Nebivolol ameliorates sepsis-evoked kidney dysfunction by targeting oxidative stress and TGF-β/Smad/p53 pathway

Sepsis is a potential fetal organ destruction brought on through an overzealous immunologic reaction to infection, causing severe inflammation, septic shock, and damage to different organs. Although there has been progress in the identification and controlling of clinical sepsis, the fatality rates are still significant. This study, for the first time, intended to examine the possible ameliorative impact of Nebivolol, a β1-adrenergic antagonist antihypertensive drug, against nephrotoxicity resulted from cecal ligation and puncture (CLP)-induced sepsis in rats, on molecular basis. Sixty male Wistar albino rats were chosen. Oxidative stress indicators and biochemical markers of kidney activity were evaluated. Inflammatory mediators, fibrosis- and apoptosis-related proteins and gene expressions were investigated. Moreover, renal histopathological investigation was performed. CLP-induced nephrotoxicity characterized by markedly elevated serum levels of creatinine, blood urea nitrogen, uric acid, and renal malondialdhyde. On the other hand, it decreased serum total protein level, renal superoxide dismutase activity and reduced glutathione level. Additionally, it significantly elevated the renal inflammatory mediators (tumor necrosis factor-alpha, ilnerlukin (IL)-6, and IL-1β) and Caspase-3 protein, reduced IL-10 level, amplified the expression of transforming growth factor-beta 1 (TGF-β1), p-Smad2/3 and alpha-smooth-muscle actin proteins, downregulated the B cell lymphoma-2 (Bcl-2) gene and elevated the transcription of Bcl-2-associated X-protein (Bax), p53 and Nuclear factor-kappa B (NF-κB) genes. Furtheremor, kidney tissues exhibited significant histopathological changes with CLP. On the contrary, Nebivolol significantly improved all these biochemical changes and enhanced the histopathological alterations obtained by CLP. This research showed, for the first time, that Nebivolol effectively mitigated the CLP-induced kidney dysfunction via its antioxidant, antifibrotic and anti-apoptotic activity through modulation of oxidative stress, TGF-β/NF-κB and TGF-β/Smad/p53 signaling pathways.

Sepsis is a frequent consequence of trauma and injuries, which remains a burdensome public health problem globally.Aggressive immune defense against infection, severe inflammation, sharp drop in blood pressure, and tissue and organ damage are the hallmarks of sepsis, a potentially fatal organ failure 1,2 .With the support of the Surviving Sepsis Campaign (SSC), the mortality rate from sepsis has decreased; nevertheless, the total number of deaths from sepsis has increased due to the increasing number of new cases 3 .Around 2017, 48.9 million instances were reported, with approximately 1.7 million new cases annually and nearly 11 million deaths, which is almost 20% of total global deaths, with the highest load on nations with moderate and low incomes, especially Africa 4 .The known methods for handling sepsis, including antibiotics use, organ support therapy, or fluid resuscitation, have poor predicting influence on patient health with sepsis.Nowadays, sepsis is deemed to be the leading cause of death, surpassing even cancer or coronary diseases, and raises the expense of medical care in the intensive care units 5 .

Experimental model
As previously secribed, the CLP model was used to provoke sepsis 31 .Briefly, a combination of xylazine (10 mg/ kg bw) and ketamine (80 mg/kg bw) was injected intraperitoneally to sedate the rats.Rats' abdomen walls were cleansed and shaved using a 10% povidone-iodine solution 32 .An incision took place in the bottom left region of the abdomen.The cecum was exteriorized and tied regularly with 0.3 mm silk medical thread at 75% of its length.The ligated part of the cecum was twice punctured through and through using an 18-gauge injection needle.The identical interventions without CLP were performed to the sham rats.

Animals and the experimental schedule
Seven-week-old, sixty healthy Wistar male albino rats weighting 180-200 g were obtained from the National Research Center in Giza, Egypt and kept in an experimental animal laboratory with temperature control, free from the pathogen with an unrestricted supply of regular laboratory food and water.The experiment was accepted and executed in compliance with the Care and Use of Laboratory Animals standard operating procedures approved by the Research Ethics Committee, Minia University (Project code: MPEC (2301202)).
Following a week of housing, rats were randomly placed into six groups of ten each.Group I (Sham group): rats were given two intraperitoneal injections: one of normal saline (i.p.) and the other of 1% tween 80 in distilled water (i.p.).After one hour, the same CLP surgical procedure was performed without CLP induction.
Group II (Nebivolol 10): rats obtained Nebivolol (10 mg/kg) i.p., as a single dose.Group III (CLP): Rats obtained a single intraperitoneal injection of normal saline.After one hour, a CLP operation was performed.
Group VI (CLP/Vitamin C): an hour following the CLP procedure, rats obtained a single intraperitoneal (i.p.) dosage of Vitamin C 38 .This group was used as a positive control group.
Rats were given isoflurane anesthesia twenty-four hours after the CLP surgery.Blood was extracted from the jugular vein by slaughtering the rats, and serum was collected to analyze the biological markers.After being removed, the kidneys were cleaned in cold saline and divided into four sections.For histological investigation, one part was immersed in 10% formaldehyde.The second part was homogenized in 1X PBS, pH 7.4, to quantify

Quantitative real-time polymerase chain reaction
In accordance with the manufacturer's guidelines, kidney tissues were treated for the total RNA extraction using a direct-zol™ RNA Miniprep Plus kit (#R2072, Zymo research crop, USA), and the quality of the extracted RNA was evaluated through the use of a Thermo Fisher Scientific, USA, Nanodrop 8000 spectrophotometer.The SuperScript IV One-Step RT-PCR kit (#12594100, Thermo Fisher Scientific, Waltham, MA, USA) was employed to perform quantitative polymerase chain reactions (qPCR) using equivalent quantities of total RNA in duplicate.The kit allows c-DNA synthesis and PCR amplification to be performed in a single reaction tube, as the supplier recommends, on a Step One Real-Time PCR system (Applied Biosystems, Foster, USA).Melt curve studies proved the selectivity of the amplification.The endogenous reference gene β-actin, was used to standardize the target genes' cycle threshold (Ct) values.The 2 −∆∆Ct method was then applied to divide the expression in each sample by the sham sample to determine the fold change.
The precise primer sequences used with their accession numbers for Bax, Bcl-2, p53, NF-κB and β-actin genes are displayed in Table 1.

Western blotting analysis
Kidney homogenate samples with equal protein portions (20 µg) were isolated using 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and placed onto a PVDF membrane.After treating the membranes with block ACE (DS Pharma Biomedical Co. Ltd, Japan) at ambient temperature for four hours, primary antibodies were added to the membranes and incubated overnight at 4 °C with primary antibodies for TGF-β1 (#SC-130348, Santa Cruz Biotechnology Inc., USA), total Smad2/3 (#3102, Cell Signaling Technology, USA), p-Smad2/3 (#8828, Cell Signaling Technology, USA), α-SMA (#NB300-978, Novus biologicals LLC., USA), and β-actin (#F1819, Santa Cruz Biotechnology, USA).The gout anti-rabbit immunoglobulin G (Novus Table 1.Primer sequences and their accession numbers used in qRT-PCR.www.nature.com/scientificreports/Biologicals LLC., USA) secondary antibody coupled with horseradish peroxidase was used to detect the primary antibodies, and an enhanced chemiluminescence system (Clarity TM western ECL substrate, #170-5060, Bio-Rad Inc., CA, USA) was employed to display the results.A CCD-camera-based imager was used to record these signals.After normalizing target protein bands against β-actin bands (housekeeping protein), band density was estimated using ChemiDoc MP imaging system software (Bio-Rad Inc., Hercules, CA.USA).Both primary and secondary antibodies were employed at a dilution of 1:1000 in 5% BSA in PBST.

Histopathological examination of kidney tissues
The kidney tissues were prepared for light microscopic study to create paraffin blocks by fixing them in 10% formaldehyde utilizing the tissue-embedding center (LEICA EG 1160, Wetzlar, Germany).Staining with hematoxylin and eosin (H&E) was performed on five-micrometer slices after being deparaffinized with xylene and rehydrated, as previously described 39 .Light microscopy and a digital slide scanner (Leica APERIO LV1 microscope) were used to examine the sections.

Statistical analysis
Every statistic point was displayed as mean ± SEM.GraphPad Prism® 9 (GraphPad Software Inc., San Diago, USA) was used for a one-way ANOVA test.The Tukey-Kramar test was then used to examine the findings and determine whether significant variations between the groups existed.Any difference was deemed significant if its probability (p) value < 0.05.

Ethical approval
The study was conducted in accordance with ARRIVE guidelines and executed in compliance with the Care and Use of Laboratory Animals standard operating procedures approved by the Research Ethics Committee, Minia University (Project code: MPEC (2301202)).

Effect of nebivolol on kidney injury indices
Sepsis induced by CLP has a powerful injurious effect on the kidney, and its action has been affirmed by different markers 39 .CLP substantially (p < 0.001) increased serum Cr, BUN, and uric acid levels contrasted to sham group (Fig. 1).Additionally, it resulted in a substantial (p < 0.001) drop in the levels of serum total protein.Compared www.nature.com/scientificreports/with the CLP group, the treated groups with Neb.4,Neb.10,or Vit C drastically (p < 0.001) reduced the changes in levels of Cr, BUN, uric acid, and total protein produced by CLP in the serum.

Impact of nebivolol on renal oxidative stress
As shown in Fig. 2, following induction of CLP, the kidney's MDA content was found to be notably (p < 0.001) higher than that in sham rats, whereas GSH level and SOD activity were significantly (p < 0.001) reduced.It's interesting to observe that when administrated in conjunction with CLP, Nebivolol (4 or 10 mg/kg) or vitamin C dramatically (p < 0.001) reduced the amount of MDA in kidney homogenate and increased renal GSH level and SOD activity in comparison to CLP rats.In addition, administration of 10 mg/kg Nebivolol in CLP + Neb10 group significantly increased the renal GSH level and SOD activity (p < 0.001 and p < 0.01, respectively) when compared with those of CLP + Neb4 group.
The present study utilized ELISA to assess the protein levels of the pro-inflammatory (TNF-α, IL-6, and IL-1β), the anti-inflammatory (IL-10) cytokines, and the pro-apoptotic mediator caspase-3 in CLP and treated groups.As illustrated in Fig. 3, CLP induction, compared to sham, drastically (p < 0.001) elevated the renal protein amounts of TNF-α, IL-6, IL-1β, and caspase-3.Furthermore, it markedly (p < 0.001) reduced the IL-10 level in kidney tissues.The administration of drugs of interest (Nebivolol (4 or 10 mg/kg) or Vit C) with CLP surgery significantly reduced the pro-inflammatory mediators and the caspase-3 levels while significantly raising the anti-inflammatory IL-10 level in comparison to CLP rats.

Effect of nebivolol on Bax, Bcl-2, p53, and NF-κB genes expression
Overactivation of pro-fibrotic and pro-apoptotic agents, such as Bax, p53, and NF-κB, and suppression of the anti-apoptotic mediator Bcl-2 in the kidney contributes to its apoptosis and fibrosis.Therefore, we measured the impact of Nebivolol on mRNA levels of Bax, Bcl-2, p53, and NF-κB to estimate its potency at different dosages as a protective molecule against kidney apoptosis and fibrosis induced by CLP.As Fig. 4 illustrates, Bax, p53, and NF-κB genes all exhibit markedly (p < 0.001) increased expression in response to CLP.In addition, it notably (p < 0.001) decreased Bcl-2 gene expression contrasted with sham rats.However, both Nebivolol doses and Vitamin C demonstrated a significant reduction in the expression of the Bax, p53, and NF-κB genes while boosting the expression of the Bcl-2 gene compared to the CLP group.

Effect of nebivolol on TGF-β1, p-Smad2/3, and α-SMA proteins expression
To check the protective impact of Nebivolol on kidney fibrosis, we assessed the blocking effect of Nebivolol on TGF-β1/Smad/α-SMA signaling cascade triggered by CLP via western blotting.Figure 5 illustrates after normalization of the intensities of targeted bands to β-actin, CLP exhibited an enormously (p < 0.001) enhanced up-regulation of TGF-β1, p/t-Smad2/3, and α-SMA proteins in the kidneys as compared to sham rats.Treated groups with Nebivolol (4 or 10 mg/kg) or Vit C showed a significant down-regulation of all proteins compared to septic rats in the CLP group.

Effect of nebivolol on histological changes of kidney tissues
When compared with kidneys of the sham group rats that displayed standard renal tubules, glomeruli, and vasculature, the kidneys of the CLP group rats displayed collapsed glomeruli and dilated Bowman's capsule, degraded tubules, and regions of diffuse hemorrhage.The rats of CLP/Nebivolol 4 group revealed a few renal glomeruli with glomerular capillary collapse and expanded Bowman's capsule.Cytoplasmic vacuolations and fuzzy edema were seen in the tubules.In addition, the CLP/Nebivolol 10 group showed near-normal renal glomeruli, minor tubular cytoplasmic vacuolation, and healthy vasculature.However, the CLP/Vitamin C group showed mild tubular cytoplasmic vacuolation and degeneration, as illustrated in Fig. 6 and Table 2.

Discussion
Sepsis, a clinical syndrome of having an extreme systemic inflammatory reaction syndrome (SIRS) to the pathogen, is considered the most frequent causal factor concerning AKI progression.AKI is strongly correlated with weak prognostic consequences involving CKD, heart events, and even demise.While the exact etiology of S-AKI is unknown, sepsis is thought to be responsible for 45-70% of AKI episodes in patients with serious conditions 6,7 .
Australia had approximately 11.7% incidence of S-AKI, and the frequency in Beijing was roughly 48.1%, with a 55% overall fatality rate 41 .
It is difficult to improve outcomes in S-AKI because individuals may exhibit clinical or subclinical AKI, and AKI linked to sepsis may manifest with distinct phenotypes and prognoses 42 .Several aspects of S-AKI are poorly  www.nature.com/scientificreports/understood, including its epidemiology, pathophysiology, biomarker's role in risk assessment and treatment protocols, and the impact of new treatments on patient outcomes.Rats' CLP operation was shown to cause sepsis with additional kidney damage and renal failure, mirroring human illness 43 .Various approaches, using synthetic or natural candidates, have been applied to relief the renal toxicity generated from CLP-induced sepsis by targeting oxidative stress, apoptosis, and fibrosis in damaged kidney tissues [43][44][45][46][47] .Nebivolol administration was recently reported to diminish ischemia-reperfusion, contrast media, and drug-induced kidney failure through its anti-oxidant and anti-inflammatory effects [48][49][50] .To the best of our information, no previous research has looked into the potential preventive role of Nebivolol against sepsis-evoked organ damage.This study is the first one to assess the possible potential signaling mechanism of Nebivolol and its protective effect on kidney fibrosis resulted from CLP-induced sepsis.
For the first time in CLP-induced nephrotoxicity, the finding of this study showed that pretreatment with Nebivolol, in a dose-dependent manner, reduced the elevated levels of uric acid, Cr, and BUN serum levels and increased the level of serum total protein.Moreover, Nebivolol reduced renal MDA content, protein levels of TNF-α, IL-1β, IL-6, and caspase-3, mRNA expression of Bax, p53, and NF-κB, and the protein expression of TGF-β1, p-Smad2/3, and α-SMA.In addition, it increased SOD activity, GSH and IL-10 levels, and up-regulated the Bcl-2 mRNA renal expression.
In the present study, induction of sepsis through CLP surgery developed a marked deterioration in kidney function through increasing the serum levels of its markers, including Cr, BUN and uric acid.In addition, a notable reduction in the total protein content was observed.These biochemical results matched the histological alterations displayed in the septic rats.The pathophysiology of the harmful effect of sepsis on the kidney involves several pathways, including the aggravation of oxidative stress, inflammation, and immune function deterioration 51 .Sepsis-related oxidative stress stimulates the body's adaptive response to tissue hypoxia, apoptosis and further fibrosis 52 .Therefore, the lipid peroxidation (oxidative stress) marker, MDA, as well as the anti-oxidant markers, including SOD and GSH were assessed in kidney homogenates.In this study, the renal tissues of CLPintoxicated rats had a significant increase MDA content.Additionally, there was a marked decrease in their SOD activity and GSH level in contrast with the normal rats.It disclaimed that an elevation in ROS generates sepsispersuaded kidney damage by decreasing the host's anti-oxidant defense mechanisms, leading to cell damage 51,53 .Bars are represented as mean ± SEM.The Tukey-Kramar test was used to analyze the significant differences between groups after conducting a one-way ANOVA test, where ### p < 0.001, compared to the sham group, **p < 0.01, and ***p < 0.001, compared to the CLP group, $$ p < 0.01, and $$$ p < 0.001, compared to CLP + Neb4 group.

D C B A
Numerous pro-inflammatory and pro-fibrotic signaling networks, including TGF-β1, NF-κB, p53, and MAPK family members, are activated more strongly by ROS and oxidative stress [53][54][55][56] .It was implicated that TGF-β1 has a crucial function in generating EMT and is the driver of kidney fibrogenesis through TGF-β1/Smad/p53 signaling pathways [57][58][59][60] .During sepsis, active TGF-β1 is released and attached to its receptor (TGFβI/II) resulting in the phosphorylation and activation for Smad2/3, which binds with Smad4, leading to the formation of an active complex of Smad2/3/4.At the same time, TGF-β1 can stimulate the non-Smad pathway, leading to activation and phosphorylation of p53-dependant tubular epithelial G2/M arrest 61 , leading to the assembly of p53-Smad complex.This complex is translocated in the nucleus and binds to target genes responsible for the induction of EMT, inflammation and renal fibrosis 57,62,63 .α-SMA is a positive EMT marker for myofibroblast, as it activates fibroblast, which is deemed a central cell of renal injury and fibrogenesis 64 .Additionally, TGF-β1 can induce TAK1/IKK/IkBα phosphorylation and subsequently stimulate the phosphorylation of the downstream NF-κB inflammatory transcriptional factor, which translocates and accumulates in the nucleus to facilitate the transcription of multiple inflammatory cytokines, including TNF-α, IL-1β and IL-6, while also diminishing the anti-inflammatory cytokine, IL-10 57,65,66 .This study revealed that CLP-induced sepsis can evoke renal fibrosis through EMT activation, affirmed by the elevation of α-SMA marker in septic rats contrasted to the sham group.We demonstrated that CLP stimulates EMT by dramatically upregulating the protein expression of α-SMA.(B-D) Expressions of TGF-β1, p-Smad2/3/t-Smad2/3, and α-SMA proteins were represented densitometrically using bands in (A).Bars are represented as mean ± SEM.The Tukey-Kramar test was used to analyze the significant differences between groups after conducting a one-way ANOVA test, where ### p < 0.001, compared to the sham group, *p < 0.05,**p < 0.01, and ***p < 0.001, compared to CLP group, $$ p < 0.01, and $$$ p < 0.001, compared to CLP + Neb4 group.Furthermore, the protein expressions of renal TGF-β1, p-Smad2/3, and also the expression of p53 and NF-kB genes were assessed in this study to illuminate the probable signaling pathway by which CLP can stimulate EMT, renal fibrosis, and inflammation.Our results supported that CLP resulted in an increase in TGF-β1 and Smad2/3 proteins expression, as well as an increase in the expression of p53 and NF-kB genes, which further up-regulated the inflammatory mediators TNF-α, IL-6, and IL-1β and inhibited the anti-inflammatory cytokine, IL-10.In such a manner, the present study illustrated that sepsis affects the onset of EMT, renal fibrosis, and inflammation by modulating TGF-β1/Smad/p53 and TGF-β1/NF-κB signaling cascades.
The Bcl-2 family has a significant role in transducing apoptotic signals within cells.Apoptosis is directly linked to the pro-and anti-apoptotic Bax and Bcl-2 genes; the ratio of Bax to Bcl-2 expression is a cell death button that controls a cell's sensitivity to an apoptotic stimulus, determining whether it will proliferate or die 67 .The overexpression of Bax and diminished Bcl-2 leads to the release of cytochrome c in the cytoplasm, which triggers caspases (caspase 3/7) and results in irreversible apoptosis and cell death 68 .It was found that activation of TGF-β1/NF-κB pathway triggered apoptosis by targeting the mitochondrial pathway.That was accomplished by lowering the level of Bcl-2 and raising the amounts of Bax and caspase-3.Consistently, kidney tissues of septic rats in this study showed an elevation in the expressions of Bax gene and Caspase-3 protein and diminished expression of Bcl-2 gene concurrently with the previous studies 69,70 , suggesting significant apoptosis in these rats.
ROS is known to contribute to the renal disease onset and progression through the induction of TGF-β1.Therefore, targeting ROS and TGF-β1 expression may be valuable in treating renal fibrosis 14,71 .As a result, we assumed that candidates with an anti-oxidant perspective or that target TGF-β1 may inhibit sepsis-evoked kidney fibrosis.Recently, Nebivolol was revealed to inhibit inflammation in ischemia-reperfusion and drug-induced www.nature.com/scientificreports/toxicity in various organs due to its potentiality to target and reduce oxidative stress 35,36,[48][49][50][72][73][74] . These iscoveries suggest that Nebivolol may protect kidneys against CLP-evoked sepsis as demonstrated in this study.Nebivolol dramatically reduced kidney damage and restored renal function in the current trial by significantly lowering uric acid, creatinine, and BUN serum levels while powerfully restoring serum total protein levels.In addition, the histological examination, which validated the recovery in all morphological alterations of kidney construction, supported these findings.According to our study, administering Nebivolol to septic rats results in a concentration-dependent reduction in lipid peroxidation, MDA content, a significant increase in anti-oxidant status, GSH levels and SOD activity, and a decrease in renal damage brought on by oxidative stress.These results are consistent with those of Gandhi et al., who found that Nebivolol, in injured kidneys by ischemia-reperfusion, exhibits anti-oxidant activity through the reduction of MDA content as well as the elevation of SOD activity and GSH levels 75 .
Moreover, for CLP-intoxicated rats, treatment with Nebivolol significantly suppressed the renal upsurge in protein expression of TGF-β1, p-Smad2/3, and α-SMA, as well as reduced the gene expression of p53, suggesting that Nebivolol could inhibit renal fibrosis in septic rats through the attenuation of TGF-β1/Smad2/3/p53 signaling pathway which stimulates EMT process.Additionally, in the present study, Nebivolol was found to suppress the gene expression of NF-κB and further its downstream inflammatory negotiators, TNF-α, IL-1β, IL-6, and upregulate the anti-inflammatory cytokine IL-10, indicating that Nebivolol has anti-inflammatory activity.Furthermore, it implied an anti-apoptotic effect by significantly suppressing the expession of Bax gene and caspase-3 protein and enhancing the expression of Bcl-2 gene.
Thus, the current data clearly showed the nephroprotective effect of Nebivolol, which might be a novel therapeutic drug for ameliorating sepsis-evoked kidney dysfunction through targeting oxidative stress and TGF-β1 canonical and non-canonical pathways.

Conclusions
This study, for the first time, demonstrated that Nebivolol can successfully exert a promising protective potentiality against kidney dysfunction resulted from CLP-induced sepsis in rats.This protective effect was associated with suppressing the Cr, BUN, and serum uric acid levels, and increasing the serum total protein level.Furthermore, it alleviated oxidative stress and inflammatory cytokines by reducing MDA, TNF-α, IL-6, and IL-1β levels.On the other hand, it increased the antioxidant protective mediators, including SOD activity, GSH and IL-10 levels.
Furthermore, it attenuated the kidney fibrosis by suppressing the protein levels of TGF-β1, p-Smad2/3, and α-SMA and the expression level of p53 gene.Additionally, Nebivolol inhibited kidney apoptosis as it repressed the expressions of NF-κB, Bax genes and caspase-3 protein levels, enhancing the expression of the anti-apoptotic Bcl-2 gene.Although further studies are needed to assess its efficacy and safety, our study concluded that Nebivolol can be a novel candidate for mitigating the sepsis-induced kidney dysfunction and its protective potentiality is achieved by inhibiting oxidative stress, inflammation, apoptosis, and fibrosis through targeting TGF-β1/Smad2/3/ p53 and TGF-β1/NF-κB signaling cascades (Supplementary Information S1).

Figure 1 .
Figure1.Effect of Nebivolol on serum creatinine (A), BUN (B), uric acid (C), and total protein (D) levels after induction of CLP in rats.Bars are represented as mean ± SEM.The Tukey-Kramar test was used to analyze the significant differences between groups after conducting a one-way ANOVA test, where ### p < 0.001, compared to the sham group, ***p < 0.001, compared to the CLP group.

Figure 2 .
Figure 2.Effect of Nebivolol on oxidative stress status of kidney tissues, MDA content (A), GSH level (B), and SOD activity (C).Bars are represented as mean ± SEM.The Tukey-Kramar test was used to analyze the significant differences between groups after conducting a one-way ANOVA test, where ### p < 0.001, compared to the sham group, ***p < 0.001, compared to the CLP group, $$ p < 0.01 and $$$ p < 0.001, compared to CLP + Neb4 group.

Table 2 .
Renal tissue scoring representation throughout the different groups.0 represents no change, 1 denotes a minor change, 2 is a moderate change, and 3 denotes a severe change.