Requirement of Na⁺/H⁺ Exchanger NHE1 for Vasopressin-Induced Osteogenic Signaling and Calcification in Human Aortic Smooth Muscle Cells

Medial vascular calcification is considered an important cause of cardiovascular diseases [1-7]. In chronic kidney disease, compromised renal phosphate excretion leads to hyperphosphatemia, which in turn stimulates vascular calcification at least partly by inducing osteo-/chondrogenic reprogramming of vascular smooth muscle cells (VSMCs) [1-3, 6]. Osteo-/chondrogenic reprogramming involves upregulation of the osteogenic and chondrogenic transcription factors such as core-binding factor α-1 (CBFA1), msh homeobox 2 (MSX2), and SRY-Box 9 (SOX9) with subsequent stimulation of tissue-nonspecific alkaline phosphatase (ALPL) expression and activity [8-11]. The enzyme contributes to calcium-phosphate precipitation and tissue calcification by degradation of the calcification inhibitor pyrophosphate [8-11].

VSMCs are a target for vasopressin [12]. Vasopressin is a powerful stimulator of vascular calcification promoting osteo-/chondrogenic reprogramming of VSMCs [13, 14], which is upregulated by insufficient hydration [15, 16]. Mechanisms involved in the procalcific effects of vasopressin include activation of the serum- and glucocorticoid-inducible kinase 1 (SGK1) [17] with subsequent upregulation of store-operated Ca²⁺ entry [14, 15, 18-20]. SGK1-induced downstream targets include the Na⁺/H⁺ exchanger NHE1 [21-28], which has been shown to participate in the regulation of calcification in osteoblasts [29, 30]. Aldosterone is an activator of SGK1 and upregulates Na⁺/H⁺ exchange in VSMCs [31]. In hypertensive rats, NHE1 is upregulated in VSMCs [32]. Mice with reduced NHE1 expression are protected from abdominal aortic aneurysm induced by angiotensin-II [33]. Furthermore, NHE1 may play a role in atherogenesis [34]. At least in endothelial cells, vasopressin stimulates Na⁺/H⁺ exchange activity [35]. NHE1-dependent cellular functions also include induction of oxidative stress [36, 37]. Thus, the present study explored whether in human aortic smooth muscle cells (HAoSMCs), NHE1 and ROS generation are upregulated by vasopressin and required for stimulation of osteogenic signaling.

HAoSMCs (Thermo Fisher Scientific) were cultured in Medium 231 (Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (Gibco, Grand Island, NE, USA) and 1% penicillin/streptomycin in a humidified atmosphere at 37°C and 5% CO₂. HAoSMCs were grown to confluency and used in all experiments from passages 4 to 10. Where indicated, the cells were exposed to 100 nM vasopressin (Sigma, Steinheim, Germany) [13], to SGK1 inhibitor GSK-650394 (1 μM; Sigma) [38], and/or to NHE1 inhibitor cariporide (10 μM; Sigma-Aldrich, St. Louis, MO, USA) [24]. Vasopressin was dissolved in Medium 231, while GSK-650394 and cariporide were dissolved in DMSO. Medium pH was 7.289 ± 0.024 (control), 7.294 ± 0.022 (cariporide), 7.292 ± 0.019 (vasopressin), and 7.295 ± 0.018 (vasopressin and cariporide) after a 24-h incubation. Medium bicarbonate concentration was 18.84 ± 0.96 mM (control), 19.04 ± 0.89 mM (cariporide), 18.97 ± 0.78 mM (vasopressin) and 19.09 ± 0.74 mM (vasopressin and cariporide) after a 24-h incubation. The values were not significantly different between the absence or presence of vasopressin and/or cariporide.

HAoSMCs were transfected with 10 nM SGK1 siRNA (s740; Thermo Fisher Scientific, Karlsruhe, Germany) [17] or with 10 nM negative control siRNA (4390843; Thermo Fisher Scientific) using Lipofectamine 2000 transfection agent (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions. HAoSMCs were collected after a 48-h transfection, and silencing efficiency was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR).

To determine transcript levels, total RNA was extracted by using TriFast reagent (Peqlab, Erlangen, Germany) according to the manufacturer’s protocol. After DNAse digestion, reverse transcription of total RNA was carried out using Oligo(dT)₁₅ primer (Promega, Hilden, Germany) and GoScript^TM Reverse Transcriptase (Promega). RT-PCR amplification of the respective genes was set up in a total volume of 15 μL using 100 ng of cDNA, 500 nM forward and reverse primers, and 2× GoTaq® qPCR Master Mix (Promega) according to the manufacturer’s instructions. Cycling conditions were as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s. RT-PCR amplifications were performed on a CFX96 Real-Time System (Bio-Rad, Munich, Germany). The primers used in this study for amplification are listed in Table 1.

Specificity of PCR products was confirmed by the analysis of a melting curve. All PCRs were performed in duplicate and relative mRNA expression, which was calculated by the 2^−ΔΔCt method using GAPDH, β-actin, or B2M as an internal reference normalized to the control group.

Protein abundance of NHE1, phosphorylated and thus activated SGK1, and total SGK1 was determined by Western blotting. The harvested cells were centrifuged for 5 min at 1,000 rpm and 4°C. The pellet was washed twice with ice-cold phosphate-buffered saline (PBS) and suspended in 40 μL ice-cold RIPA lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing Halt Protease and Halt Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Danvers, MA, USA). Protein concentration was quantified using the Bradford assay (Bio-Rad Laboratories). Equal amounts of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel in Glycine-Tris buffer, electro-transferred onto polyvinylidene difluoride membranes and blocked with 5% nonfat milk in TBST at room temperature. The membranes were incubated with primary anti-SGK1 (phospho S422) antibody (1:1,000; Abcam, Cambridge, UK), anti-SGK1 antibody (1:1,000; Cell Signaling Technology), anti-NHE1 antibody (1:1,000; Sigma-Aldrich) [39], and anti-GAPDH antibody (1:1,000; Cell Signaling Technology) at 4°C overnight. After washing (TBST), the blots were incubated with secondary anti-rabbit antibody conjugated with horseradish peroxidase (1:2,000; Cell Signaling Technology) for 2 h at room temperature. Protein bands were detected after additional washes (TBST) with an ECL detection reagent (Thermo Fisher Scientific, Waltham, MA, USA). For densitometry image analysis, Western blots were scanned and analyzed by ImageJ software (NIH, Bethesda, MD, USA), and the results are shown as the ratio of total protein to GAPDH. To assign the right protein size, a protein marker (Thermo Fisher Scientific) was used.

HAoSMCs were treated with 100 nM vasopressin in the absence and presence of NHE1 blocker cariporide. Harvested HAoSMCs were washed and incubated at a final concentration of 10 μM 2′,7′-dichlorofluorescein diacetate (Sigma-Aldrich) in the dark for 30 min at 37°C. Then, cells were washed three times with PBS and subsequently resuspended in 500 μL PBS. ROS production was immediately measured by flow cytometry (BD Biosciences, Heidelberg, Germany). Fluorescence intensity of 2′,7′-dichlorofluorescein diacetate was detected and measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm. The geometric mean was calculated using FlowJo_V10 software (Version V10; TreeStar, California, USA).

HAoSMCs were exposed to 100 nM vasopressin for 7 days, in the absence or presence of SGK1 silencing, SGK1 inhibitor GSK-650394, or NHE1 inhibitor cariporide at the concentrations indicated in the figure legends. Fresh media with agents were added every 2–3 days. Alkaline phosphatase (ALP) activity in VSMCs was determined using the ALP colorimetric assay kit (Abcam) according to the manufacturer’s protocol. The results are shown normalized to total protein concentration as assessed by the Bradford assay (Bio-Rad Laboratories).

HAoSMCs were treated with 100 nM vasopressin for 14 days. Then, HAoSMCs were decalcified in 0.6 M HCl at 4°C for 24 h. Calcium content was evaluated colorimetrically by the QuantiChrom Calcium assay kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s protocol. HAoSMCs were lysed with 0.1 M NaOH/0.1% sodium dodecyl sulfate. Calcium content was normalized to total protein concentration as assessed by the Bradford assay (Bio-Rad Laboratories). To visualize calcification, HAoSMCs were exposed to 100 nM vasopressin and 1 mM CaCl₂ (Sigma-Aldrich) for 14 days for alizarin red staining. Fresh media with agents were added every 2–3 days. VSMCs were fixed with 4% paraformaldehyde and stained with 1% Alizarin Red (pH 4.0). The calcified areas are shown as red staining.

Data are provided as means ± SD, and n represents the number of experiments. Statistical analysis was performed using statistic package for social science (SPSS; Version 22.0) and GraphPad Prism (Version 8.0.2). All data were tested for significance using unpaired t test (Student’s t test) or two-way ANOVA followed by post hoc Tukey’s test for multiple comparisons. A p < 0.05 was considered statistically significant.

To investigate the effect of vasopressin on the expression of SGK1, HAoSMCs were exposed to vasopressin (100 nM). DMSO was used as the vehicle. A 24-h incubation of HAoSMCs with vasopressin elevated the transcript levels of SGK1 (Fig. 1a). Moreover, the protein expression of phospho-SGK1 (S422) (Fig. 1b, c; online suppl. Fig. 1; for all online suppl. material, see www.karger.com/doi/10.1159/000524050) normalized to GAPDH was significantly upregulated. However, the protein expression of phospho-SGK1 (S422) (Fig. 1b, d; online suppl. Fig. 1) normalized to total-SGK1 was unchanged.

In order to determine whether vasopressin modifies the expression of Na⁺/H⁺ exchanger 1 (NHE1) in VSMCs, HAoSMCs were incubated without or with vasopressin (100 nM), and NHE1 transcript levels and NHE1 protein abundance were subsequently determined by using qRT-PCR and Western blotting. As shown in Figure 2a and b and online supplementary Figure 2, vasopressin treatment significantly increased the NHE1 transcript levels and NHE1 protein abundance in HAoSMCs. In order to explore whether the effect of vasopressin on NHE1 expression involved SGK1, experiments were performed in the absence or presence of the SGK1 inhibitor GSK-650394 (1 μM). As illustrated in Figure 2c and d and online supplementary Figure 3, GSK-650394 significantly blunted the effect of vasopressin treatment on NHE1 transcript levels and NHE1 protein abundance.

To explore a role of NHE1 in oxidative stress, HAoSMCs were treated without or with vasopressin (100 nM) in the absence and presence of the NHE1 inhibitor cariporide (10 μM). As shown in Figure 3, vasopressin stimulated ROS accumulation in HAoSMCs, an effect significantly blunted by inhibition of NHE1 with cariporide.

In order to explore whether the SGK1-sensitive NHE1 regulation by vasopressin is relevant for the effects of vasopressin on osteogenic signaling, the effects of vasopressin (100 nM) on the transcript levels of osteogenic transcription factors CBFA1, MSX2, and SOX9 as well as the osteogenic enzyme ALPL were quantified after treatment of HAoSMCs for 24 h with vasopressin in the absence or presence of the SGK1 inhibitor GSK-650394 (1 μM) or NHE1 inhibitor cariporide (10 μM). As illustrated in Figure 4, each GSK-650394 and cariporide significantly blunted the effects of vasopressin treatment on CBFA1, MSX2, SOX9, and ALPL transcript levels.

As illustrated in Figure 5, vasopressin further significantly increased ALP activity in HAoSMCs, an effect significantly blunted in the presence of GSK-650394 (1 μM) or cariporide (10 μM). As shown in Figure 6, vasopressin significantly enhanced the formation of calcium deposits, an effect again significantly blunted in the presence of GSK-650394 (1 μM) or cariporide (10 μM).

Similar to pharmacological inhibition, silencing of the SGK1 gene in HAoSMCs disrupted vasopressin-induced NHE1 expression and osteo-/chondrogenic signaling. SGK1 mRNA level was significantly decreased after transfection with SGK1 siRNA (10 nM), as compared to negative control siRNA-transfected HAoSMCs (Fig. 7a). Vasopressin (100 nM) treatment upregulated SGK1 transcription in negative control siRNA-transfected HAoSMCs (Fig. 7a). The vasopressin-induced NHE1 transcript levels (Fig. 7b) and NHE1 protein abundance (Fig. 7c, d; online suppl. Fig. 4) were significantly blunted in SGK1 siRNA-transfected HAoSMCs. Furthermore, the vasopressin-induced transcript levels of CBFA1, MSX2, SOX9, and ALPL were significantly blunted in SGK1 siRNA-transfected HAoSMCs (Fig. 8a–d). Also, the increased ALP activity and the enhanced calcium deposition by vasopressin were reversed by SGK1 knockdown (Fig. 8e, f).

The transcript levels of SGK1 and NHE1 as well as CBFA1, MSX2, SOX9, and ALPL were compared not only to GAPDH but also to β-actin or B2M as an internal reference. Importantly, no pseudogenes were found in B2M [40]. As shown in online supplementary Figure 5, vasopressin significantly upregulated the transcript levels of SGK1 and NHE1 as well as CBFA1, MSX2, SOX9, and ALPL normalized to β-actin or B2M.

Similar to what has been shown in earlier studies [13, 14], the present observations demonstrate the ability of vasopressin to upregulate the expression of osteogenic transcription factors CBFA1, MSX2, and SOX9 as well as the osteogenic enzyme ALPL in VSMCs. As a result, vasopressin promotes calcium deposition in VSMCs [13, 14]. During the formation of vascular calcification, many cells, including VSMCs, transform into cells with some osteoblast-like or chondrocytes-like properties [41]. This transformation contributes to an active calcification process [14, 20]. ALP activity is an important marker for early osteoblast differentiation, which cleaves the calcification inhibitor pyrophosphate and subsequently alters the local environment to allow for calcification [41, 42]. Thus, upregulation of ALP is considered to precede and allow for vascular calcification [43, 44].

The present study further reveals the involvement of the Na⁺/H⁺ exchanger NHE1 in the upregulation of osteogenic signaling and calcium deposition in VSMCs by vasopressin. Vasopressin-induced osteogenic signaling and calcium deposition are virtually abolished in the presence of the NHE1 inhibitor cariporide. Mechanisms linking NHE1 activity to osteogenic signaling may include luminal pH in intracellular vesicles. NHE1 is inserted into phagosomal membranes and may contribute to luminal acidification of intracellular vesicles [45]. In VSMCs, luminal acidification may be involved in phosphate-induced calcification [46] and calcium-phosphate-induced cell death [47]. NHE1 may further be required for the generation of oxidative stress [48-52], which has in turn been shown to participate in the orchestration of osteogenic signaling [53-57]. In addition, NHE1 activity may be linked to production of inflammatory cytokines [58], which may contribute to osteogenic mechanisms in VSMCs [59]. However, the exact mechanisms mediating the procalcific effects of NHE1 in VSMCs require further study.

As NHE1 is tightly regulated by cytosolic pH and turned off by alkaline cytosolic pH [60], enhanced NHE1 expression counteracts cytosolic acidification but does not affect alkaline cytosolic pH. In the absence of NHE1, H⁺ generation or entry leads to cytosolic acidification. The effects of vasopressin on NHE1 expression were significantly blunted in the presence of SGK1 inhibitor GSK-650394 or SGK1 silencing. According to the present observations, vasopressin upregulates SGK1 transcription and phospho-SGK1 (S422) protein abundance in HAoSMCs. Furthermore, pharmacological inhibition or genetic silencing of SGK1 suppressed vasopressin-induced osteogenic signaling and calcium deposition.

The present paper did not elucidate the signaling mediating the upregulation of SGK1 by vasopressin. As vasopressin stimulates Ca²⁺ entry [14, 61], which in turn upregulates SGK1 [62-64], the signaling could involve increase of cytosolic Ca²⁺. SGK1 upregulates the store-operated Ca²⁺ channel Orai1 [18]; at least in theory, Ca²⁺ entry through Orai1 could further enhance cytosolic Ca²⁺, thus augmenting the effect of vasopressin on SGK1 and NHE1.

Ample experimental evidence points to a critical role of both, vasopressin and SGK1, in the pathophysiology of dehydration [15]. According to earlier studies [17], SGK1 is decisive for stimulation of osteogenic signaling by phosphate. SGK1 has further been shown to participate in the effects of vasopressin on VSMCs [14]. SGK1 has been shown to mediate upregulation of NHE1 expression [22-26, 28]. Thus, SGK1-dependent upregulation of NHE1 may contribute to calcification of VSMCs. SGK1-dependent upregulation of NHE1 may further contribute to the regulation of diverse functions in several cell types, such as aldosterone-induced renal salt retention, hypertension and cardiac remodeling [22, 23, 27, 65, 66], cell proliferation and apoptosis [24, 67], activation of dendritic cells with bacterial lipopolysaccharides or dexamethasone [21, 68], endothelial cell swelling and brain edema [28], as well as endothelial leukocyte recruitment [26]. SGK1-sensitive upregulation of NHE1 with subsequent cell swelling and cytosolic alkalinization may further contribute to stimulation of cell proliferation and counteraction of suicidal cell death [24, 67, 69-71].

The present observations may be important for vascular calcification in chronic kidney disease. A role for vasopressin in vascular calcification has already been suggested in patients with renal insufficiency. The concentrations of copeptin, considered a marker for vasopressin release [72], are related to coronary artery calcification score in patients with chronic kidney disease [73]. In type 1 diabetes, serum copeptin concentrations are associated with coronary atherosclerosis and diabetic kidney disease [74]. Furthermore, in the general population, higher copeptin levels are linked to vascular stiffness [75]. Copeptin also has been connected to mortality in elderly people [76]. Interfering with the vasopressin-calcification pathway may ameliorate vascular calcification and reduce cardiovascular risk.

Vasopressin treatment increases NHE1 expression and ROS generation, an effect requiring SGK1 and participating in the upregulation of osteogenic signaling and calcification of VSMCs.

The authors gratefully acknowledge the meticulous preparation of the manuscript by Lejla Subasic.

An ethics statement was not required for this study type, and no human or animal subjects or materials were used.

The authors state that they have no conflicts of interest.

Xuexue Zhu, Kuo Zhou, and Jibin Liu are supported by the Chinese Scholarship Council. The sponsor(s) had no role in study design, the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

Xuexue Zhu, Ke Ma, Kuo Zhou, Xia Pan, and Jibin Liu performed experiments and analyzed data; Florian Lang, Jakob Voelkl, Ioana Alesutan, and Bernd Nürnberg designed research; and Florian Lang drafted and wrote the manuscript.

All data generated or analyzed during this study are included in this published article and its online supplementary material. Further enquiries can be directed to the corresponding author.