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To clarify the specific molecular events of progressive tubular damage in chronic renal failure (CRF), we conducted microarray analyses using isolated proximal tubules from subtotally nephrectomized (Nx) rats as a model of CRF. Our results clearly demonstrated time-dependent changes in gene expression profiles localized to proximal tubules. The expression of mitosis-specific genes Cyclin B2 and Cell division cycle 2 (Cdc2) was significantly and selectively increased in the proximal tubules during the compensated period but decreased to basal level in the end-stage period. Administration of everolimus, a potent inhibitor of mammalian target of rapamycin, markedly reduced compensatory hypertrophy and hyperplasia of epithelial cells, which was accompanied by complete abolishment of the expression of Cyclin B2 and Cdc2 enhancement; renal function was then severely decreased. Treatment with the Cdc2 inhibitor 2-cyanoethyl alsterpaullone clearly decreased epithelial cell hyperplasia, based on staining of phosphorylated histone H3 and Ki-67, while hypertrophy was not inhibited. In conclusion, we have demonstrated roles of Cyclin B2 and Cdc2 in the epithelial hyperplasia in response to Nx. These results advance the knowledge of the contribution of cell cycle regulators, especially M phase, in pathophysiology of tubular restoration and/or degeneration, and these two molecules are suggested to be a marker for the proliferation of proximal tubular cells in CRF. [ABSTRACT FROM AUTHOR]

During cholestasis, bile acids are mainly excreted into the urine, but adaptive renal responses to cholestasis, especially molecular mechanisms for renal secretion of bile acids, have not been well understood. Organic anion transporters (OAT1 and OAT3) are responsible for membrane transport of anionic compounds at the renal basolateral membranes. In the present study, we investigated the pathophysiological roles of OAT1 and OAT3 in terms of renal handling of bile acids. The Eisai hyperbilirubinemic rats (EHBR), mutant rats without multidrug resistance-associated protein 2, showed higher serum and urinary concentrations of bile acids, compared with Sprague-Dawley (SD) rats (wild type). The protein expression level of rat OAT3 was significantly increased in EHBR compared with SD rats, whereas the expression of rat OAT1 was unchanged. The transport activities of rat and human OAT3, but not OAT1, were markedly inhibited by various bile acids such as chenodeoxycholic acid and cholic acid. Cholic acid, glycocholic acid, and taurocholic acid, which mainly increased during cholestasis, are transported by OAT3. The plasma concentration of β-lactam antibiotic cefotiam, a specific substrate for OAT3, was more increased in EHBR than in SD rats despite upregulation of OAT3 protein. This may be due to the competitive inhibition of cefotiam transport by bile acids via OAT3. In conclusion, the present study clearly demonstrated that OAT3 is responsible for renal secretion of bile acids during cholestasis and that the pharmacokinetic profile of OAT3 substrates may be affected by cholestasis. [ABSTRACT FROM AUTHOR]

Human organic cation transporter 2 (OCT2/SLC22A2), which is specifically expressed in the kidney, plays critical roles in the renal secretion of cationic compounds. Tissue expression and membrane localization of OCT2 are closely related to the tissue distribution, pharmacological effects, and/or adverse effects of its substrate drugs. However, the molecular mechanisms underlying the kidney-specific expression of OCT2 have not been elucidated. In the present study, therefore, we examined the contribution of DNA methylation of the promoter region for the OCT2 gene to its tissue-specific expression using human tissue samples. In vivo methylation status of the proximal promoter region of OCT2 and that of OCT1, a liver-specific organic cation transporter, were investigated by bisulfite sequencing using human genomic DNA extracted from the kidney and liver. All CpG sites in the OCT2 proximal promoter were hypermethylated in the liver, while hypomethylated in the kidney. On the other hand, the promoter region of OCT1 was hypermethylated in both the kidney and liver. The level of methylation of the OCT2 promoter was especially low at the CpG site in the E-box, the binding site of the basal transcription factor upstream stimulating factor (USF) 1. In vitro methylation of the OCT2 proximal promoter dramatically reduced the transcriptional activity, and an electrophoretic mobility shift assay showed that methylation at the E-box inhibited the binding of USF1. These results indicate that kidney-specific expression of human OCT2 is regulated by methylation of the proximal promoter region, interfering with the transactivation by USF1. [ABSTRACT FROM AUTHOR]

A H+/organic cation antiporter (multidrug and toxin extrusion 1: MATE1/SLC47A1) plays important roles in the tubular secretion of various clinically important cationic drugs such as cimetidine. We have recently found that the regulation of this transporter greatly affects the pharmacokinetic properties of cationic drugs in vivo. No information is available about the regulatory mechanisms for the MATE1 gene. In the present study, therefore, we examined the gene regulation of human (h) and rat (r) MATE1, focusing on basal expression. A deletion analysis suggested that the regions spanning -65/-25 and -146/-38 were essential for the basal transcriptional activity of the hMATE1 and rMATE1 promoter, respectively, and that both regions contained putative Sp1-binding sites. Functional involvement of Sp1 was confirmed by Sp1 overexpression, a mutational analysis of Sp1-binding sites, mithramy-cin A treatment, and an electrophoretic mobility shift assay. Furthermore, we found a single nucleotide polymorphism (SNP) in the promoter region of hMATE1 (G-32A), which belongs to a Sp1-binding site. The allelic frequency of this rSNP was 3.7%, and Sp1-binding and promoter activity were significantly decreased. This is the first study to clarify the transcriptional mechanisms of the MATE1 gene and to identify a SNP affecting the promoter activity of hMATE1. [ABSTRACT FROM AUTHOR]

Chloride ion has a stimulatory effect on the transport of organic anions across renal basolateral membranes. However, the exact mechanisms at molecular levels have been unclear as of yet. Human organic anion transporters hOAT1 and hOAT3 play important roles in renal basolateral membranes. In this study, the effects of Cl- on the activities of these transporters were evaluated by using HEK293 cells stably expressing hOAT1 or hOAT3 (HEK-hOAT1 or HEK-hOAT3). The uptake of p-[14C]aminohippurate by HEK-hOAT1 and [³H]estrone sulfate by HEK-hOAT3 was greater in the presence of Cl than in the presence of SO2-4 or gluconate. Additionally, the uptake of various compounds by HEK-hOAT1 and HEK-hOAT3 was significantly higher in the Cl--containing medium than the gluconate-containing medium, suggesting that the influences of Cl- are not dependent on substrate and that Cl- directly stimulates the functions of hOAT1 and hOAT3. The substitution of gluconate with Cl- did not change the Km value for the uptake of p-[14C]aminohippurate by HEK-hOAT1 but caused an approximately threefold increase in the maximal uptake rate ~ value. On the other hand, replacement of gluconate with Cl- decreased the Km value for the uptake of [³H]estrone sulfate and cefotiam by HEK-hOAT3 to about one-third, while it did not change the Vmax value. In summary, Cl- upregulates the activities of both hOAT1 and hOAT3, but its effects on transport kinetics differ between these transporters. It was suggested that Cl- participates in the trans-location process for hOAT1, and the substrate recognition process for hOAT3. [ABSTRACT FROM AUTHOR]

Human organic anion transporter 1 (OAT1, SLC22A6), which is localized to the basolateral membranes of renal tubular epithelial cells, plays a critical role in the excretion of anionic compounds. OAT1 is regulated by various pathophysiological conditions, but little is known about the molecular mechanisms regulating the expression of OAT1. In the present study, we investigated the transcriptional regulation of OAT1 and found that hepatocyte nuclear factor (HNF)-4α markedly trans-activated the OAT1 promoter. A deletion analysis of the OAT1 promoter suggested that the regions spanning -1191 to -700 base pairs (bp) and -140 to -79 bp were essential for the transactivation by HNF-4α. These regions contained a direct repeat separated by two nucleotides (DR-2), which is one of the consensus sequences binding to HNF-4α, and an inverted repeat separated by eight nucleotides (IR-8), which was recently identified as a novel element for HNF-4α, respectively. An electrophoretic mobility shift assay showed that HNF-4α bound to DR-2 and IR-8 under the conditions of HNF-4α overexpression. Furthermore, under normal conditions, HNF-4α bound to IR-8, and a mutation in IR-8 markedly reduced the OAT1 promoter activity, indicating that HNF-4α regulates the basal transcription of OAT1 via IR-8. This paper reports the first characterization of the human OAT1 promoter and the first gene in the kidney whose promoter activity is regulated by HNF-4α. [ABSTRACT FROM AUTHOR]

Recently, we have isolated the rat (r) H+/organic cation antiporter multidrug and toxin extrusion 1 (MATE1) and reported its tissue distribution and transport characteristics. Functional characterization suggested that an oppositely directed H+ gradient serves as a driving force for the transport of a prototypical organic cation, tetraethylammonium, by MATE1, but there is no direct evidence to prove this. In the present study, therefore, we elucidated the driving force of tetraethylammonium transport via rMATE1 using plasma membrane vesicles isolated from HEK293 cells stably expressing rMATE1 (HEK-rMATE1 cells). A 70-kDa rMATE1 protein was confirmed to exist in HEK-rMATE1 cells, and the transport of various organic cations including [14C]tetraethylammonium was stimulated in intracellular acidified HEK-rMATE1 cells bitt not mock cells. The transport of [14C]tetraethyl-ammonium in membrane vesicles from HEK-rMATE1 cells exhibited the overshoot phenomenon only when there was an outwardly directed H+ gradient, as observed in rat renal brush-border membrane vesicles. The overshoot phenomenon was not observed in the vesicles from mock cells. The stimulated [14C]tetraethylammonium uptake by an H+ gradient [intravesicular H+ concentration ([H+]in) > extravesicular H+ concentration ([H+]out)] was significantly reduced in the presence of a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). [14Cjtetraethylammonium uptake was not changed in the presence of valinomycin-induced membrane potential. These findings definitively indicate that an oppositely directed H+ gradient serves as a driving force of tetraethylammonium transport via rMATE1, and this is the first demonstration to identify the driving force of the MATE family. The present experimental strategy is very useful in identifying the driving force of cloned transporters whose driving force has not been evaluated. [ABSTRACT FROM AUTHOR]

Focuses on the upregulation of H[sup +]-peptide cotransporter PEPT2 in rat remnant kidney. Progression of renal damage resulting from reduced nephron mass; Reabsorption of peptides and D-glucose across the renal proximal tube; Alteration of PEPT1 and PEPT2 and Na[sup +]-D-glucose cotransporters in chronic renal failure.

Investigates the expression of the sodium-L-carnitine cotransport system and its role in transport of tetraethylammonium in a kidney epithelial cell line LLC-PK... Characteristics of L-carnitine uptake in LLC-PK... cells; Effect of L-carnitine on transcellular transport and accumulation of tetraethylammonium.

PEPT1 and PEPT2 are H+-coupled peptide transporters expressed preferentially in the intestine and kidney, respectively, which mediate uphill transport of oligopeptides and peptide-like drugs such as beta-lactam antibiotics. In the present study, we have compared the recognition of beta-lactam antibiotics by LLC-PK1 cells stably transfected with PEPT1 or PEPT2 cDNA. Cyclacillin (aminopenicillin) and ceftibuten (anionic cephalosporin without an alpha-amino group) showed potent inhibitory effects on the glycylsarcosine uptake in the PEPT1-expressing cells. Other beta-lactams, such as cephalexin, cefadroxil, and cephradine (aminocephalosporins), inhibited modestly the PEPT1-mediated glycylsarcosine uptake. Except for ceftibuten, these ß-lactams showed much more potent inhibitions on the glycylsarcosine uptake via PEPT2 than via PEPT1. Comparison of the inhibition constant (Ki) values between cefadroxil and cephalexin suggested that the hydroxyl group at the NH2-terminal phenyl ring increased affinity for both PEPT1 and PEPT2. It is concluded that PEPT2 has a much higher affinity for beta-lactam antibiotics having an alpha-amino group than PEPT1 and that substituents at the NH2-terminal side chain of these drugs are involved in the recognition by both peptide transporters. [ABSTRACT FROM AUTHOR]

We have previously shown that the p-aminohippurate (PAH) transport system in OK kidney epithelial cell line is under the regulatory control of protein kinase C. Parathyroid hormone (PTH) could activate protein kinase C, as well as protein kinase A, in OK cells. In the present study, the effect of PTH on PAH transport was studied in OK cells. PTH inhibited the transcellular transport of PAH from the basal to the apical side, as well as the accumulation of PAH in OK cells. Basolateral PAH uptake was inhibited by PTH in a dose- and time-dependent manner. Protein kinase A activators did not affect the transcellular transport or the accumulation of PAH. The PTH-induced inhibition of the accumulation of PAH was blocked by a protein kinase C inhibitor staurosporine. These results suggest that PTH inhibits the PAH transport in OK cells and that the messenger system mediated by protein kinase C, not protein kinase A, plays an important role in the regulation of PAH transport by PTH. [ABSTRACT FROM AUTHOR]