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.

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.

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.