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2084 DIFFERENTIAL EFFECTS OF OXALATE ON EXPRESSION OF GENES RELATED TO NEPHROSIS
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THE JOURNAL OF UROLOGY姞
2084 DIFFERENTIAL EFFECTS OF OXALATE ON EXPRESSION OF GENES RELATED TO NEPHROSIS Sweaty Koul, Lakshmipathi Khandrika, Randall Meacham, Hari Koul*, Aurora, CO INTRODUCTION AND OBJECTIVES: Kidney stones are a major cause of morbidity and increased urinary oxalate is an established risk factor for nephrolithiasis. Oxalate interactions with the renal cells results in a plethora of changes, including cell growth, death and altered gene expression as shown in previous studies. We are the first group to report global changes in gene expression following oxalate exposure to human renal cells. In this follow up study we investigated the effects of oxalate on changes in nephrosis related genes that undergo changes in detail. METHODS: Results from this study shows potential pathways associated with oxalate nephrotoxicity. Our analysis of genes show that many genes that are potentially involved in nephrosis and injury to the kidney were identified to be differentially expressed in HK-2 cells upon exposure to oxalate for as little as 4 hours. Of interest groups of genes associated with cell proliferation and anti-apoptosis are down regulated, while groups of genes associated with apoptosis and glomerular injury are upregulated by oxalate exposure. Specific nephrosis related genes that are upregulated include AOX1, SLC44A2, POU2F3, TOM1L2, TLN2 and SAFB2. These genes were up-regulated early on at least within 4 hr of oxalate exposure. A set of nephrosos related genes that showed up-regulation at 24 hrs after oxalate exposure include SAMSN1, CAPTM5, OLA1, CALB2, SPEF2, NUP214, ARMC2, KRT9 and KRT7. RESULTS: Results from this study shows potential pathways associated with oxalate nephrotoxicity. Our analysis of genes show that many genes that are potentially involved in nephrosis and injury to the kidney were identified to be differentially expressed in HK-2 cells upon exposure to oxalate for as little as 4 hours. Of interest groups of genes associated with cell proliferation and anti-apoptosis are down regulated, while groups of genes associated with apoptosis and glomerular injury are upregulated by oxalate exposure. Specific nephrosis related genes that are upregulated include AOX1, SLC44A2, POU2F3, TOM1L2, TLN2 and SAFB2. These genes were up-regulated early on at least within 4 hr of oxalate exposure. A set of nephrosos related genes that showed up-regulation at 24 hrs after oxalate exposure include SAMSN1, CAPTM5, OLA1, CALB2, SPEF2, NUP214, ARMC2, KRT9 and KRT7. CONCLUSIONS: The results of this study also provide first direct demonstration of activation of differential sets of nephrosos related genes as early and late responders in response to oxalate, a metabolic end product that is excreted by the kidney and is associated with nephrolithiasis. Source of Funding: NIH-RO1-DK-54804(H. Koul, PI); Department of Surgery and School of Medicine Academic Enrichment Funds (H. Koul)
2085 MACROPHAGE-COLONY STIMULATING FACTOR (M-CSF) IS A NOVEL PREVENTIVE AGENT AGAINST RENAL STONE DISEASE BY INDUCING ANTI-INFLAMMATORY MACROPHAGES Kazumi Taguchi*, Atsushi Okada, Yasuhiro Fujii, Kazuhiro Niimi, Shuzo Hamamoto, Masahito Hirose, Yasunori Itoh, Takahiro Yasui, Keiichi Tozawa, Kenjiro Kohri, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Macrophages are related to metabolic syndrome, and considered as promotional factor. There are some reports macrophages play not only an inflammatory role but also anti-inflammatory role. We have reported that macrophages were related to stone formation in a microarray study and an interesting phenomenon involving the spontaneous elimination of renal crystals. We speculated there were some correlation between this phenomenon and macrophages. We investigated renal macrophage
Vol. 187, No. 4S, Supplement, Wednesday, May 23, 2012
functions in order to clarify their anti-inflammatory effect for stone formation using macrophage-colony stimulating factor (M-CSF)-deficient mice. METHODS: We divided Eight-week-old male M-CSF-deficient mice into 3 groups: wild type (⫹/⫹), homozygous (op/op), and homozygous injected with M-CSF (op⫹CSF). They were administered 80mg/kg glyoxylate by daily intra-abdominal injection, and the kidneys were extracted to examine crystal formation, at days 3, 6, 9, 12, 15. We performed CD68 and CD163 staining to evaluate the expression of renal macrophages. CD68 was used for detection of inflammatory macrophages whereas CD163 for anti-inflammatory ones. Both crystal and macrophage formations were evaluated with scanning electron microscopy (SEM) and transmitted electron microscopy (TEM). Expression of inflammation-related genes was examined by immunohistochemistry (IHC) and quantitative reverse transcriptase polymerase chain reaction (qPCR). RESULTS: The number of renal crystals in op/op was markedly higher composed to ⫹/⫹. Crystal formations were detected in the cortical-medulla region in ⫹/⫹ whereas crystals were detected in the papilla in op/op. SEM showed crystals were rough and larger size in op/op than ⫹/⫹. IHC and qPCR showed high expression of osteopontin and CD44 but low expression of CD163 in op/op. After injection of M-CSF, the amount of stones in op⫹CSF was markedly decreased than op/op and it depended on the M-CSF concentration. Each gene expression in op⫹CSF returned to same level as in ⫹/⫹. Furthermore, TEM revealed crystals were phagocytosed by antiinflammatory macrophages in the cortical-medulla region, except for op/op. CONCLUSIONS: Our study suggests that anti-inflammatory macrophages play a major role in defense against crystal formation by elimination in the renal interstitial space. We indicate that M-CSF could become a novel medicine for prevention of stone disease by inducing anti-inflammatory macrophages. Source of Funding: None
2086 RENAL MACROPHAGES COULD ENGLOBE CALCIUM OXALATE CRYSTALS DURING KIDNEY STONE FORMATION IN VITRO AND IN VIVO MODELS Atsushi Okada*, Takahiro Yasui, Kazumi Taguchi, Yasuhiko Hirose, Kazuhiro Niimi, Yasuhiro Fujii, Shuzo Hamamoto, Masahito Hirose, Yasunori Itoh, Keiichi Tozawa, Kenjiro Kohri, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Our recent investigation indicated that mice have strong renoprotective function against kidney stone formation and macrophage activity could be responsible for it. In the present study, macrophage phagocytosis of calcium oxalate crystals was investigated by conducting an in vitro study of calcium oxalate crystal phagocytosis and an in vivo study by immunohistochemistry and ultrastructural observation of mouse models with crystal-framed kidneys. METHODS: Experiment 1: Using cultured mouse macrophage cells (J774.1) and mouse renal tubular cells (M1), a phagocytosis model for calcium oxalate monohydrate (COM) crystals was established. The COM crystal phagocytosis rates of each cell group after exposure to the COM crystals (12.5 and 62.5 g/cm2) were evaluated. To establish a phagocytosis-inhibited group, cytochalasin B (CB), with a final concentration of 10 g/mL (20 M), was administered to similar experimental systems. Experiment 2: The 8-week-old male C57BL/6 mice were administered glyoxylate, an oxalate precursor, by daily intra-abdominal injections for 15 days, and kidney specimens were extracted every 3 days. Renal crystals were detected by Pizzolato staining for calcium oxalate crystals and polarized light optical microphotography. The amount of renal crystal formation was quantified by an image analysis software (ImagePro Plus®). Renal macrophage migration was detected by immunohistochemistry for F4/80, a mouse macrophage surface marker, and the number of macrophages with the F4/80 gene
THE JOURNAL OF UROLOGY姞
2084 DIFFERENTIAL EFFECTS OF OXALATE ON EXPRESSION OF GENES RELATED TO NEPHROSIS Sweaty Koul, Lakshmipathi Khandrika, Randall Meacham, Hari Koul*, Aurora, CO INTRODUCTION AND OBJECTIVES: Kidney stones are a major cause of morbidity and increased urinary oxalate is an established risk factor for nephrolithiasis. Oxalate interactions with the renal cells results in a plethora of changes, including cell growth, death and altered gene expression as shown in previous studies. We are the first group to report global changes in gene expression following oxalate exposure to human renal cells. In this follow up study we investigated the effects of oxalate on changes in nephrosis related genes that undergo changes in detail. METHODS: Results from this study shows potential pathways associated with oxalate nephrotoxicity. Our analysis of genes show that many genes that are potentially involved in nephrosis and injury to the kidney were identified to be differentially expressed in HK-2 cells upon exposure to oxalate for as little as 4 hours. Of interest groups of genes associated with cell proliferation and anti-apoptosis are down regulated, while groups of genes associated with apoptosis and glomerular injury are upregulated by oxalate exposure. Specific nephrosis related genes that are upregulated include AOX1, SLC44A2, POU2F3, TOM1L2, TLN2 and SAFB2. These genes were up-regulated early on at least within 4 hr of oxalate exposure. A set of nephrosos related genes that showed up-regulation at 24 hrs after oxalate exposure include SAMSN1, CAPTM5, OLA1, CALB2, SPEF2, NUP214, ARMC2, KRT9 and KRT7. RESULTS: Results from this study shows potential pathways associated with oxalate nephrotoxicity. Our analysis of genes show that many genes that are potentially involved in nephrosis and injury to the kidney were identified to be differentially expressed in HK-2 cells upon exposure to oxalate for as little as 4 hours. Of interest groups of genes associated with cell proliferation and anti-apoptosis are down regulated, while groups of genes associated with apoptosis and glomerular injury are upregulated by oxalate exposure. Specific nephrosis related genes that are upregulated include AOX1, SLC44A2, POU2F3, TOM1L2, TLN2 and SAFB2. These genes were up-regulated early on at least within 4 hr of oxalate exposure. A set of nephrosos related genes that showed up-regulation at 24 hrs after oxalate exposure include SAMSN1, CAPTM5, OLA1, CALB2, SPEF2, NUP214, ARMC2, KRT9 and KRT7. CONCLUSIONS: The results of this study also provide first direct demonstration of activation of differential sets of nephrosos related genes as early and late responders in response to oxalate, a metabolic end product that is excreted by the kidney and is associated with nephrolithiasis. Source of Funding: NIH-RO1-DK-54804(H. Koul, PI); Department of Surgery and School of Medicine Academic Enrichment Funds (H. Koul)
2085 MACROPHAGE-COLONY STIMULATING FACTOR (M-CSF) IS A NOVEL PREVENTIVE AGENT AGAINST RENAL STONE DISEASE BY INDUCING ANTI-INFLAMMATORY MACROPHAGES Kazumi Taguchi*, Atsushi Okada, Yasuhiro Fujii, Kazuhiro Niimi, Shuzo Hamamoto, Masahito Hirose, Yasunori Itoh, Takahiro Yasui, Keiichi Tozawa, Kenjiro Kohri, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Macrophages are related to metabolic syndrome, and considered as promotional factor. There are some reports macrophages play not only an inflammatory role but also anti-inflammatory role. We have reported that macrophages were related to stone formation in a microarray study and an interesting phenomenon involving the spontaneous elimination of renal crystals. We speculated there were some correlation between this phenomenon and macrophages. We investigated renal macrophage
Vol. 187, No. 4S, Supplement, Wednesday, May 23, 2012
functions in order to clarify their anti-inflammatory effect for stone formation using macrophage-colony stimulating factor (M-CSF)-deficient mice. METHODS: We divided Eight-week-old male M-CSF-deficient mice into 3 groups: wild type (⫹/⫹), homozygous (op/op), and homozygous injected with M-CSF (op⫹CSF). They were administered 80mg/kg glyoxylate by daily intra-abdominal injection, and the kidneys were extracted to examine crystal formation, at days 3, 6, 9, 12, 15. We performed CD68 and CD163 staining to evaluate the expression of renal macrophages. CD68 was used for detection of inflammatory macrophages whereas CD163 for anti-inflammatory ones. Both crystal and macrophage formations were evaluated with scanning electron microscopy (SEM) and transmitted electron microscopy (TEM). Expression of inflammation-related genes was examined by immunohistochemistry (IHC) and quantitative reverse transcriptase polymerase chain reaction (qPCR). RESULTS: The number of renal crystals in op/op was markedly higher composed to ⫹/⫹. Crystal formations were detected in the cortical-medulla region in ⫹/⫹ whereas crystals were detected in the papilla in op/op. SEM showed crystals were rough and larger size in op/op than ⫹/⫹. IHC and qPCR showed high expression of osteopontin and CD44 but low expression of CD163 in op/op. After injection of M-CSF, the amount of stones in op⫹CSF was markedly decreased than op/op and it depended on the M-CSF concentration. Each gene expression in op⫹CSF returned to same level as in ⫹/⫹. Furthermore, TEM revealed crystals were phagocytosed by antiinflammatory macrophages in the cortical-medulla region, except for op/op. CONCLUSIONS: Our study suggests that anti-inflammatory macrophages play a major role in defense against crystal formation by elimination in the renal interstitial space. We indicate that M-CSF could become a novel medicine for prevention of stone disease by inducing anti-inflammatory macrophages. Source of Funding: None
2086 RENAL MACROPHAGES COULD ENGLOBE CALCIUM OXALATE CRYSTALS DURING KIDNEY STONE FORMATION IN VITRO AND IN VIVO MODELS Atsushi Okada*, Takahiro Yasui, Kazumi Taguchi, Yasuhiko Hirose, Kazuhiro Niimi, Yasuhiro Fujii, Shuzo Hamamoto, Masahito Hirose, Yasunori Itoh, Keiichi Tozawa, Kenjiro Kohri, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Our recent investigation indicated that mice have strong renoprotective function against kidney stone formation and macrophage activity could be responsible for it. In the present study, macrophage phagocytosis of calcium oxalate crystals was investigated by conducting an in vitro study of calcium oxalate crystal phagocytosis and an in vivo study by immunohistochemistry and ultrastructural observation of mouse models with crystal-framed kidneys. METHODS: Experiment 1: Using cultured mouse macrophage cells (J774.1) and mouse renal tubular cells (M1), a phagocytosis model for calcium oxalate monohydrate (COM) crystals was established. The COM crystal phagocytosis rates of each cell group after exposure to the COM crystals (12.5 and 62.5 g/cm2) were evaluated. To establish a phagocytosis-inhibited group, cytochalasin B (CB), with a final concentration of 10 g/mL (20 M), was administered to similar experimental systems. Experiment 2: The 8-week-old male C57BL/6 mice were administered glyoxylate, an oxalate precursor, by daily intra-abdominal injections for 15 days, and kidney specimens were extracted every 3 days. Renal crystals were detected by Pizzolato staining for calcium oxalate crystals and polarized light optical microphotography. The amount of renal crystal formation was quantified by an image analysis software (ImagePro Plus®). Renal macrophage migration was detected by immunohistochemistry for F4/80, a mouse macrophage surface marker, and the number of macrophages with the F4/80 gene