Sudhir V. Shah

Mechanisms of neutrophil damage to human alveolar extracellular matrix: The role of serine and metalloproteases☆

Many syndromes of lung injury are associated with accumulation of neutrophils within the pulmonary parenchyma. These neutrophils have the capacity to produce lung injury by products including proteases and reactive oxygen species (ROS). We examined the ability of activated neutrophils to solubilize human alveolar extracellular matrix (ECM), and by use of scavengers and inhibitors, evaluated the role of ROS and proteases in this process. Supernatants of phorbol myristate acetate-activated neutrophils routinely solubilized 10.2% +/- 0.8% (n = 30) of collagen in human alveolar ECM, as measured by hydroxyproline release. Scavengers of ROS had no significant effect on ECM solubilization. Inhibitors of metalloproteases partially inhibited ECM solubilization (38.5% +/- 4.6% inhibition by ethylenediaminetetraacetic acid [n = 6], and 37.0% +/- 14.7% by 1,10-phenanthroline [n = 6]; p less than 0.05). Inhibitors of the neutrophil serine proteases, elastase and cathepsin G, markedly inhibited ECM solubilization (100.9% +/- 3.7% by alpha 1-protease inhibitor [alpha 1-PI] [n = 6] and 81.9% +/- 0.1% by soybean trypsin inhibitor [n = 6]; p less than 0.01). Since alpha 1-PI completely inhibited solubilization, metalloprotease activity appeared to be related to serine protease activity. This finding was confirmed by the observation that addition of a metalloenzyme activator, p-aminophenylmercuric acetate, in the presence of alpha 1-PI, restored solubilization to the same level as that inhibited by metal chelators. We conclude that human neutrophil metalloproteases and serine proteases directly solubilize human alveolar ECM. Furthermore, neutrophil serine proteases activate latent metalloproteases. However, ROS were not demonstrated to play a major role in ECM solubilization in our system.

The cysteine proteinase inhibitor, E-64, reduces proteinuria in an experimental model of glomerulonephritis

Proteinuria is a major manifestation of glomerular disease (glomerulonephritis, GN). We examined the effect of trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a specific and irreversible cysteine proteinase inhibitor, on urinary protein excretion in a complement- and neutrophil-independent model of antiglomerular basement membrane (GBM) antibody disease. A single injection of rabbit antirat-GBM IgG produced a marked increase in urinary protein excretion 24hr after injection. In two separate studies using different pools of antiGBM IgG, administration of E-64 (5mg every 6h starting 2hr prior to induction of GN) reduced proteinuria (-45 +/- 7%, and -41 +/- 14%, Mean +/- SEM, n = 6; P less than 0.001) in the 24 hour period following induction of the disease. This reduction in urinary protein excretion was accompanied by a marked decrease in the specific activity of the cysteine proteinases cathepsins B and L in glomeruli (B: -97%; L: -84%) and renal cortex (B: -87%; L: -75%) isolated from the same E-64-treated rats compared to same saline-treated controls. These data, combined with the specificity of E-64 for cysteine proteinases, suggest a potential role for cysteine proteinases in the increased GBM permeability and proteinuria in this experimental model of glomerular disease.

Oxidant mechanisms in glomerulonephritis

Several lines of evidence derived from both in vitro and in vivo studies suggest an important role for reactive oxygen metabolites and glomerular pathophysiology. Both leukocytes and glomeruli have been shown to increase the generation of reactive oxygen metabolites in response to a wide variety of stimuli. Reactive oxygen metabolites generated enzymatically or by stimulated neutrophils have been shown to affect several biological processes of potential importance in glomerular injury, including glomerular basement degradation, eicosanoid synthesis, cyclic nucleotide metabolism, and glomerular ADPase activity. Scavengers of reactive oxygen metabolites have been shown to reduce proteinuria in several leukocyte-dependent and leukocyte-independent models of glomerular disease. Taken together, these studies suggest an important role of reactive oxygen metabolites in glomerular disease.

The role of aspartic and cysteine proteinases in albumin degradation by rat kidney cortical lysosomes

We have investigated the degradation of 125I-labeled bovine serum albumin by lysates of rat kidney cortical lysosomes. Maximal degradation of albumin occurred at pH 3.5-4.2, with approximately 70% of the maximal rate occurring at pH 5.0. Degradation was proportional to lysosomal protein concentration (range 100-600 micrograms) and time of incubation (1-5 h). Dithioerythritol (2 mM) stimulated albumin degradation 5- to 10-fold. Albumin degradation was not inhibited by phenylmethanesulfonyl fluoride (1 mM) or EDTA (5 mM), indicating that neither serine nor metalloproteinases are involved to a significant extent. Pepstatin (5 micrograms/ml), an inhibitor of aspartic proteinases, inhibited albumin degradation by approximately 50%. Leupeptin (10 microM) and N-ethylmaleimide (10 mM), inhibitors of cysteine proteinases, decreased albumin degradation by 34 and 65%, respectively. Combinations of aspartic and cysteine proteinase inhibitors produced nearly complete inhibition of albumin degradation. Taken together, these data indicate that aspartic and cysteine proteinases are primarily responsible for albumin degradation by renal cortical lysosomes under these conditions. In keeping with the above data, we have measured high activities of the cysteine proteinases, cathepsins B, H, and L, in cortical tubules, the major site of renal protein degradation. Using the peptidyl 7-amino-4-methylcoumarin (NHMec) substrates (Z-Arg-Arg-NHMec, for cathepsin B; Arg-NHMec for cathepsin H; and Z-Phe-Phe-CHN2-inhibitable hydrolysis of Z-Phe-Arg-NHMec corrected for inhibition of cathepsin B activity for cathepsin L) values obtained were (means +/- SE, mU/mg protein, 1 mU = production of 1 nM product/min, n = 6): cathepsin B, 2.1 +/- 0.34; cathepsin H, 1.35 +/- 0.19; cathepsin L, 14.49 +/- 1.26. In comparison, the activities of cathepsins B, H, and L in liver were: 0.56 +/- 0.03, 0.28 +/- 0.04, and 1.27 +/- 0.16, respectively.

Vasopressinase and Diabetes Insipidus of Pregnancy

Excerpt To the Editor:Recent studies have begun to delineate the pathophysiologic basis for the syndrome of pregnancy-associated diabetes insipidus that begins during gestation and remits after del...

Degradation of glomerular basement membrane by a neutral metalloproteinase(s) present in glomeruli isolated from normal rat kidney

Incubation of glomerular homogenates (200 micrograms protein) with glomerular basement membrane (GBM, 30-35 micrograms hydroxyproline) at pH 7.5 for 36 h at 37 degrees C resulted in significant GBM degradation as measured by hydroxyproline release (40 +/- 6%, n = 17). GBM degradation increased with increasing incubation time (12-48 h) and glomerular protein concentration (50-250 micrograms). GBM degradation was not significantly decreased by inhibitors of serine or cysteine proteinases or the inhibitor of bacterial metalloproteinases, phosphoramidon. In contrast GBM degradation by glomerular homogenates was markedly inhibited by the metal chelators 10mM EDTA (-95 +/- 3%, n = 7) and 2mM 1,10-phenanthroline (-96 +/- 2%, n = 4). Preincubation of glomerular homogenates with trypsin (followed by soya bean trypsin inhibitor) markedly stimulated GBM degradation (+103 +/- 20%, n = 11). These results document the presence of a GBM-degrading, neutral metalloproteinase(s) in glomeruli suggesting an important role for this enzyme in glomerular pathophysiology.

Role of Cathepsin B and L in Anti-Glomerular Basement Membrane Nephritis in Rats

We have examined the potential role of the cysteine proteinases, cathepsin B and L, in renal tubular protein degradation and increased permeability of the glomerular basement membrane (GBM) which occurs in a neutrophil- and complement-independent model of anti-GBM antibody disease. The specific activity of cathepsin L, but not cathepsin B, was significantly increased (157%, p greater than 0.01) in cortical homogenates (85-90% tubules) prepared from anti-GBM-treated rats compared to saline-treated controls. Using highly purified cathepsin B and L, we documented the ability of these proteinases to degrade albumin in vitro (Km 5.92 and 0.22 microM for B and L, respectively). In two separate studies, treatment of rats with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, (E-64), a specific and irreversible inhibitor of cysteine proteinases, significantly reduced proteinuria (-45 and -41%, p less than 0.01) in the 24-hour period following injection of the anti-GBM IgG. Taken together, these data suggest an important role for cysteine proteinases in the increased tubular protein degradation which occurs in response to increased filtered protein loads and in the increased GBM permeability (proteinuria) characteristic of glomerular disease.

Concentration-activity profile of the modulation of cyclooxygenase product formation by reduced glutathione in microsomal fractions from the goat lung

Age-related changes in pulmonary formation of arachidonic acid (AA) metabolites are thought to play an important role in regulating cardiopulmonary function. This study addresses the potential role of reduced glutathione (GSH) in modulating cyclooxygenase product formation in the developing lung. Prostaglandin H2 (PGH2) metabolism was studied in microsomal fractions isolated from the lungs of unventilated fetal, neonatal and adult goats. GSH-dependent PGH2 to PGE2 isomerase activity in microsomal fractions from the perinatal (fetal and neonatal) goat lung was not saturable with respect to GSH and can respond to changes in GSH concentration over the range of 0.01 to 30 mM, which encompasses the full range the intracellular GSH levels reported in the literature. However, in fractions from the adult, a lower rate of PGE2 formation is observed at higher GSH concentrations. In addition, the tissue levels of GSH exhibited developmental stage-related differences with fetal being higher than neonatal or adult. The present observations may have physiologic relevance, in that decreases in pulmonary GSH levels after birth may contribute to decreases in plasma PGE2 levels by decreasing pulmonary PGE2 synthesis, thereby contributing to closure of the ductus arteriosus; conversely, increased GSH levels associated with hyperoxia may contribute to persistence of ductal patency. Formation of 6-keto-PGF1 alpha and of TXB2 (the stable metabolites of prostacyclin and TXA2) was decreased when PGE2 formation was increased by GSH activation of PGE2 isomerase in fractions isolated from all three developmental stages. A similar pattern of product formation was observed when AA was employed as substrate. These data suggest the possibility that changes in GSH concentration may modulate eicosanoid formation in cells that contain GSH-dependent PGE2 isomerase, as well as either or both prostacyclin or thromboxane synthase(s).