Regulation of Thick Ascending Limb Transport by Vasopressina
Authors: MARK A. KNEPPER, GHEUN-HO KIM, PATRICIA FERNÁNDEZ-LLAMA, CAROLYN A. ECELBARGER
Journal: Journal of the American Society of Nephrology (1999)

Body fluid tonicity is controlled largely through the regulation of renal water excretion mediated by the peptide hormone vasopressin (1). Renal water conservation occurs as a result of the combined functions of the loop of Henle and the collecting duct, both of which are regulated by vasopressin. The loop of Henle is responsible for countercurrent multiplication, a process that concentrates the interstitial fluid of the renal medulla relative to the systemic plasma. The collecting duct is the site at which water permeability is regulated by vasopressin to result in variable osmotic equilibration with the hypertonic medullary interstitium. In this brief review, we summarize current knowledge regarding the role of the thick ascending limb in the regulation of renal water excretion by vasopressin. Based on a lecture given at the 1998 ASN Gottschalk Symposium on Vasopressin-Regulated Transporters by Mark A. Knepper. Countercurrent Multiplication by Henle's Loops The role of the loop of Henle in the generation of a concentrated urine was originally proposed in Switzerland by physical chemist Werner Kuhn and his colleagues (2,3,4). These workers hypothesized that the loop of Henle generates a corticomedullary solute concentration gradient in the kidney through a process called countercurrent multiplication. The concept of countercurrent multiplication originated as a result of consideration of industrial processes that separate and concentrate economically useful products such as distillation and counter-current extraction. In these processes, a single stage or "effect" (given an appropriate energy input) is capable of modest concentration of a given component of a mixture. However, the modest concentrating effect of a single stage ("single effect") can be multiplied by successive applications of the effect, resulting in an overall amplification of the concentrating process. In the kidney, such an amplification was proposed to occur as a result of counterflow between the ascending limb and descending limb of Henle's loop, resulting in progressively higher osmolality toward the deepest part of the renal medulla. The initial experimental evidence for countercurrent multiplication was provided in 1951 by Wirz, Hargitay, and Kuhn, who showed that the inner medullary tissue of antidiuretic hamsters had an osmolality that was remarkably in excess of plasma osmolality (5). Nevertheless, the countercurrent multiplier hypothesis was not widely accepted until the classic paper of Gottschalk and Mylle (6), in which the newly reinvented micropuncture technique was used to show directly that the tubule fluid of the inner medullary loops of Henle was hypertonic to plasma. Kuhn and Ramel were the first to propose that the single effect for countercurrent multiplication could be provided by solute transport out of the ascending limb (3). Such a transport process would lower the luminal osmolality relative to the surrounding medullary tissue. Initial evidence for this possibility came from micropuncture studies. It was found that tubule fluid exiting the loop of Henle in rodents is always hypotonic (as predicted by the countercurrent multiplier hypothesis), regardless of the osmolality of the urine (6,7). Additional micropuncture studies demonstrated that the hypotonicity of the fluid in the early distal tubule is chiefly a consequence of a low luminal NaCl concentration (8). Lassiter, Mylle, and Gottschalk (9) demonstrated net reabsorption of water by the loop of Henle of nondiuretic rats, leading to the conclusion that the hypotonicity of the fluid in the early distal tubule is a consequence of solute exit from the loop of Henle rather than water entry. Tubule fluid hypotonicity is maintained throughout the distal tubule and collecting duct system during water diuresis, owing to the low osmotic water permeability of the collecting ducts in the absence of circulating vasopressin. During antidiuresis, i.e., when circulating levels of vasopressin are high, extensive net water absorption occurs in the collecting duct system (9). In the medulla, the osmotic driving force responsible for water absorption from the collecting duct lumen is provided by the high concentration of solutes in the renal medullary interstitium generated by countercurrent multiplication (5,6). When Burg and his colleagues invented the isolated perfused tubule technique (10), it became possible to study transport processes in the thick ascending limb of Henle's loop directly. Studies in isolated perfused thick ascending limbs of Henle's loops from rabbits directly demonstrated the mechanism of luminal dilution (11,12). NaCl is rapidly absorbed by active transport, lowering the luminal NaCl concentration and osmolality below levels in the peritubular fluid. Because the osmotic water permeability of the thick ascending limb is very low, water transport does not occur despite the high transepithelial osmotic gradient. Water excretion is regulated by vasopressin in part because of its action to increase the water permeability of the collecting ducts. The high osmotic water permeability of the collecting duct allows osmotic equilibration with the hypertonic medullary interstitium. The cellular mechanism of this response is discussed in considerable detail elsewhere in this symposium. In addition to its effect on the water permeability in the collecting duct system, vasopressin also enhances water conservation by stimulating increases the rate of active NaCl absorption in the medullary thick ascending limb of Henle's loops in rodents (13,14,15), enhancing countercurrent multiplication. Regulation of Ion Transport in the Thick Ascending Limb by Vasopressin Vasopressin Receptors in the Thick Ascending Limb The medullary and cortical segments of the thick ascending limb play central roles in the regulation of water excretion. The medullary portion (MTAL) actively absorbs NaCl, which drives the countercurrent multiplication process responsible for concentrating the urine. The cortical portion (CTAL) also absorbs NaCl, diluting the luminal fluid and allowing the excretion of a dilute urine when circulating vasopressin levels are low. The pioneering microdissection studies of Morel, Imbert, and Chabarde[Combining Acute Accent]s (16,17) demonstrated the presence of vasopressin-sensitive adenylyl cyclase in both the MTAL and CTAL of rodents and rabbits, thus providing the first identification of these segments as sites for vasopressin action. These findings have been confirmed and extended in subsequent studies in which vasopressin has been demonstrated to increase the intracellular level of cAMP in microdissected thick ascending limb segments (18,19). These presence of V2 vasopressin receptors in the thick ascending limb has been directly demonstrated by binding studies in microdissected rat CTAL and MTAL segments (20) and by immunohistochemistry (21). Furthermore, V2 receptor mRNA has been detected in the thick ascending limb by in situ hybridization histochemistry (22,23) and by reverse transcription combined with PCR (RT-PCR) (24,25). Thus, the major effects of vasopressin in the thick ascending limb are believed to be mediated by the V2 vasopressin receptor. Aside from the V2 vasopressin receptor, several other receptors expressed in the thick ascending limb are coupled to adenylyl cyclase in rodents (26). Ligands for these receptors include glucagon, calcitonin, and parathyroid hormone. Thus, the regulatory actions of several hormones on the thick ascending limb may overlap with those of vasopressin. Although the physiologic importance of the V2 vasopressin receptor in the thick ascending limb of Henle's loop is well established, the possible role(s) of V1 receptors in this segment is unclear. It has been observed that vasopressin, at concentrations >1 nM, can trigger a transient increase in intracellular Ca2+ in thick ascending limb cells (27,28,29,30). In addition, studies in mouse MTAL suspensions have revealed that arginine vasopressin, but not the V2-selective vasopressin analog deamino-8-D-arginine vasopressin (dDAVP), evokes an increase in inositol trisphosphate production (31). These results point to an ability of vasopressin to activate the phosphoinositide signaling pathway in thick ascending limb cells, a characteristic usually associated with V1a and V1b receptors, and not the V2 receptor. Binding studies in isolated MTAL cells from rat are also consistent with the presence of a V1-like receptor (32). In contrast, studies using quantitative RT-PCR (24) or in situ hybridization (22,23) have not detected V1a receptor mRNA in thick ascending limbs of rat. However, another study, using a nonquantitative RT-PCR approach, reported a small quantity of V1a receptor mRNA in microdissected thick ascending limbs (25). Thus, the evidence, considered as a whole, suggests that the V1a receptor gene may be expressed in the thick ascending limb, but at low levels. What processes it regulates is currently uncertain. Whether some of the effects described above may be attributable to the possible expression of the V1b receptor in the thick ascending limb has also not been reported. Mechanism of Ion Transport in the Thick Ascending Limb Vasopressin acts acutely (within seconds or minutes) to increase the rate of NaCl absorption in the medullary thick ascending limb of rodents (13,14,15,33). A consideration of the mechanism of NaCl absorption in the thick ascending limb (Figure 1) reveals several ion transporters that could be targets for regulation by vasopressin. Key features of the model shown in Figure 1 were demonstrated by the pioneering studies of Greger and colleagues (34,35,36). NaCl transport across the apical plasma membrane occurs via a furosemide-inhibitable Na+-K+-2Cl- cotransporter. The potassium ions that enter the cell on the cotransporter are largely recycled into the lumen via apical potassium channels. Patch-clamp studies have demonstrated that there are two such K+ channels in the apical plasma membrane with unit conductances of 72 and 30 pS (37). Sodium ions are actively transported from the cell into the interstitium by the basolateral Na+-K+-ATPase. The chloride ions that enter the cell via the Na+-K+-2Cl- cotransporter exit across the basolateral plasma membrane via a chloride channel and/or by K-Cl cotransport. Overall electroneutrality is maintained by paracellular sodium ion movement from the lumen driven by a lumen-positive voltage across the epithelium. The net process results in the transfer of equal numbers of sodium and chloride ions from lumen to interstitium. In addition, Good (38) has demonstrated the presence of a Na-H exchanger in the apical plasma membrane of the rat thick ascending limb, which is responsible for sodium-dependent proton secretion. This Na-H exchanger provides an alternative means of sodium entry into thick ascending limb cells.Figure 1: . Cell model of thick ascending limb epithelial transporters. Complementary DNA for several of these proteins have been cloned, facilitating their study on a molecular level.Short-Term Actions of Vasopressin To Increase Thick Ascending Limb Transport Theoretically, vasopressin could act at any of the sites demonstrated in Figure 1 to evoke an increase in NaCl absorption. Studies, using the isolated perfused tubule technique, have provided evidence that the transport activity of the bumetanide-sensitive Na-K-2Cl cotransporter of the thick ascending limb is directly and rapidly stimulated by vasopressin (39,40,41). The mechanism of this action is as yet unknown, although it has been well established that a closely related bumetanide-sensitive Na-K-2Cl cotransporter, which is responsible for fluid secretion in various tissues, is regulated by phosphorylation (42,43). Immunoelectron microscopic studies have demonstrated that the Na-K-2Cl cotransporter is abundant in both the apical plasma membrane of thick ascending limb cells and in intracellular vesicles of MTAL cells (44), raising the possibility that vasopressin could increase plasma membrane Na-K-2Cl transporter activity through regulation of trafficking as has been described for vasopressin-mediated regulation of aquaporin-2 (see above). Another key transporter, the Na-K-ATPase, has been reported to be activated by vasopressin in the thick ascending limb (45). However, another group has reported the opposite effect, a decrease in Na-K-ATPase activity in the thick ascending limb in response to vasopressin (46). Additional studies are needed to clarify this controversy. Schlatter and Greger have provided evidence that vasopressin, acting through cAMP, directly increases basolateral chloride conductance in the mouse medullary thick ascending limb (47). As of this writing, the mechanism of this action is unknown, although the effect seems likely to be associated with regulation of the ClC-K chloride channel, which has been recently localized to the basolateral plasma membrane of thick ascending limb. Studies by two groups of investigators (37,48) support the idea that the apical potassium conductance is regulated by vasopressin. They used the patch-clamp technique to demonstrate that the apical plasma membrane of the rat thick ascending limb contains a 30-pS ATP-sensitive K+ channel whose activity is increased by application of cAMP or vasopressin. The cDNA for a renal medullary potassium channel (ROMK) responsible for the apical K+ conductance in the thick ascending limb has recently been cloned (49). Subsequent studies have demonstrated that ROMK is a substrate for phosphorylation by protein kinase A (50). In summary, the work published thus far suggests that the short-term regulation of NaCl absorption by vasopressin in the thick ascending limb of Henle's loop is multifactorial. There may be a hierarchy of effects that provide differing degrees of control. Alternatively, one of the transporters discussed above may emerge from future studies as the predominant site of regulation. Short-Term Effects of Vasopressin on Acid-Base Transport in the Thick Ascending Limb Isolated perfused tubule studies have demonstrated that, in addition to its effect on NaCl transport, vasopressin also markedly decreases the rate of luminal acidification in the rat MTAL through downregulation of the apical Na-H exchanger (51). This action, like the effect on NaCl cotransport, is mediated by cAMP through activation of the V2 receptor (51,52). Using micropuncture techniques, it was demonstrated that infusion of the V2-selective vasopressin agonist dDAVP into rats does not alter fractional delivery of total CO2 to the early distal tubule (53), suggesting a lack of an overall effect on loop of Henle bicarbonate transport. However, when measures were taken to lower plasma levels of other hormones that stimulate adenylyl cyclase in thick ascending limbs (glucagon, parathyroid hormone, and calcitonin), dDAVP administration inhibited the fractional absorption of bicarbonate in loops of Henle of superficial nephrons (53). In contrast to the demonstrated effect of vasopressin to decrease Na-H exchange across the apical plasma membrane, vasopressin has been demonstrated to increase the rate of Na-H exchange across the basolateral plasma membrane (54). These two Na-H exchange processes are mediated by two different molecular Na-H exchangers, i.e., NHE-3 in the apical plasma membrane (55) and NHE-1 in the basolateral plasma membrane (56). The studies of Yun et al. (57) have demonstrated that inhibition of NHE-3-mediated transport by cAMP is dependent on the presence of regulatory proteins E3KARP and NHERF, which bind to NHE-3. Despite the fact that V2 receptor-mediated regulation in the thick ascending limb of Henle's loop has been thoroughly studied in rodents and rabbits, some doubts remain about the physiologic importance of vasopressin action in the regulation of human thick ascending limb function. Studies by Chabarde[Combining Acute Accent]s et al. (58) and Ruggles et al. (59) both demonstrated the absence of measurable vasopressin-stimulated adenylyl cyclase activity in thick ascending limbs dissected from human kidneys. It is unknown whether the lack of vasopressin responsiveness of the thick ascending limb is present in vivo or could be an artifact of the special conditions associated with the harvest and preservation of human kidneys. Interestingly, the rate of active ammonium absorption by the isolated perfused rat MTAL is unaffected by vasopressin (51). Because this transport process results from substitution of NH4+ for K+ on the apical bumetanide-sensitive Na-K-2Cl cotransporter (60), the absence of an effect of vasopressin on active ammonium absorption suggests the absence of a direct effect of vasopressin on the apical cotransporter. Nevertheless, ammonium accumulation in the rat renal medulla (driven by active transport of ammonium from the MTAL) is augmented during antidiuresis (61). This augmentation could be due largely to a decrease in medullary water content during antidiuresis (62,63). Alternatively, an increase in MTAL ammonium absorption may occur as a result of an increase in the luminal NH4+ concentration owing to increased osmotic water absorption from the thin descending limb. Short-Term Action of Vasopressin To Regulate Divalent Cation Transport in the Thick Ascending Limb Micropuncture studies have demonstrated that vasopressin enhances the reabsorption of the divalent cations Ca2+ and Mg2+ by the loop of Henle (33). Isolated perfused tubule studies have demonstrated that vasopressin augments the rate of absorption of Ca2+ and Mg2+ by the CTAL but not the MTAL (64). Long-Term Actions of Vasopressin To Regulate Thick Ascending Limb Transport In addition to the short-term actions of vasopressin on the thick ascending limb, the MTAL can undergo structural and functional changes as a result of long-term alterations in circulating vasopressin level or water intake. Bankir and her colleagues (65,66) infused vasopressin systemically for 5 to 6 wk in normal rats and Brattleboro rats and measured morphometric characteristics of the MTAL epithelium. They found evidence for marked hypertrophy of the medullary thick ascending limbs and thickening of the inner stripe of the outer medulla. In addition, long-term water restriction in Wistar rats resulted in hypertrophy of the MTAL segment (67). The hypertrophy of the MTAL in vasopressin-treated Brattleboro rats was accompanied by increases in hormone-sensitive adenylyl cyclase activities and in Na,K-ATPase activity (68). Furthermore, administration of vasopressin to Brattleboro rats for 10 to 21 d approximately doubled the spontaneous rate of active chloride absorption in isolated perfused medullary thick ascending limbs dissected from these animals (69). Thus, long-term increases in circulating vasopressin in rats causes an increase in MTAL NaCl transport rate, which increases urinary concentrating ability due to enhancement of countercurrent multiplication. An adaptive increase in NaCl transport rate could be a result of changes in expression of any of the ion transporters depicted in Figure 1. Recently, we (70) found that moderate water restriction of rats markedly increased the abundance of the Na-K-2Cl cotransporter protein in the outer medulla. We observed a similar effect with infusion of the V2-selective vasopressin analog dDAVP (70) in Brattleboro rats. Thus, the long-term adaptation of transepithelial NaCl transport in the MTAL is apparently mediated in part by an increase in the number of cotransporters per cell. We suggest that the upregulation of cotransporter abundance is likely to be a result of elevated levels of intracellular cAMP. Consistent with this possibility are findings of Igarashi et al. (71), who have recently cloned and sequenced a large portion of the 5′ flanking region of the mouse NKCC2 gene, which codes for the apical Na-K-2Cl cotransporter of the thick ascending limb. Analysis of the sequence revealed the presence of a cAMP regulatory element that could potentially be a mediator of cAMP-mediated transcriptional regulation. Figure 2 depicts the signaling pathway by which we propose vasopressin regulates expression of the NKCC2 gene. Arginine vasopressin (AVP) binds to the V2 receptor which then couples to adenylyl cyclase through the heterotrimeric G protein, Gs. The adenylyl cyclase isoform expressed in the thick ascending limb is AC-VI, a calcium-inhibitable isoform (72). Activation of adenylyl cyclase increases cAMP in the cell, which in turn activates protein kinase A. This in turn phosphorylates various proteins in the cell including transcription factors. Recently, we carried out studies of Na-K-2Cl cotransporter expression in mice in which the α-subunit of the heterotrimeric G protein Gs was expressed at 50% of normal levels due to a targeted knockout mutation in one allele. Thick ascending limbs from these mice produced half the normal amount of cAMP, and Na-K-2Cl cotransporter expression was reduced by about 50%. These data further strengthen the conclusion that there is a causal link between V2-mediated signaling and Na-K-2Cl cotransporter expression. Furthermore, recent evidence suggests that prostaglandin E2 has a long-term inhibitory effect on Na-K-2Cl cotransporter expression (73). Prostaglandin E2 binds to the EP3 receptor in the thick ascending limb, which couples to adenylyl cyclase in an inhibitory manner via the heterotrimeric G protein, Gi. It was found that prostaglandin synthase inhibitors indomethacin and diclofenac substantially increase the expression of the Na-K-2Cl cotransporter (73).Figure 2: . V2-receptor/adenylyl cyclase signaling pathway in the rat kidney thick ascending limb. Vasopressin increases intracellular cAMP levels via the V2 receptor, which is coupled to adenylyl cyclase VI through the heterotrimeric G protein, Gs. Increased cellular levels of cAMP may activate transcription factors via protein kinase A-mediated phosphorylation events.In an earlier study (74), AVP infusion or a 48-h water deprivation did not significantly increase Na-K-2Cl cotransporter expression. This raises the possibility that occupation of V1a receptors in the thick ascending limb may oppose the upregulatory effects of V2 receptor occupation. Furthermore, extracellular fluid volume depletion induced by complete water deprivation may activate other signaling mechanisms, which could counter the effects of vasopressin acting on the V2 receptor. Long-term vasopressin administration has also been reported to increase the expression of GLUT-4-facilitated glucose transporter mRNA in the thick ascending limb, providing a possible augmentation of the supply of glucose for ATP generation (75). In our studies (70), we found no change in expression for the apically expressed Na-H (type 3) exchanger or the basolateral Na-K-ATPase in the outer medulla. The expression of Tamm-Horsfall protein, an apically expressed protein in the thick ascending limb of unknown function, decreases as a result of the long-term infusion of dDAVP (70) or AVP (74). Acknowledgments Support for the work described herein was derived in part from the intramural budget of the National Heart, Lung, and Blood Institute (Project no. Z01-HL-01282).