Publications (23)42.94 Total impact

  • [Show abstract] [Hide abstract] ABSTRACT: This study examined the potential therapeutic effects of a combination therapy consisting of 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR) and N-acetyl cysteine (NAC) to attenuate ischemia-reperfusion (I/R) injury in a canine model of autologous renal transplantation. Male mongrel dogs (15-20 kg) underwent left nephrectomy followed by flushing and static preservation of the kidney in University of Wisconsin (UW) solution for 48 hr. The treatment group received AICAR (50 mg/kg) plus NAC (100 mg/kg) intravenously before the left nephrectomy. The compounds were added to the UW solution as well. All dogs underwent right nephrectomy 48 hr later followed by autotransplantation of the preserved left kidney. Treated dogs received a second dose of AICAR and NAC before implantation of the renal autograft. The treated dogs had excellent urine output posttransplant, with peak serum creatinine of 7.26 mg/dL on postoperative day (POD) 3 that normalized after 14 days. The control group were anuric and developed clinical symptoms of uremia on POD 1. Morphologic evaluation supported the protective effects of combination therapy. Immunohistochemical analysis revealed decrease of tumor necrosis factor-alpha, interferon-gamma, and inducible nitric oxide synthase; and TUNEL assay showed decreased apoptosis in the treated group. Combination therapy with AICAR and NAC attenuates renal I/R injury and improves the outcome of the transplanted kidney after prolonged cold preservation.
    No preview · Article · Oct 2004 · Transplantation
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    A Mehta · C P S Sekhon · S Giri · J K Orak · AK Singh
    [Show abstract] [Hide abstract] ABSTRACT: Ischemia followed by reperfusion has a number of clinically significant consequences. A number of pathophysiological processes appear to be involved in ischemia/reperfusion (I/R) injury. The mitogen activated protein kinases (MAPK) are integral components of the parallel MAP kinase cascades activated in response to a variety of cellular stress inducing ischemia/ATP depletion and inflammatory cytokines. Many studies suggest that members of the MAP kinase family in particular Jun N-terminal kinase (JNK) are activated in kidney following ischemia/reperfusion of this tissue. The present study underlines the therapeutic potential of the combination of N-acetyl cysteine (NAC), a potent antioxidant, sodium nitroprusside (SNP), a nitric oxide donor and phosphoramidon (P), an endothelin-1 converting enzyme inhibitor in ameliorating the MAPK induced damage during renal ischemia/reperfusion injury. Our previous results showed that 90 min of ischemia followed by reperfusion caused very severe injury and that the untreated animals had 100% mortality after the 3rd day whereas there was improved renal function and 100% survival of animals in the three drug combination treatment group. The present study, mainly on tissue sections, further supports the protection provided by the triple drug therapy. A higher degree of expression of all the three classes of MAPK, i.e. JNK, P38 MAP kinases and P-extracellular signal regulated kinases (ERKs) can be seen in kidneys subjected to ischemia/reperfusion insult. Pretreatment with a combination of N-acetyl cysteine, sodium nitroprusside, and phosphoramidon completely inhibits all three classes of MAPK and ameliorates AP-1 whereas individual or a combination of any two drugs is not as effective.
    Preview · Article · Dec 2002 · Molecular and Cellular Biochemistry
  • K Dobashi · B Ghosh · J K Orak · I Singh · AK Singh
    [Show abstract] [Hide abstract] ABSTRACT: Reactive oxygen species (ROS; O2-, H2O2, and OH), normal by-products of cellular metabolic processes, are kept in control by antioxidant enzymes, such as catalase, glutathione peroxidase (GPX) and superoxide dismutases (SODs). To understand the role of antioxidant enzymatic defenses against ROS injury following ischemia-reperfusion, we examined the effect on kidney exposed to varying periods (30, 60 or 90 min) of ischemia followed by different periods of reperfusion. The enzymatic activities and protein levels of catalase, GPX, CuZnSOD and MnSOD were relatively unaffected at 30 min of ischemia followed by 0, 2 or 24 h reperfusion. However, 60 or 90 min of ischemia followed by 0, 2 or 24 h of reperfusion resulted in a decrease in activities and protein levels which paralleled the duration of ischemic injury. MnSOD activity tended to recover towards normal during reperfusion. Examination of the mRNA levels of these antioxidant enzymes demonstrated a severe decrease in mRNA levels of catalase and GPX at a time point of minimal ischemic injury (30 min of ischemia followed by reperfusion) suggesting that loss of mRNA of catalase and GPX may be the first markers of alterations in cellular redox in ischemia-reperfusion injury. Greater loss of mRNA for catalase, GPX and CuZnSOD was observed following longer periods (60 or 90 min) of ischemia. The mRNA for MnSOD was upregulated at all time points of ischemia-reperfusion injury. Actually, the greater decrease in mRNAs for catalase, GPX and CuZnSOD in the acute phase (within 24 h) subsequently showed a further decrease in these enzyme activities in the subacute phase (72 or 120 h after ischemia). These enzyme activities in the 30 min ischemia group, (but not in the 90 min group), already showed tendencies for normalization at 120 h after ischemia. To understand the molecular basis of the loss of mRNA of these antioxidant enzymes during ischemia-reperfusion injury, we examined the rate of transcription by nuclear run-on assays. The similar rates of transcription in control and kidney exposed to ischemia-reperfusion indicates that the loss of mRNA for catalase, GPX and CuZnSOD is possibly due to the increased rate of turnover of their mRNAs. These studies suggest that expression of antioxidant genes during ischemia-reperfusion are not coordinately expressed and that the differential loss of antioxidant enzymes may be the contributing factor(s) towards the heterogeneous renal tissue damage as a result of ischemia-reperfusion induced oxidative stress.
    No preview · Article · Mar 2000 · Molecular and Cellular Biochemistry
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    [Show abstract] [Hide abstract] ABSTRACT: We report that endotoxin treatment results in decreased amounts of peroxisomes as well as changes in structure and function of peroxisomal membranes. Peroxisomes isolated from the liver of control and treated animals showed a marked decrease in total protein, but no significant alteration in the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein profile. However, the Western blot study of the peroxisomal beta-oxidation enzymes and catalase showed an increase in those enzymes in the peroxisomal peak of normal density in endotoxin-treated rats. Disintegration of peroxisomal membranes by carbonate treatment from endotoxin-treated liver and change in the fluidity of peroxisomal membranes suggests alterations in peroxisomal membrane structure. No such alterations were found in mitochondrial or microsomal membranes of endotoxin-treated livers. The lipid analysis of these organelles showed that the only organelle affected was the peroxisome, with a significant decrease in the phospholipid and cholesterol concentrations. To understand the mechanism of endotoxin-mediated alterations in peroxisomes, we studied the possible role of Kupffer cell secreted soluble factors (tumor necrosis factor alpha [TNF-alpha]) on the peroxisomal structure/function. Inactivation/elimination of Kupffer cells by gadolinium chloride before endotoxin treatment did not normalize the overall peroxisomal protein amount and the lipid composition of isolated peroxisomes. However, the levels of individual protein amount in remaining peroxisomes were normalized. Endotoxin also decreased peroxisomal beta-oxidation, and this was partially restored with gadolinium treatment. These results clearly show that peroxisomes are severely affected by endotoxin treatment and suggest that the damage to this organelle may contribute, at least in part, to endotoxin-induced hepatic cytotoxicity.
    Full-text · Article · Feb 2000 · Hepatology
  • [Show abstract] [Hide abstract] ABSTRACT: Plasma and urine concentrations of protein S were measured in five children with steroid-resistant nephrotic syndrome. It was found that plasma free protein S was reduced in three out of the five patients studied. Thus, acquired free protein S deficiency does occur in children with nephrotic syndrome and is one of many factors which may place them at risk for a thromboembolic event.
    No preview · Article · May 1996 · Annals of clinical and laboratory science
  • [Show abstract] [Hide abstract] ABSTRACT: Earlier, we reported that rat liver peroxisomes contain Cu-Zn superoxide dismutase (J. Biol. Chem. 267, 6870), thereby suggesting a new antioxidant role for this organelle in free radical metabolism. In this study, we report for the first time that mammalian peroxisomes also contain glutathione peroxidase. Using highly purified rat liver peroxisomes isolated by Nycodenz gradient, we found that peroxisomes contain glutathione peroxidase which shows enzymatic activity with different substrates such as hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide. This activity could be inhibited in vitro by mercaptosuccinate. Western blot analysis revealed that peroxisomes from control and ciprofibrate-treated livers show immunoreactive bands with antibodies raised against glutathione peroxidase. The intraperoxisomal distribution of glutathione peroxidase was investigated by using peroxisomal membrane and matrix proteins. The results revealed that glutathione peroxidase is a matrix enzyme. The presence of glutathione peroxidase in peroxisomes provides an alternate enzyme system responsible for the degradation of organic peroxides and the degradation of H2O2 under conditions in which catalase is inactivated (e.g., ischemia-reperfusion and endotoxemia). These findings suggest that glutathione peroxidase in peroxisomes may play a novel role in the cellular antioxidant responses to various oxidative stress conditions.
    No preview · Article · Jan 1995 · Archives of Biochemistry and Biophysics
  • J K Orak · A K Singh · P R Rajagopalan · I Singh
    No preview · Article · Mar 1994 · Transplantation Proceedings
  • No preview · Article · Mar 1994 · Transplantation Proceedings
  • [Show abstract] [Hide abstract] ABSTRACT: We have previously demonstrated that ischemic injury results in the loss of peroxisomal functions (e.g., inhibition of catalase activity and fatty-acid beta-oxidation activity). To understand the molecular mechanism leading to the loss of peroxisomal beta-oxidation in ischemic tissue, we examined the levels of individual enzyme activities and proteins of the peroxisomal beta-oxidation system and overall fatty-acid oxidation in peroxisomes isolated from kidney exposed to ischemia-reperfusion injury. The peroxisomal beta-oxidation decreased with an increase in time of ischemic injury (53% and 43% of the control in kidneys exposed to 60 and 90 min ischemia, respectively). In vivo inactivation of catalase with aminotriazole and exposure of isolated peroxisomes to H2O2 resulted in inhibition of peroxisomal beta-oxidation system suggesting that this enzyme system is labile to excessive H2O2 produced during ischemic injury. The enzyme activities of lignoceroyl-CoA ligase, acyl-CoA oxidase, bifunctional enzymes and acyl-CoA thiolase (individual peroxisomal beta-oxidation enzymes) after 90 min of ischemia were 87, 80, 87 and 85% of the control, respectively. This decrease in enzyme activities was more pronounced following reperfusion (28, 11, 23 and 35% of the control, respectively). Immunoblot analysis of these enzymes indicated that the major loss of these enzyme activities during ischemia was due to their inactivation, whereas during reperfusion, proteolysis also contributed toward the observed loss of these activities. In summary, these results demonstrated that loss of peroxisomal beta-oxidation in ischemia-reperfusion injury was due to inactivation and proteolysis of beta-oxidation enzymes. Acyl-CoA oxidase was more sensitive to ischemia-reperfusion injury compared to other enzymes, and the overall loss of peroxisomal beta-oxidation may be a reflection of the loss of acyl-CoA oxidase activity, a rate-limiting enzyme.
    No preview · Article · Nov 1993 · Biochimica et Biophysica Acta
  • [Show abstract] [Hide abstract] ABSTRACT: Exogenously supplied catalase, a peroxisomal enzyme, has been found to be of therapeutic value in ischemic injury. Therefore, we examined the effect of ischemic-reperfusion injury on the structure and function of kidney peroxisomes. Ischemic injury changed the density of peroxisomes from 1.21 g/cm3 (peak I) to a lighter density of 1.14 g/cm3 (peak II). The number of peroxisomes moving from the normal density population (peak I) to a lower density population (peak II) increased with an increase in ischemic injury. Latency experiments indicated both populations of peroxisomes to be of intact peroxisomes. Immunoblot analysis with antibodies against peroxisomal matrix and membrane proteins demonstrated that after 90 min of ischemia a significant number of matrix proteins were lost in the peak II population, suggesting that functions of these peroxisomes may be severally affected. Reperfusion following ischemic injury resulted in loss of peroxisomal matrix proteins in both peaks I and II, suggesting that peroxisomal functions may be drastically compromised. This change in peroxisomal functions is reflected by a significant decrease in peroxisomal catalase activity (35%) and beta-oxidation of lignoceric acid (43%) observed following 90 min of ischemia. The decrease in catalase activity was more pronounced in reperfused kidneys even after a shorter term of ischemic injury. Reperfusion restored the normal peroxisomal beta-oxidation in kidneys exposed up to 60 min of ischemia. However, 90 min of ischemia was irreversible as there was a further decrease in beta-oxidation upon reperfusion. The decrease in catalase activity during ischemia alone was due to the formation of an inactive complex, whereas during reperfusion, following 90 min of ischemia, inactivation and proteolysis or decreased synthesis of catalase contributed equally toward the injury. The observed changes in the structure and function of peroxisomes as a result of ischemic-reperfusion injury and the ubiquitous distribution of peroxisomes underlines the importance of this organelle in the pathophysiology of vascular injury in general.
    No preview · Article · Jun 1992 · Archives of Biochemistry and Biophysics
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    G S Dhaunsi · S Gulati · A K Singh · J K Orak · K Asayama · I Singh
    [Show abstract] [Hide abstract] ABSTRACT: In this study, by using highly purified rat liver peroxisomes, we provide evidence from analytical cell fractionation, Western blot, and immunocytochemical analysis that Cu-Zn superoxide dismutase is present in animal peroxisomes. Treatment with ciprofibrate, a peroxisome proliferator, increased the peroxisomal superoxide dismutase activity by 3-fold with no effect on mitochondrial activity but a marked decrease in cytosolic superoxide dismutase activity, further supporting that besides cytosolic and mitochondrial localization, Cu-Zn superoxide dismutase is present in peroxisomes also. Demonstration of superoxide dismutase in peroxisomes suggests a new role for this organelle in pathophysiological conditions, such as ischemia-reperfusion injury.
    Preview · Article · May 1992 · Journal of Biological Chemistry
  • [Show abstract] [Hide abstract] ABSTRACT: Acute peritoneal dialysis in unstable infants is at times plagued by early catheter malfunction secondary to omental plugging in both rigid acute catheters and conventional Tenckhoff catheters. This problem is inherent to the design of catheters using sideports for outflow and is enhanced by the tenacity of the omentum in this population in walling off foreign bodies. We have modified and utilized a non-luminal, channeled surgical drain for acute peritoneal dialysis in infants to avoid this problem. Five infants ranging in age from 2 days to 7 months were dialyzed acutely in a Pediatric Intensive Care Unit setting for periods ranging from 5 to 34 days utilizing this modified catheter. The infants ranged in weights from 1.96 to 8 Kg. Catheters were placed by a surgeon and peritoneal dialysis was initiated using a Y-setup. In none of the patients was there loss of catheter function secondary to omental plugging. Three patients subsequently died of their underlying illness and two recovered renal function. Two acute catheters were subsequently changed to conventional Tenckhoff catheters when it became apparent that dialysis would need to be performed for a prolonged time. The acute catheter which was used has a four channel cloverleaf appearance when cut in cross section with no central lumen. There is a transition to a luminal catheter outside the peritoneal cavity. The advantage of the cloverleaf configuration is the ability to exchange fluid along its entire intraperitoneal length, thereby excluding a defined area of catheter sideports where omentum can occlude the system causing a ball valve phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)
    No preview · Article · Feb 1992 · Advances in peritoneal dialysis. Conference on Peritoneal Dialysis
  • G S Dhaunsi · A K Singh · J Orak · I Singh
    [Show abstract] [Hide abstract] ABSTRACT: We have investigated lipid peroxidation and oxidation of lignoceric acid in response to oral thioridazine administration, to better understand the effects of phenothiazines, which are one of the more commonly used therapeutic agents. Measurements at different time intervals showed that levels of lipid peroxides in rat kidney were markedly decreased after thioridazine feeding, however, the oxidation of lignoceric acid was found to be elevated immediately after the start of thioridazine treatment. These biochemical changes were noted to be associated with mitochondrial proliferation and lipid accumulation in renal epithelial cells. The observed renal biochemical and morphological changes following thioridazine feeding return to the normal levels after two weeks of withdrawal of the drug. This study suggests that phenothiazines could be beneficial in reducing cellular injury by reducing the levels of lipid peroxides during pathological conditions like ischemia.
    No preview · Article · Feb 1990 · Journal of experimental pathology
  • [Show abstract] [Hide abstract] ABSTRACT: The fluorescence polarization technique with 1,6-Diphenyl-1,3,5-hexatriene as a probe, was used to determine the lipid rotational mobility (LRM) measured by fluorescence anisotropy of isolated whole mitochondria of the rat kidney following normothermic ischemia of 30, 45, 60 and 90 minutes and upon reperfusion for 24 hours. The LRM of mitochondrial membrane lipids of the ischemic kidney decreased steadily with increasing ischemic times (0.1590 vs. 0.1705, 0.01 less than P less than 0.001 at 60 minutes). Following 24 hours reflow, there were no significant differences in the LRM of mitochondria between ischemic and control groups up to 45 minutes of ischemia, (0.1688 vs. 0.1705, 0.5 less than P less than 0.6). However, when kidney was subjected to ischemic periods longer than 60 minutes, the decreased LRM remained fixed even after reperfusion (0.1783 vs. 0.1738, 0.5 less than P less than 0.6). This suggests that 60 minutes of ischemia probably produces irreversible damage to the mitochondrial membrane whereas lesser degrees of ischemic injury is reversible upon reperfusion.
    No preview · Article · Feb 1990 · Journal of experimental pathology
  • [Show abstract] [Hide abstract] ABSTRACT: The ability of renal tissue to synthesize ATP was examined in adult Sprague Dawley Rats immediately following normothermic ischemia of 30, 45, 60 and 90 minutes and upon reperfusion for 24 hours. Following ischemia the rate of ATP synthesis decreased progressively. It was 64.5% of the control at 45 minutes and 10.4% after 90 minutes of ischemia. Reperfusion of the ischemic kidneys for 24 hours restored ATP biosynthesis to control, nonischemic levels in kidneys subjected to ischemia up to 45 minutes (101.8 +/- 13.9% vs 64.5 +/- 2.5% p less than 0.02). However, after 60 minutes of ischemia, reperfusion had no effect (59.3 +/- 4.4% vs 51.7 +/- 7.5%) and reperfusion following 90 minutes of ischemia was associated with decrease ATP synthesis (10.4 +/- 2.2% vs 3.3 +/- 0.9% p less than .001). We conclude that mitochondrial function is restored by reperfusion when normothermic ischemic interval is 45 minutes or less. However, ischemic intervals longer than 45 minutes produce non-reversible impairment of ATP synthesis and the marked reduction following 90 minutes of ischemia signifies possible transition to a non-viable state.
    No preview · Article · Feb 1989 · Journal of experimental pathology
  • [Show abstract] [Hide abstract] ABSTRACT: The increase in free fatty acids in the ischemic tissue is a consistent observation and these free fatty acids are considered to play a role in the cellular toxicity. To elucidate the cause of higher levels of free fatty acids in ischemic tissue, we examined the catabolism of fatty acids. The beta-oxidation of lignoceric (24:0), palmitic (16:0) and octanoic (8:0) acids and the peroxidation of fatty acids were measured at different times of renal ischemia in whole kidney homogenate. The enzymatic activities for the oxidation of fatty acids decreased with the increase in ischemia time. However, the lipid peroxide levels increased 2.5-fold of control with ischemic injury. Sixty min of ischemia reduced the rate of oxidation of octanoic, palmitic and lignoceric acids by 57, 59 and 69%, respectively. Almost similar loss of fatty acid oxidation activity was observed in the peroxisomes and mitochondria. These data suggest that loss of mitochondrial and peroxisomal fatty acid beta-oxidation enzyme activities from ischemic injury may be one of the factors responsible for the higher levels of free fatty acids.
    No preview · Article · Oct 1988 · Lipids
  • E H Garin · J K Orak · K L Hiott · S E Sutherland
    [Show abstract] [Hide abstract] ABSTRACT: We have conducted a controlled trial on the efficacy of cyclosporine in eight patients with steroid-resistant nephrotic syndrome (four with idiopathic minimal lesion nephrotic syndrome and four with focal segmental glomerulosclerosis). Patients were randomly allocated to a cyclosporine (5 mg/kg/d) or a control group. After eight weeks of therapy and one month without cyclosporine therapy, patients in the control group were given cyclosporine for eight weeks and those in the cyclosporine group became controls. Before the initiation of treatment, there was no difference between the groups with regard to proteinuria and serum albumin levels. Proteinuria remained unchanged in the cyclosporine group, while there was a significant increase in proteinuria in the control group. There were no significant changes in serum albumin levels in either group during the trial. This study does not support the use of cyclosporine at the dose of 5 mg/kg/d in patients with steroid-resistant minimal lesion nephrotic syndrome or focal segmental glomerulosclerosis.
    No preview · Article · Oct 1988 · American journal of diseases of children (1960)
  • J K Orak · E H Garin
    [Show abstract] [Hide abstract] ABSTRACT: RN represents a significant cause of chronic renal failure. Once established, the entity seems to follow a progressive course, regardless of therapeutic modalities. The association of glomerular lesions and hypertension may exacerbate the deterioration of renal function. Since, in humans, RN is almost always associated with urinary tract infections, treatment and prevention of infections is of paramount importance.
    No preview · Article · Jun 1986 · Seminars in urology
  • [Show abstract] [Hide abstract] ABSTRACT: Eighteen infants, children, and adolescents were trained in the techniques of continuous ambulatory peritoneal dialysis (CAPD) as a therapy for end stage renal disease (ESRD) at the University of Florida. Fourteen patients successfully continued CAPD 4–24 months, for a total of 193 patient-months. Uremic symptoms were well controlled with blood urea nitrogen concentration (BUN) decreased to between 60 and 80mg/dl. Parathyroid hormone levels increased but roentgenographic evidence of osteodystrophy improved in most patients. The rate of peritonitis was one episode in 7.7 patient-months. Blood transfusion requirements decreased for patients transferred from in-center hemodialysis to CAPD with no significant decrease in average hematocrits. Caloric intake was adequate and anorexia was usually not a major problem. Children who were evaluated for growth were under 15 years of age, with bone ages less than 12 years, and were using CAPD for longer than 6 months. Their mean growth velocity was 74.7±20.4% (SD) of the predicted velocity.
    No preview · Article · Sep 1984 · European Journal of Pediatrics
  • [Show abstract] [Hide abstract] ABSTRACT: An association between chronic renal failure and skeletal deformities in the adolescent patient was first documented by Lucas in 1883. Since then it has been established that the kidneys play a major role in the regulation of calcium, phosphate, and parathyroid hormone, and that chronic renal failure is characterized by profound alterations in the normal metabolic homeostasis of the human body. With the hyperphosphatemia of uremia, compensatory hyperparathyroidism is also a well known complication. Due to these factors, loss of normal renal function ultimately leads to derangement in mineral and bone metabolism resulting in severe skeletal deformities. Reports in the English literature suggest that the changes of renal osteodystrophy are much more pronounced in the pediatric patient, as compared to those in the adult. In the last two decades, renal transplantation has come to be recognized as a satisfactory modality for controlling renal failure and its complications. This procedure is often not available as an option, however, in small patients, especially those under three years of age. The pediatric nephrologist is often forced to manage these patients for long periods with conservative therapy, in an attempt to control the ravages of renal osteodystrophy. The problem becomes unmanageable when the compensatory hyperparathyroidism proceeds to autonomy. When this occurs, despite maintenance of normal serum calcium levels, the renal osteodystrophy progresses rapidly, producing pain, deformities and growth retardation. At this point, the condition is often refractory to medical management and resection of parathyroid tissue remains the only satisfactory modality for control.(ABSTRACT TRUNCATED AT 250 WORDS)
    No preview · Article · Sep 1984 · Journal of Pediatric Surgery