Effect of embelin on lithium-induced nephrogenic diabetes insipidus in albino rats

Transcription

1 Asian Paifi Journal of Tropial Disease (23)S729-S733 S729 Contents lists available at SieneDiret Asian Paifi Journal of Tropial Disease journal homepage: Doument heading Effet of embelin on lithium-indued nephrogeni diabetes insipidus in albino rats Ashish K Sahu,2, MK Gautam 2, Pradeep T Deshmukh, Lokendra S Kushwah, Narendra Silawat, Zafar Akbar, MS Muthu 2 Department of Pharmaology and Toxiology, BR Nahata College of Pharmay, Mandsaur-458, India 2 Department of Pharmaology, Faulty of Mediine, Institute of medial sienes, Banaras Hindu University, Varanasi-225, India ARTICLE INFO Artile history: Reeived 5 April 2 Reeived in revised form 27 April 2 Aepted 28 June 2 Available online 28 June 2 Keywords: Embelia ribes Embelin Lithium Nephrogeni diabetes insipidus Nephrotoxiity ABSTRACT Objetive: To evaluate the nephroprotetive and anti-polyuri role of embelin on lithium indued nephrogeni diabetes insipidus (NDI) in albino rats. Methods: NDI indued by lithium hloride (4 meq/kg/day, i.p. for 6 days) whih leads to huge amount of urine exretion. After indution of NDI, embelin (5 and mg/kg) was administered orally, one daily for 2 day in rats and N-aetyl ysteine (mg/kg, twie daily, i.p.) was used as a standard drug for treatment of NDI. The body weight, urine protein, urine reatinine, plasma reatinine, blood urea nitrogen were assessed at, 7, 4 and 2 day. At the end of the study glutathione (GSH) ontent in kidney was assessed and histopathology of kidney was performed. Results: Embelin 5 and mg/ kg showed inrease in the body weight and derease in plasma and urine reatinine, blood urea nitrogen levels, and urine protein level. Embelin ats as a potent antioxidant; it inreases the level of glutathione in kidney. Histopathologial examination of the kidney indiated that embelin 5 and mg/kg were redued the vasular degeneration of tubules as well as slight degeneration and dilatation of renal tubules, however N-atyl ysteine (NAC) treated rats showed normal glomeruli and renal tubule with slight degeneration. Conlusions: Embelin seemed to be effetive in NDI by its predominant effet on promoting antioxidant status and derease the urine exretion may be due to the bloking of sodium hannels.. Introdution Diabetes insipidus is a syndrome in whih huge amount of urine output, low urine-speifi gravity, high plasma osmolality and high plasma sodium. It is produe when the kidney is not able to ontrol plasma osmolality due to a defet in the ation of arginine vasopressin (AVP)[]. Nephrogeni diabetes insipidus (NDI) is a type of diabetes insipidus, manifested by a lak of response of the olleting dut to irulating anti-diureti hormone (ADH) and ause frequent and exessive urination (Polyuria). Prolonged polyuria of any ause an result in partial NDI as a onsequene of dissolution of the intrarenal solute gradient that failitates renal water resorption. NDI results when the kidneys are unable to respond to ADH. Polyuria is a frequent ompliation in long-term use of lithium therapy. The Corresponding author: Dr. Pradeep T Deshmukh, Department of Pharmaology and Toxiology, BR Nahata College of Pharmay, Mandsaur-458, India Tel: Fax: sahuaship@gmail.om kidney ability to respond to ADH an be impaired by drugs like lithium and by hroni disorders inluding polyysti kidney disease, sikle ell disease, kidney failure, partial blokage of the ureters, and inherited geneti disorders[2]. Polyuria is a frequent ompliation in long-term use of lithium therapy. Lithium is a frequent therapeuti agent used to treat patients with various mani depressive illness. However, this mediation has a narrow therapeuti index and, therefore, frequent side effets. It has been assoiated with several forms of renal injury, whih is impaired urinary onentrating ability. It is estimated to be present in at least 5% individuals on hroni lithium therapy. Initially, the dereased urinary onentrating ability is largely reversible after essation of lithium. However, with ontinued treatment, this defet translates into irreversible polyuria and polydipsia in up to 2% of population, whih is resistant to the ations of arginine vasopressin (AVP). This funtional lesion is assoiated with a hroni foal interstitial fibrosis predominantly in the medullary region of the kidney, whih may be progressive, leading to renal failure[3, 4]. Embelin (3-undeyl 2,5-dihydroxy,,4-benzoquinone) is an naturally ourring alkyl substituted hydroxy

2 S73 Ashish K Sahu et al./asian Paifi Journal of Tropial Disease (23)S729-S733 benzoquinone obtained from dried fruits of Embelia ribes (Myrsinaeae), is reported to be an effetive analgesi and anti-fertility agent. Embelin is reported to posses antibaterial, wound healing, free radial savenging and antioxidant ativity, anti-fertility, anti-implantation, antitumour, anti-inflammatory and analgesi and anti-uler property[5, 6]. It is also reported to impair the inflammatory signaling though inhibition of nulear fator kappa B (NFkappa B) ativity and benefiiary effet on aeti aidindued olitis model[7]. 2. Materials and methods 2.. Drugs and hemials Lithium hloride and embelin purhased from Loba hemie, Mumbai and Natural Remedies, Benguluru, India, respetively. All other hemial and reagents used were of analytial grade Animals Male albino rats (5-2 g) of wistar strain were proured from animal house, BRNCP, Mandsaur and maintained under laboratory ondition of 2 h light/2 h dark yle, 25 暲 3 曟 and 35%-6% humidity. The rats were maintained at standard how diet and water ad libitium. Animals desribed as fasted had been deprived of food for 6 hr but has been allowed to free aess to water. Priniples of laboratory animal are (NIH publiation no , revised 985) guidelines were followed. Approval from the Institutional Animal Ethial Committee was taken prior to the experimental (2/MPh/9/ IAEC/BRNCP/8-9/Mandsaur) Indution of NDI and treatment protool Lithium hloride (4 meq/kg/day or mg/kg/day, one a day) dissolved in normal saline and injeted intraperitonialy for 6 days[6]. Animals with profound polyuria were seleted for study. Daily urine output was assessed for onfirmation of NDI. The drug treatment was started after 6th day of lithium hloride administration. Normal ontrol rats were reeived normal saline (i.p.), Lithium treated ontrol rats were reeived normal saline (i.p.), other NDI animals treated with embelin 5 and mg/kg dose in % tween 8 vehile, orally[6]. Standard ontrol rats were reeived N-aetyl ysteine (mg/kg i.p. twie a day)[8] Sample olletion Blood was olleted by retro-orbital method from the eye. The withdrawn blood was entrifuged with 5 r/min for min by using entrifuge and plasma was olleted and used for the determination of reatinine and blood urea nitrogen. Urine was olleted with the help of metaboli ages in graduated tubes and volume was measured. Biohemial parameters like urine protein and urine reatinine were assessed. After 4 days of the treatment the animals were euthanized by over dose of ether anaesthesia and the kidney was olleted for the determination of GSH ontent and histopathology Effet on body weight after indution of NDI The above parameters were measured on the, 7, 4 and 2 day of the experiment. Eah rat was individually weighed using standard rat weighing mahine and their respetive weights were noted down Biohemial parameters Plasma reatinine and blood urea nitrogen (Transasia Biomedials); urine protein and urine reatinine were measured using a ommerial kit (Span Diagnostis) Estimation of glutathione level in kidney Kidney was isolated and mixed with saline (2 ml), further it was homogenized in 5 ml of old KCl (.5%) and preipitated with trihloroaeti aid (.5 ml). From this,.5 ml of supernatant was taken and in this 2 ml phosphate buffer (ph 8.9) was added. Subsequently,. ml of DTNB (.4%) solution was added and absorbane was taken against blank prepared at 42 nm[9] Statistial analysis Statistial omparison was performed using either unpaired t test or one way analysis of variane (ANOVA) and for multiple omparisons versus ontrol group was done by Dunnett s test. 3. Results 3.. Effet of embelin on body weight after indution of NDI Little or no hange was observed on body weight between the lithium-treated and embelin/n-aetyl ysteine treated animals from day to 2 day of study treatments Effet of embelin on plasma reatinine and blood urea nitrogen after indution of NDI The effet of embelin on plasma reatinine and blood urea nitrogen in lithium hloride indued NDI is presented in Table and 2 respetively. The result showed that the lithium hloride inrease the plasma reatinine and blood urea nitrogen in lithium ontrol animals while, treatment with embelin 5 and mg/kg was signifiantly derease (P<.) plasma reatinine and blood urea nitrogen on 4 day ompared to lithium ontrol N-aetyl ysteine also derease the level (P<.) on 4 day (Table and 2) Effet of embelin on urine volume, urine protein and urine reatinine after indution of NDI Urine volume was inreased after lithium treatment in all groups of the animal ompared to normal ontrol group and after treatment with embelin/n-aetyl ysteine urine volume was signifiantly dereased (P<.) after 4 days treatment (Figure ). Urine protein and urine reatinine level signifiantly inrease in lithium treated rats. After treatment

3 Ashish K Sahu et al./asian Paifi Journal of Tropial Disease (23)S729-S733 S73 Table. Effet of embelin on plasma reatinine (mg/dl) after indution of NDI in rats. Groups Before treatment After treatment Normal ontrol.4478 暲 暲 暲 暲.63 Lithium ontrol.425 暲.34.7 暲.3.23 暲.5.46 暲. Lithium + Embelin 5 mg/kg.4767 暲.2.42 暲.8.77 暲.6.7 暲.6 Lithium + Embelin mg/kg.4833 暲.5.7 暲.7.7 暲.6.7 暲.5 Lithium + N-aetyl ysteine mg/kg.437 暲.5.53 暲.5.74 暲..7 暲. Results are mean 暲 SEM (n=6). P<. ompared to respetive normal ontrol group and P<. ompared to respetive lithium ontrol Table 2. Effet of embelin on blood urea nitrogen (mg/dl) after indution of NDI in rats. Groups Before treatment After treatment Normal ontrol 暲 暲 暲 暲.665 Lithium ontrol 暲 暲 暲 暲.7 Lithium + Embelin 5 mg/kg 暲 暲 暲 暲.3 Lithium + Embelin mg/kg 23.4 暲 暲 暲 暲.3 Lithium + N-aetyl ysteine mg/kg 暲 暲 暲 暲.3 Results are mean 暲 SEM (n=6). P<. ompared to respetive normal ontrol group and P<. ompared to respetive lithium ontrol group 6.4 Urine volume(ml/24 hour) b Normal ontrol Lithium ontrol Lithium+Embelin 5mg/kg Lithium+Embelin mg/kg Lithium+N-aetyl ysteine mg/kg Figure. Effet of embelin on urine volume (ml/24 hour). Results are mean 暲 SEM (n=6), P<. ompared to respetive normal ontrol group and b P<., P<. ompared to respetive lithium ontrol Urine protein(gm/24 hour) Urine reatinine(mg/dl) Normal ontrol Lithium ontrol Lithium+Embelin 5mg/kg Lithium+Embelin mg/kg Lithium+N-aetyl ysteine mg/kg Figure 2. Effet of embelin on urine reatinine (mg/dl). Results are mean 暲 SEM (n=6), P<. ompared to respetive normal ontrol group and P<. ompared to respetive lithium ontrol Glutathione(umol/gm) Normal ontrol Lithium ontrol Lithium+Embelin 5mg/kg Lithium+Embelin mg/kg Lithium+N-aetyl ysteine mg/kg Figure 3. Effet of embelin on urine protein (gm/24 hour). Results are mean 暲 SEM (n=6), P<. ompared to respetive normal ontrol group and P<. ompared to respetive lithium ontrol with embelin/n-aetyl ysteine, the level of urine protein Normal ontrol Lithium+Embelin 5mg/kg Lithium+N-aetyl ysteine mg/kg Lithium ontrol Lithium+Embelin mg/kg Figure 4. Effet of embelin on glutathione level ( 毺 mol/gm). Results are mean 暲 SEM (n=6), P<. ompared to respetive normal ontrol group and P<. ompared to respetive lithium ontrol and urine reatinine was signifiantly dereased (P<.)

4 S732 Ashish K Sahu et al./asian Paifi Journal of Tropial Disease (23)S729-S733 on 7 and 4 day (Figure 2 and 3) Effet of embelin on kidney glutathione after indution of NDI Kidney glutathione level was derease in lithium indued NDI rats while, treatment with embelin 5 and mg/kg glutathione level was inreases signifiantly (P<.) in kidney tissue (Figure 4). Figure 5. Photomirographs of rat kidney (x magnifiations) A: Histopathology of normal rat kidney showing normal renal tubule (showed by hollow arrow) and glomeruli (showed by solid blak arrow); B: Histopathology of lithium ontrol group showing renal tubular damage (showed by hollow arrow) haraterized by dilatation of renal tubules and foal flattening of renal tubular epithelial ells (showed by solid blue arrow); C: Histopathology of embelin 5 mg/kg treated rats showing vasular degeneration of tubules (showed by hollow arrow) is redued; D: Histopathology of embelin mg/kg treated rats showing slight degeneration of renal tubules (showed by hollow arrow) and slight dilatation of renal tubules; E: Histopathology of N-aetyl ysteine treated rats showing normal glomeruli (showed by solid blak arrow) and renal tubule (showed by hollow arrow) with slight degeneration. 4. Disussion Prolonged use of lithium therapy may deteriorate the renal funtion even after disontinuing lithium treatment and ould indue renal failure in a slowly progressive manner and its rate of progression is related to the both duration and umulative dose of the drug. NDI is the most ommon side effet of lithium therapy, possibly resulting from down regulation of aquaporin-2 expression in the olleting

5 Ashish K Sahu et al./asian Paifi Journal of Tropial Disease (23)S729-S733 S733 dut. With long-term use, hroni tubulointerstitial nephritis and tubular miroysts are identified[]. Lithium aumulates within the olleting tubule ells, after entering the ells through sodium hannels in the luminal membrane, lithium then interferes with ability of ADH. The predominant form of renal disease assoiated with lithium therapy is tubulointerstitial nephropathy; eletron mirosope examinations have demonstrated severe lesions in the mitohondria and endoplasmi retiulum in renal tissue of patients with lithium nephrotoxiity, impliating ishaemia as one of the pathogeneti mehanism. Under suh irumstanes, any other o-morbid onditions suh as diabetes or a derease in renal blood flow may result in a further redution in tissue oxygenation and augment lithium- mediated toxiity[, 2]. In our present study treatment with lithium aused polyurea relative to the normal ontrol. Embelin 5 and mg/kg and N-aetyl ysteine showed no hange in body weight of the rats in omparison to disease ontrol group treated with lithium hloride. Administration of lithium hloride showed a signifiant rise in plasma reatinine, blood urea nitrogen, urine protein, urine reatinine and urine volume with respet to the vehile ontrol, whih learly indiates intrinsi aute renal failure[8]. From our study embelin at a dose of 5 and mg/kg rendered signifiant protetion against lithium indued nephropathy whih was evident by the dereased plasma reatinine, blood urea nitrogen levels, and urine protein. Also embelin 5 and mg/kg showed an inhibitory effet on polyuria indued by lithium. This inhibitory ation on polyuria may be due to the sodium hannel bloking ation of embelin. Cellular antioxidants are protetive against the free radials whih are deleterious for the tissue milieu. This antioxidant system was deranged after treatment with Lithium hloride. Oxidative stress and subsequently ellular damage due to lithium ould derease the glutathione level[3]. Renal medullary ells are exposed to the stress of long-term hyperosmolality and respond to this stress by aumulating ompatible organi osmolytes. Renal vasoonstrition is may be due to inreased oxidative stress as a result inreased loal prodution of superoxide hene derease in nitri oxide bioavailability[8,4]. Embelin in a dose of mg/kg ats as potent antioxidant or free radial savenger whih indues the glutathione level. The histopathologial findings of kidney shows that rats treated with lithium hloride aused diffused tubular nerosis and several dilated tubuli with intraluminal tubular ells fragments. Histopathology renal study of rat showed that embelin in different dosages signifiantly proteted the glomerular and tubular injury in omparison to negative ontrol Embelin 5 mg/kg and mg/kg in lithium treated rats redues the vasular degeneration of tubules as well as slight degeneration and dilatation of renal tubules; however N-aetyl ysteine treated rats showed normal glomeruli and renal tubule with slight degeneration. Embelin seemed to be effetive in NDI by its predominant effet on promoting antioxidant status and derease the urine exretion may be due to the bloking of sodium hannels. Further researh is needed in order to reveal moleular mehanism and mode of ation of embelin in NDI. Conflit of interest statement We delare that we have no onflit of interest. Aknowledgments The authors express sinere thanks to BR Nahata College of Pharmay, Mandsaur, India, for providing the best failities and funding for this researh work. Referenes [] Kassebaum N, Hairr J, Goldsmith W, Barwise J, Pandharipande P. Diabetes insipidus assoiated with propofol anesthesia. J Clin Anesth 28; 2: [2] Spanakis E, Milord E, Gragnoli C. AVPR2 Variants and Mutations in Nephrogeni Diabetes Insipidus: Review and Missense Mutation Signifiane. J Cellular Physiol 28; [3] Bedford JJ, Weggery S, Ellis G, MDonald FJ, Joye PR, Leader JP, Walker RJ. Lithium-indued nephrogeni diabetes insipidus: renal effets of amiloride. Clin J Am So Nephrol 28; 3(5): [4] Trepiione F, Christensen BM. Lithium-indued nephrogeni diabetes insipidus: new linial and experimental findings. J Nephrol 2; 23(6): [5] Mahendran S, Thippeswamy BS, Veerapur VP, Badami S. Antionvulsant ativity of embelin isolated from Embelia ribes. Phytomed 2; 8: [6] Thippeswamy BS, Nagakannan P, Shivasharan BD, Mahendran S, Veerapur VP, Badami S. Protetive effet of embelin from Embelia ribes Burm. against transient global ishemia-indued brain damage in rats. Neurotox Res 2; 2: [7] Thippeswamy BS, Mahendran SB, Biradar MI, Raj P, Srivastava K, Badami S, Veerapur VP. Protetive effet of embelin against aeti aid indued ulerative olitis in rats. Euro J Pharmaol 2; 654: -5. [8] Hwang GS, Yang JY, Ryu DH, Kwon TH. Metaboli profiling of kidney and urine in rats with lithium-indued nephrogeni diabetes insipidus by H-NMR-based metabonomis. Am J Physiol Renal Physiol 2; 298: [9] Ahmad M, Elnakady Y, Farooq M, Wadaan M. Lithium indued toxiity in rats: blood serum hemistry, antioxidative enzymes in red blood ells and histopathologial studies. Biol Pharm Bull 2; 34(2): [] Loh AHL, Cohen AH. Drug-indued kidney disease - pathology and urrent onepts. Ann Aad Med Singapore 29; 38: [] Bedford JJ, Leader JP, Jing R, Walker LJ, Klein JD, Sands JM, Walker RJ. Amiloride restores renal medullary osmolytes in lithium-indued nephrogeni diabetes insipidus. Am J Physiol Renal Physiol 28; 294: [2] Grünfeld JP, Rossier BD. Lithium nephrotoxiity revisited. Nature Reviews Nephrol 29; 5: [3] Khairova R, Pawar R, Salvadore G, Juruena MF, de Sousa RT, Soeiro-de-Souza MG, Salvador M, Zarate CA, Gattaz WF, Mahado-Vieira R. Effets of lithium on oxidative stress parameters in healthy subjets. Mol Med Report 22; 5(3): [4] Andreazza AC, Kauer-Sant Anna M, Frey BN. Effets of mood stabilizers on DNA damage in an animal model of mania. J Psyhiatry Neurosi 28; 33: