Modifiable Risk Factors for Increased Arterial Stiffness in Outpatient Nephrology

<div><p>Arterial stiffness, as measured by pulse wave velocity (PWV), is an independent predictor of cardiovascular events and mortality. Arterial stiffness increases with age. However, modifiable risk factors such as smoking, BP and salt intake also impact on PWV. The finding of modifiable risk factors may lead to the identification of treatable factors, and, thus, is of interest to practicing nephrologist. We have now studied the prevalence and correlates of arterial stiffness, assessed by PWV, in 191 patients from nephrology outpatient clinics in order to identify modifiable risk factors for arterial stiffness that may in the future guide therapeutic decision-making. PWV was above normal levels for age in 85/191 (44.5%) patients. Multivariate analysis showed that advanced age, systolic BP, diabetes mellitus, serum uric acid and calcium polystyrene sulfonate therapy or calcium-containing medication were independent predictors of PWV. A new parameter, Delta above upper limit of normal PWV (Delta PWV) was defined to decrease the weight of age on PWV values. Delta PWV was calculated as (measured PWV) - (upper limit of the age-adjusted PWV values for the general population). Mean±SD Delta PWV was 0.76±1.60 m/sec. In multivariate analysis, systolic blood pressure, active smoking and calcium polystyrene sulfonate therapy remained independent predictors of higher delta PWV, while age, urinary potassium and beta blocker therapy were independent predictors of lower delta PWV. In conclusion, arterial stiffness was frequent in nephrology outpatients. Systolic blood pressure, smoking, serum uric acid, calcium-containing medications, potassium metabolism and non-use of beta blockers are modifiable factors associated with increased arterial stiffness in Nephrology outpatients.</p></div

Correlations between quantitative variables and delta PWV that are significant in the univariate analysis and remained significant in the multivariate analysis.

<p>Correlations between quantitative variables and delta PWV that are significant in the univariate analysis and remained significant in the multivariate analysis.</p

Correlations between mean PWV (m/sec) and quantitative variables in univariate analysis.

<p>Only statistically significant (p <0.05) results are shown. There was a trend towards a significant correlation for serum 25(OH)D (coefficient -0.1347, p = 0.0854), serum free T3 (-0.1703, p = 0.0500) and serum potassium (0.1425, p = 0.0504).</p><p>Correlations between mean PWV (m/sec) and quantitative variables in univariate analysis.</p

Correlations between quantitative variables and PWV that are significant in the univariate analysis and remained significant in the multivariate analysis.

<p>Correlations between quantitative variables and PWV that are significant in the univariate analysis and remained significant in the multivariate analysis.</p

Proximal tubular cell exosomes contain OPG.

<p><b>A)</b> Exosomes from HK-2 conditioned serum-free cell culture media. Representative Western blots of TRAIL, TWEAK and exosome markers. Each lane contains 5 µg exosomal protein. <b>B)</b> OPG is observed in HK-2-derived exosomes when reducing, denaturizing conditions are applied. Each lane contains 10 µg exosomal protein. <b>C)</b> OPG expression in HK-2-derived exosomes detected by ELISA. Results expressed as pg/µg of total protein. Mean+SEM of 3 independent experiments. <b>D)</b> OPG analysis by selected reaction monitoring (SRM) in a LC-(QQQ)-MS/MS showing two different transitions corresponding to the same precursor peptide which coelute in time. The mass and charge of the precursor and its fragments are shown. A single peptide (<u>YLHYDEETSHQLL</u>) and a single precursor were measured under two different charge state (1025.9624+2 and 684.3107+3), each of them with its own fragment (1230.5783+ and 1138.4687+, respectively), thus yielding two different peaks or transitions.</p

OPG immunohistochemistry in human kidneys.

<p>Control, CKD and autosomal dominant polycystic kidney disease kidneys samples were studied. For ADPKD a cortical cyst wall is shown. <b>A)</b> OPG mainly stained the basolateral aspect of proximal tubules in control kidneys (arrowheads). <b>B)</b> In CKD whole tubular cells are intensely stained in the cortex (arrows). <b>C)</b> A similar pattern of intense whole cell staining is observed in cyst epithelial lining (arrows). Original magnification ×200.</p

Clinical characteristics.

<p>Summary data expressed as mean±SD.</p><p>sCr: serum creatinine, eGFR: estimated glomerular filtration rate, UProt: urinary protein.</p

Relationships between OPG and proteins identified in tubular cell-derived exosomal-like vesicles by LC-MS/MS as evidenced by the STRING database.

<p>STRING map of predicted associations for the sub-set of proteins compiled in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072387#pone-0072387-t002" target="_blank">Table 2</a>. The network nodes are proteins and the edges represent the predicted functional associations based on evidence which are represented by different color lines: green line, neighborhood in the genome; blue line, co-occurrence across genomes; purple line, experimental evidence; yellow line, text mining evidence; light blue line, database evidence; black line, co-expression evidence. The highest confidence filter was applied. NID1: Nidogen 1; COL4A1/2: Colagen 4 A1/2; THBS1: Thrombospondin-1; FBLN1: Fibulin 1, FN1: Fibronectin, TNFRSF11B: Osteoprotegerin; LGALS3BP: Galectin-3 Binding Protein, TGFBI: TGF-β-induced protein ig-h3, GC: Vitamin D-binding protein; C3: Complement C3.</p

Exosomes from human urine contain OPG.

<p><b>A)</b> Transmission electron microscopy representative images showing microvesicles purified from human urine. i) Scale bar = 200 nm; ii) Scale bar = 50 nm; iii) Scale bar = 200 nm; iv)Scale bar = 50 nm. ii&iv) show amplified areas from i&iii). <b>B)</b> Western blot. Representative images. Urine: whole urine collected from healthy donors. Supernat.: Urine supernatant from the last exosome isolation step. 5 µg protein were loaded per well.</p

Characterization of exosomal-like vesicles isolated by serial centrifugation from cultured human proximal tubular epithelial cells.

<p>Microvesicles from tubular cell conditioned serum-free media present exosomal features. <b>A)</b> Transmission electron microscopy shows vesicles have a diameter 50–100 nm, consistent with exosomes. i) Scale bar = 200 nm, ii) Scale bar = 100 nm, iii & iv) Scale bar = 50 nm. Arrows point to exosomes. <b>B)</b> Representative Western blot for the exosome marker CD63. Each lane contains 20 µg protein obtained from the pellet of the following sequential centrifugation steps: P1 1,100×g, P2 1,100×g, P3 6,000×g, P4 17,000×g, and P5 100,000×g supernatant. Exo: exosomes present in the 200,000×g pellet. <b>C)</b> Standard exosome markers were present in exosomes secreted by HK2 cells (Western blot). Each lane contains 6 µg of exosomal proteins. <b>D)</b> CD63 expression assessed by latex bead flow cytometry. Black peak: control beads. White peak: beads+PE–conjugated anti-CD63. Grey peak: beads+PE–conjugated anti-CD63+5 µg exosomes. FLH3: fluorescence intensity.</p