Through a Glass Darkly: Salt Transport by the Distal Tubule

Abstract

The distal convoluted tubule (DCT) plays a central role in blood pressure and potassium homeostasis, as evidenced by diseases that occur when its function is modified. The paper by van der Lubbe and colleagues in this issue of Kidney International makes clear that angiotensin II itself increases the activity and abundance of the thiazide-sensitive Na-Cl cotransporter (NCC), independent of changes in circulating aldosterone. This commentary provides additional perspective on that work.

These are exciting times for the distal convoluted tubule (DCT), or at least for those who study it. During the golden age of micropuncture, this short nephron segment was studied widely. Later, however, attention shifted to other nephron segments, owing to ease of study and the belief that NaCl transport along the DCT is determined ‘in large part by delivered load’, with only ‘equivocal’ evidence for regulatory modulation¹. New molecular tools and techniques, coupled with exciting insights into genetic hypertension and salt wasting, however, now identify the DCT as a key site for regulated NaCl transport. As with any field that is moving rapidly, however, emerging results often raise confusing questions. Our understanding of DCT transport remains inchoate, but the paper by van der Lubbe and colleagues (page XXX) helps to bring some clarity.

During the past fifteen years, evidence has accumulated that aldosterone increases sodium reabsorption along the DCT². More recently, arginine vasopressin (AVP) has also been shown to enhance sodium reabsorption along this segment³. Aldosterone and AVP have long been known to stimulate Na transport along the cortical collecting duct, by acting on the epithelial Na channel, ENaC; AVP also increases water permeability of this segment (via aquaporin-2), where both mineralocorticoid (MR) and vasopressin type 2 receptors (V2R) are expressed. Yet DCT cells also expresses MR⁴ and V2R⁵. These receptors probably mediate direct hormonal effects in DCT, as aldosterone increases the activity² and abundance⁶ of the thiazide-sensitive Na-Cl cotransporter (NCC), as does AVP^{3, 7, 8}.

The dominant NaCl transport pathway of the DCT is NCC. To transport NaCl, NCC must move (‘traffic’) to, and be inserted into, the apical plasma membrane; it is also phosphorylated along its amino terminal cytoplasmic domain, enhancing activity (see Figure 1). WNKs are intracellular kinases that modulate NCC activity by altering both trafficking and phosphorylation. WNK4 reduces NCC movement to the apical membrane⁹ from sites where it is synthesized (endoplasmic reticulum) and processed (golgi apparatus), at least in part, by targeting it to lysosomes, where it can be degraded^{10, 11}; the effects of WNK4 may be modulated by angiotensin II (see below). In contrast, WNK3 increases NCC abundance and activity^12-14. Thus, some WNKs are predominantly inhibitory, while others are predominantly stimulatory, at least with respect to NCC. Little is known about how NCC is removed from the apical membrane, although the process does not appear to involve clathrin-mediated endocytosis ^{11, 15}.

Figure 1.

Simplified scheme of regulation of thiazide-sensitive Na-Cl cotransporter (NCC) regulation. NCC is synthesized and then glycosylated (green fork) within the golgi apparatus (not shown, for clarity). The NCC then moves to and into the apical membrane, where it exists as a dimer. To be full active, NCC undergoes phosphorylation along its amino terminal cytoplasmic domain, mediated largely by SPAK, thereby permitting NaCl transport. Little is know about mechanisms of removal from the membrane. Arginine vasopressin (AVP), aldosterone (Aldo), and angiotensin II (Ang II) all stimulate NCC activity. Trafficking may be a rapid effect, modulated predominantly by AVP and Ang II. Phosphorylation may occur within the membrane, and is enhanced by all three factors.

As noted, NCC is also activated by phosphorylation (Figure 1). Phosphorylation activates NCC without changing its membrane abundance, at least when it is expressed heterologously in Xenopus oocytes ¹⁶. The major kinase that phosphorylates and activates NCC appears to be SPAK^{17, 18}. SPAK, which is expressed along the distal nephron¹⁹, can itself be phosphorylated and activated by WNK kinases, so that WNK, SPAK, and NCC comprise a signaling pathway²⁰. Nevertheless, although kinase domains of the several WNKS are homologous, all WNKs do not appear to have the same effects on NCC. As noted, WNK4 appears to act as an inhibitor of NCC^{9, 21}, at least under some conditions²², whereas WNK1 phosphorylates SPAK to activate NCC¹⁸. In HeLa cells, WNK1, but not WNK4, activated SPAK and caused a large shift in electrophoretic mobility²³; thus, details of how WNK kinases modulate NCC, remain confusing.

Angiotensin II is another component of the renin/angiotensin/aldosterone system that stimulates Na transport along the DCT ²⁴. This effect is also likely to be direct, owing to the presence of AT1 receptors along DCT ²⁵. Genetic deletion of AT1a receptors reduces the abundance of NCC ²⁶, and infusion of angiotensin II for 8 days increases the abundance and phosphorylation of NCC ²⁷; thus, angiotensin II and aldosterone appear to have similar effects on NCC activity. Gamba and colleagues reported that angiotensin II relieved the inhibitory effect of WNK4 on NCC, in a SPAK-dependent manner ²².

Angiotensin II increases NCC activity, in part, by increasing the abundance of NCC at the apical plasma membrane. This effect occurs rapidly, with short-term angiotensin II infusions increasing the ratio of apical to sub-apical NCC ²⁸. In cultured mpkDCT cells, angiotensin II also increases SPAK and NCC phosphorylation, suggesting that acute exposure to angiotensin II also activates the transporter allosterically²⁷. Longer-term effects, induced by dietary NaCl restriction²⁹ or angiotensin II infusions²⁷ also stimulate NCC activity, increase NCC abundance, and increase its phosphorylation; in these situations, however, the effects may be direct, from AT1 receptor activation, or indirect, via aldosterone stimulation.

Talati and colleagues concluded, based on inhibitor studies, that long-term effects of angiotensin II on NCC are mediated by aldosterone²⁷, and suggested therefore that aldosterone is the predominant NCC regulatory factor. T paper by van der Lubbe and colleagues in this issue of Kidney International (page XXX) shows clearly that angiotensin II itself increases NCC abundance and phosphorylation, even during chronic exposure; the authors used the definitive approach of performing adrenalectomy, and then infusing hormones chronically, to fix adrenal steroid concentrations. The results are clear; angiotensin II increases NCC abundance and phosphorylation even when serum aldosterone levels are fixed. Several additional points, derived from their data, however, deserve emphasis.

First, figure 2, redrawn from data in the paper, shows that aldosterone, but not angiotensin II, increased αENaC abundance substantially. This pattern of hormonal effect on ENaC contrasts with effects on NCC, in which both angiotensin II and aldosterone increase NCC abundance and phosphorylation. These results help to explain how aldosterone, a single hormone, can generate either NaCl retention or Na/K exchange, depending on the stimulatory signal (an effect termed the ‘aldosterone paradox’³⁰). Thus, when aldosterone secretion is stimulated by angiotensin II (such as occurs when the extracellular fluid volume is depleted), Na reabsorption will be stimulated along much of the nephron (including the proximal and distal tubule by angiotensin II, and the distal tubule and collecting duct by aldosterone). These effects will restore extracellular fluid volume both because proximal segments reabsorb NaCl, and because Na delivery to the distal, K secretory sites, will be limited. In contrast, when aldosterone secretion is stimulated by hyperkalemia, in the absence of changes in angiotensin II, Na reabsorption will only be stimulated distally, favoring the exchange of Na for K. Although other mechanisms are likely to be involved, the patterns of angiotensin II and aldosterone effect on Na transport along the nephron certainly reflect physiologically adaptive processes.

Figure 2.

Redrawn from data in the paper by van der Lubbe. Bar plot shows the effects of aldosterone (Aldo), angiotensin II (Ang II) and pressor dose Ang II (Ang II High) on abundance of the alpha subunit of ENaC, SPAK, and phosphorylated NCC. Note that all three interventions increase phosphorylated NCC, whereas only aldosterone increases ENaC abundance. Please see text for more details.

Second, while NCC stimulation by either angiotensin II or aldosterone is associated with increases in SPAK abundance and SPAK phosphorylation, when animals received higher doses of angiotensin II, NCC appeared to be stimulated, even though SPAK (and phosphorylated SPAK) were at baseline levels; even though this effect did not quite reach statistical significance, it raises the possibility that other kinases can activate NCC.

Finally, while comparisons of protein abundance do not necessarily reflect changes in transporter activity, the ability of aldosterone to increase NCC abundance is quite impressive, in comparison with its ability to increase ENaC abundance. Many, if not most, introductory texts for medical and graduate students describe effects of aldosterone on ENaC, but omit effects on NCC ³¹. The accumulating data suggest that it is time to break old paradigms, and include NCC as a crucial aldosterone-regulated transport protein, when introducing students to the effects of adrenal steroids on the kidney.

Lest the current data are believed to clear all confusion, several questions remain. As noted, two groups^{7, 8} have shown that AVP increases trafficking and phosphorylation of NCC. In the study by van der Lubbe and colleagues, the abundance of aquaporin 2 was increased by both aldosterone and angiotensin II infusion. This suggests either that these peptides stimulated AVP secretion or that angiotensin II activated V2R directly; there is some evidence in support of the second model³². From a physiological standpoint, of course, the striking similarity of effects of aldosterone and AVP on distal transporters is hard to reconcile with effects on whole animal balance. Aldosterone and AVP stimulate both stimulate ENaC and NCC. Yet, hyperaldosteronism typically presents with hypertension, owing to sodium chloride retention, while the syndrome of inappropriate ADH secretion presents with hyponatremia, owing to effects on aquaporin 2, but without NaCl retention. This suggests either that the potency of stimulatory effects on Na transport, or the escape mechanisms that supervene, are different, or that other factors come into play. One possible factor is V1a receptors; most studies of AVP actions utilize the V2-receptor-specific agonist desmopressin (dDAVP). V1a-receptors, a second target of the native hormone arginine vasopressin, can increase natriuresis³³.

Finally, the roles played by WNK kinases in modulating or mediating effects of angiotensin II and/or aldosterone remain intriguing, but are not fully elucidated. In view of the phenotype that results when WNK kinases are mutated, familial hyperkalemic hypertension (pseudohypoaldosteronism type II or Gordon syndrome), it seems clear that these kinases help to determine whether aldosterone is primarily kaliuretic or NaCl retentive. Yet changes in WNK4 were not observed in the experiments reported by van der Lubbe and colleagues, and data concerning WNK1 or WNK3 are not reported. WNK kinases may play a crucial role in determining NCC membrane abundance and states of phosphorylation, but the roles of the individual players, and their integration, remain poorly understood. Further, it seems likely that effects of WNK kinases, or at least WNK4, are modulated by circulating (or local) levels of angiotensin II²², as noted above. Much remains to be learned about the interactions between WNKs, SPAK, NCC, and the renin/angiotensin/aldosterone system. Yet, the possibility that small molecule WNK modulators might provide novel ways to ‘turn down’ the distal nephron means that this pathway is an attractive target for drug development.