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S V Shah

Publications and source records attributed to S V Shah.

At least 37 records · Page 2Linked to original sources
PubMed1997

Recent advances in understanding mechanisms of renal tubular injury.

Study of cell death has emerged as an important and exciting area of research in cell biology. In this review, we first discuss two kinds of cell death, apoptosis and necrosis, then describe the evidence suggesting a role of endonuclease activation in renal tubular injury. The pathway that is followed by the cell is dependent on both the nature and the severity of the insults and may evolve from the apoptotic to the necrotic form of cell death. It is also conceivable that there are some common pathways that are shared and regulated in the two modes of cell death. Along these lines, interleukin 1 beta-converting enzyme (ICE) and ICE-like proteases, generally associated with the apoptotic mode of cell death, recently have been shown to be important in hypoxic injury. We describe the studies documenting an important role of endonuclease in renal tubular injury and some of the important mechanisms by which it may be regulated. A large body of evidence supports a role of protooncogenes in the regulation and modification of apoptotic cell death. Many of these protooncogenes and related proteins have been shown to be altered in models of acute renal failure, suggesting their potential importance. Therapeutic opportunities may lie in prevention of cell injury or enhancing recovery by inhibiting endonucleases and ICE and ICE-like proteases as well as by induction of certain oncogenes and related proteins.

Apoptosis↗
PubMed1996

Role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity to LLC-PK1 cells.

The current study was designed to test the role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity, and to determine whether it may serve as a source of catalytic iron. Hydrogen peroxide led to iron release (as measured by iron/bathophenanthroline complex) from the microsomes prepared from LLC-PK1 cells. Cimetidine, which inhibits cytochrome P-450 by interacting with the heme iron, significantly blocked iron release, whereas ranitidine, which has a similar structure as cimetidine but is a weak inhibitor of P-450, did not have an effect (H2O2, 0.42 +/- 0.04; H2O2 + cimetidine, 0.23 +/- 0.02 nmol/mg protein; N = 4, P < 0.01). Exposure of LLC-PK1 cells to hydrogen peroxide (2.5 mM) resulted in a significant increase in the bleomycin-detectable iron (iron capable of catalyzing free radical reactions) content that was prevented by cimetidine, but not ranitidine. We then examined the effect of the inhibitors of cytochrome P-450 on cell death (as measured by Trypan blue exclusion) after exposure of LLC-PK1 cells to 2.5 mM hydrogen peroxide for 120 minutes. Inhibition of cytochrome P-450 by cimetidine significantly reduced the cell death; the effect was observed with 0.05 mM and was concentration dependent with 1 mM affording almost complete protection (H2O2, 59 +/- 1.3% vs. H2O2 + cimetidine, 11 +/- 0.7%; N = 5, P < 0.01). In contrast, ranitidine did not show any protection. We confirmed that the protective effect of cimetidine was not related to scavenging hydrogen peroxide or hydroxyl radicals or chelating iron. A second inhibitor of cytochrome P-450, piperonyl butoxide, had a similar dose-dependent beneficial effect against hydrogen peroxide-induced cell injury. Our data thus indicate an important role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity to LLC-PK1 cells and suggest that cytochrome P-450 may serve as a source of catalytic iron.

Animals↗
PubMed1996

Endonuclease induced DNA damage and cell death in chemical hypoxic injury to LLC-PK1 cells.

Hypoxia is considered to result in a necrotic form of cell injury. We examined the role of endonuclease activation, considered a feature of apoptosis, in DNA damage and cell death in hypoxic injury in LLC-PK1 cells. Hypoxia in LLC-PK1 cells was induced using a combination of glucose deprivation and a mitochondrial inhibitor, antimycin A (10 microM). Chemical hypoxia caused DNA damage as measured by the alkaline unwinding assay and internucleosomal DNA fragmentation that preceded cell death. Incubating protein extract of cells subjected to chemical hypoxia with calf thymus DNA resulted in oligonucleosome length fragments, which were prevented by an endonuclease inhibitor, aurintricarboxylic acid. Chemical hypoxia resulted in an increased DNA degrading activity with a molecular mass of approximately 15 kDa. Endonuclease inhibitors, aurintricarboxylic acid and Evans blue, prevented antimycin A-induced DNA strand breaks, fragmentation and cell death. We conclude that endonuclease activation plays an important role in chemical hypoxic injury to LLC-PK1 cells.

Animals↗
PubMed1996

Evidence for cytochrome P-450 as a source of catalytic iron in myoglobinuric acute renal failure.

Iron has been implicated to play an important role in several models of tissue injury, including myoglobinuric acute renal failure. In this model, myoglobin released from the injured muscle is generally accepted as a source of iron. In the present study we measured the bleomycin-detectable iron (iron capable of catalyzing free radical reactions) in the kidneys and examined the role of cytochrome P-450 as a source of catalytic iron in glycerol-induced model of myoglobinuric acute renal failure. Rats were injected with 50% glycerol (8 ml/kg) i.m. after overnight water deprivation and sacrificed 24 hours later. There was a marked and a specific increase in the bleomycin-detectable iron content accompanied by a marked decrease in the cytochrome P-450 content in the kidneys of glycerol treated rats. We then examined the effects of two different cytochrome P-450 inhibitors, cimetidine (with ranitidine as a control) and piperonyl butoxide. Cimetidine, but not ranitidine, significantly prevented the increase of bleomycin-detectable iron in the kidneys of glycerol-treated rats. The loss of cytochrome P-450 content was substantially blocked by both inhibitors, cimetidine and piperonyl butoxide, but not by ranitidine. Both the inhibitors of cytochrome P-450 provided functional (as measured by BUN and creatinine) and histological protection against glycerol-induced acute renal failure. Our data thus demonstrate a marked increase in bleomycin-detectable iron in the kidneys of glycerol-treated rats. Our data also indicate that inhibitors of cytochrome P-450 provide protection against glycerol-induced acute renal failure and that cytochrome P-450 may be a significant source of this iron in this model of acute renal failure.

Acute Kidney Injury↗
PubMed1996

Role of 'catalytic' iron in an animal model of minimal change nephrotic syndrome.

Treatment of minimal change disease, like most glomerulonephritides, is empirical because underlying mechanisms that cause glomerular injury are not known. We examined a pathogenic role of 'catalytic' iron in a model of minimal change nephrotic syndrome induced by injection of puromycin aminonucleoside (7.5 mg/100 g body wt) to rats. Although there was no significant change in non-heme iron content in glomeruli, the bleomycin-detectable iron (capable of catalyzing free radical reactions) was markedly increased in glomeruli from nephrotic rats when compared to control. In contrast, despite a marked and significant increase in the non-heme iron content in tubules, there was no significant change in the bleomycin-detectable iron in tubules from nephrotic rats. In a separate in vivo study, the iron chelator, deferoxamine, prevented the increase in the bleomycin-detectable iron in glomeruli and provided complete protection against proteinuria. Taken together, our data suggest an important pathogenetic role for glomerular catalytic iron in the puromycin aminonucleoside-induced minimal change nephrotic syndrome.

Animals↗
PubMed1996

Role of reactive oxygen metabolites in DNA damage and cell death in chemical hypoxic injury to LLC-PK1 cells.

Hypoxia is considered to result in a necrotic form of cell injury. We have recently demonstrated a role of endonuclease activation, generally considered a feature of apoptosis, to be almost entirely responsible for DNA damage in hypoxic injury to renal tubular epithelial cells. The role of reactive oxygen metabolites in endonuclease-induced DNA damage and cell death in chemical hypoxic injury has not been previously examined. LLC-PK1 cells exposed to chemical hypoxia with antimycin A resulted in enhanced generation of intracellular reactive oxygen species as measured by oxidation of a sensitive fluorescent probe, 2',7'-dichlorofluorescin diacetate. Superoxide dismutase, a scavenger of superoxide radical, significantly reduced the fluorescence induced by antimycin A and provided significant protection against chemical hypoxia-induced DNA strand breaks (as measured by the alkaline unwinding assay). Pyruvate, a scavenger of hydrogen peroxide, provided significant protection against chemical hypoxia-induced DNA strand breaks and DNA fragmentation (as measured by agarose gel electrophoresis). The interaction between superoxide anion and hydrogen peroxide in the presence of a metal catalyst leads to generation of other oxidant species such as hydroxyl radical. Hydroxyl radical scavengers, dimethylthiourea, salicylate, and sodium benzoate, and two metal chelators, deferoxamine and 1,10-phenanthroline, also provided marked protection against DNA strand breaks and DNA fragmentation. These scavengers of reactive oxygen metabolites and metal chelators provided significant protection against cell death as measured by trypan blue exclusion and lactate dehydrogenase release. Taken together, these data indicate that reactive oxygen species play an important role in the endonuclease activation and consequent DNA damage, as well as cell death in chemical hypoxic injury to renal tubular epithelial cells.

Animals↗
PubMed1996

Kidney iron status in passive Heymann nephritis and the effect of an iron-deficient diet.

In the study presented here, the iron status in the kidney in passive Heymann nephritis, a complement-dependent model of membranous nephropathy, was examined. To examine whether the effect of immune injury on iron status has a pathogenic role, the effect of an iron-deficient diet was also determined. Injection of the anti-Fx1A antibody (10 mg/100 g body wt) in Sprague-Dawley rats resulted in no change in the serum iron level, a marked increase in the urinary excretion rate of iron, a marked increase in non-heme iron content of kidney cortex, and a marked increase in the non-heme iron level in tubules. These increases in iron were prevented by feeding the rats an iron-deficient diet. In the rats fed a normal iron diet and injected with anti-Fx1A-lgG, there was no significant change in the non-heme iron level in glomeruli. However, an iron-deficient diet resulted in a significant decrease in the non-heme iron level in glomeruli, compared with its respective control. In addition, an iron-deficient diet significantly reduced urinary protein excretion rate (Day 5: iron-replete, 68 +/- 12 mg/24 h, N = 12; iron-deficient, 36 +/- 11, N = 10, P < 0.05) in the complement-dependent immune phase of the glomerular injury. Taken together, these data indicate a marked alteration in the iron status in the kidney and suggest an important role of iron in glomerular injury of passive Heymann nephritis.

Animals↗
PubMed1995

Activation of a 15-kDa endonuclease in hypoxia/reoxygenation injury without morphologic features of apoptosis.

Hypoxia/reoxygenation is an important cause of tissue injury in a variety of organs and is classically considered to be a necrotic form of cell death. We examined the role of endonuclease activation, considered a characteristic feature of apoptosis, in hypoxia/reoxygenation injury. We demonstrate that subjecting rat renal proximal tubules to hypoxia/reoxygenation results in DNA strand breaks and DNA fragmentation (both by an in situ technique and by agarose gel electrophoresis), which precedes cell death. Hypoxia/reoxygenation resulted in an increase in DNA-degrading activity with an apparent molecular mass of 15 kDa on a substrate gel. This DNA-degrading activity was entirely calcium dependent and was blocked by the endonuclease inhibitor aurintricarboxylic acid. The protein extract from tubules subjected to hypoxia/reoxygenation cleaved intact nuclear DNA obtained from normal proximal tubules into small fragments, which further supports the presence of endonuclease activity. Despite unequivocal evidence of endonuclease activation, the morphologic features of apoptosis, including chromatin condensation, were not observed by light and electron microscopy. Endonuclease inhibitors, aurintricarboxylic acid and Evans blue, provided complete protection against DNA damage induced by hypoxia/reoxygenation but only partial protection against cell death. Taken together, our data provide strong evidence for a role of endonuclease activation as an early event, which is entirely responsible for the DNA damage and partially responsible for the cell death that occurs during hypoxia/reoxygenation injury. Our data also indicate that in hypoxia/reoxygenation injury endonuclease activation and DNA fragmentation occur without the morphological features of apoptosis.

Animals↗
PubMed1994

An old enzyme with a new function: purification and characterization of a distinct matrix-degrading metalloproteinase in rat kidney cortex and its identification as meprin.

We have purified to homogeneity the enzyme in the kidney cortex which accounts for the vast majority of matrix-degrading activity at neutral pH. The purified enzyme has an apparent molecular mass of 350 kD by gel filtration and of 85 kD on SDS-PAGE under reducing conditions; and it degrades laminin, type IV collagen and fibronectin. The enzyme was inhibited by EDTA and 1,10-phenanthroline, but not by other proteinase inhibitors. The enzyme was not activated by organomercurials or by trypsin and was not inhibited by tissue inhibitors of metalloproteinases indicating that it is distinct from the other matrix-degrading metalloproteinases. Unexpectedly, the amino acid sequence of the NH2-terminal and two internal peptides of the enzyme showed complete homology to those alpha subunits of rat meprin, an enzyme previously shown to degrade azocasein and insulin B chain but not known to degrade extracellular matrix components. Immunoprecipitation studies, Western blot analyses and other biochemical properties of the purified enzyme confirm that the distinct matrix-degrading enzyme is indeed meprin. Our data also demonstrate that meprin is the major enzyme in the renal cortex capable of degrading components of the extracellular matrix. The demonstration of this hitherto unknown function of meprin suggests its potential role in renal pathophysiology.

Amidohydrolases↗
PubMed1994

Role of cytosolic calcium-independent plasmalogen-selective phospholipase A2 in hypoxic injury to rabbit proximal tubules.

Although the activation of calcium-independent phospholipase A2 (PLA2) enzymes has been described in the heart, the pathogenetic role of this enzyme(s) in hypoxic cell injury has not been previously examined in any tissue. Therefore, we characterized the time course of activation of calcium-independent PLA2 using both plasmalogen and diacylglycerophospholipid substrates during hypoxia in rabbit proximal tubules and examined whether inhibition of calcium-independent PLA2 activity is associated with a cytoprotective effect. Subjecting rabbit proximal tubules to hypoxia for 5 min resulted in at least a threefold increase in cytosolic calcium-independent PLA2, which was selective for plasmalogen substrates (control 444 +/- 69 vs hypoxia 1,675 +/- 194 pmol.mg protein-1.min-1, n = 5). In contrast, no changes in PLA2 activity were observed in the presence of 4 mM EGTA in the membrane fraction using plasmenylcholine substrates. 20 min of hypoxia resulted in an increase in arachidonate from 3 +/- 1 to 28 +/- 4 ng/mg protein and lactate dehydrogenase release from 7.5 +/- 2% to 38 +/- 5%, n = 4. Pretreatment of proximal tubules with 10 microM Compound I, a specific inhibitor of calcium-independent PLA2, resulted in reduction in the magnitude of both hypoxia-induced arachidonic acid release (11 +/- 3 ng/mg protein) and lactate dehydrogenase release (18 +/- 4%). Our data indicate that a significant fraction of PLA2 activity in the proximal tubule is calcium-independent and selective for plasmalogen substrates. Furthermore, the activation of this enzyme plays an important role in the pathogenesis of membrane injury during hypoxia in the proximal tubule.

Animals↗
PubMed1994

Activation of potassium channels contributes to hypoxic injury in proximal tubules.

The mechanisms responsible for the loss of cell potassium during renal ischemia are poorly understood. The present studies examined the hypothesis that potassium channels are activated as an early response to hypoxia and contribute to potassium loss independent from an inhibition of active K+ uptake. Potassium flux in suspensions of freshly isolated rat proximal tubules was measured using an ion-selective electrode. Exposure of the tubules to hypoxia for only 2.5 min resulted in a rise in the passive leak rate of K+ but no decrease in active K+ uptake. The passive leak of K+ was associated with a 40% decrease in cell ATP content. The passive K+ efflux was inhibited by 5 mM Ba2+ (95%) and by 15 mM tetraethylammonium (85%) suggesting that K+ channels were the primary route of K+ movement. The effects of K+ channel blockade on the development of hypoxic injury were also examined. Tetraethylammonium and glibenclamide, an inhibitor of ATP-sensitive K+ channels, reduced hypoxic injury as assessed by the release of lactate dehydrogenase or measurement of DNA damage. These results suggest that activation of K+ channels is an early response to hypoxia and contributes to hypoxic renal injury.

Adenosine Triphosphate↗
PubMed1994

Presence of a distinct extracellular matrix-degrading metalloproteinase activity in renal tubules.

Renal tubular homogenates incubated with [3H]laminin (2 micrograms, 10(5) cpm) at 37 degrees C resulted in time- and protein-dependent laminin degradation (e.g., at 24 h, control = 6,533 +/- 771; experimental = 27,610 +/- 1,023 cpm +/- SE; N = 20). Gel chromatography confirmed that laminin (800 to 900 kd) was degraded to 20- to 50-kd fragments. Laminin degradation was not significantly decreased by serine or cysteine protease inhibitors. In contrast, metal chelators produced marked inhibition (EDTA, 93 +/- 3%; 1,10-phenanthroline, 99 +/- 1%) indicating that, at neutral pH, metalloproteinases were responsible for the laminin degradation. Laminin-degrading activity in renal tubules was not inhibited by the tissue inhibitor of metalloproteinase and was present in an active form. This activity was also present in high concentrations in the renal cortex and medulla but was only minimal in the liver. Further studies of the renal cortex revealed a similar metalloproteinase activity against type IV collagen (11,075 +/- 305; N = 6) and gelatin (41,026 +/- 1,373; N = 6), and this activity was membrane associated (97 +/- 1%; N = 4). Taken together, the characteristics of this renal metalloproteinase indicate that it is distinct from classic matrix-degrading metalloproteinases. The release of this distinct metalloproteinase from damaged renal tubular epithelial cells during injury may result in the production of fragments of laminin or other extracellular matrix components with biologic effects relevant to renal regeneration.

Animals↗
PubMed1994

Apoptosis.

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Animals↗
PubMed1993

Increase in bleomycin-detectable iron in ischaemia/reperfusion injury to rat kidneys.

Iron has been shown to be important in ischaemic, immune and toxic forms of tissue injury in various organs. Although it is generally accepted that iron participates in the generation of powerful oxidant species (e.g. hydroxyl radicals) there has not been any direct evidence that iron capable of catalysing free-radical reactions is increased in tissues in these models of injury. In the present study we demonstrate that ischaemia/reperfusion injury to the kidney results in no significant change in total, nonhaem or ferritin iron levels, but there is a marked and specific increase in bleomycin-detectable iron (capable of catalysing free-radical reactions) in the kidney. The increase in bleomycin-detectable iron is observed only after reperfusion but not during the ischaemic period. In a separate study we demonstrate that despite a drastic reduction in the iron content in the kidney, as a result of feeding an iron-deficient diet, there is a similar and a marked increase in the bleomycin-detectable iron in kidneys accompanied by a lack of protection against ischaemia/reperfusion injury.

Acute Kidney Injury↗
PubMed1993

Glomerular thromboxane A2/prostaglandin H2 receptors: characterization and effect of adriamycin-induced nephrotic syndrome.

We have characterized the thromboxane (TX) A2/prostaglandin (PG) H2 receptor in glomeruli isolated from the rat using the agonist radioligand [125I]-BOP. Binding of [125I]-BOP was highly specific, stereoselective, and to a single class of high affinity binding sites (Kd = 1.16 +/- 0.22 nM and Bmax = 348 +/- 32 fmol/mg protein; n = 6). Binding of [125I]-BOP was competed for by the agonist ONO11113 (Kd = 50.8 +/- 8.0 nM; n = 4) and the antagonists SQ29548 (Kd = 15.8 +/- 1.0 nM; n = 3), L657925 (Kd = 12.1 +/- 2.2 nM; n = 3) and L657926 (Kd = 1642 +/- 135 nM; n = 3). I-BOP also produced a TXA2/PGH2 receptor-mediated rise in [Ca2+]i in isolated glomeruli In adriamycin-induced nephrotic syndrome in the rat, the development of proteinuria is reported to be dependent on increased renal TXA2 production. We therefore examined whether or not changes in glomerular TXA2/PGH2 receptors occur between control and nephrotic rats. No changes in expression or affinity of either glomerular or platelet TXA2/PGH2 receptors were observed. Kd and Bmax values for isolated glomeruli were 1.45 +/- 0.24 nM and 406 +/- 72 fmol/mg for controls and 1.22 +/- 0.25 nM and 321 +/- 62 fmol/mg for nephrotic rats (n = 6).

Animals↗