1 Department of Internal Medicine and 4 Department of Pathology, Hallym University Hangang Sacred Heart Hospital, 2 Department of Internal Medicine, Seoul National University, Clinical Research Institute of Seoul National University Hospital, 3 Department of Internal Medicine, Eulji Medical College, Seoul, South Korea and 5 Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Tokyo, Japan
Correspondence and offprint requests to: Gheun-Ho Kim, MD, PhD, Department of Internal Medicine, Hallym University Hangang Sacred Heart Hospital, 94-200, Yeongdeungpo-dong, Yeongdeungpo-gu, Seoul 150-020, South Korea. Email: gheunho{at}hanmail.net
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. Semi-quantitative immunoblotting and immunohistochemistry were carried out in kidneys from male SpragueDawley rats using a polyclonal peptide-derived antibody to OAT1. Furosemide (12 mg/day/rat, n = 6), hydrochlorothiazide (3.75 mg/day/rat, n = 6) or vehicle (1.7% ethanolamine, n = 6) were infused subcutaneously for 7 days using osmotic minipumps. Experimental and vehicle-control rats were pair-fed, and two bottles of drinking water were provided, one containing tap water and the other containing a solution of 0.8% NaCl with 0.1% KCl.
Results. Overt diuretic responses were observed to both furosemide and hydrochlorothiazide infusions. There were no differences in body weight or creatinine clearance between the experimental and control rats. Although OAT1 protein abundance in cortical homogenates was increased by furosemide infusion (271 ± 35 vs 100 ± 15%, P < 0.05), Na-K-ATPase 1 subunit protein abundance was not affected (113 ± 14 vs 100 ± 8%, P = 0.42). Immunohistochemical localization in tissue sections confirmed a strong increase in OAT1 expression in the basolateral membrane of the S2 segment of proximal tubule. OAT1 protein abundance in cortical homogenates was also increased by hydrochlorothiazide infusion (181 ± 25 vs 100 ± 7%, P < 0.01), whereas Na-K-ATPase
1 subunit protein abundance was not affected (105 ± 4 vs 100 ± 4%, P = 0.34).
Conclusion. Chronic furosemide or hydrochlorothiazide infusion caused increases in OAT1 protein abundance in rat kidney. These results suggest that OAT1 may be up-regulated in vivo by substrate stimulation at the protein level.
Keywords: furosemide; hydrochlorothiazide; organic anion transporter 1; immunoblotting; immunohistochemistry; protein abundance
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recently, many organic anion transporter molecules have been cloned. Two different groups simultaneously have identified the multispecific organic anion transporter 1 (OAT1) from rat kidney using a functional expression cloning method [4,5]. This transporter shows characteristics that correspond to PAH transport across basolateral membrane, such as PAH/dicarboxylate exchange [6], and is localized in the basolateral membrane of S2 segments of proximal tubule in rat kidney [7].
Loop diuretics and thiazides are widely used for the clinical management of hypertension and oedema. They are weak organic acids, having a common chemical characteristic (sulfonamide diuretics), and hence are secreted by the renal organic anion transport system from the blood into the tubular lumen to reach their principal sites of action [8]. Previous studies suggest that OAT1 is probably involved in the renal tubular secretion of both thiazides and loop diuretics. Bartel et al. [9] have shown that thiazide and loop diuretics inhibit in vitro tubular uptake of PAH in single S2 segments from the proximal tubule of rabbit kidney. Recently, Uwai et al. [10] also reported that PAH uptake by OAT1-expressing Xenopus laevis oocytes was inhibited in the presence of thiazide and loop diuretics.
The molecular cloning of OAT1 provides new tools for investigating the regulation of renal tubular secretion of organic compounds at a molecular level. Despite considerable advances in understanding the basic transport pathways and mechanisms involved in the tubular secretion of organic compounds, there is still relatively little information about the regulation of this transport [11]. In addition, diuretic resistance is often encountered in clinical practice, and an interaction between diuretics and the renal organic anion transport system may play a role in the adaptation to long-term diuretic use. This study was undertaken to investigate whether OAT1 is regulated in vivo by chronic administration of diuretics at the protein level. To achieve this, we examined the effects of chronic administration of furosemide or hydrochlorothiazide on the abundance of OAT1 protein in rat kidney by using semi-quantitative immunoblotting and immunohistochemistry.
![]() |
Subjects and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For the furosemide infusion studies, six rats were anaesthetized with enflurane (Choongwae Pharma Corp., Seoul, Korea), and osmotic minipumps (model 2ML1; Alzet, Palo Alto, CA) were implanted subcutaneously to deliver 12 mg/day of furosemide (Handok Inc., Seoul, Korea). Furosemide was dissolved in a 1.7% ethanolamine solution. Six control rats were implanted with minipumps containing vehicle (ethanolamine) alone.
The hydrochlorothiazide infusion studies were carried out in the same way as the furosemide osmotic minipump infusion protocol. Rats were infused with either 3.75 mg/day of hydrochlorothiazide (YuHan Corp., Seoul, Korea) (n = 6) or vehicle (n = 6) for 7 days.
The animals were placed in metabolism cages 3 days prior to the beginning of the study. Control and treated rats were chosen randomly, and all were provided with a daily fixed amount of finely ground regular rat chow (18 g/200 g BW/day) and two separate bottles of drinking water, one containing 0.8% NaCl and 0.1% KCl, and the other containing tap water. All the rats ate the entire amount of the offered rat chow and showed a steady increase in body weight throughout the study period.
The next experiment was performed to test whether the abundance of OAT1 protein is affected by the increase in urine volume per se. To achieve this, rats were divided into water-loaded (n = 6) and water-restricted (n = 6) groups. Water-loaded rats were provided with finely ground rat chow (18 g/200 gBW/day) plus 600 mM sucrose as the drinking fluid. For water-restricted rats, the same rat chow was made into a paste by the addition of water (18 ml/200 g BW/day). This protocol was effective in producing large differences in water intake and urine output.
Urinary measurements
Daily urine samples were collected to measure urine volume and osmolality. Urine osmolality was measured with a cryoscopic osmometer (Osmomat 030-D-M; Gonotec, Berlin, Germany). Urinary sodium concentration was measured using an ion-selective method (System E4A; Beckman Coulter Inc., Fullerton, CA). Furosemide concentration in urine from the furosemide-infused rats was measured using high-performance liquid chromatography (HPLC).
Semi-quantitative immunoblotting
After 7 days of infusion, the rats were killed by decapitation, and kidneys were rapidly removed for dissection of the renal cortex. The cortices were placed in 10 ml of ice-cold isolation solution, containing 250 mM sucrose, 10 mM triethanolamine (Sigma, St Louis, MO), 1 µg/ml leupeptin (Sigma) and 0.1 mg/ml phenylmethylsulfonyl fluoride (Sigma) titrated to pH 7.6, and the mixture was homogenized at 15 000 r.p.m. with three strokes for 15 s using a tissue homogenizer (PowerGun 125; Fisher Scientific, Pittsburgh, PA). After homogenization, total protein concentration was measured using the bicinchoninic acid protein assay reagent kit (Sigma) and was adjusted to 2 µg/µl with isolation solution. The samples were then stabilized by adding 1 vol. of 5x Laemmli sample buffer per 4 vols of sample and by heating to 60°C for 15 min.
Initially, loading gels were performed for each sample set. A 5 µg aliquot of protein from each sample was loaded into individual lanes, electrophoresed on 12% SDSpolyacrylamide minigels using a Mini PROTEAN® III electophoresis apparatus (Bio-Rad, Hercules, CA), and then stained with Coomassie blue dye (G-250, Bio-Rad; 0.025% solution made in 4.5% methanol and 1% acetic acid). Selected bands from these gels were scanned (GS-700 Imaging Densitometry, Bio-Rad) to determine the density (Molecular Analyst version 1.5, Bio-Rad) and relative amounts of protein loaded in each lane. Finally, protein concentrations were corrected to reflect these measurements.
For immunoblotting, the proteins were transferred electrophoretically from unstained gels to nitrocellulose membranes (Bio-Rad). After blocking with 5% skim milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween-20, pH 7.5) for 30 min, membranes were probed overnight at 4°C with their respective primary antibodies consisting of a rabbit polyclonal peptide-derived antibody against rat renal OAT1 [7,12] and a commercial mouse monoclonal antibody for the Na-K-ATPase 1 subunit (Upstate Biotechnology Inc., Lake Placid, NY). The secondary antibodies were goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP; Pierce, Rockford, IL) for OAT1 and goat anti-mouse IgG conjugated to HRL (Pierce) for the Na-K-ATPase
1 subunit.
Antibodyantigen reaction sites were viewed using enhanced chemiluminescence substrate (ECLTM RPN 2106; Amersham Pharmacia Biotech, Buckinghamshire, UK) before exposure to X-ray film (Hyperfilm; Amersham Pharmacia Biotech). Relative quantification of resulting immunoblot band densities was carried out by densitometry using a laser scanner (GS-700 Imaging Densitometry, Bio-Rad) and ImageQuaNT software (Molecular Analyst version 1.5, Bio-Rad).
In all cases, to confirm equality of loading among lanes, electrophoresis was run initially for the entire set of samples in a given experiment on a single 12% SDSpolyacrylamide gel, which was then stained with Coomassie blue dye (G-250, Bio-Rad; 0.025% solution made in 4.5% methanol and 1% acetic acid). These loading gels established that the above immunoblots were uniformly loaded.
Immunohistochemistry
The kidneys were perfused by retrograde perfusion via the abdominal aorta with 1% phosphate-buffered saline (PBS) to remove blood and then with periodatelysineparaformaldehyde (PLP; 0.01 M NaIO4, 0.075 M lysine, 2% paraformaldehyde, in 0.0375 M Na2HPO4 buffer, pH 6.2) for 3 min to produce the kidney fixation. After completion of perfusion, each kidney was cut into 5 mm thick slices and immersed in 2% PLP solution overnight at 4°C. Each slice was dehydrated with a graded series of ethanol and embedded in polyester wax. The embedded pieces of kidney slices were sectioned to 5 µm thickness on a microtome (RM 2145; Leica Instruments GmbH, Nussloch, Germany).
The sections were dewaxed with a graded series of ethanol and treated with 3% H2O2 for 30 min to eliminate endogenous peroxidase activity. They were blocked with 6% normal goat serum (S-1000; Vector Laboratory, Burlington, CA) for 15 min. They were then incubated overnight at 4°C with their respective primary antibodies diluted in PBS. After incubation, they were washed with PBS and incubated for 30 min in biotinylated goat anti-rabbit IgG (BA-1000; Vector Laboratory) at room temperature. Next, the peroxidase standard vectastatin ABC kit (PK-4000; Vector Laboratory) was added for 3060 min at room temperature. The sections were washed with PBS and incubated in a 3,3'-diaminobenzidine (DAB) substrate kit (SK-4100; Vector Laboratory). The sections were counter-stained with haematoxylin and the slides were mounted with Canadian balsam.
Statistics
Values are presented as means ± SEM. Quantitative comparisons between groups were made by MannWhitney U-tests (Statview software; Abacus Concepts Inc., Berkeley, CA). To facilitate comparisons from the semi-quantitative immunoblotting, we normalized the band density values by dividing by the mean value for the control group. Thus the mean for the control group is defined as 100%. P-values < 0.05 were considered statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Responses to hydrochlorothiazide infusion
Hydrochlorothiazide infusion significantly increased urine volume, although this increase was not as remarkable as that induced by furosemide. Urine volume began rising on the third day of hydrochlorothiazide infusion and remained 2- to 3-fold higher than vehicle-infused controls throughout the infusion period (Figure 5A). Urine osmolality was also decreased by hydrochlorothiazide infusion by the third day and remained lower throughout the 7 day infusion period (Figure 5B). Urinary sodium excretion measured on the last day of hydrochlorothiazide infusion was significantly greater in hydrochlorothiazide-infused rats than in vehicle-infused controls (7.5 ± 0.9 vs 3.3 ± 0.6 meq/day, P < 0.05).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We demonstrated that both furosemide and hydrochlorothiazide induced a powerful diuresis in parallel with increases in OAT1 protein abundance in rat kidney. This effect appeared to result from the diuretic agent itself, because sucrose water loading, which increased urine volume, failed to increase OAT1 protein abundance. In fact, the sucrose water loading caused decreases rather than increases in OAT1 abundance. These decreases in OAT1 in sucrose water-loaded rats were unexpected and may be related to their large increases in body weight compared with water-restricted rats. Nevertheless, it is clear that increases in OAT1 abundance induced by furosemide or hydrochlorothiazide infusion were not due to the increase in urine volume. Our results support a role for OAT1 in the renal tubular secretion of thiazides and loop diuretics at the protein level, and suggest that in vivo substrate stimulation up-regulates OAT1 protein.
Substrate stimulation is one of the important factors regulating OAT1 [11]. Hirsch and Hook [14,15] were the first to demonstrate that prior injection of penicillin (twice daily for 3 days) in 2-week-old rabbit pups led to an increase in the uptake of PAH by kidney cortical slices. This phenomenon was thought to be a result of increased biosynthesis of transport proteins, since pre-treatment with penicillin leads to greater incorporation of leucine and glutamine into the slice proteins and to enhancement of protein content in the microsomal fraction, whereas administration of the protein synthesis inhibitor cycloheximide to nursing rats prevents substrate stimulation of PAH uptake by renal slices [16]. Thus, substrate stimulation may occur as a result of increased synthesis of the transporter protein involved in the secretion of organic compounds, and we showed that OAT1 protein abundance is increased by chronic diuretic infusion in adult rats.
In clinical practice, diuretics typically provide effective treatment for oedema when used judiciously. However, some patients become resistant to their effects. Up-regulation of OAT1 protein abundance induced by chronic administration of furosemide or thiazide diuretics may potentially counteract diuretic resistance.
An additional important finding was that increases in OAT1 in response to chronic diuretic infusion were not accompanied by generalized increases in transporter protein expression in the proximal tubule. Transport of organic anions across the basolateral membrane of renal proximal tubular cells is energetically uphill and is accomplished by a tertiary active process. The Na-K-ATPase maintains an inwardly directed (blood-to-cell) Na+ gradient. This Na+ gradient in turn drives an Na+dicarboxylate co-transporter, sustaining an outwardly directed dicarboxylate gradient that is utilized by a PAH/dicarboxylate antiporter (OAT1) to move the organic anion substrate into the cell [17]. This cascade of events indirectly links OAT1 to metabolic energy and to the Na+ gradient, allowing entry of a negatively charged substrate against both its chemical concentration gradient and the electrical potential of the cell. Therefore, it is possible that OAT1 activity may change in parallel with that of Na-K-ATPase.
We found, however, that Na-K-ATPase 1 subunit protein abundance did not change in response to chronic diuretic infusion. When using a newborn rabbit model, Hook and co-workers [18] found that Na-K-ATPase was not affected by penicillin pre-treatment despite an increase in PAH uptake. Recently, an in vitro microperfusion study showed that steviol, a metabolite of the natural sweetener stevioside, inhibited PAH transport at the basolateral membrane of isolated S2 segments of rabbit renal proximal tubules but had no effect on Na-K-ATPase activity [19]. Taken together with the present data, these findings suggest that furosemide and hydrochlorothiazide may have a direct stimulatory effect on OAT1 protein synthesis.
In addition to OAT1, both OAT2 and OAT3 have been identified, and these proteins are expressed in the kidney [20]. Whereas OAT2 is localized in the luminal membranes of the thick ascending limb and the collecting duct, OAT3 is localized in the basolateral membrane of the proximal tubule [20]. Further studies are warranted to verify whether other organic anion transporters, such as rOAT2 and rOAT3, are also regulated at the protein level in response to diuretic administration.
![]() |
Acknowledgments |
---|
Conflict of interest statement. None declared.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|