Pomegranate juice recuperates N ’-Nitrosodiethylamine-induced kidney injury: evidence from biochemical and histological approaches

Background Pomegranate is considered as one of the oldest elixirs having various properties. Renal fibrosis is a preliminary sign of pathological degradation in most ailments related to kidney. Several efforts have been made for the discovery of cost-effective and safe therapeutics for the alleviation of renal diseases. There is a major dearth of studies on the action of pomegranate juice (PGJ) against NDEA-instigated kidney injury. This study investigates the protective and antifibrotic action of PGJ in restricting the occurrence of experimental renal fibrosis in Wistar rats. Renal injury was generated by a single intraperitoneal dose of 10 ml kg −1 b.wt. (1% NDEA stock), while fresh PGJ (i.p.) in doses of 2 ml kg −1 b.wt was administered thrice a week on alternate days for two weeks to observe amelioration. The renal function indices (blood urea, creatinine, and uric acid), SOD, CAT, LPO levels and renal anatomy (H&E, MT, Picrosirius and SEM) were investigated. Results The assessment of renal function demonstrates augmented levels of blood urea, creatinine and uric acid in NDEA-administered groups in comparison with controls. SOD, CAT declined significantly in NDEA Day- 7-and Day-14-treated animals, while the MDA levels raised by ~ 70.5% and ~ 76.3% in these groups, respectively. However, supplementation of PGJ provided recuperation from these elevated levels in injured groups. H&E staining of the controls exhibited normal renal structure with intact glomerulus and Bowman’s capsule, while NDEA generated congestion of glomerular tuft, convoluted tubules with cloudy swelling and multiple subsidence of the renal tissue. Noticeable presence of collagen fibers in the interstitium of cortex region of kidney was observed by MT staining along with gross ultrastructural deterioration in NDEA-administered animals by electron microscopy. PGJ supplementation exhibited restoration of renal anatomy and physiology. Conclusions Pomegranate may be considered as a potent nutraceutical to prevent NDEA-induced renal damage and may be included as a daily dietary supplement.

on gene expression (Husain et al., 2018;Zarfeshany & Asgary, 2014).Studies have shown that they have several compounds beneficial for human beings that may possibly lower the risk of various diseases such as cancer, cardiovascular diseases, skin, osteoarthritis, rheumatoid arthritis, malaria, and wound healing (Wang et al., 2018;Zarfeshany & Asgary, 2014).Due to the presence of hydrolysable tannins, anthocyanins and ellagic acid derivatives, higher antioxidant activity was exhibited by Pomegranate juice (PGJ) in comparison with that of green tea and red wine (Gil et al., 2000).Flavonoids present in PGJ have the ability of scavenging the reactive oxygen radicals and show antioxidant activity via inhibi ting inflammatory markers like TNF-α (Moneim et al., 2013;Zarfeshany & Asgary, 2014;Colombo et al., 2013).Further, PGJ displayed a protective effect on the liver and showed the antifibrotic power in restraining the prevalence of liver fibrogenesis in rodents (Husain et al., 2018;Ibrahim et al., 2016).
Fibrosis is a reactive phenomenon, and it advances in reciprocation to unrestricted epithelial injury and inflammation, which leads to activation of myofibroblast and an accumulation of extracellular matrix (ECM) (Zhang et al., 2017).The progressive fibrosis is one of the best examples, which escorts all chronic renal diseases.Kidney fibrogenesis is a typical histological manifestation of functional downfall in most cases of end-stage kidney diseases.Histologically, the end-stage kidney disease exhibits itself as fibrotic lesions affecting each compartment; producing glomerulosclerosis, vascular sclerosis and tubulointerstitial fibrosis (Hewitson, 2012).The magnitude of tubulointerstitial fibrosis has been correlated with decline of renal function in animals and humans (Efstratiadis et al., 2009).It is observed that energy balance, innate immune system and neuroendocrine management of organ function are few of the biological processes that are immensely integrated.However, they are hampered by deteriorated kidney function and trigger precarious clinical phenotypes such as inflammation, malnutrition, altered autonomic and central nervous system, cardiopulmonary, vascular and bone disease (Zoccali et al., 2017).Globally, around two million people are suffering from chronic kidney disease (CKD) today (Ling & Kuo, 2018).A review of published studies suggests that complementary but different mechanisms are responsible for renal diseases or fibrosis.A large number of biochemical signaling routes have been suggested to be related to the progress of kidney fibrosis (Liu, 2006).Perhaps the most discerning attempts on reprogramming of myofibroblasts have focused on pattern of DNA methylation in human cells collected from diseased versus normal kidney (Krata et al., 2018;Duffield, 2014).Recent thorough studies of fibroblasts, their precursors, as well as findings from human clinical trials have propelled uncertainty on the central importance of TGF-β (Leaf et al., 2017).It has been proposed that one of the crucial factors of kidney fibrosis is the hypoxia inducible factor (HIF) stimulation.Thus, hypoxia may also be a key player in fibrosis formation that induces the expression of transcriptional genes, which are associated with the ECM (Efstratiadis et al., 2009).
Nitrosamines such as Nitrosodiethylamine (NDEA) are supposedly present in an enormous variety of items such as non-fat dry milk; cheese; smoked, dried and salted fish; cured meat and alcoholic beverages (Scanlan, 1983).Endogenous formation of nitrosamines in humans is the consequence of the reaction between the nitrite ion and secondary/tertiary amines at low pH (Mukherjee et al., 2015).NDEA is thought to sustain metabolic stimulation by cytochrome P450 enzymes to form reactive electrophores which cause oxidative stress resulting in peroxidative damage to the cell, cellular toxicity, mutagenicity and carcinogenicity (Bansal et al., 2005;Pashmforoosh et al., 2015).Furthermore, biotransformation of NDEA mediated by NADPH reductase may also lead to generation of ROS, thus leading to oxidative stress (Kujawska et al., 2016).Nephrotoxicity is an undesirable effect of some drugs and compounds and can be more intense in cases already having some kidney impairment (Alimoradian et al., 2017).The susceptibility of kidneys to injury from xenobiotics is because of greater perfusion and accretion of expelled compounds that happen in renal tubular cells during the process of absorption and secretion (Cekmen et al., 2013).Many attempts have been made to discover economical and risk-free alternative medicine for the cure of kidney, for example, using materials derived from plants.PGJ is also reported to exert protective effect on unilateral ureteral obstruction (UUO)-induced renal injury in rodents by lowering oxidative stress (Otunctemur et al., 2015).PGJ exhibited protective effect against the renal damage induced by steroids, leading to increase in serum creatinine level (Ali et al., 2017).PGJ demonstrated a protective and therapeutic effect against ethylene glycol (EG)-induced crystal deposition in renal tubules (Tugcu et al., 2008).Further, the consumption of pomegranate can upsurge antioxidant enzyme, which has ability to improve oxidative stress, and defends the hepatic and renal tissues in diabetic rats (Aboonabi et al., 2014;Sreekumar et al., 2014).In cisplatin-induced nephrotoxicity, pomegranate pre-treatment lowered the elevated level of serum creatinine and BUN (Karwasra et al., 2016).However, there is a major dearth of studies on the action of PGJ against NDEA-induced kidney toxicity.Here, the prophylactic effect of pomegranate juice (PGJ) against the NDEA-induced kidney injury in rats has been studied (Figs. 1,2,3,4,5,6,7).

Animals
For this experimental study, 6-7-week-old adult male rats of Wistar strain (Rattus norvegicus) were used.The approximate weight of all the thirty five rats belonging to the 7 groups ranged 140 ± 10 gm.The environment and conditions of the animals included standard illumination and temperature (25 ± 2 °C) in which they were contained in wire mesh topped polycarbonate cages.The conditions were maintained as hygienically cleaned as possible by cleansing the cages daily.These animals were freely allowed to feed sterilized diet and water daily.All the experiments conform the procedure of Institutional Ethical Committee (IEC).

Pomegranate juice preparation
Fresh Punica granatum fruits were purchased from local fruit market, identified for their taxonomic position by experts at Botany Department.Approximately two kilograms was used for each set of experiments in the study.The processing of the fruits was done by giving them a thorough wash, and manual peeling was carried out in the laboratory.Arils were properly separated for the juice from all the pomegranates.A commercial blender was used for the extraction of pomegranate juice Fig. 1 Alterations in A blood urea levels (mg/dL), B creatinine levels (mg/dL) and C uric acid levels (mg/dL) in control, NDEA-treated and PG supplemented groups.Group-1: Saline Control; Group-2: PGJ Control; Group-3: NDEA-treated (Day-7); Group-4: NDEA-treated and supplemented with PGJ (Day-7); Group-5: NDEA-treated (Day-14); Group-6: NDEA-treated and supplemented with PGJ (Day-14).All the values were taken as the mean ± standard error of replicate samples.*p < 0.05 and **p < 0.01 were considered statistically significant Ultimately, the pomegranate juice was used in the experiments and stowed away carefully at −20 °C for 14 days for other experiments and analysis (Faria et al., 2007).

Experiment protocol and treatment schedule
Throughout the study each set of experiments included seven groups of five healthy rats each in which the animals are segregated.
Group-1: Control, containing rats that were supplied with normal saline only along with the sterilized diet for a time of 14 days.Group-2: Positive controls in which animals were provided with PGJ (i.p.) in doses of 2 ml kg −1 b.wt.(Pirinççioğlu et al., 2014) thrice a week on alternate days for a week and were sacrificed eventually after the 14th day.Group-3 & 4: These groups comprised of animals that were administered with NDEA (i.p.) with one-time dose of 10 ml kg −1 b.wt. of NDEA (1% solution) on the first day (Group-3) (Mukherjee & Ahmad, 2015).After 2 h of gap PGJ was given at 2 ml kg −1 b.wt on the first day.Further repeated supplementation of PGJ thrice a week on alternate days was provided at the same dose for a week (Group-4) and subsequently the animals were sacrificed.
Group-5 & 6: Experimental animals were first treated with a single dose of NDEA (i.p.) for two weeks (Group-5).After 2 h of gap PGJ was given at 2 ml kg −1 b.wt. on the first day.Further repeated supplementation of PGJ thrice a week on alternate days was provided at the same dose for two weeks and the animals were sacrificed after two weeks (Group-6), respectively.

Induction of renal injury
The induction of renal injury was attained by the administration of N'-Nitrosodiethylamine (NDEA) intraperitoneally in animals.The animals were given a single dose of 1% NDEA (i.p.) solution at 10 ml kg −1 b.wt (diluted with sterile NaCl) (Mukherjee & Ahmad, 2015).The injections to all the animals belonging to different groups were given according to the treatment schedule only on the first day.After two weeks of NDEA administration, renal biochemistry and anatomy were assessed.

Administration of pomegranate juice
The animals of four groups, i.e., Group-2, -4, and -6, were provided with pomegranate juice (PGJ) intraperitoneally thrice a week on alternate days.However, the treatment on the first day for Group-4 and -6 was rather different as these groups were also administered with NDEA (i.p.).Hence, for the animals of these groups there was a lag of approximately 2 h after the administration of NDEA and subsequently they were supplemented with PGJ.

Sera collection and tissue sampling
Blood collection from the animals was done by cardiac puncture on the 7th and 14th days after the start of the experiment.Sera were extricated by following the steps of the established protocol (Ahmad & Ahmad, 2014).For the excision of kidney from the sacrificed rats, all the scissors and forceps that were used were thoroughly sterilized.Further, removal of tissue debris from the surface of the excised kidney was done by using phosphate-buffered saline/PBS (50 mM, pH 7.0) and ash-free filter paper was used for blotting the kidneys individually.The collected kidney tissues were homogenized in Tris-HCl buffer (1:3 w/v, 50 mM, pH 7.5) unless stated peculiarly.The crude homogenates made to undergo centrifugation at the speed of 8000 rpm under cooling (4 °C) for 15 min for procuring clear supernatants.

Estimation of total proteins
Estimation of total protein content of sera and kidney homogenates was performed using Folin-Ciocalteu's phenol color reagent (Lowry et al., 1951).Extrapolation of unspecified concentrations of proteins was done against the known standard values of bovine serum albumin (BSA) by measuring optical densities at 660 nm on UV-visible spectrophotometer.

Renal function indices
Blood urea, creatinine and uric acid were analyzed following routine procedures using commercial kits (Transasia Bio-Medicals) in all the samples belonging to the 7 groups.

Analysis of lipid peroxides
Determination of lipid peroxides generation was done by thiobarbituric acid (TBA) reaction and expressed denominations of malondialdehyde (MDA) by the protocol of Ohkawa et al. (1979).The optical density of the organic layer was then recorded at 532 nm.The calculation of MDA was done using the extinction coefficient of 1.56 × 10 5 /M/cm and presented in terms of nmoles of MDA g −1 wet wt. of tissue.

Superoxide dismutase estimation
The estimation of superoxide dismutase (SOD) enzyme was done following the step-wise protocol already reported (Marklund & Marklund, 1974), and the calculation of the value was performed according to Nandi and Chatterjee (1987).SOD enzyme activity was estimated in a reaction mixture prepared by 0.1 ml of EDTA (1 mM), 2.75 ml of phosphate buffer PO 4 (50 mM pH 8.5) and kidney homogenate in volumes of 50 µl each.This reaction mixture was kept in incubation for approximately 20 min at 25 °C.Under dark conditions of illumination, 0.1 ml of 0.2 M pyrogallol was added to the mix and absorbance was recorded simultaneously at 420 nm after every 60 s for a precise duration of 3 min.Superoxide dismutase specific activity in reducing pyrogallol was calculated by its auto-oxidation and expressed in terms of Units mg −1 protein min −1 .

Estimation of catalase activity
The established method of Aebi (1974) was used to determine the activity of catalase enzyme.The attribute of the catalase enzyme by which it causes a breakdown of hydrogen peroxide (H 2 O 2 ) was utilized during this process.Fresh kidney tissue extricated after sacrifice was mixed and dissolved with phosphate buffer (50 mM, pH 7.0) in the specified ratio of 1:10 (w/v), respectively.The readings of the optical densities were taken at 60 s.gap at 240 nm for a precise duration of 3 min.

Assessment of kidney anatomy
Tissue pieces of the dimension 1 cm 3 were collected and kept in 10% formalin.The tissues were then processed by serial dehydration, clearing and embedding in xylene and paraffin wax, respectively.Tissue sections of the size of ~ 5 μm were stained by routine hematoxylin-eosin (H-E) and Masson's trichrome (MT) separately.Picrosirius red was used to stain the tissue section to visualize the presence of collagen under fluorescence at 475-500 nm.Photomicrographs were taken on Zeiss Axioscope A1 with Jenoptik Prog Res (C5 camera model) and Zeiss Axioscope 40.

Scanning electron microscopy
The tissue sections for scanning electron microscopy were prepared to the size of 1 mm 3 .These pieces were gently fixed in 2.5% glutaraldehyde (prepared in 0.1 M sodium phosphate buffer; pH, 7.4) for overnight.Staining was performed in 1% osmium tetroxide at least for an hour.These samples were step-wise dehydrated with ethanol gradient at an interval of 10 min and allowed to vacuum dry for overnight.The mounted stubs were sputter-coated with thin layer of gold and examined at 10 kV using the field emission scanning electron microscope (FE-SEM, JEOL JSM6510LV).

Statistical analysis
Statistical assessments of all the biochemistry parameters between all the groups were carried out using the student's t-test on SPSS software.A range of probability (P value) < 0.05 was taken into consideration as being statistically significant.All the values are expressed in terms of mean ± standard deviation.

Renal function indices-blood urea, creatinine and uric acid
The assessment of kidney function which includes blood urea, creatinine and uric acid represents renal anomalies demonstrate augmented levels in the NDEAadministered groups in comparison with the controls.The supplementation of PGJ in the rats attenuated and restored the levels back in the range close to normal groups in a time-and dose-dependent manner.Blood urea levels increased in both the NDEA Day-7 and Day-14 groups (~ 56% and ~ 74.7%) as compared to the controls, respectively.The redressal in these levels was brought by the supplementation of PGJ in both the groups of NDEA + PGJ at Day-7 and Day-14 (~ 24% and ~ 51.7%), respectively.Creatinine levels when compared to control were also increased (~ 58.4% and ~ 66.4%) in both NDEA Day-7 and Day-14 groups, respectively.In these levels the abrogation of ~ 42.4% and ~ 48.3% was found prominent in NDEA + PGJ Day-7 and NDEA + PGJ Day-14 groups, respectively.Similarly, uric acid also exhibited the same trend as there was elevation of ~ 51.3% and ~ 56.9% in Day-7 and Day-14 NDEA-administered groups, respectively, in comparison with control levels.The supplementation of PGJ provided recuperation from these elevated levels in both NDEA + PGJ Day-7 and NDEA + PGJ Day-14 groups by ~ 43.4% and ~ 30.7%, respectively.The control and positive control values showed non-significant differences.

Superoxide dismutase activity
The estimation of superoxide dismutase enzyme demonstrated a significant decline in both the NDEA Day-7 and NDEA Day-14 groups (~ 19.1% and ~ 28.8%), respectively, as compared to the control groups (P < 0.05).Refurbishment of the activity was notably achieved by the supplementation of PGJ in both NDEA + PGJadministered groups at Day-7 and Day-14 by ~ 11.1% and ~ 24.3%, respectively, within two weeks.The saline control and positive control groups showed no significant difference in the enzyme activity.

Lipid peroxidation levels
Determination of lipid peroxide generation in the kidney was done by analyzing the levels of malondialdehyde (MDA).Administration of NDEA increased the MDA levels significantly in a time dependent manner in approximately two weeks.The MDA levels exhibited elevation of ~ 70.5% and ~ 76.3% in both Day-7 and Day-14 NDEA-administered groups, respectively.Subsequently, the abatement by PGJ in the NDEA + PGJtreated groups at Day-7 and Day-14 was expressed by ~ 26.3% and ~ 30.5%, respectively.

Catalase activity
Estimation of catalase activity exhibited significant reduction in the NDEA-administered groups (~ 32.3%-Day-7 and ~ 47.6%-Day-14; P < 0.01).The attenuation by PGJ increased these levels by ~ 28.8% in NDEA + PGJ Day7 group and by ~ 37.5% in NDEA + PGJ Day-14 group.There was non-significant difference between the control group and PGJ positive control group.

Structural changes observed in kidney by histopathology
The histopathological evaluation of kidney tissue sections by hematoxylin and eosin staining in the rats belonging to control groups exhibited normal tissue structure, intact glomerulus and Bowman's capsule.The NDEA treatment affected the kidney architecture in a retrograde manner by damaging it.The kidney tissue sections belonging to NDEA Day-7 and NDEA Day-14 groups showed congestion of glomerular tuft, convoluted tubules with cloudy swelling and multiple subsidence of the renal tissue.The supplementation of PGJ evidently demonstrated abrogation as sections belonging to the groups in which PGJ was administered with NDEA for 7 days and 14 days show recovery by the decline in the spaces between renal corpuscle and renal tubules.The renal tissue sections of the PGJ positive control rats displayed normal kidney architecture similar to the saline control groups.The staining by Masson's trichrome is used to display collagen fibers specifically.The tissue sections belonging to rat kidney of the saline control group exhibited normal renal anatomy and absence of any blue stained collagen fibers.Administration of NDEA in both Day-7 and Day-14 groups exhibited evident deterioration of architecture and prominent presence of blue colored collage fibers in the interstitium of renal cortex.The groups Day-7 (NDEA + PGJ) and Day-14 (NDEA + PGJ) kidney tissue sections demonstrated with lesser amount of collagen content is indicative of recuperation by pomegranate juice.
The observation under bright-field microscopy demonstrated yellow colored tissue sections with bright red colored collagen fibers.Hence, this Picrosirius staining is also collagen fibers specific that definitely assists greatly in the assessment of fibrosis.The magnitude of accumulation of collagen fibers was observed clearly in red color.The rat kidney sections of saline control groups showed intact tissue architecture with normal renal corpuscle and convoluted tubules.NDEA treatment in both the groups, i.e., Day-7 and Day-14, demonstrated profound deterioration as the sections showed presence of aggregation and vacuolated renal tubules along with prominent red colored collagen fibers.The PGJ positive control rat group kidney sections showed unaltered kidney architecture.Abatement of the kidney organizational structure in rats was evident in groups administered with NDEA along with PGJ for 7 days and 14 days both.The renal corpuscle demonstrated proper glomerulus and Bowman's capsule with lesser shrinkage as compared to damaged sections.

Ultrastructural changes in kidney by scanning electron microscopy
Control group exhibit architectural preservation as the glomeruli (arrow) and surface tissues are intact.The kidney tissue sections belonging to groups NDEA Day-7 and Day-14 show deterioration in the structure by shrunken glomerular tuft surrounded with thick fibers in the latter section.PGJ supplementation in groups NDEA + PGJ (Day-7 and Day-14) demonstrates lesser damaged convoluted tubules with a minimal amount of collagen fibers surrounding them.The positive control section also shows the presence of intact normal convoluted tubules.

Discussion
The appropriate consumption of phytoextract and nutraceuticals in accordance with suggested dose has shown their health advantages and medicinal values (Husain et al., 2018).Tests of renal function have efficacy in detecting the incidence of renal disorder, monitoring the reaction of kidneys to therapy, and ascertaining the advance of renal disorder (Gounden & Jialal, 2018).NDEA-treated animals in the present investigation exhibited renal injury as evidenced by the substantial mount in circulating creatinine, urea and uric acid levels.These findings corroborate previously published reports where augmented sera levels of creatinine, urea and uric acid are documented in DEN/PB-induced nephrotoxicity in rats (Ahmed et al., 2015;Mahmoud et al., 2015;Pashmforoosh et al., 2015;Rezaie et al., 2013).However, the control and positive control values showed non-significant differences.The supplementation of PGJ exhibited nephroprotective effect in the rats and attenuated the levels back in the range close to normal groups in a time and dose-dependent way.
Superoxide dismutase (SOD) is the enzyme which sustains protuberant protective mechanism to counter superoxide radicals (Mukherjee & Ahmad, 2019).NDEA significantly declines SOD activity within 14 days of its treatment.The activity of SOD exhibited a significant (P < 0.05) decline in the kidney of NDEA-injured rats when compared with the controls.This waning in SOD activity is possibly due to the oxidation of cysteine residues from the enzyme complex by the NDEA that eventually generates deformed enzymatic conformation and production of superoxide radicals (Shaban et al., 2013).However, the control saline and positive control groups showed no significant difference in the enzyme activity.The low and high doses of PGJ significantly ameliorated the activity of renal SOD in Day-7 and Day-14 NDEA + PGJ-administered groups.
Malondialdehyde (MDA), an indicator of lipid peroxidation, has long been used as fundamental biomarker of oxidative impairment and the escalation of MDA echoes the enhancement of LPO (Lykkesfeldt, 2007).The kidney damage may be attributed to ROS which tempt mesangial cells contraction, fluctuating the filtration surface area and amending the ultrafiltration coefficient factors that decline the glomerular filtration rate (Podkowińska & Formanowicz, 2020).In this study, the NDEA administration deleteriously increased the kidney MDA.Our results confirm previous reports that found the level of MDA was significantly enhanced in NDEA administration to rats in comparison with control groups (Husain et al., 2018;Zhang et al., 2017).NDEA-induced rats displayed a remarkable surge in levels of MDA, representing a severe injury to renal tissue (Ahmed et al., 2015).Nakae et al. (1997) reported that NDEA could intercalate with the membrane-bound lipids and produces ROS, which alleviate generation of lipid peroxides.Our study divulges that PGJ overturned the NDEA-induced lipid peroxide generation which deliberately supports other published studies exhibiting the nephroprotective effect of pomegranate juice in declining renal damage.
Catalase (CAT) is universally present in all aerobic cell types and is a primary constituent in antioxidant defense mechanism (Rana & Soni, 2008).Estimation of catalase activity was carried over which exhibited significant reduction in the NDEA-administered groups relative to the controls.Lesser effect on CAT activity observed on stress induction in test group signifies the improved protective efficacy by pomegranate extract.Decreased activities of CAT specify that stress induction by NDEA restricts with antioxygenic potential, thus subsequently cause an increase in ROS generation and leads to peroxidative damage (Latief et al., 2019).The reduced CAT activity in rats dispensed with NDEA may be owing to inactivation of CAT as superoxide anions have been revealed to decline CAT activity (Bansal et al., 2005).
It has been documented that the instigation of antioxidant enzymes is modulated by the detachment of nuclear erythroid 2-related factor 2 (Nrf2) from the Kelch-like ECH-associated protein-1 (Keap1) in the cytoplasm.This detached Nrf2 subsequently attaches with the nuclear antioxidant response element (ARE) which ultimately emanates the defensive action by increasing the activity of antioxidant enzymes (Husain et al., 2018).Renal injury induced by NDEA was further confirmed by the noted histological alterations such as including congestion and atrophy of glomerular tuft, thickening of the blood vessels, cloudy swelling in convoluted tubules, fibrotic and inflammatory cells infiltration.Based on histological observations, end-stage kidney disease exhibit itself as fibrotic lesions affecting each compartment; glomerulo-sclerosis, vascular sclerosis and tubule-interstitial fibrosis (Hewitson, 2012).Present results based on H&E, Masson's trichrome and Picrosirius-red staining further document gross anomalies and alterations in the anatomy of fibrotic kidneys.
The histopathological assessment of kidney tissue sections by hematoxylin and eosin staining in the rats belonging to control groups exhibited normal kidney structure, intact glomerulus and Bowman's capsule.The NDEA treatment affected the renal architecture in a regressive manner by damaging it.The sections belonging to renal tissue displayed congestion of glomerular tuft, convoluted tubules with cloudy swelling and multiple subsidence.The staining by Masson's trichrome is used to show collagen fibers specifically.Administration of NDEA in both groups exhibited evident deterioration of architecture and prominent presence of collagen fibers in the interstitium region of renal cortex.Picrosirius staining is collagen fibers specific that helps significantly in the assessment of kidney fibrosis.The kidney sections of saline control groups showed intact tissue architecture with normal renal corpuscle and convoluted tubules.NDEA treatment in both the groups reveal acute deterioration as the sections displayed incidence of aggregation and vacuolated of renal tubules along with flagrant collagen fibers.On the other hand, treatment of the NDEA-administered rats with doses of PGJ protected against the NDEA-induced nephrotoxicity and histological alterations.Our findings are in agreement with previous studies in which treatment with NDEA triggered kidney damage apparent by the histological changes and injuries involving dysplastic renal tubules with karyomegaly, atrophy of glomerular tuft, inflammatory cells infiltration, protein cast in the lumen and vacuolation of renal tubules (Ahmed et al., 2015;Husain et al., 2021).
In our study, the scanning electron microscopy also displayed the presence of collagen accumulation in the kidney sections intoxicated by NDEA, which is a clear indication of kidney inflammation and fibrosis.Control group exhibit architectural maintenance as the glomeruli and surface tissues are intact.The kidney tissue sections of NDEA-administered groups display depreciation in the structure by shrunken glomerular tuft surrounded with thick fibers in the latter section.PGJ supplementation in both the groups Day-7 and Day-14 NDEA + PGJ-treated demonstrate lesser damaged convoluted tubules with a minimal amount of collagen fibers surrounding them.The positive control section also shows presence of intact normal convoluted tubules.

Conclusions
The present study investigates the nephroprotective action of PGJ in restricting the occurrence of renal damage induced by NDEA in Wistar rats.The renal function indices (blood urea, creatinine and uric acid), superoxide dismutase activity, lipid peroxidation levels, catalase activity, histopathology of renal tissue (H&E, MT, Picrosirius and SEM) were investigated.It was revealed that NDEA is a puissant nephrotoxicant which leads to altered renal biochemistry, oxidative stress and subsequently causes renal injury.The administration of pomegranate juice (PGJ) significantly ameliorated the renal damage within two weeks and restored the kidney architecture and physiology.It appears that PGJ induces its beneficial effect which is exerted via its various bioavailable constituents and metabolites on gene expression.Although this study recommends PGJ consumption to prevent NDEA-induced nephrotoxicity, the effect of individual compound present in the PGJ against renal fibrosis needs to be elucidated.It is anticipated that future clinical trials on nephroprotective activity of PGJ will reveal its beneficial effects on human health ().

Fig. 2 A
Fig. 2 A Superoxide dismutase (SOD, U/mg of protein/min); B malondialdehyde (MDA, nmoles/g tissue); C catalase (U/mg protein/min) of rats treated with NDEA and NDEA + PGJ, along with their respective controls.Groups were same as explained in the legend of Fig. 1.Each bar represents the mean ± SD value (n = 5) of experiments performed in triplicates (*P < 0.05; **P < 0.01)

Fig. 7
Fig. 7Color contrast enhanced scanning electron micrographs (SEM) of rat kidney as given in Fig.6to demonstrate structural variations.Sequence of Panels is same as in Fig.6