LOH-bx, loop of Henle from your biopsy; LOH-h, loop of Henle from healthy kidney; PT-bx, proximal tubule from your biopsy; PT-h, proximal tubule from healthy kidney. Comparison of scRNA-seq and single-nucleus RNA-seq datasets has been shown to be valid after normalizing to reduce method-specific differences.29 Using a computational approach allowing for integrated analysis of multiple datasets across techniques,30 we compared proximal tubule, loop of Henle, and collecting duct cells from our biopsy dataset with the same cell types from our healthy adult kidney single-nucleus RNA-seq dataset. kidney transplant biopsy core by single-cell RNA-sequencing. Unsupervised clustering analysis of the biopsy specimen was performed to identify 16 unique cell types, including all of the major immune cell types and most native kidney cell types, in this biopsy specimen, for which the histologic go through was mixed rejection. Results Monocytes created two subclusters representing a nonclassical CD16+ group and Eltrombopag Olamine a classic CD16? group expressing dendritic cell maturation markers. The presence of both monocyte cell subtypes was validated by staining of impartial transplant biopsy specimens. Comparison of healthy kidney epithelial transcriptomes with biopsy specimen counterparts recognized novel segment-specific proinflammatory responses in rejection. Endothelial cells created three unique 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. for 5 minutes, resuspended in inDrops cell suspension buffer (9% Optiprep), and strained through a 40-(CD16) distinguishes monocyte 1 from monocyte 2, whereas is usually expressed in monocyte 2 but not monocyte 1. MSR1 is usually expressed in both clusters. (F) Immunohistochemistry for or on normal, mixed rejected, or real antibody-mediated rejection (ABMR) transplant kidney biopsies. Distinct monocyte 1 and 2 cell types can be seen. Upper and lower panels are serial sections. Scale bar, 50 (CD16) and was most much like CD16-positive, proinflammatory, nonclassic monocytes.17 Of notice, CD16++ cells are strongly associated with allograft rejection.18,19 Monocyte 2 seems to be a classic or intermediate monocyte population (Determine 2A, Supplemental Table 4). Interestingly, and (Physique 2D). We recognized unique marker genes for each monocyte cluster (Physique 2E) and performed immunohistochemistry on impartial transplant biopsies, with histologic diagnoses of no disease, mixed rejection, or ABMR. There was Eltrombopag Olamine sparse interstitial staining for both the Eltrombopag Olamine monocyte 1 marker (FCGR3A or CD16) and the monocyte 2 marker (FCN1) in biopsies with no disease. By contrast, there was strong staining for both monocyte subsets in mixed rejection, with smaller infiltration in real ABMR (Physique 2F, Supplemental Physique 5). Costaining by immunofluorescence analysis confirmed that this monocyte subtypes are individual populations (Physique 2G). The presence of these monocyte subsets in all six impartial biopsies with mixed rejection or ABMR validates the use of scRNA-seq to identify novel cell types associated with kidney rejection. Ligand-receptor analysis revealed expression of 14 receptors (excluding collagens) for which we could detect expression of their cognate ligands Rabbit polyclonal to IL18 (Physique 2B). These ligands were detected in all cell types, emphasizing the integration of signals between multiple kidney and leukocyte cell types. Pericytes, fibroblasts, and myofibroblasts expressed the chemoki and is expressed on mast cells in the biopsy (Physique 2B).25 Stem cell factor promotes mast cell migration, adhesion, proliferation, and survival. Mast cells transcripts correlate strongly with allograft biopsy fibrosis.26 These results suggest the unexpected hypothesis that collecting duct epithelia actively coordinate mast cell infiltration during rejection. Consistent with an important role for mast cells in kidney injury, a recent study showed that mast cell ablation in the early phases of renal injury is sufficient to reduce subsequent fibrosis by decreasing the inflammatory response.27,28 Activation of Epithelial, Endothelial, and Stromal Cells in Allograft Rejection We next compared epithelial transcriptomes from your biopsy with their healthy counterparts. Multiple attempts at scRNA-seq of healthy nephrectomy tissue failed to generate libraries; however, we were successful in generating adult human kidney single-nucleus RNA-sequencing (RNA-seq) data. We sequenced 4259 nuclei to a similar depth as the biopsy Eltrombopag Olamine and recognized six unique epithelial cell clusters, including podocytes, proximal tubule, loop of Henle, distal tubule, principal cells, and intercalated cells (Physique 3, ACC). The absence of stromal or leukocyte populations presumably displays either dissociation bias and/or a cell frequency below our limit of detection. Open in a separate window Physique 3. Comparison of epithelia from single-cell RNA-sequencing of healthy adult kidney with transplant biopsy discloses activated and proinflammatory cell says. (A) Unsupervised clustering recognized six unique cell types in human adult kidney. These types.