Renal Ischemia-reperfusion In Rats Research Articles

Moexipril Improves Renal Ischemia/Reperfusion Injury in Adult Male Rats

Objective: The goal of this study is to see if moexipril can protect rats against renal ischemia/reperfusion injury. Methods: Overall twenty-eight males of rats were divided randomly into four groups (7 rat each group). Sham group: Except for ischemiainduction, these rats underwent IP anesthesia and surgery. Induced group: This group rats were anesthetized and given a midlinelaparotomy to induce bilateral renal ischemia for 30 min and 2 hours of reperfusion. DMSO group: Rats received DMSO IP injection 30 minbefore ischemia and subjected to 30 min bilateral ischemia and reperfusion for 2 hours, DMSO is a vehicle of moexipril and considered ascontrol. Moexipril group (pretreated group): moexipril was given in a dose 0.3 mg/kg I.P. injection 30 min before ischemia. Results: Renal IRI as indicated by a significant increase (P < 0.05) in urea, creatinine, NF-KB P65, IL-1β, and caspase-3 level, while GSH,SOD, and Bcl-2 levels significantly (P < 0.05) reduced in Renal tissues of rats in the induced group compared to sham group. Moexiprilpretreatment significantly (P < 0.05) ameliorate RIRI as suggested from significant lowering in urea, creatinine, and inflammatory markers(NF-KB P65 and IL-1β). The renal level of oxidative marker (SOD and GSH) and anti-apoptotic marker Bcl-2 were significant decreased(P < 0.05) and also significantly increase (P < 0.05) in caspase-3 level with moexipril group in comparison to induced group. Conclusion: By inhibiting oxidative stress, inflammation, and the apoptotic pathway, moexipril significantly protect from renal ischemiareperfusion in rats.

Cell and rat serum, urine and tissue metabolomics analysis elucidates the key pathway changes associated with chronic nephropathy and reveals the mechanism of action of rhein

BackgroundRhein can significantly delay the progression of chronic nephropathy. However, its mechanism of action has not been adequately elaborated, which hinders its extensive clinical application. In this work, the effects of rhein on models of TGF-β-induced NRK-49F cellular fibrosis and rat renal ischemia–reperfusion fibrosis were evaluated using metabolomics and western blotting.MethodsThe metabolic profiles of NRK-49F cells and rat urine, serum, and kidney tissues in the control, model, and rhein groups were investigated using UPLC-QTOF-MS. The levels of p-P65, p-IKK, p-AKT, p-P38, p-JNK and AP-1 in NRK-49F cells were measured using western blotting and immunofluorescence methods. Molecular docking and network pharmacology methods were employed to explore the relationship between the potential targets of rhein and key proteins in the NF-κB and MAPK signaling pathways.ResultsVarious potential metabolites, including sphingolipids, ceramides, phosphatidylcholine, and lysophosphatidylcholine,14-hydroxy-E4-neuroprostane E, and 5-HPETE, were present in the cell, tissue, urine, and serum samples; however, few metabolites matches exactly among the four type of biological samples. These differential metabolites can effectively differentiated between the control, model, and rhein groups. Pathway enrichment analysis of differential metabolites unveiled that sphingolipid metabolism, arachidonic acid metabolism, and glycerophospholipid metabolism were closely related to nephropathy. Phosphorylation levels of AKT, IKK, P65 and AP-1 in NRK-49F cells was reduced by rhein treatment. Network pharmacology and molecular docking showed that the potential targets of rhein might regulated the expression of MAPK and AKT in the NF-κB and MAPK signaling pathways.ConclusionIn brief, rhein might delays the progression of chronic nephropathy via the metabolic pathways, NF-κB and MAPKs signaling pathways, which provides the foundation for its development and clinical application.

Sevoflurane participates in the protection of rat renal ischemia-reperfusion injury by down-regulating the expression of TRPM7.

To investigate the protective effect of sevoflurane preconditioning on renal ischemia-reperfusion injury (renalischemiareperfusionmodel, RIRI) and its related mechanism. Eighty healthy adult male SD rats were randomly divided into control group (Sham group), model group (RIRI group), sevoflurane pretreatment group (Sev group) and TRPM7 inhibitor combined with sevoflurane pretreatment group (T + Sev group), 20 animals in each group. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of renal tissue, and the levels of creatinine and urea nitrogen in each group were detected. Deoxyribonucleic acid terminal transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect renal cell apoptosis, and Western blottingwas used to detect the expression of apoptotic proteins cleaved-caspase-3, bax, Bcl-2, and TRPM7 in renal tissue; Detection of oxidative stress-related index levels in renal tissue and levels of inflammatory factors in renal tissue and serum. Compared with the Sham group, the renal tissue pathological damage was aggravated, the levels of creatinine and blood urea nitrogen were increased, and the apoptosis was increased in the RIR group and the Sev group. Death, malondialdehyde (MDA) levels and inflammatory factors were increased, and superoxide dismutase (SOD) levels were decreased (all p < .05); The scores, apoptosis rate, MDA level, and relative expression of inflammatory factor levels were decreased, and SOD levels were increased (all p < .05). Compared with the Sev group, the renal tissue pathological damage in the T + Sev group was aggravated, creatinine, blood urea nitrogen levels increased, apoptosis increased, apoptosis-related proteins cleaved-caspase-3, bax, Bcl-2 showed increased apoptosis, malondialdehyde (MDA) levels, inflammatory factor levels increased, ultrahigh The levels of oxide dismutase (SOD) were decreased (all p < .05). Therefore, we believe that sevoflurane is involved in the protection of rat renal ischemia-reperfusion injury by downregulating the expression of TRPM7.

The Effect of Thyme Essential Oil on Liver Injuries Caused by Renal Ischemia-Reperfusion in Rats.

Liver damage occurs following renal ischemia-reperfusion (RIR) that can cause inflammation and inflammatory cytokines activated after kidney injury. In this study, thyme essential oil (TE) with antioxidant and anti-inflammatory properties was used to reduce liver damage induced by renal IR. 32 male rats were randomly divided into 4 equal groups: (1) control, (2) RIR, (3) RIR+TE, and (4) TE. Rats received TE as a pretreatment at a dose of 0.5 ml/kg for one week. Then, under anesthesia for 45 minutes for ischemia, the kidneys of the animals were closed with clamps, and reperfusion was performed for 24 hours. Animal serum was isolated to evaluate alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) parameters. The liver of rats was examined for the measurement of malondialdehyde (MDA), nitric oxide (NO), glutathione (GSH), glutathione peroxidase (GPX), catalase (CAT), and expression of genes such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and caspase-3. ALP, AST, ALT, MDA, NO, IL-6, TNF-α, and caspase-3 increased significantly in the RIR group compared to the control group (p < 0.05). GSH, GPX, and CAT decreased significantly in the RIR group compared to the control group (p < 0.05). TE caused a decrease in ALP, AST, ALT, MDA, NO, IL-6, and TNF-α compared to the RIR group and caused an increase in the amount of GSH, GPX, and CAT in the RIR group (p < 0.05). This study showed that TE has antioxidant and anti-inflammatory properties that reduce liver damage induced by RIR.

MP36-19 PROSTACYCLIN (TREPROSTINIL) THERAPY IMPROVES RENAL HEMODYNAMICS DURING RAT RENAL ISCHEMIA-REPERFUSION INJURY

You have accessJournal of UrologyCME1 May 2022MP36-19 PROSTACYCLIN (TREPROSTINIL) THERAPY IMPROVES RENAL HEMODYNAMICS DURING RAT RENAL ISCHEMIA-REPERFUSION INJURY Frances Kazal, Meiwen Ding, Dicken Ko, Reginald Gohh, and Nisanne Ghonem Frances KazalFrances Kazal More articles by this author , Meiwen DingMeiwen Ding More articles by this author , Dicken KoDicken Ko More articles by this author , Reginald GohhReginald Gohh More articles by this author , and Nisanne GhonemNisanne Ghonem More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002590.19AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Renal ischemia-reperfusion injury (IRI) is a crucial factor causing acute kidney injury (AKI). To date, no pharmacological treatments are available for the prevention of I/R-induced renal injury. Here, we investigate the renal hemodynamics and reno-protective effects of the prostacyclin analog, treprostinil, against renal IRI in vivo. METHODS: Male Sprague Dawley rats were subjected to bilateral renal ischemia (45 minutes) followed by reperfusion (1–48 hour). Treprostinil (100 ng/kg/min) was administered subcutaneously either (A) at the time of ischemia (IRI-immT) or (B) approximately 18-24 hours before renal IRI (IRI-preT). RESULTS: Serum and urine creatinine were elevated at 24 hours post-reperfusion in the IRI group vs. sham (P <0.001), which both the IRI-immT and IRI-preT reduced the peak of serum creatinine at 24 hours after renal IRI (P <0.001). Also, medullary and cortical renal blood flow (RBF) were significantly reduced in the IRI group vs. sham (P <0.001), whereas the IRI-immT (P <0.01) and IRI-preT (P <0.05) groups improved RBF at 90 minutes post-reperfusion and maintained throughout 120 minutes. In parallel, animals in the IRI injury group experienced a decreased eGFR, increased urinary sodium (UNa 2+) excretion and proteinuria during the first 24 hours post-reperfusion vs. sham (P <0.01). Meanwhile, treprostinil reduced the UNa 2+ and proteinuria and completely restored the eGRF to baseline by 48 hours post-reperfusion and, compared to the IRI group (P <0.01). CONCLUSIONS: This study demonstrates the efficacy of treprostinil in improving renal hemodynamics and kidney function early post-IRI in a clinically relevant rat model of AKI. Specifically, our data show that treprostinil remarkably increased renal blood flow and GFR, and reduced sodium excretion and proteinuria post-reperfusion. Importantly, these data suggest that treprostinil has the potential to serve as a therapeutic agent to protect the kidney against renal IRI. Source of Funding: Funding support for this project was provided in part by the Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health, which funds the University of Rhode Island Core Lab (P20GM103430). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e605 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Frances Kazal More articles by this author Meiwen Ding More articles by this author Dicken Ko More articles by this author Reginald Gohh More articles by this author Nisanne Ghonem More articles by this author Expand All Advertisement PDF DownloadLoading ...

Ischemic preconditioning-induced protective effect for promoting angiogenesis in renal ischemia-reperfusion injury by regulating miR-376c-3p/HIF-1α/VEGF axis in male rats

ObjectiveIschemic preconditioning (IPC) is defined as a well-established phenomenon in which brief exposure to sublethal episodes of ischemia and reperfusion induces a tolerance to injurious effects of prolonged ischemia by exploiting intrinsic defence mechanisms. The present study was performed to determine the protective effect of IPC on the rat renal ischemia-reperfusion injury (IRI) via miR-376c-3p/HIF-1α/VEGF axis. MethodsIn vivo, these male Sprague-Dawley rats were treated by IRI and IPC. Meanwhile, these rats from different treatment groups were also injected subcutaneously with 2 nmol agomir-376c-3p and/or 10 nmol recombinant rat HIF-1α. At 72 h after reperfusion, serum samples were respectively collected for renal function. Besides, kidney tissues were harvested to observe renal morphology changes. Subsequently, the expression levels of CD31, HIF-1α and VEGF in the kidney tissues were measured using immunohistochemical staining, quantitative real-time PCR, as well as Western blotting analysis at the indicated time points after reperfusion. In vitro, HK-2 cells were used to detect the cell activity by CCK-8 and transfection of mir-376c-3p mimic in Hypoxia/Reoxygenation (H/R) group. ResultsIn vivo, the pathological changes were significantly relieved in the rats with IPC group, compared to the IRI group. Rats which were treated IPC significantly reduced the levels of blood urea nitrogen (BUN), serum creatinine (Scr) at 24 h after operation, compared to IRI group. After IPC treatment, the expression level of CD31 was obviously decreased, compared to IRI group. A total of differently expressed microRNAs were screened out by microRNA microarray assay in rat renal ischemia tissue, especially showing that miR-376c-3p was selected as the target miRNA. Compared to IRI group, the expression level of miR-376c-3p were obviously higher in IPC-treated group. Double-luciferase reporter assay demonstrated that miR-376c-3p directly targeted HIF-1α. In vitro, IPC significantly increased cell viability of HK-2, and promoted the angiogenesis via up-regulating miR-376c-3p/HIF-1α/VEGF axis. Furthermore, the expression level of HIF-1α was apparently decreased in HK-2 treated with H/R after miR-376c-3p mimic transfection respectively, as well as the increased expression level of VEGF. ConclusionsOur study provided a novel insight for investigating the protective effect of IPC on renal IRI. Consequently, miR-376c-3p played an important role in renal IRI by promoting angiogenesis via targeting HIF-1α/VEGF pathway in male rats.

Treprostinil alleviates hepatic mitochondrial injury during rat renal ischemia-reperfusion injury

BackgroundRenal ischemia-reperfusion injury (IRI) causes acute kidney injury as well as liver injury. Renal IRI depletes hepatic antioxidants, promotes hepatic inflammation and dysfunction through Tlr9 upregulation. There is no treatment available for liver injury during renal IRI. This study examines the hepatoprotective role of treprostinil, a prostacyclin analog, during renal IRI. MethodsMale Sprague-Dawley rats were divided into four groups: control, sham, IRI-placebo, or IRI-treprostinil and subjected to bilateral ischemia (45 min) followed by reperfusion (1–72 h). Placebo or treprostinil (100 ng/kg/min) was administered subcutaneously via an osmotic minipump. ResultsTreprostinil significantly reduced peak serum creatinine, BUN, ALT and AST levels vs. IRI-placebo. Treprostinil also restored hepatic levels of superoxide dismutase, glutathione, catalase, and Gclc expression to baseline, while reducing lipid peroxidation vs. IRI-placebo. Additionally, treprostinil significantly reduced elevated hepatic Tlr9, Il-1β, Ccl2, Vcam1, and Serpine1 mRNA expression. Renal IRI increased hepatic apoptosis which was inhibited by treprostinil through reduced cytochrome c and cleaved caspase-3 protein expression. Treprostinil enhanced hepatic ATP concentrations and mitochondrial DNA copy number and improved mitochondrial dynamics by restoring Pgc-1α expression and significantly upregulating Mfn1, Mfn2, and Sirt3 levels, while reducing Drp-1 protein vs. IRI-placebo. Non-targeted semi-quantitative proteomics showed improved oxidative stress indices and ATP subunits in the IRI-treprostinil group. ConclusionsTreprostinil improved hepatic function and antioxidant levels, while suppressing the inflammatory response and alleviating Tlr9-mediated apoptotic injury during renal IRI. Our study provides evidence of treprostinil’s hepatoprotective effect, which supports the therapeutic potential of treprostinil in reducing hepatic injury during renal IRI.

Treprostinil reduces mitochondrial injury during rat renal ischemia-reperfusion injury

BackgroundRenal ischemia-reperfusion injury (IRI) is a major factor contributing to acute kidney injury and it is associated with a high morbidity and mortality if untreated. Renal IRI depletes cellular and tissue adenosine triphosphate (ATP), which compromises mitochondrial function, further exacerbating renal tubular injury. Currently, no treatment for IRI is available. This study investigates the protective role of treprostinil in improving mitochondria biogenesis and recovery during rat renal IRI. MethodsMale Sprague Dawley rats were randomly assigned to groups: control, sham, IRI-placebo or IRI-treprostinil and subjected to 45 min of bilateral renal ischemia followed by 1–72 h reperfusion. Placebo or treprostinil (100 ng/kg/min) was administered subcutaneously via an osmotic minipump. ResultsTreprostinil significantly reduced peak elevated serum creatinine (SCr) levels and accelerated normalization relative to IRI-placebo (p < 0.0001). Treatment with treprostinil also inhibited IRI-mediated renal apoptosis, mitochondrial oxidative injury (p < 0.05), and the release of cytochrome c (p < 0.01) vs. IRI-placebo. In addition, treprostinil preserved renal mitochondrial DNA copy number (p < 0.0001) and renal ATP levels (p < 0.05) to nearly those of sham-operated animals. Non-targeted semi-quantitative proteomics showed reduced levels of ATP synthase subunits in the IRI-placebo group which were restored to sham levels by treprostinil treatment (p < 0.05). Furthermore, treprostinil reduced renal IRI-induced upregulated Drp1 and pErk protein levels, and restored Sirt3 and Pgc-1α levels to baseline (p < 0.05). ConclusionsTreprostinil reduces mitochondrial-mediated renal apoptosis, inhibits mitochondria fission, and promotes mitochondria fusion, thereby accelerating mitochondrial recovery and protecting renal proximal tubules from renal IRI. These results support the clinical investigation of treprostinil as a viable therapy to reduce renal IRI.

Overexpression of SP1 restores autophagy to alleviate acute renal injury induced by ischemia-reperfusion through the miR-205/PTEN/Akt pathway

BackgroundAcute kidney injury (AKI) is a major kidney disease with poor clinical outcome. SP1, a well-known transcription factor, plays a critical role in AKI and subsequent kidney repair through the regulation of various cell biologic processes. However, the underlying mechanism of SP1 in these pathological processes remain largely unknown.MethodsAn in vitro HK-2 cells with anoxia-reoxygenation injury model (In vitro simulated ischemic injury disease) and an in vivo rat renal ischemia-reperfusion injury model were used in this study. The expression levels of SP1, miR-205 and PTEN were detected by RT-qPCR, and the protein expression levels of SP1, p62, PTEN, AKT, p-AKT, LC3II, LC3I and Beclin-1 were assayed by western blot. Cell proliferation was assessed by MTT assay, and the cell apoptosis was detected by flow cytometry. The secretions of IL-6 and TNF-α were detected by ELISA. The targeted relationship between miR-205 and PTEN was confirmed by dual luciferase report assay. The expression and positioning of LC-3 were observed by immunofluorescence staining. TUNEL staining was used to detect cell apoptosis and immunohistochemical analysis was used to evaluate the expression of SP1 in renal tissue after ischemia-reperfusion injury in rats.ResultsThe expression of PTEN was upregulated while SP1 and miR-205 were downregulated in renal ischemia-reperfusion injury. Overexpression of SP1 protected renal tubule cell against injury induced by ischemia-reperfusion via miR-205/PTEN/Akt pathway mediated autophagy. Overexpression of SP1 attenuated renal ischemia-reperfusion injury in rats.ConclusionsSP1 overexpression restored autophagy to alleviate acute renal injury induced by ischemia-reperfusion through the miR-205/PTEN/Akt pathway.

Dexmedetomidine Preconditioning Protects Rats from Renal Ischemia-Reperfusion Injury Accompanied with Biphasic Changes of Nuclear Factor-Kappa B Signaling.

Acute kidney injury (AKI) is one of the most common and troublesome perioperative complications. Dexmedetomidine (DEX) is a potent α2-adrenoceptor (α2-AR) agonist with anti-inflammatory and renoprotective effects. In this study, a rat renal ischemia–reperfusion injury (IRI) model was induced. At 24 h after reperfusion, the IRI-induced damage and the renoprotection of DEX preconditioning were confirmed both biochemically and histologically. Changes in nuclear factor-kappa B (NF-κB), as well as its downstream anti-inflammatory factor A20 and proinflammatory factor tumor necrosis factor-α (TNF-α), were detected. Atipamezole, a nonselective antagonist, was then added 5 min before the administration of DEX to further analyze DEX's effects on NF-κB, and another anti-inflammatory medicine, methylprednisolone, was used in comparison with DEX, to further analyze DEX's effects on NF-κB. Different concentrations of DEX (0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM) were applied to preincubated human renal tubular epithelial cell line (HK-2) cells in vitro. After anoxia and reoxygenation, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay and enzyme-linked immunosorbent assay (ELISA) were performed to evaluate the levels of NF-κB downstream anti-inflammatory cytokines. The results showed that, unlike methylprednisolone, DEX preconditioning led to a time-dependent biphasic change (first activation then inhibition) of NF-κB in the rat renal IRI models that were given 25 μg/kg i.p. It was accompanied by a similarly biphasic change of TNF-α and an early and persistent upregulation of A20. In vitro, DEX's cellular protection showed a concentration-dependent biphasic change which was protective within the range of 0 to 100 nM but became opposite when concentrations are greater than 1 μM. The changes in the A20 and NF-κB messenger RNA (mRNA) levels were consistent with the renoprotective ability of DEX. In other words, DEX preconditioning protected the rats from renal IRI via regulation biphasic change of NF-κB signaling.

Scintigraphic evaluation of renoprotective effects of coenzyme Q10 in a rat renal ischemia-reperfusion injury.

Renal ischemia-reperfusion injury (RIRI) may occur secondary to several reasons leading to renal failure. Coenzyme-Q10 (CoQ10) is a well-known antioxidant. However, the effects CoQ10 against RIRI have not been evaluated. Our aim was to evaluate protective effects of CoQ10 to renal ischemia-reperfusion by biochemical, immunohistochemical and scintigraphic findings. Thirty Wistar-albino rats were randomly separated into groups of 10; Group Sham; Group ischemia-reperfusion (IR) had left renal pedicle clamping; Group CoQ10+IR had IR and CoQ10. Twenty-four hours later after reperfusion, scintigraphy was performed and after that, rats were sacrificed. To demonstrate effects of RIRI, serum urea and creatinine levels and tissue levels oxidative stress markers were evaluated. Both kidneys were subjected to histopathological evaluation and to confirm RIRI-induced immunohistochemical aspects of apoptosis, terminal-deoxynucleotidyl-transferase mediated-deoxyuridine-triphosphate-nick-end-labeling assay and caspase-3 were assessed. Tissue oxidative stress, histopathologic changes, apoptosis scores and quantitative scintigraphic parameters were significantly higher in Group IR compared with Group Sham. Although tissue oxidative stress levels and histopathologic changes were not significant, quantitative scintigraphic parameters of contralateral kidney of Group IR were significantly increased. Compared with Group IR, Group CoQ10+IR presented decreased tissue oxidative stress levels; decreased scores of histopathology and apoptosis; and decreased quantitative scintigraphic parameters with increased split renal function in ischemic kidney. Our results suggest that other than its antioxidant properties, CoQ10 shows antiperoxidative, antiapoptotic and antiinflammatory potential in protecting renal functioning of ischemic kidney. Furthermore, our results show that renal scintigraphy is a feasible method to detect early changes in renal functioning after RIRI.

Astragaloside IV protects rat renal ischemia-reperfusion injury by regulating Th1/Th2 imbalance

Objective To investigated the protective effect and immunology mechanism of Astragaloside Ⅳ(Ast) on rat renal ischemia-reperfusion injury model. Methods The SD rats were randomly divided into four group as the control, sham-operation, renal ischemia-reperfusion injury, and Ast treatment groups. Rat serum and urine were collected and detective for kidney function and interleukin cytokines. The kidney tissue was collected for histology exam. The rats in astragaloside group were intraperitoneally injected with 100 mg/kg astragaloside, and the other three groups were intraperitoneally injected with an equal volume of normal saline. The models of renal ischemia-reperfusion injury were prepared to generate in model and astragaloside groups, after 30 minutes of astragaloside injection. The rats with renal ischemia-reperfusion injury model were sacrificed after 24 hours, and the level of blood-urine creatinine, neutrophil gelatinase-associated lipocalin and Kidney damage molecule-1 were determined. The level of Th1 type cytokines (TNF-α, IFN-γ, IL-2) and Th2 type cytokines (IL-4, IL-5, IL-10) in serum were measured by using ELISA. The protein and gene expression of TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-10 in renal tissue were tested by western blot and PCR, respectively. The pathological changes and apoptosis of renal tissue in each group were detected by HE staining and TUNEL staining, respectively. The expression of CD20 protein in renal tissue was determined by immunohistochemistry. Results Compared with the model group, Ast treatment reduced serum creatinine (58.74 ± 9.44 μmol/L vs. 85.03 ± 23.48 μmol/L), increased creatinine clearance rate (0.81 ± 0.13 ml/min vs. 0.37 ± 0.08 ml/min), and reduce urine neutrophil gelatinase-associated lipocalin (NGAL) (579.34 ± 11.70 pg/ml vs. 827.60 ± 14.48 pg/ml), kidney injury molecule-1 (KIM-1) (105.06 ± 2.10 pg/ml vs. 151.67 ± 3.06 pg/ml) (P<0.05). Compared with the model group, Ast treatment alleviated renal tubular epithelial cell injury and significantly decreased the apoptosis (14.36 ± 1.36% vs. 28.63 ± 2.03%) (P < 0.05), and significantly decreased the serum TNF-α (361.44 ± 9.66 pg/ml vs. 515.93 ± 10.61 pg/ml), IFN-γ (64.11 ± 1.21 pg/ml vs. 93.51 ± 2.15 pg/ml), IL-2 (388.33 ± 1.21 pg/ml vs. 557.82 ± 15.29 pg/ml), IL-4 (60.89 ± 1.21 pg/ml vs. 95.56 ± 2.75 pg/ml), IL-5 (94.02 ± 2.81 pg/ml vs. 147.07 ± 3.50 pg/ml), and IL-10 (52.62 ± 2.51 pg/ml vs. 78.22 ± 3.24 pg/ml) (P<0.01). Compared with the model group, Ast treatment significantly decreased the kidney TNF-α mRNA (1.89 ± 0.59 vs. 2.87 ± 0.97), IFN-γ mRNA (3.11 ± 1.02 vs. 5.98 ± 1.52), IL-2 mRNA (1.68 ± 0.44 vs. 4.09 ± 1.65), IL-4 mRNA (2.41 ± 0.81 vs. 4.69 ± 1.62), IL-5 mRNA (1.56 ± 0.19 vs. 2.92 ± 0.55), IL-10 mRNA (1.45 ± 0.14 vs. 2.85 ± 0.32) (P<0.01). The ratio of IL-4 to IFN-γ was basically restored to the level of sham operation group (1.05 ± 0.02 vs. 1.02 ± 0.06) (P<0.01), and CD20 cells in renal tissue was reduced. Conclusions The Th1 and B lymphocytes play an important role in renal ischemia reperfusion injury, and Th2 cells play a protective role. Astragaloside can regulate the imbalance of Th1/Th2 in the early stage after acute renal injury, and alleviate renal tubular injury. Key words: Reperfusion injury; Astragaloside Ⅳ; Th1-Th2 balance; Antigens, CD20; Rats

MiRNA profiling of urinary exosomes to assess the progression of acute kidney injury

Because exosomes have gained attention as a source of biomarkers, we investigated if miRNAs in exosomes (exo-miRs) can report the disease progression of organ injury. Using rat renal ischemia-reperfusion injury (IRI) as a model of acute kidney injury (AKI), we determined temporally-released exo-miRs in urine during IRI and found that these exo-miRs could reliably mirror the progression of AKI. From the longitudinal measurements of miRNA expression in kidney and urine, we found that release of exo- miRs was a regulated sorting process. In the injury state, miR-16, miR-24, and miR-200c were increased in the urine. Interestingly, expression of target mRNAs of these exo-miRs was significantly altered in renal medulla. Next, in the early recovery state, exo-miRs (miR-9a, miR-141, miR-200a, miR-200c, miR-429), which share Zeb1/2 as a common target mRNA, were upregulated together, indicating that they reflect TGF-β-associated renal fibrosis. Finally, release of exo-miRs (miR-125a, miR-351) was regulated by TGF-β1 and was able to differentiate the sham and IRI even after the injured kidneys were recovered. Altogether, these data indicate that exo-miRs released in renal IRI are associated with TGF-β signaling. Temporal release of exo-miRs which share targets might be a regulatory mechanism to control the progression of AKI.

Study on acute kidney injury model induced by renal ischemia-reperfusion in rats

In this study,in-depth systematic evaluation of rat of acute kidney injury(AKI) caused by renal arteriovenous ligation was conducted to better master and apply this model for drug research. Male SD rats of 2-3 months old were employed in this study.The left kidney was removed,and the right kidney received ligation for 40 min and reperfusion for 24 h. Serum creatinine(Crea),urea nitrogen(BUN) and the renal tissue sections were assayed as the basic indicators to evaluate their renal function. The mRNA expression of inflammatory necrosis factors and apoptotic factors was used to evaluate the mechanism of molecular pathophysiological changes. The results showed that the serum Crea and BUN caused by ligation of both renal arteries and veins were significantly higher than those of rats with renal artery ligation. After renal arteriovenous ligation for 40 min and reperfusion for 24 h in rats,the serum Crea of the rats varied from less than 100 μmol·L-1 to more than 430 μmol·L-1. Among them,5 rats showed less than 100 μmol·L-1 serum Crea,20 rats with 100-200 μmol·L-1 serum Crea and 12 rats with more than 430 μmol·L-1. Rats with serum Crea between 300-430 μmol·L-1 accounted for 66.3%(122/184) of the total number of the experiment rats. After 72 h reperfusion,serum Crea in the group of Crea 370-430 μmol·L-1 continued to increase,while the serum Crea in the group of Crea 200-300 μmol·L-1 and the group of Crea 300-370 μmol·L-1 recovered quickly. No matter serum Crea was elevated or decreased,the renal tubules showed pathological changes such as vacuolar degeneration or even necrosis. The mRNA expression levels of Toll-like receptor(TLR4),tumor necrosis factor(TNF-α) and interleukin(IL-6) in renal tissueswere significantly up-regulated,and the effect was most obvious in the group of serum Crea 370-430 μmol·L-1. The study indicated that the model for AKI caused by renal arteriovenous ligation and reperfusion is easy to operate,and the serum Crea and BUN have the characteristics of continuous increase,beneficial to the observation of drug effects. This acute kidney injury is mainly related to the pathophysiological response of inflammatory necrosis.

Characterization of urinary exosomal release of aquaporin-1 and -2 after renal ischemia-reperfusion in rats.

Acute kidney injury (AKI) is an important risk factor for the development of chronic kidney disease (CKD), and an alteration in renal water handling has been observed during the transition of AKI to CKD. Urinary exosomal release of aquaporin-1 (AQP1) and AQP2, important proteins for renal water handling, has recently been reported to predict their levels of renal expression. Therefore, we examined the patterns of urinary exosomal release of AQP1 and AQP2, and the exosomal marker proteins tumor susceptibility 101 protein (TSG101) and ALG-2 interacting protein X (Alix), in the acute and chronic phases following induction of AKI by renal bilateral ischemia/reperfusion (I/R) in rats. Blood tests and histological examinations indicated that AKI occurred before at 7 days after renal I/R ( day 7) and that renal fibrosis developed progressively thereafter. Immunoblotting demonstrated significant decreases in the urinary exosomal release of AQP1 and AQP2 during severe AKI. Urinary exosomal release of Alix and TSG101 was significantly increased on day 7. These data were also confirmed in rats with unilateral renal I/R causing more serious AKI. Urinary exosomal release of either the Ser-256- or Ser-269-phosphorylated form of AQP2, both of which are involved in apical trafficking of AQP2, was positively correlated with that of total AQP2. These results suggest that urinary exosomal release of AQP1 and AQP2 is reduced in I/R-induced AKI, whereas that of Alix and TSG101 is increased in the initial phase of renal fibrosis. Furthermore, apical trafficking of AQP2 appears to be related to urinary exosomal release of AQP2.

Effects of Platelet-Rich Plasma (PRP) on a Model of Renal Ischemia-Reperfusion in Rats.

Renal ischemia-reperfusion injury is a major cause of acute renal failure, causing renal cell death, a permanent decrease of renal blood flow, organ dysfunction and chronic kidney disease. Platelet-rich plasma (PRP) is an autologous product rich in growth factors, and therefore able to promote tissue regeneration and angiogenesis. This product has proven its efficacy in multiple studies, but has not yet been tested on kidney tissue. The aim of this work is to evaluate whether the application of PRP to rat kidneys undergoing ischemia-reperfusion reduces mid-term kidney damage. A total of 30 monorrenal Sprague-Dawley male rats underwent renal ischemia-reperfusion for 45 minutes. During ischemia, PRP (PRP Group, n = 15) or saline solution (SALINE Group, n = 15) was administered by subcapsular renal injection. Control kidneys were the contralateral organs removed immediately before the start of ischemia in the remaining kidneys. Survival, body weight, renal blood flow on Doppler ultrasound, kidney weight, kidney volume, blood biochemistry and histopathology were determined for all subjects and kidneys, as applicable. Correlations between these variables were searched for. The PRP Group showed significantly worse kidney blood flow (p = 0.045) and more histopathological damage (p<0.0001). Correlations were found between body weight, kidney volume, kidney weight, renal blood flow, histology, and serum levels of creatinine and urea. Our study provides the first evidence that treatment with PRP results in the deterioration of the kidney’s response to ischemia-reperfusion injury.

The protective effects of prostaglandin E1 on lung injury following renal ischemia–reperfusion in rats

For the purposes of the present study, the protective effect of prostaglandin E1 (PGE1) on lung injury following renal ischemia-reperfusion (RIR) was investigated. Adult male rats were divided into four groups, namely, (I) control rats given physiological saline; (II) rats given PGE1 (20 μg/kg, intravenously); (III) rats subjected to RIR; and (IV) rats subjected to RIR given PGE1 30 min prior to ischemia and just before reperfusion. The right nephrectomy was performed in the RIR model. The left renal pedicle was occluded for 60 min to induce ischemia and then the left kidney was subjected to reperfusion for 60 min. The lungs of rats were used for microscopic and biochemical analyses. Although rats subjected to RIR did not exhibit heavy degenerative alterations in the lung structure, they possessed pulmonary interstitial edema. Lung glutathione levels and catalase, superoxide dismutase, glutathione peroxidase, and tissue factor (TF) activities were decreased in rats subjected to RIR, while lung lipid peroxidation, myeloperoxidase (MPO), xanthine oxidase and serum lactate dehydrogenase (LDH) activities, and blood urea and serum creatinine levels were increased in these rats when compared with the control group. PGE1 treatments resulted in the regression of oxidative stress via induction of antioxidant system, the decreased MPO and LDH activities, the reduced urea and creatinine levels, and the induced TF activity in rats subjected to RIR, while edema still remained permanent. We conclude that PGE1 may be useful in preventing lung injury with the exception of edema that occurred as a result of RIR in rats.

Intermedin protects against renal ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress.

BackgroundIntermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide family. Endoplasmic reticulum stress (ERS) has been implicated in the pathology of renal ischemia/reperfusion (IRI). In the present study, we investigated whether IMD could reduce ERS damage after renal ischemia.MethodsThe kidneys of SD rats were subjected to 45 min of warm ischemia followed by 24 h of reperfusion. The hypoxia/reoxygenation(H/R) model in NRK-52E cells consisted of hypoxia for 1 h and reoxygenation for 2 h. IMD was over-expressed in vivo and in vitro using the vector pcDNA3.1-IMD. The serum creatinine concentration and lactate dehydrogenase (LDH) activity in the plasma were determined. Histologic examinations of renal tissues were performed with PAS staining. Real-time PCR and Western blotting were used to determine the mRNA and protein levels, respectively. Additionally, ER staining was used to detect the ERS response.ResultsIn the rat renal IRI model, we found that IMD gene transfer markedly improved renal function and pathology and decreased LDH activity and cell apoptosis compared with the kidneys that were transfected with the control plasmid. IMD significantly attenuated the ERS stress parameters compared with IRI group. Indeed, IMD down-regulated glucose-regulated protein 78 (GRP78), C/EBP homologous protein(CHOP), and caspase 12 protein and mRNA levels. Moreover, in the NRK-52E cell H/R model, IMD overexpression prevented the apoptosis induced by H/R. Furthermore, IMD ameliorated the ER structural changes and concomitantly decreased the levels of GRP78, CHOP and caspase-12.ConclusionThis study revealed that IMD protects against renal IRI by suppressing ERS and ERS-related apoptosis.

Effect of isoflurane preconditioning on cell apoptosis during renal ischemia-reperfusion in rats

Objective To evaluate the effect of isoflurane preconditioning on cell apoptosis during renal ischemia-reperfusion (I/R) in rats. Methods Thirty male Sprague-Dawley rats, aged 12-14 weeks, weighing 300-320 g, were randomly divided into 3 groups (n = 10 each) using a random number table: sham operation group (group S); I/R group; isoflurane preconditioning plus I/R group (group Iso+ I/R). The rats were anesthetized with intraperitoneal pentobarbital sodium 30 mg/kg.To establish the model of renal I/R injury, the right kidney was removed, and the left renal pedicle was occluded for 30 min with atraumatic mini-clamp for 30 min, followed by 2 h reperfusion.In Iso+ I/R group, 1.5% isoflurane was inhaled for 1 h, followed by 30 min of washout before I/R.In S and I/R groups only oxygen 2 L/min was inhaled for 1 h. Arterial blood samples were taken at 2 h of reperfusion to determine the concentrations of serum creatinine (Cr), blood urea nitrogen (BUN) and cystatin C (CysC). The animals were then sacrificed, and left kidneys were sampled for determination of the cell apoptosis (by TUNEL), expression of p53, Bax and Bcl-2 mRNA (using real-time fluorescent quantitative PCR), and expression of Bax, Bcl-2, and caspase-3 (by Western blot analysis). The Bcl-2/Bax ratio was calculated. Results Compared with group S, the serum Cr, BUN, and CysC concentrations were significantly increased, the Bcl-2/Bax ratio was decreased, Bcl-2 mRNA, Bax mRNA, Bcl-2, Bax and caspase-3 expression was up-regulated, and AI was increased in group I/R.Compared with group I/R, the serum Cr, BUN, and CysC concentrations were significantly decreased, the Bcl-2/Bax ratio was increased, Bcl-2 mRNA, Bax mRNA, Bax and caspase-3 expression was down-regulated, and AI was decreased in group Iso+ I/R.There was no significant difference in p53 mRNA expression among the three groups. Conclusion Regulating the balance between Bcl-2 and Bax and inhibiting apoptosis in kidney cells are involved in the mechanism by which isoflurane preconditioning reduces renal I/R injury in rats. Key words: Isoflurane; Ischemic preconditioning; Apoptosis; Kidney; Reperfusion injury

Effect of remifentanil on Toll-like receptor 2 mRNA expression during renal ischemia-reperfusion in rats

Objective To evaluate the effect of remifentanil on Toll-like receptor 2 (TLR2) mRNA expression during renal ischemia-reperfusion (I/R) in rats. Methods Fifty-four male Sprague-Dawley rats, weighing 200-250 g, were randomly divided into 3 groups (n= 18 each) using a random number table: sham operation group (group S), I/R group and remifentanil group (group R). Renal I/R injury was produced by clamping the bilateral renal arteries for 45 min followed by reperfusion in I/R and R groups.Bilateral renal arteries were only exposed but not clamped in group S. Remifentanil 1.0 μg·kg-1·min-1 was infused via the tail vein starting from 15 min before ischemia until 30 min of reperfusion in group R, while the equal volume of normal saline was given instead in S and I/R groups.The animals were sacrificed at 15 min before ischemia and 6 and 24 h of reperfusion, and the renal specimens were obtained for examination of the pathological changes (with light microscope) and for determination of the contents of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) (by ELISA) and expression of TLR2 mRNA (by RT-PCR) and cell apoptosis (by double staining and flow cytometry). The apoptotic rate was calculated. Results Compared with group S, TLR2 mRNA expression was significantly up-regulated, and the contents of TNF-α and IL-6 and apoptotic rate were increased at 6 and 24 h of reperfusion in I/R and R groups.Compared with group I/R, TLR2 mRNA expression was significantly down-regulated, and the contents of TNF-α and IL-6 and apoptotic rate were decreased at 6 and 24 h of reperfusion in group R. The pathological changes were significantly attenuated in group R as compared with group I/R. Conclusion The mechanism by which remifentanil reduces renal I/R injury is related to down-regulation of TLR2 expression and decrease in TLR2 activity and inhibition of inflammatory responses in renal tissues and cell apoptosis in rats. Key words: Piperidines; Reperfusion injury; Kidney; Toll-like receptor-2