Mononuclear Phagocyte System Depletion Blocks Interstitial Tonicity-Responsive Enhancer Binding Protein/Vascular Endothelial Growth Factor C Expression and Induces Salt-Sensitive Hypertension in Rats
We showed recently that mononuclear phagocyte system (MPS) cells provide a buffering mechanism for salt-sensitive hypertension by driving interstitial lymphangiogenesis, modulating interstitial Na+ clearance, and increasing endothelial NO synthase protein expression in response to very high dietary salt via a tonicity-responsive enhancer binding protein/vascular endothelial growth factor C regulatory mechanism. We now tested whether isotonic saline and deoxycorticosterone acetate (DOCA)-salt treatment leads to a similar regulatory response in Sprague-Dawley rats. Male rats were fed a low-salt diet and received tap water (low-salt diet LSD), 1.0% saline (high-salt diet HSD), or DOCA+1.0% saline (DOCA-HSD). To test the regulatory role of interstitial MPS cells, we further depleted MPS cells with clodronate liposomes. HSD and DOCA-HSD led to Na+ accumulation in the skin, MPS-driven tonicity-responsive enhancer binding protein/vascular endothelial growth factor C–mediated hyperplasia of interstitial lymph capillaries, and increased endothelial NO synthase protein expression in skin interstitium. Clodronate liposome MPS cell depletion blocked MPS infiltration in the skin interstitium, resulting in unchanged tonicity-responsive enhance binding protein/vascular endothelial growth factor C levels and absent hyperplasia of the lymph capillary network. Moreover, no increased skin endothelial NO synthase protein expression occurred in either clodronate liposome–treated HSD or DOCA-salt rats. Thus, absence of the MPS-cell regulatory response converted a salt-resistant blood-pressure state to a salt-sensitive state in HSD rats. Furthermore, salt-sensitive hypertension in DOCA-salt rats was aggravated. We conclude that MPS cells act as onsite controllers of interstitial volume and blood pressure homeostasis, providing a local regulatory salt-sensitive tonicity-responsive enhancer binding protein/vascular endothelial growth factor C–mediated mechanism in the skin to maintain normal blood pressure in states of interstitial Na+ and Cl− accumulation. Failure of this physiological extrarenal regulatory mechanism leads to a salt-sensitive blood pressure response.
Traditionally, researchers focus on the kidney, the brain, the heart, the adrenal gland, or the blood vessels to explain the pathogenesis of hypertension.1 A recent dietary intervention study suggested that refractory hypertension is eminently salt sensitive.2 Salt-sensitive hypertension is commonly believed to be a “renal affair.” The prevailing view on the causal relationship among salt intake, total body Na+ and fluid balance, and blood pressure (BP) follows the basic notion that extracellular bodily fluids are in equilibrium,3–5 that the kidney controls total body Na+ content and thereby the extracellular volume exclusively, and that increases in extracellular volume lead to increased BP.6,7 The underlying concept of extracellular Na+, volume, and BP homeostasis controlled by the kidney relies on the idea that interstitial Na+ is readily equilibrated with plasma Na+, can be easily mobilized into the bloodstream, and that the kidneys control interstitial Na+ and volume indirectly by means of blood purification.
In earlier studies, we questioned the widely accepted view that extracellular fluids are in equilibrium. We found instead that Na+ is stored in the skin, with higher concentrations compared to blood, in animals fed a high-salt diet (HSD).8–11 Recently, we showed that interstitial mononuclear phagocyte system (MPS) cells act as such extrarenal regulators of interstitial Na+ and water homeostasis.12 We experimentally induced hypertonic Na+ storage in the skin and interstitial MPS infiltration. In response to Na+-mediated osmotic stress, MPS cells activated tonicity-responsive enhancer binding protein (TonEBP), which binds to the vascular endothelial growth factor (VEGF) C (VEGF-C) gene. By commanding VEGF-C expression, the MPS response to osmotic stress via TonEBP results in increased density and hyperplasia of the lymph-capillary network in the interstitium and in increased protein expression of endothelial NO synthase (eNOS) in interstitial cells.
In our earlier study, we induced hypertonic Na+ accumulation, which activates the TonEBP/VEGF-C–driven regulatory cascade in MPS cells by subjecting rats to an 8% salt diet plus 0.9% sodium chloride in drinking water. Other than the compensatory MPS-driven changes in lymph capillary structure and eNOS expression, such massive dietary salt loading led to salt-sensitive hypertension in Sprague-Dawley rats. Additional experimental depletion of the MPS cells or blockade of VEGF-C with its receptors VEGF receptor 3 and/or VEGF receptor 2 blocked the regulatory response of MPS cells to interstitial Na+ accumulation and augmented salt-sensitive hypertension, suggesting that MPS cells provide a buffering mechanism for hypertension.12
Here we asked whether our initial findings were restricted to an experimental approach with massive dietary salt loading only or whether more moderate dietary salt loading with 1.0% saline in drinking water, a challenge that does not usually increase BP in Sprague-Dawley rats, leads to MPS infiltration and activation of the TonEBP/VEGF-C regulatory axis in the skin. Furthermore, we speculated that blockade of this compensatory interstitial MPS response might convert the rats from a salt-resistant to a salt-sensitive state. Finally, this study represents the first test of role of MPS on volume and BP homeostasis in rats with deoxycorticosterone actetate (DOCA) treatment and 1.0% saline in drinking water. In earlier studies, we had shown that this model features salt-sensitive hypertension associated with Na+ storage in the skin.9,13,14
Local government authorities approved the studies and the experiments, according to internationally accepted criteria. We randomly assigned male Sprague-Dawley rats aged 8 to 9 weeks to 6 groups: groups 1 and 2 were assigned to a low-salt diet (LSD; <0.1% NaCl) and tap water. Groups 3 through 6 were also fed a low-salt chow (<0.1% NaCl) but received salt loading by 1.0% saline water to drink (HSD). Groups 5 and 6 were additionally treated with 100-mg DOCA acetate pellets SC (DOCA-HSD). To assess the role of MPS cells in the control of volume and BP homeostasis, groups 2, 4, and 6 received clodronate liposomes (Clod) IP every 72 hours for MPS depletion.12,15 Two days before the end of the experiment, we placed the rats in a metabolic cage and sampled urine for 24 hours. At the end of both rat experiments after 2 weeks on their specified diets, we anesthetized the rats with 1.5% to 2.0% isoflurane anesthesia and catheterized the right femoral artery. We connected arterial lines to MLT0380/A transducers and a PowerLab 8/30 data acquisition system (ADInstruments) and measured arterial BP in conscious animals kept in a restrainer 2 hours after the operation; thereafter, blood samples were taken, the animals were killed, and skin and ear samples were taken for histology and assessment of protein and gene expression. We analyzed arterial blood gases with a clinical blood gas analyzer (Radiometer Copenhagen), including Na+, K+, and Cl− measurements by ion-selective electrodes. Chemical analysis of the carcasses included Na+, K+, Cl−, and water measurements after dry ashing of the different tissues as reported previously.8,10 We calculated the Cl− space as a measure of the extracellular volume from the tissue Cl− content and the serum Cl− concentration. The Cl−-free water space was calculated as a measure of the intracellular volume. Detailed information on the ashing protocol is provided in the online Data Supplement (please see http://hyper.ahajournals.org).
Immunohistochemistry and Immunofluorescence Staining of MPS Cells and Lymphcapillaries
We performed all of the staining using the Avidin/Biotin Blocking kit (Vector Laboratories) and horseradish peroxidase super staining kit (ID Laboratories) according to the manufacturer’s instructions (please see the online Data Supplement). After staining with specific antibodies, lymph capillaries (anti-podoplanin antibody, from D. K.), MPS cells (anti-CD68 antibody, MCA1029, AbD Serotec), and VEGF-C–positive cells (anti–VEGF-C, ab9546, Abcam) were counted throughout the whole diameter of a crosswise cut rat ear. We counted lymphcapillaries in 5 consecutive sections and MPS and VEGF-C–positive cells in 6 consecutive sections and normalized capillary and cell counts per field.
Immunofluorescence of Endothelial NO Synthase Expression
We performed indirect immunofluorescence for expression of endothelial NO synthase (eNOS) in the ear with a rabbit polyclonal anti-eNOS antibody (PA1–037, Affinity BioReagents), as reported previously.12 We analyzed specimens using a Zeiss Axioplan-2 imaging microscope with the digital image processing program AxioVision 4.3 (Zeiss).
TonEBP, VEGF-C, and Tumor Necrosis Factor-α Gene and Protein Expression
We quantified mRNA expression in skin samples by real-time PCR, as reported previously (please see the online Data Supplement for details on primer sequences).12 VEGF-C protein expression in skin samples was quantified by Western blot analysis (VEGF-C–specific antibody, ab9546, Abcam). Serum tumor necrosis factor (TNF)-α levels were measured by ELISA, according to the manufacturer’s instructions (Abcam).
Comparison of means of data from animal experiments was calculated by multivariate or univariate analysis using the generalized linear measurements procedure. We tested for the effect of diet, DOCA treatment, and Clod treatment. All of the data in the article are presented as average±SD. Statistical analysis was performed with the SPSS software (version 17.0).
Experimental MPS Depletion Induces Salt Sensitivity in Rats
BP in the rats did not increase with HSD (Figure 1A). However, BP increased when HSD rats were given Clod; the BP increase was composed of ≈10 mm Hg. In LSD rats, Clod did not increase BP. These findings suggest that MPS depletion with Clod indeed blocked a compensatory mechanism to maintain basal BP in HSD-treated rats. DOCA-HSD rats developed salt-sensitive hypertension that was paralleled by increased skin Cl− content and extracellular volume (ECV) retention in the skin (Figure 1A and 1B). Adding Clod increased BP and Cl− and extracellular volume retention in the skin further. These findings suggest that salt-sensitive hypertension in DOCA-HSD rats was paralleled by electrolyte and volume retention in the rats and that MPS depletion with Clod further augmented hypertension and volume retention in the skin.
Other than the dramatic changes in body electrolyte and water composition, DOCA treatment is known to induce an inflammatory state. Accordingly, DOCA-HSD and DOCA-HSD-plus-Clod–treated rats exhibited less weight gain (Table) and showed tissue Na+ retention, water retention, and tissue K+ loss (Table), as well as hypokalemia, metabolic alkalosis (Table), increased serum TNF-α levels, and increased TNF-α mRNA expression in the skin (Figure 1C). This inflammatory response was paralleled by proteinuria in the DOCA-HSD rats, which was aggravated by Clod. In contrast to DOCA-HSD hypertension, the de novo salt sensitivity induced in HSD rats by Clod treatment was paralleled solely by a modest increase in urinary protein excretion (Table) and no increase in TNF-α expression.
Salt Loading Induces MPS/TonEBP/VEGF-C–Driven Lymphocapillary Network Hyperplasia and Increases eNOS Expression
HSD and DOCA-HSD increased interstitial MPS and VEGF-C–positive cells in the cutaneous interstitium of the rat ear (Figure 2A and Figure S2 in the online Data Supplement, please see http://hyper.ahajournals.org). The MPS accumulation with HSD and with DOCA-HSD was accompanied by slight Na+ accumulation without a detectable increase in water content in the skin in HSD rats and by pronounced Na+ and water retention in the skin in DOCA-HSD rats (Table). Skin Na+ accumulation in rats with HSD alone and in DOCA-HSD rats led to an increased Na+:water ratio in the skin (Table) and was paralleled by increased mRNA expression of TonEBP in the skin (Figure 2B), where the (Na++K+):water ratio was significantly higher than in plasma (Table). This state of affairs suggests that osmotic stress is a critical feature in this cutaneous interstitium microenvironment accumulating salt. TonEBP induces VEGF-C expression. Consequently, both HSD and DOCA-HSD increased VEGF-C mRNA (Figure 2B) and the VEGF-C protein expression in the skin (Figure 3). Activation of the TonEBP/VEGF-C regulatory axis was paralleled by increased podoplanin mRNA expression, which is a marker protein of lymph endothelial cells (Figure 2B), and was associated with hyperplasia of the lymph-capillary network in HSD and DOCA-HSD rats (Figure 2A and Figure S3), featuring increased interstitial lymph capillary count in rat ear samples. Furthermore, increased TonEBP/VEGF-C levels were paralleled by increased eNOS protein expression in the skin (Figure 4A), which did not colocalize with the lymph capillary network (Figure 4B), indicating that the lymph endothelium was not a source of increased capillary eNOS expression, suggesting that increased eNOS protein expression with HSD or DOCA-HSD originated from blood capillary endothelial cells.
Experimental MPS Depletion Prevents the Interstitial Compensatory Response to Dietary Salt
Clod effectively depleted MPS cells into the cutaneous interstitium (Figure 2A and Figure S2). In both HSD rats and DOCA-HSD rats, experimental MPS depletion with Clod blocked infiltration of VEGF-C–positive MPS cells into the cutaneous interstitium (Figure 2A). In parallel, MPS depletion in HSD and DOCA-HSD rats reduced TonEBP and VEGF-C mRNA expression (Figure 2B), as well as VEGF-C protein expression (Figure 3) in the skin, to control levels. In the absence of the TonEBP/VEGF-C response in HSD and DOCA-HSD rats with MPS depletion, the rats no longer showed hyperplasia of the lymph capillary network in the skin (Figure 2A and Figure S3), and eNOS protein expression was reduced to the control level (Figure 4). In DOCA-HSD rats, absence of hyperplasia of the lymphocapillary network was paralleled by further interstitial Cl− (Figure 1A) and volume retention (Figure 1B), as well as further BP increase. These findings suggest that MPS cells are required to maintain BP at low levels in states of NaCl accumulation. MPS cells either induce hyperplasia of the lymph capillary network, which facilitates mobilization of interstitial volume, or increase eNOS protein expression in response to isotonic dietary salt loading and provide a buffering mechanism for hypertension via a TonEBP/VEGF-C regulatory axis.
The important finding in our study is that we demonstrate conversion from a salt-resistant BP state to a salt-sensitive BP state by depleting MPS cells from the body with Clod. Clod with LSD had no influence on BP. We also found that the DOCA-HSD model (without uninephrectomy) was made even more salt sensitive by depleting MPS cells. Our new findings in animals given oral 1% saline support the physiological relevance of our recent contention that MPS cells act as regulators of volume and BP homeostasis via a TonEBP/VEGF-C regulatory mechanism. We contend that this mechanism buffers the development of salt-sensitive hypertension, either by increased lymph capillary transport capacity of interstitial fluid or by compensatory increases in eNOS expression.
We have proposed such a regulatory mechanism previously; however, the induction of salt-sensitive hypertension and the regulatory MPS response in that study had been induced by massive hypertonic salt administration that possibly had little relevance to established models.12 We now found that more moderate salt loading by oral 1.0% saline results in TonEBP expression, which acts as an “osmoprotective” gene in skin MPS cells.16,17 The ratio of (Na++K+):water, representing >95% of the potentially osmotically effective cations in the body, was 20% to 26% higher in skin than in plasma. The finding seems peculiar; however, we have made similar observations in other models previously.8–11,13,14 Furthermore, an increase in the skin Na+:water ratio occurred without any changes in plasma electrolyte concentrations in rats given 1% saline to drink. This finding indicates that the skin interstitium, in which MPS cells reside, represents a separate tissue-specific, extracellular microenvironment that is not necessarily represented by changes in serum electrolyte concentrations. Furthermore, this state of affairs suggests that extracellular fluids in the intravascular and interstitial compartments are not invariably in equilibrium. In the absence of detectable changes in serum Na+ and K+ concentrations, an increased skin Na+:water ratio was paralleled by altered gene expression of the osmoprotective transcription factor, TonEBP, in skin MPS cells. This finding indicates that skin Na+ accumulation leads to interstitial osmotic stress that initiates a local, MPS-driven, regulatory response. Our finding that skin Na+ accumulation creates an interstitial microenvironment where osmotic stress is a critical feature of MPS cell function receives support from Go et al,18 who found that osmotic stress is a critical feature for lymphatic function. Their data suggest that immune cells residing in the interstitial space, even without dietary salt loading, confront a microenvironment that is characterized by hypertonicity compared with plasma. We are aware that we must develop accurate novel methods to measure osmolality in these compartments in vivo. However, in vitro cell-based experiments render support for our conclusion.12,19
We showed earlier that activation of the TonEBP/VEGF-C regulatory axis induced hyperplasia of the lymph capillary network in the skin and increased eNOS protein expression in interstitial endothelial cells.12 We were able to corroborate those findings here. In addition, we could show that the increase in skin eNOS protein expression is not explained by increased eNOS expression in the lymph capillary network, because eNOS protein and podoplanin protein as a marker for lymph-endothelial cells did not colocalize with eNOS expression. We conclude that increased eNOS protein expression, which we have shown previously to be MPS and VEGF-C dependent,12 does not originate from increased lymph endothelial cell mass in the lymph capillary network. This finding is important, because lymph endothelial cells have the potential to express eNOS in vitro and in vivo.20–22
MPS cells are diverse and develop into morphological and functional distinct cell types in response to the tissue microenvironment. Although MPS cells are uniformly found in hypertensive target-organ damage lesions, the relative contribution of the MPS cell subsets to the “inflammatory” response in atherosclerosis is complex.23–26 Tipping the balance of macrophage polarization into more proatherogenically activated M1 subtypes or rather more antiatherogenic programs in M2 subtypes is considered to affect pathogenesis, evolution, and complications of target organ damage.25 Our data provide preliminary evidence that MPS cells may act as regulators of volume and BP homeostasis in response to dietary salt loading without inducing a concomitant inflammatory response. The findings support the idea that MPS/TonEBP/VEGF-C activity might represent an “M2 feature” of MPS cell function. TNF-α is a typical indicator for M1-type polarization of MPS cells. In our rats, MPS cell infiltration into the skin in response to HSD induced the TonEBP/VEGF-C regulatory axis without concomitant TNF-α gene and protein expression from the MPS. Furthermore, the BP increase after nonselective MPS blockade with Clod did not increase TNF-α in HSD rats. In contrast, interstitial MPS infiltration in HSD rats with additional DOCA treatment, which induces salt-sensitive hypertension and target organ damage in the long term in the rats, was paralleled not only by increased TNF-α expression but also led to proteinuria and reproducibly leads to stunted growth with decreased tissue weights, as shown in the Table and in our previous studies in DOCA-salt rats.9,13,14 These findings suggest that DOCA-HSD treatment induces a chronic inflammatory state. Interestingly, the TNF-α gene in DOCA-HSD rats remained highly expressed after additional nonselective MPS depletion with Clod. Moreover, proteinuria worsened with BP increase, whereas the TonEBP/VEGF-C regulatory response of the lymph capillary network and eNOS protein expression were blocked. These findings suggest that MPS cells contribute to prevent salt-induced hypertension and that interstitial MPS infiltration may be beneficial by providing a net protective effect against hypertension-induced target organ damage.
We suggest expanding the horizon of salt-sensitive BP research to include a third compartment: novel molecular regulators, MPS involvement, and local lymph and blood capillary participation. We present a novel, albeit unconventional, schema featuring MPS cells that modulate BP via TonEBP/VEGF-C and a vascular bed hitherto fore largely ignored, namely, the lymphocapillary network. It remains to be investigated whether the salt-sensitive BP increase that we found by the acute method of BP measurement in restrained animals is present in chronically instrumented animals as well. Radiotelemetric BP measurements may answer this question and also may provide information on the time course of the BP increase after experimental MPS depletion. Furthermore, it is tempting to speculate that animals with VEGF-C overexpression are protected from developing hypertension. Vice versa, it may be speculated that selective VEGF receptor 3 blockade, a favorite concept in cancer therapy, may increase BP by blocking MPS regulatory activity in volume and BP homeostasis. Finally, the fact has not escaped us that human subject research is required in this area. Microdialysis techniques and magnetic resonance–based 23Na methods should be helpful and are areas that we are pursuing.
We thank Elke Prell, Birgit Hausknecht, Monika Klewer, and Esther Ermeling for their technical assistance.
Sources of Funding
This study was supported by grants from the German Research Foundation (Ti345/2) and the Federal Ministry of Economics and Technology/German Aerospace Center (BMWi/DLR; 50WB0920) to J.T. D.N.M. was supported by a Helmholtz Fellowship.
- Received September 18, 2009.
- Revision received October 7, 2009.
- Accepted January 11, 2010.
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