Sci. Aging Knowl. Environ., 18 June 2003
Vol. 2003, Issue 24, p. pe15
[DOI: 10.1126/sageke.2003.24.pe15]

PERSPECTIVES

Urine-Concentrating Ability in the Aging Kidney

Jeff M. Sands

The author is in the Renal Division, in the Department of Medicine and Department of Physiology, at Emory University School of Medicine, Atlanta, GA 30322, USA. E-mail: jsands{at}emory.edu

http://sageke.sciencemag.org/cgi/content/full/sageke;2003/24/pe15

Key Words: aquaporin • water • urea • vasopressin • antidiuretic hormone • kidney

Introduction

One of the physiological changes that occurs during aging is a decrease in maximal urine-concentrating ability (1-3). The Baltimore Longitudinal Study of Aging measured this function in healthy people aged 20 to 39, 40 to 59, and 60 to 79 years by assessing the following parameters: (i) maximum urine osmolality, which is a measure of the kidney's ability to reabsorb (or conserve) water after overnight (12 hours) water deprivation; (ii) minimal urine flow over a 12-hour period; and (iii) the ability to conserve solute, which is a measure of the kidney's ability to reabsorb (or conserve) NaCl and/or urea. Compared to the two younger age groups, individuals aged 60 to 79 had an approximately 20% reduction in maximum urine osmolality, a 100% increase in minimal urine flow rate, and a 50% decrease in the ability to conserve solute (1). These changes could not be explained by a decrease in the rate of filtration by the glomerulus (1), the filtering unit of the kidney. The glomerular ultrafiltrate contains water, solute, and other small molecules and electrolytes that are normally found in plasma but does not contain plasma proteins. Glomerular filtration rate is a measure of the ability of the glomerulus to filter substances that are neither reabsorbed nor secreted by the renal tubules, such as inulin or creatinine, and is the primary measurement used to assess renal function. Thus, the urine-concentrating defect cannot be explained by a decrease in renal function in healthy older individuals.

Several possible explanations for why aged individuals display a reduced ability to concentrate urine have been proposed and subsequently disproven. One proposed explanation was that older individuals drink excessive quantities of water. However, aged individuals actually have diminished thirst. Although the precise mechanism is not known, studies have suggested that this effect may be caused by a reduced sensation of mouth dryness, previous strokes, and/or reduced levels of angiotensin II, a hormone that causes an increase in thirst and vasoconstriction and increases blood pressure. Another proposed explanation is that plasma osmolality fails to increase vasopressin (also called antidiuretic hormone) secretion in older individuals. Vasopressin is the primary hormone involved in maintaining plasma osmolality within the normal range and is secreted from the posterior pituitary in response to an increase in plasma osmolality. However, the relationship between plasma osmolality and vasopressin is preserved and may even be enhanced in older individuals (3). Thus, an abnormality in vasopressin secretion does not seem to be the mechanism responsible for the decrease in urine-concentrating ability in the aged (4). In the past decade, the genes encoding many of the key water- and solute-transport proteins and the vasopressin receptor, all of which are involved in the urine-concentrating mechanism, have been cloned [reviewed in (5-7)], thereby making possible the recent animal studies designed to delve into the molecular mechanisms that underlie the reduction in urine-concentrating ability that occurs during aging.

Urine-Concentrating Mechanism

The kidney is composed of three main regions: the cortex, the outer medulla, and the inner medulla (Fig. 1). The medulla is responsible for the generation of concentrated or dilute urine (Fig. 1). To concentrate urine, the portions of the nephron that make up the loops of Henle must generate a hypertonic state in the interstitial space of the medulla, and the collecting duct must be permeable to water. The loop of Henle is a hairpin-shaped structure that consists of a descending limb, made up of the proximal straight tubule (or pars recta) and the thin descending limb, and an ascending limb, made up of the thin ascending limb and the thick ascending limb. A hypertonic medullary interstitium is created by magnifying the small osmotic gradient generated at any level of the medulla down its length by countercurrent multiplication (see below). The osmotic gradient is generated by the following processes. In the outer medulla, NaCl is actively reabsorbed across the thick ascending limb of the loop of Henle, which has the NKCC2/BSC1 Na-K-Cl cotransporter protein in its apical membrane. Potassium, which is also reabsorbed via the NKCC2/BSC1 protein, is secreted back into the lumen of the kidney via the potassium secretory channel ROMK, resulting in the net reabsorption of NaCl into the interstitial space of the medulla. Countercurrent multiplication is the critical mechanism for generating concentrated urine and is accomplished by the nephron segments that make up the loop of Henle. A small osmolality difference can be generated between fluid that flows in the ascending and descending limbs of the loop of Henle at each level of the medulla. Due to the hairpin, or countercurrent, configuration of the loop of Henle, the small osmolality difference is increased by countercurrent multiplication, resulting in a large osmolality difference between the kidney cortex and the tip of the inner medulla (also called the papillary tip).



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Fig. 1. Transport proteins involved in the urinary-concentrating mechanism. Active NaCl reabsorption via NKCC2/BSC1 in the thick ascending limb (yellow-brown) generates a hypertonic medullary interstitium (see text), which concentrates NaCl in the proximal straight tubule (or pars recta, dark green) and the thin descending limb (light green) fluid by osmotically removing water via the AQP1 water channels. In the inner medulla, passive NaCl reabsorption exceeds urea secretion from the thin ascending limb (orange). In the presence of vasopressin [AVP, also known as antidiuretic hormone (ADH)], water is reabsorbed from the collecting duct (purple) via AQP2 in the apical membrane and AQP3 and AQP4 in the basolateral membrane. Water reabsorption also concentrates urea in the collecting duct lumen until the fluid reaches the urea-permeable terminal inner medullary collecting duct (dark purple), where urea is recycled into the inner medullary interstitium via the UT-A1 and/or UT-A3 urea transporters.

 
In the inner medulla, NaCl is passively reabsorbed across the thin ascending limb of the loop of Henle, provided that the interstitial urea concentration is sufficiently high. The fluid within the lumen of the thin ascending limb has a higher concentration of NaCl and a lower concentration of urea than does the inner medullary interstitial fluid, thereby establishing chemical gradients that favor passive NaCl reabsorption from, and urea secretion into, the thin ascending limb. The thin ascending limb has a higher permeability to NaCl than to urea, so NaCl reabsorption exceeds urea secretion, thereby resulting in dilution of the luminal fluid in the thin ascending limb as it ascends toward the outer medulla.

Not only does NaCl reabsorption in both ascending limb portions of the loop of Henle result in a hypertonic medullary interstitium, but because the ascending limb segments are water-impermeable, the fluid that exits the thick ascending limb into the distal convoluted tubule is dilute relative to plasma. In the absence of vasopressin, the collecting duct is impermeable to water and a dilute urine is excreted. However, in the presence of vasopressin, the collecting duct becomes highly permeable to water, and water is reabsorbed if hypertonic fluid is present in the interstitial space of the medulla to create the osmotic gradient necessary for water reabsorption.

Aquaporins

Water reabsorption via aquaporins (or water channels) across the collecting duct is regulated by vasopressin binding to the vasopressin V2 receptor and stimulation of cyclic adenosine monophosphate (cAMP) production [reviewed in (5, 8)]. Although all of the steps have not been elucidated, stimulation of protein kinase A ultimately results in the phosphorylation of a serine residue at position 256 of aquaporin-2 (AQP2) molecules and in the insertion of these channels into the apical membrane of collecting duct principal cells (Fig. 2). Phosphorylation at serine-256 is necessary for vasopressin-mediated trafficking of AQP2 (8). The water that is reabsorbed through AQP2 exits the principal cell and enters the medullary interstitium through AQP3 and AQP4 in the basolateral membrane. When the vasopressin stimulus ends, water reabsorption is stopped by the endocytosis of AQP2 back into the cell, where it is recycled in endosomes until the next vasopressin stimulus. One potential mechanism for the aging-related reduction in urine-concentrating ability is a reduction in the number of V2 receptors in the aged kidney. However, studies have not found a decrease in the abundance of V2 receptor protein or mRNA, or in vasopressin-stimulated cAMP production in the aged rat kidney (9-11).



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Fig. 2. Water reabsorption by principal cells in the collecting duct. Vasopressin (AVP and ADH) binds to V2 receptors in the basolateral membrane, stimulates adenylyl cyclase (AC) to produce cAMP, activates protein kinase A (PKA), phosphorylates AQP2 on serine-256, and ultimately inserts AQP2 water channels into the apical membrane. The result is a marked increase in apical membrane water permeability, and transcellular water reabsorption occurs as water exits the cell via AQP3 and AQP4 in the basolateral membrane. When the vasopressin stimulus ends, AQP2 is endocytosed and reenters subapical vesicles until the next vasopressin stimulus occurs.

 
In contrast, studies do show that the abundance of some of the aquaporins is reduced in the aged rat kidney (9, 12), which could be a mechanism that contributes to the reduction in urine-concentrating ability. AQP2 protein abundance is reduced in 30-month-old rats, as compared to 10-month-old rats, in both the inner and outer medulla (9, 12). The abundance of AQP2 that is phosphorylated at serine-256 is also markedly reduced in the older rats (12). In addition, the concentration of AQP3 protein is reduced in the inner medulla of the 30-month-old rats but not in the outer medulla (9, 12). The reductions in AQP2 and AQP3 are likely to result in a decrease in water reabsorption in the collecting duct of aged rats. In addition, these changes appear to be specific, because there is no difference in the concentrations of the AQP4 and AQP1 proteins in 10- and 30-month-old rats (9, 12).

A key question that needs to be answered is what happens when aged rats are water-restricted? Normally, water restriction results in an increase in plasma osmolality, which yields an increase in vasopressin secretion from the posterior pituitary. Vasopressin stimulates the insertion of the AQP2 protein into the apical membrane of the collecting duct, thereby increasing water reabsorption in order to return plasma osmolality to the normal range and prevent hypernatremia (a higher than normal osmolality of the blood). We compared the ability of 4- and 30-month-old rats to respond to 3 days of water restriction (13). Both 4- and 30-month-old rats lost 8% of their body weight and had similar increases in their red blood cell count, indicating a similar degree of dehydration in both age groups. However, only the older rats showed a significant increase in blood osmolality (hypernatremia), indicating a reduced ability to conserve water and restore plama osmolality to the normal range (13). Urine osmolality and AQP2 protein abundance increased significantly in the 4-month-old rats but not in the 30-month-old rats (Fig. 3) (13, 14). Interestingly, AQP2 protein abundance did not increase in dehydrated 15-month-old rats, which was similar to the response in the 30-month-old rats (14), and AQP2 mRNA abundance increased in dehydrated 2-month-old rats but not in 7-month-old rats (10). Thus, the age at which rats begin to lose urine-concentrating ability may be younger than 30 months.



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Fig. 3. Effect of 3 days of water restriction on young and old rats. Left panel: Western blot showing that AQP2 protein abundance increases significantly in dehydrated 4-month-old rats but not in dehydrated 30-month-old rats (the AQP2 protein is detected as a nonglycosylated 29-kD band and a glycosylated smear from 35 to 45 kD). H, hydrated; D, dehydrated. The migration positions of the molecular size markers are shown in kilodaltons on the right side of the left panel. Right panel: densitometic summary showing that AQP2 protein abundance increases in dehydrated 4-month-old rats but not in dehydrated 15- or 30-month-old rats. Asterisk indicates P < 0.05 by analysis of variance. Data are from (13, 14).

 
The preceding studies suggest that a decreased renal response to vasopressin, in terms of a failure to increase AQP2 abundance, may be an important mechanism that contributes to the reduction in urine-concentrating ability during aging. Further support for this hypothesis comes from studies in which supraphysiological concentrations of dDAVP (Desmopressin), a selective V2 receptor agonist that does not increase blood pressure, were given to 10- and 30-month-old rats (15). dDAVP caused a similar increase in urine osmolality and decrease in urine flow rate in 10- and 30-month-old rats, although the older rats were unable to achieve the same maximum urine osmolality as the younger rats (15). dDAVP also increased the abundances of both the AQP2 and AQP3 proteins in both young and old rats (15), suggesting that the reduced urine osmolality in the aged rats is related, at least in part, to the reduced concentration of these water channel proteins.

Urea Transporters

Urea is another important factor in generating concentrated urine (16-18). Individuals with protein malnutrition are unable to concentrate their urine maximally (19-23), and elderly individuals, especially those on fixed incomes, are at risk for protein malnutrition. During the past decade, the genes that encode two urea transporters (UT-A and UT-B) were cloned (6, 24, 25). The human UT-A gene is approximately 68 kb, contains 20 exons, and is located on chromosome 18 (24). Several mRNA isoforms derived from the UT-A gene have been identified and shown to give rise to multiple forms of the UT-A protein [reviewed in (6, 26)]. UT-A1, the largest isoform, is expressed in the apical membrane of the inner medullary collecting duct, and vasopressin rapidly increases both UT-A1 phosphorylation and urea transport (24, 27, 28). UT-A1 protein abundance is markedly reduced in 30-month-old rats as compared to 10-month-old rats (12). dDAVP increases urine osmolality and UT-A1 protein abundance (15), suggesting that the reduced urine osmolality in the aged rats may be related to the reduced level of UT-A1, in addition to the changes in the concentrations of aquaporins. A reduction in UT-A1 abundance could decrease urea reabsorption across the inner medullary collecting duct, thereby reducing inner medullary interstitial urea accumulation, which would in turn reduce urine-concentrating ability.

The decrease in UT-A1 abundance with aging may be mediated by an increase in the concentration of glucocorticoids. Recently, Combet and colleagues showed that 30-month-old rats have elevated plasma corticosterone levels as compared to 10-month-old rats (15). Earlier, we showed that glucocorticoids decrease UT-A1 protein abundance by suppressing its transcription via the glucocorticoid receptor, which functions in this context as a transcriptional repressor (29, 30). Taken together, these findings suggest that the increased concentration of glucocorticoids in aged rats may lead to a reduction in amounts of UT-A1 protein, but they raise the question of why glucocorticoid concentrations are elevated in aged rats.

UT-B is expressed in erythrocytes and in the descending vasa recta, which, along with the ascending vasa recta, constitutes the capillary network surrounding the loop of Henle. The blood supply to the medulla is provided by the descending and ascending vasa recta. Analogous to the loops of Henle, the vasa recta are arranged in a hairpin configuration. A reduction in UT-B abundance could decrease intrarenal urea recycling and/or reduce the efficiency of countercurrent exchange, both of which would reduce urine-concentrating ability. UT-B is also the Kidd blood group antigen, and people who lack the Kidd antigen on their red blood cells are unable to concentrate their urine to normal levels (31). Similarly, UT-B knockout mice are unable to appropriately concentrate their urine (32). Thus, the production of maximally concentrated urine requires UT-B protein expression in red blood cells and perhaps in the descending vasa recta (31, 33, 34). UT-B protein abundance is markedly reduced in the kidneys of aged rats (12), and dDAVP increases its abundance (15). Thus, the reduced urine osmolality observed in aged rats may also be related to reduced concentrations of UT-B.

Summary

Aged people and rats have reduced maximal urine-concentrating ability. Aged rat kidneys have reduced levels of water channel (AQP2, phosphorylated AQP2, and AQP3) and urea transporter (UT-A1 and UT-B) proteins. Aged rats do not increase AQP2 protein concentration at the appropriate cell surface after water restriction, even though their ability to secrete vasopressin is intact. However, a supraphysiologic dose of dDAVP does increase urine osmolality as well as AQP2, AQP3, UT-A1, and UT-B protein concentrations in aged rats.

Despite the recent progress in understanding some of the molecular changes that occur during aging in proteins that are part of the urine-concentrating mechanism, many questions remain unanswered. First, why are the concentrations of some water channel and urea transporter proteins reduced with aging? Are these changes related to glucocorticoids? Are there other hormonal changes during aging that affect the concentrating mechanism? Second, are the abundances of the sodium transport proteins changed during aging? Third, why doesn't AQP2 protein abundance increase after water restriction, given that the number of V2 receptors and their ability to generate cAMP are intact? The answers to these and other important questions will require further studies in animals and people.


June 18, 2003
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  35. Supported by NIH grants R01-DK41707 and R01-DK63657.
Citation: J. M. Sands, Urine-Concentrating Ability in the Aging Kidney. Sci. SAGE KE 2003, pe15 (18 June 2003)
http://sageke.sciencemag.org/cgi/content/full/sageke;2003/24/pe15








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