Renal Failure, 14(3), 401-405 (1992)
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Inflammatory Mediators in Glomerular Injury Luca De Nicola, MD, Francis B. Gabbai, MD, Lili Feng, MD, Winson W. Tang, MD, Curtis B. Wilson, MD, and Roland C. Blantz, MD Division of Nephrology-Hypertension University of California, San Diego School of Medicine Veterans Affairs Medcal Center La Jolla, California; and Research Institute of Scripps Clinic La Jolla, California
(1-3, 10). Membranous nephropathy is notable for the absence of either cellular proliferation or infiltration of glomerular capillaries with circulating inflammatory cells (4, 7-9). Administration of antibodies to the thymocyte-1 antigen on mesangial cells is characterized by initial lysis of mesangial cells followed thereafter by vigorous proliferation of mesangial cells ( 5 , 14). In spite of the diversity of histologic expression of glomerular immune injury among these various experimental models of glomerulonephritis, the early, and even later, glomerular hernodynamic responses to immune injury with the various site-directed antibodies are remarkably similar and characterized by major reductions in the glomerular ultrafiltration coefficient (LpA), an influence partially compensated by a significant increase in the glomerular hydrostatic pressure gradient (AP) (1, 5 , 14). The histologic correlates of the reductions in LpA are remarkably different in the acute phases of immune injury in (a)proliferative anti-GBM antibody disease (3), (b) membranous nephropathy due to antibodies to FX 1A (7), and (c) mesangiocapillary glomerulonephritis as a result of anti-thymocyte-1 antibodies (14). Immediately after administrationof anti-GBM antibody, polymorphonuclear leukocytes (PMN) appear within
Antibodies directed to antigens located on specific cellular and membrane sites within the glomerulus have permitted creation of experimental animal models of glomerular immune injury which largely duplicate forms of glomerulonephritisencountered in clinical practice (1); proliferative nephritis [anti-glomerular basement membrane (GBM) antibody disease] (2, 3), membranous nephropathy (anti-FX 1A) (4) and mesangiocapillary or membranoproliferative glomerulonephritis (anti-thymocyte-1 antibody) ( 5 , 6). Creation of such experimenal models of glomerular immune injury have permitted: (a) evaluation of glomerular hernodynamic alterations in early and late stages of disease (2, 3,7), (6)assessment of the role of immune mediators in these events (8-ll), (c) evaluation of factors which dictate the rate and extent of progression of renal dysfunction and glomerulosclerosis (12), and (d)examination of factors which dictate the molecular basis of glomerular scarring and accumulation of extracellular matrix which constitute sclerosis (13). These experimental models differ significantly histologically. The anti-GBM antibody model is characterized by endocapillary infiltration, initially with polymorphonuclear leukocytes and later with mononuclear cells and some degree of endothelial cell separation and swelling 401 Copyright 0 1992 by Marcel Dekker, Inc.
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glomerular capillaries, endothelial cells are detached and these leukocytes are adherent to the underlying GBM (3). One could logically attribute the reduction in LpA to the detachment of endothelial cells and the mechanical effects of the PMN acting to partially obstruct capillaries, thereby diverting plasma flow away from these segments, resulting in net loss of filtering surface area (A) (10). However, recent studies have also suggested that leukomenes, potentially generated by polymorphonuclear leukocytes, contribute to the reduction in LpA, or Kf by mechanisms as yet to be elucidated (15). The events after anti-GBM Ab infusion are highly complement dependent. Clearly, the attraction of PMNs is complement dependent via anaphylatoxins, but the vasoconstriction observed with high doses of antibody was also ameliorated by complement depletion (10). We have demonstrated that C5a exerts vasoconstrictor effects when infused directly into the kidney (16). Immune events are firmly established within 1 h after anti-GBM Ab infusions (3), but with administration of anti-FX1A antibody (passive Heymann nephritis), 5 full days are required to establish the glomerular hemodynamic changes and produce proteinuria (7). In part, this temporal delay must relate to lesser access of the antiFXlA antibody to the glomerular epithelial cell located on the outer aspect of the glomerular capillary. However, this is not the total explanation, since other antibodies to the epithelial slit diaphragm act within 1 h to produce reductions in LpA (17). Reductions in 4 A and development of proteinuria 5 days after anti-FX1A are not correlated with absolute antibody deposition but rather with fusion of epithelial cell foot processes (7). This may relate to “capping and shedding” phenomena (18) in which discharge of antigen-antibody complexes from the surface of glomerular epithelial cells correlate with loss of tertiary structure of epithelial podocytes. These eventsthat is, fusion of podocytes, proteinuria,, and reductions in LpA-are largely ameliorated and temporally delayed by complement depletion (8, 9). Complement depletion, therefore, prevents or ameliorates the reduction in LpA in both experimental immune models (9, 10) and are therefore complement dependent, although by entirely different mechanisms. Since LpA is decreased and A P increased by angiotensin 11 (AII) we have also examined the acute and chronic effects of angiotensin-converting enzyme inhibitors, and found that although nephron filtration rate was (SNGFR) improved by increases in nephron plasma flow with ACE inhibitor pretreatment, the LpA remained below normal and glomerular capillary histology was not improved.
De Nicola et al.
Antibodies directed to the thymocyte-1-antigen present on glomerular mesangial cells produce a quite different picture of glomerular immune injury ( 5 , 6, 14). Within hours the mesangial cell is damaged and mesangial cell lysis is well established at 24 h. The rapid time course is therefore similar to that observed following anti-GBM antibody administration and is undoubtedly due to the ready access of antibody to the mesangial space which is separated from the glomerular capillary lumen only by fenestrated endothelial cells. In this model, glomerular epithelial and endothelial cells and the basement membrane are not affected. However, during mesangial cell lysis, cellular constituents and plasma can be observed transiting the mesangial cell space and capillary lumina are larger. These findings may reflect the absence of mesangial cell “tethering” of the GBM by dysfunctional mesangial cells (6, 14). At this juncture, LpA is reduced and nephron plasma flow rate modestly increased. The “shunting” of plasma flow through the mesangial space and capillary enlargement may have decreased LpA by excluding a significant portion of glomerular plasma flow from contact with the GBM, the ultrafiltering surface. Studies during mesangial lysis have demonstrated a role for the mesangial cell in the regulation of glomerular capillary hydrostatic pressure (PG)and efferent arteriolar resistance (20). During the transition from chronic NaCl depletion to acute plasma expansion, PG normally remains constant, but after mesangial lysis, PG rises significantly due to high efferent arteriolar resistances and an absence of efferent arteriolar dilation after volume expansion. Five to six days after anti-thymocyte-1 antibody the mesangial cells undergo proliferation, and SNGFR is reduced due to modest reductions in plasma flow and major decreases in LpA (14). The latter effect may be the result of compression of glomerular capillaries, but functional mechanisms may also play a role. Both mesangial cell lysis and proliferation. are complement dependent events with evidence of participation of membrane attack complex (MAC) components (C6-C9), but polymorphonuclear leukocytes are not a requirement for mesangial cell injury (6, 21). Therefore, although glomerular hemodynamic alterations are similar to those observed in other experimental models of glomerular immune injury (i.e., reduction in LpA), the mechanisms and structural alterations differ markedly after anti-thymocyte antibody administration. There is considerable interest in the factors affecting the course and progression of the renal lesion after acute immune injury. The mesangial hypercellularity which
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403
occurs after anti-thymocyte antibody tends to resolve completely within several weeks; however, both membranous nephropathy and anti-GBM antibody diseases progress and develop glomerulosclerosis. The antiGBM antibody model has been examined at 14-16 weeks and at 10 weeks after anti-GBM antibody administration (12, 22). At these stages there is residual glomerular hypercellularity and a distinct pattern of glomerulosclerosis characterized by diffuse thickening of the GBM. There are significant issues which relate to factors influencing development of sclerosis: (a) what are the nonimmune factors which influence the rate and extent of glomrulosclerosis? (b) What are the molecular contributions to extracellular matrix accumulation? From a clinical perspective, it is clear that concurrent systemic hypertension is a major factor which accelerates the progressive decline in glomerular function in renal disease. Initial studies from our laboratory examined glomerular hemodynamics and glomerular histology 14- 16 weeks after administration of anti-GBM antibody, the induction of two-kidney, one-clip hypertension and the combination of these models ( 1 2). Moderate hypertension alone produced a focal form of glomerulosclerosis and the superimposition of hypertension upon anti-GBM disease resulted in both focal and diffuse sclerosis. GFR was reduced in glomerulonephritic kidneys by the addition of hypertension. In more recent studies we have exmained the effects of a m r e severe form of clip hypertension on anti-GBM disease and further investigated the
influence of treatment with an ACE inhibitor (quinapril) in (a) clipped hypertensive rats, (b) glomerulonephritic rats, and (c) in the hypertensive glomerulonephritic rats (22). ACE inhibition (i) normalize blood pressure and the glomerular capillary hydrostatic pressure and pressure gradient (A P),(ii) eliminated proteinuria, and (iii) greatly diminished the segmental glornerulosclerosis in clipped hypertensive rats (Table 1). In rats with glomerulonephritis alone, animals exhibiting a normal blood pressure, ACE inhibitors slightly improved the diffuse glomerulosclerosis and diminished proteinuria, PG , and AP without altering systemic blood pressure. In rats with both glomerulonephritis and hypertension, ACE inhibitory therapy diminished systemic blood pressure, but not to control levels, and did not normalize AP. It is of interest that in this group, ACE inhibitors did not significantly improve the difhse form of glomerulosclerosis, but the focal and segmental type, characteristic of hypertension alone, was significantly ameliorated. These studies demonstrate that ACE inhibition normalized blood pressure, PG and AP and largely prevented glomerulosclerosis in hypertensive rats, but identical doses of ACE inhibitor were less effective in normalizing glomerular hemodynamics, systemic blood pressure, and in completely preventing glomerulosclerosis in rats with a combination of systemic hypertension and glomerulonephritis. The concurrence of these two clinial events somehow confers a selective resistance to this normally effective form of therapy.
Table 1 Effects of Anti-GBM Antibody Glomerulonephritis (G) and Clip Hypertension (H) on Renal Hemodynamics: Treatment with Angiotensin-Converting Enzyme Inhibitors (CEI)
UP"
SBP
Untreated rats Control H G
G+H Rats treated with CEI Control-CEI H-CEI G-CEI G + H-CEI
SNGFR
99 f 2 161 f 2# 109 2 153 f 4t#
*
11 52 165 207
f f f f
96 104 98 129
f3 f 3* f3 f 3*t
9 13 104 165
f 2* f 17* f 14t
1
9 16# 12t#
f 1
AP
PCi
42 f 1 66 f 2# 43 f 2 40 f 2
53 62 55 58
*1 * 2# *2 *2
35 44 41 40
f2 f4 f 2*
49 49 49 55
f2 f 1* f 2* f 3t#
33 f 1 34 f 1* 36 f 1* 40 f 2t#
44 73 32 46
f 3t
f 1 f 1# f 2# f 2#
Abbreviations: SBP, awake systolic BP (mm Hg); UpV, urinary protein excretion (mg/24 h); SNGFR, nephron filtration rate (nL/min); PG,glomerular capillary hydrostatic pressure (mHg); A P , glomerular hydrostatic pressure gradient (mm Hg). *p < .05 versus untreated group. t p < .05 versus G-CEI. #p < .05 versus control.
De Nicola et al.
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Since thickening of the GBM and glomerulosclerosis are events which contribute to progression of renal dysfunction in anti-GBM Ab disease, we have examined the temporal relationship among a variety of factors which could influence fibrin formation and deposition of extracellular matrix (23). The mRNA for interleukin-1P (IL-lb), transforming growth factor beta (TGF-P), the plasminogen activators (u-PA and t-PA) and plasminogen activator inhibitor (PAI-1) and glomerular fibrin deposits were evaluated in pooled glomeruli at 6 h and at 1, 3, 6, and 17 days after induction of anti-GBM Ab glomerulonephritis. In addition, since endotoxin contaminants can induce IL-10, probably by affecting glomerular fibrinolysis, experiments were also performed in normal rats given 10 mg of endotoxin. mRNA expression was evaluated by RNase protection assay, quantitated with the AMBIS system and factored relative to GAPDH mRNA using rat riboprobes. Studies revealed that endotoxin given to normal rats increased IL- 10 and PAI- 1 mRNA expression transiently, but values returned to control levels by 2.5 h. In the antiGBM Ab glomerulonephritic rats, IL-1 mRNA expression peaked at 6 h and declined by day 3. PAI-1 mRNA expression was observed as early as 6 h and peaked at day 1, and although a modest decline in expression was observed at day 3, elevated mRNA expression for PAL1 persisted through day 17 (23). In contrast, u-PA and tPA mRNA declined slightly through day 1 , with a slight increase in t-PA at day 6. TGF-P mRNA was observed to increase at day 1, was maximal on day 6, and fell slightly by day 17. Quantities of glomerular fibrin deposited correlated with the increase in PAI-1 mRNA and the relative absence of t-PA and u-PA responses. Glomerular fibrin deposits decreased in parallel with declining PAI- 1 mRNA levels. Although several factors undoubtedly contribute to progressive histologic alterations after the initial immune insult in anti-GBM Ab glomerulonephritis: (a) increased matrix production, (b) decreased degradation, and (c) inadequate remodeling of matrix and basement membrane, the significant thickening of the GBM observed on day 17 may be due, at least in part, to PAI-1-induced inhibition of the enzymes which are normally contributing to remodeling of the extracellular matrix. After a variety of glomerular immune insults, there is a cascade of events which usually involve complement activation leading to activation of other inflammatory mediators. The early glomerular hemodynamic changes are uniformly characterized by reductions in the glomerular ultrafiltration coefficient in spite of the fact that histologic and structural changes differ significantly
among experimenal immune models. The progression to glomerulosclerosis after the initial immune insult can be conditioned by concurrent systemic hypertension. Prevention of glomerulosclerosis with normal therapy may be difficult in glomerulonephritis if hypertension persists. The factors which link the initial immune insult to the eventual deposition of extracellular matrix and basement membrane thickening have not been fully defined. Activation of inhibitors of enzymes which normally degrade and remodel extracellular matrix may be critical to this linkage and the evolution of glomerulosclerosis. Address reprint requests to: Roland C. Bantz, MD (91 1 IH), University of California, San Diego, School of Medicine, La Jolla, CA 92093-91 I 1. These studies were supported by grants from the National Institutes of Health (DK28602, DK40251, DK30043 and DK32353). funds provided by the Research Service of the Department of Veterans Affairs, and a grant from Parke Davis. Dr. De Nicola was supported by a fellowship provided by the National Kidney Foundation of Southern California. This is publication No. 7068-IMM from the Department of Immunology, The Scripps Research Institute. La Jolla, CA.
REFERENCES 1. Wilson CB, Blantz RC: Nephroimmunopathology and pathophysiology. Am J Physiol 248:F319-F331, 1985.
2 . Allison M, Wilson CB, Gottschalk CW: Pathophysiology of experimental glomerulonephritis in rats. J Clin Invest 53: 1402-1423. 1974. 3. Blantz RC, Wilson CB: Acute effects of antiglomerular basement membrane antibody on the process of glomerular filtration in the rat. J Clin Invea 58:899-911, 1976. 4. Heymann W, Hackel DB, Harwood S et al: Production of the nephrotic syndrome in rats by Freund’s adjuvants and rat kidney suspension. Proc Soc Erp Med 100:660-664, 1959. 5. Yamamoto T, Yamamoto K, Kawasaki K et al: Immunoelectron microscopic demonstration of Thy-I antigen on the surfaces of mesangial cells in the rat glomerulus. Nephron 43:293-298, 1987. 6. Wilson CB, Yaniamoto T, Moullier Pet al: Selective glomerular cell immune injury-anti-mesangial cell antibodies. In Davis AM, Briggs JD, Gren R et al. (eds), Proc Xth Int Congress of Neptirol, London, UK,Balliere-Tindall, 1988, pp. 509-522. 7. Gabbai F, Gushwa L, Wilson CB et al: An evaluation of the development of experimental membranous nephropathy , Kidney Inr 31:1267-1278, 1987. 8. Salant DJ, Belok S, Madiao MP et al: A new role for complement in experimental membranous nephropathy in rats. J CIin Invest 66: 1339-1350, 1980. 9. Gabbai FB, Mundy CA, Wilson CB et al: An evaluation of the role of complement depletion in experimental membranous nephropathy in the rat. Lob Invest 58539-544, 1988. 10. Blantz RC, Tucker JB, Wilson CB: The acute effects of antiglomerular basement membrane antibody on the process of glomerular filtration in the rat: The influence of dose and complement depletion. J CIin Invest 61:910-921, 1978.
Inflammatory Mediators in Glomerular Injury 11. Takahashi K, Schreiner GF,Yamashita K et al: Predominant func-
18. Matsuo S, Caldwell PRB, Brentjens JR et al: In vivo interaction
tional roles for thromboxane A2 and prostaglandin E2 during late nephrotoxic serum glomerulonephritis in the rat. J Clin Invesr 85:1974-1982, 1990. Blantz RC, Gabbai F, Gushwa LC et al: ?he influence of concomitant experimental hypertension and glomrulonephritis in the rat. Kidney In, 32:652-663, 1987. Okuda S, Languino LR, Ruoslahti E et al: Elevated expression of transforming growth factor-B and proteoglycan production in experimental gbmerulonephritis: Possible role in expansion of the mesangial extrscellular matrix. J clin Invest 86:453-462, 1990. Yamamoto T, Mundy CA, Wilson CB, Blantz RC: Effect of mesangial cell iysis and proliferationon glomerular hemodynamics in the rat. Kidney Inr In press, 1991. Badr KR, Schrkner GF, Wasserman M et al: Preservation of the glomerular capillary ultrafiltration coefficient h i n g rat nephrotoxic serum nephritis by a specific leukotriene D4 receptor antagonist. J Clin Invest 81:1702-1709, 1988. Pelayo JC, Chenoweth DE, Hugli TE et al: Effects of the anaphylatoxin, C5a on renal and glomerular hemodynamicsin the rat. Kidney Inr 30:62-67, 1986. Blantz RC, Gabbai F, Peterson 0,Shimizu F, Yamamoto T, Wilson C: Gbmerular hemodymrc consequences of monoclonal antibody (MoAb) direned to an antigen on glomeruhr epithelial cell slit diaphragm (ESD) and foor processes (EFP). Submitted to 24th Annual Mtg., American Society of Nephrobgy, November 17-20, 1991.
of antibodies with cell surface antigens. A mechanism responsible for in siru formation of immune deposits in the zona pellucida in rabbit ococytes. J Clin Invest 75:1369-1380, 1985. Gabbai FB,Wilson CB, Blantz RC: Role of angiotensin I1 in experimental menhanous nephropathy Am J Physiol23:F500-F506, 1988. Blantz RC, Gat!bai FB, Wilson CB: CZomerular hernodynamic (GH) response ro changes in volume sratus after lysis of mesangial cells (MC).Presented at the 23rd Annual Meeting Am SOCNephrol, Washington, Dc,December 1990 [ J A m SocNephrol1:661, 1990 (abstr)] . Yamamoto T, Wilson CB: Quantitative and qualitative studies of antibody-induced mesangial cell damage in the rat. Kidney Inr 32:514-525, 1987. Gabbai FB, De Nicola L, Thomson SC, Peterson OW, Tucker BJ, Keiser JA, Wilson CB,Blantz RC: Efectofchronic convem'ng eninhibitor (CEI) in rats wirh chronic glomerulonephritis with (GC)and wirholrt hypertension (G). Submitted to 24th Annual Mtg., American Society of Nephrology, November 17-20, 1991. Feng L, Tang WW, Su Q, Wilson CB: mRNA analysis of dysfunctional glomerular (G)jibrinolysisin rmn'-glomerularbasement membrane antibody (aGBMAb) glomerulonephritis (GN) in rats. Presented as Abstract, American Society of Nephrology Meeting, 1991.
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13.
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15.
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16.
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Inflammatory Mediators in Glomerular Injury Luca De Nicola, MD, Francis B. Gabbai, MD, Lili Feng, MD, Winson W. Tang, MD, Curtis B. Wilson, MD, and Roland C. Blantz, MD Division of Nephrology-Hypertension University of California, San Diego School of Medicine Veterans Affairs Medcal Center La Jolla, California; and Research Institute of Scripps Clinic La Jolla, California
(1-3, 10). Membranous nephropathy is notable for the absence of either cellular proliferation or infiltration of glomerular capillaries with circulating inflammatory cells (4, 7-9). Administration of antibodies to the thymocyte-1 antigen on mesangial cells is characterized by initial lysis of mesangial cells followed thereafter by vigorous proliferation of mesangial cells ( 5 , 14). In spite of the diversity of histologic expression of glomerular immune injury among these various experimental models of glomerulonephritis, the early, and even later, glomerular hernodynamic responses to immune injury with the various site-directed antibodies are remarkably similar and characterized by major reductions in the glomerular ultrafiltration coefficient (LpA), an influence partially compensated by a significant increase in the glomerular hydrostatic pressure gradient (AP) (1, 5 , 14). The histologic correlates of the reductions in LpA are remarkably different in the acute phases of immune injury in (a)proliferative anti-GBM antibody disease (3), (b) membranous nephropathy due to antibodies to FX 1A (7), and (c) mesangiocapillary glomerulonephritis as a result of anti-thymocyte-1 antibodies (14). Immediately after administrationof anti-GBM antibody, polymorphonuclear leukocytes (PMN) appear within
Antibodies directed to antigens located on specific cellular and membrane sites within the glomerulus have permitted creation of experimental animal models of glomerular immune injury which largely duplicate forms of glomerulonephritisencountered in clinical practice (1); proliferative nephritis [anti-glomerular basement membrane (GBM) antibody disease] (2, 3), membranous nephropathy (anti-FX 1A) (4) and mesangiocapillary or membranoproliferative glomerulonephritis (anti-thymocyte-1 antibody) ( 5 , 6). Creation of such experimenal models of glomerular immune injury have permitted: (a) evaluation of glomerular hernodynamic alterations in early and late stages of disease (2, 3,7), (6)assessment of the role of immune mediators in these events (8-ll), (c) evaluation of factors which dictate the rate and extent of progression of renal dysfunction and glomerulosclerosis (12), and (d)examination of factors which dictate the molecular basis of glomerular scarring and accumulation of extracellular matrix which constitute sclerosis (13). These experimental models differ significantly histologically. The anti-GBM antibody model is characterized by endocapillary infiltration, initially with polymorphonuclear leukocytes and later with mononuclear cells and some degree of endothelial cell separation and swelling 401 Copyright 0 1992 by Marcel Dekker, Inc.
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glomerular capillaries, endothelial cells are detached and these leukocytes are adherent to the underlying GBM (3). One could logically attribute the reduction in LpA to the detachment of endothelial cells and the mechanical effects of the PMN acting to partially obstruct capillaries, thereby diverting plasma flow away from these segments, resulting in net loss of filtering surface area (A) (10). However, recent studies have also suggested that leukomenes, potentially generated by polymorphonuclear leukocytes, contribute to the reduction in LpA, or Kf by mechanisms as yet to be elucidated (15). The events after anti-GBM Ab infusion are highly complement dependent. Clearly, the attraction of PMNs is complement dependent via anaphylatoxins, but the vasoconstriction observed with high doses of antibody was also ameliorated by complement depletion (10). We have demonstrated that C5a exerts vasoconstrictor effects when infused directly into the kidney (16). Immune events are firmly established within 1 h after anti-GBM Ab infusions (3), but with administration of anti-FX1A antibody (passive Heymann nephritis), 5 full days are required to establish the glomerular hemodynamic changes and produce proteinuria (7). In part, this temporal delay must relate to lesser access of the antiFXlA antibody to the glomerular epithelial cell located on the outer aspect of the glomerular capillary. However, this is not the total explanation, since other antibodies to the epithelial slit diaphragm act within 1 h to produce reductions in LpA (17). Reductions in 4 A and development of proteinuria 5 days after anti-FX1A are not correlated with absolute antibody deposition but rather with fusion of epithelial cell foot processes (7). This may relate to “capping and shedding” phenomena (18) in which discharge of antigen-antibody complexes from the surface of glomerular epithelial cells correlate with loss of tertiary structure of epithelial podocytes. These eventsthat is, fusion of podocytes, proteinuria,, and reductions in LpA-are largely ameliorated and temporally delayed by complement depletion (8, 9). Complement depletion, therefore, prevents or ameliorates the reduction in LpA in both experimental immune models (9, 10) and are therefore complement dependent, although by entirely different mechanisms. Since LpA is decreased and A P increased by angiotensin 11 (AII) we have also examined the acute and chronic effects of angiotensin-converting enzyme inhibitors, and found that although nephron filtration rate was (SNGFR) improved by increases in nephron plasma flow with ACE inhibitor pretreatment, the LpA remained below normal and glomerular capillary histology was not improved.
De Nicola et al.
Antibodies directed to the thymocyte-1-antigen present on glomerular mesangial cells produce a quite different picture of glomerular immune injury ( 5 , 6, 14). Within hours the mesangial cell is damaged and mesangial cell lysis is well established at 24 h. The rapid time course is therefore similar to that observed following anti-GBM antibody administration and is undoubtedly due to the ready access of antibody to the mesangial space which is separated from the glomerular capillary lumen only by fenestrated endothelial cells. In this model, glomerular epithelial and endothelial cells and the basement membrane are not affected. However, during mesangial cell lysis, cellular constituents and plasma can be observed transiting the mesangial cell space and capillary lumina are larger. These findings may reflect the absence of mesangial cell “tethering” of the GBM by dysfunctional mesangial cells (6, 14). At this juncture, LpA is reduced and nephron plasma flow rate modestly increased. The “shunting” of plasma flow through the mesangial space and capillary enlargement may have decreased LpA by excluding a significant portion of glomerular plasma flow from contact with the GBM, the ultrafiltering surface. Studies during mesangial lysis have demonstrated a role for the mesangial cell in the regulation of glomerular capillary hydrostatic pressure (PG)and efferent arteriolar resistance (20). During the transition from chronic NaCl depletion to acute plasma expansion, PG normally remains constant, but after mesangial lysis, PG rises significantly due to high efferent arteriolar resistances and an absence of efferent arteriolar dilation after volume expansion. Five to six days after anti-thymocyte-1 antibody the mesangial cells undergo proliferation, and SNGFR is reduced due to modest reductions in plasma flow and major decreases in LpA (14). The latter effect may be the result of compression of glomerular capillaries, but functional mechanisms may also play a role. Both mesangial cell lysis and proliferation. are complement dependent events with evidence of participation of membrane attack complex (MAC) components (C6-C9), but polymorphonuclear leukocytes are not a requirement for mesangial cell injury (6, 21). Therefore, although glomerular hemodynamic alterations are similar to those observed in other experimental models of glomerular immune injury (i.e., reduction in LpA), the mechanisms and structural alterations differ markedly after anti-thymocyte antibody administration. There is considerable interest in the factors affecting the course and progression of the renal lesion after acute immune injury. The mesangial hypercellularity which
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Inflammatory Mediators in Glomerular Injury
403
occurs after anti-thymocyte antibody tends to resolve completely within several weeks; however, both membranous nephropathy and anti-GBM antibody diseases progress and develop glomerulosclerosis. The antiGBM antibody model has been examined at 14-16 weeks and at 10 weeks after anti-GBM antibody administration (12, 22). At these stages there is residual glomerular hypercellularity and a distinct pattern of glomerulosclerosis characterized by diffuse thickening of the GBM. There are significant issues which relate to factors influencing development of sclerosis: (a) what are the nonimmune factors which influence the rate and extent of glomrulosclerosis? (b) What are the molecular contributions to extracellular matrix accumulation? From a clinical perspective, it is clear that concurrent systemic hypertension is a major factor which accelerates the progressive decline in glomerular function in renal disease. Initial studies from our laboratory examined glomerular hemodynamics and glomerular histology 14- 16 weeks after administration of anti-GBM antibody, the induction of two-kidney, one-clip hypertension and the combination of these models ( 1 2). Moderate hypertension alone produced a focal form of glomerulosclerosis and the superimposition of hypertension upon anti-GBM disease resulted in both focal and diffuse sclerosis. GFR was reduced in glomerulonephritic kidneys by the addition of hypertension. In more recent studies we have exmained the effects of a m r e severe form of clip hypertension on anti-GBM disease and further investigated the
influence of treatment with an ACE inhibitor (quinapril) in (a) clipped hypertensive rats, (b) glomerulonephritic rats, and (c) in the hypertensive glomerulonephritic rats (22). ACE inhibition (i) normalize blood pressure and the glomerular capillary hydrostatic pressure and pressure gradient (A P),(ii) eliminated proteinuria, and (iii) greatly diminished the segmental glornerulosclerosis in clipped hypertensive rats (Table 1). In rats with glomerulonephritis alone, animals exhibiting a normal blood pressure, ACE inhibitors slightly improved the diffuse glomerulosclerosis and diminished proteinuria, PG , and AP without altering systemic blood pressure. In rats with both glomerulonephritis and hypertension, ACE inhibitory therapy diminished systemic blood pressure, but not to control levels, and did not normalize AP. It is of interest that in this group, ACE inhibitors did not significantly improve the difhse form of glomerulosclerosis, but the focal and segmental type, characteristic of hypertension alone, was significantly ameliorated. These studies demonstrate that ACE inhibition normalized blood pressure, PG and AP and largely prevented glomerulosclerosis in hypertensive rats, but identical doses of ACE inhibitor were less effective in normalizing glomerular hemodynamics, systemic blood pressure, and in completely preventing glomerulosclerosis in rats with a combination of systemic hypertension and glomerulonephritis. The concurrence of these two clinial events somehow confers a selective resistance to this normally effective form of therapy.
Table 1 Effects of Anti-GBM Antibody Glomerulonephritis (G) and Clip Hypertension (H) on Renal Hemodynamics: Treatment with Angiotensin-Converting Enzyme Inhibitors (CEI)
UP"
SBP
Untreated rats Control H G
G+H Rats treated with CEI Control-CEI H-CEI G-CEI G + H-CEI
SNGFR
99 f 2 161 f 2# 109 2 153 f 4t#
*
11 52 165 207
f f f f
96 104 98 129
f3 f 3* f3 f 3*t
9 13 104 165
f 2* f 17* f 14t
1
9 16# 12t#
f 1
AP
PCi
42 f 1 66 f 2# 43 f 2 40 f 2
53 62 55 58
*1 * 2# *2 *2
35 44 41 40
f2 f4 f 2*
49 49 49 55
f2 f 1* f 2* f 3t#
33 f 1 34 f 1* 36 f 1* 40 f 2t#
44 73 32 46
f 3t
f 1 f 1# f 2# f 2#
Abbreviations: SBP, awake systolic BP (mm Hg); UpV, urinary protein excretion (mg/24 h); SNGFR, nephron filtration rate (nL/min); PG,glomerular capillary hydrostatic pressure (mHg); A P , glomerular hydrostatic pressure gradient (mm Hg). *p < .05 versus untreated group. t p < .05 versus G-CEI. #p < .05 versus control.
De Nicola et al.
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Since thickening of the GBM and glomerulosclerosis are events which contribute to progression of renal dysfunction in anti-GBM Ab disease, we have examined the temporal relationship among a variety of factors which could influence fibrin formation and deposition of extracellular matrix (23). The mRNA for interleukin-1P (IL-lb), transforming growth factor beta (TGF-P), the plasminogen activators (u-PA and t-PA) and plasminogen activator inhibitor (PAI-1) and glomerular fibrin deposits were evaluated in pooled glomeruli at 6 h and at 1, 3, 6, and 17 days after induction of anti-GBM Ab glomerulonephritis. In addition, since endotoxin contaminants can induce IL-10, probably by affecting glomerular fibrinolysis, experiments were also performed in normal rats given 10 mg of endotoxin. mRNA expression was evaluated by RNase protection assay, quantitated with the AMBIS system and factored relative to GAPDH mRNA using rat riboprobes. Studies revealed that endotoxin given to normal rats increased IL- 10 and PAI- 1 mRNA expression transiently, but values returned to control levels by 2.5 h. In the antiGBM Ab glomerulonephritic rats, IL-1 mRNA expression peaked at 6 h and declined by day 3. PAI-1 mRNA expression was observed as early as 6 h and peaked at day 1, and although a modest decline in expression was observed at day 3, elevated mRNA expression for PAL1 persisted through day 17 (23). In contrast, u-PA and tPA mRNA declined slightly through day 1 , with a slight increase in t-PA at day 6. TGF-P mRNA was observed to increase at day 1, was maximal on day 6, and fell slightly by day 17. Quantities of glomerular fibrin deposited correlated with the increase in PAI-1 mRNA and the relative absence of t-PA and u-PA responses. Glomerular fibrin deposits decreased in parallel with declining PAI- 1 mRNA levels. Although several factors undoubtedly contribute to progressive histologic alterations after the initial immune insult in anti-GBM Ab glomerulonephritis: (a) increased matrix production, (b) decreased degradation, and (c) inadequate remodeling of matrix and basement membrane, the significant thickening of the GBM observed on day 17 may be due, at least in part, to PAI-1-induced inhibition of the enzymes which are normally contributing to remodeling of the extracellular matrix. After a variety of glomerular immune insults, there is a cascade of events which usually involve complement activation leading to activation of other inflammatory mediators. The early glomerular hemodynamic changes are uniformly characterized by reductions in the glomerular ultrafiltration coefficient in spite of the fact that histologic and structural changes differ significantly
among experimenal immune models. The progression to glomerulosclerosis after the initial immune insult can be conditioned by concurrent systemic hypertension. Prevention of glomerulosclerosis with normal therapy may be difficult in glomerulonephritis if hypertension persists. The factors which link the initial immune insult to the eventual deposition of extracellular matrix and basement membrane thickening have not been fully defined. Activation of inhibitors of enzymes which normally degrade and remodel extracellular matrix may be critical to this linkage and the evolution of glomerulosclerosis. Address reprint requests to: Roland C. Bantz, MD (91 1 IH), University of California, San Diego, School of Medicine, La Jolla, CA 92093-91 I 1. These studies were supported by grants from the National Institutes of Health (DK28602, DK40251, DK30043 and DK32353). funds provided by the Research Service of the Department of Veterans Affairs, and a grant from Parke Davis. Dr. De Nicola was supported by a fellowship provided by the National Kidney Foundation of Southern California. This is publication No. 7068-IMM from the Department of Immunology, The Scripps Research Institute. La Jolla, CA.
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2 . Allison M, Wilson CB, Gottschalk CW: Pathophysiology of experimental glomerulonephritis in rats. J Clin Invest 53: 1402-1423. 1974. 3. Blantz RC, Wilson CB: Acute effects of antiglomerular basement membrane antibody on the process of glomerular filtration in the rat. J Clin Invea 58:899-911, 1976. 4. Heymann W, Hackel DB, Harwood S et al: Production of the nephrotic syndrome in rats by Freund’s adjuvants and rat kidney suspension. Proc Soc Erp Med 100:660-664, 1959. 5. Yamamoto T, Yamamoto K, Kawasaki K et al: Immunoelectron microscopic demonstration of Thy-I antigen on the surfaces of mesangial cells in the rat glomerulus. Nephron 43:293-298, 1987. 6. Wilson CB, Yaniamoto T, Moullier Pet al: Selective glomerular cell immune injury-anti-mesangial cell antibodies. In Davis AM, Briggs JD, Gren R et al. (eds), Proc Xth Int Congress of Neptirol, London, UK,Balliere-Tindall, 1988, pp. 509-522. 7. Gabbai F, Gushwa L, Wilson CB et al: An evaluation of the development of experimental membranous nephropathy , Kidney Inr 31:1267-1278, 1987. 8. Salant DJ, Belok S, Madiao MP et al: A new role for complement in experimental membranous nephropathy in rats. J CIin Invest 66: 1339-1350, 1980. 9. Gabbai FB, Mundy CA, Wilson CB et al: An evaluation of the role of complement depletion in experimental membranous nephropathy in the rat. Lob Invest 58539-544, 1988. 10. Blantz RC, Tucker JB, Wilson CB: The acute effects of antiglomerular basement membrane antibody on the process of glomerular filtration in the rat: The influence of dose and complement depletion. J CIin Invest 61:910-921, 1978.
Inflammatory Mediators in Glomerular Injury 11. Takahashi K, Schreiner GF,Yamashita K et al: Predominant func-
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tional roles for thromboxane A2 and prostaglandin E2 during late nephrotoxic serum glomerulonephritis in the rat. J Clin Invesr 85:1974-1982, 1990. Blantz RC, Gabbai F, Gushwa LC et al: ?he influence of concomitant experimental hypertension and glomrulonephritis in the rat. Kidney In, 32:652-663, 1987. Okuda S, Languino LR, Ruoslahti E et al: Elevated expression of transforming growth factor-B and proteoglycan production in experimental gbmerulonephritis: Possible role in expansion of the mesangial extrscellular matrix. J clin Invest 86:453-462, 1990. Yamamoto T, Mundy CA, Wilson CB, Blantz RC: Effect of mesangial cell iysis and proliferationon glomerular hemodynamics in the rat. Kidney Inr In press, 1991. Badr KR, Schrkner GF, Wasserman M et al: Preservation of the glomerular capillary ultrafiltration coefficient h i n g rat nephrotoxic serum nephritis by a specific leukotriene D4 receptor antagonist. J Clin Invest 81:1702-1709, 1988. Pelayo JC, Chenoweth DE, Hugli TE et al: Effects of the anaphylatoxin, C5a on renal and glomerular hemodynamicsin the rat. Kidney Inr 30:62-67, 1986. Blantz RC, Gabbai F, Peterson 0,Shimizu F, Yamamoto T, Wilson C: Gbmerular hemodymrc consequences of monoclonal antibody (MoAb) direned to an antigen on glomeruhr epithelial cell slit diaphragm (ESD) and foor processes (EFP). Submitted to 24th Annual Mtg., American Society of Nephrobgy, November 17-20, 1991.
of antibodies with cell surface antigens. A mechanism responsible for in siru formation of immune deposits in the zona pellucida in rabbit ococytes. J Clin Invest 75:1369-1380, 1985. Gabbai FB,Wilson CB, Blantz RC: Role of angiotensin I1 in experimental menhanous nephropathy Am J Physiol23:F500-F506, 1988. Blantz RC, Gat!bai FB, Wilson CB: CZomerular hernodynamic (GH) response ro changes in volume sratus after lysis of mesangial cells (MC).Presented at the 23rd Annual Meeting Am SOCNephrol, Washington, Dc,December 1990 [ J A m SocNephrol1:661, 1990 (abstr)] . Yamamoto T, Wilson CB: Quantitative and qualitative studies of antibody-induced mesangial cell damage in the rat. Kidney Inr 32:514-525, 1987. Gabbai FB, De Nicola L, Thomson SC, Peterson OW, Tucker BJ, Keiser JA, Wilson CB,Blantz RC: Efectofchronic convem'ng eninhibitor (CEI) in rats wirh chronic glomerulonephritis with (GC)and wirholrt hypertension (G). Submitted to 24th Annual Mtg., American Society of Nephrology, November 17-20, 1991. Feng L, Tang WW, Su Q, Wilson CB: mRNA analysis of dysfunctional glomerular (G)jibrinolysisin rmn'-glomerularbasement membrane antibody (aGBMAb) glomerulonephritis (GN) in rats. Presented as Abstract, American Society of Nephrology Meeting, 1991.
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