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Observational Study
. 2021 Apr 13:12:650424.
doi: 10.3389/fimmu.2021.650424. eCollection 2021.

Impaired ATG16L-Dependent Autophagy Promotes Renal Interstitial Fibrosis in Chronic Renal Graft Dysfunction Through Inducing EndMT by NF-κB Signal Pathway

Zeping Gui  1   2 , Chuanjian Suo  1 , Zijie Wang  1 , Ming Zheng  1 , Shuang Fei  1 , Hao Chen  1 , Li Sun  1 , Zhijian Han  1 , Jun Tao  1 , Xiaobin Ju  1 , Haiwei Yang  1 , Min Gu  2 , Ruoyun Tan  1
Affiliations
Observational Study

Impaired ATG16L-Dependent Autophagy Promotes Renal Interstitial Fibrosis in Chronic Renal Graft Dysfunction Through Inducing EndMT by NF-κB Signal Pathway

Zeping Gui et al. Front Immunol. .

Abstract

Chronic renal graft dysfunction (CAD) is caused by multiple factors, including glomerular sclerosis, inflammation, interstitial fibrosis and tubular atrophy (IF/TA). However, the most prominent elements of CAD are IF/TA. Our studies have confirmed that endothelial-mesenchymal transition (EndMT) is an important source to allograft IF/TA. The characteristic of EndMT is the loss of endothelial marker and the acquisition of mesenchymal or fibroblastic phenotypes. Autophagy is an intracellular degradation pathway that is regulated by autophagy-related proteins and plays a vital role in many fibrotic conditions. However, whether or not autophagy contributes to fibrosis of renal allograft and how such mechanism occurs still remains unclear. Autophagy related 16 like gene (ATG16L) is a critical autophagy-related gene (ARG) necessary for autophagosome formation. Here, we first analyzed kidney transplant patient tissues from Gene Expression Omnibus (GEO) datasets and 60 transplant patients from our center. Recipients with stable kidney function were defined as non-CAD group and all patients in CAD group were histopathologically diagnosed with CAD. Results showed that ATG16L, as one significant differential ARG, was less expressed in CAD group compared to the non-CAD group. Furthermore, we found there were less autophagosomes and autolysosomes in transplanted kidneys of CAD patients, and downregulation of autophagy is a poor prognostic factor. In vitro, we found out that the knockdown of ATG16L enhanced the process of EndMT in human renal glomerular endothelial cells (HRGECs). In vivo, the changes of EndMT and autophagic flux were then detected in rat renal transplant models of CAD. We demonstrated the occurrence of EndMT, and indicated that abundance of ATG16L was accompanied by the dynamic autophagic flux change along different stages of kidney transplantation. Mechanistically, knockdown of ATG16L, specifically in endothelial cells, reduced of NF-κB degradation and excreted inflammatory cytokines (IL-1β, IL-6 and TNF-α), which could facilitate EndMT. In conclusion, ATG16L-dependent autophagic flux causing by transplant showed progressive loss increase over time. Inflammatory cytokines from this process promoted EndMT, thereby leading to progression of CAD. ATG16L served as a negative regulator of EndMT and development of renal graft fibrosis, and autophagy can be explored as a potential therapeutic target for chronic renal graft dysfunction.

Keywords: ATG16L; EndMT; autophagy; chronic renal graft dysfunction; inflammatory cytokines; renal interstitial fibrosis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Pathological and morphological changes in kidney tissues from the non-CAD and CAD patients. (A) Representative kidney sections from the non-CAD and CAD patients were stained with HE and Masson trichrome (×100, scale bar: 50 μm, ×200, scale bar: 20 μm). (B) Semi-quantitative analyses of the degree of fibrosis in 4 non-CAD patients’ and 4 clinical CAD patients’ kidney sections stained with Masson trichrome were performed. Each patient selected 5 different sites of transplanted kidney. (***P< 0.001, CAD vs. the non-CAD group, Student t test). (C, E, G) Distributions and expressions of FN, α-SMA and CD31 in the CAD and non-CAD group were assessed by IHC staining assays (×400, scale bar: 10 μm). (D, F, H) Percentage of the relative abundance of proteins were presented as the mean ± SD values of five independent experiments. Representative images of the kidney tissues of the non-CAD group (n = 4) and CAD group (n = 4) were shown. Each patient selected 5 different sites of transplanted kidney. (***P < 0.001, CAD vs. the non-CAD group, Student t test).
Figure 2
Figure 2
ATG16L expressions in CAD and the non-CAD patients. (A) The diagram of the dataset recruitment workflow (B) Volcano plot of differential gene expression analysis between non-CAD (n=21) and CAD (n=25) kidney tissues from GEO profiles (GSE9493) (C) Heatmap indicating the differential expressions of the 20 selected ARGs. Expression values were presented in red and green to indicate expression upregulation and downregulation, respectively. (D) Representative western blot assay results of the protein expressions of ATG16L and FN in normal, non-CAD and CAD patient kidney tissues from our center. (E) Representative IHC images of ATG16L expressions in normal, non-CAD and CAD tissue (n=4) (×100, ×200, scale bar: 25 μm). Each patient selected 5 different sites of transplanted kidney. Semi-quantitative analyses of IHC staining were reported (***P < 0.001, vs. the non-CAD group, Student t test). (F) The correlation analysis between ATG16L and area of renal interstitial fibrosis (r=0.613, P < 0.01), (G) The correlation analysis between ATG16L and FN (r=0.372, P =0.105), (H) The correlation analysis between ATG16L and α-SMA (r=0.681, P < 0.01), (I) The correlation analysis between ATG16L and CD31(r=0.537, P < 0.05) were determined using Pearson correlation analysis. (n = 4, biological independent samples. Each patient selected 5 different sites of transplanted kidney).
Figure 3
Figure 3
Autophagy, MAP1LC3 expression and prognosis relationship in CAD patients. (A) Representative TEM images of autophagic vacuoles (red double arrow) were shown in glomerulus cells from normal, non-CAD and CAD patients (n=4) (scale bar: 10 μm and 500 μm). (B) Qualitative analysis of the number of autophagic vacuoles from three groups under TEM were shown. (***P < 0.001, Student t test.) (C) Representative western blot assay results of the protein expressions of SQSTM1 and LC3BI/II in normal, non-CAD and CAD patient kidney tissues were shown. (n=4) (D) Semi-quantitative analysis of Western blot assay results of SQSTM1 and LC3BI/II in normal, non-CAD and CAD patient kidney tissues. (***P < 0.001, Student t test.) (E) Serum concentrations of MAP1LC3 by ELISA assay in 30 non-CAD patients and 30 CAD patients with different degree. (***P < 0.001, **P < 0.01, vs. the non-CAD group, Student t test.) (F) Survival analysis of different MAPLC3 serum concentrations in 30 CAD patients based on data from ELISA assay. (P< 0.05, Student t test).
Figure 4
Figure 4
Effects on autophagy and progression of EndMT in HRGECs after knockdown of ATG16L. (A) Representative TEM images of autophagic vacuoles (red arrow) were shown in shNC group and shATG16L group (n=5) (scale bar: 10 μm, 2 μm and 500 μm). (B) Qualitative analysis of the number of autophagic vacuoles from two groups under TEM were shown. (***P < 0.001, Student t test.) (C) Western blot assay results of the ATG16L, SQSTM1 and LC3BI/II proteins in HRGECs transfected with shNC or shATG16L. (n=5) (D) Expressions of FN, α-SMA and CD31 protein in HRGECs transfected with shNC or shATG16L. (n=5) (E) Expressions of ATG16L, FN, α-SMA and CD31 protein in HRGECs overexpressed of ATG16L or not. (n=5) (F) Representative images of IF staining of FN and CD31 in HRGECs transfected with shNC or shATG16L. Scale bar: 20 μm (n=5).
Figure 5
Figure 5
Changes of renal interstitial fibrosis, urine protein, renal function and EndMT in chronic rejection rat model. (A) Representative images of HE and Masson’s trichrome staining from kidney tissues of syn and allo groups (scale bar: 25 μm). (B) The statistical analyses of tubulointerstitial damage scores in syn and allo group. Data were expressed as the mean ± SD of each group. (n = 5, ***P < 0.001, **P < 0.01, vs. the same weeks syn group, Student t test.) (C) Semi-quantitative analyses of positive areas of renal interstitial fibrosis were performed from Masson staining assay results. Data were expressed as the mean ± SD of each group (n = 5, ***P < 0.001, **P < 0.01, vs. the same weeks syn group, Student t test.). (D) Renal function parameters (serum Cr and BUN) and 24h urine protein quantitation in syn and allo group. Values represent the mean ± SD, (n = 5, ***P < 0.001, *P < 0.05, vs. the same weeks syn group, Student t test.) (E) Representative IHC staining images of EndMT markers (FN, α-SMA and CD31) in kidney sections from syn group and 16 weeks allo group rats (scale bar: 25 μm). (F) Representative western blot analysis results of EndMT markers in rat kidney tissues from syn group and different allo group. (n=5).
Figure 6
Figure 6
Changes of autophagy activity in chronic rejection rat models. (A) Representative images of cortical kidney sections obtained from syn and different periods after kidney transplanted rats injected with AAV-mRFP-GFP-LC3. Arrows indicated the glomerular site. Scale bar: 20 μm (B) Semi-quantification analyses of the percentage of autolysosomes (red puncta/total puncta) in the images. The data were presented as mean ± SD (n = 5, ***P < 0.001, *P < 0.05, vs. the syn group). More than 100 cells were quantified for each image in each experiment. (C) Representative IHC staining images of ATG16L expressions in syn group, 8 weeks and 16 weeks allo group rats. (n=5) (D) Representative western blot assay results of ATG16L and LC3BI/II levels in kidney tissue from syn and allo group rats. (n=5).
Figure 7
Figure 7
NF-κB pathway activation and effect on EndMT in HRGECs after knockdown of ATG16L (A) The RNA sequencing results of differential genes in HRGECs transfected with shNC or shATG16L were plotted by volcano plot. (n=5) (B) KEGG pathway analyses of the top 20 KEGG enriched gene pathways. (C) GSEA plot of the correlation between knockdown of ATG16L and NF-κB enrichment gene signatures in the RNA sequencing results. (D) Representative images of p65 expression and distribution by indirect immunofluorescence staining assay in HRGECs transfected with shNC or shATG16L under confocal microscopy. Scale bar: 50 μm. (n=5) (E) Representative western blot results of the effects of NF-κB inhibitor (QNZ) on NF-κB nuclear translocation and EndMT markers (FN, α-SMA and CD31) expressions in HRGECs transfected with shATG16L or not. (n=5) (F) Representative western blot results of p65 and p-p65 (Ser536) expressions after knocking down ATG16L for different time. (n=5) (G) Representative western blot results of p65 and p-p65 (Ser536) expressions with CHX treatment for different time in shNC and shATG16L group. (n=5) (H) The mRNA levels of p65 in HRGECs transfected with shNC or shATG16L. (n=5, NS, not significant, vs. the shNC group, Student t test.) (I) Representative images of p65 expression and distribution in the non-CAD and CAD allograft renal tissues via indirect immunofluorescence staining assay (scale bar: 25 μm). (J) Semi-quantitative analysis results for indirect immunofluorescence staining assay of p65 in the non-CAD and CAD allograft renal tissue (n = 4, ***P < 0.001, vs. the non-CAD group, Student t test).
Figure 8
Figure 8
Cytokines induced by NF-κB pathway activation and effects on progression of EndMT in HRGECs. (A) The graphs of the results of mRNA abundance of IL-1β, IL-6 or TNF‐α after knocking down ATG16L with or without QNZ. (n = 5, ***P < 0.001, **P < 0.01, vs. the shNC group, Student t test.) (B) Representative western blotting results of the expressions of FN, CD31, α-SMA and GAPDH in HRGECs after IL-1β, IL-6 or TNF-α treatment for different times or dosage. (n=5) (C) Representative western blot results of FN, CD31, α-SMA and GAPDH expressions treated with or without Anakinra, Tocilizumab and Infliximab in HRGECs transfected with shATG16L or not. (n=5) (D) A model is proposed to illustrate the mechanisms involved in EndMT induced by loss of ATG16L in the pathogenesis of CAD and transplanted renal interstitial fibrosis.
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