Angiotensin II increases vasopressin-stimulated facilitated urea permeability in rat terminal IMCDs

Akihiko Kato, Janet D. Klein, Chi Zhang, and Jeff M. Sands

Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Angiotensin II receptors are present along the rat inner medullary collecting duct (IMCD), although their physiological role is unknown. Because urea is one of the major solutes transported across the terminal IMCD, we measured angiotensin II's effect on urea permeability. In the perfused rat terminal IMCD, angiotensin II had no effect on basal urea permeability but significantly increased vasopressin-stimulated urea permeability by 55%. Angiotensin II, both without and with vasopressin, also increased the amount of 32P incorporated into urea transporter (UT)-A1 in inner medullary tissue exposed to these hormones ex vivo. Because angiotensin II activates protein kinase C, we tested the effect of staurosporine (SSP). In the absence of angiotensin II, SSP had no effect on vasopressin-stimulated urea permeability in the perfused terminal IMCD. However, SSP completely and reversibly blocked the angiotensin II-mediated increase in vasopressin-stimulated urea permeability. SSP and chelerythrine reduced the angiotensin II-stimulated 32P incorporation into UT-A1 in inner medullary tissue exposed ex vivo. We conclude that angiotensin II increases vasopressin-stimulated facilitated urea permeability and 32P incorporation into the 97- and 117-kDa UT-A1 proteins via a protein kinase C-mediated signaling pathway. These data suggest that angiotensin II augments vasopressin-stimulated facilitated urea transport in the rat terminal IMCD and may play a physiological role in the urinary concentrating mechanism by augmenting the maximal response to vasopressin.

inner medullary collecting duct; protein kinase C; urine-concentrating mechanism; phosphorylation; hyperosmolality; urea transporter


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE RENIN-ANGIOTENSIN SYSTEM may play an important role in the urinary concentrating mechanism because infusing angiotensin II into the renal artery increases urine osmolality in rats (9), whereas subcutaneous injection of an angiotensin-converting enzyme (ACE) inhibitor for 5 days reduces urine osmolality in mice (27). In addition, knockout of the gene for angiotensinogen, ACE, or angiotensin II receptors impairs urine-concentrating ability (7, 8, 15, 19, 26, 27). In some of these knockout mice (angiotensinogen, ACE), the decrease in urine-concentrating ability results from anatomic abnormalities in the renal medulla. However, mice lacking the type 1A angiotensin II receptor or ACE (ACE.2 mice) have a relatively normal medulla and still have a urine-concentrating defect (8, 27).

The preceding findings suggest that angiotensin II may have a functional role in nephron segments involved in generating the hypertonic medullary gradient that is needed to concentrate the urine maximally, i.e., the medullary thick ascending limb (mTAL) and inner medullary collecting duct (IMCD). Angiotensin II receptors are present in the mTAL, and angiotensin II stimulates Na+-K+-2Cl- cotransport (1). Both RT-PCR and in situ hybridization studies show that the mRNA for angiotensin II receptors is present in the IMCD (16, 23, 38), and radioligand binding studies show that angiotensin II receptors are present (24). However, no studies have been performed to determine whether angiotensin II has a functional role in the IMCD.

Urea is one of the major solutes transported across the terminal IMCD and is important for the generation of the hypertonic medulla needed to concentrate the urine maximally [reviewed in (30)]. In rats consuming a standard diet, the terminal IMCD is the only nephron segment in which vasopressin stimulates facilitated urea transport (18, 33). In the present study, we evaluated whether angiotensin II can regulate urea transport in the terminal IMCD by testing its effect on basal and vasopressin-stimulated facilitated urea permeability. We also tested whether any effects of angiotensin II occurred via protein kinase C (PKC) because angiotensin II can activate it (1, 17, 22).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparation. All animal protocols were approved by the Emory University Institutional Animal Care and Use Committee. Tubules were obtained from pathogen-free male Sprague-Dawley rats (National Cancer Institute, Frederick, MD). Rats were kept in filter-top cages with autoclaved bedding and received free access to water and a standard diet (NIH-31, Ziegler Brothers, Gardner, PA). Kidneys were placed into chilled (17°C), isotonic, dissecting solution to isolate the terminal IMCD (5, 6). The terminal IMCD was identified by dissecting between 50 and 70% of the distance between the inner-outer medullary border (0%) and the papillary tip (100%) as measured by using an eyepiece micrometer (32, 33).

The dissecting solution was gassed with 95% O2-5% CO2 and contained (in mM) 118 NaCl, 25 NaHCO3, 2 CaCl2, 2.5 K2HPO4, 1.2 MgSO4, 5.5 glucose, and 4 creatinine. Tubules were transferred into a bath that was continuously exchanged and bubbled with 95% O2-5% CO2 gas and perfused by using standard techniques (18, 33). Solution and urine osmolalities were measured by vapor pressure osmometry (model 5500, Wescor, Logan, UT).

Urea measurement. The urea concentration in perfusate, bath, and collected fluid was measured by using a continuous-flow ultramicrofluorometer as described (18, 32, 33). This assay is capable of resolving differences of 4% or greater in urea concentration (32). Urea flux (Jurea) was calculated as: Jurea = CoVo - ClVl, where Co is the urea concentration in perfusate, Cl is the urea concentration in collected fluid, Vo is the perfusion rate per unit length of tubule, and Vl is the collection rate per unit length of tubule.

To study facilitated urea permeability, tubules were perfused with perfusate and bath solutions, the compositions of which were identical to the dissection solution (described above) except that 5 mM urea was added to the bath solution and 5 mM raffinose was added to the perfusate solution to create a 5 mM bath-to-lumen urea gradient without any imposed osmotic gradient (18, 33). Urea permeability was calculated from the urea flux as described (18, 33).

The urea concentration of three to four collections was measured, after which the following compounds (Sigma, St. Louis, MO) were added to the bath: 1) 10 nM arginine vasopressin (AVP; 18, 33); 2) 100 pM or 100 nM angiotensin II (39, 41); or 3) 100 nM staurosporine (SSP) (3, 13, 37); three to four additional collections then were obtained. In some experiments, the osmolality of the perfusate and bath solutions was increased from 290 to 690 mosmol/kgH2O by adding NaCl (10, 34). Twenty minutes were allowed between any solution change and the beginning of measurements.

Phosphorylation of UT-A1. Both inner medullas from a single rat (~50-75 mg) were dissected as previously described (20, 25), cut into small pieces (~5 mg/piece), washed with phosphate (P)-free DMEM, then incubated in P-free DMEM containing 0.1 mCi/ml [32P]orthophosphate for 3 h at 37°C and gassed with 5% CO2. Angiotensin II (100 nM), vasopressin (10 nM), SSP (100 nM), chelerythrine (10 µM), or vehicle was added at the end of the 3-h incubation. Unincorporated 32P was removed from the tissue by six successive washes with P-free DMEM. Tissue was then homogenized in 0.5 ml of isolation buffer (20 mM triethanolamine, 250 mM sucrose, 1 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride, pH 7.5). An equal volume of radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris, pH 7.4, 2.5 mM EDTA; 50 mM NaF; 1 mM Na4P2O7 · 10H2O; 1% Triton X-100; 10% glycerol; 1% deoxycholate, 1 µg/ml aprotinin, 0.18 mg/ml phenylmethylsulfonyl fluoride, and 0.18 mg/ml orthovanadate) was added to the homogenate, and the sample was sheared with a 26-G needle. Samples were centrifuged to remove insoluble particulates, then incubated overnight with our polyclonal anti-urea transporter (UT)-A1 (25) at 4°C with gentle mixing. Immune complexes were precipitated with protein A agarose (Pierce, Rockford, IL) for 2 h at 4°C, then the pelleted beads were washed seven times with RIPA. Washes were counted to ensure complete removal of unbound radiolabeled material. Laemmli SDS-PAGE sample buffer was added directly to the pellets, samples were boiled for 1 min, and proteins were size-separated on 10% SDS-polyacrylamide gels.

Western blot analysis was performed as previously described (20, 25). Briefly, immunoprecipitated proteins were size-separated by SDS-PAGE on 10% Laemmli gels, electroblotted to polyvinylidene difluoride membranes (Gelman Scientific, Ann Arbor, MI), and incubated for 30 min at room temperature with blocking buffer: 5% nonfat dry milk suspended in Tris-buffered saline (TBS: 20 mM Tris · HCl, 0.5 M NaCl, pH 7.5) (20, 25). The Western blots were incubated with our affinity-purified antibody "C" to rat UT-A1 (20, 25) overnight at 4°C, then washed 3× in TBS-Tween. Blots were then incubated with horseradish peroxidase-linked goat anti-rabbit IgG at a dilution of 1:5,000 (Amersham, Arlington Heights, IL) for 2 h at room temperature, washed 2× with TBS-Tween, and the bound secondary antibody was visualized by using an enhanced chemiluminescence kit (Amersham). Laser densitometry was used to quantitate the enhanced chemiluminescence signal. In all cases, parallel gels were stained with Coomassie blue and showed uniformity of loading (data not shown).

Statistics. All data are presented as means ± SE, and n = number of rats. Data from three to four collections were averaged to obtain a single value from each experimental phase in each tubule. To test for statistical significance between two groups, Student's t-test was used. To test more than two groups, an ANOVA was used, followed by Tukey's protected t-test (36) to determine which groups were significantly different. The criterion for statistical significance was P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
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Effect of angiotensin II. Adding either 100 pM or 100 nM angiotensin II to the bath had no effect on basal (no vasopressin) facilitated urea permeability (Fig. 1, solid line). In contrast, either 100 pM or 100 nM angiotensin II significantly increased vasopressin (AVP:10 nM)-stimulated facilitated urea permeability (Fig. 1, dashed line).


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Fig. 1.   Effect of ANG II and vasopressin (AVP; 10 nM) on facilitated urea permeability in the perfused rat terminal inner medullary collecting duct (IMCD). Solid line: without vasopressin, ANG II has no effect on urea permeability (n = 3, P = NS). Dashed line: with 10 nM vasopressin, ANG II increases vasopressin-stimulated urea permeability (n = 5). * P < 0.01 vs. control (vasopressin only).

Phosphorylation of UT-A1. We divided the inner medullas from two rats (4 inner medullas) into tip and base samples, incubated them with [32P]orthophosphate, and immunoprecipitated the UT-A1 proteins to test whether either the 117- or 97-kDa UT-A1 protein was a phosphoprotein. The Western blot (Fig. 2A) verified that the inner medullary tip contains both the 117- and 97-kDa glycoprotein forms of UT-A1 whereas the inner medullary base contains only the 97-kDa isoform (29, 31). Both the 117- and 97-kDa UT-A1 proteins incorporated 32P (Fig. 2B). To verify that UT-A1 was specifically being immunoprecipitated, the precipitating antibody was preadsorbed with the immunizing peptide prior to the onset of immunoprecipitation. Preadsorption of the antibody resulted in greater than a 90% decrease in immunoprecipitated UT-A1 (Fig. 2C, left and middle lanes). To confirm that the precipitated proteins were not the result of a nonspecific association with the protein A bead system, a parallel inner medullary tissue sample was incubated with an unrelated polyclonal antibody, rabbit anti-actin. The Western blot, probed with anti-UT-A1, showed no recognition of 117- or 97-kDa isoforms in this sample (Fig. 2C, right lane).


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Fig. 2.   Phosphorylation of urea transporter (UT)-A1 inner medullary tip and base. A: Western blot of rat inner medulla tip and base probed with anti-UT-A1 showing both the 97- and 117-UT-A1 proteins in the inner medullary tip and only the 97-kDa protein in the base. The tissue samples were incubated with [32P]orthophosphate for 3 h, then immunoprecipitated with an antibody to the COOH terminus of UT-A1. Immunoprecipitated proteins were separated by SDS-PAGE and electroblotted to polyvinylidene difluoride (PVDF) membranes that were probed with anti-UT-A1. B: autoradiogram of an identical gel showing that both the 117- and the 97-kDa proteins are phosphorylated, regardless of inner medullary tissue location. C: Western blot of rat inner medulla immunoprecipitated with (from left to right): anti-UT-A1 [control (Ctl)], anti-UT-A1 preadsorbed with immunizing peptide (Preads), and anti-actin (alpha -actin). Preadsorption of anti-UT-A1 reduced the amount of immunoprecipitated UT-A1 proteins by 90%. No UT-A1 protein was detected when alpha -actin was used as the immunoprecipitating antibody.

Rather than pool tissue from two rats, subsequent experiments used inner medullas that were not divided into base and tip. We incubated inner medullary tissue with [32P]orthophosphate and angiotensin II (100 nM). Figure 3A shows that a similar amount of protein was precipitated from each inner medullary sample. Treatment with angiotensin resulted in an increase in 32P incorporation into both the 117- and 97-kDa UT-A1 proteins (Fig. 3B). Next, we incubated the inner medullary tip with [32P]orthophosphate and angiotensin II (100 nM) and vasopressin (10 nM), which resulted in a marked increase in 32P incorporation into both the 117- and 97-kDa UT-A1 proteins (Fig. 3C).


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Fig. 3.   Phosphorylation of UT-A1 is increased by ANG II. A: Western blot of rat inner medulla probed with anti-UT-A1 showing equal amounts of the 97- and 117-UT-A1 proteins (arrows) in both lanes. The tissue samples were incubated with [32P]orthophosphate for 3 h, then treated with ANG II (100 nM) for 0 (Ctl) or 15 min (ANG II). Immunoprecipitated proteins were separated by SDS-PAGE and electroblotted to PVDF membranes that were probed with anti-UT-A1. B: autoradiogram of an identical gel showing that ANG II increases 32P-incorporation into both the 97- and 117-kDa UT-A1 proteins. The blot is representative of 3 similar, independent results. C: autoradiogram of 32P-labeled immunoprecipitates of inner medullary tissue treated with (from left to right): no additions (Ctl), 100 nM ANG II for 30 min, and 30 min with 100 nM ANG II and 10 nM vasopressin [ANG II + arginine vasopressin (AVP)]. ANG II increased the 32P incorporation into both the 97- and 117-kDa UT-A1 proteins (arrows) whereas incubation with ANG II and vasopressin increased 32P incorporation further. Each gel is representative of 3 similar, independent results. Accompanying Western blots confirmed that a comparable amount of UT-A1 was present in each of the precipitated samples (data not shown).

Effect of SSP. We used the PKC inhibitor SSP to test whether angiotensin II increases vasopressin-stimulated urea permeability through a PKC-mediated pathway. SSP (100 nM) had no effect on vasopressin-stimulated facilitated urea permeability but blocked the increase in vasopressin (AVP)-stimulated facilitated urea permeability due to angiotensin II [AVP: 57 ± 14, AVP + SSP: 66 ± 12, AVP + SSP + angiotensin II (100 pM): 82 ± 11 × 10-5 cm/s, P = NS, n = 6-10, Fig. 4]. Removing SSP restored the angiotensin II-induced increase in facilitated urea permeability (AVP + angiotensin II: 183 ± 26 × 10-5 cm/s, P < 0.01 vs. AVP, AVP + SSP, and AVP + SSP + angiotensin II, n = 4, Fig. 4).


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Fig. 4.   Effect of staurosporine (SSP: 100 nM) and ANG II (100 pM) on vasopressin (AVP: 10 nM)-stimulated urea permeability in the perfused rat terminal IMCD. SSP has no effect on AVP-stimulated urea permeability [n = 6, P = not significant (NS)] but blocks the ANG II-induced increase in AVP-stimulated urea permeability (n = 10, P = NS vs. AVP and AVP + SSP). After SSP is washed out, the ANG II-induced increase in AVP-stimulated urea permeability is restored (n = 4). * = P < 0.01 vs. AVP, AVP + SSP, and AVP + SSP + ANG II.

Incubating inner medullary tissue with [32P]orthophosphate and 100 nM SSP resulted in a 30% reduction in the basal phosphorylation of the UT-A1 protein (data not shown). SSP also blocked the angiotensin II-stimulated increase in UT-A1 phosphorylation (Fig. 5A). SSP is known to be somewhat nonspecific, so to confirm the participation of PKC in the phosphorylation of UT-A1, we incubated inner medullary tissue with angiotensin II and 10 µM chelerythrine chloride (Fig. 5B). Chelerythrine is a more specific inhibitor of PKC with an inhibition constant value of 0.66 µM (4, 14); the constant value for inhibition of the nearest alternate substrate [protein kinase A (PKA)] is 170 µM. As with SSP, chelerythrine caused a decrease in basal phosphorylation and a blockade of the angiotensin II-stimulated increase in UT-A1 phosphorylation. In each experiment, parallel Western blots confirmed that the amount of immunoprecipitated UT-A1 was comparable (data not shown).


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Fig. 5.   Inhibition of UT-A1 phosphorylation by protein kinase C (PKC) inhibitors. A: autoradiogram of 32P-labeled immunoprecipitates of inner medullary tissue (2 inner medullae/sample lane) treated with (from left to right): no additions (Ctl), 100 nM ANG II for 15 min, and 15 min with 100 nM SSP followed by 15 min with ANG II (ANG II + SSP). ANG II increased the 32P incorporation into both the 97- and 117-kDa UT-A1 proteins (arrows) whereas SSP reduced both basal and ANG II-stimulated 32P-incorporation. B: autoradiogram of 32P-labeled immunoprecipitates of inner medullary tissue treated with (from left to right): no additions (Ctl), 100 nM ANG II for 15 min, and 15 min with 10 µM chelerythrine (Chel) chloride followed by 15 min with ANG II (ANG II + Chel). ANG II increased the 32P incorporation into both the 97- and 117-kDa UT-A1 proteins (arrows) whereas Chel reduced both basal and ANG II-stimulated 32P incorporation. Each gel is representative of 3 similar, independent results. Accompanying Western blots confirmed that a comparable amount of UT-A1 was present in each of the precipitated samples (data not shown). Note: the uppermost band in the middle lane of B was not a consistent finding, and its identity is unknown.

Hyperosmolality-stimulated facilitated urea transport. Hyperosmolality increases facilitated urea permeability independently of vasopressin in the rat terminal IMCD (10, 34) via an increase in intracellular calcium (11). Because signaling pathways that change intracellular calcium often involve PKC, we used SSP to determine whether PKC may be involved in mediating hyperosmolality's stimulation of facilitated urea transport.

In the presence of vasopressin and SSP, raising perfusate and bath osmolality from 290 to 690 mosmol/kgH2O by adding NaCl significantly increased facilitated urea permeability (290 mosmol/kgH2O: 70 ± 15, 690 mosmol/kgH2O: 109 ± 31 × 10-5 cm/s, P < 0.01, n = 4, Fig. 6). However, after SSP was washed out, facilitated urea permeability increased further (135 ± 32 × 10-5 cm/s, P < 0.01 vs. AVP + SSP at 290 or 690 mosmol/kgH2O, n = 4, Fig. 6). Thus SSP partially blocked hyperosmolality's stimulation of facilitated urea permeability.


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Fig. 6.   Effect of SSP (100 nM) on hyperosmolality-stimulated urea permeability in the perfused rat terminal IMCD. In the presence of vasopressin (AVP: 10 nM) and SSP, raising perfusate and bath osmolality from 290 to 690 mosmol/kgH2O by adding NaCl significantly increased urea permeability (n = 4). After SSP is washed out, AVP + hyperosmolality-stimulated urea permeability increased further (n = 4). * P < 0.01 vs. AVP + SSP at 290 mosmol/kgH2O, + = P < 0.01 vs. AVP + SSP at 690 mosmol/kgH2O.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The main results in this study are that 1) both the 117- and 97-kDa UT-A1 proteins are phosphoproteins; and 2) angiotensin II increases both vasopressin-stimulated facilitated urea permeability in the rat terminal IMCD and the amount of [32P]orthophosphate incorporated into the 117- and 97-kDa UT-A1 proteins. These results show that angiotensin II does affect tubular transport in the terminal IMCD, presumably by activating angiotensin II receptors present in the IMCD (16, 23, 24, 38) and by increasing the phosphorylation of UT-A1. The angiotensin II-mediated increase in facilitated urea permeability was blocked by SSP, suggesting that angiotensin II increases the maximal response to vasopressin by activating PKC.

Effect of angiotensin II on facilitated urea permeability. In rats fed a standard diet, vasopressin-stimulated facilitated urea transport is present only in the terminal IMCD (33). It plays an important role in the urinary concentrating mechanism by permitting urea to be rapidly transported down its concentration gradient, via the UT-A1 urea transporter protein, to increase urea delivery to the inner medullary interstitium. In the present study, we measured facilitated urea transport by imposing a bath-to-lumen urea gradient across the terminal IMCD. Previous studies showed that perfusing a rat terminal IMCD with a bath-to-lumen or with a lumen-to-bath urea gradient yielded the same value for urea permeability (32). However, we did not measure active (or net) urea transport, i.e., transport of urea in the absence of an imposed urea concentration gradient, in the present study.

When an animal becomes volume depleted, both the renin-angiotensin system and vasopressin release are stimulated in an effort to conserve salt and water and restore blood pressure. Thus the angiotensin II-induced increase in vasopressin-stimulated facilitated urea permeability (present study) could augment urea reabsorption into the deepest portions of the inner medullary interstitium where it is needed to maintain the osmotic gradient and aid in water reabsorption.

Role of PKC. Several studies show that angiotensin II modulates tubular transport processes by activating PKC (1, 22, 40). Even in the absence of angiotensin II, PKC inhibits vasopressin-stimulated osmotic water permeability in rabbit cortical collecting ducts (CCDs) (2, 3), whereas inhibiting PKC with SSP results in an increase in both basal- and vasopressin-stimulated osmotic water permeability in the rat IMCD (28).

Although SSP is not completely specific (like most inhibitors), it did not alter vasopressin-stimulated facilitated urea permeability, suggesting that the concentration of SSP we used in this study had no effect on the PKA pathway. Breyer and colleagues (3, 13, 37) used 100 nM SSP to inhibit PKC effects on vasopressin-stimulated osmotic water permeability in the perfused rabbit CCD. The results of the present study suggest that angiotensin II increases vasopressin-stimulated facilitated urea permeability via a PKC-mediated pathway.

In Chinese hamster ovary cells, angiotensin II can potentiate vasopressin-dependent cAMP production through the activation of PKC (21). Thus it is possible that the additive effect of angiotensin II on vasopressin-stimulated facilitated urea permeability could be due to an effect of angiotensin II on intracellular cAMP production through activation of PKC. The present studies do not determine whether PKC is acting directly or indirectly through changes in intracellular cAMP.

UT-A1 contains consensus phosphorylation sites for both PKC and PKA (35). Thus it is possible that either protein kinase could phosphorylate UT-A1 and increase its activity. The fact that SSP and chelerythrine block the phosphorylation of UT-A1 by angiotensin II suggest that angiotensin II is somehow stimulating the activity of PKC. However, the fact that these PKC inhibitors reduce, but do not eliminate, the basal phosphorylation of UT-A1 suggests that another protein kinase (perhaps PKA) may also be involved in regulating the activity of UT-A1. Future studies will be needed to test this hypothesis.

The rat IMCD also expresses a dual angiotensin II/vasopressin receptor that is coupled to adenylyl cyclase (12). This receptor can be stimulated by either angiotensin II or vasopressin (12). Because we observed no effect of angiotensin II on facilitated urea permeability in the absence of vasopressin, it is unlikely that angiotensin II is working through this dual angiotensin II/vasopressin receptor.

Role of PKC in hyperosmolality-stimulated facilitated urea permeability. We previously found that hyperosmolality acutely stimulates facilitated urea permeability in the rat terminal IMCD via increases in intracellular calcium but has no effect on cyclic AMP production (11). Increasing osmolality by adding NaCl (or mannitol) increases facilitated urea permeability, even in the absence of vasopressin (34). In addition, hyperosmolality and vasopressin have additive stimulatory effects on facilitated urea permeability, and this stimulation is stable over the time period needed to perform the present studies (10, 34). Thus hyperosmolality is an independent stimulator of facilitated urea transport.

The results of the present study suggest that the effect of hyperosmolality on facilitated urea transport may be mediated, in part, through activation of PKC. Hyperosmolality also activates the mitogen-activated protein kinase cascade in mouse IMCD cells and rat inner medulla (42). Future studies will be needed to confirm and elucidate the role of PKC and other pathways in hyperosmolality's stimulation of urea transport.


    ACKNOWLEDGEMENTS

The authors thank Drs. Douglas C. Eaton and William E. Mitch (Emory University) for their critical reading of this manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-41707 and P01-DK-50268.

Portions of this work have been published in abstract form (J Am Soc Nephrol 9: 20A, 1998, Proceedings of the XVth International Congress of Nephrology, Buenos Aires, Argentina, May, 1999, and J Am Soc Nephrol. In press.) and presented at the 31st Annual Meeting of the American Society of Nephrology, October 25-28, 1998, Philadelphia, PA, and the XVth International Congress of Nephrology, May 2-6, 1999, Buenos Aires, Argentina.

Present address of A. Kato: First Department of Medicine, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-3192, Japan.

Address for reprint requests and other correspondence: J. M. Sands, Emory Univ. School of Medicine, Renal Div., WMRB 338, 1639 Pierce Dr., NE, Atlanta, GA 30322 (E-mail: jsands{at}emory.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3 March 2000; accepted in final form 15 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Amlal, H, Legoff C, Vernimmen C, Soleimani M, Paillard M, and Bichara M. ANG II controls Na+-K+(NH+4)-2Cl- cotransport via 20-HETE and PKC in medullary thick ascending limb. Am J Physiol Cell Physiol 274: C1047-C1056, 1998[Abstract/Free Full Text].

2.   Ando, Y, Jacobson HR, and Breyer MD. Phorbol myristate acetate, dioctanoylglycerol, and phosphatidic acid inhibit the hydroosmotic effect of vasopressin on rabbit cortical collecting tubule. J Clin Invest 80: 590-593, 1987[ISI][Medline].

3.   Ando, Y, Jacobson HR, and Breyer MD. Phosphatidates inhibit vasopressin-induced water transport via protein kinase C activation. Am J Physiol Renal Fluid Electrolyte Physiol 257: F524-F530, 1989[Abstract/Free Full Text].

4.   Barg, J, Belcheva MM, and Coscia CJ. Evidence for the implication of phosphoinositol signal transduction in µ-opioid inhibition of DNA synthesis. J Neurochem 59: 1145-1152, 1992[ISI][Medline].

5.   Clapp, WL, Madsen KM, Verlander JW, and Tisher CC. Intercalated cells of the rat inner medullary collecting duct. Kidney Int 31: 1080-1087, 1987[ISI][Medline].

6.   Clapp, WL, Madsen KM, Verlander JW, and Tisher CC. Morphologic heterogeneity along the rat inner medullary collecting duct. Lab Invest 60: 219-230, 1989[ISI][Medline].

7.   Esther, CR, Jr, Howard TE, Marino EM, Goddard JM, Capecchi MR, and Bernstein KE. Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility. Lab Invest 74: 953-965, 1996[ISI][Medline].

8.   Esther, CR, Jr, Marrero MB, Howard TE, Machaud A, Corvol P, Capecchi MR, and Bernstein KE. The critical role of tissue angiotensin-converting enzyme as revealed by gene targeting in mice. J Clin Invest 99: 2375-2385, 1997[Abstract/Free Full Text].

9.   Faubert, PF, Chou SY, Porush JG, and Byrd R. Regulation of papillary plasma flow by angiotensin II. Kidney Int 32: 472-478, 1987[ISI][Medline].

10.   Gillin, AG, and Sands JM. Characteristics of osmolarity-stimulated urea transport in the rat IMCD. Am J Physiol Renal Fluid Electrolyte Physiol 262: F1061-F1067, 1992[Abstract/Free Full Text].

11.   Gillin, AG, Star RA, and Sands JM. Osmolarity-stimulated urea transport in rat terminal IMCD: role of intracellular calcium. Am J Physiol Renal Fluid Electrolyte Physiol 265: F272-F277, 1993[Abstract/Free Full Text].

12.   Gonzalez, CB, Herrera VLM, and Ruiz WG. Renal immunocytochemical distribution and pharmacological properties of the dual angiotensin II/AVP receptor. Hypertension 29: 957-961, 1997[Abstract/Free Full Text].

13.   Hebert, RL, Jacobson HR, and Breyer MD. PGE2 inhibits AVP-induced water flow in cortical collecting ducts by protein kinase C activation. Am J Physiol Renal Fluid Electrolyte Physiol 259: F318-F325, 1990[Abstract/Free Full Text].

14.   Herbert, JM, Augereau JM, Gleye J, and Maffrand JP. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 172: 993-999, 1990[ISI][Medline].

15.   Ito, M, Oliverio MI, Mannon PJ, Best CF, Maeda N, Smithies O, and Coffman TM. Regulation of blood pressure by the type 1A angiotensin II receptor gene. Proc Natl Acad Sci USA 92: 3521-3525, 1995[Abstract].

16.   Kakinuma, Y, Fogo A, Inagami T, and Ichikawa I. Intrarenal localization of angiotensin II type 1 receptor mRNA in the rat. Kidney Int 43: 1229-1235, 1993[ISI][Medline].

17.   Karim, Z, Defontaine N, Paillard M, and Poggioli J. Protein kinase C isoforms in rat kidney proximal tubule: acute effect of angiotensin II. Am J Physiol Cell Physiol 269: C134-C141, 1995[Abstract/Free Full Text].

18.   Kato, A, Naruse M, Knepper MA, and Sands JM. Long-term regulation of inner medullary collecting duct urea transport in rat. J Am Soc Nephrol 9: 737-745, 1998[Abstract].

19.   Kihara, M, Umemura S, Sumida Y, Yokoyama N, Yabana M, Nyui N, Tamura K, Murakami K, Fukamizu A, and Ishii M. Genetic deficiency of angiotensinogen produces an impaired urine concentrating ability in mice. Kidney Int 53: 548-555, 1998[ISI][Medline].

20.   Klein, JD, Price SR, Bailey JL, Jacobs JD, and Sands JM. Glucocorticoids mediate a decrease in the AVP-regulated urea transporter in diabetic rat inner medulla. Am J Physiol Renal Physiol 273: F949-F953, 1997[ISI][Medline].

21.   Klingler, C, Ancellin N, Barrault MB, Buhler JM, Elalouf JM, Clauser E, Lugnier C, and Corman B. Angiotensin II potentiates vasopressin-dependent cAMP accumulation in CHO transfected cells. Mechanisms of cross-talk between AT1A and V2 receptors. Cell Signal 10: 65-74, 1998[ISI][Medline].

22.   Liu, FY, and Cogan MG. Role of protein kinase C in proximal bicarbonate reabsorption and angiotensin signaling. Am J Physiol Renal Fluid Electrolyte Physiol 258: F927-F933, 1990[Abstract/Free Full Text].

23.   Miyata, N, Park F, Li XF, and Cowley AW, Jr. Distribution of angiotensin AT1 and AT2 receptor subtypes in the rat kidney. Am J Physiol Renal Physiol 277: F437-F446, 1999[Abstract/Free Full Text].

24.   Mujais, SK, Kauffman S, and Katz AI. Angiotensin II binding sites in individual segments of the rat nephron. J Clin Invest 77: 315-318, 1986[ISI][Medline].

25.   Naruse, M, Klein JD, Ashkar ZM, Jacobs JD, and Sands JM. Glucocorticoids downregulate the rat vasopressin-regulated urea transporter in rat terminal inner medullary collecting ducts. J Am Soc Nephrol 8: 517-523, 1997[Abstract].

26.   Okubo, S, Niimura F, Matsusaka T, Fogo A, Hogan BLM, and Ichikawa I. Angiotensinogen gene null-mutant mice lack homeostatic regulation of glomerular filtration and tubular reabsorption. Kidney Int 53: 617-625, 1998[ISI][Medline].

27.   Oliverio, MI, Delnomdedieu M, Best CF, Li P, Morris M, Callahan MF, Johnson A, Smithies O, and Coffman TM. Abnormal water metabolism in mice lacking the type 1A receptor for ANG II. Am J Physiol Renal Physiol 278: F75-F82, 2000[Abstract/Free Full Text].

28.   Rouch, AJ, and Kudo LH. Indomethacin and staurosporine reverse alpha 2 inhibition of water transport in rat IMCD. Kidney Int 52: 1351-1358, 1997[ISI][Medline].

29.   Rouillard, P, Klein JD, Timmer RT, and Sands JM. Glycosylated forms of UT-A urea transporters present in kidney medulla (Abstract). J Am Soc Nephrol 9: 25A, 1998.

30.   Sands, JM. Regulation of renal urea transporters. J Am Soc Nephrol 10: 635-646, 1999[Abstract/Free Full Text].

31.   Sands, JM, Flores FX, Kato A, Baum MA, Brown EM, Ward DT, Hebert SC, and Harris HW. Vasopressin-elicited water and urea permeabilities are altered in the inner medullary collecting duct in hypercalcemic rats. Am J Physiol Renal Physiol 274: F978-F985, 1998[Abstract/Free Full Text].

32.   Sands, JM, and Knepper MA. Urea permeability of mammalian inner medullary collecting duct system and papillary surface epithelium. J Clin Invest 79: 138-147, 1987[ISI][Medline].

33.   Sands, JM, Nonoguchi H, and Knepper MA. Vasopressin effects on urea and H2O transport in inner medullary collecting duct subsegments. Am J Physiol Renal Fluid Electrolyte Physiol 253: F823-F832, 1987[Abstract/Free Full Text].

34.   Sands, JM, and Schrader DC. An independent effect of osmolality on urea transport in rat terminal IMCDs. J Clin Invest 88: 137-142, 1991[ISI][Medline].

35.   Sands, JM, Timmer RT, and Gunn RB. Urea transporters in kidney and erythrocytes. Am J Physiol Renal Physiol 273: F321-F339, 1997[Abstract/Free Full Text].

36.   Snedecor, GW, and Cochran WG. Statistical Methods. Ames, IA: Iowa State Univ. Press, 1980, p. 217-236.

37.   Snyder, HM, Fredin DM, and Breyer MD. Muscarinic receptor activation inhibits AVP-induced water flow in rabbit cortical collecting ducts. Am J Physiol Renal Fluid Electrolyte Physiol 260: F929-F936, 1991[Abstract/Free Full Text].

38.   Terada, Y, Tomita K, Nonoguchi H, and Marumo F. PCR localization of angiotensin II receptor and angiotensin mRNAs in rat kidney. Kidney Int 43: 1251-1259, 1993[ISI][Medline].

39.   Tojo, A, Tisher CC, and Madsen KM. Angiotensin II regulates H+-ATPase activity in rat cortical collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 267: F1045-F1051, 1994[Abstract/Free Full Text].

40.   Wang, T, and Giebisch G. Effects of angiotensin II on electrolyte transport in the early and late distal tubule in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 271: F143-F149, 1996[Abstract/Free Full Text].

41.   Weiner, ID, New AR, Milton AE, and Tisher CC. Regulation of luminal alkalinization and acidification in the cortical collecting duct by angiotensin II. Am J Physiol Renal Fluid Electrolyte Physiol 269: F730-F738, 1995[Abstract/Free Full Text].

42.   Wojtaszek, PA, Heasley LE, and Berl T. In vivo regulation of MAP kinases in Ratus norvegicus renal papilla by water loading and restriction. J Clin Invest 102: 1874-1881, 1998[Abstract/Free Full Text].


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