A novel role for type 1 angiotensin receptors on T lymphocytes to limit target organ damage in hypertension

Abstract

Rationale: Human clinical trials using type 1 angiotensin (AT(1)) receptor antagonists indicate that angiotensin II is a critical mediator of cardiovascular and renal disease. However, recent studies have suggested that individual tissue pools of AT(1) receptors may have divergent effects on target organ damage in hypertension.

Objective: We examined the role of AT(1) receptors on T lymphocytes in the pathogenesis of hypertension and its complications.

Methods and results: Deficiency of AT(1) receptors on T cells potentiated kidney injury during hypertension with exaggerated renal expression of chemokines and enhanced accumulation of T cells in the kidney. Kidneys and purified CD4(+) T cells from "T cell knockout" mice lacking AT(1) receptors on T lymphocytes had augmented expression of Th1-associated cytokines including interferon-γ and tumor necrosis factor-α. Within T lymphocytes, the transcription factors T-bet and GATA-3 promote differentiation toward the Th1 and Th2 lineages, respectively, and AT(1) receptor-deficient CD4(+) T cells had enhanced T-bet/GATA-3 expression ratios favoring induction of the Th1 response. Inversely, mice that were unable to mount a Th1 response due to T-bet deficiency were protected from kidney injury in our hypertension model.

Conclusions: The current studies identify an unexpected role for AT(1) receptors on T lymphocytes to protect the kidney in the setting of hypertension by favorably modulating CD4(+) T helper cell differentiation.

Figure 1. Verification of T cell-specific deletion of the AT_1A receptor in T cell KO mice

A, Representative histology of thymus, spleen, and kidney in CD4-Cre⁺ mT/mG and Control (CD4 Cre^- mT/mG) mice. Green fluorescence indicates the presence of CD4 Cre expression whereas red fluorescence indicates the absence of CD4 Cre expression. Blue fluorescence in kidney is a nuclear DAPI stain. B, Splenocytes were harvested from T cell WT and T cell KO littermates and sorted into 3 subpopulations. Agtr1a mRNA expression was quantitated in these purified immune cell populations and in kidney and heart from T cell WT and T cell KO groups (n≥6) and normalized to the T cell WT sample in each tissue. “CD4+ T” and “CD8+ T” refer to CD4⁺ and CD8⁺ T cells, respectively. *P<0.00001 vs. T cell WT. C, mRNA expression for AT_1A receptor on T lymphocyte subsets in T cell WT mice. Agtr1a expression was similar on all T cell subsets analyzed except in double negative (DN) CD3⁺CD4^-CD8^- T cells, in which Agtr1a expression was significantly lower than in CD4⁺ or CD8⁺ T lymphocytes. Treg = CD4⁺CD25⁺ T regulatory cells. *P=0.04 vs. CD4⁺ T cells, P=0.005 vs. CD8⁺ T cells. N=6 per group.

Figure 1. Verification of T cell-specific deletion of the AT_1A receptor in T cell KO mice

A, Representative histology of thymus, spleen, and kidney in CD4-Cre⁺ mT/mG and Control (CD4 Cre^- mT/mG) mice. Green fluorescence indicates the presence of CD4 Cre expression whereas red fluorescence indicates the absence of CD4 Cre expression. Blue fluorescence in kidney is a nuclear DAPI stain. B, Splenocytes were harvested from T cell WT and T cell KO littermates and sorted into 3 subpopulations. Agtr1a mRNA expression was quantitated in these purified immune cell populations and in kidney and heart from T cell WT and T cell KO groups (n≥6) and normalized to the T cell WT sample in each tissue. “CD4+ T” and “CD8+ T” refer to CD4⁺ and CD8⁺ T cells, respectively. *P<0.00001 vs. T cell WT. C, mRNA expression for AT_1A receptor on T lymphocyte subsets in T cell WT mice. Agtr1a expression was similar on all T cell subsets analyzed except in double negative (DN) CD3⁺CD4^-CD8^- T cells, in which Agtr1a expression was significantly lower than in CD4⁺ or CD8⁺ T lymphocytes. Treg = CD4⁺CD25⁺ T regulatory cells. *P=0.04 vs. CD4⁺ T cells, P=0.005 vs. CD8⁺ T cells. N=6 per group.

Figure 1. Verification of T cell-specific deletion of the AT_1A receptor in T cell KO mice

A, Representative histology of thymus, spleen, and kidney in CD4-Cre⁺ mT/mG and Control (CD4 Cre^- mT/mG) mice. Green fluorescence indicates the presence of CD4 Cre expression whereas red fluorescence indicates the absence of CD4 Cre expression. Blue fluorescence in kidney is a nuclear DAPI stain. B, Splenocytes were harvested from T cell WT and T cell KO littermates and sorted into 3 subpopulations. Agtr1a mRNA expression was quantitated in these purified immune cell populations and in kidney and heart from T cell WT and T cell KO groups (n≥6) and normalized to the T cell WT sample in each tissue. “CD4+ T” and “CD8+ T” refer to CD4⁺ and CD8⁺ T cells, respectively. *P<0.00001 vs. T cell WT. C, mRNA expression for AT_1A receptor on T lymphocyte subsets in T cell WT mice. Agtr1a expression was similar on all T cell subsets analyzed except in double negative (DN) CD3⁺CD4^-CD8^- T cells, in which Agtr1a expression was significantly lower than in CD4⁺ or CD8⁺ T lymphocytes. Treg = CD4⁺CD25⁺ T regulatory cells. *P=0.04 vs. CD4⁺ T cells, P=0.005 vs. CD8⁺ T cells. N=6 per group.

Figure 2. Activation of AT_1A receptors on T lymphocytes protects from hypertensive kidney injury

A, Baseline blood pressures were measured by radiotelemetry for 3 days. Then experimental mice were infused for 28 days with angiotensin II (1000ng/kg/min) via subcutaneously implanted osmotic minipump. Mean arterial pressures are depicted for T cell WT (n=11) and T cell KO (n=12) littermates. B, Urine samples were collected by placing experimental mice into metabolic cages after 25 days of saline or Ang II infusion. Urinary albumin excretion was quantitated per methods. ^#P<0.001 vs. Saline T cell WT; *P<0.0001 vs. Saline T cell KO; ^†P<0.009 vs. Ang II T cell WT. C-E, Staining of glomerular podocytes with WT1 antibody following 4 weeks of Ang II. Podocytes stain bright green. Representative images of (C) T cell WT and (D) T cell KO glomeruli. (Magnification 40×) E, number of podocytes per glomerulus in T cell WT and KO kidneys (6 mice per group). *P=0.02 vs. T cell WT. F, Renal NGAL mRNA expression in saline- or Ang II-infused T cell WT or T cell KO mice. ^#P<0.02 vs. Saline T cell WT; *P=0.005 vs. Saline T cell KO; ^†P=0.001 vs. Ang II T cell WT.

Figure 2. Activation of AT_1A receptors on T lymphocytes protects from hypertensive kidney injury

A, Baseline blood pressures were measured by radiotelemetry for 3 days. Then experimental mice were infused for 28 days with angiotensin II (1000ng/kg/min) via subcutaneously implanted osmotic minipump. Mean arterial pressures are depicted for T cell WT (n=11) and T cell KO (n=12) littermates. B, Urine samples were collected by placing experimental mice into metabolic cages after 25 days of saline or Ang II infusion. Urinary albumin excretion was quantitated per methods. ^#P<0.001 vs. Saline T cell WT; *P<0.0001 vs. Saline T cell KO; ^†P<0.009 vs. Ang II T cell WT. C-E, Staining of glomerular podocytes with WT1 antibody following 4 weeks of Ang II. Podocytes stain bright green. Representative images of (C) T cell WT and (D) T cell KO glomeruli. (Magnification 40×) E, number of podocytes per glomerulus in T cell WT and KO kidneys (6 mice per group). *P=0.02 vs. T cell WT. F, Renal NGAL mRNA expression in saline- or Ang II-infused T cell WT or T cell KO mice. ^#P<0.02 vs. Saline T cell WT; *P=0.005 vs. Saline T cell KO; ^†P=0.001 vs. Ang II T cell WT.

Figure 2. Activation of AT_1A receptors on T lymphocytes protects from hypertensive kidney injury

A, Baseline blood pressures were measured by radiotelemetry for 3 days. Then experimental mice were infused for 28 days with angiotensin II (1000ng/kg/min) via subcutaneously implanted osmotic minipump. Mean arterial pressures are depicted for T cell WT (n=11) and T cell KO (n=12) littermates. B, Urine samples were collected by placing experimental mice into metabolic cages after 25 days of saline or Ang II infusion. Urinary albumin excretion was quantitated per methods. ^#P<0.001 vs. Saline T cell WT; *P<0.0001 vs. Saline T cell KO; ^†P<0.009 vs. Ang II T cell WT. C-E, Staining of glomerular podocytes with WT1 antibody following 4 weeks of Ang II. Podocytes stain bright green. Representative images of (C) T cell WT and (D) T cell KO glomeruli. (Magnification 40×) E, number of podocytes per glomerulus in T cell WT and KO kidneys (6 mice per group). *P=0.02 vs. T cell WT. F, Renal NGAL mRNA expression in saline- or Ang II-infused T cell WT or T cell KO mice. ^#P<0.02 vs. Saline T cell WT; *P=0.005 vs. Saline T cell KO; ^†P=0.001 vs. Ang II T cell WT.

Figure 2. Activation of AT_1A receptors on T lymphocytes protects from hypertensive kidney injury

A, Baseline blood pressures were measured by radiotelemetry for 3 days. Then experimental mice were infused for 28 days with angiotensin II (1000ng/kg/min) via subcutaneously implanted osmotic minipump. Mean arterial pressures are depicted for T cell WT (n=11) and T cell KO (n=12) littermates. B, Urine samples were collected by placing experimental mice into metabolic cages after 25 days of saline or Ang II infusion. Urinary albumin excretion was quantitated per methods. ^#P<0.001 vs. Saline T cell WT; *P<0.0001 vs. Saline T cell KO; ^†P<0.009 vs. Ang II T cell WT. C-E, Staining of glomerular podocytes with WT1 antibody following 4 weeks of Ang II. Podocytes stain bright green. Representative images of (C) T cell WT and (D) T cell KO glomeruli. (Magnification 40×) E, number of podocytes per glomerulus in T cell WT and KO kidneys (6 mice per group). *P=0.02 vs. T cell WT. F, Renal NGAL mRNA expression in saline- or Ang II-infused T cell WT or T cell KO mice. ^#P<0.02 vs. Saline T cell WT; *P=0.005 vs. Saline T cell KO; ^†P=0.001 vs. Ang II T cell WT.

Figure 3. Role of the AT_1A receptor on T cells in regulating infiltration of mononuclear cells into the kidney during Ang II-dependent hypertension

Embedded kidney sections were obtained from Ang II-infused T cell WT and KO mice and stained with anti-CD3 or anti-F4/80 antibody. A–B, representative images of T lymphocyte staining in kidneys from Ang II-infused (A) T cell WT mice and (B) T cell KO mice. C, proportion of renal vessels from Ang II-infused T cell WT and KO groups surrounded by 0–10, 11–20, or >20 CD3⁺ T cells, respectively. *P<0.0001 vs. T cell WT (Fisher's exact test across all 3 tertiles). D–E, representative images of macrophage staining in kidneys from Ang II-infused (D) T cell WT and (E) T cell KO mice.

Figure 3. Role of the AT_1A receptor on T cells in regulating infiltration of mononuclear cells into the kidney during Ang II-dependent hypertension

Embedded kidney sections were obtained from Ang II-infused T cell WT and KO mice and stained with anti-CD3 or anti-F4/80 antibody. A–B, representative images of T lymphocyte staining in kidneys from Ang II-infused (A) T cell WT mice and (B) T cell KO mice. C, proportion of renal vessels from Ang II-infused T cell WT and KO groups surrounded by 0–10, 11–20, or >20 CD3⁺ T cells, respectively. *P<0.0001 vs. T cell WT (Fisher's exact test across all 3 tertiles). D–E, representative images of macrophage staining in kidneys from Ang II-infused (D) T cell WT and (E) T cell KO mice.

Figure 3. Role of the AT_1A receptor on T cells in regulating infiltration of mononuclear cells into the kidney during Ang II-dependent hypertension

Embedded kidney sections were obtained from Ang II-infused T cell WT and KO mice and stained with anti-CD3 or anti-F4/80 antibody. A–B, representative images of T lymphocyte staining in kidneys from Ang II-infused (A) T cell WT mice and (B) T cell KO mice. C, proportion of renal vessels from Ang II-infused T cell WT and KO groups surrounded by 0–10, 11–20, or >20 CD3⁺ T cells, respectively. *P<0.0001 vs. T cell WT (Fisher's exact test across all 3 tertiles). D–E, representative images of macrophage staining in kidneys from Ang II-infused (D) T cell WT and (E) T cell KO mice.

Figure 4. Deficiency of AT_1A receptors on T lymphocytes permits enhanced renal expression of chemokines and inflammatory cytokines in Ang II-dependent hypertension

A–B, Expression of chemokines in kidney tissues from saline- or Ang II-infused T cell WT and KO mice. A, CCL5 (RANTES) mRNA expression. ^‡P<0.02 vs. Saline T cell WT; *P<0.05 vs. Saline T cell KO; ^#P=0.03 vs. Ang II T cell WT. B, MCP-1 mRNA expression. ^‡P=0.009 vs. Saline T cell WT; *P<0.009 vs. Saline T cell KO; ^#P<0.02 vs. Ang II T cell WT. C, Expressions of IFN-γ, TNF-α, and IL-6 mRNA in kidneys from Ang II infused-T cell WT and KO mice. *P=0.04 vs. T cell WT; ^†P<0.04 vs. T cell WT.

Figure 4. Deficiency of AT_1A receptors on T lymphocytes permits enhanced renal expression of chemokines and inflammatory cytokines in Ang II-dependent hypertension

A–B, Expression of chemokines in kidney tissues from saline- or Ang II-infused T cell WT and KO mice. A, CCL5 (RANTES) mRNA expression. ^‡P<0.02 vs. Saline T cell WT; *P<0.05 vs. Saline T cell KO; ^#P=0.03 vs. Ang II T cell WT. B, MCP-1 mRNA expression. ^‡P=0.009 vs. Saline T cell WT; *P<0.009 vs. Saline T cell KO; ^#P<0.02 vs. Ang II T cell WT. C, Expressions of IFN-γ, TNF-α, and IL-6 mRNA in kidneys from Ang II infused-T cell WT and KO mice. *P=0.04 vs. T cell WT; ^†P<0.04 vs. T cell WT.

Figure 4. Deficiency of AT_1A receptors on T lymphocytes permits enhanced renal expression of chemokines and inflammatory cytokines in Ang II-dependent hypertension

A–B, Expression of chemokines in kidney tissues from saline- or Ang II-infused T cell WT and KO mice. A, CCL5 (RANTES) mRNA expression. ^‡P<0.02 vs. Saline T cell WT; *P<0.05 vs. Saline T cell KO; ^#P=0.03 vs. Ang II T cell WT. B, MCP-1 mRNA expression. ^‡P=0.009 vs. Saline T cell WT; *P<0.009 vs. Saline T cell KO; ^#P<0.02 vs. Ang II T cell WT. C, Expressions of IFN-γ, TNF-α, and IL-6 mRNA in kidneys from Ang II infused-T cell WT and KO mice. *P=0.04 vs. T cell WT; ^†P<0.04 vs. T cell WT.

Figure 5. T cell KO mice have normal maturation of T lymphocytes in the thymus

Thymocytes were isolated from T cell WT and KO animals and labeled for CD4 and CD8. A, Representative dot-plots of single positive CD4⁺, CD8⁺, double positive, and double negative T cell proportions in thymus from T cell WT and KO animals. (n=4). B, Summary data for thymocyte proportions showing similar percentages of CD4⁺, CD8⁺, DP (CD4⁺CD8⁺), and DN (CD4⁻CD8⁻) thymocytes in T cell WT and KO animals.

Figure 5. T cell KO mice have normal maturation of T lymphocytes in the thymus

Thymocytes were isolated from T cell WT and KO animals and labeled for CD4 and CD8. A, Representative dot-plots of single positive CD4⁺, CD8⁺, double positive, and double negative T cell proportions in thymus from T cell WT and KO animals. (n=4). B, Summary data for thymocyte proportions showing similar percentages of CD4⁺, CD8⁺, DP (CD4⁺CD8⁺), and DN (CD4⁻CD8⁻) thymocytes in T cell WT and KO animals.

Figure 6. T cell KO mice have diminished proportions of CD4⁺ T lymphocytes residing in spleen during Ang II-dependent hypertension

T cell proportions in spleen from saline- and Ang II-infused T cell WT and KO mice (n≥6). A, Representative dot plots of CD4⁺ and CD8⁺ T cell subsets in spleen following Saline or Ang II infusion in T cell WT and KO groups. B, Summary data for CD3⁺ splenocyte distribution showing diminished proportions of CD4⁺ T cells with a reciprocal increase in CD8⁺ T cell proportions in the spleens of T cell KO animals following Ang II infusion. *P<0.006 vs. Ang II T cell WT; ^†P<0.03 vs. Ang II T cell WT.

Figure 6. T cell KO mice have diminished proportions of CD4⁺ T lymphocytes residing in spleen during Ang II-dependent hypertension

T cell proportions in spleen from saline- and Ang II-infused T cell WT and KO mice (n≥6). A, Representative dot plots of CD4⁺ and CD8⁺ T cell subsets in spleen following Saline or Ang II infusion in T cell WT and KO groups. B, Summary data for CD3⁺ splenocyte distribution showing diminished proportions of CD4⁺ T cells with a reciprocal increase in CD8⁺ T cell proportions in the spleens of T cell KO animals following Ang II infusion. *P<0.006 vs. Ang II T cell WT; ^†P<0.03 vs. Ang II T cell WT.

Figure 7. Stimulation of the AT_1A receptor on T lymphocytes constrains Th1 differentiation in the setting of hypertension

CD4⁺ T cells were isolated via fluorescent cell sorting from the spleens of Ang II-infused T cell WT and KO animals and characterized for Th1 vs. Th2 phenotype. A, mRNA expressions of IFN-γ, TNF-α, IL-4, and TGF-β in CD4⁺ T lymphocytes from T cell WT and KO mice (n≥6). *P<0.03 vs. T cell WT; ^†P=0.03 vs. T cell WT. B, concentrations of IFN-γ, TNF-α, and IL-4 proteins in supernatants of activated splenic lymphocytes from Ang II-infused T cell WT and KO mice (n=6). *P=0.05 vs. T cell WT; ^#P<0.005 vs. T cell WT. C, ratio of mRNA expression of T-bet/GATA3 in purified CD4⁺ T lymphocytes from T cell WT and KO mice (n=5–6). *P=0.05 vs. Ang II T cell WT. D, co-expression of PD-1 and TIM-3 measured by flow cytometry is similar on splenic CD4⁺ T cells from T cell WT and KO mice (n≥6). Representative dotplots are shown. Summary data are presented in the text.

Figure 7. Stimulation of the AT_1A receptor on T lymphocytes constrains Th1 differentiation in the setting of hypertension

CD4⁺ T cells were isolated via fluorescent cell sorting from the spleens of Ang II-infused T cell WT and KO animals and characterized for Th1 vs. Th2 phenotype. A, mRNA expressions of IFN-γ, TNF-α, IL-4, and TGF-β in CD4⁺ T lymphocytes from T cell WT and KO mice (n≥6). *P<0.03 vs. T cell WT; ^†P=0.03 vs. T cell WT. B, concentrations of IFN-γ, TNF-α, and IL-4 proteins in supernatants of activated splenic lymphocytes from Ang II-infused T cell WT and KO mice (n=6). *P=0.05 vs. T cell WT; ^#P<0.005 vs. T cell WT. C, ratio of mRNA expression of T-bet/GATA3 in purified CD4⁺ T lymphocytes from T cell WT and KO mice (n=5–6). *P=0.05 vs. Ang II T cell WT. D, co-expression of PD-1 and TIM-3 measured by flow cytometry is similar on splenic CD4⁺ T cells from T cell WT and KO mice (n≥6). Representative dotplots are shown. Summary data are presented in the text.

Figure 7. Stimulation of the AT_1A receptor on T lymphocytes constrains Th1 differentiation in the setting of hypertension

CD4⁺ T cells were isolated via fluorescent cell sorting from the spleens of Ang II-infused T cell WT and KO animals and characterized for Th1 vs. Th2 phenotype. A, mRNA expressions of IFN-γ, TNF-α, IL-4, and TGF-β in CD4⁺ T lymphocytes from T cell WT and KO mice (n≥6). *P<0.03 vs. T cell WT; ^†P=0.03 vs. T cell WT. B, concentrations of IFN-γ, TNF-α, and IL-4 proteins in supernatants of activated splenic lymphocytes from Ang II-infused T cell WT and KO mice (n=6). *P=0.05 vs. T cell WT; ^#P<0.005 vs. T cell WT. C, ratio of mRNA expression of T-bet/GATA3 in purified CD4⁺ T lymphocytes from T cell WT and KO mice (n=5–6). *P=0.05 vs. Ang II T cell WT. D, co-expression of PD-1 and TIM-3 measured by flow cytometry is similar on splenic CD4⁺ T cells from T cell WT and KO mice (n≥6). Representative dotplots are shown. Summary data are presented in the text.

Figure 7. Stimulation of the AT_1A receptor on T lymphocytes constrains Th1 differentiation in the setting of hypertension

CD4⁺ T cells were isolated via fluorescent cell sorting from the spleens of Ang II-infused T cell WT and KO animals and characterized for Th1 vs. Th2 phenotype. A, mRNA expressions of IFN-γ, TNF-α, IL-4, and TGF-β in CD4⁺ T lymphocytes from T cell WT and KO mice (n≥6). *P<0.03 vs. T cell WT; ^†P=0.03 vs. T cell WT. B, concentrations of IFN-γ, TNF-α, and IL-4 proteins in supernatants of activated splenic lymphocytes from Ang II-infused T cell WT and KO mice (n=6). *P=0.05 vs. T cell WT; ^#P<0.005 vs. T cell WT. C, ratio of mRNA expression of T-bet/GATA3 in purified CD4⁺ T lymphocytes from T cell WT and KO mice (n=5–6). *P=0.05 vs. Ang II T cell WT. D, co-expression of PD-1 and TIM-3 measured by flow cytometry is similar on splenic CD4⁺ T cells from T cell WT and KO mice (n≥6). Representative dotplots are shown. Summary data are presented in the text.

Figure 8. Chronic stimulation of AT₁ receptors on T cells suppresses Th1 response in vitro

mRNA expressions of IFN-γ and TNF-α in T cell WT and KO lymphocytes subjected to 7 days of persistent T cell receptor stimulation with anti-CD3 +/− Ang II in in vitro culture. “Veh” = vehicle. ^#P≤0.02 vs. T cell WT+anti-CD3; *P≤0.003 vs. T cell WT+anti-CD3+AngII.