SH inhibition of NRK52E cell glucotoxicity depends on the AMPK-SIRT1-PGC1alpha axis and inflammatory signals.

<p>NRK52E cells were stimulated by HG with or without SH and analyzed of phosphorylated AMPK, Sirt1, PGC1α and Cldn1 expressions (A and B). Andinflammatory molecules such as phosphorylated p38 and phosphorylated JNK changes were analyzed in HG with or without SH cultured NRK52E cells (C and D). LG, 5.5 mM D-glucose; HG, 30 mM D-glucose; SH, sarpogrelate hydrochloride. Values shown are mean ± SEM. ^*<i>p</i> < 0.05 vs. LG, ^†<i>p</i> < 0.05 vs. HG.</p

Sarpogrelate hydrochloride ameliorates diabetic nephropathy associated with inhibition of macrophage activity and inflammatory reaction in <i>db/db</i> mice

<div><p>The aim of this study was to evaluate the effects of sarpogrelate hydrochloride (SH), a selective serotonin 2A receptor antagonist, on diabetic nephropathy in a type 2 diabetes mouse model. We treated <i>db/m</i> and <i>db/db</i> mice with SH (30 mg/kg/day) for 12 weeks. Rat renal proximal tubule cells (NRK-52E) and mouse macrophages (Raw 264.7) were stimulated by high glucose (30 mM glucose) or LPS (100 ng/ml) with or without SH (20 μM). We found that SH treatment increased serum adiponectin level and decreased urinary albumin, macrophage infiltration to glomeruli, and renal inflammatory and fibrosis signals, which were highly expressed in diabetic mice. Proximal tubule cells treated with high glucose (30 mM) also showed increased inflammatory and fibrosis signals. However, SH (20 μM) treatment reduced these changes. Moreover, SH treatment inhibited LPS-stimulated macrophage migration and activation. These findings suggest that SH ameliorates diabetic nephropathy not only by suppressing macrophage infiltration, but also by anti-inflammatory and anti-fibrotic effects.</p></div

Effects of SH on the progression of diabetic nephropathy.

<p>After the 24 h urine collected by metabolic cages, albuminuria determined (A). Representative examples of glomerular hematoxylin and eosin (H&E)-stained sections (B). Differences of glomerular volume among four groups (C). Representative transmission electron microscope image of renal ultra stuctures (D). The differences of slit pore numbers (E) and glomerular basement membrane (GBM) thickness (F) among four groups. Original magnification: H&E staining, 400× and transmission electron microscopy, 30K×. NC, normal mice group; NC+SH, normal mice with sarpogrelate hydrochloride treatment group; DB, diabetic mice group; DB+SH, diabetic mice with sarpogrelate hydrochloride treatment group; UAE, urine albumin excretion. Values shown are mean ± SEM. ^*<i>p</i> < 0.05 vs. NC, ^†<i>p</i> < 0.05 vs. DB. <i>n</i> = 7 per group.</p

Influences of SH on physiological parameters in non-diabetic and diabetic mice.

<p>Influences of SH on physiological parameters in non-diabetic and diabetic mice.</p

Western blot analysis of the renal cortex after treatment with sarpogrelate hydrochloride.

<p>The renal expression of nephrin and VEGF was analyzed by Western blotting (A). The relative expression of nephrin (B) and VEGF (C) were analyzed by ImageJ software. The expression of renal Cldn1 and Sirt1 was analyzed by IHC (D) and western blotting (E).The difference renal nephrin and VEGF levels among four groups. NC, normal mice group; NC+SH, normal mice with sarpogrelate hydrochloride treatment group; DB, diabetic mice group; DB+SH, diabetic mice with sarpogrelate hydrochloride treatment group. Values shown are mean ± SEM. ^*<i>p</i> < 0.05 vs. NC, ^†<i>p</i> < 0.05 vs. DB. <i>n</i> = 6 per group.</p

Comparison of biochemical characteristics of experimental animals.

<p>Comparison of biochemical characteristics of experimental animals.</p

SH reduces macrophage migration and activation.

<p>The differences of cell migration rate (A and C) and morphological change (B) by LPS with or without SH in Raw264.7 cells. (B) Change of phosphorylated p38 and MCP1 expressions by SH treatment in macrophage cultured media stimulated NRK52E cells (D). CON, control; LPS, lipopolysaccharides; SH, sarpogrelate hydrochloride; CM, conditioned medium. Values shown are mean ± SEM. ^*<i>p</i> < 0.05 vs. CON, ^†<i>p</i> < 0.05 vs. LPS.</p

SH ameliorates renal fibrosis in DN.

<p>Collagen accumulation in glomeruli was analyzed by picrosirius red staining in the kidney sections (A) and fibrosis-related molecules such as β-catenin, snail, and TGF-β (B, C, D and E) were analyzed by western blotting. NC, normal mice group; NC+SH, normal mice with sarpogrelate hydrochloride treatment group; DB, diabetic mice group; DB+SH, diabetic mice with sarpogrelate hydrochloride treatment group. Values shown are mean ± SEM. ^*<i>p</i> < 0.05 vs. NC, ^†<i>p</i> < 0.05 vs. DB. <i>n</i> = 6 per group.</p

SH inhibits the 5HT-2A receptor and MCP1 in RAW264.7 cells.

<p>Representative immunofluorescence staining of 5HT-2A receptor (A) and western blotting of 5HT-2A receptor and MCP1 levels (B and C)in Raw264.7 cells. CON, control; LPS, lipopolysaccharides; SH, sarpogrelate hydrochloride. Values shown are mean ± SEM. ^*<i>p</i> < 0.05 vs. CON, ^†<i>p</i> < 0.05 vs. LPS.</p

Lower Serum Creatinine Is Associated with Low Bone Mineral Density in Subjects without Overt Nephropathy

<div><p>Background</p><p>Low skeletal muscle mass is associated with deterioration of bone mineral density. Because serum creatinine can serve as a marker of muscle mass, we evaluated the relationship between serum creatinine and bone mineral density in an older population with normal renal function.</p><p>Methods</p><p>Data from a total of 8,648 participants (4,573 men and 4,075 postmenopausal women) aged 45–95 years with an estimated glomerular filtration rate >60 ml/min/1.73 m2 were analyzed from the Fourth Korea National Health and Nutrition Examination Survey (2008–2010). Bone mineral density (BMD) and appendicular muscle mass (ASM) were measured using dual-energy X-ray absorptiometry. Receiver operating characteristic curve analysis revealed that the cut points of serum creatinine for sarcopenia were below 0.88 mg/dl in men and 0.75 mg/dl in women. Subjects were divided into two groups: low creatinine and upper normal creatinine according to the cut point value of serum creatinine for sarcopenia.</p><p>Results</p><p>In partial correlation analysis adjusted for age, serum creatinine was positively associated with both BMD and ASM. Subjects with low serum creatinine were at a higher risk for low BMD (T-score ≤ –1.0) at the femur neck, total hip and lumbar spine in men, and at the total hip and lumbar spine in women after adjustment for confounding factors. Each standard deviation increase in serum creatinine was significantly associated with reduction in the likelihood of low BMD at the total hip and lumbar spine in both sexes (men: odds ratio (OR) = 0.84 [95% CI = 0.74−0.96] at the total hip, OR = 0.8 [95% CI = 0.68−0.96] at the lumbar spine; women: OR = 0.83 [95% CI = 0.73–0.95] at the total hip, OR=0.81 [95% CI = 0.67–0.99] at the lumbar spine).</p><p>Conclusions</p><p>Serum creatinine reflected muscle mass, and low serum creatinine was independently associated with low bone mineral density in subjects with normal kidney function.</p></div