Atypical Hemolytic Uremic Syndrome and C3 Glomerulopathy

Atypical Hemolytic Uremic Syndrome and C3 Glomerulopathy
KDIGO 2016 Controversies Conference Atypical Hemolytic Uremic Syndrome and C3 Glomerulopathy This presentation is based on: Goodship T. et al., Kidney Intl (2017) 91:

Supported by The content of this presentation reflects the position of the authors of the original paper. It does not reflect the official opinion of Alexion or Achillion and should not be viewed as a position endorsed by them. Responsibility for the information and views expressed in this presentation lies entirely with the authors. Disclaimer: Eculizumab is not currently approved for the treatment of C3G.

Part 1: Introduction

The Roles of Complement
Ricklin D, et al. Nat Rev Nephrol 12: , 2016.

Atypical Hemolytic Uremic Syndrome (aHUS)
Ultra-rare disease characterized by acute kidney injury (AKI), thrombocytopenia, and microangiopathic hemolytic anemia. At least 50% of aHUS patients have an underlying inherited and/or acquired complement abnormality. Eculizumab, a humanized mAb against C5, makes it possible to control aHUS and prevent development of ESRD in the majority of patients. The reported incidence of aHUS is approximately 0.5 per million per year. The complement abnormality in aHUS leads to dysregulated activity of the alternative pathway (AP) at the endothelial cell surface. However, non-complement inherited abnormalities, such as mutations in DGKE, can also result in an aHUS phenotype.

C3 Glomerulopathy (C3G) C3G comprises a group of kidney diseases driven by uncontrolled activation of the complement cascade that leads to C3 deposition within the glomerulus. The dysregulation of C3 convertase is driven by genetic and/or acquired defects. A biopsy is required to make the diagnosis. Two major subtypes are recognized: dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). Most frequently, dysregulation occurs at the level of the C3 convertase of the alternative pathway in the fluid phase. Broad inter-individual variability gives rise to two major subtypes of disease that are resolved by characteristic findings on renal biopsy: dense deposit disease (DDD) C3 glomerulonephritis (C3GN)

Complement Dysregulation in C3G & aHUS
Mastellos DC, et al. Trends in Immunology 38: , 2017

Atypical Hemolytic Uremic Syndrome (aHUS)
SECTION A Atypical Hemolytic Uremic Syndrome (aHUS)

Part 2: Renal Pathology: aHUS

aHUS Pathology aHUS is a thrombotic microangiopathy (TMA).
Pathology resembles tissue responses to endothelial injury. The presence of C5b-9 staining is not a reliable indicator of aHUS. In general, it is not possible to determine etiology from morphology. In some biopsies, overt thrombosis as evidenced by intraluminal fibrin or fibrin-platelet plugging is not seen. Nonthrombotic features include endothelial swelling and denudation, mesangiolysis, double contours of the glomerular basement membrane, and subendothelial accumulation of electron-lucent, flocculent material. In arteries and arterioles, intramural fibrin, myxoid intimal thickening and concentric myointimal proliferation (onion-skinning) may occur. It is important for the pathologist to provide a differential diagnosis, especially in patients with severe hypertension, where attributing changes to hypertension alone may lead to failure to identify other specific causes such as complement dysfunction.

Morphological Features in Microangiopathy
Active Lesions Chronic Lesions Glomeruli Thrombi Endothelial swelling or denudation Fragmented red blood cells Subendothelial flocculent material by EM Mesangiolysis Microaneurysms Double contours of peripheral capillary walls by LM, with variable mesangial interposition New subendothelial basement membrane by EM Widening of the subendothelial zone by EM Arterioles Intramural fibrin Intimal swelling Myocyte necrosis Hyaline deposits Arteries Myxoid intimal swelling Fibrous intimal thickening with concentric lamination (onion skin)

aHUS Case Study 30-y.o. male, 2-week history of general malaise, presented with AKI (plasma creatinine (Cr) 250 µmol/L) and microangiopathic hemolytic anemia (hemoglobin (Hb) 9.5 g/dL and fragmented cells on a peripheral blood film). Platelet count 130 x 109/L. Renal biopsy showed endothelial swelling and denudation, mesangiolysis, double contours of the glomerular basement membrane and subendothelial accumulation of electron-lucent, flocculent material. No evidence of intraluminal thrombus. Patient’s maternal grandmother had developed kidney failure after the delivery of her second child.

aHUS Case Study Which of the following are correct?
The renal biopsy appearances are not compatible with a diagnosis of aHUS. Screening for secondary causes of a renal thrombotic microangiopathy should be undertaken. Genetic screening for inherited complement abnormalities should be undertaken. The family history is not relevant. Treatment with high-dose steroids should be commenced. Both B and C. Both D and E. The correct answer is F (both B and C). The absence of intraluminal thrombus does not exclude a diagnosis of aHUS. Screening for secondary causes of a renal thrombotic microangiopathy and complement genetic abnormalities should be undertaken in all individuals presenting with features compatible with a diagnosis of aHUS. The family history may indeed be relevant. There is no evidence for the use of high-dose steroids.

Part 3: Clinical Phenotype and Assessment: aHUS

aHUS: Presentation Current classifications of aHUS reflect a better understanding of disease mechanisms, including the impact of genetic background and etiologic triggers. Precipitating factors include autoimmune conditions, transplants, pregnancy, infections, drugs, and metabolic conditions. The time course and persistence of an aHUS episode are not well understood. Many patients appear to be at life-long risk for the recurrent acute presentation of aHUS. Disease penetrance for an acute episode of aHUS in carriers of known pathogenic mutations increases with age. A small percentage (3-5%) of patients carry more than one pathogenic genetic variant, supporting a relationship between mutation burden and penetrance. Presentation in later life is consistent with the need for an environmental trigger. Discordance between the pathological and clinical manifestations of the disease is sometimes seen, e.g., a thrombotic microangiopathy can sometimes be present on renal biopsy in the absence of thrombocytopenia.

aHUS: Extrarenal Manifestations
Extrarenal manifestations are reported in up to 20% of patients. It is unclear whether these manifestations are a direct consequence of complement activation, TMA, or other factors such as severe hypertension and uremia. (Suppl Table 1) Despite sharing many of the same rare genetic variants in CFH21 and CFI22 described in age-related macular degeneration (AMD), drusen formation is not commonly reported in aHUS.

aHUS: Extrarenal Manifestations
Digital gangrene, skin Cerebral artery thrombosis/stenosis Extracerebral artery stenosis Cardiac involvement/myocardial infarction Ocular involvement Neurologic involvement Pancreatic, gastrointestinal involvement Pulmonary involvement Intestinal involvement

aHUS: Laboratory Analysis
Investigations should focus on determining the underlying etiology and excluding other diagnoses. Measure ADAMTS13 activity to diagnose or exclude thrombotic thrombocytopenic purpura (TTP). Because the incidence of TTP is much lower in children than in adults, expert opinion recommends that in children, treatment with eculizumab should not be delayed while ADAMTS13 activity is being determined; however, signs of nonresponse should be carefully monitored. Investigation for STEC-HUS should be routine in all patients with presumed aHUS. Since the incidence of TTP is much lower in children than in adults, expert opinion recommends that in children, treatment with eculizumab should NOT be delayed while ADAMTS13 activity is being determined; however, signs of non-response should be carefully monitored. In adults, measuring ADAMTS13 activity is recommended PRIOR to eculizumab initiation. Approximately 5% of STEC-HUS cases have no prodromal diarrhea while 30% of complement-mediated aHUS cases do have concurrent diarrhea or gastroenteritis.

aHUS: Laboratory Analysis
Serum/plasma levels of complement proteins should be measured in all patients with primary aHUS prior to plasma therapy. C3 levels will be low in 30-50% of aHUS cases. Low C3 levels are also noted in acute STEC-aHUS and pneumococcal aHUS. Complement split products and assays of complement function can also be obtained, although the significance of some of these assays requires further study. Since the incidence of TTP is much lower in children than in adults, expert opinion recommends that in children, treatment with eculizumab should NOT be delayed while ADAMTS13 activity is being determined; however, signs of non-response should be carefully monitored. In adults, measuring ADAMTS13 activity is recommended PRIOR to eculizumab initiation. Approximately 5% of STEC-HUS cases have no prodromal diarrhea while 30% of complement-mediated aHUS cases do have concurrent diarrhea or gastroenteritis.

aHUS Case Study 24-y.o. female presented with: AKI (Cr 500 µmol/L)
thrombocytopenia (platelet count 50 x 109/L); and a microangiopathic haemolytic anaemia, following a 2-week history of intermittent diarrhea.

aHUS Case Study Which of the following investigations should be undertaken to elucidate the cause of illness? Measure serum levels of C3 and C4. Measure ADAMTS13 activity. Screen for STEC infection. Screen for factor H autoantibodies. Screen for anticardiolipin antibodies. All of the above. None of the above. The correct answer is F: all of the above. The clinical history is compatible with many causes of a renal thrombotic microangiopathy, including complement abnormalities, thrombotic thrombocytopenic purpura, STEC HUS and antiphospholipid antibody syndrome. The history of intermittent diarrhea is not specific for STEC HUS.

Part 4: Genetic and Acquired Drivers of Disease: aHUS

aHUS: Genetic Drivers of Disease
Studies of hundreds of aHUS patients have provided an excellent understanding of genetic drivers of disease, leading to the development of individualized care. Genetic screening and molecular diagnostics, with expert interpretation of the results, should inform therapeutic decisions. aHUS Delays in obtaining results from genetic/molecular diagnostic studies should NOT prevent a clinical diagnosis or postpone treatment, as early anti-complement treatment is crucial to preserve renal function and avoid irreversible sequelae. C3G The knowledge gap can be addressed by screening large numbers of C3G patients, studying the effects of disease-associated variants on function, and correlating these data with clinical outcomes. Genetic results may assist in treatment decisions (e.g., anti-complement therapy vs. immunosuppression) and should be undertaken in familial cases and when there is suspicion of a genetic defect (e.g., low FH or FI levels, parental consanguinity or Cypriot ancestry).

aHUS: Genetic Testing The minimum set of genes that should be screened includes CFH, CD46, CFI, C3, CFB, THBD, CFHR1, CFHR5, and DGKE. Genetic testing should also include the risk haplotypes CFH- CFHR3 and MCPggaac as they modify disease penetrance and severity. Delays in obtaining results from genetic or molecular diagnostic studies should not prevent a clinical diagnosis or postpone treatment, as early anticomplement treatment is crucial to preserve renal function and avoid irreversible sequelae. Technologies to detect copy number variation, hybrid genes and other complex genomic rearrangements in the CFH/CFHRs genomic region must be included in the genetic testing. The identification of a pathogenic genetic variant in an aHUS patient reinforces the diagnosis and establishes with accuracy the cause of the disease, facilitating patient management, effective treatment and genetic counseling. In C3G, however, present knowledge is insufficient except in cases of CFHR rearrangements leading to fusion genes (like CFHR5 nephropathy), FH or FI deficiency, or with C3 mutations.

aHUS: Genetic Testing Genetic analysis is essential in living-related kidney donor transplantation. Transplantation from living-related kidney donors should only be considered if causative genetic or acquired factors are clearly identified in the recipient and the related donor is free of these factors. Genetic testing is recommended for patients in whom discontinuation of eculizumab is being considered. The general recommendation in aHUS is that transplantation from living-related kidney donors should only be considered if causative genetic (or acquired) factors are clearly identified in the recipient and the related donor is free of these factors. the presence in the donor of CFH or MCP aHUS risk haplotypes is not a contraindication to donation.

Understanding Genetic Variants
Genetic variants should be classified as “benign,” “likely benign,” “variant of uncertain significance (VUS),” “likely pathogenic,” or “pathogenic,” following international guidelines. Pathogenic variants compromise protection of host endothelial cells and platelets from complement damage/activation. Genetic makeup also influences disease progression, response to therapies, and recurrence after transplantation. The combination of different pathogenic variants and/or the combination of pathogenic variants and common risk variants in CFH and MCP determine overall individual risk/predisposition to aHUS. It is highly recommended that genetic results be interpreted by a laboratory with expertise in aHUS and C3G.

aHUS: Acquired Drivers of Disease
Acquired drivers of disease are autoantibodies to complement proteins or protein complexes that impair normal function. The best-studied acquired drivers are FH autoantibodies, which are usually seen in association with deletion of the CFHR3 and CFHR1 genes. The deletion of CFHR3 and CFHR1 is a common copy number variation that can be identified on genetic testing. The finding of FH autoantibodies should be confirmed in a second sample at least 4 weeks after the initial sample. Testing should also be performed in the pre-renal transplant period. In pediatric patients, FH autoantibody assays should be performed following consensus guidelines: at diagnosis and if positive, at day 7, 14, 28, monthly and one year. Relapses of anti-FH associated HUS occur in about 20-25% patients.

aHUS: Acquired Drivers of Disease
Testing should also be performed in the pre-renal transplant period. In pediatric patients, FH autoantibody assays should be performed following consensus guidelines: at diagnosis and if positive, at day 7, 14, 28, monthly and one year. Relapses of anti-FH associated HUS occur in about 20-25% patients.

aHUS Case Study 35-y.o. female presented with AKI, thrombocytopenia, and a microangiopathic hemolytic anemia. Treated with eculizumab and made a full recovery. On genetic screening, she was found to carry a heterozygous CFH/CFHR1 hybrid gene. Family history: Both parents alive. Two brothers and 3 sisters. Two children aged 6 and 2.

Other members of the family are not at risk of developing aHUS. There is no need to offer screening to other family members for the CFH/CFHR1 hybrid gene. The finding of the CFH/CFHR1 hybrid gene in an unaffected family member has no implications for that individual. Family members should be offered genetic counselling. aHUS can present at any age in unaffected carriers of the CFH/CFHR1 hybrid gene. Both A and C. Both D and E. The correct answer is G: both D and E. Other family members, if they also carry the CFH/CFHR1 hybrid gene, are at risk of developing aHUS. Family members should be informed of this risk and given the opportunity to undergo genetic counselling and potentially screening for the CFH/CFHR1 hybrid gene. Within families, aHUS can present at any age in individuals carrying the underlying genetic abnormality.

Part 5: Treatment Strategies: aHUS

aHUS: Treatment All patients with a clinical diagnosis of primary aHUS are eligible for treatment with eculizumab. The dosing schedule reported in the trials that led to the approval of eculizumab should be followed. Treatment duration is controversial as there is no evidence to support life-long therapy in all aHUS patients. The treatment of FH autoantibody-driven aHUS involves the use of anti-cellular therapy and is guided by antibody titer. Interruption of anti-complement therapy during intercurrent illness, a time of high-risk for aHUS relapse, is not recommended unless an infection with an encapsulated organism is suspected or documented.

aHUS: Treatment Two options for long-term dosing have been considered:
The minimal dose required to achieve complement blockade. A discontinuation dosing schedule. No data exist to support either option, and both require monitoring of complement activity. There are no data to inform the frequency of testing Dose reduction or discontinuation require ongoing monitoring of complement activity. In patients who have undergone transplant, especially patients who have lost previous allografts, discontinuation is not recommended. The treatment of FH autoantibody-driven aHUS involves the use of anti-cellular therapy and is guided by antibody titer. Interruption of anti-complement therapy during intercurrent illness, a time of high-risk for aHUS relapse, is not recommended unless an infection with an encapsulated organism is suspected or documented.

aHUS: Treatment If access to eculizumab is unavailable, plasma therapy can be used. The use of plasma exchange when eculizumab is available may be associated with some improvement, but delaying use of eculizumab may lead to a suboptimal therapeutic outcome. Eculizumab increases the risk of meningococcal infection. Patients should receive vaccination against meningococcus (including Type B); however, vaccination should not delay the start of eculizumab therapy. Antibiotic prophylaxis is mandated during the first 2 weeks. Plasma exchange should also be considered for anti-FH positive aHUS and in the emergency treatment of critically ill patients with severe TMA (e.g., coma, convulsions) and a strong presumption of TTP until evidence of residual ADAMTS13 activity >10%. Discontinuation of plasma therapy or complement inhibition is feasible at least in some aHUS patients. The consensus favored a minimal period of treatment to allow optimal renal recovery without early relapse. Controversy remains whether vaccination is efficacious in patients with acute kidney injury, chronic kidney disease, and/or during immunosuppression. It is also unknown whether anti-meningococcal antibodies are protective in the setting of complement blockade; therefore it is recommended that antibiotic prophylaxis be maintained for the treatment duration and up to 2-3 months after discontinuation.

aHUS Treatment: Transplant
Kidney transplantation should be delayed for at least 6 months after the start of dialysis as limited renal recovery is possible several months after starting eculizumab. Living-related kidney donation carries a risk for recurrence in the recipient and a risk of de novo disease in the donor should the donor carry an at-risk genetic variant. Liver transplant remains an option in patients with liver- derived complement protein abnormalities, in particular for renal transplant recipients with uncontrolled disease activity despite eculizumab therapy. The resolution of hematological TMA features and extrarenal manifestations is a prerequisite for transplantation. The decision to use anti-complement therapy during transplantation should be based on recurrence risk. Potential donors with evidence of abnormal alternative complement pathway activity, even in the absence of detected complement gene mutations, should be excluded. If the potential living-related donor does not carry a pathogenic variant in a complement gene and has no evidence of abnormal complement activation, donation is feasible.

Abnormal titer depends on the testing laboratory;
Clinical diagnosis of aHUS High titer FH autoantibody1 Plasma therapy2 Simultaneous start of anticellular therapy3 Periodic monitoring of FH autoantibody level5 Discontinue therapy when antibody titer falls below a pathogenic titer for at least 6 months6 Continue plasma therapy indefinitely4 Eculizumab Continue eculizumab therapy indefinitely4 Treatment of complement factor H autoantibody-mediated aHUS. There are no prospective controlled studies in patients with aHUS due to anti-FH antibodies and thus the proposed management is based on a pediatric consensus. Abnormal titer depends on the testing laboratory; The decision to use plasma therapy versus eculizumab will be based on patient age and local resource availability; Cyclophosphamide, rituximab or mycophenolate mofetil; The decision to continue anti-complement therapy indefinitely is not informed by data; The interval may be monthly or quarterly and is based on local resources; This recommendation is based on limited retrospective case reviews.

aHUS Case Study 44-y.o. male on hemodialysis for 15 years.
Primary renal disease is aHUS. Received two cadaver renal transplants, both of which were lost within 3 months of transplantation to recurrent disease despite treatment with plasma exchange. Genetic screening showed a heterozygous mutation in factor H, which has been reported previously in six other patients with aHUS. Being considered for a third transplant.

Prophylactic use of eculizumab immediately before and then long-term after the transplant is unlikely to prevent disease recurrence in the transplanted kidney. Prophylactic plasma exchange immediately before and then long-term after the transplant is highly likely to prevent disease recurrence after the transplant. A living-related transplant should not be considered under any circumstances. If he receives prophylactic eculizumab, he should be vaccinated against meningococcus. The correct answer is D. Use of prophylactic eculizumab in this clinical setting is highly likely to prevent disease recurrence. A living-related transplant could be considered if the donor is screened and not found to carry the same factor H mutation Meningococcal vaccination should be undertaken in all patients receiving eculizumab.

aHUS Research Recommendations: Summary
A comparative study of biopsies from patients with well-documented malignant hypertension and patients with well-documented alternative complement pathway disease. A longitudinal study of patients with features of chronic microangiopathy on biopsy but without a history of acute presentation. Clinical studies Define how complement biomarkers correlate with current or impending aHUS relapse and/or renal involvement. Identify risk factors for relapse upon cessation of anti-complement therapy. Identify alternative anti-complement therapeutics.

SECTION B C3 Glomerulopathy (C3G)

Part 2: Renal Pathology: C3G

C3G Pathology The C3G disease spectrum is caused by abnormal control of complement activation, deposition or degradation that results in predominant glomerular C3 fragment deposition. A renal biopsy using immunofluorescence (IF) is required to diagnose C3G. Electron microscopy (EM) is used to sub-classify C3G as DDD or C3GN. Based on EM appearance, C3G may be subclassified as dense deposit disease (DDD; dense osmiophilic intramembranous deposits) or C3 glomerulonephritis (C3GN; light dense, amorphous mesangial, paramesangial, subendothelial and subepithelial deposits).

Morphological Features
Light Microscopy Active lesions Mesangial expansion with or without hypercellularity Endocapillary hypercellularity including monocytes and/or neutrophils Capillary wall thickening with double contours (the combination of capillary wall thickening and mesangial increase is referred to as a membranoproliferative pattern) Necrosis Cellular/fibrocellular crescents Chronic lesions Segmental or global glomerulosclerosis Fibrous crescents Immunofluorescence Microscopy Typically dominant C3 staining Electron Microscopy DDD: Dense osmiophilic mesangial and intramembranous electron dense deposits C3GN: Amorphous mesangial with or without capillary wall deposits including subendothelial, intramembranous and subepithelial electron dense deposits Sub-epithelial ‘humps’ may be seen in both DDD and C3GN

C3G Pathology: Controversies
Correlations between renal biopsy appearances, etiology, and clinical outcome are ill-defined. IF staining is subjective and semiquantitative. Well-defined for dense deposit disease (DDD). Not clear if characteristic for C3 glomerulonephritis (C3GN). Distinguishing DDD and C3GN by EM can be difficult. It is possible to identify different C3 breakdown products in glomeruli by IF8 or mass spectrometry after laser capture microdissection. This methodology can also be used to detect other complement components (e.g., factor H-related proteins, C5 through C9). It is not known whether some of these complement components in specific tissue compartments (e.g., C5b-9) might identify a subset of patients likely to benefit from a specific type of therapy Detailed IF studies using well characterized antibodies will be valuable in guiding therapy.

C3G Case Study 7-y.o.male with 3-day h/o macroscopic hematuria 4-wk after being culture-positive for Group A β-hemolytic Strep pharyngitis. Normal blood pressure and urine output, no edema. Urinalysis: numerous RBCs; RBC casts; 4+ protein; negative leukocytes and nitrites. Serum Cr 1.0 mg/dL (normal mg/dL); normal electrolytes and albumin. ASO elevated (1240 IM/mL [normal <150 IU/mL]); ANA negative; C3 39 mg/dL (normal mg/dL). FH: negative for renal disease. It is possible to identify different C3 breakdown products in glomeruli by IF8 or mass spectrometry after laser-capture microdissection. This methodology can also be used to detect other complement components (e.g., factor H-related proteins, C5 through C9). It is not known whether some of these complement components in specific tissue compartments (e.g., C5b-9) might identify a subset of patients likely to benefit from a specific type of therapy. Detailed IF studies using well-characterized antibodies will be valuable in guiding therapy.

C3G Case Study Diagnosed with APSGN and monitored as an outpatient. One month later, creatinine has improved to 0.7 mg/dL, UPC ratio is 0.7 (normal <0.2), and C3 is 22 mg/dL. What would you do now? Get a renal biopsy. Continue to monitor and get a renal biopsy 3 months after onset if C3 has not improved. Start an oral course of prednisone at 2 mg/kg/day. Get genetic testing. Get comprehensive complement studies. It is possible to identify different C3 breakdown products in glomeruli by IF8 or mass spectrometry after laser-capture microdissection. This methodology can also be used to detect other complement components (e.g., factor H-related proteins, C5 through C9). It is not known whether some of these complement components in specific tissue compartments (e.g., C5b-9) might identify a subset of patients likely to benefit from a specific type of therapy. Detailed IF studies using well-characterized antibodies will be valuable in guiding therapy. The correct answer is B. Wait 3 months after an episode of APSGN for C3 levels to normalize. If they have not normalized and the UPC ratio remains elevated, consider a renal biopsy at that time.

Part 3: Clinical Phenotype and Assessment: C3G

C3G: Presentation C3G generally follows a chronic, indolent course with persistent AP activation resulting in a 10-year renal survival of approximately 50%. There are, however, cases of C3G that present as a rapidly progressive GN.

C3G: Extrarenal Manifestations
Acquired partial lipodystrophy (APL) and retinal drusen are reported and appear to be direct consequences of complement activation. APL is most commonly seen in individuals with C3 nephritic factors (C3Nefs). Factor D, required for formation of the C3 convertase, is highly expressed in adipocytes, which undergo C3Nef-induced complement-dependent lysis. Drusen, the accumulation of lipids and complement-rich proteins between Bruch's membrane and the retinal pigment epithelium, is commonly seen in AMD but occurs at an earlier age in C3G.

C3G: Laboratory Analysis
Serum/plasma levels of complement proteins should be measured in all patients with C3G prior to plasma therapy. C3 levels will be low in 30-50% of aHUS cases and up to 75% of C3G cases. Complement split products and assays of complement function can also be obtained, although the significance of some of these assays requires further study.

C3G Case Study 33-y.o.female with a diagnosis of DDD.
Ophthalmologic examination showed drusen. Complement labs showed: C3, <0.15 g/L (normal g/L); C3c, 3.3mg/L (normal <2.0 mg/L) B, 23.9 mg/L (normal mg/dL); Bb, 2.3 mg/L (normal <2.2mg/L) C5, 12 mg/dL (normal mg/dL); soluble C5b-9, 0.25mg/L (normal <0.3mg/L) CH50, <5 U/ml (normal U/ml); alternative pathway functional assay (APFA) 0% (normal %)

C3G Case Study What do these results mean?
There is no ongoing complement activity. The ongoing complement activity is primarily at the level of the alternative pathway. The ongoing complement activity is primarily at the level of the classical pathway. The ongoing complement activity is primarily at the level of the terminal pathway. The correct answer is B. Complement activation is ongoing as indicated by the decreased C3. The terminal pathway is not grossly dysregulated, as indicated by the normal biomarkers the indicate terminal pathway activity. Both the CH50 and APFA are low because serum C3 is so low.

Part 4: Genetic and Acquired Drivers of Disease: C3G

C3G: Genetic Drivers of Disease
Understanding of the genetics of C3G is not yet comparable to that of aHUS. There is no clear benefit to performing genetic analysis in all cases of C3G. C3G The knowledge gap can be addressed by screening large numbers of C3G patients, studying the effects of disease-associated variants on function, and correlating these data with clinical outcomes. Genetic results may assist in treatment decisions (e.g., anti-complement therapy vs. immunosuppression) and should be undertaken in familial cases and when there is suspicion of a genetic defect (e.g., low FH or FI levels, parental consanguinity or Cypriot ancestry).

C3G: Genetic Testing The minimum set of genes that should be screened includes CFH, CD46, CFI, C3, CFB, THBD, CFHR1, CFHR5, and DGKE. Technologies to detect copy-number variation, hybrid genes, and other complex genomic rearrangements in the CFH/CFHRs genomic region must be included in the genetic testing. The identification of a pathogenic genetic variant in an aHUS patient reinforces the diagnosis and establishes with accuracy the cause of the disease, facilitating patient management, effective treatment and genetic counseling. In C3G, however, present knowledge is insufficient except in cases of CFHR rearrangements leading to fusion genes (like CFHR5 nephropathy), FH or FI deficiency, or with C3 mutations.

C3G: Genetic Testing Genetic analysis is essential in living-related kidney donor transplantation. All planned recipients of a living-related kidney should be screened. If a genetic abnormality is found, the donor should be tested to exclude that genetic abnormality. In C3G, the donor is found to carry the same genetic abnormality as the recipient, current evidence would suggest that while this finding may not an absolute contraindication to donation, each case should be evaluated on an individual basis by experts in this area, taking into account the family history and specific genetic abnormality.

Understanding Genetic Variants
Genetic variants should be classified as “benign,” “likely benign,” “variant of uncertain significance (VUS),” “likely pathogenic,” or “pathogenic,” following international guidelines. C3G appears mechanistically more complex than aHUS. There is limited information about genotype/phenotype correlations to distinguish different C3G subtypes, inform prognosis and/or recommend treatment. Genetic makeup also influences disease progression, response to therapies, and recurrence after transplantation. The combination of different pathogenic variants and/or the combination of pathogenic variants and common risk variants in CFH and MCP determine overall individual risk/predisposition to aHUS. C3G involves massive C3 activation in plasma and complement dysregulation on surfaces, including the glycocalyx overlying the glomerular endothelial pores. It is highly recommended that genetic results be interpreted by a laboratory with expertise in aHUS and C3G.

C3G: Acquired Drivers of Disease
Acquired drivers of disease are autoantibodies to complement proteins or protein complexes that impair normal function. In C3G, the most common autoantibodies are to C3 convertase, a serine protease formed from C3b and Bb. These autoantibodies are called C3Nefs. They stabilize C3 convertase and prolong its half-life. Other antibodies in C3G include FH autoantibodies, C4Nefs and C5Nefs. In older adults, serum free light chains (FLC) should be assayed. The results of autoantibody assays require expert interpretation, with their relevance to disease interpreted in the context of the results for all other complement assays and genetic screens.

C3G Case Study 24-y.o.male with a diagnosis of DDD on hemodialysis.
Planning living-related donor transplant from sibling. Family history: Cousin also on dialysis. Genetic testing identified: An ultra-rare variant in the complement factor H gene, CFH c.3229T>C, which substitutes Cys at position 1077 for Arg (p.Cys1077Arg); this is classified as a VUS (variant of uncertain significance). A common copy-number variant (CNV) in which one copy of the complement factor H–related 3 and –related 1 genes are deleted (delCFHR3-CFHR1). It is possible to identify different C3 breakdown products in glomeruli by IF8 or mass spectrometry after laser-capture microdissection. This methodology can also be used to detect other complement components (e.g., factor H-related proteins, C5 through C9). It is not known whether some of these complement components in specific tissue compartments (e.g., C5b-9) might identify a subset of patients likely to benefit from a specific type of therapy. Detailed IF studies using well-characterized antibodies will be valuable in guiding therapy.

C3G Case Study What do these results mean and what would you do?
These results can be dismissed. He should not receive a kidney transplant because factor H is synthesized by the liver, and his disease will recur. The donor sibling should be screened for the complement factor H gene variant and, if found, he should be excluded from donating. The donor sibling should be screened for the complement factor H gene variant and, if found, he should be cleared to donate. It is possible to identify different C3 breakdown products in glomeruli by IF8 or mass spectrometry after laser-capture microdissection. This methodology can also be used to detect other complement components (e.g., factor H-related proteins, C5 through C9). It is not known whether some of these complement components in specific tissue compartments (e.g., C5b-9) might identify a subset of patients likely to benefit from a specific type of therapy. Detailed IF studies using well-characterized antibodies will be valuable in guiding therapy. The correct answer is C. The variant in CFH is ultra-rare and although it is classified as a VUS, in the absence of definitive functional studies determining its effect on complement control, if a potential family member wishes to donate and carries this genetic variant, that person should be excluded from donation. Ideally, the cousin should be studied in greater detail to determine the cause of renal failure. These genetic studies could be very informative.

Part 5: Treatment Strategies: C3G

C3G Treatment: All Patients
Optimal blood pressure control (suggested blood pressure below the 90% in children and ≤120/80 in adults) Priority agents include angiotensin converting enzyme inhibitors and angiotensin receptor blockers Optimal nutrition for both normal growth in children, healthy weight in adults Lipid control Moderate Disease Description Urine protein over 500 mg/24 hours despite supportive therapy OR Moderate inflammation on renal biopsy Recent increase in serum creatinine suggesting risk for progressive disease Recommendation Prednisone Mycophenolate mofetil Severe Disease Description Urine protein over 2000 mg/24 hours despite immunosuppression and supportive therapy Severe inflammation represented by marked endo- or extracapillary proliferation with or without crescent formation despite immunosuppression and supportive therapy Increased serum creatinine suggesting risk for progressive disease at onset despite immunosuppression and supportive therapy Methylprednisolone pulse dosing as well as other anti-cellular immune suppressants have had limited success in rapidly progressive disease Data are insufficient to recommend eculizumab as a first-line agent for the treatment of rapidly progressive disease RCT results are limiting, given the change in terminology and disease characterization and the potential confounding effect on trial stratification. The conference attendees acknowledged published reports that support the effectiveness of plasma therapy in the setting of C3G induced specifically by pathogenic variants in factor H; however, this approach appears to be beneficial to only a select subgroup of C3G patients.

C3G: Treatment A retrospective study supports the effectiveness of mycophenolate mofetil in C3GN patients. No specific recommendation can be made for plasma therapy or rituximab (an anti-CD20 antibody). Since the pathogenesis of C3G is due to dysregulation and hyperactivity of the alternative pathway of complement, eculizumab has been tried in a limited number of patients with varied results. RCT results are limiting, given the change in terminology and disease characterization and the potential confounding effect on trial stratification. The conference attendees acknowledged published reports that support the effectiveness of plasma therapy in the setting of C3G induced specifically by pathogenic variants in factor H; however, this approach appears to be beneficial to only a select subgroup of C3G patients.

C3G Treatment: Transplant
No specific data are available to inform decisions surrounding transplantation in C3G. Recommendations reflect expert opinion and limited case reports. C3G recurs in allografts at a high rate, leading to graft loss in ~50% of patients.

C3G Case Study 32-y.o. male with C3GN diagnosed 2 years ago.
On ACEs and ARBs with good control of HTN but increasing proteinuria and decreasing renal function. CKD G3 with eGFR 45 mL/min/1.73 m2 Complement studies: No rare genetic variants identified in CFH, CD46, CFI, C3, CFB, THBD, CFHR1, CFHR5, and DGKE C3, <0.55g/L (normal g/L; C3c, 2.2mg/L (normal <2.0 mg/L); C5, 8.5 mg/dL (normal mg/dL); soluble C5b-9, 5.45 mg/L (normal <0.3 mg/L); CH50, <26 U/mL (normal U/mL); APFA (alternative pathway functional assay 3% (normal %) C3Nef +2; C5Nef +4 It is possible to identify different C3 breakdown products in glomeruli by IF8 or mass spectrometry after laser-capture microdissection. This methodology can also be used to detect other complement components (e.g., factor H-related proteins, C5 through C9). It is not known whether some of these complement components in specific tissue compartments (e.g., C5b-9) might identify a subset of patients likely to benefit from a specific type of therapy. Detailed IF studies using well-characterized antibodies will be valuable in guiding therapy.

C3G Case Study What would you do next? Add rituximab.
Begin plasma exchange. Add mycophenolate mofetil. Add eculizumab. It is possible to identify different C3 breakdown products in glomeruli by IF8 or mass spectrometry after laser-capture microdissection. This methodology can also be used to detect other complement components (e.g., factor H-related proteins, C5 through C9). It is not known whether some of these complement components in specific tissue compartments (e.g., C5b-9) might identify a subset of patients likely to benefit from a specific type of therapy. Detailed IF studies using well-characterized antibodies will be valuable in guiding therapy. The correct answer is C. Although there is no disease-specific treatment for C3G, some studies suggest that MMF is effective in patients with C3GN. These patients typically have massive dysregulation of the terminal pathway and C5 convertase, as is clear from the biomarker levels in this patient.

C3G Research Recommendations: Summary
A multicenter study analyzing biopsies to define the relationship of morphology to etiology, clinical course, and response to therapy. Comprehensive genetic testing to fill the knowledge gap in establishing robust phenotype-genotype correlations. Clinical studies Assess the value of proximal (at the level of the AP) anti-complement therapy. Development and trial of novel complement inhibitors. Determine value of complement biomarkers to inform clinical outcome in C3G patients and stratify them into targeted treatment groups.

Conclusions While there are knowledge gaps in both aHUS and C3G, the evidence base for the management of patients with C3G lags behind that of aHUS; addressing this disparity should be a priority. Although these two diseases are presented as distinct entities, there is substantial overlap in their pathogenesis and clinical presentation.

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Atypical Hemolytic Uremic Syndrome and C3 Glomerulopathy

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Atypical Hemolytic Uremic Syndrome and C3 Glomerulopathy

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