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OPEN
Received: 17 January 2017 Accepted: 13 June 2017 Published online: 24 July 2017
Toll-like Receptor 4 Deficiency Reduces Oxidative Stress and Macrophage Mediated Inflammation in Hypertensive Kidney Sathnur Pushpakumar1, Lu Ren1, Sourav Kundu2, Alejandra Gamon3, Suresh C. Tyagi1 & Utpal Sen1 Oxidative stress and inflammation are integral to hypertension-induced renal injury. A unifying feature for the two components is Toll-like receptors (TLR), which are key regulators of the innate immune system. Recent studies implicate TLR4 activation and oxidative stress in cardiovascular diseases and also as a link between inflammation and hypertension. However, its role in hypertension induced renal injury remains unexplored. In the present study, we investigated whether TLR-4 deficiency reduces Ang-II-induced renal injury and fibrosis by attenuating reactive oxygen species (ROS) production and inflammation. C3H/HeOuJ mice with normal TLR-4 and C3H/HeJLps-d with dysfunctional TLR4 (TLR4 deficiency) were treated without or with Ang-II. In response to Ang-II, TLR4 deficient mice had reduced renal resistive index and increased renal cortical blood flow compared to mice with normal TLR4. Further, TLR4 deficiency reduced oxidative stress and increased antioxidant capacity (MnSOD, CuSOD and Catalase activity). TLR4 deficiency was also associated with reduced inflammation (MCP-1, MIP-2, TNF-α, IL-6 and CD68), decreased accumulation of bone marrow-derived fibroblasts and TGF-β expression. Our data suggests that in C3H/HeJLps-d mice, deficiency of functional TLR4 reduces oxidative stress and macrophage activation to decrease TGF-β-induced extracellular matrix protein deposition in the kidney in Ang-II induced hypertension. Hypertension is the second leading cause of chronic kidney disease (CKD) in the world. At the subcellular level, oxidative stress and inflammation are two critical components in the pathogenesis of hypertension-induced organ damage1. Reactive oxygen species (ROS) are by-products of oxidative phosphorylation in the mitochondria and other oxidoreductase reactions and commonly include superoxide (O2−•), hydrogen peroxide (H2O2) and hydroxyl anions (OH−•)2. Excess ROS can result in impairment of redox signaling pathways leading to cellular damage and dysfunction. The deleterious effects of ROS are normally countered by an effective anti-oxidant system. An imbalance in the production of ROS and its breakdown known as oxidative stress can be a cause and consequence of hypertension. In the kidney, the interaction between superoxide and nitric oxide causes damage to the vascular endothelium promoting vasoconstriction3. Further, high levels of Angiotensin-II (Ang-II) increase ROS via upregulation of NADPH oxidase system which result in infiltration of inflammatory cells causing glomerular and tubular damage1. The inflammatory response can precede or follow the onset of hypertension and is directly related to oxidative stress. Cross-sectional studies have shown that inflammatory markers are elevated in the serum of pre-hypertensive subjects and patients with established hypertension4, 5. Recent studies have shown that macrophages play an important role in hypertension induced kidney injury6, 7. In spontaneously hypertensive rats, accumulation of macrophages and lymphocytes has been documented in the kidneys even before the onset of
1 Department of Physiology, University of Louisville School of Medicine, Louisville, KY-40202, USA. 2Institute of Advanced Study in Science and Technology, Guwahati, Assam, 781035, India. 3University of California, San Diego, CA, 92093, USA. Correspondence and requests for materials should be addressed to U.S. (email: [email protected])
ScIentIfIc RepOrtS | 7: 6349 | DOI:10.1038/s41598-017-06484-6
1
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Figure 1. TLR4 deficiency mice exhibit blunted response to Ang-II induced hypertension. Time course of systolic, diastolic and mean blood pressure changes following Ang-II treatment in C3H/HeOuJ (C3HeOuJ, normal TLR4) and C3H/HeJ (C3HeJ, TLR4 deficiency) mice. n = 7/group tested by ANOVA followed by Student’s t-test. *p
OPEN
Received: 17 January 2017 Accepted: 13 June 2017 Published online: 24 July 2017
Toll-like Receptor 4 Deficiency Reduces Oxidative Stress and Macrophage Mediated Inflammation in Hypertensive Kidney Sathnur Pushpakumar1, Lu Ren1, Sourav Kundu2, Alejandra Gamon3, Suresh C. Tyagi1 & Utpal Sen1 Oxidative stress and inflammation are integral to hypertension-induced renal injury. A unifying feature for the two components is Toll-like receptors (TLR), which are key regulators of the innate immune system. Recent studies implicate TLR4 activation and oxidative stress in cardiovascular diseases and also as a link between inflammation and hypertension. However, its role in hypertension induced renal injury remains unexplored. In the present study, we investigated whether TLR-4 deficiency reduces Ang-II-induced renal injury and fibrosis by attenuating reactive oxygen species (ROS) production and inflammation. C3H/HeOuJ mice with normal TLR-4 and C3H/HeJLps-d with dysfunctional TLR4 (TLR4 deficiency) were treated without or with Ang-II. In response to Ang-II, TLR4 deficient mice had reduced renal resistive index and increased renal cortical blood flow compared to mice with normal TLR4. Further, TLR4 deficiency reduced oxidative stress and increased antioxidant capacity (MnSOD, CuSOD and Catalase activity). TLR4 deficiency was also associated with reduced inflammation (MCP-1, MIP-2, TNF-α, IL-6 and CD68), decreased accumulation of bone marrow-derived fibroblasts and TGF-β expression. Our data suggests that in C3H/HeJLps-d mice, deficiency of functional TLR4 reduces oxidative stress and macrophage activation to decrease TGF-β-induced extracellular matrix protein deposition in the kidney in Ang-II induced hypertension. Hypertension is the second leading cause of chronic kidney disease (CKD) in the world. At the subcellular level, oxidative stress and inflammation are two critical components in the pathogenesis of hypertension-induced organ damage1. Reactive oxygen species (ROS) are by-products of oxidative phosphorylation in the mitochondria and other oxidoreductase reactions and commonly include superoxide (O2−•), hydrogen peroxide (H2O2) and hydroxyl anions (OH−•)2. Excess ROS can result in impairment of redox signaling pathways leading to cellular damage and dysfunction. The deleterious effects of ROS are normally countered by an effective anti-oxidant system. An imbalance in the production of ROS and its breakdown known as oxidative stress can be a cause and consequence of hypertension. In the kidney, the interaction between superoxide and nitric oxide causes damage to the vascular endothelium promoting vasoconstriction3. Further, high levels of Angiotensin-II (Ang-II) increase ROS via upregulation of NADPH oxidase system which result in infiltration of inflammatory cells causing glomerular and tubular damage1. The inflammatory response can precede or follow the onset of hypertension and is directly related to oxidative stress. Cross-sectional studies have shown that inflammatory markers are elevated in the serum of pre-hypertensive subjects and patients with established hypertension4, 5. Recent studies have shown that macrophages play an important role in hypertension induced kidney injury6, 7. In spontaneously hypertensive rats, accumulation of macrophages and lymphocytes has been documented in the kidneys even before the onset of
1 Department of Physiology, University of Louisville School of Medicine, Louisville, KY-40202, USA. 2Institute of Advanced Study in Science and Technology, Guwahati, Assam, 781035, India. 3University of California, San Diego, CA, 92093, USA. Correspondence and requests for materials should be addressed to U.S. (email: [email protected])
ScIentIfIc RepOrtS | 7: 6349 | DOI:10.1038/s41598-017-06484-6
1
www.nature.com/scientificreports/
Figure 1. TLR4 deficiency mice exhibit blunted response to Ang-II induced hypertension. Time course of systolic, diastolic and mean blood pressure changes following Ang-II treatment in C3H/HeOuJ (C3HeOuJ, normal TLR4) and C3H/HeJ (C3HeJ, TLR4 deficiency) mice. n = 7/group tested by ANOVA followed by Student’s t-test. *p
