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RECOMBINANT OXALATE DECARBOXYLASE YVRK DEGRADES OXALATE IN A DOSE- AND TIME-DEPENDENT MANNER
THE JOURNAL OF UROLOGY®
Vol. 181, No. 4, Supplement, Wednesday, April 29, 2009
Stone Disease: Research/New Technology (I) Moderated Poster 61 Wednesday, April 29, 2009
8:00 am - 10:00 am
1826 GENOME-WIDE ASSOCIATION STUDY IDENTIFIES NEW SUSCEPTIBILITY LOCI FOR UROLITHIASIS. Masayuki Usami*, Yasunori Itoh, Keiichi Tozawa, Nagoya, Japan; Koichi Matsuda, Yusuke Nakamura, Tokyo, Japan; Kenjiro Kohri, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Urolithiasis is a worldwide problem, sparing no geographical, cultural, or racial groups. This disease affects many individuals, and its prevalence is continuously increasing in many countries, including Japan. Approximately 80% of stones are composed of calcium oxalate (CaOx) and calcium phosphate (CaP), 10% of struvite (magnesium ammonium phosphate produced during infection with bacteria that possess the enzyme urease), 9% of uric acid (UA), and the remaining 1% are composed of cystine or ammonium acid urate or are diagnosed as drug-related stones. Among those, idiopathic urolithiasis is mainly composed of CaOx and CaP, and is a common disease with complex genetic factors. Recently, there has been increasing evidence that genome-wide association studies represent a powerful approach to identify candidate genes involved in common human diseases. METHODS: We carried out a two-stage genome-wide association study using single nucleotide polymorphism (SNP) markers (stage 1: 195,300 SNPs in 194 cases and 1,542 controls; stage 2: 8,403 SNPs in 1,217 cases and 1,460 controls) in Japanese individuals with urolithiasis and unrelated controls. RESULTS: We detected two associated SNPs (rs1860300: 17p13.1 and rs10520320: 4q34.2) that were replicated in independent Japanese populations (1,411 cases and 3,002 controls). Combined analysis (4,065 cases and 4,045 controls) showed that these two SNPs were strongly associated with urolithiasis [rs1860300, dominant model: P=3.04×10-8; odds ratio (OR) =1.31, and rs10520320, additive model: P=7.76×10-8; OR=1.28]. Dense mapping revealed that several other SNPs in the same linkage disequilibrium (LD) blocks with rs1860300 and rs10520320 were strongly associated with urolithiasis, and GPM6A (glycoprotein M6A) was included in the LD block with rs10520320, which is located on GPM6A intron 1. CONCLUSIONS: Our report is the first result of a genome-wide association study of urolithiasis. This result shows that GPM6A is a new candidate gene for urolithiasis, and the polymorphism of this gene influences susceptibility to urolithiasis.
659
1827 IN VIVO ACTIVATION OF P38 MITOGEN ACTIVATED PROTEIN KINASE SIGNALING CASCADE IN A HYPEROXALURIC RAT MODEL Lakshmipathi Khandrika, Binod Kumar, Sweaty Koul, Randall B Meacham, Hari K Koul*, Aurora, CO INTRODUCTION AND OBJECTIVES: Nephrolithiais is a multifactorial disorder and increased urinary oxalate excretion is associated with sub-sets of this disorder. Though stone disease has been extensively studied, the molecular events that trigger crystal deposition are not completely understood. In previous studies, we observed oxalate induced activation of p38 MAP kinase plays an important role in oxalate induced changes in renal epithelial cells in tissue culture setting. The objective of the present studies was to evaluate the effects of hyperoxaluria on activation of p38 MAP kinase pathway in renal epithelial cells in vivo using an animal model of hyperoxaluria. METHODS: Male Sprague-Dawley rats were fed 0.75% Ethylene Glycol in drinking water. 24 hour urine was collected and analyzed for oxalate, calcium levels and creatinine clearance. After each week, animals were sacrificed and the kidneys dissected out for tissue histochemistry and protein analysis. Deposition of crystals in the kidneys was analyzed by polarized light microscopy. RESULTS: Our results show that the rats have mild hyperoxaluria within a week after starting on 0.75% ethylene glycol. The amount of urinary oxalate is increased in ethylene glycol fed rats within a week while calcium excretion was greatly reduced. There were progressive changes to the renal tubular epithelium as early as one week after hyperoxaluria, and crystal deposition was observed in tubules by polarized light microscopy by 3 weeks of hyperoxaluria. By four weeks the crystals also accumulated at the papillary tips. Hyperoxaluria was associated with increased activity of p38 Mitogen activated Protein Kinase (MAPK) as determined by ration of phospho p38 MAPK to total MAPK within a week. These results were also confirmed by western blot analysis of tissue lysate prepared from the dissected kidneys. These data for the first time offer a direct demonstration of activation of p38 Map kinase signaling cascade in vivo in response to hyperoxaluric challenge and are in agreement with our in vitro data. CONCLUSIONS: Our study shows that p38 MAPK is activated in renal tubular epithelial cells by hyperoxaluric exposure and activation of this pathway precedes crystal retention in the renal tubules. Our studies suggest that this signaling pathway might play an important role in the pathophysiology of crystal retention in response to hyperoxaluria. Source of Funding: NIH-RO1-DK-54084
1828 RECOMBINANT OXALATE DECARBOXYLASE YVRK DEGRADES OXALATE IN A DOSE- AND TIME-DEPENDENT MANNER Peter A Cadieux*, Lee Goneau, Maaike Vanjecek, John McCormick, John D Denstedt, London, ON, Canada
Source of Funding: None
INTRODUCTION AND OBJECTIVES: Approximately 80% of kidney stones are calcium oxalate and hyperoxaluric patients are at significantly increased risk for stone formation. Previous studies have shown that decreasing intestinal oxalate may reduce hyperoxaluria, potentially preventing stone formation. YvrK is an oxalate decarboxylase from Bacillus subtilis that degrades oxalate to formate in a single step. The objective of this study was to generate a recombinant YvrK that could be purified for potential use as a treatment for hyperoxaluric patients. METHODS: The gene encoding YvrK with an added histidine tag was cloned into the pDEST14 expression vector and transformed in E. coli BL21 cells. Cultures were grown to mid-log phase and YvrK expression induced with arabinose. Cell pellets were sonicated for cell wall disruption and protein release. Supernatants were tested for oxalatedegrading activity and Yvrk purified using Nickel columns. RESULTS: DNA sequencing and SDS-PAGE confirmed that YvrK was successfully cloned and expressed from E. coli under arabinose control. Supernatants containing YvrK strongly degraded oxalate in both a time- and dose-dependent manner. For example, pure supernatant
660
THE JOURNAL OF UROLOGY®
mixed 1:1 with 2mM oxalate completely degraded all of it in <30 minutes, and as little as 10% supernatant degraded over 80% in <90 minutes (p<0.01). Nickel-column chromatography successfully purified YvrK via the C-terminal his-tag, as confirmed by SDS-PAGE. CONCLUSIONS: This proof-of-principle study demonstrated the ability to generate an active, recombinant form of oxalate decarboxylase YvrK using an E. coli expression system. Future work will involve testing the strain and enzyme in a rat model of intestinally-induced hyperoxaluria. Source of Funding: None
1829 YVRK GENE RECOMBINANT E. COLI REDUCE THE CONCENTRATION OF URINE OXALATE IN TRANSIENT HYPEROXALURIA RAT MODEL Byong Chang Jeong*, Deok Hyun Han, Seong Il Seo, Seong Soo Jeon, Hyun Moo Lee, Han Yong Choi, Yong Hyun Park, Hyeon Hoe Kim, Seoul, Republic of Korea INTRODUCTION AND OBJECTIVES: Recently the whole DNA sequence of Bacillus subtilis has been identified, resulting in the new existence of YvrK gene encoding 43 KD-sized oxalate decarboxylase (OXDC) which degrades oxalate by a simple pathway. The objective of the study is to develop recombinant Escherichia coli (E. coli) expressing Yvrk gene from Bacillus subtilis and identify its oxalate-degrading activity in animal model METHODS: After the extraction of total DNA from B. subtiluis, YvrK gene was cloned using polymerase chain reaction. The cloned DNA encoding OXDC was inserted into the pBAD/gIII-A vector, downstream of L-arabinose promotor. The plasmid vector transformed into TOP 10 E. coli and selected with ampicillin. The recombinant E. coli, named pBy was then analyzed using DNA sequencing and Western blot. To evaluate oxalatedegrading function of pBy, the oxalate-degrading activity of homogenate of pBy was evaluated. The homogenate of pBy or not with sodium oxalate was given orally to male Sprague-Dawley rats in which suprapubic catheter was inserted. The hour urine was collected before and after oxalate intake and the urinary oxalate concentration was checked. RESULTS: DNA sequencing showed the successful transformation of YvrK gene into TOP 10 E. coli. Western blot analyses showed pBy expressed OXDC. pBy removed oxalate during the overnight culture in oxalate containing LB broth and the homogenate of pBy degraded 90% of oxalate under acidic condition. In vivo test, rats orally given pBy and sodium oxalate showed less urinary oxalate level than those given only sodium oxalate. CONCLUSIONS: A recombinant E. coli expressing YvrK gene was successfully produced. The bacteria showed the potent oxalatedegrading activity in vivo and in vitro study. The results of the study will provide a solution in the treatment of calcium oxalate stone and hyperoxaluria in which there has been few medical treatment modalities Source of Funding: None
1830 BYPASSING THE UROTHELIAL BARRIER: THE EFFECT OF PROTAMINE SULFATE ON URETERAL INTRALUMINAL DRUG ADMINISTRATION AND PERISTALSIS Stanislaw Shelkovnikov, Donald L Pick*, Michael K Louie, Adam G Kaplan, Elspeth M McDougall, Ralph V Clayman, Anaheim, CA INTRODUCTION AND OBJECTIVES: Relaxing the ureter prior to endourological procedures with the intraluminal administration of pharmacological agents would ease access and decrease procedural morbidity. The urothelium, however, presents a barrier to the passage of medications into the ureteral smooth muscle. Accordingly, we quantified the effect of protamine, on the permeability of this barrier. We use our novel ex-vivo ureteral apparatus, described elsewhere, with which we can control the intraluminal and extraluminal ureteral environments, and measure peristalsis before and after drug administration. METHODS: Freshly harvested 4 cm porcine ureteral segments
Vol. 181, No. 4, Supplement, Wednesday, April 29, 2009
were placed in a novel organ bath with gassed Krebs solution. Each ureter had both ends cannulated. A pressure transducer was placed in-series at the inflow end of each ureter. Outflow drained by gravity. Intraluminal flow (1 ml/min) was maintained by a constant pressure, gravity fed reservoir. For extraluminal and intraluminal administration, agents were added to the organ bath and the elevated reservoir, respectively. 10 μM phenylephrine was added to the external bath to induce peristalsis. After this, nifedipine was added to the intraluminal reservoir or to the external bath. The concentration of nifedipine required to block peristalsis was measured. Protamine sulfate (10 mg/ml, 20 min) was then used to denude the urothelium and the study repeated. RESULTS: Nifedipine blocked ureteral peristalsis; however, effective intraluminal concentrations were 10 times higher (p<0.0001) than extraluminal concentrations. Protamine sulfate significantly (p< 0.0001) reduced the intraluminal nifedipine concentrations needed by 10 fold (Figure 1). Protamine sulfate alone did not block peristalsis. CONCLUSIONS: Pretreatment of the intraluminal surface with protamine sulfate significantly decreased the concentration of nifedipine needed to block peristalsis by nearly tenfold. Protamine sulfate appears to increase urothelial permeability; clinical applications of this observation may be deserving of further study.
Source of Funding: None
1831 HEPARAN SULFATE (HSGP2, PERLECAN) GENE POLYMORPHISM IN CALCIUM OXALATE NEPHROLITHIASIS Metin Onaran*, Akin Yilmaz, Ilker Sen, Mehmet Ali Ergun, Ahmet Camtosun, Bora Kupeli, Sevda Menevse, Ibrahim Bozkirli, Ankara, Turkey INTRODUCTION AND OBJECTIVES: Calcium oxalate (CaOx) nephrolithiasis has a complex pathogenic mechanism with both cellular and extracellular factors. Besides environmental factors, genetic factors also have influence on stone formation. HSPG2 or perlecan is a member of heparan sulfate proteoglycan subgroup which was shown on the mesangium, Bowman’s capsule, the tubuler basement membrane and in tubuli located in the papillary area of the kidney in previous studies. This study represents the effects of this perlecan gene polymorphism which shows a single nucleotide polymorphism (SNP), for determining the risk of urolithiasis. METHODS: We investigated 143 CaOx stone formers with 158 healthy individuals as a control group. Genomic DNA extracted from the peripheral blood leukocytes were used to determine the BamHI restriction site polymorphism located in intron 6 of the HSPG gene using the polymerase chain reaction - restriction fragments length polymorphism (PCR-RFLP) method. RESULTS: After digestion with BamHI, the polymorphism was assumed to cause three genotypes according to the banding types as GG (242 bp), GT (242, 144 and 98 bp) and TT (144 and 98 bp) (Figure 1). According to the genotype frequencies between the stone-formers
Vol. 181, No. 4, Supplement, Wednesday, April 29, 2009
Stone Disease: Research/New Technology (I) Moderated Poster 61 Wednesday, April 29, 2009
8:00 am - 10:00 am
1826 GENOME-WIDE ASSOCIATION STUDY IDENTIFIES NEW SUSCEPTIBILITY LOCI FOR UROLITHIASIS. Masayuki Usami*, Yasunori Itoh, Keiichi Tozawa, Nagoya, Japan; Koichi Matsuda, Yusuke Nakamura, Tokyo, Japan; Kenjiro Kohri, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Urolithiasis is a worldwide problem, sparing no geographical, cultural, or racial groups. This disease affects many individuals, and its prevalence is continuously increasing in many countries, including Japan. Approximately 80% of stones are composed of calcium oxalate (CaOx) and calcium phosphate (CaP), 10% of struvite (magnesium ammonium phosphate produced during infection with bacteria that possess the enzyme urease), 9% of uric acid (UA), and the remaining 1% are composed of cystine or ammonium acid urate or are diagnosed as drug-related stones. Among those, idiopathic urolithiasis is mainly composed of CaOx and CaP, and is a common disease with complex genetic factors. Recently, there has been increasing evidence that genome-wide association studies represent a powerful approach to identify candidate genes involved in common human diseases. METHODS: We carried out a two-stage genome-wide association study using single nucleotide polymorphism (SNP) markers (stage 1: 195,300 SNPs in 194 cases and 1,542 controls; stage 2: 8,403 SNPs in 1,217 cases and 1,460 controls) in Japanese individuals with urolithiasis and unrelated controls. RESULTS: We detected two associated SNPs (rs1860300: 17p13.1 and rs10520320: 4q34.2) that were replicated in independent Japanese populations (1,411 cases and 3,002 controls). Combined analysis (4,065 cases and 4,045 controls) showed that these two SNPs were strongly associated with urolithiasis [rs1860300, dominant model: P=3.04×10-8; odds ratio (OR) =1.31, and rs10520320, additive model: P=7.76×10-8; OR=1.28]. Dense mapping revealed that several other SNPs in the same linkage disequilibrium (LD) blocks with rs1860300 and rs10520320 were strongly associated with urolithiasis, and GPM6A (glycoprotein M6A) was included in the LD block with rs10520320, which is located on GPM6A intron 1. CONCLUSIONS: Our report is the first result of a genome-wide association study of urolithiasis. This result shows that GPM6A is a new candidate gene for urolithiasis, and the polymorphism of this gene influences susceptibility to urolithiasis.
659
1827 IN VIVO ACTIVATION OF P38 MITOGEN ACTIVATED PROTEIN KINASE SIGNALING CASCADE IN A HYPEROXALURIC RAT MODEL Lakshmipathi Khandrika, Binod Kumar, Sweaty Koul, Randall B Meacham, Hari K Koul*, Aurora, CO INTRODUCTION AND OBJECTIVES: Nephrolithiais is a multifactorial disorder and increased urinary oxalate excretion is associated with sub-sets of this disorder. Though stone disease has been extensively studied, the molecular events that trigger crystal deposition are not completely understood. In previous studies, we observed oxalate induced activation of p38 MAP kinase plays an important role in oxalate induced changes in renal epithelial cells in tissue culture setting. The objective of the present studies was to evaluate the effects of hyperoxaluria on activation of p38 MAP kinase pathway in renal epithelial cells in vivo using an animal model of hyperoxaluria. METHODS: Male Sprague-Dawley rats were fed 0.75% Ethylene Glycol in drinking water. 24 hour urine was collected and analyzed for oxalate, calcium levels and creatinine clearance. After each week, animals were sacrificed and the kidneys dissected out for tissue histochemistry and protein analysis. Deposition of crystals in the kidneys was analyzed by polarized light microscopy. RESULTS: Our results show that the rats have mild hyperoxaluria within a week after starting on 0.75% ethylene glycol. The amount of urinary oxalate is increased in ethylene glycol fed rats within a week while calcium excretion was greatly reduced. There were progressive changes to the renal tubular epithelium as early as one week after hyperoxaluria, and crystal deposition was observed in tubules by polarized light microscopy by 3 weeks of hyperoxaluria. By four weeks the crystals also accumulated at the papillary tips. Hyperoxaluria was associated with increased activity of p38 Mitogen activated Protein Kinase (MAPK) as determined by ration of phospho p38 MAPK to total MAPK within a week. These results were also confirmed by western blot analysis of tissue lysate prepared from the dissected kidneys. These data for the first time offer a direct demonstration of activation of p38 Map kinase signaling cascade in vivo in response to hyperoxaluric challenge and are in agreement with our in vitro data. CONCLUSIONS: Our study shows that p38 MAPK is activated in renal tubular epithelial cells by hyperoxaluric exposure and activation of this pathway precedes crystal retention in the renal tubules. Our studies suggest that this signaling pathway might play an important role in the pathophysiology of crystal retention in response to hyperoxaluria. Source of Funding: NIH-RO1-DK-54084
1828 RECOMBINANT OXALATE DECARBOXYLASE YVRK DEGRADES OXALATE IN A DOSE- AND TIME-DEPENDENT MANNER Peter A Cadieux*, Lee Goneau, Maaike Vanjecek, John McCormick, John D Denstedt, London, ON, Canada
Source of Funding: None
INTRODUCTION AND OBJECTIVES: Approximately 80% of kidney stones are calcium oxalate and hyperoxaluric patients are at significantly increased risk for stone formation. Previous studies have shown that decreasing intestinal oxalate may reduce hyperoxaluria, potentially preventing stone formation. YvrK is an oxalate decarboxylase from Bacillus subtilis that degrades oxalate to formate in a single step. The objective of this study was to generate a recombinant YvrK that could be purified for potential use as a treatment for hyperoxaluric patients. METHODS: The gene encoding YvrK with an added histidine tag was cloned into the pDEST14 expression vector and transformed in E. coli BL21 cells. Cultures were grown to mid-log phase and YvrK expression induced with arabinose. Cell pellets were sonicated for cell wall disruption and protein release. Supernatants were tested for oxalatedegrading activity and Yvrk purified using Nickel columns. RESULTS: DNA sequencing and SDS-PAGE confirmed that YvrK was successfully cloned and expressed from E. coli under arabinose control. Supernatants containing YvrK strongly degraded oxalate in both a time- and dose-dependent manner. For example, pure supernatant
660
THE JOURNAL OF UROLOGY®
mixed 1:1 with 2mM oxalate completely degraded all of it in <30 minutes, and as little as 10% supernatant degraded over 80% in <90 minutes (p<0.01). Nickel-column chromatography successfully purified YvrK via the C-terminal his-tag, as confirmed by SDS-PAGE. CONCLUSIONS: This proof-of-principle study demonstrated the ability to generate an active, recombinant form of oxalate decarboxylase YvrK using an E. coli expression system. Future work will involve testing the strain and enzyme in a rat model of intestinally-induced hyperoxaluria. Source of Funding: None
1829 YVRK GENE RECOMBINANT E. COLI REDUCE THE CONCENTRATION OF URINE OXALATE IN TRANSIENT HYPEROXALURIA RAT MODEL Byong Chang Jeong*, Deok Hyun Han, Seong Il Seo, Seong Soo Jeon, Hyun Moo Lee, Han Yong Choi, Yong Hyun Park, Hyeon Hoe Kim, Seoul, Republic of Korea INTRODUCTION AND OBJECTIVES: Recently the whole DNA sequence of Bacillus subtilis has been identified, resulting in the new existence of YvrK gene encoding 43 KD-sized oxalate decarboxylase (OXDC) which degrades oxalate by a simple pathway. The objective of the study is to develop recombinant Escherichia coli (E. coli) expressing Yvrk gene from Bacillus subtilis and identify its oxalate-degrading activity in animal model METHODS: After the extraction of total DNA from B. subtiluis, YvrK gene was cloned using polymerase chain reaction. The cloned DNA encoding OXDC was inserted into the pBAD/gIII-A vector, downstream of L-arabinose promotor. The plasmid vector transformed into TOP 10 E. coli and selected with ampicillin. The recombinant E. coli, named pBy was then analyzed using DNA sequencing and Western blot. To evaluate oxalatedegrading function of pBy, the oxalate-degrading activity of homogenate of pBy was evaluated. The homogenate of pBy or not with sodium oxalate was given orally to male Sprague-Dawley rats in which suprapubic catheter was inserted. The hour urine was collected before and after oxalate intake and the urinary oxalate concentration was checked. RESULTS: DNA sequencing showed the successful transformation of YvrK gene into TOP 10 E. coli. Western blot analyses showed pBy expressed OXDC. pBy removed oxalate during the overnight culture in oxalate containing LB broth and the homogenate of pBy degraded 90% of oxalate under acidic condition. In vivo test, rats orally given pBy and sodium oxalate showed less urinary oxalate level than those given only sodium oxalate. CONCLUSIONS: A recombinant E. coli expressing YvrK gene was successfully produced. The bacteria showed the potent oxalatedegrading activity in vivo and in vitro study. The results of the study will provide a solution in the treatment of calcium oxalate stone and hyperoxaluria in which there has been few medical treatment modalities Source of Funding: None
1830 BYPASSING THE UROTHELIAL BARRIER: THE EFFECT OF PROTAMINE SULFATE ON URETERAL INTRALUMINAL DRUG ADMINISTRATION AND PERISTALSIS Stanislaw Shelkovnikov, Donald L Pick*, Michael K Louie, Adam G Kaplan, Elspeth M McDougall, Ralph V Clayman, Anaheim, CA INTRODUCTION AND OBJECTIVES: Relaxing the ureter prior to endourological procedures with the intraluminal administration of pharmacological agents would ease access and decrease procedural morbidity. The urothelium, however, presents a barrier to the passage of medications into the ureteral smooth muscle. Accordingly, we quantified the effect of protamine, on the permeability of this barrier. We use our novel ex-vivo ureteral apparatus, described elsewhere, with which we can control the intraluminal and extraluminal ureteral environments, and measure peristalsis before and after drug administration. METHODS: Freshly harvested 4 cm porcine ureteral segments
Vol. 181, No. 4, Supplement, Wednesday, April 29, 2009
were placed in a novel organ bath with gassed Krebs solution. Each ureter had both ends cannulated. A pressure transducer was placed in-series at the inflow end of each ureter. Outflow drained by gravity. Intraluminal flow (1 ml/min) was maintained by a constant pressure, gravity fed reservoir. For extraluminal and intraluminal administration, agents were added to the organ bath and the elevated reservoir, respectively. 10 μM phenylephrine was added to the external bath to induce peristalsis. After this, nifedipine was added to the intraluminal reservoir or to the external bath. The concentration of nifedipine required to block peristalsis was measured. Protamine sulfate (10 mg/ml, 20 min) was then used to denude the urothelium and the study repeated. RESULTS: Nifedipine blocked ureteral peristalsis; however, effective intraluminal concentrations were 10 times higher (p<0.0001) than extraluminal concentrations. Protamine sulfate significantly (p< 0.0001) reduced the intraluminal nifedipine concentrations needed by 10 fold (Figure 1). Protamine sulfate alone did not block peristalsis. CONCLUSIONS: Pretreatment of the intraluminal surface with protamine sulfate significantly decreased the concentration of nifedipine needed to block peristalsis by nearly tenfold. Protamine sulfate appears to increase urothelial permeability; clinical applications of this observation may be deserving of further study.
Source of Funding: None
1831 HEPARAN SULFATE (HSGP2, PERLECAN) GENE POLYMORPHISM IN CALCIUM OXALATE NEPHROLITHIASIS Metin Onaran*, Akin Yilmaz, Ilker Sen, Mehmet Ali Ergun, Ahmet Camtosun, Bora Kupeli, Sevda Menevse, Ibrahim Bozkirli, Ankara, Turkey INTRODUCTION AND OBJECTIVES: Calcium oxalate (CaOx) nephrolithiasis has a complex pathogenic mechanism with both cellular and extracellular factors. Besides environmental factors, genetic factors also have influence on stone formation. HSPG2 or perlecan is a member of heparan sulfate proteoglycan subgroup which was shown on the mesangium, Bowman’s capsule, the tubuler basement membrane and in tubuli located in the papillary area of the kidney in previous studies. This study represents the effects of this perlecan gene polymorphism which shows a single nucleotide polymorphism (SNP), for determining the risk of urolithiasis. METHODS: We investigated 143 CaOx stone formers with 158 healthy individuals as a control group. Genomic DNA extracted from the peripheral blood leukocytes were used to determine the BamHI restriction site polymorphism located in intron 6 of the HSPG gene using the polymerase chain reaction - restriction fragments length polymorphism (PCR-RFLP) method. RESULTS: After digestion with BamHI, the polymorphism was assumed to cause three genotypes according to the banding types as GG (242 bp), GT (242, 144 and 98 bp) and TT (144 and 98 bp) (Figure 1). According to the genotype frequencies between the stone-formers