PMC8334176_01
Before application of MSCs (March 2019): chronic active C4d negative antibody-mediated rejection with preserved tubules (A), focal glomerulitis and peritubular capillaritis (B). According to Banff 2017 criteria, transplant kidney biopsy was consistent with chronic active C4d negative antibody mediated rejection: 12% glomerulitis with double contour formation (g1, cg3) in glomeruli, diffuse severe peritubular capillaritis (ptc3), chronic active vascular rejection (v1, cv3) in interlobular arteries without signs of thrombotic microangiopathy, diffuse more than 50% interstitial fibrosis and tubular atrophy with mild mononuclear cell inflammation consisting of lymphocytes, macrophages, and rare plasma cells (i-IFTA 3, ci3, ct3). There was no associated tubulitis. Peritubular capillary basement membrane multilamellation (ptcbm3) was seen by electron microscopy. There were no deposits in tubular basement membrane. Immunofluorescence revealed IgA deposits indicating IgA nephropathy recurrence. 60% of glomeruli were globally sclerotic. 1 of 25 glomeruli showed pseudocrescent formation without PAS positive droplets in podocytes. After application of MSCs (July 2019): glomerular TMA with mesangiolysis (C), diffuse pseudocrescent formation in glomeruli with marked podocytes injury (D), vascular TMA (E) and huge tubular injury with resorptive droplets consisted with mottled lysosomes on EM (F). Glomeruli showed advanced double contour formation and segmental sclerosis without apparent glomerulitis, but mesangiolysis was seen in some glomeruli. 20% of glomeruli showed pseudocrescent formation with PAS positive droplets "in podocytes indicating huge podocytes injury". Peritubular capillaritis was mild and focal. Tubulitis was absent. Tubules show signs of severe tubular injury (attenuation of tubular epithelium with coarse vacuolization, loss of brush border, loss of nuclei) and were filled with large PAS-positive droplets in line mottled lysosomes on electron microscopy. Electron optic dense deposits in tubular basement membrane were detected by EM. Amount of interstitial fibrosis, tubular atrophy, and interstitial inflammation was similar as in previous biopsy. In fibrotic areas, there was mild mononuclear cell inflammation consisting of lymphocytes 75%, macrophages 15%, and plasma cells 10%. No CD105+, CD73+ nor CD90+ cells were found. In small arteries and arterioles, thrombotic microangiopathy with obliteration of vascular lumens, fragmentation of erythrocytes, and fibrinoid necrosis was present. Peritubular capillary basement membrane multilamellation (ptcbm3) was similar as in previous biopsy.
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F1
The rejection was treated with high-dose steroids, antithymocyte globulin, plasmapheresis, intravenous immunoglobulins (IVIg), and rituximab. After 3 years of stable kidney function with sCr in the range between 150 and 180 mumol/L, we observed a progressive deterioration of kidney function with a sCr value of 240 mumol/L and a 24-h proteinuria of 3.4 g/day before entering the study protocol. The kidney biopsy revealed chronic active AMR (Figures 1A,B). Patohistologic analysis of the explanted kidney was consistent with advanced AMR, glomerular and vascular thrombotic microangiopathy (TMA), and extensive tubular injury (Figures 1C-F).
(A) Kidney graft function and serum concentrations of lymphocyte populations before and after application of mesenchymal stem cells. Three consecutive applications of mesenchymal stem cells are marked with arrows. eGFR, estimated glomerular filtration rate; mGFR, measured glomerular filtration rate (by Cr-EDTA); conc, concentration; Treg, regulatory T cells. (B) Cytokine concentrations after mesenchymal stem cells application (in U/mL for soluble interleukin 2 receptor and in pg/mL for other cytokines). Time frame of corticosteroid treatment is marked with arrows; Legend: TNF, tumor necrosis factor; IL, interleukin; s-IL-2-R, soluble interleukin 2 receptor; IFN, interferon.
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F2
This was associated with a slight deterioration in kidney function, which we attributed to prerenal causes (Figure 2A). Cytokine assessment after MSCs treatment (at the time of onset of symptoms) showed increased levels of IL-2R and IL-8, while other cytokines, including TNF-alpha and IL-6, were within near normal ranges and not consistent with a cytokine storm (Figure 2B, for details see Supplementary Table 2). B lymphocyte subsets before and after MSC therapy showed a transient decrease in all lymphocyte subsets with an increase in the ratio of activated T lymphocytes that occurred concomitantly with worsening of symptoms and the appearance of pancytopenia (Figure 2A). Bone marrow examination was consistent with trilineage dysplasia and a secondary myelodysplastic syndrome attributable to potentiated immunosuppressive therapy. After the transplant nephrectomy, the cell counts recovered almost instantly (Figure 2A).
Molecular microscope diagnostic report (MMDx) for kidney transplant biopsy before (A) and after (B) application of mesenchymal stem cells showing fully developed antibody-mediated rejection with extensive microvascular inflammation, chronic glomerulopathy, and atrophy-fibrosis molecular features.
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F3
Transcriptomic analysis by the molecular microscope diagnostic system (MMDx) of the subsequent kidney biopsy showed persistence of AMR, although the molecular classifiers of inflammation and AMR were lower compared to the biopsy before application of MSCs (inflammation score of 1.06 vs. 3.19, and AMR score of 0.63 vs. 0.94, respectively; Figure 3).