Glomerular C4d Staining Research Articles

The relationship of microvascular inflammation with antibody-mediated rejection in kidney transplantation

Microvascular inflammation (MVI) is a key diagnostic feature of antibody-mediated rejection (AMR); however, recipients without donor-specific antibodies (DSA) defy etiologic classification using C4d staining of peritubular capillaries (C4dptc) and conventional DSA assignment. We evaluated MVI ≥ 2 (Banff g + ptc ≥ 2) using Banff 2019 AMR (independent of MVI ≥ 2 but including C4dptc) with unconventional endothelial C4d staining of glomerular capillaries (C4dglom) and - arterial endothelium and/or intima (C4dart) using tissue immunoperoxidase, shared-eplet and subthreshold DSA (median fluorescence intensity, [MFI] 100-499), and capillary ultrastructure from 3398 kidney transplant samples for evidence of AMR. MVI ≥ 2 (n = 202 biopsies) from 149 kidneys (12.4% prevalence) correlated with DSA+, C4dptc+, C4dglom+, Banff cg, i, t, ti scores, serum creatinine, proteinuria, and graft failure compared with 202 propensity score matched normal controls. The laboratory reported DSA− MVI ≥ 2 (MFI ≥500) occurred in 34.7%; however, subthreshold (28.6%), eplet-directed (51.4%), and/or misclassified anti-Human leukocyte antigen (HLA) DSA (12.9%) were identified in 67.1% by forensic reanalysis, with vascular C4d+ staining in 67.1%, and endothelial abnormalities in 57.1%, totaling 87.1%. Etiologic analysis attributed 62.9% to AMR (77.8% for MVI with negative reported DSA [DSA− MVI ≥2] with glomerulitis) and pure T cellular rejection in 37.1%. C4dptc−DSA− MVI ≥ 2 was unrecognized AMR in 48.0%. Functional outcomes and graft survival were comparable to normal controls. We concluded that DSA− MVI ≥ 2 frequently signified a mild “borderline” phenotype of AMR which was recognizable using novel serologic and pathological techniques.

Utility of C4d Immunohistochemistry as an Adjunct Stain in Diagnostic Renal Pathology of Glomerular Diseases.

C4d is a byproduct of the activation of the classic and lectin complement pathways. Being routinely used as a marker for antibody-mediated rejection, the significance of C4d in native kidney disease is currently being widely studied. We evaluated glomerular and extraglomerular C4d staining in 82 biopsies of proliferative and nonproliferative glomerulonephritis diagnosed in our institution. The staining pattern of C4d was tabulated in various glomerular diseases. All biopsies of membranous nephropathy including membranous lupus nephritis (Class V) and immune complex-mediated membranoproliferative glomerulonephritis (MPGN) consistently showed C4d deposits along glomerular basement membrane mirroring the location of immunoglobulin and complement in these conditions. Conversely, other glomerular diseases like IgA nephropathy, postinfectious glomerulonephritis, focal segmental glomerulosclerosis, minimal change disease, and diabetic nephropathy showed variable mesangial and capillary wall C4d deposits. To summarize, the consistent pattern of C4d staining in membranous nephropathy (primary and secondary)and immune complex-mediated MPGN can be used as a valuable adjunct tool in establishing the diagnosis, especially when immunofluorescence findings are limited by inadequate sampling.C4d reactivity in other glomerular diseases are variable and may not aid as a diagnostic tool in renal biopsy evaluation.

The level of urinary C4d is associated with disease progression in IgA nephropathy with glomerular crescentic lesions: a cohort study.

Positive glomerular C4d staining, representative of lectin pathway activation, has been proven to be associated with unfavorable outcomes in immunoglobulin A nephropathy (IgAN). Our previous study suggested that urinary C4d correlated positively with an increase in crescents while the relationship between urinary C4d and disease severity and progression remains unelucidated. In this study we enrolled 168 patients diagnosed with IgAN with varying proportions of crescent formation at the time of biopsy. An independent cohort of 107 IgAN patients was enrolled for validation. Kidney biopsy specimens were stained using immunohistochemistry. Urinary C4d levels at renal biopsy were measured by enzyme-linked immunosorbent assay. The primary endpoint was end-stage kidney disease (ESKD). Higher urinary C4d/creatinine levels were associated with a lower estimated glomerular filtration rate (eGFR); massive proteinuria; hypertension and severe Oxford M, E, T and C scores. After a median follow-up of 19 months (interquartile range 9-27), 53 (31.5%) participants reached ESKD. High urinary C4d/creatinine levels were independently and significantly associated with a risk of developing ESKD [hazard ratio per standard deviation increment of log-transformed C4d/creatinine 7.623 (95% confidence interval 4.117-14.113)]. The urinary C4d/creatinine level is a potential useful biomarker that was associated with disease severity and progression in patients with IgAN and crescents.

POS-373 THE LEVEL OF URINARY C4d IN PATIENTS WITH IgA NEPHROPATHY IS ASSOCIATED WITH DISEASE PROGRESSION: A COHORT STUDY

Positive glomerular C4d staining, representative of lectin pathway activation, has been proven to be associated with unfavorable outcomes in IgA nephropathy (IgAN). Our previous study suggested that urinary C4d correlated positively with an increase in crescents. While the relationship between urinary C4d and disease severity and progression remains unelucidated. In this study, we enrolled 168 patients diagnosed of IgAN with varying proportions of crescents formation at the time of biopsy. An independent cohort of 107 IgAN patients were enrolled for validation. Kidney biopsy specimens were stained using immunohistochemistry. Urinay C4d levels at renal biopsy were measured by enzyme-linked immunosorbent assay. The primary endpoint was end-stage kidney disease (ESKD). Levels of urinary C4d/Urine creatine(C4d/Ucr) demonstrated the same tendency as the stepwise increase in glomerular C4d positivity (p for trend <0.001). Higher urinary C4d/Ucr levels were associated with a lower estimated glomerular filtration rate (eGFR); massive proteinuria; hypertension; and severe Oxford-M, Oxford-E, Oxford-T and Oxford-C scores. After a median follow-up of 19 months (interquartile range, 9–27 months), 53 (31.5%) participants reached end-stage of kidney disease (ESKD). High urinary C4d/creatinine ratio levels were independently and significantly associated with a risk of developing ESKD (hazard ratio [HR], per standard deviation increment of log transformed C4d/creatinine of 7.623; 95% confidence interval [CI]: 4.117–14.113). Urinary C4d/creatinine ratio was a potential useful biomarker that was associated with disease severity and progression in patients with IgAN and crescents.

MO264COMPLEMENT PATHWAY MIGHT HAVE A ROLE IN FOCAL SEGMENTAL GLOMERULOSCLEROSIS: A RETROSPECTIVE SINGLE CENTER STUDY OF 15 YEARS

Abstract Background and Aims The role of complement in focal segmental glomerulosclerosis (FSGS) is an area of interest and C4d staining could indicate complement related renal damage. We investigated detailed C4d staining properties in native kidney biopsies and possible relation to clinical features. Method We retrospectively evaluated the renal biopsies of 114 patients diagnosed with FSGS within last 15 years. C4d expressions examined in glomeruli (mesangial and/or capillary wall, vascular pole, sclerotic areas), tubular region (basement membrane, adsorbtion droplet) and vascular areas (arteriols and arteries) via immunohistochemistry. A novel glomerular C4d score (G-C4d-S) was achieved on the basis of the localization, pattern, extent and intensity of the C4d expression (min-max, 0-13). Results Of the patients (56 females, mean age 43±14 years) with a median follow-up time of 35±3 months, mean proteinuria, eGFR, and albumin level were 4984 mg/day, 72.2 ml/min/1.73m2, 3.56 g/dL, respectively. Median G-C4d-S was 6 (IQR, 4-7) and it was negatively correlated with serum creatinine at diagnosis (r=-0.21, p=0.02). Glomerular staining (both focal and diffuse, higher than moderate in intensity) was positive in 78 (68.4%) of the patients. C4d on glomerular sclerotic area was positive in 43 (37%) patients and it was associated with lower eGFR at diagnosis. Forty five patients achieved remission during the follow-up. Among the pathological features only glomerular C4d staining was associated to remission (p=0.02). There were 20 (18.7%) patients who need renal replacement theraphy (RRT) and 7 deaths (6.1%) at the end of the cohort. Lower rate of C4d staining on tubular adsorption droplets and arteriols/arteries were found to be associated need for RRT (p=0.013, p=0.012, respectively). There were no significant relationship between mortality and C4d staining features. Conclusion In conclusion, we noted that significant number of patients had positivie C4d on glomeruli, arteiroles and tubular area. We indicated that C4d staining at diagnosis could help to distinguish active glomerulonephritis. Additionally, it seems to be essential to examine non-glomerular area of native kidney biopsies, as well.

Prognostic Value of Mesangial C4d Staining in IgA Nephropathy: A Tunisian Study.

Immunoglobulin A nephropathy (IgAN) is the most common primary glomerular disease. The main challenge in this disease is the evaluation of prognostic factors for end-stage renal disease (ESRD). The aim of our study was to assess the clinical and prognostic implications of C4d staining in primary IgAN. This was a retrospective study, including adults with primary IgAN. The study was conducted over a period of 10 years. Renal biopsies were scored according to the Oxford classification. C4d immunohistochemical staining was performed. We included 44 patients with a sex ratio of 2.6. The average age was 35.1 ± 11 years. Twenty-two patients (57%) had hypertension (HTN). The median proteinuria was 1.92 g/day. The median of the glomerular filtration rate was 47.66 mL/min/1.73 m2. According to the Oxford classification, mesangial proliferation, endocapillary proliferation, glomerulosclerosis, interstitial fibrosis and/or tubular atrophy and crescents were present in 41%, 36%, 86%, 34%, and 25 % of cases, respectively. We found positive glomerular C4d staining in 25 renal biopsies (57%). Age at diagnosis, mean arterial pressure, HTN, and baseline glomerular filtration rate were not correlated with C4d staining. On the other hand, proteinuria was significantly higher in patients with C4d-positive renal biopsy. The median follow-up duration was 30.5 months. Ten patients (23%) reached ESRD. At univariate analysis, positive C4d staining in more than 25% of glomeruli in patients without C1q deposition in the immunofluorescent study was associated with ESRD. Our study confirms the prognostic value of C4d staining in primary IgAN.

Relevance of glomerular C4d deposition in pediatric patients with Henoch-Schönlein Purpura compared to IgA nephritis

Introduction: IgA nephropathy (IgAN) is the most common primary glomerulonephritis (GN) in western countries and Henoch-Schonlein purpura nephritis (HSPN) is the most common form of vasculitis in childhood. Renal biopsy findings in both nephropathies are often similar and are characterized by mesangioproliferative GN with mesangial or mesangiocapillary IgA and C3c deposits. Objectives: The aim of this study was to investigate the significance of glomerular C4d-deposition as a discriminating factor between pediatric HSPN and IgAN. Patients and Methods: We retrospectively analyzed patient records and renal biopsies from 53 pediatric patients from one single center with a median age of 10.5 years (range 2.3-18 years). Twenty-two patients suffered from IgAN and 31 from HSPN. Work-up of all renal biopsies was performed using standard protocols including immunohistochemistry for C4d. Results: Pediatric IgAN patients presented significantly more often with gross hematuria, higher serum creatinine, lower glomerular filtration rate, lower serum C3 and proteinuria and on histology less endocapillary hypercellularity compared to HSPN patients. However, the rate of glomerular C4d-positivity was not different between IgAN (36%) and HSPN (42%). Comparing all cases with positive versus negative glomerular C4d-staining, pediatric patients with glomerular C4d-positivity showed significantly lesser gross hematuria and received significantly more often cyclophosphamide. This was in line with a tendency towards more proteinuria, hypertension and renal insufficiency at last follow-up in C4d-positive compared to C4d-negative patients. Conclusion: In conclusion, in our monocentric study glomerular C4d does not differ between pediatric HSPN and IgAN, but was associated with a tendency to a more severe course of the disease that needs to be confirmed in larger multicentric studies.

P1670GLOMERULAR C4D IN POST-TRANSPLANT IG-A GLOMERULONEPHRITIS IS ASSOCIATED WITH DECREASED ALLOGRAFT SURVIVAL

Abstract Background and Aims Post transplant (pTX) glomerulonephritis (GN) is an important factor contributing to early and late allograft failure. De novo or recurrent IgA GN are the most frequent pTX-GN forms, however little is known about the optimal risk and therapeutic assessment. Recently, immunopathological evidence of glomerular C4d staining has been suggested as a prognostic tool in native glomerular kidney disease, in particular IgA GN. While peritubular capillary C4d is an accepted diagnostic criterion for antibody mediated rejection (ABMR), the role of glomerular C4d in pTX-GN is unkown. The primary hypothesis was that the finding of immunohistochemical glomerular C4d may be associated renal graft survival in post TX IgA GN. Method This large retrospective cohort study included all indication biopsies performed for cause in 885 kidney allografts from 1999-2006. Cases were screened for biopsy verified de novo or recurrent pTX-GN. GN cases were re-assessed by a single nephropathologist (NK). Primary endpoint was death-sensored graft survival followed until 01.01.2017. Results The prevalence of pTX-GN was 9.6% (n=85/885). De novo or recurrent IgA-GN was the most common pTX GN form with 40%. Of the 34 cases with pTX IgA GN, 27 had adequate material for interpretation of immunohistochemical glomerular C4d in the biopsy specimen. Eighteen of the 27 cases (66%) with pTx-IgA GN showed positive immunohistochemical C4d staining along glomerular capillary walls. There was no difference in renal function at biopsy or after one year comparing glomerular C4d positive vs. C4d negative pTX IgA GN. In univariate KM analysis glomerular C4d positive pTX-IgA GN showed significantly worse renal allograft survival rates (28%) compared to glomerular C4d negative pTX IgA GN (50%) or cases without GN (66%), p&amp;lt;0.001, respectively. In a multivariate Cox-Regression analysis including baseline covariates as well C4d+ABMR, Borderline lesion and TCMR, C4d+pTX IgA-GN remained independently associated with death censored graft-loss (HR 2.92, 95% CI 1.51-5.64, p=0.001). Conclusion Glomerular C4d staining in post transplant IgA glomerulonephritis is an independent risk factor for worse allograft survival and may provide a valuable risk assessment tool for prognostic and therapeutic decisions in post transplant glomerulonephritis.

Epidemiology and Pathophysiology of Glomerular C4d Staining in Native Kidney Biopsies

IntroductionRoutine C4d staining in renal transplantation has stimulated its use in kidney biopsies with glomerulonephritis (GN). Methodical description on staining patterns in the native kidney is not available.MethodsWe retrospectively evaluated C4d staining in formalin-fixed paraffin-embedded sections from 519 native kidney biopsies (bx) with and without glomerular disease.ResultsStrong C4d staining was consistently present in immune-complex GN, including lupus nephritis (LN) (n = 68), membranous GN (n = 24), membranoproliferative glomerulonephritis (MPGN) pattern (n = 22), fibrillary GN (n = 3), and proliferative GN with monoclonal IgG (n = 3). C4d stained all cases of postinfectious GN (n = 7) amyloidosis (n = 20) and C1q GN (n = 3). In contrast, IgA nephropathy (IgAN) (n = 34), was negative in 62% of bx, with the rest staining variably. The E1 Oxford classification score correlated with capillary wall C4d staining (P = 0.05). C4d marked the glomerular and arteriolar lesions in thrombotic microangiopathy (TMA; n = 16), the glomerular sclerotic segments in focal segmental glomerulosclerosis (FSGS; n = 77), and marked areas of necrosis in crescentic GN (n = 21). In diabetic glomerulopathy (n = 70), C4d marked advanced insudative lesions but was negative otherwise. C4d weakly stained the mesangium, or was negative in normal biopsies (n = 13), minimal change disease (MCD; n = 21), thin basement membrane disease (n = 20), Alport (n = 3), IgM nephropathy (n = 2), C3 glomerulopathy (n = 5), acute interstitial nephritis (n = 12), acute tubular necrosis (n = 22), ischemic glomerulopathy/nephrosclerosis (n = 23), and other miscellaneous processes (n = 14). Staining in tubular basement membranes and peritubular capillaries was most common in lupus.ConclusionBased on reliable staining in lupus and membranous GN, C4d staining is potentially useful as a screening and diagnostic tool, if only paraffin-embedded tissue is available. Knowledge of C4d staining patterns in normal and pathological tissues enhances its diagnostic value.

Glomerular C4d Staining Does Not Exclude a C3 Glomerulopathy

IntroductionC4d, an early product in the classical/lectin complement pathway has shown potential in the evaluation of C3 glomerulopathy where its absence would support an alternative pathway abnormality. As autoimmune/genetic complement testing is not readily available to most parts of the world, glomerular C4d staining may serve as a useful additional step toward the diagnosis.MethodsTo test this hypothesis, C4d staining was performed on a large cohort of C3 glomerulopathy. Archival cases from 2011 to 2017 were reviewed and immunohistochemistry for C4d was performed, scored (scale of 0 to 3+), and correlated with the immunofluorescence and ultrastructural findings. Paraffin immunofluorescence was performed in cases of “discordant C4d” to unmask Igs.ResultsTwenty-seven cases of dense deposit disease (DDD) and 14 cases of C3 glomerulonephritis (C3GN) were retrieved. C4d demonstrated a range of staining intensities with negative/traces in only 22% of DDD and 64% of C3GN. Lower-intensity C4d staining (1 to 2+) was mostly concordant with similar amounts of Igs/C1q. Discordant 3+ staining was noted in approximately 50% of cases of DDD and 20% of cases of C3GN. Among them, paraffin immunofluorescence unmasked polyclonal Igs in 2 of 5 cases of DDD and 1 of 3 cases of C3GN.ConclusionThis observational study suggests that the presence of glomerular C4d should not exclude a C3 glomerulopathy. In lower intensities, it appears to represent overlying classical/lectin pathway activation with concordant Ig/C1q deposits. A subset of cases, however, displays intense and discordant C4d staining, which raises the possibility of an associated lectin pathway abnormality, a potential future area of study.

Pattern of Glomerular Staining for C4d Immunohistochemistry is Useful for the Diagnosis of Immune Complex Mediated Glomerulonephritis in Renal Allograft Biopsies

Introduction Performance of the C4d stain is required for the adequate evaluation of kidney transplant biopsies (KTBx). Positive C4d staining in the renal peritubular capillaries is very useful for the diagnosis of antibody mediated allograft rejection (AMR) and correlates with poorer graft outcomes. Glomerular C4d staining is common in the chronic phases of AMR but this is not diagnostically useful due to inconsistent distribution and intensity of staining. Deposition of the C4d complement component in immune complex mediated (ICM) glomerulonephritis (GN) is expected but this has not been systematically studied in KTBx. Materials and Methods C4d stain (immunohistochemical method) was applied to 2432 consecutive, paraffin embedded KTBx, over a period of 5 years. In KTBx due to abrupt increase in proteinuria or history of GN in the native kidney, immunofluorescence and electron microscopy studies for standard evaluation of GN were also used for diagnosis. Results and Discussion In this unselected biopsy cohort, 33 KTBx from 27 patients showed recurrent or primary GN: 12 membranous glomerulopathy (MGN), 4 lupus nephritis (SLE), 9 IgA nephropathy and 8 focal segmental glomerulosclerosis (FSGS), Distinctive and consistent glomerular C4d stainining was noted in patients with MGN with a pattern paralleling the IF and EM findings. SImilarly, in recurrnet SLE, glomerular C4d staining corresponded to the histological lupus Class determined by light microscopy, IF and EM. The C4d staining pattern in these GNs was clearly different from that seen in transplant glomerulopathy. C4d staining in IgA nephropathy was inconsistent, mostly mesangial and did not correlate clearly with the strength of IF IgA staining. Only the segmental sclerotic lesions in FSGS were highlighted by the C4d staining in a manner that appeared nonspecific. Conclusions C4d staining is routinely evaluated in transplant biopsies and this stain consistently highlights immune complex deposits in MGN and SLE. Recognition of this distinctive staining pattern is helpful in the diagnosis of these processes and should prompt complete evaluation with IF and EM if these studies were not initially available. The findings in this study are based on C4d stain evaluation of paraffin embedded tissue sections. Since C4d staining corresponds to the classical pathway of complement activation, similar findings are expected with the IF staining method in fresh frozen tissue sections.

A Probabilistic Approach to Histologic Diagnosis of Antibody-Mediated Rejection in Kidney Transplant Biopsies.

Histologic diagnosis of antibody-mediated rejection (ABMR) in kidney transplant biopsies uses lesion score cutoffs such as 0 versus >0 rather than actual scores and requires donor-specific antibody (DSA); however, cutoffs lose information, and DSA is not always reliable. Using microarray-derived molecular ABMR scores as a histology-independent estimate of ABMR in 703 biopsies, we reassessed criteria for ABMR to determine relative importance of various lesions, the utility of equations using actual scores rather than cutoffs, and the potential for diagnosing ABMR when DSA is unknown or negative. We confirmed that the important features for ABMR diagnosis were peritubular capillaritis (ptc), glomerulitis (g), glomerular double contours, DSA and C4d staining, but we questioned some features: arterial fibrosis, vasculitis, acute tubular injury, and sum of ptc+g scores. Regression equations using lesion scores predicted molecular ABMR more accurately than score cutoffs (area under the curve 0.85-0.86 vs. 0.75). DSA positivity improved accuracy, but regression equations predicted ABMR with moderate accuracy when DSA was unknown. Some biopsies without detectable DSA had high probability of ABMR by regression, although most had HLA antibody. We concluded that regression equations using lesion scores plus DSA maximized diagnostic accuracy and can estimate probable ABMR when DSA is unknown or undetectable.

C4 Glomerulopathy: A Disease Entity Associated With C4d Deposition

Complement-mediated glomerulonephritis, which includes C3 glomerulopathy, is characterized by dominant staining of C3 with minimal or no immunoglobulin deposits on immunofluorescence studies. We describe a new entity of complement-mediated glomerulonephritis that is characterized by bright C4d staining but with no or minimal C3 or immunoglobulin deposits on immunofluorescence studies. We label this entity as C4 glomerulopathy. C4 glomerulopathy includes C4 dense deposit disease and C4 glomerulonephritis. C4 dense deposit disease is characterized by bright C4d staining and dense deposits along glomerular basement membranes. C4 glomerulonephritis is characterized by bright C4d staining and many mesangial electron-dense deposits, with or without rare intramembranous electron-dense deposits. We describe clinical featuresand kidney biopsy results in a short series of 3 patients to highlight these findings. All 3 patients presentedwith proteinuria, and 2 patients also had hematuria. Kidney function was preserved in 2 patients, whereas 1 patient presented with declining kidney function. Evaluation for autoimmune disease, infection, and paraprotein yielded negative results in all patients. Complement levels were normal, although 1 patient had borderline low C4 levels. Kidney biopsy showed mesangial proliferative or membranoproliferative glomerulonephritis with bright C4d staining and absent or minimal C1q, C3, and immunoglobulin. Laser microdissection and mass spectrometry of glomeruli in all 3 patients showed large to moderate numbers of spectra matching C4. Furthermore, analysis of amino acid sequences showed that they were localized to the C4d portion of C4, consistent with immunofluorescence findings. Further studies are required to determine the underlying cause. In summary, we describe a novel complement-mediated glomerulonephritis that is characterized by bright glomerular C4d staining with minimal or absent staining for C1q, C3, and immunoglobulin.

IgA nephropathy: association of C4d with clinical and histopathological findings and possible role of IgM

Background: In patients with IgA nephropathy (IgAN) lectin and alternative pathways of the complement can be activated. Our aim was to analyze the association of glomerular and extraglomerular C4d staining—the representative of lectin pathway—with demographic, clinical and histopathological findings in primary IgAN patients. Design: Seventy-three patients were enrolled and after re-evaluation 37 of them were included in this study. Biopsies were analyzed for staining with anti-C4d primary monoclonal antibody by immunohistochemistry. Patients were classified as positive and negative groups based on their glomerular C4d deposition. Groups were compared for their baseline clinical and histopathological findings. Results: Sixteen (43.2%) of 37 patients were C4d-positive. Glomerular C4d-staining was associated with more severe proteinuria (2906 mg/day vs. 1091 mg/day; p = 0.002), lower GFR (54.87 mL/min vs. 95 mL/min; p = 0.023), higher blood pressure (p = 0.022), more severe endocapillary hypercellularity (p < 0.001) and more severe tubular atrophy (p < 0.01). Mesangial IgM deposition was found to be associated with glomerular C4d staining and nephrotic range proteinuria. Conclusions: Glomerular C4d deposition was found to be associated with more unfavorable histopathological and clinical findings at the time of diagnosis. Association of mesangial IgM deposition with the activation of lectin pathway is a novel finding. Mesangial IgM deposition in our patients may reflect the genetic heterology of IgAN between diverse populations. However, since these data are about association, a cause-and-effect about IgM and IgAN cannot be proven solely with these findings.

C4d as a Diagnostic Tool in Proliferative GN.

Proliferative GN is classified as immune complex-mediated or complement-mediated (C3 glomerulopathy). Immune complex-mediated GN results from glomerular deposition of immune-complexes/Ig and C3; the C3 is derived from activation of the classical and/or lectin pathways of complement. C3 glomerulopathy results from deposition of C3 and other complement fragments with minimal or no deposition of immune complexes/Ig; the C3 is derived from activation of the alternative pathway of complement. C4d is a byproduct of activation of the classic and lectin pathways. Although widely used as a marker for antibody-mediated rejection, the significance of C4d in C3 glomerulopathy is undetermined. We studied glomerular C4d staining in 18 biopsy specimens of immune-complex GN, 30 biopsy specimens of C3 GN, and 13 biopsy specimens of postinfectious GN. All specimens of immune complex-mediated GN, except two specimens of IgA nephropathy and one specimen of sclerosing membranoproliferative GN, showed bright (2-3+) C4d staining. The staining pattern of C4d mirrored the staining patterns of Ig and C3. Conversely, C4d staining was completely negative in 24 (80%) of 30 specimens of C3 glomerulopathy, and only trace/1+ C4d staining was detected in six (20%) specimens. With regard to postinfectious GN, C4d staining was negative in six (46%) of 13 specimens, suggesting an abnormality in the alternative pathway, and it was positive in seven (54%) specimens. To summarize, C4d serves as a positive marker for immune complex-mediated GN but is absent or minimally detected in C3 glomerulopathy.

Renal Arteriolar C4d Deposition

The mechanism of kidney injury in hematopoietic stem cell transplantation (HSCT)-associated thrombotic microangiopathy (TA-TMA) is not completely understood. Renal C4d staining is a marker of classic complement activation and endothelial injury and has been described in preliminary reports of HSCT recipients with TA-TMA. Our objective was to evaluate complement in the pathogenesis of small vessel injury in children receiving HSCT. We hypothesized that kidney tissue from children with TA-TMA would more frequently show C4d deposition compared with HSCT recipients without histologic TA-TMA. We reviewed kidney specimens (biopsy or autopsy) from children who had undergone HSCT at a single center. Using histologic criteria alone, subjects were divided into TA-TMA (n = 8) and non-TA-TMA (control) groups (n = 12). C4d staining was performed by immunohistochemistry and evaluated on arterioles, peritubular capillaries, glomeruli, and tubular basement membranes. Diffuse or focal renal arteriolar C4d staining was more common in subjects with histologic TA-TMA (75%) compared with controls (8%). Rare peritubular capillary C4d staining was present in 50% of TA-TMA samples and was absent in controls. Glomerular C4d staining was seen at a similar frequency in cases and controls, whereas tubular basement membrane staining was less frequently observed and only in subjects with TA-TMA. Arteriolar C4d deposition may be a pathologic marker of TA-TMA, implicating localized complement fixation in HSCT recipients with kidney disease secondary to small vessel injury. Further studies to better characterize the preferential arteriolar C4d staining may identify a renal compartment of injury, possibly explaining the dramatic hypertension seen in TA-TMA.

Glomerular C4d Staining Can Be an Indicator of Disease Activity in Lupus Nephritis

Background: Abnormalities in complement activation and clearance of immune complexes by erythrocytes are the central pathogenic mechanisms in systemic lupus erythematosus (SLE). Serum C4d level, which is a degradation product of complement factor C4, was found to be a sensitive indicator of SLE activity. Our aim was to determine whether glomerular C4d staining could be a useful marker of disease activity in patients with lupus nephritis. Methods: This retrospective study included all consecutive patients who underwent a renal biopsy at our center between January 2005 and December 2009. A total of 29 patients with IgA nephritis were enrolled, and renal biopsy specimens of 24 patients have been evaluated. We evaluated baseline age, sex, hypertension, serum creatinine level, glomerular filtration rate (GFR), urine protein, and glomerular C4d staining. The primary endpoint of this study was the onset of end-stage renal disease (ESRD) in the course of study. Results: Fourteen (58%) patients were C4d+ and 10 (42%) patients C4d–. Urinary protein excretion was more elevated in C4d+ group (p = 0.0001). The renal biopsy showed that activity index score >12 was a higher proportion in C4d+ patients. The patients were followed up for 3.5 years. Four patients in the C4d+ group evolved to ESRD in the follow-up, but none of the patients in the C4d– group (p = 0.064). Discussion: We found a relationship between glomerular C4d staining and activity of lupus nephritis. C4d staining may be a useful marker to predict the prognosis of lupus nephritis.

Glomerular C4d Immunoreactivity in Acute Rejection Biopsies of Renal Transplant Patients

In renal transplant patients, glomerulitis may be present in all types of acute rejection, often accompanied by diffuse C4d staining of peritubular capillaries: C4d3 positivity in more than 50% of peritubular capillaries. It may progress to chronic transplant glomerulopathy, characterized by capillary basement membrane multilayering, proteinuria, and progressive loss of renal function. While C4d3 is a recognized marker of an antibody-mediated reaction, the significance of glomerular C4d (GlC4d) staining is unknown. The aim of this study was to evaluate GlC4d immunoreactivity and its correlation with C4d3 in acute rejection biopsies. Paraffin-embedded acute rejection biopsies from 90 renal transplant patients were evaluated according to the Banff classification. Biopsies showing C4d-positive endothelial cells in more than 50% of glomeruli were considered GlC4d-positive. C4d3-positive staining prevalence was 23%. GlC4d-positive staining showed an 89% concordance rate (r = 0.81, P < .0001; Cohen's k = 0.80, P < .0001). GlC4d detection sensitivity was 0.80 and specificity 0.97. C4d3 and GlC4d immunoreactivity was significantly associated with glomerulitis (P < .006 and P < .03, respectively) and with proteinuria at the time of biopsy (P < .03 and P < .01, respectively). Interestingly, GlC4d positivity correlated better than C4d3 positivity with the presence of posttransplant circulating anti-human leukocyte antigen alloantibodies (P < .04 and P = .7, respectively). Patients with C4d3- or GlC4d-positive acute rejections underwent graft loss due to interstitial fibrosis and tubular atrophy more frequently than those with C4d0- or GlC4d-negative rejections (P < .0001 and P < .005, respectively), whereas no differences were observed in graft loss due to death. In conclusion, C4d3 and GlC4d stains showed a high correlation rate. Compared with C4d3, GlC4d staining demonstrated good sensitivity and excellent specificity. Our results suggested that GlC4d staining may indicate glomerular endothelial damage and be of prognostic value.

Renal C4D Deposition Is a Marker of Hematopoietic Stem Cell Transplant (SCT)-Associated Thrombotic Microangiopathy (TMA)

C4d is a marker of classical complement activation by tissue specific antibodies (abs) or endothelial damage. Currently, peritubular capillary (PTC) C4d deposition aids in the diagnosis (dx) of ab mediated kidney transplant rejection (AMR). An uncontrolled case series reported PTC and glomerular C4d staining in patients with SCT-TMA (Mii et al, Path Int 2011). Expanding on these findings, we hypothesized that renal tissue from patients with SCT-TMA would more frequently show C4d deposition compared to controls. Retrospective analysis of all renal biopsy and autopsy specimens from pediatric patients undergoing SCT at our institution. Using histological criteria alone, patients were divided into TMA and non-TMA (control) groups. C4d staining was performed by immunohistochemistry on formalin-fixed, paraffin-embedded tissue and was evaluated on arterioles, PTCs, glomeruli, and tubular basement membranes. Specifically, rabbit anti-human C4d polyclonal ab was applied to sections and C4d was independently graded (diffuse: 50-100%; focal: 5-50%; rare: 1-5%; negative: 0%) by two pathologists, blinded to each subject's underlying dx. 20 total specimens were identified (19 subjects). Of these, 8 specimens (7 subjects) had histologic evidence of TMA (5 biopsy, 3 autopsy). 1 TMA subject had both a biopsy and autopsy with similar findings, therefore only the autopsy findings are included. The 12 SCT recipient autopsies without TMA served as controls. Diffuse and focal arteriolar C4d staining was far more common in TMA specimens compared to controls (71.4% versus 8.3%, p = 0.01). PTC C4d staining was present in just under half of TMA samples and was absent in controls (p = 0.04). Glomerular C4d staining was similar in both groups and tubular basement membrane staining was rare.TableC4d Deposition in TMAHistology proven TMA (n = 7)Control (n = 12)p-value∗from Fisher’s exact test for categorical variables and Wilcoxon Rank sum for continuous variables; data expressed as n (%) or median (25th-75th percentile) [range].Pathology Results Arteriolar C4d+5/7 (71.4%)1/12 (8.3%)0.01 PTC C4d+3/7 (42.9%)0/12 (0%)0.04 Glomerular capillary C4d+4/7 (57.1%)10/12 (83.3%)0.30 Tubular basement membrane C4d+2/7 (28.6%)0/12 (0%)0.12Clinical Results Age (years)3.6 (1.7-4.5) [0.6-4.9]3.2 (1.4-17.1) [0.9-28.1]0.55 Gender (male)3/7 (42.9%)8/12 (66.7%)0.38 Specimen day post-SCT237 (43 - 631) [19 - 2281]172 (78 - 253) [9 - 887]0.50 Malignancy5/7 (71.4%)2/12 (16.7%)0.03 Immunodeficiency1/7 (14.3%)9/12 (75.0%)0.03 Bone marrow failure1/7 (14.3%)1/12 (8.3%)0.03 Allogeneic2/7 (28.6%)11/12 (91.7%)0.01 Autologous5/7 (71.4%)1/12 (8.3%)0.01 GVHD in those at risk (allogeneic)1/2 (50%)3/11 (27.3%)1.00 Viral infection4/7 (57.1%)4/12 (33.3%)0.38 Dialysis6/7 (85.7%)5/12 (41.7%)0.15∗ from Fisher’s exact test for categorical variables and Wilcoxon Rank sum for continuous variables; data expressed as n (%) or median (25th-75th percentile) [range]. Open table in a new tab This is the first report of arteriolar C4d deposition in SCT-TMA, implicating localized complement fixation due to as yet unidentified abs or ab-independent direct endothelial damage in the pathogenesis of TMA. PTC C4d staining was found only in TMA, while glomerular staining was non-specific as it was similar in both groups. Although the preferential arteriolar C4d staining is incompletely understood, it may identify a renal site of injury, explaining the dramatic hypertension often seen in TMA. Future research should assess if patients with SCT-TMA and renal C4d deposition will benefit from therapies currently used to treat kidney transplant AMR.

Prospective assessment of C4d deposits on circulating cells and renal tissues in lupus nephritis: a pilot study

Complement activation plays a key role in the pathogenesis of lupus nephritis (LN), a severe complication of systemic lupus erythematosus (SLE). We prospectively evaluated 15 LN subjects and two control groups: 13 non-SLE renal subjects (control A) and 239 SLE subjects without LN (control B). All had C4d levels on circulating erythrocytes (E-C4d), reticulocytes (R-C4d) and platelets (P-C4d) measured by flow cytometry, while C4d deposition in renal tissue was semiquantitatively assessed in LN subjects and control A using immunoperoxidase staining. Compared with control A, LN biopsies had higher glomerular-C4d scores (p = 0.003), which were associated with more frequent granular glomerular immunofluorescence staining and electron dense deposits (p < 0.001). Compared with control A and B groups, LN subjects had higher E-C4d (p = 0.002 and p = 0.005) and R-C4d levels (p = 0.002 and p = 0.008), respectively. LN subjects were more likely to have P-C4d compared with control A (p = 0.016). In LN, only E-C4d correlated with National Institutes of Health (NIH) activity index (r = 0.55, p = 0.04). In conclusion, LN biopsies showed frequent glomerular-C4d staining associated with immune complex deposits. LN subjects had higher E-C4d and R-C4d levels compared with both control groups. E-C4d levels also correlated with NIH activity index. These findings suggest a potential role of C4d on circulating cells as a biomarker for lupus nephritis.