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. 2017 Nov;221(3):204-220.
doi: 10.1111/apha.12899. Epub 2017 Jun 27.

Acute toll-like receptor 4 activation impairs rat renal microvascular autoregulatory behaviour

J P Van Beusecum  1   2 , S Zhang  1   2 , A K Cook  1   2 , E W Inscho  1   2
Affiliations

Acute toll-like receptor 4 activation impairs rat renal microvascular autoregulatory behaviour

J P Van Beusecum et al. Acta Physiol (Oxf). 2017 Nov.

Abstract

Aim: Little is known about how toll-like receptor 4 (TLR4) influences the renal microvasculature. We hypothesized that acute TLR4 stimulation with lipopolysaccharide (LPS) impairs afferent arteriole autoregulatory behaviour, partially through reactive oxygen species (ROS).

Methods: We assessed afferent arteriole autoregulatory behaviour after LPS treatment (1 mg kg-1 ; i.p.) using the in vitro blood-perfused juxtamedullary nephron preparation. Autoregulatory behaviour was assessed by measuring diameter responses to stepwise changes in renal perfusion pressure. TLR4 expression was assessed by immunofluorescence, immunohistochemistry and Western blot analysis in the renal cortex and vasculature.

Results: Baseline arteriole diameter at 100 mmHg averaged 15.2 ± 1.2 μm and 12.2 ± 1.0 μm for control and LPS groups (P < 0.05) respectively. When perfusion pressure was increased in 15 mmHg increments from 65 to 170 mmHg, arteriole diameter in control kidneys decreased significantly to 69 ± 6% of baseline diameter. In the LPS-treated group, arteriole diameter remained essentially unchanged (103 ± 9% of baseline), indicating impaired autoregulatory behaviour. Pre-treatment with anti-TLR4 antibody or the TLR4 antagonist, LPS-RS, preserved autoregulatory behaviour during LPS treatment. P2 receptor reactivity was normal in control and LPS-treated rats. Pre-treatment with Losartan (angiotensin type 1 receptor blocker; (AT1 ) 2 mg kg-1 ; i.p.) increased baseline afferent arteriole diameter but did not preserve autoregulatory behaviour in LPS-treated rats. Acute exposure to Tempol (10-3 mol L-1 ), a superoxide dismutase mimetic, restored pressure-mediated vasoconstriction in kidneys from LPS-treated rats.

Conclusion: These data demonstrate that TLR4 activation impairs afferent arteriole autoregulatory behaviour, partially through ROS, but independently of P2 and AT1 receptor activation.

Keywords: endotoxemia; inflammation; oxidative stress; renal microcirculation; toll-like receptor 4.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest in relation to the publication of the present study.

Figures

Figure 1
Figure 1. TLR4 is expressed in renal vascular smooth muscle cells and LPS treatment increases renal TLR4 expression
TLR4 is expressed in renal vascular smooth muscle cells, and LPS treatment increases renal TLR4 expression. (a): DAB (TLR4) staining of kidneys from control (left panels) and LPS (1 mg kg−1; i.p.; right panels) treated groups. (b): Data are expressed as a percentage of total kidney area stained for TLR4 from control (white bars) and LPS treatment (grey bars). Each bar represents the mean ± SE. n=4 per group. (c): Immunofluorescence staining of TLR4 in OCT frozen whole kidney sections. Representative images show nuclear staining (blue; Hoechst), vascular smooth muscle α-actin (green), TLR4 (red), and co-localization of TLR4 and vascular smooth α-actin (yellow). Images were taken at 40x magnification. Bar = 20 μm. * P<0.05 vs. control TLR4 expression.
Figure 2
Figure 2. TLR4 blockade with LPS-RS preserves autoregulatory behaviour
Co-treatment with the TLR4 antagonist, LPS-RS, preserves afferent arteriole autoregulatory behaviour in LPS treated kidneys. (a): Effect of renal perfusion pressure changes on afferent arteriole diameter in kidneys from control (black diamonds), LPS (1 mg kg−1; i.p.; grey squares), and LPS + LPS-RS (5 mg; i.p.; blue triangles) treated groups. (b): Data are expressed as a percent of the baseline diameter at 100 mmHg. Each data point represents the mean ± SE. n=6 per group. * P<0.05 vs. baseline diameter in the same group. Ϯ P<0.05 vs. control group for the same perfusion pressure. # P<0.05 vs. LPS-RS for the same perfusion pressure.
Figure 3
Figure 3. Pre-treatment with anti-TLR4 antibody preserved afferent arteriole autoregulatory behaviour during acute LPS treatment
Pre-treatment with anti-TLR4 antibody preserves afferent arteriole autoregulatory behaviour in kidneys from LPS treated rats. (a): Effect of renal perfusion pressure changes on afferent arteriole in kidneys from control (black diamonds), LPS (1 mg kg−1; grey squares), LPS + anti-TLR4 antibody (1 μg; blue triangles), and LPS + anti-IgG2a antibody (red circles). (b): Data are expressed as a percent of the baseline diameter at 100 mmHg. Each data point represents the mean ± SE. n=6 per group. * P<0.05 vs. baseline diameter in the same group. Ϯ P<0.05 vs. control group for the same perfusion pressure. # P<0.05 vs. anti-TLR4 antibody for the same perfusion pressure.
Figure 4
Figure 4. Afferent arteriole reactivity to ATP, β,γ-mATP, and UTP are normal in acute LPS treated rats
Afferent arteriole responses to ATP, β,γ-mATP, and UTP in acute LPS treated rats. (a): Afferent arteriole response to superfusion of ATP was assessed in control (black diamonds) and LPS (1 mg kg−1; i.p.; grey squares). (b): Afferent arteriole response to superfusion of β,γ-mATP was assessed in control (black diamonds) and LPS (grey squares). (c): Afferent arteriole response to superfusion of UTP was assessed in control (black diamonds) and LPS (grey squares). Data are expressed as a percent of the baseline diameter at 100 mmHg. Each data point represents the mean ± SE. n=6 per group. * P<0.05 vs. baseline diameter in the same group.
Figure 5
Figure 5. Impairment of afferent arteriole autoregulatory behaviour is independent of AT1 receptor activation during acute LPS treatment
(a): Effect of renal perfusion pressure changes on afferent arteriole diameter in control (black diamonds), LPS (1 mg kg−1; i.p.; grey squares), LPS + Losartan (2 mg kg−1; i.p.; blue triangles), and control + Losartan (red circles). (b): Data are expressed as a percent of the baseline diameter at 100 mmHg during the equilibration period. Control and LPS data are included from Figure 2 for reference. Each data point represents the mean ± SE. n=6 per group. * P<0.05 vs. baseline diameter in the same group. Ϯ P<0.05 vs. control for the same perfusion pressure. # P<0.05 vs. control + Losartan for the same perfusion pressure. & P<0.05 vs. LPS + Losartan for the same perfusion pressure.
Figure 6
Figure 6. Acute superfusion of Tempol restores afferent arteriole autoregulatory behaviour in LPS treated rats
Acute superfusion of Tempol (10−3 mol L−1) restores afferent arteriole autoregulatory behaviour in LPS treated kidneys. Control and LPS groups are the same as shown in Figure 2. (a): Effect of renal perfusion pressure changes on afferent arteriole diameter in control (black diamonds), LPS (1 mg kg−1; i.p.; grey squares), and LPS + Tempol (blue triangles) kidneys. (b): Data are expressed as a percent of the baseline diameter at 100 mmHg. Each data point represents the mean ± SE. n=6 per group. * P<0.05 vs. baseline diameter in the same group. Ϯ P<0.05 vs. control group for the same perfusion pressure. # P<0.05 vs. Tempol for the same perfusion pressure.
Figure 7
Figure 7. Blockade of TLR4 during LPS treatment prevents upregulation of renal vascular TLR4 protein expression
Treatment with anti-TLR4 antibody (1 μg; i.p.) or LPS-RS (5 mg i.p.) decreases TLR4 protein expression in small intra-renal arteries in LPS (1 mg kg−1; i.p.) treated rats. (a): Representative image of typical gel. (b): Densitometry data normalized against β-actin. Each bar represents the mean ± SE. n=5 per group. * P<0.05 vs. control TLR4 expression. # P<0.05 vs. LPS TLR4 expression.
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