Proximal tubule ACE2 mediates early responses to hypertension by regulating intrarenal RAS and sodium homeostasis

Figures

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  Figure 1. Proximal tubule-specific deletion of ACE2. (A) PT ACE2–KO mice were generated by crossing mice expressing Cre-recombinase downstream of the PEPCK promoter with ACE2-floxed mice. (B–D) Ace2 mRNA expression, ACE2 protein abundance and Coomassie brilliant blue loading control, and ACE2 activity in kidney cortex and medulla of PT ACE2–KO and Control mice. (E) IHC for ACE2 in the proximal tubule in Control kidneys. (F) ACE2 expression is decreased in PT ACE2–KO mice (representative images; n = 3 mice per group). (G) ACE2 is absent in Global KO mice. Hoechst DNA stain in blue, Lotus Tetragonolobus Lectin (LTL) proximal tubule marker in green, and ACE2 in magenta. Scale bar: 200 μm. Numeric data expressed as mean ± SEM and analyzed as a 2-way ANOVA with Šídák’s multiple-comparison post hoc analysis. n = 3 mice per group, **P < 0.01, ****P < 0.0001.

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  Figure 2. Proximal tubule ACE2 attenuates early hypertension and cardiac hypertrophy. (A) Mean arterial pressures (MAP) at baseline (day –3) and with continuous AngII infusion (1,000 ng/kg/min) at day 5. Day 5 MAP values are also presented in Figure 3A. (B) Ratio of heart weight to tibia length (HW/TL) in mice at baseline and treated with AngII for 5 days (n = 5–6 for baseline groups; n = 19–23 for AngII treated groups; figure displays aggregated data across multiple experiments). (C) Representative anterior-view and mid-ventricular cross-sections of hearts from Control and PT ACE2–KO mice after 5 days of AngII treatment (n = 8–10 mice per group). (D) Echocardiographic calculated left ventricular (LV) mass at diastole (n = 8–10 mice per group). Data expressed as mean ± SEM. MAP and HW/TL data were analyzed using a 2-way ANOVA with Tukey’s multiple-comparisons post hoc analysis. Echocardiography data was analyzed by mixed-effects analysis with Fisher’s LSD pairwise comparison post hoc analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  Figure 3. Proximal tubule ACE2 stabilizes response to AngII by mitigating increases in intrarenal RAS activity. (A) Mean arterial pressure (MAP) curve at baseline and with continuous AngII infusion (1,000 ng/kg/min) for 15 days. Day 5 MAP values are also presented in Figure 2A. (B) Renal AngII levels from Control and PT ACE2–KO mice. (C) Urinary ACE2 activity. (D) Immunoblots of angiotensinogen (n = 5), ACE1 (n = 6), and ACE2 (n = 6) protein abundance at baseline and with 5 days of AngII infusion. All lanes of the angiotensinogen immunoblot were run on the same gel but were noncontiguous. (E–G) Quantification of renal angiotensinogen, ACE1, and ACE2 abundance. (H and I) Immunoblots depicting urinary abundance of full-length ACE2 (FL-ACE2) and soluble ACE2 (sACE2) at day 6 and 14 days of AngII treatment. Coomassie brilliant blue stain was used to demonstrate similar loading for urine blots (56, 62). Data expressed as mean ± SEM. MAP (n = 16) was analyzed by mixed-effects analysis with Fisher’s LSD pairwise comparison post hoc analysis. Renal AngII (n = 3–10), urinary ACE2 activity (n = 6), and renal protein abundance were analyzed using a 2-way ANOVA with Tukey’s post hoc multiple…

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  Figure 4. Alterations in sodium transporters along the proximal tubule and medullary thick ascending limb. (A) Immunoblots depicting key sodium transporter abundance at baseline and with 5 days of AngII. (B) Sodium hydrogen exchanger 3 (NHE3). (C) Sodium-glucose cotransporter 2 (SGLT2). (D) Claudin 2. (E) Aquaporin 1 major channel (AQP1-35 kDa). (F) Medullary NHE3. (G) Medullary sodium-potassium-chloride cotransporter (mNKCC2). (H) Phosphorylated mNKCC2. (I) Na+/K+ ATPase (NKA), α 1 subunit. (J) NKA β 1 subunit. All lanes of the immunoblots for mNHE3, mNKCC2, mNKCC2p, mNKA α1, and mNKA β1 were run on the same gel but were noncontiguous (n = 5–6 mice per group). Transporter abundance was analyzed as a 2-way ANOVA with Tukey’s multiple-comparison post hoc analysis. *P < 0.05, ***P < 0.001.

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  Figure 5. Alterations in sodium transporters along the more distal segments of the nephron. (A) Immunoblots depicting key sodium transporter abundance at baseline and with 5 days of AngII. (B) Sodium-potassium-chloride cotransporter (NKCC2). (C) Phosphorylated NKCC2. (D) Sodium-chloride cotransporter (NCC). (E) Phosphorylated NCC. (F) Na+/K+ ATPase (NKA), α1 subunit. (G) NKA β1 subunit. (H) Claudin 8 (Cldn 8). (I) Aquaporin 2 (AQP2), major band (37 kDa). (J) Phosphorylated AQP2 major band. Transporter abundance was analyzed as a 2-way ANOVA with Tukey’s multiple-comparison post hoc analysis (n = 5–6 mice per group). **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  Figure 6. Inappropriate regulation of ENaCγ and Na,K-ATPase activity impairs pressure natriuresis in PT ACE2–KO mice. (A) Immunoblots depicting epithelial sodium channel (ENaC) abundance at baseline and with 5 days of AngII. (B) Full-length ENaCα (ENaCα - FL) (C) Cleaved ENaCα (ENaCα – cl). (D) ENaCβ (ENaC β). (E) Full-length ENaCγ (ENaCγ-FL). (F) Cleaved ENaCγ (ENaCγ-cl). (G) K+-Dependent p-nitrophenyl phosphatase (pNPPase), a surrogate for Na,K-ATPase activity. Data expressed as mean ± SEM; transporter abundance was analyzed as a 2-way ANOVA with Tukey’s multiple-comparison post hoc analysis. pNPPase activity was analyzed by mixed-effects analysis with Fisher’s LSD pairwise comparison post hoc analysis (n = 5–6 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  Figure 7. Transcriptional changes in the kidney in response to treatment with AngII for 5 days. (A) UMAP plots of all mouse kidney datasets integrated with FindIntegrationAnchors. PTS1; S1 segment of proximal tubule; PTS2, S2 segment of proximal tubule; PTS3, S3 segment of proximal tubule; dTL, descending limb of Loop of Henle; TAL, thick ascending limb of Loop of Henle; MD, macula densa; DCT1, early segment of distal convoluted tubule; DCT2, late segment of distal convoluted tubule; CNT, connecting tubule; PC, principal cells; ICA, type A intercalated cells; ICB, type B intercalated cells; Podo, podocytes; PEC, parietal epithelial cells; EC, endothelial cells; Fib, fibroblasts; Mes, mesangial cells; Macro, macrophages; B & T, B and T lymphocytes; Uro, uroepithelial cells. (B) Dot plot displaying gene expression patterns of cluster-enriched markers. (C and D) Number of differentially expressed genes per cluster in saline- and AngII-treated groups. (E) Intrarenal RAS gene expression in proximal tubule segments 1–3, contractile cells, mesangial cells, and macrophages.

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  Figure 8. Differential pathway enrichment in PT ACE2–KO and Control mice. (A) Enrichment of pathways found significant in PT ACE2 KO Saline versus Control Saline. (B) Enrichment of pathways found significant in PT ACE2 KO AngII versus Control AngII.