Development of water transport in the collecting duct

【摘要】  The ability of the immature kidney to concentrate urine is lower than in adults. This can lead to severe water and electrolyte disorders, especially in premature babies. Resistance to AVP and lower tonicity of the medullary interstitium seem to be the major factors limiting urine concentration in newborns. AVP-stimulated cAMP generation is impaired. This is the result of inhibition of the production by PGE 2 acting through EP3 receptors and increased degradation by phosphodiesterase IV. The expression of aquaporin-2 (AQP2) in the immature kidney is low; however, under conditions of water deprivation and after stimulation with DDAVP, it rises to adult levels. The expression of AQP3 and AQP4 is intact at birth and does not seem to contribute to the hyporesponsiveness to AVP. Low sodium transport by thick ascending loops of Henle, immaturity of the medullary architecture, and adaptations in the transport of urea contribute to the lower tonicity of the medullary interstitium. This paper reviews the alterations in the AVP signal transduction pathway in the immature kidney.

【关键词】  immature kidney arginine vasopressin aquaporins prostaglandins

WATER CONSERVATION IS ONE of the most important functions of the kidney. Before birth, the ability of the organism to conserve water is not critical as the placenta controls fluid balance in the fetus. The event of birth prompts abrupt changes in the infant's environment, rendering the newborn responsible for keeping fluid and electrolyte homeostasis. Although nephrogenesis in full-term human infants is completed before birth, tubular development continues during the first few years of life. This limits the ability of the immature kidney to reabsorb water, which usually reaches adult levels by 1 yr of age. As early as 1941, McCance and Young ( 72 ) observed that the urine of a group of infants from 7 to 14 days of age was always hypotonic ( 72 ). Studies in young rats (first 3 wk of life) subjected to 8 h of dehydration showed that the maximal osmolality achieved was close to 1,000 mosmol/kgH 2 O, which was only 50% of the levels reached by adult rats subjected to the same stimulus ( 32 ).

In infants, total body water is higher at birth ( 45, 91 ). Under normal physiological conditions, the kidneys have to excrete this load of water during the first week of life ( 91 ). Therefore, maximal abilities to concentrate urine are not considered necessary at birth. However, this limitation places premature infants, who frequently have other associated medical problems, at greater risk for serious imbalances in water and electrolyte homeostasis ( 20, 101, 104 ). For years, scientists have tried to identify the factors mediating the limitation in urine concentration observed in the immature kidney. The major aim of this paper is to review the progress in investigating the development of water transport in the collecting duct (CD) and its response to AVP.

NORMAL AVP PHYSIOLOGY

AVP exerts its antidiuretic effect in the CD via V 2 receptors localized in the basolateral membrane of principal and inner medullary CD cells ( 42, 96, 110 ). The V 2 receptor is coupled to a stimulatory guanine-nucleotide binding protein (G s ). Therefore, binding of AVP to the receptor results in activation of adenylyl cyclase and an increase in intracellular cAMP levels. These events lead to activation of PKA, which phosphorylates the cytoplasmic COOH terminus at serine 256 of aquaporin-2 (AQP2) protein ( 38, 39, 80 ). The phosphorylated AQP2, localized in intracellular vesicles, is delivered to the apical plasma membrane ( 46, 65, 70, 78, 80 ). These vesicles fuse with the apical membrane of CD epithelial cells, leaving AQP2 channels inserted in the membrane. Water flows inside the cell through AQP2 and leaves through aquaporin-3 (AQP3) and aquaporin-4 (AQP4) channels localized in the basolateral membrane. Once the AVP stimulus is removed, AQP2 channels are retrieved from the apical membrane back to intracellular vesicles. This process depends on the presence of a hypertonic medullary interstitium, which drives water from the luminal fluid across the tubular epithelium ( 66 ).

AVP SECRETION

Although early in life the levels of AVP stored in the rat posterior pituitary are low ( 48, 49 ), the circulating levels of the hormone do not seem to be a limiting factor in the development of urine-concentrating abilities. Various studies have demonstrated that the ability of fetuses and infants to secrete AVP in response to diverse stimuli is intact. The concentration of AVP in cord blood samples from human infants is increased ( 17, 89 ). AVP levels are also elevated during labor, with higher levels observed in infants born by vaginal delivery (vs. cesarean section without labor), which is consistent with an enhancement in AVP secretion by the fetus in response to increases in intracranial pressure or hypoxia ( 44 ). After prolonged dehydration, young rats are able to secrete AVP to levels comparable to those in older rats ( 32, 35 ). Using the chronically catheterized fetal sheep model, Weitzman and colleagues ( 117 ) demonstrated that the ability of the pituitary gland to secrete AVP in response to an infusion of hypertonic saline is fully developed during intrauterine life. Other investigators have shown appropriate pituitary secretion of AVP in response to hemorrhage and hypotension ( 35 ). Despite this, during the first 3 wk of life there is no correlation between the serum concentration of AVP and plasma or urinary osmolality ( 89 ). From these observations, we can conclude that if AVP levels are adequate, the diminished concentrating capacity of the immature kidney is the result of resistance of the CD to AVP.

RESPONSE TO AVP

Resistance of the immature kidney to AVP has been demonstrated by several investigators ( 104 ). Using the chronic catheterized fetal sheep model, Robillard and Weitzman ( 90 ) observed that fetuses of <112-day gestation had a diminished response to an infusion of AVP compared with 112-day-old and older fetuses. Studies in humans have shown similar results. To determine the response of the immature kidney to AVP, Heller ( 47 ) injected a dose of pituitary extract intramuscularly into a group of newborn infants. An equivalent dose was injected into adult controls. There was a rapid increase in urine osmolality in adults but virtually no response in infants, even after a 10-fold increase in dose ( 47 ). Winberg ( 118 ) demonstrated several years ago that the response of infants to AVP improves with age. Similar results were reported by Svenningsen and Aronson ( 107 ), who also demonstrated that concentrating capacity is even lower in infants who have sustained neonatal asphyxia. During the last several years, our laboratory, as well as many other investigators, has tried to pinpoint the alterations in the AVP signal transduction pathway in the immature kidney. This paper portrays the findings at the different steps in the signaling pathway, from the receptor level to the final step of water absorption into the medullary interstitium.

ROLE OF THE AVP RECEPTOR

Rajerison et al. ( 87 ) showed that specific binding sites for AVP are present on kidney medullopapillary membranes from newborn rats. Moreover, using the technique of in situ hybridization, Otrowski et al. ( 85 ) demonstrated that V 2 receptor mRNA is present in rats as early as day 16 of gestational age in cells of developing medullary and cortical CD. After birth, V 2 receptor mRNA was also observed in cells of differentiating thick ascending limbs of the loops of Henle, papillary epithelium, macula densa, and short distal nephron segments. The developmental renal expression of V 1a receptor mRNA followed a similar pattern in the developing cortex. After birth, V 1a receptor transcripts were also observed in vascular elements, in developing medullary CD cells, and in the mesangial cells of deep glomeruli. Interestingly, hepatic expression of the V 1a receptor was not observed until birth. These data demonstrate that the V 2 receptor is present very early in gestation. In rats, the number of receptors does not change during the first 2 wk of postnatal life. There is a sharp increase after 20 days, reaching adult levels by the fifth week of life ( 2 ). Thus expression of the V 2 receptor does not seem to be involved in the low response of the immature kidney to AVP.

ROLE OF cAMP GENERATION

It seems clear that AVP-stimulated cAMP in the CD is developmentally regulated. The appearance of specific binding sites for AVP slightly precedes the onset of adenylyl cyclase responsiveness ( 87 ). We, as well as other investigators, have found that basal levels of intracellular cAMP, the second messenger of the respone to AVP, are independent of the developmental stage ( 9, 87 ). However, AVP-stimulated cAMP generation is markedly lower during the neonatal period in different species, including rats, rabbits, and dogs ( 9, 40, 69, 98 ). Studies in anesthetized piglets demonstrated that the development of urine-concentrating abilities in response to AVP is paralleled by an increase in nephrogenous cAMP formation ( 36 ). In a group of isolated, microdissected rabbit cortical CD (CCD), we showed that AVP-stimulated cAMP generation is only about one-third of the cAMP levels observed in adult CCD ( Fig. 1 ) ( 9 ). Our laboratory further investigated the mechanisms involved in the cAMP response. To bypass the V 2 receptor, we used NaF, a direct stimulator of the G protein-adenylyl cyclase enzyme complex. NaF failed to stimulate maximal cAMP generation in the immature CCD. To evaluate the intrinsic function of the adenylyl cyclase enzyme, the effect of forskolin, which does not require a functional G protein to stimulate the enzyme, was evaluated. Direct stimulation of adenylyl cyclase by forskolin also failed to elicit maximal cAMP generation in the immature CCD.

Fig. 1. Basal cAMP generation in the immature cortical collecting duct (CD; hatched bars) is not different from mature cortical CD (filled bars). However, AVP-stimulated cAMP generation is significantly decreased in immature cortical CD. * P < 0.0001. (Adapted from Ref. 9 )

Nonetheless, although AVP-stimulated cAMP production is low in the immature kidney, the adenylyl cyclase enzyme system is not the only factor limiting water absorption during development. We demonstrated in isolated, microperfused rabbit CCD that tubular stimulation with 8-chlorophenylthiol-cAMP, a cAMP analog, failed to increase hydraulic permeability ( L p ) in immature CCD (85.6 ± 51.3 x 10 -7 cm·atm -1 ·s -1 ) from the levels observed during stimulation with AVP (46.7 ± 10.0 x 10 -7 cm·atm -1 ·s -1, Fig. 2 ) ( 11 ). Siga and Horster ( 100 ) observed similar results in the inner medullary CD. They demonstrated that forskolin failed to stimulate maximal water permeability in microperfused immature inner medullary CD ( 100 ). These observations strongly suggest that the alteration in AVP response is localized, at least in part, at a step distal to cAMP generation ( 11 ).

Fig. 2. A : hydrosmotic permeability ( L p; 10 -7 cm·atm - 1·s -1 ) observed in the immature cortical CD in response to AVP, cAMP analog (8-chloro-phenylthiol-cAMP), or AVP in the presence of indomethacin (INDO; AVP+INDO) was not significantly different. B : response of individual tubules demonstrating no significant difference in the L p response. (Adapted from Ref. 11 )

ROLE OF PHOSPHODIESTERASES

cAMP generated after stimulation by AVP is rapidly degraded by phosphodiesterase. High levels of cAMP phosphodiesterase activity have been found in renal homogenates from newborn rats, with the highest levels observed on the first day of life ( 40 ). This would suggest that degradation of cAMP rather than decreased formation could be responsible for the blunted response of the immature CD to AVP. Although this sounds like a logical explanation, it had not been investigated formally. Some of the studies, including ours, that had shown a blunted AVP-stimulated cAMP generation in immature CCD had been performed in the presence of IBMX, a phosphodiesterase inhibitor. Therefore, it had been assumed that increased phosphodiesterase activity was not a significant factor limiting the response of the CD to AVP. Recently, Quigley et al. ( 86 ) confirmed previous observations of elevated intracellular levels of phosphodiesterase in neonatal CCD. Furthermore, they were able to show that rolipram, a specific inhibitor of phosphodiesterase IV, not only decreased phosphodiesterase activity in the immature CCD to adult levels but, more significantly, abolished the blunted AVP-stimulated osmotic water permeability in isolated, microperfused immature CCD ( 86 ). This suggests a key role for phosphodiesterases in inhibiting the AVP response in the immature CD.

ROLE OF PROSTAGLANDINS

For years, it was speculated that the diminished response of the immature kidney to AVP was the result of antagonism by excessive prostaglandin activity. High levels of prostaglandins have been observed in the urine of fetal lambs ( 117 ). Inhibition of endogenous prostaglandin synthesis by indomethacin increases urinary osmolality and nephrogenous cAMP excretion in newborn piglets, suggesting that the renal resistance to AVP could be mediated by prostaglandins ( 55 ). In isolated, microdissected CD, Schlondorff et al. ( 97 ) demonstrated lower PGE 2 synthesis in tubules derived from neonatal rabbits. In human infants, results from studies measuring urinary PGE 2 have been inconsistent. Some studies have shown lower urinary prostaglandins in infants ( 41, 106 ). Conversely, Joppich et al. ( 57 ) showed that the levels of urinary PGE 2 were higher in preterm than in full-term infants. Decreasing PGE 2 levels in the urine of full-term infants have been associated with increasing cAMP excretion and urinary osmolality ( 57 ). Melendez et al. ( 73 ) found that the renal cortex of newborn rats has not only higher rates of PGE 2 synthesis but also a prominently higher affinity for PGE 2 than the cortex from adults. Therefore, the immature kidney appears to be more sensitive to prostaglandins because of increased affinity for the receptor ( 73 ).

The role of prostaglandins in the CCD and its interaction with AVP are complex. Alone, PGE 2 stimulates cAMP generation and elicits a modest increase in water permeability, but in the presence of AVP it inhibits AVP-stimulated cAMP generation and water absorption ( 13, 16, 42, 75, 76, 82, 102, 112 ). Additionally, PGE 2 synthesis is stimulated by AVP ( 54, 62 ). These different responses of the CD to PGE 2 are the result of binding to different receptors.

To evaluate the role of endogenous prostaglandins in the lower cAMP synthesis observed in the immature CCD, we studied the effect of the cyclooxygenase inhibitor indomethacin. We observed that when immature CCD were preincubated with indomethacin, AVP stimulated a significant increase in cAMP generation to levels that were not significantly different from the ones observed in mature CCD pretreated with indomethacin. A similar response was observed when a group of immature CCD was preincubated with pertussis toxin, an inhibitor of the inhibitory G protein (G i ) ( Fig. 3 ). Pretreatment of the immature CCD with both agents (pertussis toxin and indomethacin) simultaneously did not produce an additive effect, suggesting that both were acting through the same mechanism, that is, prostaglandins inhibiting AVP-stimulated cAMP generation in the immature CCD by activating G i.

Fig. 3. Effect of inhibition of G i in AVP-stimulated cAMP generation. Although inhibition of G i did not induce any change in mature cortical CD (filled bars), it abolished the defect in immature cortical CD (hatched bars). Addition of INDO did not have an additive action on the effect of pertussis toxin. Numbers within bars denote the number of tubules studied in each group. * P < 0.0001. (Adapted from Ref. 9 )

At least four different receptors for PGE 2 have been identified in the kidney: EP 1, EP 2, EP 3, and EP 4 ( 75 ). Each of these receptors activates a different intracellular signaling system ( 4, 37, 77 ). The EP 3 subtype receptor is coupled to G i; therefore, its activation inhibits cAMP generation ( 77 ). The EP 3 receptor has been localized to the cortical CD and inner medullary CD ( 12, 108, 109 ). In this nephron segment, PGE 2 acts through the EP 3 receptor to inhibit cAMP generation by activating G i. To determine the role of this receptor in inhibiting cAMP production in the immature CCD, we examined the expression and localization of EP 3 receptor mRNA in the immature rabbit kidney ( 8 ). We observed that the expression of the EP 3 subtype PGE 2 receptor gene is developmentally regulated. Relative quantitation of EP 3 mRNA levels by three different techniques (PCR with an internal standard, competitive PCR, and an RNase protection assay) demonstrated consistently higher levels of EP 3 in the immature rabbit kidney. The levels of expression of EP 3 receptor mRNA increase rapidly during the first 2 wk of life. After the second week, the renal expression of EP 3 decreases dramatically, reaching lower adult levels between 8 and 10 wk of postnatal life ( Fig. 4 ). These findings are consistent with our previous observation that prostaglandins mediate the defect in AVP-stimulated cAMP generation in the immature CCD via activation of G i. Our finding of the upregulation of renal EP3 expression during development also agrees with reports from Melendez and colleagues ( 73 ), who reported that the immature kidney is more sensitive to prostaglandins.

Fig. 4. Postnatal developmental expression of mRNA for EP 3 receptor in kidney as determined by RNase protection assay. A : total kidney RNA was isolated from rabbits between 0 (1st day of life) to 10 wk of age. B : analysis of bands by densitometry showing higher EP 3 mRNA expression during early postnatal life. Maximal expression was observed at 2 wk of age, followed by progressive decline in EP 3 signal. Band density is expressed as percentage of the expression observed at 2 wk. Twelve micrograms of total RNA were used for each reaction. (Adapted from Ref. 8 )

Although these data would suggest a major role for prostaglandins in mediating resistance to AVP in the immature kidney, studies by Matson and colleagues ( 71 ) showed that administration of indomethacin to chronically catheterized fetal sheep failed to increase urine osmolality. Moreover, using the technique of in vitro microperfusion of isolated tubules, we demonstrated that pretreatment with indomethacin failed to induce maximal AVP-stimulated hydraulic permeability in the immature CCD ( Fig. 2 ) ( 11 ). We believe that PGE 2 has an important role in inhibiting AVP-stimulated cAMP generation in the immature kidney. Nonetheless, there is still a developmental alteration in the AVP transduction pathway distal to cAMP, which is not mediated by prostaglandins.

ROLE OF AQUAPORINS

Aquaporins are a family of water channel proteins involved in transcellular water transport. There are three aquaporins expressed in the CD (AQP2, AQP3, AQP4) ( 24, 65, 80, 114 ). AQP2 is localized in subapical vesicles and in the apical plasma membrane of principal, connecting duct, and inner medullary duct cells ( 63, 79 ). This channel is the main target of AVP action in the CD, because it is required for water absorption in this nephron segment ( 22, 25 ). AQP3 and AQP4 are localized in the basolateral membrane of principal cells, where they permit the exit of water from the cell to the tubulointerstitium ( 1, 27 - 29, 53 ). AQP3 is the main basolateral water channel in the CCD, whereas AQP4 is localized primarily in the medullary CD ( 65 ).

Because our previous data showed that a cAMP analog did not restore maximal water absorption in isolated, microperfused immature CD, we hypothesized that during development the response to AVP was disrupted at a step distal to cAMP, probably at the level of AQP2. To define the role of AQP2, we studied the developmental expression of this water channel in rats. We observed that AQP2 levels (mRNA and protein) were lower during early postnatal life, reaching maximal expression at 10 wk of age ( Fig. 5 ) ( 7 ). Concurrently, urine osmolality increased from 242 ± 60 to 1,267 ± 311 mosmol/kgH 2 O ( Fig. 6 ). A similar pattern of development of AQP2 was reported by other investigators ( 119, 120 ), who reported AQP2 signal in ureteric buds of rat kidney as early as embryonic day 18 ( 5, 119 ).

Fig. 5. Immunoblot analysis of aquaporin-2 (AQP2) protein postnatal developmental expression in rats between 0 and 10 wk of age. A : 2 major bands representing nonglycosylated (29 kDa) and glycosylatyed (between 36 and 50 kDa) proteins were observed at each developmental stage. Ten micrograms of protein were loaded per lane. B : densitometric analysis of bands, expressed as percentage of values observed in 10-wk-old rats. Values are means ± SE; n = 5/developmental stage. By ANOVA, the differences among the groups were not statistically significant. *Significantly lower AQP2 expression at 0 and 1 wk than at 10 wk ( P < 0.01 by unpaired t -test). (Adapted from Ref. 7 )

Fig. 6. Development of urine-concentrating abilities. Urine osmolalities were measured in rats from 0 to 10 wk on ad libitum fluid intake. Values are means ± SE. Differences among the groups were not statistically significant ( P 0.05 by ANOVA). (Adapted from Ref. 7 )

To study the regulation of AQP2, immature and adult rats were kept on ad libitum intake or were water deprived. Under normal conditions, AQP2 levels in the immature rat were significantly lower (52.3 ± 5.8%, P < 0.01) than in the adult. However, after dehydration the expression increased to adult levels. Interestingly, the increase in AQP2 observed in the immature kidney was not accompanied by a proportional increase in urine osmolality. To rule out a potential alteration in AQP2 trafficking, the transport of this water channel from intracellular reservoir to plasma membrane was investigated using the technique of subcellular fractionation of protein samples. For this, a group of rats was subjected to dehydration, treated with desmopressin acetate (DDAVP), or water loaded. Dehydration and DDAVP stimulated translocation of AQP2 from intracellular vesicles in both adult and immature animals ( Fig. 7 ). We observed that although AQP2 expression and trafficking in the immature kidney were appropriately stimulated by water deprivation and DDAVP, the urine osmolality remained low. Yasui and colleagues ( 120 ) demonstrated that the expression of AQP2 in the neonatal kidney is also regulated by glucocorticoids. A single injection of betamethasone increased AQP2 mRNA and protein in infant, but not adult, kidney ( 120 ). Urinary excretion of AQP2, which is a marker of AVP action in the CD ( 33, 116 ), is not different in premature or full-term infants compared with adults ( 113 ). This is consistent with data from various investigators who have reported no significant alteration in the expression of AQP2 after birth ( 23, 92 ). Thus we can conclude from these studies that although AQP2 expression may contribute to the development of urine-concentrating abilities, it does not seem to be a limiting step. Moreover, there is still a significant impairment distal to AQP2. Regarding AQP3 and AQP4, existent data show that their expression does not change significantly after birth ( 5, 60, 119 ). Therefore, they do not seem to play a role in the maturation of water transport in the CD.

Fig. 7. Translocation of AQP2 measured by immunoblots of high-speed (H) and low-speed (L) centrifugation fractions. A : after water loading of kidneys for 24 h, AQP2 expression in immature (baby) and adult kidney was higher in the intracellular vesicle fraction (H). B : overnight water deprivation (dehydration) resulted in shifting of AQP2 to the plasma membrane fraction (L). C : administration of DDAVP stimulated translocation of AQP2 to the plasma membrane fraction (L) in both adult and immature kidneys. (Adapted from Ref. 7 )

ROLE OF TONICITY OF THE MEDULLARY INTERSTITIUM

Water absorption in the CD requires the presence of an interstitium with a very high osmolality. This is primarily the result of high concentrations of sodium and urea ( 94, 95 ). The concentration of osmotically active solutes in the urine is low during the first few weeks of life ( 18, 31 ). This has been ascribed to several factors. First, the typical infant fed with maternal milk receives a diet with very high water and low protein content. In addition, it has been suggested that because of the high anabolic state of infants, the ingested protein is used for body growth rather than urea synthesis. All these factors together may contribute to the low concentration of urea in the urine of immature mammals. Other factors such as low sodium transport by thick ascending loops of Henle ( 26, 50, 51, 88 ), immaturity of the medullary architecture with shorter loops of Henle ( 15, 67, 68, 84, 103 ), and alterations in the transport of urea ( 10, 36 ) have been suggested. More recently, developmental changes in the expression of aldose reductase, which is critical for the generation of intracellular osmolytes, have also been reported ( 58, 99 ).

Studies in rats showed that the activity of Na-K-ATPase in the thick ascending limb of Henle increases after birth, with the most pronounced increase in activity between the second and third week of life. This correlates with the increase in urine-concentrating capacity ( 26, 88, 121 ). The increased ability to reabsorb sodium in this nephron segment is associated with an increase in the area of the cell basolateral membrane ( 67 ), which is stimulated by corticosteroids ( 26, 88 ). The loops of Henle elongate and penetrate the medulla, forming tubulovascular units ( 103 ). This process is completed by the fourth week of postnatal life in rats. The net result is increased capacity of the loop of Henle to transfer solutes from the urinary lumen to the medullary interstitium.

Urea, the major end product of protein metabolism in mammals, is synthesized by the liver and excreted in the urine, where it constitutes 50% of urinary solutes in a person consuming a normal protein diet ( 3 ). The ability of the kidney to recycle urea is critical for the process of water absorption in the CD. The tonicity of the medullary interstitium is maintained by a complexity of processes that involves transport of urea between different nephron segments and the vasa recta. These processes are highly regulated by AVP and dietary protein intake. Recent evidence has demonstrated that the renal handling of urea is facilitated by a group of proteins that transports urea across plasma membranes ( 93 ). The vasopressin-regulated urea transporter (UTA-1) is responsible for delivering large amounts of urea to the deeper portions of the inner medulla. This transporter, which has been localized to the apical membrane and intracellular vesicles of the terminal inner medullary CD ( 81 ), is regulated by AVP on a short-term basis and by dietary protein on a long-term basis ( 111 ).

The medulla/cortex urea ratio is low at birth in rabbits and increases markedly from birth until 21 days of age ( 36 ). This observation suggests a potential adjustment in the expression and function of urea transporters during development. To investigate this alteration, we performed immunoblots of renal tissue derived from rats between 1 and 10 wk of postnatal life using an antibody against UTA-1. We observed that the expression of UTA-1 is developmentally regulated, with lower levels observed during the first 2 wk of life ( Fig. 8 ) ( 10 ). Kim and colleagues ( 61 ) have reported similar results.

Fig. 8. Immunoblot analysis of urea transporter UTA-1 postnatal developmental expression in rats between 0 and 10 wk of age. Two major bands at 97 and 116 kDa are observed. UTA-1 expression is low at 0 (1st day of life) and 1 wk of age and increases markedly at week 2, with maximal expression observed at 10 wk of age.

UTA-2, the second isoform of the UTA family, is localized in thin descending limbs of the loop of Henle. Two other isoforms of UTA (UTA-3 and UTA-4) have been identified in rat renal medulla. These isoforms may be regulated by AVP ( 59 ). UTB, an erythrocyte-facilitated urea transporter, has been cloned ( 83 ). Two isoforms of this family (UTB-1 and UTB-2) have been identified in rat vasa recta. These transporters prevent the loss of urea from the medulla into the circulation ( 83 ). Data from rats show that after birth there is a striking increase in the number of UT-B-positive descending vasa recta in association with the formation of vascular bundles ( 61 ). Thus the development of the tonicity of the medullary interstitium seems to be limited, at least in part, by the immaturity of urea transporters.

High-protein diets or urea supplementation increases urinary urea excretion in newborn infants, with no significant change in the concentration of other solutes ( 30, 31 ). This increase in urinary solutes is accompanied by a significant improvement in their abilities to concentrate urine. This is consistent with a major role for dietary protein intake in the development of urine osmoregulation. Multiple studies have demonstrated that urine-concentrating capacity is dependent on dietary protein intake ( 6, 34, 64 ). However, the effects of changes in dietary protein in the expression of urea transporters and aquaporins during renal development have not been studied yet.

In summary, the inability of the young kidney to concentrate urine at maximum is the result of the immaturity of the AVP-signal transduction pathway at multiple steps ( Fig. 9 ). First, AVP-stimulated cAMP generation is inhibited by PGE 2 due to upregulation of the G i -coupled EP3 receptor. In addition, the cAMP formed is rapidly degraded because of the high activity of phosphodiesterase IV. Although the levels of AQP2 increase during the first few weeks of life, the expression of CD water channels (AQP2, AQP3, and AQP4) does not play a key role in the low response of the immature kidney to AVP. Finally, low concentrations of urea and sodium in the medullary interstitium, most likely resulting from low dietary protein intake, low expression of urea transporters, and low rates of sodium absorption, limit the ability of the immature kidney to reabsorb water.

Fig. 9. Mechanisms inhibiting AVP-mediated water transport in the immature collecting duct. 1, Inhibition of cAMP generation by PGE 2 through EP3 receptor; 2, rapid degradation of formed cAMP resulting from increased phosphodiesterase activity; 3, low expression of AQP2 during early postnatal life; 4, low expression of UTA-1 during early postnatal life; 5, low concentration of urea and sodium in the medullary interstitium resulting from low rates of sodium transport, low dietary protein intake, and low expression of urea transporters.

GRANTS

These studies were supported in part by a Minority Scientist Development Award from the American Heart Association, a Clinical Scientist Award from the National Kidney Foundation, Research Center for Minority Institution Grant G12 RR-03051, and Minority Biomedical Research Support from the National Institutes of Health.

ACKNOWLEDGMENTS

The author thanks mentors and collaborators in the microperfusion experiments for continuous support and Drs. V. M. Vehaskari and L. Lee Hamm, as well as Drs. Jeff Sands and Mark Knepper, for providing UTA-1 and AQP2 antibodies, respectively.

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作者单位：Department of Pediatrics, University of Puerto Rico-Medical Sciences Campus, San Juan, Puerto Rico 00936-5067