Abstract
Premature senescence is a key process in the progression of diabetic nephropathy (DN). Premature senescence of renal tubular epithelial cells (RTEC) in DN may result from the accumulation of damaged mitochondria. Mitophagy is the principal process that eliminates damaged mitochondria through PTEN-induced putative kinase 1 (PINK1)-mediated recruitment of optineurin (OPTN) to mitochondria. We aimed to examine the involvement of OPTN in mitophagy regulation of cellular senescence in RTEC in the context of DN. In vitro, the expression of senescence markers P16, P21, DcR2, SA-β-gal, SAHF, and insufficient mitophagic degradation marker (mitochondrial P62) in mouse RTECs increased after culture in 30 mM high-glucose (HG) conditions for 48 h. Mitochondrial fission/mitophagy inhibitor Mdivi-1 significantly enhanced RTEC senescence under HG conditions, whereas autophagy/mitophagy agonist Torin1 inhibited cell senescence. MitoTempo inhibited HG-induced mitochondrial reactive oxygen species and cell senescence with or without Mdivi-1. The expression of PINK1 and OPTN, two regulatory factors for mitophagosome formation, decreased significantly after HG stimulation. Overexpression of PINK1 did not enhance mitophagosome formation under HG conditions. OPTN silencing significantly inhibited HG-induced mitophagosome formation, and overexpression of OPTN relieved cellular senescence through promoting mitophagy. In clinical specimens, renal OPTN expression was gradually decreased with increased tubulointerstitial injury scores. OPTN-positive renal tubular cells did not express senescence marker P16. OPTN expression also negatively correlated with serum creatinine levels, and positively correlated with eGFR. Thus, OPTN-mediated mitophagy plays a crucial regulatory role in HG-induced RTEC senescence in DN. OPTN may, therefore, be a potential antisenescence factor in DN.
Highlights
Diabetic nephropathy (DN) is the leading cause of endstage renal disease[1]
Slanting lines on images indicate the areas assessed for fluorescence intensity. d Line scan data of fluorescence intensity in the corresponding images to show the degree of co-localization between TOMM20 and LC3II. e Electron microscopy examination of mitochondria and mitophagosomes in renal tubular epithelial cells (RTEC)
The effect of HG stimulation on mitochondria in RTECs was further confirmed by electron microscopy evaluation
Summary
Diabetic nephropathy (DN) is the leading cause of endstage renal disease[1]. Tubulointerstitial injury is crucial in promoting the initiation and progression of DN2. Highglucose (HG)-induced accelerated senescence of renal tubular epithelial cells (RTECs) is an important cellular. (see figure on previous page) Fig. 1 HG induces premature senescence and mitochondrial dysfunction in mouse RTECs. a Western blot analysis of P16, P21, DcR2 expression in mouse RTECs treated with NG, Mannitol, or HG for 0–72 h. F Drp[1] expression in RTECs after HG stimulation for 48 h. G Analysis of Drp[1] fluorescence intensity. H Mfn[2] expression in RTECs after HG stimulation for 48 h. I Analysis of Mfn[2] fluorescence intensity. K Analysis of TMRM fluorescence intensity demonstrated that HG-induced cellular senescence is associated with increased reactive oxygen species (ROS) production[5]. Accelerated senescence of RTECs as a result of mitochondrial dysfunction has been implicated in pathogenesis of DN6
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