Urocortin ameliorates diabetic nephropathy in obese db/db mice

Abstract

Background and purpose: Hyperglycaemia induces overproduction of mitochondrial reactive oxygen species (ROS) in endothelial cells, which is believed to be a major molecular mechanism underlying complications of diabetes, including diabetic nephropathy. Impairment of endothelium-dependent vasodilatation is found in type 2 diabetes. Urocortin is a 40 amino-acid peptide related to the corticotrophin-releasing factor (CRF) family, which suppresses production of ROS in endothelial cells and sustains endothelium-dependent relaxations of rat coronary artery. However, it is not clear if urocortin has any effect on diabetic nephropathy.

Experimental approach: Possible mechanisms underlying the effects of urocortin on diabetic nephropathy were investigated in db/db mice and cultured rat mesangial cells.

Key results: Urocortin decreased body weight, plasma levels of advanced glycation end-products, blood urea nitrogen and creatinine levels. However, food intake, plasma insulin and glucose levels remained unaffected. Superoxide dismutase activity was increased markedly, whereas malonaldehyde levels in kidney homogenate and sorbitol concentrations in red blood cells were decreased significantly in urocortin-treated mice. Urocortin significantly decreased glomerular extracellular matrix expansion and accumulation in kidney. Moreover, urocortin inhibited the overexpression of transforming growth factor-beta 1 and connective tissue growth factor in rat mesangial cells induced by 25 mM glucose. All the effects of urocortin, except sorbitol accumulation, were abolished by the non-selective CRF receptor blocker, astressin.

Conclusion and implications: Urocortin could significantly ameliorate diabetic nephropathy and this effect was mediated via the CRF receptor.

Figure 1

Effects of urocortin on body weight and food intake in db/db mice. (a) Body weight of db/db mice was decreased by urocortin (7 μg kg⁻¹, ip). Pretreatment with astressin (35 μg kg⁻¹, ip) prevented the effect of urocortin. (b) Food intake was not affected either by treatment with urocortin or by urocortin+astressin. Control, nondiabetic control; model, db/db mice=diabetic control; urocortin, diabetic mice (db/db mice) treated with urocortin (7 μg kg⁻¹, ip); urocortin+astressin, diabetic mice (db/db) treated with urocortin and (7 and 35 μg kg⁻¹ respectively, ip). ^*P<0.05, ^**P<0.01 compared with model.

Figure 2

Effects of urocortin on plasma insulin, blood glucose, BUN, creatinine, AGE level and RBC sorbitol concentrations in db/db mice. Plasma insulin level (a) and blood glucose level (b) were not affected by urocortin (7 μg kg⁻¹, ip) and urocortin (7 μg kg⁻¹, ip)+astressin (35 μg kg⁻¹, ip); Plasma BUN (c) and creatinine (d) level of db/db mice were decreased by urocortin; Plasma AGE level (e) and RBC sorbitol concentration (f) were decreased by urocortin. Pretreatment with astressin abolished the effect of urocortin on AGE, BUN and creatinine but not its effect on sorbitol. Control, nondiabetic control; model, db/db mice=diabetic control; urocortin, diabetic mice (db/db mice) treated with urocortin (7 μg kg⁻¹, ip); urocortin+astressin, diabetic mice (db/db) treated with urocortin and (7 and 35 μg kg⁻¹ respectively, ip).^*P<0.05, ^**P<0.01 compared with model; ^#P<0.05 compared with urocortin.

Figure 3

Urocortin-induced changes in SOD activity and MDA level in kidney homogenates of db/db mice. SOD activity (a) was increased and MDA level (b) was decreased by urocortin. Astressin abolished the effect of urocortin on SOD activity and MDA level. Control, nondiabetic control; model, db/db mice=diabetic control; urocortin, diabetic mice (db/db mice) treated with urocortin (7 μg kg⁻¹, ip); urocortin+astressin, diabetic mice (db/db) treated with urocortin and (7 and 35 μg kg⁻¹ respectively, ip). ^*P<0.05, ^**P<0.01 compared with model; ^#P<0.05 compared with urocortin.

Figure 4

Protective effect of urocortin on glomerular histology in db/db mice. (a) Pathology score; (b) Per cent mesangial area; Representative micrographs of H&E-stained paraffin sections showed effect of urocortin on glomerular histology in db/db mice (c–f; magnification × 400). (c) Glomeruli of db/m mice; (d) Glomeruli of db/db mice; (e) Glomeruli of db/db mcie treated with urocortin (7 μg kg⁻¹); (f) Glomeruli of db/db mice treated with urocortin (7 μg kg⁻¹)+astressin (35 μg kg⁻¹); Representative micrographs of PAS-stained paraffin sections showed effect of urocortin on ECM expansion and accumulation (g–j; magnification × 400). (g) Glomeruli of nondiabetic control mice; (h) Glomeruli of db/db mice; (i) Glomeruli of db/db mice treated with urocortin (7 μg kg⁻¹); (j) Glomeruli of db/db mice treated with urocortin (7 μg kg⁻¹)+astressin (35 μg kg⁻¹). Treatment with urocortin clearly decreased the ECM expansion and accumulation. Although astressin treatment did not result in statistically significant reversal of the effect of urocortin, the degree of ECM expansion and accumulation was more severe. Control, nondiabetic control; model, db/db mice=diabetic control; urocortin, diabetic mice (db/db mice) treated with urocortin (7 μg kg⁻¹, ip); urocortin+astressin, diabetic mice (db/db) treated with urocortin and (7 and 35 μg kg⁻¹ respectively, ip). ^*P<0.05; ^**P<0.01 compared with model. ▵, expanded mesangial area; ▴, accumulated ECM.

Figure 5

Urocortin-induced reduction in transforming growth factor-β1 (TGF-β1) expression in mesangial cells (MC). Expression of TGF-β1 was detected by immunocytochemistry (magnification, × 200). MC were cultured in serum-free Dulbecco's modified Eagle's medium (DMEM) containing 5 mM glucose or 25 mM glucose with urocortin (10⁻⁹ M) or urocortin (10⁻⁹ M) + astressin (5 × 10⁻⁹ M). (a) Normal DMEM; (b) DMEM contained 25 mM glucose; (c) DMEM contained 25 mM glucose and urocortin; (d) DMEM contained 25 mM glucose and urocortin+astressin; (e) Negative control. Overexpression of TGF-β1 was inhibited by urocortin and pretreatment with astressin abolished this effect.

Figure 6

Urocortin-induced reduction in connective tissue growth factor (CTGF) expression in rat mesangial cells (MC). Expression of CTGF was detected by immunocytochemistry (magnification, × 200). MC were cultured in serum-free DMEM containing 5 mM glucose or 25 mM glucose with urocortin (10⁻⁹ M) or urocortin (10⁻⁹ M)+astressin (5 × 10⁻⁹ M). (a) Normal DMEM. (b) DMEM contained 25 mM glucose. (c) DMEM containing 25 mM glucose and urocortin. (d) DMEM containing 25 mM glucose and urocortin+astressin. (e) Negative control. The overexpression of CTGF stimulated by the high glucose medium was inhibited by urocortin and pretreatment with astressin reversed this effect.