Renal tubular injury is increasingly being recognized as an early characteristic of diabetic nephropathy (DN). Mitochondrial dynamic alterations and redox protein p66Shc-mediated oxidative stress are both critical for ensuing diabetic tubular cell injury and apoptosis; whether these two processes are interlinked remains unclear. In the present study, we observed changes in mitochondrial morphology and expression of associated proteins in tubules of patients with DN. We demonstrated mitochondrial fragmentation as an important pathogenic feature of tubular cell injury that is linked to oxidative stress and p66Shc up-regulation. In renal proximal tubular cells, alterations in mitochondrial dynamics and expression of fission-fusion proteins were observed under high glucose (HG) ambience, along with p66Shc Ser36 phosphorylation. Gene ablation of p66Shc alleviated HG-induced mitochondrial fragmentation, down-regulated Fis1 and reduced p66Shc-Fis1 binding, increased Mfn1 expression, and disrupted interactions between Mfn1 and proapoptotic Bak. Overexpression of p66Shc exacerbated these changes, whereas overexpression of dominant-negative p66Shc Ser36 mutant had a marginal effect under HG, indicating that p66Shc phosphorylation as a prerequisite in the modulation of mitochondrial dynamics. Disrupted mitochondrial dynamics and enhanced Mfn1-Bak interactions modulated by p66Shc led to loss of mitochondrial voltage potential, cytochrome release, excessive ROS generation, and apoptosis. Taken together, these results link p66Shc to mitochondrial dynamic alterations in the pathogenesis of DN and unveil a novel mechanism by which p66Shc mediates HG-induced mitochondrial fragmentation and proapoptotic signaling that results in oxidative injury and apoptosis in the tubular compartment in human diabetic nephropathy.

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BackgroundCompared with traditional diagnostic methods (TDMs), rapid diagnostic methods for infectious diseases (IDs) are urgently needed. Metagenomic next-generation sequencing (mNGS) has emerged as a promising diagnostic technology for clinical infections.MethodsThis retrospective observational study was performed at a tertiary hospital in China between May 2019 and August 2022. The chi-square test was used to compare the sensitivity and specificity of mNGS and TDMs. We also performed a subgroup analysis of the different pathogens and samples.ResultsA total of 435 patients with clinical suspicion of infection were enrolled and 372 (85.5%) patients were finally categorized as the ID group. The overall sensitivity of mNGS was significantly higher than that of the TDMs (59.7% vs. 30.1%, P < 0.05). However, there was no significant difference in the overall specificity between the two methods (83.3% vs. 89.6%, P = 0.37). In patients with identified pathogens, the positive rates of mNGS for detecting bacteria (88.7%), fungi (87.9%), viruses (96.9%), and Nontuberculous mycobacteria (NTM; 100%) were significantly higher than those of TDMs (P < 0.05). The positive rate of mNGS for detecting Mycobacterium tuberculosis was not superior to that of TDMs (77.3% vs. 54.5%, P = 0.11). The sensitivity rates of mNGS for pathogen identification in bronchoalveolar lavage fluid, blood, cerebrospinal fluid, pleural fluid, and tissue were 72.6%, 39.3%, 37.5%, 35.0% and 80.0%, respectively.ConclusionWith the potential for screening multiple clinical samples, mNGS has an overall advantage over TDMs. It can effectively identify pathogens, especially those that are difficult to identify using TDMs, such as NTM, chlamydia, and parasites.