NLRP3 inflammasome as a novel target for docosahexaenoic acid metabolites to abrogate glomerular injury

Abstract

The nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome has been implicated in podocyte injury and glomerular sclerosis during hyperhomocysteinemia (hHcys). However, it remains unclear whether the NLRP3 inflammasome can be a therapeutic target for treatment of hHcys-induced kidney injury. Given that DHA metabolites-resolvins have potent anti-inflammatory effects, the present study tested whether the prototype, resolvin D1 (RvD1), and 17S-hydroxy DHA (17S-HDHA), an intermediate product, abrogate hHcys-induced podocyte injury by targeting the NLRP3 inflammasome. In vitro, confocal microscopy demonstrated that 17S-HDHA (100 nM) and RvD1 (60 nM) prevented Hcys-induced formation of NLRP3 inflammasomes, as shown by reduced colocalization of NLRP3 with apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) or caspase-1. Both DHA metabolites inhibited Hcys-induced caspase-1 activation and interleukin-1β production. However, DHA had no significant effect on these Hcys-induced changes in podocytes. In vivo, DHA lipoxygenase metabolites substantially inhibited podocyte NLRP3 inflammasome formation and activation and consequent glomerular sclerosis in mice with hHcys. Mechanistically, RvD1 and 17S-HDHA were shown to suppress Hcys-induced formation of lipid raft redox signaling platforms and subsequent O₂^·- production in podocytes. It is concluded that inhibition of NLRP3 inflammasome activation is one of the important mechanisms mediating the beneficial action of RvD1 and 17S-HDHA on Hcys-induced podocyte injury and glomerular sclerosis.

Keywords: inflammation; kidney; lipid mediators; lipid rafts; lipoxygenase; nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3; podocyte; ω-3 fatty acid.

Fig. 1.

Effects of DHA metabolites, via LOX, on NRLP3 inflammasome formation in podocytes. Podocytes were cultured for 24 h with Hcys (40 μM) in the absence or presence of 17S-HDHA (17HDHA; 100 nM), RvD1 (60 nM), or DHA (20 μM) as indicated. A: Representative images and summarized data of the colocalization between NLRP3 (green) and ASC (red) in different podocytes, which were cultured and treated on different days (n = 6). B: Representative images and summarized data of the colocalization between NLRP3 (green) and caspase-1 (red) in different podocytes which were cultured and treated on different days (n = 6). *P < 0.05 versus Ctrl-Vehl group, #P < 0.05 versus Hcys-Vehl group. Ctrl, control; Vehl, vehicle; 17S-HDHA, 17-hydroxy docosahexaenoic acid; DHA, ω-3 fatty acid docosahexaenoic acid.

Fig. 2.

Blockade of NLRP3 inflammasome activation by DHA metabolites in podocytes. A: Representative Western blot gel document and summarized data showing the protein expression of pro-caspase-1 and active caspase-1 in different podocytes that were cultured and treated on different days (n = 7). B: Caspase-1 activity in different podocytes that were cultured and treated on different days (n = 8). C: IL-1β production in different podocytes that were cultured and treated on different days (n = 9). *P < 0.05 versus Ctrl-Vehl group, #P < 0.05 versus Hcys-Vehl group. Ctrl, control; Vehl, vehicle.

Fig. 3.

DHA LOX metabolites prevented Hcys-induced podocyte injury. A: Representative images and summarized data showing the expression of podocin in different podocytes that were cultured and treated on different days (n = 6). B: Representative images and summarized data showing the expression of desmin in different podocytes that were cultured and treated on different days (n = 6). C: Images showing the expression of F-actin fiber in different podocytes that were cultured and treated on different days (n = 5). D: Summarized data showing production of VEGF in different podocytes that were cultured and treated on different days (n = 4). *P < 0.05 versus Ctrl-Vehl group, #P < 0.05 versus Hcys-Vehl group. Ctrl, control; Vehl, vehicle.

Fig. 4.

DHA metabolites by LOX blocked NLRP3 inflammasome formation and activation in podocytes of hyperhomocysteinemic mice. A: Representative images and summarized data of the colocalization between NLRP3 (green) and ASC (red) in glomeruli from mice with different genotypes and treatments (n = 6). B: Representative images and summarized data of the colocalization between NLRP3 (green) and caspase-1 (red) in glomeruli from mice with different genotypes and treatments (n = 6). C: Immunohistochemical staining of IL-1β production in glomeruli from mice with different genotypes and treatments (n = 5). D: Immunohistochemical staining of IL-18 production in glomeruli from mice with different genotypes and treatments (n = 5). E: IL-1β production in isolated glomeruli from WT mice with different treatments (n = 4). F: IL-18 production in isolated glomeruli from WT mice with different treatments (n = 4). *P < 0.05 versus WT-ND-Vehl group, #P < 0.05 versus WT-FF-Vehl group, $P < 0.05 versus WT-FF-DHA group. Vehl, vehicle.

Fig. 5.

DHA metabolites by LOX protected mouse glomeruli from hHcys-induced dysfunction and injury. A: Summarized data showing the urinary protein from mice with different genotypes and treatments (n = 7). B: Summarized data showing the urinary albumin from mice with different genotypes and treatments (n = 7). C: Images showing the glomerular collagen deposition (picrosirius red staining) from mice with different genotypes and treatments (n = 6). D: Representative images and summarized data of the glomerular morphological changes (periodic acid-Schiff staining) from mice with different genotypes and treatments (n = 6). E: Representative images showing immunostained glomeruli for podocin from mice with different genotypes and treatments (n = 6). F: Representative images showing immunostained glomeruli for desmin from mice with different genotypes and treatments (n = 6). *P < 0.05 versus WT-ND-Vehl group, #P < 0.05 versus WT-FF-Vehl group, $P < 0.05 versus WT-FF-DHA group. Vehl, vehicle.

Fig. 6.

Contribution of LR redox signaling platform formation and activation to the protective effects of DHA metabolites. Podocytes were cultured as described above. A: Representative images and summarized data of the colocalization between LT marker, CTXB (green), with NOX subunit, gp91 (red), in different podocytes that were cultured and treated on different days (n = 6). B: Representative images and summarized data of the colocalization between CTXB (green) with NOX subunit, p47 (red), in different podocytes that were cultured and treated on different days (n = 6). C: Summarized data of SOD-dependent O₂^·− production in different podocytes that were cultured and treated on different days (n = 6). *P < 0.05 versus Ctrl-Vehl group, #P < 0.05 versus Hcys-Vehl group. Ctrl, control; Vehl, vehicle.