Glomerular basement membrane thickness - a comparison of two methods of measurement in patients with unexplained haematuria.

BACKGROUND: Thin glomerular basement membranes may be an important cause of microscopic haematuria. Unfortunately measurements are often not made because of the complicated methods currently employed. METHODS: A simplified method of measurement of glomerular basement membrane thickness, involving only 16 selected measurements on a single glomerulus, was compared with the accepted, but time-consuming, orthogonal intercept technique. Thirty-one needle biopsies from patients with renal haematuria unexplained by conventional histology and immunofluorescence were studied. Measurements were made on the same ultrathin sections. RESULTS: The new method was found to give much lower values (mean (SD) 202+/-51 versus 282+/-52 nm) with limits of agreement of -131 to -30 nm compared with the orthogonal intercept method. The coefficient of repeatability was 39 nm for the orthogonal intercept method and 56 nm for the new method. However, using two glomeruli the new method had limits of agreement of -120 to -41 nm with a coefficient of repeatability of 38 nm. CONCLUSIONS: Provided two glomeruli are measured the new technique is sufficiently accurate for the diagnosis of thin membrane nephrology, in appropriate cases, and is much simpler and cheaper than the orthogonal intercept method.

Fractionation of perforin and granzymes by immobilized metal affinity chromatography (IMAC).

Cytotoxic lymphocytes and natural killer cells kill their targets by releasing pore-forming granules or by Fas ligand-Fas initiated death. The granules contain the pore-forming protein perforin, proteoglycan and multiple serine proteases termed granzymes. In this paper we describe two options for isolating perforin and granzymes. Both options separate the proteins by their ability to bind to immobilized metal affinity chromatography (IMAC) columns. The first option, with Cu2+ as the metal (Cu2+-IMAC), separates both perforin and granzymes while the second, with Co2+ as the metal (Co2+-IMAC), separates only perforin. After Cu2+-IMAC perforin is > 20-fold enriched with excellent recovery of lytic activity. Only two proteins are substantial contaminants. After Cu2+-IMAC, the perforin is dilute and requires concentration before additional steps of purification. The second option, with Co2+ as the metal Co2+-IMAC), yields perforin that is concentrated in a sharp peak. The concentrated perforin is immediately suitable for further purification. The first option, with Cu2+, isolates the granzymes while the second option, Co2+-IMAC, does not. After isolation, the perforin lytic and granzyme activities are stable for weeks at 4 degrees C, an advantage to previous isolation methods for these proteins. The excellent recoveries of perforin and granzymes also indicate that these proteins are less than 4% and 15% of the total lymphocyte granule protein, respectively.

The function of lymphocyte proteases. Inhibition and restoration of granule-mediated lysis with isocoumarin serine protease inhibitors.

To kill other cells, lymphocytes can exocytose granules that contain serine proteases and pore-forming proteins (perforins). We report that mechanism-based isocoumarin inhibitors inhibited the proteases and inactivated lysis. When inhibited proteases were restored, lysis was also restored, indicating that the proteases were essential for lysis. We found three new lymphocyte protease activities, "Asp-ase,""Met-ase," and "Ser-ase," which in addition to ly-tryptase and ly-chymase, comprise five different protease activities in rat RNK-16 granules. The general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inhibited all five protease activities. Essentially all protease molecules were inactivated by DCI before lysis was reduced, as determined from DCI's second order inhibition rate constants for the proteases, the DCI concentrations, and the times of pretreatment needed to block lysis. The pH favoring DCI inhibition of lysis was the pH optimum for protease activity. Isocoumarin reagents acylate, and may sometimes secondarily alkylate, serine protease active sites. Granule proteases, inhibited by DCI acylation, were deacylated with hydroxylamine, restoring both the protease and lytic activities. Hydroxylamine does not restore alkylated proteases and did not restore the lytic activities after inhibition with 4-chloro-7-guanidino-3-(2-phenylethoxy)-isocoumarin, a more alkylating mechanism-based inhibitor designed to react with tryptases. It is improbable that isocoumarin reagents directly inactivated pore-forming proteins because 1) these reagents require protease activation, 2) their nonspecific effects are alkylating, and 3) alkylated proteins are not restored by hydroxylamine. We conclude that serine proteases participate in lysis when lysis is mediated by the complete assembly of granule proteins.