Renoprotective Effect of Pitavastatin against TAA-Induced Renal Injury: Involvement of the miR-93/PTEN/AKT/mTOR Pathway

This research investigated if pitavastatin (Pita) might protect rats' kidneys against thioacetamide (TAA). By altering the PTEN/AKT/mTOR pathway, pitavastatin may boost kidney antioxidant capacity and minimize oxidative damage. Statins have several benefits, including antioxidant and anti-inflammatory characteristics. The principal hypothesis of this study was that Pita can regulate the miR-93/PTEN/AKT/mTOR pathways, which is thought to be responsible for its renoprotective effects. The experiment divided male rats into four groups. Group 1 included untreated rats as the control. Group 2 included rats which received TAA (100 mg/kg intraperitoneally thrice a week for two weeks) to destroy their kidneys. Groups 3 and 4 included rats which received Pita orally at 0.4 and 0.8 mg/kg for 14 days after TAA injections. Renal injury increased BUN, creatinine, and MDA levels and decreased glutathione (GSH) levels. Pitavastatin prevented these alterations. TAA decreased PTEN and increased miR-93, Akt, p-Akt, mTOR, and Stat3 in the kidneys. Pitavastatin also regulated the associated culprit pathway, miR-93/PTEN/Akt/mTOR. In addition, TAA induced adverse effects on the kidney tissue, which were significantly ameliorated by pitavastatin treatment. The findings suggest that pitavastatin can attenuate renal injury, likely by regulating the miR-93/PTEN/Akt/mTOR pathway. This modulation of the pathway appears to contribute to the protective effects of pitavastatin against TAA-induced renal injury, adding to the growing evidence of the pleiotropic benefits of statins in renal health.


Introduction
Acute kidney injury (AKI) is a clinical illness characterized by abruptly impaired renal function, retention of metabolites, and abnormalities of the water, electrolyte, and acidbase balance [1].AKI is a severe condition that causes over 2 million fatalities globally each year and is linked to higher morbidity and mortality rates [2].Prior research has shown that patients with AKI frequently experience incomplete recovery of their renal function.In addition, AKI is a substantial risk factor for the development of both chronic kidney disease (CKD) and end-stage renal disease (ESRD) [3].Infammation and oxidative stress are intricately interconnected, leading to a detrimental cycle.Oxidative stress initiates the process of infammation, which, in response, further triggers oxidative stress by generating reactive oxygen species (ROS).Tese harmful occurrences collectively contribute to tissue damage by inducing necrosis, apoptosis, and fbrosis [4].AKI lacks specifc treatments beyond standard supportive care and renal replacement therapy, which signifcantly contributes to the unfavorable short-term outlook experienced by AKI patients [4,5].Despite substantial research on AKI, the molecular pathways underlying renal damage have not yet been thoroughly explored.In order to treat or stop the progression of AKI to CKD, new medications or mechanisms must be developed.Despite extensive research on AKI, there remains a lack of comprehensive understanding regarding the molecular processes underlying renal injury.Consequently, it is essential to explore new medications or mechanisms to efectively manage or prevent the progression of AKI to CKD [6].
MicroRNAs (miRs) are short RNA molecules, typically consisting of 18-25 nucleotides, which control protein translation by binding to complementary regions of mRNA.Tis binding typically occurs in the 3′ untranslated region of the mRNA [7].In studies involving preclinical AKI models, numerous miR variations have demonstrated promising therapeutic capabilities [8].For instance, in cases of experimental acute kidney injury (AKI) caused by ischemiareperfusion (I/R), administering miR-17-5p mimic via intravenous injection has been shown to hinder histologic ischemic damage.It also leads to a decrease in blood urea nitrogen and serum creatinine levels.
In addition, when miR-21 is overexpressed in mice, it ofers protection against ischemic kidney injury by reducing apoptosis [9][10][11].In addition, Liao and colleagues [12] provided evidence that miR-140-5p enhanced the recovery from cisplatin-induced AKI in mice.Tis efect was achieved by reducing oxidative stress through the activation of the Nrf2/ARE pathway.Moreover, recent research has unveiled the protective properties of miRNAs in diferent renal cell types, as they efectively inhibit apoptosis and infammatory responses [13,14].
Phosphatase and tensin homolog (PTEN), located on chromosome 10, acts as a tumor suppressor by exhibiting the bispecifc phosphatase activity.Tis activity leads to the dephosphorylation of PI3P, converting it into PI2P.Consequently, the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is deactivated [15].PI3K becomes activated, leading to the phosphorylation of PIP2 and its conversion into PIP3.Consequently, Akt is activated by PIP3 [16].Furthermore, there is a notable rise in the levels of PTEN, leading to the suppression of the Akt/mTOR signaling pathway [17].Akt stimulates mTOR, leading to the initiation of downstream protein translation through the phosphorylation of 4EBP1 and P70S6K [18].All renal tissues express PTEN.However, renal tubular cells are where it is most abundant [19].
Statins are a traditional medication utilized to address hyperlipidemia in obese and diabetic patients.Teir mode of action involves the inhibition of the hydroxyl-3-methylglutaryl-coenzyme A (HMG-CoA) reductase enzyme, which not only leads to a direct reduction in endogenous cholesterol production but also exhibits additional benefcial efects known as pleiotropic efects, as demonstrated in both laboratory experiments (in vitro) and studies involving living organisms (in vivo) [20].In addition to reducing oxidative stress, infammation, and apoptosis, in streptozotocin-induced type 1 diabetes models in rats, statins may improve pancreatic and renal function [21].In addition, the benefcial impacts of statins on kidney dysfunction were observed in rats with diet-induced obesity and patients diagnosed with type 2 diabetes (T2DM) [22][23][24][25][26]. Te kidney-protective benefts of atorvastatin, simvastatin, and rosuvastatin have been proven [27].Statins demonstrate advantageous impacts in various animal model kidney diseases [28].Despite numerous research papers focusing on statins' impact on AKI, this study had a distinct objective of examining pitavastatin (Pita)'s potential in mitigating thioacetamide-(TAA-) induced renal injury in rats.Te main hypothesis of this investigation was that Pita's renoprotective properties are attributed to its regulation of miR-93/ PTEN/AKT/mTOR pathways.

Chemicals.
From Sigma-Aldrich Co. (St.Louis, MO, USA), thioacetamide (TAA) was purchased.From Western pharmaceutical industries, in Egypt, pitavastatin was acquired.Every other chemical that was used in the experiments was of the highest purity and analytical grade.Freshly suspended Pita was orally administered at a dose of 0.4 mg/ kg and 0.8 mg/kg in a 1% Tween 80 solution [29].

Experimental Design.
Te rats were divided into four groups (6 rats/group).Rats in group 1 (negative control group) were given an intraperitoneal (ip) injection of saline three times a week for two consecutive weeks.Rats in group 2 (TAA group) received an intraperitoneal (ip) injection of TAA (100 mg/kg) three times a week for two consecutive weeks to cause renal injury [30].Rats in groups 3 and 4 were given Pita orally (0.4 and 0.8 mg/kg) [31] every day for two weeks following TAA injections.

Preparation of Serum Samples.
After completing the experiment, the rats were deprived of food overnight.Teir blood was then drawn, and the serum was extracted by spinning the samples for fve minutes at 3000 rpm and 4 °C.Te obtained serum was then stored at −80 °C for later use in measuring blood urea nitrogen (BUN) and creatinine levels.

Collecting Renal Tissues.
A cold saline solution was used to rinse the kidneys.Tey were then split into three portions.
To enable mRNA extraction and the evaluation of miRNA 93 2 Advances in Pharmacological and Pharmaceutical Sciences and AKT gene expression, the frst portion was promptly frozen in liquid nitrogen and kept at a temperature of −80 °C.
After homogenizing the second portion with 0.5% potassium chloride (1 mL for every 100 mg of tissue), it was centrifuged for 10 min at 4 °C and 3000 rpm.Te resultant supernatant was separated and stored at 80 °C to measure renal glutathione (GSH) and malondialdehyde (MDA) using colorimetric kits, as well as AKT, p-AKT, mTOR, p-mTOR, and PTEN using ELISA.At the same time, the second portion was utilized to estimate the renal content of miRNA 93 and the AKT RNA content.Finally, the last part was fxed in 10% bufered formalin for subsequent histopathological and immune-histochemical investigations.

Renal Function Tests.
To assess the renal function, we measured serum BUN and creatinine levels using commercially available kits.BUN levels were determined colorimetrically with the BioVision kit (Catalog# K375-100, Milpitas Boulevard, Milpitas, USA).Te principle of this is a kit comprised of the hydrolysis of urea by urease to produce ammonia and CO 2 .Te ammonia that is produced then combines with hypochlorite and a phenolic chromogen to generate a complex that is green in color.Te color intensity that develops is closely correlated with the urea content of the sample and vice versa.Besides, creatinine levels were measured colorimetrically using the BioVision kit (Catalog# E4370-100, Milpitas Boulevard, Milpitas, USA).Te principle of this kit comprised the modifed kinetic Jafe methodology for the determination of creatinine involving a protein-free fltrate and a reaction with picric acid in an alkaline solution, a surfactant, and other ingredients to minimize protein and carbohydrate interferences [32].Te assays were performed following the manufacturer's instructions.

Renal Oxidative Stress Markers.
For the evaluation of renal oxidative stress, we measured MDA levels and the GSH content.Te MDA level was assessed colorimetrically with the BioVision kit (Catalog# K739-100, Milpitas Boulevard, Milpitas, USA) that is based on the reaction of MDA in the sample with thiobarbituric acid (TBA) to generate the MDA-TBA adduct which can be easily quantifed colorimetrically (OD 532 nm).Te GSH content was determined colorimetrically using the BioVision kit (Catalog# K464-100, Milpitas Boulevard, Milpitas, USA) based on an enzymatic cycling method in the presence of GSH and a chromophore.Te reduction of the chromophore produces a stable product, which can be followed kinetically at 450 nm.Te procedures were conducted in accordance with the manufacturer's instructions to ensure accuracy and reliability.

Immune-Histochemical Examination of STAT3
Expression.Te other parafn section from each group was used for immunohistochemical detection of the expression of Stat3 in various experimental groups using avidin-biotinperoxidase according to the method described in [33].Kidney slices were treated with monoclonal antibodies for Stat3 (Abcam, Cambridge, MA, USA) at a dilution of 1 : 200 and (Vactastain ABC peroxidase kit, Vector Laboratories, Newark, USA) for the aim of identifying antigen-antibody complexes.Each marker's expression was visualized using chromagen 3,3-diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical Co.).Te positive brown region of each marker's expression was assessed using seven high-power microscopic felds and image analysis software (ImageJ, 1.46a, NIH, Bethesda, USA).
Advances in Pharmacological and Pharmaceutical Sciences 2.12.Statistical Analysis.Te outcomes are represented as the "means ± S.E.".Data were processed by "one-way ANOVA followed by the Tukey-Kramer post hoc test.""GraphPad Prism software (version 9, USA)" was used to conduct the statistical analysis and create the graphs shown.Te signifcance level was set to "p < 0.05" for all statistical tests [34].

Efect of Pitavastatin on Renal Function
Tests.Te serum levels of creatinine and BUN signifcantly increased after the injection of TAA.Pita administration successfully reduced serum creatinine and BUN levels in both treatment groups, as indicated in Figure 1.

Efect of Pitavastatin on the Oxidative Stress Status.
In the TAA group, compared to the control group, there was a signifcantly higher level of renal MDA (p < 0.05) and a signifcantly lower level of renal GSH (p < 0.05).As indicated in Figure 2, Pita treatment successfully reduced the renal MDA content in both treated groups (p < 0.05) and enhanced the GSH content when compared to the TAA group (p < 0.05).

Efect of Pitavastatin on the PTEN/AKT/mTOR Pathway.
Te level of PTEN and the downstream variables AKT, p-AKT, mTOR, and p-mTOR were examined in order to clarify further the mechanism underlying the protective efect of Pita against renal injury because these factors are crucial in the development of renal injury.PTEN levels were signifcantly decreased, and AKT, p-AKT, mTOR, and p-mTOR levels were signifcantly increased by TAA-induced renal injury compared to the control group (p < 0.05).In contrast, PTEN levels were signifcantly decreased, and AKT, p-AKT, mTOR, and p-mTOR levels were signifcantly increased by the Pita treatment groups (p < 0.05, Figures 3(a)-3(e)).

Efect of Pitavastatin on Renal miR-93 and AKT Gene
Expression.In the TAA model group (p < 0.05), as compared to the control group, real-time qRT-PCR analysis revealed a signifcantly increased expression of miR-93 and AKT.Comparing the gene expression of miR-93 and AKT in both Pita-treated groups to TAA-renal damaged rats, Figure 4 demonstrates a signifcant decrease in both genes' expression (p < 0.05).

Histopathological Findings.
Pita markedly prevented the adverse efects of TAA on the kidney tissue.Normal histological structures of cortical and medullary elements, as well as the renal pelvis, were clearly noticed under light microscopic examination of the kidney tissues of control rats (Figure 5(a)).Whilst TAA administration had an adverse efect on kidney tissue, particularly in the cortex, which showed variation in the tinctorial intensity of the renal tubules with marked dilatation and congestion of the interstitial blood vessels with interstitial edema and few pockets of hemorrhages (Figure 5(b)).Te renal tubules showed epithelial lining swelling, degeneration, some pyknotic nuclei, necrosis, and sloughing of a few cells.Other tubules were cystically dilated with an attenuated epithelium (Figure 5(c)).Some cellular debris was observed in the lumen of a few tubules.Occasional granular casts were present in medullary collecting ducts and tubules (Figure 5(d)).Most of the glomeruli showed membrane thickening of the glomerular tuft with few cellular infltrates and few others appeared with atrophied tufts.Daily treatment with Pita in rats for two weeks prior to being administrated TAA showed marked protection of their kidney tissues against the harmful efect of TAA, particularly in the high-dose administrated rats.Rats administrated Pita at a low dose (Figures 5(e) and 5(f )) showed mild degenerative changes of the renal tubular epithelium with scares glomeruli appearing with atrophied tuft.While at the high dose of Pita (Figures 5(g) and 5(h)), mild degenerative changes were noticed in some tubular epithelium with the appearance of scattered foci of regenerated tubules.
Pita reduced TAA's kidney damage.Under light microscopy, the kidney tissues of control rats showed the normal histological structure of cortical, medullary, and renal pelvic components (Figure 5(a)).TAA treatment damaged the kidney tissue, notably in the cortex, which demonstrated variance in renal tubule tinctorial intensity, signifcant dilatation and congestion of interstitial blood vessels, interstitial edema, and rare hemorrhages (Figure 5(b)).Te renal tubules revealed epithelial lining enlargement, degeneration, some pyknotic nuclei, necrosis, and sloughing of a few cells.Other cystically dilated tubules have attenuated epithelium (Figure 5(c)).Few tubules had cellular debris.Medullary collecting ducts and tubules sometimes have granular deposits (Figure 5(d)).Most glomeruli had membrane thickening and minimal cellular infltrates, whereas a few had atrophied tufts.Pita given daily to rats for two weeks before TAA administration protected their kidney tissues, especially in high-dose animals.Low-dose Pita-treated rats (Figures 5(e) and 5(f )) demonstrated modest renal tubular epithelial degeneration with scarred glomeruli and atrophied tuft.At high Pita doses (Figures 5(g) and 5(h)), some tubular epithelium showed modest degeneration and scattered foci of regenerated tubules.

Discussion
Statins work by blocking cholesterol synthesis, leading to a reduction in blood lipids.Te recent focus on the pleiotropic efects of statins arises from numerous clinical and preclinical investigations that highlight the additional benefts of statins beyond cholesterol control.Tese studies reveal advantages that are not directly related to cholesterol levels [35].Pita stands as an innovative statin with remarkable pharmacokinetic and pharmacodynamic qualities.Unlike other statins, it undergoes minimal metabolism by CYP3A4 (cytochrome P450), reducing the likelihood of drug interactions with substances metabolized by this enzyme.Additional research has revealed several advantageous attributes of Pita, including safeguarding endothelial function and suppressing vascular infammation and oxidative stress [36].Tioacetamide (TAA), an organosulfur compound, is commonly used as a fungicide [37].TAA is converted in vivo to the free radical derivatives TAA sulfoxide and TAA-S, Sdioxide.Tis causes enhanced lipid peroxidation, which leads to the generation of reactive oxygen species (ROS), which causes damage to the liver [38][39][40][41].Te metabolites produced can afect amine lipids and proteins, increasing systemic oxidative stress, releasing cytokines, and altering kidney function since they are dispersed laterally throughout various organs, including the liver, kidney, adrenals, bone marrow, and other tissues [42][43][44].
While previous studies have shown the nephroprotective efects of statins, including pitavastatin [45][46][47][48][49][50][51], the specifc involvement of the miR-93/PTEN/Akt/mTOR pathway in mediating these efects has not been elucidated in vivo in the context of xenobiotic-induced kidney injury.Our study is the frst to demonstrate that pitavastatin attenuates TAAinduced kidney damage in rats by regulating this essential signaling cascade.We used the TAA model because it is a well-established approach for studying mechanisms of kidney injury due to oxidative stress and xenobiotic toxicity.
Our fndings signifcantly advance understanding of the mechanisms underlying statins' pleiotropic benefts in the kidney.We show for the frst time the integral role of miR-93 and PTEN regulation in activating downstream Akt/mTOR * compared to the normal control group, @ to the TAA group, and # to pita (0.4 mg/kg) at p < 0.05.Pita: pitavastatin and TAA: thioacetamide.

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Advances in Pharmacological and Pharmaceutical Sciences signaling to mitigate renal injury.Tese novel insights expand the evidence for the multifaceted protective properties of statins and support their potential therapeutic use to preserve kidney function in contexts of toxicant exposure or oxidative damage.Our study also suggests that the miR-93/ PTEN/Akt/mTOR axis could be a promising target for future nephroprotective strategies.
In our research, we observed notable disruption in kidney function among rats treated with TAA, as indicated by increased levels of BUN and creatinine in the Advances in Pharmacological and Pharmaceutical Sciences bloodstream.Prior studies had already indicated the potential impact of TAA on measures of the kidney function [52].Pita normalized the levels of BUN and creatinine in a dose-dependent manner.Our fndings are in accordance with [53].Te histopathological examination revealed considerable tissue damage and loss of structure in the kidneys of the group treated with TAA.However, the group treated with Pita showed a noteworthy decrease in tissue damage, along with improved preservation of cellular nuclei and organelles.
In alignment with prior research, it was observed that alongside abnormal kidney function and histological changes, there was an elevation in MDA levels and a decrease in the antioxidant content (specifcally GSH).Tese results correspond with earlier studies [54,55].TAA can cause a signifcant amount of ROS to be produced, which can hinder the antioxidant defense system.GSH and other thiols, which are components of the intracellular antioxidant system, may not be adequate to repair this damage [37].Statins have been reported to block the generation of reactive oxygen and nitrogen species, remove harmful free radicals, and boost the synthesis of vascular endothelial nitric oxide [56].Accordingly, Pita was found to reestablish the oxidantantioxidant equilibrium in the renal tissue.
Importantly, the efects of miR-93/AKT/mTOR signaling pathways and the link to Pita in the pathophysiology of renal tissue injury have never been elucidated in vivo through the context of xenobiotic toxicity.
MicroRNAs (miRNAs) play a signifcant role in various biological processes, including kidney injury and repair.One specifc miRNA that has been studied in the context of kidney injury is miR-93.In addition, PTEN (phosphatase and tensin homolog) is a critical protein involved in regulating cell growth and survival [57].Studies have shown that miR-93 can target PTEN, leading to its downregulation.Tis downregulation of PTEN, in turn, can contribute to kidney injury by promoting cell proliferation, survival, and infammation.During kidney injury, there can be an upregulation of miR-93, leading to decreased PTEN levels and subsequent alterations in cell mir-93/AKT/mTOR signaling pathways.Tese changes may impact the overall injury and repair processes in the kidney [58].It is worth noting that the study of miRNAs in kidney injury is an active area of research, and ongoing investigations continue to shed light on their specifc roles and potential therapeutic implications.Our fndings are consistent with the data presented, showing that miR-93 is linked to the activation of the AKT/mTOR pathway, resulting in kidney injury.On the contrary, the administration of Pita treatment in the current study successfully halted the miR-93 level, leading to the upregulation of the PTEN level.Tis, in turn, suppressed the AKT/mTOR pathway in the kidney, leading to the improvement and resolution of kidney injury.
A crucial transcription factor known as Stat3 controls cytokine-dependent infammation and immunity [59,60].Cytokine receptors stimulate the Janus kinase (JAK)-Stats signaling cascade and phosphorylate Stat3.As a result, Stat3 either pairs up with other Stat proteins to form stable homodimers or heterodimers.Tese activated Stats then react to cytokines, creating a feed-forward infammation loop within the afected microenvironment [61].Studies using experimental models of kidney diseases have examined the role of Stat3 in terms of pathophysiology and the protective efects of Stat3 inhibition [62].Stat3 is the target of miR-93 [63].As shown by the decreased Stat3 expression generated by TAA in the renal tissue, Pita was able to lessen Stat3 activation in this case, protecting against renal injury.Te observed results are consistent with the investigation of Al-Shabrawey et al. [64], who demonstrated that simvastatin could protect against the early signs of diabetic retinopathy by preventing NADPH oxidase-mediated activation of Stat3.
Further research is needed utilizing genetic manipulation of miR-93 to conclusively demonstrate its direct interactions with target genes like PTEN and precise impacts on Akt/mTOR signaling in this context.While beyond the scope of the current study, such molecular examination through gain and loss of function experiments represents an important future direction to complement our fndings.

Conclusions
In this study, we demonstrate for the frst time that pitavastatin attenuates xenobiotic-induced kidney injury in rats by regulating the miR-93/PTEN/Akt/mTOR signaling pathway.Specifcally, we show that thioacetamide causes marked impairment in renal function and histopathological damage associated with oxidative stress, infammation, and apoptosis.Pitavastatin treatment mitigated these harmful efects and preserved normal kidney structure and function.Mechanistically, our central fnding is that the renoprotective impact of pitavastatin involves the suppression of miR-93 expression and subsequent activation of PTEN.Tis shifts the balance of Akt/mTOR signaling to ameliorate molecular processes driving kidney toxicity.Taken together, these novel results substantially advance understanding of the pleiotropic benefts of statins in the kidney by defning a specifc molecular cascade-the miR-93/PTEN/Akt/mTOR axis-underlying their protective capacities.
Overall, this research highlights the therapeutic potential of pitavastatin in contexts of toxicant-mediated oxidative injury and infammation in the kidney.Our work suggests that the modulation of miR-93 and associated signaling could be a promising pharmacological approach to attenuate the progression of acute kidney disease.Tese fndings merit further investigation of the clinical applicability of statins to preserve renal function in patient populations at high risk of kidney damage due to xenobiotic exposures or conditions causing oxidative stress.

Data Availability
Te data used to support the fndings of this study are included within the article.

Disclosure
Te funders had no role in the study design, data collection and analysis, the decision to publish, or preparation of the manuscript.
Stat3 was noticed in the kidney tissues of the rats' group administrated TAA (Figure 6(b)) compared to the control group (Figure 6(a)).Meanwhile, Pita (0.4 and 0.8 mg/kg) treatment following TAA administration displayed a dose-related reduced Stat3 expression compared to the TAA group (Figures 6(c) and 6(d)).Te quantitative analysis of the positive brown color of Stat3, displayed as a staining score, demonstrated that the TAA rats signifcantly (p < 0.05) overexpressed Stat3 in comparison to the other treated groups.4Advances in Pharmacological and Pharmaceutical Sciences TAA-treated rats had signifcantly higher Stat3 expression in their renal tissues (Figure 6(b)) than controls (Figure 6(a)).Pita (0.4 and 0.8 mg/kg) therapy after TAA lowered Stat3 expression compared to the TAA group (Figures 6(c) and 6(d)).TAA rats overexpressed Stat3 based on the Stat3 staining score.

Figure 2 :Figure 1 :Figure 3 :Figure 4 :
Figure 2: Efect of pitavastatin on renal (a) MDA and (b) GSH.A bar shows the mean and standard error of six rats: compared * to the normal control group and @ to the TAA group at p < 0.05.Pita: pitavastatin and TAA: thioacetamide.
minutes at 45 °C for reverse transcription, 2 minutes at 98 °C for RT inactivation, and initial denaturation by 40 cycles of 10 s at 98 °C, 10 s at 55 °C, and 30 s at 72 °C for the amplifcation step.Data were expressed as cycle threshold (Ct) for the target genes and housekeeping gene following the RT-PCR run.Using the mean critical threshold (CT) expression levels of the housekeeping gene GAPDH, the target genes' expression was normalized for variation using the ΔΔCt method.
Renal Tissues via Real-Time qRT-PCR.Termo Fisher Scientifc, Waltham, Massachusetts, USA, supplied the Su-perScript IV One-Step RT-PCR kit for reverse transcription of extracted RNA and PCR.Te following thermal profle was created using a StepOne instrument (Applied Biosystem, USA): 10 2.9.ELISA Assay.Te protein concentrations of p-AKT were measured using commercially available ELISA kits (Catalog# PEL-AKT-S473-T, Ray Biotech, Norcross, USA), mTOR or p-mTOR (Catalog# SL1350Ra and SL5329R SunLong Biotech Co., Zhejiang, China), and AKT or PTEN (Catalog# MBS3807575 and MBS452729, MyBioSource, San Diego, USA) in renal tissue homogenate.Each ELISA kit's manufacturer's instructions were followed.2.10.Histopathological Examination.Te renal tissues were fxed for 24 h in 10% bufered formalin.After that, it was dried in various alcohol concentrations, cleaned in xylene, and fnally embedded in parafn wax.Te parafn slices (4 mm) were stained with hematoxylin and eosin (H&E) stain.Te cells were inspected under a light microscope by a blinded pathologist to prevent prejudice.