In vitro studies reveal antiurolithic effect of Terminalia arjuna using quantitative morphological information from computerized microscopy.

PURPOSE: For most cases, urolithiasis 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 (CaOx) deposition occurs in the kidney tissue. The present study is aimed to exam the antilithiatic potency of the aqueous extract (AE) of Terminalia arjuna (T. arjuna). MATERIALS AND METHODS: The antilithiatic activity of Terminalia arjuna was investigated in vitro nucleation, aggregation and growth of the CaOx crystals as well as the morphology of CaOx crystals using the inbuilt software 'Image-Pro Plus 7.0' of Olympus upright microscope (BX53). Antioxidant activity of AE of Terminalia arjuna bark was also determined in vitro. RESULTS: Terminalia arjuna extract exhibited a concentration dependent inhibition of nucleation and aggregation of CaOx crystals. The AE of Terminalia arjuna bark also inhibited the growth of CaOx crystals. At the same time, the AE also modified the morphology of CaOx crystals from hexagonal to spherical shape with increasing concentrations of AE and reduced the dimensions such as area, perimeter, length and width of CaOx crystals in a dose dependent manner. Also, the Terminalia arjuna AE scavenged the DPPH (2, 2-diphenyl-1-picrylhydrazyl) radicals with an IC50 at 13.1µg/mL. CONCLUSIONS: The study suggests that Terminalia arjuna bark has the potential to scavenge DPPH radicals and inhibit CaOx crystallization in vitro. In the light of these studies, Terminalia arjuna can be regarded as a promising candidate from natural plant sources of antilithiatic and antioxidant activity with high value.

Urolithiasis: phytotherapy as an adjunct therapy.

Role of herbal drugs and medicinal plant extracts in the successful treatment of urolithiasis, classified as the third most common urinary tract diseases is well documented. Ayurvedic plants and their components mediate antilithogenic effects by altering ionic composition of urine, being diuretic, antioxidant or having antimicrobial activity. Therapeutic peptides and proteins have unique place in pharmaceutical biotechnology due to their critical roles in cell biology. The innovation in antilithiatic proteins is that they are anionic, rich in acidic amino acids which make oxalate unavailable by interacting with calcium and have EF Hand domain which is a characteristic feature of various calcium binding protein like calgranulin, osteopontin. The review provides a background on the pathogenesis of urolithiasis and medical treatments. It focusses on the present research evaluating the scientific basis of antilithiatic potential of various plants and role of plant proteins as therapeutic agents thus opening new vista in the management of urolithiasis. Further investigations are required to fully decipher the mode of action of the potent biomolecules so as to exploit their preventive and therapeutic potential.

2D map of proteins from human renal stone matrix and evaluation of their effect on oxalate induced renal tubular epithelial cell injury.

PURPOSE: Proteins constitute a major portion of the organic matrix of human calcium oxalate (CaOx) renal stones and the matrix is considered to be important in stone formation and growth. The present study evaluates the effect of these proteins on oxalate injured renal epithelial cells accompanied by a 2D map of these proteins. MATERIALS AND METHODS: Proteins were isolated from the matrix of kidney stones containing CaOx as the major constituent using EGTA as a demineralizing agent. The effect of more than 3kDa proteins from matrix of human renal (calcium oxalate) CaOx stones was investigated on oxalate induced cell injury of MDCK renal tubular epithelial cells. A 2D map of >3kDa proteins was also generated followed by protein identification using MALDI-TOF MS. RESULTS: The >3kDa proteins enhanced the injury caused by oxalate on MDCK cells. Also, the 2D map of proteins having MW more than 3kDa suggested the abundance of proteins in the matrix of renal stone. CONCLUSION: Studies indicate that the mixture of >3kDa proteins in the matrix of human renal stones acts as promoter of calcium oxalate crystal nucleation and growth as it augments the renal epithelial cell injury induced by oxalate. The effect of promoters masks the inhibitors in the protein mixture thereby leading to enhanced renal cell injury. 2D map throws light on the nature of proteins present in the kidney stones.

Cytoprotective role of the aqueous extract of Terminalia chebula on renal epithelial cells.

PURPOSE: Kidney stone is one of the most prevalent diseases worldwide. Calcium oxalate (CaOx) has been shown to be the main component of the majority of stones formed in the urinary system of the patients with urolithiasis. The present study evaluates the antilithiatic properties of Terminalia chebula commonly called as ″harad ″ which is often used in ayurveda to treat various urinary diseases including kidney stones. MATERIALS AND METHODS: The antilithiatic activity of Terminalia chebula was investigated on nucleation and growth of the calcium oxalate crystals. The protective potency of the plant extract was also tested on oxalate induced cell injury of both NRK-52E and MDCK renal epithelial cells. RESULTS: The percentage inhibition of CaOx nucleation was found 95.84 % at 25µg/mL of Terminalia chebula aqueous extract which remained almost constant with the increasing concentration of the plant extract; however, plant extract inhibited CaOx crystal growth in a dose dependent pattern. When MDCK and NRK-52E cells were injured by exposure to oxalate for 48 hours, the aqueous extract prevented the injury in a dose-dependent manner. On treatment with the different concentrations of the plant extract, the cell viability increased and lactate dehydrogenase release decreased in a concentration dependent manner. CONCLUSION: Our study indicates that Terminalia chebula is a potential candidate for phytotherapy against urolithiasis as it not only has a potential to inhibit nucleation and the growth of the CaOx crystals but also has a cytoprotective role.

Diminution of oxalate induced renal tubular epithelial cell injury and inhibition of calcium oxalate crystallization in vitro by aqueous extract of Tribulus terrestris.

PURPOSE: Recurrence and persistent side effects of present day treatment for urolithiasis restrict their use, so an alternate solution, using phytotherapy is being sought. The present study attempted to evaluate the antilithiatic properties of Tribulus terrestris commonly called as "gokhru" which is often used in ayurveda to treat various urinary diseases including urolithiasis. MATERIALS AND METHODS: The activity of Tribulus terrestris was investigated on nucleation and the growth of the calcium oxalate (CaOx) crystals as well as on oxalate induced cell injury of NRK 52E renal epithelial cells. RESULTS: Tribulus terrestris extract exhibited a concentration dependent inhibition of nucleation and the growth of CaOx crystals. When NRK-52E cells were injured by exposure to oxalate for 72 h, Tribulus terrestris extract prevented the injury in a dose-dependent manner. On treatment with the different concentrations of the plant, the cell viability increased and lactate dehydrogenase release decreased in a concentration dependent manner. CONCLUSION: The current data suggests that Tribulus terrestris extract not only has a potential to inhibit nucleation and the growth of the CaOx crystals but also has a cytoprotective role. Our results indicate that it could be a potential candidate for phytotherapy against urolithiasis.

Rickettsial diseases in Haryana: not an uncommon entity.

Rickettsioses have not been reported from the plains of North India and Haryana in particular. Here we are reporting three cases of scrub typhus and one cases of Indian tick typhus in the state of Haryana, all of which presented with fever and multi organ dysfunction, rash and without eschar. All were successfully treated with doxycycline.

In vitro inhibition of calcium oxalate crystallization and crystal adherence to renal tubular epithelial cells by Terminalia arjuna.

Urolithiasis is a multifactorial disease and remains a public health problem around the world. Of all types of renal stones, calcium oxalate (CaOx) is the most common composition formed in the urinary system of the patients with urolithiasis. The present study is aimed at evaluating the antiurolithiatic properties of the Tris-Cl extract (TE) of Terminalia arjuna (T. arjuna). The antilithiatic activity of TE of T. arjuna was investigated on nucleation, aggregation, and growth of the CaOx crystals, as well as its protective potency was tested on oxalate-induced cell injury of NRK-52E renal epithelial cells. Also, in vitro antioxidant activity of TE T. arjuna bark was also determined. The TE of T. arjuna exhibited a concentration-dependent inhibition of nucleation and growth of CaOx crystals. Inhibition of aggregation of CaOx crystals remains constant. When NRK-52E cells were injured by exposure to oxalate for 48 h, the TE prevented the cells from injury and CaOx crystal adherence resulting in increased cell viability in a dose-dependent manner. The TE also scavenged the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals with an IC50 at 51.72 µg/mL. The results indicated that T. arjuna is a potential candidate for phytotherapy against urolithiasis as it attains the ability to inhibit CaOx crystallization and scavenge DPPH free radicals in vitro along with a cytoprotective role.

Role of a protein inhibitor isolated from human renal stone matrix in urolithiasis.

The role of biomolecule(s) from renal stone matrix in urolithiasis was investigated. The ability of a particular fraction (> 10 kDa fraction) isolated from the EDTA extract of powdered human renal stones to influence calcium oxalate monohydrate (COM) crystal growth was studied. The most potent inhibitor of COM crystal growth obtained from > 10 kDa fraction was purified by various chromatographic techniques and SDS-PAGE, etc. and was found to have a molecular mass of 36 kDa. The urine and serum samples obtained from normal persons were found to be more potent in inhibiting the growth of COM crystals as compared to the kidney-stone patients. Polyclonal antibodies were raised against this inhibitor and were employed to determine the concentration of 36 kDa inhibitor in urine and serum samples of normal persons and kidney-stone patients.

Mg2+: a potent inhibitor of collagen-induced in vitro mineralization.

At physiological concentrations, Mg2+ has been found to be a potent inhibitor of collagen-induced in vitro mineralization. Mg2+ inhibits mineralization by competing with Ca2+ for specific phosphate independent Ca2+ binding sites of the catalytic matrix. Matrix bound Mg2+ subsequently reacts with HPO4(2-) to form MgHPO4 complex which can not be further converted to the matrix bound mineral phase. The matrix, as well as the mineral phase associated with the matrix, influence the rate of mineralization.

Effect of nucleoside-5'-phosphates on collagen-induced in vitro mineralization.

Nucleoside triphosphates (NTPs) at 4-10 microM concentrations were found to inhibit the rates of collagen-induced in vitro mineralization and ion exchange reactions. The sequential removal of the terminal phosphate groups caused a step-wise decrease in their inhibitory potency. The results suggest that NTPs inhibit the rates of ion uptake and exchange reactions at concentrations much lower than their intracellular physiological concentrations. Thus NTPs may be involved in the control of biological mineralization and the tissues which mineralize under physiological conditions develop a system to locally convert NTPs to NDPs and NMPs.

In vitro Evaluation of Terminalia arjuna on Calcium Phosphate and Calcium Oxalate Crystallization.

Urinary stones are one of the oldest and the most common afflictions in humans. This disease has tormented humans since the earliest records of civilization. Ten percent of men and 3 % of women have a stone during their adult lives. Calcium containing stones are the most common comprising about 75 % of all urinary calculi, which may be in the form of pure calcium oxalate (50 %) or calcium phosphate (5 %) or a mixture of both (45 %). A number of plants have been mentioned in the Indian ayurvedic system, which plays a vital role in the inhibition of kidney stones. In the present study, the inhibitory potency of crude extracts or fractions of successive solvent extractions of Terminalia arjuna bark was evaluated on various stages of formation of calcium phosphate and on the growth of calcium oxalate monohydrate crystals in vitro. Results obtained indicated that Terminalia arjuna bark has the potential to inhibit the formation of both calcium phosphate and calcium oxalate crystals in vitro. Butanol fraction of Terminalia arjuna extract was the most effective in inhibiting formation of calcium phosphate and calcium oxalate crystals in vitro.

In vitro studies reveal antiurolithic effect of Terminalia arjuna using quantitative morphological information from computerized microscopy

ABSTRACT Purpose: For most cases, urolithiasis 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 (CaOx) deposition occurs in the kidney tissue. The present study is aimed to exam the antilithiatic potency of the aqueous extract (AE) of Terminalia arjuna (T. arjuna). Materials and Methods: The antilithiatic activity of Terminalia arjuna was investigated in vitro nucleation, aggregation and growth of the CaOx crystals as well as the morphology of CaOx crystals using the inbuilt software ‘Image-Pro Plus 7.0’ of Olympus upright microscope (BX53). Antioxidant activity of AE of Terminalia arjuna bark was also determined in vitro. Results: Terminalia arjuna extract exhibited a concentration dependent inhibition of nucleation and aggregation of CaOx crystals. The AE of Terminalia arjuna bark also inhibited the growth of CaOx crystals. At the same time, the AE also modified the morphology of CaOx crystals from hexagonal to spherical shape with increasing concentrations of AE and reduced the dimensions such as area, perimeter, length and width of CaOx crystals in a dose dependent manner. Also, the Terminalia arjuna AE scavenged the DPPH (2, 2-diphenyl-1-picrylhydrazyl) radicals with an IC50 at 13.1µg/mL. Conclusions: The study suggests that Terminalia arjuna bark has the potential to scavenge DPPH radicals and inhibit CaOx crystallization in vitro. In the light of these studies, Terminalia arjuna can be regarded as a promising candidate from natural plant sources of antilithiatic and antioxidant activity with high value.

Oxalate-mediated oxidant-antioxidant imbalance in erythrocytes: role of N-acetylcysteine.

The present in-vivo study was to observe the effect of N-acetylcysteine (NAC) on oxalate-induced oxidative stress on rat erythrocytes. A total of 15 Wistar rats were divided into three groups. The control group received normal saline by single intraperitoneal injection. Hyperoxaluria was induced by single intraperitoneal (i.p.) dose of sodium oxalate (70 mg/kg body weight in 0.5 mL saline) to a second group. The third group was administered single i.p. dose of NAC according to 200 mg/kg body weight dissolved in 0.5 mL saline, half an hour after oxalate dose. NAC administration normalized antioxidant enzyme activities (superoxide dismutase and catalase) and reduced malondialdehyde content (indicator of lipid peroxidation) in hyperoxaluric rat's red blood cell (RBC) lysate. NAC administration also resulted in a significant improvement of thiol content in RBC lysate via increasing reduced glutathione content and maintaining its redox status. Oxalate-caused alteration of cholesterol/phospholipid ratio (determining membrane fluidity) was also rebalanced by NAC administration. Further, after NAC administration, electron microscopy showed improved cell morphology presenting its prophylactic properties. Above results indicate that NAC treatment is associated with an increase in plasma antioxidant capacity and a reduction in the susceptibility of erythrocyte membranes to oxidation. Thus, the study presents positive pharmacological implications of NAC against oxalate-mediated impairment of erythrocytes.

2D map of proteins from human renal stone matrix and evaluation of their effect on oxalate induced renal tubular epithelial cell injury

Purpose Proteins constitute a major portion of the organic matrix of human calcium oxalate (CaOx) renal stones and the matrix is considered to be important in stone formation and growth. The present study evaluates the effect of these proteins on oxalate injured renal epithelial cells accompanied by a 2D map of these proteins. Materials and Methods Proteins were isolated from the matrix of kidney stones containing CaOx as the major constituent using EGTA as a demineralizing agent. The effect of more than 3kDa proteins from matrix of human renal (calcium oxalate) CaOx stones was investigated on oxalate induced cell injury of MDCK renal tubular epithelial cells. A 2D map of >3kDa proteins was also generated followed by protein identification using MALDI-TOF MS. Results The >3kDa proteins enhanced the injury caused by oxalate on MDCK cells. Also, the 2D map of proteins having MW more than 3kDa suggested the abundance of proteins in the matrix of renal stone. Conclusion Studies indicate that the mixture of >3kDa proteins in the matrix of human renal stones acts as promoter of calcium oxalate crystal nucleation and growth as it augments the renal epithelial cell injury induced by oxalate. The effect of promoters masks the inhibitors in the protein mixture thereby leading to enhanced renal cell injury. 2D map throws light on the nature of proteins present in the kidney stones. .

Reversal of hyperoxaluria-induced alteration in rat liver by administration of N-acetylcysteine.

The current work was designed to study the potential of N-acetylcysteine (NAC) in modulating hyperoxaluric manifestations induced by acute oxalate dose in rat liver. Hyperoxaluric conditions were induced by giving a single dose of sodium oxalate (70 mg/kg body weight) in one group, and in the other group, hyperoxaluric rats were administered NAC (200 mg/kg body weight) after 30 min of the oxalate dose. After 12 h of the above treatment, blood was taken from the orbital sinus for testing serum oxalate, and animals were sacrificed. To exploit the potential of NAC, various oxidative stress parameters [lipid peroxidation (LP) and activity of antioxidant enzymes], lipid content, and histologic analysis of rat liver were performed. The increased level of LP and activities of superoxide dismutase and catalase in hyperoxaluric rats were restored after NAC treatment. Not only the decreased amount of total lipids and phospholipids but also the increased ratio of cholesterol/phospholipid (showing decreased membrane fluidity) in hyperoxaluric rats were balanced by NAC treatment. Further restored histologic changes of liver tissue confirmed the protective antioxidant effects of the given drug. Thus, N-acetylcysteine being an extraneous antioxidant showed curative properties toward hyperoxaluric manifestations in liver.

Cytoprotective role of the aqueous extract of Terminalia chebula on renal epithelial cells

PURPOSE: Kidney stone is one of the most prevalent diseases worldwide. Calcium oxalate (CaOx) has been shown to be the main component of the majority of stones formed in the urinary system of the patients with urolithiasis. The present study evaluates the antilithiatic properties of Terminalia chebula commonly called as "harad" which is often used in ayurveda to treat various urinary diseases including kidney stones. MATERIALS AND METHODS: The antilithiatic activity of Terminalia chebula was investigated on nucleation and growth of the calcium oxalate crystals. The protective potency of the plant extract was also tested on oxalate induced cell injury of both NRK-52E and MDCK renal epithelial cells. RESULTS: The percentage inhibition of CaOx nucleation was found 95.84% at 25µg/mL of Terminalia chebula aqueous extract which remained almost constant with the increasing concentration of the plant extract; however, plant extract inhibited CaOx crystal growth in a dose dependent pattern. When MDCK and NRK-52E cells were injured by exposure to oxalate for 48 hours, the aqueous extract prevented the injury in a dose-dependent manner. On treatment with the different concentrations of the plant extract, the cell viability increased and lactate dehydrogenase release decreased in a concentration dependent manner. CONCLUSION: Our study indicates that Terminalia chebula is a potential candidate for phytotherapy against urolithiasis as it not only has a potential to inhibit nucleation and the growth of the CaOx crystals but also has a cytoprotective role.

Diminution of oxalate induced renal tubular epithelial cell injury and inhibition of calcium oxalate crystallization in vitro by aqueous extract of Tribulus terrestris

PURPOSE: Recurrence and persistent side effects of present day treatment for urolithiasis restrict their use, so an alternate solution, using phytotherapy is being sought. The present study attempted to evaluate the antilithiatic properties of Tribulus terrestris commonly called as “gokhru” which is often used in ayurveda to treat various urinary diseases including urolithiasis. MATERIALS AND METHODS: The activity of Tribulus terrestris was investigated on nucleation and the growth of the calcium oxalate (CaOx) crystals as well as on oxalate induced cell injury of NRK 52E renal epithelial cells. RESULTS: Tribulus terrestris extract exhibited a concentration dependent inhibition of nucleation and the growth of CaOx crystals. When NRK-52E cells were injured by exposure to oxalate for 72 h, Tribulus terrestris extract prevented the injury in a dose-dependent manner. On treatment with the different concentrations of the plant, the cell viability increased and lactate dehydrogenase release decreased in a concentration dependent manner. CONCLUSION: The current data suggests that Tribulus terrestris extract not only has a potential to inhibit nucleation and the growth of the CaOx crystals but also has a cytoprotective role. Our results indicate that it could be a potential candidate for phytotherapy against urolithiasis.

Inhibitors of in vitro mineralization from flexor tendons of rabbits and their role in biological mineralization.

Studies demonstrate that flexor tendons contain loosely associated biomolecules which inhibit its mineralization under physiological conditions. Based upon their molecular weights, these inhibitory biomolecules, could be classified into two categories, having molecular weights less than and greater than 13,000 daltons. The main inhibitory biomolecule was found to be an acidic polypeptide having molecular weight of 12,400 daltons.

Inhibitors of in vitro mineralization from rabbit aorta and their role in biomineralization.

Mineralization of aorta is known to occur late in life and appears to be a pathological phenomenon. In vitro studies revealed that the matrix prepared from the thoracic aorta pieces after their extraction with 3% Na2HPO4 and 0.1 mM CaCl2 were mineralized under physiological conditions of temperature, pH, and ionic strength of the media to form matrix-bound mineral phase resembling hydroxyapatite in nature. However, the matrix identically prepared from the unextracted rabbits aortae failed to mineralize under identical assay conditions. The addition of the aorta extract in the assay system inhibited the above mineralization process. Standard biochemical techniques, e.g., dialysis, ion exchange, and molecular sieve chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid analysis by high-performance liquid chromatography were employed to isolate, purify, and characterize the potent inhibitory biomolecules from the aorta extract. The inhibitory activity of the aorta extract was found to be primarily due to the presence of three biomolecules having molecular weights of 66, 45, and 27-29 kDa. The above inhibitory biomolecules loosely associated with aorta may be involved in the control of calcification associated with arteriosclerosis.