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, 87 (2), 350-8

Renal Denervation Prevents Long-Term Sequelae of Ischemic Renal Injury

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Renal Denervation Prevents Long-Term Sequelae of Ischemic Renal Injury

Jinu Kim et al. Kidney Int.

Abstract

Signals that drive interstitial fibrogenesis after renal ischemia reperfusion injury remain undefined. Sympathetic activation manifests even in the early clinical stages of chronic kidney disease and is directly related to disease severity. A role for renal nerves in renal interstitial fibrogenesis in the setting of ischemia reperfusion injury has not been studied. In male 129S1/SvImJ mice, ischemia reperfusion injury induced tubulointerstitial fibrosis as indicated by collagen deposition and profibrotic protein expression 4 to 16 days after the injury. Leukocyte influx, proinflammatory protein expression, oxidative stress, apoptosis, and cell cycle arrest at G2/M phase were enhanced after ischemia reperfusion injury. Renal denervation at the time of injury or up to 1 day post injury improved histology, decreased proinflammatory/profibrotic responses and apoptosis, and prevented G2/M cell cycle arrest in the kidney. Treatment with afferent nerve-derived calcitonin gene-related peptide (CGRP) or efferent nerve-derived norepinephrine in denervated and ischemia reperfusion injury-induced kidneys mimicked innervation, restored inflammation and fibrosis, induced G2/M arrest, and enhanced TGF-β1 activation. Blocking norepinephrine or CGRP function using respective receptor blockers prevented these effects. Consistent with the in vivo study, treatment with either norepinephrine or CGRP induced G2/M cell cycle arrest in HK-2 proximal tubule cells, whereas antagonists against their respective receptors prevented G2/M arrest. Thus, renal nerve stimulation is a primary mechanism and renal nerve-derived factors drive epithelial cell cycle arrest and the inflammatory cascade causing interstitial fibrogenesis after ischemia reperfusion injury.

Conflict of interest statement

DISCLOSURE

All the authors declared no completing interests.

Figures

Figure 1
Figure 1. Renal denervation inhibits collagen deposition, and neutrophil and macrophage influx during a period of interstitial fibrosis after IRI
Two days after denervation in left kidneys of mice, IRI or sham operation (S) in the left kidneys was carried out. (A) Collagen deposition using Sirius red stain on denervated or intact kidney sections at 16 days after IRI. Scale bars indicate 50 μm. (B) Percentage of Sirius red-positive area on kidney sections. (C and D) Neutrophil infiltration represented by the number of PMN-positive cells on immunohistochemically stained kidney sections. (C and E) Macrophage infiltration represented by the percentage of F4/80-positive area on immunohistochemically stained kidney sections. Error bars represent SD (n = 5). #P<0.05 versus intact. Scale bars indicate 50 μm.
Figure 2
Figure 2. Exogenous norepinephrine or calcitonin gene-related peptide enhances interstitial fibrosis and inflammation induced by IRI in denervated kidneys
(A–C) Two days after denervation in left kidneys of mice, IRI or sham operation (S) in the left kidneys was carried out. (A) Kidney expression of tyrosine hydroxylase using Western blot analysis at 16 days after IRI or sham. Anti-β-actin antibody served as a loading control. (B) Levels of norepinephrine and CGRP in the kidneys using ELISA kits. (C) Immunohistochemical staining for CGRP on the kidney sections at 2 days after IRI. Scale bars indicate 50 μm. (D–F) Denervation in left kidneys of mice was carried out; 2 days after the onset, norepinephrine, CGRP or vehicle was continuously infused into the denervated kidneys via mini-osmotic pump; and the kidneys were subjected to 30 min of ischemia followed by 16 days of reperfusion. (D) Percentage of Sirius red-positive area in kidneys sections. (E) Neutrophil infiltration represented by the number of PMN-positive cells on immunohistochemically stained kidney sections. (F) Macrophage infiltration represented by percentage of F4/80-positive area on immunohistochemically stained kidney sections. Error bars represent SD (n = 4). *P<0.05 versus sham, #P<0.05 versus intact, and $P<0.05 versus vehicle.
Figure 3
Figure 3. α2-AR antagonist and CGRP receptor antagonist diminish interstitial fibrosis and inflammation induced by IRI
(A–C) Mice were continuously treated with doxazosin (α1-AR antagonist, 12 mg/kg/d), atipamezole (α2-AR antagonist, 2.4 mg/kg/d), pronethalol (β-AR antagonist, 2.4 mg/kg/d), or 10% DMSO in PBS (vehicle) via an intraperitoneal implantation of mini-osmotic pump immediately before 30 min of left kidney ischemia and 16 days of reperfusion. (A) Percentage of Sirius red-positive area on the kidney sections. (B) Neutrophil infiltration represented by counting PMN-positive cells on immunohistochemically stained kidney sections. (C) Macrophage infiltration represented by percentage of F4/80-positive area on immunohistochemically stained kidney sections. (D–F) Mice were continuously treated with the CGRP receptor antagonist (CGRP(8-37), 120 μg/kg/d) or 0.9% saline (vehicle) via an intraperitoneal implantation of mini-osmotic pump immediately before 30 min of left kidney ischemia and 16 days of reperfusion. (D) Percentage of Sirius red-positive area on the kidney sections. (E) Neutrophil infiltration represented by the number of PMN-positive cells on immunohistochemically stained kidney sections. (F) Macrophage infiltration represented by percentage of F4/80-positive area on immunohistochemically stained kidney sections. Error bars represent SD (n = 5). $P<0.05 versus vehicle.
Figure 4
Figure 4. Norepinephrine and CGRP signaling contribute to kidney injury during a period of interstitial fibrosis after IRI
(A and D) Two days after denervation in left kidneys of mice, IRI or sham in the left kidneys was carried out (n = 5). (C, E and F) Denervation or intact in left kidneys of mice was carried out; 2 days after the denervation, norepinephrine, CGRP or vehicle was continuously infused into the denervated kidneys via mini-osmotic pump; and the kidneys were subjected to 30 min of ischemia followed by 16 days of reperfusion (n = 4). (A) Tubular damage represented by PAS stain on the kidney sections at 16 days after IRI. Scale bars indicate 50 μm. (B and C) Tubular injury score measured on PAS-stained kidney sections. (D and E) Glomerular filtration rate (GFR, creatinine clearance) was measured in mice housed in metabolic cages. (F) Kidney weight obtained from mice at 16 days after IRI or sham. Error bars represent SD. #P<0.05 versus intact and $P<0.05 versus vehicle.
Figure 5
Figure 5. Renal denervation reduces oxidative stress in a norepinephrine and CGRP-dependent manner in kidneys after IRI
(A) Two days after denervation in left kidneys of mice, IRI or sham in the left kidneys was carried out; then the kidneys were harvested at 16 d after reperfusion (n = 5). (B) Denervation in left kidneys of mice was carried out; 2 d after the onset, norepinephrine, CGRP or vehicle was continuously infused into the denervated kidneys via mini-osmotic pump; and the kidneys were subjected to 30 min of ischemia followed by 16 d of reperfusion (n = 4). (A and B) Lipid peroxidation represented by level of lipid hydroperoxide in kidneys. Error bars represent SD. #P<0.05 versus intact and $P<0.05 versus vehicle.
Figure 6
Figure 6. Renal denervation inhibits tubular cell cycle arrest in a norepinephrine and CGRP-dependent manner in kidneys after IRI
(A and B) Two days after denervation in left kidneys of mice, IRI or sham in the left kidneys was carried out (n = 5). (C) Two days after denervation in left kidneys of mice, norepinephrine, CGRP or vehicle was continuously infused into the denervated kidneys via mini-osmotic pump and the kidneys were subjected to 30 min of ischemia followed by 16 d of reperfusion (n = 4). (A) Immunohistochemial staining for phosphorylated histone H3 (p-H3) on kidney sections. Scale bars indicate 50 μm. (B and C) The number of p-H3-positive tubular cells on immunohistochemically stained kidney sections. Error bars represent SD. #P<0.05 versus intact and $P<0.05 versus vehicle.

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