Concentration and Dilution of Urine

Renal Mechanism for Concentration and Dilution of Urine

The concentration and dilution of urine involve several transport mechanisms along the different segments of the nephrons ( Fig. 102.1 ). The thick ascending limb of Henle loop, located in the outer medulla, plays a key role in diluting and concentrating mechanisms. This segment of the nephron has a very low hydraulic conductivity and can reabsorb NaCl in the relative absence of water. The result is dilution of fluid remaining in the tubular lumen and an increase in the interstitial concentration of NaCl surrounding the thick limbs and collecting ducts. Isotonic fluid from the proximal tubule enters the descending loop of Henle at the level of the outer medulla and is exposed to increased interstitial osmolality. Descending loop segments have a high hydraulic conductivity, but a low sodium permeability. As a consequence, water in the descending limb moves osmotically into the interstitium, and fluid in the lumen of this segment becomes more concentrated.

Urine flowing through the thick ascending limb becomes progressively dilute by virtue of the relative impermeability of this segment to water. It then enters the distal convoluted tubule, a cortical segment in which water and salt reabsorption and equilibration with blood produce a largely isotonic urine entering the outer medullary collecting duct. In a hypertonic outer medulla, water is osmotically removed from the tubule lumen, and solutes in the collecting duct become increasingly concentrated. Urea movement in distal tubules is limited, but water reabsorption is substantial, so that cortical collecting tubules receive urine with a high urea concentration. This segment has low urea permeability in both the presence and absence of ADH, so that a high urea concentration is maintained in the urine entering the outer medullary collecting duct. This segment has also a low permeability to sodium and probably does not transport NaCl actively. Thus it does not contribute to the high interstitial salt content.

In contrast to the more proximal collecting duct, the inner medullary collecting duct is permeable to urea in the presence of ADH. ^, Urea diffuses down a chemical gradient to enter the medullary interstitium. Urea can also enter the descending limb, and its concentration actually rises in this segment. Accumulation of urea in the interstitium provides an osmotic driving force that further abstracts water from medullary portions of the descending loop. This increases the luminal concentration of NaCl and urea. The urine begins to become progressively less concentrated, and NaCl accumulates in the interstitium. The presence of this added interstitial NaCl abstracts more water from urine in the inner medullary collecting duct as this segment courses through the interstitium, a step that further concentrates the final urine. In addition, urea can reenter the lumen of the thin ascending limbs to recycle back to the collecting tubule.

Role of Short-Loop Nephrons and Urea

Recycling of urea (to maintain a high medullary urea content) is critical for maximal efficiency of the concentrating process and is facilitated by short-looped nephrons that do not descend deep into the inner medulla. ^, These nephrons reabsorb a large portion of filtrate, reducing the volume of fluid and solute that needs to be concentrated. Short-looped nephrons also exhibit permeability and anatomic characteristics that place them in a good position to receive urea and facilitate its recycling to the inner medulla. The inter-tubular recycling of urea is likely accomplished through three major routes. First, urea in the ascending thin limb can remain in the tubule lumen, travel through the distal nephron to the collecting ducts, and then recycle to the interstitium. Second, urea in thin ascending limbs can reach the thick ascending limb, a segment that, in the outer medulla, is permeable to urea and is in close relationship to the proximal straight tubules (and therefore the descending limbs) of both long- and short-loop nephrons. At this site, urea can return to the inner medulla directly via proximal straight tubules and descending limbs of long-loop nephrons, or it can cycle through loops and collecting ducts of short nephrons to reenter the medulla. Third, urea can leave the inner medullary interstitium through ascending vasa recta and reenter the descending limbs of short-loop nephrons. This urea can similarly be carried back through superficial distal nephrons and transported to collecting ducts in the inner medulla.

The role of urea is complex. Urea had been thought to cross cell membranes solely by passive diffusion, but rapid urea transport rates in some tissues suggested a facilitating transport mechanism. As reviewed, several urea transporter isoforms (UTs) derived from the UT-A gene have been identified in renal tissue that can facilitate transmembrane movement of urea and can help generate the hypertonic medulla. UT-A1 and UT-A3 are restricted to the terminal portion of the inner medullary collecting duct. UT-A2 is localized in the lower part of the thin descending limb of short-loops of Henle in the inner stripe of the outer medulla, and also in the thin descending limb of long-loop nephrons in the inner medulla under more prolonged antidiuretic conditions. The UT-B gene produces the UT-B1 transporter protein located in the endothelial cells of medullary descending vasa recta. UT-B1 allows urea leaving the ascending vasa recta to reenter the descending vasa recta, become trapped in the medulla, and maintain a high medullary urea concentration. This transporter protein is down-regulated by ADH and may serve different functions (favoring nitric oxide over urea synthesis and limiting urea production in endothelial cells when a very high inner medullary content of urea is already present). ^,

Finally, animal and human studies suggest that urea is not an effective osmole when vasopressin acts in fed mammals (to allow for water conservation). Nevertheless, when the urine is electrolyte-poor, and in the context of water dehydration, urea becomes an effective osmole to ensure a safe minimum urine flow rate.

Role of Vasa Recta

Vasa recta are blood vessels coursing through the interstitium. Vasa recta provide substrate for, and removal of end products of, metabolic reactions. In addition, due to their high permeability, they play an important role in salt and water balance. The vessels descend into the medulla to varying degrees, break up into small capillaries that course through localized areas of interstitium, and rejoin to ascend toward the cortex ( Fig. 102.2 ). Ascending vasa recta are fenestrated and highly permeant. Descending vasa recta have continuous endothelial cells and pericytes (contractile smooth muscle remnants). They also contain the urea transporter UT-2 as well as aquaporin (AQP)-1. Descending vasa recta are well situated adjacently in vascular bundles and in deeper portions of the medulla to receive NaCl and urea from ascending vasa recta and to deliver water toward the ascending vessels down concentration gradients. ^, Accordingly, solutes such as NaCl and urea found in high concentration in ascending vasa recta can enter descending vasa recta that contain a fluid of lower solute concentration, whereas water can move in the opposite direction.

The ability of the vasa recta to maintain the medullary interstitial gradient is flow dependent. A substantial increase in vasa recta blood flow dissipates the medullary gradient. Alternatively, decreased blood flow reduces oxygen delivery to the nephron segments within the medulla.

Role of the Renal Pelvis

The renal pelvis may contribute to urinary concentration. Epithelium covering the inner medulla and papilla and facing the pelvic space is similar to that of papillary collecting ducts. During water diuresis, these cells are closely apposed and intercellular spaces are narrow. During antidiuresis, individual cells are more distinct, and intercellular spaces are widely dilated. This may reflect enhanced transepithelial movement of fluid. Pelvic extensions in some species allow the urinary space to surround vascular bundles and much of the papillae and may reach the cortex. Increased urine concentration has been related to an increase in contact surface area between papilla and pelvic urine. ^,

Role of Aquaporins

AQPs are a family of highly selective transmembrane channels that mainly transport water across the cell and some facilitate low-molecular-weight solutes. To date, nine AQPs, including AQP1, AQP2, AQP3, AQP4, AQP5, AQP6, AQP7, AQP8, and AQP11, have been identified in different segments and various cells of the kidney to maintain normal urine concentration function. ^,

Some features of AQPs 1 to 3 are summarized in Table 102.1 .

AQP-1 is found in renal proximal tubules, long-loop thin descending limbs of Henle, and non-fenestrated endothelium of descending vasa recta. AQP-1 imparts high osmotic water permeability. In nephron segments, this protein is identified in both apical and basolateral membranes, including both basal and lateral infoldings, but not to any substantial degree in cytoplasmic vesicle and vacuole membranes. Accordingly, AQP-1 is poised to facilitate transcellular water movement in segments responsible for reabsorbing a major portion of the glomerular filtrate, but it does not appear to require hormonal regulation and therefore is not the ADH-sensitive water channel. This protein is also abundant in red blood cells, where it is thought to increase membrane permeability. Although individuals without functional AQP channel-forming integral protein (CHIP) water channels do not appear to have clinical abnormalities, such as polyuria, rare individuals lacking AQP-1 have a urinary concentrating defect in response to ADH or to water deprivation. The role of AQP-1 in maintaining urinary concentrating ability is appreciated from studies of transgenic knockout mice lacking AQP-1. These mice are polyuric and do not tolerate water deprivation. Moreover, perfused proximal tubules and descending thin limbs from such mice have reduced osmotic water permeability. Thus AQP-1 is necessary for maximal concentrating ability. This is likely related to the need for rapid equilibration of water across the thin descending limb of Henle in helping to establish the countercurrent multiplication process.

AQP-2 is the most important ADH-regulated water channel and mediates the short-term renal response to the hormone. This conclusion is supported by the following cumulative observations. Vasopressin binds to a basolateral membrane receptor and initiates a cyclic adenosine monophosphate (cAMP)-mediated chain of signaling events leading to the insertion of water channels and increased osmotic water permeability of the collecting duct apical membrane. ^, AQP-2 protein is present primarily in cytoplasmic vesicles and in apical plasma membranes of collecting duct principal cells. Furthermore, some staining for AQP-2 occurs in basolateral plasma membranes of inner medullary collecting-duct principal cells. After stimulation by ADH, apical membrane staining for AQP-2 intensifies, but it decreases in the subapical vesicles (i.e., AQP-2 redistributes from vesicles to membrane). ^, Therefore there is a reservoir of cellular AQP-2 protein capable of actually recycling between intracellular cytoplasmic vesicles and the plasma membrane. Despite inhibition of protein synthesis in LLC-PK1 epithelial cells—cells that express many features of renal proximal tubule epithelia—AQP-2 staining was primarily localized to intracellular vesicles in nonstimulated cells and quickly redistributed (within 10 minutes) to the plasma membrane after exposure to ADH. The pattern was reversible on removal of ADH.

There is also evidence that AQP-2 plays a major role in the long-term adaptation to pathophysiologic stimuli known to alter urinary concentrating ability. For example, lithium, a drug used in affective disorders, induces an ADH-resistant concentrating defect characterized by the down-regulation of AQP-2 expression in rat inner medullary membranes that coincides with the development of polyuria. ^, The ADH-resistant urinary concentrating defect occurring after the release of bilaterally obstructed ureters is also associated with a marked down-regulation of inner medullary AQP-2 expression that correlates with polyuria. In addition, the slow recovery is marked by persistence of decreased AQP-2 expression.

Chronic hypokalemia may lead to ADH-resistant nephrogenic diabetes insipidus. When hypokalemia was induced by potassium deprivation for 11 days, polyuria correlated with the down-regulation of AQP-2 in both the cortex and the inner medulla of rat kidneys. Polyuria expression of AQP-2 corrected within a week of potassium repletion. Hypercalcemia is another electrolyte disorder in which a concentrating defect and polyuria have been associated with the down-regulation of AQP-2. ^, Thirsting increases expression of AQP-2 in rat collecting ducts, and in Brattleboro rats, a model of central diabetes insipidus, expression of AQP-2 in collecting ducts is reduced in the basal state and is significantly increased on exposure to ADH. ^, The decreased urinary-concentrating ability associated with protein-depleted or malnourished subjects is thought to relate to a decrease in the deep medullary urea content. However, rats kept on a low-protein diet for 2 weeks, without malnutrition, demonstrate decreased maximal urine osmolality after water deprivation, reduced ADH-stimulated osmotic water permeability, and decreased expression of AQP-2 protein in terminal portions of the inner medullary collecting ducts.

In both acute and chronic renal failure, the contribution of dysregulation of several AQPs in defining a nephrogenic diabetes insipidus state has been described. In a renal artery clamp model of ischemic acute renal failure, decreased collecting ducts AQP-2, -3, and -4 have been documented, in association with impaired components of the countercurrent concentrating mechanism, which generates the hyperosmotic driving force for water transport through the AQPs water channels. In a 5/6 nephrectomy rat model of chronic renal failure, several defects have been identified, including absence of the arginine vasopressin (V2) receptor mRNA as well as decreased AQP-2. A rat model of nephrotic syndrome (using puromycin aminonucleoside) is also another example of nephrogenic diabetes insipidus associated with dysfunction of collecting duct AQPs and a significant decrease of both AQP-2 and -3 in the inner medulla. Finally, in the hypothyroid rat a significant diminution in renal concentrating capacity has been documented. This defect appeared multifactorial and in part due to a decrease in AQP-2 expression and trafficking to the apical membrane of the collecting duct.

These examples of limitations in urinary concentrating ability associated with long-term alterations in AQP-2 are of particular interest considering the presence of AQPs in fetal and neonatal renal tissues (see later discussion). Moreover, AQP-2 appears to be necessary for ADH-dependent concentration of urine in humans, as evidenced by a patient with autosomal recessive nephrogenic diabetes insipidus who had two mutations in the gene encoding AQP-2. Expression of the defective proteins in Xenopus oocytes showed nonfunctional water channel proteins that failed to increase osmotic water permeability. Such abnormalities may become definable by analyzing urine from such patients. ^,

Previous studies have also revealed a non–ADH-mediated regulation of AQP-2.

In animal models decreased prostaglandin E ₂ (PGE ₂ ) production, as induced by cyclooxygenase inhibition, results in reduced endocytosis of AQP-2 and therefore increased abundance of AQP-2 in the plasma membrane with associated water reabsorption. Effective osmolality/tonicity has also been associated with increased expression of AQP-2.

As proven by both animal and human studies, several drugs may modulate AQP-2 expression or regulation, and this effect may be different in infant versus adult kidney. In infant but not in adult rats, a single injection of betamethasone induces an increase in urine osmolality, renal medullary AQP-2 mRNA, and protein levels in the 24 hours following treatment. This finding has been confirmed in human preterm neonates, as well.

In patients with chronic heart failure, furosemide administration increased the vasopressin level and stimulated water reabsorption via the AQP-2 water channels.

In human healthy adults, ibuprofen administration increased urinary AQP-2 excretion without changes in ADH, urinary output, or urinary osmolality—this effect being mediated via the inhibition of renal prostaglandin synthesis. These effects have not been proven during the first months of life in very preterm neonates treated by ibuprofen and presenting with oligo/anuria.

AQP-3 is expressed along the connecting tubule and entire length of the collecting duct (in principal cells) from the cortex and the outer and inner medulla, especially the base, rather than the tip, of the inner medulla. ^, The protein is found primarily in the lateral and basal infoldings of the basolateral membrane and is regulated by thirst and aldosterone. Under long-term influence of ADH, AQP-3 is integrally involved in urinary concentration Transgenic knockout mice lacking AQP-3 have a concentrating defect and polyuria. AQP-3 also appears to conduct small nonelectrolyte solutes, such as urea.

AQP-4 expression is found primarily in inner medullary collecting duct principal cells. It is more prominent in the inner medullary base than in the papillary tip and is found in basolateral (both basal and lateral domains) rather than apical membranes. Little staining is found in intracellular vesicles. When rats were either water restricted for 48 hours or infused with ADH for 5 days, no increase in expression of AQP-4 over a baseline water-loaded state was observed. ^, These findings are consistent with a role for AQP-4 as a basolateral exit pathway for transcellular movement of water in the inner medulla, perhaps in response to osmotic gradients, and not in response to the secretion of ADH. The role of AQP-4 has been further assessed in transgenic knockout mice lacking AQP-4. Such mice had a mild urinary concentrating defect, and their perfused inner medullary collecting ducts exhibit reduced ADH-stimulated osmotic water permeability.

Four other AQPs have been identified in kidney tissue, but their physiologic roles are unclear. ^, Procino and colleagues first reported that AQP5 is expressed in type-B intercalated cells in the collecting duct system of the rat, mouse, and human kidney, making the hypothesis that AQP5 may serve an osmosensor for the composition of the fluid coming from the thick ascending limb.

AQP-6, which has minimal water permeability, is expressed in collecting-duct intercalated cells from cortex to inner medulla. One study from Agre suggests that this AQP could be implicated in the urine acidification process. The protein appears to be present in intracellular vesicles, not plasma membranes, and may represent an intracellular water and ion channel. AQP-7 is present in the proximal tubular brush border, particularly in the S3 segment, ^, and effects metabolism by regulating the transportation of glycerol. AQP-8 is found in proximal tubule and collecting duct cells intracellular domains as well as other tissues. AQP-11 has also been identified in mammalian kidney at low abundance by Northern blot and reverse transcriptase-polymerase chain reaction techniques, but its functional significance has not been clarified yet. ^, However, AQP-11 knockout mice have been shown to have polycystic kidneys and to develop uremia.

Role of Chloride Channels

Rapid abstraction of NaCl down its concentration gradient occurs in medullary thin ascending limbs and is an integral part of the countercurrent multiplication process. This segment is particularly permeant to chloride. Chloride channels would be poised to facilitate transmembrane movement of solute and, in fact, such channels (e.g., ClC-K1) have been identified in the inner medullary thin ascending limbs of Henle in the rat kidney. The channel was localized to both apical and basolateral membranes in one study and primarily on the basolateral membrane in another. Although this channel is also found in other, more distal segments in both cortex and medulla, its location in the thin ascending limb underscores its role in concentrating and diluting urine. In this regard, dehydration was shown to increase expression of the ClC-K1 channel in cortical and medullary segments. Moreover, knockout mice without ClC-K1 showed clinical nephrogenic diabetes insipidus, a defect that was attributed to impaired generation of inner medullary hypertonicity rather than decreased collecting duct water permeability. ^, The role of chloride channels in urinary concentration as well as other ion channels along the nephron has been reviewed. ^,

To summarize, due to the countercurrent relationships of the loops of Henle, vasa recta, and proximal straight and collecting tubules, several cycles of solute transfer appear to take place more or less simultaneously. At all levels of the medulla (outer and inner stripes of the outer medulla as well as the inner medulla), but particularly in the inner stripe, in which the vessels located within the vascular bundle are separated only by a thin layer of interstitium, solute has the potential to leave the ascending vasa recta and enter the descending vasa recta. Some of this solute can be taken up by the descending loops of Henle from short-loop nephrons that are also within the bundle. This solute can ascend to the cortex and then move to the collecting tubules. When deep in the medulla, the solute can be transferred to the ascending vasa recta or to the ascending loops of Henle and can then reenter the descending loop of Henle to recycle in the medulla. In the outer stripe of the outer medulla, fluid in the ascending loop, as well as the ascending vasa recta, may also be able to enter the proximal straight tubules of the short- and long-looped nephrons. These relationships provide an ongoing transfer of solute between vessels and tubule segments to maintain sodium chloride and urea gradients in the medulla, allowing extraction of water from collecting ducts when there is need to concentrate urine. Urine leaving the renal papillary collecting ducts enters the renal pelvis and may be able to circulate in the pelvic spaces to bathe portions of the outer medulla. There is the potential for urea to recycle back to the medulla and contribute further to urinary concentration. Functional water channels appear to be amply present in both apical and basolateral membranes and are neatly poised to reclaim water by processes that are dependent on, as well as independent of, ADH.

Maturation of Fetal Water Reabsorption

A slight increase in urinary concentration occurs during fetal development. ^, At a time when plasma solute concentrations are stable, fetal sheep late in gestation (130 days of a normal 145-day gestation) have a urine osmolality significantly higher than that in younger fetuses of less than 130 days. Both urea and nonurea urinary solutes are seen to increase, whereas some investigators ^, found that intrarenal urea and salt gradients were present in fetal lamb medulla by mid-gestation ( Fig. 102.3 ), the actual contribution of urea to total urinary osmolality was relatively small. During late gestation, urine flow rates decrease, but osmolar clearance remains unchanged. Therefore free water clearance, defined as urine flow rate minus osmolar clearance, is reduced, indicating that free water is more effectively separated (reabsorbed) from solute in the older fetal kidney. Although the degree of urinary concentration in late gestation is unimpressive, the increase in water reabsorption is consistent with a heightened response to endogenous levels of ADH in the older fetus.

Availability of and Responsiveness to Vasopressin

Although fetal pituitary content at term is much less than that of the adult, ADH has been identified in the human pituitary by week 12 of gestation, with a demonstrable increase over the ensuing weeks. Neurosecretory granules and material are found in hypothalamic nuclei, the hypothalamohypophysial tract, and the infundibular process of the neurohypophysis by 16 weeks’ gestation in the human fetus. Clearly, ADH is produced at an early fetal age. Vasopressin mediates its tubular effect on water permeability by stimulating the generation of cAMP. Compared with adults, fetal ADH causes a smaller increase in cAMP from renal medullary tissue obtained from the early fetus. To achieve osmotic equilibrium (i.e., osmolality approximately 300 mOsm/kg H ₂ O), the fetal kidney needs higher plasma levels of ADH (approximately 5 μU/mL) than the adult, who requires only approximately 0.7 μU/mL. Moreover, maximal urine osmolality is achieved in both the fetus and adult at similar plasma levels of ADH, 4 to 6 μU/mL, but adult urine is much more concentrated. Therefore the fetal nephron appears to be less sensitive than the adult to vasopressin.

Exogenous Vasopressin

The fetus responds to exogenous ADH by increasing urine osmolality, a response linearly related to age over the third trimester of the fetal ewe ( Fig. 102.4 ). The human fetal nephron responds to ADH as shown in previous studies carried out in an isolated perfused human medullary collecting duct obtained from a 5.5-month male abortus with trisomy 13. This tubule increased transmembrane water flow from 2.1 to 12.0 μL/cm ² /Osm/min following exposure to peritubular ADH. Cells swelled with conspicuous dilation of intercellular spaces, indicating outward net flow of water through intercellular spaces and through cell membranes. Thus collecting duct receptors to ADH are well developed functionally in prenatal life.