PAGE 1

University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 1 Oxalobacter formigenes does not Alter slc26a6 Protein Abundance in the Large Intestine of C57BL/6 Mice Shannon A. Moore College of Medicine University of Florida The study of the intestinal bacteria Oxalobacter formigenes wild rat strain began when it was isolated from both human and animal feces and was found to have sole substrate specificity for oxalate. Oxalate is a component of the most common type of kidney stones, and hyperoxaluria is a major risk factor for kidney stone disease. Previous studies showed that in the presence of OXWR oxalate is excreted from the blood stream into the large intestine, while in the absence of OXWR oxalate is absorbed from the large inte stine into the blood stream with the help of certain membrane transport proteins encoded by genes in the slc26 gene family. In the present study, the question that was addressed was whether OXWR colonization of mice changes the expression of the Putative Anion Transporte r 1 (PAT 1) protein on the epithelial cells of the large intestine in order to excrete oxalate i nto the large intestine. Protein was isolated from intestinal tissues, and PAT 1 abundance was detected using Western Blot analysis with quantification using Image J. The results showed that there was no statistically significant difference in the PAT 1 pr otein abundance in the proximal colon, the distal colon, or the caecum due to the presence of Oxalobacter formigenes It is possible that oxalate transport proteins other than PAT 1 are responsible for the Oxalobacter effects on intestinal oxalate movements. INTRODUCTION Hyperoxaluria is a major risk factor for kidney stone disease, and there is no pharmaceutical treatment available (8 13, 15, 16). In 1980, an anaerobic bacterium called Oxalobacter formigenes was isolated from rumen contents that had sole substrate specificity for oxalate, and later it was isolated from both human and numerous other animal feces (1, 2). Subsequent studies demonstrated that the lack of intestinal Oxalobact er sp. activity was a potential risk factor in kidney stone disease (8 13, 15, 16). It was found that stone forming patients who were Oxalobacter negative had significantly higher urinary oxalate excretion (8 13, 15, 16), and recurrent kidney stone episodes in stone forming patients appeared to correlate with the lack of Oxalobacter colonization within the intestinal tract (8 13, 15, 16). In 2002, a study showed that a single oral dose of Oxalobacter reduced the urinary oxalate in four human subjects followi ng an oxalate load (9). Hoppe et al. also reported results of sizeable but transient reductions in urinary oxalate excretion when small studies of pH patients were administered Oxalobacter in the form of a paste/capsule (7). In animal studies, Oxalobacter has been found in the feces of various wild animals, but it is generally not found in laboratory rats or mice (1). When animals were exposed to increasing amounts of dietary oxalate, such animals showed increased rates of oxalate degradation which suggest s that oxalate tolerance can be developed in animals, and intestinal absorption can be reduced by robust Oxalobacter activity (5). Also, when rats were given dietary supplementation of oxalate, orally administering whole cells or oxalate degrading enzymes from Oxalobacter reduced urinary oxalate excretion (5). The PAT 1 protein is considered to be one of the primary candidates involved in intestinal oxalate transport. This transport protein is located on the apical membrane in the epithelial cells and media tes secretion of oxalate in mouse intestines (4).The goal of the present study was to determine whether the Oxalobacter induced changes in oxalate transport (5) could be correlated with changes in the abundance of PAT 1 in the mouse large intestine. MATERI ALS AND METHODS Protein was extracted from mucosal scraping of the proximal colon, distal colon, and caecal tissues removed from C57BL/6 mice (purchased from the Jackson Laboratory ME) that were colonized with Oxalobacter For comparison, non colonized C57 BL/6 mice were used as controls. Protein was isolated from the organic phase following Trizol extraction of RNA according to the manufacturer's recommendation (Invitrogen, Carlsbad, CA). Briefly, protein was precipitated by incubation with isopropanol for 10 min at room temperature. The precipitate was sedimented by centrifugation at 12,000 g for 10 min and sequentially washed four times in 0.3 M guanidine hydrochloride in 95% ethanol followed by two washes in ethanol. Washing steps included sonication of the protein pellet, incubation at room temperature for 20 min and

PAGE 2

SHANNON A M OORE University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 2 centrifugation at 7,500 g for 5 min. The final protein pellet was re suspended in 5% SDS and the protein concentrations were determined using the BCA protein assay (Pierce, Rockford, IL). For the Western Blot assay, each of the samples was prepared for gel electrophoresis by placing 100 g of protein from each sample into a 0.5 ml tube and adding 8 l of 6X sample buffer into each tube. The samples were boiled for 5 min, loaded into a 5% SD S gel, and electrophoresed for 30 min. The proteins, separated by gel electrophoresis, were electrophoretically transferred onto a Hybond ECL nitrocellulose membrane at 60V for 3 hours at 4C. After the transfer was complete, the membranes were prepared by blocking with non fat dry milk blocking solution to eliminate the non specific binding sites. The membranes were probed with a custom made PAT 1 primary antibody (Alpha Diagnostic International; San Antonio, TX) diluted 1:250 in 5% Blotto and incubated ov ernight at 4C. The membranes were washed six times for 5 min each in TBS T. The membranes were then incubated for 30 min in 5% Blotto containing a 1:13,333 dilution of a goat anti rabbit secondary antibody conjugated to horseradish peroxidase (HRP; Amersh am) followed by 6 subsequent washes in TBS T and a final 5 min wash in TBS (no Tween). For detection, the membranes were reacted for one minute with chemiluminescence reagent (Amersham, Piscataway, NJ) and exposed to autoradiographic hyperfilm ECL (Amer sham). The intensity of the resulting band appearing on the film was quantified using Image J. The membranes were stripped of antibodies using Restore Western Blot Stripping Buffer (Pierce) by incubating for an hour at 37C. The membranes were then washed with TBS T at room temperature prior to re probing with glyceraldehyde 3 phosphate dehydrogenase (GAPDH) as described above using 1:4,000 dilution of primary antibody (Ambion) for 1 hour and 1:13,333 dilution of sheep anti mouse HRP conjugated secondary an tibody. STATISTICS Statistical analysis of the data derived from Image J quantification was performed using an unpaired t test for the comparison to two means. Differences were considered RESULTS The primary question addressed in this study was whether any of the Oxalobacter induced changes in intestinal oxalate transport that were previously reported (5) could be correlated with alterations in the abundance of mucosal PAT 1 in the colonized large intestine. Accordingly, the relati ve abundance of PAT 1 protein was measured in mucosal scrapings from the distal colon, proximal colon, and caecum and these results are presented in Figures 1 3. At least two bands were visible between molecular weight markers 75 and 50 kD in all of th e mouse samples; however, a single band at ~65 kD was apparent in the control sample which is a pooled sample of rat distal colon scrapings routinely loaded in lane 10. In earlier studies validating the specificity of the PAT 1 antibody in our hands, the upper band corresponding to ~65 kD disappeared when blots were incubated with control peptide (specific competition); consequently, this upper band was the band quantified. As shown in Figure 1, no significant differences were observed in the expression of PAT 1 protein in the distal colon when colonized tissues were compared to non colonized controls. In addition, comparable expression of GAPDH in all lanes indicated consistent loading of the samples onto the gels. As shown in Figure s 2 and 3, similar resu lts were obtained for the proximal colon and caecum, respectively, indicating no affect of OXWR colonization on PAT 1 expression in any segment of the mouse large intestine. It is notable, however, that these studies have confirmed the presence of PAT 1 in all segments of the mouse large intes tine.

PAGE 3

O XALOBACTER FORMIGENE S IN INTESTINAL OXALAT E TRANSPORT University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 3 Figure 1. Western Blot analysis of PAT 1 and GAPDH in the Distal Colon of colonized (C) and non colonized (NC) C57BL/6 mice. The non colonized and colonized wild type mice were loaded next to each other on the gel in lanes 2 through 9. Lane 1 was the ladder and lane 10 was a control sample pool (P) from wild type rat distal colon. There were 7 non colonized and 8 colonized sampl es. 100 g of protein was loaded into each well. The PAT 1 bands are ~65 kD and the GAPDH bands are ~25kD. When the bands were quantified, the average mean showed no statistically significant difference in the abundance of protein between the colonized and non colonized samples. The same membranes probed for PAT 1 were also probed for GAPDH. GAPDH was probed as a control to determine the consistency of the amount of protein that was loaded into each well.

PAGE 4

SHANNON A M OORE University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 4 Figure 2: Western blot analysis of PAT 1 and GAPDH in the caecum of colonized (C) and non colonized (NC) C57BL/6 mice. The non colonized and colonized wild type mice were loaded next to each other on the gel in lanes 2 through 9. Lane 1 was the ladder and lane 10 wa s a control sample pool (P) from wild type rat distal colon. There were 7 colonized and 7 non colonized samples. The amount of protein loaded into each well was 100 g. The PAT 1 bands are ~65 kD and the GAPDH bands are ~25 kD. The quantified bands showed no statistically significant difference in the abundance of protein between the colonized and non colonized samples. The same membranes were used for the probing of both the PAT 1 and the GAPDH proteins. GAPDH was the control used to show the consistency o f the amount of protein that was loaded into each well.

PAGE 5

O XALOBACTER FORMIGENE S IN INTESTINAL OXALAT E TRANSPORT University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 5 Figure 3: Western Blot analysis of PAT 1 and GAPDH in the proximal colon of colonized (C) and non colonized (NC) C57BL/6 mice. The non colonized and colonized wild type mice were loaded next to each other on the gel in lanes 2 through 9. Lane 1 was the ladder and lane 10 was a control sample pool (P) from wild type rat distal colon. There were 6 non colonized and 8 colonized sampl es. Each well was loaded with 100 g of protein, and quantification of the bands showed no statistically significant difference in protein abundance between the colonized and non colonized samples. The PAT 1 bands are ~65 kD and GAPDH bands are ~25 kD. The same membranes were used for the probing of both the PAT 1 and the GAPDH proteins. GAPDH was probed as a control to determine the consistency of the amount of protein that was loaded into each well. DISCUSSION Based on Oxalobacter colonization studies in rats (5), we proposed that a physiological interaction between the bacteria and the transporting mucosal cells promoted intestinal elimination of oxalate leading to significant reductions in urinary oxalate. The focus of the present study was to determine if the Oxalobacter induced changes in intestinal oxalate transport were due to a change in the relative abundance of an important apical oxalate transporter in the slc26a gene family, PAT 1. PAT 1 is a multifunctional tr ansporter that mediates apical oxalate efflux in the mouse small intestine (4). Although PAT 1 expression is reported to be relatively more abundant in the small intestine than in the colon (17, 18), expression of PAT 1 was certainly confirmed in all segme nts of the large intestine in the present study. While we did not detect any changes in the relative abundance of PAT 1 correlating with the changes in oxalate transport in colonized tissues, the functional activity of the transporter may have increased in the apical membrane nonetheless. It is also possible that changes in trafficking of PAT 1 to and from

PAGE 6

SHANNON A M OORE University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 6 the membrane may underlie functional changes in oxalate transport and future studies will address this aspect by comparing total cellular PAT 1 protein with apical membrane bound PAT 1. In addition, other oxalate transporters in the slc26a gene family, such as slc26a1, slc26a2, and slc26a3, are potential candidates for future study in this regard. Finally, it is possible that the changes in vectorial oxal ate transport observed are primarily dependent upon the magnitude and direction of counterion gradients in vivo as was demonstrated in in vitro studies conducted by us using Caco 2 monolayers (3). The latter study showed that vectorial transport of oxalate mediated specifically by PAT 1 is more dependent on the magnitude and direction of counterion gradients than an intrinsic property of the protein. Clearly, a better understanding of the prevailing counterion driver gradients in addition to the relative a ffinities of the ions transported by these anion exchangers, is required. REFERENCES 1. Allison MJ, Dawson KA, Marberry WR, Foss JG. Oxalobacter formigenes gen. nov., sp. nov.: oxalate degrading anaerobes that inhabit the gastrointestinal tract. Arch Microbiol 141(1): 1 7, 1985. 2. Dawson KA, Allison MJ, Hartman PA. Characteristic of anaerobic oxalate degrading enrichment cultures from the rumen. Appl Environ Microbiol 40: 840 846, 1980. 3. Freel RW, Morozumi M, Hatch M. Parsing apical oxalate exchange in Caco 2BBe1 monolayers: siRNA knockdown of SLC26a6 reveals the role and properties of PAT 1. Am J Physiol Gastrointest Liver Physiol 297: G918 G929, 2009. 4. Freel R W, Hatch M Green M, Soleimani M. Ileal oxalate absorption and urinary oxalate excretion are enhanced in Slc 26a6 null mice. Am J Physiol Gastrointest Liver Physiol 290: G719 G728, 2006. 5. Hatch M, Cornelius J, Allison M Sidhu H, Peck A, Freel RW. Oxalobacte r sp. Reduces urinary oxalate excretion by promoting enteric oxalate secretion. Kidney International 69: 691 698, 2005. 6. Hatch M, Freel RW. Intestinal transport of an obdurate anion: oxalate. Urol Res 33: 1 16, 2005. 7. Hoppe B, Beck B, Gatter N, von Unruh G, Tischer A, Hesse A, Laube N, Kaul P, Sidhu H. Oxalobacter formigenes : a potential tool for the treatment of primary hyperoxaluria type 1. Kidney Int 70(7): 1305 1311, 2006. 8. Kleinschmidt K, Mahlmann A, Hautmann R. Microbial degradation of dietary oxalate in the human gut and urinary oxalate concentrations in patients with calcium oxalate urolithiasis and control persons. Investig Urol (Berlin) 5: 222 224, 1994. 9. Kumar R, Mukherjee M, Bhandari M et al Role of Oxalobacter formigenes in calcium oxalate stone di sease: a study from North India. Eur Urol 41: 318 322, 2002. 10. Kwak C, Kim HK Kim EC et al Urinary oxalate levels and the enteric bacterium Oxalobacter formigenes in patients with calcium oxalate urolithiasis. Eur Urol 44: 475 481, 2003. 11. Mikami K, A kakura K, Takei K et al Association of absence of intestinal oxalate degrading bacteria with urinary calcium oxalate stone formation. Int J Urol 10: 293 296, 2003. 12. Mittal RD, Kumar R, Mittal B et al Stone composition, metabolic profile and the presen ce of the gut inhabiting bacterium Oxalobacter formigenes as risk factors for renal stone formation. Med Princ Pract 12: 208 213, 2003. 13. Neuhaus TJ, Belzer T, Blau N et al Urinary oxalate excretion in urolithiasis and nephrocalcinosis. Arch Dis Child 82: 322 326, 2000. 14. Sidhu H, Allison MJ, Chow JM et al Rapid reversal of hyperoxaluria in a rat model after probiotic administration of Oxalobacter formigenes J Urol 166: 1487 1491, 2001. 15. Sidhu H, Hoppe B, Hesse A et al Absence of Oxalobacter for migenes in cystic fibrosis patients: a risk facter for hyperoxaluria. Lancet 352: 1026 1029, 1998. 16. Sidhu H, Schmidt ME, Cornelius JG et al Direct correlation between hyperoxaluria/oxalate stone disease and the absence of the gastrointestinal tract dwe lling bacterium Oxalobacter formigenes : possible prevention by gut recolonization or enzyme replacement therapy. J Am Soc Nephrol 10(Suppl 14): S334 S340, 1999. 17. Simpson JE, Schweinfest CW, Shull GE, Gawenis LR, Walker NM, Boyle KT, Soleimani M, Clarke LL. PAT 1 (Slc26a6) is the predominant apical membrane Cl/HCO3 exchanger in the upper villous epithelium of the murine duodenum. Am J Physiol Gastrointest Liver Physiol 292: G1079 G1088, 2007. 18. Wang Z, Petrovic S, Mann E, Soleimani M. Identification o f an apical Cl/HCO3 exchanger in the small intestine. Am J Physiol Gastrointest Liver Physiol 282: G573 G579, 2002.


Summer Focus on Medical Research : Oxalobacter formigenes does not Alter slc26a6 Protein Abundance in the Large Intestin...
ALL VOLUMES CITATION THUMBNAILS PDF VIEWER PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00091523/00600
 Material Information
Title: Summer Focus on Medical Research : Oxalobacter formigenes does not Alter slc26a6 Protein Abundance in the Large Intestine of C57BL/6 Mice
Series Title: Journal of Undergraduate Research
Physical Description: Serial
Language: English
Creator: Moore, Shannon
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011
 Subjects
Genre: serial   ( sobekcm )
 Notes
Abstract: The study of the intestinal bacteria Oxalobacter formigenes wild rat strain began when it was isolated from both human and animal feces and was found to have sole substrate specificity for oxalate. Oxalate is a component of the most common type of kidney stones, and hyperoxaluria is a major risk factor for kidney stone disease. Previous studies showed that in the presence of OXWR oxalate is excreted from the blood stream into the large intestine, while in the absence of OXWR oxalate is absorbed from the large intestine into the blood stream with the help of certain membrane transport proteins encoded by genes in the slc26 gene family. In the present study, the question that was addressed was whether OXWR colonization of mice changes the expression of the Putative Anion Transporter 1 (PAT-1) protein on the epithelial cells of the large intestine in order to excrete oxalate into the large intestine. Protein was isolated from intestinal tissues, and PAT-1 abundance was detected using Western Blot analysis with quantification using Image J. The results showed that there was no statistically significant difference in the PAT-1 protein abundance in the proximal colon, the distal colon, or the caecum due to the presence of Oxalobacter formigenes. It is possible that oxalate transport proteins other than PAT-1 are responsible for the Oxalobacter effects on intestinal oxalate movements.
 Record Information
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: sobekcm - UF00091523_00600
System ID: UF00091523:00600

Downloads

This item has the following downloads:

( PDF )


Full Text




.Oxalobacter formigenes does not Alter slc26a6 Protein

Abundance in the Large Intestine of C57BL/6 Mice

Shannon A. Moore

College of Medicine, University of Florida

The study of the intestinal bacteria Oxalobacterformigenes wild rat strain began when it was isolated from both human and animal
feces and was found to have sole substrate specificity for oxalate. Oxalate is a component of the most common type of kidney stones,
and hyperoxaluria is a major risk factor for kidney stone disease. Previous studies showed that in the presence of OXWR oxalate is
excreted from the blood stream into the large intestine, while in the absence of OXWR oxalate is absorbed from the large intestine into
the blood stream with the help of certain membrane transport proteins encoded by genes in the slc26 gene family. In the present study,
the question that was addressed was whether OXWR colonization of mice changes the expression of the Putative Anion Transporter 1
(PAT-1) protein on the epithelial cells of the large intestine in order to excrete oxalate into the large intestine. Protein was isolated
from intestinal tissues, and PAT-1 abundance was detected using Western Blot analysis with quantification using Image J. The results
showed that there was no statistically significant difference in the PAT-1 protein abundance in the proximal colon, the distal colon, or
the caecum due to the presence of Oxalobacter formigenes. It is possible that oxalate transport proteins other than PAT-1 are
responsible for the Oxalobacter effects on intestinal oxalate movements.


INTRODUCTION
Hyperoxaluria is a major risk factor for kidney stone
disease, and there is no pharmaceutical treatment available
(8-13, 15, 16). In 1980, an anaerobic bacterium called
Oxalobacter formigenes was isolated from rumen contents
that had sole substrate specificity for oxalate, and later it
was isolated from both human and numerous other animal
feces (1, 2). Subsequent studies demonstrated that the lack
of intestinal Oxalobacter sp.activity was a potential risk
factor in kidney stone disease (8-13, 15, 16). It was found
that stone forming patients who were Oxalobacter-negative
had significantly higher urinary oxalate excretion (8-13, 15,
16), and recurrent kidney stone episodes in stone-forming
patients appeared to correlate with the lack of Oxalobacter
colonization within the intestinal tract (8-13, 15, 16). In
2002, a study showed that a single oral dose of
Oxalobacter reduced the urinary oxalate in four human
subjects following an oxalate load (9). Hoppe et al. also
reported results of sizeable but transient reductions in
urinary oxalate excretion when small studies of pH patients
were administered Oxalobacter in the form of a
paste/capsule (7).
In animal studies, Oxalobacter has been found in the
feces of various wild animals, but it is generally not found
in laboratory rats or mice (1). When animals were exposed
to increasing amounts of dietary oxalate, such animals
showed increased rates of oxalate degradation, which
suggests that oxalate tolerance can be developed in animals,
and intestinal absorption can be reduced by robust
Oxalobacter activity (5). Also, when rats were given


dietary supplementation of oxalate, orally administering
whole cells or oxalate-degrading enzymes from
Oxalobacter reduced urinary oxalate excretion (5).
The PAT-1 protein is considered to be one of the
primary candidates involved in intestinal oxalate transport.
This transport protein is located on the apical membrane in
the epithelial cells and mediates secretion of oxalate in
mouse intestines (4).The goal of the present study was to
determine whether the Oxalobacter-induced changes in
oxalate transport (5) could be correlated with changes in
the abundance of PAT-1 in the mouse large intestine.

MATERIALS AND METHODS

Protein was extracted from mucosal scraping of the
proximal colon, distal colon, and caecal tissues removed
from C57BL/6 mice (purchased from the Jackson
Laboratory ME) that were colonized with Oxalobacter. For
comparison, non-colonized C57BL/6 mice were used as
controls.
Protein was isolated from the organic phase following
Trizol extraction of RNA according to the manufacturer's
recommendation (Invitrogen, Carlsbad, CA). Briefly,
protein was precipitated by incubation with isopropanol for
10 min at room temperature. The precipitate was
sedimented by centrifugation at 12,000 g for 10 min and
sequentially washed four times in 0.3 M guanidine
hydrochloride in 95% ethanol, followed by two washes in
ethanol. Washing steps included sonication of the protein
pellet, incubation at room temperature for 20 min, and


University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 I Summer 2011
1





SHANNON A. MOORE


centrifugation at 7,500 g for 5 min. The final protein pellet
was re-suspended in 5% SDS, and the protein
concentrations were determined using the BCA protein
assay (Pierce, Rockford, IL).
For the Western Blot assay, each of the samples was
prepared for gel electrophoresis by placing 100 ig of
protein from each sample into a 0.5 ml tube and adding 8
pl of 6X sample buffer into each tube. The samples were
boiled for 5 min, loaded into a 5% SDS gel, and
electrophoresed for 30 min. The proteins, separated by gel
electrophoresis, were electrophoretically transferred onto a
Hybond ECL nitrocellulose membrane at 60V for 3 hours
at 40C. After the transfer was complete, the membranes
were prepared by blocking with non-fat dry milk blocking
solution to eliminate the non-specific binding sites. The
membranes were probed with a custom-made PAT-1
primary antibody (Alpha Diagnostic International; San
Antonio, TX) diluted 1:250 in 5% Blotto and incubated
overnight at 40C. The membranes were washed six times
for 5 min each in TBS-T. The membranes were then
incubated for 30 min in 5% Blotto containing a 1:13,333
dilution of a goat anti-rabbit secondary antibody
conjugated to horseradish peroxidase (HRP; Amersham),
followed by 6 subsequent washes in TBS-T and a final 5-
min wash in TBS (no Tween). For detection, the
membranes were reacted for one minute with
chemiluminescence reagent (Amersham, Piscataway, NJ)
and exposed to autoradiographic hyperfilm ECL
(Amersham). The intensity of the resulting band appearing
on the film was quantified using Image J.
The membranes were stripped of antibodies using
Restore Western Blot Stripping Buffer (Pierce) by
incubating for an hour at 370C. The membranes were then
washed with TBS-T at room temperature prior to re-
probing with glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) as described above using 1:4,000 dilution of
primary antibody (Ambion) for 1 hour and 1:13,333
dilution of sheep anti-mouse HRP-conjugated secondary
antibody.


STATISTICS

Statistical analysis of the data derived from Image J
quantification was performed using an unpaired t-test for
the comparison to two means. Differences were considered
significant ifp < 0.05.

RESULTS

The primary question addressed in this study was
whether any of the Oxalobacter-induced changes in
intestinal oxalate transport that were previously reported
(5) could be correlated with alterations in the abundance of
mucosal PAT-1 in the colonized large intestine.
Accordingly, the relative abundance of PAT-1 protein was
measured in mucosal scrapings from the distal colon,
proximal colon, and caecum, and these results are
presented in Figures 1-3. At least two bands were visible
between molecular weight markers 75 and 50 kD in all of
the mouse samples; however, a single band at -65 kD was
apparent in the control sample, which is a pooled sample of
rat distal colon scrapings routinely loaded in lane 10. In
earlier studies validating the specificity of the PAT-1
antibody in our hands, the upper band corresponding to
-65 kD disappeared when blots were incubated with
control peptide (specific competition); consequently, this
upper band was the band quantified. As shown in Figure 1,
no significant differences were observed in the expression
of PAT-1 protein in the distal colon when colonized tissues
were compared to non-colonized controls. In addition,
comparable expression of GAPDH in all lanes indicated
consistent loading of the samples onto the gels. As shown
in Figures 2 and 3, similar results were obtained for the
proximal colon and caecum, respectively, indicating no
affect of OXWR colonization on PAT-1 expression in any
segment of the mouse large intestine. It is notable, however,
that these studies have confirmed the presence of PAT-1 in
all segments of the mouse large intestine.


University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 I Summer 2011
2





OX4LOBACTER FORAMGENES IN INTESTINAL OXALATE TRANSPORT


Gel# 1
1 2 3 4 5 6 7 8 9 10
PAT-I -65 kD M -
1 2 3 4 5 6 7 8 9 10
GAPDH 25 kD -00"




colonized/non-colonized NC C NC C NC C NC C P


Gel #2
1 2 3 4 5 6 7 8 9 10
PAT-I -65kD _ m -
1 2 3 4 5 6 7 8 9 10
GAPDH 25 kD "_



colonized/non-colonized
NC C NC C NC C NC C P


Quantification of Bands
Protein Loaded
# of Samples Average Mean
(Ig)
PAT-1
NC n 7 100 0.33 .08
C n 8 100 0.39 .05
GAPDH
NC n 7 100 0.50 .02
C n 8 100 0.49 .02


Figure 1. Western Blot analysis of PAT-1 and GAPDH in the Distal Colon of colonized (C) and non-colonized (NC)
C57BL/6 mice. The non-colonized and colonized wild-type mice were loaded next to each other on the gel in lanes 2
through 9. Lane 1 was the ladder and lane 10 was a control sample pool (P) from wild-type rat distal colon. There were
7 non-colonized and 8 colonized samples. 100 pg of protein was loaded into each well. The PAT-1 bands are -65 kD
and the GAPDH bands are ~25kD. When the bands were quantified, the average mean showed no statistically
significant difference in the abundance of protein between the colonized and non-colonized samples. The same
membranes probed for PAT-1 were also probed for GAPDH. GAPDH was probed as a control to determine the
consistency of the amount of protein that was loaded into each well.






















University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 I Summer 2011
3





SHANNON A. MOORE


Gel# 1
1 2 3 4 5 6 7 8 9
PAT-1 -65kD j -b -


GAPDH 25 kD




coloniz ed/non-coloniz ed


NC C NC C NC C NC C P


Gel #2


1 2 3 4 5 6 7 8 9 10

PAT-1 -65kD 3 4 -8 9 -1
1 2 3 4 5 6 7 8 9 10


GAPDH 25 kD



colonized/non-colonized


NC C NC C NC C NC C P


Quantification of Bands
Protein Loaded
# of Samples Average Mean
(Ig)
PAT-1
NC n 7 100 0.40 .08
C n 7 100 0.36 .03
GAPDH
NC n 7 100 0.57 .03
C n 7 100 0.57 .05


Figure 2: Western blot analysis of PAT-1 and GAPDH in the caecum of colonized (C) and non-colonized (NC) C57BL/6 mice.
The non-colonized and colonized wild-type mice were loaded next to each other on the gel in lanes 2 through 9. Lane 1 was
the ladder and lane 10 was a control sample pool (P) from wild-type rat distal colon. There were 7 colonized and 7 non-
colonized samples. The amount of protein loaded into each well was 100 pg. The PAT-1 bands are -65 kD and the GAPDH
bands are -25 kD. The quantified bands showed no statistically significant difference in the abundance of protein between the
colonized and non-colonized samples. The same membranes were used for the probing of both the PAT-1 and the GAPDH
proteins. GAPDH was the control used to show the consistency of the amount of protein that was loaded into each well.























University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 I Summer 2011
4





OXALOBACTER FORAEGENES IN INTESTINAL OXALATE TRANSPORT


Gel# 1
1 2 3 4 5 6 7 8 9 10
PAT-I 65 kD ---___


GAPDH 25 kD -- W
NC C NC C NC C NC C P


2 3 4 5 6 7 8 9 10
colonized/non-colonized
NC C NC C NC C NC C P


Gel #2
1 2 3 4 5 6 7 8 9 10
PAT-I -65kD llle


GAPDH 25 kD


colonized/non-colonized


2 3 4 5 6 7 8 9 10
NC C NC C NC C NC C P


Quantification of Bands
Protein
# of Samples Loadedg) Average Mean
Loaded (pug)

PAT-1
NC n 6 100 0.74 .07
C n 8 100 0.85 .10
GAPDH
NC n 6 100 0.76 .09
C n 8 100 0.97 .09


Figure 3: Western Blot analysis of PAT-1 and GAPDH in the proximal colon of colonized (C) and non-colonized (NC)
C57BL/6 mice. The non-colonized and colonized wild-type mice were loaded next to each other on the gel in lanes 2
through 9. Lane 1 was the ladder and lane 10 was a control sample pool (P) from wild-type rat distal colon. There were
6 non-colonized and 8 colonized samples. Each well was loaded with 100 pg of protein, and quantification of the bands
showed no statistically significant difference in protein abundance between the colonized and non-colonized samples.
The PAT-1 bands are -65 kD and GAPDH bands are -25 kD. The same membranes were used for the probing of both
the PAT-1 and the GAPDH proteins. GAPDH was probed as a control to determine the consistency of the amount of
protein that was loaded into each well.


DISCUSSION

Based on Oxalobacter colonization studies in rats (5),
we proposed that a physiological interaction between the
bacteria and the transporting mucosal cells promoted
intestinal elimination of oxalate leading to significant
reductions in urinary oxalate. The focus of the present
study was to determine if the Oxalobacter-induced changes
in intestinal oxalate transport were due to a change in the
relative abundance of an important apical oxalate
transporter in the slc26a gene family, PAT-1. PAT-1 is a


multifunctional transporter that mediates apical oxalate
efflux in the mouse small intestine (4). Although PAT-1
expression is reported to be relatively more abundant in the
small intestine than in the colon (17, 18), expression of
PAT-1 was certainly confirmed in all segments of the large
intestine in the present study. While we did not detect any
changes in the relative abundance of PAT-1 correlating
with the changes in oxalate transport in colonized tissues,
the functional activity of the transporter may have
increased in the apical membrane nonetheless. It is also
possible that changes in trafficking of PAT-1 to and from


University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 I Summer 2011
5





SHANNON A. MOORE


the membrane may underlie functional changes in oxalate
transport, and future studies will address this aspect by
comparing total cellular PAT-1 protein with apical
membrane-bound PAT-1. In addition, other oxalate
transporters in the slc26a gene family, such as slc26al,
slc26a2, and slc26a3, are potential candidates for future
study in this regard. Finally, it is possible that the changes
in vectorial oxalate transport observed are primarily
dependent upon the magnitude and direction of counterion


REFERENCES


1. Allison MJ, Dawson KA, Marberry WR, Foss JG. Oxalobacter formigenes
gen. nov., sp. nov.: oxalate-degrading anaerobes that inhabit the
gastrointestinal tract. Arch Microbiol 141(1): 1-7, 1985.

2. Dawson KA, Allison MJ, Hartman PA. Characteristic of anaerobic oxalate-
degrading enrichment cultures from the rumen. Appl Environ Microbiol 40:
840-846, 1980.

3. Freel RW, Morozumi M, Hatch M. Parsing apical oxalate exchange in Caco-
2BBel monolayers: siRNA knockdown of SLC26a6 reveals the role and
properties of PAT-1. Am J P: ... *. Gastrointest Liver P-F ... .' 297: G918-
G929, 2009.

4. Freel RW, Hatch M Green M, Soleimani M. Ileal oxalate absorption and
urinary oxalate excretion are enhanced in Slc 26a6 null mice. Am J :.., .
Gastrointest Liver : ... .,' 290: G719-G728, 2006.

5. Hatch M, Cornelius J, Allison M, Sidhu H, Peck A, Freel RW. Oxalobacter sp.
Reduces urinary oxalate excretion by promoting enteric oxalate secretion.
Kidney International 69: 691-698, 2005.

6. Hatch M, Freel RW. Intestinal transport of an obdurate anion: oxalate. Urol
Res 33: 1-16, 2005.

7. Hoppe B, Beck B, Gatter N, von Unruh G, Tischer A, Hesse A, Laube N,
Kaul P, Sidhu H. Oxalobacterformigenes: a potential tool for the treatment of
primary hyperoxaluria type 1. KidneyInt 70(7): 1305-1311, 2006.

8. Kleinschmidt K, Mahlmann A, Hautmann R. Microbial degradation of dietary
oxalate in the human gut and urinary oxalate concentrations in patients with
calcium oxalate urolithiasis and control persons. Investig Urol (Berhn) 5: 222-
224, 1994.

9. Kumar R, Mukherjee M, Bhandari M et al. Role of Oxalobacterformigenes in
calcium oxalate stone disease: a study from North India. Eur Urol 41: 318-
322, 2002.


gradients in vivo as was demonstrated in in vitro studies
conducted by us using Caco-2 monolayers (3). The latter
study showed that vectorial transport of oxalate mediated
specifically by PAT-1 is more dependent on the magnitude
and direction of counterion gradients than an intrinsic
property of the protein. Clearly, a better understanding of
the prevailing counterion driver gradients, in addition to
the relative affinities of the ions transported by these anion
exchangers, is required.


10. Kwak C, Kim HK Kim EC et al. Urinary oxalate levels and the enteric
bacterium Oxalobacter formigenes in patients with calcium oxalate
urolithiasis. Eur Urol 44: 475-481, 2003.

11. Mikami K, Akakura K, Takei K et al. Association of absence of intestinal
oxalate degrading bacteria with urinary calcium oxalate stone formation. Int J
Urol 10: 293-296, 2003.

12. Mittal RD, Kumar R, Mittal B et al. Stone composition, metabolic profile and
the presence of the gut-inhabiting bacterium Oxalobacter formigenes as risk
factors for renal stone formation. MedPrincPract 12: 208-213, 2003.

13. Neuhaus TJ, Belzer T, Blau N et al. Urinary oxalate excretion in urolithiasis
and nephrocalcinosis. Arch Dis Child 82: 322-326, 2000.

14. Sidhu H, Allison MJ, Chow JM et al. Rapid reversal of hyperoxaluria in a rat
model after probiotic administration of Oxalobacterformigenes. J Urol 166:
1487-1491, 2001.

15. Sidhu H, Hoppe B, Hesse A et al. Absence of Oxalobacter formigenes in
cystic fibrosis patients: a risk facter for hyperoxaluria. Lancet 352: 1026-1029,
1998.

16. Sidhu H, Schmidt ME, Cornelius JG et al. Direct correlation between
hyperoxaluria/oxalate stone disease and the absence of the gastrointestinal
tract-dwelling bacterium Oxalobacterformigenes: possible prevention by gut
recolonization or enzyme replacement therapy. J Am Soc Nephrol 10(Suppl
14): S334-S340, 1999.

17. Simpson JE, Schweinfest CW, Shull GE, Gawenis LR, Walker NM, Boyle
KT, Soleimani M, Clarke LL. PAT-1 (Slc26a6) is the predominant apical
membrane Cl /HCO3 exchanger in the upper villous epithelium of the
murine duodenum. Am J ::; ... .,' Gastrointest Liver _-:; ... .,' 292: G1079-
G1088,2007.

18. Wang Z, Petrovic S, Mann E, Soleimani M. Identification of an apical
Cl HCO3 exchanger in the small intestine. Am JP :, ... .,' Gastrointest Liver
F .'i.. .,' 282: G573-G579, 2002.


University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 I Summer 2011
6




University of Florida Home Page
© 2004 - 2011 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated May 24, 2011 - Version 3.0.2 - mvs