doi: 10.1681/ASN.2021111518. Epub 2022 Oct 17.
Anatomical Evidence for Parasympathetic Innervation of the Renal Vasculature and Pelvis
Xiaofeng Cheng 1 2, Yongsheng Zhang 1 2, Ruixi Chen 1 2, Shenghui Qian 1 2, Haijun Lv 1 2, Xiuli Liu 1 2, Shaoqun Zeng 1 2
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- PMID:36253054
- PMCID: PMC9731635
- DOI: 10.1681/ASN.2021111518
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Anatomical Evidence for Parasympathetic Innervation of the Renal Vasculature and Pelvis
Xiaofeng Cheng et al. J Am Soc Nephrol.2022 Dec.
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Abstract
Background: The kidneys critically contribute to body homeostasis under the control of the autonomic nerves, which enter the kidney along the renal vasculature. Although the renal sympathetic and sensory nerves have long been confirmed, no significant anatomic evidence exists for renal parasympathetic innervation.
Methods: We identified cholinergic nerve varicosities associated with the renal vasculature and pelvis using various anatomic research methods, including a genetically modified mouse model and immunostaining. Single-cell RNA sequencing (scRNA-Seq) was used to analyze the expression ofAChRs in the renal artery and its segmental branches. To assess the origins of parasympathetic projecting nerves of the kidney, we performed retrograde tracing using recombinant adeno-associated virus (AAV) and pseudorabies virus (PRV), followed by imaging of whole brains, spinal cords, and ganglia.
Results: We found that cholinergic axons supply the main renal artery, segmental renal artery, and renal pelvis. On the renal artery, the newly discovered cholinergic nerve fibers are separated not only from the sympathetic nerves but also from the sensory nerves. We also found cholinergic ganglion cells within the renal nerve plexus. Moreover, the scRNA-Seq analysis suggested that acetylcholine receptors (AChRs) are expressed in the renal artery and its segmental branches. In addition, retrograde tracing suggested vagus afferents conduct the renal sensory pathway to the nucleus of the solitary tract (NTS), and vagus efferents project to the kidney.
Conclusions: Cholinergic nerves supply renal vasculature and renal pelvis, and a vagal brain-kidney axis is involved in renal innervation.
Keywords: acetylcholine; arteries; gene transcription; kidney anatomy.
Copyright © 2022 by the American Society of Nephrology.
Figures
Cholinergic nerve fibers around the renal artery. (A) Distribution ofChat-tdTomato positive nerve fibers (red) around the renal artery. The green dashed box in the schematic diagram indicates the imaging region. Scale bar, 200μm. (B) Representative images of renal arteries from adultChat-ires-Cre:Ai14 mice, stained for ChAT. Note the tdTomato (red) specifically expressed in ChAT-immunoreactive nerve fibers (green). Scale bars, 10μm. (C) Representative images of renal arteries from adultChat-ires-Cre:Ai14 mice, stained for VAChT. Note the tdTomato (red) specifically expressed in VAChT-immunoreactive nerve fibers (green). The white box regions are magnified and shown in the lower-left corner. Scale bars, 10μm.
Anatomic relationship between cholinergic, sensory, and sympathetic nerve fibers around the renal artery. (A) Representative images of renal arteries from adultChat-ires-Cre:Ai14 mice, stained for CGRP. Scale bars, 20μm. (B) Higher-magnification images of the light blue box regions in (A). Scale bars, 10μm. (C) Higher-magnification images of the white box regions in (A). Scale bars, 10μm. Note the separation between tdTomato-positive (red) and CGRP-immunoreactive (green) nerve fibers. (D) Representative images of renal arteries from adultChat-ires-Cre:Ai14 mice, stained for TH. Scale bars, 100μm. (E) Higher-magnification images of the light blue box regions in (D). Scale bars, 10μm. (F) Higher-magnification images of the white box regions in (D). Scale bars, 10μm. Note the separation between tdTomato-positive (red) and TH-immunoreactive (green) nerve fibers. (G) Comparison of the densities of tdTomato-positive and TH-immunoreactive nerve fibers surrounding the renal artery (n=10 arteries from five female adult mice, two-sided unpairedt test, mean±SEM).
Cholinergic ganglion cells within renal nerve plexus. (A) A schematic representation of the location of cholinergic ganglia and nerve tracts around the renal artery. (B) A confocal image shows the tdTomato-positive cells located in a renal nerve bundle. White arrows indicate tdTomato-positive (red) cells. Nuclei were labeled with Hoechst 33258 (blue). The white dashed line delineates the outline of the renal artery. The white box region was magnified and shown in the lower-left corner. Scale bars, 50μm. (C) Representative images of renal ganglia from adultChat-ires-Cre:Ai14 mice, stained for ChAT. Scale bars, 20μm. (D) Representative images of renal ganglia from adultChat-ires-Cre:Ai14 mice, stained for VAChT. Scale bars, 20μm. (E) Representative images of renal ganglia from adultChat-ires-Cre:Ai14 mice, stained for TH (green). Scale bars, 20μm.
Cholinergic nerve varicosities are associated with the segmental renal artery and pelvis. (A) Cross-sectional view of a segmental renal artery fromChat-ires-Cre:Ai14 mouse. Stained for synapsin 1 (green). The green box in the schematic diagram indicates the imaging region. Scale bars, 20μm. (B) Higher-magnification images of the white box regions in (A). Scale bars, 20μm. Arrows indicate tdTomato and synapsin double-positive varicosities. Colocalization analysis result of varicosities is shown on the right. (n=4 male mice, mean±SEM). (C) Representative images of the renal pelvises fromChat-ires-Cre:Ai14 mice, stained for synapsin 1 (green). Scale bars: whole-mount image, 200μm; higher-magnification images, 20μm. (D) Higher-magnification images of the white box regions in (C). Scale bars, 20μm. Arrows indicate tdTomato and synapsin double-positive varicosities. Colocalization analysis result of varicosities is shown on the right. (n=4 male mice, mean±SEM).
Expression ofAChRs in the renal artery and its segmental branches. (A) Several renal arteries and their segmental branches for scRNA-seq. Scale bar, 1 mm. (B) Workflow of scRNA-seq. Renal arteries and their segmental branches were digested to the single cell suspension. After barcoded cDNA library construction, sequencing was performed on a 10× Genomics platform, followed by the unsupervised clustering analysis. (C) T-SNE representation of 6354 cells. (D) Violin plots showing the expression distribution of selected marker genes across cell subpopulations. (E) Treemap showing the composition of arterial cells analyzed by unsupervised clustering analysis. (F) T-SNE plot showing the biased expression ofmAChR3 in ECs. (G) Heatmap showing relative expression of 16 AChRs in seven major cell types of the renal artery and its segmental branches.
Assessment of the brain-kidney vagal afferent pathway. (A) Representative images of renal sections at 7 days after the sham procedure (left) and RDN (right), stained for TH (white). Scale bars, 500μm. The green box regions are magnified and shown below, respectively. Scale bars, 50μm. (B) Densities of TH-immunoreactive nerve fibers in the renal cortex of sham-operated or RDN mice. (n=5 male adult C57BL/6J mice, two-sided unpairedt test with Welch’s correction, mean±SEM). (C) Cartoon illustrates the scheme for retrograde AAV tracing. (D) TdTomato-positive neurons (red) in ipsilateral nodose/jugular complex from sham-operated and RDN Ai14 mice, infected with AAVretro-hSyn-Cre. Scale bars, 100μm. (E) Statistical results for (D) (n=5 male heterozygous Ai14 mice for each group, two-sided unpairedt test, mean±SEM). (F) TdTomato-positive neurons (red) in the ipsilateral nodose/jugular complex from Ai14 mice infected with AAVretro-hSyn-Cre, stained for VGluT2 (green). Scale bars, 50μm. (G) Statistical results for (F) (n=5 male mice, mean±SEM). (H) TdTomato-positive axons (red) distributed in NTS. White lines delineate outlines of brain regions. Scale bar, 200μm. A higher-magnification image of the white box region is shown on the right. Scale bar, 20μm.
Assessment of the brain-kidney parasympathetic connection. (A) Cartoon illustrates the scheme for retrograde PRV tracing with sdVx or RDN. CTB tracing was used to confirm the successful vagotomy. (B) Workflow of the whole brain three-dimensional imaging. Scale bars, 2 mm. (C) Whole-brain three-dimensional view of the distribution of infected neurons after PRV-CMV-GFP injection. Modeled digital spots were derived from the GFP-positive neurons, and the spots located in the DMX were rendered in red. The surfaces of the DMX, paraventricular hypothalamic nucleus, and paragigantocellular reticular nucleus (PGRN) were rendered in light blue. Scale bar, 2 mm. A, anterior; P, posterior; D, dorsal; V, ventral; L, lateral; M, medial. (D) Numbers of GFP-positive neurons in DMX of the sdVx and sham-operated mice (n=5 male mice for each group, two-sided unpairedt test with Welch’s correction, mean±SEM). (E) Frequencies of GFP-positive neurons in left DMX of the renal denervated and sham-operated mice (n=5 male mice, two-sided Mann–Whitney test, mean±SEM). (F) PRV-infected cholinergic neurons in DMX. Scale bars, 50μm. Arrows indicateChat-tdTomato (red) and PRV-GFP (green) double-positive neurons. Dashed lines delineate the outlines of the DMX. Statistical result was shown on the right (n=5 male mice, mean±SEM). (G) The absence of PRV-labeled cells within the sacral 4. Scale bars, 200μm. (H) The cell count results ofChat-tdTomato and PRV-GFP double-positive neurons (red lines) and all PRV-GFP-positive neurons (black lines) in representative coronal slices from multiple spinal cord levels (n=2 male mice).
Comment in
- Optogenetics supports to identify the function of renal autonomic system.Zhou H, Liu H, Li D, Xu Y, Xiao P, Yin Y.Zhou H, et al.Hypertens Res. 2023 Oct;46(10):2439-2441. doi: 10.1038/s41440-023-01379-8. Epub 2023 Aug 7.Hypertens Res. 2023.PMID:37550358No abstract available.
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