Perirenal Adipose Tissue Displays an Age-Dependent Inflammatory Signature Associated With Early Graft Dysfunction of Marginal Kidney Transplants

Abstract

Background: Better understanding of the contribution of donor aging and comorbidity factors of expanded criteria donors (ECD) to the clinical outcome of a transplant is a challenge in kidney transplantation. We investigated whether the features of donor-derived stromal vascular fraction of perirenal adipose tissue (PRAT-SVF) could be indicative of the deleterious impact of the ECD microenvironment on a renal transplant. Methods: A comparative analysis of cellular components, transcriptomic and vasculogenic profiles was performed in PRAT-SVF obtained from 22 optimal donors and 31 ECD deceased donors. We then investigated whether these parameters could be associated with donor aging and early allograft dysfunction. Results: When compared with the PRAT-SVF of non-ECD donors, ECD PRAT-SVF displayed a lower proportion of stromal cells, a higher proportion of inflammatory NK cells. The global RNA sequencing approach indicated a differential molecular signature in the PRAT-SVF of ECD donors characterized by the over-expression of CXCL1 and IL1-β inflammatory transcripts. The vasculogenic activity of PRAT-SVF was highly variable but was not significantly affected in marginal donors. Periorgan recruitment of monocytes/macrophages and NK cells in PRAT-SVF was associated with donor aging. The presence of NK cell infiltrates was associated with lower PRAT-SVF angiogenic activity and with early allograft dysfunction evaluated on day 7 and at 1 month post-transplant. Conclusions: Our results indicate that human NK cell subsets are differentially recruited in the periorgan environment of aging kidney transplants. We provide novel evidence that PRAT-SVF represents a non-invasive and timely source of donor material with potential value to assess inflammatory features that impact organ quality and function.

Keywords: endothelial inflammation; kidney allograft dysfunction; kidney transplantation; marginal kidney donors; natural killer cells; perirenal adipose tissue.

Figure 1

The distribution of cell subsets composing the PRAT-SVF was determined using flow cytometry and compared between the non-ECD and ECD donors: (A) CD45⁺ leukocytes were comparable in the two groups (B) mesenchymal stem/stromal cells were significantly lowered in the ECD vs. non-ECD group (C) pericytes and transitional cells (D) and endothelial cells were not statistically different between the two groups. ECD, extended criteria donors. Non ECD, non-extended criteria donors. Results on the graphs are reported as box and whiskers plots representative of median values, and 25–75 interquartile ranges (Boxes) and error bars indicative of 10-90 percentile ranges. Dots indicates values out of the 10–90% quartile range.

Figure 2

(A) Representative experiment of capillary tube formation by SVF from ECD or non-ECD. A total of 20,000 cells/well were seeded on growth factor reduced Matrigel. Images were recorded at 72 h with a phase-contrast microscope. Original magnification x5; upper panel (scale bars, 300 μm) correspond to the total image of the well while the lower panels were zooms of previous images (scale bars, 200 μm). White arrows identify initial cell clustering; red arrows marked the tip cells while yellow arrows identified branching. (B) Quantitative analysis of number of clusters, percentage of clusters with tip and stalk cells, number of branching points. Data are expressed as means ± SEM of independent experiments performed in triplicate using PRAT-SVF obtained in 14 ECD and 10 non-ECD donors (C) Representative experiment of 3D in vitro angiogenic assay with collagen gel-embedded spheroids of SVF from ECD or non-ECD (original magnification x20; scale bars, 100 μm). Imaging of Vascular sprouts was obtained after merging of actin staining (phalloidin in green) and nuclei staining (DAPI, blue) as detailed in Supplementary Figure 2 (D) Quantitative analysis of number of sprouts, branch points, and total network length per spheroid as well as average sprout length was compared in PRAT-SVF from the ECD and non ECD donors. For each experiment, at least 10 spheroids were analyzed.

Figure 3

Comparative Transcriptomic analysis of SVF from ECD (n = 5) and non-ECD (n = 5) patients. (A) Volcano plot of differentially expressed genes from SVF from ECD and non-ECD. Log2 Fold Change value obtained by RNAseq plotted against the –log10 of P-value. Genes with a fold change > |1.5| and P < 0.05 were deemed to be differentially expressed. P = 0.05 is indicated by horizontal lines. Positive and negative fold change values are reflective of down-regulated (green) or up-regulated (red) genes compared with non-ECD condition, respectively. (B) Fold enrichment over chance for the Gene Ontology Biological process of the Down (gray) and Up (black) gene lists using DAVID (fold change > |1.5| and P < 0.01). (C) Fold enrichment over chance for the KEGG Pathway of the Down (gray) and Up (black) gene lists using DAVID (fold change > |1.5| and P < 0.01). (D) qRT-PCR validation of selected genes expressed a relative levels of specific transcripts detected per 10⁶ GAPDH transcripts [median (25–75% quartile)].

Figure 4

The distribution of CD45⁺ hematopoietic cells composing the PRAT-SVF of ECD and non-ECD donors was analyzed using flow cytometry according to the gating strategy illustrated in Supplementary Figure 1. Results on the graphs are reported as box and whiskers plots representative of median values, and 25–75 interquartile ranges (Boxes) and error bars indicative of 10–90 percentile ranges. (A) CD14+ Monocyte/Macrophage subset. (B) CD14-neutrophil subset. (C) CD3+ T lymphocytes. (D) CD3−CD56+ NK cells. (E) Histograms illustrate the gating and % of CD3+ T cells and CD3-CD56+ NK Cells observed when analyzing PRAT-SVF of a non-ECD donor (0.43% of NK Cells) or (F) PRAT-SVF from an ECD donor (9.47% of NK cells).

Figure 5

Analysis of perirenal adipose tissue stromal vascular fraction (PRAT-SVF) in young vs. aged donors. PRAT-SVF was analyzed according to donor age when stratified in aging donors (≥59 years, n = 29) and younger donors (<59 years, n = 24). Percentages in stromal and endothelial cells were not different between the aging and younger donors. However, the aging donors presented a trend for increased representation of the CD45+ CD14+ monocyte macrophage cell subset and a significantly higher percentage of T and NK cells.

Figure 6

Analysis of perirenal adipose tissue stromal vascular fraction (PRAT-SVF) with lower recovery of graft persisting at M1 post-transplant. Donors were split into two groups according to graft recovery persisting at M1 post-transplant: lower recovery (eGFR M1 < 45) and normal recovery (eGFR > 45). The proportion of cell subsets was analyzed according to this splitting. The eGFR M1 < 45 presented a significantly higher percentage of monocyte/macrophage subset and NK cells. Other cells were similarly distributed between the two groups.