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. 2022 Nov 15;13(11):957.
doi: 10.1038/s41419-022-05394-4.

IL-18 deficiency ameliorates the progression from AKI to CKD

Junjun Luan  1 , Jingqi Fu  2 , Congcong Jiao  1 , Xiangnan Hao  1 , Zixuan Feng  1 , Lingzi Zhu  1 , Yixiao Zhang  3 , Guangyu Zhou  1 , Hongyu Li  4 , Wei Yang  4 , Peter S T Yuen  5 , Jeffrey B Kopp  6 , Jingbo Pi  7 , Hua Zhou  8
Affiliations

IL-18 deficiency ameliorates the progression from AKI to CKD

Junjun Luan et al. Cell Death Dis. .

Abstract

Inflammation is an important factor in the progression from acute kidney injury (AKI) to chronic kidney disease (CKD). The role of interleukin (IL)-18 in this progression has not been examined. We aimed to clarify whether and how IL-18 limits this progression. In a folic acid induced renal injury mouse model, we studied the time course of kidney injury and renal IL-18 expression. In wild-type mice following injection, renal IL-18 expression increased. In parallel, we characterized other processes, including at day 2, renal tubular necroptosis assessed by receptor-interacting serine/threonine-protein kinase1 (RIPK1) and RIPK3; at day 14, transdifferentiation (assessed by transforming growth factor β1, vimentin and E-cadherin); and at day 30, fibrosis (assessed by collagen 1). In IL-18 knockout mice given folate, compared to wild-type mice, tubular damage and necroptosis, transdifferentiation, and renal fibrosis were attenuated. Importantly, IL-18 deletion decreased numbers of renal M1 macrophages and M1 macrophage cytokine levels at day 14, and reduced M2 macrophages numbers and macrophage cytokine expression at day 30. In HK-2 cells, IL-18 knockdown attenuated necroptosis, transdifferentiating and fibrosis.In patients with tubulointerstitial nephritis, IL-18 protein expression was increased on renal biopsies using immunohistochemistry. We conclude that genetic IL-18 deficiency ameliorates renal tubular damage, necroptosis, cell transdifferentiation, and fibrosis. The renoprotective role of IL-18 deletion in the progression from AKI to fibrosis may be mediated by reducing a switch in predominance from M1 to profibrotic M2 macrophages during the process of kidney repair.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. IL-18 deletion attenuates acute and chronic kidney injury in folic acid-induced mice.
A The temporal changes of serum creatinine (Scr) and B renal mRNA of IL-18 expression following folic acid (FA) injection to wild type mice (WT). C, D kidney injuries on PAS staining and the semi-quantification of the injuries (C) and immunohistochemistry staining of IL-18 and its semi-quantification (D). E Double immunofluorescence staining of IL-18 (green) and AQP-1 (red) in the relative normal renal cortex tubular cells in WT mice at day 30 after FA injection. F Blood urea nitrogen (BUN) and Serum interferon-γ (IFN-γ) in IL-18 knockout mice (IL-18 KO) compared to WT mice. G PAS staining and its semi-quantification in renal cortex and outer medulla (OSOM). Red arrow indicates IL-18 positive staining. $Normal brush border, &loss of brush border and dilation of tubular lumen, *Protein cast, #Detachment of tubular epithelial cells from tubular basement membrane and debris in tubular lumen. Yellow arrows indicate the infiltration of inflammatory cells. Magnification, 400 × bar = 50 um. Data represents Mean ± SD. (n = 6, #p < 0.05, other groups vs. day 0; *p < 0.05, KO vs. WT).
Fig. 2
Fig. 2. IL-18 deletion ameliorated renal necroptosis in folic acid-injected mice.
A Renal protein expression of RIPK1 and RIPK3, two necroptosis biomarkers, peaked at day 2, analyzed by Western blotting and the density of the blots. B TUNEL staining and semi- quantitative analysis in renal cortex and outstrip of outer medulla (OSOM). Magnification, 200× bar = 100 um. Data represent Mean ± SD (n = 6, #p < 0.05, other groups vs. day 0; *p < 0.05, KO vs. WT).
Fig. 3
Fig. 3. IL-18 deletion suppressed tubular cell transdifferentiation at day 14 after folic acid injection to mice.
A Renal mRNA of TGF-β1, and Vimentin. B Renal proteins of TGF- β1, vimentin, and E-cadherin analyzed by Western blotting and the density of blots. C Immunofluorescent (IF) staining of TGF-β1 and immunohistochemistry (IHC) staining of vimentin (red arrows) and semi quantitative analysis in renal cortex. Magnification, 400×, bar = 50 um. Data represent Mean ± SD (n = 6, #p < 0.05, other groups vs. day 0; *p < 0.05, KO vs. WT).
Fig. 4
Fig. 4. IL-18 deletion inhibited renal fibrosis induced by folic acid.
A The kidney size started shrinking from day 14 and became much smaller at day 30 after folic acid injection. B Renal mRNA expression of COL-1. C Renal protein expression of COL-1 analyzed by Western blotting and its semi-quantification. D Immunohistochemistry (IHC) staining and semi-quantification of COL-1 (red arrows) in renal cortex. Magnification, 400×, bar = 50 um. Data represent Mean ± SD (n = 6, #p < 0.05, other groups vs. day 0; *p < 0.05, KO vs. WT).
Fig. 5
Fig. 5. IL-18 deletion reduced the renal infiltration of total macrophages after folic acid injection.
A mRNAs of total macrophages indicated by CD68 and F4/80 progressively increased after the folic acid injection. B Protein expression of F4/80 analyzed by Western blotting and its semi-quantification. C Immunohistochemistry (IHC) staining of F4/80 and semi-quantification in renal cortex. Magnification, 400×, bar = 50 um. Data represent Mean ± SD (n = 6, #p < 0.05, other groups vs. day 0; *p < 0.05, KO vs. WT).
Fig. 6
Fig. 6. IL-18 deletion decreased renal infiltration of M1 and M2 macrophages triggered by folic acid injection.
A CD11c, a M1 biomarker, peaked at day 14 and thenafter decreased at day 30. B, C M1 secretory factors such as iNOS, and CXCL10 showed similar change directions to CD11c. D Immunohistochemistry (IHC) staining of CD11c (red arrow) and its semi-quantification in renal cortex. E IL-4, an inducer of M2 from macrophage, F CD206, a M2 biomarker, responded same as IL-4. G CCL26, a M2 secreted cytokine were progressively increased and peaked at day 30 after folic acid injection analyzed by qPCR. H IHC staining of CD206 protein (red arrows) and its semi-quantification in renal cortex. Magnification, 400×, bar = 50 um. Data represent Mean ± SD (n = 6, #p < 0.05, other groups vs. day 0; *p < 0.05, KO vs. WT).
Fig. 7
Fig. 7. IL-18 knockdown attenuated necroptosis, transdifferentiation, and fibrosis in HK-2 cells.
A The mRNA expression of IL-18 in HK-2 cells with hTGF-β stimulation by qPCR. B The protein expression of IL-18 HK-2 cells treated with hTGF-β and additional transfected with IL-18 siRNA and its scramble analyzed by Western blotting. C The protein expression of necroptosis indicated by RIPK1 and RIPK3 at 12 h after the transfection of IL-18 siRNA on Western blotting. D The expression of transdifferentiation marked by TGF- β1, vimentin, and E-cadherin at 24 h after the transfection of IL-18 siRNA on Western blotting. E The expression of fibrotic COL-1 at 48 h after the transfection of IL-18 siRNA on Western blotting (n = 3, #p < 0.05, other groups vs. NC; *p < 0.05, IL-18 siRNA vs. IL-18 scramble).
Fig. 8
Fig. 8. IL-18 increased in renal biopsies from patients with interstitial nephritis (TIN).
A PAS staining. B Masson staining. C IHC staining of IL-18 (red arrow). Magnification, 200×, bar = 200 um.
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