Discussion
Recently, much has been learned about the pathophysiology of obstructive nephropathy and many novel biomarkers have been investigated for diagnostic and prognostic purposes. Some biomarkers offer promising results, such as TGF-β1, epidermal growth factor, kidney injury molecule 1.17 LPS can promote the progression of renal interstitial fibrosis in unilateral ureteral occlusion (UUO) by activating the TGF-β1 signaling pathway. In this retrospective study, 13 patients with CHn had urinary tract infections before surgery. The major pathogenic bacteria that cause urinary tract infections are Gram-negative bacteria. It has been demonstrated that LPS in the cell wall of Gram-negative bacteria can promote inflammatory injury and renal interstitial fibrosis in the renal tissues of rats with obstructive hydronephrosis (UUO).1 The serum levels of hs-CRP, PCT, IL-6, and IFN-γ in these children with CHn were elevated to varying degrees during urinary tract infection. IL-6 and IFN-γ have been shown to promote renal fibrosis.18 Masson staining showed that the patients with CHn had advanced to severe renal interstitial fibrosis at the end stage. Conclusion These clinical trial data confirm that LPS is an important factor promoting the progression of renal interstitial fibrosis in CHn.
Relieving inflammatory reactions and tissue damage is an important method to address renal fibrosis. Studies have shown that increasing the expression of SIRT1 inhibits inflammatory reactions and reduces fibrosis in a variety of kidney diseases.19 LPS can promote inflammation by inhibiting SIRT1 expression in kidney tissue.20 Specific agonists of SIRT1 can antagonize LPS, reduce the release of inflammatory factors, alleviate tissue damage, and slow fibrosis progression.21 In this study, IHC results showed that the expression of SIRT1 protein decreased in CHN-affected kidney tissue, and in vitro cell experiments showed that LPS inhibited SIRT1 expression in HK-2 cells, and the SIRT1-specific agonist SRT1720 could antagonize the inhibitory effect of LPS. Therefore, we speculate that the mechanism of LPS promoting the progression of renal interstitial fibrosis in CHn may be that LPS inhibits SIRT1 expression in renal tubular epithelial cells, leading to a decline in the ability of SIRT1 to inhibit inflammation and thus promoting the progression of renal interstitial fibrosis.
The active GSDMD-N-terminus can bind to the lipid components on the cell membrane to form 10–15 nm pores through which mature IL-1β and IL-18 can pass.7 It was reported that necrosulfonamide (NSA), an inhibitor of GSDMD-N, inhibited LPS-activated pyroptosis and reduced the inflammatory reaction.22 The results of this study showed that the expression of GSDMd-N-terminal protein was increased in CHN-affected kidney tissues and was mainly expressed in tubular epithelial cells, while there was almost no positive expression in control kidney tissues. At the same time, the expression of caspase-4 protein in patients with CHn was increased. The cell assay results showed that LPS promoted the expression of caspase-4 in HK-2 cells, cleaved caspase-1, NLRP3, and cleaved GSDMD, promoted the release of IL-1β and IL-18 in HK-2 cells, and induced cytokinesia. These results suggest that LPS-activated caspase-4/GSDMD non-canonical pyroptosis may be involved in promoting the progression of renal interstitial fibrosis in CHn. The mechanism may be that LPS directly recognizes and binds caspase-4 in renal tubular epithelial cells. After binding, caspase-4 becomes oligomeric and activated and then cleaves GSDMD to form the GSDMd-N end, inducing cell scorching, releasing IL-1β and IL-18 to trigger an inflammatory cascade, and then promoting the progression of renal interstitial fibrosis.23 Studies have confirmed that pyroptosis can promote the progression of fibrosis in heart and liver tissues. The mechanism may be that pyroptosis promotes inflammation and activates the profibrosis signaling pathway.24 25 Therefore, LPS is an important mechanism that promotes pyroptosis in renal interstitial fibrosis in CHn by promoting pyroptosis.
Recent studies show that the agonist melatonin of SIRT1 can inhibit the expression of the LPS-promoted NLRP3 inflammasome and reduce the release of IL-1β.26 Overexpression of SIRT1 inhibits pyroptosis of liver cells and reduces inflammatory reactions and cell injury in ischemic stress mice.27 SIRT1 agonists can inhibit the activation of the NLRP3 inflammasome in cardiomyocytes.28 Gardeniasin can activate the expression of SIRT1 to reduce the incidence of NLRP3/cleaved caspase-1 and GSDMD-N-mediated pyroptosis and alleviate inflammatory reactions and injury in renal tissue.29 SIRT1 can reduce the incidence of pyroptosis in human tubular epithelial cells with the help of deacetylation.30 The results are consistent with our findings that overexpression of SIRT1 inhibited pyroptosis of HK-2 cells and reduced the release of the inflammatory factors IL-18 and IL-1β. At the same time, we found that promoting SIRT1 expression could inhibit the NLRP3/caspase-1 classical cell pyrodeath pathway and inhibit the caspase-4 non-classical cell pyrodeath pathway, which is our new discovery. Therefore, we speculate that promoting SIRT1 expression in CHN-affected renal tissues can reduce inflammation by inhibiting pyroptosis, which may be an important mechanism by which SIRT1 alleviates renal interstitial fibrosis in children with CHn. In addition, excessive death of renal tubule epithelial cells caused by cell pyrosis is also one of the factors promoting the progression of renal interstitial fibrosis. SRT1720 can protect the proliferation ability of renal tubular epithelial cells by promoting SIRT1 expression, and maintaining cell proliferation ability is also a possible mechanism by which SIRT1 improves renal fibrosis.
It was found that nuclear factor kappa B (NF-κB) p65 could promote the expression of typical pyroptosis activated by NLRP3/cleaved caspase-1.31 More reactive oxygen species (ROS) in vivo could promote the expression of caspase-1 in cells, induce pyroptosis, and help release inflammatory factors.32 Previous studies have demonstrated that deacetylation of SIRT1 can inhibit the expression and activity of NF-κB p65 and ROS, inhibit the release of inflammatory factors, and relieve inflammatory injury in kidney tissues.33 Combined with the results of this study, we hypothesized that SIRT1 is an important target in the pyrodeath process of LPS-activated cells and that promoting SIRT1 expression can inhibit the pyrodeath of LPS-activated cells by inhibiting NF-κB p65 and ROS.
The LPS-activated caspase-4 non-classical pyroptosis pathway may play an important role in clinical experiments, which was also confirmed by cytological results. The results of clinical experiments showed no significant difference in NLRP3/caspase-1 classic pyroptosis pathway expression, and cytological experiments showed that LPS could also promote the expression of NLRP3 and caspase-1. The possible reason for this phenomenon was that after LPS activated caspase-4 non-classical pyroptosis pathway, activated caspase-4 could activate the NLRP3 inflammasome, thus promoting caspase-1 activation, accelerating IL-18 and IL-1β maturation, and inducing pyroptosis.34 This may also be due to the small number of experimental specimens leading to statistical bias. Alternatively, the paracancerous tissue of Wilms tumors cannot completely replace normal kidney tissue because the current research results suggest that activating pyrogenesis can inhibit the proliferation and migration of malignant tumor cells, and pyrogenesis has the potential to treat malignant tumors.35
In conclusion, LPS can inhibit SIRT1 expression and promote the occurrence of caspase-4/GSDMD non-classical cell scorching in renal tubular epithelial cells when urinary tract infection caused by Gram-negative bacteria occurs in children with CHn, thus promoting the progression of renal interstitial fibrosis. SIRT1 is an important target of LPS-activated pyrodeath cells. SRT1720 can promote SIRT1 to inhibit LPS-activated pyrodeath cells, reduce the release of IL-18 and IL-1β, and alleviate the progression of renal interstitial fibrosis. This conclusion is innovative to some extent, which is a new explanation of the progression mechanism of renal interstitial fibrosis in children with CHn and provides a new idea for the comprehensive treatment of children with CHn. The SIRT1-specific agonist SRT1720 has the potential to improve renal fibrosis and salvage renal function in children with CHn.
In addition, this study also found that SIRT1 inhibited the NLRP3/caspase-1-mediated classical pyrodeath pathway and inhibited non-classical pyrodeath pathway mediated by caspase-4. This is also an innovative finding of this study. Although the results of this study are encouraging, there are still many deficiencies in the experimental design, such as the small number of clinical specimens and the lack of relevant animal experiments. In the future, prospective studies are needed to evaluate the clinical usefulness of urinary biomarkers in the diagnosis and follow-up of children with obstructive nephropathy. We detected the expression levels of IL-1β and IL-18 in amniotic fluid and the urine of newborn hydronephros. We will further confirm pyroptosis as a potential therapeutic target in animal models of CHn.