Protective Action of Anandamide and Its COX-2 Metabolite against l-Homocysteine-Induced NLRP3 Inflammasome Activation and Injury in Podocytes

Abstract

Recent studies have demonstrated that l-homocysteine (Hcys)-induced podocyte injury leading to glomerular damage or sclerosis is attributable to the activation of the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome. Given the demonstrated anti-inflammatory effects of endocannabinoids, the present study was designed to test whether anandamide (AEA) or its metabolites diminish NLRP3 inflammasome activation and prevent podocyte injury and associated glomerular damage during hyperhomocysteinemia (hHcys). AEA (100 μM) inhibited Hcys-induced NLRP3 inflammasome activation in cultured podocytes, as indicated by elevated caspase-1 activity and interleukin-1β levels, and attenuated podocyte dysfunction, as shown by reduced vascular endothelial growth factor production. These effects of AEA were inhibited by the cyclooxygenase-2 (COX-2) inhibitor celecoxib (CEL). In mice in vivo, AEA treatment attenuated glomerular NLRP3 inflammasome activation induced by hHcys accompanying a folate-free diet, on the basis of inhibition of hHcys-induced colocalization of NLRP3 molecules and increased interleukin-1β levels in glomeruli. Correspondingly, AEA prevented hHcys-induced proteinuria, albuminuria, and glomerular damage observed microscopically. Hcys- and AEA-induced effects were absent in NLRP3-knockout mice. These beneficial effects of AEA against hHcys-induced NLRP3 inflammasome activation and glomerular injury were not observed in mice cotreated with CEL. We further demonstrated that prostaglandin E2-ethanolamide (PGE2-EA), a COX-2 product of AEA, at 10 μM had a similar inhibitory effect to that of 100 μM AEA on Hcys-induced NLRP3 inflammasome formation and activation in cultured podocytes. From these results, we conclude that AEA has anti-inflammatory properties, protecting podocytes from Hcys-induced injury by inhibition of NLRP3 inflammasome activation through its COX-2 metabolite, PGE2-EA.

Fig. 1.

Protective action of AEA at different concentrations against Hcys-induced NLRP3 inflammasome activation and podocyte injury via its COX-2 metabolites. Podocytes were cultured for 24 hours with 40 μM l-homocysteine (Hcys) in the absence (vehicle only, Vehl) or presence of increasing anandamide (AEA) concentrations as indicated. In one group, the cells were cotreated with celecoxib (1 μM). (A) Caspase-1 activity in different groups of podocytes (n = 6). (B) IL-1β production in different groups of podocytes (n = 6). (C) VEGF production in different groups of podocytes (n = 6). *p < 0.05 versus Control group, ^#p < 0.05 versus Vehl-Hcys group. ^&p < 0.05 versus AEA 100 μM-Hcys group.

Fig. 2.

COX-2-dependent protection of glomerular podocytes by AEA against hHcys-induced NLRP3 inflammasome formation and activation in mice. (A) Images showing the colocalization between NLRP3 (green) with ASC (red) in glomeruli of mice receiving either normal diet or folate-free diet and the different treatments shown with summarized data (n = 6). (B) Images showing the colocalization between NLRP3 (green) with caspase-1 (red) in glomeruli from different groups and summarized data (n = 6). (C) Images showing IL-1β immunoreactivity in glomeruli from different groups of mice and summarized data (n = 6). *p < 0.05 versus WT-Vehicle (Vehl)-ND group, ^#p < 0.05 versus WT-Vehl-FF group.

Fig. 3.

Prevention by AEA of hHcys-induced glomerular damage in mice. (A) Summarized data showing the urinary protein excretion rate from different groups of mice (n = 6). (B) Summarized data showing the urine albumin excretion rate from different groups of mice (n = 6). (C) Images showing the glomerular morphologic changes from different groups of mice and summarized data (n = 6). *p < 0.05 versus WT-Vehicle (Vehl)-ND group, ^#p < 0.05 versus WT-Vehl-FF group.

Fig. 4.

Amelioration by AEA of hHcys-induced podocyte injury in mice. (A) Images showing immunostained glomeruli for podocin from different groups of mice. (B) Images showing immunostained glomeruli for desmin from different groups of mice. (C) Summarized data for podocin (left) and desmin (right) (n = 6). *p < 0.05 versus WT-Vehicle (Vehl)-ND group, ^#p < 0.05 versus WT-Vehl-FF group.

Fig. 5.

Inhibition of Hcys-induced NLRP3 inflammasome formation by PGE2-EA in podocytes. Podocytes were cultured for 24 hours with Hcys (40 μM) in the absence or presence of PGE2-EA (10 μM) or AEA (10 μM). (A) Images showing colocalization of NLRP3 (Alexa 488, green color) with ASC (Alexa 555, red color) in podocytes and summarized data showing the fold changes in Pearson correlation coefficient for the colocalization of NLRP3 with ASC (n = 6). (B) Images showing colocalization of Nlrp3 (Alexa 488, green color) with caspase-1 (Casp; Alexa 555, red color) in podocytes and summarized data showing the fold changes in correlation coefficient for the colocalization of NLRP3 with Casp (n = 6). *p < 0.05 versus Control ^#p < 0.05 versus corresponded Hcys group. PE2, prostamide E2.

Fig. 6.

Blockade by PGE2-EA of Hcys-induced NLRP3 inflammasome activation in podocytes. (A) Caspase-1 activity in different groups of podocytes (n = 6). (B) IL-1β production in different groups of podocytes (n = 6). *p < 0.05 versus Control group; ^#p < 0.05 versus Vehicle (Vehl)-Hcys group.

Fig. 7.

Protective effect of PGE2-EA against Hcys-induced podocyte injury. (A) Images showing the expression of F-actin fiber in different groups of control or treated podocytes and summarized data (n = 6). (B) Summarized data showing production of VEGF in different groups of podocytes (n = 6). *p < 0.05 versus Control group; ^#p < 0.05 versus Vehicle (Vehl)-Hcys group.

Fig. 8.

Protective effect of PGE2-EA against Hcys-induced podocyte injury. (A) Images showing the expression of podocin in different groups of podocytes and summarized data (n = 6). (B) Images showing the expression of desmin in different groups of podocytes and summarized data (n = 6). *p < 0.05 versus Control group; ^#p < 0.05 versus Vehicle (Vehl)-Hcys group.