Candesartan augments compensatory changes in medullary transport proteins in the diabetic rat kidney

Abstract

Volume depletion due to persistent glucosuria-induced osmotic diuresis is a significant problem in uncontrolled diabetes mellitus (DM). Angiotensin II receptor blockers (ARBs), such as candesartan, slow the progression of chronic kidney disease in patients with DM. However, mice with genetic knockout of components of the renin-angiotensin system have urine concentrating defects, suggesting that ARBs may exacerbate the volume depletion. Therefore, the effect of candesartan on UT-A1, UT-A3, NKCC2, and aquaporin-2 (AQP2) protein abundances was determined in control and 3-wk DM rats. Aldosterone levels in control rats (0.36 ± 0.06 nM) and candesartan-treated rats (0.34 ± 0.14 nM) were the same. DM rats had higher aldosterone levels (1.48 ± 0.37 nM) that were decreased by candesartan (0.97 ± 0.26 nM). Western analysis showed that UT-A1 expression was increased in DM rats compared with controls in inner medullary (IM) tip (158 ± 13%) and base (120 ± 25%). UT-A3 abundance was increased in IM tip (123 ± 11%) and base (146 ± 17%) of DM rats vs. controls. UT-A3 was unchanged in candesartan-treated control rats. In candesartan-treated DM rats, UT-A3 increased in IM tip (160 ± 14%) and base (210 ± 19%). Candesartan-treated DM rats had slightly higher AQP2 in IM (46%, P < 0.05) vs. control rats. NKCC2/BSC1 was increased 145 ± 10% in outer medulla of DM vs. control rats. We conclude that candesartan augments compensatory changes in medullary transport proteins, reducing the losses of solute and water during uncontrolled DM. These changes may represent a previously unrecognized beneficial effect of type 1 ARBs in DM.

uncontrolled diabetes mellitus (DM) results in an osmotic diuresis (due to glucosuria) and a reduction in urine concentrating ability. In patients with uncontrolled DM, this frequently results in a serious level of volume depletion. Surprisingly, the volume depletion is rarely severe enough that these patients present with shock and cardiovascular collapse. Previously, we identified a renal compensatory mechanism that may partially explain why these patients do not present in shock: the major transport proteins responsible for the production of concentrated urine, the UT-A1 urea transporter, the Na-K-2Cl cotransporter NKCC2/BSC1, and the aquaporin-2 (AQP2) water channel, are upregulated in rats during uncontrolled DM induced by streptozotocin injection (3). Upregulation of these major transport proteins involved in the urine concentrating mechanism will tend to decrease the losses of solute and water, despite the persistent glucosuria.

Angiotensin receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) are widely used to slow the progression of renal disease and treat hypertension in patients with diabetes (2, 9). However, two previous studies raise the possibility that an ARB or ACEI may interfere with the compensatory upregulation of medullary transport proteins that we found in diabetic rats: 1) UT-A1 and NKCC2/BSC1 protein abundances are reduced in ACE knockout mice (5); and 2) candesartan, an ARB, reduces UT-A1 and UT-A3 protein abundances in normal rats (14).

There are three major transport proteins that work in combination to produce an osmotic gradient that is necessary to produce concentrated urine. In the outer medulla, NaCl is the main constituent of the osmotic gradient and the Na-K-2Cl cotransporter (NKCC2/BSC1), located in the medullary thick ascending limb, is chiefly responsible for the absorption of NaCl. The urea transporters UT-A1 and UT-A3 are present in the inner medulla and are responsible for the reabsorption of urea to increase the hypertonicity of the interstitium. AQP2, located in the apical plasma membrane of the inner medulla, is responsible for absorption of water (10). The hypertonic environment created by the NKCC2 and urea transporters, therefore, promotes the absorption of water by AQP2 which results in a more concentrated urine. If an ARB or an ACEI reduced or prevented the upregulation of these major transport proteins involved in the urine concentrating mechanism in DM rats, then these medications could potentially be deleterious during uncontrolled DM. Therefore, the purposes of this study were to determine first, the effect of DM on the UT-A3 urea transporter protein abundance (as this has not been measured previously); and second, the effect of candesartan on UT-A1, UT-A3, NKCC2/BSC1, and AQP2 protein abundances in diabetic rats.

RESEARCH DESIGN AND METHODS

Animals.

All animal protocols were approved by the Emory University Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), weighing 100–150 g, received free access to water and standard rat chow (Purina) containing 23% protein. Rats were made diabetic by injection of streptozotocin (STZ; 65 mg/kg) into the tail vein. Hyperglycemia was verified 24–48 h after injection using a Lifescan Ultra II glucometer. Candesartan (2 mg·kg⁻¹·day⁻¹; generous gift from Astra Zenaca) was started 14 days after STZ injection. Candesartan was dissolved in drinking water containing 0.4% splenda. The dose per animal was delivered in 10 ml of this solution and after the rat consumed the full dose, the regular water bottle was provided for ad libitum water for the remainder of that 24-h period. This was repeated each day for 7 days.

Sample preparation.

Kidneys were removed and dissected into outer medulla (OM), base of the inner medulla (IM), and tip of the IM. Tissues were placed into ice-cold isolation buffer (10 mM triethanolamine, 250 mM sucrose, pH 7.6, 1 μg/ml leupeptin, and 40 μg/ml PMSF) and homogenized with glass homogenizers. SDS was added to a final concentration of 1%, and the samples were sheared with a 25-gauge needle. Homogenates were centrifuged at 8,000 g for 15 min, and the protein in the supernatant fractions was measured by a modified Lowry method (DC Protein Assay Kit; Bio-Rad, Hercules, CA). Urine was collected for assessment of osmolality (Wescor vapor pressure osmometer, Logan, UT).

Aldosterone assay.

Plasma was collected for assessment of aldosterone. Aldosterone levels were determined by radioimmunoassay according to the manufacturer's instructions (DPC, Los Angeles, CA).

Western blot analysis.

Proteins (20 μg/lane) were size separated by SDS-PAGE by using 10% gels and then electroblotted to polyvinylidene difluoride membranes (Imobilon, Millipore, Bedford, MA). Blots were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS; 20 mM Tris·HCl, 0.5 M NaCl, pH 7.5) at room temperature for 1 h and then incubated with primary antibody overnight at 4°C. The primary antibodies used were polyclonal antibodies to the COOH terminal of UT-A1 (6) and NH₂ terminal of UT-A1 and UT-A3 (1). Blots were washed three times in TBS with 0.5% Tween-20 (TBS/Tween) and then incubated with Alexa Fluor 680-linked anti-rabbit IgG (Molecular Probes, Eugene, OR). Blots were washed two times with TBS/Tween, and then the bound secondary antibody was visualized using infrared detection with the Licor Odyssey protein analysis system.

Statistics.

All data are presented as mean percent of control ± SE. To test more than two groups, we used an ANOVA, followed by Fisher's least significant difference (protected t-test) (12) to determine which groups are significantly different. The criterion for statistical significance is P < 0.05.

RESULTS

Blood and urine chemistries.

Rats became hyperglycemic 24 to 48 h after STZ injection and remained elevated during the entire 3-wk study. Table 1 provides physiologic parameters of the DM rats, with and without candesartan administration, determined from samples collected at the time of death. All DM groups were significantly different (P < 0.01) from all control groups. Neither control nor DM groups were significantly different from their candesartan-treated counterparts. No change in body weight exceeded 15% of total. The decrease in urine osmolality developed during the first week after injection of STZ and stabilized at the final value after ∼10 days. Treatment with candesartan for the last 7 days of the protocol had no apparent effect on either blood glucose. Urine osmolality on day 6 of the candesartan treatment revealed an improvement in the candesartan-treated diabetic rats compared with untreated diabetic rats. There was no effect of candesartan treatment on the urine osmolalities of the nondiabetic animals (Table 1).

Table 1.

Effects of candesartan administration on control and diabetic rats

Sample	Final Weight, g	Weight Change, g	Blood Glucose, mg/dl	Urine Osmolality #, mosmol/kgH2O	N
Control	320±7	14±6	78±3	1,576±123	9
Control + candesartan	298±10	17±4	72±5	1,491±99	9
Diabetic	240±11*	−18±5*	388±41*	1,039±18	12
Diabetic + candesartan	256±12*	−40±9*	342±12*	1,240±48†	12

Data are means ± SE.

^*P < 0.05 vs. controls.

^†P < 0.005 vs. untreated diabetics.

^#Determined on day 6 of candesartan treatment.

We measured serum aldosterone levels to verify that the candesartan was exerting an effect. Aldosterone levels in the control rats (0.36 ± 0.06 nM) and the control rats given candesartan were not different (0.34 ± 0.14 nM) while the DM rats had a significantly higher aldosterone level (1.48 ± 0.37 nM) than either control group. Treatment of the diabetic rats lowered the aldosterone level (0.97 ± 0.26 nM). The diabetic rat blood pressures, measured by tail cuff, were not different from control rats. Treatment with candesartan did not alter either group's blood pressures (data not shown).

UT-A3 in DM rats.

While UT-A1 is known to be increased in the IM of diabetic rats, the response of UT-A3 has not previously been reported (3). UT-A3 protein abundance increases 132 ± 12% in the IM tip from DM rats compared with control rats. In the IM base, UT-A3 protein abundance increases 181 ± 24% in DM rats vs. control rats (Fig. 1).

Fig. 1.

Representative Western blot of inner medullary (IM) tip (A) and base (B) from control and diabetic rats probed with NH₂-terminal UT-A antibody that recognizes UT-A1 and UT-A3. Bar graphs: densitometry results of 4 separate experiments with a total of 9 animals per group presented as percent of control. *P < 0.05 vs. control (Ctrl). Ctrl (white bar), control animals; diabetes mellitus (DM; black bar), diabetic animals.

Effect of candesartan on concentrating mechanism transport proteins in DM rats.

UT-A1 increased to 158 ± 13% of control levels in the IM tip of DM rats (Fig. 2A), consistent with previous findings (3). Candesartan treatment did not change UT-A1 protein abundance in either control or DM rats in the IM tip.

Fig. 2.

Representative Western blot of IM tip (A) and IM base (B) from candesartan-treated control and diabetic rats probed for UT-A1. Bar graphs: densitometry results of 3 combined experiments presented as percent of untreated control animals. Ctrl (light gray bar), control animals; Ctrl + Cand (black bar), candesartan-treated control animals; DM (white bar), diabetic animals; DM + Cand (dark gray bar), candesartan-treated diabetic animals. n = 14 animals per group; *P < 0.05 vs. control.

In the IM base, UT-A1 protein abundance increased in DM rats, with a greater relative increase in the abundance of the 117-kDa glycoprotein form of UT-A1 (Fig. 2B), consistent with previous findings (4, 8). Candesartan treatment did not change UT-A1 protein abundance in either control or DM rats.

In contrast to UT-A1, candesartan treatment significantly increased UT-A3 protein abundance in both the IM tip (to 160 ± 15%; Fig. 3A) and IM base (to 210 ± 19%; Fig. 3B) in DM rats. These Western blots were probed with our NH₂ terminal UT-A antibody that recognizes UT-A1 and UT-A3. Candesartan treatment did not change UT-A3 protein abundance in control rats in either the IM tip or base.

Fig. 3.

Representative Western blot of IM tip (A) and IM base (B) from candesartan-treated control and diabetic rats probed with NH₂-terminal UT-A antibody that recognizes UT-A1 and UT-A3. Bar graphs: densitometry results of 4 separate experiments with a total of 8 animals per group presented as percent of untreated control. n = 8 animals per group, *P < 0.05 vs. control; §P < 0.05 vs. untreated diabetic.

AQP2 protein abundance increased in both the IM tip (Fig. 4A) and IM base (Fig. 4B) of DM rats compared with control rats, consistent with previous findings (3). Candesartan treatment did not change AQP2 protein abundance in either control or DM rats in either the IM tip or base.

Fig. 4.

Representative Western blot of IM tip (A) and IM base (B) from candesartan-treated control and diabetic rats probed for aquaporin-2 (AQP2). Bar graphs: densitometry results of 3 combined experiments presented as percent of untreated control. n = 14 animals per group, *P < 0.05 vs. control.

NKCC2/BSC1 protein abundance increased (145 ± 10%) in the DM rats compared with control rats, consistent with previous findings (4). NKCC2/BSC1 protein abundance was further increased in the candesartan-treated DM rats, compared with untreated DM rats or with control rats (Fig. 5).

Fig. 5.

Representative Western blot analysis of outer medulla from candesartan-treated control and diabetic rats probed for NKCC2/BSC1 (top). Bar graph: densitometry results of 2 combined experiments presented as percent of control (bottom). n = 10 animals per group, *P < 0.05 vs. control; §P < 0.05 vs. untreated diabetic.

DISCUSSION

The major results of the present study are 1) UT-A1, UT-A3, AQP2, and NKCC2/BSC1 proteins are upregulated in DM rats; 2) candesartan treatment of DM rats results in a further increase in UT-A3 and NKCC2/BSC1 protein abundances; and 3) candesartan treatment of DM rats did not decrease UT-A1 or AQP2 protein abundances. While the finding that UT-A1, AQP2, and NKCC2/BSC1 proteins are upregulated in DM rats confirms our previous study (3), UT-A3 had not been studied previously. UT-A1 is expressed in the apical membrane of inner medullary collecting ducts (7, 11), while UT-A3 is primarily located in the basolateral membrane of inner medullary collecting ducts (1, 13). However, following vasopressin administration, UT-A3 is detected in both the apical and basolateral membranes, suggesting that upregulation of both UT-A1 and UT-A3 is needed for maximal urea reabsorption and urine concentrating ability (1, 13). The present findings suggest that during uncontrolled DM, both UT-A1 and UT-A3 are upregulated in an attempt to minimize solute and water losses during the ongoing glucose-induced osmotic diuresis.

Since ARBs are routinely given to diabetic patients to control blood pressure, we looked at the blood pressures of the diabetic rats relative to the control rats and then determined whether there was any effect of the candesartan on the blood pressures. There are conflicting reports in the literature about whether diabetes alters rodent blood pressure. In our hands, there was no difference in the blood pressures of the STZ-treated rats compared with control rats. The ARBs did not alter the blood pressure in either group.

Next, we examined the effect of an ARB (candesartan) on the abundance of these transport proteins in DM rats. The rationale was that previous studies show that genetic knockout or inhibition of the renin-angiotensin system in control rats reduces several of the medullary transport proteins that are upregulated during uncontrolled diabetes (5, 14). If candesartan reduced these proteins in DM rats, that would reduce the compensatory response and reduce the conservation of solute and water during uncontrolled diabetes.

In the present study, candesartan did not alter UT-A1 or UT-A3 protein abundance in control rats, while Wang et al. (14) showed that candesartan decreased both UT-A1 and UT-A3 protein abundances in control rats. While we cannot explain the difference in the effect of candesartan on UT-A1 and UT-A3 protein abundances in the present study compared with Wang et al.'s study, one possibility is that the duration of candesartan treatment was longer in the present study. Regardless, if candesartan either reduces or prevents an upregulation of UT-A1 and UT-A3, or of NKCC2/BSC1 or AQP2, protein abundances in DM rats, then it could potentially be deleterious during uncontrolled diabetes mellitus.

The present study found that in DM rats, candesartan treatment did not decrease the DM-induced increases in either UT-A1 or AQP2 protein abundances and further increased UT-A3 and NKCC2/BSC1 protein abundances above the levels in untreated DM rats. Thus, candesartan augmented the compensatory changes in medullary transport proteins in DM. These changes will tend to further reduce the losses of solute and water during uncontrolled DM. We conclude that these changes may represent a previously unrecognized beneficial blockade of the renin-angiotensin system in DM.