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Cimetidine, an H₂ receptor antagonist, has been used to investigate the tubular secretion of organic cations in human kidney. We report a systematic comprehensive analysis of the inhibition potency of cimetidine for the influx and efflux transporters of organic cations [human organic cation transporter 1 (hOCT1) and hOCT2 and human multidrug and toxin extrusion 1 (hMATE1) and hMATE2-K, respectively]. Inhibition constants (K(i)) of cimetidine were determined by using five substrates [tetraethylammonium (TEA), metformin, 1-methyl-4-phenylpyridinium, 4-(4-(dimethylamino)styryl)-N-methylpyridinium, and m-iodobenzylguanidine]. They were 95 to 146 μM for hOCT2, providing at most 10% inhibition based on its clinically reported plasma unbound concentrations (3.6-7.8 μM). In contrast, cimetidine is a potent inhibitor of MATE1 and MATE2-K with K(i) values (μM) of 1.1 to 3.8 and 2.1 to 6.9, respectively. The same tendency was observed for mouse Oct1 (mOct1), mOct2, and mouse Mate1. Cimetidine showed a negligible effect on the uptake of metformin by mouse kidney slices at 20 μM. Cimetidine was administered to mice by a constant infusion to achieve a plasma unbound concentration of 21.6 μM to examine its effect on the renal disposition of Mate1 probes (metformin, TEA, and cephalexin) in vivo. The kidney- and liver-to-plasma ratios of metformin both were increased 2.4-fold by cimetidine, whereas the renal clearance was not changed. Cimetidine also increased the kidney-to-plasma ratio of TEA and cephalexin 8.0- and 3.3-fold compared with a control and decreased the renal clearance from 49 to 23 and 11 to 6.6 ml/min/kg, respectively. These results suggest that the inhibition of MATEs, but not OCT2, is a likely mechanism underlying the drug-drug interactions with cimetidine in renal elimination.

The substrate specificities of human (h) multidrug and toxin extrusion (MATE) 1 and hMATE2-K were examined to find functional differences between these two transporters by the transfection of the cDNA of hMATE1 and hMATE2-K into HEK293 cells. Western blotting revealed specific signals for hMATE1 and hMATE2-K consistent with a size of 50 and 40kDa, respectively, in the transfectants as well as human renal brush-border membranes under reducing conditions. In the presence of oppositely directed H(+)-gradient, the transport activities of various compounds such as tetraethylammonium, 1-methyl-4-phenylpyridinium, cimetidine, metformin, creatinine, guanidine, procainamide, and topotecan were stimulated in hMATE1- and hMATE2-K-expressing cells. In addition to cationic compounds, anionic estrone sulfate, acyclovir, and ganciclovir were also recognized as substrates of these transporters. Kinetic analyses demonstrated the Michaelis-Menten constants for the hMATE1-mediated transport of tetraethylammonium, 1-methyl-4-phenylpyridinium, cimetidine, metformin, guanidine, procainamide, topotecan, estrone sulfate, acycrovir, and ganciclovir to be (in mM) 0.38, 0.10, 0.17, 0.78, 2.10, 1.23, 0.07, 0.47, 2.64, and 5.12, respectively. Those for hMATE2-K were 0.76, 0.11, 0.12, 1.98, 4.20, 1.58, 0.06, 0.85, 4.32, and 4.28, respectively. Although their affinity for hMATE1 and hMATE2-K was similar, the zwitterionic cephalexin and cephradine were revealed to be specific substrates of hMATE1, but not of hMATE2-K. Levofloxacin and ciprofloxacin were not transported, but were demonstrated to be potent inhibitors of these transporters. These results suggest that hMATE1 and hMATE2-K function together as a detoxication system, by mediating the tubular secretion of intracellular ionic compounds across the brush-border membranes of the kidney.

This study was initiated to determine if there are differences in the recognition of beta -lactam antibiotics as substrates between intestinal and renal peptide transporters, PEPT 1 and PEPT 2. Reverse transcription-coupled polymerase chain reaction and/or Northern blot analysis have established that the human intestinal cell line Caco-2 expresses PEPT 1 but not PEPT 2, whereas the rat proximal tubule cell line SKPT expresses PEPT 2 but not PEPT 1. Detailed kinetic analysis has provided unequivocal evidence for participation of PEPT 2 in SKPT cells in the transport of the dipeptide glycylsarcosine and the aminocephalosporin cephalexin. The substrate recognition pattern of PEPT 1 and PEPT 2 was studied with cefadroxil (a cephalosporin) and cyclacillin (a penicillin) as model substrates for the peptide transporters constitutively expressed in Caco-2 cells (PEPT 1) and SKPT cells (PEPT 2). Cyclacillin was 9-fold more potent than cefadroxil in competing with glycylsacosine for uptake via PEPT 1. In contrast, cefadroxil was 13-fold more potent than cyclacillin in competing with the dipeptide for uptake via PEPT 2. The substrate recognition pattern of PEPT 1 and PEPT 2 was also investigated using cloned human peptide transporters functionally expressed in HeLa cells. Expression of PEPT 1 or PEPT 2 in HeLa cells was found to induce H(+)-coupled cephalexin uptake in these cells. As was the case with Caco-2 cells and SKPT cells, the uptake of glycylsarcosine induced in HeLa cells by PEPT 1 cDNA and PEPT 2 cDNA was inhibitable by cyclacillin and cefadroxil. Again, the PEPT 1 cDNA-induced dipeptide uptake was inhibited more potently by cyclacillin than by cefadroxil, and the PEPT 2 cDNA-induced dipeptide uptake was inhibited more potently by cefadroxil than by cyclacillin. It is concluded that there are marked differences between the intestinal and renal peptide transporters in the recognition of beta -lactam antibiotics as substrates.

Based on the involvement of zinc in hydrolysis of peptides, we hypothesized that Zn2+ may also play a role in peptide transport. To investigate this hypothesis, kidney brush border membrane vesicles (BBMV) were incubated for 30 min with different concentrations of ZnSO4 before use in uptake studies. This incubation increased by twofold the overshoot uptake of 3H-Gly-L-Gln, D-Leu-125I-Tyr and 3H-cephalexin (all high-affinity substrates for the oligopeptide/H+ symporter) without affecting passive and/or facilitated diffusion of these substrates. Zinc had no effect on the uptake of either glutamine or glucose by kidney BBMV. Among a group of metal ions (cobalt, iron, copper, cadmium, and manganese), only manganese and copper substantially stimulated the activity of the oligopeptide/H+ symporter. DTPA (a complexing agent) inhibited dipeptide uptake, which was reversed by the addition of zinc to the BBMV. Zinc treatment of BBMV reduced the EC50 value of inhibition of 3H-Gly-L-Gln uptake by unlabeled Gly-L-Gln by twofold (90 +/- 8 vs. 45 +/- 4 microM). Similarly, zinc treatment of BBMV reduced the EC50 value for inhibition of D-Leu-125I-Tyr uptake by bestatin from 80 +/- 4 to 40 +/- 3 mM. In conclusion, the data show that zinc has a selective effect on transport of nutrients into kidney BBMV. It stimulates uphill transport of oligopeptides by a modification of their affinity for the binding site of the membrane transporter.

This study was designed to determine whether beta-lactam antibiotics (cephalosporins and penicillins) are all substrates for the renal oligopeptide/H+ symporter and, if so, whether the transport system discriminates among the numerous beta-lactam antibiotics. We used [3H]glycylglutamine, [3H]cephalexin, and [3H]-ampicillin as probes for the transport of oligopeptides, cephalosporins, and penicillins in kidney brush border membrane vesicles, respectively. Among the beta-lactam antibiotics, only those with an alpha-amino group in the phenylacetamido moiety were found to interact with the oligopeptide/H+ symporter. Aminocephalosporins displayed high affinities (KiS generally < 250 microM), whereas aminopenicillins displayed low affinities (Ki 0.78-3.03 mM). These differences in affinities appeared to be a consequence of conformational features of the substrates, especially the sterical location of the carboxy group. The affinities of aminolactams for the oligopeptide/H+ symporter were, furthermore, related to the hydrophobicity of the phenylglycyl chains and the substituents attached to the thiazolidine and dihydrothiazine ring. In sharp contrast to the uptake of [3H]glycylglutamine and [3H]cephalexin, the uptake of [3H]ampicillin was not dependent on a pH gradient and was inhibited by various beta-lactam antibiotics, whether or not they contained an alpha-amino group. Our data suggest that: (a) the transport of aminocephalosporins is largely mediated by the oligopeptide/H+ symporter, which is highly influenced by the substrate structure; and (b) penicillins are transported by another system, which is less discriminative with respect to substrate structure.

Carrier-mediated transport of aminocephalosporin antibiotics by renal brush-border membrane vesicles has been studied in relation to the transport systems for dipeptides and amino acids. Dipeptides such as L-carnosine (beta-alanyl-L-histidine) and L-phenylalanylglycine competitively inhibited the uptake of cephalexin, but amino acids did not. Cephalexin uptake was stimulated by the countertransport effect of L-carnosine in the normal and papain-treated vesicles, and by the effect of L-phenylalanylglycine only in the papain-treated vesicles. In the papain-treated vesicles, the hydrolysis of dipeptides was markedly decreased, and the specific activity for cephalexin transport was increased approx. 2-fold because of the partial removal of membrane proteins. These results suggest that carrier-mediated transport of cephalexin can be transported by the system for dipeptides in renal brush-border membranes.

The pharmacokinetic distribution of 10 cephalosporin compounds, cephalothin, cephaloridine, cephaloglycine, cephalexin, cefazolin, cephapirin, cephradine, cephacentrile, cefoxitin, and cefamandole, in patients with various degrees of renal function was reviewed. The mean serum half-life (t1/2) of each cephalosporin compound was calculated from reported data in patients grouped according to their degree of renal function, as determined by the rate of creatinine clearance. Mean t1/2 values in patients with normal renal function were compared with t1/2 values for the same cephalosporin in patients with moderate renal failure and severe renal azotemia. The effect of hemodialysis and peritoneal dialysis on the mean serum t1/2 of each cephalosporin was also determined. The mean serum t1/2 of each cephalosporin progressively increased as the rate of creatinine clearance decreased. Hemodialysis decreases the t1/2 of cephaloridine, cephalexin, cefazolin, and cephacetrile but only minimally influences the t1/2 of cephalothin and cefamandole. Peritoneal dialysis reduces the t1/2 of cephalothin and cephaloridine but only minimally influences the t1/2 of cefazolin and cefamandole.

A renal clearance method based on a computer analysis after administration of a single dose of drug was developed for measuring the renal handling of several drugs in rabbits, dogs, and humans. Secretion and reabsorption of sulfamethizole, sulfanilamide, cephalexin, and ampicillin in the nephron were analyzed quantitatively using the plasma concentration and the urinary excretion rate of the drugs. The validity of the proposed model was demonstrated. It appears that tubular secretion of sulfamethizole, cephalexin, and ampicillin depend on the active transport system which is described by Michaelis-Menten kinetics; the tubular reabsorption of these drugs is expressed by first-order kinetics. The maximum velocity of renal secretion per unit weight of these drugs was much higher in rabbits than in dogs or humans. Reabsorption showed similar values in dogs and humans. These findings suggest that an analysis of the renal handling of drugs in dogs might provide useful information when considering the appropriate therapeutic dose in humans.

The effect of protein binding on the disposition of cephalexin (CEX) and cofazolin (CEZ) was investigated in a simultaneous perfusion system of rat liver and kidney. In the present study, we used bovine serum albumin (BSA) or human serum albumin (HSA) as plasma protein to control the degree of perfusate protein binding of drugs. Total clearance (CLt) of CEX perfused with BSA (0.70 +/- 0.27 ml/min) was slightly smaller than that with HSA (0.89 +/- 0.08 ml/min), corresponding to the unbound fraction of the drug in the perfusate plasma. On the other hand, CLt of CEZ perfused with BSA (0.90 +/- 0.20 ml/min) was significantly larger than that with HSA (0.32 +/- 0.10 ml/min). The unbound fraction of CEZ to BSA (0.703 +/- 0.052) was much larger than that to HSA (0.253 +/- 0.017) and the clearance of the unbound drug did not differ significantly between two kinds of albumin perfusate (1.30 +/- 0.40 ml/min for BSA and 1.26 +/- 0.40 ml/min for HSA). These results suggest that plasma protein binding is an important factor determining the biliary clearance as well as the urinary clearance of drugs.

Cimetidine reduces the renal clearances of the organic cations procainamide and n-acetylprocainamide in humans and in vitro preparations by inhibition of renal cationic proximal tubular secretion. The aim of this study was to investigate in humans the selectivity of cimetidine in altering the renal handling of three different organic ions as drug substances: anion (cephalothin), cation (ranitidine) and zwitterion (cephalexin). The study was conducted in six healthy subjects who received the above drugs as single doses with and without chronic cimetidine administration. Cimetidine had no statistically significant (P greater than .05) effect on cephalothin disposition including renal clearance, but significantly reduced the renal clearance of ranitidine by over 40% between 4 and 12 hr after administration, with a concomitant increase in ranitidine plasma concentrations and elimination half-life prolongation. In addition, the renal clearance of cephalexin was reduced by cimetidine by 27% between 1 and 2 hr, but there was no change in cephalexin plasma concentrations and elimination half-life. These findings confirmed the hypothesis that cimetidine-mediated inhibition of renal drug clearance in humans is selective for a common cationic secretory transport mechanism in the proximal tubule of the kidney, rather than a nonspecific action on renal function.

A quantitative analytical method for studying renal handling of drugs in dogs with mild renal impairment is described. Renal damage was induced experimentally by pretreatment with mercuric chloride or neomycin. Analytical results of renal handling in those animals indicated a reduction in maximum transport of secretion, while the affinity of drugs to secretion site and reabsorption showed slight or no change. These results were consistent qualitatively with other renal function test values which demonstrated the state of glomerular or proximal renal tubular function. Evidence for the applicability of the proposed analytical method for quantitative validation of functional changes in the nephron in renally impaired animals, as well as the precise determination of the site of damage, was demonstrated. This work holds considerable promise for the study of dosage adjustments in patients with renal disease.