Lionel C. Clement

Podocyte-secreted angiopoietin-like-4 mediates proteinuria in glucocorticoid-sensitive nephrotic syndrome

The main manifestations of nephrotic syndrome include proteinuria, hypoalbuminemia, edema, hyperlipidemia and lipiduria. Common causes of nephrotic syndrome are diabetic nephropathy, minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) and membranous nephropathy. Among the primary glomerular diseases, MCD is usually sensitive to glucocorticoid treatment, whereas the other diseases show variable responses. Despite the identification of key structural proteins in the glomerular capillary loop which may contribute to defects in ultrafiltration, many of the disease mechanisms of nephrotic syndrome remain unresolved. In this study, we show that the glomerular expression of angiopoietin-like-4 (Angptl4), a secreted glycoprotein, is glucocorticoid sensitive and is highly upregulated in the serum and in podocytes in experimental models of MCD and in the human disease. Podocyte-specific transgenic overexpression of Angptl4 (NPHS2-Angptl4) in rats induced nephrotic-range, and selective, proteinuria (over 500-fold increase in albuminuria), loss of glomerular basement membrane (GBM) charge and foot process effacement, whereas transgenic expression specifically in the adipose tissue (aP2-Angptl4) resulted in increased circulating Angptl4, but no proteinuria. Angptl4−/− mice that were injected with lipopolysaccharide (LPS) or nephritogenic antisera developed markedly less proteinuria than did control mice. Angptl4 secreted from podocytes in some forms of nephrotic syndrome lacks normal sialylation. When we fed the sialic acid precursor N-acetyl-D-mannosamine (ManNAc) to NPHS2-Angptl4 transgenic rats it increased the sialylation of Angptl4 and decreased albuminuria by more than 40%. These results suggest that podocyte-secreted Angptl4 has a key role in nephrotic syndrome.

Hyperinsulinaemia triggered by dietary conjugated linoleic acid is associated with a decrease in leptin and adiponectin plasma levels and pancreatic beta cell hyperplasia in the mouse

Aims/hypothesisDietary supplementation with conjugated linoleic acids (CLA) has a fat-reducing effect in various species, but induces severe hyperinsulinaemia and hepatic steatosis in the mouse. This study aimed to determine the causes of the deleterious effects of CLA on insulin homeostasis.MethodsThe chronology of adipose and liver weight, hepatic triglyceride accumulation and selected blood parameters, including lipids, insulin, leptin and adiponectin, was determined in C57BL/6J female mice fed a 1% isomeric mixture of CLA for various periods of time ranging from 2 to 28 days. Insulin secretion was measured in 1-h static incubations of pancreatic islets, and pancreas morphometric parameters were determined in mice fed CLA for 28 days.ResultsPlasma levels of leptin and adiponectin sharply decreased after 2 days of CLA feeding, although adipose tissue mass only decreased after day 6. Hyperinsulinaemia developed at day 6 and consistently worsened up to day 28, in parallel with increases in hepatic lipid content. Islets from CLA-fed mice displayed three- to four-fold increased rates of glucose-stimulated insulin secretion, both in the absence and presence of isobutyl methylxanthine or carbachol. The increased insulin-releasing capacity of islets from CLA-fed mice was explained by an increase in beta cell mass and number.Conclusions/interpretationThe data suggest that CLA supplementation induces a profound reduction of leptinaemia and adiponectinaemia, followed by hyperinsulinaemia due to the increased secretory capacity of pancreatic islets, leading, in turn, to liver steatosis. These observations cast doubt on the safety of dietary supplements containing CLA.

New insights into the fatty acid-binding protein (FABP) family in the small intestine

The fatty acid-binding protein (FABP) superfamily is constituted by 14–15 kDa soluble proteins which bind with a high affinity either long-chain fatty acids (LCFAs), bile acids (BAs) or retinoids. In the small intestine, three different FABP isoforms exhibiting a high affinity for LCFAs and/or BAs are expressed: the intestinal and the liver-type (I-FABP and L-FABP) and the ileal bile acid-binding protein (I-BABP). Despite of extensive investigations, their respective physiological function(s) are not clearly established. In contrast to the I-FABP, L-FABP and I-BABP share several common structural features (shape, size and volume of the hydrophobic pocket). Moreover, L-FABP and I-BABP genes are also specifically regulated by their respective preferential ligands through a very similar molecular mechanism. Although, they exhibit differences in their binding specificities and location along the small intestine supporting a specialization, it is likely that L-FABP and I-BABP genes exert the same type of basic function(s) in the enterocyte, in contrast to I-FABP.

Luminal Lipid Regulates CD36 Levels and Downstream Signaling to Stimulate Chylomicron Synthesis

The membrane glycoprotein CD36 binds nanomolar concentrations of long chain fatty acids (LCFA) and is highly expressed on the luminal surface of enterocytes. CD36 deficiency reduces chylomicron production through unknown mechanisms. In this report, we provide novel insights into some of the underlying mechanisms. Our in vivo data demonstrate that CD36 gene deletion in mice does not affect LCFA uptake and subsequent esterification into triglycerides by the intestinal mucosa exposed to the micellar LCFA concentrations prevailing in the intestine. In rodents, the CD36 protein disappears early from the luminal side of intestinal villi during the postprandial period, but only when the diet contains lipids. This drop is significant 1 h after a lipid supply and associates with ubiquitination of CD36. Using CHO cells expressing CD36, it is shown that the digestion products LCFA and diglycerides trigger CD36 ubiquitination. In vivo treatment with the proteasome inhibitor MG132 prevents the lipid-mediated degradation of CD36. In vivo and ex vivo, CD36 is shown to be required for lipid activation of ERK1/2, which associates with an increase of the key chylomicron synthesis proteins, apolipoprotein B48 and microsomal triglyceride transfer protein. Therefore, intestinal CD36, possibly through ERK1/2-mediated signaling, is involved in the adaptation of enterocyte metabolism to the postprandial lipid challenge by promoting the production of large triglyceride-rich lipoproteins that are rapidly cleared in the blood. This suggests that CD36 may be a therapeutic target for reducing the postprandial hypertriglyceridemia and associated cardiovascular risks.

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

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.

New Insights Into Human Minimal Change Disease: Lessons From Animal Models

The pathogenesis of minimal change disease (MCD), considered to be the simplest form of nephrotic syndrome, has been one of the major unsolved mysteries in kidney disease. In this review, recent landmark studies that have led to the unraveling of MCD are discussed. A recent study now explains the molecular basis of major clinical and morphologic changes in MCD. Overproduction of angiopoietin-like 4 (ANGPTL4) in podocytes in MCD causes binding of ANGPTL4 to the glomerular basement membrane, development of nephrotic-range selective proteinuria, diffuse effacement of foot processes, and loss of glomerular basement membrane charge, but is not associated with changes shown by light microscopy in the glomerular and tubulointerstitial compartments. At least some of this ability of ANGPTL4 to induce proteinuria is linked to a deficiency of sialic acid residues because oral supplementation with sialic acid precursor N-acetyl-d-mannosamine improves sialylation of podocyte-secreted ANGPTL4 and significantly decreases proteinuria. Animal models of MCD, recent advances in potential biomarkers, and studies of upstream factors that may initiate glomerular changes also are discussed. In summary, recent progress in understanding MCD is likely to influence the diagnosis and treatment of MCD in the near future.

ZHX Proteins Regulate Podocyte Gene Expression during the Development of Nephrotic Syndrome

Transcriptional regulation of podocyte gene expression in primary glomerular disease is poorly understood. In this study, we demonstrate a prominent role of members of the ZHX (zinc fingers and homeoboxes) family of proteins in regulating podocyte gene expression during the development of nephrotic syndrome. While studying mechanisms of glomerular disease, rat ZHX3 was cloned from a down-regulated gene fragment; its cellular localization, DNA binding, and transcriptional repressor properties were characterized; and its ability to influence podocyte gene expression directly or via ZHX1 and ZHX2 was studied. In eukaryotic promoters, ZHX3 bound to the CdxA binding motif. ZHX proteins were mostly sequestered in the non-nuclear compartment in the normal in vivo podocyte by virtue of heterodimer formation, and loss of heterodimerization was associated with entry into the nucleus. In experimental minimal change disease, ZHX3 was transiently down-regulated prior to the onset of proteinuria, and recovery of expression was associated with migration of ZHX3 protein into the nucleus and the development of proteinuria. This expression pattern mirrored the increased nuclear ZHX3 expression noted in vivo in the podocytes in human minimal change disease biopsies. In vitro, migration of ZHX3 protein into the nucleus during recovery from transient ZHX3 knockdown reproduced the gene expression profile of in vivo minimal change disease. Severe sustained knockdown of ZHX3 caused down-regulation of genes involved in focal sclerosis, including WT1, mediated mostly by increased nuclear entry of ZHX2 and ZHX1. In summary, ZHX proteins are major transcriptional mediators of podocyte disease.

A new hypotensive polyunsaturated fatty acid dietary combination regulates oleic acid accumulation by suppression of stearoyl CoA desaturase 1 gene expression in the SHR model of genetic hypertension

Polyunsaturated fatty acids (PUFA) are known to repress SCD‐1 gene expression key enzyme of monounsaturated fatty acid biosynthesis. Alterations of the monounsaturated/saturated fatty acids ratio have been implicated in various diseases related to the metabolic syndrome including hypertension. We previously evidenced that lipogenesis end‐products accumulated in spontaneously hypertensive rats (SHR) and that a dietary combination of n‐6/n‐3 PUFA had hypotensive effects. Our present objective was to test the hypothesis that these SHR liver lipid disorders might be modulated in response to this hypotensive combination by changes in SCD‐ 1 expression and activity. So we studied in hepatocytes SCD‐1 transcription by Northern blotting as well as plasma and liver fatty acid composition by gas–liquid chromatography. Liver SCD‐1 gene expression was suppressed by 50% and in different lipid classes relative abundance of stearic and oleic acids decreased. Consequently the Δ9 desaturation index calculated from the ratio of oleic vs. stearic acids decreased. In addition the level of circulating saturated fatty acids decreased when one of oleic acids increased. These data provided evidence that the tested hypotensive PUFA combination reverses the high monounsaturated/saturated fatty acids ratio associated to hypertension in SHR via a regulation monounsaturated fatty acid relative abundance by repression of SCD‐1 gene.

Angiopoietin-like 4 based therapeutics for proteinuria and kidney disease

Current drugs used to treat proteinuric disorders of the kidney have been borrowed from other branches of medicine, and are only partially effective. The discovery of a central, mechanistic role played by two different forms of the secreted glycoprotein angiopoietin-like 4 (Angptl4) in human and experimental glomerular disease has opened new treatment avenues. Localized upregulation of a hyposialylated form (lacks sialic acid residues) of Angptl4 secreted by podocytes induces the cardinal morphological and clinical manifestations of human minimal change disease, and is also being increasingly recognized as a significant contributor toward proteinuria in experimental diabetic nephropathy. Oral treatment with low doses of N-acetyl-D-mannosamine, a naturally occurring precursor of sialic acid, improves sialylation of Angptl4 in vivo, and reduces proteinuria by over 40%. By contrast, a sialylated circulating form of Angptl4, mostly secreted from skeletal muscle, heart and adipose tissue in all major primary glomerular diseases, reduces proteinuria while also causing hypertriglyceridemia. Intravenous administration of recombinant human Angptl4 mutated to avoid hypertriglyceridemia and cleavage has remarkable efficacy in reducing proteinuria by as much as 65% for 2 weeks after a single low dose. Both interventions are mechanistically relevant, utilize naturally occurring pathways, and represent new generation therapeutic agents for chronic kidney disease related to glomerular disorders.

Dietary Lipids Modify Intestinal Lipid-Binding Protein RNA Abundance in Diabetic and Control Rats

Background: Lipid-binding proteins have been identified in the enterocyte, including the cytosolic intestinal and liver fatty acid binding proteins (I-FABP and L-FABP, respectively) as well as the brush border membrane fatty acid transporter (FAT). It is unclear whether variations in the type of dietary lipids or diabetes modify the RNA abundance of these proteins. Diabetes is associated with an increased intestinal lipid uptake, and the lipid uptake is greater in rats fed a semisynthetic saturated fatty acid (SFA) as compared with a polyunsaturated fatty acid (PUFA) diet. Methods: Male Sprague-Dawley rats were injected with streptozotocin or control vehicle and fed chow or either SFA or PUFA for 2 weeks. Northern blotting was performed on RNA isolated from jejunal and ileal tissues. Results: In controls, feeding SFA as compared with PUFA reduced the jejunal abundance of I-FABP and L-FABP RNA. In diabetic rats, feeding SFA increased the ileal FAT RNA. Feeding PUFA reduced jejunal L-FABP and ileal FAT RNA in diabetic rats as compared with controls. Conclusions: The enhanced lipid uptakes reported with feeding an SFA diet or with diabetes were not associated with parallel alterations in lipid-binding proteins. We speculate that these lipid-binding proteins act as a storage mechanism for lipids in enterocytes and are not directly involved in lipid uptake.