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Arid2-IR promotes NF-κB-mediated renal inflammation by targeting NLRC5 transcription

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Abstract

Increasing evidence shows that long non-coding RNAs (lncRNAs) play an important role in a variety of disorders including kidney diseases. It is well recognized that inflammation is the initial step of kidney injury and is largely mediated by nuclear factor Kappa B (NF-κB) signaling. We had previously identified lncRNA-Arid2-IR is an inflammatory lncRNA associated with NF-κB-mediated renal injury. In this study, we examined the regulatory mechanism through which Arid2-IR activates NF-κB signaling. We found that Arid2-IR was differentially expressed in response to various kidney injuries and was induced by transforming growth factor beta 1(TGF-β1). Using RNA sequencing and luciferase assays, we found that Arid2-IR regulated the activity of NF-κB signal via NLRC5-dependent mechanism. Arid2-IR masked the promoter motifs of NLRC5 to inhibit its transcription. In addition, during inflammatory response, Filamin A (Flna) was increased and functioned to trap Arid2-IR in cytoplasm, thereby preventing its nuclear translocation and inhibition of NLRC5 transcription. Thus, lncRNA Arid2-IR mediates NF-κB-driven renal inflammation via a NLRC5-dependent mechanism and targeting Arid2-IR may be a novel therapeutic strategy for inflammatory diseases in general.

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Abbreviations

UUO:

Unilateral ureteral obstruction

mTEC:

Primary mouse tubular epithelial cells

DKD:

Diabetic kidney disease

IRI:

Ischemia Reperfusion Injury

SSC:

Saline Sodium Citrate

PBS:

Phosphate-buffered saline

Flna:

Filamin A

NLRC5:

NLR family CARD domain containing 5

qPCR:

Quantitative real-time PCR

RIP:

RNA immunoprecipitation

DEGs:

Differentially expressed genes

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Acknowledgements

This study was supported by the following Grants: Guangzhou Science, Technology and Innovation Commission (201806010123), Guangdong Basic and Applied Basic Research Foundation(2020A1515010247), Kelin Young Talents Program of the First Affiliated Hospital of Sun Yat-sen University (Y50179) National Key R&D Program of China (2016YFC0906101), Operational Grant of Guangdong Provincial Key Laboratory(2017B030314019), Guangdong Provincial Programme of Science and Technology(2017A050503003), Guangdong Provincial Programme of Science and Technology(2017B020227006), Guangzhou Municipal Programme of Science and Technology(201704020167), The Research Grants Council of Hong Kong (14163317, 14117418, 14104019, R4012-18F, and C7018-16G). The Health and Medical Research Fund of Hong Kong (HMRF 05161326, 14152321); The Guangdong-Hong Kong-Macao-Joint Labs Program from Guangdong Science and Technology Department (2019B121205005).

Author information

Author notes

  1. Puhua Zhang, Chaolun Yu, and Jianwen Yu contributed equally to this work.

Authors and Affiliations

  1. Department of Nephrology, The First Affiliated Hospital, Sun Yat-Sen University, Zhongshan Road II, Guangzhou, 510080, Guangdong, China

    Puhua Zhang, Jianwen Yu, Zhijian Li & Qin Zhou

  2. National Health Commission Key Laboratory of Nephrology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, 510080, Guangdong, China

    Puhua Zhang, Jianwen Yu, Zhijian Li & Qin Zhou

  3. Guangdong Provincial Key Laboratory of Nephrology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, 510080, Guangdong, China

    Puhua Zhang, Jianwen Yu, Zhijian Li & Qin Zhou

  4. Department of Endocrinology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510080, Guangdong, China

    Chaolun Yu

  5. Department of Medicine and Therapeutics, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, 999077, China

    Hui-yao Lan

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  1. Puhua Zhang
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Contributions

HL and QZ designed the study and revised the manuscript. PZ and CY performed the experiments and wrote the manuscript. JY collected data and did the statistical analysis. ZL prepared figures. All authors have read and approved the final submitted manuscript.

Corresponding author

Correspondence to Qin Zhou.

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Supplementary Table 1. All Arid2-IR-related DEGs identified by RNA-seq (XLSX 36 kb)

Supplementary Table 2. GO analysis of all significant Arid2-IR-related DEGs (FDR<0.05) (XLSX 27 kb)

Supplementary Table 3. KEGG analysis of all Arid2-IR-related DEGs (FDR<0.05) (XLSX 11 kb)

18_2020_3659_MOESM4_ESM.xlsx

Supplementary Table 4. Arid2-IR associated proteins or peptides identified by mass spectrometry in mTECs untreated or treated by IL-1β (10 ng/mL) for 30min (XLSX 30 kb)

18_2020_3659_MOESM5_ESM.xlsx

Supplementary Table 5. Top ten significant GO catalogs of potential Arid2-IR interacting proteins identified by mass spectrometry (XLSX 17 kb)

18_2020_3659_MOESM6_ESM.xlsx

Supplementary Table 6. Top fifteen significant KEGG pathways of potential Arid2-IR interacting proteins identified by mass spectrometry (XLSX 14 kb)

Supplementary Table 7. All the primers, probes and siRNA sequences used in this study (XLSX 11 kb)

Supplementary File 1. Sequences of Arid2-IR full length (DOCX 13 kb)

18_2020_3659_MOESM9_ESM.ab1

Supplementary File 2a. DNA sequencing results of pGL3-NLRC5-promoter. The inserted nucleotides were sequenced by RV3 primers. (AB1 271 kb)

18_2020_3659_MOESM10_ESM.ab1

Supplementary File 2b. DNA sequencing results of pGL3-NLRC5-promoter. The inserted nucleotides were sequenced by GLp2 primers (AB1 269 kb)

18_2020_3659_MOESM11_ESM.png

Supplementary Figure 1. QPCR was used to detect relative level of NLRC5 in Arid2-IR siRNA-1, siRNA-2, siRNA-3 or NC siRNA transfected mTECs. ***p < 0.001 versus NC siRNA, ##p < 0.01 Arid2-IR siRNA-1versus Arid2-IR siRNA-2.A si-1, 2, 3 were short for Arid2-IR siRNA-1, 2, 3 (PNG 7 kb)

18_2020_3659_MOESM12_ESM.png

Supplementary Figure 2. Statistical of Flna and NLRC5 protein level in UUO(A) and IRI(B) kidney respectively. * p < 0.05 versus control mice (PNG 344 kb)

18_2020_3659_MOESM13_ESM.pdf

Supplementary Figure 3. (A) Statistical of the protein level of phosho-IKK, IKKβ, phosho-p65, p65, phosho-IκB, IκB in IL-1β (10 ng/mL) treated mTECs at different time points. *p < 0.05, ***p < 0.001 versus 0h. (B)Statistical of NLRC5 protein level in NC or Arid2-IR siRNA-1 transfected mTECs. MTECs were then treated by IL-1β (10 ng/mL) at different time points. *p < 0.05, **p < 0.01, ***p < 0.001 versus 0h. (C) Statistical of NLRC5 protein level in NLRC5 or NC siRNA transfected mTECs. **p < 0.01 versus con; ###p < 0.001 versus NC. (D) Statistical of the protein level of phosho-IKK, IKKβ, phosho-p65, p65, phosho-IκB, IκB in IL-1β (10 ng/mL) treated mTECs at 0, 5m, 30m. **p < 0.01, ***p < 0.001 versus 0h; ##p < 0.01, ###p < 0.001 versus NC siRNA at same time point; &&p < 0.01, &&&p < 0.001 versus Arid2-IR siRNA-1 at same time point (PDF 160 kb)

18_2020_3659_MOESM14_ESM.png

Supplementary Figure 4. (A)Statistical of Flna protein level in NC or Flna siRNA treated mTECs. **p < 0.01 versus con; ##p < 0.01 versus NC. (B)The apoptosis rate of mTECs had no significantly change after transfecting with transfected with Arid2-IR siRNA-1 or NC siRNA (n = 3 for each group) (PNG 39 kb)

18_2020_3659_MOESM15_ESM.png

Supplementary Figure 5. Immunofluorescence of E-cadherin (green, left panel) and a-SMA (green, right panel) of mTECs, Over 95% cells were E-cadherin positive and a-SMA negative. DAPI staining is blue. Magnification: ×200 (PNG 379 kb)

18_2020_3659_MOESM16_ESM.png

Supplementary Figure 6. Pearson’ s correlation analysis was performed base on transcript abundance (RPKM) of the gene in Arid2-IR siRNA-1 (n=3) and NC (n=3) siRNA transfected groups (PNG 133 kb)

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Zhang, P., Yu, C., Yu, J. et al. Arid2-IR promotes NF-κB-mediated renal inflammation by targeting NLRC5 transcription. Cell. Mol. Life Sci. 78, 2387–2404 (2021). https://doi.org/10.1007/s00018-020-03659-9

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