Glomerular C4d Immunoperoxidase in Chronic Antibody-Mediated Rejection and Transplant Glomerulopathy.

Introduction: The diagnosis of late antibody-mediated rejection (AMR) is compromised by frequent absence of C4d in peritubular capillaries (C4dptc), termed "C4d-negative" AMR. We hypothesized that glomerular capillary C4d (C4dglom) reflected endothelial interaction with antibody and could improve immunologic classification of transplant glomerulopathy (TG). Methods: We evaluated C4d using immunoperoxidase in 3524 consecutive, kidney transplant biopsies from a single center. Results: C4dglom was detected in 16.5% and C4dptc in 9.9% of biopsies. C4dglom occurred in 60.3% of TG (n = 174) and was absent in normal glomeruli. Epidemiologic risk factors for C4dglom were younger, female, living-donor recipients with early AMR, prior treated rejection, and late presentation using multivariable analysis. Semiquantitative C4dglom score correlated with donor specific antibody (DSA) level, C4dptc, microvascular inflammation (MVI), Banff cg scores, renal dysfunction, and proteinuria. Principal component analysis colocalized C4dglom with histologic AMR. Multivariable analysis of TG found DSA, C4dptc, and post-transplant time associated with C4dglom. Addition of C4dglom into Banff chronic AMR schema improved its diagnostic sensitivity for TG (verified by electron microscopy [EM]) from 22.2% to 82.4% and accuracy from 59.6% to 93.9%, compared with Banff 2019 using only C4dptc. Tissue C4dglom and chronic AMR diagnosis incorporating C4dglom were associated with death-censored allograft failure in TG (P < 0.001), independent of the severity of glomerulopathy and chronic interstitial fibrosis. Conclusion: C4dglom is a promising diagnostic biomarker of endothelial interaction with antibody which substantially improved test performance of the Banff schema to correctly classify TG by pathophysiology and prognosticate graft loss. We recommend routine C4d immunoperoxidase to minimize underdiagnosis of late AMR in TG.

The Clinical and Pathologic Phenotype of Antibody-Mediated Vascular Rejection Diagnosed Using Arterial C4d Immunoperoxidase.

Introduction: The diagnosis of antibody-mediated vascular rejection (AM-VR) should be reliable and accurate. We hypothesized that arterial C4d (C4dart) immunoperoxidase deposition represents endothelial interaction with antibody. Methods: From 3309 consecutive, kidney transplant biopsies from a single center, 100 vascular rejection (VR) cases were compared against rejection without arteritis (n = 540) and normal controls (n = 1108). The clinical utility of C4dart for diagnosis and classification of AM-VR was evaluated against an independent reference test. Results: C4dart occurred in 20.4% of acute, 11.0% of subclinical, and 46% of VR episodes. Semiquantitative C4dart score significantly correlated with immunodominant donor-specific antibodies (DSAs) (rho = 0.500, P < 0.001), peritubular capillary C4d (C4dptc), microvascular inflammation, and Banff v scores. Banff v3 arteritis suggested AM-VR. Addition of C4dart to Banff antibody-mediated rejection (AMR) schema increased diagnostic sensitivity for AM-VR from 57.9% to 93.0%, accuracy 74.0% to 92.0%, and specificity 95.4% to 90.2% versus Banff 2019 (using C4dptc). Death-censored graft failure was associated with C4dart AM-VR criteria using Cox regression (adjusted hazard ratio [HR] 4.310, 95% CI 1.322-14.052, P = 0.015). VR was then etiologically classified into AM-VR (n = 57, including 36 mixed VR) or "pure" (TCM-VR, n = 43). AM-VR occurred within all post-transplant periods, characterized by greater total, interstitial, and microvascular inflammation, arterial and peritubular C4d, DSA levels, and graft failure rates compared with TCM-VR. Mixed VR kidneys had the greatest inflammatory burden and graft loss (P < 0.001). Conclusion: C4dart is a suggestive biomarker of the humoral alloresponse toward muscular arteries. Inclusion of C4dart into the Banff schema improved its diagnostic performance for detection of AM-VR and etiologic classification of arteritis.

The Pathological and Clinical Diversity of Acute Vascular Rejection in Kidney Transplantation.

BACKGROUND: Vascular rejection (VR) is characterized by arteritis, steroid resistance, and increased graft loss but is poorly described using modern diagnostics. METHODS: We screened 3715 consecutive biopsies and retrospectively evaluated clinical and histological phenotypes of VR (n = 100) against rejection without arteritis (v0REJ, n = 540) and normal controls (n = 1108). RESULTS: Biopsy sample size affected the likelihood of arterial sampling, VR diagnosis, and final Banff v scores ( P < 0.001). Local v and cv scores were greatest in larger arteries (n = 258). VR comprised 15.6% of all rejection episodes, presented earlier (median 1.0 mo, interquartile range, 0.4-8 mo) with higher serum creatinine levels and inferior graft survival, versus v0REJ ( P < 0.001). Early VR (≤1 mo) was common (54%) and predicted by sensitization, delayed function, and prior corticosteroid use, with associated acute dysfunction and optimal therapeutic response, independent of Banff v score. Late VR followed under-immunosuppression in 71.4% (noncompliance 38.8%, iatrogenic 32.6%), and was associated with chronic interstitial fibrosis, incomplete renal functional recovery and persistent inflammation using sequential histopathology. The etiology was "pure" antibody-mediated VR (n = 21), mixed VR (n = 36), and "pure" T cell-mediated VR (n = 43). Isolated VR (n = 34, Banff i < 1 without tubulitis) comprised 24 T cell-mediated VR and 10 antibody-mediated VR, presenting with mild renal dysfunction, minimal Banff acute scores, and better graft survival compared with inflamed VR. Interstitial inflammation influenced acute renal dysfunction and early treatment response, whereas chronic tubulointerstitial damage determined long-term graft loss. CONCLUSIONS: VR is a heterogenous entity influenced by time-of-onset, pathophysiology, accompanying interstitial inflammation and fibrosis. Adequate histological sampling is essential for its accurate diagnostic classification and treatment.

Renal tubular cell binding of ß-catenin to TCF1 versus FoxO1 is associated with chronic interstitial fibrosis in transplanted kidneys.

ß-Catenin is an important co-factor which binds multiple transcriptional molecules and mediates fibrogenic signaling pathways. Its role in kidney transplantation is unknown. We quantified binding of ß-catenin within renal tubular epithelial cells to transcription factors, TCF1 and FoxO1, using a proximity ligation assay in 240 transplanted kidneys, and evaluated their pathological and clinical outcomes. ß-Catenin/FoxO1 binding in 1-month protocol biopsies inversely correlated with contemporaneous chronic fibrosis, subsequent inflammation. and inflammatory fibrosis (P < .001). The relative binding of ß-catenin/TCF1 versus ß-catenin/FoxO1 (TF ratio) was the optimal biomarker, and abnormal in diverse fibrotic transplant diseases. A high 1-month TF ratio was followed by greater tubular atrophy and interstitial fibrosis scores, cortical inflammation, renal impairment, and proteinuria at 1 year (n = 131, all P < .001). The TF ratio was associated with reduced eGFR (AUC 0.817), mild fibrosis (AUC 0.717), and moderate fibrosis (AUC 0.769) using receiver operating characteristic analysis. An independent validation cohort (n = 76) confirmed 1-month TF was associated with 12-month moderate fibrosis (15.8% vs. 2.6%, P = .047), however, not with other outcomes or 10-year graft survival, which limits generalizabilty of these findings. In summary, differential binding of ß-catenin to TCF1 rather than FoxO1 in renal tubular cells was associated with the fibrogenic response in transplanted kidneys.

Pharmacologic targeting of renal ischemia-reperfusion injury using a normothermic machine perfusion platform.

Normothermic machine perfusion (NMP) is an emerging modality for kidney preservation prior to transplantation. NMP may allow directed pharmacomodulation of renal ischemia-reperfusion injury (IRI) without the need for systemic donor/recipient therapies. Three proven anti-IRI agents not in widespread clinical use, CD47-blocking antibody (αCD47Ab), soluble complement receptor 1 (sCR1), and recombinant thrombomodulin (rTM), were compared in a murine model of kidney IRI. The most effective agent was then utilized in a custom NMP circuit for the treatment of isolated porcine kidneys, ascertaining the impact of the drug on perfusion and IRI-related parameters. αCD47Ab conferred the greatest protection against IRI in mice after 24 hours. αCD47Ab was therefore chosen as the candidate agent for addition to the NMP circuit. CD47 receptor binding was demonstrated by immunofluorescence. Renal perfusion/flow improved with CD47 blockade, with a corresponding reduction in oxidative stress and histologic damage compared to untreated NMP kidneys. Tubular and glomerular functional parameters were not significantly impacted by αCD47Ab treatment during NMP. In a murine renal IRI model, αCD47Ab was confirmed as a superior anti-IRI agent compared to therapies targeting other pathways. NMP enabled effective, direct delivery of this drug to porcine kidneys, although further efficacy needs to be proven in the transplantation setting.

The Pathophysiology and Impact of Inflammation in Nonscarred Renal Interstitium: The Banff i Lesion.

BACKGROUND: Interstitial inflammation (i-INT) is the driver of T-cell-mediated rejection. Its causes, pathophysiology, kinetics, and outcomes are poorly documented. METHODS: The role of i-INT was evaluated in 2055 biopsies from 775 renal transplant recipients. RESULTS: i-INT was present in 374 (18.2% prevalence) from acute and subclinical rejection (67.4%); interstitial fibrosis and tubular atrophy (14.4%); BK virus nephropathy (BKVAN) 9.9%; and acute tubular necrosis (ATN with i-INT) in 5.9% of cases. i-INT was predicted by prior T-cell-mediated rejection and BKVAN, human leukocyte antigen mismatch, cyclosporine therapy, and indication biopsy for dysfunction. It correlated with tubulitis, arteritis, and antibody markers within concurrent histology (P < 0.001). After treatment, renal functional recovery was best with histological ATN, milder i-INT, and early posttransplant biopsy times. The initial histological improvement of inflammation depended on baseline i-INT severity. Complete resolution to Banff i0 was predicted by early biopsy time, antilymphocyte therapy, recipient age, and medication compliance (all P < 0.001). Clearance i-INT was followed by delayed resolution of tubulitis (P < 0.001). i-INT was associated with histological ATN, renal dysfunction, and increased incident fibrosis on sequential pathology. Progressive fibrosis following related-rejection i-INT was dependent on tubulitis using multivariable analysis. In contrast, fibrogenesis after BKVAN or ATN was unrelated to inflammation. i-INT cases were followed by recurrent rejection in 35.3%, increased graft loss, and greater patient mortality. Multiple complementary outcome analyses determined the optimal lower diagnostic threshold for inflammation was Banff i1 score. CONCLUSIONS: i-INT is a heterogeneous pathological phenotype that results in adverse functional and structural outcomes, for which active and robust therapy should be considered.

Brief Normothermic Machine Perfusion Rejuvenates Discarded Human Kidneys.

Normothermic machine perfusion (NMP) may allow resuscitation and improved assessment of kidneys before transplantation. Using discarded human kidneys, we investigated the mechanistic basis and translational potential of NMP compared with cold static storage (CS). METHODS: Discarded deceased donor kidneys (n = 15) underwent 1-hour NMP following CS. Renal perfusion, biochemical, and histologic parameters were recorded. NMP was directly compared with CS in paired donor kidneys using simulated transplantation with allogeneic whole blood, followed by assessment of the aforementioned parameters, in addition to RNA sequencing. RESULTS: Kidneys were successfully perfused, with improved renal blood flows and resistance over the course of perfusion, and evidence of urine output (median 21 mL), in all but one kidney. NMP completely resolved nonperfused regions in discarded donation after circulatory death kidneys. In paired kidneys (n = 4 pairs), transcriptomic analyses showed induction of stress and inflammatory pathways in NMP kidneys, with upregulation of pathways promoting cell survival and proliferation. Furthermore, the NMP pairs had significantly better renal perfusion (1.5-2 fold improvement) and functional parameters, and amelioration of cell death, oxidative stress, and complement activation. CONCLUSIONS: In this pilot preclinical study using simulated transplantation of paired kidneys, NMP of discarded marginal kidneys demonstrated some significant mechanistic benefits in comparison to CS alone. NMP may have potential to reduce organ discards and enhance early graft function in such kidneys.

Does tubulitis without interstitial inflammation represent borderline acute T cell mediated rejection?

Tubulitis without interstitial inflammation (Banff i0), termed "isolated tubulitis" (ISO-T), has been controversially included within the Banff "borderline" category of acute T cell mediated rejection (TCMR). This single-center, retrospective, observational study of 2055 consecutive biopsies from 775 recipients, determined the clinical significance of ISO-T. ISO-T prevalence was 19.1%, comprising mild tubulitis (i0t1) in 97.2%. Independent clinical predictors of tubulitis were HLA mismatch, prior TCMR and antibody-mediated rejection, pulse corticosteroids, and BKVAN (P = .006 to P < .001 by multivariable analysis). Histological associations of tubulitis included interstitial inflammation, peritubular capillaritis, tubular atrophy, and SV40T (P = .005 to <.001). The dominant pathological diagnoses in ISO-T (n = 393) were interstitial fibrosis/tubular atrophy (IF/TA, 44.5%) or normal/minimal (31.8%). Subanalysis of ISO-T from indication biopsies (n = 107) found acute tubular injury (37.4%), IF/TA (28.0%), normal/minimal (12.1%), acute rejection (9.3%, vascular or antibody), chronic-active TCMR (2.8%), and BKVAN (5.6%). Allograft function of ISO-T frequently improved, affected by early biopsy timing and underlying disease diagnosis. Subsequent histology of 1197 ISO-T biopsy-pairs was generally benign. The 1- and 5-year death-censored graft survivals of ISO-T were 98.8% and 92.7%. In summary, tubulitis without inflammation does not represent borderline TCMR. We suggest its removal from the borderline category, and reinstatement of i1 as the diagnostic threshold.

The clinical and pathological significance of borderline T cell-mediated rejection.

The pathological diagnosis of borderline rejection (BL-R) denotes possible T cell-mediated rejection (TCMR), but its clinical significance is uncertain. This single-center, cross-sectional cohort study compared the functional and histological outcomes of consecutive BL-R diagnoses (n = 146) against normal controls (n = 826) and acute TCMR (n = 55) from 551 renal transplant recipients. BL-R was associated with the following: contemporaneous renal dysfunction, acute tubular necrosis, and chronic tubular atrophy (P < .001); progressive tubular injury with fibrosis by longitudinal sequential histology (45.3% at 1 year); increased subsequent acute rejection (39.4%), allograft failure (P < .001), and patient mortality (P = .007). BL-R detected by biopsy indicated for impaired function was followed by suboptimal functional recovery (46.3%), persistent inflammation (27.2%), and acute rejection episodes (50.0%) despite antirejection treatment in 83.3%. By 1 year after BL-R, the incidence of new-onset microvascular inflammation (9.3%), C4d staining (22.3%), transplant glomerulopathy (13.3%), and de novo donor-specific antibodies (31.5%) exceeded normal controls (P < .05-.001). BL-R inflammation in protocol biopsy persisted in 28.0% and progressed to acute rejection in 32.6%; however, it resolved in 61.6% of the untreated cases. In summary, BL-R is a heterogeneous diagnostic grouping, ranging from mild inconsequential inflammation to clinically significant TCMR, which is capable of immune-mediated tubular injury resulting in inferior functional, immunological, and histological consequences.