And You Thought the Age of Anatomic Discovery Was Over
Authors: Vivette D’Agati
Journal: Journal of the American Society of Nephrology (2005)

In this modern age of molecular biology, it is hard to imagine that any significant new discoveries remain to be made in the field of renal anatomy. Just thumb through any textbook of nephrology and you will find the classic references falling into predictable historical eras. Anatomic advances tend to cluster in the first half of the 20th century. The introduction of micropuncture in the 1960s set the stage for investigations into renal physiology. And since the 1980s, we have all witnessed a dizzying explosion of knowledge in molecular biology, molecular genetics, and their applications to nephrology. Despite these trends, the application of electron microscopy to the study of glomerular structure has provided a window of discovery that continues to shed new and exciting insights. It is humbling to remember that the mesangial cell was discovered as a distinct cell type by the ultrastructural studies of Farquhar and Palade as recently as 1962 (1). Not before the wondrous scanning electron microscopic images of the podocyte in the 1970s could the complex cytoarchitecture of this cell be fully appreciated (2). The ultrastructural discovery of the “anatomic zipper” of the slit diaphragm by Rodewald and Karnovsky in 1974 was a seminal advance in our understanding of slit diaphragm structure (3). Investigation into the structure and function of podocytes has reached its apogee in the current age, with approaches that span whole animal experiments, immunocytochemistry, cell culture, and molecular biologic approaches. The article by Neal et al. in this issue of the Journal of the American Society of Nephrology renews our faith in the power of structural observations as it explores new, as yet uncharted frontiers in the field of podocyte microanatomy (4). The subpodocyte cell space has been a difficult region to study because it is hidden from view in scanning electron micrographs of whole glomeruli. It has been impossible to appreciate by transmission electron microscopy because of the inability of two-dimensional microscopy to depict a complex three-dimensional space that is bounded by the overhangs of podocyte cell bodies, primary processes, and foot processes. The authors overcome these limitations by using an old-fashioned approach—three-dimensional reconstruction of an anatomic compartment by serial sections. Although tedious and labor-intensive, the technique has the potential for great reward. Three-dimensional reconstructions of a series of two-dimensional images can depict the intricacies of tortuous anatomic spaces that no single snap-shot can capture. The same technique was exploited back in the 1970s to determine how the lobules of the glomerular capillary bed are formed by a series of dichotomous branchings connected by interanastamoses (5,6). Here, the technique has been used to define the space that exists under the podocyte cell bodies, a region first described by Gautier in 1950 (7). We knew that the subpodocyte space can be altered in disease long before we knew much about its normal dimensions. The concept of the subpodocyte space first entered the collective consciousness of nephrologists with the seminal work by Nagata and Kriz on the pathways to focal sclerosis mediated by adaptive responses following uninephrectomy in young rats (8). Nagata and Kriz showed that glomerular growth following uninephrectomy leads to podocyte hypertrophy, without a corresponding increase in podocyte cell number. As the podocyte cell bodies stretch to cover a larger area of the glomerular tuft, they thin out into cytoplasmic sheets. Increased filtrate is delivered into the subcell body space, producing pseudocysts that promote podocyte apposition to Bowman’s capsule, the first step in formation of the tuft adhesions that eventuate in segmental sclerosis. Thus, in this experimental model, expansion and ballooning of the subpodocyte cell space was identified as a critical step in the cascade of events leading to capsular synechiae and segmental glomerulosclerosis. The article by Neal et al. defines three structurally distinct compartments of the urinary (or Bowman’s) space (4). The first is the one we are all familiar with—the large open space that forms a broad shell delimited by Bowman’s capsule on the outside and the glomerular globe on the inside. The second is the interpodocyte space that forms an anastamosing, branching region between individual podocytes. Third is the subpodocyte space, defined as the space bounded by the glomerular basement membrane (GBM) and foot processes below, and by the underside of the podocyte cell bodies and their processes above. A sizeable area, some 60% of the total filtration surface of the glomerulus, is actually covered by podocyte cell bodies at any one time, yet until now this space has not been factored into mathematical models of glomerular ultrafiltration. In these regions, the glomerular filtrate cannot enter the open urinary space without passing through the more restricted subpodocyte space, which serial sections depict as a long, narrow, and tortuous space serviced by small exit pores. Individual exit pores measure <0.4 μm2. In one reconstruction there were 22 pores (measuring in aggregate 8.6 μm2) draining an area of GBM as large as 210 μm2 (4). In other words, to reach Bowman’s space, the filtrate entering into the subpodocyte space must pass through tiny exit pores that occupy only 4% of the corresponding glomerular filtration area. (This is an astounding statistic and one that any commuter into New York City who must cross the George Washington Bridge during the peak flow of morning rush hour can well appreciate.) Moreover, the exit pore width was found to be 50% wider in immersion-fixed than perfusion-fixed glomeruli, indicating an even greater restriction to flow under physiologic conditions (4). What are the physiologic implications of the subpodocyte cell space for glomerular ultrafiltration? The old adage “structure predicts function” makes startling mathematical predictions. According to Poiseuille’s law, the system of small exit pores located at great distance from the point where filtrate crosses the glomerular filtration barrier defines a high-resistance space. The authors estimate that the relative resistance to flow presented by the subpodocyte space and exit pores is on the order of 900- to 14,500-fold. According to Starling’s laws, the ultrafiltration pressure equals the hydrostatic pressure difference across the glomerular filtration barrier less the oncotic pressure difference. It is known that the pressure in Bowman’s space averages about 20 mmHg depending on the species and measurement techniques (9,10). The authors estimate that the hydrostatic pressure in the subpodocyte space is significantly higher, ranging from 27 to 35 mmHg. Accordingly, they calculate that the ultrafiltration pressure for portions of the glomerular filter opening onto the subpodocyte space ranges from +3 mmHg to −5 mmHg, allowing for reverse filtration to occur in the negative range. Transient reversal of flow is more likely to occur at the lower extreme of pulse pressure, when glomerular capillary hydrostatic pressure falls. Although the possibility of flow reversal across the glomerular filtration barrier remains to be shown in vivo, the implications are intriguing and provocative. First, the findings suggest that glomerular ultrafiltration is much more complex and spatially heterogeneous than previously recognized. Transient flow reversal could contribute to the hypothetical mechanism of self-cleansing or unclogging of the glomerular filter proposed by Kanwar et al. years ago (11). Glomerular back-leak might explain the conundrum of how substances secreted by the podocyte (such as vascular endothelial growth factor, TGF-β, and other soluble factors) theoretically reach receptors on other glomerular intracapillary cells (endothelial or mesangial) in health and disease (12,13). Cross-talk between podocytes and other glomerular cell types could be achieved if soluble podocyte-derived factors are intermittently reabsorbed back across the GBM, instead of continually being flushed out into the urinary space by convective forces. Because podocytes are dynamic, motile cells (14), it is likely that the subpodocyte space is constantly being remodeled, producing local differences in resistance to ultrafiltration that may change over time and across regions of the capillary bed. As podocytes migrate across the filtration surface, eventually all regions of the capillary bed could be subject to these effects. Because podocytes are endowed with a contractile apparatus (including actin, myosin, vinculin, and talin) and receptors for a variety of vasoactive amines, individual podocytes may regulate the pressures in this space by their state of contraction and relaxation (15). Finally, it remains to be seen how the filtration dynamics of the subpodocyte space may be altered in disease states such as nephrotic syndrome, where foot process effacement may reduce the dimensions of this space and the access to exit pores. The cytoarchitecture of the podocyte is becoming ever more complex. The article by Neal et al. (4) gives us new appreciation for the underbelly of the beast and the unique anatomic compartment that is the subpodocyte space.