Upregulation of Interleukin-33 in obstructive renal injury

https://doi.org/10.1016/j.bbrc.2016.04.010Get rights and content

Highlights

  • Interleukin (IL)-33 was upregulated in obstructed kidneys.

  • Interstitial myofibroblasts expressed IL-33 after UUO-induced renal injury.

  • Deficiency of IL33 reduced interstitial fibrosis and promoted tubular cell proliferation.

Abstract

Interstitial fibrosis and loss of parenchymal tubular cells are the common outcomes of progressive renal diseases. Pro-inflammatory cytokines have been known contributing to the damage of tubular cells and fibrosis responses after renal injury. Interleukin (IL)-33 is a tissue-derived nucleus alarmin that drives inflammatory responses. The regulation and function of IL-33 in renal injury, however, is not well understood. To investigate the involvement of cytokines in the pathogenesis of renal injury and fibrosis, we performed the mouse renal injury model induced by unilateral urinary obstruction (UUO) and analyze the differentially upregulated genes between the obstructed and the contralateral unobstructed kidneys using RNA sequencing (RNAseq). Our RNAseq data identified IL33 and its receptor ST2 were upregulated in the UUO kidney. Quantitative analysis confirmed that transcripts of IL33 and ST2 were upregulated in the obstructed kidneys. Immunofluorescent staining revealed that IL-33 was upregulated in Vimentin- and alpha-SMA-positive interstitial cells. By using genetically knockout mice, deletion of IL33 reduced UUO-induced renal fibrosis. Moreover, in combination with BrdU labeling technique, we observed that the numbers of proliferating tubular epithelial cells were increased in the UUO kidneys from IL33-or ST2-deficient mice compared to wild type mice. Collectively, our study demonstrated the upregulation of IL-33/ST2 signaling in the obstructed kidney may promote tubular cell injury and interstitial fibrosis. IL-33 may serve as a biomarker to detect renal injury and that IL-33/ST2 signaling may represent a novel target for treating renal diseases.

Introduction

Renal interstitial fibrosis and parenchymal tubular cell loss are common outcomes of chronic renal diseases and remains the predictor of disease progression [1], [2]. Fibrosis results from accumulation of extracellular matrix proteins with replacement of normal tissue with scar tissues and the fibrotic response is broadly considered irreversible in renal disease [1], [2]. The animal model of unilateral ureteral obstruction (UUO) has been widely used to study the mechanisms of renal diseases [3]. Obstructive renal injury leads to the formation of atubular glomeruli, proximal tubular cell loss, immune cells infiltration, collecting ducts remodeling, and interstitial fibrosis [4]. Cytokines are involved in the progression of renal fibrosis and tissue damage [5]. Expression of pro-inflammatory (e.g., TNF-α, IFN-γ, IL-1β, IL-6, IL-17, IL-23) and anti-inflammatory (e.g., IL-4, TGF-β, IL-10) cytokines by resident and infiltrated immune cells are important mediators of the injury and repair responses [6].

Interleukin (IL)-33 is an IL-1 family cytokine and expressed in the nucleus of endothelial cells, fibroblasts, and epithelial cells in various tissues as a nuclear alarmin [7], [8], [9]. In response to mechanical stress or cellular injury, IL-33 is released to the extracellular space and elicits inflammatory responses [10], [11]. Elevated IL-33 levels have been linked with tissue fibrosis in the lung and liver [12], [13], [14]. Recent study revealed that IL-33 exacerbates cisplatin-induced acute renal injury, which suggested a pro-inflammatory role of IL-33 in the kidney diseases [15]. However, the regulation and the function of IL-33 in renal injury remain unclear.

IL1RL1 gene encodes the IL-33 receptor ST2 [7]. At least two ST2 isoforms are identified; the membrane form ST2L and the soluble form sST2 [16]. ST2L binds to IL-33 and activates intracellular signals, whereas sST2 functions as a decoy receptor [8]. Serum sST2 levels have been considered as a useful biomarker for acute and chronic inflammatory diseases [17], [18], [19], [20].

This study aimed to investigate the involvement of the key factors in the progression of renal injury in the mouse model of UUO. We performed RNA sequencing analysis and identified that IL33 and its receptor ST2 were dramatically upregulated in the obstructed kidney at day 4 after UUO. By using immunofluorescent staining, we confirmed that IL-33 protein was upregulated in the interstitial myofibroblasts and peritubular vascular endothelial cells. Moreover, mice with deficiency of IL33 showed preserved parenchymal proximal tubule, inhibited renal interstitial fibrosis, and increased the number of proliferating tubular epithelial cells in the obstructed kidneys. Our study suggested that IL-33/ST2 axis represented a novel mediator of renal injury and a potential target for treating progressive renal diseases.

Section snippets

Animals

All animal experiments were conducted in accordance with the Guide for the Use and Care of Laboratory Animals and approved by the IACUC Committee of Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan. IL33 fl/fl mice and ST2 fl/fl mice were kindly provided by Dr. Richard T. Lee (Brigham & Women's Hospital, Boston, MA, USA) and maintained on the C57BL/6 background as previous described [11].

The IL33−/− and ST2−/− were generated from IL33 fl/fl and ST2 fl/fl mice by crossing the mice to

Unilateral ureteral obstruction (UUO)-induced renal interstitial fibrosis

To determine whether IL-33 is involved in the progression of obstructive nephropathy, we generated the unilateral ureteral obstruction (UUO) model in mice and compared the pathological changes and gene expressions between the obstructed and contralateral control kidneys. The MR imaging analysis showed that UUO induced renal cortical and medullar thinning, and reduced the parenchymal thickness in UUO kidney but not in contralateral kidneys (Fig. 1A and B). Massons' Trichrome staining (MTS)

Discussion

Our study demonstrated that (1) gene expressions of IL33 and its receptor ST2 are induced in the kidneys in response to obstructive injury; (2) IL-33 protein is upregulated and expressed in Vimentin+/αSMA+ interstitial cells and CD31+ peritular vascular endothelial cells in the injured kidneys; (4) Disruption of IL33 but not ST2 reduced UUO-induced interstitial fibrosis; (5) Deficiency of IL33 or ST2 attenuated UUO-induced loss of parenchymal tubules and increased proliferation of tubular

Acknowledgement

This study was supported by Grants from Chang Gung Medical Foundation (CMRPG8D0231, CMRPG8D0232, CMRPG8D0661, CMRPG8D0662, CMRPG8E0761, CMRPG8B0573), and the Ministry of Science and Technology of Taiwan (104-2320-B-182A-014-, 103-2320-B-182A-017-, 103-2320-B-182A-011-MY3). We thank Ms. Wan-Chen Wang, Ms. Ya-Ting Chang, Ms. Ru-Fang Li, and Mr. Lo-Hsin Dai at ITRBM, Kaohsiung Chang Gung Memorial Hospital for their technical assistance in this study. The C57BL/6-Tg (UBC-Cre) 8Narl (RMRC13217) mice

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