A novel assay provides sensitive measurement of physiologically relevant changes in albumin permeability in isolated human and rodent glomeruli
Authors: Sara Desideri, Karen L. Onions, Yan Qiu, Raina D. Ramnath, Matthew J. Butler, Christopher R. Neal, Matthew L.R. King, Andrew E. Salmon, Moin A. Saleem, Gavin I. Welsh, C. Charles Michel, Simon C. Satchell, Andrew H.J. Salmon, Rebecca R. Foster
Journal: Kidney International (2018)

Increased urinary albumin excretion is a key feature of glomerular disease but has limitations as a measure of glomerular permeability. Here we describe a novel assay to measure the apparent albumin permeability of single capillaries in glomeruli, isolated from perfused kidneys cleared of red blood cells. The rate of decline of the albumin concentration within the capillary lumen was quantified using confocal microscopy and used to calculate apparent permeability. The assay was extensively validated and provided robust, reproducible estimates of glomerular albumin permeability. These values were comparable with previous in vivo data, showing this assay could be applied to human as well as rodent glomeruli. To confirm this, we showed that targeted endothelial glycocalyx disruption resulted in increased glomerular albumin permeability in mice. Furthermore, incubation with plasma from patients with post-transplant recurrence of nephrotic syndrome increased albumin permeability in rat glomeruli compared to remission plasma. Finally, in glomeruli isolated from rats with early diabetes there was a significant increase in albumin permeability and loss of endothelial glycocalyx, both of which were ameliorated by angiopoietin-1. Thus, a glomerular permeability assay, producing physiologically relevant values with sufficient sensitivity to measure changes in glomerular permeability and independent of tubular function, was developed and validated. This assay significantly advances the ability to study biology and disease in rodent and human glomeruli. Increased urinary albumin excretion is a key feature of glomerular disease but has limitations as a measure of glomerular permeability. Here we describe a novel assay to measure the apparent albumin permeability of single capillaries in glomeruli, isolated from perfused kidneys cleared of red blood cells. The rate of decline of the albumin concentration within the capillary lumen was quantified using confocal microscopy and used to calculate apparent permeability. The assay was extensively validated and provided robust, reproducible estimates of glomerular albumin permeability. These values were comparable with previous in vivo data, showing this assay could be applied to human as well as rodent glomeruli. To confirm this, we showed that targeted endothelial glycocalyx disruption resulted in increased glomerular albumin permeability in mice. Furthermore, incubation with plasma from patients with post-transplant recurrence of nephrotic syndrome increased albumin permeability in rat glomeruli compared to remission plasma. Finally, in glomeruli isolated from rats with early diabetes there was a significant increase in albumin permeability and loss of endothelial glycocalyx, both of which were ameliorated by angiopoietin-1. Thus, a glomerular permeability assay, producing physiologically relevant values with sufficient sensitivity to measure changes in glomerular permeability and independent of tubular function, was developed and validated. This assay significantly advances the ability to study biology and disease in rodent and human glomeruli. The ability to measure glomerular permeability is essential in the detection and assessment of glomerular disease. Albuminuria is the standard measure; however, it does not accurately reflect glomerular permeability. This is due to confounding changes in local hemodynamics, as well as tubular reabsorption of filtered albumin.1Ciarimboli 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 For experimental studies of human glomerular permeability, measurements of albuminuria are not possible. Therefore, the development of a reliable, physiologically relevant assay to directly measure glomerular permeability using isolated glomeruli would be very useful and independent of confounding variables. We have previously developed a reliable assay of water permeability in isolated glomeruli2Salmon A.H. Neal C.R. Bates D.O. et al.Vascular endothelial growth factor increases the ultrafiltration coefficient in isolated intact Wistar rat glomeruli.J Physiol. 2006; 570: 141-156Crossref PubMed Scopus (31) Google Scholar and here we describe the development of an albumin permeability assay. In 1992, Savin et al. modified an isolated glomerular oncometric assay, originally developed to assess hydraulic conductivity,3Savin V.J. Terreros D.A. Filtration in single isolated mammalian glomeruli.Kidney Int. 1981; 20: 188-197Abstract Full Text PDF PubMed Scopus (68) Google Scholar to measure glomerular reflection coefficients4Savin V.J. Sharma R. Lovell H.B. et al.Measurement of albumin reflection coefficient with isolated rat glomeruli.J Am Soc Nephrol. 1992; 3: 1260-1269Crossref PubMed Google Scholar and further modified the assay in 2015 for higher thoughput.5Fan F. Chen C.C. Zhang J. et al.Fluorescence dilution technique for measurement of albumin reflection coefficient in isolated glomeruli.Am J Physiol Renal Physiol. 2015; 309: F1049-F1059Crossref PubMed Scopus (14) Google Scholar However, this assay is limited to measuring relative changes in permeability. Daniels et al. developed an ex vivo method to quantify the apparent diffusion of fluorophores across individual glomerular capillaries in cross-section,6Daniels B.S. Deen W.M. Mayer G. et al.Glomerular permeability barrier in the rat. Functional assessment by in vitro methods.J Clin Invest. 1993; 92: 929-936Crossref PubMed Scopus (67) Google Scholar yet permeability values were significantly higher than values previously measured in vivo.7Huxley V.H. Curry F.E. Adamson R.H. Quantitative fluorescence microscopy on single capillaries: alpha-lactalbumin transport.Am J Physiol. 1987; 252: H188-H197PubMed Google Scholar Using similar principles we developed an assay to measure apparent, diffusive glomerular albumin permeability in isolated glomeruli deriving physiologically relevant values. We aimed to develop and characterize an assay that could sensitively measure glomerular albumin permeability and demonstrate its utility under different pathophysiological conditions, in different species. The pathophysiological variables tested were: (i) disruption of endothelial glycocalyx,8Jeansson 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, 9Jeansson 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, 10Dane M.J. van den Berg B.M. Avramut M.C. et al.Glomerular endothelial surface layer acts as a barrier against albumin filtration.Am J Pathol. 2013; 182: 1532-1540Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar (ii) exposure to plasma from patients with recurrence of nephotic syndrome posttransplant,11Savin V.J. Sharma R. Sharma M. et al.Circulating factor associated with increased glomerular permeability to albumin in recurrent focal segmental glomerulosclerosis.N Engl J Med. 1996; 334: 878-883Crossref PubMed Scopus (625) Google Scholar, 12Pudur S. Srivastava T. Sharma M. et al.Serum glomerular albumin permeability activity: association with rapid progression to end-stage renal disease in focal segmental glomerulosclerosis.SpringerPlus. 2016; 5: 432Crossref PubMed Scopus (3) Google Scholar (iii) early diabetes,13Nieuwdorp 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, 14Broekhuizen 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 (iv) rescue of glomerular permeability in diabetes using angiopoietin-1.15Dessapt-Baradez C. Woolf A.S. White K.E. et al.Targeted glomerular angiopoietin-1 therapy for early diabetic kidney disease.J Am Soc Nephrol. 2014; 25: 33-42Crossref PubMed Scopus (75) Google Scholar, 16Salmon A.H. Neal C.R. Sage L.M. et al.Angiopoietin-1 alters microvascular permeability coefficients in vivo via modification of endothelial glycocalyx.Cardiovas Res. 2009; 83: 24-33Crossref PubMed Scopus (75) Google Scholar The abdominal aorta of rats and mice were cannulated to perfuse both kidneys (Figure 1a). Kidneys were perfused first with mammalian 4% Ringer bovine serum albumin (BSA) to flush out red blood cells (which interfere with analysis). They were then perfused with octadecyl rhodamine B chloride (R18, a lipophilic membrane stain) in 4% Ringer BSA, to label cell membranes red in order to distinguish the glomerular capillary walls. This was followed by perfusion with Alexa Fluor 488 (AF488; Thermo Fisher Scientific, Waltham, MA) labeled 4% BSA. Perfused glomeruli were isolated by sieving, then incubated in AF488-BSA with or without treatments. A single glomerulus was then placed in an adapted petri dish (Figure 1b) on a confocal microscope in AF488-BSA. This was trapped to micro-occlude the afferent and efferent arterioles using a glass micropipette, thereby sealing the glomerulus so that any movement of molecules could only occur across the capillary walls by diffusion. Regions of interest (ROI) were chosen within circular capillary loops (ROI1) and external to the glomerulus (ROI2, Figure 1ci). The 4% AF488-BSA was exchanged for iso-oncotic unlabeled 4% BSA, and the initial change in fluorescence intensity was measured over time in both ROI (Figure 1cii). The fluorescence intensity and concentration of AF488-BSA were linearly related (Figure 1d), the fluorescence intensity of AF488 was not impacted by time (Figure 1e), and AF488 free dye at 0.07% or below had no impact on fluorescence intensity (Figure 1f). The decline in fluorescence intensity in ROI1 was measured over 1 minute, which was the minimum length of time necessary to give sufficient data points to represent the initial decline (Figure 1g). To demonstrate that macromolecules were effectively restricted by glomeruli in this assay, 2 different molecular weight dextrans conjugated with fluorescein isothiocyanate (FITC) were used. There was a linear relationship between low molecular weight (LMW) and high molecular weight (HMW) dextran concentration and fluorescence intensity (Supplementary Figure S1A). Isolated glomeruli were significantly more permeable to LMW FITC-dextran (4 kDa) compared with HMW FITC-dextran (500 kDa), as predicted (Supplementary Figure S1B). However, baseline values for FITC-500 kDa dextran were much higher than anticipated. This is likely due to the overestimation of permeability of dextran due to the rapid photobleaching of FITC in our system (Supplementary Figure S1Ci and Cii). Although these data suggest that FITC is inferior to AF488 in our assay, the data nevertheless confirm lower glomerular permeability to a higher–molecular weight tracer. The apparent glomerular albumin permeability (Ps'alb) assay was used to demonstrate that physiologically relevant permeability values could be measured in glomeruli from different species, including humans. The cellular components of the glomerular filtration barrier contribute substantially to macromolecular permeability control.6Daniels B.S. Deen W.M. Mayer G. et al.Glomerular permeability barrier in the rat. Functional assessment by in vitro methods.J Clin Invest. 1993; 92: 929-936Crossref PubMed Scopus (67) Google Scholar Therefore intact rodent and human glomeruli were compared with partially decellularized glomeruli to demonstrate that the assay could measure changes in glomerular permeability associated with loss of the cellular components in a similar manner to that used by Daniel's et al.6Daniels B.S. Deen W.M. Mayer G. et al.Glomerular permeability barrier in the rat. Functional assessment by in vitro methods.J Clin Invest. 1993; 92: 929-936Crossref PubMed Scopus (67) Google Scholar Confocal fluorescence imaging confirmed removal of cell membranes from glomeruli (by loss of R18 staining) although there was some residual nuclear material (Figure 2a and b). Ps'alb values were significantly higher for partially decellularized glomeruli than intact glomeruli in all species examined (Figure 2c–e). Human, rat, and mouse Ps'alb values were all of a similar magnitude to those previously reported in systemic vessels in vivo (and ex vivo) (Figure 2f, Table 1). Glomerular permeability is more often measured by glomerular sieving coefficients (GSC) in vivo. To understand how Ps'alb values related to previously published GSC values in rat, mouse and human glomeruli, the membrane pore model of permeability was used to convert Ps'alb to equivalent values of GSC (Table 2) (see "Calculation of GSC values" in Supplementary Methods for details). Human-, rat-, and mouse-converted GSC values (0.00182, 0.0027, and 0.002, respectively) were very similar to those previously reported (Figure 2g, Table 3). Importantly, we have shown for the first time that human Ps'alb can be measured directly and with physiologically relevant values. Methodology varied between rodent and human glomeruli preparations, because it was impractical to perfuse whole human kidneys with fluorophores, although all kidneys were perfused with a physiological salt solution. There was also a 24-hour time delay in processing human kidneys. In 1 set of experiments, rat and mouse glomeruli were therefore also incubated ex vivo with fluorophores in the same way as human glomeruli. Glomeruli treated in this way still had significantly lower permeability than the human glomeruli, confirming that the different methods for applying fluorophores did not account for species differences. Furthermore, there were no significant differences in Ps'alb between these 2 methodologies in either rats or mice (Supplementary Figure S2A and B). Glomeruli that were isolated for up to 4 hours before measurements did not have significantly different Ps'alb, suggesting that up until this point glomerular permeability is stable (Supplementary Figure S2C). All our previous observations on rodent glomeruli were performed within this time frame. In another set of experiments, mouse kidneys were kept on ice for 24 hours before glomeruli were isolated to compare with the delayed processing time frame of human kidneys. There was a significant increase in Ps'alb in mouse glomeruli processed after 24 hours (Supplementary Figure 2D), indicating that the prolonged cold storage of human organs inherent in the retrieval process may provide some of the explanation for higher permeability values. Ps'alb was significantly higher for human glomeruli than these mouse glomeruli, indicating a species difference or other residual differences (Figure 2h). Of note, mice in these experiments were aged to 1 year to relate to aged human kidneys (from donors 72 ± 3.1 years of age).Table 1Ps'alb values in previously described modelsSpeciesMethodPs'alb valueStudy1FrogIn vivo mesenteric vessel cannulation4e-006 cm/s (15 cm H2O capillary pressure)Huxely et al., 19877Huxley V.H. Curry F.E. Adamson R.H. Quantitative fluorescence microscopy on single capillaries: alpha-lactalbumin transport.Am J Physiol. 1987; 252: H188-H197PubMed Google Scholar2RatEx vivo isolated glomeruli1.36e-0.006 cm/sDaniels et al., 19936Daniels B.S. Deen W.M. Mayer G. et al.Glomerular permeability barrier in the rat. Functional assessment by in vitro methods.J Clin Invest. 1993; 92: 929-936Crossref PubMed Scopus (67) Google Scholar345PigEx vivo, coronary microvessel, isolated and cannulated2.85e-006 cm/s3.92e-006 cm/s (11 cm H2O capillary pressure)4.0e-007cm/s (15.3 cm H2O arteriole pressure)Yuan et al., 200046Yuan S.Y. Ustinova E.E. Wu M.H. et al.Protein kinase C activation contributes to microvascular barrier dysfunction in the heart at early stages of diabetes.Circ Res. 2000; 87: 412-417Crossref PubMed Scopus (87) Google ScholarYuan et al., 199347Yuan Y. Chilian W.M. Granger H.J. et al.Permeability to albumin in isolated coronary venules.Am J Physiol. 1993; 265: H543-H552PubMed Google ScholarHuxley et al., 200048Huxley V.H. Williams D.A. Role of a glycocalyx on coronary arteriole permeability to proteins: evidence from enzyme treatments.Am J Physiol Heart Circ Physiol. 2000; 278: H1177-H1185Crossref PubMed Google Scholar6RatIn vivo venular microvessels0.92e-006 cm/s (20 cm H2O microvessel pressure)Adamson et al., 201449Adamson R.H. Clark J.F. Radeva M. et al.Albumin modulates S1P delivery from red blood cells in perfused microvessels: mechanism of the protein effect.Am J Physiol Heart Circ Physiol. 2014; 306: H1011-H1017Crossref PubMed Scopus (46) Google Scholar7RatIn vivo mesenteric vessel cannulation0.82e-006 cm/sCai et al., 198550Cai B. Fan J. Zeng M. et al.Adhesion of malignant mammary tumor cells MDA-MB-231 to microvessel wall increases microvascular permeability via degradation of endothelial surface glycocalyx.J Appl Physiol (1985). 2012; 113: 1141-1153Crossref PubMed Scopus (35) Google ScholarPs'alb, apparent glomerular albumin permeability. Open table in a new tab Table 2GSC converted from Ps'alb for human rat and mouse glomeruliCoefficientMouseRatHumanP albumin (cm/s)4.3 x 10–74.5 x 10–78.942 x 10–7Lp cm/(sec dyne) x 10–82.11.6292.735Single pore population, pore radius, R (nm)3.7423.693.776Single pore: θ (GSC)0.00260.000680.0016Two-pore population: RL = 11 nm,27Lund U. Rippe A. Venturoli D. et al.Glomerular filtration rate dependence of sieving of albumin and some neutral proteins in rat kidneys.Am J Physiol Renal Physiol. 2003; 284: F1226-F1234Crossref PubMed Scopus (120) Google Scholar Equivalent θ, (GSC)0.0020.00270.00182GSC, glomerular sieving coefficient; Ps'alb, apparent glomerular albumin permeability.See explanation in Supplementary Materials and Methods. Open table in a new tab Table 3GSC values in previously described modelsSpeciesMethodPs'albStudy12RatIn vivo, confocal, glomerular0.00270.0004Stolte et al., 197951Stolte H. Schurek H.J. Alt J.M. Glomerular albumin filtration: a comparison of micropuncture studies in the isolated perfused rat kidney with in vivo experimental conditions.Kidney Int. 1979; 16: 377-384Abstract Full Text PDF PubMed Scopus (44) Google ScholarTanner et al., 200952Tanner G.A. Glomerular sieving coefficient of serum albumin in the rat: a two-photon microscopy study.Am J Physiol Renal Physiol. 2009; 296: F1258-F1265Crossref PubMed Scopus (73) Google Scholar3MouseIn vivo, multiphoton imaging, glomerular0.0025Nakano et al., 201253Nakano D. Kobori H. Burford J.L. et al.Multiphoton imaging of the glomerular permeability of angiotensinogen.J Am Soc Nephrol. 2012; 23: 1847-1856Crossref PubMed Scopus (99) Google Scholar4MouseCooled isolated perfused kidney0.0023Jeansson et al., 20038Jeansson 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 Scholar5HumanFanconi syndrome, HPLC, and MALDI-TOF MS urine analysis0.00008Norden et al., 200434Norden A.G. Sharratt P. Cutillas P.R. et al.Quantitative amino acid and proteomic analysis: very low excretion of polypeptides >750 Da in normal urine.Kidney Int. 2004; 66: 1994-2003Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar6RatFractional micropuncture0.0006Tojo and Endou, 199254Tojo A. Endou H. Intrarenal handling of proteins in rats using fractional micropuncture technique.Am J Physiol. 1992; 263: F601-F606PubMed Google Scholar7RatLabeled normal and neutral albumin0.0006Bertolatus and Hunsicker, 198555Bertolatus J.A. Hunsicker L.G. Glomerular sieving of anionic and neutral bovine albumins in proteinuric rats.Kidney Int. 1985; 28: 467-476Abstract Full Text PDF PubMed Scopus (62) Google Scholar8RatIntravital 2-photon microscopy0.034Russo et al., 200756Russo L.M. Sandoval R.M. McKee M. et al.The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: retrieval is disrupted in nephrotic states.Kidney Int. 2007; 71: 504-513Abstract Full Text Full Text PDF PubMed Scopus (329) Google ScholarHPLC, high-performance liquid chromatography; MALDI-TOF MS, Matrix-assisted laser desorption ionization-time of flight mass spectrometry; Ps'alb, apparent glomerular albumin permeability. Open table in a new tab Ps'alb, apparent glomerular albumin permeability. GSC, glomerular sieving coefficient; Ps'alb, apparent glomerular albumin permeability. See explanation in Supplementary Materials and Methods. HPLC, high-performance liquid chromatography; MALDI-TOF MS, Matrix-assisted laser desorption ionization-time of flight mass spectrometry; Ps'alb, apparent glomerular albumin permeability. We took a number of approaches to test that the glomerular Ps'alb assay could effectively measure changes in glomerular macromolecular permeability. eGLX is situated on the luminal side of glomerular and other endothelial cells17Luft J.H. Fine structures of capillary and endocapillary layer as revealed by ruthenium red.Fed Proc. 1966; 25: 1773-1783PubMed Google Scholar, 18Weinbaum 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 is the first (most "upstream") component of the glomerular filtration barrier to encounter albumin.19Arkill K.P. Knupp C. Michel C.C. et al.Similar endothelial glycocalyx structures in microvessels from a range of mammalian tissues: evidence for a common filtering mechanism?.Biophys J. 2011; 101: 1046-1056Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Damage to eGLX decreases glomerular filtration barrier albumin permselectivity as demonstrated by fractional clearance of labeled Ficoll and albumin in cooled isolated perfused mouse kidneys.8Jeansson 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, 9Jeansson 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 To test the physiological sensitivity of the assay, eGLX dysfunction was induced with an i.v. bolus of hyaluronidase and chondroitinase 30 minutes before killing. Representative electron micrographs are shown to demonstrate systemic eGLX removal by GAG-degrading enzymes in coronary and glomerular microvessels (Figure 3a). While eGLX depth was not significantly decreased, vessel coverage by eGLX was (Figure 3b), suggesting a "patchy" coverage as demonstrated in the micrographs. Of note, we have previously shown that both vessel coverage by eGLX and eGLX depth are inversely related to permeability.20Betteridge K. Arkill K. Neal C. 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 The enzymes induced a significant increase in Ps'alb as anticipated (Figure 3c). No significant differences were found in podocyte foot process width, foot process length, number of foot processes per μm GBM, slit diaphragm length, number of endothelial fenestration per μm GBM, and GBM width (data not shown), suggesting that effects of these enzymes on the glomerular filtration barrier are limited to the eGLX. To test the use of clinical samples on this assay, plasma was used from individuals with SRNS that had undergone plasma exchange during periods of posttransplant recurrence, or subsequent remission, with the assumption being that in recurrence the "permeability factors" were present and in remission they were not.21Harris J.J. McCarthy H.J. Ni L. et al.Active proteases in nephrotic plasma lead to a podocin-dependent phosphorylation of VASP in podocytes via protease activated receptor-1.J Pathol. 2013; 229: 660-671Crossref PubMed Scopus (54) Google Scholar Rats were perfused with R18 and AF488-BSA, and glomeruli were isolated and incubated in 10% plasma from SRNS patients for 1 hour. SRNS recurrence plasma significantly increased Ps'alb in rat glomeruli compared with remission plasma, which was very similar to control conditions (no plasma) (Figure 3d). To further investigate the sensitivity of the Ps'alb assay, a rat model of early glomerular damage was utilized. Rats given streptozotocin (STZ, a type 1 model of diabetes) were significantly hyperglycemic at 1 week after injection, which was sustained (Figure 4a), confirming diabetes. Weight gain in diabetic rats was significantly reduced as anticipated (Figure 4b). Diabetic rats showed a modest, but significant, increase in urinary albumin-to-creatinine ratio (1.7-fold increase) compared with control rats 4 weeks after injection (Figure 4c), suggesting a modest increase in glomerular leak of albumin. It is important to note that albuminuria is independent of STZ-induced renal toxicity in this rat model (although total proteinuria is not).22Palm F. Ortsater H. Hansell P. et al.Differentiating between effects of streptozotocin per se and subsequent hyperglycemia on renal function and metabolism in the streptozotocin-diabetic rat model.Diabetes Metab Res Rev. 2004; 20: 452-459Crossref PubMed Scopus (72) Google Scholar Mesangial matrix expansion was assessed using periodic acid–Schiff staining, demonstrating a mild but significant increase in glomerular fibrosis in diabetic rats (Figure 4di and ii). Electron microscopy was used to analyze glomerular ultrastructure. No significant changes were observed (representative images Figure 4ei–iv, data not shown). Together these data confirm that these diabetic rats have early signs of glomerular damage rather than established diabetic nephropathy. Diabetes induced a significant increase in Ps'alb, which was ameliorated by the paracrine growth factor, angiopoietin-1 (Ang1), to a level comparable with nondiabetic glomeruli (Figure 4f), supporting previously published results.15Dessapt-Baradez C. Woolf A.S. White K.E. et al.Targeted glomerular angiopoietin-1 therapy for early diabetic kidney disease.J Am Soc Nephrol. 2014; 25: 33-42Crossref PubMed Scopus (75) Google Scholar Ang1 alone had no effect compared with sham, suggesting no effect on otherwise healthy capillaries under nondiabetic conditions. eGLX is damaged in models of diabetes.23Oltean 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 We have previously shown that Ang1 acts on glomerular endothelial cells24Satchell S.C. Anderson K.L. Mathieson P.W. Angiopoietin 1 and vascular endothelial growth factor modulate human glomerular endothelial cell barrier properties.J Am Soc Nephrol. 2004; 15: 566-574Crossref PubMed Scopus (148) Google Scholar and that it can increase eGLX depth and reduce microvascular permeability in a different capillary bed.16Salmon A.H. Neal C.R. Sage L.M. et al.Angiopoietin-1 alters microvascular permeability coefficients in vivo via modification of endothelial glycocalyx.Cardiovas Res. 2009; 83: 24-33Crossref PubMed Scopus (75) Google Scholar Therefore, we investigated whether the protective effect of Ang1 was associated with restoration of eGLX damage. Glomerular eGLX depth and coverage was significantly reduced in diabetic rats compared with control rats, but was restored by Ang1 (Figure 4gi–iii). Together these data confirm that the Ps'alb assay can sensitively measure early changes in glomerular barrier function associated with eGLX dysfunction. We have developed a glomerular albumin permeability assay that gives physiologically relevant permeability values and allows human Ps'alb measurements for the first time. We demonstrated utility of the assay and confirmed that it was sufficiently sensitive to measure early decline in glomerular permeability and could be used in conjunction with clinical samples. Ps'alb measurements were reassuringly close to those measured previously in vivo7Huxley V.H. Curry F.E. Adamson R.H. Quantitative fluorescence microscopy on single capillaries: alpha-lactalbumin transport.Am J Physiol. 1987; 252: H188-H197PubMed Google Scholar, 8Jeansson 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 and even lower than previously measured Ps'alb values ex vivo.6Daniels B.S. Deen W.M. Mayer G. et al.Glomerular permeability barrier in the rat. Functional assessment by in vitro methods.J Clin Invest. 1993; 92: 929-936Crossref PubMed Scopus (67) Google Scholar This demonstrates the strength of using permeability values compared with using relative changes in permeability, as used previously.4Savin V.J. Sharma R. Lovell H.B. et al.Measurement of albumin reflection coefficient with isolated rat glomeruli.J Am Soc Nephrol. 1992; 3: 1260-1269Crossref PubMed Google Scholar It allows us to directly compare with previously reported values and to interpret our data in the context of health and disease. Knowing that the permeability of glomeruli are within the physiological range confirms the rel