High glucose enhanced the expression of genes that regulate cell survival and structure in ERoSHK.

<p><b>a</b> Scatter plot was created using Illumina BeadStudio, to display the 6813 genes expressed by both ERoSHK_LG and ERoSHK_HG and their average detection signal on logarithmic scales. Dashed lines represent 2.0-fold difference boundaries and grey dots represent the 185 genes up-regulated by ERoSHK_HG. <b>b</b> The 185 up-regulated genes were uploaded onto Ingenuity Pathway Analysis, and over-represented in 23 molecular and cellular functions with <i>P</i><0.05 (dashed line). The top 5 most significant functions are represented by black bars. <b>c</b> Expression levels of 10 randomly selected genes, 2 from each of the top 5 most significant functions, as determined by quantitative PCR. Transcript levels are first normalized to <i>Actb</i> endogenous control, and then to those of ERoSHK_LG. Data are presented as mean ± s.d.; n = 3.</p

HG inhibited population growth by increased apoptosis with no significant effect on rate of cell division.

<p><b>a</b> Cells were cultured in LG or HG conditions and total number of viable cells was quantified each day as determined by Guava Viacount assay. Data are normalized to the number of cells seeded on day 0, and represented as mean ± s.d.; n = 3. *, <i>P</i><0.001. <b>b</b> ERoSHK cells pretreated with LG and HG for 6 days were stained with CFSE and the rate of loss of fluorescence signal was monitored over 72 h by flow cytometry. The number of divisions per 24 h during the 72 h period after 6 days of LG and HG treatment is graphically represented (mean ± s.d.; n = 3). <b>c </b><i>Left panel</i>, ERoSHK_LG and ERoSHK_HG were stained with phycoerythrin-conjugated Annexin V and dye fluorescence was measured by flow cytometry. The gates on scatter plots of side-scatter (SSC) versus phycoerythrin fluorescence intensity represent Annexin V-positive cells. <i>Right panel</i>, percentages of Annexin V-positive cells were assessed daily over a period of 72 h and graphically represented. Data are presented as mean ± s.d.; n = 3. *, <i>P</i><0.001. <b>d </b><i>Upper panel</i>, ERoSHK_LG and ERoSHK_HG were fixed, stained with propidium iodide (PI) and dye fluorescence was measured by flow cytometry. Histogram plot of cell number versus PI fluorescence intensity was deconvoluted using ModFit LT software. Percentage of cells in each phase of the cell cycle is depicted above their respective peaks. <i>Lower panel</i>, the experiment was further repeated 24, 48 and 72 h post treatment and percentages of cells in the different cell cycle phases are graphically represented. Data are presented as mean ± s.d.; n = 3. *, <i>P</i><0.05; **, <i>P</i><0.005; ***, <i>P</i><0.001.</p

High glucose induced sustained ERK phosphorylation in ERoSHK.

<p><b>a </b><i>Left panel</i>, relative levels of phosphorylated ERK1/2 total ERK1/2 in ERoSHK_LG and ERoSHK_HG were analyzed by western blotting. <i>Right panel</i>, ERoSHK_LG and ERoSHK_HG were pre-incubated in KRBH buffer containing 0.1% BSA for 2, 4 and 8 h before being treated with or without a 10 mM glucose stimulation for 10 min. Relative levels of phosphorylated ERK1/2 and total ERK1/2 were then analyzed by western blotting. <b>b</b> 3 independent ERK phosphorylation assays were performed on ERoSHK_LG and ERoSHK_HG serum-starved for 2 h. The relative intensities of phosphorylated (P) and total ERK protein bands were quantified by densitometry and expressed as a ratio (P-ERK/ERK), normalized to that of unstimulated ERoSHK_LG cells. Data are presented as mean ± s.d.; n = 3. *, <i>P</i><0.05.</p

Inhibition of ERK phosphorylation in ERoSHK_HG restored cell-cell contact and colony morphology.

<p><b>a</b> ERoSHK cells were cultured in LG or HG in the absence and presence of 25 µM of PD98059 for 6 days. Cells were fixed, immunostained for E-cadherin (green) and counterstained with phalloidin (red) for F-actin and DAPI (blue) for nuclei. White arrowheads indicate sites of membrane separation. Parts of the merged image (box with white dashed lines) are magnified in the right panels to clearly visualize the co-localization of E-cadherin and cortical actin along the cell periphery, as well as gaps between adjacent cells. Scale bars, 10 µm. <b>b</b> Representative phase contrast images of ERoSHK_LG and ERoSHK_HG treated with or without PD98059 to show changes in colony morphology. Scale bars, 100 µm. <b>c</b> ERoSHK_LG and ERoSHK_HG treated with or without PD98059 were stained with phycoerythrin-conjugated Annexin V and dye fluorescence was measured by flow cytometry. Percentages of Annexin V-positive cells are assessed and graphically represented. Data are presented as mean ± s.d.; n = 3. <b>d</b> Insulin secretion of ERoSHK_LG and ERoSHK_HG treated with or without PD98059 in response to 2.8 mM (basal) and 16.7 mM (stimulated) glucose, expressed as a percentage of total insulin content. Data are presented as mean ± s.d.; n = 3. *, <i>P</i><0.05; **, <i>P</i><0.005; ***, <i>P</i><0.001.</p

Glomerulosclerosis was reduced by CM treatment.

<p>On PAS-stained sections, glomeurosclerosis was reduced after CM treatment (A). Normal (B), segmental (C) and totally sclerotic are shown (D). Black arrows indicate sclerotic areas. 2K-NCM (n = 6); 2K-CM (n = 6); CKD-NCM (n = 13); CKD-CM (n = 13). *P<0.05: CKD vs. 2K, †P<0.05: CKD-CM vs. CKD-NCM.</p

Inflammatory cytokine expression in healthy and CKD kidneys.

<p><b>A: Cytokine array in array.</b> Striped bars = 2K-CM (n = 6); dotted bars = 2K-NCM (n = 6); black bars = CKD-NCM (n = 7); white bars = CKD-CM (n = 4). IL-8 = interleukin 8; sICAM = soluble Inter-Cellular Adhesion Molecule; L-selectin = leucocyte cell-adhesion molecule; RANTES = Regulated upon Activation, Normal T-cell Expressed, and Secreted; MIG = Monokine Induced by Gamma-Interferon; MIP1α = Macrophage Inflammatory Protein-1 alpha; MIP3α = Macrophage Inflammatory Protein-3 alpha; Timp-1 = Tissue Inhibitor of Metalloproteinase 1; VEGF = Vascular Endothelial Growth Factor; IL-1RA = Interleukin 1 Receptor Antagonist. <b>B: Fractalkine mRNA in healthy and CKD kidneys.</b> Striped bars = 2K-CM (n = 6); dotted bars = 2K-NCM (n = 6); black bars = CKD-NCM (n = 7); white bars = CKD-CM (n = 4). *P<0.05: CKD vs. 2K.</p

Tubulo-interstitial damage on PAS-stained sections was reduced after CM treatment in CKD rats.

<p>CM treatment decreased tubular fibrosis and atrophy (A). Differences between CKD (B) and healthy (C) tubular kidney tissue are shown. White arrow indicates infitration, black arrows showing proteincasts. 2K-NCM (n = 6); 2K-CM (n = 6); CKD-NCM (n = 13); CKD-CM (n = 13). *P<0.05: CKD vs. 2K, †P<0.05: CKD-CM vs. CKD-NCM.</p

CM stimulates in vitro angiogenesis (A) and wound closure (B).

<p>Average tube length was increased after CM treatment compared to NCM (C+D). In a scratch wound assay, CM treatment increased wound (indicated by black arrow in E) closure compared to NCM treatment. *P<0.05 CM vs. NCM.</p

Terminal kidney function measurements.

<p>Mean±SD *P<0.05: 2K vs. CKD.</p><p>MAP = mean arterial pressure. RBF = renal blood flow. RVR = renal vascular resistance. FF = filtration fraction. FeNa = fractional excretion of sodium. FeK = fractional excretion of potassium.</p

CM treatment increased the number of glomerular endothelial cells (A, C-D), but did not increase tubular endothelial cell number (B, E-F) shown by a JG12 staining.

<p>2K-NCM (n = 6); 2K-CM (n = 6); CKD-NCM (n = 13); CKD-CM (n = 13). *P<0.05: CKD vs. 2K, †P<0.05: CKD-CM vs. CKD-NCM.</p