Circulating angiopoietin-like 4 links proteinuria with hypertriglyceridemia in nephrotic syndrome

Abstract

The molecular link between proteinuria and hyperlipidemia in nephrotic syndrome is not known. We show in the present study that plasma angiopoietin-like 4 (Angptl4) links proteinuria with hypertriglyceridemia through two negative feedback loops. In previous studies in a rat model that mimics human minimal change disease, we observed localized secretion by podocytes of hyposialylated Angptl4, a pro-proteinuric form of the protein. But in this study we noted high serum levels of Angptl4 (presumably normosialylated based on a neutral isoelectric point) in other glomerular diseases as well. Circulating Angptl4 was secreted by extrarenal organs in response to an elevated plasma ratio of free fatty acids (FFAs) to albumin when proteinuria reached nephrotic range. In a systemic feedback loop, these circulating pools of Angptl4 reduced proteinuria by interacting with glomerular endothelial αvβ5 integrin. Blocking the Angptl4-β5 integrin interaction or global knockout of Angptl4 or β5 integrin delayed recovery from peak proteinuria in animal models. But at the same time, in a local feedback loop, the elevated extrarenal pools of Angptl4 reduced tissue FFA uptake in skeletal muscle, heart and adipose tissue, subsequently resulting in hypertriglyceridemia, by inhibiting lipoprotein lipase (LPL)-mediated hydrolysis of plasma triglycerides to FFAs. Injecting recombinant human ANGPTL4 modified at a key LPL interacting site into nephrotic Buffalo Mna and Zucker Diabetic Fatty rats reduced proteinuria through the systemic loop but, by bypassing the local loop, without increasing plasma triglyceride levels. These data show that increases in circulating Angptl4 in response to nephrotic-range proteinuria reduces the degree of this pathology, but at the cost of inducing hypertriglyceridemia, while also suggesting a possible therapy to treat these linked pathologies.

Figure 1

Elevated circulating Angptl4 levels are required for the development of hypertriglyceridemia in nephrotic syndrome. (a) Plasma ANGPTL4 levels measured by ELISA in subjects with nephrotic syndrome due to primary glomerular disease. The numbers of subjects analyzed in each group are shown in parentheses. (b) Plasma Angptl4 levels measured by ELISA at prenephrotic and nephrotic stages in PHN (n = 4 rats per group), Buffalo Mna (B. Mna; n = 9 rats per group) and single-dose intravenous PAN (n = 4 rats per group). OD₄₅₀, optical density at 450 nm. The timing of Angptl4 levels is days after disease induction in PHN and PAN and age in months in B. Mna rats. (c) Proteinuria (left), plasma triglyceride levels (middle) and post-heparin LPL activity (right) in PHN. The timing of the results is days after disease induction. (d) Proteinuria (left), plasma triglyceride levels (middle) and post-heparin LPL activity (right) in Buffalo Mna rats. (e) Proteinuria (left), plasma triglyceride levels (middle) and post-heparin LPL activity (right) in PAN rats. (f) Plasma triglyceride levels in wild-type Sprague Dawley rats (n = 6 rats), adipose tissue–specific Angptl4-overexpressing rats (aP2-Angptl4; n = 6 rats) and 3-month-old podocyte-specific Angptl4-overexpressing rats (NPHS2-Angptl4; n = 6 rats). (g) Post-heparin LPL activity in wild-type, aP2-Angptl4 transgenic and NPHS2-Angptl4 transgenic rats. (h) Plasma triglyceride levels in wild-type and Angptl4^−/− mice (n = 4 mice per reading) 48 h after the induction of nephrotic syndrome using NTS. All error bars, s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-way t test.

Figure 2

The source of circulating Angptl4 in nephrotic syndrome. (a–c) Multiorgan Angptl4 mRNA expression relative to control (n = 6 templates per organ per time point) in PHN (a), Buffalo Mna rats (b) and PAN (c). Sk., skeletal. (d) Representative two-dimensional gel electrophoresis and western blot of plasma showing circulating Angptl4 levels in wild-type Sprague Dawley and proteinuric NPHS2-Angptl4 transgenic rats before and after the induction of low-dose PAN. (e) Densitometry analysis of the two-dimensional gels (n = 3 readings per value) in d. (f) Two-dimensional gel electrophoresis and western blot of plasma from NPHS2-Angptl4 transgenic rats with PAN to look for V5-tagged transgene-expressed Angptl4 in the circulation. (g) Plasma triglyceride levels 6 d after the induction of PAN in wild-type Sprague Dawley, aP2-Angptl4 transgenic and NPHS2-Angptl4 transgenic rats (n = 4 readings per group). (h) Post-heparin LPL activity corresponding to the plasma triglyceride levels shown in g. In g and h, the black bars correspond to the data from Figure 1f,g, which is included for comparison. All error bars, s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-way t test. In g and h, statistical significance is shown for the difference between transgenic rats and the corresponding wild-type controls. P < 0.001 for each rat type before and after induction of PAN. In a–c, the threshold for significance was a threefold change (horizontal line).

Figure 3

Mechanisms of Angptl4 upregulation in peripheral organs in nephrotic syndrome. (a) Angptl4 levels (n = 5 rats per group; left), plasma triglyceride levels (n = 5 rats per group; middle) and peripheral organ Angptl4 mRNA expression (n = 6 templates per reading; right) in Normal Sprague Dawley and Nagase analbuminemic rats. (b) Plasma ratio of FFAs to albumin in a subset of individuals with MCD, FSGS or CG and age- and sex-matched control subjects. The numbers of subjects analyzed are indicated in parentheses. (c) Plasma ratio of FFAs to albumin in Sprague Dawley rats with puromycin nephrosis (n = 4 rats per group). (d) Plasma ratio of FFAs to albumin in Buffalo Mna rats (1 month, n = 5 rats; 2 months, n = 5 rats; 4.5 months, n = 14 rats; 9 months, n = 9 rats) rats per time point). (e) Plasma FFA levels before and after affinity absorption of albumin from two patients with MCD (far left and right) and two patients with CG (middle). (f) Changes in the mRNA expression (n = 4 templates per organ per time point) of Ppar-δ (left), Ppar-γ (middle) and Ppar-α (right) in liver, heart, skeletal muscle, WAT and BAT during the peripheral phase of PAN and in Buffalo Mna rats. (g) Plasma ratio of FFAs to albumin in Buffalo Mna rats (n = 4 rats per group) 5 h after the first dose of normal saline, Intralipid or oleic acid. (h) Change in plasma Angptl4 levels after 3 d of treatment. (i) Proteinuria at baseline and after 3 d of treatment. All error bars, s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-way t test. In f, the threshold for significance was a threefold change (horizontal line).

Figure 4

Effect of circulating Angptl4 on proteinuria. Red arrows indicate the time points at which an antibody or recombinant protein was injected. (a) Proteinuria after induction of high-dose PAN in wild-type Sprague Dawley and aP2-Angptl4 transgenic rats (n = 4 rats per group). (b) The effect of depleting circulating Angptl4 using an Angptl4-specifc antibody on proteinuria in Sprague Dawley rats with intermediate-dose PAN (n = 3 rats per group). (c) Proteinuria in Buffalo Mna rats after injecting recombinant rat Angptl4 or supernatant from a control cell line (n = 4 rats per group). (d) Proteinuria in severe anti-Thy1.1 nephritis with injection of recombinant rat Angptl4 or supernatant from a control cell line (n = 4 rats per group). (e) Schematic representation of wild-type and mutant human ANGPTL4 proteins showing mutations in areas that are important for LPL interaction (amino acid 40 and the adjacent amino acid 39) and protein cleavage (amino acids 161–164). (f) Western blot of recombinant tagged proteins using mouse V5-specific antibody to demonstrate the expected size of the intact protein and reduced cleavage in the mutant proteins (arrows). (g) Effect of injecting 55 µg of wild-type (8525) or mutant (8501 and 8515) human ANGPTL4 in Buffalo Mna rats (n = 3 rats per group) on plasma levels of the recombinant protein (left), proteinuria (middle) and plasma triglyceride levels (right). (h) Effect of injecting a lower dose (15 µg) of wild-type (8525) or mutant (8496 and 8520) human ANGPTLl4 in ZDF rats (n = 4 rats per group) on plasma levels of the recombinant protein (left), proteinuria (middle) and plasma triglyceride levels (right). All error bars, s.e.m. *P < 0.05, **P < 0.01 determined by two-way t test, except in b and c, in which one-way t test was used. In g (middle), black asterisks are shown where all three study groups were individually different from the control injected rats. In g and h, colored asterisks are shown where individual values were significantly different from the corresponding baseline values. The pound symbol in g (right) indicates P < 0.05 in the rats injected with mutant ANGPTL4 compared to the group injected with wild-type protein.

Figure 5

Circulating Angptl4 reduces proteinuria through its interaction with glomerular endothelial α_vβ₅ integrin. Red arrows indicate the time points at which β₅ integrin–specific antibody or preimmune serum was injected. (a) Confocal images of glomeruli from an aP2-Angptl4 transgenic rat (left) and a wild-type mouse injected with recombinant rat Angptl4 (right) to demonstrate co localization of adipose tissue–secreted (left) or injected (right) Angptl4-V5 (V5-specific antibody; red) with glomerular endothelium (von Willebrand factor–specific antibody; green). (b) Immunogold electron micrograph of a glomerulus from an aP2-Angptl4 transgenic rat using V5-specific antibody to show glomerular endothelial cell surface localization (arrows) of adipose tissue–secreted Angptl4-V5. Podo, podocyte; GBM, glomerular basement membrane; Endo, endothelial cell. (c) Effect of normosialylated recombinant rat Angptl4 on apoptosis induced in cultured rat glomerular endothelial cells using H₂O₂-induced oxidative stress (n = 3 readings per group). (d) Proteinuria during the peripheral phase of Angptl4 secretion after injection of NTS in wild-type and Itgb5^−/− mice (n = 5 mice per group). (e) Multiorgan mRNA expression profile for Angptl4 7 d after injection of NTS (n = 6 templates per organ) in the wild-type mice in d. The threshold for significance was a threefold change (horizontal line). (f) Recovery from peak proteinuria in Sprague Dawley rats with low-dose PAN (n = 4 rats per group) after blocking the endothelial β₅ integrin–Angptl4 interaction using a β₅ integrin–specific antibody. (g) Plasma Angptl4 levels in the rats in f. (h) Recovery from peak proteinuria in aP2-Angptl4 transgenic rats with full-dose PAN (n = 4 rats per group) after blocking the endothelial β₅ integrin–Angptl4 interaction using a β₅ integrin–specific antibody. (i) Recovery from peak proteinuria in wild-type and Angptl4^−/− mice injected with NTS (n = 7 mice per group). All error bars, s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-way t test. Scale bars, 8 µm (a); 0.2 µm (b).

Figure 6

Pathobiology of circulating Angptl4 in nephrotic syndrome. (a) Diagram representation of the production of circulating Angptl4 protein and its biological effects. The circulating, normosialylated form of Angptl4 is secreted from peripheral organs (mostly skeletal muscle, heart and adipose tissue) in MCD, MN, FSGS and CG. In addition, podocytes in MCD secrete a hyposialylated form of the protein that remains restricted to the kidney and induces proteinuria and a normosialylated form that enters the circulation. Circulating Angptl4 binds to glomerular endothelial α_vβ₅ integrin to reduce proteinuria or inactivates endothelium-bound LPL in skeletal muscle, heart and adipose tissue to reduce the hydrolysis of plasma triglycerides to FFA, resulting in hypertriglyceridemia. Some Angptl4 and LPL are lost in the urine. (b) Schematic illustration of negative feedback loops in the link between proteinuria, hypoalbuminemia and hypertriglyceridemia that are mediated by Angptl4 and FFA (unesterified fatty acids with a free carboxylate group). Plasma FFAs are noncovalently bound to albumin, and because of the preferential loss of albumin with low FFA content during proteinuria, albumin with higher FFA content is retained in circulation. As glomerular disease progresses and proteinuria increases, hypoalbuminemia develops, and the combination of high albumin-FFA content and lower plasma albumin levels increases the plasma ratio of FFAs to albumin. This increased available FFA enters the skeletal muscle, heart and adipose tissue to induce upregulation of Angptl4, mediated at least in part by Ppar proteins. Angptl4 secreted from these organs participates in two feedback loops. In the systemic loop, it binds to glomerular endothelial α_vβ₅ integrin and reduces proteinuria. In a local loop, it inhibits LPL activity in the same organs from which it is secreted to reduce the uptake of FFAs, thereby curtailing the stimulus for its own upregulation.