Experimental TET2 Clonal Hematopoiesis Predisposes to Renal Hypertension Through an Inflammasome-Mediated Mechanism

Abstract

Background: Hypertension incidence increases with age and represents one of the most prevalent risk factors for cardiovascular disease. Clonal events in the hematopoietic system resulting from somatic mutations in driver genes are prevalent in elderly individuals who lack overt hematologic disorders. This condition is referred to as age-related clonal hematopoiesis (CH), and it is a newly recognized risk factor for cardiovascular disease. It is not known whether CH and hypertension in the elderly are causally related and, if so, what are the mechanistic features.

Methods: A murine model of adoptive bone marrow transplantation was employed to examine the interplay between Tet2 (ten-eleven translocation methylcytosine dioxygenase 2) clonal hematopoiesis and hypertension.

Results: In this model, a subpressor dose of Ang II (angiotensin II) resulted in elevated systolic and diastolic blood pressure as early as 1 day after challenge. These conditions led to the expansion of Tet2-deficient proinflammatory monocytes and bone marrow progenitor populations. Tet2 deficiency promoted renal CCL5 (C-C motif ligand 5) chemokine expression and macrophage infiltration into the kidney. Consistent with macrophage involvement, Tet2 deficiency in myeloid cells promoted hypertension when mice were treated with a subpressor dose of Ang II. The hematopoietic Tet2^-/- condition led to sodium retention, renal inflammasome activation, and elevated levels of IL (interleukin)-1β and IL-18. Analysis of the sodium transporters indicated NCC (sodium-chloride symporter) and NKCC2 (Na⁺-K⁺-Cl^- cotransporter 2) activation at residues Thr53 and Ser105, respectively. Administration of the NLRP3 (NLR family pyrin domain containing 3) inflammasome inhibitor MCC950 reversed the hypertensive state, sodium retention, and renal transporter activation.

Conclusions: Tet2-mediated CH sensitizes mice to a hypertensive stimulus. Mechanistically, the expansion of hematopoietic Tet2-deficient cells promotes hypertension due to elevated renal immune cell infiltration and activation of the NLRP3 inflammasome, with consequences on sodium retention. These data indicate that carriers of TET2 CH could be at elevated risk for the development of hypertension and that immune modulators could be useful in treating hypertension in this patient population.

Keywords: clonal hematopoiesis; hypertension; inflammation; kidney; sodium.

Figure 1.. A subpressor dose of Ang II induces hypertension in mice with Tet2-mediated CH.

(A) Experimental design. (B) Flow cytometric analysis of donor CD45.2⁺ white blood cells (WT n=5, Tet2^−/− n=8, WT+ Ang II n=6, and Tet2^−/− + Ang II n=9), and (C) total white blood cell count in the peripheral blood of WT and Tet2^−/− CH mice following infusion with either vehicle or Ang II for 1 day (WT n=12, Tet2^−/− n=10, WT+ Ang II n=5, and Tet2^−/− + Ang II n=12). (C) Radiotelemetry measurements of systolic blood pressure, diastolic blood pressure and heart rate during the dark phase (6PM to 6AM) and light phase (6AM to 6PM) in WT and Tet2^−/− CH mice following infusion with Ang II (N = 11 per group). Statistical analysis was performed with 2-way ANOVA with Tukey’s multiple-comparisons tests for A and B. For C, results were analyzed with repeated measures 2-way ANOVA. Data are presented as mean ± SEM.

Figure 2.. A subpressor dose of Ang II promotes expansion of peripheral Ly6C^hi pro-inflammatory monocytes and renal Tet2^−/− monocyte/macrophage infiltration in Tet2⁻mediated CH.

Flow cytometric analysis of (A) proinflammatory monocytes (CD115⁺LY6CG⁻Ly6C^hi; WT n=13, Tet2^−/− n=13, WT+ Ang II n=10, and Tet2^−/− + Ang II n=12), and (B) renal macrophages (CD45⁺CD11b⁺F4/80⁺) in mice transplanted with WT or Tet2^−/− donor cells following infusion with either vehicle or Ang II (WT n=7, Tet2^−/− n=6, WT+ Ang II n=6, and Tet2^−/− + Ang II n=6, C; WT n=5, Tet2^−/− n=5, WT+ Ang II n=4, and Tet2^−/− + Ang II n=5). (C) Quantification of donor CCR2⁺ macrophages (F4/80⁺ CCR2⁺CD45.2⁺) within the kidney of mice transplanted with WT or Tet2^−/− donor cells following infusion with either vehicle or Ang II (WT n=5, Tet2^−/− n=5, WT+ Ang II n=4, and Tet2^−/− + Ang II n=5). (D) Experimental design for E) systolic blood pressure measured by tail-cuff of myeloid-specific Tet2 homozygous deficient (Lyz2^Cre/+Tet₂^fl/fl) mice and littermate controls (Lyz2^+/+TET2^fl/fl) at 1 day following Ang II infusion (n=4). For (A), results are expressed as peripheral blood chimerism of the percentage of donor cells (CD45.2⁺). For B and C, the absolute number of cells are expressed per mg of kidney tissue. Statistical analysis was performed with 2-way ANOVA with Tukey’s multiple-comparisons tests for A-B. In B and C, the absolute number per mg of kidney tissue is shown. For C and E, non-parametric Kruskal-Wallis followed by Dunn’s multiple comparison tests were performed.

Figure 3.. CCL5 mediates renal infiltration of Tet2^−/− monocytes and consequent hypertension during Tet2-mediated CH.

(A) CCL5 chemokine expression measured by qPCR in kidneys from WT or Tet2^−/− CH mice treated with either vehicle or Ang II for 1 day (WT n=8, Tet2^−/− n=12, WT+ Ang II n=9, and Tet2^−/− + Ang II n=11). (B) Immunohistochemical renal localization of CCL5 in WT and Tet2−/− CH mice following Ang II infusion. Representative images of CCL5 immunohistochemistry staining (3,3′-Diaminobenzidine, brown) in kidney sections from WT and Tet2^−/− CH mice treated with vehicle or Ang II for 1 day (n=5). Arrows show positive staining on the proximal tubule (PT), distal tubule (DT) and renal corpuscle (RC). Scale bar 100 μm. (C) Experimental design for parts D-F. Flow cytometric analysis of the ratio of CD45.2 (donor) to CD45.1 (recipient) for (D) kidney CD45⁺, CD11b⁺ myeloid cells, and (E) F4/80⁺ CD11b⁺ Ly6G⁻ renal macrophages after administration of met-CCL5 (25μg per mouse) or vehicle to Tet2^−/− CH mice infused with Ang II for 1 day. (F) Systolic blood pressure measured by tail-cuff after administration of met-CCL5 or vehicle in Tet2^−/− CH mice treated with Ang II for 1 day. Statistical analysis was performed with 2-way ANOVA with Tukey’s multiple-comparisons test for A or non-parametric 2-tailed Mann-Whitney U test for panels D, E, and F (all groups n=4).

Figure 4.. Ang II infusion promotes renal NLRP3 expression, activation, and release of IL-1β during

Tet2-mediated CH. Protein expression of (A) NLRP3 (WT n=4, Tet2^−/− n=4, WT+ Ang II n=5, and Tet2^−/− + Ang II n=5), (B) pro-caspase-1 (WT n=6, Tet2 n=5, WT+ Ang II n=7, and Tet2^−/− + Ang II n=11), (C) p20-caspase-1 (WT n=6, Tet2^−/− n=6, WT+ Ang II n=6, and Tet2^−/− + Ang II n=10) by quantified Western Blot in kidney homogenates from WT and Tet2^−/− CH mice infused with either vehicle or Ang II for 1 day. (D) IL-1 β protein expression quantified by Western blot (WT n=8, Tet2^−/− n=8, WT+ Ang II n=6, and Tet2^−/− + Ang II n=12) and (E) transcript expression of pro-IL-1β (WT n=8, Tet2^−/− n=10, WT+ Ang II n=8, and Tet2^−/− + Ang II n=10) by RT-PCR in kidney homogenates from WT and Tet2^−/− CH mice infused with either vehicle or Ang II for 1 day. (F) Protein quantification of IL-1β in serum from WT or Tet2^−/− mediated CH mice infused with vehicle or Ang II for 1 day (WT n=7, Tet2^−/− n=6, WT+ Ang II n=5, and Tet2^−/− + Ang II n=7). For Western blot analysis, β-actin or Na⁺,K⁺-ATPase α1 were measured to verify uniform protein loading. Quantification of target proteins is expressed as relative abundance ratio between the target and housekeeping protein (Na⁺ATPase or β-actin) with expression of the mean WT vehicle group expressed as 1.0. Representative blots are displayed and uncropped blots are displayed in Supplemental Material. Data are expressed as mean ± SEM. 2-way ANOVA with Tukey’s multiple-comparisons tests statistical analysis were performed for C and E, or non-parametric Kruskal-Wallis followed by Dunn’s multiple comparison tests for A, B, D, and F.

Figure 5.. Sodium homeostasis is perturbed in the Tet2⁻mediated CH model following Ang II infusion.

Mice were housed in metabolic cages to record (A) urinary Na⁺ excretion expressed as μmol/24 hr/g body weight (WT n=5, Tet2^−/− n=9, WT+ Ang II n=5, and Tet2^−/− + Ang II n=14), and (B) changes in urine volume in WT and Tet2^−/− mediated CH mice following infusion with either vehicle or Ang II for 1 day (WT+ Ang II n=4, and Tet2^−/− + Ang II n=6). Renal mRNA expression of (C) sodium phosphate cotransporter 2 (NaPi₂) (WT n=8, Tet2^−/− n=10, WT+ Ang II n=8, and Tet2^−/− + Ang II n=5), and sodium-hydrogen exchanger 3 (NHE3) (WT n=6, Tet2^−/− n=7, WT+ Ang II n=9, and Tet2^−/− + Ang II n=6) in WT and Tet2^−/− mediated CH mice following infusion with either vehicle or Ang II for 1 day. Immunoblotting and quantification of (D) total NCC and NCC phosphorylated at Threonine 53 (WT n=7, Tet2^−/− n=7, WT+ Ang II n=7, and Tet2^−/− + Ang II n=9), and (E) total NKCC2 and NKCC2 phosphorylated at Threonine 105 (WT n=7, Tet2^−/− n=5, WT+ Ang II n=5, and Tet2^−/− + Ang II n=8) in the kidneys of WT and Tet2^−/− mediated CH mice infused with either vehicle or Ang II for 1 day. Changes in urine excretion were recorded over the entire 24-hour period following Ang II infusion and are expressed as the difference (D) in urine excretion in the 24 hours before and 24 hours after Ang II infusion. Quantification of phosphorylated proteins are expressed as relative abundance ratio between total and phosphorylated protein (n=5–9) with expression of the mean WT vehicle group expressed as 1.0. Representative blots are displayed and uncropped blots are displayed in Supplemental Material. Data are expressed as mean ± SEM. P<0.05 vs other group. Statistical analyses were performed with non-parametric Kruskal-Wallis followed by Dunn’s multiple comparison tests for A, C (NaPi₂), and E, non-parametric 2-tailed Mann-Whitney U test for B, or 2-way ANOVA with Tukey’s multiple-comparisons tests for C (NHE3), and D.

Figure 6.. The inflammasome inhibitor, MCC950, blunts hypertension, sodium retention, sodium transporter upregulation and activation in the Tet2-mediated CH mouse model following Ang II infusion.

Effects of MCC950 on (A) SBP measured by the tail-cuff method (all groups n=8 except WT+Ang II n=6), (B) urinary Na⁺ excretion expressed in micromolar Na⁺(mM) (WT n=12, Tet2^−/− n=16, WT+MCC950 n= 8, Tet2^−/−+ MCC950 n=8, WT+ Ang II n=8,Tet2^−/− + Ang II n=10, WT+Ang II+ MCC950 n=8, and Tet2^−/−+Ang II+MCC950 n=6), and (C) changes in urine volume (WT+MCC950 n=8, Tet2^−/−+ MCC950 n=8, WT+ Ang II n=8,Tet2^−/− + Ang II n=11, WT+Ang II+ MCC950 n=8, and Tet2^−/−+Ang II+MCC950 n=7) in the WT and Tet2^−/− mediated CH mouse model following infusion with either vehicle or Ang II. The effects of MCC950 on renal mRNA expression of (D) NaPi₂ (WT+ Ang II n=13,Tet2^−/− + Ang II n=8, WT+Ang II+ MCC950 n=4, and Tet2^−/−+Ang II+MCC950 n=6), and (E) NHE3 (WT+ Ang II n=14,Tet2^−/− + Ang II n=12, WT+Ang II+ MCC950 n=5, and Tet2^−/−+Ang II+MCC950 n=6) in the WT and Tet2^−/− mediated CH mouse model following infusion with Ang II. (F, H) Representative immunoblotting and quantification (WT+Ang II+ MCC950 n=7, and Tet2^−/−+Ang II+MCC950 n=8 ) of protein for total NCC and NCC phosphorylated at Thr 53, and (G, I) NKCC2 and NKCC2 phosphorylated at Thr 105 (WT+Ang II+ MCC950 n=5, and Tet2^−/−+Ang II+MCC950 n=8 ) within the kidneys of the WT and Tet2^−/− mediated CH mouse model treated with Ang II ± MCC950 for 1 day. Quantification of immunoblots are expressed as relative abundance ratio between total and phosphorylated protein with expression of the mean WT group expressed as 1.0. Representative blots are displayed. Statistical analysis was performed with 2-way ANOVA with Tukey’s multiple-comparisons tests for A, B, C, 2-tailed unpaired Student t test for H, non-parametric Kruskal-Wallis followed by Dunn’s multiple comparison tests for D, E, and non-parametric 2-tailed Mann-Whitney U test for I.