Department of Cellular Biology and Anatomy, Medical College of Georgia at Augusta University, Augusta, GA, USA.
Research Department, Charlie Norwood VA Medical Center, Augusta, GA, USA.
Autophagy. 2023 Jan;19(1):256-277. doi: 10.1080/15548627.2022.2072054. Epub 2022 May 18.
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 (autophagy related 7) knockout (iRT--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--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 - or -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. 3-MA: 3-methyladnine; ACTA2/α-SMA: actin alpha 2, smooth muscle, aorta; ACTB/β-actin: actin, beta; AKI: acute kidney injury; 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.
在急性肾损伤（AKI）之后，肾小管细胞可能以旁分泌的方式刺激成纤维细胞，导致间质纤维化，但是在这种情况下，旁分泌因子及其调节仍然难以捉摸。在这里，我们确定了管状细胞中依赖于巨自噬/自噬的 FGF2（成纤维细胞生长因子 2）的产生。诱导后，FGF2 作为关键的旁分泌因子，激活成纤维细胞发生肾纤维化。在小鼠缺血性 AKI 后，肾小管细胞中的自噬激活持续数周。在可诱导的、肾小管特异性的（自噬相关 7）敲除（iRT--KO）小鼠中，AKI 后诱导的自噬缺陷抑制了管状细胞中的促纤维化表型，并减少了纤维化。在主要细胞因子中，iRT--KO 小鼠的管状自噬缺陷特异性降低了 FGF2。自噬抑制也降低了 TGFB1/转化生长因子，β 1（TGF-β1）处理的肾小管细胞中的 FGF2 表达。与旁分泌作用一致，TGFB1 处理的管状细胞的培养基刺激肾成纤维细胞，而这种作用被 FGF2 中和抗体以及管状细胞中的 - 或 - 缺失所抑制。与非 AKI 相比，来自 AKI 后患者的肾活检标本中肾小管细胞的自噬和 FGF2 水平更高，与肾纤维化有显著相关性。这些结果表明，AKI 后持续的自噬诱导管状细胞发生促纤维化表型转化，导致 FGF2 的表达和分泌，从而在适应性肾脏修复期间激活成纤维细胞发生肾纤维化。3-MA：3-甲基腺嘌呤；ACTA2/α-SMA：肌动蛋白 α 2，平滑肌，主动脉；ACTB/β-肌动蛋白：肌动蛋白，β；AKI：急性肾损伤；ATG/: 自噬相关；BUN：血尿素氮；CCN2/CTGF：细胞通讯网络因子 2；CDKN2A/p16：周期蛋白依赖性激酶抑制剂 2A；CKD：慢性肾病；CM：条件培养基；COL1A1：胶原，类型 I，α 1；COL4A1：胶原，类型 IV，α 1；CQ：氯喹；ECM：细胞外基质；eGFR：估计肾小球滤过率；ELISA：酶联免疫吸附测定；FGF2：成纤维细胞生长因子 2；FN1：纤连蛋白 1；FOXO3：叉头框 O3；GAPDH：甘油醛-3-磷酸脱氢酶；HAVCR1/KIM-1：肝炎 A 病毒细胞受体 1；IHC：免疫组织化学；IRI：缺血再灌注损伤；ISH：原位杂交；LTL：荷花 Tetragonolobus 凝集素；MAP1LC3B/LC3B：微管相关蛋白 1 轻链 3β；MTOR：雷帕霉素靶蛋白激酶；PDGFB：血小板衍生生长因子，B 多肽；PPIB/cyclophilin B：肽基脯氨酰异构酶 B；RT-qPCR：实时定量 PCR；SA-GLB1/β-gal：衰老相关半乳糖苷酶，β 1；SASP：衰老相关分泌表型；sCr：血清肌酐；SQSTM1/p62：自噬体相关蛋白 1；TASCC：TOR-自噬空间偶联隔室；TGFB1/TGF-β1：转化生长因子，β 1；VIM：波形蛋白。
