The glomerular endothelial cell coat is essential for glomerular filtration
2011; Elsevier BV; Volume: 79; Issue: 12 Linguagem: Inglês
10.1038/ki.2011.58
ISSN1523-1755
AutoresVincent Fridén, Eystein Oveland, Olav Tenstad, Kerstin Ebefors, Jenny Nyström, Ulf Nilsson, Börje Haraldsson,
Tópico(s)Trauma, Hemostasis, Coagulopathy, Resuscitation
ResumoThe endothelial cell surface layer (ESL) is believed to contribute to the glomerular barrier, and the nature of its molecular structure is still largely unknown. The ESL consists of the membrane-bound glycocalyx and the loosely attached endothelial cell coat (ECC). A brief injection of hypertonic sodium chloride into the left renal artery was used to displace, elute, and collect non-covalently bound components of the renal ESL in rats. This procedure increased the fractional clearance of albumin 12-fold without detectable morphological changes as assessed by electron microscopy compared with the control group injected with isotonic saline. Mathematical modeling suggested a reduced glomerular charge density. Mass spectrometry of the renal eluate identified 17 non-covalently bound proteins normally present in the ECC. One of these proteins, orosomucoid, has previously been shown to be important for capillary permselectivity. Another protein, lumican, is expressed by glomerular endothelial cells and likely contributes to maintaining an intact barrier. Thus, the absence of one or more of these proteins causes proteinuria and illustrates the importance of the ECC in glomerular permselectivity. The endothelial cell surface layer (ESL) is believed to contribute to the glomerular barrier, and the nature of its molecular structure is still largely unknown. The ESL consists of the membrane-bound glycocalyx and the loosely attached endothelial cell coat (ECC). A brief injection of hypertonic sodium chloride into the left renal artery was used to displace, elute, and collect non-covalently bound components of the renal ESL in rats. This procedure increased the fractional clearance of albumin 12-fold without detectable morphological changes as assessed by electron microscopy compared with the control group injected with isotonic saline. Mathematical modeling suggested a reduced glomerular charge density. Mass spectrometry of the renal eluate identified 17 non-covalently bound proteins normally present in the ECC. One of these proteins, orosomucoid, has previously been shown to be important for capillary permselectivity. Another protein, lumican, is expressed by glomerular endothelial cells and likely contributes to maintaining an intact barrier. Thus, the absence of one or more of these proteins causes proteinuria and illustrates the importance of the ECC in glomerular permselectivity. Accumulation of proteins in the urine is a key feature of renal disease and a consequence of an impaired glomerular filtration barrier. The glomerular filtration barrier is a biological membrane that includes a unique type of the fenestrated endothelium mainly without diaphragms covered with a glycoprotein surface layer, a glomerular basement membrane (GBM), and podocytes with foot processes bridged by slit diaphragms.1.Haraldsson B. Nystrom J. Deen W.M. Properties of the glomerular barrier and mechanisms of proteinuria.Physiol Rev. 2008; 88: 451-487Crossref PubMed Scopus (608) Google Scholar The permselectivity of this barrier has been considered to reside principally in the GBM and in the podocyte slit diaphragm, whereas fenestrated endothelial cells and the endothelial cell surface layer (ESL) have attracted lesser attention. Nevertheless, the classical view of the GBM as a major contributor to the permselectivity of the glomerular barrier has been challenged,2.Goldberg S. Harvey S.J. Cunningham J. et al.Glomerular filtration is normal in the absence of both agrin and perlecan-heparan sulfate from the glomerular basement membrane.Nephrol Dial Transplant. 2009; 24: 2044-2051Crossref PubMed Scopus (84) Google Scholar, 3.Harvey S.J. Jarad G. Cunningham J. et al.Disruption of glomerular basement membrane charge through podocyte-specific mutation of agrin does not alter glomerular permselectivity.Am J Pathol. 2007; 171: 139-152Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 4.Chen S. Wassenhove-McCarthy D.J. Yamaguchi Y. et al.Loss of heparan sulfate glycosaminoglycan assembly in podocytes does not lead to proteinuria.Kidney Int. 2008; 74: 289-299Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 5.van den Hoven M.J. Wijnhoven T.J. Li J.P. et al.Reduction of anionic sites in the glomerular basement membrane by heparanase does not lead to proteinuria.Kidney Int. 2008; 73: 278-287Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar and a decade of extensive research regarding the podocytes has given important information about numerous proteins of the slit diaphragm that can cause proteinuria when defective.6.Roselli S. Gribouval O. Boute N. et al.Podocin localizes in the kidney to the slit diaphragm area.Am J Pathol. 2002; 160: 131-139Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 7.Ruotsalainen V. Ljungberg P. Wartiovaara J. et al.Nephrin is specifically located at the slit diaphragm of glomerular podocytes.Proc Natl Acad Sci USA. 1999; 96: 7962-7967Crossref PubMed Scopus (608) Google Scholar, 8.Tryggvason K. Patrakka J. Wartiovaara J. Hereditary proteinuria syndromes and mechanisms of proteinuria.N Engl J Med. 2006; 354: 1387-1401Crossref PubMed Scopus (444) Google Scholar Simultaneously, experimental evidence has accumulated for the presence of a permselective cell surface layer covering the luminal face of glomerular endothelial cells.9.Ciarimboli G. Hjalmarsson C. Bokenkamp A. et al.Dynamic alterations of glomerular charge density in fixed rat kidneys suggest involvement of endothelial cell coat.Am J Physiol Renal Physiol. 2003; 285: F722-F730Crossref PubMed Scopus (15) Google Scholar, 10.Deen W.M. What determines glomerular capillary permeability?.J Clin Invest. 2004; 114: 1412-1414Crossref PubMed Scopus (121) Google Scholar, 11.Haraldsson B. Sorensson J. Why do we not all have proteinuria? An update of our current understanding of the glomerular barrier.News Physiol Sci. 2004; 19: 7-10Crossref PubMed Scopus (96) Google Scholar, 12.Ohlson M. Sorensson J. Haraldsson B. Glomerular size and charge selectivity in the rat as revealed by FITC-ficoll and albumin.Am J Physiol Renal Physiol. 2000; 279: F84-F91PubMed Google Scholar, 13.Pries A.R. Secomb T.W. Gaehtgens P. The endothelial surface layer.Pflugers Arch. 2000; 440: 653-666Crossref PubMed Scopus (667) Google Scholar In the end of the 1980s, perfusion of the rat kidneys with cationic, anionic, or neutral ferritin demonstrated that cationic ferritin bound to, and visualized, a protein coat that covered the glomerular endothelium.14.Avasthi P.S. Koshy V. The anionic matrix at the rat glomerular endothelial surface.Anat Rec. 1988; 220: 258-266Crossref PubMed Scopus (35) Google Scholar, 15.Avasthi P.S. Koshy V. Glomerular endothelial glycocalyx.Contrib Nephrol. 1988; 68: 104-113Crossref PubMed Google Scholar, 16.Koshy V. Avasthi P.S. The anionic sites at luminal surface of peritubular capillaries in rats.Kidney Int. 1987; 31: 52-58Abstract Full Text PDF PubMed Scopus (9) Google Scholar Rostgaard and Qvortrup,17.Rostgaard J. Qvortrup K. Electron microscopic demonstrations of filamentous molecular sieve plugs in capillary fenestrae.Microvasc Res. 1997; 53: 1-13Crossref PubMed Scopus (143) Google Scholar, 18.Rostgaard J. Qvortrup K. Sieve plugs in fenestrae of glomerular capillaries–site of the filtration barrier?.Cells Tissues Organs. 2002; 170: 132-138Crossref PubMed Scopus (76) Google Scholar using a different method of vascular perfusion fixation, found evidence for an extensive protein coat on the surface and in the fenestrae of the glomerular endothelium. Furthermore, we revealed a 200-nm-thick ESL stretching into the capillary lumen.19.Hjalmarsson C. Johansson B.R. Haraldsson B. Electron microscopic evaluation of the endothelial surface layer of glomerular capillaries.Microvasc Res. 2004; 67: 9-17Crossref PubMed Scopus (81) Google Scholar In mice treated with adriamycin, the thickness of this layer was markedly reduced, an effect coinciding with the development of proteinuria and impaired synthesis of certain components of the ESL.20.Jeansson 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 Another study in mice demonstrated that infusion of enzymes that specifically degrade glycosaminoglycans reduced the thickness of the ESL and increased fractional clearance for albumin.21.Jeansson 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 Others have performed in vitro studies of the passage of albumin across monolayers of human glomerular endothelial cells and have observed an increased albumin flux after treatment with human heparanase.22.Singh 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 There is also clinical evidence that suggests that damage to the glomerular endothelium can cause proteinuria in pre-eclampsia and hemolytic uremic syndrome.23.Besbas N. Karpman D. Landau D. et al.A classification of hemolytic uremic syndrome and thrombotic thrombocytopenic purpura and related disorders.Kidney Int. 2006; 70: 423-431Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 24.Karumanchi S.A. Maynard S.E. Stillman I.E. et al.Preeclampsia: a renal perspective.Kidney Int. 2005; 67: 2101-2113Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar The ESL has two components. First, there is the glycocalyx, which is composed of membrane-bound proteoglycans (PGs). Second, attached to the glycocalyx, there is the endothelial cell coat (ECC), composed of secreted PGs, glycosaminoglycans, glycoproteins, and plasma proteins.1.Haraldsson B. Nystrom J. Deen W.M. Properties of the glomerular barrier and mechanisms of proteinuria.Physiol Rev. 2008; 88: 451-487Crossref PubMed Scopus (608) Google Scholar, 25.Van Teeffelen J.W. Brands J. Stroes E.S. et al.Endothelial glycocalyx: sweet shield of blood vessels.Trends Cardiovasc Med. 2007; 17: 101-105Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 26.Weinbaum 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 The abundant, negatively charged PGs and glycosaminoglycans are believed to capture circulating plasma proteins and to produce a thick mesh with anionic properties in a gel-like structure.27.Sorensson J. Ohlson M. Haraldsson B. A quantitative analysis of the glomerular charge barrier in the rat.Am J Physiol Renal Physiol. 2001; 280: F646-F656PubMed Google Scholar, 28.Sorensson J. Ohlson M. Lindstrom K. et al.Glomerular charge selectivity for horseradish peroxidase and albumin at low and normal ionic strengths.Acta Physiol Scand. 1998; 163: 83-91Crossref PubMed Scopus (50) Google Scholar In previous studies, we have shown that the plasma protein orosomucoid is required for maintaining normal glomerular permeability in rats.29.Haraldsson B.S. Johnsson E.K. Rippe B. Glomerular permselectivity is dependent on adequate serum concentrations of orosomucoid.Kidney Int. 1992; 41: 310-316Abstract Full Text PDF PubMed Scopus (74) Google Scholar, 30.Johnsson E. Haraldsson B. Addition of purified orosomucoid preserves the glomerular permeability for albumin in isolated perfused rat kidneys.Acta Physiol Scand. 1993; 147: 1-8Crossref PubMed Scopus (30) Google Scholar The aim of this study was to provide more information on the ECC, including its permselective properties and its composition. Our approach was to establish a mild in vivo method for displacing non-covalently bound ECC components and to subsequently collect them for further characterization. A brief intrarenal high-salt (HS) perfusion through the renal artery was performed to displace anionic non-covalently bound components according to the principles of ion-exchange chromatography. A similar principle was previously used in an isolated perfused rat kidney system using low- and high-ionic strength in which we could modulate fractional clearances of albumin in a very specific and reversible manner.27.Sorensson J. Ohlson M. Haraldsson B. A quantitative analysis of the glomerular charge barrier in the rat.Am J Physiol Renal Physiol. 2001; 280: F646-F656PubMed Google Scholar, 28.Sorensson J. Ohlson M. Lindstrom K. et al.Glomerular charge selectivity for horseradish peroxidase and albumin at low and normal ionic strengths.Acta Physiol Scand. 1998; 163: 83-91Crossref PubMed Scopus (50) Google Scholar Morphological examination with electron microscopy of kidney sections obtained from animals perfused with 0.15 mol/l NaCl (normal salt, NS) and 1 mol/l NaCl (HS) confirmed that the overall architecture of the glomerular filtration barrier remained morphologically normal during treatment (Figure 1). In addition, to quantitatively assess the condition of the filtration barrier, GBM thickness, podocyte foot process width, and width of filtration slits were measured in glomerular capillaries from animals perfused with NS and HS. The morphometric measurements revealed small but statistically significant differences between the two experimental groups (Table 1).Table 1Summary of morphometric measurements of various structures in the glomerular filtration barrierNSHSGlomerular basement membrane thickness (nm)168.57±1.31174.14±1.15**P<0.01,Podocyte foot process width (nm)301.49±7.54307.38±7.58Podocyte filtration slit width (nm)40.63±0.3738.23±0.41***P<0.001, n=52 glomeruli from 4NS animals, n=60 glomeruli from 4 HS animals.Endothelial cell surface layer thickness (nm)123.42±8.06123.81±8.49Abbreviations: HS, high salt; NS, normal salt.Data are means±s.e.m.** P<0.01,*** P<0.001, n=52 glomeruli from 4 NS animals, n=60 glomeruli from 4 HS animals. Open table in a new tab Abbreviations: HS, high salt; NS, normal salt. Data are means±s.e.m. When measured 10 and 20 min after HS perfusion, the glomerular filtration rate (GFR) was significantly reduced by 34±8.7% and 23±7.7% (P <0.001, P <0.01, n=10), respectively, relative to the pre-perfusion mean. GFR then started to recover toward the pre-perfusion level. No changes were seen in the NS group; for further details, see Figure 2. The absolute mean values of pre-perfusion points (-20 and -10 min) were 1.21±0.089 and 1.19±0.073 for the NS group and 0.96±0.058 and 1.05±0.053 for the HS group (ml/min per g kidney wet weight). In rats perfused with HS, the fractional clearance for albumin increased ∼12-fold relative to both baseline and NS when measured 10 min after perfusion (Figure 3). This was followed by a slow but significant recovery over the subsequent 90 min (P<0.05, log-linear regression analysis), but the fractional albumin clearance remained significantly higher in the HS group throughout the 100-min observation period. The fractional albumin clearance remained normal in the NS group throughout the measurement period (n=7–10). The fractional clearance for Ficoll 35.5 Å (uncharged, size corresponding to albumin) was measured in HS and NS rats, and there were no significant alterations after salt perfusion (Figure 4). The fractional clearance for Ficoll 55 Å (a measure of the large pore fraction) was significantly increased 4-fold in HS-perfused rats 10 min after perfusion compared with the pre-perfusion value (P <0.001, n=8, Figure 5). Ficoll 55 Å clearance was unaffected in NS-perfused rats.Figure 5Analysis of the fractional clearance of Ficoll 55 Å before and after salt perfusion in rats. On the x axis, -10 is a pre-perfusion point and the zero point is where perfusion occurs, followed by two post-perfusion points. Statistical analysis was made by comparing the post-perfusion fractional Ficoll clearance in HS (high salt, 1 mol/l NaCl) rats with the pre-perfusion value. Data represent means±s.e.m., n=8 in HS rats and n=8 in NS (normal salt, 0.15 mol/l NaCl) rats, except for 20 min (n=7). ***P <0.001.View Large Image Figure ViewerDownload (PPT) Alterations to the barrier were analyzed using two separate models, and similar results were found with both models. The most advanced model is the heterogeneous charged fiber model according to which there was a 56% reduction in fiber net charge (P <0.001, n=8) after a short perfusion with HS compared with NS (Table 2).1.Haraldsson B. Nystrom J. Deen W.M. Properties of the glomerular barrier and mechanisms of proteinuria.Physiol Rev. 2008; 88: 451-487Crossref PubMed Scopus (608) Google Scholar, 21.Jeansson 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 The number of large pores increased five-fold (P <0.05, n=8), whereas the reduction in fiber volume fraction was non-significant. The gel membrane model showed that the kidneys of HS-perfused rats had an increase in large pores (P <0.01, n=8) and a reduced charge density (P <0.001, n=8); see Table 3.31.Ohlson M. Sorensson J. Haraldsson B. A gel-membrane model of glomerular charge and size selectivity in series.Am J Physiol Renal Physiol. 2001; 280: F396-F405PubMed Google ScholarTable 2Summary of mathematical modeling using the heterogeneous charged fiber matrix modelNormal saltHSP-valueFiber radius (Å)5.00±0.005.00±0.00NSFiber volume fraction (%)5.21±0.084.91±0.14NSLarge pore LpS (%)0.020±0.0030.100±0.038<0.05Fiber net charge (C/m2)-0.375±0.03-0.166±0.02<0.001Meas/calc albumin (%)25±1149±13NSAbbreviations: C/m2, Coulomb/meter squared; HS, high salt; LpS, total hydraulic conductance; Meas/calc, measured/calculated; NS, not significant.Data are means ± s.e.m., n=8. Open table in a new tab Table 3Summary of mathematical modeling using the gel membrane modelNormal saltHSP-valueSmall pore radius (Å)49.7±0.3650.2±1.25NSLarge pore radius (Å)79.2±2.4881.8±7.33NSLarge pore LpS (%)0.50±0.111.35±0.78<0.01Charge density (mEq/l)86.8±3.0664.8±3.61<0.001Abbreviations: HS, high salt; LpS, total hydraulic conductance; NS, not significant.Data are means±s.e.m., n=8. Open table in a new tab Abbreviations: C/m2, Coulomb/meter squared; HS, high salt; LpS, total hydraulic conductance; Meas/calc, measured/calculated; NS, not significant. Data are means ± s.e.m., n=8. Abbreviations: HS, high salt; LpS, total hydraulic conductance; NS, not significant. Data are means±s.e.m., n=8. The thickness of ESL was estimated using Intralipid droplets as indirect markers (Figure 6).20.Jeansson 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 Measurement of the distance between lipid droplets and the endothelial cell membrane in rats perfused with HS and NS demonstrated that there was no significant difference of the thickness of the ESL between the groups (Table 1). To examine displaced proteins from the ESL, eluates from both groups were immunodepleted and analyzed with liquid chromatography-mass spectrometry using an LTQ-Orbitrap. Two independent runs, using different settings but the same original material identified in total 67 proteins in the NS eluate, 93 proteins in the HS eluate, and 93 proteins in the rat plasma. The total number of unique peptides was 1993, 1847, and 2933 in the HS eluate, NS eluate, and plasma, respectively. The overlap of identified proteins is shown in a Venn diagram (Figure 7). Peptide counting was used as a semi-quantitative method. Proteins were selected from the total number of identified proteins in Figure 7 according to the following inclusion criteria and are presented in Tables 4, 5, 6: proteins found in the HS eluate in both runs, in which the number of identified peptides assigned to the protein was at least 2 times higher compared with the NS eluate (Table 4) (8 proteins out of 63); proteins found in the HS eluate and the plasma in both runs, but not found in the NS eluate (Table 5) (3 proteins out of 11); and proteins found exclusively in the HS eluate and in both runs (Table 6) (6 proteins out of 19). The molecular weight range of proteins displayed in Table 4, Table 5, Table 6, Figure 8 was 5–190 kDa. An absolute quantification was performed with unique peptides for lumican using selected reaction monitoring with respective AQUA peptides. The peptide ITNIPDEYFNR produced an HS/NS ratio of 2.3 based on peak area when compensating for the spike in the AQUA peptide (7278/1174781)/(2967/1089557), and the peptide FTGLQYLR produced an HS/NS ratio of 3.2 (15915/1356854)/(4610/1155172) (Figure 8). A third peptide (ILGPLSYSK) produced an HS/NS ratio of 3.4 without AQUA peptide compensation, resulting in an average HS/NS ratio for lumican of 3.0. Supplementary Figure S1 online illustrates the spectrum signatures of the AQUA peptides (A, B) and the quantification of the ILGPLSYSK peptide (C). Two other examples for selected reaction monitoring quantification without using AQUA peptides are shown for glutathione peroxidase 3 and gelsolin, which produced HS/NS ratios of 3.7 and 1.2, respectively (see Supplementary Table S1 online).Table 4Proteins found in the HS eluate in two runs, in which the number of identified peptides assigned to the protein was at least two times higher compared with the NS eluateNameAccession numbernP_HSnP_NSnP_plasmanP_HS/np_NSLumicanP51886113—3.7Glutathione peroxidase 1P0404141—3.7Thymosin β-4P6232931—2.814-3-3 protein zeta/δP6310252—2.5Peptidyl-prolyl cis-trans isomerase AP1011173—2.3Glutathione peroxidase 3P23764223217.3GelsolinQ68FP1124173.0Complement C4P0864984622.0Abbreviations: HS, high salt; nP, number of unique peptides assigned to displayed proteins; NS, normal salt.Merged plasma, merged HS, and merged NS eluate from three rats processed and run on an LTQ-Orbitrap, followed by analysis by Proteome Discoverer (Thermo Fisher Scientific), ProteinCenter, and MS Excel 2007 (Microsoft AB, Kista, Sweden). Open table in a new tab Table 5Proteins found in the HS eluate and the plasma in two runs, not found in the NS eluateNameAccession numbernP_HSnP_plasmaα-1-Acid glycoprotein (orosomucoid)P0276459Complement component C9Q62930510Hepatic triacylglycerol lipaseP0786735Abbreviations: HS, high salt; nP, number of unique peptides assigned to displayed proteins; NS, normal salt.Merged plasma, merged HS, and merged NS eluate from three rats processed and run on an LTQ-Orbitrap followed by analysis by Proteome Discoverer, ProteinCenter, and MS Excel 2007. Open table in a new tab Table 6Proteins found in the HS eluate in two runs, not found in the NS eluate or the plasmaNameAccession numbernP_HSPeroxiredoxin-1Q637164α-Actinin-1Q9Z1P23Heat-shock cognate 71-kDa proteinP630173Profilin-1P629623Cytochrome b–c1 complex subunit 2P325512Annexin A5P146682Abbreviations: HS, high salt; nP, number of unique peptides assigned to displayed proteins; NS, normal salt.Merged plasma, merged HS, and merged NS eluate from three rats processed and run on an LTQ-Orbitrap, followed by analysis by Proteome Discoverer, ProteinCenter, and MS Excel 2007. Open table in a new tab Figure 8Absolute quantification of lumican using selected reaction monitoring (SRM) assay. Two peptides unique for lumican were quantified in merged HS (high salt, 1 mol/l NaCl) and merged NS (normal salt, 0.15 mol/l NaCl) eluates using SRM assay designed on the basis of respective AQUA peptide standards. The unique SRM spectra are shown in the upper right corners of the figure and the corresponding SRM spectra from the AQUA peptides are shown in Supplementary Figure S1 online. The most abundant transition was used for quantification based on the peak area and the other transitions for peptide genuineness and assay verification. (a) The peptide ITNIPDEYFNR with m/z=691.4 (z=2) and its transition to m/z=940.5 (z=1) was used for quantification, and five other transitions (m/z=720.4, 843.4, 1053.6, 1167.6, and 1268.7) were used for verification. (b) FTGLQYLR, with m/z=499.3 (z=2) to m/z=749.5 (z=1), was used for quantification, and three other transitions (m/z=579.4, 692.5, and 850.5) were used for verification. AQUA, absolute quantification; counts, peptide counts; HS, high salt; m/z, mass to charge; NS, normal salt; RT, retention time.View Large Image Figure ViewerDownload (PPT) Download .jpg (.05 MB) Help with files Supplementary Figure S1 Download .doc (.03 MB) Help with doc files Supplementary Table S1 Abbreviations: HS, high salt; nP, number of unique peptides assigned to displayed proteins; NS, normal salt. Merged plasma, merged HS, and merged NS eluate from three rats processed and run on an LTQ-Orbitrap, followed by analysis by Proteome Discoverer (Thermo Fisher Scientific), ProteinCenter, and MS Excel 2007 (Microsoft AB, Kista, Sweden). Abbreviations: HS, high salt; nP, number of unique peptides assigned to displayed proteins; NS, normal salt. Merged plasma, merged HS, and merged NS eluate from three rats processed and run on an LTQ-Orbitrap followed by analysis by Proteome Discoverer, ProteinCenter, and MS Excel 2007. Abbreviations: HS, high salt; nP, number of unique peptides assigned to displayed proteins; NS, normal salt. Merged plasma, merged HS, and merged NS eluate from three rats processed and run on an LTQ-Orbitrap, followed by analysis by Proteome Discoverer, ProteinCenter, and MS Excel 2007. To confirm that lumican is present in the glomerular capillary endothelium, we performed immunohistochemical analysis in cryosections of the human kidney tissue. For additional evidence about the localization, we performed colocalization studies with the endothelial-specific lectin rhodamine Ulex europaeus agglutinin I. There was a clear colocalization of lumican with this endothelial-specific marker in the glomerular capillary endothelium (Figure 9). The main contribution of this study is the identification of novel components of the ECC, which seem to be required for normal glomerular barrier function. The tool for acquiring the data was a new in vivo method, developed for studying the ECC without using any noxious chemical agents. The method, consisting of a short perfusion with 1 mol/l NaCl, uses the basic principles of ion-exchange chromatography. Non-covalently and loosely attached components of the ESL are displaced when the kidney is perfused briefly with the high-ionic strength NaCl solution. In this study, we focused on medium-sized plasma proteins and small PGs as the perfusion time was too short to allow for diffusion of large proteins or large PGs. HS perfusion gave rise to an increased fractional albumin clearance, indicating a reduced charge selectivity of the glomerular filtration barrier. Size selectivity also was affected to some extent. The proteinuria following HS perfusion was reversible, as seen by the 50% recovery of fractional albumin clearance in the first hour after perfusion, but the fractional albumin clearance did not return to the pre-perfusion level during the observation period. The observed recovery of the fractional albumin clearance in HS-perfused rats could depend on re-enrichment of circulating plasma proteins in the ESL when the ionic strength normalizes. Potentially re-enriched proteins in the ESL are those that we found in the HS eluate and plasma (Table 5), including the last three proteins in Table 4. One of these proteins, orosomucoid, has previously been shown to be essential for the maintenance of normal permeability in peripheral capillaries.29.Haraldsson B.S. Johnsson E.K. Rippe B. Glomerular permselectivity is dependent on adequate serum concentrations of orosomucoid.Kidney Int. 1992; 41: 310-316Abstract Full Text PDF PubMed Scopus (74) Google Scholar, 30.Johnsson E. Haraldsson B. Addition of purified orosomucoid preserves the glomerular permeability for albumin in isolated perfused rat kidneys.Acta Physiol Scand. 1993; 147: 1-8Crossref PubMed Scopus (30) Google Scholar, 32.Haraldsson B. Rippe B. Orosomucoid as one of the serum components contributing to normal capillary permselectivity in rat skeletal muscle.Acta Physiol Scand. 1987; 129: 127-135Crossref PubMed Scopus (98) Google Scholar, 33.Haraldsson B. Rippe B. Serum factors other than albumin are needed for the maintenance of normal capillary permselectivity in rat hindlimb muscle.Acta Physiol Scand. 1985; 123: 427-436Crossref PubMed Scopus (35) Google Scholar, 34.Curry F.E. Rutledge J.C. Lenz J.F. Modulation of microvessel wall charge by plasma glycoprotein orosomucoid.Am J Physiol. 1989; 257: H1354-H1359PubMed Google Scholar A reason for the fractional albumin clearance remaining high 1 h after HS injection could be that components lost from the ECC need to be synthesized and secreted by glomerular endothelial cells. The PG lumican may be one such comp
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