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1Microvascular Research Laboratories, Bristol Heart Institute, Department of Physiology, University of Bristol, 2Academic Renal Unit, Clinical Science at North Bristol, University of Bristol, Paul O'Gorman Lifeline Centre, Southmead Hospital, Westbury-on-Trym, Bristol, United Kingdom; and 3Departments of Physiology, Biophysics, and Medicine, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California
Submitted 18 April 2007 ; accepted in final form 23 August 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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450 Da), accumulated in SPS-covered regions of the GFB, compared with GFB regions not covered by SPS ("naked regions"). Net lucifer yellow flux (taken to indicate fluid flux) through identifiable SPS regions was calculated to be 66–75% of that occurring through naked regions. These observations indicate both ultrafiltration and hydraulic resistance imparted by the SPS, demonstrating the potential physiological contribution of the SPS to glomerular permeability. glomerulus; permeability; podocyte; multiphoton
These three major components interact to generate the overall permeability coefficients of the GFB. Isolated defects in any one of the three layers of the barrier can result in alterations in permeability, e.g., digestion of glomerular endothelial glycocalyx with lumen-restricted enzymes (14), selective deletion of the important GBM component laminin β-1 (22), and loss of critical slit diaphragm components, including nephrin (16). These defects can result in glomerular nephropathies. Defects in the GBM are recognized in conditions such as Alport's syndrome and thin basement membrane nephropathy (30), whereas mutations in molecules at the slit diaphragm such as nephrin result in congenital nephropathies, e.g., of the Finnish type (16). Endothelial dysfunction, e.g., in diabetes or preeclampsia, also results in increased glomerular permeability and proteinuria (31). Nevertheless, no layer in isolation is considered to dominate either the hydraulic resistance or the permselective properties of the barrier (5), and both its anatomic characteristics (5) and its molecular constituents (9, 28) ensure interdependence of all layers of this barrier.
In 2005, the anatomic pathway dictating the movement of primary ultrafiltrate from the abluminal aspect of the podocyte filtration slits to the proximal tubule was reevaluated (20). What had hitherto been described as "Bowman's space" (12, 17) was shown to comprise three distinct urinary spaces, based on the identification of focal narrowings within Bowman's space. The first focal narrowing was identified between highly attenuated, tortuous urinary space regions beneath podocyte cell bodies or processes and long, narrow channels coursing through the glomerular tuft. This narrowing therefore separated these regions into distinct urinary spaces: the subpodocyte space (SPS) and interpodocyte space (IPS), respectively, and the narrowing itself was termed the "subpodocyte space exit pore" (SEP). The second narrowing was demonstrated between the IPS and the shell-like urinary space between the glomerular tuft and Bowman's capsule, thereby distinguishing the IPS from the "peripheral urinary space" (PUS). The SPS was found to cover between 50 and 65% of the GFB, irrespective of whether the GFB was on the outer aspect of the glomerular tuft or deep within it (20). In a subsequent anatomic study of SPS dimensions, in which fixation conditions required for electron microscopy were approximated as closely as possible to those found during glomerular perfusion in vivo (21), the mean SPS height was a mere 340 nm (approximately the same width as the entire underlying GFB), and the average distance from GFB to the nearest SPS exit pore was 6,700 nm (see Fig. 1A). Mathematical modeling, using anatomic information from the aforementioned study and based on a circular flow model, indicated a hydraulic resistance of the SPS to be about three to four times that of the GFB (21). These observations led to the suggestion that the movement of fluid across the SPS might be substantially lower than that occurring across regions of the GFB that were not covered by SPS ("naked regions"). In addition, the prevalence of SPS coverage of the GFB would render this differential fluid flux highly physiologically significant. We therefore set out to test the hypothesis that molecular movement across SPS-covered GFB would differ from that occurring across naked regions of the GFB. This study used multiphoton fluorescence imaging of isolated, microperfused glomeruli and the intact kidney in vivo since this technology can section optically through an entire glomerulus with high resolution and without causing damage to the living tissue (15).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Southern California. Animals [female New Zealand rabbits, 500 g (Irish farms, Norco, CA); n = 6] were housed in a temperature-controlled room with a 12:12-h light-dark cycle, with water and standard chow available ad libitum. Anesthetized rabbits were euthanized by decapitation, and a left nephrectomy was performed immediately postmortem. Transverse slices of renal tissue (2 mm thick) were immediately placed into cooled (4°C) dissection media [DMEM/F12 (D8900, Sigma, St. Louis, MO); 14.28 mM NaHCO3; (bubbled with 95% O2-5% CO2 for 
30 min); 3 g/dl FBS; pH adjusted to 7.40 ± 0.02 with 1 M NaOH]. Arcuate artery preparations that retained separated lobules of renal cortical tissue were further dissected (Carl Zeiss Stemi-2000CS stereomicroscope), to reveal afferent arteriole-glomerulus preparations, from which redundant tubular tissue was carefully removed, with care taken not to stretch the afferent arteriole. The afferent arteriole was then severed at its base with a 30-gauge needle, and the afferent arteriole-glomerulus preparation was transferred to a thermo-regulated Lucite chamber on the experimental microscope stage. Once secured, fresh mammalian modified Krebs-Ringer solution within the perfusion bath [in mM: 115 NaCl, 5 KCl; 0.24 Na2HPO4; 0.96 NaH2PO4; 25 NaHCO3; 1.2 MgSO4; 2 CaCl2; 5.5 D(+) glucose, as well as 100 µM L-arginine (Sigma), pH 7.40 ± 0.02 (95% O2-5% CO2)] was continuously exchanged, and the temperature was gradually elevated to 36 ± 2°C. The afferent arteriole was cannulated with a series of mounted concentric pipettes (Vestavia Scientific, Vestavia, AL) containing Krebs-Ringer solution (as above) supplemented with 10 mg/ml BSA, and successful glomerular capillary perfusion was marked by the longitudinal movement of red blood cells within the glomerular capillaries and their expulsion from the efferent arteriole. Additional details of the afferent arteriole-attached glomerulus microperfusion technique have been described earlier (23, 24).
In Vivo Imaging of the Intact Kidney
Munich-Wistar-Fromter male rats (200 g, Harlan, Madison, WI, n = 3) were anesthetized with thiobutabarbital (Inactin; 130 mg/kg body wt). The left kidney was prepared for multiphoton imaging as described before (15). Briefly, after adequate anesthesia was ensured, the trachea was cannulated to facilitate breathing. The left femoral vein and artery were cannulated for dye infusion and blood pressure measurements, respectively. Subsequently, a 10- to 15-mm dorsal incision was made under sterile conditions and the kidney was exteriorized. The animal was placed on the stage of an inverted microscope with the exposed kidney placed in a coverslip-bottomed heated chamber bathed in normal saline, and the kidney was visualized from below using a HCX PL APO 63X/1.4-numerical aperture oil CS objective (Leica). During all procedures and imaging, core body temperature was maintained with a homeothermic table. All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Southern California.
Imaging System
Images were obtained via a x63 oil-immersion objective attached to a Leica DM IRE2 inverted microscope (Leica Microsystems, Heidelberg, Germany) with a number of internal, external, and transmitted light detector photomultipliers and collected in time (xyt) or z-section (xyz) series with Leica LCS imaging software. Conventional one-photon excitation was achieved with a Leica TCS SP2 AOBS MP confocal microscope system, powered by a series of lasers: red (HeNe 633 nm/10 mW); orange (HeNe 594 nm/2 mW); green (HeNe 543 nm/1.2 mW); and blue (Ar 458 nm/5 mW; 476 nm/5 mW; 488 nm/20 mW; 514 nm/20 mW). Multiphoton excitation was achieved with an infrared (710–920 nm) combined photo-diode pump laser and mode-locked titanium:sapphire laser (Mai-Tai, Spectra-Physics). Further details of the imaging system are given elsewhere (24).
Dyes and Tracers
Dyes. Cell nuclei were stained with 3 µM Hoechst 33342 (Molecular Probes, Eugene, OR). Cell cytoplasm staining was obtained with 25 µM quinacrine (Sigma). Dyes were added to the perfusate solution in combination and applied to the lumen of the perfused afferent arteriole and glomerular capillaries for 20–30 min. During this loading time, an appropriate section of glomerular capillaries was selected for subsequent experiments. In in vivo experiments, Hoechst 33342 was used (10 µl of a 10 mg/ml stock in iv bolus) to label cell nuclei, and a 500-kDa dextran-fluorescein conjugate (neutral, 20 µl of a 10 mg/ml stock in iv bolus, Molecular Probes) labeled the circulating plasma.
Tracers. Lucifer yellow [LY; 0.3 mg/ml (MW 457 Da)] or 40 µg/ml 3-kDa dextran-Alexa 488 and 40 µg/ml 10-kDa dextran-rhodamine (all from Molecular Probes) served as low- and intermediate-molecular weight markers of solute movement across the GFB, respectively. These relatively low-molecular-weight markers were chosen since both should move across the GFB with little (dextran) or no (LY) selective retention (32). Because the ionic charge of fluorophores affects glomerular filtration characteristics, neutral conjugates of dextran were preferentially used. Although the fluid marker LY is an anionic compound, it is freely filtered due to its small size (0.45 kDa). Following dye loading, tracers were applied individually to the isolated, perfused glomerulus preparations. An xyt series of the initiation, perfusion, and washout of each tracer (perfusion: n = 6 for each tracer) was obtained in three glomeruli; in one glomerulus, LY and 10-kDa dextran-rhodamine were perfused sequentially. In some experiments the 3- and 10-kDa dextran were perfused simultaneously. Individual images in the xyt-series were separated by <0.8-s intervals.
Hoechst 33342 (emission between 400 and 460 nm), quinacrine (emission at 510 nm), dextran-Alexa 488, and dextran-fluorescein (emission at 520 nm), LY (emission at 540 nm), and dextran-rhodamine (emission at 590 nm) were all excited at 860 nm. The emitted, nondescanned fluorescent light was detected by two external photomultipliers [green (including blue) and red channels] with the help of a FITC/TRITC filter block (Leica).
Image Analysis
Regions of interest. For xyt series analysis, Leica LCS imaging software was used. Spatial regions of interest were selected, and the position was verified in all images comprising the series (Fig. 1B). Spatial regions were selected only on the outer aspect of peripheral capillary loops, where abluminal cell bodies were taken to represent podocytes. GFB regions were distinguished by the absence (naked regions, Fig. 1Bi) or presence ("covered" regions, Fig. 1Bii) of peripheral abluminal cell (podocyte) coverage. Covered regions were taken to represent GFB with overlying SPS, as indicated by electron microscopic studies (20), and the results are reported herein. Any minor shift in the location of the capillary wall region of interest in the multiphoton image between perfusion periods was accommodated by duplicating the analysis region when necessary and ensuring that measurements were taken from exactly the same portion of the GFB in all images comprising the xyt series. Two (paired) regions of interest were studied in all cases: one comprising GFB with or without SPS coverage, and one within an adjacent glomerular capillary lumen.
Fluorescence intensity per unit area (If) of the dyes (dye If: indicating cellular structures) and tracers (tracer If: representing solute molecules) were determined in the region of interest in every frame of the series. Dye If was carefully monitored in all frames of the xyt series, and constant values were taken to indicate constancy of the anatomy of the region of interest. Baseline tracer If values were subtracted from all subsequent tracer If measurements to facilitate comparison between tracer If at any given time in the capillary lumen and capillary wall. Tracer If values were plotted against time.
Analysis of solute flux through SPS-covered regions. Linear regression lines were applied to tracer If measurements in the time period encompassed by the beginning and end of the stable values of tracer If achieved in both the capillary lumen and adjacent capillary wall regions. The slopes of these lines describe the change in tracer If per unit time [d(tracer If)/dt]. Any change in d(tracer If)/dt in the capillary wall that is not reflected by a comparable change in d(tracer If)/dt in the capillary lumen indicates ultrafiltration of the tracer by the capillary wall. d(tracer If)/dt within the capillary lumen and capillary wall regions were compared with ANCOVA (Prism, GraphPad Software).
d(tracer If)/dt in the capillary lumen region [d(tracer If)/dt]lumen was subtracted from d(tracer If)/dt in the adjacent capillary wall region [d(tracer If)/dt]wall for each pair of regions studied, to eliminate any potential apparent change in tracer If in the wall region attributable to a change in tracer If in the perfusate. These subtracted paired measurements for all SPS-covered GFB regions and all naked GFB regions were compared with one-way ANOVA with Bonferroni's correction.
Analysis of fluid flux rate through SPS-covered regions. The absence of a rise in LY If measurements in either covered or naked GFB regions see (Fig. 4) indicates the absence of ultrafiltration of LY, as predicted by its molecular weight. LY flux was therefore taken as a surrogate marker of solvent flux. Solvent flux through naked regions was taken as the reference value. Calculation of solvent flux through the SPS, relative to that occurring through naked GFB regions, is described in RESULTS.
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RESULTS |
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Anatomic measurements of SPS were obtained from xyz series of quinacrine-loaded isolated, perfused glomeruli. SPS were identified as nonfluorescent regions bounded by GFB on one side and by cell body (or cellular processes extending from the cell body) of abluminal cells (Fig. 1B). To avoid confusion between podocytes and mesangial cells, only abluminal cells overlying the outer aspect of peripheral capillary loops were considered. In the first set of studies using three glomeruli (from 3 different animals), five SPS were unequivocally identified by this method. SPS dimensions (means ± SE) were 2.3 ± 0.6-µm depth x 4.0 ± 0.5-µm width x 0.8 ± 0.1-µm height. SPS heights are comparable to those obtained in the electron microscopic study of Neal et al. (20) under similar perfusion conditions.
Isolated glomeruli were loaded with quinacrine and perfused with LY or fluorescently labeled dextrans of different sizes (10 and 3 kDa). Figure 2 demonstrates the feasibility of detecting the dynamics of solute filtration with high-speed scanning and simultaneously resolving the anatomy of naked and SPS-covered GFB regions with multiphoton imaging. The microperfused solutes quickly appeared in the lumen of glomerular capillary loops and then filtered into Bowman's capsule. Within 5–10 s, fluorescence intensity of solutes reached a maximum and stabilized in the capillary lumen and Bowman's space. Filtration of solutes was clearly visible across both naked and SPS-covered regions of the GFB (Fig. 2).
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10-kDa dextran (larger solute). Representative plots of dextran If vs. time are given in Fig. 3. The fluorescence intensity rises as the solute moves into the region and after the filling artifact stays constant in the lumen and in naked GFB regions, i.e., GFB not covered by SPS {[d(dextran If)/dt]naked 0.01 units/s; [d(dextran If)/dt]lumen 0.04 units/s, P > 0.65, ANCOVA, Fig. 3A}, indicating that perfusion concentration is constant and flux has reached a steady state. In contrast, dextran If rose during the period of stable tracer perfusion in the SPS-covered GFB region (Fig. 3B), indicating accumulation of 10-kDa dextran-rhodamine within the SPS {[d(dextran If)/dt]SPS 0.82 units/s; [d(dextran If)/dt]lumen 0.13 units/s, P < 0.0001, ANCOVA}.
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Changes in tracer If in the capillary wall region during the tracer perfusion period may be attributable to selective retention of the tracer by the components of the capillary wall, or to changes in the concentration of tracer that is perfused. To eliminate any influence of the latter [d(tracer If)/dt]lumen was subtracted from [d(tracer If)/dt]wall for each pair of wall and adjacent lumen regions for each experimental replicate. [d(tracer If)/dt]wall – [d(tracer If)/dt]lumen values (means ± SE) are given in Table 1 and plotted in Fig. 5. Only [d(dextran If)/dt]wall – [d(dextran If)/dt]lumen for 10-kDa dextran-rhodamine in SPS-covered GFB regions was significantly different from zero (P < 0.0001, 1-sample t-test vs. 0; P > 0.25, P > 0.55, and P > 0.75 for all other tracers/regions), indicating that 10-kDa dextran-rhodamine but not LY accumulated only in capillary wall regions containing SPS. [d(tracer If)/dt]wall – [d(tracer If)/dt]lumen for 10-kDa dextran-rhodamine in SPS-covered regions was significantly different from the same parameter for 10-kDa dextran-rhodamine in naked regions and was also significantly different from LY values in both SPS and naked capillary wall regions (P < 0.001 for 10-kDa dextran-rhodamine in SPS regions vs. all other tracers/regions, 1-way ANOVA with Bonferroni's correction).
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Of interest was the observation that the intensity at which a steady state was reached with LY appeared to be closer to the luminal level in naked capillary wall regions (Fig. 4A) compared with covered capillary wall regions (Fig. 4B). The mean ratio of LY If in naked regions (nakIf) to that in adjacent luminal regions (nakIf) was 0.65 ± 0.07. The comparable ratio in covered regions (covIf/lumIf) was 0.46 ± 0.06.
Since LY is freely filtered, LY If in the nonrestrictive urinary space outside naked capillary wall regions is equal to that in the capillary lumen. The contents of the naked capillary wall regions are the GFB (height from multiphoton images: 1.02 ± 0.04 µm) and this nonrestrictive urinary space (height 0.25 ± 0.12). The 65% reduction in mean nakIf/lumIf must therefore be attributable to the fraction of the GFB from which dye is excluded (podocyte foot processes, endothelial cytoplasm, and unavailable regions of GBM: 44% of total GFB volume; see Fig. 4).
Covered GCW regions contain GFB, SPS, and podocyte cell bodies/processes (total mean height 1.55 µm). The observation that mean covIf/lumIf (0.46 ± 0.06) is considerably below mean nakIf/lumIf (0.65 ± 0.07) may be attributable to complete dye exclusion from the fraction of covered regions occupied by podocyte cell bodies/processes or may be due to reduced solvent flux through SPS (assuming that the 450-Da molecule LY is a surrogate marker for fluid). Values for SPS height have been taken from the literature [mean height under comparable perfusion conditions with low/no perfusate oncotic pressure: 0.7 µm (20); mean height with physiological perfusate oncotic pressure: 0.35 µm]. The height of the cell body component of covered regions is taken as the total covered region height less both GFB height and SPS height. Assuming that 44% of GFB volume excludes LY (as for naked regions), the fraction of the total volume of covered regions that is available to LY is in the range 0.70 (SPS height 0.7 µm) to 0.62 (SPS height 0.35 µm). These fractions are considerably higher than mean covIf /lumIf (0.46 ± 0.06), indicating that there are fewer LY molecules per unit available volume in SPS than in the adjacent capillary lumen. This indicates that solvent flux through SPS is in the range 66–75% of that occurring across naked GFB regions.
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DISCUSSION |
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It has not been possible to distinguish the exact mechanism by which the SPS restricts the movement of water and an intermediate-molecular-weight solute. The dominant resistance to molecular flux across the SPS is unlikely to reside at the entrance to the space, since there is a large filtration surface area into each individual SPS (20), and there do not appear to be any differences in the morphology of the underlying filtration barrier in SPS-covered, compared with naked, regions. Mathematical modeling indicates that the subpodocyte exit pores, whose infrequency determines that the area that is available for efflux from the SPS is 25-fold lower than that available for influx into each space (20), provide an important contribution to the hydraulic resistance of the SPS (21). The importance of these exit pores on solute ultrafiltration remains to be determined experimentally, but mathematical modeling (21) indicates that the SPS exit pore may be the primary site of restriction to fluid flow. An alternative explanation to restriction imposed by SPS exit pores is that the flux of molecules across the SPS cavity may be the dominant form of restriction. The presence of a glycocalyx, which appears to line the interior of the SPS (11), may restrict solute and/or water movement (1, 33). Finally, solute molecules may be selectively taken up by an active process within the SPS, such as macropinocytosis or endocytosis (29, 35), although such phenomena have not been demonstrated to occur over a period of seconds, as observed in our studies.
The interpretations of the experimental results outlined above rely on a number of assumptions. First, tracer If was examined in regions of the glomerular capillary wall that lay immediately beneath abluminal cell bodies or processes on the outer aspect of peripheral capillary loops, which we have assumed to represent SPS. It was not possible to identify SPS regions in living tissue with as ultra-high resolution as is possible with electron microscopy. However, we did combine a high temporal resolution for physiological studies with simultaneous high-spatial-resolution images with the multiphoton technique to give as much confidence as possible that we were measuring flux in an SPS (Figs. 1–2, 7). Secondly, we have assumed that the rise in dextran If that is observed in SPS regions during glomerular perfusion is not due to the plane of imaging gradually shifting from a cell-rich wall region into a solute-rich capillary lumen. Two major arguments can be raised against this possibility: 1) dye If remained approximately constant during the perfusion period, indicating that even if the plane of imaging did move, then it did so into an equivalently cell-rich region; and 2) a similar rise was absent in tracer If in naked capillary wall regions adjacent to the SPS in which dextran If rose, or a similar effect for LY If in SPS regions.
Finally, the calculations of relative solvent flux through SPS-covered and naked GFB regions require three assumptions. 1) The permeability coefficients of the GFB with and without SPS coverage are the same. Although there is no a priori reason to think that this is not the case, the permeability values have not been determined. At the present time, there has been no systematic investigation of the ultrastructure of the GBM, podocyte slit diaphragms, or endothelial fenestrations underneath the SPS compared with naked regions, but this needs to be carried out. 2) The solute and solvent fluxes were in steady state. The finding that LY If was stable shortly after initiation of perfusion indicates a steady-state condition. 3) The height of SPS that is used in the calculation of Jv across the SPS is representative. Conservative estimates of SPS height were employed in the calculations of filtration rate (Jv) across the SPS (0.7 and 0.35 µm) compared with SPS height measured from the multiphoton images (mean 0.8 µm), thus ensuring that estimates of Jv across SPS are likely to be conservative in magnitude.
Anatomically distinct nonfluorescent regions bounded by the GFB and abluminal cell bodies/processes, taken to represent SPS, were observed less frequently with multiphoton microscopy compared with electron microscopy (20). In addition, while the mean SPS height observed with confocal microscopy is comparable to the mean SPS height under nonphysiological perfusion conditions (20), multiphoton-derived heights were significantly greater than those obtained with electron microscopic studies using physiologically relevant perfusion conditions (21). It was not possible to identify most of the narrow but extensive portions of SPS that are so readily evident using electron microscopy (see Fig. 1), and this lack of spatial resolution would account for the apparent reduction in SPS size and frequency. Alternatively, SPS dimensions may vary with the magnitude of Starling's forces (20).
Our observations indicate two parallel pathways for solute and water movement across the GFB: a higher-resistance pathway through SPS-covered regions and a relatively low-resistance pathway through areas devoid of SPS. The concept of heterogeneous molecular flux across the GFB is consistent with those of Drumond and Deen (6). These authors used mathematical modeling to demonstrate low rates of flux across portions of the GBM immediately adjacent to endothelial cell or podocyte cytoplasm and significant hydrostatic pressure gradients acting within the GBM. While the parallel pathway implication of our results acts on a different scale, they are consistent with the concept of heterogeneous molecular movement across the GFB as proposed by Drumond and Deen (6). Nevertheless, the observations reported in this and a companion paper (21) need to be reconciled with the plethora of physiological studies reporting GFB hydraulic conductivity-area products (LpA) (3) and macromolecular sieving coefficients (4). In terms of water flux, our observations indicate that the hydraulic resistance of the GFB (glycocalyx-endowed fenestrated endothelial cells, GBM, and podocyte filtration slits) is lower than previously estimated and that LpA values obtained from studies involving micropuncture of glomerular capillaries and proximal tubules that were assumed to represent GFB LpA are in fact based on the composite of fluid flux across a lower-resistance naked GFB pathway summated with the proportion of fluid flux that traverses SPS. Significant retention of 10-kDa dextran by SPS also indicates that the degree of sieving that is imposed by the GFB (as defined above) is lower than previously estimated and that SPS make an important contribution to the composition of primary ultrafiltrate. Whether SPS make a similar contribution to the sieving of macromolecules of other sizes (e.g., albumin) is of interest.
It is not yet known whether the SPS is altered in disease states, or whether the SPS can contribute to glomerular dysfunction. A number of conditions are associated with proteinuria and yet have minor, or minimal, changes in the glomerular architecture on light microscopy and modest to moderate abnormalities on electron microscopy. These include conditions such as hypertension, minimal change nephrotic syndrome, and the early glomerular lesions of diabetes (31). Closer examination of the SPS structure may reveal abnormalities not previously described. Foot process effacement and extensive podocyte architecture disruption may lead to increased macromolecular flux through the SPS, leading to proteinuria. Alternatively, an increase in resistance through the SPS may result in hyperfiltration through uncovered regions. A dysfunction of this fourth layer of the GFB may therefore contribute to proteinuria.
In summary, we have shown, using an isolated, perfused glomerulus and an in vivo multiphoton imaging technique, that the movement of the intermediate-weight molecule 10-kDa dextran-rhodamine was retarded in regions of the glomerular capillary wall that are endowed with SPS. In contrast, the movement of the low-molecular-weight substance LY (450 Da) or 3-kDa dextran was not retarded across SPS-covered GFB. These observations indicate a size-selective barrier downstream of podocyte foot processes. There was no retardation in the movement of either solute across regions of the capillary wall that were devoid of SPS ("naked regions"). In addition, substantially less solvent traversed SPS-covered GFB regions compared with naked GFB regions, and conservative estimates indicate that this reduction in Jv is on the order of 66–75%. These observations indicate that the SPS does indeed impart additional resistance to water movement across the GFB, as postulated by Neal et al. (20).
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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, broken line) and capillary wall (
, P < 0.001, 1-way ANOVA with Bonferroni correction; n = 6}.
