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 (CVD). 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 CVD. It is not known whether CH and hypertension in the elderly are causally related and, if so, what are the mechanistic features.

Methods and Results:

A murine model of adoptive bone marrow transplantation was employed to examine the interplay between Tet2 CH and hypertension. In this model, a subpressor dose of angiotensin II (Ang 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 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 and NKCC2 activation at residues Thr53 and Ser105, respectively. Administration of the NLRP3 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.

Graphical Abstract

INTRODUCTION

Hypertension is difficult to treat due to its heterogeneous etiology and complex mechanistic basis. The complications of uncontrolled hypertension, including stroke, heart failure, and kidney disease, are associated with substantial morbidity and mortality.¹ It is widely appreciated that systolic blood pressure levels rise steadily and continuously with age in both men and women. According to the US National Health and Nutrition Examination Survey (NHANES), 70% of elderly adults (≥65 years) have hypertension, and this is expected to increase by 20% by 2050.^2–5 Though hypertension can be secondary to other pathologies, a majority of patients have primary hypertension which lacks a definitive cause. As such, further mechanistic work is warranted to better understand this heterogenous and complex disease.

A growing body of evidence suggests that chronic inflammation is one of the leading causes of hypertension in the aged population.^{1, 6–11} Clinical evidence has shown that elevations in nonspecific markers of inflammation and oxidative injury in plasma and urine from middle-aged, are found in hypertensive patients.^{12, 13} Common secondary causes of hypertension, such as obstructive sleep apnea, chronic kidney disease, and renal artery stenosis, are all associated with inflammation and are highly prevalent in the elderly.¹⁴ Experimental studies in murine models have revealed that the ablation of myeloid cells abrogates the rise of blood pressure in response to a pressor dose of Ang II.¹⁵ Furthermore, Zhang et al. have demonstrated that proinflammatory Ly6C^hi monocytes and macrophages infiltrating the kidneys promote sodium reabsorption via activation of the Na⁺-K⁺-Cl⁻ (NKCC2) cotransporter in the nephron through an IL-1 receptor-mediated mechanism.¹⁶ Despite these advances, there is an incomplete understanding of how aging, inflammation and the pathophysiology of hypertension are linked.

Recent studies have revealed the prevalent occurrence of somatic mutations in hematopoietic stem and progenitor cells (HSPC) that give rise to clonal expansions that can be detected in progeny leukocytes.¹⁷ This condition, referred to as age-related clonal hematopoiesis (CH), or CH of indeterminate potential (CHIP), can result from mutations in “driver genes”, including ten eleven translocation methylcytosine dioxygenase (TET2), that are recurrently mutated in hematologic malignancies and confer a competitive advantage to the HSPC. CH is a precancerous state that increases with age, and this condition is associated with morbidity and mortality.¹⁷ Numerous experimental studies indicate that CH can causally contribute to multiple disease processes, typically thorough promotion of proinflammatory processes.¹⁸

Epidemiological studies indicate a complex interplay between CH and several of the traditional cardiovascular disease risk factors. For example, smoking, obesity and unhealthy lifestyle are positively associated with an increased incidence of CH.^19–22 It has also been reported that there is an association between CH and diabetes,^{23, 24} and studies in mice have shown that Tet2-mediated CH can promote insulin resistance.²⁵ A number of epidemiological studies indicate a potential association of hypertension with CH.^26–30 However, issues of causality and directionality are difficult to discern from epidemiological studies, and hypertension could either be a common comorbidity in many of these conditions or independent epiphenomena of the biological aging process. Thus, the current study was designed to test whether a causal relationship exists between CH and hypertension and to define the mechanistic connections between these two risk factors.

METHODS

Data availability

The detailed methods are available in the Data Supplement. Also see the Major Resources Table in the Data Supplement. The supporting data are available from the corresponding author upon reasonable request.

Animals

Wild-type mice (Tet2^+/+), Tet2-deficient mice (Tet2^−/−: B6(Cg)-Tet2tm1.2Rao/J), and Pep Boy mice (B6.SJL-Ptprca Pepcb/BoyJ) were sourced from Jackson Laboratory (Stock #: 000664, 023359, 002014, respectively). Tet2^−/− and Tet2^+/+ mice were used for the test group (Tet2^−/− mediated CH) and the wild-type group, respectively. Mice with myeloid-restricted Tet2 ablation were generated by crossing Tet2-floxed mice (Tet2^fl/fl, stock 017573) with Lyz2-Cre mice (stock 004781). All mice are in a C57BL/6J genetic background. Male mice were used for the in vivo experiments. Mice were housed in a specific pathogen–free animal facility and given food and water ad libitum on a 12-hour light/12-hour dark schedule. Experimental mice were randomly assigned to vehicle or Ang II groups. The institutional Animal Care and Use Committee at the University of Virginia approved the study protocol.

Bone marrow cell isolation

Approximately 100 million unfractionated bone marrow cells were obtained from Tet2^+/+ and Tet2^−/− donor mice. Six bones were collected from a single donor mouse of each genotype (two femurs, two tibias, and two humeri). Isolated bones were centrifuged at 10,000 × g for 1 minute and 30 s at 4 °C to collect the bone marrow. After centrifugation, the bone marrow pellets were resuspended in 1 mL of ice-cold PBS and then transferred to a 50 mL conical tube by passing them through a 70 μm cell strainer. The bone marrow cell suspensions were centrifuged at 500 × g for 6 min at 4 °C. The supernatants were resuspended with serum-free RPMI media, counted and 5 million cells were injected into each recipient mouse (as below).

Bone marrow transplantation in nonconditioned mice

A non-myeloablative bone marrow transplantation was employed.^31–33 Briefly, 6- to 8-week-old Pep Boy mice were transplanted with suspensions of bone marrow cells (as described above) from either Tet2^+/+ mice or Tet2^−/− mice for the WT and Tet2^−/− groups, respectively. Unfractionated bone marrow cells (5 × 10⁶ cells per day) were injected via retro-orbital vein into non-irradiated recipients over 3 consecutive days (total: 1.5 × 10⁷ cells).

Blood pressure recordings by radiotelemetry

Radio telemeters for blood pressure monitoring were implanted at 6–8 weeks of age. For this, mice were anesthetized with isoflurane, and a catheter connected to a radio telemetry device (model PA-C10, Data Sciences International, St. Paul, MN) was inserted into the right carotid artery for blood pressure evaluation in conscious animals. After a 7-day recovery phase, baseline blood pressure levels were recorded and at one, two and three months after adoptive transfer. After 3.5 months, Ang II was infused at 200 ng/kg/min with implanted osmotic minipumps and systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) were recorded continuously in dark phase (6 PM to 6 AM) and light phase (6 AM to 6 PM) by radiotelemetry.

Interventions

At the 3.5 month time point post-adoptive transplant, a small subcutaneous pocket was made through an incision at the nape neck and osmotic minipumps (Alzet model 1002; Cupertino, CA) containing either a subpressor dose of angiotensin II (Ang II; 200 ng/kg/min, diluted in sterile saline, Sigma-Aldrich) or saline (vehicle) were implanted into the pocket. Mice were anesthetized with isoflurane during the entire surgical procedure and the wounds were closed with wound clips. Osmotic minipumps were primed in PBS at 37°C for 24 hours before implantation, and they remained in place for 10 days (radio telemetry studies) post-implantation. In this model of low-dose Ang II, the slow pressor response leads to renal hypertension characterized by no elevation of SBP in conscious mice by day 6, but an elevation in blood pressure appears by days 10 to 13.³⁴ For the one-day Ang II experiments, osmotic minipumps (Alzet model 1002; Cupertino, CA) containing Ang II (200ng/kg/min) pumps were implanted at 3.5 months post-non-myeloablative bone marrow transplantation. For the inflammasome inhibition experiment, osmotic minipumps (Alzet model 1002; Cupertino, CA) containing MCC950 (10 mg/kg/day, diluted in sterile PBS, Cat# S7809, Selleck Chemicals) or PBS,³⁵ were implanted 4 days before infusion of Ang II (200ng/kg/min). For met-CCL5 studies, recombinant human met-CCL5 (25μg per mouse i.p., #335-RM, R&D systems) or vehicle³⁶ was administered 6 hours prior to Ang II infusion. The surgical interventions were performed by an investigator blinded to the genotype (WT or Tet2^−/−) of the animals.

Blood pressure recordings by tail-cuff

Blood pressure was recorded by tail-cuff using the CODA^® High Throughput System Noninvasive Blood Pressure System (Kent Scientific, CT). Briefly, values were obtained from conscious-trained mice on six consecutive morning sessions by the same research assistant. Mice were placed on a heating platform at 33°C in an acrylic restrainer, and a tail-cuff with a pulse sensor was placed along the tail. Results were calculated as the mean from the valid values of 25 measurements in each session and expressed in mmHg. Based on our experience, although tail-cuff measurements do not provide nighttime blood pressure, 24-hour average mean arterial pressure, or DBP, when mice are appropriately trained, tail-cuff readings are very reproducible and provide a reliable and cost-effective approach to assess SBP.

Statistical analyses

All statistical analyses were performed with GraphPad Prism 10 (GraphPad Software Inc., San Diego, CA). The Shapiro-Wilk test was used to evaluate the normality distribution in all cases with a sample size of 6 or greater. For normally distributed data with one experimental variable, statistical analyses were performed by parametric analysis: unpaired (2-tailed) Student t test for 2 groups (Fig 6H). The data with 2 independent variables were evaluated by 2-way ANOVA with post hoc Tukey multiple comparison tests (Fig 1B, 2A, 2B, 3A, 4C, 4E, 5C–NHE3, 5D, 6A, 6B, 6C, SF1A, SF1B, SF1D, SF1E, SF2C, SF2D, SF4, SF9, SF11, SF12A, and SF14 A–C). For these tests, data are presented as mean ± SEM. For those experiments having a sample size of 5 or less, non-parametric analyses were employed: Mann-Whitney U test for two groups (Fig 3D–E, 5B, 6I, SF12B) or a Kruskal-Wallis test followed by a Dunn’s multiple comparison tests for 3 groups or more (Fig 2C, 2E, 3F, 4A, 4B, 4D, 4F, 5A, 5C–Napi₂, 5E, 6D–E, SF1C, SF1E (Lym and Mono), SF2A–B, SF2E, SF5B, SF6A–D, SF7B, and SF8D). For telemetry blood pressure measurements over time, 2-way ANOVA with repeated measures was used (Fig 1C). For SF3, the Friedman test followed by Dunn’s post-test for non-parametric distribution was used. For all results, data are presented as mean ± SEM. Mice were only excluded in the case of an identified human error during the experiment. Specifically, two mice were excluded due to the creation of an open suture at the osmotic pump wound. While a priori power analyses were not performed, estimates of sample sizes to obtain statistically significant results were made based on our previous experimental findings with these models^31–33.

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.

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.

RESULTS

A mouse model of Tet2 clonal hematopoiesis enhances the blood pressure response to Ang II

To avoid the confounding effects of myeloablation, an experimental system of clonal hematopoiesis was established by employing the adoptive transfer of bone marrow to mice (Figure 1A). In this model, CD45.2 antigen-positive cells, either wild-type or Tet2-deficient, were transplanted into nonconditioned CD45.1 recipient mice. Consistent with prior uses of this Tet2-CH model,^{25, 31–33, 37} the delivery of bone marrow from Tet2-deficient mice to non-conditioned mice led to the selective significant expansion of the CD45.2 cell fraction in white blood cells, as assessed by flow cytometry, attaining a level of 40% chimerism in white blood cells (WBC) over a 3.5 month period (Figure 1B), that is consistent with the variant allelic fraction for TET2 mutations detected in some individuals by conventional next generation sequencing.¹⁷ Notably, the expansion of Tet2-deficient cells did not significantly affect total WBC number (Figure 1B), consistent with the clinical paradigm of clonal hematopoiesis.

The analysis of the CD45.2 allelic fraction was also evaluated in the different populations of blood leukocytes and bone marrow populations of progenitor cells. In the peripheral blood leukocytes, the condition of hematopoietic Tet2-deficiency was associated with a significant time-dependent elevation of chimerism in monocytes, neutrophils, B cells and T cells to varying degrees (Figure S1A–D), as noted previously,^{33, 35, 37} but there was no significant effect on the total numbers of these cells (Figure S1E). To evaluate the expansion of Tet2-deficient HSPC, bone marrow cells were examined by flow cytometry (Figure S2). Using the immunophenotypic definitions of murine multipotent progenitor (MPP) cells elaborated by Challen et al.,³⁸ Tet2-deficiency was found to elevate chimerism in hematopoietic stem cells (HSC) (Flk2^–/CD48^–/CD150⁺/Lin⁻/Sca1⁺/Kit⁺, LSK (Lin⁻/ c-Kit⁺/ Sca-1⁺), MPP (CD150⁻/CD48⁻/Flk2⁻/ Sca1⁺/Kit⁺/Lin⁻), MPP^G/M (CD150⁻/Cd48⁺/Flk2⁻/ Sca1⁺/Kit⁺/Lin⁻), and MPP^LY (CD150⁻/CD48⁺/Flk2⁺/ Sca1⁺/Kit⁺/Lin⁻) fractions (Figure S2A–E).

Under baseline conditions, 1 week before, and at 4, 9, 11, and 14 weeks after adoptive transfer, mice transplanted with WT or Tet2^−/− bone marrow displayed similar basal SBP, DBP, MAP and heart rate as assessed by radiotelemetry (Figure S3A–D). At the 14-week post-adoptive transfer timepoint, mice were challenged with a subpressor dose of Ang II (200ng/kg/min). While baseline blood pressure and heart rate levels were similar before Ang II pump implantation, the Tet2-CH model showed a significant increase in SBP of 21 and 17 mmHg at light and dark phases, respectively, on day 1 compared to the WT group and 18 and 21 mmHg through day 9, following the challenge with the subpressor dose of Ang II (Figure 1C). Similarly, the Tet2-CH mouse model displayed significantly increased DBP (20 and 22 mmHg at dark and light phases, respectively, on day 1, and 17 and 20 mmHg at day 9) compared to the WT group after Ang II infusion (Figure 1C). There were no significant differences in heart rate between the two groups following Ang II infusion (Figure 1C).

A subpressor dose of Ang II increases Tet2-mutant proinflammatory Ly6C^hi monocyte expansion

Further experiments explored the impact of low-dose Ang II administration on the expansion of Tet2-deficient hematopoietic cells. At the one-day timepoint of Ang II infusion, a significant expansion of Tet2 chimerism was observed in the proinflammatory Ly6C^hi monocytes (defined as CD45⁺CD115⁺Ly6C^hi) in peripheral blood compared to mice that received wild-type bone marrow or the Tet2-CH experimental group treated with vehicle (Figure 2A). In contrast, the chimerism of Tet2-deficient cells showed trends of an increase but were not significantly altered in Tet2^−/− monocytes (total), neutrophils, and B cells populations (Figure S1A–D) after 1 day of Ang II-treatment. Consistent with the clonal hematopoiesis paradigm, the total numbers of white blood cells, lymphocytes, or monocytes did not display statistically significant changes resulting from the Tet2-CH model condition after the Ang II challenge (Figure S1E).

To evaluate potential sources Tet2-deficient hematopoietic cells that contribute to the Ang II-stimulated expansion of circulating Ly6C^hi monocytes, flow cytometric analyses were performed on the bone marrow and spleen. At the 1 day time point, the subpressor dose of Ang II was found to selectively expand Tet2-deficient bone marrow populations of MPP Granulocytes/Monocytes (MPP^G/M) and LSK cells (Figure S2A,B), which give rise to the myeloid population. These data are consistent with the prior observation that Tet2-deficient hematopoietic cells display a competitive growth advantage and a myeloid bias in the adoptive transfer model.³³ Nonsignificant trends of Tet2-deficient cell expansion were also observed in the HSC, MPP and MPP-^LY populations in the bone marrow after Ang II administration (Figure S2C–E). Furthermore, analysis of the spleen by flow cytometry revealed a population of Tet2-deficient Ly6C^hi monocytes under baseline conditions that were rapidly reduced after Ang II infusion (Figure S4), consistent with their rapid release to circulation.³⁹

Macrophages infiltrate the kidney and have an essential role in the hypertensive response to Ang II Infusion

As the renal system plays a critical role in blood pressure regulation, immune cell infiltration of the kidney was assessed by flow cytometry at 1 day after Ang II infusion. At this early timepoint, there was a significant increase in the absolute numbers of Tet2-deficient kidney macrophages (defined as CD45.2⁺, CD11b⁺, F4/80^hi) compared with the wild-type or vehicle conditions (Figure 2B, Figure S5A). When macrophages (F4/80^hi, CD45.2⁺) were gated for expression of the C-C motif chemokine receptor 2 (CCR2), it was revealed that the CCR2-positive and negative populations displayed marked elevation in the kidney in the Tet2-CH model in response to Ang II stimulation compared with the control groups (Figure 2C, Figure S5B). Collectively, these data indicate that experimental Tet2-CH leads to increased infiltration of Tet2-deficient monocytes and activation of the innate immune system.

To further examine the role of myeloid cells in the development of hypertension, myeloid-specific Tet2-deficient mice (Lyz2^Cre/⁺Tet2^fl/fl) were infused with a subpressor dose of Ang II or vehicle for 1 day (Figure 2D). SBP was significatively higher in the myeloid, Tet2 knockout mice compared to Lyz2^+/+Tet2^fl/fl control mice after challenge with Ang II (Figure 2E). Thus, Tet2-deficiency in myeloid cells plays a key role in the early onset hypertension in this model.

Kidney chemokine expression and role of CCL5 in clonal hematopoiesis-promoted hypertension

A diverse set of chemokines coordinate the movement of immune cells from the circulation into tissues.⁴⁰ Thus, an examination of how these trafficking molecules recruit inflammatory cells into the kidney in the Tet2-CH model was warranted. A qPCR analysis of various chemokines in the kidney was performed. Kidneys from the Tet2-CH model exhibited a significantly increased CCL5 expression after 1 day of Ang II administration compared to the WT group (Figure 3A). In contrast, no statistically significant changes in the expression of CCL2, CCL7, CCL8 or CX3CR1 transcripts were observed (Figure S6A–D). Thus, immunohistochemistry was conducted on kidney sections to investigate the source of CCL5. These findings indicate that cells in the proximal (PT) and distal tubules (DT) and renal corpuscle (RC) display significantly increased CCL5 expression in the Tet2^−/− group treated with Ang II (Figure 3B). To determine whether IL-1β, a cytokine elevated in Tet2^−/− myeloid cells,³⁵ could induce CCL5 expression, the SV40 mesangial cell-1 line was treated with this cytokine. IL-1β was sufficient to induce a significant 5.7-fold increase of CCL5, while the expression of CCL5 was not altered by Ang II stimulation (Figure S7A). Flow cytometry analysis did not detect a significant increase in renal T-cell infiltration in mice in any experimental group (Figure S7B). These results suggest that IL-1β, likely released by circulating Tet2^−/− monocytes, leads to an increase in CCL5 expression in the kidney, and this could mediate Tet2^−/− monocyte infiltration under these conditions.

To explore the pathophysiological link between CCL5 mediated by renal monocyte infiltration and hypertension in the Tet2-CH model, mice were treated with Ang II in the presence or absence of met-CCL5, a CCL5 receptor antagonist (Figure 3C). Co-treatment with met-CCL5 significantly decreased the degree of donor-derived Tet2^−/− myeloid cell infiltration (CD45.2⁺,CD11b⁺) and reduced the macrophage content in kidney of the Tet2-CH model treated with Ang II (Figure 3D,E). Furthermore, the CCL5 receptor antagonism significantly blunted the increased SBP found in the Tet2-CH/Ang II group (Figure 3F). These data indicate the prominent role of CCL5-mediated immune cell infiltration in the pathogenesis of CH-induced hypertension.

Kidney inflammasome activation in the Tet2-clonal hematopoiesis model

Previous studies have shown that Tet2-deficiency in the hematopoietic system leads to macrophage upregulation of the NLRP3 inflammasome, inducing associated inflammatory pathways in the cardiovascular system.^{35, 41} As indicated above, Tet2-deficient myeloid cells have significantly higher expression of IL-1β, and concordantly elevated IL-1β is been detected in individuals with TET2 clonal hematopoiesis.⁴² When activated, the subunits of the NLRP3 inflammasome oligomerize, resulting in cleavage and activation of caspase-1 and cleavage of pro-IL-1β and pro-IL-18 into their active forms.^{43, 44} Consistently, caspase 1 activity was significantly elevated in Tet2-deficient bone marrow-derived macrophages (BMDM) (Figure S8A), whereas the addition of Ang II did not further increase caspase 1 activity (Figure S8B).

No studies have examined NLRP3 involvement downstream of CH in the kidney. Western immunoblot analysis of the kidney revealed that NLRP3 expression was significantly upregulated 1.7-fold compared with WT after 1 day of Ang II infusion in the Tet2-CH model, but Ang II had a lesser effect on control mice (Figure 4A). Western immunoblot analysis of kidney also showed that the full-length and the p20 proteolytically active subunit of caspase-1 were increased in the Tet2-CH model (~2.1-fold; and ~1.8-fold, respectively) compared to control after treatment with the subpressor dose of Ang II (Figure 4B,C). Consistent with the NLRP3 induction and caspase-1 activation, Ang II-treatment significantly increased levels of the pro-IL-1β mRNA and cleaved IL-1β (2.2 and ~1.4-fold, respectively; Figure 4D,E) in the Tet2-CH model. Elevation of cleaved IL-18 (~1.5-fold) was also detected in the kidneys of the Tet2-CH mice after Ang II infusion (Figure S9). Consistent with these protein and transcript data, serum IL-1β levels, measured by ELISA, were found to be increased after 1 day of Ang II infusion in the Tet2-CH model compared with control mice (Figure 4F).

After inflammasome activation, the adapter protein apoptosis-associated speck-like protein containing a CARD (ASC) assembles into a large protein complex “ASC speck.” To further elucidate the pathological contribution of Tet2^−/− macrophages, immunofluorescence staining was performed to analyze ASC speck formation and IL-1β co-localization following stimulation with Ang II. Compared with WT, there was an increase in intrinsic ASC speck formation and IL-1β co-localization in the Tet2-deficient BMDMs (Figure S10), suggesting that Tet2^−/− macrophages have elevated inflammasome activity and consequential IL-1β production following stimulation with Ang II. Collectively, these findings support the notion that following administration of Ang II, Tet2-deficient myeloid cells release IL-1β, which in turn induces renal CCL5 expression. CCL5 released from the kidney consequently recruits Tet2^−/− pro-inflammatory monocytes, and upon infiltration, these cells amplify IL-1β levels within the kidney.

Anti-natriuretic responses in the Tet2-clonal hematopoiesis model

It has been shown that the IL-1 receptor mediates sodium excretion in an Ang II model of hypertension.¹⁶ Thus, to determine whether the pro-hypertensive effects seen in the Tet2-CH model were related to renal sodium retention, test and control mice with metabolic cages were analyzed to quantify daily sodium excretion and urine volume. As expected, urinary sodium excretion and sodium intake in food were indistinguishable in the control and the Tet2-CH groups prior to the infusion of Ang II (Figure 5A, Figure S11). However, within 1 day of Ang II infusion, the Tet2-CH model displayed significantly decreases in urinary sodium excretion and urine volume while maintaining the same sodium intake in food (Figure 5A,B and Figure S11). To corroborate and extend these findings, we tested whether the myeloid-specific Tet2-deficient mice exhibited a similar anti-natriuretic response as the pan-hematopoietic Tet2-CH mouse model. Under these experimental conditions, deficiency of Tet2 in myeloid cells led to a significant decrease in sodium excretion and urine volume after Ang II infusion, demonstrating that the anti-natriuretic response is mediated, at least in part, by myeloid cells (Figure S12A,B).

Because the Tet2-CH mouse model excretes less sodium than control mice in response to the subpressor dose of Ang II, we tested whether the renal transporters involved in sodium retention are dysregulated under these conditions. Previous studies have shown that changes in Na⁺ transporter expression and their phosphorylation status are associated with the natriuretic response.⁴⁵ Sodium hydrogen exchanger isoform 3 (NHE3) is the primary transporter responsible for sodium reabsorption in the proximal tubule,⁴⁶ and studies conducted on cultured kidney cells indicate that Ang II can rapidly increase NHE3 abundance and activity in the plasma membrane.⁴⁷ NHE3 and Na⁺/Pi cotransporter 2 (NaPi₂) are coordinately regulated in vivo by many stimuli including the acute and prolonged response to Ang II,^48–50 parathyroid hormone,⁵¹ high-salt diet,⁵² and in the spontaneously hypertensive rat.⁵³ Correspondingly, in the Tet2-CH model, the proximal tubule transporters NaPi₂ and NHE3 were transcriptionally upregulated (1.7-fold and 1.6-fold, respectively; Figure 5C,D) and expressed as protein (Figure S13) after one day of Ang II infusion compared with the WT group treated with Ang II. In contrast, no significant changes were found in the transcript expression of the α, β, and g subunits of the epithelial sodium channel (ENaC) (Figure S14A).

In addition to changes in NHE3 and NaPi2 transcript levels that are associated with regulation of the natriuretic response,⁴⁵ the NKCC2 cotransporter and the Na⁺-Cl⁻ cotransporter (NCC) regulate the retention of sodium by acting in the medullary and cortical loop of Henle.⁵⁴ No significant differences were detected in NKCC2 or NCC transcript expression comparing control and Tet2-CH mice after Ang II administration (Figure S14 B,C). However, these cotransporters are regulated by phosphorylation leading to their activation and the retention of sodium.^55–58 Therefore, western immunoblot analyses of NCC and NKCC2 were performed to assess their phosphorylation status. When exposed to the subpressor dose of Ang II, the Tet2-CH group displayed significantly increased NCC phosphorylation at threonine residue 53 (NCCpT53) (Figure 5D) and NKCC2 phosphorylation at threonine residue 105 (NKCC2pT105), consistent with the observed renal sodium retention under these conditions, whereas the control group was unchanged (Figure 5E). To further document that inflammatory cytokines promote NCC phosphorylation, the 209/MDCT cell line, derived from renal distal convoluted tubule cells,⁵⁹ were stimulated with IL-1β or IL-18, resulting in an increase in NCC phosphorylation at Thr53 after 1 hour of IL-1β stimulation and 3 hours after IL-18 induction (Figure S15). However, NKCC2 was undetectable in this cell line.

NLRP3 inhibition reverses hypertension in the Tet2-CH model

Collectively, the results suggest that the Tet2-CH model is sensitized to the regulation of Na⁺ transporters by Ang II, thereby leading to the rapid retention of sodium. The results also show kidney inflammasome activation under these conditions. Thus, to directly test whether NLRP3 activation contributes to the sensitization of mice to hypertensive stimuli, the effects of intervening with the NLRP3 inflammasome inhibitor MCC950 were evaluated. Consistent with the radiotelemetry data, the infusion of Ang II led to a significant elevation of SBP in the Tet2-CH model at the 1-day time point, as assessed by tail cuff analysis (Figure 6A). Co-administration of MCC950 significantly abrogated this increase in blood pressure. Treatment with MCC950 also returned sodium excretion and urine volume to baseline levels in the Tet2-CH condition (Figure 6B,C). Furthermore, treatment with MCC950 significantly reversed the Tet2-mediated increase in renal expression of NaPi2 and NHE3 transcript levels (Figure 6D,E) and blunted the activating phosphorylation of NKCC2 and NCC (Figure 6F–I). Collectively, these results suggest that the sensitization to hypertension observed in the Tet2-CH model involves the overactivation of the NLRP3 inflammasome in the kidney and the activation of cotransporters that regulate sodium homeostasis.

DISCUSSION

Hypertension represents one of the most prevalent and potentially modifiable risk factors for CVD. Notably, older adults account for the bulk of hypertension-related morbidity and mortality, and this is mainly due to the dramatically greater prevalence of hypertension among the elderly.⁶ As individuals age, they also increasingly exhibit the condition of CH, that has been causally linked to atherosclerotic cardiovascular disease^{28, 35} and heart failure.^{37, 41} The fact that CH and hypertension share risk factors, and frequently co-occur,^26–30 suggests that overlapping pathophysiological mechanisms play prominent roles in both conditions. While some epidemiological data indicate an association between CH and hypertension in the elderly population,^26–30 the hypertension observed in these studies could reflect a consequence of advanced age or other CVD conditions. Furthermore, mechanistic features that potentially link CH and hypertension have not been evaluated.

To elucidate the causal and directional nature of the relationships between CH and hypertension, a nonmyeloablative bone marrow transplantation model was employed that mimics features of native hematopoiesis and avoids the confounding effects induced by irradiation on the hematopoietic niche, cardiovascular tissues, and kidney.^31–37 In this model, adoptively transferred Tet2-deficient HSPCs underwent a selective expansion that is reflected by the time-dependent increase of the mutant allele in peripheral leukocytes. While the Tet2-CH mouse model displayed no detectable phenotype at baseline, they were hypersensitive to stimulation with Ang II. In response to a subpressor dose of Ang II, these mice showed marked elevations in SBP and DBP at the 1-day timepoint that was not observed in control mice. In contrast, control mice displayed a gradual increase in blood pressure during a 10-day time course, as reported previously.³⁴ Consistent with a renal hypertension mechanism, the Tet2-CH condition led to the rapid and selective infiltration of Tet2-deficient macrophages into the kidney and a corresponding anti-natriuretic response. Thus, while many studies have shown that CH promotes CVD risk independently of traditional risk factors,¹⁷ the data presented herein raise the possibility that CH can also contribute to cardiovascular pathology by promoting the risk from hypertension. This relationship may be of particular significance in the elderly because CVD risk from hypertension increases with age while the risk from other factors, such as hyperlipidemia and smoking decline.⁶⁰

While these data provide evidence that age-related CH can promote Ang II-induced hypertension, this study also revealed that Ang II can also promote the expansion of Tet-deficient hematopoietic cells as well as the release of proinflammatory Tet2^−/− monocytes from spleen. While Tet2-deficient hematopoietic cells underwent expansion more rapidly than Tet2-sufficient clones at baseline in the adoptive transfer model, treatment with a subpressor dose of Ang II significantly accelerated Tet2-deficient donor cell expansion in subpopulations of peripheral blood, hematopoietic stem, and progenitor cells. Specifically, Ang II accelerated the expansion of Tet2-deficient LSK and MPP^G/M cells in the bone marrow compartment that would contribute to the increase in proinflammatory Ly6C^hi monocytes in peripheral blood. Collectively, the selective expansion of Ly6C^hi monocytes, and their release from the spleen, likely contributes to the increased infiltration of Tet2-mutant macrophages in the kidney through increased CCL5 expression induced by systemic IL-1β. Thus, the associations between CH and hypertension observed in many epidemiological studies could be partly due to the stimulation of these mechanisms by hypertensive conditions that are prevalent in the elderly. Furthermore, in view of the independent disease-promoting effects of CH on the cardiovascular system, the stimulation of CH by hypertensive stimuli could serve as a positive feedback loop that exacerbates CVD in the elderly hypertensive patient.

Studies with rodent models have revealed that proinflammatory macrophages infiltrate the kidney in response to chemokine production,^{40, 61} and that proinflammatory CCR2⁺ macrophages have the capacity to induce hypertension by promoting kidney injury and impairing the excretion of sodium.^{62, 63} In the model of Tet2-CH, a low dose of Ang II stimulated the upregulation of CCL5 that was associated with recruitment of Tet2- macrophages into the kidney. Direct macrophage involvement in the hypertensive phenotype was indicated by the finding that homozygous Tet2-deficiency in myeloid cells are sufficient to confer Ang II-hypersensitivity in the Tet2-CH model. Furthermore, treatment with the CCL5 receptor antagonist met-CCL5 blocks myeloid cell infiltration into the kidney and abrogates the hypertensive effects caused by Tet2-mutant hematopoietic cells. Thus, elevated immune cell infiltration of the kidney by CCL5 is likely to be a key feature by which hematopoietic Tet2-deficient cells promotes a hypertensive state.

A growing body of experimental and epidemiological evidence suggests that activation of the NLRP3 inflammasome and elevated cytokine expression represents a common feature shared by many clonal hematopoiesis driver genes.^{17, 29} In response to stimuli, components of the NLRP3 inflammasome complex undergo ‘priming’, leading to the generation and release of IL-1β and IL-18 in their active forms.^{43, 44} Previous studies have shown that the NLRP3 inflammasome plays a pivotal role in the chronic inflammatory state that is present in hypertension.^{64, 65} The current study found that renal NLRP3 inflammasome activation was increased following one day of Ang II infusion in the Tet2-CH model and that Tet2-deficient macrophages are a potential source of these elevated NLRP3 inflammasome components. In particular, this was evidenced by increases in the protein levels of active caspase 1, ASC, and IL-1β in Tet2-deficient BMDM. To test the involvement of the NLRP3 inflammasome in the development of hypertension, the NLRP3 inflammasome inhibitor MCC950, that prevents the processing of IL-1β and IL-18 to their active forms,⁶⁶ was administered to the Tet2-CH and control mice. This treatment effectively reversed the Tet2-mediated increase in SBP but had no effect on baseline blood pressure in control mice treated with Ang II or in the mice that were not exposed to Ang II. These data suggest that inflammasome activation and the elevated cytokine production is critical for the enhanced sensitization to hypertension under conditions of CH.

In the current study, the Tet2-CH model displayed increased sodium retention during the development of hypertension. The kidney is an important organ that regulates blood pressure by controlling the extracellular fluid through renal transporters that reabsorb sodium to the circulation.⁴⁵ The NHE3 and NaPi2 transporters are primarily responsible for apical sodium transport in the proximal tubules,⁴⁵ whereas NKCC2 is responsible for sodium transport in thick ascending limb of the loop of Henle.⁴⁵ The distal nephron fine-tunes salt reabsorption through various transporters, including NCC.⁴⁵ In the Tet2-CH model, a subpressor dose of Ang II led to elevations NHE3 and NaPi2 transcript expression, and previous studies have implicated an increased abundance of these transporters in the Ang II model.⁴⁸ These conditions also led to an increase in the phosphorylation of NKCC2 and NCC.⁴⁸ These regulatory features can contribute to the increased sensitivity of the Tet2-CH model to hypertensive stimuli. For example, in the Dahl salt-sensitive rat model of hypertension, enhanced baseline NKCC2 phosphorylation precedes the development of hypertension,⁵⁵ and it has been reported that the administration of the calcineurin inhibitor, cyclosporine A, enhances NKCC2 and NCC phosphorylation, salt retention, and hypertension in Wistar rats.⁶⁷

The combined regulation of NHE3 and NaPi₂ at the transcript level and the increased activating phosphorylation of NKCC2 and NCC, could be contributing to the enhanced sensitivity to hypertensive stimuli in the Tet2-CH model. Consistent with this hypothesis, administration of the NLRP3 inflammasome inhibitor MCC950 was found to effectively reverse the anti-natriuretic effect, diminish NaPi2 and NHE3 transcript expression, and reverse the activating phosphorylation of NCC and NKCC2. Collectively, these data suggest that renal inflammasome activation and the resulting cytokine production is secondary to increased sensitivity of CH to hypertensive stimuli. Consistent with our findings, inflammatory cytokines, interferon-γ and IL-17A, have been shown to positively regulate NHE3 abundance and promote NKCC2 and NCC activation by phosphorylation in a model of Ang II-induced hypertension.⁶⁸ Furthermore, Zhang et al. demonstrated that genetic IL-1R1 deficiency or pharmacological blockade with Anakinra limits blood pressure elevation in response to Ang II infusion, and this effect was attributed in part to the modulation of sodium reabsorption via activation of NKCC2 via phosphorylation.¹⁶

We acknowledge that this study has certain limitations. First, while the data is consistent with the hypothesis that kidney transporters play a crucial role in the development of hypertension in the CH model, we cannot rule out the possibility that Tet2-mutant myeloid cells infiltrate other tissues, such as the brain or the mesenteric beds, and thereby also contribute to the hypertensive phenotype. Second, although our data supports the involvement of the proinflammatory monocytes/macrophages axis in the onset of hypertension, we cannot rule out the possibility that other immune cell populations contribute to hypertension under these conditions. However, it is worth noting that myeloid cells are the primary population that undergoes clonal expansion in the Tet2-CH model of hypertension and other models of CH-mediated diseases.^{17, 33} Furthermore, the current study is consistent with findings that have shown that Tet2-mutant macrophages are the primary source of IL1-β and IL-18 cytokines in different disease models.^{17, 35}

In summary, we provide evidence that the condition of Tet2-CH contributes to an inflammatory state in the kidney. As a result, the inflammatory environment created by CH reduces the threshold for the onset of hypertension triggered by a hypertensive stimulus. Furthermore, we show that the stimulus of low-dose Ang II will also promote the expansion of mutant Tet2 Ly6C^hi cells and stimulate their release from the spleen, likely contributing to the preferential infiltration of macrophages to the kidney. The hypertension-sensitizing effect of the Tet2-CH model, including renal inflammation and the anti-natriuretic phenotype, could be reversed by treatment with an inflammasome inhibitor. Notably, there is considerable interest in developing NLRP3 inflammasome inhibitors for the treatment of chronic disorders.⁶⁹ The current work suggests that inflammasome inhibitors may be preferentially effective for preventing the onset of hypertension in patients with CH. Further, we speculate that inflammasome inhibition may be particularly effective in salt sensitive hypertension due to the apparent renal contribution to these conditions.

Novelty and Significance.

What Is Known?

• The incidence of hypertension increases with age.

• Clonal hematopoiesis also increases with age, but the cause-and-effect relationship between clonal hematopoiesis and hypertension is unknown.

What New Information Does This Article Contribute?

• Tet2-inactivation in hematopoietic cells contributes to the development of hypertension.

• A subpressor dose of Ang II leads to the selective expansion of Tet2-deficiency in proinflammatory Ly6C^hi monocytes and progenitor populations that give rise to myeloid lineage cells.

• Experimental Tet2 clonal hematopoiesis promotes macrophage infiltration in the kidney through a CCL5-dependent mechanism.

• Activation of the NLRP3 inflammasome, facilitated by Tet2-clonal hematopoiesis, leads to the expression of intra-renal IL-1β and IL-18, sodium retention and hypertension in mice.

• These data indicate that anti-inflammatory therapies may have utility in treating hypertension in elderly clonal hematopoiesis carriers.

Our study examined the relationship between age-related clonal hematopoiesis (CH) and hypertension. A murine model of Tet2 CH developed elevated blood pressure when exposed to a subpressor dose of angiotensin II. This effect was due to increased kidney inflammation caused by the infiltration of proinflammatory Tet2-deficient monocytes. These cells elevated levels of the proinflammatory cytokine IL-1β, leading to increased sodium retention. The elevated sodium retention was mediated by modulation of specific kidney transporters, including NaPi2, NHE3, NCC, and NKCC2. This study also showed that an NLRP3 inhibitor could reverse hypertension in this model. These findings suggest that individuals with CH may be at increased risk for hypertension and that anti-inflammatory therapies may be particularly efficacious in this patient population.

NONSTANDARD ABBREVIATIONS AND ACRONYMS

Ang II

angiotensin II

ASC

adapter protein apoptosis-associated speck-like protein containing a CARD

BMDM

bone-marrow derived macrophages

CCR2

C-C motif chemokine receptor 2

CH

clonal hematopoiesis

CHIP

CH of indeterminate potential

CVD

cardiovascular disease

DBP

diastolic blood pressure

ENaC

epithelial sodium channel

HR

heart rate

HSC

hematopoietic stem cells

HSPC

hematopoietic stem and progenitor cells

Interleukin-1beta

IL (interleukin)-1β

LSK

Lin−Sca-1⁺c-Kit⁺

MAP

mean arterial pressure

MCC950

CP-456,773, CRID3

MPP

multipotent progenitors

MPP^G/M

MPP Granulocytes/Monocytes

NaPi₂

Na⁺/Pi cotransporter 2

NCC

Na⁺-Cl⁻ cotransporter

NCCpT53

NCC phosphorylation at threonine residue 53

NHANES

National Health and Nutrition Examination Survey

NHE3

sodium hydrogen exchanger isoform 3

NKCC2

Na⁺-^K+-Cl⁻

NKCC2pT105

NKCC2 phosphorylation at threonine residue 105

NLR

Nucleotide-binding domain, leucine-rich repeat

NLRP3

NLR family pyrin domain containing 3

PBS

phosphate buffered saline

SBP

systolic blood pressure

TET2

ten eleven translocation methylcytosine dioxygenase 2

WBC

white blood cells