Mechanistic Insights into the Antilithiatic Proteins from Terminalia arjuna: A Proteomic Approach in Urolithiasis.

Amisha Mittal,Surender Kumar Singla,Simran Tandon,Chanderdeep Tandon,Utpal Sen

doi:10.1371/journal.pone.0162600

Abstract

Kidney stone formation during hyperoxaluric condition is inherently dependent on the interaction between renal epithelial cells and calcium oxalate (CaOx) crystals. Although modern medicine has progressed in terms of removal of these stones, recurrence and persistent side effects restricts their use. Strategies involving plant based agents which could be used as adjunct therapy is an area which needs to be explored. Plant proteins having antilithiatic activity is a hitherto unexplored area and therefore, we conducted a detailed identification and characterization of antilithiatic proteins from Terminalia arjuna (T. arjuna). Proteins were isolated from the dried bark of T. arjuna and those having molecular weights > 3 kDa were subjected to anion exchange chromatography followed by gel filtration chromatography. Four proteins were identified exhibiting inhibitory activity against CaOx crystallization and crystal growth kinetics The cytoprotective and anti-apoptotic efficacy of these purified proteins was further investigated on oxalate injured renal epithelial cells (MDCK and NRK-52E) wherein, injury due to oxalate was significantly attenuated and led to a dose dependent increase in viability of these cells. These proteins also prevented the interaction of the CaOx crystals to the cell surface and reduced the number of apoptotic cells. Identification of these 4 anionic proteins from the bark of T. arjuna was carried out by Matrix-assisted laser desorption/ionization-time of flight Mass spectrometry (MALDI-TOF MS). This was followed by database search with the MASCOT server and sequence similarity was found with Nuclear pore anchor, DEAD Box ATP-dependent RNA helicase 45, Lon protease homolog 1 and Heat shock protein 90–3. These novel proteins isolated from T. arjuna have the potential to inhibit CaOx crystallization and promote cell survival and therefore, offer novel avenues which need to be explored further for the medical management of urolithiasis.

Highlights

The urinary tract is prone to a number of adverse conditions which impact its functioning
In vitro studies have suggested that exposure of renal epithelial cells to oxalate and/or calcium oxalate (CaOx) crystals causes an increase in expression of immediate early genes (c-myc, Egr-1, c-jun and nur-77) [33,34,35] and production of urinary macromolecules (Tamm-Horsfall protein, Osteopontin, Prothrombin fragment-1, Bikunin and inter-α-inhibitor, α1-Microglobulin, CD44, Calgranulin, Heparan sulfate, Osteonectin, Fibronectin, Matrix Gla Protein), modulating CaOx crystallization and crystal retention in the kidneys, as an adaptive responses of cells to oxalate [36]
In this study we identified 4 novel proteins namely, Nuclear pore anchor, DEAD Box ATPdependent RNA helicase 45, Lon protease homolog 1 and Heat shock protein 90–3, as anionic inhibitors of CaOx crystallization from the bark of Terminalia arjuna

Summary

Introduction

The urinary tract is prone to a number of adverse conditions which impact its functioning. One prominent urinary tract disease is urolithiasis, which is the third most frequent urological affliction in humans [1]. Stone disease affects 2–20% population worldwide [2] with a prevalence rate of 15% in India [3]. The occurrence of CaOx kidney stone is the net effect of a panoply of factors which manifests themselves both within the cells as well as the environment in which the cells are present. The composition of the urinary fluid in terms of various ions, the propensity of these ions to form crystals and their further growth, as well as the presence of macromolecules in the fluid, are some examples of extracellular factors which occur in the tubular lumen and pelvis of the kidney. Cellular events/triggers which take place in the renal epithelial and interstitial cells, comprise of the interaction of oxalate and/or CaOx crystals on renal epithelial cells and how these cells respond to high oxalate and/or CaOx crystals [4]

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Discussion

Conclusion