Hypertension and chronic kidney disease (CKD) are two major risk factors for cardiovascular (CV) disease ^[1]. CKD patients often experience varying degrees of elevated blood pressure; its incidence accounts for about 19.6% to 57.7% of all kidney diseases, and a few patients can develop malignant hypertension ^[2]. Once hypertension occurs, it can further damage the renal function and encourage blood pressure to continue to rise, forming a vicious circle ^[3]. A survey from developed countries shows that 47% of hypertensive patients over the age of 20 have uncontrolled blood pressure ^[3], and the control rate is even lower in developing countries.

Therefore, effective blood-pressure control in hypertensive patients with chronic kidney disease is of great significance to prevent the progression and deterioration of renal disease ^[4]. The coexistence of hypertension and CKD makes it more difficult to control blood pressure levels. Salt sensitivity is an independent risk factor for cardiovascular disease after hypertension. About half of the hypertensive population and one-quarter of the normotensive population exhibit blood pressure (BP) sensitivity to salt ^[5]. The pathogenesis of hypertension in CKD is complex, and its occurrence and development are affected by many factors. Sodium-imbalance-increased sympathetic nervous system (SNS) activity and changes in the renin-angiotensin system (RAAS) are some of the key pathogenic mechanisms ^[6]. An important target of these mechanisms is NCC. NCC activity is highly regulated by a complex signaling network, and these kinases appear to be sensitive to changes in hormonal and physiological environments ^[7]. They regulate Na-Cl cotransporter (NCC) in three main ways, namely, by regulating the level of total protein synthesis and transport and the phosphorylation level of NCC. The change in the total protein level of NCC is a chronic reaction of the body, which can be caused by long-term sodium and chloride imbalances in the body. Transportation and phosphorylation levels are rapid responses, and the two mechanisms are independent of each other. The phosphorylation state of NCC is the main form of its function, and only the phosphorylated NCC expressed on the cell membrane can function ^[8]. The regulatory mechanisms of NCC phosphorylation are the most frequently studied, such as protein phosphatase-4 to dephosphorylate NCC; serum and glucocorticoid-regulated kinase 1(SGK1), aldosterone can promote NCC phosphorylation (Susa et al., 2012), and so on. It was also reported that NCC phosphorylation is also regulated by NCC ubiquitination, that is, a decrease in NCC ubiquitination leads to an increase in NCC phosphorylation and an increase in NCC phosphorylation downregulates NCC ubiquitination ^[9].

2. NCC Is Regulated by a Variety of Kinases and Proteins

The total length of the distal convoluted tubule (DCT) of the kidney is less than 0.6 mm, and it can reabsorb 5 to 10% of the glomerular filtrate. The DCT is divided into two parts: the c-initial phase, expressing NCC exclusively in the apical membrane, and the late phase ^[10], which is the transition part between the DCT1 and the connecting tubule/collecting duct (CNT/CD). The DCT2 apical membrane contains NCC, epithelial sodium channel (ENaC), and renal outer medullary potassium channel (ROMK) ^[11]. The thiazide-sensitive NCC derived from the bladder of winter flounders is the first electrically neutral co-transporter protein to have been identified at the molecular level ^[12]. DCT enables different functions of each fragment through specific regulators, such as Ste20-related Proline Alanine-Rich Kinase (SPAK), Cullin 3(CUL3), Kelch-like protein 3 (KLHL3), and neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4-2). The dysfunction of NCC proteins triggers a series of water–sodium imbalances and blood-pressure abnormalities; therefore, NCC’s activity plays an important role in cardiovascular physiology and pathophysiology, and as a microregulator of renal sodium excretion, the relationship between NCC function and the pathological process of hypertension has received continuous attention.

2.1. WNK

With-no-lysine kinase (WNK) (rat WNK1) was first cloned by the PCR cloning technique when identifying new members of the mitogen-activated protein kinase kinase (MEK) family ^[13]. As the main regulator of NCC, its importance is self-evident. WNK kinases play important roles in the control of salt homeostasis and blood pressure. Each of the WNK kinases can interact at the protein level ^[14]. After the identification of WNK1 from rat kidneys, it was soon observed that the WNK kinases consist of WNK1, WNK2, WNK3, and WNK4 ^[15]. In the kidneys, WNK1 is expressed in two forms: kidney-specific-WNK1 (KS-WNK1) and long (kinase-active) WNK1 (L-WNK1). L-WNK1 whose transcription starts in exon 1, contains the kinase domain and is expressed at low levels in the kidney. The KS-WNK1 is due to its kidney-specific expression starting at the polypeptide sequence encoded by exon 5. KS-WNK contains no kinase domain ^[16]. L-WNK1 activates epithelial sodium channels and NCC, thus activating sodium reabsorption. KS-WNK1 is thought to inhibit L-WNK1 kinase activity and its effect on NCC through a dominant-negative effect ^[16]. However, given the recent studies suggesting a role for KS-WNK1 in the response to hypokalemia, KS-WNK1 knockout mice may have a potassium-loss phenotype-induced NCC activation that can be compensated by WNK4 ^[17]. Furthermore, in X. laevis oocytes, KS-WNK1 expression encourages NCC activation despite the lack of a kinase domain ^[16]. WNK4 appears to be a negative regulator of NCC in some cases. However, sometimes, it is a positive regulator in others ^[14]. Wild-type WNK4 inhibits NCC activity by interfering with the forward transport of NCC towards the cell membrane; thus, the lysosomal pathway, rather than the clathrin-mediated endocytic pathway, degrades NCC to inhibit the expression of NCC on the cell membrane and reduces the activity of NCC ^[18]. NCC interacts with the adaptor protein complex 3(AP-3), AP-3 is involved in the transport of lysosomes, and the participation of WNK4 can enhance the interaction between the two, which also indicates that WNK4 can stimulate the interaction between NCC-AP3; therefore, it also supports the transport of NCC to the lysosome ^[19]. As shown by Mu et al. ^[20], exogenous catecholamine inhibits histone deacetylases containing negative glucocorticoid-responsive elements (nGRE). This stimulates the β2- AR and acetylates the histones. Thus, WNK4/SPAK transcription is reduced and NCC activity is activated, leading to hypertension. In a study by San-Cristobal et al. ^[15], the authors used Xenopus laevis oocytes with clones and mutagenesis to show that angiotensin II converts WNK4 from an inhibitor to an activator of NCC. In normal or enlarged intravascular volume, the renin–angiotensin system is suppressed, and WNK4 reduces the amount of NCC in the plasma membrane by inhibiting NCC translocation. In the context of reduced intravascular volume, RAAS is activated and angiotensin II signaling alleviates the inhibition of NCC by WNK4, resulting in increased NCC activity. In their study, O’Reilly et al. ^[21] used male C57BL/6 mice with a specific Na-and-K diet and excess aldosterone via minipump or adrenalectomization to abolish aldosterone production and showed that renal WNK4 expression was upregulated on a high-potassium diet and downregulated on a low-sodium diet, while its mRNA level was not significantly increased with aldosterone treatment, suggesting that WNK4 acts as a regulator to adapt to changes in K⁺ and Na⁺ balance. Among other WNK isoforms, WNK3 is also expressed in the kidneys and only in the aldosterone-sensitive distal nephron, whereas WNK2 is not expressed, and the regulation of WNK2-expression levels is currently poorly understood ^[22]. Compared with the inhibitory activity of WNK4, kinase-activated WNK3 is a potent activator of Na-K-Cl cotransporter 2 (NKCC2) and NCC, and kinase-inactivated WNK3 is a potent inhibitor of NKCC2 and NCC activity ^[23]. The evidence for the effect of WNK3 on these transporters and their co-expression in renal epithelial cells suggests that WNK3 is associated with NaCl, water, and blood-pressure stabilization ^[23]. NCC and NKCC are related kidney-specific transporters that mediate apical NaCl reabsorption in the thick ascending limb and distal convoluted tubule, respectively. WNK3 regulates the activity of these transporters by altering their expression at the plasma membrane ^[23]. WNK kinases not only directly regulate the ion-transport pathway but also form a signaling complex with each other, and WNK3 can modulate the effects of WNK1 and WNK4 on NCC activity ^[14]. Increased WNK4 abundance can increase the WNK4/WNK3 molar ratio, thus inhibiting NCC activity both directly (by the effect of WNK4 on NCC) and indirectly (through the effect of WNK4 on WNK3) ^[24]. As a controllable variable resistor amplifier, the WNK kinase complex can adjust the activity of NCC in a cascade according to physiological needs ^[18]. In their study, Cary R. et al. ^[25] showed that the WNK bodies that were created by potassium imbalance in DCT were unique KS-WNK1-dependent structures of WNK signaling complexes. They proposed that WNK body formation is associated with the normal physiology of the distal nephron, which is an evolutionarily conserved manifestation of the renal response to potassium stress. Martin N. et al. ^[26] used genetically engineered mice carrying mutations of WNK-SPAK/OSR1-pathway proteins with varying-K⁺-and-NaCl-content diets to provide evidence that WNK bodies play a key functional role during changes in plasma K⁺ concentration via WNK4-induced SPAK/OSR1 activation.

2.2. Ubiquitination

Kinases induce protein phosphorylation at specific threonine, serine, or tyrosine residues. Several membrane proteins, including transporters and channels in the kidneys, are regulated by ubiquitination. The regulation of NCC activity by ubiquitination is an emerging area of interest for researchers, and these ubiquitin (Ub) ligases may have a greater impact on NCC than WNK ^[8]. For example, Ishizawa et al. ^[27] demonstrated in low-potassium-fed mice that KLHL3/CUL3-based ubiquitin ligases are involved in low K⁺-mediated NCC activation, a physiological adaptation that reduces distal electrical Na⁺ reloading absorption, preventing the further loss of K⁺ by the kidneys but promoting high blood pressure. The in vivo and in vitro findings in Penton et al.’s study ^[28] showed that cyclic adenosine monophosphate (cAMP) increased NCC phosphorylation through the protein kinase A (PKA)-dependent phosphorylation of protein phosphatase 1 inhibitor–1 (I1) and subsequent protein phosphatase 1(PP1) inhibition, and the PKA-mediated phosphorylation of KLHL3 at S433 reduced KLHL3-dependent the ubiquitination and degradation of WNK4, a pathway that may be involved in the physiological regulation of renal sodium processing by cAMP-elevating hormones and may contribute to salt-sensitive hypertension in patients with endocrine dysregulation or sympathetic hyperactivity. In addition to KLHL3 and CUL3, Nedd4-2 can also stimulate the ubiquitination of NCC. Ronzaud et al. ^[29] provided evidence that the inactivation of Nedd4L exons 6 to 8 in adult mouse Nedd4L-KO (Nedd4LPax8/LC1) renal tubules do not result in the Liddle-syndrome phenotype associated with elevated ENaC activity; rather, it results in a salt-sensitive Pseudohypoaldosteronism Type II (PHAII)-like syndrome that is characteristic of the upregulation of NCC, increasing blood pressure and hypercalciuria. NEDD4-2 is not important for the regulation of renal ENaC, as low plasma aldosterone leading to the reduced proteolytic cleavage of αENaC may be sufficient to counteract the increased abundance of β- and γENaC in Nedd4LPax8/LC1, and, based on these results, NEDD4-2 appears to target NCC primarily.

2.3. Regulation by Several Other Proteins and Kinases

In addition to the regulation of kinases and ubiquitinated proteins, NCC is also regulated by several other proteins and kinases. Ueda K. et al. ^[30] showed that ENaC and NCC, but not NKCC2, were activated in kidney-specific corticosteroid 11-β-dehydrogenase isozyme 2(Hsd11b2) knockout mice, suggesting that hypokalemia-induced NCC activation augments the renal-ENaC-activation-induced elevation of BP. HSD11B2 converts glucocorticoids to their inactive form and maintains the sensitivity of MR to mineralocorticoids. in the apparent mineralocorticoid excess syndrome Hsd11b2 has a specific variation. In their animal model of salt-dependent hypertension, the antihypertensive effect of potassium supplementation was attributable to urinary sodium excretion ^[30]. These results strengthen the hypothesis that impaired renal function, rather than vascular dysfunction, contributes to the development of salt-dependent hypertension caused by kidney-specific deletion of the Hsd11b2 gene. Gholam et al. ^[31] used Mouse DCT15 cells to confirm that Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) not only indirectly regulates NCC through filamin A but also directly phosphorylates and regulates NCC activity, and the binding between CaMKII and NCC may weaken the interaction between NCC and NEDD4-2 effect, thereby reducing NCC degradation. The binding between filamin A and NCC may also enhance the protein’s interaction with other signaling proteins, thereby further regulating sodium and chloride transduction in DCT2. The phosphorylation of filamin A by CaMKII results in a reorganization of the actin cytoskeleton and low basal NCC activity at the luminal membrane of a DCT2 cell. A deficiency in CaMKII or the pharmacological inhibition of CaMKII by KN93 blocks the phosphorylation of filamin A, resulting in a dense actin cytoskeleton and an accumulation of the active phosphorylated form of the NCC at the luminal membrane. Tokonami et al.’s ^[32] data support the notion of a role for uromodulin in functional heterogeneity along with the DCT: Uromodulin (UMOD)-/-mice show less phosphorylation NCC (pNCC) in DCT1 and increased pNCC in DCT2 concomitantly with DCT2 extension. When exposed to chronic distal salt loads (furosemide), UMOD-/- mice displayed a severely diminished ability to increase NCC phosphorylation and lacked the ability to upregulate the pNCC in DCT1 and the structure in DCT2 regions compared to UMOD+/+ mice. Various studies indicate the substantial plasticity of the DCT. Lalioti et al. ^[33] used the RPCI-22 female 129S6/ SvEvTac mouse that was screened by the hybridization to radiolabeled PCR-amplified probes of the mouse Wnk4 gene to demonstrate that the DCT plasticity was essentially driven by the activity of NCC, with corresponding alterations in electrolyte and blood-pressure control. Loffing et al. ^[34] used adult male Wistar rats to investigate whether cell proliferation contributes to the salt-load-induced hypertrophy of distal tubules. The data demonstrated that the DNA synthesis rate markedly increased in the DCT and the following segments in vivo varied in parallel with changes in their salt-transport activity and increased DNA synthesis; thus, cellular proliferation is probably a component of the structural response of nephron segments following increased salt-transport activity. The question of whether these structural changes result from the lack of uromodulin acting as a trophic factor for the DCT1 or reflect changes in NCC activity requires further investigation.