In 1997, a new aging suppressor gene was identified and named after the purported Greek Moira Klotho,1 1 2
Aging is characterized by age-related deterioration in health. Hypertension is a common aging-related disorder. The prevalence of hypertension increases with age.3 3 4 5 – 9 8 10 et al. 11
Monocyte chemotactic protein-1 (MCP-1), belonging to the C-C chemokine family, can be produced by many cell types in response to inflammatory stimuli, such as TNF-α , IL-1, and IFN-γ .12 13 14 – 16 16 , 17 12 18 H 1 and TH 2 cells.19 , 20
The progressive renal recession is also a common phenomenon in the aging process. The association of the age-related nephron loss and renal function decline has been confirmed, even after excluding other confounding factors.21 , 22 1 23 11 11
Results
One-Half Klotho Deficiency (+/−) Caused Spontaneous Hypertension and Increased Salt Sensitivity
Western blot analysis indicated that klotho protein expression in kidneys of KL(+/−) mice is about one half of that of wild-type (WT) mice (Supplemental Figure 1). Systolic BP began to increase spontaneously around 15 weeks of age (Figure 1A ). Systolic BP was significantly and persistently elevated in KL(+/−) mice from the age of 16 weeks and remained elevated thereafter. Therefore, one-half klotho deficiency causes spontaneous hypertension.
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.
Systolic BP was also remarkably increased in KL(+/−) mice after receiving 2% saline intake (Figure 1B ). Although BP was slightly elevated in KL(+/−) mice in response to 1% saline, it did not reach a significant level. High-salt (HS) intake (1% or 2%) had no effects on BP in WT mice. The results revealed, for the first time, that one-half klotho deficiency increased salt sensitivity and caused salt-sensitive hypertension.
Blockade of CCR2 Abolished HS-Induced Hypertension in KL(+/−) Mice
Daily intraperitoneal injection of INCB3284 (15 mg/kg), a CCR2-selective antagonist,24 Figure 1C ). INCB3284 did not affect BP in KL(+/−) mice receiving regular water. INCB3284 had no effects on systolic BP of WT mice in the absence or presence of 2% saline. Blockade of CCR2 by another CCR2-selective antagonist,25 , 26
HS Loading Further Upregulated MCP-1 in Kidneys of KL(+/−) Mice
The protein expressions of MCP-1 and TNF-α in kidneys were upregulated in KL(+/−) mice (Figure 2, A–D ). Remarkably, they were further increased by HS loading, suggesting that the HS loading exacerbated klotho deficiency–induced inflammatory activation in kidneys. In contrast, the HS loading did not alter the expression of MCP-1 and TNF-α significantly in WT mice (Figure 2, A–D ). Blockade of CCR2 by INCB3284 did not affect the expression of MCP-1 or TNF-α in either KL(+/−) or WT mice, regardless of HS or regular water intake. One-half klotho deficiency or INCB3284 did not affect CCR2 protein expression in kidneys (Figure 2, E and F ).
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.
HS Further Increased Infiltration of Macrophages and T Cells in Kidneys of KL(+/−) Mice, Which Can Be Abolished by INCB3284
The immunostaining showed infiltration of macrophages (CD68) (Figure 3A ) and T cells (CD4 and CD8) (Figure 4, A and C ) in kidneys. The numbers of CD68+ , CD4+ , and CD8+ cells were increased in kidneys of KL(+/−) mice versus WT mice (Figures 3B and 4, B and D ), indicating that one-half klotho deficiency increased infiltration of macrophages and T cells in kidneys. INCB3284 abolished macrophage (CD68+ ) infiltration in kidneys of KL(+/−) mice (Figure 3B ), suggesting effective blockade of CCR2. Therefore, klotho deficiency–induced macrophage infiltration was mediated by CCR2. Although INCB3284 significantly attenuated infiltration of T lymphocytes (CD4+ and CD8+ ) in KL(+/−) mice, it did not decrease it to the WT level (Figure 4 ). The results suggest that klotho deficiency–induced T-cell infiltration was largely, but not completely, mediated by the MCP-1/CCR2 pathway.
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 μ m. 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 μ m. 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.
HS exacerbated infiltration of inflammatory cells in kidneys of KL(+/−) mice (Figures 3 and 4 ). INCB3284 abolished the HS-induced increase in T cells and macrophages in kidneys of KL(+/−) mice (Figure 3B and 4, B and D ). This result suggests that the HS-induced infiltration of inflammatory cells was fully CCR2-dependent.
Blockade of CCR2 by INCB3284 Abolished HS-Induced Upregulation of the Serum Glucocorticoid-Regulated Kinase 1–Thiazide-Sensitive NaCl Cotransporter Signaling and ATP Synthase β in Kidneys of KL(+/−) Mice
One-half klotho deficiency or INCB3284 did not affect mineralocorticoid receptor (MR) protein expression in kidneys (Figure 5, A and B ). The protein expressions of serum glucocorticoid-regulated kinase 1 (Sgk1), thiazide-sensitive NaCl cotransporter (NCC), and ATP synthase β were upregulated in kidneys of KL(+/−) mice versus WT mice (Figure 5, C–H ). Interestingly, protein expressions of these signaling molecules, remarkably, were further increased by HS loading in KL(+/−) mice but not WT mice.
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.
Blockade of CCR2 by INCB3284 significantly attenuated the upregulation of Sgk1, NCC, and ATP synthase β in KL(+/−) mice, although the expression of Sgk1 and NCC in KL(+/−)-INCB mice remained higher than that of WT mice (Figure 5, C–H ). Notably, INCB3284 abolished the HS-induced increases in protein expression of Sgk1, NCC, and ATP synthase β in KL(+/−) mice (Figure 5, C–H ).
Blockade of CCR2 by INCB3284 Abolished HS-Induced Renal Structural Damage in KL(+/−) Mice
Tubular dilation and atrophy, as well as tubular collapse, were found in some medullary regions of KL(+/−) mice, which were exacerbated by HS loading in KL(+/−) mice (Figure 6A ). In contrast, these pathologic changes were not seen in age-matched WT and WT-HS mice. The results showed that one-half klotho deficiency caused renal tubular damage, which was further deteriorated by HS loading. INCB3284 attenuated tubular structural damage and abolished HS-induced exacerbation in KL(+/−) mice (Figure 6A ).
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 μ m. *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.
The Masson trichrome staining showed more collagen deposition in renal tubular interstitium in KL(+/−) mice versus WT mice (Figure 6, B and C ), indicating that one-half klotho deficiency caused tubulointerstitial fibrosis. HS loading exacerbated fibrotic damage in KL(+/−) mice. INCB3284 significantly, but not completely, rescued tubulointerstitial fibrosis in KL(+/−) mice. INCB3284 completely abolished HS-induced exacerbation of fibrotic formation.
Therefore, CCR2-mediated inflammation was involved in klotho deficiency–induced and HS-exacerbated renal tubular damage and tubulointerstitial fibrosis.
Blockade of CCR2 by INCB3284 Abolished HS-Induced Renal Functional Impairment in KL(+/−) Mice
Hematoxylin and eosin (HE) staining in kidneys showed tubular cast (protein) formation, a sign of severe kidney damage, in some local medullary regions of KL(+/−) mice but not in WT mice (Figure 7, A and B ). Tubular cast formation, remarkably, was further increased by HS loading in KL(+/−) mice. INCB3284 attenuated tubular cast formation and eliminated HS-induced exacerbation of cast formation in KL(+/−).
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 μ m. *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.
Renal glomerular impairment in KL(+/−) mice was evidenced by significant increases in urine albumin levels, which were worsened by HS (Figure 7C ). INCB3284 significantly decreased urine albumin levels in KL(+/−) and KL(+/−)-HS mice, suggesting improved glomerular function. Plasma urea concentrations were increased in KL(+/−) mice, which were abolished by INCB3284 (Figure 7D ). HS tended to further increase plasma urea concentrations in KL(+/−) mice, which were attenuated by INCB3284 treatments, although the data did not reach significance levels (Figure 7D ). Blockade of CCR2 by another CCR2-selective antagonist, RS102895, abolished the HS-induced increase in plasma urea in KL(+/−) mice (Supplemental Figure 3B). Serum creatinine was increased by HS, which could be significantly attenuated by RS102895 (Supplemental Figure 3A). These results indicated that MCP-1/CCR2-mediated inflammation may be involved in impaired renal function in KL(+/−) mice.
Discussion
This study revealed that systolic BP was elevated spontaneously and persistently in KL(+/−) mice after the age of 16 weeks (Figure 1A ), suggesting that the klotho gene plays an important role in the maintenance of normal BP. Klotho is an aging suppressor gene.1 , 2 11 3 3 27 in vitro .27 in vivo klotho gene delivery prevented the progression of hypertension in spontaneous hypertensive rats.28 et al. 29
Interestingly, BP of KL(+/−) mice was further elevated by HS loading, which however, did not affect BP in WT mice (Figure 1B ). Thus, this study showed, for the first time, that klotho deficiency increased salt sensitivity and elicited salt-sensitive hypertension. In humans, the klotho level is decreased,11 5 , 6 , 8 Figure 1C , Supplemental Figure 2), suggesting that the CCR2-mediated inflammation may play an important role in klotho deficiency–induced salt-sensitive hypertension.
The expression of MCP-1 and infiltration of macrophages and T cells were upregulated in kidneys of KL(+/−) mice (Figures 2 –4 ). Notably, these inflammatory responses were further markedly increased by HS loading. Although CCR2 expression was not different between KL(+/−) and WT mice and not affected by HS loading (Figure 2E ), MCP-1 expression was further upregulated in kidneys of KL(+/−) mice by HS loading (Figure 2A ). MCP-1 activates the chemotaxis activity by binding to CCR2.16 , 17 Figures 3 and 4 ), suggesting that the upregulation of MCP-1 may mediate klotho deficiency–induced renal inflammation by CCR2. Nevertheless, how klotho deficiency causes the upregulation of MCP-1 is incompletely understood. The in vitro studies showed that exogenous addition or overexpression of klotho suppressed TNF-α –stimulated activation of NF-κB and subsequent production of cytokines, such as MCP-1, by inhibition of RelASer536 phosphorylation.30 , 31 32 32
The infiltration of inflammatory cells in kidneys was found in salt-sensitive hypertension.33 , 34 et al. 35 36
It is noted that, although blockade of CCR2 by RS102895 abolished HS-induced hypertension (Supplemental Figure 2) and improved kidney function (Supplemental Figure 3), it did not alter vascular relaxing responses to acetylcholine or sodium nitroprusside in KL(+/−) mice on HS diet (Supplemental Figure 5). This result suggests that the antihypertensive effect of blockade of CCR2 may not be caused by vasorelaxant effects of this compound. Klotho is predominately expressed in kidneys. There is no convincing data showing that klotho is expressed in vascular cells.
The control of sodium excretion is achieved by regulating sodium transport in renal tubules. NCC is the predominant apical sodium transporter located in the distal convoluted tubule, which has the highest sodium-potassium-ATPase activity among all renal tubule segments.37 38 39 40 β were upregulated in KL(+/−) mice and further enhanced by HS loading, which can be effectively attenuated by INCB3284 (Figure 5 ). Nevertheless, this study cannot assess the functional significance of increased expression of NCC and Sgk1. It is expected that the upregulation of NCC/Sgk1 may lead to increased Na reabsorption and positive salt balance through their known function in Na reabsorption, which could be attenuated by blockade of CCR2. This hypothesis, however, needs to be validated by measuring urinary Na excretion.
The Sgk1-NCC pathway has been established as an important downstream target of aldosterone/MR.41 , 42 Figure 5 ). This result suggests that the CCR2-mediated inflammatory process interfered with the negative feedback regulation of the MR-dependent cascades in response to HS loading, although the underlying mechanism remains to be elucidated. Several studies indicated that some cytokines, such as TNF-α and IL-6, were capable of activating Rac1, a member of the Rho family of GTPases.43 , 44 45
Although the infiltration of inflammatory cells and the upregulation of the Sgk1-NCC signaling in kidneys of KL(+/−) mice were attenuated or abolished by INCB3284 (Figures 3 –5 ), the elevated BP level in KL(+/−) mice was not decreased by INCB3284. The results suggest that the spontaneous elevation of BP in KL(+/−) mice may be mediated by a mechanism other than inflammation in kidneys. The spontaneous elevation of BP was investigated in our recent study, which revealed that plasma level of aldosterone was increased in KL(+/−) mice.32 32 42 46 47 48 49
Arginine vasopressin (AVP) has been reported to play an important role in the development of deoxycorticosterone acetate salt hypertension.50 51 52
Another interesting finding was that one-half klotho deficiency caused renal structural damage and renal functional impairments, which were exacerbated by salt excess (Figures 6 and 7 ). Klotho deficiency–induced renal damage was largely attributed to the CCR2-mediated inflammatory cell infiltration, because it can be attenuated by INCB3284. Notably, INCB3284 significantly improved renal damage in KL(+/−) mice, although it did not decrease the elevated BP to the control level, suggesting that klotho deficiency–induced kidney damage may be independent of hypertension. This notion is supported by our recent finding that klotho gene delivery abolished kidney damages in spontaneous hypertensive rats, whereas BP remains elevated.28 28 , 53 28
In summary, this study showed, for the first time, that klotho deficiency caused salt-sensitive hypertension by activating the CCR2-mediated inflammatory process. The inflammatory cytokine activation and subsequent inflammatory cell infiltration upregulated the Sgk1-NCC signaling in kidneys in response to HS loading and resulted in renal structural injury and renal functional impairment. Therefore, klotho supplement and inflammation suppression may be potential therapeutic strategies for the treatment of salt-sensitive hypertension and kidney damage in elderly patients.
Concise Methods
Animal Study Protocols
KL(+/−) mice in 129Sv background were provided by Makoto Kuro-o.1 ad libitum . Details are in Supplemental Material.
In total, 16 KL(+/−) mice and 16 WT mice (9 weeks; n =16) were used. BP was measured weekly from the age of 9–23 weeks. After BP was stabilized (5 months), each strain of mice was divided into two groups. One group of each strain received 1% saline followed by 2% saline as drinking fluid, whereas the remaining group of each strain received regular tap water. BP and body weight were measured two times per week. After the BP level was stable again, each group was further divided into two subgroups at the age of 6 months (n =4). Under each diet condition in each strain, one subgroup received INCB3284 (15 mg/kg per day intraperitoneally; Tocris Bioscience, Bristol, UK), whereas the other subgroup received an equal dose of vehicle (DMSO) and served as a control. BP and body weight were measured two times per week. Urine was collected three times for assessing renal function (urea, creatinine, and albumin) during the treatment. After 10 days of the treatment, animals were euthanized (halothane). Blood was collected for measuring urea and creatinine concentrations. Animals were then perfused transcardially using heparinized saline. One kidney was collected and embedded in paraffin for histologic and immunohistochemical analysis. The other kidney was saved in −80°C for molecular assays.
Measurements of BP
BP was measured by the volume-pressure recording (VPR) tail-cuff method with slight warming (28°C) but not heating of the tail using a CODA 6 BP Monitoring System (Kent Scientific). This method has been validated by using a telemetry system.54 , 55 28 , 56 – 58
Morphologic and Immunohistochemical Investigations
Paraffin-embedded kidney sections (5 μ m) were processed for the following staining. At least three random fields of each section were observed and analyzed (15 sections).
Immunohistochemical Staining
Staining was performed as we described previously.28 α (H-160) (1:100; Santa Cruz Biotechnology). Subsequently, the sections were incubated with secondary antibody, including goat anti-mouse, goat anti-rabbit, and chicken anti-rat IgG- horseradish peroxidase (1:1000–1:2000; Santa Cruz Biotechnology) for 1 hour. The sections stained without the primary antibody served as negative controls. The sections were examined and photographed at equal exposure conditions and magnification using a Nikon Eclipse Ti-U microscope coupled with a digital color camera. The numbers of CD68+ , CD4+ , and CD8+ cells infiltrated in kidneys were directly counted under the microscope at equal magnification (×400).
HE Staining
HE staining was performed in kidney sections for histologic structure examination. Images for each section were randomly collected at equal magnification (×400) under a Nikon Eclipse Ti-U microscope. Tubular cast formation was defined as the red deposition of proteinaceous material in renal tubules. The semiquantitative analysis of relative tubular cast area fraction in medulla (percentage of cast area over the total area in a given field) was measured using NIS-Elements BR 3.0 software (Nikon, Melville, NY).
Masson Trichrome Staining
Trichrome staining was performed in kidney sections for detecting renal fibrosis. The blue staining indicated collagen deposition. The semiquantitative analysis of relative collagen area fraction (percentage of blue-stained collagen area over the total area in one field) was measured using NIS-Elements BR 3.0 software.
Measurements of Renal Function
Plasma urea level was detected with a quantichrom urea assay kit (DIUR-500; BioAssay Systems, Hayward, CA) according to the manufacturer’s instruction. Urine creatinine level was detected with a quantichrom creatinine assay kit (DICT-500; BioAssay Systems) according to the manufacturer’s instruction. Urine albumin concentration was measured with a mouse-specific microalbuminuria ELISA kit (Albuwell M; Exocell, Philadelphia, PA) according to the manufacturer’s instruction. Urine albumin excretion was normalized to urine creatinine.
Western Blotting Analyses
Western blotting analysis was performed as described previously.28 α (1 μ g/ml; Abcam, Inc.), CCR2 (1 μ g/ml; Abcam, Inc.), MR (1:250; Abcam, Inc.), Sgk1 (1:500; Santa Cruz Biotechnology), NCC (1:1000; EMD Millipore, Billerica, MA), ATP synthase β (1:20,000; BD Transduction Laboratories Inc., Mississauga, ON, Canada), and β -actin (1:7500; Abcam, Inc.). Goat anti-mouse or goat anti-rabbit with horseradish peroxidase (1:2000–1:15,000; Santa Cruz Biotechnology) was used as a secondary antibody and incubated for 1 hour at room temperature. Proteins were visualized by enhanced chemiluminescence, exposed to an x-ray film, and developed with an x-ray processor (SRA-101A; Canon). The relative protein expression of each sample was first normalized to β -actin and then calculated as fold change of the controls (WT).
Statistical Analyses
BP was analyzed using one-way ANOVA repeated in time. All other data were analyzed by one-way ANOVA. The unpaired t test was used for comparisons between two groups. Tukey multiple-comparison tests were used to assess the significance of differences between means. Significance was set at a 95% confidence limit.
Disclosures
None.
This work was supported by National Institutes of Health Grants R01-DK093403, R01-HL105302, R01-HL102074, and R01-HL118558. This publication was made possible by National Institutes of Health Grant 9P20GM104934-06 from the Center of Biomedical Research Excellence Program of the National Institute of General Medical Sciences.
Published online ahead of print. Publication date available at www.jasn.org
This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2013101033/-/DCSupplemental
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