Protective Effects of Thalidomide on High-Glucose-Induced Podocyte Injury through In Vitro Modulation of Macrophage M1/M2 Differentiation

Abstract

Objective. It has been shown that podocyte injury represents an important pathological basis that contributes to proteinuria and eventually leads to kidney failure. High glucose (HG) activates macrophage polarization, further exacerbating HG-induced podocyte injury. Our previous study on diabetic nephropathy rats indicated that thalidomide (Tha) has renoprotective properties. The present study explored the effects of Tha on mRNA and protein expressions of inducible nitric oxide synthase (iNOS), tumor necrosis factor- (TNF-) α, mannose receptor (CD206), and arginase- (Arg-) 1 in HG-activated macrophages. iNOS and TNF-α are established as markers of classically activated macrophage (M1). CD206 and Arg-1 are regarded as markers of alternatively activated macrophages (M2). During the experiment, the supernatants of (HG)-treated and (Tha)-treated macrophages, designated as (HG) MS and (Tha) MS, were simultaneously collected and processed. TNF-α and interleukin- (IL-) 1β levels as well as protein expressions of nephrin and podocin in HG, (HG) MS, and (Tha) MS-cultured podocytes were evaluated. The results showed that compared to the 11.1 mM normal glucose (NG), the 33.3 mM HG-cultured RAW 264.7 cells exhibited upregulated iNOS and TNF-α mRNAs and protein expressions, and downregulated CD206 and Arg-1 expressions significantly (p < 0.05). Tha 200 μg/ml suppressed iNOS and TNF-α, and promoted CD206 and Arg-1 expressions significantly compared to the HG group (p < 0.05). Furthermore, (HG) MS-treated podocytes showed an increase in TNF-α and IL-1β levels and a downregulation in nephrin and podocin expression significantly compared to NG-treated and HG-treated podocytes (p < 0.05). The (Tha 200 μg/ml) MS group exhibited a decrease in TNF-α and IL-1β level, and an upregulation in nephrin and podocin expressions significantly compared to the (HG) MS group (p < 0.05). Our research confirmed that HG-activated macrophage differentiation aggravates HG-induced podocyte injury in vitro and the protective effects of Tha might be related to its actions on TNF-α and IL-1β levels via its modulation on M1/M2 differentiation.

Figure 1

Effects of high glucose on nitric oxide production in RAW 264.7 cells. Values were expressed as the mean ± standard error of the mean (n = 6). ^#p < 0.05; LPS versus DMSO after 12, 24, and 48 hours' treatment separately. ^∗p < 0.05; 33.3 and 44.4 mM glucose versus 11.1 mM glucose after 12, 24, and 48 hours' treatment separately. ^{^}p < 0.05; 33.3 and 44.4 mM glucose versus LPS after 12 hours' treatment. ^&p < 0.05, 11.1 mM glucose versus DMSO after 48 hours' treatment. Abbreviations: LPS: lipopolysaccharide; DMSO: dimethyl sulfoxide, the solvent control of LPS.

Figure 2

Effects of thalidomide on cell viability of podocyte and macrophage in 33.3 mM glucose. Values were expressed as the mean ± standard error (n = 6). ^∗p < 0.05; versus 11.1 mM glucose. Abbreviations: Tha25: 25 μg/ml thalidomide; Tha50: 50 μg/ml thalidomide; Tha100: 100 μg/ml thalidomide; Tha200: 200 μg/ml thalidomide.

Figure 3

Effects of thalidomide on iNOS, CD206, Arg-1 and TNF-α protein expression in 33.3 mM glucose-induced macrophage. (a) iNOS and CD206 protein expressions. (d) Arg-1 protein expression. (f) TNF-α protein expression. The results of iNOS, CD206, Arg-1, and TNF-α were represented in (b), (c), (e), and (g), respectively. All results were expressed as a ration with respect to control and represented as the mean ± SD in triplicates. ^∗p < 0.05; versus 11.1 mM glucose. ^#p < 0.05; versus 33.3 mM glucose. ^&p < 0.05; versus Tha100. ^{^}p < 0.05; versus Tha50. Abbreviations: LPS: lipopolysaccharide; Tha50: 50 μg/ml thalidomide in 33.3 mM glucose; Tha100: 100 μg/ml thalidomide in 33.3 mM glucose; Tha200: 200 μg/ml thalidomide in 33.3 mM glucose; iNOS: inducible nitric oxide synthase; CD206: mannose receptor; TNF-α: tumor necrosis factor-α; Arg-1: arginase-1.

Figure 4

Effects of thalidomide on iNOS, CD206, Arg-1, and TNF-α mRNA expressions in 33.3 mM glucose-induced macrophage. (a) iNOS mRNA expression. (b) CD206 mRNA expression. (c) CD206 mRNA expression. (d) TNF-α mRNA expression.All the results were represented as the mean ± SD in triplicates ^∗p < 0.05; versus 11.1 mM glucose. ^#p < 0.05; versus 33.3 mM glucose. ^&p < 0.05; versus Tha100. ^{^}p < 0.05; versus Tha50. Abbreviations: LPS: lipopolysaccharide; Tha50: 50 μg/ml thalidomide in 33.3 mM glucose; Tha100: 100 μg/ml thalidomide in 33.3 mM glucose; Tha200: 200 μg/ml thalidomide in 33.3 mM glucose; iNOS: inducible nitric oxide synthase; CD206: mannose receptor; TNF-α: tumor necrosis factor-α; Arg-1: arginase-1.

Figure 5

Effects of thalidomide on TNF-α and IL-1β level in podocyte. (a) TNF-α level. (b) IL-1β level. Values were expressed as the mean ± standard error of the mean (n = 6). ^∗p < 0.05; versus 11.1 mM glucose. ^#p < 0.05; versus (33.3 mM glucose) MS. ^&p < 0.05; versus 33.3 mM glucose. Abbreviations: (33.3 mM glucose) MS: the supernatant from 33.3 mM glucose-treated macrophage; (Tha50) MS: supernatant from 50 μg/ml thalidomide and 33.3 mM glucose-treated macrophage; (Tha100) MS: supernatant from 100 μg/ml thalidomide and 33.3 mM glucose-treated macrophage; (Tha200) MS: supernatant from 200 μg/ml thalidomide and 33.3 mM glucose-treated macrophage.

Figure 6

Effects of thalidomide on nephrin and podocin protein expressions in podocyte. (a) Nephrin expression. (c) Podocin expression. The results of nephrin and podocin were represented in (b) and (d). ^∗p < 0.05; versus 11.1 mM glucose. ^#p < 0.05; versus (33.3 mM glucose) MS. ^&p < 0.05; versus 33.3 mM glucose. Abbreviations: (33.3 mM glucose) MS: the supernatant from 33.3 mM glucose-treated macrophage. (Tha50) MS: supernatant from 50 μg/ml thalidomide and 33.3 mM glucose-treated macrophage; (Tha100) MS: supernatant from 100 μg/ml thalidomide and 33.3 mM glucose-treated macrophage; (Tha200) MS: supernatant from 200 μg/ml thalidomide and 33.3 mM glucose-treated macrophage.