Progressive renal failure inability of podocytes to replicate and the consequences for development of glomerulosclerosis

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1 Nephrol Dial Transplant (1996) 11: Special Feature IMephrology Dialysis Transplantation Progressive renal failure inability of podocytes to replicate and the consequences for development of glomerulosclerosis Institut fur Anatomie und Zellbiologie der Universitat, Heidelberg, Germany Introduction The irreversible decrease of renal function in chronic renal failure results from progressing nephron loss. When studied by histopathology, the nephron loss follows a pattern of degeneration designated focal segmental glomerulosclerosis. We investigated this degenerative process in several animal models [1 4]. In agreement with others [5] we have come to the conclusion that the progressive nature of this disease is ultimately based on the inability of podocytes to replicate. Replication characteristics of podocytes During nephrogenesis, podocytes first develop as a distinct cell layer at the stage of the so-called S-shaped bodies. In this developmental stage, the presumptive podocytes appear as simple cuboidal cells which rapidly divide by mitotic cell division. At the transition to the 'capillary loop stage' podocytes stop proliferating, but develop into their differentiated phenotype of octopuslike cells adhering to the outer aspect of glomerular capillaries by a complex system of processes [6]. Along with this transition, podocytes irreversibly lose the ability to divide [7]. At this moment, in humans during prenatal life, the ultimate number of podocytes per glomerulus is established. The evidence for the inability of differentiated podocytes to replicate comes, first, from studies showing that the number of podocytes does not increase with normal postnatal or with hypertrophic kidney growth [1,5] and, second, from studies [3] showing that sustained mitogenic stimulation of podocytes in the adult animal causes podocytes to enter cell division without being capable of completing it. In the adult, podocytes may undergo mitosis (nuclear division), but are unable to undergo cytokinesis (cell division) resulting in bi-(multi)-nucleated podocytes. The consequences are far reaching: first, when podocytes are lost for any Correspondence and offprint requests to: Professor Dr Wilhelm Kriz, Institut fur Anatomie und Zellbiologie, Im Neuenheimer Feld Heidelberg, Germany. reason, they cannot be replaced; second, the only way for podocytes to cope with an increase in workload is by cell hypertrophy; multinucleated cells represent an extreme form of hypertrophy. Podocyte injury culminates in detachment from the glomerular basement membrane The renal glomerulus is a highly organized structure. A major challenge to the glomerulus appears to be the maintenance of its highly differentiated architecture in the face of high hydrostatic pressure gradients across glomerular capillary walls. These gradients, necessary to sustain filtration, tend to blow up the glomerulus and to destroy glomerular architecture. The supportive structures counteracting these expanding forces are (i) the mesangial cells and (ii) the podocytes [6,8]. By interacting with the GBM, mesangial cells play the major role in maintaining capillary architecture, whereas capillary diameters are stabilized by podocytes [8,9]. In the course of glomerular disease or in response to increased pressures, if either cell type fails the other will inevitably be affected. Thus, whenever the glomerulus is seriously damaged, podocytes will be affected. When exposed to any kind of strain, podocytes are unable to maintain their normal differentiated phenotype. They change their appearance in a fairly stereotyped manner [3,10]. To explain such a uniform reaction pattern we propose that mechanical strain and pressure are always involved, either as elevated pressure sui generis or as normal strain acting on altered structures in diseased glomeruli. The changes one observes comprise foot process effacement, cell body attenuation, pseudocyst formation, cytoplasmic overload with reabsorption droplets, and finally detachment from the GBM. If detachment is preceded or followed by podocyte cell death, repair of such defects is only possible by hypertrophy of adjacent intact podocytes. It is obvious that such a compensatory mechanism is quite limited. Detachment of podocytes from peripheral capillaries is a crucial process in the evolution of segmental glomerulosclerosis: denuded peripheral capillaries strongly tend to attach to Bowman's capsule and to 1996 European Renal Association-European Dialysis and Transplant Association

2 Progressive renal failure inability of podocytes to replicate and resulting consequences for development of glomerulosclerosis cause tuft adhesions. Deprived of their mesangial support peripheral capillaries lose their centripetal attachment and will extend towards Bowman's capsule. Centrifugal expansion and ballooning of these capillaries will be further promoted by the loss of bandaging support of podocytes, terminating eventually in the apposition of a 'naked' capillary to Bowman's capsule. Development of segmental sclerosis encroach onto the tuft by moving along the denuded GBM to neighbouring capillaries. Thus the former capillaries lose contact with parietal cells and are pushed into the centre of the lesion. As a consequence the adhesion enlarges in width and depth (Figure 2). Inside such an adhesion, capillaries will simply collapse or become occluded, either by deposition of hyaline material (hyalinosis) or by microthrombosis. To a large extent podocytes degenerate and disappear. Apart from variable numbers of macrophages, most of the cells encountered inside an adhesion appear to be mesangial cells. However, mesangial cell proliferation and de novo matrix synthesis do not occur [1,3]. Development of segmental sclerosis is a process of collapse and not a process of proliferation. Segmental sclerosis finally consists of an adherent tuft area with collapsed capillaries, disappearance of cellular elements, accumulation of hyaline material, but little if any deposition of collagen to form a scar [3,12]. The eventual fate of a glomerulus with segmental sclerosis is the extension of the sclerotic process onto the entire tuft followed by subsequent organization by cortical fibroblasts with deposition of fibrous Fig. 1. Adhesion of a single glomerular capillary loop to Bowman's capsule in chronic Masugi nephritis, as seen by transmission EM. An accompanying drawing helps to recognize the essential features. The adhesion consists of a capillary loop, the afferent and efferent limbs (asterisks) of which are seen. In the drawing the GBM and the mesangium are shown in black, capillary lumina are cross-hatched. The parietal epithelium (densely stippled in the drawing) is attached to this capillary loop from both sides (arrows). In between these attachment sites a denuded portion of GBM directly contacts the parietal basement membrane (loosely stippled in the drawing) which is expanded in thickness exhibiting its multilayered structure. Towards the cortical interstitium the parietal basement membrane is separated by an almost continuous layer of thin sheet-like processes of cortical fibroblasts (indicated by a hatched line in the drawing). Podocytes (stars) adhering to the flanks of the adhesion show severe maladaptive changes including microvillous transformation, accumulation of absorption droplets, foot process effacement ('fusion') with partial disconnection of the 'fused' area from the GBM (small arrows). From a cooperative study with Isao Shirato [4]; magnification x ~2000. When a denuded segment of the GBM comes in contact with parietal cells, the latter are apparently triggered to attach to the GBM. Thereby a 'beachhead' of parietal epithelium is established on the tuft (Figure 1) which represents the nidus for the further development of a synechia, i.e. of segmental sclerosis [1-3,11]. Adhesions appear to have a strong tendency to enlarge. For unknown reasons (inadequate mechanical strain due to hindrance of free fluctuation in the glomerular filtrate may be involved [1]) podocytes which are located at the flanks of an adhesion, will degenerate. This allows the parietal epithelium to 1739

3 1740 tissue. Figure 3 summarizes the chain of events terminating in glomerulosclerosis. Patterns of distribution of segmental sclerosis The segmental character of sclerosis depends on the location of the initial lesion leading to a tuft adhesion. In some models of renal damage, this initial event is predominantly seen near the vascular pole, in others it is randomly found at any site of the tuft circumference [13]. The former pattern is characteristic of models with presumed high glomerular pressure, which may directly overstrain and subsequently damage the podocytes, atfirstthose that serve thefirstcapillary branches of the afferent arteriole. The latter pattern is thought to be the result of random damage to podocytes anywhere on the tuft surface. Basically, however, the pathogenetic mechanism is the same in both instances: the initial event is adhesion of a capillary loop to Fig. 2. Segmental glomerulosclerosis in the kidney of a rat 3 months after uninephrectomy at young age, as seen by transmission EM. An accompanying drawing helps to recognize the essential features. The segmental sclerotic area comprises a crescent-like cap on top of a fairly intact tuft portion. The tuft and the sclerotic area appear to be quite separated from each other, but actually are in continuity with each other through two gaps in the parietal epithelium (parietal cells are densely stippled in the drawing). The two gaps are confined by the attachment of parietal cells to the GBM (each marked by two large arrows). Toward the cortical interstitium the sclerotic area is delineated by an almost continuous layer of extremely thin, sheet-like processes of cortical fibroblasts (in the drawing shown by a hatched line). Thus the sclerotic tuft remnants (shown in solid black) are located inside a space (loosely stippled in the drawing) which corresponds to extremely expanded 'intrabasement membrane spaces' of the parietal basement membrane. The sclerotic remnants consist of collapsed and hyalinized capillaries and/or mesangial areas; two capillaries (cross-hatched) are still patent. Podocytes appear to have completely disappeared from the sclerotic area. Cell nuclei inside the collapsed or hyalinized structures appear to belong mostly to mesangial cells. Cells seen outside the GBM but within the sclerotic area appear to be parietal cells. The 'intact' tuft portion protruding into Bowman's space consists of patent capillary loops (cross-hatched in the drawing) connected to a mesangium of normal appearance (shown in black in the drawing). Podocytes (small arrows; not shown in the drawing) exhibit maladaptive changes; three of them (stars) appear to be quite seriously damaged by overload with lysosomal elements. From a cooperative study with Michio Nagata [1]. Magnification x ~700.

4 Progressive renal failure inability of podocytes to replicate and resulting consequences for development of glomerulosclerosis 1741 Fig. 3. Development of segmental sclerosis illustrated in a series of sequential schematics, drawn by Rolf Nonnenmacher. The visceral epithelium layer (podocytes) is shown in red, the parietal epithelium in yellow, the endothelium in blue, the cortical fibroblasts in hazel, the glomerular basement membrane (GBM) in black, the parietal basement membrane in grey. Mesangial cells are not drawn, (a) Normal glomerulus. (b) A denuded peripheral capillary apposes to the parietal epithelium, (c) Parietal cells attach to the GBM of the denuded capillary loop associated with the formation of a gap in the parietal epithelium, (d) and (e) The parietal epithelium moves to neighbouring capillary loops along the denuded basement membrane. Capillaries inside the adhesion collapse or are occluded by hyalinosis (shown in dark grey) or microthrombosis (not shown), (f) Further enlargement of the adhesion. Note that the sclerotic tuft remnants, i.e. collapsed or hyalinized capillaries and mesangium are situated in a space which corresponds to extremely expanded intrabasement membrane spaces of the parietal basement membrane (shown in light grey), (g) The adhesion has reached the vascular pole. Cortical fibroblasts have arranged in an almost continuous layer of thin processes delineating the sclerotic area from the cortical interstitium. (h) The sclerotic process jumps, via the vascular pole, to a neighbouring lobule. In even later stages, cortical fibroblasts will invade the sclerotic area, resulting in fibrous organization.

5 1742 Bowman's capsule, followed by the encroachment onto further tuft areas, terminating in segmental sclerosis. Relevance of mesangial cell proliferation It has been almost a dogma for more than a decade that exuberant mesangial proliferation and matrix synthesis are of major pathogenetic relevance for the development of segmental sclerosis. In our view, mesangial proliferation is not directly involved in this process. Mesangial cell proliferation may often be seen to occur prior to or in the early stages of rapidly progressing glomerular injury in parallel with the development of sclerosis. However, direct histopathological evidence documenting progression of mesangial proliferation to glomerular scarring has never been presented. In contrast, recent work has uncovered a strong potential of mesangial cells to undergo mesangial remodelling in the event of an 'overshoot' of proliferation [14,15]. Mesangial proliferation basically represents a repair process aiming at re-establishing mesangial cell-to-gbm connections, e.g. mesangial support to glomerular capillaries. In the frequent case in which glomerular damage is associated with mesangial injury, widespread mesangial proliferation may be encountered as an early response, generally followed somewhat later by development of extensive sclerosis. Thus development of mesangial proliferation and of sclerosis may well be correlated with each other, since the magnitude of both processes depends on the severity of the initial insult. Such correlation by no means implies, however, that the two processes are causally related. Initiation and progression of segmental sclerosis depend on the formation and growth of an adhesion of a glomerular tuft to Bowman's capsule. This process is not necessarily preceded nor followed by mesangial cell proliferation. Conclusion The progressive character of chronic renal failure is based on the inability to replace worn out podocytes. If in the course of glomerular disease a podocyte undergoes cell death, the only option for the glomerulus to compensate for loss of its function is hypertrophy of remaining podocytes. When this process exceeds certain limits, hypertrophy of podocytes will clearly increase their vulnerability to any further challenge. Thereby the basis for a vicious cycle is estab- lished, and onset of chronic renal failure will be its result. A recent article in this journal [16] raised the question: 'Podocyte a neglected player in glomerular injury?'. The answer is that the podocyte is not merely a neglected player, it is the most effective player in glomerular injury. References 1. Nagata M, Kriz W. Glomerular damage after uninephrectomy in young rats. II. Mechanical stress on podocytes as a pathway to sclerosis. Kidney Int 42: 1992; Kretzler M, Koeppen-Hagemann I, Kriz W. Podocyte damage is a critical step in the development of glomerulosclerosis in the uninephrectomised-desoxycorticosterone rat. Virchows Arch. A 452: 1994; Kriz W, Hahnel B, Rosener S, Elger M. Long-term treatment of rats with FGF-2 results in focal segmental glomerulosclerosis. Kidney Int 48: 1995; Shirato I, Sakai T, Kimura K, Tomino Y, Kriz W. Cytoskeletal changes in podocytes associated with foot process effacement in Masugi nephritis. Am J Pathol 1996; 148: Fries JW, Sandstrom DJ, Meyer TW, Rennke HG. Glomerular hypertrophy and epithelial cell injury modulate progressive glomerulosclerosis in the rat. Lab Invest 60: 1989; Mundel P, Kriz W. Structure and function of podocytes: an update. Anat. Embryo! 192: 1995; Nagata M, Yamaguchi Y, Ito K. Loss of mitotic activity and the expression of vimentin in glomerular epithelial cells of developing human kidneys. Anat Embryol 187: 1993; Kriz W, Elger M, Mundel P, Lemley KV. Structure-stabilizing forces in the glomerular tuft. J Am Soc Nephrol 5: 1995; Kriz W, Hackenthal E, Nobiling R, Sakai T, Elger M. A role for podocytes to counteract capillary wall distension. Kidney Int 45: 1994; Kriz W, Elger M. Nagata M et al. The role of podocytes in the development of glomerular sclerosis. Kidney Int 45 [Suppl. 45]: 1994; S-64-S Kondo Y, Akikusa B. Chronic Masugi nephritis in the rat. An electron microscopic study on evolution and consequences of glomerular capsular adhesions. Ada Pathol Jpn 32: 1982; Rennke H. How does glomerular epithelial cell injury contribute to progressive glomerular damage? Kidney Int 45 (Suppl. 45): 1994; S-58-S Howie AJ, Kizaki T, Beaman M et a!. Different types of segmental sclerosing glomerular lesions in six experimental models of proteinuria J Pathol 157: 1989; Savill J, Johnson RJ. Glomerular remodelling after inflammatory injury. Exp Nephrol 3: 1995; Baker AJ, Mooney A. Hughes J, Lombardi D, Johnson RJ, Savill J. Mesangial cell apoptosis: The major mechanism for resolution of glomerular hypercellularity in experimental mesangial proliferative glomerulonephritis. J Clin Invest 94: 1994; Pavenstadt H. The podocyte a neglected player in glomerular injury? Nephrol Dial Transplant 10: 1995;