Nod-like receptor protein 3 (NLRP3) inflammasome activation and podocyte injury via thioredoxin-interacting protein (TXNIP) during hyperhomocysteinemia

Abstract

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.

Keywords: Glomerulosclerosis; Homocysteine; Inflammation; Kidney; Podocyte; Redox Signaling; Thioredoxin.

FIGURE 1.

TXNIP and NLRP3 recruitment to the high molecular weight inflammasome fractions upon stimulation with Hcys. A, SEC chromatogram illustrating the elution curves of a typical standard and podocyte protein sample. B, representative Western blot gel documents depicting the shift of NLRP3 and TXNIP protein during Hcys treatment, which was prevented during TXNIP inhibition. C, summarized quantification of either NLRP3 or TXNIP protein residing in the inflammasome fractions (n = 4). mAu, milli-absorbance units; ST, standard; Ctrl, control; Vehl, vehicle; TXNIPsi, TXNIP siRNA. *, p < 0.05 versus control; #, p < 0.05 versus Hcys.

FIGURE 2.

TXNIP inhibition prevented Hcys-induced NLRP3 inflammasome formation. A, confocal microscopic detection of NLRP3 (green) with ASC (red) and NLRP3 (green) with TXNIP (red) and their colocalization together (yellow), indicative of the inflammasome formation. B and C, summarized data showing the quantification of the extent of colocalization between NLRP3 with ASC and NLRP3 with TXNIP (n = 4–6). Ctrl, control; Vehl, vehicle; TXNIPsi, TXNIP siRNA. *, p < 0.05 versus vehicle-control; #, p < 0.05 versus vehicle-Hcys.

FIGURE 3.

Attenuation of Hcys-induced NLRP3 inflammasome activation by TXNIP blockade. A, effect of TXNIPsi and verapamil on Hcys-induced NLRP3 inflammasome activation, shown as the fold change of caspase-1 activation versus vehicle control (Vehl-Ctrl) (n = 7–8). B, measured in the supernatant of cultured podocytes, TXNIP inhibition suppressed Hcys-induced IL-1β production (n = 8). C, tunicamycin-treated cells displayed no change in caspase-1 activity, indicating no significant effect of ER stress on inflammasome activation in podocytes (n = 5–6). D, no change in IL-1β production in the supernatant of tunicamycin-treated podocytes validated caspase-1 activation results (n = 5–6). E, measurement of [Ca²⁺]_i demonstrated that verapamil inhibition of Hcys-induced NLRP3 inflammasome activation was not due to its calcium channel blocker properties (n = 4). Ctrl, control; Vehl, vehicle; Tuni, tunicamycin; TXNIPsi, TXNIP siRNA. *, p < 0.05 versus vehicle control; #, p < 0.05 versus vehicle Hcys.

FIGURE 4.

Inhibition of TXNIP preserved podocytes integrity. A, immunofluorescence staining of podocyte-specific marker podocin and podocyte injury marker desmin, where Hcys-induced decrease in podocin and increase in desmin expression were attenuated by TXNIPsi and verapamil. B, summarized data showing the relative intensity of podocin and desmin staining (n = 6). C, VEGF was measured in the supernatant of podocytes and used as an indicator of podocyte functionality, where Hcys-damaged podocytes suppressed VEGF secretion, which was restored upon TXNIP inhibition (n = 6). D, Hcys treatment increased the population of annexin V⁺ podocytes, indicative of increased cell death, which was prevented upon verapamil administration (n = 6). Ctrl, control; Vehl, vehicle; TXNIPsi, TXNIP siRNA. *, p < 0.05 versus vehicle control; #, p < 0.05 versus vehicle Hcys.

FIGURE 5.

Efficiency of local in vivo transfection of TXNIP shRNA into the renal cortex by the ultrasound microbubble technique. A, images taken 3, 7, and 14 days post-transfection by an in vivo imaging system daily confirmed transfection efficiency. B, ex vivo image 4 days after transfection of hemidissected kidney demonstrated successful localized gene expression. C, real time RT-PCR data quantifying mRNA silencing efficiency of the plasmid after 4 weeks of maintenance on either a ND or FF diet (n = 6). D, immunofluorescent staining of TXNIP in the glomeruli of transfected mice. Luci, luciferase; TXNIPsh, TXNIP shRNA. *, p < 0.05 versus luciferase-ND.

FIGURE 6.

In vivo inhibition of TXNIP and its effect on NLRP3 inflammasome formation. A, confocal microscopy demonstrated the colocalization between NLRP3 (green) with ASC (red) and NLRP3 (green) with TXNIP (red) in the glomeruli of luciferase (Luci), TXNIP shRNA (TXNIPsh), and verapamil-treated mice maintained on either a ND or FF diet. B and C, summarized data showing the quantification of the extent of colocalization between NLRP3 with ASC and NLRP3 with TXNIP (n = 6). D, co-immunoprecipitation studies demonstrated robust in vivo TXNIP-NLRP3 binding in mice with hHcys, which was not evident after TXNIP inhibition (n = 5). E, plasma Hcys concentrations (Conc.) of all mice fed a FF diet were all elevated and were unaffected by in vivo TXNIP inhibition either by shRNA transfection or verapamil administration (n = 4–6). *, p < 0.05 versus luciferase ND; #, p < 0.05 versus luciferase FF.

FIGURE 7.

In vivo TXNIP shRNA transfection and verapamil treatment blocked caspase-1 activation and IL-1β production. A, caspase-1 activity, shown as fold change versus luciferase ND, in luciferase, TXNIP shRNA, and verapamil-treated mice with FF diet-induced hHcys (n = 5–6). B, in vivo TXNIP inhibition prevented hHcys-induced IL-1β production (n = 6). Luci, luciferase; TXNIPsh, TXNIP shRNA. *, p < 0.05 versus luciferase ND; #, p < 0.05 versus luciferase FF.

FIGURE 8.

Amelioration of hHcys-induced glomerular damage by in vivo TXNIP inhibition. A and B, hHcys-induced increases in proteinuria and albuminuria were diminished by TXNIP shRNA transfection and verapamil administration (n = 6–8). C, observation of glomerular morphology in periodic acid-Schiff-stained slides revealed severe pathological changes in the glomeruli of luciferase FF mice, which were prevented in TXNIP inhibited mice. D, pathological changes in the glomeruli were semiquantitatively scored and summarized as the glomerular damage index (n = 5). E and F, in vivo immunofluorescent staining of podocin and desmin to assess glomerular podocyte condition (n = 6). BW, body weight; Luci, luciferase; TXNIPsh, TXNIP shRNA. *, p < 0.05 versus luciferase ND; #, p < 0.05 versus luciferase FF.