Apelin promotes diabetic nephropathy by inducing podocyte dysfunction via inhibiting proteasome activities

Abstract

Podocyte injuries are associated with progression of diabetic nephropathy (DN). Apelin, an adipocyte-derived peptide, has been reported to be a promoting factor for DN. In this study, we aim to determine whether apelin promotes progression of DN by inducing podocyte dysfunction. kk-Ay mice were used as models for DN. Apelin and its antagonist, F13A were intraperitoneally administered for 4 weeks, respectively. Renal function and foot process proteins were analysed to evaluate the effects of apelin on kk-Ay mice and podocytes. Apelin increased albuminuria and decreased podocyte foot process proteins expression in kk-Ay mice, which is consistent with the results that apelin receptor (APLNR) levels increased in glomeruli of patients or mice with DN. In cultured podocytes, high glucose increased APLNR expression and apelin administration was associated with increased permeability and decreased foot process proteins levels. All these dysfunctions were associated with decreased 26S proteasome activities and increased polyubiquitinated proteins in both kk-Ay mice and cultured podocytes, as demonstrated by 26S proteasome activation with cyclic adenosine monophosphate (cAMP) or oleuropein. These effects seemed to be related to endoplasmic reticulum (ER) stress, as apelin increased C/EBP homologous protein (CHOP) and peiFα levels while cAMP or oleuropein reduced it in high glucose and apelin treated podocytes. These results suggest that apelin induces podocyte dysfunction in DN through ER stress which was induced by decreased proteasome activities in podocytes.

Keywords: APLNR; ER stress; apelin; diabetic nephropathy; podocyte; proteasome.

Figure 1

Increased APLNR level in mice and human kidney. (A) Immunostating of APLNR in mice kidney. (B) The APLNR levels in the kidney of type 2 diabetic mice (kk-Ay mice) was significantly increased by 2.7-fold compared with that of control mice (C57BL). The data are expressed as the means ± SD (n = 6, *p < 0.01 versus control group). (C) APLNR level in the kidney of type 2 diabetic patients was significantly increased by 1.9-fold compared with that of control patients. The data are expressed as the means ± SD (n = 6, *p < 0.01 versus control group).

Figure 2

Apelin aggravated renal dysfunction. (A) Apelin significantly increased the albuminuria both in kk-Ay and control mice while F13A reversed it in kk-Ay mice (n = 10). (B) Apelin decreased Ccr in kk-Ay mice while F13A reveresed it (n = 10). (C) Representative photographs of kidney section from different groups. Apelin significantly increased the thickening of the GBM and podocyte effacement in kk-Ay mice while F13A reversed it. (D) Scoring of the severity of the deposits in PAS staining of mice in every group. The score represents the mean percentage of the total of 50 glomeruli affected (grade 0–4; n = 12). (E) Glycaemic levels of the different groups of mice before and after apelin or F13A treatment (n = 12). *p < 0.01 versus C57BL mice of 12 weeks old, ^#p < 0.01 versus C57BL mice of 16 weeks old, ^Δp < 0.01 versus kk-Ay mice of 16 weeks old.

Figure 3

The effects of apelin on foot process proteins in kk-Ay mice. (A) Representative images of immunostaining for synapotopodin in kidneys. (B) Representative images of western blotting for foot process proteins. (C) Graphs show densitometry of the ratio of nephrin, podocin, ZO-1, WT-1 and synapotopodin to GAPDH in mouse kidney (mean ± SD, n = 3, *p < 0.05).

Figure 4

The effects of apelin on permeability and levels of foot process proteins in podocytes. (A) Albumin permeability was increased by HG and aggravated by apelin while reversed by F13A (n = 6). (B) Representative images of western blotting for foot process proteins in podocytes. (C) Graphs show densitometry of the ratio of nephrin, podocin, ZO-1, WT-1 and synapotopodin to GAPDH in podocytes (n = 3; mean ± SD, *p < 0.05).

Figure 5

Apelin impairs proteasome peptidase activities in kk-Ay mice. (A) Polyubiquitinated proteins were increased in kk-Ay mice and aggravated by apelin while reversed by F13A (n = 3). (B) 26S proteasome activities are indicated as percentages of those in C57BL mice. All proteasome activities decreased in kk-Ay mice compared with C57BL mice and aggravated by apelin while reversed by F13A (n = 8; mean ± SD, *p < 0.05 versus control, ^#p < 0.05 versus kk-Ay mice).

Figure 6

Apelin impairs proteasome peptidase activities in podocytes. (A) Polyubiquitinated proteins were increased in high glucose (HG:25mM D-glucose) treated podocytes and aggravated by apelin while reversed by F13A (n = 3). (B) 26S proteasome activities are indicated as percentages of those in normal glucose (NG: 5.5mM D-glucose). All proteasome activities decreased in HG treated podocytes compared with NG and aggravated by apelin while reversed by F13A (n = 6). (C) Immunosubunits of proteasome were decreased in HG treated podocytes and further decreased by apelin while reversed by F13A (n = 3; mean ± SD, *p < 0.05 versus control group, #p < 0.05 versus HG treated group).

Figure 7

Apelin aggravated podocytes injuries through inhibiting proteasome activities. (A) Apelin increased the albumin permeability of podocytes treated with HG, which was inhibited by cAMP (n = 6). (B) Representative images of western blotting for foot process proteins in podocytes. (C) Graphs show densitometry of the ratio of nephrin, podocin, ZO-1, WT-1 and synapotopodin to GAPDH in podocytes (n = 3). (D) Representative images of immunostaining for synapotopodin in podocytes (mean ± SD, *p < 0.05).

Figure 8

Apelin stimulated endoplasmic reticulum stress in podocytes, which was inhibited by cAMP or oleuropein. (A and C) Representative images of western blotting for eif2α and CHOP. (B and D) Graphs show densitometry of the ratio of eIF2α and CHOP to GAPDH in podocytes (mean ± SD, n = 3, *p < 0.05 versus control group, #p < 0.05 versus HG treated group).