Ultrasound prevents renal ischemia-reperfusion injury by stimulating the splenic cholinergic anti-inflammatory pathway

Abstract

AKI affects both quality of life and health care costs and is an independent risk factor for mortality. At present, there are few effective treatment options for AKI. Here, we describe a nonpharmacologic, noninvasive, ultrasound-based method to prevent renal ischemia-reperfusion injury in mice, which is a model for human AKI. We exposed anesthetized mice to an ultrasound protocol 24 hours before renal ischemia. After 24 hours of reperfusion, ultrasound-treated mice exhibited preserved kidney morphology and function compared with sham-treated mice. Ultrasound exposure before renal ischemia reduced the accumulation of CD11b(+)Ly6G(high) neutrophils and CD11b(+)F4/80(high) myeloid cells in kidney tissue. Furthermore, splenectomy and adoptive transfer studies revealed that the spleen and CD4(+) T cells mediated the protective effects of ultrasound. Last, blockade or genetic deficiency of the α7 nicotinic acetylcholine receptor abrogated the protective effect of ultrasound, suggesting the involvement of the cholinergic anti-inflammatory pathway. Taken together, these results suggest that an ultrasound-based treatment could have therapeutic potential for the prevention of AKI, possibly by stimulating a splenic anti-inflammatory pathway.

Figure 1.

Prior exposure to US alone prevents IRI in naive mice. (A) Mice were infused (intravenously) with the microbubble (MB) contrast agent (or saline) and exposed to US 24 hours before 26 minutes of kidney ischemia, followed by 24 hours of reperfusion (IRI). The preservation of kidney function (as assessed by plasma creatinine) depends on the US application alone. (B) Preservation was replicated in animals without intravenous infusion. (C) Renal morphology (as assessed in H&E-stained tissue section) confirms prevention of IRI in animals exposed to US 24 hours before IRI. n=5–16. (D) Mice were exposed to US 1, 2, 3, 5, or 7 days before IRI. IRI was reduced in animals exposed to US 2 days before injury. n=5. *P<0.001 compared with groups without US; **P<0.005 compared with animals exposed to US 7 days before IRI. Scale bars, 100 μm in low-power image, 50 μm in the inset. Data in bar graphs are shown as mean ± SEM.

Figure 2.

The preservation of tissue morphology by prior US exposure is observed 4 weeks after IRI. Mice were exposed to US 24 hours before mild (25 minutes) IRI and were maintained for 4 weeks. Mice receiving US before IRI had (A) less tubulointerstitial fibrosis (quantified using Masson trichrome staining) and (B) >85% reduction in renal mRNA expression of profibrotic genes. n=3. Data in B are the mean ± SEM for ∆CT (change in cycle threshold) values calculated on the basis of tissue glyceraldehyde 3-phosphate dehydrogenase expression. αSMA, α-smooth muscle actin; Col1, collagen 1; Col3, collagen 3; Vim, vimentin.

Figure 3.

The spleen is required for US-induced protection from IRI. (A) Insonation of both sides of the animal 24 hours before IRI or the left side alone prevented IRI. n=3–8. (B) Splenic weight correlates with kidney function (data are compiled from mice from experiments shown in Figure 1D). (C) US does not prevent IRI in mice splenectomized 7 days before US and IRI. n=5–7. *P=0.03, **P=0.007, ***P=0.003. Data presented as mean ± SEM. SPLX, splenectomized.

Figure 4.

Splenic CD4⁺ T cells mediate the protective effect of US. (A) US does not prevent IRI in mice deficient of functional T and B lymphocytes (Rag1^−/−). n=4–11. (B) Reconstitution of Rag1^−/− mice with 0.5–2×10⁶ wild-type CD4⁺ T cells (intravenously) 10 days before US restores the protective effect of US in mice challenged with IRI. n=5–7. (C) Splenectomy (7 days before T cell administration) removes the restoration of US protection in Rag1^−/− mice receiving CD4⁺ T cells. (D) Representative H&E image of mice from C. n=4–5. *P=0.003, **P<0.001, ***P=0.002. Scale bars, 100 μm in low-power image, 50 μm in the inset. Data presented as mean ± SEM. SPLX, splenectomized.

Figure 5.

The cholinergic anti-inflammatory pathway provides a potential mechanism for US-mediated protection from IRI. Activation of the adrenergic splenic nerve results in the release of norepinephrine, which binds to adrenergic receptors on nearby CD4⁺ T cells. This stimulates the production of acetylcholine, which binds to α7nAChRs on splenic myeloid cells (macrophages) and results in reduced inflammation and IRI. Cytisine, an α7nAChR agonist, mimics the effect of acetylcholine, whereas α-bungarotoxin, an α7nAChR antagonist, blocks the effect of endogenous acetylcholine. BT, α-bungarotoxin. Adapted from reference 65.

Figure 6.

US-induced protection depends on the α7nAChR. (A) Administration of the α7nAChR agonist cytisine (100 ng/g, intravenously) 1 hour before IRI prevents kidney injury in a spleen-dependent manner, n=4–8. (B) Prior administration of the α7nAChR antagonist α-bungarotoxin (30 ng/g, intravenously) or (C) use of α7 knockout mice (Chrna7^−/−) prevents the US-induced protection from IRI. n=4–7. *P=0.007, **P=0.001, ***P<0.001. Data presented as mean ± SEM. SPLX, splenectomized.