Blocking matrix metalloproteinase-mediated syndecan-4 shedding restores the endothelial glycocalyx and glomerular filtration barrier function in early 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. 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. Endothelial glycocalyx dysfunction in disease: albuminuria and increased microvascular permeability.J Pathol. 2012; 226: 562-574Crossref PubMed Scopus (204) Google Scholar,7Satchell S. The role of the glomerular endothelium in albumin handling.Nat Rev Nephrol. 2013; 9: 717-725Crossref PubMed Scopus (89) Google Scholar The endothelial glycocalyx is a hydrated poly-anionic gel present on the luminal surface of all endothelial cells and is composed principally of proteoglycans.8Reitsma S. Slaaf D.W. Vink H. et al.The endothelial glycocalyx: composition, functions, and visualization.Pflugers Arch. 2007; 454: 345-359Crossref PubMed Scopus (1212) Google Scholar Proteoglycans consist of a core protein, such as a syndecan (SDC) and glycosaminoglycan side chains, such as heparin sulfate (HS). SDC1 and -4 in particular are prominent in the kidney.9Pyke C. Kristensen P. Ostergaard P.B. et al.Proteoglycan expression in the normal rat kidney.Nephron. 1997; 77: 461-470Crossref PubMed Scopus (39) Google Scholar, 10Chen S. Wassenhove-McCarthy D. Yamaguchi Y. et al.Podocytes require the engagement of cell surface heparan sulfate proteoglycans for adhesion to extracellular matrices.Kidney Int. 2010; 78: 1088-1099Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 11Adembri C. Sgambati E. Vitali L. et al.Sepsis induces albuminuria and alterations in the glomerular filtration barrier: a morphofunctional study in the rat.Crit Care. 2011; 15: R277Crossref PubMed Scopus (60) Google Scholar, 12Rops A.L. Gotte M. Baselmans M.H. et al.Syndecan-1 deficiency aggravates anti-glomerular basement membrane nephritis.Kidney Int. 2007; 72: 1204-1215Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar SDC4 has a short cytoplasmic domain and an ectodomain that carries 3 to 5 HS chains13Manon-Jensen T. Itoh Y. Couchman J.R. Proteoglycans in health and disease: the multiple roles of syndecan shedding.FEBS J. 2010; 277: 3876-3889Crossref PubMed Scopus (237) Google Scholar, 14Multhaupt H.A. Yoneda A. Whiteford J.R. et al.Syndecan signaling: when, where and why?.J Physiol Pharmacol. 2009; 60: 31-38PubMed Google Scholar, 15Couchman J.R. Transmembrane signaling proteoglycans.Annu Rev Cell Dev Biol. 2010; 26: 89-114Crossref PubMed Scopus (297) Google Scholar and is the most abundant among the SDC family 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 Endothelial glycocalyx has multiple roles in vascular homeostasis,17Weinbaum S. Tarbell J.M. Damiano E.R. The structure and function of the endothelial glycocalyx layer.Annu Rev Biomed Eng. 2007; 9: 121-167Crossref PubMed Scopus (821) Google Scholar and its disruption contributes to several vascular diseases including diabetes.6Salmon A.H. Satchell S.C. Endothelial glycocalyx dysfunction in disease: albuminuria and increased microvascular permeability.J Pathol. 2012; 226: 562-574Crossref PubMed Scopus (204) Google Scholar, 7Satchell S. The role of the glomerular endothelium in albumin handling.Nat Rev Nephrol. 2013; 9: 717-725Crossref PubMed Scopus (89) Google Scholar, 8Reitsma S. Slaaf D.W. Vink H. et al.The endothelial glycocalyx: composition, functions, and visualization.Pflugers Arch. 2007; 454: 345-359Crossref PubMed Scopus (1212) Google Scholar,18Singh A. Friden V. Dasgupta I. et al.High glucose causes dysfunction of the human glomerular endothelial glycocalyx.Am J Physiol Renal Physiol. 2011; 300: F40-F48Crossref PubMed Scopus (97) Google Scholar Our group and others have shown that the glomerular endothelial glycocalyx contributes to the barrier to albumin permeability in cultured cells18Singh A. Friden V. Dasgupta I. et al.High glucose causes dysfunction of the human glomerular endothelial glycocalyx.Am J Physiol Renal Physiol. 2011; 300: F40-F48Crossref PubMed Scopus (97) Google Scholar, 19Singh A. Ramnath R.D. Foster R.R. et al.Reactive oxygen species modulate the barrier function of the human glomerular endothelial glycocalyx.PLoS One. 2013; 8e55852Crossref PubMed Scopus (115) Google Scholar, 20Singh A. Satchell S.C. Neal C.R. et al.Glomerular endothelial glycocalyx constitutes a barrier to protein permeability.J Am Soc Nephrol. 2007; 18: 2885-2893Crossref PubMed Scopus (207) Google Scholar and in vivo.21Jeansson M. Haraldsson B. Morphological and functional evidence for an important role of the endothelial cell glycocalyx in the glomerular barrier.Am J Physiol Renal Physiol. 2006; 290: F111-F116Crossref PubMed Scopus (164) Google Scholar, 22Salmon A.H. Ferguson J.K. Burford J.L. et al.Loss of the endothelial glycocalyx links albuminuria and vascular dysfunction.J Am Soc Nephrol. 2012; 23: 1339-1350Crossref PubMed Scopus (173) Google Scholar, 23Jeansson M. Haraldsson B. Glomerular size and charge selectivity in the mouse after exposure to glucosaminoglycan-degrading enzymes.J Am Soc Nephrol. 2003; 14: 1756-1765Crossref PubMed Scopus (115) Google Scholar, 24Desideri S. Onions K.L. Qiu Y. et al.A novel assay provides sensitive measurement of physiologically relevant changes in albumin permeability in isolated human and rodent glomeruli.Kidney Int. 2018; 93: 1086-1097Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Loss of glycosaminoglycan within the glomerular capillary wall is seen in albuminuric diabetic rats25Satoh M. Kobayashi S. Kuwabara A. et al.In vivo visualization of glomerular microcirculation and hyperfiltration in streptozotocin-induced diabetic rats.Microcirculation. 2010; 17: 103-112Crossref PubMed Scopus (30) Google Scholar and diabetic mice.26Jeansson M. Granqvist A.B. Nystrom J.S. et al.Functional and molecular alterations of the glomerular barrier in long-term diabetes in mice.Diabetologia. 2006; 49: 2200-2209Crossref PubMed Scopus (43) Google Scholar,27Oltean S. Qiu Y. Ferguson J.K. et al.Vascular endothelial growth factor-A165b is protective and restores endothelial glycocalyx in diabetic nephropathy.J Am Soc Nephrol. 2015; 26: 1889-1904Crossref PubMed Scopus (101) Google Scholar In humans, systemic glycocalyx dimensions are reduced in diabetes.28Nieuwdorp M. Mooij H.L. Kroon J. et al.Endothelial glycocalyx damage coincides with microalbuminuria in type 1 diabetes.Diabetes. 2006; 55: 1127-1132Crossref PubMed Scopus (307) Google Scholar,29Broekhuizen L.N. Lemkes B.A. Mooij H.L. et al.Effect of sulodexide on endothelial glycocalyx and vascular permeability in patients with type 2 diabetes mellitus.Diabetologia. 2010; 53: 2646-2655Crossref PubMed Scopus (267) Google Scholar Loss of systemic and glomerular glycocalyx is associated with the development of microalbuminuria in diabetes, suggesting that glycocalyx dysfunction contributes to the pathogenesis of this condition.6Salmon A.H. Satchell S.C. Endothelial glycocalyx dysfunction in disease: albuminuria and increased microvascular permeability.J Pathol. 2012; 226: 562-574Crossref PubMed Scopus (204) Google Scholar,30Satchell S.C. Tooke J.E. What is the mechanism of microalbuminuria in diabetes: a role for the glomerular endothelium?.Diabetologia. 2008; 51: 714-725Crossref PubMed Scopus (251) Google Scholar Glycocalyx components are cleaved from the cell surface by "sheddases" including matrix metalloproteinase (MMPs).13Manon-Jensen T. Itoh Y. Couchman J.R. Proteoglycans in health and disease: the multiple roles of syndecan shedding.FEBS J. 2010; 277: 3876-3889Crossref PubMed Scopus (237) Google Scholar,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 Gelatinases MMP2 and -9 can be activated by membrane type 1 MMP, also known as MMP14. We and others have shown that diabetic conditions induce upregulation of endothelial MMP9,16Ramnath 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 MMP2,29Broekhuizen L.N. Lemkes B.A. Mooij H.L. et al.Effect of sulodexide on endothelial glycocalyx and vascular permeability in patients with type 2 diabetes mellitus.Diabetologia. 2010; 53: 2646-2655Crossref PubMed Scopus (267) Google Scholar and urinary MMP1431Lauhio A. Sorsa T. Srinivas R. et al.Urinary matrix metalloproteinase -8, -9, -14 and their regulators (TRY-1, TRY-2, TATI) in patients with diabetic nephropathy.Ann Med. 2008; 40: 312-320Crossref PubMed Scopus (38) Google Scholar; their enzymatic activities are elevated in diabetic human31Lauhio A. Sorsa T. Srinivas R. et al.Urinary matrix metalloproteinase -8, -9, -14 and their regulators (TRY-1, TRY-2, TATI) in patients with diabetic nephropathy.Ann Med. 2008; 40: 312-320Crossref PubMed Scopus (38) Google Scholar,32Romanic A.M. Burns-Kurtis C.L. Ao Z. et al.Upregulated expression of human membrane type-5 matrix metalloproteinase in kidneys from diabetic patients.Am J Physiol Renal Physiol. 2001; 281: F309-F317Crossref PubMed Google Scholar and mouse33Qing-Hua G. Ju-Ming L. Chang-Yu P. et al.The kidney expression of matrix metalloproteinase-9 in the diabetic nephropathy of Kkay mice.J Diabetes Complications. 2008; 22: 408-412Crossref PubMed Scopus (15) Google Scholar kidneys. The dysregulation of MMP2 and -9 activities has been associated in the pathophysiology of several diabetic comorbidities.34Derosa G. Avanzini M.A. Geroldi D. et al.Matrix metalloproteinase 2 may be a marker of microangiopathy in children and adolescents with type 1 diabetes mellitus.Diabetes Res Clin Pract. 2005; 70: 119-125Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 35Chung A.W. Hsiang Y.N. Matzke L.A. et al.Reduced expression of vascular endothelial growth factor paralleled with the increased angiostatin expression resulting from the upregulated activities of matrix metalloproteinase-2 and -9 in human type 2 diabetic arterial vasculature.Circ Res. 2006; 99: 140-148Crossref PubMed Scopus (117) Google Scholar, 36Thrailkill K.M. Bunn R.C. Moreau C.S. et al.Matrix metalloproteinase-2 dysregulation in type 1 diabetes.Diabetes Care. 2007; 30: 2321-2326Crossref PubMed Scopus (90) Google Scholar Urinary MMP2 and -9 concentration and activity are increased in type 1 diabetic patients with albuminuria.36Thrailkill K.M. Bunn R.C. Moreau C.S. et al.Matrix metalloproteinase-2 dysregulation in type 1 diabetes.Diabetes Care. 2007; 30: 2321-2326Crossref PubMed Scopus (90) Google Scholar, 37Diamant M. Hanemaaijer R. Verheijen J.H. et al.Elevated matrix metalloproteinase-2 and -9 in urine, but not in serum, are markers of type 1 diabetic nephropathy.Diabet Med. 2001; 18: 423-424Crossref PubMed Scopus (28) Google Scholar, 38Li S.Y. Huang P.H. 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