Decay-accelerating factor (DAF or CD55) and CD59 are regulators that protect self cells from C3b deposition and C5b-9 assembly on their surfaces. Their relative roles in protecting glomeruli in immune-mediated renal diseases in vivo are unknown. We induced nephrotoxic serum (NTS) nephritis in Daf1−/−, CD59a−/−, Daf1−/−CD59a−/−, and wild-type (WT) mice by administering NTS IgG. After 18 h, we assessed proteinuria, and performed histological, immunohistochemical, and electron microscopic analyses of kidneys. Twenty-four mice in each group were studied. Baseline albuminuria in the Daf1−/−, CD59a−/−, and Daf1−/−CD59a−/− mice was 82, 83, and 139 as compared with 92 μg/mg creatinine in the WT controls (p > 0.1). After NTS, albuminuria in CD59a−/− and WT mice (186 ± 154 and 183 ± 137 μg/mg creatinine, p > 0.1) was similar. In contrast, Daf1−/− mice developed severe albuminuria (378 ± 520, p < 0.05) that was further exacerbated in Daf1−/−CD59a−/− mice (577 ± 785 μg/mg creatinine, p < 0.05). Glomerular histology showed essentially no infiltrating leukocytes in any group. In contrast, electron microscopy revealed prominent podocyte foot process effacement in Daf1−/− mice with more widespread and severe damage in the double knockouts compared with only mild focal changes in CD59a−/− or WT mice. In all animals, deposition of administered (sheep) NTS Ig was equivalent. This contrasted with marked deposition of both C3 and C9 in Daf1−/−CD59a−/− and Daf1−/− mice, which was evident as early as 2 h post-NTS injection. The results support the proposition that in autoantibody-mediated nephritis, DAF serves as the primary barrier to classical pathway-mediated injury, while CD59 limits consequent C5b-9-mediated cell damage.

Because the binding of opsonic C3b fragments and lytic C5b-9 complexes to biological membranes is indiscriminate and can occur on self cells as well as foreign targets, self cells must be protected from injury that could result from activation of the cascade on their surfaces (reviewed in Refs. 1 and 2). Three complement regulators, decay-accelerating factor (DAF3 or CD55), membrane cofactor protein (MCP or CD46), and CD59 function intrinsically in the membranes of self cells to provide this protection (3, 4, 5). They are expressed ubiquitously on the surfaces of all cells that can come into contact with complement (6, 7, 8). DAF inactivates any C3 (C4b2a and C3bBb) and C5 (C4b2a3b and C3bBb3b) convertases that assemble on self cells by accelerating the decay of these enzymes (9, 10). MCP serves as a cofactor for the cleavage of (nonconvertase-associated) cell-bound C4b and C3b by the serum protease factor I (11). CD59 interferes with the uptake of C9 by cell-bound C5b-8 and subsequent insertion/polymerization of C9, thus preventing the formation of lytic C5b-9 membrane attack complexes (MAC) that bring about lysis (12, 13). In patients with the hemolytic disorder paroxymal nocturnal hemoglobinuria, affected blood cells that lack DAF and CD59 exhibit heightened uptake of C3b on their surfaces in vivo (14, 15) and undergo lysis intravascularly, documenting the physiological importance of these two regulators (16, 17).

Recently, for purposes of investigating the respective protective roles of DAF and CD59 activities in vivo in various disease states, DAF (18, 19) and CD59 (20, 21) knockout mice have been prepared. Differently from humans, mice have two DAF (Daf1 and Daf2) (22) and two CD59 (CD59a and CD59b) genes (23, 24). The Daf1 gene product is predominantly GPI anchored and widely expressed on all tissues (22), while the Daf2 gene product is predominantly transmembrane anchored (22) and is constitutively expressed only in testis (18, 25) and splenic dendritic cells (18). A similar pattern of expression pertains for the two CD59 genes (26). Although mice have an MCP gene, its expression is limited to testis, like the Daf2 and CD59b genes. Systemically in its place, they express another regulator termed complement receptor-related protein Y (Crry), but knockout of this gene can only be achieved on an alternative pathway deficient (C3−/−) background (see Discussion). Thus, for the knockouts, the Daf1 and CD59a genes, considered to be counterparts of DAF and CD59 genes in humans, were targeted.

Nephrotoxic serum (NTS)-induced nephritis is a widely used model of Ab-induced glomerular disease (reviewed in Ref. 27). In the passive heterologous form of the disorder using low doses of administered complement-fixing Ab, renal injury evidenced by albuminuria occurs in the first 18 h and is almost entirely complement dependent (28, 29, 30, 31). Analyses of the complement involvement in this and other animal models of Ab-mediated glomerular disease have implicated terminal pathway components in the renal damage (32, 33, 34, 35, 36, 37, 38).

In a previous study (39), we showed that when given low dosages of NTS, Daf1 mice suffer more profound proteinuria, markedly more C3b deposition in their glomeruli, and much more severe podocyte damage compared with wild-type (WT) controls. The pathological changes are Ab and complement dependent, as no evidence of leukocyte involvement is evident. In view of previous studies implicating the MAC in glomerular injury in this model, an understanding of the relative roles of DAF and CD59 in circumventing complement-mediated injury to glomerular cells is important. To address this question, in the present study, we took advantage of the availability of Daf1−/− mice, CD59a−/− mice, and Daf1−/−CD59a−/− double knockout mice, and compared the changes that occur in each knockout with those that occur in WT controls.

Daf1 knockout mice were prepared, as previously described (18). Briefly, murine GK129 embryonic stem cells were used and knockout was achieved by Cre/LoxP-mediated deletion. CD59a−/− mice were generated by replacing exon 3 of the CD59a gene with Neo using 6 kb of homologous sequence (20). In both cases, chimeric mice were bred four generations with the C57BL/6 strain. Daf1−/−CD59a−/− double knockout mice were prepared by breeding Daf1−/− and CD59a−/− mice with each other. Single and double knockouts were typed by flow cytometric analyses of their erythrocytes (Emo) following staining with anti-DAF mAb 2C6 (40) and anti-CD59 mAb MEL-4 (41). The mice were maintained in the Animal Resource Center of Case Western Reserve University, and experiments were performed according to an approved protocol of the Institutional Animal Care and Use Committee. All mice were studied at 8–10 wk of age.

The γ1 fraction of IgG was purified (by DEAE-Sephacel chromatography) from NTS raised by immunizing sheep with rat glomeruli (39). An i.v. administered dose providing for injury to mouse glomeruli that is maximally complement dependent (30, 31) was used. FITC-labeled goat anti-mouse C3 was obtained from ICN Biochemicals (Aurora, OH). Rabbit anti-rat C9 antiserum (42), which cross-reacts with mouse C9, was used for detection of the MAC. FITC-conjugated anti-rabbit IgG were purchased from ICN Biochemicals, FITC-labeled rabbit anti-sheep IgG were obtained from Zymed Laboratories (San Francisco, CA), and FITC-conjugated rabbit IgG and FITC-conjugated goat IgG were bought from Jackson ImmunoResearch Laboratories (West Grove, PA).

Glomerulonephritis was induced, as described previously (39). Briefly, 500 μg of NTS was injected into the tail vein, and mice were placed in metabolic cages. Urine samples collected after 18 h were analyzed for creatinine and albumin concentrations on a Hitachi/Roche 917 autoanalyzer (Hitachi Roche Diagnostics, Mannheim, Germany). The amount of excreted albumin was normalized for the amount of excreted creatinine, i.e., μg albumin/mg creatinine. All averages are given as means.

At the time of urine collection, i.e., 18 h after NTS injection (in some cases 2 h), kidneys from mice were harvested. For immunofluorescence staining, kidney samples were snap frozen in liquid nitrogen and cut (at 5 μm) on a cryostat. Cryostat sections were labeled with either FITC-conjugated goat anti-mouse C3 Ab (1/5000) or FITC-conjugated rabbit anti-sheep IgG Ab (1/1000) or with the same concentrations of FITC-conjugated goat IgG or FITC-conjugated rabbit IgG as controls. For detection of deposited C9, sections were blocked with goat Ig and stained with 1/600 dilution of rabbit anti-rat C9 antiserum or with the same dilution of normal rabbit serum. After washing, slides were incubated with FITC-labeled goat anti-rabbit IgG. In all cases, stained sections were examined with an Olympus OM6 fluorescence microscope.

Alternative samples of the same kidneys were fixed in 10% buffered Formalin, embedded in Tissue Prep (Fisher Scientific, Fair Lawn, NJ), and sectioned at 5 μm. Sections were stained with H&E, followed by periodic acid Schiff reagent, and examined with an Olympus BH 2 microscope.

Samples of kidneys were fixed in 2.0% glutaraldehyde and 0.1 M sodium cacodylate, postfixed in 1% osmium tetroxide, and embedded in Spurr’s epoxy. Ultrathin (silver-blue) sections were picked up on nickel grids, stained with uranyl acetate/lead citrate, and examined with a JEOL 101C microscope. At least six (×6700) fields from each of at least four glomeruli per mouse were photographed.

One million Emo from Daf1−/−, CD59a−/−, and Daf1−/−CD59a−/− mice or their WT littermates were incubated for 30 min on ice with 10 μg/ml rat anti-mouse DAF mAb 2C6, 10 μg/ml rat anti-mouse Crry mAb 5D5 (43), or 10 μg/ml rat anti-mouse CD59 mAb MEL-4, and with the same concentrations of their corresponding nonrelevant controls. Following washing, the cells were secondarily incubated for 30 min on ice with 5 μg/ml FITC-labeled anti-rat IgG, and, after washing, the stained cells were analyzed on a FACScan flow cytometer (BD Biosciences, San Jose, CA).

Total RNA from kidneys of WT and Daf1 knockout mice was extracted using TRIzol (Life Technology, Rockville, MD). For semiquantitative PCR, identical amounts of total RNA were used to synthesize cDNAs using an oligo(dT) primer for reverse transcription, and PCR was performed for 30 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 60 s using the synthesized cDNAs as templates. Mouse Crry cDNA was amplified with primer P1 (5′-GAGAATGGCTTGGTACATGTAC-3′) and P2 (5′-GTTCTCAGACAGCATGACTGCA-3′); mouse CD59a cDNA was amplified with primer P3 (5′-GCTTCTGGCTGTGTTCTGTTCC-3′) and P4 (5′-ATGGCCACCAGAACCGAGGTCC-3′). CD59b cDNA was amplified with primers P5 (5′-GAGGGGACTCATCTTACTCC-3′) and P6 (5′-AAGAGGAAGTTTCTGCGTTG-3′). Mouse actin cDNA was amplified concurrently as a control using primers PA1 (5′-GTGGGCCGCTCTAGGCACCA-3′) and PA2 (5′-TGGCCTTAGGGTGCAGGGGG-3′). Daf1 and Daf2 cDNAs were amplified using primers P7 (5′-ATGATCCGTGGGCGGGCGCCT-3′), P8 (5′-CACTGGAAACTCATCGTCCCA3′), P9 (5′-CCTCAAAACAGCTCCGGCCAA-3′), and P10 (5′-TGATTTTCCTGAGAGTGAAGGTTGTTT-3′).

As a first indicator of the in vivo importance of DAF and CD59 activities in protecting glomeruli following NTS administration, urines were collected from Daf1−/−, CD59a−/−, Daf1−/−CD59a−/−, and WT mice 18 h after injection of the Ab, and proteinuria was quantitated. Six independent experiments with four animals in each group were conducted, and the data were combined into one set. Baseline albuminuria in the Daf1−/−, CD59a−/−, and Daf1−/−CD59a−/− mice was 82, 83, and 139 as compared with 92 μg/mg creatinine in the WT controls (p > 0.1). As shown in Fig. 1, compared with WT controls, the NTS caused markedly increased proteinuria in Daf1−/− mice (378 ± 520 vs 186 ± 154 μg albumin/mg creatinine, p < 0.05), as previously reported (39). Unexpectedly, it did not result in a significant difference between the CD59a−/− and WT groups (186 ± 154 vs 183 ± 137 μg albumin/mg creatinine, p = 0.45). In contrast, the injection caused ∼1.6-fold higher proteinuria levels (577 ± 783 μg albumin/mg creatinine) in the Daf1−/−CD59a−/− double knockout mice compared with the selective Daf1 knockouts (p < 0.05).

FIGURE 1.

Proteinuria levels in wild-type (WT), Daf1−/−, CD59a−/−, and Daf1−/−CD59a−/− (DKO) 18 h after injection of the γ1 fraction of NTS IgG.

FIGURE 1.

Proteinuria levels in wild-type (WT), Daf1−/−, CD59a−/−, and Daf1−/−CD59a−/− (DKO) 18 h after injection of the γ1 fraction of NTS IgG.

Close modal

To compare the pathological changes in glomeruli of the four mouse groups, immunofluorescence, histological, and EM analyses of kidneys were performed. Three replicate experiments were done. As shown in Fig. 2,A for representative specimens at the (above) 18-h time point, immunofluorescent staining revealed markedly increased C3b deposition in glomeruli of both Daf1−/− and Daf1−/−CD59a−/− mice, which was similar in intensity, compared with little or no deposition in the WT controls. In accordance with the proteinuria measurements, minimal deposition of C3b was evident in the CD59a−/− mice as in the WT mice, consistent with normal DAF function. Similar to the C3 staining, staining for deposited C9, as shown in Fig. 2,B, revealed markedly increased amounts of the protein not only in the Daf1−/−CD59a−/− double knockouts, but also in the selective Daf1 knockouts, albeit in lesser quantities. As with the C3 staining, minimal C9 staining was evident in the CD59a−/− mice similar to that in the WT controls. In glomeruli of all of the groups, staining for the administered NTS IgG showed equivalent intensity and distribution of deposited sheep Ig (Fig. 2 C). There was no C3, C9, or sheep Ig staining in glomeruli of any of the knockouts or in WT mice that were not given NTS.

FIGURE 2.

Immunofluorescent staining for mouse C3 (A), C9 (B), and sheep Ig (C) in glomeruli from each group of mice 18 h after injection of sheep NTS IgG.

FIGURE 2.

Immunofluorescent staining for mouse C3 (A), C9 (B), and sheep Ig (C) in glomeruli from each group of mice 18 h after injection of sheep NTS IgG.

Close modal

EM analyses at 18 h of representative glomeruli for each group are given in Fig. 3. Daf1−/− mice, as previously found (39), showed effacement and diffuse flattening of podocyte foot processes, whereas Daf1−/−CD59a−/− double knockouts showed more severe damage to epithelial cell architecture and structure. In contrast, CD59a−/− knockouts, similar to the WT mice, showed only minimal segmental changes. Characteristic of the heterologous phase of the model, there were no electron-dense deposits. There also was no evidence of leukocyte margination.

FIGURE 3.

EM examination of glomeruli 18 h after NTS treatment of WT mice (A) shows generally intact epithelial podocytes, with only segmental flattening and fusion. Glomeruli from CD59a−/− mice show a similar pattern (C). In contrast, epithelial podocytes from Daf1−/− mice (B) show extensive flattening and fusion and focal lipid vacuole formation, while podocytes from Daf1−/−CD59a−/− (D) mice show the most severe damage (original magnification ×20,000).

FIGURE 3.

EM examination of glomeruli 18 h after NTS treatment of WT mice (A) shows generally intact epithelial podocytes, with only segmental flattening and fusion. Glomeruli from CD59a−/− mice show a similar pattern (C). In contrast, epithelial podocytes from Daf1−/− mice (B) show extensive flattening and fusion and focal lipid vacuole formation, while podocytes from Daf1−/−CD59a−/− (D) mice show the most severe damage (original magnification ×20,000).

Close modal

The comparative histologic appearance of representative glomeruli from each group of mice is shown in Fig. 4. As seen, no gross changes were visible in any of the groups. Immunoperoxidase staining for mouse neutrophils at 18 h showed no positive cells in glomeruli or in tubular interstitium. Moreover, at 18 h, counting of cells in 10 glomeruli from each animal showed <1 leukocyte per glomerulus in any of the Daf1−/−, CD59a−/−, Daf1−/−CD59a−/− mice or the WT controls.

FIGURE 4.

By light microscopy, after injection of NTS IgG, glomeruli from WT mice (A) appear entirely normal. Glomeruli from Daf1−/− (B), CD59a−/− (C), and Daf1−/−CD59a−/− (D) mice do not show significant differences.

FIGURE 4.

By light microscopy, after injection of NTS IgG, glomeruli from WT mice (A) appear entirely normal. Glomeruli from Daf1−/− (B), CD59a−/− (C), and Daf1−/−CD59a−/− (D) mice do not show significant differences.

Close modal

To determine how rapidly the complement deposition and glomerular damage occur in the disease, kidneys were examined 2 h post-NTS injection. As seen in Fig. 5,A, both C3 and C9 deposition in the Daf1−/− and Daf1−/−CD59a−/− double knockouts was clearly evident, although less intense than at 18 h. As at 18 h, no deposition of either component at 2 h was observed (data not shown) in the WT and CD59a−/− mice. As seen in Fig. 5 B, substantial podocyte injury in the two knockouts, although less severe, also was already clearly present.

FIGURE 5.

A, Immunofluorescent staining for mouse C3 and C9 in representative glomeruli from WT, CD59a−/−, Daf1−/−, and Daf1−/−CD59a−/− mice 2 h after injection with sheep NTS IgG. B, EM analyses of glomeruli from each group of mice show initial changes similar to, but less severe than, those apparent at 18 h (Fig. 3).

FIGURE 5.

A, Immunofluorescent staining for mouse C3 and C9 in representative glomeruli from WT, CD59a−/−, Daf1−/−, and Daf1−/−CD59a−/− mice 2 h after injection with sheep NTS IgG. B, EM analyses of glomeruli from each group of mice show initial changes similar to, but less severe than, those apparent at 18 h (Fig. 3).

Close modal

FACS analyses of Emo from each knockout verified that, in each case, normal expression levels of all nontargeted regulatory proteins were retained, i.e., Crry, a second C3 convertase regulator (see Discussion) and CD59 in the case of Daf1−/− mice, DAF and Crry in the case of CD59a−/− mice, and Crry in the case of Daf1−/−CD59a−/− double knockouts. To confirm that the levels of the nontargeted complement regulators in kidney tissue likewise were not altered, semiquantitative PCR using RNA extracted from kidneys of the animals was performed. PCR additionally was performed to confirm that there was no compensatory expression of DAF or CD59 protein deriving from the Daf2 and CD59b genes. As seen in Fig. 6, these analyses, as with flow cytometry, showed no significant alterations in nontargeted regulators, and no compensatory change in minimally detectable Daf2- or CD59b-derived message (data not shown).

FIGURE 6.

A, Semiquantitative RT-PCR analysis of Daf1, CD59, and Crry mRNA expression in representative kidneys from WT, Daf1−/−, CD59a−/−, and DKO mice before and 18 h after NTS injection. Mouse actin mRNAs were amplified as a control. No PCR product was observed for Daf2 either before or after NTS in any of the groups (data not shown). An identical low intensity PCR product for CD59b was observed in all of the groups that did not differ before or after NTS (data not shown). Systematic analyses using both polyclonal and monoclonal anti-CD59b Abs have shown no CD59b expression in the kidney (26 ). B, FACS analyses of erythrocytes from WT, Daf1−/−, CD59a−/−, and DKO mice using 2C6 anti-DAF, MEL-4 anti-CD59a, and 5D5 anti-Crry mAbs.

FIGURE 6.

A, Semiquantitative RT-PCR analysis of Daf1, CD59, and Crry mRNA expression in representative kidneys from WT, Daf1−/−, CD59a−/−, and DKO mice before and 18 h after NTS injection. Mouse actin mRNAs were amplified as a control. No PCR product was observed for Daf2 either before or after NTS in any of the groups (data not shown). An identical low intensity PCR product for CD59b was observed in all of the groups that did not differ before or after NTS (data not shown). Systematic analyses using both polyclonal and monoclonal anti-CD59b Abs have shown no CD59b expression in the kidney (26 ). B, FACS analyses of erythrocytes from WT, Daf1−/−, CD59a−/−, and DKO mice using 2C6 anti-DAF, MEL-4 anti-CD59a, and 5D5 anti-Crry mAbs.

Close modal

We and others previously demonstrated that complement activation in the glomeruli of mice with NTS-induced nephritis is tightly regulated at the level of C3, such that there is only minimal complement-mediated injury as long as DAF and Crry are present (28, 29, 30, 39). In our previous studies in DAF-deficient mice (39), we found markedly increased pathology. The mechanism of glomeruli injury and the extent to which pathology would be increased in the absence of CD59, which protects against autologous C5b-9 assembly, however, remained unclear. In the present study, we used Daf1−/−, CD59a−/−, and Daf1−/−CD59a−/− double knockouts to characterize the respective roles of the two regulators. By all criteria, we found that Daf1−/−CD59a−/− double knockouts suffered the greatest damage. These mice showed the highest proteinuria level, greatest C3 and C9 deposition in glomeruli, and most extensive podocyte damage by EM, the collective results thus documenting that both regulators are critical in protection. Surprisingly, we did not detect a significant difference between selective CD59a knockouts and WT controls. There was no significant increase in proteinuria and no significant difference in epithelial cell damage as discernible by EM. Our RT-PCR and FACS analyses verified that there were no compensatory changes in any of the regulators. Taken together, the findings indicate that while the importance of the activity of DAF is readily evident, the activity of CD59 becomes clearly apparent in the absence of DAF, and thus that the activities of both regulators are essential.

The findings that, in the absence of DAF regulation in NTS nephritis, CD59 deficiency leads to increased glomerular C9 and renders mice more susceptible to Ab-mediated injury strongly implicate the C5b-9 MAC in mediating glomerular injury. This is consistent with the observation that C6-deficient rabbits are partly protected from NTS-induced nephritis (35). Although this model has been regarded historically as a model of antiglomerular basement membrane nephritis, in addition to reactivity to type IV collagen, NTS identifies several podocyte cell surface Ags (44). These cell surface Ags serve as targets for nephritogenic Abs and lead to activation of the classical pathway, assembly of C5b-9, and complement-mediated cytotoxicity. In this regard, the mechanisms of injury, in principle, should be similar to other models of C5b-9-mediated glomerular cell injury (45), including experimental membranous nephropathy (32, 33, 37, 46) and renal microvascular injury that is induced by Ab to glomerular endothelial cells (38). Although our staining for C9 provides evidence that damage in all cases is, in fact, MAC mediated, the data, taken together, argue that DAF regulation is the principal barrier to activation of C5 and to subsequent cell surface assembly of C5b-8, the point at which CD59 exerts its activity.

The importance of intrinsic regulators in protecting self tissues from autologous complement-mediated injury is widely established. It is highlighted in two diseases. In the hemolytic disorder paroxysmal nocturnal hemoglobinuria (1), the absence of DAF and CD59 on affected blood elements renders them susceptible to heightened surface uptake of autologous C3b and intravascular lysis in vivo, eventuating not only in anemia, but, in a large proportion of patients, life-threatening or mortal thrombosis. Rare individuals with genetic deficiency of MCP constitute one subset of patients who develop the frequently fatal hemolytic uremic syndrome (47).

A number of studies have shown that Crry (48), the rodent analog of MCP, is important in protecting the kidney in NTS nephritis. Renal injury is diminished in Crry transgenics (29) or if Crry-Ig is administered to WT animals (30). Our findings in this study argue that Crry is unable to compensate for deficiency in DAF when complement-mediated damage is initiated by the classical pathway. In vitro and in vivo studies have provided evidence that Crry may be more important for controlling the alternative pathway (49, 50).

The mechanism of proteinuria in acute NTS nephritis is uncertain, but is most likely due to sublethal MAC-mediated podocyte injury with subsequent loss of the barrier function of the glomerular capillary wall. By analogy to experimental membranous nephropathy, the loss may eventuate from perturbations in intracellular calcium, activation of phospholipases and stress proteins, ATP depletion, and alterations in the actin cytoskeleton, all culminating in structural abnormalities of podocyte foot processes and possibly in disruption of slit diaphragms (46, 51, 52, 53).

A second widely studied form of NTS-induced nephritis is the autologous phase of the disease (reviewed in Ref. 54). In this model, animals are preimmunized with sheep Ig and after the elaboration of anti-sheep Ig Ab, NTS is administered. The pathology in this model is due largely to the influx of leukocytes and release from the cells of enzymes, cytokines, and other cellular mediators of inflammation. Studies of this latter phase of the disease, in other work, have yielded results similar to ours. In Daf1 knockouts, there is markedly increased polymorphonuclear cell infiltration and massive glomerular destruction with crescent formation (55), most likely in large part resulting from the generation of chemotactic C5a anaphylatoxin (56). In CD59a−/− mice, about half of the animals suffer more profound kidney damage evidenced by proteinuria and EM, detectable damage to epithelial cells, but not increased polymorphonuclear influx (57), consistent with the expected absence (due to intact DAF activity) of an increase in C5 cleavage/C5a generation. Studies in the autologous model have not yet been performed with Daf1−/−CD59a−/− double knockouts.

In summary, our studies, taken together with previous and recent work by others, highlight the importance of intrinsic regulators in the kidney, in particular glomerular epithelium, in protecting against complement-mediated damage in immune and inflammatory glomerulopathies. They show that DAF is critical in preventing the induction of the pathway and provide further evidence that injury in the acute model is mediated principally by the MAC.

We thank Denise Hatala at the Case Western Reserve University Visual Science Research Center histology core lab (National Institutes of Health EY11373) for section preparation, Beth Ann Benetz in the visual science photography lab for help in preparation of the figures, and Ramona Kim for preparation of the manuscript.

1

This work was supported by National Institutes of Health Grants AI23598 (to M.E.M.), DK62945 (to F.L.), and DK30932 (to D.J.S.).

3

Abbreviations used in this paper: DAF, decay-accelerating factor; Crry, complement receptor-related protein Y; EM, electron microscopy; MAC, membrane attack complex; MCP, membrane cofactor protein; NTS, nephrotoxic serum; WT, wild type; DKO, double knockout.

1
Yomtovian, R., G. M. Prince, M. E. Medof.
1993
. The molecular basis for paroxysmal nocturnal hemoglobinuria.
Transfusion
33
:
852
.
2
Kirschfink, M..
1997
. Controlling the complement system in inflammation.
Immunopharmacology
38
:
51
.
3
Medof, M. E., T. Kinoshita, V. Nussenzweig.
1984
. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes.
J. Exp. Med.
160
:
1558
.
4
Seya, T., J. P. Atkinson.
1989
. Functional properties of membrane cofactor protein of complement.
Biochem. J.
264
:
581
.
5
Holguin, M. H., L. R. Fredrick, N. J. Bernshaw, L. A. Wilcox, C. J. Parker.
1989
. Isolation and characterization of a membrane protein from normal human erythrocytes that inhibits reactive lysis of the erythrocytes of paroxysmal nocturnal hemoglobinuria.
J. Clin. Invest.
84
:
7
.
6
Medof, M. E., E. I. Walter, J. L. Rutgers, D. M. Knowles, V. Nussenzweig.
1987
. Identification of the complement decay-accelerating factor (DAF) on epithelium and glandular cells and in body fluids.
J. Exp. Med.
165
:
848
.
7
McNearney, T., L. Ballard, T. Seya, J. P. Atkinson.
1989
. Membrane cofactor protein of complement is present on human fibroblast, epithelial, and endothelial cells.
J. Clin. Invest.
84
:
538
.
8
Meri, S., H. Waldmann, P. J. Lachmann.
1991
. Distribution of protectin (CD59), a complement membrane attack inhibitor, in normal human tissues.
Lab. Invest.
65
:
532
.
9
Fujita, T., T. Inoue, K. Ogawa, K. Iida, N. Tamura.
1987
. The mechanism of action of decay-accelerating factor (DAF): DAF inhibits the assembly of C3 convertases by dissociated C2a and Bb.
J. Exp. Med.
166
:
1221
.
10
Hourcade, D. E., L. M. Mitchell, M. E. Medof.
1999
. Decay acceleration of the complement alternative pathway C3 convertase.
Immunopharmacology
42
:
167
.
11
Brodbeck, W. G., C. Mold, J. P. Atkinson, M. E. Medof.
2000
. Cooperation between decay accelerating factor and membrane cofactor protein in protecting cells from autologous complement attack.
J. Immunol.
165
:
3999
.
12
Harada, R., N. Okada, T. Fujita, H. Okada.
1990
. Purification of 1F5 antigen that prevents complement attack on homologous cell membranes.
J. Immunol.
144
:
1823
.
13
Davies, A., D. L. Simmons, G. Hale, R. A. Harrison, H. Tighe, P. J. Lachmann, H. Waldmann.
1989
. CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells.
J. Exp. Med.
170
:
637
.
14
Pangburn, M. K., R. D. Schreiber, J. S. Trombold, H. J. Muller-Eberhard.
1983
. Paroxysmal nocturnal hemoglobinuria: deficiency in factor H-like functions of the abnormal erythrocytes.
J. Exp. Med.
157
:
1971
.
15
Merry, A. H., V. I. Rawlinson, M. Uchikawa, M. R. Daha, R. B. Sim.
1989
. Studies on the sensitivity to complement-mediated lysis of erythrocytes (Inab phenotype) with a deficiency of DAF (decay accelerating factor).
Br. J. Haematol.
73
:
248
.
16
Medof, M. E., T. Kinoshita, R. Silber, V. Nussenzweig.
1985
. Amelioration of the lytic abnormalities of paroxysmal nocturnal hemoglobinuria with decay-accelerating factor.
Proc. Natl. Acad. Sci. USA
82
:
2980
.
17
Motoyama, N., N. Okada, M. Yamashina, H. Okada.
1992
. Paroxysmal nocturnal hemoglobinuria due to hereditary nucleotide deletion of HRF20 (CD59) gene.
Eur. J. Immunol.
22
:
2669
.
18
Lin, F., Y. Fukuoka, A. Spicer, R. Ohta, N. Okada, C. L. Harris, S. N. Emancipator, M. E. Medof.
2001
. Tissue distribution of products of the mouse decay-accelerating factor (DAF) genes: exploitation of a Daf1 knock-out mouse and site-specific monoclonal antibodies.
Immunology
104
:
215
.
19
Sun, X., C. D. Funk, C. Deng, A. Sahu, J. D. Lambris, W. C. Song.
1999
. Role of decay-accelerating factor in regulating complement activation on the erythrocyte surface as revealed by gene targeting.
Proc. Natl. Acad. Sci. USA
96
:
628
.
20
Holt, D. S., M. Botto, A. E. Bygrave, S. M. Hanna, M. J. Walport, B. P. Morgan.
2001
. Targeted deletion of the CD59 gene causes sponteneous intravascular hemolysis and hemoglobinuria.
Blood
98
:
442
.
21
Miwa, T., L. Zhou, B. Hilliard, H. Molina, W. C. Song.
2002
. Crry, but not CD59 and DAF, is indispensable for murine erythrocyte protection in vivo from spontaneous complement attack.
Blood
99
:
3707
.
22
Spicer, A. P., M. F. Seldin, S. J. Gendler.
1995
. Molecular cloning and chromosomal localization of the mouse decay-accelerating factor genes: duplicated genes encode glycosylphosphatidylinositol-anchored and transmembrane forms.
J. Immunol.
155
:
3079
.
23
Qin, X., T. Miwa, H. Aktas, M. Gao, C. Lee, Y. M. Qian, C. C. Morton, A. Shahsafaei, W. C. Song, J. A. Halperin.
2001
. Genomic structure, functional comparison, and tissue distribution of mouse Cd59a and Cd59b.
Mamm. Genome
12
:
582
.
24
Qian, Y. M., X. Qin, T. Miwa, X. Sun, J. A. Halperin, W. C. Song.
2000
. Identification and functional characterization of a new gene encoding the mouse terminal complement inhibitor CD59.
J. Immunol.
165
:
2528
.
25
Miwa, T., X. Sun, R. Ohta, N. Okada, C. L. Harris, B. P. Morgan, W. C. Song.
2001
. Characterization of glycosylphosphatidylinositol-anchored decay accelerating factor (GPI-DAF) and transmembrane DAF gene expression in wild-type and GPI-DAF gene knockout mice using polyclonal and monoclonal antibodies with dual or single specificity.
Immunology
104
:
207
.
26
Harris, C. L., S. M. Hanna, M. Mizuno, D. S. Holt, K. J. Marchbank, B. P. Morgan.
2003
. Characterization of the mouse analogues of CD59 using novel monoclonal antibodies: tissue distribution and functional comparison.
Immunology
109
:
117
.
27
Salant, D. J., A. V. Cybulsky.
1988
. Experimental glomerulonephritis.
Methods Enzymol.
162
:
421
.
28
Hebert, M. J., T. Takano, A. Papayianni, H. G. Rennke, A. Minto, D. J. Salant, M. C. Carroll, H. R. Brady.
1998
. Acute nephrotoxic serum nephritis in complement knockout mice: relative roles of the classical and alternate pathways in neutrophil recruitment and proteinuria.
Nephrol. Dial. Transplant.
13
:
2799
.
29
Quigg, R. J., C. He, A. Lim, D. Berthiaume, J. J. Alexander, D. Kraus, V. M. Holers.
1998
. Transgenic mice overexpressing the complement inhibitor crry as a soluble protein are protected from antibody-induced glomerular injury.
J. Exp. Med.
188
:
1321
.
30
Quigg, R. J., Y. Kozono, D. Berthiaume, A. Lim, D. J. Salant, A. Weinfeld, P. Griffin, E. Kremmer, V. M. Holers.
1998
. Blockade of antibody-induced glomerulonephritis with Crry-Ig, a soluble murine complement inhibitor.
J. Immunol.
160
:
4553
.
31
Schrijver, G., K. J. M. Assmann, M. J. J. T. Bogman, J. C. M. Robben, R. M. W. de Waal, R. A. P. Koene.
1988
. Antiglomerular basement membrane nephritis in the mouse.
Lab. Invest.
59
:
484
.
32
Groggel, G. C., S. Adler, H. G. Rennke, W. G. Couser, D. J. Salant.
1983
. Role of the terminal complement pathway in experimental membranous nephropathy in the rabbit.
J. Clin. Invest.
72
:
1948
.
33
Baker, P. J., R. F. Ochi, M. Schulze, R. J. Johnson, C. Campbell, W. G. Couser.
1989
. Depletion of C6 prevents development of proteinuria in experimental membranous nephropathy in rats.
Am. J. Pathol.
135
:
185
.
34
Wang, Y., Q. Hu, J. A. Madri, S. A. Rollins, A. Chodera, L. A. Matis.
1996
. Amelioration of lupus-like autoimmune disease in NZB/WF1 mice after treatment with a blocking monoclonal antibody specific for complement component C5.
Proc. Natl. Acad. Sci. USA
93
:
8563
.
35
Groggel, G. C., D. J. Salant, C. Darby, H. G. Rennke, W. G. Couser.
1985
. Role of terminal complement pathway in the heterologous phase of antiglomerular basement membrane nephritis.
Kidney Int.
27
:
643
.
36
Cybulsky, A. V., H. G. Rennke, I. D. Feintzeig, D. J. Salant.
1986
. Complement-induced glomerular epithelial cell injury: the role of the membrane attack complex in rat membranous nephropathy.
J. Clin. Invest.
77
:
1096
.
37
Kerjaschki, D., M. Schulze, S. Binder, R. Kain, P. P. Ojha, M. Susani, R. Horvat, P. J. Baker, W. G. Couser.
1989
. Transcellular transport and membrane insertion of the C5b-9 membrane attack complex of complement by glomerular epithelial cells in experimental membranous nephropathy.
J. Immunol.
143
:
546
.
38
Nangaku, M., A. C., A. C. J. Pippin, S. J. Shankland, K. Kurokawa, S. Adler, R. J. Johnson, W. G. Couser.
1997
. Renal microvascular injury induced by antibody to glomerular endothelial cells is mediated by C5b-9.
Kidney Int.
52
:
1570
.
39
Lin, F., S. N. Emancipator, D. J. Salant, M. E. Medof.
2002
. Decay accelerating factor confers protection against complement-mediated podocyte injury in acute nephrotoxic nephritis.
Lab. Invest.
82
:
563
.
40
Spiller, O. B., C. L. Harris, B. P. Morgan.
1999
. Efficient generation of monoclonal antibodies against surface-expressed proteins by hyperexpression in rodent cells.
J. Immunol. Methods
224
:
51
.
41
Harris, C. L., N. K. Rushmere, B. P. Morgan.
1999
. Molecular and functional analysis of mouse decay accelerating factor (CD55).
Biochem. J.
341
:
821
.
42
Jones, J., I. Laffafian, B. P. Morgan.
1990
. Purification of C8 and C9 from rat serum.
Complement Inflamm.
7
:
42
.
43
Li, B., C. Sallee, M. Dehoff, S. Foley, H. Molina, V. M. Holers.
1993
. Mouse Crry/p65: characterization of monoclonal antibodies and the tissue distribution of a functional homologue of human MCP and DAF.
J. Immunol.
151
:
4295
.
44
Chugh, S., H. Yuan, P. S. Topham, S. A. Haydar, V. Mittal, G. A. Taylor, R. Kalluri, D. J. Salant.
2001
. Aminopeptidase A: a nephritogenic target antigen of nephrotoxic serum.
Kidney Int.
59
:
601
.
45
Savin, V. J., R. J. Johnson, W. G. Couser.
1994
. C5b-9 increases albumin permeability of isolated glomeruli in vitro.
Kidney Int.
46
:
382
.
46
Cybulsky, A. V., T. Takano, J. Papillon, A. Khadir, J. Liu, H. Peng.
2002
. Complement C5b-9 membrane attack complex increases expression of endoplasmic reticulum stress proteins in glomerular epithelial cells.
J. Biol. Chem.
277
:
41342
.
47
Richards, A., E. J. Kemp, M. K. Liszewski, J. A. Goodship, A. K. Lampe, R. Decorte, M. H. Muslumanoglu, S. Kavukcu, G. Filler, Y. Pirson, et al
2003
. Familial hemolytic uremic syndrome and mutations in membrane cofactor protein (MCP; CD46) of the complement system (abstract). In Workshop on Animal Models of Complement Diseases-Greece.
Mol. Immunol.
40
:
192
.
48
Holers, V. M., T. Kinoshita, H. Molina.
1992
. The evolution of mouse and human complement C3-binding proteins: divergence of form but conservation of function.
Immunol. Today
13
:
231
.
49
Kraus, D., J. M. Guthridge, H. C. Marsh, Jr, V. M. Holers.
2000
. A direct comparison of complement inhibitory capacities of the GPI- and transmembrane forms of mouse DAF to mouse Crry and human rsCR1.
Immunopharmacology
49
:
64
. (Abstr.).
50
Xu, C., D. Mao, V. M. Holers, B. Palanca, A. M. Cheng, H. Molina.
2000
. A critical role for murine complement regulator Crry in fetomaternal tolerance.
Science
287
:
498
.
51
Cybulsky, A. V., D. J. Salant, R. J. Quigg, J. Badalamenti, J. V. Bonventre.
1989
. Complement C5b-9 activates phospholipase in glomerular epithelial cells.
Am. J. Physiol. (Renal Fluid Electrolyte Physiol.)
257
:
F826
.
52
Topham, P. S., S. A. Haydar, R. Kuphal, J. D. Lightfoot, D. J. Salant.
1999
. Complement-mediated injury reversibly disrupts glomerular epithelial cell actin microfilaments and focal adhesions.
Kidney Int.
55
:
1763
.
53
Yuan, H., E. Takeuchi, G. A. Taylor, M. McLaughlin, D. Brown, D. J. Salant.
2002
. Nephrin dissociates from actin, and its expression is reduced in early experimental membranous nephropathy.
J. Am. Soc. Nephrol.
13
:
946
.
54
Nangaku, M., Jr, W. G. Couser.
1997
. Glomerulonephritis and complement regulatory proteins.
Exp. Nephrol.
5
:
345
.
55
Sogabe, H., M. Nangaku, Y. Ishibashi, T. Wada, T. Fujita, X. Sun, T. Miwa, M. P. Madaio, W. C. Song.
2001
. Increased susceptibility of decay-accelerating factor deficient mice to anti-glomerular basement membrane glomerulonephritis.
J. Immunol.
167
:
2791
.
56
Marceau, F., C. Lundberg, T. E. Hugli.
1987
. Effects of the anaphylatoxins on circulation.
Immunopharmacology
14
:
67
.
57
Turnberg, D. M. B., J. Warren, B. P. Morgan, M. J. Walport, H. T. Cook.
2003
. CD59a deficiency exacerbates accelerated nephrotoxic nephritis in mice.
J. Am. Soc. Nephrol.
14
:
2271
.