Activation of COX-2/mPGES-1/PGE2 Cascade via NLRP3 Inflammasome Contributes to Albumin-Induced Proximal Tubule Cell Injury

Chronic kidney disease (CKD) is a kind of renal disease with progressive loss of renal function and structural damage caused by various primary and secondary insults over a period of months or years. In the past decades, the prevalence of CKDs is increasing with the rapid growth of hypertension, diabetes and other metabolic disorders [1]. Among the insults resulting in the CKD progression, proteinuria is proven as an independent risk factor [2]. Although a number of studies have been performed to explore the pathogenic mechanisms of proteinuria-mediated kidney injury, we still lack the effective target for the treatment of proteinuria-caused renal injury.

Recently, NOD-like receptor family, pyrin domain containing3 (NLRP3) inflammasome was characterized as a contributor of CKDs [3]. More interestingly, Fang et al. found that the activation of NLRP3 inflammasome was positively correlated with the severity of proteinuria in patients, and the NLRP3 inflammasome was directly activated by albumin in the cultured renal epithelial cells [4]. These findings highly suggested that NLRP3 might serve as an important pathogenic factor in mediating proteinuria-induced renal tubular damage. However, the mechanisms mediating the pathogenesis of NLRP3 inflammasome in proteinuria-induced renal tubular damage remains elusive. Recently, our group reported that COX-2/mPGES-1/PGE2 could be activated by NLRP3 inflammasome in thick ascending limb to downregulate NKCC2 under the proteinuric condition [5], while the role of this cascade in proteinuria-induced renal tubular cell injury was not defined.

Among five major prostaglandins (PGE2, PGD2, PGI2, PGF2α, and TXA2) produced in kidney [6], PGE2 has the established roles in mediating the fluid metabolism [7-10], and kidney injury [11-13]. PGE2 is generated through a sequential enzyme cascade of COX/PGE2 synthases (PGES) [14]. By now, two COXs (COX-1 and COX-2) and three PGESs (mPGES-1, mPGES-2, and cPGES) were cloned. COX-1 constitutively expressed in platelets, gastric epithelial cells, and renal collecting ducts and arteriolar endothelial cells, regulates blood pressure (BP) and renal hemodynamics [15, 16]. COX-2 has low basal expression in various organs but is highly inducible by a number of pathological stimuli [15-17]. In kidney, COX-2 distribution has been localized in the macula densa, cortical thick ascending limb of Henle, and interstitial cells [18]. The stimulation of COX-2 in proximal tubules possibly contributes to renal injury [17, 19, 20]. Among three PGESs, mPGES-1 is best identified as a PGE2 synthetic enzyme [14, 21]. In contrast, genetic deletion of mPGES-2 and cPGES had no effect on lowering PGE2 levels in mice [22, 23], which argues their property of PGE2 synthases. Recently, a number of reports demonstrated that COX-2/mPGES-1/PGE2 cascade is of importance in promoting renal injury in CKD and AKI [19, 24]. In the present study, we investigated: 1) whether albumin could activate COX/mPGES-1/PGE2 cascade in proximal tubular cells; 2) whether COX/mPGES-1/PGE2 cascade served as the downstream signaling of NLRP3 inflammasome in this pathological process; and 3) whether COX/mPGES-1/PGE2 cascade contributes to albumin-induced tubular cell injury.

Immortalized mouse proximal tubular cells (mPTCs) from Sciencell Research Laboratory (Cat #: M4100) were cultured in serum-free keratinocyte medium supplemented with bovine pituitary extract, epidermal growth factor (Wisent, Canada), and penicillin/streptomycin at 37°C with 5% CO₂, and subcultured at 50-80% confluence using 0.25% trypsin-0.02% EDTA (Invitrogen). Delipidated albumin (Sigma, St Louis, MO) was dissolved in the medium with no penicillin/streptomycin to stimulate mPTCs. In brief, albumin was firstly dissolved in culture medium at the concentration of 0.25g/ml which was further diluted to the final concentrations (2.5, 5, 10, and 20 mg/ml) used in the experiments. The control cells were administered with same amount of culture medium. Because the dose-dependent experiment showed that albumin at 10mg/ml caused striking induction of tubular injury marker KIM-1, 10mg/ml albumin was used in other experiments of the study.

mPTCs were cultivated to 50-60% confluence in culture medium with no penicillin or streptomycin. SiNlrp3 and scrambled siRNA were synthesized by the company of GenePharma. Then the cells were transfected with siRNAs using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. In brief, the cells were transfected with 500 nM NLRP3 siRNA or scrambled siRNA 24 h before albumin treatment. siNLRP3: 5´-CGGCCUUACUUCAAUCUGUTT-3´, 5´-ACAGAUUGAAGUAAGGCCGTT-3´; scrambled siRNA: 5´-UUCUCCGAACGUGUCACGUTT-3´, 5´-ACGUGACACGUUCGGAGAATT-3´.

Total RNA was extracted by using the TRIzol reagent (Invitrogen). Oligonucleotides were designed using Primer3 software (available at http://frodo.wi.mit.edu/) and synthesized by Invitrogen company. The sequences of the primer pairs are shown in Table 1. Reverse transcription was performed using a reaction kit (Promega Reverse Transcription System) according to the manufacturer’s protocol. Real-time PCR amplification was performed using the ABI 7500 real-time PCR detection system (Foster City, CA, USA) with SYBR Green PCR Master Mix (Applied Biosystems). The cycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The mRNA levels were normalized to GAPDH as a control and calculated using the comparative cycle threshold (∆∆Ct) method.

mPTCs were lysed using a protein lysis buffer containing 50 mM Tris, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 200 mM sodium fluoride, and 4 mM sodium orthovanadate as a protease inhibitor (pH 7.5). Immunoblotting was then performed with primary antibodies against NLRP3(1: 500), COX-2 (Cayman Chemical, USA, 1: 500), mPGES-1 (Cayman Chemical, USA, 1: 500), and β-actin (1: 1000), followed by adding HRP-labeled secondary antibodies. The blots were visualized using the Amersham ECL detection system (Amersham, Little Chalfont, UK). Densitometric analysis was performed using Quantity One software (BioRad).

The concentration of PGE2 in the medium was examined using the commercial EIA kits purchased from Cayman Chemical.

Following the treatment, mPTCs were collected in suspension by 0.25% trypsin-0.02% EDTA and were washed with PBS. Then the cells were double stained with annexin V-fluorescein isothiocyanate and propidium iodide (Annexin V: FITC Apoptosis Detection Kit, BD Biosciences, San Diego, CA) according to the manufacturer’s instructions. Quantification was performed by flow cytometry. There were 6 samples for each group.

Apoptotic cell death was determined using TUNEL staining with an In Situ Cell Death Detection Kit (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacturer's protocol. mPTCs undergoing apoptosis were detected by counterstaining with Hoechst 33258. The slides were examined by confocal microscopy. There were 6 samples for each group and 6 fields were randomly taken in each slide.

All data are presented as means ± standard deviation (SD). The statistical analysis was performed using ANOVA followed by Bonferroni’s test or unpaired Student’s t-test with SPSS 13 statistical software. P < 0.05 was considered significant.

Application of NLRP3 siRNA significantly reduced protein and mRNA expressions of NLRP3 in mPTCs (Fig. 1A-C). Interestingly, the tubular injury marker of KIM-1 was remarkably induced by albumin in dose- and time-dependent manners (Fig. 2A&B), and such an induction was significantly blunted by NLRP3 siRNA treatment (Fig. 2C). Meanwhile, the elevation of inflammatory cytokines of IL-1β, IL-18, IL-6, and ICAM-1 following albumin treatment was markedly blocked by NLRP3 siRNA, as determined by qRT-PCR (Fig. 3A-D). In line with the inhibition of KIM-1 and inflammatory cytokines, albumin-induced cell apoptosis was also attenuated by siNLRP3 (Fig. 4).

After treating the mPTCs with albumin at the dose of 10mg/ml for 24h, the mRNA expressions of COX-2 and mPGES-1, but not COX-1, mPGES-2, and cPGES, were selectively up-regulated as determined by qRT-PCR (Fig. 5A-E). By application of NLRP3 siRNA, albumin-induced upregulation of COX-2 and mPGES-1 mRNA expressions were significantly blunted (Fig. 5A&E) without affecting COX-1 and other PGE2 synthases (Fig. 5B-D). By Western blotting, we further confirmed the induction of COX-2 and mPGES-1 at protein levels (Fig. 6A-C), which was remarkably blocked by NLRP3 silencing. (Fig. 6A-C). In line with the activation of COX-2/mPGES1 pathway, the PGE2 release in the cell culture medium was also significantly elevated in response to the albumin treatment for 24 h (Fig. 6D). Such an increase of PGE2 was completely abolished by silencing NLRP3 (Fig. 6D).

Following the activation of NLRP3 inflammasome by albumin, the mRNA levels of IL-1β, IL-18, IL-6, and ICAM-1 were all stimulated in mPTCs (Fig. 7A-D). Inhibition of COX-2 by a specific COX-2 inhibitor of NS398 robustly abolished the induction of these inflammatory cytokines (Fig. 7A-D). At the same time, albumin-induced KIM-1 expression and cell apoptosis were also diminished by NS398 in parallel with reduced PGE2 production (Fig. 8A-D). These novel findings highly suggested that COX-2/mPGES-1/PGE2 cascade served as a downstream signaling of NLRP3 inflammasome in albumin-induced renal tubular cell injury.

Proteinuria is not only a common phenomenon in CKD patients but also a direct insult promoting the renal injury [2, 25, 26]. By now, there is no effective target for treating proteinuria-associated kidney injury possibly due to the incomplete understanding of the mechanisms mediating the proteinuria-induced tubular damage. Inflammation is a common feature of various CKDs and is highly involved in the development and progression of CKD. NLRP3 inflammasome is one of the best characterized inflammasomes [27-30]. Recently, accumulating evidence demonstrated that NLRP3 inflammasome contributes to the CKDs [3, 31, 32] and can be activated by the albumin in human proximal tubule cells [4]. In present study, by blocking NLRP3 via a siRNA strategy, the tubular injury marker of KIM-1, cell apoptosis and inflammatory cytokines were all suppressed, suggesting a critical role of NLRP3 in mediating albumin-caused tubular cell injury possibly via augmenting the inflammatory response.

Among the inflammatory mediators, PGE2 has a known role in mediating the inflammatory response in various kidney injuries [11, 12, 33, 34]. Blockade of PGE2 production by inhibiting its synthetic enzymes of COX-2 or mPGES-1 could improve many kidney diseases in animals [19, 24, 35-37], suggesting a pathogenic role of COX-2/mPGES-1/ PGE2 pathway in kidney injury. In the present study, we found that albumin significantly up-regulated COX-2/mPGES-1/PGE2 cascade in mPTCs. By blocking NLRP3 via a siRNA approach, the induction of this prostaglandin cascade was largely normalized, indicating that NLRP3 inflammasome could be responsible for mediating the albumin effect on activating COX-2/mPGES-1/PGE2 cascade.

To evaluate whether activation of COX-2/mPGES-1/PGE2 signaling contributes albumin-induced cell injury, a specific COX-2 inhibitor of NS398 was applied. Strikingly, inhibition of COX-2 reversed KIM-1 induction and cell apoptosis caused by albumin challenge. Meantime, the PGE2 release was also blunted. These results highly supported that COX-2/PGE2 cascade served as a detrimental factor in albumin-induced tubular cell injury.

Among three PGE2 synthases, only mPGES-1 has been proved to have the ability in producing PGE2 in vivo [14, 21], and is thought as the most promising target of next generation of anti-inflammatory drugs [38, 39]. As a downstream enzyme of COX, it is possible that the mPGES-1 inhibitors may preserve less adverse effects than the COX inhibitors in treating the inflammatory diseases. Proteinuria is known as an insult of renal tubulointerstitial inflammation resulting in the subsequent renal lesions in morphology and function [25]. In agreement with this notion, we observed an obvious elevation of mPGES-1 at mRNA and protein levels in this experimental setting, suggesting that mPGES-1-derived PGE2 might be a pathogenic contributor of proteinuria-associated renal injury.

In summary, the present study demonstrated a critical role of NLRP3 in mediating the albumin effect on activating COX-2/mPGES-1/PGE2 cascade in renal tubular cells. COX-2/ mPGES-1/PGE2 cascade activation could contribute to albumin-induced tubular cell injury. By now, there are no effective clinical strategies in treating proteinuria-related kidney injury. These findings could shed new light on the understanding and treatment of proteinuria-caused renal damage.

This work was supported by grants from the National Key Research and Development Program (no. 2016YFC0906103), the National Natural Science Foundation of China (Nos. 81325004, 81300591, 81270797, 81670647, 81570616, and 81270785), the Natural Science Foundation of Jiangsu Province (No. BK2012001), and the Program for New Century Excellent Talents in University (No. NCET-12-0738).

There is no conflict of interests to disclose.

Y. Zhuang, F. Zhao and J. Liang contributed equally this work.