Upregulation of H+-peptide cotransporter PEPT2 in rat remnant kidney

Kazushige Takahashi1, Satohiro Masuda1, Nobuhiko Nakamura1, Hideyuki Saito1, Takahiro Futami1, Toshio Doi2, and Ken-Ichi Inui1

1 Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto 606 - 8507; and 2 Department of Laboratory Medicine, School of Medicine, University of Tokushima, Tokushima 770 - 8503, Japan


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First published August 21, 2001; 10.1152/ajprenal.0346.2001.---The progression of renal damage resulting from reduced nephron mass has been extensively studied in the 5/6 nephrectomized rat. However, reabsorption of small peptides and D-glucose across the renal proximal tubule in this model remains poorly understood. In this study, we examined the alterations of H+-peptide cotransporters (PEPT1 and PEPT2) and Na+-D-glucose cotransporters (SGLT1 and SGLT2) in chronic renal failure. Two weeks after surgery, H+-dependent [14C]glycylsarcosine uptake by the renal brush-border membrane vesicles isolated from 5/6 nephrectomized rats was significantly increased compared with that from sham-operated controls. Kinetic analysis revealed that the maximum velocity value for [14C]glycylsarcosine uptake by the high-affinity-type of peptide transporter was increased threefold by 5/6 nephrectomy, without significant changes in the apparent Michaelis-Menten constant value. Competitive PCR analyses indicated that the expression of PEPT2 mRNA was markedly increased in the remnant kidney, but PEPT1, SGLT1, and SGLT2 mRNA levels showed no significant changes. These findings indicated that the high-affinity-type H+-peptide cotransport activity is upregulated by 5/6 nephrectomy, accompanied by the increased expression of PEPT2. The upregulation of PEPT2 expression would result in an increase in reabsorption of small peptides and peptide-like drugs across the brush-border membranes in chronic renal failure.

5/6 nephrectomy; renal ablation; renal failure; renal tubular reabsorption; peptide-like drug; high-affinity H+-peptide cotransporter


    INTRODUCTION
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IN THE KIDNEY, THE PROXIMAL tubule is the primary site of reabsorption of filtered small peptides and D-glucose, both of which are actively transported by specific transporters across the brush-border membranes of renal epithelial cells.

It has been suggested that there are at least two distinct oligopeptide transporters in the renal brush-border membranes (2, 20). Two distinct H+-peptide cotransporters, designated as PEPT1 (low-affinity type) and PEPT2 (high-affinity type), have been cloned and characterized (5, 21, 22). Both transporters are expressed in the brush-border membranes of the renal proximal tubules and recognize peptide-like drugs such as beta -lactam antibiotics and angiotensin-converting enzyme (ACE) inhibitors (11, 12, 17). Recently, we demonstrated that beta -lactam antibiotics interact predominantly with PEPT2 rather than PEPT1 at therapeutic concentrations (27). Similarly, to the oligopeptide transporters, two distinct types of Na+-D-glucose cotransporters (SGLT1 and SGLT2) have been cloned and characterized (8, 31). D-Glucose transport across the brush-border membranes of renal epithelial cells is mediated by SGLT1 and SGLT2 (9). Although there have been a number of studies of the functional characteristics of these transporters, no information is available about their physiological and pharmacological significance in chronic renal failure (CRF).

The 5/6 nephrectomized rat has been widely used to study the progression of renal damage resulting from reduction of nephron mass. There have been various reports about the mechanisms of glomerular dysfunction in remnant nephrons (7, 10). Renal ablation results in proteinuria, functional hypertrophy, and progressive kidney disease in the rat (10). Recently, Kwon et al. (14) reported that the levels of expression of aquaporin water channels (AQP1, AQP2, and AQP3) were decreased in CRF. In addition, it was indicated that there were significant decreases in total kidney levels of proximal tubule sodium transporters such as the type 3 Na+/H+ exchanger (NHE-3) in rats with CRF (15). However, mechanisms of tubular transport dysfunction, including the reabsorption of nutrients and the secretion of xenobiotics, have not been reported. Because filtered small peptides and peptide-like drugs are reabsorbed by PEPT1 and PEPT2, both transporters should be responsible for nutritional homeostasis and the effects of drug therapy. Therefore, understanding of the functional and molecular changes in both transporters in CRF would be useful for treatment of patients with progressive renal failure.

In the present study, we examined whether PEPT1 and/or PEPT2 is altered in CRF induced by 5/6 nephrectomy compared with SGLT1 and SGLT2.


    METHODS
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INTRODUCTION
METHODS
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Animals. For ablation of renal mass, male Wistar albino rats (200-220 g) were anesthetized with pentobarbital sodium (40 mg/kg), and the kidneys were exposed under aseptic conditions via a ventral abdominal incision. The right kidney was removed, and the posterior and anterior apical segmental branches of the left renal artery were individually ligated as described (26). In sham-operated animals used as controls, the peritoneal cavity was exposed, both kidneys were gently manipulated, and the abdominal incision was closed with 4-0 silk sutures. After surgery, all animals were allowed free access to water and standard rat chow. The animal experiments were performed in accordance with the Guidelines for Animal Experiments of Kyoto University.

Renal functional and morphological studies. Samples of aortic blood and bladder urine were obtained for functional data determination. The blood urea nitrogen (BUN) was determined by the urease/indophenol method. The levels of creatinine and glucose in serum and urine were determined by the Jaffé reaction and the o-toluidine/boric acid method, respectively. For measurement, we used assay kits from Wako Pure Chemical Industries (Osaka, Japan). The concentration of urinary albumin was measured using an ELISA kit (Nephrat II, Exocell, Philadelphia, PA).

Kidneys were fixed in ethyl Carnoy's solution and stained with periodic acid-Schiff's reagent (PAS) (26). As samples for morphological studies, viable portions of remnant kidneys were obtained.

Preparation of renal brush-border membrane vesicles. The renal brush-border membranes were isolated from the renal cortex of sham-operated and 5/6 nephrectomized rats by the Mg2+/EGTA precipitation method as described previously (27). The brush-border membranes from remnant kidneys were prepared only from the viable parts of these kidneys. The isolated membranes were suspended in an experimental buffer to give a final protein concentration of 3.5 mg/ml. The experimental buffer for glycylsarcosine uptake consisted of 100 mM mannitol, 100 mM potassium gluconate, and 10 mM HEPES (pH 7.5), and the experimental buffer for D-glucose uptake consisted of 300 mM mannitol and 10 mM HEPES (pH 7.5). The protein content was determined by the method of Bradford (1), using a protein assay kit (Bio-Rad, Richmond, CA) with bovine gamma -globulin as the standard.

Uptake studies. [14C]glycylsarcosine (1.78 GBq/mmol) was obtained from Daiichi Pure Chemicals (Ibaraki, Japan). D-[3H]glucose (566.1 GBq/mmol) was purchased from Moravek Biochemicals, (Brea, CA). [14C]glycylsarcosine and D-[3H]glucose uptake by brush-border membrane vesicles were measured by a rapid filtration technique as described (27). The uptake of glycylsarcosine and D-glucose was initiated by the addition of 180 µl of buffer containing 22.2 µM [14C]glycylsarcosine at 37°C and 20 µl of buffer containing 100 µM D-[3H]glucose at 25°C to 20 µl of membrane suspension, respectively. The incubation was stopped by diluting the reaction mixture with an ice-cold stop solution composed of either 150 mM KCl and 20 mM HEPES-Tris (pH 7.5) for glycylsarcosine or 150 mM NaCl, 20 mM HEPES-Tris (pH 7.5), and 0.1 mM phlorizin for D-glucose. The radioactivity of [14C]glycylsarcosine and D-[3H]glucose trapped in membrane vesicles was determined using ACS II (Amersham Pharmacia Biotech, Uppsala, Sweden) by liquid scintillation counting.

Western blotting. The same batch of renal brush-border membranes used for uptake studies was subsequently analyzed for expression of PEPT1 and PEPT2 proteins by immunoblotting as reported previously (27). Rabbit anti-SGLT1 antibody (a gift of Prof. Kasahara) was used according to the method of Takata et al. (28).

Competitive PCR analysis. The PEPT1, PEPT2, SGLT1, SGLT2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) competitor DNAs were constructed according to the method of Masuda et al. (19) with some modifications, using a v-erbB retrovirus cDNA fragment (Takara, Shiga, Japan) as neutral DNA. The PCR-amplified PEPT or SGLT competitor DNA was gel purified using a QIAEX kit (Qiagen, Heidelberg, Germany) and ligated into PCR-Script SK(+) plasmid vector (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The orientation and nucleotide sequences of the subcloned DNAs were confirmed by the chain-termination method, using a fluorescence 373A DNA sequencer (Applied Biosystems, Foster, CA). Rat kidney total RNA (1 µg) was reverse transcribed (the final volume of the reaction was 20 µl) with random hexamers (100 ng/reaction), using Superscript II RT (GIBCO BRL, Grand Island, NY), followed by RNase H (GIBCO BRL) digestion. After 10-fold dilution of the reaction with diethylpyrocarbonate-treated water (final volume of 200 µl), 5-µl aliquots of the diluted reactions were used for each subsequent 20-µl PCR. After denaturation of the first-strand DNA at 95°C for 3 min, PCR was performed according to the following profile: 94°C for 1 min, 65°C for 1 min (PEPT and SGLT), or 60°C for 1 min (GAPDH), 72°C for 1 min, 35 cycles for PEPT and SGLT, or 32 cycles for GAPDH, with dilutions of competitor DNAs ranging from 0.1 to 1 amol (1.0 × 10-18 mol)/reaction. Primer sets specific for PEPT1, PEPT2, SGLT1, SGLT2, and GAPDH were used, as shown in Table 1. The expected sizes of amplified products derived from mRNA (competitor DNA) were as follows: PEPT1, 735 bp (607 bp); PEPT2, 543 bp (604 bp); SGLT1, 499 bp (626 bp); SGLT2, 460 bp (582 bp); and GAPDH, 594 bp (671 bp). The amplified PCR products were separated by electrophoresis on 1.5% agarose gels and stained with ethidium bromide. The reactive amounts of bands in each reaction were determined densitometrically using NIH Image 1.61 (National Institutes of Health, Bethesda, MD). Quantification was performed according to the method of Siebert and Larrick (25). To verify the quality of total cellular RNA extracted, competitive PCR for GAPDH was performed with the same batch of single-strand DNA to detect renal PEPT1, PEPT2, SGLT1, and SGLT2 mRNA, and the densitometry data were normalized for each batch of RNA by determining the amount of GAPDH as an internal control.

                              
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Table 1.   The nucleotide sequences of the primers used in competitive PCR analysis


    RESULTS
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INTRODUCTION
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Renal functional and morphological studies. The functional parameters in each group of rats are summarized in Table 2. Two weeks after renal ablation, the body weight of the nephrectomized rats was lower than that of sham-operated controls. Serum creatinine and BUN levels were significantly higher in 5/6 nephrectomized rats compared with those in sham-operated controls. Urinary creatinine concentration was reduced, and the urinary albumin-to-creatinine ratio was markedly increased in 5/6 nephrectomized rats. Figure 1 shows paraffin-embedded sections with PAS staining. Light microscopy revealed that the tubular diameter was slightly increased in nephrectomized rats at 2 wk. Simultaneously, glomeruli from nephrectomized rats displayed a wide range of morphological abnormalities. However, sham-operated rats demonstrated none of these morphological abnormalities.

                              
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Table 2.   Body weight and serum and urinary functional data in sham-operated and 5/6 nephrectomized rats



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Fig. 1.   Glomerular and tubular periodic acid-Schiff staining of rat kidney tissue obtained from sham-operated (A) and 5/6 nephrectomized (NR; B) rats at 2 wk after surgery. Magnification: ×200.

Oligopeptide and D-glucose transport studies. To investigate whether oligopeptide transport activity was altered in the 5/6 nephrectomized rat kidney, the uptake rate of glycylsarcosine by renal brush-border membrane vesicles was examined and compared with the Na+-D-glucose cotransport activity. Figure 2 shows glycylsarcosine and D-glucose uptake by renal brush-border membrane vesicles isolated from the nephrectomized and sham-operated rat kidney cortex at 2 wk after surgery. [14C]glycylsarcosine uptake was examined in the presence of both an inward H+-gradient and an interior negative membrane potential. The uptake of [14C]glycylsarcosine was significantly enhanced in the renal brush-border membrane vesicles from the 5/6 nephrectomized rats (Fig. 2A). The rate of D-[3H]glucose uptake in the presence of an inward Na+ gradient by the renal brush-border membrane vesicles isolated from the nephrectomized rats was not significantly altered compared with those from sham-operated controls (Fig. 2B).


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Fig. 2.   H+ gradient-dependent [14C]glycylsarcosine (GLY-SAR; A) and Na+ gradient-dependent D-[3H]glucose (B) uptake by renal brush-border membranes isolated from sham-operated (Sham; open circle ) or 5/6 NR () rats. A: membrane vesicles (20 µl) isolated from kidneys of Sham (open circle ) or NR () rats and suspended in 100 mM mannitol, 100 mM potassium gluconate, and 10 mM HEPES (pH 7.5) were incubated at 37°C with a substrate mixture (180 µl) comprising 100 mM mannitol, 100 mM sodium gluconate, 22.2 µM [14C]glycylsarcosine, 10 mM HEPES (pH 6.0), and 0.4% ethanol in the presence of 3 mg/ml valinomycin. B: membrane vesicles (20 µl) isolated from kidneys of Sham (open circle ) or NR () rats and suspended in 300 mM mannitol and 10 mM HEPES (pH 7.5) were incubated at 25°C with a substrate mixture (20 µl) comprising 100 mM mannitol, 100 mM sodium chloride, 100 µM D-[3H]glucose, and 10 mM HEPES (pH 7.5). Each point represents the mean ± SE of 3 separate experiments performed in triplicate. Each experiment was performed with 2 to 3 Sham and 7 to 8 NR rats. *P < 0.05, **P < 0.01: significantly different from Sham controls.

Next, the initial uptake rate of glycylsarcosine (10 s) was examined as a function of the glycylsarcosine concentration. As illustrated in Fig. 3A, the glycylsarcosine uptake was curvilinear with saturable components. The Eadie-Hofstee plots of the uptake data after correction for the linear component showed that the glycylsarcosine uptake consisted of two saturable components (Fig. 3A, inset). The kinetic parameters of this uptake were calculated using nonlinear least squares regression analysis with the sum of two Michaelis-Menten equations (Km) for two transport systems and a diffusional component (Table 3). The maximum velocity (Vmax) value for PEPT2 in the 5/6 nephrectmized rats was markedly increased compared with that of sham-operated rats with no changes in Km value. In contrast, there was no difference in the Vmax or Km value for PEPT1 between the sham-operated and 5/6 nephrectomized rats. In addition, the curvilinear plot and the Eadie-Hofstee plot of the D-glucose uptake showed the involvement of two saturable components in D-glucose uptake in the rat renal brush-border membrane vesicles isolated from sham-operated and 5/6 nephrectomized kidney (Fig. 3B). The Vmax value for SGLT2 was significantly decreased in the brush-border membrane vesicles of the 5/6 nephrectomized rats compared with that of sham-operated controls. However, there were no differences in the Vmax or Km value for SGLT1 between 5/6 nephrectomized rats and sham-operated controls.


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Fig. 3.   Concentration dependence of initial uptake of [14C]glycylsarcosine (A) and D-[3H]glucose (B) by renal brush-border membrane vesicles isolated from Sham (open circle ) or 5/6 NR () rats. Membrane vesicles were incubated for 10 s with various concentrations of [14C]glycylsarcosine (A) and D-[3H]glucose (B). Each value represents the mean ± SE of 3 separate experiments performed in duplicate. Each experiment was performed using 3 Sham and 5-7 NR rats, respectively. Insets: Eadie-Hofstee plots of the uptake after correction for the nonsaturable component. V, initial uptake rate (nmol · mg protein-1 · 10 s-1); S, substrate concentration (mM).


                              
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Table 3.   Kinetic analysis of [14C]glycylsarcosine and D-[3H]glucose uptake by renal brush-border membranes isolated from sham-operated and nephrectomized rats

Western blotting. Western blotting was performed to detect the expression of rat PEPT1, PEPT2, and SGLT1 proteins in the same batch of renal brush-border membranes as used for transport studies. As shown in Fig. 4, the expression level of PEPT2 protein in the brush-border membranes isolated from nephrectomized rats was significantly increased at 2 wk after renal ablation. In contrast, the expression levels of PEPT1 and SGLT1 in the brush-border membranes isolated from nephrectomized rats were moderately decreased compared with those from sham-operated controls (Fig. 4).


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Fig. 4.   Western blotting analysis of renal brush-border membranes from Sham or 5/6 NR rats for H+-peptide transporter (PEPT1), PEPT2, and Na+-D-glucose transporter (SGLT1). A: membranes (20 µg) from each tissue were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 10%) and blotted onto Immobilon membranes. Antisera specific for PEPT1, PEPT2, and SGLT1 (1:500-1,000 dilution) were used as primary antibodies, respectively. A horseradish peroxidase-conjugated antirabbit IgG antibody was used for the detection of bound antibodies, and strips of blots were visualized by chemiluminescence on X-ray film. The arrowheads indicate the positions of each transporter. B: each column represents the mean ± SE of 3 separate experiments. Each experiment was performed using the brush-border membranes isolated from 3 Sham and 5-7 NR rats, respectively. *P < 0.05, significantly different from Sham controls.

Competitive PCR analysis. To obtain quantitative information about the expression levels of PEPT1, PEPT2, SGLT1, and SGLT2 in the kidney cortex, we carried out competitive PCR analysis. The data obtained by competitive PCR amplification using primer sets specific for each transporter (Table 1) were normalized with the data of competitive PCR for GAPDH in each tissue (data not shown). As shown in Fig. 5, the expression level of PEPT2 mRNA in the remnant kidney was markedly increased, compared with that in sham-operated controls (1.56 ± 0.27 amol/µg total RNA from 5/6 nephrectomized rat kidney vs. 0.67 ± 0.13 amol/µg total RNA from sham-operated rat kidney, P < 0.05). However, mRNA expression levels of SGLT1, SGLT2, and PEPT1 in the remnant kidney were not significantly different from those in the sham-operated controls.


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Fig. 5.   Quantification of levels of PEPT1, PEPT2, SGLT1, and SGLT2 mRNA expression in total RNA isolated from Sham and 5/6 NR rat kidneys. A: PCR amplification was carried out as described in METHODS. These photographs show the results of representative experiments. B: dots indicate the levels of PEPT1, PEPT2, SGLT1, and SGLT2 mRNA expression in kidneys of Sham (open circle ) or 5/6 NR () rats determined densitometrically using NIH Image 1.61. Each bar represents the mean ± SE of 5-6 rats. *P < 0.05, significantly different from Sham controls.


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The biochemical abnormalities associated with renal failure may affect pharmacological efficacy due to altered renal excretion, resulting in heightened pharmacodynamic responses (24). Therefore, the dosage of drugs must be adjusted properly in patients with renal dysfunction to avoid adverse reactions and to ensure safety and efficacy. However, the detailed mechanisms of renal excretion of drugs in CRF have not been elucidated. In the present study, we found that the oligopeptide transport activity and the expression of PEPT2 were upregulated in CRF with no morphological changes in tubules (Figs. 2, 4, and 5). However, the Vmax value for the low-affinity-type transport of glycylsarcosine and the expression of PEPT1 were maintained by the 5/6 nephrectomy (Table 3). In addition, the Vmax value of the high-affinity-type peptide transport system was significantly increased in the nephrectomized rats without changing the Vmax value for the low-affinity-type peptide transport system (Fig. 3A and Table 3). Our previous report demonstrated that the Km value of high- or low-affinity uptake of glycylsarcosine in the rat renal brush-border membrane vesicles was comparable with the Km value of PEPT2 or PEPT1 for glycylsarcosine in the stable transfectant, respectively (27). Therefore, the enhanced oligopeptide transport activity in the renal brush-border membrane vesicles isolated from nephrectomized rats would be due to the upregulation of PEPT2. To our knowledge, this is the first report demonstrating the upregulation of renal PEPT2.

Turner and Moran (30) reported that there were two distinct D-glucose transport systems with Km values of 0.35 and 6 mM in the rat renal brush-border membrane vesicles. Lee et al. (16) reported that the Km value for the 3-O-methyl-D-glucose uptake of the rat SGLT1 expressed in oocytes was 397 µM, and You et al. (32) demonstrated that the Km value for the 3-O-methyl-D-glucose uptake of the rat SGLT2 expressed in oocytes was 3 mM. In the present study, we confirmed the involvement of the high- and low-affinity Na+-D-glucose cotransport systems in the rat renal brush-border membrane vesicles isolated from sham-operated and 5/6 nephrectomized rats, corresponding to SGLT1 and SGLT2, respectively (Fig. 3B). Although the Vmax value for the low-affinity-type Na+-D-glucose cotransport activity was significantly decreased in the nephrectomized rats compared with that in sham-operated controls (Table 3), the mRNA expression level of SGLT2 was maintained in the nephrectomized rat kidneys. The discrepancy between the decreased Vmax value for the low-affinity-type D-glucose transport system and the maintained mRNA expression level of SGLT2 in nephrectomized rats should be clarified in the future by determining the expression level of SGLT2 protein using a specific antibody.

There have been some reports concerning upregulation of PEPT1 and SGLT1 in the small intestine. Tanaka et al. (29) reported that the intestinal SGLT1 mRNA levels were markedly reduced in 5-fluorouracil-treated rats, whereas the level of PEPT1 mRNA expression was increased. It has been suggested that the resistance of oligopeptide transport activity to 5-fluorouracil-induced intestinal injury was attributable to increased biosynthesis of PEPT1 (29). In the present study, the mRNA expression level of PEPT2, but not of PEPT1, was significantly increased in the remnant kidney (Fig. 5). These findings indicate that there might be tissue-specific mechanisms for preventing tissue damage by regulating the expression of intestinal PEPT1, and renal PEPT1 or PEPT2.

In regard to substrate-induced regulation, intestinal SGLT1 is upregulated in rats by a high carbohydrate diet (6). It was also reported that a high dietary protein level upregulates oligopeptide transport activity in the rat small intestine (4). In addition, Shiraga et al. (23) reported that upregulation of intestinal dipeptide transport activity by high dietary protein levels is due to transcriptional activation of the PEPT1 gene. In the present study, both urinary albumin/creatinine ratio and the level of PEPT2 mRNA expression were significantly increased in nephrectomized rats 2 wk after renal ablation (Table 2, Fig. 5). These findings and the present results suggest that the enhancement of renal PEPT2 mRNA level might be comparable with the urinary concentration of albumin.

PEPT2 is expressed preferentially in the kidney, but not in the small intestine (22). The present results indicate that the expression of PEPT2 was sensitive to 5/6 nephrectomy (Table 3, Figs. 4 and 5). Therefore, these findings suggest that reabsorption of small peptides and peptide-like drugs across the brush-border membranes was stimulated in CRF. Zhu et al. (33) reported that the oligopeptide transporters PEPT1 and PEPT2 recognize ACE inhibitors (peptide-like drugs). Enalapril, an ACE inhibitor, has been reported to reduce glomerular hypertrophy and protect against the progression of renal lesions after subtotal nephrectomy (3, 13). In addition, meta-analysis of the results of clinical trials indicated that ACE inhibitors reduce renal injury in patients with kidney disease (18). Considering the clinical implications of PEPT2 for treatment of patients with renal injury, the upregulation of PEPT2 at both functional and molecular levels might contribute to prevention of the urinary loss of ACE inhibitors by enhanced reabsorption, preventing progression of renal failure.

In conclusion, we found that the tubular PEPT2 was selectively upregulated in CRF by 5/6 nephrectomy, but PEPT1, SGLT1, and SGLT2 were not. These findings would provide useful information for clarification of the renal handling of peptide-like drugs including beta -lactam antibiotics and ACE inhibitors in CRF.


    ACKNOWLEDGEMENTS

We thank Dr. M. Kasahara, Laboratory of Biophysics, School of Medicine, Teikyo University, for providing the rabbit anti-SGLT1 antibody.


    FOOTNOTES

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan for research on metabolic disorders from the Yamanouchi Foundation.

Address for reprint requests and other correspondence: K. Inui, Dept. of Pharmacy, Kyoto University Hospital, Sakyo-ku, Kyoto 606-8507, Japan (E-mail address: inui{at}kuhp.kyoto-u.ac.jp).

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.

First published August 21, 2001; 10.1152/ajprenal.0346.2001

Received 21 December 2000; accepted in final form 6 August 2001.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 281(6):F1109-F1116
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