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

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.

Everolimus Alleviates Renal Allograft Interstitial Fibrosis by Inhibiting Epithelial-to-Mesenchymal Transition Not Only via Inducing Autophagy but Also via Stabilizing IκB-α

“…In our previous study, we proved the increase of EMT in rat allograft kidney after kidney transplantation ( 24 ). Here, we explored the role of EVR in ameliorating the progression of renal allograft EMT after transplantation.…”

“…The clinical utility of EVR is dependent on its ability to inhibit the mTOR pathway activation, which is frequently involved in protein synthesis, cell cycle and growth, angiogenesis, and autophagy. In our previous study, we discovered that autophagic flux generated by kidney transplantation gradually decreased over time ( 24 ). Therefore, we hypothesized that autophagy was involved in the effect of EVR on the progression of EMT and allograft renal interstitial fibrosis.…”

“…For tissue indirect immunofluorescence staining, the staining method was the same as in ( 24 ). For HK2 cell indirect immunofluorescence staining, a total of 5 × 10 5 cells were seeded into laser confocal dishes with a preplaced glass slide.…”

“…Total RNAs were extracted from tissues and cells with the RNA Extraction Kits (TIANGEN, Beijing, China). Detailed steps are described in ( 24 ). Gene expressions were measured by real-time PCR assay (Vazyme, Nanjing, China) and a DNA Engine Opticon 2 System (BioRad Laboratories, Hercules, CA, USA).…”

“…Our research group previously revealed that ATG16L-dependent autophagic flux decreased gradually over time following kidney transplantation. In that study, we found that autophagy was downregulated in CAD patients and decreased LC3 predicted poor prognosis in CAD patients ( 24 ). Additionally, autophagy and EMT are two critical regulators of organ fibrosis progression and are closely related according to recent evidence ( 25 ).…”