Where does antidiuretic hormone ADH exert its effects to promote water reabsorption?

Regulation of water and electrolyte balances

Hormones control tubular reabsorption to regulate body fluid volumes and solute concentrations. A hormone is a substance that is secreted from an endocrine gland or gonad and transported through the blood to the site of action. Aldosterone acts on the collecting tubule and duct cells to increase Na+ reabsorption and H+ and K+ secretion. Angiotensin II acts on the proximal, distal, and collecting tubule cells to increase Na+ reabsorption and H+ secretion. ADH acts on the collecting tubule and collecting duct cells to increase water and urea reabsorption (Guyton and Hall, 2006).

The Proximal Tubule Reabsorbs an Isosmotic Solution

The reabsorption of nutrients, water, and salt from the proximal tubule was described in Chapter 7.4 and is summarized again in Figure 7.5.2. The Na+,K+-ATPase provides the motive force for all of the cotransport processes by establishing a favorable electrochemical gradient for Na+ entry into the cell at the apical membrane. This favorable gradient powers the movement of a large number of solutes including glucose, amino acids, phosphate, lactate, sulfate, and indirectly through Na+–H+ exchange, HCO3−. Water and urea reabsorption passively follow the movement of osmotically active solutes so that the fluid that remains at the end of the proximal tubule is isosmotic with plasma. At this point, all of the nutrients are reabsorbed but the concentration of some secreted materials is higher. This fluid is presented to the loop of Henle.

12.3 Tubule Reabsorption/Secretion

Tubule reabsorption is the process by which molecules from the glomerular filtrate are returned back to the plasma. This occurs along the entire nephron unit. Metabolically important molecules are nearly completely reabsorbed, whereas wastes are reabsorbed to some extent, with the majority of waste molecules making it into the urine. By looking at the relative percent reabsorbed of various molecules, it is possible to draw some conclusions about tubule reabsorption (Table 12.1). Metabolically important molecules (water, ions, and organic molecules) are completely reabsorbed so that there is no need to constantly ingest (or produce) these molecules. Waste products are not fully reabsorbed so that they can be removed from the body. It is important to note that the reabsorption of most organic compounds (e.g., glucose) is not regulated and is typically very high. Therefore, under normal conditions, none of these compounds is found within the urine. For these compounds, it can be considered that the kidneys do not exist, because the kidneys have no effect on their plasma concentration. However, the reabsorption of most non-organic metabolically important molecules (e.g., water and ions) is tightly regulated but is also very high under normal conditions. You can prove this by drinking a few 64-oz double-big sodas from your local gas station in under 10 minutes. It is guaranteed that you will be visiting the bathroom soon to remove the excess water from your system. However, if you eat a few candy bars with a lot of sugars in them, the amount of sugar in your urine does not increase, but is stored for later use.

Table 12.1. Average Reabsorption Values for Various Metabolically Important Compounds

There are two main processes that account for the reabsorption of compounds into the peritubular capillaries. Some substances can be reabsorbed by diffusion and others involve some receptor mediated transport. Diffusion typically occurs across the tight junctions of the tubule epithelial cells, whereas the receptor mediated transport occurs through the epithelial cells themselves. For example, the reabsorption of urea occurs by diffusion. However, because the composition of the glomerular filtrate is the same as the plasma composition, there should be no concentration gradient driving force for the movement of urea. Early on within the proximal tubule system, water is removed from the filtrate (via receptor-mediated transport). With the removal of water, the effective concentration of urea increases within the tubule lumen, and therefore a concentration gradient is formed between the nephron and the peritubular capillaries. Urea can then diffuse down its concentration gradient from the filtrate into the plasma at a distal location along the nephron. The reabsorption of the majority of lipid soluble compounds occurs in this manner, and is therefore dependent on the early reabsorption of water, to effectively increase the molecular concentration within the nephron.

For a material to be reabsorbed via receptor-mediated transport, the molecule must first diffuse to the wall of the nephron tubule. The molecule must then cross the luminal wall into the tubule epithelial cells. The molecule could then diffuse across the tubule epithelial cell to the basolateral cell membrane. The molecule then crosses this cell membrane into the peritubular capillaries. It is not necessary for the molecule to be actively transported across both of the cell membranes, and typically the molecule would move down its concentration gradient when crossing one of the barriers. For example, sodium can diffuse into the tubule epithelial cells, but it is then actively transported across the epithelial cells basolateral membrane to enter the bloodstream. If the transport of a molecule is active across at least one barrier, then it falls into the category of receptor-mediated transport.

It is interesting to note that the reabsorption of many molecules is coupled to sodium movement across the tubule epithelial cells. This type of movement is mediated by a co-transporter, which in this case utilizes the energy derived from the movement of sodium in the direction of its electrochemical gradient to drive the movement of another molecule (for example, glucose, many organic compounds, and some inorganic ions) against its electrochemical gradient. The activity of co-transporters is classified by the amount of molecules that can be transported in a unit time. Under most normal conditions, the maximum rate of transport is never reached by the co-transporters. However, if the nephron concentration of a particular compound becomes so large that all of the binding sites on every transporter are occupied, then the maximum transportation rate is reached (i.e., the transporters are saturated) and this compound may enter the urine. As an example, under diabetic conditions, it is possible for the glomerular filtrate glucose concentration to exceed the maximum transportation rate, and then glucose enters the urine and is excreted. There is actually an old legend that before the age of modern medicine, diabetes mellitus would be diagnosed by determining how “sweet” a patient’s urine was. How much truth is in this legend is up for debate, but regardless, diabetic patients can excrete glucose in urine, whereas under normal conditions, all of the glucose within the glomerular filtrate is reabsorbed into the plasma.

Tubule secretion is the process by which molecules from the peritubular capillaries move into the tubule lumen. Similar to reabsorption, secretion can occur via diffusion or receptor-mediated transport. You may be wondering why the peritubular capillaries secrete compounds into the lumen. There are a variety of reasons, based on the particular compound. Many toxins or foreign compounds are secreted to be fully removed from the plasma. Hydrogen ions are secreted to regulate the pH of the blood. Secretion mediated by diffusion occurs similarly to the diffusion associated with tubule reabsorption, except that it occurs in the opposite direction. Interestingly, the receptor-mediated secretion of molecules is typically coupled to sodium reabsorption. Therefore, the electrochemical gradient of sodium drives the movement of other compounds against their electrochemical gradients. These types of transports are typically called anti-porters because the two molecules move in opposite directions. Hydrogen ion secretion makes use of an anti-porter coupled to sodium.

It is important to note before we discuss specific examples of reabsorption and secretion, what components of the nephron perform what functions during urine formation. The primary function of the proximal tubule is to reabsorb large quantities of water and other solutes within the glomerular filtrate. This helps to form the concentration gradient which will be used in later segments of the nephron to drive the reabsorption and/or secretion of particular compounds. The Loop of Henle also functions to reabsorb large quantities of solutes and small quantities of water. The proximal tubule system is also responsible for the secretion of the majority of compounds, except for potassium. This early movement of solutes and water within the proximal tubule and the Loop of Henle are by bulk processes, where the major goal of these processes is to get the plasma/urine solute concentration close to its acceptable level. The distal convoluted tubule and the collecting duct system are primarily responsible for fine-tuning the concentrations of the solutes and determining the final excreted concentration and the final plasma concentration. Therefore, it should be intuitive that the majority of the mechanisms that exert control on the nephron (e.g., hormones) and affect urine concentration act on the distal convoluted tubule and the collecting duct. As a summary, the amount of a compound that is excreted can be calculated by measuring the amount filtered, secreted, and reabsorbed, as follows:

AmountExcreted=AmountFiltered+AmountSecreted −AmountReabsorbed

WATER REABSORPTION – DISTAL AND COLLECTING TUBULES – 1.

Water reabsorption in the distal convoluted tubules and the collecting ducts depends on (1) the permeability of the tubules to water, and (2) the osmotic pressure of the interstitial fluid surrounding the tubules.

The function of the EARLY distal convoluted tubule (first two thirds) differs from that of the last third, called the LATE distal tubule.

The late distal tubule and the collecting tubules are made permeable to water by the presence in the circulation of antidiuretic hormone (ADH) released from the posterior pituitary gland (p. 212). The early distal tubule is not permeable to water and its permeability is not changed by ADH.

The osmotic pressure of the interstitial fluid which surrounds the tubules throughout the cortex is isosmotic or the clinical term isotonic (300 mosmol/kg H2O, the same as inside the cells). In the medulla there is a gradient of osmotic pressure in the interstitial fluid. It increases from 300 mosmol/kg H2O at the cortico-medullary junction to 1400 mosmol/kg H2O at the tip of the papilla. The gradient is formed by the counter-current mechanism in the loops of Henle (pp. 175, 176).

When ADH is PRESENT in circulation:

ADH increases intracellular cAMP which causes the insertion of water channels into the membrane of the cells, making them permeable to water.

The Sodium-Monocarboxylate Cotransporter (SMCT; SLC5A8)

Lactate reabsorption in proximal tubules occurs via sodium-monocarboxylate cotransport as shown by early studies with rat (17) and rabbit renal brush-border membrane vesicles (250) and with the intact rat kidney (Fig. 1) (388). Half-maximal transport rates were observed at 1.7 mM D-lactate in situ (388). Moreover, the transporter handled aliphatic monocarboxylates with two (acetate) and up to eight (octanoate) carbons (389). Substitutions at position 2 and 3 of propionate were tolerated (e.g., pyruvate, lactate, 2-mercaptopropionate). Aromatic monocarboxlyates (i.e., benzoate and its meta- and para-substituted analogues) also interacted with the lactate transporter in rat kidney in vivo (391). In these experiments, nicotinate and pyrazinoate were good inhibitors, whereas PAH, urate, and taurocholate were without affect. In rabbit renal brush-border membrane vesicles, unlabeled lactate, pyruvate, acetate, propionate, butyrate, acetoacetate, and β-hydroxybutyrate inhibited Na+-dependent uptake of labeled lactate (250). Thus, the brush-border membrane of proximal tubule cells is endowed with a transporter for monocarboxylates that include lactate, short-chain fatty acids, and intermediates of the fatty-acid β-oxidation.

The molecular nature of the transporter involved in renal Na+-dependent lactate transport remained elusive for a long time. In 2004, two groups independently reported that a member of the sodium-glucose cotransporter family, SLC5A8, is a sodium monocarboxylate cotransporter (68, 99). This transporter was first cloned from a human cDNA library as a protein related to the thyroid gland sodium-iodide cotransporter NIS (288). The gene was located on chromosome 12q23. Later, the same protein was described as a human sodium transporter that is silenced in colon tumor cells by hypermethylation of GpC-rich regions in exon 1 (203). Whereas the human SLC5A8 was found to be expressed at the apical membrane of thyroid follicle epithelial cells, transcripts of mouse SLC5A8 were detected in small intestine, colon, and kidneys (99). In the mouse kidney, message was detected along the complete proximal tubule.

Following heterologous expression, human (68, 231) and mouse (99) SLC5A8 exhibited Na+-dependent transport of aliphatic monocarboxylates ranging from acetate to octanoate and D- and L-lactate. Therefore, the transporter was named sodium monocarboxylate cotransporter (SMCT). The uptake of monocarboxylates together with sodium ions by the murine SMCT generated an inward current that decreased in the order lactate > pyruvate > propionate, acetate > butyrate > pentanoate and longer monocarboxylates (99). Similar inward currents were reported for human SMCT with half-maximal values at 81 μM butyrate, 127 μM propionate, 235 μM L-lactate, and 2460 μM for acetate (231). Interestingly, the coupling ratio between Na+ and monocarboxlates appeared to depend on the substrate: It was two Na+ for one lactate, but four Na+ for one propionate (99). In another report, the stoichiometry was reported to be 2:1 for propionate (68), the reason for the different results unclear at this point.

α-Cyano-4-hydroxycinnamate, a typical inhibitor of H+-coupled monocarboxylate transporters, did not inhibit SMCT (68, 99). Probenecid exerted a weak, and ibuprofen a strong, inhibition of monocarboxylate-induced inward currents, but did not produce themselves currents, indicating that these compounds are not translocated by SMCT (68).

The physiological role of SMCT/SLC5A8 in proximal tubule is the reabsorption of filtered lactate. Part of the lactate taken up by SMCT may exchange for urate via the URAT1 that is also located in the brush-border membrane (Fig. 3). The antiuricosuric, pyrazinoate, is also taken up into proximal tubule cells by SMCT and subsequently exchanged for urate. Among clinical drugs, the uricosuric probenecid and the analgesic ibuprofen have been found to inhibit SMCT. At present it is not clear whether other anionic drugs interact with SMCT.

CONTROL OF PROXIMAL TUBULE MAGNESIUM REABSORPTION

Fractional Excretion of Lithium

Markers of proximal fluid reabsorption can be used to assess proximal tubular sodium reabsorption (11). Such a marker should be freely filtered at the glomerulus, should be reabsorbed in the same fashion as sodium and water in the proximal tubule, should not be reabsorbed beyond the proximal tubule, should not be secreted, and its plasma levels should not fluctuate significantly in response to hormones and changes in ECFV. Lithium fits this profile with the exception that it is reabsorbed in the loop of Henle as well as in the proximal tubule.

To assess proximal fluid reabsorption, the fractional clearance of exogenous lithium can be used (11). To do so, it is necessary to give lithium either acutely or for several days prior to measuring lithium clearance or its fractional clearance. The fractional excretion of lithium (FELi) is approximately 20% in healthy controls and below 10% in prerenal disease regardless of diuretic therapy (164). In ATN, FELi is typically higher than 25% (164).

FELi is a better index than FENa in differentiating prerenal states from ATN, especially in patients receiving concomitant diuretics (11). The use of lithium, however, has its own disadvantages. Its use is limited by the necessity of administering exogenous lithium, by the expertise needed to detect lithium levels in urine and by the acute changes in tubular electrolyte handling induced by lithium loading (130, 164). Moreover, concern has been raised regarding the possible reabsorption of lithium in the thick ascending limb of the loop of Henle, which could be inhibited by loop diuretics (57, 105). For clinical purposes, lithium clearance is not used but it offers valuable information in clinical physiology studies aimed at assessing segmental sodium handling through the nephron. It should be noted, however, that initial studies showed that lithium was reabsorbed almost entirely in the proximal tubule in a fashion similar to that of sodium (57), thus making lithium the marker of choice for determining proximal reabsorption. However, further studies uncovered a number of problems with using lithium clearance for assessing proximal reabsorption (12, 58, 86, 172, 173). First, lithium undergoes significant reabsorption in the loop of Henle, and to a lesser extent in the distal tubule and collecting duct (11, 57). Amiloride can be used to block the reabsorption of lithium in the distal tubule and collecting duct (21). The reabsorption of lithium in Henle's loop, however, cannot be inhibited pharmacologically. A second problem with the use of lithium is that in the presence of mineralocorticoid-induced volume expansion and with the administration of inhibitors of prostaglandin synthesis, lithium reabsorption in Henle's loop is increased (29, 125). In the aggregate, lithium clearance is the best available method to assess the delivery of sodium and water out of the proximal tubule, and is reasonably accurate in the steady state. In states of volume and hormonal perturbations, however, lithium clearance is much less reliable.

BIOARTIFICIAL RENAL TUBULE

The efficiency of reabsorption, which depends on natural physical forces governing fluid movement across biologic as well as synthetic membranes, requires specialized epithelial cells to perform vectorial solute transport. A population of cells residing in the adult mammalian kidney has retained the capacity to proliferate and morphogenically differentiate into tubule structures in vitro [33] and can be used as the key cellular element of a tissue-engineered renal tubule device.

Implementation of any device based on cell therapy, including a tissue-engineered renal tubular device, requires a steady and predictable supply of tissues from which cells may be isolated and cultured. Currently, these cells must be procured through the harvest of animal or human tissue. Until stem cells can be isolated and induced to differentiate into organ-specific cell types, the supply of cells available for cell therapy will be constrained.

The bioartificial renal tubule can be readily conceived as a combination of living cells supported by polymeric substrata, using epithelial progenitor cells cultured on water- and solute-permeable membranes seeded with various biometric materials so that expression of differentiated vectorial transport as well as metabolic, endocrine, and immunologic function is attained (Figure 9.3). With appropriate membranes and biomatrices, immunoprotection of cultured progenitor cells has been achieved concurrent with long-term functional performance as long as conditions support tubule cell viability. This bioartificial tubule has been shown to transport salt and water effectively along osmotic and oncotic gradients [44].

The bioartificial proximal tubule satisfies the major requirement of reabsorbing a large volume of filtrate to maintain salt and water balance within the body. The need for additional tubule segments to replace other nephronal functions, such as the loop of Henle to perform more refined homeostatic elements of the kidney (including urine concentration or dilution), may not be necessary. Patients with moderate renal insufficiency lose the ability to finely regulate salt and water homeostasis because they are unable to concentrate or dilute, yet they are able to maintain reasonable fluid and electrolyte homeostasis due to redundant physiologic compensation via other mechanisms. Thus, a bioartificial proximal tubule—which reabsorbs isoosmotically the majority of the filtrate—may be sufficient to replace required tubular function and sustain fluid electrolyte balance in a patient with ESRD.

5 Conclusion

Inhibition of renal glucose reabsorption by SGLT2 inhibitors and subsequent glucose excretion into urine is a unique mechanism of action to lower blood glucose levels. Recent clinical data demonstrate that this potential new insulin-independent antidiabetic therapy not only can reduce HbA1c levels as effectively well as existing therapeutic agents but also confers other beneficial features, such as body weight loss and low propensity for causing hypoglycemia. Overall, the available data show that SGLT2 inhibitors have demonstrated good benefit-risk profiles in human clinical trials. The U.S. Food and Drug Administration accepted a New Drug Application for dapagliflozin for review in March, 2011. It is hoped that dapagliflozin and other SGLT2 inhibitors will become important treatment options for type 2 diabetic patients.

Proximal Tubule

Micropuncture experiments indicate that net reabsorption occurs over most of the proximal tubule so that ∼50% of the filtered potassium is delivered to the end of the PCT (Malnic et al., 1964). Similar amounts of water are also reabsorbed and the proximal tubule does not sustain a large lumen-to-blood concentration gradient for potassium.

Two major mechanisms account for potassium reabsorption in the proximal tubule, shown in Figure 4. First, solvent drag and a low reflection coefficient mean that potassium transport is strongly dependent upon transepithelial water movement (Malnic et al., 2013). Second, in vivo micropuncture indicates that a small concentration gradient supports significant paracellular diffusion of potassium from luminal to peritubular fluid, influenced by the transepithelial potential difference (Shirley et al., 1998). These effects are particularly significant in the S2 proximal convoluted tubule, which has a lumen positive transepithelial potential difference. If this potential difference is absent, or reversed, reabsorption of potassium is impaired (Shirley et al., 1998).

There is inferential support for a third mechanism for potassium reabsorption in the proximal tubule. The underlying molecular mechanism is not defined: H,K-ATPase mediates potassium reabsorption in the distal nephron (see below) but there is no evidence for such activity in the mammalian PCT (Doucet, 1997).

Potassium channels have been identified in both apical and basolateral membranes and serve multiple physiological functions. The channels stabilize membrane potential and thus maintain the electrical driving force moving charged solutes into, and out of the cell (Hamilton and Devor, 2012). This is critical in the apical membrane to offset the depolarizing effect of electrogenic sodium-coupled transport. The KCNQ1 channel and KCNE1 accessory protein may mediate this effect: both are localized to the apical membrane of the PCT (Sugimoto et al., 1990; Vallon et al., 2001) and Kcne1 null mice have an increased urinary excretion of sodium and glucose (Vallon et al., 2001). Potassium channels in both membranes are activated by cell swelling and mediate volume regulation of proximal tubule cells.

The basolateral membrane expresses potassium channels and a K–Cl cotransporter (Hamilton and Devor, 2012), both of which permit ‘recycling’ across the basolateral membrane of potassium ions that enter via Na,K-ATPase. These exit pathways enable the cell to maintain a constant intracellular potassium concentration in the face of fluctuating transcellular sodium flux. For example, increased transcellular sodium transport necessitates increased activity of the Na,K-ATPase. By varying the magnitude of basolateral potassium recycling in proportion to changes in pump rate, renal cells, including those of the proximal tubule, maintain physiological cytosolic potassium concentrations (Beck et al., 1994). Several mechanisms, including cytosolic ATP concentration, pH and calcium may contribute to the coupling between potassium recycling and basolateral Na,K-ATPase activity (Figure 5).

The proximal tubule is the main site of potassium reabsorption and, being ‘coupled’ to sodium and fluid movement, potassium transport is influenced by factors (such as angiotensin II and dopamine) that influence the movement of sodium. Diminished potassium reabsorption may contribute to the increased potassium excretion that occurs following a high potassium diet (Brandis et al., 1972) but most of this excretion reflects processes in the distal nephron: reabsorption of potassium by the proximal tubule does not play a major role in the physiological regulation of potassium homeostasis.