Macrophage-derived tumor necrosis factor-α mediates diabetic renal injury

Abstract

Monocyte/macrophage recruitment correlates strongly with the progression of diabetic nephropathy. Tumor necrosis factor-α (TNF-α) is produced by monocytes/macrophages but the direct role of TNF-α and/or macrophage-derived TNF-α in the progression of diabetic nephropathy remains unclear. Here we tested whether inhibition of TNF-α confers kidney protection in diabetic nephropathy via a macrophage-derived TNF-α-dependent pathway. Compared to vehicle-treated mice, blockade of TNF-α with a murine anti-TNF-α antibody conferred kidney protection in Ins2(Akita) mice as indicated by reductions in albuminuria, plasma creatinine, histopathologic changes, kidney macrophage recruitment, and plasma inflammatory cytokine levels at 18 weeks of age. To assess the direct role of macrophage-derived TNF-α in diabetic nephropathy, we generated macrophage-specific TNF-α-deficient mice (CD11b(Cre)/TNF-α(Flox/Flox)). Conditional ablation of TNF-α in macrophages significantly reduced albuminuria, the increase in plasma creatinine and blood urea nitrogen, histopathologic changes, and kidney macrophage recruitment compared to diabetic TNF-α(Flox/Flox) control mice after 12 weeks of streptozotocin-induced diabetes. Thus, production of TNF-α by macrophages plays a major role in diabetic renal injury. Hence, blocking TNF-α could be a novel therapeutic approach for treatment of diabetic nephropathy.

Figure 1. Effects of TNF-α inhibition on renal function in Ins2^Akita mice

Ins2^Akita and their wild type littermate mice were treated with TNF-α inhibitor, captopril or vehicle for 9 weeks. Urine and plasma were collected for measurement of urine albumin/creatinine ratio (A) and plasma creatinine (B) at 18 wk of age. Data are presented as mean ± SEM. *p<0.0001 compared to normal. #p<0.01, ##p<0.0001 compared to Ins2^Akita+vehicle.

Figure 2. Effects of TNF-α inhibition on macrophage recruitment and histological changes in Ins2^Akita mice

A) Immunohistochemical staining for Mac-2 positive macrophages in glomeruli and PAS-stained sections at 18 wk of age. Images are representative of 7-15 mice in each group. B) Summary data for macrophages/glomerulus. C) Summary data for glomerular PAS score. Data are presented as mean ± SEM. *p<0.01, **p<0.0001 compared to normal. Scale bar: 10 μm.

Figure 3. Effects of TNF-α inhibition on inflammatory cytokines in Ins2^Akita mice

Ins2A^kita and their wild type littermate mice were treated with TNF-α inhibitor, captopril or vehicle for 9 weeks. Plasma levels of GMCSF, KC, TNF- α and MCP-1 were determined at 18 wks of age. Data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 compared to normal.

Figure 4. Effects of TNF-α inhibition on kidney TNF receptors expression in lns2^Akita mice

RT-PCR was performed on whole mouse kidney total RNA at 18 wk of age. TNFR1 (A) and TNFR2 (B) mRNA expression were normalized with GAPDH mRNA. Results are means ± SEM. *p<0.01 compared to normal; #p<0.05, ##p<0.01 compared to Ins2^Akita±vehicle.

Figure 5. Characterization of CD11b^Cre/TNF-α^Flox/Flox mice

Bone marrow derived (A) or peritoneal (B) macrophages were isolated from TNF-α^Flox/Flox and CD11b^Cre/TNF-α^Flox/Flox mice after treatment with LPS or vehicle for 24 hours. RNA was isolated from cells for qRT-PCR quantitation of TNF-α. Results are means ± SEM. Open bar, non-stimulated macrophages; black-filled bar, LPS-stimulated macrophages. *p<0.01, **p<0.0001 compared to non-stimulated macrophages TNF-α^Flox/Flox; #p<0.01 ##p<0.0001 compared to LPS-stimulated macrophages TNF-α^Flox/Flox.

Figure 6. Selective TNF-α depletion in macrophages reduces renal dysfunction in diabetic mice

Mouse urinary albumin/creatinine ratio (A), plasma creatinine (B), and BUN (C) were determined after 12 weeks of STZ-induced type 1 diabetes. Results are means ± SEM. Open bar, normal group; black-filled bar, diabetic group. *p<0.05, **p<0.01 compared to normal TNF-α^Flox/Flox; #p<0.05, ##p<0.01 compared to diabetic TNF-α^Flox/Flox.

Figure 7. Selective TNF-α depletion in macrophages reduces macrophage recruitment and histological changes in diabetic mice

A) Immunohistochemical staining for Mac-2 positive macrophages in glomeruli and PAS-stained sections after 12 weeks of STZ-induced type 1 diabetes. Images are representative of 7-12 mice in each group. B) Summary data for macrophages/glomerulus. C) Summary data for glomerular PAS score. Data are presented as mean ± SEM. *p<0.01 compared to normal; #p<0.05 compared to diabetes TNF-α^Flox/Flox. Scale bar: 10 μm.

Figure 8. Selective TNF-α depletion in macrophages prevented the increase in kidney TNF-α, TNFR1 and TNFR2 expression in diabetic mice

RT-PCR was performed on whole mouse kidney total RNA after 12 weeks following diabetes. TNF-α (A), TNFR1 (C), TNFR2 (E) and MCP-1 (G) mRNA expression were normalized with GAPDH mRNA. Kidney TNF-α (B), TNFR1 (D) and TNFR2 (F) proteins were determined using Elisa kits according to manufacturer's protocol. Open bar, normal group; black-filled bar, diabetic groups. Results are means ± SEM. *p<0.05; **p<0.01 compared to normal; #p<0.05; ##p<0.01 compared to diabetic TNF-α^Flox/Flox.