Reno-Cerebral Reflex Activates the Renin-Angiotensin System, Promoting Oxidative Stress and Renal Damage After Ischemia-Reperfusion Injury

Abstract

Aims: A kidney-brain interaction has been described in acute kidney injury, but the mechanisms are uncertain. Since we recently described a reno-cerebral reflex, we tested the hypothesis that renal ischemia-reperfusion injury (IRI) activates a sympathetic reflex that interlinks the renal and cerebral renin-angiotensin axis to promote oxidative stress and progression of the injury.

Results: Bilateral ischemia-reperfusion activated the intrarenal and cerebral, but not the circulating, renin-angiotensin system (RAS), increased sympathetic activity in the kidney and the cerebral sympathetic regulatory regions, and induced brain inflammation and kidney injury. Selective renal afferent denervation with capsaicin or renal denervation significantly attenuated IRI-induced activation of central RAS and brain inflammation. Central blockade of RAS or oxidative stress by intracerebroventricular (ICV) losartan or tempol reduced the renal ischemic injury score by 65% or 58%, respectively, and selective renal afferent denervation or reduction of sympathetic tone by ICV clonidine decreased the score by 42% or 52%, respectively (all p < 0.05). Ischemia-reperfusion-induced renal damage and dysfunction persisted after controlling blood pressure with hydralazine.

Innovation: This study uncovered a novel reflex pathway between ischemic kidney and the brain that sustains renal oxidative stress and local RAS activation to promote ongoing renal damage.

Conclusions: These data suggest that the renal and cerebral renin-angiotensin axes are interlinked by a reno-cerebral sympathetic reflex that is activated by ischemia-reperfusion, which contributes to ischemia-reperfusion-induced brain inflammation and worsening of the acute renal injury. Antioxid. Redox Signal. 27, 415-432.

Keywords: acute kidney injury; brain; kidney; renin-angiotensin system; sympathetic nervous system.

FIG. 1.

Overactivation of intrarenal RAS, sympathetic nervous system, and oxidative stress in mice after IRI. (A) Representative photos of intrarenal RAS expression showed by immunohistochemical staining of AGT and Ang II (A1), semi-quantitative data of AGT (A2) and Ang II (A3). (B) The concentration of Ang II in renal homogenates. (C) Expression of intrarenal AGT measured by Western blot. (D) The level of urinary AGT excretion. (E) Concentration of renal norepinephrine. (F) Expression of intrarenal Nox2 (F1) and Nox4 (F2). Data are expressed as mean ± SD (Each experiment has been replicated three times. For each time, each study group consists of six mice.). *p < 0.05 versus sham mice at the same time point. The unedited blots have been provided in Supplementary Figure S7. AGT, angiotensinogen; Ang II, angiotensin II; IRI, ischemia-reperfusion injury; RAS, renin-angiotensin system.

FIG. 2.

Localization of activated RAS in renal tubules in mice after IRI. (A) Representative photos of intrarenal AGT expression determined by double staining with antibodies against AGT and the markers of renal tubular segments. (B) Location of Ang II determined by double staining with antibodies against Ang II and the markers of renal tubular segments. AQP-1, aquaporin 1 (proximal tubule, and descending thin limb of Henle's loop); AQP-2, aquaporin 2 (collecting duct); NCCT, thiazide-sensitive NaCl cotransporter (distal convoluted tubule); THP, Tamm-Horsfall protein (ascending thick limbs of Henle's loop).

FIG. 3.

Activation of brain RAS in mice after IRI. (A) Schematic drawings of study area (in gray) and light-field photomicrograph of coronal sections of SFO (A1) and hippocampus (Hippo) CA3 (A2). (B) Representative photos of immunohistochemical staining of AGT and angiotensin (Ang) II in SFO and Hippo CA3 (B1), semi-quantitative data of AGT (B2), mRNA level of AGT (B3), semi-quantitative data of Ang II (B4), and the concentration of Ang II in homogenates of SFO and Hippo (B5). (C) Localization of central AGT or Ang II determined by double staining with antibodies against AGT or Ang II (green) and an antibody recognizing NSE (red). Data are expressed as mean ± SD (Each experiment has been replicated three times. For each time, each study group consists of six mice.). *p < 0.05 versus sham mice at the same time point. Hippo, hippocampus; NSE, neuron-specific enolase; SFO, subfornical organ.

FIG. 4.

Overexpression of brain TH, Noxs, and inflammation in mice after IRI. (A) Enhanced expression of TH (brown) in activated neurons (c-fos positive, red) in the RVLM: representative photographs (A1) and quantitative data (A2). (B) Expression of Nox2 (B1) and Nox4 (B2) in SFO and Hippo CA3 assessed by Western blot. (C) Expression of GFAP: representative photos of GFAP expression in SFO or Hippo CA3 and their surrounding area, corpus callosum, and cerebral cortex (C1) and semi-quantitative data (C2 and C3). Data are expressed as mean ± SD (Each experiment has been replicated three times. For each time, each study group consists of six mice.). *p < 0.05 versus sham mice at the same time point. The unedited blots have been provided in Supplementary Figure S7. GFAP, glial fibrillary acidic protein; RVLM, rostral ventrolateral medulla; TH, tyrosine hydroxylase.

FIG. 5.

Blockade of central RAS or oxidative stress or renal deafferentation inhibits central TH and Noxs expression in mice after IRI. (A) Central administration of Tem or renal deafferentation with capsaicin downregulates expression of central RAS in SFO and Hippo: representative photos of immunohistochemistry staining (A1), semi-quantitative data of AGT (A2) and angiotensin (Ang) II (A3). (B) The concentration of Ang II in homogenates of SFO and Hippo. (C) Central administration of Los or Tem or renal deafferentation with capsaicin downregulates central Nox2 (C1) and Nox4 (C2). (D) Central administration of Los or Tem or renal deafferentation with capsaicin decreases the expression of TH in c-fos-positive neurons in RVLM. Data are expressed as mean ± SD (Each experiment has been replicated three times. For each time, each study group consists of six mice.). *p < 0.05 versus mice with renal IRI. The unedited blots have been provided in Supplementary Figure S7. aCSF, artificial cerebrospinal fluid; Clo, clonidine; Hyd, hydralazine; ICV, intracerebroventricular; IG, intragastric; Los, losartan; Tem, tempol.

FIG. 6.

Blockade of central RAS, sympathetic outflow, oxidative stress, or renal deafferentation inhibits central inflammation in mice after IRI. (A) Central administration of Los or Tem or renal deafferentation with capsaicin downregulates expression of GFAP: representative photos of GFAP expression in SFO or Hippo CA3 and their surrounding area, corpus callosum, and cerebral cortex (A1). Semi-quantitative data of GFAP staining (A2 and A3). Data are expressed as mean ± SD (Each experiment has been replicated three times. For each time, each study group consists of six mice.). *p < 0.05 versus mice with renal IRI.

FIG. 7.

Blockade of central RAS, sympathetic outflow, oxidative stress, or renal deafferentation inhibits activation of intrarenal RAS in mice after IRI. (A) Central administration of Los or Tem or renal deafferentation with capsaicin downregulates overexpression of renal RAS: representative photos (A1) and semi-quantitative analysis (A2 and A3). (B) Expression of intrarenal AGT analyzed by Western blot. (C) The concentration of Ang II in renal homogenates. (D) The level of urinary AGT excretion. Data are expressed as mean ± SD (Each experiment has been replicated three times. For each time, each study group consists of six mice.). *p < 0.05 versus mice with ischemic AKI given vehicle. The unedited blots have been provided in Supplementary Figure S7. AKI, acute kidney injury; PBS, phosphate-buffered saline.

FIG. 8.

Blockade of central RAS, sympathetic outflow, oxidative stress, or renal deafferentation attenuates renal injury in mice after IRI. (A) Central administration of Los or Tem or renal deafferentation with capsaicin inhibits acute tubular injury in mice after ischemic-reperfusion: representative photos of ischemic renal injury showed by HE staining (A1) and semi-quantitative data (A2). (B) Changes in mRNA level of KC. (C) Changes in serum creatinine. (D) Concentration of renal norepinephrine. (E) Expression of intrarenal Nox2 (E1) and Nox4 (E2). Data are expressed as mean ± SD (Each experiment has been replicated three times. For each time, each study group consists of six mice.). *p < 0.01 versus mice with ischemic AKI given vehicle. The unedited blots have been provided in Supplementary Figure S7. HE, hematoxylin-eosin; KC, keratinocyte-derived cytokine.

FIG. 9.

Schematic diagram summarizing co-activation of oxidative stress and RAS in the ischemic kidney and brain linked via the renal afferent and efferent sympathetic nerves in a positive feedback mode.