Neural and central mechanisms of kidney fibrosis after relief of ureteral obstruction

Figures

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  Figure 1. Fibrosis in the post-obstructed kidney is accompanied by activated Ang II signaling in PVN and enhanced SNS discharge to the kidney (A) Outline of experimental design: male C57BL/6J mice were subjected to 2-day or 5-day UUO, after which the obstruction was relieved and animals were followed up for a further 28 days. (B) Fibrosis visualized by Masson trichrome staining in the post-obstructed kidney from mice at various time points after relief of UUO: representative images (left) and quantitative data (right). Scale bar, 50 μm. Bule areas indicate fibrosis. (C) Level of collagen I (Co I) mRNA in homogenates of the post-obstructed kidney. (D) Immunostaining for angiotensinogen (AGT) in paraventricular nucleus (PVN, Bregma −0.94 mm): representative images (left) and quantitative data (right). Scale bar, 50 μm. (E) Level of AGT mRNA in PVN homogenates. (F) Immunostaining for angiotensin II (Ang II) in PVN (Bregma −0.94 mm): representative images (left) and quantitative data (right). Scale bar, 50 μm. (G) Concentration of Ang II in PVN homogenates. (H) Sympathetic nerve activity (SNA) in the post-obstructed kidney: representative raw records (left) and quantitative data (right). Scale…

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  Figure 2. Activated Ang II signaling in PVN enhances SNS discharge that promotes fibrosis in the post-obstructed kidney (A) Outline of experimental design: PVN-specific deletion of Ang II type 1a receptor (AT1a) was achieved by injecting AAV-Cre into PVN of AT1afl/fl mice at the time of relief of 5-day UUO. Blockade of sympathetic outflow was achieved by denervation of the post-obstructed kidney (kdn). (B) Immunostaining of Cre protein and in situ hybridization of AT1a mRNA in PVN. Scale bar, 50 μm. (C) Level of AT1a mRNA in PVN homogenates. (D) Sympathetic nerve activity (SNA) in the post-obstructed kidney: representative raw records (left) and quantitative data (right). Scale bar, 1 s. (E) Immunostaining for collagen I (Co I) in the post-obstructed kidney: representative images (left) and quantitative data (right). Scale bar, 50 μm. (F) Level of Co I mRNA in homogenates of the post-obstructed kidney. (G) Norepinephrine (NE) level in homogenates of the post-obstructed kidney. Error bars, mean ± standard deviation (n = 6 in each group). ns, no significant. ∗, p < 0.01. One-way analysis of variance or t test with Bonferroni correction. Ctrl = control.

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  Figure 3. Afferent nerve signals from the post-obstructed kidney activate Ang II signaling in PVN (A) Outline of experimental design: kidney deafferentation was conducted by capsaicin (Cap) treatment in the post-obstructed kidney in mice at the time of relief of 5-day UUO. (B) Expression of AGT and Ang II in PVN: representative images (left) and quantitative data (right). Scale bar, 50 μm. ∗, p < 0.01. (C) Level of AGT mRNA and Ang II protein in PVN homogenates. (D) Sympathetic nerve activity (SNA) in the post-obstructed kidney: representative raw records (left) and quantitative data. Scale bar, 1 s ∗, p < 0.01. (E) Fibrosis visualized by Masson trichrome staining in the post-obstructed kidney: representative images (left) and quantitative data (right). Bule areas indicate fibrosis. Scale bar, 50 μm. (F) Amounts of CREB-CRTC1 complex in PVN homogenates. ∗, p < 0.01. Error bars, mean ± standard deviation (n = 6 in each group). One-way analysis of variance or t test with Bonferroni correction.

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  Figure 4. Ang II signaling in PVN activates the PVN-RVLM pathway that enhances SNS discharge to the post-obstructed kidney (A) Outline of experimental design: PVN-specific deletion of Ang II type 1a receptor (AT1a) was achieved by injecting AAV-Cre into PVN of AT1afl/fl mice at the time of relief of 5-day UUO. (B) Rostral ventrolateral medulla (RVLM)-projecting PVN neurons were labeled with rAAV-CaMKIIα-EGFP. (C) Representative images showing AT1a mRNA expression in EGFP-labeled PVN neurons by in situ hybridization in normal mice. (D) Expression of c-fos was detected in EGFP-labeled PVN neurons: representative images and summary of percentage of c-fos+ cell in EGFP+ cells. Scale bar, 50μm. ∗, p < 0.01. (E) Schematic of the coronal brain section indicating the RVLM, and immunostaining of c-fos and tyrosine hydroxylase (TH) in RVLM: representative images and summary of percentage of c-fos+ cell in TH+ cells. Scale bar, 50 μm. ∗, p < 0.01. (F) Optical silence of RVLM-projecting PVN neurons with simultaneous recording of sympathetic nerve activity (SNA) in post-obstructed kidney. (G) Changes of SNA in the post-obstructed kidney after the optical silence in mice at day 28 post UUO relief. SNA…

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  Figure 5. Enhanced SNS discharge induces persistent autophagy and promotes fibrosis in post-obstructed kidney (A) Expression of LC3II in the post-obstructed kidney of mice after UUO relief. ∗, p < 0.01 versus sham. (B) Autophagosomes observed by transmission electron microscopy in the post-obstructed kidney in mice after UUO relief. The yellow arrow indicates autophagosome. (C) Kidney denervation (kdn) or PVN-specific deletion of AT1a reversed the upregulation of LC3II in the post-obstructed kidney in mice after relief of 5-day UUO. ns, not significant; ∗, p < 0.01. (D and E) Exposure of human proximal tubular cells to norepinephrine (NE) elevated the expression of LC3II and collagen I (Co I) (D), and increased the number of mRFP-LC3 puncta in cells transfected with mRFP-GFP-LC3 adenoviral vectors (E). Alpha2-adrenergic receptor blocker reversed the NE-induced responses in tubular cells. Scale bar, 25 μm. Error bars, mean ± standard deviation (n = 6 in each group). #, p < 0.01 versus NE. One-way analysis of variance or t test with Bonferroni correction. Ctrl = control; anti-α2 = α2-adrenergic receptor blocker.

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  Figure 6. Increased tyrosine hydroxylase expression and enhanced autophagy in human fibrotic post-obstructed kidney (A) Fibrosis visualized by Masson trichrome staining in human kidney samples: representative images (left) and quantitative data (right). Scale bar, 50 μm. Blue Areas indicate fibrosis. (B) Immunostaining for tyrosine hydroxylase (TH) in human kidney samples: representative images (left) and quantitative data (right). Scale bar, 50 μm. (C) Immunostaining for LC3 in human kidney samples: representative images (left) and summary of percentage of autophagic tubular cells (right). Scale bar, 50 μm. (D) Autophagosomes and autolysosomes observed by transmission electron microscopy in human kidney samples. The yellow arrow indicates autophagosome, and the white arrow indicates autolysosome. (E) Immunostaining for Nox4 protein in human kidney samples. Green, Nox4. Scale bar, 50 μm. Error bars, mean ± standard deviation (n = 10 in each group). ∗, p < 0.01 versus normal kidney samples. t test.

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  Figure 7. Schematic diagram summarizing that sustained enhancement of afferent nerve inflow from the post-obstructed kidney in severe UUO mice activates Ang II signaling in RVLM-projecting PVN neurons to drive SNS discharge back to the kidney This causes the kidney to undergo fibrosis with loss of function.