LpX plasma clearance and glomerular upake <i>in vivo.</i>

<p>LCAT deficiency markedly decreases LpX plasma clearance. WT and <i>Lcat</i>^-/- mice were injected with lissamine rhodamine B PE-tagged LpX and plasma samples were taken at the indicated times. (A) Plasma-associated fluorescence. Each data point represents the total fluorescence of pooled mouse plasma samples (mean ± S.D.; n = 3). (B) Agarose gel electrophoresis of pooled mouse plasma lipoprotein PE fluorescence (same samples as in (A)). LpX cleared from WT plasma by 240 min, whereas <i>Lcat</i>^-/- LpX remained elevated at all times. HDL-associated fluorescence was increased in WT plasma. W<i>hite line</i> indicates origin. (C) Fluorescent LpX retention in renal glomeruli is markedly increased in <i>Lcat</i>^-/- mice. Representative confocal maximum projection images of 10 μm fixed frozen kidney sections 4 hrs after injection of fluorescent-PE tagged LpX in mice chronically treated with 3 mg/wk synthetic LpX. Note the markedly increased retention of LpX in <i>Lcat</i>^-/- mice glomeruli. (D) Electron microscopic analysis of LpX in renal glomerular capillaries. Representative TEM of renal glomerular capillaries in WT (<i>left panels</i>) and <i>Lcat</i>^-/- mice (<i>right panels</i>). Endogenous multilamellar structures with features of LpX particles were occasionally present in the capillaries of (-) LpX <i>Lcat</i>^-/-, but not (-) LpX WT mice. Synthetic LpX particles resembling endogenous LpX were frequently observed in renal capillaries of both (+) LpX WT and (+) LpX <i>Lcat</i>^-/- mice. Both endogenous and exogenous synthetic LpX were often seen to be engulfed by endothelial cell processes (<i>insets</i>). Exogenous LpX in the capillary lumen bound to red blood cells in LpX-treated WT and <i>Lcat</i>^-/- mice. GBM: Glomerular Basement Membrane; PFP: Podocyte Foot Process. Scale bars = 500nm. Inset scale bars = 250 nm (WT+LpX); 100 nm (<i>Lcat</i>^-/- ± LpX).</p

Electron microscopic analysis of LpX movement through renal glomerular compartments.

<p>Circulating LpX particles (small arrows in (A, B)) bind to endothelial cell lamellipodia in (A) WT and (B) <i>Lcat</i>^-/- mouse glomerular capillaries (arrowheads), are internalized (long arrows in (A), and degraded (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s003" target="_blank">S3 Fig</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s004" target="_blank">S4 Fig</a>). LpX bound to the cell surface (B1), is partially (B2), small arrows in inset) and then completely engulfed (B3). LpX penetrates the glomerular basement membrane (GBM) in WT (C) and <i>Lcat</i>^-/- mice ((D) and, inset in (D), arrowheads), markedly disrupting its structure (C, D; asterisks). The typical intramembranous lesion as found in the peripheral GBM of human FLD is seen in the inset in D, displaying a characteristic lamellar structure within a lucent lacuna in <i>Lcat</i>^-/- mice. In (D), several lamellipodia (arrows) engulf an LpX particle in the GBM. LpX penetrates the glomerular urinary space of both WT (E, G) and <i>Lcat</i>^-/- (F, H) mice. LpX binds to podocyte cell bodies (PCBs) and foot processes (PFPs) at multiple sites (E, F: small arrows; H: arrowheads), and was internalized into PCBs (F; large arrow). Large vacuoles (G, H; large arrows) containing partially degraded LpX particles (G, H; small arrows) as well as numerous small unilamellar vesicles are often observed, consistent with cell-mediated LpX degradation. (I) In WT mice, LpX did not accumulate in the mesangial matrix and occasional foamy mesangial cells were observed. (J) Mesangial cells near the sites of LpX deposition engulf LpX particles. (K) Marked retention of LpX in <i>Lcat</i>^-/- mouse mesangial matrix. The regions near large arrows 1 & 2 in (K) are shown enlarged in K1&2. LpX binds to the mesangial cell prior to engulfment. Scale bars: A, B1, F, H, J = 200 nm; B2, D (inset), K1, K2 = 250 nm; B–E, G, I, K = 500 nm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s003" target="_blank">S3 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s004" target="_blank">S4 Fig</a>, for additional examples.</p

LpX metabolism <i>in vivo.</i>

<p>(A) Blood samples from <i>Lcat</i>^-/- and WT mice were collected prior to (“basal”) and, at 1 and 24 hrs after LpX injection. Plasma samples from WT (n = 6) and <i>Lcat</i>^-/- (n = 6) mice were pooled and lipoproteins were separated by FPLC. Phospholipid (PL), Total Cholesterol (TC), and Free Cholesterol (FC) were measured in collected fractions. Prior to LpX injection, TC, PL and FC were abundant in HDL in WT mice, whereas they were absent in <i>Lcat</i>^-/- mice, which have only small amounts of lipids in VLDL/LpX and small HDL. One hour after injection in <i>Lcat</i>^-/- mice, LpX PL and FC are clearly present in a large peak in the VLDL region, whereas in WT mice, the peak is reduced, consistent with our findings using fluorescent PE-tagged LpX (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.g001" target="_blank">Fig 1B</a>). One hour after LpX administration, a new peak in the HDL region (25 ml elution volume) appeared in WT mice; in <i>Lcat</i>^-/- mice, this peak was observed prior to LpX administration and was increased at 1 hr post-injection. At this time, the PL and FC content of the <i>Lcat</i>^-/- peak was increased compared to the <i>Lcat</i>^-/- pre-injection peak, as well as to the WT peak. (B) Characterization of particles eluted at 25 ml using native gradient gel electrophoresis 1 hr post-injection. Native gradient gel electrophoresis confirmed that lipid-containing particles were present in the 25 ml fraction in the 7–8 nm size range. (C) SDS-PAGE (16% acrylamide gel) apoA-I immunoblot of small HDL particles (25 ml elution volume) generated by LpX at I hr. ApoA-I immunostaining confirmed the presence of apoA-I in these particles, which suggests that in the presence of apoA-I, LpX-derived PL, and to a lesser extent FC, increased the pool size of small HDL particles. These findings <i>in vivo</i> are consistent with the apoA-I and LCAT-dependent remodeling of LpX that we observed in vitro (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.g001" target="_blank">Fig 1B–1D</a>). The peak is still visible 24 hours after injection in WT mice, while in <i>Lcat</i>^-/- mice, it returns to basal levels (Fig 4A).</p

LpX remodeling <i>in vitro.</i>

<p>(A) TEM analysis of synthetic LpX particles. <i>Left panel</i>: Low magnification image (Scale bar; 500 nm<i>)</i>. <i>Middle panel</i>: High magnification (Scale bar; 100 nm). <i>Right panel</i>: LpX particle size distribution. Size categories (nm): I (0–50), II (50–100), III (100–150), IV (150–200), and V (200–245). Small unilamellar vesicles as well as small, medium and, large multivesicular vesicles are seen. (B) LpX remodeling by LCAT and apoA-I in vitro. Agarose gel electrophoresis of LpX labeled with both fluorescent PE <i>(red)</i> and cholesterol (<i>blue</i>) incubated with Alexa 647-tagged apoA-I (<i>green</i>) and/or LCAT in vitro and scanned. Colocalization of LpX PE and cholesterol fluorescence is seen as <i>magenta</i> (merged image). Lane 1: ApoA-I; Lane 2: LpX; Lane 3: LpX + ApoA-I; Lanes 4–6: LpX + ApoA-I + 2, 4, or, 6 mg LCAT, respectively; Lane 7: LpX + 6 mg LCAT. (C) FPLC analysis of dual fluorescent PE- and cholesterol-tagged LpX incubated without (<i>left</i>) or with apoA-I and 6 mg LCAT <i>(right</i>). Fractions were analyzed for rhodamine (PE) fluorescence (<i>upper panels</i>) and TopFluor cholesterol fluorescence (<i>lower panels</i>). Note the additional peak (<i>arrows</i>) after incubation with apoA-I and LCAT. (D) LpX is converted to plasma HDL in vitro. Fluorescent PE-tagged LpX was incubated overnight with pooled human plasma. Agarose gels were scanned for PE fluorescence and then stained with Sudan Black. Lane 1: Fluorescent LpX. Lane 2: Pooled human plasma. Lane 3: Pooled human plasma + fluorescent LpX. <i>Arrows</i> indicate origin.</p

Effect of LpX on glomerular cell function <i>in vitro.</i>

<p>(A) LpX compromises the integrity of endothelial cell monolayers. Confluent HUVEC cell monolayers were incubated with PBS alone, or with PBS containing HDL (1 mg/ml), LDL (1 mg/ml), or LpX 5 mg/ml). Following a transient artifactual increase in impedance after the change in medium, LpX markedly decreased impedance. (B,C) LpX alters podocyte cytoskeletal actin organization. Representative confocal images of phalloidin-stained immortalized human podocytes incubated in vitro in the absence (B) or presence of (C) synthetic LpX. Scale bar: 100 μm.(D) LpX endocytosis stimulates mesangial cell IL-6 cytokine secretion in vitro. Confluent cultured mesangial cells were treated with 200 μg/ml fluorescent PE-tagged synthetic LpX, in the absence or presence of 5 μM amiodarone for 18 hrs, and then IL-6 was quantified by ELISA assay. Note that amiodarone further increased IL-6 secretion with or without LpX treatment. *p <0.05; **p = 0.01; unpaired two-tailed t-test.</p