Gymnemic Acid Ameliorates Pancreatic β-Cell Dysfunction by Modulating Pdx1 Expression: A Possible Strategy for β-Cell Regeneration

BACKGROUND@#Endogenous pancreatic β-cell regeneration is a promising therapeutic approach for enhancing β-cell function and neogenesis in diabetes. Various findings have reported that regeneration might occur via stimulating β-cell proliferation, neogenesis, or conversion from other pancreatic cells to b-like cells. Although the current scenario illustrates numerous therapeutic strategies and approaches that concern endogenous β-cell regeneration, all of them have not been successful to a greater extent because of cost effectiveness, availability of suitable donors and rejection in case of transplantation, or lack of scientific evidence for many phytochemicals derived from plants that have been employed in traditional medicine. Therefore, the present study aims to investigate the effect of gymnemic acid (GA) on β-cell regeneration in streptozotocin-induced type 1 diabetic rats and high glucose exposed RIN5-F cells. @*METHODS@#The study involves histopathological and immunohistochemical analysis to examine the islet’s architecture.Quantitative polymerase chain reaction (qPCR) and/or immunoblot were employed to quantify the β-cell regeneration markers and cell cycle proliferative markers. @*RESULTS@#The immunoexpression of E-cadherin, β-catenin, and phosphoinositide 3-kinases/protein kinase B were significantly increased in GA-treated diabetic rats. On the other hand, treatment with GA upregulated the pancreatic regenerative transcription factor viz. pancreatic duodenal homeobox 1, Neurogenin 3, MafA, NeuroD1, and β-cells proliferative markers such as CDK4, and Cyclin D1, with a simultaneous downregulation of the forkhead box O, glycogen synthase kinase-3, and p21 cip1 in diabetic treated rats. Adding to this, we noticed increased nuclear localization of Pdx1 in GA treated high glucose exposed RIN5-F cells. @*CONCLUSION@#Our results suggested that GA acts as a potential therapeutic candidate for endogenous β-cell regeneration in treating type 1 diabetes.

Mesoporous silica incorporated PCL/Curcumin nanofiber for wound healing application.

Electrospinning, a recent fast-emerging technique highly applicable in the production of nanofibers has gained vast recognition owing to its explicit applications in various domains. Amongst which, the production of nanoscaffolds for wound healing applications has been focused recently due to advantages over conventional wound healing methods. In the present research, a composite nanoscaffold comprising SBA-15 (Santa Barbara Amorphous), amine functionalized SBA-15 polycaprolactone (PCL) and curcumin was investigated for its potentiality in wound healing therapeutics. The high biocompatibility and cell adhesion of amine functionalized SBA-15 and the widely explored antimicrobial properties of curcumin added benefit for the wound healing target. The prepared highly interconnected electrospun fibers with porous structure were characterized through various studies such as FTIR, XRD, SEM and EDAX. Further, antibacterial studies against both Gram positive (Bacillus subtilis) and Gram negative (Escherichia coli) strains revealed an improved zone of inhibition. Major invitro studies such as cell migration, proliferation, bio-compatibility was experimented through cell adhesion and live and dead assay using Swiss 3T6 cell lines. In vivo studies on female Wister rats using the fabricated nanofibers incorporated with curcumin and amine functionalized SBA-15 showed 99% scar-less wound healing within 21 days. Re-epithelization of tissue, collagen deposition and formation of granulation tissue were observed from the results of Hematoxylin-Eosin and Masson's tri-chrome staining. From the observations, it can be concluded that the fabricated nanoscaffold could be an effective substrate for wound healing therapeutics.

A Rare Genetic Mutation in a Stone Former.

A 30-year-old woman with history of passage of stones since childhood presented with oliguria and pedal edema for 10 days. She had hypertension with a creatinine of 4.1 mg/dL. Evaluation showed presence of bilateral multiple renal calculi with features of chronicity of kidney disease. Metabolic work-up for nephrolithiasis turned out to be negative and eventually renal biopsy revealed features of chronic interstitial nephritis with greenish brown refractile crystals in the tubular lumen and interstitium. The possibility of dihydroxy adenine crystalline nephropathy was considered. Spectrophotometry of RBC lysates revealed decreased activity of Adenine phosphoribosyl-transferase enzyme. Gene amplification by PCR and sequential analysis identified a missense mutation in exon 3 region of APRT gene in the patient and her family members. This case report highlights the need to contemplate the diagnosis of DHA crystalline nephropathy in young patients with nephrolithiasis and the identification of a rare genetic mutation, which is being reported for the first time in India.

Targeting the Nrf2/ARE Signalling Pathway to Mitigate Isoproterenol-Induced Cardiac Hypertrophy: Plausible Role of Hesperetin in Redox Homeostasis.

Cardiac hypertrophy is the underlying cause of heart failure and is characterized by excessive oxidative stress leading to collagen deposition. Therefore, understanding the signalling mechanisms involved in excessive extracellular matrix deposition is necessary to prevent cardiac remodelling and heart failure. In this study, we hypothesized that hesperetin, a flavanone that elicits the activation of Nrf2 signalling and thereby suppresses oxidative stress, mediated pathological cardiac hypertrophy progression. A cardiac hypertrophy model was established with subcutaneous injection of isoproterenol in male Wistar rats. Oxidative stress markers, antioxidant defense status, and its upstream signalling molecules were evaluated to discover the impacts of hesperetin in ameliorating cardiac hypertrophy. Our results implicate that hesperetin pretreatment resulted in the mitigation of oxidative stress by upregulating antioxidant capacity of the heart. This curative effect might be owing to the activation of the master regulator of antioxidant defense system, known as Nrf2. Further, analysis of Nrf2 revealed that hesperetin enhances its nuclear translocation as well as the expression of its downstream targets (GCLC, NQO1, and HO-1) to boost the antioxidative status of the cells. To support this notion, in vitro studies were carried out in isoproterenol-treated H9c2 cells. Immunocytochemical analysis showed augmented nuclear localization of Nrf2 implicating the action of hesperetin at the molecular level to maintain the cellular redox homeostasis. Thus, it is conceivable that hesperetin could be a potential therapeutic candidate that enhances Nrf2 signalling and thereby ameliorates pathological cardiac remodelling.

LOX-1, the Common Therapeutic Target in Hypercholesterolemia: A New Perspective of Antiatherosclerotic Action of Aegeline.

BACKGROUND: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is the major receptor for oxidized low-density lipoprotein (Ox-LDL) in the aorta of aged rats. Ox-LDL initiates LOX-1 activation in the endothelium of lipid-accumulating sites of both animal and human subjects of hypercholesterolemia. Targeting LOX-1 may provide a novel diagnostic strategy towards hypercholesterolemia and vascular diseases. HYPOTHESIS: This study was planned to address whether aegeline (AG) could bind to LOX-1 with a higher affinity and modulate the uptake of Ox-LDL in hypercholesterolemia. STUDY DESIGN: Thirty-six Wistar rats were divided into six groups. The pathology group rats were fed with high-cholesterol diet (HCD) for 45 days, and the treatment group rats were fed with HCD and aegeline/atorvastatin (AV) for the last 30 days. In vivo and in vitro experiments were carried out to assay the markers of atherosclerosis like Ox-LDL and LOX-1 levels. Histopathological examination was performed. Oil Red O staining was carried out in the IC-21 cell line. Docking studies were performed. RESULTS: AG administration effectively brought down the lipid levels induced by HCD. The lowered levels of Ox-LDL and LOX-1 in AG-administered rats deem it to be a potent antihypercholesterolemic agent. Compared to AV, AG had a pronounced effect in downregulating the expression of lipids evidenced by Oil Red O staining. AG binds with LOX-1 at a higher affinity validated by docking. CONCLUSION: This study validates AG to be an effective stratagem in bringing down the lipid stress induced by HCD and can be deemed as an antihypercholesterolemic agent.

Morinda citrifolia mitigates rotenone-induced striatal neuronal loss in male Sprague-Dawley rats by preventing mitochondrial pathway of intrinsic apoptosis.

OBJECTIVES: Parkinson disease (PD) is a neurodegenerative disorder affecting mainly the motor system, as a result of death of dopaminergic neurons in the substantia nigra pars compacta. The present scenario of research in PD is directed to identify novel molecules that can be administered individually or co-administered with L-Dopa to prevent the L-Dopa-Induced Dyskinesia (LID) like states that arise during chronic L-Dopa administration. Hence, in this study, we investigated whether Morinda citrifolia has therapeutic effects in rotenone-induced Parkinson's disease (PD) with special reference to mitochondrial dysfunction mediated intrinsic apoptosis. METHODS: Male Sprague-Dawley rats were stereotaxically infused with rotenone (3 µg in both SNPc and VTA) and co-treated with the ethyl acetate extract of Morinda citrifolia and levodopa. RESULTS: The results revealed that rotenone-induced cell death was reduced by MCE treatment as measured by decline in the levels of pro-apoptotic proteins. Moreover, MCE treatment significantly augmented the levels of anti-apoptotic Bcl2 and blocks the release of cytochrome c, thereby alleviating the rotenone-induced dopaminergic neuronal loss, as evidenced by tyrosine hydroxylase (TH) immunostaining in the striatum. DISCUSSION: Taken together, the results suggest that Morinda citrifolia may be beneficial for the treatment of neurodegenerative diseases like PD.

Beneficial antioxidative effect of the homeopathic preparation of Berberis vulgaris in alleviating oxidative stress in experimental urolithiasis.

BACKGROUND: Oxidative stress is a major mediator in the pathophysiology of several kidney diseases. The cellular damage is mediated by an alteration in the antioxidant status, which increases the concentration of reactive oxygen species (ROS) in the stationary state (oxidative stress). Therefore, interventions favoring the scavenging and/or depuration of ROS should attenuate or prevent the oxidative stress, thereby safeguarding the kidneys against damage. In this sense, this study attempts to evaluate the extent of oxidative stress in experimental urolithiasis by measuring some parameters of oxidant stress and antioxidant defenses in rat kidneys, before and after Berberis vulgaris homeopathic preparation supplementation, and to assess the role, if any, of homeopathic treatment in mitigating free radical toxicity in kidney stone disease. METHODS: Rat model of urolithiasis was established by administering 0.75% ethylene glycol (EG) in drinking water, and the effects of a homeopathic preparation of B. vulgaris root bark (HPBV) on the renal antioxidative defense system as well as on potent markers of free radical activities were investigated. RESULTS: HPBV brought about an augmentation in the activities of enzymatic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase and improved the nonenzymatic antioxidants, e.g., tocopherol, ascorbic acid, and glutathione. HPBV ameliorated the malondialdehyde and protein carbonyl levels and restored renal thiols almost completely. CONCLUSION: Thus, it is shown that HPBV acts as a renoprotective remedy in alleviating the renal calculi-associated oxidative damage by upregulating the antioxidant status.

Preliminary investigation on ultra high diluted B. vulgaris in experimental urolithiasis.

PURPOSE: The study focuses on the anti-urolithiasis potential of ultra-diluted homeopathic potency of Berberis vulgaris (B. vulgaris) root bark, commonly used in homeopathic system to treat renal calculi. METHODOLOGY: B. vulgaris root bark (200c, 20 µl/100 g body weight/day, p.o, for 28 days) was tested in an animal model of urolithiasis. Urolithiasis was induced in male Wistar rats by adding 0.75% ethylene glycol (EG) to drinking water. Urine and serum samples were analyzed for calcium, magnesium, phosphorus, uric acid and creatinine. Enzymic makers of renal damage (alkaline phosphatase, lactate dehydrogenase, leucine aminopeptidase and γ-glutamyl transpeptidase) were assessed in kidney and urine. Renal tissues were analyzed for oxalate content. RESULTS: Administration of EG to rats increased the levels of the stone-forming constituents calcium, phosphorus and uric acid, in urine. Levels were normalized by B. vulgaris treatment. The decrease in the urolithiasis inhibitor magnesium in urine was prevented by treatment with B. vulgaris. Serum creatinine levels were largely normalized by B. vulgaris treatment. Hyperoxaluria induced renal damage was evident from the decreased activities of tissue marker enzymes and an apparent escalation in their activity in the urine in control animals; this was prevented by B. vulgaris treatment. CONCLUSION: Homeopathic B. vulgaris root bark has strong anti-urolithiasis potential at ultra-diluted dose.

Neuroprotective potential of epigallo catechin-3-gallate in PC-12 cells.

Oxidative stress is a major player in aging and neurodegenerative disorders. Macromolecular damage occurs as a result of oxidative stress that affects the mitochondria. Mitochondrial damage leads to cell death by apoptosis or necrosis. EGCG is a tea polyphenol that protects the cells against oxidative stress. Neuroprotective potential of EGCG was tested against H(2)O(2) induced oxidative stress in PC-12 cells. PC-12 cells were grown in tissue culture flasks. Oxidative stress was induced by adding H(2)O(2) to the cells. EGCG was also added and the cell death was assessed using MTT assay. Oxidative stress was assessed by protein carbonyl and thiol status. Mitochondrial membrane potential was studied using JC-1 staining. TNF-α levels were assessed using ELISA. H(2)O(2) increased the protein carbonyl content and reduced the thiol status in the PC-12 cells. Cell death was increased in H(2)O(2) treated cells as shown by MTT assay. Mitochondrial membrane potential was also decreased along with increase in TNF-α level in H(2)O(2) treated cells. EGCG brought about an increase in the cellular thiol status and decreased the protein carbonyl content in the PC-12 cells. Cell death was attenuated by EGCG treatment along with an increase in mitochondrial membrane potential and decrease in TNF-α level. EGCG conferred its antioxidant potential to PC-12 cells as evident by decreased protein damage. Mitochondrial membrane potential was improved along with a decrement in the cell death in PC-12 cells. EGCG acts as a good neutraceutical antioxidant to render neuroprotectivity to PC-12 cells.

Impact of epigallo catechin-3-gallate on acetylcholine-acetylcholine esterase cycle in aged rat brain.

Neurotransmission plays an important role in communication of messages in brain. Cholinergic alterations during aging are associated with learning and memory. Neurotransmitters and enzymes that influence these neurotransmitters are significant in age-associated memory. Neurotransmitters like acetylcholine, serotonin and dopamine levels were studied. Kinetics of acetylcholine esterase was studied. There was an alteration in km and Vm values which was brought back to near-normalcy by EGCG. Behavioural changes were assessed by radial maze experiment. EGCG, a good neuroprotective drug proved to alleviate the behavioural alterations in aged rat brain. Acetylcholine esterase was partially purified from rat brain and assayed in vitro. Several modifiers like EGCG and donepezil were added in silico and the activity of the enzyme was calculated. EGCG increased the activity when compared to negative control, donepezil. Using bioinformatics tools EGCG, acetylcholine and donepezil were docked with acetylcholine esterase. EGCG formed a good docking-complex with the enzyme. Thus, it shall be hypothesized that the neuroprotective activity of EGCG might be due to its influence on cholinergic neurotransmission thereby improving the cognitive functions of the brain.

Attenuation of the inflammatory changes and lipid anomalies by epigallocatechin-3-gallate in hypercholesterolemic diet fed aged rats.

Epidemiological studies suggest that even in the absence of other risk factors advanced age itself significantly increases cardiovascular morbidity. Age aggravated inflammatory activity further plays a central role in the pathogenesis of atherosclerosis and its complications. EGCG, a major flavonoid present in green tea extract has been proved to be useful in lowering cholesterol levels thereby slowing down the progression of cardiovascular diseases in young animals. Thus, the endeavor of this study was to assess the impact of high-cholesterol diet on aging and vice versa and to exploit the potential of EGCG to combat age-associated hypercholesterolemia and mitigate inflammation. Male albino rats of Wistar strain (3 months-young and 24 months old-aged) were used in this study. Hypercholesterolemia was induced by the diet comprising of the normal rat chow supplemented with 4% cholesterol and 1% cholic acid. EGCG (100 mg/kg body weight/day) was given orally for 30 days. The results revealed abnormally elevated lipid levels, marker enzymes and inflammatory markers in serum of aged hypercholesterolemic rats when compared to young hypercholesterolemic rats, while treatment with EGCG partially reversed these aberrations. The present work demonstrates the inflammatory responses in hypercholesterolemic atherogenesis during aging and further underscores the salubrious role played by the EGCG in attenuating the inflammatory and lipid anomalies.

Mitochondrial alterations in aging rat brain: effective role of (-)-epigallo catechin gallate.

Aging is a multi-factorial process which involves deprivation in body's metabolism. Brain mitochondria are prone to oxidative damage owing to their high metabolic rate. The decline in antioxidant system during aging augments the neuronal damage to mitochondrial components like antioxidant system, Kreb's cycle enzymes and electron transport chain complexes. Since brain is an organ rich in fatty acids, lipid peroxidation products like hydroxynonenal are predominant. Those lipid peroxidation products conjugate with amino acids to form adducts which alter their structural and functional properties. Epigallo catechin gallate is a potent antioxidant which is rich in green tea extract. This study elucidated the antioxidant potential of epigallo catechin gallate to counteract the mitochondrial oxidative damage in brain. The study comprised of young (3-4 months old; 150+/-20 g) and aged (above 24 months; 420+/-20 g) male albino rats of Wistar strain in Groups I and II. Groups III and IV comprised of young and aged rats supplemented with epigallo catechin gallate (2mg/kg body weight) for 30 days. Antioxidants, Kreb's cycle enzymes and electron transport chain complexes were assayed in the mitochondrial fraction. Hydroxynonenal expression was carried out using immunohistochemical analysis. Epigallo catechin gallate supplementation decreased the expression of hydroxynonenal in aged brain, up-regulated the antioxidant system and augmented the activities of Kreb's cycle enzymes and electron transport chain complexes in aged brain mitochondria thus proving its antioxidant potential at the level of mitochondria.

Senescence mediated redox imbalance in cardiac tissue: Antioxidant rejuvenating potential of green tea extract.

OBJECTIVE: The activities and capacities of antioxidant systems of tissue cells are declined during aging, leading to the gradual loss of pro-oxidant/antioxidant balance and accumulation of oxidative damage. Hence, the present study evaluated the role of green tea extract (GTE), rich in polyphenols, in combating age-associated macromolecular damage in rat cardiac tissue. METHODS: The antioxidant defense system, lipid peroxidation, protein oxidation, and redox status in heart tissue were studied using young and aged rats. RESULTS: Significant increases in lipid peroxidation, protein carbonyls, and marked decreases in glutathione redox status, protein thiols, and activities of enzymatic and non-enzymatic antioxidants were observed in aged rats compared with young rats. Supplementation of GTE (100 mg/kg of body weight per day) orally for 30 days ameliorated these changes significantly. CONCLUSION: This study accredits GTE's antioxidant rejuvenating potency and its role in the amelioration of senescence-mediated redox imbalance in aged rat cardiac tissue.

Attenuation of senescence-induced oxidative exacerbations in aged rat brain by (-)-epigallocatechin-3-gallate.

Aging is a complex biological phenomenon which involves free radicals and oxidative stress. Brain is more susceptible and vulnerable to oxidative damage due to its high-polyunsaturated fatty acid content and high rate of aerobic metabolism. Since the antioxidant defense system is diminished during aging, antioxidant supplementation might be a protective strategy against age-associated oxidative damage. The present study evaluates the antioxidant potential of (-)-epigallocatechin-3-gallate (EGCG), a major polyphenol present in green tea against age-associated oxidative damage in rat brain. Male albino rats of Wistar strain were used in the study. Group I (young) and Group II (aged) rats received saline alone orally for 30 days. Group III (young) and Group IV (aged) rats received EGCG (2mg/kg body weight/day) orally for 30 days. Antioxidant status and oxidative damage were assessed. EGCG brought about an augmentation in the activities of enzymic antioxidants like superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and improved the non-enzymic antioxidants like tocopherol, ascorbic acid and glutathione. EGCG ameliorated the malondialdehyde and protein carbonyl levels. Thus, EGCG has emerged out as a good antioxidant neutraceutical and a neuroprotective agent in alleviating the age-associated oxidative damage in aged rat brain.

Repletion of antioxidant status by EGCG and retardation of oxidative damage induced macromolecular anomalies in aged rats.

Ageing is defined as the loss of ability to maintain cellular homeostasis of an organism associated with the free radical-elicited oxidative damage to cellular macromolecules. The aim of this study was to evaluate the efficacy of epigallocatechin-3-gallate (EGCG), the key component of green tea catechins on attenuating the age associated oxidative perturbances by assessing the redox status in mitotic (liver) and post-mitotic (skeletal muscle) tissues of young and aged rats. From the results, we observed an increase in the marker for oxidative damage such as lipid peroxidation and protein carbonyl content in aged rats, when compared to young rats. This was accompanied with decreased levels of both enzymic and non-enzymic antioxidants and redox index. On supplementation with EGCG (100 mg/kg body weight by oral gavage for 30 days), the levels of lipid peroxidation and protein carbonyl content were significantly decreased in aged rats, possibly by enhancing the GSH redox status, and both enzymic and non-enzymic antioxidants status. In conclusion, this study supports the beneficial effect of EGCG in alleviating oxidative disturbances in ageing and retard the age associated derangements in both mitotic and post-mitotic tissues.

Transcriptional repression mediated by 45-kDa calcium oxalate monohydrate binding protein.

BACKGROUND: This study was done to investigate the DNA binding ability of a diagnostic biomarker, 45-kDa calcium oxalate monohydrate (COM) binding protein, isolated from human kidney and its effect on transcription. METHODS: The 45-kDa COM binding protein was isolated and purified from human kidney. The subcellular localization of the protein and the amino acid composition of the protein were analyzed. Oxalate-binding activity in the presence or absence of DNA was determined. The possibility of forming DNA-protein adducts was checked by diethylaminoethyl (DEAE)-Sephadex column chromatography. The effect of the protein on in vitro transcription was also studied. RESULTS: The isolated 45-kDa protein was found to be basic in nature and its AACompIdent analysis showed it to be related to known transcription factors. The protein was found to be present in kidney cytosol and nucleus. The decreased oxalate binding activity in the presence of the DNA, and the shift in the DEAE-Sephadex elution profile established the DNA-binding ability of the protein. The in vitro transcription assay demonstrated the repression effect of the protein on gene expression during hyperoxaluria. CONCLUSIONS: Transcriptional repression by the 45-kDa COM binding protein in an in vitro transcription assay system was reduced in the presence of oxalate. Hence, altered expression of certain genes involved in the prognosis of urolithiasis might be mediated by this 45-kDa protein.

Investigation on the early events of apoptosis in senescent erythrocytes with special emphasis on intracellular free calcium and loss of phospholipid asymmetry in chronic renal failure.

BACKGROUND: The pathophysiological link between increased blood concentrations of factors responsible for the derangement and erythrocyte membrane functions in chronic renal failure (CRF) patients are not thoroughly elucidated. We studied the erythrocyte characteristics and phospholipid asymmetry loss in CRF patients with different grades of uremia and also examined the involvement of intracellular free Ca(2+) in early events of apoptosis in uremic erythrocytes. METHODS: The studied population consisted of 90, age and sex matched control subjects (Group I) and 238 CRF cases divided into 3 groups (Group II, III and IV) according to urea concentrations and complexity of secondary complications. Erythrocyte membrane fluidity determined by binding of MC540. Intracellular free Ca(2+) concentration was determined by the 2-wavelength method by using fluorescent calcium-sensitive probe FURA-2AM. Measurement of erythrocyte phosphatidylserine exposure by flow cytometry using Annexin V-FITC. RESULTS: Cholesterol shedding increased with increasing severity of uremic complications. Erythrocytes from Group II show mild echinocyte or formation of spicules on the erythrocyte membrane surface whereas in Group III and IV they were echinocytic. Binding of MC540 was significantly higher with progression of uremic complications. Surface charge of uremic erythrocyte membrane was significantly reduced when compared with control subjects. Intracellular free Ca(2+) was positively correlated with binding of MC540 and surface hydrophobicity. The phosphatidylserine exposure of erythrocytes was significantly higher (p<0.001) in uremic patients when compared with controls. CONCLUSIONS: Phosphatidylserine (PS) exposure erythrocytes were significantly increased in uremic patients when compared with controls. Uremic complications predisposes to membrane damages in erythrocytes.

Counteracting adriamycin-induced oxidative stress by administration of N-acetyl cysteine and vitamin E.

Adriamycin (ADR), a cytotoxic antineoplastic drug, is used in the treatment of various solid tumors. However, its efficacy continues to be challenged by significant toxicities including nephrotoxicity. In the present study, the effects of N-acetyl cysteine (NAC) and vitamin E, known antioxidants, were investigated on ADR-induced peroxidative damage in rat kidney. Adult male albino rats of Wistar strain were administered ADR as a single dose (10 mg/kg body weight, i.v.). Histopathological studies indicated that ADR-treated kidney sections show focal tubular necrosis and casts. ADR-injected rats showed a significant decline in the activities/levels of enzymic antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione-S-transferase) and non-enzymic antioxidants (thiols, vitamin C and vitamin E) with high malondialdehyde levels. The extent of nephrotoxicity was evident from the increased activities of urinary marker enzymes (alkaline phosphatase, lactate dehydrogenase and gamma-glutamyltransferase). Treatment with NAC and vitamin E (50 mg/kg b.w., i.p.) 1 day prior to ADR administration maintained near normal activities of the enzymes, significantly reduced lipid peroxidation and prevented the necrosis caused by ADR, thereby proving to be an effective thiol replenishing agent and antioxidant.

Oral L-arginine supplementation ameliorates urinary risk factors and kinetic modulation of Tamm-Horsfall glycoprotein in experimental hyperoxaluric rats.

BACKGROUND: Oral supplementation of l-arginine (l-arg) is found to be beneficial in many kidney disorders. We determined whether l-arg supplementation safeguards the renal epithelial cell damage induced by hyperoxaluria with excretion of urinary marker enzymes and lithogenic salts with special reference to Tamm-Horsfall glycoprotein (THP). METHODS: Hyperoxaluria was induced by 0.75% ethylene glycol (EG) in drinking water. l-Arg was co-supplemented at the dose of 1.25 g/kg b.w. orally for 28 days. At the end of experimental period, 24-h urine samples were collected in all the experimental groups. Isolation and purification of THP was carried in rat urine and were subjected to spectrophotometric crystallization assay and calcium-(14)C-oxalate binding studies. Determination of the lithogenic risk factors like calcium, oxalate, phosphorus, citrate, and marker enzymes such as lactate dehydrogenase (LDH) and gamma-glutamyltransferase (gamma-GT) were carried out in the collected urine sample. RESULTS: Urinary excretion of calcium and oxalate was significantly increased in EG-treated rats. In l-arg supplemented hyperoxaluric rats, these concentrations were significantly (p<0.001) decreased when compared to that of hyperoxaluric rats, and were moderately elevated from that of control rats. The activities of urinary marker enzymes, both LDH and gamma-GT were 2-fold increased in EG-treated rats, when compared to control rats, but these values were maintained near normal in l-arg supplemented EG-treated rats. Citrate excretion was enhanced in the l-arg co-supplemented hyperoxaluric rats. In spectrophotometric crystallization assay system, l-arg supplemented rat THP showed inhibition in nucleation and aggregation phases, whereas EG-treated rat THP showed promotion of both calcium oxalate nucleation and aggregation phases. In calcium-(14)C-oxalate binding assay, THP derived from hyperoxaluric rats exhibited 2-fold increase (p<0.001) in the Ca*Ox binding when compared to control and l-arg supplemented animals. CONCLUSIONS: l-Arg could act as a potent antilithic agent, by increasing the level of citrate in the hyperoxaluria-induced rats and decreasing calcium oxalate binding to the THP. l-Arg also effectively prevents the deposition of calcium oxalate crystals by curtailing the renal epithelial damage and protein oxidation as evidenced by the normal activities of urinary marker enzymes in l-arg supplemented hyperoxaluric rats.

Beneficial effect of vitamin E supplementation on the biochemical and kinetic properties of Tamm-Horsfall glycoprotein in hypertensive and hyperoxaluric patients.

BACKGROUND: This study aimed to assess the therapeutic efficacy of oral vitamin E supplementation on the biochemical and kinetic properties of Tamm-Horsfall glycoprotein (THP) in hypertensive and hyperoxaluric patients. METHODS: Newly detected hypertensives (n = 200) and stone formers (n = 200) were each subdivided into two groups. One group (n = 100) was administered the antioxidant vitamin E at 400 mg/day given as an oral supplement along with standard therapeutic drugs for hypertension and hyperoxaluria and the patients were followed for a period of 9 months. The other group (n = 100) did not receive vitamin E (placebo controls). Age and sex-matched controls (n = 100) were monitored simultaneously. THP was isolated from 24 h urine samples before and at the end of every third month during a period of 9 months from the vitamin E-treated hypertensive and hyperoxaluric groups. THP samples were also collected from control subjects, and at the end of the ninth month from placebo controls. The isolated protein was assessed for purity by SDS-PAGE. The purity-checked proteins were subjected to spectrophotometric crystallization assay, calcium oxalate (CaOx) crystal interaction studies, and biochemical analysis of sialic acid, thiol and carbonyl content. Plasma superoxide, hydroxyl radical, hydrogen peroxide and vitamin E levels as well as superoxide dismutase and catalase activities were also monitored. RESULTS: The THP from the hypertensive and hyperoxaluric subjects exhibited a significant promoting effect on the nucleation and aggregation phases and caused a concomitant increase in CaOx crystal interaction. The altered kinetic properties of THP in these subjects were strongly associated with increased carbonyl content and with decreased thiol and sialic acid contents. Oral administration of vitamin E to these patients caused near normalization of these biochemical alterations and satisfactorily restored the kinetic properties of THP to near normal activity. At the end of 9 months, THP isolated from placebo controls (hypertensive and hyperoxaluric) showed highly aggregated calcium oxalate monohydrate crystals as observed by light microscopy. In contrast, vitamin E-supplemented patients showed CaOx dihydrate crystals that were similar to control THP. There was an imbalance in the oxidant and antioxidant levels. For the oxidants, superoxide, hydrogen peroxide and hydroxyl radical levels were increased, and for the antioxidants, there was loss of antioxidant enzyme activities and a decline in plasma vitamin E level in both hypertensive and hyperoxaluric patients. Supplementary antioxidant (vitamin E) corrected this imbalance to near normal conditions. CONCLUSION: We hypothesize that the loss of THP inhibitory activity in the hypertensive and hyperoxaluric patients in a crystallizing medium is mediated primarily by oxidative damage to this protein. The possible occurrence of renal stones in essential hypertensive subjects, and the risk of recurrence in hyperoxaluric subjects, may be explained by oxidative damage to renal tissues that remained unchecked by standard drug therapies. The normalization of the kinetic properties of THP following vitamin E supplementation is in support of our hypothesis.