Characterization of mouse urea transporters UT-A1 and UT-A2

Fenton, Robert A.; Stewart, Gavin; Carpenter, Brian; Howorth, A.; Potter, Elizabeth A.; Cooper, Gordon J.; Smith, Craig P.

doi:10.1152/ajprenal.00263.2001

Cited by 78 publications

(98 citation statements)

References 29 publications

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“…5A), compatible with the small size (148 bp) of the deleted exon. The approximate sizes of the transcripts observed, 4.0, 3.0, and 2.1 kb, are consistent with those reported for full-length UT-A1, UT-A2, and UT-A3, respectively (21). When an equivalent probe was hybridized to testis RNA, a strong signal representing UT-A5 was observed at 1.4 kb in wild-type mice, but signals centered at 0.6 kb, representing truncated mRNA transcripts, were observed for UT-A1͞3 Ϫ/Ϫ mice (Fig.…”

Section: Resultssupporting

confidence: 88%

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Urinary concentrating defect in mice with selective deletion of phloretin-sensitive urea transporters in the renal collecting duct

Fenton

Chou

Stewart

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

227

230

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To investigate the role of inner medullary collecting duct (IMCD) urea transporters in the renal concentrating mechanism, we deleted 3 kb of the UT-A urea transporter gene containing a single 140-bp exon (exon 10). Deletion of this segment selectively disrupted expression of the two known IMCD isoforms of UT-A, namely UT-A1 and UT-A3, producing UT-A1͞3 ؊/؊ mice. In isolated perfused IMCDs from UT-A1͞3 ؊/؊ mice, there was a complete absence of phloretin-sensitive or vasopressin-stimulated urea transport. On a normal protein intake (20% protein diet), UT-A1͞ 3 ؊/؊ mice had significantly greater fluid consumption and urine flow and a reduced maximal urinary osmolality relative to wildtype controls. These differences in urinary concentrating capacity were nearly eliminated when urea excretion was decreased by dietary protein restriction (4% by weight), consistent with the 1958 Berliner hypothesis stating that the chief role of IMCD urea transport in the concentrating mechanism is the prevention of ureainduced osmotic diuresis. Analysis of inner medullary tissue after water restriction revealed marked depletion of urea in UT-A1͞3 ؊/؊ mice, confirming the concept that phloretin-sensitive IMCD urea transporters play a central role in medullary urea accumulation. However, there were no significant differences in mean inner medullary Na ؉ or Cl ؊ concentrations between UT-A1͞3 ؊/؊ mice and wild-type controls, indicating that the processes that concentrate NaCl were intact. Thus, these results do not corroborate the predictions of passive medullary concentrating models stating that NaCl accumulation in the inner medulla depends on rapid vasopressin-regulated urea transport across the IMCD epithelium.UT-A ͉ isolated perfused tubule ͉ vasopressin ͉ concentrating mechanism F or survival remote from water sources, terrestrial animals require effective water-conservation mechanisms. In birds and mammals, water conservation depends on specialized urinary concentrating mechanisms that reduce water excretion while maintaining solute excretion. This concentrating function is carried out in the renal medulla. In mammals, the medulla is divided into two regions, the outer medulla and the inner medulla (IM), both of which manifest increased tissue osmolality relative to the blood plasma (1). In these regions, high interstitial osmolality draws water from the renal collecting duct via aquaporin water channels, resulting in a concentrated final urine. In the outer medulla, the interstitial space is concentrated through the classic countercurrent multiplication mechanism (2) in which water and solute are separated by active NaCl transport from the water-impermeable thick ascending limb of Henle (3, 4). However, in the IM the ascending portion of Henle's loop is incapable of high rates of active transport (5, 6), implying that solute concentration in the IM occurs by a different, yet unknown mechanism.One important feature of the IM that distinguishes it from the outer medulla is its ability to accumulate large amounts of urea. Berliner ...

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Section: Resultssupporting

confidence: 88%

“…Mutant PCR fragments were subcloned into full-length UT-A1 or UT-A3 cDNAs, replacing the corresponding region, and sequenced. Expression of the mutant clones, termed UT-A1 (10Ϫ) and UT-A3 (10Ϫ) , in Xenopus oocytes was performed as described (21).…”

Section: Methodsmentioning

confidence: 99%

Urinary concentrating defect in mice with selective deletion of phloretin-sensitive urea transporters in the renal collecting duct

Fenton

Chou

Stewart

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

227

230

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“…Multiple cDNAs (Figure 2) that encode urea transporters have been isolated and characterized (18 -25). UT-A1 is expressed exclusively in IMCD cells (Figure 3) (19,26). UT-A1 activity is regulated acutely by AVP (27), but the long-term effects of AVP on UT-A1 abundance are still a subject of controversy.…”

Section: Urea Transportersmentioning

confidence: 99%

“…UT-A1 activity is regulated acutely by AVP (27), but the long-term effects of AVP on UT-A1 abundance are still a subject of controversy. UT-A2 is expressed in the inner stripe of the outer medulla, where it is localized to the lower portions of the thin descending limbs (tDL) of short loops of Henle (Figures 1 and 3) (19,28,29) and, under prolonged antidiuretic conditions, in the inner medulla, where it is localized to the tDL of long loops of Henle (29). AVP increases UT-A2 abundance (19,29,30), and recent studies have determined that UT-A2-mediated urea transport can be regulated acutely by cAMP (31).…”

Section: Urea Transportersmentioning

confidence: 99%

Urea and Renal Function in the 21st Century

Fenton¹,

Knepper²

2007

Journal of the American Society of Nephrology

115

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Since the turn of the 21st century, gene knockout mice have been created for all major urea transporters that are expressed in the kidney: the collecting duct urea transporters UT-A1 and UT-A3, the descending thin limb isoform UT-A2, and the descending vasa recta isoform UT-B. This article discusses the new insights that the results from studies in these mice have produced in the understanding of the role of urea in the urinary concentrating mechanism and kidney function. Following is a summary of the major findings: (1) Urea accumulation in the inner medullary interstitium depends on rapid transport of urea from the inner medullary collecting duct (IMCD) lumen via UT-A1 and/or UT-A3; (2) as proposed by Robert Berliner and colleagues in the 1950s, the role of IMCD urea transporters in water conservation is to prevent a urea-induced osmotic diuresis; (3) the absence of IMCD urea transport does not prevent the concentration of NaCl in the inner medulla, contrary to what would be predicted from the passive countercurrent multiplier mechanism in the form proposed by Kokko and Rector and Stephenson; (4) deletion of UT-B (vasa recta isoform) has a much greater effect on urinary concentration than deletion of UT-A2 (descending limb isoform), suggesting that the recycling of urea between the vasa recta and the renal tubules quantitatively is less important than classic countercurrent exchange; and (5) urea reabsorption from the IMCD and the process of urea recycling are not important elements of the mechanism of protein-induced increases in GFR. In addition, the clinical relevance of these studies is discussed, and it is suggested that inhibitors that specifically target collecting duct urea transporters have the potential for clinical use as potassium-sparing diuretics that function by creation of urea-dependent osmotic diuresis.

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“…UT-A1 and UT-A3 are located in the inner medullary collecting duct (IMCD), whereas UT-A2 has been localized to type 1 and type 3 thin descending limbs (2,12,21). In terms of subcellular localization, UT-A1 has been located to the apical membrane of IMCD cells (9), while UT-A2 is found on both apical and basolateral membranes of the thin descending limbs (2). In contrast, UT-A3 has been localized to the basolateral membrane of IMCD cells in both the mouse (18) and rat (8,13), and also apically in the rat (20).…”

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confidence: 99%

Acute regulation of mUT-A3 urea transporter expressed in a MDCK cell line

Stewart

King²,

Potter³

et al. 2007

American Journal of Physiology-Renal Physiology

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Renal facilitative urea transporters play a vital role in the urinary concentrating mechanism. UT-A3 is a phloretin-sensitive urea transporter that in the mouse is expressed on the basolateral membrane of renal inner medullary collecting duct (IMCD) cells. In this study, we engineered a Madin-Darby canine kidney (MDCK) I cell line that stably expresses mouse UT-A3 (MDCK-mUT-A3). Immunoblotting using the UT-A-targeted antibody ML446 detected a ∼40-kDa signal in MDCK-mUT-A3 protein that corresponds to mUT-A3. Using cultured epithelial monolayers, radioactive 14C-urea flux experiments determined that basolateral urea transport was no different between MDCK-mUT-A3 and control MDCK-FLZ cells under basal conditions [not significant (NS), ANOVA]. However, exposure to arginine vasopressin (AVP) significantly stimulated basolateral urea flux in MDCK-mUT-A3 monolayers ( P < 0.05, ANOVA), while it had no effect in control MDCK-FLZ monolayers (NS, ANOVA). The AVP-stimulated basolateral urea transport in MDCK-mUT-A3 was inhibited by 1,3 dimethyl urea ( P < 0.05, ANOVA) or phloretin ( P < 0.05, ANOVA), both known inhibitors of facilitative urea transporters. MDCK-mUT-A3 basolateral urea flux was also stimulated by increasing intracellular levels of cAMP, via forskolin ( P < 0.05, ANOVA), or intracellular calcium, via ATP ( P < 0.05, ANOVA). Finally, 1-h preincubation with a specific PKA inhibitor, H89, significantly inhibited the increase in urea transport produced by AVP ( P < 0.05, ANOVA). In conclusion, we have produced the first renal cell line to stably express the mUT-A3 urea transporter. Our results indicate that mUT-A3 is acutely regulated by AVP, via a PKA-dependent pathway. These findings have important implications for the regulation of urea transport in the renal IMCD and the urinary concentrating mechanism.

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Characterization of mouse urea transporters UT-A1 and UT-A2

Cited by 78 publications

References 29 publications

Urinary concentrating defect in mice with selective deletion of phloretin-sensitive urea transporters in the renal collecting duct

Urinary concentrating defect in mice with selective deletion of phloretin-sensitive urea transporters in the renal collecting duct

Urea and Renal Function in the 21st Century

Acute regulation of mUT-A3 urea transporter expressed in a MDCK cell line

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