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Kidney Week

Abstract: SA-PO996

Impact of Uremia on Regulatory T Lymphocytes Proliferation and Phenotype

Session Information

  • CKD: Pathobiology - II
    November 05, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

  • 2203 CKD (Non-Dialysis): Mechanisms

Authors

  • Valentini, Nicolas, Hopital Maisonneuve-Rosemont Centre de Recherche, Montreal, Quebec, Canada
  • Raymond, Maxime, Hopital Maisonneuve-Rosemont Centre de Recherche, Montreal, Quebec, Canada
  • Lamarche, Caroline, Hopital Maisonneuve-Rosemont Centre de Recherche, Montreal, Quebec, Canada
Background

Chronic kidney disease (CKD) patients have a dysfunctional immune system that is chronically and non-specifically activated, leading to low grade inflammation. Inflammation is now considered both a risk factor and a consequence of reduced kidney function and is highly associated with cardiovascular disease, which is the leading cause of mortality on dialysis.
Regulatory T cells (Tregs) are important inhibitors of proinflammatory responses. While Tregs numbers are decreased in patients with CKD, their state and effectiveness remain poorly understood. We aim to investigate the impact of uremia on those cells.

Methods

Tregs and conventional CD4+ cells from hemodialysis (HD) patients and healthy donors were fluorescence-activated cell sorting (FACS)-sorted and serum was collected. We analyzed the Treg transcriptome using single cell RNA sequencing (scRNA-seq). Tregs and conventional CD4+ T cells from healthy donors were also expanded in vitro with media (IL-2 500U/mL) containing healthy donors’ or HD patients’ serum. After 7 and 12 days in culture, we used flow cytometry to compare changes in phenotype, viability, apoptosis and assess their function.

Results

We identified 11 Treg clusters from the scRNA seq data. From those, one is more present in HD patients, irrespective of donor’s sex (FDR <0.05 & abs(Log2FD)>0.26) and 3 less present in HD patients. In vitro cultures show decreased Tregs number in uremic serum after 7 days compared to healthy donors (fold expansion 11.35 vs 19.27; p<0.001) and no significant impact on conventional CD4+ T cells (fold expansion 17.88 vs 19.52; p=0.34). They were no significant differences in Treg expansion or function, as assessed by a suppression assay, after 12 days (p>0.99). Staining for apoptosis with Annexin V/PI indicate no difference in viability or apoptosis between serums after 7 and 12 days. Markers associated with Tregs functions and activation such as CTLA4, GARP, LAP, CD69 and CD71 were expressed similarly in HD and healthy donors’ serum after 7 days (p=0.82, 0.77, 0.73, 0.23 and 0.44 respectively).

Conclusion

Serum from HD patients selectively delays Tregs proliferation in vitro and not conventional CD4+ T cells, with no impact on cell death, activation and Tregs markers’ expression. HD patients also have different Treg cluster composition. Further studies are needed to identify the causes and impacts of those changes.