Autophagy Regulates TGF-b Expression and Suppresses Kidney Fibrosis Induced by Unilateral Ureteral Obstruction

Autophagy is an evolutionarily conserved process that cells use to degrade and recycle cellular proteins and remove damaged organelles. During the past decade, there has been a growing interest in defining the basic cellular mechanism of autophagy and its roles in health and disease. However, the functional role of autophagy in kidney fibrosis remains poorly understood.Here, usingGFP-LC3 transgenicmice, we show that autophagy is induced in renal tubular epithelial cells (RTECs) of obstructed kidneys after unilateral ureteral obstruction (UUO). Deletion of LC3B (LC3mice) resulted in increased collagen deposition and increasedmature profibrotic factor TGF-b levels in obstructed kidneys. Beclin 1 heterozygous (beclin 1) mice also displayed increased collagen deposition in the obstructed kidneys after UUO.We also show that TGF-b1 induces autophagy in primary mouse RTECs and human renal proximal tubular epithelial (HK-2) cells. LC3 deficiency resulted in increased levels of mature TGF-b in primary RTECs. Under conditions of TGF-b1 stimulation and autoinduction, inhibition of autolysosomal protein degradation by bafilomycin A1 increased mature TGF-b protein levels without alterations in TGF-b1 mRNA. These data suggest a novel intracellular mechanism by which mature TGF-b1 protein levels may be regulated in RTECs through autophagic degradation,which suppresses kidney fibrosis inducedbyUUO. The dual functions of TGF-b1, as an inducer of TGF-b1 autoinduction and an inducer of autophagy and TGF-b degradation, underscore the multifunctionality of TGF-b1. J Am Soc Nephrol 25: ccc–ccc, 2014. doi: 10.1681/ASN.2013101068 In the kidney, fibrosis is responsible for chronic progressive kidney failure, and the prevalence of CKD is increasing worldwide.1,2 Extracellular matrix (ECM) protein production and progressive accumulation are hallmarks of renal tubulointerstitial fibrosis in progressive kidneydisease.Collagens are themaincomponents of the ECM in the kidney, and type I collagen (Col-I) is the major type associated with disease states.3,4 The cellular mechanisms that facilitate tubulointerstitial fibrosis after injury remain incompletely defined. Recent lineage tracing or genetic fate mapping studies have strongly challenged the theory that renal tubular epithelial cells (RTECs) traverse the tubular basementmembrane to becomemyofibroblasts in a process of epithelial-to-mesenchymal transition (EMT), but rather, that interstitial pericytes/perivascular fibroblasts are the myofibroblast progenitor cells.5–7 It also has been proposed that profibrotic factors, such as TGF-b1, are upregulated in the tubular interstitial area on injury, leading to kidney fibrosis.8 TGF-b1 induces production of ECM proteins, including fibronectin and collagens, and inhibits degradation of Received October 11, 2013. Accepted April 1, 2014. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Mary E. Choi, Weill Cornell Medical College, Division of Nephrology and Hypertension, 525 East 68th Street, Box 3, New York, NY 10065. Email: [email protected] Copyright © 2014 by the American Society of Nephrology J Am Soc Nephrol 25: ccc–ccc, 2014 ISSN : 1046-6673/2512-ccc 1 ECM proteins mainly by matrix metalloproteinases.9–11 Given the recent evidence that casts doubts about the role of EMT in vivo, how RTECs contribute to the development of renal tubulointerstitial fibrosis is not entirely clear. TGF-b is synthesized as a single polypeptide precursor that includes a preregion signal peptide, which is removed by proteolytic cleavage, and pro–TGF-b, containing a proregion called the latency-associated peptide and a mature TGF-b, and it converts to homodimeric pro–TGF-b through disulfide bonds.12 After cleavage by proprotein convertases, such as furin, latency-associated peptide remains noncovalently associated with the dimeric form of mature TGF-b as the small latent complex (SLC).13 SLC formation occurs in the Golgi apparatus, and mature TGF-b is secreted as part of SLC and associated with latent TGF-b–binding protein to form TGF-b large latent complex, which interacts with ECM. On stimulus, the dimeric form of mature TGF-b is dissociated from large latent complex and becomes the bioactive mature TGF-b ligand, which can then bind TGF-b receptors to trigger downstream Smad-dependent or -independent signaling pathways.12,13 Thus, the availability of mature TGF-b is the limiting factor of TGF-b activity and not TGF-b synthesis per se, because the body generates more pro–TGF-b than necessary. Whereas TGF-b/TGF-b receptor downstream signaling pathways have been extensively investigated, the regulation of TGF-bmaturation and bioavailability has not been well studied but may serve as an important target for fibrotic diseases that alter TGF-b signaling. Macroautophagy, hereafter referred to as autophagy, is a fundamental cellular homeostatic process that cells use to degrade and recycle cellular proteins and remove damaged organelles. The process of autophagy involves the formation of double membrane–bound vesicles called autophagosomes that envelop and sequester cytoplasmic components, includingmacromolecular aggregates and cellular organelles, for bulk degradation by a lysosomal degradative pathway.14 Autophagy can be induced in response to either intracellular or extracellular factors, such as amino acid or growth factor deprivation, hypoxia, low cellular energy state, endoplasmic reticulum stress or oxidative stress, organelle damage, and pathogen infection.15–22 To date, over 30 genes involved in autophagy have been identified in yeast, and they have been termed autophagy-related genes (Atgs). Themammalian ortholog of Atg8 is comprised of a family of proteins known as microtubule-associated protein 1 light chain 3 (LC3) that functions as a structural component in the formation of autophagosomes.23 LC3B (herein referred to as LC3) is the best characterized form and the most widely used as an autophagicmarker. The conversion of the cytosolic formof LC3 (LC3-I) to lipidated form (LC3-II) indicates autophagosome formation. In contrast to LC3, Beclin 1, encoded by the beclin 1 gene, is the mammalian ortholog of yeast Atg6 that is required for the initiation of autophagy through its interaction with Vps34. Homozygous deletion of beclin 1 (beclin 1) exhibits early embryonic lethality, whereas heterozygous deletion (beclin 1) results in increased incidence of spontaneous tumorigenesis, abnormal proliferation of mammary epithelial cells and germinal center B lymphocytes, and increased susceptibility to neurodegeneration.24–27 We previously reported that autophagy promotes intracellular degradation of Col-I induced by TGF-b1 in glomerular mesangial cells.28 In the present study, we explored the functional role of autophagy in an in vivo model of progressive kidney fibrosis induced by unilateral ureteral obstruction (UUO) in autophagy-deficient LC3 null (LC3) and heterozygous (beclin 1) mice and green fluorescent protein (GFP)-LC3 transgenic mice. We also performed functional studies in primary cultured mouse RTECs and human renal proximal tubular epithelial (HK-2) cells.We hypothesized that induction of autophagy in RTECs promotes TGF-b degradation and thereby reduces TGF-b secretion and suppresses development of kidney fibrosis.