The Role And Mechanism Of SIRT1-Mediated Pyroptosis And Senescence In Cadmium-Induced Kidney Injury In Rats

Cadmium(Cd)is a toxic,non-essential metal that poses a health risk to both humans and animals.The kidney is the main target organ for Cd accumulation,and the renal tubules are the leading target site for Cd nephrotoxicity.Both cellular pyroptosis and senescence are closely associated with non-microbial(aseptic)inflammatory responses.Still,the occurrence mechanisms of cellular pyroptosis and senescence during Cd-induced kidney injury and the regulatory relationships between them are unclear.Therefore,the present study aimed to investigate the mechanism of Cd-induced cellular pyroptosis and senescence in rat kidney,using rat proximal tubular cells as a model in vivo and vitro experiments.In addition,a SIRT1 overexpression model was established in rat kidney cells to further investigate the role of SIRT1 in this process.The main research contents are as follows:1.Effect of cadmium exposure on pyroptosis of rat proximal tubular cellsNRK-52E and rPT cells were used as models for this study,the CCK8 method was used to detect the effect of different concentrations of cadmium chloride(CdCl2)on the cell proliferation viability;morphological changes of rPT cells were observed by scanning electron microscopy;Western Blot and qRT-PCR assays were used to detect the effects of different concentrations of CdCl2 on the expression levels of pyroptosis-related proteins and genes;the effect of CdCl2 on the release of inflammatory factors by ELISA;different concentrations of CdCl2 were used to detect the effects on the levels of lactate dehydrogenase release by spectrophotometric method.In addition,to further investigate the role of NLRP3 inflammasome in Cd-induced pyroptosis,NLRP3 siRNA was used to suppress the expression of NLRP3 in NRK-52E and rPT cells.Western Blot and qRT-PCR assays were used to detect the effects of inhibition of NLRP3 protein expression or activity on the levels of pyroptosis-related proteins and genes.The effect of inhibition of NLRP3 protein expression or activity on the level of lactate dehydrogenase release was detected by spectrophotometric assay.The results showed that with increasing Cd concentration,cell proliferation viability was significantly reduced(P<0.05 or P<0.01),cell steric structure collapsed,cell membrane integrity was lost and pores the size of inflammatory contents could be released were formed;compared with the control group,the Cd-exposed groups pyroptosis-related protein and gene expression levels(NLRP3,ASC,Caspase-1,GSDMD and GSDME)were significantly higher(P<0.05 or P<0.01),LDH release levels were significantly higher in the Cd-exposed group(P<0.05 or P<0.01)and the secretion levels of pro-inflammatory factors IL-6,IL-18,1L-1β and TNF-α were significantly increased(P<0.05 or P<0.01).When NLRP3 protein activity was inhibited,the levels of pyroptosis-related protein and gene expression were significantly reduced(P<0.05 or P<0.01),and LDH release levels were also significantly inhibited(P<0.05 or P<0.01).These results suggest that Cd induces NLRP3-dependent pyroptosis in rat proximal tubular cells.2.Effect of cadmium exposure on the senescence of rat rat proximal tubular cellsNRK-52E cells were selected as the research model.The number of βgalactosidase-positive cells was counted after different times of CdCl2 treatment;the intracellular reactive oxygen content and mitochondrial reactive oxygen content were detected by flow cytometry;Western Blot and qRT-PCR assay were used to detect the indexes related to senescence and mitochondrial function in NRK-52E cells.The results showed that the positive area of β-galactosidase,a marker of cellular senescence,increased significantly with prolonged Cd exposure compared with the control group,and the mRNA transcript levels of senescence associated secretory phenotypes(SASP)IL-1β,IL-6 and TNF-α increased significantly(P<0.05 or P<0.01);the expression levels of senescence-related proteins(P53 and P21)were significantly increased in Cd-exposed group(P<0.05 or P<0.01);the mRNA transcript levels of mitochondrial function-related genes(UCP2,COXIV,ATP5α,TFAM and NRF1)were significantly decreased in Cd-exposed group(P<0.05 or P<0.01);the expression levels of PGC-1α and PGC-1β were significantly reduced(P<0.05 or P<0.01).At the same time,Cd exposure significantly inhibited the activity of SIRT1(P<0.05 or P<0.01).These results suggest that Cd accelerates the aging process of rat renal tubular epithelial cells.3.Effects of cadmium exposure on kidney injury and cellular pyroptosis and cellular senescence in ratsSD rats were used in vivo experiments,and Cd exposure was carried out through free drinking water(50 or 75mg/L CdCl2).At the end of the test at 4,12,24 and 48 weeks,blood and kidney tissues were collected and preserved according to the needs of subsequent tests.Indicators related to renal injury such as morphological changes and renal function were detected;indicators related to pyroptosis,premature senescence and mitochondrial function were detected by qRTPCR and Western blot;indicators related to renal fibrosis in rats were detected by Masson staining and immunohistochemistry.The results showed that the expression of SIRT1 protein was significantly decreased(P<0.05 or P<0.01),and the expression of NF-κB activity and pyroptosis-related proteins were significantly increased(P<0.05 or P<0.01)in rats at 4,12,24 and 48 weeks.The protein expression of P53 and P21 was significantly increased in the rats in Cd group(P<0.05);the mRNA transcript levels of SASPs were significantly increased(P<0.05 or P<0.01);the protein expression levels of PGC-la and PGC-1β were significantly decreased(P<0.05 or P<0.01);Masson staining showed that Cd-exposed rats had a significant increase in renal fibrosis and collagen deposition;TGF-β,Collagen I and α-SMA immunopositive areas were significantly increased;α-SMA,Collagen I,MMP2 and TGF-β gene transcript levels were significantly increased(P<0.05 or P<0.01).The above studies suggest that Cd exposure induces cellular pyroptosis and senescence,and SIRT1/ac-RelA/p65 may be involved in regulating pyroptosis.4.Effect of NAC on cadmium-induced kidney injury,pyroptosis and senescence in ratsThe antioxidant NAC was used to interfere with Cd-induced pyroptosis and senescence in rat proximal tubular cells in vivo and in vitro.The effects of NAC on reactive oxygen species and cell cycle distribution in Cd-treated cells were detected by flow cytometry.qRT-PCR and Western blot were used to detect the effects of NAC on the expression levels of indicators related pyroptosis,premature senescence and renal fibrosis;the effects of NAC on the release of lactate dehydrogenase from Cd-treated cells were detected by spectrophotometry;Masson staining and immunohistochemistry were used to detect the effects of NAC on the expression levels of renal fibrosis related-indicators.In addition,the cells were treated with SIRT1 activator and inhibitor,and the expression levels of phosphorylated and acetylated NF-κB were detected by Western blot.The results showed that in vivo and in vitro experiments,compared with the Cd-exposed group,NAC significantly decreased the expression levels of pyroptosis-related proteins(P<0.05 or P<0.01),and lactate dehydrogenase release levels were inhibited(P<0.05 or P<0.01);NAC significantly down-regulated p53 and p21 protein expression levels(P<0.05 or P<0.01),reduced SASP factor gene transcription levels(P<0.05 or P<0.01),inhibited β-galactosidase activity and alleviated cell cycle arrest;NAC significantly down-regulated the expression levels of mitochondrial biogenesis proteins PGC-1αand PGC-1β(P<0.05 or P<0.01)and decreased the transcription levels of mitochondrial function-related genes(P<0.05 or P<0.01);NAC alleviated renal fibrosis and collagen deposition and reduced TGF-β,Collagen Ⅰ and α-SMA-positive regions.In addition,SIRT1 activator significantly reduced levels of phosphorylated and acetylated NF-κB protein expression(P<0.05 or P<0.01).The above study showed that NAC significantly reduced Cd-induced oxidative stress and renal injury,and delayed oxidative stress-induced pyroptosis and senescence in rat renal tubular epithelial cells,in which the SIRT1/NF-κB axis played an important role.5.Role of SIRT1 in cadmium-induced renal cell pyroptosis and senescenceRat renal SIRT1 overexpression model was established in vivo,and cells were treated with SIRT1 activator and inhibitor in vitro.qRT-PCR and Western blot were used to detect the effects of pyroptosis,premature senescence and renal fibrosisrelated protein and gene expression levels;the effect of cellular lactate dehydrogenase release level was detected by spectrophotometry;the morphological and structural changes of cellular mitochondria were observed by transmission electron microscopy;the effect of Masson staining and immunohistochemistry on renal fibrosis-related indexes in rats was detected.The results showed that in vivo and in vitro experiments,SIRT1 overexpression significantly decreased the expression levels of pyroptosis-related proteins(P<0.05 or P<0.01),lactate dehydrogenase release levels were reduced(P<0.05 or P<0.01);SIRT1 overexpression significantly downregulated p53 and p21 protein expression levels(P<0.05 or P<0.01),decreased SASP factor gene transcription levels(P<0.05 or P<0.01),inhibited β-galactosidase activity and alleviated cell cycle arrest;SIRT1 overexpression significantly down-regulated the expression levels of mitochondrial biogenesis proteins PGC-1α and PGC-1β(P<0.05 or P<0.01)and decreased the transcription levels of mitochondrial function-related genes(P<0.05 or P<0.01);SIRT1 overexpression alleviated renal fibrosis and collagen deposition and reduced TGF-β,Collagen Ⅰ and α-SMA-positive regions.The above results indicate that inhibition of pyroptosis by overexpression of SIRT1 significantly reduces the accumulation of Cd-induced senescent cells and renal tissue fibrosis.In conclusion,Cd can cause oxidative stress in rat kidney,which leads to kidney injury,pyroptosis,premature senescence and renal fibrosis;SIRT1 regulates Cd-induced pyroptosis of rat proximal tubular cells by deacetylating NF-κB(RelA/p65);Inhibition of pyroptosis by SIRT1 overexpression can significantly reduce Cd-induced senescence and renal fibrosis in rat proximal tubular cells.