PMC8083054_01
Course of Patient 1's DSA and serum creatinine after the diagnosis of chronic active AMR. (A) HLA phenotype results of the patient 1 and his donor. (B) Flow-CDC results before and after the second treatment of chronic AMR. Donor lymphocytes incubated with neither the patient's serum nor rabbit complement (cells only) and cells incubated with rabbit complement alone (cells+complement) served as negative controls. (C) changes in MFI values of donor-specific anti-DQ7 antibody (1:3 or 1:6 diluted serum) during the first round of treatment (a) and the second round of treatment (b). (D) Changes of serum creatinine levels before and after the diagnosis/treatment of chronic active AMR.
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The donor-recipient HLA-A, -B, -DR, -DQ mismatch grade was 4 ( Figure 1A ). AMR: rituximab (200 mg), three sessions of double-filtration plasmapheresis (DFPP)/IVIG (25g each time), followed by three sessions of PP/IVIG (25g each time). After the treatment, DSA detection showed no obvious decease in anti-DQ7 antibody MFI (13,576) ( Figure 1C-a ). Flow-CDC testing using isolated fresh donor lymphocytes as target cells showed a weakly positive result (4.93%) as compared to the negative controls (cells only: 1.27%; cells + complement: 3.09%) ( Figure 1B ). Therefore, the patient was hospitalized again and received his second round of treatment: eight PP/IVIG sessions in combination with splenic irradiation (10 times, 50 cGy each time, 500 cGy in total) within a 42-day course ( Figure 1C-b ). MFI value of the DSA demonstrated a more obvious reduction (from 18,914 to 8,405) ( Figure 1C-b ). Meanwhile, the Flow-CDC result decreased to 3.76%, which was very close to the negative control value. His serum creatinine remained relatively stable for the first 10 months (248-255 mumol/L) ( Figure 1D ). However, from the 11th month of follow-up, the serum creatinine gradually increased to >300 mumol/L ( Figure 1D ), the proteinuria increased (5,346 mg/24h), and the MFI of the DQ7-DSA rebounded to 14,923.
Histological features of chronic active AMR in Patient 1 before and after treatment. (A) Typical histological findings of chronic AMR in Patient 1 before the first round of treatment and their Banff 2017 Scores. (B) Histological findings of renal allograft in Patient 1 at the 14th month after the second round of treatment and their Banff 2017 Scores. Panel A1 and B1 (hematoxylin and eosin, 400x magnification) show glomerulitis. Panel A2 and B2 (hematoxylin and eosin, 400x magnification) show peritubular capillaritis (arrow). Panel A3 and B3 (immunohistochemical staining, 400x magnification) show peritubular capillary C4d deposition (arrow). Panel A4 and B4 (Masson's trichrome, 400x magnification) show transplant glomerulopathy, characterized by focal glomerular basement membrane double contours. Panel A5 (electron microscopy, 2000x magnification) shows 5-7 circumferential layers of peritubular capillary basement membrane multilayering (ptcml, arrow). Panel B5 (electron microscopy, 3000x magnification) shows 8-10 circumferential layers of peritubular capillary basement membrane multilayering (ptcml, arrow).
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The pathology results showed moderate glomerulitis (g2), severe peritubular capillaritis (ptc3), mild peritubular capillary C4d deposition (C4d1), focal glomerular basement membrane double contours (cg2), peritubular capillary basement membrane multilayering (electron microscopy), and positive IgA deposition (++), which were diagnosed as mild chronic active AMR (Banff 2017 Schema), transplant glomerulopathy (TG), and recurrent IgA nephropathy ( Figure 2A ). Results showed that the acute tissue injury was alleviated (g1 and ptc2) as compared with earlier examinations, and the C4d deposition in peritubular capillaries had become negative, but the chronic tissue lesions were slightly worse ( Figure 2B ).
PMC8083054_02
Course of Patient 2's DSA and serum creatinine after the diagnosis of chronic active AMR. (A) HLA genotype results of the patient 2 and his donor. (B) Flow-CDC results before and after the treatment of chronic AMR. Donor lymphocytes incubated with neither the patient's serum nor rabbit complement (cells only) and cells incubated with rabbit complement alone (cells +complement) served as negative controls. (C) changes in MFI values of donor-specific anti-DR12 and anti-DQA6 antibodies (1:3 diluted serum) during the treatment. (D) Changes of serum creatinine levels after the diagnosis and treatment of chronic active AMR.
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The donor-recipient HLA-A, -B, -C, -DR, -DQA, -DQB, -DP mismatch grade of allele-level genotypes was 9 of 14 ( Figure 3A ). The Flow-CDC test using isolated fresh donor lymphocytes as target cells showed a mildly positive result (8.87%) that was higher than that of the negative controls (cells only: 3.92%; cells + complement: 3.04%) ( Figure 3B ). AMR, the patient was given rituximab (200 mg), eight PP/IVIG (20g each time) sessions (every 2-4 days), and splenic irradiation (10 times, 50 cGy each time, 500 cGy in total) ( Figure 3C ). DSA significantly decreased (1:3 diluted serum; DR12-MFI: 1,742; DQA6-MFI: 1,222) and the Flow-CDC result became negative (3.74%) ( Figure 3B ). At the same time, the second renal biopsy showed that both the glomerulitis and peritubular capillaritis were significantly improved (g0-1, ptc0-1) ( Figure 4B ). During the course of the treatment, the patient had no anorexia, fatigue, or other abnormal symptoms, as well as no abnormal changes in liver function or routine blood test results. The renal function remained quite stable during the whole follow-up period ( Figure 3D ).
Histological features of chronic active AMR in Patient 2 before and after treatment. (A) Typical histological findings of chronic AMR in Patient 2 before the treatment and their Banff 2017 Scores. (B) The pathology results of a second renal biopsy performed after 4 weeks of treatment and their Banff 2017 Scores. (C) The pathology results of a third renal biopsy performed at the tenth month after the treatment and their Banff 2017 Scores. Panel A1, B1 and C1 (hematoxylin and eosin, 400x magnification) show different degrees of glomerulitis. Panel A2, B2 and C2 (hematoxylin and eosin, 400x magnification) show different degrees of peritubular capillaritis (arrow). Panel A3, B3 and C3 (immunohistochemical staining, 400x magnification) show no C4d deposition in peritubular capillaries (asterisk). Panel A4, B4 and C4 (Masson's trichrome, 400x magnification) show different degrees of transplant glomerulopathy, characterized by focal glomerular basement membrane double contours (arrow). Panel A5 (electron microscopy, 2500x magnification) and Panel C5 (electron microscopy, 4000x magnification) show 5-7 circumferential layers of peritubular capillary basement membrane multilayering (ptcml, arrow).
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The results showed moderate glomerulitis (g2) and peritubular capillaritis (ptc2), focal glomerular basement membrane double contours (cg1), peritubular capillary basement membrane multilayering (electron microscopy), IgA deposition (++), and focal glomerular C4d deposition, but no peritubular capillary C4d deposition (C4d0), all of which indicated mild chronic active AMR and recurrent IgA nephropathy ( Figure 4A ). DSA significantly decreased (1:3 diluted serum; DR12-MFI: 1,742; DQA6-MFI: 1,222) and the Flow-CDC result became negative (3.74%) ( Figure 3B ). At the same time, the second renal biopsy showed that both the glomerulitis and peritubular capillaritis were significantly improved (g0-1, ptc0-1) ( Figure 4B ). During the course of the treatment, the patient had no anorexia, fatigue, or other abnormal symptoms, as well as no abnormal changes in liver function or routine blood test results. The pathology results showed that both glomerulitis and peritubular capillaritis remained mild (g1 and ptc1), and the C4d deposition was still negative (C4d0), but there was some progression in the chronic renal lesions (focal glomerular basement membrane double contours, mild interstitial fibrosis, and tubule atrophy; Banff 2017 scores: cg2, mm2, ci1, ct1;  Figure 4C ).