Renal-protective effect of thalidomide in streptozotocin-induced diabetic rats through anti-inflammatory pathway

Abstract

Background: Diabetic nephropathy (DN) is a major microvascular complication in diabetes. An increasing body of evidence has shown that DN is related to chronic inflammation, kidney hypertrophy, and fibrosis. While thalidomide has been shown to have anti-inflammatory and antifibrotic effects, the effects of thalidomide on the pathogenesis of DN are unclear. This study was undertaken to explore whether thalidomide has renal-protective effects in diabetic rats.

Methods: Male Sprague Dawley rats were injected intraperitoneally with 50 mg/kg streptozotocin to induce diabetes. Diabetic rats were treated with thalidomide (200 mg/kg/d) for 8 weeks, and then blood and urine were collected for measurement of renal function-related parameters. Histopathology, immunohistochemistry, enzyme-linked immunosorbent assay, and Western blot analyses were performed to assess renal proinflammatory cytokines, fibrotic protein, and related signaling pathways.

Results: Diabetic rats exhibited obvious renal structural and functional abnormalities, as well as renal inflammation and fibrosis. Compared with diabetic control rats, those treated with thalidomide showed significantly improved histological alterations and biomarkers of renal function, as well as reduced expression of renal inflammatory cytokines, including NF-κB and MCP-1. Furthermore, renal fibrotic proteins, such as TGF-β1, TβRII, TβRI, smad3, collagen IV, and fibronectin were also remarkably suppressed. Treatment with thalidomide markedly stimulated the phosphorylation of AMPKα.

Conclusion: In this study, thalidomide suppressed the inflammatory and fibrotic processes in DN. These effects were partly mediated by the activation of AMPKα, and inhibition of the NF-κB/MCP-1 and TGF-β1/Smad signaling pathways. These results suggest that thalidomide may have therapeutic potential in diabetic renal injury through the anti-inflammatory pathway.

Keywords: AMPK; NF-κB; TGF-β1; diabetic nephropathy; thalidomide.

Figure 1

Renal histology after treatment with or without thalidomide. Notes: (A) Representative micrographs of H&E, PAS, and Masson staining. Original magnification: 400×. (B) Quantitative analyses of mesangial area and Masson staining of glomerular and tubulointerstitial lesions. Values are expressed as mean ± SEM (n=5). ^#P<0.01 versus NC group; **P<0.01 versus DC group. Abbreviations: DC, diabetic control; H&E, hematoxylin and eosin; NC, normal control; PAS, periodic acid-Schiff; SEM, standard error of the mean; Thd, thalidomide.

Figure 2

Expression of TGF-β1, MCP-1, and NF-κB in kidneys. Notes: (A) Immunohistochemistry of TGF-β1, MCP-1, and NF-κB. Original magnification 400×. (B) TGF-β1, MCP-1, and NF-κB expression were semiquantitatively analyzed. Values are expressed as mean ± SEM (n=5). ^#P<0.01 versus NC group; **P<0.01 versus DC group. Abbreviations: DC, diabetic control; NC, normal control; SEM, standard error of the mean; Thd, thalidomide.

Figure 3

Expression of collagen IV and fibronectin in kidneys. Notes: (A) Immunohistochemistry of collagen IV and fibronectin. Original magnification 400×. (B) Collagen IV and fibronectin expression were semiquantitatively analyzed. Values are expressed as mean ± SEM (n=5). ^#P<0.01 versus NC group; *P<0.05 versus DC group; **P<0.01 versus DC group. Abbreviations: DC, diabetic control; NC, normal control; SEM, standard error of the mean; Thd, thalidomide.

Figure 4

Effects of thalidomide on the expression of TGF-β1, Smad3, and p-Smad3 by Western blot analysis. Notes: (A) TGF-β1, Smad3, and p-Smad3 expression in kidneys. (B) Band density analysis of TGF-β1, Smad3, and p-Smad3. Values are expressed as mean ± SEM (n=5). ^#P<0.01 versus NC group; *P<0.05 versus DC group; **P<0.01 versus DC group. Abbreviations: DC, diabetic control; NC, normal control; p-Smad3, phospho-Smad3; SEM, standard error of the mean; TGF-β1, transforming growth factor β1; Thd, thalidomide.

Figure 5

Effects of thalidomide on the expression of TβRII and TβRI by Western blot analysis. Notes: (A) TβRII and TβRI expression in kidneys. (B) Band density analysis of TβRII and TβRI. Values are expressed as mean ± SEM (n=5). ^#P<0.01 versus NC group; *P<0.05 versus DC group; **P<0.01 versus DC group. Abbreviations: DC, diabetic control; NC, normal control; SEM, standard error of the mean; TβRII, transforming growth factor-β receptor II; TβRI, transforming growth factor-β receptor I; Thd, thalidomide.

Figure 6

Effects of thalidomide on the expression of NF-κB, AMPKα, and p-AMPKα by Western blot analysis and the level of p-AMPKα by ELISA. Notes: (A) NF-κB expression in kidneys. (B) Band density analysis of NF-κB. (C) AMPKα and p-AMPKα expression in kidneys. (D) Band density analysis of p-AMPKα/AMPKα. (E) p-AMPKα expression in kidneys by ELISA. Values are expressed as mean ± SEM (n=4). ^#P<0.01 versus NC group; *P<0.05 versus DC group; **P<0.01 versus DC group. Abbreviations: AMPKα, AMP-activated protein kinase α; DC, diabetic control; NC, normal control; NF-κB, nuclear factor-κB; p-AMPKα, phospho-AMPKα; SEM, standard error of the mean; Thd, thalidomide.

Figure 7

Schematic representation of the proposed mechanism by which thalidomide reduces ECM accumulation through the activation of AMPKα and suppression of NF-κB/MCP-1 and TGF-β1/Smad3 signaling pathways. Abbreviations: AMPKα, AMP-activated protein kinase α; ECM, extracellular matrix; MCP-1, monocyte chemoattractant protein-1; NF-κB, nuclear factor-κB; TGF-β1, transforming growth factor β1.