the term acute renal failure ( arf ) is currently substituted by acute kidney injury ( aki ) .
aki is a reversible condition in which there is a sudden decline in renal function , manifested by hourly / daily / weekly elevation in serum creatinine and blood urea nitrogen ( bun ) .
different organizations such as acute dialysis quality initiative ( adqi ) , acute kidney injury network ( akin ) and kidney disease international global outcome ( kdigo ) have provided different definitions for acute kidney injury . among these definitions of aki
, the most acceptable one is that of kdigo : aki is any of the followingincrease in serum creatinine ( scr ) by 0.3  mg / dl ( 26.5 
orincrease in scr to 1.5 times baseline , which is known or presumed to have occurred within the prior 7 days 
orurine volume < 0.5  ml / kg / h for 6  h  .
 increase in serum creatinine ( scr ) by 0.3  mg / dl ( 26.5 
mol / l ) within 48  h  or  increase in scr to 1.5 times baseline , which is known or presumed to have occurred within the prior 7 days  or  urine volume < 0.5  ml / kg / h for 6  h  .
the incidence of aki in the community is 2147 and 4085 per million populations per year ( pmp ) in developing and developed nations.4 , 5 recent reports in the developed world indicate that aki is seen in 3.29.6% of hospital admissions with overall mortality of 20%50% in icu patients.6 , 7 aki demanding renal replacement therapy is 56% with a high in - hospital mortality rate of 60% .
it is estimated that nearly 2 million people die of aki every year globally.8 , 9 those who survive aki are at a greater risk for later development of chronic kidney disease ( ckd ) .
the causes of aki could be pre - renal , renal and post - renal ones .
pre - renal failure , the commonest form of acute renal failure , is due to decrease in renal blood flow primarily as a result of hypovolemia .
it is reversible if the cause of the decreased renal blood flow can be identified and rectified before kidney damage occurs .
intrinsic renal causes of aki include diseases that affect the renal parenchyma which can be divided based on the compartment of the kidney that is affected like tubular injury , tubulointerstitial diseases , diseases of the renal microcirculation and glomeruli and diseases of larger renal vessels .
post - renal causes of aki are diseases associated with urinary tract obstruction which account for 5% of renal failures .
the management of arf is mainly supportive and in refractory patients renal replacement therapy ( rrt ) may be the best option .
rrt refers to life supporting measures which include hemodialysis , peritoneal dialysis , hemofiltration and renal transplantation . in spite of rrt ,
mortality in patients with aki remains high and this may be due to the severity of the disease or the adverse effects of rrt . as there is no effective pharmacotherapy for aki ,
more than 221 plants have been screened for nephroprotective activity both in acute and chronic renal failure models . some of the medicinal plants such as ocimum sanctum , aerva lanata , aegle marmelos , pongamia pinnata , salviae radix , ginkgo biloba , allium sativum , cassia auriculata , nigella sativa , drynaria fortune , tribulus terrestris and others are reported to have nephroprotective activity .
azima tetracantha ( at ) has been traditionally used for many diseases including renal disease .
it belongs to the family , salvadoraceae and known as mulchangu in siddha and kundali in ayurvedha .
its common names are needle brush ( english ) , kantagurkamai ( hindi ) and cankakiranam ( tamil ) . at
has been reported to have antimicrobial , antifungal , anti - inflammatory , analgesic , antioxidant , antipyretic , antiulcer , anticancer , anti - snake venom , diuretic and hepato - protective activities.19 , 20 , 21 , 22 , 23 though at is traditionally used for nephroprotective effect , it has not been evaluated by either preclinical or clinical studies .
hence this study was undertaken to evaluate its action in animal models of renal failure .
root and fruit : glycosides  n - methoxy-3-indolylmethyl - glucosinolate , iodole glucosinolate and n - methoxy indole 3- corbinol .
root and stem : flavonoids  myricetin , quercetin , rutin , isorhamnetin , rhamnetin and rhamnazin .
the objectives were to evaluate the nephroprotective activity of ethanolic extract of a. tetracantha root in glycerol - induced acute renal failure in wistar albino rats and to find out the probable mechanism .
chemicals for in  vitro and in  vivo antioxidants assays were purchased from sigma aldrich , new delhi , india .
the plant was collected from tindivanam , villupuram district , tamil nadu , india and it was identified and authenticated by dr .
narasimhan , associate professor of botany , madras christian college , chennai , tamil nadu .
one kg of roots was thoroughly washed with water and air dried under shade at room temperature for 30 days and milled into a coarse powder .
the dried powder obtained was subjected to continuous extraction with 95% v / v ethanol in soxhlet apparatus for 15 cycles .
thirty healthy male wistar albino rats weighing between 150 and 250  g were obtained from the central animal house of chettinad hospital and research institute .
the antioxidant activity of atr ethanolic extract was evaluated by 2,2-diphenyl-1-picrylhydrazyl ( dpph ) assay , nitric oxide ( no ) assay , hydrogen peroxide ( h2o2 ) and ferric reducing antioxidant power ( frap ) assay .
all assays were performed using spectrophotometer and the values were calculated by the following formula.percentageinhibition=absorbanceofcontrolabsorbanceoftest100absorbanceofcontrol the atr extract was prepared in increasing concentrations from 25 to 1600  g / ml for all the antioxidant assays .
one ml of atr extract solution was mixed with 1  ml of ethanolic solution of dpph ( 200  m ) .
another 1  ml dpph solution was mixed with 1  ml of ethanolic solution of vitamin- c ( 200  g / ml ) .
a mixture of 1  ml of ethanolic solution of dpph ( 200  m ) and 1  ml of ethanol served as control . after mixing ,
all the solutions were incubated in dark for 2  min and absorbance was measured at 517  nm .
the experiments were carried out in triplicate and the average percentage of inhibition was calculated by the above formula .
three ml of 10  mm sodium nitroprusside in phosphate buffer is added to 2  ml of atr extract and the reference compound .
. 5  ml of the incubated sample and 5  ml of griess reagent ( 1% sulphanilamide , 0.1% naphthyl ethylenediamine dihydrochloride in 2% phosphorous acid ) were added and absorbance of the chromophore formed was measured at 540  nm .
two ml atr extract was mixed with 0.6  ml of 4  mm hydrogen peroxide solution prepared in phosphate buffer ( 0.1  m ph 7.4 ) and incubated for 10  min .
the assay mixture contained 2.5  ml of 300  mm acetate buffer at ph 3.6 , 0.25  ml of 10  mm 2 , 4,6-tripyridyl - s - triazine solution in 40  mm hcl , 0.25  ml of 20  mm ferric chloride and atr extract at various concentrations in 0.1  ml of methanol .
frap values were measured by comparing the absorption change in the test mixture with that obtained from increasing concentrations of fe .
female rats ( 150200  g ) , nulliparous and non - pregnant were randomly distributed to four groups ( 14 ) of 3 each .
the rats were fasted overnight and given orally 5 , 50 , 300 and 2000  mg / kg of atr extract .
they were observed continuously for the first 2  h and hourly for the next 6  h and at 24 , 48 , and 72  h for general behavior , convulsions and mortality .
no mortality or toxicity was observed up to the dose of 2000  mg / kg .
based on the acute toxicity studies 250 and 500  mg / kg of the ethanolic extract were used for the current study .
induction of arf : arf was induced by intramuscular injection of single dose of 50% hypertonic glycerol ( 8  ml / kg ) into both the hind limbs in equal volume as per the standard methods prescribed.30 , 31 rats were divided into 5 groups of 6 animals in each .
group 1 was treated with normal saline ( 10  ml / kg ) per oral , group 2 with single dose of hypertonic glycerol ( 8  ml / kg ) by intramuscular injection into both the hind limbs , group 3 with glycerol and atr extract 250  mg / kg , group 4 with glycerol and atr extract 500  mg / kg and group 5 was treated with atr extract 500  mg / kg alone .
the atr extract was given by oral route 60  min prior to glycerol injection .
urine output was measured for all the animals for 24  h. after 24  h , the blood samples were collected by retro orbital sinus puncture and tested for serum creatinine , blood urea nitrogen , total proteins and albumin .
the antioxidants , superoxide dismutase ( sod ) , reduced glutathione ( gsh ) , glutathione reductase ( gr ) , glutathione peroxidase ( gpx ) , and catalase ( cat ) were determined using standard methods prescribed .
kidneys were rinsed with normal saline and then fixed in 10% neutral buffered formalin solution , embedded in paraffin and used for histopathological examination .
the sections were 2  m thick , deparaffinized , hydrated and stained with haematoxylin and eosin .
the renal sections were examined for the extent of damage to glomeruli , tubules and interstitium as well as for capillary congestion and hemorrhage .
one way analysis of variance ( anova ) traced by tukey 's multiple range test was applied to calculate the statistical significance among different groups .
the results from hptlc finger print scanned at wavelength 254  nm for ethanolic extract of atr showed 15 polyvalent phytoconstituents ( graph ) and corresponding retarding fraction ( rf ) , height , area and lambda max  ( table  1 ) .
ethanolic extract of a. tetracantha root in graded concentrations was tested for antioxidant activity in four different in  vitro methods and compared with the standard antioxidant , vitamin c. percentage of inhibition of dpph radicals by different concentrations of atr extract was 46% at 25  g / ml and 89% at 1600  g / ml .
standard drug vitamin- c showed 50 % inhibition at 25  g / ml and 95% at 1600  g / ml ( table  2 ) .
the percentage inhibition of no by atr extract was 24% at 25  g / ml and 91% at 1600  g / ml .
vitamin - c showed 28 % and 95% inhibition at 25 and 1600  g / ml respectively  ( table  3 ) .
atr extract showed 23% and 88% at 25 and 1600  g / ml and vitamin c at the same concentration showed 28 and 99% of inhibition  ( table  4 ) .
atr extract had shown 22% inhibition at 25  g / ml and 87% inhibition at 1600  g / ml .
vitamin - c showed 28 % and 98% inhibition at 25 and 1600  g / ml respectively  ( table  5 ) .
nephroprotective activity was assessed by the levels of serum creatinine , blood urea nitrogen , total proteins , albumin and urine output as well as histopathological changes ( table  6 ) .
increased levels of serum creatinine , bun and decreased total proteins , albumin and urine output in glycerol treated animals were seen ( group 2 ) compared with normal saline treated animals ( group 1 ) confirming renal failure . in atr
extract treated groups there was a significant decrease ( p  <  0.05 ) in serum creatinine , blood urea nitrogen and increase in total proteins , albumin and urine output at both the doses of 250 and 500  mg / kg . the group treated with atr extract alone ( group 5 ) did not show any change in the above parameters .
sod , gsh , gr , gpx and cat levels were significantly ( p  <  0.05 ) increased in group 3 and 4 compared with group 2 .
atr extract at 500  mg / kg had shown higher antioxidant levels than 250  mg / kg ( fig .  2 , in  vivo antioxidant activity ) .
rat kidney in the control group had shown normal glomeruli with intact bowman 's capsule , normal proximal convoluted and distal convoluted tubules and there was no capillary congestion or hemorrhage ( group 1 ) .
severe distortion of glomeruli , capillary congestion , hemorrhages and apical blebbing were seen in glycerol treated group indicating renal damage . in group 3 and 4 ,
treated with 250 and 500  mg / kg of atr extract showed markedly reduced capillary congestion , tubular damage and glomerular distortion compared to glycerol treated group .
the protective effect was found to be better with 500  mg / kg than 250  mg / kg .
group 5 animals were not sacrificed as they did not show abnormalities in biochemical parameters .
the present study has evaluated the effect of a. tetracantha root extract in glycerol - induced acute renal failure and its antioxidant potential both in  vitro and in  vivo .
glycerol - induced acute renal failure is one of the commonest animal models used for evaluation of nephroprotective activity.30 , 31 the mechanisms involved in glycerol - induced renal failure include oxidative stress , inflammation and apoptosis38 , 39 which lead to rhabdomyolysis resulting in myoglobinemia , myoglobinuria and cast formation .
rhabdomyolysis causes cytokine activation and release of inflammatory mediators such as il-1 , icam-1 and tnf. these pro - inflammatory cytokines mediate the inflammatory reaction causing afferent and efferent renal arteriolar vasoconstriction eventually leading to renal failure . in general , about 1040% of patients with rhabdomyolysis develop arf .
as oxidative stress and inflammatory damage have been mainly attributed to the development of acute kidney injury , any compound or drug having antioxidant & anti - inflammatory activity will have a protective action against renal damage . in our study
atr extract has shown a protective effect evidenced by its inhibition of alteration of renal biochemical parameters and reversal of histopathological changes .
prior administration of atr extract has caused reduction in serum creatinine , blood urea nitrogen and increase in total proteins and albumin .
the histopathological examination has shown significant reduction in cellular infiltration , capillary congestion , glomerular damage , tubular necrosis and cast formation induced by glycerol .
the possible mechanisms for the nephroprotective effect of atr extract could be due to the antioxidant and anti - inflammatory actions .
the root extract has shown antioxidant activity both in  vitro and in  vivo assays .
the in  vivo antioxidant effect of ethanolic extract atr on the levels of sod , gsh , gpx , gr and cat is shown in fig .  2 .
glycerol treatment has reduced the levels of these antioxidants , which get utilized in scavenging the free radicals generated during glycerol - induced oxidative damage of renal parenchyma .
atr extract has reduced the oxidative damage as evidenced by the dose dependent increase in the level of the antioxidants .
the importance of protecting the antioxidants pool in preventing renal damage has been emphasized by singh et  al .
the in  vitro antioxidant effect exhibited in different assays has further supported the antioxidant property of atr extract .
the phytochemicals found to be present in the root extract are the flavonoids , terpinoids , alkaloids , tannins , saponins & glucosinolates.45 , 46 among them tannins , triterpinoids , flavonoids and saponins could be responsible for antioxidant property as these phytoconstituents are already reported to have antioxidant activity .
the anti - inflammatory activity could be due to the presence of flavonoids which are well known to have anti - inflammatory action .
this could be a reason for using atr extract as a diuretic agent in traditional systems .
hence terpinoids , tannins and flavonoids present in the root extract may have contributed to the protection from renal damage induced by glycerol by their anti - inflammatory , antioxidant and vasodilatory as well as diuretic actions .
freidelin has been established to have antioxidant , free radical scavenging , hepato - protective ant - inflammatory , analgesic and antipyretic activities .
beta sitosterol , also a terpinoid has been reported to have antipyretic and anti - inflammatory activity .
the glucosinolate and n - hydroxy-3-indolylmethyl - glucosinolate were detected in atr and not in the other parts of the plant .
glucosinolates have vasodilatory effect like tannins and flavonoids.51 , 52 renal vasodilatation by these components can improve renal blood flow , reduce the ischemic changes and also increase urine output .
thus the in  vitro and in  vivo findings suggest that protection from oxidative damage , anti - inflammatory and vasodilatory actions of the various phytochemicals present in atr extract could have contributed to the nephroprotective actions of a. tetracantha root extract in acute renal failure . between the 2 doses
administered , 250 and 500  mg / kg , 500  mg / kg is found to have significant nephroprotective action .
the ethanolic extract of atr is found to have nephroprotective effect in glycerol - induced acute renal failure in wistar albino rats .
atr extract is found to have significant action at 500  mg / kg compared to 250  mg / kg . the possible mechanisms could be the antioxidant , anti - inflammatory , vasodilatory and diuretic activity of its phytochemical constituents such as flavonoids , terpinoids , tannins , glucosinolate and saponins .
 the gravity of the impact of renal failure on human health is well known and as there is no specific pharmacotherapy for renal failure , the current study was undertaken to evaluate the effect of root extract of azima tetracantha , an ancient medicinal plant used in siddha and ayurvedhic systems of medicine.the experiment was done in glycerol - induced acute renal failure in wistar albino rats . 
 thirty rats were divided into five groups . 
 group 1 was given normal saline ( 10  ml / kg ) per oral , group 2 with single dose of hypertonic glycerol ( 8  ml / kg ) by intramuscular injection into the hind limbs , group 3 with glycerol and ethanolic extract of a. tetracantha root ( atr ) 250  mg / kg , group 4 , glycerol and atr 500  mg / kg and group 5 , 500  mg / kg atr . 
 extract was given orally 60  min prior to glycerol injection . 
 24  h urine output , serum creatinine , blood urea nitrogen , total proteins and albumin were measured for all the groups . 
 kidneys were examined for histopathological changes.the antioxidant activity of the extract was tested in  vitro and in  vivo . 
 rats treated with atr showed significant improvement in biochemical parameters and histopathological changes compared to glycerol treated group . 
 the protective effect was highly significant at 500  mg / kg . 
 both in  vitro and in  vivo assays showed significant antioxidant activity . 
 the in  vitro activity was comparable to vitamin-c.the ethanolic extract of atr has nephroprotective effect in glycerol - induced acute renal failure and the mechanism of action could be the antioxidant effect . 
Introduction
Objectives
Materials and methods
Results
Discussion
Conclusion
Conflicts of interest