A Cluster of Proteins Implicated in Kidney Disease Is Increased in High-Density Lipoprotein Isolated from Hemodialysis Subjects

Cardiovascular disease is the leading cause of death in end-stage renal disease (ESRD) patients treated with hemodialysis. An important contributor might be a decline in the cardioprotective effects of high-density lipoprotein (HDL). One important factor affecting HDL’s cardioprotective properties may involve the alterations of protein composition in HDL. In the current study, we used complementary proteomics approaches to detect and quantify relative levels of proteins in HDL isolated from control and ESRD subjects. Shotgun proteomics analysis of HDL isolated from 20 control and 40 ESRD subjects identified 63 proteins in HDL. Targeted quantitative proteomics by isotope-dilution selective reaction monitoring revealed that 22 proteins were significantly enriched and 6 proteins were significantly decreased in ESRD patients. Strikingly, six proteins implicated in renal disease, including B2M, CST3, and PTGDS, were markedly increased in HDL of uremic subjects. Moreover, several of these proteins (SAA1, apoC-III, PON1, etc.) have been associated with atherosclerosis. Our observations indicate that the HDL proteome is extensively remodeled in uremic subjects. Alterations of the protein cargo of HDL might impact HDL’s proposed cardioprotective properties. Quantifying proteins in HDL may be useful in the assessment of cardiovascular risk in patients with ESRD and in assessing response to therapeutic interventions

A Cluster of Proteins Implicated in Kidney Disease Is Increased in High-Density Lipoprotein Isolated from Hemodialysis Subjects

Cardiovascular disease is the leading cause of death in end-stage renal disease (ESRD) patients treated with hemodialysis. An important contributor might be a decline in the cardioprotective effects of high-density lipoprotein (HDL). One important factor affecting HDL’s cardioprotective properties may involve the alterations of protein composition in HDL. In the current study, we used complementary proteomics approaches to detect and quantify relative levels of proteins in HDL isolated from control and ESRD subjects. Shotgun proteomics analysis of HDL isolated from 20 control and 40 ESRD subjects identified 63 proteins in HDL. Targeted quantitative proteomics by isotope-dilution selective reaction monitoring revealed that 22 proteins were significantly enriched and 6 proteins were significantly decreased in ESRD patients. Strikingly, six proteins implicated in renal disease, including B2M, CST3, and PTGDS, were markedly increased in HDL of uremic subjects. Moreover, several of these proteins (SAA1, apoC-III, PON1, etc.) have been associated with atherosclerosis. Our observations indicate that the HDL proteome is extensively remodeled in uremic subjects. Alterations of the protein cargo of HDL might impact HDL’s proposed cardioprotective properties. Quantifying proteins in HDL may be useful in the assessment of cardiovascular risk in patients with ESRD and in assessing response to therapeutic interventions

Conservation of Apolipoprotein A‑I’s Central Domain Structural Elements upon Lipid Association on Different High-Density Lipoprotein Subclasses

The antiatherogenic properties of apolipoprotein A-I (apoA-I) are derived, in part, from lipidation-state-dependent structural elements that manifest at different stages of apoA-I’s progression from lipid-free protein to spherical high-density lipoprotein (HDL). Previously, we reported the structure of apoA-I’s N-terminus on reconstituted HDLs (rHDLs) of different sizes. We have now investigated at the single-residue level the conformational adaptations of three regions in the central domain of apoA-I (residues 119–124, 139–144, and 164–170) upon apoA-I lipid binding and HDL formation. An important function associated with these residues of apoA-I is the activation of lecithin:cholesterol acyltransferase (LCAT), the enzyme responsible for catalyzing HDL maturation. Structural examination was performed by site-directed tryptophan fluorescence and spin-label electron paramagnetic resonance spectroscopies for both the lipid-free protein and rHDL particles 7.8, 8.4, and 9.6 nm in diameter. The two methods provide complementary information about residue side chain mobility and molecular accessibility, as well as the polarity of the local environment at the targeted positions. The modulation of these biophysical parameters yielded new insight into the importance of structural elements in the central domain of apoA-I. In particular, we determined that the loosely lipid-associated structure of residues 134–145 is conserved in all rHDL particles. Truncation of this region completely abolished LCAT activation but did not significantly affect rHDL size, reaffirming the important role of this structural element in HDL function

Plasma membrane protein signatures of myeloid cells identify unique cell functions.

<p>Gene ontology analysis of plasma membrane proteins enriched in M1 macrophages, M2 macrophages, all macrophage types (BmM, M1, and M2), and BmDCs identifies functional categories of proteins enriched in each cell type (<i>p</i><0.05 with Benjamini-Hochberg correction). The top three functional annotations are presented for each cell type along with three representative proteins.</p

Mass spectrometric and immunohistochemical staining of thioglycolate-elicited peritoneal cells (eMPCs), polarized macrophages, and DCs.

<p><b><i>Panels A–B:</i></b> Hierarchical cluster analysis of eMPCs. Cells were harvested from the peritoneal cavity of C57BL/6J-<i>Ldlr^tm1Her</i> mice 5 days after intraperitoneal injection with thioglycolate. Isolated plasma membrane proteins detected by LC-MS/MS analysis of eMPCs were subjected to hierarchical cluster analysis, using the 107 proteins identified as differentially expressed by myeloid cells generated <i>in vitro</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033297#pone-0033297-g002" target="_blank"><b>Fig. 2</b></a>). <b><i>Panel B:</i></b> Relationships among eMPCs, M1 cells, M2 cells, BmMs, and DCs, as determined by cluster analysis. <b><i>Panel C:</i></b> Protein expression in eMPCs, M1 cells, M2 cells, BmMs, and BmDCs. Protein levels were quantified by MS/MS and spectral counting. Data are presented as means and SDs. <b><i>Panels D–E:</i></b> Flow cytometric analysis of CD11b, CD11c, and F4/80 in eMPCs. Results are presented as contour plots with 10% probability increments. <b><i>Panel F:</i></b> qRT-PCR analysis of M1 marker genes (<i>Nos2</i>, <i>Il12b</i>, <i>Tnfa</i>) in M1 macrophages, BmDCs, and eMPCs. Results (means and SEMs; N = 6) were standardized to 18S levels and expressed relative to M1 macrophages. <b><i>Panel G:</i></b> Immunostaining of eMPCs. Cells were stained with antibodies (red channel) to plasma membrane proteins differentially expressed by BmDCs (MBC2, FER1L), BmMs (ITGA6, STAB1), M2 cells (TFRC, ITGB5), and M1 cells (CD11a, CD40). Nuclei were visualized by DAPI staining (blue channel). Immunostaining and microscopy were performed on the same day and with identical microscope settings to experiments presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033297#pone-0033297-g003" target="_blank"><b>Fig. 3D</b></a> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033297#pone.0033297.s003" target="_blank">Fig. S3</a></b>. Results obtained for flow cytometry, qRT-PCR, and immunocotyochemistry are representative of 3 independent analyses.</p

The plasma membrane proteome of bone marrow-derived dendritic cells (DCs).

<p><b><i>Panel A:</i></b> Bone marrow-derived dendritic cells (BmDCs) were obtained by culturing bone marrow cells with GM-CSF. <b><i>Panel B:</i></b> Flow cytometric analysis of CD11c and F4/80 expression in BmDCs and BmMs. Results are presented as a contour plot with 10% probability increments. <b><i>Panel C:</i></b> Cell-surface exp ression of MHC-II by BmDCs and BmMs as assessed by flow cytometry. <b><i>Panel D:</i></b> The plasma membrane proteome of DCs. <i>Upper Panel:</i> Proteins expressed at similar levels by DCs and either M1 cells, M2 cells, or BmMs. <i>Lower Panel:</i> Proteins differentially expressed by DCs relative to M1 cells, M2 cells, and BmMs (<i>G-</i>test>1.5 or <−1.5 and <i>t-</i>test: <i>p</i><0.05). Red, upregulated; green, downregulated. <b><i>Panel E:</i></b> Examples of proteins expressed at similar levels by DCs and either M1 cells, or M2 cells. Results (N = 6 per group) are means and SDs. <b><i>Panel F:</i></b> Examples of plasma membrane proteins differentially expressed by DCs. Flow cytometry experiments are representative of 3 independent analyses.</p

The plasma membrane proteome of macrophages.

<p><b><i>Panel A:</i></b> Bone marrow-derived macrophages (BmM) were derived from bone marrow precursor cells of C57BL/6 mice cultured with M-CSF. Classically activated macrophages (M1) and alternatively activated macrophages (M2) were derived from BmMs by treatment with IFN-γ and LPS or with IL-4. <b><i>Panel B:</i></b> qRT-PCR of markers used to detect M1 and M2 macrophages. Results (means and SEMs, N = 6) were standardized to 18S, expressed relative to the cell type with the highest expression of each gene, and are representative of 3 independent analyses. <b><i>Panel C:</i></b> LC-ESI-MS/MS analysis of plasma membrane proteins isolated from differentially activated macrophages. Proteins were quantified by spectral counting (total number of peptides identified for a given protein) and subjected to sequential criteria to identify 192 plasma membrane proteins that were reproducibly detected with high confidence. <b><i>Panel D:</i></b> Quantification of the membrane proteomes of M1 and M2 macrophages. Differentially expressed proteins (red, upregulated; green, downregulated; gray, not significantly different) were identified based on <i>t-</i>test and <i>G-</i>test statistics. Significance cutoffs (dashed lines; <i>p</i><0.05 and <i>G-</i>statistic >1.5 or <−1.5) were determined based on permutation analysis (estimated FDR<5%). <b><i>Panel E:</i></b> Quantification of the membrane proteomes of M1 macrophages and BmMs. Proteins differentially expressed by M1 cells relative to both BmMs and M2 cells are indicated with colored dots (red, upregulated; green, downregulated). Proteins differentially expressed by M1 and M2 cells (<i>Panel D</i>) but not differentially expressed by M1 and BmMs are indicated by gray dots. <b><i>Panel F:</i></b> Examples of proteins that distinguish M1 cells from both BmM and M2 cells (CSF1R, ITGAL). Results (N = 6 per group) are means and SDs. <b><i>Panel G:</i></b> Examples of proteins that fail to distinguish M1 cells from both BmM and M2 cells (CD14, ITGAV). <b><i>Panel H:</i></b> Plasma membrane proteins differentially expressed by M1 cells (36 proteins), M2 cells (35 proteins), and BmMs (17 proteins).</p

Immunocytochemical detection of plasma membrane protein markers.

<p>Expression levels of widely used plasma membrane protein markers (<b><i>Panels A–B</i></b>) and newly identified markers (<b><i>Panels C–D</i></b>) of M1 cells, M2 cells, BmMs, and BmDCs were assessed by mass spectrometry (<b><i>Panels A,C</i></b>) and immunocytochemistry (<b><i>Panels B,D</i></b>). For MS/MS, proteins were quantified by spectral counting and expressed relative to the cell type with the highest expression level for each protein. Results are means and SDs. Cells were stained with antibodies specific to each protein (red channel), counterstained with DAPI to visualize nuclei (blue-channel), and examined by confocal microscopy. Immunostaining and microscopy were performed on the same day with identical microscope settings. Results are representative of 3 independent analyses.</p

Analysis of eMPCs harvested from wild-type and GM-CSF-deficient (<i>Csf2−/−</i>) mice.

<p>eMPCs isolated from wild-type (<i>wt</i>) and <i>Csf2</i>−/− mice were interrogated for cell number, function, and protein expression. <b><i>Panel A–B:</i></b> Accumulation of eMPCs 3 days (<i>Panel A</i>) and 5 days (<i>Panel B</i>) following intraperitoneal injection with thioglycolate. Results (N = 6) are means and SEMs. <b><i>Panel C:</i></b> Plasma membrane proteomic analysis of eMPCs isolated from <i>Csf2</i>−/− and wild-type mice. Differentially-expressed proteins were identified using the <i>t-</i>test and <i>G-</i>test (<i>p</i><0.05 and <i>G-</i>statistic >1.5) and quantified using the spectral index. <b><i>Panel D:</i></b> Proteins differentially expressed by eMPCs isolated from <i>Csf2</i>−/− mice (see <i>Panel C</i>) were measured in BmMs and BmDCs and quantified using the spectral index. <b><i>Panel E:</i></b> Cell surface CD11c and F4/80 expression on eMPCs was assessed by flow cytometry. Results are presented as contour plots with 10% probability increments. <b><i>Panel F:</i></b> Phagocytosis of fluorescein-labeled <i>E. coli</i> by eMPCs. Results (arbitrary units, AU; N = 4) are means and SEMs. <b><i>Panel G–H:</i></b> Antigen cross-presentation by eMPCs. Ovalbumin (0.2 mg/mL)-treated eMPCs were incubated with CFSE-labeled spleen cells isolated from OT-I transgenic mice. Levels of CFSE were assessed in OT-I T cells selected by flow cytometry and expression levels of CD8 and Vb5 (<i>Panel G</i>). The division index was calculated using FlowJo software. Results (N = 4) are means and SEMs (<i>Panel H</i>). Where applicable, <i>p</i>-values were derived using a two-tailed Student's <i>t-</i>test. Results obtained for eMPC quantification, flow cytometry, phagocytosis and antigen cross-presentation are representative of 3 independent analyses.</p

The TLR-2/TLR-4/MyD88 pathway is dispensable in macrophage activation in kidney fibrosis, but important in mesenchyme cell activation.

<p>(<b>A–C</b>) <i>Csf1R-icre; Myd88^fl/fl</i> mice or respective controls were subjected to U-IRI and kidney harvested for tissue analysis 5 days later. Q-PCR (A) for different inflammatory transcripts, (<b>B</b>) pro-fibrotic transcripts, collagen1a1 (<i>col1a1</i>) and alpha smooth muscle actin (<i>Acta2</i>), and the tubule injury marker, kidney injury molecule-1 (<i>Kim-1</i>) from whole kidney day 5 after U-IRI. (<b>C</b>) Representative fluorescent images (left) and quantitative graphs (right) showing+αSMA (red) cells and+F4/80 cells (green). (<b>D–E</b>) Primary pericytes were isolated from <i>Myd88^−/−</i> and <i>Tlr2–4^−/−</i> mice and stimulated <i>in vitro</i> for 8h with kidney DAMPs. (<b>D</b>) Graph showing <i>Il-6</i> and <i>Col1a1</i> transcript expression by Q-PCR. (<b>E</b>) Graph showing IL-6 and MCP-1 concentration in supernatant by ELISA. (*P<0.05, n = 5–7/group, 3 independent experiments; ns, p is not significant; Bar = 50 µm; Q-PCR results were normalized to wild type control).</p