Phosphorylation of S6RP in peritubular capillaries of kidney grafts and circulating HLA donor-specific antibodies

Abstract

Antibody-mediated rejection (ABMR) caused by donor-specific HLA-antibodies (DSA) is a mediator of allograft loss after kidney transplantation (KT). DSA can activate microvascular endothelium damage through the mTOR pathway. In this study we assessed the mTOR pathway activation by DSA in KT with ABMR (ABMR + DSA+) compared to controls (ABMR-DSA-), biopsies with ABMR changes without DSA (ABMR + DSA-) and DSA without ABMR changes (ABMR-DSA+), and the potential modulation by mTOR inhibitors (mTORi). We evaluated 97 biopsies: 31 ABMR + DSA+, 33 controls ABMR-DSA-, 16 ABMR + DSA-, and 17 ABMR-DSA+ cases. Regarding immunosuppression of full ABMR + DSA+ and controls, 21 biopsies were performed under mTORi treatment (11 of them ABMR + DSA+ cases) and 43 without mTORi (20 of them ABMR + DSA+) so as to explore its effect on the mTOR pathway. Biopsies were stained for C4d, Ki67, and phosphorylated (p) S6RP, ERK, and mTOR by immunohistochemistry. Labeling was graded according to peritubular capillary staining. ABMR biopsies showed significantly higher C4d, p-S6RP, and Ki67 staining in peritubular capillaries (PTC) compared to controls, and light differences in p-ERK or p-mTOR. mTORi treatment did not modify p-S6RP, p-mTOR, and p-ERK staining. Diffuse p-S6RP in PTC in the biopsies significantly associated with circulating HLA-DSA independently of graft rejection, and with worse death-censored graft survival. These findings suggest that activation of endothelium through the mTOR pathway evidence different mechanisms of damage in ABMR + DSA+ and ABMR + DSA- despite similar histological injury.

Keywords: antibody mediated allograft rejection; donor specific antibodies; mammalian target of rapamycin (mTOR); peritubular capillaries; phosphorylation; ribosomal protein S6 (S6RP); transplantation—kidney.

FIGURE 1

Immunohistochemistry staining of p-S6RP, p-ERK, and p-mTOR in kidney transplant biopsies (40×). Representative images of different staining scores for each protein are shown.

FIGURE 2

Staining of p-S6RP in peritubular capillaries is increased in ABMR biopsies. (A) Staining score (upper graphs) and percentage of positive staining (lower graphs) of C4d, p-S6RP, p-ERK, and p-mTOR in PTC of renal allografts biopsies with ABMR + DSA+ (n = 31) compared to ABMR–DSA– (n = 33). Mann Whitney and Fisher exact tests were used to assess significant differences between these two groups. (B) Staining of the proliferation marker Ki67 was analyzed in biopsies with and without ABMR. Plots show the means (bars), standard deviation (error bars), and individual values (dots) for each sample. Mann Whitney and Fisher exact tests were used to assess significant differences between groups.

FIGURE 3

Death censored graft survival in patients with biopsies with p-S6RP positive and negative staining. (A) Kaplan-Meier survival curve representing death censored graft survival in patients with positive or negative p-S6RP staining in PTC. (B Left), Kaplan-Meier survival curve representing death censored graft survival in patients with and without ABMR diagnosis according to the p-S6RP staining in the biopsy. P-value between ABMR+ cases with positive or negative p-S6RP staining is p = 0.21. (B Right), Kaplan-Meier survival curve representing death censored graft survival in patients with and without DSA diagnosis according to the p-S6RP staining in the biopsy. P-value between DSA+ cases with positive or negative p-S6RP staining is p = 0.043.

FIGURE 4

Potential modulation of C4d, p-S6RP, p-mTOR, and p-ERK staining by mTORi immunosuppression. Staining score (A) and percentage of positive staining (B) of C4d, p-S6RP, p-ERK, and p-mTOR in PTC of renal allografts biopsies with ABMR + DSA+ treated (n = 11) or not (n = 20) with mTORi, and ABMR–DSA– treated (n = 4) or not (n = 7) with mTORi. Plots of staining score show the means (bars), standard deviation (error bars), and individual values (dots) for each sample. Kruskal-Wallis test was used to assess significant differences among groups for staining score and Fisher exact test for proportions of staining. (C) Percentage of positive staining for C4d, p-S6RP, p-ERK, and p-mTOR in PTC of renal allografts biopsies with active and chronic ABMR, compared to no ABMR, receiving or not mTORi immunosuppression. Fisher exact test was used to assess significant differences among groups.

FIGURE 5

P-S6RP in peritubular capillaries is associated with HLA-DSA. (A) Comparison of p-S6RP staining score in biopsies compatible with ABMR with DSA (n = 31) and with no detectable HLA-DSA (n = 16). (B) Comparison of p-S6RP staining score in biopsies with DSA and ABMR (n = 31) with biopsies with HLA-DSA but not ABMR (n = 18). Plots show the means (bars), standard deviation (error bars), and individual values (dots) for each sample. Mann Whitney test was used to assess significant differences between groups. (C) Percentage of positive staining (bars) for p-S6RP, p-mTOR, and p-ERK on biopsies with DSA class I, II, or I&II compared to biopsies with no DSA and no rejection (red dotted line).