Permeation of macromolecules into the renal glomerular basement membrane and capture by the tubules

Abstract

How the kidney prevents urinary excretion of plasma proteins continues to be debated. Here, using unfixed whole-mount mouse kidneys, we show that fluorescent-tagged proteins and neutral dextrans permeate into the glomerular basement membrane (GBM), in general agreement with Ogston's 1958 equation describing how permeation into gels is related to molecular size. Electron-microscopic analyses of kidneys fixed seconds to hours after injecting gold-tagged albumin, negatively charged gold nanoparticles, and stable oligoclusters of gold nanoparticles show that permeation into the lamina densa of the GBM is size-sensitive. Nanoparticles comparable in size with IgG dimers do not permeate into it. IgG monomer-sized particles permeate to some extent. Albumin-sized particles permeate extensively into the lamina densa. Particles traversing the lamina densa tend to accumulate upstream of the podocyte glycocalyx that spans the slit, but none are observed upstream of the slit diaphragm. At low concentrations, ovalbumin-sized nanoparticles reach the primary filtrate, are captured by proximal tubule cells, and are endocytosed. At higher concentrations, tubular capture is saturated, and they reach the urine. In mouse models of Pierson's or Alport's proteinuric syndromes resulting from defects in GBM structural proteins (laminin β2 or collagen α3 IV), the GBM is irregularly swollen, the lamina densa is absent, and permeation is increased. Our observations indicate that size-dependent permeation into the lamina densa of the GBM and the podocyte glycocalyx, together with saturable tubular capture, determines which macromolecules reach the urine without the need to invoke direct size selection by the slit diaphragm.

Keywords: Alport's syndrome; Pierson's syndrome; gel permeation; gold nanoparticles; slit diaphragm.

Fig. 1.

The GBM/plasma ratio decreases as molecular size increases in general agreement with Ogston’s equation; filtered proteins are captured by the proximal tubule, but some reach the urine. (A–C) Confocal images at appropriate wavelengths of a glomerulus in a paraformaldehyde-fixed kidney of a male mouse killed 2 min after tail-vein injection of parvalbumin (488 nm fluorophore), ovalbumin (555 nm), and BSA (647 nm). The overall brightness of the images has been adjusted so that the fluorescences for the three fluorophores are equal in a capillary that contains plasma (dotted square boxes). Many capillaries are devoid of plasma (asterisks) for reasons detailed in Fig. S2. The brush border of a proximal tubule is indicated with an arrow. The basement membrane of Bowman’s capsule is indicated with an arrowhead. (D) Time course of the urinary concentration of the three proteins relative to their concentrations in the injected mixture. (E) Logarithmic plot of the GBM/plasma fluorescence intensities, measured in whole-mount unfixed kidneys, for four proteins and three dextrans versus the simple function, (R + 0.75)², of their hydrodynamic radii (R) expected from Ogston’s equation (2, 4); molecular masses and Stokes/Einstein hydrodynamic radii are listed in the Inset.

Fig. S1.

Confocal images at two PMTs of a whole-mount preparation of a kidney from mice injected with fluorescent-tagged ovalbumin, BSA, or 200-kDa dextran. (A and D) Confocal images of a glomerular capillary taken at PMTs of 700 and 800 V, respectively, showing the localization of plasma in a capillary after coinjection of fluorescent 200-kDa dextran (pseudocolored yellow), and fluorescent ovalbumin (pseudocolored white). A thin layer of plasma is apparent between the GBM and the packed red cells or endothelial nuclei, which occupy most of the capillary space (Fig. S2), emphasizing the need to restrict the area used in assessing the fluorescence of the GBM. (B and C) Two glomerular capillaries imaged at a PMT of 700 V after the injection of fluorescent BSA. At this PMT, fluorescence of the plasma areas (outlined in yellow) is within the optimal range of our instrument although GBM fluorescence is too low to be measured with precision. (E and F) The same capillaries imaged at a PMT of 800 V. The plasma is now too fluorescent to be measured (indicated in red), but fluorescence in the GBM is within the optimal range. Imaging at two PMTs allows nonsaturating measurements of fluorescence in both the plasma and the GBM; restricting the areas (yellow outlines) reduces inadvertent inclusion of fluorescence from the plasma when measuring fluorescence in the GBM. The use of different PMTs required a correction factor to normalize intensities taken at different voltages to a chosen standard voltage (in our case 700 V). To determine the correction factors, we prepared 1:4 serial dilutions of all of our conjugate solutions and imaged each of them at four different PMT voltages with at least one PMT setting overlapping the adjacent higher and lower dilutions. Table S1 lists the correction factors, determined from these measurements, that allowed us to normalize all subsequent measurements to that which would have been obtained had the measurement been taken at a PMT of 700 V. For example, if the fluorescence intensity of BSA-647 was determined at a PMT of 800 V, the normalized reading at 700 V would be obtained by multiplying the reading by 0.42.

Fig. S2.

Plasma is expelled from glomerular capillaries during tissue preparation. (A) Confocal image of Cy5 fluorescence in a section of a fixed kidney from a mouse that had received Cy5-labeled BSA. Similar images were seen whether the kidney was fixed or not (whole mount). Note the presence of Cy5 fluorescence in the GBM, but its absence in the lumen of most of the capillaries. (B) A pseudophase image of a section from the same kidney after staining cell nuclei with DAPI. The capillary lumen is filled with either packed red cells or the cell bodies of podocytes, endothelial cells, or mesangial cells.

Fig. 2.

Permeation of gold-tagged albumin and nanoparticles into the GBM and accumulation in a layer close to the bases of podocyte foot processes. (A) Enhanced TEM image of a glomerular capillary fixed immediately after retrogradely injecting gold-tagged mouse serum albumin into the superior mesenteric artery of a mouse under sublethal avertin anesthesia; the tagged albumin is plentiful in the plasma (asterisk); no albumin is observed in the GBM (g). (B) Enhanced image after injecting NaBH₄ gold nanoparticles comparable in size with ovalbumin; many particles (arrows) have accumulated within the GBM. (C) Higher magnification unenhanced image after injecting NaBH₄ nanoparticles; the particles within the GBM have actually accumulated close to the podocyte foot processes (f) though the podocyte associated particles (PAPs) are some distance from the podocyte plasma membrane. (D) Comparable magnification unenhanced image after injecting NaSCN oligoclusters; the oligoclusters also associate with the podocytes. (E, Insets) Histograms of sizes of particles in capillary plasma (outlined in red in the representative image behind the Inset) and of the particles associated with the podocytes (outlined in blue in the representative image); larger particles are overrepresented in the PAPs. (F) PAPs appear as a line (blue arrows) when viewed in cross-section although they appear as a layer when viewed in oblique sections (red arrows).

Fig. S3.

Heparin inhibits/displaces the binding of nanoparticles to the GBM. (A) Enhanced TEM image of the GBM and adjacent portion of Bowman’s capsule fixed 1 min after i.v. injection of NaBH₄ nanoparticles having a hydrodynamic size comparable with that of ovalbumin. Numerous particles (white arrows) have accumulated close to the podocyte foot processes (f). (B) A comparable region from a mouse that had received 400 I.U. of heparin before the nanoparticles. Particles have no longer accumulated close to the podocytes although they have still permeated into the GBM. (C) A comparable region from a mouse that received heparin after the nanoparticles. The accumulated particles were displaced. Asterisks, plasma; r, a red blood cell.

Fig. 3.

When concentrated particles are injected, clusters of PAPs accumulate at the glomerular slits well upstream of the slit diaphragm, and the clusters can persist for minutes to hours. (A) Unenhanced cross-section of part of a glomerulus 5 min after arterially injecting concentrated small NaBH₄ nanoparticles. In vivo formed clusters of PAPs are seen at the podocyte slits (black arrows) and along the bases of the foot processes (white arrow). (B) En face view from the same kidney showing clusters along the slits. (C) Enlargement of B showing that the number of particles in the clusters is variable. (D) Cross-section 1 h after injecting small NaBH₄ nanoparticles. A large cluster of particles (white ellipse) is seen at this slit in a plane well upstream of the slit diaphragm (white arrow). (E) A neighboring slit in which a few individual particles appear in the same plane (white ellipse) well upstream of the slit diaphragm (white arrow). No particles are close to the slit diaphragm.

Fig. 4.

Size discrimination by the GBM. (A) An unenhanced TEM image from a kidney fixed immediately after intraarterially injecting fraction 22 to 24 NaSCN oligoclusters into a heparinized mouse. The hydrodynamic size of these particles is comparable with that of IgG dimers. (B) GBM from a heparinized mouse that had received fraction 28 NaSCN oligoclusters approximately the size of IgG monomers. (C) GBM from a heparinized mouse that received fraction 30 and 31 NaSCN oligoclusters approximately the size of albumin. (D–F) Distributions of the three sizes of particle across the GBM from the endothelium to the bases of the podocytes; n, the number of particles of each size counted in the GBM. (A–C) Asterisks, plasma; f, podocyte foot process; g, GBM.

Fig. 5.

The GBM is grossly and irregularly thickened but more permeable in mouse models of Pierson’s and Alport’s syndromes. (A) Unenhanced image of the GBM and adjoining plasma in the kidney of a heparinized WT mouse fixed 2 min after tail-vein injection of NaSCN oligoclusters comparable in size with IgG dimers. The GBM (g) is uniform in thickness. (B) Unenhanced image, at the same magnification as in A, of the GBM and adjoining plasma from a heparinized mouse model of Pierson’s syndrome. The GBM is grossly and irregularly thickened up to three times that in WT. (C) An equivalent image from a heparinized mouse model of Alport’s syndrome; the GBM shows the same gross and irregular thickening of the GBM as in the Pierson’s model. (D) The distribution of particles across the GBM from the endothelium to the bases of the podocytes in the WT mouse. A region where the number of particles is at a minimum, the lamina densa, is clear. (E) The distribution in the Pierson’s model; a region of low particle density is not present. (F) The distribution in the Alport’s model. As in the Pierson’s model, there is no region of low permeation. (G) A plot of the ratio of the number of particles per unit area in the GBM to the number of particles per unit area in the plasma (GMB/P) at 20 different locations along the GBM in a Pierson-model mouse; permeation increases in direct proportion to GBM thickness (R² = 0.53). (A–C) Asterisks, plasma; f, podocyte foot process; g, GBM; n, number of particles counted.

Fig. 6.

Capture of filtered particles by the proximal tubule is saturable. (A) Enhanced image of a proximal tubule fixed 10 min after i.v. injection of NaBH₄ nanoparticles comparable in size with ovalbumin. Many filtered particles have been captured by the proximal tubule microvilli (m) and concentrated into intracellular vesicles (v). (B) A proximal tubule after injecting one-ninth the number of particles used for A. The number of captured particles and their location is similar to that in the higher dose mouse. (C) A collecting duct from a higher dose mouse. Many particles are in the urinary space (u), indicating that the capacity of the upstream proximal tubules to capture particles was saturated. (D) A collecting duct from the lower dose mouse. Almost no particles are in the collecting duct urinary space, indicating that the capacity of the upstream proximal tubules was not saturated.

Fig. S4.

Capture of filtered particles by unusual cells. (A) An enhanced image of a portion of a collecting duct from a mouse whose kidney was harvested ∼5 min after i.v. injection of a concentrated preparation of GSH-coated NaBH₄ nanoparticles, which readily traverse the glomerulus. Two intercalated cells in this image (i) have captured the particles, either from the urine or from the interstitial fluid. Less heavily labeled intercalated cells were seen in other sections. (B) An unusual proximal tubule cell (t) in the same kidney with microvilli (m) that seem to have captured more particles than the microvilli of neighboring tubule cells. (C) A higher magnification image of the same cell.

Fig. S5.

The slit diaphragm has a component that binds very small gold nanoparticles. (A) A conventional cross-sectional view of the GBM (g) and podocyte foot processes (f) from a heparinized mouse given a heterogeneous preparation of NaBH₄ nanoparticles with an average size comparable with ovalbumin. All sizes of particles have permeated into the GBM. Some of the smallest particles appear at or below the level of the slit diaphragm (white ovals). (B) An oblique view of a similar region in the same kidney. This view shows that the small particles are actually regularly spaced in single file (white arrows). (C) En face view of two glomerular slits. This grazing incidence section has captured the plane in which the bound particles reside. Asterisks, plasma; e, the fenestrated endothelial lining of the capillary.