Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Observational Study
. 2022 Mar 17;17(3):e0265393.
doi: 10.1371/journal.pone.0265393. eCollection 2022.

Peripheral blood mononuclear cell gene expression and cytokine profiling in patients with intermittent claudication who exhibit exercise induced acute renal injury

Pasha Normahani  1   2 , Joseph J Boyle  3 , Luke Cave  3 , Paul Brookes  4 , Kevin J Woollard  5 , Usman Jaffer  1   2
Affiliations
Observational Study

Peripheral blood mononuclear cell gene expression and cytokine profiling in patients with intermittent claudication who exhibit exercise induced acute renal injury

Pasha Normahani et al. PLoS One. .

Abstract

Background: Intermittent claudication (IC) is a common manifestation of peripheral arterial disease. Some patients with IC experience a rise in Urinary N-acetyl-β-D-Glucosaminidase (NAG)/ Creatinine (Cr) ratio, a marker of renal injury, following exercise. In this study, we aim to investigate whether peripheral blood mononuclear cells (PBMC) from patients with IC who exhibit a rise in urinary NAG/ Cr ratio following exercise exhibit differential IL-10/ IL-12 ratio and gene expression compared to those who do not have a rise in NAG/ Cr ratio.

Methods: We conducted a single center observational cohort study of patients diagnosed with IC. Blood and urine samples were collected at rest and following a standardised treadmill exercise protocol. For comparative analysis patients were separated into those with any rise in NAG/Cr ratio (Group 1) and those with no rise in NAG/Cr ratio (Group 2) post exercise. Isolated PBMC from pre- and post-exercise blood samples were analysed using flow cytometry. PBMC were also cultured for 20 hours to perform further analysis of IL-10 and IL-12 cytokine levels. RNA-sequencing analysis was performed to identify differentially expressed genes between the groups.

Results: 20 patients were recruited (Group 1, n = 8; Group 2, n = 12). We observed a significantly higher IL-10/IL-12 ratio in cell supernatant from participants in Group 1, as compared to Group 2, on exercise at 20 hours incubation; 47.24 (IQR 9.70-65.83) vs 6.13 (4.88-12.24), p = 0.04. 328 genes were significantly differentially expressed between Group 1 and 2. The modulated genes had signatures encompassing hypoxia, metabolic adaptation to starvation, inflammatory activation, renal protection, and oxidative stress.

Discussion: Our results suggest that some patients with IC have an altered immune status making them 'vulnerable' to systemic inflammation and renal injury following exercise. We have identified a panel of genes which are differentially expressed in this group of patients.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Schematic of workflow within Partek.
Blue boxes, processes as indicated. Blue circles, data types as indicated.
Fig 2
Fig 2. Representative plots from flow cytometric analysis.
A) CD3+/16-/56- cells (gate E), CD16+/56+/3- cells (gate G); B) CD45+/14+ cells (gate I); C) CD14+/64+ cells (gate L); D) CD19+/3- cells (gate J). We gated for the whole population from the Lymphoprep isolation; forward and side scatter plots is available in the S1 Fig.
Fig 3
Fig 3. Percentage composition of PBMC in claudicants pre- and post-exercise separated by those who experiences a rise in urinary NAG/ Creatinine ratio (Group 1) vs. no rise on exercise (Group 2).
Fig 4
Fig 4
A) IL-10 (pcg/ ml), * p = 0.045 B) IL-12 (pcg/ ml), C) IL-10/IL-12 ratio from cell medium supernatant of PBMC cultured from patients with (Group 1) and without (Group 2) rise in urinary NAG/Creatinine ratio following exercise, * p = 0.04. Statistical comparisons made using Mann Whitney U test.
Fig 5
Fig 5
A) hierarchical cluster analysis. Upregulated genes (red) and downregulated genes (green) as indicated. There is a clear separation between the groups with (POS) and without (NEG) urinary NAG/Creatinine rise following exercise. The group with a rise urinary NAG/ Creatinine has a cluster of upregulated genes to the right-hand side of the diagram. B) principal component analysis. There is a clear separation between the groups with (blue, POS) and without (red, NEG) urinary NAG/ Creatinine rise following exercise. The groupings are shown with the superimposed polygons.
Fig 6
Fig 6. List of top 24 genes by fold up regulation.
Fig 7
Fig 7
A) Y-axis, important GO categories of Molecular Function; X-axis, probability expressed as negative log of the false discovery rate (FDR). B) Y-axis, important GO categories of Cellular Function; X-axis, expressed as negative log of the false discovery rate (FDR).
Fig 8
Fig 8
A) overview of gene network. Blue, nodes in the network. Grey lines, known connections between nodes. Some nodes are very highly connected. B) alternate overview of gene network, redrawn to maximally display connections and the most highly connected nodes. Blue, nodes in the network. Grey lines, known connections between nodes. Some nodes are very highly connected. C) graph of node connections. X-axis, number of connections per node; Y-axis, number of nodes.
Fig 9
Fig 9. STRING network summary.
The summary data are shown at the top and summarise the graphs in Fig 7.
Fig 10
Fig 10
A) list of predicted Transcription Factor binding sites (PASTAA) ranked by P-value (generated using the Max Planck Institute for Molecular Genetics Database), B) X-axis, score calculated as an aggregate of rankings of common transcription factor binding sites prediction programs (OPPOSUM using JASPAR, JASPAR CORE, X2K using databases ENCODE, ChEA2015, JASPAR and TRANSFAC, ChEA2016, archs4, ENRICHR, ChEA3, PASTAA).
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Song P, Rudan D, Zhu Y, Fowkes FJI, Rahimi K, Fowkes FGR, et al.. Global, regional, and national prevalence and risk factors for peripheral artery disease in 2015: an updated systematic review and analysis. Lancet Glob Heal. 2019. Aug;7(8):e1020–30. - PubMed
    1. Robless P, Mikhailidis DP, Stansby GP. Cilostazol for peripheral arterial disease. Cochrane database Syst Rev. 2007. Jan;(1):CD003748. doi: 10.1002/14651858.CD003748.pub2 - DOI - PubMed
    1. Brevetti G, Silvestro A, Di Giacomo S, Bucur R, Di Donato A, Schiano V, et al.. Endothelial dysfunction in peripheral arterial disease is related to increase in plasma markers of inflammation and severity of peripheral circulatory impairment but not to classic risk factors and atherosclerotic burden. J Vasc Surg. 2003. Aug;38(2):374–9. doi: 10.1016/s0741-5214(03)00124-1 - DOI - PubMed
    1. Gokce N, Keaney JF, Hunter LM, Watkins MT, Nedeljkovic ZS, Menzoian JO, et al.. Predictive value of noninvasively determined endothelial dysfunction for long-term cardiovascular events in patients with peripheral vascular disease. J Am Coll Cardiol. 2003. May;41(10):1769–75. doi: 10.1016/s0735-1097(03)00333-4 - DOI - PubMed
    1. Silvestro A, Scopacasa F, Ruocco A, Oliva G, Schiano V, Zincarelli C, et al.. Inflammatory status and endothelial function in asymptomatic and symptomatic peripheral arterial disease. Vasc Med. 2003. Nov;8(4):225–32. doi: 10.1191/1358863x03vm503oa - DOI - PubMed

Publication types

-