S. K. Singla

In vivo efficacy of Trachyspermum ammi anticalcifying protein in urolithiatic rat model.

ETHNOPHARMACOLOGICAL RELEVANCE Many medicinal plants have been employed during ages to treat urinary stones though the rationale behind their use is not well established. Recently, we have successfully purified an anticalcifying protein from the seeds of Trachyspermum ammi (L.) Sprague ex Turril (Umbelliferae) using oxalate depletion assay and deciphered its inhibitory activity against calcium oxalate crystal growth. AIM In this report, the antilithiatic activity of Trachyspermum ammi anticalcifying protein (TAP) was studied in urolithiatic rat model. METHODOLOGY Urolithiasis was induced by exposure of 0.4% ethylene glycol (EG) and 1.0% ammonium chloride (NH(4)Cl) for 9 days. The efficacy of TAP was studied in another group given same dose of EG and NH(4)Cl in addition to 2mg/kg body weight of TAP. Further, we evaluated ability of TAP to inhibit the attachment of calcium oxalate (CaO(x)) crystal in kidney tissue and studied the consequences of CaO(x) adhesion on renal functioning and tissue integrity. RESULTS The antilithiatic potential of TAP was confirmed by its ability to maintain renal functioning, reduce renal injury and decrease crystal excretion in urine and retention in renal tissues. CONCLUSIONS Thus, the present investigation suggests the potential of TAP in preventing calcium oxalate deposition and forms the basis for the development of antilithiatic drug interventions against urolithiasis.

Modulatory effects of N-acetylcysteine on hyperoxaluric manifestations in rat kidney

Hyperoxaluria is a condition where excessive oxalate is present in the urine. Many reports have documented free radical generation followed by hyperoxaluria as a consequence of which calcium oxalate deposition occurs in the kidney tissue. The present invivo study was designed to investigate the potential of N-acetylcysteine in modulating hyperoxaluric manifestation induced by sodium oxalate in the rat kidneys. Male wistar rats in one group were administered single dose of sodium oxalate (70mg/kg body weight) intraperitoneally to induce hyperoxaluric conditions and in the other group, rats were injected N-acetylcysteine (NAC) (200mg/kg body weight) intraperitoneally, half an hour after sodium oxalate dose. The treatment is for a period of 24h. N-acetylcysteine significantly reduced hyperoxaluria caused oxidative stress by reducing lipid peroxidation, restoring antioxidant enzymes activity in kidney tissue, followed by reduction in impairment of renal functioning. In addition, NAC administration reduced the number of calcium oxalate monohydrate (COM) crystals in the urine as observed under polarization microscope. Histological analysis depicted that NAC treatment decreased renal epithelial damage, inflammation and restored normal glomeruli morphology. Thus, it shows that use of an extraneous antioxidant may prove beneficial for combating the conditions of oxidative stress produced by hyperoxaluria.

A novel antilithiatic protein from Tribulus terrestris having cytoprotective potency.

Adhesion of calcium oxalate (CaOx) crystals to kidney cells is a key event in kidney stones associated with marked hyperoxaluria. As the propensity of stone recurrence and persistent side effects are not altered by surgical techniques available, phytotherapeutic agents could be useful as an adjuvant therapy. The present study is aimed at examining the antilithiatic potency of the protein biomolecules of Tribulus terrestris, a plant which is a common constituent of herbal marketed preparations to treat urolithiasis. Various biochemical methods with mass spectrometry were used to purify and characterize the purified protein. The protective potency of the protein was tested on the oxalate induced injury on renal epithelial cell lines (NRK 52E). An antilithiatic protein having molecular weight of ~ 60kDa was purified. This purified protein showed similarities with Carotenoid cleavage dioxygenase 7 (CCD7) of Arabidopsis thaliana after matching peptide mass fingerprints in MASCOT search engine. An EF hand domain was identified in CCD7 by SCAN PROSITE. Presence of an EF hand domain, a characteristic feature of calcium binding proteins and a role in the synthesis of retinol which is transported by retinol binding protein, a protein found in kidney stone matrix; of CCD7 support the role of TTP as an antilithiatic protein. The protective potency of TTP on NRK 52E was quite comparable to the aqueous extract of cystone. Our findings suggest that this purified protein biomolecule from Tribulus terrestris could open new vista in medical management of urolithiasis.

The Restrained Expression of NF-kB in Renal Tissue Ameliorates Folic Acid Induced Acute Kidney Injury in Mice

The Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) represent family of structurally-related eukaryotic transcription factors which regulate diverse array of cellular processes including immunological responses, inflammation, apoptosis, growth & development. Increased expression of NF-kB has often been seen in many diverse diseases, suggesting the importance of genomic deregulation to disease pathophysiology. In the present study we focused on acute kidney injury (AKI), which remains one of the major risk factor showing a high rate of mortality and morbidity. The pathology associated with it, however, remains incompletely known though inflammation has been reported to be one of the major risk factor in the disease pathophysiology. The role of NF-kB thus seemed pertinent. In the present study we show that high dose of folic acid (FA) induced acute kidney injury (AKI) characterized by elevation in levels of blood urea nitrogen & serum creatinine together with extensive tubular necrosis, loss of brush border and marked reduction in mitochondria. One of the salient observations of this study was a coupled increase in the expression of renal, relA, NF-kB2, and p53 genes and proteins during folic acid induced AKI (FA AKI). Treatment of mice with NF-kB inhibitor, pyrrolidine dithio-carbamate ammonium (PDTC) lowered the expression of these transcription factors and ameliorated the aberrant renal function by decreasing serum creatinine levels. In conclusion, our results suggested that NF-kB plays a pivotal role in maintaining renal function that also involved regulating p53 levels during FA AKI.

Purification and Characterization of an Anticalcifying Protein from the Seeds of Trachyspermum ammi (L.)

Till date various plants extract have been studied to reduce the incidence of urolithiasis but the identification of naturally occurring calcium oxalate (CaOx) inhibitory biomolecules from plants was hampered in past by limitation in identification method. The present study is aimed at examining the efficacy of Trachyspermum ammi on CaOx crystallization in vitro and further by combining conventional biochemical methods with recent advances in mass spectrometry, a novel calcium oxalate (CaOx) crystal growth inhibitor was purified from the seeds of Trachyspermum ammi. An anticalcifying protein from the seeds of Trachyspermum ammi was purified by three step purification scheme; ammonium sulphate fractionation, anion exchange chromatography and molecular sieve chromatography based on its ability to inhibit calcium oxalate crystallization in vitro. An anticalcifying protein having molecular weight 107 kDa and isolectric point 6.2 was isolated. Amino acid analysis of Trachyspermum ammi anticalcifying protein (TAP) showed abundant presence of acidic amino acids (Asp and Glu). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry of TAP showed similarities with an unnamed protein product of Vitis vinifera (CAO23876) after matching peptide mass fingerprints in MASCOT search engine. Two EF hand domains were identified in unnamed protein product of Vitis vinifera (CAO23876) by SMART normal module. Due to a significant similarity of TAP with unnamed protein product of Vitis vinifera, presence of two EF hand domains in TAP was anticipated, signifying its calcium binding properties which is a feature of most kidney stone inhibitory proteins.

The most potent antilithiatic agent ameliorating renal dysfunction and oxidative stress from Bergenia ligulata rhizome.

ETHNOPHARMACOLOGICAL RELEVANCE The rhizome of Bergenia ligulata is referred by the Ayurvedic system for the treatment of kidney stone since decades and a few, in vitro and in vivo studies also support it. To identify the main phytochemical constituent(s) responsible for antilithiatic activity of its rhizome. MATERIALS AND METHODS In order to identify the most potent antilithiatic metabolite, the crude extract of rhizome was fractionated using in vitro Calcium oxalate (CaOx) crystal growth inhibitory activity guided fractionation followed by its characterization via LC-MS, FTIR and NMR. Further, the antioxidant potential of purified molecule was assessed using in vitro assays (FRAP and H2O2 scavenging). In vivo activity of the metabolite was evaluated in hyperoxaluric rats given 0.4% ethylene glycol (EG) and 1.0% ammonium chloride (NH4Cl) for 9 days. RESULTS Activity guided fractionation led to the isolation of most potent antilithiatic metabolite from the rhizome of Bergenia ligulata and spectroscopic analysis revealed it as bergenin. Bergenin showed reducing ability and H2O2 scavenging activity comparable with commercially available anitioxidant, α-tocopherol. At a dose of 10mg/kg body weight of the treated rat, it protected against deleterious effects of lithogenic treatment including weight loss, impaired renal function and oxidative stress, manifested as increased malondialdehyde, reduced redox ratio and decreased antioxidant enzyme activities in the kidneys of hyperoxaluric rats. The creatinine clearance and kidney damage were more improved by bergenin as compared to crude extract of rhizome. CONCLUSIONS Since, bergenin maintained oxidant/antioxidant balance in hyperoxaluric rats, thus mechanistic insight of its antilithiatic activity was attributed to the antioxidant capability of bergenin. The results of the present study provide significant evidence that bergenin is an active component present in the rhizome of Bergenia ligulata for managing CaOx calculi.

Role of mitochondria and NADPH oxidase derived reactive oxygen species in hyperoxaluria induced nephrolithiasis: therapeutic intervention with combinatorial therapy of N-acetyl cysteine and Apocynin.

The interactions between the main cellular sources of ROS, such as mitochondria and NADPH oxidase, are known to play an imperative role in the pathogenesis of hyperoxaluria-induced nephrolithiasis. The present study was designed to investigate the protective effect of a combinatorial therapy based on the attenuation of oxidative stress with antioxidant (N-acetyl cysteine), and NADPH oxidase inhibitor (apocynin), that might be required to effectively eliminate hyperoxaluric manifestations. Hyperoxaluria was induced in male Wistar rats by administering 0.4% ethylene glycol with 1% ammonium chloride in drinking water for 9 days. Hyperoxaluria accentuated renal oxidative stress in terms of increased ROS production and lipid peroxidation. Mitochondrial dysfunction, a central deleterious event in renal stone crystallization, was evident by decreased activities of electron transport chain complex I, II and IV, augmented mitochondrial ROS, reduced GSH/GSSG ratio, which resulted in the mitochondrial permeability transition pore (mPTP) opening as indicated by increased mitochondrial swelling in hyperoxaluric rats. Furthermore, NADPH oxidase activity was significantly increased, with raised expression of NOX1, NOX2, NOX4, p38MAPK and MnSOD, in the renal tissue of hyperoxaluric rats compared to control. However, combinatorial therapy with N-acetyl cysteine (50mg/kg/day) and apocynin (200mg/kg/day), intraperitoneally, significantly improved renal functions in hyperoxaluric rats and considerably ameliorated mitochondrial dysfunction. NAC with apocynin was also found to be effective in reducing the redundant activity of NADPH oxidase in renal tissue of hyperoxaluric rats. Hence, our investigation provides novel mechanistic insights that combinatorial approaches using targeted modulators of ROS offer therapeutic benefits in hyperoxaluria-induced nephrolithiasis.

Prophylactic effect of coconut water (Cocos nucifera L.) on ethylene glycol induced nephrocalcinosis in male wistar rat

PURPOSE Many medicinal plants have been employed during ages to treat urinary stones though the rationale behind their use is not well established. Thus, the present study was proposed to evaluate the effect of coconut water as a prophylactic agent in experimentally induced nephrolithiasis in a rat model. MATERIALS AND METHODS The male Wistar rats were divided randomly into three groups. Animals of group I (control) were fed standard rat diet. In group II, the animals were administrated 0.75% ethylene glycol in drinking water for the induction of nephrolithiasis. Group III animals were administrated coconut water in addition to ethylene glycol. All the treatments were continued for a total duration of seven weeks. RESULTS AND CONCLUSION Treatment with coconut water inhibited crystal deposition in renal tissue as well as reduced the number of crystals in urine. Furthermore, coconut water also protected against impaired renal function and development of oxidative stress in the kidneys. The results indicate that coconut water could be a potential candidate for phytotherapy against urolithiasis.

Rottlerin, a polyphenolic compound from the fruits of Mallotus phillipensis (Lam.) Müll.Arg., impedes oxalate/calcium oxalate induced pathways of oxidative stress in male wistar rats.

BACKGROUND Oxalate and/or calcium oxalate, is known to induce free radical production, subsequently leading to renal epithelial injury. Oxidative stress and mitochondrial dysfunction have emerged as new targets for managing oxalate induced renal injury. HYPOTHESIS Plant products and antioxidants have gained tremendous attention in the prevention of lithiatic disease. Rottlerin, a polyphenolic compound from the fruits of Mallotus phillipensis (Lam.) Müll.Arg., has shown free radical scavenging, antioxidant activity and has been reported to interfere in signaling pathways leading to inflammation and apoptosis. In this study, the potential role of rottlerin, in rats exposed to hyperoxaluric environment was explored. METHODS Hyperoxaluria was induced by administering 0.4% ethylene glycol and 1% ammonium chloride in drinking water to male wistar rats for 9 days. Rottlerin was administered intraperitoneally at 1mg/kg/day along with the hyperoxaluric agent. Prophylactic efficacy of rottlerin to diminish hyperoxaluria induced renal dysfunctionality and crystal load was examined along with its effect on free radicals generating pathways in hyperoxaluric rats. RESULTS 0.4% ethylene glycol and 1% ammonium chloride led to induction of hyperoxaluria, oxiadtive stress and mitochondrial damage in rats. Rottlerin treatment reduced NADPH oxidase activity, prevented mitochondrial dysfunction and maintained antioxidant environment. It also refurbished renal functioning, tissue integrity and diminished urinary crystal load in hyperoxaluric rats treated with rottlerin. CONCLUSIONS Thus, the present investigation suggests that rottlerin evidently reduced hyperoxaluric consequences and the probable mechanism of action of this drug could be attributed to its ability to quench free radicals by itself and interrupting signaling pathways involved in pathogenesis of stone formation.

Bergenin attenuates renal injury by reversing mitochondrial dysfunction in ethylene glycol induced hyperoxaluric rat model.

Bergenin, isolated from Bergenia ligulata is a potent antioxidant and antilithiatic agent. Present work was designed to establish the biochemical role of bergenin on mitochondrial dysfunction in the ethylene glycol induced hyperoxaluric rat model. Bergenin was administrated at a dose of 10mg/kg body wt i.p. from 14th day of establishing the 28 days hyperoxaluria rat model. α-Tocopherol was given as positive control at a dose of 100mg/kg body wt i.p. Mitochondrial dysfunction was studied by evaluating the activities of respiratory chain complexes, mitochondrial membrane potential and reactive oxygen species. Histopathological analysis of the kidney tissue was done after Pizzolato staining. Also, expression of monocyte chemoattractant protein -1(MCP-1) and kidney injury marker protein (KIM-1) were studied and the levels of IL-1β were evaluated in kidney tissue homogenate. Mitochondrial dysfunction during stone crystallization was evident by decreased activities of electron transport chain complexes I, II and IV and augmented mitochondrial oxidative stress in hyperoxaluric rats. Bergenin treatment significantly (P<0.05) restored the activities of these complexes. Moreover, it curtailed the lipid peroxidation and up regulated antioxidant levels, ameliorating the state of mitochondrial dysfunction. The protective role of bergenin was also reinforced by reducing IL-1β production and expression of KIM-1 and MCP-1 in the renal tissue. The findings of the present study provide evidence that bergenin exerted protective effects in hyperoxaluria through mitochondrial protection that involves attenuation of oxidative stress. Hence, it presented itself as an effective remedy in combating urolithiasis.