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Kidneys from 5 normal fetuses and 3 with congenital nephrotic syndrome gestational age of 13 to 24 weeks, were studied under a scanning electron microscope. Differentiation of the visceral epithelial cells was observed in normally developing glomeruli. The originally cuboidal, smooth epithelial cells first flattened and acquired cytoplasmic buds on the cell margin. The buds gradually grew longer and branched out to form regular interdigitating foot processes. In the most mature glomeruli the majority of the visceral epithelial cells were trabeculated and the cytoplasmic processes were tightly wound around the glomerular capillaries. In the glomeruli of specimens from fetuses with congenital nephrotic syndrome the epithelial differentiation was different. The individual epithelial cells varied in size and shape and formed irregular cell groups. The cytoplasmic processes of visceral epithelial cells were shortened and flattened. Also, there was a marked loss of interdigitation of adjacent processes. The findings confirm previous transmission electron microscopic observations of pathological foot processes in glomeruli of fetuses with congenital nephrotic syndrome and show that the differentiation of the visceral epithelial cells is abnormal prior to the formation of the final interdigitating processes.

Effects of high glucose and TGF-beta1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes.The podocyte takes center stage in the pathogenesis of glomerular basement membrane (GBM) thickening and proteinuria in diabetic glomerulopathy. In part, GBM thickening may occur when the podocyte synthesizes increased amounts of collagen IV. Proteinuria may develop if the podocyte secretes excessive amounts of vascular endothelial growth factor (VEGF), which may increase the glomerular permeability to macromolecules. The augmented production of collagen IV and VEGF may be caused by metabolic mediators of diabetes such as hyperglycemia and transforming growth factor-beta (TGF-beta).The effects of high glucose and exogenous TGF-beta1 were examined on a mouse podocyte cell line that retains its differentiated phenotype. The gene expression and protein production of certain alpha chains of collagen IV, the major isoforms of VEGF, and components of the TGF-beta system were assayed. An inhibitor of TGF-beta signaling was used to determine whether some of the high glucose effects might be mediated by the TGF-beta system.Compared with normal glucose (5.5 mmol/L), high glucose (HG, 25 mmol/L) for 14 days stimulated [3H]-proline incorporation, a measure of collagen production, by 1.8-fold, and exogenous TGF-beta1 (2 ng/mL) for 24 hours stimulated proline incorporation by 2.4-fold. Northern analysis showed that exposure to HG for 14 days increased the mRNA level of alpha1(IV) collagen by 51% and alpha5(IV) by 90%, whereas treatment with TGF-beta1 (2 ng/mL) for 24 hours decreased the mRNA level of alpha1(IV) by 36% and alpha5(IV) by 40%. Consistent with these effects on mRNA expression, Western blotting showed that HG increased alpha1(IV) protein by 44% and alpha5(IV) by 28%, while TGF-beta1 decreased alpha1(IV) protein by 29% and alpha5(IV) by 7%. In contrast to their opposing actions on alpha1 and alpha5(IV), both HG and exogenous TGF-beta1 increased alpha3(IV) collagen and VEGF, with TGF-beta1 having the greater effect. An inhibitor of the TGF-beta type I receptor (ALK5) was able to prevent the stimulation of alpha3(IV) and VEGF proteins by HG. Unlike in other renal cell types, HG did not increase TGF-beta1 mRNA or protein in the podocyte, but HG did induce the expression of the ligand-binding TGF-beta type II receptor (TbetaRII). Because HG had up-regulated TbetaRII after two weeks, the addition of physiological-dose TGF-beta1 (0.010 ng/mL) for 24 hours stimulated the production of alpha3(IV) and VEGF proteins to a greater extent in high than in normal glucose. Up-regulation of TbetaRII in the podocyte was corroborated by immunohistochemistry of the kidney cortex in the db/db mouse, a model of type 2 diabetes.High glucose and exogenous TGF-beta1 exert disparate effects on the expression of alpha1 and alpha5(IV) collagen. However, high glucose and TGF-beta1 coordinately induce the production of alpha3(IV) collagen and VEGF in the podocyte. The HG-induced increases in alpha3(IV) collagen and VEGF proteins are mediated by the TGF-beta system. By increasing the expression of TbetaRII, high glucose may augment the response of the podocyte to ambient levels of TGF-beta1.

The simplification of the foot processes of the glomerular epithelial cells is the structural hallmark of proteinuria in nephrotic man and animals. The increased shear force in a filtrate funnelled within the lamina densa to simplified slit pores may decrease the friction of macromolecules which are prolate ellipsoids (cigar-shaped). The permeability of rigid long narrow proteins such as albumin, transferrin and immunoglobulin G may selectively increase when the slit pore is converted from a wide serpentine to a straight narrow space.

Orthogonal arrays of small intramembranous particles characterize freeze-fractured astrocytic plasma membranes. The normal variation of assemblies in plasma membranes of subpial astrocytic processes in mature and developing rats was established and compared with assemblies in plasma membranes of reactive astrocytes. In mature rats, subpial astrocytic processes had the greatest number of assemblies. As the parenchyma was approached, this number decreased in each successive layer of astrocytic processes. The advent of assemblies within the plasma membrane is a new criterion of astrocytic differentiation. The foot processes of subpial astrocytes in foetal rats began to acquire assemblies between day 19 and 20 and continued to mature postnatally by a constant addition and rearrangement of assemblies. In contrast to the paucity of assemblies in deeper laminae of the normal brain, reactive astrocytes comprising the lower lamellae in glial scars had an increased number of assemblies while the most striking feature of the superficial, astrocytic processes within the scar was a rearrangement of assemblies. Although the function of these intramembranous particles is still unknown, it appears from our measurements that they are localized primarily in the outermost astrocytic foot process and to a progressively lesser degree in the underlying astrocytic lamellae.

Enzyme histochemistry on endothelia/glial co-cultures showed γ-glutamyltranspeptidase induction in those endothelial cells which were in direct contact with a glial cell or a glial foot process. All other endothelial cells (free of glial contacts) remained negative for the enzyme.

The width of individual glomerular epithelial foot processes appears very different on electron micrographs. A method for obtainining distributions of the true width of foot processes from that of their apparent width on electron micrographs has been developed based on geometric probability theory pertaining to a specific geometric model. Analyses of foot process width in humans and rats show a remarkable interindividual invariance implying rigid control and therefore great biological significance of foot process width or a derivative thereof. The very low inter-individual variation of the true width, shown in the present paper, makes it possible to demonstrate slight changes in rather small groups of patients or experimental animals.

Podocyte loss plays a key role in the progression of glomerular disorders towards glomerulosclerosis and chronic kidney disease. Podocytes form unique cytoplasmic extensions, foot processes, which attach to the outer surface of the glomerular basement membrane and interdigitate with neighboring podocytes to form the slit diaphragm. Maintaining these sophisticated structural elements requires an intricate actin cytoskeleton. Genetic, mechanic, and immunologic or toxic forms of podocyte injury can cause podocyte loss, which causes glomerular filtration barrier dysfunction, leading to proteinuria. Cell migration and cell division are two processes that require a rearrangement of the actin cytoskeleton; this rearrangement would disrupt the podocyte foot processes, therefore, podocytes have a limited capacity to divide or migrate. Indeed, all cells need to rearrange their actin cytoskeleton to assemble a correct mitotic spindle and to complete mitosis. Podocytes, even when being forced to bypass cell cycle checkpoints to initiate DNA synthesis and chromosome segregation, cannot complete cytokinesis efficiently and thus usually generate aneuploid podocytes. Such aneuploid podocytes rapidly detach and die, a process referred to as mitotic catastrophe. Thus, detached or dead podocytes cannot be adequately replaced by the proliferation of adjacent podocytes. However, even glomerular disorders with severe podocyte injury can undergo regression and remission, suggesting alternative mechanisms to compensate for podocyte loss, such as podocyte hypertrophy or podocyte regeneration from resident renal progenitor cells. Together, mitosis of the terminally differentiated podocyte rather accelerates podocyte loss and therefore glomerulosclerosis. Finding ways to enhance podocyte regeneration from other sources remains a challenge goal to improve the treatment of chronic kidney disease in the future.

We measured the thickness of glomerular basement membrane in 7 patients in familial hematuria and compared with that in control group, 2 cases of orthostatic albuminuria, 1 case of urolithiasis and 1 case of hematuria with stomatitis. Needle biopsies were performed on the 7 patients with familial hematuria. 2 patients had a third renal biopsy. The measurements of glomerular basement membrane were taken from photographs and at intervals of 1 from cytoplasmic membrane of the endothelial cells to that of epithelial foot process. Comparing with control group, the thickness of glomerular basement membrane in familial hematuria is remarkable thin. (Table 1 and Table 2)

To the Editor:— For seven years I have been interested in the ultrastructure of the kidney and my work has been rewarded with a finding that I would like to share with the readers ofThe Journal. I have discovered why the cytoplasmic extensions of the epithelial cells in the glomeruli are called foot processes. One can see this clearly from the electron micrograph (Figure). In future work I shall endeavor to find the rest of the lady.