Mesangiolysis in Pathological Chronic Active Antibody-Mediated Rejection in Kidney Transplant Biopsies

Chronic active antibody-mediated rejection (CAABMR) has been reported to be the major reason of allograft loss [1]. Transplant glomerulopathy is a pathological feature mainly caused by antibody-mediated rejection [2]. A previous Banff meeting report stated that mesangiolysis (MGLS) was often detected in patients with transplant glomerulopathy, and may lead to advanced sclerotic lesions [3]. However, the etiology of mesangiolysis in CAABMR has not been fully elucidated. MGLS is classified into two groups, including primary lesions due to direct mesangial injury and secondary lesions due to endothelial injury [4]. MGLS was observed in cases of antibody-mediated rejection showing endothelial injury [5]. MGLS may also result from nonimmune etiologies, via endothelial injuries caused by diabetes mellitus [4], hypertension [4, 6], and calcineurin inhibitors (CNIs) [7]; however, the associations between these clinical findings and MGLS in CAABMR cases are unknown.

The Banff 2013 classification defined CAABMR as the combination of: (1) chronic tissue injury of cg ≥1 or new-onset arterial intimal fibrous thickening (cv ≥ 1); (2) evidence of antibody interaction on glomerular or peritubular endothelium (g + ptc ≥2 or C4d deposition in ptc) by immunofluorescence on frozen section; and (3) serological evidence of donor-specific antibodies (DSA) [8]. The Banff 2015 classification suggested that pathological chronic injury and evidence of antibody interaction on the microvascular endothelium or evidence of DSA, but not both, was suspicious lesion of CAABMR [9]. In a recent study, the Banff acute and chronic pathological scores showed no difference between DSA-positive and -negative (suspicious for CAABMR in Banff 2015 classification) groups [10]. We recently defined biopsy-proven acute/active ABMR regardless of DSA positivity as “pathological” acute/active ABMR, and the acute and chronic Banff scores showed no significant difference among groups with pathological acute/active ABMR with different levels of DSA mean fluorescence intensity [11]. The relationship between acute or chronic pathological findings in biopsy-proven “pathological” CAABMR (P-CAABMR) and the formation of MGLS still remains unclear. In the present study, we aimed to explore the immune and nonimmune clinical factors related to MGLS in P-CAABMR, and to elucidate the associations of acute and chronic pathological findings of MGLS in P-CAABMR.

A total of 670 biopsies were obtained from 374 kidney transplant recipients from January 2016 to December 2019, excluding implantation biopsies. We recently defined Banff 2013 pathological acute/active antibody-mediated rejection regardless of DSA positivity (mean fluorescence intensity ≥1,000) as pathological acute/active ABMR, diagnosed by light microscopy (LM) and C4d deposition, but not by electron microscopy (EM) [11]. In the present study, we determined P-CAABMR by LM and C4d deposition, regardless of the DSA and EM findings, with reference to our previous report [11]. We analyzed 41 biopsies from 41 recipients diagnosed with P-CAABMR. We compared the clinical characteristics and histological findings between the MGLS-positive and -negative groups. Hypertension was defined as blood pressure at the time of biopsy ≥140/90 mm Hg, according to the Japanese Society of Hypertension guidelines for the management of hypertension, or by the administration of antihypertensive therapy. Hypertension grade was defined based on the blood pressure at biopsy as follows: grade 0, <140/90 without use of antihypertensive agents; grade 1, <140/90 with use of antihypertensive agents; and grade 2, ≥140/90 with or without use of antihypertensive agents. DSA positivity was defined by a peak mean fluorescence intensity ≥1,000 according to our institution’s criteria [11]. Data on clinical factors including the use of anti-immunosuppressive agents (mycophenolate mofetil, tacrolimus/cyclosporine, everolimus), diabetes, hypertension, hypertension grade, estimated glomerular filtration rate (eGFR), and urinary protein were obtained at the time of biopsy.

Multivariate analysis was performed using three models: clinical model 1 included covariates of conventional clinical risk factors (type of CNI, DSA, presence of diabetes, and hypertension grade); clinical model 2 included the same covariates as clinical model 1 plus statistically significant clinical factors evaluated by univariate analysis; the histological model included covariates of acute inflammation score (g and ptc scores), and statistically significant histological factors evaluated by univariate analysis.

Banff scores were determined according to the Banff meeting reports [3, 8, 12]. A recent consensus definition by the Renal Pathology Society described MGLS as partial or complete mesangial matrix dissolution using LM detected by periodic acid-Schiff or silver diminished staining [13], and ballooning of the glomerular capillary and nodular lesions was reported to be subsequent with MGLS [4]. In the present study, we defined MGLS as reduced mesangial matrix staining mainly evaluated by periodic acid-Schiff staining with or without glomerular capillary ballooning or nodular lesions. The mesangial matrix increased during the healing process after MGLS [4], and the mm score, defined as an increase in mesangial matrix in previous Banff classification [3], was therefore not evaluated considering the interaction with MGLS. We further evaluated six electron microscopic findings regarding endothelial injury, including new criteria, in addition to the criteria based on previous reports [11, 14]: endothelial swelling (endothelial cytoplasm covering the glomerular basement membrane by ≥3/4 circumference of at least three glomerular capillaries); absence of endothelial fenestration (absence of fenestration in at least half of the capillary circumference in ≥3 glomerular capillaries); thickening of endothelial cytoplasm (most thickening endothelial cytoplasm exceeded lamina densa of glomerular basement membrane in ≥3 glomerular capillaries); widening of subendothelial space (widest subendothelial space exceeded lamina densa of glomerular basement membrane in ≥3 glomerular capillaries); early neo-intima (early neo-intima observed in ≥3 glomerular capillaries); and resolution of mesangial matrix (resolution in >50% of mesangial cell area in one mesangial region).

We analyzed the data using SPSS version 23.0 (IBM, Japan) in the present study. Data were expressed as mean ± standard deviation, and a p value <0.05 was judged to be statistically significant. Continuous variables with a normal distribution were compared using Student’s t test, and variables with a nonnormal distribution were compared using the Mann-Whitney U test. Dichotomous variables were compared using χ² or Fisher’s test. Univariate and multivariate analyses were performed by logistic regression to explore the clinical and pathological factors related to MGLS, and multivariate logistic analysis was performed by the forward selection method. Correlations were determined by Spearman’ correlation test. Death-censored graft survival was analyzed by log-rank test comparing the MGLS-positive and -negative groups.

MGLS was observed in 15 cases (36.6%) of P-CAABMR. The mean duration after transplantation was 103.2 months in all P-CAABMR cases. Table 1 shows the baseline clinical characteristics in the MGLS-positive and negative groups. Duration after transplantation was significantly longer, prevalence of hypertension, hypertension grade, and urine protein were significantly higher and eGFR was significantly lower in the MGLS-positive group compared with the MGLS-negative group. There was no significant difference in any of the other clinical parameters (Table 1).

Table 2 shows the LM findings in the MGLS-positive and -negative groups. Representative light microscopic and EM images are shown in Figure 1. The prevalence of FSGS, g score, cg score, ah score, and aah score, and prevalence of ballooning of glomerular capillary were all significantly higher in the MGLS-positive group. Table 3 shows the electron microscopic findings in the two groups. There were no significant differences between the groups.

Table 4 shows the clinical and pathological factors related to MGLS using univariate logistic analysis. Duration after transplantation, hypertension grade, and eGFR were the clinical factors significantly related to MGLS, while the prevalence of FSGS, and g, cg, ah and aah scores were the significant histological factors related to MGLS. Table 5 shows the result of multivariate logistic analysis. Hypertension grade was significantly related to MGLS in clinical model l, including type of CNI (TAC/CYA), DSA positivity, prevalence of diabetes, and hypertension grade. In clinical model 2, we performed multivariate analysis using four variables from clinical model 1, with addition of eGFR and duration after transplantation significantly related to MGLS by univariate analysis. Hypertension grade was thus a significant factor related to MGLS.

Both ah and aah scores were significant histological factors related to MGLS (Table 4), with high correlation between the two scores (r = 0.960, p < 0.001). We therefore put the covariate of ah and aah score separately into the pathological model for multivariate analysis. In the histological model including acute inflammation score (g and ptc scores) and FSGS, cg score, and aah score, cg score was the only independent factor related to MGLS (Table 5). In the multivariate model including g, ptc, FSGS, and cg and ah score, cg score was also significantly related to MGLS (odds ratio 3.808, 95% confidence interval 1.624–8.928, p = 0.002) (data in this multivariate model including ah score are not shown in Table 5). There was no significant difference in death-censored graft survival between the MGLS-positive and -negative groups (p = 0.547) (Fig. 2). Table 6 demonstrates the correlation of cg score and the clinicopathological findings. Hypertension grade, duration after transplantation, g, ah, and aah score were significantly correlated with cg score.

In this study, we identified MGLS in 36.6% of cases with P-CAABMR. Graft function was significantly lower and proteinuria was higher in patients with P-CAABMR with MGLS. Multivariate analysis showed that Banff cg score was independently associated with the formation of MGLS in P-CAABMR in a histological model, and hypertension grade was also independently related to MGLS in clinical models. The Banff cg score was correlated with hypertension grade, the duration after transplantation, and Banff g, ah, and aah scores.

The Banff cg score was evaluated by duplicated glomerular basement membrane [3], and the glomerular basement membrane duplication is the main feature of transplant glomerulopathy [2]. In transplant glomerulopathy, the new basement membrane formation of glomeruli developed with endothelial injury in the electron microscopic findings of longitudinal protocol biopsies study [15]. The pathogenesis of MGLS due to endothelial injury involves endothelial swelling and subendothelial widening with electron-lucent material, and the subendothelial and mesangial areas are connected, and the mesangial area changes loose forming secondary MGLS leading to the possible formation of a capillary aneurysm [4]. In the current study, Banff cg lesions were moderate to severe (mean cg score 2.40 in the MGLS-group). These findings implied that advanced cg lesions due to endothelial injury caused loose changes in the mesangial matrix.

Our data also showed that the Banff cg score was correlated with both immune and nonimmune factors including glomerulitis, time after transplant, arteriolar lesions, and hypertension. The aah score is used to evaluate CNI arteriolopathy [12], and transplant glomerulopathy is similar to the pathological feature of thrombotic microangiopathy [2], and MGLS was also found in patients with thrombotic microangiopathy with CNI nephrotoxicity [7]. In addition, hypertension grade was correlated with cg lesion in this study. Malignant hypertension was the etiology of secondary thrombotic microangiopathy in 4% of 531 cases [16]. Taken together, long-term exposure of hypertension, as well as CNI nephrotoxicity and glomerulitis may cause thrombotic microangiopathy, leading to the formation of MGLS. MGLS in P-CAABMR was thus considered to be caused from the cg lesion related to both immune and nonimmune factors.

Our data showed discrepancies between the LM and EM evaluations of mesangial matrix resolution as shown in Table 3. Because the glomeruli observed in LM and EM were different, we considered that this discrepancy might have been largely due to sampling bias. Furthermore, there were no significant differences in EM findings other than mesangial matrix resolution between the LM-identified MGLS-positive and -negative groups. Some of the criteria for endothelial injury were based on the previous reports, but these studies included acute and early chronic antibody-mediated rejection evaluated by EM [11, 14]. In the present study, we considered that all the definitions of endothelial injury determined by EM were basically mild lesions. The mean cg score of P-CAABMR evaluated by LM was 1.66 ± 1.06, indicating that P-CAABMR in this study included the histological finding of relatively advanced P-CAABMR. This may account for the lack of difference in endothelial injury evaluated by electron microscopy between the two groups.

This study had some limitations. The number of cases of MGLS in P-CAABMR was too small to accurately determine the significant factors using multivariate analysis. In addition, the clinicopathological factors and MGLS in P-CAABMR were analyzed basically using a cross-sectional approach, and the clinical significance of interventions for risk factors of MGLS in P-CAABMR were not evident from this study. Further longitudinal study is needed to elucidate suitable clinical intervention methods to prevent MGLS in P-CAABMR related to severe allograft dysfunction.

In summary, MGLS occurred in 36.6% of late P-CAABMR cases. Reduced graft dysfunction and higher proteinuria were observed in the presence of MGLS in P-CAABMR. Banff cg score was significantly related to MGLS in multivariate analysis. Sustained glomerulitis, CNI nephrotoxicity, and hypertension may promote the formation of advanced Banff cg lesions, leading to MGLS in P-CAABMR.

We appreciate Dr. Yutaka Yamaguchi of Yamaguchi’s Pathology Laboratory for the evaluation of pathological findings. We also thank Susan Furness, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

This study was approved by Toho University Omori Medical Center Ethics Committee (Approval No. M21290 21128). By opt-out method, patients were given the chance to reject joining the present study.

The authors have no conflicts of interest to declare.

None.

Yuki Suto designed the study, collected data, conducted analysis, and wrote the manuscript. Hideyo Oguchi designed the study, performed pathological evaluation, conducted analysis, and wrote the manuscript. Naobumi Tochigi and Tetuo Mikami contributed the methodology of pathological evaluation and performed the pathological evaluation. Kazunobu Shinoda contributed the methodology, data interpretation, and revised the paper. Kazuho Honda contributed the methodology of pathological evaluation and data interpretation. Noriyuki Kounoue contributed data interpretation and revised the paper. Junya Hashimoto contributed the methodology and data interpretation. Masaki Muramatsu, Yoshihiro Itabashi, and Ken Sakai contributed the data interpretation and revised the paper.

Yuki Suto and Hideyo Oguchi contributed equally to the present study.

Any question of the present study can be asked to the corresponding author with reason.