Tubular cells produce FGF2 via autophagy after acute kidney injury leading to fibroblast activation and renal fibrosis

Abstract

ABSTRACT Following acute kidney injury (AKI), renal tubular cells may stimulate fibroblasts in a paracrine fashion leading to interstitial fibrosis, but the paracrine factors and their regulation under this condition remain elusive. Here we identify a macroautophagy/autophagy-dependent FGF2 (fibroblast growth factor 2) production in tubular cells. Upon induction, FGF2 acts as a key paracrine factor to activate fibroblasts for renal fibrosis. After ischemic AKI in mice, autophagy activation persisted for weeks in renal tubular cells. In inducible, renal tubule-specific atg7 (autophagy related 7) knockout (iRT-atg7-KO) mice, autophagy deficiency induced after AKI suppressed the pro-fibrotic phenotype in tubular cells and reduced fibrosis. Among the major cytokines, tubular autophagy deficiency in iRT-atg7-KO mice specifically diminished FGF2. Autophagy inhibition also attenuated FGF2 expression in TGFB1/TGF-β1 (transforming growth factor, beta 1)-treated renal tubular cells. Consistent with a paracrine action, the culture medium of TGFB1-treated tubular cells stimulated renal fibroblasts, and this effect was suppressed by FGF2 neutralizing antibody and also by fgf2- or atg7-deletion in tubular cells. In human, compared with non-AKI, the renal biopsies from post-AKI patients had higher levels of autophagy and FGF2 in tubular cells, which showed significant correlations with renal fibrosis. These results indicate that persistent autophagy after AKI induces pro-fibrotic phenotype transformation in tubular cells leading to the expression and secretion of FGF2, which activates fibroblasts for renal fibrosis during maladaptive kidney repair. Abbreviations: 3-MA: 3-methyladnine; ACTA2/α-SMA: actin alpha 2, smooth muscle, aorta; ACTB/β-actin: actin, beta; AKI: acute kidney injury; ATG/Atg: autophagy related; BUN: blood urea nitrogen; CCN2/CTGF: cellular communication network factor 2; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; CKD: chronic kidney disease; CM: conditioned medium; COL1A1: collagen, type I, alpha 1; COL4A1: collagen, type IV, alpha 1; CQ: chloroquine; ECM: extracellular matrix; eGFR: estimated glomerular filtration rate; ELISA: enzyme-linked immunosorbent assay; FGF2: fibroblast growth factor 2; FN1: fibronectin 1; FOXO3: forkhead box O3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HAVCR1/KIM-1: hepatitis A virus cellular receptor 1; IHC: immunohistochemistry; IRI: ischemia-reperfusion injury; ISH: in situ hybridization; LTL: lotus tetragonolobus lectin; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PDGFB: platelet derived growth factor, B polypeptide; PPIB/cyclophilin B: peptidylprolyl isomerase B; RT-qPCR: real time-quantitative PCR; SA-GLB1/β-gal: senescence-associated galactosidase, beta 1; SASP: senescence-associated secretory phenotype; sCr: serum creatinine; SQSTM1/p62: sequestosome 1; TASCC: TOR-autophagy spatial coupling compartment; TGFB1/TGF-β1: transforming growth factor, beta 1; VIM: vimentin