Apelin retards the progression of diabetic nephropathy

Abstract

Apelin and its receptor APJ have pleiotropic effects in mice and humans and play a protective role in cardiovascular diseases at least partially by inhibiting oxidative stress. Our objective was to study the effect of apelin on the progression of kidney disease in mice with established type 1 diabetes. Ove26 mice with type 1 diabetes received daily subcutaneous injections of apelin for 2 or 14 wk. APJ localizes in the glomeruli and blood vessels of kidneys. Renal APJ expression was reduced in diabetic mice but increased after treatment with apelin. Apelin treatment did not affect glycemia, body weight, or blood pressure in diabetic mice. Whole kidney and glomerular hypertrophy, as well as renal inflammation, including monocyte chemoattractant protein 1 and vascular cell adhesion molecule 1 expression, NF-κB activation, and monocyte infiltration, was inhibited after short and long treatment with apelin. Apelin administration significantly reduced albuminuria at 6 mo. Short treatment with apelin was sufficient to reverse the downregulation of the antioxidant enzyme catalase. Expression of angiotensin II and angiotensin type 1 receptor (AT1) in kidneys from diabetic mice treated was not affected by apelin. These findings show for the first time that apelin exerts a protective effect on the diabetic kidney. Short administration is sufficient to reduce kidney and glomerular hypertrophy as well as renal inflammation, but prolonged treatment is required to improve albuminuria. This effect was independent of the activation of the renin angiotensin system but correlated with upregulation of the antioxidant catalase. Apelin may represent a novel tool to treat diabetic nephropathy.

Fig. 1.

Experimental design. Diabetic Ove26 mice and control littermates were treated with 1 daily subcutaneous injection of apelin (150 μg·kg⁻¹·day⁻¹) at 10 wk (2.5 mo) of age. Mice were euthanized at 12 wk (3 mo) and 24 wk (6 mo) of age.

Fig. 2.

Regulation of APJ in kidneys from diabetic mice. A: renal expression of APJ was studied by immunohistochemistry on sections of paraffin-embedded kidneys from 3-mo-old mice. Arrows point to glomeruli. Top, middle, and bottom: represent 3 different mice. B: APJ protein was measured by immunoblot on kidney cortex homogenates from 3-mo-old mice. Bottom: combined data obtained on kidneys from 5 individual mice for each experimental condition. C: APJ mRNA was measured by RT-quantitative (q)PCR on total RNA extracted from kidney cortex and normalized using GAPDH expression; n = 5 mice per group. Numbers inside brackets represent P values calculated by ANOVA; con, control.

Fig. 3.

Effect of apelin on renal hypertrophy. A: kidney weight was measured at the time of death. B: renal hypertrophy was defined as increased kidney-to-body weight ratio; n = 5 mice per group. Numbers inside brackets represent P values calculated by ANOVA; ns, not significant.

Fig. 4.

Effect of apelin on glomerular hypertrophy. A: renal histology was studied by periodic acid Schiff staining on section of paraffin-embedded kidneys. B: glomerular tuft area was traced and measured using the Image Pro Plus software. A minimum of 30 glomeruli per mouse were counted; n = 5 mice per group. Numbers inside brackets represent P values calculated by ANOVA; ns, not significant.

Fig. 5.

Effect of apelin on albuminuria. A: 24-h urine collection was performed on the eve of death; n = 5 mice per group. B: podocytes were identified as WT1⁺ nuclei in the glomeruli. WT1 was detected by immunohistochemistry on sections of frozen kidneys from 6-mo-old mice. Top, middle, and bottom: represent 3 different mice. C: WT1⁺ nuclei were counted by a blinded investigator. Numbers inside brackets represent P values calculated by ANOVA; ns, not significant. Megalin expression was assessed by immunohistochemistry on sections from paraffin-embedded kidneys (D) and by immunoblot on kidney cortex homogenates (E) from 6-mo-old mice; n = 5 mice per group.

Fig. 6.

Effect of apelin on moncyte infiltration. A: CD68⁺ cells were detected by immunohistochemistry on sections of paraffin-embedded kidneys from 3 and 6-mo-old mice. CD68⁺ nuclei were counted by a blinded investigator in glomeruli (C) and the tubulo-interstitial compartment (D). Numbers inside brackets represent P values calculated by ANOVA; ns, not significant.

Fig. 7.

Effect of apelin on mediators of renal inflammation. Monocyte attractant protein 1 (MCP1; A) and vascular cell adhesion molecule 1 (VCAM1; D) were detected by immunohistochemistry on sections of paraffin-embedded kidneys from 6-mo-old mice. Top, middle, and bottom: represent 3 different mice. MCP1 protein (B) and VCAM1 protein (E) was measured by immunoblot on kidney cortex homogenates from 6-mo-old mice. Bottom: combined data obtained on kidneys from 5 individual mice for each experimental condition. MCP1 mRNA (C) and VCAM1 mRNA (F) were measured by RT-qPCR on total RNA extracted from kidney cortex and normalized using GAPDH expression; n = 5 mice per group. Numbers inside brackets represent P values calculated by ANOVA.

Fig. 8.

Effect of apelin on NF-κB activation. A: nuclear translocation of the p65 subunit of NF-κB was assessed by immunoblot on nuclear fractions purified from kidney cortex from 6-mo-old mice. Loading was assessed by measuring expression of lamin B1. B: ratio of p65/lamin B1 in nuclear extracts. C: ratio of p-p65/p65 in nuclear extracts. D: binding of p65 to VCAM1 promoter by ChIP; n = 5 mice per group. Numbers inside brackets represent P values calculated by ANOVA.

Fig. 9.

Effect of apelin on catalase expression. A: catalase protein was detected by immunoblotting on kidney cortex homogenates from 6-mo-old mice. Bottom: densitometric analysis of catalase expression, normalized for loading using actin. B: catalase mRNA was measured by RT-qPCR on total RNA extracted from kidney cortex and normalized using GAPDH expression; n = 5 mice per group. Numbers inside brackets represent P values calculated by ANOVA.

Fig. 10.

Effect of apelin on ANG II and AT₁ receptor activation. A: ANG II content was measured by ELISA on kidney cortex homogenates from 6-mo-old mice. B: AT₁ activation was measured by immunoblot using a conformation-specific antibody under nondenaturating conditions. membranes were stripped and reprobed with antibodies directed against AT₁. C: densitometric analysis of AT₁ expression, corrected for actin. D: densitometric analysis of AT₁ activation, corrected for AT₁ expression; n = 5 mice per group.