Proximal Tubule

Approximately 50% of filtered NaCl and 70-90% of filtered NaHC03 are reabsorbed in the proximal tubule. The proximal tubule is subdivided into three segments, the most proximal S1; the middle S2, and the terminal S3 segment. In

Diuretic Agents: Clinical Physiology and Pharmacology

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Proximal Tubule

general, Na + transport mechanisms are qualitatively similar in these segments, but differ quantitatively. Transport rates in Si are most rapid, slower in S2, and slowest in S3. The proximal tubule can also be divided into an early proximal convoluted tubule which includes Si and part of S2 and is convoluted and a proximal straight tubule which includes the remainder of S2 and S3 and is straight (Fig. 1).

Figure 2 shows a general model of a proximal tubule cell containing the most important transporters that are key to NaCl and NaHC03 reabsorption. A number of these transport proteins have been purified and/or cloned. The

Lumen

Interstitium h+

Base-

2K"1

hco;

FIGURE 2. Proximal tubule Na + and H + transporters. Active transport mechanism; O, passive transporter; =, channel.

Na/K ATPase includes two subunits, a and ¡3. All of the transport functions are mediated by the a subunit. The (3 subunit is heavily glycosylated and may play a role in trafficking of the Na/K ATPase to the basolateral membrane. Three isoforms of the a subunit and two isoforms of the (3 subunit have been identified. The predominant isoforms expressed in the kidney are a 1 and /? 1, but this varies between nephron segments and among species.

Five Na/H antiporter isoforms have been cloned. These all have a similar structure with an amino terminal domain containing 10-12 membrane spanning regions and a large carboxyterminal cytoplasmic regulatory domain. The predominant isoform encoding the proximal tubule apical membrane Na/H antiporter is NHE-3. In some segments of the proximal tubule, there is also a basolateral membrane Na/H antiporter, which is encoded by NHE-1. This is not shown in Fig. 2 because basolateral Na/H exchange does not contribute to transepithelial Na + or H + transport, but rather contributes to cellular housekeeping functions. The Na/H antiporter is inhibited by the diuretic amiloride. However, the concentrations of amiloride achieved clinically are too low to inhibit this transporter. Thus, clinically amiloride is without effect on the proximal tubule.

Acidification

Reabsorption of filtered NaHC03 from the proximal tubule luminal fluid is mediated by H+ secretion, as shown in Fig. 3. H+ secreted into the luminal fluid neutralizes OH ~ ions causing luminal acidification. Luminal carbonic an-hydrase then catalyzes the conversion of HC03" —> C02 and OH"; the C02 then diffuses out of the lumen. C02 inside the cell, now catalyzed by intracellular carbonic anhydrase, combines with OH " and forms HCO, . OH ~ and H + are formed from H20 within the cell. The H+ is secreted into the lumen while the HC03" exits across the basolateral membrane. As can be seen, both luminal and cellular carbonic anhydrase play key roles in this scheme. Luminal carbonic anhydrase activity is encoded by the membrane-bound isoform, type IV carbonic anhydrase. Cytoplasmic activity may be encoded by the membrane bound type IV or by cytosolic type II carbonic anhydrase.

Lumen

Interstitium

FIGURE 3. Proximal tubule NaHC03 reabsorption. H+ is secreted into the proximal tubule lumen by an Na/H antiporter and an H ATPase. OH ~ generated within the cell by apical membrane H + secretion reacts with C02 to form HCO-r and C032-, which exit with a Na + on the basolateral membrane Na+/HC03/C03 cotransporter. Na+ absorbed by the Na/H antiporter exits the cell on the basolateral membrane NaK ATPase and the Na/HC03/C03 cotransporter. K + which enters the cell on the NaK ATPase exits on a basolateral membrane K + channel. Carbonic anhydrase catalyzes the conversion of HC03~ to C02 and OH ~ in the lumen and the reverse reaction in the cell. Elec-trogenic H+ secretion generates a small lumen positive voltage which generates a current flow across the paracellular pathway. This comprises mostly Na + efflux and CI ~ influx into the lumen. •, Active transport mechanism; o, passive transporter; =, channel. C.A., carbonic anhydrase.

FIGURE 3. Proximal tubule NaHC03 reabsorption. H+ is secreted into the proximal tubule lumen by an Na/H antiporter and an H ATPase. OH ~ generated within the cell by apical membrane H + secretion reacts with C02 to form HCO-r and C032-, which exit with a Na + on the basolateral membrane Na+/HC03/C03 cotransporter. Na+ absorbed by the Na/H antiporter exits the cell on the basolateral membrane NaK ATPase and the Na/HC03/C03 cotransporter. K + which enters the cell on the NaK ATPase exits on a basolateral membrane K + channel. Carbonic anhydrase catalyzes the conversion of HC03~ to C02 and OH ~ in the lumen and the reverse reaction in the cell. Elec-trogenic H+ secretion generates a small lumen positive voltage which generates a current flow across the paracellular pathway. This comprises mostly Na + efflux and CI ~ influx into the lumen. •, Active transport mechanism; o, passive transporter; =, channel. C.A., carbonic anhydrase.

As will be discussed later, carbonic anhydrase inhibitors block proximal tubule HCO, absorption because they prevent the formation of C02 and OH~ from HC03" in the luminal fluid and the conversion of C02 and OH" to HCO3 within the cell. In the absence of carbonic anhydrase, secreted H + would react with luminal HC03~ to form H2C03, which could then slowly dehydrate to form C02 and H20. The C02 would then diffuse into the cell where it could form H2C03 and then slowly dissociate to H+ and HC03~. However, rates of proximal tubule HCO , absorption observed in the presence of carbonic anhydrase inhibitors are greater than would be predicted from the uncatalyzed rates of H2C03 dehydration and C02 hydration. The explanation for the high rate of carbonic anhydrase independent HCO . ~ absorption likely resides in H2C03 recycling. Following inhibition of carbonic anhydrase, luminal H2C03 concentration rises and cytoplasmic H2C03 concentration falls. This generates an increased gradient for nonionic diffusion of H2C03 which, together with a high apical membrane H2C03 permeability, allows H2C03 recycling to support a limited but significant rate of luminal acidification. Thus in the presence of carbonic anhydrase inhibitors, rates of NaHC03 absorption are significantly slowed, but are not as slow as would be predicted from uncatalyzed rates of H2C03 dehydration and C02 hydration.

In general, apical membrane H + secretion in the proximal tubule occurs by two distinct mechanisms, electroneutral transcellular NaHC03 absorption, and electrogenic HC03~ absorption. Approximately two-thirds of HCO , absorption is mediated by a transcellular electroneutral mechanism, while one-third is mediated by an electrogenic mechanism. These mechanisms will now be discussed.

1. Electroneutral Transcellular NaHC03 Absorption (Fig. 3).

In this mode, the basolateral membrane Na/K ATPase utilizes the energy of ATP hydrolysis to transport Na+ out of the cell and K+ into the cell. K+ then diffuses out across the basolateral K+ conductance, generating a cellnegative voltage. The low cell [Na + ] provides a driving force for the apical membrane Na/H antiporter and allows Na + to enter passively while actively extruding H+. The transporter functions with a 1:1 Na+:H+ stoichiometry. Base generated within the cell exits either as HC03 per se, or as C032- on the Na/HC03/C03 cotransporter. C032- is formed from HC03~ according to the reaction: 2HCCV ->C02 + C032" + H20. The Na/HC03/C03 cotransporter carries two negative charges. The electrochemical driving force for Na + is in the direction of cell entry. However, because of the high cell-negative voltage there is a large favorable electrochemical gradient for efflux of HC03~ and CO ,2 , which drives the transporter.

If one considers three turnovers of the Na/H antiporter, three H + enter the luminal fluid leading to the generation of three HC03~ in the cell. This is accompanied by the movement of three Na + from the lumen into the cell. The Na/HC03/C03 cotransporter mediates the exit of one HC03 and one C032", equivalent to three HC03", with one Na+. The Na/K ATPase would then have to turn over 2/3 times to effect absorption of two Na + and entry of 4/3 Kthe latter exiting via the basolateral membrane K + conductance. In sum, these processes lead to no net charge transfer (the number of cations and anions crossing each membrane are equal), such that there is no requirement for paracellular transport. Net transport across the apical membrane involves movement of three Na + into the cell and three H + into the lumen, resulting in zero net charge transfer. Transport across the basolateral membrane results in absorption of three Na+, one HCO, , and one C032", with recycling of K + , once again resulting in no net charge transfer.

A second mechanism of transport involves apical membrane electrogenic H+ secretion. Here, H+ is actively secreted into the luminal fluid driven by metabolism of ATP. HC03~ and CO ,2 formed in the cell then exit across the basolateral membrane on the Na/HC03/C03 cotransporter, once again as described above. This must be accompanied by Na+ efflux on the basolateral membrane cotransporter and thus requires entry of an Na + across the apical membrane, likely by Na+-coupled transport of solutes such as glucose, amino acids, phosphate, and mono- and dicarboxylic acids. This mode of transport is electrogenic in that for every three H + secreted into the lumen and three negatively charged base equivalents exiting to the interstitium, only one Na + is absorbed. Thus, this mechanism will generate a lumen positive voltage (approximately +1 mV) which will drive a passive paracellular current that could be outward cation diffusion or inward anion diffusion. Based on the measured properties of the proximal tubule tight junction, it will likely involve some combination of Na+ diffusion out of the lumen (equivalent to net NaHC03 absorption) and CI~ diffusion into the lumen (equivalent to net HC1 secretion or Cl~/HC03_ exchange).

NaCl Absorption

There are three general mechanisms of NaCl absorption in the proximal tubule, electroneutral transcellular NaCl absorption, electrogenic Na + absorption, and passive paracellular NaCl absorption (Fig. 4).

1. Electroneutral Transcellular NaCl Absorption (Fig. 4).

Transcellular Na+ absorption may result, as described above, from the coordinated activities of the basolateral membrane Na/K ATPase and K + channel,

Lumen

Interstitium

FIGURE 4. Proximal tubule NaCl absorption. Three modes of NaCl absorption are shown. Elec-troneutral transcellular NaCl absorption is mediated by parallel apical membrane Na/H and Cl/ base exchangers, with the protonated base recycling across the apical membrane. Electrogenic Na + absorption is mediated by a Na +-coupled cotransporter which carries a number of different solutes (designated X). Na+ which enters the cell across an apical membrane transporter, exits the cell on the basolateral membrane Na/K ATPase. K + which enters the cell on the Na/K ATPase exits on a basolateral membrane K + channel. The mechanism of basolateral membrane Cl " efflux is not fully understood but may involve a Cl" conductance, a KC1 cotransporter, or a Na(HC03)2/Cl exchanger. Electrogenic Na + absorption establishes a lumen negative voltage which drives a paracel-lular current. In addition, active transport of NaHC03 and organic solutes generates a high luminal Cl" concentration which drives paracellular Cl" absorption and generates a lumen + voltage that drives paracellular Na+ absorption (passive paracellular NaCl absorption). Active transport mechanism; o, passive transporter; =, channel; B ", base; HB, protonated base.

Voltage +1 mV

FIGURE 4. Proximal tubule NaCl absorption. Three modes of NaCl absorption are shown. Elec-troneutral transcellular NaCl absorption is mediated by parallel apical membrane Na/H and Cl/ base exchangers, with the protonated base recycling across the apical membrane. Electrogenic Na + absorption is mediated by a Na +-coupled cotransporter which carries a number of different solutes (designated X). Na+ which enters the cell across an apical membrane transporter, exits the cell on the basolateral membrane Na/K ATPase. K + which enters the cell on the Na/K ATPase exits on a basolateral membrane K + channel. The mechanism of basolateral membrane Cl " efflux is not fully understood but may involve a Cl" conductance, a KC1 cotransporter, or a Na(HC03)2/Cl exchanger. Electrogenic Na + absorption establishes a lumen negative voltage which drives a paracel-lular current. In addition, active transport of NaHC03 and organic solutes generates a high luminal Cl" concentration which drives paracellular Cl" absorption and generates a lumen + voltage that drives paracellular Na+ absorption (passive paracellular NaCl absorption). Active transport mechanism; o, passive transporter; =, channel; B ", base; HB, protonated base.

and the apical membrane Na/H antiporter. However, if in addition, an apical membrane Cl/base exchanger operates at a rate equal to that of the apical membrane Na/H antiporter the net result is NaCl absorption. Secretion of H + and a negatively charged base (B") at equal rates leads to generation of the neutral acid, HB, which is lipophilic and is thought to recycle across the apical membrane. It is also possible that a specific HB transporter exists. Thus, with this mode of transport there is no net H + secretion and thus no luminal acidification or bicarbonate absorption. Na + and Cl - enter the cell across the apical membrane at equal rates.

The nature of the base exchanged with Cl " is not totally settled but appears to include OH -, formate ~, and oxalate ". Na + which enters the cell on the Na/H antiporter exits on the basolateral membrane Na/K ATPase. Possible mecha nisms of basolateral membrane CI" efflux include: (i) a CI" conductance; (ii) an electroneutral KC1 transporter where K+ exits with CI rather than across the K+ conductance; and (iii) a Na(HC03)2/Cl exchanger where Na + and 2HCCV enter the cell in exchange for CI". The Na+ and HC03" can then leave the cell on the Na/HC03/C03 cotransporter. With all of these mechanisms, the net result is efflux of equal amounts of Na + and Cl ~ with recycling of various ions. Thus, transcellular NaCl absorption is electroneutral, requiring no paracellular transport.

2. Electrogenic Na + Cotransport (Fig. 4).

Na+ can also cross the apical membrane on a cotransporter which carries Na+ and another solute such as sugars, amino acids, phosphate, sulfate, or organic anions such as citrate. Na + then exits across the basolateral membrane on the Na/K ATPase. The other solute exits the basolateral membrane by a variety of mechanisms including facilitated diffusion. If the number of Na + ions exceeds the number of negative charges on the cotransported solute, transport is electrogenic and will generate a lumen-negative voltage. This will then serve to drive either paracellular Cl " absorption (resulting in net NaCl absorption) or paracellular Na + secretion (resulting in Na + recycling). Once again, the relative magnitudes of these passive processes are determined by the ratio of Na + and Cl " permeabilities of the tight junction.

3. Passive Paracellular NaCl Absorption (Figs. 4 and 5).

The last mechanism of NaCl absorption is passive paracellular NaCl absorption. Because the proximal tubule is highly permeable to water (see below), the total concentration of solutes in the luminal fluid remains relatively constant along the length of the tubule. Thus, the active reabsorption of organic solutes

Length of the Proximal Tubule

hco;

: Organic solute

FIGURE 5. Profile of solute concentrations along the length of the proximal tubule. The ratio of tubular fluid to plasma solute concentration (TF/P) is plotted as a function of length along the proximal tubule. Active reabsorption of organic solutes with secondary water reabsorption leads to decreases in luminal organic solute concentrations and small increases in the luminal concentrations of Na + and Cl-. Active reabsorption of NaHCO 3 with secondary water reabsorption leads to a decline in luminal HCO,~ concentration and a further increase in luminal Cl" concentration.

from the luminal fluid leads to water reabsorption which results in a modest increase in the concentration of Na + and Cl ~ in the lumen (Fig. 5). In addition, the rapid rate of NaHC03 absorption in the early proximal tubule causes water reabsorption, resulting in a decrease in luminal HC03~ concentration and an increase in luminal CI"" concentration (Fig. 5). The net result is a small concentration gradient for passive Na + absorption and large concentration gradients for passive Cl~ absorption, HCO . ~, and organic solute secretion. Because proximal tubule paracellular permeability for CI greatly exceeds that for HCOand organic solutes, passive CI" absorption predominates. This leads to a lumen-positive voltage which drives passive Na + absorption. The net result is passive paracellular NaCl absorption.

Regulation of NaCl and NaHC03 Absorption (Table 1)

The majority of filtered NaCl and NaHC03 reabsorption takes place in the proximal tubule, and this nephron segment is also an important site of regulation, by modulation of both transcellular and paracellular ion fluxes. Because of the key role of the apical membrane Na/H antiporter in proximal salt retrieval from the filtrate, this transporter is a key site of regulation. As pointed out above, the Na/H antiporter mediates 2/3 of transcellular NaHC03 absorption and all of transcellular NaCl absorption. In addition, by mediating NaHC03 absorption, it also contributes to the high luminal Cl" concentration which provides the driving force for passive paracellular NaCl absorption (Fig. 5). In many conditions, the activity of the Na/HC03/C03 cotransporter is regulated in parallel with that of the Na/H antiporter. This allows significant changes in the rates of NaHC03 and NaCl transport, while cell pH remains close to normal.

TABLE 1 Regulation of Proximal Tubular NaCl and NaHC03 Absorption

Glomerulotubular balance

Luminal pH and HC03 concentration

Direct effect of luminal flow rate on the Na/H antiporter

Chronic adaptation in the Na/H antiporter and the Na/HC03/C03 cotransporter Decreases in intracellular pH (metabolic acidosis, K + deficiency) Effective arterial volume Physical factors Angiotensin II Endothelin

Renal nerves («2 adrenergic, dopamine) Miscellaneous PTH

In general there are three physiologically important regulatory determinants of proximal tubule NaCl and NaHC03 absorption. First, increases in glomerular filtration rate (GFR) cause proportional increases in NaCl and NaHC03 absorption. Because augmentation of GFR increases the filtered loads of these solutes, parallel stimulation in reabsorption is required to prevent massive bi-carbonaturia and salt wasting. This phenomenon is referred to as glomerulotu-bular balance. Three mechanisms have been identified to explain this phenomenon. First, increases in GFR and thus luminal flow rate lead to increases in luminal pH and HCO, concentration. In the presence of higher luminal flow rates, a given rate of H + secretion will lower the luminal HCO, concentration and thus pH to a lesser degree. The net result is a higher luminal pH and HCO r which causes the rate of cellular H + secretion to increase. Second, acute increases in luminal flow rate cause a rapid activation of Na/H antiporter activity. The mechanism responsible for this effect is not yet elucidated but may involve flow dependent mixing of an unstirred layer in the lumen, or may involve activation of a signaling pathway by cell stretch. Last, chronic elevations of GFR lead to adaptive increases in the activities of the Na/H antiporter and the Na/ HCO3/CO3 cotransporter.

A second major regulator is cell pH. Decreases in cell pH, which occur in metabolic acidosis and K + deficiency, increase the activities of the Na/H antiporter and the Na/HC03/C03 cotransporter, leading to increased rates of NaHC03 reabsorption. However, in metabolic acidosis there is significant inhibition of passive paracellular NaCl absorption. This occurs because the concentration of NaHC03 in the filtrate is low, and thus the ability of NaHC03 absorption to decrease luminal HCO , concentration and increase luminal Cl ~ concentration is limited. The net result is inhibition of passive NaCl absorption.

One of the most important regulators of proximal tubule transport is effective arterial volume. Decreases in effective arterial volume increase net NaHC03 and NaCl absorption. This is of prime importance in the regulation of the effective arterial volume and blood pressure, and is likely important in mediating the effect of volume contraction to maintain metabolic alkalosis. Contraction of effective arterial volume has been shown to lower tight junction and paracellular permeability which can decrease the backleak of HCO, into the lumen and thus increases net HC03~ absorption. However, given that some NaCl is absorbed across the paracellular pathway, a decrease in permeability cannot explain the observed increase in NaCl absorption. Thus, decreased effective arterial volume also directly stimulates transcellular NaCl and NaHC03 absorption.

Stimulation of transcellular NaCl absorption in response to decreased effective arterial volume is related in part to coordinated effects of increased peritubular capillary protein concentration and decreased peritubular capillary hy drostatic pressure (so-called physical factors). Decreases in effective arterial volume lead to efferent arteriolar vasoconstriction which results in a decrease in peritubular capillary hydrostatic pressure and an increase in filtration fraction, which secondarily results in higher peritubular capillary oncotic pressures. Both of these physical factors would be expected to increase the rate of fluid movement into the peritubular capillary, but it is not clear how this leads to an increase in tubular NaCl transport. The above changes in glomerular hemodynamics are likely related to changes in the levels of hormones such as angiotensin II, autocrine/paracrine factors such as endothelin-1 and endoth-elin-3, or increased renal nerve activity.

In addition, these factors have been demonstrated to regulate directly proximal tubule transport, independent of changes in hemodynamics. Angiotensin II, endothelins, and a2 adrenergic catecholamines increase apical membrane Na/H antiporter activity, whereas dopamine inhibits Na/H exchange. In addition, dopamine lowers Na + reabsorption by inhibiting basolateral Na/K ATPase activity. PTH (parathyroid hormone) is also a potent inhibitor of the proximal tubular Na/H antiporter, but the physiologic significance of this is unclear.

Potassium Transport

The proximal tubule reabsorbs 50-70% of filtered K + . The magnitude of K + absorption parallels the magnitudes of Na + and volume absorption. This relationship is due to the fact that the majority of K + absorption occurs passively and is indirectly coupled to volume absorption. Volume absorption causes an increase in luminal K + concentration which provides a driving force for K+ diffusion across the paracellular pathway. There may also be some convective K + absorption (solvent drag) which would be more directly coupled to volume absorption.

The proximal tubule cell has K + conductances on its apical and basolateral membranes. The basolateral K+ conductance, mediated by ATP-inhibitable K + channels, is physiologically important because it plays a key role in the generation of the cell-negative potential. The latter is a major driving force for passive movement of positively charged solutes across the apical brush border membranes. Under certain experimental conditions small active transcellular K + fluxes can be demonstrated, but these fluxes are physiologically insignificant. Thus, the majority of proximal tubule K+ absorption is passive and coupled directly and indirectly to volume absorption.

In the S3 proximal tubule some passive K + secretion into the luminal fluid has been reported. This flux is driven by high medullary interstitial K + concentrations. Such passive secretion, which continues in the thin descending limb, is a component of potassium recycling and is discussed below.

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