To our knowledge, this is the first study to demonstrate the renoprotective effect of PE extract to prevent the development of renal dysfunction and pathological destruction as assessed by biochemical and markers of oxidative stress in a rat model of CI-AKI. Prophylaxis with PE extract at doses of 250 and 500 mg/kg/day markedly suppressed renal tubular injuries and improved antioxidant activity in CI-AKI rats.
The nephrotoxic effects of radiocontrast media have been reported in many experimental studies [21, 24, 29] and we followed the same methodology as reported elsewhere [20, 21, 30]. The histopathological studies confirmed significant acute tubular necrosis, vacuolization, loss of brush borders, proteinaceous cast formation and interstitial edema were demonstrated similar to previous studies in this nephropathy model [20, 21, 24, 30]. We also found that serum BUN and Cr levels increased significantly in the CM group compared with the control group, supporting the nephrotoxicity of radiocontrast media to renal function. Pretreatment with PE extract demonstrated clearly its renoprotetctive effect by attenuating the severity of renal pathological damage and improving renal function with the dose dependent renoprotective effect of PE extract.
Oxidative stress has been proposed as one of the most important mechanisms in the pathogenesis of CI-AKI . In vitro and in vivo studies demonstrate clearly that iodinated contrast media administration enhances hypoxia and increases the production of ROS within the kidney [21, 24, 32]. Lipid peroxidation is initiated as a result of the ROS induced abstraction of hydrogen in cellular membranes, which results in the formation of relatively stable compounds such as MDA. The production of free radicals is blocked by endogenous antioxidant systems such as TAC, SOD and CAT enzymes [21, 24]. Thus, oxidative stress induced by contrast media during AKI is a relative excess of oxidants caused by increased free radical production and/or decreased antioxidant defense systems [20, 21, 24]. In the present study, increased MDA and decreased TAC, SOD and CAT enzyme activities in serum and renal tissues exposed to CM suggests that these enzymes were consumed due to increased oxidative stress. Decreased SOD and CAT scavenger activities results in higher H2O2 concentration and explains the change in the level of MDA during CI-AKI. The higher of antioxidant enzyme SOD and CAT activities and lower MDA in the PE extract pretreatment might be a response for renal injury after contrast administration. Thus, rats that receive PE extract before CM had decreased MDA and increased SOD and CAT activities in renal tissues could protected against CI-AKI from ROS. Moreover, the increased MDA and decreased TAC in rat plasma that exposed to CM reflect an increase in lipid peroxidation and decrease antioxidation in the systemic response. Thus, decreasing oxidative stress injury following PE extract application has been associated with antioxidant effects in circulation system.
Current recommendations to decrease the incidence of CI-AKI in high risk patients are hydration with saline, use of iso-osmolar contrast agents, minimum volume of contrast media, and avoiding nephrotoxic drugs [6, 33]. Recent studies try to use antioxidants as protective remedies against CI-AKI have yielded conflicting results [34–36]. Antioxidant effect of PE was demonstrated in vitro and vivo studies by increased the concentration of antioxidant enzymes SOD and CAT and decreased MDA in lipid peroxidation [37–39]. The present study demonstrated that antioxidant effect of PE extract could decrease MDA in both plasma and renal tissues. Moreover, PE extract preserved plasma TAC and renal tissues TAC, SOD and CAT activities. These effects correlated with the attenuation of histopathological injury from contrast media administration. The dose dependent effect of PE extract started at dose 250 mg/kg/d and had the additional effect at dose 500 mg/kg/d similar to the antioxidant effect of vitamin E in the experimental study  and clinical trials [25, 40]. Thus, the renoprotective effect of PE extract could prevent CI-AKI through antioxidant property.
Phyllanthus emblica is widely used in Thai traditional medicine and Ayurvedic Medicine for treatment of various diseases. The fruit of PE is known as a rich source of ascorbic acid [10, 11], gallic acid , and also contains a mixture of phenolic compounds . The major substrate which acts to be antioxidant in PE is gallic acid, vitamin C and others depending on the technique that separates the compound from PE [41, 42]. From the process to prepared PE extract in the present study, plant was dried by oven at 50°C before grinded and boiled in water. In general, vitamin C was destroyed by heat that could be loss from the preparing process . In addition, PE extract by HPLC analysis in the present study showed the main substrate was gallic acid. Gallic acid is a polyphenolic compound with multiple hydroxyl groups which are able to donate its proton to break the chain reaction of free radicals consequently as a lipid peroxidation inhibitor [44, 45]. Moreover, gallic acid was demonstrated as an excellent antioxidant with high free radical scavenging effect [45, 46]. From the experimental studies, gallic acid was demonstrated to prevent or attenuate the severity of brain, heart and liver damage from ischemic or toxic substances injury [47–49]. Furthermore, the reno-protective effect of gallic acid from antioxidant and antiinflammation properties was demonstrated in many experimental studies in AKI against lindane , ferric nitriloacetic acid , and sodium fluoride  and chronic kidney disease . By our HPLC analysis, gallic acid was present in 6% of the PE extract, corresponding to doses of 15 and 30 mg/kg/d in the 250 and 500 mg/kg/d of PE extracts, respectively. These doses of gallic acid could be the basis of human studies. Thus PE extract administration could prevent CI-AKI in rats with antioxidant effect from gallic acid. These finding could be assessed in patients with high risk CI-AKI in the future clinical trials.
The plasma levels of PE extract and the accumulation of PE extract on renal tissues with the repeated daily dose may be higher than the single dose administration. In experimental study, the bioavailability of gallic acid from grape seed polyphenol extract is improved by repeated dosing in rats . The single dose with oral administration demonstrated that the intestinal absorption of gallic acid is poor (<2%). While repeated exposure to the extract has shown the absorption of gallic acid was significantly higher than single dose treatment and reached to tissues level at day 10. Moreover, two gallic acid-derived compounds isolated from Casearia sylvestris leaves could reverse NK cell cytolysis which was suppressed from tumor growth when mice were treated with these compounds for 4 days . In contrast, a single dose administration with a large volume and high concentration of any compound via oral route may cause adverse effects and could not reach to the therapeutic levels in the tissues. From these informations, we make the decision to give PE extract orally as presented in the protocol. However, a high dose administration of PE extract for 2 or 3 days before contrast administration should be evaluated for the preventing CI-AKI.
Monitoring of urine output and/or novel biomarker in plasma or urine is a much better indicator for early diagnosis of CI-AKI. However, we have measured only serum BUN and Cr levels. Also, we have only H&E and PAS staining for histopathologic examination of renal tissues. Therefore, the immunohistochemistry staining such as Tunnel or PCNA and the evaluation of protein and gene expression in apoptosis or inflammatory pathway should be prepared to confirm CI-AKI.