HSPN as the most serious complication of HSP, because the specific pathophysiology of its pathogenesis is not yet clear, its incidence has not decreased over time29. The results of this study showed that the expressions of TRPC6 and NLRP3 inflammasome in kidney tissues of children with HSPN were significantly higher than those in the normal control group (P<0.05). The increased expression of TRPC6 in kidney tissues of HSPN children was correlated with the increased expression of NLRP3, ASC, and IL-18. The expression of IL-1β and IL-18 was correlated with the expression of NLRP3, ASC. It indicated that the increased expression of TRPC6 and the activation of NLRP3 inflammasome do participate in the process of HSPN kidney injury.
TRPC6 is one of the newly discovered podocytes slit diaphragm proteins (SD). It interacts with a variety of SD molecules such as Podocin, Nephrin, and CD2AP to form a signaling complex to maintain the structural and functional integrity of glomerular podocytes30, 31. TRPC6 expression is increased in a variety of acquired proteinuria glomerular diseases, which may be caused by regulating calcium influx32, 33. However, it is not very clear how TRPC6 is specifically involved in the pathogenesis of podocyte injury. Studies have shown that the upregulation of TRPC6 can be detected in various glomerular diseases such as minimal change nephropathy, membranous nephropathy, and FSGS34. Another study has found that TRPC6 is found in some secondary glomerular diseases such as diabetic nephropathy (DN)35. It also significantly increased the expression of glomerular podocyte TRPC6 in children with nephrotic syndrome36. Combined with the results of this study, it is not difficult to see that TRPC6 plays an important role in the progression of glomerular disease, and taking measures against it will greatly help the diagnosis and prevention of kidney disease. NLRP3 inflammasome is one of the members of the intracellular inflammatory protein complex family, which is composed of NLRP3 protein, adaptor ASC (an apoptosis-related speck-like protein containing CARD domain) and Caspase-1 precursor9. In normal cells, these protein complexes exist in an inactive form. After activation of the inflammatory body, the precursor protein of Caspase-1 is cleaved into functional Caspase-1. Its main function is to convert the inactive and intracellular pro-inflammatory cytokines pro-IL-1β and pro-IL-18 It is converted into active IL-1β and IL-18 and released to the outside of the cell, causing local tissue inflammatory damage or systemic inflammatory response37, 38. Zhang used confocal microscopy to observe the expression of NLRP3 and ASC in glomerular podocytes in vitro39. At the same time, studies have found that the amount of NLRP3 protein and its mRNA will be significantly increased in kidney diseases, such as IgA nephropathy40, hypertensive nephropathy41, crescentic nephritis42, and acute tubular necrosis43. Although few studies have reported on the role of NLRP3 inflammasome in the occurrence and development of HSPN. Combining the above research results with this research results, we can see that the NLRP3 inflammasome is a key protein complex of kidney inflammation and is inextricably linked to kidney disease.
Studies have shown that Ang Ⅱ can quickly activate the TRPC6 channel of podocytes and mediate calcium influx, and at the same time cause a significant upregulation of TRPC6 activity and expression22, 23. Ang Ⅱ can increase the level of TRPC6 channel protein expression, increase podocyte calcium influx, activate calcineurin and stimulate NFAT dephosphorylation, at the same time upregulate the expression of cytokines, stimulate extracellular matrix (ECM) production, activate multiple cells internal signal pathway leads to cell damage35, 44. The NLRP3 inflammasome is one of the intracellular inflammasomes. When the body senses an external stimulus, the stimulus signal enters the cell, activates NLRP3, recruits ASC, and Pro-Caspase1 to form the NLRP3 inflammasome, generates activated Caspase-1, cleaves the inflammatory factors precursor and activated inflammatory factors IL-1β and IL18 are produced, and cell efflux leads to inflammation. It is currently believed that various NLRP3 inflammasome stimulants can cause mitochondrial dysfunction, leading to the release of reactive oxygen species ROS and mtDNA, thereby activating the NLRP3 inflammasome. Ca2+ released into the cytoplasm under endoplasmic reticulum stress can be taken up by the mitochondrial unidirectional transporter, which in turn reduces the normal negative mitochondrial transmembrane potential, activates the mitochondrial outer membrane voltage-gated channel, promotes related metabolites and ion transport and produce ROS, activates the NLRP3 inflammasome. When the voltage on the outer membrane of the mitochondria is knocked down, the generation of ROS is reduced, and the activation of the NLRP3 inflammasome is impaired45. A report46 believed that oxidized mtDNA released by dysfunctional mitochondria is also associated with the activation of NLRP3 inflammasome. They found that the α7-nicotinic acetylcholine receptor signaling pathway can inhibit the activation of the NLRP3 inflammasome by blocking the release of mtDNA. Recently, more and more researchers have begun to pay attention to the role of Ca2+ in the activation of NLRP3 inflammasome. Extracellular Ca2+ influx or Ca2+ release from the intracellular calcium pool to the cytoplasm and cause mitochondrial dysfunction is a common pathway for multiple activators to activate the NLRP3 inflammasome26. Based on the results of this study, it is reasonable to infer that: TRPC6 channel-mediated calcium influx may release ROS and mtDNA through mitochondrial damage and participate in the activation of NLRP3 inflammatory bodies, producing free IL-1β and IL-18, and participating in HSPN kidney damage process (Figure 4).
To our knowledge, this is the first time to investigate the relationship between expression levels of TRPC6 and NLRP3 inflammasome in kidney tissue of children with HSPN. Some limitations should be considered. First, the sample size of this study recruiting 33 cases and 6 controls was small, with the statistical power is limited. Second, demographic characteristics as well as other covariates were not abstracted. Other indicators including Ang Ⅱ and ROS expression levels were not detected because of limited samples and funding. Finally, only the statistical association between TRPC6 and NLRP3 was observed. The specific mechanism pathway should be further explored by animal experiments and in vitro cell experiments. This study also did not delve into the effects of changes in the molecular structure of TRPC6 and NLRP3 inflammasome on the disease. The main objective was to propose a hypothesis based on findings of this study, to provide the guidelines for our subsequent cell experimental research.