544
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[59]
[59] E i c o s a n o i d B i o c h e m i s t r y in C u l t u r e d G l o m e r u l a r M e s a n g i a l Cells B y M I C H A E L S . SIMONSON
and MICHAEL J. DUNN
Eicosanoids play an important role in the autocrine and paracrine regulation of glomerular hemodynamics.l-3 Vasodilatory prostaglandins (PGE2, PGI2) function as negative feedback signals to attenuate vasoconstruction in the glomerular arterioles and mesangium. In contrast, PGF2,, TxA2, and the peptidoleukotrienes (LTC4, LTD4) are potent vasoconstrictors in the renal vasculature, and these eicosanoids probably mediate inflammatory reactions in glomerular disease.4 Glomerular mesangial cells play a central role in the kidney's synthesis of and response to eicosanoids. Techniques are available for isolating and culturing mesangial cells in vitro, and cultured mesangial cells provide a useful model for investigating eicosanoid biochemistry and cell biology (see Refs. 1-9 for review). This chapter focuses on (/) the use of mesangial cells in eicosanoid research; and (ii) basic techniques for culturing, characterizing, assessing viability, and measuring contraction of mesangial cells. Structure and Function of Glomerular Mesangium In addition to endothelial and epithelial cells, the glomerulus contains a perivascular, smooth musclelike mesangial cell. i0 The mesangial cell and its surrounding matrix comprise the central portion of the glomerular tuft 1 M. J. Dunn, in "Contemporary Nephroiogy" (S. Klahr and S. Massry, eds.), pp. 133. Plenum, New York, 1987. 2 B. Brenner, L. D. Dworkin, and I. Ichikawa, in "The Kidney" (B. M. Brenner and F. C. Rector, eds.), pp. 124. Saunders, Philadelphia, PA, 1986. 3 M. J. Dunn, in "Renal Endocrinology" (M. J. Dunn, ed.), p. 1. Williams and Wilkins, Baltimore, 1983. 4 L. Scharschmidt, M. S. Simonson, and M. J. Dunn, Am. J. Med. 81($2B), 30 (1986). s p. Mene', M. S. Simonson, and M. J. Dunn, Physiol. Rev. 69, 1347 (1989). 6 j. I. Kreisberg and A. Hassid, Miner. Electrolyte Metab. 12, 25 (1986). 7 D. Schlondorff, FASEB J. 1, 272 0987). 8 G. E. Striker and L. J. Striker, Lab. Invest. 53, 122 (1985). 9 p. Mene', M. S. Simonson, and M. J. Dunn, Am. J. Physiol. 256, F375 (1989). to H. Latta, in "Handbook of Physiology" ( J. Orloff, R. W. Berliner, and S. R. Geiger, eds.). pp. I. Williams and Wilkins, Baltimore, MD, 1973.
METHODSIN ENZYMOLOGY.VOL. 187
Copyright© 1990by AcademicPress,Inc. All fightsof reproductionin any formreserved.
[59]
EICOSANOIDS AND MESANGIAL CELL CULTURE
545
between the capillary loops. Mesangial cells serve several specialized functions including synthesis and assembly of the mesangial matrix, endocytosis and processing of plasma macromolecules, control of glomerular hemodynamics via mesangial contraction, and mesangial synthesis and release of vasoactive autacoids, especially prostaglandins and thromboxane. !"4"5"7'8 Current evidence suggests that eicosanoids have diverse effects in the mesangium, but the best-studied role of mesangial eicosanoids is the regulation of glomerular hemodynamics. 1,3.4 Cultured Mesangial Cells as Model Systems for Eicosanoid Research Selected examples illustrating the use of mesangial cells in eicosanoid research are highlighted below. A detailed account is available in several reviews.~'4-9 Glomerular epithelial cells synthesize smaller amounts of eicosanoids ~'2"6'8 and are not considered here. Eicosanoid Biosynthesis
Rat mesangial cells in vitro express abundant cyclooxygenase activity, yielding PGE2 and PGF2~, and lesser amounts of poorly characterized lipoxygenase and cytochrome P-450 enzymatic products. 5'9 Of cyclooxygenase products, rat cells in culture synthesize mainly PGE2 > PGF2~ ~> TxA2 ~ PGI2. By contrast, human mesangial cells in culture display modest cyclooxygenase activity with PGI2 or PGE2 representing the major metabolite.l~,12 Mesangial prostaglandin synthesis is enhanced by both physiological and pathophysiological stimuli. Most agents that activate the phosphoinositide cascade, which, in turn, elevates cytosolic-free Ca 2+ concentration and activates protein kinase C, also stimulate prostaglandin synthesis. These agents include angiotensin II (ANG II), bradykinin, arginine vasopressin, serotonin, platelet-activating factor, TxA2 mimetics, platelet-derived growth factor, and endothelin. 5"7"9 Peptidoleukotrienes are a notable exception as they are compounds that evoke the phosphoinositide cascade but fail to increase prostaglandin synthesis in human or rat cells. 12A3 Other stimuli of prostaglandin synthesis include Ca 2+ ionophores, melittin, Escherichia coli lipopolysaccharide, and endocytosis •of opsinized zymosan as well as immune complexes. 5,7'9 In addition, the ~t N. Ardaillou, J, Hagege, M. P. Nivez, R. Ardaillou, and D. Schlondorf, Am. J. Physiol. 7.48, F240 (1985). t2 M. S. S i m o n s o n , P. Mene, G. R. D u b y a k , and M. J. D u n n , Am. J. Physiol. 255, C771 (1988). t3 R. Barnett, P. G o l d w a s s e r , L. A, S c h a r s c h m i d t , and D. Schlondorff, Am. J. Physiol. 250, F838 ~ 1986).
546
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[59]
cytokines interleukin 1 and tumor necrosis factor cause a delayed (->6 hr) increase in prostaglandin synthesis that requires RNA and protein synthesis. T M Mesangial cells are also useful for studying the inhibition of prostaglandin synthesis by nonsteroidal antiinflammatory drugs (NSAIDs) and cyclooxygenase inhibitors. For example, blockade of PGE2 synthesis by NSAIDs greatly amplifies basal- and vasoconstrictor-induced mesangial cell contraction.4"5"~5Eicosanoids attenuate mesangial cell contraction by a cAMP-dependent mechanism. ~5
Eicosanoid Receptors Functional evidence suggests that mesangial cells contain receptors for PGE2, PGF2~, TxAz, and PGI2. PGE2, PGF2,~, and TxA2 activate phospholipase C, whereas PGE2 and PGI2 stimulate adenylate cyclase.5"9 However, radioligand binding studies in mesangial cells have not been performed to directly evaluate prostaglandin receptors and their coupling to phospholipase C or adenylate cyclase. We recently demonstrated that human mesangial cells express a single class of saturable, specific binding sites for [ 3 H ] L T D 4 (KD = 12.0 nM, Bm~ = 987 fmol/mg protein) coupled to phospholipase C but not to adenylate cyclase or phospholipase A2. ~2The regulation of eicosanoid receptor affinity and number in mesangial cells, as well as GTP-binding protein coupling, remains unknown.
Transmembrane Signaling In mesangial cells, eicosanoids evoke several pathways of transmembrane signaling to produce diverse second messengers. 5'9 Eicosanoids activate the phosphoinositide and adenylate cyclase cascades, and electroneutral, amiloride-inhibitable Na+/H ÷ exchange. It remains unknown whether eicosanoids affect membrane potential or tyrosine kinase activity in the mesangium. Ion-exchange chromatography and HPLC techniques have been employed in mesangial cells to study radiolabeled phosphoinositide turnover. 16,17Furthermore, mesangial cell monolayers on plastic coverslips are well-suited for fluorescent probes (i.e., fura-2, and BCECF) to measure receptor-triggered Caz+ signaling and changes in intracellular pH.12,17
14 L. Baud, J. Perez, G. Friedlander, and R. Ardaillou, FEBS Lett. 239, 50 (1988). 15 p. Men6 and M. J. Dunn, Circ. Res. 62, 916 (1988). 16 j. V. Bonventre, K. Skorecki, J. I. Kreisberg, and J. Y. Cheung, Am. J. Physiol. 251, F94 (1986). 17 p. Men6, G. R. Dubyak, H. E. Abboud, A. Scarpa, and M. J. Dunn, Am. J. Physiol. 255, F1059 (1988).
[59]
EICOSANOIDS AND MESANGIAL CELL CULTURE
547
Mesangial Cellular Functions Eicosanoids help control diverse mesangial cell functions including contraction, 3"5 mitogenesis, 5"9 and matrix accumulation. 18 Techniques to study cell contraction by computer-aided microscopy, 4"5 mitogenesis by [3H]thymidine incorporation and measurements of cell number in 96-well plates, 19 and fibronectin accumulation by immunoprecipitation and immunocytochemical staining 2° have been published using cultured mesangial cells. Protocols for Mesangial Cell Culture Isolation of glomeruli by selective sieving is the first step in mesangial cell culture. Mesangial cell strains are then cultured from the outgrowth of explanted glomerular "cores" (Figs. 1 and 2) formed when isolated glomeruli are partially digested with bacterial collagenase. 5'8 The " c o r e s " consist mostly of mesangium and capillary loops. Described below is a protocol for culturing mesangial cells from isolated rat glomeruli; with small changes this protocol applies to other species including dog, 2~ mouse, 2z guinea pig, 23 and humans. 8 Isolation of glomeruli is performed on ice using standard aseptic techniques.
Primary Mesangial Cell Culture Procedure. Kidneys are removed from four ether-anesthetized, exsanguinated Sprague-Dawley rats (150-200 g) and placed in Hanks' balanced salt solution (HBSS) containing 100 U/ml penicillin and 100/xg/ml streptomycin at 4°. The kidneys are decapsulated and halved sagitally, the medulla dissected out with iridectomy scissors, and the cortex minced with a sharp razor blade in a petri dish on ice. If the kidney is wellblanched, the medulla is easily identified by its dark red color in contrast to the light-colored cortex. The cortical paste is pressed through a 106 p~m 18 p. Klotman, L. Bruggeman, J. Hassell, E. Horigan, G. Martin, and Y. Yamada, Kidney lnt. 35, 294A (1989). 19 M. S. Simonson, S. Wann, P. Men6, G. R. Dubyak, M. Kester, Y. Nakazato, J. R. Sedor. and M. J. Dunn, J. Clin. Invest. 83, 708 (1988). 2o M. S. Simonson, L. A. Culp, and M. J. Dunn, Exp. Cell Res. 184, 484 (1989). z~ S. R. Holdsworth, E. F. Glasgow, R. C. Atkins, and N. H. Thomson, Nephron 22, 454 (1978). zz j. S. Hunt, A. E. Jackson, W. A. Day, and A. R. McGiven, Br. J. Exp. Pathol. 62, 52 (1981). z3 T. D. Oberley, J. E. Murphy-Ullrich, B. W. Steinert, and J. VicMuth, Am. J. Pathol. 104, 181 (1981).
548
[59]
CELL MODELS OF LIPID MEDIATOR PRODUCTION
I. Mince 2. 105,+ 74 ~m sieve
Kidney
Isolated Glomeruli
Collogenase
I. Wash Mesangial /'~'/'~ Cell ~ /_v,/ ~")~' Monoloyers
2. Culture
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[59]
[59] E i c o s a n o i d B i o c h e m i s t r y in C u l t u r e d G l o m e r u l a r M e s a n g i a l Cells B y M I C H A E L S . SIMONSON
and MICHAEL J. DUNN
Eicosanoids play an important role in the autocrine and paracrine regulation of glomerular hemodynamics.l-3 Vasodilatory prostaglandins (PGE2, PGI2) function as negative feedback signals to attenuate vasoconstruction in the glomerular arterioles and mesangium. In contrast, PGF2,, TxA2, and the peptidoleukotrienes (LTC4, LTD4) are potent vasoconstrictors in the renal vasculature, and these eicosanoids probably mediate inflammatory reactions in glomerular disease.4 Glomerular mesangial cells play a central role in the kidney's synthesis of and response to eicosanoids. Techniques are available for isolating and culturing mesangial cells in vitro, and cultured mesangial cells provide a useful model for investigating eicosanoid biochemistry and cell biology (see Refs. 1-9 for review). This chapter focuses on (/) the use of mesangial cells in eicosanoid research; and (ii) basic techniques for culturing, characterizing, assessing viability, and measuring contraction of mesangial cells. Structure and Function of Glomerular Mesangium In addition to endothelial and epithelial cells, the glomerulus contains a perivascular, smooth musclelike mesangial cell. i0 The mesangial cell and its surrounding matrix comprise the central portion of the glomerular tuft 1 M. J. Dunn, in "Contemporary Nephroiogy" (S. Klahr and S. Massry, eds.), pp. 133. Plenum, New York, 1987. 2 B. Brenner, L. D. Dworkin, and I. Ichikawa, in "The Kidney" (B. M. Brenner and F. C. Rector, eds.), pp. 124. Saunders, Philadelphia, PA, 1986. 3 M. J. Dunn, in "Renal Endocrinology" (M. J. Dunn, ed.), p. 1. Williams and Wilkins, Baltimore, 1983. 4 L. Scharschmidt, M. S. Simonson, and M. J. Dunn, Am. J. Med. 81($2B), 30 (1986). s p. Mene', M. S. Simonson, and M. J. Dunn, Physiol. Rev. 69, 1347 (1989). 6 j. I. Kreisberg and A. Hassid, Miner. Electrolyte Metab. 12, 25 (1986). 7 D. Schlondorff, FASEB J. 1, 272 0987). 8 G. E. Striker and L. J. Striker, Lab. Invest. 53, 122 (1985). 9 p. Mene', M. S. Simonson, and M. J. Dunn, Am. J. Physiol. 256, F375 (1989). to H. Latta, in "Handbook of Physiology" ( J. Orloff, R. W. Berliner, and S. R. Geiger, eds.). pp. I. Williams and Wilkins, Baltimore, MD, 1973.
METHODSIN ENZYMOLOGY.VOL. 187
Copyright© 1990by AcademicPress,Inc. All fightsof reproductionin any formreserved.
[59]
EICOSANOIDS AND MESANGIAL CELL CULTURE
545
between the capillary loops. Mesangial cells serve several specialized functions including synthesis and assembly of the mesangial matrix, endocytosis and processing of plasma macromolecules, control of glomerular hemodynamics via mesangial contraction, and mesangial synthesis and release of vasoactive autacoids, especially prostaglandins and thromboxane. !"4"5"7'8 Current evidence suggests that eicosanoids have diverse effects in the mesangium, but the best-studied role of mesangial eicosanoids is the regulation of glomerular hemodynamics. 1,3.4 Cultured Mesangial Cells as Model Systems for Eicosanoid Research Selected examples illustrating the use of mesangial cells in eicosanoid research are highlighted below. A detailed account is available in several reviews.~'4-9 Glomerular epithelial cells synthesize smaller amounts of eicosanoids ~'2"6'8 and are not considered here. Eicosanoid Biosynthesis
Rat mesangial cells in vitro express abundant cyclooxygenase activity, yielding PGE2 and PGF2~, and lesser amounts of poorly characterized lipoxygenase and cytochrome P-450 enzymatic products. 5'9 Of cyclooxygenase products, rat cells in culture synthesize mainly PGE2 > PGF2~ ~> TxA2 ~ PGI2. By contrast, human mesangial cells in culture display modest cyclooxygenase activity with PGI2 or PGE2 representing the major metabolite.l~,12 Mesangial prostaglandin synthesis is enhanced by both physiological and pathophysiological stimuli. Most agents that activate the phosphoinositide cascade, which, in turn, elevates cytosolic-free Ca 2+ concentration and activates protein kinase C, also stimulate prostaglandin synthesis. These agents include angiotensin II (ANG II), bradykinin, arginine vasopressin, serotonin, platelet-activating factor, TxA2 mimetics, platelet-derived growth factor, and endothelin. 5"7"9 Peptidoleukotrienes are a notable exception as they are compounds that evoke the phosphoinositide cascade but fail to increase prostaglandin synthesis in human or rat cells. 12A3 Other stimuli of prostaglandin synthesis include Ca 2+ ionophores, melittin, Escherichia coli lipopolysaccharide, and endocytosis •of opsinized zymosan as well as immune complexes. 5,7'9 In addition, the ~t N. Ardaillou, J, Hagege, M. P. Nivez, R. Ardaillou, and D. Schlondorf, Am. J. Physiol. 7.48, F240 (1985). t2 M. S. S i m o n s o n , P. Mene, G. R. D u b y a k , and M. J. D u n n , Am. J. Physiol. 255, C771 (1988). t3 R. Barnett, P. G o l d w a s s e r , L. A, S c h a r s c h m i d t , and D. Schlondorff, Am. J. Physiol. 250, F838 ~ 1986).
546
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[59]
cytokines interleukin 1 and tumor necrosis factor cause a delayed (->6 hr) increase in prostaglandin synthesis that requires RNA and protein synthesis. T M Mesangial cells are also useful for studying the inhibition of prostaglandin synthesis by nonsteroidal antiinflammatory drugs (NSAIDs) and cyclooxygenase inhibitors. For example, blockade of PGE2 synthesis by NSAIDs greatly amplifies basal- and vasoconstrictor-induced mesangial cell contraction.4"5"~5Eicosanoids attenuate mesangial cell contraction by a cAMP-dependent mechanism. ~5
Eicosanoid Receptors Functional evidence suggests that mesangial cells contain receptors for PGE2, PGF2~, TxAz, and PGI2. PGE2, PGF2,~, and TxA2 activate phospholipase C, whereas PGE2 and PGI2 stimulate adenylate cyclase.5"9 However, radioligand binding studies in mesangial cells have not been performed to directly evaluate prostaglandin receptors and their coupling to phospholipase C or adenylate cyclase. We recently demonstrated that human mesangial cells express a single class of saturable, specific binding sites for [ 3 H ] L T D 4 (KD = 12.0 nM, Bm~ = 987 fmol/mg protein) coupled to phospholipase C but not to adenylate cyclase or phospholipase A2. ~2The regulation of eicosanoid receptor affinity and number in mesangial cells, as well as GTP-binding protein coupling, remains unknown.
Transmembrane Signaling In mesangial cells, eicosanoids evoke several pathways of transmembrane signaling to produce diverse second messengers. 5'9 Eicosanoids activate the phosphoinositide and adenylate cyclase cascades, and electroneutral, amiloride-inhibitable Na+/H ÷ exchange. It remains unknown whether eicosanoids affect membrane potential or tyrosine kinase activity in the mesangium. Ion-exchange chromatography and HPLC techniques have been employed in mesangial cells to study radiolabeled phosphoinositide turnover. 16,17Furthermore, mesangial cell monolayers on plastic coverslips are well-suited for fluorescent probes (i.e., fura-2, and BCECF) to measure receptor-triggered Caz+ signaling and changes in intracellular pH.12,17
14 L. Baud, J. Perez, G. Friedlander, and R. Ardaillou, FEBS Lett. 239, 50 (1988). 15 p. Men6 and M. J. Dunn, Circ. Res. 62, 916 (1988). 16 j. V. Bonventre, K. Skorecki, J. I. Kreisberg, and J. Y. Cheung, Am. J. Physiol. 251, F94 (1986). 17 p. Men6, G. R. Dubyak, H. E. Abboud, A. Scarpa, and M. J. Dunn, Am. J. Physiol. 255, F1059 (1988).
[59]
EICOSANOIDS AND MESANGIAL CELL CULTURE
547
Mesangial Cellular Functions Eicosanoids help control diverse mesangial cell functions including contraction, 3"5 mitogenesis, 5"9 and matrix accumulation. 18 Techniques to study cell contraction by computer-aided microscopy, 4"5 mitogenesis by [3H]thymidine incorporation and measurements of cell number in 96-well plates, 19 and fibronectin accumulation by immunoprecipitation and immunocytochemical staining 2° have been published using cultured mesangial cells. Protocols for Mesangial Cell Culture Isolation of glomeruli by selective sieving is the first step in mesangial cell culture. Mesangial cell strains are then cultured from the outgrowth of explanted glomerular "cores" (Figs. 1 and 2) formed when isolated glomeruli are partially digested with bacterial collagenase. 5'8 The " c o r e s " consist mostly of mesangium and capillary loops. Described below is a protocol for culturing mesangial cells from isolated rat glomeruli; with small changes this protocol applies to other species including dog, 2~ mouse, 2z guinea pig, 23 and humans. 8 Isolation of glomeruli is performed on ice using standard aseptic techniques.
Primary Mesangial Cell Culture Procedure. Kidneys are removed from four ether-anesthetized, exsanguinated Sprague-Dawley rats (150-200 g) and placed in Hanks' balanced salt solution (HBSS) containing 100 U/ml penicillin and 100/xg/ml streptomycin at 4°. The kidneys are decapsulated and halved sagitally, the medulla dissected out with iridectomy scissors, and the cortex minced with a sharp razor blade in a petri dish on ice. If the kidney is wellblanched, the medulla is easily identified by its dark red color in contrast to the light-colored cortex. The cortical paste is pressed through a 106 p~m 18 p. Klotman, L. Bruggeman, J. Hassell, E. Horigan, G. Martin, and Y. Yamada, Kidney lnt. 35, 294A (1989). 19 M. S. Simonson, S. Wann, P. Men6, G. R. Dubyak, M. Kester, Y. Nakazato, J. R. Sedor. and M. J. Dunn, J. Clin. Invest. 83, 708 (1988). 2o M. S. Simonson, L. A. Culp, and M. J. Dunn, Exp. Cell Res. 184, 484 (1989). z~ S. R. Holdsworth, E. F. Glasgow, R. C. Atkins, and N. H. Thomson, Nephron 22, 454 (1978). zz j. S. Hunt, A. E. Jackson, W. A. Day, and A. R. McGiven, Br. J. Exp. Pathol. 62, 52 (1981). z3 T. D. Oberley, J. E. Murphy-Ullrich, B. W. Steinert, and J. VicMuth, Am. J. Pathol. 104, 181 (1981).
548
[59]
CELL MODELS OF LIPID MEDIATOR PRODUCTION
I. Mince 2. 105,+ 74 ~m sieve
Kidney
Isolated Glomeruli
Collogenase
I. Wash Mesangial /'~'/'~ Cell ~ /_v,/ ~")~' Monoloyers
2. Culture
