Thick Ascending Limb of Henle

The thick ascending limb of Henle reabsorbs 20% to 30% of the filtered sodium chloride load. Sodium and chloride enter the thick ascending limb cell via the Na+-K+- 2Cl- cotransporter, which is inhibited by loop diuretics. Because sodium and chloride concentrations in urine are much higher than potassium, for the transporter to operate maximally there must be a mechanism present for potassium to recycle back into the tubular lumen. A ROMK potassium channel in the luminal membrane mediates potassium recycling. Sodium leaves the cell via the Na+-K+-ATPase and chloride via a chloride channel.

The rate of NaCl reabsorption in this segment is load dependent. The higher the delivered NaCl load the higher the reabsoprtion. Sodium reabsorption is increased by activation of the sympathetic nervous system and ^-adrenergic agonists, AVP in some species, parathyroid hormone, calcitonin, and glucagon. Prostaglandin E2 inhibits sodium reabsorption.

Distal Convoluted Tubule

The DCT reabsorbs 5% to 10% of the filtered sodium load. Sodium and chloride enter the DCT cell via the thiazide- sensitive Na+-Cl- cotransporter (NCC) and sodium exits via the Na+-K+-ATPase. For mineralocorticoids to play a role in the regulation of sodium transport in any nephron segment, that segment must also express the min- eralocorticoid receptor and type 2 11/i-hydroxysteroid dehydrogenase (HSD). The mineralocorticoid receptor is expressed in the entire DCT, while type 2 11/i-HSD is expressed in the later half (DCT2) of the DCT. DCT2 also contains the epithelial sodium channel (ENaC). Type 2 11/i-HSD degrades cortisol to the inactive cortisone in mineralocorticoid target tissues. This is required to maintain mineralocorticoid specificity, given the facts that the mineralocorticoid receptor binds glucocorticoids and that glucocorticoids circulate at much higher concentrations than mineralocorticoids.

Genetic studies of a rare monogenic disorder provide insight into NCC regulation. Familial hyperkalemic hypertension (FHH), also known as pseudohypoaldoste- ronism type II (PHA II), is an autosomal dominant disease characterized by hypertension, hyperkalemia, and extreme sensitivity to thiazide diuretics. Mutations in 2 members of the WNK (with no lysine [K]) kinase family, WNK1 and WNK4, cause the disease. Three members of this gene family WNK1 (also known as long WNK1), 2, and 4 are expressed in kidney. In addition, an alternatively spliced isoform of WNK1, kidney-specific WNK1 (KS-WNK1) is also expressed. Their expression pattern varies: long WNK1 (L-WNK1)—all along the distal nephron; KS-WNK1—in the DCT and decreases gradually in the connecting tubule; WNK3—along the entire nephron; and WNK4—DCT1 to the collecting duct. WNK4 reduces expression of NCC in the cell membrane. It does this via a kinase-dependent mechanism that does not involve changes in the synthesis or processing of NCC. Mutations in WNK4 lead to NCC overactivity via a loss of function mechanism of WNK4. Recently, other kinases including SPAK (Ste20p-related proline-alanine rich kinase) and OSR1 (oxidative stress response) have been identified as intermediates in the pathway between WNK4 and NCC. WNK4 inhibits the ROMK potassium channel. ROMK inhibition is not dependent on WNK4 kinase activity but occurs through clathrin-dependent endocytosis of the channel. Mutations result in a gain of function of this process with further increases in endocy- tosis from the luminal membrane.

In collecting duct, WNK4 increases claudin phosphorylation, resulting in increased paracellular chloride transport and stimulation of ENaC activity. FHH mutations further increase both of these processes and augment NaCl reabsorption. Interestingly, the WNK4 mutations of

FHH increase NCC activity but decrease ROMK activity. This not only explains the hypertension and hyperkalemia of FHH but also shows that WNK4 can differentially regulate NCC and ROMK.

Aldosterone production is stimulated by both hypovolemia and hyperkalemia. In hypovolemia the distal nephron must reabsorb sodium but not increase potassium secretion, whereas in hyperkalemia the goal is to secrete potassium without an effect on sodium homeostasis. How 1 hormone can mediate 2 apparently disparate functions has been termed the aldosterone paradox. When aldosterone concentrations are elevated, how does the distal nephron know whether to reabsorb sodium (stimulate NCC and inhibit ROMK) or excrete potassium (stimulate ROMK and inhibit NCC)? WNK4 may be the master switch that regulates the balance between NaCl reabsorption and potassium secretion in the distal nephron. With hypovolemia both AII and aldosterone are stimulated. AII even in the presence of WNK4 stimulates NCC. Experimental studies have also shown that AII stimulates the phosphorylation of SPAK and NCC. AII thereby activates NCC in DCT1 directly and in DCT2 indirectly via aldosterone. AII also inhibits ROMK activity through WNK4 dependent and independent mechanisms. The combined interaction of AII with aldosterone favors the electroneutral reabsorption of sodium with chloride.

By contrast, in hyperkalemia, aldosterone is stimulated but AII is not. WNK4-mediated inhibition of NCC in DCT1 is maintained because type II 11/i-HSD is not expressed in DCT1. This inhibition of sodium reabsorption in DCT1 results in sodium delivery further downstream to the connecting segment and collecting duct where sodium reabsorption via an electrogenic process (ENaC) can stimulate potassium secretion. S1169 phosphorylation mediated by Sgk1 (serum and glucocorticoid- regulated kinase) releases WNK4 inhibition of ENaC and ROMK in the connecting segment and collecting duct. In addition, Sgk1 phosphorylates Nedd4-2 on 3 motifs. This creates binding sites for the 14-3-3 protein that blocks interaction of Nedd4-2 with ENaC and prevents its ubiq- uitinization and subsequent removal from the luminal membrane.

L-WNK1 is expressed in a variety of chloride-transporting epithelia, including kidney, colon, sweat ducts, pancreas, and bile ducts. L-WNK1 does not appear to bind NCC but rather interacts with WNK4 and inhibits its ability to downregulate NCC. In FHH, mutations in L-WNK1 increase its expression and further augment

Simplified model of DCT sodium transport and FHH

FIGURE 2-3. Simplified model of DCT sodium transport and FHH. The FHH phenotype is caused by mutations in both WNK4 and WNK1. WNK4 impairs delivery of the NCC to the luminal membrane by shunting the protein to a lysosomal compartment and mutations that decrease its activity increase NCC expression in the cell membrane. L-WNK1 interacts with WNK4 and decreases its activity. KS-WNK1 interacts with L-WNK1 and decreases its activity.

its ability to inhibit WNK4, resulting in increased NCC activity. KS-WNK1 is stimulated by aldosterone and antagonizes the effects of L-WNK1. WNK3 increases NCC activity and inhibits the activity of ROMK and KCl cotransporters in the distal nephron and may also play a role in hypovolemia. In the model of DCT sodium transport shown in Figure 2.3 , delivery of NCC to the luminal membrane is inhibited by WNK4, while L-WNK1 inhibits the activity of WNK4, and KS-WNK1 inhibits L-WNK1. Mutations in either L-WNK1 or WNK4 result in increased NCC activity and the FHH phenotype.

 
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