JN 2000; Vol.13 N°4: 260-266

Review

JNEPHROL 2000; 13: 260-266

Urea: new questions about an ancient solute

Serena M. Bagnasco - Department of Pathology, Emory University School of Medicine, Atlanta, Georgia - USA

ABSTRACT: Urea recycling and counter-current exchange within the renal tubular, vascular and interstitial compartments help maintain high levels of this solute in the renal medulla, that are crucial for the production of concentrated urine. The role of urea in physiological and pathological conditions is still unclear, although new information is becoming available. Several urea transporters have been identified that mediate facilitated transport of urea across biological membranes in the mammalian kidney, in amphibians, and in elasmobranchs. Evidence that urea transporters may be expressed in other mammalian organs is also beginning to emerge. The mechanisms involved in the regulation of urea transport are incompletely understood. In this respect, the structural and functional characterization of individual transporters is providing the basis to identify specific regulatory factors. Urea can be viewed as a perturbing osmolyte in the renal inner medulla, and the mechanisms of adaptation of renal cells to high concentration of this destabilizing solute are being investigated. Urea-specific signaling pathways have been identified, that could contribute to clarify how cells handle urea.

Key words: Urea, UT-A, UT-B, Transporter, Kidney

This article focuses on some recent research trends broadly related to renal handling of urea, and urea transporters in physiological and pathologic conditions. The reader is referred to other recent reviews on the topic for areas not covered here (1-3).

Physiological role of urea in the kidney

In mammals urea is synthesized in the liver as the end product of protein catabolism. The mammalian kidney can eliminate large amounts of urea in concentrated urine and minimize loss of water. This process requires an axial osmolality gradient in the renal medulla. Concentrations of both Na⁺ and urea are high in the renal inner medulla, where osmolarity is higher than in any other organ. Na⁺ is concentrated by the countercurrent multiplication system, driven mostly by the active transport of NaCl in the thick ascending limb of Henle's loop. High medullary levels of urea are maintained by pathways of urea recycling through the medullary interstitial and tubular compartments, and through the countercurrent exchange in the vasa recta (Fig. 1).

bagnasco f1

Fig. 1 - Pathways of urea recycling in the renal medulla. The distribution of known urea transporters is shown. Parentheses indicate uncertain localization. Question marks refer to carriers that have not been identified.

Though urea can diffuse across biological membranes, carrier-mediated transport in the kidney and in red blood cells allows rapid transfer between cellular and extracellular compartments. Some of the physiological and biophysical aspects of urea transport and its regulation have been reviewed in depth elsewhere (2,3). In the medulla, urea diffuses from the ascending vasa recta (AVR) into the interstitium, and can enter adjacent descending thin limbs of Henle (TDL) and descending vasa recta (DVR). The permeability of TDL and DVR to urea is consistent with the presence of facilitated transport in these structures.
Efflux from the inner medullary collecting duct (IMCD) is the major source of urea in the inner medullary interstitium. The high urea permeability of this tubular segment is, again, consistent with the presence of facilitated transport, and recent experimental evidence supports the existence of active transport in IMCD (4,5). In addition, facilitated transport of urea from erythrocytes has been described, by which these cells can rapidly lose the urea acquired during their passage through the DVR (2).

Urea transporters

In the past few years several urea transporters have been cloned and characterized in mammals, amphibians, elasmobranchs, and bacteria (Tab. I).

TABLE I - DIFFERENT TYPES OF UREA TRANSPORTERS CLONED IN VARIOUS SPECIES

Name	Species	Tissue	GenBank/EMBL
rUT-A1 (UT1)	Rat	Kidney	U77971
oUT-A2 (oUT2)	Rabbit	Kidney	U10358
rUT-A2 (rUT2)	Rat	Kidney	U09957
hUT-A2 (hUT2)	Human	Kidney	X96969
rUT-A3	Rat	Kidney	AF041788
rUT-A4	Rat	Kidney	AF042167
hUT-B1 (HUT11)	Human	Erythrocyte	L36121
rUT-B2 (rUT11)	Rat	Brain	X98399
rUT-B1 (UT3)	Rat	Kidney	U81518
fUT	Frog	Urinary bladder	Y12784
ShUT	Shark	Kidney	Not provided
DUR3	Yeast	.	L19875
URE I	Bacteria	.	AJ239719

Two types of mammalian urea transporters have been identified: the renal urea transporter UT-A, and the erythrocyte urea transporter UT-B. UT-A is expressed in the renal medulla, and four isoforms are known: UT-A1 (6), UT-A2 (7), UT-A3 (8), and UT-A4 (8). UT-B is expressed in red blood cells (9), and corresponds to the Kidd blood antigen (10). UT-B expression has been reported in the brain (11), kidney, and testis (12).
Urea transporters have also been identified in the elasmobranch Squalus achantias (ShUT) (13), and in the frog Rana esculenta (fUT) (14). The elasmobranch ShUT peptide appears to be 66% identical the UT-A2 transporter (13). The amphibian fUT peptide is 63% identical to UT-A2, and 61% identical to UT-B (14). Whether these newly identified urea transporters belong to yet another family, distinct from UT-A and UT-B, remains to be clarified.
In addition to facilitated transport, active transport of urea may occur in the mammalian kidney (4, 5). Although the molecular carrier responsible for the renal active transport of urea has not been identified, active urea transporters exist in yeast, and one (DUR3) has been cloned in Saccharomycescerevisiae. Bacterial urea transporters have also been cloned in Actinobacillus pleuropneumoniae, and in Helicobacter pylori (15). In the latter, urea is transported by an H⁺-gated channel, and the process is linked to bacterial cytoplasmic urease activity.
As more urea transporters are identified, more details are needed of their substrate specificity and kinetics, and of the mechanisms involved in the short- and long-term regulation of their expression, to classify the different types and to understand their physiological roles in the kidney and other organs.

Distribution of the expression of urea transporters

The distribution of known urea transporters is illustrated in Figure 1. In the kidney, the highest basal urea permeability, which is significantly increased by vasopressin, hypertonic NaCl or mannitol, is in the terminal portion of the IMCD (16-18). Several studies have analyzed the correlations between experimental measurements of renal tubular urea permeability and the distribution of facilitated urea transporters in the mammalian kidney. Expression of UT-A1 mRNA in rat kidney was detected by RT-PCR, only in the IMCD, while expression of UT-A2 was detected in distal, descending limb of short loops of Henle, and in the inner medullary portion of descending limbs of long loops of Henle (19).
Similar results were obtained by immunocytochemical analysis of UT-A1 and UT-A2 distribution in the rat kidney (20). In the same study, immunoelectron microscopy showed that the expression of UT-A protein appeared restricted to the apical aspect of renal tubular cells. The existence of a basolateral urea carrier has been postulated, but no such transporter has been identified so far. By Northern hybridization, expression of the UT-A2 mRNA isoform is highest in outer medulla, UT-A1, UT-A2, and UT-A3 mRNA are detected in the inner medulla (Fig. 2B),

bagnasco f2

Fig. 2 - A. Schematic representation of the rat UT-A cDNA isoforms: UT-A1, UT-A2, UT-A3, and UT-A4. Similar color/pattern indicates identical sequence regions in individual cDNAs.
B. Northern analysis of expression of UT-A mRNA isoforms in rat kidney, using a full cDNA probe for UT-A1. Molecular sizes are indicated on the right. Different regions of the kidney are indicated on the top (C: cortex, O.M.: outer medulla, I.M.: inner medulla).

while UT-A4 is detectable only by RT-PCR in the inner medulla (8). The exact localization of UT-A3 and UT-A4 expression along the nephron, and their intracellular distribution, remain to be determined.
The erythrocyte urea transporter UT-B is also expressed in the kidney. By in situ hybridization and immunocytochemistry, UT-B expression has been detected in the endothelial cells of the medullary vasa recta (12, 21, 22).
While relatively abundant levels of urea transporters may be expected in the kidney, there is increasing evidence of substantial expression of UT-A and UT-B isoforms in other organs as well. Couriaud et al first detected a rat UT-B mRNA in brain and testis (11). UT-B expression in testis was confirmed subsequently by others (12). We recently observed the expression of two UT-A mRNA isoforms in the rat testis (8). By immunocytochemistry and Western analysis, we detected the expression of UT-A proteins in rat seminiferous tubules (23). These novel UT-A isoforms need to be further characterized. Complex transport processes occur in the testicular efferent ductules, epididymus and vas deferens, which affect the composition of the seminal fluid (24-27). Whether urea transport plays a significant role in sperm maturation and function needs to be established.
The liver is the major site of urea synthesis in mammals, and facilitated transport of urea out of the liver cells may be required to lower the intracellular urea concentration. Phloretin-inhibitable urea transport has been detected in rat hepatocytes (28); this transport may be mediated by a novel member of the aquaporin family, AQP9, which has broad selectivity for neutral solutes, including urea, glycerol, sorbitol, and adenine (29). The possibility of a 49 kD UT-A protein isoform mediating phloretin-inhibitable urea transport in the liver has been proposed on the basis of Western analysis (30), although no liver-specific UT-A cDNA isoform has been cloned yet. Interestingly, the same study found a significantly higher abundance of this putative 49 kD UT-A isoform in livers of uremic rats compared to controls, and this might constitute an adaptive response of hepatocytes to uremia.

Regulation of the expression of urea transporters

Several factors and conditions have been defined that regulate the renal expression and/or function of some, but not all, urea transporters in vivo. They are shown in Table II, and include: vasopressin (18,31,32) hyperosmolarity (17), hydration (33), low-protein diet (34), glucocorticoids (35), and have been recently reviewed (2,3). Functional analysis in heterologous expression systems suggests that the activity of three of the four UT-A transporter isoforms may be stimulated by vasopressin through cAMP and protein kinase A (PKA) (6,8). The four UT-A isoforms have consensus sites for phosphorylation by both PKA and PKC (8), while the UT-B transporter lacks PKA consensus sites, and there is no evidence that UT-B activity is stimulated by vasopressin or cAMP.
The significance of different intracellular signaling pathways in the short-term regulation of the individual UT-A and UT-B transporter activity in the kidney still needs to be elucidated. The long-term regulation of urea transporter expression, and which mechanisms it involves, is also incompletely understood. In vivo studies have examined the relative abundance of UT-A1 and UT-A2 mRNA in the renal medulla in different states of hydration and after vasopressin. UT-A2, but not UT-A1, increased in water-deprived rats and in rats treated with vasopressin (31,33), suggesting that the expression of individual UT-A isoforms may be regulated independently (Tab. II).

TABLE II - LONG-TERM REGULATION OF UT-A MRNA ISOFORMS IN RAT INNER MEDULLA

In vivo treatment/Rat strain	UT-A1	UT-A2	Reference
Low-protein diet/SD(UT1)	No change	Increase	Ashkar, AJP, 1995
"	Increase	No change	Smith, JCI, 1995
Adrenalectomy/SD	No change	No change	Naruse, JASN, 1997
Water diuresis/SD	Decrease	No change	Smith, JCI, 1995
"	No change	Decrease	Promeneur, JASN, 1996
Water restriction/SD	Decrease	Increase	Smith, JCI, 1995
"	No change	Increase	Promeneur, JASN, 1996
DDAVP or AVP/Brattle.	Increase	Increase	Promeneur, JASN, 1998
DDAVP/SD	No change	Increase	Promeneur, JASN, 1996

We have extended these earlier observations to include the rat UT-A3 isoform, and used Northern hybridization to evaluate UT-A1, UT-A2, and UT-A3 expression in cortex, outer medulla, and inner medulla of water - deprived and water - loaded rats. Compared to controls, the water - deprived rats had a significantly greater mRNA abundance of UT-A2 and UT-A3 in the inner medulla, while UT-A1 was not significantly changed, supporting differential, independent regulation of the long-term expression of each UT-A isoform (unpublished observations).
This result is particularly intriguing in light of new information obtained by our group on the genomic organization of the UT-A transporter. The four UT-A cDNAs are highly homologous (Fig. 2A), which supports our suggestion that they are all encoded by a single gene. UT-A1, UT-A3 and UT-A4 have the same transcription start site, distinct from that of UT-A2, and two different promoters may control each transcription start site (36). The difference in UT-A1 and UT-A3 mRNA abundance in response to dehydration suggests that transcriptional and post-transcriptional mechanisms may participate in regulating the differential expression of individual UT-A isoforms.
Expression of at least one UT-A isoform has been reported in the human kidney (37). UT-A1 may also be expressed in human renal inner medulla, to achieve efficient excretion of concentrated urine, but the human homologue of UT-A1 has not been cloned yet, and no information is available on the structure of the human UT-A gene. By in situ hybridization using the hUT-A2 cDNA, Olives et al assigned the UT-A and UT-B genes both to chromosome 18q12.1-q21.1 (37), suggesting that gene duplication may have occurred in the evolution of mammalian urea transporters.
The human Kidd/UT-B gene has been cloned, and mutations have been identified that result in the expression of altered, non-functional urea transporter proteins in JK_null individuals, who do not express the Kidd blood antigen (38). Only limited defects in the ability to concentrate urine have been detected in JK_null patients (39). Whether, compared to UT-B, deficient function of the UT-A transporter proteins has more severe consequences in humans, remains to be established. There is abundant evidence of a urinary concentrating defect in patients with protein or protein-calorie malnutrition, uncontrolled diabetes mellitus, and in patients with papillary necrosis due to sickle cell anemia or analgesic nephropathy. Specific regulatory pathways may become active in these settings that affect the expression and/or activity of the UT-A transporter, contributing to the impairment of urinary concentration. In addition, states involving abnormal levels of urea such as renal failure or familial hyperazotemia with normal renal function (40), and congenital defects of urea metabolism, may also result in unexpected effects on the regulation of the expression and function of the UT-A transporter in organs other than the kidney.
Considerable new information on urea transporters has still not fully clarified their roles, which may be diverse, or the possible impact of their functional deficit and dysregulation, which may be far-reaching.

Cellular adaptation to urea and urea toxicity

Levels of urea are similar in the intra- and extracellular compartments in the renal medulla (41), and urea can be viewed as one of many organic solutes, or osmolytes, that help maintain the internal osmolarity of inner medullary cells, in the face of high extracellular tonicity (42, 43). However, unlike the non-perturbing osmolytes, urea at concentrations similar to those normally present in the kidney, can destabilize essential enzymatic activities (44, 45). Urea deleterious effects on macromolecular functions may be counteracted by methylamines such as trimethylamine oxide (TMAO), betaine, and glycerophosphorylcholine (46, 47), which are present in relatively high concentrations in the mammalian renal medulla (48). Other mechanisms may protect renal cells from the untoward effects of urea, since cultured renal epithelial cells can grow in the presence of up to 300 mM urea, without addition of methylamines (49-51). However, higher concentrations result in cell death, and cellular exposure to a high concentration of either NaCl or urea can lead to apoptosis (52, 53).
How renal cells adapt to concentrations of urea higher than everywhere else in the body needs further investigation. Although the mechanisms involved in urea's toxicity are incompletely understood, the possibility that urea-induced generation of free radicals and oxidative stress leads to significant cellular damage has been advanced (54). Whether renal inner medullary cells, which are adapted to a urea-rich environment are more resistant to urea-induced oxidative stress has not been clarified. Urea may activate intracellular signaling pathways distinct from those activated by non-permeant solutes like NaCl (55-59). Investigations in this area could help elucidate urea-specific signal transduction and disclose novel regulatory effects of urea on gene expression and protein function in the kidney and other organs. They could also improve our current understanding of the physiological role of urea, and its toxicity in pathological states.

Reprint requests to: Serena M. Bagnasco, M.D. - Department of Pathology, Emory University School of Medicine, WMB Room 7105-A - 1639 Pierce Dr. - Atlanta, GA 30322, USA sbagnas@emory.edu

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Received: February 29, 2000 Revised: April 03, 2000 Accepted: May 29, 2000

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