Klotho gene deficiency causes salt-sensitive hypertension via monocyte chemotactic protein-1/CC chemokine receptor 2-mediated inflammation

Abstract

Klotho (KL) is a newly discovered aging suppressor gene. In mice, the KL gene extends the lifespan when overexpressed and shortens the lifespan when disrupted. This study investigated if KL deficiency affects BP and salt sensitivity using KL mutant heterozygous (+/-) mice and wild-type (WT) mice (9 weeks of age, 16 mice per group). Notably, systolic BP in KL(+/-) mice began to increase at the age of 15 weeks, reached a peak level at the age of 17 weeks, and remained elevated thereafter, whereas systolic BP remained consistent in WT mice. High salt (HS) intake further increased BP in KL(+/-) mice but did not affect BP in WT mice. Blockade of CC chemokine receptor 2 (CCR2), involved in monocyte chemotaxis, by a specific CCR2 antagonist (INCB3284) abolished the HS-induced increase in BP in KL(+/-) mice. Furthermore, HS loading substantially increased the expression of monocyte chemotactic protein-1 and the infiltration of macrophages and T cells in kidneys in KL(+/-) mice, and treatment with INCB3284 abolished these effects. Treatment of KL(+/-) mice with INCB3284 also attenuated the increased renal expressions of serum glucocorticoid-regulated kinase 1, thiazide-sensitive NaCl cotransporter, and ATP synthase β along with the renal structural damage and functional impairment induced by HS loading. In conclusion, KL deficiency caused salt-sensitive hypertension and renal damage by CCR2-mediated inflammation.

Keywords: cardiovascular; hypertension; monocyte chemotactic protein-1.

Copyright © 2015 by the American Society of Nephrology.

Figures

Figure 1.

One-half klotho deficiency (+/−) causes spontaneous hypertension and increases salt sensitivity. (A) The time course of BP change in KL(+/−) and WT mice. (B) The HS intake further increased BP in KL(+/−) mice. Each strain of mice was divided into two groups after BP was stabilized. One group in each strain received 1% saline followed by 2% saline as drinking fluid, whereas the remaining groups received regular tap water. (C) Inhibition of CCR2 by INCB3284 abolished HS-induced elevation of BP in KL(+/−) mice. Each group was further divided into two subgroups when they were over the age of 6 months. The two subgroups were treated with INCB3284 (CCR2 antagonist) daily (15 mg/kg per day intraperitoneally) and DMSO, respectively, after stable BP was obtained. Data=means±SEMs (n=16 mice/group). *P<0.05 versus KL(+/−); +P<0.05, ++P<0.01 versus WT; #P<0.05 versus KL(+/−)-HS-INCB.

Figure 2.

HS loading further upregulated MCP-1 in kidneys of KL(+/−) mice. (A) Representative Western blots and (B) quantitative analysis of MCP-1 in kidneys. (C) Representative Western blots and (D) quantitative analysis of TNF-α protein expression in kidneys. (E) Representative Western blots and (F) quantitative analysis of CCR2 protein expression in kidneys. Western blots analyses were performed when animals were euthanized at 10 days after treatment. The relative protein expression was first normalized to β-actin and then calculated as fold changes of the controls (WT). Data=means±SEMs (n=4 mice/group). *P<0.05 versus KL(+/−); +P<0.05, ++P<0.01, +++P<0.001 versus WT; #P<0.05 versus KL(+/−)-HS.

Figure 3.

HS further increased infiltration of macrophages in kidneys of KL(+/−) mice, which can be abolished by INCB3284. (A) Representative photomicrographs of CD68 immunostaining in kidney sections (arrows indicate CD68+ cells). (B) Semiquantitative analysis of infiltration of CD68+ cells in kidney sections. Immunostaining was performed when animals were euthanized at 10 days after treatment with INCB3284. Scale bars, 50 μm. Data=means±SEMs (n=4 mice/group). ***P<0.001 versus KL(+/−); +++P<0.001 versus WT; ###P<0.001 versus KL(+/−)-HS.

Figure 4.

HS further increased infiltration of T cells in kidneys of KL(+/−) mice, which can be abolished by INCB3284. (A) Representative photomicrographs of CD4 immunostaining in kidney sections (arrows indicate CD4+ T cells). (B) Semiquantitative analysis of infiltration of CD4+ T cells in kidneys. (C) Representative photomicrographs of CD8 immunostaining in kidney sections (arrows indicate CD8+T cells). (D) Semiquantitative analysis of infiltration of CD8+ T cells in kidneys. Immunostaining was performed when animals were euthanized at 10 days after treatment with INCB3284. Scale bars, 50 μm. Data=means±SEMs. ***P<0.001 versus KL(+/−); +P<0.05, ++P<0.01, +++P<0.001 versus WT; ###P<0.001 versus KL(+/−)-HS.

Figure 5.

Blockade of CCR2 by INCB3284 abolished HS-induced upregulation of Sgk1-NCC signaling and ATP synthase β in kidneys of KL(+/−) mice. (A) Representative Western blots and (B) quantitative analysis of MR in kidneys. (C) Representative Western blots and (D) quantitative analysis of Sgk1 in kidneys. (E) Representative Western blots and (F) quantitative analysis of thiazide-sensitive NCC in kidneys. (G) Representative Western blots and (H) quantitative analysis of ATP synthase β in kidneys. Western blots analysis was performed when animals were euthanized at 10 days after treatment with INCB3284. Data=means±SEMs. The relative protein expression was first normalized to β-actin and then calculated as fold changes of the controls (WT). *P<0.05, **P<0.01 versus KL(+/−); +P<0.05, ++P<0.01, +++P<0.001 versus WT; #P<0.05, ##P<0.01 versus KL(+/−)-HS.

Figure 6.

Blockade of CCR2 by INCB3284 abolished HS-induced renal structural damage in KL(+/−) mice. (A) Representative photomicrographs of HE-stained kidney sections. Tubular damages, including dilation and atrophy of tubules, as well as loss of tubular structure (indicated by arrows), were observed in some local regions of medulla in KL(+/−) mice. These structural damages were exacerbated by HS in KL(+/−) mice. INCB3284 attenuated these local medullary tubular injuries. (B) Representative photomicrographs of Masson trichrome–stained kidney sections (blue staining indicates collagen deposition). (C) Semiquantitative analysis of collagen staining in tubular interstitium. Both stainings were performed when animals were euthanized at 10 days after treatment with INCB3284. Scale bars, 50 μm. *P<0.05, **P<0.01, ***P<0.001 versus KL(+/−); +P<0.05, +++P<0.001 versus WT; ###P<0.001 versus KL(+/−)-HS.

Figure 7.

Blockade of CCR2 by INCB3284 abolished HS-induced renal functional impairment in KL(+/−) mice. (A) Representative photomicrographs of HE-stained kidney sections. Tubular cast (protein) formations (indicated by arrows) were observed in some local medullary regions in KL(+/−) mice, which were more pronounced in KL(+/−)-HS mice. INCB3284 attenuated tubular cast formation. (B) Semiquantitative analysis of tubular cast formation in medulla. (C) Urine albumin (normalized to urine creatinine). (D) Plasma urea concentration. HE staining and plasma/urine measurements were performed when animals were euthanized at 10 days after treatment with INCB3284. Scale bars, 50 μm. *P<0.05, ***P<0.001 versus KL(+/−); +P<0.05, ++P<0.01, +++P<0.001 versus WT; #P<0.05, ###P<0.001 versus KL(+/−)-HS.