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S V Shah

Publications and source records attributed to S V Shah.

At least 55 records · Page 3Linked to original sources
PubMed1993

Evidence suggesting that iron and calcium are interrelated in oxidant-induced DNA damage.

The effect of iron chelators and agents that buffer cytosolic-free calcium ([Ca2+]i) on hydrogen peroxide-induced DNA strand breaks in LLC-PK1 cells has not been previously examined. In addition, the interrelationship between iron and calcium in the pathogenesis of DNA damage has not been studied in any model of tissue injury. Exposure of LLC-PK1 cells to 1 mM hydrogen peroxide resulted in marked DNA damage, as measured by the alkaline unwinding assay (residual intact double stranded DNA at 10 min, control: 88 +/- 1%; hydrogen peroxide-treated cells: 17 +/- 3%, N = 8). The iron chelators, 1,10-phenanthroline and deferoxamine, and agents which buffer [Ca2+]i, BAPTA and quin-2, provided highly significant protection against hydrogen peroxide-induced DNA strand breaks. We then examined the effect of iron chelators on hydrogen peroxide-induced rise in [Ca2+]i in LLC-PK1 cells. Both 1,10-phenanthroline and deferoxamine prevented the marked and sustained rise in [Ca2+]i induced by exposure of LLC-PK1 cells to 1 mM hydrogen peroxide ([Ca2+]i at 15 min, control 100 +/- 3 nM; hydrogen peroxide 195 +/- 14 nM; 1,10-phenanthroline + hydrogen peroxide 100 +/- 4 nM; deferoxamine + hydrogen peroxide 106 +/- 4 nM; N = 4). We excluded the possibility that the iron chelators were directly chelating calcium by performing experiments using a cell free system. We also confirmed that BAPTA and quin-2, in concentrations used in our study, chelate calcium but not iron or copper.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1993

Gentamicin-induced mobilization of iron from renal cortical mitochondria.

Iron, presumably by participating in generation of hydroxyl radical or other oxidant species or initiation of lipid peroxidation, has been shown to play an important role in several models of tissue injury, including acute renal failure induced by the antibiotic gentamicin. However, the sources of iron remain unknown. Rat renal mitochondria incubated at 37 degrees C with gentamicin resulted in a time- (15-60 min) and a dose-dependent (0.01-5 mM) iron release as measured by formation of iron-bathophenanthroline sulfonate complex FeII-(BPS)3 [at 60 min, control: 1.2 +/- 0.1 nmol/mg protein, n = 7; gentamicin (5 mM): 5.1 +/- 0.4 nmol/mg protein, n = 7]. No formation of FeII(BPS)3 complex was detected in the absence of mitochondria or when incubations were carried out at 0 degrees C. Similar results were obtained when 2,2'-dipyridyl, another iron chelator, was used for measurement of iron release. On the basis on our previous study that gentamicin enhances generation of hydrogen peroxide by renal cortical mitochondria, we examined whether effect of gentamicin on iron release is mediated by hydrogen peroxide. Catalase (which decomposes hydrogen peroxide), but not heat-inactivated catalase, as well as pyruvate, a potent scavenger of hydrogen peroxide, prevented gentamicin-induced iron mobilization. Superoxide dismutase, a scavenger of superoxide anion, or hydroxyl radical scavengers (dimethylthiourea or sodium benzoate) had no effect. Taken together, the data with scavengers indicate that gentamicin-induced iron mobilization from mitochondria is mediated by hydrogen peroxide.

2,2'-Dipyridyl↗
PubMed1993

Intracellular calcium mediates the cytotoxicity of lipid A in LLC-PK1 cells.

Acute renal failure is a frequent and serious complication of endotoxemia. However, the biochemical events that might ultimately lead to renal cell injury have not been examined previously. The renal tubular epithelial cell line LLC-PK1 was used to evaluate the role of intracellular-free calcium ([Ca++]i) in lipid A-induced cytotoxicity. Lipid A, considered to be the active component of bacterial endotoxin, produced a time- and concentration-dependent cytotoxicity as assessed by loss of trypan blue exclusion (38 +/- 3% cell deaths in 120 min with 50 micrograms/ml, n = 4). Chelation of [Ca++]i by quin 2 and inhibition of Ca++ release by 8-(N,N-dimethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride each protected against lipid A (50 micrograms/ml) cytotoxicity whereas removal of extracellular Ca++ was without effect. Fura-2 fluorescence was used to demonstrate that lipid A induces a rise in [Ca++]i in a concentration-dependent manner. Extracellular Ca++ was not required for the rise in [Ca++]i. Lipid A (50 micrograms/ml) produced a similar rise in [Ca++]i in the presence of 1 mM Ca++ (144 +/- 3 nM above basal) or in the absence of added Ca++ (155 +/- 28 nM, n = 4). The peak rise in [Ca++]i occurred rapidly (within 30 sec), clearly preceding cell death. The observations that lipid A induces a rise in [Ca++]i that precedes cell death, and that quin 2 and 8-(N,N-dimethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride protect against cell death suggest release of intracellular Ca++ mediates the cytotoxicity of lipid A in this renal tubular epithelial cell line.

Aminoquinolines↗
PubMed1992

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.

Basement Membrane↗
PubMed1992

Effect of selenium-deficient diet in experimental glomerular disease.

We examined the effect of a selenium-deficient diet on two experimental models of glomerular disease, the puromycin aminonucleoside (PAN)-induced nephrotic syndrome, a model of minimal change disease, and passive Heymann nephritis, a complement-dependent and neutrophil-independent model that resembles membranous nephropathy. The specific activity of selenium-dependent glutathione peroxidase was markedly reduced in the liver, the kidney cortex, and in glomeruli in weanling male Sprague-Dawley rats placed on a selenium-deficient diet for 6 wk compared with rats fed a selenium-replete diet, with no significant differences in the specific activities of superoxide dismutase or catalase. PAN-injected selenium-deficient rats had a marked and significantly greater proteinuria throughout the course of the experiment compared with PAN-injected selenium-replete rats with no significant histological differences. In the passive Heymann nephritis model induced by injecting anti-Fx1A immunoglobulin G, rats fed a selenium-deficient diet had significantly higher urinary protein (day 5: 91 +/- 16 mg/24 h, n = 10) compared with rats fed a selenium-replete diet (52 +/- 5 mg/24 h, n = 11) with no differences in the amount of antibody deposited in the kidney. The most likely explanation for the effect of a selenium-deficient diet is that selenium deficiency resulted in a marked reduction of glutathione peroxidase, thus indicating an important role of glutathione peroxidase in these models of glomerular injury.

Animals↗
PubMed1992

Role of intracellular calcium in hydrogen peroxide-induced renal tubular cell injury.

Both reactive oxygen metabolites and calcium have been implicated in ischemic and toxic renal tubular cell injury. However, the role of calcium in oxidant injury to renal tubular cells has not been previously examined. In the present study we examined the role of intracellular free Ca2+ ([Ca2+]i) in H2O2-mediated injury to LLC-PK1 cells, a renal tubular epithelial cell line. H2O2 induced a significant rise in [Ca2+]i within 1 min after exposure of cells to 5 mM H2O2, with a sustained rise in [Ca2+]i during the course of experiments, reaching a value of 1.3 microM at 60 min (n = 10). The rise in [Ca2+]i preceded sublethal cell injury as measured by [3H]adenine release or irreversible cell injury as determined by trypan blue exclusion. Buffering [Ca2+]i with quin-2 (50 microM) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 50 microM) was highly protective against the H2O2-induced cell injury. For example, at 120 min after exposure of cells to 5 mM H2O2, irreversible cell injury was reduced from 45 +/- 8 to 9 +/- 1% (n = 3) by quin-2. The acetoxymethyl ester of quin-2 (quin-2/AM) and BAPTA/AM did not interfere with the trypan blue exclusion assay or scavenge H2O2. Preventing mobilization of Ca2+ from intracellular storage sites using 8-(N,N-dimethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8, 10(-4) M) significantly reduced the rise in [Ca2+]i and thus prevented H2O2-mediated cytotoxicity to LLC-PK1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Buffers↗
PubMed1992

Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells.

Hydrogen peroxide (H2O2)-induced DNA damage and cell death have been attributed to the direct cytotoxicity of H2O2 and other oxidant species generated from H2O2. We examined the possibility that oxidants activate endonucleases leading to DNA damage and cell death in renal tubular epithelial cells, similar to that described for apoptosis. Within minutes, H2O2 caused DNA strand breaks in a dose-dependent manner, followed by cell death. DNA fragmentation was demonstrated both by the release of [3H]thymidine in 27,000-g supernatant as well as the occurrence of low molecular weight DNA fragments on agarose gel electrophoresis, characteristic of endonuclease cleavage. Endonuclease inhibitors, aurintricarboxylic acid, Evans blue, and zinc ion prevented H2O2-induced DNA strand breaks, fragmentation, and cell death. Inhibitors of protein or mRNA synthesis had only minor protection against H2O2-induced DNA damage in contrast to complete protection reported in apoptotic thymocytes. Micrococcal endonuclease induced similar DNA strand breaks in LLC-PK1 cells, and the endonuclease inhibitors prevented the events confirming the ability of endonucleases to induce DNA damage. The protective effect of aurintricarboxylic acid was not due to the prevention of the rise in intracellular free calcium. We conclude that endonuclease activation occurs as an early event leading to DNA damage and cell death in renal tubular epithelial cells exposed to oxidant stress and, in contrast to apoptotic thymocytes, does not require macromolecular synthesis.

Animals↗
PubMed1992

Reactive oxygen metabolites in toxic acute renal failure.

We have examined the role of reactive oxygen metabolites (ROM) in gentamicin nephrotoxicity and in glycerol-induced acute renal failure, a model for myoglobinuric acute renal failure. Several agents which affect mitochondrial respiration have been shown to enhance the generation of hydrogen peroxide. Based on gentamicin's ability to alter mitochondrial respiration both in vitro and in vivo we postulated that gentamicin may enhance the generation of ROM by renal cortical mitochondria. Gentamicin, in a dose-dependent fashion, enhanced hydrogen peroxide production by rat renal cortical mitochondria as measured by the decrease in scopoletin fluorescence. At the highest concentration of gentamicin tested (4.0 mM), the rate of hydrogen peroxide generation was markedly increased from 0.17 +/- 0.02 to 6.21 +/- 0.67 nmol/mg/min. We next demonstrated that hydroxyl radical scavengers and an iron chelator provide a marked functional and histological protection in gentamicin-induced acute renal failure in rats. Hydroxyl radical scavengers and the iron chelator deferoxamine also protected renal function in glycerol-injected rats, a model for acute renal failure due to muscle injury. Although these data suggest that ROM may be important mediators of toxic renal injury, in vivo generation of ROM by kidney in normal and pathological states has not been previously examined. Aminotriazole (AT) irreversibly inactivates catalase only in the presence of hydrogen peroxide and previous studies have shown that AT-mediated inhibition of catalase in a sensitive measure of in vivo changes in the hydrogen peroxide generation. Using this method, we have demonstrated the in vivo generation of hydrogen peroxide under normal conditions and enhanced generation of hydrogen peroxide in rats treated with gentamicin or glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Kidney Injury↗
PubMed1991

Role of glutathione in an animal model of myoglobinuric acute renal failure.

In a previous study we have shown a role for reactive oxygen metabolites in glycerol-induced acute renal failure, a well-established model for myoglobinuric acute renal failure. In the present study we examined the role of glutathione in this model of acute renal failure. Administration of 50% (vol/vol) glycerol at a dose of 10 ml/kg of body weight to rats intramuscularly resulted in significant renal failure associated with depletion of total kidney glutathione (GSH) from 2.6 +/- 0.1 mumol/g (mean +/- SEM control level) to 1.7 +/- 0.1 mumol/g after 6 hr (P less than 0.001). If GSH were important in glycerol-induced acute renal failure, one would anticipate that exogenously administered GSH should afford protection, while injury should be potentiated if endogenous GSH is depleted. We examined the effect of i.p. administration of L-buthionine-(S,R)-sulfoximine (BSO) at 2 mmol/kg (which results in depletion of kidney GSH) and the effect of increasing renal GSH by i.v. administration of reduced GSH (2 mmol/kg every 3 hr) on kidney function in glycerol-treated rats. Glycerol-injected rats treated with BSO showed significantly worse renal failure than did rats given glycerol alone, while administration of GSH resulted in significant amelioration of glycerol-induced acute renal failure [glycerol treatment alone, blood urea nitrogen (BUN) = 96 +/- 10 and creatinine = 2.5 +/- 0.4 mg/dl; BSO + glycerol treatment, BUN = 123 +/- 7 and creatinine = 3.5 +/- 0.1 mg/dl (n = 9, P less than 0.05); GSH + glycerol treatment, BUN = 78 +/- 10 and creatinine = 1.25 +/- 0.2 mg/dl (n = 8, P less than 0.05)]. In separate experiments 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) [which interferes with the enzyme GSH reductase and prevents recycling of oxidized GSH (GSSG) into GSH] resulted in worsening of glycerol-induced acute renal failure similar to that produced by BSO. These functional differences between GSH-depleted and GSH-repleted rats were further substantiated by significant histological differences in tubular injury. Taken together, these results provide evidence for an important role of GSH in glycerol-induced acute renal failure.

Acute Kidney Injury↗
PubMed1991

Evidence suggesting a role for cathepsin L in an experimental model of glomerulonephritis.

We have utilized specific, irreversible inhibitors of cysteine proteinases to examine the role of renal cathepsin B and cathepsin L in the proteinuria which occurs in an experimental model of human glomerular disease. Administration of trans-epoxysuccinyl-L-leucylamido-(3-methyl)butane (Ep475) a specific, irreversible inhibitor of cysteine proteinases, including cathepsins B and L, significantly reduced proteinuria in rats with experimentally induced, neutrophil-independent, anti-GBM antibody disease (controls: 10 +/- 1 mg/24 h, N = 8; anti-GBM antibody disease: 203 +/- 30 mg/24 h, N = 8; anti-GBM antibody disease + Ep475: 112 +/- 13 mg/24 h, mean +/- SEM, N = 6, P less than 0.05). There was a marked reduction in the activity of both cathepsin B and cathepsin L in renal cortices obtained from Ep475-treated rats compared to either saline-treated controls or rats treated with anti-GBM IgG only. Administration of Z-Phe-Tyr(O-t-butyl)CHN2, a specific, irreversible cysteine proteinase inhibitor with a high degree of selectivity toward cathepsin L, also caused a reduction in anti-GBM antibody-induced proteinuria (90 +/- 18 mg/24 h, N = 6, P less than 0.05). This reduction in proteinuria was accompanied by a marked decrease (-84%) in the specific activity of renal cortical cathepsin L in Z-Phe-Tyr(O-t-butyl)CHN2-treated rats. However, cathepsin B activity was unchanged. There was no significant change in the renal anti-GBM antibody uptake, plasma urea nitrogen, or plasma creatinine values in the Z-Phe-Tyr(O-t-butyl)CHN2-treated rats compared to rats treated with anti-GBM IgG only or saline-treated controls. These data document the ability of cysteine proteinase inhibitors to decrease the proteinuria which occurs in a neutrophil-independent model of human anti-GBM antibody disease and suggest an important role for cathepsin L in the pathophysiology of the proteinuria which occurs in this model.

Animals↗
PubMed1991

Hydrogen peroxide cytotoxicity in LLC-PK1 cells: a role for iron.

Reactive oxygen metabolites have been postulated to play an important role in both toxic and ischemic forms of acute renal tubular epithelial injury. In the present study, we examined the effect of enzymatically generated hydrogen peroxide on LLC-PK1 cells, a renal proximal tubule cell line. Exposure of LLC-PK1 cells to glucose and glucose oxidase (GO; which generates hydrogen peroxide) resulted in cytotoxicity (as measured by trypan blue exclusion) which was dose dependent and increased linearly over time to 81 +/- 5% at 180 minutes (8 +/- 1% at time 0; mean +/- SEM, N = 3 to 7). Catalase (which decomposes hydrogen peroxide) completely prevented the cytotoxicity, confirming that the toxicity was due to hydrogen peroxide production. To assess whether the hydrogen peroxide toxicity was a direct effect or mediated by other toxic oxygen metabolites, several scavengers of reactive oxygen metabolites and iron chelators were used. Superoxide dismutase (a scavenger of superoxide) had no effect. Deferoxamine (DFO), an iron chelator, provided marked protection (GO alone 45.9 +/- 4.4%; GO + DFO 13.0 +/- 2.0%; control 7.1 +/- 1.2%; N = 15 to 17, P less than 0.001). Pretreatment with DFO (1 hr, then 2 washes to remove DFO before GO addition) also markedly inhibited the cytotoxicity, suggesting that DFO's effect was due to iron chelation. Two other metal chelators (dihydroxybenzoic acid and 1,10-phenanthroline) also significantly decreased the GO-induced cytotoxicity. However, three of four hydroxyl radical scavengers used (mannitol, dimethyl sulfoxide, sodium benzoate) did not significantly decrease cell death. Only dimethylthiourea provided protection.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Receptor-mediated increase in cytosolic calcium in LLC-PK1 cells by platelet activating factor and thromboxane A2.

Several studies indicate an important role of platelet activating factor (PAF) and thromboxane A2 (TXA2) in glomerular pathophysiology. However, the potential role of PAF or TXA2 in renal tubular pathophysiology has received little attention, and the presence of functional receptors for these autacoids in renal tubular epithelium has not been previously studied. We examined the effects of PAF and the TXA2 analogue, ONO11113, on the cytosolic free calcium concentration [( Ca2+]i) in cultured LLC-PK1 cell line using a fluorescent probe, fura-2. In these cells, the addition of PAF or ONO11113 caused a significant increment in [Ca2+]i in a dose-dependent manner: both agonists (10(-7) M) increased [Ca2+]i from 148 +/- 16 to 288 +/- 39 nM and from 130 +/- 8 to 240 +/- 18 nM, with the values of EC50 for PAF and ONO11113 being 17 +/- 4 and 17 +/- 2 nM, respectively. These effects were both rapid and transient, returning to baseline in two minutes. The effect of PAF was selectively blocked by PAF receptor antagonist BN50730, but not by TXA2 receptor antagonist L657925. Similarly ONO11113 response was abolished by L657925, but not by BN50730. PAF- or ONO11113-challenged cells did not respond to a second addition of the same agent and showed heterologous desensitization to the other agonist. The initial peaks of [Ca2+]i as well as the sustained elevations in [Ca2+]i induced by PAF or ONO11113 were reduced following the chelation of extracellular Ca2+ by 10 mM ethylene glycol-bis(beta-aminomethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Effects of dietary protein intake on muscle protein synthesis and degradation in rats with gentamicin-induced acute renal failure.

In the study presented here, the muscle protein synthesis and degradation in gentamicin-induced acute renal failure were examined in rats fed a low (7%)-, normal (22%)-, and high (35%)-isocaloric protein diet. Male Sprague-Dawley rats were fed equivalent amounts of these diets for 10 days and then received daily subcutaneous injections of either 1 mL of sterile isotonic saline or 100 mg/kg of gentamicin for 7 consecutive days. The rats were sacrificed the following day, and epitrochlearis muscles were obtained for measurement of protein turnover. The serum creatinine in each of the gentamicin-treated groups were significantly higher than that in the saline-treated controls but were no different from each other. Muscle protein synthesis (calculated from the incorporation of radiolabeled (U-14C) phenylalanine) was slightly but not significantly decreased in gentamicin-treated rats as compared with that of the corresponding saline controls in each of the dietary groups. Net protein degradation (the rate of tyrosine release into media) in the 7 and 22% gentamicin-treated groups was similar to that in the corresponding saline controls. In contrast, net protein degradation was significantly greater in the 35% gentamicin group of rats when compared with that in the 7 and 22% gentamicin groups and its own control. In the 7 and 22% saline- and gentamicin-treated protein groups, there was a reduction in net protein degradation in response to insulin. In contrast, the net protein degradation continued to remain significantly elevated in the 35% gentamicin-treated group, despite addition of insulin, when compared with that in the 7 and 22% gentamicin groups and its own control.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Kidney Injury↗
PubMed1990

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).

6-Ketoprostaglandin F1 alpha↗
PubMed1990

Glomerular basement membrane degradation by endogenous cysteine proteinases in isolated rat glomeruli.

Recent in vitro and in vivo studies suggest that cysteine proteinases may play an important role in degradation of the glomerular basement membrane (GBM) by renal glomeruli. However, little information is available concerning the cysteine proteinases present in glomeruli, the distribution of cysteine proteinases in other areas of the kidney, or the potential role of endogenous glomerular cysteine proteinases in GBM degradation. Using well characterized fluorogenic substrates, we have documented the presence of the cysteine proteinases, cathepsins B, H, and L, in glomeruli (0.45 +/- 0.06, 0.39 +/- 0.05, and 0.66 +/- 0.14 mU/mg protein, mean +/- SEM, N = 8) and other fractions prepared from normal rat kidney. The presence of cysteine proteinases in glomeruli was verified by fluorescence microscopy. For each proteinase, the activity was: proportional to the amount of tissue protein and time of incubation; dependent on the presence of exogenously added dithiothreitol; and completely inhibited by the specific cysteine proteinase inhibitor, E-64. The pH optimum for cathepsin B (substrate: Z-Arg-Arg-HNMec) and L (substrate: Z-Phe-Arg-HNMec in the presence of Z-Phe-Phe-CHN2) was approximately pH 6.0 for both glomeruli and renal cortex; while that for cathepsin H (substrate: Arg-HNMec) was approximately 6.5. Incubation of sonicated glomeruli with 3H-GBM under conditions optimal for cysteine proteinase activity (pH 4.5, 1 mM EDTA, and 1 mM dithiothreitol, 37 degrees C) resulted in significant GBM degradation as measured by the release of non-sedimentable (10,000 x g, 10 min) radioactivity or hydroxyproline.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗