Compensatory increase in AQP2, p-AQP2, and AQP3 expression in rats with diabetes mellitus

Lene N. Nejsum1, Tae-Hwan Kwon1,2, David Marples3, Allan Flyvbjerg4, Mark A. Knepper5, Jørgen Frøkiær6, and Søren Nielsen1

1 Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C; 2 Department of Physiology, School of Medicine, Dongguk University, Kyungju 780 - 714, Korea; 3 School of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, United Kingdom; 4 Laboratory M (Diabetes and Endocrinology), Aarhus University Hospital, DK-8000 Aarhus C; 5 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892; and 6 Department of Clinical Physiology, Institute of Experimental Clinical Research, Aarhus University Hospital, DK-8200 Aarhus N, Denmark


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Diabetes mellitus (DM) is associated with osmotic diuresis and natriuresis. At day 15, rats with DM induced by streptozotocin (n = 13) had severe hyperglycemia (27.1 ± 0.4 vs. 4.7 ± 0.1 mM in controls) and had a fivefold increase in water intake (123 ± 5 vs. 25 ± 2 ml/day) and urine output. Semiquantitative immunoblotting revealed a significant increase in inner medullary AQP2 (201 ± 12% of control rats, P < 0.05) and phosphorylated (Ser256) AQP2 (p-AQP2) abundance (299 ± 32%) in DM rats. Also, the abundance of inner medullary AQP3 was markedly increased to 171 ± 19% of control levels (100 ± 4%, n = 7, P < 0.05). In contrast, the abundance of whole kidney AQP1 (90 ± 3%) and inner medullary AQP4 (121 ± 16%) was unchanged in rats with DM. Immunoelectron microscopy further revealed an increased labeling of AQP2 in the apical plasma membrane of collecting duct principal cells (with less labeling in the intracellular vesicles) of DM rats, indicating enhanced trafficking of AQP2 to the apical plasma membrane. There was a marked increase in urinary sodium excretion in DM. Only Na+/H+ exchanger NHE3 was downregulated (67 ± 10 vs. 100 ± 11%) whereas there were no significant changes in abundance of type 2 Na-phosphate cotransporter (128 ± 6 vs. 100 ± 10%); the Na-K-2Cl cotransporter (125 ± 19 vs. 100 ± 10%); the thiazide-sensitive Na-Cl cotransporter (121 ± 9 vs. 100 ± 10%); the alpha 1-subunit of the Na-K-ATPase (106 ± 7 vs. 100 ± 5%); and the proximal tubule Na-HCO3 cotransporter (98 ± 16 vs. 100 ± 7%). In conclusion, DM rats had an increased AQP2, p-AQP2, and AQP3 abundance as well as high AQP2 labeling of the apical plasma membrane, which is likely to represent a vasopressin-mediated compensatory increase in response to the severe polyuria. In contrast, there were no major changes in the abundance of AQP1, AQP4, and several major proximal and distal tubule Na+ transporters except NHE3 downregulation, which may participate in the increased sodium excretion.

aquaporins; polyuria; sodium transport; urinary concentrating mechanism


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DIABETES MELLITUS (DM) is a common endocrine disease and is characterized by metabolic abnormalities associated with long-term complications involving kidneys, blood vessels and other organs. Characteristic features observed in early phase of DM are a distinct glomerular hyperfiltration, osmotic diuresis and polyuria, and a significant elevation of plasma vasopressin levels (3, 17, 18, 39, 53), which may be due to a relative dehydration resulting from increased water loss associated with the glycosuria and polyuria (1, 18). The mechanisms leading to the altered glomerular hemodynamics have not been delineated; however, renal vasodilation or reduced vascular resistance of afferent arterioles may be a cause of diabetic glomerular hyperfiltration (17). Moreover, an increase in plasma vasopressin levels in DM is well documented (1, 18). Consistent with this, it was suggested that elevated plasma vasopressin levels may play a role in the development of glomerular hyperfiltration in an experimental DM model using Brattleboro rats (3). The alterations of renal tubular reabsorption of water and sodium in response to the increased glomerular filtration in DM is also poorly understood. DM is well known to be associated with significant natriuresis. However, several lines of evidence suggest that the reabsorption of fluid and sodium in the proximal tubule is increased in the early phase of insulin-dependent DM in humans (15), as well as in rats with streptozotocin (STZ)-induced experimental DM (2). These observations suggest that the high concentration of filtered glucose may lead to enhanced proximal tubule water and sodium reabsorption, which may be mediated by enhanced activity of the Na+-glucose cotransporter (2, 51) and/or the Na+/H+ exchanger (16). Regarding the collecting duct, little is known about the altered tubular function in DM.

The aquaporins (AQPs) are a family of membrane proteins that function as water channels. AQP1 is highly abundant in the proximal tubule and descending thin limb, and the critical role of AQP1 in urinary concentration was confirmed in transgenic knockout mice lacking AQP1. At least three aquaporins (AQP2, AQP3, and AQP4) are known to be expressed in kidney collecting duct principal cells. As described, AQP2 (14) is the apical water channel of collecting duct principal cells and is the chief target for vasopressin regulation of collecting duct water permeability (40). A series of studies have shown that altered expression and apical targeting of AQP2 play a significant role in multiple hereditary and acquired water balance disorders (36, 41). Water transport across the basolateral plasma membrane of collecting duct principal cells is thought to be mediated by AQP3 (11) and AQP4 (49). Consistent with this view, transgenic mice lacking AQP3 are markedly polyuric (32), and inner medullary collecting ducts (IMCD) from AQP4-deficient mice have a significant reduction in vasopressin-stimulated water permeability (6). High plasma vasopressin levels have been shown to increase the expression of both AQP2 and and AQP3 (8, 11, 36, 40, 41, 50). Therefore, it is possible that there may be a compensatory upregulation of collecting duct aquaporins in DM with glycosuria and increased renal water loss, caused by increased plasma vasopressin levels.

Recently, we also demonstrated that altered expression of major renal sodium transporters is associated with deranged urinary concentration and urinary sodium excretion in several experimental models showing water and sodium balance disorders (13, 28, 29). In proximal tubule, type 3 Na+/H+ exchanger (NHE3) and type II sodium-phosphate cotransporter (NaPi-2) are both expressed apically (4, 5, 21), whereas the Na-K-ATPase and the electrogenic Na-HCO3 cotransporter rkNBC1 are heavily expressed in the basolateral membrane of the renal tubule cells (20, 47) and are responsible for sodium reabsorption. The loop of Henle generates a high osmolality in renal medulla by driving the countercurrent multiplier, which is dependent on the NaCl absorption by the thick ascending limb (TAL) (26). The apically expressed Na-K-2Cl cotransporter [rat type 1 bumetanide-sensitive cotransporter (BSC-1 or NKCC2)] and NHE3, in conjunction with basolaterally expressed Na-K-ATPase, are mainly responsible for sodium reabsorption by the TAL (24). In the distal convoluted tubule, the thiazide-sensitive Na-Cl cotransporter (TSC or NCC) is involved in apical sodium reabsorption (23, 46). Therefore, DM may be associated with alterations in the expression of proximal and distal tubule sodium transporters. Potentially, reduced or maintained (i.e., lack of upregulation) expression of sodium transporters may participate in the increased urinary sodium excretion, or, alternatively, an increased expression might reflect secondary compensatory changes to conserve water and sodium in this pathological state of polyuria and natriuresis.

In the present study we examined 1) whether experimental DM is associated with changes in the abundance of kidney AQP levels; 2) whether DM is associated with changes in the subcellular localization/targeting of collecting duct AQP2; 3) whether DM is associated with changes in the abundance of major renal sodium transporters; and, finally, 4) whether the changes in the abundance of AQPs and sodium transporters are associated with alterations in urinary concentration and urinary sodium excretion in rats with DM. Thus the overall purpose was to establish the compensatory or secondary changes in the expression and targeting of renal aquaporins and in the expression of sodium transporters to compensate for DM-induced osmotic diuresis/polyuria and natriuresis.


    METHODS
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METHODS
RESULTS
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Experimental Animals

Adult female Wistar rats (M & B, Eiby, Denmark), with a mean body weight of 155 g, were studied. The animals had free access to standard rat chow (Altromin, Lage, Germany) and tap water throughout the experiment. The animals were randomized into two groups matched for body weight: saline-injected nondiabetic animals as the control group (n = 7) and STZ-injected diabetic animals as the experimental group (n = 13). DM was induced by intravenous injection of STZ (50 mg/kg body wt) in acidic 0.154 M NaCl (pH 4.5) after 12 h of food deprivation. The rats were maintained in metabolic cages for 2 wk for the measurement of water intake and urine output. Tail-vein blood glucose was determined by hemoglucotest 1-44 and Reflolux II reflectance meter (Boehringer Mannheim, Mannheim, Germany). On day 15, the animals were anaesthetized with halothane, blood was drawn from the retroorbital venous plexus, and serum was stored at -20°C for later analyses. The kidneys were rapidly removed and washed with normal saline, weighed, snap-frozen in liquid nitrogen, and stored at -80°C until they were prepared for immunoblotting analyses.

Membrane Fractionation for Immunoblotting

Whole kidneys or inner medulla were homogenized (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, and containing the protease and phosphatase inhibitors 8.5 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 100 nM okadaic acid, 1 mM sodium orthovanadate, and 25 mM sodium fluoride) by using an ultra-turrax T8 homogenizer (IKA Labortechnik, Staufen, Germany) at maximum speed for 30 s, and the homogenate was centrifuged in an Eppendorf centrifuge at 4,000 g for 15 min at 4°C to remove whole cells, nuclei, and mitochondria (27). The supernatant was then centrifuged at 200,000 g for 1 h, producing a pellet containing membrane fractions enriched for both plasma membranes and intracellular vesicles. Gel samples (Laemmli sample buffer containing 2% SDS) were made of this membrane preparation.

Electrophoresis and Immunoblotting

Samples of membrane fractions from either total kidney or inner medulla were run on 12 or 6-16% gradient polyacrylamide minigels (Bio-Rad Mini Protean II). For each gel, an identical gel was run in parallel and subjected to Coomassie staining to ensure identical loading. The other gel was subjected to immunoblotting. After transfer by electroelution to nitrocellulose membranes, blots were blocked with 5% milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated with primary antibodies (see below) overnight at 4°C. The labeling was visualized with horseradish peroxidase (HRP)-conjugated secondary antibodies (P447 or P448, diluted 1:3,000, DAKO, Glostrup, Denmark) by using the enhanced chemiluminescence system (ECL; Amersham Pharmacia Biotech, Buckinghamshire, UK).

Primary Antibodies

For semiquantitative immunoblotting and immunocytochemistry, we used previously characterized monoclonal and polyclonal antibodies as summarized below.

AQP1 (CHIP serum or LL266AP). Immune serum or an affinity-purified polyclonal antibody to AQP1 has previously been characterized (44, 50).

AQP2 (LL127 serum or LL127AP). Immune serum or an affinity-purified polyclonal antibody to AQP2 has previously been characterized (33, 40).

p-AQP2 (AN244-pp-AP). An affinity-purified rabbit polyclonal antibody to phosphorylated AQP2, which is phosphorylated in the protein kinase A phosphorylation consensus site (S256) has previously been characterized (7).

AQP3 (LL178AP). An affinity-purified polyclonal antibody to AQP3 has previously been characterized (11).

AQP4 (LL182AP). An affinity-purified polyclonal antibody to AQP4 has previously been characterized (49, 50).

NHE3 (LL546AP). An affinity-purified rabbit polyclonal antibody to NHE3 has previously been characterized (21).

NaPi-2 (LL696AP). An affinity-purified polyclonal antibody to NaPi-2, which was raised against the final 24 amino acids of the COOH-terminal sequence (4), has previously been characterized (30).

Na-K-ATPase. A monoclonal antibody against the alpha 1-subunit of Na-K-ATPase has previously been characterized (20).

rkNBC1. An affinity-purified polyclonal antibody to the basolateral rat kidney Na-HCO3 cotransporter has previously been characterized (38).

BSC-1 (LL320AP). An affinity-purified polyclonal antibody to the apical Na-K-2Cl cotransporter of the TAL has previously been characterized (12, 22, 42).

TSC (LL573AP). An affinity-purified polyclonal antibody to the apical thiazide-sensitive Na-Cl cotransporter of the distal convoluted tubule has previously been characterized (23).

Semiquantitation of Kidney Abundance of AQP and Major Na+ Transporters

ECL films with bands within the linear range were scanned by using an AGFA scanner (ARCUS II) and Corel Photopaint software to control the scanner (34). The labeling density was quantitated with blots, whereby samples from kidneys of rats with DM were run on each gel with samples from control kidneys. The labeling density was corrected by densitometry of Coomassie-stained gels.

Immunocytochemistry

The kidneys from rats with DM (n = 3) and control rats (n = 3) were fixed by retrograde perfusion via the abdominal aorta with 4% paraformaldehyde with 0.1% glutaraldehyde in 0.1 M cacodylate buffer (31, 52). Kidneys were postfixed for 1 h, and tissue blocks were infiltrated for 30 min with 2.3 M sucrose containing 2% paraformaldehyde, mounted on holders, and rapidly frozen in liquid nitrogen. For light microscopy, the frozen tissue blocks were cryosectioned (0.8-1 µm, Reichert Ultracut S Cryoultramicrotome, Reichert, Vienna, Austria), and sections were incubated with anti-AQP2. For immunoperoxidase microscopy, labeling was visualized with HRP-conjugated secondary antibody (P448 1:100, DAKO, Glostrup, Denmark), followed by incubation with diaminobenzidine. For immunofluorescence microscopy, labeling was visualized by using goat anti-rabbit IgG (Z0421, 1:50, DAKO) and FITC-conjugated rabbit anti-goat antibody (F250, 1:50, DAKO).

Immunoelectron Microscopy

The frozen samples were freeze-substituted in a Reichert Auto Freeze-Substitution Unit (Reichert) (37, 43, 52). Briefly, the samples were sequentially equilibrated over 3 days in methanol containing 0.5% uranyl acetate at temperatures that were gradually increased from -80 to -70°C and then rinsed in pure methanol for 24 h while the temperature was increased from -70 to -45°C. At -45°C, the samples were infiltrated with Lowicryl HM20 and methanol 1:1, 2:1, and, finally, pure Lowicryl HM20 before ultraviolet polymerization for 2 days at -45°C and 2 days at 0°C. For electron microscopy, immunolabeling was performed on ultrathin Lowicryl HM20 sections (60-80 nm), which were incubated overnight at 4°C with anti-AQP2 antibodies. The labeling was visualized with goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles (GAR.EM10, BioCell Research Laboratories, Cardiff, UK) diluted 1:50. The sections were stained with uranyl acetate and lead citrate before examination in Philips CM100 or Philips 208 electron microscopes.

Statistical Analyses

Values are presented as means ± SE. Comparisons between groups were made by unpaired t-test. P values < 0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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DM Was Associated With Altered Renal Water and Sodium Handling

DM induced by STZ treatment was characteristically associated with high blood glucose levels as well as elevated glomerular filtration rate (GFR) measured by creatinine clearance (P < 0.05, Table 1). This is consistent with findings in the early phase of DM. Diabetic rats showed a significant increase in urine output compared with control rats: 368 ± 30 in diabetic rats vs. 56 ± 4 µl · min-1 · kg body wt-1 in control rats at day 15 after STZ injection (P < 0.05, Table 1). In parallel, the increase in urine output in diabetic rats was accompanied by a significant increase in water intake (457 ± 20 vs. 101 ± 7 µl · min-1 · kg body wt-1 in control rats, P < 0.05). Consistent with this, diabetic rats had reduced urine osmolality and urine-to-serum osmolality ratio values compared with control rats, suggesting that DM is associated with a reduction in urinary concentration (Table 1). However, solute-free water reabsorption (TcH2O)1 was markedly increased in diabetic rats (Table 1), probably due to elevated osmolar excretion (Cosm: 1,231 ± 68 vs. 246 ± 6 µl · min-1 · kg body wt-1, P < 0.05, Table 1), consistent with previous observations in Long-Evans rats (3).

                              
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Table 1.   Functional changes in diabetic rats

Diabetic rats also had a significant increase in urinary sodium excretion (UNaV): 18.3 ± 2.3 in diabetic rats vs. 9.8 ± 0.4 mmol · day-1 · kg body wt-1 in control rats at day 15 (P < 0.05, Table 1). Moreover, fractional excretion of sodium (FENa) was significantly increased in diabetic rats: 1.23 ± 0.07 vs. 0.89 ± 0.04% in control rats at day 15 (P < 0.05, Table 1). This suggests that DM is associated with significant natriuresis.

Changes in AQP2, AQP3, But Not AQP1 and AQP4, in rats With DM

To test whether DM is associated with changes of the abundance of the collecting duct water channels AQP2, AQP3, and AQP4 and the proximal nephron water channel AQP1, semiquantitative immunoblotting of membrane fractions from the kidney inner medulla (AQP2, AQP3, and AQP4) or from whole kidney (AQP1) was performed. DM was associated with a significant increase in the inner medullary abundance of AQP2 to 201 ± 12% of control levels (100 ± 15%, P < 0.05, Fig. 1, Table 2). In parallel, inner medullary abundance of the basolateral collecting duct water channel AQP3 was also increased to 171 ± 19% of control levels (100 ± 4%, P < 0.05, Fig. 2, Table 2).


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Fig. 1.   Semiquantitative immunoblotting of membrane fractions of kidney inner medulla. A: immunoblots were reacted with anti-aquaporin-2 (AQP2) and revealed 29- and 35- to 50-kDa AQP2 bands, representing nonglycosylated and glycosylated forms of AQP2, respectively. CON, control; DM, diabetes mellitus. B: densitometric analyses revealed a marked increase in inner medullary AQP2 protein abundance in diabetic rats compared with control rats. *P < 0.05.


                              
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Table 2.   Changes of abundance of renal AQPs and major sodium transporters



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Fig. 2.   Semiquantitative immunoblotting of membrane fractions of kidney inner medulla. A: immunoblots were reacted with anti-AQP3 and revealed 27- and 33- to 40-kDa AQP3 bands, representing nonglycosylated and glycosylated forms of AQP3, respectively. B: densitometric analyses revealed a marked increase in inner medullary AQP3 protein abundance in diabetic rats, compared with control rats. *P < 0.05.

Moreover, semiquantitative immunoblotting with antibodies that selectively recognize AQP2 (p-AQP2), which is phosphorylated in the protein kinase A phosphorylation consensus site (Ser256) (7), demonstrated that phosphorylated AQP2 was also dramatically increased in diabetic rats (Fig. 3). As shown in Fig. 3, densitometric analysis of immunoblots revealed an increase in inner medullary p-AQP2 abundance to 299 ± 32%, compared with control rats (100 ± 20%, P < 0.05). This is consistent with the increased targeting of AQP2 to the apical membrane domains of the collecting duct principal cells in diabetic rats as demonstrated by immunoelectron microscopy (see below). Therefore, this suggests that the inner medullary abundance of AQP2 and AQP3 as well as targeting of AQP2 to the apical plasma membrane of the collecting duct principal cells were markedly increased, probably to compensate for the increased renal water loss due to glycosuria. The increase in abundance and targeting is likely to be caused by the increased plasma vasopressin levels demonstrated in previous studies (1, 3, 18).


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Fig. 3.   Semiquantitative immunoblotting of membrane fractions of kidney inner medulla. A: immunoblots were reacted with anti-AQP2 antibodies that selectively recognize AQP2 (p-AQP2), which is phosphorylated in the protein kinase A (PKA) phosphorylation consensus site (Ser256). This revealed 29- and 35- to 50-kDa AQP2 bands, representing nonglycosylated and glycosylated forms of AQP2, respectively. B: densitometric analyses revealed a marked increase in inner medullary p-AQP2 abundance in diabetic rats, compared with control rats. *P < 0.05.

In contrast, in diabetic rats whole kidney abundance of the proximal nephron water channel AQP1 and of the IMCD water channel AQP4 was not altered but was maintained at control levels (Figs. 4 and 5). At day 15 after STZ injection, the abundance of the whole kidney AQP1 was maintained at 90 ± 3% of control levels [100 ± 4%, not significant (NS), Fig. 4], and the inner medullary abundance of AQP4 was 121 ± 16% of control levels (100 ± 7%, NS, Fig. 5). Table 2 shows the summarized data of changes in the abundance of AQPs and sodium transporters.


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Fig. 4.   Semiquantitative immunoblotting of membrane fractions of whole kidneys of rats with DM and control rats. A: immunoblot was reacted with anti-AQP1 and revealed 29-kDa band (35- to 50-kDa AQP1 band; not shown). B: densitometric analysis revealed that whole kidney AQP1 abundance was not changed in rats with DM.



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Fig. 5.   Semiquantitative immunoblotting of membrane fractions of kidney inner medulla. A: immunoblots were reacted with anti-AQP4 and revealed an ~32- to 34-kDa band as well as a higher molecular weight band representing oligomeric AQP4. B: densitometric analyses revealed that inner medullary AQP4 protein abundance was not changed in rats with DM.

Abundance of Major Renal Sodium Transporters in Kidneys of Rats with DM

Semiquantitative immunoblotting, using membrane fractions prepared from the whole kidney of rats with DM and control rats, revealed that DM was not associated with altered abundance of major renal proximal and distal tubule sodium transporters except NHE3 (Table 2). In the proximal tubule, NHE3 is mainly involved in sodium reabsorption and hydrogen ion secretion, and rkNBC1 is mainly responsible for bicarbonate reabsorption. Moreover, transport of Pi through the proximal apical membrane is largely performed by NaPi-2.

In DM rats, whole kidney abundance of NHE3 was reduced (67 ± 10 vs. 100 ± 11%, P < 0.05), whereas rkNBC1 (98 ± 16 vs. 100 ± 7%, NS, Table 2, Fig. 6) and NaPi-2 (128 ± 6 vs. 100 ± 10%, NS, Fig. 7) were not significantly altered compared with controls (Table 2). Moreover, in rats with DM, whole kidney Na-K-ATPase levels were not significantly different from the levels in control rats (106 ± 7 vs. 100 ± 5%, NS, Table 2).


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Fig. 6.   Semiquantitative immunoblotting of membrane fractions of whole kidneys of rats with DM and control rats. A: immunoblot was reacted with anti-electrogenic Na-HCO3 cotransporter (rkNBC1) and revealed an ~140-kDa band. B: densitometric analysis revealed that whole kidney rkNBC1 protein abundance was not changed in rats with DM.



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Fig. 7.   Semiquantitative immunoblotting of membrane fractions of whole kidneys of rats with DM and control rats. A: immunoblot was reacted with anti-type II Na-Pi cotransporter (NaPi-2) and revealed an ~85-kDa band. B: densitometric analysis revealed that whole kidney NaPi-2 protein abundance was not changed in rats with DM.

Next, we examined the whole kidney abundance of BSC-1 in the thick ascending limb and TSC in the distal convoluted tubule in DM and control rats. The whole kidney abundance of BSC-1 (125 ± 19 vs. 100 ± 10%, NS) and TSC (121 ± 9 vs. 100 ± 10%, NS) were not significantly changed in rats with DM (Table 2). This suggested that there are apparently no compensatory increases in most sodium transporter expression to compensate for the renal sodium loss.

Immunocytochemistry Revealed Significant Trafficking of AQP2 to the Apical Plasma Membrane of IMCD Principal Cells in Rats With DM

Immunoperoxidase (not shown) and immunoflourescence (Fig. 8) labeling of AQP2 using semithin sections of the IMCD revealed strong AQP2 labeling in IMCD principal cells of diabetic kidney compared with control rats (Fig. 8, A and B). Moreover, AQP2 labeling in diabetic kidney was predominantly associated with apical plasma membrane domains of IMCD principal cells, consistent with maintained or enhanced trafficking of AQP2, in addition to the increased abundance. Immunoelectron microscopy further demonstrated that AQP2 immunogold labeling was mainly associated with apical plasma membrane domains of IMCD principal cells (Fig. 9, A and B), suggesting AQP2 trafficking from intracellular vesicles to the apical plasma membrane was maintained or enhanced in DM. This would be consistent with the increased plasma vasopressin levels in rats with experimental DM, as demonstrated previously (1, 3, 18).


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Fig. 8.   Immunofluorescent localization of AQP2 in 0.8- to 1-µm semithin cryosections of kidney inner medulla from control rat (A) and rats with DM (B). A: AQP2 labeling was associated with apical plasma membrane domains (arrows) and intracellular vesicles in the inner medullary collecting duct principal cells from control rats. B: in a kidney from rat with DM, there was a marked increase in overall AQP2 labeling, and the labeling is mainly associated with apical plasma membrane domains of inner medullary collecting duct principal cells (arrows). Magnification: ×1,000 (A and B).



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Fig. 9.   Immunoelectron microscopy of inner medullary collecting duct principal cell. A: in a control rat, AQP2 labeling was seen at both apical plasma membrane (arrows) and subapical intracellular vesicles (arrowheads). B: in a rat with DM, AQP2 labeling in the inner medullary collecting duct principal cell was mainly observed at the apical plasma membrane (arrows), whereas AQP2 labeling at the intracellular vesicles (arrowhead) was reduced. G, Golgi apparatus; N, nucleus, Magnification: ×63,000 (A and B).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have demonstrated that the abundance of kidney collecting duct water channels AQP2 and AQP3, as well as p-AQP2, was significantly increased after 2 wk of insulin-dependent DM induced by STZ administration in rats having marked polyuria and natriuresis. This suggests that there might be a vasopressin-mediated compensatory upregulation in the abundance of AQP2, p-AQP2, and AQP3 in response to the severe water loss induced by glycosuria. Immunoperoxidase, immunofluorescence microscopy, and immunoelectron microscopy further demonstrated that trafficking of AQP2 to the apical plasma membrane was markedly enhanced. In contrast, the abundance of proximal nephron water channel AQP1 and collecting duct AQP4 was not changed but was maintained at control levels. Moreover, the expression of several major renal sodium transporters (NaPi-2, Na,K-ATPase, BSC-1, and TSC) was unchanged except NHE3. Thus, in conclusion, the results suggest that there is a marked increase in collecting duct AQP2, p-AQP2, and AQP3 abundance and AQP2 targeting in response to DM with polyuria and glycosuria. This likely represents a compensatory phenomenon to minimize water loss due to the osmotic diuresis. The absence of a compensatory increase in TAL and distal tubule sodium transporter expression, which is seen in other salt-losing conditions, may participate in the increased urinary sodium excretion in DM.

Water Balance in Rats with DM

Diabetic rats induced by STZ treatment had severe polydipsia and polyuria, consistent with diabetic conditions seen in cases of patients with uncontrolled DM. Consistent with polyuria, diabetic rats had urine osmolality values and urine-to-serum osmolality ratios that were significantly lower than those seen in control rats (Table 1). In contrast, TcH2O was markedly higher in DM rats than in control rats (Table 1), consistent with previous studies in Long-Evans rats (3). Moreover, it is well established that vasopressin secretion and plasma vasopressin levels are significantly increased in DM (1, 3, 18). Hence, these findings together suggest that there might be vasopressin-mediated compensatory changes in renal tubular function, particularly of the collecting duct principal cells in response to the osmotic diuresis.

Increased Abundance of Collecting Duct Water Channels AQP2 and AQP3 in DM, But Unchanged Abundance of AQP1 and AQP4

We demonstrated that the abundance of the vasopressin-regulated collecting duct water channels AQP2, AQP3, as well as p-AQP2, was significantly increased 2 wk after induction of DM. Moreover, immunoelectron microscopy indicated that trafficking of AQP2 to the apical plasma membrane of collecting duct was enhanced, with less labeling of intracellular vesicles (Fig. 9). However, in contrast to the increased abundance of AQP2 and AQP3, semiquantitative immunoblotting revealed that whole kidney abundance of proximal nephron water channel AQP1 and the abundance of inner medullary water channel AQP4 were not altered but maintained.

The collecting duct represents the final site for the control of water excretion into the urine. Water permeability of the collecting duct is tightly regulated, under the control of vasopressin, which causes a dramatic increase in collecting duct water permeability, allowing reabsorption of water from the tubular fluid down an osmotic gradient (25, 26). AQP2 is abundant in the collecting duct principal cells and is the chief target for vasopressin to regulate collecting duct water reabsorption (40, 41). Acute regulation involves vasopressin-regulated trafficking of AQP2 between an intracellular reservoir and the apical plasma membrane. In addition, AQP2 is involved in adaptational long-term regulation of body water balance achieved through regulation of AQP2 abundance. Importantly, multiple studies have now emphasized a critical role of AQP2 in several inherited and acquired water balance disorders, including nephrogenic diabetes insipidus (36, 41). Thus the compensatory increase in AQP2 and p-AQP2 is consistent with the increase in plasma vasopressin in DM (as previously demonstrated; see Refs. 1, 3, 18). It is curious that there is no vasopressin escape associated with this condition, unlike in other conditions with maintained high levels of vasopressin {endogenous or exogenous administration of the vasopressin V2-receptor-selective agonist [desamino-Cys1,D-Arg8]vasopressin (dDAVP)}. Water-loaded rats that were continuously treated with dDAVP in osmotic minipumps exhibited a marked downregulation of AQP2 levels [i.e., representing vasopressin escape (9, 10)]. Also, a similar escape has been seen in experimental cirrhosis (19). The reason that this does not take place in DM rats is unclear but is likely to be due to the glycosuria. The results also demonstrate that in an experimental setting with enhanced diuresis there is no downregulation of AQP2 expression. This is consistent with previous data using 1-5 days of furosemide treatment, which also induced a marked polyuria. In these settings, there was no downregulation of AQP2 (33, 35).

Our data presented here also demonstrate significant upregulation of AQP3 in kidneys of diabetic rats. The mechanisms underlying regulation of AQP3 expression are presently not well understood. AQP3 is localized in the basolateral plasma membrane domains of collecting duct principal cells (11). Immunoelectron microscopy demonstrated a predominant labeling of AQP3 in the basolateral plasma membranes with little labeling of intracellular vesicles (11), suggesting that AQP3 is not regulated by vesicular trafficking (in contrast to the findings with AQP2). Immunoblotting evidence has shown that thirsting of rats for 48 h (11) or dDAVP treatment of Brattleboro rats for 5 days (50) induces a marked increase in AQP3 abundance. Moreover, after dDAVP administration or water deprivation, AQP3-null mice are able to increase their urine concentrating ability to ~30% of that in wild-type mice (32). Thus this evidence suggests that AQP3 regulation is related to changes in vasopressin and water balance. However, there are several examples in which there is a decoupling of AQP2 and AQP3 expression (10, 45, 48), indicating that more needs to be learned about the mechanisms controlling AQP3 expression. Moreover, the signaling mechanisms involved in the maintained expression of another collecting duct water channel, AQP4, are presently not understood, but the data presented here indicate that the mechanisms are distinct from those regulating AQP2 and AQP3 expression. Consistent with this, for lithium-induced severe nephrogenic diabetes insipidus, where the abundance of kidney AQP2 and AQP3 was dramatically reduced, we recently demonstrated that there was no major reduction in AQP4 labeling in the basal plasma membrane domains of IMCD principal cells as determined by immunoelectron microscopy (30).

Abundance of Proximal Tubule Sodium Transporters NaPi-2, Na-K-ATPase, and rkNBC1 Was Not Altered in DM

In proximal tubule, NHE3 and NaPi-2 are both expressed apically, whereas Na-K-ATPase and rkNBC1 are heavily expressed in the basolateral membrane of renal tubule cells, and these transporters play a major role in proximal tubule sodium reabsorption (4, 20, 21). Our data presented here demonstrate that the abundances of the major renal sodium transporters in the proximal tubule, NHE3, was reduced, indicating a potential role in increased sodium excretion. However, NaPi-2, Na-K-ATPase, and rkNBC1 were not significantly altered in polyuric conditions induced by osmotic diuresis in DM.

Previous studies demonstrated that absolute and fractional reabsorption of water and sodium up to the early distal tubule were increased in diabetic rats (2, 51). Interestingly, treatment with phlorizin, an inhibitor of Na+-glucose cotransport, elicited a significant reduction in the marked increase in absolute and fractional reabsorption of water and sodium in diabetic rats, hence resulting in a larger increase in early distal tubular concentration of sodium (51). Therefore, this may suggest that the Na+-glucose cotransporter may be stimulated by highly filtered glucose concentration in proximal tubular fluid. Thus it could be possible that this may partially contribute to the increased reabsorption of sodium (and hence fluid) in this segment, despite the absence in increased abundance of AQP1, NHE-3, NaPi-2, Na,K-ATPase, and rkNBC1. However, further studies are needed to determine the potential role of the Na+-glucose cotransporter in the reabsorption of fluid and sodium in the proximal tubule in DM.

Abundance of BSC-1 in the TAL and TSC in the Distal Convoluted Tubule Was Not Altered in DM

The loop of Henle generates a high osmolality in renal medulla by driving the countercurrent multiplier, which is dependent on the NaCl reabsorption by TAL cells. BSC-1 and NHE3, in conjunction with basolaterally expressed Na-K-ATPase, are responsible for sodium reabsorption by the TAL (24). In the distal convoluted tubule, TSC is also involved in apical sodium reabsorption (23).

In diabetic rats, we demonstrated that the abundance of BSC-1 in the TAL and of TSC in the distal convoluted tubule were not altered. This is in contrast to what has been observed in other conditions with increased urinary sodium excretion, e.g., chronic renal failure (28) and high sodium intake(12). Several factors have now been demonstrated to regulate the abundance of BSC-1 in kidney. An increase in the delivery of NaCl to the loop of Henle by chronic oral saline loading is known to upregulate BSC-1 abundance (12). Moreover, abundance of BSC-1 in the TAL is also known to be regulated by vasopressin (shown by use of dDAVP, a V2-selective agonist), and this regulation may be significantly involved in the long-term regulation of the countercurrent multiplication system (22).

The underlying causes for the unaltered abundance of BSC-1 in kidneys of rats with DM, where the plasma vasopressin levels are high (1, 3, 18), are not known. However, it could be that the delivery of water and sodium to the distal nephron may be relatively reduced (fraction of filtered load) in severe, uncontrolled DM, possibly due to the increased reabsorption of water and sodium in the proximal tubule. Hence this factor may contribute to the maintained levels of sodium transporter in the distal nephron including BSC-1 and TSC. However, further studies are needed to determine the mechanisms involved in the regulation of BSC-1, TSC expression, and sodium reabsorption in diabetic rats.

Summary

We demonstrated that the abundance of the vasopressin-regulated collecting duct water channels AQP2, AQP3, as well as p-AQP2 increased significantly 2 wk after induction of DM. Moreover, trafficking of AQP2 to the apical plasma membrane of collecting duct principal cells was markedly enhanced. This strongly suggests that there is a vasopressin-mediated compensatory increase in expression in response to the severe polyuria. In contrast, there were no changes in the abundance of AQP1, AQP4, and several major proximal and distal tubule Na+ transporters. The reduction in NHE3 and the absence of an increased abundance of the TAL and distal tubule Na+ transporters may participate in the enhanced urinary sodium excretion in DM.


    ACKNOWLEDGEMENTS

The authors thank Helle Høyer, Mette Vistisen, Zhila Nikrozi, Inger Merete Paulsen, and Gitte Christensen for expert technical assistance.


    FOOTNOTES

Support for this study was provided by the Karen Elise Jensen Foundation, the Danish Diabetes Foundation, the Eva and Henry Frænkels Memorial Foundation, the Novo Nordic Foundation, the Danish Medical Research Council, the University of Aarhus Research Foundation, the University of Aarhus, the Dongguk University, the Commission of the European Union (EU-Biotech Program and EU-TMR Program), and the intramural budget of the National Heart, Lung, and Blood Institute.

1 TcH2O = Cosm - V = V[(Uosm/Sosm- 1], where TcH2O is solute-free water clearance, Cosm is osmolal clearance, V is total daily urine ouput, Uosm, is urine osmolality, and Sosm is serum osmolality.

Address for reprint requests and other correspondence: S. Nielsen, Dept. of Cell Biology, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus C, Denmark (E-mail: sn{at}ana.au.dk).

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 24 July 2000; accepted in final form 7 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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