Cellular Cholesterol Transport Proteins in Diabetic Nephropathy

<div><p>Background</p><p>Lipid accumulation has been shown to accelerate renal injury, and the intracellular accumulation of lipids may be caused by alterations in synthesis as well as lipid uptake and efflux. We have investigated the role of cellular cholesterol transport proteins including adenosine triphosphate binding cassette transporter A1 (ABCA1), G1 (ABCG1) and scavenger receptor class B type I (SR-BI) in diabetic nephropathy.</p><p>Methods</p><p>Protein expression and the ability to mediate cholesterol efflux of ABCA1, ABCG1 and SR-BI was determined in human renal mesangial cells and proximal tubular epithelial cells cultured under normal or high glucose conditions. Renal expression of these cholesterol transporters was examined in a murine model of streptozotocin-induced type 1 diabetes.</p><p>Results</p><p>ABCA1, ABCG1 and SR-BI were expressed in both human renal mesangial cells and proximal tubular epithelial cells, and mediated cholesterol efflux to apolipoprotein AI and HDL. <i>In vitro</i>, hyperglycemia reduced the expression and the ability to mediate cholesterol efflux of all three cholesterol transporters (<i>p</i><0.05). <i>In vivo</i> studies showed that intra-renal accumulation of lipids was increased in diabetic mice, particularly in mice with nephropathy. This was associated with a significant reduction in the expression of ABCA1, ABCG1 and SR-BI in the kidneys. These changes were already seen in diabetic mice without nephropathy and preceded the development of nephropathy. Diabetic mice with nephropathy had the lowest level of these cholesterol transporters.</p><p>Conclusion</p><p>Inducing diabetes with streptozotocin significantly reduced renal expression of ABCA1, ABCG1 and SR-BI. Defects in cholesterol export pathway in renal cells could therefore promote cholesterol accumulation and might contribute to the development of diabetic nephropathy.</p></div

Expression of cholesterol transporters under different concentrations of glucose.

<p>Human mesangial cells (A) and HK-2 cells (B) were cultured under different conditions of glucose concentrations. Relative target protein band intensities were normalized against its <i>β</i>-actin and were presented as means + SD by densitometric analysis from 3 separate experiments. *<i>p</i><0.05 and **<i>p</i><0.01 vs. cells with 5 mM glucose in culture medium.</p

Immunohistochemical images of cholesterol transporters in different groups of mice.

<p>Representative images of cholesterol transporters ABCA1, ABCG1, and SR-BI in renal tissues from control, diabetic (DM), and diabetic nephropathy (DN) mice. Original magnification x400 (A). Image-based computer assisted analysis was performed to semi-quantify the amount of ABCA1, ABCG1 and SR-BI in the glomeruli and tubulo-interstitium of control, DM and DN mice (B). Results were presented as mean + SD of data obtained from 5 mice per group. **<i>p</i><0.01 or ***<i>p</i><0.001, control vs. DM; ^###<i>p</i><0.001, control vs. DN; ^§<i>p</i><0.05 or ^§§<i>p</i><0.01, DM vs. DN.</p

Oil Red O staining of cholesterol in renal tissues.

<p>Oil Red O staining of cholesterol in renal tissues from control, diabetic (DM), and diabetic nephropathy (DN) mice. Original magnification x400. Box area is enlarged to compare cholesterol accumulation (denoted by arrows) between control and diabetic mice (lower panels).</p

Effect of inhibition of ADAM10 on constitutive (A) and insulin-induced shedding of RAGE (B) in THP-1 macrophages.

<p>Cell surface receptors were biotinylated and cell-conditioned media was immunoprecipitated with anti-biotin agarose, electrophoresed and immunobloted with anti-RAGE antibody or streptavidin-HRP (STP-HRP) antibody. Biotinylated RAGE in cell-conditioned media (cRAGE) was significantly reduced by addition of GM6001 (*p<0.05) and GI254023X (**p<0.01) vs control cells (A). Cells were incubated with insulin (10mIU/ml) for 24 h in the presence or absence of inhibitors and biotinylated RAGE in cell-conditioned media was quantified. Data represent the mean ± SEM. ^#p< 0.01 vs insulin-treated cells (B).</p

Serum Level of Soluble Receptor for Advanced Glycation End Products Is Associated with A Disintegrin And Metalloproteinase 10 in Type 1 Diabetes

<div><p>Background</p><p>The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of diabetic complications, and soluble forms of the receptor (sRAGE) can counteract the detrimental action of the full-length receptor by acting as decoy. Soluble RAGE is produced by alternative splicing [endogenous secretory RAGE (esRAGE)] and/or by proteolytic cleavage of the membrane-bound receptor. We have investigated the role of A Disintegrin And Metalloproteinase 10 (ADAM10) in the ectodomain shedding of RAGE.</p><p>Methods</p><p>Constitutive and insulin-induced shedding of RAGE in THP-1 macrophages by ADAM10 was evaluated using an ADAM10-specific metalloproteinase inhibitor. Serum ADAM10 level was measured in type 1 diabetes and control subjects, and the association with serum soluble RAGE was determined. Serum total sRAGE and esRAGE were assayed by ELISA and the difference between total sRAGE and esRAGE gave an estimated measure of soluble RAGE formed by cleavage (cRAGE).</p><p>Results</p><p>RAGE shedding (constitutive and insulin-induced) was significantly reduced after inhibition of ADAM10 in macrophages, and insulin stimulated ADAM10 expression and activity. Diabetic subjects have higher serum total sRAGE and esRAGE (p<0.01) than controls, and serum ADAM10 was also increased (p<0.01). Serum ADAM10 correlated with serum cRAGE in type 1 diabetes (r = 0.40, p<0.01) and in controls (r = 0.31. p<0.01) but no correlations were seen with esRAGE. The association remained significant after adjusting for age, gender, BMI, smoking status and HbA1c.</p><p>Conclusion</p><p>Our data suggested that ADAM10 contributed to the shedding of RAGE. Serum ADAM10 level was increased in type 1 diabetes and was a significant determinant of circulating cRAGE.</p></div

Correlation between ADAM10 and cRAGE in type 1 diabetes (A) and control (B).

<p>Correlation between ADAM10 and cRAGE in type 1 diabetes (A) and control (B).</p

Clinical characteristics and serum levels of ADAM 10 and soluble RAGE isoforms in controls and type 1 diabetic patients.

<p>Values are mean ± SD, or median (interquartile range) or percentage.</p><p>*p<0.05</p><p>** p<0.01 vs controls.</p><p>Clinical characteristics and serum levels of ADAM 10 and soluble RAGE isoforms in controls and type 1 diabetic patients.</p

Effect of insulin on ADAM10 protein expression (A) and activity (B) and shedding of RAGE (C) in THP-1 macrophages.

<p>THP-1 macrophages were incubated with increasing concentrations of insulin (0 to 50 mIU/ml) or blank medium as control for 24 hours. ADAM10 protein in whole cell lysate was then measured by Western blot (A) and cellular ADAM10 activity was measured by fluorimetric assay (B). Data represent the mean ± SEM. *p<0.05, **p<0.01 vs control. Cell-conditioned media was harvested for quantification of cRAGE and experiments were repeated with the addition of specific ADAM10 inhibitor (C). *p<0.05 vs control, ^##p<0.01 vs corresponding insulin-treated cells.</p