Urinary excretion of uric acid, allantoin, and 8-OH-Deoxyguanosine in uricase-knockout mice.

Although uricase-knockout (Uox KO) mice are reported to develop uric acid (UA) nephropathy, those that mature without severe nephropathy could be useful for research into purine metabolism in humans. In this study, we measured the urinary excretion of creatinine, UA, allantoin, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) collected from Uox KO mice housed in metabolic cages. UA and allantoin were determined using liquid chromatography-mass spectrometry and creatinine and 8-OHdG were measured with a commercial kit. Uox KO mice excreted significantly higher levels of UA than wild-type mice (C57BL/6), while the excretion of allantoin was significantly lower. Urinary allantoin was detected in Uox KO mice despite a lack of uricase, which is the same as in humans. In contrast to the elevated levels of UA, the daily excretion of 8-OHdG, an oxidative stress marker, was lower in Uox KO mice. UA is thought to act as an anti-oxidizing agent in humans; thus, these results show that Uox KO mice are potential animal models for research into human purine metabolism.

The effect of testosterone upon the urate reabsorptive transport system in mouse kidney.

It is hypothesized that hyperuricemia in males is caused by androgen-induced urate reabsorptive transport system in the kidney. The expression of urate transporter 1 (Urat1), sodium-coupled monocarboxylate transporter 1 (Smct1) and glucose transporter 9 (Glut9) were investigated in orchiectomized mice with or without testosterone replacement. Testosterone enhanced mRNA and protein levels of Smct1 while those of Glut9 were attenuated. Although the mRNA level of Urat1 was enhanced by testosterone, the corresponding levels of Urat1 protein remained unaffected. Thus, the induction of Smct1 by testosterone is a candidate mechanism underlying hyperuricemia in males.

Establishment and analysis of SLC22A12 (URAT1) knockout mouse.

In order to elucidate the mechanisms of post-exercise acute renal failure, one of the complications of hereditary renal hypouricemia, we have targeted the mouse Slc22a12 gene by the exchange of exons 1-4 with pMC1neo-polyA. The knockout mice revealed no gross anomalies. The concentration ratio of urinary urate/creatinine of the knockout mice was significantly higher than that of wildtype mice, indicating an attenuated renal reabsorption of urate. The plasma levels of urate were around 11 muM and were similar among the genotypes. Although the fractional excretion of urate of knockout mice was tend to higher than that of wildtype mice, the urate reabsorption ability remained in the kidney of knockout mice, indicating a urate reabsorptive transporter other than Urat1.

Development of LC-MS method for detection of mutant uromodulin protein.

Mutations in the uromodulin gene cause the autosomal disorders familial juvenile hyperuricemic nephropathy (FJHN) and medullary cystic kidney disease type 2 (MCKD2). However, methods to detect the mutant form of the uromodulin protein have not been developed. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) method for detection of the mutated uromodulin peptide (C148W). Our method can distinguish the mutant peptide, GWHWE, from wildtype peptide, GWHC*E. Using MS/MS analysis with a selected reaction monitoring (SRM) mode, peptide-specific fragment ions (m/z 714 --> 381, 471, 567, and 679 for GWHWE and m/z 688 --> 381, 445, 541, and 653 for GWHC*E) were detected.

Age and origin of the G774A mutation in SLC22A12 causing renal hypouricemia in Japanese.

Renal hypouricemia is an inherited disorder characterized by impaired tubular uric acid transport. Impairment of the function of URAT1, the main transporter for the reabsorption of uric acid at the apical membrane of the renal tubules, causes renal hypouricemia. The G774A mutation in the SLC22A12 gene encoding URAT1 predominates in Japanese renal hypouricemia. From data on linkage disequilibrium between the G774 locus and the 13 markers flanking it (12 single nucleotide polymorphisms and 1 dinucleotide insertion/deletion locus), we here estimate the age of this mutation at approximately 6820 years [95% confidence interval (CI) 1860-11,760 years; median = 2460 years]. This indicates that the origin of the G774A mutation dates back from between the time when the Jomon people predominated in Japan and the time when the Yayoi people started to migrate to Japan from the Korean peninsula. These data are consistent with a recent finding that this G774A mutation was also predominant in Koreans with hypouricemia and indicate that the mutation originated on the Asian continent. Thus, this mutation found in Japanese patients was originally brought by immigrant(s) from the continent and thereafter expanded in the Japanese population either by founder effects or by genetic drift (or both).

A Turkish case with molybdenum cofactor deficiency.

Molybdenum cofactor deficiency (MIM 252150) is a rare progressive neurodegenerative disorder with about 100 cases reported worldwide. We have identified a male with molybdenum cofactor deficiency and analyzed the molybdenum cofactor synthesis (MOCS)1 gene, MOCS2 gene, MOCS3 gene and GEPH gene. We homozygously identified the CGA insertion after A666 of the MOCS1 gene which produces arginine insertion at codon 222 of MOCS1A. The parents, his brother and his sister who did not have any symptoms were heterozygous for the same mutation. This region was highly conserved in various species. The N-terminal part of MOCS1 a protein is suggested to form the central core of the protein and be composed of an incomplete [(alpha/beta)6] triosephosphate isomerase (TIM) barrel with a lateral opening that is covered by the C-terminal part of the protein. The insertion is located in the loop connecting the fifth beta strand to the sixth alpha helices of the TIM barrel structure. This arginine insertion would induce the conformation change and the lack of the activity.

The effect of dexamethasone on defective nephrin transport caused by ER stress: a potential mechanism for the therapeutic action of glucocorticoids in the acquired glomerular diseases.

The mechanism by which glucocorticoids govern antiproteinuric effect in nephrotic syndrome remains unknown. Present study examined the protective role of dexamethasone (DEX) in the intracellular trafficking of nephrin under endoplasmic reticulum (ER) stress. Human embryonic kidney-293 cell line expressing a full-length human nephrin was cultured in mediums containing 5.5 or 25 mM glucose with or without DEX. The result revealed that glucose starvation evoked a rapid ER stress leading to formation of underglycosylated nephrin that was remained in the ER as a complex with calreticulin/calnexin. DEX rescued this interfered trafficking through binding to its receptor and stimulating the mitochondrial transcripts and adenosine 5' triphosphate (ATP) production, leading to synthesis of fully glycosylated nephrin. These results suggest that ER-stress in podocytes may cause alteration of nephrin N-glycosylation, which may be an underlying factor in the pathomechanism of the proteinuria in nephrotic syndrome. DEX may restore this imbalance by stimulating expression of mitochondrial genes, resulted in the production of ATP that is essential factor for proper folding machinery aided by the ER chaperones.

Characterization of ochratoxin A transport by human organic anion transporters.

The purpose of this study was to investigate the characteristics of ochratoxin A (OTA) transport by multispecific human organic anion transporters (hOAT1 and hOAT3, respectively) using the second segment of proximal tubule (S2) cells from mice stably expressing hOAT1 and hOAT3 (S2 hOAT1 and S2 hOAT3). S2 hOAT1 and S2 hOAT3 exhibited a time- and dose-dependent, and a saturable increase in uptake of [3H]-OTA, with apparent Km values of 0.42 microM (hOAT1) and 0.75 microM (hOAT3). These OTA uptakes were inhibited by several substrates for the OATs. Para-aminohippuric acid (PAH), probenecid, piroxicam, octanoate and citrinin inhibited [3H]-OTA uptake by hOAT1 and hOAT3 in a competitive manner (Ki = 4.29-3080 microM), with the following order of potency: probenecid > octanoate > PAH > piroxicam > citrinin for hOAT1; probenecid > piroxicam > octanoate> citrinin > PAH for hOAT3. These results indicate that hOAT1, as well as hOAT3, mediates a high-affinity transport of OTA on the basolateral side of the proximal tubule, but hOAT1- and hOAT3-mediated OTA transport are differently influenced by the substrates for the OATs. These pharmacological characteristics of hOAT1 and hOAT3 may be significantly related with the events in the development of OTA-induced nephrotoxicity in the human kidney.

Characterization of organic anion transport inhibitors using cells stably expressing human organic anion transporters.

The organic anion transport system is involved in the tubular excretion of various clinically important drugs. The purpose of this study was to characterize the effects of various organic anion transport inhibitors on organic anion transport using proximal tubule cells stably expressing human organic anion transporter 1 (human-OAT1) and human-OAT3, which are localized to the basolateral membrane of the proximal tubule. Organic anion transport inhibitors including betamipron, cilastatin, KW-3902 (8-(noradamantan-3-yl)-1,3-dipropylxanthine) and probenecid significantly inhibited human-OAT1- and human-OAT3-mediated organic anion uptake in a dose-dependent manner. Kinetic analyses revealed that these inhibitions were competitive. The Ki values of betamipron, cilastatin, KW-3902 and probencid for human-OAT1 were 23.6, 1470, 7.82 and 12.1 microM, whereas those for human-OAT3 were 48.3, 231, 3.70 and 9.0 microM. These results suggest that betamipron and probenecid could inhibit both human-OAT1- and human-OAT3-mediated organic anion transport in vivo, whereas cilastatin could inhibit only human-OAT3-mediated one. In contrast, KW-3902 did not exert the effects of significance, whereas KW-3902 was the most potent.

Developmental changes in multispecific organic anion transporter 1 expression in the rat kidney.

BACKGROUND: The cDNA of the multispecific organic anion transporter 1 (OAT1) responsible for the tubular secretion of organic anions was recently isolated. In the current study, we investigated the developmental changes in OAT1 expression in the rat kidney. METHODS: Ontogenic expression of rat OAT1 was investigated by Northern blot, in situ hybridization, Western blot, and immunohistochemical analysis. In addition, para-aminohippurate (PAH) accumulation was measured using fetal, neonatal, and adult rat kidney slices. RESULTS: In Northern blot analysis, OAT1 was detected as early as on embryonic day 18 in the fetal kidney. The expression level of OAT1 mRNA increased remarkably just after birth (postnatal day 0). In situ hybridization revealed OAT1 expression on embryonic day 19. In both the fetal and neonatal kidneys, OAT1 mRNA was localized in a relatively deep region in the cortex. Western blot analysis detected OAT1 protein on embryonic day 20, and the expression level increased after birth. Immunohistochemical analysis did not reveal OAT1 staining in the fetal kidneys. A faint signal of OAT1 protein was detected on postnatal day 0; thereafter, the expression level increased. In the functional study using kidney slices, low but definite probenecid-sensitive PAH accumulation was noted in fetal rat kidney on embryonic day 20. After birth, probenecid-sensitive PAH uptake was increased. CONCLUSIONS: The present study consistently demonstrates the remarkable increase of OAT1 expression after birth, and the immature excretory capacity of the proximal tubules of the neonatal kidney can be attributed, at least in part, to the low expression level of OAT1.

Hydrogen peroxide downregulates human organic anion transporters in the basolateral membrane of the proximal tubule.

Hydrogen peroxide (H2O2) is known to be involved in drug-induced and ischemic proximal tubular damage. The purpose of this study was to elucidate the effects of hydrogen peroxide on organic anion transport mediated by human organic anion transporters 1 and 3 (hOAT1 and hOAT3), which are localized at the basolateral side of the proximal tubule. For this purpose, we established and utilized the second segment of the proximal tubule cells from mice stably expressing hOAT1 or hOAT3 (S2 hOAT1 or S2hOAT3, respectively). H2O2 induced a dose- and a time-dependent decrease in organic anion transport mediated by hOAT1 and hOAT3. Kinetic analysis revealed that H2O2 decreased the Vmax, but not Km of organic anion transport both in S2hOAT1 and S2hOAT3. The effects of gentamicin, known to induce proximal tubular damage via the production of H2O2, on the organic anion transporters were also examined. Gentamicin induced a significant decrease in organic anion transport in S2hOAT1 but not S2hOAT3. H2O2-induced decrease in organic anion transport was significantly inhibited by pretreatment with pyruvate as well as catalase, whereas the gentamicin-induced decrease was significantly inhibited by pretreatment with pyruvate but not with catalase. In conclusion, these results suggest that H2O2, which is produced during tubular injuries, downregulates organic anion transport mediated by both hOAT1 and hOAT3, leading to further modulation of pathophysiology.

Role of organic anion transporter 1 (OAT1) in cephaloridine (CER)-induced nephrotoxicity.

UNLABELLED: Role of organic anion transporter 1 (OAT1) in cephaloridine (CER)-induced nephrotoxicity. BACKGROUND: Cephaloridine (CER) has been used to elucidate the mechanisms of cephalosporin antibiotic-induced nephrotoxicity. Organic anion transporters have been thought to mediate CER uptake by the proximal tubule. The purpose of this study was to elucidate the possible involvement of organic anion transporter 1 (OAT1) in CER-induced nephrotoxicity. METHODS: A mouse terminal proximal straight tubule (S3) cell line stably expressing rat OAT1 (S3 rOAT1) was established and used in this study. The cellular uptake of [14C]-para-aminohippuric acid (PAH), a prototype organic anion, and that of [14C]-CER were measured. The effects of CER on the viability of the cells and the amount of lipid peroxidation were estimated. RESULTS: S3 rOAT1 expressed a functional organic anion transporter in the cytoplasmic membrane, and exhibited CER uptake activity. CER treatment resulted in a more significant decrease in the viability and a more significant increase in the amount of lipid peroxidation in S3 rOAT1 than in S3 cells transfected with an expression vector lacking the rOAT1 insert. Probenecid, an inhibitor of organic anion transport, and probucol, an antioxidant, significantly suppressed the decrease in viability and increase in the amount of lipid peroxidation in S3 rOAT1 treated with CER. The effects of various cephalosporin antibiotics on the uptake of [14C]PAH were correlated significantly with the effects of these drugs on cell viability. CONCLUSIONS: These results suggest that rOAT1 is, at least in part, responsible for the cellular uptake of CER and therefore CER-induced nephrotoxicity.

Immunohistochemical localization of multispecific renal organic anion transporter 1 in rat kidney.

Renal proximal convoluted tubules have an important role, i.e., to excrete organic anions, including numerous drugs and endogenous substances. Recently, multispecific organic anion transporter 1 (OAT1) was isolated from rat kidney. In this study, the cellular and subcellular localization of OAT1 in rat kidney was investigated. Kidneys from normal rats were perfused and fixed with periodate-lysine-paraformaldehyde solution and were then processed for immunohistochemical analysis using the labeled streptavidin-biotin method, preembedding horseradish peroxidase method, and immunogold method. Light microscopic examination revealed immunostaining for OAT1 in the middle portion of the proximal tubule (S2 segment), but not in the initial portion of the proximal convoluted tubule, next to the glomerulus. Nephron segments other than the S2 segment and the renal vasculature were not stained with antibody to OAT1. Electron-microscopic observation using a preembedding method revealed that OAT1 was exclusively expressed in the basolateral membrane of S2 segments of proximal tubules. The immunogold method showed no labeling for OAT1 in the cytoplasmic vesicles, suggesting that OAT1 may not move together with organic anions into the cells. These results are consistent with previous physiologic data showing that organic anions, including para-aminohippurate, are taken up by the basolateral Na+-independent organic anion/dicarboxylate exchanger and excreted at S2 segments. In conclusion, OAT1 was localized to the basolateral membrane of S2 segments of proximal tubules in rat kidneys.

Molecular cloning and functional expression of a multispecific organic anion transporter from human kidney.

Recently, we isolated the multispecific organic anion transporter (OAT1) from the rat kidney, which plays important roles in the renal elimination of endogenous and exogenous organic anions including clinically important drugs. In the present study, we cloned and characterized human OAT1. Two cDNA clones, hOAT1-1 cDNA and hOAT1-2 cDNA, were isolated from a human kidney cDNA library, whose amino acid sequences were 86.0% and 87.8% identical to that of rat OAT1, respectively. When expressed in Xenopus laevis oocytes, hOAT1 mediated sodium-independent uptake of p-aminohippurate (PAH) (Km = 9. 3 +/- 1.0 microM). hOAT1-mediated PAH uptake was inhibited by bulky inorganic anions, various xenobiotics, and endogenous substances, including benzylpenicillin, furosemide, indomethacin, probenecid, phenol red, urate, and alpha-ketoglutarate. Northern blot analysis revealed that hOAT1 mRNA is strongly expressed in human kidney; transcripts of different sizes are expressed in skeletal muscle, brain, and placenta. Immunohistochemical analysis using rabbit IgG antibody against the carboxy-terminal 14 peptides of hOAT1 revealed that hOAT1 is expressed at the basolateral membrane of the proximal tubule. hOAT1 gene was located on human chromosome 11q13.1 by fluorescent in situ hybridization analysis. These results indicate that hOAT1 is a multispecific organic anion transporter on the basolateral membrane of the proximal tubule in human kidney.

Cloning, functional characterization, and localization of a rat renal Na+-dicarboxylate transporter.

We report here the isolation, functional characterization, tissue distribution, and membrane localization of rat renal Na+-dicarboxylate transporter (rNaDC-1). rNaDC-1 consists of 2,245 nucleotides, and the deduced amino acid sequence showed 73% and 75% identity to rabbit and human NaDC-1, respectively. When expressed in Xenopus laevis oocytes, rNaDC-1 mediated sodium-dependent uptake of di- and tricarboxylates. Substrates of rNaDC-1 evoked inward currents in oocytes expressed with rNaDC-1; succinate, alpha-ketoglutarate, and glutarate were relatively high-affinity substrates, and citrate was a low-affinity substrate of rNaDC-1. The coupling ratio of citrate to charge was determined to be 1:1 at pH 7.4; influx of one positive charge per citrate molecule suggests a symport of three Na+ with a divalent citrate. Expression of rNaDC-1 mRNA was detected in the kidney and the small and large intestines. Immunohistochemistry using polyclonal antibodies raised against the 14 amino acids at the COOH terminus of rNaDC-1 revealed that rNaDC-1 is localized exclusively in the luminal membrane of S2 and S3.

Expression cloning and characterization of a novel multispecific organic anion transporter.

Numerous drugs and endogenous compounds are efficiently excreted from the renal proximal tubule via carrier-mediated pathways. Transepithelial excretion of organic anions occurs via their accumulative transport from the blood into the proximal tubule cells across the basolateral membrane and subsequent secretion into the urine through the apical membrane. Here we report on the isolation of a novel complementary DNA from rat kidney that encodes a 551-amino acid residue protein (OAT1) with 12 putative membrane-spanning domains. When expressed in Xenopus laevis oocytes, OAT1 mediated sodium-independent para-aminohippurate (PAH) uptake (Km = 14.3 +/- 2.9 microM). The uptake rate of PAH was increased by the outwardly directed dicarboxylate gradient, consisting with the idea that OAT1 is an organic anion/dicarboxylate exchanger. OAT1 displayed remarkably wide substrate selectivity, covering endogenous substrates such as cyclic nucleotides, a prostaglandin and uric acid, and a variety of drugs with different structures (e.g. antibiotics, a nonsteroidal anti-inflammatory drug, diuretics, an antineoplastic drug, and a uricosuric drug). The Northern blot analysis and in situ hybridization revealed that OAT1 is exclusively expressed in the particular segment of the proximal tubule in the kidney. These data suggest that OAT1 is a multispecific organic anion transporter at the basolateral membrane of the proximal tubule. Isolation of OAT1 will facilitate elucidation of the molecular basis of drug kinetics and the development of new drugs lacking unwanted side effects.

Cisplatin-induced toxicity in immortalized renal cell lines established from transgenic mice harboring temperature sensitive SV40 large T-antigen gene.

We established renal cell lines from definite nephron segments which were microdissected from kidneys of transgenic C57BL/6 mice, harboring the large T-antigen gene of temperature-sensitive mutant simian virus 40, pSVtsA58(ori-). Cell culture was under a humidified atmosphere of 5% CO2 in air, on collagen-coated dishes, and in RITC80-7 medium with 5% fetal bovine serum, 10 micrograms/ml transferrin, 1 microgram/ml insulin, 10 ng/ml recombinant human EGF, penicillin and streptomycin. Cell line which kept contact inhibition character was established from each segment. Cells derived from distal tubule, cortical and outer medullary collecting duct possessed their cyclic AMP response to arginine-vasopressin, like their original nephron segment. On the other hand, cells derived from terminal proximal tubules (S3 segment) formed a cobblestone-like confluent monolayer, and did not respond to arginine-vasopressin like their fresh segments. Since cisplatin, a well-known nephrotoxic substance, damages proximal tubules (especially S3) rather than collecting ducts, we assayed cell number, protein content, and ATP content of cultured S3 cells at various times after addition of 0.2 mM cisplatin. Decrease of cell number, total protein content and total ATP content of culture cells occurred after 10 h incubation with 0.2 mM cisplatin. The 50% lethal dose (LD50) of cisplatin in S3 cells was 4 x 10(-5) M after 20 h incubation and 8.5 x 10(-6) M after 40 h incubation. Outer medullary collecting duct (OMCD) cells were damaged 30% maximally after 20 h incubation with cisplatin, and LD50 in them became 2.5 x 10(-5) M after 40 h incubation. We could show that the LD50 of cisplatin in the OMCD cell line was three times higher than that in the S3 cell line. Thus, these cell lines are the first in the kidney to definite the segmental origin and to maintain some differentiated unique functions. They are valuable for studies on intrarenal site-specific actions and possible mechanisms of action of pharmacological and toxic substances.

Establishment of vasopressin-responsive early proximal tubular cell lines derived from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene.

A little is known about vasopressin receptor in early proximal tubule (S1). The purpose of this study is to establish the vasopressin-responsive S1 cell line derived from transgenic mice harboring temperature-sensitive(ts) simian virus (SV) 40 large T-antigen gene. The cells showed a temperature-sensitive cell growth characteristic of encoding tsSV40. The S1 cells retained a unique morphology specific to proximal tubule. The cells showed the vasopressin-induced increase in intracellular calcium concentration ([Ca2+]i) mediated by both V1a and the putative Vp receptor. Of nineteen clonal cell lines established from the parental cells, three expressed only V1a receptor, and five retained both V1a and Vp receptor. In conclusion, these immortalized S1 cell lines may be useful for studying vasopressin receptor subtypes in S1.