Abstract
Apoptosis of renal tubular epithelial cells is a key feature of the pathogenicity associated with tubulointerstitial fibrosis and other kidney diseases. One factor that regulates important cellular processes like apoptosis and cell proliferation is HRD1, an E3 ubiquitin ligase that acts by promoting ubiquitylation and degradation of its target protein. However, the detailed mechanisms by which HRD1 acts as a regulator of apoptosis in renal tubular epithelial cells have not been established. In our previous liquid chromatography-tandem mass spectrometry (LC-MS/MS) study (Mol Endocrinol. 2016;30:600–613), we demonstrated that one substrate of HRD1 was eIF2α, a critical protein in the PERK-eIF2α-ATF4-CHOP signaling pathway of endoplasmic reticulum (ER) stress. Here, we show that eIF2α expression was increased and HRD1 expression decreased when apoptosis was induced in HKC-8 cells by palmitic acid (PA) or high glucose (HG). HRD1 expression was also lower in kidney tissues from mice with diabetic nephropathy (DN) than in control mice. Forced expression of HRD1 also inhibited apoptosis in HKC-8 cells, while HRD1 overexpression decreased the expression of phosphorylated eIF2α and eIF2α. Further analysis indicated that HRD1 interacted with eIF2α and promoted its ubiquitylation and degradation by the proteasome. Moreover, the HRD1 protection of PA-treated HKC-8 cells was blunted by transfection with Myc-eIF2α. Thus, eIF2α ubiquitylation by HRD1 protects tubular epithelial cells from apoptosis caused by HG and PA, indicating a novel upstream target for therapeutic prevention of renal tubulointerstitial injury.
Highlights
Renal tubular epithelial cells are the primary targets of a variety of kidney injury regardless of the initial insults
Flow cytometry measurements showed that palmitic acid (PA) treatment significantly induced HKC-8 cell apoptosis (Fig. 1e), as confirmed by the numbers of apoptotic cells (Fig. 1f)
Protein ubiquitylation is a sequential three-step enzymatic reaction consisting of ubiquitin activation by a single ubiquitin activating enzyme (E1), transfer to ubiquitin-conjugating enzymes (E2) that act as ubiquitin carrier proteins by forming a thioester bond with the E2ubiquitin (E2-Ub) complex, and subsequent recruitment by substrate-ubiquitin E3 ligases and transfer of the activated ubiquitin linked to the target for its degradation by the ubiquitin-proteasome system (UPS)[22]
Summary
Renal tubular epithelial cells are the primary targets of a variety of kidney injury regardless of the initial insults. Accumulating evidence indicates that the apoptosis of tubular epithelial cells is a crucial step in the. For this reason, treatments that can decrease apoptosis, such as addition of bone morphogenetic protein-74 or an angiotensin receptor blocker[5], are beneficial and can prevent the progression of fibrosis. Previous work has shown that high-glucose levels (HG) are an initiating factor that promotes the generation of reactive oxygen species and subsequent apoptosis in tubular epithelial cells. This apoptosis induced by HG has been verified by the morphological changes observed during the development of diabetic nephropathy (DN)[6]. Many studies have reported the Official journal of the Cell Death Differentiation Association
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