Renal Artery Stenosis Alters Gene Expression in Swine Scattered Tubular-Like Cells

Figures

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  Figure 1. Characterization of Scattered tubular-like cells (STCs) isolated from pig kidneys. (A) Representative immunofluorescence staining (original magnification 40X) for the STC surface markers CD133 (red) and CD24 (green) in isolated swine STCs. (B) Flow cytometry analysis of isolated STCs showing that 97% of cells expressed CD133, 95% CD24, and 98% were double-positive for CD133 and CD24 (n = 3 each).

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  Figure 2. mRNAseq analysis. (A) Correlation between messenger RNA sequencing data derived from scattered tubular-like cells (STCs), showing well-defined Normal and renal artery stenosis (RAS) groups. (B) Hierarchical clustering showing the pattern of expression of Normal- and RAS-STCs (n = 3 each). (C) Three-dimensional plots of the first three components of STCs showing complete separation between samples from Normal and RAS pigs (n = 3 each).

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  Figure 3. messenger RNA (mRNA) differentially expressed in renal artery stenosis (RAS)-scattered tubular-like cells (STCs). (A) Heat map and scatter plot showing 1,430 genes upregulated and 315 genes downregulated in RAS- versus Normal-STCs (n = 3 each). (B) Expression of selected candidate genes followed the same patterns as the mRNA sequencing findings, where Dachsous Cadherin-Related 1 (DCHS1), Olfactomedin 4 (OLFM4), and Pleckstrin Homology Like Domain Family A Member 3 (PHLDA3) were upregulated and Centromere Protein F (CENPF), Claspin (CLSPN), and RNA Binding Motif Protein 41 (RBM41) were downregulated in RAS-STCs (n = 3 each). * p < 0.05 vs. Normal-STCs.

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  Figure 4. Genes upregulated in renal artery stenosis (RAS)-scattered tubular-like cells (STCs). Protein Analysis Through Evolutionary Relationships (PANTHER) analysis of cellular component (A) and molecular function (B) of genes upregulated in RAS-STCs. (C) Database for Annotation, Visualization, and Integrated Discovery (DAVID) 6.8 functional annotation analysis of messenger RNAs upregulated in RAS-STCs.

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  Figure 5. Venn diagram analysis (Venny 2.1) indicated that 133 cell adhesion (A) 50 extracellular matrix remodeling (B) and 23 kidney development (C) genes were upregulated in renal artery stenosis (RAS)-scattered tubular-like cells (STCs). Search Tool for the Retrieval of Interacting Genes (STRING) was used to predict associations between these genes.

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  Figure 6. Genes downregulated in renal artery stenosis (RAS)-scattered tubular-like cells (STCs). Protein Analysis Through Evolutionary Relationships (PANTHER) analysis of cellular component (A) and molecular function (B) of genes downregulated in RAS-STCs. (C) Database for Annotation, Visualization, and Integrated Discovery (DAVID) 6.8 functional annotation analysis of messenger RNAs downregulated in RAS-STCs.

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  Figure 7. Venn diagram analysis (Venny 2.1) indicated that 85 cell cycle (A) and 73 cytoskeletal (B) genes were downregulated in RAS-STCs. Search Tool for the Retrieval of Interacting Genes (STRING) was used to predict associations between these genes.

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  Figure 8. Scattered tubular-like cells (STC) activation and proliferation. (A) Representative immunofluorescence staining (40X and 100X) for the STC surface markers CD133 (green) and CD24 (pink), and the tubular cell marker phytohemagglutinin (PHA)-E (red) showing CD133+/CD24+ cells in renal proximal tubules. (B) Flow cytometry analysis showing that the percentage of CD24+/CD133+ STCs from dissociated kidney cells was higher in RAS versus Normal pigs (n = 3 each). (C) Cell proliferation, measured in percent phase object confluence unit, was impaired in RAS-STCs (Repeated-measures analysis of variance (ANOVA), n = 3 each). * p < 0.05 vs. Normal-STCs.