Vasopressin regulates apical targeting of aquaporin-2 but not of UT1 urea transporter in renal collecting duct.
In the renal inner medullary collecting duct (IMCD), vasopressin regulates two key transporters, namely aquaporin-2 (AQP2) and the vasopressin-regulated urea transporter (VRUT). Both are present in intracellular vesicles as well as the apical plasma membrane. Short-term regulation of AQP2 has been demonstrated to occur by vasopressin-induced trafficking of AQP2-containing vesicles to the apical plasma membrane. Here, we have carried out studies to determine whether short-term regulation of VRUT occurs by a similar process. Cell surface labeling with NHS-LC-biotin in rat IMCD suspensions revealed that vasopressin causes a dose-dependent increase in the amount of AQP2 labeled at the cell surface, whereas VRUT labeled at the cell surface did not increase in response to vasopressin. Immunoperoxidase labeling of inner medullary thin sections from Brattleboro rats treated with 1-desamino-8-D-arginine vasopressin (DDAVP) for 20 min revealed dramatic translocation of AQP2 to the apical region of the cell, with no change in the cellular distribution of VRUT. In addition, differential centrifugation of inner medullary homogenates from Brattleboro rats treated with DDAVP for 60 min revealed a marked depletion of AQP2 from the low-density membrane fraction (enriched in intracellular vesicles) but did not alter the quantity of VRUT in this fraction. Finally, AQP2-containing vesicles immunoisolated from a low-density membrane fraction from renal inner medulla did not contain immunoreactive VRUT. Thus vasopressin-mediated regulation of AQP2, but not of VRUT, depends on regulated vesicular trafficking to the plasma membrane. (+info)
Urinary excretion of aquaporin-2 in rat is mediated by a vasopressin-dependent apical pathway.
Clinical studies have shown that aquaporin-2 (AQP2), the vasopressin-regulated water channel, is excreted in the urine, and that the excretion increases in response to vasopressin. However, the cellular mechanisms involved in AQP2 excretion are unknown, and it is unknown whether the excretion correlates with AQP2 levels in kidney or levels in the apical plasma membrane. The present study was undertaken to clarify these issues. Immunoblotting of rat urine samples revealed significant excretion of AQP2, whereas AQP3, being a basolateral aquaporin in the same cells, was undetectable. Thus, there was a nonproportional excretion of AQP2 and AQP3 (compared with kidney levels), indicating that AQP2 is excreted predominantly via a selective apical pathway and not by whole cell shedding. Urinary AQP2 was associated with small vesicles, membrane fragments, and multivesicular bodies as determined by immunoelectron microscopy and negative staining techniques. In rats with normal water supply, daily urinary excretion of AQP2 was 3.9+/-0.9% (n = 6) of total kidney expression. Treatment with desmopressin acetate subcutaneously caused a fourfold increase in urinary excretion of AQP2 during 8 h. Forty-eight hours of thirsting, known to increase endogenous vasopressin secretion, resulted in a three-fold increase in kidney AQP2 levels but urinary excretion increased ninefold to 15+/-3% (n = 6) of AQP2 in kidney of thirsted rats. Moreover, rats that were thirsted for 48 h and subsequently allowed free access to water for 24 h produced a decrease in urinary AQP2 excretion to 38+/-15% (n = 6) of that during thirsting. In Brattleboro rats or lithium-treated normal rats completely lacking vasopressin action, and hence having extremely low levels of AQP2 in the apical plasma membrane, AQP2 was undetectable in urine. Thus, conditions with known altered vasopressin levels and altered levels of AQP2 in the apical plasma membrane were associated with corresponding major changes in AQP2 urine excretion. In contrast, in such conditions, kidney AQP2 levels and urinary AQP2 excretion did not show a proportional relationship. (+info)
Regulation of cyclooxygenase-2 expression in renal medulla by tonicity in vivo and in vitro.
Renal medullary prostaglandins are believed to exert an important functional role in antagonizing vasopressin effects in dehydration. Studies were undertaken to determine the effect of hyperosmolality on cyclooxygenase (COX) isoform expression in the renal medulla. COX-1 and COX-2 mRNA and protein levels were determined by RT-PCR or Western blotting in Sprague-Dawley rats on varying water intakes, in Brattleboro rats and in Long-Evans controls. Over a wide range of urinary tonicity, COX-2 expression correlated closely with urine osmolality levels (R = 0.872). COX-1 levels did not vary. Immunolocalization showed that the stimulation of COX-2 expression by dehydration occurred predominantly in the collecting duct. Hypertonicity caused by addition of NaCl produced a dose- and time-dependent stimulation of COX-2 expression in mIMCD-K2 cells as well as in MDCK cells. COX-1 was unaffected. In the same cell lines, mannitol, sucrose, and raffinose also had a stimulatory effect. The tonicity-stimulated COX-2 expression in mIMCD-K2 cells was almost completely blocked by a tyrosine kinase inhibitor, genistein at 100 microM. In MDCK cells transfected with a 2.7-kb COX-2 promoter and lacZ reporter construct, NaCl induced a twofold increase in beta-galactosidase activity. Using mIMCD-K2 cells, hypertonic NaCl (600 mosmol/kgH(2)O for 24 h) induced a 33-fold increase in PGE(2) release determined by enzyme immunoassay, an effect completely blocked by 3 microM indomethacin or the COX-2-specific blocker N-(2-cyclohexy-4-nitrophenyl)methanesulfonamide (NS-398). We conclude that in inner medulla, COX-2 but not COX-1 is upregulated by hyperosmolality. (+info)
Lack of vasopressin-independent upregulation of AQP-2 gene expression in homozygous Brattleboro rats.
Arginine vasopressin (AVP) plays an important role in the expression of aquaporin (AQP-2) in the collecting duct. The present study was undertaken to determine whether there is an AVP-independent regulation of AQP-2 gene expression in homozygous Brattleboro rats in which endogenous AVP is absent. Exogenous administration of 1-deamino-8-D-AVP produced an antidiuresis and expressed AQP-2 mRNA and AQP-2 protein in the renal medulla of the homozygous Brattleboro rats. Twelve hours of water deprivation produced severe dehydration in the homozygous Brattleboro rats, such that urinary osmolality increased from 200 to 649 mosmol/kgH(2)O. However, no increase in AQP-2 mRNA expression was observed after this dehydration, and the medullary tissue content and urinary excretion of AQP-2 also remained unchanged. Increases in AQP-2 mRNA expression and AQP-2 protein were evident in Long-Evans rats after 64 h of water deprivation, with a severity of dehydration almost equal to the 12-h dehydrated, homozygous Brattleboro rats. These results indicate the lack of an AVP-independent mechanism for upregulating AQP-2 mRNA expression in renal collecting duct cells. (+info)
Vasopressin contributes to hyperfiltration, albuminuria, and renal hypertrophy in diabetes mellitus: study in vasopressin-deficient Brattleboro rats.
Diabetic nephropathy represents a major complication of diabetes mellitus (DM), and the origin of this complication is poorly understood. Vasopressin (VP), which is elevated in type I and type II DM, has been shown to increase glomerular filtration rate in normal rats and to contribute to progression of chronic renal failure in 5/6 nephrectomized rats. The present study was thus designed to evaluate whether VP contributes to the renal disorders of DM. Renal function was compared in Brattleboro rats with diabetes insipidus (DI) lacking VP and in normal Long-Evans (LE) rats, with or without streptozotocin-induced DM. Blood and urine were collected after 2 and 4 weeks of DM, and creatinine clearance, urinary glucose and albumin excretion, and kidney weight were measured. Plasma glucose increased 3-fold in DM rats of both strains, but glucose excretion was approximately 40% lower in DI-DM than in LE-DM, suggesting less intense metabolic disorders. Creatinine clearance increased significantly in LE-DM (P < 0.01) but failed to increase in DI-DM. Urinary albumin excretion more than doubled in LE-DM but rose by only 34% in DI-DM rats (P < 0.05). Kidney hypertrophy was also less intense in DI-DM than in LE-DM (P < 0.001). These results suggest that VP plays a critical role in diabetic hyperfiltration and albuminuria induced by DM. This hormone thus seems to be an additional risk factor for diabetic nephropathy and, thus, a potential target for prevention and/or therapeutic intervention. (+info)
Thiazide induces water absorption in the inner medullary collecting duct of normal and Brattleboro rats.
The reduction of urinary volume after the use of thiazide in the treatment of diabetes insipidus (DI) is known as the "paradoxical effect." Since enhanced proximal solute and water reabsorption only partially account for the reduction in urinary volume, an additional diuretic effect on nephron terminal segments was postulated. Thus the aim of our work was to investigate the effect of hydrochlorothiazide (HCTZ) on water transport in the inner medullary collecting duct (IMCD) of normal and Brattleboro rats. Osmotic water permeability (P(f)) and diffusional water permeability (P(dw)) were studied at 37 degrees C and pH 7.4 by the in vitro microperfusion technique. In the absence of antidiuretic hormone (ADH), HCTZ (10(-6) M) added to the perfused fluid enhanced P(f) from 6.36 +/- 0. 56 to 19.08 +/- 1.70 micro(m)/s (P < 0.01) and P(dw) from 38.01 +/- 4.52 to 52.26 +/- 4.38 x10(-5) cm/s (P < 0.01) in normal rats and also stimulated P(f) in Brattleboro rats from 3.53 +/- 1.41 to 11.16 +/- 1.13 micro(m)/s (P < 0.01). Prostaglandin E(2) (PGE(2)) (10(-5) M) added to the bath fluid inhibited HCTZ-stimulated P(f) (in micro(m)/s) as follows: control, 16.93 +/- 2.64; HCTZ, 29.65 +/- 5.67; HCTZ+PGE(2), 10.46 +/- 1.84 (P < 0.01); recovery, 16.77 +/- 4.07. These data indicate that thiazides enhance water absorption in IMCD from normal rats (in the absence of ADH) and from Brattleboro rats and that the HCTZ-stimulated P(f) was partially blocked by PGE(2). Thus we may conclude that the effect of thiazide in the treatment of DI occurs not only in the Na(+)-Cl(-) cotransport in the distal tubule but also in the IMCD. (+info)
Localization and regulation of PKA-phosphorylated AQP2 in response to V(2)-receptor agonist/antagonist treatment.
Phosphorylation of Ser(256), in a PKA consensus site, in AQP2 (p-AQP2) appears to be critically involved in the vasopressin-induced trafficking of AQP2. In the present study, affinity-purified antibodies that selectively recognize AQP2 phosphorylated at Ser(256) were developed. These antibodies were used to determine 1) the subcellular localization of p-AQP2 in rat kidney and 2) changes in distribution and/or levels of p-AQP2 in response to [desamino-Cys(1),D-Arg(8)]vasopressin (DDAVP) treatment or V(2)-receptor blockade. Immunoelectron microscopy revealed that p-AQP2 was localized in both the apical plasma membrane and in intracellular vesicles of collecting duct principal cells. Treatment of rats with V(2)-receptor antagonist for 30 min resulted in almost complete disappearance of p-AQP2 labeling of the apical plasma membrane with only marginal labeling of intracellular vesicles remaining. Immunoblotting confirmed a marked decrease in p-AQP2 levels. In control Brattleboro rats (BB), lacking vasopressin secretion, p-AQP2 labeling was almost exclusively present in intracellular vesicles. Treatment of BB rats with DDAVP for 2 h induced a 10-fold increase in p-AQP2 labeling of the apical plasma membrane. The overall abundance of p-AQP2, however, was not increased, as determined both by immunoelectron microscopy and immunoblotting. Consistent with this, 2 h of DDAVP treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that AQP2 phosphorylated in Ser(256) is present in the apical plasma membrane and in intracellular vesicles and that both the intracellular distribution/trafficking, as well as the abundance of p-AQP2, are regulated via V(2) receptors by altering phosphorylation and/or dephosphorylation of Ser(256) in AQP2. (+info)
Long-term regulation of urea transporter expression by vasopressin in Brattleboro rats.
Regulation of urea concentration in the renal medullary interstitium is important for maintenance of hypertonicity and therefore the osmotic driving force for water reabsorption. Studies in Sprague-Dawley rats showed that restriction of water intake for 3 days results in upregulation of urea transporter (UT) mRNA in the inner stripe of outer medulla of the kidney (2.9-kb UT2) but not in the inner medulla (4.0-kb UT1). The present study was performed to investigate the role of vasopressin in long-term regulation of UT1 and UT2 in neurogenic diabetes insipidus (Brattleboro) rats treated with a 7-day continuous infusion of [Arg(8)]-vasopressin (AVP), [deamino-Cys(1), D-Arg(8)]-vasopressin (dDAVP) or vehicle. Northern analysis showed that water restriction alone had no effect on the level of UT2 mRNA in vehicle-treated Brattleboro rats but UT2 mRNA markedly increased and UT1 mRNA modestly decreased after treatment with dDAVP. In situ hybridization further demonstrated that the UT2 signal is upregulated and spread along the descending thin limbs of loops of Henle and that UT1 signal is downregulated in the inner medullary collecting ducts in vasopressin-treated rats, with a greater response for dDAVP compared with the AVP-treated group. Immunocytochemistry studies revealed that the UT1 and UT2 proteins are also modified in the same pattern as the transcript changes. Our studies reveal the role of vasopressin in long-term regulation of UT1 and UT2 expression during water restriction. (+info)