mitochondria, which in turn induces oxidative stress. 42,43PKCβ agonists can reverse the effects of Syk inhibitors in vitro.In the present study, oxidative stress and apoptosis were inhibited by PKC inhibitor treatment, and Syk inhibitor treatment ameliorated tubular oxidative stress and apoptosis in DKD by downregulating the PKCβ/P66shc pathway.We also found that SP1 silencing inhibited oxidative stress and apoptosis induced by high glucose and oxLDL.This result provides direct evidence that modulation of Syk promoter methylation may have therapeutic potential for DKD through targeting the PKCβ pathway.
There were several limitations to this study.First, we cannot exclude the possibility that other molecules downstream of Syk may also contribute to the regulation of cell function.For instance, studies have demonstrated that Syk is involved in JNK-dependent NLRP3 inflammasome activation in high-glucose-induced HK2 cells. 16Therefore, the role of JNK signaling, and that of other potential downstream targets epigenetically regulated by Syk in HK2 cells, should be clarified.Second, although our data suggest an important effect of Syk inhibition on renal tubular cells in this model, we did not attempt to explore the role of Syk-expressing glomeruli in the pathogenesis of DKD.Future studies should reveal the detailed mechanisms and crosstalk between tubules and glomeruli when using drugs that act on renal tubules to alleviate glomerular injury.
In conclusion, our study provides new insights into the molecular mechanisms underlying DKD and first highlights the crucial role of Syk in the pathogenesis of DKD.Targeting Syk may be a promising therapeutic strategy for the treatment of DKD.ethical standards of the and its later amendments.

F I G U R E 1
Upregulation of Syk, PKCβ, and P66shc in renal biopsies from patients with diabetic kidney disease (DKD).(A) Representative immunofluorescence staining and quantification of Syk expression in kidneys from DKD patients with grades I and II (DN I/II), DKD patients with grades III and IV (DN III/IV), and controls (CON).(B) Representative immunohistochemical and quantitative analysis of PKCβ and P66shc expression in kidneys from CON, DN I/II, and DN III/IV.Scale bars = 100 μm.Results are expressed as mean ± SE (n = 10); *p < .05versus control.

A
chromatin immunoprecipitation assay was performed using the Millipore EZ ChIP Assay (17-371, merck millipore).Chromatin was sheared by sonication to fragments of ~500 base pairs in length.The supernatant was precleared by incubation with Protein G Agarose and incubated overnight at 4°C with either anti-Sp1 antibody or nonimmune IgG.The resulting complexes were recovered from protein G magnetic beads, the cross-links were F I G U R E 2 Expression of Syk and Syk promoter methylation in different groups of mice.(A) Representative immunofluorescence images showed the expression of p-Syk in the kidneys of different groups of mice.Scale bars = 50 μm.(B) Representative western blot gel documents and summarized data showing the relative protein level of p-Syk.(C) Amplification and sequencing after bisulfite treatment of DNA showed methylation levels of the Syk promoter region in the kidneys of different groups of mice.(D) Real-time quantitative PCR analyses of Syk expression.Results are expressed as mean ± SE (n = 6) *p < .05versus control and ApoE −/− mice.

2. 16 |
Reactive oxygen species or JC-1 detection ROS accumulation was quantified using the DCFH-DA kit (D-1002, US Everbright Inc) or JC-1 (J6004, US Everbright Inc) according to the manufacturer's instructions.The medium was removed and DCFH-DA or JC-1 working solution was added to cover the cell surface and placed in a 37°C cell incubator and incubated for 20 min, the staining solution was removed and the cells were washed once with PBS buffer or medium.The culture medium was added again.The live cells were observed and analyzed with fluorescence microscope.F I G U R E 3 Expression of Syk and Syk promoter methylation in HK2 cells stimulated with different conditions.(A) Representative western blot gel documents and summarized data showing the relative protein levels of p-Syk in HK2 cells with different treatments.(B) Amplification and sequencing after bisulfite treatment of DNA showed methylation levels of the Syk promoter region in HK2 cells with different treatments.(C) Real-time quantitative PCR analyses of Syk expression in HK2 cells with different treatments.Results are expressed as mean ± SE (n = 6) *p < .05versus control and oxLDL groups.

F I G U R E 4
Expression of Sp1 in mice from each group.(A) Chip-qPCR was performed to detect the enrichment of antibody Sp1 in the promoter region of Syk.(B) Representative immunohistochemistry images showed the expression of Sp1 in the kidneys of different groups of mice.Scale bars = 50 μm.(C) Western blot gel documents and summarized data showing the expression of Sp1 protein.(D) Real-time quantitative PCR analyses of SP1 expression in different groups.Results are expressed as mean ± SE (n = 6) *p < .05versus control and ApoE −/− mice.
Thus, Sp1 enrichment was significantly increased in ApoE −/− DM F I G U R E 5 Silencing SP1 in HK2 cells downregulated syk expression.(A) Chip qPCR detected Sp1 enrichment at the promoter region in HK2 cells.(B) Representative immunofluorescence staining and quantification of Sp1 in HK2 cells with different treatments.Scale bars = 50 μm.(C) Real-time quantitative PCR analyses of SP1 expression in HK2 cells with different treatments.(D) Representative western blot gel documents and summarized data showing the expression of Sp1 and p-Syk protein in HK2 cells with different treatments.Results are expressed as mean ± SE (n = 6).*p < .05versus control and oxLDL groups.

F
I G U R E 6 Silencing SP1 decreased oxidative stress and apoptosis of HK2 cells under oxLDL combined with high-glucose stimulation.(A) The activity levels of GSH, NOX, and COX1 were detected by kits.(B) Representative western blot gel documents and summarized data showing the protein levels of NOX, NDUFS1, and GPX.(C) Representative western blot gel documents and summarized data showing the expression levels of Bcl-2, Bax, and cleaved caspase-3 protein in HK2 cells.Results are expressed as mean ± SE (n = 6) *p < .05versus control and oxLDL groups.

F I G U R E 7
Histopathology and ultrastructural changes of mice in each group.(A) Representative HE staining, PAS staining, and Masson staining images shows pathological and morphological changes in the kidneys of each group.Black arrow shows mesangial expansion in ApoE −/− DM mice.White arrow shows glomerular basement membrane thickening in ApoE −/− DM mice.(B) Scanning electron microscopy shows the structure of glomerular surface; white lines frame the fusion of the foot processes.(C) Transmission electron microscopy reveals the glomerular basement membrane (GBM) thickness, foot process width, the number of foot processes, tubular mitochondria, and abundant mitochondria in tubules in different groups of mice.Red arrow indicates the foot process fusion and blue arrow indicates significant GBM thickening in ApoE −/− DM mice.(D) Representative immunohistochemical and quantitative analysis of Ngal in different groups of mice.Results are expressed as mean ± SE (n = 6) *p < .05versus control and ApoE −/− mice.Scale bars = 50 μm.

F I G U R E 8
Effects of Syk inhibitor on oxidative stress and apoptosis in diabetic mice.(A) The levels of NOX, GSH, and COX1 activities were measured by kits.(B) Representative western blot gel documents and summarized data showing the relative protein levels of NOX4, NDUFS1, and GPx in different groups of mice.(C) TUNEL assay for tissue apoptosis.Scale bars = 50 μm.(D) Representative western blot gel documents and summarized data showing the expression of Bcl-2, Bax, and cleaved Caspase-3in the kidneys of different groups of mice.Results are expressed as mean ± SE (n = 6) *p < .05versus control and ApoE −/− mice.

F I G U R E 9
Effects of Syk inhibitor on oxidative stress and apoptosis in HK2 cells with different treatments.(A) Representative western blot gel documents and summarized data showing the relative protein levels of NDUFS1, NOX, and GPX in HK2 cells with different treatments.(B) Representative western blot gel documents and summarized data showing the relative protein levels of Bcl-2, Bax, and cleaved caspase-3 in HK2 cells with different treatments.(C) Detection of mitochondrial membrane potential by JC-1in HK2 cells with different treatments.(D) DCFH-DA labeled reactive oxygen species in HK2 cells with different treatments.Results are expressed as mean ± SE (n = 6) *p < .05versus control and oxLDL groups.Scale bars = 50 μm.

F I G U R E 1 0
Syk inhibitors modulate oxidative stress and apoptosis in diabetic nephropathy through the PKCβ/P66 pathway.(A) Representative western blot gel documents and summarized data showing the expression of PKCβ and P66shc in the kidneys of different groups.Results are expressed as mean ± SE (n = 6).*p < .05versus control and ApoE −/− mice.(B) Representative western blot gel documents and summarized data showing the relative protein levels of NOX, NDUFS1, and GPX in HK2 cells with different treatments.(C) Representative western blot gel documents and summarized data showing the relative protein levels of Bcl-2, Bax, and cleaved caspase-3 in HK2 cells.Results are expressed as mean ± SE (n = 6) *p < .05versus control and oxLDL groups.
Correlation analysis of Syk, PKC, and P66shc expression with clinicopathological parameters in diabetic kidney disease patients.
T A B L E 1Note: A two-tailed p < .05 was considered statistically significant.Abbreviations: ACR, albumin/creatinine ratio; eGFR, estimated glomerular filtration rate; FBG, fasting blood sugar; IFTA, interstitial fibrosis and tubular atrophy.

2.17 | Statistical analyses
Data are presented as the mean ± SD (unless stated otherwise).SPSS software (version 21, IBM Corp., NY, USA) was used for statistical analysis.Comparisons among groups were analyzed by one-way analysis of variance (ANOVA).The chi-square test was used for analyzing categorical variables.Pearson or Spearman correlation analysis was performed for clinical and renal pathological indexes.Differences with a p value <.05 were considered statistically significant.