Hazard Ratios (and 95% CIs) for Death per Standard Deviation of FGF23 and Klotho levels and according to the level tertiles.

<p>*Model 1 = demographics: adjusted for age, gender (male) and by dialysis center clustering.</p>†<p>Model 2 = dialysis specific risk factors and comorbid conditions: adjusted for covariates in Model 1 plus dialysis vintage, systolic and diastolic blood pressure, body-mass index, vascular access on study enrolment (fistula, graft, catheter), coexisting conditions listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100688#pone-0100688-t001" target="_blank">Table 1</a> (coronary artery disease, valvular heart disease, atrial fibrillation, pulmonary hypertension, implantable cardioverter defibrillator carrier; diabetes mellitus, peripheral vascular disease, stroke, vasculitis, malignoma, chronic obstructive pulmonary disease), cause of renal failure (diabetic nephropathy, hypertensive nephropathy, glomerulonephritis, polycystic kidney disease, others/unknown), medication use listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100688#pone-0100688-t001" target="_blank">Table 1</a> (phosphate binders, vitamin D replacement, angiotensin converting enzyme inhibitors or angiotensin receptor blockers, beta blockers, aspirin, anticoagulant or clopidogrel, statin), pooled Kt/V.</p>‡<p>Model 3 = fully adjusted model: adjusted for covariates in Model 2 plus parathyroid hormone, 25(OH)vitamin D, phosphate, calcium, albumin, hemoglobin, C-reactive protein, cholesterol.</p>§<p>Patients were categorized according to Klotho level tertiles at enrolment (1^st tertile <286 pg/ml, 2^nd tertile 286–392 pg/ml, 3^rd tertile >392 pg/ml).</p>||<p>Patients were categorized according to FGF23 level tertiles at enrolment (1^st tertile <118 RU/ml, 2^nd tertile 118–468 RU/ml, 3^rd tertile >468 RU/ml).</p><p>Abbreviations: FGF23, fibroblast growth factor 23; HR, hazard ratio; R, reference.</p

Association of Klotho levels with the presence of atrial fibrillation.

<p>*Model 1: adjusted for age and gender (male).</p>†<p>Model 2: adjusted for covariates in Model 1 plus cardiovascular comorbidities and anuria.</p>‡<p>Model 3: adjusted for covariates in Model 2 plus laboratory results for mineral metabolism, calcium dialysate, potassium serum and dialysate, inflammation, cholesterol, hemoglobin and TSH.</p>§<p>per standard deviation.</p><p>Abbreviations: Ca, Calcium; CAD, coronary artery disease; CI, confidence interval; CRP, C-reactive protein; DM, diabetes mellitus; FGF23, fibroblast growth factor 23; Hb, hemoglobin; K, potassium; PAD, peripheral artery disease; TSH, thyroid stimulating hormone.</p><p>OR, odds ratio; VHD, valvular heart disease.</p

PLD inhibitors trigger autophagosome formation.

<p>(A) PLD inhibitors induced autophagy in a time-dependent way. Left panel: PKD cells were treated by either 10 µM PLD1 inhibitor or 20 µM PLD2 inhibitor for 24 h and 48 h. Right panel: PKD cells were plated for 24 h and then shifted to medium containing 0.5% serum. 1% 1-BtOH or 1% <i>tert</i>-BtOH was added for 2 h, 20 µM honokiol was added for 20 h. Western blots analyzing the expression of caspase-3, LC3B and GAPDH. (B) Transmission electron microscopy analysis of PLD1 and PLD2 inhibitor-treated HAK and OX161 cells (10 µM and 20 µM respectively, 48 h) showing intensive vacuolization and autophagosome-like vesicles. The lower panels show the magnification of the black frame area in the upper panels for each type of cell. The scale bar in the upper and lower panels represents 10 µm and 5 µm respectively. (C) Immunofluorescence microscopy analyzing autophagosome distribution after PLD1-/2- inhibitor (10 µM and 20 µM) treatment for 48 h. Cells were stained with anti-LC3B antibody. Scale bar represents 20 µm. Representative of three independent experiments.</p

Regression analysis for Klotho tertiles with the absence of atrial fibrillation.

<p>*Model 1: adjusted for age and gender.</p>†<p>Model 2: adjusted for covariates in Model 1 plus diabetes mellitus, coronary artery disease, valvular heart disease, peripheral vascular disease, stroke and anuria.</p>‡<p>Model 3: adjusted for covariates in Model 2 plus parathyroid hormone, fibroblast growth factor 23, calcium, phosphate, albumin, calcium dialysate, potassium serum and dialysate, hemoglobin C-reactive protein, cholesterol and thyroid stimulating hormone.</p><p>Abbreviations: CI, confidence interval; OR, odds ratio.</p

Elevated mTOR and PLD activity in PKD cells.

<p>(A) Western blots and densitometry analyzing the endogenous activity of the mTOR in PKD and control cells. (B) The endogenous level of PLD activity was determined in PKD and control cells by the Amplex Red PLD assay kit. Honokiol (20 µM) treated OX161 for 20 h as a positive control. An experiment which is representative of three independent experiments is shown. Data are expressed as mean ± SD and were analyzed by one-way ANOVA. * p< 0.05, ** p< 0.01, *** p< 0.001.</p

PLD and mTOR pathway in PKD.

<p>(A) Elevated PLD activity stimulates the mTORC1, mTORC2 and other relative pathways and promotes cell proliferation in PKD cells. (B) Inhibition of PLD reduces phosphorylation of downstream targets of mTORC1 and mTORC2, and induces autophagy in PKD cells.</p

PLD inhibitors block mTOR signaling pathway and exogenous PA abrogates the effect of PLD inhibitors on mTOR signaling and proliferation of PKD cells.

<p>(A) PLD inhibitors affect mTOR signaling in a dose-dependent way. Western blots analyzing the expression of phospho-Akt, Akt, phospho-S6K, S6K, phospho-S6, S6, phospho-4EBP1, 4EBP1 and GAPDH either upon treatment with the indicated concentration of PLD1-/2- inhibitor for 48 h or without treatment. (B) PKD cells were plated for 24 h and then shifted to medium containing 0.5% serum. 1% 1-BtOH or 1% <i>tert</i>-BtOH was added for 2 h, 20 µM honokiol was added for 20 h. Western blots analyzing the expression of phospho-S6K, S6K, phospho-Akt and Akt. (C, D) Exogenous PA stimulated mTOR signaling in a time-dependent way, but not PLD activity. Human PKD cells were cultured under either normal culture medium (5% FBS) or starvation culture medium (0.5% FBS). After 24 h starvation, cells were treated with 100 µM PA for 20, 40 and 60 minutes. Whole cell lysates were analyzed by (C) western blots to detect the expression of phospho-mTOR, mTOR, phospho-S6K, S6K, phospho-Akt, Akt and GAPDH. (C) Whole cell lysates were determined by Amplex Red PLD kit to measure the PLD activity. (E) Exogenous PA impairs the effect of PLD inhibitors on cell growth. Cells were pretreated with either 10 µM PLD1 inhibitor or 20 µM PLD2 inhibitor for 48 h, and then with 100 µM PA for the indicated time. DNA synthesis was measured by using the BrdU assay. An experiment which is representative of three independent experiments is shown. Data are expressed as mean ± SD and were analyzed by one-way ANOVA. * p< 0.05, ** p< 0.01.</p

Klotho and FGF23 levels in patients with and without atrial fibrillation.

<p>Klotho and FGF23 levels in patients with and without atrial fibrillation.</p

Cumulative survival by tertiles of Fibroblast growth factor 23 (FGF23).

<p>Patients were stratified by their FGF23 levels according to the tertiles. Kaplan-Meier analysis with long-rank test approached a significant difference between groups (P = 0.05).</p

PLD inhibitors impair cell growth and block PLD activity in PKD cells.

<p>Effect of PLD1-/2-inhibitors on (A) the cell viability, (B) DNA synthesis ability and (C) cell proliferation were determined by MTS, BrdU and cell counting, respectively. All types of cells were exposed to various concentrations of either PLD1 or PLD2 inhibitor for 48 h. An experiment which is representative of three independent experiments is shown.</p