Inhibition of the renin-angiotensin system causes concentric hypertrophy of renal arterioles in mice and humans

Figures

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  Figure 1. Reninnull cells have different transcriptomic profiles. (A) Pathological findings of Ren1c-KO mice. Immunohistochemistry for α-SMA showed hypertrophic arterioles in Ren1c-KO mouse kidneys. Scale bars: 20 μm. Kidney sections from Ren1c-KO Ren1c-Cre Confetti mice showed multiple colors in the hypertrophic arterioles, suggesting the multiclonality of Reninnull cells. Scale bars: 20 μm. Left: WT mice showed normal architecture of periglomerular arterioles with a single layer of smooth muscle cells (SMCs). Right: Ren1c-KO mice showed concentric hypertrophy of arteriolar smooth muscle. Scale bars: 10 μm. (B) Schematic of scRNA-Seq of WT renin cells and Reninnull cells. Ren1c-YFP mice have a transgene consisting of the YFP genes driven by the Ren1c gene regulatory region. YFP-positive kidney cells from Ren1c+/+ Ren1c-YFP mice and Ren1c–/– Ren1c-YFP mice were isolated by FACS. Single cells were captured with the Fluidigm C1 System, and scRNA-Seq was performed. Scale bars: 50 μm. (C) The UMAP with all the cells after normalization. (D) Volcano plot showing the differentially expressed genes between WT renin cells and Reninnull cells. (E) Heatmap analysis with differentially expressed…

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  Figure 2. scRNA-Seq of Reninnull cells identifies specific TFs and secreted proteins. (A) Heatmap of the TF genes differentially expressed between WT renin cells and Reninnull cells. (B) PPI enrichment analysis using differentially expressed TF genes. The genes higher in WT renin cells and those higher in Reninnull cells are shown in blue and red, respectively. (C) Heatmap of the secreted protein genes differentially expressed between WT renin cells and Reninnull cells. (D) PPI enrichment analysis using differentially expressed secreted protein genes. The genes higher in WT renin cells and those higher in Reninnull cells are shown in blue and red, respectively. PPI, protein-protein interaction; scRNA-Seq, single-cell RNA sequencing; WT, wild-type.

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  Figure 3. Reninnull cells secrete proteins underlying the vascular abnormalities. (A) Violin plots and validation by ISH of selected secreted protein genes upregulated in the Reninnull cells. The expression of the genes was detected in the hypertrophic arterioles at higher levels in Ren1c-KO than WT. Scale bars: 50 μm. (B) ELISA for osteopontin during disease development (n = 4, Student’s t test). All data are reported as means ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ISH in situ hybridization, Ren1c-KO, Ren1c gene knockout; scRNA-Seq, single-cell RNA sequencing; WT, wild-type.

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  Figure 4. Reninnull mural cells comprise 2 phenotypically different groups of cells. (A) Schematic of scRNA-Seq of Reninnull cells and other vascular SMCs. Fluorescent cells from Ren1c–/– Myh11-CreERT2 tdTomato Ren1c-YFP mouse kidneys were isolated by FACS. scRNA-Seq was performed with the 10x Genomics Chromium System. (B) The UMAP with all the cells after normalization and volcano plots and feature plots for YFP and Akr1b7. Respectively, 0.95% and 20.9% of the cells were positive for YFP and Akr1b7. (C) Violin plots of representative DEGs between Akr1b7-negative cells and Akr1b7-positive cells. (D) Heatmap analysis with DEGs between Akr1b7-negative cells and Akr1b7-positive cells. (E) Violin plots from C1 scRNA-Seq and ISH for Klk1 and Mafb mRNA in WT and the Ren1c-KO kidneys. Arrows indicate hypertrophic afferent arterioles. Scale bars: 50 μm. DEGs, differentially expressed genes; FACS, fluorescence-activated cell sorting; GO, Gene Ontology; ISH, in situ hybridization; Mafb, v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B; Ren1c-KO, Ren1c gene knockout; scRNA-Seq, single-cell RNA sequencing; SMCs, smooth muscle cells; UMAP, uniform manifold approximation and projection;…

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  Figure 5. Phenotypical transition from SMCs to endocrine Reninnull cells. (A) UMAP with unbiased clustering indicated 2 clusters in scRNA-Seq of Reninnull cells and other vascular SMCs. (B) Violin plots of representative DEGs between clusters 0 and 1. (C) Heatmap analysis with DEGs between clusters 0 and 1. (D) The 10 most enriched categories identified by GO analysis on DEGs between clusters 0 and 1. (E) Unbiased trajectory analysis of cells of Ren1c-KO mice. DEGs, differentially expressed genes; FACS, fluorescence-activated cell sorting; GO, Gene Ontology; ISH, in situ hybridization; scRNA-Seq, single-cell RNA sequencing; SMCs, smooth muscle cells; UMAP, uniform manifold approximation and projection; WT, wild-type.

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  Figure 6. Deletion of integrin β1 in renin lineage cells inhibits arteriolar hypertrophy in Ren1c-KO mice. (A) Violin plots and ISH for Itgb1 mRNA in Reninnull cells. Scale bars: 50 μm. (B) Immunofluorescence staining for α-SMA and integrin β1 in kidneys from Ren1c-KO control and Ren1c-KO with Itgb1-cKO mice. Arrows indicate the α-SMA–positive cells at the JG area. Integrin β1 was not observed at the JG area of the Ren1c-KO Itgb1-cKO mice. Scale bars: 20 μm. (C and D) Arteriolar hypertrophy was prevented in Ren1c-KO Itgb1-cKO mice. Immunohistochemistry for α-SMA showed decreased thickness of the walls of afferent arterioles in Ren1c-KO Itgb1-cKO mice compared with Ren1c-KO control (C). Scale bars: 50 μm. The mean wall thickness of afferent arterioles at the JG area was significantly smaller in Ren1c-KO Itgb1-cKO mice (n = 6, Student’s t test) (D). Relative frequency distribution histograms show that the distribution curve corresponding to the Ren1c-KO Itgb1-cKO mice is displaced to the left of the control mice (D). (E) ISH for Akr1b7 mRNA in kidneys from Ren1c-KO Itgb1-c-KO and Ren1c-KO control mice. The number of Akr1b7 mRNA-positive cells in Ren1c-KO Itgb1-cKO mice is much smaller than…

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  Figure 7. Long-term inhibition of RAS induces arteriolar hypertrophy in mice. (A) Schematic of the treatments of BPN/3 and BPH/2 mice. After 6 months of treatment with captopril in the drinking water, BUN (B, n ≥ 4), Ren1 mRNA (C, n ≥ 3), and plasma renin (D, n ≥ 5) were significantly increased in both BPN/3 and BPH/2 mice (2-way ANOVA followed by Tukey’s multiple comparison test). (E) The mean blood pressure was significantly decreased by the captopril treatment in both BPN/3 and BPH/2 mice (n ≥ 4, 2-way ANOVA followed by Tukey’s multiple comparison test). Arteriolar hypertrophy induced by renin cells with long-term captopril treatment in BPN/3 mice (F) and BPH/2 mice (G) shown by PAS staining, Masson’s trichrome staining, and immunohistochemistry for renin. Arrows indicate hypertrophic afferent arterioles. Scale bars: 50 μm. Immunohistochemistry for α-SMA showed increased thickness of the walls of afferent arterioles in kidneys from BPN/3 mice (H) and BPH/2 mice (I). The mean wall thickness of afferent arterioles at the JG area was significantly larger in mice with long-term captopril treatment (n = 4, each, Student’s t test). Relative frequency distribution histograms show that the…

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  Figure 8. Long-term inhibition of RAS induces arteriolar hypertrophy in human patients. (A) Representative images of periodic acid–methenamine silver (PAM) with Masson’s trichrome staining of kidney sections from healthy control individuals, patients with benign nephrosclerosis without RASi, and patients with benign nephrosclerosis with long-term usage of RASi. Patients with long-term usage of RASi showed marked hypertrophy in afferent arterioles. (B) Mean thickness of afferent arterioles at the JG area. Patients with long-term usage of RASi (n = 6) showed significantly thicker renal arteriolar walls compared with controls (n = 4) and patients with nephrosclerosis without RASi (n = 6) (1-way ANOVA followed by Tukey’s multiple comparisons test). (C) Representative images of immunohistochemistry for renin in kidney sections. Patients with long-term usage of RASi showed extensive renin-positive regions in the JG area. (D) Mean renin-positive area at the JG area. Patients with long-term usage of RASi (n = 6) showed significantly larger areas compared with controls (n = 4) and patients with nephrosclerosis without RASi (n = 6) (1-way ANOVA followed by Tukey’s multiple comparisons test). (E)…