Angiotensin II-induced hypertensive renal inflammation is mediated through HMGB1-TLR4 signaling in rat tubulo-epithelial cells

Exp Cell Res. 2015 Jul 15;335(2):238-47. doi: 10.1016/j.yexcr.2015.05.011. Epub 2015 May 29.

Abstract

Background and purpose: Angiotensin II is a vaso-constrictive peptide that regulates blood pressure homeostasis. Even though the inflammatory effects of AngII in renal pathophysiology have been studied, there still exists a paucity of data with regard to the mechanism of action of AngII-mediated kidney injury. The objective of this study was to elucidate the mechanistic role of HMGB1-TLR4 signaling in AngII-induced inflammation in the kidney.

Experimental approach: Rat tubular epithelial cells (NRK52E) were treated with AngII over a preset time-course. In another set of experiments, HMGB1 was neutralized and TLR4 was knocked down using small interfering RNA targeting TLR4. Cell extracts were subjected to RT-PCR, immunoblotting, flow cytometry, and ELISA.

Key results: AngII-induced inflammation in NRK52E cells increased gene and protein expression of TLR4, HMGB1 and key proinflammatory cytokines (TNFα and IL1β). Pretreatment with Losartan (an AT1 receptor blocker) attenuated the AngII-induced expression of TLR4 and inflammatory cytokines. TLR4 silencing was used to elucidate the specific role played by TLR4 in AngII-induced inflammation. TLR4siRNA treatment in these cells significantly decreased the AngII-induced inflammatory effect. Consistent observations were made when the Ang II treated cells were pretreated with anti-HMGB1. Downstream activation of NFκB and rate of generation of ROS was also decreased on gene silencing of TLR4 and exposure to anti-HMGB1.

Conclusions and implications: These results indicate a key role for HMGB1-TLR4 signaling in AngII-mediated inflammation in the renal epithelial cells. Our data also reveal that AngII-induced effects could be alleviated by HMGB1-TLR4 inhibition, suggesting this pathway as a potential therapeutic target for hypertensive renal dysfunctions.

Keywords: Angiotensin II; HMGB1; Hypertensive renal injury; Inflammation; Reactive oxygen species; TLR4.

MeSH terms

  • Angiotensin II
  • Animals
  • Antihypertensive Agents / pharmacology
  • Cell Line
  • Epithelial Cells / immunology
  • Epithelial Cells / metabolism*
  • HMGB1 Protein / metabolism*
  • Hypertension / chemically induced
  • Hypertension / complications
  • Hypertension / metabolism*
  • Kidney Failure, Chronic / etiology
  • Kidney Failure, Chronic / immunology
  • Kidney Failure, Chronic / metabolism*
  • Kidney Tubules / pathology
  • Losartan / pharmacology
  • NF-kappa B / metabolism
  • Rats
  • Signal Transduction
  • Toll-Like Receptor 4 / metabolism*

Substances

  • Antihypertensive Agents
  • HMGB1 Protein
  • Hbp1 protein, rat
  • NF-kappa B
  • Tlr4 protein, rat
  • Toll-Like Receptor 4
  • Angiotensin II
  • Losartan