Sabena M. Conley

Nod-like Receptor Protein 3 (NLRP3) Inflammasome Activation and Podocyte Injury via Thioredoxin-Interacting Protein (TXNIP) during Hyperhomocysteinemia *

Background: Hyperhomocysteinemia (hHcys) contributes to glomerular injury by activating NLRP3 inflammasomes in response to increased oxidative stress. Results: Thioredoxin-interacting protein (TXNIP) aggregated with NLRP3 inflammasomes, and blocking TXNIP prevented inflammasome activation during hHcys. Conclusion: TXNIP uniquely links changes in oxidative stress to hHcys-induced NLRP3 inflammasome activation. Significance: Glomerular injury related to hHcys can be prevented by TXNIP inhibition. NADPH oxidase-derived reactive oxygen species (ROS) have been reported to activate NLRP3 inflammasomes resulting in podocyte and glomerular injury during hyperhomocysteinemia (hHcys). However, the mechanism by which the inflammasome senses ROS is still unknown in podocytes upon hHcys stimulation. The current study explored whether thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of the antioxidant thioredoxin and ROS sensor, mediates hHcys-induced NLRP3 inflammasome activation and consequent glomerular injury. In cultured podocytes, size exclusion chromatography and confocal microscopy showed that inhibition of TXNIP by siRNA or verapamil prevented Hcys-induced TXNIP protein recruitment to form NLRP3 inflammasomes and abolished Hcys-induced increases in caspase-1 activity and IL-1β production. TXNIP inhibition protected podocytes from injury as shown by normal expression levels of podocyte markers, podocin and desmin. In vivo, adult C57BL/6J male mice were fed a folate-free diet for 4 weeks to induce hHcys, and TXNIP was inhibited by verapamil (1 mg/ml in drinking water) or by local microbubble-ultrasound TXNIP shRNA transfection. Evidenced by immunofluorescence and co-immunoprecipitation studies, glomerular inflammasome formation and TXNIP binding to NLRP3 were markedly increased in mice with hHcys but not in TXNIP shRNA-transfected mice or those receiving verapamil. Furthermore, TXNIP inhibition significantly reduced caspase-1 activity and IL-1β production in glomeruli of mice with hHcys. Correspondingly, TXNIP shRNA transfection and verapamil attenuated hHcys-induced proteinuria, albuminuria, glomerular damage, and podocyte injury. In conclusion, our results demonstrate that TXNIP binding to NLRP3 is a key signaling mechanism necessary for hHcys-induced NLRP3 inflammasome formation and activation and subsequent glomerular injury.

Inhibition of Hyperhomocysteinemia-Induced Inflammasome Activation and Glomerular Sclerosis by NLRP3 Gene Deletion

Background/Aims: Hyperhomocysteinemia (hHcys) has been reported to initiate Nod-like receptor protein 3 (NLRP3) inflammasome formation and activation in podocytes, leading to glomerular dysfunction and sclerosis. However, it remains unknown whether Nlrp3 gene is critical for the formation and activation of inflammasomes in glomeruli of hHcys mice. Methods: Plasma homocysteine concentration was estimated utilizing HPLC, inflammasome formation and immunofluorescence expression from confocal microscopy, IL-1β production from ELISA. Results: Uninephrectomized Nlrp3 knockout (Nlrp3-/-) and wild type (Nlrp3+/+) and intra renal Nlrp3 shRNA-transfected wild type mice (Nlrp3 shRNA) were fed a folate free (FF) diet or normal chow (ND) for 4 weeks to produce hHcys. The plasma Hcys levels were significantly elevated in both Nlrp3-/- and Nlrp3+/+ mice fed a FF diet compared to ND fed mice. The FF diet significantly increased the colocalization of Nlrp3 with apoptosis-associated speck-like protein (ASC) or caspase-1, caspase-1 activity and IL-1β production in glomeruli of Nlrp3+/+, but not in Nlrp3-/- mice and local Nlrp3 shRNA transfected mice. Correspondingly, the glomerular damage index (GDI) and urinary protein excretion were significantly higher in Nlrp3+/+ mice compared to ND fed mice. However, the hHcys-induced increase in GDI and proteinuria were significantly lower in Nlrp3-/- and local Nlrp3 shRNA transfected mice than in Nlrp3+/+ mice. Immunocytochemical analysis showed that hHcys decreased expression of podocin and nephrin, but increased desmin expression in glomeruli of Nlrp3+/+ mice compared to Nlrp3-/- mice. Conclusion: Nlrp3 gene is an essential component of Nlrp3 inflammasomes and that targeting Nlrp3 may be important therapeutic strategy to prevent inflammasome activation and thereby protect podocytes and glomeruli from hHcys-induced injury.

Mesenchymal stem cell transplantation inhibited high salt-induced activation of the NLRP3 inflammasome in the renal medulla in Dahl S rats

Inflammasomes activate caspase-1 to produce interleukin (IL)-1β. Activation of the NLRP3 inflammasome is involved in various renal pathological conditions. It remains unknown whether the NLRP3 inflammasome activation participates in the abnormal renal response to high-salt (HS) diet in Dahl salt-sensitive (S) rats. In addition, our lab recently showed that transplantation of mesenchymal stem cells (MSCs) attenuated HS-induced inflammation in the renal medulla in Dahl S rat. However, it is unclear whether the anti-inflammatory action of MSCs is associated with inhibition of the NLRP3 inflammasome. The present study determined the response of the NLRP3 inflammasome to HS intake and the effect of MSC transplantation on the NLRP3 inflammasome in the renal medulla in Dahl S rats. Immunostaining showed that the inflammasome components NLRP3, ASC, and caspase-1 were mainly present in distal tubules and collecting ducts. Interestingly, the renal medullary levels of these inflammasome components were remarkably increased after a HS diet in Dahl S rats, while remaining unchanged in normal rats. This HS-induced activation of the NLRP3 inflammasome was significantly blocked by MSC transplantation into the renal medulla in Dahl S rats. Furthermore, infusion of a caspase-1 inhibitor into the renal medulla significantly attenuated HS-induced hypertension in Dahl S rats. These data suggest that HS-induced activation of the NLRP3 inflammasome may contribute to renal medullary dysfunction in Dahl S rats and that inhibition of inflammasome activation may be one of the mechanisms for the anti-inflammatory and anti-hypertensive effects of stem cells in the renal medulla in Dahl S rats.

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

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

Inflammasome Activation in Chronic Glomerular Diseases

BACKGROUND The intracellular multiprotein complex termed the inflammasome functions as a platform of pro-inflammatory cytokine production such as IL-1β and IL-18. Under certain conditions, however, the inflammasome produces non-canonical effects such as induction of cell death, pyroptosis and cell metabolism alterations. OBJECTIVE In mammalian cells, several types of inflammasomes were identified, but the most widely studied one is the inflammasome containing NOD-like receptor with pyrin domain 3 (NLRP3), which has recently been reported as a central pathogenic mechanism of chronic degenerative diseases. Many activators or risk factors exert their actions through the activation of the NLRP3 inflammasome to produce a variety of functional changes in different cells including inflammatory, metabolic or survival responses. Several molecular signaling pathways are shown to mediate the activation of the NLRP3 inflammasome, and they are related to the modifications in K+ efflux, increased lysosome leakage and activation of cathepsin B or enhanced reactive oxygen species (ROS) production. In the kidney, inflammation is believed to mediate or promote the progression of glomerular sclerotic pathologies resulting in end-stage renal disease (ESRD). NLRP3 inflammasome activation may turn on glomerular inflammation and other cell damages, contributing to the onset of glomerular injury and ESRD. This inflammasome activation not only occurs in immune cells, but also in residential cells such as endothelial cells and podocytes in the glomeruli. SUMMARY This review briefly summarizes current evidence of NLRP3 inflammasome activation and related molecular mechanisms in renal glomeruli. The possible canonical and non-canonical effects of this inflammasome activation and its potential implication in the development of different glomerular diseases are highlighted.

Contribution of guanine nucleotide exchange factor Vav2 to NLRP3 inflammasome activation in mouse podocytes during hyperhomocysteinemia

Abstract NADPH oxidase (NOX)‐derived reactive oxygen species (ROS) have been demonstrated to mediate the activation of NOD‐like receptor protein 3 (NLRP3) inflammasomes in podocytes in response to elevated levels of homocysteine (Hcys). However, it remains unknown how NLRP3 inflammasome activation is triggered by NOX. The present study tested whether the guanine nucleotide exchange factor Vav2 mediates Rac1‐mediated NOX activation in response to elevated Hcys leading to NLRP3 inflammasome activation in podocytes and consequent glomerular injury. In a mouse model of hyperhomocysteinemia (hHcys), we found that mice with hHcys (on the FF diet) or oncoVav2 (a constitutively active form of Vav2) transfection in the kidney exhibited increased colocalization of NLRP3 with apoptosis‐associated speck‐like protein (ASC) or caspase‐1 and elevated IL‐1&bgr; levels in glomeruli, indicating the formation and activation of the NLRP3 inflammasome. This glomerular NLRP3 inflammasome activation was accompanied by podocyte dysfunction and glomerular injury, even sclerosis. Local transfection of Vav2 shRNA plasmids significantly attenuated hHcys‐induced NLRP3 inflammasome activation, podocyte injury, and glomerular sclerosis. In cultured podocytes, Hcys treatment and oncoVav2 transfection were also found to increase NLRP3 inflammasome formation and activation, which were all inhibited by Vav2 shRNA. Furthermore, Vav2 shRNA prevented Hcys‐induced podocyte damage as shown by restoring Hcys‐impaired VEGF secretion and podocin production. This inhibitory action of Vav2 shRNA on Hcys‐induced podocyte injury was associated with reduction of Rac1 activity and ROS production. These results suggest that elevated Hcys levels activate Vav2 and thereby increase NOX activity leading to ROS production, which triggers NLRP3 inflammasome activation, podocyte dysfunction and glomerular injury. Graphical abstract Figure. No Caption available. HighlightsVav2 is essential for hHcys‐induced Rac‐1 activation and inflammasome activation.Overexpression of Vav2 enhanced inflammasome activation in glomeruli of hHcys mice.Vav2 alone trigger inflammasome activation and contribute to glomerular injury.