GSTK1 and RETREG1/FAM134B-mediated reticulophagy attenuates tubular injury in diabetic nephropathy through endoplasmic reticulum stress and apoptosis

Reticulophagy is a key process to recovery from endoplasmic reticulum (ER) stress and for maintaining ER homeostasis by selectively removing damaged ER and its components. However, its precise mechanisms in diabetic nephropathy (DN) remain unclear. Here, we found that the expression of RETREG1/FAM134B (reticulophagy regulator 1) was decreased in the tubular cells in DN patients and animal models, which was positively correlated with estimated glomerular filtration rate (eGFR) and negatively associated with tubulointerstitial damage. Proximal tubule-specific knockout of <i>Retreg1</i> exacerbated reticulophagy abnormalities in diabetic mice induced by high-fat diet (HFD) combined with streptozotocin (STZ), which was accompanied by increased ER stress, apoptosis of tubular cells and tubulointerstitial fibrosis. <i>In vitro</i>, overexpression of RETREG1 notably restored reticulophagy, and alleviated ER stress and apoptosis in HK-2 cells, a human proximal tubular cell line, treated with high glucose. Mechanistically, immunoprecipitation coupled with mass spectrometry (IP-MS) suggested that RETREG1 could interact with GSTK1 (glutathione s-transferase kappa 1). Silencing of GSTK1 further aggravated the reduction of reticulophagy and tubular injury both <i>in vivo</i> and <i>in vitro</i>. These effects in <i>in vitro</i> were partially blocked by overexpressing RETREG1. Collectively, these findings suggest that GSTK1 and RETREG1 exert a protective role in tubular injury through restoring reticulophagy and mitigating ER stress of tubular cells in DN.<b>Abbreviation</b>: ACTB: actin beta; cCASP3: cleaved caspase 3; CANX: calnexin; CASP: caspase; Co-IP: co-immunoprecipitation; DDIT3: DNA damage-inducible transcript 3; DN: diabetic nephropathy; ER: endoplasmic reticulum; FN1: fibronectin 1; GSTK1: glutathione S-transferase kappa 1; HFD: high-fat diet; HG: high glucose; HK-2: human tubular cell; HSPA5: heat shock protein family A (Hsp70) member 5; IHC: immunohistochemistry; IF: immunofluorescence; IP MS: immunoprecipitation coupled with mass spectrometry; LIR: LC3-interacting region; LTL: Lotus tetragonolobus lectin; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PACS2: phosphofurin acidic cluster sorting protein 2; PTCs: proximal tubular cells; PT: proximal tubule; RETREG1/FAM134B: reticulophagy regulator 1; RHD: reticulon homology domain; RT-qPCR: real time-quantitative PCR; SQSTM1/p62: sequestosome 1; STZ: streptozotocin; TECs: tubular epithelial cells; TEM: transmission electron microscopy; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling; UACR: urine albumin creatine ratio; UPR: unfolded protein response.