The Protective Role of Angiotensin AT2 Receptors in Obesity-Linked Inflammation and Renal Injury

Obesity is an independent risk factor for the development of chronic kidney disease (CKD). Non-resolving inflammation is a key mechanism that initiates and amplifies progressive renal injury in the setting of obesity. Recently, Toll-like receptor 4 (TLR4) has been identified as a molecular link between the innate immune system and obesity-linked chronic inflammation. Up-regulation of TLR4 on various tissues has been documented in obese patients and its activation by endogenous ligands, such as free fatty acids, may represent an important inflammatory mechanism that contributes to end-organ damage in obesity. We found that TLR4 expression was also up-regulated in the kidneys of obese rats, suggesting their potential involvement in mediating renal injury in obesity. Angiotensin AT2 receptors (AT2R) are up-regulated in the kidneys from obese rats. AT2R stimulation has been shown to exert anti-inflammatory, renoprotective effects in diverse models of renal injury. However, whether AT2R activation can attenuate TLR4-induced inflammation, which occurs in obesity, is not clear. The present study was designed to characterize the anti-inflammatory, renoprotective role of AT2R stimulation in obesity-linked inflammation and renal injury using a combination of in vivo and in vitro experimental models. First, obesity was induced in AT2R knock-out (AT2KO) mice by placing them on a high-fat diet (HFD) for a period of 16 weeks. Subsequently, plasma and renal pro- and anti-inflammatory cytokines were measured and renal morphology was assessed. Compared to wild-type (WT) mice on the HFD, AT2KO mice on the same diet had increased systemic and renal levels of pro-inflammatory cytokine TNF-α while anti-inflammatory IL-10 was markedly lower. This was associated with increased renal injury in HFD-fed AT2KO mice. Also, stimulation of AT2R for 2 weeks in pre-hypertensive obese Zucker rats (OZR) was found to attenuate plasma and renal pro-inflammatory TNF-α and IL-6. This was associated with decreased renal macrophage infiltration and mesangial matrix expansion (MME). Conversely, AT2R antagonist treatment led to decreased renal IL-10 and worsening of MME. These findings suggested that AT2R stimulation may favor the production of anti-inflammatory cytokine IL-10, while down-regulating pro-inflammatory cytokine production, thus affording renoprotection in obesity. Next, a series of in vitro experiments using proximal tubule epithelial cells (PTEC), glomerular epithelial cells and macrophages was conducted to clarify the cellular mechanisms that orchestrate the anti-inflammatory response to AT2R stimulation. AT2R agonist was found to attenuate TLR4-induced increase in pro-inflammatory cytokine production via increased IL-10 production in PTEC and macrophage cell-lines. Furthermore, IL-10 was increased in PTECs, but not in macrophages, by AT2R stimulation in the absence of TLR4 activation. The increase in IL-10 was mediated by NO/cGMP signaling in PTECs. The altered cytokine profile of PTECs was found to inhibit the activation of macrophages in response to the TLR4 ligand, lipopolysaccharide (LPS).
Lastly, to demonstrate the anti-inflammatory role of AT2R stimulation in vivo, a mouse model of LPS-induced systemic inflammation and acute kidney injury was used. Mice pre-treated with AT2R agonist had lower renal pro-inflammatory cytokine levels. This was associated with preserved renal function and structural integrity compared to LPS-treated mice. Furthermore, renal TLR4 expression in AT2R agonist pre-treated mice was also lower than LPS-treated controls. Overall, these findings demonstrate a protective role for AT2R stimulation in obesity-linked inflammation and renal injury. This protective effect is mediated via increased IL-10 production and cross-talk between renal epithelial cells and infiltrating macrophages, which can attenuate local renal inflammation. In the present study, we also identify signaling pathways, including NO/cGMP signaling and sustained ERK1/2 phosphorylation as potential mechanisms involved in promoting IL-10 production. Our results indicate that AT2R stimulation may be beneficial in TLR4-induced acute renal injury as well as obesity-linked chronic kidney disease.