JOURNAL CLUBJOURNAL CLUB

Barbara Labban, M.D.Barbara Labban, M.D.

Complement SystemComplement System

Crucial component of the innate Crucial component of the innate immune system.immune system.

- Host defenseHost defense- Clearance of immune complexes and Clearance of immune complexes and

dying cellsdying cells- Adjuvant role in immune responseAdjuvant role in immune response

Classical Pathway

Lectin Pathway

Alternative Pathway

Immune ComplexesNon- Immune Complexes

C1

C4

C4b

C2

C2a

C4b2a(C3 Convertase)

C1-INHC4bpfI

(-)

(-)

C3 C3b

C4b2a3b(C5 Convertase)

C5 C5b+C6+C7+C8+C9

C5b-9 (MAC)

EndotoxinC3

C3b Factor B Properdin

f D

C3bBb(C3

Convertase)C3b

C3bBb3b(C5 Convertase)

C3

Classical PathwayClassical PathwayActivationActivation

Ab-Ab-dependentdependent Ag-IgM, Ag-IgG Ag-IgM, Ag-IgG complexescomplexes

Ab-Ab-IndependentIndependent polyanions, polyanions, protamine, DNA, protamine, DNA, RNA of apoptotic RNA of apoptotic cells, G- Bacteria, cells, G- Bacteria, CRPCRP

C1 +

+ C4

+ C2

+ C3

C 4b2a3b

C1-INH ( - )

( - )

Classical Pathway

Immune ComplexesNon- Immune Complexes

C1

C4

C4b

C2

C2a

C4b2a(C3 Convertase)

C1-INHC4bpfI

(-)

(-)

C3 C3b

C4b2a3b(C5 Convertase)

C5 C5b+C6+C7+C8+C9

C5b-9 (MAC)

Lectin PathwayLectin Pathway

Ab-Independent Ab-Independent

C4

Activation

Microbial Carbohydrates

(+)

MBL +(MASP-1, MASP-2)

Alternative PathwayAlternative Pathway

Continuous state of low-level Continuous state of low-level activationactivation

Continuous generation of C3b in Continuous generation of C3b in plasmaplasma

Active regulation is achieved by 2 Active regulation is achieved by 2 plasma proteins: Factor H & Factor plasma proteins: Factor H & Factor I.I.

Alternate PathwayC3

C3b

C3a

LPS

+H2OC3(H2O)+

f B

C3(H2O)Bf D

+f B

C3bB

f D

IgG-C3bBb-Properdin(C3 Convertase)

C3(H2O)Bb(C3 Convertase)

IgG-C3bB-Properdin

n C3

n C3b

IgG-C3b-Properdin+f B

i C3b

+C3f

C3dg + C3c

H

Factor IFactor I

H

MCPCD46CR1

C3NeF Stabilized, C3 Convertase.Auto-antibody attaches to convertase

H

H

AmplificationLoop

H

Factor HFactor H Single chain glycoprotein. Produced by Single chain glycoprotein. Produced by

the liver.the liver. Composed of 20 Short Consensus Composed of 20 Short Consensus

Repeat (SCR) domains.Repeat (SCR) domains. Each SCR composed of 60 amino-acids. Each SCR composed of 60 amino-acids. N-terminal SCR domains: Regulation of N-terminal SCR domains: Regulation of

fluid-phase C3 activation. fluid-phase C3 activation. C-terminal domains: Regulation of C-terminal domains: Regulation of

surface bound C3b (surface surface bound C3b (surface recognition function, depends on the recognition function, depends on the chemical composition of the surface).chemical composition of the surface).

Factor HFactor H

- Regulates the alternative Pathway :- Regulates the alternative Pathway : Inhibits the formation of the C3 Convertases Inhibits the formation of the C3 Convertases

of the alternative pathway by binding to C3bof the alternative pathway by binding to C3b Promotes dissociation of the C3 Convertases Promotes dissociation of the C3 Convertases

(Decay acceleration activity)(Decay acceleration activity) Cofactor to Factor I- mediated proteolytic Cofactor to Factor I- mediated proteolytic

conversion of C3b to inactive C3b (i C3b).conversion of C3b to inactive C3b (i C3b).- Deficiency of Factor H - Deficiency of Factor H Uncontrolled Uncontrolled

alternative pathway activation with alternative pathway activation with secondary depletion of C3, Factor B and secondary depletion of C3, Factor B and properdin.properdin.

Factor H and Renal Factor H and Renal DiseaseDisease

Factor H gene mutation is associated Factor H gene mutation is associated with MPGN type II and atypical HUS.with MPGN type II and atypical HUS.

Mutations located in the C terminal Mutations located in the C terminal aHUS. Rarely result in low complement aHUS. Rarely result in low complement or low Factor H plasma levels. or low Factor H plasma levels.

Mutations located in the N- terminal Mutations located in the N- terminal (complement regulatory domains) or (complement regulatory domains) or complete factor H deficiency complete factor H deficiency MPGN MPGN 2. 2.

- Caprioli et al. The molecular basis of familial hemolytic uremic syndrome: mutation analysis of factor H gene reveals a hot spot in short consensus repeat 20. J Am Soc Nephrol 2001; 12:297-307

- Licht C., et al. Deletion of Lys224 in regulatory domain 4 of Factor H reveals a novel pathomechanism for dense - Licht C., et al. Deletion of Lys224 in regulatory domain 4 of Factor H reveals a novel pathomechanism for dense deposit disease (MPGN II). Kidney Int. 2006;70:42–50)deposit disease (MPGN II). Kidney Int. 2006;70:42–50)

MPGN type II (Dense Deposit MPGN type II (Dense Deposit Disease)Disease)

MPGN II: intramembranous electron-MPGN II: intramembranous electron-dense material along the GBMdense material along the GBM

Positive staining for C3, C5 & C9. Positive staining for C3, C5 & C9. Cleavage fragments of C3b such as Cleavage fragments of C3b such as

C3c and C3dg are found in the plasma C3c and C3dg are found in the plasma of patients with MPGN2. of patients with MPGN2.

Complete Factor H deficiency in Complete Factor H deficiency in humans, pigs & mice results in humans, pigs & mice results in uncontrolled Alternative pathway uncontrolled Alternative pathway activation leading to MPGN2.activation leading to MPGN2.

Appel et al. Membranoproliferative Glomerulonephritis Type II (Dense Deposit Disease) An update. J Am Soc Nephrol 16: 1392-1404, 2005. doi: 10.1681

MPGN type II (Dense Deposit MPGN type II (Dense Deposit Disease)Disease)

Case Report: Patient with Autoantibody to factor H Case Report: Patient with Autoantibody to factor H

MPGN2MPGN2 Meri S. et al. Activation of the alternative pathway of complement by monoclonal lambda light Meri S. et al. Activation of the alternative pathway of complement by monoclonal lambda light

chains in membranoproliferative glomerulonephritis. J. Exp. Med. 1992;chains in membranoproliferative glomerulonephritis. J. Exp. Med. 1992;175175:939–950) :939–950)

Case Report: Mutation of factor H complement regulatory Case Report: Mutation of factor H complement regulatory

domains domains MPGN2 MPGN2 Licht C., et al. Deletion of Lys224 in regulatory domain 4 of Factor H reveals a novel Licht C., et al. Deletion of Lys224 in regulatory domain 4 of Factor H reveals a novel

pathomechanism for dense deposit disease (MPGN II). Kidney Int. 2006;70:42–50pathomechanism for dense deposit disease (MPGN II). Kidney Int. 2006;70:42–50 ))

Hogasen et al. Hereditary porcine membranoproliferative glomeulonephritis type II is caused by Factor H Hogasen et al. Hereditary porcine membranoproliferative glomeulonephritis type II is caused by Factor H deficiency. The American Society for Clinical Investigation.Vol 95, March 1995, 1054-1061deficiency. The American Society for Clinical Investigation.Vol 95, March 1995, 1054-1061

Hogasen et al studied 13 piglets affected Hogasen et al studied 13 piglets affected by MPGN type II to investigate the cause of by MPGN type II to investigate the cause of complement activation .complement activation .

Plasma transfusion resulted inPlasma transfusion resulted in- increase in C3 levels- increase in C3 levels- increase in median and maximum survival - increase in median and maximum survival

( 82 and 375 d vs 37 and 72 d)( 82 and 375 d vs 37 and 72 d)- MPGN was less proliferative compared to - MPGN was less proliferative compared to

untreated pigs untreated pigs Conclusion: The deficient regulatory Conclusion: The deficient regulatory

protein was present in plasma.protein was present in plasma.

Hogasen et al. Hereditary porcine membranoproliferative glomerulonephritis type II is caused by Factor H Hogasen et al. Hereditary porcine membranoproliferative glomerulonephritis type II is caused by Factor H deficiency. The American Society for Clinical Investigation.Vol 95, March 1995, 1054-1061deficiency. The American Society for Clinical Investigation.Vol 95, March 1995, 1054-1061

Fractionation of normal plasma, and Fractionation of normal plasma, and administration of single proteins with administration of single proteins with monitoring of the C inhibitory activity in monitoring of the C inhibitory activity in vivo vivo

Conclusion: Inhibitory activity is confined Conclusion: Inhibitory activity is confined to one single protein. to one single protein.

- 150 kDa - 150 kDa - Cofactor to factor I in vitro- Cofactor to factor I in vitro - NH2-terminal amino acid sequence was - NH2-terminal amino acid sequence was

homologous with human, murine and homologous with human, murine and rabbit FHrabbit FH

Pickering et al. Pickering et al. Uncontrolled C3 activation causes membranoproliferative glomerulonephritis in micedeficient in complement factor H. Nature genetics • volume 31 • august 2002

Pickering et al produced Factor H Pickering et al produced Factor H deficient mice and compared cohorts deficient mice and compared cohorts of Cfh-/-, Cfh+/- and wild-type mice of Cfh-/-, Cfh+/- and wild-type mice for 8 monthsfor 8 months

At 8 months:At 8 months:- 23 % mortality in Cfh -/- mice23 % mortality in Cfh -/- mice- Cfh -/- mice developed light Cfh -/- mice developed light

microscopic features of MPGN IImicroscopic features of MPGN II

Light microscopy: the glomerulus of Cfh–/– mice shows hypercellularity, mesangial expansion and thickening of the capillary walls with double contours.

Immunoflurescence microscopy capillary wall and mesangial deposition of C3 and C9 occurs in Cfh–/– mice. No deposition of C3 or C9 is seen in wild-type mice.

Glomerular C3 deposition preceded Glomerular C3 deposition preceded the appearance of GBM dense deposit the appearance of GBM dense deposit which preceded the histological light which preceded the histological light microscopic appearance of MPGN.microscopic appearance of MPGN.

MPGN II was totally dependent on C 3 MPGN II was totally dependent on C 3 activation.activation.

Generation of mice deficient in factors Generation of mice deficient in factors H and factor B prevented C3 H and factor B prevented C3 activation and prevented the activation and prevented the development of MPGNdevelopment of MPGN

Capillary wall deposition of C3 can be seen in Cfh–/–Bf+/– mice but is absent in Cfh–/–Bf–/– littermates. Age-matched healthy Cfh–/– mice show capillary wall deposition of C3 and C9. Before the developmentof MPGN, no IgG deposition is evident in the kidneys of Cfh–/– mice.

Pickering et al. Prevention of C5 activation ameliorates spontaneous and experimental Pickering et al. Prevention of C5 activation ameliorates spontaneous and experimental glomerulonephritis in factor H-deficient mice. Proc Natl Acad Sci U S A. 2006 June 20; glomerulonephritis in factor H-deficient mice. Proc Natl Acad Sci U S A. 2006 June 20; 103(25): 9649–9654.103(25): 9649–9654.

In another study, Pickering et al. In another study, Pickering et al. prevented C5 activation by crossing prevented C5 activation by crossing factor H- deficient mice with C 5 factor H- deficient mice with C 5 deficient animals.deficient animals.

No prevention in the development of No prevention in the development of the GBM deposits.the GBM deposits.

Reduction in glomerular cellularity, Reduction in glomerular cellularity, serum creatinine levels and serum creatinine levels and mortality.mortality.

Reduction in glomerular cellularity, serum creatinine levels and mortality Reduction in glomerular cellularity, serum creatinine levels and mortality in Cfh-/- C5-/- mice.in Cfh-/- C5-/- mice.

Uncontrolled activation of C3 in Uncontrolled activation of C3 in plasma is essential for the plasma is essential for the development of MPGN IIdevelopment of MPGN II

Factor H deficiencyFactor H deficiency IgG autosomal Ab (IgG autosomal Ab (C3NeF): binds to ): binds to

and stabilizes the Alternative and stabilizes the Alternative Pathway C3 Convertase Pathway C3 Convertase enhanced enhanced Alternative Pathway activation. Alternative Pathway activation.

Inherited dysfunctional C3 Inherited dysfunctional C3 molecules: C3 convertase are molecules: C3 convertase are resistant to inhibiton by factor H resistant to inhibiton by factor H Enhanced AP activation. Enhanced AP activation. - Linshaw M.A., et al. Hypocomplementemic glomerulonephritis in an infant and mother. Evidence for an abnormal form of C3. Am. J. Nephrol. 1987;7:470–477 - Schena FP et al. Biological significance of the C3 Nephritic factor in membranoproliferative Glomerulonephritis. Clin Nephrol 1982; 18:240-6

Alternative PathwayC3

C3b

C3a

LPS

+H2OC3(H2O)+

f B

C3(H2O)Bf D

+f B

C3bB

f D

IgG-C3bBb-Properdin(C3 Convertase)

C3(H2O)Bb(C3 Convertase)

IgG-C3bB-Properdin

n C3

n C3b

IgG-C3b-Properdin+f B

i C3b

+C3f

C3dg + C3c

H

Factor IFactor I

H

MCPCD46CR1

C3NeF Stabilized, C3 Convertase.Auto-antibody attaches to convertase

H

H

AmplificationLoop

H

Factor I Factor I

88 k-Da serine protease 88 k-Da serine protease Function: inactivation of C3bFunction: inactivation of C3b Inactivation of C3b in 2 steps:Inactivation of C3b in 2 steps:- Step 1Step 1: Factor I cleaves the : Factor I cleaves the αα-chain of -chain of

C3b at 2 sites, releasing C3f and forming C3b at 2 sites, releasing C3f and forming i C3b.i C3b.

Cofactors: Factor H, MCP, CD 46, CR1. Cofactors: Factor H, MCP, CD 46, CR1.

- - Step 2:Step 2: Factor I further cleaves iC3b to Factor I further cleaves iC3b to produce C3dg & C3c. produce C3dg & C3c.

Appel et al. Membranoproliferative Glomerulonephritis Type II (Dense Deposit Disease) An update. J Am Soc Nephrol 16: 1392-1404, 2005. doi: 10.1681

Factor I inhibits C3b Factor I inhibits C3b inhibits the C3 inhibits the C3 convertase of the alternative pathway convertase of the alternative pathway limits the alternative pathway amplification. limits the alternative pathway amplification.

Factor I deficiencyFactor I deficiency: Uncontrolled AP : Uncontrolled AP activation activation

Depletion of C3, Factor B & Properdin Depletion of C3, Factor B & Properdin High C3bHigh C3b Reduction of Factor H levels (due to Reduction of Factor H levels (due to

formation of factor H- C3b complexes)formation of factor H- C3b complexes)

NEVER ASSOCIATED WITH MPGN 2 ! NEVER ASSOCIATED WITH MPGN 2 !

Study PurposeStudy Purpose

- MPGN2 has never been reported in MPGN2 has never been reported in individuals with Factor I deficiency individuals with Factor I deficiency

To determine why uncontrolled C3 To determine why uncontrolled C3 activation in the context of factor I activation in the context of factor I deficiency does not result in MPGN deficiency does not result in MPGN II. II.

Cf i -/- mice were produced Cf i -/- mice were produced (disrupted the gene encoding factor (disrupted the gene encoding factor I, by deleting exon 4 of the Cfi gene I, by deleting exon 4 of the Cfi gene with a gene-targeting replacement with a gene-targeting replacement vector). vector).

ResultsResultsComplement analysis Complement analysis C3 levelC3 levelCfi -/- : Low Cfi -/- : Low Cfi +/ - : normalCfi +/ - : normalCfi +/+ : normalCfi +/+ : normal

Factor H levelFactor H levelCfi -/- : Low Cfi -/- : Low Cfi +/ - : normalCfi +/ - : normalCfi +/+ : normalCfi +/+ : normal

Factor B levelFactor B levelCfi -/- : Low Cfi -/- : Low Cfi +/ - : normalCfi +/ - : normalCfi +/+ : normalCfi +/+ : normal

Complement analysis in Cfi-/- mice Complement analysis in Cfi-/- mice

Plasma C3, factor Plasma C3, factor H and factor B H and factor B levels in levels in Cfi Cfi deficient, deficient, CfiCfi+/–, +/–, and wild-type and wild-type mice. Horizontal mice. Horizontal bars denote bars denote median values. median values.

Western blot to determine the nature of plasma Western blot to determine the nature of plasma C3: C3:

C3 C3 αα chain 115 kDa chain 115 kDa

C3b C3b αα’ chain 110 kDa’ chain 110 kDa

ß chain 55-60 kDaß chain 55-60 kDa Cfi -/- : HMW bands ( aggregates of C3b) Cfi -/- : HMW bands ( aggregates of C3b)

presentpresent

C3c not detectableC3c not detectable Cfi +/+: HMW bands ( aggregates of C3b) Cfi +/+: HMW bands ( aggregates of C3b)

absentabsent

C3c DetectableC3c Detectable

Western blot from wild-type Western blot from wild-type and and CfiCfi–/– mice: –/– mice:

(D) α′-chain(C3b) derived (D) α′-chain(C3b) derived from from CfiCfi–/– plasma ran at –/– plasma ran at approximately 110 kDa.approximately 110 kDa.

α-chain(C3) derived from α-chain(C3) derived from wild-type plasma 115 kDawild-type plasma 115 kDa

(E) C3c was detectable only (E) C3c was detectable only in plasma derived from wild-in plasma derived from wild-type mice, as evident by the type mice, as evident by the presence of 130-kDa bands . presence of 130-kDa bands .

MPGN2 developmentMPGN2 development

Monitored cohorts of wild type, Cfi+/- and Monitored cohorts of wild type, Cfi+/- and

Cfi-/- mice over an 8 month period at which Cfi-/- mice over an 8 month period at which time all were sacrificied and renal function time all were sacrificied and renal function and histology assessed.and histology assessed.

No difference in plasma urea levels or No difference in plasma urea levels or albuminuriaalbuminuria

No difference in glomerular hypercellularity. No difference in glomerular hypercellularity. No capillary wall double contour. No capillary wall double contour.

More mesangial expansion and deposition of More mesangial expansion and deposition of nodular hyaline material.nodular hyaline material.

Light microscopic appearances of mesangial hyalinosis in 8-month-old Light microscopic appearances of mesangial hyalinosis in 8-month-old CfiCfi–/– mice. –/– mice. PAS-stained glomerular PAS-stained glomerular sections from 8-month-old wild-type (sections from 8-month-old wild-type (AA) and ) and CfiCfi–/– mice (–/– mice (BB and and CC). The glomerulus from the wild-type ). The glomerulus from the wild-type mouse had normal appearances. In contrast, areas of mesangial expansion with a nodular hyaline mouse had normal appearances. In contrast, areas of mesangial expansion with a nodular hyaline appearance were evident in the appearance were evident in the CfiCfi–/– mice (arrows). Original magnification, ×40 (–/– mice (arrows). Original magnification, ×40 (BB); ×100(); ×100(AA and and CC).).

Glomerular C3 staining in Glomerular C3 staining in Cfi-/- mice :Cfi-/- mice :

Increased glomerular C3 staining in Increased glomerular C3 staining in Cfi-/- mice but MESANGIAL in Cfi-/- mice but MESANGIAL in distribution.distribution.

Linear capillary wall staining pattern Linear capillary wall staining pattern in age matched Cfh-/- animals.in age matched Cfh-/- animals.

Glomerular Ig G staining did not differ Glomerular Ig G staining did not differ between Cfi-/- and wild type mice.between Cfi-/- and wild type mice.

Glomerular C3 staining in 8-month-old Glomerular C3 staining in 8-month-old CfiCfi–/– mice. –/– mice. ((AA) Increased glomerular C3 staining in ) Increased glomerular C3 staining in CfiCfi–/– mice, with mesangial in distribution. –/– mice, with mesangial in distribution. (B) linear capillary wall staining pattern in (B) linear capillary wall staining pattern in CfhCfh–/– mice.–/– mice.(C) Glomerular C3 staining in (C) Glomerular C3 staining in CfiCfi+/– mice is identical to the wild-type animals (+/– mice is identical to the wild-type animals (DD). ). (E) Quantification of the glomerular C3 staining confirmed significantly increased glomerular C3 in the (E) Quantification of the glomerular C3 staining confirmed significantly increased glomerular C3 in the Cfi deficient miceCfi deficient mice

Cfi-/- mice developed increased Cfi-/- mice developed increased mesangial C3 staining but not mesangial C3 staining but not deposition of C3 along GBMdeposition of C3 along GBM

Factor I is an absolute requirement Factor I is an absolute requirement for GBM C3 deposition and the for GBM C3 deposition and the development of MPGN II in Cfh-/- micedevelopment of MPGN II in Cfh-/- mice

Glomerular staining patterns in Cfi-/- Glomerular staining patterns in Cfi-/- mice with either heterozygous or mice with either heterozygous or homozygous factor H deficiencyhomozygous factor H deficiency

Glomerular C3 staining in Glomerular C3 staining in CfiCfi–/– mice with normal (–/– mice with normal (CfhCfh+/+), heterozygous +/+), heterozygous ((CfhCfh+/–), and homozygous (+/–), and homozygous (CfhCfh–/–) factor H genotypes. A mesangial staining –/–) factor H genotypes. A mesangial staining pattern was evident in the glomeruli of the pattern was evident in the glomeruli of the CfiCfi–/– mice regardless of factor H –/– mice regardless of factor H genotype.genotype.

Factor I deficiency prevented Factor I deficiency prevented accumulation of C3 along the GBM accumulation of C3 along the GBM in Cfh-/- mice.in Cfh-/- mice.

Light Microscopy: No evidence of Light Microscopy: No evidence of MPGN2MPGN2

in 8 month old Cfh-/- Cfi-/- mice.in 8 month old Cfh-/- Cfi-/- mice.

Factor H deficient mice (cfh -/-)Factor H deficient mice (cfh -/-)

- Florid C3 deposition along GBM/ - Florid C3 deposition along GBM/ MPGN2 occurredMPGN2 occurred

Factor I deficient mice (cfi -/-)Factor I deficient mice (cfi -/-)

Uncontrolled AP Uncontrolled AP

-- Did Did notnot develop C3 deposition /MPGN2 develop C3 deposition /MPGN2 Factor H & Factor I deficient (cfh -/-, cfi Factor H & Factor I deficient (cfh -/-, cfi

-/-)-/-)

Uncontrolled APUncontrolled AP

-- Did Did notnot develop C3 deposition/MPGN2 develop C3 deposition/MPGN2

Plasma C3 (Plasma C3 (BB) and factor B () and factor B (CC) levels in ) levels in CfiCfi–/– mice with heterozygous and homozygous factor H –/– mice with heterozygous and homozygous factor H deficiency. C3 level is lowest in Cfh-/- mice. Factor B level is equally low in all types. deficiency. C3 level is lowest in Cfh-/- mice. Factor B level is equally low in all types.

Plasma analysis:Plasma analysis:(cfi -/-): C3b(cfi -/-): C3b(cfh -/-) : C3b cleavage products(cfh -/-) : C3b cleavage products(cfi -/- , cfh -/-): C3b(cfi -/- , cfh -/-): C3b Addition of Factor I: Addition of Factor I: (cfi-/-),(cfi-/-,cfh-/-) + Factor I (cfi-/-),(cfi-/-,cfh-/-) + Factor I C3b C3b

fragments in plasma.fragments in plasma. Plasma analysis Plasma analysis

Western blot for C3 from Western blot for C3 from CfiCfi–/– and –/– and CfiCfi–/––/–CfhCfh–/– mice under reducing conditions show α′-–/– mice under reducing conditions show α′-chain and HMW bands. Fragments were only present in the chain and HMW bands. Fragments were only present in the CfhCfh–/– EDTA plasma . –/– EDTA plasma . Sera from Sera from CfiCfi–/– and –/– and CfiCfi–/––/–CfhCfh–/– mice before and after incubation with murine sera –/– mice before and after incubation with murine sera deficient in C3 (as a source of autologous factors I and H) show complete cleavage of the α′-deficient in C3 (as a source of autologous factors I and H) show complete cleavage of the α′-chain with the concomitant appearance of the α′-chain fragment chain with the concomitant appearance of the α′-chain fragment

Under nonreducing conditions C3c was detectable in EDTA Under nonreducing conditions C3c was detectable in EDTA plasma from plasma from CfhCfh–/– and wild-type mice, but not from –/– and wild-type mice, but not from CfiCfi–/– –/–

mice.mice.

Following injections of sera from Cfh–/–C3–/– mice (used as a source of autologous factor I) to Following injections of sera from Cfh–/–C3–/– mice (used as a source of autologous factor I) to mice with combined deficiency of factor H and factor I. plasma C3 levels fell rapidly to levels mice with combined deficiency of factor H and factor I. plasma C3 levels fell rapidly to levels comparable with those seen in mice with factor H deficiency alonecomparable with those seen in mice with factor H deficiency alone

Infusion of serum containing factor I ( sera from Cfh-/-C3-/- mice) to mice with combined deficiency of Infusion of serum containing factor I ( sera from Cfh-/-C3-/- mice) to mice with combined deficiency of factor H and factor I. factor H and factor I.

Appearance of α chain fragments in the circulation.Appearance of α chain fragments in the circulation.

Florid glomerular C3 staining identical to that seen in unmanipulated Florid glomerular C3 staining identical to that seen in unmanipulated CfhCfh–/– mice was –/– mice was present in the reconstituted animals, in marked contrast to the mesangial C3 staining present present in the reconstituted animals, in marked contrast to the mesangial C3 staining present in the noninjected in the noninjected CfhCfh–/––/–CfiCfi–/– control mice .–/– control mice .

The The CfhCfh–/– mice with heterozygous factor I deficiency had identical glomerular C3 staining –/– mice with heterozygous factor I deficiency had identical glomerular C3 staining to that seen in to that seen in CfhCfh–/– animals. Also had markedly reduced C3 levels (median 17.8) –/– animals. Also had markedly reduced C3 levels (median 17.8) comparable with mice deficient in Cfh-/- alone.comparable with mice deficient in Cfh-/- alone.

Factor I is critically required for GBM C3 Factor I is critically required for GBM C3 deposition in Cfh-/- mice.deposition in Cfh-/- mice.

The C3 deposited along the GBM in factor The C3 deposited along the GBM in factor H deficient mice derives from the H deficient mice derives from the circulation: circulation:

Examined glomerular staining 6 weeks post Examined glomerular staining 6 weeks post kidney transplant:kidney transplant:

- wild-type mice into Cfh-/- recipients- wild-type mice into Cfh-/- recipients- Cfh-/-,C3-/- mice into Cfh-/- recipientsCfh-/-,C3-/- mice into Cfh-/- recipients- Cfh-/- mice into Cfh-/- recipientsCfh-/- mice into Cfh-/- recipients- wild-type mice into wild-type recipientswild-type mice into wild-type recipients

(A), (B) & (D): Linear capillary wall C3 staining. kidneys transplanted (A), (B) & (D): Linear capillary wall C3 staining. kidneys transplanted into into CfhCfh–/– animals. –/– animals. (C): No abnormal glomerular C3 staining. (C): No abnormal glomerular C3 staining.

ConclusionConclusion In In CfiCfi–/– mice, factor H is not –/– mice, factor H is not

responsible for the lack of GBM C3 responsible for the lack of GBM C3 deposition. deposition.

Factor I is a critical requirement for Factor I is a critical requirement for GBM C3 deposition and subsequent GBM C3 deposition and subsequent MPGN2 in MPGN2 in CfhCfh–/– mice. –/– mice.

During uncontrolled alternative pathway During uncontrolled alternative pathway activation, it is the nature of the plasma activation, it is the nature of the plasma C3 activation product that determines C3 activation product that determines whether GBM C3 deposition develops. whether GBM C3 deposition develops.

ConclusionConclusion The important difference between factor I and The important difference between factor I and

factor H deficiency is the fate of plasma C3b: factor H deficiency is the fate of plasma C3b: - Factor I deficiency: No Cleavage productsFactor I deficiency: No Cleavage products No MPGNNo MPGN- Factor H deficiency: Cleavage products presentFactor H deficiency: Cleavage products present MPGN developsMPGN develops

Factor I–mediated cleavage of C3b to form Factor I–mediated cleavage of C3b to form iC3b, C3c, and C3dg in Factor H deficiency iC3b, C3c, and C3dg in Factor H deficiency occurs as a consequence of alternative factor I occurs as a consequence of alternative factor I cofactors in the circulation (CR1 on cofactors in the circulation (CR1 on erythrocytes, plasma factor H–like protein 1 erythrocytes, plasma factor H–like protein 1 and plasma factor H–related protein 5). and plasma factor H–related protein 5).

ConclusionConclusion

There remains no definitive therapy for There remains no definitive therapy for MPGN2. MPGN2.

Prevention of C5 activation ameliorated but Prevention of C5 activation ameliorated but did not prevent the development of MPGN2 did not prevent the development of MPGN2 in in CfhCfh–/– mice.–/– mice.

GBM C3 deposition secondary to alternative GBM C3 deposition secondary to alternative pathway dysregulation could be prevented, pathway dysregulation could be prevented, regardless of etiology, by strategies that regardless of etiology, by strategies that either prevent proteolytic cleavage of C3b or either prevent proteolytic cleavage of C3b or sequester its metabolites in the circulation. sequester its metabolites in the circulation.