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Enzyme-induced hypoxia leads to inflammation in urothelial cells in vitro

  • Urology - Original Paper
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Abstract

Purpose

To determine the contributions of different durations of hypoxia to NLRP3 inflammasome activation in urothelial cells and how ischemic changes in bladder tissues is an important chemical que that leads to pathological changes seen in BOO.

Methods

A rat urothelial cell line (MYP3) was exposed to either a short duration (2 h) or long duration (6 h) of enzyme-induced hypoxia. Following exposure to a short duration of hypoxia, NO and ATP concentrations were measured from supernatant media and caspase-1 levels were measured from cell lysates. In a separate experiment, cells were fixed following hypoxia exposure and immunostained for HIF-1α stabilization.

Results

Although short exposure of low oxygen conditions resulted in a hypoxic response in MYP3 cells, as indicated by HIF-1α stabilization and increased NO activity, NLRP3 inflammasome activation was not observed as caspase-1 activity remained unchanged. However, exposure of MYP3 cells to a longer duration of hypoxia resulted in an increase in intracellular caspase-1 activity. Furthermore, treatment with antioxidant (GSH) or TXNIP inhibitor (verapamil) attenuated the hypoxia-induced increase in caspase-1 levels indicating that hypoxia primarily drives inflammation through a ROS-mediated TXNIP/NLRP3 pathway.

Conclusion

We conclude that hypoxia induced bladder damage requires a duration that is more likely related to elevated storage pressures/hypoxia, seen in later stages of BOO, as compared to shorter duration pressure elevation/hypoxia that is encountered in normal micturition cycles or early in the BOO pathology where storage pressures are still normal.

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Data availability

The data that support this study are available upon request from the corresponding author, JN.

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Acknowledgements

The authors thank the members of the Harcum Cell-Culture Optimization Lab at Clemson University for use of their blood gas analyzer, specifically Tom Caldwell and Dr. Sarah Harcum. In addition, the authors thank the Nagatomi Mechanobiology Laboratory and lab members Cody Dunton, Julia Lunt, and Rachael Christensen. Funding was provided by NIH DK103435 and GM103444. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. 2139915.

Funding

National Institute of Diabetes and Digestive and Kidney Diseases, DK103435, J. Todd Purves, National Institute of General Medical Sciences, GM103444, Jiro Nagatomi, National Science Foundation, 2139915, Britney Hudson

Author information

Authors and Affiliations

  1. Department of Bioengineering, 301 Rhodes Engineering Research Center, Clemson University, Clemson, SC, 29634-0905, USA

    Britney N. Hudson, J. Todd Purves, Francis M. Hughes & Jiro Nagatomi

  2. Department of Surgery, Division of Urology, Duke University Medical Center, Durham, NC, USA

    J. Todd Purves & Francis M. Hughes

  3. Department of Pediatrics, Duke University Medical Center, Durham, NC, USA

    J. Todd Purves

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  1. Britney N. Hudson
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Contributions

BNH, JTP, FMH and JN conceived the project; BNH, JTP, FMH and JN designed experiments; BNH performed experiments; BNH analyzed data; BNH, JTP, FMH and JN interpreted results of experiments; BNH prepared figures; BNH and JN drafted manuscript; BNH, JTP, FMH and JN edited and revised manuscript. BNH, JTP, FMH and JN approved the final version of the manuscript.

Corresponding author

Correspondence to Jiro Nagatomi.

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Supplementary Information

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11255_2023_3900_MOESM1_ESM.docx

Supplementary file 1 Fig. 1 Glucose Levels of GOX/CAT Media. There was no difference in glucose concentrations of the 0 µg/mL GOX media and the 0.5 µg/mL GOX media. Data are the mean ± SEM analyzed using two-way ANOVA. Each group is compared to the control. N=2. Fig. 2 Nitric Oxide Levels After Treatment with L-NAME. MYP3 cells were pre-treated with 16 mM of L-NAME for 1 h before hypoxic exposure. During hypoxic exposure via GOX/CAT media, L-NAME remained in the media. Treatment with L-NAME was able to attenuate the hypoxia induced increase in NO levels at hour 1. Data are mean ± SEM and compared to the normoxic control (one-fold). Data was analyzed using two-way ANOVA. N=3. *p<0.05, **p<0.01. Fig. 3 Caspase-1 Levels After Treatment with L-NAME. MYP3 cells were pre-treated with 16 mM of L-NAME for 1 h before hypoxic exposure. L-NAME remained in the enzyme media for the duration of hypoxic exposure. Treatment with L-NAME didn’t result in any changes in intracellular caspase-1 activity. Data are mean ± SEM and compared to the normoxic control (one-fold). Data was analyzed using one-way ANOVA. N=3.

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Hudson, B.N., Purves, J.T., Hughes, F.M. et al. Enzyme-induced hypoxia leads to inflammation in urothelial cells in vitro. Int Urol Nephrol 56, 1565–1575 (2024). https://doi.org/10.1007/s11255-023-03900-x

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