Role of ultrastructural determinants of glomerular permeability in ultrafiltration function loss

Abstract

The epithelial filtration slit is a crucial component of the glomerular capillary membrane, which is essential for maintaining glomerular filtration function. Though chronic kidney diseases are an immense clinical problem, the mechanisms through which structural alterations reduce glomerular water filtration have not yet been understood completely. To investigate the mechanisms underlying filtration function loss, we studied rats with spontaneously occurring progressive kidney disease, either treated with angiotensin II antagonist or untreated, combining high-resolution electron microscopy of the glomerular capillary wall with theoretical water filtration modeling. Under pathological conditions, epithelial filtration pores and the extension of the subpodocyte space were larger than in normal controls. Numerical analyses indicated that these ultrastructural changes increased hydraulic resistance of the glomerular capillary wall by extending coverage of the filtration barrier by the subpodocyte space, with the changes in hydrodynamic forces acting on podocytes likely being responsible for their detachment. Angiotensin II inhibition normalized the subpodocyte space's hydraulic resistance, restored mechanical podocyte load, and preserved CD151-α3 integrin complex assembly, improving podocyte adherence and survival. Our results show that ultrastructural changes in podocytes are major determinants of the hydraulic resistance of the glomerular capillary wall and highlight the mechanism of podocyte loss in kidney disease progression, as well as the mechanisms underlying angiotensin II inhibition.

Keywords: Chronic kidney disease; Nephrology.

Figure 1. ACEi treatment reduced mean pore radius in MWF rats.

Distribution of slit pore sizes of Wistar (A–C), MWF (D–F), and MWF rats treated with lisinopril (G–I), as measured by digital morphometrical analysis and the best fit lognormal probability distribution of pore radii. Distribution histograms of fractional pore area (%) for Wistar (B), MWF (E), and MWF rats treated with lisinopril (H). Representative scanning electron photomicrographs of filtration pore ultrastructure in Wistar (C) and MWF rats at 60 weeks of age (F) and MWF rats treated with lisinopril from 40–60 weeks of age (I). Scale bars: 100 nm. n = 3 rats per group were used, and 600 pores were quantified per group. The blue-shaded areas highlight the subpopulation of larger pores, with area >2.50 x 10³ nm² (as indicated on the x axis by the the blue dotted vertical lines).

Figure 2. ACEi reduced SPS in proteinuric MWF animals.

(A) Quantification of SPS area. *P < 0.05 vs. Wistar; ANOVA with Tukey’s post hoc test. lis, lisinopril. (B) Representative images from Wistar and MWF rats at 20 weeks and in MWF rats treated with lisinopril between 10 and 20 weeks of age. The SPS is highlighted in yellow. Scale bars: 500 nm. Images are representative of 3 rats per group. In proteinuric MWF animals, podocytes are lost by detaching from the GBM as viable cells. Immunoperoxidase staining for WT-1 (C and E) and toluidine blue–stained sections in MWF rats (D and F) showing detached podocytes with viable-appearing nuclei (arrowheads) in the Bowman’s space (C and D) and in the tubular lumen (E and F). Images are representative of 3 rats per group, and 10–12 glomeruli were analyzed per rat. Scale bars: 20 μm for C, E, and F; 10 μm for D.

Figure 3. 3D model of the unit cell of GFB based on a single FS.

(A) Representative scanning electron photomicrographs of FS ultrastructure at low (left, scale bar: 1 μm) and high magnification (middle, scale bar: 200 nm); high-resolution transmission electron photomicrograph of glomerular capillary membrane (right, scale bar: 250 nm). n = 3 rats per group. (B) The model consists of 3 layers: ENL, GBM, and EPL; the cell is periodic in the x and y directions, while filtration occurs in the positive z direction (left). Insets show details of ENL and EPL pores (right). (C) Top and bottom views showing EPL pore and ENL fenestra size and distribution (left). 3D view of the computational model: the blue box represents the fluid domain of width (W) × length (L) × height (H) dimensions (middle). Section view of the fluid mesh used for CFD simulation (right). For the definitions and values of geometrical parameters, see Table 4.

Figure 4. The higher hydraulic resistance of the glomerular capillary wall in MWF rats is due to a greatly increased resistance of the SPS.

Total hydraulic resistance as function of SEP diameter b (upper panels) and as function of SEP length δ (lower panels). Arrows indicate the extremities of the ε range presented in Table 4.

Figure 5. MWF rats exhibited reductions in CD151, α3 integrin, and their colocalization; and lisinopril restored CD151–α3 integrin interaction in proteinuric MWF animals.

(A) Representative images of immunofluorescence staining for CD151 (red) and α3 integrin (green) and their colocalization in Wistar, MWF, and MWF rats treated with lisinopril. DAPI (blue) stains nuclei. Scale bars: 50 μm. (B) Expression of CD151 and α3 integrin (score) and their colocalization in Wistar, MWF, and MWF rats treated with lisinopril. Quantification of CD151–α3 integrin colocalization is represented as Pearson’s and Manders’s coefficients. *P < 0.01 vs. Wistar, ^†P < 0.05 vs. MWF, ^#P < 0.01 vs. MWF; ANOVA with Tukey’s post hoc test. For all staining, n = 3 rats per group, and 10 glomeruli were quantified per rats.

Figure 6. Schematic representation of the cascade of events that lead to changes in glomerular capillary wall function as a consequence of podocyte changes.

(A) In untreated MWF rats, the hemodynamic stress induced by expanded SPS exposes podocytes to an increase in the hydrodynamic detaching forces that tend to detach podocytes from the GBM, disrupting CD151–α3 integrin complex assembly, with changes that reduce water filtration and facilitate albumin filtration. (B) In ACE inhibitor–treated MWF rats, CD151–α3 integrin complex assembly is preserved and SPS expansion is reduced, so the physical forces acting on podocytes through filtered water are lowered and podocyte loss is limited, preserving the permselective properties of the glomerular capillary wall to albumin filtration. (C) Schematic diagram of the pathogenesis of progressive glomerular disease toward glomerulosclerosis. Initial podocyte changes activate a vicious cycle in the glomerular capillary membrane, wherein disruption of CD151–α3 integrin complex assembly leads to podocyte detachment and the expansion of SPS, which in turn causes an increase in SPS hydraulic resistance and in the resulting detaching force, thereby perpetuating podocyte depletion and glomerulosclerosis.