Blocking matrix metalloproteinase-mediated syndecan-4 shedding restores the endothelial glycocalyx and glomerular filtration barrier function in early diabetic kidney disease
Authors: Raina D. Ramnath, Matthew J. Butler, Georgina Newman, Sara Desideri, Amy Russell, Abigail C. Lay, Chris R. Neal, Yan Qiu, Sarah Fawaz, Karen L. Onions, Monica Gamez, Michael Crompton, Chris Michie, Natalie Finch, Richard J. Coward, Gavin I. Welsh, Rebecca R. Foster, Simon C. Satchell
Journal: Kidney International (2019)

The endothelial glycocalyx is a key component of the glomerular filtration barrier. We have shown that matrix metalloproteinase (MMP)-mediated syndecan 4 shedding is a mechanism of glomerular endothelial glycocalyx damage in vitro, resulting in increased albumin permeability. Here we sought to determine whether this mechanism is important in early diabetic kidney disease, by studying streptozotocin-induced type 1 diabetes in DBA2/J mice. Diabetic mice were albuminuric, had increased glomerular albumin permeability and endothelial glycocalyx damage. Syndecan 4 mRNA expression was found to be upregulated in isolated glomeruli and in flow cytometry-sorted glomerular endothelial cells. In contrast, glomerular endothelial luminal surface syndecan 4 and Marasmium oreades agglutinin lectin labelling measurements were reduced in the diabetic mice. Similarly, syndecan 4 protein expression was significantly decreased in isolated glomeruli but increased in plasma and urine, suggesting syndecan 4 shedding. Mmp-2, 9 and 14 mRNA expression were upregulated in isolated glomeruli, suggesting a possible mechanism of glycocalyx damage and albuminuria. We therefore characterised in detail the activity of MMP-2 and 9 and found significant increases in kidney cortex, plasma and urine. Treatment with MMP-2/9 inhibitor I for 21 days, started six weeks after diabetes induction, restored endothelial glycocalyx depth and coverage and attenuated diabetes-induced albuminuria and reduced glomerular albumin permeability. MMP inhibitor treatment significantly attenuated glomerular endothelial and plasma syndecan 4 shedding and inhibited plasma MMP activity. Thus, our studies confirm the importance of MMPs in endothelial glycocalyx damage and albuminuria in early diabetes and demonstrate that this pathway is amenable to therapeutic intervention. Hence, treatments targeted at glycocalyx protection by MMP inhibition may be of benefit in diabetic kidney disease. The endothelial glycocalyx is a key component of the glomerular filtration barrier. We have shown that matrix metalloproteinase (MMP)-mediated syndecan 4 shedding is a mechanism of glomerular endothelial glycocalyx damage in vitro, resulting in increased albumin permeability. Here we sought to determine whether this mechanism is important in early diabetic kidney disease, by studying streptozotocin-induced type 1 diabetes in DBA2/J mice. Diabetic mice were albuminuric, had increased glomerular albumin permeability and endothelial glycocalyx damage. Syndecan 4 mRNA expression was found to be upregulated in isolated glomeruli and in flow cytometry-sorted glomerular endothelial cells. In contrast, glomerular endothelial luminal surface syndecan 4 and Marasmium oreades agglutinin lectin labelling measurements were reduced in the diabetic mice. Similarly, syndecan 4 protein expression was significantly decreased in isolated glomeruli but increased in plasma and urine, suggesting syndecan 4 shedding. Mmp-2, 9 and 14 mRNA expression were upregulated in isolated glomeruli, suggesting a possible mechanism of glycocalyx damage and albuminuria. We therefore characterised in detail the activity of MMP-2 and 9 and found significant increases in kidney cortex, plasma and urine. Treatment with MMP-2/9 inhibitor I for 21 days, started six weeks after diabetes induction, restored endothelial glycocalyx depth and coverage and attenuated diabetes-induced albuminuria and reduced glomerular albumin permeability. MMP inhibitor treatment significantly attenuated glomerular endothelial and plasma syndecan 4 shedding and inhibited plasma MMP activity. Thus, our studies confirm the importance of MMPs in endothelial glycocalyx damage and albuminuria in early diabetes and demonstrate that this pathway is amenable to therapeutic intervention. Hence, treatments targeted at glycocalyx protection by MMP inhibition may be of benefit in diabetic kidney disease. see commentary on page 858 see commentary on page 858 Translational StatementManipulating glycocalyx, through matrix metalloproteinase (MMP) inhibition, provides an attractive therapeutic target in diabetic kidney disease. MMP inhibitor treatment is realistic in the clinical setting: tetracyclines, antibiotic agents, inhibit MMPs at subantibiotic doses and have been shown to have benefits in human disease,1Dziembowska M. Pretto D.I. Janusz A. et al.High MMP-9 activity levels in fragile X syndrome are lowered by minocycline.Am J Med Genet A. 2013; 161A: 1897-1903Crossref PubMed Scopus (122) Google Scholar,2Chang J.J. Kim-Tenser M. Emanuel B.A. et al.Minocycline and matrix metalloproteinase inhibition in acute intracerebral hemorrhage: a pilot study.Eur J Neurol. 2017; 24: 1384-1391Crossref PubMed Scopus (40) Google Scholar and low-dose doxycycline (MMP inhibitor) is licensed for the treatment of periodontitis.3Gu Y. Walker C. Ryan M.E. et al.Non-antibacterial tetracycline formulations: clinical applications in dentistry and medicine.J Oral Microbiol. 2012; 4 (Accessed March 9, 2019)https://doi.org/10.3402/jom.v4i0.19227Crossref PubMed Scopus (65) Google Scholar The development of more specific MMP inhibitors promises to reduce the adverse effects associated with broad-spectrum MMP inhibitors seen in some clinical trials. Shed glycocalyx components, including syndecan-4, warrant further investigation as potential biomarkers for diabetic kidney disease and associated cardiovascular complications. Manipulating glycocalyx, through matrix metalloproteinase (MMP) inhibition, provides an attractive therapeutic target in diabetic kidney disease. MMP inhibitor treatment is realistic in the clinical setting: tetracyclines, antibiotic agents, inhibit MMPs at subantibiotic doses and have been shown to have benefits in human disease,1Dziembowska M. Pretto D.I. Janusz A. et al.High MMP-9 activity levels in fragile X syndrome are lowered by minocycline.Am J Med Genet A. 2013; 161A: 1897-1903Crossref PubMed Scopus (122) Google Scholar,2Chang J.J. Kim-Tenser M. Emanuel B.A. et al.Minocycline and matrix metalloproteinase inhibition in acute intracerebral hemorrhage: a pilot study.Eur J Neurol. 2017; 24: 1384-1391Crossref PubMed Scopus (40) Google Scholar and low-dose doxycycline (MMP inhibitor) is licensed for the treatment of periodontitis.3Gu Y. Walker C. Ryan M.E. et al.Non-antibacterial tetracycline formulations: clinical applications in dentistry and medicine.J Oral Microbiol. 2012; 4 (Accessed March 9, 2019)https://doi.org/10.3402/jom.v4i0.19227Crossref PubMed Scopus (65) Google Scholar The development of more specific MMP inhibitors promises to reduce the adverse effects associated with broad-spectrum MMP inhibitors seen in some clinical trials. Shed glycocalyx components, including syndecan-4, warrant further investigation as potential biomarkers for diabetic kidney disease and associated cardiovascular complications. Diabetic kidney disease (DKD) is a serious complication of diabetes developing in approximately 1 in 3 people with diabetes. The cost of treating diabetic complications, including DKD, is expected to rise from the current total of £7.7 billion to £13.5 billion by fiscal year 2036 in the United Kingdom.4Hex N. Bartlett C. Wright D. et al.Estimating the current and future costs of type 1 and type 2 diabetes in the UK, including direct health costs and indirect societal and productivity costs.Diabet Med. 2012; 29: 855-862Crossref PubMed Scopus (563) Google Scholar Despite this, renin-angiotensin system blockade remains the only established treatment in slowing the progression of disease and delaying renal failure. New treatments specifically targeting key steps in disease evolution are desperately needed. The glomerular endothelium is emerging as a key player in diabetes and other glomerular diseases.5Satchell S.C. The glomerular endothelium emerges as a key player in diabetic nephropathy.Kidney Int. 2012; 82: 949-951Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar The glomerular endothelial cell (GEC) glycocalyx in particular plays an important role in glomerular barrier function.6Salmon A.H. Satchell S.C. 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Yang A.H. et al.Matrix metalloproteinase-9 deficiency attenuates diabetic nephropathy by modulation of podocyte functions and dedifferentiation.Kidney Int. 2014; 86: 358-369Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar We have previously shown that in response to tumor necrosis factor-α (TNF-α), an inflammatory mediator that is increased in diabetic milieu, SDC4 was specifically and significantly upregulated among other SDCs in human GEC in vitro.16Ramnath R. Foster R.R. Qiu Y. et al.Matrix metalloproteinase 9-mediated shedding of syndecan 4 in response to tumor necrosis factor alpha: a contributor to endothelial cell glycocalyx dysfunction.FASEB J. 2014; 28: 4686-4699Crossref PubMed Scopus (82) Google Scholar TNF-α treatment caused a disruption of the GEC glycocalyx through MMP9-mediated shedding of SDC4 and HS, and this was accompanied by an increase in protein permeability across GEC monolayers.16Ramnath R. Foster R.R. Qiu Y. et al.Matrix metalloproteinase 9-mediated shedding of syndecan 4 in response to tumor necrosis factor alpha: a contributor to endothelial cell glycocalyx dysfunction.FASEB J. 2014; 28: 4686-4699Crossref PubMed Scopus (82) Google Scholar Here we seek to determine whether this mechanism is important in glomerular disease in diabetes in vivo. We hypothesize that GEC glycocalyx dysfunction in diabetes is caused by MMP-mediated shedding of SDC4 and that this is amenable to therapeutic intervention. DBA/2J mice became hyperglycemic 2 weeks after the first of 5 daily streptozotocin STZ injections (Figure 1ai). There was no significant change in body weight in the STZ-treated mice when compared with baseline, prior to STZ injection (Figure 1aii). However, the diabetic mice gained less weight than control mice did, resulting in a 1.2-fold lower body weight after week 8 post-STZ (Figure 1aii). The mice became significantly albuminuric after week 6 post-STZ, and this persisted until the mice were killed with a 4.9-fold increase in albuminuria (Figure 1bi) at 8 week post-STZ. Compromised glomerular capillary wall integrity was confirmed by a 2-fold increase in glomerular albumin permeability (Ps'alb) (Figure 1bii). Permeability changes were associated with disruption of the glomerular glycocalyx. There was a 2-fold decrease in endothelial glycocalyx depth measured by electron microscopy but no significant change in glycocalyx coverage (Figure 1c and d). Moreover, a decrease in podocyte glycocalyx depth was observed, but there was no significant change in other ultrastructural features, including glomerular basement membrane (GBM) thickness, slit diaphragm, and foot process width (Supplementary Figure S1A–E). Moreover, picrosirius red staining showed no significant collagen deposition in diabetes when compared with control (Supplementary Figure S1F and G), confirming the electron microscopy results, suggesting no change in GBM structure. Marasmium oreades agglutinin (MOA) lectin bound specifically to the endothelial glycocalyx, on the luminal surface of the GEC (determined using the membrane label R18) (Figure 1ei). We have applied our peak-to-peak measurement technique, previously used in vivo,39Betteridge K.B. Arkill K.P. Neal C.R. et al.Sialic acids regulate microvessel permeability, revealed by novel in vivo studies of endothelial glycocalyx structure and function.J Physiol. 2017; 595: 5015-5035Crossref PubMed Scopus (71) Google Scholar,40Butler M.J. Ramnath R. Kadoya H. et al.Aldosterone induces albuminuria via matrix metalloproteinase-dependent damage of the endothelial glycocalyx.Kidney Int. 2019; 95: 94-107Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar for the first time on fixed kidney tissue (Figure 1eii). Peak-to-peak measurement of MOA labeling, an index of glycocalyx thickness, demonstrated a 1.5-fold reduction in endothelial glycocalyx thickness in diabetes that is consistent with the reduction in endothelial glycocalyx depth observed by electron microscopy (Figure 1eiii). Expression of the glomerular glycocalyx-related genes Sdcs and glycocalyx modifying genes Mmps relative to glyceraldehyde-3-phosphate dehydrogenase in untreated mice was determined. Sdc44 mRNA expression was significantly higher than other Sdc and Mmp mRNAs (Figure 2A), in line with Sdc expression in human GEC.16Ramnath R. Foster R.R. Qiu Y. et al.Matrix metalloproteinase 9-mediated shedding of syndecan 4 in response to tumor necrosis factor alpha: a contributor to endothelial cell glycocalyx dysfunction.FASEB J. 2014; 28: 4686-4699Crossref PubMed Scopus (82) Google Scholar The effect of diabetes on the expression of different components of the glycocalyx was investigated in isolated glomeruli using a custom-designed TaqMan quantitative polymerase chain reaction (qPCR) array focused on glycocalyx-related genes (Figure 2b, Supplementary Table S1) similar to that used previously.16Ramnath R. Foster R.R. Qiu Y. et al.Matrix metalloproteinase 9-mediated shedding of syndecan 4 in response to tumor necrosis factor alpha: a contributor to endothelial cell glycocalyx dysfunction.FASEB J. 2014; 28: 4686-4699Crossref PubMed Scopus (82) Google Scholar Expression Suite software (Thermo Fisher Scientific, Waltham, MA) and 2−ΔΔCT method followed by Student t test were used to analyze the array data and both methods showed a significant increase in Sdc1, Sdc4, and Mmp14 mRNA expression among other significantly modulated gene expressions (Figure 2b, Supplementary Table S2). Sdc1, -3, and -4, and Mmp14 were independently validated by real-time qPCR, demonstrating significant upregulation in all of them (Figure 2c). Although there was no significant increase in Mmp2 and -9 mRNA expression with the TaqMan qPCR array, because of previous data suggesting their importance they were further examined by individual qPCR, which showed significant increase in Mmp2 and -9 (Figure 2c). In contrast, Sdc2 was significantly downregulated (Figure 2c). Expression of Sdc4 and -1 mRNA specifically in GEC was determined in cells isolated by flow cytometry (Supplementary Figure S2). Sdc4 mRNA expression was upregulated 2.5-fold (Figure 3a), whereas Sdc1 mRNA expression was not significantly changed in diabetes (Figure 3b). To quantify endothelial glycocalyx SDC4 protein expression, an anti-SDC4 antibody was injected intravenously and shown to localize predominantly to GEC (Supplementary Figure S3). To specifically quantify the SDC4 on the luminal surface of the endothelial cells, we have used R18 labeling to highlight the endothelial membrane and enable separation of luminal from nonluminal labeling. Peak-to-peak measurement demonstrated a 2.8-fold reduction in glomerular endothelial glycocalyx SDC4 in diabetes (Figure 3c). Similarly, SDC4 expression by enzyme-linked immunosorbent assay (ELISA) was reduced 1.6-fold in diabetic glomeruli, suggesting glomerular SDC4 shedding (Figure 3d), whereas SDC1 glomerular protein expression was not changed in diabetes (Figure 3e). There was a 1.8-fold (Figure 3f) and an 11.8-fold (Figure 3g) increase in plasma and urine SDC4 concentrations, respectively, again consistent with systemic SDC4 shedding in diabetes. We measured the sheddases MMP2 and -9 and found that active MMP2 concentration was increased by 2-fold in kidney cortex lysate, 1.5-fold in plasma, and 3-fold in urine (Figure 4a–c) and active MMP9 concentration was increased by 1.8-fold in kidney cortex lysate, 1.8-fold in plasma, and 2.5-fold in urine (Figure 4d–f).Figure 4Matrix metalloproteinase (MMP) activity is altered in multiple compartments in early diabetic kidney disease (DKD). Renal cortex lysates from control and diabetic mice were used to determine (a) MMP2 (control, 5.462 ± 0.3652, n = 4 mice; diabetes, 9.797 ± 1.753, n = 6 mice) and (d) MMP9 (control, 41.34 ± 3.85, n = 4 mice; diabetes, 73.12 ± 9.85, n = 6 mice) activities using MMP2 and MMP9 Biotrak Activity Assays (GE Healthcare Life Sciences, Buckinghamshire, UK). The data were then normalized to total protein content at week 8 post-streptozotocin (STZ); **P = 0.0095, Mann-Whitney test, and **P = 0.037, unpaired Student t test were carried out, respectively. Systemic circulation of (b) plasma MMP2 (control, 2.232 ± 0.2873, n = 11 mice; diabetes, 3.285 ± 0.3760, n = 10 mice; *P = 0.0365), (c) urine MMP2 (control, 0.03142 ± 0.01251, n = 6 mice; diabetes, 0.1002 ± 0.01577, n = 10 mice; **P = 0.0091), (e) plasma MMP9 (control, 1.053 ± 0.1014, n = 6 mice; diabetes, 1.920 ± 0.2262, n = 6 mice; **P = 0.0058), and (f) urine MMP9 (control, 0.1264 ± 0.03644, n = 5; diabetes, 0.3196 ± 0.03456, n = 10 mice; **P = 0.0080 Mann-Whitney test) activities were determined in DKD. Urine MMP2 and MMP9 were normalized to urine creatinine at week 8 post-STZ. Each dot or square on the graph represents a mouse. Data are expressed as the mean ± SEM, and unpaired t test was used for statistical analysis unless specified.View Large Image Figure ViewerDownload Hi-res image Download (PPT) An MMP2 and -9 inhibitor (MMPI) was given therapeutically, after the onset of albuminuria at week 6 post-STZ, for 21 days (Figure 5a). Blockade of MMP had no significant effect on glycemia and body weight (Figure 5b and c) but significantly attenuated the diabetes-induced increase in urinary albumin creatinine ratio by 2.6-fold (Figure 5d and Supplementary Table S4). MMPI treatment reduced the diabetes-mediated increase in Ps'alb by 2-fold, restoring the permeability barrier of the glomerular capillaries (Figure 5e). MMPI also restored diabetes-induced endothelial glycocalyx loss, evidenced by a 1.6-fold increase in endothelial glycocalyx depth and a 2-fold increase in endothelial glycocalyx coverage (Figure 5f and g). MMPI treatment also resulted in a significant increase in podocyte glycocalyx depth, but there was no significant effect on GBM thickness, nor podocyte foot process and slit diaphragm width (Supplementary Figure S4A–E). MMPI treatment restored the decrease in SDC4 labeling by peak-to-peak measurement 2.2-fold (Figure 6a). It attenuated the increase in Sdc4 mRNA expression observed in diabetes by 1.5-fold (Figure 6b) and restored the loss of glomerular SDC4 protein expression by 1.8-fold (Figure 6c). MMPI significantly attenuated diabetes-induced plasma SDC4 shedding by 2-fold but not urine SDC4 shedding (Figure 6d and e). Therapeutic treatment with MMPI blocked diabetes-induced increase in plasma MMP2 and -9 activities by 2- and 1.2-fold, respectively (Figure 6f and g), confirming that this inhibitor reduces the activity of both MMPs in vivo. In this study, we investigated our hypothesis that GEC glycoca