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ENDOSTATIN IS UPREGULATED IN URETERIC OBSTRUCTION AND ENDOSTATIN IS PROFIBROTIC THROUGH INHIBITION OF HEPATOCYTE GROWTH FACTOR
564
THE JOURNAL OF UROLOGY®
stone matrix is much more complex than previously thought. Matrix extracted from both calcium oxalate and calcium phosphate stones FRQWDLQV D ODUJH QXPEHU RI LQÀDPPDWRU\ SURWHLQV VLJQLI\LQJ HLWKHU D causal or secondary role in calcium stone pathogenesis. Source of Funding: B.K.C. is supported by a Foundation Research Scholar Grant from the AUA and a Biomedical Genomics Seed Grant from the University of Minnesota.
1651 OSTEOPONTIN HAS A CRUCIAL ROLE IN ULTRASTRUCTURAL CONVERSION OF CALCIUM OXALATE CRYSTALS INTO MATRIXINVOLVING KIDNEY STONES Atsushi Okada*, Shuzo Hamamoto, Masahito Hirose, Yasunori Itoh, Takahiro Yasui, Keiichi Tozawa, Kenjiro Kohri. Nagoya Aichi, Japan. INTRODUCTION AND OBJECTIVE: The starting point of kidney stone formation is the conversion of retained intratubular crystals into concrete stones. We previously reported a strong expression of osteopontin (OPN) on renal tubular cells in the stone-forming kidney, suggesting that OPN plays important roles in crystal-cell interaction and VWRQH IRUPDWLRQ ,Q WKH SUHVHQW VWXG\ ZH DQDO\]HG SDWKRORJLFDO DQG ultrastructural differences of generated kidney crystal deposition, and determined the role of OPN in the morphological changes of calcium oxalate crystals using OPN knockout mice. METHODS: Saline and glyoxylate (80 or 100 mg/kg) were intraabdominally injected into wild-type mice (WT) and OPN knockout mice (KO) for a week, and kidneys and 24hr urine samples were taken on day 7 after the initiation of glyoxylate administration. OPN expression ZDV GHWHFWHG E\ TXDQWLWDWLYH 573&5 LQ VLWX K\EULGL]DWLRQ DQG immunohistochemical staining. Urine biochemistry was determined from 24h urine. Kidney crystal formation was estimated by light microscopy DQGPRUSKRORJLFDOREVHUYDWLRQZDVSHUIRUPHGE\SRODUL]HGOLJKWRSWLFDO microphotography and scanning electron microphotography. Crystal components were detected by X-ray diffraction. 5(68/76 8ULQH R[DODWH H[FUHWLRQ VKRZHG QR VLJQL¿FDQW difference between WT and KO. Kidney crystal depositions were detected in WT administered with 80 and 100mg/kg glyoxylate and KO administered 100mg/kg glyoxylate, but there were no crystals in KO with 80mg/kg administration. The number of kidney crystals generated in the NLGQH\RI.2DGPLQLVWHUHGPJNJJO\R[\ODWHZDVVLJQL¿FDQWO\IHZHU than that of WT administered 100mg/kg glyoxylate. OPN expression was detected in WT mouse kidney, but not KO kidneys, and OPN protein was contained in WT crystals by observation of immunohistochemical VWDLQLQJ 3RODUL]HG OLJKW RSWLFDO PLFURSKRWRJUDSK\ DQG 6(0 VKRZHG large rosette-shaped crystals growing in renal tubules in WT, whereas small and disorderly scattered crystals in KO. The crystal component of both genotypes was calcium oxalate monohydrate. CONCLUSIONS: Osteopontin has a crucial role in ultrastructural conversion of calcium oxalate crystals into matrix-involving kidney stones.
Source of Funding: None
1652 COLLAGEN XVIII/ENDOSTATIN IS UPREGULATED IN URETERIC OBSTRUCTION AND ENDOSTATIN IS PROFIBROTIC THROUGH
Vol. 179, No. 4, Supplement, Tuesday, May 20, 2008
INHIBITION OF HEPATOCYTE GROWTH FACTOR Barry B McGuire*, R William G Watson, John M Fitzpatrick, Neil G Docherty. Dublin, Ireland. INTRODUCTION AND OBJECTIVE: Acute ureteric obstruction (UO) leads to hydronephrosis and can progress to tubulointerstitial ¿EURVLV 3UHYLRXV *HQH FKLS DQDO\VLV RI DFXWH 82 LQ UDWV LGHQWL¿HG XSUHJXODWLRQRI&ROODJHQ;9,,,Į7KHFDUER[\OWHUPLQDORI&ROODJHQ;9,,, contains a region sensitive to protease cleavage releasing fragments with anti-angiogenic properties (Endostatin). Hepatocyte Growth Factor +*) LV DQWL¿EURWLF WKURXJK LQKLELWLRQ RI 6PDG SKRVSKRU\ODWLRQ preventing upregulation of Connective Tissue Growth Factor (CTGF). Endostatin prevents excessive outgrowth of the developing ureteric bud during embryogenesis by opposing HGF. METHODS: In vivo: Left ureters of male rats were obstructed for 3 or 10 days. The expression of Collagen XVIII and Endostatin was compared in the obstructed (Ob.) and non-obstructed (N.O.) kidneys by Western Blotting, RT-PCR and Immunohistochemistry. MMP activity and H[SUHVVLRQZDVH[DPLQHGXVLQJ=\PRJUDSK\DQG573&5UHVSHFWLYHO\ In vitro:+XPDQ.LGQH\FHOOV +. ZHUHWUHDWHGZLWK7*)ȕIRU hours. Supernatant was analysed for Endostatin via Western Blotting, and cellular MMP upregulation via RT-PCR. The relationship between (QGRVWDWLQ UHOHDVH DQG SURWHRO\WLF HQ]\PHV ZDV H[DPLQHG ZLWK SDQSURWHRO\WLF HQ]\PH LQKLELWLRQ 7*)ȕ &HOOV ZHUH WUHDWHG ZLWK Recombinant HGF and/or Endostatin for 48 hours to examine the pro- or DQWL¿EURWLFSRWHQWLDORI(QGRVWDWLQ RESULTS: Collagen XVIII mRNA and protein expression is upregulated in Ob. kidney and there is increased generation of a 28kD Endostatin fragment. Immunohistochemistry demonstrates &ROODJHQ;9,,,ORFDOL]DWLRQLQWXEXOHVDQGQHJDWLYHLQJORPHUXOL7KHUH LVXSUHJXODWLRQRI(QGRVWDWLQUHOHDVLQJHQ]\PHVLQ2ENLGQH\In-vitro DQDO\VLVGHPRQVWUDWHVWKDW7*)ȕWUHDWHG+.FHOOVGRQRWXSUHJXODWH &ROODJHQ ;9,,, KRZHYHU 7*)ȕ FDXVHV XSUHJXODWLRQ RI (QGRVWDWLQ UHOHDVLQJHQ]\PHVDQGFRQFRPLWDQW(QGRVWDWLQGHWHFWLRQLQVXSHUQDWDQW ,QKLELWLQJSURWHRO\WLFHQ]\PHVSUHYHQWV(QGRVWDWLQGHJUDGDWLRQ$GGLWLRQ RI+*)WR7*)ȕWUHDWHGFHOOVSUHYHQWVXSUHJXODWLRQRI&7*)DQGWKH addition of Endostatin opposes HGF action resulting in upregulation of CTGF. CONCLUSIONS: UO leads to increased generation of Collagen XVIII and a 28kDa Endostatin fragment. HK-2 cells, when WUHDWHG ZLWK 7*)ȕ UHOHDVH (QGRVWDWLQ LQWR WKH VXSHUQDWDQW DQG degradation of Endostatin is via proteolytic activity. Endostatin is pro¿EURWLFE\XSUHJXODWLQJ&7*)WKURXJKDQWDJRQLVPRI+*) Source of Funding: British Urological Foundation, Mater Misericordiae Hospital.
1653 OSTEOPONTIN AND TAMM-HORSFALL PROTEIN ARE FUNCTIONALLY SYNERGISTIC IN PREVENTING RENAL CALCIFICATION Lan Mo, Lucy Liaw, Andrew P Evan, Andre J Sommer, John C Lieske, Xue-Ru Wu*. New York, NY, Scarborough, ME, Indianapolis, IN, Oxford, OH, and Rochester, MN. INTRODUCTION AND OBJECTIVE: A key requirement for the kidney to maintain homeostasis is to prevent often supersaturated mineral salts from forming insoluble crystals. This property has been ascribed at least in part to the urinary presence of macromolecules, although the exact role(s) of these proteins in vivo are only beginning to be unraveled. Here we use knockout mouse models to study the effects of the loss of osteopontin (OPN) and Tamm-Horsfall protein (THP), either alone or in a combination, in spontaneous and chemically LQGXFHGUHQDOFDOFL¿FDWLRQ METHODS: Mice lacking OPN and THP were intercrossed and back-crossed to yield four major genotypes: wild-type, OPN knockout (KO), THP KO and OPN/THP double KO mice. Spontaneous renal FDOFL¿FDWLRQZDVDVVHVVHGE\YRQ.RVVDDQG
THE JOURNAL OF UROLOGY®
stone matrix is much more complex than previously thought. Matrix extracted from both calcium oxalate and calcium phosphate stones FRQWDLQV D ODUJH QXPEHU RI LQÀDPPDWRU\ SURWHLQV VLJQLI\LQJ HLWKHU D causal or secondary role in calcium stone pathogenesis. Source of Funding: B.K.C. is supported by a Foundation Research Scholar Grant from the AUA and a Biomedical Genomics Seed Grant from the University of Minnesota.
1651 OSTEOPONTIN HAS A CRUCIAL ROLE IN ULTRASTRUCTURAL CONVERSION OF CALCIUM OXALATE CRYSTALS INTO MATRIXINVOLVING KIDNEY STONES Atsushi Okada*, Shuzo Hamamoto, Masahito Hirose, Yasunori Itoh, Takahiro Yasui, Keiichi Tozawa, Kenjiro Kohri. Nagoya Aichi, Japan. INTRODUCTION AND OBJECTIVE: The starting point of kidney stone formation is the conversion of retained intratubular crystals into concrete stones. We previously reported a strong expression of osteopontin (OPN) on renal tubular cells in the stone-forming kidney, suggesting that OPN plays important roles in crystal-cell interaction and VWRQH IRUPDWLRQ ,Q WKH SUHVHQW VWXG\ ZH DQDO\]HG SDWKRORJLFDO DQG ultrastructural differences of generated kidney crystal deposition, and determined the role of OPN in the morphological changes of calcium oxalate crystals using OPN knockout mice. METHODS: Saline and glyoxylate (80 or 100 mg/kg) were intraabdominally injected into wild-type mice (WT) and OPN knockout mice (KO) for a week, and kidneys and 24hr urine samples were taken on day 7 after the initiation of glyoxylate administration. OPN expression ZDV GHWHFWHG E\ TXDQWLWDWLYH 573&5 LQ VLWX K\EULGL]DWLRQ DQG immunohistochemical staining. Urine biochemistry was determined from 24h urine. Kidney crystal formation was estimated by light microscopy DQGPRUSKRORJLFDOREVHUYDWLRQZDVSHUIRUPHGE\SRODUL]HGOLJKWRSWLFDO microphotography and scanning electron microphotography. Crystal components were detected by X-ray diffraction. 5(68/76 8ULQH R[DODWH H[FUHWLRQ VKRZHG QR VLJQL¿FDQW difference between WT and KO. Kidney crystal depositions were detected in WT administered with 80 and 100mg/kg glyoxylate and KO administered 100mg/kg glyoxylate, but there were no crystals in KO with 80mg/kg administration. The number of kidney crystals generated in the NLGQH\RI.2DGPLQLVWHUHGPJNJJO\R[\ODWHZDVVLJQL¿FDQWO\IHZHU than that of WT administered 100mg/kg glyoxylate. OPN expression was detected in WT mouse kidney, but not KO kidneys, and OPN protein was contained in WT crystals by observation of immunohistochemical VWDLQLQJ 3RODUL]HG OLJKW RSWLFDO PLFURSKRWRJUDSK\ DQG 6(0 VKRZHG large rosette-shaped crystals growing in renal tubules in WT, whereas small and disorderly scattered crystals in KO. The crystal component of both genotypes was calcium oxalate monohydrate. CONCLUSIONS: Osteopontin has a crucial role in ultrastructural conversion of calcium oxalate crystals into matrix-involving kidney stones.
Source of Funding: None
1652 COLLAGEN XVIII/ENDOSTATIN IS UPREGULATED IN URETERIC OBSTRUCTION AND ENDOSTATIN IS PROFIBROTIC THROUGH
Vol. 179, No. 4, Supplement, Tuesday, May 20, 2008
INHIBITION OF HEPATOCYTE GROWTH FACTOR Barry B McGuire*, R William G Watson, John M Fitzpatrick, Neil G Docherty. Dublin, Ireland. INTRODUCTION AND OBJECTIVE: Acute ureteric obstruction (UO) leads to hydronephrosis and can progress to tubulointerstitial ¿EURVLV 3UHYLRXV *HQH FKLS DQDO\VLV RI DFXWH 82 LQ UDWV LGHQWL¿HG XSUHJXODWLRQRI&ROODJHQ;9,,,Į7KHFDUER[\OWHUPLQDORI&ROODJHQ;9,,, contains a region sensitive to protease cleavage releasing fragments with anti-angiogenic properties (Endostatin). Hepatocyte Growth Factor +*) LV DQWL¿EURWLF WKURXJK LQKLELWLRQ RI 6PDG SKRVSKRU\ODWLRQ preventing upregulation of Connective Tissue Growth Factor (CTGF). Endostatin prevents excessive outgrowth of the developing ureteric bud during embryogenesis by opposing HGF. METHODS: In vivo: Left ureters of male rats were obstructed for 3 or 10 days. The expression of Collagen XVIII and Endostatin was compared in the obstructed (Ob.) and non-obstructed (N.O.) kidneys by Western Blotting, RT-PCR and Immunohistochemistry. MMP activity and H[SUHVVLRQZDVH[DPLQHGXVLQJ=\PRJUDSK\DQG573&5UHVSHFWLYHO\ In vitro:+XPDQ.LGQH\FHOOV +. ZHUHWUHDWHGZLWK7*)ȕIRU hours. Supernatant was analysed for Endostatin via Western Blotting, and cellular MMP upregulation via RT-PCR. The relationship between (QGRVWDWLQ UHOHDVH DQG SURWHRO\WLF HQ]\PHV ZDV H[DPLQHG ZLWK SDQSURWHRO\WLF HQ]\PH LQKLELWLRQ 7*)ȕ &HOOV ZHUH WUHDWHG ZLWK Recombinant HGF and/or Endostatin for 48 hours to examine the pro- or DQWL¿EURWLFSRWHQWLDORI(QGRVWDWLQ RESULTS: Collagen XVIII mRNA and protein expression is upregulated in Ob. kidney and there is increased generation of a 28kD Endostatin fragment. Immunohistochemistry demonstrates &ROODJHQ;9,,,ORFDOL]DWLRQLQWXEXOHVDQGQHJDWLYHLQJORPHUXOL7KHUH LVXSUHJXODWLRQRI(QGRVWDWLQUHOHDVLQJHQ]\PHVLQ2ENLGQH\In-vitro DQDO\VLVGHPRQVWUDWHVWKDW7*)ȕWUHDWHG+.FHOOVGRQRWXSUHJXODWH &ROODJHQ ;9,,, KRZHYHU 7*)ȕ FDXVHV XSUHJXODWLRQ RI (QGRVWDWLQ UHOHDVLQJHQ]\PHVDQGFRQFRPLWDQW(QGRVWDWLQGHWHFWLRQLQVXSHUQDWDQW ,QKLELWLQJSURWHRO\WLFHQ]\PHVSUHYHQWV(QGRVWDWLQGHJUDGDWLRQ$GGLWLRQ RI+*)WR7*)ȕWUHDWHGFHOOVSUHYHQWVXSUHJXODWLRQRI&7*)DQGWKH addition of Endostatin opposes HGF action resulting in upregulation of CTGF. CONCLUSIONS: UO leads to increased generation of Collagen XVIII and a 28kDa Endostatin fragment. HK-2 cells, when WUHDWHG ZLWK 7*)ȕ UHOHDVH (QGRVWDWLQ LQWR WKH VXSHUQDWDQW DQG degradation of Endostatin is via proteolytic activity. Endostatin is pro¿EURWLFE\XSUHJXODWLQJ&7*)WKURXJKDQWDJRQLVPRI+*) Source of Funding: British Urological Foundation, Mater Misericordiae Hospital.
1653 OSTEOPONTIN AND TAMM-HORSFALL PROTEIN ARE FUNCTIONALLY SYNERGISTIC IN PREVENTING RENAL CALCIFICATION Lan Mo, Lucy Liaw, Andrew P Evan, Andre J Sommer, John C Lieske, Xue-Ru Wu*. New York, NY, Scarborough, ME, Indianapolis, IN, Oxford, OH, and Rochester, MN. INTRODUCTION AND OBJECTIVE: A key requirement for the kidney to maintain homeostasis is to prevent often supersaturated mineral salts from forming insoluble crystals. This property has been ascribed at least in part to the urinary presence of macromolecules, although the exact role(s) of these proteins in vivo are only beginning to be unraveled. Here we use knockout mouse models to study the effects of the loss of osteopontin (OPN) and Tamm-Horsfall protein (THP), either alone or in a combination, in spontaneous and chemically LQGXFHGUHQDOFDOFL¿FDWLRQ METHODS: Mice lacking OPN and THP were intercrossed and back-crossed to yield four major genotypes: wild-type, OPN knockout (KO), THP KO and OPN/THP double KO mice. Spontaneous renal FDOFL¿FDWLRQZDVDVVHVVHGE\YRQ.RVVDDQG