Graphical abstract

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   [31]Open in a new tab

Highlights

     * •
       High expression of ANP32E is associated with the progression of AKI
     * •
       The deficiency of ANP32E alleviates the damage to renal tubular
       epithelial cells
     * •
       Inhibiting ANP32E plays a protective role in AKI through the AMPK
       pathway
     __________________________________________________________________

   Molecular biology; Cell biology

Introduction

   Acute kidney injury (AKI) is a clinical syndrome characterized by a
   rapid decrease in kidney function (within 48 h) induced by injury to
   renal structure or function caused by a variety of factors, such as
   renal hypoperfusion, trauma, sepsis, and toxic
   drugs.[32]^1^,[33]^2^,[34]^3 Kidney ischemia-reperfusion (I/R) damage
   is a frequent cause of AKI[35]^4 and a primary trigger of kidney
   transplant dysfunction.[36]^5 Despite tremendous efforts in examining
   the mechanisms underlying the pathophysiology of AKI, there are no
   valid therapeutic measures for treating or preventing AKI.[37]^6
   Therefore, identifying the mechanism of AKI is critical for
   pharmaceutical discovery and improving the quality of life of patients
   with acute kidney damage.

   I/R injury is a significant cause of AKI and is associated with
   increased morbidity, mortality, and extended
   hospitalization.[38]^7^,[39]^8 This condition is characterized by
   unusual regional tissue function after the abrupt interruption of the
   blood supply to an organ or a large area of the body, resulting in
   severe tissue injury after blood flow restoration and reoxygenation.
   Acute ischemia results in ATP depletion, tubular epithelial damage, and
   hypoxic cell death. Reperfusion further aggravates injury by increasing
   the generation of reactive oxygen species and inducing leukocyte
   activation, infiltration, and
   inflammation.[40]^9^,[41]^10^,[42]^11^,[43]^12 I/R-induced organ or
   tissue injury can occur in other medical conditions, such as myocardial
   infarction,[44]^13^,[45]^14 chemic stroke,[46]^15 and organ
   transplantation.[47]^16^,[48]^17^,[49]^18

   Acidic nuclear phosphoprotein 32 family member E (ANP32E) is a member
   of the histone chaperone family with leucine-rich repeats that removes
   the histone variant H2A.Z from chromatin. These proteins play
   significant roles in numerous cellular processes, such as chromatin
   remodeling and modification, the regulation of gene expression, protein
   phosphorylation, and intracellular transport
   regulation.[50]^19^,[51]^20^,[52]^21 ANP32E was first identified in the
   cerebellum as a member of the ANP32E family of protein phosphatase 2
   (PP2A) inhibitors,[53]^19 where it regulated cerebellar synaptogenesis
   by inhibiting PP2A activity at synapses.

   AMP-activated protein kinase (AMPK) activation can facilitate
   autophagy, whereas mTOR activation can repress autophagy. Since mTOR
   modulates autophagy negatively, downregulation of mTOR by AMPK is an
   important regulatory mechanism that maintains cell survival in the
   context of cellular energy depletion. Phosphorylated AMPK can directly
   suppress the phosphorylation of mTORC1 and promote autophagy.

   In this study, we investigated ANP32E accumulation in AKI and clarified
   its correlation with ANP32E. Alleviating apoptosis and inflammation by
   silencing ANP32E suppressed the progression of AKI by promoting the
   AMPK/autophagy pathway.

Results

Identification of DEGs in mice subjected to I/R and validation of ANP32E in
different kidney cell types and ATN patient specimens

   Single-cell RNA sequencing data from healthy human organs in the Human
   Protein Atlas ([54]http://www.proteinatlas.org/) showed that ANP32E was
   present in all the tissues with low tissue specificity ([55]Figure 1A),
   and the single-cell resolution data indicated that the expression of
   ANP32E was higher in tubular cells than in other cells ([56]Figure 1B).
   The translating ribosome affinity purification sequencing (TRAP-seq)
   validation dataset [57]GSE192532
   (adj: p < 0.05) confirmed the increased expression of ANP32E in kidney
   after I/R injury ([58]Figure 1C). We then collected renal biopsy
   tissues from patients with acute tubular necrosis (ATN). Paracancerous
   tissues from patients who underwent nephrectomy were used as controls.
   H&E staining revealed the loss of epithelial cell brush borders of
   tubules, necrosis, tubular dilatation, and cast deposition in the ATN
   group compared to the control group ([59]Figures 1D and 1E). TUNEL
   staining showed that the cells underwent apoptosis ([60]Figures 1F and
   1G). We used immunohistochemical staining to confirm the upregulation
   of ANP32E in renal tissue from patients with ATN compared to that in
   paracancerous renal tissue obtained from patients without other renal
   diseases who underwent nephrectomy ([61]Figures 1H and 1I).

Figure 1.

   [62]Figure 1
   [63]Open in a new tab

   Bioinformatics analysis of ANP32E expression in different renal cells
   and patients with ATN

   (A and B) The average expression level of ANP32E in human organs and
   single-cell type data of kidney samples in the Human Protein Atlas.

   (C) The expression level of ANP32E after renal I/R injury in the
   TRAP-seq validation dataset ([64]GSE192532
   ).

   (D) Representative images of H&E staining. Bar, 50 μm.

   (E) Pathological score indicating tubular damage.

   (F) TUNEL staining of apoptosis-positive cells. Bar, 50 μm.

   (G) Quantification of TUNEL-positive cells in the two groups.

   (H) Immunohistochemical staining showing the expression of ANP32E in
   patients with ATN. Bar, 50 μm.

   (I) Statistical charts quantifying the immunohistochemical staining of
   ANP32E.

   ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

ANP32E expression is upregulated and positively correlated with renal injury

   We constructed a bilateral renal I/R mouse model. Serum creatinine
   ([65]Figure 2A) and urea nitrogen ([66]Figure 2B) were used to assess
   the degree of kidney damage; renal structure was analyzed by H&E
   staining and revealed tubular deposition and tubular dilation after I/R
   ([67]Figures 2C and 2D). TUNEL staining indicated that apoptosis
   occurred in the model ([68]Figures 2E and 2F). We further confirmed the
   upregulation of ANP32E in the kidneys of mice with renal I/R by
   quantitative reverse-transcription PCR (RT-qPCR) ([69]Figure 2G),
   western blotting ([70]Figures 2H–2J), and immunohistochemical analysis
   ([71]Figures 2K and 2L). To determine the tubular segment specificity
   of ANP32E expression in the kidney, we performed dual immunostaining
   for ANP32E (red) and proximal tubular markers (green) in the kidney
   ([72]Figures 2M and 2N). ANP32E was expressed in proximal tubules after
   kidney I/R.

Figure 2.

   [73]Figure 2
   [74]Open in a new tab

   ANP32E is upregulated in the kidneys of mice with renal I/R and is
   positively correlated with kidney injury

   (A) Scr concentrations in the two groups.

   (B) BUN levels in the two groups.

   (C) Representative images of hematoxylin and eosin (H&E) staining. Bar,
   50 μm.

   (D) Pathological score indicating tubular damage.

   (E and F) Representative images of TUNEL staining and the
   quantification of TUNEL-positive cells in the two groups. Bar, 50 μm.

   (G) The mRNA levels of ANP32E in the two groups.

   (H–J) Western blotting and statistical analysis of the expression of
   ANP32E and NGAL in the two groups.

   (K and L) Representative images and statistical analysis of
   immunohistochemical staining of ANP32E in the different groups. Bar, 50
   μm.

   (M) Representative immunofluorescence staining of ANP32E in the renal
   proximal tubules in the sham group and the I/R group. Bar, 50 μm.

   See also [75]Figure S7. n = 6. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and
   ∗∗∗∗p < 0.0001.

ANP32E is upregulated in hypoxia/reoxygenation-stimulated HK-2 cells

   To further examine ANP32E expression in vitro, we established two
   models of AKI by treating HK-2 cells with hypoxia/reoxygenation and
   CoCl[2]. We first examined ANP32E protein expression in HK-2 cells
   exposed to different durations of reoxygenation following hypoxia
   ([76]Figures 3A–3C). RT-qPCR and immunofluorescence staining revealed
   that ANP32E expression levels were increased in the
   hypoxia/reoxygenation group compared to the control group ([77]Figures
   3D and 3E). Subsequently, HK-2 cells were treated with increasing
   concentrations of CoCl[2] (100, 250, and 500 μM) for 24 h, and cells in
   the control group were not treated with any CoCl[2]. Western blotting
   ([78]Figures 3F–3H), RT-qPCR ([79]Figure 3I), and immunofluorescence
   analysis ([80]Figure 3J) demonstrated that the expression of ANP32E in
   mRNA level and protein level was increased compared with the control
   group. Overexpression of ANP32E ([81]Figures 3K and [82]S2A)
   upregulated NGAL in HK-2 cells ([83]Figures 3L and 3M). Overexpression
   of ANP32E increased the overall percentage of cell death and the number
   of apoptotic and dead cells, as determined by flow cytometry
   ([84]Figures S2B and S2C). In addition, we found that the
   hypoxia/reoxygenation-induced increase in the levels of proinflammatory
   mediators was enhanced by the overexpression of ANP32E in HK-2 cells
   ([85]Figures S2D and S2E). Taken together, these results suggest that
   ANP32E may play an important role in facilitating AKI.

Figure 3.

   [86]Figure 3
   [87]Open in a new tab

   ANP32E is upregulated in hypoxia/reoxygenation-stimulated HK-2 cells
   and promotes damage

   (A–C) Western blotting and statistical analysis of the expression of
   ANP32E and NGAL in response to different reoxygenation durations after
   hypoxia and in the control group.

   (D) The mRNA levels of ANP32E in the four groups.

   (E) Representative immunofluorescence staining of ANP32E in HK-2 cells
   in the four groups. Bar, 50 μm.

   (F–H) Western blotting and statistical analysis of the expression of
   ANP32E and NGAL in cells treated with increasing concentrations of
   CoCl[2] for 24 h and in the control group.

   (I) The mRNA levels of ANP32E in the four groups.

   (J) Representative immunofluorescence staining of ANP32E in HK-2 cells
   in the four groups. Bar, 50 μm.

   (K) Western blotting analysis of ANP32E in HK-2 cells transfected with
   the empty vector or lentivirus plasmids.

   (L and M) Western blotting and densitometric analysis of NGAL in HK-2
   cells transfected with the empty vector or lentivirus plasmids.

   See also [88]Figure S2. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p
   < 0.0001.

Knockdown of ANP32E modulates apoptosis and inflammatory responses in
I/R-induced AKI

   We examined whether knockdown of ANP32E protected against AKI induced
   by I/R. The experimental design is shown in [89]Figure 4A. ANP32E
   expression was inhibited by the lentivirus, and the efficiency of
   ANP32E inhibition was examined by western blotting ([90]Figures S1A and
   S1B). Next, we tested whether the inhibition of ANP32E could protect
   tubule cells from injury and restore some of the function of the
   kidney. As shown by H&E staining, there was no significant difference
   in morphology in the sham group. The renal tubular epithelial cells
   showed vacuolar degeneration and cell tube type formation and
   inflammatory cell infiltration when the mice undergo I/R. However,
   inhibiting ANP32E reduced tubular damage and reduced tube structure
   ([91]Figures 4D and 4E). Immunohistochemical staining of KIM-1, a
   tubular cell damage marker, showed that inhibiting ANP32E alleviated
   I/R-induced impairment of kidney function in AKI mice ([92]Figures 4F
   and 4G). Inhibition of ANP32E also alleviated the increase in serum
   creatinine and urea nitrogen concentrations ([93]Figures 4B and 4C). We
   next examined cellular apoptosis. Consistent with our hypothesis, in
   I/R-induced mice, LV-Anp32e abrogated the increase in the expression of
   cleaved caspase-3, which is a key marker of apoptosis ([94]Figures
   4H–4J). TUNEL staining also showed that LV-ANP32E blocked apoptosis
   ([95]Figures 4K and 4L). Furthermore, RT-qPCR was used to measure tumor
   necrosis factor alpha (TNF-α) and interleukin (IL)-1β levels
   ([96]Figures 4M and 4N), and the results indicated that LV-Anp32e
   reduced the inflammatory response in the kidneys of mice subjected to
   I/R.

Figure 4.

   [97]Figure 4
   [98]Open in a new tab

   Knockdown of ANP32E ameliorates renal I/R injury in mice

   (A) A schematic of the experimental design.

   (B and C) Scr and BUN levels in the four groups.

   (D) Representative images of hematoxylin and eosin (H&E) staining. Bar,
   50 μm.

   (E) Pathological score indicating tubular damage.

   (F and G) Representative images and statistical analysis of
   immunohistochemical staining of KIM-1 in the different groups. Bar, 50
   μm.

   (H–J) Western blotting and statistical analysis of the expression of
   cleaved caspase-3 and NGAL in the four groups.

   (K and L) Representative images of TUNEL staining and quantification of
   TUNEL-positive cells. Bar, 50 μm.

   (M and N) The mRNA levels of TNF-α and IL-1β in the four groups.

   See also [99]Figures S1 and [100]S7. n = 6. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p
   < 0.001, and ∗∗∗∗p < 0.0001.

Silencing ANP32E ameliorates apoptosis and inflammatory responses in vitro

   In vitro, we successfully constructed ANP32E-targeted siRNA sequences
   ([101]Figures 5A–5C). Silencing ANP32E reduced the protein levels of
   cleaved caspase-3, which is a major target of apoptosis ([102]Figures
   5D and 5E). ANP32E protein and mRNA expression levels were decreased
   after treatment with ANP32E-targeted siRNA sequences ([103]Figures 5F
   and 5G). The results also indicated that the overall percentage of cell
   death and the number of apoptotic and dead HK-2 cells were reduced by
   ANP32E silencing under hypoxia/reoxygenation conditions, as determined
   by flow cytometry ([104]Figures 5H and 5I). Silencing ANP32E inhibited
   the hypoxia/reoxygenation-induced increases in the levels of
   proinflammatory mediators ([105]Figures 5J and 5K).

Figure 5.

   [106]Figure 5
   [107]Open in a new tab

   Silencing ANP32E alleviates disease progression by affecting
   hypoxia/reoxygenation-induced inflammation and apoptosis in HK-2 cells

   (A–C) Western blotting, statistical analysis, and RT-qPCR were used to
   examine the silencing efficiency of ANP32E.

   (D–F) The protein levels of ANP32E and cleaved caspase-3 in the six
   groups.

   (G) The mRNA levels of ANP32E in the six groups.

   (H and I) Flow cytometric analysis of cell apoptosis and death.

   (J and K) The influence of ANP32E on the mRNA levels of proinflammatory
   mediators in HK-2 cells exposed to hypoxia/reoxygenation conditions.

   ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Knockdown of ANP32E promotes autophagy during I/R-induced AKI

   We then examined how ANP32E controls AKI progression. The results of
   our analysis are summarized in volcano plots, which included 547 genes
   that were upregulated and 323 genes that were downregulated following
   ANP32E silencing in patients with AKI ([108]Figure 6A). Gene Ontology
   analysis of the differentially expressed genes (DEGs) revealed
   functions, bioprocesses, and expression levels ([109]Figure 6B). KEGG
   (Kyoto Encyclopedia of Genes and Genomes) and heatmap enrichment
   analysis revealed that autophagy and AMPK pathway were enriched in the
   siRNA-ANP32E + AKI group compared to the NC + AKI group ([110]Figures
   6C and 6D). Western blotting analysis revealed that knockdown of ANP32E
   increased the protein levels of Beclin 1 and decreased the expression
   of P62 in vivo ([111]Figures 6E–6G), which was further confirmed by
   immunofluorescence and immunohistochemical analyses of LC3
   ([112]Figures 6H–6J).

Figure 6.

   [113]Figure 6
   [114]Open in a new tab

   Knockdown of ANP32E promotes autophagy in AKI induced by I/R

   (A) Volcano plots showing the DEGs (DEGs: |log2FC|>1, adjusted p <
   0.05).

   (B) Loopcircos of the DEGs.

   (C) Heatmap of molecules related to autophagy.

   (D) KEGG enrichment analysis of the AMPK signaling pathway.

   (E–G) Western blotting and statistical analysis of the expression of
   Beclin 1 and P62 in the four groups.

   (H and I) Representative images and statistical analysis of
   immunohistochemical staining of LC3 in the different groups. Bar, 50
   μm.

   (J) Representative immunofluorescence staining of LC3 in vivo. Bar, 50
   μm.

   See also [115]Figures S4 and [116]S7. n = 6. ∗p < 0.05, ∗∗p < 0.01,
   ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Silencing ANP32E promotes AMPK pathway activity

   After confirming that ANP32E affected the progression of AKI by
   inhibiting autophagy in vivo, we examined whether this effect also
   occurred in vitro. Western blotting analysis revealed decreased protein
   levels of Beclin 1 and decreased expression of P62 in HK-2 cells
   ([117]Figures 7A–7C), which was further confirmed by immunofluorescence
   analyses of LC3 ([118]Figure 7D). The RNA sequencing (RNA-seq) data
   indicated that the AMPK pathway was affected by ANP32E silencing in the
   AKI cell model. Western blotting analysis revealed a marked change of
   key molecules of the AMPK pathway, such as P-AMPK and P-mTOR
   ([119]Figures 7E–7G). Then, we tested AMPK pathway-related molecules in
   vivo; the results showed that the expression of P-AMPK was increased
   after ANP32E knockdown in the I/R group, while the expression of P-mTOR
   was further reduced ([120]Figures S4A–S4C), indicating that ANP32E
   knockdown could exert a renal protective effect by activating the AMPK
   signaling pathway. Pathway activation was decreased after the AMPK
   inhibitor compound C (CC) was used to inhibit AMPK in the AKI model
   ([121]Figures 7H and 7I). The autophagic flux showed an increase of RFP
   in hypoxia/reoxygenation-induced HK-2 cells. The signal intensity was
   increased in HK-2 cells transfected with si-ANP32E compared to the
   hypoxia/reoxygenation group, while exposure to the AMPK inhibitor CC
   reduced autophagy ([122]Figure S5A). These results indicate that
   silencing ANP32E promotes AMPK pathway activity. Subsequently, we used
   mTOR inhibition to stimulate HK-2 cells after the overexpression of
   ANP32E. The results indicated that the addition of rapamycin reduces
   the expression level of the NGAL ([123]Figures S6A and S6B). This
   suggests that the highly expression of ANP32E can partly damage renal
   tubular epithelial cells through mTOR.

Figure 7.

   [124]Figure 7
   [125]Open in a new tab

   Silencing ANP32E promotes HK-2 cell AMPK activity and autophagy in
   vitro

   (A–C) The protein levels and statistical analysis of Beclin 1 and P62
   in the four groups.

   (D) Representative images and statistical analysis of
   immunofluorescence staining of LC3. Bar, 50 μm.

   (E–G) Western blotting images and corresponding quantification about
   the molecules of the AMPK pathway.

   (H) Western blotting images and corresponding quantification in HK-2
   cells treated with an AMPK inhibitor.

   See also [126]Figures S3, [127]S5, and [128]S6. ∗p < 0.05, ∗∗p < 0.01,
   ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

   Briefly, our study has established how ANP32E affects AKI.
   Downregulation of ANP32E can activate AMPK according to the RNA-seq.
   When AMPK is activated, it can inhibit the phosphorylation of mTOR,
   leading to the lipidation of LC3 and activation of Beclin 1.[129]^22
   Autophagy suppresses apoptosis by reducing the abundance of
   pro-apoptotic proteins in the cytoplasm and regulates NLRP3
   inflammasome activation as well as IL-1β and IL-18 secretion
   negatively[130]^23 through the Toll-like receptor and Nod-like receptor
   signaling pathway ([131]Figure 8).

Figure 8.

   [132]Figure 8
   [133]Open in a new tab

   The mechanism described in this study

   In this study, I/R increased the expression of ANP32E in renal tubular
   epithelial cells, and ANP32E mediated the development of kidney
   inflammation and promoted cell apoptosis, which was associated with the
   regulation of the AMPK/autophagy pathway.

Discussion

   Among innate renal cells and infiltrating cells, renal tubular
   epithelial cells are critical contributors to the development of AKI
   and are the most susceptible to kidney ischemia.[134]^24 Pathological
   analysis reveals the loss of tubular epithelial cells by apoptosis and
   necrosis, and renal tubular epithelial cell death and injury are
   significant causes of kidney dysfunction.[135]^25 DNA damage has been
   reported to be involved in I/R injury in a variety of vital organs,
   including the testis, brain, liver, and
   heart.[136]^26^,[137]^27^,[138]^28^,[139]^29 During renal I/R injury,
   DNA fragmentation and damage occurs as early as 12 h after reperfusion
   in the renal tubules and increases within 24 h.[140]^30 The present
   study used in vivo and in vitro models of ischemic injury and examined
   the underlying molecular mechanism.

   Autophagy is an evolutionarily conserved multistep process involving
   the degradation of intracellular organelles, proteins, and other
   macromolecules by lysosomal hydrolases.[141]^31^,[142]^32^,[143]^33 It
   has many features, including the regulation of programmed cell death,
   inflammation, and other biological processes.[144]^34 Pharmacological
   and genetic inhibition studies have indicated that autophagy plays a
   nephroprotective role in AKI.[145]^35 The occurrence of cellular
   autophagy requires the involvement of multiple proteins. These protein
   interactions regulate the occurrence of autophagy. In this process, the
   post-translational modification behaviors of proteins such as
   phosphorylation, acetylation, ubiquitylation, and succinylation play an
   important role in regulating the process of autophagy.[146]^36 A study
   of histone lactate showed that histone lactation promotes the
   transcription of rubicon-like autophagy enhancer, which promotes
   autophagosome maturation through interaction with Beclin 1. Inhibition
   of histone milk acidification has a therapeutic efficiency comparable
   to that of autophagy inhibitors.[147]^37 Studies have shown that
   methylation of histone H3K4 regulates the expression and activity of
   two key transcription factors HLH-30 and PHA-4,[148]^38 while the two
   molecules can regulate autophagy by controlling the expression of many
   autophagy-related genes.[149]^39^,[150]^40 Moreover, histone
   acetylation also affects a variety of cellular and physiological
   processes, including transcription, neural function, autophagy, and
   differentiation.[151]^41 However, autophagy is a double-edged sword. In
   general, it can maintain normal cellular homeostasis. However, when the
   stimulation is too strong, autophagy can play the opposite
   role.[152]^42 AMPK is an energy-sensitive kinase that is involved in
   regulating bioenergetic metabolism.[153]^43 Under conditions of AKI,
   the expression of phosphorylated AMPK is significantly increased in
   kidney tissue, and activated AMPK can promote autophagy by inhibiting
   the mTOR signaling pathway.[154]^44^,[155]^45 In our study, we found
   that silencing ANP32E inhibited inflammation and apoptosis during AKI
   progression by further activating the AMPK/autophagy pathway.

   ANP32E is a protein with leucine-rich repeats that functions as a
   chaperone of H2A.Z histone and regulates the expression of downstream
   genes[156]^46 and other cellular processes, including chromatin
   modification and remodeling, gene regulation, protein phosphorylation,
   and cell apoptosis.[157]^47 It has been reported that ANP32E is not
   only essential for cerebellar development and synaptogenesis but is
   also involved in tumorigenesis. In triple-negative breast cancer,
   ANP32E induces tumorigenesis by increasing the expression of the
   transcription factor E2F1.[158]^48 ANP32E activates AKT/mTOR/HK2
   signaling and glycolysis in thyroid cancer.[159]^49 In gastric cancer,
   overexpression of ANP32E can cause abnormal NUF2 expression, which
   promotes cancer progression.[160]^50 We assessed ANP32E expression in
   the GEO dataset and found increased expression of ANP32E in I/R-induced
   AKI. Our results suggested that the ANP32E protein was upregulated in
   the renal tubular cells of patients with ATN and I/R mice and in
   cultured proximal tubular cells exposed to hypoxia/reoxygenation. We
   examined the function and mechanism of ANP32E by conducting a series of
   in vivo and in vitro experiments. The results indicated that ANP32E
   knockdown alleviated the inflammatory response and apoptosis in
   I/R-induced mice and hypoxia/reoxygenation-stimulated HK-2 cells. KEGG
   pathway enrichment analysis was used to screen the AMPK signaling
   pathway and showed that ANP32E could participate in AKI progression by
   regulating autophagy through the AMPK signaling pathway.

   Studies have shown that knockdown of ANP32E can reduce AKI damage by
   further activating the AMPK signaling pathway. However, the specific
   mechanism by which ANP32E regulates the AMPK signaling pathway remains
   unclear. Previous reports have suggested the colocalization of ANP32E
   with the catalytic subunit of PP2A and the inhibition of PP2A
   activity.[161]^51 PP2A plays an important role in protecting against
   renal injury,[162]^52 and the involvement of PP2A in renal I/R injury
   has been demonstrated.[163]^53 Renal tubular epithelial cells have
   extremely high energy requirements, and AMPK is a key regulator of
   energy production. AMPK activity can be regulated by PP2A.[164]^54
   These theories suggest that ANP32E can regulate the AMPK pathway by
   regulating the localization of PP2A in the cytoplasm. However, this
   hypothesis requires further research.

   In summary, we found that silencing ANP32E further activated autophagy
   through the AMPK pathway and ameliorated I/R-induced AKI. These
   findings will provide new ideas and targets for the treatment of AKI.

Limitations of the study

   Although we found that ANP32E participates in the progression of AKI,
   our study still has many limitations. The progression of injury in mice
   is very similar to the progression of human AKI injury, but the mouse
   model cannot represent the complexity of human kidney fully. Moreover,
   the pathogenesis of I/R-induced AKI is complex, involving the
   involvement and crosstalk of multiple links such as apoptosis,
   inflammation, and autophagy, and this study needs to further explore
   the molecular mechanism interaction of the aforementioned links.

Resource availability

Lead contact

   Further information and requests for resources and reagents should be
   directed to the lead contact, Xiangdong Yang (yxd@email.sdu.edu.cn).

Materials availability

   This study did not generate new unique reagents. Antibodies, reagents,
   and cell lines used were obtained from commercial or other sources as
   outlined in the [165]key resources table.

Data and code availability

     * •
       Data from this study can be provided by the [166]lead contact upon
       request.
     * •
       No code was generated or analyzed during the current study.
     * •
       Any additional information is available from the [167]lead contact
       upon request.

Acknowledgments