Matrix metalloproteinase-9 expression is enhanced in renal parietal epithelial cells of zucker diabetic Fatty rats and is induced by albumin in in vitro primary parietal cell culture.

As a subfamily of matrix metalloproteinases (MMPs), gelatinases including MMP-2 and MMP-9 play an important role in remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether and how MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. In situ zymography, immunostaining and Western blot for renal MMP gelatinolytic activity and MMP-9 protein expression were performed in Zucker lean and Zucker diabetic rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of diabetic rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells (PECs) expressing claudin-1 in the diabetic kidneys. Interestingly, increased parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin) in the sclerotic area of affected glomeruli in diabetic rats. Additionally, urinary excretion of podocyte marker proteins was significantly increased in association with the levels of MMP-9 and albumin in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-9, primary cultured rat glomerular PECs were incubated with rat serum albumin (0.25 - 1 mg/ml) for 24 - 48 hrs. MMP-9 mRNA levels were significantly increased following albumin treatment. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein and activity in culture supernatants of PECs. Moreover, albumin activated p44/42 mitogen-activated protein kinase (MAPK) in PECs. Inhibition of p44/42 MAPK suppressed albumin-induced MMP-9 secretion from glomerular PECs. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a loss of adjacent podocytes in progressive diabetic nephropathy. Albumin overload may induce MMP-9 expression and secretion by PECs via the activation of p44/42 MAPK pathway

Focally increased gelatinolytic activity and MMP-9 protein in the glomeruli of Zucker diabetic fatty rats.

<p>A and B: Hematoxylin-eosin (A) and Masson’s trichrome (B) stains of paraffin kidney sections show normal glomeruli of Zucker lean (Normal), and progressive glomerulosclerosis in 20 to 28-week-old Zucker diabetic fatty rats (Diabetic-20 wk and 28 wk). C: In situ zymography shows gelatinolytic activity (bright green) in individual glomerular cells of normal rats. In diabetic rats, focally increased gelatinolytic activity was observed at the periphery of glomerulus (Diabetic-20 wk, white arrow) and in continuity onto the capillary tuft (Diabetic-28 wk, white arrow). D: Individual MMP-9 positive cells (red) are shown throughout the glomerular tuft in a normal glomerulus. Enhanced MMP-9 signal is seen in the sclerotic lesions in glomeruli of diabetic rats. E: Dual labeling for FITC-conjugated DQ-gelatin (green) and MMP-9 (red) shows a colocalization of gelatinolytic activity and MMP-9 protein in rat glomeruli.</p

Effect of albumin overload on MMP-9 production by primary glomerular PECs.

<p>Rat serum albumin (RSA 0.25–1 mg/ml) administration resulted in a dose-dependent increase in MMP-9 protein (A) and activity (B) in culture supernatants of primary rat glomerular PECs. Values are mean±SEM. An n of 4–6 epithelial cultures were treated for each condition; *<i>P</i><0.05 vs. untreated control group.</p

Inhibition of p44/42 MAPK blocks albumin-induced MMP-9 in glomerular PECs.

<p>Western blot (A) and gelatin zymography (B) analyses show that pre-treatment of PECs with U0126, a selective p44/42 MAPK inhibitor, significantly reduced MMP-9 protein and activity in culture supernatants of PECs treated with rat serum albumin (0.25 mg/ml) for 12 and 24 hrs.</p

Effects of high glucose and TGF-β1 on MMP-9 secretion by glomerular PECs.

<p>(A) MMP-9 protein and activity in culture supernatants of PECs treated with normal glucose (NG), high glucose (HG) or mannitol (Man) for 24 or 48 hrs. (B) MMP-9 protein and activity in culture supernatants of PECs treated with recombinant TGF-β1 (1 or 2 ng/ml) for 24 or 48 hrs.</p

Representative confocal images of multiple labeling for MMP-9 and mesangial or parietal epithelial cell marker proteins in the glomeruli of Zucker rats.

<p>A: In normal rat kidney tissue, MMP-9 (green) is mainly present in desmin (red)-positive mesangial cells (A-Normal). In the diabetic glomeruli, increased MMP-9 green signal was often observed in the area with low desmin staining (A-Diabetic). B: In the diabetic rats, enhanced MMP-9 (red) signal is present in parietal epithelial cells expressing claudin-1. C: Triple staining for MMP-9 (red), claudin-1 (blue) and nephrin (green) in a glomerulus of diabetic rats. D: Dual labeling for nephrin (green) and claudin-1 (red) in the diabetic kidney.</p

Increased urinary excretion of MMP-9 and podocyte marker proteins in Zucker diabetic rats.

<p>(A) Western blot analysis of renal cortical MMP-9 protein in 20-week-old Zucker lean controls and diabetic rats. (B) Representative Western blot images show a significant increase in urinary MMP-9 and podocyte marker proteins, nephrin and synaptopodin, in association with albuminuria in 20-week-old Zucker diabetic rats compared to normal controls. (C) Gelatin zymography analysis confirms a massive increase in urinary MMP-9 and MMP-2 activities in the diabetic rats.</p

Expression and localization of MMP-9 in glomerular PECs.

<p>Dual labeling using antibodies specific for MMP-9 (green) and α–tubulin (red) reveals a vesicular staining pattern that MMP-9 localizes on most microtubules (A) in rat glomerular PECs. Western blot analysis shows that primary PECs express high-level of claudin-1 and low-level of synaptopodin (B). Taqman real-time PCR analysis shows a dose-dependent increase in MMP-9 mRNA when the PECs were incubated with rat serum albumin (RSA, 0.25–1 mg/ml) for 24 hrs (C), whereas MMP-2 mRNA level was not affected. Values are mean±SEM. An n of 4–6 epithelial cultures were treated for each condition; *<i>P</i><0.05 vs. untreated control group.</p

Representative confocal images of dual labeling for MMP-9 and podocyte marker protein in the glomeruli of Zucker rats.

<p>Normal glomerulus presents nephrin (A-normal: green) and podocalyxin (B-normal: red) positive staining covering the entire capillary tuft. In the glomeruli of diabetic kidneys, neither nephrin (A-diabetic) nor podocalyxin (B-diabetic) was expressed in sclerotic lesions, where was often populated by MMP-9 positive cells. The podocyte markers, nephrin and podocalyxin, are preserved along the remaining intact portion of the glomerular tuft (white arrows). Dual labeling for nephrin and WT-1 shows that WT-1 positive cells are within nephrin-positive area in the glomerular tuft (A). A significant decrease in the number of WT-1 positive cells was observed in the diabetic glomeruli (A-Diabetic).</p

Albumin activates p44/42 MAP kinase in glomerular PECs.

<p>(A) Representative Western blots show protein levels of phospho- and total p44/42 and p38 MAP kinases in PECs in the absence or presence of rat serum albumin (RSA, 0.25 mg/ml) for 1.5, 3 or 6 hrs. (B) Quantitative data show activation of p44/42 MAPK in response to albumin stimulation. Values are mean±SEM. An n of 4 epithelial cultures were treated for each condition; *<i>P</i><0.05 vs. untreated normal control.</p