scholarly journals Single Cell Transcriptomics of Human Native Kidney and Kidney Allografts and New Insights into the Mechanism of Fibrosis

2020 ◽  
Author(s):  
Hemant Suryawanshi ◽  
Hua Yang ◽  
Michelle Lubetzky ◽  
Pavel Morozov ◽  
Mila Lagman ◽  
...  
Keyword(s):  
Single Cell ◽  
Successful Treatment ◽  
Kidney Biopsy ◽  
Cell Types ◽  
Transplant Recipients ◽  
Hla Antibodies ◽  
Kidney Allograft ◽  
Antibody Mediated Rejection ◽  
Native Kidney ◽  
Allograft Kidney

AbstractBackgroundSingle-cell RNA-sequencing (scRNA-seq) provides unique opportunity to study cell types and cell states at a hitherto unavailable level of precision. We tested the hypothesis that scRNA-seq and computational analysis of human kidney allograft biopsies will reveal new cell types and cell states and yield insights to personalize the care of transplant recipients.MethodsWe selected 3 kidney biopsies from 3 individuals for scRNA-seq using the 10x Chromium Single Cell platform; (i) HK: native kidney biopsy from a living kidney, (ii) AK1: allograft kidney biopsy with transplant glomerulopathy, fibrosis, and worsening kidney function but with undetectable circulating anti-HLA antibodies, and (iii) AK2: allograft kidney biopsy after successful treatment of active antibody-mediated rejection but with persistent circulating donor-specific anti-HLA antibodies.ResultsWe generated 7,217 high-quality single cell transcriptomes. Taking advantage of the recipient-donor sex mismatches, we determined that in the AK1 biopsy with fibrosis, more than half of the kidney allograft fibroblasts were—unexpectedly—recipient-derived and therefore likely migratory and graft infiltrative, whereas in the AK2 biopsy without fibrosis, all the fibroblasts were donor-derived. Furthermore, tubular progenitor cells that overexpress profibrotic extracellular matrix genes potentially contributing to fibrosis, were enriched in AK1 biopsy. Eight months after successful treatment of antibody-mediated rejection, AK2 biopsy contained endothelial cells that expressed mRNA for T-cell chemoattractant cytokines. In addition to these key findings, our analysis revealed unique cell types and cell states in the kidney.ConclusionsAltogether, single cell transcriptomics complemented histopathology and yielded novel mechanistic insights for individualizing the care of transplant recipients.

scholarly journals Detection of Infiltrating Fibroblasts by Single-Cell Transcriptomics in Human Kidney Allografts

2021 ◽  
Author(s):  
Hemant Suryawanshi ◽  
Hua Yang ◽  
Michelle Lubetzky ◽  
Pavel Morozov ◽  
Mila Lagman ◽  
...  
Keyword(s):  
Single Cell ◽  
Successful Treatment ◽  
Graft Function ◽  
Human Kidney ◽  
Cell Types ◽  
Kidney Allograft ◽  
Antibody Mediated Rejection ◽  
Allograft Kidney ◽  
Distinct Cell ◽  
Unique Cell

Abstract We tested the hypothesis that single-cell RNA-sequencing (scRNA-seq) analysis of human kidney allograft biopsies will reveal distinct cell types and states and yield insights to decipher the complex heterogeneity of alloimmune rejection. We selected 3 kidney biopsies from 3 individuals for scRNA-seq and processed them fresh using an identical protocol on the 10x Chromium platform; (i) HK: native kidney biopsy from a living donor, (ii) AK1: allograft kidney with transplant glomerulopathy and tubulointerstitial fibrosis, and worsening graft function, and (iii) AK2: allograft kidney after successful treatment of active antibody-mediated rejection. We generated 7217 high-quality single cell transcriptomes. Taking advantage of the recipient-donor sex mismatches, we determined that in AK1 with fibrosis, more than half of the kidney allograft fibroblasts were—unexpectedly—recipient-derived and therefore likely migratory and graft infiltrative, whereas in the AK2 without fibrosis, all the fibroblasts were donor-derived. Furthermore, AK1 was enriched by tubular cells that overexpressed profibrotic extracellular matrix genes. AK2, eight months after successful treatment of rejection, contained endothelial cells that expressed T-cell chemoattractant cytokines. In addition to these key findings, our analysis revealed unique cell types and cell states. Altogether, single cell transcriptomics yielded novel mechanistic insights for individualizing the care of transplant recipients.

scholarly journals The determinants, biomarkers, and consequences of microvascular injury in kidney transplant recipients

2019 ◽  
Vol 316 (1) ◽  
pp. F9-F19 ◽  
Author(s):  
Alice Doreille ◽  
Mélanie Dieudé ◽  
Heloise Cardinal
Keyword(s):  
Kidney Disease ◽  
Kidney Transplant ◽  
Interstitial Fibrosis ◽  
Ischemia Reperfusion ◽  
Transplant Recipients ◽  
Kidney Transplant Recipients ◽  
Hla Antibodies ◽  
Microvascular Injury ◽  
Antibody Mediated Rejection ◽  
Peritubular Capillaries

Independent of the initial cause of kidney disease, microvascular injury to the peritubular capillary network appears to play a central role in the development of interstitial fibrosis in both native and transplanted kidney disease. This association is explained by mechanisms such as the upregulation of profibrotic genes and epigenetic changes induced by hypoxia, capillary leakage, endothelial and pericyte transition to interstitial fibroblasts, as well as modifications in the secretome of endothelial cells. Alloimmune injury due to antibody-mediated rejection and ischemia-reperfusion injury are the two main etiologies of microvascular damage in kidney transplant recipients. The presence of circulating donor-specific anti-human leukocyte antigen (HLA) antibodies, histological findings, such as diffuse C4d staining in peritubular capillaries, and the extent and severity of peritubular capillaritis, are commonly used clinically to provide both diagnostic and prognostic information. Complement-dependent assays, circulating non-HLA antibodies, or evaluation of the microvasculature with novel imaging techniques are the subject of ongoing studies.

scholarly journals MO065DIAGNOSTIC CRITERIA OF HUMORAL REJECTION IN RENAL TRANSPLANT RECIPIENTS WITH PRE-EXISTING ANTI-HLA ANTIBODIES BASED ON SENTINEL SKIN FLAP BIOPSY

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Denis Sadouski ◽  
Aleh Kalachyk ◽  
Alexadr Nosik ◽  
Aliaksei Narbin ◽  
Anna Startsava ◽  
...  
Keyword(s):  
Renal Transplant ◽  
Vascular Bundle ◽  
Renal Allograft ◽  
Skin Flap ◽  
Renal Transplant Recipients ◽  
Transplant Recipients ◽  
Humoral Rejection ◽  
Hla Antibodies ◽  
Kidney Allograft

Abstract Background and Aims Twenty years of transplantation of composite vascularised allografts have revealed the high immunogenicity of the constituent parts, especially the skin.Several researchers have proposed the use of vascular stalk flaps, which allow to performe skin biopsy and diagnose rejection without biopsy of the transplanted solid organ.Pre-existing anti-HLA antibodies represent a serious immunological barrier to successful kidney transplantation.We are not aware of any studies on the diagnosis of antibody-associated acute kidney allograft rejection from deceased donors in recipients with pre-existing antibodies, based on morphological examination of a sentinel skin flap on a vascular stalk. The Aim: To determine the morphological features of humoral rejection in renal transplant recipients with pre-existing anti-HLA antibodies based on sentinel skin flap biopsy. Method Three skin-kidney allografts recipients underwent skin flap biopsy on 2nd day after transplantation. A kidney allograft biopsy was performed on day 7, 30, 60, 90.The results of morphological studies are presented using the Banff classification of renal allograft and skin allograft pathology. All recipients were female; 35, 44, and 57 years old. Two patients were re-transplanted and the main cause of CKD was chronic glomerulonephritis. The third patient, with congenital abnormality of the urinary tract, received her first graft.Pre-existing anti-HLA antibody levels before transplantation were 50%, 60%, 80%, respectively. Results All recipients showed signs of humoral rejection of the skin flap with thrombosis of the feeding vascular bundle, phlebitis, predominantly intimal arteritis with median necrosis, detachment and areactive necrosis of epidermis and epithelium of skin appendages. Clinical rejection, similar to the algorithm proposed by Etra J.W. et al. to assess preclinical skin rejection in an animal model (2019), was interpreted as G2 in two cases and G3 in one case. The Banff classification of the skin flap offers a qualitative assessment of certain biopsy parameters, while the kidney graft has qualitative-quantitative criteria for assessing rejection. When comparing the two classifications, a quantitative gradation of pathological changes in the skin flap according to the parameter of intimal arteritis (v) and immunoreactivity of the C4d marker similar to renal rejection was possible. In histological examination of skin flap biopsies, the degree of vasculitis was assessed in a large feeding artery: in all three cases this parameter was equal to v3. C4d expression was analyzed in the endothelium of microcirculatory blood vessels of the dermis and hypodermis: C4d1 in one case and C4d3 in the other two. The analysis of renal allograft biopsies revealed signs of humoral rejection (v1) only at day 30 in two recipients whose C4d expression in the skin and hypodermis was strong (C4d3). Conclusion Antibody-mediated rejection of a vascularized sentinel skin flap in recipients with combined skin-kidney transplantation is characterized by vasculitis affecting a core vascular bundle in the form of endarteritis with necrosis of media,phlebitis and associated thrombosis.Further studies are required to determine the feasibility of using a vascular stalked skin flap in the diagnosis of humoral rejection after renal transplantation.

Association of vimentin antibody and other non-HLA antibodies with treated antibody mediated rejection in heart transplant recipients

2020 ◽  
Vol 81 (12) ◽  
pp. 671-674
Author(s):  
Xiaohai Zhang ◽  
Ryan Levine ◽  
Jignesh K. Patel ◽  
Michelle Kittleson ◽  
Lawrence Czer ◽  
...  
Keyword(s):  
Heart Transplant ◽  
Transplant Recipients ◽  
Hla Antibodies ◽  
Antibody Mediated Rejection ◽  
Heart Transplant Recipients

scholarly journals Non-HLA Antibodies and Epitope Mismatches in Kidney Transplant Recipients With Histological Antibody-Mediated Rejection

2021 ◽  
Vol 12 ◽  
Author(s):  
Marta Crespo ◽  
Laura Llinàs-Mallol ◽  
Dolores Redondo-Pachón ◽  
Carrie Butler ◽  
Javier Gimeno ◽  
...  
Keyword(s):  
Kidney Transplant ◽  
De Novo ◽  
Transplant Recipients ◽  
Kidney Transplant Recipients ◽  
Serum Samples ◽  
Hla Antibodies ◽  
Aortic Endothelial Cells ◽  
Antibody Mediated Rejection ◽  
Donor Specific Antibodies

BackgroundCorrelation between antibody-mediated rejection (ABMR) and circulating HLA donor-specific antibodies (HLA-DSA) is strong but imperfect in kidney transplant (KT) recipients, raising the possibility of undetected HLA-DSA or non-HLA antibodies contributing to ABMR. Detailed evaluation of the degree of HLA matching together with the identification of non-HLA antibodies in KT may help to decipher the antibody involved.MethodsWe retrospectively assessed patients with transplant biopsies scored following Banff’15 classification. Pre- and post-transplant serum samples were checked for HLA and non-HLA antibodies [MICA-Ab, angiotensin-II type-1-receptor (AT1R)-Ab, endothelin-1 type-A-receptor (ETAR)-Ab and crossmatches with primary aortic endothelial cells (EC-XM)]. We also analyzed HLA epitope mismatches (HLA-EM) between donors and recipients to explore their role in ABMR histology (ABMRh) with and without HLA-DSA.ResultsOne-hundred eighteen patients with normal histology (n = 19), ABMRh (n = 52) or IFTA (n = 47) were studied. ABMRh patients were HLA-DSApos (n = 38, 73%) or HLA-DSAneg (n = 14, 27%). Pre-transplant HLA-DSA and AT1R-Ab were more frequent in ABMRh compared with IFTA and normal histology cases (p = 0.006 and 0.003), without differences in other non-HLA antibodies. Only three ABMRhDSAneg cases showed non-HLA antibodies. ABMRhDSAneg and ABMRhDSApos cases showed similar biopsy changes and graft-survival. Both total class II and DRB1 HLA-EM were associated with ABMRhDSApos but not with ABMRhDSAneg. Multivariate analysis showed that pre-transplant HLA-DSA (OR: 3.69 [1.31–10.37], p = 0.013) and AT1R-Ab (OR: 5.47 [1.78–16.76], p = 0.003) were independent predictors of ABMRhDSApos.ConclusionsIn conclusion, pre-transplant AT1R-Ab is frequently found in ABMRhDSApos patients. However, AT1R-Ab, MICA-Ab, ETAR-Ab or EC-XM+ are rarely found among ABMRhDSAneg patients. Pre-transplant AT1R-Ab may act synergistically with preformed or de novo HLA-DSA to produce ABMRhDSApos but not ABMRhDSAneg. HLA epitope mismatch associates with ABMRhDSApos compared with ABMRhDSAneg, suggesting factors other than HLA are responsible for the damage.

CRISPR/Cas9-Engineered HLA-Deleted Glomerular Endothelial Cells as a Tool to Predict Pathogenic Non-HLA Antibodies in Kidney Transplant Recipients

2021 ◽  
Vol 32 (12) ◽  
pp. 3231-3251
Author(s):  
Baptiste Lamarthée ◽  
Carole Burger ◽  
Charlotte Leclaire ◽  
Emilie Lebraud ◽  
Aniela Zablocki ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Kidney Transplant ◽  
Transplant Recipients ◽  
Kidney Transplant Recipients ◽  
Hla Antibodies ◽  
Specific Antibodies ◽  
Antibody Mediated Rejection ◽  
Biopsy Specimens ◽  
Glomerular Endothelial Cells ◽  
Donor Specific Antibodies

BackgroundAfter kidney transplantation, donor-specific antibodies against human leukocyte antigen donor-specific antibodies (HLA-DSAs) drive antibody-mediated rejection (ABMR) and are associated with poor transplant outcomes. However, ABMR histology (ABMRh) is increasingly reported in kidney transplant recipients (KTRs) without HLA-DSAs, highlighting the emerging role of non-HLA antibodies (Abs).MethodsW e designed a non-HLA Ab detection immunoassay (NHADIA) using HLA class I and II–deficient glomerular endothelial cells (CiGEnCΔHLA) that had been previously generated through CRISPR/Cas9-induced B2M and CIITA gene disruption. Flow cytometry assessed the reactivity to non-HLA antigens of pretransplantation serum samples from 389 consecutive KTRs. The intensity of the signal observed with the NHADIA was associated with post-transplant graft histology assessed in 951 adequate biopsy specimens.ResultsW e sequentially applied CRISPR/Cas9 to delete the B2M and CIITA genes to obtain a CiGEnCΔHLA clone. CiGEnCΔHLA cells remained indistinguishable from the parental cell line, CiGEnC, in terms of morphology and phenotype. Previous transplantation was the main determinant of the pretransplantation NHADIA result (P<0.001). Stratification of 3-month allograft biopsy specimens (n=298) according to pretransplantation NHADIA tertiles demonstrated that higher levels of non-HLA Abs positively correlated with increased glomerulitis (P=0.002), microvascular inflammation (P=0.003), and ABMRh (P=0.03). A pretransplantation NHADIA threshold of 1.87 strongly discriminated the KTRs with the highest risk of ABMRh (P=0.005, log-rank test). A multivariate Cox model confirmed that NHADIA status and HLA-DSAs were independent, yet synergistic, predictors of ABMRh.ConclusionThe NHADIA identifies non-HLA Abs and strongly predicts graft endothelial injury independent of HLA-DSAs.

scholarly journals Single-Cell Transcriptomics of a Human Kidney Allograft Biopsy Specimen Defines a Diverse Inflammatory Response

2018 ◽  
Vol 29 (8) ◽  
pp. 2069-2080 ◽  
Author(s):  
Haojia Wu ◽  
Andrew F. Malone ◽  
Erinn L. Donnelly ◽  
Yuhei Kirita ◽  
Kohei Uchimura ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Biopsy Specimen ◽  
Kidney Biopsy ◽  
Human Kidney ◽  
Cell Types ◽  
Biopsy Specimens ◽  
Single Cell Rna Sequencing ◽  
Transplant Biopsy

Background Single-cell genomics techniques are revolutionizing our ability to characterize complex tissues. By contrast, the techniques used to analyze renal biopsy specimens have changed little over several decades. We tested the hypothesis that single-cell RNA-sequencing can comprehensively describe cell types and states in a human kidney biopsy specimen.Methods We generated 8746 single-cell transcriptomes from a healthy adult kidney and a single kidney transplant biopsy core by single-cell RNA-sequencing. Unsupervised clustering analysis of the biopsy specimen was performed to identify 16 distinct cell types, including all of the major immune cell types and most native kidney cell types, in this biopsy specimen, for which the histologic read was mixed rejection.Results Monocytes formed two subclusters representing a nonclassical CD16+ group and a classic CD16− group expressing dendritic cell maturation markers. The presence of both monocyte cell subtypes was validated by staining of independent transplant biopsy specimens. Comparison of healthy kidney epithelial transcriptomes with biopsy specimen counterparts identified novel segment-specific proinflammatory responses in rejection. Endothelial cells formed three distinct subclusters: resting cells and two activated endothelial cell groups. One activated endothelial cell group expressed Fc receptor pathway activation and Ig internalization genes, consistent with the pathologic diagnosis of antibody-mediated rejection. We mapped previously defined genes that associate with rejection outcomes to single cell types and generated a searchable online gene expression database.Conclusions We present the first step toward incorporation of single-cell transcriptomics into kidney biopsy specimen interpretation, describe a heterogeneous immune response in mixed rejection, and provide a searchable resource for the scientific community.

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