Ultrastructure of the glomerular basement membrane of rats with proteinuria due to subtotal nephrectomy.

Subtotal nephrectomy in the rat gives rise to progressive proteinuria, glomerular hypertrophy, and glomerulosclerosis. The proteinuria antedates significant ultrastructural lesions demonstrable by conventional techniques; its mechanism is obscure. In this article it is shown that proteinuria in this system is not accompanied by evident changes in the ultrastructural distribution of anionic sites in the glomerular basement membrane, as determined by organ perfusion with two cationic probe molecules. It is suggested that the proteinuria may reflect a change in the steric aspect of glomerular ultrafilration, in which the loss of renal mass imposes a significant rise in capillary pressure in a structure whose capillaries are peculiar in the high pressures they normally sustain and in their lack of mechanical support from the interstitium. It is suggested that a sustained rise in capillary pressure overcomes the rigidity of the glomerular basement membrane, so that its pore size increases and proteinuria results from basement membrane failure.

Total-field electron-microscopic examination of thin tissue slices via surface-embedding and multiple-mesa technics.

Total-field electron-microscopic examination of tissue slices of 50--200 micrometers thickness, used in various prestaining methods, can be accomplished by combining the following two technics: surface-embedding, a process in which the thin tissue slice remains on one plane during embedding, so that its entire cross section can be obtained; multiple-mesa technic, a trimming process to obtain multiple targets for examination on the same field. The two technics are described in detail.

Humoral inhibitors of the immune response in uremia. II. Further characterization of an immunosuppressive factor in uremic serum.

The serum of Lewis rats with chronic renal insufficiency (induced by subtotal nephrectomy) contains a nondialyzable inhibitor of the mixed lymphocyte reaction. Fractionation of uremic serum by gel filtration on Sephadex G-200 shows that the inhibitory activity elutes with an apparent molecular weight of greater than 200,000 daltons. To establish the possible relationship of the uremic inhibitor to known immunosuppressive components of normal serum, uremic serum was further fractionated on a DEAE cellulose column. The inhibitory activity elutes in 10 mM sodium phosphate at pH 8.0. This fraction contains both alpha-macroglobulin and IgG. The inhibitory activity of this fraction is completely inactivated by treatment with 2-mercaptoethanol, indicating that the inhibitor is a protein. The inhibitory activity is partially inactivated by periodate treatment, suggesting that it may be a glycoprotein. To determine whether or not the inhibitory factor is an immunoregulatory alpha-macroglobulin, or an immune complex, the uremic serum was fractionated by affinity chromatography procedures, which do not induce artifactual inhibitory properties in control serum. The alpha-macroglobulin was removed by affinity chromatography on a column of Con A-Sepharose; its removal had no effect on the inhibitory activity of serum in the mixed lymphocyte reaction. To examine the possibility that immune complexes may be the uremic inhibitor, the serum was fractionated by affinity chromatography on Protein A-Sepharose or by adsorption to a suspension of Staphylococcus aureus, cowan I. Neither of the two latter procedures had any effect on the inhibitory activity of uremic serum. So far all of our findings indicate that the immunosuppressive factor of uremic serum is distinct from two major immunoregulatory factors, alpha-macroglobulin and immune complexes.

Embedding in large plastic blocks. Diagnostic light and potential electron microscopy on the same block.

Specimens destined for light and electron microscopy were fixed in a modified buffered formalin, postosmicated, dehydrated, and embedded in a mixture of epoxy resins (Epon-araldite) in large plastic molds. These blocks were sectioned at 0.5 to 1 micron on a JB-4 microtome and stained with a combined nuclear and cytoplasmic stain (Paragon). The sections were examined by light microscopy for diagnostic evaluation. If ultrastructural examination was also desired, the selected area was isolated using the "mesa" technique. The trimmed block was then sectioned on an ultramicrotome, picked up on grids, stained, and examined in the electron microscope. We think these techniques offer the diagnostic pathologist the potential of viewing 1-micron sections at a light microscopy level with the option of subsequent electron microscopy of the same area of the same block.