Combination of Peritubular C4d and Transplant Glomerulopathy Predicts Late Renal Allograft Failure

Abstract

The histologic associations and clinical implications of peritubular capillary C4d staining from long-term renal allografts are unknown. We identified 99 renal transplant patients who underwent an allograft biopsy for renal dysfunction at least 10 yr after transplantation, 25 of whom were C4d-positive and 74 of whom were C4d-negative. The average time of the index biopsy from transplantation was 14 yr in both groups. Compared with C4d-negative patients, C4d-positive patients were younger at transplantation (29 ± 13 versus 38 ± 12 yr; P < 0.05) and were more likely to have received an allograft from a living donor (65 versus 35%; P < 0.001). C4d-positive patients had more inflammation, were more likely to have transplant glomerulopathy, and had worse graft outcome. The combined presence of C4d positivity, transplant glomerulopathy, and serum creatinine of >2.3 mg/dl at biopsy were very strong predictors of rapid graft loss. C4d alone did not independently predict graft loss. Retrospective staining of historical samples from C4d-positive patients demonstrated C4d deposition in the majority of cases. In summary, these data show that in long-term renal allografts, peritubular capillary staining for C4d occurs in approximately 25% of biopsies, can persist for many years after transplantation, and strongly predicts graft loss when combined with transplant glomerulopathy.

Advances in understanding immunologic mechanisms underlying acute renal allograft rejection have enabled the development of efficient diagnostic tools and therapeutic strategies directed against early immune-mediated graft loss. This led to an increase of 1-yr graft survival rates to >90%; however, long-term graft survival has not improved to a similar degree.^1,2 A steady decline of renal function over years is still the rule in the majority of cases after renal transplantation. Multiple factors can influence graft outcome in the late posttransplantation setting, including acute and or chronic rejection; patient compliance with immunosuppressive therapy; and other medical conditions, such as cardiovascular disease, hypertension, diabetes, infections, drug toxicities, and recurrent disease. Evaluation of renal biopsies may reveal changes related to calcineurin inhibitor toxicity, immune-mediated injury, BK nephropathy, thrombotic microangiopathy, hypertension, diabetes, and recurrent disease, which can help guide appropriate therapy.

There is a growing awareness of the contribution of chronic immune-mediated injury and alloantibodies in late renal allograft dysfunction. Immunohistochemical detection of the complement degradation product C4d in peritubular capillaries (PTCs) of renal allograft biopsies has gained considerable attention because of its diagnostic and prognostic importance in early acute antibody-mediated rejection (AMR). Detection of C4d is regarded as indirect evidence (a “footprint”) of a host's antibody response to a renal allograft and is one of the key criteria used to diagnose AMR.³ C4d deposition has also been implicated in more chronic immune injury of allograft kidneys. Transplant glomerulopathy (TG), a cause of renal dysfunction in longstanding renal allografts, has an estimated prevalence of 1.6 to 12% in renal transplant populations.^4–9 TG is often associated with evidence of AMR, such as the presence of donor-specific antibodies (DSAs) and positive PTC C4d staining, and is considered to be a hallmark of chronic AMR.^4,9–13 The combination of circulating alloantibodies, glomerular and PTC basement membrane multilamination, PTC C4d, and duplication of the glomerular basement membrane has been termed the “ABCD tetrad” of late AMR by Halloran et al.⁴

Many reports have diffused that PTC C4d deposition predicts poor graft survival in both early (<1 yr) and late (>1 yr) posttransplantation periods.^12,14–23 Several reports also have shown C4d staining has little impact on graft survival. Satoskar et al.²⁴ examined 80 cases of late allograft rejection that occurred >1 yr after transplantation. They followed patients for 20 mo and found no difference in outcome between C4d⁺ and C4d⁻ groups. Nickeleit et al.²⁵ analyzed 400 transplant biopsies, at a median of 38 d after transplantation (range 7 to 5646 d) and found no differences between C4d⁺ and C4d⁻ groups in serum creatinine or graft survival at 1 yr of follow-up. A European study including protocol biopsy samples from early and late time points found no reduction in allograft survival in C4d⁺ versus C4d⁻ biopsy samples.¹³ Recently, a study by Haas et al.²⁶ of ABO-incompatible renal allografts showed that diffuse PTC C4d deposition without histologic evidence of AMR or cellular rejection in their initial protocol biopsies was associated with a lower risk for scarring at 1 yr. Several factors may explain the conflicting conclusions in these studies, including limitations as a result of highly selected patient groups,¹² short follow-up,^24–26 and the inclusion of both early and late allograft biopsies.^13,22,23,25

In this study, we identified a unique cohort of renal transplant recipients with allografts surviving ≥10 yr after transplantation with biopsies performed for evaluation of graft dysfunction with or without proteinuria at these later time points. The aim of this study was to assess the prevalence of C4d staining and to clarify the clinical and pathologic significance of C4d positivity in long-term renal allografts.

Results

Renal Biopsy Findings

We identified 99 kidney transplant recipients who underwent allograft renal biopsy for cause ≥10 yr after transplantation. The prevalence of C4d staining was 25% in this population. Two of 25 patents in our C4d⁺ cohort had focal PTC C4d staining (C4d2, 10 to 50%) in their index biopsies; one of them had a working graft, whereas the other lost his graft. No clear conclusion can be drawn on the basis of the small number of cases with focal C4d staining. The remaining 23 patients had diffuse positive staining for C4d (C4d3, >50%) as defined by Banff 2007.²⁷ The C4d⁺ patients compared with C4d⁻ patients had statistically higher scores of total interstitial inflammation (2.04 ± 0.23 versus 1.26 ± 1.26; P = 0.002) and active inflammation (1.28 ± 0.22 versus 0.23 ± 0.07; P < 0.0001), tubulitis (0.72 ± 0.14 versus 0.16 ± 0.05; P < 0.0001), glomerulitis (1.12 ± 0.23 versus 0.15 ± 0.06; P < 0.0001), and PTC congestion with leukocytes (1.28 ± 0.16 versus 0.22 ± 0.06; P < 0.0001; Table 1). Reflective of these findings, C4d positivity was seen more often in the setting of acute cellular rejection (2.92 ± 0.06 versus 0.08 ± 0.03; P < 0.0001). C4d⁺ patients also had more frequent TG (1.64 ± 0.29 versus 0.34 ± 0.10; P < 0.0001). Of note, the overall incidence of TG in this study was 20% and was present in 15 (60%) of 25 C4d⁺ patients but was also present in 10 (14%) of 74 C4d⁻ patients. Other glomerular lesions included FSGS, IgA nephropathy, lupus nephritis, diabetic nephropathy, and one case of membranous nephropathy (Table 2).

Table 1.

Histologic findings of 99 late renal allograft biopsies for cause as evaluated using Banff 1997 and 2007 criteria

Histologic Feature	C4d+(n = 25)	C4d−(n = 74)	P
Total inflammation	2.04 ± 0.23	1.26 ± 0.12	0.002
Active inflammation	1.28 ± 0.22	0.23 ± 0.07	<0.0001
Tubulitis	0.72 ± 0.14	0.16 ± 0.05	<0.0001
Arteritis	0.28 ± 0.12	0.05 ± 0.04	0.03
Glomerulitis	1.12 ± 0.23	0.15 ± 0.06	<0.0001
Peritubular congestion	1.28 ± 0.16	0.22 ± 0.06	<0.0001
Glomerulopathy (cg)	1.64 ± 0.29	0.34 ± 0.10	<0.0001
C4d score	2.92 ± 0.05	0.00 ± 0.00	<0.0001
Mesangial matrix expansion	1.48 ± 0.26	0.74 ± 0.13	0.01
Glomerulosclerosis (%)	31.44 ± 4.18	35.55 ± 2.70	0.4
Interstitial fibrosis (ci)	1.64 ± 0.21	1.54 ± 0.12	0.7
Tubular atrophy (ct)	1.60 ± 0.19	1.54 ± 0.11	0.8
Fibrous intimal thickening (cv)	1.48 ± 0.18	1.19 ± 0.09	0.1
Arteriolar hyalinosis	0.80 ± 0.08	0.82 ± 0.04	0.8
Acute tubular injury	0.60 ± 0.10	0.18 ± 0.04	<0.0001
Acute cellular rejection	2.92 ± 0.06	0.08 ± 0.03	<0.0001
Chronic score (cg + ci + ct + cv)	6.36 ± 0.73	4.59 ± 0.32	0.01
Chronic score (ci + ct + cv)	4.72 ± 0.51	4.26 ± 0.28	0.4

Data are means ± SEM.

Table 2.

Other histologic features as noted in 36 biopsy samples on analysis of 99 late renal allograft biopsy samples for cause

Glomerular Lesion	C4d+ (n = 11)	C4d− (n = 25)
FSGS	6 (54%)	12 (48%)
FSGS collapsing	2 (18%)	2 (8%)
DN	0 (0%)	7 (28%; 4 had primary diagnosis of DN)
IgA nephropathy	1 (9%)	3 (12%)
Lupus nephritis	1 (9%)	1 (4%)
Membranous nephropathy	1 (9%)	0 (0%)

DN, diabetic nephropathy.

The degree of chronic tubular, interstitial, and vascular sclerosing injury and arteriolar hyalinosis were similar between the groups (Table 1). The mean chronic sum score (comprising the sum of Banff indices for TG, interstitial fibrosis, tubular atrophy, and arterial intimal fibrosis) was significantly higher in the C4d⁺ group (6.36 ± 0.73 versus 4.59 ± 0.32; P = 0.01) and was solely dependent on the presence of TG (Table 1). Removing TG from the chronicity score eliminated any statistical difference in the chronic injury score (4.72 ± 0.51 versus 4.26 ± 0.28; P = 0.4).

Historically Stained Samples

Review of the index patient's records revealed that 16 of 25 C4d⁺ patients and 31 of 74 C4d⁻ patients had previous biopsies preformed at our institution, which were available for retrospective review. All of these biopsies were performed for cause, both the number and the timing of the biopsies varied between the patients. Sixteen C4d⁺ patients had a total of 45 previous biopsies (range 1 to 9 per patient), performed as early as 10 d and up to 16 yr after transplantation. Tissue was available from 37 of these 45 (total number of previous biopsies) for retrospective staining for C4d using immunoperoxidase technique. Retrospectively stained biopsy specimens from 14 of 16 available C4d⁺ patients were positive for C4d. The intensity of staining varied from 25% (focal) to >50% (diffuse), with at least one biopsy per patient with diffuse staining. The longest interval between the first C4d⁺ biopsy and the index biopsy was 16 yr. The histologic findings in the previous biopsies ranged from no significant pathologic abnormalities to a variety of findings, including acute tubular injury, acute cellular rejection ranging from mild to severe, and vascular rejection. Previous biopsies were negative for C4d in two of 16 available patients, one of whom had single biopsy that consisted predominantly of medulla, and the other had weak staining (25%) in the first biopsy done at 4 wk and two subsequent negative biopsies, all without evidence of significant pathologic abnormalities. TG was noted in seven of 16 these patients. On the basis of review of available previous biopsies, the time interval between C4d⁺ and the diagnosis of TG was 10 yr on average (range 7 to 14 yr; median 10 yr).

For the 74 C4d⁻ patients, 31 (52 biopsies) had tissue available for restaining with C4d. These biopsies were performed at 8 d to 11 yr after transplantation. Whereas the majority of these previous biopsies were negative for C4d, seven patients had previous biopsies that showed positive staining for C4d. These seven patients had two to nine biopsies per patient with the earliest available positive biopsy performed at 8 d after transplantation. The intensity of the C4d staining ranged from very strong in the earliest biopsy (as early as 8 d after transplantation) to focal or negative at later time points (focal staining detected up to 10 yr after transplantation) in available samples. These patients received transplants on average 11.1 ± 0.6 yr previously, six patients were male, six received simultaneous kidney-pancreas transplants, and none received living-donor transplants. None of the earliest biopsies from these patients showed acute rejection; therefore, none of them were specifically treated at that time. None of these seven patients developed TG in the subsequent biopsies. Of the seven patients, five survived and two died with functioning grafts. The survivors had the last available creatinine of 2.28 ± 0.32 g/dl and proteinuria of <100 mg/dl, with average follow-up of 46.1 ± 0.6 mo after the last biopsy.

As mentioned already, 10 of 74 C4d⁻ patients had TG. Only three of these 10 patients had previous biopsy available for review. Two of them had a relatively recent biopsy, already showing TG with no C4d staining. The third biopsy, however, done 3 mo after transplantation (15 yr before the index biopsy showed mild acute cellular rejection and 25% of PTCs positive for C4d (by immunoperoxidase).

Clinical Associations

Complete clinical data were available for 85 of the 99 patients: 23 of 25 C4d⁺ and 62 of 74 C4d⁻ patients. The demographic and clinical details of the patients studied are outlined in Tables 3 and 4. Patients who were C4d⁺ had their renal transplants performed at a significantly younger age (29 ± 13 versus 37 ± 12 yr) and more often were the recipients of living-donor transplants (15 [65%] of 23 versus 14 [23%] of 62, C4d⁺ versus C4d⁻, respectively). Living-donor transplants all were living related, and none was from an HLA-identical sibling. All of these renal biopsies were performed for cause (raised creatinine and/or proteinuria) on average 14 yr after transplantation. Both groups had similar nadir creatinine after transplantation (1.5 ± 0.4 versus 1.3 ± 0.3 mg/dl) and had no difference in creatinine at biopsy (2.6 ± 0.7 versus 2.4 ± 1.7 mg/dl; Table 4).

Table 3.

Patient characteristics: Demographic features of patients with full clinical follow-up available

Feature	C4d+(23 Patients)	C4d−(62 Patients)	P
Male gender (n [%])	15 (65)	32 (53)	NS
Age at transplantation (yr; mean ± SD)	29.4 ± 13.0	37.5 ± 12.0	0.01
Race (n [%])
white	21 (91)	57 (91)	NS
black	1 (0)	1 (0)	NS
other	1 (0)	4 (0)	NS
Transplant type (n [%])
living donor	15 (65)	14 (23)	0.0002
deceased donor	8 (35)	48 (77)	0.0002
kidney transplant alone	17 (74)	47 (76)	NS
previous transplantation	4 (17)	4 (0)	NS
Type of immunosuppression
cyclosporine	16	48	NS
tacrolimus	5	9	NS
azathioprine	12	33	NS
mycophenolate	4	18	NS
prednisone	22	55	NS
Cause of ESRD
diabetes	9	22	NS
glomerulonephritis	11	19	NS
reflux	2	4	NS
hereditary/congenital	1	3	NS
other		14	NS

Table 4.

Clinical features of patients and graft outcome: Clinical features of patients with clinical follow-up available

Feature	C4d+(23 Patients)	C4d−(62 Patients)	P
Biopsy details
biopsy time (yr after transplantation; mean ± SD)	14.2 ± 5.4	13.2 ± 5.5	0.4
creatinine nadir after transplantation	1.5 ± 0.4	1.3 ± 0.3	NS
creatinine at biopsy	2.6 ± 0.7	2.4 ± 1.5	0.5
Immunologic risk
PRA before transplantation (%)	3 ± 5	15 ± 28	NS
HLA mismatch	3.0 ± 1.4	2.7 ± 2.0	NS
Graft outcome
follow-up since biopsy (mo)	15 ± 13	32 ± 16	NS
total graft loss (n [%])	11 (48)	15 (24)	0.0001
death-censored graft loss (n [%])	10 (45)	9 (16)	0.0002
time to graft loss from biopsy (mo; mean ± SD)	10.2 ± 9.6	26.6 ± 14.7	0.01
follow-up since biopsy graft survived (mo; mean ± SD)	24.0 ± 14.0	29.6 ± 16.5	NS
creatinine in survivors (mg/dl; mean ± SD)	1.8 ± 0.6	1.8 ± 0.8	NS

Details of graft outcome in both groups are shown. PRA, panel-reactive antibody.

In an attempt to define the role of DSAs in these patients, we tested samples that were available for 18 of the 25 C4d⁺ patients. Antibodies were found in 15 (83%) of 18 patients tested: Anti-HLA class I in two patients, anti-HLA class II in seven patients, and both classes I and II in six patients. There was no predominance of any specific anti-HLA antibodies. There seemed to be correlation between the development of TG and anti-HLA class I. There was no correlation between DSA class and graft outcome. DSAs were not checked at the time of biopsy in patients who were C4d⁻; however, stored sera were available for seven of the C4d⁻ cases. DSA testing was negative in all seven cases.

Graft Outcome

A striking finding in this study was the observation that C4d alone was not an independent predictor of graft outcome (Figures 1 and 2; Table 5). Figure 1 shows Kaplan Meier survival curves for death-censored graft outcome in patients, showing graft loss in 45% (10 of 22) of C4d⁺ patients compared with 16% (nine of 56) of C4d⁻ patients (P = 0.0002; Figure 1A), TG graft loss in 68% (13 of 19) of patients with TG compared with 12% (seven of 59) patients without TG (P < 0.0001), and the powerful interaction between TG and C4d⁺ (Figure 1C). TG was seen in all 10 cases with C4d⁺ graft loss. Patients who died with a functioning graft were censored at the time of their death for this analysis (one patient in C4d⁺ group, six patients in the C4d⁻ group). Patients with C4d⁺ staining and features of rejection were treated for AMR with combinations of antibody-depleting therapy (nine patients) or changes to conventional immunosuppression (14 patients). Treatment had no impact on outcome in patients with TG.

Figure 1.

Kaplan Meier curves showing graft outcome after biopsy. (A) C4d and graft failure. (B) TG and graft failure. (C) C4d interacts with glomerulopathy to be the strongest predictor of graft outcome.

Figure 2.

Interactions with C4d predict graft failure and Kaplan Meier survival curve in predictive model. Cox proportional hazard model was used to identify significant independent variables between groups. A scoring system was developed by approximating ratios of the RR values of the significant variables from the Cox proportional hazard model. Serum creatinine >2.3 mg/dl was assigned 1 point, TG 1 point, and TG and C4d positivity 2 points, reflective of RR values 5.3, 3.7, and 9.3, respectively.

Table 5.

Clinical and pathologic factors associated with graft loss

Factors	Univariable Analysis		Interactions of C4d+ve with Other Pathological Features		Multivariable Analysis
	RR Graft Loss	P	RR Graft Loss	P	RR Graft Loss (MV)	P
C4d+ve	4.40	0.001			n/a	Ns
Age	0.96	0.06			n/a	Ns
Creatinine at biopsy >2.3 mg/dl	6.60	<0.0001			5.30	0.01
Total inflammation	3.30	0.03	None	0.4		Ns
Active inflammation	3.90	0.002	None	0.4		Ns
Arteritis	2.00	0.1	None	0.7		Ns
Glomerulitis	4.30	0.01	None	0.7		Ns
Peritubular congestion	4.40	0.001	None	0.2		Ns
Glomerulopathy	7.70	<0.0001	n/a	n/a	3.70	0.01
Glomerulopathy with C4d+ve	n/a	n/a	9.30	<0.0001	9.30	0.0001
Mesangial matrix expansion	5.90	<0.0001	8.50	<0.0001	n/a	Ns
Global glomerulosclerosis	1.01	0.2	None	0.2		Ns
Interstitial fibrosis	1.40	0.6	None	0.5		Ns
Tubular atrophy	3.10	0.02	None	1.0		Ns
Fibrous intimal thickening	1.70	0.5	None	0.5		Ns
Arteriolar hyalinosis	2.80	0.4	None	0.4		Ns
Acute tubular injury	0.80	0.02	None	0.2		Ns
Acute cellular rejection	3.10	0.008	None	1.0		Ns

Clinical and pathologic factors associated with graft loss in late renal allograft biopsies as determined using univariable and mulitvariable Cox proportion hazard models. Independent predictors of graft outcome were assessed in multivariable analysis, showing C4d alone is not an independent predictor of graft outcome but requires TG to affect outcome. As seen, C4d had no effect on grafts with interstitial fibrosis, inflammation, or arterial fibrosis (P = 0.5, 0.4, and 0.4, respectively). n/a, not available; Ns, not significant.

Cox proportional hazard model was used to identify significant independent variables between groups and demonstrated a strong interaction, showing the combination of C4d⁺ staining with TG and associated mesangial matrix expansion were very strong predictors of graft loss (relative risk [RR] 9.3 and 8.5, respectively; P < 0.0001). C4d⁺ staining had no effect on grafts with interstitial fibrosis, inflammation, or arterial fibrosis (P = 0.5, 0.4, and 0.4, respectively; Table 5). The three most powerful independent predictors of graft loss were creatinine at biopsy >2.3 mg/dl (RR 6.6; P < 0.0001), TG (RR 7.7; P < 0.0001), and the combination of C4d with TG as mentioned. We developed a scoring system by approximating ratios of the RR values of the significant variables. We assigned 1 point to serum creatinine >2.3 mg/dl, 1 point to TG, and 2 points for TG and C4d⁺. A receiver operating characteristic (ROC) curve was developed for the presence of graft failure by logistic regression with a ROC score of 0.83. This analysis showed that in patients who underwent an allograft renal biopsy for cause >13 yr after transplantation, a score of 3 (1 point TG, 1 point creatinine >2.3 g/dl, and 2 points combined presence of TG and C4d⁺) suggests a >80% chance for graft loss at 1 yr.

Discussion

The findings in this study help to define and clarify the clinical and pathologic significance of C4d staining in late renal allograft biopsy samples. We found that in this unique cohort of renal allograft recipients ≥10 yr after transplantation, identified on review of our pathology database, 25% were C4d⁺, which is similar to the prevalence of C4d positivity in allograft biopsies performed at earlier times.^21,28 Diffuse positive C4d staining (C4d3 >50%) as defined by Banff 2007²⁷ was seen in 23 patients; two patients had focal PTC C4d staining (C4d2 10 to 50%) in their index biopsies. Evaluation of historical samples, retrospectively stained for C4d, revealed that the great majority of pervious biopsies from C4d⁺ patients were also positive. In some patients, the time between the first and the last available positive biopsy was as long as 16 yr. Because the historical biopsies were performed before C4d staining was introduced as standard evaluation of renal transplants, none of these patients was specifically treated for AMR. Thus, in some patients, early and persistent PTC C4d staining is not associated with adverse graft outcome.

To define possible pathogenic prognostic factors in this setting, we examined the histologic associations in our late biopsy samples using the Banff criteria,^27,29 Positive C4d staining in this cohort of patient biopsies was significantly associated with inflammation and cellular rejection (Table 1). C4d positivity also correlated well with circulating DSAs (80% of tested patients), a finding considered to be one of the most important diagnostic criteria for AMR.^27,29 Thus, similar to findings from transplant patients who underwent biopsies in earlier periods after transplantation, C4d positivity was often associated with an active rejection process^27,29,30; however, when C4d positivity is associated with TG, the risk for graft failure is high.

Of particular interest in this study was this association of C4d deposition with TG. A recent study of nonhuman primates demonstrated that chronic AMR progresses through four defined clinicopathologic stages: Development of DSA, deposition of C4d, TG, and finally rising creatinine/renal allograft failure.³¹ As described already, overall, 25 (20%) patients in our study had TG, 15 (60%) of 25 of whom were C4d⁺ and 10 (14%) of 74 of whom were C4d⁻. The findings of our study are consistent with previous studies that also found a strong association between TG and C4d positivity.^4,10,12 We attempted to time the development of TG as we calculated the time interval between C4d⁺ and the diagnosis of TG (10 yr on average; range 7 to 14 yr; median 10 yr); however, on the basis of these data, we cannot determine the timing of development of TG, because the biopsies are too widely separated in time. We also found patients with TG and no concurrent or previous evidence of C4d deposition. Similar observations were made in other studies.^32–34 Together, our results and the findings of others suggest that many but not all TG are antibody mediated, and additional processes, such as T cell–medicated events or drug toxicities, may also contribute to the heterogeneous cause of TG.³⁴

Having defined the prevalence of C4d positivity and its histologic associations, we next determined the clinical relevance of positive C4d staining. Positive staining was significantly more often in younger patients who received living-donor transplants. This finding may reflect either the inherent survival advantage of living-donor renal allografts^35,36 or early graft loss of C4d⁺ deceased-donor grafts. Graft loss was higher in the C4d⁺ group (45%) compared with the C4d⁻ group (16%), consistent with previous reports of poor outcome in the presence of C4d staining,^12,20–23 and our study includes larger numbers and extends these results to later time points than previously examined; however, in our study, the median follow-up time was relatively short, with a median of 28 mo after index biopsy (Table 4).

Because 55% of C4d⁺ grafts did not suffer graft loss despite persistent positive staining, we examined which additional or interacting factors predict allograft outcome. To our surprise, C4d positivity alone did not predict graft outcome, but C4d positivity interacted strongly with TG to predict graft loss. The interaction of TG with C4d⁺ staining was a strong predictor of graft loss with RR 9.3. Indeed, the most powerful predictors of rapid graft loss were the combination of TG, TG and C4d positivity together, and creatinine >2.3 mg/dl at biopsy (RR 3.7, 9.3, and 5.3, respectively). The “CTC” scoring system we described (creatinine >2.3 mg/dl 1 point, TG 1 point, and combined C4d⁺ and TG 2 points), whereby a score of ≥3 predicts rapid graft loss in 80%, may be useful for clinicopathologic correlation and treatment of patients with long-term renal allografts. As seen in Figures 1 and 2, C4d⁺ staining alone in the absence of TG has favorable graft outcome compared with the combination of C4d positivity, TG, and raised creatinine. This finding may explain in part previous conflicting reports of the clinical relevance of C4d staining in later renal allograft biopsies.^12,20–24

A question that arises from our observations is why only some patients with positive C4d staining develop TG and other patients tolerate persistent C4d deposition for years without developing TG or graft failure. We found no association between the presence of anti–class I or II HLA antibodies and TG. Similarly, there was no difference in maintenance immunosuppressive medication or in the use of any other medications (Table 3). The higher prevalence of living-donor transplants in the C4d⁺ group may suggest that C4d deposition in living-donor transplants is more likely to be benign. This observation, if confirmed, may provide insight into the additional factors that influence whether C4d deposition progresses to TG and graft failure. Stable “accommodation” may explain some of our findings.^37,38 This term was coined from the observation that some transplants across an ABO barrier are tolerated in the presence of recipient antibodies directed against the graft. Studies of ABO-incompatible grafts have revealed PTC C4d deposition in 25 to 80% of protocol biopsies, with evidence of AMR in only 4 to 12%.^26,39,40 In addition, ABO-incompatible renal allografts with C4d positivity but without histologic evidence of AMR or cellular rejection have a lower risk for scarring at 1 yr.²⁶ Thus, diffuse PTC C4d staining is an indicator of alloantibodies, but it does not always portend AMR. Additional factors may determine whether the deposited alloantibodies and complement evoke an inflammatory response and cause injury. Elucidation of the precise factors that determine the fate of alloantibody and C4d deposition will be of great use in the clinical treatment of allograft recipients.

Our study also uncovered a group of seven patients who were historically positive on first renal transplant biopsy and, without specific treatment, cleared C4d staining on subsequent biopsies. None of these patients developed TG, and because of the retrospective nature of this study, we were not able to determine whether these patients had DSAs at the time of their C4d⁺ biopsies. This observation provides further insight into the natural history of C4d positivity and indicates that some individuals may show transient positivity after transplantation without any adverse consequences.

This study expands our understanding of the clinicopathologic associations of PTC C4d in the late renal allograft biopsy. Our findings demonstrate that in renal allografts, on average 14 yr after transplantation, biopsied for a cause, C4d deposition may be found in as many as 25% of cases. PTC C4d is strongly associated with TG and is more commonly seen in patients who received a living-donor transplant at a younger age. In some patients, C4d positivity may persist for several years without the development of TG and adverse graft outcome. We conclude that C4d alone is not an independent predictor of graft outcome; however, the combination of C4d positivity with histologic and clinical evidence of renal allograft dysfunction (TG and serum creatinine >2.3 mg/dl at biopsy) is a very powerful predictor of rapid graft loss. In our study, 40% of C4d⁺ patients did not develop TG and were protected from graft loss. The biopsy and clinical findings from these patients indicate that, in addition to alloantibodies and C4d deposition, other factors are necessary to cause clinically relevant graft dysfunction.

Concise Methods

Kidney Biopsy Selection

With the approval from the institutional review board of the University of Washington, we searched the database of the Department of Pathology for kidney allograft biopsies performed at ≥10 yr after transplantation, which were received during the study period from April 1, 2003 (date of the introduction of C4d staining by immunofluorescence [IF] technique), until October 30, 2007. According to local practice, these biopsies were not performed on a prospective protocol basis but were based on the clinical parameters (e.g., deterioration of kidney function and/or proteinuria). We identified 99 kidney transplant recipients and divided them according the presence or absence of C4d staining in PTCs. No eligible biopsy specimens were excluded from the study. All biopsies were evaluated by one pathologist (J.K.), who was blinded to the clinical and IF data. Evaluation of the biopsies was based on the Banff '07 acute and chronic indices,^27,29 including total interstitial inflammation, active interstitial inflammation, presence of glomerulitis, TG, mesangial matrix expansion (based on silver stain), degree of PTC congestion, extent of interstitial fibrosis (based on trichrome stain) and tubular atrophy, vascular pathology, and deposition of C4d. Biopsies had at least seven glomeruli per section and at least one artery, fulfilling the Banff adequacy criteria.²⁹

Glomeruli were also evaluated for the percentage of global glomerulosclerosis and assessed for additional lesions, including focal segmental sclerosis, and other recurrent or de novo glomerular diseases. The presence of acute tubular injury was also recorded. We retrospectively analyzed all of the previous transplant biopsies in our database for the study patients.

These biopsies were reviewed for the presence or absence of C4d deposition and allograft pathology. Because the majority of the previous biopsies were preformed before the introduction of C4d staining in our institution (April 2003), all of the available previous biopsies were retrospectively stained for C4d using the immunoperoxidase technique on formalin-fixed, paraffin-embedded biopsy tissue. Banff 2007 guidelines were followed to score these samples.²⁷

Tissue Processing

All renal biopsies were processed by standard techniques for light and IF microscopy. For light microscopy evaluation, 2-μm histologic sections prepared from formalin-fixed and paraffin-embedded tissue were stained with hematoxylin and eosin, periodic acid-Schiff reagent, and Jones methenamine silver stain. For IF studies, 3-μm cryostat sections were stained with FITC-conjugated anti-human IgG, IgM, IgA, C3, C1q, κ and λ light chain, fibrinogen, and albumin (Dako, Carpinteria, CA), as described previously.⁴¹ At our institution, all of the transplant biopsies are evaluated for deposition of C4d using IF (cat. no. A213; Quidel, San Diego, CA; dilution 1:100) and, when fresh-frozen tissue is not available, on formalin-fixed, paraffin-embedded tissue by immunoperoxidase technique (cat. no. 01-B1-RC4D; ARP, Belmont, MA; dilution 1:50, followed by secondary anti-rabbit antibodies, and Envision, Dako enhancing system).

Patient Clinical Characteristics

Patient demographics and clinical information including age, gender, race, transplant source and date, cause of ESRD, panel-reactive antibody titers, degree of matching of transplant, serum creatinine, proteinuria, immunosuppressive therapy, and current graft function were obtained from hospital charts and laboratory record. The study and data collection were reviewed and approved by the institutional review board of the University of Washington. Most patients received transplants in the early 1990s. All patients received ABO-compatible grafts. Graft failure was defined as return to dialysis.

Detection of Anti-HLA Antibodies

Donor-specific HLA alloantibodies were detected with FlowPRA Class I and II single-antigen beads (SABs; One Lambda, Canoga Park, CA). Briefly, 3 μl of relevant beads (beads coated with HLA representing mismatched donor antigens) were incubated with 20 μl of serum, incubated, washed, and then stained with FITC-conjugated goat anti-human IgG. After a final wash, the SABs were analyzed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Results were deemed positive when the SABs incubated with recipient serum were visibly and quantitatively right-shifted, when compared with negative serum incubated with SABs.⁴²

Statistical Analysis

Continuous variables are given as the means ± SEM unless otherwise stated, and categorical variables are presented as proportions. A nonparametric test (Mann-Whitney test) as appropriate was used for comparing continuous variables, and the Fisher exact test was used for comparing categorical variables. The survival curves were calculated by Kaplan-Meier survival analyses and compared by the log-rank test. Cox proportional hazard model was used to identify significant independent variables between groups. A scoring system was developed by approximating ratios of the RR values of the significant variables from the Cox proportional hazard model. From the resulting scores for each patient, an ROC curve was developed for the presence of graft failure by logistic regression. The statistical software package JMP 7.0.2 (SAS Institute, Cary, NC) was used. P < 0.05 was considered significant.

Disclosures

None.