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ABSTRACT: The renal urea transporter gene (UT-A) produces different transcripts in the inner medullary collecting ducts (UT-A1) and thin descending limbs of Henle's loop (UT-A2), coding for distinct proteins. Peptide-directed rabbit polyclonal antibodies were used to identify the UT-A2 protein in renal medulla of mouse and rat. In the inner stripe of outer medulla, an antibody directed to the COOH terminus of UT-A recognized a membrane protein of 55 kDa. The abundance of this 55-kDa protein was strongly increased in response to chronic infusion of the vasopressin analog 1-deamino-[8-D-arginine]vasopressin (DDAVP) in Brattleboro rats, consistent with previous evidence that UT-A2 mRNA abundance is markedly increased. Immunofluorescence labeling with the COOH-terminal antibody in Brattleboro rats revealed labeling in the lower portion of descending limbs from short-looped nephrons (in the aquaporin-1-negative portion of this segment). This UT-A labeling was increased in response to DDAVP. Increased labeling was also seen in descending limbs of long-looped nephrons in the base of the inner medulla. These results indicate that UT-A2 is expressed as a 55-kDa protein in portions of the thin descending limbs of Henle's loop and that the abundance of this protein is strongly upregulated by vasopressin.
American journal of physiology. Renal physiology 02/2000; 278(1):F52-62. · 3.68 Impact Factor
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ABSTRACT: Cyclooxygenase inhibitors, such as indomethacin and diclofenac, have well-described effects to enhance renal water reabsorption and urinary concentrating ability. Concentrating ability is regulated in part at the level of the thick ascending limb of Henle's loop, where active NaCl absorption drives the countercurrent multiplication mechanism. We used semiquantitative immunoblotting to test the effects of indomethacin and diclofenac, given over a 48-h period, on the expression levels of the ion transporters responsible for active NaCl transport in the thick ascending limb. Both agents strongly increased the expression level of the apical Na-K-2Cl cotransporter in both outer medulla and cortex. Neither agent significantly altered outer medullary expression levels of other thick ascending limb proteins, namely, the type 3 Na/H exchanger (NHE-3), Tamm-Horsfall protein, or alpha1- or beta1-subunits of the Na-K-ATPase. Administration of the EP3-selective PGE(2) analog, misoprostol, to indomethacin-treated rats reversed the stimulatory effect of indomethacin on Na-K-2Cl cotransporter expression. We conclude that cyclooxygenase inhibitors enhance urinary concentrating ability in part through effects to increase Na-K-2Cl cotransporter expression in the thick ascending limb of Henle's loop. This action is most likely due to elimination of an EP3-receptor-mediated tonic inhibitory effect of PGE(2) on cAMP production.
The American journal of physiology 09/1999; 277(2 Pt 2):F219-26.
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ABSTRACT: Transport processes along the nephron are regulated in part by hormone stimulation of adenylyl cyclases mediated by the heterotrimeric G protein G(s). To assess the role of this pathway in the regulation of Na-K-2Cl cotransporter abundance in the renal thick ascending limb (TAL), we studied mice with heterozygous disruption of the Gnas gene, which codes for the alpha-subunit of G(s). Outer medullary G(s)alpha protein abundance (as assessed by semiquantitative immunoblotting) and glucagon-stimulated cAMP production were significantly reduced in the heterozygous G(s)alpha knockout mice (GSKO) relative to their wild-type (WT) littermates. Furthermore, Na-K-2Cl cotransporter protein abundance in the outer medulla was significantly reduced (band density, 48% of WT). In addition, GSKO mice had a significantly reduced (72% of WT) urinary osmolality in response to a single injection of 1-deamino-[8-D-arginine]vasopressin (DDAVP), a vasopressin analog. In contrast, outer medullary protein expression of the type 3 Na/H exchanger (NHE-3) or Tamm-Horsfall protein did not differ between the GSKO mice and their WT littermates. However, abundance of type VI adenylyl cyclase was markedly decreased in the outer medullas of GSKO mice, suggesting a novel feed-forward regulatory mechanism. We conclude that expression of the Na-K-2Cl cotransporter of the TAL is dependent on G(s)alpha-mediated hormone stimulation, most likely due to long-term changes in cellular cAMP levels.
The American journal of physiology 09/1999; 277(2 Pt 2):F235-44.
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ABSTRACT: Previously, we demonstrated that escape from vasopressin-induced antidiuresis ("vasopressin escape") in rats is associated with a large, selective decrease in whole kidney expression of aquaporin-2, the vasopressin-regulated water channel. Here, we show that isolated perfused inner medullary collecting ducts (IMCDs) from vasopressin-escape rats desamino-[D-arginine]vasopressin (DDAVP)/water-loaded have dramatically reduced vasopressin-dependent osmotic water permeabilities [46% of control rats (DDAVP alone)], which coincides with a fall in inner medullary aquaporin-2 protein abundance as measured by immunoblotting in the opposite kidney. Furthermore, we demonstrate in IMCD suspensions that cAMP accumulation in response to DDAVP is substantially reduced in the vasopressin-escape rats both in the presence and absence of the phosphodiesterase inhibitor IBMX. By immunoblotting, we show that the abundance of two proteins important in cAMP generation: the stimulatory heterotrimeric G protein subunit Gs and adenylyl cyclase type VI, do not change. We conclude that vasopressin escape is associated with relative vasopressin resistance of the collecting duct cells manifested by decreased intracellular cAMP levels. The decreased cAMP levels can contribute to the demonstrated decrease in collecting duct water permeability in two ways: 1) by causing a decrease in aquaporin-2 expression and 2) by limiting the acute action of vasopressin to increase collecting duct water permeability.
The American journal of physiology 07/1998; 274(6 Pt 2):F1161-6.
