Endothelin receptor-A mediates degradation of the glomerular endothelial surface layer via pathologic crosstalk between activated podocytes and glomerular endothelial cells

Emerging evidence of crosstalk between glomerular cells in pathological settings provides opportunities for novel therapeutic discovery. Here we investigated underlying mechanisms of early events leading to filtration barrier defects of podocyte and glomerular endothelial cell crosstalk in the mouse models of primary podocytopathy (podocyte specific transforming growth factor-β receptor 1 signaling activation) or Adriamycin nephropathy. We found that glomerular endothelial surface layer degradation and albuminuria preceded podocyte foot process effacement. These abnormalities were prevented by endothelin receptor-A antagonism and mitochondrial reactive oxygen species scavenging. Additional studies confirmed increased heparanase and hyaluronoglucosaminidase gene expression in glomerular endothelial cells in response to podocyte-released factors and to endothelin-1. Atomic force microscopy measurements showed a significant reduction in the endothelial surface layer by endothelin-1 and podocyte-released factors, which could be prevented by endothelin receptor-A but not endothelin receptor-B antagonism. Thus, our studies provide evidence of early crosstalk between activated podocytes and glomerular endothelial cells resulting in loss of endothelial surface layer, glomerular endothelial cell injury and albuminuria. Hence, activation of endothelin-1-endothelin receptor-A and mitochondrial reactive oxygen species contribute to the pathogenesis of primary podocytopathies in experimental focal segmental glomerulosclerosis. Emerging evidence of crosstalk between glomerular cells in pathological settings provides opportunities for novel therapeutic discovery. Here we investigated underlying mechanisms of early events leading to filtration barrier defects of podocyte and glomerular endothelial cell crosstalk in the mouse models of primary podocytopathy (podocyte specific transforming growth factor-β receptor 1 signaling activation) or Adriamycin nephropathy. We found that glomerular endothelial surface layer degradation and albuminuria preceded podocyte foot process effacement. These abnormalities were prevented by endothelin receptor-A antagonism and mitochondrial reactive oxygen species scavenging. Additional studies confirmed increased heparanase and hyaluronoglucosaminidase gene expression in glomerular endothelial cells in response to podocyte-released factors and to endothelin-1. Atomic force microscopy measurements showed a significant reduction in the endothelial surface layer by endothelin-1 and podocyte-released factors, which could be prevented by endothelin receptor-A but not endothelin receptor-B antagonism. Thus, our studies provide evidence of early crosstalk between activated podocytes and glomerular endothelial cells resulting in loss of endothelial surface layer, glomerular endothelial cell injury and albuminuria. Hence, activation of endothelin-1-endothelin receptor-A and mitochondrial reactive oxygen species contribute to the pathogenesis of primary podocytopathies in experimental focal segmental glomerulosclerosis. Translational StatementOur findings provide compelling evidence that podocyte activation and pathologic crosstalk with endothelial cells via endothelin-1 results in dysfunction and loss of endothelial surface layer (glycocalyx), and this event may underlie early albuminuria in early glomerular disease. Our findings provide compelling evidence that podocyte activation and pathologic crosstalk with endothelial cells via endothelin-1 results in dysfunction and loss of endothelial surface layer (glycocalyx), and this event may underlie early albuminuria in early glomerular disease. Microalbuminuria represents an early biological marker of endothelial dysfunction, and endothelial cell dysfunction can drive progression of kidney disease.1Stehouwer C.D. Smulders Y.M. Microalbuminuria and risk for cardiovascular disease: analysis of potential mechanisms.J Am Soc Nephrol. 2006; 17: 2106-2111Crossref PubMed Scopus (336) Google Scholar, 2Fioretto P. Stehouwer C.D. Mauer M. et al.Heterogeneous nature of microalbuminuria in NIDDM: studies of endothelial function and renal structure.Diabetologia. 1998; 41: 233-236Crossref PubMed Scopus (88) Google Scholar The glomerular endothelium is covered with an endothelial surface layer (ESL) that consists of a membrane bound glycocalyx and a "cell coat." The glycocalyx is composed of a negatively charged network of proteoglycans, glycoproteins, and glycolipids anchored to the endothelium and a more loosely attached cell coat that aid in maintaining the charge selective glomerular barrier function and prevent proteinuria.3Haraldsson B. Nystrom J. The glomerular endothelium: new insights on function and structure.Curr Opin Nephrol Hypertens. 2012; 21: 258-263Crossref PubMed Scopus (65) Google Scholar, 4Jeansson M. Bjorck K. Tenstad O. et al.Adriamycin alters glomerular endothelium to induce proteinuria.J Am Soc Nephrol. 2009; 20: 114-122Crossref PubMed Scopus (131) Google Scholar, 5Singh 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 Loss of the ESL may contribute to endothelial dysfunction in renal and cardiovascular disease progression.6Satchell S. The role of the glomerular endothelium in albumin handling.Nat Rev Nephrol. 2013; 9: 717-725Crossref PubMed Scopus (89) Google Scholar, 7Rabelink T.J. de Zeeuw D. The glycocalyx—linking albuminuria with renal and cardiovascular disease.Nat Rev Nephrol. 2015; 11: 667-676Crossref PubMed Scopus (111) Google Scholar Transforming growth factor-β (TGF-β) and its signal transducers play important roles in the development of various kidney diseases and are important mediators of renal injury and fibrosis in progressive chronic kidney disease.8Okuda S. Languino L.R. Ruoslahti E. et al.Elevated expression of transforming growth factor-beta and proteoglycan production in experimental glomerulonephritis: possible role in expansion of the mesangial extracellular matrix.J Clin Invest. 1990; 86: 453-462Crossref PubMed Scopus (531) Google Scholar, 9Yamamoto T. Nakamura T. Noble N.A. et al.Expression of transforming growth factor beta is elevated in human and experimental diabetic nephropathy.Proc Natl Acad Sci U S A. 1993; 90: 1814-1818Crossref PubMed Scopus (813) Google Scholar TGF-βexpression in podocytes is a stress response signal associated with sclerosis lesions,10Kim J.H. Kim B.K. Moon K.C. et al.Activation of the TGF-beta/Smad signaling pathway in focal segmental glomerulosclerosis.Kidney Int. 2003; 64: 1715-1721Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 11Casalena G. Bottinger E. Daehn I. TGFbeta-induced actin cytoskeleton rearrangement in podocytes is associated with compensatory adaptation of mitochondrial energy metabolism.Nephron. 2015; 131: 278-284Crossref PubMed Scopus (11) Google Scholar apoptosis, and podocyte depletion.12Schiffer M. Bitzer M. Roberts I.S. et al.Apoptosis in podocytes induced by TGF-beta and Smad7.J Clin Invest. 2001; 108: 807-816Crossref PubMed Scopus (544) Google Scholar, 13Schiffer M. Mundel P. Shaw A.S. et al.A novel role for the adaptor molecule CD2-associated protein in TGF-beta-induced apoptosis.J Biol Chem. 2004; : 37004-37012Crossref PubMed Scopus (180) Google Scholar In a mouse model of focal segmental glomerulosclerosis (FSGS) where podocyte-specific TGF-β receptor 1 (TGF-βR1) activation induces progressive glomerular disease and renal failure, we have earlier demonstrated release of endothelin-1 (Edn1) by podocytes, followed by increased endothelin receptor-A (EdnrA)–mediated mitochondrial oxidative stress and dysfunction of adjacent glomerular endothelial cells (GECs), which in response, release factor(s) that mediate damage and depletion of adjacent podocytes.14Daehn I. Casalena G. Zhang T. et al.Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis.J Clin Invest. 2014; 124: 1608-1621Crossref PubMed Scopus (189) Google Scholar A similar stressed endothelial-to-podocyte crosstalk could also underlie segmental lesions in diabetic kidney disease (DKD).15Qi H. Casalena G. Shi S. et al.Glomerular endothelial mitochondrial dysfunction is essential and characteristic of diabetic kidney disease susceptibility.Diabetes. 2017; 66: 763-778Crossref PubMed Scopus (122) Google Scholar Here, we provide evidence of early GEC injury and ESL degradation in response to podocyte TGF-βR1 signaling activation. Podocyte-derived Edn1 interacted with increased GEC EdnrA expression resulting in mitochondrial reactive oxygen species (ROS) and activation of ESL degradation and remodeling pathways. Together, these crosstalking events could underlie increase in glomerular permeability to albumin in kidney disease. We examined the effects of podocyte-specific doxycycline (DOX)-inducible activation of TGF-βR1 signaling in PodTgfbr1 transgenic mice14Daehn I. Casalena G. Zhang T. et al.Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis.J Clin Invest. 2014; 124: 1608-1621Crossref PubMed Scopus (189) Google Scholar using scanning electron microscopy. In this mouse model, canonical TGF-βR1 signaling was demonstrated to be specific to podocytes.14Daehn I. Casalena G. Zhang T. et al.Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis.J Clin Invest. 2014; 124: 1608-1621Crossref PubMed Scopus (189) Google Scholar Glomeruli from DOX-treated PodTgfbr1 showed distinct morphological changes in capillary vessels starting at day 4 including cytoplasmic protrusions, ridges (Figure 1a), and overall decreased number of fenestrae compared with glomeruli from control mice. Semiquantitative analysis using the endothelial morphology index15Qi H. Casalena G. Shi S. et al.Glomerular endothelial mitochondrial dysfunction is essential and characteristic of diabetic kidney disease susceptibility.Diabetes. 2017; 66: 763-778Crossref PubMed Scopus (122) Google Scholar confirmed significantly increased lesions at 4 days with prominent lesions at days 7 to 14 of DOX treatment (Figure 1a and c). Widening and effacement of podocyte foot processes were evident at days 7 to 14 of DOX treatment (Figure 1b). Compared with control mice, there was no change in the podocyte foot processes numbers at day 4, but significant reductions were detected at days 7 to 14 in DOX-treated mice (Figure 1d). Analysis by transmission electron microscopy at day 4 of DOX-treated PodTgfbr1 shows GECs with blebs protruding into capillary lumens (Figure 1e), while there was a normal pattern of podocyte foot processes (arrows) and normal glomerular basement membrane. As previously reported,14Daehn I. Casalena G. Zhang T. et al.Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis.J Clin Invest. 2014; 124: 1608-1621Crossref PubMed Scopus (189) Google Scholar podocyte TGF-βR1activation resulted in significantly increased albumin-to-creatinine ratio after 4 to 14 days (Figure 1f), and urine 8-Oxo-2′-deoxyguanosine (an oxidative stress marker15Qi H. Casalena G. Shi S. et al.Glomerular endothelial mitochondrial dysfunction is essential and characteristic of diabetic kidney disease susceptibility.Diabetes. 2017; 66: 763-778Crossref PubMed Scopus (122) Google Scholar) after days 7 to 14 of DOX treatment (Figure 1g). DOX treatment induced segmental glomerulosclerosis by day 7, and global glomerulosclerosis with tubulointerstitial fibrosis by day 14 (Supplementary Figure S1A–D). These observations suggest endothelial injury and dysfunction mediated by podocyte activation through TGF-βR1 signaling. Interestingly, significant albuminuria was detected at day 4 of DOX treatment without any visible podocyte defects. We hypothesize that podocyte-GEC crosstalk results in GEC injury and concomitant albuminuria. To understand the GEC-specific signaling in response to podocyte TGF-βR1 activation in mice, we next performed transcriptomic analysis of isolated GECs.Figure 1Transforming growth factor-β receptor 1 signaling activation in podocytes induces ultrastructural changes in endocapillaries preceding ultrastructural changes in podocytes. (a) Glomerular capillary structure of PodTgfbr1 mice was examined by scanning electron microscopy in untreated control mice and after transforming growth factor-β receptor 1 signaling activation in podocytes with doxycycline (DOX) at days 4, 7, and 14. Representative images of capillary vessels at original magnification ×10,000 upper panel, ×30,000 lower panel (white arrows show endothelial ridges, white asterisks depict endothelial without fenestrations). (b) Representative images of podocytes from glomeruli in (a). (c) A semiquantitative endothelial morphology index (EMI) at ×10,000 magnification was applied: 0 = normal fenestrations, no visible irregularities; 1 = partly disrupted endothelial cell pattern and fenestrations, 10% to 30% of area; 2 = endothelial cells containing few visible fenestrations, 30% to 70% of area; 3 = no fenestrations present and irregular protrusions. Mean ± SEM of at least 125 capillary vessels of glomeruli from 5 mice per group. (d) The number of podocyte foot processes (PFP) per length (μm) of PodTgfbr1 with or without DOX at different times (n = 700 PFP counted per group, 3 mice per group. Mean ± SEM PFP/μm per capillary). (e) Transmission electron microscopy of glomeruli at day 4 of DOX treatment (black arrows show normally arranged PFP; black asterisks depict endothelial cell protrusions). Functional data from PodTgfbr1: (f) albumin-to-creatinine ratio (ACR) and (g) urinary 8-Oxo-2'-deoxyguanosine (8-oxo-dG) excretion of day 0 in control mice and days 4 to 14 in DOX-treated mice (± SEM, n = 4–9 mice at each time point). Error bars represent SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. Bars = 2 μm and 1 μm in (a) and 5 μm in (b) and (e). To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We examined the early transcriptome of GECs by crossing PodTgfbr1 mice with Flk1::H2B-EYFP mice, and these mice allow for detection and isolation of GECs through enhanced yellow fluorescent protein (EYFP) expressed under the control of an Flk1 (vascular endothelial growth factor receptor-2) promoter.16Fraser S.T. Hadjantonakis A.K. Sahr K.E. et al.Using a histone yellow fluorescent protein fusion for tagging and tracking endothelial cells in ES cells and mice.Genesis. 2005; 42: 162-171Crossref PubMed Scopus (70) Google Scholar We generated PodTgfbr1-Flk1 triple transgenic mice (Figure 2a) on an FVB background. EYFP-positive nuclei in glomeruli from PodTgfbr1-Flk1 mice were associated with cytoplasmic CD31 staining (Figure 2b) and were not associated with podocytes.17Fan Y. Li X. Xiao W. et al.BAMBI elimination enhances alternative TGF-beta signaling and glomerular dysfunction in diabetic mice.Diabetes. 2015; 64: 2220-2233Crossref PubMed Scopus (47) Google Scholar EYFP-positive GECs (gated P4 in Figure 2c) were isolated from PodTgfbr1-Flk1 no-DOX control mice (triple transgenic, Ttg), Pod-Flk1+4dDOX (double transgenic, Dtg) control mice, to PodTgfbr1-Flk1+4dDOX–treated mice. The GECs were pooled from 3 to 6 mice per sample run for sequencing (n = 2 Ttg-DOX, n = 2 Dtg+4dDOX, n = 2 Ttg+4dDOX). Among the top differentially expressed genes were pathways involving carbohydrate and glycosaminoglycan (GAG) metabolism, dysregulation of N-glycan/chondroitin/keratin/heparan sulfate biosynthesis, and concomitant GAG degradation (Supplemental Figure S2A), suggesting loss and turnover of the ESL reservoir. The heat-map of differentially expressed genes for GAG metabolism and validated expression of selected genes in sorted GECs is shown in Figure 2d and Supplemental Figure S2B. To confirm that there was loss and turnover of ESL in glomerular capillaries on TGF-βR1 activation in podocytes, we next measured ESL in PodTgfbr1 mice ±DOX by intralipid infusions.18Friden V. Oveland E. Tenstad O. et al.The glomerular endothelial cell coat is essential for glomerular filtration.Kidney Int. 2011; 79: 1322-1330Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar Representative transmission electron microscopy images of capillary vessels showing the intralipids (Figure 3a, arrow) depicting indirectly the thickness of the ESL showed a striking and significant loss of ESL thickness at day 4 of DOX treatment that declined further with treatment time (Figure 3a and b). We used isolectin B4 (IB4) to label glycoconjugates, which make up a large portion of the ESL.19Sieve I. Munster-Kuhnel A.K. Hilfiker-Kleiner D. Regulation and function of endothelial glycocalyx layer in vascular diseases.Vascul Pharmacol. 2018; 100: 26-33Crossref PubMed Scopus (97) Google Scholar Figures 3c and 4c and d show a significant reduction of IB4 staining intensity per glomerulus at day 4 of DOX treatment, declining further with time of treatment.Figure 4Transforming growth factor-β receptor 1 signaling in podocytes results in the loss of the endothelial surface layer (ESL) mediated by endothelin receptor-A (EdnrA) and mitochondrial reactive oxygen species. (a) Isolectin B4-positive staining intensity of day 0 control mice and mice treated for 14 days with doxycycline (DOX) with or without MitoTEMPO (mTe; a mitochondrial reactive oxygen species scavenger, 1 mg/kg per d) or BQ-123 (BQ) (an endothelin receptor-A inhibitor; 0.1 nM/kg per d), 4 to 5 mice per group. (b) BALB/c control mice and mice + Adriamycin (AD), with or without mTe, or with or without BQ-123, 5 to 8 mice per group. (c–j) Isolectin B4 glomerular staining in glomerular endothelial cells from control PodTgfbr1 mice (c) and mice treated with DOX for 14 days (d), DOX + mTe (e), and DOX + BQ-123 (f); and BALB/c control (g), BALB/c treated with AD, AD + mTe (i), and AD + BQ-123 (j). (k) Albumin-to-creatinine ratio (ACR) and (l) serum creatinine of control mice and mice with 4 to 14 days of DOX treatment with or without BQ-123; 5 to 8 mice per group. Error bars represent SEM and *P < 0.05, **P < 0.01, ***P < 0.001. M, mol/l. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) EdnrA inhibitors have been shown to prevent glycocalyx loss in mice with DKD.20Boels M.G. Avramut M.C. Koudijs A. et al.Atrasentan reduces albuminuria by restoring the glomerular endothelial glycocalyx barrier in diabetic nephropathy.Diabetes. 2016; 65: 2429-2439Crossref PubMed Scopus (83) Google Scholar, 21Garsen M. Lenoir O. Rops A.L. et al.Endothelin-1 induces proteinuria by heparanase-mediated disruption of the glomerular glycocalyx.J Am Soc Nephrol. 2016; 27: 3545-3551Crossref PubMed Scopus (76) Google Scholar We have reported an increase in EdnrA expression specifically in GECs of experimental FSGS and DKD mice, and EdnrA antagonism prevented GEC mitochondrial oxidative damage, albuminuria, and podocyte loss.14Daehn I. Casalena G. Zhang T. et al.Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis.J Clin Invest. 2014; 124: 1608-1621Crossref PubMed Scopus (189) Google Scholar, 15Qi H. Casalena G. Shi S. et al.Glomerular endothelial mitochondrial dysfunction is essential and characteristic of diabetic kidney disease susceptibility.Diabetes. 2017; 66: 763-778Crossref PubMed Scopus (122) Google Scholar We next examined EdnrB expression. In control glomeruli, EdnrB is expressed mainly in podocytes (Supplementary Figure S3A, arrows) and in large vessels (Supplementary Figure S3A, arrows open arrows), as previously reported.22Lenoir O. Milon M. Virsolvy A. et al.Direct action of endothelin-1 on podocytes promotes diabetic glomerulosclerosis.J Am Soc Nephrol. 2014; 25: 1050-1062Crossref PubMed Scopus (71) Google Scholar, 23Wendel M. Knels L. Kummer W. Koch T. Distribution of endothelin receptor subtypes ETA and ETB in the rat kidney.J Histochem Cytochem. 2006; 54: 1193-1203Crossref PubMed Scopus (66) Google Scholar EdnrB expression in glomeruli decreased with TGF-βR1 signaling activation in PodTgfbrI mice (Supplementary Figure S3A). A temporal loss of synaptopodin staining after days 7 to 14 of DOX treatment as podocyte density decreases, verifying our previous findings.14Daehn I. Casalena G. Zhang T. et al.Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis.J Clin Invest. 2014; 124: 1608-1621Crossref PubMed Scopus (189) Google Scholar Gene expression in isolated glomeruli from PodTgfbrI mice, compared with control mice, demonstrated a transient and significant increase in EdnrA and a decrease of EdnrB expression (Supplementary Figure S3B). Next, we examined Edn1 expression in isolated glomerular cells from PodTgfbr1-Flk1 mice. Supplementary Figure S2C shows that EYFP-negative cells (non-GEC glomerular cells), had increased Edn1 mRNA expression after day 4 of DOX treatment, while EdnrA levels were low and unchanged after day 4 of DOX treatment. As expected, EYFP-negative cells had high expression levels of TGF-βR1transgene, undetectable levels of E-cadherin, and higher vimentin expression than EYFP-positive GECs (Supplementary Figure S1D). These results confirm that, on TGF-βR1activation of podocytes by DOX in vivo, there is a local increase in Edn1 expression by podocytes potentially interacting with the increased EdnrA by GECs, which is consistent with our previous findings.14Daehn I. Casalena G. Zhang T. et al.Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis.J Clin Invest. 2014; 124: 1608-1621Crossref PubMed Scopus (189) Google Scholar Treatment of 14-day DOX PodTgfbrI mice treated mice with BQ-123, a specific EdnrA antagonist,24Eguchi S. Hirata Y. Ihara M. et al.A novel ETA antagonist (BQ-123) inhibits endothelin-1-induced phosphoinositide breakdown and DNA synthesis in rat vascular smooth muscle cells.FEBS Lett. 1992; 302: 243-246Crossref PubMed Scopus (86) Google Scholar or MitoTEMPO a mitochondrial-specific ROS scavenger, rescued the IB4-positive glomerular staining (Figure 4a and c–f). BQ-123 also reduced DOX induced albumin-to-creatinine ratio significantly at days 7 to 14 and prevented increased serum creatinine detected at days to -14 (Figure 4k and l). These data suggest that Edn1-EdnrA mediates ESL loss of GECs, possibly through increased mitochondrial ROS,14Daehn I. Casalena G. Zhang T. et al.Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis.J Clin Invest. 2014; 124: 1608-1621Crossref PubMed Scopus (189) Google Scholar which in turn could result in the early albuminuria observed at day 4 of DOX treatment (Figures 1f and 4k). We examined the intensity of glycoconjugates labeling by IB4 in Adriamycin (AD)-treated BALB/c mice, another model of podocyte injury, and experimental FSGS.25Zheng Z. Schmidt-Ott K.M. Chua S. et al.A Mendelian locus on chromosome 16 determines susceptibility to doxorubicin nephropathy in the mouse.Proc Natl Acad Sci U S A. 2005; 102: 2502-2507Crossref PubMed Scopus (83) Google Scholar We confirmed albuminuria and FSGS in these mice 6 days post AD injection reported in Daehn et al.14Daehn I. Casalena G. Zhang T. et al.Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis.J Clin Invest. 2014; 124: 1608-1621Crossref PubMed Scopus (189) Google Scholar Interestingly at 6 days post AD injection, there was a significant decrease in glomerular IB4 staining that was ameliorated by treatment with BQ-123 and MitoTEMPO (Figure 4b and g–j). Both MitoTEMPO and BQ-123 treatment also significantly reduced albumin-to-creatinine ratio (from 6685 ± 1401 to 5175 ± 853 and 2535 ± 789 nmol/l/mg [nM/mg], respectively) and serum creatinine (from 0.87 ± 0.074 to 0.56 ± 0.111 and 0.65 ± 0.075 μg/ml, respectively).14Daehn I. Casalena G. Zhang T. et al.Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis.J Clin Invest. 2014; 124: 1608-1621Crossref PubMed Scopus (189) Google Scholar Podocytes produce Edn1 on TGF-β1 activation in vivo (Supplementary Figure S2C) and in vitro as we have previously shown via canonical SMAD2/3 signaling pathways.14Daehn I. Casalena G. Zhang T. et al.Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis.J Clin Invest. 2014; 124: 1608-1621Crossref PubMed Scopus (189) Google Scholar We hypothesize that Edn1 is a key mediator of GEC stress via EdnrA; therefore, we next examined the effect of Edn1 on cell surface expression of both Edn1 receptors of conditionally immortalized murine GECs (mGEC)26Akis N. Madaio M.P. Isolation, culture, and characterization of endothelial cells from mouse glomeruli.Kidney Int. 2004; 65: 2223-2227Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar using fluorescence-activated cell sorting. Exposure to Edn1 for 48 hours resulted in an increase in EdnrA expression (Figure 5a), as well as further upregulation of Edn1 itself in mGEC, presumably reflecting an autocrine effect of Edn1 (Supplementary Figure S4). In contrast, the levels of EdnrB were unchanged (Figure 5a). To test the effect of crosstalking factors from activated podocytes on receptor expression on mGECs, we coincubated mGECs with supernatant from PodTgfbr1 podocytes with DOX (DoxSN) or without (CtrlSN) for 48 hours. EdnrA and EdnrB mRNA expression in CtrlSN-treated mGECs was the same as in untreated control GECs, however, only EdnrA was significantly increased in DoxSN coculture (Figure 5a), and we observed an increase in Edn1 mRNA (Supplementary Figure S4). We used atomic force microscope (AFM) elastography on mGECs to measure the in vitro 3-dimensional biomechanical properties of the ESL through direct contact without exposing cultured cells to fixation or staining procedures that may alter the fragile ESL structure.27Azeloglu E.U. Costa K.D. Atomic force microscopy in mechanobiology: measuring microelastic heterogeneity of living cells.Methods Mol Biol. 2011; 736: 303-329Crossref PubMed Scopus (51) Google Scholar, 28Wiesinger A. Peters W. Chappell D. et al.Nanomechanics of the endothelial glycocalyx in experimental sepsis.PLoS One. 2013; 8e80905Crossref PubMed Scopus (113) Google Scholar Supplementary Figure S5A to C illustrates how the ESL may influence the pre- and postcontact interactions with the AFM probe. We examined the shallow postcontact region within the 50 nm proximity of the cell membrane (i.e., postcontact segment of the force-depth response curves [Supplementary Figure S5D]) using the depth-dependent pointwise modulus method.27Azeloglu E.U. Costa K.D. Atomic force microscopy in mechanobiology: measuring microelastic heterogeneity of living cells.Methods Mol Biol. 2011; 736: 303-329Crossref PubMed Scopus (51) Google Scholar Figure 5b shows that the ESL stiffness within the first 50 nm of contact (50% effective dose [ED50]) was reduced significantly (14.2 ± 0.3 to 11.4 ± 0.4 kPa) after a 2-hour treatment with hyaluronidase + heparanase (Hpse)-III (Hyal+Hpse), compared with control mGECs. Additionally, we examined the effect of Edn1 on mGEC ESL and the effect of specific inhibition of EdnrA or EdnrB with BQ-123 (1 ng/ml) or BQ-788 (15 ng/ml), respectively. Edn1 reduced ESL stiffness significantly compared to controls (control mGECs to Edn1: 14.1 ± 0.3 to 12.4 ± 0.3 kPa), and this reduction was partly prevented by BQ-123 (Edn1 to Edn1+BQ-123: 12.4 ± 0.3 to 13.5 ± 0.4 kPa) but not with BQ-788. To determine whether Edn1 secreted by podocytes on activation can impact the ESL of mGECs in culture, we examined the effect of PodTgfbr1 podocyte supernatants without or with TGF-βR1activation (CtrlSN and DoxSN, respectively), as shown in Figure 5c. Compared with CtrlSN, DoxSN exhibited significantly reduced ESL stiffness (CtrlSN to DoxSN: 22.4 ± 1.2 to 15.1 ± 0.3 kPa). Here, the DoxSN effect on mGEC ESL was significantly ameliorated by BQ-123 (DoxSN to DoxSN+BQ-123: 15.1 ± 0.3 to 18.6 ± 0.5 kPa) but not by BQ-788. These data suggest that podocyte-derived Edn1 reduces mGEC ESL stiffness specifically via EdnrA. Studies have shown that low expression of heparan sulfate (HS), the main polysaccharide component of the glomerular glycocalyx, is inversely correlated with the degree of proteinuria.29Garsen M. Rops A.L. Rabelink T.J. et al.The role of heparanase and the endothelial glycocalyx in the development of proteinuria.Nephrol Dial Transplant. 2014; 29: 49-55Crossref PubMed Scopus (75) Google Scholar A reduction of the glycocalyx in diabetic apolipoprotein E knockout mice was mediated by increased glomerular expression of Hpse, which cleaves the glycosidic bonds of HS.20Boels M.G. Avramut M.C. Koudijs A. et al.Atrasentan reduces albuminuria by restoring the glomerular endothelial glycocalyx barrier in diabetic nephropathy.Diabetes. 2016; 65: 2429-2439Crossref PubMed Scopus (83) Google Scholar To better understand the mechanism for ESL degradation, we examined the expression of Hpse and Hyal of mGECs and PodTgfbr1 podocytes treated with Edn1. After 48-hour incubation with Edn1, there was a significant increase in Hpse and Hyal expression in mGECs but not in podocytes, in contrast to other report