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

Nirlep Chhibera, Tanzeer Kaurb, Surinder Singlaa,*

ABSTRACT

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 1 mg/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.

Keywords: Hyperoxaluria, PKCδ, Rottlerin, NADPH oxidase, Renal function

Introduction

Renal stones are believed to be a form of pathological calcification, which is triggered by reactive oxygen species (ROS) and development of oxidative stress (Khan 2014). High oxalate, as well as crystals of calcium oxalate and calcium phosphate generates ROS, leading to injury, inflammation, alteration in gene expression and mitochondrial dysfunction in renal epithelial cells (Jonassen et al.2005). There is sufficient clinical and experimental data to support that ROS is involved in the formation of calcium oxalate kidney stones (Khan 2014). In case of calcium oxalate nephrolithiasis, an increased production of a variety of crystallization modulating macromolecules takes place. Presence of such macromolecules and ROS induced damage to proteins, lipids, carbohydrates and nucleotides, lead to renal injury and inflammation during stone formation (Khan 2004).
The main contributors of ROS at cellular levels are mitochondria and NADPH oxidase (Meimaridou et al. 2006; Khan et al. 2010). NADPH oxidase is considered as a major source of ROS in kidneys (Geiszt et al. 2000). Oxalate and/or CaOx crystals induced ROS in renal tissue is also primarily produced by NADPH oxidase (Joshi et al. 2013) and mitochondria (Khand et al. 2002). In the view of oxidative imbalance caused by calcium oxalate crystals, a number of studies have pointed out the potential role of antioxidants towards its management. Since, targeting the contributors of ROS is the best strategy, therefore many studies have explored potential of apocynin (inhibitor of NADPH oxidase) (Li et al. 2009) and N-acetyl cysteine (Sharma et al. 2015) to impart protection against hyperoxaluria. Recently PKCδ activation is also suggested to be an important contributor of ROS and rottlerin being its direct/indirect inhibitor showed to protect LLC-PK1 cells from oxalate induced oxidative stress (Thamilselvan et al. 2009).
The plant Mallotus phillipensis (Lam.) Müll.Arg. is well recognised to be an antiurolithiatic plant and is being traditionally used in kidney stone treatment in Indian folklore (Gillespie and Stapleton, 2004). In a study by Mohandas et al. (2015), the vidangadi churna, which contains Mallotus phillipensis (Lam.) Müll.Arg. as one of its constituents, has shown to possess significant antiurolithiatic property. Rottlerin is the major phytochemical isolated from the fruit coverings of this plant and has shown prophylactic properties in other pathological conditions (Maioli et al., 2012). Although initially suggested as PKCδ inhibitor, lately it was revealed that rottlerin is a mitochondrial uncoupler as it depolarizes the mitochondrial membrane potential and reduces cellular ATP levels. Being an uncoupler of mitochondria, rottlerin affects ROS production and interestingly it has both proapoptotic and anti-apoptotic abilities (Soltoff 2007). Moreover, rottlerin inhibits ROS synthesis imposed by various NADPH oxidase isoforms irrespective of directly blocking PKCδ activity (Jagnandan et al. 2007). The antioxidant potential of rottlerin was established in MCF-7 cells as a result of its hydrogen donating ability and free radical scavenger activity (Maioli et al. 2009). Being itself an antioxidant, it is imperative to explore the effect of rottlerin in animals exposed to hyperoxaluria. The present study was designed to investigate the efficacy of rottlerin in combating oxalate induced renal injury and pathways of ROS generation in rats.

Materials and methods

Chemicals

All the chemicals used were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, USA), Merck (Mumbai, India) and Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). Rottlerin (Pubchem CID: 5281847) was purchased from Merck Biosciences (Germany). Primary antibody for PKCδ was purchased from Sigma-Aldrich (St.Louis,MO).

In-vitro redox ability assay

Total redox capability by Fe3+-Fe2+ transformation at different concentrations of rottlerin and hydrogen peroxide scavenging capacity of rottlerin was measured (Aggarwal et al. 2014). Briefly, the ability to reduce potassium ferricyanide [K3Fe(CN)6] was measured at 700 nm. Higher absorbance at 700 nm of the reaction mixture indicated greater reducing power. The hydrogen peroxide scavenging ability was estimated by measuring hydrogen peroxide concentration at 230 nm using the molar extinction coefficient for H2O2 (81 mol−1cm−1). The hydrogen peroxide scavenging ability was calculated by the formula: % scavenging = (1–Ae/Ao) x 100, where Ao = absorbance without sample,and Ae = absorbance with sample. As a positive control, ascorbic acid was used.

Animals and treatment schedule

Healthy male wistar rats weighing between 150 and 200 g of equivalent age groups were obtained from central animal house of Panjab University, Chandigarh, India. The procedures followed were approved by the Institutional Animal Ethics Committee and were in accordance with the Guidelines for Humane Use and Care of Laboratory Animals (PU/IAEC/S/14/41).
To induce CaOx crystal formation, rats were exposed to 0.4% ethylene glycol (EG) with 1.0% ammonium chloride (NH4Cl) in drinking water for 9 days. All rats were randomly divided into the groups having 5-7 rats each. Normal (NRM) rats were provided with standard animal feed and water ad libitum for 9 days. Hyperoxaluric group (HYO) of rats were given 0.4% EG (v/v) with 1.0% NH4Cl (w/v) in drinking water for 9 days. Rottlerin treated (HYR1) rats were administered an intraperitoneal dose of 1mg/kg/day in addition to hyperoxaluric dose of 0.4% EG with 1.0% NH4Cl in their drinking water for 9 days. ROT1 group rats were given intraperitoneal dose of 1 mg/kg/day alone for 9 days. The standardization of the hyperoxaluric rat model was already done in the lab from previous studies (Aggarwal et al. 2014).

Sample collection

At the end of treatment period, rats were placed in metabolic cages and urine was collected for 24 h period having 20 µl of 20 % sodium azide as preservative. A drop of freshly obtained urine was spread on a glass slide and visualized under polarized light using Leica DM 3000 light microscope. Rats were anaesthetized with diethyl ether and sacrificed by decapitation on day 10. Before sacrificing, the blood was taken from orbital sinus into a centrifuge tube to collect serum. After dissection both kidneys were removed and transverse sections were fixed in formaldehyde for histological analysis. The paraffin embedded sections were cut and stained in Delafield’s Hematoxylin and eosin staining and viewed using Leica DM 3000 light microscope.

Biochemical assays in urine and serum

Urinary oxalate level was quantified by the colorimetric method (Hodgkinsons and Williams 1972). Concentration of creatinine was estimated by commercially available kit using manufacturer’s instructions (Erba diagnostics Mannheim, Germany). Alkaline phosphatase (ALP) in serum was determined using commercially available kit (Recombigen laboratories).Urinary lactate dehydrogenase (LDH) was measured by decrease in absorbance at 340 nm resulting from the oxidation of NADH. Creatinine clearance was calculated according to standard clearance formula C = U/S × V, where U = urinary concentration of creatinine, S = concentration of creatinine in the serum and V = urinary volume.

Isolation of mitochondria

The kidney was washed in normal saline at 4 ºC, trimmed of adipose and connective tissues, weighed, and homogenized, (10 % w/v) in buffer containing 0.25 M sucrose, 5 mM HEPES, 1 mM EDTA, and 0.1 % bovine serum albumin pH 7.2. The homogenate was centrifuged at 1000 x g for 5 min to remove the nuclear fraction and cell debris. Mitochondrial pellet was obtained by centrifuging the post-nuclear supernatant at 14,000 x g for 20 min. The pellet was washed thrice with 1.15 % potassium chloride solution and finally suspended in 0.25 M sucrose solution. The purity of mitochondrial preparation was checked by measuring the activity of citrate synthase (Spinazzi et al. 2012).

Measurement of oxidant/antioxidant status in renal tissue and renal mitochondria

Oxidant/antioxidant status in whole renal tissue was determined by assaying malondialdehyde (MDA) content, superoxide dismutase (SOD), catalase (CAT) and redox ratio; and in renal mitochondria by measuring redox ratio, glutathione peroxidase (GPx) and glutathione reductase (GR) by the methods as described previously (Veena et al. 2008; Aggarwal et al. 2014)

Respiratory chain complexes

Activity of respiratory chain complexes I, II and IV was measured by the methods as described previously (Veena et al. 2008).

NADPH oxidase assay

NADPH oxidase assay was performed by the method of Kumar et al. 2002. NADPH has absorbance maxima at 340 nm, and the reduction in absorbance at 340 nm is proportional to the decrease in NADPH through its consumption by the NADPH oxidase. NADPH has an absorption coefficient of 6.22 mM-1cm-1, which was used to calculate the amount of NADPH consumed during the assay.

Immunoblotting of PKCδ

Briefly, cells were broken in ice-cold 10 mM HEPES, pH 7.4, 5 mM MgCl2, 40 mM KCl, 1 mM phenylmethylsulfonyl fluoride and centrifuged at 200 x g to pellet nuclei. Supernatants were centrifuged at 10,000 x g to pellet the heavy membrane fraction containing mitochondria, and the resulting liquid phase was further centrifuged at 100,000 x g to pellet plasma membranes and supernatant represented the cytosolic fraction.
A sample containing 50 µg protein was separated by 12% SDS-polyacrylamide gel electrophoresis along with prestained protein marker (Real Biotech Corporation,Taiwan) at 25 mA. The protein from the gel was transferred onto the Nitrocellulose membrane with a constant current at 200 mA in transfer buffer (25 mM Tris HCl, 192 mM glycine, and 20% (v/v) methanol, pH 8.3) for 2 h. After the electrophoretic transfer, nonspecific binding was blocked by incubating the membrane with 2% BSA in Tris-buffered saline containing Tween (TBST; 25 mM Tris, pH 8.0, 150 mM NaCl, and 0.5% (v/v) and Tween 20) for 3 h at room temperature. The membrane was then probed with primary antibody for PKCδ diluted 1:1,000 in 1% BSA in TBST, with gentle shaking overnight at room temperature. After being washed with TBST, membranes were incubated with the corresponding alkaline phosphatase-conjugated secondary antibody diluted 1:10,000 in 1% BSA/TBST for 2 h. The membranes were washed with TBST, and the proteins were detected with NBT/BCIP. The reaction was stopped by washing the membrane briefly with water and then TBS. The membrane was photographed, and relative intensity was quantitated using Image J software. A ratio of expression of PKCδ in membrane and mitochondria to that of cytoplasm was calculated with respect to expression of β-actin.

Statistical analysis

Data were analyzed by one-way ANOVA and the Tukey’s test for multiple comparisons using GraphPad Prism (version 5.0; San Diego, USA). They are expressed as mean ± SD. Results were considered significant if P ˂ 0.05.

Results

The present study was designed to investigate the efficacy of rottlerin to combat hyperoxaluric manifestations (especially focusing on oxidative imbalance) and to further explore the possible targets of ROS generators in the renal tissue of hyperoxaluric male wistar rats. Ethylene glycol supplemented with ammonium chloride induces hyperoxaluria and calcium oxalate crystals formation in male wistar rats, ethylene glycol metabolizes to oxalate ions and ammonium chloride causes urine acidification, which makes a favorable environment for calcium oxalate crystal formation (Fan et al. 1999). We intended to study the efficacy of rottlerin administered concurrently with EG and NH4Cl. The dosage of rottlerin was selected as 1 mg/kg/day and the said dose was chosen after comparing two dosages 1 mg/kg/day and 2 mg/kg/day for their ability to decrease both the activity of LDH (Suppl. Fig. 1A), and the extent of lipid peroxidation (Suppl. Fig. 1B) in hyperoxaluric rats.

Antioxidant potential of rottlerin

Our foremost aim was to study the ability of rottlerin to act as an antioxidant, for this, we analyzed whether rottlerin- as a molecule, has reducing power (Fig. 1A), and/or hydrogen peroxide scavenging ability (Fig. 1B), which is a feature of most antioxidants. The direct antioxidant ability of rottlerin was compared with known antioxidant ascorbic acid and antioxidant potential of rottlerin was found to be equivalent to ascorbic acid. Results of our in vitro studies and previous studies by various authors assert its antioxidant potential.
Even though rottlerin has in-vitro potential as an antioxidant, to possess similar abilities in in vivo conditions, it was investigated in hyperoxaluria induced oxidative imbalance. To investigate the consequences of rottlerin on oxidative stress induced by EG exposure in renal tissue, we determined the peroxidative injury by measuring malondialdehyde level, redox ratio determined by reduced and oxidized glutathione content in renal tissue and activity of the antioxidant enzymes SOD and CAT. Oxidative imbalance determined by MDA levels (Fig. 2A) and redox ratio (Fig. 2B) were significantly refurbished by rottlerin treatment in hyperoxaluric rats. Activities of antioxidant enzymes (viz. CAT and SOD) which serve to maintain the redox balance of cells were found diminished in HYO group possibly due to excessive oxidative insult (Figs. 2C and D). Interestingly, rottlerin treatment enhanced their activities in HYR1 group.

Amelioration of renal injury and crystal load by rottlerin

Consistent with previous studies by our group (Sharma et al. 2015; Aggarwal et al. 2014) administration of EG and NH4Cl led to induction of hyperoxaluria and renal dysfunction in male wistar rats, as characterized by increased urinary excretion of oxalate and impaired creatinine clearance, respectively (Table 1). Concomitant administration of rottlerin to hyperoxaluric rats maintained creatinine clearance and urinary excretion of calcium and free oxalate ions, but there was no significant change in alkaline phosphatase level in serum.
The histo-morphological analysis of kidney tissue from different groups revealed the presence of extensive calcium oxalate crystal aggregates in the tubules of hyperoxaluric rats (Fig. 3C). Moreover, renal histological analysis of hyperoxaluric rats presented signs of renal injury as revealed by shrunken glomeruli, tubular dilation and increased urinary space (Fig. 3B) as compared to the NRM rats (Fig. 3A). Rottlerin supplementation improved (Fig. 3D) the renal histological architecture and none of the rats in HYR1 showed crystal deposition. The renal histology of ROT1 rats (Fig. 3E) was similar to NRM rats. Furthermore, the urine of all HYO rats (Fig. 4B) showed presence of calcium oxalate crystals (bipyramidal calcium oxalate dihydrate and dumbbell shaped calcium oxalate monohydrate) whereas in the urine of rottlerin treated hyperoxaluric rats (Fig. 4C) fewer crystals and that too mainly calcium oxalate dihydrate were spotted. NRM (Fig. 4A) and ROT1 (Fig. 4D) group of rats presented no crystalluria.

Impact of rottlerin on pathways of ROS generation

Hyperoxaluria induced renal injury is associated with oxidative burst involving production of superoxide radicals and other reactive oxygen species (Khan 2014) . NADPH oxidase is one of the major sources of oxidative stress in hyperoxaluria as it produces superoxide radicals and promotes inflammation resulting in calcium oxalate deposition in the renal tissue (Joshi et al. 2013). To further unravel the mechanism underlying the effects of rottlerin on hyperoxaluric rats, the effect of rottlerin on NADPH oxidase activity was studied. Simultaneous treatment of the hyperoxaluric rats with rottlerin led to a significant reduction in the activity of NADPH oxidase enzyme (Fig. 5).
Another major source of ROS is mitochondrial electron transport chain (ETC) and we hypothesized that another mechanism by which oxalate induces free radical generation, might be due to faulty electron transport chain. The respiratory complexes of ETC in mitochondria are responsible for the leakage of reactive oxygen species and on investigating the activity of these respiratory complexes, the HYO group showed (Table 2), altered activity of all three complexes studied (viz. Complex I, Complex II, Complex IV), and rottlerin treatment lead to an improved activation of mitochondrial complex I and IV, whereas complex II activity could not be restored by rottlerin treatment in HYR1 group. Recently we have reported that hyperoxaluria induced ROS oxidizes mitochondrial redox environment (reflected by oxidation state of GSH and glutathione disulphide redox pair) and antioxidant N-acetyl cysteine can normalize it (Sharma et al. 2015). To determine whether rottlerin-being an antioxidant, has an ability to maintain mitochondrial redox environment, ratio of GSH/GSSG and activity of glutathione reductase and peroxidase enzymes were observed. It was found that rottlerin could significantly stimulate mitochondrial redox environment (Fig. 6A) as compared to hyperoxaluric rats. Moreover, activities of glutathione reductase (Fig. 6B) and glutathione peroxidase (Fig. 6C) enzymes that are vital for maintaining redox environment were also significantly restored by rottlerin dosage.
Recent investigations have supported the crucial role of PKCδ in hyperoxaluria induced ROS, therefore its translocation from cytosol to membrane was studied. Although rottlerin is not a specific inhibitor of PKCδ ,but an increasing number of studies report it to affect PKCδ translocation. Similarly, in our study the dosage of 1 mg/kg/day rottlerin significantly inhibited PKCδ translocation to membrane (Figs. 7A and B). Furthermore, translocation of PKCδ to mitochondria from cytosol was also analysed (Figs. 7A and C), which revealed that translocation to mitochondria was also evident in hyperoxaluria and to our revelation, rottlerin at the given dose inhibited this translocation significantly as well.

Discussion

Rottlerin, has exhibited antioxidant properties in a diverse array of pathological conditions but the underlying molecular mechanism by which rottlerin suppresses oxidative burden has been dubious. Initially considered as direct inhibitor of PKCδ, its property of being a mitochondrial uncoupler was actually responsible for inhibiting kinases. Rottlerin interferes in oxidative and inflammatory pathways, modulating expression of several enzymes like DNA methyltransferase, cyclooxygenase, lipoxygenase and transcription factors like NF-κB (Torricelli et al. 2008; Maioli et al. 2012). Moreover, rottlerin- as a molecule exhibits free radical scavenging properties. Rottlerin has hydrogen donating groups, which is a common feature of antioxidant molecules (Longpre et al. 2008). In our study we found that rottlerin also has reducing potential and hydrogen peroxide scavenging ability comparable with ascorbic acid. The antioxidant potential of rottlerin may be accredited to the presence of five hydroxyl groups in its structure, which scavenge free radicals like other naturally occuring plant derived polyphenols such as genistein, quercetin, curcumin, and resveratrol (Maioli et al. 2009).
Oxalate and/or calcium oxalate crystals exposure causes ROS generation which further lead to disruption of renal epithelial cells and consequently results in stone deposition in renal tissue. Several antioxidants like vitamin E, N-acetylcysteine and phycocynin have been used to ameliorate oxidative burden caused by oxalate exposure in renal tissue (Thamilselvan et al. 2005, Bijarnia et al. 2009 and Farooq et al. 2004). In our study, the antioxidant property of rottlerin in hyperoxaluric rats strengthens the concept that rottlerin is a ROS quencher. Moreover, rottlerin also ameliorated renal cell injury (depicted by increased leakage of LDH and ALP into urine and serum) resulted by oxidative burden in hyperoxaluric rats. ROS generation in hyperoxaluric condition is regulated by a number of signalling pathways and here we studied the role of rottlerin in the cascade that mediates oxidative stress.
Although rottlerin is not a direct inhibitor of PKCδ but still it has the ability to inhibit PKCδ with an IC50 of 3-6 µM (Gschwendt et al. 1994). Being a mitochondrial uncoupler, it reduces ATP production and thus blocks phosphorylation of kinases (Soltoff 2007). Translocation of PKCδ from cytosol to membrane is required for its function and we found that hyperoxaluria enhances this translocation, which is markedly hindered by rottlerin. It is suggested that, rottlerin inhibits PKCδ translocation by inhibiting PKCδ tyrosine phosphorylation and/or causes PKCδ cleavage by caspase-3 activation. PKCδ, as a regulator of the NADPH oxidase, is required for complex assembly of the enzyme’s components and is one of the essential mechanisms responsible for peroxidative cell injury in hyperoxaluria, which paves way for calcium oxalate adhesion, aggregation and growth of kidney stones (Thamilselvan et al. 2009).
As stated earlier, NADPH oxidase is a major contributor of superoxide (O2−∙) radicals in both renal cortex and medulla. Inhibiting NADPH oxidase by apocynin or atrovastatin treatment reduced the production of ROS and renal deposition of CaOx crystals in hyperoxaluric rats (Li et al.2009). PKCs, specifically PKCδ, are involved in activation of NADPH oxidase by phosphorylating its cellular subunit p47phox and inducing its membranous translocation. Rottlerin interferes in p47phoxtranslocation from the cytosol to the membrane in ATPγS stimulated A549 cells (Cheng et al. 2013). Another possible reason for reduction in NADPH oxidase activity, could be attributed to inhibition of PKCδ, which acts upstream of NADPH oxidase. Additionally, according to a report by Byun et al (2008), rottlerin inhibits NADPH oxidase activity through the down-regulation of GTPbound Rac1, and subsequent suppression of mitochondrial O2- production.
Mitochondrion is regarded as another source of ROS in hyperoxaluric rats. Kohda et al. (2005) hypothesized that the proteins of mitochondrial respiratory chain complexes become phosphorylated by the transolcated PKCδ, resulting in enhancement of the mitochondrial ROS production, leading to leakage of electrons from the ETC. Accordingly, in our study, there was a decrease in the activities of electron transport chain enzymes and in redox environment of mitochondria, which was replenished by rottlerin.
Taken together, rottlerin may represent a potential phytochemical in combating the predominant pathways (mitochondrial dysfunction and oxidative abuse) involved in the pathogenesis of calcium oxalate stone formation. The phytotherapeutic actions of rottlerin could be attributed to its polyphenolic structure and its ability to interfere in the signalling pathways operating in a hyperoxaluric environment.Our study for the first time confirms that in hyperoxaluric animals, rottlerin treatment leads to reduced renal cellular injury, diminished crystalluria, reduced NADPH oxidase activity as well as a decline in translocation of PKCδ from cytoplasmic fraction to the membrane and mitochondrial fraction, suggesting an inhibition of PKCδ activity .

References

Aggarwal, D., Kaushal, R., Kaur, T., Bijarnia, R.K., Puri, S. and Singla, S.K., 2014. The most potent antilithiatic agent ameliorating renal dysfunction and oxidative stress from Bergenia ligulata rhizome. J Ethnopharmacol. 158, 85-93.doi:10.1016/j.jep.2014.10.013.
Aggarwal, K., and Varma, R., 2012. Some ethnomedicinal plants of Bhopal district used for treating stone diseases. Int J Pharm Life Sci. 3(1) : 1356-1362.
Bijarnia, R.K., Kaur, T., Singla, S.K. and Tandon, C., 2009. Oxalate-mediated oxidant–antioxidant imbalance in erythrocytes: role of N-acetylcysteine. Hum Exp Toxicol. 28(4), 245-251.doi: 10.1177/0960327109104825.
Byun, H.S., Won, M., Park, K.A., Kim, Y.R., Choi, B.L., Lee, H., Hong, J.H., Piao, L., Park, J., Kim, J.M. and Kweon, G.R., 2008. Prevention of TNF-induced necrotic cell death by rottlerin through a Nox1 NADPH oxidase. Exp Mol Med. 40(2), 186-195. doi: 10.3858/emm.2008.40.2.186.
Cheng, S.E., Lee, I.T., Lin, C.C., Wu, W.L., Hsiao, L.D. and Yang, C.M., 2013. ATP mediates NADPH oxidase/ROS generation and COX-2/PGE2 expression in A549 cells: role of P2 receptordependent STAT3 activation. PLoS ONE. 8(1), p.e54125.doi: 10.1371/journal.pone.0054125.
Fan, J., Glass, M.A. and Chandhoke, P.S., 1999. Impact of ammonium chloride administration on a rat ethylene glycol urolithiasis model. Scanning Microsc.13(2-3), 299-306.
Farooq, S.M., Asokan, D., Kalaiselvi, P., Sakthivel, R. and Varalakshmi, P., 2004. Prophylactic role of phycocyanin: a study of oxalate mediated renal cell injury. Chem Biol Interact. 149(1), 1-7. doi:10.1016/j.cbi.2004.05.006.
Geiszt, M., Kopp, J.B., Várnai, P. and Leto, T.L., 2000. Identification of renox, an NAD (P) H oxidase in kidney. Proc. Natl. Acad. Sci. USA. 97(14), 8010-8014. doi: 10.1073/pnas.130135897
Gillespie, R.S., and Stapleton, F. B., 2004. Nephrolithiasis in children. Pediatr Rev, 25(4 ); 131139
Gschwendt, M., Muller, H.J., Kielbassa, K., Zang, R., Kittstein, W., Rincke, G. and Marks, F., 1994. Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun. 199(1), 9398.doi: 10.1006/bbrc.1994.1199.
Hodgkinson, A. and Williams, A., 1972. An improved colorimetric procedure for urine oxalate. Clin Chim Acta. 36(1), 127-132. doi:10.1016/0009-8981(72)90167-2.
Jagnandan, D., Church, J.E., Banfi, B., Stuehr, D.J., Marrero, M.B. and Fulton, D.J., 2007. Novel mechanism of activation of NADPH oxidase 5 calcium sensitization via phosphorylation. J Biol Chem. 282(9), 6494-6507. doi:10.1074/jbc.M608966200.
Jonassen, J.A., Kohjimoto, Y., Scheid, C.R., Schmidt, M., 2005. Oxalate toxicity in renal cells. Urol Res. 33(5), 329-339. doi: 10.1007/s00240-005-0485-3.
Joshi, S., Peck, A.B. and Khan, S.R., 2013. NADPH oxidase as a therapeutic target for oxalate induced injury in kidneys. Oxi Med Cell Longev. 2013, 1-18. doi:10.1155/2013/462361.
Khan, S.R., 2004. Crystal-induced inflammation of the kidneys: results from human studies, animal models, and tissue-culture studies. J Clin Exp Nephrol. 8(2), 75-88. doi: 10.1007/s10157-004-02920.
Khan, S.R., Khan, A. and Byer, K.J., 2010. Temporal changes in the expression of mRNA of NADPH oxidase subunits in renal epithelial cells exposed to oxalate or calcium oxalate crystals. Nephrol Dial Transplant. p.gfq692. doi: 10.1093/ndt/gfq692.
Khan, S.R., 2014. Reactive oxygen species, inflammation and calcium oxalate nephrolithiasis. Transl Androl Urol. 3(3), 256-276. doi:10.3978/j.issn.2223-4683.2014.06.04.
Khand, F.D., Gordge, M.P., Robertson, W.G., Noronha-Dutra, A.A. and Hothersall, J.S., 2002. Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells. Free Radic Biol Med. 32(12), 1339-1350. doi: 10.1016/S0891-5849(02)00846-8.
Kohda, Y. and Gemba, M., 2005. Cephaloridine Induces Translocation of Protein Kinase C. DELTA. Into Mitochondria and Enhances Mitochondrial Generation of Free Radicals in the Kidney Cortex of Rats Causing Renal Dysfunction. J Pharmacol Sci. 98(1), 49-57. doi:10.1254/jphs.FP0040926.
Kumar, P., Pai, K., Pandey, H.P. and Sundar, S., 2002. NADH-oxidase, NADPH-oxidase and myeloperoxidase activity of visceral leishmaniasis patients. J Med Microbiol. 51(10), 832-836. doi: 10.1099/0022-1317-51-10-832.
Li, C.Y., Deng, Y.L. and Sun, B.H., 2009. Effects of apocynin and losartan treatment on renal oxidative stress in a rat model of calcium oxalate nephrolithiasis. Int Urol Nephrol. 41(4), 823-833. doi: 10.1007/s11255-009-9534-0.
Longpre, J.M. and Loo, G., 2008. Protection of human colon epithelial cells against deoxycholate by rottlerin. Apoptosis. 13(9), 1162-1171.doi: 10.1007/s10495-008-0244-3
Maioli, E., Greci, L., Soucek, K., Hyzdalova, M., Pecorelli, A., Fortino, V., Valacchi, G. and Bandiera, S.M., 2009. Rottlerin inhibits ROS formation and prevents NFB activation in MCF-7 and HT-29 cells. J Biomed Biotechnol. 2009, 1-7.doi:10.1155/2009/742936.
Maioli, E., Torricelli, C. and Valacchi, G., 2012. Rottlerin and curcumin: a comparative analysis. Ann. N.Y. Acad. Sci. 259(1), 65-76. doi: 10.1111/j.1749-6632.2012.06514.x.
Meimaridou, E., Lobos, E. and Hothersall, J.S., 2006. Renal oxidative vulnerability due to changes in mitochondrial-glutathione and energy homeostasis in a rat model of calcium oxalate urolithiasis. Am J Physiol Renal Physiol. 291(4), F731-F740. doi: 10.1152/ajprenal.00024.2006.
Mohandas, S., Nizar, S., Radhika, P., 2015. Evaluation of in vitro antiurolithiatic activity of Vidangadi Churna. World J Pharm Pharm Sci. 4(10), 800-807.
Sharma, M., Kaur, T. and Singla, S.K., 2015. Protective effects of N-acetylcysteine against hyperoxaluria induced mitochondrial dysfunction in male wistar rats. Mol Cell Biochem. 405(12),105-114. doi: 10.1007/s11010-015-2402-6.
Soltoff, S.P., 2007. Rottlerin: an inappropriate and ineffective inhibitor of PKCδ. Trends Pharmacol Sci. 28(9), 453-458. doi:10.1016/j.tips.2007.07.003.
Spinazzi, M., Casarin, A., Pertegato, V., Salviati, L. and Angelini, C., 2012. Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nat Protoc. 7(6), 1235-1246. doi:10.1038/nprot.2012.058.
Thamilselvan, S. and Menon, M., 2005. Vitamin E therapy prevents hyperoxaluria‐induced calcium oxalate crystal deposition in the kidney by improving renal tissue antioxidant status. BJU Int. 96(1), 117-126. doi: 10.1111/j.1464-410X.2005.05579.x.
Thamilselvan, V., Menon,M. and Thamilselvan, S.,2009. Oxalate induced activation of PKC-alpha and -delta regulates NADPH oxidase-mediated oxidative injury in renal tubular epithelial cells. Am J Physiol Renal Physiol. 297, F1399–F1410. doi: 10.1152/ajprenal.00051.2009.
Torricelli, C., Fortino, V., Capurro, E., Valacchi, G., Pacini, A., Muscettola, M., Soucek, K. and Maioli, E., 2008. Rottlerin inhibits the nuclear factor κB/Cyclin-D1 cascade in MCF-7 breast cancer cells. Life Sci. 82(11), 638-643. doi:10.1016/j.lfs.2007.12.020.
Veena, C. K., Josephine, A., Preetha, S. P., Rajesh, N. G., and Varalakshmi, P., 2008. Mitochondrial dysfunction in an animal model of hyperoxaluria: a prophylactic approach with fucoidan. Eur J Pharamacol. 579, 330–336. doi: 10.1016/j.ejphar.2007.09.044.