Endothelin A receptor blockade reduces diabetic renal injury via an anti-inflammatory mechanism

Abstract

Endothelin (ET) receptor blockade delays the progression of diabetic nephropathy; however, the mechanism of this protection is unknown. Therefore, the aim of this study was to test the hypothesis that ET(A) receptor blockade attenuates superoxide production and inflammation in the kidney of diabetic rats. Diabetes was induced by streptozotocin (diabetic rats with partial insulin replacement to maintain modest hyperglycemia [HG]), and sham rats received vehicle treatments. Some rats also received the ETA antagonist ABT-627 (sham+ABT and HG+ABT; 5 mg/kg per d; n = 8 to 10/group). During the 10-wk study, urinary microalbumin was increased in HG rats, and this effect was prevented by ET(A) receptor blockade. Indices of oxidative stress, urinary excretion of thiobarbituric acid reactive substances, 8-hydroxy--deoxyguanosine, and H2O2 and plasma thiobarbituric acid reactive substances were significantly greater in HG rats than in sham rats. These effects were not prevented by ABT-627. In addition, renal cortical expression of 8-hydroxy--deoxyguanosine and NADPH oxidase subunits was not different between HG and HG+ABT rats. ETA receptor blockade attenuated increases in macrophage infiltration and urinary excretion of TGF-beta and prostaglandin E2 metabolites in HG rats. Although ABT-627 did not alleviate oxidative stress in HG rats, inflammation and production of inflammatory mediators were reduced in association with prevention of microalbuminuria. These observations indicate that ETA receptor activation mediates renal inflammation and TGF-beta production in diabetes and are consistent with the postulate that ETA blockade slows progression of diabetic nephropathy via an anti-inflammatory mechanism.

Figure 1

Urinary ET-1 (A) and microalbumin (B) excretion in Sham, Sham rats treated with ABT-627 (Sham+ABT), HG, and HG rats treated with ABT-627 (HG+ ABT). Values are means ± SE. * p< 0.05 vs sham, † p<0.05 vs HG+ABT, n=4–6.

Figure 2

Mean arterial pressure and heart rate in conscious Sham, Sham+ABT, HG, and HG+ABT rats during the 10-wk course of study. Values are means for 24 hour periods, reported every 5 days. * p< 0.05 vs sham, † p<0.05 ABT vs untreated, n=4–6.

Figure 3

Representative images of renal histology of Sham (A, D, and G), HG (B, E, and H) and HG+ABT rats (C, F, and I) rats after 10 weeks of treatment (original magnification = 40X). Periodic Acid-Schiff stain (A, B, and C), Gomori’s trichrome stain (D, E, and F), and picro Sirius red staining (G, H, and I).

Figure 4

Indices of oxidative stress in Sham, Sham+ABT, HG and HG+ABT rats (n=5–11). Urinary excretion of (A) hydrogen peroxide and (B) TBARS (MDA equivalents) were measured at 2, 6, and 10 weeks after injection of STZ or vehicle. (C) Plasma TBARS and 8-hydroxy-2’-deoxyguanosine (8-OHdG) excretion measured at the 10 wk time point. Typical immunohistochemical localization of 8-OHdG is provided for the renal cortex of (D) HG and (E) HG+ABT. (F) Western blot analysis of renal cortical expression of the NADPH oxidase subunits p22phox, p47phox, and p67phox in each group, normalized to β–actin. * p< 0.05 vs sham

Figure 5

Pro-fibrotic and inflammatory markers in Sham, Sham+ABT, HG, and HG+ABT rats. (A) Urinary excrretion of TGF-β (n=4–5; * p<0.05 vs Sham, † p<0.05 vs HG+ABT). (B) Quantification of ED-1⁺ cells in the renal cortex.

Figure 6

Prostaglandin excretion by Sham, Sham+ABT, HG, and HG+ABT rats. (A) Time course of PGEM excretion. (B) 6-keto PGF_1α and TxB₂ excretion at the 10 week time point. (n=4–5; * p<0.05 vs Sham, † p<0.05 vs HG)

Figure 7

Renal cortical expression of cyclooxygenase-2.after 10 weeks of treatment. (A) Representative Western Blot. (B) Densitometric analysis (normalized to β-actin). n=5–6. Immunohistochemical localization of cyclooxygenase-2 in the renal cortex of Sham (C), HG (D) and HG+ABT rats (E) after 10 weeks of treatment. Panel F is a negative control (no primary antibody).