Molecular heterogeneity in prostate cancer and the role of targeted therapy.

Data collected from large-scale studies has shown that the incidence of prostate cancer globally is on the rise, which could be attributed to an overall increase in lifespan. So, the question is how has modern science with all its new technologies and clinical breakthroughs mitigated or managed this disease? The answer is not a simple one as prostate cancer exhibits various subtypes, each with its unique characteristics or signatures which creates challenges in treatment. To understand the complexity of prostate cancer these signatures must be deciphered. Molecular studies of prostate cancer samples have identified certain genetic and epigenetic alterations, which are instrumental in tumorigenesis. Some of these candidates include the androgen receptor (AR), various oncogenes, tumor suppressor genes, and the tumor microenvironment, which serve as major drivers that lead to cancer progression. These aberrant genes and their products can give an insight into prostate cancer development and progression by acting as potent markers to guide future therapeutic approaches. Thus, understanding the complexity of prostate cancer is crucial for targeting specific markers and tailoring treatments accordingly.

Arsenicum album Induces Cell Cycle Arrest and Apoptosis, and Inhibits Epithelial-Mesenchymal Transition in Hormone-Dependent MCF7 Breast Cancer Cells.

BACKGROUND: Arsenic trioxide (As2O3) has been in therapeutic use since the 18th century for various types of cancers including skin and breast; however, it gained popularity following FDA approval for its use against acute promyelocytic leukemia. This present work was designed to evaluate the anti-cancer potential of a homeopathic potency of arsenic trioxide (Arsenicum album 6C) in hormone-dependent breast cancer. METHODS: Breast cancer cells (MCF7) were treated with Arsenicum album (Ars 6C) to evaluate its anti-proliferative and apoptotic potential. We examined the effect of Ars 6C on the cell cycle, wound healing, reactive oxygen species (ROS) generation, and modulation of expression of key genes which are aberrant in cancer. RESULTS: Treating breast cancer cells with Ars 6C halted the cell cycle at the sub-G0 and G2/M phases, which could be attributed to DNA damage induced by the generation of ROS. Apoptotic induction was associated with upregulation of Bax expression, with concurrent downregulation of the Bcl-2 gene. Ars 6C was also seen to reverse epithelial to mesenchymal transition and reduce the migration of breast cancer cells. CONCLUSION: The findings suggest that Ars has significant anti-proliferative and apoptotic potential against breast cancer cells. Further studies are required to elucidate the mechanism by which Ars exerts its effect in the in vivo setting.

Computational and In Vitro Approaches to Elucidate the Anti-cancer Effects of Arnica montana in Hormone-Dependent Breast Cancer.

BACKGROUND: Breast cancer is the most common cancer in women worldwide. Use of homeopathic medicines for the treatment of cancers has increased in the last several years. Arnica montana is an anti-inflammatory homeopathic medicine used in traumatic conditions and because of this property we performed investigations for its potential as a chemotherapeutic agent against breast cancer. METHODS: An ethanolic extract of Arnica montana (mother tincture, MT), prepared according to the Homoeopathic Pharmacopoeia of India, was characterized by gas chromatography-mass spectroscopy (GC-MS), followed by computational (in silico) analysis using molecular docking, to identify specific compounds that can bind and modulate the activity of key proteins involved in breast cancer survival and progression. To validate the in silico findings, in a controlled experiment breast cancer cells (MCF7) were treated in vitro with Arnica montana and the cytotoxic effects assessed by flowcytometry, fluorescence microscopy, scratch assay, clonogenic potential and gene expression analysis. RESULTS: Phytochemical characterization of ethanolic extract of Arn MT by GC-MS allowed identification of several compounds. Caryophyllene oxide and 7-hydroxycadalene were selected for molecular docking studies, based on their potential drug-like properties. These compounds displayed selective binding affinity to some of the recognized target proteins of breast cancer, which included estrogen receptor alpha (ERα), progesterone receptor (PR), epidermal growth factor receptor (EGFR), mTOR (mechanistic target of rapamycin) and E-cadherin. In vitro studies revealed induction of apoptosis in MCF7 cells following treatment with Arn MT. Furthermore, treatment with Arn MT revealed its ability to inhibit migration and colony forming abilities of the cancer cells. CONCLUSION: Considering the apoptotic and anti-migratory effects of Arnica montana in breast cancer cells in vitro, there is a need for this medicine to be further validated in an in vivo model.

4-PBA rescues hyperoxaluria induced nephrolithiasis by modulating urinary glycoproteins: Cross talk between endoplasmic reticulum, calcium homeostasis and mitochondria.

AIM: Urinary glycoproteins such as Tamm Horsfall Protein (THP) and Osteopontin (OPN) are well established key regulators of renal stone formation. Additionally, recent revelations have highlighted the influence of Endoplasmic Reticulum (ER) and mitochondria of crucial importance in nephrolithiasis. However, till date conclusive approach highlighting the influence of ER stress on urinary glycoproteins and chaperone in nephrolithiasis remains elusive. Therefore, the present study was focussed on deciphering the possible effect of 4-PBA mitigating ER stress on urinary glycoproteins and calnexin (chaperone) with emphasis on interlinking calcium homeostasis in hyperoxaluric rats. MATERIAL AND METHODS: Post 9 days of treatment, animals were sacrificed, and renal tissues were investigated for urinary glycoproteins, calnexin, calcium homeostasis, ER environment, redox status, and mitochondrial linkage. KEY FINDINGS: 4-PBA appreciably reversed the altered levels of THP, OPN, and calnexin observed along with curtailing the disrupted calcium homeostasis when assessed for SERCA activity and intra-cellular calcium levels. Additionally, significant improvement in the perturbed ER environment as verified by escalated ER stress markers, disturbed protein folding-aggregation-degradation (congo red assay) pathway, and redox status was found post 4-PBA intervention. Interestingly, linkage of ER stress and mitochondria was established under hyperoxaluric conditions when assessed for protein levels of VDAC1 and GRP75. SIGNIFICANCE: 4-PBA treatment resulted in rectifying the repercussions of ER-mitochondrial caused distress when assessed for protein folding/aggregation/degradation events along with disturbed calcium homeostasis. The present study advocates the necessity to adopt a holistic vision towards hyperoxaluria with emphasis on glycoproteins and ER environment.

Insights into the cytoprotective potential of Bergenia ligulata against oxalate-induced oxidative stress and epithelial-mesenchymal transition (EMT) via TGFß1/p38MAPK pathway in human renal epithelial cells.

Oxalate exposure to human renal epithelial cells triggers a vicious cycle of oxidative stress leading to cellular injury and deposition of calcium oxalate crystals on the injured cells. This results in further oxidative damage causing inflammation and loss of cell-cell adhesion factors, ultimately leading to irreparable kidney damage. However, these events can be attenuated or prevented by plants rich in antioxidants used in the traditional system of medicine for treatment of kidney stones. To delineate the mechanism by which Bergenia ligulata extract exerts its cytoprotective role in oxalate-induced injury we designed this study. Our results revealed that oxalate-injured HK2 cells cotreated with ethanolic extract of Bergenia ligulata displayed increased viability, reduced oxidative stress due to lowered production of intracellular reactive oxygen species (ROS) and decreased apoptosis. We also observed lowered markers of inflammation, along with increased expression of epithelial marker E-cadherin and decreased expression of mesenchymal markers Vimentin, F-actin, Transforming growth factor beta 1 (TGF-ß1) and EMT-related proteins in renal tubular epithelial cells through immunocytochemistry, real-time PCR and western blotting. Our findings collectively suggest that by reducing oxidative stress, modulating crystal structure and preventing crystal-cell adhesion, B. ligulata inhibits the EMT pathway by downregulating the various mediators and thereby exerts its cytoprotective effect.

Stem cell therapy: A paradigm shift in breast cancer treatment.

As per the latest Globocan statistics, the high prevalence rate of breast cancer in low- and middle-income countries has led to it becoming the most common cancer to be diagnosed, hence posing a major public health challenge. As per this data, more than 11.7% of the estimated new cancer cases in 2020 were due to breast cancer. A small but significant subpopulation of cells with self- renewing ability are present in the tumor stroma and have been given the nomenclature of cancer stem cells (CSCs). These cells display a high degree of plasticity owing to their ability to transition from the slowly cycling quiescent phase to the actively proliferating phenotype. This attribute of CSCs allows them to differentiate into various cell types having diverse functions. Breast CSCs have a pivotal role in development, metastasis, treatment resistance and relapse of breast cancers. This review focuses on the pathways regulating breast CSC maintenance and the current strategies that are being explored for directing the development of novel, targeted, therapeutic approaches for limiting and eradicating this aberrant stem cell population.

Downregulation of inflammatory mediators by ethanolic extract of Bergenia ligulata (Wall.) in oxalate injured renal epithelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: In the Indian traditional system of medicine, Bergenia ligulata (Wall.) Engl. has been used for treatment of urolithiasis. Its efficacious nature has led to its incorporation in various commercial herbal formulations such as Cystone and Neeri which are prescribed for kidney related ailments. AIM OF THE STUDY: To assess whether ethanolic extract of B. ligulata can mitigate the cascade of inflammatory responses that cause oxidative stress and ultimately cell death in renal epithelial cells exposed to hyperoxaluric conditions. MATERIAL AND METHODS: Bioactivity guided fractionation using solvents of varying polarities was employed to evaluate the potential of the extracts of B. ligulata to inhibit the crystallization process. Modulation of crystal morphology was visualized through Scanning electron microscopy (SEM) analysis. Cell death was assessed using flow cytometry based assays. Alteration in the inflammatory mediators was evaluated using real time PCR and immunocytochemistry. Phytochemical characterization of the ethanolic extract was carried out using FTIR, LC-MS and GC-MS. RESULTS: Bioactivity guided fractionation for the assessment of antilithiatic activity revealed dose dependent inhibition of nucleation and aggregation process of calcium oxalate crystals in the presence of various extracts, however ethanolic extract showed maximum inhibition and was chosen for further experiments. Studies on renal epithelial NRK-52E cells showed, cytoprotective efficacy of B. ligulata extract against oxalate injury. SEM anaysis further revealed the potential of the extract to modulate the crystal structure and adhesion to renal cell surface. Exposure of the renal cells to the extract led to conversion of the calcium oxalate monohydrate (COM) crystals to the less injurious calcium oxalate dihydrate (COD) form. Expression analysis for oxidative stress and inflammatory biomarkers in NRK-52E cells revealed up-regulation of Mitogen activated protein kinase (MAPK), Osteopontin (OPN) and Nuclear factor- ĸB (NF-ĸB), in response to calcium oxalate insult; which was drastically reduced in the presence of B. ligulata extract. Flow cytometric evaluation pointed to caspase 3 mediated apoptotic cell death in oxalate injured cells, which was attenuated by B. ligulata extract. CONCLUSION: Considering the complex multifactorial etiology of urolithiasis, ethanolic extract from B. ligulata can be a promising option for the management of kidney stones, as it has the potential to limit inflammation and the subsequent cell death.

Emerging role of exosomes in arterial and renal calcification.

Exosomes are small, cell-derived vesicles of 30-100 nm that participate in cell-to-cell communication. They are released by many cells, such as dendritic cells (DC), lymphocytes, platelets, epithelial cells, endothelial cells (EC), and are found in most body fluids, including blood, saliva, urine, and breast milk. The exosomes released from cells within the cardiovascular system may contain either inhibitors of calcification in normal physiological conditions or promoters in the pathological environment [atherosclerosis (AS), and Chronic Kidney Disease (CKD)]. The exosomes of the vascular smooth muscle cells (VSMCs) are novel players in vascular repair processes and calcification. Several studies have shown that the cytoplasmic contents of exosomes are rich in a variety of proteins, nucleic acids, and lipids. Currently, exosomal micro RNAs and proteins are increasingly being recognized as biomarkers for the diagnosis of several diseases, including those of kidney and liver, as well as different types of cancer. In this review, we summarize recent advances in the role of exosomes in vascular calcification and their potential applications as diagnostic markers as well as a brief overview of the role of stem cell-derived exosomes in cardiovascular diseases.

An update on vascular calcification and potential therapeutics.

Pathological calcification is a major cause of cardiovascular morbidities primarily in population with chronic kidney disease (CKD), end stage renal diseases (ERSD) and metabolic disorders. Investigators have accepted the fact that vascular calcification is not a passive process but a highly complex, cell mediated, active process in patients with cardiovascular disease (CVD) resulting from, metabolic insults of bone fragility, diabetes, hypertension, dyslipidemia and atherosclerosis. Over the years, studies have revealed various mechanisms of vascular calcification like induction of bone formation, apoptosis, alteration in Ca-P balance and loss of inhibition. Novel clinical studies targeting cellular mechanisms of calcification provide promising and potential avenues for drug development. The interventions include phosphate binders, sodium thiosulphate, vitamin K, calcimimetics, vitamin D, bisphosphonates, Myoinositol hexaphosphate (IP6), Denosumab and TNAP inhibitors. Concurrently investigators are also working towards reversing or curing pathological calcification. This review focuses on the relationship of vascular calcification to clinical diseases, regulators and factors causing calcification including genetics which have been identified. At present, there is lack of any significant preventive measures for calcifications and hence this review explores further possibilities for drug development and treatment modalities.

Stem Cell Based Preclinical Drug Development and Toxicity Prediction.

Stem cell based toxicity prediction plays a very important role in the development of the drug. Unexpected adverse effects of the drugs during clinical trials are a major reason for the termination or withdrawal of drugs. Methods for predicting toxicity employ in vitro as well as in vivo models; however, the major drawback seen in the data derived from these animal models is the lack of extrapolation, owing to interspecies variations. Due to these limitations, researchers have been striving to develop more robust drug screening platforms based on stem cells. The application of stem cells based toxicity testing has opened up robust methods to study the impact of new chemical entities on not only specific cell types, but also organs. Pluripotent stem cells, as well as cells derived from them, can be evaluated for modulation of cell function in response to drugs. Moreover, the combination of state-of-the -art techniques such as tissue engineering and microfluidics to fabricate organ- on-a-chip, has led to assays which are amenable to high throughput screening to understand the adverse and toxic effects of chemicals and drugs. This review summarizes the important aspects of the establishment of the embryonic stem cell test (EST), use of stem cells, pluripotent, induced pluripotent stem cells and organoids for toxicity prediction and drug development.

Human kidney stone matrix proteins alleviate hyperoxaluria induced renal stress by targeting cell-crystal interactions.

Increased levels of urinary oxalate also known as hyperoxaluria, increase the likelihood of kidney stone formation through enhanced calcium oxalate (CaOx) crystallization. The management of lithiatic renal pathology requires investigations at the initial macromolecular stages. Hence, the current study was designed to unravel the protein make-up of human kidney stones and its impact on renal cells' altered proteome, induced as the consequence of CaOx injury. CaOx kidney stones were collected from patients; stones were pooled for entire cohort, followed by protein extraction. Immunocytochemistry, RT-PCR and flow-cytometric analysis revealed the promising antilithiatic activity of kidney stone matrix proteins. The iTRAQ analysis of renal cells showed up-regulation of 12 proteins and down-regulation of 41 proteins due to CaOx insult, however, this differential expression was normalized in the presence of kidney stone matrix proteins. Protein network analysis revealed involvement of up-regulated proteins in apoptosis, calcium-binding, inflammatory and stress response pathways. Moreover, seven novel antilithiatic proteins were identified from human kidney stones' matrix: Tenascin-X-isoform2, CCDC-144A, LIM domain kinase-1, Serine/Arginine receptor matrix protein-2, mitochondrial peptide methionine sulfoxide reductase, volume-regulated anion channel subunit-LRRC8A and BMPR2. In silico analysis concluded that these proteins exert antilithiatic potential through crystal binding, thereby inhibiting the crystal-cell interaction, a pre-requisite to initiate inflammatory response. Thus, the outcomes of this study provide insights into the molecular events of CaOx induced renal toxicity and subsequent progression into nephrolithiasis.

In Vitro Assessment of Homeopathic Potencies of Hydrastis canadensis on Hormone-Dependent and Independent Breast Cancer.

BACKGROUND: Breast cancer is the second leading cause of cancer-related deaths in women. Conventional treatment such as chemotherapy, hormonal therapy and radiotherapy has decreased the mortality rate among cancer patients but has also revealed long-term side effects. Drug resistance and toxicity to normal cells compound the problems associated with the use of modern medicines. Hence, complementary or alternative treatment options are being explored. The current study, using different homeopathic potencies of Hydrastis canadensis, was conducted to distinguish between any effects they might have on hormone-dependent and independent breast cancer. MATERIALS AND METHODS: The cytotoxic effect of homeopathic medicine Hydrastis on hormone-dependent (MCF 7) and hormone-independent (MDA-MB-468) breast cancer cells was assessed using viability and colony-forming assays after 48 or 72 hours of treatment. Flow cytometry-based Annexin V-PI (propidium iodide), caspase 3 and cell cycle analysis was performed following treatment of cells with mother tincture or various potencies of Hydrastis (1C, 2C, 30C, 200C). RESULTS: Different potencies of Hydrastis displayed selective cytotoxic effects against MCF 7 cells, but only marginal effects against MDA-MB-468. The maximum cytotoxicity was established in the case of 1C following 72 hours of treatment. Treatment of breast cancer cells revealed an increase in the G0/G1 cell population, along with an increase in the caspase 3 levels and induction of apoptosis. CONCLUSION: Hydrastis may have a selective cytotoxic effect against hormone-dependent breast cancer MCF 7 cells, leading to cell cycle arrest in the G0/G1 phase, which could be the plausible reason for the induction of apoptosis. The results need to be validated in vivo.

Delving into the Antiurolithiatic Potential of Tribulus terrestris Extract Through -In Vivo Efficacy and Preclinical Safety Investigations in Wistar Rats.

Modern treatment interventions for kidney stones are wrought with side-effects, hence the need for alternative therapies such as plant-based medicines. We have previously documented through in vitro studies that statistically optimized aqueous extract of Tribulus terrestris (Zygophyllaceae family) possesses antiurolithic and antioxidant potential. This provides strong scientific foundation to conduct in vivo efficacy and preclinical safety studies to corroborate and lend further proof to its ability to prevent and cure kidney stones. The preventive and curative urolithiatic efficacy in experimentally induced nephrolithiatic Wistar rats, along with preclinical toxicity was evaluated following oral administration of statistically optimized aqueous extract of T. terrestris. Treatment showed augmented renal function, restoration of normal renal architecture and increase in body weight. Microscopic analysis of urine revealed excretion of small sized urinary crystals, demonstrating that treatment potentially modulated the morphology of renal stones. Tissue enzymatic estimation affirmed the antioxidant efficacy of treatment with reduced free radical generation. Significant upregulation of p38MAPK at both the gene and protein level was noted in hyperoxaluric group and interestingly treatment reversed it. Acute oral toxicity study established the Median Lethal Dose (LD50) to be greater than 2000 mg/kg body weight (b.wt.) No observed adverse effect level (NOAEL) by repeated oral toxicity for 28 days at 750 mg/kg b.wt. was noted. This study lends scientific evidence to the safe, preventive and curative potential of statistically optimized aqueous extract of T. terrestris at a dose of 750 mg/kg b.wt. and suggests that the extract shows promise as a therapeutic antiurolithic agent.

Role of DHH superfamily proteins in nucleic acids metabolism and stress tolerance in prokaryotes and eukaryotes.

DHH superfamily proteins play pivotal roles in various cellular processes like replication, recombination, repair and nucleic acids metabolism. These proteins are important for homeostasis maintenance and stress tolerance in prokaryotes and eukaryotes. The prominent members of DHH superfamily include single-strand specific exonuclease RecJ, nanoRNases, polyphosphatase PPX1, pyrophosphatase, prune phosphodiesterase and cell cycle protein Cdc45. The mutations of genes coding for DHH superfamily proteins lead to severe growth defects and in some cases, may be lethal. The members of superfamily have a wide substrate spectrum. The spectrum of substrate for DHH superfamily members ranges from smaller molecules like pyrophosphate and cyclic nucleotides to longer single-stranded DNA molecule. Several genetic, structural and biochemical studies have provided interesting insights about roles of DHH superfamily members. However, there are still various unexplored members in both prokaryotes and eukaryotes. Many aspects of this superfamily associated with homeostasis maintenance and stress tolerance are still not clearly understood. A comprehensive understanding is pre-requisite to decipher the physiological significance of members of DHH superfamily. This article provides the current understanding of DHH superfamily members and their significance in nucleic acids metabolism and stress tolerance across diverse forms of life.

Role of Matrix Metalloproteinases in Degenerative Kidney Disorders.

Matrix metalloproteinases (MMPs) are members of calcium dependent-zinc containing endopeptidases that play a pivotal role in extracellular matrix (ECM) remodeling. MMPs are also known to cleave non-matrix proteins, including cell surface receptors, TNF-α, angiotensin-II, growth factors, (especially transforming growth factor-ß1, ΤGF- ß1) plasminogen, endothelin and other bioactive molecules. The tissue inhibitors of metalloproteinases (TIMPs) inhibit the activity of MMPs and decrease ECM degradation. Various patho-physiological conditions have been linked with the imbalance of ECM synthesis and degradation. Numerous studies have reported the significance of MMPs and TIMPs in the progression of kidney pathologies, including glomerulonephritis, diabetic nephropathy, renal cancer, and nephrolithiasis. Although dysregulated activity of MMPs could directly or indirectly lead to pathological morbidities, their contribution in disease progression is still understated. Specifically, MMP activity in the kidneys and it's relation to kidney diseases has been the subject of a limited number of investigations. Therefore, the aim of the present review is to provide an updated insight of the involvement of MMPs and TIMPs in the pathogenesis of inflammatory and degenerative kidney disorders.

Human kidney stone matrix: Latent potential to restrain COM induced cytotoxicity and inflammatory response.

Kidney stone disease is a multi-factorial disorder resulting from the interplay of various risk factors including lifestyle, environment and genetics along with metabolic activities inside the body. However, it is difficult to determine how these factors converge to promote stone disease. Extensive investigations of kidney stone composition at the molecular level have been carried out however; its impact on the complex mechanism of stone formation is still obscure. Hence, an in vitro study was designed to investigate the attenuation of calcium oxalate toxicity by human kidney stone matrix proteins on NRK-52E cells using flowcytometry, Western blotting, RT-PCR and immunofluorescence assays. Morphological alterations in cell-crystal interaction were assessed using scanning electron microscopy. Microscopic studies showed profound impairment of COM crystal structure as a consequence of protein-crystal interactions. RT-PCR analysis and immunocytochemistry of NRK-52E cells revealed the up-regulation of inflammatory and stress biomarkers OPN and HSP-70, respectively, in response to COM toxicity; which diminished significantly in the presence of kidney stone matrix proteins. The results of present study propose that the mechanism undertaken by matrix proteins to attenuate COM induced cytotoxicity could be attributed to the modulation of crystal structure, which subsequently restraint the inflammatory response and apoptotic cell death. The inference drawn through this study could provide better understanding of the intricate process of kidney stone formation.

Effect of endoplasmic reticulum stress inhibition on hyperoxaluria-induced oxidative stress: influence on cellular ROS sources.

PURPOSE: Hyperoxaluria-induced calcium oxalate crystallisation is associated with the generation of reactive oxygen species (ROS) via mitochondria and NADPH oxidase. Endoplasmic reticulum (ER) has emerged as an organelle which could influence mitochondrial functioning and ROS generation. Plugging an upstream pathway of mitochondrial and NADPH oxidase-induced ROS generation may have better prophylaxis. Therefore, we propose to investigate the linkage of hyperoxaluria-induced ROS generation with ER stress by inhibiting the later with 4-Phenylbutyric acid (4-PBA). METHODS: Male wistar rats were divided into three groups: a normal control group, an ethylene glycol with ammonium chloride-induced hyperoxaluric group (EA) and a third group which has hyperoxaluric animals given 4-PBA at a dose of 300 mg/kg. After 9 days of treatment, animals were sacrificed and renal tissues were analysed for histopathological examination, ROS, mitochondrial dysfunction, ER stress markers, inflammatory markers and NADPH oxidase subunits expression. RESULTS: Hyperoxaluric rats exhibited a significant increase in the levels of ROS, subsequently up-regulated levels of ER stress markers, inflammatory indicators, NADPH oxidase subunits and compromised mitochondrial functioning. However, ER stress amelioration appreciably curtailed the alterations caused by hyperoxaluric abuse. CONCLUSIONS: Therefore, suggesting the major role of ER in hyperoxaluric manifestations thereby providing an opportunity to target ER stress for future therapeutic interventions.

Response surface methodology based extraction of Tribulus terrestris leads to an upsurge of antilithiatic potential by inhibition of calcium oxalate crystallization processes.

Tribulus terrestris has significant antilithiatic efficacy established via both in vitro as well as in vivo studies and is used in numerous anti-urolithiatic herbal formulations viz. Cystone, Uriflow, Uritone and Neeri. However, to fully utilize its antilithiatic potential, the influence of different extraction parameters on antilithiatic ability of T. terrestris aqueous extract needs elucidation. Thus, the current study was undertaken using statistically optimized extraction conditions for aqueous extract preparation. Response surface methodology was employed to observe the influence of three variables i.e. temperature (°C), time (h) and solid: liquid ratio (S: L) on the extraction yield (%) and protein content (mg/g) of T. terrestris aqueous extract. RSM results revealed that the high S:L ratio, low temperature and reduced incubation time were optimal conditions for aqueous extraction. Under such extraction conditions the protein content reached the value of 26.6±1.22 mg/g and the obtained extraction yield was 27.32±1.62%. The assessment of antilithiatic activity of 4 selected extracts (AE1-4), revealed enhanced nucleation and aggregation inhibition of calcium oxalate crystals with AE1 and AE2, which in addition significantly altered the size and morphology of calcium oxalate monohydrate (COM) crystals compared to AE3 and AE4. In vitro cell culture based studies on renal epithelial cells (MDCK, NRK-52E and PK 15) proved that the AE1 showed higher cytoprotective potency by increasing cell viability as compared to the oxalate treated group. The free radical scavenging activity of aqueous extract lowered the reactive oxygen specie's induced damage and potentially reduced the signals of programmed cell death due to oxalate injury. In addition, modulation of the COM crystal morphology was enhanced by AE1 as compared to AE2. The FTIR and GC-MS analysis of AE1, showed the presence of biomolecules which could aid in the attenuation of lithiatic process. In the light of these results the utility of the RSM approach to fully optimize the antilithiatic potential of T. terrestris cannot be undermined.

Implication of hyperoxaluria on osteopontin and ER stress mediated apoptosis in renal tissue of rats.

Hyperoxaluria is a stress that leads to calcium oxalate crystal deposition which further causes inflammation and renal cell necroptosis. Many studies have linked osteopontin expression with apoptosis and inflammation but so far its association with apoptosis with regard to hyperoxaluria is undiscovered. Moreover, a recent report has suggested that osteopontin induces endoplasmic reticulum stress and subsequently apoptosis in myocytes. In this study, the impact of hyperoxaluria on the modulation of osteopontin expression and endoplasmic reticulum (ER) stress mediated apoptosis in rats is explored. Hyperoxaluria was induced in rats by three different doses viz. ethylene glycol alone, ethylene glycol and ammonium chloride together and third group were fed with hydroxyl-l-proline. After hyperoxaluria induction rats were sacrificed and renal tissue was analysed for crystal depositions, osteopontin expression, inflammation, ER stress and subsequent unfolded protein response intermediates (UPR). Altered histoarchitecture of renal tissue and elevated levels of reactive oxygen species (ROS) along with the presence of calcium oxalate crystals were observed in the hyperoxaluric groups. As expected, inflammation and apoptosis was significantly high in all hyperoxaluria groups. Osteopontin expression showed significant up-regulation following hyperoxaluria. Further, a similar trend between expression of osteopontin and elevated ER stress level was observed. Moreover, UPR intermediates expression was also concurrent with osteopontin levels. It is observed that the extent of calcium oxalate crystal deposition is directly associated with the expression of osteopontin, inflammation and ER stress. The results advocate possible association of osteopontin with ER stress, thus suggesting that the ER could be a new target for developing therapeutic regimes for kidney stones.

Cytoprotective and anti-apoptotic role of Terminalia arjuna on oxalate injured renal epithelial cells.

Urolithiasis is one of the painful multifactorial disorders caused by metabolic abnormalities influencing the composition of body fluids and urine. The bark of Terminalia arjuna (T. arjuna), very well known in Ayurveda for the treatment of cardiovascular diseases, possesses antioxidant and diuretic activity. The present study was undertaken to investigate the antiurolithiatic efficacy of aqueous extract of bark of T. arjuna on oxalate-induced injury to renal tubular epithelial cells. Madin-Darby canine kidney (MDCK) cells were exposed to 2 mM oxalate for 48 h to evaluate the protective effect of T. arjuna aqueous extract on cell viability, CaOx crystal adherence and apoptotic changes caused by oxalate. The results confirmed that oxalate injured MDCK cells were protected by T. arjuna extract. On treatment with a range concentrations, the cell viability increased in a concentration dependent manner. Moreover, the extract prevented the interaction of the calcium oxalate (CaOx) crystals with the cell surface and reduced the number of apoptotic cells. The current data suggests that T. arjuna bark confers a cytoprotective role and based on our results it could be a potential candidate from natural plant sources against urolithiasis.