(T1130-04-19) Thioredoxin Interacting Protein (TXNIP) Inhibitor Protects Against Nicotine-Induced Podocyte Injury and Glomerular Sclerosis

Purpose: Cigarette smoking is a well-established risk factor responsible for a broad spectrum of diseases and is the leading cause of preventable deaths globally. Cigarette smoking induces renovascular resistance and downregulation of glomerular function that results in chronic kidney disease (CKD) onset and eventually culminates to end-stage renal disease. Although studies have enumerated the cigarette smoking induced phenotypic alterations responsible for CKD, the cellular and molecular mechanisms governing the same remains poorly understood. The activation and formation of Nod-like receptor family pyrin domain containing 3 (Nlrp3) inflammasomes in podocytes are implicated in the development of nicotine-induced podocyte injury. Recently, we demonstrated that NADPH oxidase dependent O₂^•− production (ROS) mediates the nicotine-induced Nlrp3 inflammasome activation and thereby results in podocyte injury. However, it remains unknown how NADPH oxidase-derived ROS is sensed by Nlrp3 inflammasomes in podocytes upon nicotine stimulation. Hence, the current study tested whether thioredoxin-interacting protein (TXNIP) mediates nicotine-induced NLRP3 inflammasome activation and consequent podocyte injury.

Methods: Inflammasome formation and immunofluorescence expressions were quantified by confocal microscopy. Interleukin (IL)-1β production was measured using ELISA. Caspase-1 activity was measured by colorimetric assay and cell permeability assay was determined by microplate reader.

Results: Confocal microscopic analysis showed that nicotine treatment significantly increased the colocalization of Nlrp3 with apoptosis associated speck-like protein containing a caspase recruitment domain (Asc) (Figure 1) or Nlrp3 with caspase-1 or Nlrp3 with TXNIP in podocytes compared to control cells. Pretreatment with TXNIP inhibitor verapamil abolished the nicotine-induced colocalization of Nlrp3 with Asc (Figure 1) or Nlrp3 with caspase-1 or Nlrp3 with TXNIP. Correspondingly, nicotine treatment significantly increased the caspase-1 activity and IL-1β production compared to control cells. However, prior treatment with TXNIP inhibitor verapamil significantly attenuated the nicotine-induced caspase-1 activity and IL-1β production. Further immunofluorescence analysis showed that nicotine treatment significantly decreased the podocin (Figure 2) and nephrin expression (indicative of podocyte damage) compared to control cells. However, pretreatment with TXNIP inhibitor verapamil attenuated the nicotine-induced podocin (Figure 2) and nephrin reduction. In addition, confocal and biochemical analysis showed that nicotine treatment significantly increased the desmin expression (Figure 3) and cell permeability compared to control cells. However, prior treatment with TXNIP inhibitor verapamil significantly attenuated the nicotine-induced desmin expression (Figure 3) and cell permeability.

Conclusion: Our results demonstrate that TXNIP binding to Nlrp3 is a key signaling mechanism necessary for nicotine induced NLRP3 inflammasome formation and activation and subsequent podocyte damage and glomerular sclerosis.

Acknowledgements:

Funding: supported by NIH grant, R01HL148711.


TXNIP specific pharmacological inhibition (with verapamil) attenuates nicotine-induced increase in Nlrp3/Asc colocalization.


TXNIP specific inhibition (with verapamil) attenuates nicotine-induced decrease in podocin expression.


TXNIP targeted inhibition (with Verapamil) prevents nicotine-induced upregulation of desmin expression.