*3.3. Na+–K+–2Cl*<sup>−</sup> *Cotransporter1 (NKCC1)*

NKCC1 located on vascular smooth muscle cells functions to accumulate intracellular Cl− [20,21]. The most compelling evidence of the functional role of NKCC1 in the regulation of vascular tone and arterial blood pressure comes from studies on NKCC1 knockout mice: the systolic blood pressure was significantly reduced in NKCC1 knockout mice compared to wild-type mice [94], and treatment with bumetanide, an inhibitor of NKCC1 [95], inhibited the vascular contractile activity and lowered mean arterial blood pressure in wild-type mice, with the effects being lost in NKCC1 knockout mice [94,96]. Thus, theoretically, an increase in the activity of the vascular smooth muscle NKCC1 could augment vascular contractility and subsequently lead to enhanced blood pressure, and this is indeed the case in several types of hypertensive rats.

In some experimental models of hypertensive rats, including SHRs [97–99], Milan hypertensive rats [100] and deoxycorticosterone acetate (DOCA) salt hypertensive rats [101], increase in the activity of NKCC in vascular smooth muscle cells has been reported (Table 3). Interestingly, Lee et al. reported that the mRNA and protein expression levels of NKCC1 were epigenetically upregulated in the aorta of SHRs due to *Nkcc1* gene promoter hypomethylation [102], and the *Nkcc1* gene promoter hypomethylation resulted from the decreased activity of DNA methyltransferase 3B [103] (Table 3). Likewise, an epigenetic up-

regulation of NKCC1 via histone modifications was reported in the aorta of Ang II-induced hypertensive rats [104] (Table 3).

**Figure 3.** Upregulation of endothelial TMEM16A impairs endothelial function in hypertension. In hypertension, the expression and function of vascular endothelial Ca2+-activated Cl<sup>−</sup> channel TMEM16A are increased. Endothelial stimulation with agonists and shear stress increases the intracellular Ca2+concentration, which subsequently activates endothelial small-conductance Ca2+-activated K+ channels (SKCas), intermediate-conductance KCa (IKCa) and TMEM16A simultaneously. The endothelium-dependent hyperpolarization (EDH) through the activation of both SKCa and IKCa is reduced by the opposing membrane depolarization evoked by the activation of TMEM16A. In addition, activation of TMEM16A may facilitate the generation of reactive oxygen species (ROS) through Nox2-containing NADPH oxidase, leading to reduced bioavailability of nitic oxide (NO). Impaired EDH and/or NO could be at least partly responsible for the blood pressure rise in hypertension.

**Table 3.** Alterations in vascular smooth muscle NKCC1 during hypertension.


NKCC1, Na+–K+–2Cl<sup>−</sup> cotransporter 1; SHRs, spontaneously hypertensive rats; DOCA, deoxycorticosterone acetate; Ang II, angiotensin II.

In addition to the epigenetic upregulation of NKCC1, another factor may also contribute to the increase in the activity of NKCC1 during hypertension. In fact, some studies have suggested the possible positive regulation of vascular NKCC1 by with-no-lysine kinase (WNK) and sterile-20-related praline–alanine-rich kinase (SPAK): heterozygous WNK1 knockout mouse aorta exhibited reduced phosphorylation of downstream SPAK and NKCC1, leading to decreased responses to vasoconstrictive stimuli [105]. Similarly, the aorta of SPAK knockout mice exhibited reduced phosphorylation of NKCC1 and decreased NKCC1-mediated vascular constriction, and the SPAK knockout mice had low blood pressure [106]. Moreover, activation of the WNK3/SPAK/NKCC1 pathway has been shown to be involved in both the Ang II-induced aortic constriction and Ang II-induced blood pressure rise in mice [107].

These observations suggest that the WNK/SPAK signaling pathway positively regulates the vascular NKCC1 toward vasoconstriction and hypertension. Interestingly, mutations of WNK have been found in patients with familial hyperkalemic hypertension, a form of monogenic hypertension [108]. Nevertheless, there is no evidence to date that demonstrates changes in the WNK/SPAK pathway in animal models of polygenic hypertension such as SHRs or in human essential hypertension.

The studies mentioned above have demonstrated that the expression and/or the function of NKCC1 are upregulated in vascular smooth muscle cells of hypertensive rats. Then, the question arises how the upregulation of NKCC in hypertension contributes to the augmented vascular contractility and increased blood pressure. It has been reported that the intracellular concentration of Cl− is increased in vascular smooth muscle cells of DOCA salt hypertensive rats because of the increase in the activity of NKCC [101].

The increase in the intracellular Cl− concentration would increase the driving force for Cl− efflux via Cl− channels such as CaCCs upon vasoconstrictor stimulation, and the increase in Cl− efflux would make the membrane potential more depolarized [20], which in turn would enhance the open probability of voltage-gated L-type Ca2+ channels, leading to an increase in vascular tone. In support of this notion, we have shown that the inhibition of the NKCC with bumetanide, an inhibitor of NKCC1 [95], significantly reduced the CaCC-mediated membrane depolarization and constriction in vascular smooth muscle of SHRs [86]. Since, as discussed in the previous section, CaCCs are also functionally upregulated in the vasculature of hypertensive rats, we propose that the enhanced activities of NKCC1 and CaCCs act additively and sequentially to increase vascular contractility and hence blood pressure in hypertension (Figure 4).

**Figure 4.** Upregulation of smooth muscle Na+–K+–2Cl<sup>−</sup> cotransporter 1 (NKCC1) and TMEM16A additively

and sequentially increases vascular contractility in hypertension. In hypertension, the intracellular concentration of Cl− is increased in vascular smooth muscle cells because of the increased activity of NKCC1. The increase in the intracellular Cl− concentration then increases the driving force for Cl− efflux via the Ca2+-activated Cl<sup>−</sup> channel TMEM16A when TMEM16A is activated by intracellular Ca2+ rise upon stimulation with vasoconstricting agonists, which in turn induces membrane depolarization. TMEM16A might be regulated by a local Ca2+ increase that could be generated by IP3R channels on the sarcoplasmic reticulum (SR) and/or transient receptor potential (TRP) channels. The membrane depolarization would then enhance the open probability of voltage-gated L-type Ca2+ channels, leading to an increase in vascular contractility and blood pressure.

#### **4. Clinical Perspectives**

While many animal studies suggest that the upregulation of TMEM16A and NKCC1 could contribute to the increased vascular contractility and elevated blood pressure in hypertension as mentioned in the preceding sections, there is very little information concerning their possible involvement in the pathogenesis of hypertension in humans. Interestingly, however, two independent population-based studies reported that some genetic variants of TMEM16A were significantly associated with hypertension in humans [57,109]. Further exploration of the functional impact of the SNP in the TMEM16A coding region could provide a clue to understand the pathophysiological role of TMEM16A in human hypertension.

In addition, it has been reported that there was a positive association between hyperchloremia and in-hospital mortality in hospitalized patients [110]. Moreover, in a recent study in patients with chronic kidney disease, hyperchloremia was an independent predictor of hypertension and proteinuria [111]. Taking these observational studies together, it might be possible to speculate that hyperchloremia might lead to blood pressure elevation and hence to poor prognosis. By contrast, in outpatients with hypertension [112] or chronic heart failure [113], hypochloremia was a predictor of mortality [112,113] but the level of Cl− was not associated with the level of blood pressure [112]. Thus, while these findings indicate that serum Cl− alterations are associated with poor prognosis in patients with elevated cardiovascular risk, the ability of changes in serum Cl− concentration to affect blood pressure is not clear. Further studies are needed to clarify the role of serum Cl− concentrations on blood pressure regulation and its association with long-term prognosis in patients with elevated cardiovascular risk.

#### **5. Conclusions**

Accumulating experimental evidence suggests that Cl− plays an important role in the regulation of vascular tone through its ability to depolarize vascular smooth muscle cells, and the increased contribution of Cl− to arterial constriction appears to be associated with the development and progression of hypertension. Of note, there is a growing body of evidence that the upregulation of CaCC TMEM16A in the vasculature contributes to the increased vascular contractility and elevated blood pressure in genetically hypertensive rats. In addition, the increased activity of NKCC1 may also promote hypertension as the result of a robust accumulation of intracellular Cl− in vascular cells.

Nevertheless, much remains to be determined about the precise molecular mechanisms underlying the increased activity of TMEM16A and NKCC1 as well as their interactions with other signaling pathways during hypertension, and most importantly the pathophysiological roles of these molecules in human hypertension. Further exploration of the arterial tone regulation by Cl− may facilitate a better understanding of the pathogenesis of hypertension, which may help to develop a novel therapeutic strategy to tackle hypertension and hypertension-associated cardiovascular diseases.

**Author Contributions:** K.G. was responsible for the conceptualization, literature research, and writing of the manuscript. T.K. assisted in the writing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by JSPS KAKENHI grant number JP21K11023. The APC was funded by JSPS KAKENHI grant number JP21K11023.

**Acknowledgments:** This work was supported by JSPS KAKENHI Grant Number JP21K11023.

**Conflicts of Interest:** The authors declare no conflict of interest.
