urate ( uric acid ) is the major inert end product of purine degradation in humans and higher primates in contrast to most other mammals because of the genetic silencing of hepatic enzyme uricase , which is oxidized to water soluble allantoin1 ) , and serum urate concentration is determined by the balance between production and elimination2 ) .
the kidney plays a dominant role in urate elimination because it excretes approximately 70% of the daily urate production .
particularly , the renal proximal tubule has an important role in transport of organic anions including urate3 ) .
renal handling of uric acid in the proximal tubule seems bidirectional , apical absorption via the urate / anion exchanger and basolateral uptake via organic anion transporters4 ) .
the organic anion transporter type 1 ( oat1 ) is expressed at the basolateral membrane of proximal tubular cells and functions as an organic anion / dicarboxylate exchanger that takes up organic anions from the plasma into proximal tubular cells .
the organic anion transporter type 3 ( oat3 ) is also localized at the basolateral membrane of the proximal tubular cells , and may be involved in the transport of endogenous and exogenous organic anions5 ) .
also , oat3 has been identified as an organic anion / dicarboxylate exchanger similarly to oat16 ) .
in addition , urat1 is a urate - anion exchanger regulating blood urate levels and has been reported to be expressed in the apical membrane of proximal tubular cells .
. however , the physiologic roles of these urate transporters in regulation of uric acid transport in the kidney are not clearly defined8 ) .
hyperuricemia and gout in humans have important clinical implications because of their pathogenic roles in atherosclerosis and other cardiovascular diseases9 ) .
this study was undertaken to investigate in rat kidneys whether the protein abundance of urat1 , oat1 and oat3 is affected by the increase in uric acid intake .
we used specific pathogen - free male sprague - dawley rats ( orient bio co. , korea ) weighing 180 - 220 g. the rats were randomly divided into control ( n=6 ) and uric acid - supplemented ( n=6 ) groups .
the control rats were fed regular rat chow 15 g per 180 g bw per day and the uric acid - supplemented rats were additionally given 0.75 g of uric acid ( sigma , st louis , mo ) per 180 g body weight per day for 8 days .
all the rats ate the entire amount of the offered rat chow and showed a steady increase in body weight throughout the study period .
after the animal experiment , kidneys were harvested and semi - quantitative immunoblotting was carried out from cortical homogenates using polyclonal peptide - derived antibodies to urat1 , oat1 , and oat3 .
when the rats were killed , kidneys were rapidly removed for dissection of the cortices .
the cortices were placed in 10 ml of ice - cold isolation solution containing 250 mm sucrose , 10 mm triethanolamine ( sigma ) , 1 ug / ml leupeptin ( sigma ) , and 0.1 mg / ml phenylmethylsulfonyl fluoride ( sigma ) titrated to ph 7.6 .
the mixture was then homogenized at 15,000 r.p.m . with three strokes for 15 sec using a tissue homogenizer .
after homogenization , total protein concentration was measured using the bicinchoninic acid protein assay regent kit ( sigma ) and was adjusted to 2 g / ul with isolation solution .
the samples were then stabilized by adding 1 volume of 5 laemmli sample buffer per 4 volumes of sample and by heating to 60 for 15 min . initially , ' loading gels ' were performed for each sample set to allow fine adjustment of loading amount so as to guarantee equal loading on subsequent immunoblots .
a 10 g aliquot of protein form each sample was loaded into individual lanes , electrophoresed on 12% sds - polyacryl - amide minigels using a mini protean iii electrophoresis apparatus ( bio - rad , herculs , ca ) and then stained with coomassie blue dye ( g-250 , bio - rad ; 0.025% solution made in 4.5% methanol and 1% acetic acid ) .
after destaining , selected bands from these gels were scanned ( gs-700 imaging densitometry , bio - rad ) to determine the density and relative amounts of protein loaded in each lane .
finally , protein concentrations were ' corrected ' to reflect these measurements . for immunoblotting ,
the proteins were transferred eletrophoretically 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 percent tween-20 , ph 7.5 ) for 30 min , membranes were probed overnight at 4 with the respective primary antibodies . for probing blots ,
all antibodies were diluted into a solution containing 150 mm nacl , 50 mm sodium phosphate , 10 mg / dl sodium azide , 50 mg / dl tween 20 , and 0.1 g / dl bovine serum albumin ( ph 7.5 ) .
the secondary antibody was goat anti - rabbit igg conjugated to horseradish peroxidase ( 31458 ; pierce , rockford , il ) diluted to 1:5,000 .
antibody - antigen reaction sites were viewed using an enhanced chemiluminescence substrate ( ecl rpn 2106 ; amersham pharmacia biotech , buckinghamshire , uk ) .
relative quantification of resulting immunoblot band densities was carried out by densitometry using a laser scanner and imagequant software ( molecular analyst version 1.5 , bio - rad , hercules , ca ) .
for semiquantitative immunoblotting , we used rabbit polyclonal peptide - derived antibodies against rat oat1 ( roat1 ) , rat oat3 ( roat3 ) , and mouse urat1 ( murat1 ) .
quantitative comparisons between the groups were made by the mann - whitney u test ( statview software ; abacus concepts inc . ,
berkeley , ca ) . to facilitate comparisons in the semiquantitative immunoblotting , we normalized the band density values by dividing by the mean value for the normal control group .
we used specific pathogen - free male sprague - dawley rats ( orient bio co. , korea ) weighing 180 - 220 g. the rats were randomly divided into control ( n=6 ) and uric acid - supplemented ( n=6 ) groups .
the control rats were fed regular rat chow 15 g per 180 g bw per day and the uric acid - supplemented rats were additionally given 0.75 g of uric acid ( sigma , st louis , mo ) per 180 g body weight per day for 8 days .
all the rats ate the entire amount of the offered rat chow and showed a steady increase in body weight throughout the study period .
after the animal experiment , kidneys were harvested and semi - quantitative immunoblotting was carried out from cortical homogenates using polyclonal peptide - derived antibodies to urat1 , oat1 , and oat3 .
when the rats were killed , kidneys were rapidly removed for dissection of the cortices .
the cortices were placed in 10 ml of ice - cold isolation solution containing 250 mm sucrose , 10 mm triethanolamine ( sigma ) , 1 ug / ml leupeptin ( sigma ) , and 0.1 mg / ml phenylmethylsulfonyl fluoride ( sigma ) titrated to ph 7.6 .
the mixture was then homogenized at 15,000 r.p.m . with three strokes for 15 sec using a tissue homogenizer .
after homogenization , total protein concentration was measured using the bicinchoninic acid protein assay regent kit ( sigma ) and was adjusted to 2 g / ul with isolation solution .
the samples were then stabilized by adding 1 volume of 5 laemmli sample buffer per 4 volumes of sample and by heating to 60 for 15 min . initially , ' loading gels ' were performed for each sample set to allow fine adjustment of loading amount so as to guarantee equal loading on subsequent immunoblots .
a 10 g aliquot of protein form each sample was loaded into individual lanes , electrophoresed on 12% sds - polyacryl - amide minigels using a mini protean iii electrophoresis apparatus ( bio - rad , herculs , ca ) and then stained with coomassie blue dye ( g-250 , bio - rad ; 0.025% solution made in 4.5% methanol and 1% acetic acid ) .
after destaining , selected bands from these gels were scanned ( gs-700 imaging densitometry , bio - rad ) to determine the density and relative amounts of protein loaded in each lane . finally , protein concentrations were ' corrected ' to reflect these measurements . for immunoblotting ,
the proteins were transferred eletrophoretically 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 percent tween-20 , ph 7.5 ) for 30 min
, membranes were probed overnight at 4 with the respective primary antibodies . for probing blots ,
all antibodies were diluted into a solution containing 150 mm nacl , 50 mm sodium phosphate , 10 mg / dl sodium azide , 50 mg / dl tween 20 , and 0.1 g / dl bovine serum albumin ( ph 7.5 ) .
the secondary antibody was goat anti - rabbit igg conjugated to horseradish peroxidase ( 31458 ; pierce , rockford , il ) diluted to 1:5,000 .
antibody - antigen reaction sites were viewed using an enhanced chemiluminescence substrate ( ecl rpn 2106 ; amersham pharmacia biotech , buckinghamshire , uk ) .
relative quantification of resulting immunoblot band densities was carried out by densitometry using a laser scanner and imagequant software ( molecular analyst version 1.5 , bio - rad , hercules , ca ) .
for semiquantitative immunoblotting , we used rabbit polyclonal peptide - derived antibodies against rat oat1 ( roat1 ) , rat oat3 ( roat3 ) , and mouse urat1 ( murat1 ) .
quantitative comparisons between the groups were made by the mann - whitney u test ( statview software ; abacus concepts inc . ,
berkeley , ca ) . to facilitate comparisons in the semiquantitative immunoblotting , we normalized the band density values by dividing by the mean value for the normal control group .
physiologic parameters measured after the animal experiments are summarized in table 1 . in response to uric acid supplementation for 8 days , serum uric acid level showed an increasing tendency as compared with the control rats ( p=0.055 ) .
serum uric acid concentrations were 2.600.27 mg / dl in uric acid - supplemented rats and 1.970.29 mg / dl in the control rats , respectively . however , the urinary uric acid excretion was not significantly different between the uric acid - supplemented rats ( 3.270.40 mg / d ) and the control rats ( 2.610.34 mg / d ) .
the antibody to urat1 detected a band that corresponded to the utat1 protein , with a molecular mass of approximately -75 kd , in the renal cortex .
uric acid supplementation for 8 days resulted in an insignificant change in urat1 protein abundance in the renal cortex .
relative densitometry revealed that the urat1 protein abundance in the uric acid - supplemented rats was 13214% ( p=0.078 , fig .
the antibody to oat1 detected a specific band that corresponded to the oat1 protein , with a molecular mass of approximately -70 kd , in the renal cortex .
uric acid supplementation for 8 day resulted in a significant increase in oat1 protein abundance in the cortex .
oat1 protein abundance was significantly ( p=0.037 ) increased in the uric acid - supplemented rats ( 14813% ) compared with the control rats ( 1008% ) .
oat3 protein abundance was not significantly different between the uric acid - supplemented rats ( 13112% ) and control rats ( 10017% ) . throughout the study period ,
both the control and uric acid - supplemented rats showed a steady increase in body weight : from 1982 g to 2534 g in the control rats and from 1992 g to 2404 g in the uric acid - supplemented rats .
thus , the amount of weight gain was significantly lower in the uric acid - supplemented rats than in the controls ( p<0.05 ) . although the daily urine output was not significantly different between the two groups ( table 1 ) , it was significantly larger in the uric acid - supplemented rats than in the controls ( 55.06.9 vs. 38.73.9
l / g / d , p<0.05 ) when it was expressed per g body weight .
this relative difference in urinary concentration led us to carry out immunoblotting against water channels aquaporin 1 and aquaporin 2 .
4 revealed that both aquaporin 1 and aquaporin 2 protein abundances had no significant differences between the control and uric acid - supplemented rats .
we examined the effects of uric acid supplementation on the abundance of oat1 , oat3 , and urat1 proteins in the kidney using semi - quantitative immuoblotting .
the results showed that oat1 protein abundance in the renal cortex was significantly increased by uric acid supplementation whereas urat1 and oat3 protein abundances were not significantly affected .
although urate may have beneficial effects in conjunction with genetic or environmental factors , such as scavenging potential harmful radicals in our body12 , 13 ) , it can cause significant health problems , including complications associated with urate crystals such as kidney stones and gout .
humans are the only mammals in whom gout is known to develop spontaneously , probably because hyperuricemia only commonly develops in humans14 ) .
the absence of uricase , combined with extensive reabsorption of filtered urate , results in urate levels in human plasma that are approximately 10 times those of most other mammals15 ) .
however , hyperuricemia can be detrimental in human , as demonstrated by its proven pathogenetic roles in gout and nephrolithiasis and by its putative roles in hypertension and other cardiovascular disorders16 ) .
thus , we tried our animal model of urate excess to investigate renal adaptation on the level of urate transporters .
there is general agreement that virtually all urate transport occurs in the renal epithelial cells of the proximal convoluted tubule .
since transport is bidirectional , urate anions must be capable of moving both from lumen to blood and from blood to lumen at this site17 ) .
although reabsorption and secretion may occur simultaneously within a single cell under specific conditions , there is direct evidence that net reabsorption occurs in the early part of the proximal convoluted tubule ( s1 region ) , net secretion in the s2 region and post - secretory reabsorption , if it exists , in the later part ( s3 region ) of this section of the nephron18 , 19 ) .
the roat1 , which is mainly located in s2 , constitutes a specific mechanism for the secretion of urate from the blood to the lumen at the proximal tubules17 ) .
our result of increased abundance of roat1 in response to uric acid supplementation suggests the contributory role of roat1 to enhance urate secretion to relieve the state of urate excess . on the other hand ,
since it is distributed over s1 , s2 , and s3 segments , the contributions of roat1 and roat3 in the basolateral uptake of organic anions may differ . as a new member of the oat family , urat1 , may act for urate reabsorption7 ) in the kidney , where the protein is located at the apical ( luminal ) membrane of the epithelium of proximal tubules .
the urat1 has been demonstrated to be located on the brush borders of apical membranes of the proximal tubule and may constitute a specific mechanism for the reabsorption of urate from the lumen ( extracellular ) to the cytosol ( intracellular ) at the proximal tubule .
initially we hypothesized that urat1 might be down - regulated in response to uric acid supplementation .
however , our results showed that the urat1 was not altered by uric acid supplementation at least on the level of transporter protein abundance . in conclusion , among the uric acid transporters in the rat kidney , oat1 may have a regulatory role in response to the increase in uric acid intake in the rat kidney .
the up - regulation of oat1 would exert to stimulate urinary uric acid excretion and might contribute to protection from hyperuricemia .
therapeutics that are designed to modify oat1 activity for enhancing secretion of uric acid might be useful in treating pathologies that are associated with hyperuricemia and gout .
 renal handling of uric acid mainly occurs in the proximal tubule , and bidirectional transport of urate may involve apical absorption via the urate - anion exchanger ( urat1 ) and basolateral uptake via organic anion transporters ( oat1 and oat3 ) . in rat kidneys , we investigated whether the protein abundance of urat1 , oat1 , and oat3 is affected by the increase in uric acid intake . 
 male sprague - dawley rats were randomly divided into control and uric acid - supplemented groups , and uric acid - supplemented rats were given 0.75 g of uric acid per 180 g body weight per day for 8 days . 
 after the animal experiment , kidneys were harvested and semi - quantitative immunoblotting was carried out from cortical homogenates using polyclonal peptide - derived antibodies to urat1 , oat1 , and oat3 . 
 serum uric acid level showed an increasing tendency in the uric acid - supplemented rats compared with control rats , whereas urinary uric acid excretion was not significantly different between the uric acid - supplemented rats and control rats . 
 urat1 protein abundance in cortical homogenates was not significantly different between the uric acid - supplemented rats and control rats . 
 however , oat1 protein abundance was significantly increased in the uric acid - supplemented rats compared with the control rats . 
 oat3 protein abundance was not significantly different between the uric acid - supplemented rats and control rats . in conclusion 
 , oat1 may have a regulatory role in response to the increase in uric acid intake in the rat kidney . 
 the up - regulation of oat1 would exert stimulation of urinary uric acid excretion and might contribute to protection from hyperuricemia . 
Introduction
Materials and Methods
1. Animals and experimental protocols
2. Semiquantitative immunoblotting
3. Primary antibodies
4. Statistical analysis
Results
Discussion