GRAPHICAL ABSTRACT

Highlights

• PUM2 expression decreased and HDAC9 increased in DM-CIAKI.

• PUM2 knockdown or HDAC9 upregulation aggravated kidney injury in DM-CIAKI.

• PUM2 inhibited the stability of HDAC9 mRNA and reduced HDAC9 expression.

• PUM2 regulated oxidative stress and autophagy in DM-CIAKI by reducing HDAC9.

INTRODUCTION

With the wide application of clinical contrast media (CM)-related examination techniques such as enhanced computed tomography and vascular diagnosis, the incidence of contrast-induced acute kidney injury (CIAKI) has been increasing and has gradually attracted clinical attention [1,2]. Currently, CIAKI is reported as the third reason for hospital-acquired renal failure [3]. After receiving iodinated contrast agents, about 30% of patients developed acute kidney injury (AKI) [4]. Furthermore, there was evidence supporting that diabetes mellitus (DM) was a risk factor for CIAKI [5]. It has been reported that patients with kidney injury, especially those with DM-associated nephropathy, have a significantly increased incidence of CIAKI [6]. However, the pathogenesis of CIAKI is relatively complex, and current interventions have very limited effects [7]. Thus, the underlying molecular mechanism of CIAKI companied with DM deserves further exploration.

The gene of pumilio RNA binding family member 2 (PUM2) could encode a protein, which belongs to an RNA-binding protein (RBP) [8]. PUM2 was reported to be implicated in various diseases, such as osteoporosis, glioma and acute ischemic kidney injury [9-11]. Wang et al. [10] suggested that PUM2 expression was evidently decreased in acute ischemic kidney injury and PUM2 overexpression exerted protective effects on renal tubules via improving mitochondrial quality, which results indicated PUM2 probably play a crucial role in CIAKI companied with DM. At present, the role of PUM2 in AKI, especially CIAKI companied with DM, is still in a blank stage, and it is urgent to further elucidate the role of PUM2 and the molecular regulatory mechanism behind it in CIAKI companied with DM.

RBPs, which were widely investigated in multiple diseases, could mediate various post-transcriptional features, such as affecting mRNA stability, function, and cellular localization to participate in the progression of diseases [12]. For instance, PUM1, a RBP, mediated a negative impact on the stability of p21 expression and the expression of p21 in colorectal cancer [13]. As previously documented, PUM2 decreased mitochondrial fission factor (Mff) expression through interacting with 3’ untranslated region (3’ UTR) of Mff mRNA in acute ischemic kidney injury [10]. Besides, PUM2 could negatively regulate distal-less homeobox 5 (DLX5) expression through binding to DLX5 mRNA, thereby mediating osteoporosis progression [11]. Therefore, we are eager to find the targeted gene, which could bind to RBP PUM2 to affect gene expression, participating in the progression in CIAKI companied with DM.

Histone deacetylases (HDACs) deacetylate histones, change the static properties of chromatin, and facilitate gene suppression [14]. Previous research suggested that HDACs played multiple roles in kidney development and pathogenesis of kidney disease [15]. Currently, HDACs are grouped into types I, II, III, IV, and histone deacetylase 9 (HDAC9), a member of the II HDACs, was implicated in tumors, inflammation, atherosclerosis, and diabetic nephropathy through affecting targeted genes [16-18]. Some HDACs such as HDAC2 and HDAC5 were reported to play a harmful role in septic AKI [19]. In addition, there was evidence supporting HDAC9 inhibition exerted a suppressing role in diabetic nephropathy [20], implying HDAC9 is probably implicated in CIAKI companied with DM. Thus, we desire to provide more evidence about the role and underlying mechanism of HDAC9 in CIAKI companied with DM.

Oxidative stress and autophagy were reported to be intimately related to CIAKI [21]. Some studies supported PUM2 and HDAC9 could regulate oxidative stress and autophagy in diseases [22-25]. Therefore, standing on these backgrounds, we put forward our hypothesis that PUM2 attenuates oxidative stress and stimulates autophagy to alleviate renal injury of DM-CIAKI through interacting with HDAC9 mRNA and reducing HDAC9 mRNA stability and HDAC9 expression. Our findings will provide more targeted genes for CIAKI companied with DM.

METHODS

Mouse models of diabetes

C57BL/6J male mice (4 weeks) were acquired from Hunan Slack King Experimental Animal Company (Changsha, China). To establish a DM model, a high-fat diet was used for feeding mice for 4 weeks. Afterwards, 50 mg/kg streptozotocin (STZ) was intraperitoneally injected into mice for 5 consecutive days. After 3 days, if blood sugar was above 200 mg/dL, the mice were diagnosed with diabetes. STZ-induced mice were kept for an additional 4 to 5 weeks before administration of contrast medium. Of note, animal experiments in this study were approved by the ethics committee of the Second Xiangya Hospital (approval no.: 20240280).

CIAKI model

To achieve different experimental purposes, the mice were divided into several groups and treated differently. (1) Mice were divided into four groups (six mice/each group) including control, DM, CIAKI, and DM-CIAKI. (2) PUM2 knockout (PUMKO) mice were purchased from Cyagen (catalog number: S-KO-18016; Suzhou, China, https://www.cyagen.cn/micebank/S-KO-18016). PUM2-wild type (WT) and PUM2-KO mice were fallen into PUM2-WT, PUM2-KO, DM-CIAKI+PUM2-WT, and DM-CIAKI+PUM2-KO (six mice/each group). (3) Mice were divided into five groups (six mice/each group) including control, DM, CIAKI, DM-CIAKI, and DM-CIAKI+HDCAi. (4) Mice were divided into three groups (six mice/each group) including control, DM-CIAKI+small interfering negative control (si-NC), and DM-CIAKI+si-HDAC9. As described previously [26], CIAKI model in mice was established. Shortly, after 16 hours without water, NG-nitro-L-arginine methyllester (L-NAME, 10 mg/kg intraperitoneally) was injected into mice to inhibit nitric oxide synthase. After 15 and 30 minutes, the prostaglandin synthesis inhibitor (indomethacin, 10 mg/kg intraperitoneally) was injected into the mice, and the low-permeability monoiodized contrast agent Omnipaque (iodine 3.0 g/kg; GE Healthcare Inc., Marlborough, MA, USA) was injected into the mice through the tail vein, respectively. The mice in control group were not given Omnipaque (3.0 g iodine/kg) but were replaced with saline. Other treatments were consistent with the experimental group. After iohexol or saline injection, mice foraged and drank freely for 24 hours. To investigate the influences of BRD4354, an HDAC9 inhibitor (HDACi), after 20 minutes of intraperitoneally injection of L-NAME and indomethacin, BRD4354 was intraperitoneally injected into mice at a dose of 10 mg/kg. In addition, to silence HDAC9, a total of 100 μL lentivirus (1×10⁵ transduction units [TU]/μL) carrying si-HDAC9 was injected into renal tissues of mice. Finally, contrast agent Omnipaque was injected into the mice. After 48 hours, the needed samples were collected.

Immunofluorescence assay

Tissue samples and human kidney-2 (HK-2) cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. After blocking with 5% bovine serum albumin, the primary antibodies against PUM2 (PA5-56436, Thermo Fisher Scientific, Waltham, MA, USA) and light chain 3B (LC3B; PA5-32254, Thermo Fisher Scientific) were applied to incubate samples overnight at 4°C. The secondary antibodies were added, and 4ʹ,6-diamidino-2-phenylindole (DAPI) stained cell nucleus. The images were pictured using a fluorescence microscope and fluorescence intensity was analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA).

Intracellular reactive oxygen species detection

To evaluate reactive oxygen species (ROS) levels in renal tissues of mice, fluorescent probe dihydroethidium (DHE) was used in this study. In short, the renal tissues from mice were sectioned into 2 μm thick frozen slices. These sections were then incubated with a 10 μM solution of the fluorescent probe DHE at 37°C for 1 hour in the dark. Following incubation, the stained renal tissues were examined and visualized using a fluorescent microscope (excitation: 515 nm; emission: 585 nm).

To assess ROS levels in HK-2 cells, a 2ʹ,7ʹ-dichloroflfluorescein diacetate (DCFH-DA) (Biosharp, Seattle, WA, USA) was used for ROS detection. HK-2 cells were cultured on 6-well plates overnight. The cells were collected and stained with 10 μM DCFH-DA in the dark for 30 minutes. Fluorescence intensity (excitation: 488 nm; emission: 525 nm) was measured using a fluorescent microscope.

Cell treatment

HK-2 cells, a human kidney cell line, were purchased from American Type Culture Collection (Manassas, VA, USA). All cells were cultured in DMEM/F12 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 100 U/mL penicillin at 37°C in a humidified atmosphere of 5% CO₂.

To induce a cell model of DM, different concentrations of 5.5, 30, and 50 mM D-glucose were used to treat HK-2 cells for 48 hours. To induce a cell model of AKI with iohexol, 150 mg/mL iohexol [27] was treated HK-2 cells for 0, 2, 4, and 6 hours. To inhibit the effect of HDAC9 on HK-2 cells, 2 μmol/L BRD4354 [28] was used to treat HK-2 cells for 2 hours. To suppress autophagy, 5 mM 3-methyladenine (3-MA) was added into HK-2 cells for 24 hours.

Cell transfection

Small interfering RNA targeting HDAC9 (si-HDAC9), overexpressing plasmid of PUM2 (ov-PUM2) or HDAC9 (ov-HDAC9) and short hairpin targeting PUM2 (sh-PUM2) along with correspondent controls (si-NC, ov-NC, sh-NC) were purchased from GenePharma (Shanghai, China). HK-2 cells were implanted onto 6-well plates and incubated overnight. HK-2 cells were transfected with plasmids for 48 hours using Lipo-fectamine 3000 (Invitrogen, Waltham, MA, USA) following the instructions. Of note, the sequences of sh-PUM2, si-HDAC9, sh-NC, and si-NC were presented in Supplementary Table 1.

RNA immunoprecipitation assay

A magna RNA immunoprecipitation (RIP) RNA-Binding Protein Immunoprecipitation Kit (Millipore, Burlington, MA, USA) was employed to conduct RIP assay. HK-2 cells (about 1×10⁷ cells) were lysed with RIP Lysis Buffer for 5 minutes. The supernatants were obtained after centrifugation. The magnetic beads conjugated with antibodies anti-PUM2 (ab92390, Abcam, Cambridge, UK) or immunoglobulin G (IgG; ab172730, Abcam) were added into the supernatants and incubated overnight at 4°C. IgG acted as the control. Immune-precipitated HDAC9 mRNA was determined using real-time quantitative reverse transcription polymerase chain reaction and the sequences of primers were shown in Supplementary Table 2.

RNA pull-down assay

Approximately 4×10⁷ HK-2 cells were collected and lysed in 1 mL of radio-immunoprecipitation assay (RIPA) buffer. The lysates were centrifuged at 13,000 rpm for 10 minutes at 4°C. Biotinylated RNA probes specific for HDAC9 were incubated with the lysates for 4 hours at 37°C. Subsequently, the lysate was incubated with streptavidin-coated magnetic beads (Invitrogen) at 4°C overnight. After washing the beads five times with wash buffer, The PUM2 enrichment by the biotinylated RNA probe was examined by Western blot.

Statistical analysis

All data were expressed as mean±standard error of the mean and data of this study were analyzed using SPSS version 16.0 statistical software (SPSS Inc., Chicago, IL, USA). Student’s t-test was utilized for comparison between groups. The comparisons of equal to or greater than three groups were analyzed by one-way analysis of variance. P<0.05 was considered statistically significant. Each experiment was repeated at least three times. Some methods were described in the Supplementary Methods.

RESULTS

PUM2 expression was abnormally downregulated in DMCIAKI mice while HDAC9 expression was abnormally elevated

CIAKI was reported to achieve a worse prognosis in DM patients [29]. To investigate crucial molecules in DM-CIAKI, we established a model of CIAKI in STZ-induced mice and in normal mice. The detailed groups were control, DM, CIAKI, and DM-CIAKI. As presented in Supplementary Fig. 1A, compared with control mice, the ratio of kidney weight/body weight (KW/BW) and the levels of serum creatinine (SCr), 24-hour urinary protein, blood urea nitrogen (BUN), fasting blood glucose (FBG), and glycated serum protein (GSP) were markedly higher in DM or CIAKI or DM-CIAKI mice, especially in DM-CIAKI mice. In addition, hematoxylin and eosin (HE) staining exhibited the obvious vacuolar degeneration, fragmented cells and dilatation of lumen in kidney tissue sections of DM or CIAKI or DM-CIAKI mice, especially in DM-CIAKI mice (Supplementary Fig. 1B). Encouragingly, PUM2 expression was evidently reduced while HDAC9 was apparently enhanced in DM, CIAKI, and DM-CIAKI mice. It was noted that the reduction of PUM2 expression and the elevation of HDAC9 expression were most obvious in DM-CIAKI mice (Supplementary Fig. 1C and D). Taken together, PUM2 and HDAC9 probably play crucial roles in DM-CIAKI.

PUM2-KO enhanced oxidative stress in DM-CIAKI mice

To further probe the influences of PUM2 on DM-CIAKI, PUM2-KO mice were applied. Firstly, immunofluorescence assay revealed that PUM2 expression in mice with PUM2-KO was greatly declined compared to WT mice (Fig. 1A), indicating a successful PUM2 knockdown in mice. Then, mice with/without PUM2-KO mice were grouped into PUM2-WT, PUM2-KO, DM-CIAKI+PUM2-WT, and DM-CIAKI+PUM2-KO. Fig. 1B indicated that PUM2 expression was declined in PUM2-KO, DM-CIAKI+PUM2-WT, and DM-CIAKI+PUM2-KO groups relative to PUM2-WT and was lowest in DM-CIAKI+PUM2-KO group among groups. Compared to DM-CIAKI+PUM2-WT group, the ratio of KW/BW and the levels of SCr, 24-hour urinary protein, BUN, FBG, and GSP in mice were dramatically enhanced by PUM2-KO (Fig. 1C). Besides, PUM2-KO further aggravated the injury of kidney tissues in DM-CIAKI+PUM2-WT group (Fig. 1D). In addition, DM-CIAKI induction could promote the levels of malondialdehyde (MDA) and ROS and decrease the levels of glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) in PUM2-WT mice, which were further strengthened by PUM2-KO (Fig. 1E and F). Moreover, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) displayed more cell apoptosis in DM-CIAKI+PUM2-WT group compared to PUM2-WT, which was further elevated by PUM2-KO (Fig. 1G). Notably, as presented in Fig. 1C-G, PUM2-KO failed to change the ratio of KW/BW and the levels of SCr, 24-hour urinary protein, BUN, FBG, and GSP, renal morphology, the levels of MDA, ROS, GSH-PX, and SOD, cell apoptosis in mice of PUM2-WT group. Collectively, PUM2-KO could further deteriorate injury and oxidative stress in DM-CIAKI mice.

HDAC9 inhibitor or HDAC9 knockdown suppressed oxidative stress and alleviated the injury in DM-CIAKI mice

Here, to explore the impacts of HDAC9 on DM-CIAKI. DM-CIAKI mice were prepared and then administrated with BRD-4354, an inhibitor of HDAC9 or lentivirus carrying si-HDAC9. As indicated in Fig. 2A and B, Supplementary Fig. 2A, HDACi treatment or HDAC9 knockdown partially reversed DM-CIAKI-induced elevation of HDAC9 expression in mice. Besides, DM-CIAKI-induced enhancement of the ratio of KW/BW and the levels of SCr, 24-hour urinary protein, BUN, FBG, and GSP in mice was compromised by HDACi treatment or HDAC9 knockdown (Fig. 2C, Supplementary Fig. 2B). As expected, HDACi treatment or HDAC9 knockdown could improve morphological injury of kidney tissues in DM-CIAKI mice (Fig. 2D, Supplementary Fig. 2C). Moreover, elevated levels of MDA and ROS and decreased levels of GSH-PX and SOD in DM-CIAKI mice were abolished by HDACi treatment or HDAC9 knockdown (Fig. 2E and F, Supplementary Fig. 2D and E). Furthermore, HDACi treatment or HDAC9 knockdown attenuated cell apoptosis in DM-CIAKI mice (Fig. 2G, Supplementary Fig. 2F). In total, HDAC9 inhibitor or HDAC9 knockdown alleviated kidney injury and inhibited oxidative stress and tissue apoptosis in DM-CIAKI mice.

HDAC9 silencing improved high glucose and CM treatments-induced oxidative stress and apoptosis of HK-2 cells

Subsequently, HK-2 cells were subjected to a series of high glucose (HG) concentrations (5.5, 30, and 50 mM). As presented in Fig. 3A, HG treatments promoted HDAC9 expression in a concentration-dependent form. In addition, under 50 mM glucose condition, iohexol was used to treat HK-2 cells for 2, 4, and 6 hours. We found that iohexol (represented by CM in images) also elevated HDAC9 expression in HG-induced HK-2 cells in a time-dependent approach (Fig. 3B). Furthermore, HK-2 cells underwent CM (iohexol), HG+CM and HG+CM+HDACi (BRD4354) treatments. As shown in Fig. 3C, HG or/and CM apparently decreased cell viability and the combination of HG and CM acquired the most inhibition. However, HDACi partially reversed the suppressing influence caused by HG and CM treatments. Moreover, HG or/and CM evidently enhanced ROS level in HK-2 cells, especially the combination of HG and CM, but HDACi compromised the elevated ROS level caused by the combination of HG and CM treatments (Fig. 3D). Afterwards, si-HDAC9-1, si-HDAC9-2, and si-HDAC9-3 was used to knock down HDAC9 expression in HK-2 cells. si-HDAC9-3 transfection resulted in the most obvious downregulation of HDAC9 expression (Fig. 3E) and it was selected as subsequent experiments. si-HDAC9-3-treanfected HK-2 cells were treated with the combination of HG and CM. Fig. 3F displayed that HDAC9 knockdown abolished the combination of HG and CM treatments-mediated enhancement of HDAC9 expression. As expected, decreased cell viability, increased ROS production and cell apoptosis caused by the combination of HG and CM were compromised by HDAC9 knockdown (Fig. 3G-I). Collectively, HDAC9 knockdown alleviated oxidative stress and decreased cell apoptosis in HG and CM-treated HK-2 cells.

PUM2 attenuated stability of HDAC9 mRNA and reduced HDAC9 expression

From the above results, PUM2 and HDAC9 were implicated in DM-CIAKI. Therefore, we suspected that there may be some interaction between the two. PUM2 is an RBP, which was reported to regulate the mRNA stability of targeted genes [30,31]. Further experiments including RIP and RNA-pulldown validated the interaction between PUM2 and HDAC9 (Fig. 4A and B). In detail, PUM2 antibody successfully enriched HDAC9 mRNA and HDAC9 probe successfully pulled down PUM2 protein (Fig. 4A and B). Besides, PUM2 knockdown led to increased HDAC9 expression and PUM2 overexpression acquired opposite result (Fig. 4C and D). Furthermore, PUM2 knockdown promoted the stability of HDAC9 mRNA and PUM2 overexpression attenuated the stability of HDAC9 mRNA (Fig. 4E). Taken together, PUM2 overexpression suppressed HDAC9 mRNA stability and reduced HDAC9 expression through interacting with each other.

HDAC9 overexpression abolished PUM2 upregulation-mediated alleviation of cell injury and suppression of oxidative stress in HG and CM treatments-induced HK-2 cells

To probe the role of PUM2/HDAC9 axis in DM-CIAKI, HK-2 cells were subjected to ov-PUM2 or/and ov-HDAC9 under HG and CM treatments. The detailed groups were control, HG+CM+ov-NC, HG+CM+ov-PUM2, HG+CM+ov-HDAC9, and HG+CM+ov-PUM2+ov-HDAC9. As for PUM2 and HDAC9 expression, PUM2 overexpression promoted PUM2 expression and inhibited HDAC9 expression in HG and CM treatmentsinduced HK-2 cells. However, HDAC9 overexpression merely increased HDAC9 expression and partially reversed the inhibitory effect of PUM2 overexpression on HDAC9 expression (Fig. 5A and B). In addition, PUM2 overexpression resulted in increased cell viability and proliferation and HDAC9 overexpression acquired opposite results in HG and CM treatmentsinduced HK-2 cells. It was noted that HDAC9 overexpression abolished PUM2 overexpression-induced promoting influences on cell viability and proliferation (Fig. 5C and D). Furthermore, ROS production and cell apoptosis were inhibited by PUM2 overexpression in HG and CM treatments-induced HK-2 cells. However, HDAC9 overexpression obtained contrary results and compromised PUM2 overexpression-mediated suppressing impacts on ROS production and cell apoptosis (Fig. 5E and F). In total, PUM2 upregulation improved cell injury and suppressed oxidative stress in HK-2 cells upon HG and CM treatments through decreasing HDAC9 expression.

The dysregulation of autophagy was observed in DMCIAKI mice and was regulated by HDAC9 and PUM2

Growing evidence has demonstrated autophagy was closely related to AKI including contrast-induced AKI [32]. Therefore, we detected the expression of LC3BII/I and p62, which were identified as indicators of autophagy, in various groups including control, DM, CIAKI, DM-CIAKI, and DM-CIAKI+HDACi. Our results exhibited that, in DM, CIAKI, DM-CIAKI groups, the ratio of LC3BII/I was evidently decreased while p62 expression was apparently increased, especially in DM-CIAKI group. However, HDACi treatment could abolish DM-CIAKI-mediated the reduction of LC3BII/I ratio and elevation of p62 expression in mice (Fig. 6A). Besides, lentivirus carrying si-HDAC9 was injected into DM-CIAKI mice and lentivirus carrying si-HDAC9 increased the ratio of LC3BII/I and decreased p62 expression (Fig. 6B). Subsequently, we investigated how PUM2 influences the indicators of autophagy. PUM2-WT and PUM2-KO mice received DM-CIAKI treatments. PUM2 knockdown could further strengthen DM-CIAKI-mediated the reduction of LC3BII/I ratio and elevation of p62 expression in mice (Fig. 6C). In total, autophagy was suppressed in DM-CIAKI mice, which was regulated by HDAC9 and PUM2 in vivo.

HDAC9 downregulation inhibited oxidative stress and apoptosis caused by HG and CM induction in HK-2 cells through promoting autophagy

Here, some experiments were designed on the effects of autophagy on HDAC9 downregulation-mediated cell viability, oxidative stress and apoptosis in HG and CM treatments-induced HK-2 cells. HK-2 cells were transfected with si-HDAC9 and followed HG, CM and 3-MA (an inhibitor of autophagy) treatments. The detailed groups were control, HG+CM+si-HDAC9, and HG+CM+si-HDAC9+3-MA. Our results indicated that HG and CM treatments suppressed LC3BII/I ratio and enhanced p62 expression, whereas HDAC9 knockdown reversed these alterations. However, 3-MA could abolish HDAC9 knockdown-mediated elevation of LC3BII/I ratio and reduction of p62 expression in HG and CM treatments-induced HK-2 cells (Fig. 6D). As expected, 3-MA offset HDAC9 knockdown-mediated promotion of LC3B expression (Fig. 6E). Furthermore, HDAC9 knockdown promoted cell viability and inhibited ROS production and cell apoptosis in HG and CM treatments-induced HK-2 cells, which were offset by 3-MA treatment (Fig. 6F-H). Taken together, HDAC9 silencing suppressed oxidative stress and apoptosis in HG and CM treatments-induced HK-2 cells via promoting autophagy.

DISCUSSION

Various mechanisms were related to renal toxicity of CM, including renal medullary hypoxia, oxidative stress, direct toxicity of contrast agents, inflammation, apoptosis, immunity and autophagy [33-35]. At present, mounting evidence has linked oxidative stress and autophagy to AKI including CIAKI in the context of DM [36,37]. Rapamycin, an autophagy inducer, could suppress inflammatory response and oxidative stress and alleviate renal injury in iodixanol-induced DM rats [37]. In this study, our findings suggested that PUM2 inhibited oxidative stress and promoted autophagy in mice with DM-CIAKI and in HG and CM treatments-induced HK-2 cells through weakening HDAC9 mRNA stability and reducing HDAC9 expression, thereby alleviating AKI.

PUM2 is a member of FBF (PUF) family, which is extensively investigated in multiple biological activities including cell viability, apoptosis, oxidative stress and autophagy [22,23,38]. For example, PUM2 participated in cell viability and apoptosis of various types of cancers [38,39]. It was found that PUM2 overexpression improved the behavioral and cognitive changes of subarachnoid hemorrhage (SAH) mice by inhibiting SAH-induced oxidative stress and neuronal apoptosis [23]. D’Amico et al. [22] indicated that PUM2 mediated mitophagy in aging. More importantly, PUM2 was reported to affect mitochondrial quality in acute ischemic kidney injury through inhibiting Mff expression [10]. Besides, massive investigations have demonstrated that promoting autophagy exerted a protective role in contrast-induced AKI [32,40]. For instance, αKlotho protein alleviated contrast-induced AKI through suppressing NLR family pyrin domain containing 3 (NLRP3) inflammasome-mediated pyroptosis and motivating autophagy [32]. However, how PUM2 affects oxidative stress and autophagy in DMCIAKI has not been expounded. In this study, we firstly established cell and mouse models of DM-CIAKI and then we observed that PUM2 expression was evidently inhibited in models of DM-CIAKI. In addition, DM-CIAKI induced oxidative stress and inhibited autophagy and PUM2-KO could further strengthen DM-CIAKI-mediated these effects in mice. Moreover, HG and CM treatments elevated oxidative stress and attenuated cell autophagy in HK-2 cells; however, PUM2 overexpression attenuated HG and CM treatments-generated influences on oxidative stress and autophagy. Our findings first elaborated the effects of PUM2 on oxidative stress and autophagy in DM-CIAKI.

It was worth noting that PUM2, a RBP, can bind to the 3’UTR of a specific targeted mRNA to block the formation of the translation initiation complex, thereby inhibiting the expression of target genes [30,31]. Therefore, in this study, PUM2 is considered to be a transcriptional suppressor. To date, more than 1,000 mRNAs have been identified to harbor PUM2-binding moieties that contain mRNAs associated with autophagy, suggesting that PUM2 was a post-transcriptional regulator of these genes [22,41]. As previous documented, Janus kinase 2 (JAK2), Runt-related transcription factor 2 (RUNX2), insulinoma-associated protein 1 (INSM1), and peroxiredoxin 6 (PRDX6) were identified as targeted genes of PUM2 [42-44]. Tao et al. [43] revealed that PUM2 facilitated INSM1 mRNA degradation to reduce INSM1 expression, thereby participating in malignant features of breast cancer. In current study, we predicted that PUM2 had the binding site on 3’UTR of HDAC9 and we further validated the interaction between PUM2 and HDAC9. Further experiments exhibited that PUM2 overexpression accelerated degradation of HDAC9 mRNA and reduced HDAC9 expression, while PUM2 silencing achieved opposite results. Thus, our results indicated that HDAC9 was a targeted gene of PUM2.

HDACs were reported to be implicated in AKI [45]. A review revealed that class IIa HDACs (4, 5, 7, 9) inhibition alleviated AKI through inhibiting renal tubular cell apoptosis and promoting autophagy [46]. Wang et al. [47] proposed that HDAC2/4/5 expression was enhanced in kidneys of STZ-induced rats, diabetic db/db mice and diabetic patients. Besides, HDAC9 upregulation contributed to podocyte injury and developed glomerulosclerosis in diabetic nephropathy mice [17]. Furthermore, Gene Expression Omnibus (GEO) data revealed that HDAC9 expression was enhanced in renal tissues of diabetic nephropathy patients [20]. In our study, HDAC9 expression was apparently enhanced in mice with DM-CIAKI and in HG and CM treatments-induced HK-2 cells. Besides, HDAC9 inhibitor and HDAC9 silencing obviously attenuated oxidative stress and promoted autophagy in mouse and cell models of DM-CIAKI. However, HDAC9 overexpression could enhanced oxidative stress and cell apoptosis in HG and CM treatments-induced HK-2 cells. Furthermore, HDAC9 overexpression could offset PUM2 overexpression-mediated oxidative stress inhibition and autophagy promotion in HG and CM treatments-induced HK-2 cells. Notably, HDAC9 downregulation-mediated oxidative stress inhibition and autophagy promotion could be abolished by 3-MA, an inhibitor of autophagy in HG and CM treatments-induced HK-2 cells. Taken together, HDAC9 could participate in DM-CIAKI development.

In conclusion, PUM2 expression was abnormally declined and HDAC9 expression was abnormally enhanced in models including cells and mice of DM-CIAKI. Furthermore, PUM2 suppressed oxidative stress and promoted autophagy to improve kidney injury caused by DM-CIAKI through attenuating HDAC9 mRNA stability and reducing HDAC9 expression. Our findings will provide new targets for DM-CIAKI.

SUPPLEMENTARY MATERIALS

NOTES

CONFLICTS OF INTEREST

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

AUTHOR CONTRIBUTIONS

Conception or design: W.C., H.L., L.H.

Acquisition, analysis, or interpretation of data: W.D., H.L., Y.C.

Drafting the work or revising: G.L.

Final approval of the manuscript: L.H.

FUNDING

This work was supported by the Natural Science Foundation of China (No.82470759 and 82000697), the Natural Science Foundation of Hunan Province (No.2024JJ3022), the Scientific Research Fund of Hunan Provincial Health Commission (B2023 03056777), the Hunan Provincial Natural Science Foundation for Outstanding Youth (No. 2022JJ10093).

ACKNOWLEDGMENTS

None

Fig. 1.

Pumilio RNA binding family member 2 (PUM2) knockdown enhanced oxidative stress in diabetes mellitus (DM)-contrast- induced acute kidney injury (CIAKI) mice. (A) PUM2 expression in kidney tissues was measured in PUM2-knockout (KO) mice and PUM-wild type (WT) mice using immunofluorescence assay. (B-G) PUM2-KO mice and PUM2-WT mice were treated with streptozotocin and iohexol. The detailed groups were as follows: PUM2-WT, PUM2-KO, DM-CIAKI+PUM2-WT, and DM-CIAKI+PUM2-KO. (B) PUM2 expression was examined in kidney tissues using real-time quantitative reverse transcription polymerase chain reaction. (C) The ratio of kidney weight/body weight (KW/BW) and the levels of serum creatinine, 24-hour urinary protein, blood urea nitrogen (BUN), fasting blood glucose (FBG), and glycated serum protein (GSP) were detected. (D) The morphology of kidney tissues was examined by hematoxylin and eosin staining. (E) Glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), and malondialdehyde (MDA) in kidney tissues were detected using commercial kits. (F) Reactive oxygen species production was evaluated using dihydroethidium (DHE) probe. (G) Cell apoptosis in kidney tissues was investigated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). ^aP<0.05, ^bP<0.01, ^cP<0.001.

Fig. 2.

Histone deacetylase 9 (HDAC9) knockdown suppressed oxidative stress and alleviated the injury in diabetes mellitus (DM)- contrast-induced acute kidney injury (CIAKI) mice. Mice were treated with streptozotocin and iohexol and then received lentivirus carrying small interfering (si) RNA targeting HDAC9 (si-HDAC9). The detailed groups were control, DM-CIAKI, and DMCIAKI+ si-HDAC9. (A, B) HDAC9 expression was detected using real-time quantitative reverse transcription polymerase chain reaction and Western blot. (C) The ratio of kidney weight/body weight (KW/BW) and the levels of serum creatinine, 24-hour urinary protein, blood urea nitrogen (BUN), fasting blood glucose (FBG), and glycated serum protein (GSP) were detected. (D) The morphology of kidney tissues was examined by hematoxylin and eosin staining. (E) Glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), and malondialdehyde (MDA) in kidney tissues were detected using commercial kits. (F) Reactive oxygen species production was evaluated using dihydroethidium (DHE) probe. (G) Cell apoptosis in kidney tissues was investigated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). NC, negative control. ^aP<0.05, ^bP<0.01, ^cP<0.001.

Fig. 3.

Continued. Histone deacetylase 9 (HDAC9) silencing improved high glucose (HG) and contrast media (CM) treatments-induced oxidative stress and apoptosis of human kidney-2 (HK-2) cells. (A) HDAC9 expression was measured in HK-2 cells with 5.5, 30, and 50 mM glucose using Western blot. Iohexol was used to incubate HK-2 cells for 2, 4, 6 hours, which was added with 50 mM glucose. (B) HDAC9 expression was measured using real-time quantitative reverse transcription polymerase chain reaction (RTqPCR). HK-2 cells were subjected to 50 mM glucose or/and 150 mg/mL iohexol and then received HDAC9 inhibitor (HDACi) treatment. (C) Cell viability was examined using Cell Counting Kit-8 (CCK-8). (D) Reactive oxygen species (ROS) production was evaluated using 2ʹ,7ʹ-dichloroflfluorescein diacetate (DCFH-DA) method. (E) HDAC9 expression was measured in HK-2 cells with small interfering HDAC9 (si-HDAC9)-1, 2, and 3 transfection using Western blot. HK-2 cells were transfected with si-HDAC9-3 and followed treatment with the combination of HG and CM. (F) HDAC9 expression was examined using RT-qPCR. (G) Cell viability was evaluated using CCK-8. (H) ROS production was evaluated using DCFH-DA method. (I) Cell apoptosis was investigated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). NC, negative control. ^aP<0.05, ^bP<0.01, ^cP<0.001.

Fig. 4.

Pumilio RNA binding family member 2 (PUM2) attenuated stability of histone deacetylase 9 (HDAC9) mRNA and reduced HDAC9 expression. (A, B) The interaction between PUM2 and HDAC9 was validated in human kidney-2 (HK-2) cells by RNA immunoprecipitation and RNA pull-down. Human kidney-2 (HK-2) cells were transfected with overexpressing plasmid of PUM2 (ov-PUM2) or short hairpin targeting PUM2 (sh-PUM2). (C, D) HDAC9 expression was evaluated using real-time quantitative reverse transcription polymerase chain reaction and Western blot. (E) HDAC9 mRNA stability was detected in HK-2 cells after actinomycin D using real-time quantitative reverse transcription polymerase chain reaction. IgG, immunoglobulin G; NC, negative control. ^aP<0.05, ^bP<0.01, ^cP<0.001.

Fig. 5.

Histone deacetylase 9 (HDAC9) overexpression abolished pumilio RNA binding family member 2 (PUM2) upregulationmediated alleviation of cell injury and suppression of oxidative stress in high glucose (HG) and contrast media (CM) treatmentsinduced human kidney-2 (HK-2) cells. HK-2 cells were transfected with overexpressing plasmid of PUM2 (ov-PUM2) or/and ov- HDAC9 and then followed with HG and CM treatments. (A, B) PUM2 and HDAC9 expression was evaluated using real-time quantitative reverse transcription polymerase chain reaction and Western blot. (C) Cell viability was evaluated using Cell Counting Kit-8 (CCK-8). (D) Cell proliferation was detected using 5-ethynyl-2ʹ-deoxyuridine (EdU). (E) Reactive oxygen species (ROS) production was evaluated using 2ʹ,7ʹ-dichloroflfluorescein diacetate (DCFH-DA) method. (F) Cell apoptosis was investigated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). NC, negative control. ^aP<0.05, ^bP<0.01, ^cP<0. 001.

Fig. 6.

Histone deacetylase 9 (HDAC9) downregulation inhibited oxidative stress and apoptosis caused by high glucose (HG) and contrast media (CM) induction in human kidney-2 (HK-2) cells through promoting autophagy. Mice were grouped into control, diabetes mellitus (DM), contrast-induced acute kidney injury (CIAKI), DM-CIAKI, and DM-CIAKI+HDAC9 inhibitor (HDACi). (A) Light chain 3B (LC3B) II/I and p62 expression in kidney tissues was evaluated using Western blot. DM-CIAKI mice were injected with lentivirus carrying small interfering HDAC9 (si-HDAC9). The detailed groups were control, DM-CIAKI+si-negative control (NC), and DM-CIAKI+si-HDAC9. (B) LC3BII/I and p62 expression in kidney tissues was evaluated using Western blot. Mice were fallen into pumilio RNA binding family member 2 (PUM2)-wild type (WT), PUM2-knockout (KO), DMCIAKI+ PUM2-WT, and DM-CIAKI+PUM2-KO. (C) LC3BII/I and p62 expression in kidney tissues was measured using Western blot. (D-G) HK-2 received indicated treatments and the detailed groups were control, HG+CM+si-NC, HG+CM+si-HDAC9, and HG+CM+si-HDAC9+3-methyladenine (3-MA). (D) LC3BII/I and p62 expression was measured using Western blot. (E) LC3B expression was investigated using immunofluorescence assay. (F) Cell viability was evaluated using Cell Counting Kit-8 (CCK-8). (G) Reactive oxygen species (ROS) production was evaluated using dihydroethidium (DHE) probe. (H) Cell apoptosis was investigated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). ^aP<0.05, ^bP<0.01, ^cP<0.001.

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About this article

Chen W, Lu H, Dai W, Li H, Chen Y, Liu G, He L. PUM2 Lowers HDAC9 mRNA Stability to Improve Contrast-Induced Acute Kidney Injury through Attenuating Oxidative Stress and Promoting Autophagy. Diabetes Metab J. 2025 Sep 10. doi: 10.4093/dmj.2024.0396. Epub ahead of print.

Received: Jul 18, 2024; Accepted: May 21, 2025

DOI: https://doi.org/10.4093/dmj.2024.0396.

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