ESTABLISHMENT OF NOVEL URINARY KIDNEY DISEASE NEW BIOMARKERS AND THERAPEUTIC EFFECT OF METHANOL FRACTION OF TERMINALIA ARJUNA ON ACETAMINOPHEN INDUCED KIDNEY DISEASE IN RATS

Objective: There were main two objectives, first was the identification of best biomarkers for early screening of kidney diseases whether plasma urea and creatinine or novel urinary low molecular weight protein biomarkers Interleukin-18 (IL-18), kidney injury molecule-1 (KIM-1), and cystatin-C. Second was the therapeutic efficacy of methanol fraction of Terminalia arjuna (MFTA) on urinary novel biomarkers. Methods: A total of 35 adult male rats were divided into three Groups (n=5), Group 1 was fed normal food, Group 2, normal food with administration of acetaminophen (APAP) for 5 days, 10 days, and 15 days, and Group 3, normal food with administration of APAP and coadministration of MFTA for 5 days, 10 days, and 15 days. All rats were sacrificed at 15th day of the experiment. Results: Results showed 5 days, 10 days, and 15 days administrations of APAP increased novel urinary biomarkers as IL-18, KIM-1 near two-folds and cystatin-C near six-folds increased than old biomarkers plasma urea and plasma creatinine. Administration of APAP with coadministration of MFTA represented the protective effect by decreasing old and new novel biomarkers with superoxide dismutase and catalase but malondialdehyde level increased. Sodium dodecyle sulphate-polyacrylamide gel electrophoresis showed new low molecular weight urinary protein bands in APAP administration rats, the protective effect of MFTA presents no band at this molecular level as normal rats. Conclusion: MFTA is the most potent nephroprotective agent, and urinary low molecular proteins are the best thing diagnostic tools for early detection of kidney disease over common plasma urea and creatinine.


INTRODUCTION
Chronic kidney disease (CKD) is an irreversible progressive disorder of structural and functional changes in kidney parenchyma into five stages on the presence of decreased kidney function and measured by the concentration of serum creatinine and estimated glomerular filtration rate [1]. However, the use of serum creatinine to detect kidney disease which is poor in assay specificity and sensitivity to delayed diagnosis and treatment timing. Kidney disease becomes more fatal and severe with comorbid disorders, reactive oxygen species, diabetes mellitus, hypertension, etc. [2]. From the earlier background of our research, different anti-uremic and renoprotective plant extract such as phytocompounds from Terminalia arjuna (TA) [3], Asparagus racemosus [4], and Withania somnifera [5] which efficiently played as a remedy on experimentally induced uremic and oxidative stress rats. Alternative therapy such as alpha lipoic acid [6] and probiotic [7] shown brilliance nephroprotective action against the administration of acetaminophen (APAP)-induced renal failure male rats in our laboratory [8]. In early research anti-uremic and anti-oxidative activity of TA (Family -Combretaceae) is already established [9]. Therefore, the aim of the study was to evaluate early urinary biomarkers such as kidney injury molecule-1 (KIM-1), Interleukin-18 (IL-18), and cystatin-C at an early stage of kidney diseases. KIM-1 appears in urine by specific mild renal tubular disorder [10], IL-18 for proximal tubular necrosis [11], and cystatin-C for kidney tissue macrophages cell in collecting duct [12] after duration dependent induction of kidney disease by peritoneal administration of acetaminophen in rats and also evaluate the urinary protein band by protein gel electrophoresis that was best result of protein marker would be sequence and identified in future.

Giri et al.
in the department of botany. Preparation of MFTA was done as earlier modified work in our laboratory [2].

Animal model and administration of MFTA
The study was performed on 35 healthy adult male albino rats of Wister strain (Supplied from Saha Enterprise, 386/2, Nilachal, Birati, Kolkata, West Bengal 700051, Regd. No.:1828/PO/BT/S/15/Committee for the Purpose of Control and Supervision of Experiments on Animals [CPCSEA]) having a body weight of 200±15 g. They were acclimatized to laboratory condition for 2 weeks before experimentation. Animals were housed five rats/cage in a temperature-controlled room (22±2°C) with 12-12 h dark-light cycles (8.00-20.00 h light, 20.00-8.00 h dark) at a humidity of 50±10%. They were provided with standard food and water ad libitum throughout the experimental period. Animal care was provided according to the guiding principle for the care and use of animals (Olfert et al. 1993) [13]. This experiment was approved by our Institutional Animal Ethics Committee (Regd. No.: 1905/PO/ Re/S/2016/CPCSEA), guidelines followed by CPCSEA.
A total of 35 rats were divided into three Groups (n=5). Group 1; five animals were normal animals without inducing kidney injury and fed a normal diet with ad libitum water. Group 2; 15 animals were divided into three subgroups (A-5, A-10, and A-15); each subgroup was with five rats, APAP at the concentration of 500 mg 5 mL de-ionized water/kg of body weight/day was injected with peritoneal route at zero "0" day, at 5 th day, at 10 th day for 14 days (A-15, at "0" day denotes 15 days treated; A-10, at 5 th day for 10 days treated and A-5, at 10 th day for 5 days treated by APAP) to achieve uremia and feed normal diet with ad libitum water. Group 3; 15 animals were subjected same as Group 2 with coadministered MFTA (TA-15, at zero "0" day denotes 15 days treated; TA-10, at 5 th day for 10 days treated and TA-5, at 10 th days for 5 days treated by APAP), at the dose of 40 mg/kg body weight by orally and feed normal diet with ad libitum water for 14 days daily at 11.00 A.M. before giving the food. APAP-induced uremia was established in our earlier research [8].
Animal scarification to collect plasma, organs, and urine All rats were sacrificed on the 15 th day. Blood was collected from the aorta and was centrifuged with heparin anticoagulant to collect the plasma. Plasma and kidneys were stored in −20°C for different biochemical analysis. Urine samples were collected directly from the bladder using a needle through the syringe. Collected sample was stored at −20°C. Urine samples were centrifuged using a micro centrifuge operating 10,000 rpm for 10 min, supernatants were separated [14]. A small part of the kidney was fixed in Bouin's fixative for histology.

Biochemical
analysis by semi autoanalyzer, UV-VIS Spectrophotometer, and ELISA Plate Reader Biochemical estimation of urea and creatinine level of plasma and urine were measured by commercially available standard kit with standard protocol for photometric determination by semi autoanalyzer [6,8]. SOD, catalase, MDA level of kidney tissue measured in UV-VIS Spectrophotometer by standard protocol [9,15,16]. IL-18 [17,18], KIM-1 [18,19], and cystatin-C [20] were measured by commercially available standard kit with standard protocol for kit literature by an ELISA plate reader.

Polyacrylamide gel electrophoresis (PAGE)
Denaturing sodium dodecyl sulfate (SDS)-PAGE was made after over 14 day's period of either APAP administration or coadministration of MFTA in rats with normal feeding rats. Urine sample was centrifuged at 10,000 rpm for 10 min, and the supernatant was discarded. Pellet was mixed with an equal volume of ×2 electrophoresis sample buffer (0.125 m Tris-Hydrochloride, 4% SDS, 20% v/v glycerol, 0.2 M Dithiothreitol, and 0.02% bromophenol blue, pH 6.8), 5 µg protease cocktail and heated at 95°C for 5 min, was analyzed by SDS-PAGE according to the method of Laemmli using 15% acrylamide resolving gels and 10% stacking gel. Electrophoresis was performed on Tarson electrophoresis apparatus. After washing in water for 5 min, the gels were stained with Coomassie Brilliant Blue R250 (0.1% in 25% methanol, and 10% acetic acid). The distaining step was done with Coomassie Brilliant Blue by incubation in 30% acetic acid distinguished with blue stain for protein bands [21,22].

Histological examination
Kidney fixed in Bouin's fixative for 24 h. Then kidney tissues were dehydrated with an elevated degree of ethanol and embedded in paraffin for preparing paraffin blocks. Sections from kidney tissue (4-5 mm) were stained with hematoxylin-eosin. The sections were examined and photographed by a Magnus MLX PLUS microscope (Nagano, Japan) [23].

Statistical Analysis
Data were analyzed using the origin software package (version 9.1). Analysis of variance (ANOVA) was followed by a multiple two-tail "t"test at the significance level (p<0.05) and expressed as SEM (n=5) [24].

Changes of old kidney disease biomarkers versus new kidney disease biomarkers of progression of kidney disease by APAP administration
APAP administration for 5 days in Group 2 (A-5) rats rises plasma urea 38.39% and plasma creatinine 51.61% significantly (p<0.05) than untreated Group 1 rat, while IL-18, KIM-1 and cystatin-C level in urine raised in 75.41%, 93.50%, and 307.69%, respectively, then Group 1. All biomarkers in plasma and urine significantly increased (p<0.05) in Group 2 (A-5) than Group 1 ( Table 1, Fig. 1 G-2a and G-2b).
APAP administration for 10 days in Group 2 (A-10) elevates plasma urea 112.11% and plasma creatinine 96.77% significantly (p<0.05) than untreated Group 1 rats, while IL-18, KIM-1, and cystatin-C level of Table 1: Changes of new biomarkers either APAP administration or co-administration of MFTA with APAP on plasma, urine, and kidney tissue. Data are expressed as SEM (n=5). ANOVA followed by multiple two-tail t-test and data with different superscripts letters (a, b, c, d, e, f, and g) revealed significant difference from each other (P<0.05). Group 1, normal rats without inducing kidney injury. Group 2, APAP administration rats groups (A-5, A-10, and A-15, respectively, APAP administration for 5 days, 10 days, and 15 days), Group 3: APAP with MFTA coadministration rats (TA-5, TA-10, and TA-15, respectively, APAP with MFTA coadministration for 5 days, 10 days, and 15 days) Effect of APAP administration for 5 days, 10 days, and 15 days created a vast difference between plasma old biomarkers and urinary proteoimmuno new biomarkers. KIM-1 and cystatin-C are low molecular weight signaling proteins molecules. IL-18 is synthesized from tissue macrophages in epithelial cells of the proximal tubule.

Effect of MFTA on SOD, Catalase, and MDA (oxidative biomarkers)
Effect of APAP in co administration with MFTA on kidney tissue levels of SOD, catalase and MDA were shown in Table 2. Quantification level of SOD and catalase significantly low (P<0.05) in kidney tissue of A-5, A-10, and A-15 rats was compared to other two groups, but MDA level is significantly high (P<0.05) in compare to Group 1, TA-5, TA-10, and TA-15. MFTA coadministration with APAP causes improvement of oxidative stress in kidney tissues.

Histological examination
The histological architecture of kidney indicated (Fig. 5) necrosis and atrophy in proximal tubule (black arrow in Fig. 5b) and glomerulus (black circle in Fig. 5b) in sub-group of Group 2 (APAP treated) compares

Giri et al.
to Group 1. Group 1 shows a normal proximal tubule (black arrow in Fig. 5a) and glomerulus (black circle in Fig 5a). Peritubular congestion and epithelial desquamation (black star in Fig. 5b) increased in APAP treated group in compared to MFTA coadministration group (Group 3) and improved or prevented injuries by regenerating normal glomerulus with light Bowmans degeneration and renal tubules (black circle in Fig. 5c, black arrow in Fig. 5c).

Old biomarkers versus new biomarkers on duration dependent study
Our study shown by line diagrams, increased percentage of old biomarkers (urea and creatinine) is less than the increased level of new biomarkers depends on the duration of the study. IL-18 and plasma urea level of Group 2 when compared with Group 1, the result proves the enriched level of IL-18 increased by 93.50%, 161.51%, and 230.52%, respectively, in 5 th , 10 th , and 15 th days but while plasma urea level increased in only 38.39%, 112.11%, and 200.00%, respectively, in 5 th , 10 th , and 15 th days for low sensitivity and protectivity ( Fig. 1 G-2a). Furthermore, KIM-1, cystatin-C, and plasma creatinine when compared with Group 1, there the result shows KIM-1 level in 75.41%, 134.76%, and 194.14% and cystatin-C level in 307.69%, 423.07%, and 507.69% increased, respectively, in 5 th , 10 th , and 15 th days but plasma creatinine shows 51.61%, 96.77%, and 145.16%, respectively, in 5 th , 10 th , and 15 th days for poor sensitivity and protectivity in kidney injury interstitium [25]. The study demonstrated that APAP administration in rats raises plasma urea 38.39% for 5 days, 112.11% for 10 days, and 200.00% for 15 days while urinary IL-18 raise 75.41% for 5 days, 161.51% for 10 days, and 230.52% for 15 days. Urinary biomarker is IL-18 (24 kDa), a proinflammatory and immunomodulatory cytokine in distal convoluted tubule and collecting tubule in kidney [26]. A mild injury or stress in distal and collecting tubule, elevation of IL-18 occurs invasive amount [27]. Plasma creatinine, end product of muscle protein breakdown, showed high in the late destructive stage of kidney [28]. We elaborate APAP administration in rats elevates plasma creatinine 51.61% for 5 days, 96.77% for 10 days, and 145.16% for 15 days even as urinary KIM-1 lifts up 75.41% for 5 days, 134.76% for 10 days, and 194.14% for 15 days. KIM-1, which found in proximal tubular epithelium transmembrane glycoprotein (90 kDa) absent in normal kidney tissue or urine. KIM-1 increased and excreted in urine a huge amount by mild tubular stress [29]. A low molecular weight protein (13 kDa) cystatin-C is prepared in all organs in our body while oxidative damage based necrosis of cells and tissues. It is completely assured by normal nephrons. It is contained in urine when any part of nephrons gets damaged [30]. First 5 days APAP administration increases urinary cystatin-C level 307.69%, thereafter, 423.07% for 10 days, 507.69% for 15 days. However, it is distinctly clear urinary IL-18, KIM-1, and cystatin-C showing a higher level in duration dependent APAP administration than plasma urea and plasma creatinine. In our study, coadministrated MFTA with APAP to another groups of rats which was effective in reducing old kidney biomarkers plasma urea, creatinine, and new kidney biomarkers urinary IL-18, KIM-1, and cystatin-C. The authors speculate that antioxidant enzymes, SOD, and catalase ( Fig. 1 G-2b). The percentage levels in Group 3 compared to Group 2 of IL-18, and plasma urea was decreased at an equal level at the presence of MFTA, 36.83%, 42.51%, and 44.87% and 10.57%, 35.90%, and 53.52%, respectively, in 5 th , 10 th , and 15 th days ( Fig. 1 G-3a). Identical same result was also found with KIM-1(40.75%, 50.84%, and 63.19%, respectively, in 5 th , 10 th , and 15 th days), cystatin-C (16.98%, 50.00%, and 58.22%, respectively, in 5 th , 10 th , and 15 th days), and plasma creatinine (19.14%, 32.78%, and 48.68%, respectively, in 5 th , 10 th , and 15 th days) ( Fig. 1 G-3b).

DISCUSSION
Our earlier investigation, acetaminophen-induced nephrotoxicity [8], MFTA having three major polyphenols details published earlier by corresponding author [2] showed nephroprotective activity on old biomarkers those were urea and creatinine [6] and also MFTA established as a powerful antioxidant evidenced on SOD, catalase, and MDA on kidney tissue [5]. Plasma urea is synthesized in liver, and plasma creatinine is a breakdown product of muscle protein, both were expected in late stages of kidney disease [29]. Plasma urea is screened high at late stages of kidney diseases. Used of plasma urea old biomarker at late stage patients suffer irreversible destruction and damage of kidney  letters (a, b, c, d, e, f, and g) revealed significant difference from each other (P<0.05). Group 1, normal rats without inducing kidney injury. Group 2, APAP administration rats groups (A-5, A-10, and A-15, respectively, APAP administration for 5 days, 10 days, and 15 days), and Group 3: APAP with MFTA coadministration rats (TA-5, TA-10, and TA-15, respectively, APAP with MFTA coadministration for 5 days, 10 days, and 15 days).  in kidney tissues significantly enhanced and significantly diminished MDA by MFTA feeding orally [6]. It is difficult to conclude with the exact mechanism by which MFTA controls the kidney biomarkers. It should be hypothesized that three major compounds (earlier published, [2]) have a significant main effect on renal tubules.
Evidence from correlation study in between plasma urea and urine IL-18 in untreated rats, APAP administration for 5 days and 10 days, there was no significant relation of urea and IL-18 while significantly related to 15 days APAP administration. Plasma creatinine was not significantly correlated with urinary KIM-1 where there were strongly significant correlated with APAP administration for 5 days. MFTA causes negative correlation with plasma creatinine and urinary KIM-1.
Urinary IL-18, KIM-1, and cystatin-C are low molecular protein measured by ELISA kit method which was strong evidenced with the presence of extra band around 25-30 kDa of APAP administration by SDS-PAGE. Histological observations showed according to Abhirama et al., tubular degeneration, desquamation, necrosis, intertubular hemorrhage, and hyaline in tubules with congestion and swelling in glomerulus evaluated due to nephrotoxicity in animals [31]. However, coadministration of MFTA caused marked histological protection on tissue injuries including tubular necrosis, capillary congestion, and epithelial desquamation against APAP administration in Group 3. Cirevalli et al. suggested that CKD patient with low-income also against the provision of various drugs to reduce the financial burden on pharmaceutical costs not only for the patient but also to caregivers to their family [32].

CONCLUSION
It can be possible to conclude that the use of these urinary low molecular weight proteins biomarkers could be used to monitor at risk in kidney disease patients. These biomarkers will allow to diagnose kidney disease at a much earlier stage. MFTA possess anti-uremic and urinary biomarkers suppressive capacity. The kidney protective effect in this study provides a mechanistic evidence for why tribal people in our Jangalmahal area of Paschim Medinipur and Jhargram, state of West Bengal, India found it useful for treating kidney ailments as well as being used to cure other health disorders.

Deblina Giri had contributed in definition of intellectual content,
literature search, data acquisition, data analysis, manuscript preparation, and manuscript editing. 2. Koushik Das had contributed in concept, design, manuscript preparation, and manuscript editing. 3. Dilip Kumar Nandi had contributed in concept, statistical analysis, and data acquisition.

CONFLICTS OF INTEREST
All the authors have an equal contribution in the manuscript and declare no conflicts of interest.