Matrix metalloproteinase 9 Contributes to Glomerulosclerosis by Causing Profibrotic Changes in Podocytes and Glomerular Endothelial Cells

Research Article

Austin J Nephrol Hypertens - Volume 8 Issue 1 - 2021

Matrix metalloproteinase 9 Contributes to Glomerulosclerosis by Causing Profibrotic Changes in Podocytes and Glomerular Endothelial Cells

Qiao X1,2#, Guo J1,3#, Chen J1#, Loron MC1,4, Zhao Y1,5, Rao P1, Cao Q1, Wang Y1, Harris DCH1 and Zheng G1*

1Centre for Transplant and Renal Research, The Westmead Institute for Medical Research, the University of Sydney, Sydney, NSW, Australia

2Department of Nephrology, Second Hospital of Shanxi Medical University, Shanxi Kidney Disease Institute, Taiyuan, Shanxi, People’s Republic of China

3Department of Pathophysiology, Shanxi Medical University, Taiyuan, Shanxi, People’s Republic of China

4Department of Nephrology, Rouen University Hospital, University of Paris, Paris, France

5The School of Biomedical Sciences, Chengdu Medical College, Chengdu, People’s Republic of China

#Contributed Equally to this Work and are co-First Authors

*Corresponding author: Zheng G, Centre for Transplantation and Renal Research, The Westmead Institute for Medical Research, University of Sydney, Sydney, NSW 2145, Australia

Received: May 18, 2021; Accepted: June 29, 2021; Published: July 06, 2021


Background: Glomerulosclerosis is characterized by progressive (myo) fibroblast accumulation and collagen deposition involving profibrotic changes of podocytes and endothelial cells. A profibrotic role of MMP-9 in interstitial fibrosis has been reported. Whether MMP-9 plays a role in glomerulosclerosis is not clear yet.

Methods: Mouse glomerulosclerosis model [Adriamycin Nephropathy (AN) model] was induced by a single adriamycin injection (10.2mg/kg, with physiological saline for controls) through tail vein in MMP-9-/- and wild-type control mice of BALB/c background. All animals were sacrificed at 4 weeks after injection. Albuminuria (albumin to creatinine ratio) and calculated GFR were measured. Gomori Trichrome (GT) and Sirius Red (SR) staining were used for assessment of glomerular fibrosis. Profibrotic changes of podocytes or glomerular endothelial cells were examined by confocal microscopy using immunofluorescence staining (IF) of desmin or a-SMA with P-cadherin or VEcadherin.

Results: Albuminuria was reduced while GFR was increased in MMP-9-/- AN mice compared with those of wild-type mice. Confocal microscopy showed a significant decrease in podocytes double-stained with P-cadherin and desmin, demonstrating that MMP9-/- AN mice were protected from profibrotic changes in podocytes and glomerular endothelial cells. Glomerulosclerosis was significantly reduced in MMP9-/- AN mice compared to that of WT, as demonstrated by GT and SR staining.

Conclusions: MMP-9 contributes to glomerulosclerosis at least in part by causing profibrotic changes in podocytes and glomerular endothelial cells.

Keywords: Matrix metalloproteinase 9; Glomerulosclerosis; Podocyte; Endothelial cell


CKD: Chronic Kidney Disease; EMT: Epithelial-Mesenchymal Transition; EMT: Endothelial-Mesenchymal Transition; EndMT: Podocyte and Endothelial-Mesenchymal Transition; GBM: Glomerular Basement Membrane; VE-cadherin: Vascular Endothelial–Cadherin; MMP-9: Matrix Metalloproteinase 9; AN: Adriamycin Nephropathy; GFR: Glomerulus Filtration Rate; SR: Sirius Red; GT: Gomori Trichrome


Glomerulosclerosis is a hallmark of Chronic Kidney Disease (CKD) [1,2]. It is characterized by the accumulation of myofibroblasts and excessive deposition of extracellular matrix components. Myofibroblasts are the key effectors in glomerulosclerosis. Both Epithelial-Mesenchymal Transition (EMT) of podocyte and endothelial-Mesenchymal Transition (EndMT) are major sources of myofibroblasts formation in kidney fibrosis [3,4].

Podocytes are terminally differentiated visceral epithelial cells that are critical components of the glomerular barrier and play an important role in selective permeability of the glomerular filtration barrier. Previous study indicated that podocyte damage leads to glomerulosclerosis [5]. Accumulating evidence indicates that in response to injurious stimuli, podocytes may undergo an EMT process, lose their epithelial surface markers such as P-cadherin, and express mesenchymal markers such as desmin. Podocytes were rendered motile after EMT, resulting in detachment from the Glomerular Basement Membrane (GBM) and podocyte loss, not apoptosis, and finally leading to a defective glomerular filtration, proteinuria and glomerulosclerosis [6].

Renal endothelial cells, especially glomerular endothelial cells, contribute to fibroblast formation in kidney by EndMT [7]. EndMT may be a notable source of activated fibroblasts or myofibroblasts [8]. During EndMT, endothelial cells eliminate endothelial markers, such as vascular endothelial–cadherin (VE-cadherin), and acquire mesenchymal markers, such as a-Smooth Muscle Actin (a-SMA) [9]. Previous studies have indicated that glomerular sclerosis were related to EndMT, and inhibition of EndMT could prevent glomerular sclerosis [10].

Matrix Metalloproteinase 9 (MMP-9) has been proven to cause kidney interstitial fibrosis [11]. However, whether it plays a role in glomerulosclerosis is not clear yet. We have demonstrated that MMP-9 induced EndMT in mouse peritubular endothelial cells downstream of TGF-β1 [11], indicating that it may also contribute to glomerulosclerosis. In the present study, we aim to investigate the role of MMP-9 in the development of glomerulosclerosis. This study hypothesized that MMP-9 may induce EMT process of podocytes and EndMT process of glomerular endothelial cells, thereby leading to glomerular sclerosis.

Materials and Methods

Animals and adriamycin-induced nephropathy model

Mouse Adriamycin Nephropathy (AN) was induced by a single injection of adriamycin (10.2 mg/kg, with physiological saline for controls) through tail vein in MMP-9 knockout (MMP-9-/-, BALB/c background) and wild-type control mice. All animals were sacrificed at 4 weeks after injection. Experiments were carried out in accordance with the protocols approved by the Animal Ethics Committee of Western Sydney Local Health District.

Urinary proteinuria concentration and kidney function

Urinary albumin to creatinine ratio was used to evaluate proteinuria. Urinary albumin concentration was determined by nephelometric method as reported [12]. Urinary creatinine concentration was determined by enzymatic method. Blood samples were taken from mice before sacrifice.

For calculated GFR detecting, mice were acclimatised to the metabolism cages for 48 h prior to 24 h urine collection. Urine samples were collected in metabolism cages 24 h before sacrifice. Serum creatinine levels were determined using the creatinine assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. Calculated GFR was evaluated by creatinine clearance using the standard formula.

Histological analysis

Four weeks after adriamycin treatment, paraffin-embedded kidney sections (4 μm) were deparaffinised with xylene and rehydrated through a descending ethanol gradient. Histological sections were examined following Sirius Red (SR) or Gomori Trichrome (GT) staining. Quantification of pulmonary and kidney fibrosis was performed as we described previously [13]. All scoring was performed in a blinded manner.

Immunofluorescence analysis

Frozen kidney blocks were cut into 7 μm sections and fixed with ice-cold acetone for 10 min at -20°C and blocked with 2% BSA for 1 h. Double immunofluorescence staining was performed using combinations of antibodies of P-cadherin and desmin, VE-cadherin and a-SMA, respectively. Tissue sections were then incubated with its corresponding fluorescence-conjugated secondary antibody. After washing with PBS, sections were counterstained with DAPI for 5 min before mounting with the fluorescence mounting medium. Images were obtained using a confocal microscope (Olympus FV1000) at ×40 magnification. For quantitative analysis, the percentage of the area stained positive for P-cadherin, desmin, or VE-cadherin, a-SMA, were counted on High Power Fields (HPFs) in a blinded manner.

Statistical analysis

Results were expressed as Mean ± SEM. Statistical significance was evaluated using unpaired two-tailed t-test for comparison between two groups. The level of significance was set at p<0.05.


MMP-9 knockout reduces albuminuria and improves kidney function in mice

The urinary albumin/creatinine ratio and GFR were not different between wild-type control mice and MMP-9-/- controls. Adriamycin injection resulted in massive proteinuria in MMP-9 wild-type mice. However, it induced less pronounced albuminuria in MMP-9-/- mice (Figure 1A). Mice treated with adriamycin had markedly decreased GFR compared with normal animals. MMP-9-/- mice were protected from developing renal impairment, their GFR was significantly higher than wild-type AN mice (Figure 1B).