Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 26 (10), 2470-81

Ultrasound Modulates the Splenic Neuroimmune Axis in Attenuating AKI

Affiliations

Ultrasound Modulates the Splenic Neuroimmune Axis in Attenuating AKI

Joseph C Gigliotti et al. J Am Soc Nephrol.

Abstract

We showed previously that prior exposure to a modified ultrasound regimen prevents kidney ischemia-reperfusion injury (IRI) likely via the splenic cholinergic anti-inflammatory pathway (CAP) and α7 nicotinic acetylcholine receptors (α7nAChR). However, it is unclear how ultrasound stimulates the splenic CAP. Further investigating the role of the spleen in ischemic injury, we found that prior splenectomy (-7d) or chemical sympathectomy of the spleen with 6-hydroxydopamine (6OHDA; -14d) exacerbated injury after subthreshold (24-minute ischemia) IRI. 6-OHDA-induced splenic denervation also prevented ultrasound-induced protection of kidneys from moderate (26-minute ischemia) IRI. Ultrasound-induced protection required hematopoietic but not parenchymal α7nAChRs, as shown by experiments in bone marrow chimeras generated with wild-type and α7nAChR(-/-) mice. Ultrasound protection was associated with reduced expression of circulating and kidney-derived cytokines. However, splenocytes isolated from mice 24 hours after ultrasound treatment released more IL-6 ex vivo in response to LPS than splenocytes from sham mice. Adoptive transfer of splenocytes from ultrasound-treated (but not sham) mice to naïve mice was sufficient to protect kidneys of recipient mice from IRI. Ultrasound treatment 24 hours before cecal ligation puncture-induced sepsis was effective in reducing plasma creatinine in this model of AKI. Thus, splenocytes of ultrasound-treated mice are capable of modulating IRI in vivo, supporting our ongoing hypothesis that a modified ultrasound regimen has therapeutic potential for AKI and other inflammatory conditions.

Keywords: acute renal failure; cholinergic anti-inflammatory pathway; inflammation; neuroimmune; spleen.

Figures

Figure 1.
Figure 1.
SPLX exacerbates kidney IRI. Mice underwent SPLX or sham surgery 7 days before a subIRI (24 minutes of ischemia, 24 hours of reperfusion), which was not sufficient alone to cause AKI. Prior SPLX exacerbated subthreshold injury and resulted in elevated plasma creatinine (A) and robust ATN (B) (scored from hematoxylin and eosin samples) (C). ATN, acute tubular necrosis; SPLX, splenectomy; subIRI, subthreshold ischemia-reperfusion injury. n=6–13. *P≤0.001. Scale bar is 100 μm in the main panel and 50 μm in the inset.
Figure 2.
Figure 2.
Prior SPLX increases renal inflammation after subIRI. (A) Leukocyte infiltration (CD45+ cells as a percent of total kidney cells) measured in kidneys by flow cytometry 24 hours after subIRI was higher in mice with prior SPLX. (B) Neutrophil infiltration was highest in the outer medulla (upper left quadrant of large panel) and sparse in the inner medulla (lower right region) and other areas of kidney after subIRI. White box indicates area sampled for photographs in smaller panels at right. Neutrophils (green, FITC-7/4 immunofluorescence) were densely sequestered in the outer medulla of kidneys from mice with prior SPLX. Blue, DAPI-labeled nuclei. Scale bar is 50 μm. (C) Renal mRNA expression of CXCL1 and IL-6 was significantly greater in mice with SPLX plus subIRI compared with subIRI alone. Sham, sham ischemia-reperfusion injury surgery; SPLX, splenectomy; SPLX+subIRI, splenectomy plus subthreshold ischemia-reperfusion injury; subIRI, sham splenectomy plus subthreshold ischemia-reperfusion injury. n=4–11. *P<0.001; **P=0.003.
Figure 3.
Figure 3.
Splenic sympathectomy exacerbates subIRI. Mice received splenic injections of saline (60 μl) or 6-OHDA (120 μg in 60 μl saline) 14 days before subIRI. Mice with splenic 6-OHDA injections had elevated plasma creatinine. n=3–12. *P<0.001.
Figure 4.
Figure 4.
Prior ultrasound (US) exposure improves survival after kidney IRI. Mice were exposed to a modified ultrasound regimen 24 hours before bilateral IRI (26 minutes of ischemia, 24 hours of reperfusion). Mice were maintained for a week, and survival was determined using log-rank Kaplan–Meier survival analysis. n=8. P=0.004 (IRI versus US plus IRI).
Figure 5.
Figure 5.
Protective effect of prior ultrasound exposure is abolished by splenic sympathectomy. Mice received splenic injections of saline or 6-OHDA (as in Figure 3) 14 days before the ultrasound (US) and IRI (26 minutes of ischemia) treatments. Splenic injections of 6-OHDA abolished the protective effects of US as determined by (A) plasma creatinine and (B) histologic assessment (hematoxylin and eosin) of acute tubular necrosis. Scale bar is 100 μm in the main panel and 50 μm in the inset. n=5. *P<0.001.
Figure 6.
Figure 6.
Hematopoietic α7nAChRs are required for the protective effects of prior ultrasound (US) exposure. α7nAChR–/– or WT mice were lethally irradiated, reconstituted with bone marrow from either WT or α7nAChR–/– (KO) mice, and 8–10 weeks later were exposed to ultrasound and IRI (26 minutes of ischemia). Only mice with WT bone marrow reconstitution (WT→WT or WT→KO), regardless of the parenchymal phenotype, benefitted from the ultrasound treatment, as determined by (A) plasma creatinine and (B) hematoxylin and eosin analysis of morphology. Scale bar is 100 μm in the main panel and 50 μm in the inset. n=4−7. *P<0.001.
Figure 7.
Figure 7.
Ultrasound (US) blunts the IRI-induced increases in renal mRNA expression of IL-6 and MIP2. Mice were exposed to US 24 hours before IRI (26 minutes of ischemia). Then 24 hours after IRI, kidney samples were assayed for mRNA expression of key chemokines/cytokines by real-time PCR. Only IL-6 and MIP2 (CXCL2) expression (levels relative to sham) differed between the IRI and US plus IRI groups. n=13–14. $P=0.003; #P=0.02.
Figure 8.
Figure 8.
Splenocytes from ultrasound-treated mice have heightened IL-6 production when stimulated with LPS. Splenocytes were isolated 24 hours after ultrasound or sham treatment. Splenocytes were cultured (1×105 cells/well) and stimulated with LPS for 18 hours. The supernatants were then assayed for IL-6 by ELISA. n=3. *P=0.02; **P<0.001.
Figure 9.
Figure 9.
Adoptive transfer of splenocytes from ultrasound (US)-treated mice confers protection from IRI. Splenocyte single-cell suspensions were isolated from mice 24 hours after US or sham (no US) treatment and were injected (2×105 or 1×107 cells/mouse, intravenously) into naïve mice 24 hours before IRI (26 minutes of ischemia). Splenocytes from US-treated mice reduced plasma creatinine in a dose-dependent manner. n=4–5. ***P=0.04; *P<0.001.
Figure 10.
Figure 10.
Prior ultrasound protects mice from AKI in a CLP model of sepsis but does not protect in α7nAChR–/– mice. Mice were exposed to ultrasound (US) 24 hours before CLP and were euthanized 24 hours after CLP. (A) US reduced plasma creatinine in WT mice but α7nAChR–/– mice were resistant to the protective effects. **P<0.001. (B) Hematoxylin and eosin staining of kidney sections from WT mice. Scale bar is 100 μm. (C) Kim-1 mRNA expression in WT kidney relative to glyceraldehyde 3-phosphate (GAPDH). P=0.09. n=8–9 from two experiments. Circles and squares refer to the 2 groups, CLP and US+CLP, respectively.

Similar articles

See all similar articles

Cited by 26 PubMed Central articles

See all "Cited by" articles

Publication types

Feedback