Endocytosis of Albumin Induces Matrix Metalloproteinase-9 by Activating the ERK Signaling Pathway in Renal Tubule Epithelial Cells

Abstract

Matrix metalloproteinase-9 (MMP-9) is dysregulated in chronic kidney diseases including diabetic nephropathy. This study was performed to examine the expression of MMP-9 in renal tubule epithelial cells (TECs) under diabetic conditions and its regulatory mechanisms. We characterized MMP-9 protein in diabetic animals and primary cultured rat TECs exposed to exogenous albumin and high glucose. We also used specific inhibitors to determine if internalization of albumin and/or extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation were required for MMP-9 secretion. Immunostaining of kidney sections revealed enhanced MMP-9 signal in the damaged proximal tubules in Zucker diabetic fatty (ZDF) rats. ZDF rats also exhibited an albuminuria-related and age-dependent increase in MMP-9 excretion, which was prevented by rosiglitazone. In primary cultured rat TECs, high glucose exposure did not increase MMP-9 secretion. In contrast, administration of rat serum albumin (RSA, 0.1-0.5 mg/mL) resulted in a dose-dependent increase in MMP-9 expression and secretion by TECs, which was abolished in the presence of an ERK1/2-specific inhibitor, U0126. Simvastatin, an inhibitor of albumin endocytosis, also prevented MMP-9 secretion. Taken together, these results demonstrate that endocytosis of albumin stimulates MMP-9 secretion by TECs through the ERK signaling pathway.

Keywords: MMP-9; U0126; albuminuria; chronic kidney disease; diabetes; simvastatin.

Figure 1

Albumin aggregation in proximal tubule epithelial cells of diabetic kidneys. Dual labeling using antibodies specific for rat albumin (green) and aquaporin-1 (AQP-1, red) reveals an accumulation of albumin in the AQP-1-positive tubules of 20-week-old Zucker diabetic fatty (ZDF) rats compared to Zucker lean (ZL) normal controls. Cell nuclei were stained by DAPI (blue).

Figure 2

Increased tubular expression and urinary excretion of MMP-9 protein in ZDF rats. (A) Cell nuclei were stained by DAPI (blue). Double immunofluorescence staining shows that MMP-9 (green) signal was enhanced on the basolateral membrane of damaged tubules with strong kidney injury molecule-1 (KIM-1) (red) staining in 20-week-old ZDF rats. As expected, KIM-1 staining was absent in normal ZL kidneys. Weak linear MMP-9 staining was detected along normal tubules; (B) Representative Western blotting images of MMP-9 and Ponceau red staining show a gradual increase in MMP-9 protein in the urine collected from nine- to 20-week-old ZDF rats, whereas MMP-9 was not detectable in the urine of ZL rats; (C) Urinary albumin levels were determined in 24-h urine collected from five- to 20-week-old ZL and ZDF rats. Values are mean ± SEM; n = 4–7; * p < 0.01 vs. age-matched ZL controls.

Figure 3

Effect of rosiglitazone on blood glucose, urinary albumin, and body weight in Zucker rats. Rosiglitazone reduced blood glucose (A) and urinary albumin (B) levels but increased body weight (C) in ZDF rats. Values are mean ± SEM. n = 4–7 rats; * p < 0.05 and ** p < 0.01 compared with their ZL controls (ZDF vs. ZL or Ros-ZDF vs. Ros-ZL); ^# p < 0.05 compared to vehicle-treated ZDF group (Ros-ZDF vs. ZDF).

Figure 4

Effect of rosiglitazone on urinary gelatinolytic activity and MMP-9 protein in ZDF rats. (A) Representative gelatin zymograph shows a decrease in urinary MMP-9 and MMP-2 activity in ZDF rats following rosiglitazone treatment; (B) Western blot confirms a decrease in urinary MMP-9 protein in rosiglitazone-treated ZDF rats compared to vehicle-treated ZDF ones.

Figure 5

Albumin endocytosis and megalin expression in primary rat TEC. Rat TECs were incubated with 100 µg/mL FITC-BSA at 37 °C for 1 h (A: 1-h) or 3 h (A: 3-h) and then washed and fixed. Fluorescence was visualized by confocal microscopy. Representative Western blot images show megalin protein expression in primary TECs (B).

Figure 6

Albumin increased MMP-9 protein expression and secretion by primary rat TECs. Rat serum albumin (RSA 0.1–0.5 mg/mL) administration for 48 h resulted in a dose-dependent increase in MMP-9 protein in culture supernatants (A,C) and cell homogenates (A,D) of primary cultured rat TECs; (B) Representative confocal image shows MMP-9 signal is predominantly present on the cell surface of TECs. Values are mean ± SEM. n = 4; * p < 0.05 vs. untreated control group.

Figure 7

MMP-9 activity was increased in culture supernatants of primary rat TECs upon albumin stimulation. Gelatin zymography analysis confirms a dose-dependent increase in MMP-9 activity in the culture supernatants when the cells were incubated with rat serum albumin (RSA, 0.1–0.5 mg/mL) for 24 h (A) or 48 h (A,B), whereas MMP-2 activity was not altered. Values are mean ± SEM. n = 4; * p < 0.05 vs. untreated control group.

Figure 8

Effect of high glucose on secretion TGF-β1 and MMP-9 by primary rat TECs. (A) Representative Western blotting images show TGF-β1 protein in culture supernatants of TECs treated with normal glucose (NG), high glucose (HG) and mannitol (M) for 24 h. Ponceau red staining was used as loading control; (B) Gelatin zymograph shows MMP-2 and MMP-9 activities in culture supernatants of TECs treated with normal glucose, high glucose, or mannitol for 24 h.

Figure 9

Albumin-induced MMP-9 was inhibited by U0126 in primary rat TECs. The cells were exposed to RSA (0.5 mg/mL) for 24 h in the absence or presence of U0126. (A) U0126 abolished albumin-induced MMP-9 protein in TECs; (B) MMP-9 activity was increased in culture supernatant of RSA-treated TECs, which was suppressed in the presence of U0126. Values are mean ± SEM. n = 4; * p < 0.05 vs. unstimulated normal control; ^# p < 0.05 vs. vehicle-treated albumin-stimulated cells.

Figure 10

Albumin-induced MMP-9 production was attenuated by simvastatin treatment in primary rat TECs. (A) Representative confocal images show decreased FITC-BSA signal in simvastatin-treated TECs compared to DMSO vehicle controls; (B) Western blot analysis of MMP-9 protein in TECs treated with RSA (0.5 mg/mL) for 24 h in the absence or presence of simvastatin (10 µM); (C) Gelatin zymograph of MMP-2 and MMP-9 activities in the culture supernatants of TECs. Values are mean ± SEM, n = 4; * p < 0.05 vs. unstimulated normal control; ^# p < 0.05 vs. vehicle-pretreated albumin-stimulated cells.