Pathogenic Crosstalk Between Mitochondria And The NLRP3Inflammasome Mediates Proteinuria-Induced Renal Tubular Injury

Proteinuria, as a well-known marker of kidney disease, has also been well recognized as an independent factor leading to the renal tubular epithelial cell injury and tubulointerstitial inflammation, which further promotes the progression of kidney injury and the loss of renal function. In the past decades, a number of reports roughly suggested some potential pathogenic mechanisms of proteinuria-associated renal injury including the phenotypic changes of proximal tubular cells and PKC-8-mediated cell apoptosis. However, the detailed molecular mechanism remains unclear, which results in the absence of an effective therapeutic strategy in clinic by controlling a downstream target of proteinuria production. Many clinical studys showed that despite of the severity of renal diseases, severe proteinuria was one of the most important factor in the process of renal diseases. So, to study the mechanism of proteinuria induced-renal diseases is necessary for protecting kidney.Renal proximal tubule is vulnerable to mitochondrial dysfunction from various insults such as genetic mitochondrial cytopathy, toxic xenobiotics, and ischaemia reperfusion injury. Mitochondria is a complex intracellular organelle and is involved in many metabolic circuitries and signaling transduction including the energy production, reactive oxygen species (ROS) generation, calcium homeostasis, and regulation of cell death pathways. Elif Erkan et al. reported that mitochondria abnormality is associated with an albumin-induced apoptosis in human proximal tubular cells. However, this report did not clearly define whether mitochondrial dysfunction serves as a cause or a result during such a pathological process.Inflammasomes as the intracellular multi-protein complexes, play a vital role in the release of inflammatory cytokines. Inflammasomes can be activated by many danger signals, e.g. oxidative stress, potassium efflux, and monosodium urate crystals, and mediate type2diabetes, atherosclerosis, gout, Alzheimer’s disease and other metabolic and immune diseases. Among the inflammasomes, NLRP3inflammasome is one of the best characterized ones., which is composed of NLRP3, ASC and caspasel, and is inactive in cells. When cells are stimulated by special materials, NLRP3combining with ASC recruits pro-caspasel to from the multi-protein complexes. After caspasel is acitivated, also called IL-1beta converting enzyme, inflammasome activation triggers the maturation of proinflammatory cytokines such as interleukin (IL)-1β and IL-18to initiate innate immune defenses and subsequent cellular injury. NLRP3inflammasome not only promotes the release of inflammatory cytokines, but also a key role in the initiation of the local inflammation. Growing evidence indicated that NLRP3inflammasome plays an important role in the progression of kidney diseases. In unilateral ureteral obstruction (UUO) and ischemia reperfusion injury (IRI), NLRP3KO mice were significantly resistant to injury, possibly through inhibition of the inflammatory response. The study found a significant upregulation of NLRP3in renal tissues of patients with chronic kidney disease (CKD), such as systemic lupus erythematosus (SLE), IgA nephropathy; and membranous nephropathy, and also in renal tissues of patients with AKI, such as crescentic nephritis and acute renal tubular necrosis. The NLRP3gene or protein exppressing was closely related to renal function. But the role of NLRP3inflammasome is unclear in albumin overload model.Reports have showed mitochondrial dysfunction plays an important role in the activation of NLRP3inflammasome. Studys have reported that mitochondrial ROS production and mitochondrial DNA (mtDNA) copy number can activate the NLRP3inflammasome. Reports have demonstrated that mitochondrial autophagy inhibits the activation of NLRP3inflammasome. The mitochondrial antiviral protein MAVS promotes the mitochondrial localization of NLRP3and inflammasome activation. However, all these reports are focus on the effect of mitochondrial dysfunction on NLRP3inflammasome activation, none of these reports involves the effect of NLRP3inflammasome activation on mitochondrial dysfunction.Proteinuria as the maker and an independent factor of renal diseases, induces renal tubular injury via promoting the release of inflammatory cytokines, and then participates in the progress of kidney diseases. The activation of NLRP3inflammasome promotes the release of inflammatory cytokines, and then participates in the progress of kidney diseases. So far only one report has shown that proteinuria can activate the NLRP3inflammasome. Inflammatory cytokines as the common approach of proteinuria and NLRP3inflammasome to promote the progress of kidney diseases, may be a link between proteinuria with NLRP3inflammasome in kidney diseases associated with proteinuria. We speculate that NLRP3inflammasome plays an important role in with kidney disease associated with proteinuria, and the mechanism should be further explored Part1The expression of NLRP3in renal tissue of patients with chronic kidney disease and its relationship with proteinuria Objective:To explore the expression of NLRP3in kidneys of patients with chronic kidney disease. Methods:We choose18cases of patients with chronic kidney diseases,11male (61%) and7female (39%), with an average age of9years and6months. The types of these CKD involved:8cases of nephritis,3cases of IgA nephropathy,2cases of IgM nephropathy,2cases of lupus nephritis,1case of focal segmental glomerulosclerosis,1case of membranous nephropathy,1case of mesangial proliferative glomerulonephritis. According to the degree of proteinuria, the patients were divided into three groups:mild proteinuria group (urinary protein<1.0g/24h,6cases), moderate proteinuria group (urinary protein1-3g/24h,6cases) and severe proteinuria group (urinary protein>3.0g/24h,6cases). The normal renal tissue, was from the6cases of children with normal urinary protein, who received the kidney tumor resection. We examined NLRP3protein expression in renal biopsy specimens by immunohistochemistry, and analyze its correlation with proteinuria. Result:Immunohistochemical staining showed that NLRP3expression of kidney tissue biopsies in children with CKD significantly increased compared with normal kidney tissue, and NLRP3expression was strongest in children with severe proteinuria in renal biopsy tissue. NLRP3expression was mainly located in the renal tubular, companied with little expression in glomerular. NLRP3protein expression gradually increased according to the severity of proteinuria. A regression analysis of urinary protein and NLRP3also indicated that tubular NLRP3expression was positively correlated with the patients’proteinuria levels (r2=0.8215, P<0.01). Conclusion:NLRP3expression in renal tissue of chlidren with chronic kidney disease was significant increased and was positively correlated with the degree of proteinuria. Part2The role of NLRP3inflammasome activation in albumin-induced mPTCs injuryObjective:To explore the role of NLRP3inflammasome activation in albumin-induced mouse proximal tubular epithelial cells (mPTCs) injury.Methods:To evaluate the dose and time effect of albumin on mPTCs, mPTCs were treated with albumin at the concentration of0,2,5,10,20mg/ml and at different time points of0,2,4,8,12,24,48h, respectively. In vivo, we built the albumin overload model via intraperitoneal injection of albumin in Nhp3";" and caspasel-/-mice, and divided mice into four groups:wild-type control, albumin group in wild type mice, NLRP3-/-control, albumin group in NLRP3-/-mice. After treated with albumin for12days, mice were killed. Renal pathological changes were observed by light microscopy after Periodic acid Schiff (PAS) staining. Tubular morphology was observed by electromicrograph morphometry. The makers related to mPTC injury and NLRP3inflammasome activation were detected. We applied the same methods for preparing albumin load model and experimental groups in caspasel-/-mice. The expression of E-cadherin, a-SMA, vimentin and the makers related to NLRP3inflamasome activation (NLRP3, caspasel, IL-1β and IL-18) were examed by real-time PCR and western blotting. mPTC apoptosis was assessed by Hoechst33258staining and annexin V/flow cytometry detection. Renal tubular apoptosis in kidney tissue was examed by TUNEL.Result:(1) Albumin induced mPTCs injury dose and time dependently, evidenced from the apoptosis of mPTC and phenotypic transition of mPTC, including the downregulation of E-cadherin, and the upregulation of a-SMA and vimentin. The results of annexin V/flow cytometry detection showed that5mg/ml albumin began to induce mPTC apoptosis (8%), and20mg/ml albumin induced11.5%of mPTC apoptosis. Albumin significantly downregulated E-cadherin mRNA and protein levels, and upregulated a-SMA and vimentin mRNA and protein levels after mPTCs were treated by albumin for12h.(2) Albumin induced the activation of NLRP3inflammasome in a dose and time dependent, evidenced from the increase of NLRP3, caspasel, IL-Iβ and IL-18in a dose and time dependent. NLRP3inflammasome was activated after mPTCs were treated by albumin for8h. The time that albumin induced NLRP3inflammasome activation was early than the time that albumin induced mPTC injury (12h), suggest that albumin could induce mPTC injury via activating NLRP3iflammasome.(3) NLRP3siRNA inhibited NLRP3inflammasome activation, manifested by NLRP3siRNA downregulating caspasel, IL-Iβ and IL-18by albumin. At the same time, NLRP3siRNA blocked mPTC injury which including cellular phenotypic changes involving E-cadherin, a-SMA, and vimentin expression, and mPTC apoptosis induced by albumin. In vivo, albumin induced-renal injury was ameliorated in NLRP3-/-and caspasel-/-mice. Compared with wide type (WT) group, the loss of tubular brush border and reduced tubular atrophy were significantly reduced in NLRP3-/-and caspasel-/-mice observed by light microscopy after PAS staining. A complete brush border was maintained in NLRP3-/-and caspasel-/-mice observed by electromicrograph morphometry. Tubular apoptosis detected by TUNEL was significantly decreased in NLRP3-/-and caspasel-/-mice compared with WT mice. From the level of mRNA and protein, E-cadherin, a-SMA and vimentin were restored in NLRP3-/-and caspasel-/-mice. At the same time, the activation of NLRP3inflammasome induced by albumin was inhibited in NLRP3-/-and caspasel-/-mice, evidenced from the downregulation of caspasel, IL-1β and IL-18which are the downstream molecules of NLRP3.Conclusion:Albumin-induced mPTCs injury via NLRP3inflammasome activation Part3The role of mitochondrial dysfunction in the activation of NLRP3inflammasome induced by albuminObjective:To explore the relationship between mitochondrial dysfunction and the activation of NLRP3inflammasome.Methods:mPTCs were treated with different dose (0,2,5,10,20mg/ml) and time (0,2,4,8,12,24h) of albumin, or pretreated with MnTBAP (100μM), CsA (0.5μg/ml) for30min. We built the albumin overload model via intraperitoneal injection of albumin in mice from129strain, and18mice were divided mice into three groups:control, albumin overload group, albumin overload group treated with MnTBAP. After treated with albumin for12days, mice were killed. Cellular ROS production was determined by DCFDA fluorescence; The ATP content was measured by using a luciferase-based luminescence assay kit and the mitochondrial membrane potential was examined by JC-1staining; mtDNA copy numbers were determined by real-time PCR. Cytochrome C was detected by western blotting. Mitochondrial morphology was observed by electromicrograph morphometry. The expression of E-cadherin, a-SMA, vimentin and the makes related to NLRP3inflamasome activation (NLRP3, caspasel, IL-1β and IL-18) were examed by real-time PCR and western blotting. mPTC apoptosis was assessed using Hoechst33258staining and annexin V/flow cytometry detection. Renal tubular apoptosis in kidney tissue was examed by TUNEL. Renal pathological changes was observed by light microscopy after PAS staining. Tubular morphology was observed by electromicrograph morphometry.Result:(1) Albumin induced ROS production and reduced MMP, ATP production and the mtDNA copy number in a dose-dependent manner with a noticeable effect at5mg/ml and a maximal effect at20mg/ml.(2) In a time-dependent study, albumin-induced ROS production was noticeable at30min (by310%, P<0.01), maximal at1h (by487%, P<0.01), and sustained for4h. In contrast, MMP, ATP production and the mtDNA copy number were reduced in a time-dependent manner. But albumin induced-injury occurred after24h albumin treatment, later than albumin induced mitochondrial dysfunction.(3) In vitro, MnTBAP and CsA blocked mPTC injury induced by albumin, evidenced from inhibiting mPTC apoptosis and phenotypic changes; and at the same time, MnTBAP and CsA blocked mitochondrial dysfunction, evidenced from the decrease of ROS generation, the increase of MMP, ATP and mtDNA copy number. In vivo, albumin overload mice treated with MnTBAP showed the loss of tubular brush border and reduced tubular atrophy were significantly blocked. A complete brush border was maintained in albumin overload mice treated with MnTBAP observed by electromicrograph morphometry. Also tubular apoptosis detected by TUNEL was significantly decreased. The expression of E-cadherin, a-SMA and vimentin were restored. At the same time, MnTBAP blocked mitochondrial dysfunction, evidenced from the normal mitochondrial morphology observed by electromicrograph morphometry, the little release of cytochrome C detected by western blotting, the increase of mtDNA copy number, and the upregulation of ND1and ATP synthase expression.(4) MnTBAP and CsA blocked the activation of NLRP3inflammasome evidenced from reducing the expression of NLRP3and the release of active caspasel, IL-1β and IL-18. Also albumin overload mice treated with MnTBAP showed no activation of NLRP3inflammasome.(5) NLRP3siRNA inhibited mitochondrial dysfunction evidenced from reducing ROS production and increased MMP, ATP production and the mtDNA copy number. In vivo, Nlrp3-/-and caspasel-/-mice showed mitochondrial dysfunction was almost blocked. Albumin group has swollen mitochondria with disorganized and fragmented cristae, In contrast, typical mitochondria with normal intact cristae and dense matrices were observed in albumin treated with MnTBAP group. The release of cytochrome C was less in albumin treated with MnTBAP group detected by western blotting. The increase of mtDNA copy number, and the upregulation of ATP synthase expression were observed. Conclusion:Albumin induced mPTC injury via NLRP3inflammasome activation induced by mitochondrial dysfunction, and the inhibition of NLRP3inflammasome activation blocked mitochondrial dysfunction.