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Role of phospholipase A2S in hydrogen peroxide-induced arachidonic acid release in murine mesangial cell
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TENTH ANNUAL CLINICAL NEPHROLOGY MEETING ABSTRACTS
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31
ROLE OF PHOSPHOLIPASE A2S IN HYDROGEN PEROXIDEINDUCED ARACHIDONIC ACID RELEASE IN MURINE MESANGIAL CELL. Won K. Han, Adam Sapirstein, and Joseph V. Bonventre. Massachusetts General Hospital and Departments of Medicine and Anesthesia, Harvard Medical School, Charlestown, Massachusetts. In this study, we addressed the potential for cross-talk between cytosolic (cPLAa) and secretory phospholipase A 2 (sPLA2) during H202-indneed arachidonic acid (AA) release. We used two types of murine mesangial ceils (MC): i) MC +t+, which lack group HA and V sPLA2s and ii) MC 4", which lack group HA and V sPLA2 and cPLA v To evaluate potential interactions among PLA2s, cPLA2 and sPLA2 were expressed in MC 4" using recombinant adenovirus vectors expressing group V-sPLA2 (Ad-VsPLA2) and cPLA2 (Ad-cPLA2). We found that H202-induced A A releas e was greater in the MC +~÷ versus MC 4", Expression of cPLA z in H2Oz-treated MC 4" increased AA release to levels approaching that of HzO2-treated MC */+. AA release was further increased in the Ad-VsPLA2-infected MC ~a÷. AdVsPLA2 had no effect, however, on MC 4". Thus the effect of group VsPLA 2 on AA release was dependent upon the presence of cPLA2. Secondly, ERK 1/2 enhances cPLA2 activity. Ad-V sPLA 2 increased phosphorylation of ERKI/2 in MC. H202-indnced AA release was significantly reduced by the MEK-1 inhibitor, U0126, in Ad-
PROTEINS CAN REDUCE THE OSMOTIC WATER PERMEABILITY OF PLASMA MEMBRANES. 3~arren G. Hill, Marcia A. Kaetzel*, Ge Zhou °, Xiangpeng Kong °, T-T. Sun °, John R. Dedman*, Maik L. Zeidel. Renal Div., Dept. of Medicine, Univ. &Pittsburgh, PA; *Dept. of Molecular and Cellular Physiology, Univ. of Cincimtati, OH; ~NYU School of Medicine, New York, NY. Epithelial cells can reduce their plasma membrane permeability by creating bilayers which contain high amounts o f cholesterol and sphingolipids in the outer leaflet [Hill & Zeidel (2000) J. BioL Chem. 275, 30176]. The role of proteins in regulating membrane permeability has, however, not been explored. We hypothesized that (1) uroplakins which are found embedded in the luminal membrane of bladder epithelial cells may contribute to the barrier function of that organ, and (2) armexin IV, a protein which reversibly binds to the inner leaflet of plasma membranes, and which is found associated with the apical membrane in the kidney collecting duct. may reduce membrane permeability under appropriate conditions. When purified bovine uroplakins were reconstituted into proteoliposomes, water permeability was reduced by 40% compared to control liposomes (P<0.05), Freezefracture EM revealed large areas of the surface were covered by closely packed arrays of 16 nm uroplakin particles. These data support a role for uroplakins in contributing to the bladder's permeability barrier. Exogenous armexin 1V was shown to bind the outer leaflet of liposomal membranes in response tn elevated Ca z+ by a llposome aggregation assay and the binding was accompanied by a 30% reduction in osmotic water permeability (P<0.001) which Was reversed by EDTA. These preliminary data represent the first known examples of protein interactions with lipids re~ulting in reductions to passive water permeabihry and introduce a new paradigm for how cells may regulate membrane permeability.
VsPLA2-infected MC +f÷but had less of an effect in MC 4". We conclude that the H202-induced A A release in murine MC is cPLA2 dependent and that group V-sPLA2 enhances the activity of cPLA2 via the ERKI/2 pathway.
30 Renal functional reserve (RFR) in pregnant and non-pregnant women. Heguil6n R*; Lapidus A#; Paz C+; Mulld O#; Bellnsci D+; Vote L#; Bemaseoni A*. *Departments of Nephrology, # Obstetrics and +Laboratory. Hospital Juan A Femfindez. Buenos Aires. Argentina Background: ILFR refers to the capability of the kidney to increase its operational functional level when an appropriated stimulus is applied. In healthy women (GC), both, GFR and RPF increase steadily throughout the pregnant state due to a marked decrease in renal vascular resistance. This might be assumed as a temporary arrest of RFR, so no further increases in renal hemodynamic could be elicited. The aim of this study was to evaluate the renal response to an acute protein load and whether RFR is still present in pregnant women (GP) without evidence of renal disease. Material and methods: 5 GP and 8 GC were evaluated. Procedure: After fasting overnight, all the subjects received an oral water load of 20 ml/kg BW and urinary volume was then replaced orally with equal volumes o f water. After two 30-rain equilibration periods, 80 g of proteins, (cooked red meat) was provided. Creatinine clearence (CCr) was measured, every 30 rain, from 1 h before and for 8 h following the protein load (PL). Participants remained in recumbent position along the study. Bladder emptiness was assessed by ultrasoundmonitoring immediately after each miemriton. Basal GFR was assumed as the average of the two 30-min periods before PL and peak GFR as the maximal CCr recorded thereafter. All data are expressed as mean :~ SEM. Paired and unpaired t test and linear regression were used for data analysis. A p value < 0.05 was considered significant. Results: Both groups were similar with regard to age, weight, height or BSA. The median gestafional age for GP was 15 weeks. Basal GFR (100.4 ± 3.52 [GC] and 128.01 ± 6.56 rnl/mird1.73 m 2 [CAP]p< 0.01) increased attar protein load to a peak of 148.84 ~=3.7 (GC) and to 215.16 ± 23.l-ml/min/1.73 m 2 (GP) p< 0.001. RFR was 48.64 ± 2.97 and 82.27 ± 21.3 for GC and GP respectively (p: NS). There were not significant differences between ~onps with regard to die time to peak GFR: 135 ± 10.6 (GC) and 108 ± 26.1 (GP) [p: NS] and ( % ) peak to basal GFR (49.36 + 5.6 vs. 61.9 ± 17.8) GC vs. GP respectively. Peak GFR correlated positively with basal GFR (r: 0.74 p<0.01 ) in both groups. Conclusion: RFR is still present in GP without evidence of renal disease.
32 Cell Proliferation and Gone Expression in Cells from Diabetic (D) Patients (pts) Were Altered by Early Nonenzymatically Glycated Proteins C l-luang, Y Kim, MLA Caramori, AJ Fish, M Manor, University of Minnesota, Minneapolis, MN Recent studies have examined the effects of early nouensymatically glycated proteins (NGP) (Amaduri glucose adducts) on single cell lines (e~g.human glomerular mesangial or endothelial cetls), suggesting significant effects of NGP on cell behaviors which may contribute to the development of diabetic nephropathy (DN). However, it is not clear whether there are variable responses to NGP in cells from D pts, which may be related to individual risk factor for DN. In this study, we investigated the responses of cultured skin fibroblasts (SF) to determine if there are variabilities in the cellular responses to NGP from a large group of type 1 D patients, which would allow testing of the relationship of these responses to DN risk. SF from 15 D pts were seeded at the density of 104 cells/cm 2 on 75 cm2 flask and cultured in DMEM with 10% FCS and 25rnM glucose for 24 hours. Cells were synchronized and then incubated for 72 hours in DMEM with 25ram glucose and 3% human serum, which was glycated or non-glycated. Human serum was glyeated at 25°C with 28 mM glucose for 5 days. Harvested cells were counted by Coulter Counter and total RNA isolated. Expression ofmRNA for ctltype IIl collagen (COL 3) and sodium hydrogen exchanger isofomr 1 (NHE-1 ) were measured by realtime RT-PCR and competitive RT-PCR. The values of roRNA expression per gg total RNA were normal/zed to a reference standard of pooled mRNA from normal SF. SF number was lower in glyeated (t 0.9±3.8x105) vs non-glycated semm (14.6±5.2x10~; P<0.01). The expression of NHE-I mRNA in cells cultured with glycated serum was higher (9362:t.2655 copies) than with nonglycated serum (5901~2376 copies; P<0.01). SF cultured with glycated serum had higher expression of COL 3 mRNA (11203~-3267 copies) than with nonglyeated serum (8882~3336 copies; P<0.01). For each parameter, the percentage of change from the non-glycated baseline among these pts was at least 33% (medium, 23%; range, 3%-98%). Thus, human glycated serum inhibits cell proliferation and enhances gone expressions for COL 3 arAdNHE-1 in SF from individual D pts. Although the directions of these changes are similar to those observed in single cell lines, there are also individual variabilities in responses to NGP among D pts, suggesting the feasibility of studies to determine if the magnitude of these responses are related to DN risk.
TENTH ANNUAL CLINICAL NEPHROLOGY MEETING ABSTRACTS
29
31
ROLE OF PHOSPHOLIPASE A2S IN HYDROGEN PEROXIDEINDUCED ARACHIDONIC ACID RELEASE IN MURINE MESANGIAL CELL. Won K. Han, Adam Sapirstein, and Joseph V. Bonventre. Massachusetts General Hospital and Departments of Medicine and Anesthesia, Harvard Medical School, Charlestown, Massachusetts. In this study, we addressed the potential for cross-talk between cytosolic (cPLAa) and secretory phospholipase A 2 (sPLA2) during H202-indneed arachidonic acid (AA) release. We used two types of murine mesangial ceils (MC): i) MC +t+, which lack group HA and V sPLA2s and ii) MC 4", which lack group HA and V sPLA2 and cPLA v To evaluate potential interactions among PLA2s, cPLA2 and sPLA2 were expressed in MC 4" using recombinant adenovirus vectors expressing group V-sPLA2 (Ad-VsPLA2) and cPLA2 (Ad-cPLA2). We found that H202-induced A A releas e was greater in the MC +~÷ versus MC 4", Expression of cPLA z in H2Oz-treated MC 4" increased AA release to levels approaching that of HzO2-treated MC */+. AA release was further increased in the Ad-VsPLA2-infected MC ~a÷. AdVsPLA2 had no effect, however, on MC 4". Thus the effect of group VsPLA 2 on AA release was dependent upon the presence of cPLA2. Secondly, ERK 1/2 enhances cPLA2 activity. Ad-V sPLA 2 increased phosphorylation of ERKI/2 in MC. H202-indnced AA release was significantly reduced by the MEK-1 inhibitor, U0126, in Ad-
PROTEINS CAN REDUCE THE OSMOTIC WATER PERMEABILITY OF PLASMA MEMBRANES. 3~arren G. Hill, Marcia A. Kaetzel*, Ge Zhou °, Xiangpeng Kong °, T-T. Sun °, John R. Dedman*, Maik L. Zeidel. Renal Div., Dept. of Medicine, Univ. &Pittsburgh, PA; *Dept. of Molecular and Cellular Physiology, Univ. of Cincimtati, OH; ~NYU School of Medicine, New York, NY. Epithelial cells can reduce their plasma membrane permeability by creating bilayers which contain high amounts o f cholesterol and sphingolipids in the outer leaflet [Hill & Zeidel (2000) J. BioL Chem. 275, 30176]. The role of proteins in regulating membrane permeability has, however, not been explored. We hypothesized that (1) uroplakins which are found embedded in the luminal membrane of bladder epithelial cells may contribute to the barrier function of that organ, and (2) armexin IV, a protein which reversibly binds to the inner leaflet of plasma membranes, and which is found associated with the apical membrane in the kidney collecting duct. may reduce membrane permeability under appropriate conditions. When purified bovine uroplakins were reconstituted into proteoliposomes, water permeability was reduced by 40% compared to control liposomes (P<0.05), Freezefracture EM revealed large areas of the surface were covered by closely packed arrays of 16 nm uroplakin particles. These data support a role for uroplakins in contributing to the bladder's permeability barrier. Exogenous armexin 1V was shown to bind the outer leaflet of liposomal membranes in response tn elevated Ca z+ by a llposome aggregation assay and the binding was accompanied by a 30% reduction in osmotic water permeability (P<0.001) which Was reversed by EDTA. These preliminary data represent the first known examples of protein interactions with lipids re~ulting in reductions to passive water permeabihry and introduce a new paradigm for how cells may regulate membrane permeability.
VsPLA2-infected MC +f÷but had less of an effect in MC 4". We conclude that the H202-induced A A release in murine MC is cPLA2 dependent and that group V-sPLA2 enhances the activity of cPLA2 via the ERKI/2 pathway.
30 Renal functional reserve (RFR) in pregnant and non-pregnant women. Heguil6n R*; Lapidus A#; Paz C+; Mulld O#; Bellnsci D+; Vote L#; Bemaseoni A*. *Departments of Nephrology, # Obstetrics and +Laboratory. Hospital Juan A Femfindez. Buenos Aires. Argentina Background: ILFR refers to the capability of the kidney to increase its operational functional level when an appropriated stimulus is applied. In healthy women (GC), both, GFR and RPF increase steadily throughout the pregnant state due to a marked decrease in renal vascular resistance. This might be assumed as a temporary arrest of RFR, so no further increases in renal hemodynamic could be elicited. The aim of this study was to evaluate the renal response to an acute protein load and whether RFR is still present in pregnant women (GP) without evidence of renal disease. Material and methods: 5 GP and 8 GC were evaluated. Procedure: After fasting overnight, all the subjects received an oral water load of 20 ml/kg BW and urinary volume was then replaced orally with equal volumes o f water. After two 30-rain equilibration periods, 80 g of proteins, (cooked red meat) was provided. Creatinine clearence (CCr) was measured, every 30 rain, from 1 h before and for 8 h following the protein load (PL). Participants remained in recumbent position along the study. Bladder emptiness was assessed by ultrasoundmonitoring immediately after each miemriton. Basal GFR was assumed as the average of the two 30-min periods before PL and peak GFR as the maximal CCr recorded thereafter. All data are expressed as mean :~ SEM. Paired and unpaired t test and linear regression were used for data analysis. A p value < 0.05 was considered significant. Results: Both groups were similar with regard to age, weight, height or BSA. The median gestafional age for GP was 15 weeks. Basal GFR (100.4 ± 3.52 [GC] and 128.01 ± 6.56 rnl/mird1.73 m 2 [CAP]p< 0.01) increased attar protein load to a peak of 148.84 ~=3.7 (GC) and to 215.16 ± 23.l-ml/min/1.73 m 2 (GP) p< 0.001. RFR was 48.64 ± 2.97 and 82.27 ± 21.3 for GC and GP respectively (p: NS). There were not significant differences between ~onps with regard to die time to peak GFR: 135 ± 10.6 (GC) and 108 ± 26.1 (GP) [p: NS] and ( % ) peak to basal GFR (49.36 + 5.6 vs. 61.9 ± 17.8) GC vs. GP respectively. Peak GFR correlated positively with basal GFR (r: 0.74 p<0.01 ) in both groups. Conclusion: RFR is still present in GP without evidence of renal disease.
32 Cell Proliferation and Gone Expression in Cells from Diabetic (D) Patients (pts) Were Altered by Early Nonenzymatically Glycated Proteins C l-luang, Y Kim, MLA Caramori, AJ Fish, M Manor, University of Minnesota, Minneapolis, MN Recent studies have examined the effects of early nouensymatically glycated proteins (NGP) (Amaduri glucose adducts) on single cell lines (e~g.human glomerular mesangial or endothelial cetls), suggesting significant effects of NGP on cell behaviors which may contribute to the development of diabetic nephropathy (DN). However, it is not clear whether there are variable responses to NGP in cells from D pts, which may be related to individual risk factor for DN. In this study, we investigated the responses of cultured skin fibroblasts (SF) to determine if there are variabilities in the cellular responses to NGP from a large group of type 1 D patients, which would allow testing of the relationship of these responses to DN risk. SF from 15 D pts were seeded at the density of 104 cells/cm 2 on 75 cm2 flask and cultured in DMEM with 10% FCS and 25rnM glucose for 24 hours. Cells were synchronized and then incubated for 72 hours in DMEM with 25ram glucose and 3% human serum, which was glycated or non-glycated. Human serum was glyeated at 25°C with 28 mM glucose for 5 days. Harvested cells were counted by Coulter Counter and total RNA isolated. Expression ofmRNA for ctltype IIl collagen (COL 3) and sodium hydrogen exchanger isofomr 1 (NHE-1 ) were measured by realtime RT-PCR and competitive RT-PCR. The values of roRNA expression per gg total RNA were normal/zed to a reference standard of pooled mRNA from normal SF. SF number was lower in glyeated (t 0.9±3.8x105) vs non-glycated semm (14.6±5.2x10~; P<0.01). The expression of NHE-I mRNA in cells cultured with glycated serum was higher (9362:t.2655 copies) than with nonglycated serum (5901~2376 copies; P<0.01). SF cultured with glycated serum had higher expression of COL 3 mRNA (11203~-3267 copies) than with nonglyeated serum (8882~3336 copies; P<0.01). For each parameter, the percentage of change from the non-glycated baseline among these pts was at least 33% (medium, 23%; range, 3%-98%). Thus, human glycated serum inhibits cell proliferation and enhances gone expressions for COL 3 arAdNHE-1 in SF from individual D pts. Although the directions of these changes are similar to those observed in single cell lines, there are also individual variabilities in responses to NGP among D pts, suggesting the feasibility of studies to determine if the magnitude of these responses are related to DN risk.