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

Abstract

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 ...

Quantitative phosphoproteomics of vasopressin-sensitive renal cells: Regulation of aquaporin-2 phosphorylation at two sites

“…The two isoforms, UT-A1 and UT-A3, expressed in collecting duct show increases in urea uptake in response to agents that increase intracellular cAMP (forskolin ϩ 3-isobutyl-1-methylxanthine) when expressed in Xenopus oocytes (23). They are produced by alternative splicing from a single precursor RNA and share the same N-terminal 459 aa (24).…”

Urea and Ammonia Metabolism and the Control of Renal Nitrogen Excretion

“…Vasopressin increases urea transport, urea transporter phosphorylation, and apical plasma membrane accumulation through two cAMP-dependent pathways: protein kinase A and exchange protein activated by cAMP (28) ( Figure 6). Genetically engineered mice lacking UT-A1 and UT-A3 have reduced urine concentrating ability, have reduced inner medullary interstitial urea content, and lack vasopressinstimulated urea transport in their IMCDs (29).…”

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

“…UT-A1 and UT-A3 transporters localized in the IMCD regulate urea reabsorption into the interstitium, under the control of arginine vasopressin (AVP) (3). In rat perfused, isolated IMCD, AVP has been shown to increase intracellular levels of cAMP and calcium, as well as stimulating trans-epithelial phloretin-sensitive urea transport (17,22).…”