Vagus nerve stimulation mediates protection from kidney ischemia-reperfusion injury through α7nAChR+ splenocytes

Abstract

The nervous and immune systems interact in complex ways to maintain homeostasis and respond to stress or injury, and rapid nerve conduction can provide instantaneous input for modulating inflammation. The inflammatory reflex referred to as the cholinergic antiinflammatory pathway regulates innate and adaptive immunity, and modulation of this reflex by vagus nerve stimulation (VNS) is effective in various inflammatory disease models, such as rheumatoid arthritis and inflammatory bowel disease. Effectiveness of VNS in these models necessitates the integration of neural signals and α7 nicotinic acetylcholine receptors (α7nAChRs) on splenic macrophages. Here, we sought to determine whether electrical stimulation of the vagus nerve attenuates kidney ischemia-reperfusion injury (IRI), which promotes the release of proinflammatory molecules. Stimulation of vagal afferents or efferents in mice 24 hours before IRI markedly attenuated acute kidney injury (AKI) and decreased plasma TNF. Furthermore, this protection was abolished in animals in which splenectomy was performed 7 days before VNS and IRI. In mice lacking α7nAChR, prior VNS did not prevent IRI. Conversely, adoptive transfer of VNS-conditioned α7nAChR splenocytes conferred protection to recipient mice subjected to IRI. Together, these results demonstrate that VNS-mediated attenuation of AKI and systemic inflammation depends on α7nAChR-positive splenocytes.

Figure 1. Optimization of VNS.

BP and HR were recorded while mice underwent left or right VNS at constant frequency (5 Hz), but with varied current (10, 30, and 50 μA). Average change in mean arterial BP (A) and HR (B) during vagal stimulation compared with conditions without vagal stimulation. 50 μA stimulation decreased HR significantly. (C and D) Recording of the right vagus efferent nerve during the left VNS. (C) Representative example of right vagus efferent nerve activity (VNA) during left VNS (5 Hz, 1 ms, 50 μA, 10 minutes). Representative data of 3 independent experiments. rVNA, rectified vagus efferent nerve activity. (D) Stimulus-triggered rectified vagus efferent nerve activity was averaged (3,000 sweeps). Arrow indicates stimulation. The latency of the evoked potential is about 20 ms. (E) Mice underwent VNS or sham stimulation (no VNS) surgery 24 hours prior to LPS (10 μg/ml infused at the rate of 10 μl/h for 3 hours) or saline administration, and blood was collected at the end of the infusion period. VNS treatment 24 hours before LPS administration suppressed the LPS-induced increase in circulating TNF. n = 6 each in A and B and n = 5 in E. Data in A and B were analyzed using 2-way ANOVA, and data in E were analyzed using 1-way ANOVA. Means were compared by post hoc multiple-comparison test (Tukey’s). ***P < 0.001.

Figure 2. Kidneys are not protected from IRI when VNS is applied 10 minutes before IRI.

Mice underwent left VNS (5 Hz, 1 ms, 50 μA for 10 minutes) or sham stimulation surgery 10 minutes prior to IRI (26 minutes ischemia, 24 hours reperfusion) or sham IRI surgery. VNS applied 10 minutes before IRI did not protect kidneys from IRI, as shown by plasma creatinine (A), tissue morphology (B; representative H&E staining of kidney sections), and ATN (C; scored from H&E samples). n = 4 each. Data were analyzed using 1-way ANOVA. Means were compared by post hoc multiple-comparison test (Tukey’s). ***P < 0.001. Scale bars: 100 μm; 50 μm (inset).

Figure 3. VNS 24 hours before IRI protects kidneys from injury.

Mice underwent either VNS(i), VNS(a), VNS(e), or sham stimulation surgery (all on the left vagus nerve) 24 hours before IRI or sham IRI surgery. Prior VNS protected kidneys and reduced the IRI-induced increase in plasma creatinine (A), Kim1 expression (RNA from whole kidney) (B), and ATN (C, representative H&E staining of kidney sections; D, scored from H&E samples). n = 5–11. Data were analyzed using 1-way ANOVA. Means were compared by post hoc multiple-comparison test (Tukey’s) *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 100 μm; 50 μm (inset).

Figure 4. VNS 24 hours before IRI suppresses IRI-induced increases in circulating TNF-α.

Mice underwent VNS or sham stimulation surgery 24 hours prior to IRI or sham IRI surgery. VNS applied 24 hours before IRI suppressed IRI-induced increases in circulating TNF. n = 6. Data were analyzed using 1-way ANOVA. Means were compared by post hoc multiple-comparison test (Tukey’s). *P < 0.05.

Figure 5. More than half of the cytokines evaluated are upregulated in kidney by IRI and suppressed by prior VNS.

Mice underwent VNS or sham stimulation surgery 24 hours prior to IRI or sham IRI surgery. RNA was isolated from whole kidneys, and qPCR was performed. Relative gene expressions compared with sham-sham group were calculated (raw data in Supplemental Figure 1), and clustering was performed to generate a heat map. n = 5–11.

Figure 6. Protection against IRI by VNS requires the spleen.

Splenectomy (SPLX) or sham surgery was performed 7 days before VNS or sham VNS treatment. Twenty-four hours after VNS treatment, mice were subjected to IRI or sham IRI operation. The protective effect by VNS was eliminated or reduced by prior splenectomy, as demonstrated by plasma creatinine (A) and ATN (B, representative H&E staining of kidney sections; C, scored from H&E samples). n = 6 each. Data were analyzed using 1-way ANOVA. Means were compared by post hoc multiple-comparison test (Tukey’s). *P < 0.05; **P < 0.01. Scale bars: 100 μm; 50 μm (inset).

Figure 7. Adoptive transfer of splenocytes from VNS-treated mice confers protection from kidney injury after IRI in naive recipient mice.

Donor mice underwent VNS or sham VNS treatment, and 24 hours later, splenocytes isolated from donor mice were injected i.v. into recipient mice. The recipient mice were subjected to IRI 24 hours after splenocyte transfer, and plasma creatinine was evaluated 24 hours after IRI. (A) In pilot studies, the protective effect of donor splenocytes was proportional to the number of cells transferred, and the greatest difference between splenocytes from sham- and VNS-treated donors was observed with 1 million cells. (B) Using transfer of 1 million cells, significant protection was seen in mice that received splenocytes from VNS-treated donor mice compared with administration of PBS (i.v.) or splenocytes from sham-treated donor mice. n = 3 each in A and n = 6 each in B. Data in B were analyzed using 1-way ANOVA. Means were compared by post hoc multiple-comparison test (Tukey’s). ***P < 0.001.

Figure 8. Protection against IRI by VNS is absent in α7 KO mice.

WT (progeny control), Chrna7^+/– (α7HE), and α7KO mice underwent VNS 24 hours prior to IRI. The protective effect by VNS was lost in Chrna7^+/– and α7KO mice, as demonstrated by plasma creatinine (A) and ATN (B, representative H&E staining of kidney sections; C, scored from H&E samples). n = 5. Data were analyzed with 1-way ANOVA. Means were compared by post hoc multiple-comparison test (Tukey’s). *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 100 μm; 50 μm (inset).

Figure 9. Protective effect of adoptively transferred splenocytes from VNS-treated mice requires α7nAChRs.

α7KO mice and WT (progeny controls) were used as donor mice. Donor mice underwent VNS or sham VNS treatment 1 day before splenocyte transfer. Twenty-four hours later, 1 × 10⁶ splenocytes from donor mice were injected i.v. into the recipient mice (WT). The recipient mice were subjected to IRI 24 hours after the transfer, and plasma creatinine was evaluated 24 hours later. The recipient mice that received splenocytes from VNS-treated WT mice were protected against IRI, but this protection was abolished when the mice received splenocytes from VNS-treated α7KO mice. n = 5 each (n = 4 for PBS-treated group). Data were analyzed using 1-way ANOVA. Means were compared by post hoc multiple-comparison test (Tukey’s). ***P < 0.001.

Figure 10. Prior VNS does not change the number of macrophages infiltrating the kidney, but changes their phenotype.

α7KO mice and WT (progeny controls) mice underwent IRI 24 hours after VNS or sham VNS treatment. Mice were euthanized after 0, 4, or 24 hours of reperfusion, and the number of macrophages/monocytes and granulocytes in the kidney was evaluated by flow cytometry (gating strategy in Supplemental Figure 2). (A and B) The number of macrophages/monocytes infiltrating the kidney increased with time after IRI in WT (A) and α7KO mice (B), but prior VNS did not change the number. (C and D) The number of granulocytes infiltrating the kidney increased with time after IRI in WT (C) and α7KO mice (D), and this increase was suppressed 24 hours after IRI in VNS-treated WT mice (C), but not in α7KO mice (D). (E and F) qPCR was performed using FACS-sorted macrophages/monocytes from the kidney of WT (E) and α7KO mice (F) (raw data in Supplemental Figure 3). Relative gene expressions compared with control group were calculated, and clustering was performed. Data were analyzed using 2-way ANOVA. Means were compared by post hoc multiple-comparison test (Tukey’s). *P < 0.05. n = 3 in A–D. n = 3 for control (untreated) and n = 6 for sham-IRI and VNS-IRI (E and F).

Figure 11. CAP in protection from kidney IRI.

VNS applied 24 to 48 hours before an ischemic episode protects the kidney from IRI. Stimulation of vagal afferents or efferents protects the kidneys through α7nAChR-positive (α7-positive)splenocytes. Vagal afferent stimulation also protects kidney, but through a different and unidentified pathway.