DEF6 regulates renal ischemia reperfusion injury through suppressing the WWP2 mediated ubiquitination of PARP1

doi:10.21203/rs.3.rs-4867731/v1

Download PDF

Research Article

DEF6 regulates renal ischemia reperfusion injury through suppressing the WWP2 mediated ubiquitination of PARP1

https://doi.org/10.21203/rs.3.rs-4867731/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Renal ischemia-reperfusion injury (RIRI) stands as an unavoidable complication arising from kidney surgery, profoundly intertwined with its prognosis. The role of differentially expressed in FDCP 6 homolog (DEF6) in RIRI remains elusive, despite its confirmation as a potential therapeutic target for diverse diseases. Here, we investigated the mechanism by which DEF6 regulated RIRI.

Methods

RNA sequencing data and IP-MS were used to identify the expression and potential targets of DEF6 through bioinformatics analysis. To elucidate the impact of DEF6 on RIRI, both an in vivo model of RIRI in mice and an in vitro model of kidney cell hypoxia/reoxygenation were established. Biochemical and histological analyses were used to investigate the influence of DEF6 on kidney damage mediated by RIRI.

Results

We confirmed that DEF6 was upregulated during RIRI and had a close correlation with RIRI-related inflammation and apoptosis. Moreover, inhibition of DEF6 could mitigate RIRI-induced kidney damage, inflammation, and apoptosis. Through our comprehensive mechanistic investigation, we revealed that DEF6 interacts with poly ADP-ribose polymerase 1 (PARP1) and suppresses the ubiquitination of PARP1. Inhibition of DEF6 resulted in reduced cleaveage of PARP1, leading to a marked suppression of PARP1-mediated apoptosis activation. The aggravation effect on inflammation and apoptosis achieved through DEF6 was nullified by the inhibition of NF-κB and Bax/Bcl2 signaling activation through PARP1 deletion.

Conclusions

The findings from our study indicate that DEF6 suppressed the WWP2 mediated ubiquitination of PARP1 and modulates the activation of NF-κB and Bax/Bcl2 pathway, thus involved in RIRI-induced inflammation and apoptosis. These results suggest that DEF6 holds promise as a potential therapeutic target for mitigating RIRI.

renal ischemia reperfusion injury

DEF6

PARP1

WWP2

ubiquitination

Renal ischemia-reperfusion (I/R) injury is a pivotal clinical problem associated with acute kidney injury (AKI), which significantly contributes to morbality and prolonged hospital stays in different patient populations^[1], particularly in those undergoing renal transplantation, cardiac surgery, or being affected by shock^[2–4]. Renal ischemia-reperfusion injury(RIRI) stems from an initial restriction of blood supply to the kidney (ischemia), followed by the subsequent restoration of perfusion and reoxygenation (reperfusion). The onset of ischemia halts the delivery of oxygen and nutrients, crucial for cellular metabolism and function, leading to tissue hypoxia and cellular damage. The reintroduction of blood supply should theoretically restore normal function; however, paradoxically, reperfusion leads to a cascade of inflammatory and apoptotic reactions that further damage the renal tissue^[5]. The inflammatory response is a hallmark of I/R injury. Ischemia leads to the activation of endothelial cells and the subsequent expression of adhesion molecules^[6], which facilitate the sequestration and infiltration of neutrophils and macrophages into the renal parenchyma^[7]. These immune cells release cytokines, chemokines, and further reactive oxygen species(ROS), intensifying renal injury^[8]. Several key molecular pathways and mediators are implicated in the exacerbation of renal I/R injury. These include the activation of pro-apoptotic signaling pathways, such as the c-Jun N-terminal kinase (JNK) and p53 pathways^[9], and the activation of transcription factors such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) which promote inflammation^[10].Therefore, the mechanism of inflammation and apoptosis in kidney injury triggered by renal I/R requires further investigation.

Rho GTPase is a subgroup of the Ras family. It functions as a molecular switch, cycling between two conformational states: an inactive GDP-bound state and an active GTP-bound state. In the active state, Rho GTPase is able to interact with downstream effector proteins to induce cellular responses^[11].Differentially expressed in FDCP 6 homolog(DEF6), also known as IRF4-binding protein (IBP) or SWAP70-like articulator of T cells (SLAT), is a unique guanine nucleotide exchange factor (GEF). It contains a PH-DH like domain, in which the PH domain receives upstream signals (e.g. inositol triphosphate) to activate and enable the DH like domain to exert regulatory effects downstream.. As an upstream activator of the Rho-GTPase family, DEF6 can activate the small GTPases of the Ras family, such as RhoA, Rac1, or Cdc42^[12].It showed that DEF6 recruited and translocated small GTPases to make them functional. Recent studies on DEF6 have focused on autoimmune diseases, orthopaedics, and tumours, with no researches on RIRI^[13–16]. However, some researches have shown that the signalling pathways acted by DEF6 are correlated with the mechanism of RIRI.A recent report showed that DEF6 regulated pathological cardiac hypertrophy via MEK-ERK signalling pathway^[17].And MEK-ERK signalling pathway belongs to MAPK pathway. Therefore, It is worthwhile to investigate whether and how DEF6 is able to regulate I/R-induced renal injury.

ADP-ribosylation is a post-translational modification of proteins that is widespread in a variety of physiological and pathological processes, and refers to the enzyme-catalysed binding of one or more ADP-ribose units to a protein-specific site^[18].Poly(ADP-ribose) polymerase 1(PARP1) is the first member of the ADP-ribosylation enzyme superfamily, which consists of proteins that share homology with PARP1. The PARP1 protein can be broadly divided into three main regions: the first is the DNA damage-sensing region, the second is the upstream regulatory region and the third is the downstream functional region. Due to its structure, PARP1 plays an important enzyme in the cellular dynamics of DNA repair and cellular stress responses, exerting a significant influence on cell viability, gene expression, and genomic integrity^[19]. Remarkably, PARP1 induces a specific form of apoptosis called parthanatos, which allows it to play the opposite role from the one it would otherwise play in the process of injury onset^[20]. During parthanatos, PARP1 is cleaved into two different parts, hindering DNA repair and promoting cell death^[21]. This allows PARP1 to upregulate inflammation and apoptosis in pathological state^[22]. And this phenomenon is reversed when PARP1 cannot be cleaved^[23]. However, the role of PARP1 in RIRI has not been elucidated.

WWP2,a E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. POU5F1 polyubiquitinated by 'Lys-63'-linked conjugation and promotes it to proteasomal degradation; in embryonic stem cells the ubiquitination is proposed to regulate POU5F1 protein level^[24]. Ubiquitinates EGR2 and promotes it to proteasomal degradation^[25].WWP2 ubiquitinates PARP1 in a variety of disease models including cardiac hypertrophy and endothelial damage^{[26, 27]}.Therefore,WWP2 is expected to be an intermediate site for regulation of PARP1 in RIRI.

This study reveals the involvement of DEF6 in the regulation of inflammation and apoptosis during RIRI. Moreover, DEF6 exhibited interaction with PARP1, suppressing the ubiquitination of PARP1 through WWP2, thereby influencing the downstream NF-κB and Bax/Bcl2 signaling pathways. Overall, our findings offer a promising treatment strategy for addressing RIRI.

Animals and treatment

Mice were provided with a standard diet and housed in a controlled environment with a temperature of 22 to 24°C, air filtration, and light regulation. The room maintained humidity levels between 40% and 70%. The RIRI model was performed on the designated mice according to the established procedure^[28], and kidney and serum samples were collected on the basis of experimental requirements. All animal experimental protocols were approved by the Animal Care and Use Committee at the Renmin Hospital of Wuhan University, following the standard guidelines of the Animal Experiment Center of Wuhan University.

Production of DEF6-KO mice

The CRISPR online design tool (http://chopchop.cbu.uib.no/) was used to predict the guiding sequence as follows: guideRNA1 target site: 5′-CCTCTTGCTCCCAGGTAGAGTA − 3′, guideRNA2 target site: 5′- TTGGATGAGCTCCTGGTAGACT-3′, which resulted in a 2 kb deletion in total. The GALNT4-sgRNA expression vector was constructed on the BsaI restriction site of pUC57-sgRNA (Addgene 51132). The products of the Cas9 expression vector pST1374-Cas9 (Addgene 44758) and the sgRNA expression vector were mixed and microinjected into C57BL/6J zygotes with the FemtoJet 5247 microinjection system. F0 generation mice were obtained after 19–21 days, and were identified by PCR and sequencing DNA was extracted from the ear tissue of 2-week-old mice and assessed using the primers below: DEF6-check F1: 5′-TTGCATTTCCTGAAACCGGA-3′, DEF6-check R1: 5′- GCTGAGAATGGCAACCAAGT-3′ .

Cell culture and treatment

HK2 is a cell line originating from human renal tubular epithelial cells obtained from American Type Culture Collection (ATCC, Manassas, VA). The cells were maintained in Dulbecco's Modifed Eagle Medium (DMEM) (Gibco, Rockville, MD) supplemented with 10% fetal bovine serum (FBS) (HyClone, Salt Lake City, UT) and 1% penicillin-streptomycin (Sigma, St. Louis, MO, USA) at 37℃ in a controlled humidifed atmosphere with 5% CO₂. 2×10⁵ cells were plated in 6-well plates and incubated. The culture medium was replaced once every 2 days, and the cells were passaged by 1% trypsin when they reached 80–90% confuence. the experimental cells were divided into 2 groups, the control group and the H/R group. For the control group, cells were maintained at 37°C in 5% CO₂ and 20% O₂. For the H/R group, cells were exposed to hypoxia (1% O₂, 5% CO₂ and 94% N₂) for 24 h, followed by replacement with fresh medium equilibrated at 21% O₂, 5% CO₂ and 74% N₂ and incubation for 4 h.

Histological staining

The kidney was fixed with 4% paraformaldehyde and subse- quently embedded in paraffin. Sections of 4 µm in thickness were prepared, followed by dewaxing, hydration, and staining with hematoxylin and eosin (H&E). The assessment of necrotic area was performed by 2 experienced pathologists and quantified using ImageJ software.

Immunofluorescence

Paraffin sections were subjected to dewaxing and hydration procedures. Subsequently, the sections were permeabilized with 0.5% Triton X-100 for 10 min at room temperature. Following a phosphate-buffered saline wash, the sections were blocked with 10% goat serum for 1 h at room temperature. They were then incubated overnight at 4°C in a refrigerator with primary antibodies against CD11b (53-0112-82, Invitrogen; 1:100). On the following day, the sections were incubated with corresponding secondary antibodies in the dark at room temperature for 1 h. Before observation, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min.

Terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling

The assessment of apoptotic cells in renal tissue was performed using a TUNEL Assay Kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. The average count of TUNEL-positive cells was determined in randomly selected fields of view at 400× magnification.

Transfection

In vitro, DEF6 overexpression was induced in HK2 cells by exposing them to an lentivirus carrying human DEF6 (GenePharma, Shanghai) with a multiplicity of infection of 50 for 6 h in serum-free and penicillin–streptomycin-free DMEM. Similarly, knockdown of DEF6 and PARP1 were induced cells by exposing them to lentivirus carrying short hairpin RNA against DEF6 and PARP1. Subsequently, the cells were incubated in DMEM supplemented with 10% fetal bovine serum for 72 h. All carriers were constructed and packaged by GenePharma (Shanghai, China) and were used for cells 1 weeks before inducing H/R. As a control, we exposed cells to a blank vector.

Quantitative real-time PCR

Total RNA was extracted using the RNAiso Plus kit (TaKaRa Biotechnology). Reverse transcriptase reactions were carried out using the SuperScript First-Strand Synthesis System (Invitrogen). Real-time PCR reactions were conducted using glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) as the internal control. The gene expression levels were presented as fold change rela- tive to the control, calculated using the 2 − ΔΔCT method. The primers utilized for quantitative real-time PCR analysis were listed in Supplementary Table.

Western blotting

Radioimmunoprecipitation assay buffer with protease inhibitor cocktail tablet were used to extract the total proteins. Western blotting assay was performed with anti-DEF6(PA5-113458, Invitrogen; 1:1000), anti-p-inhibitor of NF-κBkinase β (IKKβ) (#2697, Cell Signaling Technology; 1:1000), anti-IKKβ(#8943,Cell Signaling Technology; 1:1000), anti-NF-κB inhibitor α (IKBα) (10268- 1-AP, Proteintech; 1:5000), anti-p-p65 (82335-1-RR, Proteintech; 1:2000), anti-p65 (80979-1-RR, Proteintech; 1:5000), anti-Bax (50599-2-Ig, Proteintech; 1:2000), anti-Bcl-2 (26593-1-AP, Proteintech; 1:1000), anti-Caspase 3/C-Caspase 3 (19677-1-AP, Proteintech; 1:1000), anti-PARP1 (PA5-34803, Invitrogen; 1:1000), anti-C-PARP1 (PA5-77850, Invitrogen; 1:1000), anti-GAPDH (10494-1-AP, Proteintech; 1:5000), anti-Flag (GB11938, Servicebio; 1:1000), anti-Myc(PA1-981,Invitrogen; 1:1000), and anti-HA (GB12939, Servicebio; 1:1000).

Immunoprecipitation

Transfected cells were lysed using IP buffer [20 mMtris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 12 mM glycerophosphate, 10 mM sodium fluoride, 5 mM EGTA, 2 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluroride, aprotinin (2 µg/ml), and leupeptin (2 µg/ml)]. Cell lysates were centrifuged at 12,000g for 10 min at 4°C. The result- inglysates were subjected to overnight IP at 4°C using the des- ignated antibodies. Protein A/G agarose beads were introduced and incubated with agitation for 2 h at 4°C. The proteins bound to the beads were eluted and subsequently analyzed through immunoblotting using the specified antibodies.

Statistical analysis

GraphPad Prism software (version 8.0, USA) was used for sta- tistical analysis. All values are presented as the means ± SD. One-way analysis of variance (ANOVA) and Tukey–Kramer test were used to compare the differences among the groups. Statistical significance was determined at P < 0.05.

DEF6 is upgraded in experimental models of RIRI

Through literature research, we found that DEF6 has a regulatory role in abnormal immune response, cardiac hypertrophy and other conditions. However, its role in the regulation of RIRI is not clear. Therefore, we examined the expression changes of DEF6 in RIRI. In Fig. 1A, a Western Blot analysis also showed that the protein level of DEF6 increased significantly following I/R surgery. Furthermore, in vitro the protein level of DEF6 increased after H/R treatment(Fig. 1B). Consistent with the results of WB, a qPCR analysis showed that DEF6 mRNA levels were upgraded in human renal cortex proximal tubule epithelial cells(human kidney 2, HK2)(Fig. 1C). The above findings suggested that DEF6 may play a role in RIRI.

Inhibiting DEF6 alleviated RIRI-induced inflammation and apoptosis in vivo

To further explore the regulation of RIRI around DEF6 in vivo, DEF6 global knockout (DEF6-KO) mice were used to conﬁrm the function of DEF6 in the I/R model treated by surgery. The DEF6 protein was conﬁrmed not to be expressed in the DEF6-KO mice(Fig. 2A). I/R or sham operation was performed on DEF6-KO and WT mice. 24 hours later, we found no differences in renal function between WT sham group and DEF6-KO sham group. However, in the I/R surgery group with different reperfusion duration, the serum creatinine and urea nitrogen in the DEF6-KO mice were significantly lower than those in WT mice (Fig. 2B, C). Additionally, H&E staining showed a lower extent of tubular injury and kidney dysfunction in DEF6-KO mice(Fig. 2D).

Significantly, our RNA sequencing assay uncovered a robust correlation between DEF6 and RIRI-related molecular events, especially inflammation(left) and apoptosis(right) processes(Fig. 2E, F). In following qPCR, WB and immunofluorescence staining, markers of pro-inflammation and pro-apoptosis, such as p-IkKβ, p-p65, Bax, C-caspase 3, were lower in DEF6-KO mice than those in WT mice. On the contrary, markers of anti-inflammation and anti-apoptosis, such as IkBα and Bcl2, were higher in DEF6-KO mice(Fig. 2G-L). From the above results, we concluded that the absence of DEF6 relieved the level of RIRI.

DEF6 regulated inflammation and apoptosis of HK2 cells induced by hypoxia/reoxygenation in vitro

Above we have demonstrated that DEF6 regulates RIRI in vivo. To confirm the effect of DEF6 on RIRI in vitro, DEF6 expression was increased or suppressed following infection of HK2 cells with a lentiviral DEF6(DEF6-OE) or a lentiviral short hairpin RNA targeting DEF6 (shDEF6)(Fig. 3A, E). Besides, we chose to treat HK2 cells with reoxygenation 4h after hypoxia 24h to simulate I/R in vivo. Consistently, relative protein expression of inflammation and apoptosis was proven to be same as that in vivo(Fig. 3B, C). Additionally, we examined the mRNA expression and found that partial markers corresponded with that in vivo(Fig. 3D). Equally, the opposite trend of inflammation and apoptosis was confirmed in shDEF6 group(Fig. 3E-G). From the above results, we concluded that the overexpression or knockdown of DEF6 promoted or suppressed the level of RIRI induced by hypoxia/reoxygenation in vitro.

DEF6 regulated inflammation and apoptosis of HK2 cells through targeting PARP1

Initially, to understand the downstream targets of DEF6 in RIRI, we utilized immunoprecipitation and mass spectrometry(IP-MS) of DEF6. The protein abundance and independent peptide number of PARP1 were found to be higher among the proteins interacting with DEF6. Consequently, after preliminary literature research on PARP1^[18-23], we selected it as a reliable downstream target due to its function. Afterward, we determined the changes in PARP1 expression in different cell models. PARP1 expression was found to be upregulated when DEF6 was overexpressed and vice versa(Fig. 4A-D). Interestingly, the expression trend of cleaved PARP1(C-PARP1) was identical to that of PARP1. This suggested that C-PARP1 is the activated form of PARP1 in the H/R model and DEF6 synergised with PARP1. And the C-PARP1 caused parthanatos-dominant apoptosis.Accordingly, we examined the protein expression content of inflammation and apoptosis after knockdown of PARP1 in the DEF6-OE group. As expected, knockdown of PARP1 almost reversed the upregulation of inflammation and apoptosis caused by DEF6 overexpression(Fig. 4E-G). The above results led us to believe that DEF6 did affect inflammation and apoptosis through targeting PARP1.

DEF6 upregulated protein expression of PARP1 via competitively inhibiting the ubiquitination of WWP2

To explore the mechanism by which DEF6 upregulates PARP1 protein expression, we queried the literature related to PARP1 expression. We then found that PARP1 is regulated by ubiquitination in a variety of disease models, such as vascular injury and breast cancer^{[29, 30]}. However DEF6 has not been identified as a protein with ubiquitination. Therefore, we tried to find an intermediate molecule to regulate PARP1 expression. In several researches, WWP2 was to be an E3 ubiquitin ligase capable of ubiquitinating PARP1 degradation^{[26, 27]}. Thereafter, we set up a hypothesis that DEF6 can upregulate PARP1 expression by competitively inhibiting the interaction of WWP2 with PARP1.

Initially, we verified the interaction of DEF6 with PARP1 by Co-immunoprecipitation(Co-IP)(Fig. 5A, B). And we constructed three mutants of PARP1 protein(PARP1^{△P1(1-360AA)}, PARP1^{△P2(361-645AA)}and PARP1^{△P3(646-1015AA)}, AA means amino acid), which each removed a part of the PARP1 structural domain. Protein interactions indicated that interactions remained strong only when P1 was deleted(Fig. 5C). In other words, both P2 and P3 are essential for PARP1 to interact with DEF6. Immediately thereafter, to verify whether WWP2 was involved in the above interactions as an intermediate molecule, we performed interactions of WWP2 with DEF6 and PARP1. And the above results showed that WWP2 interacted with both proteins(Fig. 5D-G). Afterwards, we utilized ubiquitination interaction experiments of WWP2 on PARP1. The ubiquitination degradation of PARP1 by WWP2 was confirmed(Fig. 5H). Ultimately, competitive interactions experiments showed that DEF6 inhibited the interactions between WWP2 and PARP1(Fig. 5I, J). In conclusion, the above results suggest that DEF6 attenuates the ubiquitination of PARP1 by WWP2 by inhibiting the interactions between WWP2 and PARP1, thus upregulating the protein expression of PARP1.

Renal ischemia-reperfusion injury (RIRI) is a complex, multifactorial pathological process that plays a significant role in determining the prognosis of kidney surgeries and influences their outcomes considerably^[31]. Following comprehensive functional studies, we have recognized DEF6 as a promising therapeutic target for RIRI treatment. Through our investigation, we discovered that DEF6 is involved in inhibiting the ubiquitination of PARP1 by WWP2, resulting in the activation of NF-κB and Bax/Bcl2 signaling and aggravation of renal injury, inflammation, and cell death induced by RIRI. By demonstrating that DEF6 depends on PARP1 to regulate RIRI-induced kidney injury, we confirmed DEF6 as a therapeutic target.

Cell apoptosis and inflammatory responses are primary factors contributing to the development and clinical manifestations of RIRI^[32]. As an important guanine nucleotide exchange factor, upregulation of DEF6 has been observed in various cancer types, and its overexpression has been linked to poor prognosis^[33]. Recently, there has been a growing exploration of the mechanisms by which DEF6 regulated various diseases. DEF6 endogenous type-I interferon responses in osteoblasts and suppresses osteogenesis^[15]. It was reported that DEF6 was involved in the dendritic cell differentiation and maturation^[13]. Through our study, we have verified the regulatory capacity of DEF6 in RIRI, extending its functional scope to encompass kidney inflammation and cell death. This finding remarkably broadens our understanding of DEF6’s role in the domain of acute organ injury. Although the direct impact of DEF6 on the prognosis of RIRI in patients remains to be established, these findings at least suggested that DEF6 was closely linked to RIRI, and targeting DEF6 to improve RIRI was a promising therapeutic approach.

According to our bioinformatics analysis and literature review, we have identified the NF-κB and Bax/Bcl2 signaling pathway as the primary mechanism by which DEF6 regulates RIRI-related molecular events. Anomalous activation of NF-κB and Bax/Bcl2 etc., and their upstream regulator,p65 and caspase-3, can induce cell inflammation and apoptosis during RIRI^[34]. As a result, clinical applications were focused on NF-κB and Bax/Bcl2 upstream signals. Here, we elucidated the direct interaction between DEF6 and PARP1, establishing a connection between DEF6 function and the activation of parthanatos. Notably, PARP1 has been identified as a safe and effective therapeutic target for kidney diseases^[35]. Under physiological conditions, PARP1 remained inactive and stable, but with pathological stress, it was rapidly cleaved and activated as a inhibitor of DNA repair^[36]. Previous study has shown that PARP1 was crucial for parthanatos-dominant cell death in HeLa cells^[21]. Besides, that noncleavable PARP1 downgraded the inflammation response showed an opposite result against cleavage,which indicated that preventing PARP1 from being cleaved is a potential target for suppressing I/R-induced inflammation^[23].Our in-depth mechanism study revealed that DEF6 interacted with PARP1 and WWP2, modifying the ubiquitination of PARP1 by competitive suppressing WWP2, thus regulating inflammation and apoptosis in RIRI. These findings highlighted the therapeutic potential of DEF6 in RIRI and suggested novel strategies for modifying the PARP1 protein to regulate damage.

In conclusion, our findings provide compelling evidence for the crucial role of DEF6 in the context of RIRI. DEF6 suppressed the WWP2 induced ubiquitination of PARP1 and upregulated kidney inflammation and apoptosis induced by I/R injury through targeting NF-κB and Bax/Bcl2 signaling. Hence, the targeted inhibition of DEF6 in kidney cells holds promise as a strategy to alleviate RIRI and enhance the prognosis of kidney surgery.

Acknowledgements

Thanks to the members of our laboratory for their contributions.

Funding

Financial support for this study was provided by the National Natural Science Foundation of China (grant nos. 81870067 and 82170664).

Ethics Approval and Consent to participate

This research was approved by the Medical Ethics Committee of the Wuhan University.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Availability of data and Materials

All the data in this paper are available from the corresponding author on reasonable request.

Author’s Contributions

H-H: Conceptualization, Methodology, Software, Investigation, Formal Analysis, Writing - Original Draft;

Y-L: Methodology, Data Curation, Investigation, Writing - Original Draft;

S-H: Methodology, Resources, Data Curation, Investigation;

J-G: Visualization, Investigation;

T-Q and J-Z:Conceptualization, Funding Acquisition, Resources, Supervision, Writing - Review & Editing.

KAI S, JOHN A K. AKI in the ICU: definition, epidemiology, risk stratification, and outcomes [J]. Kidney Int, 2011, 81(9).
STEPHANIE F S, SARAH A H, MICHAEL L N. Ischemia-reperfusion injury in renal transplantation: 3 key signaling pathways in tubular epithelial cells [J]. Kidney Int, 2019, 95(1).
FENG W, GUANGYUAN Z, ZEYUAN L, et al. Antithrombin III/SerpinC1 insufficiency exacerbates renal ischemia/reperfusion injury [J]. Kidney Int, 2015, 88(4).
JOHAN M L, SANDOR B, THOMAS T. Regulation of cardiac and renal ischemia-reperfusion injury by microRNAs [J]. Free Radic Biol Med, 2013, 64(0).
LUIS A J, PATRICK M W, KIANOUSH K. Pathophysiology of Acute Kidney Injury in Critical Illness: A Narrative Review [J]. Compr Physiol, 2022, 12(4).
KORMANN R, KAVVADAS P, PLACIER S, et al. Periostin Promotes Cell Proliferation and Macrophage Polarization to Drive Repair after AKI [J]. Journal of the American Society of Nephrology : JASN, 2020, 31(1): 85-100.
KARASAWA K, ASANO K, MORIYAMA S, et al. Vascular-resident CD169-positive monocytes and macrophages control neutrophil accumulation in the kidney with ischemia-reperfusion injury [J]. Journal of the American Society of Nephrology : JASN, 2015, 26(4): 896-906.
KNOPPOVA B, REILY C, MAILLARD N, et al. The Origin and Activities of IgA1-Containing Immune Complexes in IgA Nephropathy [J]. Frontiers in immunology, 2016, 7: 117.
ALAAELDIN R, BAKKAR S, MOHYELDIN R, et al. Azilsartan Modulates HMGB1/NF-κB/p38/ERK1/2/JNK and Apoptosis Pathways during Renal Ischemia Reperfusion Injury [J]. Cells, 2023, 12(1).
HU X, DING C, DING X, et al. Inhibition of myeloid differentiation protein 2 attenuates renal ischemia/reperfusion-induced oxidative stress and inflammation via suppressing TLR4/TRAF6/NF-kB pathway [J]. Life sciences, 2020, 256: 117864.
NETHE M, HORDIJK P. The role of ubiquitylation and degradation in RhoGTPase signalling [J]. Journal of cell science, 2010, 123: 4011-8.
MAVRAKIS K, MCKINLAY K, JONES P, et al. DEF6, a novel PH-DH-like domain protein, is an upstream activator of the Rho GTPases Rac1, Cdc42, and RhoA [J]. Experimental cell research, 2004, 294(2): 335-44.
JELENA P, INGA W, ALESSANDRA P, et al. Control of GM-CSF-Dependent Dendritic Cell Differentiation and Maturation by DEF6 and SWAP-70 [J]. J Immunol, 2020, 205(5).
BENJAMIN F, MAUD T, MARINE V, et al. DEF6 deficiency, a mendelian susceptibility to EBV infection, lymphoma, and autoimmunity [J]. J Allergy Clin Immunol, 2020, 147(2).
ZHONGHAO D, COURTNEY N, KAZUKI I, et al. Def6 regulates endogenous type-I interferon responses in osteoblasts and suppresses osteogenesis [J]. Elife, 2020, 9(0).
ZHEN-PENG Z, LAN-RUO L, TONG-DE L, et al. High expression levels of DEF6 predicts a poor prognosis for patients with clear cell renal cell carcinoma [J]. Oncol Rep, 2020, 44(5).
YAN S, CHANGLU X, ZHONGXIU J, et al. DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1 [J]. Cell Death Dis, 2023, 14(7).
SUSKIEWICZ M, PROKHOROVA E, RACK J, et al. ADP-ribosylation from molecular mechanisms to therapeutic implications [J]. Cell, 2023, 186(21): 4475-95.
ALEMASOVA E, LAVRIK O. Poly(ADP-ribosyl)ation by PARP1: reaction mechanism and regulatory proteins [J]. Nucleic acids research, 2019, 47(8): 3811-27.
HUANG P, CHEN G, JIN W, et al. Molecular Mechanisms of Parthanatos and Its Role in Diverse Diseases [J]. International journal of molecular sciences, 2022, 23(13).
MASATO M, MAYU O, ARINA U, et al. The 89-kDa PARP1 cleavage fragment serves as a cytoplasmic PAR carrier to induce AIF-mediated apoptosis [J]. J Biol Chem, 2020, 296(0).
JING K, FAN Z, YANWEN L, et al. MiR-124 Negatively Regulated PARP1 to Alleviate Renal Ischemia-reperfusion Injury by Inhibiting TNFα/RIP1/RIP3 Pathway [J]. Int J Biol Sci, 2021, 17(8).
VIRGINIE P, ZDENKO H, PAUL O H, et al. Noncleavable poly(ADP-ribose) polymerase-1 regulates the inflammation response in mice [J]. J Clin Invest, 2004, 114(8).
HUIMING X, WEICHENG W, CHUNLIANG L, et al. WWP2 promotes degradation of transcription factor OCT4 in human embryonic stem cells [J]. Cell Res, 2009, 19(5).
AN C, BEIXUE G, JINGPING Z, et al. The HECT-type E3 ubiquitin ligase AIP2 inhibits activation-induced T-cell death by catalyzing EGR2 ubiquitination [J]. Mol Cell Biol, 2009, 29(19).
NAIJIN Z, YING Z, YONG C, et al. BAF155 promotes cardiac hypertrophy and fibrosis through inhibition of WWP2-mediated PARP1 ubiquitination [J]. Cell Discov, 2023, 9(1).
NAIJIN Z, YING Z, WEI M, et al. An unexpected role for BAG3 in regulating PARP1 ubiquitination in oxidative stress-related endothelial damage [J]. Redox Biol, 2022, 50(0).
QINGQING W, ZHENG D. Mouse model of ischemic acute kidney injury: technical notes and tricks [J]. Am J Physiol Renal Physiol, 2012, 303(11).
NAIJIN Z, YING Z, BOQUAN W, et al. Deacetylation-dependent regulation of PARP1 by SIRT2 dictates ubiquitination of PARP1 in oxidative stress-induced vascular injury [J]. Redox Biol, 2021, 47(0).
XIAOXIANG S, HUANYIN T, YU C, et al. Loss of the receptors ER, PR and HER2 promotes USP15-dependent stabilization of PARP1 in triple-negative breast cancer [J]. Nat Cancer, 2023, 4(5).
T C S, E K V D A, J N M I, et al. Improving the outcome of kidney transplantation by ameliorating renal ischemia reperfusion injury: lost in translation? [J]. J Transl Med, 2016, 14(0).
XIRUI L, JUN L, XIAOJUN S, et al. Human urine-derived stem cells protect against renal ischemia/reperfusion injury in a rat model via exosomal miR-146a-5p which targets IRAK1 [J]. Theranostics, 2020, 10(21).
PHUI-LY L, CHIH-YEU F, YU-CHIEH L, et al. DEF6 expression in ovarian carcinoma correlates with poor patient survival [J]. Diagn Pathol, 2016, 11(1).
SHAO G, HE J, MENG J, et al. Ganoderic Acids Prevent Renal Ischemia Reperfusion Injury by Inhibiting Inflammation and Apoptosis [J]. International journal of molecular sciences, 2021, 22(19).
DEVALARAJA-NARASHIMHA K, SINGARAVELU K, PADANILAM B. Poly(ADP-ribose) polymerase-mediated cell injury in acute renal failure [J]. Pharmacological research, 2005, 52(1): 44-59.
MEZA-SOSA K, MIAO R, NAVARRO F, et al. SPARCLE, a p53-induced lncRNA, controls apoptosis after genotoxic stress by promoting PARP-1 cleavage [J]. Molecular cell, 2022, 82(4): 785-802.e10.

No competing interests reported.

SupplementaryMaterial.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

DEF6 regulates renal ischemia reperfusion injury through suppressing the WWP2 mediated ubiquitination of PARP1

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials and Methods

Animals and treatment

Production of DEF6-KO mice

Cell culture and treatment

Histological staining

Immunofluorescence

Terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling

Transfection

Quantitative real-time PCR

Western blotting

Immunoprecipitation

Statistical analysis

Results

DEF6 is upgraded in experimental models of RIRI

Inhibiting DEF6 alleviated RIRI-induced inflammation and apoptosis in vivo

DEF6 regulated inflammation and apoptosis of HK2 cells induced by hypoxia/reoxygenation in vitro

DEF6 regulated inflammation and apoptosis of HK2 cells through targeting PARP1

DEF6 upregulated protein expression of PARP1 via competitively inhibiting the ubiquitination of WWP2

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1