DKD is the primary cause of chronic kidney disease. Effective treatment of DKD and exploration of new therapeutic targets has always been a major topic of research. In addition to abnormal blood glucose and lipid metabolism, abnormal activation of inflammasomes also plays a key role in the development of DKD [10, 11]. Pyroptosis is a form of inflammatory programmed cell death, and NLRP3 inflammasome activation plays a central role in this process. In our study, we found that NLRP3 and GSDMD( key protein of pyroptosis) are primarily expressed in human renal tubular epithelial cells. Their expression in kidney tissue of patients with DKD is significantly higher than that in normal human kidney tissues, and the expression of GSDMD is positively correlated with that of NLRP3. It is therefore speculated that NLRP3 cooperates with GSDMD to participate in the development and progression of DKD.
Inflammasomes are multi-protein complexes assembled by pattern recognition receptors in the cytoplasm. They can recognize a variety of stimuli such as pathogen-associated molecular patterns or damage-associated molecular patterns. By recruiting and activating caspase-1, they induce mature inflammatory factors such as IL-1β and IL-18. NLRP3 is the most widely studied inflammasome, which can be activated through classical or non-classical pathways and participates in the pathogenesis of acute kidney injury and chronic kidney disease . The activation of NLRP3 inflammasome in DKD has attracted widespread attention. The activated NLRP3 inflammasome promotes secretion of IL-1β and IL-18, which in turn promotes the progression of DKD . Researchers have observed upregulation of NLRP3 and caspase-1 expression in endothelial cells and podocytes in the kidneys of mice and patients with DKD . In vitro cell culture and animal models have confirmed the role of NLRP3 in DKD [15–17]. In this study,we stimulated HK-2 cells with high concentrations of glucose and observed increased expression of NLRP3, caspase-1 p20, IL-1β, and IL-18, suggesting that high glucose concentration activates HK-2 cell inflammasome and causes the release of inflammatory factors.That consistent with previous studies.
Although pyroptosis was initially considered a unique feature of immune cells, recent studies have shown that it also plays a role in non-immune cells [6, 18]. In contrast-induced acute kidney injury and renal ischemia-reperfusion injury, renal tubular epithelial cell pyroptosis is an indispensable process [19, 20].The activated NLRP3 activates caspase-1, cleaves GSDMD, breaks self-inhibition, and produces an N-terminal fragment. GSDMD-N targets the cell membrane to form pores and causes water influx, so that the ion gradients across the cell membrane disappear, and the cells undergo swelling and osmotic lysis, eventually leading to cell pyroptosis [21, 22].In our study,we also observed that renal tubular epithelial cells showed obvious pyroptosis with the increase expression of NLRP3 after stimulated by high glucose. In addition, for the first time, we observed through TEM that the HK-2 cells in the high-glucose groups (15 mmol/L and 30 mmol/L) showed ultrastructural changes that are typical of pyroptosis, such as cell membrane damage, discontinuity, cytoplasmic content overflow, and chromatin margination and adhesion to the nuclear membrane. However, the cells in the control group did not undergo pyroptosis. Further, we found through western blotting that compared with that in the normal control group, the expression of NLRP3, caspase-1 p20, GSDMD-N, IL-1β, and IL-18 in the high-glucose groups (15 mmol/L, 30 mmol/L, and 45 mmol/L) increased in a concentration-dependent manner, which also indicated that high glucose concentration promotes the occurrence of cell pyroptosis, and that NLRP3-caspase-1 may be related to GSDMD-mediated pyroptosis. Although the expression of pyroptosis-related proteins in the 45 mmol/L group was higher than that in the other groups, and TEM showed that the cells in this group were significantly swollen with part of cells appearing large number of myeloid structures and vacuolated mitochondria. This phenomenon indicated that the cells were seriously damaged, but there were no ultrastructural characteristics typical of pyroptosis. We speculated that the cells in the 45 mmol/L group have other complex cell damage patterns, such as autophagy.
In conclusion,our study confirmed that high glucose concentration can induce pyroptosis of human renal tubular epithelial cells. We stimulated human renal tubular epithelial cells with different concentrations of glucose, which showed a series of changes in NLRP3 inflammasomes, pyroptosis-related proteins, and inflammatory factors, suggesting that high glucose concentration affects the activation of NLRP3 inflammasomes in the autoimmune system, leading to the occurrence of pyroptosis and release of inflammatory factors. This provides a basis for further animal and clinical experiments. With the elucidation of the relevant mechanisms of action, the targets in the pyroptosis-related signaling pathways are expected to become a new hotspot in the treatment of DKD, and targeting pyroptosis through inflammasome assembly, caspase activation, GSDMD-mediated nuclear pore formation, and other unknown upstream or downstream pathways may be a new way to treat DKD.