SIRT1 Blocks NLRP3 Inflammatory Body Activation And Podocyte Injury Through Mitochondrial Autophagy

Podocytes are terminally differentiated and highly specialized cells and the foot processes that extend from the podocyte wrap themselves aroud the capillaries of the glomerulus to form the filtration slits. Podocyte injury is a key component of progressive glomerulosclerosis, growing evidence showed that in variety of glomerular disease, such as membranous nephropathy (MN), minimal change disease (MCD), focal segmental glomerulonephritis (FSGS) and Diabetic nephropathy (DN), podocyte injury leads to proteinuria and aggravate progressive glomerulosclerosis, and eventually progresses to chronic kidney disease (CKD). Therefore, to fully understand the mechanism of podocyte injury, finding the control method of podocyte injury, is the key to block the development of CKD.Inflammasome firstly proposed by Tschopp in 2002, are complex that formed by NOD like receptors (NLRs) protein family members. On the basis of NLR protein in different inflammasomes, which can be divided into NLRP1 (previously also known as NALP1), NLRP3, NLRC4, AIM2 and other inflammasomes. In those inflammasomes, NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome is the best characterized one. Its basic structure unit is by NLRP3, ASC (apoptosis speck protein with card) and caspase-1 and is inactive in cells. When cells are exposed to a specific stimulus, NLRP3 combined with ASC recruits pro-caspase-1 to form complex, and make it become biologically active caspase-1, also called interleukin hormone (IL)-1 beta invertase. Inflammasome activation triggers the maturation of proinflammatory cytokines such as IL-18 to initiate innate immune defenses and subsequent cellular injury. It is reported that the NLRP3 inflammasome participates in various kidney diseases, such as renal fibrosis. But the role in the podocyte injury needs to be further studied.Mitochondrial autophagy (Mitophagy) in renal injury also attracted attention. Mitochondria are essential intracellular organelles that have a major role in energy production by ATP synthesis through oxidative phosphorylation (OXPHOS), and involved in many cellular activities, which determine cell death. Mitochondria are easy to be damaged by external factors, leading to mitochondrial dysfunction. The damaged or unwanted mitochondria must be effective clear, to ensure the normal life activities of cells. Mitophagy is a process of removing damaged or unnecessary mitochondrial by selective mechanism. There is a close relationship between the abnormality of autophagy with neurodegenerative diseases, diabetes and cancer. It has been reported in macrophages that mitophagy can inhibit the production of NLRP3 inflammasome, but not in podocytes.SIRT1, silent mating type information regulation 2 homolog 1, is one of seven mammalian homologs of Sir2 that catalyzes NAD+-dependent protein deacetylation. In mammals, SIRT1 primarily localized in the nuclei and most effects of SIRT1 depend on its deacetylation activity. In kidney, SIRT1 is documented to have renoprotective functions and SIRT1 regulates glucose or lipid metabolism, maintenance of glomerular barrier function, and regulation of mitochondria function and energy metabolism, anti-fibrosis effects. Moreover, SIRT1 could regulate the activity of transcription factor FOXO (forkhead box-containing protein), that is involved in regulating autophagy. This pathway plays a key role in many diseases, such as diabetes, tumor, and obesity etc.We therefore speculate that Aldo could worsen podocyte damage by activating NLRP3 inflammasome. SIRT1 could promote mitophagy, prevent mitochondrial dysfunction and inhibit the activation of NLRP3.Part1 The role of NLRP3 inflammasome activation in Aldo-induced podocyte injuryObjective:To explore the role of NLRP3 inflammasome activation in Aldo-induced mouse podocyte injury.Methods:Immunohistochemical staining was used to detect the NLRP3 expression in CKD, including MCD and FSGS. To evaluate the dose and time effect of Aldo on podocytes, podocytes were treated with Aldo at the concentration of 25,50,100, 200nmol/L and at different time points of 0,2,4,8,12,24,48 h, respectively. To explore the role of NLRP3 in podocyte damage, podocytes were transfected with NLRP3 siRNA, and divided into four groups:NC, NLRP3 siRNA, NC+Aldo, NLRP3 siRNA+Aldo. To examine the relationship between mitochondrial dysfunction and NLRP3 inflammasome, we treated podocytes with CsA and MnTBAP. We applied real-time PCR, western blot and ELSA to detect the activation of NLRP3 inflammassome (NLRP3, caspase-1 and IL-18). Caspase-3,-8 and -9 activities were measured using caspase-activity assay kits. In vivo, WT mice were implanted subcutaneously with 14-day-realease pellets containing Aldo. Aldo-infused mice were sacrificed at dayl,3,5,7,14, respectively. We also built the same model in Nlrpp3-/- mice, and divided mice into four groups:wild-type control, Aldo group in wild type mice, NLRP3-/- control, Aldo group in NLRP3-/- mice. After treated with Aldo for 14 days, mice were sacrificed. The proteinuria was evaluated by urine protein/urine creatinine. Periodic acid Schiff (PAS) staining was performed to observe renal pathological changes. The podocyte ultramicrostructure was investigated by transmission electron microscopy. The makers related to podocyte injury and NLRP3 inflammasome activation were detected by real-time PCR and western blot.Results:(1) Immunohistochemical staining and IOD rate showed that NLRP3 expression of kidney tissue biopsies in children with MCD and FSGS significantly increased compared with normal kidney tissue. The secretion of IL-18 in urine of patients with primary nephropathy syndrome also significantly increased compared with the control group. (2) Real-time PCR, western blot and ELISA showed that Aldo induced NLRP3 inflammasome activation dose dependently, evidenced from the upregulation of NLRP3, caspase-1 and IL-18. NLRP3 and IL-18 began to increase at the dose of 50nM for 24h, and reached the peak at 100nM. Aldo also time dependently induced the activation of NLRP3 and IL-18; IL-18 secretion was increased at 100nM Aldo for 4h, and continued to 24h. (3) In Aldo-infused mice, western blot and immunofluorescence showed NLRP3 expression increased after 3 days of Aldo trearment (mainly expressed in podocytes), the day 5 further increased and lasted to day 14. Caspase-1 increased at day 7 and reached the peak at day 14. IL-18 increased at day 5 and continued up to day 14. Nephrin expression decreased after 5 days of Aldo treatment (about 37% reduction) and continued to 14 days. (4) NLRP3 siRNA inhibited NLRP3 inflammasome activation, manifested by NLRP3 siRNA downregulating caspase-1 and IL-18 induced by Aldo. At the same time, NLRP3 siRNA blocked podocyte injury which including podocyte apoptosis, nephrin expression, and caspase-3,-8,-9 activities induced by Aldo. (5) In vivo, Aldo induced-renal injury was ameliorated in NLRP3-/- mice. Aldo treatment resulted in glomerular enlargement and increased mesengial area, and NLRP3-/- restored the normal structure of the glomeruli. Albuminuria was significantly reduced in NLRP3-/- mice. Extensive fusion of foot processes was evident in Aldo-treated mice in electron micrographs. In contrast, treatment of the Aldo in NLRP3-/- mice maintained the normal shape of the foot processes. NLRP3-/- restored nephrin and podocin mRNA and protein levels. (6) CsA blocked the opening of mPTP, and blocked mitochondrial dysfunction, evidenced from the increase of MMP and mtDNA copy number. At the same time, CsA and MnTBAP blocked the activation of NLRP3 inflammasome evidenced from reducing the expression of NLRP3 and the release of active caspase-1 and IL-18.Conclusion:Aldo induces mitochondrial dysfunction, and then activates NLRP3 inflammasome to promote podocyte injury.Part2 The role of SIRT1 in the activation of NLRP3 inflammasome induced by AldoObjective:To explore the role of SIRT1 in the activation of NLRP3 inflammasome induced by Aldo.Methods:Immunohistochemical staining to detect the SIRT1 expression in CKD, including MN, MCD and FSGS. To explore the role of SIRT1 in NLRP3 inflammasome activation, podocytes were transfected with SIRT1 siRNA, and divided into three groups:control group, vehicle group, and SIRT1 siRNA group. Podocytes were also transfected with SIRT1 plasmid, and divided into four groups: vehicle group, SIRT1 overexpression group, vehicle+Aldo group and SIRT1+Aldo group. DCFDA fluorescence was used to determine cellular reactive oxygen species (ROS). JC-1 staining was used to examine mitochondrial membrane potential. Mitochondrial DNA (mtDNA) copy numbers were determined by real-time PCR. Podocyte apoptosis was assessed using annexin V/flow cytometry detection. Real-time PCR and western blot examined the expressions of nephrin and podocin. Caspase-3,-8 and -9 activities in podocytes were measured using caspase-activity assay kits. In vivo, podocyte conditional knock out SIRT1 mice were made, namely SIRT1flox/flox NPHS2cre(+),and the control is SIRT1flox/flox NPHS2cre(-). Those mice were implanted subcutaneously with 14-day-realease pellets containing Aldo. Mice were sacrificed at day 14. The proteinuria was evaluated by urine protein/urine creatinine. Periodic acid Schiff (PAS) staining was performed to observe renal pathological changes. The podocyte ultramicrostructure was investigated by transmission electron microscopy. Immunofluorescence was used to observe the expression of NLRP3 and WT1. The makers related to podocyte injury and NLRP3 inflammasome activation were detected by real-time PCR and western blot.Results:(1) The results of immunohistochemistry showed high expression of SIRT1 in normal renal tissue, decrease in renal tissues of children with MN, MCD, and almost no expression in FSGS patients. SIRT1 protein expression gradually decreased according to the severity of proteinuria. A regression analysis of urinary protein and SIRT1 also indicated that SIRT1 expression was negatively correlated with the patients’proteinuria levels. (2) SIRT1 siRNA can induce mitochondrial dysfunction and performance for an increase of ROS, and reduction in membrane potential JC-1 and mtDNA copy number. SIRTl siRNA also induce podocyte injury, performance for podocyte apoptosis increase, activity of caspase-3 and caspase-9 increased and the expression of nephrin reduce. At the same time, Knockdown of SIRT1 significantly promoted the expression of NLRP3and the secretion of IL-18. (3) The prepared SIRT1flox/flox NPHS2cre(+) mice were identified. Immunofluorescence detected the expression of SIRT1 in glomerular, and the results showed that SIRT1 expression was significantly reduced. Extraction of primary glomerular podocytes, identified its purity by immunofluorescence staining with WT1, and results showed that podocyte purity proposed by>95%. Western blot and real-time PCR showed that SIRT1flox/flox NPHS2cre(+)mice almost had no expression of SIRT1. The SIRT1flox/flox NPHS2cre(+) mice had no difference with the control group SIRT1flox/flox NPHS2cre(-)mice under physiological condition. Evidence from:no proteinuria, normal glomerular morphology, and normal expression of podocyte specific marker WT1. (4) In vivo, Aldo induced-renal injury was worsen in SIRT1flox/flox NPHS2cre (+) mice evidence from increased 24h urinary protein excretion, increased glomerular volume and mesangial cells. Real-time PCR and western blot showed SIRT1flox/flox NPHS2cre(+) mice had more reduction in nephrin and podocin induced by Aldo. Under the electron microscope, SIRT1flox/flox NPHS2cre (+) mice infused with aldosterone appear more extensive foot process fusion. The activation of NLRP3 inflammasome was also promoted in SIRT1flox/flox" NPHS2cre (+) mice infused with Aldo. The expression of NLRP3 increased, as well as the secretion of IL-18 in serum. (5) Overexpression of SIRT1, inhibited NLRP3 inflammasome activation, mitochondrial dysfunction and podocyte injury induced by aldosterone. Performance:Real-time PCR and western blot results showed that compared with Aldo treatment group, overexpression of SIRT1 with Aldo stimulated group, the expression of NLRP3 and caspase-1 decreased significantly. SIRT1 also blocked the secretion of inflammatory cytokines IL-18 induced by aldosterone. At the same time, the overexpression of SIRT1 blocked the reduction of membrane potential, mtDNA copy number, and the expression of nephrin and podocin.Conclusion:SIRT1 could inhibit mitochondrial dysfunction, and then inhibit the activation of NLRP3 inflammasome to protect podocyte.Part3 SIRT1/BNIP3 axis blocked NLRP3 inflammasome activation induced by AldoObjective:To explore SIRT1/BNIP3 axis in the activation of NLRP3 inflammasome induced by Aldo.Methods:In vivo, use the model prepared in Part2. Real-time PCR and weatern blot detected the expression of mitophagy related genes (BNIP3, Beclin-1, Atg5 and LC3). In vitro, cultured podocytes were transfected with BNIP3 plasmid for 24h, and then stimulated with Aldo. They were divided into four groups:Vehi group, BNIP3 overexpression group, Vehi+Aldo group, overexpression of BNIP3+Aldo group. Podocyte apoptosis was labelled by TUNEL assay. The expression of nephrin, podocin and NLRP3 was determined by real-time PCR and western blot. Mitochondrial ROS levels were determined by MitoSOX staining. Mitochondrial DNA (mtDNA) copy numbers were determined by real-time PCR and the mitochondrial membrane potential was examined by JC-1 staining.Results:(1) Without Aldo infusion, podocyte specific knockout SIRT1 had no effect on mitophagy. After Aldo infusion, the expression of BNIP3, Beclin-1, Atg5 and LC3II was increased, but not in podocyte specific knockout SIRT1 group. These results indicated that podocyte specific knockout SIRT1 inhibited mitophagy after Aldo infusion. (2) Overexpression of BNIP3, inhibited mitochondrial dysfunction, NLRP3 inflammasome activation, and reduced podocyte injury induced by Aldo. Performance:TUNEL staining showed that overexpression of BNIP3 can block the apoptosis of podocytes induced by Aldo. Real-time PCR and western blot results showed that compared with Aldo treatment group, overexpression of BNIP3 with Aldo stimulated group, the expression of nephrin and podocin increased significantly. BNIP3 also blocked the expression of NLRP3 and the secretion of inflammatory cytokines IL-18 induced by aldosterone. At the same time, overexpression of BNIP3 blunted the mitochondrial dysfunction with increase of membrane potential, mtDNA copy number.Conclusion:Mitochondrial autophagy mediated by BNIP3 is involved in the regulation of SIRT1 on mitochondrial function and NLRP3 inflammasome activation.