Zinc oxide nanoparticles regulate NF-kB expression and restrict inflammation response in doxorubicin-induced kidney injury in rats

Doxorubicin (DOX) is an anthracycline drug used for cancer treatment. However, its treatment is contiguous with renal toxic effects. This study aims to investigate the therapeutic effects of zinc oxide nanoparticles (ZnONPs) on doxorubicin-induced nephrotoxicity. ZnONPs were prepared by the hydrothermal microwave method and characterized by XRD, SEM (combined with EDX) and HRTEM. To explore the possible nephrotoxicity and antioxidant effect of ZnONPs, rats were grouped as follows: control group, ZnONPs-treated group, doxorubicin group, and ZnONPs-Doxorubicin-treated group. DOX treatment increases renal tissue oxidative stress markers, while lowering antioxidant enzymes in tissue along with degenerative alterations in the renal tissue compared to control rats. Upon treatment with ZnONPs, a significant alteration was observed in the activities of superoxide dismutase, glutathione peroxidase, malondialdehyde, catalase, and the levels of kidney function, albumin, albuminuria, immune nuclear factor kappa B (NF-kB), and interleukin-6 (IL-6) compared to the doxorubicin group and control group. ZnONPs' administration to the doxorubicin group showed eminent renal injury control and restoration of the biochemical profile. This increases their active role in controlling kidney functions in order to improve nephrotoxicity and inflammatory responses. Histopathological and immunohistochemical observations provided context for these findings. In addition, ZnONPs alone did not show any undesirable effects on the renal parameters. However, its administration improves the renal functions. We concluded that the administration of ZnONPs ameliorated nephrotoxicity in rats caused by doxorubicin through its antioxidant and anti-inflammatory and antiapoptotic properties. This shows the therapeutic application of ZnONPs as a safe anti-inflammatory and might serve as a potential adjuvant that avoids DOX-induced nephrotoxicity.


Introduction
The kidney is commonly regarded as an organ that responsible for the removal of metabolic wastes.It is involved in many complex processes like blood homeostasis, acid-base balance, bone integrity, electrolyte levels and blood pressure.Kidney disease is defined as changes in kidney structure, function or both and may be acute or chronic injury [1].Acute kidney injury (AKI) is defined by a sudden loss of excretory kidney function.AKI is part of a variety of functional kidney conditions, which are summarized as acute kidney disease and disorders (AKD), and can range from mild and self-limiting to severe and persistent [2].AKD can occur without ever meeting the criterion of rapid onset of AKI, in which slow deterioration of kidney function or persistent kidney dysfunction is associated with an irreversible loss of kidney cells and nephrons [3].
When kidney dysfunction does not resolve or when structural damage to the kidney persists.By definition, AKD persisting for > 3 months is referred to as chronic kidney disease (CKD) [4].
Conversely, CKD also plays an important role in AKI.Patients with CKD may suffer higher risk of transient decreases in renal function consistent with AKI [5].The underlying mechanism that results in acute renal dysfunction 1 3 may involve decreased GFR, increased proteinuria, renal auto-regulation failure, and drug side effects [6].
There is increasing recognition that AKI and CKD are closely linked and are therefore regarded as an integrated clinical syndrome [7].Key biological processes such as cell death, cell proliferation, inflammation, and fibrosis, as well as common biomarkers, are detected in both kinds of nephropathy [8,9].Generally, tubular cell death, which includes necrosis, apoptosis, or necroptosis, is the main histological feature in early stage AKI, whereas fibrosis tends to occur under CKD.An increasing number of studies have shown that AKI is a major risk factor that can accelerate CKD progression [10].Clinical observations have also found a strong relationship between AKI and CKD.Compared to patients with no history of AKI or CKD, AKI patients are more likely to develop new CKD or end-stage renal disease (ESRD) [5,7].Further the syndrome is quite common among patients without critical illness and it is essential that health care professionals, particularly those without specialization in renal disorders, detect it easily.
Acute kidney injury (AKI) and chronic kidney disease (CKD) are linked to high morbidity and mortality.AKI is regarded as a rapid and reversible decline in renal function and is associated with accelerated CKD [11].Many risk factors such as drugs/toxins, sepsis, and ischemia-reperfusion (IR) commonly result in AKI and lead to reduced glomerular filtration rate (GFR) as well as acute tubular cell death [12].CKD is a significant medical problem globally, with a rapid increase in incidence due to the rise in hypertension and diabetes [13].
The underlying mechanisms that result in acute renal dysfunction may involve decreased GFR, increased proteinuria, renal autoregulation failure, and drug-induced nephrotoxicity [6].Doxorubicin (DOX), which is a member of the anthracycline family of cytotoxic antibiotics and one of the most potent and commonly used chemotherapeutic agents for the treatment of various types of cancer [14].The antitumor activity of doxorubicin is attributed to its ability to intercalate into the DNA helix and/or bind covalently to proteins involved in DNA replication and transcription, resulting in inhibition of DNA, RNA, and protein synthesis, leading finally to cell death [15].Doxorubicin-based chemotherapy for the treatment of a wide spectrum of both hematological and solid tumors.It is also associated with toxicities to different organs like the heart, kidney, liver, and testis [16].The exact mechanism of DOX-induced nephrotoxicity is not yet known.Furthermore, it has been suggested by many investigators that DOX can induce the generation of reactive oxygen species (ROS), which cause damage to membranes and macromolecules through lipid peroxidation, oxidative stress, DNA fragmentation, and protein oxidation [17].The increase in oxidative stress and depletion of endogenous antioxidants trigger the apoptotic pathway in the kidney [18,19].Available laboratory evidence shows that DOX induces nephropathy via its detrimental effects on renal tissue, including increased glomerular capillary permeability and glomerular atrophy [20], and increased serum levels of creatinine and BUN decreased serum albumin, as well as an elevated level of albuminuria [21].Therefore, administering drugs with anti-inflammatory and antioxidant effects could mitigate the side effects of DOX [22].
Nanoparticles are used in a variety of preclinical and clinical applications due to their unique properties, such as cellular uptake and delivery efficiency to biological systems.In the nanoscale dimension, nanoparticles (NPs) have different catalytic properties than larger particles of the same composition [23].
Nanoparticles, particles with a diameter less than 50 nm, have a lot of applications, including as drug carriers, which are important in the management of several disorders [24,25].Zinc oxide nanoparticles (ZnONPs) are one of the important nanoparticles that are commonly used in sunscreens, biosensors, pigments, food additives, and medicine [26].Although several studies reported that ZnONPs have cytotoxic effects [27], other studies reported that they have antioxidant effects [28] and antibacterial effects [29].Furthermore, ZnONPs inhibit the expression of several cytokines at the mRNA and protein levels, including IL6, IL-1, iNOS, COX-2, and TNF)tumor necrosis factor( [29].Furthermore, it has anti-diabetic properties, as ZnONPs reduce oxidative stress in diabetic rats [30].Due to the inherent toxicity of ZnONPs, they possess strong inhibitory effects against cancerous cells and bacteria by inducing intracellular ROS generation and activating the apoptotic signaling pathway, which makes ZnONPs a potential candidate as anticancer and antibacterial agents [31].Although many studies have shown that ZnONPs have cytoprotective effects in various tissues [28][29][30].To the best of our knowledge, the effect of ZnONPs against renal IRI is the first one.Thus, the current study was designed to examine the effect of ZnONPs on renal I/R injury (Ischemia/reperfusion injury) status and antioxidant profile.Then, we explored the effects on the expression of immune nuclear factor-kappa B (NF-kB) and interleukin-6 (IL-6) and kidney function in addition to the histopathological picture of the kidney.This is all to provide evidence that will support therapeutic efforts to treat renal Ischemia followed by reperfusion (I/R) injury with ZnONPs.This study aims to indicate the ZnONPs effect in acute kidney injury induced by DOX.
The present experiment was intended to delineate the protective perspective of ZnONPs against DOX-prompted renal injuriousness and oxidative trauma in rats.We hypothesized that ZnONPs could preclude DOX-induced nephrotoxicity due to its antioxidant properties.We studied the effect on the antioxidant status, biochemical alterations, renal function tests, antioxidant enzymes of renal tissue, and oxidative stress.Then, we explored the effects on the expression of immune nuclear factor-kappa B (NF-kB) and interleukin-6 (IL-6) and kidney function in addition to the histopathological picture of the kidney.This is all to provide evidence that will support therapeutic efforts to treat renal I/R injury with ZnONPs.

Chemicals
Creatinine and urea kits were purchased from (Diamond Diagnostics Company).Albumin, ALT, and AST kits were measured spectrophotometrically in serum using biodiagnostic kits (BioMed Diagnostics Company, Badr, Egypt).Damietta, Egypt).All chemicals were purchased from Sigma-Aldrich: St. Louis, MO, U.S.A.

Preparation of dispersible ZnO nanoparticles (ZnONPs)
ZnO nanoparticles (ZnONPs) manufactured as follows: 40 mmol of zinc sulfate (ZnSO4) and 40 mmol of urea (CO(NH 2 ) 2 ) dissolved in 1 L of deionized water with vigorous stirring.A 3.0 M sodium hydroxide (NaOH) solution added stirring constantly until the pH reached 12.The stirring continued for 30 min at room temperature.The solution transferred to a Teflon container open to the atmosphere and heated in microwave oven (2.45 GHz) at 800 watts of power for 10 min.After cooling to room temperature, they produced ZnO nanoparticles (ZnONPs) centrifuged and washed with deionized water.Part of the ZnO nanoparticles (ZnONPs) dried at 95 °C for XRD analysis, and the remaining part dispersed in deionized water and kept in the refrigerator for use, according to Torabi et al. [32].Then, ZnONPs suspended in saline (0.9%) before use and dispersed by ultrasonication to avoid particle aggregation.

Characterization of ZnONPs
In this study, the average particle size of ZnONPs was less than 50 nm.X-ray diffraction (XRD) is used to identify the nature and size of ZnONPs crystalline phases.In order to confirm size and identify morphology and crystalline qualities, ZnONPs examined through HRTEM (Tecnai G2 20 STWIN, Philips, the Netherlands), and ZnONPs particle sizes inspected by scanning electron microscope (SEM, Hitachi S4800).

Experimental animals
The present study included fifty adult male albino rats aging 6 weeks, with an average body weight of 100-120 g purchased from Holding Company for Biological Products and Vaccines, Cairo, Egypt.The rats were housed in groups in metal cages for bedding on a standard rat pellet diet and tap water ad libitum under controlled conditional of temperature (23.0 ± 1.0 °C) and humidity at 55 ± 10% with a 12 h light/dark cycle, respectively, for one week before and during the experiment.Animal treatments in this study were performed in compliance with the guidelines of the Guide for the Care and Use of Laboratory Animals and approved by Suez Canal University animal care committee, code number REC25/2022.

Animal grouping: experimental design
After an acclimatization period of one week, the rats were randomly distributed into five equal groups (n = 10).The experimental study period was four weeks, and the studied groups were as follows: In the control group (CNT group), the rats were intraperitoneally (IP) injected with three equal injections at 4-day intervals over a period of two weeks, starting on the first day of the second week, with 500 µl physiological saline.The rats in the nephrotoxic control group (DOX treated) were IP injected three times at four-day intervals for two weeks, beginning on the first day of the second week, with 500 µl of DOX dissolved in physiological saline at a dose of 5 mg/kg body weight, for a total cumulative dose of 15 mg/kg body weight.In ZnONP's normal treated group, the rats were administered with 200 µl of ZnONPs solution, respectively, at a dose of 10 mg/kg body weight, through IP (injected every day over a period of two weeks).The nephrotoxic group was treated with ZnONPs (DOX-ZNONPs group), and the rats were given both DOX (as mentioned in the DOX group) at 5 mg/kg body weight and 200 µl of ZnONPs dissolved in physiological saline, respectively, at a dose of 10 mg/kg BW via IP, injected every day for two weeks (two weeks after DOX injection).

Collection of urine samples
Urine samples were collected from all rats in each group, which were individually housed in metabolic cages to allow for 24 h urine collection.Rats continued to have free access to water and a standard diet.Total urine volume was measured; 1 ml was collected from a 24 h urine sample, stored at − 80 °C, and used for the determination of urinary albumin excretion.

Biochemical assessment
Blood samples were collected for measurement of creatinine using creatinine kits, Jaffé, colorimetric method-end point (Diamond Diagnostics Company, Hannover, Germany) according to manufacture instruction [33]; blood urea nitrogen (BUN) using urea kits, Berthelot enzymatic colorimetric method (Diamond Diagnostics Company, Hannover, Germany) according to manufacture instruction [34]; and albumin, ALT, and AST were measured spectrophotometrically in serum using biodiagnostic kits (BioMed Diagnostics Company, Badr, Egypt) according to manufacture instruction [35]; and urinary albumin excretion (g/mL) was also measured using microalbuminuria kits (ABC Diagnostic, New Damietta, Egypt) and a rapid colorimetric method according to the manufacturer's instructions [36] Albuminuria was calculated as g/24 h by taking the urine volume (ml) in 24 h into account.

Assessment of oxidative stress and inflammatory markers
The right kidney of all rats was collected and homogenized in phosphate buffer (pH 7.4) to assess the level of oxidative stress markers (malondialdehyde "MDA," reduced glutathione "GSH," catalase "CA," and superoxide dismutase "SOD") and the inflammatory marker "IL-6" using specific kits according to the manufacturer's instructions.Kits

Histopathological assessment
All rats' left kidneys were perfused with neutral buffered formalin (10%) and processed in 5 m paraffin sections for histopathological examination.All kidney tissues were stained with hematoxylin and eosin (H&E), periodic acid Schiff (PAS), and Masson Trichrome.Changes in kidney structure were analyzed to assess the severity of the morphologic changes (glomerulosclerosis, interstitial inflammation, interstitial fibrosis tubular atrophy, "IFTA," and vascular lesions), and then, each type of lesion was graded semi-quantitatively as described previously [37].

Immunohistochemistry (IHC(
NF-kB expression was evaluated on paraffin-embedded tissues by a standard immunostaining assay.Briefly, xylene and a graded alcohol series were used for deparaffinization and rehydration, respectively.Then, the slides were incubated for 30 min in a blocking reagent containing 1.5% hydrogen peroxide in methanol.Antigens were retrieved and placed on slides using a microwave protocol.They were then incubated in serum for 30 min and immunostained with NF-kB (cat.no.sc-48,366; Santa Cruz Biotechnology, Inc.) primary antibodies for 1 h at room temperature.Afterward, the slides were washed with PBS three times and then incubated with secondary antibodies for 30 min.The sections were stained using 3,30-diaminobenzidine (Dako liquid DAB color solution), and the slides were then counterstained with hematoxylin.An Olympus BX51 microscope (Olympus, Tokyo, Japan) was used to visualize the results.Five different microscopic fields were randomly selected from each slide, and positive staining within each slide was measured using Image-Pro Plus 6.1.Finally, quantitative analysis was performed in a blinded manner [38].

Statistical analysis
Data analysis was expressed as mean ± SEM.Difference between groups was detected using one-way ANOVA followed with post hoc Tukey test using SPSS software version 21.Values at P < 0.05 were considered statically significant.

XRD analysis of ZnONPs
The phase of the as-synthesized ZnONPs into the product is determined using XRD, as shown in Fig. 1a.XRD analysis shows that the sample is a pure ZnONPs in the stage of the hexagonal ZnONPs phase (wurtzite-type structure, space group: P63mc (186).All the spread peaks indexed to the (100), (002), (101), (102), (110), (103), (200), and (211) of the pure hexagonal wurtzite structure.No contamination was detected in these products.

SEM microstructure analysis
The morphology of the samples examined using SEM. Figure 1b shows the SEM micrographs.The SEM images indicate that samples agglomerate to some extent and have an irregular spherical form with particle size.A composition of the samples estimated using the EDX spectra received in conjunction with the SEM micrographs.Figure 1c explains the resultant EDX spectrum, wherever Zn and O peaks clearly visible with no other peaks which bear any similarity to other elements.

TEM analysis of ZnONPs
The TEM remarks explained that ZnONPs showed almost spherical geometry with diameter about 30-50 nm. Figure 1d.

Effects of ZnONPs on kidney and liver functions of experimental groups
The treatment effect of ZnONPs on kidney dysfunction induced by doxorubicin is presented in Table1 and indicated by analyzing serum creatinine, BUN, albumin, AST and ALT.In doxorubicin-treated group, serum creatinine, BUN  were a highly significant increase when compared with the normal control group.While treated group with ZnONPs (10 mg/kg body weight/day) showed a highly significant reduction in serum creatinine, BUN levels when compared with the doxorubicin-treated group (Table 1).While there was no significant change in serum creatinine, BUN levels in group treated with ZnONPs (10 mg/kg body weight/day) when compared with the normal control group.There was a highly significant decrease in serum albumin level in doxorubicintreated group when compared with the normal control group (Table 1).While there was no significant change in serum albumin level in group treated with ZnONPs (10 mg/kg body weight/day) when compared with the normal control group.While there was a highly significant increase in serum albumin level in treatment group with ZnONPs (10 mg/kg body weight/day when compared with the doxorubicin-treated group (Table 1).Moreover, the effect of DOX resulted in a highly significant increase in serum AST and ALT levels compared to the normal control group.While there was no significant change in serum AST and ALT levels in the group treated with ZnONPs (10 mg/kg body weight/day) when compared to the normal control group, While there was a highly significant decrease in serum AST and ALT levels in the treatment group with ZnONPs (10 mg/kg body weight/day) when compared with the doxorubicin-treated group,

Effects of ZnONPs on albuminuria of experimental groups
Figure 2 shows the serial changes of albuminuria (g/24 h) in the experimental groups.Albuminuria was significantly higher in the doxorubicin-treated group than in the normal control group.Therapy with ZnONPs led to a variable and significant decrease in albuminuria compared to the doxorubicin-treated group.While there was no significant change in albuminuria in the group treated with ZnONPs (10 mg/kg body weight/day) when compared with the normal control group, Values are expressed as mean ± SEM (n = 9-10/group); statistics were done using one-way ANOVA followed by post hoc Tukey test (P value ≤ 0.05).

Effect of ZnONPs on renal oxidative stress and antioxidant markers
Renal MDA, SOD, CAT, and GSH were performed for evaluation of the reno-protective effect of ZnONPs (Figs. 3a-d) on doxorubicin treatment induced a highly significant increase in MDA and decreases in SOD, CAT, and GSH when compared to the normal control.While there was no significant change in MDA, SOD, CAT, or GSH in group treated with ZnONPs (10 mg/kg body weight/day) when compared with the normal control group.Interestingly, the concentration of MDA was highly significant (P 0.001), while that of SOD, CAT, and GSH were significantly elevated (P 0.001) in the ZnONPs treated group when compared with the doxorubicin-treated group.

Interleukin-6 (IL-6) levels in renal tissue of experimental groups
Renal IL-6 levels were significantly elevated in the doxorubicin-treated group compared to the normal control group.When compared to the doxorubicin-treated group, the ZnONPs (10 mg/kg body weight/day) group showed a highly significant decreased in renal IL-6 levels.While there was no significant change in renal IL-6 level in the group treated with ZnONPs (10 mg/kg body weight/day) when compared with the normal control group (Fig. 4),

Histopathological investigations
Histopathological investigations of kidneys are shown in (Fig. 5A-C).Normal histology of H&E stained renal tissue was observed in control group and group received ZnONPs.Various histopathological changes were illustrated in the H&E stained renal sections from Dox group including: dilated Bowman's space with thickened glomerular basement membrane, mononuclear cells infiltration in interstitial tissue, congestion, interstitial fibrosis, tubular cast formation, endothelial cell swelling.ZnONPs effectively reduced the histopathological changes induced by Dox.
The PAS stained renal sections showed no excess glycogen deposition (magenta-purple color) in glomeruli and tubules of control group and group received ZnONPs.The PAS stained renal sections from Dox group showed significantly increased deposition in glomerular tuft, glomerular and tubular basement membranes.The renal sections from treated group with ZnONPs showing decreased deposition of PAS + ve material in basement membranes.

GSH
The MT stain displayed no collagen deposition in interstitial tissue in control group and group received ZnONPs.MT stained renal sections from Dox group displayed excess blue stained collagen deposition in interstitial tissue indicating induced fibroplasia.MT stained renal sections from Dox + ZnONPs group displayed very mild blue stained collagen deposition in interstitial tissue.

Immunohistochemical staining
The immunostained renal sections against NF-kB showed negative NF-kB expression in tubular cells.The immunostained renal sections from Dox group showed marked positive brown staining against NF-kB in many tubules.The positive staining against NF-kB markedly decreased in the immunostained renal sections from Dox + ZnONPs group (Fig. 5D).

Discussion
DOX is used to treat multiple solid tumors but has severe adverse effects on the kidneys [38][39][40].The mechanisms by which DOX causes glomerular toxicity are not yet fully understood.However, previous reports have shown that reactive oxygen species and free radicals are the most significant contributors to DOX-induced nephrotoxicity [40][41][42].DOX exerts direct toxic damage to the glomerular base membrane, podocytes, and glomerular endothelial cells, inducing tubular interstitial inflammation and fibrosis [43].Inflammation increases ROS production, oxidative stress, apoptosis, and a decrease in antioxidant enzymes in the kidney, all of which may be underlying causes of DOX-induced renal injury [44].As a result, renal function is compromised following DOX administration; in turn, serum urea and creatinine concentrations increase while serum albumin decreases, ultimately leading to extreme proteinuria [45].Because rat intrarenal enzyme dissemination is similar to that of humans, drug-induced improvements in kidney function and tubular lesions can be easily evaluated [46].The current study was proposed to discern the potential of ZnONPs to prevent the chemotherapeutic drugs' induced renal toxicity, which might enhance the antitumor efficacy of DOX.In the current research, we used a cumulative dose of DOX injections to induce an experimental nephrotic syndrome model.This model was characterized by increased serum levels of creatinine and BUN (two significant indicators of renal function) and decreased serum albumin [21], as well as an elevated level of urine albumin (one of the general indicators of kidney injury) [47], all of which are associated with increased oxidative stress and inflammatory factors.The toxic effects of DOX administration observed in the current study are consistent with the findings of previous studies in which renal function parameters including serum urea and creatinine increased [48,49].However, administering ZnONPs before, during, and after DOX injections yielded improvements in renal function parameters, including decreased serum urea, increased creatinine, decreased urine albumin, and increased serum albumin.These improvements in DOXinduced renal dysfunction have been confirmed by histological and biochemical findings [50].For example, DOX has been shown to increase oxidative stress in the kidneys, as indicated by changes in antioxidant status.
The decline of SOD, CAT, and GR activities, accompanied by a decrement in GSH content, was unveiled after DOX injection, resulting in the reduced ability of the kidney to scavenge toxic H 2 O 2 and lipid peroxides.These findings were also noticed by other researchers [21].In the present investigation, ZnONPs were able to significantly restore the above-mentioned antioxidant enzyme activities in kidney tissues.Reno-protective actions of ZnONPs against DOXinduced nephrotoxicity.
Renal MDA was found to be increased after DOX inoculation, among other oxidative stress inducers.Outcomes of the present research also indicate that there was a significant increase in MDA and H 2 O 2 quantity in the renal tissue of rats that received DOX alone in contrast to the control group.These results were in accord with preceding studies, which demonstrated an increase in lipid peroxidation and subsidence of the antioxidant defense system in the kidney after DOX treatment [51].
The findings show that ZnONPs can reverse renal injury caused by DOX treatments and confirm the importance of ZnONPs antioxidant properties in preventing drug-induced nephrotoxicity, specifically by enhancing the antioxidant defense system consistently [51,52].NF-kB is a transcription factor that regulates a wide variety of genes involved in developing renal disease [53,54].NF-kB activation plays a pivotal role in the pathogenesis of DOX-induced renal inflammation [55].TNF-, IL-1, and IL-6 expression is mediated by NF-kB, which is responsible for inflammatory reactions [56,57].
Furthermore, serum IL-6 levels were found to be higher in DOX-treated rats.The anti-inflammatory effects of ZnONPs have been described in multiple studies [58][59][60].Several investigators reported that ZnONPs had an antiinflammatory effect by downregulating the expression of interleukin (IL)1, IL6, and TNF [48].Consistent with these observations, ZnONPs co-therapy reduced the extent to which IL-6 levels were altered by DOX.These results suggest that ZnONPs have anti-inflammatory effects and can reduce the harm done by DOX in harvested tissues.Dysregulation of inflammation and apoptosis is involved in DOX-induced renal injury.NF-kB is involved in the signaling pathways of inflammation and apoptosis.It is reported that DOX could induce NF-kB activation both in vivo and in vitro [61,62].However, little evidence is currently available on the relationship between DOX-induced cell apoptosis and NF-kB activation.We examined the involvement of NF-kB signaling molecules that have been implicated as stimulators of inflammation and apoptosis [63].In our study, we observed that DOX-induced NF-kB activation was prevented by ZnONPs.It was demonstrated that ZnONPs pretreatment decreased the expression of NF-kB.Our results with ZnONPs show that ZnONPs reduce NF-kB expression.
The beneficial effects of ZnONPs were further illustrated by a histological evaluation using H&E staining, periodic acid Schiff (PAS), and Masson Trichrome.It was assumed that histopathological changes are related to the absorption capacity of renal tubules, which causes functional congestion of nephrons and subsequent kidney dysfunction [60].ZnONPs' treatment significantly ameliorates the damaging effects of DOX on kidney morphology.By acting as a potent quencher of radical species in the kidney, it prevents toxic influences at both the histological and biochemical levels [61].These findings supported the hypothesis that ZnONPs could alleviate the biochemical and histological changes caused by DOX treatment.Therefore, ZnONPs might be a significant candidate, providing an excellent protective effect in combination with chemotherapy due to their antioxidant and radical species quenching potential.Our results suggest that ZnONPs alleviate DOX-induced nephrotic syndrome in rats, likely owing to its antioxidant and anti-inflammatory properties.These findings indicate that ZnONPs could be used as a therapeutic agent for DOX-induced nephrotoxicity.

Conclusion
The present study indicates that the treatment effect of ZnONPs on DOX provoked kidney damage in rats.The outcomes revealed the ameliorating potential of ZnONPs against DOX-induced kidney injury via suppression of the effect of DOX-mediated oxidative deterioration in renal tubular cells, preservation of kidney function biomarkers, and prevention of DNA damage.ZnONPs offered added protection against DOX-induced kidney damage.The mechanism of nephroprotective action of the ZnONPs fraction could be due to the antioxidant and free radical scavenging activities of its active metabolites.The present results suggest that ZnONPs might be potentially therapeutic in preventing the nephrotoxic and inflammatory effects of DOX.

Fig. 1
Fig. 1 Characteristics of ZnONPs nanoparticles: a XRD pattern, b SEM image, c EDX analysis and d HRTEM image

Fig. 4 Fig. 5 A
Fig.4 The effects of DOX and/or ZnONPs on interleukin-6 (IL-6) of renal tissues of different groups.Values are expressed as mean ± SEM

Table 1
The effects of DOX and/or ZnONPs on Kidney and Liver functions of experimental groups Values are expressed as mean ± SEM; statistics were done using one-way ANOVA followed by post hoc Tukey test) P value ≤ 0.05 ( a Highly significant in comparison with normal control group b Highly significant in comparison with doxorubicin-treated group; NS, not significant