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Targeted Ablation of Distal Cerebrospinal Fluid-Contacting Nucleus Alleviates Renal Fibrosis in Chronic Kidney Disease

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Targeted Ablation of Distal Cerebrospinal Fluid-Contacting Nucleus Alleviates Renal Fibrosis in Chronic Kidney Disease

Minzi Qiu et al. Front Physiol.

Abstract

The potential function of distal cerebrospinal fluid-contacting nucleus (dCSF-CNs) in chronic kidney disease (CKD) development is poorly understood. We hypothesized that dCSF-CNs might affect the renin-angiotensin system (RAS) in kidney injury progression, with dCSF-CNs ablation potentially alleviating local RAS and renal fibrosis in rats after five-sixths nephrectomy (5/6Nx). Part of rats were randomly administered artificial cerebrospinal fluid (aCSF) intracerebroventricularly (icv), followed by 5/6Nx or sham operation; and other part of rats were administered Cholera toxin B subunit conjugated with saporin (CB-SAP) for dCSF-CNs lesion before 5/6Nx. The effect of CB-SAP on dCSF-CNs ablation was confirmed by double immunofluorescence staining. RAS component, NOX2 and c-fos levels in the subfornical organ (SFO), hypothalamic paraventricular nucleus (PVN) and hippocampus, as well as tyrosine hydroxylase (TH) and c-fos positive cells in rostral ventrolateral medulla (RVLM) were assessed. Next, the levels of RAS components (angiotensinogen [AGT], angiotensin-converting enzyme [ACE], Ang II type 1 receptor [AT1R], angiotensin-converting enzyme 2 [ACE2], and Mas receptor), NADPH oxidases (NOX2 and catalase), inflammatory cytokines (monocyte chemotactic protein 1 [MCP-1] and IL-6), and fibrotic factors (fibronectin and collagen I) were assessed. Less CB-labeled neurons were found in dCSF-CNs of CB-SAP-treated rats compared with 5/6Nx animals. Meanwhile, CB-SAP downregulated AGT, Ang II, AT1R, NOX2, catalase, MCP-1, IL-6, fibronectin, and collagen I, and upregulated ACE2 and Mas receptor, compared with CKD rats. More TH and c-fos positive cells were found in RVLM of 5/6Nx rats but the number decreased after dCSF-CNs ablation. Targeted dCSF-CNs ablation could alleviate renal inflammation and fibrosis in chronic kidney injury by inhibiting cerebral and renal RAS/NADPH oxidase.

Keywords: NADPH oxidase; chronic kidney disease; dCSF-CNs; inflammation; renal fibrosis; renin-angiotensin system; sympathetic nervous system.

Figures

FIGURE 1
FIGURE 1
Schematic illustration of CB treatment and confirmation. (A) Schematic time-line of the CB treatment experiment. (B) Representative images of dCSF-CNs in the brainstem by CB-immunoreactive immunofluorescence after an intracerebroventricular injection of CB-SAP (magnification ×200; scale bar = 100 μm). CB (green) double-staining with the antibody against CB and the antibody-recognized Neun (red). Aq, ventral mesencephalic aqueduct; 2/3Nx, two-third of the left kidney was removed by surgical resection; UNX, unilateral nephrectomy.
FIGURE 2
FIGURE 2
Overexpression of the central RAS in 5/6Nx rats. Representative photographs of immunohistochemistry staining of AGT (A1), Ang II (B1), and AT1R (C1) in SFO, PVN, and hippocampus, respectively [(A1–C1) magnification ×100; scale bar = 100 μm]. Semiquantitative data of AGT (A2), Ang II (B2), and AT1R (C2). #P < 0.05 versus sham in the respective group. P < 0.05 versus icv aCSF+5/6Nx in the respective group.
FIGURE 3
FIGURE 3
Expression of central TH and c-fos are increased in 5/6Nx rats. Representative photographs of c-fos in SFO, PVN and hippocampus (A), and semiquantitative data (B). Double labeling of TH (brown cytoplasmic) and c-fos (red nuclear) staining in RVLM (C). Arrows indicate the TH- and c-fos positive cells in RVLM. The semiquantitative data are expressed as the mean ± SEM (D). (A) Magnification ×100; scale bar = 100 μm. (C) Magnification ×200; scale bar = 100 μm. #P < 0.05 versus sham in the respective group. P < 0.05 versus icv aCSF+5/6Nx in the respective group.
FIGURE 4
FIGURE 4
Physiological parameters of 5/6Nx rats after targeted ablation of dCSF-CNs. (A) Serum creatinine, (B) blood urea nitrogen (BUN), (C) plasma Ang II concentration, (D) systolic blood pressure (SBP), (E) heart rate, and (F) urinary albumin. Results are represented as mean ± SEM (n = 6). Data were analyzed using one-way ANOVA followed by least significant difference (LSD) test. #P < 0.05 versus sham in the respective group. P < 0.05 versus icv aCSF+5/6Nx in the respective group.
FIGURE 5
FIGURE 5
Overexpression of the renal RAS was inhibited by targeted ablation of dCSF-CNs. (A) Representative photographs of RAS component expression by immunohistochemistry (magnification ×200; scale bar = 100 μm). (B) Expression of RAS components detected by western blot. (C) Semiquantitative data of RAS expression by immunohistochemistry. (D) Renal tissue Ang II concentration. (E) AT1 receptor mRNA level analyzed by real-time PCR. (F) Semiquantitative data of RAS component expression by western blot. #P < 0.05 versus sham in the respective group. P < 0.05 versus icv aCSF+5/6Nx in the respective group.
FIGURE 6
FIGURE 6
Expression of catalase, NOX2, and inflammatory cytokines in 5/6Nx rats. Representative photographs of protein levels of catalase and NOX2 (A) and inflammatory cytokines (MCP-1, IL-6) (B) in rats. Semiquantitative data of catalase and NOX2 (C), as well as that of MCP-1 and IL-6 (D). #P < 0.05 versus sham in the respective group. P < 0.05 versus icv aCSF+5/6Nx in the respective group.
FIGURE 7
FIGURE 7
Renal inflammation and fibrosis in 5/6Nx rats was inhibited by targeted ablation of dCSF-CNs. (A) Representative photographs of renal fibrosis shown by H&E, PAS, and Sirius red-staining (magnification ×200; scale bar = 100 μm). Quantitative analysis of interstitial macrophage (B), glomerulosclerosis index (C), and tubulointerstitial fibrosis score (D). #P < 0.05 versus sham in the respective group. P < 0.05 versus icv aCSF+5/6Nx in the respective group.
FIGURE 8
FIGURE 8
Expression of fibronectin and collagen I in 5/6Nx rats. Representative photographs of expression levels of fibronectin and collagen I in renal cortex (A,B). (A) Magnification ×200; scale bar = 100 μm. Semiquantitative data of fibronectin and collagen I in renal cortex (C,D). #P < 0.05 versus sham in the respective group. P < 0.05 versus icv aCSF+5/6Nx in the respective group.
FIGURE 9
FIGURE 9
Schematic illustration of the modulation served by dCSF-CNs in kidney injury progression. The dCSF-CNs might exert a link between the cerebral fluid and the brain. The dCSF-CNs might act as the sensor of circulating Ang II and other unknown humoral factors in the cerebrospinal fluid, communicating signal to brain centers. The signal is then transferred from the cerebral fluid to the blood-brain barrier integrated circumventricular organs; this results in activation of cerebral- and renal-RAS/ROS axes consequently.

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