Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the laminae rarae by cationic probes.

Abstract

Cationized ferritin (CF) of narrow pI range (7.3-7.5) and the basic dye ruthenium red (RR) have been used as cationic probes to partially characterize anionic sites previously demonstrated in the glomerular basement membrane (GBM). When CF was given i.v. to normal rats and the left kidney was fixed by perfusion 15 min thereafter, clusters of CF molecules were found throughout the lamina rara interna (LRI), lamina rara externa (LRE), and mesangial matrix distributed at regular (-60 nm) intervals. When kidneys were perfused with aldehyde fixative containing RR, small (20 nm) RR-stained particles were seen in the same locations distributed with the same 60 nm repeating pattern, forming a quasiregular, lattice-like arrangement. Fine (-3 nm) filaments connected the sites and extended between them and the membranes of adjoining endothelial and epithelial cells 9 When CF was given i.v. followed by perfusion with RR in situ, both probes localized to the same sites 9 CF remained firmly bound after prolonged perfusion with 0.1-0 9 M KCI or NaCI. It was displaced by perfusion with buffers of high ionic strength (0 9149 M KCI) or pH (<3.0 or >10.0). CF also bound (clustered at -60 nm intervals) to isolated GBM's, and binding was lost when such isolated GBM's were treated with buffers of high ionic strength or pH. These experiments demonstrate the existence of a quasi-regular, lattice-like network of anionic sites in the LRI and LRE and the mesangial matrix 9 The sites are demonstrable in vivo (by CF binding), in fixed kidneys (by RR staining), and in isolated GBM's (by CF binding). The results obtained with CF show that the binding of CF (and probably also RR) to the laminae rarae is electrostatic in nature since it is displaced by treatment with buffers of high ionic strength or pH. With RR the sites resemble in morphology and staining properties the proteoglycan particles found in connective tissue matrices and in association with basement membranes in several other locations 9 KEY WORDS cationized ferritin red proteoglycans 9 ionic interaction isolated GBM's 9 ruthenium Recent studies have shown that the glomerular capillary wall contains fixed negatively charged sites which may be important in maintaining nor- J. CELL BIOLOGY 9The Rockefeller University Press 9

Basement membrane heparan sulfate proteoglycans are concentrated in the laminae rarae and in podocytes of the rat renal glomerulus.

“…We conclude (29) (26) of this approach for localizing antigens within the layers of the GBM. We believe that the more consistent, restricted localization of HSPG to the laminae rarae obtained by the direct immunoperoxidase and frozen thin-section techniques more accurately reflects the sites where the core proteins are concentrated; moreover, it corresponds to the site of localization of the heparan sulfate side chains as detected with cationic probes (3,8).…”

“…Previous work from this laboratory (7) established that HSPG isolated from the rat GBM are relatively small (mean Mr 130,000) and possess approximately four heparan sulfate side chains (Mr 26,000). By cytochemical techniques the heparan sulfate side chains of the HSPG were found to be concentrated in regularly spaced 60-nm anionic sites in the inner and outermost layers (lamina rara interna and externa) of the GBM (3,8). The location of the core protein of these HSPG has not been resolved because to date no specific antibodies have been generated against HSPG from the GBM, and immunocytochemical studies utilizing antibodies raised against HSPG derived from other basement membrane sources have provided conflicting evidence on the localization of HSPG in the GBM (9,10).…”

Polycation-siRNA nanoparticles can disassemble at the kidney glomerular basement membrane

“…5 B-D). Most objects were localized to the lamina rara interna with some of the smaller objects localized to the limina rara externa, both are locations of anionic sites within the GBM (25). Additionally, some objects were observed within the glomerular endothelial fenestrations.…”

Proteinuria with and without Renal Glomerular Podocyte Effacement

“…The GBM acts as a physical filter and also as a charger barrier. Molecules Ͼ10 nm in diameter do not cross the GBM, and the negatively charged proteins of Ͼ70 kD can minimally cross GBM (17).…”