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1Renal Division, Department of Medicine, and 2Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322
Submitted 26 December 2002 ; accepted in final form 10 April 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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urea; water; urea transporter; aquaporin; sodium
The goal of this study was to test this hypothesis using rats made diabetic by streptozotocin (STZ) injection. The STZ-treated rat is a commonly used animal model of type I diabetes. These rats rapidly develop hyperglycemia and polyuria, although they do not develop ketoacidosis. Because the renal medulla is responsible for the production of concentrated or dilute urine, we hypothesized that any compensatory mechanism to conserve water and solute may involve changes in the abundance of the medullary transport proteins involved in the urinary concentrating mechanism. Therefore, we measured the abundance of the UT-A1 and UT-B urea transporters, the aquaporin-2 (AQP2) water channel, the NKCC2/BSC1 Na+-K+-2Cl– cotransporter, and the ROMK K+ channel from rats made diabetic for 5, 10, 14, or 20 days.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Western blot analysis. The kidney medulla was dissected into three regions: outer medulla, base of the inner medulla, and tip of the inner medulla, as previously described (9, 13, 20). Tissue from liver or the pooled tissue from both kidneys of a single rat was placed into an ice-cold isolation buffer (10 mM triethanolamine, 250 mM sucrose, pH 7.6, 1 µg/ml leupeptin, and 0.1 mg/ml PMSF), homogenized, and diluted 1:1 with 1% SDS for Western blot analysis of total cell lysate (9, 13, 20). Total protein in each sample was measured by the Bradford method (Bio-Rad, Richmond, CA). Proteins (10 µg/lane) were size separated by SDS-PAGE using 7.5, 10, or 15% polyacrylamide gels. Proteins were blotted to polyvinylidene difluoride membranes (Gelman Scientific, Ann Arbor, MI), and Western blot analysis was performed as described previously (9). Blots were quantified using an Imaging Densitometer GS670 and Molecular Analyst software (Bio-Rad Laboratories, Hercules, CA). Where multiple bands were observed resulting from multiple glycosylated forms of a single protein (UT-A1: 110–120 kDa; UT-B: 41–54 kDa; AQP2: 35–50 kDa), all bands in the group were measured together and designated the molecular mass of the major form. In all cases, parallel gels were stained with Coomassie blue to confirm uniformity of loading (data not shown). Results are expressed as arbitrary units per microgram of protein.
Deglycosylation of UT-A1 protein in inner medullary homogenates. A
sample of inner medullary (IM) homogenate (45µg) containing 0.5% SDS and 1%
-mercaptoethanol was denatured by heating to 100°C for 10 min. After
addition of NP-40 detergent to 1% and addition of 2,500 U of peptide
N-glycosidase F (PNGase F; catalog no. 704S, New England Biolabs,
Beverly, MA), the mixture was incubated at 37°C for 60 min. The reaction
was quenched by the addition of an equal volume of 2x Laemmli sample
buffer. These samples were heated again to 60°C for 15 min before SDS-PAGE
and immunoblotting.
Antibodies. Western blot analyses were probed with antibodies (diluted in TBS/Tween) to the following proteins: 1) UT-A1 and UT-A2 (13); 2) AQP2 (8, 15); 3) UT-B (22); 4) NKCC2/BSC1 (6, 7); and 5) ROMK (generous gift from Dr. M. A. Knepper, National Institutes of Health) (4).
Statistics. Data are presented as means ± SE (n), where n indicates the number of rats studied. To test for statistically significant differences between two groups, a paired Student's t-test was used. To test for statistically significant differences among three groups, an ANOVA was used followed by a multiple comparison, protected t-test (21).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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50 and 20% of the levels found in control rats, respectively, with no
further decrease to 20 days. Urinary volume, total solute excretion, and urea
excretion per 100 g body wt gradually increased with the duration of diabetes,
but the average percentage of urea in total urinary solute was constant in
both control (45%) and diabetic (18%) rats. Urinary glucose concentration and
the percentage of glucose in total urinary solute were relatively constant
during the 20 days of diabetes. Plasma vasopressin levels were measured in
5-day (control: 1.7 ± 0.2; DM: 2.2 ± 0.5 pg/ml) and 20-day
(control: 2.2 ± 0.8; DM: 3.0 ± 0.9 pg/ml) animals. The
vasopressin levels were not statistically different in diabetic animals
compared with controls at either duration of DM.
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UT-A1. The abundance of UT-A1 protein in IM tip of control and diabetic rats is shown in Fig. 1. UT-A1 exists as two distinct glycoproteins with molecular masses of 117 and 97 kDa (3). To determine whether the 117- and 97-kDa proteins that increase in the diabetic rats are the same glycoproteins as in control rats and represent glycosylated forms of the same base UT-A1 protein, kidney lysates from both control and 20-day diabetic rats were treated with PNGase F. Deglycosylation of UT-A1 protein from diabetic and control rats revealed the identical 88-kDa nonglycosylated base protein (Fig. 2). When reporting on the total UT-A1 protein abundance differences, we make the assumption that the antibody recognizes both glycoproteins equally. However, we are also providing information about shifts in the relative abundances of each of the two forms to better characterize the changes in UT-A1 that occur in the diabetic animal (reviewed in Ref. 19). The functional difference between these two glycoproteins, if any, is not known.
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Compared with control rats, the abundance of UT-A1 in 5-day diabetic rats was significantly decreased to 55% of control rats in the IM tip. The decrease of UT-A1 in the IM tip was mainly due to a decrease in the abundance of the 97-kDa UT-A1 protein (to 26% of control) rather than a change in the abundance of the 117-kDa UT-A1 protein (87% of control). In contrast, the abundance of UT-A1 was significantly increased in the IM tip of rats made diabetic for 10, 14, or 20 days: UT-A1 increased to 170% of control at 10 days; 220% of control at 14 days; and 280% of control at 20 days. The amount of the 117-kDa UT-A1 protein relative to total UT-A1 protein in diabetic rats also increased with time to 56, 66, and 70% at 10, 14, and 20 days, respectively. At each time point, six control and six diabetic rats were used and the results are representative of four experiments.
The abundance of UT-A1 protein in the IM base of control and diabetic rats is shown in Fig. 3. In contrast to the IM tip, the abundance of UT-A1 in the IM base was significantly increased to 325% of control levels by 5 days of diabetes and did not increase further at 10, 14, or 20 days (300, 390, and 340% of control, respectively). The increase in UT-A1 protein abundance in the IM base of diabetic rats was mainly due to an increase in the 117-kDa UT-A1 protein (to 2,000% of control) rather than an increase in the 97-kDa UT-A1 protein (135–190% of control). The percentage of 117-kDa UT-A1 protein to total UT-A1 protein (62, 55, 56, and 55% at 5, 10, 14, and 20 days, respectively) did not vary with the duration of diabetes in the IM base.
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UT-B. UT-B is normally expressed in the IM tip, base, and outer medulla (22). It is found exclusively in red blood cells and descending vasa recta but not in tubules (reviewed in Ref. 19). There was no significant difference in UT-B protein abundance in any of these kidney regions at 20 days of diabetes compared with control rats (Fig. 4) nor at 5 or 10 days of diabetes (data not shown).
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AQP2 in the IM tip and base. AQP2 protein abundance in the IM tip (Fig. 5A) was unchanged at 5 days, but significantly increased to 150% of control at 10 days of diabetes and remained elevated at 20 days. In the IM base (Fig. 5B), AQP2 protein abundance was significantly increased to 290% of control at 5 days of diabetes and remained elevated at 10 and 20 days. At each time point, six control and six diabetic rats were used and the results are representative of four experiments.
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NKCC2/BSC1 and ROMK abundance in the outer medulla. At 5 or 10 days of diabetes, there was no significant difference in the abundance of either NKCC2/BSC1 or ROMK protein between control and diabetic rats (data not shown). At 20 days of diabetes, NKCC2/BSC1 was significantly increased to 245% of control levels (Fig. 6). In contrast, ROMK was not significantly increased at 20 days of diabetes (Fig. 7).
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UT-A in liver. UT-A is found in several extrarenal tissues including the liver (11), the site of ureagenesis. We previously reported that the liver form of UT-A is regulated by uremia and acidosis (10, 11). The 49-kDa UT-A protein was significantly increased to 400% of control in liver from 20-day diabetic rats but not in liver from rats with diabetes for 5 or 10 days (Fig. 8). The abundance of the 36-kDa protein was not significantly different at any time point. The abundance of the 36-kDa protein was also unchanged in the liver from uremic rats (11).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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The diabetic rats have reduced urinary osmolality, but urinary osmolality
does not vary between 5 and 20 days, even though urinary volume progressively
increases (Table 1). The
polyuria of diabetes results from nonreabsorbable glucose in the tubule lumen.
Theoretically, 300 mosmol of nonreabsorbable solute will retain about 1 liter
of water in the tubular lumen and reduce urinary osmolality to 300
mosmol/kgH2O. However, the diabetic rats were able to maintain
their urinary osmolality at 850–950 mosmol/kgH2O. The present
findings suggest that the increases in UT-A1, AQP2, and NKCC2/BSC1 proteins
play a role in maintaining urinary osmolality above isotonicity. If urinary
osmolality had continued to decrease as urinary volume increased with the
longer duration of diabetes, it is likely that the rats would have lost more
water and solute.
UT-A1. The findings in the present study appear to resolve some discrepancies between previous studies of the effect of diabetes on UT-A1 abundance. We previously showed that at 3 days of diabetes, UT-A1 protein abundance is downregulated in the IM tip, compared with control rats (9), and the present study shows that UT-A1 protein is downregulated in the IM tip at 5 days. In contrast, Bardoux and colleagues (1) showed that at 21 days post-STZ treatment, diabetic rats show an increase in UT-A1 mRNA and protein in the IM base. In particular, they showed that the 117-kDa band was increased without a change in the 97-kDa band (1). Consistent with Bardoux and colleagues (1), the present study shows that the 117-kDa form of UT-A1 is consistently upregulated in the IM base from 5 to 20 days of diabetes and also shows that it is increased in the IM tip from 10 to 20 days. Thus both previous studies (1, 9) were correct, but neither recognized that there are temporal and IM regional changes in UT-A1 protein during the first 3 wk after STZ.
AQP2. The findings in the present study also appear to resolve some discrepancies between previous studies on the effect of diabetes on AQP2 abundance. We previously showed that at 3 days of diabetes, AQP2 protein abundance is unchanged in the IM tip, compared with control rats (9), and the present study shows that AQP2 protein is unchanged in the IM tip at 5 days. In contrast, Nielsen and colleagues (14) showed that AQP2 and phospho-AQP2 protein abundances increase in the whole IM (base + tip) of diabetic rats at 15 days post-STZ. Bardoux and colleagues (1) also show that AQP2 protein is increased in the IM at 21 days post-STZ. Consistent with these latter studies (1, 14), the present study shows that AQP2 is consistently upregulated in the IM base from 5 to 20 days of diabetes and also shows that it is increased in the IM tip from 10 to 20 days. Thus all previous studies (1, 9, 14) were correct, but again they did not recognize that there are temporal and IM regional changes in AQP2 protein during the first 3 wk after STZ. The present study does not address whether diabetic rats have an abnormality in the regulated trafficking of AQP2 nor whether there is a change in the abundance of AQP3 or AQP4 (located in the basolateral membrane of the collecting duct), and future studies will be needed to test these possibilities.
NKCC2/BSC1. Nielsen and colleagues (14) found no significant change in NKCC2/BSC1 protein at 15 days post-STZ. The present study shows that NKCC2/BSC1 is unchanged at 10 days but increases at 20 days. However, ROMK protein was unchanged at all time points.
Interestingly, UT-A1, AQP2, and NKCC2/BSC1 protein abundances are decreased in 6-mo-old, obese Zucker rats, a model of type II diabetes (2). Thus more prolonged periods of diabetes or type II (vs. type I) diabetes may have different effects on these transporters. Regardless, the present and previous (1, 9, 14) findings suggest a complex compensatory response in which UT-A1, AQP2, and NKCC2/BSC1 are upregulated at different times after STZ injection in different medullary regions, but all of these changes will tend to limit the loss of water and solute during uncontrolled diabetes.
Possible mechanisms. Several metabolic and hormonal abnormalities present in diabetes could contribute to the changes in medullary transport protein abundances. In normal rats, we showed that glucocorticoids downregulate UT-A1 protein abundance in the IM tip and lower basal and vasopressin-stimulated facilitated urea permeability in rat terminal IMCDs (13) by decreasing the transcription of UT-A promoter I (17). Rats with uncontrolled DM induced by STZ have increased corticosterone production and urea excretion at 3–6 days (12). We previously showed that glucocorticoids mediate the downregulation of UT-A1 protein abundance in rats with uncontrolled diabetes at 3 days (9). Thus the decrease in UT-A1 at 3–5 days after STZ treatment is likely to be mediated by repressive effect of glucocorticoids on UT-A1 transcription.
What about the upregulation of UT-A1, AQP2, and NKCC2/BSC1 at the later time points? Vasopressin upregulates AQP2 protein long term by a transcriptional mechanism (reviewed in Ref. 16). Vasopressin also upregulates NKCC2/BSC1 and ROMK proteins (4, 5, 7). A previous study showed that diabetic rats have polyuria despite elevated plasma vasopressin levels (23). However, we did not find a significant change in vasopressin levels in the present study. In addition, the difference in time course for AQP2 between IM base and tip, and the lack of change in ROMK, suggests that factors other than vasopressin may play a role.
Regardless of the mechanism, the increase in UT-A1 protein is likely to promote the production of a more concentrated urine. Given the sustained increase in urinary volume, and presumably urinary flow rates, the increase in UT-A1 protein may be an important compensatory mechanism to maintain urea delivery to the IM interstitium. Vasopressin does mediate the rapid increase in urea reabsorption by phosphorylating UT-A1 (24). Because vasopressin levels are not suppressed in diabetic rats, vasopressin is likely to result in UT-A1 phosphorylation and an increase in urea transport per UT-A1 molecule, potentially compensating for the reduced time for urea transport due to the increase in urinary flow rate. Consistent with this hypothesis, the percentage of urea in total urinary solute excretion remained constant from 5 to 20 days of diabetes (Table 1).
Summary. The abundance of the major medullary transport proteins involved in the urinary concentrating mechanism varies with time and kidney region after rats are made diabetic by STZ. These findings tend to support the hypothesis that increases in UT-A1, AQP2, and NKCC2/BSC1 proteins during uncontrolled diabetes are compensatory changes that prevent a progressive decline in urinary concentrating ability despite the continuing osmotic diuresis. Future studies in which knockout mice lacking one or more of these transport proteins are made diabetic may be very useful in defining the importance of each transporter to the compensatory response. However, if similar changes in these medullary transport proteins occur in patients with uncontrolled type I diabetes, they would tend to lessen the degree of volume depletion that occurs in these patients.
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DISCLOSURES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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