Thick Ascending Limb

The thick ascending limb (TAL) begins at the junction between the inner and the outer medulla for deep nephrons and at the tip of the loop of Henle for more superficial nephrons. The thick ascending limb includes medullary and cortical segments and ends at the juxtaglomerular apparatus, where the nephron abuts against its own glomerulus.

Figure 7 shows a cell model that includes mechanisms for NaCl and NaHC03 absorption in the thick ascending limb. The mechanism of NaCl absorption begins, once again, with an NaK ATPase in the basolateral membrane. K + which enters the cell on the Na/K ATPase exits on a basolateral membrane K+ channel. The resulting decrease in cell Na+ concentration provides the driving force for the apical membrane electroneutral Na/K/2C1 cotransporter. When considered on the basis of electrochemical gradients, there is a large passive electrochemical gradient for Na+ entry on this transporter, including a significant chemical concentration gradient as well as the cell negative voltage. This provides the driving force for the transport of K + and CI" against their passive electrochemical gradients. Potassium which enters the cell across the apical membrane mostly recycles back into the lumen across the apical membrane K + channel. Some K + may exit across a basolateral membrane K + channel, leading to K+ absorption. As noted above, the Na+ which enters the cell exits on the Na/K ATPase. CI" which enters the cell from the lumen exits through a basolateral membrane CI" channel. There may also be some contribution from basolateral membrane KC1 cotransport.

The Na/K/2C1 cotransporter has been well characterized. Na + first binds to the transporter, promoting the binding of K+ and CI . Binding of K+ then promotes the binding of a second CI". This second CI" binding site appears to also be the binding site for loop diuretics. Competition with CI" for binding at this site provides the mechanism by which loop diuretics inhibit NaCl absorption in the thick ascending limb. The affinity of this transporter for Na + is very high, with half maximal transport rates achieved at concentrations less than 10 mM Na +. Similarly, K+ affinity is very high. Given that the transporter has two CI" binding sites, it likely has two separate CI" affinities. One of these affinities, probably that related to binding of the second CI" ion is lower, allowing changes in luminal CI" concentration to affect transporter rates. Once the transporter is fully occupied with substrates, the complex translocates to the internal surface of the cell membrane where substrates are released.

The cDNA encoding the thick ascending limb Na/K/2C1 cotransporter has recently been cloned by two groups of investigators, based on its homology to the flounder Na/Cl cotransporter and the ubiquitous Na/K/2C1 cotransporter. The clone has been referred to as NKCC2 (referring to the second Na/K/2C1

Lumen Interstiiium

apical membrane on the Na/K/2C1 cotransporter, driven by the low cell Na+ concentration. A significant fraction of K + which enters on the cotransporter recycles across an apical membrane K + conductance. CI" which enters on the cotransporter exits the cell on a basolateral membrane CI ~ channel. H + is secreted into the lumen by an Na/H antiporter. OH ~ generated by apical membrane H+ secretion reacts with C02 to form HC03" and C032", which exit with a Na+ on the Na/HC03/ C03 cotransporter. Na+ which enters the cell across apical membrane transporters exits the cell on the basolateral membrane Na/K ATPase and the Na/HC03/C03 cotransporter. K+ which enters the cell on the Na/K ATPase exits across a basolateral membrane K + channel. Because transcellular NaCl absorption is electrogenic, a lumen + voltage is generated which drives a paracellular current.

Active transport mechanism; O, passive transporter; =, channel.

cotransporter isoform cloned) or as BSC1 (referring to the first bumetanide sensitive cotransporter cloned). In both cases, the cDNAs are similar and encode a protein of predicted size of 120 kDa, with 12 transmembrane domains, a large extracellular domain, and large cytoplasmic aminoterminal and carboxy-

terminal domains. It is likely that this isoform is expressed in the kidney only on apical membranes. A second isoform, referred to as BSC2 or NKCC1 encodes a more ubiquitously expressed Na/K/2C1 cotransporter, which may mediate such functions as cell volume regulation.

Patch clamp studies have extensively characterized K + and CI" channels in this epithelia. The apical membrane of the thick ascending limb has been found to contain a number of K+ channels. A "maxi-K+" channel has been found in cultured cells of the TAL. This channel is Ca2+-activated, has a single channel conductance of 140-150 pS, and is inhibited by barium and stimulated by depolarization of the apical cell membrane. At physiologic apical membrane voltage and cell Ca2+ concentration, its open probability is too low to account for the observed K+ conductance of the apical membrane. It has been suggested that this maxi-K + channel may play a role in cell volume regulation.

Two additional K+ channels with lower single channel conductances, of —30 or 70 pS, respectively, have also been defined. Their open channel probability is much higher; they are highly K+ selective (high PK:PNa) and slightly inwardly rectifying. The open probability of these channels is regulated by the cytoplasmic ATP: ADP ratio, with increases in the ratio inhibiting the K + channel. These K + channels are voltage insensitive and inhibited by low cytosolic pH and barium. The 30 pS K + channel is similar to the secretory K + channel in the apical membrane of cortical collecting duct (CCD) cells and will be described in more depth later. The 30 pS K + channel in the TAL and the CCD are encoded by similar genes, ROMK1 and ROMK2 (rat outer medullary K channel), and their function has been explored in heterologous expression systems such as the oocyte membrane.

Basolateral membrane CI " channels have also been characterized by application of the patch clamp technique. Two groups of investigators have found 40 pS channels which are highly Cl " selective. The open probability of the CI" channel is regulated by the concentrations of Cl" both inside and outside the cell. However, regulation by intracellular Cl" concentration occurs at lower concentrations (0-50 mM), while regulation by extracellular Cl" concentration occurs at higher concentrations (50-300 mM). Regulation by intracellular Cl" concentration is key in that increases in Cl" entry across the apical membrane on the Na/K/2C1 cotransporter will raise intracellular Cl" concentration and increase the open probability of the basolateral membrane Cl" channel.

To understand the net result of the activity of the individual transporters in the cell model shown in Fig. 7, let us consider one turnover of the Na/K ATPase molecule. Three Na + enter the cell with three turnovers of the Na/K/2C1 co-transporter, and exit with one turnover of the NaK ATPase. K + transported into the cell on the apical and basolateral membrane both recycle across their respective membranes on K+ channels. The six Cl" which enter the cell exit through the basolateral membrane Cl " conductance. The net result is the trans-

epithelial absorption of six CI" ions and only three Na+ ions. Thus, NaCl transport is electrogenic and generates a lumen-positive voltage. This lumenpositive voltage can then drive a paracellular current which could involve either passive absorption of three Na + or passive secretion of three Cl ~~ to complete the circuit. Because the tight junction of the thick ascending limb is highly Na + selective (PNa/Pa = 2-6), most of the paracellular current involves passive Na + absorption. Furthermore, the thick ascending limb is impermeable to water so that active NaCl absorption lowers luminal [NaCl] to values below those of plasma and interstitial fluid. This concentration gradient generates an additional lumen positive diffusion voltage because the tight junction is Na+ selective. As will be discussed below, lumen positive voltage in the thick ascending limb provides a pathway for passive paracellular absorption of other cations including K + , Ca2+, Mg2+, and NH4+. Thus, loop diuretics which inhibit NaCl absorption and the lumen positive voltage, inhibit absorption of these cations.

The thick ascending limb also absorbs 5-10% of the filtered load of NaHC03. The mechanism for this appears to be similar to that in the proximal tubule (Fig. 7). Apical membrane H+ secretion is mediated by an Na/H antiporter that is encoded by NHE-3. HC03~ generated in the cell exits on an Na/ HC03/C03 cotransporter. This allows some Na+ efflux, but the majority of basolateral membrane Na + efflux occurs on the NaK ATPase. The possibility of other basolateral membrane transport mechanisms, including a K/HC03 co-transporter, has been raised.

NaCl absorption is regulated in this segment (Table 2). Thick ascending limb NaCl absorption is an important component of urinary concentration, and thus it is not surprising that regulators of urinary concentration regulate NaCl absorption in this segment. Arginine vasopressin stimulates NaCl absorption in the medullary thick ascending limb, and hypertonicity inhibits NaCl absorption. PGE-2, also known to inhibit urinary concentration, inhibits NaCl transport. Increases in delivery of NaCl to the thick ascending limb increase the rate of NaCl absorption (glomerulotubular balance). NaCl absorption is also regu-

TABLE 2 Regulation of Thick Ascending Limb NaCl Absorption

Factors which regulate urinary concentration Arginine vasopressin Hypertonicity PGE-2

Glomerulotubular balance

Effective arterial volume

Renal nerves (beta catecholamines)

Mineralocorticoids

PGE-2

lated by effective arterial volume, such that volume contraction enhances NaCl absorption and volume expansion inhibits NaCl absorption in the thick ascending limb. These effects are mediated by a number of factors. Beta adrenergic catecholamines increase NaCl absorption, providing a possible mechanism for similar effects of renal nerves. In addition, mineralocorticoids increase NaCl absorption in the thick ascending limb. Last, as noted above, PGE-2, whose levels increase in volume expansion, inhibits NaCl absorption.

NaHC03 absorption in the thick ascending limb is also regulated. AVP, glucagon, and hyperosmolality inhibit NaHC03 absorption in this segment. The physiologic significance of these effects is unclear. Last, chronic metabolic acidosis increases the rate of NaHC03 absorption in the medullary thick ascending limb.

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