Date of Award

5-1-1990

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Anatomy and Cell Biology

Abstract

Real glomerular basement membranes (GBMs) exhibit a charge-selective barrier, consisting of anionic sites, that restricts the passage of anionic molecules into the urine. These sites are located primarily in the laminae rarae interna (LRI) and externa (LRE) of the GBM and consist of heparan sulfate proteoglycan (HSPG). Previous efforts to localize the HSPG core protein within various layers of the GBM have been contradictory. Furthermore, attempts to correlate proteinuria in several disease states with a decrease in anionic sites or HSPG core protein have yielded conflicting results. In the present study when rat renal cortex blocks were treated by immersion with the cationic probe, polyethyleneimine (PEI), GBMs exhibited anionic sites concentrated primarily in the LRE and more irregularly within the LRI and lamina densa. All sites were heparitinase sensitive indicating that PEI positive sites represent negatively charged groups associated with heparan sulfate.In order to gain information of the distribution of the HSPG protein core, antibodies to HSPG from the EHS tumor matrix (anti-(EHS) HSPG) and GBMs (anti-(GBM) HSPG) were used together with immunogold to label thin sections of Lowicryl embedded kidney cortex. Immunolabeling with anti-(GBM) HSPG suggested a distribution of HSPG which was restricted to the laminae rarae, whereas labeling with anti-(EHS) HSPG indicated that the protein core penetrates through all layers of the GBM.When anti-(GBM) HSPG and anti-(EHS) HSPG were used to label renal tissues from puromycin aminonucleoside nephrotic (PAN) rats, immunolabeling results indicated that a portion of the protein core recognized by anti-(EHS) HSPG was significantly reduced, resulting in decreased binding of immunogold. When glomeruli from PAN rats were immunolabeled with anti-(GBM) HSPG a small reduction in the number of gold particles was detected in early PAN. At later stages, however, staining with this antibody was similar to controls. Anionic sites within the LRE remained unaltered throughout the course of PAN. Based on these results we conclude that although the HSPG core protein is significantly altered in PAN, the heparan sulfate rich anionic sites of the LRE are unaffected, and therefore may not be responsible for proteinuria associated with this disease.

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